Food Microbiology and Food Safety

Research and Development

Vijay K. Juneja
Hari P. Dwivedi
John N. Sofos Editors

Microbial
Control
and Food
Preservation
Theory and Practice
Food Microbiology and Food Safety

Series Editor:
Michael P. Doyle

More information about this series at http://www.springer.com/series/7131

 Food Microbiology and Food Safety Series

The Food Microbiology and Food Safety series is published in conjunction with
the International Association for Food Protection, a non-profit association for
food safety professionals. Dedicated to the life-long educational needs of its
Members, IAFP provides an information network through its two scientific jour-
nals (Food Protection Trends and Journal of Food Protection), its educational
Annual Meeting, international meetings and symposia, and interaction between
food safety professionals.

Series Editor

Michael P. Doyle, Regents Professor of Food Microbiology (Retired), Center for

Food Safety, University of Georgia, Griffith, GA, USA

Editorial Board

Francis F. Busta, Director, National Center for Food Protection and Defense,
University of Minnesota, Minneapolis, MN, USA
Patricia Desmarchelier, Food Safety Consultant, Brisbane, Australia
Jeffrey Farber, Food Science, University of Guelph, ON, Canada
Vijay Juneja, Supervisory Lead Scientist, USDA-ARS, Philadelphia, PA, USA
Manpreet Singh, Department of Food Sciences, Purdue University, West Lafayette,
IN, USA
Ruth Petran, Vice President of Food Safety and Pubic Health, Ecolab, Eagan,
MN, USA
Elliot Ryser, Department of Food Science and Human Nutrition, Michigan State
University, East Lansing, MI, USA
Vijay K. Juneja • Hari P. Dwivedi • John N. Sofos
Editors

Microbial Control and Food

Preservation
Theory and Practice
Editors
Vijay K. Juneja Hari P. Dwivedi
Residue Chemistry and Predictive BioMerieux, Inc.
Microbiology Research Unit Hazelwood, MO, USA
Eastern Regional Research Center
USDA-Agricultural Research Service
Wyndmoor, PA, USA

John N. Sofos
Department of Animal Sciences
Center for Meat Safety and Quality
Colorado State University
Fort Collins, CO, USA

Food Microbiology and Food Safety

ISBN 978-1-4939-7554-9    ISBN 978-1-4939-7556-3 (eBook)
https://doi.org/10.1007/978-1-4939-7556-3

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Contents

1 Food Preservation and Safety ����������������������������������������������������������������    1

Azlin Mustapha and Jee Hye Lee
2 Principles of Food Preservation��������������������������������������������������������������   17
Sudarsan Mukhopadhyay, Dike O. Ukuku, Vijay K. Juneja,
Balunkeswar Nayak, and Modesto Olanya
3 Application of Omics Technologies and Computational
Approaches for Control of Foodborne Pathogens in Foods ����������������   41
Jayanthi Gangiredla, Xianghe Yan, Isha R. Patel,
and Mark K. Mammel
4 Natural Food Antimicrobials of Animal Origin������������������������������������   55
Elba Verónica Arias-Rios, Elisa Cabrera-Díaz,
Mayra Márquez-González, and Alejandro Castillo
5 Antimicrobials of Plant Origin ��������������������������������������������������������������   85
Dinesh Babu, Kalpana Kushwaha, Shalini Sehgal,
and Vijay K. Juneja
6 Natural Food Antimicrobials of Microbial Origin�������������������������������� 101
Shalini Sehgal and Vasudha Sharma
7 Antimicrobial Peptides and Polyphenols: Implications
in Food Safety and Preservation������������������������������������������������������������ 117
Amardeep Singh Virdi and Narpinder Singh
8 Delivery Systems for Introduction of Natural
Antimicrobials into Foods ���������������������������������������������������������������������� 153
Shalini Mishra and Kanika Bhargava
9 Microbial Resistance to Antimicrobials������������������������������������������������ 173
Sean Pendleton and P. Michael Davidson

v
vi Contents

10 Interventions for Fresh Produce������������������������������������������������������������ 199

Govindaraj Dev Kumar, Sadhana Ravishankar,
and Vijay K. Juneja
11 Preservation Methods for Meat and Poultry���������������������������������������� 225
Jarret D. Stopforth
12 Microbial Control of Milk and Milk Products�������������������������������������� 255
Mustafa Guzel and Yesim Soyer
13 Microbial Fermentation in Food Preservation�������������������������������������� 281
Ilenys M. Pérez-Díaz, Evrim Gunes Altuntas,
and Vijay K. Juneja
14 Non-thermal Methods for Food Preservation �������������������������������������� 299
Lynette E. Orellana, María de Lourdes Plaza,
Fernando Pérez, Yarilyn Cedeño, and Oscar Perales
15 Antimicrobial Gases for Food Application�������������������������������������������� 327
David Kasler and Ahmed E. Yousef
16 Current State of the Art and Recent Innovations
for Antimicrobial Food Packaging �������������������������������������������������������� 349
Tony Z. Jin
17 Consumer Perception of Food Preservation Techniques���������������������� 373
Christine M. Bruhn
18 Statistical Derivation of Sampling Plans
for Microbiological Testing of Foods������������������������������������������������������ 381
Ursula Gonzales-Barron and Vasco Cadavez
19 Antimicrobials and Food Preservation:
A Risk Assessment Approach������������������������������������������������������������������ 413
Daniele F. Maffei, Bernadette D.G.M. Franco,
and Donald W. Schaffner

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Contributors

Evrim Gunes Altuntas Ankara University, Biotechnology Institute Central

Laboratory, Ankara, Turkey
Elba Verónica Arias-Rios Department of Nutrition and Food Science, Texas
A&M University, College Station, TX, USA
Dinesh Babu Earthbound Farm, The WhiteWave Foods Company, San Juan
Bautista, CA, USA
Kanika Bhargava Department of Human Environmental Sciences, University of
Central Oklahoma, Edmond, OK, USA
Christine M. Bruhn Center for Consumer Research, Cooperative Extension
Specialist Emerita, University of California, Davis, CA, USA
Elisa Cabrera-Díaz Departamento de Salud Pública, Centro Universitario de
Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Zapopan,
Jalisco, Mexico
Vasco Cadavez CIMO Mountain Research Centre, School of Agriculture (ESA)
of the Polytechnic Institute of Braganza (IPB), Bragança, Portugal
Alejandro Castillo Department of Animal Science, Texas A&M University,
College Station, TX, USA
Yarilyn Cedeño Department of Engineering Science and Materials College of
Engineering, University of Puerto Rico at Mayaguez, Mayagüez, Puerto Rico
P. Michael Davidson Department of Food Science and Technology, University of
Tennessee, Knoxville, TN, USA
Govindaraj Dev Kumar Department of Plant Science & Landscape Architecture,
University of Maryland, College of Agriculture and Natural Resources, College
Park, MD, USA

vii
viii Contributors

Bernadette D.G.M. Franco Food Research Center, Department of Food and

Experimental Nutrition, School of Pharmaceutical Sciences, University of Sao
Paulo, Sao Paulo, SP, Brazil
Jayanthi Gangiredla Division of Molecular Biology, U.S. Food and Drug
Administration, Center for Food Safety and Applied Nutrition, Office of Applied
Research and Safety Assessment, Laurel, MD, USA
Ursula Gonzales-Barron CIMO Mountain Research Centre, School of Agriculture
(ESA) of the Polytechnic Institute of Braganza (IPB), Bragança, Portugal
Mustafa Guzel Department of Food Engineering, Hitit University, Corum, Turkey
Tony Z. Jin Residual Chemistry and Predictive Microbiology Research Unit,
Eastern Regional Research Center, USDA-Agricultural Research Service,
Wyndmoor, PA, USA
Vijay K. Juneja Residue Chemistry and Predictive Microbiology Research Unit,
Eastern Regional Research Center, USDA-Agricultural Research Service,
Wyndmoor, PA, USA
David Kasler Department of Food Science and Technology, The Ohio State
University, Columbus, OH, USA
Kalpana Kushwaha Food Safety Professional, Gilroy, CA, USA
Jee Hye Lee Department of Food and Nutrition, University of Ulsan, Ulsan,
Republic of Korea
María de Lourdes Plaza Program of Food Science and Technology, University of
Puerto Rico at Mayagüez, Mayagüez, Puerto Rico
Daniele F. Maffei Food Research Center, Department of Food and Experimental
Nutrition, School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo,
SP, Brazil
Mark K. Mammel Division of Molecular Biology, U.S. Food and Drug
Administration, Center for Food Safety and Applied Nutrition, Office of Applied
Research and Safety Assessment, Laurel, MD, USA
Mayra Márquez-González Food Science and Technology Department, Zamorano
University, Tegucigalpa, Honduras
Shalini Mishra Department of Agricultural and Biosystems Engineering, South
Dakota State University, Manteca, CA, USA
Sudarsan Mukhopadhyay Residue Chemistry and Predictive Microbiology
Research Unit, Eastern Regional Research Center, USDA-Agricultural Research
Service, Wyndmoor, PA, USA
Contributors ix

Fernando Pérez Agricultural and Biosystems Engineering Department, University

of Puerto Rico at Mayagüez, Mayagüez, Puerto Rico
Azlin Mustapha Food Science Program, University of Missouri, Columbia, MO,
USA
Balunkeswar Nayak Food Science and Human Nutrition, School of Food &
Agriculture, University of Maine, Orono, ME, USA
Modesto Olanya Food Safety Intervention Technologies Research Unit, Eastern
Regional Research Center, USDA-Agricultural Research Service, Wyndmoor, PA,
USA
Lynette E. Orellana Program of Food Science and Technology, University of
Puerto Rico at Mayagüez, Mayagüez, Puerto Rico
Isha R. Patel Division of Molecular Biology, U.S. Food and Drug Administration,
Center for Food Safety and Applied Nutrition, Office of Applied Research and
Safety Assessment, Laurel, MD, USA
Sean Pendleton Fort Worth, TX, USA
Oscar Perales Department of Engineering Science and Materials, University of
Puerto Rico at Mayaguez, Mayaguez, Puerto Rico
Ilenys M. Pérez-Díaz United States Department of Agriculture, Agricultural
Research Service, Food Science Research Unit, Raleigh, NC, USA
Sadhana Ravishankar School of Animal & Comparative Biomedical Sciences,
University of Arizona, Tucson, AZ, USA
Donald W. Schaffner Department of Food Science, School of Environmental and
Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick,
NJ, USA
Shalini Sehgal Department of Food Technology, Bhaskaracharya College of
Applied Sciences, University of Delhi, New Delhi, India
Vasudha Sharma Department of Food Technology, Jamia Hamdard (Hamdard
University), New Delhi, India
Narpinder Singh Department of Food Science & Technology, Guru Nanak Dev
University, Amritsar, Punjab, India
Yesim Soyer Department of Food Engineering, Middle East Technical University
(ODTU), Ankara, Turkey
Jarret D. Stopforth Chobani, LLC, New York, NY, USA
Dike O. Ukuku Food Safety Intervention Technologies Research Unit, Eastern
Regional Research Center, USDA-Agricultural Research Service, Wyndmoor,
PA, USA
x Contributors

Amardeep Singh Virdi Department of Food Science & Technology, Guru Nanak
Dev University, Amritsar, Punjab, India
Xianghe Yan U.S. Department of Agriculture, Environmental Microbial and Food
Safety Laboratory, USDA Agricultural Research Service (ARS), Beltsville, MD,
USA
Ahmed E. Yousef Department of Food Science and Technology, The Ohio State
University, Columbus, OH, USA
About the Editors

Vijay K. Juneja Dr. Vijay K. Juneja is a lead scientist (microbiologist, GS-15) of

a research project on predictive microbiology at the Eastern Regional Research
Center, ARS, USDA, Wyndmoor, PA, USA. He has developed a nationally and
internationally recognized research program on foodborne pathogens, with empha-
sis on microbiological safety of minimally processed foods and predictive microbi-
ology. His research program has been highly productive, generating over 170
peer-reviewed journal articles and 45 book chapters, and he is a coeditor of 8 books.
Currently, Dr. Juneja serves as an editor of LWT-Food Science and Technology. He
served as a coeditor of the International Journal of Food Microbiology until
December 2011 and as an associate editor for the “Food Microbiology Section” of
the Journal of Food Science from 2002 to 2007. A certified food scientist (2013), Dr.
Juneja is a fellow of the IFT (2008), American Academy of Microbiology (2013),
and IAFP (2107).

Hari P. Dwivedi Before his current position as senior manager of clinical affairs
at bioMerieux, Inc., in Hazelwood, MO, Hari P. Dwivedi was a US R&D food man-
ager supporting the research and development activities on microbial quality indica-
tors and pathogen detection in food. Hari is a veterinarian by training and did his
PhD at North Carolina State University while working on the molecular methods
for foodborne pathogen detection. Hari is an active member of the International
Association for Food Protection (IAFP), where he most recently served as the chair
of the Applied Laboratory Methods Professional Development Group. Hari is also a
current member of the Food Safety and Technology Council of United Fresh and a
past member of the executive committee of the Indian Society for Veterinary
Medicine (ISVM). He serves as a member of the editorial board of the International
Journal of Food Microbiology and Food Protection Trends.

John N. Sofos John N. Sofos is a distinguished professor emeritus of Colorado

State University. He retired in 2015, after serving on 103 graduate committees and
working with 38 scholars. He taught food processing, food microbiology, etc. His
research interests included ecology, detection, resistance, and control of bacterial

xi
xii About the Editors

pathogens. He authored 324 papers, 10 books, 72 book chapters, 462 abstracts, and
380 miscellaneous publications, and participated in 210 invited lectures worldwide.
He was elected fellow of five scientific societies and received awards from the
International Association for Food Protection (IAFP), the American Meat Science
Association, the Institute of Food Technologists (IFT), etc. He served as editor of
the Journal of Food Protection for 18 years, as chair of the Biological Hazards Panel
of the European Food Safety Authority, and as president of the council of the
Agricultural University of Athens, Greece.
Chapter 1
Food Preservation and Safety

Azlin Mustapha and Jee Hye Lee

Abstract Food preservation has evolved from primitive, simple methods like sun
drying to sophisticated techniques like high pressure processing. Although the tech-
niques employed in food preservation vary drastically, the goal is one and the same,
which is to increase the safety and shelf-life of food. This, in turn, results in a con-
tinuous supply of nutritious, safe and palatable food for humans and animals glob-
ally. Food preservation can be achieved via chemical, physical or biological
treatments or modification of foods. This chapter will briefly introduce the concept
and history of food preservation, important considerations to be taken, its benefits
and some examples of food preservation techniques. Because food is marketed to
consumers, consumer perspectives of various food preservation techniques will also
be covered. Because of the fluidity of agricultural systems, global markets, climate
changes and market trends, the science and technology of food preservation will
continue to evolve.

Keywords Food safety • Shelf life • Chemical preservation • Physical preservation

• Biological preservation

1 Introduction

Food preservation has been an essential process in human civilization because it

allows for the production of a varied supply of food with longer shelf lives. Food
preservation methods have been developed alongside the evolution of civilization
and with the development of the food industry. Preservation methods aim to inhibit
the growth of microorganisms, maintain the appearance of the food and slow down
chemical reactions that result in product deterioration. In this chapter, the purpose,

A. Mustapha (*)
Food Science Program, University of Missouri, Columbia, MO, USA
e-mail: [email protected]
J.H. Lee
Department of Food and Nutrition, University of Ulsan, Ulsan, Republic of Korea
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 1

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_1
2 A. Mustapha and J.H. Lee

significance and history of food preservation will first be introduced. Secondly,

some traditional and modern preservation methods will be briefly reviewed; and
finally, consumers’ perspectives on food preservation will be introduced.

2 What Is Food Preservation?

The practice of preserving food began in the prehistoric age when ancient humans
hunted animals and ate fresh animal meat. Acquired foods were sometimes stored
to prevent famine and different food preservation methods for improving food qual-
ity were attempted. For example, grains were sun-dried to extend their storage
period and fresh food were stored in cool, dry caves. About 400,000 years ago,
Peking Man (Homo erectus pekinensis) may have utilized cooking methods using
fire, such as grilling food over a fire to improve its flavor (Table 1.1). The main

Table 1.1 Historic events in food preservation (from Noh et al. (2009))
Year Summary
Before Natural drying agents, such as fire, wind, sun, seawater, and halite were used for
1500 preservation, along with cooking, grilling, roasting, and curing with salt or sugar; and
natural cool storage, such as caves, were mostly used. In 1492, American Indians were
using fire for drying pemmican
1600s A low-heat air-drying room for using the hot air artificial drying method was
developed
1780 In the UK, preconditioning with hot water for drying vegetables was performed
1795 In France, hot air was used for drying vegetables
1800s In 1804, Nicolas Appert (France) invented a canning method using wide-mouth
bottles. In 1810, Peter Durand (UK) discovered the tin can. In 1819, Thomas Kensett
(UK) started to can shrimp, salmon, and oysters
1875 Carl von Linde invented a compression refrigerating machine using ammonia for
making ice
1900s Canning was more developed in this period
1916 Rudolph Planck proposed a theoretical quick-freezing technique
1922 Franklin Kidd and Cyril West (England) began studying methods of gas storage
1929 Clarence Birdseye (USA) invented a quick-freezing system and began to make frozen
food
1942 During WWII, freezing, cooling, drying, canning, and bottling processing techniques
were further developed
1950 Vacuum concentration of liquid food and lyophilization of dehydrated food became
available
1953 In the US, studies into the application of radiation to food began
After Until 1950, processing techniques and equipment were being improved side-by-side,
1960 and through the incorporation of food chemicals and additives, the quality of
processed food similarly improved. Also, through the development of petro-chemistry,
many types of plastic products were developed for use as packaging materials or
containers. Electronic engineering, such as microwave ovens, thermoelectric cooling,
supersonic machining, etc. are applied to food processing
1 Food Preservation and Safety 3

functions of food preservation are to prevent food quality deterioration and to extend
the time between production and consumption so that the food could be stored for
longer periods. Food preservation aims to minimize the physical and chemical
changes that occur during storage, while at the same time, ensure food safety. Some
food preservation considerations (Bowers 1992; VanGarde and Woodburn 1994) are
as follows:
• Good food preservation must maintain the food’s acceptability. Food preserva-
tion is useless if adequate appearance, odor or taste of the food is not
maintained.
• Good food preservation must keep the food safe. Properly monitoring the food
during storage and evaluating it before consumption are good ways to ensure
food safety.
• Good food preservation must retain nutrients. Ensuring a minimal loss of nutri-
ents, including protein, carbohydrates, fibers, minerals, and vitamins during stor-
age is important.
• Good food preservation must be an asset to the food distribution chain. From an
operator’s viewpoint, practical uses of packaging materials are helpful for effec-
tiveness of food distribution.
• Good food preservation must be feasible and applicable. Food preservation is
practiced not only by food manufacturers but also by consumers, thus, a simple,
yet effective preservation technique, such as fermentation, is useful.
• Good food preservation must conserve available energy resources. Food preser-
vation methods should be evaluated based on the appropriateness of the preser-
vation process for a specific food and environmental condition.

3 Why Food Preservation Is Important

Food preservation techniques can be grouped into three categories: physical tech-
niques (e.g. drying, pasteurization, or freezing), chemical techniques (e.g. addition
of salt or sugar), and biological techniques (e.g. microbial fermentations) (Lück and
Jager 1997).
The objectives of food preservation are:
• to preserve quality.
• to eliminate pathogens.
• to eliminate or reduce spoilage-causing microorganisms.
• to extend the shelf-life of the food.
The outcomes of effective food preservation are:
• to overcome the restrictions of time and region (e.g. seasonality).
• to improve the palatability of the food (e.g. flavor, appearance, or texture).
• to improve nutrient availability in the food (e.g. protein).
4 A. Mustapha and J.H. Lee

The benefits gained from food preservation (Noh et al., 2009) are discussed
below:

3.1 Planned Manufacturing and Distribution

Food preservation allows for the planned production, processing and movement of
food according to seasonal, climatic and distribution demands because food can be
mass-produced without fear of the surplus decomposing.

3.2 Economically Favorable Containment and Distribution

The use of proper sizes and types of food containers in the food industry maximizes
the efficiency of food processing and distribution.

3.3 Prevention of Loss in Food Quality

Undesirable changes and decay of food are prevented by proper preservation pro-
cesses. These processes can preserve the quality of the food in terms of its nutri-
tional and organoleptic qualities, leading to greater economic benefits.

3.4 Stabilization of Price

Considering the nature of agriculture, stockbreeding, and fishing, some variations in

food supplies are expected, even with proper planning. A big harvest or haul results
in a decrease in price whilst a poor harvest or haul causes the opposite. Thus, main-
taining supplies of preserved food helps keep the price stable over time.

4 Food Preservation Methods

Current food preservation methods are designed to minimize undesirable changes in

the wholesomeness, sensorial qualities, and nutritional composition of food through
efficient and effective ways of controlling the growth of microorganisms. Food
preservation can be addressed via chemical, biological, and physical methods.
Chemical preservation methods can be applied by adding substances, such as sugar,
salts, acids, or smoke to food. Biological preservation is based on the principles of
1 Food Preservation and Safety 5

alcoholic or acidic fermentation. Physical preservation is accomplished by control-

ling the food products’ temperature and water activity (Aw), and by the use of pack-
aging. Physical methods are commonly used in commercial food preservation
around the world (Jang 2015).

5 Current Food Preservation Methods

5.1 Dehydration/Drying

Drying is one of the first techniques for preserving foods developed by humans.
Fresh foods become less perishable when their Aw is decreased. A decrease in Aw
usually translates to a delay in microbial growth and chemical reactions. The mini-
mum Aw limit for growth of most bacteria is 0.86 and that for most molds and yeast
is 0.61 (Fontana 2007). The principle of drying is the most simple, in that available
water is removed, which can be accomplished using natural methods, such as sun,
fire, or wind. Around 12,000 B.C., the Middle Eastern and Asian cultures com-
monly dried foods using the sun (Nummer 2002).
Various types of drying methods exist: sun drying, roast drying, hot air drying,
heated-surface drying, and vacuum drying. Sun drying is a method which is still
being widely used around the world. Roast drying is a process by which heat is
applied to dry foodstuffs and food are stirred as they are roasted to heat evenly. Hot
air drying is a method where water in the food is evaporated by hot air. Various types
of dryers can be used for hot air drying: bin dryer, cabinet dyer, conveyor dryer,
fluidized bed dryer, kiln dyer, spray dryer, tunnel dryer, and pneumatic dryer (Shinh
et al. 2009). When heat is applied to dry food using a heated surface, the process is
referred to as heated-surface drying. This process has a high thermal efficiency and
can be done without oxygen. On the other hand, vacuum drying for solid foods is a
process by which heat is applied to a sealed container with a pressure of 5–30 kPa
at 35–80 °C (Brennan 1994; Oakley 1997).
The application of drying preservation techniques results in changes to the
food, including a movement of water molecules from the internal parts of the
food to the surface, shrinkage of food volume, surface hardening, and loss of
vitamins (Lee 2013).

5.2 Chilling/Low Temperature Storage

Low temperature is also effective for the extension of food storage periods. The
deep parts of caves, dark cellars, and cool streams are still being used as cool stor-
age places in some countries (Kim et al. 2000). The refrigerator, which was invented
in the 1800s (Burstall 1963), is a major milestone in the development of food
6 A. Mustapha and J.H. Lee

preservation techniques because it effectively prevents the growth of microorgan-

isms, inhibits post-harvest metabolic activities in plant tissues and post-slaughter
metabolic activities in animal tissues, as well as deteriorative chemical reactions,
including enzyme-catalyzed oxidative browning or oxidation of lipids, autolysis in
fish, and other chemical changes that affect food quality (Shinh et al. 2009). Further,
refrigeration reduces the loss of nutrients and moisture, and helps satisfy consum-
ers’ needs for health and freshness.
Although the growth of thermophilic and mesophilic organisms can be inhibited
during low temperature storage, psychrotrophic bacteria can still proliferate. For
example, Vibrio and Acinetobacter can grow in fish, and Pseudomonas, Micrococcus
and Lactobacillus can grow in meat products during low temperature storage (Shinh
et al. 2009). Low temperature storage can also result in undesirable changes in food
quality after a certain amount of time. One of the examples is ‘chilling injuries’
which can occur in fresh foods, such as fruit and vegetables, if appropriate tempera-
tures are not maintained (Wang 1994).

5.3 Freezing

Ancient freezing methods were useful only in places with an appropriate climate,
viz. cold weather. In the late nineteenth century, Clarence Birdseye discovered that
freshly caught fish that was instantly frozen under thick ice, tasted fresh when
thawed, a condition referred to now as “quick freezing” (Birdgeye and Fitzgerald
1932). Freezing preserves food at a very low temperature, and prevents the growth
of microorganisms as well as changes caused by chemical reactions in the food.
During the freezing process, however, physical and chemical changes can still occur
in the food, including recrystallization, oxidation of lipids, enzymatic browning,
destruction of vitamin C, and rapid insolubility of proteins (Noh et al. 2009). Slow
freezing and thawing are more effective at decreasing microbial survival rates than
are quick freezing and thawing (Geigeis 1996).

5.4  ontrolled Atmosphere (CA) and Modified Atmosphere

C
(MA) Storage

Food preservation can be achieved through controlled and modified atmospheres of

the storage environment. An inhibition of microbial growth and a decrease in respi-
ration of fresh fruits and vegetables occur when the oxygen density in the storage
environment air is decreased and carbon dioxide concentration is increased (Brenna
and Day 2006). There are three ways to change the composition of air in a storage
environment (Shinh et al. 2009): (1) Controlled atmosphere storage (CAS) involves
regulation of oxygen, carbon dioxide, and ethylene gas, (2) Modified atmosphere
1 Food Preservation and Safety 7

storage (MAS) is when the storage environment is sealed so that the air is changed
by the natural food respiration, and (3) Modified-atmosphere packaging (MAP) is
done by sealing the product to change the composition of the air.

6 Emerging Food Preservation Methods

Emerging food preservation methods that use newer technologies have been devel-
oped to meet greater food market requirements for foods with better quality and
sensorial attributes, and containing fewer additives while maintaining a longer shelf
life. Some of these methods are discussed below.

6.1  igh Pressure Processing/High Hydrostatic Pressure/

H
Ultra High Pressure

High pressure processing (HPP), also called high hydrostatic pressure (HHP) or
ultra high pressure (UHP), has been used to kill bacteria since 1895 (Hite 1989;
Patterson et al. 2006). High pressures of 5–6 GPa or less have been applied in com-
mercial food applications (Crossland 1995). HPP at 100–800 MPa is usually
employed for liquid and solid foods without packaging within a range of tempera-
tures (<0 °C to >100 °C) for more than 20 min (Smith and Hui 2004). Pressure kills
various microorganisms, including spoilage-causing and pathogenic microorgan-
isms. Previous studies supported the antimicrobial effects of HPP on selected
microorganisms, such as Aspergillus awamori and Saccharomyces cerevisiae in
Satsuma mandarin juice (Ogawa et al. 1992), Listeria innocua in minced beef mus-
cle (Carlez et al. 1993), and Listeria monocytogenes, Vibrio parahaemolyticus, and
Salmonella Typhimurium in phosphate butter at pH 7.0 (Metrick et al. 1989;
Patterson et al. 1995; Styles et al. 1991). Although spore-forming bacteria, such as
Clostridium botulinum, can be inactivated by HPP at 500–700 MPa and 90-110 °C,
some types of C. botulinum spores) can survive (Smith and Hui 2004). HPP contrib-
utes to the retention of freshness and sensorial and nutritional attributes of the prod-
ucts, but it is quite costly at the initial establishment stage (Aleman et al. 1994;
Deplace 1995).

6.2 Ultrasound Treatment

Ultrasound is an oscillating pressure produced by ultrasonic sound waves at fre-

quencies of 20 kHz and higher (McClements 1995). In the food industry, ultrasound
treatment has been combined with other technologies. Low energy, high frequency
8 A. Mustapha and J.H. Lee

ultrasound (<1 W/cm2; >100 kHz) confers an antimicrobial effect in a broad range
of food matrices, including fats, milk, bread, fruits and sauces, while high energy,
low frequency ultrasound (10–10,001 W/cm2; 20 ~ 100 kHz) is employed to improve
the efficiency of various food processes (Torley and Bhandari 2007).
Ultrasound treatment is an emerging method that inactivates microorganisms by
disrupting cellular structural and functional components using ultrasonic waves
(Smith and Hui 2004). It has been shown to have antimicrobial effects on bacteria,
spores, yeasts, fungi, and viruses, but the sensitivity of microorganisms to ultra-
sound treatment differs, and some spores or viruses are less sensitive than Gram
positive and Gram negative bacteria to ultrasound (Earnshaw 1998; Earnshaw et al.
1995; Manas and Pagan 2005; Nakano et al. 1989; Raso and Barbosa-Canovas
2003; Ross et al. 2003; Palacios et al. 1991; Piyasena et al. 2003). Ultrasound treat-
ment at low temperature is less effective. In a study by Palacios et al. (1991),
Bacillus stearothermophilus spores were not affected by ultrasound treatment at
12 °C and C. botulinum spores showed the same results in a study by Nakano et al.
(1989).

6.3 Pulsed Electric Field

Pulsed electric field processing is an emerging non-thermal preservation method in

which a food is placed between two electrodes in a high-voltage electric field, typi-
cally 20–80 kV/cm−1 (Yang et al. 2004). This method reduces the metabolic activi-
ties of microorganisms but minimizes undesirable changes of sensorial properties of
the treated food, such as flavor, taste, and color, as well as nutrients (Mertens and
Knorr 1992; Knorr 1999; Ayhan et al. 2001, 2002) and functionality (Li et al. 2003).
The inactivation effects of pulsed electric field on food enzymes have also been
evaluated by previous studies. For example, Ho et al. (1997) showed that after a
specific pulsed electric field treatment, conducted at 13–87 kV/cm, 0.5 Hz, 2 s
pulse width and 30 pulses, significant reductions of lipase, glucose oxidase, and
amylase in foods occurred. The inactivation effect of pulsed electric field on sev-
eral microorganisms showed reductions of S. cerevisiae in orange juice at a field
strength of around 6.5 kV/cm−1, E. coli in milk at a field strength of around 21 kV/
cm−1, and Salmonella Dublin in milk at a field strength of around 18 kV/cm−1
(Sitzmann 1995).

7 Natural Antimicrobial Preservatives

Increasing consumer concerns about the use of chemical preservatives in foods

have generated greater demand for “naturally produced antimicrobial agents” and
“minimally processed” food (Cleveland et al. 2001). Thus, the food industry and
food scientists alike are currently expending more efforts to utilize useful
1 Food Preservation and Safety 9

antimicrobial agents from natural sources. Some promising options come from the
natural defense mechanisms or competitive abilities of certain plants and microor-
ganisms (Rahman 2007).
Two components of milk, lactoperoxidase and lactoferrin, possess antimicrobial
properties. Lactoperoxidase has been shown to have a bactericidal effect on Listeria
and Staphylococcus (Beumer et al. 1985a, b, 1988; Bibi and Bachmann 1990; Borch
et al. 1989; Denis and Ramet 1989; Earnshaw and Banks 1989; El-Shenawy et al.
1990; Kamau et al. 1990a, b; Siragusa and Johnson 1989; Wray and McLaren 1987),
and lactoferrin has an antibacterial effect on E. coli K-12 (Visca et al. 1990). Nisin,
which is produced by Lactococcus lactis, is a well-known antimicrobial protein.
Nisin has an antimicrobial effect on a wide range of Gram-positive bacteria and
some Gram-negative bacteria (Stevens et al. 1991, 1992). In addition, it also has a
similar effect on spores (Hall 1966).
Generally, bacterial starter cultures, such as Lactococcus, Leuconostoc,
Pediococcus and Lactobacillus have their own bactericidal effects on other organ-
isms. For example, Pediococcus FBB-61 inhibits strains of Bacillus cereus,
Clostridium perfringens, L. monocytogenes, Listeria spp., and S. aureus (Spelhaug
and Harlander 1989). Starter cultures are effective at inhibiting Gram-positive spoil-
age and pathogenic bacteria (Ray and Daeschel 1994). Dillon and Board (1994)
emphasized four desirable characteristics of starter cultures as biopreservatives.
First, they should be effective against many Gram-positive food spoilage-causing
and pathogenic bacteria, as well as spores. Second, they should be effective regard-
less of different food environments, and thirdly, they should provide economic
value but should not affect food quality.
Spices and essential oils also possess antimicrobial properties. Allicin, a diallyl
thiosulfinate in garlic, exhibits antimicrobial effects on a broad range of bacteria,
including Enterobacter, Escherichia, Klebsiella, Proteus, Pseudomonas,
Salmonella, Serratia, and Shigella (Srivastava et al. 1982; Stoll and Seebeck 1949).
Thyme and oregano have an inhibitory effect on the growth of V. parahaemolyticus
(Beuchat 1976; Deans and Ritchie 1987).
In addition, various antimicrobial compounds are found in food plants and veg-
etable oils. For example, the antibiotic influence of anthocyanins, leucoanthocyani-
dins, and phenolic acids on bacteria, such as Salmonella Typhi, Shigella spp., and E.
coli have been tested (Powers 1964). An antimicrobial effect of phenolic compounds
from olive extracts on Lactobacillus plantarum, S. aureus, and Salmonella
Enteritidis has been proven by several researchers (Etchells et al. 1966; Radford
et al. 1991; Ruiz-Barba et al. 1990, 1991; Nychas et al. 1990).

8 Consumer Perspectives on Food Preservation Methods

In the twenty-first century, modern food preservation techniques provide numerous

benefits to consumers, including an improvement in food safety, an extension of
food storage periods, prolonged shelf-life, availability of various food products
10 A. Mustapha and J.H. Lee

throughout the year, and convenient access to food products from different regions.
Further, food preservation techniques have advanced in response to consumer
trends. Consumer demands can be considered from two perspectives: (1) improved
food quality and (2) consumer health (Fox et al. 2002; Gayán et al. 2014; Grebitus
et al. 2013; Haugaard et al. 2014; Junqueira-Gonçalves et al. 2010; Zink 1997).
Consumers prefer fresh, attractive and hygienic qualities, including good color, fla-
vor, and odor. Consumers’ perception and evaluation of food quality (color and
shelf life) are important factors in purchasing decisions. In a study by Grebitus et al.
(2013), consumers preferred a bright red meat color and showed a willingness to
pay more for such meat. Modified atmosphere packaging with the inclusion of car-
bon monoxide (CO-MAP) contributes color stabilization to meat. Thus, a brighter
red color of fresh red meat is possible. Interestingly, however, this study showed that
consumers had a lower willingness to pay for red ground beef when they are given
more detailed information about CO-MAP technology. It might be that consumers
perceive the new technology as having its own health risks since most people have
been warned about the dangers of carbon monoxide in other situations.
In addition, consumers’ concerns for health show up in a number of different
ways. A demand for minimal use of chemical additives in food products is one of
them. In a study by Haugaard et al. (2014), consumers showed positive attitudes and
purchase intentions toward new preservation techniques that use natural preserva-
tives in food products.
Many irradiated food products, including meats, have been approved or
endorsed by several authorities, including the FDA, USDA, WHO, and FAO, and
scientists agree about the safety of irradiated food (CAST 1986). However, in a
study by Kwon et al. (1992), more than two-thirds of 600 Korean participants
exhibited fear about irradiated foods and were confused about the difference
between irradiated and radioactively contaminated food. In a survey conducted
by Junqueira-Gonçalves et al. (2010), the respondents perceived irradiated foods
as radioactive foods.
Novel food preservation technologies are developed and introduced in order to
satisfy consumers and food markets. However, the introduction of new food preser-
vation technologies for commercialized foods evokes fear that consumption might
carry possible risks. This concern is often seen in the case of irradiated food prod-
ucts (Fox et al. 2002; Gunes and Tekin 2006), which consumers believe will cause
short- or long-term health effects when consumed (Cardello 2003; Deliza et al.
2003; Cardello et al. 2007). Ample research pointed out that providing more infor-
mation about the process and advantages of irradiation resulted in positive attitudes
(Deliza et al. 2003; Ornellas et al. 2006; Gunes and Tekin 2006). The positive rela-
tionship between a higher level of knowledge and a tendency to buy irradiated food
was supported by Crowley et al. (2002). As the conceptual model (Fig. 1.1) shows,
food industry marketers should try to provide accurate information about new pres-
ervation technologies and promote their benefits since media exposure has a critical
influence on consumers’ attitudes.
1 Food Preservation and Safety 11

Knowledge

Characteristics of Risk and benefit

consumer perceptions

Communication Attitude toward

new technique

Acceptance or
rejection

Fig. 1.1 Conceptual framework for attitude formation toward new preservation techniques
(Haugaard et al. 2014)

9 Concluding Remarks

Food preservation and food safety are intricately intertwined. Although food preser-
vation is concerned with prolonging the shelf life of food by way of preventing the
growth of spoilage organisms or chemical reactions, often times, the same processes
also confer a safety aspect to foods by inhibiting pathogens from growing or elimi-
nating them altogether. Many preservation methods used by humans in ancient
times are still being used today. However, modern time food processing systems
have evolved into a high technology science. Because of the ever-changing way of
how we grow, process, market, transport, and consume our food, the science and art
of food preservation and food safety will continue to evolve to ensure safe and con-
sumable food for the world’s population.

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Chapter 2
Principles of Food Preservation

Sudarsan Mukhopadhyay, Dike O. Ukuku, Vijay K. Juneja,

Balunkeswar Nayak, and Modesto Olanya

Abstract Food preservation is an action or method used to maintain foods at certain

desirable properties or quality to obtain maximum benefit. A good method of food pres-
ervation is one that slows down or prevents altogether the action of the agents of spoilage
without damaging the food. To achieve this, certain basic methods are applied depending
on the food types. Food preservation has been an essential activity throughout human
history. The cycle of seasons brings periods of shortage and abundance of various foods
at different times of the year. Preservation makes it possible to consume some of these
foods during off seasons, throughout the year. Food preservation usually involves con-
trolling or preventing growth of microrganisms or minimizing the quality degradation
due to microbial spoilage or unwanted chemical changes in foods such as rancidity due
to oxidation of fats over time. Preservation of foods is no longer simple and straightfor-
ward today; it has evolved to a highly inter-disciplinary field of science. In recent years,
many new sophisticated preservation techniques have developed to extend the quality
and shelf-life, minimize risk, protect the environment, and improve functional, sensory,
and nutritional properties. Many of emerging preservation technologies have already

Mention of trade names or commercial products in this article is solely for the purpose of providing
specific information and does not imply recommendation or endorsement by the U.S. Department
of Agriculture. USDA is an equal opportunity provider and employee.
S. Mukhopadhyay (*) • V.K. Juneja
Residue Chemistry and Predictive Microbiology Research Unit, Eastern Regional Research
Center, USDA-Agricultural Research Service, Wyndmoor, PA, USA
e-mail: [email protected]; [email protected]
D.O. Ukuku • M. Olanya
Food Safety Intervention Technologies Research Unit, Eastern Regional Research Center,
USDA-Agricultural Research Service, Wyndmoor, PA, USA
e-mail: [email protected]; [email protected]
B. Nayak
Food Science and Human Nutrition, School of Food & Agriculture, University of Maine,
Orono, ME, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 17

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_2
18 S. Mukhopadhyay et al.

reached commercial adoption in specific applications while many others remain promis-
ing. Development of suitable equipment, especially for continuous processing for a vari-
ety of foods and standardization of the process parameters for easy regulatory approval
will pave the way for improved food preservation. The objective of this chapter was to
examine the science and technology involved in the manipulation of conventional as
well as sophisticated emerging preservation methods.

Keywords Thermal preservation • Advance thermal technology • Emerging

nonthermal preservation • Pulsed light • Membrane technology

1 Introduction

Preservation of foods involves controlling or preventing the growth of microorganisms

such as bacteria, fungi and yeasts, although in some instances food preservation can be
achieved by introducing benign microorganisms to the food. Preservation is also
achieved by retarding the oxidation of fats that cause rancidity. Processes that inhibit
visual deterioration of foods, for example the browning of apples due to enzymatic
reaction once apples are cut is also considered as food preservation. In some cases food
preservation involves a number of different food methods each of which independently
as a separate preservation technique. Preparation of Jam or Jelly, for example, involves
steps as heat treatment or boiling to lower the moisture content of fruit and to kill
microbes, etc., addition of sugar as preservative to control the re-growth of organisms
and lastly sealing of the food within an airtight container in order to stop recontamina-
tion of the final product. Many old preservation methods which been used traditionally
in the past have been proven to be less energy intensive and have lower carbon footprint
compared to modern intentions (Preserving food without freezing or canning 1999).
Quality maintenance, for example preservation of color, flavor, texture, micronutrients
and overall nutritional quality in an integral part of food preservation.

2 Advantage of Food Preservation

Food preservation has many advantages. It makes possible to prevent the spoilage
foods due to the action of inherent microorganisms and enzymes. Preservation
prolongs the duration of safe storage of foodstuffs and thereby creates its avail-
ability throughout the season and makes up for the deficiencies. Preservation also
makes food transportation much easier.
2 Principles of Food Preservation 19

3 Principles of Food Preservation

There are three main principles by which food preservation is achieved:

Controlling microorganisms. Microbial contamination may cause change that ren-
ders food unfit for human consumption. Primary sources of contamination are irri-
gation water, air, soil, animals, feed, and food processing equipments. Foods may be
contaminated by bacteria yeasts or molds at any stages of pre-harvest to post-har-
vest continuum. Inactivation or control of microbial population is achieved by
increasing or decreasing food temperature, adding salt, sugar or acid to food or by
removing the water or air from the food. Removing air from food inhibits oxygen
dependant enzymatic and chemical reactions and inhibits growth of aerobic micro-
organisms. Removal of moisture from produce such as leafy greens causes leaves to
dry which limits microbial growth.
Controlling enzymes. Enzymes play important role in living tissues of food. But
it catalyzes spoilage reactions following harvest or slaughter of plant or animal
based foods. In case of plant foods, certain enzymes remain active in the tissue
cells of fruits and vegetables following harvest and may continue to catalyze the
biochemical processes of ripening of these foods which may eventually lead to
rotting, as can be observed in many fruits. Oxidative enzymes in produce such
as banana or other fruits may continue to maintain cellular respiration by metab-
olizing sugar with oxygen for energy and may shorten the shelf life of fresh
fruits leading to spoilage. Respiration may be controlled by refrigerated storage
or modified-atmosphere packaging. Respiration may be controlled by refriger-
ated storage or modified-­atmosphere packaging.

3.1  ontrolling Insects, Rodents, Birds and Other Physical

C
Causes of Food Deterioration

Insects grow in humid warm environments and the types of foods subject to pest
attack are cereal grains and products derived from cereal grains, legumes, dairy
products, dried fruits, dried and smoked meats and nuts. The presence of insects and
insect excreta causes loss of food nutritional value, promotes decay of food and cre-
ates off -flavors and this makes food unsalable, causing considerable economic loss.
Mice and rats carry disease and are frequently associated with food-borne infections
in humans. Proper sanitation in food processing and storage areas is very important
in the fight against rodents, since all packaging materials, except metal and glass
containers, can be attacked by rats and mice.
20 S. Mukhopadhyay et al.

4 Methods of Food Preservation

Food preservation is achieved by three main methods as inactivation of microorganisms

and/or enzymes, inhibition of microbial growth or deterioration of quality and avoid-
ance of recontamination of food at all steps from pre- to post-harvest continuum. Based
on the principle of action, the majority of food preservation techniques can be classified
as (a) thermal and (b) emerging nonthermal processing (Preserving food without freez-
ing or canning 1999; Ravishankar and Maks 2007). These techniques cause inactivation
of pathogenic microorganisms assuring food safety and control growth of spoilage
microorganisms extending the shelf life.

5 Thermal Processing for Food Preservation

Thermal processing refers to the application of heat energy. Thermal processing is an

effective way of preserving food because the great majority of harmful pathogens are
killed at temperatures close to the boiling point of water. Heating of food has long been
at the heart of food processing. Thermal treatment is not only an important method of
preserving foods but also a means of developing texture, flavor and color. An important
challenge for thermal processing is the design and development of effective application
of thermal technologies to achieve these objectives without altering the desirable sen-
sory, functional and nutritional qualities of food (Rahman 2007; Richardson 2001).
Depending on their strength, thermal preservation can be classified as pasteuriza-
tion or sterilization processes. Pasteurization is a thermal treatment which can destroy
vegetative cells but has almost no effect on spores (U.S. Food and Drug Admin 2006;
U.S. Dept. of Health and Human Services 2009; Walstra et al. 1999). Sterilization on
the other hand, is designed for inactivating all forms of microorganisms, including
spores (Dion and Parker 2013). In terms of kinetics of thermal inactivation of micro-
organisms, the decimal reduction time or log-linear model is generally accepted. The
model assumes the behavior of thermal destruction of microorganisms is that of first-
order kinetics. The relationship between the decimal reduction time and the tempera-
ture is also assumed to be log-linear, leading to the concept of important kinetic
parameters as D and Z values which helps in thermal calculation and defining rate of
thermal lethality of the treatment The D value is a measure of the heat resistance of a
microorganism. It is the time in minutes at a given temperature required to destroy 1
log cycle (90%) of the target microorganism. In an actual process, all other organisms
that are less heat tolerant are destroyed to a greater extent. The Z value reflects the
temperature dependence of thermal destruction and is defined as the temperature
change needed to change the D value by a factor of 10.
Both the first-order kinetics and the Arrhenius-type effect of temperature are con-
tested. The lethality of a thermal process is expressed in terms of its F0 value. The F0
value of a thermal sterilization process is the number of minutes of heating at 121 °C
required to achieve the same thermal destruction ratio of a specified target microorgan-
ism, having a z value of 10 °C. Process optimization is the selection of thermal pro-
2 Principles of Food Preservation 21

cessing conditions that results minimal damage to food quality while meeting the
required food safety criteria. These “heat and kill” sterilization systems include high-
temperature, short-time pasteurization (HTST) and ultra-high t­ emperature (UHT) pro-
cessing designed for heating liquid foods, and retorting or canning for processed solid
foods (Stabel and Lambertz 2004).

6 Advance Thermal Technologies for Food Preservation

Thermal processing technologies for treatment of foods have been around for many
years. In recent years, there has been some new development in the area of source of heat
supply which are considered emerging or novel. These emerging thermal technologies
for food preservation include microwave, radio frequency, ohmic and infrared heating.

6.1 Microwave and Radio Frequency Preservation Process

Microwave and radio frequency electric field heating refers to use of electromagnetic
(EM) waves of certain frequencies to generate heat in a material. Microwave (MW)
has EM range of 300–300,000 MHz, while electromagnetic wave for radio frequency
(RF) ranges from 0.003–300 MHz. Microwave is simply a part of the RF spectrum
that has become quite popular due to the large number of its possible uses. Microwaves
can also be used to transmit power from one point to another. Both MW and RF work
by generating heat energy in foods, dielectric media, through dipole rotation and
ionic polarization (Metaxas and Meredith 1993). Water in the food is the primary
dipolar component responsible for the dielectric heating. In an alternating current
electric field, the polarity of the field is varied at the rate of microwave frequency and
molecules attempt to align themselves with the changing field. Heat is generated
rapidly as a result of internal molecular friction. The second major mechanism of
heating with microwaves is through the polarization of ions as a result of the back
and forth movement of the ionic molecules trying to align themselves with the oscil-
lating electric field. Microwave heating is also affected by the state of the constitu-
ents, whether they are bound or free, e.g., bound ions have much lower microwave
absorbing capability (Decareau and Peterson 1986; Von Hippel 1954). Critical fac-
tors affecting the effectiveness of microwave heating are the size, shape, composition
(electrolytes, moisture, salt, etc.), multiple components (frozen meal), and liquidity/
solidity of the food being treated. Also the power level, time of heating, cycling,
presence of hot water or air around the food, dimension, shape and other electromag-
netic properties of the oven are also important. Microwave heating for pasteurization
and sterilization may be preferable to conventional heating processes because they
require less time to rise to the desired process temperature, particularly for solid and
semi-solid foods. Microwave heating has the advantage of overcoming the limitation
imposed by the slow thermal diffusion process of conventional heating (Meredith
1998). The volumetric heat generated by microwaves can significantly reduce the
22 S. Mukhopadhyay et al.

total heating time and severity at the elevated temperatures required for commercial
sterilization whereby bacterial destruction is enhanced, but thermal degradation of
the desired components is reduced (Decareau 1985).
RF heating shortens process times by approximately one-third compared to the
conventional method. RF energy inactivates heat-resistant spores in foods and the
quality of RF treated food is usually better than conventionally processed foods.
Computer models based on finite element methods have been developed to predict RF
energy in packaged foods. Commercial application of RF systems, to provide uniform
EM field patterns remains a major challenge (Wang et al. 2003; Chan et al. 2004;
Luechapattanaporn et al. 2004; Luechapattanaporn et al. 2005).
Microwave pasteurization and sterilization systems and its practical indus-
trial applications have been reported over the past few decades. However, radio
frequency heating systems for the purpose of food pasteurization or sterilization
are not yet fully commercialized. Scientists have conducted several studies into
radio frequency electric fields (RFEF) technology, subjecting liquid foods to its
high electric fields, and found it to be both efficient for pathogen inactivation
and cost-­effective. A pasteurization process that uses RFEF for inactivation of
bacteria in foods was developed (Geveke et al. 2002; Geveke and Brunkhorst
2004). In these studies, a set of RFEF operating parameters that achieved
99.999% (5 logs) reduction of E. coli in apple cider was determined, and the
kinetics of bacterial inactivation established. The effect of RFEF treatment on
bacterial membrane was investigated.

6.2 Infrared Heating

Like microwave and radiofrequency, the infrared (IR) radiation (wavelength

0.78–1000 μm) transmits thermal energy in the form of electromagnetic (EM)
waves. IR encompasses a section of the EM spectrum that borders between visi-
ble light and microwaves. By exposing food materials to infrared (IR) radiation,
the heat energy generated by IR can be absorbed by the food. Infrared uses elec-
tromagnetic radiation generated from the vibrational and rotational energy of
molecules of a hot source (quartz lamp, quartz tube or metal rod). Thermal energy
is formed following the absorption of radiating energy. The IR heating system
contains a radiator and a reflector. The radiator or the source which radiated the
heat energy to the surrounding environment while the reflector directs the heat
energy to the food surface. The flux of energy of the radiator usually ranges from
a long (50 kW.m−2) to short (4000 kW.m−2) wave range. The radiator is usually
heated by gas or electric power. A polished metal surface or quartz tube with low
absorptivity and high emission capability to reflect the infrared energy, are used
as reflector (Regier et al. 2010). The characteristics of IR heating are the high heat
transfer capacity and the direct penetration of heat into the food. This is fast pro-
cess with no heating of surrounding air. Infrared heating is safer and economical
as compared to the conventional heating process (Vicente and Castro 2007).
2 Principles of Food Preservation 23

In IR, a desired levels of heating, both at the surface and also at the core of
the food, can be achieved. This is not possible with the microwave heating
process. IR energy, both long (5 μm) and short (1 μm) waves, is a popular
method for drying foods as compared to the conventional drying process due to
its rapid drying characteristics with lower energy consumption and due to a
uniform homogeneous distribution of temperature (Tewari 2007). Infrared is a
newer technology and its application remains limited to the experimental stage.
It is an accepted and popular method of drying, heating, baking, cooking and
drying of food (Tewari 2007). IR heating has also been used for the surface
pasteurization of bread, thawing of foods and for the decontamination of pack-
aging materials. When applied to bread, IR heating has been reported to control
the bread crumb thickness, texture, and color (Regier et al. 2010).

6.3 Ohmic Heating Technology

Ohmic heating, sometimes referred to as Joule heating, electrical resistance heating

or electro conductive heating, is the process of passing an electric currents through
foods or other materials to heat them. Ohmic heating is an advanced thermal process
where food acts as an electrical resistor. The experimental design usually consists of
electrodes that contact the food, whereby electricity is passed through the substance
using a variety of voltage and current combinations. The food is heated by the dissi-
pation of electrical energy. In comparison to the conventional mode of heating, where
heat is conducted from the outside using a hot surface, ohmic heating conducts heat
throughout the entire mass of the food uniformly. The defining characteristics of
ohmic heating compared to other electrical methods, such as microwave and radio-
frequency heating, lie in the frequency and waveforms of the electric field, and that
the electrodes contact the material. The success of ohmic heating depends on the rate
of heat generation in the system, the electrical conductivity of the food, and the
method by which the food flows through the system (Leizerson and Shimoni 2005a).
The inactivation of pathogens by ohmic heating is thermal in nature. Microbial inac-
tivation curves of ohmic heating are similar to conventional heating curves except for
a difference in the slope, which can most likely be explained by the presence of the
electric field (Food and Drug Administration-Center for Food Safety and Applied
Nutrition (FDACFSAN) 2000). Some literature reported non-­thermal effects of
ohmic heating on microbial inactivation. Research indicates that a mild electropora-
tion mechanism occurs during ohmic heating (Alberts et al. 2002). The principal
reason for the additional effect of ohmic treatment may be its low frequency (50–
60 Hz), which allows cell walls to build up charges and form pores. This is in contrast
to high-frequency methods such as radio or microwave frequency heating, where the
electric field is essentially reversed before sufficient charge buildup occurs at the cell
walls. The ohmic heating method has been applied successfully to a variety of foods
such as fruits and vegetables, juices, meats, seafood, soups, crèmes, and pasta
(Bengtsson et al. 2010). Additionally, reports also indicate that the suitability of
24 S. Mukhopadhyay et al.

ohmic heating for sterilization to produce high-quality shelf-stable low-acid foods

such as ready-prepared meals and high-acid foods. Research indicated that ohmic
heating processed foods usually retain the quality attributes such as nutrient profile,
texture, color, and flavor, far better compared to traditional thermally processed
foods (Leizerson and Shimoni 2005b).

7 Emerging Nonthermal Food Preservation Technologies

Although thermal technologies are being used extensively for processing and pres-
ervation of foods, recent consumer awareness about good nutrition and increasing
demand for fresher tasting food have paved the way for emerging nonthermal food
processing technologies. Non-thermal food processing/preservation methods
cause minimal impact on the nutritional and sensory qualities of foods, and yet are
capable of extending shelf life of foods by inhibiting or killing spoilage microor-
ganisms. Nonthermal treatments do not require a heat source and hence are con-
sidered to be more energy efficient and capable of providing better quality
preserved foods compared to conventional thermal based treatments. The emerg-
ing non-thermal technologies also meet the need of industry as it offers value-
added products, new market opportunities and added safety margins. Recent
exploitation of these technologies in inactivating pathogens and background
microflora for preservation of food has been considered in this chapter.

7.1 Ultraviolet Light Treatment

Ultraviolet (UV) radiation is a U.S. Food and Drug Administration (FDA) approved
non-thermal microbial intervention technology that finds increased applications in
surface decontamination of food and food contact surfaces (US-FDA 2001). UV
light’s potential in destroying bacteria, viruses and parasites have been documented
by many researchers (Bintsis et al. 2000; Chang et al. 1985; Demirci 2002; Dunn
et al. 1995; Harm 1980; Huang and Toledo 1982; Rowan et al. 1999; Shechmeister
1983; Sommers et al. 2010; Mukhopadhyay et al. 2014). Like many of its counter-
part decontamination technologies, UV light has a destructive effect on bacteria. UV
light has the ability to inhibit the bacteria’s replication process by dimerization of
their thymine bases in their DNA strands (Demirci 2002; Bank et al. 1991; Sinha and
Hader 2002). Membrane destruction due to localized overheating was hypothesized
as the reason for microbial death (Fine and Gervais 2004). Krishnamurthy (2006)
suggested photo physical effects on the bacterial structure as the main reason when
UV was applied as high intensity pulses. Because the UV light systems vary in
design and depending on whether the UV light is applied continuously or through
pulsed power technology and depending on the specific wavelengths used in the
power source, it is hypothesized that a combination of these factors contributes to the
inactivation of pathogens. Electromagnetic wavelengths of the range 220–280 nm
2 Principles of Food Preservation 25

(most common: 254 nm) and several application doses (mW cm−2) were shown to
deliver the optimum effect. While the UV dose is used to represent the total absorbed
dose, UV fluence in units of μJ cm−2 describes incident UV radiation on non-flat
surfaces. Researchers have reported varied power levels, process times, UV light
source-product distances, product thicknesses to achieve varied inactivation levels in
many pathogens. Many difficulties and subtleties are involved in measuring UV dose
(Chang et al. 1985). Hence, it is difficult to compare and standardize the process
conditions. Bolton and Linden (2003) developed a protocol for bench-­scale UV light
experiments with specifications for construction, determination of fluence (UV
dose) given to microorganisms and microbiological testing.
The effect of UV light (254 nm) on inactivation of Salmonella Senftenberg on
surfaces of tryptic soy agar (TSA), pork skin and pork muscle was evaluated
using UV intensities from 20 to 1000 μW cm−2 (Wong et al. 1998). Intensities
were varied by adjusting the distance (10–30 cm) between the UV lamp and the
treatment surface. It was found that the maximum log reduction was achieved at
UV intensities of 80 μW cm−2 or higher for all surfaces. While >7 logs inactiva-
tion was possible on the agar surface, only 2–3 logs in pork skin and 1–2 logs in
pork muscle was possible at 80 μW cm−2. Greater inactivation rate at 100 μW cm−2
as indicated by lower DUV-value was reported for TSA (15 s), followed by pork
skin (595 s) and pork muscle (1163 s). Similar results were also reported for
beef steak (Stermer et al. 1987), chicken halves (Wallner-Pendleton et al. 1994),
poultry skin (Sumner et al. 1995a), and chicken meat (Kim et al. 2002). In their
study (UV: 250–500 μW cm−2; 3 min) on chicken meat, peptone water and stain-
less steel, Kim et al. (2002) reported 5-log reduction of Salmonella Typhimurium
in peptone water and stainless steel compared to only 1-log and 0.36-log reduc-
tions in chicken meat with and without skin, respectively. The source of UV
light used was a bench lamp (Model UVG-54 of Ultra-violet products, Inc.
USA). Aluminium foil was used to cover the sample tray. Report indicates 7-logs
(99.9%) of Salmonella Typhimurium could be inactivated on agar plates but
only 89.5% reduction was obtained on poultry skin (UV: 3120 μW cm−2)
(Sumner et al. 1995a). These results indicate that UV irradiation is less effective
on irregular surfaces.
UV light was used in combination (hurdle approach) with other technologies and
antimicrobials more commonly to reap the maximum benefit. This minimizes the
negative impact of single technologies, when used alone, especially on product qual-
ity (Raso and Barbosa-Canovas 2003; Ross et al. 2003). UV light in combination with
antimicrobials (Mukhopadhyay et al. 2015; Uesagi and Moraru 2009), hydrogen per-
oxide (Hadjok et al. 2008) pulsed electric field (Gachovska et al. 2008), were a few of
the recent studies reported for the combined use of UV light with other inactivation
methods to inactivate pathogens and maintain quality for food preservation.
Hadjok et al. (2008) combined UV light with H2O2 to inactivate Salmonella and
a number of other pathogens inoculated on lettuce, spinach, cauliflower, broccoli,
onion and tomato. UV light acts as the oxidizing agent inducing the formation of
highly reactive hydroxyl radicals from H2O2 which react with the cellular
­components of the pathogens to induce their inactivation (Rosenfeldt et al. 2006).
This radical formation and subsequent reaction on cell components is positively
26 S. Mukhopadhyay et al.

influenced by temperature and the optimal temperature is 50 °C (Hadjok et al.

2008; Yaun et al. 2004). A combination treatment of UV light (37.8 mJ
cm−2/~0.63 mW cm−2) and H2O2 (1.5%) at 50 °C reduced surface Salmonella of
lettuce by >4 logs and internalized Salmonella by 2.84 log10CFU/mL (Hadjok et al.
2008). H2O2 at 1.5% v/v concentration performed better than 2.0% and the reason
was attributed to neutralization of excess radicals (Reidmiller et al. 2003). UV
light—H2O2 was reported to be more effective in leafy vegetables such as lettuce
than produce such as cauliflower, broccoli, onion and tomatoes (Hadjok et al.
2008). The reason for this was reported to be the inability of reactive radicals to
reach deeper into the interior bacterial locations on the latter vegetables.
Mukhopadhyay et al. (2015) reported >4.7 logs reduction of a bacterial composite
made of three serotypes of S. enterica (S. Newport H1275, S. Stanley H0558, and
S. Montevideo G4639) by a combined a low dose (0.6 kJ/m2) treatment of UV light
(253.7 nm) followed by 2 min immersion in a novel antimicrobial formulation
‘HEN’ composed of the antimicrobials hydrogen peroxide, nisin and a surfactant
EDTA at room temperature (22 °C).
Photo reactivation is the light-dependent DNA repairing ability of organisms. In
the presence of UV-A and visible light, the enzyme photolyase reverses the pyrimi-
dine dimers to facilitate the repair (Sinha and Hader 2002).Techniques of applying
UV light in cycles of light and dark as well as UV radiation plus photo reactivation
did not show any significant difference with UV treatment alone (Kuo et al. 1997).
Comparing the sensitivity of Salmonella against other pathogens to UV treatment
is difficult as there are many variables involved and the available data are mixed.
While Salmonella Typhimurium (Kim et al. 2002) and Salmonella Montevideo
(Hadjok et al. 2008) were reported to be more resistant than E. coli O157:H7,
Salmonella Senftenberg was reported more sensitive than E. coli (Wong et al. 1998)
to UV irradiation. In general, most of the vegetative bacteria exhibited similar resis-
tance to UV light and required less than 3–15 times of the UV dose requirements for
inactivation of viruses, bacterial spores and amoebic cysts (Chang et al. 1985).

7.2 Pulsed Light Technology

UV radiation could be applied either continuously or as high intensity short duration

pulses (Krishnamurthy and Demirci 2007; McDonald et al. 2000). Pulsed light (PL)
covers a broader spectrum (100–1100 nm) consisting of UV, visible and infrared
radiation. Pulsed Light (PL) is a novel, non-thermal decontamination technology for
food products which uses short time, high frequency pulses of an intense broad
spectrum of light. The spectrum of light for pulsed light treatment includes wave-
lengths in the ultraviolet (UV) to the near infrared region (Krishnamurthy and
Demirci 2007). The material to be sterilized is exposed to at least 1 pulse of light
(typically 1–20 flashes per s) with a duration range from 1 μs to 0.1 s (Dunn et al.
1991). For most applications, a few flashes provide a high level of microbial inacti-
vation. In terms of mechanism of its operation, UV-C light is effective in blocking
microorganisms’ development by altering their DNA. As a physical preservation
2 Principles of Food Preservation 27

method, UV irradiation has a positive consumer image. The US Food and Drug
Administration (FDA) and US Department of Agriculture (USDA) have concluded
that the use of UV irradiation is safe. In 2000, the FDA approved UV-light as alter-
native treatment to thermal pasteurization up to a cumulative dosage of 12 J/cm2 by
FDA Code 21CFR179.41 (FDA (Food and Drug Administration) 2015). The perfor-
mance criterion defined by FDA for fruit and vegetable juice processing is a 5-logs
reduction in the number of the target pathogen of concern (FDA (Food and Drug
Administration) 2015). In addition to the germicidal effect of UV, application of
high intensity pulses also results in rise of food surface temperature temperatures
(Dunn et al. 1991). Due to the physical and thermal effects associated with pulsed
application of UV light, microbial inactivation efficiency is 4–6 times more than
that of continuous application (Fine and Gervais 2004; Dunn 1996).
Levy et al. (2012) demonstrated that pulsed UV light is efficient in achieving more
than 5 log reduction for a range of spore-forming bacteria, for vegetative cells of non-
spore-forming bacteria, and for yeasts spread on agar media. Abshire and Dunton
(1981) found that some species (Pseudomonas aeruginosa) were more sensitive than
others (Micrococcus radiodurans and Candida albicans). The inactivation effect of
intense pulsed light (IPL) on Micrococcus roseus, an irradiation resistant bacterium
has been investigated by Kim et al. (2013). Approximately 6.6 log CFU/ml reduction
in the cell viability for irradiation resistant M. roseus was achieved by 3 min treatment
of 1000 V intensity of PL. Various factors govern the efficacy of PL. A number of
physical factors (dose, input voltage and the UV content of PL), biological factors
(microbial strains, spores, vegetative cells) and environmental factors (surface mor-
phology/quality) have been investigated for the efficacy of pulsed light in microbial
decontamination (Levy et al. 2012). Ozer and Demirci (2006) demonstrated about one
log reduction of E. coli O157:H7 or L. monocytogenes could be achieved by 60-s
treatment at a distance of 8 cm from the UV strobe without affecting the quality.
Nicorescu et al. (2013) compared the inactivation kinetics of pulsed UV light treat-
ment in the dry and liquid state and highlighted that 8 log reduction of B. subtilis
vegetative cells could be achieved in the liquid state compared to only 1 log reduction
in the dry state the same intensity of treatment. Empirical models as a function of
distance, layer thickness and treatment time have been developed for predicting the
population of Escherichia coli O157:H7 during pulsed light UV treatment (Sharma
and Demirci 2003) for eliminating pathogens from alfalfa seeds. Similar models for
deactivation of fungal spores of Aspergillus niger have been developed by Jun et al.
(2003) by varying the treatment time, voltage input and distance from the UV strobe.
Hillegas and Demirci (Hillegas and Demirci 2003) also developed models for pulsed
UV light sterilization system for the inactivation of C. sporogenes in honey. The num-
ber of pulses, the distance between the food product and lamp and depth of the honey
were varied to enhance the inactivation of C. sporogenes by 90%. Krishnamurthy
et al. (Krishnamurthy and Demirci 2007) varied the flow rate (20, 30, and 40 mL/min),
number of passes (1, 2, and 3 times) and distance of the sample from the source (5, 8,
and 11 cm) to optimize Staphylococcus aureus inactivation in milk using the pulsed
UV system. They reported complete inactivation (>7 log10CFU/mL) in only two cases.
Sample distance from the UV source window was the only variable reported by these
researchers that had a significant (P < 0.05) impact on the outcome.
28 S. Mukhopadhyay et al.

Studies on microbial decontamination of food powders (Fine and Gervais 2004)

using pulsed UV light revealed that 58 J/cm2 fluence was sufficient to decrease the
microbial population of S. cerevisiae by 7 logs on glass beads or dried powdered prod-
ucts. Pulsed UV light has been applied to strawberries, blueberries and raspberries at
varying UV doses and times (Bialka and Demirci 2007; Bialka and Demirci 2008).
Maximum reductions of E. coli O157:H7 and Salmonella were reported to be 3.9 and
3.4 log10 CFU/g at 72 and 59.2 J/cm−2 respectively on raspberries. Whereas on the
surface of strawberries, maximum reductions of 2.1 and 2.8 J/cm−2 log10 CFU/g at 25.7
and 34.2 J/cm−2 respectively have been recorded with no visible damage to the fruits.
On blueberries, 4.3 and 2.9 log10 CFU/g reduction was reported. PL treatment has also
been proved to be effective as an intervention strategy to prevent cross-contamination
between equipment and the final product and founding effective when applied along
meat processing lines for decontamination of L. monocytogenes and Escherichia coli
O157:H7 on the surface of a stainless steel meat slicing knife (Rajkovic et al. 2010).
Pulsed UV light has also been used to inactivate semi-opaque and solid systems.
Bialka et al. (2008) studied the efficacy of pulsed UV light technology in solid model
systems. Inactivation and energy penetration data obtained from the treatment of agar
(clear solid medium) and whey protein (opaque solid medium) gels after treatment
with pulsed UV light were used to construct several models to estimate the amount of
energy penetrating the sample at a given depth and the inactivation of E. coli K12. It
was found that pulsed UV light can penetrate opaque materials up to 10 mm deep with
decreasing energy levels and Weibull type model can be used to model the inactivation.
Decontamination studies of Penicillium roqueforti in agar media, bread and preformed
pizza (Mohammadbeygy 2013) revealed that 400 V, 1200 pulses at a distance of 5 cm
from the UV strobe resulted in complete inactivation of P. roqueforti. Overall, pulsed
UV light was found to have a potential use for the decontamination of spoilage micro-
organisms in a variety of products.
Pulsed light has been demonstrated to extend the shelf-life and improve the qual-
ity of several fruits and vegetables (Aguilo-Aguayo et al. 2013; Charles et al. 2013;
Gomez et al. 2012; Klein 1987; Oms-Oliu et al. 2010) and has been used in dairy
(Krishnamurthy and Demirci 2007), egg (Kuo et al. 1997) and meat (Ozer and
Demirci 2006; Rajkovic et al. 2010; Pollock 2007; Sumner et al. 1995b) products. At
low doses (0.25–8.0 kJ/m2) UV radiation from pulsed light can induce beneficial
reactions in the plant, such as a defense response against pathogens attack via a phe-
nomenon known as hormesis (Gardner and Shama 2000). Many studies have dem-
onstrated that at low doses, the technology can control various kind of storage rots of
fruit and vegetables, delay fruit ripening during postharvest storage and simulate the
synthesis of enzymes or compounds with antimicrobial activity (Klein 1987).
From a nutritional point of view, post-harvest processing generally reduces the nutri-
tional value of fruits and vegetables. As a result, processed fruits and vegetables are
assumed to have lower health promoting capacity when compared to fresh produce (Klein
1987; Nicoli et al. 1999). Few studies have been carried out to determine the use of pulsed
light to enhance the nutritional quality of food materials. The enhancement of antioxidant
content of elderberry fruit by pulsed UV light was studied by Murugesan (2010). They
observed upto 50% increase in the total phenolic content of elderberry fruit by 1.1 J/cm2/
pulse treatment for a 10 seconds treatment time. Charles et al. (2013) studied the impact of
2 Principles of Food Preservation 29

pulsed light treatment on the physical and nutritional quality of fresh-cut “Kent” mangoes.
Pulse light treatment with a total fluence of 8 J/cm−2 maintained the firmness, the color and
the carotenoid content of the fresh-cut mangoes. It was also found to maintain the phenyl-
alanineammonialyase, (PAL) activity and increased polyphenoloxydase, (PPO) activity
after 3 days. For the nutritional aspect, pulsed light maintained phenol and total ascorbic
acid contents similar to that of the control. The effect of pulsed light on surface decontami-
nation (natural and inoculated microorganisms), physical (color, texture and weight) and
nutritional quality (ascorbic acid and major carotenoids) was investigated in red-ripe toma-
toes during 15 days of storage at 20 °C by Aguilo-­Aguayo et al. (2013). They reported that
PL treatment of fresh tomato would result in a reduction in microbiological contaminants
without compromising the nutritional value. However, some appearance defects were
noted on the treated tomatoes. Literature reports on shelf-life studies using pulsed light
treatments is scarce, although pulsed light treatment in combination with low temperature
storage (4 °C) has the potential to extend the shelf-life of various products. In an earlier
work, pulsed UV light treated cold smoked salmon products stored for 30 days exhibited
microbial free products without compromising sensory quality (Pollock 2007) at
4 °C. However, a moderate temperature of 25 °C showed growth of L. monocytogenes and
thus moderate temperatures were found to be problematic for pulsed-­light treated salmon.
The effect of pulsed light combined with an anti-browning pretreatment (ascorbic acid
plus CaCl2 solution) on the quality and shelf-life stability of fresh cut apples was studied
by Gomez et al. (2012). The anti-browning dipping solution was effective in inhibiting
browning on apple surfaces exposed to a PL dose of 71.6 J/cm−2. The results of the work
done by Oms-Oliu et al. (2010) suggested that the application of pulsed light at doses of
4.8 J/cm−2 could extend the shelf-life of fresh-cut mushrooms without appreciable changes
in the texture and antioxidant properties.

7.3 Ultrasound Processing

Ultrasound has applications in many areas of food processing and food analysis.
These include, extraction of aroma and other plant materials (Cocito et al. 1995), food
freezing (Sigfusson et al. 2004; Zheng and Sun 2006), inspection of food containers
(Garcia-Gonzalez et al. 2007; Griffin et al. 2001; Pascall et al. 2002), temperature
monitoring (Haeggstrom and Luukkala 2000), food characterization and analysis
(Kulmyrzaev et al. 2000; McClements 1995; Saggin and Coupland 2001), inactivation
of microbes (Piyasena et al. 2003; Seymour et al. 2002; USDA 2000) and enzymes
(De Gennaro et al. 1999), to name a few. A number of published review papers are
available on these aspects (Hauptmann et al. 2002; Knorr et al. 2004; McClements
1995; Mason et al. 1996; Piyasena et al. 2003; Povey 1998; Samari 1994; Zheng and
Sun 2006; Withers 1996). Its application in microbial inactivation gained importance
in 1990s like any other non-thermal technologies (Sherba et al. 1991). The very sparse
available literature on its application to Salmonellae inactivation is limited to liquid
foods such as water, milk, liquid whole egg and minimally processed fruits and veg-
etables (Seymour et al. 2002; Wrigley and Llorca 1992). This may be due to ultra-
sound’s limitations in reaching deeper areas of solid foods.
30 S. Mukhopadhyay et al.

Mechanical vibrations applied at frequencies higher than 15 kHz generate

ultrasound waves (Raso et al. 1998). When applied to any liquid foods, sonic
waves create alternate compression and expansion cycles inside the liquid.
These lead to the development of bubbles. Generated bubbles grow in size till
the point when it is no longer sufficient to withstand the pressure from outside.
The bubbles collapse (implosion) causing collision between liquid molecules.
This process, called cavitation, along with the resulting shear and rise in tem-
perature (approximately 5500 °C) and pressure (approximately 50 MPa) are
responsible for disrupting the cell structure and subsequently the cell’s death
(Butz and Tauscher 2002; Neppiras 1980; Suslick 1988). Scherba et al. (1991)
suggested that transient cavitation (short violent bursts) may be the reason for
microbial inactivation at low frequency range rather than stable cavitation (less
violent with vibrating gaseous bodies). More the number of bubbles and higher
the duration/intensity of cavitation, better is the microbial inactivation. This
mechanical effect could be improved by increasing the amplitude of the sonic
waves and by applying this under pressure (manosonication) (Pagan et al.
1999; Raso et al. 1998). Though ultrasound alone can be successful in many
food preservation applications, high treatment intensities may have adverse
effects on food quality (Lee et al. 1989; Wrigley and Llorca 1992). Application
of ultrasound combined with pressure will improve the lethality of a less-inten-
sive treatment. Raso et al. (1998) reported exponential reduction in decimal
reduction time (DUS-values, time required to reduce the initial microbial popu-
lation by 90% by ultrasound) from 4 to 0.3 min by increased amplitude from 21
to 150 μm at 20 kHz frequency. The influence of pressure was also reported to
increase with increase of amplitude. Application of temperature (thermosoni-
cation) has a negative effect on cavitation (Pagan et al. 1999; Raso et al. 1998).
Also, heat and ultrasound are reported to act independently (Raso et al. 1998).
Thermomanosonication is the application of temperature and pressure toward
specific advantages.
Lethality of ultrasonic treatment, as expressed by its D-value is influenced by the
wave’s amplitude, pressure and temperature. Inactivation rate increased exponen-
tially with increase in amplitude of sonic waves. General equations describing their
influence were developed and reported by Pagan et al. (1999) and Raso et al. (1998).
Manas et al. (2000) investigated the resistance of three Salmonella serovars namely,
S. Enteritidis, S. Typhimurium and S. Senftenberg 775 W which are common in
liquid egg white to pressured ultrasonic waves at varied temperatures.
Salmonella Senftenberg, the most resistive of the three, exhibited greater
resistance to heat treatment than manosonication and the resistance is much
higher in liquid whole egg. Also, the resistance of all three serovars were similar
in manosonication suggesting that a uniform, milder treatment would be suffice
for making liquid white egg safe from Salmonella. Manothermosonication at
60 °C reduced Salmonella Senftenberg 775 W by 3 logs compared to 0.5 log by
heat at 60 °C. This technology is also more effective on gram-negative than
gram-positive species (Pagan et al. 1999). In general, gram-positive microorgan-
isms are considered more tolerant of the effect of intervention technologies due
to the presence of outer peptidoglycan layer. However, Scherba et al. (1991), in
2 Principles of Food Preservation 31

their quantitative assessment of the germicidal efficacy of ultrasonic energy

(26 kHz at various exposure levels) using various bacteria of different structural
property indicated that the main target of ultrasonic damage may be the inner
cytoplasmic membrane and not the outer presence or absence of the peptidogly-
can layer. It was concluded that the percentage kill by ultrasonic energy was not
influenced by the morphological feature of the microorganisms.
The potential application of ultrasound in effectively inactivating Salmonella
spp. in high sugar chocolate was investigated by Lee et al. (1989). A 4-log reduction
was reported for Salmonella Eastbourne (DUS-value: 3 min) and Salmonella Anatum
(DUS-value: 2.1 min). These DUS values were based on first order kinetics. High
sugar (solids) and high viscosity likely inhibits microbial inactivation because of
reduced cavitation (Hulsen 1999; Wrigley and Llorca 1992).
Seymour et al. (2002) demonstrated a reduction of 99.8% of Salmonella
Typhimurium on cut iceberg lettuce by using power ultrasound (32–40 kHz) and
chlorine (25 ppm) together. This was one log more than that of chlorine (1.7 logs)
or ultrasound (1.6 logs) used independently and two logs more than that of water dip
without agitation (0.7 logs). Similar results were reported by Kim et al. (1999) in
their experiments with chlorinated water (99% reduction), ozonated water (99.5%)
when compared to only 90 per cent of ultrasound treatment. The improved reduc-
tion by combining chlorine and ultrasound was supposed to be due to the ultra-
sound’s cavitation effect in detaching the microbial cells from the produce surface
rendering them susceptible to the sanitizing effect of chlorine. Sonication aiding the
effect of chlorine in Salmonellae inactivation on broiler breast skin was highlighted
by Lillard (1993, 1994). The irregular skin surface of the broiler skin providing
physical protection to the Salmonella spp. was indicated as the possible reason for
the decreased less effect of sonication when used alone (Sams and Feria 1991).

7.4 Bacteriophage Application

With the fresh-cut industry looking for an alternative to chlorine and chlorine based
compounds, potential use of safe, clean and novel biocontrol agents such as lytic
bacteriophages is gaining importance. As the bacteria became resistant to antibiot-
ics, medical researchers, especially in the erstwhile Soviet Union and Eastern
European countries started using bacteriophages for treating diseases due to patho-
gens including Salmonella (Alisky et al. 1998). Bacteriophages are viruses capable
of lysing specific bacteria by penetrating their cell membrane and disrupting their
metabolic processes. . Ubiquitous presence of these phages and their flexibility in
preparation to act against specific strain or serotype of any pathogenic bacteria
makes it a promising alternative to treat minimally processed food produce.
Leverentz et al. (2001) examined the application of a phage cocktail mixture
with a specificity for Salmonella Enteritidis on fresh-cut honeydew melon (pH5.8)
and apple slice (pH4.2) surfaces. They also evaluated the effect of these bacterio-
phages at various storage temperatures (5, 10 and 20 °C) in comparison to standard
chemical sanitizers. The main limitation of this application is the development of
32 S. Mukhopadhyay et al.

resistance to phages by the target pathogens, the use of cocktail of phages to take
care of mutants is recommended. The mixture was found to be more effective than
chemical sanitizers and reduced the inoculated (1 × 106 CFU/mL) Salmonella popu-
lations by 2.5 to 3.5 logs on honeydew melon slices. High temperature storage
(20 °C) resulted in less reduction and no significant reduction was observed in apple
slices. This result was hypothesized to be the result of high acidity (pH4.2) of apple
slices inactivating the phages along with the bacteria. Higher concentrations of bac-
teriophages and development of low-pH tolerant phages may make this technology
suitable for all types of fruit and vegetables.
The ratio of host cells and phages, described as ‘multiplicity of infection
(MOI)’, ‘multiplicity of adsorption (MOA)’ and PFU/CFU ratio by various
researchers was considered to be important for the successful application of this
technology (Kasman et al. 2002; O’Flynn et al. 2004; Whichard et al. 2003).
Bigwood et al. (2009) conducted experiments to determine the influence of host
and bacteriophage concentrations on Salmonella inactivation by a Salmonella
phage (P7). It was reported by the authors that the host cell concentration did not
influence the inactivation efficiency for a given phage concentration. However,
increase in phage concentration from 1.8 × 104 to >5 × 198 PFU/mL yielded
increased inactivation with the lowest concentration showing no inactivation to
the highest concentration showing almost complete inactivation.

7.5 Membrane Processing

Membrane processing is a unique method for liquid foods which is driven by pres-
sure difference across a microporous membrane. It is an attractive alternative to
energy intensive thermal processing. In addition, r membrane processed foods are
superior in terms of nutritive value and sensory quality. Membrane processing does
not require application of heat and provides the ‘cold pasteurization’ option by sim-
ply removing the harmful bacteria, spores and other microbial contaminants from
food. It can also be used to concentrate dilute liquid foods as milk, juice, etc.
Microfiltration is a pressure-driven membrane process which can separate fine par-
ticles, microorganisms and other contaminants from fluids such as liquid foods.
Membranes used in microfiltration process have a microporous structure that
separates particulate matters in size ranges from 0.1 to 10 μm. In cross-flow MF, the
fluid to be filtered flows parallel to the membrane surface, which reduces pore block-
age and hence facilitates a quasi-steady filtrate flow for a longer time compared to
standard filtration processes. Cross-flow microfiltration has been explored as a means
to remove natural microflora from milk. Guerra and coworkers observed a high spore
reduction in the range of 104–105 spores/mL of Bacillus cereus and lactate ferment-
ing Clostridium spores from skim milk by cross-flow MF using a 1.0 micron ceraflo
ceramic membrane (Guerra et al. 1997). In microfiltration, selection of the appropri-
ate membrane and process conditions not only provides rapid removal of pathogens
and spores from food but also minimize the loss of nutrients and quality functional
2 Principles of Food Preservation 33

properties of the food. Thus, from a safety and functionality and consumer accept-
ability point of view, cross-flow microfiltration processing is advantageous over con-
ventional thermal processing.
The performance of a membrane process is determined by the amount and the
stability of the permeate flow through a unit area of membrane in a second and is
called “permeate flux,” which is influenced by feed concentration, temperature and
feed velocity. Permeate flux increases with transmembrane pressure (TMP), which
is the pressure differential across the membrane between the feed and permeate.
Performance of a microfiltration processing usually declines over time mainly due
to membrane fouling and other factors. Microfiltration processing has been success-
fully applied for cold pasteurization of milk (Cheryan 1998; Saboya and Maubois
2000; Tomasula et al. 2011) and eggs (Mukhopadhyay et al. 2009). Reports on pro-
cessing of eggs in the form of liquid egg describes removal of natural background
microflora (Mukhopadhyay et al. 2009), human pathogens such as Salmonella
Enteritidis (Mukhopadhyay et al. 2010) and spores of Bacillus anthracis
(Mukhopadhyay et al. 2011) using a ceramic GP membrane. The author in this
study reported no significant (p < 0.05) change in liquid eggs protein composition
and individual egg protein distribution due to microfiltration processing.

8 Conclusions

Many of the reviewed preservation technologies are extensively studied and await-
ing commercial adoption. Commercialization is hampered by the variation in
research results due to non-standard process equipment, procedures, test conditions,
microbial strains and their stress tolerance to the various processes. Any small
increase in the processing cost would deter the conservative food industry from suc-
cessful adoption of these technologies. Joint research initiatives among academia,
industry and regulatory authorities should clear the path for commercialization of
many of these technologies. Combination or integration of compatible preservation
technologies (hurdle approach), both conventional and novel should be identified
and research should be explored vigorously for improved efficacy of microbial inac-
tivation but minimal effect on the quality of food.

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Chapter 3
Application of Omics Technologies
and Computational Approaches for Control
of Foodborne Pathogens in Foods

Jayanthi Gangiredla, Xianghe Yan, Isha R. Patel, and Mark K. Mammel

Abstract Control of Foodborne Pathogens is an important health-related quality of

life topic due to the global burden of foodborne pathogen diseases. The strategies to
destroy or control of foodborne pathogens probably starts with the recognition and
avoidance of foodborne pathogens that are naturally present in our environment.
The foodborne illness could be caused by pathogen itself (e.g. viruses, bacteria,
parasites, prions) or biological toxins directly produced by microorganisms in the
food; such foods become unsafe for human consumption. The newly emerging or
evolving pathogens, along with relatively low minimum infectious doses and/or
infection with multidrug resistant pathogens have been increasingly reported and is
a new challenge. Control of Foodborne Pathogens need to be closely monitored at
various stages throughout the complex food supply chain: food production, food
processing, and food consumption. For monitoring of contaminant levels of food-
borne pathogens, one must adhere to Hazard Analysis and Critical Control Point
(HACCP) principles, an official systematic approach of identifying, evaluating and
controlling food safety hazards, including biological foodborne pathogens. The
importance of HACCP principles in contributing to protecting our food supply has
been recognized by food safety professionals since the 1970s. In this chapter,
HACCP principles were followed, discussing traditional and recent advances in
some basic concepts and terminology which can be reasonably used for detection

This chapter reflects the views of the authors and should not be construed as official or reflecting
the views of the US Department of Health and Human Services or the US Food and Drug
Administration.
J. Gangiredla • I.R. Patel • M.K. Mammel (*)
Division of Molecular Biology, U.S. Food and Drug Administration, Center for Food Safety
and Applied Nutrition, Office of Applied Research and Safety Assessment, Laurel, MD, USA
e-mail: [email protected]; [email protected];
[email protected]
X. Yan
U.S. Department of Agriculture, Environmental Microbial and Food Safety Laboratory,
USDA Agricultural Research Service (ARS), Beltsville, MD, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 41

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_3
42 J. Gangiredla et al.

and control of foodborne pathogens. The traditional computational approaches

mainly rely on information systems, data management, surveillance networking,
predictive and computational modeling; whereas the modern computational
approaches integrate multiple molecular features of foodborne pathogens and other
innovative technologies, including omics technologies.

Keywords Foodborne pathogen • Outbreak detection system • Predictive modeling

• Metagenomics • Whole genome sequencing

1 Introduction

Control of foodborne pathogens is an important health-related quality of life topic

due to the global burden of diseases caused by these microbes. Strategies to eliminate
or control these pathogens start with elimination of those that are naturally present in
our environment. The origin of foodborne pathogen related illness could be caused
by the pathogen itself (e.g. viruses, bacteria, parasites, prions) or biological toxins
produced by microorganisms in the food which becomes unsafe for human consump-
tion. The emerging or evolving pathogens, along with relatively low minimum infec-
tious doses and/or combined with multidrug resistance, have also been increasingly
reported and have become a new challenge (Critzer and Doyle 2010; Doyle 2015).
Control of foodborne pathogens needs to be closely monitored at various stages
along with the following complex food supply chain: food production, processing,
and consumption. The importance of Hazard Analysis and Critical Control Point
(HACCP) principles, an official systematic approach of identifying, evaluating and
controlling hazards and biological foodborne pathogens in contributing to food safety
has been recognized by professionals in this field since the 1970s (Sneed et al. 2004).
In this chapter, we will follow these HACCP principles discussing traditional and
recent advances of some basic concepts and terms which can be reasonably used for
detection and control of foodborne pathogens. The traditional computational
approaches rely on information systems, data management, surveillance networking,
predictive and computational modeling; whereas the modern computational
approaches integrate multiple molecular features of pathogens and other innovative
technologies, such as omics technologies.

2  raditional Approaches for Control of Foodborne

T
Pathogens

Traditionally and the most common computational approaches for controlling food safety
include: (1) Surveillance System (FoodNet (https://www.cdc.gov/foodnet)); (2) Outbreak
Detection System (PulseNet (https://www.cdc.gov/pulsenet)); (3) Response System
3 Application of Omics Technologies and Computational Approaches for Control… 43

(OutbreakNet Enhanced (https://www.cdc.gov/foodsafety/outbreaknetenhanced); (4):

Predictive Microbiology Information Portal (United States Department of Agriculture
(https://portal.errc.ars.usda.gov)); (5) Predictive Modeling (USDA); and (6): Foodrisk.org
and FDA-iRISK.

2.1 Surveillance System (FoodNet)

The Foodborne Diseases Active Surveillance Network (FoodNet) was established in

July 1995. It is multi-state, multi-Federal agency effort to obtain reports of infections
diagnosed across the United States. In addition, it consists of active surveillance of
foodborne diseases and related epidemiologic studies. FoodNet investigators iden-
tify and collect information on all laboratory confirmed cases of selected foodborne
illnesses. Infections under surveillance include: Campylobacter, Cryptosporidium,
Cyclospora, Listeria, Salmonella, Shiga-toxin-producing Escherichia coli O157,
Shiga-toxin-producing Escherichia coli (STEC) non-O157, Shigella, Vibrio, and
Yersinia infections among residents of the catchment area. In addition to laboratory
surveillance, FoodNet conducts active surveillance of pediatric hemolytic uremic
syndrome (HUS) through a network of nephrologists in the catchment area.
FoodNet serves the purposes to (1) determine the burden of foodborne illness in
the United States; (2) monitor trends in the burden of specific foodborne illness over
time; (3) attribute the burden of foodborne illness to specific foods and settings; and
(4) disseminate information that can lead to improvement in public health practice
and the development of interventions to reduce the burden of foodborne illness. This
system allows local and state health departments to implement active surveillance
and epidemiologic studies designed to help health officials to better understand the
epidemiology of foodborne diseases in the United States. Data are voluntarily trans-
mitted electronically to CDC (Centers for Disease Control and Prevention) from all
FoodNet collaborators which are then analyzed. This is the major difference from
the passive surveillance systems that rely upon reporting of foodborne diseases by
clinical laboratories to state health departments, which then report) to CDC. In
2015, “FoodNet received reports of 20,107 confirmed cases, 4531 hospitalizations,
and 77 deaths. FoodNet also received reports of 3112 positive culture-independent
diagnostic tests (CIDTs) without culture-confirmation – a rate more than double
that of 2012–2014” (https://www.cdc.gov/foodnet/reports/prelim-data-intro.html).

2.2 Outbreak Detection System (PulseNet)

PulseNet (https://www.cdc.gov/pulsenet/index.html) is another national network of

public health and food regulatory agency laboratories initiative coordinated by the
CDC. PulseNet International was born from the success of PulseNet USA and now it
spans more than 80 countries, including Canada, Europe, Latin America and the
44 J. Gangiredla et al.

Caribbean, the Asia Pacific region, the Middle East, and Africa. PulseNet plays a
critical role in providing the best techniques to identify an outbreak in early detection
and rapid response during the food safety crisis. Currently, PulseNet monitors at the
CDC collect fingerprints generated by using molecular subtyping tools such as
pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem
repeat analysis (MLVA), and whole genome sequencing (WGS). Some of associated
networks, including CaliciNet (https://www.cdc.gov/norovirus/reporting/calicinet/)
and CryptoNet (https://www.cdc.gov/parasites/crypto/cryptonet.html) have similar
functions for norovirus and Cryptosporidium parasites.

2.3 Response System (OutbreakNet Enhanced)

OutbreakNet Enhanced (https://www.cdc.gov/foodsafety/outbreaknetenhanced/index.

html) is an enhanced form and reporting platform of the old CDC OutbreakNet program,
started in August 2015. It provides support and access to state and local health depart-
ments to improve their capacity to detect, investigate, control, and respond to foodborne
disease outbreaks. Currently, “OutbreakNet Enhanced sites work together and with the
CDC to share experiences and insights to improve enteric disease outbreak response.
OutbreakNet Enhanced activities focus on improving detection and rapid interviewing
of cases of Salmonella, Shiga-toxin producing Escherichia coli (STEC), and Listeria as
well as any cases of enteric disease with pathogens that demonstrate antimicrobial resis-
tance”. The purpose of OutbreakNet Enhanced Team is to ensure rapid, coordinated
detection and response to multi-state outbreaks of enteric diseases and promote compre-
hensive outbreak surveillance. Communication networks are key components in order
for public health agencies to responsd to an outbreak in a timely matter.
Besides above systems, the FDA coordinates with state health departments, IT lead-
ers, universities, and other federal agencies, such as the CDC and the USDA to establish
the Electronic Laboratory Exchange Network (eLEXNET), an integrated Web-based
data exchange system (https://www.elexnet.com/elex/) for “FDA on issues of informa-
tion management, vocabulary and messaging standards and data exchange at food and
feed testing laboratories” (https://www.aphl.org/programs/food_safety/laboratory-
accreditation/Pages/eLEXNET.aspx). The implementation of eLEXNET will facilitate
the investigation and greatly reduce the duplicated efforts among agencies to compare
and coordinate findings of laboratory analyses. Public mailing lists play critical roles in
response to outbreaks. FSNET (­http://www.foodsafetynetwork.ca/) and Promed (http://
www.promedmail.org) are two of the examples of the public mailing lists that can be used
to disseminate information about foodborne and emerging infectious diseases. The World
Health Organization has established a Global Outbreak and Alert Response Network
(GOARN), which is a collaboration of existing institutions and networks, constantly alert
and ready to respond. The network pools human and technical resources for rapid identi-
fication, confirmation and response to outbreaks of international importance (http://www.
who.int/ihr/alert_and_response/outbreak-network/en/). New information distribution
approaches using social network such as: Facebook and Twitter, can also facilitate the
public awareness in a timely matter, especially in the younger generation.
3 Application of Omics Technologies and Computational Approaches for Control… 45

2.4 Predictive Microbiology Information Portal (USDA)

“The USDA Food Safety & Inspection Service (FSIS) and the USDA Agricultural
Research Service (ARS) have joined together to produce the Predictive
Microbiology Information Portal (PMIP). This portal is geared to assist food com-
panies (large and small) in the use of predictive models, the appropriate applica-
tion of models, and proper model interpretation. Our vision is that the PMIP will
be the highway to the most comprehensive websites that brings together large and
small food companies in contact with the information needed to aid in the pro-
duction of the safest foods” (https://portal.errc.ars.usda.gov/?modeCode=80-72-
05-00&modeCode=80-72-05-00). “The PMIP links users to numerous and diverse
resources associated with models (PMP), combined databases (ComBase, http://
www.combase.cc/index.php/en/about-combase), regulatory requirements, and food
safety principles”. ComBase, a free online tool and database for quantitative micro-
bial responses to food environments, was initially developed by the research groups
of Eastern Regional Research Center (ERRC) of the USDA Agricultural Research
Service (ARS), the Institute of Food Research, Norwich, UK, Food Standards
Agency, London, UK, and the Center of Excellence for Food Safety, Australia.

2.5 Predictive Modeling (PMP)

PMP was developed by USDA-ARS-ERRC in Wyndmoor, Pennsylvania. The PMP

(Ross et al. 1999; Tamplin et al. 2003; McMeekin et al. 2006) is a package of models
that can be used to predict the growth, survival, and inactivation of food-borne bacte-
ria, primarily pathogens, under various environmental conditions. These predictions
are specific to certain bacterial strains and specific environments (e.g., culture media,
food, etc.) that were used to generate the models. Since the early 1990s, the PMP has
been distributed in various forms, ranging from spreadsheets to standalone software.
New versions of the PMP (­ https://portal.errc.ars.usda.gov/PMP.aspx) are developed
with the addition of new models and/or changes in the graphic user interface.

2.6 Foodrisk.org and FDA-iRISK

Foodrisk.org is an online resource for food safety risk analysis and also provides some
unique contents through its website to the research community. Currently it offers
FDA-iRISK® 2.0, a Comparative Risk Assessment Tool for “users to compare and
rank risks from multiple foodborne microbial and chemical hazards and to predict
effectiveness of prevention and control measure” (http://foodrisk.org/exclusives/fda-
irisk-a-comparative-risk-assessment-tool/). “FoodRisk.org is operated by Joint
Institute for Food Safety and Applied Nutrition (JIFSAN) in collaboration with the
46 J. Gangiredla et al.

Center for Food Safety and Applied Nutrition from US Food and Drug Administration
(CFSAN/FDA) and the Food Safety and Inspection Services from US Department of
Agriculture (FSIS/USDA)” (http://foodrisk.org/aboutus/).

2.7 Data Mining Techniques

Data mining is an emerging multi-disciplinary field, including statistics, machine

learning, databases, information retrieval, visualization, etc. Data mining is about
data processing and applications of extracting valuable, previously unknown, and
comprehensible information from large databases and/or various resources. There
are two standard approaches for data mining: verification driven and discovery
driven. The possibilities for applying data mining for early warning in food supply
networks has been extensively reviewed (Beulens et al. 2006). For instance, existing
applications of data mining for food quality problems in the food supply chain
mainly consider specific tasks at individual stages. Due to the advances of IS, large
amounts of data about food production and processing are recorded every day from
the food supply chain. A variety of food safety measures throughout the entire food
chain are available for food quality at either the qualitative level or quantitative
level. The quality of predictive models also depends on how well the model class is
able to represent patterns in the data set. Some researchers are developing automatic
data mining techniques (Liao et al. 2012).

3 Novel Strategies for the Control of Foodborne Pathogens

Bioinformatics, an interdisciplinary field involving biology, computer science,

mathematics, and statistics, plays an important role in food safety research.
Understanding the genetic diversity among and between all foodborne pathogens
that are associated with human illnesses is not a trivial task in food safety. To be able
to understand the microorganisms and their associated potential threats, it is essen-
tial that the scientific and public health communities comprehend the genomic revo-
lution, and novel bioinformatic and computing tools. The genomic information can
be used to build several useful tools that allow food safety researchers to study how
specific microbial pathogens evolve and understand the massive compatible data in
order to study the pathogen. Advances in microbial bioinformatics offer better
insights into the emergence and spread of food borne pathogens especially in out-
break scenarios and in predicting where food contamination can occur at any step in
the “farm-to-the-fork” continuum (Fang et al. 2010).
In the era of variety in sequencing techniques, one of the fundamental roles of
bioinformatics is the prediction of function from the large amounts of sequence infor-
mation generated. By applying such high resolution molecular methods for identify-
ing and tracking microbial pathogens, the nature of foodborne outbreaks caused by
3 Application of Omics Technologies and Computational Approaches for Control… 47

these pathogens can be better understood, thereby helping to ensure the safety and
integrity of the food supply. Bioinformatics has been used in the development and
implementation of numerous “big science”-type projects that address specifically
issues applied to food safety and food microbiology. Bioinformatics is especially
successful if databases with high-quality data are available, together with structured
vocabularies that describe the content of the data.
For food safety, the term “omics” encompasses a cornucopia of different meth-
ods including genomics, transcriptomics, proteomics, and metabolomics that,
through advanced bioinformatics, provide insights relevant to reducing microbial
food safety hazards or risks (Bergholz et al. 2014). Omics approaches, other than
genomics and WGS, also are important tools and technology platforms in food
safety, but are more typically used in research applications and have not yet moved
to routine application to the same extent as WGS. Metagenomics-based character-
ization of microbial communities provides a very promising and powerful approach
in food safety testing, which allows for culture-independent identification from food
and environmental samples, clinical specimens, and non-culturable and difficult to
detect foodborne disease etiological agents.
Currently, four major research topics were identified over and about developing
Application of Omics technologies and Computational Approaches for Control of
Foodborne Pathogens in Foods.

3.1 Metagenomics Approach

Metagenomics encompass analyzing genetic information from all microbes that are
present in a particular community or environment. It can be used to investigate the
contribution of the foodborne pathogens living in specific environments (community)
to their onset and progression of their pathogenicity. Analysis of microbial genomes
in a particular community, for example, food borne disease associated microbes, can
be performed using different next-generation sequencing-based techniques such as
shotgun metagenomics and metatranscriptomics where random fragments of the
DNA or (enriched) mRNA of a given microbiome are sequenced with next-generation
sequencing (NGS) technologies. Metagenomics and metatranscriptomics techniques
are powerful, as they circumvent the need to grow microbes yet are able to determine
the gene content or gene expression of all microbes present in this community. This
provides a meaningful understanding of the molecular functions encoded by the
DNA, taxonomic assignment of that DNA fragments, or inferring similar information
for expressed mRNAs. These methods can be used in addition to sequencing of indi-
vidual isolates post purification from complex cultures. Sequencing of individual iso-
lates, however, has the advantage that comparative genomics can be done and
metabolic models can be built more straight-forwardly, provided that the isolates
under study are representative of the biodiversity present in the complex culture. In
the era of increasing heterogeneity of data sets, many bioinformatics methods and
techniques have been adapted to determine trends in typically large data sets. With the
48 J. Gangiredla et al.

help of next generation sequencing technologies, it has been demonstrated to be pro-

ficient in achieving the high-throughput and high-­quality sequencing results through
WGS of respective strains of interest. Machine learning algorithms have been devel-
oped and trained to use properties derived from samples to predict the nature of the
sample within the community.

3.2 Bioinformatics Data Analysis and NGS Technologies

Bioinformatics data analysis has been impactful in gaining a better understanding of

the community ecology of foodborne pathogens. This is a direct reflection of the
increase in NGS of large numbers of food samples and the sizeable amount of data
generated from these studies.
As an initial foray into bioinformatic analysis of such data, it is necessary to cata-
logue the diversity of genes found in a single species, therefore, large numbers of
whole genome sequences of that species need to be examined. As more isolates are
sequenced, additional genes are discovered and then added to the pangenome (Rasko
et al. 2008). However, as more isolates are sequenced, the number of core genes
(found in every isolate) decreases. The diversity of the isolates in a species that have
been sequenced can be observed by constructing a phylogenetic tree based on the
alignment of approximately 1000 core genes. Sequencing diverse representatives for
each species is vital to allow detection of varied isolates of each species in a mixed
community. In order to identify individual species in a whole genome shotgun
sequencing run of a mixed community, unique sequences can be identified that are
common to all members of a species or subspecies but are not found outside of the
species (Burall et al. 2016). This ability to identify pathogenic species in a mixed
sample of bacteria is critical to the detection and control of foodborne pathogens.
If many species of the community are not well represented in sequence databases,
a characterization of the genes present in the community can be made by matching
sequencing reads to protein databases (functional metagenomics). In this manner,
metabolic capabilities of the community can be inferred (Abubucker et al. 2012).
Study of metabolic functionality in communities may shed some light on the persis-
tence and spread of pathogenic species in different environments.

3.3 Computational Approaches

Computational approaches can be applied to elucidate the forces and mechanisms

that influence the communities in which foodborne pathogens evolve. The recent
advances in sequencing technologies and computational approaches are propelling
scientists to close the knowledge gap in regard to human-microbial interactions. As
an example, these sequence data can be retrieved, assembled, and analyzed to identify
microbial pathogens and diagnosis of diseases associated with foodborne illnesses.
3 Application of Omics Technologies and Computational Approaches for Control… 49

Traditional methods for detecting and identifying pathogens usually require culturing
of bacteria or viruses and then subsequently using the phenotypic, biochemical, or
serological tests. These methods have proven very successful for many types of
microorganisms and are standardized for most of the known human and domesticated
animal pathogens. However, one important limitation is that only a small fraction of
the estimated number of microbial species have been described, and therefore current
methods are applicable to relatively few species. Furthermore, many microorganisms
are very difficult to culture or may not grow at all, and therefore, they cannot be iden-
tified using traditional techniques. This is particularly problematic when an outbreak
associated with an unknown pathogen occurs, which demands the characterization
and identification of the microbes associated with the illnesses.
In addition to traditional methods, pan-genomic DNA microarrays are one of the
emerging research techniques used in the field of clinical microbiology for identifi-
cation of various pathogens which may be difficult to identify through the afore-
mentioned techniques. Microarrays have been proven to be an applicable technology
to detect food borne pathogens within 24 h. DNA microarrays offer a simple, rapid
and reproducible application for detection and characterization of the food borne
pathogens using gene targets as probes (Patel et al. 2016).
Next generation sequencing methods can provide the complete genome sequence
of a microbial strain of interest. This technology has supplanted many of the tradi-
tional methods due to its robustness, and high-throughput and high-quality sequenc-
ing results. The sequencing data generated are represented in FASTQ format. These
files provide, next to the raw sequence data, additional information regarding the
quality of the reads. Quality control and trimming can be applied using tools or done
manually using FASTQC computational method. Raw sequence reads from differ-
ent NGS technologies can be assembled into contigs, long stretches of DNA
sequence representing part of the genome. Most of the assembly methods are based
on alignment of sequence reads with each other (de novo assembly) or against a
reference genome (mapping assembly), thereby generating long DNA sequences
from the fragments generated by sequencing. Some examples of assembly tools are
CLC Genomics Workbench (https://www.qiagenbioinformatics.com/) and Spades
(http://bioinf.spbau.ru/spades).
Scaffolding is process that can assemble the contigs to larger, gapped, DNA
sequences. Some NGS techniques allow the synthesis of paired end (PE) or mate
pair (MP) libraries; libraries with a fixed insert size that are sequenced at both ends.
As reads span a larger DNA fragment, the matched reads pairs can be used to order
contigs, even if the sequence in between the contigs has not been assembled.
Gene functions can be predicted by comparing sequences to databases con-
taining genes with known functions with tools like MG-RAST (http://metage-
nomics.anl.gov) for the Functional annotation of these larger fragments is more
straight-­forward, but the fraction of reads that can be assembled into contigs
depends on both the complexity of the microbiome (many different microbes
with varying abundances) as well as the presence of microdiversity (many differ-
ent microbes with similar genome sequences). The development of powerful
alignment and annotation programs lead to identification of inaccurate sequence
assemblies and helped to refine the existing databases.
50 J. Gangiredla et al.

3.4 Computational Modeling

Computational modeling approaches are used to analyze the interactions between

microorganisms, as well as their impact on foodborne pathogen transmission.
Recent advances in sequencing technologies and computational approaches are
raising the possibility of determining the further understanding of microbial and
environmental interactions. Widespread use of new shotgun metagenomics
methodology could improve real-time disease surveillance, provide a better
quantitative picture of pathogen abundance, and assist scientists and medical
professionals to understand the response of the body’s natural microbiome.
Shotgun metagenomics is a derivation of conventional microbial genomics, with
the key difference being that it bypasses the requirement for obtaining pure cul-
tures for sequencing. Since the samples are obtained from communities rather
than isolated populations, metagenomics may provide opportunities to establish
hypotheses in regard to interactions between microbial community members. In
addition to identifying the microbes present in the samples, the method can also
measure the relative abundance of each microbial species and their virulence
potential, among other things.
This process begins with sample and metadata collection and proceeds to DNA
extraction, library construction, sequencing, read preprocessing, and assembly.
Community composition analysis is employed at several stages of this workflow,
and databases and computational tools are used to facilitate the analysis. Analysis
and comparison of complex metagenomic data are driving the development of a
new class of bioinformatics and visualization software. The field is moving forward
rapidly, driven by enormous improvements in sequencing technology and the avail-
ability of many complementary technologies.
Once DNA sequence data are generated, sequences must be analyzed with
special considerations in mind to facilitate accurate bacterial identification. First,
different taxonomic classifications can be used for identification, and different
species identifications may be generated depending on the taxonomic scheme.
HUMAnN2 (The HMP Unified Metabolic Analysis Network 2) is a pipeline that
was developed by the Huttenhower lab (Segata et al. 2012) for efficient and accu-
rate profiling to determine the presence/absence and abundance of microbial
pathways in a community from metagenomics or metatranscriptomic sequencing
data (typically millions of short DNA/RNA reads). This pipeline is also useful to
analyze community functional profiles stratified by known and unclassified
organisms. Gene function is typically inferred from similarity in amino acid
sequence and is then annotated. As many as half of foodborne disease outbreaks
are never attributed to a source. Those outbreaks may be caused by new infec-
tious agents, or by microbes not commonly seen in food. Metagenomics analysis
could help identify unknown microbes, potentially providing advance notice of
new outbreaks.
3 Application of Omics Technologies and Computational Approaches for Control… 51

3.5 Computational Metagenomic Approach

Currently there are over 10,000 raw genome sequences from numerous disease-­related
pathogens in the public domains, including over 1500 E. coli genomes. In 2012, “The
100 K Genome Project”, led by US scientists (http://100kgenome.vetmed.ucdavis.
edu) announced a project to sequence 100,000 bacterial and viral genomes. In several
years, there will be millions of genomes with online electronic records associated with
the source/origin and other phenotypic features of microbes available. Therefore, there
is a tremendous opportunity for computational identification of reads from NGS
sequencing of mixed samples. In this approach, scientists have to create a comprehen-
sive biological database of the most common food-borne pathogens based on a multi-
factorial combination of genetic, phenotypic, and environmental factors. As an
example, E. coli serogroup information that can be applied to identify and detect a
variety of existing, new, and emerging food-borne pathogens. This approach is par-
ticularly useful to identify the individual organisms present in the sample and to deter-
mine their relative abundance based on this comprehensive reference database.
Computationally, to cluster reference sequences of bacterial strains by similarity, one
can find the set of all k-mers (short nucleotide sequences) in each sequence and then
find the Jaccard Index. The Jaccard Index is calculated as the number of shared k-mers
divided by the total number of distinct k-mers in each pair of sequences. To cluster a
large number of sequences, the MinHash technique can reduce k-mer representations
of genome sequences into a much smaller set which can be used for distance calcula-
tions (Ondov et al. 2016). Signatures for each cluster can be obtained by finding shared
k-mers within a cluster that are not found in other clusters. Metagenomic reads can
then be identified by determining the presence of any signature k-mers (Leonard et al.
2015) Another approach, used by the MetaPhlan program, is to map reads against a
curated set of core genes from reference sequences (Segata et al. 2012).

3.6 A Case Study

For instance, serotyping provides a basis for categorization of bacterial strains and
is an important tool for outbreak detection and epidemiological surveillance. There
are approximately 187 different types of E. coli strains based on the reaction of their
surface lipopolysaccharide antigen to different antisera (serogroup) (Stenutz et al.
2006, DebRoy et al. 2016). There is also variety of virulence factors associated with
specific serotypes and presentence with different occurrence of diseases, such as
O157:H7 and non-O157 STEC. E. coli serotype O157:H7 and non-O157 STEC
serogroups, including O26, O45, O103, O111, O121, and O145 (top six) and others,
are important food-borne pathogens that cause similar illnesses (CDC Report 2011).
The classification of STEC is traditionally based on phenotypic analyses and/or
PCR-based molecular typing targeting specific biomarkers such as O-antigen and
52 J. Gangiredla et al.

stx genes (Aidar-Ugrinovich et al. 2007; Blanco et al. 2004; Monaghan et al. 2011).
These methods are time-consuming and labor-intensive because the samples from
complex clinical, food, or environmental sources must be enriched for further iden-
tification and characterization. As we have discussed in this chapter, one major
advantage of metagenomics is their selectivity and sensitivity of the enrichment/
isolation procedures for new or emerging pathogens viable but non-culturable
(VBNC) bacteria (Chen and Pachter 2005). Currently there are over 10,000 raw
genome sequences from numerous disease-related pathogens in the public domains,
including over 1500 E. coli genomes. Because of the 100 K Genome Project, there
is a tremendous opportunity here to create a comprehensive biological database of
the most common food-borne pathogens based on a multifactorial combination of
genetic, phenotypic, and environmental factors, including E. coli serogroup infor-
mation that can be applied to identify and detect a variety of existing, new, and
emerging food-borne pathogens.
In our laboratory, we use an iterative mapping approach to find the best fit
for each individual raw reads against the reference O-antigen cluster and vari-
ous factor sequences collected from various public resources. Although the
uniqueness of O-antigen clusters sequence, the presence of repeat elements,
some O-antigen genes with high levels of similarity dispersed throughout the
cluster, or the very nature of short-read assemblers, there will certainly be
assembled errors present in the mapping. Because multiple species are present
in these data sets, the pathogenicity of these data pools can only be detected as
a connectivity prediction and risk assessment with the whole “community
structure” based on the numerical output. And these outputs could be an indica-
tor for revealing the potential risks associated with food samples in this case.

4 Conclusion

In each of these areas, the first step will be to identify a number of methodological
methods but not limited to effective bioinformatics analysis approaches in order to
generate accurate metagenomics inventories for the descriptions of foodborne
pathogens and other organisms diversity. An additional step will be comprehen-
sively to compare publicly accessible databases and cross referencing to predictive
modeling, such as USDA developed PMP modeling. Finally, fill the gap between
the “foodborne pathogen focused” with their living environmental communities
and provide strategies for control foodborne pathogens as a whole community. In
the future, the only way to successfully address these new and exciting issues will
be to ensure that researchers remain open to these new concepts, facilitating valu-
able translational collaborations and exchanges between field workers including
industries and farmers, data analysts and scientists.
3 Application of Omics Technologies and Computational Approaches for Control… 53

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Chapter 4
Natural Food Antimicrobials of Animal Origin

Elba Verónica Arias-Rios, Elisa Cabrera-Díaz, Mayra Márquez-González,

and Alejandro Castillo

Abstract Various compounds found in animals possess antimicrobial activity

(Board, New methods of food preservation, Springer, New York, 1995; Phoenix
et al., Antimicrobial peptides, Wiley-VCH Verlag, GmbH & Co. KGaA, 2013).
These innate antimicrobials, known as antimicrobial peptides (AMPs), often are
part of the various immunity mechanisms of these animals. For example, the pres-
ence of endogenous peptides (natural or induced) provide effective means of defense
against pathogens. Some AMPs may be used in the food industry as preservatives to
prevent the growth of specific pathogens, including Gram-positive and Gram-
negative bacteria, viruses, parasites, yeast and molds.

Keywords Natural antimicrobials • Antimicrobial peptides • Animal AMP •

Lactoferrin • Lysozyme

E.V. Arias-Rios (*)

Department of Nutrition and Food Science, Texas A&M University,
College Station, TX, USA
e-mail: [email protected]
E. Cabrera-Díaz
Departamento de Salud Pública, Centro Universitario de Ciencias Biológicas y
Agropecuarias, Universidad de Guadalajara, Zapopan, Jalisco, Mexico
e-mail: [email protected]
M. Márquez-González
Food Science and Technology Department, Zamorano University, Tegucigalpa, Honduras
e-mail: [email protected]
A. Castillo
Department of Animal Science, Texas A&M University, College Station, TX, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 55

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_4
56 E.V. Arias-Rios et al.

1 Introduction

Various compounds found in animals possess antimicrobial activity (Board 1995;

Phoenix et al. 2013). These innate antimicrobials, known as antimicrobial peptides
(AMPs), often are part of the various immunity mechanisms of these animals. For
example, the presence of endogenous peptides (natural or induced) provide effec-
tive means of defense against pathogens. Some AMPs may be used in the food
industry as preservatives to prevent the growth of specific pathogens, including
Gram-positive and Gram-negative bacteria, viruses, parasites, yeast and molds.
AMPs are classified by (1) their source, including amphibians, fish, reptiles, and
mammals; (2) their biological function, such as antifungal, antimicrobial, antiviral,
antiparasitic, and insecticidal; (3) their nuclear magnetic resonance three-dimen-
sional structure, (4) their physicochemical properties such as charge, polarity, and
size; and (5) their covalent bonding patterns (Sadredinamin et al. 2016).
The mode of action of AMPs can be classified according to the disruption of the
cell due to interactions primarily with the cell membrane. AMPs typically have a
high concentration of hydrophobic residues containing amino acids, which provide
amphipathic characteristics. The amphipathic and cationic properties make them
more susceptible to interact with the cell membranes by disrupting it and leading to
leakage of the cell (Ayaad et al. 2012; Brandenburg et al. 2016). The non-lytic inter-
actions are still not well understood. This mechanism involves the translocation of
the AMPs to the inner membrane causing intracellular damage. These include inhi-
bition of the synthesis of DNA, proteins, cell wall, and inactivation of indispensable
enzymes (Scocchi et al. 2016).

2 Natural Antimicrobials in Milk

Milk is defined as the fluid secreted by the mammary glands of female mammals for
the nourishment of their young. Milk and dairy products are considered important
food items which contain numerous essential nutrients needed in human nutrition.
Although bovine milk is most commonly produced for human consumption, milk
from other mammal species including goat, sheep, donkey, camel and buffalo are
consumed in different regions worldwide. Bovine milk is a source of lipids, pro-
teins, amino acids, vitamins and minerals. Bovine milk also contains immunoglobu-
lins, hormones, growth factors, cytokines, nucleotides, peptides, polyamines,
enzymes, and other bioactive peptides (Haug et al. 2007; O’Mahony and Fox 2014).
Milk also contains AMPs that are active against a wide variety of microorganisms.
These include lactoferrin and several derived-peptides, lactoperoxidase, lysozyme,
and N-acetyl-ß-D-glucosaminidase, defensins, cathelicidins, RNases, and calgranu-
lins (Park and Nam 2015). Advance proteomic technologies and improvement in
bovine protein sequence databases have demonstrated the existence of more than
200 intact proteins in bovine’ milk, many of them associated with host-defense
4 Natural Food Antimicrobials of Animal Origin 57

(Wheeler et al. 2012). All together, these compounds seem to be involved in protect-
ing against mastitis and post-harvest bacterial growth, resulting in a protective effect
for the newborn and to some extent, for the consumers of processed milk. This sec-
tion presents the main characteristics and mechanisms of action of antimicrobial
compounds naturally present in milk and some of the technological applications of
these compounds.

2.1 Lactoferrin

Lactoferrin (LF) is an iron-binding glycoprotein classified as a member of the trans-

ferrin family and is present in the milk and other fluids of several mammals. The
molecule is a monomeric glycoprotein with a molecular weight of 80-kDa and a
single polypeptide chain of about 690 amino acid residues folded into homologous
N- and C-terminal lobes, each comprising two domains that enclose a conserved
iron-binding site (Baker and Baker 2005). Several biological activities of LF have
been described since this protein was first described by Sørensen and Sorensen in
1939 and isolated by Groves in 1960 (Groves 1960). Biological activities include
beneficial changes in the intestinal microbiota, antimicrobial effects on enteric
pathogens, immunomodulatory, and anticarcinogenic effects. The concentration of
this protein in the milk of food-animals is affected by several factors including
breed, age, stage of lactation, milk production, somatic cell count, and occurrence
of mastitis (Cheng et al. 2008).

2.1.1 Modification of the Intestinal Microbiota

Microbial colonization of the gut begins during birth and progressively change until
it stabilizes when infants reach 1 to 2 years of age. The intestinal microbiota is
largely diverse, includes 500 to 1000 species and is influenced by genetic factors as
hyper-immunity or immunodeficiency conditions and environmental factors like
antibiotic administration, diet, lifestyle, hygiene preferences, and competition
between bacterial populations. Anaerobic bacteria of the Firmicutes, Bacteroidetes,
and Actinobacteria phyla dominate the intestinal microbiota by establishing and
maintaining beneficial interactions with the host, thereby strengthening the intesti-
nal barrier, modulating the immune response and antagonizing pathogens (Pandeya
et al. 2012; Sommer and Backhed 2013). The oral administration of LF increases
the proportion of bacteria that are considered beneficial. The establishment of
Bifidobacterium as the dominant fecal microbiota was favored when LF was admin-
istrated to human infants at 10 mg/100 mL of the infant formula (Roberts et al.
1992). The administration of bovine transgenic milk containing recombinant human
LF to neonatal pig -used as an animal model for the human infant-, increased the
concentration of Lactobacillus and Bifidobacterium, creating an advantageous
microbiota in the gut. LF also increased the length of intestinal villi and had deeper
58 E.V. Arias-Rios et al.

Fig. 4.1 Antibacterial mechanisms of milk proteins and peptides

crypts (Cooper et al. 2014). However, in other investigations, the addition of bovine
LF to infant formulas had little effect on the fecal microbiota of infant babies.
Compounds such as immunoglobulin A, lysozyme, citrate, and bicarbonate, neces-
sary for the optimum activity of LF, are not present or occurs in very low concentra-
tions in the infant formulas (Wharton et al. 1994). In long-term adult patients, the
oral administration of LF resulted in prevention of post-antibiotic diarrhea, an
important cause of disability and death in this population (Laffan et al. 2011).

2.1.2 Antimicrobial Activity

The antibacterial activity of LF is related to mechanisms that alter the bacterial cell
physiology (Fig. 4.1). LF generates an iron-deficient environment that limits bacte-
rial growth and expression of virulence factors and damages the outer bacterial cell
membrane (Yen et al. 2009). The effect is similar to that observed for agents that
permeates the Gram-negative outer membrane, including metal chelators such as
ethylene diamine tetraacetic acid (EDTA) and polycationic compounds such as
polymyxin B and poly-l-lysine (Finnegan and Percival 2014).
Three major mechanisms explain the alteration produced by LF on the permea-
bility of the outer cell membrane: (1) the metal chelating capacity of the protein
remove stabilizing cations, (2) the bacterial membrane permeability is distorted
through production of bacterial siderophores, and (3) the direct binding of LF to
lipopolysaccharide (LPS) through an interaction with the anionic core structure
alters the normal membrane physiology. In Gram-positive bacteria, LF increases the
4 Natural Food Antimicrobials of Animal Origin 59

amount of lipoteichoic acid released from the surface of cells. In combination with
lysozyme, exert a synergistic antimicrobial effect through the binding of LF to lipo-
teichoic acid that neutralizes the negative charge of this structure and allows greater
access of lysozyme to the peptidoglycan (Leitch and Willcox 1999). In addition to
the effect on the bacterial surface, a ribonuclease activity that produces hydrolysis
of DNA and RNA molecules due to the elevated production of hydroxyl radicals via
a Fenton-type Haber-Weiss reaction has been described (Zhao and Hutchens 1994).
LF blocks the H+-ATPase during bacterial fermentation, causing an intracellular H+
accumulation and the acidification of the bacterial cytoplasm, whereas, during bac-
terial respiration, the protein blocks the ATPase complex causing accumulation of
H+ in the periplasmic space and perturbation of transmembrane proton gradient and
intracellular pH (Andres and Fierro 2010).
LF also interferes with the attachment of enteropathogenic bacteria to target
intestinal cells (Ochoa and Cleary 2009). In the presence of human and bovine LF,
the adherence of enteroaggregative Escherichia coli (EAggEC) to Caco-2 cells was
decreased and the biofilm formation was inhibited because of the degradation of
bacterial aggregative adherence fimbria (Yekta et al. 2010). Similarly, the invasive-
ness of Shigella flexneri and enteroadherent E. coli (EAEC) was reduced by the
presence of LF through mechanisms involving the disruption of the type III secre-
tory system (TTSS), a fundamental element in their pathogenesis (Ochoa and Cleary
2004; Ochoa et al. 2003). It has also been described that bovine LF blocks shiga
toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC)-mediated
actin polymerization in HEp-2 cells by degradation of the EspB protein, which is a
component of the TTSS (Ochoa and Cleary 2009).
LF exhibits a synergistic effect in the presence of other antimicrobials that
depends on the mechanism of action of each compound and the target microorgan-
ism and is also affected by the antimicrobial concentration and levels of cations in
the substrate. This synergistic effect could be useful either in therapeutic applica-
tions or in food systems helping to reduce the number of antimicrobials to be used
as part of a hurdle approach. When lysozyme and LF were individually used against
Vibrio cholerae, Salmonella Typhimurium and E. coli, the proteins showed a bacte-
riostatic effect. However, when applied in combination, the effect was bactericidal
and dose-dependent (Ellison and Giehl 1991). A synergistic effect was also described
for rifampin and is related to the ability of LF to destabilize the Gram-negative outer
membrane which becomes more permeable to this antibiotic (Ellison et al. 1988).
The minimum inhibitory concentration (MIC) of different antimicrobial agents is
reduced when they are combined with LF. Listeria monocytogenes was inhibited by
1000 μg/mL of LF and 100 UI/mL of nisin when individually tested. Only 250 μg/
mL of LF and 10 IU/mL of nisin were necessary to inhibit the pathogen when used
in combination (Murdock et al. 2007). Similar results were obtained for E. coli
O157:H7 when the MIC was reduced from 5000 to 500 μg/mL of LF and from 1000
to 250 IU/mL of nisin when the antimicrobials were individually tested or in com-
bination, respectively. For Shigella spp., 33% of 88 strains showed a twofold or
more reduction in the ampicillin MIC in presence of 10 mg/mL of bovine LF, but
only 5% of the strains showed a reduction in the trimethoprim-sulfamethoxazole
60 E.V. Arias-Rios et al.

MIC. This response was dependent on Shigella spp. because S. sonnei showed no
LF-induced decrease in ampicillin MIC, while S. flexneri, S. boydii and S. dysente-
riae did (Mosquito et al. 2012). Another synergistic effect was observed after using
LF in combination with a milk protein fraction that belongs to the RNase family,
obtaining higher reductions in E. coli O111, Cronobacter sakazakii, S. Enteritidis,
Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus,
Streptococcus mutans and Candida albicans populations in comparison to those
reductions observed for LF alone (Murata et al. 2013).
Several peptides derived from LF have demonstrated a potent antimicrobial
activity than sometimes is even more potent than that observed for LF. These pep-
tides which include lactoferricin (LFcin), lactoferrampin (LFampin), LF hydroly-
sate (LFH), L10, LF1–11 and LF chimera are produced either by enzymatic
hydrolysis with proteases in the gastrointestinal tract after milk is ingested, or they
are artificially synthesized (Sinha et al. 2013). LF-derived peptides belong to the
N-terminal half of LF and interact with negatively charged elements such as the
bacterial surface. Transmission electron microscopy analysis shows that some of
them modify the morphology of the bacterial cell membrane and cytoplasm causing
membrane “blisters”. LFcin is able to translocate across the cytoplasmic membrane
of both Gram-positive and Gram-negative bacteria and once inside the cytoplasm,
the peptide inhibits macromolecular synthesis of DNA, RNA, and proteins (Ulvatne
et al. 2004). Their antimicrobial activity is commonly dose-dependent and fre-
quently modulated by the cations Ca2+, Mg2+, and Fe3+. In some cases, the antimi-
crobial activity is influenced by hydrolysis type. For example, the hydrolysates
prepared by cleavage of LF with porcine pepsin or acid protease from Penicillium
duponti had stronger inhibition against E. coli O111 when compared to hydroly-
sates produced by trypsin, papain, or other neutral proteases (Tomita et al. 1991).
However, in the intestinal environment, the antimicrobial activity of the peptides
may be affected and ultimately determined by physiological factors of the host such
as pH, metal ions, and bile salts concentrations.
LFcin has shown antimicrobial effect against four strains of C. sakazakii and E.
coli (Wakabayashi et al. 2008), and demonstrated MIC values from 2 to 8 μM
against L. monocytogenes, E. coli, S. Salford, S. aureus, E. coli O:9, Bacillus cereus
and P. fluorescens (Hoek et al. 1997). Likewise, LFampin (van der Kraan et al.
2004) is active against Bacillus subtilis, E. coli, and P. aeruginosa but ineffective to
inhibit fermenting bacteria such as Actinomyces naeslundii, Porphyromonas gingi-
valis, Streptococcus mutans, and S. sanguis. The recently designed peptide L10
(Mishra et al. 2013) has a potent antibacterial activity with MICs ranging from 1 to
8 μg/mL against extended spectrum beta lactamases (ESBL) producing Gram-
negative bacteria, and for this reason has been proposed as a therapeutic solution for
infections caused by ESBL-producing bacteria of clinical significance.
Another peptide produced by the fusion of LFcin and LFampin is named LF
chimera and has significantly higher antimicrobial activity compared to those pep-
tides individually tested or evaluated in an equimolar mixture. The increased anti-
microbial activity of LF chimera seems to be related to its higher positive charge
that enhances bacterial membrane permeability providing entry of the peptide into
4 Natural Food Antimicrobials of Animal Origin 61

the cytoplasm to inhibit macromolecular synthesis (Haney et al. 2012; Ulvatne et al.
2004). LF chimera was very effective in inhibiting the growth of Vibrio parahaemo-
lyticus and V. cholerae O1 and non-O1 (Leon-Sicairos et al. 2009), showing a con-
centration-dependent response, and a synergistic effect with ampicillin, gentamicin,
and kanamycin. This hybrid peptide has also been effective in reducing the attach-
ment of EPEC to HEp-2 cells (Flores-Villaseñor et al. 2012a) because the molecule
blocks the EPEC-mediated actin polymerization and inhibits in a dose-dependent
manner, the attachment and A/E lesions caused by EPEC. At concentrations of 20
and 40 μM, LF chimera diminished the level of espA gene expression and signifi-
cantly blocked the hemolysis produced by EPEC. In vivo experiments using animal
models indicated the oral administration of LF chimera produced a significant
reduction in the mortality rate, sepsis, and damage in kidney and liver of mice
infected with E. coli O157:H7 (Flores-Villaseñor et al. 2012b). The pathogen con-
centration excreted in the mice feces after 48 h of infection was also reduced.
LF and LF-derived peptides used in combination with probiotic bacteria are
effective against foodborne pathogens. The growth of probiotic strains including
Lactobacillus rhamnosus, L. reuteri, L. fermentum, L. coryniformis, L. acidophilus,
Bifidobacterium infantis, B. bifidum and Pediococcus acidilactici was not affected
by LFH while the peptide inhibited foodborne pathogens including E. coli, S.
Typhimurium, S. aureus, and Enterococcus faecalis (Chen et al. 2013). However,
the antimicrobial activity observed in buffer systems or culture broths under labora-
tory conditions may be different to that observed in food systems where the antimi-
crobial activity may be affected by water activity, pH, ingredients and cation content
in the food (Bellamy et al. 1992). For example, the antimicrobial activity of LF and
LFH against S. Stanley, E. coli O157:H7, L. monocytogenes, and S. aureus was
effective when suspended in peptone yeast extract glucose broth (PYE) but very
limited in UHT milk (Murdock and Matthews 2002).
The antimicrobial effect of LF and derived peptides on foodborne parasites has
also been observed. LFcin showed a highly parasiticidal effect on Cryptosporidium
parvum and a strong reduction of the sporozoite infectivity (Carryn et al. 2012). A
giardicidal activity (DL50 of 8 μg/mL) and a decrease in trophozoite viability at
levels of 12 μg/mL was described for a bovine LF-derived peptide (Turchany et al.
1995). Amoebicidal activity against Entamoeba histolytica was observed for LFcin,
LFampin and LF chimera in a time and dose-dependent mode (López-Soto et al.
2010). At concentrations of 50 μM, LFcin and LFampin affected the viability of
50–55% of trophozoites after 24 h of culture, while only 25 and 0% of the tropho-
zoites remained viable when LF chimera was used at 50 and 100 μM, respectively.
According to this study, LF-derived peptides may be used as therapeutic agents to
substitute metronidazole, a very toxic compound for the host.
Bovine LF and its derived peptides have also revealed a significant inhibitory
effect on foodborne viruses. The antiviral effect on naked viruses including rotavi-
rus, poliovirus, coxsackievirus, enterovirus and adenovirus is related to the early
phases of viral infection preventing virus attachment to intestinal cells by binding to
viral particles, inhibiting a post adsorption step, or interfering with virus attachment
to cell membranes through competition for common glycosaminoglycan receptors
62 E.V. Arias-Rios et al.

and interaction with viral structural polypeptides (Pietrantoni et al. 2003; Seganti
et al. 2004). Bovine LF inhibits enterovirus 71 (EV71) infection by binding to viral
protein VP1 and to host cells, as demonstrated in a challenge using human rhabdo-
myosarcoma and neuroblastoma SK-N-SH cell lines (Weng et al. 2005). Peptides
produced by digestion of bovine LF with human gastric juice and duodenal juice in
a digestion model simulating physiological conditions in adults and infants were
effective in inhibiting the human echovirus 5 in a cell culture (Furlund et al. 2012).
Apparently, LF inhibits echovirus infectivity by interaction with capsid proteins that
induces alterations preventing virion uncoating and entry to the target cell
(Ammendolia et al. 2007). There is also evidence that the antiviral mechanism of LF
against rotavirus lies in the prevention of adsorption of the virus to the target cell by
binding virus particles, as determined with flow cytometry (Superti et al. 1997).
However, using LF in humans may not show similar results as laboratory experi-
ments as noted by a study where the oral supplementation of bovine LF at a dose of
70 mg/day did not show any benefits in the prevention of EV71 or rotavirus infec-
tion in healthy children (Yen et al. 2011), and the authors concluded that further
studies using higher doses of LF are required. LF and derived peptides have antifun-
gal activity by permeating the fungal membrane and suppressing mitochondrial res-
piration. These effects have been demonstrated in Candida spp. (Mishra et al. 2013).

2.1.3 Applications in Foods

There is an increasing interest in the use of LF and its derived peptides for techno-
logical applications either as nutritional dietary supplements, functional foods, anti-
microbial agents in food systems and cosmetics, as well as preventive or therapeutic
agents against foodborne pathogens, particularly because antimicrobial resistance
to antibiotics has been recognized as a worldwide problem (Table 4.1). However, as
most proteins, these compounds are fragile and structural changes produced by

Table 4.1 Technological applications of lactoferrin and its derived peptides

Application References
Nutritional supplement in capsules, powders, sticks, Wakabayashi et al. (2006)
bars
Nutritional and antimicrobial supplement in infant Yener et al. (2009), Johnston et al.
powder formulas and liquid concentrates (2015), Ke et al. (2015)
Nutritional supplement in skim milk Wakabayashi et al. (2006)
Antimicrobial agent in chitosan, alginate, whey protein, Brown et al. (2008)
starch, and amaranth edible films
Nutritional supplement in yogurt and ice cream Wakabayashi et al. (2006)
Antimicrobial spray for beef carcasses Loretz et al. (2010)
Antimicrobial additive in toothpaste and mouth washing Wakabayashi et al. (2006)
solutions
Nutritional and antimicrobial additive in feed rations for Wakabayashi et al. (2006)
calves, heifers, pigs and fish
4 Natural Food Antimicrobials of Animal Origin 63

interaction with other molecules in a commercial formulation may reduce its activ-
ity. To overcome this problem, stabilization by encapsulation into nanometre sized
vesicles to protect the bioactive protein or peptide from denaturation by proteolysis
and dilution effects has been proposed (Balcao et al. 2013). Using this approach, the
LF-encasing nanovesicles could be utilized in the formulation of antimicrobial edi-
ble films or food packing.
The high cost of LF or derived peptides production from native digestion or
chemical synthesis may represent a limitation for their use in large scale and to
overcome this, several approaches has been proposed. Some studies focus on the
expression of recombinant bovine LF-derived peptides in bacteria such as E. coli
(Garcia-Montoya et al. 2013) and Photorhabdus luminescens (Tang et al. 2010), or
in the yeast Pichia pastoris (Tang et al. 2012b). A LF-derived peptide produced via
fed-batch fermentation in recombinant strain P. pastoris (KM71) XS10 (Tang et al.
2012a) was added to the diet of weaned piglets. After 21 days, the Lactobacilli and
Bifidobacteria populations were increased in the stomach, duodenum, jejunum,
ileum, colon, and caecum. On the other hand, the advanced computer-assisted
design of enhanced variants of antimicrobial peptides has been developed to deliver
more potent, cost-effective, broad-spectrum peptides as potential next-generation
antibiotics (Fjell et al. 2012). A different approach consists in the production of milk
containing recombinant human LF (rhLF). When the milk containing rhLF obtained
from transgenic cattle was administrated to piglets, the concentrations of foodborne
pathogens throughout the intestine were reduced, and concentrations of
Bifidobacterium spp. in the ileum and of Lactobacillus spp. were increased, sug-
gesting that rhLF can modulate the intestinal microbiota in this animal model (Hu
et al. 2012; Maga et al. 2012). Commercial production of milk from genetically
engineered cattle despite its benefits is influenced by the consumer’s acceptance and
the perception of direct or indirect risks and benefits. Therefore, development of
transgenic foods with improved health benefits will likely be better received by
consumers than products that primarily benefit the grower or developer of the prod-
uct (Byrne 2006).

2.2 Lysozyme

Lysozyme (LZ) is a protein found in the milk of almost all mammals either as a free
soluble protein or within leucocytes and lysosomes (Benkerroum 2008). The con-
centration of soluble LZ in milk varies among species and within the same species
depending on the breed, stage of lactation, parturition, nutrition, udder health and
season of the year. In bovine milk, the protein concentration is much lower (0.05–
0.21 mg/L) than in human milk (224–426 mg/L) (Benkerroum 2008). Bovine’s milk
has a LZ concentration of 0.1–0.2 μg/L (Piccinini et al. 2005), and this protein
contains 129 amino acid residues, which differ from those present in human milk
and egg white LZ (Benkerroum 2008). Health benefits related to LZ include changes
in the gut microbiota, inhibition of a wide range of microorganisms, and production
of synergistic effects with other milk proteins.
64 E.V. Arias-Rios et al.

2.2.1 Modification of the Intestinal Microbiota

The intestinal microbial diversity in young piglets after feeding with goat’s milk
expressing human LZ was significantly changed (Maga et al. 2012). The levels of
clostridia declined whereas those of Bacteroidetes increased over time and the
abundance of bacteria associated with gut health including members of
Bifidobacteriaceae and Lactobacillaceae.

2.2.2 Antimicrobial Activity

The LZ molecule has an inhibitory effect on bacteria, fungi, protozoa and viruses.
In bacteria, mainly Gram-positive, the antimicrobial effect is related to the hydroly-
sis of (1 → 4)-β-linkages between N-acetylmuramic acid and N-acetyl-D-
glucosamine residues in peptidoglycan. Lysozyme can be effective against
Gram-negative bacteria in the presence of membrane destabilizing agents, such as
detergents and chelators (Boland et al. 2003). In fungi, the hydrolysis is produced in
the N-acetyl-D-glucosamine residues in chitodextrins (Safarik et al. 2007).
LZ antimicrobial action varies according to its origin. Buffalo milk LZ possesses
a sequence of 23 amino acid residues at the N-terminal end with 56.5% homology
to bovine milk LZ, and 30.4% to equine milk LZ. The specific activity of buffalo
milk LZ is ten-times that of bovine milk LZ. It is active over a wide range of pH and
its activity is strongly influenced by the molarity of the medium. Antibacterial activ-
ity of buffalo milk LZ showed that out of seven Gram-positive bacteria tested, four
(M. luteus, B. subtilis, L. lactis subsp. Lactis, E. faecalis) were inhibited, while
Gram-negative bacteria (E. coli, P. vulgaris, P. aeruginosa, S. ser. Typhi) were resis-
tant (Priyadarshini and Kansal 2002).
The amoebicidal activity of LZ is related to the damage of the amoebic mem-
brane causing lipid disruption and cell rounding as demonstrated by studies on
Entamoeba histolytica trophozoites treated with 5–20% of bovine milk; the effect
was concentration-dependent and increased in the absence of iron (Leon-Sicairos
et al. 2006).

2.2.3 Applications in Foods

LZ has been used as an antimicrobial agent on fresh fruits and vegetables, seafood,
meats, dairy products and infant formulas (Proctor and Cunningham 1988; Liburdi
et al. 2014).
As part of a hurdle approach, the synergistic effect of LZ and high-pressure
homogenization in skim milk has been proposed for L. monocytogenes control. The
interaction of high-pressure homogenization and LZ was associated with conforma-
tional changes of the protein with a consequent enhancement of its activity and a
significant reduction of the pathogen within 3 h in skim milk at 37 °C (Vannini et al.
2004). Similarly, the antimicrobial effect of high-intensity pulsed electric field
4 Natural Food Antimicrobials of Animal Origin 65

(HIPEF) may be enhanced by combining this treatment with LZ added to milk

inoculated with S. aureus. The application of 1200-μs HIPEF treatment time to milk
containing 300 IU/mL of LZ resulted in a reduction of more than 6.2 log of S.
aureus (Sobrino-López and Martín-Belloso 2008). For applications in food packag-
ing, combinations of LZ and LF have been incorporated into carboxymethyl cellu-
lose, producing 2 and 1 log cycle reduction in E. coli and L. innocua populations,
respectively (Barbiroli et al. 2012). The LF/LZ interaction is considered synergistic,
as LF inhibits L. innocua growth by binding iron, and therefore facilitating the
action of LZ. When incorporated into edible films used in salmon packaging, L.
monocytogenes was reduced 1.3 and 2.7 log CFU/g when the films contained 25 and
35 mg/g of LZ, respectively (Min et al. 2005).
The stability of LZ results very important for technological applications. Some
studies have demonstrated that LZ activity is slightly affected by HTST pasteuriza-
tion and more than 75% of the protein activity in bovine milk survives heating at
75 °C for 15 min or 80 °C for 15 s (Fox and Kelly 2006a, b). To increase the con-
centration of LZ in bovine milk, cloned cattle expressing recombinant human LZ
with similar pH and temperature stability than natural LZ has been produced (Yang
et al. 2011). However, consumption of milk from genetically modified animals may
have a low consumer’s acceptance in the near future.

2.3 Lactoperoxidase

Lactoperoxidase (LPO) is a heme-containing glycoprotein of 608 amino acids and

a molecular mass of approximately 78 kDa. LPO is one of the most prominent
enzymes in bovine milk and catalyzes the oxidation of certain molecules, mainly
thiocyanate ion (SCN−), using hydrogen peroxide to generate reactive products with
a wide antimicrobial activity. SCN− can be converted by LPO into hypothiocyanate
(SCNO−), a potent inhibitor of bacterial growth. Bovine’s milk contains a higher
concentration of LPO when compared to human milk. This difference is attributed
to the bovine diet (Hettinga et al. 2011). The activation of the LPO system (LPOs)
has a bacteriostatic effect on raw milk and extends the shelf life of this product for
7 to 8 hours at 30 °C, and for longer periods at lower temperatures. This provides
longer time to transport the raw milk from the collection point to the processing
center without refrigeration and is particularly useful in small communities.
According to the recommendations of the Food and Agricultural Organization and
the World Health Organization of the United Nations (FAO/WHO), the purpose of
using the LPOs is not to render milk safe for consumption but to preserve its initial
quality (FAO/WHO 2006). The inhibitory effect of the treatment depends on the
storage temperature of the LPOs treated milk and the initial microbial count of milk.
Therefore, good hygienic practices in milk production are critical to the efficacy of
the LPOs and to the microbiological quality of the milk.
Thiocyanate ions, in the form of sodium or potassium salt, are the substrate for
LP and are usually added to milk at a level of approximately 14 mg/L. This is followed
66 E.V. Arias-Rios et al.

by addition of peroxide, either as hydrogen peroxide (1–10 mg/L) or sodium percar-

bonate (30 mg/L). The LPOs can also be activated using a halide such as I− instead
of SCN−. LP + I− + H2O2 system can reduce the S. aureus, P. aeruginosa, and B.
cereus counts below the detection limit (1 log CFU/mL) shortly after treatment
(Fweja et al. 2008). The LPOs was found to increase the keeping quality (KQ) of
ultra-high treated milk inoculated with P. aeruginosa, S. aureus and Streptococcus
thermophilus, but had little or no effect in raw milk, presumably due to the higher
resistance of the microflora to the LP systems. The authors of this study suggest that
the LPOs is responsible for the greater KQ of milk pasteurized at 72 °C/15 s com-
pared with 80 °C/15 s and emphasizes the importance of selecting pasteurization
temperatures. The increment of the temperature during pasteurization of low quality
milk will not result in an increment on the KQ of the milk (Marks et al. 2001). The
LPOs was also tested to inhibit S. Enteritidis in tomato juice, carrot juice, milk,
liquid whole egg, and chicken skin extract under various conditions (Touch et al.
2004). The system was more effective against the organism when tested in vegetable
juices than in animal products, at lower pH, and higher temperatures. The LPOs
reduced up to 5.4 logs of S. Enteritidis in vegetable products and inhibited the
growth of the organism in animal-derived foods during 4 h incubation at
30 °C. According to this study, the LPOs could control Salmonella in minimally
processed fruit and vegetable products. However, the combination of the system
with other preservatives or treatments would be needed to effectively inhibit the
growth and the survival of the pathogen in animal products. However, more studies
challenging other Salmonella serotypes are necessary because resistance to antimi-
crobial agents may differ among serotypes.
As demonstrated with other natural antimicrobial components of milk, the LPOs
shows a synergistic effect when combined with other antimicrobials. The addition
of nisin and LPOs had a synergistic effect and resulted in L. monocytogenes counts
up to 5.6 logs lower than the control milk. When the two preservatives were added
to actively growing cells of L. monocytogenes in two steps with a 2 h interval, their
synergistic effect was enhanced. The difference in counts increased to 7.4 logs if
LPOs was added after 3 h and nisin after 5 h of growth (Zapico et al. 1998). When
100 or 200 UI/mL of nisin were added to cells already in contact with LPOs over
24 h, L. monocytogenes was not detectable after 196 and 244 h, respectively
(Boussouel et al. 2000). In the presence of lacticin 3147 and the LPOs in rehydrated
infant formula, Cronobacter spp. was inhibited for up to 12 h at 40 or 50 °C show-
ing the potential of hurdle approaches to improve the safety of infant milk formula
(Oshima et al. 2012).

2.3.1 Technological Applications

The inhibitory activity of nisin, reuterin, and the LPOs, added individually or in com-
bination, against L. monocytogenes and S. aureus was investigated in a curdled milk.
The combination of these three antimicrobial compounds acted synergistically on the
inactivation of the inoculated pathogens over 12 days at 10 °C and may be part of a
hurdle concept to control the growth of pathogenic microorganisms in non-acidified
4 Natural Food Antimicrobials of Animal Origin 67

dairy products (Arques et al. 2008). However, other studies have reported no synergis-
tic effect when a combination of bacteriocin-producing lactic acid bacteria and the
LPOs were used in refrigerated raw milk to control L. monocytogenes (Rodriguez
et al. 1997). This emphasizes the importance of validating the antimicrobial treat-
ments proposed to control foodborne pathogens in any food system.

2.4 Cathelicidins

Cathelicidins comprise a family of small, cationic peptides with antimicrobial prop-

erties found in vertebrates such as humans and farm mammals, some species of
birds, and fish. Cathelicidins contain an N-terminal cathelin-like ___domain, which is
conserved between mammals, and a diverse C-terminal cationic antimicrobial
___domain. Most cathelicidin peptides show a wide spectrum of antimicrobial activity
which includes Gram-positive and Gram-negative bacteria, enveloped viruses, and
fungi, and take part in the regulation of the adaptive immunity in the host (Brogden
et al. 2001; Das et al. 2006; Dürr et al. 2006; Gennaro et al. 1989; Kokryakov et al.
1993; Shamova et al. 1999; Scocchi et al. 1999; Uzzell et al. 2003; Xiao et al. 2006).
The bactericidal activity correlates positively with the magnitude of a gradient of
hydrophobicity along the peptide backbone and with net positive charge. These gra-
dients induce destabilization of the membrane and partial penetration of the peptide
in the bilayer membrane (Travis et al. 2000). Some cathelicidins, such as bovine
bacternecin 5 and 7 show a different mode of action according to their structure. The
homodimeric bactenecins form pores in the membrane and cause leakage of the
bacterial cell whereas monomeric peptides attack intracellular organelles. Therefore,
the mechanisms for the synthesis of the cell walls, proteins, and nucleic acids can be
disrupted (Lee et al. 2009).

3 Natural Antimicrobials in Egg

There are several antimicrobial compounds present in the egg white. Their antibac-
terial effect is mainly attributed to avian albumen which possesses alkaline pH and
proteins that inhibit microbial growth. The major albumen proteins with antimicro-
bial activity are ovotransferrin and lysozyme (Giansanti et al. 2012).

3.1 Ovotransferrin

Ovotransferrin is a glycoprotein found in egg white and belongs to the iron-binding

glycoproteins. Ovotransferrin structure is a single glycopeptide chain containing
686 amino acids, with a molecular weight of 77.90 kDa (Wu and Acero-Lopez
2012). The principal function of ovotransferrin is to promote the growth of the egg
68 E.V. Arias-Rios et al.

embryo due to its iron-binding capability (Giansanti et al. 2012). Ovotransferrin

antimicrobial activity has been associated with its ability to bind and sequester iron
(Fe+3), which is essential for bacterial growth and to membrane damage (Aguilera
et al. 2003; Beekman et al. 2007; Ibrahim et al. 2000; Seol et al. 2009; Valenti et al.
1983). Ovotransferrin can permeate E. coli outer membrane to access inner mem-
brane and cause ions permeation, inhibiting energy production by respiration and
disruption of cytoplasmic membrane function. It seems that a 92 amino acid peptide
(OTAP-92) disrupts the lipid bilayer of the cell likely by forming an ion cannel
(Ibrahim et al. 2000).
Antiviral activity against Marek’s disease virus in domestic chickens has been
reported (Giansanti et al. 2005). The antiviral activity of ovotransferrin is related to
two peptides that have sequence homology to bovine lactoferrin fragments. The
antimicrobial effect of ovotransferrin in combination with other antimicrobials on
edible coatings has been evaluated against foodborne pathogens. Seol et al. (2009)
evaluated the antimicrobial effect of edible coating films containing κ-carrageenan,
ovotransferrin, and EDTA (5 mM) on total aerobic bacteria and E. coli counts in
chicken breasts stored at 5 °C for 0, 1, 4 and 7 days. Chicken breasts with ovotrans-
ferrin edible films showed significant lower counts than controls (5.23 and 7.03 log
CFU/g after 7 days of storage for total bacteria and 1.99 and 4.65 log CFU/g for E.
coli counts for coated breasts and controls respectively).

3.2 Lysozyme

Lysozyme is defined as 1,4-β-N-acetylmuramidases, which cleave the glycosidic

bond between carbon C1 and C4 of N-acetylmuramic acid and N-acetylglucasamine
molecules present in the bacterial peptidoglycan (Jollès and Jollès 1984), result-
ing in peptidoglycan hydrolysis and cell lysis of bacteria (Masschalck and
Michiels 2003). The antimicrobial activity of chicken egg-white lysozyme is
well known as an antimicrobial protein, effective against to some Gram-positive
and Gram-negative bacteria (Ibrahim et al. 1996; Masschalck and Michiels 2003;
Pellegrini et al. 1997). Lysozyme has been reported to be effective as a preserva-
tive of milk, dairy products (Nakimbugwe et al. 2006), and beer (Makki and
Durance 1996).
Lysozyme peptides powder (LzP) is commercially available and has been evalu-
ated to be used as a potential preservative in foods. Abdou et al. (2007) evaluated
LzP in a liquid media as a food system against vegetative cells and spores of differ-
ent Bacillus spp. at a concentration of 0, 5, 10, 20, 50, and 100 μg/mL. Complete
inhibition was observed for B. subtilis, B. licheniformis, B. pumilus, B. mycoides, B.
coagulans, B. amyloliquefaciens, B. megaterium, B. polymexa, and B. macerans at
concentration of 100 μg/mL. B. cereus and B. stearothermophilus showed slight
resistance. Lysozyme decreased activity against B. stearothermophilus was attrib-
uted to prolonged incubation at 55 °C, the optimum temperature for B. stearother-
mophilus growth.
4 Natural Food Antimicrobials of Animal Origin 69

Ávila et al. (2014) studied the inhibition of lysozyme against spores and veg-
etative cells of Clostridium spp. on modified reinforced Clostridia media (mRCM)
and milk. Lysozyme inhibited the growth of vegetative cells of all Clostridium
strains in mRCM. Vegetative cells of C. tyrobutyricum showed the highest sensi-
tivity to lysozyme (MIC values <0.20–12.50 μg/mL), while spores of C. tyrobu-
tyricum showed MIC values of 1.57–400 μg/mL. C. sporogenes strains were not
inhibited even using the highest concentration of lysozyme (400 μg/mL). Higher
concentrations of lysozyme were required to inhibit the growth of C. sporogenes
in milk (50→400, 400 μg/mL).
The more useful applications of lysozyme have been proposed as active packag-
ing film in several meat and fish products (Barbiroli et al. 2012; Gill and Holley
2000; Mangalassary et al. 2008; Min et al. 2005; Nattress and Baker 2003). Min
et al. (2005) demonstrated feasibility to use whey protein films (WPI) containing
lysozyme to control L. monocytogenes on cold-smoked salmon. The coating (con-
taining 204 mg of lysozyme per g of film on a dry basis) inhibited the growth L.
monocytogenes that contaminated the surface of smoked salmon, no matter where
contamination occurred before or after the film or coating treatment. Barbiroli et al.
(2012) evaluated the effect of active paper with lysozyme on veal carpaccio (thin
slices of veal fillet, to be consumed as raw meat). After 48 h of storage at 4 °C, total
viable counts were reduced by 0.93 log cycles of CFU/g (initial population of 5.55
log CFU/g).

3.3 Regulation of Natural Antimicrobials in Egg

Egg-white lysozyme is recognized as GRAS by the US Food and Drug Administration

(US FDA) for use in preventing late blowing of cheese caused by the bacterium
Clostridium tyrobutyricum during cheese production. It is also approved for use in
casings and on ready-to-eat cooked meat and poultry products. Casings can contain
up to 2.5 mg per pound in finished products, 2.0 mg per pound on cooked meat and
poultry products (USDA FSIS 2014). In the European Community, lysozyme is
allowed to be used during cheese and ripened cheese production (Commission
Regulation (EU) 2011), beer and malt beverages (Règlement (UE) 2012), and fruit
wine (Commission Regulation (EU) 2013).

4 Natural Antimicrobials in Honey

“Honey is the natural sweet substance produced by honey bees from the nectar of
plants or from secretions of living parts of plants or excretions of plant sucking
insects on the living parts of plants, which the bees collect, transform by combining
with specific substances of their own, deposit, dehydrate, store and leave in the
honeycomb to ripen and mature” (Codex Alimentarius 2001). Honey composition
70 E.V. Arias-Rios et al.

may vary among the plants from which bees take nectar and pollen. Honey contains
an average of 17% water, and 95% of its solids are carbohydrates. The main sugars
in honey are fructose, glucose, and sucrose. Beside sugars and water, honey also
contains enzymes and acids. Enzymes found in honey are derived from the hypo-
pharyngeal glands of worker honeybees such as: invertase, glucose oxidase, and
amylase (El-Soud 2012). Other authors reported the presence of the enzyme lyso-
zyme. It is related with antibacterial defense of insects and its presence is strongly
induced when the insects are infected (Weston et al. 2000).
The antimicrobial activity of honey has been associated with several compounds
such as hydrogen peroxide (Brudzynski 2006; Taormina et al. 2001), phenolic com-
pounds (Alvarez-Suarez et al. 2010; Weston et al. 1999), lysozyme, and flavonoids.
Antimicrobial compounds in honey may vary depending upon floral source
(Alvarez-Suarez et al. 2010), heat treatment (Taormina et al. 2001), and age
(Brudzynski and Kim 2011).
Hydrogen peroxide was the one of the first antimicrobial compounds found in
honey that has been extensively reviewed (Kwakman and Zaat 2012; Molan 1992).
Hydrogen peroxide in honey is formed during the ripening process of honey by
degradation of glucose by the enzyme glucose oxidase (White 1978). Taormina
et al. (2001) studied the inhibitory effect of honey on foodborne pathogens. When
S. sonnei, L. monocytogenes, and S. aureus were exposed to 25% honey solutions in
0.1 M potassium phosphate buffer (pH 7.0) and incubated at 37 °C for 24 h, the
Montana buckwheat and blueberry honeys showed bactericidal effect on S. aureus
populations while little or no effect was showed on S. sonnei and L. monocytogenes.
In the same study honey from avocado, safflower and clover floral sources did not
show inhibitory effect against any of the pathogens tested. The honeys that showed
either little bacteriostatic or bactericidal effect against S. aureus also showed the
highest antioxidant power values (345–1009 μM expressed as ferric reducing anti-
oxidant power, FRAP). Honeys with no effect on foodborne pathogens showed
FRAP values <300 μM, supporting that antioxidant substances present may contrib-
ute to honey antibacterial activity.
An in vitro study conducted by Alnaqdy et al. (2005) reported the inhibition
effect of honey on Salmonella adherence to intestinal epithelial cells of MF1 mice.
In their study, Salmonella cells suspensions (1 × 109 CFU/mL) were pretreated by
incubation for 60 min at 37 °C in honey preparations diluted serially (ranging from
1:2 to 1:64) with phosphate-buffered saline (PBS) at pH7.2. In the absence of honey,
epithelial cells showed significantly greater adherence of bacteria (25.6 ± 6.5
­bacteria per cell) compared to that in any of the preparations from bacterial groups
pretreated with honey at dilutions of 1:2–1:8 (5–7 bacteria per cell).
There are several phenolic compounds reported in honey (Table 4.2). Phenolic
compounds and flavonoids in honey are derived from propolis (bee glue). Propolis
is a sticky dark-colored material that honeybees collect from living plants. The
source of propolis may be substances secreted by plants or exuded by plants wounds
such as mucilage, gums, and resins, among others (Martinioni and Ranzato 2015).
Bees mix these substances with wax and use in the construction and adaptation of
their nests (Bankova et al. 2000). Phenolic composition may vary upon geographi-
4 Natural Food Antimicrobials of Animal Origin 71

Table 4.2 Phenolic compounds identified in honey

Concentration
Floral source Phenolic compound reported Units Reference
Manuka Gallic acid trace - 1.4 mg/kg Stephens
(Leptospermun Abscisic acid ≤ 1.0 mg/kg et al. (2010)
scoparium) Phenyllactic acid trace - 1920 mg/kg
4-Methoxyphenyllactic acid 2.1–36.3 mg/kg
4-Methoxybenzoic acid 0.2–3.0 mg/kg
2-Methoxybenzoic acida 1.4–52.1 mg/kg
Trimethoxybenzoic acida 24–240 mg/kg
Methyl syringate 5.1–207 mg/kg
Syringic acid 0.2–2.9 mg/kg
Methylgyoxal 218–1541 mg/kg
Kanuka (Kunzea Gallic acid trace - 0.5 mg/kg Stephens
ericoides) Abscisic acid 0.4–2.9 mg/kg et al. (2010)
Phenyllactic acid 360–910 mg/kg
4-Methoxyphenyllactic acid 13.9–161 mg/kg
4-Methoxybenzoic acid trace - 5.0 mg/kg
2-Methoxybenzoic acida trace - 1.1 mg/kg
Trimethoxybenzoic acida 1–22 mg/kg
Methyl syringate 29–130 mg/kg
Syringic acid 1.0–1.9 mg/kg
Methylgyoxal trace-174 mg/kg
Clover (Trifolium Gallic acid trace mg/kg Stephens
spp.) Abscisic acid 2.8 mg/kg et al. (2010)
Phenyllactic acid 20 mg/kg
4-Methoxyphenyllactic acid 5 mg/kg
4-Methoxybenzoic acid trace mg/kg
2-Methoxybenzoic acida trace mg/kg
Trimethoxybenzoic acida 2.0 mg/kg
Methyl syringate 1.7 mg/kg
Syringic acid 0.1 mg/kg
Methylgyoxal non detectable mg/kg
Rewarewa Gallic acid 0.5 mg/kg Stephens
(Knightia excelsa) Abscisic acid 2.2 mg/kg et al. (2010)
Phenyllactic acid 80 mg/kg
4-Methoxyphenyllactic acid trace mg/kg
4-Methoxybenzoic acid non detectable mg/kg
2-Methoxybenzoic acida 0.4 mg/kg
Trimethoxybenzoic acida 27.2 mg/kg
Methyl syringate 12.7 mg/kg
Syringic acid 0.4 mg/kg
Methylgyoxal non detectable mg/kg
(continued)
72 E.V. Arias-Rios et al.

Table 4.2 (continued)

Concentration
Floral source Phenolic compound reported Units Reference
Thyme 3-Hydroxy-4-phenyl-2-­ 13.3–68.0 %b Melliou and
butanone Chinou
3,4,5-trimethoxybenzaldehyde 1.3–8.4 % (2011)
Orange tree 8-Hydroxylinalool Not determined Melliou and
(Citrus sinensis) Chinou
(2011)
Nodding thistle 8-Hydroxylinalool Not determined Wilkins et al.
(Carduus nutans) (1993)
(Salix spp.) 8-Hydroxylinalool Not determined Jerkovic and
Marijanovic
(2010)
(Acer spp.) 8-Hydroxylinalool Not determined Jerkovic
et al. (2010)
Tentative identification
a

Area percentage
b

cal region, floral origin, heat treatment, age of honey, and bee species (Martinioni
and Ranzato 2015). Manuka honey (produced from nectar of the New Zeland tree
Leptospermun scoparium) has been reported to contain high levels of methylgly-
oxal (MGO), a compound reported of having antimicrobial activity. Concentrations
of MGO in manuka honey may vary among 100–800 mg/kg in fresh honey and
1000–1500 mg/kg in matured honey (Stephens et al. 2010). Melliou and Chinou
(2011) identified 3-Hydroxy-4-phenyl-2-butanone and 8-Hydroxylinalool as anti-
microbial components in thyme and orange sources respectively from Greece honey
samples.
Propolis has been demonstrated to have bactericidal and bacteriostatic activities
against foodborne bacteria (Table 4.3) (Abdou et al. 2007; Kim and Chung 2011; Lu
et al. 2005). The antibacterial activity of propolis has been related to phenolic com-
pounds and flavonoids present, which may vary by region, season, and floral source
(Dias et al. 2012; Lu et al. 2005; Mohammadzadeh et al. 2007). Kim and Chung
(2011) studied the effect of propolis on vegetative cells and spores of B. cereus. The
authors concluded that propolis induced pores at several locations in the cell enve-
lope. In addition to the disintegration of the cell envelope, exposure to propolis
affected also binary fission leading to loss of cytoplasmic and nuclear material.
Antimicrobial compounds in honey have been reported to show quorum sensing
inhibition (QSI) capacity. Truchado et al. (2009) concluded that unifloral honeys
were able to inhibit the production of acyl-homoserine lactones (AHLs) produced
by Cromobacterium violaceum at 0.1 g/mL independently of the geographical ori-
gin. In a separate study, Bulman et al. (2011) reported that propolis inhibit quorum
sensing (QS) flavonoid catechin which was identified as a bioactive molecule that
reduces AHL QS-controlled virulence factors in Pseudomonas aeruginosa.
 4

Table 4.3 Effectiveness of antimicrobial compounds present in honey and propolis: (a) bactericidal, (b) bacteriostatic, (c) little or no effect
Inoculum Population
level (log survival (log
Microorganism tested Floral source Test conditions CFU/mL) CFU/mL) Effect Reference
S. aureus Montana buckwheat, 5% solution, 37 °C/24 h 5.8 2.2 a Taormina
Blueberry, 5.8 3.7 a et al. (2001)
MRSA Manuka 80-fold dilution, 37 °C/24 h 6.0 < 3.0 a Kwakman
B. subtilis 2.5-fold dilution, 37 °C/2 h 6.0 < 3.0 a et al. (2011)
E. coli 10-fold dilution, 37 °C/24 h 6.0 < 3.0 a
P. aeruginosa 5-fold dilution, 37 °C/24 h 6.0 < 3.0 a
MRSA Revamil 10-fold dilution, 37 °C/24 h 6.0 < 3.0 a Kwakman
B. subtilis 13.3-fold dilution, 37 °C/2 h 6.0 < 3.0 a et al. (2011)
E. coli 2.5-fold dilution, 37 °C/2 h 6.0 < 3.0 a
Natural Food Antimicrobials of Animal Origin

P. aeruginosa 2.5-fold dilution, 37 °C/2 h 6.0 < 3.0 a

S. sonnei Montana buckwheat 25% sol. in PBPa, 37 °C/24 h 4.7 6.5 c Taormina
L. monocytogenes Blueberry 25% sol. in PBP, 37 °C/24 h 6.1 6.4 b et al. (2001)
S. aureus 5.9 2.2 a
S. sonnei 4.7 7.3 c
L. monocytogenes 6.1 8.1 c
S. aureus 5.9 3.7 a
B. cereus vegetative cells 1.8 mg/mL solution, 5.5 2.0 a Kim and
B. cereus spores 37 °C/100 min 5.7 5.7 a Chung (2011)
287.5 mg/mL solution,
37 °C/30 h
MRSA methycillin resistant S. aureus
a
PBP 0.1 M potassium phosphate buffer (pH7.0)
73
74 E.V. Arias-Rios et al.

Antimicrobial activity of peptides present in honey has been reported in the last
decade (Ayaad et al. 2012; Czihal et al. 2007; Kwakman et al. 2011). The antimicro-
bial peptide present in honey: bee defensin-1 (also known as royalisin) was previ-
ously found in honeybee hemolymph, honeybee head and thoracic glands, as well
as in royal jelly (food of queen bee larvae). Bee defensin-1 has been reported to
have potent activity only against Gram-positive bacteria, for example, B. subtilis, S.
aureus, and Paenibacillus larvae (Kwakman et al. 2011).

4.1 Applications in Foods

Applications of edible films containing propolis have been studied on fresh and
minimally processed fruits and vegetables to preserve their shelf life (Ali et al.
2014; Pastor et al. 2011).

5 Antimicrobials Obtained from Crustaceans

Crustaceans, such as shrimp, crab, lobster, crayfishes, and prawns contain high con-
centrations of chitin [poly-ß-(1–4)-N-acetyl-D-glucosamine] in the shells. Chitosan
is a carbohydrate polymer obtained from the partial deacetylation of chitin (Dutta
et al. 2009).

5.1 Chitosan

Chitosan is widely used in agriculture as a biopesticide to combat fungal infections

in plants, as preservative of food, for water filtration, in medicine, and for other
industrial uses (Hajji et al. 2016). It has been classified by the US FDA as Generally
Recognized as Safe (GRAS) as food additive (Sagoo et al. 2002). Due to properties
such as biocompatibility, biodegradability, and film formation, chitosan has a wide
application in the food industry (Blanquicet et al. 2015). In some cases, when
applied as a biofilm, chitosan is mixed with other compounds such as whey, cellu-
lose, sorbitol, glycerol, ethylenglycol, starch, and gelatin to improve its hydropho-
bicity, antibacterial and/or mechanical properties (Blaquicet et al. 2015).
Chitosan has a higher inhibitory activity against Gram-positive bacteria than
Gram-negative (Jridi et al. 2014; Gómez-Estaca et al. 2011; No et al. 2002) and
against bacteria than fungi (Tsai et al. 2002). Even though it has been demonstrated
that the higher concentration of chitosan has a higher antimicrobial effect, this
inhibitory activity effect is dependent of the chitosan molecular weight (Jeon et al.
2002; No et al. 2002), degree of deacetylation (Tsai et al. 2002), the target microor-
ganism, and the delivery technology (No et al. 2002; Wang et al. 2015).
4 Natural Food Antimicrobials of Animal Origin 75

Chitosan-based composite films have also been evaluated. Tan et al. (2015)
reported that using 0.5–1.5% grapefruit seed extract in the chitosan-based film dem-
onstrated an extra 3–5 day delay of mold growth in bread when compared to pure
chitosan-based film. Incorporation of oleic acid to a chitosan film also delayed the
fungal infection on strawberries (Vargas et al. 2006). However, a synergistic antimi-
crobial effect is not always produced after combining chitosan with other com-
pounds. For instance, chitosan added with acetic acid in mayonnaise inoculated
with L. fructuvorans had a higher inactivation effect than when using chitosan added
with lemon juice (Roller and Covill 2000). Furthermore, the level of the antimicro-
bial effect obtained in vitro could not be obtained when working in vivo. When a
coating of chitosan supplemented with potassium sorbate applied onto cultures of
Rhizopus sp. and Cladosporium sp. in vitro, the fungal inhibition was improved.
When the same chitosan-potassium sorbate film was applied to inoculated strawber-
ries, no significant difference in the fungal inhibitory effect was detected. This could
be explained by the difficulty of the antifungal compounds within the coating to
reach the spores within the irregularities of the strawberry’s surface (Park et al.
2005).
Wang et al. (2015) suggested that the particle size may affect the antimicrobial
effect of chitosan. In their study, significant differences were found in the inhibition
of total viable counts in whiteleg shrimp stored at 4 °C for 10 days when using a
nanoparticle chitosan film compared with a carboxymethyl chitosan coating.

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Chapter 5
Antimicrobials of Plant Origin

Dinesh Babu, Kalpana Kushwaha, Shalini Sehgal, and Vijay K. Juneja

Abstract Antimicrobial compounds of plant origin are used as natural preserva-

tives to control spoilage and pathogenic bacteria in foods. The food safety appli-
cations of these plant derived compounds are considered as natural alternatives to
chemical antimicrobial agents, in addition to being cheaper and safer substitutes.
These bioactive compounds in plants are secondary metabolites, usually produced
and accumulated in various plant parts and their activities depend on the concen-
tration, composition, structure, and functional groups. Major examples of such
plant compounds include vitamins, antioxidants, essential oils, hydrocolloids,
proteins, aldehydes, flavonoids, and other phytochemicals. These compounds
have been documented to exhibit antimicrobial properties against various food-
borne pathogens. The food safety applications of selected plant derived antimicro-
bials in controlling survival and resistance of pathogens in foods are briefly
discussed in this chapter.

Keywords Flavonoids • Phytochemicals • Phenolics • Essential oils • Green tea

D. Babu (*)
Earthbound Farm, The WhiteWave Foods Company, 1721 San Juan Highway,
San Juan Bautista, CA, USA
e-mail: [email protected]
K. Kushwaha
Food Safety Professional, Gilroy, CA, USA
e-mail: [email protected]
S. Sehgal
Department of Food Technology, Bhaskaracharya College of Applied Sciences,
University of Delhi, New Delhi, India
e-mail: [email protected]
V.K. Juneja
Residue Chemistry and Predictive Microbiology Research Unit, Eastern Regional Research
Center, USDA-Agricultural Research Service, Wyndmoor, PA, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 85

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_5
86 D. Babu et al.

1 Introduction

Throughout ancient history, antimicrobial compounds of plant origin have been

extensively used to treat microbial infections. Historical approaches of treating
infectious diseases involved use of plant extracts as active ingredients in ancient
medicines. Ancient medical texts of Babylon, China, Egypt, India and Greeks
introduced the development of medicines. Although the use of biological therapeu-
tic agents such as antibiotics is often attributed to mid-twentieth century time
period, there is no clear record on the first official use of plants for medicinal appli-
cations. Ancient civilizations used various forms of natural antimicrobial com-
pounds such as herbs, honey, heavy metals and even animal feces to treat infections
(Keyes et al. 2003; Playfair 2004). Ancient antimicrobial agents and their exposure
or usage can also be confirmed through the paleobotanical findings and human
skeletal remains at archaeological sites. For example, ancient human skeletons of
Sudanese Nubians dating back to 350–550 CE contained traces of tetracycline
(Bassett et al. 1980; Nelson et al. 2010). Tetracycline exposure or ingestion was
also found from the histological testing of the Roman period skeletons from the
Dakhleh Oasis, Egypt (Cook et al. 1989). Many of the ancient or traditional anti-
microbial agents are used even in modern day medicines and the plant antimicro-
bial agents continue to be one of the most successful forms of therapeutic agents.
The plant derived antimicrobials are relatively lower in toxicity and also mitigate
any side effects associated with commercial antibiotics (Paulo et al. 2011).
In the case of food applications, antimicrobials of plant origin are widely used as
food preservatives to control the growth of spoilage causing bacteria and foodborne
pathogens. Along with the newer food safety and health standards, demand for natu-
ral alternatives of chemical preservatives has increased as consumers have become
more concerned about negative health issues from the chemical residues in their
foods (Smith-Palmer et al. 1998; Charu et al. 2008; Sofos and Geornaras 2010).
Further, plant derived natural antimicrobials also provide cheaper and safer alterna-
tives to synthetic food preservatives as most of them are generally regarded as safe
(GRAS) as per the US code of Federal Regulations, CFR 21, part 182, 184 (Burt
2004). In the United States, significant number of FDA approved pharmaceutical
drugs are derived from plants, but only few are used as antimicrobials when com-
pared with antimicrobials produced from bacteria and fungi. However, this does not
reflect the future applications of plant antimicrobials and their potential to address
emerging issue such as drug resistance among pathogenic bacteria.
Many of the plant products are rich sources of bioactive compounds such as vita-
mins, antioxidants, essential oils, hydrocolloids, proteins, aldehydes, flavonoids, and
other phytochemicals with proven antimicrobial properties along with potential
health benefits. For example, extracts of spices and herbs are reported as strong anti-
oxidants and some of the most recognized spices for their antimicrobial properties
include clove, cinnamon, oregano, and rosemary. Plants produce these antimicrobial
compounds as secondary antimicrobials that have also shown antibacterial activity
against a range of pathogenic microorganisms. In this chapter, we discuss some of
the food safety applications and properties of plant derived antimicrobials.
5 Antimicrobials of Plant Origin 87

2  ategories of Plant Derived Antimicrobials, Their

C
Functions and Food Safety Applications

Plant derived antimicrobials are produced as secondary metabolites by plants and

they are categorized based on the active ingredient components (Cowan 1999).
These metabolites are usually produced and accumulated in specific plant parts such
as leaves, roots, fruits, seeds, etc. and a specific medicinal or antimicrobial property
of a plant species is commonly attributed to the specific metabolite produced by that
particular plant species.
These plant derived antimicrobials or also known as “phytochemicals” are non-
nutritive and usually confer organoleptic properties. They serve as antimicrobial
agents and their antimicrobial activity depends on the concentration, composition,
structure, and functional groups. Phenolic compounds are the most effective class
among phytochemicals and they are divided into various categories such as simple
phenolic compounds, flavonoids, quinones, tannins, and coumarins, based on their
chemical structures.
Also, essential oils are the most important phytochemicals which have been used
as food preservatives since ancient times. Many different photochemical and their
antibacterial activities have been summarized by Negi 2012 (Fig. 5.1).
Various modes of action of these compounds have been identified and may
include inactivation of proteins, genes, signaling pathways, disruption of cell walls,
biofilms and so on. Several properties, classification, functions, food safety appli-
cations and modes of action of some of these natural antimicrobial compounds are
discussed below.

2.1 Phenolics and Their Antimicrobial Properties

One of the large class of plant secondary metabolites are ‘phenolic compounds’ that
are of significance for a plant’s morphological and physiological functions. These
consist of a single substituted phenolic ring (Galal 2006). The degree of toxicity to
microorganisms depends on the sites and number of hydroxyl groups on the phenolic
ring. There is an increase in toxicity with increased hydroxylation and higher oxida-
tion confers greater inhibition (Cowan 1999). Cabral et al. (2013) showed phenolic
compounds alter microbial cellular permeability, resulting in loss of macromole-
cules, and interact with membrane proteins, causing structural changes. Major func-
tions of these phenolic compounds include providing protection to the plants against
pathogens, insects and other stress conditions (Bravo 1998), growth and reproduc-
tion, color and sensorial attributes of fruits and vegetables (Alasalvar et al. 2001;
Cheynier 2012). For example, in red fruits, juices and wines, phenolic compounds
are known to provide color, enzymatic browning and flavor (Cheynier 2012).
Phenolic compounds present in fruits and vegetables are known to have antioxidant
88 D. Babu et al.

Flavonols
Flavonones
Catechins Found in tea leaves

Anthocyanins
Genistein
Flavonoids Isoflavones Found in soy
Diadzein
Flavones

Chalcones Found in onion, green tea, red wine

Quercetin Hydrolyzable tannins

Condensed tannins Found in berries, trees,
Tannic acid seaweeds, etc.
Complex tannins
Phenolic Phenolic acids Ellagic acid
compounds Vanillin

Caffeic acid
Found in fruits,
Ferulic acid vegetables and grains
Hydroxycinnamic
acids Chlorogenic acid

Curcumin Found in turmeric

Courmarins Found in citrus fruits

Lignans Found in flax and sesame

seeds

Fig. 5.1 Classification of phenolic compounds (Adapted from Maqsood et al. 2012)

properties (Parr and Bolwell 2000; Heim et al. 2002). Other benefits of phenolic
compounds include anti-inflammatory, anti-allergenic, anti-microbial, cardioprotec-
tive and chemopreventive effects (Benavente-Garcia et al. 1997; Samman et al. 1998;
Middleton et al. 2000; Puupponen-Pimia et al. 2001; Manach et al. 2005).
The phenolic compounds vary in their chemical structures from simple pheno-
lic acids to polyphenols to polymeric compounds. Regardless of diversity in their
structures, phenolic compounds are commonly known as ‘polyphenols’ mainly
due to their natural occurrence as conjugates of at least two phenolic rings linked
with mono- and polysaccharides and also as esters and methyl esters (Shahidi and
Naczk 1995; Harborne et al. 1999). The polyphenols can be grouped into three
main subclasses as flavonoids, phenolic acids and stilbenoids.

2.1.1 Flavonoids

Flavonoids are the major polyphenols isolated and are hydroxylated phenolic
structures with a C3–C6 aromatic ring linkage (Clifford 2001; Beecher 2003).
Their ability to inactivate proteins by binding and to form complexes with bacterial
5 Antimicrobials of Plant Origin 89

cell walls makes them effective against many microorganisms. Their degree of
hydroxylation does not predict the level of toxicity to microorganisms (Cowan
1999). Most flavonoids occur as glycosides and other conjugates in nature. Based on
their chemical structure and functional groups such as ketones and hydroxyls, the
prominent food flavonoids are termed as flavones found in teas, red grapes and red
wines, isoflavones of soy and legumes, isoflavanes, flavanones in citrus foods, flava-
nols, anthocyanidins in red, purple and blue berries, chalcones and dihydrochal-
cones; the flavones are further subdivided mainly into simple monomeric cathechins
and complex polymeric proanthocyanidins or tannins (Beecher 2003).

2.1.2 Phenolic Acids

Phenolic acids comprise of a phenolic ring conjugated with sugars and carboxylic
acids such as benzoic or cinnamic acids. They are produced by many plants as broad
spectrum antimicrobials against microbial infections (Van Loon 2000) and also dur-
ing rhizospheric plant-microbe interactions (Martens 2002). Major phenolic acids
are gallic acid, chlorogenic acid, caffeic acid, ferulic acid, p-coumaric acid, and
gentisic acid. The two subgroups of phenolic acids are hydroxybenzoic and hydroxy-
cinnamic acids that have C6-C1 (one carbon side chain aromatic ring) and C6-C3
(three-carbon side chain aromatic ring) structure respectively. For example, gallic
acid, p-hydroxybenzoic acids, vanillic acids are some of the widely used hydroxy-
benzoic acids and hydroxycinnamic acids class includes quinic acid, shikimic acid,
tartaric acid, ceffeic, p-courmaric and sinapic acids (Bravo 1998; Gharras 2009).

2.1.3 Stilbenoids

Stilbenoids are another major class of polyphenols that have been isolated as mono-
mers, oligomers and glycosides (Shen et al. 2009). These compounds are known for
their roles in plant resistance to fungal pathogens and their biological effects. The
stilbenoids are also known for their antimicrobial activity and as botanical supple-
ments or as active ingredients in medicinal preparations (Shen et al. 2009; Chong
et al. 2009). Stilbenoids are found in variety of plants such as grapes, berries and
some nuts. Most common stilbenoid compound is resveratrol found in grapes, red
wine, peanuts, and some berries which is known to provide several health benefits
including anti-oxidant, chemo preventive and cardio protective effects (Leonard
et al. 2003; Frankel et al. 1993; Wang et al. 2012; Igura et al. 2001).

2.1.4 Quinones

Quinones are aromatic rings with two carbonyls, providing a constant source of
free radicals. Apart from being antioxidants, they also act as antimicrobial com-
pounds. Their mode of antimicrobial action is by binding and inactivating proteins.
The 6-(4, 7-dihydroxy-heptyl) quinone isolated from the leaves of Pergularia
90 D. Babu et al.

daemia (Forsk.) is effective against food pathogens including Bacillus subtilis,

Staphylococcus aureus, and Escherichia coli (Ignacimuthu et al. 2009).

2.1.5 Tannins and Coumarins

Tannins are polymeric phenolic substances that are divided into hydrolysable and
condensed tannins. Polymerization of quinones or condensation of flavan deriva-
tives leads to the formation of tannins.Their antimicrobial mode of action of tannins
is very similar to that of quinones and they have been found to be toxic to microor-
ganisms such as bacteria, yeasts, and some fungi (Cowan 1999).Tannins are found
in many fruits, nuts, and seeds. Nile and Park (2014), reviewed the antimicrobial
activities of bioactive components from berries including flavonoids (anthocyanins,
flavonols, and catechins), phenolic acids, stilbenes, and tannins.
Coumarins are phenolic structures comprised of a fused benzene and alpha-­
pyrone ring. Venugopala et al. (2013) have reported that coumarins possess antifun-
gal, and antiviral activities apart from other therapeutic properties.

2.1.6 Alkaloids

Alkaloids are heterocyclic nitrogen compounds and have demonstrated limited

microbicidal activity. One such example is berberine (Cowan 1999). Berberine is
the main antibacterial substance of rhizoma Coptidis and cortex Phellodendri. Yu
et al. (2005) have reported that the MICs of berberine against methicillin resistant
Staphylococcus aureus (MRSA) bacteria ranges from 32 to 128 μg/mL.

2.2 Essential Oils and Their Antimicrobial Properties

Essential oils (EO) are derived from spices, herbs and medicinal plants by way of
distillation, expression or solvent extraction and the EOs or their compounds are
widely used in traditional medicine. Essential oils are also used in food preservation,
as food flavorings, in pharmaceuticals, in cosmetics as fragrances (Hussain et al.
2010). Essential oils as antimicrobial agents exhibit broad-spectrum activity, includ-
ing antifungal, antibacterial and antiviral activities. These properties of EOs are stud-
ied worldwide and the major plants used to extract the essential oils include lemon
grass (Cymbopogon citrates), Fennel (Foeniculum vulgare), peppermint (Mentha
piperita), geranium (Geranium dissectum), caraway (Carum carvi), lavender
(Lavandula officinalis), clove (Syzygium aromaticum), cinnamon (Cinnamomum
cassia) and anise (Pimpinella anisum) (Prabuseenivasan et al. 2006).
Not all essential oils exhibit antimicrobial properties. For example, when the
antimicrobial activities of 96 essential oils were tested against many food-borne
pathogens such as Campylobacter jejuni, Escherichia coli, Listeria monocyto-
genes, and Salmonella enterica, only about 27 essential oils showed antimicro-
5 Antimicrobials of Plant Origin 91

bial inhibition of these pathogens (Friedman et al. 2002). Essential oils of

oregano, thyme, bay, and clove are found to be most active against pathogenic
bacteria such as E. coli (Smith-Palmer et al. 1998; Hammer et al. 1999; Dorman
and Deans 2000). Oregano and thyme EO seem to exhibit stronger antimicrobial
properties than clove and bay (Burt and Reinders 2003; Hammer et al. 1999;
Dorman and Deans 2000).
The essential oils are present as variable mixtures of monoterpenes and sesqui-
terpenes including carbohydrates, phenols, alcohols, ethers, aldehydes and ketones
that known to be responsible for the biological activity (Fig. 5.2).

Fig. 5.2 Chemical structures of selected essential oil constituent (Adapted from Hyldgaard et al. 2012)
92 D. Babu et al.

The mechanisms of antimicrobial action of essential oils and/or their compo-

nents are a factor of their chemical composition. For example, thymol and carvacrol
with similar antimicrobial effects differ in their mechanisms of action against Gram-­
positive and Gram-negative bacteria (Nazzaro et al. 2013). The hydrophilic or
hydrophobic nature of essential oils and the type of exposed microorganisms play
major role in determining the effect. Gram-negative bacteria that have complex cell
wall, are less susceptible to the effects of EOs than Gram-positive bacteria
(Trombetta et al. 2005). However, the hydrophobic constituents of EOs may eventu-
ally enter the periplasm of Gram-negative bacteria through the porin proteins of
their outer membrane (Helander et al. 1998). Scanning electron microscopy revealed
that oregano EOs renders E. coli O157:H7 cell membranes permeable by entering
through the pores of the membrane (Lambert et al. 2001). Thus, the main antibacte-
rial mechanism of EOs seem to involve disrupting the permeability of cell mem-
branes and causing loss of cellular components or influx of other substances into the
cell, ultimately leading to cell death.

2.2.1 Polyphenol Antimicrobials in Food Safety

Polyphenols have shown both broad spectrum and targeted antimicrobial activity
against food pathogens. Major polyphenols with demonstrated antimicrobial activi-
ties include tea polyphenols (catechins, theaflavins, tannins, and flavonoids, etc.),
curcumin, resveratrol, cinnemaldehyde, p-coumaric acid, caffeic acid, eugenol, grape
seed extracts or grape polyphenols such as proanthosyanidins, anthocyanins.

2.2.2 Turmeric Extracts

The antibacterial activity of various types of polyphenols against major foodborne

pathogens have been well documented throughout the literature. For example, tur-
meric (Curcuma longa) is widely popular in in India, China and South East Asia
mainly as spice and as a food preservative along with its use in traditional medi-
cines in preventing and treatment of several diseases and health conditions is also
known to provide antimicrobial activity against several microroganisms (Aggarwal
et al. 2007). The antimicrobial activity of turmeric is attributed to the presence of
essential oil, curcumins, curcuminoids, turmeric oil, turmerol, turmerone, curlone
and veleric acid (Negi et al. 1999; Cikricki et al. 2008; Rai et al. 2008; Basniwal
et al. 2011). Curcumin is known to be active against a range of food spoilage and
pathogenic microorganisms such as Staphylococcus aureus, Bacillus subtilis,
Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger etc. (Arora and
Kaur 1999; Kapoor 1997; Roth et al. 1998; Gupta and Ravishankar 2005; Basniwal
et al. 2011; Gul and Bakht 2015). Susceptibility of Staphylococcus aureus to dif-
ferent aqueous extracts of turmeric is well documented (Mohammad et al. 2010;
Gul and Bakht 2015).
5 Antimicrobials of Plant Origin 93

2.2.3 Grape Seed Extracts

Grapes contain resveratrol that has been shown to have antibacterial properties in
addition to human health benefits (Henry-Vitrac et al. 2006). Resveratrol is mainly
present in grape skin, but also in stems and seeds at lower concentrations (Kataliníc
et al. 2010; Liu et al. 2013). The antimicrobial compounds in grape are not limited
to any one particular part of the plant, but can be found in flesh, whole fruit grape
extracts, fermented fruit, fermented pomace, skin, leaves and seeds (Xia et al. 2010).
Grape seed extract is a rich source of polyphenols such as oligomeric proanthocy-
anidins and free flavanol monomers (Burdock 2005). High concentrations of flavo-
noids and their derivatives in grape seeds and flavonoids, stilbenes, and phenolic
acids in grape stems are reported to be responsible for the antimicrobial activity
(Anastasiadi et al. 2009). More specifically, the antimicrobial properties of grapes
can be attributed to the constituent phenolics such as catechins (Tagurt et al. 2004),
the non-flavonoid caffeic acids, flavonoids such as rutin and quercetin. Combinations
of some of these antimicrobials are reported to be inhibitory to the growth of Listeria
monocytogenes (Vaquero et al. 2007) and have shown a broad spectrum of antimi-
crobial activity against various food pathogens. For example, grape skins extracts
when screened against Gram-positive (Staphylococcus aureus, Bacillus cereus) and
Gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis,
Campylobacter coli) food pathogens, showed Minimum Inhibitory Concentrations
(MICs) in the range 0.014–0.59 mg of Gallic Acid Equivalents (GAE)/mL (Kataliníc
et al. 2010). The grape seed extract (GSE) against Camylobacter jejuni showed
inhibition of 5.08–6.97 log CFU/mL demonstrating a strong antibacterial effects of
the seed extracts at 20 mg/L as Minimal Inhibitory Concentration (MIC) and a mini-
mal bactericidal concentration (MBC) of 60 mg/L (Silván et al. 2013). Reports of
using grape seed extracts to decontaminate meat and produce surfaces have given
encouraging results in various studies. Several authors have proposed washing fresh
produce with solutions of natural products such as grape seed extracts, olive extracts,
and essential oils as alternatives to chlorine based solutions (Bisha et al. 2010; Lu
and Wu 2010; Moore et al. 2011). Bisha et al., (2010) used commercial GSE prepa-
ration known as Gravinol-S at lower concentrations of 0.00015–0.125% in aqueous
solution against listeria on fresh produce. They found that GSE showed MICs of 50
and 78 μg/mL against L. monocytogenes and L. innocua strains, respectively. At
sub-MICs, GSE caused rapid permeabilization and clumping of L. innocua and also
caused 2 log reduction of L. monocytogenes on tomato at higher concentrations
(0.125%). The efficacy of GSE against food pathogens such as L. monocytogenes
appears to be lower in foods such as beef and turkey frankfurters, and requires using
in combination with other antimicrobials such as nisin (Sivarooban et al. 2007). The
combination of 1% GSE and 6400 IM of nisin (a polypeptide produced by
Lactococcus lactis spp.) was found effective against L. monocytogenes on turkey
frankfurters. This suggested that use of GSE when combined with other antimicro-
bials can be more effective in controlling pathogens on complex foods such as
meats. This is also true of many of the plant extracts that may not be effective when
applied alone. Multiple hurdle approaches involving proven interventions such as
94 D. Babu et al.

pH and thermal controls, organic acid treatments and other natural antimicrobials
may be found effective (Juneja et al. 2010; Tsigarida et al. 2000).

2.2.4 Green Tea Extracts

Green Tea Extract (GTE) is another natural antimicrobial product. Natural green tea
extracts have GRAS status and have been used in various food applications. Tea
from Camellia sinensis comes in different types as a not fermented green tea which
is pan-fried or steamed and dried, fermented black tea and semifermented oolong
tea. Black tea extractions contain lower concentrations of similar components found
in green tea, but mainly contain high molecular weight compounds such as theafla-
vins and thearubigens (Taylor et al. 2005). Oolong teas are made from special tea
cultivars of Camellia sinensis and the traditional tea making process involves with-
ering the plant or leaves under hot sun and oxidizing.
Aqueous extractions of tea leaves (Camellia sinensis) contain polyphenols such
as catechins, theaflavins, tannins, and flavonoids. The health-promoting effects of
green tea are mainly attributed to catechins and the main types of catechins are
epigallocatechin-3-gallate (EGCG), epigallocatechin, epicatechin-3-gallate and
epicatechin (Taylor et al. 2005). Catechin fractions constitute up to 30% of solid
green tea leave and most of the antimicrobial activities in addition to other biologi-
cal properties of green tea have been associated with the polyphenol catechin frac-
tions (Chacko et al. 2010; Taylor et al. 2005). The most abundant of these catechins
is EGCG which comprises of about 50–65% of the catechin pool (Chacko et al.
2010). Several reports have documented the antimicrobial properties of the major
green tea extract components against food borne pathogens such as Listeria mono-
cytogenes, Escherichia coli O157:H7, Salmonella typhimurium, Salmonella
enteriditis, Campylobacter jejuni, Staphylococcus aureus, Staphylococcus epider-
midis, Shigella flexneri, Shigella dysenteriae, and Vibrio cholera (Toda et al. 1991;
An et al. 2004; Gadang et al. 2008; Hamilton-Miller 1995). Antimicrobial activity
of green tea polyphenols is attributed to their interaction with the bacterial cell wall
phospholipid bilayer and cell membrane that destabilize cell structure, function
causing cell death (Perumalla and Hettiarachchy 2011; Shimamura et al. 2007). In
addition, the structures of the polyphenols is also a major factor in deciding the
extent of antimicrobial activities against pathogenic bacteria.

2.2.5 Natural Antimicrobial Products to Address Antibiotic Resistance

Treating and preventing bacterial infections in humans and food animals with the
use of antibiotic drugs has been a long time practice in medicine and agriculture.
The antibacterial resistance against majority of synthetic antibiotics is a global
problem impacting public health. The increasing use of synthetic antibiotics causes
a selective pressure on the pathogenic bacteria that eventually show antibiotic resis-
tance. Emergence of antibiotic resistance among the outbreak associated foodborne
5 Antimicrobials of Plant Origin 95

pathogens such as Salmonella, Listeria monocytogenes, E. coli O157:H7 has been

documented by several researchers (Babu et al. 2016).
Plant-derived antibacterial compounds may have a great potential in inhibiting
drug resistance among food pathogens. Plant derived natural antimicrobials are
mainly unique in their diverse modes of action on infectious microorganisms.
Several modes of action are proposed by which the natural antimicrobials includ-
ing their isolated components may simultaneous affect multiple targets through
synergistic actions, resistance modification and increasing susceptibility for antibi-
otics. Thus, due to the nature of phytochemicals showing their antimicrobial activ-
ity through different mechanisms than conventional antibiotics, and their potential
to act as resistance modifying agents (Abreu et al. 2012), it is suggested that their
use in the treatment of resistant bacteria could be highly effective. When natural
antimicrobials of eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate
were used against Salmonella Typhimurium SGI 1 (tet A), the resistance against
ampicillin, tetracycline, penicillin, bacitracin, erythromycin and novobiocin antibi-
otics was found to be reduced significantly with increased susceptibility towards
the drugs (Palaniappan and Holley 2010). Similar observations were also noted for
drug resistant Streptococcus pyogenes ermB, Escherichia coli N00 666,
Staphylococcus aureus blaZ, and S. pyrogenes (Palaniappan and Holley 2010). For
this reason, use of natural antimicrobials has revived interest in various plant
derived antimicrobials such as polyphenols, essential oils and bioactive compounds
of grape and green tea extracts as a possible alternative for the control of foodborne
pathogenic bacteria. Along with the other benefits from plant antimicrobials such
being natural and safe, resultant lower incidence of resistance among pathogens is
major advantage of using plant-based drugs (Spann et al. 2003).
It is clearly evident from the numerous studies conducted around the world
that there are thousands of “phytochemicals” that have inhibitory effects on sev-
eral types of microorganisms including foodborne pathogens. Several of these
compounds seem to show synergistic effects when combined with other natural
antimicrobials or other drug compounds. Extent of synergistic effects also
depends on food systems and microorganism in question (Langeveld et al. 2014).
For example, EOs containing carvacrol, cinnamaldehyde, cinnamic acid, eugenol
and thymol have shown a synergistic effect in combination with antibiotics.
Hence such combinations should be subjected to further studies to determine
their effectiveness in different food systems (Langeveld et al. 2014). It would also
be beneficial to standardize methods of extraction of plant antimicrobials as the
efficacy of these antimicrobials may also depend on the extraction methods and
the type of plant material used (Van Loo et al. 2012; Babu et al. 2013).
In conclusion, the phytochemicals show great potential as natural antimicrobials
against several food pathogens. The biological activity and antimicrobial action of
plant derived antimicrobials on foodborne pathogens remains a focal area of future
research. In particular, studies towards finding an effective combination of these
natural antimicrobials that show synergistic effects should be of major focus to
arrive at antimicrobials that also work at low concentrations without affecting food
sensorial qualities and remain effective against food pathogens.
96 D. Babu et al.

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Chapter 6
Natural Food Antimicrobials of Microbial
Origin

Shalini Sehgal and Vasudha Sharma

Abstract The use of preservatives from natural sources like plants, animals and
microbes has gained popularity, as they are more reliable, ethical and safe.
Consumer’s preference for food additives from natural sources and concerns
regarding safety of synthetic preservatives has prompted the food industry to
search for natural anti microbials. In addition to the greater stability, these preser-
vatives are also economical and readily available in nature. Natural anti microbi-
als include bacteriocins, plant extracts, enzymes, peptides, fermented ingredients.
Several LAB bacteriocins offer potential applications in food preservation, and
the use of bacteriocins in the food industry can help to reduce the addition of
chemical preservatives as well as the intensity of heat treatments, resulting in
foods which are more naturally preserved and richer in organoleptic and nutri-
tional properties. Bacteriocins can be added to foods in the form of concentrated
preparations as food preservatives, shelf-life extenders, additives or ingredients,
or they can be produced in situ by bacteriocinogenic starters, adjunct or protective
cultures. In recent years, application of bacteriocins as part of hurdle technology
has gained great attention, and they show additive or synergistic effects when
used in combination with other antimicrobial agents, and physical treatments like
high pressure processing or pulsed electric fields providing opportunities for more
effective preservation of foods.

Keywords Bacteriocins • Nisin • Preservatives • Food • Microorganisms • LAB

S. Sehgal (*)
Department of Food Technology, Bhaskaracharya College of Applied Sciences,
University of Delhi, New Delhi, India
e-mail: [email protected]
V. Sharma
Department of Food Technology, Jamia Hamdard (Hamdard University), New Delhi, India
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 101

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_6
102 S. Sehgal and V. Sharma

1 Introduction

Food spoilage and food borne illnesses are a major concern for food processors,
consumers and food safety authorities. Food safety is a very crucial global concern,
and several methodologies including thermal and low temperature exposure are
employed to kill or inactivate the micro organisms. However, these processes besides
affecting micro organisms, adversely affect the nutritional and organoleptic proper-
ties of the food. In recent years, consumer demand for high quality products which
retain nutrients and organoleptic attributes has led to considerable efforts to find
natural antimicrobials that can inhibit bacterial and fungal growth in order to improve
quality and shelf-life. Consumer awareness about the safety of synthetic preserva-
tives used in food has resulted in increasing demand for natural products that can
serve as food preservatives. This in turn has opened avenues for the use of natural
preservatives derived from a diversity of natural sources. Several antimicrobials are
approved for use in foods by international regulatory agencies. Food antimicrobials
are compounds used to extend the lag phase or kill micro organisms. Antimicrobials
that are naturally occurring can be obtained from different sources including plants,
animals, bacteria, algae and fungi, in which they form a part of the host defence
mechanisms against microbial infections (Gyawali and Ibrahim 2014). However, the
application of antimicrobial peptides from lactic acid bacteria (LAB) that target food
pathogens without toxic or other adverse effects has received great attention.

2 Antimicrobials of Microbial Origin

Micro organisms produce a wide range of components that influence the growth
of other micro organisms present in their environment. Several bacteria produce
compounds that are fermentation end products, such as organic acids, hydrogen
peroxide and diacetyl, in addition to bacteriocins and other antagonistic com-
pounds, which act against, and inhibit the growth of pathogenic and spoilage
causing micro organisms. Often these components increase the competitive edge
of the producing organisms and are an important feature of their survival and
proliferation. The most important group of micro organisms, with regards to
food preservation, which are a source of biopreservatives are the LAB. Both,
Gram positive and Gram negative bacteria produce bacteriocins. Bacteriocins
are basically, proteinaceous antibacterial compounds which constitute a heter-
ologous subgroup of ribosomally synthesized antimicrobial peptides. Production
of bacteriocins can be utilized by food processors to provide an additional bar-
rier to undesirable bacterial growth in foods. Bacteriocins have been classified
based on several criteria (Tiwari et al. 2009), for example, bacteriocins are
named after the genus, species or family of bacteria producing them, such as
lantibiotics for the bacteriocins produced by Lactobacillus species, klebicins for
bacteriocins produced by Klebsiella pneumoniae and colicins for bacteriocins
6 Natural Food Antimicrobials of Microbial Origin 103

from E.coli (Riley and Chavan 2007). In addition, these bacteriocins have also
been classified based on the mechanism by which the bacteriocins are produced
i.e. ribosomal and nonribosomal mechanisms by which bacteriocins kill microbes
i.e. pore formation or nuclease. A large number of bacteriocins have been iso-
lated and characterized from LAB, and some have acquired a status as potential
food preservatives because of their antagonistic effect on important pathogens.
Many bacteriocins are active against food borne pathogens and spoilage bacte-
ria, the major ones include nisin, diplococcin, acidophilin, plantaricin, bulgari-
can, helveticin, lactacin and pediocin. Bacteriocins, however are usually
ineffective against gram negative bacteria as they are incapable of penetrating
the bacterium’s outer membrane. The antimicrobial proteins or peptides pro-
duced by bacteria are ribosomally synthesized and kill closely related bacteria
(Klaenhammer 1993). For food use, focus has been on LAB bacteriocins, which
have been shown to be safe, and have potential as effective natural food preser-
vatives. Bacteriocins have applications in hurdle technology, which utilizes syn-
ergies of combined treatments to more effectively preserve food. Since
bacteriocins have been isolated from foods, such as meat and dairy products,
which normally contain LAB, they have unknowingly been consumed for centu-
ries. For over 50 years, nisin has been in use as a food preservative and is
approved for use in over 50 countries worldwide. However, it is not considered
natural when it is applied in concentrations that exceed what is found in food
naturally fermented with a nisin-producing starter culture. The term natural is
also compromised when the bacteriocin is produced by genetically modified
bacteria. Though nisin is currently the only bacteriocin approved for use in the
United States, many bacteriocins produced by members of the LAB have poten-
tial application in food products (Cleveland et al. 2001).

3 Classification of LAB Bacteriocins

Most of LAB bacteriocins are small (<10 kDa) cationic, heat-stable, amphiphilic
and membrane permeabilizing peptides. They can be divided into four major
classes; their classification has been constantly revised throughout the last decade
due to the extensive research in this area. Many of these bacteriocins appear to
exhibit relatively little adsorption specificity. Relatively large molecules are able
to pass through the cell wall of Gram positive bacteria. Teichoic and lipoteichoic
acids form an important part of the cell wall and are crucial in the initial interface
of anionic bacteriocins produced by Gram positive bacteria. At lower pH values
(below 5), LAB bacteriocins demonstrate a greater antibacterial activity, and
their adsorption to the cell surface of Gram positive bacteria, including the pro-
ducing cells is pH dependent. There may be amino acid sequence homologies not
only within the mature peptide, but also in the N-terminal leader region and the
associated proteins in bacteriocin secretion and processing within any class of
bacteriocin (Zacharof and Lovittb 2012).
104 S. Sehgal and V. Sharma

3.1 Class I: The Lantibiotics

Class I, the lantibiotics, are a class of peptide substances that contain the characteristic
polycyclic thioether amino acids lanthionine or methyllanthionine, as well as the
unsaturated amino acids dehydroalanine and 2-aminoisobutyric acid. They are further
divided into two types based on structural similarities.
Type A comprises of relatively elongated, screw shaped, positively charged,
amphipatic, flexible molecules. They generally act through pore formation, through
membrane depolarization, of the cytoplasmic membrane of the sensitive target spe-
cies. Nisin and lacticin 3147 are the major representatives of this group. Their
molecular mass varies between 2 and 4 kDa.
Type B lantibiotics, are globular in structure and interfere with cellular enzy-
matic reactions. Their molecular mass lies between 2 and 3 kDa and either they have
no net charge or a net negative charge.
Class I LAB bacteriocins are small (<5 kDa) heat stable peptides, which are
extensively modified after translation resulting in the formation of characteristic
thioether amino acids lanthionine (Lan) and β-methyllanthionine (MeLan). Firstly
gene encoded serine and threonine can be subject to enzymatic dehydration to
give rise to dehydroalanine (Dha) and dehydrobutyrine (Dhb). Subsequently, thiol
groups from neighbouring cysteine attack the double bond of Dha and Dhb yield-
ing both Lan and MeLan respectively. This condensation between two neighbour-
ing residues results in the formation of covalently closed rings within the formerly
linear peptide conferring both structure and functionality. These arise after a two-
step process (Deegan et al. 2006).

3.2 Class II: The Non-Lantibiotics

Class II bacteriocins are also small (<10 kDa) relatively heat stable, non-lanthionine
containing membrane active peptides. They are divided into two subclasses.
Subclass II a, pediocin-like or listeria active bacteriocins subclass possesses an
N-terminal consensus sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys. They show a high
degree of homology (40–60%) when the corresponding amino acid sequences are
aligned and they are synthesized with a leader peptide which is removed by proteo-
lytic processing, usually after a double glycine residue for example like pediocin
PA-1 or sakacin A (Patton and Van Der Donk 2005).
Subclass II b refers to two-component (two separate peptides) bacteriocins
by means that require two peptides to work synergistically in order to have anti-
microbial activity. Lactacin F and lactococcin G are members of this group
(Daw and Falkiner 1996).
6 Natural Food Antimicrobials of Microbial Origin 105

3.3 Class III: Bacteriocins

This group consists of heat labile proteins which are in general of large molecular weight
(>30 kDa). This group has not been extensively investigated. Bacteriocins representing
this group are helveticin I produced by Lactobacillus helveticus and enterolysin pro-
duced by Enterococcus faecium. Their molecular mass varies between 2 and 4 kDa.

3.4 Class IV: Complex

This group is composed of protein plus one or more chemical moiety (lipid or car-
bohydrate) e.g. Leuconosin S, and Lactococcin 27.
The most common bacteriocins produced by LAB are summarized in Table 6.1.
Table 6.1 Bacteriocins produced by lactic acid bacteria
Bacteriocin Bacteriocin producing strain
Acidocin A Lactobacillus plantarum spp.
Bacteriocin J46 Lactobacillus sake 706
Brevicin 286 Lactobacillus helveticus 481
Diacetin B Lactobacillus brevis VB286
Lactobacillus curvatus2711
Diplococcin Lactobacillus plantarum LC74
Helveticin J Leuconostoc spp.
JK,Plantaricin S Lactococcus lactis spp.
Lactacin F Lactococcus johnsonii spp.
Lactocin 705
Lactococcin 972
Lactoccin G Lactococcus casei spp.
Lactococcin MN
Leucocin H
Leucocidin R1 Lc. lactis subsp. lactis
Nisin Lactococcus lactis spp.
Nisin Lactobacillus acidophilus TK9201
Pediocin A Lc. lactis subsp. cremorisJ46
Pediocin P02 Lc. lactis subsp. lactis biovar. diacetylactis UL720
Plantaricin EF, Plantaricin W Plantaricin Lactococcus lactis var. cremoris
Plactricin LC74
Sakacin A
Sakacin X
Thermophilin ST1 Lc. lactis subsp. lactis
Thermophilin 347 Lc. lactis subsp. cremoris
Leuconostoc. paramesenteroides NM14
Streptococcus thermophilus ACA-DC0001
Streptococcus thermophilus 347
Pediococcus pentosaceus FBB61
Pediococcus acidilactici P02
106 S. Sehgal and V. Sharma

4 Bacteriocins and Food Systems

The bacteriocins produced by LAB have several advantageous properties, making

them suitable for food preservation. They are pH and heat tolerant, nontoxic and
non reactive on eukaryotic cells. Furthermore, bacteriocins are inactivated by
digestive proteases; thus, have little influence on the gut microbiota. These are
bactericidal in their mode of action, usually acting on the bacterial cytoplasmic
membrane. Bacteriocins exhibit no cross resistance with antibiotics, have a broad
antimicrobial spectrum against many food-borne pathogenic and spoilage bacteria,
and are categorized as Generally recognized as safe (GRAS).
The studies carried out in recent years clearly state that the use of bacteriocins in
food preservation offers several benefits (Thomas et al. 2000), such as (1) extend
shelf life of foods, (2) provide extra protection during temperature abuse conditions,
(3) decrease the risk for transmission of food borne pathogens through the food
chain, (4) decrease the economic losses due to food spoilage, (5) serve as an alterna-
tive to chemical preservatives, (6) non thermal method of food preservation without
compromising food safety leading to better nutritional quality of food as well as
organoleptic properties of foods, (7), help in the marketing of “novel” foods (less
acidic, with a lower salt content, and with a higher water content), and (8) serve to
satisfy industrial and consumers demands. In this respect some of the trends of the
food industry in Europe, such as the need to eliminate the use of artificial ingredi-
ents and additives, the demands for minimally-processed and fresher foods, as well
as or ready-to-eat food or the request for functional foods and nutraceuticals
(Robertson et al. 2004) could be satisfied, at least in part, by application of bacterio-
cins. The present chapter will address different aspects related to food preservation
by bacteriocins including factors influencing bacteriocin activity in food systems,
hurdle technology, and the impact of recent advances in molecular biology and the
analysis of bacterial genomes on bacteriocin studies and application.

5  actors Influencing the Efficacy of Bacteriocins in Food

F
Systems

Comparisons of data obtained in culture media with those obtained in food systems
reveal that the efficacy of bacteriocins is often much lower in the later (Schillinger
et al. 1996). Sometime, at least ten-fold higher bacteriocin concentrations must be
added to foods in order to achieve an equivalent inhibitory effect. The efficacy of
bacteriocins in foods will greatly depend on a number of food-related factors
(Table 6.2) that in most cases involve interaction with food components, precipitation,
inactivation, or uneven distribution of bacteriocin molecules in the food matrix.
As an illustrative example, application of nisin in meat products faces several
limitation derived from its interaction with phospholipids emulsifiers and other
6 Natural Food Antimicrobials of Microbial Origin 107

Table 6.2 Bacteriocin efficacy in foods: limiting factors

Food-related factors
• Food processing conditions
• Food storage temperature
• Food pH, and bacteriocin instability to pH changes
• Inactivation by food enzymes
• Interaction with food additives/ingredients
• Bacteriocin adsorption to food components
• Low solubility and uneven distribution in the food matrix
• Limited stability of bacteriocin during food shelf life
The food microbiota
• Microbial load
• Microbial diversity
• Bacteriocin sensitivity
• Microbial interactions in the food system
The target bacteria
• Microbial load
• Bacteriocin sensitivity (Gram-type, genus, species, strains)
• Physiological stage (growing, resting, starving or viable but non-culturable cells, stressed
or sub-lethally injured cells, endospores)
• Protection by physico-chemical barriers (microcolonies, biofilms, slime)
• Development of resistance/adaptation

food constituents, poor solubility at pH above 6.0, and inactivation by formation

of a nisin glutathione adduct (Henning et al. 1986; Aasen et al. 2003). However,
inactivation is lower in cooked meats due to the loss of free sulphydryl groups during
cooking as a result of the reaction of glutathione with proteins (Stergiou et al. 2006).
Foods are complex ecosystems with a range of microbial compositions. These vary
from (commercially) sterile foods to raw or fermented foods. In commercially ster-
ile foods, microorganisms from post-process contamination may easily proliferate
because of the lack of competitors. Under these conditions, the efficacy of the bac-
teriocin will depend more directly on the microbial load of the contaminant, and a
higher bacteriocin concentration will always be required in order to inactivate a
higher number of target cells. In raw foods, the autochthonous microbiota may
counteract development of potential pathogens. However; a complex food micro-
biota may frequently cause a lower efficacy of bacteriocins due to the presence of
resistant bacteria (such as Gram-negatives) and/or the higher chances for production
of inactivating enzymes (such as proteases). Sometimes, the bacteriocin may not
target the desired bacterial group (for example, bacteriocin inactivation of the starter
LAB responsible for fermentation would be clearly undesirable). In processed
foods, the microorganisms are present in a variety of physiological stages which
may greatly influence the activity of bacteriocins.
108 S. Sehgal and V. Sharma

6 Applications of Bacteriocins

At present, bacteriocins have found a wide application in the field of food preservation.
The use of bacteriocins in the food industry especially in the dairy sector, meat and
meat products, eggs and vegetables has been extensively studied. Nisin A and its natu-
ral variant nisin Z has been proven to be very effective against food borne pathogens as
well as food spoilage microorganisms. Nisin is the only bacteriocin that has been
approved for use as an additive in the food industry worldwide with GRAS status and
has the number E234 (ECCU 1983 EEC Commission Directive 8314631EEC) (Moll
et al. 1999; Deegan et al. 2006).
Nisin is the most widely used bacteriocin. It is active against Gram positive bac-
teria and its use has been approved since 1988 by FDA in cheese, heat treated chill
stored soups and pasteurized cheese spreads. Nisin belongs to the Class I lantibiot-
ics, is composed of thirty-four amino acids and has a pentacyclic structure with one
lanthionine residue (Ring A) and four β-methyllanthionine residues (rings B, C, D,
E). Nisin Z, the natural variant of nisin is different only in that the histidine mole-
cule on place 27 is replaced by asparagine. Nisin can be effective at nanomolar
concentrations depending on the target strain. Nisin is heat stable at 121 °C but on
prolonged heating, it becomes less heat stable (especially between pH 5–7).
Various methods of food preservation have been used in order to prevent food
poisoning and spoilage such as thermal treatment (pasteurization, heating steriliza-
tion), pH and water activity reduction (acidification, dehydration) and addition of
preservatives (antibiotics, organic compounds such as sorbate, benzoate, lactate,
and acetate). These methods have been proven to be highly successful but there is a
growing need for natural, microbiologically safe preservatives (Deegan et al. 2006).
Bacteriocins can be applied in two forms—purified or crude form or through the
use of a product fermented with a bacteriocin producing strain previously or incor-
porated through a bacteriocin producing strain (starter culture). However, it has been
proven that a bacteriocin alone in a food is not likely to ensure complete safety;
especially in the case of Gram negative bacteria. The use of bacteriocins needs to be
combined with other technologies that are able to alter the cellular membrane so that
bacteriocins can eliminate the pathogenic bacteria (Daw and Falkiner 1996). The
use of non-thermal treatments such as pulsed electric field (PEF) along with bacte-
riocins is one such combination. Furthermore bacteriocins could be used with other
antimicrobial compounds such as sodium acetate and sodium lactate resulting in
more effective inactivation of bacteria. Bioactive packaging is another application of
bacteriocins. Bioactive packaging can be prepared by addition of a sachet containing
the bacteriocin into the packaged food, which will be released during storage of the
food product or directly immobilizing the bacteriocin on to the food packaging. This
has an advantage over dipping and spraying foods with bacteriocins because antimi-
crobial activity may be lost or reduced due to inactivation of the bacteriocins by food
components (Paul Ross et al. 2002). There are various methods to prepare packaging
films with bacteriocins. One method is to incorporate bacteriocin directly into
polymers. Another method is to coat or adsorb bacteriocins to polymer surfaces,
6 Natural Food Antimicrobials of Microbial Origin 109

e.g., include polyethylene films with nisin methylcellulose coatings for the use on
poultry meat and adsorption of nisin on polyethylene, ethylene, vinyl acetate,
polypropylene, polyamide, and polyvinyl chloride (Deegan et al. 2006).

6.1 Bacteriocins in Combination with Chemical Preservatives

With the increasing demand for more natural and safe food products, there is a
need for new preservation techniques to avoid different forms of spoilage and
food poisoning. The US National Food Processors Association recommends the
use of combined preservation methods to create a series of ‘hurdles’ throughout the
process, each representing a barrier that must be overcome by bacteria to initiate
food spoilage (Leistner 1999). Several studies focus on combination of physical
treatments and/or chemical preservatives along with bacteriocins, creating a series
of hurdles; some of these have been discussed further.
Ananou et al. (2004) studied the control of the enterotoxigenic Staphylococcus
aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour
of the AS-48 activity in the presence of food preservatives. It was found that
Enterocin AS-48 has a bactericidal and bacteriolytic mode of action on S. aureus
CECT 976. The inhibitory effect of AS-48 was pH and temperature dependent, and
enterocin activity was higher at pH 5. The minimum bactericidal concentration
(MBC) of AS-48, decreased from 15 μg mL−1 at 37 °C to 10 μg mL−1 at 15 °C.
Sublethally injured cells showed an increased sensitivity with a MBC of 5 μg mL−1.
The study showed that Ent AS-48 has a potential for application as a protective
agent against S. aureus in foods.
Molinos et al. (2009) found increased bactericidal activity of Enterocin AS-48
(30–60 μg/g) in combination with bioactive components from essential oils, plant
extracts and food preservatives (citric and lactic acid, sucrose palmitate, sucrose
stearate, p-hydroxybenzoic methylester acid—PHBME, and Nisaplin). The essential
oils and plant extracts used in the study were carvacrol, eugenol, thymol, terpineol,
tyrosol, hydroxytyrosol, caffeic, ferulic and vanillic acid, luteolin, geranyl butyrate,
geranyl phenylacetate, pyrocatechol, hydrocinnamic acid, tert butylhydroquinone,
phenylphosphate, isopropyl methyl phenol, coumaric acid, and 2-­nitropropanol.
The combination of anti microbials significantly reduced viable counts of Listeria
monocytogenes in Russian-type salad during one week storage at 10 °C. Antilisterial
activity of AS-48 (30 μg/g) in salad was strongly enhanced by essential oils (thyme
verbena, thyme red, Spanish oregano, ajowan, tea tree, clove, and sage oils tested at
1%, as well as with 2% rosemary oil). Antilisterial activity also increased as AS-48
acted synergistically with citric, lactic acid, and PHBME. A mixed population of two
L. monocytogenes strains was markedly reduced for one week in salads treated with
AS-48 (30 μg/g) in combination with lactic acid, PHBME or Nisaplin. The increased
bactericidal activity of these combinations can be useful to improve protection
against L. monocytogenes during salad storage.
110 S. Sehgal and V. Sharma

Jamuna et al. (2005) evaluated antibacterial efficiency of bacteriocins from

Lactobacillus isolates from appam batter (LABB), vegetable pickle (LABP) and nisin
individually and in combination against several Gram-positive and Gram-­negative
food spoilage and pathogenic organisms in liquid medium by pour plate method.
When employed individually nisin was found to be a potent inhibitor of Clostridium
sporogenes while the bacteriocins from Lactobacillus isolates, LABB and LABP
were potent against Listeria monocytogenes and Staphylococcus aureus. LABB or
LABP was a better antibacterial in combination with nisin than when they were used
alone. However, the combination of LABB with nisin was more promising as there
was no growth of Pseudomonas up to 72 h of incubation. The efficacy of these bacte-
riocins was also evaluated individually and in combination against L. monocytogenes
and S. aureus in a vegetarian food model system. The bacteriocin LABB was found to
be more effective as there was considerable inhibition of both L. monocytogenes and
S. aureus as compared to either nisin or LABP while the combination of LABB with
nisin proved to be more beneficial as there was maximum inhibition of both the
organisms and no bloating of the sealed pouches was observed up to 14 days.
The researchers concluded that bacteriocins in combination may be useful to improve
the microbial quality and ultimately, the safety and shelf life of vegetarian foods.
Effect of combined physico-chemical treatments based on enterocin AS-48 for
inactivation of Gram-negative bacteria in soybean sprouts was studied by Molinos
et al. (2008). The researchers, tested for decontamination of soybean sprouts against
Gram-negative bacteria using Enterocin AS-48. Although treatment with bacterio-
cin alone had no effect on Salmonella enterica, a synergistic antimicrobial effect
was detected at pH 9.0 and in combination with moderate heat treatment. Greatest
inactivation was achieved for sprouts heated for 5 min at 65 °C in an alkaline
(pH 9.0) enterocin AS-48 solution of 25 μg/mL. Bactericidal activity against
S. enterica increased greatly when enterocin AS-48 was used in washing solutions
in combination with several chemical compounds: EDTA, lactic acid, peracetic
acid, polyphosphoric acid, sodium hypochlorite, hexadecylpyridinium chloride,
propyl-­p-­hydroxybenzoate, or hydrocinnamic acid. The combined treatment of
enterocin AS-48 and polyphosphoric acid was tested against several other Gram-
negative bacteria inoculated on sprouts. The bacteria tested showed great differ-
ences in sensitivity to polyphosphoric acid, but synergism with enterocin AS-48 was
confirmed in all cases. Combinations of enterocin AS-48 (25 μg/mL) and polyphos-
phoric acid in a concentration range of 0.1 to 2.0% significantly reduced or inhibited
growth of the populations of S. enterica, Escherichia coli O157:H7, Shigella spp.,
Enterobacter aerogenes, Yersinia enterocolitica, Aeromonas hydrophila and
Pseudomonas fluorescens in sprout samples stored at 6 °C and 15 °C. The combined
treatment could therefore be applied to reduce the risks of Gram-negative patho-
genic bacteria as well as spoilage bacteria on sprouts.
Another study by Arques et al. (2011) investigated the antimicrobial activity of
reuterin in combination with different bacteriocins from lactic acid bacteria against
food-borne pathogens in milk. They observed a strong synergistic effect of reuterin
in combination with nisin, lacticin 481 or enterocin AS-48 on Listeria monocyto-
genes. Only nisin increased the antimicrobial activity of reuterin against
Staphylococcus aureus. Bactericidal activity of reuterin towards Escherichia coli
6 Natural Food Antimicrobials of Microbial Origin 111

O157:H7, Salmonella enterica, Yersinia enterocolitica, Aeromonas hydrophila and

Campylobacter jejuni was not enhanced significantly by the addition of any of the
bacteriocins investigated. The synergism of reuterin and nisin against L. monocyto-
genes and S. aureus was also found at refrigeration temperatures, where the patho-
gens were completely inactivated. Refrigerated milk treated with both natural
antimicrobials would mean a feasible system to control pathogenic contaminants.

6.2 Bacteriocins in Combination with Physical Treatments

Combination treatments of bacteriocin alongwith novel processing technologies

have been studied for successful applications in enhanced microbial inactivation.
Successful combinations of nisin with high hydrostatic pressure (nisin-HHP) and
ultrasound with high hydrostatic pressure (US-HHP) were explored to achieve
enhanced microbial inactivation in liquid whole egg processing by Lee et al. (2003).
The HHP processing conditions were fixed to either 250 MPa for 886 s or 300 MPa
for 200 s at the treatment temperature of 5 °C, which have been determined as the
optimum HHP processing conditions considering egg protein coagulation and micro-
bial inactivation kinetics. Between the two types of combinations, the nisin-­HHP
combination showed more promising results. The addition of nisin prior to pressure
treatments significantly increased the lethal effects of HHP against Listeria seeligeri
up to 5 log cycles. Because the individual effects of each nisin or HHP used alone on
the Listeria were almost negligible, the Listeria reductions are considered to be due
to the synergistic action of nisin and HHP. However, the combination of nisin-HHP
on E. coli showed exactly the same degree of inactivation by HHP alone, which
supports protection mechanism of gram negative bacteria against the action of nisin.
The US-HHP combination caused no reductions of Listeria and only a slightly
increased inactivation of E. coli under the experimental conditions.
The combined effects of high pressure and nisin on germination and inactivation
of Bacillus spores in milk was studied by Black et al. 2008. Spores of Bacillus
subtilis and Bacillus cereus suspended in milk or buffer were treated at 100 or
500 MPa at 40 °C with or without 500 IU mL−1 of nisin. Treatment at 500 MPa
resulted in high levels of germination (4 log units) of B. subtilis spores in both milk
and buffer; this increased to >6 logs by applying a second cycle of pressure. Viability
of B. subtilis spores in milk and buffer was reduced by 2 · 5 logs by cycled HP, while
the addition of nisin (500 IU mL−1) prior to HP treatment resulted in log reductions
of 5 · 7 and 5 · 9 in phosphate buffered saline and milk, respectively. Physical dam-
age of spores of B. subtilis following HP was apparent using scanning electron
microscopy. Treating four strains of B. cereus at 500 MPa for 5 min twice at 40 °C
in the presence of 500 IU mL−1 nisin proved less effective at inactivating the spores
of these isolates compared with B. subtilis and some strain-to-strain variability was
observed. The study concluded that combinations of HP treatment and nisin may be
an appealing alternative to heat pasteurization of milk.
Alpas and Bozoglu (2000) studied the combined effect of high hydrostatic
pressure, heat and bacteriocins on inactivation of foodborne pathogens in milk
112 S. Sehgal and V. Sharma

and orange juice. The study was to combine pressure (345 MPa) with heat (50 °C),
and bacteriocins (5000 AU/mL sample) for a short time (5 min) for the inactiva-
tion of relatively pressure-resistant strains of four foodborne pathogens:
Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and
Salmonella in pasteurized milk and orange juice. Without bacteriocin addition,
5.5 log-cycle reductions was obtained for S. aureus 485 in milk whereas more
than 8 log-cycle reduction was achieved for all the other strains studied. After stor-
age of samples for 24 h at 4 °C, S. aureus 765 showed no growth on selective media
for all the other micro-­organisms assayed. Incubation of the same pressurized sam-
ples at 37 °C for 48 h showed growth of L. monocytogenes strains in addition to
S. aureus strains, where still no growth was observed for E. coli O157:H7 and
Salmonella strains in their respective selective media. For orange juice samples,
more than 8 log-cycle reduction was achieved for all the bacterial species using the
combined effect of high hydrostatic pressure, heat and bacteriocins. No growth was
seen for these species on their respective selective media agar plates after storage at
4 °C for 24 h and at 37 °C for 48 h. When a bacteriocin-based biopreservative (BP1)
was combined with pressurization, more than 8 log-cycle reductions in cell popula-
tion of the resistant strains of S. aureus and L. monocytogenes were achieved in
milk after pressurization. Milk samples were stored at 25 °C up to 30 days to test
the effect of treatment and samples showed no growth whereas all the controls
were positive.

6.3 Microgard

Microgard™, a commercially available fermented milk product containing antimicro-

bial metabolites, was found to be a potent inhibitor for Gram-negative bacteria such as
Pseudomonas, Salmonella, and Yersinia when 1% concentration was incorporated
into agar media. Gram-positive Bacillus cereus, Staphylococcus aureus, and Listeria
monocytogeneswere insensitive to Microgard™. Kluyveromyces marxianus, an
unidentified black yeast, and Penicillium expansum were partially suppressed, whereas
Aspergillus niger and a yogurt spoilage yeast were tolerant to 5% Microgard™.
Optimum activity of Microgard™ was at pH 5.3 and below; the concentration that
gave complete inhibition depended upon the number of bacteria present as well as the
genus tested. Blood agar base reversed the antagonistic activity of Microgard™
against Pseudomonas putida compared with plate count agar (Al-Zoreky et al. 1991).
Von Staszewski and Jagus (2008) studied the antimicrobial activity of Microgard™
individually or in combination with nisin against Listeria innocua in liquid cheese
whey (LCW). Microgard™ did not reduce the initial count of L. innocua during
storage at 7, 12, 20 and 25 °C, and showed a response similar to the untreated whey.
In comparison, nisin exhibited an immediate bactericidal effect that was followed by
regrowth of L. innocua. Initially, a significant antagonistic effect was detected when
Microgard™ was combined with nisin in all systems evaluated. However, during
6 Natural Food Antimicrobials of Microbial Origin 113

storage, different responses were observed. Some combinations were more effective
than single treatments, providing an adequate control of L. innocua. The combination
of Microgard™ and nisin appears to be a feasible means for extending the shelf life of
liquid cheese whey.
Zuckerman and Avraham (2002) studied the effect of bacteriocin solutions
(Microgard™ and Nisin) on reducing total microbial counts, inhibiting Listeria
monocytogenes, and prolonging the shelf-life. Listeria monocytogenes was inocu-
lated onto chilled and on frozen and thawed salmon samples. The combination of
Nisin and Microgard reduced the total aerobic bacteria populations of fresh chilled
salmon by 2 log (P < 0.05) and increased its shelf-life, at 6 °C, by 3–4 days, as
compared with the control. The above bacteriocin combination also reduced the
growth of inoculated Listeria monocytogenesin frozen–thawed salmon and increased
its shelf-life from 5 to 10 days at 6 °C. The bacteriocin treatment did not affect sur-
face pH values or color of the fish.

7 Future Outlook

7.1 Bacteriocins and Nanotechnology

In the last few years, the application of nanotechnology to food safety has attracted
the attention of many researchers due to its considerable potential for the develop-
ment of antimicrobial delivery systems (Zou et al. 2012). This technology could be
used to improve antimicrobial stability and could be applied directly or as a coating
or packaging in different food systems to inhibit the growth of foodborne patho-
gens. Applications of nanotechnology to deliver antimicrobials have been reported
in several studies. However, the study of nanoparticles as antimicrobials in food
models is very limited due to the complexity of food components. Some of the
recent studies have been effectively applied in food models using natural com-
pounds. Zou et al. (2012) demonstrated the potential use of liposomal nanoparticles
for enhancing the antimicrobial efficacy of nisin against L. monocytogenes and
S. aureus in food systems. The antimicrobial activity of free nisin and nisin loaded
solid lipid nanoparticles was also studied by Prombutara et al. (2012). The results of
their study showed stable and longer antimicrobial activity of nisin loaded nanopar-
ticles against L. monocytogenes DMST 2871 compared to free nisin, indicating that
the nisin was released from nanoparticles throughout the storage period.
Nanoparticles hold tremendous potential as an effective antimicrobial delivery
system and could be employed to incorporate natural antimicrobials that result in
safer food products. However, the use of nanotechnology in food has raised a
number of concerns over their safety to the consumers. Furthermore, safety and
health risks of natural antimicrobials need to be assessed before future applications
in food products.
114 S. Sehgal and V. Sharma

7.2 Bacteriocins and Hurdle Technology

Leistner (1999) has described over 60 potential hurdles to improve food stability and/
or quality. The application of bacteriocins as part of hurdle technology has received
great attention in recent years (Chen and Hoover 2003; Deegan et al. 2006), since
bacteriocins can be used intentionally in combination with selected hurdles in order to
increase microbial inactivation. The combination of hurdles to be applied will depend
greatly on the type of food and its microbial composition. As an example, food
acidification may select for aciduric bacteria while the heat treatment may select
thermophilics. Also, lack of microbial competition due to elimination of some bacte-
rial population may provide a more conducive environment for those present.

8 Conclusion

There is great need for food preservation in both developing and developed countries
due to increasing demand for extending shelf life and prevention of food spoilage.
Exploration of new avenues for the food preservation has gained importance due to
the emergence of new pathogens and ability of micro-organisms to undergo changes.
There is increasing demand by consumers for chemical-free and minimal processed
food products as the awareness among consumers regarding harmful effects of
chemical preservatives has increased. Bacteriocins and their application in the pres-
ervation of food has long been recognized. They are normally specific to closely
related species without affecting the growth of other microbial species.
A large number of bacteriocins from LAB have been characterized and many
researchers have indicated the potential usefulness of bacteriocins in food preserva-
tion. The combined use of novel preservation technologies such as PEF offers new
opportunities for application of bacteriocins as part of hurdle technology. However,
many other technologies (such as ultrasonication, irradiation, microwave and ohmic
heating, or pulsed light) along with bacteriocins still remain unexplored. The study of
bacterial genomes of LAB can provide valuable information on the bacteriocino-
genic potential of these microorganisms in near future.

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Chapter 7
Antimicrobial Peptides and Polyphenols:
Implications in Food Safety and Preservation

Amardeep Singh Virdi and Narpinder Singh

Abstract The concept of minimally processed and natural foods is gaining popu-
larity among the different group of consumers, and the globalization of food mar-
kets, unique new manufacturing processes, and demand for nutritional food without
preservatives has enforced research for natural antimicrobials. Food outbreaks
caused by Listeria monocytogenes, Escherichia coli O157 and eradication of
Bacillus and Clostridium spores have emerged as major challenges faced by the
modern food industry. The antimicrobial compounds have a broad range of biologi-
cally active molecules of multiple applications. These molecules could be of mul-
tiple origins, and mainly consist of carbohydrates, proteins (cationic or anionic),
lipids, essential oils or secondary metabolites (saponins, flavonoid, thymols, lin-
alool, citral, tanins, eugenols, terpene, polyphenols, phytophenols, phenolic acids
etc.). Many of the naturally occurring antimicrobials are commercialized however;
the efficacy, consumer acceptance, and regulation are still a matter of extensive
research. In the present chapter, identification, characterization, the mechanism of
action of selected natural antimicrobial peptides and polyphenols and their potential
applications in food safety and preservation have been discussed.

Keywords Antimicrobial peptides • Antimicrobial polyphenols • Food safety •

Food preservation

1 Introduction

The concept of minimally processed and natural foods is gaining popularity among
different consumers. The globalization of food markets, unique new manufacturing
processes and demand for nutritional food products without preservatives have

A.S. Virdi (*) • N. Singh (*)

Department of Food Science & Technology, Guru Nanak Dev University,
Amritsar, Punjab, India
e-mail: [email protected]; [email protected]

© Springer Science+Business Media, LLC 2017 117

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_7
118 A.S. Virdi and N. Singh

enforced research for the identification and characterization of natural antimicrobial

agents. The major food pathogens are E. coli, Salmonella, Listeria monocytogenes,
Staphylococcus aureus, Bacillus cereus, Yersinia enterocolytica, Clostridium per-
fringens, Clostridium botulinum and Campylobacter jejuni, whereas the pathogenic
toxin-producing fungi, usually related to food-borne diseases, are Aspergillus flavus
and Aspergillus paraciticus. Food outbreaks caused by L. monocytogenes, E. coli
O157 and eradication of Bacillus and Clostridium spores from preserved food have
emerged as the most challenging tasks faced by the modern food industry. Although
several modern technologies, hygiene and safety concepts such as risk assessments
and hazard analysis and critical control points (HACCP), good manufacturing prac-
tices etc., have been developed to overcome food-borne diseases, nonetheless the
cases of food outbreaks and intoxication have increased. The uses or sometimes
misuses of bactericidal/antibacterial agents or antibiotics in healthcare of human
and animals resulted in the development of resistance/tolerance in several microbial
organisms against these chemical compounds. The tremendous use of hurdles and
safety barriers in food safety and preservation also accelerated the acquisition of
resistance in different pathogenic food microorganisms. In the United States, 9.4
million incidents of foodborne illness, 55,961 hospitalizations, and 1351 deaths
have been estimated (Scallan et al. 2011). Among these cases, the causative agents
for majority (58%) of illnesses were norovirus, followed by nontyphoidal Salmonella
(11%), Clostridium perfringens (10%), and Campylobacter (9%), whereas the
majority of deaths were due to the nontyphoidal Salmonella (28%), Toxoplasma
gondii (24%), Listeria monocytogenes (19%), and norovirus (11%). The maximum
hospitalization was owed to nontyphoidal Salmonella (35%), norovirus (26%),
Campylobacter (15%), and T. gondii (8%) (Scallan et al. 2011). Therefore, the
World Health Organization (WHO) has taken a serious note of these alarming con-
ditions and declared multidrug resistance as a major public health concern (Bryan
1982; WHO 1995; Rydlo et al. 2006a). The 922 beef, pork and poultry products
showed the presence of antibacterial and multidrug resistant pathogenic E. coli, L.
monocytogenes, Campylobacter, Salmonella and Yersinia enterocolitica (Mayrhofer
et al. 2004). The uses of frankincense, myrrh, garlic, black pepper, cumin, clove,
ginger, caraway, essential oils and plant extracts as antiseptic, therapeutic agents,
natural food preservatives and flavoring agents had been a routine practice in human
civilization. Not only Hippocrates documented this in late fifth century but also in
the Bible (Cowan 1999; Hammer et al. 1999; Arora and Kaur 1999). Antibacterial,
antifungal, and other related compounds are synthesized by medicinal plants, bacte-
rial and fungal strains in self-defense as well as to cope with adverse environmental
conditions. The preservation of food by thermal denaturation of microbial patho-
gens has widely been applied. However, it has to compromise with nutrition and
organoleptic properties of food. The major bactericidal agents were isolated from
bacterial and fungal strains and commercially produced by fermentation and other
chemical techniques. Imposition of strict regulatory restrictions against the applica-
tion of high doses of antibiotics in food products led to the tremendous demands for
minimally processed food or organic food with natural food preservatives. The anti-
microbial compounds are a broad range of biologically active molecules having
7 Antimicrobial Peptides and Polyphenols… 119

multiple uses. These molecules could be of multiple origins and mainly consist of
carbohydrates, proteins (neutral, cationic or anionic), lipids, essential oils or sec-
ondary metabolites (saponins, flavonoids, thymols, linalool, citral, tannins, euge-
nols, terpene, polyphenols, phytophenols, phenolic acids etc.). Natural antimicrobial
peptides (AMPs) emerged as highly successful natural defense molecules.
Eukaryotic organisms deploy these molecules to cope with damage caused by dif-
ferent protozoans, bacteria, fungi, and viruses (Zasloff 2002). Therefore, the poten-
tial role of eukaryote-specific AMPs and polyphenols in food safety and food
preservation are discussed in this chapter.

2 Eukaryotic Antimicrobial Peptides

Lysozyme, discovered by Alexander Fleming in 1922 is recognized as the first anti-

microbial peptide (Wang 2015). The antimicrobial activity in the skin secretions of
frogs (Csordás and Michl 1970) and lymph of insects (Habermann and Jentsch
1967) was discovered in the early 80s. This led to the foundation of future research
to explore the antimicrobial properties of peptides of diverse origin and molecular
weight. Approximately 2400 AMPs (Narayana and Chen 2015) were identified
from different microorganisms, amphibians, insects, plants, mammals and sea spe-
cies (Khamis et al. 2015 and references therein). The biological activity and the
consensus of amino-acid of different AMPs are not definite and also not an evolu-
tionarily conserved feature, as depicted in many protein families. However, several
AMPs share some common structural and biochemical features, such as net charge,
hydrophobicity, and amphipathicity (Narayana and Chen 2015; Zasloff 2002). In
general, the categorization of AMPs is broadly based on the amino-acid composi-
tion, size, type of biological sources, biological function of peptides (antibacterial,
antifungal, anticancer etc.), covalent bonding pattern, connection patterns of the
polypeptide chain, and molecular targets (Wang 2015; Khamis et al. 2015). These
AMPs are grouped into two classes based on amino-acid composition and second-
ary structure. Class (1) is further subdivided into (a): linear; (b) cysteine-rich pep-
tides; and (c) AMPs with glycine, proline, arginine, or histidine amino-acids.
Whereas, class 2 is divided into four subclasses (i) α-helical (cecropin B, cecropin
P1, melittin, magainins etc.) (ii) β-sheet (2–4 disulfide bridges i.e., human defen-
sins, plectasin or protegrins), (iii) mixed α/β (human β-defensin-2) and (iv) random
coil (Wang 2015). Several public databases, such as DAMPD (Sundararajan et al.
2012), CAMP (Waghu et al. 2014), APD2 and APD3 (Wang et al. 2009) and
ANTIMIC (Brahmachary et al. 2004) etc., have been established for the identifica-
tion, characterization and structure-function prediction of existing and emerging
synthetic AMPs. Most of the AMPs are cationic in nature with few exceptions and
showed bacteriostatic and bactericidal activity, whereas the majority of anionic
AMPs (AAMPs) have been identified in vertebrates, invertebrates, and plants
(Narayana and Chen 2015) (Table 7.1).
 120

Table 7.1 Anionic antimicrobial peptides (AMP) from different eukaryotes

Accession number
Name of AMPs of Peptide Amino acid sequence of peptide Reference
Amblyomma defensin 1 Q5VJF9 FDNPFGCPADEGKCFDHCNNKAYDIG Lai et al. (2004)
YCGGSYRATCVCYRK
Amblyomma defensin 2 Q5VJF8 YENPYGCPTDEGKCFDRCND Lai et al. (2004)
SEFEGGYCGGSYRATCVCYRT
BmKa1 Q9Y0X5 EENEEGSNESGKSTEAKNTD Zeng et al. (2004)
ASVDNEDSDIDGDSD
BmKa2 Q8N0N8 MSSKTLLVLLLVGVLVSTFFTADAYPASMDNYDDAL Zeng et al. (2004)
EELDNLDLDDYFDLEPADFVLLDMWANMLESSDFDDME
Chromogranin A P05059 YPGPQAKEDSEGPSQGPASREK Strub et al. (1996)
DCD-1 L SSLLEKGLDGAKKAVGGLGKLGKD Schittek et al. (2001)
AVEDLESVGKGAVHDVKDVLDSVL
Hlsal-defensin ABO28926.1 DDSDHGFRTAHVDLVCPDNPDNCIQQCV Zhou et al. (2007)
SKGAQGGYCTNEKCTCYEKIPSATKRVRIVA
Kappacin (variant B) P02668 MAIPPKKNQDKTEIPTINTIASGEPTSTP Malkoski et al. (2001)
TTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV
Kappacin (variant B) P02668 MAIPPKKNQDKTEIPTINTIASGEPTSTPTIE Malkoski et al. (2001)
AVESTVATLEASPEVIESPPEINTVQVTSTAV
Peptide B/ enkelytin P01211 FAEPLPSEEEGESYSKEVPEMEKRYGGFMRF Goumon et al. (1998)
Scygonadin ACY78509.1 GQALNKLMPKIVSAIIYMVGQPNAGVTFLG Wang et al. (2007)
HQCLVESTRQPDGFYTAKMSCASWTHDNPIVG
EGRSRVELEALKGSITNFVQTASNYKKFTIDEVEDWIASY
Temporin-1Ja BAJ34720.1 ILPLVGNLLNDLL-NH2 Simmaco et al. (1996)
Theromyzin Q6T6C1 DHHHDHGHDDHEHEELTLEKIKEKIKDYADKTPVDQLTERVQ Tasiemski et al. (2004)
AGRDYLLGKGARPSHLPARVDRHLSKLTAAEKQELADYLLTFLH
A.S. Virdi and N. Singh
7 Antimicrobial Peptides and Polyphenols… 121

2.1 Anionic AMPs

Anionic AMPs (AAMPs) are an integral part of the innate immune system of
their hosts and localized to the vital organs (epidermis, epididymis, respiratory
tract, brain, blood and gastrointestinal tract) of the body (Narayana and Chen
2015). The amphibian AAMP are 10–13 amino-acid residues, which were iso-
lated from the European red frog Rana temporaria and Japanese brown frog,
Rana japonica and named as temporin A, temporin B, and temporin 1Ja, respec-
tively (Table 7.1). Temporins are the smallest AMPs, which showed differential
activity against Gram-­positive and Gram-negative bacteria, with varied minimum
inhibitory concentrations (MICs) (Simmaco et al. 1996). Temporin A showed
strong antibacterial activity against S. aureus and S. pyogenes with MICs of
2.3 μM and 2.0 μM, respectively. On the contrary, temporin A and B showed poor
bactericidal active against E. coli D21 with MIC of 21.0 μM and were completely
inactive against P. aeruginosu with MIC of >360 μM. These findings suggested
that both temporin A and B act specifically against Gram-positive bacteria
(Simmaco et al. 1996). AAMPs were identified from annelid (Theromyzin)
(Tasiemski et al. 2004), arachnids [Amblyomma defensin 1; Amblyomma defen-
sin 2 (Lai et al. 2004), and Hlsal-­defensin (Zhou et al. 2007)], scorpion venom
(BmKa1) and crustaceans (Scygonadin) (Wang et al. 2007) (Table 7.1).
Theromyzin from Thermyzon tessulatum demonstrated antimicrobial activity
against M. luteus with MIC < 1 μM. It was proposed that the antibacterial activity
of theromyzin may be because of histidine and aspartate rich N-terminal region.
The antibacterial activity of theromyzin was divalent ion (Zn2+) dependent
(Tasiemski et al. 2004). Amblyomma defensin 1 and Amblyomma defensin 2
from haemolymph of female ticks showed peptide activity against Gram-positive
and Gram-negative bacteria with low MICs values (Lai et al. 2004). The cleavage
of bovine caseino macropeptides resulted in small molecular weight peptides and
named as Kappacins. Kappacins were anionic in nature and have partially pH
dependent, broad-spectrum antimicrobial activity against Gram-­ positive and
Gram-negative bacteria with low MIC (Malkoski et al. 2001; Rizzello et al. 2005)
(Table 7.1). Chromacins and peptide B/enkelytin were isolated from bovine that
possess differential antimicrobial activities (Table 7.1). The antimicrobial activ-
ity of chromacins was post-translationally modulated, phosphorylation-­dependent
and was specific against Gram-positive bacteria (Strub et al. 1996), whereas, pep-
tide B/enkelytin, also showed antimicrobial activity against a wide range of
Gram-positive bacteria with low MICs (Goumon et al. 1998).
Dermicidin (DCD) is proteolytically modified, 47 amino-acid residues long
AAMP, which was isolated from human sweat. A DCD, named DCD-1, was proven
to possess antimicrobial active against E. coli, E. faecalis, S. aureus and C. albicans
over a broad pH range as well as in high salt concentrations which was observed
useful in food safety applications (Schittek et al. 2001).
122 A.S. Virdi and N. Singh

2.2 Cationic Antimicrobial Peptides

Cationic antimicrobial peptides (CAMPs) are the largest group of AMPs. CAMPs
have been identified in many plant and animal species and have been divided into
three categories. Type 1 CAMPs include short linear chain CAMPs, α-helical
CAMPs, pro-rich CAMPs, gly-rich CAMPs, his-rich CAMPs, his/gly-rich CAMPs,
and trp-rich CAMPs. Type 2 CAMPs include cyclic, disulfide-bridged CAMPs with
two or four cysteine-β-hairpins and Rana box CAMPs, six or eight cysteine amino-­
acid residues CAMPs, and ten or twelve cysteine amino-acid residues CAMPs.
Type 3 includes all the other types of CAMPs having covalent bridges and cova-
lently linked dimers (Tossi and Sandri 2002).
Defensins are amphipathic, cystein-rich, small CAMPs, which are widely dis-
tributed in plants, insects, mammals, and birds. All defensins are synthesized as
“prepropetides”, and processed at the site of expression, therefore, active defensins
are 18 to ~45 amino-acids long, hydrophobic amino-acid residues rich CAMPs with
a varied net charge ranging from +1 to +11. Defensins do not undergo post-­
translational modifications such as glycosylation or acylation (Selsted and Ouellette
2005). Defensins play a key role in the improvement of animal health and protect
them from zoonotic diseases. Two isoforms of sphenisins, namely spheniscin-1 and
spheniscin-2, were isolated from the preserved stomach content of the king penguin
Aptenodytes patagonicus. The bacteriostatic activity of both these spheniscins was
specific to Gram-negative bacteria, whereas, bactericidal activity of these sphenis-
cins against Gram-positive bacteria was ranged from pH 4–6. The MICs of sphenis-
cin-­1 and spheniscin-2 range 0.4–15 μM and 1.5–>100 μM, respectively, against
bacteria, yeast and filamentous fungi (Thouzeau et al. 2003). However, the antibac-
terial and bacteriostatic activity of spheniscin-1 and spheniscin-2 was not evaluated
in food preservation.
Human β-defensin 3 (HBD3) and synthetic cathelicidin (LL-37) (Table 7.2) were
isolated from the epidermal keratinocytes of psoriasis patients and neutrophils,
respectively. HBD3 showed broad-spectrum antibacterial activity against multidrug-­
resistant S. aureus and vancomycin-resistant Enterococcus faecium (Harder et al.
2001), whereas, the bactericidal activity of cathelicidin LL-37 was demonstrated
against Gram-positive and Gram-negative bacteria such as Pseudomonas aerugi-
nosa, S. typhimurium, E. coli, L. monocytogenes, and S. aureus (Turner et al. 1998).
Bactenecins 5 and 7, also named as Bac 5 and Bac 7, were the first cathelicidins
isolated from bovine neutrophils. Both, Bac 5 and Bac 7 were polycationic in nature
and possessed repeated proline motifs (Gennaro et al. 1989) (Table 7.2). The mono-
and dimeric forms of bactenecins showed cytolytic activity against S. aureus with
MIC of 8–16 μM. Structural analysis revealed that Bac 5 contains an intra-­molecular
disulfide bond, a β-hairpin structure with four arginine residues. As compared to
monomeric form, the homodimeric form of bactenecin showed higher antibacterial
activity with less sensitivity to salt concentration (Lee et al. 2009b). The monomeric
form of bactenecin penetrates into the bacterial cell wall, interacts with nucleic acid
and inhibits the growth of cell, synthesis of cell wall and protein metabolism,
 7

Table 7.2 Different eukaryotic cationic, neutral, and synthetic antimicrobial peptides
Accession no.
Name of the AMP (Peptide) Amino acid sequence of peptide Reference
Brevinin-1 P32423 FLPVLAGIAAKVVPALFCKITKKC Morikawa et al. (1992)
Brevinin-1E P84839 FLPLLAGLAANFLPKIFCKITRKC Simmaco et al. (1993)
Brevinin-2 P32424 GLLDSLKGFAATAGKGVLQSLLSTASCKLAKTC Morikawa et al. (1992)
Brevinin-2E P32413 GIMDTLKNLAKTAGKGALQSLLNKASCKLSGQC Simmaco et al. (1993)
Buforin I P55897 AGRGKQGGKVRAKAKTRSSRAGLQFPVGRVHRLLRKGNY Park et al. (1996)
Cecropin P1 F1LGW1 SWLSKTAKKLENSAKKRISEGIAIAIQGGPR Lee et al. (1997)
Clavanin A P80710 VFQFLGKIIHHVGNFVHGFSHVF.NH2 Lee et al. (1997)
Clavanin B P80711 VFQFLVFQFLGRIIHHVGNFVHGFSHVF.NH2 Lee et al. (1997)
Clavanin C O18493 VFHLLGKIIHHVGNFVYGFSHVF.NH2 Lee et al. (1997)
Clavanin D P80713 AFKLLGRIIHHVGNFVYGFSHVF.NH2 Lee et al. (1997)
Antimicrobial Peptides and Polyphenols…

Clavanin E O18492 FKLLGKIIHHVGNFVHGFSHVF Lee et al. (1997)

Clavanin MO O18492 FLPIIVFQFLGKIIHHVGNFVHGFSHVF-NH2 Silva et al. (2016)
Dermaseptin S4 P80280 ALWMTLLKKVLKAAAKALNAVLVGANA Mor et al. (1994)
Dicynthaurin P0C007 ILQKAVLDCLKAAGSSLSKAAITAIYNKIT Lee et al. (2001)
Esculentin-1 P32414 GIFSKLGRKKIKNLLISGLKNVGKEVGMDVVRTGIDIAGCKIKGEC Simmaco et al. (1993)
Frenatin-1 P82021 GLLDALSGILGL Raftery et al. (1996)
Gaegurin-1 P80395 SLFSLIKAGAKFLGKNLLKQGACYAACKASKQC Park et al. (1994)
Gaegurin-2 P80396 GIMSIVKDVAKNAAKEAAKGALSTLSCKLAKTC Park et al. (1994)
Gaegurin-3 P80397 GIMSIVKDVAKTAAKEAAKGALSTLSCKLAKTC Park et al. (1994)
Gaegurin-4 P80398 GILDTLKQFAKGVGKDLVKGAAQGVLSTVSCKLALTC Park et al. (1994)
Gaegurin-5 P80399 FLGALFKVASKVLPSVKCAITKKC Park et al. (1994)
Gaegurin-6 P80400 FLPLLAGLAANFLPTIICKISYKC Park et al. (1994)
Halocyntin P86415 FWGHIWNAVKRVGANALHGAVTGALS Galinier et al. (2009)
(continued)
123
Table 7.2 (continued)
124

Accession no.
Name of the AMP (Peptide) Amino acid sequence of peptide Reference
Leptoglycin P86267 GLLGGLLGPLLGGGGGGGGGLL Sousa et al. (2009)
LL-37 J3KNB4 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Turner et al. (1998)
LL-37 from Human P49913 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRT Boge et al. (2016)
CAP18
Maculatin 1.1 P82066 GLFGVLAKVAAHVVPAIAEHF Rozek et al. (1998)
Maculatin 1.2 P82067 GLFGVLAKVASHVVPAIAEHFQA Rozek et al. (1998)
Magainin P11006 GIGKFLHSAKKFGKAFVGEIMNS Zasloff (1987)
Magainin (PGLa) Q91826 GMASKAGAIAGKIAKVALKAL Zasloff (1987)
Melittin I3RJI9 GIGAVLKVLTTGLPALISWIKRKRQQ Habermann and Jentsch
(1967)
Papillosin P86416 GFWKKVGSAAWGGVKAAAKGAAVGGLNALAKHIQ Galinier et al. (2009)
Pleurocidin Q90ZY0 GWGSFFKKAAHVGKHVGKAALTHYL Cole et al. (2000)
PR-39 P80054 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP Shi et al. (1996)
Protamine P69014 MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR Moir and Dixon (1988)
Ranalexin P39084 FLGGLIKIVPAMICAVTKKC Clark et al. (1994)
Rugosin-A P80954 GLLNTFKDWAISIAKGAGKGVLTTLSCKLDKSC Suzuki et al. (1995)
Rugosin-B P80955 SLFSLIKAGAKFLGKNLLKQGAQYAACKVSKEC Suzuki et al. (1995)
Rugosin-C P80956 GILDSFKQFAKGVGKDLIKGAAQGVLSTMSCKLAKTC Suzuki et al. (1995)
Salmine-AI P69014.2 MPRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR Moir and Dixon (1988)
Spheniscin-1 P83429.1 SFGLCRLRRGFCAHGRCRFPSIPIGRCSRFVQCCRRVW Thouzeau et al. (2003)
Spheniscin-2 P83430.1 SFGLCRLRRGFCARGRCRFPSIPIGRCSRFVQCCRRVW Thouzeau et al. (2003)
A.S. Virdi and N. Singh
7 Antimicrobial Peptides and Polyphenols… 125

whereas the homodimeric form, disrupts the pathogens’ cell wall through pore
forming mechanism. Both Bac 5 and Bac 7 killed Gram-negative bacteria with
higher selectivity than Gram-positive bacteria (Lee et al. 2009b). Bac 5 bind to cell
wall localized lipopolysaccharides (a glycolipid) of Gram-negative bacteria (E. coli,
Bacillus subtilis and Pseudomonas aeruginosa) and inhibited the growth under low
as well as high salt (100 mM NaCl) concentration (Shamova et al. 1999). These
studies thus demonstrated that the cathelicidins have selective antimicrobial prop-
erty against Gram-negative bacteria.
Cecropin P1 and PR-39 were isolated from upper part of pig small intestine (Lee
et al. 1989; Agerberth et al. 1991) (Table 7.2). PR-39 showed antimicrobial activity
against, E. coli, Salmonella typhimurium, Bacillus megatrrium and Streptococcus
suis (Agerberth et al. 1991; Shi et al. 1996). The antimicrobial activity of the PR-39
was temperature-dependent and a decline of bacterial populations by 4 and 5 log
units was observed at 24 °C and 37 °C, respectively. PR-39 penetrated into the outer
membrane of E. coli, interferes with DNA/protein synthesis, and thus affected the
cell viability (Shi et al. 1996). The synthetic peptide of cecropin P1 showed high
activity against E. coli K-12, Salmonella and Acinetobacter. However, the peptide
activity of cecropin P1 was not observed against Gram-positive bacteria. A cecropin
B from Hyalophora cecropia showed cytolytic activity against Gram-negative bac-
teria (Moore et al. 1996) and Gram-positive bacteria with low MIC ranged from 0.1
to 5 μM (Chen et al. 1997). Application of cecropin XJ of the Xinjiang silkworm
(Bombyx mori) larvae resulted in enhanced shelf life of tomato juice and grape juice
(Table 7.2). An amide-modified synthetic peptide CecXJ-37 N showed lower MIC
values of 0.25–7.8 μM against Gram-negative (E. coli, Klebsiella pneumoniae and
Pseudomonas aeruginosa) and Gram-positive (B. subtilis, S. epidermidis, methicil-
lin and oxacillin sensitive and resistant S. aureus) bacteria. The amide-modification
of CecXJ-37 N was reported to improve the antibacterial activity, reduced cell lytic
activity, and appeared as a novel tool for food preservation (Liu et al. 2017).
Many species secrete mucus from mucosal epithelial cells, which protect them
from several bacterial and fungal pathogens and act as physical barrier against cli-
matic hurdles. A fish pleurocidin, isolated from the skin mucous secretions of the
winter flounder (Pleuronectes americanus) was reported to have bactericidal active
against 11 different Gram-negative and Gram-positive bacteria. The peptide activity
of pleurocidin was tolerant to salt and heat conditions but higher concentration of
magnesium and calcium inhibited the peptide activity of pleurocidin (Cole et al.
2000). Recently, a pleurocidin was incorporated into the poly(vinyl alcohol) elec-
trospun nanofiber to enhance its bioavailability and bactericidal activity. The
pleurocidin-­incorporated fiber rapidly released pleurocidin in a growth medium and
showed improved bactericidal activity against E. coli O157:H7 present in apple
cider (Wang et al. 2015).
An arginine-rich (66%) protamine showed differential bactericidal activity
against L. monocytogenes and E. coli at temperatures and pH ranging from 5 °C to
30 °C and 5.5 to 8.0, respectively (Table 7.2). The peptide activity of protamine was
influenced by the presence of ethylene diamine tetraacetic acid (0.9 mM EDTA) and
gelatin A, a positively charged protein, in culture media (Hansen and Gill 2000;
126 A.S. Virdi and N. Singh

Hansen et al. 2001). Comparison between the efficacy of native and low positively
charged protamine revealed that the modified protamine effectively inhibited the
growth of L. monocytogenes in milk and Enterobacteriaceae (Gram-negative food-­
spoilage bacteria) in ground beef better than native protamine. While the antimicro-
bial efficacies of both types of protamine in a protein rich tryptic soy broth model
system did not vary significantly. Furthermore, the interaction with L. monocyto-
genes cells was 10–20% higher at pH 7 and 8 (Potter et al. 2005). Salmine, a homo-
log of protamine and a cationic antimicrobial peptide derived from the milt of
salmon, possess potent antibacterial activity against L. monocytogenes (Cheng et al.
2015) (Table 7.2). Cold smoked salmon either covered with biopolymers, or with
agar/к-carrageenan films containing salmine, resulted in significant inhibition of L.
monocytogenes with enhanced shelf life of cold smoked salmon meat (Cheng et al.
2015). Piscidins are long linear-helical forming CAMPs, whose presence was con-
firmed in many higher teleosts (Silphaduang et al. 2006) (Table 7.2). Piscidins pos-
sess differential antibacterial activity against Gram-negative (V. vulnificus 204; A.
hydrophila BCRC 13018; V. alginolyticus; P. aeruginosa ATCC 19660) and Gram-­
positive (S. agalactiae 819; E. faecalis BCRC 10066; S. agalactiae BCRC 10787)
bacteria. Piscidin 4 showed the lowest MIC against Gram-negative and Gram-­
positive bacteria, which ranged from 0.03 to 1.05 μg/mL and 0.13 to 8.39 μg/mL,
respectively, as compared to piscidin 1, 2, 3 and 5 (Peng et al. 2012). This implies
that the piscidin 4 possess very strong antibacterial activity against a wide range of
microorganisms and may be useful in food preservation. Arenicins (arenicin 1, 2
and 3) of lugworm Arenicola marina were 21 amino-acid long peptides
(Ovchinnikova et al. 2004) (Table 7.2) with hairpin-like β-sheet or α-helical-β-sheet
mixed structures linked with disulfide bonds. Arenicins were inhibitors of Gram-­
negative (E. coli, P. aeruginosa) and Gram-positive (L. monocytogenes, S. aureus,
S. epidermidis) with MICs ranging between 2 and 8 μM (Ovchinnikova et al. 2004).
Arenicin-1 showed high levels of peptide activity at 4 °C and at 37 °C against Gram-­
negative and Gram-positive including polymyxin B-resistant Proteus mirabilis
(Andrä et al. 2008). The role of fish-specific AMPs such as pleurocidin, protamine,
salmine, piscidins and arenicin in inhibition of several food-borne and food spoilage
bacteria was characterized but the commercialization of all these AMPs were not
done. More detailed scientific investigation in a holistic view is required in future
using a different kind of food systems such as high protein and carbohydrate rich
dairy, beverages, and meat products.
Amphibians live in humid climatic habitats, near water bodies and in wet land
conditions and possess poor cell-mediated adaptive immune systems. Therefore,
amphibians are prone to a wide range of threats imposed by microorganisms and
other natural predators. To cope with such adverse challenges, the dermal glands in
the humid skin of amphibians synthesize, store and secrete AMPs, which kill nox-
ious microorganisms (Xu and Lai 2015 and references therein). Dermaseptins S
family members are divided into family A (eight members) and B (six members).
The members of both families are derived from a group of precursors, known as
prepro-dermaseptins/prepro-dermorphins. After processing, dermaseptins are
stored in the large granules of dermal glands of the skin. A dermaseptin was isolated
7 Antimicrobial Peptides and Polyphenols… 127

from the skin extract of the South American arboreal frog Phyllomedusa sauuagii
(Table 7.2). The 34 amino-acid long dermaseptin showed cytolytic activity against
wide range of microorganisms such as viruses, Gram-positive and Gram-negative
bacteria, filamentous fungi, protozoa and yeasts ((Mor et al. 1994; Xu and Lai 2015
and references therein). A 28 amino-acid residues long, analog of dermaseptin,
named K4K20S4 (P28), showed MIC of 8 μM against E. coli O157:H7. K4K20S4 main-
tained antimicrobial activity at high salt concentrations, under acidic or basic condi-
tions, as well as under extreme temperatures. Whereas, N-terminal 14 amino-acid
long analog sequence (P14) of dermaseptin displayed higher potency with 4 μM
MIC against tested pathogens (Rydlo et al. 2006b). The MIC of dermaseptin analog
was limited to a narrow range of culture conditions thus demonstrated the signifi-
cance of C-terminal region of P28 in peptide activity. On the contrary, the conjuga-
tion of amino dodecanoic acid to P14 amino-acid residue of P28 resulted in enhanced
cytolytic activity of P28 (MIC, 2 μM) under all experiment conditions (Rydlo et al.
2006b). Intriguingly, dermaseptins showed high cytolytic activity against slow
growing and non-growing bacteria, spoilage yeasts, and pathogenic food-related
bacteria, thus suggesting that these AMPs could be useful as potent food preserva-
tive agents (Rydlo et al. 2006b).
Although magainins were initially isolated from the skin of the African clawed
frog Xenopus laevis, later their presence was confirmed in the stomach as well
(Zasloff 1987; Moore et al. 1991). Magainins were 21–27 amino-acid residues long,
cationic, hydrophobic AMPs belonged to the cathelicidins family and possess bac-
tericidal activity against a wide range of bacteria. Magainins acquire an amphipa-
thic α-helix confirmation after interaction with bacterial membranes and the
cytolytic activity of magainins was reported against many bacteria, fungi, and pro-
tozoa (Zasloff et al. 1988) (Table 7.2). Magainin and its synthetic analogs were
tested against 13 food-related pathogens, which revealed that all these CAMPs have
MICs ranging from <3 to 50 μg/mL (Abler et al. 1995). Among these CAMPs and
their synthetic analogs, when subjected to different temperature regimes and growth
media, Mag2 showed reduced potency at 4 and 25 °C, and peptide activity of Mag2
was also interfered by bovine serum albumin protein (Abler et al. 1995). A 22
amino-acid residues long pexiganan, MSI-78 showed high peptide activity not only
against broad spectrum of bacteria but against multi-resistant bacterial species as
well. MSI-78 evolved from the structure-activity relationship studies, an analog of
the magainin family AMPs (Maloy and Kari 1995) (Table 7.2). It was assumed that
the application of magainins after processing of food either in the form of biofilms
or spraying on product would greatly reduce the bacterial activity.
Antibacterial peptides, brevinin-1, brevinin-2, brevinins-1E (homolog of
brevinin-1) (Morikawa et al. 1992), and brevinins-2E (homolog of brevinin-2)
(Simmaco et al. 1993) were isolated from the skin extracts of a Japanese frog Rana
brevipoda porsa. The brevinin homologs, for example, ranalexin, gaegurins (gae-
gurin 1–5), rugosins A, B and C, type-1 and type-2 brevinins, brevinin-1VLa and
brevinin-1VLc and brevinin-1BLc were isolated in later studies (Table 7.2). These
AMPs were identified from American bullfrog (R. catesbeiana), central European
frogs (R. temporaria and Lithobates vaillanti) and plains leopard frog (L. blairii)
128 A.S. Virdi and N. Singh

(Clark et al. 1994; Park et al. 1994; Suzuki et al. 1995; Conlon et al. 2009c; Won
et al. 2009; Park et al. 2000). At the gene level, the cDNA of these CAMPs encodes
a single copy of mature peptide with a signal peptide, having similar length, pro-
cessing sites and high levels of homology with the precursor of dermorphin and
deltorphin. Dermorphin and deltorphin are opioid AMPs, which are found in the
skin secretions of amphibians of the South American subfamily. Brevinin-1VLa and
brevinin-1VLc showed antibacterial activity against Gram-positive bacteria (S.
aureus) and an opportunistic yeast pathogen (Candida albicans) with MIC of
≤3 μM, whereas the E. coli showed MIC ≤50 μM in response to brevinin-1VLc
(Conlon et al. 2009c). Brevinin-1BLc showed high peptide activity against E. coli,
S. aureus and Candida albicans with MIC of 25 μM, 1.5 μM and 3 μM, respectively
(Conlon et al. 2009a). The presence of a homolog of ranatuerin-2 family, ranatuerin-­
2Ya; was also confirmed in the skin secretions of L. yavapaiensis. The MICs of
ranatuerin-2Ya against E. coli and S. aureus was 50 μM for each strain (Conlon
et al. 2009a).
Amino-acid composition analysis of different gaegurins (GGNs) revealed that
each peptide posseses two invariant cysteine residues, one at the C-terminus and
another at the seventh position from the C-terminus (NH2-C7KXXXXC1-COOH)
which is attributed to the formation of a heptapeptide. Formation of an intra-­
molecular disulfide linkage (S-S) among in heptapeptide motif is a conserved fea-
ture of AMPs derived from Rana species (brevinin-1, brevinin-2, brevinins-1E,
brevinins-2E and esculentin), therefore this heptapeptide motif was designated as
“Rana box” (Park et al. 2000). The antimicrobial activity of GGN1-GGN-5 was dif-
ferential against different bacterial strains. The antimicrobial activity of GGN-1 was
observed against several food-borne pathogens (Micrococcus luteus, S. epidermidis,
B. subtilis, Klebsiella pneumoniae, Shigella dysentariae and Pseudomonas putida)
with MIC ranged from 5 to 100 μg/mL. As compared to GGN-1, some of these
bacterial strains were more sensitive to GGN-2 – GGN-5 with reduced MICs (Park
et al. 1994). Further studies carried out by Lee et al. (1998) demonstrated that the
presence of cysteine amino-acid residues in GGN-6 not only provided conforma-
tional stability to the α-helical region but also was crucial for the antimicrobial
activity of GGN-6 (Lee et al. 1998). Rugosin A strongly inhibited the growth of S.
aureus 209P (Gram-positive bacteria) with MIC of 6.25 μg/mL. Rugosins B inhib-
ited the growth of both Gram-negative (E.coli NIHJ) and Gram-positive bacteria
with MICs of 12.5 μg/mL and 6.25 μg/mL, respectively. Rugosin C also inhibited
the growth of Gram-positive bacteria such as S. aureus (Suzuki et al. 1995).
Alyteserin (alyteserin-1, alyteserin-2) AMPs were identified from skin secretions of
the toad Alytes obstetricans and A. maurus. Both alytes toad AMPs showed homol-
ogy with the ascaphins and bombinin H6 of Leiopelmatidae and Bombinatoridae
frogs, respectively. The antibacterial property of alyteserin-1 and alyteserin-2 was
selective; former inhibited the growth of Gram-negative bacteria, whereas the later
inhibited the growth of Gram-positive bacteria (S. aureus) with low hemolytic activ-
ity (Conlon et al. 2009b; Conlon et al. 2010). The skin secretions of the Australian
frogs of the genus Litoria and Limnodynastes also showed the presence of CAMPs.
Litoria genus showed the presence of caeridins (L. splendida and L. caerulea),
7 Antimicrobial Peptides and Polyphenols… 129

­caerins (L. xanthomera and L. chloris), frenatins (L. infrafrenata) (Raftery et al.
1996) and maculatins (L. genimaculata) etc. (Rozek et al. 1998) (Table 7.2). Only
caerins showed antimicrobial activity against Gram-positive strains (Steinborner
et al. 1998). Two AMPs, 37 amino-acid residue long buforin I, and buforin II were
isolated from the stomach of the Asian toad Bufo bufo gargarizans (Park et al. 1996)
(Table 7.2). Buforins showed high levels of identity with histone H2A proteins of
Xenopus, which revealed that these peptides were evolutionary conserved. As com-
pared to buforin 1, the 16–36 amino-acid residue long peptide of buforin II strongly
inhibited cell metabolism, which was contrary to magainins, reported to kill bacte-
ria through cell lysis (Park et al. 1998). The application of these peptides in food
preservation and food industry, however, is scanty and need more attention for food
safety. The presence of ascaphins was reported in the most primitive extant anurans
tailed frogs Ascaphus montanus (ascaphin 1–8) and A. truei (ascaphin-1 M-7 M).
Tailed frogs live in rocky streams in the cascades and coastal ranges from Southern
British Columbia to Northwestern California. The antibacterial activity of ascaph-
ins was specifically highly lethal against Gram-negative bacteria (Conlon et al.
2004; Xu and Lai 2015 and references therein). Ascaphin-8 not only possess hemo-
lytic activity (LC50 = 55 μM) but also have strong antimicrobial activity against
clinical isolates of E. coli and Klebsiella pneumoniae strains (MIC, <25 μM)
(Conlon et al. 2004) (Table 7.2). Tailoring of ascaphins with reduced hemolytic
activity could serve as potent food preservation agents in the future.
Macrophages contain granule-associated AMPs, such as lysozyme, defensins,
serprocidins, and microbicidal proteins (MUMP-1, -2, and -3). Ubiquicidin was
isolated from the granule fraction of the murine macrophage cell line RAW264.7
that showed antibacterial property against L. monocytogenes EGD, S. Typhimurium
14028S, S. aureus 502A, E. coli ML-35p and an avirulent strain of Yersinia entero-
colitica (Hiemstra et al. 1999) (Table 7.2). Melittin from Apis mellifera bee venom
showed cytolytic activity against mammalian and bacterial cells (Habermann and
Jentsch 1967) (Table 7.2). The N-terminal region (residues 1–20) of melittin was
predominantly hydrophobic, whereas the C-terminal region (residues 21–26) was
hydrophilic in nature due to the presence of a chain of positively charged amino-­
acid residues. The varied cytolytic and bactericidal properties of melittin against
Gram-positive bacteria (S. aureus, E. faecalis and L. monocytogenes) were attrib-
uted to the varied N- and C-terminal regions. (Ebbensgaard et al. 2015). Tunicates
are simple marine protochordate and ancestors of early vertebrates. The hemocytes
of tunicate are phagocytic cells and mimic the leukocytes of higher vertebrates. The
presence of clavanins was confirmed in hemocytes of the invertebrates stalked sea
squirt (Styela clava). High levels of homology of clavanin A, clavanin B, clavanin C
and clavanin D were observed with magainin 1 of Xenopus laevis (Lee et al. 1997).
Clavanin A showed antibacterial activity against E. coli ML-35p and L. monocyto-
genes with MIC 1.6 μg/mL and 3.5 μg/mL, respectively (Lee et al. 1997) (Table 7.2).
Dicynthaurin, a 60 amino-acid long, α-helical forming AMP was isolated from
solitary tunicate Halocynthia aurantium and was shown to have antibacterial
activity against Gram-negative (E. coli, P. aeruginosa) and Gram-positive (M.
luteus, L. monocytogenes, S. aureus) with MIC of 140 μg/mL (Lee et al. 2001).
130 A.S. Virdi and N. Singh

Whereas, halocyntin and papillosin (Ascidian: Halocynthia papillosa), also showed

bactericidal activity against Gram-negative (E. coli, P. aeruginosa, S. typhimurium,
K. pneumoniae) and Gram-positive (M. luteus, B. megaterium, A. viridans, S.
aureus, E. facecolis) bacteria with MBC of 0.75–100 μM and 0.25–1 μM, respec-
tively (Galinier et al. 2009).

2.3 Neutral Antimicrobial Peptides

The analysis of skin secretion of Leptodactylus pentadactylus revealed the pres-

ence of neutral glycine rich/leucine-rich AMPs which were named as leptogly-
cins (Sousa et al. 2009) (Table 7.2). Leptoglycine amino-acid composition
analysis revealed the presence of high level of glycine (59.1%) and leucine
(36.4%) and an unusual central proline amino-acid residue. The leptoglycins
were 89 and 59% identical with Xenopus spp. protein 4 (bactericidal/permeabil-
ity-increasing (BPI)/lipopolysaccharide-­ binding protein protein family) and
acanthoscurrin isoforms (the long palate, lung and nasal epithelium carcinoma-
associated protein 4: Ligand-­binding protein RY2G5), respectively. Leptoglycins
inhibited the growth of Gram-­negative bacteria (Pseudomonas aeruginosa, E.
coli and Citrobacter freundii) with MICs of 8 μM, 50 μM, and 75 μM, respec-
tively, however, antimicrobial activity against Gram-positive bacteria was not
observed (Sousa et al. 2009).

2.4 Synthetic AMPs

Studies also carried out on chemically synthesize AMPs which were either not
available in a fair amount for characterization or their analogs and derivatives were
developed to enhance the antimicrobial properties by using chemical and pro-
tein engineering tools. Ahn et al. (2008) synthesized cyclic disulfide-bonded syn-
thetic peptides with various net positive charges (+2–+5) from linear peptides
originated from the alpha-helical ___domain (DAACAAHCLWR-NH2) of Tenecin
1 (VTCDILSVEAKGVKLNDAACAAHCLFRGRSGGYCNGKRVCVCR), an
insect defensin isolated from Tenebrio molitor (Moon et al. 1994). Tenecin 1 showed
homology with sapecin and sapecin C, antibacterial proteins of Sarcophaga pereg-
rina. The antibacterial activity of synthetic peptides was modulated by the net positive
charge and disulfide-bond formation. Synthetic cyclic peptides with highest positive
charge showed antimicrobial activity comparable to linear peptides, whereas cyclic
peptides with low net charge showed lower antimicrobial activity against M. luteus
ATCC 9341, S. aureus ATCC 6538, E. coli ATCC 2592, and P. aeruginosa ATCC
9027 with differential MIC (100–>200 μg/mL) for different microbial strains. On the
7 Antimicrobial Peptides and Polyphenols… 131

contrary, higher antimicrobial activity was depicted for the cyclic peptide with opti-
mum net charge (Ahn et al. 2008). A synthetic truncated peptide named 2Abz23S29,
lacking disulphide bridges, showed a broad spectrum of antibacterial and bacte-
ricidal activity against Gram-positive (S. aureus ATCC 25923, B. subtilus NCTC
10400, S. mutans NCTC 10449, E. faecalis NCTC 12697) and Gram-negative (E.
coli ATCC 25922 and P. aeruginosa ATCC 27853) bacteria with differential MIC
for different pathogenic strains. The MIC of synthetic 2Abz23S29 AMPs against S.
aureus ATCC 25923, B. subtilus NCTC 10400, S. mutans NCTC 10449 and E. faeca-
lis NCTC 12697 was 38.9 μg/mL, 26.3 μg/mL, 49.0 μg/mL, 64.5 μg/mL and 64.5 μg/
mL, respectively. Whereas, the MICs of synthetic 2Abz23S29 AMP was 37.6 μg/mL
and 73.0 μg/mL. This was observed against E. coli ATCC 25922 and P. aeruginosa
ATCC 27853, respectively. Synthetic AMP 2Abz23S29 derived from the C-terminal
beta-hairpin region of HNP-1 (human neutrophil peptide 1) (ACYCRIPACIAGE
RRYGTCIYQGRLWAFCC), α-defensin antimicrobial peptide having disulfide pair-
ings are 1–6, 2–4 and 3–5 (Lundy et al. 2008). It was proposed that substitution of G23
by the mimetic 2-aminobenzoyl (2Abz) derivative may be involved in stabilization
of the β-hairpin structure through bend forming mechanism, whereas substitution of
C29 with S to avoid disulfide bond formation resulted in an increase in solubility of
peptides (Lundy et al. 2008). Clavanin-MO possesses a short hydrophobic ___domain
(FLPII) at the N-terminal. It also showed broad-spectrum potent antibacterial activity.
Clavanin-MO showed higher lethality against methicillin-resistant S. aureus (a clini-
cal isolates), antibiotic-­resistant Gram-negative strains (KPC positive strains of E. coli
and K. pneumoniae or multidrug resistant E. coli 2,101,123), as compared to the fre-
quently recommended β-lactam (imipenem) and aminoglycoside (gentamicin) antibi-
otics (Silva et al. 2016). Two in silico designed synthetic peptides, MTP1 and MTP2
(mitochondrial-­targeted peptides 1; and 2), of 15 (KVSGVLFGTGLWVAL) and 13
(MAEAHQAKAFQDT-NH2) amino-acid residues. Which effectively inhibited the
growth of L. monocytogenes with EC50 values of 16.8 ± 1.2 μM and 109 ± 7.0 μM,
respectively (Palmieri et al. 2016). Both MPT1 and MPT2 were derived from the
N-terminal (1MAEAHQAVAFQFT13-NH2) regulatory ___domain and trans-­membrane-­2
(TM2:103NVVSGVLFGTGLWVAL118 amino-acids) region of human CPT-1a (carni-
tine palmitoyltransferase-1a), a mitochondrial outer membrane (OMM) localized pro-
tein (accession no.: P50416.2) that catalyzes the primarily regulated steps involved in
mitochondrial fatty acid oxidation. It was suggested that the TM2 membrane ___domain
may be involved in translocation of the acyl-carnitine products into mitochondria
through the formation of a channel into the OMM (Palmieri et al. 2016). The sub-
stitution of Val104 amino-acid of MPT1 with Lys (V104K) altered the positive charge
and enhanced the potential interaction of MPT1 with the outer membrane of bacteria.
Whereas, substitution of Val8 with Lys (V8K) and Phe12 with Asp (F12D) amino-acid
of MTP2 resulted in improving the polarity of the peptide without alteration of the net
charge, that affect the stabilization of α-helix of MTP2 (Palmieri et al. 2016).
132 A.S. Virdi and N. Singh

2.5 Mode of Action of AMPs

The structural diversity and broad spectrum antibacterial activity of different AMPs
rely upon the sophisticated structural architecture, the variety of biophysical proper-
ties such as size, amphipathicity, structural folding (α-helices, β-plated sheets), size,
overall net charge, and balance between polar and hydrophobic regions. These
diverse structural attributes enable AMPs to penetrate into the plasma membrane of
both Gram-negative and Gram-positive bacteria, fungi and other harmful patho-
genic microorganisms (Teixeira et al. 2012). AMPs mainly attack the cell wall of
microorganisms through electrostatic interaction, binding with glycolipids such as
lipopolysaccharides and permeabilize into the cell wall in three steps (Fig. 7.1a).
Step 1: The presence of membrane structures in the outer capsular polysaccharides
(anionic phospholipids, phosphate groups in Gram-negative bacteria and teichoic
acids and lipoteichoic acids in Gram-positive bacteria) provide net negative charge
on the bacterial surfaces (Brogden 2005). The net charge on bacterial outer surface
attracts anionic or cationic AMPs through electrostatic bond formation (Brogden
2005). After attraction with membrane structures, the AMPs must cross the capsular
polysaccharides before interaction with outer membrane and cytoplasmic mem-
brane of Gram-negative and Gram-positive bacteria, respectively. When peptides
are able to reach the cytoplasmic membrane, they can easily interact with lipid
bilayer (Brogden 2005). Step 2: At low peptide/lipid ratios, the α-helical peptides,
β-sheet peptides, and θ-defensins lean to adsorb in the lipid head groups in parallel
to lipid bilayer (Huang 2000; Yang et al. 2001). The attachment of AMPs stretches
the membrane, which thus leads to the thinning of the membrane (Chen et al. 2003).
The thinning of the membrane is AMP-specific and directly proportional to the ratio
of AMPs (Chen et al. 2003; Brogden 2005). Step 3: At high peptide/lipid ratios, the
orientation of peptides became perpendicular to the bacterial membrane and inserted
into the lipid bilayer, resulting in formation of transmembrane pores (Lee et al.
2004). Three models were proposed to explain the membrane permeabilization (1)
Barrel-stave model (Fig. 7.1b); (2) Carpet model (Fig. 7.1c) and (3) toroidal pore
model (Fig. 7.1d). In the barrel-stove model, the peptide helices form a bundle in the
membrane with a central lumen, like a barrel made up of helical peptides as the
staves (Yang et al. 2001). The hydrophobic peptide regions interact with hydrophilic
lipid core region of the bilayer, while the hydrophilic peptide regions form the inter-
nal core of the pore (Fig. 7.1b). The bundling of AMPs in the core region of lipid
bilayer results into compositional changes and imposition of stress. It tend towards
disintegration of lipid bilayer and bacterial cell death (Bahar and Ren 2013). In the
carpet model, peptides orientate parallel to the entire lipid bilayer surface (Fig. 7.1c).
The electrostatic charges on the anionic phospholipid head groups interact with pep-
tides leading to carpet like lining of the membrane by the latter and finally leading to
the formation of “micelles” (Fig. 7.1c). At critical threshold, peptide/lipid ratios, the
formation of toroidal transient holes in the lipid bilayer by peptides grant access to the
additional peptides to interact with membrane, resulting in the formation of micelles
followed by membrane disintegration and bacterial cell death (Gazil et al. 1996).
Fig. 7.1 (a) An illustration describing the attachment of antimicrobial peptide on surface of the
bacterial cell membrane. The Figs. 1a–d were re-drawn from Brogden (2005) with permission
(License Number 4050550333585). (Adapted by permission from Macmillan Publishers Ltd.:
[Nature] (Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? (Nature Revi
Microbiol Nature Rev. Microbiol. 2005;3:238–50), copyright (2005). (b) An illustration describ-
ing the barrel-stove model-mediated-lysis of the bacterial cell. Hydrophilic regions of the peptides
are shown in yellow colour and the hydrophobic regions of the peptides are shown in red colour.
(c) An illustration describing the carpet model of antimicrobial peptide-mediated-lysis of the bac-
terial cell. Hydrophilic regions of the peptides are shown in yellow colour and the hydrophobic
regions of the peptides are shown in red colour. (d) An illustration describing the toroidal-pore
model of antimicrobial peptide-mediated-lysis of the bacterial cell. Hydrophilic regions of the
peptides are shown in yellow colour and the hydrophobic regions of the peptides are shown in red
colour
134 A.S. Virdi and N. Singh

In the toroidal-pore model, peptides interject into the lipid bilayer and cause the
lipid monolayer to bend continuously through the pore until the water core is sur-
rounded by both the inserted peptides and lipid head groups (Fig. 7.1d). In the pro-
cess of toroidal pore formation, the polar faces of the peptides interact with the polar
head groups of the lipids. The lipids of outer leaflet bend into the inner leaflet from
the lamellar normal and connect from top to bottom like a toroidal hole (Ludtke
et al. 1996; Matsuzaki et al. 1996; Brogden 2005). The toroidal hole is surrounded
with peptides and polar head groups, which probably screen and mask the positive
half of the amphipathic peptides (Fig. 7.1d). Contrary to the Barrel-stave model, the
peptides always remain attached with lipid head groups not only when they are laid
horizontally on the outer surface of the lipid bilayer but also when they are perpen-
dicularly interjected into it in the toroidal-pore model (Yang et al. 2001; Brogden
2005).

3 Antimicrobial Polyphenols

Higher plants being sessile in nature have evolved several defense mechanisms to
cope with adverse climatic conditions, herbivores animals’ aggression and patho-
genic microorganisms. Polyphenols are widely synthesized in higher plants, as sec-
ondary metabolites, which act as defense molecules against these challenges, and in
response to abiotic stresses such as water logging, drought-, salt-, heat-, mechanical
and osmotic stress, and ultraviolet radiations. Based on chemical structure, poly-
phenols are broadly divided into flavonoids and nonflavonoids. Flavonoids com-
prise of a common carbon skeleton of diphenyl propanes, two benzene rings (ring A
and B) which are joined through a linear three-carbon chain. The central three-­
carbon chain forms a closed pyran ring (ring C) with A benzene ring. Flavonoids,
based on oxidation of central pyran ring, are further subdivided into flavonols, fla-
vones, flavanones, anthocyanidins, flavanols, and isoflavones. Whereas, nonflavo-
noids are subdivided into derivatives of benzoic acid (gallic acid and protocatechuic
acid) and derivatives of cinnamic acid [coumaric, caffeic, ferulic acid, stilbenes
(resveratrol) and lignans (produced by oxidative dimerization of two phenylpropane
units)]. In addition to these polyphenols, plants also synthesize quinones, saponins,
coumarins, terpenoids, alkaloids, glycosides (associated with various organic acids)
and polymerized, high molecular weight tannins (Quideau et al. 2011).
Polyphenols have antioxidant, free radical scavenging, metal chelation proper-
ties, telomerase (Naasani et al. 2003), cyclooxygenase (O’Leary et al. 2004; Hussain
et al. 2005), or lipoxygenase (Sadik et al. 2003; Schewe et al. 2001), and enzymes
inhibiting or reducing capabilities. Polyphenols also interact with signal transduc-
tion pathways and many cell receptors. These attributes enable polyphenols as
potential therapeutic tools for the prevention of cardiovascular, cancer, osteoporo-
sis, diabetes mellitus, and neurodegenerative diseases (Daglia 2012). The antimi-
crobial activity of purified polyphenols has been discussed below.
7 Antimicrobial Peptides and Polyphenols… 135

3.1 Antimicrobial Activity of Phenolic Acids

Gallic acid, ferulic acid, chlorogenic acid, caffeic acid, p-coumaric acid, hydroxy-
cinnamic acid, benzoic acid, p-hydroxybenzoic acid, succinic acid, methyl gallate,
ethyl gallate, methyl m-digallate, monomethyl ester and tyrosol are considered as
non-flavonoids or simple phenolic acids. The antimicrobial activity of these pheno-
lic acids was examined in past decades. Most of these phenolic acids have antibac-
terial as well as bacteriostatic properties against L. monocytogenes, E. coli, P.
aeruginosa, S. aureus and Lactobacillus spp., pathogenic strains alone or in com-
binations. Application of the gallic- and protocatechuic acids, gallic and caffeic
acids, rutin and quercetin combinations resulted in inhibition of growth of E. coli
ATCC 35218 without cellular death at 20 °C, whereas, gallic and caffeic acids,
rutin and quercetin showed the most effective antibacterial activity 4 °C (Rodríguez
Vaquero et al. 2010). However, it was observed that the synergistic effects of dif-
ferent phenolic acids (chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid,
and hydroxycinnamic acid) together was more pronounced against L. monocyto-
genes, as compared to when tested alone (Wen et al. 2003). The antilisterial effect
of chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, hydroxycinnamic
acids was dose dependent, modulated by pH and more effective in combinations
(Wen et al. 2003). All hydroxycinnamic acids were bactericidal at pH 4.5 and bac-
teriostatic at higher pH with MIC ranging between 0.20 and 0.27% (w/v), whereas
chlorogenic acid was ineffective at higher concentration (1.0% w/v) but the antil-
isterial activity of chlorogenic acid was observed at pH 6.5. The effect of phenolic
acids (tyrosol, gallic-, caffeic-, ferulic-, and chlorogenic acids) was less, when con-
sidered alone, against Gram-positive bacteria. The application of these phenolic
acids with streptomycin (antibiotic) appeared more lethal against Gram-negative
bacteria. It thus revealed that uses of phenolic acids with antibiotics help to reduce
the cost of food preservation and misuses of antibiotics (Saavedra et al. 2010). The
study of Cueva et al. (2010) demonstrated that the susceptibility of pathogenic
strain of E. coli (O157:H7) and non-pathogenic (ATCC 25922) strains, in response
to benzoic-, phenylacetic- and phenylpropionic acid, was differential and strain-
dependent. The growth of several lactobacilli species (L. fermentum CECT 5716
and L. fermentum LPH1) and other pathogens (C. albicans and S. aureus) was
inhibited by benzoic-, phenylacetic- and phenylpropionic acid. On the contrary, the
growth of P. aeruginosa was not affected by phenolic acids (Cueva et al. 2010).
Liang et al. (2010) demonstrated that, among gallic acid, methylgallate, ethylgal-
late, methyl m-­digallate, p-hydroxybenzoic acid, and succinic acid monomethyl
ester, only gallic acid and methyl m-digallate showed antibacterial activity against
S. aureus, S. epidermidis, Corynebacterium xerosis, Micrococcus luteus and P.
aeruginosa, respectively. Borges et al. (2013) assessed the antibacterial activity of
gallic and ferulic acids against E. coli, Pseudomonas aeruginosa, S. aureus, and L.
monocytogenes. Gallic acid inhibited the growth of P. aeruginosa, E. coli, S. aureus
and L. monocytogenes with MIC of 500 μg/mL, 1500 μg/mL, 1750 μg/mL and
2000 μg/mL, respectively. Whereas, ferulic acid showed 100 μg/mL of MIC against
136 A.S. Virdi and N. Singh

E. coli and P. aeruginosa, 1100 μg/mL and 1250 μg/mL of MIC against S. aureus
and L. monocytogenes, respectively. The minimum bactericidal concentration
(MBC) of ferulic- and gallic acid against E. coli was 2500 and 5000 μg/mL, respec-
tively. Whereas, MBC against S. aureus was 5000 μg/mL and 5250 μg/mL for
ferulic- and gallic acid, respectively. On the contrary, the MBC of ferulic- and gal-
lic acid was 5300 μg/mL and 5500 μg/mL, respectively, was reported against L.
monocytogenes. The MBC against P. aeruginosa was 500 μg/mL, which was
depicted for ferulic- and gallic acid (Borges et al. 2013). The ferulic- and gallic
acid alter hydrophobicity of the membrane (negative charge, intra and extracellular
permeability, and physicochemical properties), and causes local rupture or pore
formation in the bacterial cell membrane which ultimately result in leakage of
essential intracellular constituents followed by cell death (Borges et al. 2013).
Application of 1500 ppm ferulic acid in coleslaw resulted in a 1.5 log CFU/g
reduction in L. monocytogenes growth after 5 days incubation at 10 °C, whereas,
application of 3000 ppm ferulic acid +1% glycine/sodium acetate compound in
egg salad completely inhibited the growth of L. monocytogenes. It thus strength-
ened the potential antilisterial efficacy of ferulic acid in food preservations
(Takahashi et al. 2015). The study carried out by Suárez-­Quiroz et al. (2013) dem-
onstrated that green coffee chlorogenic acids (CGA) and dodecyl chlorogenates
(DCGA) possess anti-aspergillus activities and proved better anti-mycotoxigenic
agents for ochratoxin A than for aflatoxin B1. CGA showed antibacterial activity
against Gram-positive and Gram-negative bacteria, and the bactericidal activity
against P. fluorescens and S. aureus, whereas the antibacterial activity of DCGA
was limited to Gram-positive bacteria (Suárez-Quiroz et al. 2013).

3.2  ntimicrobial Activity of Flavonoids, Flavonols

A
and Flavan-3-ols

Flavonoids, for example, flavonol (Galangin, pyrogallol catechin, gatechol cate-

chin), flavan-3-ol (gallocatechin, epigallocatechin, catechin, epicatechin, and
theaflavin gallates), flavanones [neohesperidin, hesperetin (aglycone) neoeriocitrin,
eriodictyol (aglycone), naringin, naringenin (aglycone)], chalcones, proanthocyani-
dins were demonstrated to have broad-spectrum antibacterial and antifungal activi-
ties (Tsuchiya et al. 1993; Cushnie et al. 2007; Friedman et al. 2006; Hirasawa and
Takada 2004; Mandalari et al. 2007; Heinonen 2007; Dixon et al. 2005; Batovska
et al. 2009; Daglia 2012). The antibacterial and antifungal activity of flavan-3-ol
was observed against V. cholerae, S. mutans, C. jejuni, C. perfringes, E. coli and
Candida albicans. However, the broad-spectrum antimicrobial activity of flavonols
was reported against Gram-positive (S. aureus, Lactobacillus acidophilus,
Actinomyces naeslundii) and Gram-negative (Prevotellaoralis, Prevotella melanin-
ogenica, Porphyromonas gingivalis, and Fusobacterium nucleatum) bacteria
(Daglia 2012 and references therein). Taguri et al. (2004) examined the potential
7 Antimicrobial Peptides and Polyphenols… 137

antibacterial activity of purified polyphenols (epigallocatechin, epigallocatechin-­3-­

O-gallate, punicalagin, tannic acid, castalagin, prodelphinidin, geraniin, procyani-
dins), a theaflavin mixture of black tea and green tea polyphenols treated with loquat
polyphenol oxidase. These polyphenols were tested against S. aureus (20 strains),
some serotypes of the genus Salmonella (26 strains), E. coli (23 strains), and some
species of the genus Vibrio (27 strains). The study revealed that all polyphenols pos-
sess strong antibacterial activity against food borne pathogenic bacteria of S. aureus
and the genus Vibrio with MIC of 192 ± 91 mg/mL and 162 ± 165 mg/mL, respec-
tively. Whereas, the antibacterial activity of purified polyphenols was poor against
bacterial strains of genus Salmonella and E. coli with MICs of 795 ± 590 mg/mL
and 1519 ± 949 mg/mL, respectively. It was proposed that relatively lower mean
MIC values of epigallocatechin, epigallocatechin-3-O-gallate, castalagin and pro-
delphinidin were attributed to the presence of 3, 4, 5-trihydroxyphenyl groups
(Taguri et al. 2004). Some tea catechins flavan-3-ols such as (−)-gallocatechin-­3-­
gallate, (−)-epigallocatechin-3-gallate, (−)-catechin-3-gallate, and (−)-epicatechin-­
3-­gallate showed potent antibacterial activity against food-borne pathogenic bacteria
like B. cereus. These tea catechins show antibacterial activity in nanomolar concen-
trations, which were even better than tetracycline or vancomycin antibiotics. It was
reported that the tea catechins may act as potential natural agents against gastroin-
testinal diseases and foodborne pathogens (Friedman et al. 2006; Lee et al. 2009a).
Reygaert (2014) reported that epigallocatechin gallate (EGCG) has antibacterial
activity against both Gram-positive and Gram-negative bacteria (Enterococcus, S.
aureus, Streptococcus, Salmonella and E. coli). EGCG inhibit both the FabG and
FabI reductase steps in the fatty acid elongation which thus affect cellular fatty acid
synthesis (Zhang and Rock 2004). However, the analysis of mode of action of
EGCG against E. coli revealed that exposure of E. coli OP50 to EGCG resulted in a
profound increase in intracellular oxidative stress, which led to bacterial death
(Xiong et al. 2017). The incorporation of 10 wt% EGCG (with and without adhesive
resin) in low-density polyethylene/adhesive resin films induced morphological
changes in S. aureus and Pseudomonas sp. (with adhesive) and inhibited the growth
of S. aureus thus signify the use of EGCG in food packaging. It was proposed that
adhesive resins increased the leaching of EGCG into the medium (Moreno-Vásquez
et al. 2017).
The antibacterial activity of tyrosol, gallic acid, caffeic acid, ferulic acid, chloro-
genic acid (phenolics), phloridzin (chalcone), − (−) epicatechin (flavan-3-ol), oleu-
ropein (a secoiridoid glucoside); allylisothiocyanate, benzylisothiocyanate and
2-phenylethylisothiocyanate (hydrolysates of glucosinolate) was evaluated against
Gram-positive and Gram-negative bacteria. As compared to phenolics, all isothio-
cyanates singly or in dual combination with less efficient antibiotics showed syner-
gistic effects against examined food pathogens (Saavedra et al. 2010). The growth
of E. coli was inhibited in response to thymoquinone, rutin, (−) epicatechin and
myricetin (commercially available) with MIC <20 ppm while Salmonella was most
sensitive to (−) epicatechin with MIC of <15 ppm (Cetin-Karaca and Newman
2015). Recently, a procyanidin B5 was isolated from Chrysophyllum albidum
G. Don-Holl. (Sapotaceae), which showed antimicrobial activity against E. coli
138 A.S. Virdi and N. Singh

(ATCC 25922), S. aureus (NCTC 6571), Pseudomonas aeruginosa (ATCC 10145)

and B. subtilis (NCTC 8263). The MIC of procyanidin B5 against these bacteria was
156.25 μg/mL, 156.25 μg/mL, 625 μg/mL, and 156.25 μg/mL, respectively (Idowu
et al. 2016).
Application of flavonol galangin, an antimicrobial constituent of the traditional
medicines propolis and Helichrysum aureonitens, resulted in the aggregation and
inhibition of S. aureus NCTC 6571 several fold within 60 min of incubation
(Cushnie et al. 2007). The ethanolic extract of bergamot peel (Citrus bergamia
Risso) inhibited the growth of Gram-negative (E. coli K-12 MG1655, S. enterica
ser. Typhimurium LT2) and Gram-positive (B. subtilis ATCC 6633) bacteria
(Mandalari et al. 2007). Whereas, Tunisian quince (Cydonia oblonga Miller) peel
extract contain antibacterial activity against Gram-positive (S. aureus), Gram-­
negative bacteria (E. coli, P. aeruginosa) and yeast (C. albicans) (Fattouch et al.
2007). The MIC of ethanolic extract of bergamot peel and quince peel extract was
ranged between 0.2–0.8 mg/mL and 0.1–5.0 mg polyphenol/mL, respectively
(Mandalari et al. 2007; Fattouch et al. 2007). The key flavonoids of bergamot peel
were eriodictyol (aglycone) and naringenin that showed strong antibacterial activity
with MICs in the range of 250–800 μg/mL, and 800–1000 μg/mL, respectively
against tested microorganisms (Mandalari et al. 2007). Phenolic profiling of resins
and propolis obtained from Populus spp. revealed the presence of p-hydroxybenzoic
acid, p-coumaric acid, caffeic acid, chrysin, apigenin, quercetin, pinocembrin, pino-
banksin and galangin, which have differential strong antibacterial activity against
Gram-positive bacteria (Dimkić et al. 2016). The strong antimicrobial activity in
resins and propolis was attributed to the higher accumulation of apigenin, quercetin,
pinocembrin, pinobanksin, galangin, and caffeic acid (Dimkić et al. 2016).
Furthermore, the analysis of cherry bud phenolic acids also revealed the higher
levels of naringenin correlated with strong activity against B. subtilis and L. mono-
cytogenes (Dimkić et al. 2016).

3.3 Antimicrobial Activity of Tannins

Plants possess hydrolyzable and condensed tannins (e.g., proanthocyanidins).

Hydrolyzable tannins have been classified into four groups: Groups 1: gallotan-
nins (1, 2, 3, 4, 6-pentagalloyl-β-D-glucose); 2: hexahydroxydiphenoyl (tellima-
grandin I & II); 3: dehydrohexahydroxydiphenoyl (geraniin) and; 4:
dehydroellagitannin (chebulagic acid). Condensed tannins have antibacterial (S.
mutans, E. coli, S. aureus) and antiviral (influenza A virus type −1 herpes simplex
virus) activity (Quideau et al. 2011; Daglia 2012 and references therein). The
antibacterial activity of proanthocyanidins was shown against several food-borne
pathogenic and food-­spoilage bacteria. Proanthocyanidins, extracted from ber-
ries, inhibited the activity of several pathogenic bacteria such as E. coli (uropatho-
genic), S. mutans (cariogenic) and S. aureus (oxacillin-resistant strain) (Côté et al.
2010). Ellagitannins, isolated from cloudberry, red raspberry and strawberry,
7 Antimicrobial Peptides and Polyphenols… 139

acted as strong antimicrobial agents against a broad-spectrum of bacteria, as well

as anti-adherence compounds by preventing the colonization and infection of
many pathogens. Berry ellagitannins showed the potent inhibitory effect on the
growth of Salmonella, Staphylococcus, Helicobacter, Bacillus, Escherichia,
Clostridium and Campylobacter species, however, Lactobacillus and Listeria spe-
cies were not sensitive to berry phenolics (Heinonen 2007). On the contrary,
hydrolysable tannins showed broad-spectrum antibacterial activity against salmo-
nella (Staphylococcus), helicobacter (E. coli, Bacillus), and clostridium
(Campylobacter, Lysteria) bacteria (Buzzini et al. 2008; Daglia 2012 and refer-
ences therein). Tellimagrandin I, identified from petals of Rosa canina L. (rose
red), possess weak bactericidal activity but the application of tellimagrandin I
along with β-lactams resulted in significant decline in MIC of β-lactams against
some methicillin-resistant S. aureus OM584 strains (Shiota et al. 2000). These
findings validated the synergistic effect of tellimagrandin I against bacterial
strains. Catechins (EC, EGG, ECG, EGCG) also have weak anti-MRSA activity
but application of epicatechin gallate (ECG) along with β-lactams resulted in sig-
nificant reduction of MIC for β-lactams (Shiota et al. 1999); similar results were
also observed for corilagin, an ellagitannin isolated from Arctostaphylos uva-ursi
(Shimizu et al. 2001). Funatogawa et al. (2004) extracted 20 hydrolyzable tannins
from medicinal plants, which are used for the treatment of gastric disorders in
Asian countries. All the hydrolyzable tannins showed antibacterial activity against
H. pylori, an etiological agent in gastroduodenal disorders. Among them, EGCG
(epigallocatechin gallate) demonstrated promising antibacterial activity against
H. pylori, whereas the bactericidal activity of TG-I (Tellimagrandin I) was time-
and dose-dependent. It was also demonstrated that the membrane-damaging activ-
ity of all hydrolyzable tannins was dose-dependent (Funatogawa et al. 2004). The
role of these hydrolyzable tannins was, however, not tested against food-borne
pathogenic Gram-positive and Gram-negative bacteria.
Mango (Mangifera indica L.) kernel extract exerts some food preservative
properties as well as broad-spectrum antibacterial activity against Gram-positive
and Gram-negative bacteria. Mango kernel extract not only extended the shelf life
of fresh-type cheese and ghee but also enhanced the oxidative stability of these
products (Puravankara et al. 2000). Another study revealed that purified mango
kernel tannins (gallotannins) inhibited the growth of food spoilage, foodborne
pathogenic and Gram-negative bacteria such as B. subtilis, S. aureus and E. coli,
respectively (Engels et al. 2009). Furthermore, Gram-positive bacteria showed
more susceptibility towards mango gallotannins (penta-O-galloylglucose, hexa-
O-galloylglucose, hepta-O-galloylglucose,octa-O-galloylglucose, nona-O-gall-
oylglucose, and deca-­O-­galloylglucose), as compared to Gram-negative bacteria.
The MIC of these gallotannins against B. subtilis, B. cereus, C. botulinum, C.
jejuni, L. monocytogenes, and S. aureus was 0.2 mg/mL, as compared to entero-
toxigenic E. coli and S. enterica, which were resistant to these gallotanins and
showed a MIC of 0.5–1 mg/mL (Engels et al. 2011). The strong antibacterial
activity of gallotanins was associated with their strong affinity for iron and inacti-
vation of membrane-bound proteins.
140 A.S. Virdi and N. Singh

The skin and pulp of fruits and vegetables are a rich source of minerals, organic
acid, and phenolics and phenolics possess a wide range of broad-spectrum antimi-
crobial properties (Gyawali and Ibrahim 2014). The seeds, skins, and by products of
pomace family members such as grapes, olives, and pomegranates possess a good
quantity of antibacterial phenolic compounds. Pomegranate fruit and pulp contains
polyphenols, such as flavan-3-ol (catechin, gallocatechin), flavones (luteolin,
kaempferol), hydroxybenzoic acids (gallic acid), hydroxycinnamic acids (caffeic
acid, p-coumeric acid), anthocyanins (delphinidin, cyaniding, pelargonidin
3-­glucosides and 3, 5-diglucosides), and hydrolysable tannins (ellagitannins i.e.,
punicalagin, punicalagin gallotannins, punicalagin isomers, granatin B, tellima-
grandin I, casuarinin, granatin A, pedunculagin, punicalin, corlagin, gallagic acid,
ellagic acid, and gallagyl esters) (Akhtar et al. 2015 and references therein). The
extracts of pomegranate peel was shown to reduce the growth of foodborne patho-
gens (L. monocytogenes, S. aureus, E. coli, Y. enterocolitica, and B. cereus)
(Al-Zoreky 2009; Agourram et al. 2013). Studies carried out by Kanatt et al. (2010)
and Negi and Jayaprakasha (2003) demonstrated that lower concentrations of pome-
granate peel extracts effectively inhibited the growth of Gram-positive bacteria.
However, higher concentrations of the same extracts were required to inhibit the
growth of Gram-negative bacteria such as Pseudomonas spp. Incorporation of 0.1%
pomegranate peel extract in chicken meat products reduced the growth of coliform,
enhanced the shelf-life by 2–3 weeks at low temperature storage and also lowered
the oxidative rancidity in chicken products (Kanatt et al. 2010). The antilisterial
activity of pomegranate peel extract at low temperature was further demonstrated by
mixing of 7.5% v/w pomegranate peel extract (24.7 mg/g), which resulted in the
reduction of the growth of L. monocytogenes by 4.1 log CFU/g during 46 days of
storage at 4 °C. The antilisterial activity of pomegranate peel extract was crucial for
temperature (Hayrapetyan et al. 2012). The tannin rich fraction of pomegranate peel
extract significantly reduced the growth of different strains of L. monocytogenes
with MIC 1.25–5.0 mg/mL (Li et al. 2014). The antibacterial activity in pomegran-
ate peel was largely attributed to the presence of hydrolyzable ellagitannins. The
pomegranate peel extract contains a mixture of hydrolysable tannins (ellagic acid,
punicalagin, punicalin and gallagic acid) which possess very strong antioxidant and
antimicrobial activities. The polyphenols and tannins, after interaction with bacte-
ria, damage the cell membrane structure followed by a loss of cellular homeostasis
leading to cell death (Li et al. 2014).

3.4 Antimicrobial Activity in Phenolics of By-Products

The antimicrobial activity of seed extracts of jackfruit, papaya, plum, guava, and
tamarind was demonstrated against Gram-positive (S. aureus, B. subtilis) and Gram-­
negative bacteria (E. coli, P. aeruginosa) (Debnath et al. 2011). The presence of
sinapic acid in mustard meal also showed antimicrobial activity against Gram-­
negative (E. coli, P. fluorescens) and Gram-positive (B. subtilis) along with
7 Antimicrobial Peptides and Polyphenols… 141

anti-pathogenic activity to counter S. aureus, and L. monocytogenes (Engels et al.

2012). Similar kind of antimicrobial activity was also found in the husk of fruits and
hulls of grains such as walnut green husks, coconut husks, almond skin, hull from
buckwheat grains, Bengal gram (Cicer arietinum), and pigeon pea (Cajanus cajan)
(Guil-Guerrero et al. 2016). The aqueous extract of green husk of walnut inhibited
the growth of Gram-positive bacteria (B. cereus, B. subtilis, S. aureus etc.) with
MIC of 0.1 mg/mL, whereas 50% ethanolic extract of coconut husk inhibited the
activity of Gram-positive (B. cereus, L. monocytogenes, S. aureus) and Gram-­
negative bacteria (E. coli, S. typhimurium, V. cholera). The flavonoid-rich fractions,
extracted from almond skins, possess antimicrobial activity against L. monocyto-
genes and S. aureus with MIC of 0.25–0.5 mg/mL (Mandalari et al. 2010). The hull
of buckwheat grains are rich source of total phenolics and flavonoids which showed
potential bactericidal activity against wide range of Gram-positive and Gram-­
negative bacteria such as B. cereus, S. aureus, E. faecalis and Salmonella cholerae-
suis, E. coli, Proteus mirabilis. Hull extracts (0.1%) of Bengal gram completely
inhibited the growth of B. cereus under salt conditions whereas, the application of
0.2% hull extract of Bengal gram and pigeon pea resulted in one log reduction of S.
aureus growth (Kanatt et al. 2011). Intriguingly, split Bengal gram and pigeon pea
dhal is widely consumed in India and hull of both of the pulses is a by-product.
These results thus demonstrate that hulls of Bengal gram and pigeon pea could be a
rich source of antimicrobial compounds.
The edible part of jambolan showed the presence of 9 anthocyanins (mainly,
delphinidin, petunidin and malvidin), 9 flavonols (myricetin, laricitrin and syringe-
tin glycosides), 19 flavanonols (dihexosides of dihydromyricetin and its methylated
derivatives), 8 flavan-3-ol monomers (mainly gallocatechin), 13 gallotanins and 13
ellagitanins, together with some proanthocyanidins (highly galloylated prodelphini-
dins) and free gallic and ellagic acids (Tavares et al. 2016). These findings sug-
gested that the fruit jambolan is a rich source of almost all major polyphenols.
However, the antibacterial activity of these polyphenols against Gram-positive and
Gram-negative bacteria was not tested. A recent study of Singh et al. (2016) demon-
strated the antimicrobial properties of jambolan (Syzygium cumini) fruit polyphe-
nols against pathogenic strains (S. aureus, S. aureus [MRSA], Escherichia coli, and
Klebsiella pneumonia. The zone of inhibition and MICs of these polyphenols
ranged from 14.3 to 23.0 mm and 0.5 to 2.5 mg/mL, respectively (Singh et al. 2016).

3.5 Antimicrobial Mechanism of Polyphenols

Structurally, plant phenolics are greatly diversified groups of secondary metabo-

lites. The antimicrobial activity of phenolic compounds is attributed to the presence
of hydroxyl group (-OH) which interact with bacterial cell membrane and disrupt
membrane structures and resulted in the leakage of cellular components (Xue et al.
2013). The presence and position of -OH groups and number of double bonds in
phenolic compounds play key role in bacterial cell death (Dorman and Deans 2000;
142 A.S. Virdi and N. Singh

Cueva et al. 2010; Stojković et al. 2013). At molecular levels, interaction of -OH
groups with bacterial cell membrane resulted in the formation of proton exchangers,
which caused delocalization of electrons and reduction in gradient across the bacte-
rial membrane. Destabilization of the proton motive forces and depletion of the ATP
pool in bacterial cell membrane resulted in the collapse of membranes and cell
death (Ultee et al. 2002). The antioxidant activity of phenolic compounds was
attributed to the presence of highly active -OH groups which reduced the redox
potential of growth medium through the inhibition of the formation of reactive oxy-
gen species and scavenging of free radicals. Depletion in the redox potential of the
growth medium causes inhibition of pathogenic bacterial growth (Cueva et al. 2010;
Stojković et al. 2013). The antimicrobial activity of terpenes such as terpinen-4-ol,
α-terpineol, caffeic acid and p-coumaric acid was also greatly influenced by the
presence and number of -OH groups (Figueiredo et al. 2008; Stojković et al. 2013).

4  atural Antimicrobials: Implications in Food Preservation

N
and Safety

Reviews by Narayana and Chen (2015), Gyawali and Ibrahim (2014), Juneja et al.
(2012) and Daglia (2012) provided ample evidence to consider natural peptides and
polyphenols as natural antimicrobial agents. Several commercial scavengers of O2,
CO2 and moisture, CO2 emitters, ethanol emitters, C2H4 emitters, antimicrobial and
antioxidant release systems were developed, which are widely employed in the
preservation of dairy, meat, cereal, dry fruits, nuts, roasted nut and other food prod-
ucts (Suppakul et al. 2003 and references therein). ALTA™ 2341 or Microgard™
are trade names of commercially available bacteriocins, produced ex situ and can be
directly applied to the food matrix. Bioactive films containing bacteriocins, entero-
cins A and B (bacteriocins produced by Enterococcus faecium) were shown to have
a synergistic effect against L. monocytogenes with enhanced shelf life of cooked
ham, stored at 6 °C for 90 days (Marcos et al. 2008). Coating lactocin 705 and lac-
tocin AL705 (bacteriocins produced by Lactobacillus curvatus CRL705) on linear
low-density polyethylene resulted in inhibition of Lactobacillus plantarum CRL691
and Listeria innocua in vitro (Massani et al. 2008). Incorporation of nisin, grape
seed extract and ethylenediaminetetraacetic acid (EDTA) together in soy protein
films showed synergistic antibacterial activity against pathogenic bacterial strains
(L. monocytogenes, E. coli O157:H7, S. typhimurium) (Sivarooban et al. 2008).
Antimicrobial activity of a synthetic peptides 6K8L, derived from magainin and
E14LKK and covalently attached to polystyrene resin and low-density polyethyl-
ene, against E. coli O157:H7, L. monocytogenes, and Pseudomonas fluorescens)
(Appendini and Hotchkiss 2001; Appendini and Hotchkiss 2002). Incorporation of
antimicrobial agents in tiny microsphere/microcapsules of diameter ≥1 μM to sev-
eral hundred micrometers appear to be the valuable tool for controlled delivery of
antimicrobial agents into food systems. The microencapsulation of AMPs ensure
7 Antimicrobial Peptides and Polyphenols… 143

the protection against chemical reaction and interaction with food system simulta-
neously. The encapsulation enhances the shelf life of perishable foods, but the tex-
tural and sensory attributes of food may be affected by the size of microspheres
(Aloui and Khwaldia 2016). Sadiq et al. (2016) developed nisin loaded monolaurin
nano-micelles, which inhibited the growth of S. aureus ATCC-25923 for up to 164 h
in vitro and the effect of nisin and monolaurin was synergistic. It was also reported
that the loading of nisin does not affect the diameter of monolaurin nano-particles
and nisin was released from monolaurin nano-particles by pore forming mecha-
nisms (Sadiq et al. 2016). The lyotropic liquid crystalline structures, named cuba-
somes and hexasomes, were synthesized from cubic glycerol monooleate/water and
hexagonal glycerol monooleate/oleic acid/water systems (Boge et al. 2016). Three
AMPs namely, AP114, DPK-060, and LL-37, derived from plectasin, endogenous
human protein kininogen and C-terminal part of the human CAP18 cathelicidin
protein, respectively, were incorporated into cubosomes and hexosomes. The load-
ing of these AMPs in cubosomes and hexosomes showed differential antimicrobial
activity. AMPs AP114 and DPK-060 loaded cubasomes retained antimicrobial
activity, whereas the encapsulation of these AMPs on hexasomes showed a reduc-
tion in antimicrobial activity (Boge et al. 2016). These studies provided evidence for
the effective uses of AMPs in the enhancement of food quality, safety, and preserva-
tions. Starch-based edible films, multilayered films, whey protein films, locust bean
gum, sodium alginate-, locust bean gum-, sodium casienate-, gelatin- and carboxy-
methyle cellulose bio-polymers contained AMPs and polyphenols, were examined
for the preservation of food. However, the rate of diffusion, possible interaction with
food ingredients, impact on sensory and organoleptic attributes, need more exten-
sive investigation in in vivo and in vitro model systems. Nanoemulsions and nanol-
aminated antimicrobial delivery systems could play a key role in the preservation of
food spoilage and pathogenic bacteria, enhancement of shelf life of meat products
and dairy products and minimally processed food products such as fruits and
vegetables.

5 Conclusion

Application of natural AMPs and polyphenols in food preservation and shelf

life enhancement are taking shape in the food sector. The development of high-­
throughput technologies in genomics, proteomics, metabolomics, chemical syn-
thesis of AMPs and other chemical compounds at low price enable scientist to
characterize natural preservatives in a more speedy way. The incorporation of
pleurocidin into the poly(vinyl alcohol) electrospun nanofiber resulted in enhance-
ment of its bioavailability as well as bactericidal activity Gram-negative bacteria.
Application of purified or commercially available thymoquinone, rutin, (−) epi-
catechin myricetin, procyanidin B5, p-hydroxybenzoic acid, p-coumaric acid,
caffeic acid, chrysin, apigenin, quercetin, pinocembrin, pinobanksin, galangin,
naringenin, mango gallotannins (penta-O-galloylglucose, hexa-O-galloylglucose,
144 A.S. Virdi and N. Singh

hepta-O-galloylglucose, octa-O-galloylglucose, nona-O-galloylglucose, and deca-­

O-­galloylglucose) demonstrated the strong differential antibacterial activity against
Gram-positive and Gram-negative bacteria. These studies demonstrated the signifi-
cance of polyphenols in inhibition of the growth of food-borne pathogens and food
spoilage bacteria naturally. Incorporation of EGCG into low-density polyethylene
films provided evidence for the uses of EGCG in food packaging and enhancement
of shelf life of packaged food. It is, therefore, likely that several-fold increase in the
application of natural preservatives in food preservation and improvement of pack-
aged food products, minimally processed food, perishable fruits, and vegetables
will be evident in the coming decades.

Acknowledgements ASV is thankful to Professor Narpinder Singh, Department of Food Science

and Technology, Guru Nanak Dev University, Amritsar for providing financial assistance from J C
Bose National Fellowship (2016-2021) awarded by Department of Science & Technology,
Government of India, New Delhi.

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Chapter 8
Delivery Systems for Introduction of Natural
Antimicrobials into Foods

Shalini Mishra and Kanika Bhargava

Abstract Antimicrobials are to a large extent less active in complex food matrices
compared to their activity in model microbiological systems. Minimum inhibitory
concentration (MIC), i.e. the minimum concentration required to inhibit microbial
growth for a specific time period, generally increases when the antimicrobial is tested
in a select food system. The increases in MIC depend on the nature of the antimicro-
bial, composition and structure of the food system. Several factors governing this limi-
tation should be carefully considered when designing and selecting a suitable delivery
system for antimicrobials. A delivery system is able to protect food antimicrobials
from interfering food components and improves delivery of the food antimicrobials to
the site where they can be active. Attributes of efficient delivery systems or carriers
include Generally regarded as safe (GRAS) status, biocompatible with no side-effects,
inexpensive and stability during processing and storage. Furthermore, delivery system
should be able to (1) preserve the functionality of the embedded compounds until the
time of delivery, and (2) deliver the preserved form effectively at the target site with
predictable release profiles. Delivery systems can be made up of naturally occurring
polymers and polysaccharides, synthetic polymer or nanomaterials to tailor encapsu-
lation and delivery of antimicrobials (Janaswamy et al., Carbohydr Polym 94:209–
215, 2013; Janaswamy and Youngren, Food Funct 3:503–507, 2012; McClements
et al., Crit Rev Food Sci Nutr 49:577–606, 2009). These delivery systems present
exciting opportunity for food technologists to enhance bioavailability, stability, sus-
tained activity and shelf-life of food through encapsulation and controlled release.

Keywords Antimicrobials • Delivery systems • Microemulsions • Nanoemulsions

• Nanofibers

S. Mishra
Department of Agricultural and Biosystems Engineering, South Dakota State University,
Manteca, CA, USA
e-mail: [email protected]
K. Bhargava (*)
Department of Human Environmental Sciences, University of Central Oklahoma,
Edmond, OK, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 153

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_8
154 S. Mishra and K. Bhargava

1 Introduction

Antimicrobials are to a large extent less active in complex food matrices compared to
their activity in model microbiological systems. Minimum inhibitory concentration
(MIC), i.e. the minimum concentration required to inhibit microbial growth for a
specific time period, generally increases when the antimicrobial is tested in a select
food system. The increases in MIC depend on the nature of the antimicrobial, compo-
sition and structure of the food system. Several factors governing this limitation
should be carefully considered when designing and selecting a suitable delivery sys-
tem for antimicrobials. A delivery system is able to protect food antimicrobials from
interfering food components and improves delivery of the food antimicrobials to the
site where they can be active. Attributes of efficient delivery systems or carriers
include Generally regarded as safe (GRAS) status, biocompatible with no side-­
effects, inexpensive and stability during processing and storage. Furthermore, deliv-
ery system should be able to (1) preserve the functionality of the embedded compounds
until the time of delivery, and (2) deliver the preserved form effectively at the target
site with predictable release profiles. Delivery systems can be made up of naturally
occurring polymers and polysaccharides, synthetic polymer or nanomaterials to tailor
encapsulation and delivery of antimicrobials (Janaswamy et al. 2013; Janaswamy and
Youngren 2012; McClements et al. 2009). These delivery systems present exciting
opportunity for food technologists to enhance bioavailability, stability, sustained
activity and shelf-life of food through encapsulation and controlled release.
There are a large number of different delivery systems that could be used to deliver
functional food components. It is therefore useful to have some criteria that can be
used to distinguish between different delivery systems, so that one can select the most
appropriate system for a particular application. Some of the most important criteria
to consider when developing or selecting a delivery system are listed in Table 8.1.

2  election Criteria for Food Antimicrobials Delivery

S
Systems

2.1 Structure of Antimicrobials

Most of the antimicrobials are partially or totally lipophilic and are only sparingly
soluble in the aqueous phase. It appears that in many cases this lipophilicity is vital
to cause compounds to migrate into or through the microbial cell membranes.
However, for food manufacturers, this creates a significant application challenge
since compounds may migrate not only into the microbial cell membrane but also
into any other phase that is thermodynamically more favorable than the aqueous
phase. For example, in food products where significant amounts of other lipid
phases or interfaces are present, such as high-fat milk or cream, the lipid droplets act
as a ‘sink’ resulting in a loss of concentration and efficacy when compared to a
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 155

Table 8.1 Important criteria for developing or selecting a delivery system

Criteria Definition Characteristics of delivery systems
Loading capacity This is defined as measure of the A delivery system should have a
(LC) mass of encapsulated material (ME) high loading capacity
per unit mass of carrier material
(MC).
LC = ME/MC
Loading efficiency The loading efficiency is a measure Ideally, one would like the loading
(LE) of the ability of the delivery system efficiency to remain high (100%)
to retain the encapsulated material throughout storage
over time: LE = 100 × ME(t)/
ME(0), where ME(t) and ME(0) are
the mass of encapsulated material
at time t and 0, respectively
Delivery efficiency The delivery efficiency is a measure Ideally, one would like the delivery
(DE) of the ability of the delivery system efficiency to be high (100%)
to deliver the encapsulated material
at the required site of action:
DE = 100 × ME(I))/ME(D), where
ME(I) andME(D) are the masses of
encapsulated material in the initial
delivery system and that are
delivered to the site of action,
respectively
Delivery The delivery system may have to be
mechanism: designed so that it carries the
functional component to a
particular site-of-action and then
releases it. The release may have to
be at a controlled rate or in
response to a particular
environmental trigger (e.g., pH,
ionic strength, enzyme activity, or
temperature)
Protection against The delivery system may have to be
chemical designed to protect an encapsulated
degradation: material against some form of
chemical degradation mechanism,
e.g., oxidation, hydrolysis, etc. The
rate of these chemical degradation
reactions may be promoted by
certain factors that need to be
controlled, such as heat, light,
oxygen, or specific chemicals
Food matrix The delivery system should be
compatibility: compatible with the surrounding
food matrix, i.e., it should not
adversely affect the appearance,
texture, flavor, or stability of the
final product
(continued)
156 S. Mishra and K. Bhargava

Table 8.1 (continued)

Criteria Definition Characteristics of delivery systems
Food grade: The delivery system should be
fabricated from food-grade (GRAS)
ingredients using easily
implemented processing operations
Economic The delivery system should be
Production: capable of being economically
manufactured using inexpensive
ingredients. Ultimately, the benefits
gained from encapsulating the
functional component (e.g.
improved shelf life, enhanced
marketability, novel functionality)
should outweigh any additional
costs associated with encapsulation
Bioactivity: A delivery system should either
improve or at least not adversely
affect the bioavailability of the
encapsulated component
Adopted from: (McClements and Li 2010)

lipid-free food system (e.g. skim milk) (Rico-Muñoz and Davidson 1983). In terms
of selecting a suitable encapsulation system, polarity is thus a key criterion since the
capsules should provide a thermodynamically favorable environment for the antimi-
crobials to prevent migration to other lipid phases while not inhibiting interaction
with microbial cells. Nanoemulsions and solid lipid nanoparticles may be used for
highly hydrophobic compounds, while for amphiphilic and low-molecular-weight
hydrophobic compounds, microemulsions, nanoemulsions, and solid lipid nanopar-
ticles may be suitable.

2.2 Effect of Temperature and pH on Antimicrobial Activity

Wide varieties of antimicrobials such as organic acids require a specific pH environ-

ment in order to be effective. Their activity may also depend on temperature since
the dissociation equilibrium can be altered if temperature increases or decreases. In
high-pH environments, some antimicrobial compounds may completely lose their
efficacies. It is thus important for many antimicrobial encapsulation systems to
maintain, to the extent possible, the pH environment required for optimal antimicro-
bial activity and to protect the antimicrobial compounds from degradation upon
temperature-induced changes.
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 157

2.3 Sensory Effects of Antimicrobials

A significant limitation of many of the emerging naturally occurring antimicrobials

is their sensory threshold level. Moreover, essential oil extracts have been predomi-
nantly used as flavors, and therefore introduction of essential oil compounds in food
products will undoubtedly alter the flavor profile of the food. While some flavor
alterations can be masked, compatibility with the food matrix in terms of sensory
characteristics is nevertheless essential. Moreover, the high levels of essential oil
compounds required to inhibit or inactivate microorganisms may exceed regulatory
levels. At these levels, the health of the consumer upon repeated exposure/consump-
tion may be compromised. Therefore, two essential goals of any antimicrobial
delivery system should be (1) to reduce the potential negative sensory effects of
antimicrobials (e.g. by suppressing volatility) and (2) to not increase (or possibly
even decrease) the minimum inhibitory concentrations of antimicrobials to allow
for a reduction of required concentrations.

3 Delivery System for Food Antimicrobials

Antimicrobial agents are encapsulated to increase the compatibility with the food
matrix and increase their compatibility with food matrix to increase their efficacy to
control release to masks off-flavor/ or to increase their storage transport and utiliza-
tion. Antimicrobial encapsulation may assist in (1) stabilizing the antimicrobial
against deleterious reactions with food components; (2) stabilizing volatile antimi-
crobials against rapid evaporation; (3) reducing the rate of the antimicrobial’s release
into the food, allowing lengthened exposure of microbes to antimicrobial pressure;
and (4) protection of the antimicrobial during processing (Taylor et al. 2005).
Important delivery systems are described in this section and Fig. 8.1. These systems
are extensively studied to safely deliver the antimicrobials in food matrix.

3.1 Emulsions

An emulsion consists of at least two immiscible liquids with one of the liquids being
dispersed as small spherical droplets in the other (Friberg et al. 2003; McClements
2005). Oil droplets dispersed in an aqueous phase is called an oil-in-water (O/W)
emulsion whereas water-in-oil (W/O) emulsion is a system in which water droplets
are dispersed in an oil phase. Moreover, small water droplets trapped within larger oil
droplets dispersed in an aqueous continuous phase are termed as a W/O/W emulsion
158 S. Mishra and K. Bhargava

Oil/water Water/oil

Water phase
Oil phase
Emulsifying agent
Emulsifying agent Oil soluble
Water soluble antimicrobial
antimicrobial in oil phase
in water phase

Nanoemulsion Multilayer

Added surfactant Polyelectrolyte 1 Polyelectrolyte 2

Oil Single- Two- Three-

droplet layer emulsion layer emulsion layer emulsion

Phospholipid bilayer
Water insoluble
Nanoliposomes
antimicrobial
Water soluble
antimicrobial
Hydrophilic region

Hydrophobic region
antimicrobial

Nanocapsules Nanospheres

Nanoparticles
Solid lipid nanoparticles

Surface functionalization
Antimicrobial
Solid core
Surfactant layer

Polymeric matrix

Nanofibers

Antimicrobial

Fig. 8.1 Structure of nanoscale delivery systems used to entrap food antimicrobials (Blanco-­
Padilla et al. 2014)
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 159

(Friberg et al. 2003). Stability of the emulsion is obtained by dispersion of very fine
droplets of one liquid, called the disperse phase, through the other liquid, which is
called the continuous phase. The emulsion is stable when it can persist without
change, for long periods of time, without the droplets of the disperse phase coalescing
with each other, or rising or settling. The stability of an emulsion is controlled by
interfacial surface forces, size of the disperse phase droplets, viscous properties of the
continuous phase and density difference between the two phases.
Most of the lipid-based delivery systems are formed using emulsion technology.
Emulsions are thermodynamically unfavorable systems that tend to break down
over time. This breakdown may be due to variety of physicochemical mechanisms,
including gravitational separation, flocculation, coalescence, and Ostwald ripening
(McClements 2005). Emulsions that are kinetically stable for a reasonable period
of time can be formed by including substances known as stabilizers, e.g., emulsi-
fiers or texture modifiers. Emulsifiers are surface-active molecules that absorb to
the surface of freshly formed droplets during homogenization, forming a protective
layer that prevents the droplets from aggregating. Texture modifiers thicken or gel
the continuous phase, which improves emulsion stability by retarding or prevent-
ing droplet movement. Selection of the most appropriate stabilizer(s) is one of the
most important factors determining the shelf-life and physicochemical properties
of emulsion-based delivery systems. In the remainder of this section we will focus
on the properties of various emulsions systems, since these are the most commonly
used as delivery systems for food antimicrobials in the food industry.

3.1.1 Microemulsions

The conventional food industry approach to emulsion preparation has involved the
application of energy (e.g. mechanical, sound) to a mixture of oil, water, and emul-
sifier. The emulsifier acts to stabilize the interfacial layer between the dispersed and
continuous phase which has been created through the addition of energy to the system.
These emulsions, or macroemulsions, are turbid, have droplet sizes ranging from
0.2 to 10 μm and may remain stable for a considerable period of time; however, they
are kinetically stable, albeit thermodynamically unstable.
Microemulsions are defined as systems which comprise of a mixture of water,
hydrocarbons, and amphiphilic compounds which form thermodynamically stable,
homogeneous (heterogeneous at molecular scale), optically isotropic solutions.
They are thermodynamically stable, transparent isotropic solutions with particle
sizes ranging from 5 to 100 nm, and arise from the spontaneous self assembly of the
hydrophobic or hydrophilic parts of surfactant molecules. These self-assembled
aggregates have well-defined properties, e.g. size and charge. Their spontaneous
formation is driven by the thermodynamic tendency of the system to lower its free
energy, which can be achieved by self-association of the hydrophobic groups of the
surfactant tails, removing them from contact with the polar solvent. Organic
solvents of low solubility can be brought into solution by incorporation into micelles
(Holmberg 2003). This increase in solubility in the presence of micelles is referred
160 S. Mishra and K. Bhargava

to as solubilization. Due to the fact the mechanism is based on an uptake and inclusion
of the normally insoluble material in a micellar aggregate, the form and shape,
as well as the temperature–concentration dependence, of the micelles are a major
factor for solubilization.
Investigations on the use of microemulsions to deliver food antimicrobials and
to inhibit the growth of food borne pathogens such as E. coli and L. monocytogenes
were conducted (Gaysinsky et al. 2008). Carvacrol- and eugenol containing micro-
emulsions were prepared by dispersing the antimicrobials in micellar solutions.
UV-visible spectroscopy was used to obtain pH and temperature stability phase
diagrams; particle size was determined by dynamic light scattering, and structural
information about microemulsions was obtained by nuclear magnetic resonance
spectroscopy (NMR).

Formation of Microemulsions

The three main theories of microemulsion formation and stabilization are discussed
in brief here; first, the mixed film theory, the interfacial film is considered as a
duplux film, having different properties on the water and oil side of the interface
(Prince 1967). The solubilization theory considers microemulsions as swollen
micellar systems, i.e. solutions with solubilized water or solubilized hydrocarbon;
in effect, one-phase systems (Rance and Friberg 1977). The thermodynamic theory
proposes that the free energy of formation of microemulsion, ∆Gm, consists of dif-
ferent terms, such as interfacial free energy, energy of interaction between the drop-
lets, and an entropy term. When, ∆Gm is of a very low or slightly negative value,
microemulsion formation can be facilitated (Ruckenstein and Chi 1975). Irrespective
of the mechanism of microemulsion stabilization, the reduction of the interfacial
free energy to a very low value is critical in facilitating microemulsion formation.
In theory, the arrangement of the emulsifier molecules occurs spontaneously.
However, in some cases energy is provided to the system to speed up the rearrangement
of the surfactant molecules, or to overcome a small kinetic energy barrier. There are
three principle methods which may be used in microemulsion formation. Microemulsion
preparation can be achieved in three different low energy emulsification methods:
dilution of an oil surfactant mixture with water; dilution of a water-­surfactant mixture
with oil; and mixing all the components together in the final composition. As each of
these methods involves the spontaneous formation of microemulsions, the order of
ingredient addition may determine the formation of microemulsion.

3.1.2 Nanoemulsions

Nanoemulsions are defined as stable colloidal systems within nanometric size

(≤100 nm) formed by dispersing one liquid in another immiscible liquid using suit-
able emulsifiers (Burguera and Burguera 2012). Nanoemulsions differ appreciably
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 161

from conventional emulsions in their functional activity due to the decreased size
(Friberg et al. 2003; McClements 2005). For example, these emulsions may not
scatter light strongly in the visible region and can thus be transparent. These emul-
sions are optically transparent, demonstrating better shelf stability, and the droplet
size distribution remains after water dilution as compared to other emulsions.
Nanoemulsions can be formulated with different approaches and materials depending
on the desired structure and functionality. Broadly, high-energy methods such as high-
pressure homogenization, microfluidization, and ultrasonication and low-­ energy
methods such as solvent diffusion are the most commonly used for the preparation of
nanoemulsions (Fathi et al. 2012).
High-energy methods utilize mechanical devices to produce intense disruptive
forces, whereas in low energy methods spontaneous emulsification of all the emul-
sion ingredients is performed (Donsì et al. 2011; Ghosh et al. 2014). Nanoemulsions
can be classified into oil in water (O/W), the multiple emulsions oil-in-water-in-oil
(O/W/O) and water-in-oil-in-water (W/O/W), and the multilayer emulsions which
consist of oil droplets surrounded by nanometric size layers of different polyelectro-
lytes (Weiss et al. 2006).
These nanoemulsions can be beneficial for delivering poorly water-soluble food
antimicrobials improving physical stability of the active compound and increasing
its uniform distribution in food models. The droplets size of nanoemulsion offers
the advantage of increasing interactions of the active compounds with the cell
membrane of bacteria, affecting the stability of the lipid membrane and resulting
in leakage of bacteria intracellular constituents (Blanco-Padilla et al. 2014). This
nonspecific action mechanism decreases the development of resistant microbial
strains. Antimicrobial delivery system should enhance the mass transfer rates to
the sites of action, in order to maximize the antimicrobial activity and to use con-
centrations which are low enough to minimally alter the quality of the product, but
are sufficient to inhibit microbial growth within the limits of food regulations
(Blanco-­Padilla et al. 2014).

Formation of Nanoemulsion

Conventionally, oil-in-water emulsions are prepared by homogenizing the oil,

water, and emulsifier together using a mechanical device known as a homoge-
nizer, e.g. high-shear mixer, high-pressure homogenizer, colloid mill, sonicator
or membrane homogenizer (McClements 2005; Walstra 1993). To manufacture
nanoemulsions, mean droplet diameter should be less than 100 nm. High pres-
sure homogenization, microfluidization diverts the flow of the emulsion in the
homogenization chamber to create fluid jets that impinge in a mixing chamber,
thereby creating additional forces through cavitation and droplet disruption in
addition to pressure changes. In the microfluidizer that has no moving parts,
maintenance is typically lower.
162 S. Mishra and K. Bhargava

The factors determining the final droplet size of the nanoemulsion are given below
1. Emulsifier type and concentration,
2. Volumetric energy input during the homogenization process,
3. Composition of component phases,
4. Temperature.
In the homogenizer two processes occur simultaneously, resulting in the genera-
tion of small droplets from a premix that contains larger droplets. Firstly, the volu-
metric energy input induces droplet deformation and disruption, generating smaller
droplets with new interfaces. Normally, the larger the volumetric energy input the
smaller the droplets. The droplet disruption also depends on the viscosity ratio
between the two phases, which may depend on the temperature of the system.
Secondly, surfactants must rapidly absorb at the new interfaces, creating an inter-
facial layer that reduces the interfacial tension (thereby facilitating droplet dis-
ruption) and induces repulsive interactions (thereby retarding re-coalescence).
The droplet size thus depends on the molecular properties of the emulsifiers govern-
ing the rate of adsorption, reduction in surface tension, and degree of induced
repulsive and attractive colloid interactions, e.g. van der Waals, steric, and electro-
static (McClements 2005).
In nanoemulsions, the choice of surfactants is very critical since emulsifiers have
to rapidly cover the many new surfaces that are formed. Generally, in food emul-
sions two classes of surface-active species are used: (1) small-molecule surfactants
such as monoglycerides, sucrose esters, and others and (2) macro-molecular emulsi-
fiers such as protein or modified starches. Since small-molecule surfactants adsorb
much more rapidly to newly formed interfaces than macro-molecular surfactants,
they are typically better suited for manufacture of nanoemulsions.

Nanoemulsions as Antimicrobial Delivery System

Nanoemulsions may theoretically be designed in a variety of different ways to serve

as carriers of antimicrobials to improve the safety and quality of foods. Firstly, the
lipid phase of the nanoemulsion may be loaded with a lipophilic antimicrobial. In
this case, the delivery of the antimicrobial may occur via a mass transport of the
antimicrobial from the inside of the nanoemulsion droplets through the aqueous
phase of the food system to the membrane of food pathogens or spoilage organisms.
This process would be governed by the solubility of the lipid antimicrobial in the
aqueous phase, which is a function of temperature, the composition of the aqueous
phase, the chemical structure of the antimicrobial, and the droplet size.
Nanoemulsions, because of their small size and large curvature, can be expected to
have better activity since the driving force for the mass transport process, i.e. the
concentration difference of the antimicrobial in the vicinity of the oil droplet and in
the bulk phase, is much higher due to the Laplace effect (McClements 2005).
Secondly, antimicrobial nanoemulsions can be constructed from a surface-active
antimicrobial that stabilizes the nanoemulsion and an inert lipid. Examples of
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 163

surface-­active antimicrobials include lysozyme, nisin, and lauric arginate. Additional

emulsifiers may be required to enhance emulsion formation and to achieve the small
droplet diameter needed to produce nanoemulsions, as well as to improve stability
of nanoemulsions for breakdown of mechanisms such as coalescence or floccula-
tion. Additional emulsifiers also allow for the interaction of droplets with the micro-
organisms, which can be improved by creating an electrostatic attraction between
droplets and negatively charged microbial surfaces.
Nanoemulsions may have an advantage over conventional emulsions because the
frequency of collision with microorganisms is higher due to the smaller size.
Thirdly, antimicrobial nanoemulsions could be constructed by combining the two
approaches outlined above, i.e. using one antimicrobial that is part of the dispersed
droplet phase and a second antimicrobial that is surface active and part of the emul-
sifier layer. It should be noted though, that some antimicrobials (e.g. simple and
more complex phytophenols) may not strictly fall into either category. While anti-
microbials are typically added to the lipid phase of nanoemulsions, they have a
tendency to accumulate in higher concentrations near the vicinity of the interface.
In the case of phenolic antimicrobials, this is because the hydroxyl group facilitates
an interaction with the headgroup of the surfactant.

3.1.3 Nanostructured Multiple Emulsions

The use of multiple emulsions can create delivery systems with novel encapsulation
and delivery properties. The most common examples of this are oil-in-water-in-oil
(O/W/O) and water-in-oil in-water (W/O/W) emulsions (Garti and Benichou 2004).
For example, a nanostructured W1OW2 emulsion would consist of nanometer-sized
water droplets or reverse micelles (W1) contained within larger oil droplets (O) that
are dispersed within an aqueous continuous phase (W2). Functional food compo-
nents could be encapsulated within the inner water phase, the oil phase, or the outer
water phase, thereby making it possible to develop a single delivery system that
contains multiple functional components. This technology could be used to separate
two aqueous phase components that might adversely react with each other if they
were present in the same aqueous phase. Alternatively, it could be used to protect
and release an aqueous phase component trapped within the inner water droplets
(W1) to a specific site such as the mouth, stomach, or small intestine.

Nanostructured Multilayer Emulsions

Current studies have shown that the use of multilayer emulsions can create novel
delivery systems. These systems typically consist of oil droplets (the core) sur-
rounded by nanometer thick layers (the shell) comprised of different polyelectro-
lytes. These layers are formed using a layer-by-layer (LbL) electrostatic deposition
method that involves sequential adsorption of polyelectrolytes onto the surfaces of
oppositely charged colloidal particles. Fig. 8.2 shows an example of the LbL
164 S. Mishra and K. Bhargava

Separate Oil Primary Secondary

and Water Emulsion Emulsion

Add Emulsifier Add Biopolymer

Single-Layer Two Layers

Fig. 8.2 Schematic for formation of a number of nanolayers around particles (Weiss et al. 2006)

approach to encapsulating oil droplets in an O/W emulsion. An ionic emulsifier that

rapidly adsorbs to the surface of lipid droplets during homogenization is used to
produce a primary emulsion containing small droplets; then an oppositely charged
polyelectrolyte is added to the system, which adsorbs to the droplet surfaces and
produces a secondary emulsion containing droplets coated with a 2-layer interface.
This procedure can be repeated to form oil droplets coated by interfaces containing
three or more layers. Under certain circumstances, emulsions containing oil drop-
lets surrounded by multilayer interfaces have been found to have better stability
against environmental stresses than conventional oil-in-water emulsions with
single-­layer interfaces (Guzey and McClements 2006). In addition, it is possible to
develop smart delivery systems by engineering the properties of the nanostructured
shell around the droplets.
This interfacial engineering technology would utilize food-grade ingredients
(such as proteins, polysaccharides, and phospholipids) and processing operations
(such as homogenization and mixing) that are already widely used in the manufac-
ture of food emulsions. Therefore, this technology should be economically viable
and could be easily implemented by the food industry. A functional component
trapped within the core of a multilayer emulsion delivery system could be released
in response to a specific environmental trigger by designing the response of the shell
to the environment as in the following examples:
1. Complete Shell Dissociation: Weakening electrostatic interactionscan cause
shells to completely dissociate under specific solution conditions (pH, ionic
strength). For instance, changing the pH can cause one or more of the polyelec-
trolytes to lose its charge, or increasing the ionic strength can weaken the elec-
trostatic attraction of a polyelectrolyte to the next layer, thereby promoting
desorption.
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 165

2. Modulation of Shell Porosity: The thickness and porosity of shells can change with
exposure to pH and ionic strength. This determines the rate at which ­functional
components trapped inside the core will diffuse into the surrounding medium.
By selecting the appropriate polyelectrolytes to use and the assembly conditions,
one could design systems to release, under specific environmental triggers,
functional components smaller than some particular dimension. In principle,
one could vary the release of one or more encapsulated materials using either of
these release mechanisms, either individually or in combination (simultaneously
or sequentially).

3.1.4 Liposomes

Liposomes are spherical particles formed from polar lipids (e.g. phosphatidylcholine
or phosphatidylethanolamine) or mixtures of polar lipids with cholesterol or ergos-
terol; liposomes are also components available in abundance in nature. Their name is
derived from a combination of the words lipid and the Greek word ‘soma’ meaning
body. The size of liposomes can range from tens of nanometers to tens of micrometers
depending on the method of manufacturing (Taylor et al. 2005). In polar solvents such
as water, polar lipids generally have a tendency to self-assemble in the form of bilayer
membranes. These bilayer membranes are relatively flexible and under shear can be
forced to assume a curvature that leads to the generation of particles composed of a
thin shell of a polar lipid bilayer; this bilayer surrounds an interior compartment that
consists of the initial solvent in which the polar lipids were dispersed prior to the
application of shear. Depending on the degree of shear applied during the manufac-
turing process, liposomes may assume a variety of different structures, such as unil-
amellar liposomes (which have a single bilayer membrane shell), multilamellar
vesicles (liposomes with multiple bilayer membranes stacked in a concentric
configuration similar to the skins of an onion), and multivesicular vesicles (liposomes
that may contain other randomly sized liposomes in their interior.
Liposomes can serve as a separate microenvironment in which food antimicrobi-
als and other functional components can be incorporated and their activity main-
tained despite changes in the surrounding aqueous phase (Bouwstra et al. 1996).
Interestingly, liposomes are able to encapsulate both lipophilic and hydrophilic
functional components. In the case of lipophilic compounds, the bilayer membrane
acts as the host environment to the compounds. The compounds are solubilized in
the interior of the bilayer, which consists of the self-assembled fatty acid tails of the
polar lipids via a process also known as ad-solubilization (a combination of adsorption/
solubilization processes). Alternatively, hydrophilic compounds may be encapsulated
in the interior of the liposomes. In this case, the liposomal membrane must provide a
barrier to diffusion of compounds from the interior to the exterior. This mass transport
may be initiated when a concentration difference between the interior and exterior
of the liposomes exists. For example, after manufacturing, liposomes are typically
added to a food or beverage system where the aqueous phase environment differs
from that of the interior of the liposomes.
166 S. Mishra and K. Bhargava

Formation of Liposomes

Liposomes may be manufactured using a variety of different manufacturing proce-

dures including thin-film rehydration, freeze-drying rehydration, reverse-phase
evaporation, detergent depletion, membrane extrusion, high-pressure homogeniza-
tion or microfluidization, and ultrasonication by probe or bath. For large-scale
manufacture of liposomes with sizes below 100 nm, microfluidization has emerged
as the predominant method.

Liposomes as Antimicrobial Delivery Systems

While liposomes have been used for delivery of various bioactive compounds,
the application of liposomes for the delivery of food antimicrobials is still in its
early stages. Liposome was used to encapsulate lysozyme and nisin to prevent
spoilage of cheeses (Thapon and Brule 1986). Degnan and Luchansky (Degnan
and Luchansky 1992) reported anti-listerial effects of the bacteriocin, pediocin
AcH, in beef tallow and muscle slurries using PC liposomes. Benech et al.
(Benech et al. 2002) encapsulated nisin Z (asparagine-containing variant of
nisin) in hydrogenated phosphatidylcholine liposomes and added nisin Z con-
taining liposomes or unencapsulated nisin Z to cheese at a final concentration of
300 IU/g.

3.1.5 Nanofibers

Nanofibers are ultrathin fibers with average diameters below 100 nm and produced
from electrospinning. Electrospinning is a process that produces continuous poly-
mer fibers through the action of an external electric field imposed on a polymeric
solution or melt. Proteins, carbohydrates, and lipids-like materials can be used for
the production of nanofibers.
These fibers have been shown to possess exceptional properties that distin-
guish them from larger non-woven fibers, such as a high orientation of polymers
within the fibers that leads to mechanically superior properties, e.g. increased
tensile strength and large surface-to-mass ratio, high porosity (Frenot and
Chronakis 2003). Electrospun nanofibers exhibit properties such as high surface
area-to-volume ratios. This is the reason for great interest by food industry in this
delivery system. Mats composed of electrospun fibers find applications in food
packaging materials and edible films (Blanco-Padilla et al. 2014). Nanofibers
offer several advantages such as large surface area, high gas permeability how-
ever biopolymer solubility limits their current applications. Antibacterial activi-
ties of nanofibers has been shown against E. coli and S. aureus (Gao et al. 2014;
Jia et al. 2011).
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 167

4  pplications of Antimicrobial Delivery Systems

A
In Vitro and Food Models

Naturally occurring antimicrobials fail to show antimicrobial activity in food models

due to their lipophilic nature. Importantly, antimicrobial activities of these com-
pounds are eliminated or reduced because of interaction or binding with other food
components such as lipids and proteins. Delivery systems of naturally occurring
antimicrobials discussed in this chapter offers excellent potential to increase activity
against microbial pathogens in vitro and enhance their food based applications
(Gomes et al. 2011; Hill et al. 2013; Xiao et al. 2011). Studies on delivery systems
of antimicrobials are summarized in Table 8.2. There are several studies in vitro but
food application studies are limited. Mostly encapsulated natural antimicrobials are
applied to juices and recently on lettuce (Bhargava et al. 2015; Donsì et al. 2011).
Efficacy of antimicrobials in broth does not ensure success in food systems. New
delivery systems may fail when incorporated into a food or require significantly
higher dosages to gain efficacy.

5  oxicity Associated with Natural Antimicrobials

T
Delivery Systems

It is evident from literature that these delivery systems due to their unique physic-­
chemical and biological properties have far reaching food applications. But there is
lack of knowledge about the effects of prolonged exposure to these systems on
human health and environment. Potential toxicity can be caused by increased bio-
availability of these antimicrobials that are toxic at high levels, interference with
normal GI function and due to some of components typically used to prepare these
delivery systems (McClements and Rao 2011). The implications of these delivery
systems on health and environment need to be assessed completely before their
large scale production and application in food industry. These delivery systems can
possibly cause adverse biological effects at the cellular and subcellular levels.
Therefore, cytotoxicity and clinical studies should be conducted in research articles
exploring the industrial scale applications of these delivery systems.

6 Conclusions

Delivery systems of natural antimicrobials are fast emerging as viable solutions due
to their help in controlling the growth of pathogenic and spoilage causing organisms
on food surfaces. These systems display many advantages such as transparency and
higher physical stability but they may or may not enhance antimicrobial efficacy
and functionality. Increase in antimicrobial activity can be derived because the
168 S. Mishra and K. Bhargava

Table 8.2 Invitro and food model research studies on delivery system for natural antimicrobials
Other Target Research
Antimicrobial components Microorganism Model Reference
Eugenol Sesame oil, Escherichia coli Fruit Ghosh et al.
Miglycol 812 N O157:H7 Juice (2014)
Listeria Invitro Terjung et al.
monocytogenes (2012)
Escherichia coli C
600,
Listeria innocua
Carvacrol Miglyol 812 N Escherichia coli C invitro Terjung et al.
Sunflower oil 600, Apple and (2012)
Listeria innocua Pear Juice Donsì et al.
Lactobacillus (2012)
delbrueckii
Saccharomyces
cerevisiae
Limonene Sunflower oil Lactobacillus Apple and Donsì et al.
delbrueckii Pear Juice (2012)
Saccharomyces
cerevisiae
Escherichia coli
Cinnamaldehyde Sunflower oil Lactobacillus Apple and Donsì et al.
delbrueckii Pear Juice (2012)
Saccharomyces
cerevisiae
Escherichia coli
Basil oil Basil oil/water Escherichia coli Invitro Ghosh et al.
(2013)
Lemongrass Carnauba Escherichia coli Invitro Salvia-Trujillo
Lemongrass oil/ O157:H7 Plum et al. (2014)
alginate Salmonella Fuji Kim et al.
Typhimurium Apples (2013)
Yeast Kim et al.
(2013)
Bovine lactoferrin Lecithin and Staphylococcus NA Balcão et al.
poloxamers aureus (2013)
Listeria innocua
Candida albicans
Oregano oil Tween 80 Listeria Lettuce (Bhargava et al.
monocytogenes (2015)
Salmonella
Typhimurium
E.coli O157:H7
Eucalyptus oil Tween 80 B. cereus invitro Sugumar et al.
S. aureus (2013)
E. coli
Peppermint oil Purity gum Listeria invitro Liang et al.
monocytogenes (2012)
S. aureus
8 Delivery Systems for Introduction of Natural Antimicrobials into Foods 169

delivery system is able to deliver several antimicrobials that attack the cells by dif-
ferent antimicrobial mechanism. However, there exist several challenges in the
design of such system and application in real food matrices.
This rapidly progressing field holds considerable promise for application in food
preservation. However, the possible interactions of delivery systems with food
matrices need to be elucidated. At the same time, the studies on mechanisms of the
release of encapsulated food antimicrobials, and toxicity need to be performed
(Blanco-Padilla et al. 2014). Currently, these delivery systems are mainly applied in
pharmaceutical industries but its applications in food industry are emerging. It is
estimated that market for nanoencapsulated food additives will reach $26,208.3
million by 2018 (Blanco-Padilla et al. 2014; Sekhon 2010). However, to apply these
technologies in the food industry, low cost ingredients and simple methods should
be used. The advances in the development of delivery system of natural antimicrobi-
als will offer important challenges for both industry and government with regard to
implementation. Food industry must consider building up consumer confidence in
these systems and regulatory agencies should present guidance’s in evaluating
safety of these delivery systems with novel properties (Sekhon 2010).

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Chapter 9
Microbial Resistance to Antimicrobials

Sean Pendleton and P. Michael Davidson

Abstract Recently, there has been a growing concern across the globe that humans are
approaching the end of the antibiotic era. However, this is not a new idea; concern about
antibiotic resistance has been around for many years. As more and more microorgan-
isms become resistant to antibiotics, there are fewer and fewer options to treat infec-
tions. To develop control measures for microbial antibiotic resistance, the scientific
community must have information about the extent of the problem, the mechanism of
antibiotic resistance, and how resistance spreads. A potential contributor to the problem
is development of antibiotic resistance through exposure to food-related antimicrobials
and sanitizers. A focus of the chapter is a discussion of the research on the role of appli-
cation of antimicrobial food preservatives and environmental sanitizers in the food
industry on development of resistance to medically-­important antibiotics.

Keywords Antibiotics • Food preservatives • Sanitizers • Antimicrobial-resistance

• Microorganisms

1 Antibiotics

In 1929, Dr. Alexander Fleming published his work focused around his fortuitous
discovery of penicillin. A contaminating mold was found on a culture plate that
appeared to be inducing lysis of the Staphylococcus colonies. Further study found
that this mold was in fact producing an antibacterial substance with activity
against pyogenic cocci and bacilli of the diphtheria group (Fleming 1929). What
Fleming had discovered was a class of drugs now referred to as β-lactams. These
drugs inhibit cell wall synthesis, which leads to a cell being unable to maintain

S. Pendleton
Fort Worth, TX, USA
e-mail: [email protected]
P.M. Davidson (*)
Department of Food Science and Technology, University of Tennessee, Knoxville, TN, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 173

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_9
174 S. Pendleton and P.M. Davidson

and protect itself from the environment (Bycroft and Shute 1985). This discovery
began the era of antibiotics and coincidentally, the era of antibiotic resistance.
Recently, there has been a growing concern across the globe that humans are
approaching the end of the antibiotic era (WHO 2014). However, this is not a new
idea; concern about antibiotic resistance has been around for many years. As more
and more microorganisms become resistant to antibiotics, there are fewer and fewer
options to treat infections. As a result, there has become a greater focus on tracking
antibiotic resistance. In 1994, The World Health Organization had a meeting to dis-
cuss antimicrobial resistance and during the meeting presented recommendations
for countries to develop isolate-based databases to track antimicrobial susceptibility
on a national level (Tenover and Hughes 1995). In 1996, the CDC, FDA, and USDA
partnered to create the National Antimicrobial Resistance Monitoring System
(NARMS), which seeks “to track antimicrobial resistance in foodborne bacteria
from humans, retail meats, and food animals” (NARMS website). Also in 1996, the
Asian Network for Surveillance of Resistant Pathogens (ANSORP) was created to
surveil antimicrobial resistance in Asia. Two years later, the European Antimicrobial
Resistance Surveillance System (EARSS) was established to make a concerted
effort to control antimicrobial resistance. Its administration and coordination has
since been shifted to the European Center for Disease Prevention and Control
(ECDC) and been renamed the European Antimicrobial Resistance Surveillance
Network (EARS-Net). Together, these networks have taken on the task of tracking
the spread of antibiotic resistance and the knowledge they have generated has greatly
influenced the understanding of the issue of antibiotic resistance.

1.1 Mode of Action

To understand the development of resistance to antibiotics, it is first necessary to

explain the mechanism of action of these compounds against microorganisms.

1.1.1 Aminoglycosides

Among the aminoglycosides, the most well-known are streptomycin and gentamycin.
Streptomycin was the first of this class of antibiotics to be discovered and is therefore
the most well studied (Davis 1987). Cells treated with streptomycin exhibit inhibition
of protein synthesis and membrane damage (Jana and Deb 2006). Aminoglycosides
contain multiple positively charged amine groups, which give them an affinity to bind
to negatively charged cell surfaces (Jana and Deb 2006) (Fig. 9.1). Once bound, the
antibiotic diffuses across the cell membrane and binds to the ribosomes in such a way
as to alter their ability to proofread the proteins being synthesized. This leads to trun-
cated and missense proteins being incorporated into the cell membrane and ultimately
increased permeability of the cell membrane. Increased permeability allows more of
the antibiotic to pass through the membrane and bind to more ribosomes to the point
9 Microbial Resistance to Antimicrobials 175

OH
NH2
HO O OH
HO NH2
HO
NH OH Streptomycin
O O
CH3 O N NH2
CHO
NH2
OH CH3

OH
CH3 O NH2 CH3
NH O
Gentamicin
CH3 OH HO NH2
O O
NH2
H2 N

Fig. 9.1 Structures of common aminoglycosides

where ribosomes associated with initiating protein synthesis are bound and protein
synthesis is halted. This cascade of events ultimately leads to cell death (Davis 1987;
Jana and Deb 2006). Aminoglycosides are known to cause varying degrees of ototox-
icity, neuromuscular paralysis, and nephrotoxicity (Lietman 1990).

1.1.2 Beta-Lactams

The class of beta-lactam antibiotics is very broad and consists of carbapenems,

cephalosporins, monobactams, and penicillins. The defining structure of this drug
class is the beta-lactam ring (Fig. 9.2). Beta-lactam antibiotics inhibit cell wall syn-
thesis which leaves the cell unable maintain structure and ultimately leads to cell
death. Beta-lactams attack the transpetidases and carboxypetidases by binding to
the active sights through their beta-lactam bond, which inactivates the enzyme
(Waxman and Strominger 1983), resulting in the inability to form cross-linkages
between peptidoglycan chains. Carbapenems show activity against a wide range of
bacteria and are considered a last line of defense in many cases due to growing
levels or resistance and concerns about nephrotoxicity (Papp-Wallace et al. 2011).
While penicillins are effective against a broad range of bacteria they are also associ-
ated with allergic reactions, which make them difficult to implement in emergency
situations (Saxon et al. 1987). The main monobactam, azteronam, has not been
found to be nephrotoxic and only causes weak allergic reactions (Hellinger and
Brewer 1999). Cephalosporins have also not been associated with nephrotoxicity or
major allergic reactions like carbapenems or penicillins (Dancer 2001; Neu 1990),
making them the most useful of the beta-lactam antibiotics in modern medicine.
176 S. Pendleton and P.M. Davidson

Fig. 9.2 Structures of H

β-lactam antibiotic classes R NH
S
(adapted from Saxon et al.
1987). β-lactam rings Penicillins
O N
marked in red for each class
O
COOH

NH H
R S

O N Cephalosporins
R'
O
O OH

H R'
R
R" Carbapenems
N
O
COOH

R NH

O N Monobactams
SO3 H
O

1.1.3 Glycopeptides

Vancomycin was the first discovered glycopeptide and remains the most used of the
class, though resistant strains are beginning to become an issue around the world.
Glycopeptides consist of a seven-unit long amino acid chain with side groups arising
from aromatic rings allow for with addition of sugars, amino sugars, and other groups
(Reynolds 1989) (Fig. 9.3). As a contrast to vancomycin, teicoplanin possesses a long
fatty acid chain (Barna et al. 1984) that gives it potential to penetrate the cell mem-
brane, though it does not play a role in antimicrobial mechanism (Reynolds 1989).
The active site of glycoproteins lies within the cleft formed by the tertiary structure of
the peptide, referred to as the aglycone (Reynolds 1989). This site does not interfere
with the enzyme responsible for assembly of the cell wall, but rather the substrates,
specifically those with “acyl-D-ala-D-ala” (Reynolds 1989). The glycopeptide hydro-
gen bonds this sequence and prevents both transglycosylases and transpeptidases
from acting on the substrate. Inability to incorporate cell wall precursors results in
their inevitable build-up within the cell and as the cell is unable to grow without the
ability to incorporate new segments into its cell wall, it ultimately dies. While vanco-
mycin is an effective alternative to penicillins, it also presents concerns over toxicity.
9 Microbial Resistance to Antimicrobials 177

OH
HO
OH
O OH

NH O NH2 O HN O
NH NH NH O
NH NH
O
O O
HO OH
Vancomycin
O O
Cl Cl
O O
HO
HO O
OH
O

NH2
OH

OH
N OH
OH
HO O
O O
Cl
HO
O O O

HO O
Cl O
O NH
O NH
NH O NH NH O NH O Teicoplanin
NH O
HO
HO
NH2
O
O
OH
HO O
HO HO
O

HO
OH OH

Fig. 9.3 Structures of glycopeptides. Fatty acid side chain of teicoplanin marked in red
178 S. Pendleton and P.M. Davidson

O
H3 C CH3

HO OH
OH H3 C
CH3 CH3
H3 C N
H3 C
H5 C2 O HO Erythromycin
O O CH3

O O OCH3
CH3 CH3 OH
O
CH3

H O OH
O OH

H3 C O H OH O O

OH
Natamycin
O O

HO OH
NH2

Fig. 9.4 Structure of macrolides

One study found nephrotoxicity in 7% of patients and ototoxicity in 4% of patients

tested, which in their study was more than acceptable for the severity of the diseases
being treated (Mellor et al. 1985).

1.1.4 Macrolide

In 1952, erythromycin was the first macrolide discovered. From there various other
naturally occurring macrolides were discovered and other partially-synthetic macro-
lides were created (Mazzei et al. 1993). Macrolides are characterized by a large 12–16
carbon lactone ring with attached sugars (Tenson et al. 2003; Mazzei et al. 1993; Mao
and Wiegand 1968) (Fig. 9.4). Macrolides attack bacterial cells by interfering with
protein synthesis. Initially it was, hypothesized that macrolides bound to either ribo-
somal subunit blocking either aminoacyl-tRNA or peptidyl-tRNA from binding (Mao
and Wiegand 1968). Hypotheses were then generated that theorized that macrolides
caused the release of peptidyl-tRNA from the ribosome, which in turn causes the
9 Microbial Resistance to Antimicrobials 179

Fig. 9.5 Structures of O O

quinolones. Core
4-quinolone structures OH
marked in red Nalidixic acid
N N

O O
F
OH
Ciprofloxacin
N N
NH

interruption of protein synthesis (Mazzei et al. 1993). This hypothesis was further
explored in more detail by Tenson et al. (2003). They used X-ray crystallography to
determine binding sites of various macrolides in order to better understand their mode
of action. It was determined that macrolides bind in the tunnel where newly formed
proteins are exiting the ribosome (Tenson et al. 2003). It was also found that the depth
into the ribosomal tunnel where the antibiotic binds determines the protein length
before it is truncated (Tenson et al. 2003). As proteins are essential for the vitality of
a cell, inability to create new proteins will lead to the death of the cell. Macrolides
have one of the least amounts of adverse reactions among antibiotics and are consid-
ered safer than many others available (Periti et al. 1993; Williams 2001). Long term
usage has of erythromycin has been associated with hearing loss, but it is often
reversed after discontinuation of the antibiotic treatment, and most often the side-
effects are just nausea, abdominal pain, vomiting, and or diarrhea (Williams 2001).
One polyene macrolide-type antibiotic, natamycin is used in food preservation to
prevent mold growth on cheeses (Davidson et al. 2015).

1.1.5 Quinolones

While not a true quinolone due to its structure, nalidixic acid (Fig. 9.5) was the first
compound discovered in the class (Emmerson and Jones 2003). The 4-quinolone
structure is made up of two joined aromatic rings with one ring containing a nitrogen
substituted for one of the carbons and a ketone para to the nitrogen (Fig. 9.5). In
nalidixic acid, another carbon is substituted for nitrogen in the adjacent ring, giving
it a non-canonical structure. A fluorine in the sixth or seventh position is typical of
fluoroquinolones (Hooper and Wolfson 1988). In 1964, it was discovered that nali-
dixic acid prevented DNA synthesis, as DNA concentrations were lower in treated
cells compared to untreated cells (Goss et al. 1964). However, it wasn’t until DNA
gyrase was discovered that it was identified as one of the true targets of quinolones.
180 S. Pendleton and P.M. Davidson

DNA gyrase is tetramer of two subunits A and B and, its homologue, topoisomerase
IV has subunits C and E (Hawkey 2003). DNA gyrase and topoisomerase IV are
involved in topological changes in DNA. Gyrase can introduce negative supercoils
in DNA or relax them, as well as catenate and decatenate duplexed DNA (Reece
et al. 1991). Quinolones bind subunit A of DNA gyrase, interacting with serine 83,
and form a complex of the antibiotic, enzyme, and DNA (Hooper and Wolfson 1991;
Vashist et al. 2009). This causes a conformation change in the enzyme, which causes
stress on the DNA that results in the breakage (Drlica 1999). This process ultimately
leads to the death of the cell. The effects of quinolones were enhanced by the addi-
tion of a fluoride group to create fluoroquinolones. Ciprofloxacin was one of the first
fluoroquinolones created and has become one of the widest used. The enhanced
function of fluoroquinolones is thought to be due in part to the increased hydrophi-
licity and entry through porins rather than across the cell membrane, as with quino-
lones (Hooper and Wolfson 1988). Quinolones and fluoroquinolones are largely
well tolerated, though they do cause some side effects (Stahlmann and Lode 1999;
Lipsky and Baker 1999). Most commonly these include gastrointestinal and central
nervous system issues, as well as skin rashes (Stahlmann and Lode 1999; Lipsky and
Baker 1999). Less commonly, tendonitis, cartilage damage, phototoxicity, and slight
cardiotoxicity can occur (Stahlmann and Lode 1999; Lipsky and Baker 1999).

1.1.6 Sulfonamides

Sulfonamides were first discovered in 1935 when Gerhard Domagk published

work examining various synthetic antibacterial compounds. He found that a red
azo dye could prevent disease in mice infected with streptococci, however he found
it ineffective in vitro (Bentley 2009). It was then discovered that one of the break-
down products of the dye was responsible for the antibacterial activity, sulfanil-
amide (Bentley 2009; Anand 1975). From there, studies were undertaken to
determine the mode of action and the impacts structure has on sulfonamide activity
(Henry 1943; Seydel 1968, 1981). The core of sulfonamide structure is NH2-
benzene ring-SO2NH-­R with various substitutions in the R position and elsewhere
to change specificity (Spinks et al. 1999) (Fig. 9.6). Sulfonamides were found to
inhibit synthesis of folic acid by acting as an analogue of the substrate para-amino-
benzoic acid (PABA) within the reaction (Achari et al. 1997). This was partially
discovered early on, when PABA was found to be antagonistic to the effects of
sulfonamides (Henry 1943). Sulfonamides are competitive inhibitors of folic acid
synthesis and as a result are only bacteriostatic (Henry 1943; Anand 1975). Since
the discovery of sulfanilamide, many different sulfonamide compounds have been
created. One of the more potent side effects that was found to occur was nephrotox-
icity, which could lead to renal failure and death (Lehr 1957). This reaction was
found to occur more frequently in certain sulfonamides and their clinical use
diminished (Lehr 1957). Additionally, allergic reactions were identified which
involved a “drug fever” and skin rashes (Lehr 1957). While renal damage is less of
an issue, sulfonamide allergy still remains an issue (Stohs and Miller 2014).
9 Microbial Resistance to Antimicrobials 181

OH
H2 N Para-aminobenzoic acid
O

O
O
H2N S Sulfanilamide
NH2

O
O O N
S
NH Sulfamethoxazole

H2 N

Fig. 9.6 Structures of sulfonamides and the substrate para-aminobenzoic acid. Commonalities
between the substrate and analogues are marked in red

1.1.7 Tetracyclines

In 1948, aureomycin was discovered by Duggar (Duggar 1948) which was followed
shortly by discovery of terramycin (Finlay et al. 1950) both of which had broad
spectrum activity and came from colonies of similar color (Nelson and Levy 2011).
Once the structure for these compounds was determined, they became known as
chlortetracycline and oxytetracycline, respectively (Nelson and Levy 2011). These
were the first two compounds of the tetracycline family discovered, which were
followed shortly by tetracycline itself (Conover et al. 1953). Tetracyclines, as their
name implies, are composed of four interconnected cyclic rings (Fig. 9.7).
Tetracyclines act on bacteria by inhibiting protein synthesis (Chopra et al. 1992;
Chopra and Roberts 2001; Nelson and Levy 2011). More specifically, they bind
near the aminoacyl-tRNA acceptor site on the 30S subunit, which prevents the addi-
tion of amino acids to the peptide chain (Brodersen et al. 2000). This effect of tet-
racycline and several others of the class is bacteriostatic, while the others have
bactericidal action unrelated to ribosomal binding (Schnappinger and Hillen 1996;
Chopra et al. 1992). These other “atypical tetracyclines” interact with the cell mem-
brane causing changes that result in cell death (Chopra and Roberts 2001).
Tetracyclines are known to cause enamel damage in young children and can cross
the placental barrier, therefore they are not recommended for use in children or
pregnant women (Lietman 1990). They are also known to cause gastrointestinal
distress, specifically nausea, vomiting, and diarrhea, and while food can help miti-
gate these effects, it also inhibits tetracycline absorption (Lietman 1990).
182 S. Pendleton and P.M. Davidson

HO O OH O O
OH
NH2
Tetracycline
OH
H H
H3 C OH H C N CH
3 3

OH O O OH OH
OH
NH2
Chlortetracycline
O
H H
N H3 C OH Cl
H3 C CH3

O O O OH OH
OH
H2 N
Oxytetracycline
HO
H H
N OH H C OH
H3 C CH3 3

Fig. 9.7 Structures of tetracyclines

Fig. 9.8 General mechanism of hydrolysis of a β-lactam antibiotic by a serine β-lactamase (Touchet
et al. 2011). The β-lactam ring is broken by the β-lactamase, rendering the antibiotic inactive
9 Microbial Resistance to Antimicrobials 183

1.2 Antibiotic Resistance

Soon after the discovery of penicillin, Abraham and Chain (1940) published a note
indicating the existence of an enzyme that could inactivate penicillin. They had
initially found that some Bacterium coli cultures were not inhibited by penicillin.
After further investigation, they determined that the bacteria were producing an
enzyme that was secreted from the cell and inactivated penicillin. These enzymes
are now known as β-lactamases and they act by hydrolyzing the beta-lactam ring
(Touchet et al. 2011) (Fig. 9.8).
Resistance to antibiotics can be an evolutionary advantage for bacteria that are
exposed regularly. This regular exposure is referred to as selective pressure, mean-
ing the use of antibiotics are actually selecting for resistance in bacterial p­ opulations.
As penicillin was increasingly used for the treatment of bacterial infections, those
bacteria that could produce beta-lactamases were at an advantage to survive and
proliferate, thus increasing the resistant populations.
Acquired resistance to any compound begins with a mutation in the DNA. Mutations
happen randomly during replication of DNA or can be induced by exposure to muta-
genic compounds. As mutations are random, they can have various effects. Mutations
can be silent, meaning that they do not produce any phenotypic effect on the bacteria.
Mutations can also be missense or nonsense, where a base change results in a different
amino acid being coded for in the sequence or the results in a stop codon instead of an
amino acid, respectively. Finally, a mutation can be a sense mutation, which is the con-
version of a stop codon to an amino acid codon resulting in a longer protein sequence.
These mutations can have a wide variety of effects on the bacterium, including the
death of the cell, newly acquired resistance to a compound, or increased fitness.
Mutations conferring antibiotic resistance may result in: (1) an enzyme that can break-
down a compound, (2) a change of the target for an inhibitor, (3) an up-­regulation of efflux
mechanisms, or (4) the elimination the pathway to uptake a compound. These mutations
are not always evolutionarily advantageous. For example, up-regulation of efflux mecha-
nisms requires increased energy output from the cell and thus could reduce the “fitness”
of the cell. If the cell is not in an environment where this mutation is needed, then other
cells will out compete it and this mutation will be lost. However, if the mutation is one for
a toxic compound present in the environment, then the mutation might be selected for and
then maintained in the bacterial population, thus creating a resistant population.

1.3 Spread of Antibiotic Resistance

Once a mutation is selected for and maintained within a population, there becomes
a chance for it to be transferred between cells through horizontal gene transfer
(Fig. 9.9). There are a few different means by which this transfer can occur depend-
ing on where the gene or genes are located. If the genes are located on the chromo-
some, there are two different means by which this can be transferred to another cell.
When a bacteriophage is replicating within a host cell, it can inadvertently pick up
bacterial host cell DNA instead of viral DNA. Upon subsequent infection of anther
184 S. Pendleton and P.M. Davidson

Fig. 9.9 Horizontal gene transfer. Transduction involves the integration of bacteriophage DNA
(orange) into the recipient cell’s chromosome (yellow). Transformation involves the incorporation
of free-floating DNA (pink) into the recipient cell’s chromosome. Conjugation involves the trans-
fer of a donor cell plasmid (green) to a recipient cell

cell, the bacterial DNA can be transferred to a new cell and integrated into the
genome. This pathway is referred to as transduction. Alternatively, naked DNA can
either be excreted by a cell or released from a dying cell to be taken up by other cells
in the environment, via a mechanism called transformation.
If the resistance genes are carried on a plasmid, an extra-chromosomal genetic
element, then the genes can be passed to another cell via bacterial conjugation. The
process begins by replicating the plasmid, which allows the original cell to maintain
resistance, as well as pass it to another cell. The cell containing the plasmid, the
donor cell, can pass it on to a recipient cell. A secretory apparatus, known as a pilus,
is formed by the donor cell, which then creates a bridge between the two cells. The
plasmid is then transferred into the recipient cell, giving it the resistance genes and
any other genes that happen to be present on the plasmid.
Transposons also play a role in the transfer of genes. They are so-called “jumping
genes”, meaning that they can move from one area of DNA to another. They are genetic
elements that contain a transposase, which allows them to be excised from the DNA, a
set of genes in between repeated sequences that the transposase acts upon. The inter-
vening genes, which could encode resistance determinants, can then be removed from
one area of the DNA and either recombine in another area of the chromosome, or can
integrate into a plasmid in the cell which could then be horizontally transferred.
As resistance elements can potentially be mobile and transferred between bacteria,
this increases the opportunity for a single bacterium to collect multiple resistance ele-
ments making it multi-drug resistant (MDR). As more and more bacteria are resistant
to the antibiotics that were once effective, new antibiotics must be found to treat them.
9 Microbial Resistance to Antimicrobials 185

Of utmost concern. is development of pan-resistance or resistance to all known classes

of antibiotics, which would make an infection untreatable by current means. In late
2007, a study was published in the Journal of the American Medical Association on
methicillin-resistant Staphylococcus aureus (MRSA) infections (Klevens et al. 2007).
It was found that incidence of MRSA infections was around 32 per 100,000, which
meant they were more common than previously thought. Additionally they had a
higher incidence than invasive pneumococcal disease, invasive group A Streptococcus,
invasive meningococcal disease, and invasive H. influenzae combined (Bancroft
2007). This finding spiked MRSA-related news stories and internet inquires (Hahn
et al. 2009), making MRSA a household name and the most well-known MDR bac-
teria. MRSA is mainly associated with hospital-­acquired infections, but community-
acquired infections were found to be increasingly common as well, which augmented
the perceived threat of these bacteria by the public.
Beyond MRSA, there are other important bacterial pathogens that are becoming
increasingly multidrug-resistant. Various strains of Salmonella have been identified as
multidrug-resistant (Han et al. 2011; Rowe et al. 1997; Lu et al. 2014; Egorova et al.
2008). E. coli, including those of the O157:H7 serotype, have also been found to pos-
sess multidrug resistances which is known to cause serious illness and death (Tadesse
et al. 2012; Fitzgerald et al. 2003). Campylobacter, while not causing severe infections,
is one of the leading causes of bacterial foodborne illness and is known to possess a
multidrug efflux pump that contributes to multidrug resistance (Lin et al. 2002; 2003).
Listeria monocytogenes is another major foodborne pathogen, which can produce
severe illness and disease, and has been shown to have multidrug resistant strains
(Hadorn et al. 1993; Poyart-Salmeron et al. 1990). These pathogens are of particular
concern because they are some of the most commonly acquired foodborne illness
pathogens and can be found at all levels of food production, from the farm to fork. The
resistance in these pathogens is believed to be due, in part, to the improper use of anti-
biotics in livestock production (Singer et al. 2003). In Denmark, a correlation was
found between agricultural antibiotic usage and antibiotic resistance prevalence in
Enterococcus faecium and E. faecalis (Aarestrup et al. 2001). Because of findings like
these, there has been increasing call for new regulations to limit or eliminate the use of
antibiotics in food-producing animals in order to help control the spread of antibiotic
resistance (Paphitou 2013; Fishman 2006; Marshall and Levy 2011). Antibiotic use on
the farm has been under scrutiny, as well as the over prescription of antibiotics by phy-
sicians (Aarestrup et al. 2001; Singer et al. 2003; Fishman 2006).

2 Resistance Mechanisms

2.1 Intrinsic

Intrinsic resistance mechanisms are those that originate within the bacterium
itself. These arise from genetic mutation or are inherent to the bacteria, such as an
enzymatic alteration of the target or antimicrobial, change in permeability to the
antimicrobial, active transport of the antibiotic out of the cell.
186 S. Pendleton and P.M. Davidson

As mentioned previously, beta-lactamases were the first antibiotic resistance

mechanisms identified. Beta-lactamases, and other enzymatic modifiers, attack the
antimicrobial directly, altering their structure in such a way that they lose their
activity. Beta-lactamases break the beta-lactam ring, which causes them to be unable
to bind transpeptidase and therefore unable to inhibit cell wall synthesis.
Aminoglycosides are also subject to modification by acetyltransferases, nucleotid-
yltransferases, and phosphotransferases. These enzymes add different groups to the
antibiotics, which hinder its ability to bind the target site correctly (Jana and Deb
2006). While generally rare, macrolides can also be enzymatically modified by
esterases that break the lactone ring or phosphorylated by macrolide 2′-phos-
photransferase I (Noguchi et al. 2000; Nakajima 1999).
Intrinsic resistance can also be found for compounds used as antimicrobial food
preservatives and sanitizers. For example, some Penicillium species are capable of
degrading the antifungal food preservative sorbic acid to 1,3 pentadiene (Davidson and
Harrison 2002). Additionally there are several bacteria, including Streptococcus ther-
mophilus, Lactobacillus plantarum, and Bacillus sp., that produce the enzyme nisinase
which can neutralize the antimicrobial activity of the bacteriocin nisin, a regulatory-
approved food preservative (Davidson and Harrison 2002). The bisphenol biocide tri-
closan used in some types of food contact surfaces, was recently found to degraded by
two soil bacteria Pseudomonas putida and Achromobacter xylosoxidansdenitrificans,
though the process of degradation has not been identified (Welsch and Gillock 2011).
Catalase can offer increased resistance to hydrogen peroxide by breaking it down to
oxygen and water. P. aeruginosa biofilms produce catalase that offers them increased
protection to hydrogen-peroxide disinfection (Hassett et al. 1999; Stewart et al. 2000).
Another option for bacteria is to modify the target of the antimicrobial. This is a
fairly common trend for avoiding antibiotic attack. As many of the antibiotics dis-
cussed here focus on a single target, modification of that site can render them useless.
For aminoglycosides, methylation of the 16S rRNA at specific sites can alter the struc-
ture of the binding site enough to not allow the drug finding its target (Jana and Deb
2006; Wright 2011; Thompson et al. 1985). Beta-lactams can also be subject to target
site modification. Changes in the structure of penicillin binding proteins, such as trans-
peptidases, can cause a decrease in affinity for beta-lactam binding and thus increase
the bacterium’s resistance to the antibiotic (Hartman and Tomasz 1984; Cardenas and
Palzkill 2014). Glycopeptides are most commonly inhibited by the alteration of the
acyl-D-ala-D-ala sequence (Binda et al. 2014; Pootoolal et al. 2002). A change of the
terminal alanine to either a lactate or a serine causes marked decrease in glycopeptide
affinity (Binda et al. 2014; Pootoolal et al. 2002). Macrolide resistance in
Campylobacter spp., specifically erythromycin resistance, is mediated by ribosomal
mutations at two adenine sites (Engberg et al. 2001). The erythromycin resistance
methylase gene (erm) can be found in various gram-positive and bacteria of the
Staphylococcus and Streptococcus genus, as well as some gram-negative bacteria
(Nakajima 1999; Syrogiannopoulos et al. 2001). For quinolones, the most common
form of resistance is alteration of the target site via chromosomal mutation of the DNA
gyrase or topoisomerase IV gene (Redgrave et al. 2014; Jacoby 2005). The mutations
at Thr-86, Asp-90, and Ala-70 in gyrA of Campylobacter, Ser-83 or Asp-87 in gyrA or
9 Microbial Resistance to Antimicrobials 187

Ser-80 or Glu-84 in parC E. coli, or Ser-84 or Ser-85 of in gyrA of S. aureus all confer
fluoroquinolone resistance (Everett et al. 1996; Engberg et al. 2001; Sreedharan et al.
1990). Sulfonamides are also subject to target-site mutation associated resistance.
Changes in the folP gene in E. coli, Campylobacter, Staphylococcus, and Streptococcus
have led to decreased affinity for sulfonamides, while still maintaining sufficient affin-
ity for para-­aminobenzoic acid, thus conferring resistance to sulfonamides (Sköld
2000; Huovinen et al. 1995). While not a traditional modification of the antibiotic
binding site, some tetracycline resistance is mediated by the production of ribosomal
protection proteins, which bind the ribosome and alter conformation of the tetracy-
cline binding site (Chopra and Roberts 2001; Connell et al. 2003; Li et al. 2013).
The majority of antibiotics require entry into the cell in order to act on their targets,
which means the composition of the cell wall or outer membrane can also offer resis-
tance to antimicrobials. The presence of lipopolysaccharide (LPS) increase the nega-
tive charge of gram-negative outer cell membranes thus conferring an increased
resistance to hydrophobic antimicrobials (Delcour 2009). As mentioned earlier, hydro-
phobic quinolones are impeded by the presence of lipopolysaccharide. This was noted
by the improved quinolone activity on LPS deficient mutants of E. coli and Salmonella
Typhimurium (Hirai et al. 1986). Cellular membranes also contain various proteins.
Resistance to the biocide isothiazolinone in P. aeruginosa was found to be mediated
by a missing porin protein which facilitated transfer of compounds across the mem-
brane, and thus resulted in increased resistance to the biocide (Chapman et al. 1998).
Mycobacteria have an unusual cell wall structure as compared to most other bacteria,
which give them an increased resistance to ­antibiotics. The cell wall is much thicker
than other bacterial cells, which decreases the rate of permeation by hydrophobic anti-
microbials (Jarlier and Nikaido 1994). The cell wall also has less efficient and a fewer
number of porin proteins, which limit the permeation of hydrophilic antimicrobials
(Jarlier and Nikaido 1994). While these do increase the resistance of Mycobacteria,
they do not contribute significantly to antibiotic resistance (Jarlier and Nikaido 1994).
Similar to antibiotics, resistance to phenolic-based antimicrobial food preservatives is
generally greater in gram-­negative bacteria because of the presence of the LPS
(Davidson and Harrison 2002).
Active transport of antimicrobials out of the cell can offer extensive resistance to
sanitizers, biocides and antibiotics. Efflux pumps are nearly ubiquitous among bacte-
ria and are capable of offering resistance to multiple classes of antibiotics and some
biocides (Sun et al. 2014; McMurry et al. 1998; Romanova et al. 2006; Nikaido
1998). At their core, efflux pumps are a simple mechanism for a cell to rid itself of
potentially harmful compounds from the environment, including heavy metals, anti-
biotics produced by other bacteria, and any other compound that might upset the
homeostasis of the cellular environment (Webber and Piddock 2003). Efflux pumps
range in complexity from simple membrane transporters, common to gram-positive
and some gram-negative, to complex, multi-protein units that span both inner and
outer membranes of gram-negative bacteria (Nikaido 1998). There are multiple fami-
lies of drug efflux pumps, which include the Major Facilitator Superfamily (MFS),
the Small Multidrug Resistance (SMR) family, the Resistance-­Nodulation-­Division
family, and the ATP-binding cassette (ABC) family (Nikaido 1998). The majority, all
188 S. Pendleton and P.M. Davidson

except the ABC family, use proton motive force to expel molecules from the cell
(Nikaido 1998). Every antimicrobial compound that can pass through into the inte-
rior of the cell can potentially be expelled by an efflux pump, depending on the speci-
ficity of the pump. Many of the classes of pump mentioned are multidrug efflux
pumps, meaning that they possess sufficiently low specificity so as to expel many
different types of compounds. CmeABC, a RND family multidrug efflux pump found
in Campylobacter jejuni, is able to eliminate antibiotics of multiple classes, including
aminoglycosides, beta-lactams, cephalosporins, macrolides, quinolones, and tetrac-
ylines (Lin et al. 2002). The AcrAB-TolC efflux pumps of E. coli and Salmonella
were identified as the source of resistance to beta-­lactam, naladixic acid, tetracycline
antibiotics and others for E. coli and quinolones, tetracyclines, and others for
Salmonella (Baucheron et al. 2004; Okusu et al. 1996). Pseudomonas aeruginosa is
known for high levels of resistance and contains multiple efflux pump systems,
MexAB-OprM, MexCD-OrpJ, and MexXY-OrpM, which give P. aeruginosa resis-
tance to beta-lactams, macrolides, quinolones, tetracyclines, and the biocide triclosan
(Masuda et al. 2000; Schweizer 2001). Staphylococcus is known to possess qac
genes, which code for efflux systems specific for quaternary ammonium compounds
(Russell 1997). Efflux pumps represent a dangerous source of resistance, mainly due
to non-specificity and potential for multi-resistance.
A final method for intrinsic resistance is the biofilm. These are aggregates of
bacteria that form with extracellular polymers that bind them (Bjarnsholt 2013;
Hammer and Bassler 2003; Cvitkovitch et al. 2003; González Barrios et al. 2006).
These structures house millions of bacteria and some will be on the very interior,
away from potentially harmful chemicals, heavy metals, and antibiotics. The interior
cells can survive the antimicrobial treatments even if those on the exterior are killed
due to the inability to penetrate into the biofilm matrix. As mentioned earlier, P.
aeruginosa can form biofilms, which is another reason these bacteria are so resistant
to various agents. E. coli, Listeria, Campylobacter, Streptococcus, and Salmonella
have also been found to form biofilms, which represents a major concern for the use
of sanitizers in food industry settings (Doern et al. 2009; Pan et al. 2006; Pratt and
Kolter 1998; Joshua et al. 2006; Steenackers et al. 2012).

2.2 Acquired

Acquired resistances are those provided through horizontal gene transfer. This
occurs when foreign DNA is taken into a cell and expressed by the cell. As described
above, there are three methods of horizontal gene transfer: conjugation, transforma-
tion, and transduction. Conjugation involves transfer of a plasmid between cells.
There are various types of plasmids, but one of type is the multidrug resistance
plasmid. These plasmids harbor various genes that offer resistance to different
classes of antibiotics and biocides. Conjugation is generally limited to bacteria of
the same species or genus, which can limit the spread of resistance. Transformation
involves the uptake of naked DNA by a cell. Most DNA taken up by a cell is
degraded by restriction enzymes before it can be incorporated into the genome. This
9 Microbial Resistance to Antimicrobials 189

degradation is a common defense mechanism and means that natural transformation

requires a cell to be open to the incorporation of foreign DNA, also known as “com-
petent”. Naked DNA can come in several forms. As a cell is lysed, it releases its
cellular contents including DNA, which can be taken up by transformation compe-
tent bacteria and incorporated in their DNA; or bacteria can specifically excrete
DNA fragments for uptake by other cells nearby. The non-directed way transforma-
tion occurs leads to a less likely spread of antibiotic resistance. However it could
potentially cause a wider distribution because there is no need for a bacterium of the
same species or genus as with conjugation. Finally, transduction occurs when bac-
teriophages transfer DNA between cells. This creates an even less likely scenario
for antibiotic resistance transfer in that the phage must uptake the complete resis-
tance gene present in the host, and it must then transfer it to another bacterium that
incorporates it into its DNA. While, not the most efficient method for the spread of
antimicrobial resistance, it does present an avenue to greater resistance.
Additional factors for acquired resistance are the mobile elements: plasmids, trans-
posons, and integrons. Plasmids fall into different Inc. (incompatibility) groups
depending on their replication machinery. As one type of replication machinery can
only ensure the passage of one of the plasmids to the new cell, having multiples of the
same Inc. goup causes the eventual loss of one or more of the plasmids, until only one
is present. Additionally, there are different types of plasmids, depending on their gene
content. Fertility (F) plasmids facilitate conjugation while virulence plasmids contain
genes that allow the bacterium to become virulent. Most importantly, some plasmids
carry resistance genes (resistance (R) plasmids). While many of the genes that have
been discussed previously for intrinsic resistance are chromosomally located, they can
also be found on plasmids that can be transferred via conjugation. Even if the
R-plasmid doesn’t contain the conjugation machinery, if another plasmid in the cell
does, it can facilitate the transfer of the resistance plasmid along with itself. Transposons
contain a gene or set of genes flanked by two complementary inverted repeats, as well
as a transposase that facilitates movement between both genomes and plasmids (Frost
et al. 2005). This ability to move between genome and plasmid increases the ability for
genes to be spread through a bacterial population. Integrons are similar to transposons
in their ability to excise themselves from and relocate into DNA. However they are not
flanked by inverted repeats, but rather have contain a recombinase, a recombination
site, a 59-base element, and a set of gene cassettes, which are often antibiotic resis-
tance genes (Bennett 2004). Integrons have been found in transposons, as well as
contained on plasmids, increasing their mobility (Hall and Collis 1998).

3  otential Contribution of Food-Related Antimicrobials

P
to Antibiotic Resistance

An area which may play a role in development of antibiotic resistance is the use of
food antimicrobial preservatives and sanitizers. This area has not been as carefully
studied but could potentially create bacterial strains that have cross-resistance to
antibiotics and thus contribute to the spread of antibiotic resistance. The link between
190 S. Pendleton and P.M. Davidson

tolerance to food-related antimicrobials (preservatives and sanitizers) and antibiotic

resistance has been undertaken in some studies by looking at the resistance patterns
for various isolates against both types of compounds. Cole et al. (2003) looked at
isolates from homes where antimicrobial cleaners and products were either used or
not. They determined susceptibility for the triclosan, quaternary ammonium com-
pounds, pine oil, and para-chloro-meta-xylenol, as well as between 4 and 8 genera-
relevant antibiotics. They found no link between use of antimicrobials in a home
environment and antibiotic resistance. Beier et al. (2013) evaluated disinfectant and
antibiotic susceptibility of 344 Escherichia coli O157:H7 strains from beef cattle
carcasses, feces, and hides as well as ground beef. Fourteen percent were antibiotic
resistant, 20% were resistant to sanitizers and food preservatives (organic acids) and
some biocide-resistant strains were resistant to multiple antibiotics. However, no
link was found between antibiotic resistance and biocide resistance. Lavilla Lerma
et al. (2013) found conflicting results using principle component analysis for various
isolates in a lamb slaughterhouse. Additionally, data of sanitizer and antibiotic resis-
tance within strains was not provided, instead only resistance to sanitizers or antibi-
otics for bacterial groups was identified and analyzed. This approach makes it
impossible to infer the effects sanitizers have on antibiotic resistance. Pseudomonads
and psychrotrophs were found to be the most resistant to santizers or antibiotics of
the bacteria isolated. This is not surprising in light of the multiple intrinsic resistance
mechanisms pseudomonads carry and that the cold-adapted psychrotrophs which
are more fit to survive in a cold slaughterhouse environment. Fernández-Fuentes
et al. (2012) found 36 of 378 isolates of spoilage and pathogenic bacteria with a high
minimum inhibitory concentrations for at least one of the antimicrobials tested also
were resistant to at least one of the antibiotics tested. Thus, while less than 10% of
the isolates had dual resistance and, while concerning, does not indicate wide spread
cross-resistance. Even though these studies found isolates with dual resistance, these
studies did not fully link sanitizer tolerance to antibiotic resistance. This is because
it is impossible to determine when the antibiotic resistance was acquired. To indicate
a link between antimicrobial resistance and acquisition of an antibiotic resistance, a
study should prove that exposure leads to a new antibiotic resistance not seen prior
to antimicrobial exposure.
Studies have attempted to prove that preservative and/or sanitizer tolerance can
lead to antibiotic resistance by creating a biocide tolerant mutant and evaluating
antibiotic resistance (Alonso-Hernando et al. 2009; Apolonio et al. 2014; Braoudaki
and Hilton 2004; Capita et al. 2014; Christensen et al. 2011; Condell et al. 2012;
Fadli et al. 2014; Karatzas et al. 2007; Ledder et al. 2006; Maseda et al. 2009;
Moken et al. 1997; Molina-González et al. 2014; Potenski et al. 2003; Rakic-­
Martinez et al. 2011; Russell et al. 1998; Sanchez et al. 2005; Schwaiger et al. 2014;
Sheridan et al. 2012; Thomas et al. 2000, 2005). These studies focused mainly on
Salmonella, E. coli, Pseudomonas, and Listeria and, while not all of found antimi-
crobial exposure led to increased antibiotic resistance, some did. As mentioned
above, bacteria can acquire resistance to some compounds through efflux pumps. In
a number of studies where cross-resistance was suggested, efflux pumps were con-
sidered a highly probably reason. Potenski et al. (Potenski et al. 2003) exposed
Salmonella Enteritidis to sublethal levels of chlorine (25 ppm), sodium nitrite
9 Microbial Resistance to Antimicrobials 191

(1.0%), sodium benzoate (1.0%) or acetic acid (0.05%) and that this induced
­resistance to tetracycline, chloramphenicol, nalidixic acid, and ciprofloxacin. They
concluded that co-selection or cross-resistance did occur and was attributed the
multiple antibiotic resistance (mar) operon. The increased resistance to the antibi-
otics however was below “clinical resistance”. The mar operon conveys tetracy-
cline resistance through an efflux pump (Alekshun and Levy 1997). Karatzas et al.
(2007), Rakic-Martinez et al. (2011), and Maseda et al. (2009) all found that efflux
pump related genes were up-regulated or involved in antibiotic resistance pheno-
types. The naturally-derived antimicrobial essential oils and their components are
likely to target multiple cellular structures and in a non-specific manner. Three stud-
ies focused on the developed tolerance of bacteria to various essential oils (Apolonio
et al. 2014; Fadli et al. 2014; Moken et al. 1997). Interestingly, two of the three
studies found increased antibiotic resistance after strains had been made resistant to
the essential oils. Moken et al. (1997) and Fadli et al. (2014) both found that multi-
drug efflux pumps had been activated in the resistant strains. In E. coli exposed to
pine oil, the marA gene was found to be overexpressed, which controls expression
of the multidrug efflux pump AcrAB (Moken et al. 1997). Similarly, when E. coli
was exposed to thymol and carvacrol, activation of AcrAB-TolC was identified, as
well as a decrease in membrane proteins (Fadli et al. 2014). The authors hypothe-
sized that this decrease in membrane proteins might reflect a decrease in membrane
porins, which would decrease the access points for charged molecules to enter the
cell. The up-regulation or over expression of multidrug efflux pumps, like those
mentioned earlier, as a result of biocide exposure could very well lead to the
increase in antibiotic resistance within this mechanism for resistance.
It has yet to be definitively determined that sanitizer or preservative exposure
leads to antibiotic susceptibility modifications. Most of the studies investigating
whether preservatives or sanitizers cause antibiotic resistance, first use a step-wise
increase of sub-lethal exposure the preservative or sanitizer. If a tolerant or resistant
bacteria is created, they are then tested for potential antibiotic resistance.
Unfortunately, this brings into question the relationship of the studies a “real world”
situation. There are few, if any scenarios in the food industry where a bacterium
would be exposed to slowly increasing amounts of sub-lethal preservatives or sani-
tizers. Potenski et al. (2003) and Karatzas et al. (2007) were the only studies that
examined the long-term effect on antibiotic resistance. Both stated that each resis-
tant isolate maintained its biocide resistance, however no data was presented. It is,
therefore, difficult to discern if the antibiotic resistance is permanent or transient. If
simply transient stress adaptions, then removal of the biocide stressor should return
the bacteria to an antibiotic susceptible state.

4 Conclusions

While it is difficult to draw conclusions based on a few studies to indicate a major role
in development of antibiotic resistance for preservatives and sanitizers. However, it is
probable that exposure to antimicrobials at sub-lethal levels can induce expression of
192 S. Pendleton and P.M. Davidson

multi-drug resistance efflux pumps and thus lead to an increase in antibiotic resistance
temporarily. This scenario of antibiotic resistance acquisition should be rare in the
food industry. Only in a situation where a cells are exposed to sub-lethal concentra-
tions of food-related antimicrobials will development of antibiotic resistance be pos-
sible. Thus use of appropriate sanitizers or preservatives at appropriate concentrations
and/or combinations of preservatives, preferably compounds with different cellular
mechanisms, are the most obvious solution to combatting resistance development.
One problem area might be biofilms. If bacteria are allowed to form biofilms,
they will reduce the efficacy of the biocides being used simply by preventing the
biocides from reaching all the bacteria. In order to prevent this potential, proper
cleaning and use of anti-biofilm sanitizers should be a standard for areas that encour-
age biofilm formation (Costerton et al. 1995). Some potential novel solutions for
controlling biofilms might be use of inhibitors for quorum sensing compounds that
regulate biofilm formation. Another class of compounds to integrate into biocide
cleaning might be efflux pump inhibitors. As has been seen in the research that
investigated a link to efflux pump activity, many of biocides induce bacterial efflux
pumps. If these pumps can be inhibited or inactivated, bacteria cannot excrete the
biocides from the cells, which would increase the killing potential and decrease the
likelihood of antibiotic resistance spread.
Finally, the development of new food-related antimicrobials should also include
testing for their potential to induce antibiotic resistance. If their use can induce anti-
biotic resistance, then they should not be allowed out on the market. This one provi-
sion would essentially eliminate the risk for induced antibiotic resistance.

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Chapter 10
Interventions for Fresh Produce

Govindaraj Dev Kumar, Sadhana Ravishankar, and Vijay K. Juneja

Abstract Environmental matrices such as soil, water, and dust harbor microorgan-
isms. Many of the microorganisms found in the environment are essential for biogeo-
chemical cycles and are required for plant growth. The microbiome of the produce
production environment might also contain foodborne pathogens and spoilage causing
organisms that can be detrimental to the quality of produce and the health of the con-
sumer. While management practices and regulations are used to minimize produce
contamination by harmful microorganisms, these are seldom foolproof. Hence, post-
harvest washing and sanitizer treatment of produce are often used during hydrocooling,
comminution, or before wax application and packaging. These steps can help extend
the shelf-life and improve the microbiological safety of produce, which is essential for
commerce. Ensuring that the customer receives a safe product becomes imperative
since produce is often shipped long distances from packing houses to retail facilities.
This chapter provides information about sanitizers commonly used by produce
growers and the packaging industry. The modes of action, salient properties, and limita-
tions of some of the current sanitizers have been described. Alternative technologies
that have the potential to replace current sanitation and washing protocols have been
discussed in detail. Current trends in the industry lean towards the use of “clean labels”
while the demand for improved quality is always on the rise. Understanding the risks,
benefits and legislative boundaries of a sanitizer is imperative before implementation
into commercial practice. The optimal use of a sanitizer often becomes a confluence of
chemistry, microbiology, plant physiology, and consumer preference.

G. Dev Kumar
Department of Plant Science & Landscape Architecture, University of Maryland,
College of Agriculture and Natural Resources, College Park, MD, USA
e-mail: [email protected]
S. Ravishankar (*)
School of Animal & Comparative Biomedical Sciences, University of Arizona,
Tucson, AZ, USA
e-mail: [email protected]
V.K. Juneja
Residue Chemistry and Predictive Microbiology Research Unit, Eastern Regional Research
Center, USDA-Agricultural Research Service, Wyndmoor, PA, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 199

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_10
200 G. Dev Kumar et al.

Keywords Fresh produce • Interventions • Contamination • Antimicrobials •

Foodborne pathogens

1 Introduction

Food crops grown in fields are subject to contact with vehicles of pathogen contami-
nation. These could include soil, water, dust, rainfall, manure, biosolids, improperly
composted material, agricultural runoffs, animal intrusions, fertilizer and human
handling/processing. Increased consumption of ready-to-eat, fresh, and/or mini-
mally processed fruits and vegetables has been accompanied by an increase in out-
breaks of foodborne illnesses associated with these commodities (Fig. 10.1).
Contaminated produce could contribute to large scale cross contamination during
washing, comminuting and packaging. Foodborne pathogens are robust and can
survive in the environment for long durations of time. The possibility of survival of
foodborne pathogens on edible plant tissue during the journey from farm to fork
exists making interventions an important measure in ensuring produce safety.
Several studies have indicated that factors such as the physiological state of the
cell, adaptation mechanisms in bacteria (Fig. 10.2), wash water, the type of produce,
method of application of the intervention, and even the interval between inoculation
and application of the interventions could affect the effectiveness of the interven-
tions. In commercial settings, the reuse of water to wash multiple batches of pro-
duce is unavoidable because of environmental and economic implications. The
effectiveness of the sanitizer in a commercial setting, residual antimicrobial effect,
presence of harmful chemical residue, effect on organoleptic properties of produce,

Sources of Contamination
Manure, Composts
Sewage, Untreated Water, Reclaimed Water
Human Handling
Plant Tissue Damage
Dust
Humidity
Rainfall
Flooding
Animal Intrusion, Bird Intrusion
Parasites Hosting Foodborne Pathogens
Insects

Fig. 10.1 Sources of foodborne pathogen contamination

10 Interventions for Fresh Produce 201

Sanitizer Resistance
Strategies

Biofilms

Capsule

Efflux Pumps

Enzyme Mediated Resistance

hip (High Persister) mutants

Change in membrane fluidity

RpoH and RpoS modulation

Fig. 10.2 Strategies used by bacterial cells to counter sanitizer challenge

specificity against pathogenic bacteria and cost are all important considerations for
sanitizer selection.
Equipment design, construction and material, sprays vs. dips, foaming, pitting and
corrosion are also considerations that processors should make while selecting sanitizers.
Certain equipment such as ozone generators, UV lamps and acidified sodium chlorite
electrolysis units could have a high initial cost. Monitoring the dose and titer value of
sanitizers in the washing medium is another important consideration. Choosing the right
test method for the determination of the sanitizer concentration is essential, as the pres-
ence of impurities and organic matter in water could result in false positive results or
wrong measures.
Combination treatments of sanitizers could bridge gaps in antimicrobial effec-
tiveness that could occur during the use of a single antimicrobial agent for sanita-
tion. Currently, the applications of organic acids or oxidizing agents are common in
the produce industry. Recent foodborne outbreaks related to fresh produce suggest
a critical need for better intervention strategies to prevent outbreaks and improve the
microbiological safety of fresh produce.

2 Chlorination

History: Bleaching powder was used by the paper and textile industries of Europe
during the nineteenth century. The first use of chlorine as a sanitizer occurred in
1897, when the British scientist Sims Woodhead used bleach solution in the water
mains during a Typhoid outbreak (Pools 2007). The use of chlorine to treat water
started in 1908 in the United States (Pools 2007). Chlorination of water does not
require expensive equipment and is popular because of its low price and the ability
of the sanitizer to oxidize the outer membranes and cellular components of bacteria
202 G. Dev Kumar et al.

(Haas and Engelbrecht 1980). Chlorine is a commonly used sanitizer to wash pro-
duce. In commercial applications, after harvest, chlorinated water is used in dump
tanks, flume washes and sprays to wash many types of fresh fruit and produce
(Beuchat and Ryu 1997).
The antimicrobial efficacy of chlorine as a sanitizer depends on the availability
of free chlorine (hypochlorous acid). Efficacy of chlorine is also affected by the pH
of the environment and the organic load in the water (Wang and Ryser 2014). As a
sanitizer, chlorine is most efficacious at a neutral pH, where in an aqueous solution
it is mostly present as hypochlorous acid (HOCl). It is converted to less efficacious
forms, such as hypochlorite or gaseous chlorine, at alkaline and acidic conditions,
respectively (Luo et al. 2012). Section 173.315 of Title 21 of the Food Code by the
United States Food and Drug Administration specifies that a maximum of 0.2%
(2000 ppm) hypochlorite can be used in wash water, though this concentration is
typically used for the chemical peeling of fruits and not for washing produce
(Beuchat and Brackett 1991). In commercial settings, 50–200 ppm of chlorine is
used to wash produce (Beuchat and Brackett 1991) in dump tanks, flume washes
and other dip or spray systems. The suggested contact time of produce with chlori-
nated water is 2 min. The Good Agricultural Practices (GAPs) recommendation for
free chlorine content in tomato dump tanks by the Florida Department of Agriculture
is 150 ppm or an Oxidation Reduction Potential (ORP) of 650 mV. In the United
States and France, produce washed with chlorine has to be rinsed with water to
remove residual chlorine (Varoquaux and Mazollier 2002; Baur et al. 2004).
Bacterial tolerance to chlorine could be a consequence of a biphasic process, in
which oxidation of the surface membrane components is followed by interaction of
the disinfectant with internal cellular components, such as heat shock proteins and
redox regulons (Fig. 10.2). The effectiveness of chlorine as a sanitizer could be
affected when interactions with extrinsic barriers, such as capsule or biofilms,
diminishes the efficacy of the sanitizer by reducing the concentration of chlorine
that can react with inner membrane proteins of the cell and can cause cell damage
(Fig. 10.2). Sub-lethal concentrations of oxidizing agents could also result in intrin-
sic repair mechanisms being induced in bacterial cells which could cause chlorine
resistance in target microorganisms.

3 Hydrogen Peroxide

Hydrogen peroxide is an antimicrobial compound that is produced by catalase posi-

tive bacteria, certain anaerobic bacteria and by macrophages during the oxidative
burst to destroy bacterial cells (Juven and Pierson 1996). The antibacterial activity
of H2O2 depends on the extracellular makeup of the cell and could be attributed to
the oxidative activity against proteins, lipids and nucleic acids caused by the inter-
mediate products during breakdown (Juven and Pierson 1996). Hydrogen peroxide
might be more effective against bacterial pathogens when heated (Ukuku et al.
2004). The sanitizer is popular in the produce industry for washing because of its
10 Interventions for Fresh Produce 203

Generally Recognized As Safe (GRAS) status and its ability to break down into
hydrogen and oxygen without residual toxicity. The treatment of alfalfa seeds with
a combination of dry heat and hydrogen peroxide (2% for 10 min) reduced the
population of Salmonella up to 3.6 log CFU/g. The treatment also improved germi-
nation by 97% in comparison to untreated seeds which germinated at 79.5% (Hong
and Kang 2016). Hydrogen peroxide (0.5%) used for washing strawberries resulted
in discoloration of the fruit (Lukasik et al. 2003). The use of hydrogen peroxide at
3% concentration to wash organic leafy greens, such as spinach and lettuce, resulted
in 1 log CFU/g reduction of S. Newport populations (Moore et al. 2011). The effi-
cacy of H2O2 might be diminished due to the presence of organic matter and a con-
centration of 1% might not demonstrate antimicrobial activity (Huang and Chen
2011). In a study by Beuchat (1997) on alfalfa seeds, it was shown that hydrogen
peroxide inactivated Salmonella serotypes, with up to a 1000-fold reduction with
6% hydrogen peroxide.

4 Organic Acids

Organic acids are often byproducts of fermentation that have been used as food
additives. Organic acids can be categorized as straight chain monocarboxylic acids
and their derivatives (Ricke 2003). Organic acids have bacteriostatic and bacteri-
cidal properties as their un-dissociated forms penetrate the lipid membrane of bacte-
rial cells. The change in pH could result in the depletion of adenosine triphosphate
(ATP) in the cells due to the export of protons to maintain neutral pH in the cell.
Based on the physiology, metabolic status of the bacterial cell and the concentration
of organic acid present, different levels of inactivation can be expected (Ricke
2003). The use of lactic acid dips (1%) to wash cut iceberg lettuce resulted in a 1.5
log CFU/g reduction of Listeria monocytogenes (Akbas and Ölmez 2007). Lactic
acid (2% concentration) was considered an effective treatment for reducing E. coli
O157:H7 on spinach leaves (Neal et al. 2012). Hydrogen peroxide in combination
with lactic acid was used to treat lettuce leaves contaminated by E. coli, Salmonella
Enteritidis and L. monocytogenes (Lin et al. 2002). The sanitizer combinations were
heated to 40 °C, as studies have indicated higher efficacy with increase in tempera-
ture (Ukuku et al. 2004). The treatments resulted in a 4 log CFU/g decrease in the
populations of Gram negatives and a 3 log CFU/g decrease in the population of the
Gram positive pathogen, L. monocytogenes (Lin et al. 2002).

5 Electrolyzed Water

Electrolyzed oxidizing water (EO water) was developed in Japan through the elec-
trolysis of water in a device where the anode and the cathode are separated by a dia-
phragm. It has the efficacy of a sanitizer because of the availability of chlorine, low
204 G. Dev Kumar et al.

pH (2.7) of the water, and an oxidation reduction potential of greater than

1100 mV. These characteristics of electrolyzed water are attained by conducting elec-
trolysis of deionized water containing sodium chloride at very low concentrations
(0.1%) and collecting the water from the anode. Electrolyzed water has free chlorine
at a concentration of 10–80 ppm. Chlorine in electrolyzed water can exist in different
forms such as hypochlorous acid. The advantages of electrolyzed water include ease
in handling and better safety than traditional chlorine based sanitizers (Kim et al.
2003). Different types of electrolyzed waters have been tested against foodborne
pathogens (Guentzel et al. 2008).

6 Ozone

Ozone (O3) is a potent oxidant, formed by introducing air or pure oxygen (O2)
through a high energy input. Commercially, ozone can be generated using different
types of energies that include photochemical (i.e. ultraviolet radiation), electric dis-
charge (i.e. corona discharge) chemical, thermal, chemo nuclear, and electrolytic
methods (Emer et al. 2008; Karaca and Velioglu 2007; Novak et al. 2008). Ozone is
also a powerful oxidizing agent, recommended to reduce produce decay and extend
storage shelf-life (Skog and Chu 2001).
Ozone can be applied in gaseous form or as ozonated water for sanitizing
(Pascual et al. 2007; Perry and Yousef 2011). Ozone can be spontaneously decom-
posed into a nontoxic product, oxygen (Vurma et al. 2009), leaving no disinfectant
residues (Karaca and Velioglu 2007). Ozone was approved as a disinfectant or sani-
tizer in food processing by the FDA (Emer et al. 2008; Karaca and Velioglu 2007).
Ozone is a strong oxidant and can react with organic matter. Ozone has been typi-
cally used to treat waste water and for drinking water treatment. When passed
through wash water or water used for chilling foods and produce, it can result in
disinfection, antimicrobial activity, reduce organic matter and improve the color and
odor of the liquid (Kumar et al. 2016). Treating food surfaces with ozone can be
achieved either by adding gaseous ozone continuously or intermittently to the stor-
age atmosphere throughout the storage period or by washing or dipping in ozonated
water to prevent the spread of cross-contamination and inactivate the microbial cells
(Emer et al. 2008; Hassenberg et al. 2008; Huang et al. 2008; Mahmoud et al. 2004;
Novak et al. 2008; Park et al. 2004). Gaseous ozone concentration of 0.1 mg/L for
6 h was found to be appropriate to inactivate E. coli in whole and ground black pep-
pers without alteration of the organoleptic properties (Emer et al. 2008). The effi-
cacy of ozone in water depends on parameters such as the presence of organic matter
in the water, the bubble size for ozone dispersion, temperature of the water and
agitation. Waters at lower temperatures tend to have higher concentrations of detect-
able ozone than waters at room or higher temperature (Kumar et al. 2016). Ozone
gas can also be directly used to disinfect and reduce pathogen population from
produce. Spinach leaves treated with gaseous ozone in packaging resulted in 3–5
log reduction in E. coli O157:H7 per leaf. The treatment resulted in discoloration of
10 Interventions for Fresh Produce 205

leaf resulting in a product that was not as desirable as air vs. oxygen (before contact
with electrode for ozone generation) treated leaves (Klockow and Keener 2009).
Several studies have indicated that the use of aqueous ozone to wash produce can
result in decrease of total plate counts and coliform counts (Garcia et al. 2003).
Treatment of produce with aqueous ozone might result in sensory changes and
browning of the product depending on the type of produce being washed and stor-
age post-washing (Klockow and Keener 2009).

7 Peroxyacetic Acid

Peroxyacetic acid or peracetic acid (PAA) has been approved by the FDA for the
disinfection of fruits and vegetables. PAA is commonly used to disinfect food con-
tact surfaces because of its strong oxidizing capabilities. PAA is a popular oxidizing
sanitizer that is used in the liquid, gas and aerosolized form. PAA has been found to
be more efficacious that hydrogen peroxide and has been used for the sanitation of
surfaces that cannot be treated with heat. It belongs to a class of organic acid perox-
ides and hence, combines the action of hydrogen peroxide and acetic acid. PAA is a
colorless liquid with the smell of acetic acid. The pH of PAA is around 2 or less and
its efficacy is not affected by organic matter, unlike chlorine-based oxidizers (Baert
et al. 2009). Another significant feature of PAA is that it is more stable than hydro-
gen peroxide during storage. PAA is more efficacious against bacterial cells in com-
parison to bacterial spores and protozoan cysts. Some antiviral activity by PAA has
also been observed (Baert et al. 2009). PAA is used commercially for waste water
treatment, cooling towers, hospitals, and food and beverage industries. Its efficacy
as a sanitizer makes it a candidate of choice for the produce industry for applica-
tions such as contact surface, equipment decontamination and also for washing pro-
duce. When the efficacy of PAA was tested at different concentrations on different
types of produce, it was observed that antimicrobial efficacy depended on the con-
centration and type of produce being treated. When efficacy of PAA was evaluated
for its ability to reduce microflora in carrots, cabbage, iceberg lettuce and leeks, it
was determined that the sanitizer was the most effective against the native micro-
biota of carrots and least effective against the native microbiota of leeks
(Vandekinderen et al. 2009). PAA was capable of reducing Salmonella populations
of 2 log CFU/g to undetectable levels from the surfaces of bell peppers and cucum-
bers (Yuk et al. 2006).

8 Acidified Sodium Chlorite

Acidified sodium chlorite is a combination of citric acid and sodium chlorite in an

aqueous phase. It has been recently approved by the FDA and the EPA for the sani-
tation of fruits and vegetables through sprays and dips at a concentration of
206 G. Dev Kumar et al.

500–1200 ppm. Acidified sodium chlorite (1000 ppm) was highly effective in reduc-
ing E. coli O157:H7 from shredded carrots through addition to both tap water and
processing water (Gonzalez et al. 2004). Processing water used in this study had a
higher organic load (3500 mg/L Chemical Oxygen Demand) that was detrimental to
the activity of chlorine. The addition of acidified sodium chlorite reduced the levels
of E. coli O157:H7 below detection (>5 log CFU/mL). The study indicated that
acidified sodium chlorite was not affected by the organic content of wash waters in
conditions similar to processing facilities. Acidified sodium chlorite at 500 mg/L
was effective in reducing populations of aerobic microorganisms, E. coli and coli-
forms in fresh-cut zucchini, cucumbers, green bell peppers, potatoes, sweet pota-
toes, carrots, and radishes during refrigerated storage (Sun et al. 2012). In addition,
acidified sodium chlorite also prevented browning of fresh-cut potatoes and sweet
potatoes during refrigerated storage by inhibiting the polyphenol oxidase enzyme
activity (Sun et al. 2012). Acidified sodium chlorite treatment at 100–1000 mg/L
concentrations was effective in reducing E. coli O157:H7, total plate count, and
yeast and mold populations during 14 day storage at 5 °C (Allende et al. 2008).

9 Chlorine Gas

The gaseous form of chlorine is a strong oxidizing agent with a broad antimicrobial
spectrum, and is already applied to decontaminate various fruits and vegetables (Du
et al. 2002; Gómez-López et al. 2009). Gaseous chlorine in the form of ClO2 or bet-
ter known as chlorine dioxide, has been widely used to prevent contamination of
fresh produce. Although chlorine in the form of bleach has been used extensively,
recent outbreaks linked to fresh produce have questioned the efficacy of chlorine
itself. An approach to develop sanitizers that reduce the use of water led to the use
of gaseous chlorine as a preventive sanitizer for fresh produce. (Ölmez and
Kretzschmar 2009). The sanitizing properties of gaseous chlorine dioxide were
evaluated against common foodborne pathogens, such as Salmonella, Listeria and
E. coli O157:H7, as well as yeasts and molds on foods such as fresh cut cabbage,
lettuce, apples, peaches, carrots, tomatoes and onions (Sy et al. 2005b). Though
there was no significant reduction in the yeast and mold population, substantial
reductions in populations of the pathogens on apples, tomatoes and onions were
observed but not on cabbage, carrots and lettuce. This study also indicated that there
were no marked adverse sensory effects after treatments with the gas (Sy et al.
2005b). Kim et al. (2006) conducted a study in which they investigated the sanitiz-
ing effect of chlorine dioxide among other sanitizers against Cronobacter sakazakii
on produce such as strawberries, lettuce, cantaloupes, tomatoes and apples. Among
the other sanitizers used, with effects of storage temperatures being taken into con-
sideration, chlorine dioxide turned out to be the most effective and chlorine being
an equally good sanitizer (Kim et al. 2006). Minimally processed fruits and vegeta-
bles (MPFV) have a short shelf-life due to their metabolism and the action of spoil-
age microorganisms. Chlorine dioxide is a powerful oxidizing agent that can be
10 Interventions for Fresh Produce 207

used as a decontaminant. It does not form significant amounts of chlorinated by-­

products like chlorine (Gómez-López et al. 2009). The same authors have also
investigated the efficacy of chlorine dioxide on minimally processed foods such as
lettuce, carrots, blueberries and strawberries and have not seen any adverse effects
both in visual and sensory analyses (Gómez-López et al. 2007; Sy et al. 2005a).

10 Ionizing Radiation

Ionizing radiation represents a technique for pathogen reduction (Black and

Jaczynski 2008; Medina et al. 2009) that involves accelerating electrons almost to
the speed of light and subsequently passing them through the matrix in order to
inactivate bacteria (Jaczynski and Park 2003). The electron source is electricity, no
radioisotopes are involved, and in addition high dose rates are possible, resulting in
a short time exposure. The FDA has set a limitation of 10 kGy. For compliance with
FDA standards when treating foods, the dose of radiation can be monitored by the
use of dosimeters such as cellulose triacetate.
Radiation is known as cold pasteurization indicating its ability to destroy
pathogenic microorganisms. The treatment of foods with radiation does not ren-
der them radioactive because no contact with radioactive material takes place.
Also the dosage of radiation is not high enough to cause atomic nuclei disintegra-
tion. Radiation used in foods is about 2–7 kGy. The use of radiation might be
advantageous in produce because of the ability of foodborne pathogens to inter-
nalize into produce. The surfaces of produce are irregular; hence, providing a
challenge to completely eliminate foodborne pathogens. The use of radiation with
another hurdle might further reduce the population of foodborne pathogens in
produce (Andrews et al. 1998).
The combination of UV light and low doses of gamma radiation were used to
treat cherry tomatoes. The intensity of treatments were-UVC (253.7 nm) dose of
0.6 KJ/m2 followed by four different low doses of gamma irradiations (0.1, 0.25,
0.5, 0.75 kGy), resuling in 3–5 log reductions of E. coli O157:H7 and Salmonella
per tomato (Mukhopadhyay et al. 2013). While gamma radiation is generated from
a radioactive source, electron beams are produced through the generation of elec-
tricity (Su et al. 2004). An electron beam treatment dose of 7 kGy for 1 min resulted
in a 4 log CFU/g reduction of Salmonella on tomato seeds (Trinetta et al. 2011).

11  hyto-Antimicrobials: Essential Oils, Their Active

P
Components, Plant Extracts and Edible Films

Phytoantimicrobials have been gaining popularity in recent years, since the current
consumers prefer to have natural ingredients and processes in food production as
opposed to chemical preservatives, as well as chemical treatments during food
208 G. Dev Kumar et al.

production and processing. The present day consumers are becoming more aware of
the health risks that may arise out of chemical ingredients and processes. Plant com-
pounds are strong bactericidal agents is small quantities, and have residual activity
upon storage. Also their efficacy is not affected in the presence of organic matter
and they are biodegradable and thus, do not cause harm to the environment. Many
plant compounds are very well known for their health benefits and medicinal value.
Hence, plant compounds have been the focus of research by many investigators in
the field of food processing and production.

11.1 Essential Oils and Their Active Components

Essential oils are aromatic oily liquids obtained from various parts of a plant includ-
ing leaves, bark, flowers, buds, roots, fruits and seeds (Burt 2004). Essential oils are
very widely used in flavor and fragrance markets, due to their delicate and pleasant
flavors and to some extent are also used as flavoring ingredients in some foods.
Essential oils and their active components have shown potent antimicrobial activi-
ties against a wide spectrum of microorganisms. Cinnamaldehyde, the active com-
ponent of cinnamon oil and carvacrol, the active component of oregano oil exhibited
rapid antimicrobial activity against both antibiotic-resistant and non-resistant
Campylobacter jejuni strains, at concentrations of 0.1% and higher in phosphate
buffered saline (Ravishankar et al. 2008). Carvacrol and cinnamaldehyde were
effective against antibiotic resistant and non-resistant Salmonella serotypes in phos-
phate buffered saline at 0.1% and higher concentrations, and on celery and oysters
at 1.0% (Ravishankar et al. 2010).
The antimicrobial activities of carvacrol, cinnamaldehyde and thymol were eval-
uated against B. cereus strains in carrot broth stored at 8–16 °C and lag phase pro-
longation as well as growth inhibition of the strains was observed (Valero and
Salmeron 2003). Yossa et al. (2012) studied the effects of a 1000 ppm 1 min treat-
ment of cinnamaldehyde against E. coli O157:H7 and a Salmonella cocktail on
baby spinach. E. coli O157:H7 was not detected on the spinach leaves, whereas
there was a recovery of 1.66 log CFU/g of Salmonella after 14 days (Fig. 10.3).
Essential oils and their components were found to be effective against Salmonella
(Fig. 10.3), E. coli, Listeria monocytogenes and Campylobacter jejuni (Friedman
et al. 2002). Citron essential oil was effective against Salmonella Enteritidis, E.
coli, and L. monocytogenes in fruit based salads (Belletti et al. 2008). Essential oil
from Satureja thymbra also showed antimicrobial activity against biofilms contain-
ing Staphylococcus simulans, Lactobacillus fermentum, Pseudomonas putida, S.
enterica, and L. monocytogenes (Chorianopoulos et al. 2008). Studies have shown
that many essential oils and their components such as carvacrol, cinnamaldehyde,
have antimicrobial activity against S. enterica (Fig 10.3) in in vitro and can be
delivered though edible films or by direct addition in, edible films, apple juice, and
wine marinade (Yadegarinia et al. 2006, Mild et al. 2011, Du et al. 2009a; Friedman
et al. 2002, 2004, 2007). Tea tree and clove essential oils exhibited antimicrobial
10 Interventions for Fresh Produce 209

Fig. 10.3 Real time image of S. Newport contaminated lettuce leaf treated with cinnamon oil
emulsion using a 3 min dip process. Leaf to the left was contaminated with bioluminescent
Salmonella Newport, indicated by colored portion. Leaf to the right was imaged post-treatment
with dark regions indicating S. Newport inactivation on leaf. Image taken using AMI 2000 Imager
with an EMCCD camera, Spectral Imaging, Tucson, AZ

effects against background microflora including bacteria, yeasts and molds on

romaine lettuce leaves (Ponce et al. 2011).
The antimicrobial activities of lemongrass essential oil against S. Newport
were investigated on organic leafy greens stored for 3 days at 4 and 8 °C, and
romaine and iceberg lettuces, and mature and baby spinach samples showed
between 0.6–1.5-­log, 0.5–4.3-log, 0.5–2.5-log and 0.5–2.2-log CFU/g reductions
in S. Newport by day 3, respectively (Moore-Neibel et al. 2012). Lemongrass oil
exhibited antibacterial activity against Candida albicans, E. coli, S. Typhimurium,
Serratia marcescens, Staphylococcus aureus and other potential pathogens
in vitro (Hammer et al. 1999; Friedman et al. 2002; Maizura et al. 2007; Aiemsaard
et al. 2011) and inhibited the growth of foodborne pathogens in minced meat
products and strawberry juice (Barbosa et al. 2009; Duan and Zhao 2009).
Carvacrol, cinnamaldehyde and thymol exhibited antimicrobial activity against
Bacillus cereus strains in carrot broth (Valero et al. 2003). Citral and geraniol, the
main active components present in lemongrass oil were effective against food-
borne pathogens in apple juice (Friedman et al. 2002, 2004) and inhibited biofilm
formation (Aiemsaard et al. 2011).
Oregano and carvacrol have been accepted as food additives and flavorings in the
United States (United States Food and Drug Administration 2011). The antimicro-
bial activity of oregano oil against Salmonella Newport on organic leafy greens was
investigated and reductions of up to 5 logs (no survivors detected) were obtained at
both 4 and 8 °C (Moore-Neibel et al. 2013). Gündüz et al. (2010, 2009) studied the
effect of oregano oil (75 ppm) and myrtle oil (1000 ppm) suspended in distilled
water against S. Typhimurium on iceberg lettuce and whole tomatoes and a 20 min
exposure resulted in reductions of up to about 2.0 log CFU/g. Cinnamon oil treat-
ments of organic iceberg lettuce, romaine lettuce, baby spinach and mature spinach
showed concentration, storage time and exposure time-dependant activities against
S. Newport (Todd et al. 2013). Carvacrol and cinnamaldehyde in vapor phase were
effective against Salmonella and E. coli O157:H7 on sliced and whole tomatoes
(Obaidat and Frank 2009).
210 G. Dev Kumar et al.

Essential oils have been shown to have antifungal activity and efficacy against
spoilage organisms and thus, may aid in enhancing shelf-life of fresh produce.
Cinnamon oil stunted the hyphal growth of Colletotrichum coccodes, Cladosportium
herbarum, Aspergillus niger, Botrytis cinerea, and Rhizopus stolonifer, as well as
reduced the spore production (Tzortzakis 2009). Basil essential oil was effective
against spoilage organisms, such as Aeromonas hydrophila and Pseudomonas fluo-
rescens, on lettuce (Wan et al. 1998).
In addition, it seems that it may be more difficult for bacteria to develop resis-
tance to essential oils. For example, Becerril et al. (2012) exposed E. coli, Serratia
marcescens, Morganella morganii, Proteus mirabilis, and Pseudomonas aerugi-
nosa to cinnamon oil and then re-exposed these organisms to cinnamon oil. Each
exposure was designated as one passage. No substantial differences between the
minimum inhibitory and the minimum bactericidal concentrations against all five
bacteria were observed following up to 50 passages.

11.2 Plant Extracts

Plant extracts have been evaluated for their antimicrobial activity by numerous
investigators and have been found to have bactericidal effects. The survival of
Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes in
roselle (hibiscus) calyx aqueous (RCA) or roselle leaf aqueous (RLA) extracts over
72 h at 4, 8 and 25 °C and the bactericidal effects of roselle calyx concentrate (RCC)
and roselle tea (RT) against E. coli O157:H7 on lettuce and of RCC against
Salmonella on alfalfa sprouts were investigated (Jaroni and Ravishankar 2012). No
E. coli O157:H7 and Salmonella survivors were detected in RCA or RLA at 24 h
and at all temperatures. L. monocytogenes populations were reduced by: 5 and 3
logs in RCA and RLA, respectively, at 24 h and all temperatures; by 4–6 logs at 4
and 8 °C; and to undetectable levels at 25 C, at 48 h. At 24 h, E. coli O157:H7 and
Salmonella were not detected on RCC- or RT-treated lettuce or sprouts (Jaroni and
Ravishankar 2012). Roselle showed inhibitory effects against multidrug resistant
Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and
Acinetobacter baumanii (Liu et al. 2005) and against S. aureus, Bacillus stearother-
mophilus, Micrococcus luteus, Serratia marcescens, Clostridium sporogenes, E.
coli, K. pneumoniae, Bacillus cereus, and P. fluorescence in laboratory media
(Olaleye 2007). Roselle aqueous extract was shown to have antimicrobial effects
(3–4 log reduction) against L. monocytogenes, E. coli O157:H7, S. aureus, and B.
cereus in ground beef and apple juice (Chao and Yin 2009).
The effectiveness of olive and apple powder extracts and hibiscus aqueous extract
was evaluated against S. Newport and the background microflora on organic iceberg
lettuce, romaine lettuce, baby spinach and mature spinach stored at 4 °C for 3 days
(Moore et al. 2011). The antimicrobial activities were concentration and storage
time dependant, with olive, apple and hibiscus extracts showing 2–3, 1–2 and 1 log
reductions, respectively, while hydrogen peroxide (control) showed 1 log reduction
10 Interventions for Fresh Produce 211

(Moore et al. 2011). The antimicrobial activity of hibiscus tea, grapeseed and green
tea extracts against antibiotic resistant Salmonella Newport on organic romaine and
iceberg lettuces and bunched mature and packaged baby spinaches was investigated
(Rojas et al. 2012). Compared to the water control, the concentration-dependent
reductions in Salmonella populations by hibiscus tea, grapeseed and green tea
extracts ranged between 0.7–2.3 log CFU/g, 0.4–2.0 log CFU/g, and 0.2–1.6 log
CFU/g, respectively (Rojas et al. 2012). Clove extract has been shown to have anti-
microbial activity against Salmonella Typhimurium and E. coli O157:H7 on fresh
lettuce, with reductions of 4 and 3.7 logs, respectively, upon 10 min exposure (Kim
et al. 2011). Apple extract containing apple polyphenols was shown to reduce the
biological activity of Staphylococcus enterotoxin A (Rasooly et al. 2010).
Commercial green tea leaf and rosemary powders were effective against B. cereus,
S. aureus and L. monocytogenes in laboratory media and oriental-style rice cakes
(Lee et al. 2009).

11.3 Combination Treatments

Plant essential oils have been shown to have strong bactericidal properties; however,
the amount that can be added to foods is limited due to their pungency and strong
flavors, since this could have an adverse effect on the sensory properties of foods.
Plant extracts and powders on the other hand have limited effects on the sensory
properties of foods, since they are not as pungent as essential oils. Combining plant
essential oils with plant extracts can help lower the concentrations of each needed
to achieve the same levels of microbial inactivation when each is applied individu-
ally at a higher concentration. Since the mechanism of action of one plant com-
pound may differ from another, combination treatments may help achieve better
inactivation by targeting various component of a microbial cell. Researchers have
investigated the efficacy of essential oils in combination with plant extracts or other
plant compounds. Rada et al. (2016) investigated the efficacy of cinnamon and oreg-
ano essential oils at 0.1% in combination with 3% olive extract against S. Newport
on organic romaine and iceberg lettuce and baby and mature spinach. These inves-
tigators found that these combination treatments induced reductions in Salmonella
population of up to 3.5–4 logs and 3–4.4 logs CFU/g which were better than the
reductions obtained when individual treatments were used.
The combination of clove bud oil (0.1%) and olive extract (5%) as well as indi-
vidual treatments were investigated against S. Newport on organic romaine and
iceberg lettuces and baby and mature spinaches (Schneider et al. 2015). Reductions
from individual treatments ranged from 0.40 to 2.5 log CFU/g and were less effec-
tive than the combination treatments that yielded reductions ranging from 0.87 to
3.6 log CFU/g in Salmonella population on organic leafy greens (Schneider et al.
2015). Ganesh et al. (2010) reported reductions of 2.6–3.3 log CFU/g in
S. Typhimurium populations on spinach leaves after 14 days of storage when elec-
trostatically sprayed with a combination of 3% grape seed extract and 2% malic
212 G. Dev Kumar et al.

acid. After 14 days of storage, the combination had higher reductions compared to
when the 3% grape seed extract was sprayed individually. Lv et al. (2011) demon-
strated that combinations of oregano-basil, oregano-bergamot, basil-bergamot, and
oregano-perilla essential oils were synergistic against Staphylococcus aureus;
combinations of oregano with basil or bergamot had additive effects on Bacillus
subtilis; and combinations of oregano with basil or perilla had additive effects on
E. coli and yeast. These authors stated that essential oil combinations inhibited
bacterial growth at lower concentrations than was needed when they were used
individually.

11.4 Antimicrobial Edible Films

Antimicrobial edible films represent a unique way of applying antimicrobials, such

as essential oils onto food products. Edible films are natural products that can be
made using various plant parts such as fruits (apple), vegetables (carrot), leaves
(spinach), and flowers (hibiscus) which are pureed and essential oils are added.
These along with other ingredients are homogenized and cast into thin films that are
dried and cut into any desired shape and size. Edible films can also be made from
other natural substrates such as algin, chitosan, casein or whey proteins. Edible
films can either be used as wrappings on food products or added as ingredients in
packages such as salad bags. Edible films can have several advantages such as:
reducing the microbial load of foods; preventing cross-contamination; preventing
browning in some fruits and vegetables; preventing lipid oxidation in foods; pre-
venting loss of moisture; preventing foreign odor pick up by the food product; pre-
venting loss of volatile flavors from foods (Gennadios et al. 1997); and addition of
unique flavors to foods from the essential oils in the films. Edible films and coatings
can serve as carriers for various food additives, including antimicrobials. The incor-
poration of antimicrobial compounds into edible films or coatings provides a novel
way to control foodborne pathogen contamination (Cagri et al. 2004).
The effectiveness of antimicrobial edible films has been the subject of investiga-
tion by many researchers. Edible apple- and tomato-based films containing low lev-
els of plant essential oils (cinnamon, lemongrass, and oregano) and their major
constituents (carvacrol, citral, and cinnamaldehyde) reduced E. coli O157:H7, S.
enterica, and C. jejuni in vitro (Rojas-Graü et al. 2006, 2007; Du et al. 2008a, b).
Oregano and cinnamon oil containing edible films were active against S. enterica on
agar both by direct contact with the bacteria and indirectly by vapors emanating
from the films (Du et al. 2009a, b). Apple, carrot and hibiscus-based edible films
containing three concentrations (0.5%, 1.5% and 3%) of carvacrol or ­cinnamaldehyde
were added as ingredients in salad bags containing organic leafy greens and their
antimicrobial effectiveness against Salmonella Newport during storage at 4 °C for
7 days was evaluated (Zhu et al. 2014). All three types of 3% carvacrol films reduced
the Salmonella population by 5 log CFU/g at day 0 and 1.5% carvacrol films reduced
10 Interventions for Fresh Produce 213

Salmonella by 1–4 log CFU/g at day 7, while the films with 3% cinnamaldehyde
showed 0.5–3 log reductions on different leafy greens at day 7 (Zhu et al. 2014).
Lemongrass oil (1.0–1.5%) and oregano oil (0.5%) containing apple puree-­
alginate edible coatings exhibited strong antimicrobial activity against L. innocua,
reducing the bacterial population to below the limit of detection within 7 days
(Rojas-Graü et al. 2007). Films made of partially hydrolyzed sago starch and algi-
nate mixture containing lemongrass oil inhibited E. coli O157:H7 (Maizura et al.
2007). Garlic oil containing chitosan films showed antimicrobial activity against E.
coli, S. aureus, S. typhimurium, L. monocytogenes, and B. cereus (Pranoto et al.
2005a, b). Garlic oil in alginate films at >0.2% concentrations exhibited antibacte-
rial activity against S. aureus and B. cereus (Pranoto et al. 2005b). Oregano oil at
2% in whey protein films was effective against E. coli O157:H7, S. aureus, S.
Enteritidis, L. monocytogenes, and Lactobacillus plantarum (Seydim and Sarikus
2006). Films with a 90/10 blend ratio of chitosan and polyethylene oxide exhibited
strong antimicrobial properties (Zivanovic et al. 2007).
Cinnamon, clove, and lemongrass oils or their active compounds cinnamalde-
hyde, eugenol, and citral incorporated into alginate films reduced E. coli O157:H7
population by 4 logs on fresh-cut Fuji apples (Raybaudi-Massilia et al. 2008a).
Malic acid and essential oils as well as their main active compounds incorporated
into an alginate-based edible coating significantly reduced S. Enteritidis population
in inoculated fresh-cut melon (Raybaudi-Massilia et al. 2008b). The use of 1.5–
2.0% chitosan in the methyl cellulose coating of fresh-cut cantaloupe reduced the
growth of mesophilic aerobes, psychrotrophs, lactic acid bacteria, and total coli-
forms by 3–4 log CFU/g (Krasaekoopt and Mabumrung 2008).

11.5 Mechanisms of Action

It is difficult to determine the mechanism of action for plant essential oils since they
are composed of multiple components that can contribute to the death of the micro-
bial cell (Skandamis and Nychas 2001; Carson et al. 2002). The phenolic com-
pounds in essential oils are known to disrupt the cell membrane causing cell death
(Sikkema et al. 1995; Rasooli et al. 20062006). Studies showed that carvacrol dis-
solves in the phospholipid bilayer of cells and interacts with the cell membrane.
This would cause expansion and destabilization of the membrane and could cause
an increase in membrane fluidity and passive permeability (Ultee et al. 2000, 2002).
Carvacrol causes leakage of phosphate ions from cells of Staphylococcus aureus
and Pseudomonas aeruginosa (Lambert et al. 2001). Adenosine triphosphate release
following disruption of E. coli cell membranes by carvacrol was confirmed with the
aid of a different physical technique (Friedman 2006). On the other hand, cinnam-
aldehyde did not disintegrate the outer cell membrane. Instead, it interfered with the
activity of some enzymes. For example, Wendakoon and Sakaguchi (1995) exam-
ined the action of cinnamaldehyde against Enterobacter aerogenes and hypothe-
sized that its carbonyl group could bind to proteins, preventing the action of amino
214 G. Dev Kumar et al.

acid decarboxylases in cells. Cinnamaldehyde has also been found to decrease the
amount of cellular ATP in L. monocytogenes and E. coli O157:H7, which could lead
to problems with glucose uptake and utilization (Gill and Holley 2004, 2006a).
Kwon et al. (2003) established that cinnamaldehyde can inhibit the separation of
Bacillus cereus cells after cell division. Eugenol acts upon a cell’s cytoplasmic
membrane through non-specific permeabilization (Gill and Holley 2006a). Eugenol
may also bind and inhibit the functions of proteins and enzymes. Studies have
shown that eugenol can inhibit ATPase, amylase, protease, and histidine decarbox-
ylase (Thoroski et al. 1989; Wendakoon and Sakaguchi 1995; Gill and Holley
2006b). Aiemsaard et al. (2011) reported that the components of lemongrass oil,
citral and geraniol inhibited biofilm formation, killed preformed biofilms and could
have multiple targets on the bacterial cell.
Plant extracts, such as olive and apple extracts, are rich in polyphenols that may
contribute to the antimicrobial activity of these compounds. In olive extract, poly-
phenols constitute 12% of the total composition, with 4-hydroxytyrosol being the
most abundant (50–70%) and having the greatest biological effect (Soni et al. 2006).
In vitro studies have demonstrated the broad range of antimicrobial properties of
hydroxytyrosol against pathogenic bacteria including Salmonella typhi, Vibrio
parahaemolyticus and Staphylococcus aureus (Bisignano et al. 1999; Friedman
et al. 2011). Apple extract contains polyphenols, which have antimicrobial effects
on human pathogens including E. coli (Alberto et al. 2006).

11.6 Organic Sanitizers

Organic sanitizers have certain advantages in comparison to chlorine: their efficacy is

not affected in the presence of organic matter and hence, they can be recycled without
loss in efficacy; they are environmentally friendly since they do not form carcinogenic
compounds; they are effective at varying temperatures; and they have shown residual
activity. The efficacy of a citric acid-based organic sanitizer (organic Chico Wash™)
against S. Newport and background microflora on fresh-cut celery and leeks was
investigated (Zhu and Ravishankar 2016). Compared to the controls washed in water
at day 6, Chico Wash reduced Salmonella and background microflora by 0.7–1.5 and
1.2–2.6 log CFU/g, respectively. All three concentrations of Chico Wash showed 0.2–
1.1 log additional reductions in Salmonella population compared to 200 ppm chlorine
(Zhu and Ravishankar 2016). The efficacy of Chico Wash against S. Newport on
organic iceberg lettuce, romaine lettuce, baby spinach and mature spinach was inves-
tigated during 3 days of storage at 4 °C (Zhu et al. 2015). Reductions of up to 2.3–2.9
logs in Salmonella population were observed at day 3, with the most reduction seen in
case of iceberg lettuce (Zhu et al. 2015). In a large scale test investigating the efficacy
of Chico Wash against E. coli K12 on organic iceberg lettuce with recycling the wash
water five times, and storing the lettuce at 4 °C for 3 days, reductions of 2.9–3.5 logs
were obtained; the efficacy of ChicoWash was maintained during the five washes and
no surviving E. coli K12 were detected in the wash water (Zhu et al. 2015). Sanitizers
10 Interventions for Fresh Produce 215

approved for organic use including formulations of chlorine dioxide, calcium hypo-
chlorite, and a mixture of peroxyacetic acid and hydrogen peroxide were evaluated
against S. Newport on organic romaine and iceberg lettuces and baby and mature
spinaches, and reductions of up to 1.6 logs in Salmonella population were obtained
(Zhu and Ravishankar 2012).

12 Bacteriophages

Bacteriophages were a popular treatment for bacterial infections in the Soviet Union
before the advent of antibiotic therapies. A bacteriophage is a virus that can lyse
bacterial cells as a consequence of its life cycle. This property of bacteriophages
along with their specificity for their hosts (bacteria) make them an attractive alterna-
tive to commonly used chemical sanitizers for the sanitation of fruits and vegeta-
bles. Factors such as the use of sprays vs. dips, pH of the fruit and the presence of
magnesium could aid in better activity of the phages. Magnesium is known to
improve phage packaging and absorption into bacterial cells (Arber et al. 1983).
Specificity of bacteriophages could also serve another advantage, as they do not
eliminate native microbiota unlike chemical sanitizers. Native microbiota could
help in competitive inhibition of pathogenic bacteria and also serve as a source of
probiotics or beneficial bacteria to the consumer. A lytic phage mixture of four
phages that were specific for S. Enteritidis was used at a concentration of 2 × 108
PFU/mL for the treatment of fruit slices. The treatment was successful in causing a
3 log CFU/g reduction on honeydew melon slices over a period of 168 h but was not
effective in reducing pathogen levels in apple slices. The acidic nature of apples
could have reduced the effectiveness of the treatment (Leverentz et al. 2001). When
multiple bacterial pathogens are targeted, bacteriophage pools targeting multiple
pathogens can be used. Lytic phage cocktails LMP-103 and LMP-102 that were
specific against Listeria monocytogenes serotypes 1/2a, 1/2b, and 4b were inocu-
lated on apple and honeydew melon tissue plugs. The phage mixture resulted in a
reduction of 2.0–4.6 log units of L. monocytogenes on honeydew melons but resulted
in a reduction of 0.4 log units on apples. Apples have a pH of 3.8–4.2. Spray appli-
cation was less effective than pipet based application in reducing pathogen, though
the difference was not statistically significant (Leverentz et al. 2003).
A bacteriophage cocktail against Salmonella serotypes, Shigella and E. coli
O157:H7 was tested on strawberries, cantaloupes and broccoli. The bacteriophage
cocktail caused significant reductions of E. coli O157:H7 on cantaloupes and broc-
coli but not on strawberries when used in water with organic load. Similar results of
significant reduction of Salmonella on cantaloupes and broccoli was observed when
a bacteriophage cocktail specific for Salmonella was employed. The use of bacterio-
phage cocktail against Shigella on produce did not result in significant reductions of
the pathogen in cantaloupes, broccoli and strawberries (Magnone et al. 2013). The
reduced effectiveness of the bacteriophage cocktails in strawberries and apples
could indicate that the pH of the produce being treated could play a role in affecting
the efficacy of bacteriophage sprays, rinses and dips.
216 G. Dev Kumar et al.

13 Conclusions

Many recent produce outbreaks have indicated the possibility of microbial contami-
nation at the field level, which may be very difficult to prevent as well as control.
Hence, post-harvest sanitization of produce becomes a very critical step in inactivat-
ing pathogens that could have contaminated them during production, in order to
prevent any outbreaks or recalls. An ideal sanitizer will be one that is effective
against a wide range of pathogenic and spoilage microorganisms, is environmen-
tally and user friendly, is derived from natural sources, has residual activity and can
enable recycling of wash water, and is economical and readily available. Research
is critically needed in this area for fresh and fresh-cut produce that are consumed
raw or with minimal processing. An effective sanitizer can help improve microbio-
logical safety of fresh produce and help gain consumer confidence updatedin safe
supply of these healthful commodities.

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Chapter 11
Preservation Methods for Meat and Poultry

Jarret D. Stopforth

Abstract The transformation of animals into meat involves several operations

including: (a) handling and loading of animals on the farm, (b) transportation of
animals to the slaughterhouse, (c) off-loading and holding of animals and (d)
slaughtering of animals. The conditions under which animals are reared and slaugh-
tered determine the level, extent and type of microbial contamination that may be
introduced to the essentially sterile underlying tissue. The most likely sources of
contamination of fresh meat include the abiotic environment in contact with the
animals (air, soil, water, feed), the animal itself (skin, intestinal tract, feces) and the
slaughterhouse environment (equipment, utensils and humans).
Consumer demands and expectations for safer, higher quality meat with longer
shelf-life has driven the development of alternative and advanced preservation tech-
nologies. The more recent preservation technologies that have been developed can
be grouped into five main categories namely: (1) low temperatures (superchilling),
(2) antimicrobials, (3) biopreservation, (4) non-thermal technologies such as high
hydrostatic pressure (HHP) and irradiation and (5) packaging technologies.
This chapter provides a review of traditional preservation methods for meat and
an introduction to the advanced and developing preservation technologies that have
been or are in development for the preservation of meat.

Keywords Meat • Poultry • Preservation • Thermal • Chilling • Freezing • Drying •

Irradiation • High hydrostatic pressure • Packaging • Antimicrobials • Biopreservation

1 Introduction

For the purpose of this chapter, meat shall be defined as the flesh of domesticated
agricultural animals including cattle, swine, sheep, goats, horses and poultry (chick-
ens, turkeys, ducks and geese). Reference to the word “fresh” meat implies meat
from a recently harvested animal that has been exposed to minimal processing other

J.D. Stopforth (*)

Chobani, LLC, New York, NY, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 225

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_11
226 J.D. Stopforth

than portioning and packaging. The transformation of animals into meat involves
several operations including: (a) handling and loading of animals on the farm, (b)
transportation of animals to the slaughterhouse, (c) off-loading and holding of ani-
mals and (d) slaughtering of animals. The conditions under which animals are
reared and slaughtered determine the level, extent and type of microbial contamina-
tion that may be introduced to the essentially sterile underlying tissue. The most
likely sources of contamination of fresh meat include the abiotic environment in
contact with the animals (air, soil, water, feed), the animal itself (skin, intestinal
tract, feces) and the slaughterhouse environment (equipment, utensils and humans).
The primary constituents of meat are fat, protein, carbohydrates and water (Heinz
and Hautzinger 2007). The diverse nutrient composition of meat makes it an ideal
environment for the growth of and propagation of meat spoilage microorganisms
and foodborne pathogens (Zhou et al. 2010). The microorganisms that usually dom-
inate the initial microbiota of fresh meat are Gram-negative rods (mainly pseudo-
monads) and micrococci (mainly Staphylococcus and Kocuria spp.). Other
microorganisms that are typically present may include Gram-negative bacteria
(such as Acinetobacter spp., Alcaligenes spp., Moraxella spp. and Enterobacteriaceae),
Gram-positive bacteria (including spore-forming bacteria, lactic acid-producing
bacteria and Brochothrix thermospacta), as well as yeasts and molds in low num-
bers (Paramithiotis et al. 2009). A comprehensive list of microorganisms (bacteria,
yeasts and molds), some of which are pathogenic, that are frequently associated
with fresh meats is provided in Table 11.1. A number of interrelated factors influ-
ence the shelf-life and keeping quality of meat, specifically holding temperature,
atmospheric oxygen (O2), endogenous enzymes, moisture, light and most-­
importantly microorganisms. All of these factors, either alone or in combination,
can result in detrimental changes in the color, odor, texture and flavor of meat and
although deterioration of meat quality can occur in the absence of microorganisms,
microbial growth is by far the most important factor affecting the quality and shelf-­
life of meat (Lambert et al. 1991).
There are three main mechanisms for fresh meat spoilage including: (a) micro-
bial spoilage, (b) lipid oxidation and (c) autolytic enzymatic spoilage (Rahman
1999a). All of the physiochemical changes that occur in fresh meat are in the aque-
ous phase and there are three primary classes of substances that are used by spoilage
microflora, namely: (1) compounds involved in the glycolytic pathway (e.g., glu-
cose, glucose-6-P), (2) metabolic products (e.g., lactate) and (3) nitrogen energy
sources (e.g., amino acids, proteins) (Paramithiotis et al. 2009). Carbohydrates and
their intermediate catabolic products are preferentially used by meat microflora as a
source of energy followed by amino acid metabolism; the resulting compounds of
this substrate utilization are numerous (Dainty 1996; Garcia-Lopez et al. 1998) and
lead to pH changes, slime formation, structural component degradation, off odors
and change in appearance of the muscle tissue (Dave and Ghaly 2011). It is there-
fore critical that adequate preservation methods and technologies be applied to
maintain the safety and quality of meat. The processes used in meat preservation are
primarily concerned with inhibiting microbial spoilage although other methods of
preservation are employed to minimize the deteriorative changes associated with
11 Preservation Methods for Meat and Poultry 227

Table 11.1 Genera of microorganisms most frequently found on fresh meat and poultry (adapted
from Jay et al. 2005)
Genus Gram reaction Fresh meats Fresh poultry
Bacteria
Acinetobacter − xx xx
Aeromonas − xx x
Alcaligenes − x x
Arcobacter − x
Bacillus + x x
Brochothrix + x x
Campylobacter − xx
Carnobacterium + x
Caseobacter + x
Citrobacter − x x
Clostridium + x x
Corynebacterium + x xx
Enterobacter − x x
Enterococcus + xx x
Erysipelothrix + x x
Escherichia − x
Flavobacterium − x x
Hafnia − x
Kocuria + x x
Kurthia + x
Lactococcus + x
Leuconostoc + x
Listeria + x xx
Microbacterium + x x
Micrococcus + xx xx
Moraxella − xx xx
Paenibacillus + x x
Pantoea − x x
Pediococcus + x
Proteus − x x
Pseudomonas − xx xx
Psychrobacter − xx x
Salmonella − x x
Serratia − x x
Shewanella − x
Staphylococcus + x x
Vagococcus + xx
Weissella + x
Yersinia − x
Molds
(continued)
228 J.D. Stopforth

Table 11.1 (continued)

Genus Gram reaction Fresh meats Fresh poultry
Alternaria x x
Aspergillus x x
Aureobasidium x
Cladosporium xx x
Eurotium x
Fusarium x
Geotrichum xx x
Monascus x
Monilia x
Mucor xx x
Neurospora x
Penicillium x x
Rhizopus xx x
Sporotrichum xx
Thamnidium xx
Yeasts
Candida xx xx
Cryptococcus x x
Debaryomyces x xx
Hansenula x
Pichia x x
Rhodotorula x xx
Saccharomyces x
Torulopsis xx x
Trichosporon x x
Yarrowia xx
x known to occur, xx most frequently reported

lipid oxidation and autolytic enzymatic spoilage. The preservation methods and
technologies presented in this chapter will focus on those implemented to control
microbial spoilage of meat.
Growth and development of microorganisms on fresh meat is governed by
(Mossel et al. 1995):
(a) Intrinsic parameters of the meat including pH, water activity (aw) and redox
potential (Eh),
(b) Extrinsic factors of the environment including temperature, relative humidity
and composition of the gas atmosphere,
(c) Process-related factors (antimicrobial compounds, heat treatments, etc.),
(d) Implicit factors (those relating to the microflora present) including antagonism
and synergism and
(e) Interactive effects of the above-mentioned factors other than that expected by
their individual effect.
11 Preservation Methods for Meat and Poultry 229

Traditional methods of meat preservation rely on three primary strategies includ-

ing the use of high temperatures (heat treatments), low temperatures (chilling and/
or freezing) and drying (and further processing in addition to drying such as curing,
smoking and fermentation). Each of these control strategies applied individually
may be considered a “hurdle” while combinations of these processes, termed hurdle
technology (HT), provide additive or synergistic effects in controlling microbial
growth on meat (Leistner 2000). The application of some of these traditional strate-
gies, especially the use of high temperatures and drying transforms fresh meat into
a semi- or fully-processed state. However, there is an increasing demand by con-
sumers for high quality, safe and extended shelf-life of meat products, which cannot
solely be achieved by traditional preservation methods. Consumer demands and
expectations for safer, higher quality meat with longer shelf-life has driven the
development of alternative and advanced preservation technologies. The more
recent preservation technologies that have been developed can be grouped into five
main categories namely: (1) low temperatures (superchilling), (2) antimicrobials,
(3) biopreservation, (4) non-thermal technologies such as high hydrostatic pressure
(HHP) and irradiation and (5) packaging technologies (Zhou et al. 2010).
This chapter provides a review of traditional preservation methods for meat and
an introduction to the advanced and developing preservation technologies that have
been or are in development for the preservation of meat.

2 Traditional Preservation Methods

Historically, the most common methods for preserving meat included cooking,
chilling and freezing, salting, drying, smoking, curing, fermenting and marinating.
These preservation methods are broadly categorized into the following control
strategies:
(a) Thermal treatments—application of high temperatures to inactivate
microorganisms,
(b) Chilling and freezing—application of low temperatures to slow down or stop
microbial growth, and
(c) Drying—reduction in the aw of meat accomplished solely or in combination
with other preservation methods such as salting, curing, smoking and
fermenting

2.1 Thermal Treatments

Heating is one of the oldest methods for preserving meat and most likely first
adopted by early man as a crude culinary technique for enhancing the general palat-
ability and flavor of meat (Kerry 2011). Heating is also probably the cheapest and
230 J.D. Stopforth

most efficient preservation method aimed at destroying pathogens and spoilage

microorganisms and inactivating enzymes that may result in the spoilage reactions
within the meat. Regardless of the type of meat, the effects of heating manifest as
follows:
• Physical alteration of the meat texture through solubilization of collagen proteins
into gelatin.
• Physical alteration of the meat product shape and homogeneity through the gela-
tion of salt soluble meat proteins especially in further processed meat relying on
binding properties.
• Generation of unique cooked meat flavors and appearance.
• Increased shelf-life stability through inactivation of meat enzymes and other
reactive substances.
• Increased shelf-life and enhanced safety of the meat through destruction of meat
microflora; ultimate shelf-life and safety is determined by the time-temperature
function of the heat treatment and post-treatment handling.
Thermal processing alters the sensory and physical characteristics of the meat
and different time-temperature treatments provide differing degrees for elimination
of microorganisms and enzymes; therefore, a compromise must be reached between
food safety and quality and the desirable sensory and physical properties of the
resulting meat product.
When a thermal treatment is applied for the first time, it is calculated taking as
basis the destruction of the most harmful or most abundant microorganism present
or the enzyme(s) that need to be inactivated to prevent spoilage for a determined
period of time (Guerrero-Legarreta and Garcia-Barrientos 2012). The thermal
destruction of microorganisms is, as mentioned, a time-temperature dependent pro-
cess where vegetative cells are destroyed at temperatures higher than their maxi-
mum growth temperature whereas spores can survive much higher temperatures.
Thermal treatments such as retorting (canning) deliver processes sufficient to elimi-
nate virtually all microorganisms and their spores, especially the important patho-
genic Clostridium botulinum, to produce a thermostable food of long shelf-life
without the need for applying any other preservation method (Lawson et al. 1994).
Depending on the desired shelf-life and sensory qualities, meat may be subjected to
a varying set of thermal processing conditions which is determined by calculating a
thermal process. In order to establish a thermal process, the calculation requires two
types of basic information: (1) heat resistance of a given microorganism used as a
process indicator (for example: C. botulinum or C. perfringens) and (2) previous
handling of the meat product prior to heating (Guerrero-Legarreta and Garcia-­
Barrientos 2012). Griffis and Osaili (2009) provide a detailed description for calcu-
lating a thermal process based on the concept of decimal reduction time (D-value)
and thermal resistance constant (z-value).
Process calculations also take into account the amount of heat that is transferred
which allows calculation of the transport parameters in the system. Since heating is
a dynamic process, heat flow is proportional to the driving force and inverse to the
11 Preservation Methods for Meat and Poultry 231

resistance to flow (Guerrero-Legarreta and Garcia-Barrientos 2012). The heat trans-

fer mechanisms may be classified as follows (Kerry 2011):
(a) Conduction—this form of heating occurs when the heating source and meat are
in direct physical contact with one another. Example(s): Heated belt-direct con-
tact cooking system (belt grill),
(b) Convection—this form of heating involves the movement of a heated fluid (i.e.,
gas or liquid) from the primary heating source (heating elements, coils, etc.) to
the meat. Example(s): Free air convection, forced air convection, impingement,
water immersion systems, “sous vide” cooking and oil frying, and
(c) Radiation—this form of heating involves the transfer of electromagnetic and
radio waves from the source to the meat, however, in contrast to conduction and
convection, heat is first generated inside the meat. Example(s): Infrared, micro-
wave, radio-frequency and ohmic heating.
As previously discussed, the application of a given thermal treatment intensity
depends on the desired shelf-life and use of the meat; it can be as mild as a surface
application to render vegetative microorganisms and enzymes inactive to commer-
cial sterilization where virtually all microorganisms and spores are destroyed. Mild
heat treatments require additional preservation technologies (such as drying or
chilling/freezing) to prevent proliferation of microorganisms while retorting (can-
ning) renders the meat shelf-stable for long periods of time (Wang 2006). Although
heat transfer principles are the same for all thermal technologies, the treatments are
applied for different purposes. The varying severity of thermal treatments can be
divided into the following categories (Hanson 1990):
(a) Scalding—this treatment is usually an incomplete heating (temperatures around
65 °C) for the purpose of inactivating enzymes to prevent further changes in
color, flavor or nutritional value and is done as a pre-treatment before the meat
is chilled/frozen, dried, or retorted. Example(s): submersion of meat in heated
water.
(b) Cooking—this treatment is primarily applied to improve the sensory attributes
of meat and may be considered a preservation technique to extend the shelf-life
of meat by destroying vegetative microorganisms and inactivating enzymes.
Example(s): oven cooking, roasting, grilling, frying, boiling and steaming.
(c) Pasteurization—this treatment is primarily used to destroy vegetative cells and is
used in combination with one or more preservation techniques. It usually involves
the heating of meat in a sealed container under vacuum conditions that will subse-
quently be stored under refrigerated temperatures. Example(s): “sous vide” meats
(d) Sterilization—use of the term “sterile” implies the absence of viable organisms
and since this is practically unobtainable in meat processing, the term “com-
mercially sterile” is used to denote a product that has received a process that
inhibits the growth of vegetative cells and spores under normal storage condi-
tions. Virtually all vegetative organisms and spores of concern are inactivated
during this process rendering a meat product with a long shelf-life requiring no
other form of preservation. Example(s): retorting (canning).
232 J.D. Stopforth

It is important to note that the effectiveness of all of the above-mentioned treat-

ments in inactivating microorganisms may be affected by: (a) the type of microor-
ganism, (b) the number of microorganisms present on the meat, (c) the physiological
state of the microorganisms (stressed, stationery, exponential, etc.) and (d) the phys-
icochemical characteristics of the meat including pH, aw, Eh, and type of meat (%
fat, etc.) (Kerry 2011).

2.2 Chilling and Freezing

The use of chilling originated in early civilizations with the recognition of the
preservative effects of cool temperature storage of perishable foods such as meat
in caves where temperatures remained relatively low throughout the year. The
principles of ice formation and mechanical refrigeration in commercial-scale
operations dates back to the late 1900s (Lawrie and Ledward 2006). In many
countries, animals slaughtered for meat were and in some cases still are immedi-
ately distributed, sold and consumed making the need for further preservation
unnecessary. However, in order to satisfy increasing demand for meat, the indus-
try moved to production of surplus meat that required preservation to provide
extended shelf-life and the ability to be transported and stored for distribution.
The use of chilling and freezing of meat was thus employed to increase the stor-
age life of meat without dramatically changing its state. Microorganisms have a
range of temperature in which they can physically grow and as the environmental
temperature moves away from their preferred range, their growth rate slows until
it eventually stops (Lawrie and Ledward 2006).
Chilling, or cooling, depends on removing heat from meat and then maintaining
it at a lower temperature than its surroundings. Cooling of meat much like heating
relies on three mechanisms discussed previously for heat transfer namely: (1) con-
duction, (2) convection and (3) radiation (North and Lovatt 2012). Cooling of meat
by conduction, achieved by placing the meat into a cold liquid or gas, is uncommon
in the meat industry. Instead, chilling of meat is achieved by convection through
exposure of the meat to chilled air which results in both the reduction of the surface
temperature of the meat as well as enhancing the surface drying of the meat; both of
which reduce the growth of microorganisms (Ockerman and Basu 2004). There are
two primary means for convection chilling of meat: (1) natural-convection air chill-
ing and (2) forced-convection air chilling. Natural-convection air chilling where
refrigerant is pumped through cooling tubes is slow and largely uncontrollable
whereas forced-convection air chilling accomplished via coupled fans and con-
trolled air movement is a more efficient and effective means of meat cooling. Rapid
chilling of meat increases product yield due to lower evaporative loss from the meat
surface while simultaneously reducing microbial growth due to the surface drying
of the meat. Increasing the air velocity and/or decreasing the air temperature can
result in substantial decrease in chilling time; however, the process is limited due to
the difficulty in removing heat rapidly from the deeper internal tissues (Lawrie and
11 Preservation Methods for Meat and Poultry 233

Ledward 2006). However, many meat-processing facilities have incorporated the

use of spray-chilling, which is a combination of both conduction and convection
chilling. The contact of chilled water with the surface of the meat helps to carry the
heat content of the deeper tissues to the meat’s surface while the convection (natural
or forced) carries the heat away from the surface resulting in slightly faster chilling
of meat and reduced weight loss due to shrinkage as compared to use of air chilling
alone (Wiklund et al. 2010).
Chilling is able to slow both microbial growth and enzymatic changes in meat
but it is not able to completely inhibit the deterioration since psychrotrophic bacte-
ria, yeasts and molds are able to grow slowly under refrigerated temperatures
(Neumeyer et al. 1997). Freezing, however, is an ideal method for maintaining the
original characteristics of fresh meat for extended periods of time. Meat contains
approximately 50–75% of water (depending on the species and cut) (Heinz and
Hautzinger 2007). Freezing is the process of removing heat to convert the water in
the meat to ice; the process of phase transition from liquid to ice requires a large
amount of energy termed the “latent heat of freezing”. There are essentially three
distinct components of the freezing process: (1) reducing the temperature to the
freezing point, (2) removing the latent heat of freezing and (3) reducing the tem-
perature further to the storage temperature (Reid 1991). Meat does not start to freeze
until its surface temperature drops below the initial freezing temperature of the
meat; at that point, the meat starts to freeze from the outside toward the inside. Meat
freezing is fast and almost 75% of tissue fluid freezes at −5 °C; the rate of freezing
increases relative to the rate of temperature drop in the immediate environment with
almost 98% of water freezing at −20 °C and complete crystal formation occurring
at −65 °C (Rosmini et al. 2004). Under normal freezing conditions, the water con-
tent in meat forms discrete ice crystals around nucleation sites and the size of these
crystals is highly dependent on the rate of freezing. Fast freezing of meat results in
formation of small ice crystals while slower freezing leads to larger ice crystals
(Reid 1983). The size of the crystals has a direct impact on the quality of the meat
since these crystals grow both within and between tissue cells. The larger ice crys-
tals can cause mechanical damage to cell membranes and the concentration of sol-
utes into unfrozen portions of the meat can damage the proteins which may
eventually result in what is known as “drip loss” during subsequent thawing of meat
(Sahagian and Goff 1996).
Freezing is very effective at preserving meat from a microbiological standpoint
since microbial growth stops at −12 °C and during the freezing process, about 60%
of viable microbial populations die (Rahman 1999b). Where chilled storage of
vacuum-­packed meat at 0–2.3 °C is able to provide a shelf-life of 70–80 days, fro-
zen storage of the same vacuum-packed meats at −18 or −30 °C can provide a shelf-­
life of up to 12 or 24 months, respectively (Berkel et al. 2004). Although, deterioration
due to microbial growth is well controlled in frozen meat, physical, chemical and
biochemical reactions can still occur in tissues leading to enzymatic changes,
­oxidative rancidity and ice crystallization (Zhou et al. 2010). Frozen storage at
−55 °C has been proposed as a means to completely prevent quality changes in
fresh meat (Hansen et al. 2004).
234 J.D. Stopforth

2.3 Drying

Drying of meat for the purpose of preservation involves removal of most of the
water present in the meat by either evaporation or sublimation. In general, dried
meat products can be considered shelf-stable and are able to be stored at ambient
conditions for prolonged periods of time since the aw has been reduced to levels that
inhibit both microbial and biochemical reactions. In developing countries, drying
achieved through a natural process known as air- or sun-drying has been practiced
for centuries and is still practiced today where preservation by other methods (i.e.,
chilling and freezing) is too costly or there is no infrastructure to support it. In
developed countries, drying is more often than not only one of various other process
steps such as salting, curing, smoking and fermenting used to manufacture dried
meat products. The additional treatments during the drying process while aimed at
enhancing the organoleptic properties (such as flavor and texture) and improving the
palatability of the dried products also contribute to the overall preservation of the
meat. Drying of meat products nowadays even in developing countries is achieved
by more conventional methods than air- or sun-drying and is based on convective
drying incorporating heat in the form of solar, microwave or hot air streams
(Santchurn et al. 2012).
The principle of convection-based drying systems is the removal of water by
evaporation due to the exposure of meat to a continuous stream of hot hair (Ratti
2001). Drying decreases the water content of raw meat from 70–80% (wet basis) to
a safe level in the range of 12–15% (wet basis) when used as the sole process of
preservation and down to 38–50% when drying is combined with other methods
such as salting, curing, smoking and fermenting. During drying the aw of fresh meat
is reduced from ≥0.98 to 0.70–0.75 for intermediate moisture products (that are also
combined with other preservation methods such as fermentation) to 0.60 which ren-
ders the meat microbiologically safe. With dried meat products, aw is the single
most important factor contributing to the shelf-life and safety of the resulting prod-
ucts. Adequate drying of meats will inhibit virtually all bacteria at an aw < 0.86
(except for certain halophilic bacteria which seldom result in discernible spoilage)
and virtually all molds and yeasts at an aw < 0.80 (Jay et al. 2005).
As mentioned above, a range of different dried meats may be produced based on
use of drying solely or in combination with other preservation methods. Dried meats
can be categorized by their applications as follows (Santchurn et al. 2012):
(a) Dried meats—dried meats are simply meats that have been dried by either air-/
sun-drying or convection-based drying. Preservation of the meat is achieved
solely by lowering aw of the meat to 0.60–0.65 through evaporation. Examples
of dried meats are: Kilishi and Sharmoot (north African countries).
(b) Salted dried meats—salted dried meats are meats that are salted (either wet- or
dry-salting) prior to convection-based drying. Drying of these meats is achieved
both by evaporation and sublimation. Examples of salted dried meats: biltong
(South Africa), Sou gan (China) and Pastirma (East Mediterranean)
11 Preservation Methods for Meat and Poultry 235

(c) Cured dried meats—Cured dried meats are similar to salted dried meats in that
a mix of salt and nitrate/nitrite are added to meat prior to drying. The addition
of nitrate/nitrite as part of a dry mix dates back to the third century when nitrate
was discovered as an impurity in salt; observation of the characteristic reddish-­
pink color it gave to meat became desirable and was intentionally added in brine
mixes to develop a uniform color fix in meats (Martin 2012). Nowadays, curing
is widely used as a pre-treatment of whole meats and meat emulsions that are
subsequently fully-cooked in a diverse segment of the meat industry.
(d) Smoked dried meats—Smoked dried meats are sometimes salted (either wet- or
dry-salted) before smoking. Smoke is a mixture of multiple wood combustion
products (gases, tar, ash, carbonyls, phenols, etc.) that are visible as gases (CO2,
N2, water vapor, etc.) and carry unburned solid particles (resin, tar, ash) as they
escape the combustible heat source; the exact ratio of gases and solids within
the smoke stream is determined by type and moisture content of the wood, the
rate and temperature of heating and the rate of air flow (Herring and Smith
2012). Preservative effects of the smoke are thought to be due to the bacterio-
static activity of the resin, phenols, aldehydes and aromatic compounds (Draudt
1963). Examples of smoked dried meats: Banda (Nigeria) and Wiltshire Bacon
(UK).
(e) Fermented dried meats—Fermentation is an additional step in the processing of
salted dried meats. They are divided into two groups based on the form of meat:
(1) whole-muscle meat such as dry-cured hams and breseola, and (2) sausages
made from finely chopped meat. Whole-muscle meats are essentially salted
(usually with a mixture of salt and nitrate/nitrite and where appropriate other
seasonings), aged/ripened and dried over the course of days to months at vary-
ing temperature-time regimens (Toldra 2007). Fermented sausages (such as
chorizo and salamis) are made by blending a number of ingredients including
salt and nitrate into ground meat (lean and fat), filling the mixture into casings
and tying the casings, fermenting the tied casings using starter cultures in tem-
perature and humidity controlled conditions to a pH between 4.9 and 5.3 and
ripening the sausages for weeks to months at varying temperature-time regi-
mens (Cantoni 2007). At the end of the processes, these products are defined by
a long shelf-life at room temperature due to the acidification during fermenta-
tion and the depression of aw during ripening. The decrease in aw further results
in an increase in the ratio of salt/aw thereby protecting the product from spoilage
and pathogenic microorganisms during ripening (Cantoni 2007).

3 Advanced and Developing Preservation Technologies

A growing trend in consumer eating habits over the last 15 years reveals that there
is an increasing demand for higher quality, safer meat with extended shelf-life
(Aymerich et al. 2008). This has resulted in the development and adoption of many
novel and advanced technologies and processes in the meat industry to meet the
236 J.D. Stopforth

needs of consumers and extend value in the meat supply chain whilst maintaining
the safety and quality of products. The following section provides a brief account of
the latest advances and developments in meat preservation.

3.1 Superchilling

Superchilling, also known as deep-chilling, is not a new concept but one that is
gaining more traction in recent times as a viable commercial practice since it has the
potential to increase the shelf-life of fresh meat (Magnussen et al. 2008).
Superchilling refers to a process where a minor part of the meat’s water content is
frozen thereby acting as a refrigeration reservoir; the temperature of the meat is
lowered 1–2 °C below the initial freezing point of the meat to prevent formation of
ice crystals and thus also to prevent potential drip loss that may result from freeze/
thaw processes during transportation and distribution of meat (Bahuaud et al. 2008).
After initial surface freezing, the ice distribution equilibrates and the meat main-
tains a constant uniform temperature during subsequent storage. Superchilling
results in reduced product weight losses and lower costs in storage and transport;
however, the main reason for superchilling meat is that it is able to prolong the
shelf-life of meat for at least 1.4–4 times that achieved using traditional chilling
methods (Magnussen et al. 2008). Duun et al. (2008) clearly demonstrated the
potential for preserving the shelf-life of fresh meat longer using superchilling com-
pared to traditional chilling; meat that was superchilled at −2 °C maintained good
sensory and microbiological quality through 16 weeks of storage in comparison to
the 14 days achieved using traditional chilling at 3.5 °C. Despite the observed suc-
cesses with superchilling, it remains a challenge to implement in commercial chill-
ing operations due to the difficulty in calculating the chill time and temperature
distributions in the chill and freeze process. Until the industry is able to generate
more data in support of software development to calculate chilling times, tempera-
tures, air-flow and refrigeration loads, the use of this process will find limited com-
mercial application (Magnussen et al. 2008).

3.2 Natural Antimicrobials

The use of antimicrobials to inhibit or inactivate spoilage microorganisms and

pathogens in meat is a common practice. A number of antimicrobial compounds
have received international regulatory approval for use as direct additives to meat
including but not limited to (Drosinos et al. 2009):
• Acetic acid and acetate salts, diacetates, dehrydroacetic acid
• Lactic acid and lactate salts
• Benzoic acid and benzoate salts
11 Preservation Methods for Meat and Poultry 237

• Propionic acid and propionate salts

• Nitrates and nitrites
Most of these antimicrobials are however chemical compounds, even those stem-
ming from natural sources are typically synthesized additives. The use of these
chemical additives has become undesirable by consumers in recent times, creating
the demand for more natural means of preservation. This coupled with the need to
expand the spectrum of antimicrobial activity over that of the regulatory-approved
substances has caused the scientific community to explore the use of naturally-­
derived antimicrobials. The interest to explore natural antimicrobials is further
driven by the fact that international agencies have very stringent requirements for
the toxicological evaluation of novel direct antimicrobials and thus testing of new
synthetic compounds could take many years to be approved whereas approval of
compounds derived from natural sources have a higher chance of gaining fast
approval. Naturally-occurring antimicrobials can be classified by the source they
are derived from namely animal, plant and microbial (Davidson and Zivanovic
2003; Drosinos et al. 2009; Hayes and Brunton 2011).
Natural antimicrobials from animal sources that have or may be considered for
use in meat preservation include (Davidson and Zivanovic 2003):
• Chitosan—chitosan is derived from alkaline deacetylation of chitin, a nitrogen-­
containing polysaccharide and the principal component of shellfish exoskele-
tons. Chitosan inhibits growth of molds, yeasts and bacteria, however, minimum
inhibitory concentrations vary widely from 0.01 to 5.0% depending on polymer
characteristics, pH, and temperature (Roller and Covill 2000). It is thought that
chitosan affects microbial cells by interaction with the anionic cell wall polysac-
charides or components of the cytoplasmic membrane resulting in altered perme-
ability or prevention of transport (Tsai and Su 1999).
• Lactoferrin—lactoferrin is a globular glycoprotein derived primarily from bovine
milk and colostrum. Lactoferrin has a limited range of effectiveness; it has dem-
onstrated efficacy against Bacillus subtilus, B. stearothermophilus, Listeria
monocytogenes and Micrococcus spp. (Naidu and Bidlack 1998). Lactoferrin
acts by increasing the permeability of the outer membrane to hydrophobic com-
pounds including other antimicrobials, blocking the adhesion of some microor-
ganisms to mucosal surfaces in the gut, blocking expression of colonizing factors
of enteric pathogens, inactivating lipopolysaccharides of Gram-negative bacteria
and making iron inaccessible by binding it up (Naidu and Bidlack 1998).
Lactoferrin B also known as hydrolyzed lactoferrin is produced by acid-pepsin
hydrolysis of lactoferrin and is thought to be more active than lactoferrin (Naidu
2002).
• Lactoperoxidase—lactoperoxidase is an enzyme that is derived from bovine
milk and colostrum and reacts with thiocyanate in the presence of hydrogen per-
oxide and forms hypothiocyanate and hypothiocyanous acid. These compounds
cause leakage of potassium ions, amino acids and peptides in bacterial cells con-
sequently interfering with bacterial replication and protein synthesis. The chal-
lenge, however, with lactoperoxidase is that it requires both thiocyanate and
238 J.D. Stopforth

hydrogen peroxidase to be present to exert any antimicrobial activity; thus, it

requires exogenous addition of these reactants to be considered a viable
preservative.
• Lysozyme—lysozyme is an enzyme that is derived from mammalian milk and
avian eggs. Lysozyme catalyzes hydrolysis of glycosidic bonds between
N-­acetylmuramic acid and N-acetylglucosamine of the peptidoglycan of bacte-
rial cell walls. It is most effective against Gram-positive bacteria since the pepti-
doglycan of the cell wall is more exposed. Lysozyme has been proposed as a
surface treatment on many further processed meats to inhibit growth of the
Gram-positive pathogen, L. monocytogenes.
Natural antimicrobials from plant sources include those present as intact (pre-­
infectional) and those released (post-infectional) as part of the plant’s defense to
microorganisms and insects (Drosinos et al. 2009). Components present in intact
plants include alakaloids, dienes, flavonols, flavones, glycosides, lactones, organic
acids, phenolic compounds and protein-like compounds. Post-infection compounds
include isothiocyanates, phenolic compounds, phytoalexins and sulfoxides. The
components that have shown the most potential in food preservation are phenolics
and essential oils (Davidson and Zivanovic 2003). These compounds are typically
obtained by steam- or hydro-distillation as well as solvent extraction (e.g., ethanol)
from spices and herbs (Coma 2008). The antimicrobial activity of spices and herbs
is primarily attributed to the phenolic component of their essential oil fraction
(phyto-phenols). Essential oils mainly consist of terpenes, terpenoides and other
aromatic compounds (e.g., phenols, such as eugenol, thymol, aldehydes, esters and
alcohols) (Bakkali et al. 2008). Other plant extracts include isothiocyanate deriva-
tives (e.g., found in cabbage, horseradish, mustard, broccoli) and phenolic com-
pounds such as di- or tri-phenols, phenolic acids such as hydroxycinnamic acid and
flavonoids (Davidson and Zivanovic 2003). The majority of active components
from herbs and spices are considered to be food-grade or generally-regarded-as-safe
(GRAS). Drosinos et al. (2009) provides a comprehensive review of the effective-
ness of essential oils and/or their components including eugenol, pimento leaf, cara-
way seeds, mustard, rosemary, sage, clove, coriander, mint, thyme, oregano, garlic,
nutmeg, cilantro oil, carvacrol and cinnamaldehdye applied as preservatives of
meat. Various mechanisms of inhibition have been proposed for essential oils, most
predominantly, disrupted structural and functional properties of bacterial mem-
branes. Specifically, it is proposed that essential oils penetrate the cell envelope,
dissolve in the lipid layer of cellular membranes, bind to hydrophobic sites of mem-
brane proteins and by increasing the permeability of the cell membrane, result in
loss of vital intracellular material or inhibit nutrient intake by dissipation of pH
gradient and the electrical potential (Burt 2004; Oussalah et al. 2006). Additional
modes of action include inhibition of the oxygen uptake, inhibition of nucleic acid
synthesis and inactivation of membrane proteins or other intracellular enzymes
(Burt 2004). Gram-positive bacteria are considered to be more susceptible to essen-
tial oils than Gram-negative bacteria (Fisher and Phillips 2006).
11 Preservation Methods for Meat and Poultry 239

The performance of essential oils in meat may be affected by several factors

including: (a) certain intrinsic properties of meat such as fat, pH, salt, water and
proteins, (b) the structure of the meat (whole-muscle vs. emulsion), (c) the decom-
position of some essential oil constituents in the aqueous phase and/or their interac-
tion with hydrophilic substances such as thiols and sulphydryl or terminal amino
groups of proteins and (d) factors affecting the physiology of microorganisms such
as composition of bacterial membranes, availability of nutrients, oxygen tension
and incubation temperatures (Drosinos et al. 2009). The major limiting factor in the
effectiveness of essential oils even when they are applied at concentrations higher
than what is needed for inhibition, is their reduced solubility due to the presence of
apolar constituents in their composition. In high fat meats, highly hydrophobic con-
stituents of essential oils may have limited effectiveness since the essential oil will
partition in the lipid fraction of the meat thereby reducing the residual concentration
in the hydrophilic portion of the meat where microorganisms typically reside (Burt
2004). Furthermore, low pH may also increase the hydrophobicity of essential oils
enhancing their potential to bind onto hydrophobic portions of the membrane pro-
teins and to become more soluble in the lipid-rich membranes of the target microor-
ganisms thereby counteracting the antimicrobial effectiveness. One of the strategies
to reduce the binding of essential oils by fats in meats and to moderate any negative
sensory impact of the essential oils is to encapsulate them within edible matrices or
surfactant micelles (Gayinsky et al. 2007).
Natural antimicrobials from microbial sources are covered in the next section
under Biopreservation.

3.3 Biopreservation

Biopreservation refers to the use of non-pathogenic microorganisms and/or their

metabolites to reduce meat spoilage and/or improve food safety. Undesirable micro-
organisms such as pathogens or those causing spoilage in meat may be inhibited or
even eliminated by the action of competitors or antagonistic microbiota without the
need for chemical additives (Minei et al. 2008). Lactic acid bacteria (LAB) are a
major potential for use in biopreservation as they have a long history of safe con-
sumption and naturally dominate the microflora of meat during storage. LAB con-
stitutes a group of Gram-positive, catalase-negative cocci or rods with similar
characteristics and the ability to produce lactic acid as the main product of fermen-
tation of carbohydrates. Most LAB are considered food grade and can exert antimi-
crobial effects against bacteria, yeasts and molds in various ways including: (a)
production of volatile acids, hydrogen peroxide, carbon dioxide, diacetyl and acet-
aldehyde, (b) competitive exclusion and (c) production of bacteriocins (Settanni and
Corsetti 2008). Bacteriocins are antimicrobial peptides produced by numerous
Gram-positive and Gram-negative bacteria but those produced by LAB are of spe-
cial interest in biopreservation of meat based on their long history of safe consump-
tion, their existence as natural microflora of meat and use of LAB and/or its
240 J.D. Stopforth

metabolites is generally accepted by consumers as natural and health-promoting

(Deegan et al. 2006). Bacteriocins can be applied to meat in two ways: (1) by adding
crude, purified or semi-purified bacteriocin preparations or (2) by inoculation with
pure cultures of the bacteriocinogenic strains. Bacteriocins produced by LAB are
very attractive for use in biopreservation of meat due to: (a) production by strains
generally recognized as safe, (b) their lack of action against eukaryotic cells, (c)
inactivation by digestive proteases, which preserve the gut microbiota, showing no
cross-resistance with antibiotics, (d) tolerance to pH and heat, (e) mostly bacteri-
cidal mode of action, and (f) genetic determinants usually plasmid encoded, facili-
tating genetic manipulations (Galvez et al. 2007).
Bacteriocins may be categorized into four classes of peptides based on their bio-
chemical and genetic properties (Deegan et al. 2006):
(a) Class I peptides are lantibiotics, which are small and characterized by unusual
amino acids such as lanthionine. The major representative bacteriocin from this
class is nisin. Class I is further subdivided into class 1a bacteriocins consisting
of cationic and hydrophobic peptides and class 1b bacteriocins which are glob-
ular peptides with no net negative charge,
(b) Class II peptides are low molecular weight, heat-stable bacteriocins that do not
contain lanthionine. This class is organized into three subclasses: class IIa
pediocin-like bacteriocins, class IIb which consists of two peptides (lactococcin
G) and class IIc which are one-peptide bacteriocins requiring cysteine mole-
cules for activity expression (lactococcin B),
(c) Class III peptides which are large and thermosensitive bacteriocins (helveticin
J) and
(d) Class IV peptides are proteins containing one or more chemical moieties
required for activity (plantaracin S, leuconocin S, lactocin 27, pediocin SJ-1.
The mode of action of bacteriocins is characterized by two distinct phases: (1)
adsorption of the bacteriocin on receptors located on the cell wall of the sensitive
strains and (2) denaturation of the cytoplasmic membrane. The first step is revers-
ible and removal of the bacteriocin leaves the membrane structure intact without
damage to the bacterial cells while the second step is irreversible and damage caused
is characteristic of the particular bacteriocin(s) active (Abee et al. 1995). The cyto-
plasmic membrane is the main target of bacteriocins. As a group, bacteriocins act on
the target cells by various mechanisms as follows: (a) permeabilization of the cyto-
plasmic membrane followed by leakage of low-molecular weight cellular com-
pounds and dissipation of the proton motive force, (b) cell lysis, (c) degradation of
vital macromolecules such as DNA and RNA and (d) inhibition of biological pro-
cesses such as synthesis of protein, DNA, RNA and peptidoglycan (Motta et al.
2008). Bacteriocins from LAB are mainly active against Gram-positive close-­
related species while Gram-negative strains are more resistant given that their mem-
brane composition differs from that of the Gram-positive bacteria. Immunity of the
bacteriocin-producing strains to their own bacteriocin is regulated by the corre-
sponding gene encoding the extraction of immunity-related proteins, which deacti-
11 Preservation Methods for Meat and Poultry 241

vate the bacteriocin by preventing it from binding to receptors of the producing-strain

(Ennahar et al. 2000).

3.4 Irradiation

Irradiation is the most effective postharvest technology for inactivating microorgan-

isms in meat and meat-based products (Lim et al. 2008). Atoms are made up of
protons, neutrons and electrons that are held together by energy. When these nuclear
particles lose balance by changing the arrangement of forces, the unstable atoms
can restabilize by emitting energy to rebalance the nucleus; this emission of energy
in particles or waves is known as radiation (Halliwell and Gutteridge 1989). If radia-
tion has sufficient energy to move atoms in another material without chemical
changes it is known as non-ionizing radiation and when it has sufficient energy to
break chemical bonds it is known as ionizing radiation (Josephson and Peterson
2000). Irradiation is the process of exposing meat to a source of radiation. Meats
that are irradiated for preservation have the advantage over other preservation meth-
ods such as heating, chilling and freezing and addition of chemical antimicrobials
in that: (a) it does not significantly raise the temperature of the meat, (b) it does not
leave potentially harmful residues and (c) it can be used to treat packaged meats
which will remain protected from microbial contaminants after treatment (Blackburn
2011). For commercial irradiation of meat, three types of ionizing radiation and one
type of non-ionizing radiation may be used. The three sources of ionizing radiation
that are authorized for meat irradiation according to Codex General Standard for
Irradiated Food (Codex Alimentarius Commission 2003) are: (1) γ-rays emitted by
the radioactive elements Cobalt-60 or Cesium-137, (2) X-rays generated from
machine sources operated at or below an energy level of 5 MeV (million electron
volts) and (3) electrons generated from machine sources operated at or below an
energy level of 10 MeV (also called electron beam or e-beam). High-intensity light,
also termed pulsed broad-spectrum white light, pulsed white light, near infra-red
light or more commonly ultraviolet (UV) light describes the range of light in which
non-ionizing radiation is emitted (Green et al. 2003). In 1980, the Food and
Agriculture Organization (FAO) of the United Nations, the International Atomic
Energy Agency (IAEA), and the World Health Organization (WHO) stated that irra-
diation of any food commodity up to an overall average dose of 10 kGy presents no
toxicological hazard and thus any foods treated below that level would not require
further toxicological testing (WHO 1981). Currently, 56 countries have permitted
irradiation of food with an estimated half million tons of food irradiated annually
(Lee and Ahn 2009).
With ionizing radiation, meat is exposed to the ionizing energy source in such a
way that a precise and specific dose is absorbed; the absorbed dose is the mean
energy imparted by the ionizing radiation to the meat in a volume element divided
by the mass of the meat in the volume element. The dose of ionizing radiation or the
amount of ionizing radiation absorbed by the meat is measured in Gray (Gy) in the
242 J.D. Stopforth

SI unit; 1 Gy is equivalent to 1 joule (J) of absorbed energy per kilogram (1 kGy

equals 1000 Gy) (Badr 2012). The energy from ionizing radiation is too low to
induce radioactivity in the irradiated meat (Badr 2012). For commercial application
of ionizing radiation to meat, γ-rays and e-beam are most frequently used. γ-rays are
highly penetrable and most frequently applied as a batch treatment of meat that is
already packaged in both primary (plastic pouches and trays) and secondary pack-
aging (cases). Electron-beam is less penetrable than γ-rays and is typically used in
a continuous treatment of thin cuts of meat on a conveyor since it can only satisfac-
torily penetrate up to 35 mm of unit density material. Although X-rays have high
penetrability, they are not typically used in irradiation of foods due to the poor
conversion of accelerated electrons to X-rays. Ionizing radiation is an effective
means of preserving meat as it is able to inactivate bacteria, yeasts and molds. The
sensitivity of microorganisms to ionizing radiation in meat and meat products is,
however, influenced by a number of different factors including (Badr 2012; Olson
1998):
• The organism—radiation sensitivity of microorganisms varies with species and
even strain. In general, sensitivity to radiation decreases in the following order:
Gram-negative > Gram-positive = molds > spores = yeasts > viruses. The pres-
ence of large populations of microorganisms in or on the meat being irradiated
reduces the effectiveness of the applied dose. Furthermore, the physiological
state of bacteria has a significant effect on the sensitivity to radiation in that
exponential-phase cells are more sensitive than stationary-phase or lag-phase
cells.
• Meat composition—the high protein content of meat may protect microorgan-
isms against the destructive effect of radiation by neutralization of the free radi-
cals generated. Other meat components such as natural antioxidants can compete
for the free radicals generated by the radiolysis of the water in meat leading to
reduced radiation antimicrobial efficacy.
• Gas atmosphere—the atmosphere in packaged meat affects both the destruction
of microorganisms by irradiation as well as their post-irradiation recovery. The
presence of oxygen appears to increase the destruction of microorganisms by
irradiation.
• Temperature during irradiation—in general, freezing reduces the antimicrobial
effects of radiation by reducing the water activity in meat when water is con-
verted to ice. Freezing reduces the destructive effects of radiation by drastically
reducing the generation of free radicals from radiolysis of water as well as reduc-
ing the migration of free radicals generated to other parts of the meat.
• Irradiation dose—the destructive effect of radiation on microorganisms depends
on the total absorbed dose and thus higher doses of radiation results in higher
antimicrobial effects.
The application of ionizing radiation in the meat industry is limited partially due
to the skepticism of radioactivity by consumers but primarily due to the effects on
meat quality. Irradiation affects the quality of meat in a number of ways (Badr 2012;
Lee and Ahn 2009):
11 Preservation Methods for Meat and Poultry 243

• Physical characteristics—irradiation, especially at doses >5 kGy, may result in

decreased water-holding capacity of meat with the subsequent appearance of
purge or weep due to decreased solubility of the protein fraction.
• Oxidation—ionizing radiation generates hydroxyl radicals by splitting water
molecules. With meat containing around 70% water, a significant amount of free
radicals are formed during irradiation. These free radicals play a significant role
in lipid oxidation, especially in the presence of oxygen. In frozen meat, the
degree of lipid oxidation is much lower due to the fact that the free radicals gen-
erated are less mobile and will tend to recombine into the original substances
than diffuse through the meat.
• Color—the color changes in irradiated meat are highly dependent on irradiation
dose, animal species, muscle type and packaging atmosphere. Under aerobic
conditions, irradiation changes the red color of red meats to brown or gray; it is
thought that radicals such as hydroxyl or sulfuryl radicals react with the binding
sites of myoglobin to form metmyoglobin and sulfmyoglobin which generates
the brown/gray and green off-colors.
• Odor and taste—irradiation of meat results in a higher degree of volatile com-
pounds such as 2-methyl butanal, 3-methyl butanal, 1-hexen, 1-heptene, 1-octene,
1-nonen, hydrogen sulfide, sulfur dioxide, mercaptomehtane, dimethyl sulfide,
methyl thioacetate, dimethyl sulfide and trimethyl sulfide. Initially, it was
assumed that the resulting hydrocarbon radicals from lipid oxidation were
responsible for the characteristic off-odor associated with irradiated meat, it has
been determined that the sulfur compounds are the major volatile compounds
associated with the off-odor while the volatiles from lipids account for only a
small part of the off-odor.
Non-ionizing radiation in the form of UV light (high-intensity light) is measured
in terms of its wavelength rather than in energy absorbed as with ionizing radiation;
the wavelength is in essence the speed of light divided by the frequency. UV wave-
lengths in the range of 170—800 nm are suitable for detrimental changes in micro-
bial structures and functions (Green et al. 2003). UV light affects microbial cells by
denaturation of proteins, including modification or inactivation of functional
enzymes and disruption of the functional components of nucleoproteins. Enzyme-­
directed DNA repair in microorganisms is also negatively affected due to cleavage
by the high energy (Barbosa-Canovas et al. 2000). Much like ionizing radiation, the
antimicrobial effectiveness of non-ionizing radiation is dependent on the type and
number of microorganisms present, meat composition and the number, intensity and
speed of pulses delivered (Barbosa-Canovas et al. 2000). UV light is an effective
preservation method in that it has antimicrobial activity against bacteria, spores,
yeasts and molds. The success of UV radiation of meats as a preservative, however,
is restricted to surface applications due to the restricted penetration of high-intensity
light in opaque substances and on irregular-surfaces (Dunn et al. 1995). One of the
most likely successful applications of UV light in meat preservation is in the post-­
harvest treatment of carcass sides where the meat is hanging and UV bulbs can be
positioned and oriented to ensure maximal contact of light with the surface of the
244 J.D. Stopforth

meat. Although UV light may result in production of some off-odors, flavors and
colors (Colchin et al. 2001), the impact on sensory characteristics is less than that
compared to the use of ionizing radiation. Optimization of light conditions and
exposure times will minimize negative organoleptic impacts of UV light treatment
of meat.

3.5 High Hydrostatic Pressure (HHP)

The application of HHP to meat processing is an innovative alternative to traditional

preservation methods including heating and chemical additives that is gaining
worldwide attention in recent times due to the commercial availability of equip-
ment. The main components of an HHP system are a pressure vessel, a pressure
generation system, a temperature control device and a materials handling system
(Mertens 1995). Most pressure vessels are made from a single piece of high tensile
steel alloy and made to withstand pressures up to 700 mPa. To generate the desired
pressure, all air is removed from the vessel after which the pressure-transmitting
medium (either water or oil) is pumped from a reservoir into the pressure vessel
using a pressure intensifier. Temperature control is achieved by pumping a heating/
cooling medium through a jacket that surrounds the pressure vessel. There are
essentially two methods for processing foods in high-pressure vessels: (1) in-­
container processing and (2) bulk processing. Meat and meat products are processed
in-container with batch/semi-continuous systems. HHP treatment has the advantage
of affecting only covalent bonds of macromolecules in meat constituents, thus
resulting in minimal nutritional and sensorical changes as compared to those expe-
rienced when meat is heated. The difference between reactions affected by pressure
(activation volume) and temperature (activation energy) is what leads to meat retain-
ing its quality attributes (Pandrangi and Balasubramaniam 2005). The effectiveness
of HHP treatment is influenced by various intrinsic and extrinsic factors including:
treatment time, pressurization/decompression rate, temperature and the number of
pulses (Knorr 2001).
The effect of HHP on microorganisms has been investigated extensively (Abe
2007; Chen et al. 2006; Smelt et al. 2002). High pressure induces several changes in
the cell membrane and cell wall of microorganisms, including separation of the cell
membrane from the cell wall, contraction of the cell membrane, compression of gas
vacuoles, cell lengthening, ribosome dissociation and release of intracellular
­material. Several factors may influence the extent of inactivation including: the
pressure applied, the time of processing, meat composition, temperature, pH and aw
(Tewari et al. 1999). In general, sensitivity to HHP decreases in the following order:
molds = yeasts > Gram-negative > Gram-positive > viruses > spores. Molds and
yeasts are typically inactivated by pressures between 200 and 300 mPa and bacteria
by pressures in the range of 350–600 mPa (dependent on Gram-type and strain).
Viruses are quite resistant to pressure typically requiring >600 mPa and up to
11 Preservation Methods for Meat and Poultry 245

850 mPa for significant destruction. Spores are the most resistant microorganisms
and can resist pressures in excess of 1000 mPa.
HPP treatment may affect the following quality aspects of meat and meat prod-
ucts (Ikeuchi 2011):
• Water-holding capacity (WHC)—water-holding capacity is the ability of meat to
retain its water when external forces such as heat and pressure are applied. The
application of low pressure (100 mPa) reduces the WHC of meat most likely due
to the fact that the space able to retain water increases with the partial destruction
of the structure of myofibrils in the meat. This phenomenon is reversed when
pressure increases to 200–300 mPa due to release of divalent cations that bind to
myofibrillar proteins thereby preventing formation of salt bridges and resulting
in increased moisture retention. Further pressure increases once again result in
reduction of WHC due to the extensive denaturation of myofibrillar proteins.
• Meat color—application of pressure at or above 400 mPa result in a change of
red meat from a bright red color to slight gray most likely due to reduction in the
extractability of myoglobin, however, the color of meats stored for more than a
week prior to treatment were not affected indicating that pressure inactivates the
enzymes responsible for metmyoglobin reduction.
• Lipid oxidation—oxidation of lipids in meat, although slight when compared
with that observed when meat is heated, tends to increase with linear increases in
pressure. It is thought that the denatured ferric form of myoglobin, which occurs
during pressurization, plays a role in catalysis of the lipid oxidation reaction.
Lipid oxidation due to pressure cannot be avoided, however, the effects can be
minimized by selection of moderate temperatures for treatment of meat.
• Flavor and texture—the use of HHP treatment is thought to increase the free
amino acid content thereby adding to the overall positive development of meat
flavor. Furthermore, HHP combined with moderate temperature has been shown
to improve the tenderness of meat.
HHP processing of meat is becoming more commercialized despite the high ini-
tial investment for equipment. More and more companies are using co-­manufacturers
that have already invested in the technology to process their products. The most
predominant application of HHP is on value-added, ready-to-eat (RTE) meats for
replacement of chemical additives to extend shelf-life.

3.6 Packaging

Packaging protects meat from microbial and/or chemical contamination, provides

mechanical support to the product, and can delay biological and chemical deteriora-
tive effects such as discoloration and off-flavor and off-color development (Nychas
and Skandamis 2005). Packaging technologies have evolved in recent years to
improve the safety and shelf-life of meat; the advances in preservative technologies
246 J.D. Stopforth

associated with packaging can be categorized into three broad areas: (1) gas atmo-
sphere, (2) active packaging and (3) smart packaging.
Management of the gas atmosphere in packaging involves the use of gas atmo-
spheres different than the composition of dry air at sea level which is composed by
volume of: 78% N2, 21% O2, 0.94% argon, and 0.02–0.03% CO2 (McMillin 2008).
Important terms associated with controlled gas atmosphere packaging are:
• Vacuum packaging (VP)—VP refers to the removal of air from the headspace of
a package
• Modified atmosphere packaging (MAP)—refers to the replacement of air in a
package by a precisely-defined atmosphere
VP essentially removes O2 from the package and thereby minimizes the oxida-
tive deteriorative reactions that may occur with the meat as well as preventing aero-
bic bacterial (primarily the pseudomonads that cause slime formation) and mold
growth that may cause meat spoilage. Vacuum conditions in a package are achieved
either solely by the application of a strong vacuum to remove the atmospheric air in
the package for via a gas flush using N2 and CO2 followed by application of a strong
vacuum. The packaging material typically employed is a low O2 permeable
(<15.5 mL/m2 in 24 h at one atmosphere) film and is applied as a vacuum skin or
heat-shrunk around meat that has been placed in a barrier styrene or polystyrene
tray (Belcher 2006). MAP generally uses three major gases singly or in combination
including O2, N2 and CO2. Other gases that have had some experimental success in
inhibiting microbial growth include CO, SO2, N2O, NO, HE, H2, Ar, ethylene oxide,
propylene oxide and Cl2, however, their commercial application has been limited
due to possible safety, legal and organoleptic issues (Day 2003). MAP has the
advantage of being able to generate both high O2 and low O2 environments (Ooraikul
2003). The concept of packaging fresh meat under high O2 concentrations to retard
metmyoglobin (browning) formation is well known, however, the high O2 concen-
trations do not inhibit the growth of aerobic spoilage organisms. The growth rate of
aerobic spoilage organisms is only reduced when moderate amounts of CO2 are
added to the mixture. When CO2 is added at more than 20% to the gas mixture, it
benefits both microbiological quality and meat color. Commercially, mixtures of
60–80% O2 and 20–40% CO2 are commonly used to preserve fresh meats. The use
of high O2 MAP is suitable for meat products that will be held for a short time at
refrigerated temperatures and in which development of a bright red (oxymyoglobin)
is desirable. Low O2 MAP is employed to maximize the antimicrobial effects of CO2
on spoilage microflora and is used for meat products that will be in storage and
distribution for extended times. The mode of action of CO2 inhibition of microor-
ganisms may be due to: (a) lowering of the pH of the meat, (b) penetration of the
microbial cell followed by a decrease in the cytoplasmic pH, (c) specific destabiliz-
ing action son the cytoplasmic enzymes and (d) specific destabilizing actions on the
microbial membranes (Devlieghere et al. 1998). Low O2 MAP usually involves the
use of at least 65% CO2 combined with up to 35% N2 to prevent the package from
collapsing. Although use of this environment with red meats develops a purple color
(deoxymyoglobin), a commercial strategy called dual-layer film has been employed
11 Preservation Methods for Meat and Poultry 247

to counter this apparent drawback (Jeyamkondan et al. 2000). Dual-layer film

involves the packaging of meat cuts in pre-formed plastic trays containing the low
O2 mixture which are then sealed with a second peelable O2 impermeable film that
when removed after extended storage of the product exposes an O2 permeable film
that allows the product to bloom and regain the desirable red color. Another strategy
employed is called master packaging (Belcher 2006). In this strategy, conventional
retail packs are wrapped in gas-permeable film and then placed in a larger high-­
barrier bag filled with a low O2 gas mixture as described above. After the outer bag
is removed, the retail pack is allowed to bloom.
Active packaging (AP) refers to the incorporation of specific compounds into
packaging systems or materials that interact with the meat or the packaging environ-
ment to maintain or extend the shelf-life and product quality (Kerry et al. 2006). AP
technologies for the extension of shelf-life and improvement of meat quality may
include: moisture control, O2-permeable films, O2 scavengers or absorbers, O2 gen-
erators, CO2 controllers, odor controllers, flavor enhancers, ethylene removal, and
antimicrobial agents (Brody 2009; Coma 2008). A leading strategy for AP is the
inclusion of antimicrobial substances in what is termed antimicrobial packaging.
Antimicrobial packaging may be achieved using one or more of five techniques
(Cooksey 2005): (1) attachment of a sachet bearing volatile antimicrobial agents to
the inner surface of the packaging material and release of active volatile compounds
during storage, (2) direct incorporation of an antimicrobial agent in the packaging
material, (3) coating of the packaging material with a matrix-carrier (i.e., chitosan)
of antimicrobial agents, (4) non-food-grade polymers containing non-diffusible bio-
cides (acting only on the food surface such as chitosan and triclosan) and (5) edible
coatings containing antimicrobials that are applied directly to meats. Antimicrobial
agents used in AP may either migrate into the food through diffusion and partition-
ing or be released through evaporation into the package headspace (Coma 2008).
Potential antimicrobial agents that may be considered for use in AP are organic
acids, acid salts, acid anhydrides, para-benzoic acids, alcohol, bacteriocins, fatty
acids, fatty acid esters, chelating agents, enzymes, metals, antioxidants, antibitoics,
fungicides, sterilizing gases, sanitizing agents, polysaccharides, phenolics, plant
volatiles, plant and spice extracts and biopreservative organisms (Cutter 2006). One
of the most promising technologies for AP is the inclusion of phenolic compounds
and essential oils (primarily allyl isothiocyanate, rosemary, oregano, carvacrol, and
eugenol) into packaging films, encapsulated as bioactive edible coatings or applied
as surfactant micelles (Drosinos et al. 2009).
Smart packaging (also known as intelligent packaging) is the incorporation of
compounds into the packaging material that can monitor and transmit information
on the quality and/or spoilage status of the meat to the processor, retailer and/or
consumer (Kerry et al. 2006). The compounds built into smart packaging act as
biosensors or bioindicators for a variety of measurements including detection of
residual O2 levels, spoilage metabolites, temperature monitoring and package integ-
rity (Potter et al. 2008). The biosensors/bioindicators are primarily color-changing
and are highly correlated to the levels of the compounds they are detecting. One of
the more successful uses of smart packaging is the use of biosensors to indicate
248 J.D. Stopforth

levels of spoilage metabolites through detection of volatiles such as the concentra-

tion of amines (microbial breakdown products) (Pacquit et al. 2007). Biosensors are
not effective in monitoring growth of pathogenic microflora on meat since patho-
genic organism are very rarely responsible for deterioration of meat quality and
organoleptic properties (flavor, color and odor).

4 Hurdle Technology

Hurdle technology (HT) also known as combined processes, combination tech-

niques/strategies or barrier technology proposes the deliberate use of a combination
of preservation methods to establish a series of hurdles for microbial growth and
survival in an attempt to maintain or improve the quality and safety of meat (Leistner
2000). As described earlier, microbial growth in food is influenced by various fac-
tors classified as extrinsic (environmental) such as temperature and gas atmosphere,
intrinsic properties of the food such as pH and aw, process-related factors such as
heat treatment or antimicrobial treatment and implicit factors (related to the micro-
organism). In order to control the growth of microorganism, it is essential that at
least some of these factors be kept to sub-optimal or even lethal levels. In general, it
is not feasible to rely on a single sub-optimal or lethal factor in the processing of
meat as this affects the organoleptic qualities of the product. Leistner (2000) has
reviewed more than 60 potential hurdles that may be applied during food preserva-
tion, the most important of which include combinations of temperature (high or
low), aw, pH, oxidation-reduction potential (Eh), addition of chemical antimicrobi-
als and biopreservatives cultures such as LAB. However, with the evolution of new
technologies as presented in this chapter, the possibilities for HT increase signifi-
cantly. Of the most promising new technologies that can be considered for incorpo-
ration in HT are the use of novel and natural antimicrobial ingredients such as
volatile compounds and essential oils, non-thermal technologies such as irradiation
and HHP, biopreservation using bacteriocins and active packaging with natural anti-
microbials. The most successful application of HT in future applications will be the
combination of traditional preservation methods with the advanced and developing
methods mentioned above. Success in combining new preservation methods has
already been demonstrated:
• Use of MAP combined with oregano essential oil resulted in a longer shelf-life
of fresh meat compared to use of MAP alone (Chouliara et al. 2007)
• Use of VP combined with rosemary and oregano essential oils resulted in
increased shelf-life of cooked poultry (Ntzimani et al. 2010)
• Use of HHP combined with nisin and lactates increased the shelf-life of cooked
meat (Aymerich et al. 2005)
• Use of HHP, enterocin (bacteriocin), and refrigeration at 1 °C resulted in signifi-
cant reduction in pathogenic L. monocytogenes on cooked meat (Marcos et al.
2008)
11 Preservation Methods for Meat and Poultry 249

5 Conclusions and Future Perspectives

Technologies employing combinations of traditional and new preservation methods

to establish a series of preservative effects that limit microbial activity is an essential
approach that has tremendous potential to improve the shelf-life, quality and safety
of meat and meat products. The microorganism’s physiological responses during
storage are the basis for selection of applicable technologies. Although microbial
stress responses complicate meat preservation, the critical element of effective pres-
ervation rests with a good understanding of the homeostasis of microorganisms.
Meat preservation is most effectively achieved by disturbing the homeostatic mech-
anisms of microorganisms and the best way to accomplish this is to disturb several
of the homeostatic mechanisms simultaneously. The multi-faceted approach is the
essence of combination strategies which will allow use of different preservation
methods applied at lower intensity than when applied singly thereby not only acting
synergistically or additively but also minimizing negative impacts on the organolep-
tic properties of the meat. Future approaches should focus on technologies that have
proven effective when applied singly and in combination and those that are already
approved or in the process of being approved for use in foods. Of the technologies
described in this chapter, there are specific technologies that meet these criteria and
should be the focus of future research and development efforts to develop
commercially-­feasible applications that are effective, affordable and accepted by
consumers. The specific technologies requiring further development for targeted
applications of public and commercial importance are:
• The use of HHP in inactivating a wider range of microorganisms important to the
safety and shelf-life of meat, specifically the optimization of this technology to
inactivate spore-forming bacteria. It has been established that pressures exceed-
ing 1000 mPa are needed to inactivate these organisms. This is, however, not
feasible from a cost standpoint given the amount of energy needed to achieve
high pressures or from a product quality standpoint given the deteriorative effects
of high pressures. Research has shown that the combination with antimicrobials
may reduce the amount of pressure needed to control spores; however, there is no
current commercially-feasible solution to accomplish this in meat.
• The use of both non-ionizing and ionizing radiation either singly or in combina-
tion with refrigeration to control microorganisms of particular public health sig-
nificance (i.e., pathogenic E. coli) on fresh meat. Pathogenic E. coli is of
particular concern in non-intact fresh meat and although the industry has applied
traditional preservation methods including application of antimicrobials and
chilling/freezing to control this pathogen, it is still of major public health con-
cern especially to the young, elderly and immune-compromised. A possible
approach requiring further research and development is the use of high-intensity
light (UV light) to sanitize carcass surfaces combined with the use of γ-irradiation
of ground meat derived from these carcasses to render fresh ground meat free of
the pathogen. These technologies can further be combined with incorporation of
natural antimicrobials in the ground meat to counteract any undesirable organo-
250 J.D. Stopforth

leptic effects of irradiation while also reducing the dose of irradiation needed to
treat the meat.
• The use of natural antimicrobials such as volatile compounds and essential oils
applied as surface treatments or incorporated into packaging with the use of bac-
teriocins applied as surface technologies to ready-to-eat meats to control patho-
genic L. monocytogenes as well as improve the overall shelf-life of these
products. Both these technologies have shown promise when applied singly or in
combination with traditional preservation technologies such as heat and chilling
and thus should provide promising results when used in combination along with
traditional preservation methods.
While much research has been performed in the field of new preservation meth-
ods and hurdle technology, many key issues still need to be addressed with regards
to combining traditional and new preservation methods to be useful in the meat
industry to meet consumer demands of improved shelf-life and quality and impor-
tant public health concerns.

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Chapter 12
Microbial Control of Milk and Milk Products

Mustafa Guzel and Yesim Soyer

Abstract Milk has been the one of the main nutrient sources of human diet for
centuries. Microbial studies on milk date back to the seventeenth century, when
Kircher used a microscope, and observed the minute worms in milk. Two centuries
later, in the1850s, Pasteur proved that the spoilage of milk resulting the sour taste
was caused by microorganisms. Pasteur’s discoveries on the effect of heat on unde-
sirable microorganisms in beer and wine opened a new era in food science.
Therefore, the process was named “pasteurization”. In the following years of his
invention, pasteurization was conducted in Germany and the U.S.A. (Jay, Modern
food microbiology. Aspen Publishing, Gaithersburg, MD, 2000). Another break-
through in milk safety was refrigeration, which became popular after the 1950s.
With the advances in heat treatment and low temperature storage, shelf life of pas-
teurized milk had been increased significantly. Today, pasteurization, partial steril-
ization, refrigeration, dehydration, and fermentation are commonly used to increase
the shelf life of milk and dairy products. Besides these traditional methods, there are
also novel methods used to prevent dairy products from spoilage and pathogen con-
tamination such as high pressure processing an UV light.

Keywords Milk preservation • Thermal processes • Novel processes • Non-thermal

processes • Natural antimicrobials

1 Introduction

Milk has been the one of the main nutrient sources of human diet for centuries.
Microbial studies on milk date back to the seventeenth century, when Kircher
used a microscope, and observed the minute worms in milk. Two centuries later,

M. Guzel
Department of Food Engineering, Hitit University, Corum, Turkey
e-mail: [email protected]
Y. Soyer (*)
Department of Food Engineering, Middle East Technical University (ODTU), Ankara, Turkey
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 255

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_12
256 M. Guzel and Y. Soyer

in the1850s, Pasteur proved that the spoilage of milk resulting the sour taste was
caused by microorganisms. Pasteur’s discoveries on the effect of heat on unde-
sirable microorganisms in beer and wine opened a new era in food science.
Therefore, the process was named “pasteurization”. In the following years of his
invention, pasteurization was conducted in Germany and the U.S.A. (Jay 2000).
Another breakthrough in milk safety was refrigeration, which became popular
after the 1950s. With the advances in heat treatment and low temperature stor-
age, shelf life of pasteurized milk had been increased significantly. Today, pas-
teurization, partial sterilization, refrigeration, dehydration, and fermentation are
commonly used to increase the shelf life of milk and dairy products. Besides
these traditional methods, there are also novel methods used to prevent dairy
products from spoilage and pathogen contamination such as high pressure pro-
cessing an UV light.
Shelf-life of dairy products is directly attributed to their microbial quality. Milk
can easily be spoiled by microorganisms developing off-odors, off-colors, and off-­
flavor. Some notable spoilage agents in milk are Pseudomonas, Yersinia (Gram
negatives), Micrococcus, Clostridium, and Bacillus (Gram positives) (Whitfield
et al. 2000). Among these microorganisms, Micrococcus shows thermoresistance,
while Clostridium and Bacillus are sporeformers. Furthermore, microbial quality
is also important for public health as dairy products provide an ideal medium to
pathogens like Listeria monocytogenes, Staphylococcus aureus, and Campylobacter
jejuni (Jayaroa and Henning 2001). Moreover, it has been shown that methicillin
resistant S. aureus (MRSA) can contaminate milk (Normanno et al. 2007). In the
U.S., dairy products are the second most commonly attributed food commodity to
foodborne illnesses after fresh produce, and the most frequent reason of hospital-
izations (Painter et al. 2013). Contamination of milk may occur due to inadequate
cleanness of farm environment, cows, and milking equipment, invalid disinfection
before and between milking, and mastitis (Akam et al. 1989; Sanaa et al. 1993;
Chambers 2002). Akam et al. (1989) reported that microorganisms in farm envi-
ronment might contaminate the animal teats via dirt, and contaminate milk. In
addition, pathogens may transmit to milk via feed. Sanaa et al. (1993) highlighted
that poor quality of silage attributed to Listeria monocytogenes contamination sig-
nificantly. Similar observations were made for other common foodborne patho-
gens such as Salmonella and Escherichia coli (Davis et al. 2003; Dargatz et al.
2005). To prevent microbial contamination and growth in milk, good farming
practices, including management of animal health, feed cleanness, farm and facil-
ity hygiene, milking, milking and storage equipment are required. (Vissers and
Driehuis 2009).
In this chapter, commonly used traditional methods, as well as promising appli-
cations of those methods were evaluated mainly in terms of microbial inactivation
in milk and milk products.
12 Microbial Control of Milk and Milk Products 257

2 Microbial Control Via Thermal Processing Methods

Thermal processing of milk includes the processes thermization, pasteurization, and

sterilization. Main goals of thermal treatment are to reduce both pathogen and spoil-
age microorganisms, inactivate enzymes, and reduce chemical and physical changes
(Lewis and Deeth 2009). The effect of heat on microorganisms has been well stud-
ied in terms of reaction kinetics and principles. However, according to Langer et al.
(2012), 48 of 121 outbreaks were associated with pasteurized dairy products, and
resulted in more than 2800 cases between 1996 and 2012. Source of contamination
was able to be determined in 7 of 48 cases, and 4 of those 7 cases were associated
with post pasteurization contamination. The authors suggested that remaining cases
probably linked to temperature abuse in consumer homes. Furthermore, the most
common infectious agent was norovirus in the outbreaks associated with pasteur-
ized dairy products suggesting that poor handling was the reason of the contamina-
tion. On the other hand, in non-pasteurized dairy product outbreaks, the causative
agents were bacterial pathogens (Langer et al. 2012). The results of that study
clearly show that although the heat treatment has been applied for a long time, con-
sumption of raw milk and non-pasteurized dairy products still causes foodborne
disease outbreaks.

2.1  asteurization and Ultra High Temperature (UHT)

P
Processing

Heat treatment has been used to process safe milk and milk based products with
extended shelf life for a century. Most common heat treatments include thermiza-
tion, pasteurization, and UHT processing (Table 12.1). Thermization, also called as
subpasteurization, is a process that applied to milk to extend the shelf life before
further processing. It is carried out to inactivate pyschrotrophs such as Pseudomonas

Table 12.1 Types of heat treatment used in milk preservation (Kelly et al. 2012)
Process conditions
Process type (temperature, time) Targets
Thermization 57–68 °C, 15 s Psychrotrophs
Batch pasteurization 63 °C, 30 min Vegetative pathogens
High temperature short time 72–74 °C, 15–30 s Vegetative pathogens
(HTST)
Higher heat shorter time (HHST) 89 °C, 1 s; 94 °C, 0.1 s, Vegetative pathogens
100 °C, 0.01 s
High temperature pasteurization 120–130 °C, <1–5 s Vegetative bacteria and most
(ESL) spores
Ultra high temperature (UHT) 135–140 °C, 3–5 s All bacteria and spores
258 M. Guzel and Y. Soyer

sp. to prevent them to produce spoilage enzymes. After then milk is cold stored until
the manufacture of dairy products such as cheese and milk powder (Senyk et al.
1982; West et al. 1986).
Pasteurization is well defined in terms of target microorganisms and efficacy
indicator. Typically, most heat resistant pathogens, Coxiella burnettii and
Mycobacterium tuberculosis, are targeted, and after the pasteurization the inactiva-
tion of alkaline phosphatase enzyme is tested as this enzyme show similar kinetics
with those pathogens. Since pasteurization inactivates all pathogens except
­sporeformers (e.g., Clostridium perfringens, Bacillus cereus), it is assumed that
pasteurization following by cold storage ensures the safety of milk. However, inad-
equate process conditions, post-process contamination and temperature abuse still
cause foodborne diseases associated with dairy products (Langer et al. 2012).
Furthermore, recent studies show that L. monocytogenes may survive during the
pasteurization (Rebagliati et al. 2009; Wen et al. 2009).
UHT processing is always combined with aseptic packaging to produce com-
mercially sterile milk with a shelf-life between 3 and 12 months at room tempera-
ture (Farkas 2007; Lewis and Deeth 2009). Apart from some dormant spores, all
bacteria and spores are inactivated (Kelly et al. 2012). Post process contamination
is not a big concern in UHT processing due to aseptic packaging. Although UHT is
considered as microbiologically safe, nutrient loss and off-flavor development are
big concerns.
Recently, studies on heat treatment are focused on increasing the shelf life while
minimizing effect on nutrient and sensory quality. In this regard, another form of
pasteurization, high temperature pasteurization or extended shelf life (ESL), has
been introduced. ESL is in between HTST and UHT in terms of process conditions.
Unlike UHT, ESL milk should be stored under refrigeration temperatures because
all spores are not inactivated. ESL should be combined with either ultraclean or
aseptic packaging to eliminate/minimize the risk of post process contamination.
While aseptic packaging extends the shelf life of ESL, ultraclean packaging is gen-
erally preferred because of the cost (Lewis and Deeth 2009).
Combination of heat with non-thermal treatments (e.g., high pressure, pulsed elec-
tric field) or natural antimicrobials (e.g., bacteriocins, essential oils, endolysins) is
aimed to reduce the amount of heat, and thus decrease the effect of heat on organolep-
tic properties, or increasing the shelf life while using typical pasteurization conditions.
For example, Wirjantoro and Lewis (1996) determined that when pasteurization
(HTST) was combined with nisin, the shelf life of milk increased significantly. Further
studies on the effect of heat combinations on the microorganisms and shelf life of milk
were mentioned in the non-thermal and antimicrobials sections.

2.2 Novel Techniques

Due to quality losses in organoleptic properties of milk as a result of traditional

thermal methods, new thermal processes have been developed to minimize the
losses and saving nutritional qualities of milk while keeping the benefits of
12 Microbial Control of Milk and Milk Products 259

Table 12.2 The overview of novel thermal processes

Process type Main process parameters Reference
Microwave Frequency, t, T (915–2450 MHz) Clare et al. (2005)
Radiofrequency Frequency, t, T (13.56–40.68 MHz) Tewari and Juneja (2007)
Ohmic heating t, T de Alwis and Fryer (1990)

traditional methods (i.e., destruction of most of the spoilage bacteria). In novel pro-
cessing techniques, radiation is the main mechanism of heat transfer, whereas heat
is transferred via conduction and convection in conventional thermal processes
(Table 12.2). Furthermore, instead of fossil fuels, in novel techniques, heat is gener-
ated via electricity, which provides easier manipulation and control, while being
more environmental friendly.

2.2.1 Microwave and Radiofrequency Heating

Microwave is a form of non-ionizing, electromagnetic radiation with wavelengths

between 300 MHz and 300 GHz. In industrial applications, 915 MHz and 2450 MHz
are the most used frequencies. The mechanism of microwave relies on the rotation
of water molecules (electric dipoles), due to the microwave energy. The reorienta-
tion of water molecules causes intermolecular frictions, which generate heat.
Microwave heating has some advantages over conventional processing methods.
First, heating process with microwave is faster than traditional methods. Therefore,
it is possible to achieve same microbial reductions as with conventional methods,
while minimizing the negative effects of thermal process on nutrients, flavor, and
sensory characteristics. Furthermore, microwave pasteurization can be applied to
the packaged foods, and thus prevent the post treatment contamination. Some of the
most used packaging materials in dairy industry such as glass, paper and plastic are
suitable for microwave heating.
First published study on the microwave pasteurization of milk was conducted in
1969 (Hamid et al. 1969). Since then, many other researchers have been studied the
effects of microwave on the organoleptic properties of milk. Choi et al. (1993a, b)
studied the effect of microwave heating on Listeria monocytogenes, Yersinia entero-
colitica, and Campylobacter jejuni at 71.1 °C. It was reported that complete inacti-
vation occurred at 10, 8, and 3 min for L. monocytogenes, Y. enterocolitica, and C.
jejuni respectively (Choi et al. 1993a, b). Villamiel et al. (1996) compared the shelf
life of cow’s milk pasteurized with a continuous microwave system and conven-
tional plate heat exchanger. The results showed that microwave pasteurization not
only prolonged the shelf life, but also provided higher sensory qualities than plate
heat exchanger. Clare et al. (2005) designed a continuous microwave heating sys-
tem, and compared that system with indirect UHT. Both systems effectively inacti-
vated microorganisms and provided a long shelf life. Researchers concluded that
microwave might be an alternative processing method of long shelf life milk.
260 M. Guzel and Y. Soyer

Since each of come up and come down profile of microwave heating is much
lower than conventional methods, effect of thermal destruction on milk nutrients is
limited. However, non-uniform distribution is a big concern, as it carries the risk of
microbial survival (Ohlsson 1990). As a consequence, continues-flow systems that
designed specifically for milk have been proposed (Lopez-Fandino et al. 1996;
Villamiel et al. 1996; Clare et al. 2005).
Radiofrequency heating is another form of nonionizing radiation that has a
wavelength between 3 kHz and 300 MHz. In food applications, generally three
frequencies (13.56, 27.12, and 40.68 MHz) are used (Tewari 2007). Because of its
lower frequency, radiofrequency heating has more penetrating power than micro-
wave. Awuah et al. (2005) used a 2 kW, 27.12 MHz radiofrequency applicator to
evaluate the inactivation of surrogates Listeria innocua and Escherichia coli K12
under laminar flow. It was concluded that radiofrequency might be an alternative to
conventional heating exchangers. However, in spite of its high antimicrobial effect
and very short processing time, radiofrequency has been failed to draw attention.
Further studies are needed to rationalize the potential application of radiofrequency
heating under different flow conditions (Marra et al. 2009).

2.2.2 Ohmic Heating

Ohmic Heating, also called electroheating or joule heating, is based on heat genera-
tion in foods by application of electric resistance (de Alwis and Fryer 1990). In
ohmic heating, electrodes are in contact with food material that provides more uni-
form heat distribution compared to microwave heating. Although ohmic heating
was popular in early nineteenth century (Getchel 1935; Moses 1938), it was replaced
with heat exchangers, due to high application costs and short supply of inert materi-
als required for electrodes (Mizrahi et al. 1975; de Alwis and Fryer 1990). However,
the interest in this technology has been increased in the last three decades because
it is possible to produce higher quality products because of faster processing times
(Castro et al. 2003). Microbial inactivation with ohmic heating occurs not only
through thermal but also non-thermal destruction, namely electroporation (Vicente
et al. 2005).
One of the applications of ohmic heating is the pasteurization of milk (Fillaudeau
et al. 2006). Sun et al. (2008) compared the microbial inactivation of ohmic heating
and conventional heating in milk under same temperature conditions. Authors
reported that D values and final microbial counts for ohmic heating were signifi-
cantly lower than those obtained with conventional heating. It is highlighted that the
difference between two methods occurred due to non-thermal effects of ohmic heat-
ing (Sun et al. 2008). Pereira et al. (2007) found that with ohmic heating, the death
kinetics of E. coli in goat milk was significantly lower than conventional heating. It
was reported that observed D values for E. coli were 0.86 and 3.2 min for ohmic
heating and conventional heating respectively. Similar to previous studies, authors
reported that non-thermal effect of ohmic heating was the main reason behind the
difference.
12 Microbial Control of Milk and Milk Products 261

3 Microbial Control Via Non-thermal Processing Methods

Conventional heating methods have been used for a long time in dairy industry due
to their effectiveness in microbial control. In addition to microorganisms, heat also
deactivates enzymes that attributed to shelf-life. However, it has been shown that
heat cause undesirable changes in physical and chemical properties of milk. These
changes include, but not limited to loss of nutrients, brown color formation and
cooked flavor (Ansari and Datta 2007; Lima 2007; Marra et al. 2009). In addition to
prevent undesirable changes, and the increasing trend of awareness and consumer
demand to fresh products force the food industry to develop alternative methods to
conventional heating techniques. Non-thermal processing methods aim to provide
the same protective effect of thermal pasteurization, without damaging nutrients
and sensory characteristics (Table 12.3). Furthermore, non-thermal methods
endeavor to achieve lower cost, minimal environmental impact, increased shelf-life,
and additive free products.

3.1 Pulsed Electric Fields

Pulsed electric fields (PEF) is the application of high voltage electric fields (20–
80 kV/cm) for short durations. Contrary to ohmic heating and other electric based
thermal treatments, PEF is a non-thermal treatment that relies on the high intensity
pulses. The electric field is generated in foods in an insulated treatment chamber
with two electrodes positioned 0.1–1 cm away from each other. It has been stated
that the best application of PEF is to liquid and semi liquid foods such as milk and
yogurt as ions are needed for electric charge. The main microbial inactivation mech-
anism of PEF is a phenomenon called as “electroporation”. An external potential
difference is triggered with electric field generation. When this potential difference
is higher than the maximum potential difference that cell membrane can endure,

Table 12.3 The overview of non-thermal processes (Villamiel et al. 2009)

Process Type Main process parameters Targets
Pulsed electric fields Frequency, number of pulses, field Vegetative pathogens/No
(PEF) strength, (20–80 kV/cm) effect on spores
High pressure Pressure, t, T (13.56–40.68 MHz) Vegetative pathogens/limited
processing (HPP) effect on spores
Ultrasound (US) Frequency, t, T Limited effect on vegetative
pathogens and spores
Pulsed light Wavelength, number of pulses Vegetative pathogens/limited
effect on spores
UV radiation Wavelength Vegetative pathogens/limited
effect on spores
Microfiltration Membrane type, flow rate, pressure Vegetative pathogens and
spores
262 M. Guzel and Y. Soyer

pores are formed due to breakdown of membrane (Zimmermann 1986). At higher

electric field levels (>25 kV/cm), pore formation is irreversible (van Heesch et al.
2000). Pulse length, number of pulses, initial temperature, pulse number, composi-
tion and pH of food, type of microorganism, and electric field strength are the most
important parameters of PEF application.
In one of the first studies, Dunn and Pearlman (1987) treated Salmonella Dublin
inoculated milk with PEF, and stored for 8 days. After PEF application (36.7 kV/
cm), no S. Dublin was detected during storage period. Furthermore, while natural
microflora increased to 107 cfu/mL level in untreated control group, only reached to
4 × 102 cfu/mL level in treated samples. Evrendilek and Zhang (2005) applied PEF
(24 kV/cm) to skimmed milk, and 1.88 log reduction was observed in E. coli popu-
lation. In a similar study, Dutreux et al. (2000) applied PEF (41 kV/cm) to E. coli
and L. innocua inoculated skimmed milk, and achieved 4.5 and 4 log cfu/mL reduc-
tion, respectively. In another study, Fernandez-Molina (2001) applied PEF (50 kV/
cm) to skim milk, and observed 2.6 and 2.7 log reductions in L. innocua and P. fluo-
rescens populations respectively.
Combination of PEF with other treatments such as temperature and bacteriocins,
increases the effectiveness of the treatment. For example, Smith et al. (2002b) com-
bined PEF (80 kV/cm at 52 °C) with nisin and lysozyme to inactivate the natural
microflora. Among the combinations, PEF plus lysozyme reduced the microbial
load 3.2 log, while reduction was 5.7 log with PEF plus nisin. When lysozyme and
nisin are used together along with PEF, total reduction was more than 7 log. The
combination of antimicrobials with PEF was tested in different studies, and similar
results were reported (Fernandez-Molina et al. 2005a; Sobrino-López and Martín-­
Belloso 2006; Sobrino-López et al. 2009). Sobrino-López et al. (2009) evaluated
the synergetic effect of PEF, nisin, enterocin AS-48, and lysozyme. It was found that
nisin and enterocin AS-48 increased the effectiveness of PEF treatment. Furthermore,
higher log reduction was achieved when both antimicrobials were added prior to
PEF treatment. It was concluded that antimicrobial addition before PEF treatment
increased the sensitivity of cell membrane for potential difference.
A similar synergetic effect was observed in the combination of PEF and a mild
heat treatment. Fernandez-Molina et al. (2005b) applied PEF (30 kV/cm) following
a heat treatment (80 °C, 6 s) to skim milk and prolonged shelf-life to 30 days. In a
similar study, PEF (35 kV/cm) was applied after heat treatment (72 °C, 15 s), and
more than 60 days of shelf-life was achieved (Sampedro et al. 2007). Yeom et al.
(2004) combined PEF (30 kV/cm) with heat (60 °C, 30 s) in a yogurt based product,
and reported that combined treatment extended the shelf-life for 90 days, which was
three times higher than untreated samples.

3.2 High Pressure Processing

High pressure processing (HPP) also called as high hydrostatic pressure, relies on
an instantaneous and uniform compression from all directions (Neeto and Chen
2014). HPP is one of the most popular non-thermal processing methods since the
12 Microbial Control of Milk and Milk Products 263

early 90’s because of its potential to kill microorganisms without damaging

organoleptic properties (Jung et al. 2011). HPP has some other advantages over
other processing methods like faster process times, and in package processing
(Koutchma 2009). The main mechanism of inactivation is the breakdown of non-
covalent bonds (Tewari 2007). It is also believed that HPP affects morphology,
genetic mechanisms and disrupts the cell membrane (Casadei et al. 2002; Patterson
2005). Also, a recent study revealed that inactivation occurs due to cell wall rup-
ture, membrane damage, and DNA degradation (Yang et al. 2012). The applica-
tion pressure ranges between 100 and 1000 MPa. The efficacy of HPP depends on
pressure level, process temperature, microbial types, water activity, cell growth
phase, and time (Alpas et al. 1999; Manas and Mackey 2004; Tewari 2007). It was
reported that under room temperatures, HPP is capable to inactivate most patho-
genic and spoilage bacteria. However, bacterial spores are much more resistant to
pressure (Villamiel et al. 2009).
The first study on the effects of HPP on microorganisms in milk was conducted
more than a century ago by Hite (1899). Since then, many other researchers studied
or reviewed the pasteurization of milk with HPP (Dow and Mathews 1939; Timson
and Short 1965; Rademacher and Kessler 1996; Balci and Wilbey 1999; Datta and
Deeth 1999; Alpas and Bozoglu 2000; Lopez-Fandino 2006). Garcia-Risco et al.
(1998) reported that when milk was pasteurized with HPP at room temperature
(400 MPa for 30 min), no psychrophilic bacteria was detected. However, after
45 days of storage at 7 °C, psychrophilic bacteria count exceeded 7 log cfu/mL
level. It was proposed that microorganisms might be injured sub-lethally after the
treatment, and covered slowly during the storage (Garcia-Risco et al. 1998). In a
study, Patterson et al. (1995) treated UHT milk with HPP at 20 °C to determine the
inactivation of pressure resistant strains of pathogens E. coli O157:H7, S. aureus,
and L. monocytogenes. Only <2 and 2 log cfu/mL reductions were observed in the
population of E. coli O157:H7 and S. aureus subjected to 600 MPa for 15 min,
while <1 log cfu/mL reduction was achieved at 375 MPa for 15 min in the popula-
tion of L. monocytogenes, which was reportedly the most resistant strain among
those three pathogens. It was proposed that the low reductions in the pathogen pop-
ulations might be due to breakdown of heat sensitive anti-microbial compounds in
UHT milk, and the results might be different in raw milk (Datta and Deeth 1999).
Gallot-Lavallee (1998) reported that HPP at 450 MPa for 10 min reduced the
L. monocytogenes level by more than 5 log in goat milk cheese.
HHP can be combined with other treatment methods to increase the microbial
reduction and reduce the operational costs. Combined processes also enable milder
treatment parameters, and thus limits the nutrient losses. Also known as the pressure
assisted thermal sterilization, HPP with a mild heat treatment is the most well
known process combination. With this combination, it is possible to inactivate bac-
terial spores which is a drawback of HPP alone. Alpas et al. (2000) evaluated the
effectiveness of HPP (345 MPa) and temperature (50 °C) combination on several
pathogens in pasteurized milk. Apart from a S. aureus strain, more than 8 log reduc-
tion was observed in pathogen (L. monocytogenes, E. coli, O157:H7, and Salmonella
ssp.) populations. After 3 days of incubation period, L. monocytogenes cells were
recovered, while Gram-negative pathogens were not detected. In the same study,
264 M. Guzel and Y. Soyer

Alpas et al. (2000) also evaluated the addition of bacteriocin at the same pressure
(345 MPa) and temperature (50 °C) conditions. More than 8 log reductions were
observed in both S. aureus and L. monocytogenes populations. Furthermore, milk
was stored at 25 °C for 30 days, and no cfu was detected. It was concluded that
when combined with other treatments, HPP could effectively destroy the aforemen-
tioned pathogens in milk. Combination of HPP and bacteriocin (nisin) was also
extended the shelf-life of cheese (Ray 1992). Similar to bacteriocins, essential oils
like carvacrol (Karatzas et al. 2001), and antimicrobials like lactoperoxidase
(Garcia-Graellis et al. 2003) were combined with HPP successfully to reduce patho-
gen concentrations in milk.

3.3 UV Radiation

Although UV light has been used for equipment sanitation for a long time, it has
also been used in food industry for microbial inactivation as a non-thermal tech-
nique. Main inactivation mechanism of UV lies in nucleic acid level. UV light can
interrupt replication by forming pyrimidine dimers in DNA and RNA (Cutler and
Zimmerman 2011). Based on the wavelength, UV rays are categorized as UV-A,
UV-B, and UV-C. Among them, UV-C (200–280 nm) is used in food safety applica-
tions, while others are used for different purposes such as water disinfection. The
effect of UV light on microbial load in milk and dairy products depends on the
product type, soluble solids, type of the microorganism, and UV absorptivity.
Because of high soluble solid content and opaque color, milk is not an ideal medium
for UV treatment. However, UV light has gained interest in dairy industry because
of low initial and operational costs and easy application possibilities at ambient
temperatures.
Microbial inactivation kinetics of UV light treatment has been studied exten-
sively. Matak et al. (2005) found that a 2 s treatment of UV light at 15.8 ± 1.6 mJ/
m2 resulted in more than 5 log reduction in L. monocytogenes, E. coli, and
Cryptosporidium parvuum populations in goat’s milk. Pereira et al. (2014) inocu-
lated whole milk with several pathogens, and treated with UV light under continu-
ous flow process. After the treatment L. monocytogenes, Salmonella spp., E. coli, S.
aureus, and Streptococcus spp., reduced by 3.2, 3.7, 2.8, 3.4, and 3.4 log cfu/mL,
respectively. However, reduction in Mycobacterium smegmatis population was not
found significant. Similar observations were reported by Altic et al. (2007) in a
study, where 1 kJ/L UV light treatment reduced only 0.5 and 1 log Mycobacterium
avium subsp. paratuberculosis population in whole and semi skimmed milk, respec-
tively. It might be concluded that Mycobacterium species were resistant to UV light.
Although it is possible to achieve higher reductions with higher doses of UV light,
application dose should be 1 kJ/L at most for sensory quality (Reinemann et al.
2006). In fact, even at lower doses such as 15.8 ± 1.6 mJ/m2 sensory parameters
such as odor affected negatively (Matak et al. 2005). Oxidation of milk proteins,
formation of volatile compounds, and losses in micro nutrients were also highlighted
12 Microbial Control of Milk and Milk Products 265

as serious drawbacks of UV light treatment (Scheidegger et al. 2010; Webster et al.

2011; Guneser and Karagul Yuceer 2012).
To overcome these drawbacks, several reactors were designed. For example,
SurePure Turbulator™, a commercially available UV light applicator, aims to
negate the drawbacks associated with UV treatment. Several studies showed that
new designs might be valuable alternatives to conventional processing techniques in
milk and cheese production (Rossitto et al. 2012; Cilliers et al. 2014; Cappozzo
et al. 2015). Kim et al. (2015) developed UVC light emitting diodes (LED) to over-
come the technological limitations of common UV lamps. In addition to technical
advantages, newly developed UV LEDs showed significantly different reduction
compared to standard UV lamps. Following the UV LED treatment at 3 mJ/cm2,
L. monocytogenes, E. coli O157:H7, and Salmonella typhimurium counts reduced
by 4–5 log in sliced cheese.

3.4 Pulsed Light

Pulsed light (PL), also called as high intensity pulsed light, is an emerging technol-
ogy that inactivates microorganisms with intense, very short duration pulses
(flashes) of light from inert gas lamps. The frequency region covers a broad spec-
trum from UV light (180 nm) to infrared rays (1100 nm). The inactivation mecha-
nism relies on photophysical, photochemical, and photothermal effects (Palmieri
and Cacacea 2005; Krishnamurthy et al. 2010; Miller et al. 2012). These photo
related effects damages cells morphologically at the nucleic acid level (Palmieri and
Cacacea 2005). Although PL may harm nutrients at high doses, occurrence of nega-
tive effects can be controlled nature of the process (Demirci and Krishnamurthy
2011).
PL can be modified to use UV as the only source of light which is named as
pulsed UV light (PUVL). PUVL was found to be more than two times effective than
continuous UV light systems (Palgan et al. 2011). One of the most important fea-
tures of PUVL was reported that microorganisms did not show innate resistance,
means that unlike temperature or pressure, there is no PUVL resistant bacteria
(Dunn et al. 1995). Also, negative effects on product quality is very limited due to
very short treatment times (Demirci and Panico 2008) and limited oxidation reac-
tions (Krishnamurthy et al. 2007).
Smith et al. (2002a) treated milk with PL, and investigated fate of mesophilic
bacteria. After the PL treatment (25 J/m2) mesophilic bacteria was under detection
limits. What is more, there was no sign of recovery after 21 days as no growth was
observed. Krishnamurthy et al. (2007) studied the effect of treatment parameters
(distance from source, number of passes, and flow rate) on the effectiveness of a
continuous PL system. S. aureus inoculated milk was treated with PL at different
exposure rates. It was reported that complete inactivation achieved when the sam-
ple passed 8 cm distance from source at 20 mL/min flow rate (Krishnamurthy
et al. 2007).
266 M. Guzel and Y. Soyer

3.5 Ultrasound

Ultrasound (US), also referred as sonication and ultrasonication, is the sound waves
with the frequency above 20 kHz. Based on the intensity level, US can be catego-
rized under two groups; high intensity US with low frequency, and low intensity US
with high frequency. Although both categories have different food applications,
high intensity US is found to be more efficient in terms of antimicrobial activity
(Demirdöven and Baysal 2009). High intensity US, also called as power US, covers
the frequency range between 20 and 100 kHz. As ultrasound travels through media,
alternating compression and expansion cycles are created. In the expansion cycles,
microbubbles (cavities) are formed. These bubbles eventually implode and generate
shockwaves, turbulence, local temperature rise and pressure. The antimicrobial
activity of US comes from this phenomenon that is also called as cavitation
(Ashokkumar et al. 2004, 2010; Ashokkumar and Grieser 2007).
It was reported that Gram-positive and cocci shaped bacteria are more resistant to
ultrasound than Gram-negative and rod shaped cells (Hulsen 1999; Feng et al. 2008).
Cameron et al. (2009) demonstrated that 10 min ultrasonication reduced the popula-
tions of E. coli by 100% and L. monocytogenes by 99%, whereas Pseudomonas fluo-
rescens population reduced by 100% after 6 min, without damaging total protein and
casein content. However, it was reported that combination of ultrasound with other
treatments such as temperature and pressure generally preferred because of the
increased efficiency and reduced power as well as time consumption (Ordonez et al.
1984). In milk and other dairy products, ultrasound with temperature, called as ther-
mosonication or ultrasound assisted thermal treatment, is a well studied combination
(Earnshaw et al. 1995; Villamiel et al. 1999; Villamiel and de Jong 2000; Bermudez-
Aguirre et al. 2009; Riener et al. 2009). Bermudez-Aguirre and Barbosa-­Canovas
(2008) reported that when milk was sonicated (24 kHz) at 63 °C, growth of meso-
philic microorganisms was less than 2 log at room temperature for 16 days. In another
study, Bermudez-Aguirre et al. (2009) achieved 5 log reduction in Listeria innocua
and mesophilic bacteria count in raw milk with the same experimental setup (24 kHz
at 63 °C). Zenker et al. (2003) reported that although energy consumption remains the
same with conventional methods, microbial load reduction was achieved in lower
temperatures with thermosonication. Gera and Doores (2011) calculated the D values
for E. coli and L. monocytogenes in whole milk, skim milk and phosphate buffer. Both
E. coli (2.43 min in milk, 2.19 min in buffer) and L. monocytogenes (9.31 min in milk,
7.63 min in buffer) showed higher resistance in milk. It was suggested that milk might
show “sonoprotective effect” (Gera and Doores 2011).

3.6 Other Methods

Apart from the aforementioned applications, there are some important non-thermal
treatments worth mentioning. One of these applications is microfiltration (MF).
Microfiltration process contains a semi permeable membrane with 0.1–1 μm pores.
12 Microbial Control of Milk and Milk Products 267

Besides some technological advantages like defatting, microfiltration can also be

used to reduce microbial load in milk (Maubois 2002). Elwell and Barbano (2006)
treated milk with a MF and heat combination and achieved 5.6 log reduction in
natural microflora. In another study, Walkling-Ribeiro et al. (2011) evaluated the
effectiveness of PEF plus MF combination and compared that combination with
thermal pasteurization treatment. Thermal treatment (75 °C, 24 s) reduced the
microbial count (4.6 log) more than PEF alone (2.5 log) and MF alone (3.7 log)
applications. MF-PEF combination with different process parameters resulted in
4.1, 44, and 4.8 log reductions. However, when combination was reversed, MF was
applied after PEF, the microbial reduction was increase to 4.9, 5.3, 5.7, and 7.1 log.
Another potential application is cold plasma. Cold plasma (CP), also called as
cold atmospheric plasma or non-thermal plasma, is a novel method that causes
structural and metabolical damages on microbial cells. Song et al. (2009) showed a
potential application of cold plasma on sliced cheese. The viable L. monocytogenes
cells were reduced by 5.8 log after 120 s treatment at 125 W.

4 Microbial Control Via Natural Antimicrobials

Natural antimicrobials are the compounds that can be derived from biological
sources without alteration (Li et al. 2011). The biological source can be animal (e.g.,
lysozyme, lactoperoxidase system), plant (e.g., essential oils) or microbiological
(e.g., bacteriocins). Milk has several antimicrobials such as conglitunin, lactoferrin,
lactoferricin, lactoperoxidase system, and lysozyme. Combinations of the treat-
ments may be synergistic, additive, or antagonistic. A synergistic effect occurs
when the effect of the combination was greater than the sum of individual treat-
ments. In antagonistic interactions, combined effect is lower than individual treat-
ments, when in additive interactions the effect is equal to the sum of individual
effects (Branen and Davidson 2004).

4.1 Essential Oils and Other Plant Based Antimicrobials

There has been an increasing interest to plants and their extracts due to their effec-
tiveness against foodborne pathogens, moulds, and mycotoxins. Essential oils (EO)
and plant by products such as tannins and phenolic acids are the main antimicrobial
substances derived from plants. Although the inactivation mechanism of EOs are
not clearly understood, it has been proposed that EOs penetrate through cell mem-
brane due to their lipophilic characteristics, and show inhibitory effect on microor-
ganisms (Li et al. 2011).
Pan et al. (2014) evaluated the effectiveness of free and nanoencapsulated thy-
mol by sodium caseinate. Although minimal inhibitory concentration (MIC) and
minimal bactericidal concentration (MBC) of thymol was higher in skim milk than
268 M. Guzel and Y. Soyer

tryptic soy broth, both free and encapsulated thymol showed antilisterial activity.
Cava et al. (2012) tested the antimicrobial activity of vanillin extract and cinna-
mon—clove combination against L. monocytogenes and E. coli O157:H7 in tripti-
case soy broth, whole, and semi skim milk. MIC and MBC of the antimicrobials
were lower in broth than milk. Authors suggested that fat molecules in milk might
have showed protective effect against antimicrobials. Also, MIC and MBC values
for L. monocytogenes were higher than E. coli O157:H7 in all mediums. In a similar
study, Cava et al. (2007) reported that cinnamon bark, cinnamon leaf and clove
showed inhibitory effect on L. monocytogenes in milk. The authors suggested that
because these compounds had been used as milk flavor worldwide, the consumer
acceptance would be high for these essential oils as antimicrobial additives. Chen
et al. (2015) co-encapsulated thymol and eugenol and observed inhibitory effect
against the L. monocytogenes and E. coli O157:H7 growth. Abdalla et al. (2007)
evaluated the antimicrobial properties of mango seed kernel extract and oil, and
reported that kernel extract reduced the total bacterial counts and inhibited the
growth of coliforms in pasteurized milk. Shah et al. (2013) reported that although
free eugenol was more effective than nanoencapsulated eugenol in TSB, in milk,
antimicrobial activity of eugenol was higher in nanodispersed form compared to
free eugenol.
Essential oils can also be used as a component in combined treatments. Karatzas
et al. (2001) reported that addition of carcavrol after HP treatment showed a syner-
gistic effect on L. monocytogenes cells in semi skimmed milk, by preventing the
recovery of sub-lethality injured cells. Similarly, Pina-Perez et al. (2012) reported
that combination of cinnamon and PEF showed synergistic effect on S. typhimurium
in skim milk. In another study, Pina-Pérez et al. (2013) combined PEF with
polyphenol-­rich cocoa powder to inactivate the Cronobacter sakazakii in infant
milk formula. It was reported that cocoa powder increased the effectiveness of
PEF. The sequence of combination affected the impact of the treatment signifi-
cantly, and the maximum inactivation occurred when cocoa powder was added to
formula 4 h after PEF treatment.

4.2 Bacteriophages

Bacteriophages (phages) are considered as natural biocontrol agents due to their

ability to inactivate the target bacteria without disturbing natural microbiota (Bueno
et al. 2012). Phages are bacterial viruses that are ubiquitous in the environment,
highly host specific, and harmless to human cells. In addition, phages can be used
as a food safety tool against antibiotic resistant bacteria. At the end of the lytic
cycle, endolysins, phage encoded enzymes, are released. These enzymes degrade
the peptidoglycan layer of cell wall, and allow newly formed virions out of the cell.
When used externally, endolysins effectively destroys Gram positive bacteria due
the lack of an outer membrane.
12 Microbial Control of Milk and Milk Products 269

García et al. (2007) determined the effectiveness of phages against S. aureus dur-
ing curd manufacture. A cocktail of phages Φ88 and Φ35 reduced the S. aureus
count by 6 log in UHT milk. Also complete inactivation occurred in 1 h at 30 °C in
renneted curd, and in 4 h at 25 °C in acid curd. In another study, Bueno et al. 2012
used a bacteriophage cocktail to inactivate S. aureus in fresh and hard-type cheese.
In fresh cheese S. aureus counts were below detection limits. In the hard-type cheese
however, 1.24 log cfu/g S. aureus was found after ripening, compared to 6. 73 log
cfu/g in control samples. Similar results were reported by Jeddi et al. (2014), where
bacteriophages effectively reduced the E. coli count below detection limits in milk.
In one of the first endolysin studies in milk, Obeso et al. (2008) evaluated the
effectiveness of ΦH5, a S. aureus bacteriophage, endolysin against S. aureus. In that
study, endolysin coding gene cloned in E. coli, and after characterization, the puri-
fied protein showed significant resemblance with other hydrolyses. The purified
protein then tested in milk as an antimicrobial, and rapidly inactivated S. aureus.
The pathogen could not be detected after 4 h of incubation.
Phages and endolysins can also be used in combination with other treatments.
For instance, Tabla et al. (2012) combined HPP and phages to inactivate S. aureus
in milk. The authors determined that 400 MPa pressure combined with a phage
cocktail reduced the S. aureus count in milk below detection limits (<10 cfu/mL).
Garcia et al. (2010) combined LysH5 endolysin with nisin to inactivate S. aureus in
milk. The antimicrobials showed strong synergistic effect, MIC of LysH5 and nisin
reduced by 16- and 64-fold, respectively. On the other hand, Martínez et al. (2008)
showed that although bacteriophages and nisin showed synergistic effect in short
term, nisin adapted cells reduced the bacteriophage activity in milk. When the nisin
adapted cells were taken from the environment, phage susceptibility was restored.
The authors highlighted that the combination should be applied with care to effec-
tively reduce S. aureus in milk.

4.3 Bacteriocins

Bacteriocins are antimicrobial peptides or proteins produced by various groups of

bacteria (Jack et al. 1995). The lactic acid bacteria (LAB) produces a number of
bacteriocins that has been studied or reviewed extensively (Table 12.4). Since LAB
are naturally present in many foods, their bacteriocins are generally regarded as safe
(GRAS) by FDA. LAB bacteriocins are also pH and heat tolerant, non-toxic to
eukaryotic cells, and have a positive effect on gut microbiota (Gálvez et al. 2007).
Bacteriocins can be applied either by adding LAB as the starter culture or in isolated
purified-concentrated form. Although inactivation mechanism may differ for vari-
ous bacteriocins, dissipation of membrane chemicals is suggested as the common
mechanism (Kaur et al. 2013). For instance, the main antimicrobial mechanism of
nisin comes from the hydrophobic and electrostatic interactions between the nisin
and membrane phospholipids (Bauer and Dicks 2005).
270 M. Guzel and Y. Soyer

Table 12.4 Overview of bacteriocins produced by LAB (O’Bryan et al. 2015)

Bacteriocin Source Targets
Nisin L. lactis subsp. lactis Gram-positive
Pediocin AcH P. acidilactici Gram-positive, Listeria, LAB
Enterocin A & B E. faecium Gram-positive
Lacticin 3147 L. lactis subsp. lactis Gram-positive
Lacticin 481 L. lactis subsp. lactis Gram-positive
Lactocin 705 L. casei Listeria, LAB, streptococci
Lactacin B L. acidophilus Lactobacilli, L. lactis
Sakacin L. sakei Lactobacilli, Listeria

Fat globules in milk decrease the antibacterial effect of bacteriocins. Jung et al.
(1992) demonstrated that antilisterial effect of nisin decreased by 33% in skim milk,
and 50% in milk with 1.29% fat. Similarly, Bhatti et al. (2004) reported that nisin
was able to reduce the L. monocytogenes numbers in skim milk to <10 cfu/
mL. However, L. monocytogenes was able to regrowth in whole milk after a decline.
Authors confirmed that either raw or pasteurized milk, nisin lost the antilisterial
effect in homogenized whole milk.
Samelis et al. (2003) reported that addition of nisin to anthotyros (a traditional
Greek cheese) prevented post process contamination with L. monocytogenes, and
changed the natural microflora Gram positive to Gram negative. Ananou et al.
(2010) evaluated the effectiveness of spray dried enterocin AS-48 on L. monocyto-
genes and S. aureus in skim milk, and found that L. monocytogenes cells completely
inactivated early, while S. aureus population inhibited partially. Martinez et al.
(2016) determined the effect of free and encapsulated commercial nisin and their
combinations on L. monocytogenes and B. cereus in whole and skim milk under
refrigeration. It was reported that combination of free and encapsulated nisin
showed the greatest inhibitory effect on L. monocytogenes, while both bacteriocin
forms were effectively controlled the germination and growth of B. cereus. It was
also reported that encapsulated form protected the antimicrobial effect for 90 days.
Farías et al. (1999) highlighted that enterocin CRL35 (10,400 AU/mL) reduced the
L. monocytogenes population by 9 log in goat cheese without affecting the quality.
In another study, Lauková et al. (2001) achieved around 5 log reduction in L. mono-
cytogenes population in Saint-Paulin cheese with enterocin CCM 4231 addition.
Bacteriocins show synergetic effect with other treatment methods such as HPP
and PEF. The combination of HP (250 MPa) and lacticin 3147 reduced the L. mono-
cytogenes and S. aureus populations more than 6 log, while the log reductions were
only 2.2 log and 1 log for HP and lacticin 3147 when they used alone (Morgan et al.
2000). Black et al. (2005) combined nisin and HPP to inactivate L. innocua, E. coli,
P. fluorescens, and Lactobacillus viridescens. It was reported that 500 MPa for
5 min HP and 500 IU/mL nisin was sufficient to reduce tested strains by 8 log.
Muñoz et al. (2007) showed that the combination of enterocin CCM 4231 with a
mild heat treatment (65 °C, 5 min) reduced the Staphlyococci population below
detection limits for the first 8 h, and prevent the overgrowth in 24 h. Gallo et al. (2007)
12 Microbial Control of Milk and Milk Products 271

observed a synergistic effect against L. innocua when nisin was added to whey
before PEF. However, nisin addition after PEF treatment did not show same effect.
Authors suggested that alterations in cell envelope and medium might have reduced
the nisin activity. Combination of nisin with other antimicrobial substances increases
the antibacterial activity. Boussouel et al. (2000) added nisin, lactoperoxidase
system, and their combinations to skim milk to inactivate L. monocytogenes. When
used alone, although both substances provided bacteriostatic phase for limited times,
L. monocytogenes was able to grow. However, when lactoperoxidase system was
added 4 h later than nisin, maximum inhibitory effect was reached, and L. monocyto-
genes was below detection limits in skim milk for 15 days.
Bacteriocin like substances (BLS) are produced by bacteria and show similar
antibacterial effect with bacteriocins. Malheiros et al. (2012a) evaluated the effec-
tiveness of nanovesicle encapsulated BLS P34, an antimicrobial peptide produced
by Bacillus sp. P34, against L. monocytogenes in milk, and found that both free and
encapsulated BLS P34 showed inhibitory effect at 1600 IU/mL level. In another
study, Malheiros et al. (2012b) encapsulated nisin and BLS P34 in PC-1 liposomes
to inhibit the L. monocytogenes growth in Minas frescal cheese. Authors reported
that both substances showed increased inhibitory effect against L. monocytogenes
compared the control group.

4.4 Antimicrobials from Other Sources

Apart from the antimicrobials mentioned above, effect of some enzymes (e.g.,
lysozyme) and antimicrobials from animal origin (e.g., lactoferrin), and organic
acids have also been tested in milk.
Murdock and Matthews (2002) used lactoferrin hydrolysate with pepsin in UHT
milk to inactivate L. monocytogenes and E. coli O157:H7. Result of that study
showed that at pH 4 lactoferrin hydrolysate reduced the populations of both patho-
gens approximately by 2 log. On the other hand, at pH 7, inhibitory effect was
shown only in E. coli O157:H7 cells. It was also reported that addition of EDTA did
not increase the effectiveness of lactoferrin hydrolysate. McLay et al. (2002) com-
bined lactoperoxidase system with various lipids to inhibit the growth of E. coli
O157:H7 and S. aureus in milk. The highest inhibitory effect achieved when lacto-
peroxidase (5–200 mg/kg) combined with monolaurin (50–1000 ppm). Researchers
concluded that lactoperoxidase monolaurin combination showed synergistic effect
on the pathogens and inhibited the growth significantly (McLay et al. 2002).
Arqués et al. (2008) evaluated the antibacterial effect of combinations of reuterin
with nisin and lactoperoxidase against Gram-negative bacteria in milk. Reuterin is an
antimicrobial compound produced by Lactobacillus reuteri. While nisin did not
increase the bactericidal activity of reuterin, the combination of lactoperoxidase
system and reuterin showed synergistic effect against E. coli O157:H7, Yersinia
enterocolitica, Aeromonas hydrophila, Campylobacter jejuni, and Salmonella
enterica. In another study, Arqués et al. (2011) combined reuterin and LAB bacteriocins
272 M. Guzel and Y. Soyer

nisin, lacticin 481, and enterocin AS-48 to inactivate C. jejuni, Y. enterocolitica,

A. hydrophila, E. coli, S. enterica, L. monocytogenes and S. aureus in milk. Among the
bacteriocins, only nisin showed synergistic effect with reuterin against L. monocyto-
genes and S. aureus, and prevented the growth of the pathogens after 12 days.
Similarly, Stevens et al. (2011) reported that reuterin showed inhibitory effect
against Gram positive and Gram negative pathogens. Tsai et al. (2000) evaluated the
effectiveness of a chitosan mixture against several pathogenic and spoilage bacteria.
The mixture reduced the mesophilic and psychotropic counts by more than 3 logs
without affecting pH significantly. Moreover, the mixture inhibited the Salmonella
growth and reduced the Staphylococcus spp. counts.
Organic acids (OA), mainly lactic acid, provide antimicrobial effect in some of the
dairy products including yogurt and kefir by reducing pH. OA may also be used in com-
binations with other antimicrobials and non-thermal treatment methods. For example,
whey protein-based film containing nisin and various organic acids was tested
against L. monocytogenes in cheese. Although all of the organic acids (lactic, malic, and
citric) increased the inhibitory effect of nisin, malic acid plus nisin incorporated film
provided the best antimicrobial results against L. monocytogenes (Pintado et al. 2009).

5 Conclusion

Milk is one of the most favorable food commodity to pathogenic and spoilage bac-
teria. As a result, safety of milk and other dairy products, while keeping nutrients
and sensory quality is still a big concern. In the near future, the authors expect the
application of more than one treatment in combination will draw more attention.
The combination of applications, as called hurdle technology, was discussed in each
treatment sections.
Hurdle technology is the intentional combination of hurdles (e.g., acidity, water
activity, preservatives) to improve the microbial, sensory, and nutritional quality of
foods (Leistner 2000). Multitarget preservation is the employment of more than one
hurdles at the same time with different inactivation mechanisms or targets (e.g., cell
membrane, DNA, enzymes) (Leistner 1995). With the synergistic effect, it is possible
to use gentler version of current applications in milk and dairy products. In other
words, it is possible to reach higher microbial quality by employing more than one
application with small intensity compared to one application with high intensity.

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Chapter 13
Microbial Fermentation in Food Preservation

Ilenys M. Pérez-Díaz, Evrim Gunes Altuntas, and Vijay K. Juneja

Abstract Fermented foods are consumed worldwide representing a significant

component of the human diet. Foods preserved by fermentation are perceived as a
natural and healthy choice. The safe and palatable fermentation of foods is subjected
to basic principles of acidification, salting, water activity and oxygen availability.
Food fermentations are dependent on the activity of the type of microbes added as
starter cultures. The multiple microbially mediated metabolic conversions in food
fermentations impact bioactivity, stability, antimicrobial activity and toxicity of the
finished product. This book chapter discusses basic preservation principles, micro-
bial activity and chemistry of food fermentations.

Keywords Fermented foods • Lactic acid bacteria • Yeasts • pH • Preservatives •

Antimicrobials

1 Description of Food Preservation by Fermentation

Fermentation is mainly regarded as a practical and effective mean to preserve foods

with unique organoleptic properties, and minimal energy inputs (Daeschel et al. 1987;
Leroy and De Vuyst 2004). It has been used to prevent the spoilage of raw foods since
the Neolithic period (Bourdichon et al. 2012). While the discovery of fermentation is
associated with production of alcoholic beverages and bread in Babylon and Egypt,

I.M. Pérez-Díaz (*)

United States Department of Agriculture, Agricultural Research Service,
Food Science Research Unit, Raleigh, NC, USA
e-mail: [email protected]
E.G. Altuntas
Ankara University, Biotechnology Institute Central Laboratory, Ankara, Turkey
e-mail: [email protected]
V.K. Juneja
Residue Chemistry and Predictive Microbiology Research Unit, Eastern Regional Research
Center, USDA-Agricultural Research Service, Wyndmoor, PA, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 281

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_13
282 I.M. Pérez-Díaz et al.

today it represents a cosmopolitan industry with a billionary value in US dollars

(Scott and Sullivan 2008; Konings et al. 2000). Although, extremely beneficial to
human kind, the fermentation process remained largely uncharacterized for centuries.
Deprived of an understanding of the microbiology behind complete and desirable
fermentations, old generations use high quality cover brines or yeasts paste from fer-
mentations with desirable attributes to initiate fresh ones, a technique known as back
slopping. With the observations made by Anton van Leeuwenhoek in 1680 of living
cells, using an early version of the microscope, and those contributed by Cagnard-
Latour in 1839, fermentation was understood as a microbially induced process in
which yeasts produce ethanol and carbon dioxide from sugars (Nanninga 2010).
An industrialist in Lille, France working with Louis Pasteur, discovered the role of
lactic acid bacteria in fermentations. The problem of a reduced concentration of alcohol
and sourness in ethanol production existed. Nevertheless, the discovery permanently
changed the fermentation field. Pasteur published several papers between 1857 and 1860
documenting the replacement of the ethanol producing yeast population by microbes able
to produce lactic acid in the fermenting samples. Such documentation was the first proof
of the bacterial nature of fermentation, understood as a chemical degradation of sugars
prior to the 1830s (Nanninga 2010). The very first pure starter culture was prepared by
Joseph Lister in 1873 by diluting fermented milk. Realizing the potential impact of pure
cultures in fermentations, 15 years later, Vilhelm Storch prepared pure cultures used for
souring pasteurized cream (Knudsen 1931). The use of starter cultures for dairy fermenta-
tions was introduced around the 1890 in Copenhagen (Stiles and Holzapfel 1997).
Defined fermentative cultures were introduced commercially in New Zealand in 1934
(Cogan and Hill 1993) beginning the era of “controlled” fermentations. Today a starter
culture is defined as a microbial preparation of large numbers of cells of at least one
microorganism to be added to a raw material to accelerate and dictate the course of a food
fermentation (Leroy and De Vuyst 2004; Ayhan et al. 2005). The modern understanding
of fermented foodstuff is thus the microbial metabolic process that converts sugars to
acids, gases or alcohol to achieve long term preservation while generating desirable
organoleptic properties. It is estimated that today 600,000 tons of the baker’s yeast are
sold annually (Pretorius et al. 2015). The total commercial production of starter cultures
for large scale fermentation is estimated to be above 40,000 L annually used to inoculate
tens of thousands of tons of raw material (Hansen et al. 2015).

2  atural Acidification and Production of Preservatives

N
in Fermented Foods

In food fermentations, organic substrates, usually a carbohydrate, is incompletely

oxidized, and an organic carbohydrate acts as the electron acceptor with the release
of energy (Sahlin 1999; Chacko et al. 2010; Chojnacka 2010). Two main reactions
occur in food fermentations: conversion of sugars to ethanol and carbon dioxide by
yeasts and/or conversion of sugars to organic acids and carbon dioxide by lactic acid
bacteria. Homofermentative lactic acid bacteria convert one molecule of glucose to
13 Microbial Fermentation in Food Preservation 283

two molecules of lactic acid. Heterofermentative lactic acid bacteria produce one
molecule of lactic and acetic acids and one molecule of carbon dioxide per molecule
of glucose. Conversion of sugars to organic acids generates a decrease in the pH of
the food matrix beneficial for preservation.
pH, defined as the negative value of the logarithmic concentration of hydrogen
ions, is a critical factor in controlling the quality and safety of food fermentations.
High hydrogen concentration or an acidic pH is effective in protein hydrolysis and
denaturation, which impairs the functionality of cell proteins and the microbial
metabolic machinery (Meng et al. 2007), and impacts the physical state and texture
of foods. The intracellular pH of most microorganisms is maintained near neutrality.
Thus, an acidic pH may kill microbes intolerant to high hydrogen ion concentra-
tions. However, fermentative lactic acid bacteria can tolerate pH values as acidic as
pH 3.3 (McDonald et al. 1990). Few microorganisms can proliferate at pH values
below 4.6, which is a critical control point in the production of acidified foods in the
USA. If oxygen is present, most yeasts can proliferate at pH values between 4.5 and
6.0 at the expense of sugars or organic acids. Utilization of organic acids by yeasts
or spoilage lactic acid bacteria results in an undesired increase in pH (Ruiz-Cruz
and Gonzalez-Cancho 1969; Franco and Pérez-Díaz 2012).
Besides inducing a decrease in pH, the production of organic acids also increases
the antimicrobial activity of fermented foods. Lactic acid is well known to reduce the
populations of pathogenic microorganisms in fermented foods (Sahlin 1999). Organic
acids are more effective at controlling microbial growth as compared to inorganic
acids, partially due to the contribution of the undissociated species. The undissociated
species of organic acids diffuse through the cell membrane and dissociates intracel-
lularly resulting in: (1) cytoplasmic acidification and accumulation of anionic species
(Booth and Kroll 1989), (2) the disruption of pH homeostasis and cellular metabolism
(Beales 2004), and (3) the impairment of the proton motive force and active transport
(Sheu et al. 1972). Although, different rankings for the effectiveness of organic acids
have been published under a variety of conditions, it has been observed that lactic acid
is more effective at reducing pathogens of public health significance under compara-
ble conditions of pH, temperature, ionic strength and anaerobiosis (Lu et al. 2011).
Preservatives commonly used in acidified foods such as sorbic acid, benzoic acid and
fumaric acid are significantly more effective against pathogens, as compared to lactic
acid (Lu et al. 2011). The effectiveness of organic acids in preservation is dependent
on pH as determined by their dissociation constant. At neutral pH most organic acid
exist in their dissociated forms, meaning that they release the hydrogen ion from their
carboxilic groups. At acidic pH however, most of the organic acids exists in their
undissociated forms or associated with their corresponding hydrogen ions. For
instance, only 1.6% of the total amount of benzoic acid is present in its active form at
pH 6.0. The dissociation constant for benzoic acid is 4.2. Thus, at pH 3.5 about 83%
of the total acid concentration is active. The undissociated hydrophobic form of the
organic acids diffuse over the cell membrane, dissociates inside the cell, and release
H+ ions that acidify the cytoplasm. In essence the undissociated form of the acids
induces the collapse of the electrochemical proton gradient, causing bacterial death
(Gürakan 2007). Different organic acids are inherently more or less effective against
284 I.M. Pérez-Díaz et al.

specific microbes. In general terms, sorbic acid is more effective at controlling yeasts
than benzoic acid, however lactic acid bacteria are insensitive to sorbic acid. Thus,
sorbic acid is often incorporated in cover brines used for cucumber fermentation in
which lactic acid is the desired metabolic product.
Lactic acid bacteria often outcompete yeasts in vegetable fermentations given
their ability to grow and metabolize energy sources at a rate faster than spoilage
yeasts and to produce antifungal compounds including organic acids, hydrogen per-
oxide, carbon dioxide, diacetyl, acetaldehyde, low molecular weight antmicrobial
substances such as reuterin, 2-pyrolidone-5-carboxylic acid, bacteriocins, adhesion
inhibitors, fatty acids, hydroxylated fatty acids, phenyllactic acid, and cyclic dipep-
tides (Gürakan 2007; Ganzle 2009). Hydrogen peroxide (H2O2) is an antimicrobial
compound produced by most lactic acid bacteria by mean of flavoprotein containing
oxidases, NADH oxidases, and superoxide dismutases in the presence of oxygen.
Formation of hydroxyl radicals from hydrogen peroxide has long being considered
as one of the main antimicrobial factors in fermentations. However, with the under-
standing that phytochemicals and antioxidants may formed during fermentations, in
particular of plant derived foods, the impact of such compounds as antimicrobials is
uncertain. Diacetyl, the major aroma and flavor component of butter, is a metabolic
product of heterofermentation by some lactic acid bacteria. It has been documented
that diacetyl has a bactericidal effect on strains of Yersinia enterocolitica, Aeromonas
hyrophilia, Escherichia coli and Salmonella anatum (Naidu et al. 1999). Some
Lactobacillus strains are capable of producing reuterin which consists of an equili-
brated mixture of monomeric, hydrated monomeric and cyclic dimeric forms of
3-HPA (3-hydroxypicolinic acid). Reuterin is produced by Lactobacillus reuteri
from glycerol by starving cells under anaerobic conditions (Montiel et al. 2014).
Although lactic acid bacteria are able to produce many antimicrobial compounds,
bacteriocins are the most studied and commercially exploited to date (Leroy et al.
2003; Gürakan 2007; Altuntas et al. 2010; Altuntas et al. 2014). Even though bacte-
riocins or antimicrobial peptides were originally studied as a product of the Gram-­
negative Escherichia coli, the homolog produced by Gram-positive lactic acid
bacteria LAB are of particular interest for commercial applications given the
“Generally Recognized as Safe” status of such fermentative microbes (Fujita et al.
2007; Galvez et al. 2007; Liu et al. 2011; Cleveland et al. 2001; Deegan et al. 2006).
The molecular weight of the many bacteriocins produced by lactic acid bacteria is
below 10 KDa. Although the producing microbes are immune to the antimicrobial
activity of such peptides, they are effective against other Gram-positive bacteria
(Schillinger et al. 1996). Bacteriocins are considered natural antimicrobial com-
pounds, which can be used as food additives, incorporated in fermented products
using strains able to produce them, as starter cultures or by using a fermented food
prepared with a bacteriocin producing starter culture as an ingredient (De Vuyst and
Leroy 2007; Deegan et al. 2006). The use of purified bacteriocins is not always
attractive to the food industry given that such peptides may be degraded by proteo-
lytic enzymes in the stomach. However, bacteriocin producing probiotic strains may
produce the peptides in the gastrointestinal tract and aid in the control of pathogens.
Bacteriocinogenic lactic acid bacteria strains can be used as starter cultures, co-­
13 Microbial Fermentation in Food Preservation 285

cultures, or bioprotective cultures, to improve food safety. Bacteriocin producing

starter cultures may be applied in the making of sourdough, fermented sausages,
and cheeses to increase competitiveness, inhibit Listeria spp., and inhibit Listeria
and Clostridium spp., respectively (De Vuyst and Leroy 2007; Cosansu et al. 2010).
Anaerobic metabolism of Saccaharomyces spp. is the primary source of ethanol
production from sugars in alcoholic fermentations. Spoilage of alcoholic fermenta-
tions by lactic acid bacteria is likely the most common problem in achieving a high
yield, due to the tolerance of the former to ethanol. Although, lactic acid bacteria
can tolerate ethanol, and some produce it under certain conditions, the alcohol has
the ability to impair the physiology of lactic acid bacteria. Oenococcus oeni, is one
of the most resistant lactic acid bactetria to ethanol. The ability of O. oeni to resist
up to 10% ethanol has been attributed to the ability to biosynthesize phospholipids
and L-lactic acid to control membrane fluidity, metabolize citric acid and aggregate
(Teixera et al. 2002; Olguín et al. 2009; Elahwany 2012). Ethanol concentration
effective against non-lactic acid bacteria has been found to be around 5% (Oh and
Marshall 1993), which is half of that concentration effective against O. oeni.

3  elevant Extrinsic Parameters in the Production of Safe

R
Biopreserved Foods

The safe biopreservation of foods is achieved by controlling the natural microbiota

with starter cultures, salt, water activity, heat treatments, preservatives or the use of
protective cultures (Devlieghere et al. 2004) and assuring the complete removal of pri-
mary sugars. Although harder to control in large scale fermentations, temperature is
also a critical factor to achieve the desired fermentation.
Salt, primarily NaCl, influences the type and extent of microbial activity, helps
prevent softening of food tissues, determines the flavor of the final product, and
assists in rupturing the membranes of vegetables and fruits to be fermented, allowing
the diffusion of various components into the cover brine solutions used by microbes
for growth and metabolic activities (Pérez-Díaz et al. 2015 and references therein).
In some foods high salt concentrations significantly retard or preclude fermentation
(Pérez-Díaz et al. 2015 and references therein). Softening of vegetable tissues can be
reduced or prevented by adjusting the salt level to retard the activity of pectinolytic
enzymes derived from eukarotic cells (Bell and Etchells 1961; Bell et al. 1950).
Water activity is a measurement of the vapor pressure in a substance over that of
pure water, essentially describing whether the conditions in a fermented product are
conducive to microbial growth, primarily yeasts. Although, most brined fermented
products are not susceptible to changes in water activity, it is a critical control point
in fermented meat products. Adjustment in water activity in fermented meats is
achieved by salting and drying. The higher the salt concentration the lower the water
activity. Although, low water activity prevents microbial growth, it does not exert a
killing effect and many microbes do not tolerate levels of salt lower than those
286 I.M. Pérez-Díaz et al.

needed to achieve a low water activity. In sweetened fermented products, water

activity is controlled by the addition of sugars. Bacteria are more sensitive to sugars
as compared to yeasts, some of which can proliferate in saturated sugar concentra-
tions. Yeast species commonly found in fermented vegetables, meats and alcoholic
beverages such as Candida, Torulopsis, and Hansenula can grow at water activity of
0.87 or higher. Saccharomyces and Debaryomyces species can tolerate a water
activity as low as 0.80 (Beuchat 1983). Significantly higher concentrations of sugars
are needed to lower water activity as compared to salts. For instance, 19% sodium
chloride is needed to drop the water activity to 0.85 at 25 °C. More than 60% glu-
cose is needed to achieve a water activity of 0.85 under comparable conditions.
The microbiological nature of fermentation imposes a temperature effect to
achieve preservation (Rodgers 2001; Kaur et al. 2011). Generally, lower tempera-
tures reduce the movement of molecules and frequently prolongs the fermentative
microbes doubling time. Although, fermentative lactic acid bacteria are mesophilic
and capable of growing at temperatures between 5 and 45 °C, their optimum growth
temperature is between 30 and 40 °C (Caplice and Fitzgerald 1999). Although, opti-
mum growth temperature assist in achieving fast fermentation, it can also influence
the quick initiation of spoilage by undesired microbes. Thus, often times a variety
of extrinsic factors are used in food fermentations, to incorporate the necessary
hurdles for growth of non-desired microbes, while creating the best conditions for
the proliferation of the desired fermentative bacteria and/or yeasts. Optimum growth
temperature for yeasts varies between −2 and 45 °C (Arthur and Watson 1976),
however, one of the most studied fermentative yeast essential in winemaking, bak-
ing and brewing, Saccharomyces cerevisiae grows optimally between 30 and 35 °C
(Walsh and Martin 1977). While yeast cells are more sensitive to heating treatments
as compared to bacteria, they survive freezing under certain conditions, with viabil-
ity decreasing over time (Arthur and Watson 1976). Short time and relatively low
temperature (70 °C) heating steps are often used on fresh produce prior to fermenta-
tion to induced inactivation of softening enzymes, release simple sugars from polys-
sacharides or starch, and reduced the initial microbial load. This is particularly
relevant in the processing of vegetables, tubers and fruits.
Fermentation is regarded as an anaerobic metabolic process, meaning that oxy-
gen is not necessary for the conversion of sugars to organic acids and/or alcohol.
Lactic acid bacteria are facultative anaerobic, meaning that they do not have a
requirement for oxygen to grow but can tolerate it. Acetic acid bacteria cannot
grow in the absence of oxygen. Saccahromyces cerevisiae may grow and ferment
under aerobic or anaerobic conditions, however most yeasts have an oxygen
requirement. Fermentative yeasts can grow well in the absence of oxygen, if sugars
are available. Control of oxygen availability in a fermentation process is critical for
the achivement of the desired microbial metabolism and the inhibition of spoilage
and pathogenic microbes. In most fermentations the appropriate combination of
factors that can affect microbial growth results in safe and high quality products.
The c­ ombination of pH below 4.6 and anaerobiosis, for instance, prevents the pro-
liferation of Clostridium botulinum and corresponding toxin production, which
may occur should the pH remained above 4.6.
13 Microbial Fermentation in Food Preservation 287

4 Physical and Nutritional Changes in Fermented Foods

Aside from preservation, microbial fermentation detoxifies foods, improves accessibility

of nutrients, and improves organoleptic properties (Bourdichon et al. 2012; Caplice and
Fitzgerald 1999; Liu et al. 2011). During a fermentation process, a significant increase in
the soluble fraction of a food is observed (Sahlin 1999). The quantity as well as the qual-
ity of the water soluble vitamins and the food proteins, expressed as the biological value,
increases, while the antinutritional factors show a decline as the result of fermentation
(Sahlin 1999). Yoghurt in particular contains almost all nutrients present in milk in a
more assimilable form (Chacko et al. 2010). The toxic cyanide content of cassava is
reduce during the fermentation and acidification of the tuber by linamarase (Ikediobi and
Onyike 1982). Milk digestibility is increased in cheeses with a reduced lactose content.
The phytochemical content and antioxidant properties of fermented plant derived foods
increase as compared to the fresh vegetables and fruits (Yeo and Ewe 2015). The antioxi-
dant content contributes to the health benefits of plant foods by reducing the incidence of
age-related diseases such as heart conditions and some cancer types (Yeo and Ewe 2015).
Although the development of characteristic flavors is a critical factor in fer-
mented foods, specific mechanisms to achieve the desired flavor remain unknown
(Caplice and Fitzgerald 1999). Lactic acid bacteria have a significant role on the
composition of non-volatile and volatile compounds through the release and/or deg-
radation of free amino acids and prevent the oxidation of unsaturated free fatty acids
associated with rancidity (Fadda et al. 2010).
The microbial activity in fermented foods also results in an increment in the water
soluble exopolysaccharides, conjugated linoleic acid, and bioactive peptides content.
Exopolysaccarides or long sugar chains occasionally combined with phosphate, acetyl,
uronic acid and glycerol (Nwodo et al. 2012) are produced by microbial metabolism uti-
lizing the fermenting food matrix components (De Vuyst and Degeest 1999). The proteo-
lytic activity of lactic acid bacteria present in fermented foods, primarily in dairy products,
generates peptides that are further modified in the gastrointestinal tract (Pihlanto and
Korhonen 2015). Conjugated linoleic acid isomers are derived from rumminants and con-
sequently found in fermented meats and dairy products (Csápo and Vargas-Visi 2015).
Conjugated linoleic acid isomers are generally recognized as safe by the US Food and
Drug Administration and may be produced by fermentative lactic acid bacteria under
certain conditions. These metabolically produced compunds have been associated with
beneficial health effects and the improvement of organoleptic properties in fermented
products. Exopolysaccharides are used in finished yogurt and bread products as texturiz-
ers, viscosifiers, emulsifiers and synerisis-lowering agents given their plasticity and water
absorbing capacity (Cerning 1990). Proteolysis and peptidic systems have been impli-
cated in the texture, taste and flavor of fermented dairy foods. Multiple reviews can be
found in the literature regarding the potential functionality of bioactive peptides, exopoly-
sacharides and conjugated linoleic acids as immunomodulators, cytomodulators, anti-
cholesterole, oxidants, microbial, cancer, tumors, ulcers and genetic mutations, protectants
of the intestine, anti-diabetes, obesity and cardiovascular diseases, and mineral binders
(Duboc and Mollet 2001; Jolly et al. 2002; Ruas-Madiedo et al. 2002; Aswathy et al. 2008;
Ruas-Madiedo et al. 2006; Csápo and Vargas-Visi 2015).
288 I.M. Pérez-Díaz et al.

5  ypes of Fermented Foods and Interactions

T
of the Microorganisms Involved

Desirable fermentations occur when organisms able to preserved a food are capable
of outcompeting the microbiota naturally present in the substrate. Fermentative
organisms are intrinsic in the environment, thus production of fermented foods does
not require knowledge of the biologically mediated nature of fermentation (Scott and
Sullivan 2008). Human survival is connected to yeasts and bacteria that produce lac-
tic acid and alcohol in preserved foods (Scott and Sullivan 2008). Fermentative lactic
acid bacteria and yeasts are capable of competing for nutrients in foods, such as the
sugars, nitrogen sources, and vitamins. Robust fermentative lactic acid bacteria and
yeasts create unfavorable conditions, such as an acidic pH or high ethanol concentra-
tions to exclude competing microbes from the food matrix. Fermentative microbes
also adhere or localize near the sources of nutrients (Caplice and Fitzgerald 1999).
Fermented foods could be classified as dairy, starchy foods, cereals, meat, vegetables,
alcoholic beverages, and legumes and oil seeds (Caplice and Fitzgerald 1999; Achi 2005).
Table 13.1 describes the main types of fermented foods and microorganisms involved. In
a significant proportion of fermented foods, Enterobactereaceae initiate the fermentation
until lactic acid bacteria produce enough lactic acid to reduce the pH of the food matrix.
In some food fermentations, such as cocoa, acetic acid bacteria convert the product of
primary fermentation into acetic acid by means of membrane bound or cytoplasmic
dehydrogenases (Scott and Sullivan 2008).
As mentioned above, lactic acid bacteria are responsible for many of the microbial
transformations found in popular acidic fermented food products (Ross et al. 2002).
Lactic acid bacteria consists of 12 genera, among them; Lactococcus, Lactobacillus,
Leuconostoc, Enterococcus, Streptococcus and Bifidobacterium are the most com-
monly studied and used in commercial applications (Özer 2007). Lactic acid bacteria
are Gram-positive, catalase and oxidase negative, facultative anaerobic rods and
cocci, which generally have complex nutritional requirements especially for amino
acids, vitamins, and purines. This group of bacteria have a number of auxotrophies
and weak lipolytic activity, which defines their need to dominate in food matrixes
(Caplice and Fitzgerald 1999; Françoise 2010). While the main catabolite of homo-
fermentative lactic acid bacteria from sugars is lactic acid; heterofermentative lactic
acid bacteria convert sugars to lactic acid, acetic acid and carbon dioxide.
Even though, enterococci, lactic acid bacteria able to colonize milk and meat, are
considered by some as unacceptable for food production due to their ability to colo-
nize the instestinal tract (Khan et al. 2010). Given its ability to colonize raw meats,
E. faecium is frequently found in meat fermentation in significant numbers contribut-
ing to lactic acid production and the inhibition of Listeria monocytogenes, and the
sensory characteristics resulting from the ripening of cheese. It is understood that
E. faecium metabolic ability includes lipolysis, citric acid utilization, and the produc-
tion of aromatic volatile compounds (Hugas et al. 2003; Leroy et al. 2003; Gürakan
2007; Giraffa 2003). Bacteriocin production by E. faecium RZS C5 under acidic
conditions during logarithmic phase, as opposed to stationary phase, makes its use as
13 Microbial Fermentation in Food Preservation 289

Table 13.1 Description of the primary microbiota in selected fermented foods

Fermented Particularities of the Review
food process or microbes Participating microbes reference
Sausage Psychrophilic Lactobacillus sakei, L. curvatus Lücke (2015)
lactobacilli-pentose
utilizers
Catalase positive Staphylococcus warneri, S. xylosus,
cocci-superficial S. equorum, S. succinus, S. carnosus
fermentation
Unsmoked, superficial Penicillium nalgiovense,
ripening Debaryomyces hansenii
Fish Halophilic LAB Tetragenococcus spp. Kuda (2015)
(>10%)
L. plantarum, L. rennini, L.
acidipiscis, L. versmoldensis
Debaromyces hansenii,Pichia
anomala and superficial molds
Bread baker’s yeast Brandt (2015)
Sourdough L.sanfranciscensis, L. reuteri, L.
pontis, L. crispatus, L.
paralimentarius
Soy Inoculated Aspergillus oryzae Nout (2015)
Halophilic microbes Tetragenococcus halophilus,
(~20%) Zygosaccharomyces rouxii
Doujiang Bacillus spp., L. fermentum, L.
plantarum
Candida humilis, Kluyveromyces
lactis, Williopsis saturnus, Sac.
gallinarum
Wine Primary sugar Sac.cerevisiae Bisson and
utilization Walker (2015)
Proton motive force Oenococcus oeni, L. hilgardii, L.
as source of energy brevis, L. plantarum, L. pentosus
Utilization of organic Acetic Acid Bacteria
substrates
Ripening Aureobassidium, Cryptococcus,
Rhodosporidium, and Rhodotorula
Beer Sac. pastorianus (formerly S. Kyselová and
carlsbergensis) Brányik (2015)
Ale yeasts Sac. cerevisiae
Spoilage Megasphaera and Pectinatus spp.
Coffee Initiation of the Enterobacteriaceae Huch and Franz
fermentation (2015)
Undefined and highly Bacteria, yeasts and filamentous
variable fungi
(continued)
290 I.M. Pérez-Díaz et al.

Table 13.1 (continued)

Fermented Particularities of the Review
food process or microbes Participating microbes reference
Vegetables Initiation of the Enterobacteriaceae Medina-Pradas
fermentation et al. (2016)
Active fermentation L. plantarum, L. pentosus, L. brevis,
Pediococcus spp., Leu.
mesenteroides, Weisella spp.
Candida spp., Pichia spp. and
Saccharomyces spp.
Scum formation Debaryomyces spp., Pichia spp.,
Mycoderma
Dairy Various subspecies of Lactococcus Leroy and De
lactis and L. delbrueckii Vuyst (2004)
Leu. mesenteroides subsp. cremoris,
Streptococcus thermophilus

an adjunct culture advantageous (Leroy et al. 2003). Selected Enterococcus spp., are
used as starter cultures in dairy products and widely used as probiotics (Khan et al.
2010).
The ability of lactic acid bacteria to produce exopolysaccharides aids them to
survive under stressful conditions, including the acidic pH, and high salt (Cerning
et al. 1994; Donot et al. 2012; Harutoshi 2013) characteristic of fermented foods. It
also aids in resisting bacteriophage infections and the antimicrobial effect of preser-
vatives and toxic ions, in establishing their ecological niche through cell adhesion,
biofilm formation and quorum sensing (Ruas-Madiedo et al. 2002; Kodali et al. 2009;
Nwodo et al. 2012), and in nutrient accumulation (Patel and Prajapati 2013).
Exopolysaccahrides also help the producing bacteria to outcompete the natural
microbiota and survive in extreme habitats such as those with high or cold tempera-
tures (Nichols et al. 2005; Kim and Yim 2007; Poli et al. 2010).
Ocassionally the lactic acid produced during fermentation can be utilized as an
energy source by spoilage organisms such as Propionebacterium spp., Pichia man-
shurica, Issatchenkia occidentalis, Lactobacillus buchneri, Lactobacillus rapi and
Lactobacillus namurensis (Gonzalez-Cancho et al. 1970, 1980; Franco et al. 2012;
Medina-Pradas et al. 2016). Removal of the acid from the fermentation matrix
causes an increase in pH, which enables the growth of other spoilage and patho-
genic microbes. Thus, extrinsic factors should be appropriately applied to achieve
the stable fermentation of foods.
Competing fermentative lactic acid bacteria strains can be selected based on their
ability to grow in the food matrix of interest and outcompete and survive selected
pathogens of public health significance. Lactococcus lactis subsp. lactis and
Pediococcus acidilactici are used as biocontrol for pathogens like Salmonella
enterica, Escherichia coli O157:H7 and Listeria monocytogenes in alfalfa seeds and
sprouts (Kostrzynska and Bachand 2006; Altuntas 2013).
13 Microbial Fermentation in Food Preservation 291

The baker’s yeast, Saccharomyces cerevisiae, is well known today for being the first
to be observed under the microscope, and the first to be recognized as a living organism
able to biochemically transform bread and alcoholic beverages (Pretorius et al. 2015).
Even though it is the most frequently used yeast for fermentations; it is assisted by O.
oeni for the conversion of malic acid into lactic acid in wine making. The lactic acid
bacterium transforms the sharp apple like flavor of malic acid to the softer tasting lactic
acid (Pretorius et al. 2015). The co-existence of the baker’s yeast with O. oeni in wines is
possible due to the resistance of the former to more than 10% ethanol (Teixera et al. 2002;
Olguín et al. 2009; Elahwany 2012). However, the lactic acid bacteria may cause spoilage
at different stages of wine making and its proliferation has to be monitored and controlled
to retain quality.

6  dvantages of the Utilization of Authoctonus Starter

A
Cultures and Probiotic Cultures for Food Fermentations

Development of starter and adjunct cultures for food fermentations represents a field
heavily researched since the 1930s. Such studies have been recently accelerated by
the advances in high-throughput sequencing technology, deepening insight into com-
plex fermentation systems (Bokulich and Mills 2012; Ivey et al. 2013). Although, to
date the traditional fermentation processes rely on the natural microbiota and their
spontaneous proliferation in foodstuffs, the industrial production of fermented foods
heavily relies on the use of starter cultures (De Vuyst and Leroy 2007). Utilization of
selected starter culture aids in accelerating the initiation of fermentation, shortening
of the fermentation process and reduction of the risk of fermentation failure (Leroy
and De Vuyst 2004; Ivey et al. 2013). It has been demonstrated that food fermenta-
tions with autochthonous starter cultures are efficient and produced high quality fin-
ished products as compared to allochthonous or wild fermentation (Di Cagno et al.
2008a, b, 2009). More specifically, a faster reduction in pH and proliferation of
desired microbes proceeds in vegetables fermented with autochthonous starter cul-
tures. Longer lag phases have been observed for allochthonous starter cultures (Di
Cagno et al. 2008a, b, 2009). Quality and nutritional attributes such as color, sensory
and rheological properties, vitamins and antioxidant activity of fermented vegetables
are favorably impacted and retained when autochthonous starter cultures are used (Di
Cagno et al. 2008a, b, 2009, 2013). Autochthonous bacteria have an intrinsic ability
to produce metabolites such as bacteriocins and exopolysaccharides, that are tailored
specifically for their establishment during fermentation, and thus have a greater
chance of survival when used as starter cultures (Di Cagno et al. 2013). Selected
secondary metabolites specifically produced by allochthonous starter cultures in their
native food matrix, such as bacteriocins, exopolysaccharides and vitamins, are often
times considered added value to the final product (Di Cagno et al. 2009).
Most of the commercialized starter cultures include the group of lactic acid bacteria
and the bread making yeast Saccahromyces spp. Aside from serving as starter cultures,
292 I.M. Pérez-Díaz et al.

Bifidobacterium, Lactobacillus and to a lesser extend Saccahromyces species also serve

as supplementary probiotic cultures. Probiotic cultures are generally defined by the
Food and Agriculture Organization-World Health Organization (WHO) as live microbes
that when added in foods confer a beneficial health effect to the host (WHO 2002;
Morelli and Capurso 2012) and are generally added to 109 colony forming unit per serv-
ing size (Forssten et al. 2011). Although most fermented foods provide for the growth
of probiotic cultures, only dairy and soy derived products have been labeled to contain
probiotic cultures, mainly due to the compatibility of processing with the survival of the
microbes to the required levels (Ouwehand and Röytiö 2015). Probiotic cultures are not
generally effective fermenters and can benefit from the presence of robust compatible
starter cultures in fermented foods. Although, the most recognize health effect from
probiotic cultures is in the recovery of antibiotic treated diarrhea, they have also been
implicated in the exclusion of pathogens from the gastrointestinal tract, shortening of
colonic transit, modulation of the host immunity, and improvement of metabolism (see
Ouwehand and Röytiö 2015 for a review; Gürakan 2007).

7 New Trends in Food Fermentations

Traditional spontaneous fermentation will continue to be central to the production,

availability and supply of foods for a growing population. Current consumer trends
are aligned with the deliberables of fermented foods, such as increased bioavail-
ability and bioactivity of natural components and the demand for natural processes
to create healthy foods with reduced environmental impact. Additionally, food fer-
mentations done with standard hygienic practices and the recommended processes
very often result in safe food alternative for consumers. In a decade when, non-­
thermal preservation technologies such as high hydrostatic pressure and pulsed
electric fields, and new packaging systems with modified atmospheres are subjected
to scrutiny by regulatory agencies and offer limited production capacity, food fer-
mentation will continue to deliver.
Food fermentations in which starter cultures are added are likely to continue to
emerge as alternatives with enhanced nutrition and flavor. The use of molecular
genetic tools is expected to result in bacteriocinogenic cultures that combined with
other food additives or technologies could offer stable and savory products
(Devlieghere et al. 2004; Settanni and Corsetti 2008).
Enhanced packaging and sanitizers for food contact surfaces are under develop-
ment and expected to result in a generation of polymers and antimicrobials that could
enhance the long term stability of fermented foods (Appendini and Hotchkiss 2002).
The selection, design and development of starter cultures for fermented foods
will continue to be the main research focus in this field, more so with the availability
of new molecular genetic techniques, the multiple-omics technologies and bioinfor-
matics. Starter cultures capable of positively impacting the organoleptic properties
of foods, reduce the toxic content of foods or modify the undesired microbial activ-
13 Microbial Fermentation in Food Preservation 293

ity in the gut, and contribute to the health of consumers will continue to be desirable
(van Hylckama Vlieg et al. 2011).
Advances in genomics and physiology of lactic acid bacteria are enabling a
link between individuals genetic background and metabolic traits of starter cul-
tures to specific food quality attributes (Ganzle 2009). Selection of starter cultures
will continue to be increasingly influenced by their functionality. Development of
optimized food fermentation systems with optimal starter cultures and chemical
composition is around the corner.

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Chapter 14
Non-thermal Methods for Food Preservation

Lynette E. Orellana, María de Lourdes Plaza, Fernando Pérez,

Yarilyn Cedeño, and Oscar Perales

Abstract Consumers are looking for convenient, fresh-like, minimally processed

foods. Therefore, new trends in food processing, product development, and quality
assurance are promoting intense research on alternative methods for food preserva-
tion and the concept of non-thermal treatments was developed. These technologies
allow the food sector to meet product safety and shelf-life requirements while
reducing the impacts on food quality attributes. Foods can be non-thermally pro-
cessed by (1) cold plasma, (2) high hydrostatic pressure (HHP), (3) microwave and
radio-frequency and (4) dense phase carbon dioxide, among other techniques. This
chapter includes a brief summary of the latest developments of some of these novel
technologies. In addition, a brief discussion about the use of nanomaterials for food
applications has been included.

Keywords Cold-plasma • High hydrostatic pressure • Microwave • Radio-­

frequency • Dense phase carbon dioxide • Nanomaterials

L.E. Orellana (*) • M. de Lourdes Plaza

Program of Food Science and Technology, University of Puerto Rico at Mayagüez,
Mayagüez, Puerto Rico
e-mail: [email protected]; [email protected]
F. Pérez
Agricultural and Biosystems Engineering Department, University of Puerto
Rico at Mayagüez, Mayagüez, Puerto Rico
e-mail: [email protected]
Y. Cedeño
Department of Engineering Science and Materials College of Engineering,
University of Puerto Rico at Mayaguez, Mayagüez, Puerto Rico
e-mail: [email protected]
O. Perales
Department of Engineering Science and Materials, University of Puerto Rico at Mayaguez,
Mayaguez, Puerto Rico
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 299

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_14
300 L.E. Orellana et al.

1 Summary

Consumers are looking for convenient, fresh-like, minimally processed foods.

Therefore, new trends in food processing, product development, and quality assur-
ance are promoting intense research on alternative methods for food preservation.
Most foods are thermally processed by exposing the products to high temperatures
for a few seconds to several minutes. Thermal treatments have traditionally been used
in the food industry to provide the required safety profiles and to extend product
shelf-life. However, heat treatments lead to damage of temperature-labile compounds,
resulting in partial loss of organoleptic and nutritional properties of food products.
To overcome or minimize such disadvantages, the concept of non-thermal treatments
was developed. These technologies allow the food sector to meet product safety and
shelf-life requirements while reducing the impacts on food quality attributes. They have
the potential to replace, at least partially, the traditional food preservation techniques.
Foods can be non-thermally processed by (1) cold plasma, (2) high hydrostatic pres-
sure (HHP), (3) microwave and radio-frequency and (4) dense phase carbon dioxide,
among other techniques. This chapter includes a brief summary of the latest develop-
ments of some of these novel technologies. In addition, a brief discussion about the use
of nanomaterials for food applications has been included. Nanomaterial applications in
the food processing and storage area include nanocomposites for plastic film coatings
used in food packaging, antimicrobial nanoemulsions for applications in decontamina-
tion of food equipment, packaging, or food. Each of these techniques can be used either
alone or in combination to optimize product quality, processing time, and bacterial and
enzyme inactivation. Another advantage of non-thermal processes is that in general they
utilize less energy than thermal processes. Each non-thermal technology has specific
applications in terms of the type of food processed. Therefore, non-thermal technologies
are not applicable in processing every variety of food. Each non-thermal technology has
its merits and limitations, but in many cases, the use of a combined method or hurdle
approach is necessary. Commercial development of these technologies will be depen-
dent upon a sound understanding of the science and technology of the observed effects,
suitably scaled equipment, and economic viability.

2 Cold Plasma

The term plasma, for an ionized gas, was first introduced in 1928 by Irving Langmuir
(Langmuir 1928).When a gas is in contact with an electrical discharge, there is a glow
flow composed of very reactive chemical species such as charged particles, free radicals,
and some radiation; this is known as plasma (Bermúdez-Aguirre et al. 2013). Plasma
can be defined as the state of ionized gas consisting of positively and negatively charged
ions, free electrons, and activated neutral species (excited and radical), and are generally
classified into two types, thermal (or equilibrium) plasma and cold (or non-equilibrium)
plasma, based on the difference in characteristics (Yasushi et al. 2011). When the plasma
14 Non-thermal Methods for Food Preservation 301

is generated under mild conditions of temperature, it is referred to as cold atmospheric

plasma (Fernández and Thompson 2012). Cold atmospheric plasmas (CAP) are pro-
duced by the excitation of gas with electrical discharges at room temperature and atmo-
spheric pressure (Fernández and Thompson 2012). The temperature of cold plasma is
around 30–60 °C and this temperature is preferred by the food industry because of the
low energy required to generate it (Misra et al. 2011). Plasma is a source of different
antimicrobial substances including UV photons, charged particles, and reactive species
such as superoxide, hydroxyl radicals, nitric oxide, and ozone (Daeschlein et al. 2010;
Deng et al. 2006; Gaunt et al. 2006; Laroussi and Leipold 2004). The gas in which the
plasma is created determines which active species are formed (Moreau et al. 2005). An
air discharge, for example, can produce oxygen atoms, ozone, OH-radicals, N-radicals,
plasma electrons, photons, and other species, that are strong oxidizers that can decom-
pose inorganic and organic compounds (Deng et al. 2007b). It is believed that the free
charged particles interact with the surface of product affecting chemical bonds that lead
to sterilization (Soroka 2009).
Cold plasma is an emerging non-thermal food technology which has recently
drawn considerable interest for decontamination of foods and food processing sur-
faces (Pankaj et al. 2013). Plasma consists of highly energetic species in permanent
interaction including photons, electrons, positive and negative ions, free radicals,
and excited or non-excited molecules and atoms, which in combination are able to
inactivate microorganisms (Deng et al. 2006; Laroussi 2005; Moisan et al. 2002).
The capability of non-thermal atmospheric plasma to inactivate vegetative cells,
including Gram-negative and Gram-positive bacteria, yeast and fungi, biofilm developing
microorganisms, endospores, and biomolecules such as proteins was shown in various
studies (Brandenburg et al. 2007; Deng et al. 2007a; Laroussi 2005; Vleugels et al. 2005).
However, the emitted reagents do not only react with bacteria, but they may also react
with food components such as water, lipids, proteins, and carbohydrates that may lead to
off-flavors (Keener 2008).
The antimicrobial effect of gas plasmas is not entirely understood. Several studies
propose that one of the inactivation mechanisms is UV radiation in the 200–300 nm wave-
length range, which causes damage to DNA by inducing the formation of thymine dimers
(Boudam et al. 2006; Laroussi 2005; Muranyi et al. 2010; Sharma et al. 2005, 2009).
However, other studies suggest that UV radiation does not play a significant direct role in
the sterilization process by cold plasma because the power density of the emitted UV
radiation is very low (b50 μW/cm2) (Boudam et al. 2006; Laroussi and Leipold 2004). The
diffusion of reactive species such as free-radicals and excited molecules through the bacte-
rial cell membranes can also cause severe local damage by reacting with macromolecules
such as membrane lipids, proteins, and nucleic acids (Dobrynin et al. 2009; Gallagher
et al. 2007; Laroussi and Leipold 2004; Montie et al. 2000). Several studies have recently
been shown that CAP treatment induces the expression of DNA repair systems and the
oxidative stress response in Escherichia coli (Sharma et al. 2009). In addition, free radicals
can cause surface lesions in membranes by direct bombardment (etching) (Fernández and
Thompson 2012). The free radicals present in cold plasmas can also adsorb onto the sur-
faces of microorganisms to form volatile compounds such as carbon dioxide that are then
eliminated from the cell (Moreau et al. 2008; Sharma et al. 2005).
302 L.E. Orellana et al.

Charged particles such as electrons and ions may act in tandem with reactive species
to synergistically inactivate microorganisms (Gallagher et al. 2007). It has been shown
that bombardment of the cell wall by charged particles can induce breaking of chemical
bonds, erosion, and localized lesions in the membrane with further penetration of toxic
reactive plasma species into the cell (Gallagher et al. 2007). Both physical and chemical
mechanisms have been implicated in causing damage to the cell membrane (Fernández
and Thompson 2012). The electrostatic tensions generated by plasma electrons and ions
that accumulate on the cell surface may cause the disruption of the cell membrane
(Laroussi et al. 2003; Mendis et al. 2000; Ying et al. 2006; Yu et al. 2005). In addition,
plasma ions may catalyze oxidation and peroxidation processes within the cell and in the
external environment which would result in inactivation (Dobrynin et al. 2009). The avail-
able data seem to indicate that CAP can inactivate microorganisms by a number of differ-
ent mechanisms that are likely to act synergistically (Fernández and Thompson 2012).
The design of effective plasma treatments that ensure the safety and stability of food
products also depends on factors affecting the resistance of microorganisms to CAP. It has
long been known that microbial resistance to physicochemical agents is influenced by
many factors such as the intrinsic resistance of the microorganisms, the physiological
state of the cells, and the treatment conditions (Tomlins and Ordal 1976).
Typical plasma processes occur at elevated temperatures and low pressures (i.e.,
vacuum). Cold or non-thermal plasma, in contrast, refers to a plasma application
that takes place at atmospheric pressure and near ambient temperatures (i.e.,
30–60 °C). Energy consumption, thus degree of gas ionization, is low compared to
other plasma application such as chemical deposition and neon lighting.
An important characteristic of cold plasma is that it is strictly a surface treatment
because the active species (charged particles) cannot penetrate more than the first
few layers of the product’s surface. This has two important implications. First, the
technology must be applied previous to packaging since packaging would impair
the plasma’s access to the product. As such, packaging material will depend strictly
on the product’s requirements. Alternatively, the product can be packed with enough
headspace gas to allow plasma generation. Plastic polymers had been the preferred
choice since they are flexible enough to make good contact with the machine elec-
trodes that generate the plasma inducing electromagnetic field. Second, its food
application would be limited to products such as ready-to-eat meat and poultry
products, fruits, vegetables, nuts, and spices, where the main intent is to reduce
microbial load (as opposed to commercial sterilization). Thin liquid films could also
be treated with cold plasma. Pankaj et al. (2013) for example, used cold plasma to
study the inactivation kinetics of peroxidase enzymes from tomato.
Commercial application of cold plasma is still limited. Further studies are
required to investigate the potential generation of harmful by-products, changes
to sensory properties, and nutritional and chemical effects (Fernández and
Thompson 2012). Bermúdez-Aguirre et al. (2013) worked with E. coli inactiva-
tion on carrot, lettuce, and tomatoes using cold plasma and found no significant
changes of color parameters, but a high degree of electroporation, loss and dis-
ruption of membrane, and deformation of cell membranes after treatment.
14 Non-thermal Methods for Food Preservation 303

Significant research on cold plasma food applications has been performed at the
USDA-ARS Eastern Regional Research Center (Niemira 2009, 2012a, b, c; Niemira
and Sites 2008). Work includes various microorganisms, products, and processing
conditions, all proving the successful application of the technology. Niemira (2012c)
concludes that ‘as a dry, nonthermal process, cold plasma has the potential advan-
tages of antimicrobial reductions that can be obtained with minimal water usage
and minimal sensory impact to the treated foods’.
Successful application of cold plasma to food requires the right combination of
processing parameters, including treatment time (2 s–3 min), voltage level (>20 kV),
electric power, frequency, gas (air, oxygen, nitrogen, argon, or helium), and gas flow
rate. As an example, Deng et al. (2007b) obtained a 5-log reduction of E. coli in
almonds after a 30 s treatment at 25 kV and 2500 Hz treatment using air as the gas,
and modeled inactivation kinetics using D and z values.

3 High Hydrostatic Pressure (HHP)

High hydrostatic pressure (HHP) technology has emerged as a great alternative to

thermal treatment of foods because it allows for the inactivation of microorganisms
with minimal effect on the physicochemical characteristics of food. Due to consum-
ers’ demands for fresh-like, minimally-processed food products, today, high pres-
sure processing is being used extensively in the food industry. High pressure
processing is carried out with intense pressure in the range of 100–1000 MPa, with
or without heat, allowing most foods to be preserved with minimal effect on taste,
texture, or nutritional characteristics (Yordanov and Angelova 2010). HHP acts
instantaneously and uniformly throughout a mass of food independent of size,
shape, and food composition (Farkas and Hoover 2000).
HPP is based on two basic principles. According to the equilibrium law, changes
in pressure at constant temperature will favor changes leading to reductions in vol-
ume. This means that molecules subjected to high pressures will tend to increase
molecular ordering that result in higher density (i.e., smaller volumes for the same
mass). For complex macromolecules, like protein, HPP leads to partial denaturation
caused by damage to quaternary and tertiary structures that have weaker bonds
compared to primary and secondary structures. In fact, HPP is said not to affect the
strong covalent bonds at low temperatures (Muredzi 2012). That explains why
starch macromolecules are not as affected as protein under HPP. The main
­implication of this principle is that, since enzymes and microbial cellular walls are
made of proteins, HPP can effectively destroy them.
The second principle governing HPP is that the process is isobaric. Pressure is uni-
formly distributed by the water medium throughout the sample or product. Thus, in
contrast to heat that needs to be transferred by a combination of conduction, convection,
and radiation, HPP is said to be independent of time since pressure is transmitted as it
increases regardless of package size and shape. Another implication of this principle is
304 L.E. Orellana et al.

that, if the product contains enough moisture and no entrapped air, pressure will not
damage the product’s external configuration.
Although the main process parameter is hydrostatic pressure, HPP applications
have been designed at temperatures ranging between 0 and 100 °C. Processing at
the lower temperatures helps maintain heat sensitive components, including nutri-
ents, pigments, and aromas, while processing at higher temperatures strives for
destruction of bacterial spores with the shortest possible processing time. Usage of
such hurdle technology results in processing times ranging from seconds to several
minutes depending on product pH, water activity, and formulation. Both these
parameters, along with pressure compression, holding and decompression times,
and food factors (pH, composition, water activity, and packaging) define the sched-
uled process for a specific product (Farkas and Hoover 2000).
Inactivation kinetics of HPP has been extensively studied. Ramaswami et al. (2010)
observed the effect of pressure and combined pressure and temperature on microbial
cell membrane and concluded that the pressure effect decreases as temperature
increases and vice versa. Their work applied the traditional models to attain an equiva-
lent D-value to describe the pressure dependence of the inactivation kinetics.
Rendueles et al. (2011) explain that predictive models of microbial inactivation for
HPP require the development of two models. A primary model establishes first order
kinetic equations regarding the effects of process parameters on cell population as a
function of process time. These models are useful to determine D-values. Secondary
models are also required to account for the non-linearity of semi-­logarithmic survival
curves that often exhibit shoulders (inactivation latency due to cellular repair) and/or
tails (due to spores and/or pressure resistant sub-populations).
Hereu et al. (2012) studied the effect of HPP on ready to eat meats inoculated
with L. monocytogenes and describe the observed inactivation behavior composed
of a pressure sensitive population that decreases first, and a residual (pressure resis-
tant) population that requires a more severe treatment. Hereu et al. (2012) consid-
ered various secondary models to describe the inactivation kinetics; including a
biphasic model and a log-linear model that includes a tail to consider the remaining
pressure resistant cells. Zimmermann et al. (2013) also applied the biphasic model
to the inactivation of B. coagulans in tomato pulp with acceptable results.
One of the principal advantages of the HHP process is the expanded shelf-life
and improvement of food safety due to microbial inactivation, destroying vegetative
cells, and inactivating certain enzymes (Simpson and Gilmour 1997). The loss of
viability of microorganisms through HHP is probably the result of a combination of
injuries in the cell. The resistance of microorganisms is highly variable, depending
mainly on the type of organism and the food matrix involved, e.g., spores show
great resistance to inactivation.
In 1899, Hite observed microorganisms’ reduction after milk treatment to pressures
of 600 MPa for 1 h at room temperature (Hite 1899). Later, Hite and collaborators
(1914) observed that pressure-treated fruits (400–820 MPa) achieved commercially
sterility for about 5 years. Today, hundreds of publications can be found on a great
diversity of high pressure applications for the food industry (Considine et al. 2008;
Devlieghere et al. 2004; Farr 1990). In addition to food preservation HHP has been
14 Non-thermal Methods for Food Preservation 305

shown to achieve interesting functional effects (Leadley and Williams 1997). High
hydrostatic pressure does not alter the low-energy, covalent bonds, which have low
compressibility and do not break within the ranges of pressures normally used in food
(Rendueles et al. 2011). Therefore, the primary structure of molecules such as proteins
or fatty acids remains intact (Considine et al. 2008). The tertiary and quaternary struc-
tures of molecules, which are maintained mainly by hydrophobic and ionic interactions
are beneficially altered by high pressure above 200 MPa (Hendrickx et al. 2005)
because the ionic bonds and hydrophobic interactions, responsible for maintaining the
secondary and tertiary structure of proteins, are disrupted (Aymerich et al. 2008;
Considine et al. 2008; Heremans 1995; Ross et al. 2003). This disruption can influence:
enzyme inactivation, nutrient digestibility and bioavailability, technological and func-
tional properties (Rendueles et al. 2011). Molecules such as vitamins, amino acids,
flavor molecules or other low-molecular-weight compounds are hardly affected and as
a result, the organoleptic and nutritional properties are slightly modified (Aymerich
et al. 2008; Farkas and Hoover 2000).
Apparently, there is no single damage in a cellular structure or function (Hoover
et al. 1989; Manas and Mackey 2004; Ritz et al. 2001). Cell death is due to a mul-
tiplicity of damage accumulated in different parts of the cell (Malone et al. 2002).
On occasions, the cell is damaged but could recover if post-treatment conditions
are appropriate (Bozoglu et al. 2004; Bull et al. 2005). Because of its special fea-
tures, the cell membrane is the main target of HHP (Pagan and Mackey 2000; Ritz
et al. 2000, 2002; Russell 2002), resulting mainly in permeability modification and
functionality disruption (Pagan and Mackey 2000).
Prokaryotes are usually more resistant, compared to eukaryotes (Yuste et al. 2001).
Studies on the effectiveness of HHP in eliminating foodborne parasites show the
sensitivity of protozoa and parasites: Toxoplasma gondii (Lindsay et al. 2006),
Cryptosporidium parvum (Collins et al. 2005), Anisakis simplex (Brutti et al. 2010;
Molina-Garcia and Sanz 2002), Trichinella spiralis (Noeckler et al. 2001), and
Ascaris (Rosypal et al. 2007) in low pressure ranges (100–400 MPa). Molds and
yeasts have intermediate resistance (Palou et al. 1997, 1998; Shimoda et al. 2002).
In studies with foodborne vegetative pathogens (Alpas et al. 1999), large viability
losses (reductions in excess of 8 logs) are shown when relatively low pressures
(300 MPa) are combined with intermediate temperatures (50 °C, 5 min). Large differ-
ences among pathogenic microorganisms (Listeria monocytogenes, Staphylococcus
aureus, Escherichia coli, Salmonella typhimurium) have been reported. Also, differ-
ences between pathogenic strains belonging to the same genus or species have been
described (Alpas et al. 1999; Benito et al. 1999; Bull et al. 2009; Garcia-Graells et al.
2000). Cell membrane structure in Gram-positive and Gram-negative bacteria differs in
resistance to HHP (Russell 2002). Generally, Gram-positive bacteria are more resistant
compared to Gram-negative (Shigehisa et al. 1991). The double-layered phospholipids
which are present in the lipid membranes are packed tightly during the compression
phase, promoting the transition towards a gel state (Hazel and Williams 1990) and dur-
ing decompression the dual layer structure is lost, promoting pore formation and cyto-
plasmic material leaking (Hoover et al. 1989; Shimada et al. 1993). The membrane
must maintain the fluid state to maintain its function and properties, and fluidity is
306 L.E. Orellana et al.

mainly determined by composition and percentages of unsaturated fatty acids (FA)

(Rendueles et al. 2011). The FA in the membrane of barophiles and barotolerants have
a greater degree of unsaturation (Smelt 1998; Yano et al. 1998). Several studies observed
that psychrophiles contains high levels of polyunsaturated FA (docosahexaenoic acid)
and are generally more pressure-resistant, confirming that fluid membranes, rich in
unsaturated FA, are partially responsible for resistance to HHP lethal effects (Casadei
et al. 2002; Smelt 1998). The inactivation of the ATPase of the cell membrane or other
proteins such as efflux pumps also affects membrane function and alters the acid–base
physiology of the cell (Hoover et al. 1989; Russell 2002). In addition, ribosomes
(Kaletunc et al. 2004), protein synthesis and enzyme activity (Considine et al. 2008;
Simpson and Gilmour 1997; Wouters et al. 1998), and structure of DNA-enzyme com-
plexes, as the DNA can be degraded due to the action of endonucleases not normally in
contact with DNA (Chilton et al. 1997) are sensitive to high pressures.
Morphological and structural changes in the cell have also been observed including:
separation of the membrane from the cell wall, lengthening of the cell, compression of
gas vacuoles, (Patterson 2005) and condensation of nuclear material (Manas and Mackey
2004; Wouters et al. 1998). HHP treatments are reported to induce: cold and heat stress
and oxidative stress response, changes in the genetic regulation of quimiotaxis gene,
phosphotransferase production, flagella, and expression of genes involved in cell elon-
gation and development of septa (Aertsen et al. 2004, 2005; Malone et al. 2006).
Spores show great resistance to inactivation by high pressure and the inactivation
mechanisms are not clearly understood, but it is believed that spores are activated due
to particular pressure/temperature conditions, losing their inherent resistance, and sub-
sequently killed by other treatment conditions (Rendueles et al. 2011). During inacti-
vation, a series of events such as the release of dipicolinic acid (chelate of Ca2þ and
pyridine-2,6-dipicolinic acid or Ca-DPA) and small acid-soluble spore proteins
(SASPs), the hydrolysis of core and cortex, and the decrease of intracellular pH occur
(Ahn and Balasubramaniam 2007). Spores are sensitized by moderately high pressures
(50–300 MPa), and it has been postulated that nutrient germinant receptors are acti-
vated by low-level HHP, which in turn, provokes the release of Ca-DPA. Ca-DPA trig-
gers a cascade of later germination events (hydrolysis of spore cortex by cortex-lytic
enzymes (CLE), degradation of SASPs, and ATP ­generation) (Paidhungat et al. 2002).
The resultant germinated spores are sensitive to subsequent heat and pressure treat-
ments (Black et al. 2005, 2007). Also, high pressures (>500 MPa) can induce rapid
germination by direct release of Ca-DPA (Wuytack et al. 1998).
Viruses possess a wide range of pressure resistance, depending on their structural
diversity. Enveloped viruses are usually more sensitive to the pressure than naked
viruses and the polioviruses seem to be the most resistant virus (Rendueles et al. 2011).
The inactivation rate for viruses follows generally non-linear kinetics, fitting Weibull,
or log-logistic models (Buckow et al. 2008). Also, for viruses, HHP resistance varies
within related taxonomic groups or even strains, and differences in protein sequence
and structure are responsible (Baert et al. 2009).
HHP has been used for a wide range of commercial fruit based products and
ready-to-eat meats, all of which require special packaging considerations. Since
products are subjected to high pressures, package materials must be able to
14 Non-thermal Methods for Food Preservation 307

withstand and transfer process induced stress to the product and recover from it
at the end of the cycle. Thus, flexible plastic pouches are the obvious choice for
HHP since most multilayer materials their barrier, mechanical, volatile migra-
tion, and delamination properties. (Min and Howard-Zhang 2007).

4 Microwave and Radio-Frequency

Microwave (MW) and radio-frequency (RF) heating refers to the use of electromag-
netic waves of certain frequencies to generate heat in a material (Metaxas 1996;
Metaxas and Meredith 1983; Roussy and Pearce 1995). Frequencies of 2450 and
915 MHz are used for microwave food processing. For radio-frequency, 13.56,
27.12, and 4068 MHz could be used, however there is not much commercial use of
these frequencies for pasteurization or sterilization, although they are used in bak-
ing processes in the food industry (Datta and Davidson 2000) and in package steril-
ization of food in large packages. Sumnu and Sahin (2005) and Chandrasekaran
et al. (2013) present comprehensive reviews of the technology and food applications
for blanching, baking, drying, thawing, pasteurization, and sterilization.
Microwave techniques were developed originally for military activities. The appli-
cation of microwave energy to heat foods was patented in 1945 by Percy Spencer of
Raytheon Corporation as an offshoot of radar technology developed during World War
II (Buffler 1993). The first Radarange™ became available for foodservice use in 1947
and commercial ovens were introduced in 1955. Approximately 93% of American
households own a microwave oven, primarily for use in rewarming previously cooked,
chilled, or frozen foods (Fusaro 1994). There are additional commercial applications of
microwave heating other than cooking and reheating operations typically used by con-
sumers. Microwaves are used to inactive enzymes (Copson 1954; Pour-El et al. 1981),
blanch foods (Goldblith 1966), defrost, temper, or thaw products (Decareau 1985),
bake, pasteurize, or sterilize (Olsen 1965; Ayoub et al. 1974; Kenyon et al. 1971; Sale
1976), and evaporate, and dry and freeze-dry (Sunderland 1982). Industrial microwave
pasteurization and sterilization systems have been reported for almost 50 years (Jeppson
and Harper 1967; Kenyon et al. 1971; Mudgett and Schwartzberg, 1982; Decareau
1985; Schlegel 1992; Harlfinger 1992; Anonymous 1996). Studies with implications
for commercial pasteurization and sterilization have also appeared for many years
(Proctor and Goldblith 1951; Chandrasekaran et al. 2013).
In the food heating process, the advantages of microwaves include: speed of heat-
ing, energy saving, precise process control, and faster start-up and shutdown times
(Decareau 1985). Microwave and radio-frequency heating for pasteurization and ster-
ilization are preferred over the conventional heating for the primary reason that they
are rapid and, therefore, require less time to come up to the desired process tempera-
ture (Datta and Davidson 2000). Rapid heating allows for shorter processing times
that will provide microbial and enzyme inactivation with minimal impact to nutri-
tional or sensory quality. Microwave and radio-frequency heating may be relatively
more uniform than conventional heating, depending on the particular heating situation
308 L.E. Orellana et al.

(Datta and Hu 1992); however, heating uniformity is hard to predict. An inherent

disadvantage of MW and RF systems is the generation of hot and cold stops through
the product resulting from the uneven distribution of the electric field inside the pro-
cessing chamber and the product. As technology progresses, improvements such as
fans to increase wave distribution, rotary plates or moving conveyors, and variable
wave frequency have been incorporated to achieve uniform heating of products.
Microwave heating is caused by the ability of the materials to absorb microwave
energy and convert it into heat (Chandrasekaran et al. 2013). Production of heat occurs
during microwave heating primarily through dipolar rotation and ionic polarization
(Chipley 1980), that is caused by field oscillations. When microwave energy is absorbed
by the food, energy is converted to heat. Because of the way in which microwaves heat,
some foods require reformulation to accommodate these changes (Schiffmann 1990).
Polar molecules such as water have negatively and positive charged ends. In the pres-
ence of a microwave electric field, the water molecules attempt to line up with the field.
Since the microwave field is reversing polarity millions of times per second, the water
molecules moves first in one direction and then reverses to move in the opposite direc-
tion. The friction caused by constantly oscillating charged particles produces heat. Most
foods are composed of 50–90% water (Ohlsson 1983), and it is the unbound water that
contributes to the heating effect. In addition, ions, because of their electric charge, flow
in one direction, then in the opposite direction in an electromagnetic field producing
heat by ionic conductivity. This effect leads to a higher temperature at the surface of
water or foods containing salt. The oscillatory migration of ions in the food also gener-
ates heat under the influence of the oscillatory electric field (Tewari and Juneja 2007).
For MW heating, dipole rotation tends to be the dominant heating mechanism while
ionic depolarization is typical of RF (and ohmic) heating. As can be expected, formula-
tion (e.g., water, fat, protein, or salt content) plays an important role in heating as it defines
the product’s dielectric properties. These properties define the amount of heat absorbed,
conducted or loss, as well as the penetration depth of the electric field (Marra 2012).
The ability of microwaves to heat depends upon the dielectric properties of the food
as described by the dielectric constant and the dielectric loss factor (Schiffmann 1990).
They are also highly dependent on the frequency of the applied electric field, the moisture
content, temperature, and bulk density (Decareau 1985; Metaxas and Meredith 1983).
The dielectric constant (ε′) describes the ability of the material to store electrical energy
and varies significantly with temperature and frequency. The dielectric loss factor (ε″)
describes the ability of the material to dissipate electrical energy as heat.
When electromagnetic waves go through the package without being absorbed,
materials are said to be microwave transparent. Food packaging polymers, paper
products, and glass are microwave transparent materials. Yet, packaging for MW
and RF is mostly limited to certain plastics, typically polypropylene (PP), polysty-
rene (PS), and polyethylene terephthalate (PET). Such transparent plastics are also
referred to as passive (Robertson 2012) and the temperature resistance of the pack-
age must be carefully considered for each application.
Properties of such polymers can be enhanced by co-extrusion of several materials.
A common application is the metallization of one of the internal layers of the film by
means of vacuum deposition of an aluminum vapor. The resulting packaging material
14 Non-thermal Methods for Food Preservation 309

will absorb MW energy and transform it to heat to provide a localized heat source that
will allow the development of crisp textures in pastry products or pizza, for example.
The effectiveness of microwave technology depends on factors such as time-­
temperature history (cold and hot spots during the process), package characteristics
(shape, size, surface area, and composition), and equipment (oven geometry, power of
microwave generator, frequency of microwaves, presence of stirrers and/or turntables)
(Datta and Davidson 2000). Though there are microbial inactivation effects directly
resulting from MW or RF application, these are considered to be negligible compared
to the resulting effects of the process. As such, inactivation kinetic models are basically
the same as for thermal processes. Yet, microbial inactivation using MW or RF tech-
nologies must be carefully considered because the lack of uniformity in temperature
distribution yields cold spots that might foster microbial survival to the treatment.
Hamoud-Agha et al. (2013) developed a 3D finite elements model for the inacti-
vation of Escherichia coli K12 CIP 54.117 in a gel medium and compared the
numerical results against the experimental data. An agreement was found between
the experimental data and the model based on Maxwell’s equations on the existence
of cold spots under the conditions of the study, and Hamoud-Agha et al. (2013)
proposed that their model could be used as feedback on a closed loop control system
to optimize the MW treatment.
The energy absorption from microwaves and radio-frequency can raise the tempera-
ture of the food high enough to inactivate microorganisms for effective pasteurization or
sterilization. Several theories may explain the destruction of microorganisms or enzymes
by microwave or radio-frequency at sublethal ­temperatures including: selective heating,
electroporation, cell membrane rupture, and magnetic field coupling (Kozempel et al.
1998). Selective heating suggests that microorganisms are selectively heated due to
microwaves and reach a temperature higher than that of the surrounding fluid. This
causes the microorganisms to be destroyed more quickly (Goldblith and Wang 1967).
According to the electroporation theory, the electrical potential across the cell mem-
brane causes pores, which results in leakage of cellular materials, and the cell membrane
is ruptured due to voltage applied across the membrane (Chandrasekaran et al. 2013).
According to the magnetic field coupling theory, the internal components of the cell are
disrupted due to the coupling of electromagnetic energy with critical molecules such as
protein or DNA (Kozempel et al. 1998). Although various theories suggest the non-­
thermal effect of microwaves, it was further observed that in the absence of other stresses
such as pH or heat, microwave energy did not inactivate microorganisms. However, a
significant enhancement or magnification of thermal effects might have been caused by
microwaves (Kozempel et al. 2000). Regardless of the exact origin of the enhancement
of thermal effect, microwaves are effective in the destruction of microorganisms or inac-
tivation of enzymes (Chandrasekaran et al. 2013). Bacteria reported to be inactivated by
microwave heating include Bacilllus cereus, Campyobacter jejuni, Clostridium perfrin-
gens, pathogenic Escherichia coli, Enterococcus, Listeria monocytogenes,
Staphylococcus aureus, and Salmonella (Heddleson et al. 1994; Rosenberg and Bogl
1987; Knutson et al. 1988; Chipley 1980). The nematode Trichinella spiralis may also
be inactivated (Zimmermann, 1983). Microwave heating has been shown to be effective
in various food products including poultry, beef, fish, pork, milk, and eggs.
310 L.E. Orellana et al.

5 Dense Phase Carbon Dioxide

Today, consumers demand fresh, natural, and nutritious food, while food safety is a
big concern. For this reason, non-thermal technologies have gained increasing
importance as a valuable process to replace or complement the traditional technolo-
gies currently used for preserving foods and other biological materials (Balaban
and Ferrentino 2012). Thermal processing has a long history of use to preserve
food. Its purpose is to inactivate enzymes and microorganisms (including vegeta-
tive cells and spores). This technology involves the use of temperature (higher than
60 °C) for a specific amount of time (from a few seconds to several minutes)
depending on the product. Although thermal processing renders safe food, the
severity of the treatment affects the quality of the final product. Color, flavor, and
vitamin contents are some of the quality parameters affected. Quality loss during
thermal processing is associated to unwanted reactions that allow formation of by-
products and undesirable changes (Balaban and Ferrentino 2012).
During non-thermal processing, food is held at temperatures below normal ther-
mal processing temperatures. Therefore, the changes in organoleptic characteristics
caused by thermal processing are minimal with non-thermal processing (Palou 1997).
Fraser (1951) and Foster et al. (1962) were the first researchers who reported the effect
of CO2 under pressure on the bacterial cell. The effect was the rupture and liberation of
bacterial cell onto the environment due to decompression of CO2 used for processing.
Dense phase carbon dioxide (DP-CO2) is a cold pasteurization method that
affects microorganisms and enzymes through the effect of CO2 under pressure
below 50 MPa without affecting the fresh-like, physical, chemical, and sensory
qualities. Carbon dioxide, a natural constituent of many foods, is a non-toxic,
nonflammable, inexpensive gas and has a Generally Recognized as Safe status
(Damar and Balaban 2006).

5.1 Mechanisms of Microbial Inactivation

A number of hypotheses have been proposed to explain the effect of microbial inac-
tivation caused by DP-CO2, including cytoplasmic pH decrease, explosive cell rup-
ture due to internal pressure, modification of the cell membrane and extraction of
cell wall lipids, inactivation of key enzymes for cell metabolism, and extraction of
intracellular substances.
Cytoplasmic pH decrease or acidification has been proposed as the main mecha-
nism for microbial inactivation. In this mechanism, CO2 is dissolved in an aqueous
solution forming carbonic acid, which at a sufficient concentration is dissociated
into bicarbonate and hydrogen ions lowering the extracellular pH.
The first theory proposed for microbial inactivation was the explosive cell rup-
ture due to internal pressure. It was thought that during the rapid depressurization of
the sample, the CO2 would have rapidly expanded through the cells so that a part of
14 Non-thermal Methods for Food Preservation 311

them could have been mechanically broken. However, pictures of microbial cells
after treatment have shown that the mechanism of inactivation did not always
involve cell rupture (Spilimbergo and Bertucco 2003).
Modification of cell membranes and extraction of cell wall lipids is another
hypothesis for microbial inactivation. This mechanism is based on the lipophilic
and solvent characteristics of CO2. The cell membrane consists of a double layer
of phospholipids. The CO2 could easily penetrate into the membrane, leading to
an increase of is fluidity and permeability, altering the characteristics of the
membrane, and destroying its essential function. This mechanism is known as
the anesthesia effect. The anesthetic theory is a strong explanation for microbial
inactivation since images showed a modification of cell membrane with possible
leakage of cytoplasm, together with enlarged periplasmic space between the
walls and the cytoplasmic membranes (Spilimbergo and Bertucco 2003).
The theory of the inactivation of key enzymes for cell metabolism is based on the
interference of bicarbonate and molecular carbon dioxide on certain enzymatic and
biochemical pathways. Another proposed mechanism is the precipitation of
­intracellular calcium and magnesium carbonate ions from bicarbonate. This can
occur since there are some proteins sensitive to calcium and magnesium that could
be precipitated by carbonate.

5.2 Factors Affecting Microbial Inactivation

Factors that affect microbial inactivation using DPCD include water content within
the cell (more log reductions are achieved in wet cells than dry cells) and water activ-
ity of the food (DPCD is more effective as water activity increases). Generally, any
factor that increases levels and rate of CO2 solubility enhances microbial inactivation
caused by DP-CO2. For example, CO2 solubility increases with increasing pressure
when temperature, residence time, and CO2 concentration are equal. Generally, inac-
tivation efficiency increases with increases in pressure, temperature, and residence
time. Temperature has a complex effect on microbial inactivation. Even when the
CO2 solubility decreases with increasing temperature, inactivation of microbes by
DPCD is more effective at high temperature. Higher temperature increases the CO2
diffusion and fluidity of the cell membrane. Another effect of temperature is the
change of CO2, since its penetrating power is higher under supercritical conditions
and there is a rapid change in solubility and density when processing temperature is
near this critical region. The initial pH of the medium is another factor that affects
effectiveness of DP-CO2 microbial inactivation. Acid medium facilitates carbonic
acid penetration through the cell membrane, allowing a higher inactivation. The final
factor affecting microbial inactivation is the cell growth phase; young cells are more
sensitive than mature ones. The type of bacteria affects the DP-CO2 effectiveness.
Gram positive bacteria showed more resistance than gram negative bacteria due to
the differences in their cell membrane composition.
312 L.E. Orellana et al.

5.3 Solubility of CO2

CO2 solubility in liquid foods can be affected by pressure, temperature, and food
composition. Pressure has a direct effect on CO2 solubility: as pressure increases,
CO2 solubility increases. On the other hand, as temperature increases, solubility of
CO2 decreases. Food composition may increase or decrease the solubility of CO2
(Calix et al. 2008).

5.4 Types of Systems

Three different types of DP-CO2 equipment have been developed: batch, semi-­
continuous, and continuous.
The batch system was the first system developed. In this system, CO2 and the
food to be treated are stationary in a container during treatment. This system con-
sists of a CO2 gas cylinder, a pressure regulator, a pressure vessel, a water bath or
heater, and a CO2 release valve. The sample is placed into the pressure vessel and
temperature is set to the desired value. CO2 is then introduced into the vessel until
the sample is saturated at the desired pressure and temperature. The sample is left in
the vessel for a period of time, after which the CO2 outlet valve is opened to release
the gas. Some systems contain an agitator to decrease the time to saturate the sample
with CO2 (Damar and Balaban 2006).
A semi-continuous system allows a continuous flow of CO2 through the chamber
while a continuous system allows flow of both CO2 and the liquid food through the
system. In a continuous flow system, the liquid CO2 and the product are pumped
through the system and are mixed before entering the high pressure pump, which
allows adjustment of the pressure to the desired processing levels. The processing
temperature is controlled in holding coils. Residence time is adjusted by setting the
flow rate of the product through the coils. At the end of the process, an expansion
valve is used to release CO2 from the mixture and the treated product is collected
(Damar and Balaban 2006).

5.5 Food Applications and Effect on Quality

DP-CO2 has been applied mostly to fruit juices and beverages. The application of
DP-CO2 treatment to some fruits can cause tissue damage even at low pressures.
DP-CO2 treatment of orange juice showed that this non-thermal technology is effective
in reducing microbial load, enzyme inactivation, cloud stabilization, and maintenance
of the quality of the product.
Kincal et al. (2005) used a continuous high-pressure carbon dioxide (HPCD) system
for microbial reduction in orange juice, in which the effectiveness of the equipment in
14 Non-thermal Methods for Food Preservation 313

reducing the natural microflora of pulp-free Valencia orange juice was tested at different
pressures (38, 72, and 107 MPa), for a residence time of 10 min and CO2/juice ratios
between 0.1 and 1.0. To test the effectiveness on spoiled juice, juice with a load of
2 × 106 colony forming units per mL was prepared and subjected to sub- (25 °C) and
super-critical CO2 treatments (34.5 °C) at pressures of 38, 72, or 107 MPa, residence
time of 10 min and CO2/juice ratio of 1.0. To study the capacity of the equipment in
pathogen inactivation, untreated sterilized juice was inoculated with Salmonella
Typhymurium, E. coli O157:H7, or Listeria monocytogenes and the system was run at
pressures of 21, 38, or 107 MPa and a residence time of 10 min. A storage study was
conducted at 1.7 °C with juice processed at 107 MPa, CO2/juice ratio of 1.0 and resi-
dence time of 10 min. When the variables pressure and residence time were compared,
residence time had the greater influence on microbial reduction. Continuous high pres-
sure CO2 processing was capable of destroying the natural microflora in orange juice
and residence time and pressure had little influence on the destruction of low levels of
microorganisms. The CO2/juice ratio and temperature were shown not to be the driving
forces on microbial load reduction in this system. Kincal et al. (2005) proved that the
system was able to achieve a 5-log reduction of the natural flora in spoiled juice (38, 72,
and 107 MPa at 25 and 34.5 °C, CO2/juice ratio of 1.0 and residence time of 10 min),
and a 5-log decrease of pathogenic Salmonella typhimurium (all three pressures and
residence time of 10 min), Escherichia coli O157:H7 (pressure-related decrease), and
Listeria monocytogenes (destroyed after treatment with all pressures). During the
refrigerated storage study, an increase in the bacterial number possibly because of an
injury/repair mechanism of some of the microorganisms or due to post-contamination
was observed (Kincal et al. 2005).
In 2005, Gunes et al. studied the inactivation of yeast in grape juice using a con-
tinuous DPCD system. The processing parameters where carbon dioxide concentra-
tion (0, 80, and 170 g/kg), temperature (25 and 35 °C) and pressure (6.9, 27.6, and
48.3 MPa). Gunes et al. (2005) found that as processing parameters increased, the
inactivation rate increased. In addition, the CO2 in the supercritical state (pressure
higher than 7.34 MPa and temperature higher than 31 °C) was more effective at
inactivating yeast cells (achieved more than 6-log reduction). This can be due to the
enhanced chemical properties (better dissolving power and diffusivity) of the CO2
in this state. A preliminary sensory test showed that DPCD treated (48.3 MPa, 35 °C
and 170 g/kg) was indistinguishable from the control grape juice. A year later, inac-
tivation of E. coli (ATCC 4157) in diluted apple cider (Gunes et al. 2006) was stud-
ied. A response surface design with three factors for each processing parameter
(temperature, pressure and CO2 concentration) was used. CO2 concentration was the
primary processing parameter affecting the inactivation of E. coli in apple cider.
Kincal et al. (2006) published a work on a HPCD system for cloud and quality reten-
tion in orange juice, in which a continuous HPCD to treat pulp-free Valencia orange
juice at pressures of 38, 72, and 107 MPa, and CO2/juice (w/w) ratios from 0.10 to 1 with
a constant residence time of 10 min was used. For the storage study, orange juice was
treated at 107 MPa for 10 min, a CO2/juice ratio of 1.09 and stored it at 1.7 °C (Kincal
et al. 2006). The highest pectinesterase PE inactivation (56.0%) occurred at 72 MPa and
a residence time of 10 min followed by inactivation of 53% achieved at 107 MPa and a
314 L.E. Orellana et al.

residence time of 8.6 min. When the treatment was conducted at three different pressures
and a residence time of 10 min, the highest inactivation (46.3%) was obtained when the
pressure was 107 MPa and no heat was applied. These results demonstrate that pressure
affects PE inactivation. HPCD preserved and enhanced the cloud of the treated orange
juice in some cases. The greatest increase was found in samples treated at 38 MPa and
1.18% of CO2, and it was shown that pressure has little effect on cloud. Treatment with
continuous HPDP did not have any effect on pH and degrees Brix (°Bx), but titratable
acidity increased slightly after treatments. During the storage study, PE activity decreased
with ­storage time and cloud remained four times higher than the control during storage.
Juice color did not change drastically during storage (Kincal et al. 2006). Sensory evalu-
ations of DPCD-treated and untreated orange juice were not significantly different after
2 weeks of refrigerated storage at 1.7 °C (Balaban et al. 2008).
Lim et al. (2006) processed mandarin juice with DP-CO2. The process variables
were temperature (25, 35, and 45 °C), pressure (13.8, 27.6, and 41.4 MPa), resi-
dence time (5, 7, and 9 min) and CO2 (2, 7, and 12%). Temperature and CO2 had a
significant effect in log reduction of total aerobic plate count while pressure and
residence time were not significant. The HPCD processing reduced the total aerobic
count of natural flora in mandarin juice by about three orders of magnitude.
Maximum log reduction (3.47) was observed at the conditions of 35 °C, 41.4 MPa,
9 min, and 7% CO2. PE inactivation ranged from 6.1 to 50.7% with a maximum
inactivation achieved at 45 °C, 41.4 MPa, 7 min and 7% CO2. Cloud was not only
retained but enhanced. The highest cloud increase was 38.4% at 45 °C, 27.6 MPa,
7 min, and 2% CO2. Lightness and yellowness increased and redness decreased
after treatment. pH and °Bx did not change after treatment while titratable acidity of
treated samples was higher than the untreated juice (Lim et al. 2006).
Beer quality after pasteurization with DP-CO2 was studied. A maximum log reduc-
tion in yeast population of 7.38 logs was predicted at 26.5 MPa, 21 °C, 9.6% CO2, and
residence time of 4.8 min. The maximum haze reduction from 146 nephelometric
turbidity units (NTU) to 95.3 NTU was observed at a processing pressure of 27.6 MPa.
Aroma and flavor of beer processed under the same conditions were not significantly
different when compared to a fresh beer sample in a difference from control test.
Foam capacity and stability of beer were minimally affected by the process; however
these changes were unnoticed by consumers (Dagan and Balaban 2006).
The effects of DP-CO2 on microbial, physical, chemical, and sensorial quality of
a coconut water beverage were evaluated by Damar et al. (2009). Processing vari-
ables were: pressure (13.8, 24.1, and 34.5 MPa), temperature (20, 30, and 40 °C)
and CO2 (7, 10, 13 g CO2/100 g beverage). A constant residence time of 6 min was
used during the experiment. DPCD-treated (at 34.5 MPa, 25 °C, 13% CO2, 6 min),
heat pasteurized (74 °C, 15 s) and untreated coconut beverages were evaluated dur-
ing 9 weeks of storage at 4 °C. Results showed that pressure was not significant in
microbial reduction, whereas temperature and CO2 levels were significant. Total
aerobic bacteria of DPCD and heat-treated samples decreased while that of untreated
samples increased to >105 CFU/mL after 9 weeks. DP-CO2 increased titratable acid-
ity but did not change pH and °Bx. Likeability of DPCD-treated coconut water was
similar to the untreated one (Damar et al. 2009).
14 Non-thermal Methods for Food Preservation 315

The effect of DP-CO2 on physical and quality attributes of red grapefruit juice was
studied by Ferrentino et al. (2009). A central composite design was used with pressure
(13.8, 24.1, and 34.5 MPa) and residence time (5, 7, and 9 min) as variables. A constant
temperature of 40 °C and a CO2 level of 5.7% was used to treat the juice. A storage study
was performed with the fresh juice. °Bx, pH, titratable acidity (TA), pectinesterase (PE)
inactivation, cloud, color, hue and color density, total phenolics, antioxidant capacity,
and ascorbic acid were measured after treatment and during 6 weeks of storage at
4 °C. Five log reduction for yeasts and molds and total aerobic microorganisms occurred
at 34.5 MPa and 7 min of treatment. During storage, the DPCD-treated juice showed no
growth of total aerobic microorganisms, and yeasts and molds. Cloud increased by 91%
while PE inactivation was 69.2%. No significant (α = 0.05) differences were detected
between treated and untreated samples for °Bx, pH, and TA. Treated juice had higher
lightness and redness and lower yellowness. Slight differences were detected for the
ascorbic acid content and the antioxidant capacity (Ferrentino et al. 2009).
Ramírez-Rodrígues et al. (2011, 2012) used a continuous DPCD system to study
the effect of this technology on the microbial stability and physicochemical attributes
of a Hibiscus sabdariffa beverage. A central composite design was used with pres-
sure (13.8–34.5 MPa) and residence time (5–8 min) at a constant temperature (40 °C)
and CO2 concentration (8%). Optimum processing parameters for yeast and mold
inactivation were 34.5 MPa and 6.5 min of residence time. In conducting a storage
study (14 weeks at 4 °C) heat treatment (HTST pasteurization) was compared to
DPCD. Aerobic plate count and yeast and mold results presented the same pattern for
both treatments. Overall results demonstrate the effectiveness of the DPCD treatment
when compared to traditional heat pasteurization treatment. In addition, the effect of
DPCD processing in the Jamaican beverage (Hibiscus sabdariffa) over the phyto-
chemical, sensory attributes, and aroma stability during 14 weeks of refrigerated stor-
age was studied (Ramírez-Rodrígues et al., 2012). The antioxidant capacity and the
total phenolic content showed no significant differences between the treatments, and
the loss of anthocyanins was less than the thermally processed beverage (9% and
14%, respectively). The study showed that the DP process caused the retention of
more volatile compounds that the hot pasteurized one (75 °C for 15 min). The overall
likeability of the hibiscus beverage was not affected by storage up to week five.
Microbial reduction on guava puree was quantified as a function of pressure and resi-
dence time using 8% CO2 and a temperature of 35 °C (Plaza et al. 2009). Optimum
DPCD treatment conditions for microbial inactivation were 34.5 MPa and 6.9 min.
Quality attributes of DPCD-treated, freshly thawed and heat-pasteurized (90 °C for 60 s)
guava puree were measured and compared throughout refrigerated storage (4 °C for
14 weeks). Results showed that DPCD did not cause a change in pH or °Bx but increased
the titratable acidity and viscosity of the product. Pectinesterase (PE) was partially inacti-
vated by DPCD treatment and DPCD delayed the degradation of vitamin C during stor-
age (Plaza 2010). Flavor and aroma compounds in guava puree were identified using
GC–MS. Flavor profiles showed that heat-treated guava puree had less aroma active com-
pounds than DPCD-treated guava puree. Volatile compounds analysis showed a lower
total peak area for DPCD treated sample when compared to fresh and pasteurized sam-
ples. Differences in volatile composition were found for the three samples (Plaza 2010).
316 L.E. Orellana et al.

6 Nanomaterials for Food Packaging Applications

6.1 Basic Principles

Nanotechnology and its application is one of the rapidly developing sciences in the
new century. Nanotechnology-enabled products can be defined as products derived
or issued from materials at scales measuring less than 100 nm in at least one dimen-
sion (Gruère 2012). In general, characteristics such as particle size and distribution,
agglomeration state, shape, crystal structure, chemical composition, and surface
area, among others, may be important for understanding the properties of materials
at the nanoscale because such nanomaterials behave differently from the corre-
sponding bulk materials (Chau et al. 2007).
Applications in the food processing and storage area include nanocomposites for
plastic film coatings used in food packaging, antimicrobial nanoemulsions for
applications in decontamination of food equipment, packaging or food, in addition
to nanotechnology-based antigen detecting biosensors for identification of pathogen
contamination, among others (Salamanca-Buentello et al. 2005).
As demand for food safety, nanotechnology can contribute noticeably to the
development and improvement of existing safety processes. Some nanomaterials
can be used for this purpose due to their antimicrobial properties. A significant num-
ber of research efforts was done or continues to be done to understand the mecha-
nisms and enhance the efficiency of nanomaterials as antimicrobial agents, although
it will take more time to understand the full potential of nanomaterials in this field.

6.2 Historical Perspective

It is estimated that of the $100 billion packaging market in the US, 70% is attributed
to beverage and food production (Comstock et al. 2004). Although polymers and
co-polymers fulfill most of the technical and economical requirements for food
packaging, they are discarded to landfills and minimally recycled. Comstock et al.
(2004) has reported that less than 10% of all plastic packaging materials (not includ-
ing bottles) were recycled in the 90s. This poor recycling practice added to the non-­
biodegradability of these plastics, making the search for alternative bio-compatible,
non-toxic and bio-degradable materials an indispensable task.
Here is where polymer nanotechnology comes to play an important role. This
technology involves the research and development in the design, manufacture, pro-
cessing and application of environmental friendly polymeric materials hosting par-
ticles or devices that have one or more dimensions below 100 nm.
Novel and more efficient composites for food packaging based on nanotechnology
will provide innovative and safe alternatives to increase the coating performance, in
addition, to further adding safety, inhibition of any critical interaction with food matri-
ces and with human health, reduction in cost associated with ­production, transport and
14 Non-thermal Methods for Food Preservation 317

storage, increase of biodegradability and barrier protection to gases and light, and the
reduction of the volume of waste material to be disposed in landfills. Biopolymers,
such as calcium alginate and chitosan, represent an alternative to common, but
minimally recycled, plastics.
Previous studies demonstrated the attractiveness of antimicrobial formulations
within packaging films in the form of nanoparticles (Suyatma et al. 2004). Titanium
dioxide (TiO2), zinc oxide (ZnO), silver (Ag), carbon nanotubes, and magnesium
oxide (MgO) have been proposed as antimicrobial agents. The nanotechnology
enables a tuning in antimicrobial as function of particle size and concentration.

6.3 Mechanisms of Inhibition

The mechanisms of inhibition of some antimicrobial agents are discussed in this

section. Magnesium oxide is a promising nanomaterial with a strong antimicro-
bial effect. MgO can inactivate microbial by forming superoxide ( O•− 2 ) on its
surface or adsorbing negatively charged bacteria and spores on its positively
charged surface (Huang et al. 2005). Stoimenov et al. (2002) used E. coli as an
example of Gram-­negative bacteria, Bacillus megaterium as a representative of
Gram-positive bacteria, and Bacillus subtilis as endospores to observe the antimi-
crobial efficiency of MgO prepared by the aerogel procedure (AP). Whereas B.
subtilis had some resistance, E. coli and B. megaterium was completely inacti-
vated after 20 min by MgO (Stoimenov et al. 2002). Shi et al. (2010) found
almost the same antimicrobial effect of MgO on Gram-negative and Gram-
positive bacteria. Pathogen inactivation might depend on factors like: contact of
MgO particles with bacteria, alkaline condition of the particle surface, particle
specific surface area, and contact time and availability of active oxygen on the
surface. In their opinion, reactive oxygen species (ROS) generally attacked the
carbonyl group of peptide linkages and eventually promoted the degradation of
protein. Smaller particle size might produce more superoxide ions and accelerate
the destruction of bacteria (Shi et al. 2010).
MgO can inactivate the pathogens by forming ROS; however, the adsorption
process or direct cell membrane penetration by the particles may be the other pos-
sible ways for pathogens inactivation. These two possibilities need to be investi-
gated in more detail to comprehend the inactivation mechanisms by MgO, and
hence, optimize their use. Similarly, understanding the precise degradation mecha-
nism of protein and cell by MgO nanoparticles (NPs) is required to explain, control,
and tune the reliable inactivation process.
The antimicrobial activity of TiO2 particle is based on the inactivation of micro-
organisms under UV/solar irradiation by forming some ROS such as a hydroxyl
radical (•OH), superoxide radical ( O•−2 ), and hydrogen peroxide (H2O2). ROS are
formed by reaction of surface oxygen and water with positive cavities formed on the
TiO2 crystal lattice during UV/solar irradiation.
318 L.E. Orellana et al.

ROS generally works by destroying the cell membrane, damaging DNA and
protein, releasing hazardous ions for cell malfunction, disrupting electron transfer,
and hampering the respiration process.
On the other hand, ZnO has been proved to be a strong antimicrobial element by
exhibiting the activity in the 7.0–8.0 pH range, which is favorable for the treatment
of drinking water (Yamamoto 2001). ZnO is a semiconductor with a large band gap.
This means that when photon energy greater than the band gap is applied, holes (h+)
and free electrons are formed. The holes react with water to form •OH and electrons
and the conduction band reacts with oxygen to form • O2− and •OH. All these reac-
tions will ultimately end with the formation of H2O2. Shi et al. (2010) observed that
ZnO worked as a bacteria growth inhibitor and this effect was more on Gram-­
positive bacteria. H2O2 formation was linearly proportional to the concentration of
ZnO. Electrostatic forces might play an important role by promoting the binding of
bacteria on particle surface (Stoimenov et al. 2002).

6.4 Food Applications

Polymer nanotechnology has enabled the preparation of new food packaging based on
the dispersion of nanometric antimicrobial agents into a biodegradable polymeric
matrix. Polysaccharide-derived films (e.g., alginates and chitosan) exhibit excellent gas
permeability properties that enhance the shelf-life of the product without creating anaer-
obic environments. In addition to its capability to adsorb divalent metal ions, chitosan
also exhibits antimicrobial activity on a number of microorganisms, e.g., E. coli, among
others of similar importance in food packaging and preserving applications. The incor-
poration of antimicrobial compounds into food packaging materials can combine struc-
tural integrity and barrier properties provided by the polymeric matrix with the
antimicrobial properties of antimicrobial agents added as solid dispersoids or impregnat-
ing compounds. Conventional approaches are based in the incorporation of liquid and
solid natural antimicrobial compounds to edible films to reduce bacteria in solution, on
culture media or on muscle foods (Cutter and Sumner 2002). Although this route was
conducive to certain improvements on the food packaging efficiency, in most cases,
dispersed compounds failed to enhance the mechanical strength, flexibility, barrier pro-
tection to gases and light, and permeability properties required in food packaging.
Besides, some antimicrobial agents are extremely irritant and toxic. In some cases,
unwanted release of the antimicrobial compounds to the food was observed. Accordingly,
the formulation of new types of safe and cost-effective biocide materials is needed.
In particular, the dispersion of functional nanoparticles in the polymer matrix
will improve the packaging properties of the resulting nanocomposite while enhanc-
ing gas barrier properties, temperature/moisture stability and resistance to bacte-
rium and other microorganisms. These features coupled with outstanding bactericide
activity of specific nanoparticles and polymeric matrices will enable the d­ evelopment
of more efficient and effective materials for food preservation and protection against
human health-compromising microorganisms.
14 Non-thermal Methods for Food Preservation 319

Cedeño-Mattei et al. (2013) have worked on the development of biopolymer

nanocomposites containing MgO as dispersoids. MgO has been used in catalysis,
thermal insulating, and adsorption, among other applications (Gao et al. 2011;
Selvamani et al. 2011). Compared to others, MgO is expected to provide an
improved food packaging solution under cost-effective conditions. Also, the capa-
bility of nanoscale MgO to destructively adsorb chemical warfare agents and its
bactericidal activity even in the absence of UV illumination, coupled with its ability
to block UV light, enable this nanomaterial to be considered a promising candidate
for food protection and preserving applications.
Cedeño-Mattei et al. (2013) synthesized MgO in the aqueous phase with particle
sizes between 7 and 13 nm. A bacterial growth inhibition of 100% was obtained for
nanoparticles concentration around 1500 ppm. The substitution of Mg ions by Zn
ions to create ZnxMg1−xO solid solution exhibits an enhanced inhibitor behavior when
compared to pure MgO at lower nanoparticle concentration (e.g., 500 and 1000 ppm).
The zinc incorporation into the MgO structure should be conducive to a synergistic
effect and enable the tailoring in the corresponding antimicrobial activity.

6.5 Remarks

Nanotechnology is increasingly being employed in the areas of food production and

packaging. Some studies point that the public perception will be crucial to the realization
of these technological advances and that the nanotechnology packaging is perceived as
being more beneficial than nanotechnology foods (Siegrist et al. 2007). Although nano-
technology can be considered the latest industrial revolution, there is still a need to estab-
lish strict and well documented regulations regarding the application, discharge, and
handling conditions for candidate nanomaterials. Furthermore, environmental effects
may also affect the nanoparticles’ bioavailability and bioaccumulation in specific organ-
isms. On the other hand, the suitable handling of isolated nanomaterials in any system
will be a challenging task, unless they are immobilized inside some macroscopic, eco-
friendly, and porous matrix to ensure food safety.

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Chapter 15
Antimicrobial Gases for Food Application

David Kasler and Ahmed E. Yousef

Abstract Food processors use various decontamination methods to improve the

safety of food and extend product shelf-life. Antimicrobial gases have been used infre-
quently in food processing for many decades, but renewed interest in these agents has
been increasing lately. Food, equipment and processing environments may be decon-
taminated using alkylating, halogen-based, or oxidizing gases. Examples of these
gases are propylene oxide, chlorine dioxide, and ozone, respectively. In this chapter,
these categories of decontaminating gases are discussed in depth, including their
application to food, targeted microorganisms, and safe use in processing facility.

Keywords Antimicrobials gases • Food safety • Alkylating gases • Halogen gases •

Oxidizing gases

1 Introduction

Raw food can easily be contaminated with spoilage or disease-causing microorgan-

isms if producers and processors do not adhere strictly to good production and man-
ufacturing practices. Before processing, washing is the most likely first step for
ensuring proper food sanitation. During processing, foods often are subjected to
decontamination treatments (known in the industry as kill-steps) which vary in
stringency from mild antimicrobial treatment or pasteurization to retorting. Although
heat is the most commonly-used decontamination factor in food processing, interest
in using antimicrobial gases for this purpose is rising.
Raw agricultural commodities (e.g., raw fruits and vegetables), animal carcasses,
sea food, and eggs are examples of foods washed before further processing. Equipment
surfaces, particularly food-contact surfaces, also should be thoroughly washed before
and after use. In today’s food industry, clean food and equipment should be comple-
mented with clean and hygienic processing environment. Therefore, food handling and

D. Kasler • A.E. Yousef (*)

Department of Food Science and Technology, The Ohio State University,
Columbus, OH, USA
e-mail: [email protected]; [email protected]

© Springer Science+Business Media, LLC 2017 327

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_15
328 D. Kasler and A.E. Yousef

processing environment including walls, floors, conveyer belts, and pipes are routinely
washed. Washing operations aid in the removal of visible foreign material but viable
microorganisms may remain on washed surfaces. Suitable antimicrobial agents (i.e.,
sanitizers or disinfectants) are applied on washed surfaces to inactivate microorgan-
isms that are hard-to-remove by washing. Antimicrobial gases, such as ozone, may be
dissolved in water and used to sanitize equipment surfaces and processing environ-
ment. These gases also may be used to decontaminate processing water while being
reused during washing of raw agricultural commodities. Additionally, antimicrobial
gases such as ethylene oxide have been suggested as potentially useful to treat packag-
ing material for aseptic processing (Ansari and Datta 2003).
Sanitizers are antimicrobial agents capable of reducing populations of microor-
ganisms of public health importance to safe levels without adversely affecting the
safety or quality of treated product (CFR 2016a). Population reduction by a sanitizer,
tested under standardized condition, should be 5 log in 30 s for food-contact surfaces
or 3 log, within 30 s, for non-food contact surfaces. Some of the sanitizers can be used
on food surfaces, provided that these substances are useful during processing but do
not need to be present in the finished product (CFR 2016b; FDA 2015). This is the
case for most antimicrobial gases used on food; it is often specifically required that
they leave little or no residue on the food. Microbial inactivation achieved by treat-
ment of food with a sanitizer depends greatly on food type and its surface character-
istics. Sanitizers used in the food industry include chlorine-based compounds,
quaternary ammonium compounds, oxidizers, organic acids, among others.
In addition to sanitizers, other forms of antimicrobials are commonly used in the
food industry. Many of the food additives are used because of their antimicrobial
characteristics; ozone gas is a good example of antimicrobial food additives.
Disinfectants are similar in antimicrobial action to sanitizers but are tested and regu-
lated differently. Sterilants are applied when the goal is to eliminate all living forms
of microorganisms, including bacterial spores, which are often resistant to other
antimicrobial agents. Antimicrobials are usually applied in a food processing facil-
ity as liquids (i.e., in the aqueous state) but gaseous sanitizers and fumigants have
been in use for several decades. The demand for safer and better-preserved food,
than what is available currently, renewed the interest in research on antimicrobial
gases. In this chapter, antimicrobial gaseous that are currently-used or potentially-­
useful for food applications are reviewed.

2 Gaseous vs. Aqueous Antimicrobials

Some food antimicrobials, such as ozone and chlorine dioxide, can be used either in
the aqueous or the gaseous state. Others are inherently aqueous (e.g., hypochlorous
acid) or gaseous (e.g., propylene oxide) in nature. There are many reasons that a
food processor would choose to use a gaseous over aqueous antimicrobials, when
application of both to food is feasible.
15 Antimicrobial Gases for Food Application 329

2.1 Advantages of Gaseous Antimicrobials

1. Considering the importance of fast access of a sanitizer to microbial contaminant,

diffusion rate is orders of magnitude faster for gases than liquids (Cussler 1997).
According to Middleman (1998), diffusion rate of gas molecules is roughly
1000–10,000 times larger than that of liquid molecules.
2. Many food products contain water-soluble surface components; these are more
likely to be lost with the application of aqueous than gaseous sanitizer.
3. Most antimicrobial agents lose effectiveness after a short period of contact with
food. Water remaining, after the liquid sanitizer loses efficacy, can promote food
spoilage.
4. It is easier to adapt gaseous than aqueous applications to food at different stages
of the supply chain. During storage and shipping of fresh produce, for example,
it is possible to generate a mild ozone-containing atmosphere to prevent prema-
ture spoilage of the produce.
5. Application of sanitizers in the aqueous state increases water usage and disposal;
this increases food production costs and raises concerns about sustainability in
food processing. Additionally, the type of water is very important when sanitiz-
ing a product. Water sources may contain minerals, chemicals, or other sus-
pended compounds that can interact with sanitizers before they even reach food
microbiota. Hard water will reduce the efficacy of many sanitizers. Solution pH
also affects sanitizer effectiveness.
Despite these compelling reasons in favor of gaseous antimicrobial agents, food
processors may favor using liquid over gaseous antimicrobials.

2.2 Challenges in the Use of Gaseous Antimicrobials

Although gaseous sanitizers provide better diffusion rates, more efficacy, and mini-
mized water demand than do aqueous applications, food processors need to overcome
several hurdle before using these gases. Application of gaseous sanitizers must be done
in a closed or contained environment, often a sealable chamber that can withstand
pressures and vacuums (Gehringer 2012; Han et al. 2001; Perry et al. 2008; Vurma
et al. 2009). Many liquid sanitizers such as sodium hypochlorite can be applied in the
presence of the operator, whereas exposure to gaseous sanitizers can be hazardous to
operators and other personnel in the vicinity of the sanitization equipment. Hazards
due to off-gassing from liquid sanitizers can be controlled with proper ventilation;
however, gaseous sanitizers require expensive monitoring equipment and containment
systems, and a leak of the gas will likely require evacuation of the facility. A schematic
of a setup for antimicrobial gaseous treatment is shown in Fig. 15.1.
Setting up a gaseous treatment for food could be a challenging task. Antimicrobial
gases are instantly diluted by air present in a treatment chamber unless the air is removed
initially by applying vacuum or flushing the chamber with the applied gas. Dilution of
330 D. Kasler and A.E. Yousef

Fig. 15.1 Basic gaseous antimicrobial treatment setup

the antimicrobial gas limits its effectiveness, and it may generate a dangerous interaction
product. Ethylene oxide, for example, becomes explosive in the presence of air (Steris
2015). An additional complicating factor is some food products suffer quality loss under
heavy vacuum, and pathogenic microorganisms may be pulled from the surface into
pores in the product (i.e., internalized) during vacuum release, making these contami-
nants less accessible to the antimicrobial agent and thus more difficult to kill (Pena-
Melendez 2011). Flushing air out of a treatment chamber using the antimicrobial gas
limits these issues but the gas exchange can cause product drying and the process is
often time-consuming. Efficacy of gaseous antimicrobials depends greatly on relative
humidity in the vicinity of treated product; this factor is irrelevant when aqueous sanitiz-
ers are used. After the gaseous treatment is completed, the antimicrobial gas must be
flushed out of the chamber with air to make the environment safe for personnel to
remove the product. Similar to chamber filling, this procedure also is time-consuming.
Depending on the antimicrobial agent and the concentration used, a destruction, dis-
posal, or containment units may be required to eliminate the risk of human exposure and
to meet environmental laws for gas emissions.

2.3 Gaseous-Aqueous Interrelations

Some of the antimicrobial gases have limited solubility in water (e.g., ozone). For such
an agent, the maximum amount that can be dissolved in water depends on the gas pres-
sure and concentration in the head space over the solution. At constant pressure, tem-
perature, and shear force, concentration equilibrium can be achieved between the
gaseous and aqueous phases. Disturbance of this equilibrium, e.g., loss of pressure, can
cause a rapid release of antimicrobial from the liquid (i.e., off-­gassing). It is believed that
off-gassing from solutions may become limited if the released gas forms very small
15 Antimicrobial Gases for Food Application 331

bubbles. Several researchers reported that formation of nano-bubbles helps in stabilizing

the gas concentration in the liquid (Kaneo and Takahashi 2007; Ushikubo et al. 2010).It
was suggested that nano-bubble solutions have added antimicrobial potential due to the
generation of free radicals when bubbles are collapsing (Takahashi et al. 2007).

2.4 Diffusion

Success of any antimicrobial treatment depends on the transfer of the antimicrobial

agent to the site of microbial cells residing on, or within, the treated product, so that
an interaction between the agent and the target occurs. Diffusion laws govern the
rate and extent of this transfer. It is common knowledge that gasses diffuse much
faster than liquids. According to Middleman (1998), gases diffuse in gas media at
rates between 0.2 and 1 cm2/s, whereas a liquid (or a gas) diffuses in a liquid medium
at the rate of only 0.00001 cm2/s. Grahams law of diffusion states that the ratio of
the rates of diffusion of two gases is equal to the square root of the reciprocal of the
molar masses, M (Middleman 1998).

Rate1 M2
=
Rate2 M1

Under constant pressure and temperature, density of gasses is proportional to

their molecular weights. Therefore, the rate of diffusion is inversely proportional to
the square root of the density, ρ (Bond 1989)

1
rate ∝
ρ

Gases typically are much less dense than liquids. Air at 300 K (27 °C) and 1 atm
has a density of 1.1614 kg/m3 whereas liquid water has a density 997 kg/m3. The
inverse relationship between density and diffusion rate helps in explaining the practi-
cal aspects of gaseous treatments; larger molecules diffuse slower than small ones.
Additionally, density variation between a gas and a liquid accounts for the faster dif-
fusion of gases than liquids. Molecules are at a higher energy state and move faster in
gases than in liquids, creating larger voids between the molecules. It’s the combination
of molecular spacing and speed of motion that cause the high diffusivity of gasses.

3 Categories of Antimicrobial Gasses

Antimicrobial gases may be categorized according to the intended use as sanitizers,

disinfectants and sterilants. Brief description of these categories has been presented
earlier in the chapter. Alternatively, antimicrobial gases may be grouped based on
332 D. Kasler and A.E. Yousef

mode of action into alkylating gases, halogen-releasing agents, and oxidizers. The
mode of action of an antimicrobial agent is linked to its chemical makeup.
Antimicrobial gases with mode of action different than those just listed include
nitric oxide (NO) and sulfur dioxide (SO2). Nitric oxide is an antimicrobial gas that
inactivates microorganisms by producing reactive “nitrogen oxide species” which
causes oxidative and nitrosative damage by altering DNA, inhibiting enzyme func-
tion, and inducing lipid peroxidation (Schairer et al. 2012). Sulfur dioxide is one of
the most versatile and efficient additives used in winemaking due to its antimicro-
bial and antioxidant properties. The following is a description of the most relevant
categories to food.

4 Alkylating Gases

Alkylating agents attack proteins, nucleic acids, and other organic compounds in
microbial cell by transferring alkyl (e.g., methyl) groups. The sulfhydryl group is an
example of cell targets that are alkylated by these compounds. Alkylation disrupts a
number of functions in bacterial cells. Primary cell functions such as protein synthe-
sis and DNA and RNA replication are altered or halted due to the structural changes
that occur in enzymes. Alkylating agents include ethylene oxide, propylene oxide,
formaldehyde, and glutaraldehyde.

4.1 Ethylene Oxide

Although ethylene oxide (Fig. 15.2) was considered for long time as an insecticidal
fumigant, it has used recently as an antimicrobial agent to sterilize medical supplies
and instruments, and to decontaminate foodstuff (Bond 1989; EPA 2008). The com-
pound has a boiling point of 10.7 °C, thus it occurs in the gaseous state at ambient
temperature and pressure. The gas is colorless but it has an irritating mustard-like
odor. It is readily soluble in water and mixtures of the gas with air are flammable.
For application as a fumigant, the gas is discharged from cylinders as a 9:1-mixture
of carbon dioxide and ethylene oxide.

Fig. 15.2 Structure of

alkylating agents
15 Antimicrobial Gases for Food Application 333

4.1.1 Generation and Method of Application

Ethylene oxide can be generated from ethylene by exposing the gas to air or oxygen at
elevated temperatures. For most food and medical applications, the gas is discharged
from bottles or cylinders and is not generated on-sight. Before application, air must be
removed from the treatment chamber because the gas is explosive when diluted even
with only 3% in air, vol/vol. Air removal is commonly performed by applying vacuum
and replacing air with nitrogen or water vapor. Ethylene oxide also can be mixed with
compatible gases prior to introduction to the treatment chamber to lower the possibility
of explosion (Steris 2015). In addition to vacuum, food decontamination processes
also require mild heating; for spices a temperature of 43–49 °C is used (Leistritz 1997).

4.1.2 Measurement

Reliable measurement methods are needed for monitoring employee exposures to

ethylene oxide or validating decontamination processes based on the use of this anti-
microbial agent. Methods currently used differ in techniques of gas sampling (e.g.,
using of charcoal tube or collection bag), desorbing solvents, and equipment used in
the analysis. Common methods involve the use of charcoal tubes and sampling
pumps, followed by analysis of the samples by gas chromatograph (CFR 2016c).
In one of these methods, the gas sample is absorbed on acid-coated charcoal, extracted
using dimethylformamide and then derivatized to 2-­ bromoethylheptafluorobutyrate
where it is analyzed using the gas chromatography equipment. The method has a detec-
tion limit of 1 μg ethylene oxide/ml sample. Alternatively, a portable device can be used
to detect ethylene oxide in workplace air. The device consists of a gas chromatograph
and a photoionization detector. In this case, a gas sample is withdrawn from the source
and injected directly into the gas chromatography unit. The method’s detection limit is
2.5 pg ethylene oxide/ml sample (Liteplo et al. 2003).

4.1.3 Main Uses

Ethylene oxide is heavily used in the medical industry for sterilizing equipment and
instruments that cannot be sterilized in high temperature/high moisture environ-
ments, i.e., steam autoclaves (HICPAC 2008). In the food industry, ethylene oxide
is often used as a fumigant of packaging materials for the dairy industry, on spices
and seasonings and nuts and on equipment in the bee keeping industry (NIOSH
1981). It is also a component in pesticides sprayed on agriculture commodities
(ATSDR 1990). However, use of ethylene oxide as a sterilant for spices is banned in
the European Union (EU). The EU also implemented strict restrictions on tolerable
residues of ethylene oxide in other foods or food additives (Atungulu and Pan 2012;
EC 2002; Rossi and Silvestris 2014).
334 D. Kasler and A.E. Yousef

4.1.4 Efficacy and Mechanism of Action

Ethylene oxide is a potent antimicrobial gas that controls Bacillus larvae, the cause
of American foulbrood disease, Streptococcus pluton that causes European foul-
brood disease, Mycobacterium spp., and Pseudomonas spp. (EPA 2008). In addition
to being bactericidal, the gas is also effective against fungi (e.g., Candida albicans)
and viruses (e.g., Herpes simplex virus, and Rhinoviruses).
Ethylene oxide is thought to react with microorganisms and replace hydrogen
atoms in proteins, DNA, and RNA with alkyl groups. Alkylation of these vital cell
components prevents normal cells functions and reproduction which ultimately kills
the population and sterilizes the product. Efficacy of the gas on microorganisms is
likely to be altered by the presence of inorganic salts and organic materials. Efficacy
is also affected by how deep a microorganism is embedded inside an object, which is
true for all chemical sanitizers where direct interaction with targeted microorganisms
is required. The spores of Bacillus atrophaeus are used as a biological indicator in
ethylene oxide treatments due to their high resistance to this sterilant (HICPAC 2008).
For effective sterilization using ethylene oxide, the gas concentration, tempera-
ture, relative humidity and exposure time are all critical. Increasing temperature, gas
concentration, or humidity allows for decreases in time required to achieve steriliza-
tion. Concentrations are generally in the 450–1200 mg/L range, humidity in the
40–80% range and temperature in the 37–63 °C range, yielding 1–6 h of needed
treatment time (HICPAC 2008).

4.1.5 Hazards

Ethylene oxide is potentially hazardous to personnel operating the processing units, or

those in the vicinity of these units. The gas is a human carcinogen. Occupational expo-
sure is known to increase the risk of spontaneous abortions in pregnant women.
Inhalation has been linked to polyneuropathies, neurologic dysfunctions, cognitive
impairment, and cataracts. Acute exposure is known to cause irritation of skin, eyes,
and gastrointestinal or respiratory tracts and central nervous system depression.
Because of these hazards, OSHA limits exposure in the work place to 1 ppm for 8-h
exposure, for a 40-h weeks, and 5 ppm exposure is allowed for only 15 min. Items
sterilized with ethylene oxide can also absorb the gas and release it even after it is at a
safe level in the sterilization chamber. This requires many sterilized items to undergo
an aeration process post sterilization to make the device or food safe for use. As
described earlier, ethylene oxide also is flammable and explosive (HICPAC 2008).

4.2 Propylene Oxide

Propylene oxide (Fig. 15.2) is an insecticidal fumigant and a sterilant. It is used to

control microbial contamination and insect infestations of food products, and to
control insects in nonfood products (EPA 2006). At ambient temperature, propylene
15 Antimicrobial Gases for Food Application 335

oxide is a colorless volatile liquid (boiling point of 34 °C) that is capable of rapid
combustion. The compound is very soluble in water. Propylene oxide has an odor
that is described as sweet, alcoholic, and ether-like, with an estimated odor thresh-
old of 44 ppm (Amoore and Hautala 1983).

4.2.1 Generation and Method of Application

Propylene oxide is produced from propylene by one of two methods (OSHA

2016c). The chlorohydrin process involves reacting propylene with chlorine,
whereas the hydroperoxide process relies on the expoxidation of propylene with
an organic hydroperoxide. Commercial propylene oxide is formulated as a pres-
surized liquid and/or gas. For treating a product with propylene oxide, it is
applied in vacuum-­sealed chambers, referred to as “commercial sterilization
chambers” (EPA 2006). In the liquefied form, it may be diluted in water for aque-
ous use, or injected into a chamber for gaseous use. In gaseous treatment, it is
common to raise the temperature of the treatment chamber above the agent’s
boiling point to ensure that all propylene oxide in the chamber is in the gaseous
phase. Vacuum may be applied prior to introducing propylene oxide into the
chamber to ensure that it enters the gaseous phase. A concentration of 0.5 kg/m3
(500 ppm) was used experimentally to decontaminate Salmonella-contaminated
almonds (Danyluk et al. 2005).

4.2.2 Measurement

The following is a brief description of a method for measuring propylene oxide

in air (OSHA 2016c). Air (or the gas itself) is sampled, by drawing a specified
volume through specially-designed adsorbent tubes (charcoal tubes). The gas
sample is ideally sent to the laboratory in a refrigerated state. The entrapped gas
is desorbed with carbon disulfide and the desorbate is analyzed by gas chroma-
tography. A gas chromatograph equipped with flame-ionization detector is used
in the analysis.

4.2.3 Efficacy and Mechanism of Action

The mechanism of action of propylene oxide against microorganisms is presumed

to be similar to that of ethylene oxide; however, the antimicrobial agent is less
effective against bacterial spores than is ethylene oxide (Hoffman 1971). Based on
this assumption, the microbial cell is inactivated when its sensitive components are
alkylated with propylene oxide. Similar to ethylene oxide, the bactericidal and
sporicidal activity of propylene oxide are dependent on humidity (Bruch and
Koesterer 1961).
336 D. Kasler and A.E. Yousef

4.2.4 Main Uses

Most of the propylene oxide produced in the US is used as an intermediate for the
manufacture of polyether polyols in the production of polyurethane foams, and for
the manufacture of propylene glycol in the production of unsaturated polyester res-
ins. Small quantities of polypropylene oxide are used for sterilization of medical
equipment and for fumigating foodstuffs (OSHA 2016c). Propylene oxide is regis-
tered as a fumigant in the US. Food industry applications include using the antimi-
crobial agent on dried food ingredients such as herbs, spices, onions, and garlic.
Other foods treated with propylene oxide are cacao beans, cocoa powder and in-­
shell and processed nutmeats. The gas may be usable on figs, prunes, and raisins.
For nonfood uses, propylene oxide is used to treat cosmetic articles, gums, ores,
packaging, pigments, and pharmaceutical materials (EPA 2006).
Since 2007, almonds produced in California should be pasteurized before reaching
the consumer. This new requirement was mandated after several disease outbreaks
and products recalls were linked to contamination of almonds with Salmonella
(Federal Register 2007). Use of propylene oxide is considered an acceptable treat-
ment option which can reduce Salmonella population on almond kernels by at least 4
log. Therefore, a method to pasteurize almond kernels using propylene oxide has been
developed (Almond Board of California 2008). Briefly, the product is placed in a
chamber held at 47–51 °C and vacuum is applied to 27 in. Hg. Polypropylene oxide
gas is introduced in the chamber to a concentration of 0.5 oz./ft3. The treatment lasts
for 4 h, and then vacuum is applied to empty the chamber. The chamber is flushed
with air several times before the pasteurized almond is removed.

4.2.5 Hazards

Similar to ethylene oxide, exposure to propylene oxide could lead to carcinogenic

risks. The International Agency for Research on Cancer listed propylene oxide as a
group 2B carcinogen, meaning “possibly carcinogenic to humans.” Rats exposed to
the gas have produced both benign and cancerous tumors (NIOSH 1989). However,
the rats were exposed to high doses and it is assumed the risk is very small if safe
levels are realized in the work place. In the US, OSHA has set a time weighted aver-
age limit of 20 ppm for workplace exposure to propylene oxide (OSHA 2016c).
Based on evidence that propylene oxide is an animal carcinogen, it was recom-
mended that occupational exposure to propylene oxide be reduced to the lowest
feasible level. The current permissible exposure limit for ethylene oxide, which is
chemically similar to propylene oxide and is also an animal carcinogen, is 1 ppm.
Propylene oxide is highly flammable. It may combust at percentages as low as
1.7% in air. Propylene oxide can produce harmful by-products during use on food.
The gas may react with natural inorganic chlorine, such as NaCl, found in food and
form toxic propylene chlorohydrins (Wesley et al. 1965). Interestingly, similar
products can be formed when ethylene oxide is used to treat food. Wesley et al.
(1965) found that up to 1000 ppm of ethylene chlorohydrins remained on spices
after they were commercially treated with ethylene oxide.
15 Antimicrobial Gases for Food Application 337

5 Halogen-Based Compounds

Some halogens and halogen-based compounds exist in the gaseous state. These
are potent antimicrobial agents, but halogen-based compounds are more suitable
than elemental halogen in food applications. Although the general mechanism of
inactivation by these agents is the halogenation of microbial cell components,
oxidation by some members of this category also contributes to microbial lethal-
ity. Food processors are increasing interested in chlorine dioxide as an emerging
antimicrobial gas suitable for food applications; this compound will be addressed
in this chapter.

5.1 Chlorine Dioxide

Chlorine dioxide, ClO2, is a reactive gas with yellow-green to orange color and a
pungent, sharp odor (DHHS 1978). It is detectable to the human nose at 17 ppm in
air (EPA 1978). The compound has an oxidative potential of 0.95–1.27 V, in the
gaseous and aqueous states, respectively (Jin et al. 2006). Because of its strong
oxidative potential, chlorine dioxide is susceptible to decomposition in both gas-
eous and aqueous states. Chlorine dioxide gas breaks down to elemental chlorine
and oxygen. In water, it reacts with organic substances to form chlorite ions (ClO2−);
these further react to form free chlorine as an end product. The reactivity of chlorine
dioxide makes it a useful antimicrobial agent in both aqueous and gaseous applica-
tions. One of the advantages of chlorine dioxide in aqueous treatments is that it is
more effective than hypochlorites under alkaline conditions. Chlorine dioxide also
is much more water-soluble than chlorine. Chlorine dioxide is a gas above 11 °C,
and its solidification point is-59 °C. In the gas state, it is denser than air. Chlorine
dioxide has been used for many years in the wood pulp industry as a bleaching
agent, and in the wastewater treatment. Recently, this compound has been used as
an antimicrobial agent for food and food contact applications (NCBI 2016).When
used as a gas, its efficacy on products is greatly affected by the humidity. Park and
Kang (2015) report nearly a 4-log increase in lethality on Escherichia coli,
Salmonella and Listeria monocytogenes when relative humidity was raised from 50
to 90% (Fig. 15.3).

5.1.1 Generation

Chlorine dioxide is highly reactive, and as such it is not kept under storage condi-
tions. In the US, the transportation of gaseous chlorine dioxide is prohibited
(CFR 2016d). For this reason, chlorine dioxide must be generated on-site.
Chlorine dioxide can be produced by the reaction of sodium chlorite (NaClO2)
with chlorine gas (Cl2), or sodium chlorate (NaClO3) with hydrochloric acid
(Paulus 2005).
338 D. Kasler and A.E. Yousef

Fig. 15.3 Importance of relative humidity for the inactivation of foodborne pathogens by chlorine
dioxide (Park and Kang 2015)

5.1.2 Measurement

Chlorine dioxide, in the gaseous state, is measured commonly with spectrophotom-

eter, at a wavelength of 358 nm. A flow cell with a suitable light-path length is
needed for measuring small amounts of the gas. The correlation between absor-
bance and ClO2 concentration is determined by the Beer-Lambert Law.

A = ε cl

where A is absorbance, ε is the molar absorptivity of ClO2 (1250 M−1 L−1 cm−1), c is
the concentration (mol/L),and l is the path length.
Similarly, when chlorine dioxide is dissolved in water, its concentration could be
determined using standard spectrophotometer, set at 358 nm (EPA 1978).

5.1.3 Mechanism of Antimicrobial Action

Chlorine dioxide is presumed to inactivate microbial cell by interfering with the

functionality of critical cellular proteins. Ogata (2007) suggested that the primary
antimicrobial activity of ClO2 is due to its ability to denature proteins. The researcher
demonstrated that the denaturation is caused by covalent oxidative modification of
protein’s tryptophan and tyrosine residues. Other forms of interference of chlorine
15 Antimicrobial Gases for Food Application 339

dioxide with cell functions were investigated. Napolitano et al. (2006) demonstrated
the interaction between ClO2 and guanosine 5′-monophosphate. Based on another
study, Bakhmutova-Albert et al. (2008) suggested that cell’s NADH is the primary
target of ClO2. According to these researchers, two ClO2 molecules are needed to
oxidize NADH to NAD+ in phosphate buffer solutions.

5.1.4 Current and Potential Uses

Chlorine dioxide is used for the antimicrobial treatment of water, the disinfection of
pipelines, as a slimicide and bleaching agent in the pulp and paper industry, and as
a deodorizing treatment of sewage plant effluent (Paulus 2005). Currently, chlorine
dioxide is not approved for widespread use in the food industry, but rather for spe-
cific applications that each has specific allowable levels. For example, chlorine
dioxide may be used to disinfect poultry wash water at levels up to 3 ppm of residual
ClO2. In fresh fruit and vegetable processing, it may also be used up to 3 ppm in the
aqueous form. Residual ClO2 on the fresh produce should be removed by rinsing
with water (CFR 2016d).
Researchers have demonstrated the effectiveness of chlorine dioxide in
decontaminating vegetables, fruits and other food products. Han et al. (2000)
reported up to 6.5 log reduction in Escherichia coli O157: H7 on green peppers
when the contaminated product was treated with 1.24 mg/L ClO2 for 30 min.
When the green peppers were inoculated with Listeria monocytogenes and
treated with 3 mg/L ClO2, up to 6 log CFU/5 g reduction in the pathogen popu-
lation was observed. Injury of the pepper tissues lowers the maximum inactiva-
tion to 3.5 log CFU/5 g (Han et al. 2001). Gaseous ClO2 has been tested for
decontaminating blueberries (Zhang et al. 2015), strawberries (Han et al. 2004),
and spinach leaves (Park and Kang 2015). The aqueous form of ClO2 has been
tested on eggs (Choi et al. 2015), lettuce, and baby carrots (Singh et al. 2002).
Based on these studies, both gaseous and aqueous treatments produced good
antimicrobial effects.
Chlorine dioxide can be used in several areas of water treatment, with the
potential to be used as an additive to clarification tanks, or a primary decontami-
nant. It is beneficial that ClO2 yields less organic disinfection byproducts than
traditional chlorine treatment; however, it readily produces the inorganic disinfec-
tion byproduct, chlorite. A sizable portion (~70%) of added ClO2 becomes chlo-
rite, a compound regulated in drinking water for health reasons to a maximum of
only 1 mg/L; therefore, chlorine dioxide cannot be applied at levels greater than
1.4 mg/L (EPA 2015).
Chlorine dioxide is a promising sanitizer for hard food-contact surfaces. Epoxy-­
lined stainless surfaces, contaminated with Lactobacillus buchneri, Leuconostoc
mesenteroides, Eurotium spp., Penicillium spp., Candid spp., and Saccharomyces
cerevisiae, were sterilized with exposure to10 mg/L gaseous ClO2 for 30 min.
However, process efficacy was found sensitive to humidity and temperature. At
9–25 °C, at least 90% RH was needed to obtain sterility. When increasing tempera-
340 D. Kasler and A.E. Yousef

ture to 28 °C, the relative humidity could be decreased to 69% and still sterilize the
stainless equipment (Han et al. 1999).
Chlorine dioxide may be suitable for removing biofilms from food-contact sur-
faces. Nam et al. (2014) found it effective in eliminating Bacillus cereus biofilms on
stainless steel in 6 h. The authors also found that Bacillus cereus spores, trapped in
a biofilm on stainless equipment, could be inactivated by 5.3 log CFU when sub-
jected to 115 ppm gaseous ClO2.Other uses of chlorine dioxide on food have been
investigated. Pesticide residue removal from lettuce (Chen et al. 2014), and lower-
ing ethylene production from tomatoes to slow ripening (Guo et al. 2014) were
possible by ClO2 treatments.

5.1.5 Hazards

Short-term exposure to ClO2 gas causes irritation to the eye, nose, throat and lungs,
whereas chronic bronchitis may be caused by long-term exposure (DHHS 1978).
OSHA occupational exposure limit for an 8-h workday, 40-h workweek, is 0.1 ppm
or 0.3 g/m3. Short term Exposure for 15 min is specified by American Conference
of Governmental Industrial Hygienists as being 0.3 ppm or 0.83 mg/L (OSHA
2016a). Chlorine dioxide is corrosive (Paulus 2005). It is however less corrosive
than other chlorine-based sanitizers because it is often used at much lower concen-
trations. At concentrations higher than 10% in air, chlorine dioxide decomposes
explosively (EPA 1978).

6 Oxidizing Gases

Oxidizing gases are compounds that have a strong oxidizing power; hence, these
have great biocidal effects and an ability to decontaminate many products and
environments (Paulus 2005). The strong oxidizing power unspecifically targets
organic matter, including microbial cells. The non-selective nature of these com-
pounds makes them suitable for inactivating various categories of microorgan-
isms, i.e., Gram-positive and Gram-negative bacteria (both vegetative and
spore-forms), yeasts, fungi, algae, and viruses. These characteristics made the
oxidizing antimicrobial agents usable, on a large scale, as sanitizers in food pro-
cessing factories, hospitals, restaurants and household. Additionally, these agents
are used largely in treatment of water, including drinking water, process water,
pools, sewage and waste water effluents. The potency of these agents sometimes
is accompanied by undesirable bleaching affect and the occasionally-desirable
deodorizing effect. Examples of oxidizing gases include ozone, hydrogen perox-
ide and peroxyacetic acid. This chapter covers ozone as an emerging antimicro-
bial agent for food application.
15 Antimicrobial Gases for Food Application 341

6.1 Ozone

Ozone (O3) is a tri-atomic oxygen molecule with a bond angle of 116o. It has a melt-
ing point of −192.5 °C (Jenkins and DiPaolo 1956) and a boiling point of −111.9 °C
(Jenkins and Birdsall 1952); thus it is a gas at the ambient temperature. Ozone is a
very potent oxidizing agent, with a reduction potential of 2.07 V. At ambient tem-
perature, ozone has a characteristic odor that can be discerned at concentrations as
low as 0.02 ppm. During thunderstorms, ozone is generated in small amounts, and
is associated with the after-rain fresh and clean smell. In 1982, ozone was approved
as “Generally Recognized as Safe” (GRAS) by the FDA for use in the treatment of
bottled water at small doses (Federal Register 1982). In 1997, ozone was declared
as GRAS in food applications by a panel of scientific experts (EPRI 1997). Ozone
treatment for the decontamination of foods was approved in 2001 by the FDA for
use in both aqueous and gaseous forms (Federal Register 2001). The GRAS status
and FDA approval of ozone for use in foods has led to an abundance of research on
its antimicrobial applications.

6.1.1 Generation

Because ozone is highly reactive, it cannot be stored; thus, it must be produced at

the site of its intended use. Ozone is readily produced through three methods: corona
discharge, electrolyzing water, and ultraviolet radiation (Chawla et al. 2012). Many
current commercial ozone generators rely on the corona discharge method. In this
method, a carrier gas (air or dry oxygen) is passed through the dielectric space
between two high voltage electrodes. As electric current is passed between this gap
and through the carrier gas, the oxygen molecules are split into atomic oxygen.
Some of these atoms have enough energy to recombine as ozone. When pure oxy-
gen is used as a carrier gas instead of air, higher levels of ozone may be produced.
It is also important to use dry feed gas, as air water content above 39 ppm can cause
a significant decline in the efficiency of ozone generator (Horvath et al. 1985).
Ozone is naturally produced in the upper earth atmosphere. At high altitudes,
short range ultraviolet (UV) radiation (<240 nm) produces ozone by splitting oxygen
molecules; resulting atoms rearrange to form ozone. At lower altitudes, ozone mol-
ecules decompose into diatomic oxygen by long-range UV radiation (240–320 nm).
In a similar way, ozone may be produced on a commercial scale by exposing oxygen
to short-wave UV radiation. The most efficient UV wavelength for ozone generation
at a pressure of 1 bar is 172 nm. Production of ozone by UV is not suitable for pro-
cesses requiring a large amount of the gas. Production of ozone by UV is generally
an energy intensive method (Chawla et al. 2012; Horvath et al. 1985).
Ozone decomposes rapidly in the presence of reacting organic compounds.
Aqueous ozone has ≤30 min half-life, depending mainly on the organic load of the
water and the storage temperature (Kim et al. 2003). In the gaseous form, the half-­
life of ozone ranges between 20 and100 h, provided that it is kept in a contamination-­
342 D. Kasler and A.E. Yousef

free, non-reactive container (Horvath et al. 1985). At normal atmospheric conditions,

however, the half-life is much shorter. Ozone is relatively stable at low temperature
and pH conditions; these two parameters should be considered carefully when
designing a process using ozone. When using ozone gas experimentally or industri-
ally, any excess gas should be decomposed before it is vented into the atmosphere.
Ozone gas is commonly decomposed using a thermal destruction unit with a tem-
perature of ≥350 °C. Other options for the removal of ozone gas are adsorption onto
granular activated carbon beds and decomposition by a catalytic column.
Commercially available ozone decomposition catalysts (e.g., Carulite® 200) contain
manganese dioxide, copper oxide or other agents. Aqueous ozone may be decom-
posed by reacting it with an aqueous sodium thiosulfate solution (Carus Corporation
2016; Chawla et al. 2012; Horvath et al. 1985).

6.1.2 Measurement

There are two approaches for detecting and quantifying ozone in different media or
environments: ozone-specific and ozone non-specific methods. Ozone specific
methods rely on reactions that are completed in the presence of ozone only, whereas
ozone non-specific methods do not discriminate between ozone and other oxidizing
agents. In a given laboratory setting, the type of measurements performed depends
on the application. Although most methods are developed to measure ozone in
aqueous solutions, they can be easily adapted to measure gaseous ozone.
One of the oldest chemical methods for measuring ozone concentration is the
iodometric method. In this method, ozone-containing gas is bubbled through a 2%
solution of aqueous potassium iodide. The reaction solution is acidified with sulfu-
ric acid and then titrated with sodium thiosulfate. This procedure has been found to
be accurate within 2% of physical detection methods (Birdsall et al. 1952). The
iodometric method is an example of non-specific ozone detection methods. Other
oxidants that may be in the air, or in solution, will cause overestimation of ozone
concentration. Additionally, at low ozone concentrations, the iodometric method
does not have good reproducibility.
The current standard method for measuring ozone, in solutions, utilizes an acidi-
fied potassium indigotrisulfonate indicator solution (USPC 2012). This indigo solu-
tion absorbs light at a wavelength of 600 nm and can be measured by a
spectrophotometer. Potassium indigotrisulfonate contains a carbon-carbon double
bond that can be broken by ozone. In this method, ozone reacts with the sulfonated
indigo in a one-to-one molar ratio. The end product of this reaction is sulfonated
isatin, which does not absorb at 600 nm. The difference in absorbance at 600 nm
between the ozone and blank solutions is directly correlated to the amount of ozone.
Using the indigo method, ozone is detectable down to 0.005 μg/ml (Bader and
Hoigne 1981; Chawla et al. 2012).
Gaseous ozone is typically measured in real-time by the absorption of ultraviolet
radiation, using commercially-available ozone monitors. The absorbance is mea-
15 Antimicrobial Gases for Food Application 343

sured at a wave length of 253 nm, which is similar to the maximum absorbance
wavelength of ozone (Bocci 2005; NASA 2015).

6.1.3 Efficacy and Mechanism of Action

Ozone has been proven effective against Gram-positive and Gram-negative bacte-
ria, fungi, and viruses (Chawla et al. 2012). Kim and Yousef (2000) reported the
kinetics of inactivation of Pseudomonas fluorescens, E. coli O157:H7, Leuconostoc
mesenteroides, and Listeria monocytogenes by aqueous ozone in both batch and
continuous reactor systems. The study showed that ozone reacts very quickly with
the microorganism in suspensions.
The antimicrobial properties of ozone are mainly attributed to its oxidative nature.
Ozone may react with many of microbial cell components. The primary target is the
carbon-carbon double bond, but sulfhydryl groups within proteins also are likely key
target for ozone. Researchers traditionally have emphasized the importance of either
cell membranes or intracellular components as the site of interaction of ozone with
the cell; the following are some examples. Based on the calculation of diffusion and
reaction rate of ozone with lung-lining fluid layer, Pryor (1992) concluded that most
of ozone-cell interactions happen at the membrane level. According to Van der Zee
et al. (1987), ozone causes DNA damage both directly and through reactive interme-
diates such as hydroxyl radicals. Based on more recently findings, Patil et al. (2011)
suggested that cell lysis is not the major mechanism of microbial inactivation by
ozone, and that some intracellular ­interactions are needed for the susceptibility of the
bacterium to ozone. Other researchers highlighted the importance of both sites (cell
membranes and intracellular contents) for ozone efficacy. Komanapalli and Lau
(1996) found that short exposure of Escherichia coli to ozone compromised cell
membrane but longer-term exposure was needed for effective cell inactivation. The
researchers concluded that although membrane components are the primary targets
of ozone damage, subsequent reactions involving the intracellular components, pro-
tein and DNA, also contribute to lethality.

6.1.4 Current and Potential Uses

Ozone has been widely used in the treatment of drinking water, and water used in
food processing. It has been approved for use in bottled water since 1982 (Federal
Register 1982). Ozone has been used in the US as a direct treatment of raw fish to
reduce microbial load and improve product’s quality (Higgins 2014). Recently, use
of ozone for direct contact with fish has been approved in Canada. Ozone is usable
on fresh produce to improve product safety and quality. The aqueous form of the
gas, in sequential combination with chlorine, was used commercially in the US in
1998 (Higgins 2014). Based on the authors’ personal knowledge, several fresh pro-
duce packers use ozone at different stages of the supply chain. This includes gas
application during fresh produce transportation.
344 D. Kasler and A.E. Yousef

Use of ozone to decontaminate food has been tested experimentally, with

varying degrees of success. The gas, in combination with vacuum cooling, was
effective in eliminating Escherichia coli O157:H7 on spinach leaves (Vurma
et al. 2009). The application of bubbled ozone and heat was sufficient to elimi-
nate up to 6 log Salmonella and Escherichia coli O157:H7 in apple and orange
juices (Williams et al. 2004). The application of gaseous ozone and 75 °C water
on the surface of cantaloupes was effective at reducing the number of mesophilic
bacteria by >3 log and psychrotrophic bacteria by >5 log (Selma et al. 2008).
Eggs have been successfully treated by a combination process including mild
heat and gaseous ozone treatment (Perry et al. 2008). The sensory quality of this
product was determined to be as good as, or better than the quality of normal
whole shell eggs (Perry et al. 2011). Quality, however, is not always maintained
on food processed with ozone. The use of ozone on chicken carcasses to reduce
microbial load was not successful for quality reason; the product experienced
some rancidity due to fat oxidation by the applied ozone (Higgins 2014).

6.1.5 Hazards

Because of its high oxidation potential, ozone can pose hazards to workers who may
be exposed to it. At low concentrations, ozone is an irritant to the respiratory sys-
tem, and can be lethal at high concentrations. The US Occupational Safety and
Health Administration (OSHA) has set a Permissible Exposure Limit (PEL) for
long term exposure to ozone of 0.1 ppm in air when taken as an 8-h time weighted
average (OSHA 2016b). The National Institute for Occupational Safety and Health
(NIOSH) has determined the concentration of ozone that is immediately dangerous
to life or health to be 5 ppm (NIOSH 2015). Because of these hazards, care must be
taken in process design to limit the exposure of employees to ozone.
Another important factor in worker safety is the choice of materials used. Materials
chosen in processing must be compatible with ozone. Natural rubber is susceptible to
ozone damage and becomes brittle over time. Other compounds, such as fluorinated
polymers, have shown resistance to decomposition by ozone. For high concentrations
of ozone, using polytetrafluoroethylene (PTFE) is recommended, while polyvinyl
chloride (PVC) and polyethylene (PE) can be used safely for low concentrations of
ozone (Smedt et al. 2001).Tubing and containers that hold ozone or ozonated water
should be inspected regularly to ensure that leaks are detected in a timely manner.

References

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Chapter 16
Current State of the Art and Recent
Innovations for Antimicrobial Food Packaging

Tony Z. Jin

Abstract Antimicrobial packaging encompasses any packaging technique(s) used

to control microbial growth in a food product. Antimicrobial packaging can be in
the form of a film or coating. Films can be composite and coatings can be applied to
the surface of foods. The two purposes for using antimicrobial packaging are to
inactivate foodborne pathogens and spoilage microorganisms present in food prod-
ucts, thus enhancing their safety and extending their shelf life. An antimicrobial
packaging system consists of three components: (1) Base materials to form film or
coating; (2) Antimicrobials to reduce or inhibit microorganisms; and (3) Methods to
making and applying films or coatings. In this chapter, we review the various types
of antimicrobial food packaging from the currently published literature, summarize
the basic materials and methods used for antimicrobial packaging, and also discuss
the recent innovations in the research and development of antimicrobial food pack-
aging systems.

Keywords Antimicrobial packaging • Materials • Methods • Applications • Food

safety and shelflife

1 Introduction

The demand for minimally processed, easily prepared and ready-to-eat ‘fresh’ food
products, globalization of food trade, and distribution from a centralized processing
area pose major challenges for food packaging. Supermarkets and consumers ask
for pathogen-free as well as good quality of foods throughout the entire shelf life
period (Balev et al. 2011). The application of antimicrobial packaging including
edible films and coatings could be an effective means to achieve these goals by

T.Z. Jin (*)

Residual Chemistry and Predictive Microbiology Research Unit, Eastern Regional Research
Center, USDA-Agricultural Research Service, Wyndmoor, PA, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 349

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_16
350 T.Z. Jin

controlling the growth of pathogenic and spoilage bacteria. Food deterioration and
pathogen contamination usually starts at the food surface. Therefore, food surface
treatments and packaging after treatments are critical for protecting food quality
and safety. Food packaging materials containing antimicrobials would allow for the
antimicrobials to be released gradually from the film or coating and thus maintain a
relatively high concentration of the antimicrobials on the food surface for a longer
period of time (Ye et al. 2008b).
Additionally, as the demand for natural, biodegradable, and environmentally-­
friendly packaging materials increases, new and novel food packaging materials or
technologies have been and continue to be developed. Examples of these packaging
materials include bio-based polymers made from raw materials originating from
agricultural or marine sources. Furthermore, researchers are developing or employ-
ing various types of natural, effective and safe antimicrobial materials. Therefore,
the application of biodegradable polymers coated or incorporated with natural anti-
microbial compounds has the potential of controlling food spoilage as well as
enhancing the microbial safety of food products (Kuorwel et al. 2011).
Antimicrobial packaging encompasses any packaging technique(s) used to con-
trol microbial growth in a food product. Antimicrobial packaging can be in the form
of a film or coating. Films can be composite and coatings can be applied to the
surface of foods. The two purposes for using antimicrobial packaging are to inacti-
vate foodborne pathogens and spoilage microorganisms present in food products,
thus enhancing their safety and extending their shelf life. An antimicrobial packag-
ing system consists of three components: (1) Base materials to form film or coating;
(2) Antimicrobials to reduce or inhibit microorganisms; and (3) Methods to making
and applying films or coatings.
In this chapter, we will review the various types of antimicrobial food packaging
from the currently published literature, summarize the basic materials and methods
used for antimicrobial packaging, and also discuss the recent innovations in the
research and development of antimicrobial food packaging systems.

2 Packaging Materials/Polymers

Antimicrobial carriers or polymers used in antimicrobial packaging can be divided

into three categories: (A) Edible and biodegradable, (B) non-edible and biodegrad-
able, and (C) non-edible and non-biodegradable (Table 16.1).

2.1 Edible and Biodegradable

Edible and biodegradable antimicrobial coatings and films are protein-based,

polysaccharide-­based, or lipid-based. Most of them are based on natural resources
in the form of raw or purified materials.
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 351

Table 16.1 Polymers and application methods

Application
Polymer/Carrier method Reference
Fruit Purees Composite Du et al. (2009a, b), Mild et al. (2011),
film Ravishankar et al. (2009, 2012), and Zhu et al.
(2014)
Chitosan Double-layer Guo et al. (2013b) and Ye et al. (2008a, b)
film
Chitosan Composite Abdollahi et al. (2012) and Moreira et al. (2011,
film 2009)
Chitosan Coating on Guo et al. (2013a, b, c) and Jin and Gurtler (2012)
foods
Alginate Composite Lim et al. (2010), Marcos et al. (2007), Norajit and
film Ryu (2011) and Pranoto et al. (2005)
Alginate Coating on Datta et al. (2008) and Lu et al. (2009)
foods
Pullulan Coating on Gniewosz et al. (2013)
foods
Zein Coating on Lungu and Johnson (2005)
foods
Zein Composite Hoffman et al. (2001) and Marcos et al. (2007)
film
Sodium Caseinate Composite Audic and Chaufer (2005) and Moreira et al.
film (2009, 2011)
Whey Protein Isolate Composite Fairley et al. (1996), Fernández-Pan et al. (2012),
film Mate and Krochta (1996), McHugh and Krochta
(1994) and Min et al. (2005)
Wax Coating on Barman et al. (2011), Chiumarelli and Hubinger
foods (2012), Jo et al. (2004), Mcguire (1997) and Ochoa
et al. (2011)
Polyvinyl Alcohol Composite Marcos et al. (2007)
film
Polylactic acid Composite Jin (2010), Jin and Zhang (2008), Jin and Gurtler
film (2011), Jin et al. (2010, 2013) and Liu et al. (2009)
Polylactic acid Double-layer Guo et al. (2014)
film
Polylactic acid/Pectin Composite Jin et al. (2009a, b) and Liu et al. (2007)
film
Polylactic acid/sugar beet Composite Li et al. (2012a, b)
pulps film
Cellulose-Based Paper Double-layer Ben Arfa et al. (2007), Chalier et al. (2007) and
film Scannell et al. (2000)
Low density polyethylene Composite Emamifar et al. (2012)
film
Polyvinyl Chloride, Low Double-layer Li et al. (2009), Mauriello et al. (2005), Natrajan
density polyethylene, film and Sheldon (2000a) and Siragusa et al. (1999)
Nylon films
(continued)
352 T.Z. Jin

Table 16.1 (continued)

Application
Polymer/Carrier method Reference
Polypropylene Film Double-layer Chawengkijwanich and Hayata (2008)
film
Polyethylene/Polyamide Double-layer Scannell et al. (2000)
film film
Other Plastic Films Double-layer Neetoo et al. (2007) and Ye et al. (2008a, b)
film

Apple, tomato, carrot, and hibiscus purees have been used by a group of research-
ers to make edible antimicrobial films. Each of these purees was mixed with pectin
and glycerin to improve the moisture barriers of each of these films (Du et al. 2009a,
b; Mild et al. 2011; Ravishankar et al. 2009, 2012; Zhu et al. 2014).
Pectin is a water-soluble, hygroscopic polymer purified from various plants.
Pectin has been used as a thickening, coating and encapsulating material. It can be
used as a vehicle to carry and deliver a variety of bioactive substances (Jin et al.
2009b; Liu et al. 2007).
Methyl cellulose, hydroxypropyl methylcellulose, and carboxy methyl cellulose
are polysaccharides used to form coatings or films. These films are flexible, trans-
parent, have moderate strength, are resistant to oil and fat migration and can act as
a moderate barrier to moisture and oxygen (Kuorwel et al. 2011; Maftoonazad and
Ramaswamy 2005; Moreira et al. 2009).
Chitosan is a polysaccharide obtained from the exoskeletons of shellfish and the
cell walls of mushrooms. It is a natural polymer which is nontoxic, biodegradable,
and biocompatible (Ye et al. 2008b). It is a good choice for antimicrobial coatings
and films because of its superior film-forming properties, ability to absorb nutrients
used by bacteria, and capacity to bind water and inhibit various bacterial enzyme
systems (Darmadji and Izumimoto 1994; Young et al. 1982). Chitosan has intrinsic
antimicrobial activity; however, chitosan film alone had no inhibitory effect on the
growth of L. monocytogenes when applied to the surface of ham steaks (Ye et al.
2008a).
Alginate is a salt of alginic acid, a polymer of D-mannuronic acid and L-guluronic
acid, and is isolated from brown algae. Alginate-based coatings and films are edible
and have been widely studied. Alginate is used as an edible coating because of its
unique colloidal properties and its ability to form strong gels or insoluble polymers
upon reaction with multivalent metal cations, such as calcium (Datta et al. 2008;
Lim et al. 2010; Lu et al. 2009; Marcos et al. 2007; Natrajan and Sheldon 2000b;
Norajit and Ryu 2011; Pranoto et al. 2005).
Pullulan is a natural polysaccharide with film-forming properties and is synthe-
sized by the fungus Aureobasidium pullulans. Pullulan-based films, compared to
protein-based and lipid-based films, are colorless, shiny, flexible, transparent, highly
impermeable to oil and oxygen, and heat-stable. Furthermore, pullulan films exhibit
good properties in terms of the preservation of fruits and vegetables because of their
selective permeability to gas exchange (Gniewosz et al. 2013).
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 353

Protein-based edible polymers include those from plant sources, such as zein and
soy, and those from animal sources, such as casein and whey. Zein is a water-­
insoluble prolamine protein extracted from corn gluten. It dissolves only in organic
solvents and has Generally Recognized As Safe (GRAS) status for use in foods. It
is presently used in the food industry to coat candy, dried fruit, and nut meats
because this protein forms a barrier to oxygen penetration (Hoffman et al. 2001;
Lungu and Johnson 2005; Marcos et al. 2007). Casein is the main protein in cow
milk (Moreira et al. 2011). Sodium caseinate (SC) is obtained by acid precipitation
of casein. Caseinate presents thermoplastic and film-forming properties due to its
random coil nature and its ability to form weak intermolecular interactions
(Arvanitoyannis 1999; Audic and Chaufer 2005; Longares et al. 2005; Moreira et al.
2011). Casein-based edible films are attractive for food applications due to their
high nutritional quality, excellent sensory properties and potential to adequately
protect food products from their surrounding environment. Casein and caseinates
can readily form edible films from aqueous solutions, are more permeable to water
vapor than plastic films, and are capable of retarding water transfer to some degree
(Moreira et al. 2009; Schou et al. 2005). Whey protein isolate (WPI)-based edible
films are transparent, odorless, and tasteless, and have excellent oxygen, lipid, and
aroma barriers (Fairley et al. 1996; Fernández-Pan et al. 2012; Mate and Krochta
1996; McHugh and Krochta 1994; Min et al. 2005). Soy protein isolate was also
used for making edible films (Emiroglu et al. 2010).
Wax is a lipid-based material that has been used for a long time as a coating for
fresh fruits and vegetables. Wax-based edible coatings incorporated with antimicro-
bials were used to prevent moisture loss, extend the shelf-life, and improve the
safety of fruits (Barman et al. 2011; Chiumarelli and Hubinger 2012; Jo et al. 2004;
Mcguire 1997; Ochoa et al. 2011).

2.2 Non-edible and Biodegradable

Polylactic acid (PLA) is a biodegradable and compostable polymer with hydropho-

bic surface properties, and can be derived from renewable resources. Polymers of
PLA are of current interest not only because of the need to ultimately replace many
fossil fuel-derived polymers but also due to the growing global problems associated
with plastic waste disposal (Plackett et al. 2006). The use of PLA in food packaging
has already received wide attention (Conn et al. 1995; Frederiksen et al. 2003;
Haugaard et al. 2002; Jin and Zhang 2008; Jin and Gurtler 2011; Jin et al. 2009b,
2010; Sinclair 1996).
In addition to being used alone, PLA resins have been used with other biopoly-
mers, such as pectin and sugar beet (Jin et al. 2009b; Li et al. 2012a). Jin et al.
(2009b) found that PLA films have hydrophobic characteristics and a smooth film
surface which limits the capability to incorporate hydrophilic antimicrobials such as
nisin onto the film surface. The incorporation of pectin in the PLA film created a
rough and ragged surface, which was hydrophilic and facilitated the access and
354 T.Z. Jin

adsorption of nisin without affecting the tensile strength, flexibility and toughness
of pectin/PLA films.
Paper can be used as an antimicrobial carrier. Several studies have demonstrated
the use of biopolymer coating on paper packaging materials and their use for anti-
microbial packaging (Ben Arfa et al. 2007; Chalier et al. 2007; Khwaldia et al.
2010).

2.3 Non-edible and Non-biodegradable

Due to their thermal properties, capable of being processed to films, and excellent
barrier-forming properties towards gas and moisture, traditional plastic packaging
materials such as low density polyethylene (LDPE), polyvinyl chloride, nylon,
polypropylene, polyethylene/polyamide (PE/PA), and other plastic films are still
widely used in food packaging including antimicrobial/antioxidant packaging. Most
of these plastic films are commercially available. They can be directly used as base
films for coating to make double-layer antimicrobial packaging films, which consist
of an active layer and a base layer (Chawengkijwanich and Hayata 2008; Li et al.
2009; Natrajan and Sheldon 2000a; Neetoo et al. 2007; Scannell et al. 2000; Ye
et al. 2008a, b). When composite antimicrobial packaging films are needed, a resin
of these plastics is mixed with antimicrobials and antimicrobial films are made by
the extrusion (Emamifar et al. 2012; Liu et al. 2009; Siragusa et al. 1999) or solvent
methods (Jin and Zhang 2008; Jin et al. 2010).

3 Antimicrobials

Antimicrobials are the key component of an antimicrobial packaging system.

Table 16.2 shows the different antimicrobials which have been used in antimicrobial
packaging systems.

3.1 Essential Oils

Essential oils have been intensively studied as antimicrobial agents for food appli-
cations. An essential oil is a concentrated hydrophobic liquid containing volatile
aroma compounds from plants. Essential oils are generally extracted by steam dis-
tillation, or solvent extraction. For example, oregano is an aromatic herbal plant
native to Europe and southern and central Asia. The leaves of the oregano plant are
used to add flavor to many Greek and Italian dishes. One of the components of the
essential oil of the oregano plant is carvacrol, a phenol also present in the essential
oils of thyme, pepperwort, and wild bergamot. Cinnamon is a small evergreen tree
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 355

Table 16.2 Antimicrobials, target microorganisms and test media used for antimicrobial
packaging
Antimicrobial Medium/
Agent Microorganism Food Reference
Cinnamaldehyde Salmonella, L. Agar, Ben Arfa et al. (2007), Caillet
essential oil (EO) monocytogenes, E. coli produce, et al. (2006), Du et al. (2009a),
O157:H7, C. jejuni, S. meat Juneja et al. (2012), Mild et al.
hadar (2011), Ravishankar et al. (2009,
2012) and Zhu et al. (2014)
Carvacrol EO Salmonella, L. Agar, Ben Arfa et al. (2007), Du et al.
monocytogenes, E. coli produce, (2009b), Juneja et al. (2012),
O157:H7, C. jejuni, S. meat Kuorwel et al. (2011), Lim et al.
hadar, S. aureus (2010), Mild et al. (2011),
Ravishankar et al. (2009, 2012),
Scannell et al. (2000) and Zhu
et al. (2014)
Oregano EO Salmonella, S. aureus, Agar Fernández-Pan et al. (2012)
L. innocua, P. fragi
Caraway EO Salmonella, S. aureus, Produce Gniewosz et al. (2013)
E. coli, B. subtilis,
Aerobic Bacteria,
Yeasts and Molds
Eugenol EO L. monocytogenes, S. Agar Du et al. (2009a, b)
hadar, E. coli O157:H7
Allyl Salmonella, Listeria Agar, liquid Chen et al. (2012), Guo et al.
Isothiocyanate EO egg, produce (2013a, c), Jin and Gurtler (2011),
Li et al. (2012a, b)
Linalool, Thymol S. aureus Agar, cheese Kuorwel et al. (2011)
EO
Garlic oil Salmonella, S. aureus, Agar Pranoto et al. (2005)
E. coli, B. cereus
Orange EO Salmonella, L. Agar O’Bryan et al. (2008) and
monocytogenes Shannon et al. (2011)
Ginseng extract B. subtilis, L. Agar, broth Norajit and Ryu (2011)
monocytogenes, E.
coli, Salmonella, P.
aeruginosa
Lysozyme L. monocytogenes, Agar, broth, (Datta et al. (2008), Mastromatteo
Salmonella, Spoilage seafood, meat et al. (2010) and Min et al. (2005)
Bacteria
(continued)
356 T.Z. Jin

Table 16.2 (continued)

Antimicrobial Medium/
Agent Microorganism Food Reference
Nisin L. monocytogenes, Agar, broth. Datta et al. (2008), Franklin et al.
Salmonella, E. coli Seafood, (2004), Guo et al. (2013b),
O157:H7, Spoilage meat, liquid Hoffman et al. (2001), Jin (2010),
Bacteria, Yeasts and egg, puree, Jin and Gurtler (2011), Jin et al.
Molds, M. luteus, L. juice, cheese,(2009a, b, 2010), Juck et al.
lactis milk (2010), Lu et al. (2009), Lungu
and Johnson (2005),
Mastromatteo et al. (2010),
Mauriello et al. (2005), Natrajan
and Sheldon (2000a, b), Neetoo
et al. (2007), Scannell et al.
(2000), Siragusa et al. (1999) and
Ye et al. (2008a, b)
Enterocins A and B L. monocytogenes Meat Marcos et al. (2007)
Lacticin 3147 L. innocua, S. aureus, Agar, cheese, Scannell et al. (2000)
Spoilage Bacteria, meat
Yeasts and Molds
Potassium sorbate, L. monocytogenes, E. Seafood, Jin et al. (2010), Juck et al. (2010)
sodium benzoate coli O157:H7, Spoilage meat, puree and Ye et al. (2008a, b)
Bacteria, Yeasts and
Molds
Potassium lactate, L. monocytogenes Seafood, Jiang et al. (2011a, b), Juck et al.
sodium lactate, meat (2010), Lungu and Johnson
sodium diacetate (2005), Neetoo et al. (2010),
Stopforth et al. (2010), Ye et al.
(2008a, b, 2011)
Lauric arginate L. monocytogenes, Broth, meat, Guo et al. (2013a, b, c, 2014),
ester Salmonella seafood Hoffman et al. (2001) and
U.S. Department of Agriculture,
Food Safety and Inspection
Service (1989)
EDTA L. monocytogenes, E. Broth, meat, Hoffman et al. (2001), Lu et al.
coli O157:H7, puree, (2009) and Mastromatteo et al.
Salmonella, Spoilage seafood (2010)
Bacteria, Yeasts and
Molds
TiO2 nanoparticle Spoilage Bacteria, Agar, juice, Chawengkijwanich and Hayata
Yeasts and Molds, E. produce (2008)
coli
ZnO nanoparticle Spoilage Bacteria, Broth, liquid (Emamifar et al. (2012), Jin and
Yeasts and Molds, Egg white Gurtler (2011), Jin et al. (2009c)
Salmonella, S. aureus, and Li et al. (2009)
E. coli O157:H7,
Listeria
Liquid smoke L. monocytogenes Meat Murphy et al. (2005)
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 357

native to Sri Lanka. The tree is also grown commercially in South and Southeast
Asia, Brazil, and in parts of Africa. The bark of the tree is used as a spice in many
foods. The flavor of cinnamon is due to the presence of cinnamaldehyde, an essen-
tial oil which can be extracted from the bark by pounding it, macerating the bark in
seawater, and quickly distilling it.

3.2 Bacteriocins and Lysozyme

Bacteriocins are low molecular weight peptides or proteins consisting of 30–60

amino acids. They are synthesized ribosomally and exhibit either bactericidal or
bacteriostatic effects on bacteria of other species or of the same species
(Klaenhammer 1988). Nisin is a bacteriocin which is used successfully in more than
50 countries as a biopreservative. It was first isolated from Lactococcus lactis in
1928 (Rogers 1928). Nisin has been given GRAS status by U.S. Food and Drug
Administration (Register 1988). Nisin is the only bacterial product which is
approved for use as a biopreservative by the US FDA (Register 1988) and is com-
mercially known as Nisaplin™. Other bacteriocins such as enterocins A and B
(Marcos et al. 2007) and lacticin 3147 (Scannell et al. 2000) have also been used in
studies.
Lysozyme is another type of natural antimicrobial. Lysozyme is a 15-kDa single-­
chain protein (Min et al. 2005; Shah 2000). Lysozyme hydrolyzes the β-1,4 linkages
between N-acetylmuramic acid and 2-acetyl-amino-2-deoxy-D-glucose residues in
the peptidoglycan layer of bacterial cell walls, resulting in the lysis of microbial
cells (Shah 2000). Lysozyme is of interest for use in food systems since it is a natu-
rally occurring enzyme that has an antimicrobial activity and is produced by many
animals, including humans (Proctor and Cunningham 1988). Lysozyme is active
against a number of Gram-positive bacteria including L. monocytogenes (Walzem
et al. 2002).

3.3 Other Antimicrobials

Traditionally GRAS antimicrobials such as sodium lactate (SL), sodium diacetate

(SD), potassium sorbate (PB), sodium benzoate (SB), and EDTA (Hoffman et al.
2001; Lu et al. 2009; Mastromatteo et al. 2010) were incorporated into antimicro-
bial coatings and films (Hoffman et al. 2001; Jiang et al. 2011a, b; Jin et al. 2010;
Juck et al. 2010; Lu et al. 2009; Lungu and Johnson 2005; Mastromatteo et al. 2010;
Neetoo et al. 2010; Stopforth et al. 2010; Ye et al. 2008a, b, 2011). Several others
have used lauric arginate ester (Guo et al. 2013b; Hoffman et al. 2001; Stopforth
et al. 2010), a relatively new antimicrobial, in their films and coatings. Lauric argin-
ate (Na-lauroyl-L-arginine ethyl ester monohydrochloride; LAE) is a cationic sur-
factant derived from lauric acid, L-arginine, and ethanol and has received GRAS
358 T.Z. Jin

status for many food applications in the United States (Rodriguez et al. 2004;
Stopforth et al. 2010). Notably, it has received approval by the USDA/FSIS for
application on the surface (up to 44 ppm when applied as a “sprayed lethality in
container” without labeling and up to 200 ppm surface treatment [provided it is
labeled]) of RTE meat and poultry products (U.S. Department of Agriculture, Food
Safety and Inspection Service 1989). Recent developments in nanotechnology have
allowed for the incorporation of metal nanoparticles with antimicrobial activity
such as Titanium Dioxide (TiO2) (Chawengkijwanich and Hayata 2008) and Zinc
Oxide (ZnO) (Emamifar et al. 2012; Jin and Gurtler 2011; Li et al. 2009) in antimi-
crobial packaging.

4 Methods for Making and Applying Antimicrobial Films

Antimicrobial films can be produced by two methods. One method involves appli-
cation of the coating solution directly on the food surface. Once the coating solution
on the food surface dries, a thin and unique film is formed. The other method
involves making the film and applying the film onto the surface of food.

4.1 Direct Coating on Foods

The three ways to apply the coating solution directly onto the food surface are by
dipping, spreading, and brushing. Food samples such as carrots (Gniewosz et al.
2013), smoked salmon (Datta et al. 2008), turkey frankfurters (Lungu and Johnson
2005), or fish fillets (Lu et al. 2009), etc. can be dipped in coating solutions for one
to five minutes. Coating solutions can be spread evenly on each side of the food
sample using a sterile hockey stick (Jiang et al. 2011a; Neetoo et al. 2010; Ye et al.
2011) or a compressed air-assisted sprayer (Severino et al. 2015). Coating solutions
can also be applied evenly to the surface of the food sample using a poly form brush
(Chen et al. 2012; Min et al. 2005). The treated food surfaces are allowed to air dry
so that an antimicrobial film can be formed.

4.2 Edible Composite Film

A film solution is made of edible polymers and antimicrobials. After the film com-
ponents are mixed, film-forming solutions are cast onto flat substrates made of glass
(Fernández-Pan et al. 2012), polyester (Gniewosz et al. 2013; Norajit and Ryu 2011;
Zhu et al. 2014), polyacrylic, high density polyethylene (HDPE) (Min et al. 2005),
PET (Mild et al. 2011; Ravishankar et al. 2009, 2012), Teflon (Jin and Zhang 2008;
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 359

Lim et al. 2010; Moreira et al. 2011), and others (Du et al. 2009a, b). The solutions
are left to dry and the resulting films are peeled off before applying on foods. For
making alginate-based film, additional calcium chloride solution is needed to make
alginate solutions to form gels and then films after drying (Marcos et al. 2007;
Norajit and Ryu 2011; Pranoto et al. 2005). These films can be used to package food
with or without additional plastic packaging.

4.3 Non-edible Composite Film

A film solution is made of non-edible polymers with antimicrobials. Antimicrobials

are mixed with non-edible polymers and then films are made using solvent/casting
method or extrusion/casting method. Jin et al. (Jin and Gurtler 2011; Jin et al. 2010)
used methylene chloride to dissolve PLA resin with antimicrobial compounds.
Once the PLA resin had completely dissolved, each film solution was distributed to
Teflon petri dishes. The methylene chloride was allowed to evaporate at room tem-
perature under a chemical hood. The resulting films were peeled from the dishes and
placed in a sealed container until time of use. Others (Jin et al. 2009b; Siragusa et al.
1999) used a twin-screw extruder to make composite PLA or LDPE films with
antimicrobials.

4.4 Double Layer Film

Commercial plastic films are used as base films for antimicrobial packaging. The
antimicrobial coating solution can be cast or sprayed onto one side of the plastic
film using a thin-layer chromatography plate coater (Franklin et al. 2004; Neetoo
et al. 2007; Ye et al. 2008a, b), a bar coater (Chawengkijwanich and Hayata 2008),
a coating rod (Mauriello et al. 2005), a roller (Kuorwel et al. 2011), or a liquid atom-
izer (Natrajan and Sheldon 2000a).

4.5 Advantage and Disadvantage of Applying Methods

Composite film vs. double-layer film: There are various ways to incorporate antimi-
crobial compounds into polymers as discussed above. Antimicrobial compounds
such as volatile essential oils that can’t tolerate the high temperatures used in poly-
mer processing such as extrusion and injection molding are often either coated onto
the film material after forming or are added to cast films. The double layer method
provides several advantages over the one step method, such as: (1) Less concern of
the loss of antimicrobial activity during thermal film making, (2) Reduced amount
360 T.Z. Jin

of antimicrobial compounds used in packaging material since only the outer layer
plays an antimicrobial part, (3) Avoid “filler effect” and minimal impact on physical
or mechanical properties of base materials (Guo et al. 2014).
Nevertheless, for the development of double-layer antimicrobial films, one of the
big challenges is the binding or adhering capacity of coating solutions on base films
due to the hydrophobic surface characteristics of PLA or other plastic films. A pre-
vious study showed that the binding capacity was significantly affected by pretreat-
ment conditions of films, concentration of chitosan, and other additives in the
coating solutions (Guo et al. 2013b).
Direct coating vs. film packaging: Either method has advantages and disadvan-
tages. Antimicrobial film packaging must come into direct contact with the surface
of food to exert an antimicrobial effect. The direct coating method may be more
suitable for foods with irregular shapes or rough surfaces, where films cannot
directly contact food surfaces without vacuum-packaging. However, preparation of
coating solutions and coating/drying procedures might slow down production
speed. In contrast, antimicrobial films can be customized within current film manu-
facturing lines, pre-made by a packaging company, and used as regular packaging
materials. In a previous study (Guo et al. 2013b), there was no significant difference
in antimicrobial effectiveness between antimicrobial films and coatings. Therefore,
food processors can make a choice based on their preference, convenience, avail-
ability, and cost effectiveness.

5 Target Microorganisms

Antimicrobial efficacy of films or coatings has been evaluated for reduction or inhi-
bition of multiple kinds of microorganisms as shown in Table 16.2. Salmonella spp.,
E. coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus were mostly
tested for enhancing food safety after antimicrobial packaging treatments.
Surrogates, such as Listeria innocua and E. coli ATCC 11775, were also used in
situations where pathogens were not allowed. Spoilage microorganisms also were
determined for extending food shelf life.

6 Evaluation Method for Antimicrobial Efficacy

The antimicrobial efficacy of an antimicrobial packaging material can be evaluated

in culture media and in real food systems. The culture media method is usually used
for screen tests that require many trails while tests in real food systems are used for
further validations.
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 361

6.1 Culture Media

The two methods usually used in microbial reduction/inhibition studies in culture

media are the agar diffusion and liquid incubation methods.
The agar diffusion test is used to simulate a test for solid food packaging. Each
film sample is placed on a solid agar medium inoculated with the test microorgan-
ism. The agar plates are incubated until growth is visible. A clear zone surrounding
the film indicates antimicrobial diffusion from the film and subsequent growth inhi-
bition. The diameter of the growth inhibition zones are measured with a caliper. The
ratio of the diameter of inhibition zone to the diameter of the film specimen is used
to determine antimicrobial activity (Jin et al. 2009b).
In order to quantitatively evaluate the antibacterial activity of sample films, the
liquid culture test is used. Liquid media buffer, growth media or foods are seeded
with the target microorganisms and the antimicrobial film sample. The flasks are
incubated with mild agitation. Samples are taken over time and enumerated periodi-
cally during the incubation to obtain microbial growth profiles.

6.2 Evaluation and Application in Foods

6.2.1 Liquid Foods

Emamifar et al. (2012) studied the effect of LDPE packaging containing silver and
zinc oxide nanoparticles on the shelf life of orange juice stored at 4 °C over a 112-­
day period. Jin et al. (Jin 2010; Jin and Gurtler 2011; Jin et al. 2009b, 2010) incor-
porated antimicrobials in pectin and polylactic acid composite films against Listeria
monocytogenes, E. coli O157:H7 and Salmonella in orange juice, strawberry puree,
milk, and liquid egg albumen. Mauriello et al. (2005) also made a pouch of LDPE
film coated with nisin to inactivate M. luteus in ultra-high temperature milk and to
extend the shelf life of raw milk and pasteurized milk stored at 4 °C over a 4-day
period.

6.2.2 Produce

Zhu et al. (2014) showed that antimicrobial films containing 3% carvacrol were
effective in reducing S. newport populations in organic romaine lettuce, organic
iceberg lettuce, organic adult spinach, and organic baby spinach stored at 4 °C over
a 7-day period. (Chawengkijwanich and Hayata 2008) showed that E. coli popula-
tions in lettuce treated with TiO2-coated film bags and exposed to black-light
decreased by 1.5 log within 24 h compared to lettuce samples treated with uncoated
film bags and exposed to black light only. Pullulan-based coatings containing 10%
caraway essential oil reduced S. aureus populations in carrot by 3.6 log and reduced
362 T.Z. Jin

S. enteritidis populations by 2.4 log (Gniewosz et al. 2013). Moreira et al. (2009)
treated squash slices with chitosan film-forming solution and found it was most
effective in retaining ascorbic acid levels and in reducing total microbial counts in
butternut squash slices. Chen et al. (2012) and Jin and Gurtler (2012) developed
edible coating solutions for cantaloupe and tomato, and demonstrated that these
coating solutions significantly reduce pathogens on the surface of cantaloupe and
tomato stem.

6.2.3 Meat Products

Jiang et al. (2011b) demonstrated that the pectin-based coatings containing 0.25%
sodium diacetate, 0.25% sodium diacetate and 2% sodium lactate, 2.5% Opti.Form
PD4, and a combination of 2.5% Opti.Form PD4 and 0.1% Protect-M were the most
effective in suppressing the growth of L. monocytogenes populations in roasted tur-
key during frozen storage and chilled storage. Juck et al. (2010) also found the most
effective alginate-based coatings against L. monocytogenes populations in roasted
deli turkey over eight weeks at 4 °C were those containing 0.25% sodium diacetate
and 0.3% potassium sorbate, 2.4% sodium lactate and 0.3% potassium sorbate, and
500 IU/g nisin and 0.3% potassium sorbate. Lungu and Johnson (2005) demon-
strated that zein-ethanol-glycerol solution containing nisin and sodium diacetate,
zein-ethanol-glycerol solution containing nisin, sodium lactate, and sodium diace-
tate, zein-propylene-glycerol solution containing nisin and sodium diacetate, and
zein-propylene-glycol solution containing nisin, sodium lactate, and sodium diace-
tate reduced L. monocytogenes populations to undetectable levels in turkey frank-
furters stored at 4 °C over a 28-day period. Ye et al. (2008a) demonstrated that
chitosan-coated films containing sodium lactate reduced L. monocytogenes popula-
tions in ham steaks stored at 4 °C by 1.5 log over a twelve week period compared to
control samples.
Ravishankar et al. showed that apple-based edible films containing carvacrol
were more effective than films containing cinnamaldehyde in reducing L. monocy-
togenes populations in ham and bologna stored at 4 °C (Ravishankar et al. 2012)
and antimicrobial films containing 3% carvacrol were more effective than 3% cin-
namaldehyde films in reducing E. coli O157:H7 populations in chicken breast
stored at 23 °C and at 4 °C (Ravishankar et al. 2009). Gelidium corneum films
containing 0.6% carvacrol were more effective against L. monocytogenes popula-
tions than against E. coli O157:H7 populations in ham stored at 4 °C over a 9-day
period (Lim et al. 2010). However, Mild et al. (2011) showed that edible apple-­
based films containing cinnamaldehyde were found to be more effective with
increasing concentration levels than carvacrol against all strains of C. jejuni on
chicken breast after 72 h of treatment and incubation at 4 °C or 23 °C.
Natrajan and Sheldon (2000b) found that 0.75% agar films containing 500 μg/ml
nisin were most effective in reducing S. Typhimurium populations in chicken skin
stored at 4 °C at 96 h. B. thermosphacta counts in beef carcass samples wrapped in
plastic filled with nisin and stored at 12 °C were reduced by 2.7 log over 20 days
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 363

compared to the control samples (Siragusa et al. 1999). Franklin et al. (2004)
showed that packaging pouches coated with 1.875 g or 2.5 g nisin were effective in
inhibiting the growth of L. monocytogenes on the surface of hot dogs over a 60-day
period at 4 °C. However, Marcos et al. (2007) found that alginate-based, zein-based
films, and polyvinyl alcohol-based films containing up to 2000 AU/cm2 enterocins
were not very effective in significantly reducing L. monocytogenes populations in
cooked ham stored at 6 °C for 8- or 29-day, regardless of whether they were air-­
packed or vacuum-packed (Marcos et al. 2007).

6.2.4 Seafoods

Antimicrobial coating on cold-smoked salmon significantly inhibited L. monocyto-

genes cells, total aerobic mesophilic bacterial populations, and yeast and mold pop-
ulations (Min et al. 2005). Calcium alginate coating solutions containing lysozyme
and nisin were most effective in reducing L. monocytogenes populations and S.
anatum populations in cold-smoked salmon stored at 4 °C over a 35-day period
(Datta et al. 2008).
Chitosan-coated plastic films containing sodium lactate, sodium diacetate, potas-
sium sorbate or their combinations were effective in reducing L. monocytogenes
populations in cold-smoked salmon over an eight-week period at 4 °C (Jiang et al.
2011a; Ye et al. 2008b). Gelatin-based antimicrobial coatings were most effective in
reducing L. monocytogenes populations in cold-smoked salmon followed by
alginate-­based antimicrobial coatings and starch-based antimicrobial coatings after
twelve months of frozen storage (Neetoo et al. 2010; Ye et al. 2011). The alginate
coating solution was the most effective in reducing total mesophilic bacterial popu-
lations and total psychrophilic bacterial populations in fish fillets, extending their
shelf life (Lu et al. 2009). Sodium caseinate/chitosan films were also most effective
against the native bacterial populations of salami (Moreira et al. 2011). Chitosan
coating with allyl isothiocyanate, in combination with cryogenic freezing achieved
more than 5 log reduction of L. innocua and natural bacteria in raw shrimp (Guo
et al. 2013c), and the antimicrobial coatings achieved ca. 5.5–1 log CFU/g reduc-
tion of L. innocua on RTE shrimp, depending on the inoculated population levels
(Guo et al. 2013a).

6.2.5 Dairy Products

Moreira et al. (2011) showed that sodium caseinate/chitosan films were most effec-
tive against the native bacterial populations of cheese. Kuorwel et al. (2011) found
that antimicrobial films containing thymol, carvacrol, and linalool were effective in
reducing S. aureus populations in cheddar cheese stored at 15 °C over a 21-day
period. S. aureus populations in cheddar cheese packed in PE/PA pouches treated
with nisin and stored at 4 °C over a 12-week period decreased by 1.5 log (Scannell
et al. 2000).
364 T.Z. Jin

Jin et al. (2013) developed antimicrobial coatings for improving the microbio-
logical safety and quality of shell eggs. Chitosan coatings with 0.1, 0.5 and 1.0% of
LAE reduced Salmonella populations by 1.7, 2.5, and 5.2 log CFU/cm2, respec-
tively and significantly reduced weight loss of shell eggs during 12 weeks of storage
at 7 or 4 °C.

7  ecent Innovations in Research and Development

R
of Antimicrobial Packaging

Nanotechnology will become one of the most powerful forces for innovation in the
food packaging industry. One such innovation is biobased nanocomposite technol-
ogy, which holds the key to future advances in flexible packaging. Recent studies
have focused on the use of nanocomposites in developing new types of active pack-
aging (Bradley et al. 2011; Busolo and Lagaron 2012; Silvestre et al. 2011).
Abdollahi et al. (2012) developed an active bionanocomposite film with a synergis-
tic effect between nanoclays and natural extracts and with antioxidant and antimi-
crobial activities. For instance, essential oils (EOs) have gained interest as natural
antimicrobial agents for food preservation against foodborne pathogens and spoil-
age bacteria (Caillet et al. 2006). However, since the constituents of EOs are char-
acterized by their low solubility in water, they need to be encapsulated in an
appropriate delivery system to promote their efficiency (Weiss et al. 2009). The
encapsulation of EOs in nanoscale delivery systems was shown to offer the potential
of improving EOs bioactivity through the activation of passive mechanisms of cell
absorption, owing to their subcellular size, therefore enabling the reduction of the
dose of essential oils required to ensure antimicrobial activity in foods and minimiz-
ing their impact on aroma, flavor and taste of foods (Donsì et al. 2011, 2012).
Nanoscale encapsulation can also increase the concentration of bioactive com-
pounds in food areas where microorganisms are preferably located, for example
water-rich phases or liquid-solid interfaces (Weiss et al. 2009).
Antimicrobial film and coating of high-moisture foods puts specific constraints
on the structural integrity and flexibility of the coating material. It should be suffi-
ciently water-resistant to remain intact and should cover all parts of a heterogeneous
product adequately when applied via dipping, spreading, or brushing. For fresh
fruits, which consist of living tissues with respiratory activity, coatings should not
fully deplete O2 or lead to build-up of excessive CO2 which may trigger physiologic
deterioration, leading to anaerobic respiration and the development of off-flavors,
abnormal ripening, and spoilage. The O2 levels that cause anaerobic reactions vary
among commodities according to the permeability of the peel, respiration patterns,
storage temperature, as well as the type and thickness of coatings applied. Coatings
applied to such respiring products should allow O2 to penetrate into the package and
excessive CO2 to escape from it. As a general rule of thumb, a minimum of 1–3%
O2 is required around a product to avoid a shift from aerobic to anaerobic respiration
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 365

(Arvanitoyannis and Gorris 1999). Therefore, films formed on food surfaces after
coatings should have specific characteristics that allow for the exchange of CO2 and
O2 between the food surface and the environment to maintain the freshness of the
food. The potential approach is to make porous films with nano-scale pores and
micro-scale channels. By manipulating the size and number of pores and channels,
the gas permeability of the films can be controlled to meet the requirement for each
type of food.
Antimicrobial films and coatings consisting of porous polymers could provide
appropriate pore sizes, adequate mechanical properties, highly porous and intercon-
nected pore structures, high surface area to volume ratios, biodegradability, and
allow for surface chemistries that favor the attachment, proliferation, and differen-
tiation of cells. Porous polymers are a subset of porous materials with an advantage
of the ease of processability associated with polymers to generate monoliths, films,
and beads, often with well-defined porosities and high specific surface areas (SSAs)
(Gokmen and Du Prez 2012; Wu et al. 2012). Porous polymers are of interest for
such applications as microelectronics, biomedical devices, membrane processes,
and catalysis as well as for precursors that can be used to synthesize porous ceram-
ics or porous carbons. Depending on the nature of the colloidal system employed
(emulsions, micro emulsions, solid particles, or breath figure droplets), the charac-
teristic pore size can range from a few nanometers to hundreds of micrometers.
Porous systems that offer the ability to manipulate the porous structure can be used
for controlled release of active compounds. For example, the release time of a dye
from a stand-alone hydrogel was 10 h, while incorporating the hydrogel within a
highly porous polymer extended the release time to 10 days (Gitli and Silverstein
2011). The diffusion pathway for this bicontinuous system was modeled using the
diffusion through an assembly of polydisperse spheres (Ritger and Peppas 1987).
The surfactant reduces the interfacial tension, reducing the droplet size, increasing
the number of droplets, and reducing the wall thickness. This reduction in wall
thickness allows interconnecting holes to be formed during polymerization and/or
processing.
To make nano-scale porous coating films, the technique used to make nanoemul-
sions with polymer and emulsifier/surfactant is critical. Microfluidization has led to
good nanoemulsion-based systems in several research studies, with droplet sizes
ranging from 60 to 600 nm (Hatanaka et al. 2008, 2010; Jafari et al. 2007a, b; Qian
and McClements 2011; Rao and McClements 2011; Wooster et al. 2008). Therefore,
microfluidization can be used to produce porous polymers that will be part of a new
generation of food coatings containing natural and edible food polymers and plant
essential oils. By manipulating the number of pores and pore size, desired barrier
properties to CO2, O2, moisture, and UV light, the controlled release of active agents
can be achieved so that the safety and shelf life of fresh products such as fruits and
vegetables can be ensured and extended. The antibacterial activity of modified
chitosan-­based coatings containing nanoemulsion of essential oils (EOs) against E.
coli O157:H7 and S. Typhimurium was evaluated on inoculated green bean samples
(Severino et al. 2015).
366 T.Z. Jin

8 Future Research Direction and Challenges

Effective, natural, and safe antimicrobials will continuously be developed, evalu-

ated and applied in food packaging. Bio-based polymers from agricultural or marine
sources, particularly from by-products of these, will consist of a greater portion of
packaging materials to meet the growing demand of environmental protection.
Many of the works reported in the literature are focused on the production and
the mechanical properties of the biobased nanocomposites. Little attention has been
paid to gas permeability of biobased nanocomposites. Therefore, food packaging
with multiple functions is needed. Films or coatings with desired barrier properties
to CO2, O2, moisture, UV light as well as allowing for the controlled release of
active agents can be developed to meet the needs of different foods to improve food
quality and safety.
Researchers have focused primarily on developing new approaches and testing
new methods on model systems, but not quite as much on commercial applications
in real food products. This may be due to different challenges.
Technical challenges exist in incorporating appropriate antimicrobial agents into
packaging systems. For example, (1) The choice of an antimicrobial compound may
be limited not only by its compatibility to the type of packaging material used, but
also to the heat liability of the component during extrusion. (2) Stability of antimi-
crobial coatings and films during storage: loss of antimicrobial activities of a coat-
ing solution, or separation of the active layer from the base layer of double-layer
films during storage have not been widely studied or evaluated. There are other
challenges as well from legislative restrictions and consumer resistance.
No specific regulations exist on the use of antimicrobial packaging. Antimicrobial
packaging is subjected to traditional packaging legislation, which requires that
compounds are registered on positive lists and that the overall and specific migra-
tion limits are respected. This is more or less contradictory to the concept of some
active packaging systems in which packaging releases substances to extend shelf-­
life or improve quality. In addition, as lack of scientific certainty about the safety of
manufactured nanomaterials in food additives, ingredients and packaging, the food
industry’s main concern about introducing active components to packaging seems
to be that consumers may consider the components harmful and may not accept
them (Mikkola et al. 1997).
Antimicrobial packaging, one of active packaging, is an emerging and exciting
area of food technology which can confer many preservation benefits on a wide
range of foods (Day 1998). As more companies become aware of the economic
advantages (Smith et al. 1995) of using absorbent technology, and consumers accept
this approach, the technology will likely emerge as the preservation technology of
the twenty first century (Smith 1996).
16 Current State of the Art and Recent Innovations for Antimicrobial Food Packaging 367

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Chapter 17
Consumer Perception of Food Preservation
Techniques

Christine M. Bruhn

Abstract Food preservation techniques have enabled consumers to select a variety

of nutritional, safe and flavorful foods that can be used to create an appealing meal
in minimum time. While consumers value these attributes, many do not realize that
preserved foods retain significant nutritional value and may be as safe or safer than
their fresh equivalent. Consumers respond positively to information that empha-
sizes product benefits. A communication approach built on an understanding of
consumer concerns, utilizes consumer information sources, and delivered by trusted
sources can increase consumer acceptance of food preserved by traditional as well
as innovative methods.

Keywords Consumer attitudes • Communication • Trust • Food processing • New

technologies

1 Introduction

The quality, variety and safety of the food supply have increased markedly in
the past decades. Often the public has taken this advancement for granted.
Today people expect their food to be flavorful, safe, nutritious, convenient, and
produced in a sustainable manner. At the same time, they may be critical of the
industry that has made these advances possible. This chapter will briefly review
the current status of the US consumer’s view toward food preservation and sug-
gest an approach to increase public recognition of potential risks and benefits
of food preservation.

C.M. Bruhn (*)

Center for Consumer Research, Cooperative Extension Specialist Emerita,
University of California, Davis, CA, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 373

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_17
374 C.M. Bruhn

2 Consumer Priorities

Consumers are seeking convenient products with good flavor and health enhancing
properties. Most consumers cite good taste as the primary reason for selecting a
product (International Food Information Council Foundation 2015). Over the last
decade, price has been of secondary importance with the percentage citing it as a
factor ranging from 64 to 79% depending on the state of the economy. Healthfulness
is consistently the third in importance with 58–66% indicating it is a major factor.
Convenience is a necessity in today’s households. Meals are prepared quickly
with 19% of households estimating that they spend less than 15 min preparing din-
ner during the week. About half, 52% spend 15–44 min, with 29% indicating they
spend more than 45 min daily (International Food Information Council Foundation
2015). In response, the food industry has made available a variety of fresh, frozen,
dried, canned main dish and accompaniment items.

3 Attitudes Toward Food Processing and Preservation

While many appreciate the quality and diversity of the food supply, awareness of
current and future food processing technology is not widespread.
Consumer survey response indicates that many are excited about the idea of
futuristic food technologies that make food easier to prepare and are more
healthy. If an invention turned raw ingredients into a meal, 80% said they would
be very excited to try it (International Food Information Council Foundation
2015). Similarly, 78% were very excited to try food that was customized in
nutritional value and calories, and 69% were excited to try a 3D printer that
could make food you wanted from scratch. Consistent with this view,
Progressive Grocer reports that innovation is very important to consumers
(Progressive Grocer 2015). Consumers indicate they are willing to pay a pre-
mium for innovative goods and services. More than half of consumers indicate
that they purchased a product without fully understanding what it did or how it
worked, but solely because they thought it was “cool.”
Despite these positive views toward futuristic food technologies, when asked
to select up to four possible benefits of processed foods from a list of nine,
healthful and better safety were selected by only 18% and 16% respectively
(International Food Information Council Foundation 2015). Half of respondents
identified convenience as a benefit, and 44% noted that processed foods stay
fresher longer. Affordability was selected by 40% of respondents, variety by
30%, better taste by 25%, and less food waste by 14%. When specifically asked
about safety, only 54% believed that food processing can help improve food
safety (International Food Information Council Foundation 2014). This suggests
that about half of consumers interviewed were not aware of or did not believe
that processing increases food safety.
17 Consumer Perception of Food Preservation Techniques 375

4 Food Preservation and Perceptions of Healthfulness

In 2015, almost half of U.S. consumers indicated that they gave a lot of thought to
the healthfulness of the foods and beverages they consume (International Food
Information Council Foundation 2015). Dietary advice in the United States is cen-
tered on a graphic described as “My Plate.” This is an illustration of a plate in which
half is designated for fruits and vegetables, about a quarter for grains, another quar-
ter for protein and a circle separate from and above the plate for dairy (U.S. Department
of Agriculture 2016). About 40% of consumers indicate they are familiar with this
educational tool (International Food Information Council Foundation 2015).
A significant number of consumers do not view processed foods as nutritionally
beneficial as fresh food. Only 50% of consumers believe that processed foods con-
tain the nutrients needed for a healthful diet (International Food Information Council
Foundation 2014). Slightly more, 63%, believe that some processed foods can pro-
vide affordable, nutritious options. Consistent with this view processed fruits and
vegetables are viewed as less healthy than fresh (Produce for Better Health
Foundation 2014). Researchers report that 92% of parents considered fresh fruits
and vegetables healthy, 66% viewed pre-cut and washed fruits and vegetables
healthy, fewer still, 55 and 47% viewed dried fruits and freeze dried fruits as healthy,
and only 23% considered canned fruits or vegetables to be healthy. Further, freeze
dried products were considered not healthy by 18% of parents and canned fruit and
vegetables were viewed as not healthy by 28%.
When asked if they are changing their diet to increase healthfulness, 82% indi-
cate they are eating more fruits and vegetable and 70% are eating more whole grains
(International Food Information Council Foundation 2015). The Produce for Better
Health Foundation (2014) found that 45% of moms and 51% of dads believed their
family eats too little fruit. The primary barriers to fruit consumption included wish-
ing for new ways to prepare fruit, 52%, believing fruit was too expensive, 50% and
finding that fruit goes bad before it can be eaten, 48%. These barriers may be
reduced if consumers selected processed fruits since the food industry often includes
recipe ideas, processed products may be less expensive than the fresh equivalent,
and the processed product often has a longer and more predicable shelf life that
fresh. Consumers, however appear unaware of these positive attributes.

5 Attitudes Toward Food Safety

Consumers expect food to be safe. When specifically asked, 84% responded that they
have given the safety of foods and beverages a lot, 39% or a little, 45% thought
(International Food Information Council Foundation 2015). In 2015 about 60% of
Americans responded that they were very or somewhat confident in the safety of the
US food supply. Response to this question in the past three years indicated that confi-
dence has decreased. In 2014, 66% were very or somewhat confidence while in 2013
376 C.M. Bruhn

confidence was expressed by 70% of respondents. Older people, men and those with
a higher income were more likely to express confidence than their counterpart. When
asked to identify the most important food safety issue for themselves and their family,
36% identified chemicals in food, 24% foodborne illness from bacteria, 9% pesticide
residues, 7% animal antibiotics, and 3% identified undeclared allergens. Concern
about chemicals in food was highest among those who were not confident in the
safety of the food supply, while concern about foodborne illness from bacteria was
highest among those confident in the food supply. There was no significant difference
between concern levels for other potential hazards between those who were and who
were not confident in the food supply. This indicates that people who were not confi-
dent in the food supply have been highly sensitized to chemical concerns. Researches
have not identified which specific chemical issues were driving these concerns.
Consumers say they respond to information about food recall. If people hear in the
news that a food item they purchased has been recalled, 62% said they would check
the food in their home to verify if it was indeed recalled, 69% indicated they would
dispose of or return the item (International Food Information Council Foundation
2015). Older people, women, persons with higher income, and college graduates
were more likely to dispose of or return the recalled item. Consumers indicated that
a recall may have a broad impact on their purchase behavior with 24% indicating that
they would stop buying all similar items for a time. This indicates that a recall not
only affects the company involved, but other supplies of the same product type.

6 Consumer Attitudes Toward Newer Processing

and Preservation Methods

New food technologies enable food producers and processors to respond to con-
sumer demand for flavorful, convenient, and health enhancing and safe foods.
Consumer perception of the potential benefit and risk of newer approaches will
impact consumer acceptance. Attitudes are affected by information received as well
as the consumer’s philosophic outlook. Some consumers are skeptical of technol-
ogy and believe a low technology approach promotes health and environmental
sustainability. The introduction of a food processed by a new technology may create
concern among these individuals. The public is often unaware of methods used or
safeguards employed in processed food. Any risks associated with a new technol-
ogy may be perceived by the public as imposed by the processor and beyond the
control of the consumer. In some consumer’s mind an unfamiliar approach presents
unknown risks which could be potentially harmful.
When asked about new products, consumers indicate that the potential risk
associated with a product is the most important determinant in product use
(Cardello et al. 2007). Hicks and coworkers found 15% of volunteers from a
national sample admitted that they were fearful of new food processing technolo-
gies (Hicks et al. 2009). Consumers express concern about potentially harmful
by-products and unknown health risks.
17 Consumer Perception of Food Preservation Techniques 377

Providing information about the technology reduced concern. Focusing

consumer response on the method of processing, such as using the term “mini-
mally processed” and “fewer preservatives” however has been shown to gener-
ate a negative response from some consumers (Cardello et al. 2007).
Communicating about the product and providing information about the process
was more likely to address consumer information needs and result in product
acceptance compared to simply stating “minimally processed.”
Concern about a technology influenced flavor expectations, (Lahteenmaki et al. 2002;
Cardello 2003). Researchers found that flavor ratings were lower when people were told
the product was produced by a new processing method. Flavor ratings increased when
people actually saw the produce processed by the new technology, when statements about
safety were made, and when benefits were described (Bruhn 1995; Frever et al. 1997;
Schutz et al. 1989; Cardello 2003). Consistent with these findings, Tuorila and colleagues
(Tuorila et al. 1994) found that uncertainty associated with a novel product may introduce
a degree of expected disliking, but additional factual information reduced this uncertainty
and improved expected liking. Repeated exposure to neutral or positive information
about a technology has been shown to lower concern. While examining the effect of an
educational intervention on attitudes toward food irradiation, Schutz and colleagues
(Schultz 1994) found people expressed less concern about irradiation with repeat testing
even though they received no educational intervention. Similarly, Cardello (2003) found
post concern levels for many technologies were reduced by participating in a study in
which they sampled products described as processed by a new method.
Providing information on the benefits consumers may receive increased accep-
tance. When consumers in Hicks and colleagues sample (2009) were given a brief
explanation of high pressure process and it its benefits, 40% indicated that they would
be willing to pay more for processed products while 45% were not sure. The majority
of those willing to pay indicated they would pay $0.25 or $0.50 more per item.
The importance of perceived benefits was demonstrated in research based upon 3000
personal interviews in the United Kingdom, Germany, and France. The perception of
personal benefits and environmental friendliness were the most important factors affect-
ing likelihood to purchase products processed by high pressure (Butz et al. 2003). The
importance of improved flavor appears to differ by culture. About half of French con-
sumers indicate they would purchase a product processed by high pressure for better
quality, while only four percent of British consumers indicated that they would purchase
for this benefit (Butz et al. 2003). In contrast, almost 40% of German consumers indi-
cated they would purchase a product processed by high pressure for better health while
this was a driving factor for only 18% of French consumers (Butz et al. 2003).

7 Trust and Communication

Continuous consumer communication plays a pivotal role in acceptance.

Communication is more than advertising. Effective communication is a two way
process which involves listening, identifying, and responding to consumer ques-
tions, evaluating the impact of the communication.
378 C.M. Bruhn

Information should be presented in a variety of sources, with preferred sources

varying by age and gender of the target population. Consumers find television, web-
sites, newspaper, magazines and supermarket brochures convenient. At 44%, news
channels and websites were the most selected source of information about chemicals
in food, pesticides, and animal antibiotics (International Food Information Council
Foundation 2015). Health websites at 33% were the second most frequent source of
information followed by government agency materials/websites, 29%, family and
friends, 28%, and natural health websites, 26%. Consumer advocacy groups were
followed by 19% of survey respondents, bloggers by 13%, food producer/manufac-
turers websites by only 12%, and nonprofit organizations by 7%. More men and
younger consumers prefer web based sources than women or older persons (Li-Cohen
and Bruhn 2002; Hicks et al. 2009; Food Marketing Institute 2008).
Factors other than product attributes that can be scientifically measured often
determine if a technology is accepted (Belton 2001). When message components
were segregated, trust in the spokesperson was significantly more important in
explaining attitudes than accuracy of information (Bord and O’Conner 1989). For
example, Sapp (Sapp et al. 1994), found that word of mouth and trust in government
and industry were more important than demographic factors in predicting consumer
acceptance of irradiation.
Trust is greatest for groups perceived as knowledgeable, unbiased, and acting
with the public’s best interest in mind (Frewer et al. 1996). While no one orga-
nization is trusted by everyone, patterns of trust have emerged. The most trusted
source of food safety information, selected by 65% of consumers surveyed, was
their personal healthcare professional (International Food Information Council
Foundation 2015). Government agencies were a trusted source of 42% of respon-
dents. Other trusted sources include family and friends, 29%, a food expert on
TV, 24%, health, food and nutrition bloggers, 24% and farmers 23%. A food
company or manufacturer was trusted by only 11% of respondents. Higher
income consumers and college graduates were more likely to trust government
agencies while bloggers were more likely to be trusted by younger consumers.
Those who prefer low technology approaches to food processing appear to have
lower levels of trust in government sources. For example, compared with those
who selected conventional products, those who buy organic foods had a greater
feeling of distrust toward regulatory agencies (Williams and Hammitt 2001).
Therefore, those with a message to communicate should direct their efforts to
the news and health websites and utilize spokespersons trusted by the target
audience. The importance of family and friends should not be overlooked, espe-
cially in sight of the trust in which this source is held.
To increase trust and the likelihood that communications are understood,
educational programs should be built around what the public wants to know, as
determined by consumer research. New technology should be described using
lay terminology. Terms used by the scientists and industry persons, like GRAS
(Generally Recognized as Safe), for example are not understood by most con-
sumers. In 2015, only 10% said they had heard the term (International Food
Information Council Foundation 2015).
17 Consumer Perception of Food Preservation Techniques 379

A new method or technology may be compared to a similar or more familiar

technology. Communication should acknowledge that risks can never be completely
eliminated. Consumer benefits should be emphasized. Transparency is the operative
word, sharing what is known and not known both in regards to risk and benefits.
Consumers today are interested in the impact of a technology on worker safety and
environmental stewardship, as well as personal welfare.
Developing descriptions of products processed by new technologies should be
done systematically. Statements describing new technologies should be evalu-
ated by focus groups prior to adoption to test message clarity. Messages should
anticipate and respond to concerns special interest groups may express. When
these approaches are used, public acceptance generally increases (Bruhn 1995;
Bruhn and Mason 2002; Resurreccion et al. 1995; Johnson et al. 2004; Fox 2002;
Schutz et al. 1989; Zienkewicz and Penner 2004; Delgado-Gutlerrez and Bruhn
2008). A guide on developing and testing communication messages is available
from the US Food and Drug Administration. (Fischhoff et al. 2011).

8 Summary

Today consumers benefit from a nutritious flavorful food supply; however the food
supply is not risk free. Traditional methods of preservation are being refined and
newer approaches developed that increase quality and safety and extend shelf life.
Consumers often hear what a product does not contain rather than the positive attri-
butes and benefits of newer technologies or ingredients. Newer products and preser-
vation methods will have the greatest likelihood of success when developers address
consumer needs, respond to consumer concerns and offer tangible benefits.
Researchers have demonstrated that statements about the benefits associated with a
particular food or food processing technique will reduce concerns and increase like-
lihood of consumption. Factual information from a trusted source, clear statements
about safety and benefits, and exposure to a product that delivers quality and conve-
nience will increase the likeliness of consumer acceptance. Building consumer
knowledge of how and why food is processed will benefit the public and society by
increasing use and acceptance of beneficial technologies.

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Chapter 18
Statistical Derivation of Sampling Plans
for Microbiological Testing of Foods

Ursula Gonzales-Barron and Vasco Cadavez

Abstract Sampling food for microbial testing is a risk management strategy used
to evaluate whether a food safety system is correctly implemented. Within-batch
testing can be accomplished to comply with microbiological criteria or to assess
whether the food production process is under control. Generally, sampling plans can
be designed to meet the consumer’s and/or producer’s quality requirements, and
have been conventionally derived by borrowing notions of acceptance sampling
theory from classical quality statistics. This chapter describes and illustrates the
methodologies to derive the different types of sampling plans used for microbio-
logical criteria in foods; namely, the two-class attributes sampling plans (based on
prevalence, on concentrations, and with an enrichment step) and the variables sam-
pling plans. The chapter also discusses the weakness of the classical assumption
that the measure of the quality characteristic (i.e., log microbial concentration) is
normally distributed among food units with a variance that is approximately stable
batch to batch; and provides an insight to novel modelling trends to produce more
efficient and discriminatory sampling plans.

Keywords Microbiological criteria • Food safety objective • Microbial distribution

• Attributes • Variables • Operating characteristic curve • Within-batch •
Between-batch

1 Introduction

Foods are examined microbiologically so as to determine its microbial quality, veri-

fying if it meets established safety and quality standards, to investigate the cause of
its spoilage or the presence of pathogenic microorganisms. The presence of

U. Gonzales-Barron (*) • V. Cadavez

CIMO Mountain Research Centre, School of Agriculture (ESA) of the Polytechnic Institute
of Braganza (IPB), Bragança, Portugal
e-mail: [email protected]; [email protected]

© Springer Science+Business Media, LLC 2017 381

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_18
382 U. Gonzales-Barron and V. Cadavez

pathogens in foods needs to be controlled through a complete farm-to-fork approach

from primary production to consumption. In most food processes, there are critical
points where food can become a microbiological hazard. Once these critical points
are identified and controlled, the occurrence of pathogens in the food supply can be
monitored by the implementation of effective sampling plans. A well-designed sam-
pling plan can identify unacceptable batches of food in the supply chain, and actions
can be then taken to reduce the risk.
Both industry and government use a range of food safety tools to ensure the pro-
duction of microbiologically-safe products. At the governmental level, food safety
control for public health protection covers the range of different food chains relevant
to a certain food product, including all pertinent stakeholders within the country as
well as those importing into the country. The use of risk analysis is advocated as the
single framework for building food safety control programmes. Microbiological
Risk Assessment (MRA) is a tool used by risk managers in (inter)national govern-
mental bodies, which provides a clear understanding of the food safety issues related
to the particular pathogen from farm to fork by considering all relevant production
steps and current control measures. Based on the outcome of an MRA, a risk man-
ager can determine whether the expected risks are acceptable or not, given the ability
to implement certain mitigation options. In cases where specific risk mitigation
options need to be adopted to reduce the burden of disease, risk managers may
choose to set health protection goals, such as the “Appropriate Level of Protection”
(ALOP). An ALOP expresses, on a population level, what level of risk a society is
prepared to tolerate or is considering to be achievable (Gorris 2005). Nonetheless,
given the difficulty of using public health goals such as the ALOP to establish control
measures at the industry level, the risk-based concepts of food safety objectives
(FSOs: the maximum frequency and/or concentration of a microbiological hazard in
a food at the time of consumption) and performance objectives (POs: the maximum
frequency and/or concentration of a microbiological hazard in a food at any point in
the food chain) were introduced to provide meaningful guidance to food safety man-
agement in practice (Fig. 18.1). Within the current MRA framework, it is proposed
that, when appropriate, competent authorities can formulate a so-called FSO. It is
evident that specific targets (POs) need to be selected in the food chain that can be
linked directly to improvements in public health, such that public health goals begin
to drive the performance requirements of the food safety management chain.
At the industry operational level, the FSO and PO performance requirements
are regulated through the control measures operated by the GAP, GHP, GMP and
HACCP systems at diverse points of the food chain (Fig. 18.1). At a particular
step in the chain, one or more control measures can be implemented as part of the
product and process design to control a hazard (Gorris 2005). The performance
criterion (PC) describes the overall effect of the control measures on the hazard
level at a step, and is in general decided on by food safety managers as key points
in the design of the production of a food in a supply chain. Said otherwise, PCs
are the specific operational, supply chain measures at (a) specific step(s) that
result in meeting the objective for that step, the PO (Fig. 18.1).
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 383

National level Operational food-chain level

Primary Control
production PC measures
PO
(GAP)

PCs Control
Manufacture measures
PO (GMP, GHP,
Sampling and HACCP)
compliance
(MC)
Retail
PO

Preparation Control
PC measure
FSO
(cooking, etc)

Public health Consumption

goal (ALOP)

Fig. 18.1 Schematic diagram of the positions of the risk-based metrics at national level and operational
level along the food chain. The control measures are operated in the chain by GAP, GHP, GMP, HACCP

Currently, the link between public health goal (i.e., governmental level) and
microbiological targets (i.e., industry level)—in some cases ascertained by
microbial risk assessment models—does not exist for all food commodities.
Although PO and FSO are not intended to be enforced, they provide the indus-
try with quantitative targets to be met. These targets could lend themselves to
be verified by specific sampling and microbiological testing at the same time
that industry validates that its food safety system is capable of controlling the
hazard of concern (i.e., to provide evidence that control measures can meet the
targets). In order to harmonize and standardize microbiological testing,
European regulators established in the Commission Regulation N° 1441/2007
(Anonymous 2007) the microbiological criteria (MC) for foodstuffs. To assess
compliance with FSOs and POs, control authorities also rely on inspection
procedures to verify the adequacy of control measures adopted by industry
(Van Schothorst et al. 2009). During food production, within-­batch testing
compares the level of a microbiological hazard detected in food samples
against a pre-specified limit or microbiological limit, and can be accomplished
to comply with MC (standards written into law regulations; Fig. 18.1) or to
384 U. Gonzales-Barron and V. Cadavez

assess that the food production process is under control (i.e., process control
used voluntarily by producers as part of their quality control systems)
(Gonzales-Barron et al. 2013).
This chapter describes the theoretical considerations for the design of microbiological
sampling plans used in the food industry to meet a predefined level of safety, a perfor-
mance objective or a food safety objective. The statistical basis and assumptions of the
different sampling plans, namely, the classical two-class attributes sampling plan, the
two-class with enrichment and the variables sampling plan, are explained. Examples
illustrating the construction of operating characteristic curves are provided for each of
the types of sampling plans. Finally, the chapter addresses the weakness of the traditional
statistical assumptions, and discusses, in brief, the new modelling trends for the deriva-
tion of more efficient sampling plans.

2 Types and Components of a Microbiological Criterion

A microbiological criterion (MC; Fig. 18.1) is a set of parameters used to define the
microbiological quality of raw materials, food ingredients and end-products at any stage
in the food chain, or can be used to evaluate or compare the stringency of alternative
food control systems and product and process requirements. The establishment of an
MC is useful for verifying that food control systems are implemented correctly. Based
on regulatory consequences, three classes of MC are distinguished: standards, specifi-
cations and guidelines. ‘Standards’ are mandatory MCs that are written into law or
government regulations. ‘Specifications’ are MCs established between buyers and pro-
ducers that define product quality and safety attributes required. ‘Guidelines’ are MCs
that provide advice to industry about acceptable or expected microbial levels when the
food production process is under control. They are used by producers, to base their own
processes, and by government inspectors when conducting audits (ICMSF 2012).
Alternatively, the MC can be broadly categorised as GHP-based, hazard-based or
risk-based, with basis on the knowledge by which it is derived:
1. GHP-based MCs are developed from empirical scientific knowledge and experi-
ence, and are related to food hygiene. They are used to verify that hygiene condi-
tions have been applied.
2. Hazard-based MCs are developed from scientific knowledge of a likely level of
control of a microbiological hazard at a step or series of steps in a food chain and
can be validated as to their efficacy in hazard control. There is an expectation of
consumer protection but the actual degree of protection will be unknown. They
are for example used for the verification of the performance of HACCP systems
and for lot-by-lot acceptance.
3. Risk-based MCs are developed from risk assessment or specific knowledge of
the likely levels of consumer protection that will result. For example, they can be
used for verification that a PO has been met or by the use of an appropriate risk
assessment model to show that the ALOP has been achieved.
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 385

However, an MC should be established only when there is a need and when it can
be shown to be effective and practical for the stated purpose. Any MC should include
the ten following components:
1. the microorganism, toxins or metabolite and the reason for selection;
2. the food, process or environment to where the criterion applies;
3. a sampling plan defining the number of samples to be taken and the size of the
analytical unit;
4. the analytical method to be used to detect and/or quantify the microorganism(s)
or their toxins/metabolites;
5. the specific point(s) in the food chain where the MC should be applied;
6. the microbiological limit(s) considered appropriate to the food at the specified
point(s) of the food chain;
7. the sampling plan defining the number and size of samples to be taken;
8. the number of samples that should conform to the microbiological limit(s);
9. any indication of the statistical performance of the sampling plan; and
10. the actions to be taken when the criterion is not met.

3 Types of Sampling Plans Used for Microbiological Control

Within the risk analysis context, MCs are meant to provide a statistically-sound
means for determining whether the FSO/PO targets are being achieved. The MC for
foodstuffs, laid down in Regulation EC No 1441 (Anonymous 2007), comprises two
types of sampling plans, which are classified as by attributes and by variables in
classical acceptance sampling theory (Duncan 1986). The parameters defining each
of these types of sampling plans within the context of microbiological criteria are
presented in Table 18.1.
As in any sampling plan, two types of errors can result from decisions based on
the results from the test samples. Thus, the design of a sampling plan needs to con-
sider either or both of the following errors in order to reduce risks to a minimum, yet
without the need for excessive sampling:
1. the probability that an ‘acceptable batch’ will be rejected by the sampling scheme
(Type I or α error). This error is also called producer’s risk as it may bring about
economic losses.
2. the probability that a ‘bad batch’ is accepted (Type II or β error). This error is
also called consumer’s risk as ultimately it may involve adverse health effects.
A simple way to decide whether to accept or reject a food batch may be based
on some microbiological test performed on several sample units. For pathogens,
this will usually be a test for the presence (positive) or absence (negative) of the
microorganism. If instead concentrations of microorganisms are measured, the
result of the individual sample can be assigned to a particular attribute class by
determining whether it is above (positive) or below (negative) some preset con-
centration. The decision-making process of a two-class attributes sampling plan
386 U. Gonzales-Barron and V. Cadavez

Table 18.1 Types of acceptance sampling plans currently used to express microbiological
criteria in foods
Sampling plan Parameters Definition Uses
By attributes
a) Two-class c Maximum number of sample Stringent sampling plans to
units giving microbial assess presence of pathogens
concentrations over m for the in foods. Unsatisfactory
batch to be ‘accepted’ results should lead to batch
n Number of samples to test or rejection
sample size
m Microbiological limit, commonly
expressed as absence in certain
sample weight
b) Three-­class c Maximum number of sampling Less stringent sampling
units giving microbial plans to assess hygiene
concentrations between m and M indicators. Unsatisfactory
and still allows the batch to be results should lead to
‘accepted’ improvements in production
n Sample size hygiene
m Lower microbiological limit of an
individual sample
M Upper microbiological limit of an
individual sample. If any sample
gives results exceeding M, the
batch is rejected
By variables
n Sample size Sampling plans more
m Lower microbiological limit. If informative than by
the sample’s average is below m, attributes but of limited use.
the batch is considered of Unsatisfactory results
satisfactory hygiene should lead to improvements
M Upper microbiological limit. If in production hygiene
the sample’s average falls
between m and M, the batch is
considered of acceptable hygiene.
Beyond M, the batch is
unsatisfactory

is essentially defined by two numbers. The first, denoted as n, determines the

number of sample units that have to be draw independently and randomly from
the batch. The second number, denoted as c, is the maximum allowable number
of defective sample units. In microbiological testing, the term defective implies
that the individual sample contains more than the specified number of microor-
ganisms m, when tested by an appropriate test), or in the case of a presence/
absence test, that the target m
­ icroorganism is detected. In the former case of
grouped quantitative data, there is one microbiological limit, denoted by m,
which separates good quality from defective quality. In this case, the maximum
allowable number of sampling units exceeding this limit is given by c, which is
usually set to zero for pathogens (Jarvis 2008). Two-class attributes sampling
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 387

plans are used for more stringent microbiological criteria testing pathogens or
toxins in foods, and the microbiological limit m is commonly expressed as
‘absence in a certain sample weight’ (for instance, the safety criterion for milk
powder is based on absence of Salmonella in 25 g (m) in none of the five indi-
vidual samples taken (c = 0, n = 5)).
In classical acceptance sampling, the two-class attributes sampling plan used in
microbiological criteria in foods belongs to the category of single sampling plans by
attributes. In double and multiple sampling plans, an additional chance is given to a
questionable batch. In double sampling, if the results of the first sample are not defi-
nite in leading to acceptance or rejection, a second sample is taken which then leads
to a decision on the outcome of the batch. A natural extension of double sampling is
to allow further additional samples to be taken to achieve even more discrimination
in the disposition of the batch. Such procedures are called multiple sampling plans.
In situations where decisions are not based on results of presence-absence tests
but on quantitative analytical results, three-class attributes sampling plans can be
applied as an alternative to two-class plans working with data grouped according to
a single microbiological limit m. In a three-class sampling plan, the quality of food
batches can be divided in three groups. The marginally defective sample is defined
as one that contains a number of microorganisms lower than a specified upper limit
M but a greater number than a lower (acceptable) specified limit m. However, sam-
ple results above the upper concentration M are unacceptable (or defective), and
usually a batch is rejected if the test from any sample unit exceeds M (Dahms 2004).
Jarvis (2008) argues that a marginally defective grouping may be considered in
order to make some allowance for variation in the distribution of microorganisms in
the food, and for the imprecision associated with colony count procedures.
In a sampling plan by variables, microbiological measurements are made on a
series of samples taken from a batch. A batch is considered of acceptable quality
when the mean value of the microbial counts from the individual samples does not
exceed a microbiological limit m. As such plans make full use of microbial counts,
rather than ascribing them to categories or classes, variables plans can be more use-
ful under some conditions than attributes plans. The design of a variables sampling
plan involves several decisions to be made – such as the knowledge or assumption
of the underlying distribution of microbial concentrations within a batch; and
although formulation of the decision rule (n, m) may be more complex than for
attributes sampling plans, this procedure has the advantages of using a smaller sam-
ple size and that the definition of a conforming batch and the desired confidence in
decision making become more transparent (Dahms 2004).

4 Classical Acceptance Sampling Theory

‘Operating characteristic’ (OC) curves are used as an important tool to measure the
performance of a sampling plan allowing the assessment of the relative efficiencies
of various potential sampling plans. The construction of OC curves for both attri-
butes and variables sampling plans have been traditionally based on classical
388 U. Gonzales-Barron and V. Cadavez

1,0
α=0.10

Probability batch acceptance (Pa)

0,8

0,6

0,4

0,2

β=0.05
0,0
0,0001 AQL LQL1 LQL2 LQL3 100

Within-batch mean concentration, µ (CFU/g)

Fig. 18.2 Operating characteristic (OC) curves showing the points of interest defining a sampling
plan: the acceptable quality level (AQL) at a producer’s risk α and the limiting quality level (LQL)
at a consumer’s risk β. Sampling plans of greater discriminatory power producer steeper OC curves

acceptance sampling theory (Duncan 1986), and is a graphical representation that

relates the probability of accepting a batch, based on the number of food units
tested, to the mean quality of the batch (Fig. 18.2).
In attributes sampling plans (i.e., each unit in the sample is judged to be either
conforming or nonconforming), the OC curve plots the cumulative probability of
acceptance (Pa) conditional to the percentage of defective sample units in a batch
(the batch fraction defective). In classical acceptance sampling theory, two types
of OC curves are recognized (Schilling and Neubauer 2009): Type A, whereby
samples are taken from an individual batch, and the size of the batch N is small
compared to the sample size n; and a Type B, whereby samples are taken from a
process, and the size of the batch N is much larger than the sample size n. However,
in food microbiology, OC curves for attributes sampling plans have been tradition-
ally built on the assumption of an infinite lot or process (Type B). Moreover, they
have been produced converting the proportion defective of a batch on the x-axis to
the mean quality characteristic; this is, expressing prevalence as an estimate of the
mean concentration of microorganisms in the batch, by assuming that the underly-
ing statistical distribution of a microorganism in a batch is lognormal (Hildebrandt
et al. 1995; Dahms and Hildebrandt 1998; Legan et al. 2001; Dahms 2004; Van
Schothorst et al. 2009). In a variables sampling plan (i.e., the quality characteristic
of each unit in a sample is measured), the OC curve similarly displays the dis-
criminatory power of a sampling plan with Pa plotted against the mean microbial
concentration of the batch, nevertheless it is constructed in a different way. While
the primary advantage of variables sampling plans is that the same OC curve can
be obtained with a smaller sampling size than would be required by an attributes
sampling plans, its use as a basis for microbiological criteria is very limited,
almost certainly due to the limited research in this field.
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 389

In an OC curve belonging to either an attributes sampling plan or a variables

sampling plan (Fig. 18.2), two points of interest can be identified: (AQL, 1−α)
and (LQL, β). The acceptable quality level (AQL) defines the quality (mean
microbial concentration) of good batches that the purchaser/consumer is prepared
to accept most of the time (set at the probability P1). Hence, the producer’s risk
(type I error or α) of rejecting good quality batches is at 1−P1. The producers are
interested in determining the batch quality that would need to be achieved so that
there is a high probability that batches would be accepted and adjust their produc-
tion processes accordingly (Van Schothorst et al. 2009). The limiting quality level
(LQL) point denotes the quality of poor batches that the purchaser/consumer
wishes to reject as often as possible (at the probability P2). Hence, the consumer’s
risk (type II error or β) of accepting bad quality batches is 1−P2. It is assumed that
the producer is operating at a fall-out level that is considerably better than the
LQL. Specifications of either the AQL or LQL points and their probabilities of
acceptance make it possible to design sampling plans; and setting either the AQL
or the LQL implies the other. It is customary to define the producer’s risk as 0.05
or 0.10, indicating that a batch having a minimum mean quality AQL should be
accepted with a probability of at least 95% or 90%. The consumer’s risk is mostly
defined as 0.05, indicating that poor quality batches of mean LQL or worse should
be rejected with a probability of at least 95%.
OC curves can be used to evaluate the influence that parameters of the MC (n, m, c),
and the mean and standard deviation of the underlying batch distribution have on the
efficiency of the microbiological testing programme. An OC curve allows quantifying
the confidence that we can have that an unacceptable or defective batch will be rejected.
If one were to able to test every unit of food within the batch, the OC curve would
change from 100% probability of acceptance to a 100% probability of rejection exactly
at the quality level that distinguishes an acceptable from a defective batch. At the other
extreme, taking a single sample, particularly if negative, has virtually no ability to
discriminate between conforming and non-conforming batches. Increasing the number
of samples (n) examined is one of the primary means for increasing the ability of a
sampling plan to discriminate acceptable from defective batches. A higher sample size
will produce a steeper OC curve, indicating a higher discriminatory power of the sam-
pling plan. For the specific case shown in Fig. 18.2, notice that the higher the sample
size, the greater the discriminatory power, and the lower the LQL value.

4.1  esign of Two-Class Attribute Sampling Plans of the Types

D
(n, c), (n, c, m) and (n, c, absence in w)
4.1.1 Based on Microbial Prevalence (n, c)

In its simplest form, an OC curve can be constructed based on the true within-batch
prevalence (d) of the microorganism as the quality measure. In that case, the prob-
ability of accepting a batch (Pa) after sampling with a regime (n, c) reduces to a
cumulative binomial expression.
390 U. Gonzales-Barron and V. Cadavez

1
n=30

Probability batch acceptance (Pa)

n=20
0,8
n=10
n=5
0,6

0,4

0,2

0
0,00 0,04 0,08 0,12 0,16 0,20
True prevalence of target microorganism (d)

Fig. 18.3 Operating characteristic curves for two-class attributes sampling plans (n, c) based on
prevalence for different sample size n.

c
n
Pa = P ( x ≤ c ) = ∑   d c (1 − d )
n−c
(18.1)
c
C =0  

Recall that any result that can be classified on the basis of presence or absence is
governed by the binomial distribution; hence, the stringency of the sampling plan
will be increased if n is large, but no matter how many sample units are tested with
negative results, it can never be stated unequivocally that the batch is totally free
from the target bacterium. Yet, confidence limits can be established on the ‘zero’
result (Jarvis 2007). In stringent sampling plans, normally there is no allowance for
positive samples (i.e., the value of c is set to zero); and hence Pa becomes,

Pa = P ( x = 0 ) = (1 − d )
n
(18.2)

The OC curve is then simply the plot of the probability of accepting a batch (Pa
on the y-axis) of a given microbial prevalence (d on the x-axis) under the sampling
plan (c = 0, n). Observing the OC curves of Fig. 18.3, we can see that, if negative
results are obtained on 30 samples, and the true prevalence is 2%, the likelihood of
accepting the batch is 55% (conversely, the probability of rejection is 45%), but if
only five samples were tested, the probability of accepting the batch is very high at
90%. Likewise, if the batch contained 10% true defective units, then the likelihood
of product acceptance after testing ten sample units with negative results will be only
35% but if five samples were tested, the probability of acceptance is higher at 59%.
In setting a criterion for the number of samples to be tested, an assumption is
made about the true prevalence that is acceptable. For instance, a sampling plan
n = 30, c = 0 for Cronobacter spp. powdered infant formulae has an AQL of
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 391

d = 0.095 at a β risk of 5% (Fig. 18.3). This means that such sampling plan would
give a confidence of at least 95% of rejecting batches that have 9.5% or more defec-
tive units. For any given true prevalence in a batch, and assuming that the distribu-
tion of contaminated units in the batch is random, we can determine the sample size
that would be necessary for attaining a given confidence Pa.

log ( Pa )
n= (18.3)
log (1 − d )

Using the same binomial expression (Eq. (18.2)), it is also possible to estimate
confidence limits for the true prevalence of contamination at a given confidence
level (1−α), only when all sample units test negative (Jarvis 2007). The confidence
limits are bounded by 0 and du, where du is chosen so that if the true prevalence were
du, then the probability of the observation of no positive results P(x = 0) in n samples
is equal to α. Thus, rearranging Eq. (18.2),

du = 1 − n α (18.4)

For example, if no positive samples were found in a sample size n = 15, we can
estimate that the true prevalence of contamination lies within 0 and 0.18 (du) with a
95% confidence (α = 0.05).
From the estimate of the upper confidence limit of the true prevalence, it is also
possible to approximate an upper confidence limit for the mean microbial concen-
tration (μ) in the batch tested. For this, we must presume that the microbial cells are
randomly distributed among food units, and that the test procedure is sufficiently
sensitive to detect the microorganism if present. The probability P(x = 0) of a nega-
tive result on any sample is given by the Poisson probability of finding zero
microorganisms,

P ( x = 0 ) = exp ( −wµ ) (18.5)

where w is the sample unit’s weight. Thus, following the previous example, if we
tested 10 samples of w = 25 g with negative results (P(x = 0) = 1—du = 1–0.18 = ­0.
82), the upper limit of the mean microbial concentration estimated by Eq. (18.5)
would be 0.0079 cell/g; meaning that we have a 95% confidence that the mean
microbial concentration lies within 0–8 cells/kg.
Example 1
Suppose that you need to design a sampling plan to test Salmonella spp. in frozen
turkey, where a PO has been formulated as proportion of defectives in a batch, as: “not
more than 20% turkey carcasses in a batch may test positive for Salmonella”, and that
the confidence that a defective batch is rejected is set at 95% probability. Replacing in
Eq. (18.3), the true microbial prevalence d by 0.20 and the probability of accepting the
batch Pa by 0.05, the sample size n equals 13.4. Thus, a batch of frozen turkey will be
392 U. Gonzales-Barron and V. Cadavez

compliant to the PO if none of the 14 samples randomly withdrawn tests positive for
Salmonella. Considering that the analytical sample unit (w) is 5 g of neck skin, we can
further say that 95% of the times that a batch is labelled as compliant, the mean
Salmonella concentration (μ) in frozen turkey within a batch should not be higher than
0.044 CFU/g (Eq. (18.5)).

4.1.2 Based on Microbial Concentration (n, c, m)

When the result of a microbiological analysis is given in a quantitative manner (for

instance CFU/g or MPN/g), the batch quality may be then described by a statistical
distribution of the concentration of microorganisms among the food units produced
in a batch (instead of the less-informative true prevalence measure). Although a
sampling plan of the type (n, c, m) is still an attributes sampling plan, it is related to
microbial concentrations by using a statistical distribution of microorganisms to
establish the proportion of defective samples.
For this, an assumption must firstly be made regarding the statistical distribution of
the microorganisms in a batch (i.e., this is also referred to as within-batch distribution
of variability in contamination). Traditionally, and as a matter of convenience, the
normal distribution (of the logarithm of the microbial concentrations) has been
assumed, or said otherwise, the lognormal distribution of the microbial concentra-
tions. However, as we will discuss later, the assumption of normality is more valid for
highly contaminated batches than for low contamination levels (Gonzales-Barron and
Butler 2011a). When little or no data are available, a normal distribution of log CFU
is often assumed and a default value for the standard deviation applied. Standard devi-
ation values of 0.2 log CFU/g to describe a rather homogeneous spread of microbes in
food within a batch (e.g., for liquid food with a high degree of mixing); 0.4 log CFU/g
for food of intermediate homogeneity (e.g., ground beef); and 0.8 log CFU/g for an
inhomogeneous food (e.g., solid food) have been suggested by Van Schothorst et al.
(2009). Higher standard deviation values may be used for greater inhomogeneity
although, in such cases, it is rather advisable to consider another family of distribu-
tions such as the heterogeneous Poisson which characterise far better high microbial
clustering and high proportion of zero counts. (examples of fitting mixed distributions
to microbial load data and deriving ­sampling plans under such assumptions can be
found in Gonzales-Barron et al. (2010, 2013) and Mussida et al. (2013).
Secondly, we need to know the detection limit of the analytical procedure as its
value is normally assigned to the microbiological limit m; which is a measure that
separates a conforming sample unit (microorganisms non-detected by the microbio-
logical protocol) from a non-conforming sample unit (microorganism detected)
(Fig. 18.4). For instance, a plate counting technique able to quantify Listeria monocy-
togenes above 1 CFU/g has a m = 0 log CFU/g, while a 1/0.1/0.01 g three-tube MPN
procedure with a lower limit of detection of 3 CFU/g has a m = 0.48 log CFU/g. If the
analytical procedure was sensitive to extremely low levels of contamination, batches
that should be accepted would have samples test positive and be erroneously rejected.
Whiting et al. (2006) argues that the sensitivity of the analytical procedure must be
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 393

m
0,6

0,5
Probability density
0,4

0,3

0,2

0,1
pa pd
0,0
0 1 2 3 4 5 6

Microbial concentration (log CFU/g)

Fig. 18.4 Proportion of acceptable food units (pa) and non-conforming food units (pd) in a two-­
class sampling plan for a within-batch distribution of microbial concentrations described by a
Normal (3.0, 0.8) [log CFU/g]

positioned at an appropriate level so that a set of samples from a batch of food has a
high probability of at least one sample being positive (c = 0, 95% confidence).
Assuming that bacteria are log-normally distributed in the product and that the stan-
dard deviation is known, an OC curve can then be established by using the statistical
distribution of microbial concentration to calculate the proportion of acceptable (pa)
and defective (pd) food units in a batch (Hildebrandt et al. 1995; Legan et al. 2001;
Dahms 2004). The probability of extracting one defective sample (pd) is then used to
determine the probability of accepting the batch after testing (Pa) in the usual way by
a cumulative binomial distribution (Eq. (18.1)). For a given within-­batch microbial
mean concentration μ, the area under the probability density function above the micro-
biological limit m is used the define the value of proportion defective (pd) for a two-
class sampling plan (Fig. 18.4). The value of pd is calculated as 1- pa, where pa is the
cumulative probability of the normal distribution (μ, σ) evaluated at m. The proportion
of acceptable units pa can be calculated in Microsoft® Excel using the “Normdist” func-
tion, pa = Normdist (m, μ, σ, 1). The next step is then to calculate the probability of
accepting the batch (Pa) for the normal distribution (μ, σ) of microbial concentrations
after the sampling plan (n, c, m), assuming a binomial process where pd becomes the
probability of success (i.e., finding a positive sample). The value of Pa can be com-
puted using Eq. (18.1), replacing d by pd, or as Pa = Binomdist (pd, n, c, 1) in Microsoft®
Excel. This procedure, repeated for a range of mean microbial values μ (i.e., a range of
normal distributions with constant σ), will yield different Pa values, which coupled will
produce the OC curve of the sampling plan (n, c, m). Notice that in this way OC curves
in terms of mean concentrations are developed by fixing the standard deviation σ, and
then increasing the mean of the normal distribution through a range of values. This
implies that σ is assumed invariable for a low or highly contaminated batch.
394 U. Gonzales-Barron and V. Cadavez

1,0

Probability batch acceptance (Pa)

0,9 n=5
0,8 n=10
0,7 n=15

0,6
0,5
0,4
0,3
0,2
0,1
0,0
-2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0
Mean microbial concentration (log CFU/g)

Fig. 18.5 Operating characteristic curves for two-class attributes sampling plans of microbiologi-
cal limit m = 0 log CFU/g and different sampling size n, assuming a within-batch lognormal distri-
bution of microbial counts with standard deviation σ = 0.6 log CFU/g

From the OC curve of sampling plan of the type (n, c, m), two important measures
can be determined: the mean microbial concentration at which the batch will be
accepted with 95% probability (AQL) and the mean concentration at which the batch
will be rejected with 95% probability (LQL). Reading off the AQL and LQL values
from an OC curve may be imprecise on steeply sloping curves. The ‘goal seek’ fea-
ture of Microsoft® Excel may be used as a more precise alternative to find the mean
microbial concentrations that correspond to Pa = 0.95 and Pa = 0.05, respectively.
Figure 18.5 illustrates OC curves derived for three sampling plans with microbio-
logical limit 1 CFU/g (m = 0 log CFU/g) and different sample size n. They were built
assuming that σ = 0.6 log CFU/g. Compliance with a sampling plan n = 5, c = 0,
m = 1 CFU/g) ensures that batches with mean contamination equal or higher than
−0.058 log CFU/g (LQL = 0.87 CFU/g) have at least 95% probability of being
rejected. Batches with lower levels of contamination would have lower chance of
being rejected and greater chance of being accepted (Fig. 18.5). For comparison, a
sample size n = 15 (producing a LQL = −0.55 log CFU/g) ensures that now batches
of lower contamination level (LQL = 0.28 CFU/g) will have at least 95% chance of
being rejected (Fig. 18.5). Hence, as explained earlier, a greater sample size leads to
a more discriminatory and stringent sampling plan, which is characterised by a
steeper OC curve. Finally, notice in Fig. 18.5 that an increased sample size has a
greater effect on the LQL value than on the AQL value.
The performance of an attributes sampling plan is also dependent on the validity
of the assumed standard deviation σ. Errors in σ can greatly affect the target AQL
value at which the industry intends to operate (Fig. 18.6). For instance, if the hetero-
geneity of the microorganisms among food units was underestimated by assuming
σ = 0.6 instead of an ‘actual but unknown’ 1.0, the sampling plan (n = 5, c = 0,
m = 1.0 log CFU/g) would wrongly lead the producer to target its maximum mean
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 395

1,0

0,9 σ=0.6

Probability batch acceptance (Pa)

0,8 σ=0.8
σ=1.0
0,7

0,6

0,5

0,4

0,3

0,2

0,1

0,0
-2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0
Mean microbial concentration (log CFU/g)

Fig. 18.6 Effect of the within-batch standard deviation (σ) on the performance of the sampling
plan n = 5, c = 0, m = 1.0 log CFU/g

microbial concentration in a batch (AQL) to −0.40 log CFU/g (instead of −1.3 log
CFU/g for a σ = 1.0). At −0.40 log CFU/g, the producer would expect that 95% of
the batches will be positively accepted, when in fact, that quality level will only
provide 66% confidence (Fig. 18.6). The producer should instead aim for a mini-
mum quality level (AQL) of −1.30 log CFU/g. Thus, a higher standard deviation
means that the mean microbial concentration that must be achieved so as to not
exceed the PO/FSO must be decreased; and that a greater sample size should be
tested to assess compliance to a given safety target. Whiting et al. (2006) indicates
that taking a larger sample, homogenising or compositing samples may reduce the
variation between samples within the batch, particularly in non-homogeneous
foods. This may reduce the standard deviation of the samples and will allow a shift
in the distribution of the LQL batch to a higher batch mean and m value which may
be easier to detect. For non-homogeneous or low levels of contamination, modifica-
tions in the sampling and sample manipulation can reduce the variation. Nonetheless,
if past monitoring microbial data were available in the industry, the standard devia-
tion can be approached with more certainty, and attributes sampling plans can still
be used to assess mean microbial concentrations in batches of food.
Example 2
When designing a sampling plan from knowledge of the FSO/PO in terms of maxi-
mum microbial concentration, a second requirement is the proportion (95%, 99%,
99.9%, etc) of the distribution of possible concentrations that must satisfy the test
limit so that FSO/PO is met. Suppose that we wish to derive a sampling plan to
ensure that Listeria monocytogenes levels in a fermented meat product do not
exceed the maximum concentration at consumption (FSO) of 2.0 log CFU/g.
Assuming that from the point of manufacture to the point of consumption, the
396 U. Gonzales-Barron and V. Cadavez

m = -0.52 PO = 1.40
0,6

0,5
LQL ~ N(-0.46, 0.8)

0,4
Probability

0,3

0,2

0,1
pa pd 1%

0,0
-3 -2 -1 0 1 2 3
Microbial concentration (log CFU/g)

Fig. 18.7 Derivation of an LQL distribution of Listeria monocytogenes in a fermented meat product
from a hypothetical PO of 1.40 log CFU/g that should be met in 99% of the food units in the batch

microbial concentration may increase in up to 0.6 log CFU/g when stored at 5 °C;
the required PO would then be 1.4 (2.0–0.6) log CFU/g. Furthermore, to ensure that
the PO would be met by 99% of the food units in such “limiting quality” batch, the
mean concentration of the LQL batch should be 2.33 × σ below the calculated PO
(Fig. 18.7). Thus, assuming a within-batch standard deviation of σ = 0.8 for this
heterogeneous food product, the mean microbial concentration of the LQL batch is
1.4–2.33 × 0.8 = −0.46 log CFU/g. Batches with contamination levels higher than
LQL = Normal (−0.46, −0.8) should be rejected at least 95% of the times (Pa = 0.05).
If the three-tube MPN analytical procedure has a limit of quantification of 0.3
Listeria monocytogenes/g, the m value can be set at −0.52 log MPN/g. Then, the
next step is to calculate the proportion pa of acceptable food units in the LQL batch,
which is estimated as the cumulative probability evaluated at m. In Microsoft® Excel
notation, this is pa = Normdist (m, μ, σ,1) = Normdist (−0.52, −0.46, 0.8, 1) = 0.470.
The value of pd will then be 0.53. Replacing in Eq. (18.3), where now d = pd, n = log
Pa/(log(1-pd)) = 3.9 ~ 4. Hence, a sampling plan (n = 4, c = 0, m = −0.52 log CFU/g)
should ensure that whenever the microbial concentration in a batch exceeds the PO,
the batch will be rejected with at least 95% confidence.

4.1.3 Based on Microbial Concentration with an Enrichment Step (n, c, w)

The attributes sampling plan of the type (n, c, w) is applied when a specified number
of analytical units are cultured via enrichment and then assessed for presence/
absence rather than enumeration (Van Schothorst et al. 2009). If microbial cells
were completely evenly distributed in the samples and were present at the level of
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 397

one cell per sample unit, one would not expect every sample to be positive for
growth: some samples selected at random would contain one or more cells, and
produce a positive result, while others not. Thus, when testing absence of a micro-
organism in an analytical unit (i.e., m = absence of Salmonella in 25 g), it is neces-
sary to consider the consequences of sampling coincidences (i.e., the detection of a
cell in a set of samples even when such detection is highly improbable based on the
mean concentration of the organism in the batch) on the interpretation of the results
of analytical methods. The probability of detecting cells (Pdetect), by randomly sam-
pling from a well-mixed system can be described by a Poisson distribution, as

Pdet ect = P ( x > 0 ) = 1 − exp ( −wC ) (18.6)

This means that, if the concentration of the cells in the sample (C) is perfectly
homogeneous, our sampling and enumeration method will sometimes over-estimate
or under-estimate the concentration. As long as the distribution of microbial con-
centrations is known, it is possible to estimate the overall probability of detecting a
cell from any sample drawn from a batch. This is because the overall probability of
obtaining a positive sample (pd) is the product of the probability of that concentra-
tion occurring in the batch, and the probability of detecting a cell in the sample
based on the weight of the analytical unit (w) and the concentration of cells in the
sample (C). If the distribution of microbial concentrations can be assumed to be
lognormal, the probability of sampling any particular concentration is given by the
lognormal distribution, and is combined with the Poisson sampling process to cal-
culate the probability that a cell is present in the sample. Mathematically, this is
expressed by the (mixed) Poisson-lognormal distribution,

∞
pd = ∫ Pnormal ( log C ,,µ ,,σ )•Pdet ect d log C
−∞
∞
= ∫ Pnormal ( log C ,,µ ,,σ )·(1 − exp ( −wC ) ) d log C (18.7)
−∞

After calculating the probability of extracting one defective sample (pd) from the
batch, the probability of accepting the batch (Pa) can be computed, as usual, from the
binomial process (Pa = Binomdist (pd, n, c, 1) in Microsoft® Excel notation). However,
the Poisson-lognormal distribution does not have an explicit form from which
pd = 1−P(x = 0) can be readily estimated (as in the Poisson distribution, Eq. (18.6));
yet, an easy way to solve for Eq. (18.7) is through Monte Carlo simulation. Table 18.2
represents a spreadsheet that uses @Risk software for the construction of OC curves
for attributes sampling plans with an enrichment step, to estimate by simulation pd
values for a range of mean microbial concentrations μ. Another way to construct OC
curves for the sampling plans with enrichment (Van Schothorst et al. 2009) can be
done by using a downloadable spreadsheet file developed by ICMSF (2009). This
application also constructs OC curves and assesses the performance of the simple
two-class and three-class attributes sampling plans proposed by Legan et al. (2001).
398 U. Gonzales-Barron and V. Cadavez

Example 3
Suppose that we wish to determine an appropriate sampling plan to control
Salmonella in frozen angel cake that is compliant with an hypothetical PO of −1.4
log CFU/g (0.04 CFU/g), and that, additionally, we are constraint to sample a maxi-
mum number of units of ten per batch. Fixing n = 10, the problem reduces to find
the optimal sample weight w. For being a well-mixed product, we can assume that
the concentration of Salmonella among frozen cakes produced in a batch follows a
lognormal distribution with standard deviation σ = 0.4. Thus, OC curves for differ-
ent sample weights (w) were derived by simulation using the spreadsheet shown in
Table 18.2 to ensure that a batch of frozen cakes in which more than 1% of the units
have a concentration higher than −1.4 log CFU/g (PO) would be rejected with 95%
confidence, we first need to calculate LQL. The mean concentration of the LQL
batch is LQL = −1.4–2.33 × 0.4 = −2.33 log CFU/g, and should be rejected with at
least 95% confidence (Pa = 0.05). Analysis of the OC curves constructed by simula-
tion (Fig. 18.8) indicates that a sample weight of 50 g is the most appropriate to
ensure this level of safety (i.e., notice that for this OC curve, the AQL for Pa = 0.05
is ~2.3 log CFU/g). Whilst a sample weight of 25 g will be insufficient to assure the
PO level of safety, the sample weight of 100 g will only lead to a lower (stricter)
PO. Moreover, the defined sampling plan (n = 10, c = 0, absence in w = 50 g)
demands that the producer should target its production at a safer level (AQL = −4.15
log CFU/g) so that good batches be accepted at least 95% of the times. Said other-
wise, the producer should aim to produce frozen cakes of mean Salmonella concen-
tration levels below −4.15 log CFU/g (< 7 CFU/100 kg).

4.2 Design of Variables Sampling Plans (n, m)

There are instances where sufficient historical information is available to characterise

well the distribution of contamination. In such cases, variables sampling plans may
be more cost-effective because all the information in the test results is used to support
decision making. Variables plans are generally more flexible than three-­class sam-
pling plans, have more discriminating power, and they respond generally in a more
consistent manner to the true microbial quality of the batch. A special advantage of
the variables sampling plans is that a clear distinction can be made between decisions
for safety/quality and GMP (Kilsby et al. 1979). Nevertheless, the variables sampling
plans are useful only when the distribution of the microbial concentrations within a
batch is well known. Ideally, the distribution is assumed to be lognormal, although a
variables sampling plan can be derived with basis on other distributions such as het-
erogeneous Poisson distributions (Gonzales-Barron et al. 2012).
To design a variables sampling plan, the first decision to make is to define what
makes a batch unacceptable. A batch of food is defined as unacceptable if more than
a proportion p0 of the units produced has microbial concentrations exceeding a criti-
cal concentration C (Fig. 18.9). It is at this stage that either safety (risk-based) or
GMP specifications are set. Based on these values and information on the expected
 18

Table 18.2 An overview of a spreadsheet with @Risk add-in used to construct by simulation operating characteristic curves of attributes sampling plans (n, c, w)
with enrichment based on the Poisson-lognormal distribution
Row A B C D E F G
1 Construction of an OC curve for a sampling plan of the type (n, c, w) based on the Poisson-lognormal distribution
2
3 INPUT VALUES Weight = 10 g
4 Sigma = 0.4 log CFU/g n= 8
5 c= 0
6
7 Mean log Normal Poisson flag pa pd Pa
8 −3.0 =RiskNormal(A8,$B$4) =RiskPoisson((10^B8)*$E$3) =IF(C8 = 0,1,0) =RiskMean(D8) =1-E8 =BINOMDIST
($E$5,$E$4,F8,1)
9 −2.9 −2.184243244 0 1 0.725 0.274 0.040
10 −2.8 −2.167406964 0 1 0.669 0.330 0.018
11 −2.7 −1.644919527 0 1 0.603 0.396 0.006
12 −2.6 −1.931329466 0 1 0.594 0.405 0.005
13 −2.5 −1.841656364 1 0 0.525 0.474 0.001
14 −2.4 −1.612998431 0 1 0.404 0.595 0.000
15 …
Statistical Derivation of Sampling Plans for Microbiological Testing of Foods
399
400 U. Gonzales-Barron and V. Cadavez

1,0

0,9
w = 25 g
w = 50 g
Probability batch acceptance (Pa)
0,8
w = 100 g
0,7

0,6

0,5

0,4

0,3

0,2

0,1

0,0
-5,0 -4,5 -4,0 -3,5 -3,0 -2,5 -2,0 -1,5 -1,0
Mean microbial concentration (log CFU/g)

Fig. 18.8 Effect of the sample weight (w) on the performance of a sampling plan testing absence
of Salmonella in n = 10 frozen cake samples, assuming a within-batch standard deviation σ = 0.4

standard deviation σ within batches, the mean concentration of the limiting quality
batch (LQL) can be found as LQL = C–z(p0) × σ (Fig. 18.9). The second decision is
to choose the maximum risk to accept a defective batch (β). This corresponds to the
1−β being the desired confidence to reject a defective batch (i.e., a batch that exceeds
the tolerance criterion defined by C and p0). Thus, a batch of mean concentration
higher than LQL must be rejected with ‘1−β’ confidence. On the basis of the sample
size n, it is necessary to decide whether the percentage of potential values beyond C
in a batch is likely to exceed the value p0. The problem consists then of finding the
decision rule m (maximum mean log of the samples or microbiological limit) and n
(sample size) using the prescriptions above. When acceptable batches are tested, the
mean of the samples is expected to be lower than the limit mean concentration m
derived from C and p0. Because the within-batch distribution of the log microbial
concentrations is assumed to be normal, the distribution of the samples’ mean will
also be a normal distribution with σ’ = σ/√n. As it is on this samples’ mean distribu-
tion that the limit m is measured, the values of m and n should be chosen so that,

  σ  
Pr  Normal  LQL,  > m = 1− β (18.8)
  n 

Since different (m, n) pairs can meet the consumer’s requirement above, the equal-
ity can only be solved by keeping one variable fixed (for instance, assuming a suit-
able m) and solving for the other (n), and vice versa. Thus, for a fixed sample size n,
m can be solved as m = LQL−z(β) × σ/√n. The whole procedure can be illustrated
by the following example: Suppose that a batch should be rejected with 95% confi-
dence (β = 0.05) if more than p0 = 10% of the food units exceed a critical concentra-
tion C = 2.5 log CFU/g. For the probabilities of p0 and β, the z values are z(p0) = 1.28
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 401

LQL C
0,6

0,5

Probability density
0,4

0,3

0,2

p0
0,1

0,0
0 1 2 3 4 5 6

Microbial concentration (log CFU/g)

Fig. 18.9 A normal distribution of log microbial concentrations characterising a limiting quality
batch of LQL mean, defined as unacceptable because a proportion of the units produced in a batch
equal or higher than p0 has microbial concentrations exceeding the critical concentration C

and z(β) = 1.64. Assuming that the standard deviation of the microbial concentrations
among units is σ = 0.6, the mean of the LQL batch can be calculated as, LQL = 2.5–
1.28 × 0.6 = 1.73. Thus, fixing the sample size to n = 5, the m ­ icrobiological limit
m = 1.73–1.64 × 0.6/√5 = 1.29 log CFU/g. If the mean log of the individual results
from the individual samples exceeds that m limit, the batch should be then rejected.
Other microbiological limits can be estimated for different sample sizes.
In cases, where there is also a known requirement on the side of the producer (i.e.,
the AQL mean concentration of a batch that should be accepted with ‘1-α’ confi-
dence), solving for the decision criterion (m, n) will only lead to one solution since
now two preconditions need to be satisfied (i.e., two equations and two variables),

  σ  
Pr  Normal  AQL,  < m = 1−α
  n 
  σ  
Pr  Normal  LQL,  > m = 1− β
  n  (18.9)
The construction of OC curves for the assessment of the performance of vari-
ables sampling plans is simpler than for attributes sampling plans. Assuming that
the log microbial concentrations among food units in a batch distribute as a normal
distribution (μ, σ), given a sampling plan (n, m), the probability of batch acceptance
Pa is determined as the cumulative probability of the sample’s mean distribution
below the microbiological limit m. In Microsoft® Excel notation, this probability is
calculated as Pa = Normdist (m, μ, σ/√n, 1). The OC curve is a plot of Pa values
calculated for a range of within-batch mean concentrations μ.
402 U. Gonzales-Barron and V. Cadavez

Example 4
Figure 18.10 presents OC curves constructed in this way for a fixed microbiological
limit m = 0.4 log CFU/g and three sample sizes n = 5, 8 and 12, assuming a constant
within-batch standard deviation σ = 1.0 log CFU/g. If the safety requirement was to
reject with 95% confidence (Pa = β = 0.05) batches having more than 10% (p0) of the
food units surpassing the contamination critical limit of 2.3 log CFU/g (C), the mean
of the LQL batch can be estimated as LQL = 2.3–1.28 × 1.0 = 1.02 log CFU/g. Then,
the problem is to find a decision criterion (m, n) that meets the condition (Eq. (18.8)),

  1.0  
Pr  Normal  1.02,  < m  = 0.05
  n 

or, in other words, to find an OC curve that contains or approximately passes the
LQL point (1.02 log CFU/g). This can be done by first keeping n or m fixed.
Figure 18.10 shows OC curves constructed for a fixed microbiological limit m = 0.4
log CFU/g and different sample sizes. It can be noticed that the higher the sample
size, the higher the discriminatory power of the sampling plan, and the lower the
misclassification errors (i.e., hence the steeper the curve). The OC curve for n = 5
indicates that such sample size will not be sufficient to achieve that degree of safety
as LQL at Pa = 0.05 is 1.16 log CFU/g. Similarly, a sample size n = 12 will lead to a
conservative sampling plan with a LQL = 0.86, that in the long run will reject batches
that still meet the safety criterion. The OC curve for n = 8 presents a LQL = 0.98,
which is very close to the safety requirement at a confidence of rejection of 95%.
Figure 18.11 shows OC curves derived by fixing the sample size n = 8, and attempt-
ing different m values. It shows that the higher the microbiological limit, the less
conservative the sampling plan. Once again, this exercise leads to the sampling plan
solution of m = 0.4 log CFU/g and n = 8. At this level of consumer’s protection, the
producer will seek to produce food batches of mean microbial concentrations lower
than 0.63 CFU/g (AQL = −0.2 log CFU/g in Figs. 18.10 and 18.11).

5 Recent Concerns

The design of new sampling plans should encompass the use of past monitoring micro-
bial data, the characterisation of the spatial clustering of bacteria, and the inclusion of the
between-batch variability in microbial concentrations. The statistical methods proposed
for the design of sampling plans to test microorganisms in foods have been entirely based
on quality control statistics (Duncan 1986); the reason as to why two simplifying assump-
tions have been traditionally adopted: that the log microbial concentrations among food
units produced in a batch can be represented by a normal distribution, and that its measure
of spread (i.e., the standard deviation for a normal distribution) is constant among batches
of production. Furthermore, from these assumptions, it is implicitly understood that the
normal distribution is adequate for any contamination level (i.e., low counts or high
counts), and that the measure of spread (i.e., within-batch standard deviation) is
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 403

1,0

Probability batch acceptance (Pa)

0,9
n=5
0,8 n=8
0,7 n = 12

0,6

0,5

0,4

0,3

0,2

0,1

0,0
-1,0 -0,6 -0,2 0,2 0,6 1,0 1,4 1,8
Mean microbial concentration (log CFU/g)

Fig. 18.10 Operating characteristic curves for variables sampling plans of microbiological limit
m = 0.4 log CFU/g, and different sample size n, assuming a within-batch lognormal distribution of
microbial counts with standard deviation σ = 1.0 log CFU/g

1,0

0,9
m = 0.2 log CFU/g
Probability batch acceptance (Pa)

m = 0.4 log CFU/g

0,8
m = 0.8 log CFU/g
0,7

0,6

0,5

0,4

0,3

0,2

0,1

0,0
-1,0 -0,6 -0,2 0,2 0,6 1,0 1,4 1,8
Mean microbial concentration (log CFU/g)

Fig. 18.11 Operating characteristic curves for variables sampling plans of sample size n = 8, and
different microbiological limits m, assuming a within-batch lognormal distribution of microbial
counts with standard deviation σ = 1.0 log CFU/g

independent of the contamination level. Nevertheless, recent work has demonstrated that
in many cases the normality assumption does not hold, specially when modelling low
microbial concentrations (Gonzales-Barron et al. 2010; Gonzales-Barron and Butler
2011a), and that the within-batch measure of spread is in fact associated with the within-
batch mean concentration (Fig. 18.12) (Gonzales-Barron and Butler 2011b;
404 U. Gonzales-Barron and V. Cadavez

1000
Enterobacteriaceae on

Within-batch variance (CFU/cm 2)2

sheep carcasses
100
E. coli on beef carcasses
10

0,1

0,01

0,001
0,001 0,01 0,1 1 10 100
Within-batch mean (CFU/cm2)

Fig. 18.12 Association between mean and variance estimated from different batches for two
microbial data sets

Gonzales-Barron et al. 2012). It is highly likely that the cause of these findings is the
occurrence of a less investigated phenomenon: the clustering or agglomeration of bacte-
rial cells. The association shown in Fig. 18.12 between mean concentration (m) and vari-
ance (s2) has been also observed in population ecology for physically aggregated species,
and described by the Taylor’s power law (Taylor 1961), whereby s2 tends to increase as a
power function of m (s2 = amb), with the fitted parameter b described as an index of aggre-
gation of the population. Thus, for randomly distributed populations the Taylor’s power
law (b = 1) obeys to the Poisson property that the variance equals the mean while for
aggregated populations (b > 1) the data obtained will be over-dispersed (s2 > m). This
kind of data, generally found for microbial counts, is mathematically better represented
by heterogeneous Poisson distributions than by the normal distribution.

5.1 Microbial Clustering and Statistical Distributions

An important phenomenon recognised for some years although still little investi-
gated in food microbiology (Marks and Coleman 1998; Nauta 2005; Jarvis 2008;
ILSI 2010) is the spatial clustering of microbial cells. Especially, in or on solid
foods, a random spatial distribution of cells seems highly unlikely as cells adhere to
one another after duplication leading to the occurrence of cell clumps instead of
individually distributed cells. In principle, and spatially speaking, cell clustering
can be understood as a ‘true contagious process’, whereby each ‘favourable’ event
(presence of an individual cell) increases the probability of future favourable events
(Feller 1943). However, in practice the ___location of the organisms is of no concern,
and of more relevance to food safety management decisions is the statistical
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 405

representation of clustering by means of appropriate frequency distributions. In this

sense, the spatial distribution of cell clusters can be related to a statistical distribu-
tion for over-dispersed or clustered data (variance > mean).
Although the assumption of data normality of bacterial counts has been applied
for long time (Kilsby and Pugh 1981), and in many cases, supported by experimen-
tal evidence (Gill et al. 1996; Peleg and Horowitz 2000), some concerns have been
lately raised in that it does not allow complete absence of microorganisms in a
sample, which is of significance when modelling microorganisms present in low
concentrations such as pathogens. To address this, Gonzales-Barron et al. (2010)
proposed the use of heterogeneous Poisson distributions as a better approach to deal
with over-dispersed data and to model low microbial counts consisting of a high
proportion of zero counts. A number of heterogeneous Poisson models exist
(Johnson et al. 2005), but the assumption that the mean densities from sample to
sample are realisations of a gamma distribution gives rise to the heterogeneous
Poisson distribution (Poisson-gamma or negative binomial) with perhaps the broad-
est applicability to biological data. Gonzales-Barron and Butler (2011b) compared
the suitability of two heterogeneous Poisson distributions in different microbial data
sets and concluded that the Poisson-lognormal distribution is better suited for high
counts while the Poisson-gamma represents better low counts consisting of many
zero counts. The importance of the choice of the most appropriate distribution to
represent bacterial clustering resides on its critical effect on the acceptance proba-
bility for typical microbiological criteria and on the design of sampling plans.
Gonzales-Barron and Butler (2011b) have shown that the Poisson-gamma assump-
tion allowing for clustering produces stricter sampling plans than both the lognor-
mal and the Poisson distribution for the same level of safety (LQL or PO).

5.2 Measure of Spread Within a Batch

The simplification of constant variability stemmed from the adaptation of quality

control theories of the manufacturing industry to the food industry. Unlike the
manufacturing process of non-food items that, when under control, has been
proven to operate with only chance (or non-assignable) causes of variation that
are an intrinsic part of the process, and therefore leading to a stable variance
(Montgomery 2009), in the production of food items, the batch-to-batch vari-
ability in the microbiological quality of the food product is expected to be more
unstable due to phenomena inherent to the biological systems (i.e., microbial
growth and death, usual microbial variability in raw food materials due to envi-
ronmental factors, difference among serotypes and serovars, cross contamina-
tion, etc.). Consequently, in the development of sampling plans, the variability of
the quality of the end product has been as a norm assumed more or less constant
(Smelt and Quadt 1990), or said otherwise, independent of the level of contami-
nation of the batch (Whiting et al. 2006). Recently, in an AOAC report concern-
ing statistical process control for microbial load in foods, it is stated that the
406 U. Gonzales-Barron and V. Cadavez

100 Poisson Γ, 1/k=2

Γ, 1/k=5 Γ, 1/k=10
Γ, 1/k=20 Γ, 1/k=50
PG, 1/k=2 PG, 1/k=5
PG, 1/k=10 PG, 1/k=20
PG, 1/k=50

CV 10

1
0.001 0.01 0.1 1 10 100
μ (CFU)

Fig. 18.13 Coefficient of variation (CV = σ/μ) in relation to the mean level of contamination (μ)
determined for a Poisson, Gamma (Γ) and Poisson-gamma (PG) distribution with fixed dispersion
parameters (1/k). Adapted from Mussida et al. (2013)

correlation between means and variances often observed for plate counts data
can be removed by logarithmic transformation (AOAC 2005). While there may
be some degree of reduction in some cases, for the two microbial data sets shown
in Fig. 18.12, the association between the observed mean and observed variance
estimated from samples taken from different batches is evident in spite of the log
transformation. The dependence of the spread measure (i.e., variance) to the
mean is a phenomenon that has been observed in many microbial data sets, and
is very likely to be an effect of the physical agglomeration of bacterial cells.
Therefore, one can say that two of the factors having an effect on the within-
batch variability, i.e., the level of microbial concentration and the aggregation of
bacterial cells, may be interrelated being the former effect a manifestation of the
latter, and furthermore, modelling the dependence of the variance on the mean
will somehow take into account both factors. This is of significance in the design
and assessment of microbiological criteria since the derivation of OC curves for
more efficient sampling plans should ideally take into account the shifts in the
measure of spread for the acceptance probability calculation as we move along
the x-axis values of within-batch mean concentration. The same applies to the
spread measures of other statistical distribution. For instance, Fig. 18.13 shows
how assuming that a measure of spread (the dispersion parameter 1/k in a
Poisson-gamma distribution) is fixed at different mean concentrations, may lead
to inexact OC curves as for low concentrations the assumed Poisson-gamma dis-
tribution will converge to a Poisson distribution, while for high concentrations it
will simplify to a gamma distribution.
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 407

5.3 Variability Batch to Batch

The classical derivation of acceptance sampling plans does not consider the variability
in the concentration of the target microorganism that takes place batch to batch.
Following technical developments in risk assessment and microbiological crite-
ria, Paoli and Hartnett (2006) introduced the notion of between-batch variability
while exploring the impact of the acceptance sampling plan upon the level of
contamination in powdered infant formula, and from their model, it was deduced
that the effectiveness of a sampling plan to distinguish between batches of differ-
ent quality is affected by both the within-batch and the between-batch variability.
As a rule of thumb, if the within-batch variability is small compared to the
between-­batch variability, a relatively small sample can give a good indication of
the quality of a batch in comparison to other batches. If the within-batch vari-
ability is large in comparison to the between-batch variability, a limited size sam-
ple is highly uninformative. When the within-batch and between-batch variability
are comparable, a limited size sample can only distinguish grossly different
batches (ILSI 2010). To account for the effect of the batch-to-batch variability,
Gonzales-Barron et al. (2012) developed a novel approach to model the associa-
tion between the within-batch mean and the within-batch spread measure using a
random-effects regression model based on the Poisson-gamma distribution char-
acterising low and high microbial counts from many batches of production. A
model of such association is a joint model of within-batch and between-batch
variability in microbial counts. Thus, to derive an OC curve, the uncertainty
around the within-batch spread measure for a given within-batch mean is propa-
gated by simulation to the probability of batch acceptance. In this way, OC curves
are obtained with confidence intervals representing the uncertainty about the
spread measure arising from the between-batch variability. The OC curves
obtained as such lead to more conservative sampling plans. Notice in Fig. 18.14,
that under the classical lognormal assumption with fixed standard deviation, the
sampling plan for Enterobacteriaceae on sheep carcasses would suggest that a
LQL batch of mean concentration of 230 CFU/cm2 (LQL) has only 5% confi-
dence of being accepted. However, under the Poisson-gamma assumption, such
batch of limiting quality has a probability of acceptance of up to 15% (see 97.5th
percentile for Pa in Fig. 18.14), depending on its within-batch measure of spread.
Thus, under the Poisson-gamma assumption allowing for the between-batch vari-
ability and variable measure of spread, the effective sampling plan is derived on
the upper interval of the probability of acceptance for a predefined LQL and β
risk values. This will in practice lead to a more stringent sampling plan (greater
sample size and/or lower microbiological limit) than the classical lognormal
approach with fixed standard deviation. On the other hand, an insight of the
between-batch variability is also important in statistical process control
(Montgomery 2009), although still few developments have been done for the
derivation of control charts for monitoring microbiological contamination
(Augustin and Minvielle 2008; Gonzales-­Barron et al. 2012, 2013).
408 U. Gonzales-Barron and V. Cadavez

1,0

0,9 Poisson-gamma,

Probability batch acceptance (Pa)

m=80 CFU/cm², k
0,8
variable + 95% CI
0,7
Lognormal,
0,6
m=1.5 log
0,5 CFU/cm², σ fixed
0,4

0,3

0,2

0,1

0,0
10 100 1000
Mean microbial concentration (CFU/cm2)

Fig. 18.14 Operating characteristic curves for the variables sampling plan n = 5, m = 1.5 log CFU/
cm2 or 80 CFU/cm2 testing Enterobacteriaceae on pre-chill sheep carcasses assuming the classical
lognormal distribution with constant σ = 0.6 log CFU/cm2 and the proposed Poisson-gamma model
accounting for between-batch variability. Notice that the Poisson-gamma approach produces OC
curves with confidence intervals arising from the association between within-batch mean and
within-batch measure of spread

5.4 Use of Past Monitoring Data

In classical acceptance sampling theory, the sampling plans are designed basically on a
pre-established level of safety (that could be the AQL/LQL at a risk α/β, or alternatively
a tolerance criterion (C, p0), both in accordance to a PO/FSO), and with little information
about the process itself. Using the methods from classical acceptance sampling, the log-
normality of microbial concentrations will be presumed, and the only piece of informa-
tion from the process itself will be at most some estimate of the ‘constant’ standard
deviation, if not an assumption. Nevertheless, when there is availability of past monitor-
ing data, sampling plans should ideally be designed making use of this information, in
particular when they are derived on the producer’s risk and there is evidence that hygiene
and safety measures in the production process have long been under control. In such
case, the objective of the sampling plan would be to verify that the safety of the produc-
tion process is under control; and mathematically it should be able to discern the outlying
‘out-of-­control’ batches. As proposed in Gonzales-Barron et al. (2012, 2013), when
microbial data is available, the within-batch and between-batch variability in contamina-
tion can be modelled jointly, and this spectrum of possible batches (i.e., universe of con-
taminated batches) can be used to estimate the misclassification risks (α and β) with
confidence intervals for the all possible microbiological limits and sample sizes. Selecting
the sampling plan will be then a matter of finding the optimal trade-­off between risks and
taking into account risk management considerations. Depending on the purpose of the
sampling plan, the same methodology can be also used in a statistical process control
framework to derive microbial control charts with an expected, warning and upper limit,
18 Statistical Derivation of Sampling Plans for Microbiological Testing of Foods 409

30
Average of 10 samples (CFU/cm2)
25

20
m

15
WL
10

5
la

0
0 5 10 15 20 25
Batch

Fig. 18.15 A control chart for monitoring Enterobacteriaceae concentrations on sheep carcasses
derived on the producer’s side by means of a Poisson-gamma model that makes use of historical
data. Apart from the microbiological limit or upper limit (m) and the sample size (n), the method
allowed also the derivation of an expected microbial concentration ( λa ) and a warning limit (WL)

as illustrated in Gonzales-Barron et al. (2013) (Fig. 18.15). In this way, taking into
account the results of sample units that were created by the process itself, will result in
sampling plans that are more informative and dynamic. Sampling plans obtained using
past information are also amenable to be updated as new sampling data are obtained,
which is unarguably its better advantage. Hald (1981) details interesting methodologies
to updating attributes sampling plan parameters by means of Bayesian analysis.

6 Concluding Remarks

Microbiological testing is a common tool used to evaluate whether a food safety risk
management system provides the appropriate level of control (FSO, PO), which
compares the level of a microbiological hazard detected in food samples against a
pre-specified limit or microbiological limit, and can be accomplished to comply to
microbiological criteria (i.e., standards written into law regulations) or to assess that
the food production process is under control (i.e., process control used by produc-
ers). The sampling plans used to test microorganisms can be assigned to two groups
depending on the type of microbiological test results: two-class type (n, c, m) or the
two-class by enrichment (n, c, w) for qualitative results, and by variables (n, m/M)
for quantitative results. Despite considerable research has been devoted to attributes
410 U. Gonzales-Barron and V. Cadavez

sampling plans, little attention has been paid to variables sampling plans despite
their advantage of being more informative and requiring less sampling for the same
level of protection. The sampling plans—derived either to meet the producer’s or
consumer’s safety requirements, or both, at certain confidence level—have tradi-
tionally been designed using classical acceptance sampling theory, which assumes
that the measure of the quality characteristic (i.e., microbial log concentration) dis-
tributes in the product (i.e., food) following a normal distribution with a variance
that is approximately stable batch to batch. Recent concerns in relation to bacterial
physical agglomeration have prompted questioning as to whether these oversimpli-
fying assumptions may lead to an inefficient design of sampling plans. The aggrega-
tion of bacterial cells causes the association between means and variance, and along
with other biological and food processing factors, underpins the variability nor-
mally found in microbial levels batch to batch. To address this problem, lately there
have been some efforts in the investigation of the adequacy of other statistical dis-
tributions to better represent bacterial clustering, and a situation originated thereof,
the high proportion of zero counts in samples. It has been found that the Poisson-
gamma or negative binomial distribution is more suitable than both the lognormal
and the Poisson-lognormal distributions when dealing with clustered microbial data
consisting of many zero counts; although other compound distributions should still
be evaluated. Nevertheless, whatever the statistical distribution of microorganisms
characterising over-dispersion, modelling the between-batch variability is relevant
for making some safety allowance for the effectiveness of sampling plans. Thus, the
design of statistically-sound sampling plans that are more efficient and with better
discriminatory power should be based on historical microbial data from many pro-
duction batches; and new methodologies, if possible Bayesian, should be developed
that are based on more realistic assumptions taking into account bacterial cluster-
ing, variable measures of spread conditional to the mean contamination level, and
the natural variability among batches of production.

Acknowledgments Dr. Gonzales-Barron wishes to acknowledge the financial support provided

by the Portuguese Foundation for Science and Technology (FCT) through the award of a five-year
Investigator Fellowship (IF) in the mode of Development Grants (IF/00570).

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Chapter 19
Antimicrobials and Food Preservation:
A Risk Assessment Approach

Daniele F. Maffei, Bernadette D.G.M. Franco, and Donald W. Schaffner

Abstract A variety of physical, chemical and biological methods for food preser-
vation are used to prolong shelf life of foods and keep them safe for consumers,
without compromising the nutritional and sensory characteristics. However, food-
borne illnesses caused by pathogenic microorganisms in these products are reported
to health authorities every year, highlighting the importance of continued scientific
inquiry for identification and quantification of the risks posed by these foods, and
for establishment of proper control measures. Qualitative or quantitative risk assess-
ments are used to estimate the potential adverse health effects associated with expo-
sure of individuals or populations to hazards in foods. This chapter provides an
overview on the available information on microbiological risk assessments in foods
containing antimicrobial compounds for preservation. Data from risk assessment
studies in such products are discussed in this chapter.

Keywords Food preservation • Antimicrobials • Microbiological risk assessment

• Ready-to-eat meat products

1 Introduction

Foods are susceptible to chemical, enzymatic, microbiological, and physical

changes, which may impair quality, safety and nutritional value, as well as causing
odor, color, flavor and texture problems. Different techniques of food preservation
have been developed aiming to prolong shelf life of foods and keep them safe for
consumers, without compromising the nutritional and sensory characteristics.

D.F. Maffei (*) • B.D.G.M. Franco

Food Research Center, Department of Food and Experimental Nutrition,
School of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil
e-mail: [email protected]; [email protected]
D.W. Schaffner
Department of Food Science, School of Environmental and Biological Sciences,
Rutgers, The State University of New Jersey, New Brunswick, NJ, USA
e-mail: [email protected]

© Springer Science+Business Media, LLC 2017 413

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3_19
414 D.F. Maffei et al.

These techniques have become an increasingly important component of the food

industry and comprise a variety of physical, chemical and biological methods.
Despite application of these methods for food preservation, foodborne illnesses
caused by pathogenic microorganisms such as Salmonella spp., pathogenic
Escherichia coli, and Listeria monocytogenes, among others, are reported to health
authorities every year, in many locations, and a significant fraction of these illnesses
are linked to consumption of processed foods. The United States Centers for Disease
Control and Prevention (CDC 2015) estimates that each year, one in six Americans
get sick and approximately 3000 die of foodborne illness. These estimates highlight
the importance of continued scientific inquiry to identify, quantify and control the
risk of illnesses posed by the consumption of contaminated foods and beverages.
Good Manufacturing Practices (GMP) and the Hazard Analysis and Critical
Control Point (HACCP) system are food safety management tools used by food
processors to ensure that food products meet the public health goals set by food
safety authorities. GMPs and HACCP use specific practices and steps to reduce,
eliminate or prevent the occurrence of hazards in foods. Increasingly, however, risk
analysis and its three component parts (risk assessment, risk management and risk
communication) are seen to provide a more scientific and systematic approach for
gathering, managing and communicating information about health risks.

2 Basic Concepts of Microbiological Risk Assessment

Risk assessment has become an important tool in food safety, providing a structured
process to identify hazards and to estimate the potential adverse health effects asso-
ciated with exposure of individuals or populations to those hazards. As noted above,
risk assessment is one part of a three-part system known as risk analysis (CAC
1999; FAO/WHO 2008):
• Risk assessment: A scientific and systematic process consisting of four steps: (1)
hazard identification, (2) hazard characterization, (3) exposure assessment, and
(4) risk characterization.
• Risk management: A process for evaluating, selecting and implementing policy
alternatives for controlling risks based on outputs of a risk assessment and other
factors relevant for the health protection of consumers.
• Risk communication: The interactive exchange of information and opinions
among all interested parties (risk assessors, risk managers, consumers, industry,
academia, etc.).
The activities of these three components are integrated into a system where each
one has its own rules and processes while also informing the others (Jaykus et al.
2006). Microbiological risk assessment (MRA) has been increasingly used interna-
tionally as a tool to inform risk management decisions and to facilitate communica-
tion with risk managers regarding microbiological hazards. MRA can be qualitative
or quantitative, presenting a written description or calculating numerical probabili-
19 Antimicrobials and Food Preservation: A Risk Assessment Approach 415

ties of the risk, respectively. A MRA typically includes the following steps (CAC
1999; FAO/WHO 2003; FAO/WHO 2008; FAO/WHO 2009):
• Hazard identification: The identification of the hazard in the food and its poten-
tial health effects. For microbial agents, the purpose is to identify the microor-
ganisms or the microbial toxins of concern. The food or foods and the affected
population are also identified.
• Exposure assessment: Describes the exposure pathways. Factors such as fre-
quency of contamination of foods by the pathogen, level of contamination over
time and patterns of consumption are typically considered.
• Hazard characterization (dose-response relationship): Provides a qualitative or
quantitative description of the severity and duration of adverse effects that may
result from the ingestion of a microorganism or its toxins in a food and may
include a dose-response assessment, which links concentration to probability of
illness or adverse outcome.
• Risk characterization: Represents the integration of the three prior components
(hazard identification, hazard characterization and exposure assessment) to
obtain a risk estimate.
Scientific committees around the world have published guideline documents
addressing MRA. These documents provide valuable tools that serve as a resource
to help safeguard consumer health. An example is the MRA guideline published by
the U.S. Department of Agriculture’s Food Safety and Inspection Service and the
U.S. Environmental Protection Agency (USDA/FSIS/EPA 2012), which provide a
structured approach for microbial risk assessment addressing pathogenic microor-
ganisms with focus on the safety of food and water.

3  pplication of Microbiological Risk Assessment to Foods

A
Containing Antimicrobial Compounds

The application of antimicrobial compounds for food preservation has a long his-
tory. Food antimicrobials are defined here as chemical compounds added to or pres-
ent in foods that inhibit growth or kill spoilage and/or pathogenic microorganisms.
They are often used in combination with other food preservation procedures
(Davidson et al. 2013). Traditional antimicrobials include organic acids (e.g. acetic,
lactic, propionic, sorbic and benzoic acids), enzymes obtained from animal sources
(e.g. lysozyme, lactoferrin), bacteriocins from microbial sources (e.g. nisin), nitrates
and nitrites, among others (Davidson et al. 2013; Lucera et al. 2012).
Despite the use of these compounds, ready-to-eat (RTE) meat products (e.g.
deli meats, smoked and cured meat products) have been involved in outbreaks
caused by pathogenic microorganisms. During 1998–2008, a total of 24 confirmed
listeriosis outbreaks were reported to the CDC Foodborne Disease Outbreak
Surveillance System (FDOSS) in the United States, resulting in 359 illnesses, 215
416 D.F. Maffei et al.

hospitalizations, and 38 deaths. A food vehicle was implicated in 20 (83%) of the

confirmed outbreaks: six (25%) were attributed to deli meats and three (13%)
were associated with frankfurters (Cartwright et al. 2013). In Canada, 57 cases
(24 deaths) in a ­listeriosis outbreak occurred in 2008 were linked to contaminated
ready-to-eat meat products (delicatessen meat) from one meat processing plant;
the costs associated with the cases were estimated to be nearly $242 million
Canadian dollars (Thomas et al. 2015). Moreover, the presence of L. monocyto-
genes and Salmonella in ready-­to-­eat meat products has been reported in many
studies (Garrido et al. 2009; Gombas et al. 2003; Gomez et al. 2015; Gottlieb
et al. 2006; Meyer et al. 2012; Modzelewska-­Kapitula and Maj-Sobotka 2014;
Osaili et al. 2014; Saludes et al. 2015).
A number of MRA models have been published in the literature focusing ready-­
to-­eat products over the past years, and some of them addressed products containing
antimicrobial compounds for preservation. The studies focusing L. monocytogenes
and Salmonella spp. in RTE meat and poultry products are summarized in Table 19.1
and discussed below.

Table 19.1 Microbiological risk assessment studies on RTE foods

Microorganism Product Reference
L. monocytogenes Deli meats FDA/FSIS (2003)
L. monocytogenes Deli meats Endrikat et al. (2010)
L. monocytogenes Deli meats (ham, turkey, roast beef) Pradhan et al. (2009)
L. monocytogenes Deli meats (ham, turkey) Pradhan et al. (2010)
L. monocytogenes Deli meats (ham, turkey) Pradhan et al. (2011)
L. monocytogenes Ready-to-eat meats (deli meats, Ross et al. (2009)
sausages, pâtés)
L. monocytogenes Deli meats Tian and Liu (2009)
L. monocytogenes Deli meats Yang et al. (2006)
L. monocytogenes Smoked fish (salmon, trout) and Garrido et al. (2010)
sliced cooked ham
L. monocytogenes Smoked or gravad salmon and Lindqvist and Westöö
rainbow trout (2000)
L. monocytogenes Ready-to-eat meat products Mataragas et al. (2010)
L. monocytogenes Deli meats FDA/FSIS (2013)
S. Typhimurium Dry-cured pork sausages Alban et al. (2002)
S. Typhimurium Fresh pork sausages Gonzales-Barron and
Butler (2015)
Salmonella spp. Fresh pork sausages Mürmann et al. (2011)
Salmonella spp. Pork products (fresh and dry Giovannini et al. (2004)
sausages, cured and fresh meat)
FDA Food and Drug Administration, FSIS Food Safety and Inspection Service
19 Antimicrobials and Food Preservation: A Risk Assessment Approach 417

3.1 Listeria monocytogenes

L. monocytogenes is a bacterium, which may be found in agricultural and food pro-

cessing environments and which is the causative agent of listeriosis. This disease
has serious health consequences to susceptible individuals, including newborns,
pregnant women, older adults and immunocompromised individuals (Adams and
Moss 2008). RTE meat and poultry products appear to be an important source of
foodborne listeriosis, and a target of a considerable number of risk assessment
studies.
In an attempt to better understand the sources of L. monocytogenes in foods, the
Food and Drug Administration and the Food Safety and Inspection Service of the
United States (FDA/FSIS 2003), working collaboratively, conducted a quantitative
assessment of the risk from L. monocytogenes among 23 categories of ready-to-eat
foods in the United States. Deli meats were classified in the “very high risk” cate-
gory (>1500 cases domestically per annum), the greatest risk of listeriosis among all
RTE food categories. Endrikat et al. (2010) conducted a risk assessment using a
modified version of the FDA/FSIS L. monocytogenes model (FDA/FSIS 2003) aim-
ing to compare the risk for this bacteria in retail-sliced versus prepackaged deli
meats, formulated with and without microbial growth inhibitors. The estimated
numbers of deaths per year were 37.1 and 163.9 for products with and without
growth inhibitors, respectively. The estimated deaths were 34.1 and 166.9 for pre-
packaged and retail-sliced products, respectively, indicating the important role
growth inhibitors and slicing ___location play in calculating listeriosis risk estimates.
Pradhan et al. (2009) provided a quantitative risk assessment for L. monocyto-
genes in deli meats (ham, turkey, and roast beef) modeling contamination and
growth from production to consumption, assessing also the impact of growth inhibi-
tors (lactate and diacetate) on the number of human listeriosis cases. They con-
cluded that reductions in the number of cases because of reformulation of RTE deli
meats varied for different product types. Growth inhibitors were predicted to reduce
the number of cases by 2.5–7.8-fold, but did not reduce predicted cases to zero. In a
related study, Pradhan et al. (2010) estimated the relative risk of deaths in the elderly
population due to L. monocytogenes contamination in deli meats (ham and turkey)
formulated without and with growth inhibitors, simulating two contamination sce-
narios: (1) prepackaged deli meats with contamination originating from manufac-
ture and (2) retail-sliced deli meats with contamination originating from retail. The
estimated number of deaths/annum attributable to ham formulated without growth
inhibitors was 13.2 for product contaminated at manufacture and 42.6 to product
contaminated at retail. The estimated deaths/annum attributable to ham formulated
with growth inhibitors were 4.7 and 22.5 for prepackaged and retail-sliced ham,
respectively. For processed turkey products formulated without growth inhibitors,
the estimated number of deaths/annum was 21.3 for product contaminated at manu-
facture and 35.8 for the product contaminated at retail. Estimates for processed
turkey products formulated with growth inhibitors were 2.4 and 12.6 deaths/annum,
for prepackaged and retail-sliced turkey respectively. Reformulation with growth
418 D.F. Maffei et al.

inhibitors was estimated to reduce human listeriosis deaths linked to ham and turkey
by 2.8- and 9-fold, respectively, when contamination originated at manufacture and
by 1.9- and 2.8-fold, respectively, when contamination originated at retail.
A third study published by Pradhan et al. (2011) compared the relative public
health impact of different types of contamination events by L. monocytogenes
(product-to-product and environment-to-product) at retail level in ham and turkey
formulated without growth inhibitors. The authors concluded that cross-­
contamination of deli ham and turkey from other products was estimated to increase
the relative risk of listeriosis-associated deaths by 5.9- and 6.1-fold, respectively.
Similarly, cross-contamination from retail environment-to-product increased the
relative risk of listeriosis-associated deaths by 4.9- and 5.8-fold for deli ham and
turkey respectively.
Ross et al. (2009) developed a risk assessment model to estimate the probable
number of cases of listeriosis per year due to consumption of various categories of
RTE meat products (luncheon meats, pâtés/ liverwurst, cooked sausages) in
Australia. The predicted number of cases of listeriosis per year was 44, and the
authors concluded that processed meats could be responsible for up to 40% of cases
of listeriosis in the country. A risk model for L. monocytogenes in deli meats and
vegetable salads by Tian and Liu (2009) indicated that deli meats are also of high
risk of causing listeriosis in China, where the listeriosis cases caused by consump-
tion of deli meats and vegetable salads was estimated to be 5.4 and 0.2 cases per
million people, respectively.
Yang et al. (2006) developed a consumer phase risk assessment for L. monocyto-
genes in deli meats with a focus on handling practices in the home. Their results
showed that cross-contamination via refrigerators and hands did not substantially
increase the mean level or prevalence of L. monocytogenes, while storage (growth
at less than ideal refrigeration temperature) had a significant impact. Simulations
indicated that 0.3% of deli meats were contaminated with L. monocytogenes at lev-
els >104 CFU/serving at the time of consumption. These authors estimated mean
risk of death for the intermediate-age population to be approximately 7 deaths per
1011 servings.
Garrido et al. (2010) developed a risk assessment model from retail to home level
to assess the risk of listeriosis in the Navarra region of Spain due to the consumption
of smoked fish (salmon and trout) and sliced cooked ham (vacuum or non-vacuum-­
packed). These authors predicted that the annual number of cases of listeriosis were
less than 0.3 in smoked fish and ~1 case each in vacuum-packed ham and retail-­
packed ham. They also found that storage temperature at 4 °C through the food
chain seems to have the greatest effect on decreasing the risk of listeriosis.
A risk assessment performed by Lindqvist and Westöö (2000) for L. monocyto-
genes in packaged smoked or gravad salmon and rainbow trout in Sweden used two
different dose-response models for probability of illness per serving: an exponential
model based on German data (GR) and a flexible Weibull-Gamma model (WG).
The predicted mean numbers of annual cases per year were 168 (range 47–2779;
GR model) and 95,000 (range 34,000 to 1.6 × 106; WG high-risk model). The
authors also noted that if only a fraction (1–10%) of the strains were considered
19 Antimicrobials and Food Preservation: A Risk Assessment Approach 419

virulent, the predicted mean number of annual listeriosis cases decreased to nine
(GR model) and 5202 (WG high-risk model).
One of the functions of risk assessment is to assist risk managers in decision
making. Mataragas et al. (2010) give examples of specific risk management strate-
gies (e.g. reformulation of products, use of antimicrobials, review of shelf life, sani-
tation practices and control of temperature) in their publication of a quantitative
microbiological risk assessment model for L. monocytogenes in RTE meat products.
Similarly, a recent technical report published by the Food and Drug Administration
and the Food Safety and Inspection Service of the United States (FDA/FSIS 2013)
provides information on how the risk of listeriosis associated with consumption of
RTE meats may be impacted by sanitary and food handling practices. This report
concluded that use of antimicrobial compounds, strict control of temperature during
refrigerated storage, control of contamination in incoming products, sanitation
practices to eliminate Listeria and control of cross-contamination especially during
slicing have an important influence on reducing the predicted risk of listeriosis.

3.2 Salmonella spp.

Salmonellosis is one of the most common foodborne illnesses worldwide and is

caused by the consumption of foods and beverages contaminated with Salmonella
spp. This organism affects millions of people every year and the symptoms are usu-
ally characterized by nausea, diarrhea, abdominal pain, vomiting and fever, which
can be more severe in susceptible groups, such as the very young or older people
and those already ill (Adams and Moss 2008). The risk of salmonellosis due to the
consumption of ready-to-eat meat products contaminated with this bacterium also
has been the focus of some risk assessment studies.
Alban et al. (2002) carried out two risk assessments for S. Typhimurium DT104
associated with consumption of dry-cured sausages produced with Danish pork
(DK) and imported pork (IMP). These authors noted that when Salmonella is pres-
ent in raw pork, it is usually in low numbers and processing typically results in at
least 2 log-unit reductions. Furthermore, DT104 occurred more often in IMP sau-
sages (18%) compared to DK sausages (0.2–1.0%). They also estimated that in one
million 25 g servings of DK sausages, up to two DT104 bacteria could be found in
245 servings while in IMP sausages, up to two DT104 bacteria would be present in
19,260 servings.
Gonzales-Barron and Butler (2015) used a second-order simulation model to
estimate the risk of infection by S. Typhimurium associated with the consumption
of Irish fresh pork sausages using results from a previous exposure assessment
model and an Irish consumption database. A risk of 192.8 infections and 18.0
expected cases per year was estimated. These authors also concluded that Irish fresh
pork sausages were estimated to cause 30% of the total cases of salmonellosis
attributed to pork.
420 D.F. Maffei et al.

A quantitative risk assessment conducted by Mürmann et al. (2011) evaluated

the risk of Salmonella infection to consumers of fresh pork sausages prepared at
barbecues in Porto Alegre, Brazil. These authors estimated that the average risk of
salmonellosis when the worst case cooking time (15.6 min) was assumed was
6.24 × 10−4 per barbecue, while the risk per month was 1.61 × 10−3, with a mean of
1554.58 cases. On the other hand, when a longer cooking time was assumed
(19.2 min, with a mean internal temperature of 75.7 °C), Salmonella prevalence was
reduced by 99.9%, thus reducing the risk significantly.
Giovannini et al. (2004) carried out a survey and risk assessment to estimate the
frequency and level of contamination of pork products with Salmonella spp. in
Abruzzi, Italy. Almost 10% of 595 pork samples (58/595 or 9.7%) were contami-
nated with Salmonella spp., including 40 samples of fresh sausages, 9 samples of
dry sausages and 9 samples of fresh meat. The risk assessment showed that fresh
sausages may be an important source of salmonellosis in Abruzzi, and a high inci-
dence of salmonellosis (13.7%) was predicted by the model during the two months
of peak consumption for this product.

4 Concluding Remarks

Data from the studies developed in different countries and discussed in this chapter
highlight the importance of microbiological risk assessment as a tool to identify and
quantify the risks posed by L. monocytogenes and Salmonella spp. in foods contain-
ing antimicrobial compounds for preservation, including RTE meat products. Those
studies have shown that antimicrobials and cooking have the potential to reduce the
risks of illnesses, while post-processing operations such as slicing at manufacture or
retail, and storage temperature may increase the risks. These studies contribute use-
ful information helping risk managers to improve food safety and protect consum-
er’s health through application of proper control measures.

Acknowledgements Grants #2012/03471-1, #2013/07914-8, #2014/17017-6 and #2016/09601-­

5, Sao Paulo Research Foundation (FAPESP).

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Index

A GRAS, 357
Acyl-homoserine lactones (AHLs), 72 liquid foods, 361
Advance thermal technology meat products, 362, 363
IR heating, 22 microorganisms, 360
microwave and radio frequency non-edible and biodegradable, 353, 354
preservation, 21 non-edible and non-biodegradable, 354, 359
Ohmic heating, 23 production, 361–362
Alginate, 352 quality and safety, 350
Alginic acid, 352 research and development, 364, 365
Alkaloids, 90 seafoods, 363
Alkylating gases, 332–336 USDA/FSIS, 358
ethylene oxide (see Ethylene oxide) Antimicrobial peptides (AMPs), 122
propylene oxide (see Propylene oxide) AAMPs, 121
structure, 332 barrel-stove model-mediated-lysis, 133
Animal sources, 415 biological activity, 119
Anionic antimicrobial peptides (AAMPs), CAMPs (see Cationic antimicrobial
119–121 peptides (CAMPs))
Antibacterial mechanisms, 58 classification, 56, 119
Antibiotic resistance, 94, 95 globalization, 117
Antimicrobial agent, 355 immunity mechanisms, 56
Antimicrobial food packaging interactions, 56
advantage and disadvantage, 359, 360 mode of action, 132–134
application of, 349 modern technologies, 118
bacteriocins and lysozyme, 357 neutral antimicrobial peptides, 130
bio-based polymers, 350 resistance/tolerance, 118
categories, 350 skin secretions, 119
and challenges, 366 subclasses, 119
components, 350 synthetic AMPs, 130, 131
culture media, 361 Antimicrobial polyphenols
dairy products, 363–364 benzoic acid, 134
direct coating, 358 by-products, 140, 141
double layer film, 359 flavonoids, 134
edible and biodegradable, 350–353 gallic and ferulic acids, 135
edible composite film, 358, 359 hydrophobicity, 136
essential oils, 354–357 hydroxycinnamic acids, 135

© Springer Science+Business Media, LLC 2017 423

V.K. Juneja et al. (eds.), Microbial Control and Food Preservation, Food
Microbiology and Food Safety, https://doi.org/10.1007/978-1-4939-7556-3
424 Index

Antimicrobial polyphenols (cont.) design of, 389–398

mechanism, 141–142 double, 387
non-flavonoids, 135 OC curves, 387–389
pathogenic strain, 135 single, 387
polyphenols, 134
signal transduction pathways, 134
synergistic effects, 135 B
tannins, 138–140 Bactenecins, 122
Antimicrobial preservatives, 8, 9 Bactericidal activity, 130
Antimicrobials, 355–356 Bactericidal/permeability-increasing
food models, 167 (BPI), 130
sensory effects, 157 Bacteriocins, 103, 357, 415
structure, 154–156 antagonistic compounds, 102
temperature and pH, 156 applications, 108–113
toxicity, 167 chemical preservatives, 109–111
Antimicrobials gases E.coli, 103
advantages, 329 efficacy, 107
categorization, 331, 332 in food systems, 106–108
diffusion, 331 hurdle technology, 114
equipment surfaces, 327 LAB classifications, 103–106
food handling and processing environment, lactic acid bacteria, 105
327–328 nanotechnology, 113
food sanitation, 327 physical treatments, 111, 112
gaseous-aqueous interrelations, 330, 331 proteinaceous antibacterial
sanitizers, 328 compounds, 102
sterilants, 328 Bacteriophage application, 31, 32
treatment, 330 Beer-Lambert Law, 338
usage, 329, 330 Between-batch variability
washing operations, 328 microbial concentrations, 402
Antimicrobials of microbial origin Poisson-gamma assumption,
concentrations, 103 407, 408
micro organisms, 102 and within-batch, 407
peptides, 102 Bioinformatics, 46, 48
proteins, 103 Biological preservation, 4
Antimicrobials of plant origin
alkaloids, 90
ancient civilizations, 86 C
ancient human skeletons, 86 Caerins, 129
bioactive compounds, 86 Carboxy methyl cellulose, 352
EO, 90, 92 Casein, 353
flavonoids, 88, 89 Caseinate, 353
food applications, 86 Cathelicidins
microbial infections, 86, 89 bactericidal activity, 67
phenolics, 87–90 homodimeric bactenecins, 67
quinones, 89 N-terminal cathelin-like ___domain, 67
secondary metabolites, 87 Cationic antimicrobial peptides (CAMPs)
stilbenoids, 89 alyteserin-1, 128
Appropriate level of protection (ALOP), alyteserin-2, 128
382, 384 amide-modified synthetic peptide, 125
Arenicin-1, 126 amphibians, 126
Ascaphins, 129 AMPs, 126
Attributes, sampling plan, 384 antimicrobial activity, 126, 128
classical acceptance, 387 arenicin-1, 126
decision-making process of, 385 Bac 5 and Bac 7, 125
Index 425

bactericidal activity, 125 D

brevinin homologs, 127 Data mining techniques, 46
categories, 122 Defensins, 122
cecropin B, 125 Dehydration/drying, 5
cecropin P, 125 Delivery systems
defensins, 122 compatibility, 157
deltorphin, 128 criteria, 155, 156
dermaseptins S family, 126, 127 emulsions, 157–166
dermorphin, 128 encapsulation, 157
food preservation, 129 food antimicrobials, 158
gram-positive and gram-negative, 126 food matrix, 157
hemocytes, 129 food model research, 168
heptapeptide, 128 functional food components, 154
keratinocytes, 122 MIC, 154
magainins, 127 nanomaterials, 154
magnesium, 125 synthetic polymer, 154
mono- and dimeric forms, 122 toxicity, 167
mucosal epithelial cells, 125 Dermaseptin, 126
multi-resistant bacterial species, 127 Dermorphin, 128
N-terminal region, 129 Dicynthaurin, 129
pathogens, 127 Direct coating vs. film packaging, 360
rugosin A, 128 D-mannuronic acid, 352
skin extracts, 127 Dodecyl chlorogenates (DCGA), 136
skin secretions, 128 Dose-dependent mode, 61
synthetic peptide, 125
Center for Food Safety and Applied
Nutrition from US Food and E
Drug Administration Eastern Regional Research Center (ERRC), 45
(CFSAN/FDA), 46 Egg-white lysozyme, 69
Chemical preservatives, 109–111 Electronic Laboratory Exchange Network
Chitosan, 74, 352 (eLEXNET), 44
Chitosan-coated plastic films, 363 Emerging nonthermal preservation
Chlorine dioxide (ClO2) membrane processing, 32
antimicrobial action, 338, 339 PL, 26–29
antimicrobial agent, 337 ultrasound processing, 29–31
gaseous and aqueous states, 337 UV light treatment, 24–26
generation, 337 Emulsions, 157–166
hazards, 340 Encapsulation, 157
inactivation of foodborne pathogens, 338 Entamoeba histolytica, 61
measurement, 338 Enteroaggregative Escherichia coli
usage, 339, 340 (EAggEC), 59
Chlorohydrin process, 335 Enterobacteriaceae, 407–409
Cinnamaldehyde films, 357, 362 Enterovirus 71 (EV71), 62
Coffee chlorogenic acids (CGA, 136 EPEC-mediated actin polymerization, 61
Commercial sterilization chambers, 335 Epigallocatechin-3-gallate (EGCG), 94
Communication, 377–379 Essential oils (EOs), ), 86, 87, 90, 92, 93, 364
Composite vs. double-layer film, 359 Ethylene oxide
Computational metagenomic approach, 51 application, 333
Consumer attitudes, 376, 377 efficacy and mechanism of action, 334
Consumer perspectives, 9–11 hazards, 334
Controlled atmosphere (CA), 6–7 measurement, 333
Coumarins, 90 usage, 333
Cryptosporidium parvum, 61 Ethylenediaminetetraacetic acid (EDTA), 142
Culture-independent diagnostic tests (CIDTs), 43 Extended spectrum beta lactamases (ESBL), 60
426 Index

F computational approaches, 42
FDA-iRISK, 45–46 genomic information, 46
Fermentation, 107 genomics and WGS, 47
Flavan-3-ols, 136–138 health-related quality, 42
Flavonoids, 86, 88–90, 92–94, 134, 136–138 PulseNet, 44
Flavonols, 136–138 research topics, 47
Food and Agricultural Organization and the sequence information, 46
World Health Organization of the surveillance system, 43
United Nations (FAO/WHO), 65 FoodNet, 43
Food borne illnesses, 102 Foodrisk.org, 45
Food chain Foods
risk-based metrics, 383 microbiological criteria, 386
Food containers, 4 Freezing methods, 6
Food preservation techniques, 142, 143,
413–415
advantages, 18 G
antimicrobes, 8, 9 Gaegurins (GGNs), 128
carbon footprint, 18 Gallic acid equivalents (GAE), 93
considerations, 3 Gelatin-based antimicrobial coatings, 363
consumer perspectives, 9–11, 376, 377 Generally recognized as safe (GRAS), 86,
consumer priorities, 374 106, 154, 341
dehydration/drying, 5 GHP-based MCs, 384
factual information, 379 Global Outbreak and Alert Response Network
food safety, 375, 376 (GOARN), 44
freezing methods, 6 Grape seed extracts (GSE), 93, 94
healthfulness, 375 Green tea extract (GTE), 94
historic events, 2
market requirements, 7
methods, 4, 5, 19 H
microorganisms, 18 Halogen-based compounds, see Chlorine
objectives, 3 dioxide (ClO2)
outcomes, 3 Hazard analysis and critical control points
physical and chemical changes, 3 (HACCP), 42, 118, 414
planned production, 4 Hazard-based MCs, 384
in prehistoric age, 2 High density polyethylene (HDPE), 358
price stabilization, 4 High hydrostatic pressure (HHP), 7
principles, 18–19 High pressure processing (HPP), 7
quality, 4 High-intensity pulsed electric field (HIPEF),
techniques, 3, 11 64–65
temperature storage, 5–6 Human β-defensin 3 (HBD3), 122
trust and communication, 377–379 Hydrogen peroxide, 70
Food processing, 374 Hydroperoxide process, 335
Food quality, 4 Hydroxypropyl methylcellulose, 352
Food safety, 3, 9, 11, 118, 142, 143
Food safety & inspection service (FSIS), 45
Food safety and inspection services from US I
Department of Agriculture Infrared (IR) radiation, 22
(FSIS/USDA), 46 Insects growth prevention, 19
Food safety objectives (FSOs), 382–384, 395 Intense pulsed light (IPL), 27
Food spoilage, 102
Foodborne Disease Outbreak Surveillance
System (FDOSS), 415 J
Foodborne pathogen Joint Institute for Food Safety and Applied
bioinformatics, 46 Nutrition (JIFSAN), 45
Index 427

L M
Lactic acid bacteria (LAB) Macrophages, 129
antimicrobial peptides, 102 Magainins, 127
bacteriocins, 105 Membrane processing, 32
complex, 105, 106 Metagenomics, 47, 48
lantibiotics, 104 Methyl cellulose, 352
non-lantibiotics, 104 Methylglyoxal (MGO), 72
Lactobacillus isolates from appam batter Microbial distribution, 387, 392, 393, 397,
(LABB), 110 398, 400, 404, 405
Lactobacillus species, 102 Microbial pathogens, 118
Lactoferrampin (LFampin), 60 Microbial sources, 415
Lactoferricin (LFcin), 60 Microbiological criterion (MC), 384, 385
Lactoferrin (LF), 56 Microbiological risk assessments (MRA), 382,
antimicrobial activity, 58–62 415–420
applications in foods, 62, 63 antimicrobial compounds
concentration, 57 application of, 415
intestinal microbiota, 57, 58 FDOSS, 415
LF-derived peptides, 61 L. monocytogenes, 417–419
monomeric glycoprotein, 57 ready-to-eat products, 416
technological applications, 62 Salmonella spp., 419, 420
Lactoperoxidase (LPO) application of, 414
antimicrobial agents, 66 components, 414
heme-containing glycoprotein, 65 exposure assessment, 415
infant milk formula, 66 in food safety, 413, 414
microbial count, 65 GMP, 414
pasteurization, 66 HACCP, 414
synergistic effect, 66 hazard characterization, 415
technological applications, 66–67 risk characterization, 415
thiocyanate ions, 65 RTE meat products, 420
ultra-high treated milk, 66 scientific committees, 415
Lantibiotics, 104 United States Centers for Disease Control
Lauric arginate, 357 and Prevention, 414
Leptoglycins, 130 Microbiological testing, 384–385
LF hydrolysate (LFH), 60 ALOP, 382
L-guluronic acid, 352 AOAC report, 405
Liposomes attributes sampling plan, 396–398
as antimicrobial delivery between-batch variability, 407
systems, 166 biological and food processing factors, 410
degree of shear, 165 clustering and statistical distributions,
formation, 166 404, 405
host environment, 165 constant variability, 405
mass transport, 165 contamination level, 402
polar lipids, 165 European regulators, 383
Listeria monocytogenes, 392, 395, 396, food industry, 384
417–419 food safety risk management system, 409
Listeriosis, 417–419 historical microbial data, 410
Low density polyethylene (LDPE), 354 industry and government, 382
Lysozyme (LZ), 56, 59, 68, 69, 357 industry operational level, 382
antimicrobial activity, 64 MC (see Microbiological criterion (MC))
applications in foods, 64, 65 microbial concentration, 392–396
health benefits, 63 microbial prevalence, 389–392
intestinal microbiota, 64 microbiological control, 385–387
leucocytes, 63 monitoring data, 408, 409
lysosomes, 63 MRA, 382
428 Index

Microbiological testing (cont.) proteomic technologies, 56

pathogenic microorganisms, 381 technological applications, 57
Poisson-gamma distribution, 406 Neutral antimicrobial peptides
public health, 383 eukaryotic cationic, 123, 124
sampling theory, 387–402 skin secretion, 130
statistical methods, 402 New technologies, 376–379
Taylor’s power law, 404 Next-generation sequencing (NGS)
technical developments, 407 technologies, 47
variables sampling plans, 398–402 Nisin with high hydrostatic pressure
within-batch variability, 406, 407 (nisin-­HHP), 103, 104, 107–111
Microemulsions Non-lantibiotics, 104
definitions, 159 Nylon, 354
food industry approach, 159
formation, 160
investigations, 160 O
nanoemulsions, 160, 161 O-antigen clusters, 52
organic solvents, 159 Ohmic heating technology, 23
self-assembled aggregates, 159 Oil-in-water-in-oil (O/W/O), 161
Microfluidization, 365 Operating characteristic (OC) curve, 388–390,
Microgard™, 112, 113 393, 394, 397, 398, 401–403,
Microorganisms, 107, 114 406–408
Microwave (MW) preservation, 21 Operating characteristic (OP) curves, 384, 399
Minimum bactericidal concentration (MBC), Organic acids, 415
93, 109, 136 Outbreak detection system, 43–44
Minimum inhibitory concentration (MIC), 59, OutbreakNet program, 44
93, 154 Ovotransferrin, 67, 68
Modified atmosphere (MA) storage, 6–7 Oxidizing gases, see Ozone (O3)
Multiple-locus variable number tandem repeat Ozone (O3)
analysis (MLVA), 44 efficacy and mechanism of action, 343
Multiplicity of adsorption (MOA), 32 generation, 341, 342
Multiplicity of infection (MOI), 32 GRAS, 341
hazards, 344
measurement, 342, 343
N usage, 343, 344
N-acetyl-ß-D-glucosaminidase, 56
Na-lauroyl-L-arginine ethyl ester
monohydrochloride, 357 P
Nanoemulsions, 160, 161 Permissible exposure limit (PEL), 344
as antimicrobial delivery system, Phenolic compounds, 71, 72, 89
162, 163 Phenolics, 87–90
formation, 161, 162 Phenylalanineammonialyase (PAL), 28
Nanofibers, 166 Physical preservation, 5
Nanostructured multilayer emulsions, 163–165 Phytochemicals, 86, 87, 95
Nanotechnology, 113 Planned production, 4
National Institute for Occupational Safety and Pleurocidin-incorporated fiber, 125
Health (NIOSH), 344 Poisson-gamma (PG) distribution, 406, 407
Natural antimicrobials Poisson-lognormal distribution, 397, 399, 405
applications in foods, 74 Polyethylene/polyamide (PE/PA), 344, 354
in egg, 67–69 Polylactic acid (PLA), 353
in honey, 69–74 Polymers, 351–352
LF, 57 Polyphenoloxydase (PPO), 28
milk, 56 Polyphenols, 92, 134
propolis, 73 Polypropylene, 354
Index 429

Polytetrafluoroethylene (PTFE), 344 Sodium caseinate (SC), 353

Polyvinyl chloride (PVC), 344, 354 Sodium diacetate (SD), 357
Potassium indigotrisulfonate, 342 Sodium lactate (SL), 357
Potassium sorbate (PB), 357 Specific surface areas (SSAs), 365
Predictive Microbiology Information Portal Spectrophotometer, 338
(PMIP), 45 Stilbenoids, 89
Predictive modeling (PMP), 45 Sulfur dioxide, 332
Price stabilization, 4 Surveillance System (FoodNet), 43
Probiotic bacteria, 61
Propylene oxide
application, 335 T
efficacy and mechanism of action, 335 Tannins, 90, 138–140
hazards, 336 Taylor’s power law, 404
measurement, 335 Temperature storage (low), 5–6
usage, 336 Thermal preservation
Pullulan, 352 application, 20
Pulsed electric field (PEF), 8, 108 classification, 20
Pulsed light (PL) technology D and Z values, 20
anti-browning dipping solution, 29 design and development, 20
decontamination, 28 destruction of microorganisms, 20
FDA, 26 first-order kinetics, 20
microbial decontamination, 27 Titanium dioxide (TiO2), 358
microbial inactivation, 26 Transmembrane pressure (TMP), 33
non-thermal decontamination, 26 Trust, 377–379
post-harvest processing, 28 Turmeric extracts, 92
shelf-life studies, 28, 29 Type III secretory system (TTSS), 59
USDA, 26
UV light sterilization system, 27
Pulsed-field gel electrophoresis (PFGE), 44 U
PulseNet, 43 Ultrasound treatment, 7, 8
Ultrasound with high hydrostatic pressure
(US-HHP), 111
Q Ultraviolet (UV) radiation, 24–26
Quinones, 89 US Occupational Safety and Health
Quorum sensing inhibition (QSI), 72 Administration (OSHA), 344

R V
Ready-to-eat (RTE) meat products, 415, Viable but non-culturable (VBNC) bacteria, 52
416, 419
Response system (OutbreakNet Enhanced), 44
Risk-based MCs, 384 W
Rugosin, 128 Water-in-oil-in-water (W/O/W), 161
Wax, 353
Weibull-Gamma (WG) model, 418
S Whey protein isolate (WPI), 69, 353
Salmonella spp., 419, 420 Whole genome sequencing (WGS), 44
Serotyping, 51 bioinformatics data analysis, 48
Shelf-life, 7, 10, 11, 102 computational approaches, 48, 49
Shiga-toxin-producing Escherichia coli computational modeling, 50
(STEC), 43 food safety, 47
SK-N-SH cell lines, 62 genomic information, 46
Sodium benzoate (SB), 357 metagenomics, 47, 48
430 Index

Within-batch testing Worker safety, 344

and between-batch variability, 407 World Health Organization
vs. food production, 383 (WHO), 118
heterogeneous food product, 396
microbial concentrations, 393
OC curve, 389 Z
Pa values, 401 Zein, 353
standard deviation, 395, 400, 403 Zinc oxide (ZnO), 358