application of alternative food-preservation - Bentham Science

APPLICATION OF ALTERNATIVE FOOD-PRESERVATION 

TECHNOLOGIES TO ENHANCE FOOD SAFETY AND STABILITY 

Edited by 

Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia

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CONTENTS 

Foreward i 

Preface ii 

Contributors iii 

CHAPTERS 

1. Green Consumerism and Alternative Approaches for Food Preservation: an Introduction 01 

Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia 

2. Risk Assessment and Food Safety Objectives 04 

Antonio Bevilacqua, Barbara Speranza and Milena Sinigaglia 

3. Food spoilage and safety: Some Key-concepts 17 

Barbara Speranza, Antonio Bevilacqua and Maria Rosaria Corbo 

4. Essential Oils for Preserving Perishable Foods: Possibilities and Limitations 35 

Barbara Speranza and Maria Rosaria Corbo 

5. Enzymes and Enzymatic Systems as Natural Antimicrobials 58 

Daniela D’Amato, Daniela Campaniello and Milena Sinigaglia 

6. Antimicrobial agents of Microbial Origin: Nisin 83 

Daniela D’Amato and Milena Sinigaglia 

7. Chitosan: a Polysaccharide with Antimicrobial Activity 92 

Daniela Campaniello and Maria Rosaria Corbo 

8. Use of High Pressure for Food Preservation 114 

Antonio Bevilacqua, Daniela Campaniello and Milena Sinigaglia 

9. Alternative Non-Thermal Approaches: microwave, Ultrasound, Pulsed Electric Fields, 

Irradiation 143 

Nilde Di Benedetto, Marianne Perricone and Maria Rosaria Corbo 

10. Food shelf life and safety: challenge tests, prediction and mathematical tools 161 

Antonio Bevilacqua and Milena Sinigaglia 

APPENDIX 

11. Microencapsulation as a new approach to protect active compounds in foods 188 

Mariangela Gallo and Maria Rosaria Corbo 

12. Alternative Modified Atmospheres for Fresh Food Packaging 196 

Maria Rosaria Corbo and Antonio Bevilacqua 

Index 205

Università degli Studi della Basilicata 

Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali 

Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy 

Prof. Patrizia Romano 

Tel: +39-0971-205576 Fax +39-0971-205686, E-mail: patrizia.romano@unibas.it 

Object: Book “APPLICATION OF ALTERNATIVE FOOD-PRESERVATION TECHNOLOGIES TO 

ENHANCE FOOD SAFETY AND STABILITY” EDITED BY A. BEVILACQUA, M.R. CORBO, M. 

SINIGAGLIA 

FOREWORD 

Preservation is a continuous struggle against pathogens and spoiling microorganisms to maintain food safety at 

high quality levels; in additional, there is also a trend towards green consumerism, i.e. the consumption of foods 

with high levels of nutrients and nutraceutical compounds without chemical preservatives. 

This e-book can be considered as a suitable answer to the consumer demand, as it offers some useful alternatives 

to traditional thermal processing, focusing both on the antimicrobial effectiveness of the proposed approaches 

and a description of their effects on food structure and health. Moreover, the chapters on the key-concepts of risk 

assessment and mathematical modeling of microbiological data, along with the two appendices on the microencapsulation 

and non-conventional atmospheres, add some important topics for food microbiologists. 

It is quite impressive to note that the editors and authors have tried to capture a wide and dynamic topic in a 

series of captivating chapters, highlighting on newly emerging technologies, protocols, methodologies and 

approaches, advantages, new school of thoughts from around the world, potential future prospects and also 

negative criticism that is associated with some frontier development of green consumerism. 

I think that the e-book will be beneficial to students and researchers in different fields of food microbiology and 

technology; I wish the authors and editors great success and hope that this book will be the 1st work of a new 

editorial series. 

Potenza, 1 st February 2010 

i

ii 

PREFACE 

Nowadays, western countries are experiencing a trend of green consumerism, desiring fewer synthetic additives 

and more friendly compounds. Therefore, bacteriocins and other natural compounds (lysozyme, bacteriocins, 

fatty acids, monoglycerides and essential oils) could be considered promising bioactive molecules and their use 

might be proposed to control and/or inhibit pathogens and spoiling microorganisms. 

Thermal pasteurization and sterilization are the most important techniques to achieve safety in foods; however, 

they can result in some unfavorable changes, like protein denaturation, non-enzymatic browning and loss of 

vitamins and volatile compounds. Advances in food processing were allowed in the past to avoid some 

undesirable changes; however, thermally processed foods still lack the fresh flavour and texture 

In the light of these ideas, alternative approaches have been extensively investigated in the past 30-40 years; they 

are usually labeled as non-thermal techniques, as food is (in some cases) treated at room or refrigeration 

temperature or the rest at relatively high temperatures (e.g. 70-90 °C for high homogenization pressures) is 

limited within the time. 

Dealing with these considerations, the book will focus on the alternative approaches for prolonging food shelf 

life; in particular, the topics of the book are: 

1. use of natural compounds in food preservation (essential oils, lysozyme, lactoperoxidase system, 

lactoferrins, bacteriocins and related antimicrobial compounds) 

2. use of high hydrostatic and homogenization pressures 

3. use of non conventional atmospheres 

4. other alternative approaches (microwave, ultrasounds, pulsed electric fields, irradiation) 

5. definition of food safety objectives and mathematical modeling and challenge for shelf life 

definition and evaluation. 

The book proposes an integrated approach of shelf life extension, focusing on both the inhibition of the spoilage 

microorganisms and on the implications of the alternative approaches, in terms of quality and costs (technical 

and economical feasibility).

CONTRIBUTORS 

CHAPTER 1 

Antonio Bevilacqua (1,2) a.bevilacqua@unifg.it 

Maria Rosaria Corbo (1,2) m.corbo@unifg.it 

Milena Siniggalia (1,2) m.sinigaglia@unifg.it 

1 Department of Food Science, Faculty of Agricultural Science, University of Foggia 

2 Food Quality and Health Research Center (BIOAGROMED), University of Foggia 

CHAPTER 2 


Barbara Speranza (1,2) b.speranza@unifg.it 

Milena Sinigaglia (1,2) m.sinigaglia@unifg.it 



CHAPTER 3 






CHAPTER 4 





CHAPTER 5 

Daniela D'Amato (1,2) d.damato@unifg.it 

Daniela Campaniello (1) d.campaniello@unifg.it 




CHAPTER 6 

Daniela D'Amato (1,2) d.damato@unifg.it 




CHAPTER 7 





iii

iv 

CHAPTER 8 






CHAPTER 9 

Nilde Di Benedetto (1) n.dibenedetto@unifg.it 

Marianne Perricone (3) m.perricone@unifg.it 




3 

Department of Agro-Environmental Sciences, Chemistry and Crop Protection, Faculty of Agricultural Science, 

University of Foggia 

CHAPTER 10 





APPENDIX 1 

Mariangela Gallo (1) m.gallo@unifg.it 




APPENDIX II 




2 Food Quality and Health Research Center (BIOAGROMED), University of Foggia

Application of Alternative Food-Preservation Technologies to Enhance Food Safety & Stability, 2010, 01-03 1 

Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) 

All rights reserved - © 2010 Bentham Science Publishers Ltd. 

CHAPTER 1 

Green Consumerism and Alternative Approaches for Food Preservation: 

an Introduction 

Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia* 

Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy 

Abstract: Consumer awareness towards the use of natural compounds has increased significantly since the 

beginning of 1990s and new trend has arisen in food industry, i.e. the green consumerism. 

The green consumerism is the basis for the development of alternative approaches for food preservation, like 

the use of natural compounds (essential oils, lysozyme, nisin and other bacteriocins, chitosan) and nonthermal 

treatments (high hydrostatic pressures, homogenization, microwave, irradiation). 

These new technologies are the topics of this e-book; this chapter offers an introduction to the entire work. 

Key-concepts: what is green consumerism, why green consumerism, book structure. 

INTRODUCTION 

Generally foods are thermally treated for few seconds to minutes at temperatures ranging between 60 and 100°C 

(or higher values in some cases) to destroy pathogens and spoiling microorganisms. During these treatments a 

large quantity of energy is transferred to foods; however, this energy can cause undesirable changes in terms of 

organoleptic and nutritional properties and general appearance [1]. 

As an alternative or as an additional hurdle to thermal treatments, microbial growth is usually controlled through 

the use of chemical compounds and preservatives; due to some toxicological reports, it is well known that some 

of these molecules could have an adverse effect on human health. 

Based on these assumptions, consumer awareness towards the use of natural compounds has increased 

significantly since the beginning of 1990s and new trend has arisen in food industry, i.e. the green consumerism. 

The definition of green consumerism was introduced in 1980s, referred to a new way of producing goods and 

foods, without any adverse effect on the environment. Gradually, this concept has been introduced in food 

technology as a new approach of managing food production, through the use of lower amount of energy and 

water, the reduction of chemicals with adverse effects on human health and the addition to foods of friendly 

compounds (a friendly compound is a non toxic compound, without any negative effect on humans, available at 

low cost and environmentally safe) [2-4]. 

Green consumerism can be considered as a philosophy for managing food production; the key concepts of this 

new way are the following: 

1. all products have an impact on environment and health. A green product can be defined as a food 

with a small impact on nature and man; 

2. consumers have been asking for green products; 

3. a consumer has to realize that he/she does not just buy a product, but everything that went into its 

production and everything that will happen in the future as a result of that product. 

Some keywords of green consumerism are reported in the Table 1. 

Carried out for food technology, this philosophy means that it’s time to employ an alternative approach for food 

preservation, making a balance between the need to fight pathogens and spoiling microorganisms and preserve 

health benefit and natural appearance of foods. 

*Address correspondence to this author Milena Sinigaglia at: Department of Food Science, Faculty of Agricultural Science, University 

of Foggia, Italy; E-mail: m.sinigaglia@unifg.it

2 Application of Alternative Food-Preservation Technologies Bevilacqua et al. 

A good answer for this demand could be the use of natural compounds, as well as the employment of some notthermal 

treatments (homogenization, high hydrostatic pressure, microwave and irradiation), able to assure safety 

and quality. 

A drawback of the literature for these topics is that the most of books and reviews available are referred to data 

collected in model systems or laboratory media or, when the in vivo information is present, there is a lack on the 

practical use in foods. 

This book offers an overview of the most recent findings on the topics of natural compounds and not-thermal 

approaches, focusing on the practical application and implications of these alternative methods in foods. 

Chapters 2 and 3 report a theoretical background, useful to understand how manage the concepts of safety and 

quality (quantitative risk analysis, food safety objectives, use of microbiological criteria), along with a brief 

description of the most important pathogens and spoiling microorganisms recovered in foods and the 

biochemical changes occurring throughout food storage and spoilage. 

After these two chapters, that can be considered as a necessary introduction, there are the key chapters of the 

book, divided into two groups: in the section I (chapters 4, 5, 6 and 7) readers can find an exhaustive description 

of the most important natural compounds used for food preservation (i.e. essential oils, nisin, lysozyme and other 

enzymatic systems and chitosan); otherwise, the chapters 8 and 9 (section II) focus on the not-thermal 

approaches (high pressure, microwave and irradiation). 

Each chapter includes one or more paragraphs covering the basic aspects of the topic (mode of action, details on 

the antimicrobial activity, equipments) and then the information on the use of the proposed approach in foods, 

along with a description of food changes, if the data are available. Finally, in the case of the natural compounds, 

there is always a final paragraph covering the toxicological data and legal aspects. 

Chapter 10 proposes another theoretical and necessary background, i.e. the predictive microbiology and the 

mathematical approach for shelf life prediction and evaluation. After a brief description of the most important 

primary models (both growth and survival functions), the chapter goes on some new approaches, like the S/P 

models, along a brief synopsis of the most important secondary models. An appendix to the chapters reports 

some details on the design of experiments, focusing on the Central Composite Design and Centroid Approach. 

Finally, the book proposes two appendices, focusing on the microencapsulation of active ingredients, as a new 

way for shelf life prolonging, and the use of not-conventional atmospheres, as a convenient approach to control 

microbial growth and preserve food quality. 

In summary, we feel that this book will be a useful mean for students, researchers and people acting in food 

chain with the spectrum of current knowledge, practical implications and applications and perspectives, along 

with some provoking issues. 

We hope that it can contribute to increase consumer awareness towards some alternative approaches for food 

preservation, as well as the firm belief amongst food producers and governments that natural compounds and 

not-thermal approaches have really a practical significance and can be the future in the field of food technology, 

thus assuring safety, quality and low impact on human health and environment. 

Table 1: Keywords of green consumerism 

Keyword Why 

Health 

A sentary lifestyle combined with health impacts of environmental pollution and emissions, use and 

abuse of pesticides, antibiotics and chemicals, could have dramatic consequences. 

Energy 

Every source of energy has an environmental impact. Energy efficiency is not just technology, but also 

cutting back. 

Water Water use is increasing at twice the rate of population increase. Much can be done at individual level. 

Chemicals 

Pesticides, preservatives and other chemical hazards have long term effects on human health and wellbeing. 

Genetic 

engineering 

Natural world 

Includes many ethical and moral issues. Genetic engineering is not necessarily bad, but consumer 

should be given the choice. 

Considerable pressures are put on the natural world due to population increase and rise in consumption. 

Nowadays, it has been esteemed that ca. 40% of all plant is consumed by humans. Somewhere, 

something should stop!

An Introduction to Green Consumerism Application of Alternative Food-Preservation Technologies 3 

REFERENCES 

[1] Tiwari BK, Valdramidis VP, O’Donnell CP, Muthukumarappan K, Bourke P, Cullen PJ. Application of natural 

antimicrobials for food preservation. J Agric Food Chem 2009; 57: 5987-6000. 

[2] Burt S. Essential oils and their antibacterial properties and potential applications in foods-a review. Int J Food 

Microbiol 2004; 94: 223-253. 

[3] Bevilacqua A, Sinigaglia M, Corbo MR. Alicyclobacillus acidoterrestris: new methods for inhibiting spore 

germination. Int J Food Microbiol 2008; 125: 103-110. 

[4] Corbo MR, Bevilacqua A, Campaniello D, D’Amato D, Speranza B, Sinigaglia M. Prolonging microbial shelf life of 

foods through the use of natural compounds and non-thermal approaches-a review. Int J Food Sci Technol 2009; 44: 

223-241.

4 Application of Alternative Food-Preservation Technologies to Enhance Food Safety & Stability, 2010, 04-16 

Risk Assessment and Food Safety Objectives 

Antonio Bevilacqua*, Barbara Speranza and Milena Sinigaglia 




CHAPTER 2 

Abstract: Foodborne diseases are a global threat both in Developing and Industrialized Countries thus 

forcing Public Agencies to define some key-concepts for a correct management of the risk associated with 

foods. Food producers have always used an empirical approach; however, a new way of risk management and 

definition has been proposed since 1995 and labeled as Quantitative Risk Analysis (QRA). QRA is a 3-step 

process (risk management, risk analysis/assessment, risk communication) and results in the definition of some 

Public Goals, labeled as ALOP (Appropriate Level of Protection) and FSO (Food Safety Objectives), along 

with some intermediate parameters (performance objectives and criteria, process criteria), useful to maintain 

the risk below a certain threshold. 

The chapter proposes a brief description of the main steps of QRA, along with some-key concepts to define 

microbiological criteria for foods. 

Key-concepts: how to achieve health protection (quantitative risk analysis: QRA, risk-benefit analysis, risk 

categorization); the steps of QRA; food safety Objectives (FSO) and appropriate level of protection (ALOP); 

microbiological criteria and sampling plans 

ACHIEVING A SUFFICIENT LEVEL OF HEALTH PROTECTION 

Foodborne diseases are a global threat, due to the increase of international travel and trade, changes in human 

demographics and behaviour, as well as a result of microbial adaptation and changes in food production chain 

[1]; in fact, the consumption of foods and water contaminated with pathogens is generally considered as the 

leading cause of illness and death in less developed countries [2] and is responsible of ca. 1.9 millions deaths 

annually in the world [3]. In addition to these data, it has been estimated that up one third population in 

developed countries usually suffers a foodborne disease [3] and that bacteria (Listeria monocytogenes, 

Staphylococcus aureus, Clostridium botulinum and Cl. perfringens, Bacillus cereus, Escherichia coli, 

Salmonella sp., Yersinia enterocolitica, Campylobacter jejuni) are responsible for about 60% of illness. 

Food producers have always used either empirical or experimental approaches for the evaluation of the risk 

associated with their products, based on a simple scheme [4]: 

1. What is wrong? 

2. Who knows and what is known about the topic? 

3. What are the options of the control? 

4. Which one should be used for action, from the options? 

5. Who needs telling about our decision? 

6. What will we do? 

These questions can be considered as important issues at Country level and need to be solved, in order to achieve 

a sufficient level of health protection; therefore, since the 1980s traditional tools for determining hazard 

associated with food have been developed into a formal system with well defined stages and procedures. These 

methods, based on the SPS Agreement (Sanitary and Phitosanitary Measures Agreement) [5] (see Table 1), were 

expressed as a principle by the European Union in 2001 (precautionary principle) (see box 2.1) and can be 

grouped into 3 classes: 

Quantitave risk analysis (QRA) 

Risk or food-factory categorization 

The risk-benefit analysis 

*Address correspondence to this author Antonio Bevilacqua at: Department of Food Science, Faculty of Agricultural Science, University 

of Foggia, Italy; E-mail: a.bevilacqua@unifg.it; abevi@libero.it

Risk Assessment Application of Alternative Food-Preservation Technologies 5 

The QRA was proposed by the Codex Alimentarius, as decision process to maintain hazard below a defined 

level; it is generally used both at International and Country Levels as a mean for the definition and assessment of 

health policies, through the evaluation of the sanitary risks associated with some hazards (microorganisms, 

toxins, chemicals…) by means of epidemiological data, predictive modeling and challenge tests. It is followed 

by a phase or risk communication (from the scientists to the stakeholders and vice versa) and risk management 

(definition of standards and guidelines). The following sections of this chapter focus on this kind of approach. 

The risk categorization, known also as food-factory categorization, was introduced in the EU by the regulation 

882/2004 for food of animal origin (milk, meat, egg) and is based on the evaluation and classification of a food 

factory as a function of the risk associated. This classification is aimed to the establishment of the number of the 

official controls for each producer and for the individuation of the weak point of the food chain. 

The regulation states that a flow-chart should be assessed for each group of food-producers (or better for each 

food-producer), with the aim of combining and evaluating the risk associated with production and the 

effectiveness of the control measures. This flow chart uses 9 different parameters, as follows: 

Potential hazard (microbiological, chemical, physical…), as a function of the product. 

Food processing and raw material. 

Target (consumers using the product). 

Amount of food produced by the industry. 

Risks for the human health. 

Animal wellness. 

Manufacturing practices. 

Hygiene into the environment. 

Efficacy of the risk management system. 

The use of the flow chart results in a numerical rank, which defines: 

1. the risk associated with the particular food producers and food-product; 

2. the number of official and internal controls needed to assure a sufficient level of health protection; 

3. the weak points of the chain that should be checked. 

The risk-benefit analysis is an approach quite similar to the QRAs and is generally proposed and used for the 

evaluation of the acceptable daily intake and toxic levels of nutrients and chemicals; this kind of evaluation has 

been recently proposed by the European Food Safety Agency (EFSA) also for the microbiological hazards. 

The risk-benefit analysis is based on some simple assumptions (Fig. 1): 

Everyone has a propensity to take risks. 

This propensity varies amongst individuals. 

Perception of risk is influenced by experience of accidents. 

Individual risk taking decisions represent balancing act in which perceptions of risk are weighed 

against propensity to take risk. 

By definition accidents are a consequence of taking risk; the more risks a person takes, the greater 

will be both the rewards and losses he/she incurs. 

As regards the use of the risk-benefit analysis throughout food microbiology and food science, the evaluation of 

the hierarchy of risks is performed through a parameter called DALYs (e.g. Disability Adjusted Life), defined as 

the sum of the years loosen as a consequence of the hazards and the years affected by a “disability” [6].


Propensity 

to take risk 

Perceived 

danger 

Figure 1: Scheme of the risk-benefit analysis. 

Table 1: Focus on SPS Agreement. 

Perceptual filters 

Balancing 

behaviour 

What SPS: Sanitary and Phytosanitary Measures Agreement. 

Perceptual filters 

Rewards 

Accidents 

Where The Agreement applies to all sanitary and phytosanitary measures that may affect international trade. 

Why 1. Maintain the sovereign right of its member government to provide an appropriate level of health 

protection (ALOP) 

2. Ensure that the ALOP does not form unnecessary barriers 

How 1. Measures should be based on risk assessment standards, provided by international organizations 

(e.g. Codex Alimentarius, OIE, IPPC)* 

2. The SPS measures applied in different countries should be accepted as equivalent if they provide 

the same level of health protection 

*OIE, World Organization for Animal Health; IPPC, Secretariat of the International Plant Protection Convention of FAO 

BOX 2.1: The precautionary principle in the EU. 

1) The precautionary principle allows authorities to adopt and maintain provisional measures on the available 

pertinent information to protect human health, in situations when complete scientific information is absent 

and available data are insufficient for a comprehensive risk assessment [7]. 

2) As a prerequisite, the measure that has been set according to the principle should be revised within a 

reasonable period of time [7]. 

3) The principle was initially developed in the context of environmental policy in the 1970s and recognized 

in the Rio declaration in 1992. 

4) The EU incorporated the principle in the Treaty of the European Union, as a basic rule for European 

environmental policy. 

5) The Regulation 178/2002 of EU adopted the precautionary principle as an option open to risk managers 

when a decision on human health should be made, but the scientific information are not exhaustive or 

incomplete in some way. 

6) The Regulation stated that the principle may be adopted provisionally until a complete risk assessment. 

AN OVERVIEW ON THE QUANTITATIVE RISK ANALYSIS 

According to the Codex Alimentarius, the microbiological risk analysis, or quantitative risk analysis (QRA), is a 

complex process, consisting of risk assessment, risk management and risk communication [8] and involving a 

network amongst risk assessors, risk managers, operators and other interested parties.


Food Spoilage and Safety: Some Key-concepts 

Barbara Speranza, Antonio Bevilacqua* and Milena Sinigaglia 




CHAPTER 3 

Abstract: Several mechanisms cause deterioration of food and limit their shelf life: biochemical or microbial 

decay, chemical changes - especially oxidation (rancidity of fats, respiration of fruits and vegetables, 

discoloration, vitamin loss) -, physical deterioration (moisture migration, water loss or uptake). Therefore, 

food spoilage is a complex phenomenon, involving physical, chemical, microbiological and biochemical 

changes. As all involved factors and mechanisms operate interactively and often unpredictably, it is very 

difficult to predict shelf life of food products precisely. In addition, due to the high diversity of food products, 

there is no standard method for determining shelf life. 

The sections of this chapter try to supply some key-concepts about food spoilage and food safety with a 

particular focusing on the microbiological aspects. A brief synopsis of the bacterial pathogens mostly involved 

with foodborne outbreaks and of the main spoiling microflora of foods is given. As there are several ways to 

detect microbial spoilage in foods, - i.e. the microbiological methods, chemical/physical/physiochemical 

methods, acceptability criteria, including sensory determinations (color, texture, odor, flavor and general 

appearance)-, some details for each kind of approach are reported. 

Key-concepts: What is food shelf life, Foodborne pathogens, Food spoilage, Food structure. 

FOOD SHELF LIFE 

There is not a generally accepted definition of the term shelf life; hereby, we can report the most important 

definitions. In particular, Hine [1] defined shelf life as “the duration of that period, between packing a product 

and using it, for which the quality of the product remains acceptable to the product user”. Another interesting 

definition of the term shelf life is that reported by Labuza and Taoukis [2], i.e. “the shelf life is the period in 

which the food will retain an acceptable level of eating quality from a safety and organoleptic point of view”. 

Focusing on the definition of regulatory agencies, the Institute of Food Science and Technology (UK) proposed 

the following definition: the shelf life is “the time during which the food product will remain safe, be certain to 

retain the sensory, chemical, physical and microbiological characteristics, and comply with any label 

declaration of nutritional data” 

FAO (Food and Agricultural Organizations of the United States) and WHO (World Health Organization) 

provided a more useful definition, without referring directly to the term shelf life; in particular, they introduced 

three basic concepts: 

1. the Sell-by-Date, defined as the last date of offer for sale to the consumer after which there 

remains a reasonable storage period in the home; 

2. the Use-by-Date, defined as the recommended last consumption date; 

3. the Best-before-Use (or Date of Minimum Durability), which is the end of the period under any 

stated conditions during which the product will remain fully marketable and retain any specific for 

which tacit or explicit claims have been made; this definition complies with the generally accepted 

meaning of the term shelf life. 

The actual shelf life of a product depends generally on four factors: a) formulation; b) processing; c) packaging 

and d) storage conditions; these elements can be considered crucial, but their relative importance relies on the 

kind of foods. 

Several mechanisms cause deterioration of food and limit shelf life, i.e.: 

1. biochemical or microbial decay; 

2. chemical changes, especially oxidation (rancidity of fats, respiration of fruit and vegetables, 

discoloration, vitamin loss); 


of Foggia, Italy; a.bevilacqua@unifg.it; abevi@libero.it

18 Application of Alternative Food-Preservation Technologies Speranza et al. 

3. physical deterioration (moisture migration, water loss or uptake). 

As all stated factors and mechanisms operate interactively and often unpredictably [3], it is very difficult or even 

impossible to predict shelf life of food products precisely. Due to the high diversity of food products, there is no 

standard method for determining shelf life. However, most manufacturers have developed their own protocols 

for shelf life prediction and the main approaches are: 

1. the shelf life estimated is based on literature data; 

2. the distribution time of similar products can be used as an indication for the determination of the 

shelf life; 

3. challenge tests, with or without the inoculation of target microorganisms, under conditions 

simulating the storage and distribution; 

4. consumer claims; 

5. accelerated shelf life tests. 

The need to extend the shelf life of products arises from several reasons, as for example: 

more convenience to rely on products with longer shelf life that can be stocked at home, thus 

avoiding frequent shopping; 

increasing transportation times of fresh products due to the growing importance of economies-ofscale 

in production and to the trend to more and more exotic products; 

consumers demand seasonal products to be available throughout the year, and so on. 

However, consumers associate very long shelf lives with poor product quality [3] and expect food products with 

more sensory appeal and less additives as well as optimized minimal processing. When looking specifically at 

perishable products, their shelf life is mainly determined by the ability to control microbial growth killing the 

microorganisms (e.g. by heat or radiation) and/or limiting their growth (by reducing the temperature, reducing 

water activity or adding preservatives). The following sections of this chapter focus on some key-concepts about 

food spoilage and food safety. 

FOODBORNE PATHOGENS 

A pathogen is an organism able to cause cellular damage by establishing in tissue, which results in clinical signs 

with an outcome of either morbidity (defined by general suffering) or mortality (death) [4]. The class of the 

foodborne pathogens can be divided into 4 groups, as follows: 

1. Bacteria (Aeromonas hydrophila, Bacillus anthracis, B. cereus/subtilis/licheniformis, Brucella 

abortis/melitensis/suis, Campylobacter jejuni/coli, Clostridium perfringens/botulinum, Escherichia 

coli, Enterobacter sakazakii, Listeria monocytogenes, Mycobacterium paratubercolosis, Salmonella 

enterica, Shigella spp., Staphylococcus aureus, Vibrio cholerae/parahaemolyticus/vulnificus/ 

fluvialis, Yersinia enterocolitica); 

2. Moulds (Aspergillus spp., Fusarium spp., Penicillium spp.); 

3. Virus (Astrovirus, Hepatitis A virus, Hepatitis E virus, Norovirus, Rotavirus); 

4. Parasites (Cryptosporidium parvum, Cyclospora cayatanensis, Entamoeba histolytica, Giardia 

intestinalis/lamblia, Isospora belli, Taenia solium/saginata, Toxoplasma gondii, Trichinella 

spiralis). 

Hereby we will focus on the bacterial pathogens that are the main etiological agents of the foodborne outbreaks. 

Pathogens are responsible for food intoxication (ingestion of preformed toxin), toxicoinfection (toxin is 

produced inside the host after the ingestion of the cells) and infection (due to the ingestion of live cells). Food 

intoxication is caused by Staph. aureus, Cl. botulinum and B. cereus; toxicoinfection is caused by Cl. 

perfringens, enterotoxinogenic E. coli and V. cholerae. Finally, foodborne infection is caused by Salm. enterica, 

Camp. jejuni, enterohemorragic E. coli, Shigella spp., Y. enterocolitica, L. monocytogenes, viruses and parasites.

Food Microoganisms Application of Alternative Food-Preservation Technologies 19 

Some pathogens are referred as primary pathogens; otherwise, there are some microorganisms labeled as 

opportunistic pathogens, as they infect immuno-compromised individuals. Nevertheless, both the primary and 

the opportunistic pathogens show some basic attributes, known as “attributes of pathogenicity” (Table 1) and a 

common mechanism of pathogenesis (Table 2). 

Randell and Whitehead [5] divided food-borne pathogens and parasites into three classes as a function of the 

hazard associated with human health diffusion. The classification is the following: 

1. Category 1 (severe hazards), including Cl. botulinum types A, B, E and F, Sh. dysenteriae, Salm. 

enterica servovars Paratyphi A and B, E. coli EHEC, Hepatitis A and E viruses, Br. abortis, Br. 

suis, V. cholerae O1, V. vulnificus, T. solium. 

2. Category 2 (moderate hazards, potentially extensive spread), including L. monocytogenes, 

Salmonella sp., Shigella spp., E. coli, Streptococcus pyogenes, Rotavirus, Norwalk virus group, E. 

histolytica, Diphyllobothrium latum, Ascaris lumbricodes, C. parvum. 

3. Category 3 (moderate hazards, limitate spread), including B. cereus, Camp. jejuni, Cl. perfringens, 

Staph. aureus, V. cholerae non O1, V. parahaemolyticus, Y. enterocolitica, G. lamblia, T. sagitata. 

Many foodborne pathogens are ubiquitous and generally recovered in soil, humans, animals and vegetables; they 

are introduced into an equipment through the raw material or by humans, due to a not correct manipulation and 

low level of hygiene standards. 

A topic of great interest, reported by Bhunia [4], is the persistence of foodborne pathogens on the surfaces of 

equipments; the data available for some pathogens are the following: 

Camp. jejuni, up to 6 days; 

E. coli, 1.5h-16 months; 

Listeria spp., 24h-several months; 

Salmonella Typhi, 6h-4 weeks; 

Salmonella Typhimurium, 10 days-4.2 years; 

Shigella spp., 2 days-5 months; 

Staph. aureus, 7 days-7 months. 

Foodborne Pathogens and Indicators 

In some cases microbiological criteria (MC) for food safety propose the evaluation of indicator microorganisms, 

rather than of the pathogen of concern; for example E. coli in drinking water is the sign of fecal contamination 

and can indicate the possible presence of other pathogens [6]. 

An indicator microorganism should possess at least 7 characteristics: 

Detectable easily and in a short time. 

Distinguishable from the naturally occurring microflora. 

Always associated with the pathogen under investigation. 

Its cell number is correlated with the contamination due to pathogen. 

Growth requirements equal (or similar) to those of the pathogen. 

Growth kinetic similar to that of the pathogen. 

Absent when the pathogen is absent. 

The classical example of the indicator microorganisms is that of the fecal coliforms.




CHAPTER 4 

Essential Oils for Preserving Perishable Foods: Possibilities and Limitations 

Barbara Speranza* and Maria Rosaria Corbo 


Abstract: Since the middle ages, essential oils (EOs) have been widely used for bactericidal, virucidal, 

fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications. Nowadays, it is well known that 

EOs can enhance the shelf life of unprocessed or processed foods because of their antimicrobial nature. 

Nevertheless, very few preservation methods based on EOs utilization are implemented until now by the food 

industry. 

The aims of this chapter are: 1) to make an overview of the current knowledge on the antibacterial activity of 

EOs; 2) to describe their possible modes of action; 3) to evaluate possibilities and limitations of their use in 

the food industry. 

In vitro studies have demonstrated antibacterial activity of EOs against a wide range of spoilage and 

pathogenic bacteria. As EOs comprise a large number of components, it is likely that their mode of action 

involves several targets in the bacterial cell, but it is generally recognized that their hydrophobicity enables 

them to partition in the lipids of the cell membrane and mitochondria, rendering these membranes permeable 

and leading to leakage of cell contents. A higher concentration is generally needed to achieve the same effect 

in foods, but studies with meat, fish, milk, dairy products, vegetables and fruits have shown promising results 

at very low concentrations of EOs (

36 Application of Alternative Food-Preservation Technologies Speranza and Corbo 

canal sealers, antiseptics and feed supplements [7]. Because of the recent greater consumer awareness and 

concern regarding synthetic chemical additives, EOs (and their components) are gaining more and more ground 

as antibacterial additives for food preservation. Table 1 reports a list of some EOs, their origin (Latin name of 

plant source) and their current prevalent use. In Table 2 these oils are grouped according to the plant section 

from which they are produced. 

Table 1: Some examples of EOs with their origin (Latin name of plant source) and their current prevalent use. 

Oil of Origin Plant Current Prevalent Use 

Agar Aquilaria malaccensis Perfume industries 

Ajwain Trachyspermum copticum Digestive and antiseptic 

Angelica Angelica archangelica Medicinal applications 

Anise Pimpinella anisum Pharmaceutical industries 

Assafoetida Ferula assafoetida Pharmaceutical and food industries (as flavouring agent) 

Balsam Myroxylon pereirae Botanical medicines 

Basil Ocimum basilicum Perfume industries, aromatherapy 

Bay laurel Laurus nobilis Perfume industries, aromatherapy, flavouring in foods 

Bergamot Citrus bergamia risso Perfume industries, aromatherapy 

Black Pepper Piper nigrum Pharmaceutical industries 

Cannabis Cannabis sativa 

Caraway Carum carvi 

Flavouring in foods, primarily candies and beverages. Scent in perfumes, 

cosmetics, soaps, and candles 

Flavouring in foods. Also used in mouthwashes, toothpastes, etc. as a 

flavouring agent 

Cardamom Amonum elettaria 

Aromatherapy and other medicinal applications. Also used as a fragrance 

in soaps, perfumes, etc. 

Carrot Daucus carota Aromatherapy 

Cedarwood 

Cedrus deodara 

Cedrus libani 

Perfumes and fragrances 

Chamomile Anthemis nobilis Aromatherapy as anti-inflammatory agent 

Calamus Acorus calamus Pharmaceutical industries 

Cinnamon Cinnamomum zeylandicum Pharmaceutical and food industries (as flavouring agent) 

Citronella 

From leaves and stems of: 

Cymbopogon nardus 

Cymbopogon winterianus 

Insect repellent. Also medicinal applications 

Clary Sage Salvia sclarea 

Flavouring in foods. Also used in mouthwashes, toothpastes, etc. as a 

flavouring agent 

Clove Syzygium aromaticum Pharmaceutical and food industries (as flavouring agent) 

Coriander Coriandum sativum Cooking 

Costmary Tanacetum balsamita Medicinal applications 

Costus About 24 Costus species Pharmaceutical industries 

Cranberry 

Vaccinium erythrocarpum 

Vaccinium macrocarpon 

Vaccinium microcarpum 

Vaccinium oxycoccos 

Cosmetic industries 

Cubeb Piper cubeba Pharmaceutical and food industries (as flavouring agent) 

Cumin Cuminum cyminum 

Flavouring in foods, particularly in meat products. Also used in veterinary 

medicine 

Cypress Juniperus virginiana Used as a perfume and medicine ingredient 

Cypriol Cyperus scariosus Aromatherapy 

Curry Murraya koenigii Pharmaceutical and food industries 

Davana Artemisia pallens Used as a perfume ingredient and as a germicide 

Dill Anethum graveolens Medicinal applications 

Elecampane Inula helenium Medicinal applications 

Eucalyptus 

About 746 Eucalyptus species 

and 3 hybrids 

Historically used as a germicide. Commonly used in cough medicine, 

among other medicinal uses 

Fennel Foeniculum vulgare Medicinal applications 

Fenugreek Trigonella foenum- graecum Pharmaceutical and cosmetic industries

Essential Oils in Foods. Application of Alternative Food-Preservation Technologies 37 

Frankincense Boswellia dalzielii Aromatherapy and perfume industries 

Galangal 

Alpinia galanga 

Alpinia officinarum 

Kaempferia galanga 

Pharmaceutical and food industries (as flavouring agent) 

Galbanum 

Ferula gummosa 

Ferula rubricaulis 

Medicinal applications 

Geranium About 200 Pelargonium species Medicinal applications 

Ginger Zingiber officinale Medicinal applications 

Goldenrod Solidago virgaurea Medicinal applications 

Grapefruit Citrus x paradisi Aromatherapy 

Henna Lawsonia inermis Medicinal applications 

Helichrysum Helichrysum augustifolium Perfume industries 

Hyssop About 11 Hyssopus species Herbal remedies and culinary use 

Jasmine About 10 Jasminum species Perfume industries 

Juniper About 67 Juniperus species Pharmaceutical and food industries (as flavouring agent) 

Lavender About 39 Lavandula species Pharmaceutical and perfume industries 

Ledum (current name: 

Rhododendron) 

About 8 Rhododendrum species Herbal uses 

Lemon Citrus limon Used medicinally, as an antiseptic, and in cosmetics 

Table 1: cont.... 

Lemongrass About 55 Cymbopogon species 

It is widely used as a herb in Asian cuisine (dried and powdered or used 

fresh). It is commonly used in teas, soups, and curries. It is also suitable 

for poultry, fish, and seafood. It is often used as a tea in African and Latin 

American countries (e.g., Togo, Mexico, DR Congo). Also used as insect 

repellent 

Lemon balm Litsea cubeba Perfumes and aromatherapy 

Marjoram Origanum majorana Pharmaceutical and perfume industries. Also used as flavour 

Melissa Melissa officinalis Medicinal applications, particularly aromatherapy 

Mint Mentha arvensis 

Used in flavouring toothpastes, mouthwashes and pharmaceuticals, as well 

as in aromatherapy and other medicinal applications 

Mountain Savory Satureja montana Medicinal applications. Culinary uses 

Mugwort Artemisia vulgaris 

Mustard 

Myrrh 

Myrtle 

Brassica nigra 

Brassica juncea 

Brassica hirta 

Commiphora myrrha 

Commiphora opobalsamum 

Myrthus communis 

Myrthus nivellei 

Used in ancient times for medicinal and magical purposes. Currently 

considered to be a neurotoxin 




Neroli 

From blossom of Citrus 

aurantium 

Medicinal applications. Aromatherapy 

Nutmeg Myristica fragrans Culinary uses 

Orange Citrus x sinensis Used as a fragrance, in cleaning products and in flavouring foods 

Oregano Origanum vulgare Fungicide. Also used to treat digestive problems 

Orris oil Iris florentina Flavouring agent; also used in perfume, and medicinally 

Parsley 

Petraselinum crispum var. 

neapolitum 

Patchouli Pogostemon cablin Perfume industries 

Perilla Perilla frutescens var. japonica Medicinal applications 

Used in soaps, detergents, colognes, cosmetics and perfumes, especially 

men’s fragrances 

Pennyroyal Mentha pulegium 

Highly toxic. It is abortifacient and can even in small quantities cause 

acute liver and lung damage 

Peppermint Mentha piperita Medicinal applications. Flavouring agent 

Petitgrain 

From leaves of : 

Citrus aurantium 

Flavouring agent; also used in perfume 

Pine oil Pinus sylvestris Used as a disinfectant, and in aromatherapy 

Ravensara Ravensara aromatica Used primarily as a fragrance, but also as antiseptic and antibacteric 

Rose Rosa damascena Used primarily as a fragrance


Enzymes and Enzymatic Systems as Natural Antimicrobials 

Daniela D’Amato, Daniela Campaniello and Milena Sinigaglia* 




CHAPTER 5 

Abstract: Lysozyme is a hydrolytic enzyme which has been purified from cells, secretions and tissues of 

virtually all living organisms and viruses. Lysozyme has been shown to have antimicrobial activities towards 

bacteria, fungi, protozoan and viruses; moreover, it is essentially known for its antibacterial activity and has 

been used in food preservation. Lysozyme is currently used as a preservative in many foods, such as cheese, 

fish, meat, fruit, vegetables and wine. 

Lactoferrin is a globular multifunctional protein with antimicrobial activity. It is produced by mucosal 

epithelial cells in various mammalian species. It is found in mucosal surfaces and in biological fluids, 

including milk and saliva. It possesses a strong antimicrobial activity against a broad spectrum of bacteria, 

fungi, yeasts, viruses and parasites. Lactoferrin is used in a wide range of products including infant formulae, 

sport and functional foods. 

The lactoperoxidase system (LPS) consists of three components: enzyme lactoperoxidase, thiocyanate and 

hydrogen peroxide. LPS exerts both bacteriostatic and/or bactericidal activity; therefore, its use in dairy 

industry to preserve raw milk quality or to extend the shelf life of pasteurized milk has been extensively 

proposed in the past. LPS has also been used the stabilization cream, cheese, liquid whole eggs, ice cream, 

infant formula and for the preservation of tomato juice, mangoes and chicken. 

Key-concepts: Lysozyme, Lactoferrin, Lactoperoxidase, Mode of action and application in foods. 

LYSOZYME: STRUCTURE AND PROPERTIES 

LYSOZYME 

Lysozyme is a hydrolytic enzyme which has been purified from cells, secretions and tissues of virtually all living 

organisms and viruses. It is well known as an antimicrobial protein, used as a natural food preservative. 

Lysozyme is a 129-amino acid protein with a molecular weight of approximately 14.7 kDa, and with hydrolytic 

activity against ß (1–4) glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine in bacterial 

peptidoglycan [1]. In addition, it has been reported to have an antibacterial activity that is independent of its 

enzymatic activity [2]. Lysozyme was discovered in 1922 by Flemming in human nasal secretion and 

subsequently purified from various plant, animal, microbial (bacteria, virus and fungi) materials [3-5]. It is 

naturally present in many foods such as egg white (with a concentration ranging between 3.2 and 5.8 mg/ml), 

cow milk (ca. 0.13 mg/ml) and human colostrum (ca. 65 mg/ml) but also in several plants, such as cauliflower 

(ca. 27.6 mg/ml) and cabbage (ca. 2.3 mg/ml) [6], and widely distributed in various biological fluids and tissues 

including plant, bacteria, and animal secretions, tears, saliva, respiratory and cervical secretions, and secreted by 

polymorphonuclear leukocytes [7]. 

Lysozyme constitutes a natural defence against bacterial pathogens. Many bacteriophages also produce lysozyme 

that locally hydrolyses the peptidoglycan to facilitate penetration of the phage injection apparatus, or that 

induces cell lysis at the end of the phage replication cycle. 

Lysozyme has an extremely high isoelectric point (>10) and consequently is highly cationic at neutral or acid 

pH. In solution, it is relatively stable at pH 3-4, but when pH increases its stability decreases. 

Several classes of lysozyme have been defined on the basis of the wide variability in origin, and structural, 

antigenic, chemical and enzymatic properties of the molecules. In Table 1 some of the different types of 

lysozyme are reported. The most studied and the best known is the conventional or chicken-type (i.e. clysozyme) 

with the lysozyme derived from the egg white of domestic chicken (Gallus gallus) as the prototype 

[8]. Although, c-lysozyme is typically found in the egg white of birds, it was also purified from various tissues 

and secretions of mammals. 



Enzymes as Antimicrobials Application of Alternative Food-Preservation Technologies 59 

Table 1: Different types of lysozyme. 

Lysozyme types Provenience References 

Conventional or chicken-type 

c-lysozyme 

-egg white of domestic chicken (Gallus gallus); 

-purified from various tissues and secretions of mammals including 

milk, saliva, tears, urine, respiratory and cervical secretions 

Other types of lysozyme: 

[3, 5, 9] 

g-type lysozyme egg white of domestic goose [4, 9-11] 

h-type lysozyme Plant 

i-type lysozyme Invertebrates 

b-type lysozyme Bacteria (Bacillus) 

v-type lysozyme Viruses 

Despite the variability in the amino acid composition and sequence of lysozyme molecules, amino acids of the 

catalytic centre of the active site are well conserved [9]. 

In 1965 the structure of lysozyme was solved by X-Ray analysis with 2 angstrom resolution by David Chilton 

Phillips. In particular, lysozyme structure is characterized by an α-helix which links two domains of the 

molecule; one is mainly β-sheet in structure, and the other primarly α-helical. The hydrophilic groups are mainly 

oriented inward, with most of the hydrophilic residues on the exterior of the molecule [12]. 

It has been proposed that the enzymatic action of the molecule is dependent on its ability to change the relative 

position of its two domains and cause large conformational changes in the molecule. 

In particular, glutamic acid and aspartic acid residues are directly involved in the breakdown of the glycosidic 

bond between N-acetylglucosamine and N-acetylmuramic and their presence in the catalytic centre is thus 

crucial for the hydrolytic activity of the enzyme. However, the amino acid sequence of known lysozymes reveals 

that aspartic acid is not consistently present in the active site of lysozyme molecules [8]. In contrast, the 

substitution of glutamic acid results in a complete inactivation of the enzyme [8], thus confirming the critical 

role of this amino acid in the enzymatic activity of lysozyme [9]. 

ANTIMICROBIAL EFFECT AND MODE OF ACTION OF LYSOZYME 

Lysozyme has been recognized to possess many physiological and functional properties, including important 

roles in surveillance of membranes of mammalian cells; it enhances phagocytic activity of polymorphonuclear 

leukocytes and macrophages and stimulates proliferation and antitumor functions of monocytes [7], but its high 

microbicidal activity remains, by far, the main virtue that explains the high attention of scientists and industrial 

stakeholders for its practical applications in medicine and food industry [9]. 

The antimicrobial activity of lysozyme has been extensively demonstrated in vitro or in physiological fluids and 

secretions including milk, blood serum, saliva, and urine [13]. Although lysozyme has been shown to have 

antimicrobial activities towards bacteria, fungi, protozoan and viruses [14-16], it is essentially known for its 

antibacterial activity and has been used, on this basis, in food preservation. 

Lysozyme has been demonstrated to be active throughout a wide pH range of 4–10; however, high ionic strength 

(>0.2 M salt) was shown to have an inhibitory effect on lysozyme activity [2]. Under physiological conditions, 

only a minority of Gram positive bacteria are susceptible to lysozyme, and it has been suggested that the main 

role of lysozyme is to participate in the removal of bacterial cell walls, after the bacteria have been killed by 

antimicrobial polypeptides present in egg albumin, insect hemolymph [17] or by complement in animal serum 

[18]. This is in line with the notion that the lytic action of lysozyme does not kill susceptible bacteria under 

physiological conditions, osmotically balanced [19]. 

The bacteriostatic and bactericidal properties of lysozyme have been the subject of many studies, and over the 

last 10 years, several authors have proposed a novel antibacterial mechanism of action of lysozyme that is 

independent of its muramidase activity [20, 21]. A non-lytic bactericidal mechanism involving membrane

60 Application of Alternative Food-Preservation Technologies D’Amato et al. 

damage without hydrolysis of peptidoglycan, has been reported for c-type lysozymes, including human 

lysozyme and hen egg white lysozyme (HEWL) [7, 22, 23]. Other studies observed that the denatured lysozyme 

deprived of muramidase activity has an unique and potent microbicidal property [7, 24]. 

Antimicrobial Action Towards Gram Positive Bacteria 

Lysozyme belongs to a class of enzymes that lyses the cell walls of certain Gram positive bacteria, as it 

specifically splits the bond between N-acetylglucosamine and N-acetylmuramic acid of the peptidoglycan in the 

bacterial cell walls. Extensive hydrolysis of the peptidoglycan by exogenous lysozymes results in cell lysis and 

death in a hypo-osmotic environment but some exogenous lysozymes can also cause lysis of bacteria by 

stimulating autolysin activity upon interaction with the cell surface [23]. 

According to this effect, lysozyme affects strongly Gram positive bacteria but not Gram negative ones, which are 

shielded by the lipopolysaccharide (LPS). Nevertheless, recent studies suggest that resistance of bacteria to 

lysozyme is not exclusively related to the presence of the lipopolysaccharidic layer. In fact, Gram positive 

bacteria are generally sensitive to lysozyme because their peptidoglycan is directly exposed, but some of them 

are intrinsically resistant due to a modified peptidoglycan structure [25]. The occurrence of resistant Gram 

positive bacteria indicates that the lack of the LPS does not expose de facto the bacterium to lysozyme 

hydrolysis [22, 26-28]. Various mechanisms of resistance in Gram positive bacteria have been suggested, as the 

exact mechanism of lysozyme resistance is not fully understood and may vary according to the bacterial strain or 

species. Fig. 1 reports some of the suggested mechanisms of resistance to lysozyme in Gram positive bacteria. 

Hindrance of lysozyme 

action by surface 

attachment polymers 

Deacetylation of the 

amino group of Nacetylglucosamine 

residues 

Modification of hexosamine 

residues of the glycan 

backbone, by O-acetylation 

or N-deacetylation 

High degree of 

peptide crosslinking 

Suggested mechanisms 

of resistance in Gram- 

positive bacteria 

Production of proteininhibitors 

specific to 

lysozyme 

Figure 1: Possible mechanisms of resistance to lysozyme in Gram positive bacteria. 

Teichoic acid content 

in the cell-wall 

N-deacetylation of 

the acetamido group 

of the hexosamine 

residues 

Incorporation of Daspartic 

acid in the 

bacterial peptidoglycan 

crossbridge 

Several studies provided evidence that the modification of hexosamine residues of the glycan backbone, by Oacetylation 

or N-deacetylation, is the primary mechanism of resistance to lysozyme in Gram positive bacteria. 

Clarke and Dupont [29] were the first to suspect a possible role of O-acetylation of the peptidoglycan muramic 

acid in lysozyme resistance, and this idea was confirmed by other studies [25, 27]. 

A different bacterial strategy to evade the bactericidal action of lysozyme is the production of inhibitors. In the 

streptococci belonging to group A, a protein, identified as an inhibitor of the complement system and designated 

as SIC (streptococcal inhibitor of complement), was also shown to inhibit lysozyme [30, 31]. 

Other protein inhibitors may be produced by Gram positive bacteria, but they remain to be identified and 

characterized. Therefore, it is clear that one or more mechanisms of resistance can be involved. 

For example, deacetylation of the amino group of NAG residues appears to be a common mechanism of 

resistance in Bacillus and streptococci [26], while other bacteria (e.g., Staphylococcus aureus and lactobacilli) 

would counteract lysozyme action essentially by means of O-acetylation [28].


Antimicrobial Agents of Microbial Origin : Nisin 

Daniela D’Amato* and Milena Sinigaglia 




CHAPTER 6 

Abstract: Nisin belongs to a group of bacteriocins known as “lantibiotics”, small peptides produced by 

Gram-positive bacteria of different genera. Nisin consists of 34 amino acids and is the only commercially 

accepted bacteriocin for food preservation; it is produced by certain strains of Lactococcus lactis subsp. 

lactis. Nisin is a natural, toxicologically safe, antibacterial food preservative, characterized by an 

antimicrobial activity against a wide range of Gram-positive bacteria, but not against Gram-negative bacteria, 

yeasts or fungi. It can act against Gram-negative bacteria, in conjunction with chemically induced damage of 

the outer membrane. Nisin was labeled as GRAS (generally recognized as safe) in 1988 by FDA, and is 

currently permitted as a food additive in over 50 countries around the world. 

Nisin has found practical application as a natural food preservative in many categories of food, such as 

natural cheese (Emmental and Gouda), processed cheese (slices, spread, sauces and dips), pasteurized dairy 

products (milk, chilled desserts, clotted cream and mascarpone cheese), egg products, hot baked flour 

products (crupets), canned products, alcoholic beverages (beer and wine), salad dressing, meat and fish 

products, yogurt and pasteurized soups. 

Key-concepts: Mode of action, Safety, Food applications. 

INTRODUCTION 

Bacteria are a source of antimicrobial peptides, which have been examined for applications in microbial food 

safety. The antimicrobial proteins or peptides produced by bacteria are termed bacteriocins. They are 

ribosomally synthesized and kill closely related bacteria [1]. Many bacteriocins have a narrow host range, and 

are likely most effective against related bacteria competing for the same scarce resources. Although bacteriocins 

are produced by many Gram positive and Gram negative species, those produced by the Lactic Acid Bacteria 

(LAB) are of particular interest to the food industry, since these bacteria have generally been regarded as safe 

(GRAS status) [2]. 

Bacteriocins are used as a preservative in food due to its heat stability, wider pH tolerance and its proteolytic 

activity [3]. Bacteriocins have applications in hurdle technology, which utilizes synergies of combined 

treatments to preserve food more effectively. 

Bacteriocins have been grouped into four main distinct classes [4]. 

Class-I Lantibiotics characterized by the presence of unusual thioether amino acid which are 

generated through post translational modification. 

Class-II Bacteriocins represent small (30 kD) heat labile protein. 

Class-IV Represent complex bacteriocins that contain essential lipid, carbohydrate moieties in 

addition to a protein compared. 

NISIN: STRUCTURE AND PROPERTIES 

Nisin is the best known and widely used bacteriocin, employed as a food preservative, with a high antibacterial 

activity and a relatively low toxicity for humans. It is often used in various food system including dairy, meat 

*Address correspondence to this author Daniela D'Amato at: Department of Food Science, Faculty of Agricultural Science, University of 

Foggia, Italy; E-mail: d.damato@unifg.it

84 Application of Alternative Food-Preservation Technologies D’Amato and Sinigaglia 

and canning systems. The bacteriocin nisin (or group N inhibitory substance), discovered in England by Rogers 

and Whittier in 1928, is produced by certain strains of Lactococcus lactis subsp. lactis [5-7]. 

Nisin belongs to a group of bacteriocins known as “lantibiotics” (class-I). Lantibiotics are relatively small 

peptides produced by Gram positive bacteria of different genera as reported in the Table 1. 

Table 1: Lantibiotics produced from different bacteria. 

Producer bacteria Lantibiotics 

L. lactis Nisin (A and Z), lactacin 481 

Lactobacillus sake Lactocin S 

Staphylococcus Pep 5, epidermin, gallidermin 

Streptococcus Streptococcin A-FF22, salivaricin Av 

Bacillus Subtilin, mersacidin 

Carnobacterium Carnocin U149 

Streptomyces Duramycin 

Micrococcus varians Variacin 

The lantibiotics are a group of post-translationally modified peptide antibiotics that characteristically have cyclic 

structures formed by the rare thioether amino acids lanthionine and 3-methyl-lanthionine and often also by 

dehydroalanine (Dha) and/or dehydrobutyrine residues [8]. Nisin was the first member of this group of 

antibiotics. 

On the basis of their different ring structure, charge and biological activity, the lantibiotics are classified into two 

subgroups: type A (nisin type lantibiotics) constituted by elongated, amphiphilic peptides, and type-B (duramicin 

type lantibiotics) that are compact and globular. Nisin consists of 34 amino acids and is the only commercially 

accepted bacteriocin for food preservation. Its biosynthesis occurs during the exponential growth phase and stops 

completely when cells enter the stationary growth [9]. Nisin is a small (3.5 kDa) amphiphilic peptide that is 

cationic at neutral pH, having an isoelectric point above 8.5, and shares similar characteristics with other poreforming 

antibacterial peptides such as cationic peptides with a net positive charge and amphipathicity [5, 10]. It 

is overall positively charged (+4) and its structure possesses amphipathic properties; however, some structural 

properties make nisin rather special. Nisin is a ribosome-synthesized peptide characterized by intramolecular 

rings formed by the thioether amino acids lanthionine and 3-methyllanthionine [11, 12]. The structure of nisin, 

initially determined by chemical degradation [11] and confirmed by nuclear magnetic resonance (NMR) 

spectroscopy [13], is shown in Fig. 1; serine and threonine residues are dehydrated to become dehydroalanine 

and dehydrobutyrine. Subsequently, five of the dehydrated residues are coupled to upstream cysteines, thus 

forming the thioether bonds that produce the characteristic lanthionine rings. 

1 

Ile 

NH2 

Dhb 

COOH 

Ala 

Ile 

5 

Dha 

S 

Leu 

Ala 

Abu 

Pro 

Nisin A 

Lys Dha Val His Ile 

34 

30 

S 

Ala Lys Abu 

Ala 

Gly 

S Asn 

10 

20 

Met 

His 

S 

Lys 

Asn Ala 

Abu 

Ser Ala 

Abu Ala 

Figure 1: Structure of nisin. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, β-methyllanthionine. 

Two naturally occurring nisin variants that have similar activities, nisin A and nisin Z, have been found [12, 14]. 

Nisin A differs from nisin Z in a single amino acid residue at position 27, being a histidine in nisin A and an 

S 

Leu 

15 

Ala Met 

Gly 

25 

Gly

Nisin in Foods Application of Alternative Food-Preservation Technologies 85 

asparagine in nisin Z [14]. The structural modification has no effect on the antimicrobial activity, but it gives 

nisin Z higher solubility and diffusion characteristics compared with nisin A, which are important characteristics 

for food applications [15, 16]. 

The thioether bonds give nisin two rigid ring systems, a N-terminally and a C-terminally located; a hinge region 

(residues 20-22) separates the ring systems. Due to the ring structures, the nisin molecule is maintained in a 

screw-like conformation that possesses amphipathic characteristics in two ways: the N-terminal half of nisin is 

more hydrophobic than the C-terminal one; and the hydrophobic residues are located at the opposite side of the 

hydrophilic residues throughout the screw-like structure of the nisin molecule. 

Nisin production is affected by several cultural factors such as producer strain, nutrient composition of media, 

pH, temperature, agitation and aeration, as also by other factors, for example, substrate and product inhibition, 

adsorption of nisin onto the producer cells and enzymatic degradation [17]. 

ANTIMICROBIAL EFFECT AND MODE OF ACTION OF NISIN 

Nisin is a natural, toxicologically safe, antibacterial food preservative. It was shown to have antimicrobial 

activity against a wide range of Gram positive bacteria, including L. monocytogenes, together with different 

strains or species of streptococci, staphylococci, lactobacilli, micrococci and most spore-forming species of 

Clostridium, Bacillus and Alicyclobacillus, but not against Gram negative bacteria, yeasts or fungi. It can also act 

against Gram negative bacteria, such as Escherichia coli or Salmonella species, in conjunction with chemically 

induced damage of the outer membrane. 

Moreover, several works showed the antimicrobial activity of nisin against a number of food pathogens 

including, E. coli, Campylobacter jejuni, Cl. difficile, Helicobacter pylori, B. cereus, as well as Shigella and 

Enterococcus species [15, 18, 19]. The potent activity of nisin against a broad range of gastrointestinal pathogens 

indicates that the peptide could have therapeutic potential in the treatment of gastrointestinal infections. 

There are many hypotheses about the mechanism of action against spores and vegetative cells [20-22]. Initially, 

the antimicrobial activity of nisin was thought to be caused by reacting with sulfhydryl groups of enzymes via 

the dehydro residues [11], by inhibition of cell wall synthesis [23, 24] or by the strong adhesion to cells, causing 

leakage of cellular material and subsequent lysis, as a cationic surface-active detergent [25]. However, 

experiments with intact bacterial cells and isolated plasma membrane vesicles have shown that treatment of nisin 

resulted in rapid efflux of small cytoplasmic compounds [26, 27]. The mode of action of nisin is shown to 

involve interactions with the membrane-bound cell wall precursor lipid II (undecaprenylpyrophosphoryl- 

MurNAc-(pentapeptide)-GlcNac), concomitant with pore formation in the cytoplasmic membrane of the target 

organism [28, 29]. In particular, the C-terminal region of nisin binds to the cytoplasmic membrane of vegetative 

cells and penetrates into the lipid phase of the membrane [30], forming pores which allow the efflux of 

potassium ions, ATP, and amino acids [31-36], resulting in the dissipation of the proton motive force and 

eventually cell death [28, 37, 38]. It is now believed that the depletion of the proton motive force is the common 

mechanistic action of bacteriocins from lactic acid bacteria. Therefore, it is generally accepted that the bacterial 

plasma membrane is the target for nisin, and that nisin kills the cells by pore formation. 

Nisin’s effectiveness is concentration-dependent, in terms of both the amount of nisin added and the number of 

spores or vegetative cells that need to be inhibited or killed. Nevertheless, the effectiveness of nisin depends also 

on growth and exposure conditions, such as the temperature [33, 39, 40-42] and the pH [33, 39, 41]. In general, 

nisin is more active at lower pH values, whereas the influence of temperature on its effectiveness is 

controversial. 

Its action against vegetative cells can be either bactericidal or bacteriostatic, depending on a number of factors 

including nisin concentration, bacterial population size, physiological state of the bacteria and the condition of 

growth. 

The insensitivity of Gram negative bacteria to nisin could be due to the large size (1.8–4.6 kDa) of nisin, which 

restricts its passage across the outer membrane of Gram negative bacteria [43, 44]. The outer membrane, 

covering the cytoplasmic membrane and peptidoglycan layer of Gram negative bacteria, is composed of 

lipopolysaccharide (LPS) molecules in its outer leaflet and glycerophospholipids in the inner leaflet [45].


Chitosan: a Polysaccharide with Antimicrobial Action 

Daniela Campaniello* and Maria Rosaria Corbo 




CHAPTER 7 

Abstract: At present, discards from the world’s fisheries exceed 20 million tons. Traditionally, fisheries 

wastes are used in the production of fertilizers, fish silage or pet foods; nowadays with advances in 

bioprocess engineering technologies and novel enzymatic and microbial hydrolysis methods, processing 

wastes may serve as cheap raw materials for the generation of high-value bioactive compounds and novel 

environmental and ecological material derived from marine wastes. In particular, shellfish waste is the main 

source of biomass for chitin (and its derivatives) production. In this contest chitosan, thanks to its versatility, 

has found numerous applications as antimicrobial agents, antioxidants, additives, enzyme immobilization and 

use in the encapsulation of nutraceuticals. In addition, chitosan possesses a film-forming properties for use as 

edible films or coating. Several researchers studied chitosan, its chemical and physical characteristics and its 

applications. This chapter is an attempt to summarize these works focused on the following questions: what is 

chitosan? How does it act against microorganisms and what is its impact on food properties? 

Key-concepts: What is chitosan, How it acts against microorganisms to enhance shelf life of foods, What is its impact on 

food properties. 

BIOCHEMICAL CHARACTERISTICS, PREPARATION AND USE 

Chitosan is a natural polysaccharide, prepared by the alkaline deacetylation of chitin (found in fungi, arthropods 

and marine invertebrate); commercially, it is produced from exoskeletons of crustacean such as crab, shrimp and 

crawfish. 

Structurally chitin is a straight-chain polymer composed of β-1,4-N-acetylglucosamine (Fig. 1); chitosan, is also 

a straight-chain polymer composed of N-acetylglucosamine and glucosamine [1] and like the cellulose is one the 

most abundant natural polysaccharide on the earth [2]. 

Hydroxyl‐ group 

Amino‐ group 

Acetyl‐ group 

Figure 1: Structure of chitin, chitosan and cellulose 

A variety of processes have been proposed for the preparation of chitosan: generally, this biopolymer is prepared 

by N-deacetylation of chitin. In particular, the procedure consists of four basic steps: deproteinization (DP), 

deminarilization (DM), decolouration (DC) and deacetylation (DA) (Fig. 2), where DP and DM are 

*Address correspondence to this author Daniela Campaniello at: Department of Food Science, Faculty of Agricultural Science, 

University of Foggia, Italy; E-mail: d.campaniello@unifg.it

Chitosan in foods Application of Alternative Food-Preservation Technologies 93 

interchangeable in terms of order, depending on the proposed use of chitin. DP and DM steps produce a coloured 

product, but if a bleached chitinous product is desired, pigments can be removed with reagents such as ethanol, 

ether, sodium hypochlorite solution, absolute acetone, chloroform, hydrogen peroxide or ethyl acetate. This 

process is too expensive; however removing the DC step could reduce considerably production cost. It is 

important to point out that the use of bleaching agents reduce considerably the viscosity of the chitosan and 

sometime cause an undesirable light brown colour; therefore Youn et al. [3] studied an alternative and economic 

decolouration method, that yields decolourized chitosan with high viscosity through the use of sun drying. 

N-deacetylation involves an alkaline hydrolysis with sodium hydroxide or potassium hydroxide at elevated 

temperature under heterogeneous conditions, which can result in an incomplete N-deacetylation and in a 

depolymerization to varying extents, thus obtaining chitosans with different molecular weight (high, medium 

and low molecular weight). 

The degree of deacetylation is defined as the percentage of acetylated monomers referred on the total units; it is a 

function of alkali concentration, temperature, size of particles and reaction time. For example, it is well known 

that an ordinary reaction time (1 h) leads to a partial deacetylation (about 80%), whereas a reaction time of 48 h 

results in a complete deacetylation. However, a high degree of deacetylation (realised in drastic condition) causes 

a reduction of molecular weight of polymer. 

Yen et al. [4] prepared chitosan from shiitake (Lentinula edodes (Berkeley) Pegler) stipes, a potential source of 

fungal chitosan, usually discarded due to their tough texture. They isolated fungal chitin from stipes using 

alkaline treatment, followed by a decolourization with potassium permanganate and a N-deacetylation treatment 

with a sodium hydroxide solution. 

Aqueous base solutions 

(NaOH or KOH) are used 

for the DP step and the 

effectiveness depends on 

the ratio shell/solution, 

temperature, 

concentration of alkali and 

reaction time. 

Shellfish waste 

deproteinization 

demineralization 

decolouration 

chitin 

deacetylation 

chitosan 

DM can be achivied using 

diluted HCl (1‐8%) at 

room temperature for 1‐3 

h, or other acids such as 

acetic and sulfuric acids 

Figure 2: Simplified flowsheet for the preparation of chitosan, from shellwish waste. (modified from Shahidi et al. [5]) 

The use of chemicals throughout chitosan preparation has several disadvantages like a complicated recovery of 

shell-waste products (proteins, pigments, etc.) or the generation of large quantities of hazardous chemical waste. 

Fermentations with proteolytic or chitinolytic enzymes may be an alternative with varying levels of success: for 

example, chitin deacetylase from either Mucor rouxii or Absidia butleri and Aspergillus nidulans convert chitin 

to chitosan. Hayes et al. [6] reported a detailed review on the methods used to extract and characterize chitin, 

chitosan and glucosamine obtained through industrial, microbial and enzymatic hydrolysis of shell waste. 

The degree of deacetylation depends on both the raw material, from which chitin has been obtained, and the 

procedure influences the fraction of free amino groups, that can interact with metal ions. Chitosan is a waterinsoluble 

compound; however, when the degree of deacetylation is larger than 40-50%, chitosan becomes 

soluble in acidic media [7]. Although the distribution of acetyl groups along the chain may modify the solubility,

94 Application of Alternative Food-Preservation Technologies Campaniello and Corbo 

the solubilization occurs by protonation of the NH2 groups on the C2 position of the D-glucosamine units 

according to the equation (1). Because of the positive charge on the C2 of the glucosamine monomer at pH < 6, 

chitosan is more soluble and has a better antimicrobial activity than chitin [8]. 

Chitosan-NH2 + H3O + ↔ Chitosan-NH3 + + H2O (1) 

Due to the presence of free amino groups, chitosan (pka = 6.5) is a cationic polyelectrolyte at pH < 6.5; 

consequently, this property along with the chelating ability of amine groups of macromolecule is used for the 

most of the applications of chitosan [8]. 

Chitosan preparations commercially available possess a degree of deacetylation (DD) > 85% with molecular 

weights between 100 kDa and 1000 kDa. They are usually complexed with acids, such as acetic or lactic acids [9]. 

Different studies focused on the possibility of obtaining reproducible and straightforward depolymerization 

methods for generating low molecular weight chitosan (LMWC) from high molecular weight chitosan (HMWC), 

through enzymatic or oxidative degradation, acidic cleavage and ultrasonic degradation. Liu et al. [10] reported 

that NaNO2 showed better performances during the depolymerization of chitosan if compared to H2O2 and HCl 

and these results were confirmed by other authors [11]; however no detail on the procedure was provided. To 

obtain low molecular weight fragments, Mao et al. [12] performed a depolymerization of chitosan through an 

oxidative degradation with NaNO2, thus producing a large series of chitosan with desired molecular weights by 

changing chitosan/NaNO2 molar ratio, chitosan concentration and reaction time. 

In a recent work, Baxter et al. [13] investigated the influence of high-intensity ultrasonication on the molecular 

weight and degree of acetylation of chitosan. In particular, the aim of their research was to develop a reaction 

kinetic model as a function of ultrasonic processing parameters to predict degree of acetylation and 

polymerization of ultrasonicated product; they concluded that high-intensity ultrasound could be a convenient 

and easily controllable methodology to produce this important functional carbohydrate. They observed that in 

presence of an acidic solvent neither power level (16.5, 28.0 and 35.2 W/cm 2 ) nor sonication time (0, 0.5, 1, 1.5 

15 and 30 min at 25°C) altered the degree of deacetylation of chitosan molecules. 

Applications 

Properties such as biodegradability, low toxicity and good biocompatibility make chitosan suitable for use in 

biomedical and pharmaceutical formulations, for hypobilirubinaemic and hypocholesterolemic effects, antiacid 

and antiulcer activities, wound and burn healing properties (Fig. 3). Furthermore, applications of chitosan 

include wastewater purification, chelation of metals, coating of seeds, to improve yield and protection from 

fungal diseases and drug delivery system [13]. 

FOOD 

INDUSTRY 

removal dye, 

suspended solid 

preservative 

colour stabilization 

anticholesterol and fat 

binding 

flavour and taste 

Figure 3: Commercial applications of chitosan. 

BIOTECHNOLOGY 

enzyme immobilization 

protein separation 

cell recovery 

chromatography 

cell immobilization 

CHITOSAN 

MEDICAL 

bandage 

blood cholesterol control 

controlled release of drug 

skin burn 

contact lens 

AGRICOLTURE COSMETICS 

seed control 

fertilizer 

controlled agrochemical 

release 

moisturizer 

face, hand and body 

cream 

bath lotion 

WASTEWATER 

TREATMENT 

removal of metal ions 

flocculant/coagulant 

(protein, dye, aminoacid)


Use of High Pressure Processing for Food Preservation 

Antonio Bevilacqua, Daniela Campaniello and Milena Sinigaglia* 




CHAPTER 8 

Abstract: High pressure processing has been proposed since the beginning of the 1900, as a suitable mean 

for reducing food contamination by pathogens and spoiling microorganisms. It is defined as non-thermal 

treatment that uses the pressure (300-700 MPa, in some cases up to 1000 MPa) as the main preservation 

method. 

Based on the different ways to achieve pressure increase, we can distinguish between High Hydrostatic 

Pressure (HHP) and High Pressure Homogenization (HPH); HHP attains pressure rise through a fluid, 

whereas in HPH treatments pressure increases as a consequence of forcing product through a small valve 

(homogenizing valve). 

Both these approaches have been proposed for different kinds of foods (HHP, for chopped onions, apple 

sauce and apple sauce/fruit blends as eat-on-to-the-go single serve tubes; HPH, for milk and juices) and 

currently used in many industrial applications. 

The chapter proposes an exhaustive description of both these methods, including the mode of actions against 

the microorganisms, the modifications on foodstuffs, a possible combination with some other hurdles and 

some examples of industrial applications. 

Finally, in the case of HHP there is a report on its safety and implications on health, based on some 

publications of Public Agencies. 

Key-Concepts: High Hydrostatic Pressure, Homogenization, Effects of pressure on microorganisms, 

Equipments. 

HIGH HYDROSTATIC PRESSURE 

HIGH HYDROSTATIC PRESSURE: AN INTRODUCTION 

There are several definitions of the term high pressure processing; hereby, we will use the most simple, i.e. High 

Pressure Processing (HPP) is a non-thermal food processing, that uses the pressure (300-700 MPa, in some cases 

up to 1000 MPa) as the main preservation method [1]. Due to the fact that pressure increase is achieved through 

a fluid (for example water), this process has been also referred to as High Hydrostatic Pressure (HHP) as 

opposite to the High Pressure of Homogenization (HPH), where the increase of the pressure is obtained forcing 

the product through a small valve (homogenizing valve). 

Bert Hite was the first to use this method as an alternative approach for food preservation; he pressurized many 

kinds of foods and beverages in the late 1890s and at the beginning of the 20 th century [2]. Since these initial 

efforts, other researchers tried to use this approach, but only in the 1980s the suitability of HHP as a food 

preservation method was realized [2]. 

The first HHP-treated products (jams and jellies) appeared in 1991 in Japan; in 2001, guacamole (pressuretreated 

avocado) entered US marketplace, followed by HHP salsa and in 2004 by chopped onions, apple sauce 

and apple sauce/fruit blends as eat-on-to-the-go single serve tubes. 

In the EU a consumer research [3] reported a 67% of acceptability from consumers of three different European 

countries (France, Germany and United Kingdom), thus opening the way for the marketing of HHP-treated 

products. 

An interesting description of HHP treatment can be found in the paper of Riva [4]: We should imagine two 

elephants (10-12 tons) laid on a penny; the coin is subjected to a pressure of ca. 900 MPa. Now, we should 

image that the same pressure has been applied on a egg through water: you don’t break the egg, but you cook it 



High Pressure Processing Application of Alternative Food-Preservation Technologies 115 

without increasing the temperature, thus obtaining a “safe” and “fresh” egg, without off-odours. This is the HHP 

processing. 

This simple imagine offers a friendly description of how HHP works: hydrostatic pressure is usually applied to food 

products through a water bath that surrounds the product; it can be applied both to liquid and packed solid foods. 

Riva [4] reported that HHP is based on 5 basic principles: 

1. Le Chatelier’s principle: pressure enhances reactions leading to a volume decrease (e.g. starch 

gelatinization, protein denaturation and phase transitions). Le Chatelier’s principle can explain the 

antimicrobial effectiveness of HHP, as high pressures denatures proteins, solidifies lipids and 

destabilizes biomembranes [2]. 

2. Adiabatic heating: the increase of pressure results in a uniform increase of the temperature. This 

phenomenon can be described through the following equation: 

dT 

dP 

T 

 

C 

where T is the temperature (K); P, the pressure (Pa); α, the thermal expansion (1/K); ρ, the density 

(kg/m 3 ); Cp, the heat capacity (J/kg*K). This equation indicates that temperature increase depends 

on the characteristics of the system and the initial temperature: for example it has been evaluated 

that water temperature increases of 2.8-4.4°C/100 MPa (2.8, 3.8 and 4.4 at 20, 60 and 80°C 

respectively); otherwise the temperature of oil increases of 6-8°C/100 MPa. 

3. Isostatic rule: HHP processing is not affected neither by the volume nor by the shape of the foods; 

the pression is uniformly distributed around and throughout the product. 

4. Squeezing: pressure enhances ionization phenomena inside the system, thus resulting in little 

changes of the pH. 

5. Energy of compression: energy input required by HHP is lower than that used in the traditional 

thermal treatments; therefore, at room temperature pressure can affect only hydrogen and ionic 

bonds. In contrast, covalent bonds remain unchanged. 

Nowadays, HHP has been proposed and applied for the preservation of different product, as a suitable alternative 

to the traditional heat processing. 

In the following paragraphs, the reader will find some details on the antimicrobial effectiveness of HHP, a brief 

description of the physico-chemical modifications caused by pressure in foods, some examples of the application 

of this approach for some foods and a safety evaluation of the method. 

EFFECT OF HHP ON THE MICROORGANISMS OF FOODSTUFFS 

It is well known that HHP can be used successfully for the inactivation of the pathogens and spoiling microflora 

of foodstuffs. As regards the kind of resistance, different reports suggest the following hierarchy of resistance: 

Bacteria (cells)>fungi>protozoa-parasites and amongst the bacteria, the Gram positive are more resistant than 

Gram negative ones, thus highlighting that pressure resistance could be inversely related to cell dimension, 

although there are some exceptions to this general statement [2]. The viruses cannot be included in this scale, as 

they are characterized by a broad range of sensitivity/resistance [2]. 

Pressure treatments at 400-600 MPa for 5-20 min at various temperatures are able to inactivate the vegetative 

forms of foodborne pathogens (see Table 1); however, it is important to underline that pressure effectiveness is 

influenced strongly by the temperature, the kind of treatment (single or multi-step) and food components. A final 

consideration on the bacteria is the following: amongst Gram positive bacteria, lactic acid bacteria appeared as 

the most resistant ones. 

Despite bacteria sensitivity, HHP cannot be used to inactivate spores. In fact, bacterial spores are the most 

difficult life-forms to eliminate with hydrostatic pressure [2]; for example, Hoover et al. [2] reported that it was 

possible to detect viable spores of Bacillus spp. after a treatment at 1700 MPa for 45 min at room temperature. 

This report, along with other data available in the literature [5], suggests that HHP alone cannot be used to 

inactivate spore-formers; in contrast the use of the hurdle approach (i.e. the combination of two or more 

preserving elements) is a reliable way [2, 6]. In the case of bacterial spores, it has been suggested the 

p


combination of HHP with a mild heat treatment [6], some antimicrobials (nisin, lysozyme, essential oils) [7], 

along with the storage under refrigerated conditions, to avoid the germination of survivors [2]. 

Table 1: Effect on HHP, as single hurdle, on pathogens and spoiling microorganisms of foods. 

Pathogens 

Food Reduction Condition Reference 

Campylobacter jejuni Poultry meat slurry 5 log cfu/g 400 MPa/2 min at 15°C [18] 

Enterobacter sakazakii Infant formula 5 log cfu/ml 500 MPa/6.3 and 7.9 min at 25 and 

40°C, respectively 

[57] 

Escherichia coli 

O157:H7 

Cashew apple juice 6 log cfu/ml 400 MPa/3 min at 25°C [59] 

Listeria 

monocytogenes 

Mycobacterium avium 

subsp. 

paratubercolosis 

Alfalafa seeds 5 log cfu/g 600 MPa/2 min at 20°C 

Model cheese 5 log cfu/g 500 MPa at 20°C [60] 

Cooked smoked 

dolphinfish 

4 log cfu/g 300 MPa/15 min at 20°C [61] 

Milk 7 log cfu/ml 700 MPa/5 min at room temperature [62] 

Salmonella Enteritidis Liquid whole egg 4-8-6.0 log 

cfu/ml 

Raw almonds 1.27 log 

reduction 

Salmonella sp. Navel and Valencia 

orange juices 

Salmonella 

Typhimurium 

350-400 MPa/ up to 40 min at 25°C [63] 

6 cycles of pressurization at ca. 400 

MPa/20 s at 50°C 

Model cheese 2.84 log cfu/g 400 MPa/10 min at room temperature [64] 

5 log cfu/ml 600 MPa/300 s or 300 MPa/198-369 s 

at 20°C 

Model cheese 3.30 log cfu/g 400 MPa/10 min at room temperature [66] 

Staphylococcus aureus Cheese 8 log cfu/ml 400 MPa at room temperature [60] 

Streptococcus 

agalactiae 

Spoiling microflora 

Mesophilic count and 

filamentous fungi 

Human milk 6 log cfu/ml 400 MPa/30 min at 31°C [10] 

Human milk 6 log cfu/ml 400 MPa/6 min at 31°C [10] 

Cashew apple juice 4 log cfu/ml 400 MPa/3 min at 25°C [59] 

Lactic acid bacteria Sliced ham Prolongation of 

the lag phase 

400 MPa/15 min at room temperature [66] 

Enterococcus faecalis Meat batters 4 log cfu/g 400 MPa/60 min at 25°C [67] 

Leuc. mesenteroides Blood sausages 1 log cfu/g 600 MPa/10 min; initial temperature of 

15°C 

[68] 

Pseudomonas spp. Blood sausages 4 log cfu/g 600 MPa/10 min; initial temperature of 

15°C 

[68] 

Weissella viridescens 

Spore-formers 

Blood sausages 1 log cfu/g 600 MPa/10 min; initial temperature of 

15°C 

[68] 

Alicyclobacillus 

acidoterrestris (cells) 

Orange, apple and 

tomato juices 

[11] 

[65] 

4 log cfu/ml 350 MPa/20 min at 50°C [69] 

B. cereus Milk 6 log cfu/ml 540 MPa/ 16.8 min at 71°C [70] 

B. coagulans Tomato juice [71] 

Geobacillus 

stearothermophilus 

Meat batters 2 log cfu/g 400 MPa/60 min at 25°C [67] 

B. subtilis Various food Depending on 479 MPa/14 min at 46°C [22, 72] 

systems 

food 

constituents 

Meat batters 2.5 log cfu/g 400 MPa/60 min at 25°C [67]




CHAPTER 9 

Alternative Non-Thermal Approaches: Microwave, Ultrasound, Pulsed 

Electric Fields, Irradiation 

Nilde Di Benedetto, Marianne Perricone and Maria Rosaria Corbo* 

Department of Food Science, Faculty of Agricultural Science, University of Foggia 

Abstract: This chapter proposes a description of some non-thermal technologies (microwave, ultrasound, 

pulsed technologies, irradiation) as suitable tools to inactivate foodborne pathogens and spoiling 

microorganisms in heat-sensitive foods. 

Ultrasound (US) is defined as pressure waves with a frequencies of 20 kHz or more; pulsed electric field 

(PEF) processing involves treating foods placed between electrodes by high voltage pulses in the order of 20- 

80 kV/cm (usually for a couple of microseconds); ionizing irradiation occurs when one or more electrons are 

removed from the electronic orbital of the atom; and microwaves (MW) are defined as electromagnetic waves 

in the range of infrared (IR) and radio waves (RF) with a wavelength ranging from 1 mm to 1 m and 

operating at a frequency ranging from 300 Mhz to 300 Ghz. Each technique allows killing of vegetative 

microorganisms but fail until now, when applied alone, to destroy spores. 

This chapter reports some practical applications of the proposed approaches in food industry and also focuses 

on their drawbacks and limitations. 

Key-concepts: Microwave, Ultrasound, Pulsed electric fields, Irradiation, Food applications of non-thermal 

approaches. 

INTRODUCTION 

Modern consumers are increasingly conscious of the health benefits and risks associated with consumption of 

food. In addition, consumers demand for foods that are fresher, more natural and healthier and that at the same 

time provide a high degree of safety have increased interest in non-thermal preservation techniques for 

inactivating microorganisms and enzymes in foods. 

For these reasons, the food industry is devoting considerable resources and expertise to the production of 

wholesome and safe products, but it needs some unit operation such as scrutinizing materials, entering food 

chain, suppressing microbial growth and reducing or eliminating the microbial load. The microbial destruction is 

the principal aim to ascertain safety and stability of food. Heat treatments are traditionally applied to pasteurize 

and sterilize food, generally at the expense of its sensory and nutritional qualities. 

Microwave, high power ultrasound, irradiation, γ rays and pulsed electric field represent the alternative foodpreservation 

technologies designed to obtain safe food, while maintaining its nutritional and sensory qualities. 

Satisfactory evaluation of a new preservation technology depends on reliable estimation of its efficacy against 

pathogenic and spoilage food-borne microorganisms. Moreover, the success of these new technologies depends 

on the advances in understanding what happens to microbial cells during and after treatment. 

Microorganisms are inactivated when they are exposed to factors that substantially alter their cellular structure or 

physiological functions, such as DNA strand breakage, cell membrane breakdown or mechanical damage to cell 

envelope. Furthermore, cell functions are altered when key enzymes are inactivated or membrane selectivity is 

disabled. A preservation technology, e.g. heat, may cause cell death through multiple mechanisms, but limited 

information is available about that. 

For example, membrane structural or functional damage is, generally, the cause of cell death during exposure to 

high-voltage electric field. Whereas, ionizing and UV radiations damage microbial DNA and to a lesser extent 

denature proteins. Cells that are unable to repair their radiation-damaged DNA die. 

Microorganisms are more likely stressed or injured than killed in food processed by alternative preservation 

technologies, although adaptation of microorganisms to stress during processing constitutes a potential hazard. 

*Address correspondence to this author Maria Rosaria Corbo at: Department of Food Science, Faculty of Agricultural Science, 

University of Foggia, Italy; E-mail: m.corbo@unifg.it

144 Application of Alternative Food-Preservation Technologies Di Benedetto et al. 

Generally, bacterial spores are the most resistant to inimical processes, followed by Gram positive and Gram 

negative bacteria. Alternative preservation technologies should inactivate unusually resistant contaminants and 

prevent or minimize stress adaptation. Effective food-preservation processes eliminate hazardous pathogens and 

decrease the levels of spoilage microorganisms. In conclusion, the alternative technologies are developed to 

produce safe food with high sensory and nutritional values. The choice of a technique for industrial application 

depends on food properties and process design [1]. 

MICROWAVES 

Microwave technique is widely used for technical, medical and analytical purposes, even if the heating of food 

can be regarded as the major application. In fact, it is used in households and industry for several purposes, like: 

thawing, heating, drying, pasteurizing and decontamination of food and packaging materials [2]. 

PRINCIPLES AND PROPERTIES OF MICROWAVE 

Microwave technology is a dielectric heating approach and uses the principle of heating by electromagnetic waves, 

the term dielectric heating is used to identify technologies designed to warm bodies that are not good conductors of 

heat. This technology makes heating with a transmission of energy and not through a transmission of heat. 

In particular, microwaves (MW) are defined as electromagnetic waves in the range of infrared (IR) and radio 

waves (RF) with a wavelength ranging from 1 mm to 1 m and operating at a frequency ranging from 300 Mhz to 

300 Ghz (Fig. 1). Within this portion of the electromagnetic spectrum, there are frequencies that are used for 

cellular phone, radar and television satellite communications and for microwave heating. The frequencies most 

commonly used for MW-heating are 0.915 and 2.45 GHz [3]. 

Figure 1: Electromagnetic Spectrum 

This technique is based on the foodstuff property to be a “dipole”; in fact, the molecules of food may be regarded 

as "dipoles" (Fig. 2) and this means that at one side they have a positive electrical charge, while at the opposite 

one possess a negative charge. 

Figure 2: Dipole 

A microwave oven uses a device (magnetron), usually positioned on top of the oven, that generates a force field 

that changes direction continuously (usually at a frequency of 2450 MHz) and acts on the molecules of foods. 

The continuous change of polarity of the electromagnetic waves, made by the oven, originates vibrations 

throughout the molecules of foods, thus, leading to the heating of the system. 

The microwave penetrates into the food with a depth ranging from 2 to 4 cm in all directions, this means that the 

food cooks but other chemicals changes do not occur. This characteristic highlights the importance of the volume 

and exposure time to microwave [4].

Non-Thermal Approaches Application of Alternative Food-Preservation Technologies 145 

In order to assess the efficacy of this technology, some parameters should be taken into account: 

1. Dp = Microwave power penetration depth 

(eq. 1) 

where Dp is in centimetres, f is in GHz and ε’ is the dielectric constant, whilst ε” is the dielectric loss factor (a 

material property called the “dielectric property”, representing the material capacity to absorb microwaves). 

Simply, the higher the frequency, the less is the depth of penetration; ε’ and ε” can be dependent on both the 

frequency (f) and the temperature. This has practical consequences for batch processes [5]. 

1. Q = Rate of microwave heat generation per unit of volume at a particular location within the 

foodstuff during the microwave irradiation process 

(eq. 2) 

where E is the strength of the electric field of the wave at the location, f is the frequency of the wave (generally 

2450 MHz), ε0 is the permittivity of free space (a physical constant), and ε’’ is the dielectric loss factor [6]. It can 

be seen from this formula that the temperature could be increased by choosing a higher frequency for the 

microwave, a higher relative dielectric constant or through a larger loss factor; even if only certain frequencies 

are permitted for microwave heating in order to prevent interference in radio traffic. This formula also shows 

that air pockets in the foodstuff, which may be inevitable or are necessary for a good sensory quality of the 

product, reduce the ability of food to be heated in the microwave field [2]. 

There is another dielectric property, called the dielectric constant, εr, which affects the strength of the electric 

field inside food product; the Table 1 shows the relative dielectric constants of different materials. The dielectric 

property depends on the composition of the food product, with moisture and salt being the most significant 

determinants. The temperature increase in food depends on the duration of heating, the location of the food in the 

reactor, the convective heat transfer at the surface and the extent of evaporation of the water inside the food and 

at its surface [7]. 

Table 1: Relative dielectric constant of different foods-materials 

Material Relative ε” 

Water (0°C) 88 

Water (20°C) 81 

Ice (-20°C) 16 

Ice (0°C) 3 

Olive oil 3.01 

Air 100.059 

PASTEURIZING AND STERILIZING WITH THE MICROWAVE 

Thermal pasteurization and sterilization are predominantly used in the food industry for their efficacy and 

product safety record; however, excessive heat treatment may cause undesirable protein denaturation, nonenzymatic 

browning and loss of vitamins and volatile flavour compounds. Advances in technology allowed 

optimization of thermal processing for maximum efficacy against microbial contaminants and minimum 

deterioration of food quality [1]. 

Another advantage to pasteurizing with microwave is due to the possibility of sterilizing packed foods, acting at 

the same time on both foodstuffs and packaging. Nowadays, a new field in MW processing is the combination of 

two microwave frequencies, used successfully to extend the shelf-life of packed sour milk products; in this batch 

process, the whole product is heated with 10-30 MHz while the surface is treated with 2450 MHz. 

To avoid hot and cold spots, which imply sensory and quality defects in foods, microwave long-term treatment 

was tested with defined standing times. Even various foodstuff with different container shapes can be heated by 

computerized processing units with a microwave hybrid system [2].




CHAPTER 10 

Food Shelf Life and Safety: Challenge Tests, Prediction and Mathematical 

Tools 

Antonio Bevilacqua* and Milena Sinigaglia 


Abstract: Predictive microbiology (PM) is an interesting tool to predict the survival/growth of pathogens and 

spoiling microorganisms in foods, as well as a powerful mean for the evaluation of the shelf life and the 

effects of some hurdles in food industry. This chapter offers an overview of the most important primary 

models to fit growth (Baranyi, Gompertz and lag-exponential equations) or survival kinetics (the function of 

Bigelow, along with the equations included in the Add-in-Excel component GInaFiT). 

Then, the chapter describes some secondary models used in food microbiology (square root, cardinal, 

Arrhenius and polynomial equations), as well as a brief synopsis of a new approach, the S/P model, based on 

the simultaneous evaluation of the microflora growth and the production of an end-product or the 

consumption of a substrate. 

Another interesting tool proposed by the chapter is a summary of the approaches used for the evaluation of 

the lag phase of a microbial population, along with an appendix reporting some key-concepts of the Design of 

Experiments and a description of the indices for the evaluation of the goodness of fitting of a function. 

Key-concepts: Challenge tests, Primary model (Gompertz, Baranyi and lag-exponential equations), Inactivation 

curves, Secondary models, Growth/no growth model, Design of experiments. 

PREDICTIVE MICROBIOLOGY: AN INTRODUCTION 

Many authors cite the papers of Bigelow [1, 2] as the date of birth of predictive microbiology (PM) and the 

beginning of use of this kind of approach to predict pathogen growth and/or survival in food industry, especially 

in canning industry [3, 4]. For the next 30-40 years PM remained quiescent, due to the lack of tools to use 

practically the concepts of microbial modeling in food industry; this impasse was overcome in 1970s-1980s and 

a renaissance of PM started, due to the development and diffusion of electronic technologies, which enabled 

continual monitoring of time and temperature throughout food chain and made easier to solve mathematical 

equations quickly [4]. 

In 1983 Roberts and Jarvis published the first review in the field of PM and coined the term “predictive 

microbiology”, as an interesting tool to predict pathogen survival and growth in food. 

Since the 1980s PM has been dominated by the dichotomy between the use of kinetic equations and probability 

models to predict the probability of growth of Clostridium botulinum and other toxinogenic microorganisms in 

food; this dichotomy has resulted in the definition by Bridson and Gould [5] of two branches: the classical 

microbiology, opposed to the quantal microbiology, where uncertainty dominates [3]. 

Devlieghere et al. [6] reported a brief synopsis of the basic concepts of PM and elucidated some parameters to 

classify models for microbial growth: 

1. the approach (empirical versus mechanistic). 

2. the level (primary, secondary or tertiary models) 

3. kind of inactivation (thermal and non-thermal inactivation). 

Regarding the kind of approach, empirical models are not based on theoretical assumptions; they are built on 

the basis of the data of a challenge test and cannot be extended to other situations. Examples of empirical models 

are the Arrhenius and the polynomial equations. 


of Foggia, Italy; E-mail: a.bevilacqua@unifg.it

162 Application of Alternative Food-Preservation Technologies Bevilacqua and Sinigaglia 

On the other hand, mechanistic models are derived from basic principles and are designed to describe 

fundamental biochemical or thermodynamic phenomena. If the original model is sufficiently sophisticated, it can 

be extrapolated for other situations. They have fewer parameters than the empirical equations and the parameters 

have a biological meaning. 

As regards model level, models can be conceived as having three levels (primary, secondary and tertiary 

models). 

A primary model is a mathematical equation that describes the changes of microbial population with the time 

(e.g. cell numbers vs time) and results in the evaluation of some fitting parameters (lag time, maximal growth 

rate, maximal cell number in the stationary phase, D-value), related with microbial growth/death into the system. 

Examples of primary models are the Gompertz, Baranyi and Weibull equations. 

A second-level model is an equation describing how the parameters, evaluated through a primary model, change 

with changes of environmental or other factors, for examples temperature, pH, aw, preservatives. 

In the tertiary-level models, this process is reversed to obtain a prediction concerning a particular pathogen or 

spoiling microorganism in a defined system. The tertiary models are usually incorporated into predictive 

softwares (ComBase, SSP, Simprevius), that give a good estimation of growth/survival of pathogens and/or 

spoiling microflora for different purposes (implementation of the HACCP system and risk assessment). 

A crucial question is: why predictive microbiology? which uses? 

As reported by Legan [7], PM is an useful tool, able to meet different issues, i.e.: 

Establishing safe shelf life for a food product; 

Exploring the effects of formulation changes on shelf life (e.g. the addition of an antimicrobial 

compound); 

Establishing process lethality and optimizing process parameters; 

Establishing the probability of pathogen survival in a food and the risk of product failure; 

Understanding the behaviour of occurring microflora in a system. 

CHALLENGE TESTS 

Microbiological challenge tests (MCT) are useful tools to determine if a food can support the growth of spoiling 

and/or pathogenic microorganisms; they play a fundamental role in the definition of the effectiveness and/or 

lethality of a preserving treatment (e.g. thermal treatments, alternative approaches, addition of antimicrobials). 

Moreover, MCT are also useful for the determination of the shelf life of some foods: in this case they can be 

labeled as durability test. 

Vestergaard [8] reported that there are some crucial factors to be considered when conducting a MCT, i.e.: 

1. the selection of appropriate target microorganism, taking into account food composition and 

storage conditions; 

2. the level of inoculum; 

3. inoculum preparation and method of inoculation; 

4. duration of the study; 

5. sample analysis. 

Hereby, we report briefly some details for each factor. 

Target Microorganisms 

Knowledge of food composition, along with its technological history, is a fundamental prerequisite for the 

choice of the target of a MCT; Table 1 reports the microbial targets suggested by Vestergaard for some products.

Predictive Microbiology Application of Alternative Food-Preservation Technologies 163 

Table 1: Targets for MCT for some products [8]. 

Food Microorganisms 

Salad dressings Salmonella sp., Staphylococcus aureus 

MAP products Clostridium botulinum, Listeria monocytogenes, Escherichia coli 

Bakery items Salmonella sp., Staph. aureus 

Sauces and salsas stored at room temperature Salmonella sp., Staph. aureus 

Dairy products 

Confectionery products Salmonella sp. 

Formula with new preservatives 

Salmonella sp., Staph. aureus, Cl. botulinum, E. coli, L. 

monocytogenes 

Salmonella sp., Staph. aureus, Cl. botulinum, E. coli, L. 

monocytogenes 

Level of Inoculum 

The level of inoculum in a MCT depends on whatever the aim of the study is to determine the shelf life or to 

validate a preserving treatment. Generally, an inoculum level of 10 2 -10 3 cfu/g (or ml) is used for the evaluation 

of the stability of a formulation. Otherwise, an initial cell number of 10 6 -10 7 cfu/g is required to validate the 

lethality of a treatment: for example in the USA juice processors require for a MCT an initial level of 6 log units, 

as they used a 5D reduction as the goal to bale a processing as effective. 

Inoculum Preparation and Method of Inoculation 

Inoculum preparation is an important detail for the success of a MCT. Typically for vegetative cells 18-24 h 

cultures, revived from slants or frozen aliquots are used; for some challenge studies a preliminary adaptation step 

is required: for example, E. coli O157:H7 should be adapted with acidulants before using in acidic foods. Spores 

should be diluted in distilled water and heat-shocked before inoculation. 

The method of inoculation is another crucial factor for the success of a MCT. There are a variety of modes of 

inoculation, depending on food structure; in aqueous matrices with aw>0.96, the cells can be dispersed directly 

into the medium by mixing, using water or an appropriate diluent as a carrier. 

For individually packed products, the inoculum should be distributed using a sterile syringe through the package 

wall; otherwise, for solid foods with aw>0.96 (cooked pasta, meat) spraying is an alternative way to the syringe. 

Finally, foods or components with aw




APPENDIX I 

Microencapsulation as a New Approach to Protect Active Compounds in 

Food 

Mariangela Gallo and Maria Rosaria Corbo* 


Abstract: Microencapsulation has been defined as “the technology of packaging solid, liquid and gaseous 

active ingredients in small capsules that release their contents at controlled rates over prolonged periods of 

time”. The production of microcapsules began in 1950s, when Green and Schleicher produced microcapsules 

dyes by complex coacervation of gelatine and gum Arabic, for the manufacture of carbonless copying paper. 

In relation to their structure, the particles can be classified as: mononuclear, polynuclear and matrix type. 

There are some methods for the microcapsules production; the choice of the microencapsulation method 

relies both on the nature and characteristics of the polymeric material used and the properties of the active 

ingredients. The main encapsulation techniques are: emulsion and interfacial polymerization, coacervation, 

liposome, suspension crosslinking, spray drying, spray cooling, solvent evaporation or extraction. 

Firstly proposed in the pharmaceutical industry, today this technique is popular in agriculture, food industry, 

cosmetic and energy generation. In food industry microencapsulation is used for vitamins, flavors, enzymes 

and probiotic microorganisms. 

Key-concepts: What is microencapsulation, Method of microencapsulation, Release mechanisms, Application in 

foods. 

INTRODUCTION 

Microencapsulation has been defined as the technology of packaging solid, liquid and gaseous active ingredients 

in small capsules that release their contents at controlled rates over prolonged periods of time [1]. 

The most important feature of microcapsules is their dimension, i.e. the size, the thickness and the weight of the 

membrane, included in the following ranges: 

Size: 1 µm-2 mm 

Thickness of the membrane: 0.1-200 µm 

Weight of the membrane: 3-30% of total weight. 

In relation to their structure, the particles can be classified as: mononuclear, polynuclear and matrix types (Fig. 

1). Mononuclear types consist of a shell around the central core containing the active ingredient; the polynuclear 

capsules have many cores distribuited into shell, whereas in the matrix type the active substance is distribuited 

throughout the shell material. 

Figure 1: Types of microcapsules 

Core material 

Shell material 

MONONUCLEAR POLYNUCLEAR MATRIX 

The production of microcapsules began in 1950s, when Green and Schleicher produced microcapsules dyes by 

complex coacervation of gelatin and gum Arabic, for the manufacture of carbonless copying paper. In the 1960 it 

was proposed the microencapsulation of a cholesteric liquid crystal through the coacervation of gelatin and 



Mincroencapsulation Application of Alternative Food-Preservation Technologies 189 

acacia for the production a thermosensitive display material. Firstly proposed in the beginning for the 

pharmaceutical industry, afterwards microencapsulation, became popular in agriculture, food industry, cosmetic 

and energy generation [2]. 

There are many reasons to use encapsulation of active ingredients, the most important ones are: 

Controlled release of active compounds over the time; 

Target release of encapsulated materials into human body; 

Protection of the active substance from the environment; 

Protection of the encapsulated materials against oxidation or deactivation; 

Conversion of a liquid into solid; 

Separation of incompatible components; 

Masking of odour, taste and activity of encapsulation substance; 

Gastric irritation reduction; 

Best handlage of the product. 

MICROENCAPSULATION: METHODS 

The choice of the microencapsulation method relies both on the nature and characteristics of the polymeric 

material used and the properties of the active ingredients. 

A polymer for microencapsulation should be: 

Chemistry trifle; 

Non-toxic; 

Biocompatible; 

Autoclavable. 

Moreover, the polymer can have: 

Good hiding power; 

Good adhesion; 

Good elasticity; 

Good chemical and physical stability; 

Resistance to mechanical stress. 

Table 1 shows the most used polymers for the production of microcapsules, both of natural and of syntetic 

origin. 

Table 1: Polymers used 

NATURAL POLYMERS SYNTETIC POLYMERS 

Albumin, Alginate, Casein, Chitosan, Cellulose, Gelatin, 

Gluten, Gum Arabic, K-Carragenen, Kraft Lignin, 

Methocel, Methylcellulose, Natural Rubber, Pectin, Starch, 

Sodium Caseinates, Soy Proteins, Whey Protein 

Ethylene, Polyacrilate, Polyacrilonitrile, Polybutadiene, 

Polyethylene, Polyisoprene, Polypropylene, Polystyrene, 

Polyvinyl acetate, Polyvinyl chloride, Silicone 

Microencapsulation techniques can be divided into two categories: Chemical methods, if the starting materials 

are monomers or prepolymers and chemical reactions are involved with microsphere formation; Physical 

methods, if the starting materials are polymers and physical changers usually occur [2].

190 Application of Alternative Food-Preservation Technologies Gallo and Corbo 

Chemical Methods 

Emulsion Polymerization 

In this method a water-insoluble monomer is added dropwise into the aqueous polymerization medium, 

containing the ingredient to be encapsulated and a emulsifier. Initially, polymer molecules form primary nucleus, 

thus they growing and entrapping the core material [2]. 

The size of the particles is ca. 50-500 μm [3]. 

Interfacial Polymerization 

In this technology, the polycondensation of two complementary monomers takes place at the interface of two 

immiscible phases, that are mixed under carefully-controlled conditions to form small droplets of one phase in 

the other. For this process is it necessary to use a small amount of a suitable stabilizer to prevent droplet 

coalescence or particle coagulation. Microcapsules can be either monocore or matrix type, depending on the 

solubility of the polycondensate in the droplet phase [2]. 

This method was initially applied for the preparation of semipermeable artificial cells and then used for many 

materials: solids, aqueous solution and organic liquids. The size of microcapsules varies from 2 to 6 µm to 2000 

µm [3]. 

In-situ Polymerization 

The process involves the direct polymerization of a single monomer shell carried out on a core materials surface. 

Coating thickness is of 0.2-75 µm and the coating is uniform [4]. 

Coacervation 

It is the most promising microencapsulation technology, because the recovery is up to 99%; this method is used 

mainly to encapsulate flavour oil, fish oil, vitamins and enzymes [5]. 

This technique is carried out by preparing an aqueous polymer solution (1-10%) containing the core material at 

40-50°C; a suitable stabilizer can be added to the mixture to maintain the individuality of the microcapsules. A 

coacervating agent is gradually added to the solution, thus leading to the formation of partially desolvated 

polymer molecules, and hence their precipitation on the surface of the core particles. The coacervation mixture is 

cooled to about 5-20°C, followed by the addition of a crosslinking agent to solidify the microcapsule wall 

formed around the core particles [2]. 

The most studied and well understood coacervation system is probably the gelatin/gum acacia system, which 

presents a major drawback, due to the use of gelatin in foods. However, this issue could be solved by using an 

enzymatic crosslinking [5]. 

Liposome 

Developed in recent years, nowadays this technology is used routinely in food industry and in pharmaceutical 

area, due to: the high encapsulation efficiency, the simple production method and the good stability of 

capsules [5]. 

Liposomes or phospholipide vesicles are structures composed of a lipid vesicle bilayers and are generally large, 

irregular and unilamellar. Chemically, liposome is an amphoteric compound containing both positive and 

negative charges [6]. 

Today, the methods for liposome formation do not use the sonication or organic solvent, and allow the 

continuous production of microcapsules on a large scale. Several authors have proposed different methods for 

liposome production for microencapsulation technology [5]. 

The maximum particle size (16 μm) is obtained by using a liposome concentration of 4 g/l [6].


Alternative Modified Atmosphere for Fresh Food Packaging 

Maria Rosaria Corbo* and Antonio Bevilacqua 




APPENDIX II 

Abstract: Modified atmosphere packaging (MAP) has a long history of safe and effective use to prolong the 

shelf life of foods; it involves the change of gas composition inside the bag, mainly an increase of CO2 

content and the lowering of O2. 

In recent years, many authors have proposed the use of some non-conventional gases, like argon (Ar) and 

other noble gases, or the use of volatiles in the head space (hexanal, hexenal, essential oils from citrus) to 

improve the safety and quality of fresh products. 

This appendix offers a short overview of the most recent findings in the application of these novel approaches 

for food preservation, along with a proposal for some possible ways to improve the use of these methods for 

an effective scale up in food industry. 

Key-concepts: Noble gases , Aroma compounds, Modified atmosphere packaging, Fresh-products. 

INTRODUCTION TO MODIFIED ATMOSPHERE PACKAGING (MAP) 

Definitions and Overview of MAP 

Food packaging serves to protect products against deteriorative effects, contain the products, communicate to the 

consumers as a marketing tool and provide consumers with ease of use and convenience [1]. The display of fresh 

product in plastic materials allows consumer evaluation of the product in an attractive, hygienic and convenient 

package [2]. Therefore, food packaging now performs beyond the conventional protection properties and 

provides many functions for the container product [3]. 

Modified atmosphere packaging (MAP) is a technique used for prolonging the shelf-life of fresh or minimally 

processed foods. In this preservation technique the atmosphere surrounding the food in the package is changed to 

another composition before sealing in vapour-barrier materials [3]. MAP can be vacuum packaging (VP), which 

removes most of the air before the product is enclosed in barrier materials, or forms of gas replacement, where 

air is removed by vacuum or flushing and replaced with another gas mixture before packaging sealing in barrier 

materials. The headspace environment and product may change during storage in MAP, but there is no additional 

manipulation of the internal environment, while controlled atmosphere packaging (CAP) uses continuous 

monitoring and control of the environment to maintain a stable gas atmosphere and other conditions such as 

temperature and humidity within the package. CAP has most often been used to control ripening and spoilage of 

fruits and vegetables [4], usually in containers larger than retail-sized packages although some research has been 

conducted on packaging for individual fruits and vegetables. 

The use of MAP allows the prolonging of the initial fresh state of the perishable products like meat, fish, fruits 

and vegetables, since it slows the natural deterioration of the product. MAP is used with various types of 

products, where the mixture of gases in the package depends on the type of product, packaging materials and 

storage temperature. But fruits and vegetables are respiring products where the interaction of the packaging 

material with the product is important. If the permeability (for O2 and CO2) of the packaging film is adapted to 

the product respiration, an equilibrium modified atmosphere will establish in the package and the shelf-life of the 

product will increase. Among fresh-cut produce equilibrium modified atmosphere packaging (EMAP) is the 

most commonly used packaging technology. When packaging vegetables and fruits the gas atmosphere of 

package is not air (O2 – 21%; CO2 – 0.01%; N2 – 78%) but consists usually of a lowered level of O2 and a 

heightened level of CO2. This kind of package slows down the normal respiration of the product, thus prolonging 

the shelf life of the product. There are many factors which affect modified atmosphere packaging of fresh 

produce: 



Alternative MAPs Application of Alternative Food-Preservation Technologies 197 

1. Movement of O2, CO2, and C2H4 in produce tissues is carried out by the diffusion of the gas 

molecules under a concentration gradient. Different commodities have different amounts of 

internal air space (for example, potatoes 1–2%, tomatoes 15–20%, apples 25–30%). A limited 

amount of air space leads to increase in resistance to gas diffusion. 

2. Ethylene (C2H4), a natural plant hormone, plays a central role in the initiation of ripening, and is 

physiologically active in trace amounts (0.1 ppm). C2H4 production is reduced by about half at O2 

levels of around 2.5%. This low O2 retards ripening by inhibiting both the production and action of 

C2H4. 

3. Metabolic processes such as respiration and ripening rates are sensitive to temperature. Biological 

reactions generally increase two to three-fold for every 10°C rise in temperature. Therefore 

temperature control is important to achieve an effective work of MAP system. 

4. Film permeability also increases as temperature increases, with CO2 permeability responding more 

than O2 permeability. Low RH (relative humidity) can increase transpiration damage and lead to 

desiccation, increased respiration, and ultimately to an unmarketable product. A serious problem 

associated with high in-package humidity is condensation on the film driven by temperature 

fluctuations. A mathematical model was developed for estimating the changes in the atmosphere 

and humidity within perforated packages of fresh produce [5]. The model was based on the mass 

balances of O2, CO2, N2 and H2O vapours in the package. Also a procedure to maintain desired 

levels of O2 and CO2 inside packages that are exposed to different surrounding temperatures was 

designed and tested [6]. 

5. For most commodities light is not an important factor in their post-harvest handling. However 

green vegetables, in the presence of sufficient light, could consume substantial amounts of CO2 

and produce O2 through photosynthesis. Finally, shock and vibration leads to damage to produce 

cells which causes an increase in respiration, the release of enzymes and the beginning of 

browning reactions. 

Gases Used in MAP 

The main gases used in modified atmosphere packaging are CO2, O2 and N2. The choice of gas depends upon the 

food product being packed. Used singly or in combination, these gases are commonly used to balance safe shelflife 

extension with optimal sensorial properties of the food. Noble or ‘inert’ gases, such as argon, are used for 

products such as coffee and snack products, although their use for minimally processed apples and kiwifruit has 

been recently proposed [7, 8]. Experimental use of carbon monoxide (CO) and sulphur dioxide (SO2) has also 

been reported. 

Carbon Dioxide 

Carbon dioxide is a colourless gas with a slight pungent odour at very high concentrations. It is an asphyxiant 

and slightly corrosive in the presence of moisture. CO2 dissolves readily in water (1.57 g/kg at 100 kPa, 20°C) to 

produce carbonic acid (H2CO3) that increases the acidity of the solution and reduces the pH. This has significant 

implications on the microbiology of packed foods. 

Oxygen 

Oxygen is a colourless and odourless gas, that is highly reactive and supports combustion. It has a low solubility 

in water (0.040 g/kg at 100 kPa, 20°C) and promotes several types of deteriorative reactions in foods including 

fat oxidation, browning reactions and pigment oxidation. Most of the common spoilage bacteria and fungi 

require oxygen for growth. Therefore, the pack atmosphere should contain a low concentration of residual 

oxygen to increase shelf life of foods; however, superatmospheric O2 concentrations (> 70 kPa) have been 

proposed as an alternative to low O2 modified atmospheres in order to inhibit the growth of typical spoilage 

microorganisms of fresh-cut produce (strawberries, raspberries, pears), prevent undesired anoxic fermentation 

and maintain fresh sensory quality [9, 10, 11, 12, 13, 14]. 

Nitrogen 

Nitrogen is a relatively un-reactive gas with no odour, taste, or colour. It has a lower density than air, nonflammable 

and has a low solubility in water (0.018 g/kg at 100 kPa, 20°C) and other food constituents. Nitrogen 

does not support the growth of aerobic microorganisms and therefore inhibits the growth of aerobic spoilage 

microorganisms, without affecting the growth of anaerobic bacteria.

198 Application of Alternative Food-Preservation Technologies Corbo and Bevilacqua 

Carbon Monoxide 

Carbon monoxide is a colourless, tasteless and odourless gas, highly reactive and very inflammable. It has a low 

solubility in water but it is relatively soluble in some organic solvents. CO has been studied in the MAP of meat 

and has been licensed for use in the USA to prevent browning in packed lettuce. Commercial application has 

been limited because of its toxicity and the formation of potentially explosive mixtures with air. 

Noble Gases 

The noble gases are a family of elements characterized by their lack of reactivity and include helium (He), argon 

(Ar), xenon (Xe) and neon (Ne). These gases are being used in a number of food applications, e.g. potato-based 

snack products. 

LIMITATIONS OF MAP IN FRESH-CUT FRUIT AND VEGETABLES 

Fresh produce is more susceptible to diseases because of increase in the respiration rate after harvesting; so, the 

shelf life under ambient conditions is very limited. The respiration of fresh fruits and vegetables can be reduced 

by many preservation techniques, like low temperature, canning, dehydration, freeze-drying, controlled 

atmosphere, and hypobaric and modified atmosphere. Dehydration also controls the activity of microorganisms 

by the removal of water under controlled conditions of temperature, pressure and relative humidity. The 

controlled atmosphere packaging (CAP) is used for bulk storages. In this approach the composition of gases is 

maintained in the package, so it requires continuous monitoring of gases. Freeze-drying is a very important 

technique in which product volume remains the same as sublimation leads to direct removal of ice. But it is 2–5 

times more expensive and slower as compared to other methods. Modified atmosphere packaging technology is 

largely used for minimally processed fruits and vegetables including fresh, ‘‘ready-to-use’’ vegetables [15]. 

Fresh-cut fruit and vegetable products for both retail and food service applications have increasingly appeared in 

the market place recently. In the coming years, it is commonly perceived that the fresh-cut products industry will 

have unprecedented growth. Fresh-cut vegetables for cooking are the largest segment of the fresh-cut produce 

industry; salads are another major category, as consumers perceive them as being healthy. Fresh-cut fruit is 

growing very fast; however, processors of fresh-cut fruit products will face numerous challenges not commonly 

encountered during fresh-cut vegetable processing. The difficulties encountered with fresh-cut fruit, require a 

new and higher level of technical and operational sophistication. 

Fresh-cut processing increases respiration rates and causes major tissue disruption as enzymes and substrates, 

normally sequestered within the vacuole, become mixed with other cytoplasmic and nucleic substrates and 

enzymes. Processing also increases wound-induced C2H4, water activity and surface area per unit volume, 

which, may accelerate water loss and enhance microbial growth, since sugars become readily available, too [16, 

17, 18]. These physiological changes may be accompanied by flavour loss, cut surface discoloration, colour loss, 

decay, increased rate of vitamin loss, rapid softening, shrinkage and a shorter storage life. Increased water 

activity and mixing of intracellular and intercellular enzymes and substrates may also contribute to flavour and 

texture changes/loss during and after processing. Therefore, proper temperature management during product 

preparation, refrigeration throughout distribution and marketing is essential for maintenance of quality. 

The effect of modified atmospheres on the quality of many fresh-cut products (mushroom, apple, tomato, 

pineapple, butterhead lettuce, potato, kiwifruit, salad savoy, honeydew, mango, carrot) has been extensively 

studied and recently reviewed by Sandhya [15]. However little is reported about the safety of fresh-cut products. 

Raw and minimally processed fruits and vegetables are sold to the consumer in a ready-to-use or ready-to-eat 

form, without preservatives or antimicrobial substances and any heat processing before consumption. Therefore, 

a variety of pathogenic bacteria, such as Listeria monocytogenes, Salmonella sp., Shigella sp., Aeromonas 

hydrophila, Yersinia enterocolitica and Staphylococcus aureus, as well as some Escherichia coli strains may be 

present on fresh fruits and in the related minimally processed refrigerated products [19, 20, 21]. In fact, the 

number of documented outbreaks of human infections associated to the consumption of raw and minimally 

processed fruits and vegetables has considerably increased during the past decades [22]. 

Moreover, the inefficacy of the sanitizers used, probably due to the inability of active substances to reach 

microbial cell targets, makes difficult the decontamination of raw fruits and vegetables [19]. The presence of cut 

surfaces, with a consequent release of nutrients, the absence of treatments able to ensure the microbial stability,


A 

Appropriate Level of Protection (ALOP) 10-11 

B 

Bacillus cereus 29 

Baranyi and Roberts 165-166 

Biphasic model 170 

C 

Campylobacter jejuni 27 

Cardinal model 174 

Central Composite Design 183-184 

Centroid 185 

Challenge tests 162-163 

Chitosan derivatives 95-97 

Chitosan: antimicrobial activity 97-101 

Chitosan: food application 101-110 

Chitosan: preparation and use 92-95 

Clostridium botulinum 29 

Clostridium perfringens 28 

D 

Design of Experiments 180-182 

E 

Escherichia coli 28 

Essential oils: chemistry 39-40 

Essential oils: in vivo application 44-50 

Essential oils: mode of action 40-44 

Essential oils: production 38-39 

Essential oils: toxicology 51-52 

Exposure assessment 9 

F 

Factorial design 182-183 

Food Safety Objectives (FSO) 11-12 

Food structure 24-25 

G 

Gamma model 173 

Geeraerd models 168-169 

Gompertz equation 164-165 

Goodness of fitting 179-180 

Green consumerism 1-3 

Growth models 167 

Growth/no growth models 180 

H 

Hazard characterization 9 

Hazard identification 9 

Health protection 4 

High Hydrostatic Pressure (HHP) 114-128 

HHP: equipments 121-122 

HHP: food application 122-124 

HHP: mode of action 115-121 

HHP: principles 114-115 

INDEX 


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High Pressure Homogenization (HPH) 128-35 

HPH: equipments and mode of action 128-131 

HPH: food application 132-135 

Hurdle approach 50-51, 75-76. 124-126, 131-132 

I 

Indicators 19 

Irradiation 154-158 

L 

Lactoferrin 64-69 

Lactoferrin:toxicology 68 

Lactoperoxidase components 69-70 

Lactoperoxidase system 69-76 

Lactoperoxidase system: antifungal activity 73 

Lactoperoxidase system: bactericidal effect 71-73 

Lactoperoxidase system: food application 73-75 

Lag phase: modeling 177-178 

Lag-exponential equation 166 

Listeria monocytogenes 29 

Lysozyme 58-64 

Lysozyme: in vivo application 62-64 

Lysozyme: toxicology 61-62 

M 

Microbial spoilage of foods 22-25 

Microbiological criteria (MC) 13-15 

Microencapuslation: chemical methods 190 

Microencapuslation: physical methods 191-192 

Microencapuslation: release of active compounds 192-193 

Microencapuslation: uses 193-195 

Microwaves 144-147 

Modified atmosphere packaging (MAP) 196-199 

N 

Nisin 83-89 

Noble gases 198 

Non conventional atmospheres in food 199-204 

Non-isothermal conditions 170-172 

P 

Pathogen classification 18-19 

Pathogen persistance 19 

Polynomial equations 176 

Power ultrasound 147-150 

Precautionary Principle 6 

Pulsed Electric Fields (PEF) 150-152 

Pulsed Light Irradiation (PLI) 152-153 

Q 

Quantitative Risk Analysis (QRA) 4-12 

R 

Risk assessment 7 -10 

Risk categorization 5 

Risk characterization 9 

Risk communication 7 

Risk management 7, 10-12 

Risk-benefit analysis 5

Index Application of Alternative Food-Preservation Technologies 207 

S 

S/P models 166-167 

Salmonella sp. 27 

Salmonella Typhi and Paratyphi 28 

Sampling plans 14-15, 20 

Secondary models 172-177 

Shelf life 17-18 

Shigella dysenteriae 27 

Shoulder 168 

Spoilage 20-21 

Spoilage and food changes 21-22 

Spoilage microorganisms 32-33 

SPS Agreement 6 

Square root 173 

Staphylococcus aureus 29 

T 

Tail 168 

W 

Weibull equation 169 

Y 

Yersinia enterocolitica 29

application of alternative food-preservation - Bentham Science

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