Application of Alternative Food-Preservation Technologies to Enhance Food Safety and Stability

APPLICATION OF ALTERNATIVE FOOD-PRESERVATION TECHNOLOGIES TO ENHANCE FOOD SAFETY AND STABILITY Edited by Antonio Bevil...

Author: Bevilacqua | Antonio Corbo | Maria Rosaria Sinigaglia | Milena

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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 Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia

01

2.

Risk Assessment and Food Safety Objectives Antonio Bevilacqua, Barbara Speranza and Milena Sinigaglia

04

3.

Food spoilage and safety: Some Key-concepts Barbara Speranza, Antonio Bevilacqua and Maria Rosaria Corbo

17

4.

Essential Oils for Preserving Perishable Foods: Possibilities and Limitations Barbara Speranza and Maria Rosaria Corbo

35

5.

Enzymes and Enzymatic Systems as Natural Antimicrobials Daniela D’Amato, Daniela Campaniello and Milena Sinigaglia

58

6.

Antimicrobial agents of Microbial Origin: Nisin Daniela D’Amato and Milena Sinigaglia

83

7.

Chitosan: a Polysaccharide with Antimicrobial Activity Daniela Campaniello and Maria Rosaria Corbo

92

8.

Use of High Pressure for Food Preservation Antonio Bevilacqua, Daniela Campaniello and Milena Sinigaglia

9.

Alternative Non-Thermal Approaches: microwave, Ultrasound, Pulsed Electric Fields, Irradiation Nilde Di Benedetto, Marianne Perricone and Maria Rosaria Corbo

10. Food shelf life and safety: challenge tests, prediction and mathematical tools Antonio Bevilacqua and Milena Sinigaglia

114

143 161

APPENDIX 11. Microencapsulation as a new approach to protect active compounds in foods Mariangela Gallo and Maria Rosaria Corbo

188

12. Alternative Modified Atmospheres for Fresh Food Packaging Maria Rosaria Corbo and Antonio Bevilacqua

196

Index

205

i

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: [email protected]

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, 1st February 2010

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).

iii

CONTRIBUTORS CHAPTER 1 Antonio Bevilacqua (1,2) Maria Rosaria Corbo (1,2) Milena Siniggalia (1,2)

[email protected] [email protected] [email protected]

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 Antonio Bevilacqua (1,2) Barbara Speranza (1,2) Milena Sinigaglia (1,2)


1


2


CHAPTER 3 Barbara Speranza (1,2) Antonio Bevilacqua (1,2) Maria Rosaria Corbo (1,2)


1


2


CHAPTER 4 Barbara Speranza (1,2) Maria Rosaria Corbo (1,2)

[email protected] [email protected]

1


2


CHAPTER 5 Daniela D'Amato (1,2) Daniela Campaniello (1) Milena Sinigaglia (1,2)


1


2


CHAPTER 6 Daniela D'Amato (1,2) Milena Sinigaglia (1,2)


1


2


CHAPTER 7 Daniela Campaniello (1) Maria Rosaria Corbo (1,2)


1


2


iv

CHAPTER 8 Antonio Bevilacqua (1,2) Daniela Campaniello (1) Milena Sinigaglia (1,2)


1


2


CHAPTER 9 Nilde Di Benedetto (1) Marianne Perricone (3) Maria Rosaria Corbo (1,2)


1


2


3

Department of Agro-Environmental Sciences, Chemistry and Crop Protection, Faculty of Agricultural Science, University of Foggia CHAPTER 10 Antonio Bevilacqua (1,2) Milena Sinigaglia (1,2)


1


2


APPENDIX 1 Mariangela Gallo (1) Maria Rosaria Corbo (1,2)


1


2


APPENDIX II Maria Rosaria Corbo (1,2) Antonio Bevilacqua (1,2)


1


2


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

1

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: [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.

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 Health Energy Water Chemicals Genetic engineering Natural world

Why A sentary lifestyle combined with health impacts of environmental pollution and emissions, use and abuse of pesticides, antibiotics and chemicals, could have dramatic consequences. Every source of energy has an environmental impact. Energy efficiency is not just technology, but also cutting back. Water use is increasing at twice the rate of population increase. Much can be done at individual level. Pesticides, preservatives and other chemical hazards have long term effects on human health and wellbeing. 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] [2] [3] [4]

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. Burt S. Essential oils and their antibacterial properties and potential applications in foods-a review. Int J Food Microbiol 2004; 94: 223-253. Bevilacqua A, Sinigaglia M, Corbo MR. Alicyclobacillus acidoterrestris: new methods for inhibiting spore germination. Int J Food Microbiol 2008; 125: 103-110. 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


CHAPTER 2 Risk Assessment and Food Safety Objectives Antonio Bevilacqua*, Barbara Speranza and Milena Sinigaglia Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy 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: [email protected]; [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.

Risk Assessment


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].

Bevilacqua et al.

Propensity to take risk

Perceptual filters


Rewards

Balancing

Perceived danger

Perceptual filters

behaviour

Accidents

Figure 1: Scheme of the risk-benefit analysis. Table 1: Focus on SPS Agreement. What

SPS: Sanitary and Phytosanitary Measures Agreement.

Where

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

Why

1. 2.

How

1. 2.

Maintain the sovereign right of its member government to provide an appropriate level of health protection (ALOP) Ensure that the ALOP does not form unnecessary barriers Measures should be based on risk assessment standards, provided by international organizations (e.g. Codex Alimentarius, OIE, IPPC)* 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.

Risk Assessment


Brown [4] reported that the aim of QRA is to reduce the risk by: 

Identifying realistic microbiological hazards and characterizing them as a function of the severity of risk on consumers.



Examining the impact of raw material contamination, processing and use on the level of risk.



Communicating clearly the level of the risk to the consumer.

This aim can be achieved through different kinds of QRA; Paparella [9] reported at least 6 different models (event tree, fault tree, dynamical tree, process risk model, modular process risk model and networks), that can be used for the mathematical evaluation of the risk associated with a particular pathogen and/or food chain (Table 2). As reported above, the QRA is a complex process, that consists of three main steps, labeled as: 1.

risk management

2.

risk analysis/assessment

3.

risk communication.

Generally, risk assessment may be considered as the science-based part of risk analysis, making risk understandable, whereas risk management is developing and carrying out actions to reduce the risk when necessary [10]. As a consequence, risk management needs to take social, economic and political aspects into consideration when risks are evaluated. All communications exchanged between risk managers, risk assessors and other interested parts are termed risk communication. If we try to carry out a key-concept, able to describe easier the QRA and its three steps, we can say that risk management and risk assessment are the two bound but separate elements of QRA, where risk communication is the main combining factor [8] (Fig. 2). Table 2: Kinds of QRA [9]. Event Tree

Description of the scenario as a function of the initial event

Fault Tree

Description of the hazard throughout the chain, with the aim of pointing out the “hazard-increasing factors”

Dynamical Tree

Use of the predictive microbiology step by step, to evaluate as the exposure to a pathogen and/or a chemical hazard varies throughout the chain

Process Risk Model

Evaluation of the prevalence and cell count in the different steps of the chain, aimed to point out the critical point increasing the exposure to the pathogen

Modular Process Risk Model

The chain is divided into 6 steps (microbial growth, inactivation, mixing, portioning, reduction of the population and cross-contamination), studying the prevalence of each phase

Networks

The chains are studied as networks

Risk management

Risk analysis

Risk communication Figure 2: Connections of management, analysis and communication in the QRA.

A basic principle in the QRA is the functional separation between risk management and risk assessment, although the “essence of the interactive communication is recognized” [11].

Bevilacqua et al.


Different ways and procedures have been proposed and used at Country Level; Fig. 3 reports the scheme of Brown [4], modified by Tuominen [12] on the basis of some reports of the Codex Alimentarius. When a food safety issue has been identified, the risk management is urged to make a decision and defines the public health goals. A public health goal can be used as the basis to take a decision (i.e. preparing and issuing a regulation-for a Public Agency- or defining the kind of control in the case of the inside quality control of food producers). In some situations, social, demographic and economic factors (called stakeholders in the figure) induce an action by the risk management task force or highlight the need of a risk assessment for some hazards. The running of a risk assessment shows as a final result the definition of the appropriate levels of protection (ALOP) and food safety objectives (FSO). A basic contribution in this process is furnished by the risk assessment step, which results in some parameters (ALOP, appropriate level of protection; FSO, food safety objective) used for the decision making [13]. Identified food safety issues

STAKEHOLDERS

RISK MANAGEMENT

Public health goals

Scope, purpose, policy

RISK ASSESSMENT

Risk management decision

ALOP FSO

Regulation: actions taken by governments In-house control: actions taken by food-industry

Figure 3: Risk analysis process.

Risk Assessment Codex Alimentarius defined the risk assessment as a four-step process, consisting of hazard identification, hazard characterization, exposure assessment and risk characterization [8]; it is generally described as a process for the identification “of adverse consequences and their associated probability” [10]. In food safety, risk assessment has been labeled also a “scientific study of the risk, having roots in mathematical theories of probability and in scientific methods for identifying causal links between adverse health effects and foods” [12]. Codex Alimentarius [8] established 11 basic principles for the risk assessment, as follows: 1.

Basis on sound science

2.

Functional separation from risk management

3.

Structured format including hazard identification, hazard characterization, exposure assessment and risk characterization

4.

Clearly stated purpose and output (risk estimate)

5.

Transparency of conduct

6.

Identified constraints such as cost and resources and their consequences

7.

Description of uncertainty and the source of the uncertainty

8.

Use of such data and data collection systems that allow uncertainty of the risk estimate to be determined, and of sufficient quality and precision to minimize the uncertainty

9.

Explicit consideration of the dynamics of microbiological growth, survival and death in foods and of the complexity of the interaction between human and agent following consumption as well as the potential for further spread

Risk Assessment


10. Re-assessment over time by comparison with independent human data 11. Re-evaluation when new relevant information becomes available. The aforementioned principles underline that the importance of the risk assessment lies not only upon the ability to estimate health risk, but also in the use of this process as a framework to organize data and allocate responsibility for analysis [14]. Focusing on the 4 steps of risk assessment (hazard identification, hazard characterization, exposure assessment and risk characterization), Fig. 4 shows the paradigm proposed by Tuominen [15] for the development of an efficient network. Hazard identification

Exposure assessment

Human

Consumer (targets, portions…)

Raw materials

Microbe (product, process, initial contamination, evolution)

Products Environment

Hazard characterization Host (dose-response effects, symptoms) Microbe (resistance to biotic and abiotic elements, virulence)

PREDICTIVE MICROBIOLOGY

Risk characterization Magnitude of risk Consequences

Figure 4: Steps and connections in Risk assessment [15]: the case of foodborne pathogens.

Hazard Identification It is the identification of biological, chemical and physical agents, capable of causing adverse effects on health and which may be found in foods [8]. In the microbiological risk assessment, the hazard could be identified with a foodborne pathogen, based on epidemiological data [16]. Data may be available from the published literature or government databases [16]. Hazard Characterization This step is the qualitative and quantitative evaluation of the adverse effects on health of the hazard, along with the study of the characteristics of the hazard [8]. The evaluation of the adverse effects on health includes the identification of a possible dose-response effects, as well as the symptoms of the hazards associated with different targets (including age, immune status, gender, ethnicity, location, profession, education…). On the other hand, the study of the characteristics of the hazard includes the evaluation of the resistance of the microorganism (in the case of the microbiological risk analysis) to some biocides, to environmental and process parameters (e.g. temperature, pH, nutrients, thermal treatment…), through the use of challenge tests and/or databases developed by predictive microbiology. Exposure Assessment Exposure assessment is the qualitative and/or quantitative evaluation of the likely intake of biological, chemical and physical agents via food as well as exposure to other sources, if relevant [8]. When evaluating the exposure to a pathogen, researchers need to know the frequency of contamination (prevalence), the number of the microorganisms in foods (concentration) and the amount of food consumed. Ideally the contamination by the pathogen under investigation should be determined at the time of consumption, but foods are typically analyzed and sampled at earlier points of the food chain [16]. The concentration of pathogen, its growth and/or inactivation throughout food processing, storage and distribution can be estimated through predictive softwares (i.e. Combase, SSP…).

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Consumption data (e.g. number of servings, average size of a typical serving, demographic characteristic of consumers) are necessary to complete the exposure assessment scenario; these details are available from national surveys and Public Agencies [16]. Risk Characterization Risk characterization is the last step of the risk analysis; it includes the evaluation of attendant uncertainties, probability of occurrence and severity of known or potential adverse health effects in a given population, based on hazard identification, hazard characterization and exposure assessment [8]. This step gives as a final result a risk profile associated with a particular foodborne pathogen as a function of the kind of food and consumer type (e.g. infant, adult…). Risk Management Codex Alimentarius [17] defines risk management as the “process, distinct from risk assessment, of weighing policy alternatives, in consultation with all interested parties, considering risk assessment (when available) and other factors relevant for the health protection of the consumers and for the promotion of fair trade practices, and if needed, selecting appropriate prevention and control options”. This process can be managed at regional, Country or International level [12] and should be based on some principles [18], as follows: 1.

Protection of human health

2.

Consideration of the whole food chain

3.

Structure approach

4.

Transparency, consistency, documentation

5.

Consultation with relevant interested parties

6.

Interaction with risk assessors

7.

Consideration of regional differences (of hazard and management options)

8.

Monitoring, review (and revision).

There are four key-concepts in the risk management, i.e. the appropriate level of protection (ALOP), the food safety objectives (FSO), the performance objectives (PO) and the microbiological criteria (MC). Some details for each parameter are reported in the following sections. Appropriate Level of Protection The concept of ALOP was introduced by SPS Agreement in 1995, as the level of protection deemed appropriate by the Member establishing a sanitary or a phytosanitary measure to protect human, animal or plant life or health within its territory. In other terms, the level of risk a society is willing to accept is referred as the ALOP [19] and this parameter can be expressed as a quantitative value of the probability of an adverse public health consequence or an incidence of disease (for example, a level of listeriosis set to 3 million people per year in the world). ALOP is decided at Country level, based on some economical, scientific and technological factors (Table 3). Table 3: Factors related with the setting of the ALOP [20]. Scientific and production factors       

All available scientific evidence Relevant processes and production methods Relevant inspection, sampling and testing methods Prevalence of specific disease Existence of disease free areas Relevant ecological and environmental conditions Quarantine and other treatments

Economical factors  The potential damage in terms of loss of production or sales in the event of the disease  The costs of control and eradication in the Country  The cost-effectiveness of an eventual alternative approach to limit risks.

For example, if risk manager has set an ALOP for E. coli mortality of 10 deaths/million/year for the total population, this ALOP should attributed to the various sources (food, water, person-person) or products.

Risk Assessment


Therefore, the total number of cases (ALOP) equals the sum of the cases per source or product group (ALOPp) [21], as reported in the following equation:

ALOP   ALOPP

eq. (1)

Another important equation is the following, reporting ALOP as a function of the consumption of a particular food per year and the probability of death associated with one cell of the pathogen [21]: ALOP= deaths per million per year= =servings per million per year*probability of mortality per serving= =servings per million per year*probability of mortality for one cell*dose= = S * 10

6

*r *D

eq. (2)

where: S is the number of servings per person per year, r the probability of mortality following exposure to 1 organism and D the dose consumed. Food Safety and Performance Objectives When a government expresses public health goals relative to the incidence of a disease or the maximum level of a pathogen in a food, this does not provide food processors, producers, handlers, retailers or trade partners with information about what they need to do to reach this level of illness [19]. To be meaningful, the targets for food safety should be translated into numerical parameters useful for both government agencies and food processors. The ideas of food safety (FSO) and performance objectives (PO) have been introduced into the risk analysis to serve to this purpose. According to the definition reported into the Codex Alimentarius, a FSO is “the maximum frequency and/or concentration of a hazard in a food at the time of the consumption that provides or contributes to the appropriate level of protection”. The FSO is directly related with the ALOP, as it could be evaluated through the following equation:

ALOP  10 FSO * M * S *10 6 * r

eq. (3)

this equation has been derived from the ALOP function (eq. (1)), imposing the dose consumed as equal to the mass per serving (M) and the concentration of the pathogen (10FSO). Zwietering [21] used a simple example to show how it is possible to evaluate the FSO from the aforementioned equation. The pathogen under investigation is L. monocytogenes in raw milk cheese; assuming that the person consumption is 50 servings of 30g/year and the probability of mortality after consumption of 1 cell of L. monocytogenes is 7*10-12, the FSO can be evaluated as follows: 10 FSO *

1 * 10 6 * p 30 g 50 S death death g death * * 7.2 * 10 12 * * 10 FSO  1  0.0108 * S p * year million List year * million List year * million

eq. (4)

with S meaning serving and p person. This equation results in a FSO of 2 (i.e. 102 Listeria/g). The FSO concept, however, has been concluded to be of a limited practical use (FAO/WHO, 2006), because of “the difficulty in setting an FSO that is able to act in accordance with both intermediate risk-based food safety targets of food production and with the ALOP” [12]. The definition of FSO has been considered as useful and significant for some ready-to-eat foods [21, 22], but not for those products that are treated prior to consumption [23], thus suggesting that the definition of the Codex Alimentarius, relating with the time of consumption, is not relevant and compatible with risk assessment [24].

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Based on the results of some epidemiological studies as well as on the need of a more realistic definition compatible with the ALOP set by government agencies, a new definition of FSO has been proposed [24] as “a limit to the prevalence and the average concentration of a microbial hazard in food, at an appropriate step in the food chain at or near the point of the consumption that provides the appropriate level of protection”. In other terms, the FSO can be labeled as the maximum concentration of a pathogen or spoiling microorganism that should be in a food before consumption and/or cooking able to maintain the human health at an acceptable level. The FSO is the desired value to meet at the end of the food chain and depends on some elements, like the initial contamination (H), the reduction of the microbial population due to a preserving treatment (R), the increase of the population within the storage (G) or following a not correct handling and/or storage (re-contamination) (C). Based on the concept of FSO and on its implication of human health, microbial population in every point of the food chain should be lower than the FSO, as reported in the following equation:

H   R   G   C  FSO

eq. (5)

However, the equation reported above is a general function which does not take into account that a food chain is a complex framework, involving different steps and that each of them usually reacts with a different producer and/or handler. Therefore the concept of performance objective (PO) has been introduced in the quantitative risk analysis; a PO is equivalent to a FSO, but indicates a target at an earlier stage of the food chain. The correct definition of a PO is the following: The level (the frequency and/or concentration) of a hazard to be achieved at a specific point of the food chain [25]; in other words, a PO is the maximum level of the hazard for each step of the food chain and acts as a milestone for the definition of the ultimate FSO. The definition, as well as the obedience to the PO throughout a specific step of the food chains, is based on the setting of performance and process criteria. Performance and process criteria “belong to the risk management measures that are applied on the company level in order to carry out functional risk-based food safety management” [12]. A performance criterion is defined as the frequency and/or reduction of the microbial hazard in the food that should be achieved through a technological process (for example a 6D reduction of the L. monocytogenes population in raw milk); otherwise, a process criterion is the combination of the physical and chemical parameters that should be applied to achieve the performance criterion and then the PO (for example a thermal treatment at 71.5°C for 15 s to achieve the 6D reduction). An example of the framework FSO-PO and their connections with performance and process criteria is shown in the Fig. 5. H0 

R

0



G

0



C

0

 PO 0

Primary production Process criteria

H 1   R1   G1   C1  PO1 Food industry Performance criteria

H 2   R2   G2   C2  FSO Consumer

Figure 5: FSO and PO throughout the food chain.

Risk Assessment


MICROBIOLOGICAL CRITERIA Government agencies rely on inspection procedures to assess the compliance of a food or a food-processing with FSOs and PSOs; these procedures are based on microbiological criteria (MC). MC are used to distinguish between acceptable and unacceptable foods or food practices, as well as the safety and the quality of a food. Specifically, MC are used to assess: 

The safety of a food (absence or low level of pathogens)



Adherence of food processing to good manufacturing practices and the respect of HACCP principle (absence or low level of indicator microorganisms)



Shelf life of perishable food (level of spoiling microorganisms)



Suitability of a food or ingredient for a particular purpose.

The National Research Council (NRC) of the US National Academy of Sciences addressed the issue of the MC reporting the following definition: “a microbiological criterion should state what microorganism, group of microorganisms, or toxin produced by a microorganism is covered. The criterion should also indicate whether it can be present or is present in only a limited number of samples or a given quantity of food or food ingredient” [26]. In other words, a MC consists of several elements [27]: 1.

microorganisms of concern and/or their toxins and metabolites

2.

the food to which the criterion must be applied

3.

the specific point in the food chain where the criterion must be applied

4.

a qualitative and quantitative analytical method validated and chosen to give a sufficiently reliable estimate

5.

critical limits, based on data appropriate to foods

6.

a sampling plan, including the sampling procedure and decision criteria for a batch

7.

any action to be taken when the criterion is not met.

When and Who Establishes MC A MC should be derived only in response to a real need and when its use is practical and effective; some considerations to take into account for the definition of a MC are the following: 1.

Evidence of health hazard of the pathogen or the microorganism under investigation. This evidence should be based on epidemiological data.

2.

Food structure and characteristics and its ability to support microbial growth.

3.

Role of the natural microflora of food for keeping both quality and safety.

4.

Effect of processing throughout the food-chain on the natural microflora.

5.

Possibility of contamination throughout the food chain.

6.

Sensitivity and specificity of the test method.

7.

Randomness and efficacy of the sampling scheme.

As regards the question “who establishes MC”, different scientific organizations are involved in their definition: the most important ones are the Joint Food and Agricultural Organization and World Health Organization Codex Alimentarius Food Standards Program (briefly related as Codex Alimentarius), the International Commission on Microbiological Specifications of Foods (ICMSF) and some agencies at Country level, like the EFSA and the US National Advisory Committee on Microbiological Criteria for Foods. The World Trade Organization provides a framework in order to harmonize National Standards, as stated by the SPS Agreement and Technical Barriers to Trade. Member Countries are strongly encouraged to base their standards on the guidelines elaborated by the Codex Alimentarius, to make all regulations similar.

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Kinds of MC MC can be mandatory or advisory. A mandatory criterion must be met and foods that exceed it are required to be rejected, destroyed, reprocessed or diverted. Otherwise, an advisory criterion allows acceptability judgments to be made and may be used as an alert for deficiencies throughout the food chain [26]. For application purposes, there are 3 different kinds of MC, based on regulatory consequences [28]: 1.

standards: criteria specified by government agencies to protect public health

2.

specifications: criteria established between producers and buyers, in order to define product quality and safety attributes; failure to meet the criteria could result in the rejection of the lot or in a decrease of the price;

3.

guidelines: criteria that provide advice to industry about acceptable or expected microbial levels when the process is under control. They are used by producers to assess their own processes and by government inspectors for controls.

In addition to this classification, we can report the division of MC into two classes, as suggested by the EFSA in the Regulation of the microbiological criteria for foodstuffs [29]; this division is based on the “place of application”. Therefore, the MC related with the acceptability of foods are referred to as food safety criteria, whereas the MC concerning the contamination of foodstuffs by indicators are defined as process hygiene criteria. Relations Between the MC and FSO The MCs are derived from the risk management step, therefore there is an important relation between FSO and MC. The links between these two concepts are reported in the Table 4, modified from Stringer [30] and Tuominen [12]. Table 4: Relations betwseen FSO and MCs. FSO Aim

 A goal for food chain to achieve safe foods  Aimed at consumer protection  Applied to the food at the time of consumption or prior cooking

 Statement that defines the acceptability of a food  Used to confirm that HACCP and Good Manufacturing Process (GMP) are applied  Applied to lots of foods

 Maximum frequency and/or concentration of the hazard  Product

     

 Food Safety

 Food safety  Quality characteristics

Components

Use

MC

Microorganism of concerns and their metabolites Sampling plan Analytical unit Analytical method Limits Number of units that must conform to the limits

BOX 2.2: Focus on the sampling plans and the microbiological limits. Sampling plan: it includes both the sampling procedure and the decision criteria. Sampling plans are divided into two main categories: variables and attributes. A variable plan is based on the assumption that the microorganism under investigation is log-normally distributed: otherwise an attribute plan is used when the microorganism is not distributed homogeneously. This kind of plans are required for the pathogenic microorganisms and to monitor the performances of food producers and/or handlers in terms of GMPs. Microbiological limit: a critical level of microbial population above which an action is required. It should be: Realistic Based on the knowledge of the raw materials, effects of processing, handling storage and final use Based on epidemiological study and/or challenge tests, as well as on the goals established at Country level (FSO)

Risk Assessment


BOX 2.3: Attributes sampling plans. Two-class plan. The decision criterion is based on the critical level of microorganism (m), the numbers of lot units to be tested (n) and tolerance (c). This kind of plan is applied in the qualitative pathogen testing, in which the results are expressed as presence or absence of the microorganism under investigation. Three-class plan. This plan is based on 4 different parameters (the number of lot units to be examined-n; the tolerance-c; the minimal-m- and the maximal-M- counts) and 3 classes of decision, as follows: Desirable, cell count below the inferior limit (m) in all the lot units Acceptable, cell count ranging from m to M in c samples amongst the n lot units Unacceptable, cell count higher than the superior limit (M) in one or more lot units or cell count in the range m-M in more than c samples. Table 5: Key-concepts of the chapter. QRA (Quantitative Risk Analysis)

A risk assessment that provides numerical expressions of risk and indication of the attendant uncertainties [8]

Qualitative Risk Assessment

A risk assessment based on data which, while forming an inadequate basis for numerical risk estimations, nonetheless, when conditioned by prior expert knowledge and identification of attendant uncertainties permits risk ranking or separation into descriptive categories of risk [8]

Risk Assessment

A scientifically based process consisting of the following steps: hazard identification, hazard characterization, exposure assessment and risk characterization [8]

Risk Management

The process of weighing policy alternatives in the light of the results of risk assessment and, if required, selecting and implementing appropriate control options, including regulatory measures [8].

Risk Communication

An interactive process of exchange of information and opinion on risk among risk assessors, risk managers, and other interested parties [8].

FSO (Food Safety Objective)

The maximum frequency and/or concentration of a hazard in a food at the time of consumption that provides the appropriate level of protection

ALOP (Appropriate Level of Protection)

Level of protection that a country decides is appropriate to protect human, animal or plant life within its territory

TLR (Tolerable Level of Risk)

The level of risk adopted following consideration of public health impact, technological feasibility, economic implications, and that which society regards as reasonable in the context of and in comparison with other risks in everyday life

PO (Performance Objective)

The level (the frequency and/or concentration) of a hazard to be achieved at a specific point of the food chain

PC (Performance Criterion)

Frequency and/or concentration of a hazard in a food that must be achieved by the application of one or more control measures to provide or contribute to a performance objective or a food safety objective

Process Criteria

Combination of parameters that assure that a performance criterion is achieved

CM (Control Measure)

Any action and activity that can be used to prevent or eliminate a food safety hazard or to reduce it to an acceptable level

REFERENCES [1] [2] [3] [4] [5]

[6]

Patil SR, Cates S, Morales R. Consumer food safety knowledge, practices, and demographic differences: findings from a meta-analysis. J Food Prot 2005; 68: 1884-94. Schlundt J, Toyofuku H, Jansen J, SA Herbst. Emerging food-borne zoonoses. Rev Sci Tech 2004; 23: 513-33. Käferstein F, M Abdussalam. Food safety in the 21st century. B World Health Organ 1999; 77: 347-51. Brown MH. Quantitative microbiological risk assessment: principles applied to determining the comparative risk of salmonellosis from chicken products. Int Biodeterior Biodegradation 2002; 50: 155-60. World Trade Organization (WTO). The WTO agreement on the application of phitosanitary measures (SPS Agreement) 1995 (available online from http://www.wto.org/english/tratop_e/sps_e/spgar_e.htm), [Accessed on September 30, 2009]. Murray C, Lopez A. The Global Burden of Disease. A comprehensive Assessment of Mortality and Disability from Diseases, Injuries and Risk Factors in 1990, and Projected to 2020. Cambridge (MA): Harvard University Press 1996: vol. 1


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World Trade Organization (WTO). Report of the Appellate Body in Japan – Measures Affecting Agricultural Products, WT/DS76/AB/R, 19 March 1999. Codex Alimentarius Commission (CAC). Principles and guidelines for the conduct of a microbiological risk assessment. Rome: FAO 1999. Paparella A. In: Cocolin LS, Comi, G., Eds. La microbiologia applicata alle industrie alimentari. Rome (Italy), Aracne editrice s.r.l. 2007; pp.27-78. McKone TE. Overview of the risk analysis approach and terminology: the merging of science, judgement and values. Food Control 1996; 7: 69-76. Codex Alimentarius Commission (CAC). Principles and guidelines for the conduct of microbiological risk management. CAG-GL-63. Rome: FAO 2007. Tuominen P. Developing risk-based food safety management. Academic Dissertation. University of Helsinki (Finland) 2009. Codex Alimentarius Commission (CAC). Report on the third session of the codex ad hoc intergovernmental task force on foods derived from biotechnology. ALINORM 03/04. Rome: FAO 2002. FAO/WHO. Application of risk analysis to food standards issues. Report of the Joint FAO/WHO Expert Consultation in Geneva, Switzerland. March 13-17 1995. Tuominen P, Hielm S, Aarnisalo K, Suihko ML, Raaska L, Maijala R. Development of a risk assessment-based software tool for the evaluation of food industry HACCP plans. Poster in: Proceedings of the 4th International Meeting of the Noordwijk Food Safety and HACCP Forum, Food safety - a shared responsibility; Noordwijk, the Netherlands: March 15-16 2001. Walls I. Role of quantitative risk assessment and food safety objectives in managing Listeria monocytogenes on readyto-eat meats. Meat Sci 2006; 74: 66-75. Codex Alimentarius Commission (CAC). Proposed draft principles and guidelines for the conduct of microbiological risk management (MRM) (at step 3 of the procedure). CX/FH 03/72003. Orlando, Florida, USA: Joint FAO/WHO 2003. Codex Alimentarius Commission (CAC). Food Standards Programme Codex Committee on Food Hygiene. Houston, Texas, USA: Joint FAO/WHO 2006. International Commission on Microbiological Specifications on Foods (ICMSF). Use of epidemiological data to measure the impact of food safety control programs. Food Control 2006; 17: 825-37. de Swarte C, Donker RA. Towards an FSO/ALOP based food safety policy. Food Control 2005; 16: 825-30. Zwietering M. Practical considerations on food safety objectives. Food Control 2005; 16: 817-23. Gorris LGM. Food safety objective: an integral part of food chain management. Food Control 2005; 16: 801-9. Nauta MJ, Havelaar AH. Risk-based standards for Campylobacter in the broiler meat chain. Food Control 2008; 19: 372-81. Halevaar AN, Nauta MJ, Jansen JT. Fine-tuning food safety objectives and risk assessment. Int J Food Microbiol 2004; 93: 11-9. Codex Alimentarius Commission (CAC). Proposed draft principles and guidelines for the conduct of microbiological risk management (MRM) (at step 3 of the procedure). CX/FH/05/37/6. Buenos Aires, Argentina: Joint FAO/WHO 2005. Montvillle TJ, Matthwes KR. Food Microbiology. An introduction. 2nd ed. Washington DC, ASM Press 2008; pp. 7794. Codex Alimentarius Commission (CAC). Principles for the establishment and application of microbiological criteria for foods. CAG/GL-21. Rome: FAO 1997. International Commission on Microbiological Specifications for foods (ICMSF). Micro-organisms in foods 7. Microbiological testing in food safety management. New York: Kluwer Academic/Plenum Publishers 2002. European Commission (EC). Commission Regulation no. 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. OJEU 2005; L338/1-26. Stringer M. Food safety objectives-role in microbiological food safety management, Food Control 2005; 16, 775-94.


17

CHAPTER 3 Food Spoilage and Safety: Some Key-concepts Barbara Speranza, Antonio Bevilacqua* and Milena Sinigaglia Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy 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);

*Address correspondence to this author Antonio Bevilacqua at: Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy; [email protected]; [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.


3.

Speranza et al.

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


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.

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Foodborne Pathogens and Sampling Plans The stringency of a sampling plan for a pathogens takes into account some basic elements, i.e. the hazard to the consumer from pathogen and/or its toxin, the kind of microorganism, the consumer target to which the food is intended (adults, infants…). Based on these considerations, the ICMSF (International Committee on Microbiological Specifications of Foods) proposed a two-entry table, reporting as variables the type of hazard (no direct hazard, low or indirect, moderate and depending by a pathogen characterized by a limited spread, moderate and severe hazard) and the conditions in which the food is intended to be consumed and/or handled (reduced hazard, no change, increased hazard). The combinations are summarized in the Table 3. Table 1: Attributes of pathogenicity [4]. Pathogens must 1. enter and survive inside a host; 2. find a niche for persistence; 3. be able to avoid the host’s defense (stealth phase); 4. be able to replicate to significant numbers; 5. be transmitted to other host with high frequency; 6. be able to express specialized traits within the host. Table 2: Mechanisms of pathogenesis for foodborne pathogens [4]. Infectious dose (depending on the immunological status of the host and the natural infectivity of the microorganism) Adhesion factors (pili and fimbriae, adhesion proteins, biofilm formation) Invasion and intracellular residence (phagocytosis, invasion-mediated induced phagocytosis) Iron acquisition Motility and chemotaxis Evasion of immune system Production of toxins Table 3: Sampling plans for pathogens (from Montville and Matthews [6]; modified).

Hazard Low (indicator)

Conditions in which the food is expected to be handled and consumed Reduced hazard

No change

Increased hazard

3-class (n, 5; c, 3)**

3-class (n, 5; c, 2)

3-class (n, 5; c, 1)

Group A*

3-class (n, 5; c, 2)

3-class (n, 5; c, 1)

3-class (n, 5; c, 0)

Group B

2-class (n, 5, c, 0)

2-class (n, 10; c, 0)

2-class (n, 20; c, 0)

Group C

2-class (n, 15; c, 0)

2-class (n, 30; c, 0)

2-class (n, 60; c, 0)

*Group A, moderate hazard, limited spread; group B, moderate hazard, potentially extensive spread; group C, severe hazard. **3-class/2-class, sampling plan; n, number of lot units to be analyzed; c, tolerance.

FOOD SPOILAGE Food spoilage can be considered as any change which renders a product unacceptable for human consumption [7]; it may arise from insect damage, physical damage (bruising, freezing, drying, etc…), indigenous enzyme activity in animal or plant tissues, chemical changes (usually involving oxygen). Therefore, spoilage is a complex phenomenon, involving physical, chemical, microbiological and biochemical changes; in this section we will focus on the microbiological spoilage. The microflora that colonizes a food or a beverage depends highly by the characteristics of the product and the way it is processed and stored [8]; the parameters that affect microbial growth on foods are divided into four different groups, as follows:

Food Microoganisms




Intrinsic parameters: this group includes the physical, chemical and structural properties of foods, i.e. pH, water activity, redox potential, nutrients and natural antimicrobial compounds.



Extrinsic parameters, i.e. the factors of the environment where the food is stored (temperature, humidity, atmosphere…).



Modes of processing and preservation (heating, acidification, reduced water activity, use of preservatives, chilled storage, modified atmosphere, combination of the mentioned treatments).



Implicit parameters (influences, synergism, antagonism amongst the different microorganisms).

As regards the microbiological spoilage, an important question that arises is the following: how does it manifest itself? There are several signs of the microbiological spoilage of foods; the most important ones are the following: 1.

Visible growth: moulds produce large, pigmented colonies, which render the product unacceptable; otherwise, a visible bacterial and yeast growth is less common.

2.

Gas production (e.g. the swelling of plastic bags in mozzarella cheese due to an abnormal growth of coliforms).

3.

Slime, a typical spoiling phenomenon of meat due to pseudomonads.

4.

Diffusible pigments and enzymes, which may produce softening and rottening (proteolysis, pectolysis…).

5.

Production of off-odours and off-flavours, due to the synthesis of various compounds (alcohols, sulphur compounds, ketones, hydrocarbons, organic acids, esters, carbonyl and diamines). This is the most common kind of spoilage.

The Table 4 reports a brief synopsis of the most important kinds of spoilage, as well as the foods involved with the responsible microorganisms and the chemical/biochemical causes (compounds produced by the spoiling microflora). Table 4: Food spoilage. Microorganisms, foods and chemical causes. FLAVOUR Problem

Food

Chemical cause

Microorganism

Nitrogeneous (bad eggs)

Meat, eggs, fish

Ammonia, H2S, trymethylamines

Pseudomonas spp., Acinetobacter, Moraxella, Clostridia

Souring

Dairy products, vacuum packed meat, beer, wine

Acid production (acetic, lactic, citric, butyric)

Lactic Acid Bacteria, Bacillus spp., Acetobacter

Alcoholic

Fruit juices, mayonnaisedressed salads

Ethanol

Yeasts

Mustiness

Bread

Chloroanisole

Moulds

Pig-sty

Vegetables

p-cresole, indole, skatole

Erwinia, clostridia

Garlic

Various foods

Bis (methylthio)-methane, trimethylarsine

Unknown

Fruity

Meat

Esters of short chain fatty acids

Ps. fragi

Potato-like

Meat, eggs, milk

2-methoxy-3-isopropylpyrazine

Pseudomonas

TEXTURE Problem

Food

Chemical/biochemical cause

Microorganism

Slime

Meat

Polysaccharide production

Ps. fragi, Leuconostoc mesenteroides, B. subtilis

Ropiness

Bread

Polysaccharides

Alcaligenes

Bittierness

Milk

Holes

Hard cheese

B. cereus Gas production

Coliforms

Speranza et al.

22 Application of Alternative Food-Preservation Technologies Table 4: cont....

Softening/rotting

Fruit and vegetables

Pectinases, cellulases, xylanases

Erwinia, Clostridia, Yeasts, Moulds

Curdling

Milk, meat

Acid production

Lactic acid bacteria

VISUAL SPOILAGE Problem

Food

Microorganism

Cheese

Coliforms

Bubbles

Cottage cheese, coleslaw

Yeasts, Lactic Acid Bacteria

Fish-eyes

Olives

Bloaters

Cucumbers

Gas formation Holes

Discolouration Fluorescent

Meat, eggs

Pseudomonas

Pink

Sauerkraut, olives

Rhodotorula

Red spot

Cheese

Lactobacillus plantarum

Browning

Brined vegetables

Lact. brevis

Blackening

Dairy products

Ps. nigrifaciens

Greening

Meat

Lact. viridescens

HOW ASSESS MICROBIAL SPOILAGE OF FOODS There are several ways to detect microbial spoilage in foods, i.e. the microbiological methods, chemical/physical/physicochemical methods, acceptability criteria, including sensory determinations (colour, texture, odour, flavour and general appearance). Some details for each kind of approach are reported in the following paragraphs. Microbiological Methods As regards the microbiological methods, the evaluation of the microbial spoilage is based on two different approaches: assessment of the total viable count or evaluation of the quality-indicator microorganisms. Total viable count (TVC) is an interesting approach for assessing food quality and pointing out an incipient spoilage. Jay [9] proposed a “spoilage spectrum”, organized into 5 different groups, as follows: 

Range A (TVC, 103-106 cfu/g): microbial spoilage is generally not recognized (with the exception of milk).



Range B (TVC, 106-107 cfu/g): some foods show an incipient spoilage. In some cases (e.g. vacuum packed meat) consumers could recover off-odours.



Range C (TVC, 107-108 cfu/g): off-odours are generally associated with aerobically stored meat and vegetables.



Range D (TVC, 5x107-108 cfu/g): almost all food products display signs of spoilage; slime is generally recovered on aerobically stored meat.



Range E (TVC>5x108 cfu/g): foods show structural changes.

Despite its versatility, there some critical points that should be taken into account when using the TVC [6]: TVC determines only the live cells and many spoiled products may have a low viable count if the spoilage microorganisms have died; in addition, it seems that TVC has a little value in assessing sensory quality, because only high counts result in a significant loss of the sensorial quality. TVC is an useful tool for assessing microbial spoilage; however, it requires the knowledge of the expected population level at any point of the food chain, as well as the effects of any processing and preservation treatments and the storage conditions. It is generally accepted that an alternative approach of microbial spoilage assessment is based on the evaluation of the quality indicator microorganisms, i.e. microorganisms highly and negatively correlated with food quality and acting as the main elements of the spoiling phenomenon.

Food Microoganisms


Some examples of quality indicator microorganisms (along with the associated foods) are: Acetobacter spp. (cider), Bacillus spp. (bread), Byssochlamys spp. (canned fruits), Clostridium spp. (hard cheeses), flat-sour spores (canned vegetables), lactic acid bacteria (beers, wine), Lactococcus lactis (raw milk), Leuc. mesenteroides (sugar), Pectinatus cervisiiphilus (beer), Ps. putrefaciens (butter), Zygosaccharomyces bailii (mayonnaise, salad dressing) [9]. Metabolites Evaluation Bacterial levels in spoiled foods can be estimated through the evaluation of the amount of the metabolites produced as a result of the spoiling activity. Some metabolites, as mentioned above, are highly correlated with food spoilage, thus suggesting a possible set of indicator metabolites, associated with particular kind of foods: cadaverine and putrescine (beef packed under vacuum), diacetyl (frozen juice concentrate), histamine (canned tuna), lactic acid (canned vegetables), trimethylamine (fish), total volatile bases (seafish), total volatile nitrogen (seafish), volatile fatty acids (butter) [9]. Sensory Determinations Sensory evaluation is a key-factor in determining the shelf life of many foods; it has been defined as “the use of one or more of the five senses to judge or form an opinion on some aspects of quality. The senses in question are sight, smell, taste, touch and hearing” [10]. Initially developed for fish and fish-products, the sensory evaluation is used both as a complementary test to microbiological challenges and as the only test to assess the quality of the product. As reported for other approaches, the sensory evaluation shows both advantages and disadvantages (Table 5). Table 5: Positive and negative aspects of the sensory evaluation. Advantages

Disadvantages

Closest to what consumer perceives More rapid than most non-sensory methods Sensible Non-destructive No laboratory facilities required

Assessors can become fatigued or adapted Assessors subjected to loss of interest Long training for assessors It can be more expensive than some non-sensory methods Several assessors required for high precision The interpretation of the results is sometimes problematical and open to debate It is not easy to replace the assessors

There are two kinds of sensory assessment, the objective and subjective ones; in the objective assessment the effects of personal influence are minimized by avoiding bias and feelings of liking or disliking, whereas in the subjective evaluation (known also as hedonic assessment) personal feelings of liking, pleasure and acceptance are freely expressed. As an example, we can report an objective and a subjective evaluation for fish. In the objective assessment the description is accurate and uses statements as follows: This fish tastes of seaweed; this fish is in size grade 3; this product is very soft; this product is straw-coloured. In the subjective evaluation, the assessors describes the product with freely expression or word of liking and disliking: I don’t like the taste of this fish; I prefer the product A to product B; I would (or would not) buy this product. Another idea to keep in mind is the different use of the objective and hedonic evaluation; in fact, the objective assessment is used to test product compliance with regulatory or quality standards, as a substitute or complementary analysis to bacteriological and chemico-physical tests. Otherwise, the hedonic evaluation is used in product development and market research and is largely confined to find out what consumers think about a particular product. Hereby we will discuss on the objective sensory assessment, as a method for an indirect evaluation of product quality and microbial contamination. The starting point of the objective sensory assessment is the building of a scale of freshness or deterioration, storing the product to be examined under fixed conditions and choosing the parameter for the evaluation (texture,


Speranza et al.

colour, smell, general appearance…); the scale of freshness/deterioration is based on a score sheet [from 5 to 0 (as reported in many research papers for dairy products, vegetables and fish) or from 10 to 1 (for the evaluation of fish for commercial purpose)] and on a critical break-point (sometimes 2 or 3, for the 5-point scale, or 6, in the 10-point scale), used as the threshold to regard the product as acceptable or not-acceptable. As an example we can cite the sensory evaluation used by Bevilacqua et al. [11] for the assessment of the quality of caprese salad. These authors used a 5-point scale for the evaluation of general appearance, odour and firmness of mozzarella and tomato slices of caprese salad; the data were modeled through a negative Gompertz equation and the stability time (i.e. the time to achieve a drastic decrease of the sensory score) was assessed. The findings of these authors underlined that odour and general appearance could be used as suitable parameters for the evaluation of the shelf life of caprese salad; moreover, these sensorial parameters were highly related to pseudomonads counts and underwent to a drastic decrease when Pseudomonas spp. attained a critical value of ca. 6 log cfu/g. This approach, used extensively in many research papers, is used at regulatory level for milk (in the USA), fish and fish products. Some details are reported below. Milk Evaluation Milk is judged according to the guidelines of the American Dairy Association. Flavour scores are on a 10-point scale, with >9.0 excellent, alkyl). For example, limonene (1-methyl-4-(1-methylethenyl)- cyclohexene) is more active than pcymene [20]. c) Expression of antimicrobial activity is often very clear, but the mechanism of antimicrobial action is incompletely understood. Considering the large number of different groups of chemical compounds present in EOs, it is most likely that their antibacterial activity is not attributable to one specific mechanism because they seem to have no specific cellular targets [7]. Different modes of action have been suggested, as follows (Fig. 2):


□

Speranza and Corbo

As typical lipophiles, EOs pass through the cell wall and cytoplasmic membrane, disrupt the structure of their different layers of polysaccharides, fatty acids and phospholipids and permeabilize them. The permeabilization of the membranes is associated with loss of ions and reduction of membrane potential, collapse of the proton pump and depletion of the ATP pool. Walsh et al. [22] showed that the efflux of K+ is usually an early sign of damage, often followed by efflux of cytoplasmic constituents including ATP [13, 15, 21, 23]. Although a certain amount of leakage from bacterial cells may be tolerated without loss of viability, extensive loss of cell contents or the exit of critical molecules and ions will lead to death [7].

Some workers have reasoned that the permeabilization of cell membrane is not the sole explanation for EOs antimicrobial activity [22] and that the real events which could lead to membrane disfunction and subsequent disruption include: i) dissipation of the two components of the proton motive force in cells (the pH gradient and the electrical potential) either by changes in ion transport or depolarization through structural changes in the membrane; ii) interference with the energy (ATP) generation system; iii) enzyme inhibition preventing substrate utilization for energy production [15, 21]. □

EOs can coagulate the cytoplasm and damage lipids and proteins [7, 15]. Generally, the EOs possessing the strongest antibacterial properties against microorganisms contain a high percentage of phenolic compounds (such as carvacrol, eugenol and thymol) making reasonable that their mechanism of action would be similar to other phenolics, i.e. the disturbance of the proton motive force (PMF), electron flow, active transport and coagulation of cell contents [20, 21, 24]. Enzymes such as ATPases are known to be located in the cytoplasmic membrane and to be bordered by lipid molecules. Two possible mechanisms have been suggested whereby cyclic hydrocarbons could act on these. Lipophilic hydrocarbon molecules could accumulate in the lipid bilayer and distort the lipid–protein interaction; alternatively, direct interaction of the lipophilic compounds with hydrophobic parts of the protein is possible [7].

□

In eukaryotic cells, EOs can provoke depolarisation of the mitochondrial membranes by decreasing the membrane potential, influence ionic Ca++ cycling and other ionic channels and reduce the pH gradient, affecting (as in bacteria) the proton pump and the ATP pool. The membrane becomes abnormally permeable resulting in leakage of radicals, cytochrome C, calcium ions and proteins. Permeabilization of outer and inner mitochondrial membranes leads to cell death by apoptosis and necrosis [25-29].  Permeabilization of cell membrane  Reduction of membrane potential  Collapse of the proton pump  Coagulation of cytoplasm  Efflux of cytoplasmic constituents (metabolites and iones including ATP)  Interference with the energy (ATP) generation system in the cell  Damage to lipids and proteins  Enzyme inhibition preventing substrate utilization for energy production

Figure 2: Mechanisms proposed for EOs action in the bacterial cell.

Further investigations are required to try to give an insight into the mode of action of EOs against cells and understand how EOs components really interact with cells to cause bacteriostatic or bactericidal effect. There are some open-ways: are changes in membrane permeability the primary effect or are they a consequence of other mechanisms? Is the antimicrobial effect due to the disturbance of the energy (ATP) generation system? Or to the inhibition of substrates uptake or utilization? Moreover, is the inhibition of enzymatic reactions critical to maintenance of cellular viability and normal metabolism to cause the antibacterial effect? Or do all these mechanisms act in a synergic way?

Essential Oils in Foods.


BOX 4.1: What it has been recognized.

□

Carvacrol and thymol are able to disintegrate the outer membrane of Gram negative bacteria, releasing lipopolysaccharides (LPS) and increasing the permeability of the cytoplasmic membrane to ATP [21].

□

Studies with Bacillus cereus have shown that carvacrol can interact with the cell membrane, where it dissolves in the phospholipid bilayer and is assumed to align between the fatty acid chains [30]: this distortion of the physical structure would cause expansion and destabilisation of the membrane, increasing membrane fluidity and passive permeability [15].

□

Thymol is thought to bind to membrane proteins hydrophobically and by means of hydrogen bonding, thereby changing the permeability characteristics of the membrane [31].

□

Eugenol, the major component of clove oil, have been found to inhibit production of amylase and proteases by B. cereus and to bind to proteins, preventing enzyme action in Enterobacter aerogenes [7].

□

The biological precursor of carvacrol, p-cymene causes swelling of the cytoplasmic membrane to a greater extent than does carvacrol [15]. This compound is not an effective antibacterial when used alone [20, 24, 30], but when combined with carvacrol, its efficiency is greater: being incorporated in the lipid bilayer, it very likely facilitates transport of carvacrol across the cytoplasmic membrane [15].

□

Cell ultrastructural alterations in several compartments such as plasma membrane, cytoplasm (swelling, shrivelling, vacuolations, leakage) and nucleus of bacteria treated by some EOs have been confirmed by scanning and transmission electron microscopy observations [32-34].

□

After treatment of bacteria with some EOs, analyses of the lipid profiles by gas chromatography and of membrane showed a strong decrease in unsaturated and an increase in saturated fatty acids, as well as alterations of the cell envelopes [35].

□

The induction of membrane damages has been confirmed also by a micro-array analysis, showing that Saccharomyces cerevisiae genes involved in ergosterol biosynthesis and sterol uptake, lipid metabolism, cell wall structure and function, detoxification and cellular transport are affected by a treatment with -terpinene [36]. d) The susceptibility of microorganisms to EOs bioactivity is quite different.

Among compounds with phenolic components, the oils of clove, oregano, rosemary, thyme, sage and vanillin have been found to be the most inhibitory against Gram positive bacteria [7, 12, 37] whereas oregano, clove, cinnamon and citral have shown a good effectiveness against both Gram positive and Gram negative bacteria [7, 12, 38]. But also some non phenolic constituents of oils (such as allyl isothiocyanate or garlic oil) have shown a good inhibitory effect against Gram negative bacteria [39]. Also in this case, it is impossible to point out an univocal effect. By now the antimicrobial activity of different EOs was demonstrated against Aeromonas hydrophila, Alicyclobacillus acidoterrestris, Listeria monocytogenes, Clostridium botulinum, Enterococcus faecalis, Staphylococcus sp., Micrococcus sp., Bacillus sp., Enterobacteriaceae, Campylobacter jejuni, Vibrio parahaemolyticus, Pseudomonas fluorescens, Ps. putida, Photobacterium phosphoreum, Shewanella putrefaciens, Bacillus cereus, Shigella sp., Salmonella enterica Typhimurium and Enteritidis, and Escherichia coli as well as yeasts and moulds (Saccharomyces cervisiae, Aspergillus flavus, A. parasiticus, Penicillium sp.). The reader can find an exhaustive review of this topic in some recent papers [7, 12, 40]: a large number of studies have examined the in vitro antimicrobial activity of spices, herbs and naturally occurring compounds from other sources. Antimicrobial effects were observed by agar diffusion method (using a filter paper disc) or by the dilution method (using agar or liquid broth cultures) in most of bacteria [7, 12, 41-48], and in fungi [49-52] including yeasts [53-56]. In general: □

A greater resistance of Gram negative bacteria to these compounds was observed [7, 12, 21, 22]. This greater resistance is likely to be related to the fact that these bacteria possess an outer membrane surrounding the cell wall which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering. However, not all studies on EOs have concluded that Gram


Speranza and Corbo

negatives are more resistant: in fact, Aer. hydrophila (Gram negative) appears to be one of the most sensitive species. In attempting to explain differences in the sensitivity of Gram positive and -negative cells, differences in cell surface hydrophobicity have been also suggested as contributing factors. It is likely that the effects of differences in hydrophobicity between these two bacterial groups, with Gram negative cells having more hydrophobic surfaces, can be offset by the presence of porin proteins in the outer membrane of Gram negative cells: these can create channels large enough to allow restricted passage of small molecular mass compounds (< 200 Mw), like the substituted phenolics in EOs, allowing their access to the periplasmic space, the glycoprotein layer and the cytoplasmic membrane. In addition, the good solubility of lipopolysaccharide in phenols suggests that the differences in susceptibility of the two groups may result from the interaction of a number of different factors including differences at the cytoplasmic membrane/glycoprotein interface as well as a greater physicochemical complexity of the Gram negative cell wall [57]. □

Among the generally sensitive Gram positive bacteria, the lactic acid bacteria (LAB) are the most resistant [58]. Their ability to generate ATP by substrate level phosphorylation may contribute to this resistance. Differences in internal levels of ATP in L. monocytogenes and Lactobacillus sakei after challenge by eugenol or cinnamaldehyde support this explanation [58]. It is also possible that the greater resistance of the LAB is related to their better ability to deal with conditions of osmotic stress and respond more effectively to K+ efflux caused by many of these antimicrobials.

□

Among the Gram negative bacteria examined, pseudomonads consistently show high or often the highest resistance to these antimicrobials (see for example the papers of Careaga et al. [59], Ps. aeruginosa and Capsicum or bell pepper; Galindo-Cuspinera et al. [60], Ps. fluorescens and annatto).

□

EOs are generally more active against fungi than they are against Gram positive bacteria. Chao et al. [57] evaluated antifungal activity of 45 different plant oils against Candida albicans, A. niger and Rhizopus oligosporus. Of the 45 oils, those of coriander, cinnamon bark, lemongrass, savory and rosewood were effective against all three microorganisms. Other oils showed selective activity against the three organisms. Some of them were effective against C. albicans only. Angelica and pine oils used in this study were not effective against A. niger and R. oligosporus which have a symbiotic relationship with the mycorhizae associated with the plants from which the oils were isolated (Angelica archangelica and Pinus sylvestris), respectively. In addition to inhibition of vegetative growth, some oils also inhibited production of mycotoxins by fungi [61]. Some plant EOs have also been shown to inhibit mycelial growth and conidial germination [62].

In view of the published data on EOs bioactivity against microorganisms, the following approximate general ranking (in order of decreasing susceptibility) can be made: Fungi > Gram positive bacteria >Gram negative bacteria. APPLICATION OF EOS IN FOODS: POSSIBILITIES AND LIMITATIONS An overview of the literature of the last decade reporting the studies on the antibacterial effect of EOs or their components in foods is presented in Table 3. Despite the different methodologies and approaches, some keypoints can be derived: □

While most of the papers showed that EOs and/or their components had a significant antimicrobial activity in vitro, higher amounts are required (1–3%) to achieve the same effect in foods. These levels are often higher than those normally acceptable for the organoleptic characteristics (2-fold in semi-skimmed milk, 10-fold in pork liver sausage, 50-fold in soup and 25- to 100-fold in soft cheese) [7]. Several studies have investigated the effect of foodstuffs on the effectiveness of EOs against different microorganisms, but none quantified or explained this effect. However, some ideas have been suggested, e.g. the need for higher concentrations of oils in foods could be related to the complex nature of food: the greater availability of nutrients could enable bacteria to repair damaged cells faster [7]. Another suggestion is that the lower water content of food compared to laboratory media may hamper the progress of antibacterial agents to the target site in the bacterial cell [63].



□

The presence of fat, carbohydrates, proteins, water, salt, antioxidants, preservatives, other additives and pH reaction influence the effectiveness of these natural compounds in foods. As regards this issue, the extrinsic determinants (temperature, packaging in vacuum/gas/air, characteristics of microorganisms), along with the intrinsic properties of foods, play a crucial role [64, 65].

□

The pH is an important factor affecting the activity of oils. At low pH, the hydrophobicity of some EOs (for example, thyme oil and the phenolic oleuropein) increases due to their ability to dissolve more easily in the lipid phase of the bacterial membrane, thus enhancing the antimicrobial action [7].

□

It is generally supposed that the high levels of fat and/or protein in foodstuffs protect the bacteria from the action of EOs in some way. Some authors suggested that the fat provides a protective layer around the bacteria, or the lipid fraction could absorbe the antimicrobial agent, thus decreasing its concentration and effectiveness in the aqueous phase [63, 66]. The interaction carvacrol/ proteins has been recovered as the limiting factor in the antibacterial activity of the EO against B. cereus in milk [67]; moreover, Smith-Palmer et al. [63] showed that the protein content was the limiting factor for the antimicrobial activity of clove oil against Salmonella Enteritidis in a low-fat cheese. The effect of milk proteins was confirmed also by other studies [7, 67].

□

Carbohydrates in foods do not appear to protect bacteria from the action of EOs as much as fat and protein do [7].

□

A high water and/or salt level appears to facilitate the action of EOs [7, 64].

□

The physical structure of a food may limit the antibacterial activity of EOs. A study of the relative performance of oregano oil against Salmonella Typhimurium in broth and in gelatine gel revealed that the gel matrix dramatically reduced the inhibitory effect of the oil. This effect was attributed to the limitation of the diffusion of the EO by the structure of the gel matrix [68].

Some case studies on the application of EOs in foods are described in the following paragraphs. Case Study n° 1: Application in Meat and Meat Products Raw meat can be easily contaminated by microorganisms and support the growth of pathogens, leading to serious foodborne illnesses. Refrigeration is the most common preservation method of raw meat and meat products, but in order to extend “naturally” the shelf life time of refrigerated storage, EOs have been proposed in recent years. Since the ancient time, some spices and herbs (like thyme, oregano, rosemary, marjoram) have been used in meats as aromatic agents; therefore, this is not a surprise that most of the studies on the application of EOs in meat and meat products focused on the aforementioned oils (see Table 3). Eugenol and coriander, clove, oregano and thyme oils were found to be very effective in inhibiting autochthonous spoilage flora in meat products as well as pathogens, as they caused a marked initial reduction in the viable cell number [7, 65, 69] whilst mustard, cilantro, mint and sage oils were less effective or ineffective [7, 70, 71]. A high fat content appears to reduce markedly the action of EOs in meat products; for example, thyme oil reduced significantly bacterial population of L. monocytogenes in zero and low-fat (90 g/Kg) beef hot-dogs, but not in full-fat hot-dogs (260 g/Kg) [72]. An interesting recent application is described by Chouliara et al. [73] who investigated the combined effect of oregano EO and modified atmosphere packaging (MAP) for the prolongation of the shelf life of fresh breast chicken meat, stored at 4 °C. Oregano is a characteristic spice of the Mediterranean cuisine, obtained by drying leaves and flowers of Origanum vulgare subsp. hirtum, a plant well known for its antioxidative and antimicrobial activity due mainly to the two phenols - carvacrol and thymol- which are its major components [24]. In the cited study the effect of oregano EO (0.1% and 1% w/w) was evaluated in combination with MAP [30:70 CO2:N2 (MAP1) and 70:30 CO2:N2 (MAP2)]. The parameters monitored were: microbiological (Total Viable Count (TVC), Pseudomonas spp., lactic acid bacteria, yeasts, Brochothrix thermosphacta and Enterobacteriaceae), physico-chemical (pH, lipid oxidation, colour) and sensory (odour and taste) attributes. The initial value of TVC (day 0) for the fresh chicken meat was ca. 4.3 log cfu/g, indicative of good quality chicken meat [74]. TVC reached a value of 7 log cfu/g, considered as the upper microbiological limit for good quality fresh poultry meat, as defined by the International Commission on Microbiological Specifications for Foods (ICMSF) [75], on:


□

day 5–6 for the air packaged samples;

□

day 6–7 for the samples containing 0.1% oregano oil;

□

day 25 for the samples containing 1% oregano oil;

□

day 11–12 for the samples packaged under MAP1;

□

day 14–15 for the samples containing 0.1% oregano oil packaged under MAP1;

□

day 14–15 for the samples packaged in MAP2;

□

day 15 for the samples containing 0.1% oregano oil packaged under MAP2.

Speranza and Corbo

Samples treated with oregano oil at concentration 1% packaged under both MAPs, never reached the limit of 7 log cfu/g during the 25-day storage period. As it can be easily inferred, the combination of MAP and oregano oil had an additive effect on the inhibition of the microflora in chicken meat. A reduction of pseudomonads, LAB, Enterobacteriaceae and B. thermosphacta growth was also recovered. Yeasts population was also markedly reduced (more than 2.5 log cfu/g under MAP1 plus oregano oil 1%). The physico-chemical attributes were not considerably affected by oregano oil or by MAP: in particular, a very low degree of lipid oxidation was observed thus pointing out that modified atmosphere packaging and more surprisingly oregano oil did not significantly affect lipid oxidation. On the other hand, sensory analyses pointed out that 1% oregano oil imparted a very strong taste to the product, whereas samples containing 0.1% oregano oil gave a characteristic desirable odour and taste to chicken meat, very compatible to cooked chicken flavour. On the basis of the sensory evaluation a shelf life extension of breast chicken meat by ca. 3–4 days for samples containing 0.1% oregano oil, 2–3 days for samples under MAP and 5–6 days for samples under MAP containing 0.1% of oregano oil, was attained. Case Study n° 2: Application in Seafood Products In fish, just as in meat products, a high fat content appears to reduce the effectiveness of antibacterial EOs. For example, oregano oil at 0.05 % (v/w) was more effective against the spoilage organism Ph. phosphoreum on cod fillets than on salmon, which is a fatty fish [66]. The spreading of EO on the surface of whole fish or using EO in a coating for shrimps appeared effective in inhibiting the natural spoilage flora [76, 77]. As regards this issue, an interesting approach is the use of a combination of EOs, as proposed by Corbo et al. [78] and by Del Nobile et al. [79]. In a preliminary phase, thymol, lemon extract and Grape Fruit Seed Extract (GFSE) were identified as successful active natural preservatives to improve the microbial stability of fish burgers [80, 81], thus suggesting in a 2nd phase the use of a combination of them. In particular, a mix containing 0.11% of thymol, 0.10% of GFSE and 0.12% of lemon extract was proposed, as it resulted in an increase by 40% of the microbiological acceptability limit of fish burgers stored under refrigeration and packaged in air [78]. Based on these results, the combined effect of the EOs and MAP on the quality retention of mackerel-hake burgers stored at 4°C was evaluated, in order to identify a suitable storage strategy. In particular, samples were packaged in air and in three different gas mix compositions: 30:40:30 O2:CO2:N2, 50:50 O2:CO2 and 5:95 O2:CO2. During a 28-days storage period at 4°C, the quality decay of the fish burgers was assessed by monitoring three quality sub-indices: nutritional (percentage of fat, fatty acids composition and nutritional indices), microbiological (TVC, Pseudomonas sp., hydrogen sulphide producing bacteria and psychrotolerant and heat labile aerobic viable count) and sensorial (odour, texture, drip loss, and overall quality). The results obtained highlighted that nutritional quality of fish burgers was unaffected by MAP and active compounds. In fact, no differences emerged from analyses of moisture, fat, protein content and fatty acid composition among samples. As regards microbiological results, the proposed packaging strategies were all effective in inhibiting the TVC growth. For fresh water and marine species, the microbiological limit recommended by the ICMSF [75] for TVC, at 30°C, is 7 log cfu/g or log cfu/cm2. Since in all samples, TVC never exceeded this value, the fish burgers could be considered microbiologically acceptable under all proposed conditions, even after 28 days of storage at 4°C. It is worth noting that spoiled marine fish is characterized by the development of offensive, fishy, rotten H2S-off odours and flavours [82] which are mainly related to specific spoilage organisms (SSOs), thus the counts of Pseudomonas sp., hydrogen sulphide producing bacteria and psychrotolerant and heat labile aerobic bacteria were also performed. MAP and active compounds acted in a synergistic way also against these microbial groups. The recorded cell numbers indicated acceptable microbial quality for the blue fish burger investigated until 28 days of storage at 4°C, thus suggesting the possibility to improve the quality of these products by using very small amount of EOs (about 0.10-0.12%) in combination with a packaging system characterized by a high CO2 concentration MAP. The sensory quality loss of the investigated food products was minimized by the studied



packaging strategies, even if this sub-index was the limiting factor for burgers shelf life (estimated at 22-23 days). Case Study n° 3: Application in Vegetables and Fruits It appears that, in vegetable dishes just as for other products, the antimicrobial activity of EOs is benefited by a decrease in storage temperature and/or a decrease in the pH of the food [64]. Vegetables generally have a low fat content, which may contribute to the successful results obtained with EOs. All EOs and their components tested on vegetables appeared effective against the natural spoilage flora and food borne pathogens at levels of 0.010.1% in washing water [83]. Cinnamaldehyde and thymol were effective against Salmonella sp. on alfalfa seeds when applied in hot air at 50°C as fumigant. Increasing the temperature to 70 °C reduced the effectiveness of the treatment [84]. This could be due to the volatility of the antibacterial compounds. Oregano oil at 0.7–2.1% (w/w) was effective in the inhibition of E. coli O157:H7 reducing its final population in eggplant salad [64]. Thyme or oregano and their respective major components (thymol and carvacrol), used in a dip treatment, reduced disease development in tomatoes inoculated with Botrytis and Alternaria fungi [85]. Carvacrol and cinnamaldehyde reduced the viable count of the natural flora on kiwifruit when used at 0.10% in a dipping solution, but they appeared less effective on honeydew melon. This result could be due to the difference in pH between the fruits; the pH of kiwifruit was 3.2–3.6 and of the melon 5.4–5.5 [86]. Eugenol and carvacrol were found to be the most active compounds amongst 17 different essential oils against E. coli O157:H7 and Salmonella sp. in apple juices [87]. Finally, carvacrol reduced the growth of B. cinerea in grapes [88]. An interesting recent application of EOs in vegetables is provided by Fisher et al. [89]. In this study, a blend of citrus EOs vapour (orange and bergamot) against vancomycin resistant (VRE) and vancomycin susceptible (VSE) Ent. faecium and Ent. faecalis on lettuce and cucumber was assessed. Iceberg lettuce leaves and cucumber skin were inoculated with the studied strains (ca. 108 cfu/ml), left to dry for 20 min, placed in a vapour chamber and subjected to 0.15% air final concentration of the orange ⁄ bergamot blend vapour diffused using a heat diffuser at 25°C for 15, 30, 45 and 60 s after a 15 min accumulation of vapour within the chamber. The experiment was repeated with an initial inoculum on the food-stuff of 105 cfu/ml and a final concentration 0.30% air of the citrus antimicrobial vapour. Samples were analyzed immediately after being in the vapour chamber and after 6, 8, 10, 24 and 48 h storage. Results show that the initial load per sample was reduced by 99.9% for both VRE and VSE strains, using citrus vapour at 0.15%. When the dosage was doubled, there was no corresponding increase in the effect and the higher dosage was actually less effective. One reason might be that the vapours reached a saturation point at which they were active against the cells and so the maximum effect was seen at 0.15% air. Sensory panel testing demonstrated that there were no significant changes in taste of treated vegetables. It is worth noting that currently only two fumigants are used in the food industry: methyl bromide and phosphine. Methyl bromide is due to be phased out and the effectiveness and cytotoxicity of phosphine is under question, therefore the citrus vapour, after some development, may be a safe and effective alternative to the current technologies available. The use of EOs as antimicrobials on food has mainly been evaluated in oil form, despite EO vapours being shown to have antimicrobial properties against a range of pathogens [90-94] without affecting the organoleptic properties of the food stuff. Case Study n° 4: Other Applications The potential use of EOs as natural preservatives has also been investigated in dairy products, ready-to-eat foods and bakery products (see Table 3). With regards to dairy products, clove, cinnamon, thyme and bay oil were found to be highly effective against L. monocytogenes and Salmonella Enteritidis both in low (16%) and high fat (30%) cheeses [63]. Cheeses slices were often used for the preparation of “convenience foods”, which are products designed to save consumers time in the kitchen. These foods require minimum preparation (typically just heating), and their basic element is that they can minimize preparation, cooking, and clean-up time. Among minimally processed and ready-to-eat foods, Caprese salad is a traditional Italian dish, which contains tomatoes, cow or water buffalo mozzarella cheese slices, oregano, extra-virgin olive oil, basil, and wall rocket (arugula, Eruca sativa). Despite several benefits, including simplicity and quickness of preparation, the very short shelf life (1 to 2 days) of caprese salad hampers its presence in canteens and local markets. The possibility of prolonging the shelf life of caprese salad was evaluated using modified atmosphere packaging in combination with thymol [95]. Populations of lactic acid bacteria, yeasts, and molds were determined in sliced tomatoes, and populations of mesophilic and psychrotrophic bacteria, lactic acid bacteria, total coliforms, enterococci, and Pseudomonadaceae were determined in the mozzarella cheese. The combination of the thymol dip and the modified atmosphere used (65:30:5 N2:CO2:O2) extended the shelf life from 3.77 to 12 days and did not affect the growth kinetics of lactic


Speranza and Corbo

acid bacteria and enterococci, thus preserving the function of mozzarella cheese in the salad. Another interesting result was that thymol did not affect the sensory characteristics of caprese salad because oregano is a traditional component of this dish. Table 3: Overview of studies testing the antibacterial activity of EOs or their components in foods. FOOD

EO or COMPONENT

CONCENTRATION

BACTERIAL SPECIES

REFERENCES

Meat and Meat Products Cooked Beef

Grape seed extract (ActiVin ) Pine bark extract (Pycnogenol®) Oleoresin rosemary (Herbalox®)

1% w/w

E. coli O157:H7 L. monocytogenes Salmonella Typhimurium Aer. hydrophila

[96]

Dried Beef

Cinnamaldehyde

0.5% v/w

Inoculated Gram positive bacteria Natural Gram negative bacteria

[97]

TM

Beef fillets

Oregano oil

0.8% v/w

L. monocytogenes

[65]

Beef fillets

Oregano oil

0.8% v/w

Salmonella Typhimurium

[38]

Ground Beef

Oregano oil

0.05-1% v/w

Natural microflora

[69]

Ground Beef

Capsicum (bell pepper)

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. *Address correspondence to this author Milena Sinigaglia at: Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy; E-mail: [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.

Enzymes as Antimicrobials


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

[3, 5, 9]

Other types of lysozyme: g-type lysozyme

egg white of domestic goose

h-type lysozyme

Plant

i-type lysozyme

Invertebrates

b-type lysozyme

Bacteria (Bacillus)

v-type lysozyme

Viruses

[4, 9-11]

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


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 Teichoic acid content in the cell-wall

Suggested mechanisms of resistance in Grampositive bacteria

Production of proteininhibitors specific to lysozyme

N-deacetylation of the acetamido group of the hexosamine residues

Incorporation of Daspartic acid in the bacterial peptidoglycan crossbridge

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

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 Action Towards Gram Negative Bacteria Gram negative bacteria are generally resistant to lysozymes, due to the presence of a membrane outer the peptidoglycan, which shields the peptidoglycan from lysis. On the other hand, sensitive Gram negative bacteria have been described [20, 32], demonstrating that the presence of the outer membrane in Gram negative bacteria does not provide them with an absolute protection against the hydrolytic action of lysozyme and that other mechanisms, not hindered by LPS, may exist [3, 33, 34]. The existence of these mechanisms of resistance is well established and has been extensively evidenced by the sensitization of Gram negative bacteria upon disruption or permeabilization of their lipopolysaccharidic layer [3, 35]. Many researchers tried to enhance the antimicrobial activity of lysozyme against Gram negative bacteria by using chemical or physical treatment to disrupt the bacterial membrane [36], combining with other antimicrobial or/and chemical substances [37, 38] and modifying the structure of lysozyme [39, 40, 41]. In addition, some natural lysozymes, as well as chemically or genetically modified hen egg white lysozyme (HEWL), have been reported to be active against Gram negative bacteria also in the absence of such permeabilizers [42, 43]. Recently, it has been suggested that Gram negative bacteria could use another strategy, involving specific protein-inhibitors with high affinity to lysozyme [33]. In particular, a high affinity lysozyme inhibitor, was identified during a systematic study of orphan gene products in Escherichia coli [33], and was accordingly renamed Ivy (Inhibitor of vertebrate lysozyme). A survey on the prevalence of Ivy homologues among bacteria revealed that Ivy represents a family of protein-inhibitors occurring mostly in Gram negative bacteria [9]. At the functional level, Ivy does not substitute the LPS as the primary mechanism of resistance to lysozyme, but it plays a subsidiary role, as it is expressed only when the outer membrane becomes porous as a result of exposure to damaging agents [44, 45]. In a recent work, Callewort et al. [34] reported the discovery of a novel family of bacterial lysozyme inhibitors with widespread homologs in Gram negative bacteria. This family of protein-inhibitors comprises a lysozyme inhibitor isolated by Salmonella Enteritidis and designated as PliC (periplasmic lysozyme inhibitor of c-type lysozyme) and a membrane bound lysozyme inhibitor of c-type lysozyme (MliC), isolated from E. coli and Pseudomonas aeruginosa. Further studies, revealed the mode of action of the novel lysozyme inhibitors (PliC and MLiC) on lysozyme resistance, demonstrating that these protein-inhibitors, like Ivy, would play a subsidiary role in lysozyme resistance and act only on strains with damaged outer membrane [34]. Antimicrobial Action Towards Viruses, Yeast and Moulds Lysozyme appears to inhibit not only bacteria, but also viruses [13] and eukaryotic microorganisms including parasites [46] and fungi [47-49] despite the absence of typical peptidoglycan in their envelopes. However, the inhibition of yeast and mould has been explained by the presence of chitin as an important constituent of their cell-wall [50, 51], and by the fact that lysozyme also possesses a chitinase activity [52]. Therefore, lysozyme would not act only by its hydrolytic activity, but it would inhibit the growth of some microorganisms either by permeabilizing the plasma membrane and/or acting on intracellular components by virtue of its cationic hydrophobic nature, as described for a variety of antimicrobial peptides [14, 53, 54]. LYSOZYME: SAFETY AND TOXICITY Egg whites are well known to be allergenic to sensitive individuals, particularly children [55], and lysozyme may constitute one of the allergen in egg albumine. Moreover, allergic reaction produced by egg-white lysozyme in animals and human are less dangerous than reactions to other egg proteins such as albumin and ovalbumin, used as food ingredients. Lysozyme has been regarded as Generally Recognized As Safe (GRAS) by the FDA with its tentative final rule dated March 13, 1998. However, because of the potential for allergenicity of lysozyme, the FDA has tentatively concluded that bulk and packaged foods containing lysozyme must be labeled to indicate the presence of “eggwhite lysozyme”. On September 2006, the USDA has authorized the use of hen egg-white lysozyme as an ingredient in or on processed products labeled as “organic”. In addition, the Joint Food and Agricultural Organization/World Health


D’Amato et al.

Organization Expert Committee on Food Additives stated in its report that: “Lysozyme is obtained from edible animal tissue commonly used as food and can thus be designed as class I enzyme and regarded as a food. It was therefore considered acceptable for use in food processing when used in accordance with good manufacturing practice” [56]. In the European Union, its use in specific products such as hard cheeses (to prevent late blowing) and in wine making (to control infection) is allowed. Worldwide, lysozyme is used as a preservative in cheese, sea foods, fruit and vegetables, beer and wine, and as a component of pharmaceutical products [57]. The use of hen eggwhite lysozyme has been approved in many countries to control growth of spoilage organisms in food, but the legislation on lysozyme differs from country to country. The product must always be used in accordance with prevailing rules and regulations. Lysozyme (extracted from egg white) can be safely used in cheese production and is approved as a preservative (E1105) in the EU directive on food additives. Lysozyme is also approved for use in cheese in Austria, Belgium, Canada, Denmark, Finland, France, Germany, Italy, UK and Spain. The "Organisation Internationale du Vin" (OIV) approved the use of lysozyme in wine (up to 500 ppm) during its General Assembly held in Argentina in 1997, and its use in wines is awaiting approval in other countries. APPLICATION OF LYSOZYME Egg-white lysozyme is desiderable as a food preservative because its ease of purification in economically feasible quantities from egg white, specific activity against target bacteria and fungi, low effective usage levels, and low impact on sensory qualities of foods [58]. Commercially, lysozyme has been used primarily to prevent late blowing in semi-hard cheeses, which is caused by the fermentation of lactate by butyric acid bacteria, primarily Clostridium tyrobutyricum [1, 59]. The potential use of lysozyme as a food preservative has invoked considerable interest, particularly in Japan and in Far East [1]. Several applications have been developed and patented, including the treatment of fresh fruits, vegetables, seafood, meat, tofu, sake, butter, potato salads, custard. Lysozyme is also added to infant formulae in order to make them more closely resemble to human milk (cow's milk contains very low levels of a lysozyme enzyme). Lysozyme is currently used in other food such as frankfurters, cooked meat and poultry products [60]. Wine Lysozyme has recently been introduced to the wine industry to control malolactic fermentation in wine and has been permitted to the level of 100 mg/l [61]. The principle of using lysozyme in wine depends on its ability to control the growth of lactic acid bacteria. Lactic acid bacteria can indeed play both a positive and a negative role in wine; for example, they are responsible for the conversion of the malic acid into the lactic acid; in the same time, they can become a problem if their growth is uncontrolled, either during the early stages of the alcoholic fermentation, or after the completion of the malo-lactic fermentation, mainly because of the production of excessive volatile acidity and biogenic amines. Finally, certain spoilage lactic acid bacteria (Lactobacillus spp, Pediococcus spp) can produce particularly negative results, leading to flawed or unmarketable wines. Until recently, sulfur dioxide was used, worldwide, to control malolactic fermentation, inhibit spoilage bacteria and yeasts and provide antioxidative properties to wine during storage. Although they have been used for almost as long as wine has been made, it is widely admitted nowadays that sulphites should be utilised more rationally in wine, because there is a general trend towards the reduction of the sulphite levels in wine due to the widespread use of sulphites in other foods (fruit, vegetables, fruit juices), and the related risk of toxicity coming from a too high cumulative intake, together with the well-known allergenicity of sulphites. Moreover, sulfur dioxide is not active against the growth of lactic acid bacteria in low acid wines, and when it is used to stabilize the wine after malo-lactic fermentation, sulphites have a detrimental impact on the organoleptic qualities of the wine ("bleaching effect" on the colour and off-flavours). Several studies have shown that lysozyme enzyme can be a good alternative/complement to the use of sulphites. For example, lysozyme does not interfere with the alcoholic fermentation, because of its specific activity on lactic acid bacteria; moreover, its activity increases with the pH.



The research done on the use of lysozyme in wine has resulted in the identification of three major types of applications: 1.

The total inhibition of the malo-lactic fermentation, by the addition of 500 ppm (50 g/hl) of lysozyme to grape must.

2.

The protection of the wine during sluggish or stuck alcoholic fermentation accidents; in fact, the addition of 250 to 350 ppm (25 to 35 g/hl) can inhibit the growth of the lactic acid bacteria during the time necessary to re-start the alcoholic fermentation.

3.

The microbiological stabilization of wine after malo-lactic fermentation. In particular, an addition of 250 to 300 ppm (25 to 30 g/hl) of lysozyme allows for a dramatic reduction in the quantity of sulphites necessary to stabilise the wine, down to the minimum level required to ensure the protection against oxidation and the control of the acetic acid bacteria (not inhibited by lysozyme).

Dairy Products A patented process in the UK applies to the addition of lysozyme to butter or milk used to make Italian cheeses so as to control undesirable organisms such as Cl. tyrobutyricum [62]. Sinigaglia et al. [63] in a recent study evaluated the effectiveness of lysozyme combined with ethylenediaminetetraacetic disodium salt (Na2-EDTA) against the spoilage microorganisms of mozzarella cheese. Lysozyme is commonly used instead of nitrate to prevent late blowing in hard and semi-hard cheeses such as Gouda, Edam, Provolone and Emmentaler, in the United States and numerous other countries. Late blowing is due to the contamination of milk by a naturally occurring, spore-forming bacterium, Cl. tyrobutyricum. The origin of the contamination of milk by this bacterium lies in the widespread use of silage as a feed. The spores of Cl. tyrobutyricum are present in the soil particles, into the corn or hay used to make silage; they germinate in the silage if a rapid acidification does not take place. When the cows are fed with the contaminated silage, the spores are excreted and, if the milking is not carried out under very strict hygienic conditions, the spores can subsequently contaminate the milk. Spores of Cl. tyrobutyricum have been found to survive the normal heat treatment of milk during the production of cheese. During the ripening of salt brined, semi- and hard cheeses clostridia ferment lactate into butyric acid and large quantities of gas (CO2 and H2). The formation of gas produces undesirable effects in texture (cracks and irregular eyes) and the acids cause unacceptable tastes and smells. Late-blowing can create considerable loss of product, especially in the production of semi- hard and hard cheeses. Lysozyme is usually added to milk at 20-35 ppm, resulting in levels in cheese ranging from 200 to 400 ppm, which does not generally inhibit starter cultures [58] or affect physical or organoleptic properties of the cheese. The use of lysozyme in cheese will prevent the growth of Cl. tyrobutyricum as lysozyme is able to lyse the cell walls of the vegetative form of Cl. tyrobutyricum through the enzymatic cleavage of the bacterial cell wall. Lysozyme binds to the casein prior to clotting of the milk, remains active in the curd throughout the ripening process and can disrupt the vegetative cell walls once the spores germinate. Meat Products Lysozyme was used to improve the storage properties of parboiled sausage [64]. Lysozyme used in combination with other preservatives, reduced microbial spoilage of salami and sausages. However, it was inactivated in presence of common salt concentrations exceeding 1.2% [65]. Fish and Seafood Products Due to the inhibitory action towards clostridia, lysozyme has been proposed in smoked fish to prevent the growth and toxin formation of Cl. botulinum. Lysozyme at a concentration of 0.005–0.03% in combination with 0.25-0.35% NaCl has been used to preserve low quality salmon or trout roe. A combination of lysozyme and glycine has been reported by Eisai [66] to be an effective preservative of fish paste.


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Fruit and Vegetables Fruit and vegetables are low acid foods and very susceptible to spoilage by Cl. botulinum strains and Listeria monocytogenes. US patent obtained by Dell’Acqua et al. [67] and Ueno et al. [68] report on bacterial decontamination of vegetables using hen egg white lysozyme. Fruit and vegetables may also be contaminated by non-pathogenic bacteria and fungi that may alter the quality of the product. Fresh vegetables, tofu, kimchi, Japanese potato salad, sushi, Chinese noodles and creamed custards have been preserved using lysozyme or a combination of lysozyme and amino acids [1].

LACTOFERRIN LACTOFERRIN: STRUCTURE AND PROPERTIES Lactoferrin is a non-haem iron-binding protein that is part of the transferrin protein family, along with serum transferrin, ovotransferrin, melanotransferrin and the inhibitor of carbonic anhydrase [69], whose function is to transport iron in blood serum. Lactoferrin was first isolated by Sorensen and Sorensen from bovine milk in 1939. In 1960 it was concurrently determined to be the main iron binding protein in human milk by three independent laboratories [70]. Lactoferrin is produced by mucosal epithelial cells in various mammalian species, including humans, cows, goats, horses, dogs and several rodents. It is found on mucosal surfaces, within the specific granules of polymorphonuclear leukocytes, and in biological fluids, including milk, saliva and seminal fluid, indicating that it may play a protective role in the innate immune response. Milk is by far the most abundant source of lactoferrin togheter with colostrum (7 g/l) [71] and lactoferrin represent the second most abundant protein in milk [72], after caseins. There is a great variation in the concentration of lactoferrin in human body fluids and the lowest lactoferrin concentrations were found in blood plasma. The concentration in blood is normally very low ( C. krusei > C. albicans > C. gulliermondi > C. parapsilosis > C. glabrata, with the last species being the most resistant. As for bacteria, the anti-Candida activity of lactoferrin was initially considered as related to its ability to bind and sequester environmental iron. Later, it was observed that lactoferrin can kill both C. albicans and C. krusei by altering the permeability of the cell surface, as it does with bacteria [125]. Similar cell


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wall damage has been reported by Nikawa et al. [103], after Candida exposure to both human and bovine lactoferrin [103, 104], and it was concluded that the candidacidal activity of human lactoferrin is due to the direct interaction of the protein with the fungal cell surface, rather than to iron sequestration [126]. Further studies have confirmed lactoferrin antifungal activities. In addition to all this fundamental research carried out on Candida, it is necessary to cite also some investigations by Wakabayashi et al. [127, 128] and by Yamauchi et al. [129] on dermatophytic fungi, belonging to the genus Trichophyton. The sensitivity of these fungi to lactoferrin was tested in vitro and in vivo, in animal models and in patients, indicating another important potential usefulness of lactoferrin. Zarember et al. [88] showed that Fe3+ sequestration by neutrophil apo-lactoferrin is important for host defence against A. fumigatus. The antifungal activity of lactoferrin has also been reported to be considerably lower than the activity of commercially available antifungal drugs. However, the combined use of lactoferrin and several commercial drugs, e.g. clotrimazole, fluconazole, amphotericin B and 5-fluorocytosine, demonstrates additive or synergistic activity [105, 130]. LACTOFERRIN: SAFETY AND TOXICITY Bovine lactoferrin as a food ingredient is thought to be safe, because there is a long dietary history of its use. Several studies have also revealed the beneficial effect of lactoferrin on gut health. This protein helps maintaining optimum levels of beneficial bacteria such as bifidobacteria in the intestinal tract and so prevents gastrointestinal inflammations. In the United States, the Food and Drug Administration (FDA) granted the generally recognised as safe (GRAS) status to DMV International's milk-derived lactoferrin, in August 2001 and permitted it at levels of 65.2 mg/kg in beef. Currently, the European Union does not have a specific regulation for lactoferrin. It is considered a milk protein (provision 79/112/ EEC allows all types of milk protein to be labeled as milk protein) and hence would be allowed in foods [131]. In Japan, lactoferrin is specified in the List of Existing Food Additives, which is a list of the permitted natural additives. In addition, biotechnology organisations such as Applied Phytologics and Japan Agricultural Cooperatives are exploring the plant molecular farming potential of lactoferrin through rice crops. In South Korea, lactoferrin concentrates are also listed as authorized natural additives. In Taiwan, lactoferrin may be used in special nutritional foods under the condition “only for supplementing foods with an insufficient nutritional content and may be used in appropriate amounts according to actual requirements” [131]. APPLICATION OF LACTOFERRIN Lactoferrin is already used in a wide range of products including infant formulae, sport and functional foods. Some examples of bovine lactoferrin application in commercial products are reported in Table 3. Currently, bovine lactoferrin is used to supplement foods such as infant formulae, health supplements, functional food and drinks, yogurt and pet foods. The expected effects of these products include anti-infection, improvement of orogastrointestinal microflora, anti-inflammation and antioxidation. Infant formulae supplemented with bovine lactoferrin are already available in Japan, in Korea and Indonesia. Other fields of application are skin care and oral care products. Bovine lactoferrin containing oral care products are sometimes combined with other mucosal defense proteins such as lactoperoxidase and lysozyme. The first application of bovine lactoferrin in a commercial product was its supplementation in the infant formula ‘‘BF-L’’ by Morinaga Milk Industry in 1986. Several studies reported the employment of lactoferrin in different foods to extend the shelf life, increase the nutritional value and reduce the oxidation of the products. For example, Huang et al. [132] reported that bovine lactoferrin inhibited lipid oxidation in corn oil emulsions and lecithin liposome systems. In a recent work, Chiu and Kuo [133] reported that the addition of lactoferrin extended the shelf life and increased the nutritional value (iron) of ground pork. Ransom and Belk [134] showed that activated lactoferrin was effective in inhibiting the growth of different pathogen strains on meat products, whereas Satué-Gracia et al. [135] investigated the use of lactoferrin in a infant formulae to reduce the oxidation.



Moreover, several works evaluated the effectiveness of lactoferrin in combination with other natural products such as chitosan [136], lysozyme [137] and nisin [138]. Table 3: Example of commercial application of bovine lactoferrin. Product

Functionalities/ effect

Milk-based infant formulae

Improved resistance against pathogens and oro-gastro-intestinal microflora, antioxidant

Yogurt

Improved resistance against pathogens, anti-infection and orogastro-intestinal microflora, antioxidant

Health supplements

Aid in iron absorption, e.g. for pregnant women, immune aid

Functional food drinks

Increased iron solubility and absorption

Cosmetics

Antioxidant, hygiene

Oral care products (mouth wash, mouth gel, toothpaste), chewing gums

Improved oral hygiene, moistening

LACTOPEROXIDASE INTRODUCTION TO THE LACTOPEROXIDASE SYSTEM Refrigeration is the most commonly used method to stop or retard the deterioration of milk on its way from the farm to the dairy industry. In developed countries, the bacteriological quality of raw milk is safeguarded during collection, storage and transportation through refrigeration; whereas cooling facilities in rural areas are not available leading to the excessive multiplication of bacteria and increases in the acidity of raw milk far beyond the level acceptable for processing. The lactoperoxidase system (LPS) has been introduced as an alternative way of preserving milk: it is currently the only method (apart refrigeration) of raw milk preservation approved by Codex. It was adopted by Codex Alimentarius as a guideline in 1991 (CAC GL 13/91), following an evaluation by JECFA (Joint FAO/WHO Expert Committee on Food Additives) [139]. Although its name points out the dairy origin, lactoperoxidase is also present and active in many secretory fluids in various parts of the body: saliva, tears, nasal fluid, uterine luminal fluid and vaginal secretions. Numerous works focused on the dairy and processing dimensions of lactoperoxidase, but recently different studies have also been conducted to extend the applications of the LPS in food systems and commercial products. THE LACTOPEROXIDASE SYSTEM It consists of the enzyme lactoperoxidase (LP) and two substrates: thiocyanate ions (SCN¯) and hydrogen peroxide (H2O2). Lactoperoxidase Lactoperoxidase (LP) (EC 1.11.1.7) is a glycoprotein consisting of a single peptide chain containing 612 amino acid residues with a molecular weight of ca. 78 kDa and an isoelectric point of 9.8. Lactoperoxidase contains one haem group (protohaem 9), and about 10% carbohydrate. The haem group in the catalytic centre of the LP is a protoporphyrin IX, covalently bound to the polypeptide chain through an ester bond. The iron content of LP is 0.07%, corresponding to one iron per LP molecule, being part of the haem group. A calcium ion is strongly bound to LP, stabilising its molecular conformation and the calcium ion activity appears to be of vital importance for the structural integrity of LP [140]. This enzyme is an oxidoreductase and catalyses the oxidation of thiocyanate (SCN ¯) at the expense of H2O2 to generate intermediate products with antimicrobial properties against bacteria, fungi and viruses. Thiocyanate Ions Thiocyanate ions (SCN ¯) is present in mammary, salivary and thyroid glands and their secretions, in organs such as the stomach and kidney and in fluids such as synovial, cerebral, cervical and spinal fluids, lymph and plasma [141]. Its concentration partly depends on the feeding regime of the animal; in bovine milk, the SCN ¯ concentration reflects blood serum levels and varies with breed, species, udder health and type of feed. Fresh


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cow milk contains 1-10 mg/l of thiocyanate, which is not always sufficient to activate the LP system, thus assuming that for the activation of LPS the SCN ¯ might be assumed through the diet (the main sources are glucosinolates and cyanogenic glucosides). Vegetables and cabbage, kale, brussel sprouts, cauliflowers, turnips and rutabaga, are particularly rich in glucosinolates, which upon hydrolysis yield thiocyanate in addition to other reaction products. Cyanogenic glucosides are also found in cassava, potatoes, maize, millet, sugar cane, peas and beans [141]. Hydrogen Peroxide Hydrogen peroxide (H2O2) is not normally detected in raw milk, but it may be generated endogenously, for example, by polymorphonuclear leucocytes in the process of phagocytosis; in addition many lactobacilli, lactococci and streptococci produce sufficient H2O2, under aerobic conditions, to activate LPS. LPS is also activated artificially with an enzymatic generator, an association of glucose oxidase (GOD) and glucose as reported: Glucose O2 GOD Gluconic acid H2O2 SCN ˉ LPO H2O O SCN ˉ

Hydrogen peroxide is approved for the preservation of milk in the absence of refrigeration. It may be added at a concentration of 100-800 ppm. It is highly toxic for mammalian cells; however, at low concentrations (100µM or less) and in the presence of LP and SCN ¯ mammalian cells are protected from this toxicity [141]. REACTION MECHANISM OF THE LACTOPEROXIDASE SYSTEM In Fig. 3 the reaction mechanism of the lactoperoxidase system (LPS) is reported. The first step is the reaction of the ferric haem of the native enzyme with hydrogen peroxide, that results in the compound-I reaction intermediate, which is the highest oxidized form of the enzyme: it reacts with a substrate molecule and is converted into a secondary compound (Compound II) that has lost one equivalent [142]. Compound-I is reduced back to the native state in either two successive one-electron steps, via the intermediate compound-II, or in one two-electron step.

Figure 3: Reaction mechanism of the LPS.



In the presence of an excess of hydrogen peroxide, the intermediates compound-I and -II may also react further with hydrogen peroxide, resulting in the superoxide adduct (compound-III) [143]. Although LPS has been extensively studied and described in scientific literature, several questions remained unanswered; Ghibaudi and Laurenti [142] highlighted some controversial features tried to solve these questions and proposed a more extended mechanism, including catalase activity and a protein radical intermediate [142]. The LP enzyme catalyses the peroxidation of thiocyanate to generate products, which kill or inhibit the growth of many species of microorganisms, exerting biocidal and/or biostatic activities. By this reaction thiocyanate in the presence of H2O2 is converted in hypothiocyanous acid (HOSCN). Two different pathways have been proposed for the production of SCN ¯ ions. The first pathway is the oxidation of SCN ¯ produces thiocyanogen (SCN)2, which is rapidly hydrolysed to yield HOSCN and hence hypothiocyanite OSCN ¯(see the reactions): 2 SCN ¯ + H2O2 + 2H+

peroxidase

(SCN)2 + 2H2O

(SCN)2 + H2O → HOSCN + SCN ¯ + H+ HOSCN ↔ H+ + OSCN ¯ OSCN ¯ is in equilibrium with HOSCN and at the pH of maximal LP activity (pH 5.3) their amounts are equal. Both forms exert antibacterial activity, but there is evidence that the uncharged HOSCN is more bactericidal [140]. In fact, as reported by Tenovuo et al. [144] at low pH (≤ 5.3) the antimicrobial hypothiocyanite compound occurs as an uncharged molecule; this makes it easier to pass through the bacterial membrane, enter the cell and inhibit metabolic process. The second pathway for the production of SCN ¯ions is the direct oxidation to OSCN ¯ : SCN ¯ + H2O2

peroxidase

OSCN ¯ + H2O

The stability of OSCN ¯ is affected by many factors: pH, light, metal ions, glycerol, ammonioum sulphate as well as by the presence and removal of LP. ANTIMICROBIAL ACTIVITY OF LACTOPEROXIDASE SYSTEM Bacteriostatic and/or Bactericidal Effect The oxidation of the thiol groups (-SH) of enzymes and proteins is of crucial importance in the bacteriostatic and/or bactericidal effect of the LPS: the structural damage of microbial cytoplasmatic membranes by the oxidation of SH-groups results in a leakage of potassium ions, amino acid and peptides into the medium, thus the uptake of glucose, amino acids, purines, pyrimidines in the cell and synthesis of proteins, DNA and RNA are also inhibited [140]. On the other hand, the antimicrobial activity of LPS can be inhibited by reducing agents containing SH groups, such as cysteine, glutathione, mercapto-ethanol, dithiothreitol, and sodium hydrosulphite either by direct binding to the haem group or by scavenging OSCN ¯ [140]. The reaction of (SCN)2 or OSCN ¯ with proteins results in the oxidation of the protein –SH groups, to produce sulphenyl thiocyanate, usually followed by disulphide formation: +

(SCN)2 → R-S-SCN + SCN ¯ + H+

+

O SCN ¯ → R-S-SCN + H+

R-SH

Sulphenyl thiocyanate derivatives may be further modified to yield mainly sulphonic acid:


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R-S-SCN + H2O → R-S-OH + SCN ¯ + H+ The rate of release of SCN ¯ (important for the further oxidation of SH groups) from sulphenyl derivatives is dependent on the concentration of SCN ¯. The effect of the LPS on bacteria can be reversible or irreversible; in fact, different groups of microorganisms show a varying degree of sensitivity to the LPS: 1.

Gram negative, catalase positive organisms, such as pseudomonads, coliforms, salmonellae and shigellae, are not only inhibited by the LPS but may be killed if sufficient H2O2 is provided chemically, enzymatically or by hydrogen peroxide producing microorganism [145, 146].

2.

Gram positive, catalase negative bacteria, such as streptococci and lactobacilli are generally inhibited but not killed by the LPS [147]. The capacity of cells to recover from inhibition depends mainly on environmental conditions, e.g. temperature and pH.

3.

Yeasts, including Candida species, and moulds are strongly inhibited.

Bactericidal effect of LPS is more pronounced on bacteria during the exponential growth phase than in the stationary phase. The difference in sensitivity to the LPS can probably be explained by the differences in cell wall structure and their different barrier properties [148, 149]. The inner membrane of Gram negative bacteria appears to be more extensively damaged by LP-treatment than that of Gram positive species [150]. The activity of the LPS in vivo is not fully known. Many investigations on the antibacterial activity of the LPS have been carried out in vitro: in these experiments LP, thiocyanate and hydrogen peroxide are added to a suitable medium; in milk the system can be “activated” by addition of thiocyanate and hydrogen peroxide or an appropriate hydrogen peroxide source. Uğuz and Özdemir [151] studied the effect of different concentrations of thiocyanate-H2O2 in the medium on antibacterial properties of LPS against some pathogenic bacteria LPS and concluded that bovine LPS showed high antibacterial activity in 100 mM thiocyanate-100 mM H2O2 medium. Several reports indicated that it is better to supply H2O2 continuously by the glucose oxidase (GOD)/glucose system [152]: oxygen is one of the GOD system substrates, but its concentration was never determined for a better system efficiency. Adolphe et al. [153] used the response surface methodology (RSM) approach to determine the best concentration of GOD, glucose and LPS in order to obtain maximal SCN ¯ peroxidation (at 4 and 25°C), focusing also on the effect of each component and their interaction. At 4 and 25°C the average of the optimized concentration of the LPS (for a fixed SCN- concentration of 0.5 mmol, to work in excess of substrate for the LP) was 85.5 IU/l for GOD, 8 mmol for glucose and 3927.5 IU/l for LP at an initial pH value of 6.5. The optimized system had a bacteriostatic effect on L. monocytogenes CIP 82110T and a strong bactericidal effect against Ps. fluorescens CIP 6913T. Seifu et al. [154], studied the effect of the LPS on the activity of commercial mesophilic cheese starter cultures (Table 4) and screened for LPS resistance cultures which could be used for cheesemaking from goat’s milk preserved by activation of the LPS. Table 4: Composition of the starter cultures [154]. STARTER CULTURES

COMPOSITION OF THE STARTER CULTURES

SUPPLIERS

CHN11 CHN22 DCC240 CHN19 Flora Danica Normal (FDN)

Lactococcus lactis subsp. lactis; L. lactis subsp. cremoris; L. lactis subsp. diacetylactis; Leuc. mesenteroides subsp. cremoris.

Chr. Hansen, Denmark

LL50C

Lact. lactis subsp. lactis; Lact. lactis subsp. cremoris.

Anchor Biotechnologies (Johannesburg, South Africa)

Lact. lactis subsp. diacetylactis NCDO 176

Irene Animal Nutrition and Animal Products Institutes of the Agricultural Research Council (Irene, South Africa).



Table 4: cont....

Lact. lactis subsp. lactis NCDO 605

Irene Animal Nutrition and Animal Products Institutes of the Agricultural Research Council (Irene, South Africa).

Leuc. mesenteroides subsp. cremoris ATCC 33313

South African Institute of Medical Research.

Most of the starter cultures examined were sensitive to the LPS but varied in their susceptibility. Amongst the mixed starter cultures, the LL 50C was insensitive to the LP system, thus it could be used for cheesemaking from goat’s milk preserved by the LPS. On the other hand, the production of goat milk is a major problem, limiting its consumption, caused by the growth of both pathogenic and spoilage organisms: various bacterial infections have been linked to the consumption of raw goat milk [155]. Seifu et al. [155] determined the effect of LPS on the growth and survival of E. coli, Staph. aureus, L. monocytogenes and Brucella melitensis in the milk of Saanen and South African Indigenous goats kept at 30°C for 6 h. Activation of LPS exhibited both bacteriostatic and bactericidal effect as reported in the Table 5. Table 5: Bacteriostatic and bactericidal effect in the milk of Saanen and South African Indigenous [155] Milk source

Pathogen

LPS-microbial effect

L. monocytogenes

Bactericidal effect

Br. melitensis

Bactericidal effect

Staph. aureus

Bacteriostatic effect

E. coli


L. monocytogenes

Bactericidal effect

Br. melitensis

Bactericidal effect

Indigenous

Saanen

Staph. aureus

Bactericidal effect

E. coli


Fungal Activity of Lactoperoxidase System The antifungal activity of LPS (lactoperoxidase system) against Rhodotorula rubra, Sacch. cerevisiae, Mucor rouxii, A. niger and Byssochlamys fulva was reported. Goat milk LP-thiocyanate- H2O2 system was found to inhibit the growth and proliferation of many fungal species like A. flavus, Trichoderma spp., Corynespora cassicola, Phytophthora meadii and Corticium salmonicolor as well as Alternaria spp., Penicillium chrysogenum and Claviceps spp.; however, C. albicans and Pythium spp. were not affected by the LP system [141]. FOOD APPLICATIONS Milk and Dairy Products: Factors Limiting the LPS Activity In bovine milk, LP (lactoperoxidase) is always present in excess; buffalo’s milk, ewe’s milk and goat’s milk generally also containing high levels of LP. The factor limiting the activity of the LPS in milk from these species are the low concentrations of thiocyanate and hydrogen peroxide: they are naturally present in milk but in limited quantities (i.e. 1-15 mg/l) depending on the feed of the animal [156]; consequently, variable quantities of the two substrates should be added to the milk to make LPS more efficient. Although IDF [157] recommends that 10 ppm of thiocyanate and 10 ppm of peroxide be used to render the LPS effective, several researchers [158, 159] have recommended that for each specific area the optimal dosage of the substrates should first be determined before the LPS is applied. Furthermore, the determination of LPS components, at different time, is very important although, not specified in many studies; therefore, Althaus et al. [160] studied the effect of time elapsed from the moment of taking samples on LPS components and analyzed the activity of LP and the concentrations of SCN ¯ and H2O2 in 46 individual samples of Manchega ewe milk. Samples were maintained at 4°C until analysis, which took place at 6, 12, 24 and 48 h after extraction. They observed that: 

Higher concentrations of SCN ¯ and H2O2 were observed in samples analyzed 6 h after sampling than in samples analyzed at 12, 24 and 48 h.




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LP activity was lower when determined at 48 h compared with the analyses made 6, 12 and 24 h after sampling.

These results confirmed that the time of the analysis of the LPS components affects their concentrations and activity. Fonteh [161] analyzed the milk collected from 54 cows (23 Red Fulani, 16 Goudali and 15 Holstein) to assess the level of lactoperoxidase activity and thiocyanate and to evaluate how breed and season may influence LPS components. As reported in previous works [162, 163], significant variations in thiocyanate concentration were observed, indicating that the efficacy of LPS in preserving milk could also vary. The feed composition is, probably, the most important factor that influence the thiocyanate levels in dairy cattle; differences in thiocyanate levels between breeds could be due also to genetic factors. Gouda Gouda is one of the variety of cheeses made from goat milk: the high pH and the insufficient lactic acid content to suppress spoilage bacteria emphasize the importance of using milk of good bacteriological quality for making this cheese. As the composition of goat milk is different from cow milk (especially the casein fractions), Seifu et al. [164] focused their study to assess the suitability of goat milk preserved by the LPS for the manufacture of Gouda cheese and determined its effect on the biochemical, microbiological and organoleptic properties of Gouda cheese during a ripening period of 90 days. They concluded that: 1.

Gouda made from LP-activated goats’ milk had a significantly (p < 0.05) lower coliform and coagulase positive staphylococci as compared to cheese made from the untreated control goats’ milk.

2.

The LP treatment did not affect the overall chemical composition of the cheese.

3.

The level of proteolysis in both the control and the LP-treated goats’ milk cheeses was comparable whereas the level of lipolysis was different: in cheese made from LP-activated goats’ milk was significantly (p < 0.05) lower than that made from the control goats’ milk at the end of the ripening period; this is important in order to reduce the strong flavour associated with goats’ milk cheeses.

4.

Significantly differences (p < 0.05) in all sensory attributes were observed between cheeses made from the untreated control and LP-activated goats’ milk; the latter had a more pronounced flavour than the control.

Beef Kennedy et al. [165] used a ground beef model to assess the effect of LPS on the growth of natural population of microorganisms present in ground beef and on the growth of E. coli O157:H7, L. monocytogenes and Staph. aureus inoculated in a broad system at 37°C and in a ground beef system stored at 0, 6 and 12°C. The sensitivity hierarchy was the following: L. monocytogenes > Staph aureus > E. coli O157:H7 The numbers of bacteria increased markedly in the control over 4 h, on the other hand, growth of the microbial population was strongly inhibited by the presence of LPS. Vegetables The ability to preserve food in a state that is both appetising and nutritious is a basic requirement for health. In recent years, an increasing of foodborne microbial outbreaks was registered favoured by the quickly preparation with little or no heating. Moreover, fresh-cut fruit and vegetables and their juices are of special concern because the removal of the peel also eliminates the external barrier for invading bacteria [166]. As a consequence, outbreaks of E. coli O157 :H7, L. monocytogenes, Salmonella sp. and Sh. flexneri have been linked to products as unpasteurized apple juice and apple cider, tomatoes and tomato juice and unpasteurized orange juice [167, 168]. The LPS was used to inactivate or inhibit Salmonella Enteritidis in several juices (tomato, carrot) and animal products (milk, liquid whole egg and chicken skin) [169].



The system was more effective: 

Against the organism in vegetable juices than in animal products. The lower effectiveness of the system in animal products can be attributed to: 1) the binding of LPS to fats and proteins in the products; 2) loss of the hypothiocyanite by adsorption to food components; 3) cell repair in the rich media provided by foods of animal origin.



At low pH than at neutral pH. Salmonella Enteritidis was inoculated in carrot juice at three different pH (4.5, 5.5 and 6.5) and added with LPS. Moreover, after 4 h no viable cells could be detected in juice at pH 4.5 and 5.5 while in juice at pH 6.5 numbers of Salmonella Enteritidis has decreased by 3.7 log units. The acidic pH may itself stress the bacterial cells, increasing their sensitivity to the LPS. Moreover, it is known that at acidic pH, the major oxidation product is HOSCN: being uncharged, it could diffuse more readily through the hydrophobic bacterial membrane than OSCN ¯ to oxidize intracellular components.



At higher temperatures than at lower temperatures.

An important drawback of adding LPS to fruit juices is that ascorbic acid content is significantly reduced and, vice versa, the presence of ascorbic acid significantly reduces the antimicrobial efficiency protecting E. coli and Shigella spp. against inactivation by the LPS. HURDLE TECHNOLOGIES Hurdle technology advocates the synergistic combinations of various antibacterial techniques in order to drastically limit the growth of spoilage bacteria [170]. Combined Effects of Lactoperoxidase System and Heat Goat milk is a very nutritious food in many part of the tropics and contribute to human nutrition in many developing Countries that process the milk into various type of cheeses. The use of starters in cheese manufacture is as old as cheesemaking and their growth and relative activity may be inhibited by different factors, including LPS. Heat sensitivity of E. coli is well documented [171]. The application of heat remains one of the most important technologies used for the control of E. coli in the foods; thus, combining heat, pH and LP, Parry-Hanson et al. [172] evaluated the influence of these treatments on survival of acid-adapted and nonadapted E. coli O157:H7 in goat milk. They concluded that the combination of LP and heat treatments inhibited both acid-adapted and non-adapted E. coli O157:H7 cells at 55°C and non-adapted cells at 60°C at pH 6.9. The inhibitory effect was more pronounced at pH 5.0; however, these treatment may be insufficient to eliminate the presence of E. coli O157:H7 in acidified dairy products. When pasteurization is applied, the spoilage microflora in milk is completely different to that found in raw milk. It consists mainly of: 

post-pasteurization contaminants (PPC) (bacteria that contaminate the milk after heating);



thermoduric microorganisms (microbes that are able to withstand the pasteurization process);



spore-former bacteria.

Marks et al. [173], determined the role of LPS in inhibiting growth of Ps. aeruginosa, Staph. aureus, Strep. thermophilus and B. cereus in pasteurized milk (at 72°C/15 s and 80°C/15 s) and on the “Keeping Quality (KQ)”. An active LPS was found to greatly increase the KQ of milk inoculated with Ps. aeruginosa, Staph. aureus and Strep. thermophilus and pasteurized at 72°C, whereas no or little effect on milks pasteurized at 80°C was observed. However, pasteurization temperature had no effect on the KQ of milk challenged with B. cereus spores. Combined Effects of Lactoperoxidase System and Antimicrobial Compounds Because LPS (lactoperoxidase system) has a broad-activity spectrum inhibiting Gram positive and Gram negative bacteria, an additional hurdle to improve the safety of food preservation is represented by nisin that inhibits only Gram positive bacteria. Elotmani and Assobhei [174], tested the antimicrobial effects of nisin (25, 50, 100 and 200 IU/ml) and LPS, alone or in combination, against sardines flora to investigate the preservability of fish using these inhibitors as potential biopreservatives. The best antimicrobial activity was showed by LPS in combination with nisin (100 IU/ ml). In particular:


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

the inhibitory effect on Gram positive bacteria was enhanced when nisin and LPS were added in two steps; nisin makes bacteria more susceptible to LPS by placing additional environmental stresses (hurdle effects) on the organisms [170].



the inhibitory effect on Gram negative bacteria was enhanced as results of the addition of LPS in the first place which injuries Gram negative, thus cell supporting the effect of nisin.

Although the modes of action are different for both inhibitors, the fact that their primary cellular target is the same (cytoplasmic membrane) could explain their synergic action: it could be that LPS increases the permeability of the membrane to nisin facilitating its bactericidal action. Recently Arqués et al. [175] combined the LPS (or nisin) with reuterin in order to inactivate Gram negative bacteria pathogens in refrigerated milk. At 4°C reuterin and LPS alone was bactericidal against Salm. enterica, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica; at the same temperature, a strong synergistic bactericidal activity of reuterin in combination of LPS on E. coli O157:H7 and S. enterica was observed, whereas at 8°C all the Gram negative bacteria tested were inhibited. The mechanism of inactivation by the combination of reuterin and LPS is not known; also in this case, the structural damage of cytoplasmic membranes by oxidation of –SH groups due to LPS activation might be the primary target of the inhibition. Afterwards, injured cells could be an easier target for reuterin inhibiting DNA synthesis. Combined Effects of Lactoperoxydase System and Other Processes García-Graells et al. [176] treated a wide range of relevant food bacteria by high hydrostatic pressure in skim milk supplemented with LPS; the effects of LPS alone and combined to HHP treatment are reported in the following table (Table 6). Table 6: the effects of LPS alone and combined to HHP treatment [176]. Microorganisms SalmonellaTyphimurium E. coli LMM1010

LPS effect no effect

LPS + HHP effect at low cell concentrations (106 cfu/ml) growth inhibiting effect

no effect

no effect

no effect

E. coli MG1655 L. innocua Staph. aureus Lact. plantarum

no effect growth inhibiting effect

E. faecalis Ps. fluorescens

LPS + HHP effect at high cell concentrations (109 cfu/ml)

growth inhibiting effect growth inhibiting effect

no effect growth inhibiting effect growth inhibiting effect

bactericidal effect

no effect

As observed, the presence of LPS affected inactivation by high pressure and was influenced by cell density. Furthermore, in contrast with some reports in the literature, both the most sensitive (Ps. fluorescens) and the most resistant organisms (Salmonella Typhimurium and E. coli LMM1010) were Gram negative, demonstrating no systematic difference in LPS sensitivity between Gram negative and Gram positive bacteria. Successively Vannini et al. [177], evaluated the interactive effects of high pressure homogenization on the activity of lysozyme and/or LPS against a selected group of microorganisms (Gram positive and Gram negative bacteria) inoculated in skim milk. An instantaneous viability loss of all the species during the high pressure homogenization in the presence of the LPS was observed: this behaviour could be explained as consequence of the passage of the suspension through the narrow valve that made easier the contact between LPS and the target cells. REFERENCES [1] [2] [3]

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Lactoperoxidase: Physico-chemical properties, occurrence, mechanism of action and applications. Br J Nutr 2000; 84: S19–25. [141] Seifu E, Buys EM, Donkin EF. Significance of the lactoperoxidase system in the dairy industry and its potential applications: a review. Trends Food Sci Technol 2005; 16: 137–54. [142] Ghibaudi E, Laurenti E. Unraveling the catalytic mechanism of lactoperoxidase and myeloperoxidase. Eur J Biochem 2003; 270: 4403–12. [143] Boots JW, Floris R. Lactoperoxidase: From catalytic mechanism to practical applications. Int Dairy J 2006; 16: 1272– 6. [144] Tenovuo J, Lumikari M, Soukka T. Salivary lysozyme, lactoferrin and peroxidases: antibacterial effects of carcinogenic bacteria and clinical applications in preventive dentistry. Proc Finn Dent Soc 1991; 87: 197–208. [145] Björck L. Fox PF, Eds. Advanced Dairy chemistry proteins. London; Elsevier. 1992; vol. 1, pp. 332-8. [146] Reiter B, Marshall VME, Björck L, Rosén CG. Non specific bactericidal activity of the lactoperoxidasethiocyanatehydrogen peroxide system of milk against Escherichia coli and some Gram negative pathogens. Infect Immun 1976; 13: 800–7. [147] Oram JD, Reiter B. The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The effect of the inhibitory system on susceptible and resistant strains of group N streptococci. Biochem J 1966; 100: 373–81. [148] deWit JN, van Hooydonk ACM. Structure, functions and applications of lactoperoxidase in natural antimicrobial systems. Neth Milk Dairy J 1996; 50: 227–44. [149] Reiter B, Härnulv G. Lactoperoxidase antibacterial system: natural occurrence, biological functions and practical applications. J Food Prot 1984; 47: 724-32. [150] Marshall VM, Reiter B. Comparison of the antibacterial activity of the hypothiocyanite anion towards Streptococcus lactis and Escherichia coli. J Gen Microbiol 1980; 120: 513–6. [151] Uguz MT, Özdemir H. Purification of bovine milk lactoperoxidase and investigation of antibacterial properties at different thiocyanate mediated. Appl Biochem Microbiol 2005; 41: 349-53.


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[152] Björck L, Rosén CG, Marshall V, Reiter B. Antibacterial activity of the lactoperoxidase system against pseudomonads and other Gram negative bacteria. Appl Microbiol 1975; 30: 199-204. [153] Adolphe Y, Jacquot M, Linder M, Revol-Junelles AM, Milliér JB. Optimization of the components concentrations of the lactoperoxidase system by RSM. J Appl Microbiol 2006; 100: 1034-42. [154] Seifu E, Buys EM, Donkin EF. Effect of the lactoperoxidase system on the activity of mesophilic cheese starter cultures in goat milk. Int Dairy J 2003; 13: 953-9. [155] Seifu E, Buys EM, Donkin EF, Petzer IM. Antibacterial activity of the lactoperoxidase system against food-borne pathogens in Saanen and South African Indigenous goat milk. Food Control 2004; 15: 447–52. [156] FAO - Food and Agricultural Organization. Manual on the use of the LP-system in milk handling and preservation. Rome: Food and Agriculture Organization of the United Nations. 1999. [157] International Dairy Federation (IDF). Indigenous Antimicrobial Agents of Milk – Recent Developments. Brussels, Belgium: IDF. 1994. [158] Kamau DN, Kroger M. Preservation of raw milk by treatment with hydrogen peroxide and by activation of the lactoperoxidase (LP) system. Milchwissenschaft 1984; 39: 658–60. [159] Ridley SC, Shalo PL. Farm application of lactoperoxidase treatment and evaporative cooling for the intermediate preservation of unprocessed milk in Kenya. J Food Prot 1990; 53: 592-7. [160] Althaus RL, Molina MP, Rodríguez M. Analysis time and lactation stage influence on lactoperoxidase system components in dairy ewe milk. J Dairy Sci 2001; 84: 1829-35. [161] Fonteh FA. Influence of breed and season on lactoperoxidase system components in cow’s milk in Western Cameroon. Livestock Research for Rural Development 2006; available on line at: www.Irrd.org/Irrd18/2font18030.htm. Accessed October, 30 2009 [162] Medina M, Gaya P, Nuñez, M. The lactoperoxidase system in ewes’ milk: levels of lactoperoxidase and thiocyanate. Lett Appl Microbiol 1989; 8: 147-9. [163] Fonteh FA, Grandison AS, Lewis MJ. Variations of lactoperoxidase activity and thiocyanate content in cows’ and goats’ milk throughout lactation. J Dairy Res 2002; 69: 401-9. [164] Seifu E, Buys EM, Donkin EF. Quality aspects of Gouda cheese made from goat milk preserved by the lactoperoxidase system. Int Dairy J 2004; 14: 581–9. [165] Kennedy MJ, O’Rourke AL, McLay JC, Simmonds RS. Use of a ground beef model to assess the effect of the lactoperoxidase system on the growth of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus in red meat. Int J Food Microbiol 2000; 57:147-58. [166] Leverentz B, Conway WS, Alavidze Z, et al. Examination of bacteriophage as a biocontrol method for Salmonella on fresh-cut fruits: a model study. J Food Prot 2001; 64: 1116-21. [167] Parish ME. Public health and nonpasteurized fruit juices. Crit Rev Microbiol 1997; 23: 109-19. [168] Krause G, Terzagian R, Hammond R. Outbreak of Salmonella serotype Anatum infection associated with unpasteurized orange juice. South Med 2001; 94: 1168-72. [169] Touch V, Hayakawa S, Yamada S, Kaneko S. Effects of lactoperoxidase-thiocyanate-hydrogen peroxide system on Salmonella enteritidis in animal or vegetable foods. Int J Food Microbiol 2004; 93: 175-83. [170] Leistner L, Gorris LGM. Food preservation by hurdle technology. Trends Food Sci Technol 1995; 6: 41–6. [171] Kaur J, Ledward DA, Park RWA, Robson RL. Factors affecting heat resistance of Escherichia coli O157:H7. Lett Appl Microbiol 1998; 26: 325-30. [172] Parry-Hanson A, Jooste PJ, Buys EM. The influence of lactoperoxidase, heat and low pH on survival of acid-adapted and non-adapted Escherichia coli O157:H7 in goat milk. Int Dairy J 2009; 19: 417-21. [173] Marks NE, Grandison AS, Lewis MJ. Challenge testing of the lactoeproxidase system in pasteurized milk. J Appl Microbiol 2001; 91; 735-41. [174] Elotmani F, Assobhei O. In vitro inhibition of microbial flora of fish by nisin and lactoeproxidase system. Lett Appl Microbiol 2003; 38: 60-6. [175] Arqués JL, Rodríguez E, Nuñez M, Medina M. Inactivation of Gram negative pathogens in refrigerated milk by reuterin in combination with nisin or the lactoperoxidase system. Eur Food Res Technol 2008; 227: 77-82. [176] Garcia-Graells C, Van Opstal I, Vanmuysen SCM, Michiels CW. The lactoperoxidase system increases efficacy of high-pressure inactivation of food-borne bacteria. Int J Food Microbiol 2003; 81: 211-21. [177] Vannini L, Lanciotti R, Baldi D, Guerzoni ME. Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase. Int J Food Microbiol 2004; 94: 123– 35.


83

CHAPTER 6 Antimicrobial Agents of Microbial Origin : Nisin Daniela D’Amato* and Milena Sinigaglia Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy 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: [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.

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. 15

5 Dha Ile

1 Ile

Dhb

S

Leu Ala

Ala

Abu

Ala Pro

S

NH 2

Lys

Leu Ala

Met

Gly

Gly

Abu

Ala

Gly

Asn

S

10

Met Nisin A

S

His Asn COOH

Lys 34

Dha

Val

His

Ile

Ser

Lys Abu

Ala Abu

Ala

30

20

Ala

25

S

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

Nisin in Foods


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 (undecaprenylpyrophosphorylMurNAc-(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].



However, it has been observed that Gram negative cells, normally insensitive to the action of nisin, can be sensitized by the addition of chelating agents which disrupt the integrity of the outer membrane and allow the bacteriocin access to the cytoplasmic membrane [46, 47]. Although chelating agents show good in vitro effects in buffer systems, results in food systems are far less pronounced owing to preferred interaction between chelating agents and divalent ions in foods [48]. Other treatments that can make Gram negative bacteria susceptible to nisin include sub-lethal heat, osmotic shock and freezing. Nisin is used mainly to prevent the outgrowth of spores in a wide variety of foods. The action of nisin is predominantly, as it affects the post-germination stages of spore development, and inhibits pre-emergent swelling, and thus the outgrowth and formation of vegetative cells [25]. Nisin action against spores is caused by binding to sulfhydryl groups of protein residues [49]. Some species of spore-formers are more sensitive than others, Geobacillus stearothermophilus is more sensitive than B. cereus, B. megaterium or B. polymyxa. It is effective in preventing the outgrowth of Cl. botulinum types A, B and E, but the proteolitic types are more resistant that the non-proteolitic ones. Sensitivity increases with lower pH, increased temperature and heat shocking. Sensitivity to nisin to both vegetative cells and spores can vary between genera and even between strains of the same species [50]. SAFETY AND TOXICITY OF NISIN The suitability of nisin as a food preservative arises from the following characteristics: it is not toxic; the producer strains of L. lactis are regarded as safe; it is not used clinically, at present; there is not apparent crossresistance in bacteria that might affect antibiotic therapeutics; it is sensitive to digestive proteases (can be hydrolyzed into amino acids in the intestine by a-chymotrypsin) [9]; it does not produce changes in the organoleptic properties of the foods [51] and is heat stable at low pHs. Since 1953, nisin has been sold as a commercial preparation under the trade name Nisaplin by Aplin & Barret Ltd (UK). Nisin was labelled as GRAS (generally recognized as safe) in 1988 by FDA [52], although it had been in use in Europe for some time (the world Health Organization approved the use of nisin in 1961) and was also added to the European food additive list where it was assigned the number E234 [53] and is currently permitted as a food additive in over 50 countries around the world. The FAO/WHO Codex Committee on milk and milk products accepted nisin as a food additive for processed cheese at a concentration of 12.5 mg pure nisin per kilogram product [7]. However, there are major differences in national legislations concerning the presence and levels of nisin in various food products. For instance, nisin can be added to cheese without limit in the United Kingdom, while a maximum concentration of 12.5 mg/g in that food is allowed in Spain [54]. In several countries (eg. France, Netherlands, Australia and Argentina), the addition of nisin to processed cheese is permitted, also if with different limits. The addition of nisin to pasteurized milk is permitted only in some countries in the Middle East, where shelf life problems occur owing to the warm climate, the necessity to transport milk over long distances and poor refrigeration facilities. However, it is not permitted in the UK and other countries with temperate climate. FOOD APPLICATIONS Nisin has found practical application as a natural food preservative; Table 2 reports the main categories of food in which nisin is used, its typical action and the level of effectiveness. Natural Cheese The first application of nisin was to prevent blowing problems in some hard and semi-hard cheese, such as Emmenthal and Gouda. This is caused by contamination with anaerobic spore-formers Cl. butyricum and Cl. tyrobutyricum. Cheese can also be contaminated with Lactobacillus spp., causing off flavours and gas production and with food poisoning pathogens L. monocytogenes and Staph. aureus, all of which are susceptible to nisin. Using food-grade genetic transfer techniques of conjugation it has now been possible to develop nisinproducing and nisin-resistant starter cultures with the desired properties for cheese quality. Cheeses have been made with sufficient nisin content to provide protection against growth of Clostridium spp., Staph. aureus and L. monocytogenes.

Nisin in Foods


Processed Cheese Processed cheese products cover a wide range, including block cheese (44–46% moisture), slices (46–50% moisture), spreads (52–60% moisture), sauces and dips (56–65% moisture). All these products are heat processed and contain emulsifying salts; several factor, such as, the bacterial quality of the raw ingredients, severity of the melt process, filling temperature and shelf life requirement can affect their microbial stability and hence the requirement and level of nisin. Typical heat processing (85–105°C for 5–10 min) of the raw cheese during melting does not eliminate spores, that are often present in the ingredients used in the manufacture of these products and are able to survive the heat process. The composition of processed cheese, the relatively high pH (5.6–6.0), the moisture content and low redox potential (anaerobic conditions), can result in spore germination and growth. Spore former associated with processed cheese are Cl. butyricum, Cl. tyrobutyricum, Cl. sporogenes and Cl. botulinum. Spore outgrowth of the first three species listed may result in spoilage due to the production of gas, off odours and liquefaction of the cheese, whereas Cl. botulinum produces a potentially fatal toxin. Other Pasteurized Dairy Products Other pasteurized dairy products, such as milk, chilled desserts, clotted cream and mascarpone cheese, cannot be subjected to a full sterilization without damaging their organoleptic qualities, and are thus preserved with nisin to extend their shelf life. Egg Products Pasteurized liquid egg products can comprise the whole egg, the egg yolk and egg white, together with valueadded egg products (eg. omelettes, scrambled eggs, pancake mixes). All these products receive heat treatments (62-65°C for 2-3 min) to destroy Salmonella. However, such heat treatment is insufficient to kill bacterial spores and the more heat-resistant non-spore forming Gram positive bacteria, like Ent. faecalis. Many of the surviving bacteria are psychrotrophic, and so these products usually have a limited shelf life. Nisin also protects the eggs from the growth of the psychroduric food poisoning bacteria B. cereus and L. monocytogenes. Hot Baked Flour Products Typical products include crumpets (popular products in UK, Australia and New Zealand) and potato cakes. Crumpets have a non-acid pH (pH 6), high moisture (48–54%) and high water activity (0.95–0.97). They are lightly cooked on a hot plate during manufacture, and are traditionally toasted before eating. The product is sold at ambient temperature and has a shelf life of five days. Flour used in the manufacture of crumpets contains low numbers of B. cereus spores that are not killed during the hot plate cooking process. During the 3-5 day ambient shelf life of the product the condition are ideal for outgrowth; therefore, B. cereus can increase from undetectable levels to > 105 cfu/g – a sufficient number to cause food poisoning. The addition of nisin to prevent the growth of B. cereus has received regulatory approval in Australia and New Zealand. Canned Products Nisin is used in canned foods principally for the control of thermophilic spoilage. In most countries it is mandatory that low acid canned foods (pH between 4.5 and 7) should receive a minimum heat process to ensure the destruction of Cl. botulinum spores. Nisin addition can facilitate prolonged storage of canned vegetables at warm ambient temperatures by inhibiting spore outgrowth of some thermophilic organisms, such as, G. stearothermophilus (cause of flat sour spoilage) and Cl. thermosaccharolyticum (cause of blown cans). Nisin is used in canned peas, carrots, peppers, potatoes, mushrooms, okra, baby sweet corn and asparagus. It is also used in canned dairy puddings containing semolina and tapioca. The bacterial spoilage of canned high acid foods (pH below 4.5) is restricted to non-pathogenic, heat resistant, aciduric, spore-forming bacterial species such as Cl. pasteurianum, B. macerans, B. coagulans and A. acidoterrestris.



Alcoholic Beverages Nisin has a potential role in the production of alcoholic beverages and control the growth of acid tolerant lactic acid bacteria of the genera. The insensitivity of yeasts to nisin allows its use to control the spoilage due to Lactobacillus, Pediococcus and Leuconostoc that can spoil beer and wine. Nisin can be added to fermenters to prevent or control contamination, reduce the pasteurization regimes and increase the shelf life of unpasteurized and bottle-conditioned beers. Furthermore, nisin can be used in the pitching yeast wash, as an alternative to acid washing which affects yeast viability. Formerly, nisin cannot be used during fermentations, to avoid the inhibition of malolactic fermentation. However, this problem has been overcome by developing nisin resistant strains of Oenococcus oeni, that can grow and maintain malolactic fermentation in the presence of nisin. Salad Dressings The development of salad dressings with reduced acidity may improve the flavour of cold blended; however, the use of reduced levels of acetic acid can favour lactic acid bacterial spoilage. The addition of nisin can control the growth of these bacteria. Meat Products Nisin has been considered as an alternative preservative system to that of nitrite in processed meat products. However, several studies have shown that nisin is generally not as effective in the preservation of meat as it is in dairy products. This is thought to be due to interference by meat components such as phospholipids that limit its activity, especially where there is a high-fat content [2]. The reasons of the inadequacy of nisin as preservative system in meat could include also binding of nisin onto meat surface, poor solubility in meat system and uneven distribution. At current state only high (and uneconomic) levels of nisin, achieved good control of Cl. botulinum. However, both modified atmosphere and vacuum packaging in combination with nisin have shown promising results. Encouraging results have been obtained also in vacuum-packed cooked continental type sausages where lactic acid bacteria can cause spoilage. Nisin has better inhibitory effects against lactic acid bacteria in sausages with lower fat levels, and in sausages containing diphosphate compared to those with orthophosphate. This application has achieved regulatory approval in USA. Fish Products The potential hazard of botulism both in vacuum and modified atmosphere-packed fish led to a trial application of nisin by spray to fillets of cod, herring and smoked mackerel inoculated with Cl. botulinum Type E spores. Toxin production showed a significant delay both at 10 and 26°C. Another problem in smoked fish is the growth of the psychroduric pathogen L. monocytogenes, especially in fresh and lightly preserved products. Nisin is an effective antilisterial agent in smoked salmon, especially when packed in a carbon dioxide atmosphere. Yogurt Use of nisin as biological additive to yogurt to control sensitive pathogens and to extend the shelf life has been suggested [55]. The addition of nisin to stirred yogurt post-production has an inhibitory effect on the starter culture (a mixture of Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus strains), thereby preventing over-acidification. Thus an increase in shelf life is obtained by maintaining the flavour of the yogurt (less sour) and preventing syneresis. However, such use is limited by the sensitivity of the starter culture to nisin which may impair the fermentation depending on the concentration of nisin and the strains used. Pasteurized Soups A recent trend in soup manufacture is the production of fresh pasteurized products with relatively limited chilled shelf life. Heat resistant spores of Bacillus spp. and Alicyclobacillus spp. are able to survive pasteurization. Nisin is effective at preventing or delaying outgrowth of Bacillus and Alicyclobacillus spp. during a prolonged storage. Other Applications Several works showed different use of nisin as preservative system, in combination with other antimicrobial compounds, for example, some authors reported a synergistic effect between nisin and carvacrol against B.

Nisin in Foods


cereus cells at neutral pH [42, 56]; whereas, Natress et al. [57] showed the effectiveness of combination of nisin with lysozyme to control meat spoilage bacteria. Besides, in a recent study, Arqués et al. [58] evaluated the antimicrobial activity of nisin combined with other antimicrobial compounds, such as, reuterin and lactoperoxidase system, in a Spanish dairy product. Other researchers focused on the enhancement of the antimicrobial activity of nisin in liposome encapsulation [16] and incorporated in micro-particles of calcium alginate [59]. Table 2: Typical application of nisin in food products. Food application

Action

Level of nisin

Natural cheeses, ripened cheeses, soft white fresh cheese

To control L. monocytogenes, prevent blowing in some hard and semi-hard ripened cheeses, caused by contamination with Cl. butyricum and Cl. tyrobutyricum, control Lactobacillus spp., causing off flavours and gas production

Processed cheese (block cheese, slices, spreads, sauces, dips)

To control the outgrowth of the spores

To control non botulinic spoilage from 6.25 to 12.5 mg/kg; for antibotulinum protection 12.5 mg/kg or higher

Other pasteurized dairy products (milk, dairy desserts, clotted cream, mascarpone cheese)

To extend shelf life of the products

Nisin at levels of 0.75-1.25 mg/l can double the shelf life of milk

Pasteurized liquid egg

To extend shelf life of the products and protect egg from the growth of B. cereus and L. monocytogenes

Nisin at levels of 2.5-5 mg/l caused a significant increase in shelf life

Hot baked flour products (crumpets, potato cakes)

To control the outgrowth of Bacillus spores

Addition of nisin at concentration of 3.75 mg/kg inhibited B. cereus

Canned products

To prevent spoilage by thermophilic, heat resistant spore formers

Nisin addition between 2.5 and 5 mg/kg can be used to control spoilage in low acid canned vegetables; 1.25-2.5 mg/kg are used in high acid tomato-based products

Alcoholic beverages (beer, wine)

To control spoilage by lactic acid bacteria (Lactobacillus, Pediococcus, Leuconostoc)

Nisin at level of 0.25-2.5 mg/l was effective in both beer and wine

Salad dressing

To control the spoilage by lactic acid bacteria (LAB)

Growth of LAB has been successfully controlled by the addition of 2.5-5 mg/l of nisin

Meat products

Nisin does not perform at its full potential in meat system. Both modified atmosphere and vacuum packaging in combination with nisin have shown more promising results

Nisin results effective only at high levels (12.5 mg/kg and above)

Fish products

Few studies have been carried out on the use of nisin as a preservative hazard of fish and shellfish

Nisin at 25 mg/kg in combination of a reduced heat process achieved a Listeria reduction significantly better than either heat or nisin alone

Yogurt

The addition of nisin to stirred yogurt post production has an inhibitory effect on the starter culture, preventing over-acidification of the products

Typical addition levels for this application are 0.5-1.25 mg/kg

Pasteurized chilled soup

To prevent or delay outgrowth of spoilage Bacillus spp., during prolonged storage

Nisin at level of 2.5-5 mg/l is effective for this application

REFERENCES [1] [2] [3]

Cleveland J, Montville TJ, Nes IF, Chikindas ML. Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 2001; 71:1-20. Deegan LH, Cotter PD, Hill C, Ross P. Bacteriocins: biological tools for bio-preservation and shelf life extension. Int Dairy J 2006; 16: 1058-71. Gautam N, Sharma N. Bacteriocin: safest approach to preserve food products. Indian J Microbiol 2009; 49: 204-11.


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Klaenhammer TR. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev 1993; 12: 39-85. Breukink E, de Kruijff B. The lantibiotic nisin, a special case or not? Biochim Biophys Acta 1999; 1462: 223-34. Carr FJ, Chill D, Maida N. The lactic acid bacteria: a literature survey. Crit Rev Microbiol 2002; 28: 281-370. Ross RP, Morgan S, Hill C. Preservation and fermentation: past, present and future. Int J Food Microbiol 2002; 79: 3-16. Jung G, Sahl HG, Eds. Nisin and novel lantibiotics. Leiden: ESCOM Science Publishers 1991. Pongtharangkul T, Demirci A. Evaluation of agar diffusion bioassay for nisin quantification. Appl Microbiol Biotechnol 2004; 65: 268-72. Garcera MJ, Elferink MG, Driessen AJ, Konings WN. In vitro poreforming activity of the lantibiotic nisin. Role of proton motive force and lipid composition. Eur J Biochem 1993; 212: 417–22. Gross E, Morell JL. The structure of nisin. J Am Chem Soc 1971; 93: 4634–5. Buchman GW, Banerjee S, Hansen JN. Structure, expression, and evolution of a gene encoding the precursor of nisin, a small protein antibiotic. J Biol Chem 1988; 263: 16260–6. Chan WC, Lian LY, Bycroft BW, Roberts GCK. Confirmation of the structure of nisin by complete 1H NMR resonance assignment in aqueous and dimethyl sulfoxide solution. J Chem Soc Perkin Trans I 1989; 2359–67. Mulders JW, Boerrigter IJ, Rollema HS, Siezen RJ, de Vos W. Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur J Biochem 1991; 201: 581-4. De Vos WM, Mulders JW, Hugenholtz J, Kuipers OP. Properties of nisin Z and distribution of its genes, nisZ, in Lactococcus lactis. Appl Environ Microb 1993; 59: 213-8. Laridi R, Kheadr EE, Benech RO, Vuillemard JC, Lacroix C, Fliss I. Liposome encapsulated nisin Z: optimization, stability and release during milk fermentation. Int Dairy J 2003; 13: 325-36. Parente E, Ricciardi A. Production, recovery and purification of bacteriocins from lactic acid bacteria. Appl Microbiol Biotechnol 1999; 52: 628-38. Mota-Meira M, LaPointe G, Lacroix C, Lavoie MC. MICs of mutacin B-Ny266, nisin A, vancomycin, and oxacillin against bacterial pathogens. Antimicrob Agents Chem 2000; 44: 24–9. Bartoloni A, Mantella A, Goldstein BP et al. In vitro activity of nisin against clinical isolates of Clostridium difficile. J Chemotherapy 2004; 16: 119–21. Hansen JN. Nisin as a model food preservative. Crit Rev Food Sci 1994; 34: 69-93. Breukink E, van Heusden HE, Vollmerhaus PJ et al. Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes. J Biol Chem 2003; 278: 19898-903. Hasper HE, Kramer NE, Smith JL et al. An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II. Science 2006; 313: 1636-7. Linnett PE, Strominger JL. Additional antibiotic inhibitors of peptidoglycan synthesis. Antimicrob Agents Chem 1973; 4: 231-6. Reisinger P, Seidel H, Tschesche H, Hammes WP. The effect of nisin on murein synthesis. Arch Microbiol 1980; 127: 187-93. Davies J, Delves-Broughton J. Nisin. In: Robinson RK, Batt CA, Patel PD. Eds. Encyclopedia of Food Microbiology. London, Academic Press. 1999; pp 191-7. Ruhr E, Sahl HG. Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles. Antimicrob Agents Chem 1985; 27: 841-5. Sahl HG. In: Jung G, Sahl HG Eds. Nisin and Novel Lantibiotics. Leiden, ESCOM Science Publishers. 1991; pp. 347-58. Breukink E, Wiedemann I, van Kraaij C, Kuipers OP, Sahl H, de Kruijff B. Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 1999; 286: 2361-4. van Heusden HE, de Kruijff B, Breukink E. Lipid II induces a transmembrane orientation of the pore-forming peptide lantibiotic nisin. Biochemistry 2002; 41: 12171-8. Breukink E, van Kraaij C, Demel RA, Siezen RJ, Kuipers HG, de Kruijff B. The C-terminal region of nisin is responsible for the initial interaction of nisin with the target membrane. Biochemistry 1997; 36: 6968-76. Harris LJ, Fleming HP, Klaenhammer TR. Developments in nisin research. Food Res Int 1992; 25: 57-66. Ray B. In: Ray B, Daeschel MA Eds. Food Biopreservatives of Microbial Origin. Boca Raton, CRC Press 1992; pp. 207-64. Abee T, Rombouts FM, Hugenholtz J, Guihard G, Letellier L. Mode of action of nisin Z against Listeria monocytogenes Scott A grown at high and low temperatures. Appl Environ Microb 1994; 60: 1962-8. Delves-Broughton J, Gasson MJ. In: Dillon VM, Board RG Eds. Natural Antimicrobial Systems and Food Preservation. Wallingford, Cab International 1994; pp. 99-131. Jack RW, Tagg JR, Ray B. Bacteriocins of Gram positive bacteria. Microbiol Rev 1995; 171-200. Crandall AD, Montville TJ. Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype. Appl Environ Microb 1998; 64: 231-7. Gao FH, Abee T, Konings WN. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes. Appl Environ Microb 1991; 57: 2164-70.

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[38] [39]

[40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51]

[52] [53] [54] [55] [56] [57] [58]

[59]


Bruno MEC, Montville TJ. Common mechanistic action of bacteriocins from lactic acid bacteria. Appl Environ Microb 1993; 59: 3003-10. Thomas LV, Wimpenny JWT. Investigation of the effect of combined variations in temperature, pH, and NaCl concentration on nisin inhibition of Listeria monocytogenes and Staphylococcus aureus. Appl Environ Microb 1996; 62: 2006-12. Beuchat LR, Rocelle M, Clavero S, Jaquette CB. Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy. Appl Environ Microb 1997; 63: 1953-8. Jaquette CB, Beuchat LR. Combined effects of pH, nisin, and temperature on growth and survival of psychrotrophic Bacillus cereus. J Food Prot 1998; 61: 563–70. Pol IE, Smid EJ. Combined action of nisin and carvacrol on Bacillus cereus and Listeria monocytogenes. Lett Appl Microbiol 1999; 29: 166-70. Sahl HG, Bierbaum G. Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from Gram positive bacteria. Annu Rev Microbiol 1998; 52: 41-79. Helander IM, Mattila-Sandholm T. Permeability barrier of the Gram negative bacterial outer membrane with special reference to nisin. Int J Food Microbiol 2000; 60: 153-61. Vaara M, Plachy WZ, Nikaido H. Partitioning of hydrophobic probes into lipopolysaccharide bilayers. Biochim Biophys Acta 1990; 1024: 152-8. Ukuku DO, Fett WF. Effect of nisin in combination with EDTA, sodium lactate, and potassium sorbate for reducing Salmonella on whole and fresh-cut cantaloupe. J Food Prot 2004; 67: 2143-50. Belfiore C, Castellano P, Vignolo G. Reduction of Escherichia coli population following treatment with bacteriocins from lactic acid bacteria and chelators. Food Microbiol 2007; 24: 223-9. Delves-Broughton J. Nisin as a food preservative. Food Aust 2005; 57: 525-7. Morris SL, Walsh RC, Hansen JN. Identification and characterization of some bacterial membrane sulfhydryl groups which are targets of bacteriostatic and antibiotic action. J Biol Chem 1984; 259: 13590-4. Delves-Broughton J, Blackburn P, Evans RJ, Hugenholtz J. Applications of the bacteriocin, nisin. Antonie Van Leeuwenhoek 1996; 69: 193-202. Guerra NP, Agrasar AT, Macías CL, Bernárdez PF, Castro LP. Dynamic mathematical models to describe the growth and nisin production by Lactococcus lactis subsp. lactis CECT 539 in both batch and re-alkalized fed-batch cultures. J Food Eng 2007; 82: 103-13. Food and Drug Administration. Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Fed Reg 1988; 53:11247-51. EEC, Commission Directive 1983; Official Journal Reference: OJ L 255, 15/09/83 p.1 83/463/EEC. Sobrino-López A, Martín-Belloso O. Use of nisin and other bacteriocins for preservation of dairy products. Int Dairy J 2008; 18: 329-343. Vandenbergh RA. Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiol Rev 1993; 12: 221-38. Periago PM, Moezalaar R. Combined effect of nisin and carvacrol at different pH and temperature levels on the viability of different strains of Bacillus cereus. Int J Food Microbiol 2001; 68: 141-8. Nattress FM, Yost CK, Baker LP. Evaluation of the ability of lysozyme and nisin to control meat spoilage bacteria. Int J Food Microbiol 2001; 70: 111-9. Arqués JL, Rodríguez E, Nuñez M, Medina M. Antimicrobial activity of nisin, reuterin, and the lactoperoxidase system on Listeria monocytogenes and Staphylococcus aureus in cuajada, a semisolid dairy product manufactured in Spain. J Dairy Sci 2008; 91: 70–5. Wan J, Gordon JB, Muirhead K, Hickey MW, Coventry MJ. Incorporation of nisin in micro-particles of calcium alginate. Lett Appl Microbiol 1997; 24: 153-8.

92


CHAPTER 7 Chitosan: a Polysaccharide with Antimicrobial Action Daniela Campaniello* and Maria Rosaria Corbo Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy 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: [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.

Chitosan in foods


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. Shellfish waste 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.

deproteinization

demineralization

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

decolouration

chitin

deacetylation

chitosan 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,


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/cm2) 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].

BIOTECHNOLOGY enzyme immobilization protein separation cell recovery chromatography  cell immobilization

FOOD INDUSTRY removal dye, suspended solid preservative colour stabilization anticholesterol and fat binding  flavour and taste

CHITOSAN

AGRICOLTURE seed control fertilizer controlled agrochemical release

Figure 3: Commercial applications of chitosan.

MEDICAL bandage blood cholesterol control controlled release of drug skin burn  contact lens

WASTEWATER TREATMENT removal of metal ions flocculant/coagulant (protein, dye, aminoacid)

COSMETICS moisturizer face, hand and body cream bath lotion

Chitosan in foods


Chitosan has been also used as a dietary supplement due to its ability to bind lipids, marketed as capsules to be ingested after meals to prevent lipid adsorption [9]. In addition, chitosan has been proposed to produce biodegradable films, cell and enzyme immobilization matrices, healing-accelerating sutures and coverings, contact lenses and artificial skin. The US Food and Drug Administration approved chitosan for fruit juice clarification, protein recovery from food process waste, edible coatings and as an additive for animal feed [13]. Moreover, recently chitosan and chitosan oligomers have attracted notable interest due to their biological activities, i.e. antimicrobial, antifungal and antitumor functions. CHITOSAN AND CHITOSAN DERIVATIVES Molecular weight and DD of glucosamine-units in the chitosan polymer chain influence chitosan solubility and interaction with the cell walls of the target microorganisms; consequently, the biological activity of chitosan depends on these physico-chemical properties. Manipulation of chitosan structure alter its mechanical and chemical properties. In particular, primary amino groups can be derivatized with useful biological ligands or modified with other entities [14]. Generally, chitosan derivatives are classified as hydrophobic and hydrophilic chitosan derivatives, depending on the group introduced on polymer. Physical Modifications Chitosan is used in various forms: 1) flakes and powder and 2) gels. 1.

flakes and powder are not used as adsorbents due to their low surface area and no porosity;

2.

to obtain porous gels and/or three-dimensional sponges, chitosan solutions are frozen and then liophylised: the porosity and morphology of the final product depends on the chitosan molecular weight, on the composition and concentration of the starting solution and on the freezing temperature and freezing rate [7]. Furthermore, in order to manufacturing gel beads, an expansion of the polymer network it could be observed so that there is an improving access to internal sorption sites and consequently an enhancing of the diffusion mechanisms. It is important to pointed out that transferring this process to industrial applications results difficult, probably due to the fragility of beads (in terms of mechanical strength) and to their low stability in acidic media [7].

In addition, to obtain chitosan membranes and films, fibers and spherical beads (of different sizes and porosities) the method of the evaporation of solvent is mainly used. Comparing its different formulations, chitosan exhibits a different influence on the microbial growth. The experimental work conducted by Fernandez-Saiz et al. [15] represents an attempt to explain this behaviour: the antimicrobial activity of 50 mg of different chitosan formulations (chitosan flakes as-receveid, chitosonium acetate solution, chitosonium acetate film) against Staphylococcus aureus, was compared. Chitosan shows optimum biocide properties only in gelled or viscous acid solution forms and as chitosonium acetate films, due to the fact that the amine groups of the biopolymer are protonated or “activated”. Another study [16], demonstrated the absence of “active” carboxylate bands in the “as-receveid chitosan flakes”. These results agreed with those previously obtained by Kiskó et al. [17], who showed that chitosan powder was not able to inhibit bacterial growth in apple juice: although some “activated” amine groups were probably formed when chitosan was added into juice owing to the relatively low pH of the medium, the chitosan quantity used was insufficient to have a detectable biocide performance. Furthermore, Lopez-Caballero et al. [18] confirmed that powdered chitosan had no effect on the growth of Gram negative microorganisms, probably for a poor solubility of chitosan at neutral pH and the presence of uncharged amino groups. Chemical Modifications Chemical modifications do not change the fundamental skeleton of chitosan; on the other hand, they bring new improved properties, obtaining a wide range of chitosan derivatives (with new characteristic) for specific use in diversified areas. Chemical modifications have two aims: 1.

improving the metal adsorption properties;

2.

changing the solubility properties of chitosan in water or acidic medium.



Chitosan can easily change the conformation from the extended two-fold to other structures by salt formation with acids; this is due to the presence of free amino group on the monomer residue of chitosan. This conformational flexibility is an advantage of chitosan for its use [19]. Chemical modifications of these groups have provided numerous useful materials in different fields of applications; however, chitosan-related applications are limited by its insolubility at neutral or high pH values. One of the reason for the intractability of chitosan lies in the rigid crystalline structure and the acetamido or primary amino group residues, which are important in forming conformational features through intra and/or intermolecular hydrogen bonding. The removal of the two hydrogens of the amino groups of chitosan and the introduction of some cross-linking agents improves the solubility of chitosan in aqueous solvents: crosslinking reduces the adsorption capacity and decreases the quantities of free amino groups thus increasing the stability of the polymer. In particular, cross-linking agents are functional groups separated by some spaced molecules and structured in various forms: rings, straight chain, branched chain; the most important compounds are gluteraldehyde (GLA), ethylene glycol diglycidyl ether (EGDE), glyoxal, epichloridrin (EPI) benzoquinone, cyclodextrin (CD), etc. It is known that they are neither safe nor environmentally friendly, thus it was suggested the use of water-soluble cross-linking agents, such as sodium trimetaphosphate, sodium tripolyphosphate, or carboxylic acids [7]. However, in some cases the addition of another polymer adds an extra level of complexity to the system and might result in adverse changes of other desirable properties [14]. As the chitosan is a polyammine, its solubility may be increased through via Schiff’s base, thus obtaining quaternary ammonium salt (Fig. 4).

Figure 4: The synthesis of quaternized N-alkyl chitosan.

The “quaternization” of chitosan was investigated by Jia et al. [20]: they prepared chitosan derivatives with quaternary ammonium salt, such as N,N,N-trimethyl chitosan, N-N-propyl-N,N-dimethyl chitosan and Nfurfuryl-N,N-dimethyl chitosan using native chitosans at DD of 96% and with different MW (214, 19 e 7.8 kDa). The antibacterial activity of these derivatives against Escherichia coli was evaluated and it was observed that NN-propyl-N,N-dimethyl chitosan showed higher antimicrobial activity than N,N,N-trimethyl chitosan, thus suggesting that the alkyl chain length affects strongly the antibacterial activity of the chitosan derivatives. These results are in agreement with the studies of Kim et al. [21], who reported that the antibacterial activity of quaternized chitosan (N-N-propyl-N,N-dimethyl chitosan, particularly) was higher than that of the whole chitosan. Liu et al. [22] reported the preparation of complexes of chitosan with alkyl β-D-glucopyranoside, a non-ionic surfactant class produced on a large scale: these complexes showed a lower thermal degradation and a different crystallinity, due to the extended chain conformation and the disaggregation of chitosan to single chain. Furthermore, they studied the antimicrobial activity of these compounds against E. coli, Pseudomonas aeruginosa, Staph. aureus, Staph. epidermidis and Candida albicans and concluded that the antimicrobial activity of the complexes was stronger than that of the chitosan and alkyl β-D-glucopyranoside alone. Further investigations have been carried out to prepare functional derivatives of chitosan and to increase its solubility in water: Yang et al. [23] prepared derivatives through the reductive N-alkylation of chitosan with various mono and disaccharides (lactose, maltose and cellobiose) and they observed that these sugars enhanced

Chitosan in foods


effectively the solubility of chitosan. The reaction products of chitosan derivatives had a structural formula as follows:

where R is either lactose, maltose or cellobiose. As reported by Lin and Chou [24] these chitosan derivatives possessed a metal-chelating and antioxidative activities. Among these chitosan derivatives, maltose derivative exerted the highest antibacterial action against Staph. aureus while E. coli was the most susceptible to cellobiose derivative. Moreover, the N-alkylated disaccharide chitosan derivatives showed a higher antibacterial activity than native chitosan at pH 7.0. [23]. These data were confirmed by other authors [25, 26]. ANTIMICROBIAL ACTIVITY - MODE OF ACTION The exact mechanism of the antimicrobial action of chitosan is not clear, but several hypothesis have been proposed. The influence of chitosan on the microbial growth is due to its ability to change cell permeability through the interaction between the positively charged chitosan molecules and negatively charged microbial cell membranes. As consequence, an internal osmotic imbalance causes leakage of electrolytes and proteins, resulting in a pronounced cell disorganization and finally in the plasmolysis [27]. The molecular weight and number of primary amino groups present in chitosan and chitosan oligosaccharides (COS) are of great importance for the bioactivity of the molecules. COS, having a cationic charge, have the ability to disrupt the cell membranes, which is predominantly anionic in charge, causing leakage and eventually cell death. Similarly, the antifungal activity of chitosan involves its positive charge which interferes with the phospholipids of the fungal cell wall, causing alterations in the permeability of the membrane [28]. As regard bacteria, chitosan acts mainly on the outer surface; it acts also as a chelating agent and binds selectively trace metals, thus inhibiting the production of toxins and growth. Finally, it activates several defence processes in the host tissue, acts as a water binding agent and inhibits various enzymes. The interaction of chitosan with DNA is another mechanism proposed for the bioactivity against fungi. It has been suggested that the consequences of this interaction are the inhibition of mRNA synthesis and proteins, causing cell dysfunction and eventual cell death. Chitosan molecules itself is too large to enter cell membranes, consequently it should be hydrolyzed by host hydrolytic enzymes such as chitinase; the hydrolytic products could penetrate into the nuclei of the fungus where they could interfere with mRNA and protein synthesis or result in the chelation of metals, spore elements and essential nutrients [29]. CHITOSAN AND MICROORGANISMS The antimicrobial and antifungal activities of chitosan are well known [30, 31, 28]; however antimicrobial activity depends on several factors: type of chitosan and its different formulation, the target organisms, growth phases and initial concentration of the population. Type of Chitosan Due to its versatility, chitosan may be applied in several and heterogeneous fields, in particular as antimicrobial and antifungal natural compound; nevertheless its use is often conditioned by its molecular weight (MW), degree of polymerization (DP) and deacetylation (DD). Chien et al. [32] studied the effects of a coating with low molecular weight chitosan (LMWC, MW = 15 kDa) and high molecular weight chitosan (HMWC, MW = 357 kDa) on the decay of citrus fruit. LMWC was more effective in controlling the growth of fungus on citrus fruits caused by Penicillium digitatum and P. italicum exhibiting effective antifungal activity. These results were probably attributable to LMWC permeability which is higher than that of HMWC.



The effects of HMWC (600 kDa) and LMWC (80 kDa) (degree of acetylation, 85%) and chitosan oligosaccharides, alone and complexed with casein, were compared using milk fermentative bacteria (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Propionibacterium freudenreichii). The presence of chitosan and chitosan oligosaccharides influenced differently the growth of bacteria. In a preliminary phase, the addition of LMWC or HMWC to the culture media inhibited strongly Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus growth, whereas for chitosan oligosaccharides treatment, a 10 fold higher concentration was necessary to obtain a similar inhibition. Both HMWC and LMWC, but not chitosan oligosaccharides, inhibited P. freudenreichii growth [33]. The best performance of chitosan on the growth of microorganisms than chitosan oligomers was observed also by No et al. [34]. They studied the antibacterial activity of chitosan and chitosan oligomers with different molecular weight (MW) against four Gram negative (E. coli, Ps. fluorescens, Salmonella Typhimurium and Vibrio parahaemolyticus) and seven Gram positive bacteria (Listeria monocytogenes, Bacillus megaterium, B. cereus, Staph. aureus, Lact. plantarum, Lact. brevis and Lact. delbrueckii subsp. bulgaricus). Several results were obtained, as follows: 1.

chitosan showed higher antibacterial activities than chitosan oligomers;

2.

chitosan (0.1%) showed stronger bactericidal effect with Gram positive bacteria than Gram negative bacteria; the minimum inhibitory concentration (MIC) of chitosans ranged from 0.05% to > 0.1% depending on the bacteria and MW of chitosan;

3.

as a chitosan solvent, 1% acetic acid was effective in inhibiting the growth of most of the bacteria tested except for lactic acid bacteria that were most effectively suppressed with 1% lactic or formic acids;

4.

antibacterial activity was inversely affected by pH (4.5-5.9) with higher activity at lower pH value. This result suggests that the addition of chitosan to acidic foods will enhance its effectiveness as a natural preservative.

On the other hand, Lee et al. [35] demonstrated that chitosan oligosaccharides have considerable bifidogenic potential observing a stimulatory effect of chitosan oligosaccharides on some strains of Bifidobacterium bifidum and Lactobacillus spp. and could be used as probiotics. MW, DD and other characteristics such as particle size, density and viscosity, influence the properties of several formulation based on chitosan. As consequence, it is very difficult to postulate an univocal effect and point out a correlation antimicrobial activity of chitosan vs MW or DD. Nevertheless, by now, the best performance resulting by the application of chitosan both in vitro that in vivo, involved chitosan with high DD. This might be due to the fact that high DD generally has higher solubility and positive charges, specially in acid environment. This evidence is supported by Chang et al. [36], who evaluated the effect of chitosan with different degree of deacetylation (DD = 54, 73 and 91%) on the gel properties and shelf life of tofu. They observed that the shelf life increased with increasing DD and that the MW of chitosan decreased from 2780 kDa for 54% DD to 189 kDa for 91% DD. Due to the fact that deacetylation at elevated temperature in alkaline solution also broke down the glycosidic linkage in chitosan molecules. As a consequence the intrinsic viscosity and MW of chitosan decreased with increasing DD. The Targets The antimicrobial properties of chitosan have been reported widely in the literature, nevertheless it is impossible to point out an univocal effect against microorganisms. By now the antimicrobial activity of chitosan was demonstrated against the following bacteria: Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, B. cereus, B. licheniformis, B. megaterium, B. subtilis, B. bifidum, Brochothrix thermosphacta, Clostridium historyticum, Cl. perfringens, Colletotrichum gloesporiodes, Enterobacter aeromonas, Ent. aerogenes, Ent. sakazakii, Enterococcus faecalis, E. coli, E. coli O157:H7, Kloeckera apiculata, Lactobacillus sp., Lact. acidophilus, Lact. brevis, Lact. delbrueckii subsp. bulgaricus, Lact. curvatus, Lact. fructivorans, Lact. plantarum, Lact. sakei, Lact. sakei subsp. carnosum, Lact. viridescens, Leuconostoc sp., Leuc. mesenteroides, Listeria innocua, L. monocytogenes, Metschinikowia pulcherrima, Kokuria varians, Pediococcus acidilactici, Ped. pentosaceus, Photobacterium phosphoreum, Pseudomonas sp., Ps. aeruginosa, Ps. fluorescens, Ps. fragi, P. freudenreichii, Rhodotorula sp., Salmonella spp., Salmonella Enteritidis, Salmonella Typhimurium, Sclerotinia sclerotorium, Serratia liquefaciens, Ser. marcescens, Shigella dysenteriae, Staphylococci, Staph. aureus, Staph. epidermidis, Strep. thermophilus, V. cholerae, V. parahaemolyticus;

Chitosan in foods


the following yeasts: C. albicans, C. humicolus, C. lambica, Cryptococcus humiculus, Saccharomyces cerevisiae, Sacch. exiguus, Saccharomycodes ludwigii, Schizosacch. pombe, Torulaspora delbrueckii, Zygosacch. bailii; and the following moulds: A. fumigatus, A. niger, A. parasiticus, Botrydiplodia lecanidion, Botrytis cinerea, Byssochlamys spp., Cladosporium sp., C. cladosporioides, Fusarium oxysporum, P. aurantiogriseum, P. chrysogenum, P. digitatum, P. expansum, P. italicum, P. notatum, Rhizopus sp., R. nigricans, R. stolonifer; both in vitro and in vivo. The reader can find exhaustive review of this topic in some papers [28, 33, 37]. Bacteria and Yeasts Sagoo et al. [38] studied the antimicrobial action of chitosan combined with sodium benzoate on three spoiling yeasts (Sacch. exiguus, Sacch. ludwigii, and T. delbrueckii). In particular the aim of their study was to determine whether low concentrations of chitosan combined with benzoate could be used to enhance their antimicrobial action. The results of this work showed that chitosan (0.005%) and sodium benzoate (0.025%) acted synergistically in saline solutions against the microorganisms tested. An interesting use of chitosan was studied by Knowles and Roller [39] to remove the microbial films on foodprocessing surface. Generally, a sanitation program involving both cleaning and disinfection processes can reduce the spread of microbial contamination; nevertheless the application of a regular cleaning regime is often expansive and chemical disinfectants sometimes release toxic substance in the environmental as well as into foods. For these reasons, the actual trend is to use natural antimicrobial compounds as novel food preservatives and sanitizers. Knowles and Roller [39] studied the combined efficacy of chitosan, carvacrol and a hydrogen peroxide-based biocide (named Spor-Klenz RTU) against dried microbial films prepared from three foodborne bacteria and one yeast (L. monocytogenes, Salmonella Typhimurium, Staph. aureus and Sacch. cerevisiae, respectively). They observed that the efficacy of each single compound was species-specific: a)

in the case of microbial films prepared using listeriae and salmonellae, Spor-Llenz RTU was the most effective, followed by carvacrol and then chitosan;

b)

in the case of microbial films prepared using Staph. aureus they observed that this microorganism was sensitive to chitosan and relatively resistant to carvacrol and Spor-Klenz RTU;

c)

in the case of yeast biofilms it was observed that Sacch. cerevisiae was more sensitive to carvacrol than to chitosan.

Based on these results, they concluded that both chitosan and carvacrol may have potential for use as natural biocides. The mechanism of the antimicrobial activity of chitosan is different in the case of Gram positive and Gram negative bacteria. Zheng and Zhu [40] studied the antimicrobial activity of chitosan with MW < 305 kDa and observed that for the Gram positive Staph. aureus the antimicrobial activity increased with increasing MW, whereas for the Gram negative E. coli the antimicrobial activity increased with decreasing of MW. They explained this different mode of action suggesting two mechanisms: in the case of Staph. aureus chitosan could form a polymer membrane on the surface of the cell, thus inhibiting nutrients from entering the cell; in the second case due to low MW, chitosan entered the cell through pervasion, disturbing the metabolism. As reported by Helander et al. [38], chitosan inhibited some Gram negative bacteria (E. coli, Ps. aeruginosa, Sh. dysenteriae, Vibrio spp. and Salmonella Typhimurium) involving binding of the cation chitosan to the anionic cell surface resulting in changes in permeability. In particular, chitosan disrupts the barrier properties of the outer membrane (OM) of Gram negative bacteria; therefore, the perturbation of the OM is reflected in increased permeability to hydrophobic probes such us 1-N-phenylnaphtylamine (NPN) and increased sensitivity to the biocidal and/or inhibitory action of a range of inimical compounds (anion agent, dyes and bile acids). These results were confirmed by Liu et al. [22] who observed, through an electron micrographs, that chitosan acetate solution-treated E. coli showed altered OMs, which were disrupted and covered by an additional tooth-like layer, whereas the inner membranes (IMs) appeared to be unaffected. In micrographs of chitosan acetate solution-



treated Staph. aureus the membrane of dividing cells appeared disrupted in the constricting region with the loss of cell contents. However, cells of Staph. aureus that were not dividing were not obviously affected. This damage was likely caused by the electrostatic interaction between NH3+ groups of chitosan acetate solution and phosphoryl groups of phospholipids components of cell membranes [42]. These observations could explain the reduction of cell numbers of E. coli and Staph. aureus by about 1 log in 5 min due to the bactericidal activity of chitosan. In particular, E. coli population was completely inactivated whereas in the case of Staph. aureus a tail effect was observed. Chitosan’s ability to disrupt the permeability barrier of the OM in Gram negative bacteria could cause the access for other substances present in the food materials, sensitizing bacteria to many external agents. This is compatible with the hurdle concept, which refers to incorporating several antimicrobial measures to gain a synergistic antimicrobial net effect [43]. Chung et al. [44] reported the major susceptibility of Gram negative bacteria to chitosan on the reasoning that Gram negative bacteria possesses higher negative charge values on the cell surface. These results, supported by Devlieghere et al. [45], showed that Gram negative bacteria seemed to be generally more sensitive to chitosan solutions than Gram positive bacteria. They exposed several microorganisms (C. lambica, C. humicolus, Ph. phosphoreum, Ps. fluorescens, Ent. aerogenes, B. cereus, Broch. thermosphacta, L. monocytogenes, Lact. sakei subsp. carnosum, Lact. plantarum, Lact. curvatus, Ped. acidilactici) to chitosan concentrations varying from 40 to 750 mg/l (DD = 94% and MW 43 kDa) and they concluded that Gram negative bacteria seemed to be very sensitive for the applied chitosan, while the sensitivity of Gram positive bacteria was highly variable and that yeast showed an intermediate resistance. Fernandez-Saiz et al. [15] highlighted also, the influence of bacteria type (Staph. aureus and Salmonella spp. incubated for 24 h at 37°C) on the antimicrobial capacity of chitosonium acetate films: Gram positive bacteria are considerably more susceptible to chitosan, under the conditions used, than Gram negative bacteria. Considering the electrostatic interaction between chitosan and the cell wall, Gram positive bacteria cell wall is composed of a peptidoglycan layer and by polymers called teichoic acids; these teichoic acids are highly charged by phosphate groups with negative charge, which could establish electrostatic interaction with cationic antimicrobial compounds such as chitosan salts. Fungi Postharvest disease, often caused by fungal infection, posed serious problems to producers; consequently, numerous researchers investigated the potential use of antifungal compounds and various works acknowledge the fungistatic and fungicidal properties of chitosan against pathogens of various fruit and vegetables. In this context, chitosan has a dual function: (a) to inhibit fungal growth and (b) to activate several defence processes (accumulation of chitinases, synthesis of proteinase inhibitors and lignification and induction of callous synthesis). Bautista-Baños et al. [46] evaluated the effect of chitosan, aqueous extract of custard apple and papaya leaves and seeds on C. gloesporioides and studied the effect of these treatment on the quality of papaya fruit. C. gloesporioides was very sensitive to chitosan (both in vitro e in situ experiments); in fact, chitosan alone (2.0% and 3.0%) had a fungicidal effect on C. gloesporioides, while in combination with aqueous extract it exerted a fungistatic effect. Changes in the conidial morphology indicated that chitosan affected various stages of the development of C. gloesporioides. Molloy et al. [47], measured the antifungal activity of 0.2% (w/v) chitosan hydrolysed (number average DP = 7) against S. sclerotiorum, the most common fungal storage pathogen during monthly monitoring of cool-stored carrots. Whereas the hydrolysate did not affect radial growth of S. sclerotiorum on potato dextrose agar (PDA) plates, it reduced the frequency and size of rot compared to untreated controls when applied to carrots 3 days before inoculation. When carrots were treated at time zero with either chitosan hydrolysate or HMWC (high molecular weight chitosan), there was a decline in S. sclerotiorum infection, with the hydrolysate showing a greater effect than the high molecular weight chitosan. Previous work reported that chitosan reduce the incidence of Botrytis storage rot of table grapes [48] and blue mould of apples [49], stimulating defence responses in the respective hosts. Chitosan was shown to increase host chitinases, chitosanases and β-1,3-gluconase in strawberry fruit bell paper and tomato fruit and phenylalanine ammonia-lyase in table grapes [48].

Chitosan in foods


Bacteria in Different Growth Stages or Initial Microbial Population This issue was investigated by Fernandez-Saiz et al. [15]. The antimicrobial activity of various amount of chitosonium acetate film (from 10 to 70 mg) against Staph. aureus and Salmonella spp. at three different initial bacterial concentrations (103, 105 and 107 cfu/ml) was evaluated and it was observed a bacteriostatic effect when 50 and 70 mg samples were tested for all initial “inocula”. Nevertheless significantly lower bacterial counts were obtained for the lowest bacterial concentration. Finally, 70 mg of chitosan exerted a nearly bactericidal action when the initial inoculum was 103 cfu/ml. These observations, agreed well with the works of Tsai and Su [29] and Zivanovic et al. [50], indicating that bacterial concentration is an essential parameter to take into account for the evaluation of the antimicrobial properties of chitosan and that there is a correlation between the initial bacterial number and the biocide properties of chitosan film [50]. Another important parameter is the physiological state of the bacteria. Fernandez-Saiz et al. [15] observed that at 40 mg of chitosan, the sensitivity of Staph. aureus seemed to be higher when bacteria were inoculated in the mid-log phase instead that in the stationary and/or in the late exponential phases. Similar results were obtained by Tabak et al. [51] and Liu et al. [52] against Salmonella Typhimurium and E. coli, respectively. In contrast with these results Chen and Chou [53] observed a greater susceptibility of Staph. aureus when this microorganism was in the late-log phase in presence of water soluble lactose chitosan derivative. In addition, Tsai and Su [29] highlighted the importance of the surface electronegativity and its changes in relation at the different cell growth phases and concluded that changes in surface electronegativity seemed to depend on bacteria species. In any case, the results of Fernandez-Saiz et al. [15] suggested that the cells in stationary phase could be probably more able to implement adaptive stress responses. Fux et al. [54] suggested that the observed tolerance could be a reversible phenomenon, caused by phenotypic changes rather than the genetic alterations. FOOD APPLICATIONS Active Packaging Active packaging is defined as the packaging system possessing attributes beyond basic barrier properties that are achieved by adding active ingredients in the packaging system and/or using functional active polymers. When the packaging system acquires antimicrobial activity, the packaging system (or material) limits or prevents microbial growth by extending the lag period, reducing the growth rate or decreasing live counts of microorganisms [55]. Although somewhat expensive, biopackaging is the future for packaging, especially for a few value added food products [56]. Chitosan and its derivatives are ideal candidates as natural bioactive materials for food application, especially in view of recent outbreaks of contamination associated with food products and the negative environmental impact of packaging materials currently in use [31]. In fact, chitosan is considered a very promising biopolymer because it is environmentally friendly, due to its biodegradability, biocompatibility, antimicrobial activity, not toxicity and versatile chemical and physical properties. The Food and Drug Administration (FDA) has approved the use of chitosan for certain food applications (edible films to protect foods) and to our knowledge chitosan produced by Primex® of Norway is “Generally Recognized As Safe” (GRAS) and is recognized as functional food. Chitosan has already been approved as a food additive in some countries, for instance in Japan and Korea [57, 58]. However, chitosan has not made major inroads into American market where annual sales are in the region of US$20 million per annum [6]. Chitosan based polymeric materials can be formed into fibers, films, gels, sponges, beads or even nanoparticles. Furthermore, thanks to its excellent film-forming capacity and antimicrobial properties, it has been used widely in antimicrobial films to provide edible protective coating in dipping and spraying for foods; if compared with other bio-based food packaging materials, chitosan has the advantage of being able to incorporate functional substances (mineral, vitamin, flavouring, colouring, antioxidant and antimicrobial agents) enhancing the food value [31]. Chitosan is used also as an organic material for the preparation of immobilized enzymes. In fact, enzymes are often immobilized onto or into solid support to increase their thermostability, operation stability and recovery through chemical (where covalent bonds are formed with the enzyme) and physical methods (where weak interactions between support and enzyme exist). Thus, chitosan finds a real industrial application: for example, Çetinus and Öztop [59] immobilized catalase, an enzyme with a relativity high activity and industrial using, on a chitosan film, achieving kinetics parameters, thermal, storage and operation stabilities.



Krasaekoopt et al. [60] protected some probiotics like Lact. acidophilus and B. bifidum through the microencapsulation into low molecular weight chitosan-coated alginate beads: chitosan, formed a strong complex with alginates which became stable in the presence of Ca2+. LMWC (low molecular weight chitosan) (0.4 g) (Fluka, Sydney, Australia), dissolved in 90 ml distilled water acidified with 0.4 ml of glacial acetic acid was used as coating. Lact. acidophilus and B. bifidum were added, as free cells and microencapsulated cells, in yoghurt from UHT-and conventionally treated milk. The results of this work highlighted the influence of LMWC on the probiotic bacteria: the survival of encapsulated Lact. acidophilus and B. bifidum was higher than of free cells (ca. 1 log cycle during the storage). Edible Films and Coating Edible packagings have to be selective toward mass transfers or they have active properties or they can be both selective and active. Edible films or coatings are able to retard the organic vapours (aroma, solvents), water vapour, solute (lipids, salts, additives, pigments) and gases (oxygen, carbon dioxide, nitrogen) [31]. Why retarding these compounds is desirable? For fresh or frozen products, the water barrier efficiency of films retards their surface dehydration. In addition, the control of oxygen and other gas exchanges allows a better control of the ripening of fruits or reduces the oxidation of oxygen-sensitive foods and the rancidity of polyunsaturated fats. Organic vapour transfers may be minimised in order to retain aroma compounds in the product during storage or to prevent solvent penetration in foods, which induces toxicity or off-flavouring. Functional efficiency strongly depends on the nature of components and film composition and structure. The choice of film-forming substances and/or active additives is made based on the objective, the nature of the food product and/or the application method [31]. Bioactive edible coating or packaging materials could be used as an alternative way to control undesirable microorganisms on foods: in this case, edible coating is a thin film prepared from edible material that acts as barrier to the external elements (moisture, oil) and thus protects the product and extends its shelf life. Generally, antimicrobial agents are incorporated into the packaging and the antimicrobial activity is based on the release of the biocidal molecule. Another concept is based on edible coatings, where functional groups, that have antimicrobial activity, are immobilized on the surface of polymer films or the antimicrobial activity comes from the polymer itself, directly used as film-forming entities, leading to antimicrobial activity especially onto food surface [61]. Due to its ability to form active edible or biodegradable films, chitosan coating can be expected to limit contamination on food surface [61]. The potential of an edible antimicrobial film based on chitosan matrix against L. monocytogenes and L. innocua, was studied by Coma et al. [62]. They observed that edible chitosan coatings showed anti-listerial effect imparting a strong localized functional effect at the food surface by active packaging [62]. Sebti et al. [61] assessed the potential of chitosan coating as antifungal polymer against A. niger (an opportunistic human pathogen commonly encountered in food contamination cases) and they confirmed that chitosan films offer a great advantage, as antimicrobial coating, in preventing A. niger surface growth, even at very low concentration of chitosan. Various methods have been employed to prepare chitosan films and coating for food packaging applications. Chitosan films are prepared through: a)

cross-linking by aglycone geniposidic acid [63]: a comparative study was performed to study a chitosan film without cross-linking, a gluteraldehyde-cross-linked chitosan film and an aglycone geniposidic acid-cross-linked chitosan film and it has been concluded that the aglycone geniposidic acid-cross-linked chitosan film may be a promising material as an edible film for food packaging. [31];

b)

ternary chitosan-glucomannan-nisin: Li et al. [64] incorporated chitosan and nisin in koniac glucomannan edible film to improve not only physical properties but also the antimicrobial activity against E. coli, Staph. aureus, L. monocytogenes and B. cereus;

c)

blending of ferulic acid incorporated starch-chitosan: Mathew and Abraham [65] incorporated ferulic acid in starch-chitosan blend films, improving the barrier properties and tensile strength of

Chitosan in foods


starch-chitosan blend films (which were more compact if compared with control), and enhancing the lipid peroxide inhibition capacity and consequently extending the shelf life; d)

incorporation of garlic oil, potassium sorbate and nisin: Pranoto et al. [66], incorporated garlic oil, potassium sorbate and nisin in a chitosan film to enhance its antimicrobial activity against E. coli, Staph. aureus, Salmonella Typhimurium, L. monocytogenes and B. cereus. They concluded that garlic oil incorporated in chitosan film led to an increase in its antimicrobial efficacy;

e)

derivatives of chitosan: Li et al. [67] studied the antimicrobial ability of two types of Ocarboxymethylated chitosan/cellulose polyblended prepared by using LiCl/N,N-dimethylacetamide solution. Both the blend films exhibited a satisfactory antibacterial activity against E. coli.

Most recently, Tripathy et al. [68] have synthesised chitosan based antimicrobial films employing supercritical carbon dioxide and microwave techniques. The novelty of this method lies in achieving the film formation without addition of any cross-linker or plasticizer. Some typical preparative techniques are the following [31]: 1.

preparation of chitosan/starch films by using supercritical carbon dioxide treatment;

2.

preparation of chitin whiskers;

3.

supercritical fluid (SCF) drying;

4.

preparation of chitosan/starch films: the antibacterial activity of chitosan-starch film using microwave treatment has been carried out using agar plate diffusion method against E. coli, Staph. aureus and B. subtilis; it was found that the solution of chitosan-starch showed inhibitory effect. Salleh et al. [69] incorporated chitosan and lauric acid into starch based film and they observed that the film containing the fatty acid showed a more pronounced antimicrobial ability against B. subtilis and E. coli;

5.

Preparation of antimicrobial chitosan-potato starch films by using microwave treatment;

6.

Preparation of chitosan/starch blend films under the action of irradiation;

7.

Preparation of chitosan films enriched with oregano essential oil;

8.

Preparation of chitosan-oleic acid edible coatings;

9.

Preparation of water soluble chitosan and amylose films.

Portes et al. [70] made film exhibiting antibacterial and antioxidative properties using a protonated chitosan as film matrix, added with two smaller molecules of tetrahydrocurcuminoids (THCs), potentially released in foods. THCs may be able to offer protection against L. monocytogenes (and other Gram positive bacteria). The authors concluded that the antioxidative properties of the film results from a progressive release of THCs into the medium. Furthermore chitosan, retained its antimicrobial properties against the growth of L. innocua, when associated with THCs; THCs alone are not bioactive.



APPENDIX 1 CHITOSAN AND FOODS Table 1: Use of chitosan in bread and gluten free-pasta.

GLUTEN FREE FRESH PASTA

BREAD

FOODS

SHELF-LIFE LIMITING ELEMENTS

staling (time dependent loss in quality of flavour and texture of bread) and microbial growth

The presence of gluten sometimes causes celiac disease.

EXPERIMENTAL CONDITION

APPLICATION

RESULTS AND IMPROVEMENTS

AUTHORS

Park et al. [71]

0.5-1.0-1.5% chitosan in 1% acetic acid.

Coating

Shelf life (36 h) of 1% chitosan treated baguette was extended to 24 h compared with that of the control (12 h). Chitosan coating offered a protective barrier for moisture transfer through the bread surface thus reducing weight loss and retarding starch retrogradation and hardness.

chitosan olygomer (1% distilled water; MW = 2kDa)

Coating

Shelf life was extended to 24 h compared with that of the control (12 h).

Park et al. [72]

1-2% of chitosan (120 kDa; DD 85%) in 0.3% lactic acid

Coating

Lower bacterial count and inhibition of moulds, retarding antioxidation and retrogradation.

Ahn et al. [73]

Chitosans (MW: 1, 5, 30, 120 kDa; DD: 95, 95, 92, 85%, respectively) in 0.03% (v/v) lactic acid were added to dough at concentrations of 0.01, 0.1, 0.3, 0.5%.

Coating

Bread containing 30 or 120 kDa chitosan at 0.1% concentration showed 101-103 cfu/g viable cells whereas control 106 cfu/g after storage for 8 days at room temperature.

Lee et al. [74]

Solution

Amaranth (grain crop without prolamine and gluteline) combined with chitosan (at whatever concentrations) and MAP (specially 30:70 N2:CO2) improved quality of home-made fresh pasta extending the microbial acceptability limit of the home-made fresh pasta beyond two months.

Del Nobile et al.. [75]

in

Chitosan dissolved in 1.38% (v/v) lactic acid, was added to dough to obtain the final concentrations: 2000 mg/kg and 4000 mg/kg, combined with 3 MAP (80:20, 0:100 and 30:70, N2:CO2)

Table 2: Use of chitosan in eggs and litchi. FOODS



EGGS

1-2% chitosans (MW: 470, 746 and 1100 kDa) dissolved in 1-2% acetic acid during 5 weeks of storage at 25°C.


AUTHORS

Coating

2% chitosan coating (470 kDa) reduced weight loss obtaining more desirable albume and yolk quality without sensorial difference on external quality between coated eggs and control.

Bahle et al. [76]

Coating

Regarding sensory perception on external quality, no difference of the coated eggs and control (noncoated eggs) was observed. Chitosan coating may offer a protective barrier for moisture and gas transfer through the eggs shell, extending of two weeks the shelf life.

Caner [77]

Coating

Chitosan coating delayed changes in content of anthocyanins, flavonoids and total phenolics. It also delayed the increase in PPO activity and partially inhibited the increase in peroxidase activity.

Zhang and Quantick [78]

APPLICATION


AUTHORS

Dipping

Chitosan coating reduced the decay of strawberries compared to the control and no difference among the concentrations used was observed. Chitosan inhibited the growth of several fungi and induced chitinase (a defence enzyme), thus the control of decay in strawberries could be attributed either to the fungistatic property of chitosan or to its ability to induce chitinase and β-1,3-glucanase or a combination. Coating of intact fruits with chitosan did not stimulate chitinase, chitosanase

El Ghaouth et al. [79,80]

Weight loss, interior quality deterioration and microbial contamination. 3% chitosan dissolved in 1% acetic acid

LITCHI

APPLICATION

Lose of the red colour after harvest; rapid post-harvest browning caused by polyphenol oxidase activity, anthocyanin hydrolysis, non enzymatic polymerization of o-quinones into melanins.

1 and 2% chitosan dissolved in 2% glutamic acid

Table 3: Use of chitosan in strawberries.

STRAWBERRY

FOODS


Strawberries are highly perishable being subjected to mechanical injury, desiccation, decay and fungal infection caused mainly by B. cinerea and R. stolonifer


Chitosan solutions (1 and 1.5% in 0.25 N HCl). Intact and fresh-cut strawberries were dipped in chitosan solution.

Chitosan in foods

Application of Alternative Food-Preservation Technologies 105 or β-1,3-glucanase activities in the tissue, whereas stimulation of chitinase activities was observed on coated freshcut strawberries.

Strawberry plants were sprayed with 2, 4 and 6 g/l, chitosan solution as the fruit were turning red. A second spray was performed after 10 days. Fruits were picked 5 (pick 1) and 10 (pick 2) days after each spray. Harvested strawberries were stored at 3 and 13°C.

Three chitosan–based coating were applied to strawberries: -chitosan; -chitosan with 5% Gluconal® CAL ; -chitosan with 0.2% DL-αtocopheryl acetate

Three 1% chitosan (DD 89.8%) solutions (chitosan in 0.6% acetic acid solution; chitosan in 0.6% lactic acid solution; chitosan in 0.6% lactic acid solution + 0.2% vitamin E). 2% chitosan (DD 89.9% in 0.5% acetic acid) solution and 2% chitosan containing 0.3% potassium sorbate (PS) Fruits, dipped in chitosanlactic acid/Na-lactate solution were dried at room temperature for 15 min, packaged in Equilibrium Modified Atmosphere (3% O2; 7% CO2) and stored at 7°C. Manually sliced strawberries were treated with a solution of 1% LMWC (DD 75-85%) dissolved in 1% citric acid, packaged in modified atmosphere (MA) with low and high percentage of oxygen (MA1: 65% N2, 30% CO2 and 5%O2; MA2: 80% O2 and 20% CO2) and stored at 4, 8, 12 and 15°C.

Spraying

Chitosan spray reduced post-harvest fungal rot and maintained the quality of the fruit. The incidence of decay decreased with increasing chitosan concentrations, with storage period and temperature. Pre-harvest chitosan spray had a beneficial effect on flesh firmness, titratable acidity and in slowing the synthesis of anthocyanins in strawberries stored both at 3 and 13°C; this could be due to the formation of a chitosan film on fruit which acts as a barrier from O2 uptake thereby slowing the metabolic activity and consequently the ripening process. The second spray of chitosan extended the protective effect against decay of fruit from subsequently picks.

Bhaskara Reddy et al. [81]

Coating

The extension of the shelf life by decreasing the decay incidence and weight loss, delaying changes in colour, pH and titratable acidity during storage at 2°C, were observed. No significant difference in weight loss among 3 chitosans was observed. Adding calcium or vitamin E into chitosan-based coatings did not significantly alter their antifungal and moisture barrier functions; on the contrary, the moisture barrier properties result improved.

Han et al. [82]

Coating

1% chitosan coated resulted in no perception of astringency. Lactic acid helped increase the glossiness of coated strawberries whereas the incorporation of vitamin E in chitosan coating reduced their glossiness, affecting consumer acceptance.

Han et al. [83]

Dipping

Coating with chitosan + PS did not show any advantage over the coating with chitosan-alone in delaying of fungal growth. On the other hand, significant synergistic inhibition activity was observed in vitro testing.

Park et al. [84]

Dipping

Sensorial and microbiological characteristics were evaluated during storage. The microbiological counts on the chitosan-dipped samples were lower than undipped samples and the antimicrobial effect of chitosan was maintained over 12 days.

Devlieghere et al. [45]

Dipping

Chitosan coating could control browning and decay in strawberry fruits and in combination with other methods (low temperature and suitable packaging). Chitosan showed a high antimicrobial activity, inhibiting and/or controlling the growth of psychrotrophic, lactic acid bacteria and yeasts and did not affect the visual appearance. pH and tickness values were not changed by chitosan coating whereas colour was positively influenced by it (this effect was more evident in MA2)

Campaniello et al. [85]

APPLICATION


AUTHORS

Dipping

Chitosan is valid alternative fungicide to control post-harvest decay of table grapes. All pre-harvest treatment reduced the incidence of grey mould, as compared to the control.

Romanazzi et al. [48]

Table 4: Use of chitosan in grape and tomatoes.

GRAPE

FOODS



Infections induced by B. cinerea during pre and/or post-harvest storage in grapes.

Different concentrations of chitosan (0.1, 0.5 and 1%) were used in this work.


Colletotrichum sp. cause anthracnose in fruits during pre- and/or post-harvest.

This fruit can deteriorate rapidly due to peel browning when removed from the cold storage. Thus the major limitation in litchi marketing is the rapid lose of the red colour after harvest: rapid postharvest browning is the result of polyphenol oxidase (PPO) activity, anthocyanin hydrolysis and nonenzymatic polymerization of oquinones into melanins.

Berries treated with aqueous solution of chitosan (1 and 2.5%, w/v) were inoculated with Colletotrichum sp. and incubated at 4 and 24°C. Lesion diameters were recorder after 7 and 10 days.

Chitosan: 2% in 5% acetic acid


Dipping/spraying

Chitosan significantly reduced the lesion diameter on pre-treated berries at 24°C. However no differences were observed between the chitosan concentrations and the corresponding controls at 4°C.

Muñoz et al. [86]

Coating

Chitosan coating delayed the decrease in anthocyanin content and the increase in PPO activity. Such effects of chitosan coating were also observed with peeled litchi fruit (Dong et al., 2004), longan fruit (Jiang and Li, 2001) and fresh-cut Chinese water chestnut vegetable (Pen and Jiang, 2003).

Jiang et al. (2005)

During storage (20°C) the respiration rate increased gradually. Between 2 and 7 days, treated tomatoes had a slightly higher respiration rate than the control.

Chitosan (1-2%) was dissolved in HCl (0.25 N)

Dipping

TOMATO

Postharvest loss often due to fungal infections, physiological disorders and physical injuries.

After 10 days, untreated samples showed a higher respiration rate than chitosan-coated tomatoes. Successively, the respiration rate of 2% chitosan-coated fruits was markedly lower than that of 1% chitosan-coated fruits.

El Ghaouth et al. [87]

Chitosan-coated tomatoes were firmer, higher in titratable acidity, less decayed and they exhibited less red pigmentation. Berries treated with aqueous solution of chitosan (1 and 2.5%, w/v) were artificially inoculated with Colletotrichum sp. and incubated at 4 and 24°C. Lesion diameters were recorder after 7 and 10 days after inoculation.

After 10 days at 24°C chitosan reduced the lesion size of tomato fruits treated with 1 and 2.5%. Dipping/spraying

Anthracnoses lesions on tomatoes at 4°C were smaller than on tomatoes stored at 24°C and no influence of chitosan on lesions were observed.

APPLICATION


Muñoz et al. [86]

Table 5: Use of chitosan in carrots and mayonnaise. FOODS



AUTHORS

CARROTS

Sample immersion in the film-forming dispersions is a common way to apply coatings; nevertheless, depending on the dispersion viscosity and extensibility on the sample surface, the retention of the dispersion to form a coating is usually low.

Respiration rate and ethylene production, decay and firmness reduction

Edible coating based on HMWC, pure or combined with methylcellulose or oleic acid, were applied to fresh-cut carrots cv. Nantesa by immersion and by applying a vacuum pulse (5 kPa for 4 min).

Applied by simple immersion and vacuum impregnation

Vacuum impregnation (VI) could improve the dispersion retention and form a thicker, more effective coating. Coatings improved sample appearance, since they diminished the occurrence of the white blush during storage. In contrast, coating application with a vacuum pulse enhanced all the positive effects and resulted in a better preservation of the sample colour and mechanical response during cold storage. Differences in film composition did not significantly affect the coating behaviour

Vargas et al. [88]

Chitosan in foods


MAYONNAISE

Mayonnaise containing 3 g/l of chitosan combined with acetic acid (0.16%) or lemon juice (1.2 and 2.6%) was inoculated with 5 - 6 log cfu/g of Salmonella Enteritidis, Zygosacch. bailii or Lact. fructivorans and stored at 5 and 25°C for 8 days.

Emulsifier

Mayonnaise samples containing acetic acid were less supportive of microbial growth than those containing lemon juice. Chitosan accelerated the inactivation of salmonellae but only after 6 days at 5°C and inactivated Zygosacch. bailii, at 25°C, within the first day of incubation; this initial inactivation was followed by growth. At 5°C the number of yeasts remained constant during the storage time independently by the presence or absence of chitosan. A completely inactivation of Lact. fructivorans was obtained in mayonnaise containing 0.16% acetic acid.

Roller and Covill [89]

APPLICATION


AUTHORS

Ouattara et al. [90]

Table 6: Use of chitosan in meat. SHELF-LIFE LIMITING ELEMENTS


Organoleptic changes due to microbial growth during the storage. Meat is susceptible to lipid oxidation which leads to rapid development of rancid or warmed-over flavour.

Antimicrobial films (prepared by incorporating acetic or propionic acid into a chitosan matrix, + or lauric acid or cinnamaldehyde) were tested in combination with vacuum-packaged on processed meats (bologna, regular cooking ham or pastrami) during refrigerated storage. The antimicrobial activity was also tested against indigenous lactic acid bacteria and Enterobacteriaceae, and against Lact. sakei or S. liquefaciens, surfaceinoculated onto the meat.

Film

Chitosan had a desirable effect on the development of the red colour of beef during storage. Propionic acid was nearly completely released from the chitosan matrix within 48 h of application, whereas release of acetic acid was more limited, with 2-22% of the acid remaining in chitosan after 168 h of storage. Addition of lauric acid, but not cinnamaldehyde, to the chitosan matrix, reduced the release of acetic acid and the release was more limited onto bologna, than onto ham or pastrami. Whereas lactic acid bacteria were not affected by the antimicrobial films the growth of Enterobacteriaceae and S. liquefaciens was delayed or completely inhibited.

Pork was dipped for 60 s in chitosan solutions at different concentrations (0.1%, 0.5%, 1%) with different molecular weight (5, 30, 120 kDa) and stored at 8 and 10°C.

Dipping

Chitosan effect was influenced by its molecular weight, as the antioxidative effect was evident for 30 and 120 kDa.

Lee et al. [91]

Coating

Antilisterial efficacy of chitosan-coated plastic film, alone or incorporating nisin, sodium lactate, sodium diacetate, potassium sorbate and sodium benzoate on ham steak (stored at room temperature for 10 days), was evaluated. Chitosan-coated plastic film containing sodium lactate was the most effective antilisterial film during 12week storage at 4°C.

Ye et al. [92]

Coating

The use of the radiation processing is proposed as an alternative technology to eliminate microbial contamination in meat and meat products. The antioxidant activity of chitosan increased upon irradiation. Furthermore the results demonstrated that chitosan irradiated could prevent the spoilage.

Rhao et al. [93]

APPLICATION


AUTHORS

Solution

Chitosan (2% dissolved in water), used as clarifying agent in fruit juices, was more effective in reducing turbidity of juice than bentonite and gelatine. Chitosan has acid binding properties thanks to its partial positive charge; furthermore, it is effective in aiding the separation of colloidal and disperse particles from food processes wastes. The appearance and acceptability of the

Chatterjee et al. [94]

MEAT

FOODS

L. monocytogenes was able to survive at low temperatures and tolerate relativity high heat and high concentration of salt.

Chitosan films were prepared as follows: two grams of MMWC (SigmaHaldrich, St. Louis, MO, USA) were dissolved in 100 ml of 1% acetic acid and stirred overnight at room temperature.

Lipid peroxidation microbial spoilage

Chitosan (1% in 1% acetic acid) was irradiated at a 4kGy dose in a Cobalt-60 Gamma cell-220

and

Table 7: Use of chitosan in juices.

JUICES

FOODS


Turbidity


Water soluble chitosan, hydrolyzed with 7% acetic acid, for clarification of apple, grape, lemon and orange juices.


Campaniello and Corbo juices, after chitosan increased significantly.

Turbidity

Juices were treated with two type of chitosan (chitosan from Absidia glauca, var. paradoxa which is a fungal chitosan with DD = 86% and chitosan from shrimp shells). Chitosans were dissolved in 2% malic acid at concentrations from 0.1 to 1.0 g/l

Microbial and fungal growth

Chitosan glutamate (a derivative of chitin) (DD = 75-85%) in laboratory media and apple juice (pH 3.4) at various concentrations (0.1-10 g/l) against a total of 15 microorganisms (8 yeasts and 7 filamentous fungi)

Microbial and fungal growth

Chitosan powder was added to apple juice to give a final concentration of 0.05 and 0.1% and for challenge testing Salmonella Typhimurium or Escherichia coli O157:H7 were inoculated (104 CFU/ml).

treatment

Solution

Fungal chitosan was more effective in reducing the apple juice turbidity and yielded lighter (higher Hunter L* value) juices than chitosan obtained from shrimp. In particular, turbidity decreased gradually with increasing chitosan concentration from 0.1 to 0.7 g/l of juice but it was increased to 1.0 g/l, probably due to the saturation of the active chitosan adsorption sites.

Rungsardtho ng et al. [95]

Solution

The presence of chitosan (0.1-5 g/l) in apple juice inhibited growth of all spoilage yeasts (three strains of Sacch. cerevisiae, two strains of Zigosacch. bailii, Sacch. exiguus, Schizosacch. pombe and Sacch. ludwigii) and reduced the growth rate of Mucor racemosus at 1 g/l whilst concentrations of 5 g/l were required to completely prevent growth of three strains of Byssochlamys spp. Three strains of filamentous fungi (A. flavus, C. cladosporioides and P. aurantiogriseum) were resistant to the antifungal effects of chitosan at 10 g/l.

Roller and Covill [96]

Solution

Chitosan delayed spoilage by yeasts at 25°C for up 12 days but the effect was specie-specific: K. apiculata and Metschnikowia pulcherrima were inactivated whereas Sacch. cerevisiae and Pichia spp., multiplied slowly. The addition of chitosan in apple juice delayed spoilage by yeasts but enhanced the survival of E. coli O157:H7. Salmonella Typhimurium was unaffected by chitosan.

Kiskò et al [17]

APPLICATION


AUTHORS

Solution

Consistency of chitosan-added milk increased with increasing molecular weight and concentrations. Addition of 0.5% water-soluble chitosan to milk affected negatively its sensory quality of colour, taste and flavour, browing in colour and chemical off-flavour with both 0.2-3 kDa and 3-10 kDa chitosan and astringent taste with 10-30 kDa chitosan. However, there were no differences in the sensory quality between coffee-flavoured milk containing chitosan and the control: this may have been due to masking effect from coffee.

Lee and Lee [97]

Solution

The effect of chitosan on milk fermentative processes seemed to be dependent non only on its molecular weight and concentration, but also on the presence of casein micelles or milk fat, that, acting in a competitive manner, could prevent the inhibitory activity of these biopolymers on bacterial growth.

Ausar et al. [98]

Solution

The addition of chitosan to spreadable cheeses, thanks to its antimicrobial and hypocholesterolemic functions and its capacity to produce destabilization, improved rheological and sensorial properties, without affecting the dairy microflora.

Gammariello et al. [99]

Table 8: Use of chitosan in milk and milk-based products.

MILK AND MILK PRODUCTS

FOODS



Change of physicochemical and sensory properties of milk

Water-soluble chitosans with three different range of molecular weights (0.23, 3-10, 10-30 kDa), and different concentrations (0.5%, 1% and 1.5%) were added to milk.

Microbial growth

The growth of Lact. delbrueckii subsp. bulgaricus, Strep. thermophilus and P. freudenreichii) was tested in fermented diary products (yogurt, cultured milk and cheeses), in presence of HMWC (average MW 600 kDa) and LMWC (average MW 80 kDa); (DD = 85%) and chitosan oligosaccharides (di-tritetra-penta and hexasaccahride) alone or complexed with casein.

Rapid ripening due to the high percentage of water.

LMWC (DD = 85%, Aldrich, Milan, Italy) was put into the working milk to obtain cheeses with final chitosan concentrations of 0.012, 0.024 and 0.036% (w/v).

Chitosan in foods


Table 9: Use of chitosan in tofu. FOODS



TOFU

Six chitosans (59, 224, 470, 746, 1106 and 1671 kDa) and six oligomers (1, 2, 4, 7, 10 and 22 kDa) were placed in plastic bottle and stored at ambient temperature during the experiment.

Quite perishable due to its relatively high pH (5.8-6.2), moisture content (80-88%) and characterized by a sour taste associated with bacterial growth. The processing conditions (type and concentration of coagulants, temperature and extent of stirring during coagulation, and pressure and time applied for moulding) affect its quality.

Chitosan solutions (0.5 or 1% w/v) were prepared by dissolving chitosan in 0.53% acetic acid, or in a mixture of 1% acetic acid and 1% lactic acid. Six chitosans (7, 28, 224, 471, 746, 1106 kDa) under various treatment conditions, were examinated and the optimum processing conditions were the following: HMWC of 28 kDa; chitosan solution type, 1% chitosan/1% acetic acid; chitosan solution to soymilk ratio, 1:8; coagulation temperature 80°C; coagulation time, 15 min.

APPLICATION


AUTHORS

Solution

Chitosan exhibits a growth inhibitory effect on bacteria isolated from spoiled tofu; in particular the antibacterial activity of chitosan and chitosan oligomers varied depending on their molecular weight and specific bacterium.

No et al. [100]

Coagulant

Chitosan had minimal effect on the characteristic of tofu (yield and hardness) and whey (volume, turbidity), but the sensory quality and shelf-life of tofu was affected. They also reported that the chitosan tofu had a longer shelf life, about 3 days, than the tofu made with CaCl2.

No and Meyers [101]

The textural properties of tofu (hardness, cohesiveness, gumminess and chewiness) were not significantly changed by the addition of high viscosity chitosan.

Han and Kim [102]

APPLICATION


AUTHORS

Coating

Chitosan may retard lipid oxidation by chelating, through its amino group, ferrous ions present in the fish model system, thus eliminating prooxidant activity of ferrous ions or preventing their conversion to ferric ions. The antioxidant capacity of chitosan added to the fish muscle depended on the molecular weight (and concentration) of chitosan because in their charge state, the cationic amino groups impart intramolecular electric repulsive forces, which increase the hydrodynamic volume by extended chain conformation. This effect, probably, is responsible for lesser chelation by HMWC: among the three chitosans, 660 kDa chitosan was the most effective in preventing lipid oxidation.

Kamil et al. [103]

Coating solution

The preservative efficacy of 960 and 1800 kDa chitosans was superior to that of chitosan with 660 kDa: chitosan coating reduced significantly lipid oxidation, chemical spoilage and microorganisms in both fish compared to the uncoated samples.

Jeon et al. [104]

Incorporation of 0.2, 0.5 and 1% chitosan with various molecular weight into salmon, reduces lipid oxidation. Chitosans showed antioxidative activity in salmon reducing the lipid oxidation for 7 days of storage.

Kim and Thomas [105]

The film containing SL inhibited completely the growth of L. monocytogenes during 10 days of storage, whereas in sample packaged in

Ye et al. [106]

Table 10: Use of chitosan in fish and seafood products.

FISH AND SEAFOOD PRODUCTS

FOODS


Highly susceptible to quality deterioration due to lipid oxidation of unsaturated fatty acids, contamination and growth of microorganisms and loss of protein functionality.

Lipid oxidation of unsaturated fatty acids, contamination and growth of microorganisms and loss of protein functionality.

Human listeriosis outbreaks


Fish flesh were treated with chitosans of different MW: 660, 960 and 1800 kDa and at different concentrations (50, 100 and 200 ppm) and, with conventional antioxidants (butylated hydroxyanisole + butylated hydroxytoluene (200 ppm) and tert-butulhydroquinone (200 ppm) in cooked, comminuted flesh or herring (Clupea harengus).

The effect of 3 chitosans (660, 960 and 1800 kDa) on shelf life of fresh fillet of Atlantic cod (Gadus morhua) and herring (Clupea harengus) over 12 days storage and at 4°C, was tested. Chitosans (MW: 30, 90 and 120 kDa) at different concentrations (0.2, 0.5 and 1%) were incorporated into salmon Chitosan-coated plastic films containing five GRAS antimicrobials: nisin, sodium lactate (SL), sodium

Coating

110 Application of Alternative Food-Preservation Technologies diacetate (SD), potassium sorbate (PS) and sodium benzoate (SB).

Campaniello and Corbo the other four antimicrobial films, L. monocytogenes grew, but the increase in count was lower than in the control.

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Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin. LWT – Food Sci Technol 2008; 38: 859-65. Li Z, Zhuang XP, Liu XF, Guan YL, Yao KD. Study on antibacterial O-carboxymethylated chitosan/cellulose blend film from LiCl/N, N-dimethylacetamide solution. Polymer 2002; 43: 1541-7. Tripathy S, Mehotra GK, Dutta PK. Chitosan based antimicrobial films for food packaging applications. e-polymers 2008; 93: 1-7. Salleh E, Muhamad I, Khairuddin N. Preparation, characterization and antimicrobial analysis of antimicrobial starchbased film incorporated with chitosan and lauric acid. Asian Chitin J 2007; 3: 55-68. Portes E, Gardrat C, Castellen A, Coma V. Environmentally friendly films based on chitosan and tetrahydrocurcuminoid derivatives exhibiting antibacterial and antioxidative properties. Carbohydr Polym 2009; 76: 578-84. Park IK, Lee YK, Kim MJ, Kim SD. Effect of surface treatment with chitosan on shelf-life of baguette. J Chitin Chitosan 2002; 7: 208-13. Park IK, Lee YK, Kim MJ, Kim SD. Effect of surface treatment with chito-oligosaccharide on shelf-life of baguette. J Chitin Chitosan 2002; 7: 214-8. Ahn DH, Choi JS, Lee HY, Kim JY, Youn SK, Park SM. Effects on preservation and quality of bread with coating high molecular weight chitosan. Korean J Food Nutr 2003; 16: 430-6. Lee HY, Kim SM, Kim JY et al. Effect of addition of chitosan on improvement for shelf-life of bread. J Korean Soc Food Sci Nut 2002; 31: 445-50. Del Nobile MA, Di Benedetto N, Suriano N, Conte A, Corbo MR, Sinigaglia M. Combined effects of chitosan and MAP to improve the microbial quality of amaranth home-made fresh pasta. Food Microbiol 2009; 26: 151-6. Bhale S, No HK, Prinyawiwatkul W, Farr AJ, Nadarajah K, Meyers SP. Chitosan coating improves shelf life of eggs. J Food Sci 2003; 68: 2378-83. Caner C. The effect of edible eggshell coatings on egg quality and consumer perception. J Sci Food Agric 2005; 85: 1897-902. Zhang D, Quantick PC. Effects of chitosan coating on enzymatic browning and decay during postharvest storage of litchi (Litchi chinensis Sonn.) fruit. Postharvest Biol Technol 1997; 12: 195-202. El Ghaouth A, Arul J, Ponnampalam R, Boulet M. Chitosan coating effect on storability and quality of fresh strawberries. J Food Sci 1991; 56:1618-20, 1631. El Ghaouth A, Arul J, Grenier J, Asselin A. Antifungal activity of chitosan on two postharvest pathogens of strawberry fruits. Phytopathology 1992; 82:398-402. Baskara Reddy MV, Belkacemi K, Corcuff R, Castaigne F, Arul J. Effect of pre-harvest chitosan sprays on postharvest infection by Botrytis cinerea and quality of strawberry fruit. Postharvest Biol Technol 2000; 20: 39-51. Han C, Zhao Y, Leonard SW, Traber MG. Edible coatings to improve storability and enhance nutritional value of fresh and frozen strawberries (Fragaria × ananassa) and raspberries (Rubus ideaus). Postharvest Biol Technol 2004; 33: 67-78. Han C, Lederer C, McDaniel M, Zhao Y. Sensory evaluation of fresh strawberries (Fragaria ananassa) coated with chitosan-based edible coatings. J Food Sci 2005; 70:S172-8. Park SI, Stan SD, Daeschel MA, Zhao Y. Antifungal coatings on fresh strawberries (Fragaria × ananassa) to control mold growth during cold storage. J Food Sci 2005; 70: M202-7. Campaniello D, Bevilacqua A, Corbo MR, Sinigaglia M. Chitosan: antimicrobial activity and potential applications for preserving minimally processed strawberries. Food Microbiol 2008; 25: 992-1000. Muñoz Z, Moret A, Garces S. Assessment of chitosan for inhibition of Colletotrichum sp., on tomatoes and grapes. Crop Prot 2009; 28: 36-40. El Ghaouth A, Ponnampalam R, Castaigne F, Arul J. Chitosan coating to extend the storage life of tomatoes. HortScience 1992; 27:1016-8. Vargas M, Chiralt A, Albors A, Gonzales-Martinez C. Effect of chitosan-based edible coatings applied by vacuum impregnation on quality preservation of fresh-cut carrot. Postharvest Biol Technol 2009; 51: 263-71. Roller S, Covill N. The antimicrobial properties of chitosan in mayonnaise and mayonnaise-based shrimp salads. J Food Prot 2000; 63: 202-9.

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Ouattara B, Simard RE, Piette G, Bégin A, Holley RA. Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan. Int J Food Microbiol 2000; 62: 139-48. Lee HY, Park SM, Ahn DH. Effect of storage properties of pork dipped in chitosan solution. J Korean Soc Food Sci Nutr 2003; 32: 519-25. Ye M, Neetoo H, Chen H. Control of Listeria monocytogenes on ham steaks by antimicrobials incorporated into chitosan-coated plastic films. Food Microbiol 2008; 25: 260-8. Rhao MS, Chander R, Sharma A. Development of shelf-stable intermediate moisture meat products using active edible chitosan coating and irradiation. J Food Sci 2005; 70: M325-31. Chatterjee S, Chatterjee S, Chatterjee BP, Guha AK. Clarification of fruit juice with chitosan. Process Biochem 2004; 39:2229-32. Rungsardthong V, Wongvuttanakul N, Kongpien N, Chotiwaranon P. Application of fungal chitosan for clarification of apple juice. Process Biochem 2006; 41: 589-93. Roller S, Covill N. The antifungal properties of chitosan in laboratory media and apple juice. Int J Food Microbiol 1999; 47:67-77. Lee JW, Lee YC. The physico-chemical and sensory properties of milk with water soluble chitosan. Korean J Food Sci Technol 2000; 32: 806-13. Ausar SF, Passalacqua N, Castagna LF, Bianco ID, Beltramo DM. Growth of milk fermentative bacteria in the presence of chitosan for potential use in cheese making. Int Dairy J 2002; 12: 899-906. Gammariello D, Chillo S, Mastromatteo M, Di Giulio S, Attanasio M, Del Nobile A. Effect of chitosan on the reological and sensorial characteristics of apulia spreadable cheese. J Dairy Sci 2008; 91: 4155-63. No HK, Park NY, Lee SH, Meyers SP. Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. Int J Food Microbiol 2002; 74: 65-72. No HK, Meyers SP. Preparation of tofu using chitosan as a coagulant for improved shelf-life. Int J Food Sci Technol 2004; 39:133-41. Han JS, Kim M. Effects of chitooligosaccharide on the physicochemical, textural and sensory properties of tofu. J Texture Stud 2002; 33: 1-14. Kamil JYVA, Jeon YJ, Shahidi F. Antioxidative activity of chitosans of different viscosity in cooked comminuted flesh of herring (Clupea harengus). Food Chem 2002; 79: 69-77. Jeon YJ, Kamil JYVA, Shahidi F. Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod. J Agric Food Chem 2002; 50: 5167–78. Kim KW, Thomas RL. Antioxidative activity of chitosans with varying molecular weights. Food Chem 2007; 101: 308–13. Ye M, Neetoo H, Chen H. Effectiveness of chitosan-coated plastic films incorporating antimicrobials in inhibition Listeria monocytogenes on cold-smoked salmon. Int J Food Microbiol 2008; 127: 235-40.

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CHAPTER 8 Use of High Pressure Processing for Food Preservation Antonio Bevilacqua, Daniela Campaniello and Milena Sinigaglia* Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy 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 20th 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 *Address correspondence to this author Milena Sinigaglia at: Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italy; E-mail: [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.

High Pressure Processing


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   T  dP   C p where T is the temperature (K); P, the pressure (Pa); α, the thermal expansion (1/K); ρ, the density (kg/m3); 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


Bevilacqua et al.

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. Food

Reduction

Condition

Reference

Campylobacter jejuni Enterobacter sakazakii

Poultry meat slurry Infant formula

5 log cfu/g 5 log cfu/ml

[18] [57]

Escherichia coli O157:H7

Cashew apple juice

6 log cfu/ml

400 MPa/2 min at 15°C 500 MPa/6.3 and 7.9 min at 25 and 40°C, respectively 400 MPa/3 min at 25°C

Alfalafa seeds Model cheese

5 log cfu/g 5 log cfu/g

600 MPa/2 min at 20°C 500 MPa at 20°C

[60]

Cooked smoked dolphinfish Milk

4 log cfu/g

300 MPa/15 min at 20°C

[61]

7 log cfu/ml

700 MPa/5 min at room temperature

[62]

Liquid whole egg

4-8-6.0 log cfu/ml 1.27 log reduction 2.84 log cfu/g 5 log cfu/ml

350-400 MPa/ up to 40 min at 25°C

[63] [11]

3.30 log cfu/g

6 cycles of pressurization at ca. 400 MPa/20 s at 50°C 400 MPa/10 min at room temperature 600 MPa/300 s or 300 MPa/198-369 s at 20°C 400 MPa/10 min at room temperature

Cheese Human milk Human milk

8 log cfu/ml 6 log cfu/ml 6 log cfu/ml

400 MPa at room temperature 400 MPa/30 min at 31°C 400 MPa/6 min at 31°C

[60] [10] [10]

Mesophilic count and filamentous fungi Lactic acid bacteria

Cashew apple juice

4 log cfu/ml


[59]

Sliced ham

400 MPa/15 min at room temperature

[66]

Enterococcus faecalis Leuc. mesenteroides

Meat batters Blood sausages

Prolongation of the lag phase 4 log cfu/g 1 log cfu/g

[67] [68]

Pseudomonas spp.

Blood sausages

4 log cfu/g

Weissella viridescens

Blood sausages

1 log cfu/g

400 MPa/60 min at 25°C 600 MPa/10 min; initial temperature of 15°C 600 MPa/10 min; initial temperature of 15°C 600 MPa/10 min; initial temperature of 15°C

Alicyclobacillus acidoterrestris (cells)

Orange, apple and tomato juices

4 log cfu/ml


B. cereus

Milk

6 log cfu/ml

540 MPa/ 16.8 min at 71°C

B. coagulans

Tomato juice

Geobacillus stearothermophilus

Meat batters

2 log cfu/g


[67]

B. subtilis

Various food systems

Depending on food constituents


[22, 72]

Meat batters

2.5 log cfu/g


[67]

Pathogens

Listeria monocytogenes

Mycobacterium avium subsp. paratubercolosis Salmonella Enteritidis

Raw almonds

Salmonella sp. Salmonella Typhimurium Staphylococcus aureus Streptococcus agalactiae

Model cheese Navel and Valencia orange juices Model cheese

[59]

[64] [65] [66]

Spoiling microflora

[68] [68]

Spore-formers [69] [70] [71]



As regards the behaviour of fungi, most conidiospores and ascospores could be inactivated in pressure range of 300-450 MPa at room temperature [2]; however, the ascospores of Talaromyces macrosporus are an exception to this statement. Reyns et al. [8], in fact, reported that a treatment at 200-500 MPa at 20°C activated the dormant spores and that a processing at 700 MPa for 60 min reduced ascospores number only by 2 log cfu/ml. Generally, the effect of HHP against bacteria (and probably against fungi and parasites) is affected by some keyelements, i.e.: 1.

Pressure. It is well known that bacteria inactivation relies upon the pressure [2, 9-11]. the increase of the pressure, in fact, results in an increase of the effectiveness of the treatment. An example of the pressure dependence of the number of the survivors can be found in the paper of Chen et al. [9], who proposed the parameter Dp, defined through the following equation:

   P   a log N  D   p  N0   where N0 and N are the cell numbers before and after the treatment; P, the pressure (MPa); a, the intercept and Dp, the decimal reduction pressure, defined as the increase of pressure required to achieve a 1-log reduction in the population number. This equation describes microbial inactivation in a well-defined pressure range, referred to as range of linearity. Chen et al. [9] defined the Dp value, along with the range of linearity, for some foodborne for treatments at 21.5°C for 10 min in whole milk, as follows: Escherichia coli O157:H7, 28.7±1.5 MPa (450-690 MPa) Listeria monocytogenes, 16.3±0.9 MPa (350-450 MPa) Staphylococcus aureus, 29.9±1.9 MPa (450-690 MPa) Salmonella Enteritidis, 39.5±0.6 MPa (350-690 MPa) Salmonella Typhimurium, 31.3±2.3 MPa (350-600 MPa) Shigella flexneri, 127.0±8.8 MPa (400-650 MPa) Vibrio parahaemolyticus, 21.7±0.1 MPa (150-350 MPa) Yersinia enterocolitica, 25.4±0.7 MPa (250-450 MPa). 2.

Time. It is well known that the processing time can enhance the effectiveness of the treatment and that pressure and time are negatively related, i.e. to achieve the same reduction it is possible to increase the pressure and decrease the processing time or, alternatively, increase the time and decrease the pressure. However, there are some exceptions to this statement, as reported by Chen et al. [9]; in fact, they found that an increase of the processing time did not affect significantly the Dp value of L. monocytogenes and V. parahaemolyticus in whole milk.

3.

Temperature. Increasing the temperature throughout HHP processing generally results in a stronger effect on the microorganisms, as reported by many bibliographic sources [2, 5, 9, 12]. In particular, Erkmen [12] studied the effect of the temperature (15-45°C) on the inactivation of Salmonella Typhimurium in tryptone soy broth at four different pressures (200-250-300-350 MPa) and found a significant effect of the temperature on the inactivation rate (μ) of the microbial targets. In particular, they used a square root and an Arrhenius type equations to model μ as a function of the temperature and found a stronger dependence of Salmonella Typhimurium by the temperature at low rather than at high pressures.

4.

Kind of treatment. HHP treatments can consist of a single exposure period (single pulse or static HHP) or the application of multi-pulsed pressure in shorter periods, called pulses or cycling pressure treatments. There are many papers dealing with the multi-pulse treatments; hereby, we report the data of Buzrul et al. [13]. These authors used a multi-pulse processing (up to 10 pulses) to inactivate E. coli and L. monocytogenes in whole milk and compared its effectiveness with a traditional pressure treatment, recovering that in some cases a multi-pulse treatment resulted in a higher inactivation of both the pathogens, depending on the combination of holding time and number of pulses; in particular they recovered a significant reduction of both E. coli and L. monocytogenes through a treatment at 300 MPa for 2 min per 10 pulses (20 min of holding time, 10 min of compression time, 120 min of decompression time). Nevertheless, they suggested an optimization of holding time, number of pulses and applied pressure to reach a number of logreduction, compatible with an industrial application of the method.


Bevilacqua et al.

5.

Initial inoculum. The initial cell number could affect dramatically the effectiveness of hydrostatic pressure, as showed by Furukawa et al. [14]. In fact, they tested E. coli and spores of B. subtilis in a wide range of concentrations (104-108 cfu/ml) and found that the effect of the pressure decreased as the cell number increased; a possible hypothesis for this phenomenon could be the formation of clumps or aggregates that decrease protein regions exposed to the pressure.

6.

Shoulder and tail effects. Different authors focused on the mathematical modeling of microbial inactivation throughout the HHP-processing [9, 10, 15- 19]. Despite the different conditions and microorganisms used for the assays, there are some key-concepts that can be found in these papers: a) in some cases the inactivation kinetic can be divided into three phases: a shoulder phase (i.e. a phase where the increase of the pressure does not result in any significant reduction of cell number); a linear phase (characterized by a liner correlation of pressure vs cell number); c) a tail (i.e. the final phase of the inactivation curve where a further increase of the pressure does not result in an increase of the inactivation). A typical inactivation curve is showed in the Fig. 1. It is easy to find an explanation for the shoulder phase (i.e. below a defined threshold the changes induced by the pressure are not lethal) and there are several hypotheses for the tail effect. In particular, Chen [19] suggested that within a population a small fraction could be more resistant, due probably to a different physiological state. A practical implications of this phenomenon is that it is not convenient to increase indefinitely the pressure, because there will be a residual population surviving the treatment. The complete inactivation of the targets could be achieved through a proper combination of pressure/time/temperature. 8.00

shoulder

N (arbitary units)

7.00 6.00 5.00

linear phase

4.00

tail

3.00 2.00 1.00 0.00 0.00

100.00

200.00

300.00

400.00

500.00

Pressure (MPa)

Figure 1: Typical inactivation curve of a population subjected to hydrostatic pressure.

7.

Food ingredients. As demonstrated by many authors, foods can exert a protective effect; in particular some papers focused on the protective effect exerted by milk [13, 20, 21]. As regards this issue, Narisawa et al. [21] studied the role of skim milk and protein fractions on the inactivation of E. coli K12 through the use microscopy analysis and the DAPI/PI staining (DAPI, 4',6-diamidino-2-phenylindole dihydrochloride n-hydrate; PI, propidium iodide), thus recovering a probable contributing role of the whey fraction. Other authors studied the role of different food ingredients, like Gao et al. [22], who investigated the role of soybean and sucrose against B. subtilis; in particular, the baroprotective effect was directly proportional to the concentration of the two ingredients. The effect of the sucrose was probably related to the decrease of the water activity; water, in fact, is necessary for a significant effect of hydrostatic pressure and the baroprotective effect observed at reduced aw could be explained with cell shrinkage, which would cause a thickening in the membrane, as well as a reduction of membrane permeability and fluidity [23]. The effect of solute concentration was reported also by Smiddy et al. [24], who studied the role of some compatible solutes (L-carnitine and glycine-betaine) on the barotolerance of L. innocua. However, in contrast with the idea of Palou et al. [23], they suggested that the baroprotection at elevated osmolarity could not be simply the result of the osmotic stressor itself but a consequence of the uptake of glycine and carnitine, that would play a contributing role in maintaining the fluidity of the membrane. Finally, Hauben et al. [25] investigated the effect of some minerals on the inactivation rate of E. coli and discovered that Ca2+ increased the proportion of barotolerant cells up to 1000-fold, whereas EDTA lowered the resistance to high pressure.

8.

Effect of pressurizing fluid. To best of our knowledge, there is only the paper of Robertson et al. [26] focusing on this issue; in particular, these authors studied the effects of the pressure against the spores of 28 strains of Bacillus spp. of dairy origin and belonging to six different species. The



strains were inoculated in skim milk and pressure-processed at 600 MPa/60 s at 75°C using either a water based bath or a silicon oil. The results appeared quite surprising, as silicon oil resulted in the highest reduction for all the strains, depending probably on the coefficient of thermal expansion, density, specific heat and thermal conductivity. These properties can influence the rate of heating and cooling of the fluid, as well as its adiabatic compression and thermal diffusivity, and consequently the inactivation of spores. Mode of Action High pressure processing exerts a variety of effects on microorganisms, including decreasing membrane fluidity, altering enzyme activities and affecting transcriptional and translational processes [27]. Some literature reports suggest that the primary target of the pressure is the membrane; for example, Pilavtepe-Çelik et al. [16] studied the change of the shape of E. coli O157:H7 and Staph. aureus when pressurized (180-325 MPa/1 min at 40°C) through an image analysis quantification method and recovered an increase of the cell volume in E. coli, due probably to the liquid-to-gel transition of the lipid bilayer and denaturation of proteins; volume increase of Staph. aureus was less evident and occurred only at higher pressures. The effect of HHP on cell shape and volume of E. coli was observed also by Mañas and Mackey [28], who discovered that some changes were influenced by the physiological state of the cells. In particular, a treatment at 200 MPa/8 min caused physical perturbations in the cell envelope, along with a loss of the osmotic responsiveness and a loss of RNA and proteins in the medium. Some of these modifications were observed in the cells under the exponential phase, but not under the stationary phase, thus suggesting that the injury was irreversible if exerted in the exponentially-growing cells and reversible for the cells treated under the stationary phase. Envelope modifications were studied also by Kaletunç et al. [29]; they evaluated the structural changes induced by the pressure on Leuconostoc mesenteroides through the scanning (SEM) and transimission electron microscopy and differential scanning calorimetry (DSC). Leuc. mesenteroides usually grows in chains of cells; however, HHP processing induced de-chaining, along with the formation of blisters. In addition, TEM and DSC revealed a partial coagulation of cytoplasm and the denaturation of ribosomes. In contrast with these data, Ritz et al. [30] reported that the pressure affected cell shape of L. monocytogenes only in a low fraction of the population, as measured through the oxonol fluorescence or analytical methods. Neverthless, cells suffered some physiological and structural changes, like the formation of buds on the surface or a partial loss of membrane potential. As regards the targets on the membrane, some authors reported that the pressure could act at various levels. For example, Kawarai et al. [31] demonstrated that high pressure caused the impairment of AcrAB-TolC pump of E. coli; it is one of the most studied carrier at membrane level, as it is related with the resistance of the cell with various drugs. This carrier involves a protein on the outer membrane (TolC) and a main pump component located on the inner envelope (AcrAB). The HHP treatment caused its impairment, thus resulting in the loss of tolerance to deoxycholate in E. coli but not to tetracycline. Another target of HHP on the cell envelope treatment is the mechanosensitive channel (MS), as reported by Macdonald and Martinac [32]. MS channels act as molecular mechanoelectrical transducers by converting mechanical stimuli into electrical or chemical signals [32]; one of these MS channels is the MS channel of small conductance (MscS). Pressure affected channel kinetic but not conductance, resulting in a reversible reduction of the activity with increasing the pressure up to 90 MPa. The effect of HHP on the membranes has not been elucidated fully and it is not known what happens at sublethal pressures; some authors [33, 34] hypothesized that HHP could induce a strong change in the relative concentration of the proteins of the outer and inner membranes, determining the complete disappearance of some proteins (e.g. OmpA and OmpB-two proteins of the outer membrane- in Salmonella Typhimurium). Another issue of great interest in the field of the HHP treatments is their effect on trasductional and translational mechanism of cells. In the case of a foodborne pathogen (L. monocytogenes), Bowman et al. [35] demonstrated that the high pressure resulted in an increased expression of genes linked to cell sections damaged by the treatment and in the suppression of genes associated with cellular growth processes and virulence; in fact, HHP (400-600 MPa/5 min) induced an increased expression of genes associated with DNA repair mechanisms, transcription and translation protein complexes, the general protein translocase system, flagella assemblage and chemotaxis and lipid and peptidoglycan biosyntehic pathways; on the other hand, pressure suppressed


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carbohydrate metabolism and virulence-associated mechanisms. The suppression of genes involved in cell growth was demonstrated also in Saccharomyces cerevisiae by Fernandes et al. [36] and Iwahashi et al. [37], as well as the activation of stress-responsive element controlled genes (STRE). As regards spores, it is well known that they are greatly resistant to the pressure; however, some data are available and suggest that the SASP protein (small, acid-soluble protein) could play a significant role in spore barotolerance. Lee et al. [38] investigated the role of α/β-type SASP proteins in spores under HHP treatments; SASP proteins are synthesized during the sporulation and bind to DNA, changing it from a B-like to and A-like conformation [39]. The α/β-type SASP bind tightly to DNA in the spore core and protect it form various kinds of stress (radiation, dry heat, desiccation). Surprisingly, the data of Lee et al. [38] suggested that mild pressure levels (100-300 MPa) induced the degradation of SASP proteins, and consequently the germination of spores, whereas this degradation did not occur at higher pressure values (e.g. 600 MPa). As reported for the SASP proteins in the spores, there are other cell constituents that could play a significant role in the barotolerance; the most important are the heat shock proteins (HSP) and the trehalose content [40, 41]; in particular, trehalose appeared to increase barotolerance of Sacch. cerevisiae, whereas the role of HSP proteins is not so clear, although Aersten et al. [42] demonstrated that they mediated resistance to HHP in E. coli. Despite the reports and data available in the literature, it is not possible to say why HSP proteins can mediate barotolerance; in addition, it has been observed that barotolerance is strictly related to other kind of stresses (acidic, cold, heat and osmotic shocks) and that in some cases the exposure of a microbial population to one of this stress induces a higher resistance to pressure [43, 44, 45]. Probably, barotolerance could be an element of a more general stress response, mediated by some common signal, like the HSP; however, this is only an hypothesis, that needs to be investigated in the future. Effect on Viruses A new frontier in the field of HHP processing is the study of the effect of the pressure on foodborne viruses (rotavirus and astrovirus, noroviruses, Hepatitis A virus). The first attempt in this direction was the paper of Giddings et al. [46], who studied pressure effect on tobacco mosaic virus; they found that it was greatly resistant to the pressure and required a treatment at 920 MPa to be reduced significantly. As reported by Grove et al. [47], fortunately the most of foodborne viruses are more sensitive and can be inactivated at pressures < 450MPa. It has been reported that the extent of virus inactivation is dependent upon the pressure, the treatment duration and temperature [47]; amongst these factors, pressure variations are the most significant elements, as suggested by Jurkiewicz et al. [48]. As regards the mode of action of the pressure, many references suggest that the dissociation and protein denaturation are responsible of the inactivation of viruses; moreover, some authors hypothesized that these reactions could be enhanced by low temperatures, rather than by high ones [47]. It is not clear why this happens, but Grove et al. [47] suggested that low temperatures could promote the exposure of the non polar side chains of the proteins to water and due to the fact that the non polar parts are more compressible, they would be more sensitive to the pressure. Some examples of the application of the HHP against viruses can be found easily in the literature; hereby, we report some of them. Calci et al. [49] studied the effect of the pressure on Hepatitis A virus (HAV) inoculated on oysters and recovered a 3 log PFU reduction (PFU, plaque forming unit) at 400 MPa/1 min at 9.0°C. Similar results were recovered by Kingsley et al. [50] on mashed raspberries and sliced green onion. Kingsley et al. [51] studied the role of the temperature and pressure oscillations on the inactivation of this virus; they found surprisingly that pressure oscillations did not affect significantly the effectiveness of the treatment. As regards the effect of the temperature in contrast with the data reported for other foodborne viruses, they found that an increase of the temperature throughout the treatment could be advantageous for HAV inactivation. Focusing on the other foodborne viruses, Kingsley et al. [52] studied the inactivation of noroviruses on oysters and found 4.05 log PFU reduction at 400 MPa/5 min/5°C. The application of HHP against foodborne viruses is an interesting way in the field of food technology; however, there are some limitations, i.e.: 1.

Baert et al. [53] reported that the sensitivity of viruses towards pressure “does not agree between genetically related group or even strains”, but it relies on the protein sequence and structure. Therefore, the sensitivity of each strain should be assessed separately.



2.

The efficacy of HHP might be influenced by the ionic strength of the system, as suggested by Kingsley et al. [50]; in fact, they found an inverse correlation between ionic strength (i.e. saline concentration of the solution) and virus inactivation, thus suggesting a protective effect of food on viruses. In contrast with these data, Sheldon et al. [54] reported that in some cases T7 virus (dsFNA phage) was inactivated in foods more than in cell culture medium. The results reported suggest that the effect of food components of virus resistance towards pressure is not clear.

3.

Baert et al. [53] and Smiddy et al. [55] reported that a disadvantage of using HHP against viruses is that some viral strains could develop resistance to this technology; in particular, Smiddy et al. [55] noticed altered plaques of Qß (ss-RNA coliphage) after pressure treatment; these altered shape plaques were able to persist when sub-cultured and appeared more pressure resistant.

Effect on Prions Prions (bovine spongiform encephalopathy) are highly resistant to the pressure; Fernandez-Garcia et al. [56], in fact, reported that a treatment at 700-1000 MPa at 60°C for 2 h reduced the survival rate by 47% on infected meat. However, it is important to underline that prions are greatly resistant to any kind of preserving treatment (included the sterilization) and the combination of HHP with heat appears as the only way to reduce their infective level on meat [5], as suggested also by the results of Cardone et al. [57], who reported a reduction of the level of infectivity from 103 to 106 mean lethal (LD50) per gram of meat when a combination thermal treatment-HHP processing was used. PROCESSING PARAMETERS AND EQUIPMENTS Most pressure units for food processing use pressure in the range between 100 and 800 MPa; as regards the processing time, foods are treated for milliseconds to over 20 min, although times of 5 to 7 min are more common [2]. Another processing parameter of great importance is the temperature, that can be maintained below 0°C or above 100°C; however, it is a common practice to use the room temperature (around 20-25°C). In the case of solid or semi-solid foods, a typical processing cycle in the field of HHP technology includes the following steps: 1.

packed foods are placed in the pressure vessel;

2.

vessel is sealed and filled with the pressurizing fluid;

3.

a pump forces more fluid into the vessel, creating the hydrostatic pressure. The pressure is isostatically distributed around the food and a little temperature increase can occur;

4.

vessel pressure is maintained for a predetermined time;

5.

when the cycle is complete, the vessel is quickly depressurized and temperature returns to the starting value;

6.

vessel is opened and product is removed (single step-processing);

7.

in the case of multi-step processing, after a defined rest time the vessel is pressurized again (pulses).

As can be inferred from the reported sheet, apart from pressure, pressurizing time and temperature, other critical parameters are the come-up time to achieve the pressure, the decompression time, the initial temperature of food materials, the temperature distribution in the pressure vessel as a result of the adiabatic heating, the characteristics of the product (e.g. pH, composition and water activity), the packaging material and the kind of microorganisms that should be inactivated [73]. If pressure pulsing is used, additional process factors include frequency and pulse-pressure magnitudes [2]. As regards the kind of equipments, there are both semi-continuously systems (in-container processing) for pumpable fluid foods or batch plants, used for pre-packed solid or semi-solid foods (bulk processing). The main components of a high pressure system are: 1.

A pressure vessel and its closure: the pressure vessel is a monobloc made of high tensile strength steel alloy, that can withstand pressures of 400-600 MPa; for higher pressures, pre-stressed multilayer or wire-wound vessels are used. The vessels are sealed by threaded steel closure.


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2.

A pressure generation system: the pressure units can use either the indirect and the direct compression to generate the pressure inside the vessel. As regards the indirect compression, after removal of air a pressure transmitting medium (water or oil) is pumped from a reservoir using a pressure intensifier until the desired pressure is reached; this kind of generation system requires static pressure seals. The direct compression systems use a piston to compress the vessel; this kind of unit requires dynamic pressure seals between piston and the internal vessel.

3.

A temperature control device: temperature control is achieved pumping a cooling medium through a jacket, along with internal heat exchanger for large vessels.

4.

A material handling system: it can be either an automatic equipment (in-container processing), similar to that used to load/unload batch retorts, or a simple system consisting of pumps pipes and valves (bulk processing).

USE OF HHP IN FOODS Nowadays, there are several companies all over the world (Japan, USA, Italy, Spain, Germany, Australia) that use this technology in meat products (cooked and cured ham, precooked meals with turkey, chicken and pork cuts, mortadella, bacon, salami and other smoked or not smoked sausages), jams, fruit juices (orange, apple, strawberry and peach), apple cider, apple cubes and banana puree, fresh raw vegetables (lettuce, tomato, asparagus, onion, cauliflower, green peas), crushed vegetables (carrot, tomato, broccoli), salsas, tofu, olive and seed oils, sprout seeds, fish and fish products (oysters, mussels, seafood salads) [5, 74]. Like any other processing method, HHP cannot be applied universally to all types of foods: it can be used both for liquid and solid foods; moreover foods with a high acid content are good candidates for this kind of technology. As reported above, HHP technology cannot be used alone to inactivate spores, for example in soups, vegetables and milk product and more generally in low-acid product. However, it can be used to extend the refrigerated shelf life of these foods, usually in combination with some antimicrobials, and eliminate some foodborne pathogens. Another limitation of this approach is that food must contain water, without air entrapped; food matrices with air pockets (e.g. strawberries and marshmallows) should be crushed before the treatment, whereas the dry solids do not have a sufficient moisture to achieve a significant microbial reduction [74]. Table 2 and 3 report pressure ranges for some products and the effect on some food constituents. A new frontier for the HHP is the combination of the hydrostatic pressure with some antimicrobials (essential oils, bacteriocins, lysozyme). Table 2: Pressure ranges for some foods. Processing time (usually 5-20 min) is greatly variable and depends on the equipment, kind of treatment (single- or multi-step) and eventual combination of other hurdles (antimicrobials, heat, modified atmosphere) [74]. Food

Processing pressure (MPa)

Fruit juices Orange juice

180-800

Apple juice

150-621

Peach juice

600

Lettuce

200-400

Tomato

200-400

Vegetables

Asparagus

200-400

Onion

200-400

Cauliflower

200-400

Green peas

400-900



Table 2: cont....

Crushed/extract of vegetables

400-600

Olive and seed olis

700

Sprout seeds

250-400 100-400

Eggs Dairy products Fresh cheese

50-1000

Milk

100-600

Yogurt

200-800

Meat and related products Beef

50-1000

Datty duck liver

550

Frankfurters

300-700

Ham

300

Lamb

200

Luncheon meals

600

Pork

200-827

Poultry

350-500

Rabbit

> Lact. plantarum > Lact. brevis Finally, yeasts were more sensible than Gram positive bacteria and amongst the yeast strains tested, the following susceptibility hierarchy was proposed [149]:



R. bacarum > Sacch. bayanus > P. membranifaciens Influence of Cell Concentration Is the HPH treatment depending by cell concentration? This question has not been addressed extensively and no theory resulted exhaustive; on the other hand, each way to solve this question resulted in contrast with the others. In fact, some researchers supposed that cell concentration has no discernible influence on cell disruption over a wide range of cell concentrations and operating pressures [134, 150-152], whereas Sauer et al. [153], Middelberg et al. [154] and Kleinig et al. [155] contradicted this idea. In addition, Vachon et al. [132] treated L. monocytogenes, E. coli O157:H7 and Salmonella Enteritidis to HPH treatment at 200 MPa at 25°C in 10 mM phosphate buffer (from 104 to 109 cfu/ml): the highest degree of inactivation was obtained with the lowest initial load. Successively they replicated this test using milk instead of phosphate buffered saline and in contrast with the results of buffer, the initial bacterial concentration of the milk samples had not impact on the effectiveness of HPH. Agerkvist and Effors [151], Harrison et al. [152] and Kleinig et al. [155] suggested that the decrease in disruption with increased cell concentration was caused by the increased viscosity of the homogenate. This hypothesis was denied by Diels et al. [156] who studied the effect of HPH on E. coli suspensions at 105-108 cfu/ml. They recovered that the viscosity of cell suspensions of E. coli was not affected, thus they concluded that the effectiveness of HPH was independent by the cell concentration. Influence of the Growth Phase Few data are available on the effect of the growth phase on HPH. Harrison et al. [152] treated cells of Alcaligenes eutrophus at 60 MPa for 2 or 3 passes and found that actively growing cells were disrupted by a single pass whereas cells under stationary phase were inactivated after 2 or 3 passes. Viscosity The influence of fluid viscosity on bacterial inactivation by HPH has not been systematically studied, but this factor play an important role. Kleinig et al. [155] proposed that the increased level of HPH inactivation upon dilution of E. coli cell suspensions could be explained by the lower viscosity of diluted cell suspensions. However they did not measure viscosity and based their hypothesis on the finding of Harrison et al. [152] that the viscosity of cell suspensions increased with cell density. Successively, Miller et al. [157] determined the effect of fluid viscosity on various fluid dynamic parameters using a Computer Fluid Dynamic model (CFD) and predicted that fluid viscosity (1-5 cP) would affected some of the cell breakage mechanisms, i.e. turbulence, extensional stress and impact pressure. In recent years, Diels et al. [156] contradicted the hypothesis suggested by Kleinig et al. [155] demonstrating that the viscosity of E. coli cell suspension containing 105-108 cfu/ml was not affected and that the bacterial inactivation was inversely related to the initial fluid viscosity. In another work, Diels et al. [158] evaluated not only the influence of viscosity, but also water activity and product composition on the inactivation of E. coli subjected to HPH treatment (100-300 MPa). E. coli suspensions with a different water activity (0.953-1.000) but characterized by the same relative viscosity (1.0, 1.3, 1.7, 2.7 and 4.9) were prepared using polyethylene glycol (PEG) of different molecular weight (400, 600, 1000 and 6000). Finally, the results were validated on three systems with different components (skim milk, soy drink and strawberry-raspberry; the relative viscosity was 1.7, 2.4 and 7.2, respectively). The results were the following: 

bacterial inactivation decreased with increasing viscosity of the cell suspensions and this effect was more pronounced at higher pressures;



water activity does not influence inactivation;



the inactivation of E. coli by HPH treatment in skim milk, soy drink and strawberry-raspberry was the same as in PEG characterized by the same viscosity.

HPH: HURDLE APPROACH Pathanibul et al. [159] used the hurdle approach, in order to inactivate E. coli and L. innocua. Both the microorganisms, inoculated into apple or carrot juice (ca. 7 log cfu/ml), in combination with nisin (10 IU/ml), were treated with HPH (from 0 to 350 MPa). In particular, a 5-log reduction was achieved by HPH both for E. coli and L. innocua as required by juice HACCP regulation. In particular, L. innocua showed a stronger


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resistance to HPH than E. coli; in fact for E. coli a 5 log reduction of cells was achieved after exposure to pressures in excess of > 250 MPa, whereas for L. innocua little inactivation was observed with pressure < 250 MPa and a processing pressure of 350 MPa was required to achieve an equivalent inactivation. No additional inactivation effects of nisin where observed when combined with HPH against the Gram negative bacteria; on the contrary, an interactive effect was observed in the case of L. innocua. In a previous work, Diels et al. [158] studied the sensitisation of E. coli to antibacterial peptides and enzymes (lysozyme, nisin and lactoperoxidase) by HPH in a pressure range from 100 to 300 MPa. At 150 MPa, the time of adding of these compounds was very important. In fact two different behaviours were observed: 1.

E. coli became sensitive to lysozyme and nisin when these compounds were added before the HPH treatment;

2.

E. coli remained insensitive to lysozyme and nisin when these compounds were added after the HPH treatment.

HPH could sensitise E. coli to lysozyme and nisin by inducing a transient permeabilisation of the outer membrane immediately repaired after the process; this effect would not involve a physical disruption. No sensitisation to lactoperoxidase enzyme system was observed. FOOD APPLICATIONS Juices Fruit juices are an ideal substrate for acido-tolerant bacteria and yeasts. In particular: 

Lact. plantarum, Lact. brevis and B. coagulans are involved during the processing of acidic and acidified foods and they resist to the thermal treatment usually used by fruit juice producers. Moreover B. coagulans is responsible of the flat-sour spoilage because of the production of lactic acid without gas formation.



Saccharomyces and Pichia are responsible of the formation of a film onto the surface of juices or of the production of ethanol from sugars. In addition, Las Heras-Vazques et al. [160] and Tournas et al. [161] reported that another yeasts genus involved in juices spolage, is Rhodotorula (R. mucilaginosa, R. glutinis, R. bacarum and R. rubra).

Furthermore, in the U.S. E. coli O157:H7 and Salmonella spp. are associated with fruit juice outbreaks. The ability of foodborne pathogen to contaminate fruit and vegetables juice has led the United States Food and Drug Administration (FDA) to impose HACCP requirements on juice processors. Thermal processing has been recognized for long time as an effective method to eliminate pathogen vegetative cells in fluid foods such as juices, nevertheless they lead to undesirable effects (e.g. loss of nutrients, development of off-flavours), thus within the non thermal technologies, the homogenization could be considered a promising approach for fruit juice. Briñez et al. [162] studied the bactericidal efficacy of ultrahigh-pressure homogenization (UHPH) against L. innocua ATCC 33090 inoculated into milk and orange juice, evaluating the effect of inlet temperature on the lethality and production of sublethal injuries in this microorganism and its ability to survive, repair, and grow in refrigerated storage after UHPH treatment. Samples of juices, inoculated at a concentration of approximately 7.0 log cfu/ml were pressurized at 300 MPa on the primary homogenizing valve and at 30 MPa on the secondary valve, with inlet temperatures of 6.0 ± 1.0°C and 20 ± 1.0°C. L. innocua viable counts and injured cells were measured 2 h after UHPH treatment and after 3, 6, and 9 days of storage at 4°C for milk and after 3, 6, 9, 12, 15, 18, and 21 days of storage at 4°C for orange juice. Both the inlet temperature and the food matrix influenced significantly (P < 0.05) the inactivation of L. innocua, which was higher in whole milk at 20°C. The UHPH treatment caused few or no sublethal injuries in L. innocua. During storage at 4°C after treatments, counts increased by approximately 2 log units during 9 days in whole milk, whereas in orange juice counts diminished by approximately 2.5 log units during 18 days. The effect of HPH treatment on orange juice was evaluated also by Welti-Chanes et al. [163]. Orange juice was subjected to five pressures (0-250 MPa) and with a maximum of five passes. Less that 2.93 and 3.27 log cfu/ml of mesophiles and yeasts plus moulds, respectively, were counted in orange juice treated five times at 100 MPa. In addition, homogenization reduced pectinmethylesterase enzyme: this result is an advantage for the appearance of the product. Saldo et al. [164] applied the HPH treatment to apple juice and recovered that a treatment at 200 MPa reduced cell count to the undetectable level.



Milk Milk is a nutritious medium with a favourable environment for the growth of spoilage and pathogenic microorganisms. As raw material, milk has a short shelf life, thus for commercial use it needs to be processed and the conventional technologies are the homogenization and pasteurization. The former prevents creaming during storage due to the reduction of fat globule size, whereas pasteurization eliminates any potential pathogenic microorganisms and reduce spoilage bacteria which can create negative sensory attributes decreasing processed milk shelf life. On the other hand, thermal processes can cause changes in nutritional, organoleptic or technological properties of milk [165]. For these reasons, in recent years, new technologies are developed and the interest in HPH treatment has increased. Thanks to this new technology, Guerzoni et al. [166, 167] improved safety and microbiological quality of milk and whole liquid eggs. Their results were confirmed by Kheadr et al. [146] and Vannini et al. [168] who reported that the influence of the treatment on food components, especially proteins, leads to changes in their functional properties and activities. More specifically, HPH treatment of skim and whole milk modified the ratio of the nitrogen fraction and the soluble forms of calcium and phosphorous, improved the coagulation characteristics of milk as well as increased cheese yields. Successively, Lanciotti et al. [169] reported that when Crescenza, an Italian soft cheese, had been produced using milk HPH-treated at 100 MPa, an accelerated lypolisis was observed. In addition HPH causes cell disruption for the recovery of intracellular metabolites or enzymes and activation or deactivation of enzymes. As regard to this issue, Vannini et al. [168] reported that HPH caused an enhancement of the activity of some enzymes, such as lysozyme and lactoperoxidase, against several spoilage and pathogenic species. They attributed this effect to an increased exposure of hydrophobic region of proteins since hydrophobicity plays an important role in the enhancement of the antimicrobial action of chemically modified or heat treated lysozyme [170-172]. Temperature seems to be an additional parameter influencing on the enzyme activity: the temperature increase depends upon the pressure drop from the pressure energy transformation into thermal energy and corresponds to about 12°C each 50 MPa [173]. In a recent work, Lanciotti et al. [174] observed the activation of endogenous and microbial proteolytic enzymes in cheeses obtained from caw and goat milk treated at 100 MPa; the inlet temperature was 5-7°C, whereas the outlet temperature did not exceeded 30 ± 2°C. Successively, the potential of HPH (50, 100 and 150 MPa; 2 cycles at 50 and 100 MPa) was tested for the control and the enhancement of the proteolytic and fermentative activities of some Lactobacillus spp. [175] thus observing a little viability loss (it did not exceed 1.3 log cfu/ml) and a positive effect on the proteolytic activity of some strains. Pereda et al. [165] evaluated the effect of ultra-high pressure homogenization (UHPH) (also called high pressure homogenization – HPH) on microbial and physicochemical shelf life of milk applying 100, 200 and 300 MPa (single stage). Moreover they compared this method with high pasteurized milk (90°C for 15 s). Their results showed that UHPH treatment produced milk with a microbial shelf life between 14 and 18 days, similar to that achieved with high pasteurization. The UHPH treatment reduced the L* value of treated milk and induced a reduction in viscosity values at 200 MPa compared with high pasteurization milks. Furthermore, no creaming was observed in any UHPH treated milk. Serra et al. [176] compared the impact on acid coagulation properties of the UHPH-treated milk (15 MPa) with conventional homogenized heat-treated skim milk (90°C for 90 s) and to skim milk treated under the same thermal conditions but fortified with 3% skim milk powder; they also evaluated the effects of UHPH on skim milk yogurt. The results of their work showed that UHPH was capable to reduce skim milk particle size, thus leading to the formation of fine dispersions. HPH induced an increase of some rheological parameters (density of gel, aggregation rate and water retention). In a previous work, Serra et al. [177] studied the suitability of UHPH for the production of full-fat yogurt and concluded that yogurt produced from UHPH-treated milk ( P > 200 MPa) presented better texture and water holding capacity than yogurt produced by the conventional processes in which skim milk powder is added. Patrignani et al. [178] proposed another use of HPH and evaluated its potential for the production of fermented milk carrying probiotic bacteria. Four type of fermented milks were manufactured from HPH treated and heat treated milk with and without added probiotics (Lact. paracasei and Lact. acidophilus) and using Strep. thermophilus and Lact. delbrueckii subsp. bulgaricus as starters. HPH did not modify the viability of the probiotic cultures but increased the cell loads of the starters (ca. 1 log order) compared with traditional products. The coagula from HPH-milk were more compacted than those obtained with heat treated milk and characterized by higher values of consistency, cohesiveness and viscosity indexes. Similarly, Lanciotti et al. [179] evaluated the suitability of HPH treated milk for the production of yogurt and observed that HPH treatment influenced the growth of Strep. thermophilus. In particular, the growth of Strep. thermophilus seemed to be favoured with respect to that of Lact. delbrueckii subsp. bulgaricus.


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Vannini et al. [180] studied the effect of pre-treated milk on the yield as well as on the microbiological, lipolytic and proteolytic patterns of Pecorino cheese. Pecorino cheeses obtained from ewes’ milk previously subjected to HPH (at 100 MPa) were compared to Pecorino cheeses produced from raw and heat treated ewes’ milk. A significant increase of the cheese yield of 3-5% as a consequence of HPH treatment, was observed. This effect was due to the increase of the water binding capacity of the casein and incorporation of the whey proteins into cheese curd. In addition, HPH treatment led to a reduction of enterococci, lactococci and yeast counts; this reduction could be explained as consequence of the direct modification of the initial milk population due to different species or strains sensitiveness [129, 132, 147, 168]. Moreover, enterococci cell loads remained at the undetectable levels in HPH-treated cheeses throughout the ripening period. A marked lipolysis, an early proteolysis and a relevant modification of the volatile molecule profiles suggested that HPH treatment of milk could have a potential to differentiate Pecorino cheese or to accelerate its ripening and consequently to respond to the recent increasing demand for new varieties of dairy products. These conclusions are in agreement with several works reporting that the HPH treatment of milk is a useful tool for the dairy industry to reduce ripening span time and innovate dairy products without detrimental effects on yields and safety and substantial modification of well established flow sheets [174]. Another issue of great concern is the effect of HPH on the safety of dairy products. As regards this topics, Lanciotti et al. [181] studied the effect of HPH on biogenic amine (BA) accumulation during ripening of ovine and bovine Italian cheeses. HPH treatment of bovine and ovine milks, influenced differently the microbial ecology of Caciotta and Pecorino. In Caciotta, HPH treatment reduced significantly the presence of yeasts, Micrococcaceae and lactobacilli at the end of the ripening; on the other hand, it favoured the proliferation of yeasts in ovine cheese. As regards to BA content, HPH treatment reduced biogenic amine concentrations in both the cheeses. Surprisingly, the highest BA content was found in the thermized samples probably due to a selective pressure of the mild thermal treatment which favoured the growth of decarboxylating microbiological population. This result is in contrast to the work of Novella-Rodriguez et al. [182] who reported that the use of HPH for the sanification of milk did not show significant BA differences in cheese obtained from thermally treated and pressurised milk. BOX 8.1: Biogenic amines.

What about biogenic amine?

Biogenic amines (BA) are organic bases with aliphatic, aromatic or heterocyclic structures that can be present in several foods mainly produced by microbial decarboxylation of amino acids(except polyamines) [183].

Is the presence of BA desirable? No! They are potently toxic compounds. BA presence in foods indicates inadequate or prolonged storage; however their presence in fermented foods is unavoidable due to the diffusion of decarboxylases among lactic acid bacteria. BA contents may be variable indicating that the production of such compounds depends on a complex interactionof factors. Unfortunately, the presence of relevant amounts of BA in cheeses has been recently documented [184-188]. The main BA found were tyramine, putrescine and cadaverine [ 186, 189] and their production has been mainly attributed to the activity of non‐starter microorganisms; nevertheless starter LAB could play an indirect role in BA production.

Soymilk and Soy Yogurt Soymilk is an oil-in-water emulsion obtained through the heat treatment of the product after soaking and grinding soybeans with water. It can be used as an inexpensive source of proteins compared to meat and the increased interest by consumers about soymilk and its benefits related to health led industry to invest on this field. Cruz et al. [190] realized a preliminary study to evaluate how ultra-high pressure homogenization (UHPH or HPH) technology influenced microbiological, physicochemical, and microstructural characteristics of soymilk



and compared UHPH treated-soymilk to a UHT treated soymilk and to the base-product (BP). They found that UHPH treatments (200 and 300 MPa) reduced bacteria by 2.42 and 4.24 log cfu/ml, respectively; in addition, spores were reduced by around 2 log cfu/ml and enterobacteria were below the detection level in both treatments. As consequence of the UHPH application, an intense reduction of particle size was also observed, although a formation of aggregates was detected at 300 MPa. Colour differences between UHPH and BP or UHT soymilks were found: treated soymilk (300 MPa) showed the lowest values of L*, a* and b* coordinates and this result highlighted the best performance of UHPH than other treatments. Finally, soymilk proteins were partially denatured by 200 MPa, whereas UHPH treatment at 300 MPa showed the same extent of denaturation as UHT soymilk. Soymilk could be considered as an ideal alternative to cow’s milk especially for lactose-intolerant subjects. In fact, the production of fermented soymilk by adding Strep. thermophilus and Lact. delbreuchii subsp. bulgaricus, resolved some problems related with milk protein allergenicity, vegetarian alternatives, introduction of soy derivatives in the occidental diets, etc. Several studies reported that lactic acid bacteria fermentation provide an improvement of the volatile profile to soymilk [191, 192] processed with a traditional heat treatment. On the other hand, treatment conditions were quite extreme (121°C for 15 s, 95°C for 15 min., 80°C for 20 min.) and often lead to important changes of soy components. Cruz et al. [193] studied soymilk coagulation and demonstrated that gel characteristics of the fermented soy products obtained using UHPH technology are improved when compared to those obtained from conventionally treated milk or soymilk. Successively, Ferragut et al. [194] compared the physical characteristics during storage of soy yogurt made from UHPH soymilk. Soy yogurts were prepared from UHPH-treated soymilk pre-heated at 50°C and processed at 200 and 300 MPa; moreover, a combined treatment at 300 MPa with a retention time of 15 s was investigated. Soymilk treated at 95°C for 15 min (HT) was used as control. Results showed that soy yogurts from UHPH treated soymilk possessed improved mechanical characteristics and water holding capacity compared with yogurts made from soymilk using conventional heat treatment (95°C for 15 min). In addition, a more homogeneous and compact network structure of the UHPH soy yogurts was observed. CONCLUSIONS. HPH AND TREATMENTS

HHP:

A

COMPARISON

BETWEEN TWO

NON-THERMAL

Nonthermal treatment can be attractive alternatives to traditional heat treatment for manufacturing minimally processed, high quality, preservative-free, convenient, and safe food products. In this chapter, we focused on the HPH and HHP treatment; both these treatments are based on the application of pressure, however several difference can be found. We can try to point out the most important differences, based on some bibliographic sources (Table 7) Table 7: A comparison between HPH and HHP HPH

HHP

II is a continuous and/or semi-continuous process in which products are only in a liquid state.

It is a batch process in which products are submerged in a liquid that is subsequently pressurised

The applied pressures are from 50 to 300 MPa

The applied pressures are from 200 to 800 MPa

Microorganisms are subjected to high pressures for a very short time in the order of second or less.

Microorganisms are subjected to high pressures for a time in the order of minutes or more.

It is only applicable to homogeneous liquids or fine dispersions

It is applicable to homogeneous liquids and solid products submerged in liquid, except if they contain too much gasfilled spaces.

HPH disrupts physically cells

HHP does not physically disrupt cells

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Comparison of pressure resistance of spores of six Bacillus strains with their heat resistances. Appl Environ Microbiol 1996; 62: 3897– 900. [144] Bevilacqua A, Cibelli F, Corbo MR, Sinigaglia M. Effects of high-pressure homogenization on the survival of Alicyclobacillus acidoterrestris in a laboratory medium. Lett Appl Microbiol 2007; 45: 382-6. [145] Bevilacqua A, Corbo MR, Sinigaglia M. Use of high pressure-homogenisation, sodium-benzoate and eugenol for the inibition of Alicyclobacillus acidoterrestris spores. In: Sohier S, Leguerinel I, Eds. Proceedings of the Conference Spore-forming bacteria in food; 2009: Quemper, France: Adria Development 2009; pp. 144-146. [146] Kheadr EE, Vachon JF, Paquin P, Fliss I. Effect of dynamic high-pressure on microbiological, rheological and microstructural quality of Cheddar cheese. Int Dairy J 2002; 12: 435-46. [147] Lanciotti R, Gardini F, Sinigaglia M, Guerzoni ME. 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[149] Bevilacqua A, Costa C, Corbo MR, Sinigaglia M. Effects of high pressure of homogenization on some spoiling microorganisms, representative of fruit juice microflora, inoculated in saline solution. Lett Appl Microbiol 2009; 48: 261-7. [150] Hetherington PJ, Follows M, Dunnill P, Lilly MD. Release of protein bakers’yeast (Saccharomyces cerevisiae) by disruption in an industrial homogenizer. Chem Eng Res Des Trans I Chem 1971; 49: 142-8. [151] Agerkvist I, Enfors S. Characterization of E. coli cell disintegrates from a bead mill and high-pressure homogenizers. Biotechnol Bioeng 1990; 36: 1083-9. [152] Harrison STI, Chase HA, Dennis JS. The disruption of Alcaligenes eutrophus by high-pressure homogenization: key factors involved in the process. Bioseparation 1991; 2: 155-66. [153] Sauer T, Robinson CW, Glick BR. Disruption of native and recombinant Escherichia coli in a high-pressure homogenizer. Biotechnol Bioeng 1989; 33: 1330-42. [154] Middelberg APJ, O’Neill BK, Bogle IDL. 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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 2080 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: [email protected] Antonio Bevilacqua, Maria Rosaria Corbo and Milena Sinigaglia (Eds) All rights reserved - © 2010 Bentham Science Publishers Ltd.


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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


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) Water (20°C) Ice (-20°C) Ice (0°C) Olive oil Air

88 81 16 3 3.01 100.059

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].


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MICROWAVE EFFECT ON BACTERIAL SPECIES There are many studies concerning the reduction of microorganisms by microwave lower time-temperature profile than with conventional cooking. Some authors studied the effects of microwave on foodborne bacteria and pathogens to assess the efficacy of this alternative technology. a

Escherichia coli

Belyaev [8] studied the effect of millimetre microwaves (MMW) on cells of E. coli K12 AB1157, irradiated within the power density range of 10-20 to 10-4 W/cm2 for 0.5-70 minutes at 51.76 GHz or 41.32 GHz and recovered that the magnitude of the effects depended on the concentration of irradiated cells. Moreover, they found that MMW provoked the change of genome conformational state (GCS) of the cells of microorganism examined. Apostolou et al [9] examined the effect of short time microwave exposure on E. coli O:157-H: 7 inoculated in chicken meat and found that bacterial population decreased within the time. In this study three chickens, inoculated with the target were cooked in the microwave oven for different times. For a treatment time of 10 s, the mean surface temperature was 40±4° C and the mean concentration of E. coli O157:H7 was 106 cfu/g. After 30 s, the mean final temperature was 70±2°C and the concentration of E. coli O157:H7 was 80±70 cfu/g. Thus, the elimination of E. coli O157:H7 was observed for a treatment time of 35 s, when the surface temperature was increased to ≥ 73 °C. Yaghmaee and Durance [10] investigated the effects of 2450 MHz microwave radiation under vacuum conditions to assess the survival and injury of E. coli and search for possible non-thermal effects associated with the vacuum microwaves. They found that E. coli is sensitive to temperature change under microwave heating. b

Staphylococcus aureus

Yeo et al [11] investigated the effect of 2450 MHz radiation against Staph. aureus. This microorganism (NCTC 6571; Oxford strain), inoculated on steel discs, was exposed to microwave radiation at 2450 MHz, the extent of cell reduction was increased as the exposure time increased, with the complete inactivation at 110 s, corresponding to a temperature of 61.4°C. c

Bacillus subtilis

Celandroni et al [12] investigated the killing efficacy and effect exerted by both microwaves and conventional heating on structural and molecular components of B. subtilis. They discovered that the two treatments produced similar kinetics of spore survival, although they resulted in different effects on spore structures. It was discovered that the cortex layer of the spores heat-treated was 10 times wider than the untreated spores; in contrast, the cortex of irradiated spores did not change. They concluded that MW induce changes in the structural or molecular components of spores that differ from those due to heat. d

Campylobacter jejuni

Göksoy et al [13] exposed skinless chicken inoculated with Camp. jejuni (5-6 log cfu/cm2) to 2.45 Ghz irradiation for periods of 10, 20 and 30 s. A treatment time of 10 s resulted in a decrease in population numbers of 5.43 to 5.31 log cfu/cm2, the subsequent 20 seconds and 30 seconds gave increases of population counts of 5.48-5.62 log cfu/cm2 and 5.44-5.53 log cfu/cm2 respectively. This is, perhaps, due to the environmental factors. SOME EXAMPLES OF APPLICATION OF MW ON FOODS Yarmand et Homayouni [14] studied the effect of microwave cooking on the microstructure and quality of meat in goat and lamb. The cooking process influenced the fat content of beef, its distribution and meat tenderness. Another study focused on the drying of banana [15]. Drying is one of the oldest method of food preservation and it is a difficult food processing operation mainly due to undesirable changes in quality of the dried product. In conventional air drying, high temperature and long drying time, used to remove the water from the sugar



containing fruit material, may provoke serious damage to the flavour, colour, nutrients, reduction in bulk density and rehydration capacity of the dried food. Microwave drying is rapid, more uniform and energy efficient, although, this method is known to result in a poor quality product if not properly applied. Microwave application has been reported to improve product quality like a better aroma, faster and better rehydration, considerable saving in energy and shorter drying time compared with hot air drying alone. In order to assess if this innovative technology may be considered a suitable alternative to the traditional treatments of stabilization, Giuliani et al [16] investigated the effectiveness of microwave pasteurizing treatment on a vegetable cream (cream of asparagus). The target microorganism was Alicyclobacillus acidoterrestris; chosen for its thermal resistance and its ability of growing in food with low pH (3-4.5); moreover it is a sporeformer microorganism, able to survive the conventional stabilization treatments. In order to compare the effectiveness between the traditional and the microwave processing, the treatments were regarded as sufficient when a 2-fold reduction of spore number was observed (99% reduction of the initial population). This result was observed when the samples were heated at 90, 95 and 100°C for 75, 57 and 40 min processing time, respectively; whereas the microwave treatment could be regarded as efficient for the following combinations: 90% - 6 min, 80% - 7 min and 100% - 5 min. Moreover, microwave heating did not promote the oxidation of lipid fraction immediately after the treatment, especially when low power was applied and, during storage, microwaved samples were more stable than traditionally pasteurized ones.

POWER ULTRASOUND High power ultrasound is an efficient tool for some industrial applications, like emulsification, homogenization, extraction, crystallization, dewatering, low temperature pasteurization, degassing, defoaming, activation and inactivation of enzyme, particle size reduction and viscosity alteration [17]; moreover, it is well known that ultrasound is able to disrupt biological structures. In fact, since 1960 research has focused on understanding the mechanisms that provoked the damage of microbial cells; while, in more recent years, ultrasound technology has been investigated for its potential to cause bacterial cell inactivation. PRINCIPLES AND PROPERTIES OF ULTRASOUND Ultrasound (US) is defined as pressure waves with a frequencies of 20 kHz or more (Fig. 3). Generally, it uses frequencies from 20 Khz to 10 MHz. It can be divided into three frequency ranges: 1.

Higher-power US at lower frequencies (16-100 kHz), called “Power Ultrasound”, used in food processing to inactivate microorganism and chemicals;

2.

Low intensity US at high frequencies (from 100 kHz to 1 MHz) used in non-invasive imaging, sensing and analytical tools;

3.

Diagnostic US (1-10 MHz), used for medical imaging.

It is important to highlight that the use of the this new technology in industrial processes has two main requirements: a liquid medium, even if the liquid element forms only 5% of the overall medium, and a source of high-energy vibrations (the ultrasound) [17].

Figure 3: Ultrasound frequencies

Based on its intensity, ultrasound can be, generally, divided into two groups: destructive ultrasound (1) and nondestructive ultrasound (2 e 3). Table 2 shows the different applications of ultrasound. The effects of destructive ultrasound principally derive from acoustic cavitation.

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Table 2: Applications of ultrasound Destructive US

Non-destructive US

Sonochemistry

Flaw detection

Welding

Medical diagnosis

Cleaning

Sonar

Cell disruption

Chemical analysis

Removal of kidney stones

Study of relaxation phenomena

Nowadays ultrasound technology uses the electrostrictive transformer principle. This is based on the elastic deformation of ferroelectric materials within a high frequency electrical field, caused by the mutant attraction of the molecules polarised in the field. For the polarization of molecules a high-frequency alternating current will be transmitted via two electrodes to the ferroelectrical material; then, after the conversion into mechanical oscillation, the sound waves are transmitted to an amplifier, to the sound radiating sonotrode and finally to the treated medium [18]. The principal effect of ultrasound on a fluid medium is to impose an acoustic pressure (Pa) in addition to the hydrostatic pressure acting on the fluid. This acoustic pressure is a sinusoidal wave that depends on: time (t), frequency (ƒ) and the maximum pressure amplitude, Pa,max (eq. 3). Pa = Pa,max sin (2πƒt)

(eq. 3)

It can be seen from this formula that the maximum pressure amplitude of the wave is directly proportional to the power input of the transducer. In fact, at lower intensity the pressure wave induces motion and mixing within the fluid; this phenomenon is called acoustic streaming; while, at higher intensities the local pressure causes the formation of tiny bubbles. A further increase generates negative transient pressures within the fluid, enhancing bubble growth and producing new cavities by the tensioning effect on the fluid; then, during the compression cycle, the bubble shrinks and their contents are absorbed back into the liquid. This process of compression and rarefaction of the medium particles and the consequent collapse of the bubbles describes the well-known phenomenon of cavitation (Fig. 4). Some authors reported that the frequency is inversely proportional to the bubble size; in fact, low frequency ultrasound (power ultrasound) generates large cavitation bubbles, resulting in higher temperatures and pressures in the cavitation zone; otherwise, when the frequency increases, the cavitation zone became less violent and in the MHz range no cavitation is observed.

Figure 4: Cavitation bubble formation

APPLICATIONS IN FOOD PROCESSING The most important application of ultrasound in foods are shown in Table 3 [17].



Table 3: High power ultrasound applications in the food industry Application

Mechanism

Advantage

Extraction

Increased mass transfer to and from interfaces; Better release of cellular materials.

More efficient extraction.

Homogenization

Very efficient mixing of the two layers; Formation of fine, highly stable emulsions.

Ultrasonic emulsification process can be installed within the existing plant.

Crystallization

Nucleation crystals formation and its modifications.

Ensured the formation of small and evensized crystals.

Filtration

Particles remain in suspension leaving filter open and free for solvent elution.

Extension of filter life.

Extrusion

Excitation of a metal tube or extrusion dye

Reduced drag resistance; Improved flow rate.

Enzyme and microbial inactivation

Greater sensitivity of microorganisms due to ultrasound and increase of the temperature (over 50°C).

Improved enzyme inactivation at lower temperatures.

Fermentation

Improved mass transfer of reagents and products through the boundary layer or cellular wall and membrane.

Increase of the fermentation rate of sake, beer and wine.

Heat transfer

Cavitation strongly affects the heat transfer.

Acceleration of heating; Food cooling at low temperature.

EFFECT OF ULTRASOUND ON MICROORGANISMS Studies on ultrasound as potential method to inactivate microorganisms began in the 1960s, after the discovery that the sound waves used in anti-submarine warfare killed the fish. The mechanism of microbial inactivation is due mainly to the thinning of the cell membranes, the localized heating and the production of free radicals. Conventional methods of bacteria inactivation used thermal treatment, such as pasteurization or ultra high temperature; on the contrary, ultrasound process provokes the destruction of bacteria by the changes in pressure created by the ultrasonic waves (cavitation). During the sonication process, the sonic wave encounters a liquid medium creating longitudinal waves, that generates regions of high pressure alternating with areas of low pressure; these regions of different pressure cause cavitation and gas bubbles formation. These bubbles increase gradually their volume until they implode, creating regions of high temperature and pressure. Pressure resulting from these implosions cause the main bactericidal effect of ultrasound. The efficacy of ultrasonic treatment depends on the type of microorganism treated, in fact, for example, spores are relatively resistant to the effects of ultrasound. Moreover, Gram positive cells have been found to be more resistant to ultrasound than Gram negative cells and this may due to the structure of the cell wall. This is why ultrasound should be used in combination with treatments for high pressure (manosonication), thermal treatments (themosonication) or both (manothermosonication). Other factors that influence the antimicrobial effectiveness are the amplitude of the waves ultrasound, the time of exposure/contact, the volume of processed food, the composition product and the temperature of treatment. Furthermore, free radical formation is another proposed mode of action of ultrasound inactivation with bactericidal effects. Application of ultrasound to a liquid can lead to the formation of free radicals that provoked the breakages along the length of the DNA causing the formation of small DNA fragments [19]. a

Escherichia coli

Knorr et al [18] studied the combined effects of ultrasound and temperature on microbial inactivation with the aim to reduce the temperature and/or the process time. The target microorganism used in this study was E. coli K12 DH 5 α. In particular, treatments of 24.6 W resulted in one log cycle reduction after 300 s of treatment time, while two log cycle reductions was obtained after the same exposure-time with a treatment at 42.0 W.


b

Di Benedetto et al.

Listeria monocytogenes

The combination of sonication with an increased pressure of 200 kPa provoked a reduction of the D-value at ambient temperature of L. monocytogenes from 4.3 to 1.5 min. A further increase in pressure up to 400 kPa reduced the D-value to1.0 min. Moreover, temperature up to 50°C did not exert any significant effect, but once temperatures exceeded this threshold, a considerable enhancing effect was noted [20]. c

Salmonella spp.

Chocolate is a potential source of Salmonella spp. and the high sugar content of chocolate might increase the heat resistance of the microorganism. When Salmonella spp. was subjected to ultrasound of 160 kHz at a power of 100 W for 10 min in a peptone water, a 4-log reduction in viable cell count was observed. Furthermore, some differences were observed between the D-value of Salmonella Easbourne (3 min) and Salmonella Anatum (2.1) treated with ultrasound at 100 W and 160 kHz at 5°C. The ultrasonic treatment of chocolate inoculated with Salmonella Easbourne showed a 26% reduction after 10 min and a 74% after 30 min. This result suggested that ultrasound might be useful for the conching process in chocolate production [20]. d

Bacillus subtilis

Some studies showed that the effect of thermosonication with water as medium provoked a reduction of the heat spore resistance between 70% and 99.9% at 70-95°C. Furthermore, it was observed that manothermosonication at 110-112°C caused a 1/10 reduction of the B. subtilis spores heat resistance. Moreover, comparisons between B. subtilis spores treated with manosonication and manothermosonication showed that the heat effect due to manothermosonication made the inactivation process more effective. Instead, increasing pressure to 500 kPa in manosonication resulted in increasing microbial inactivation, but any further increase didn’t result in a greater spore inactivation [20]. FUTURE PERSPECTIVES The considerable interest in high-powered ultrasound is due to its promising effects in food processing and preservation, such as higher product yields, shorter processing time, reduced operating and maintenance costs, improved taste, texture, flavour and colour, and the reduction of pathogens at lower temperature [17]. Moreover, the future of ultrasound in the food industry for bactericidal porpouses lies in thermosonication, manosonication and manothermosonication, as they are more energy-efficient and result in a higher reduction of D-value. However, further research is required before ultrasound can become a real alternative method for food preservation [20].

PULSED TECHNOLOGIES PULSED ELECTRIC FIELD (PEF) PEF is an emerging non-thermal process for the inactivation of microorganisms in liquid and semi-liquid food, preserving the fresh flavour, colour, texture and integrity of food compounds. In general, the shelf-life of PEFtreated and thermally pasteurized foods is comparable. PEF pasteurization kills microorganisms and inactivates some enzymes and, unless the product is acidic, it requires refrigerated storage. For heat-sensitive liquid foods where thermal pasteurization is not an option (due to flavour, texture, or colour changes), PEF treatment would be advantageous [21]. Mechanism of Action 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). The electric pulses used for PEF treatment are extremely short in duration, therefore temperature does not increase during treatment. The high voltage results in an electric field that causes microbial inactivation. Part of the potential utility of PEF is that the properties of the electric field can be modified to have different effects on cells. Most plant cells can survive weak electric fields, but over around 15,000 volts per centimeter they are killed. The increase of field to 30,000 volts per centimeter results in the inactivation of bacteria and



fungi. The electric field may be applied in the form of exponentially decaying, square wave, bipolar or oscillatory pulses and at ambient, sub-ambient, or slightly above-ambient temperature. After the treatment, the food is packaged aseptically and stored under refrigeration.

Figure 5: Yeast cells (Saccharomyces cerevisiae) in apple juice. Left - untreated; Right - PEF - treated cell. [1]

The electric field enlarges the pores of the cell membranes thus killing the cells and releasing the cytoplasmatic content into the medium (Fig. 5). Pore formation is reversible or irreversible, depending on factors such as the electric field intensity, the pulse duration, and number of pulses. The membranes of PEF-treated cells become permeable to small molecules; permeation causes swelling and eventual rupture of the cell membrane. PEF treatment has lethal effects on various vegetative bacteria, moulds, and yeasts. Efficacy of spore inactivation by PEF in combination with heat or other hurdles is a subject of current research. The survival of spores and enzymes means that products should be refrigerated after passing through PEF processing in order to slow the action of the enzymes and keep pathogens from growing. Gram positive vegetative cells are more resistant to PEF than Gram negative bacteria while yeasts show a higher sensitivity than bacteria. Many papers describe the inactivation of vegetative cells by PEF, but only some reports can be found on the inactivation of spores, describing a limited effect of PEF. B. cereus spores were practically resistant (only a 1 log reduction) to a mild PEF treatment with electric field strength of 20 kV/cm and pulse numbers of 10.4 in apple juice [22]. Pagan, et al. [23] did not find any inactivation of B. subtilis spores with a PEF treatment of 60 kV/cm for 75 pulses at room temperature. On the other hand, Marquez, et al. [24] described for B. subtilis and B. cereus spores large inactivation degrees, 3.42 and 5 log units respectively, realised by high voltage PEF (50 kV/cm with 50 pulses at 25 °C) in a salt solution. Mould conidiospores showed to be very sensitive to PEF in fruit juices, but Neosartorya fischeri ascospores were resistant to PEF treatments [25]. Probably, other factors than process parameters such as the preparation of the spores and the type of medium in which the treatment was performed could influence the kind of inactivation of bacterial spores and explain therefore the high variation in the reported results. It is indeed known that the electrical conductivity of the medium influences strongly the effect of PEF [26]. PEF Applications PEF is a continuous processing method, which is not suitable for solid food products. On the other hand application of PEF technology has been successfully used for the pasteurization of some foods like juices, milk, yogurt, soups, and liquid eggs. Application of PEF processing is restricted to food products with no air bubbles and with low electrical conductivity. In fact, the use of PEF process, in liquids with gas bubble, burns some component that potentially generate unwanted materials, such as carcinogens. Any way the maximum particle size in the liquid must be smaller than the gap of the treatment region in the chamber in order to ensure proper treatment. PEF is also applied to enhance the extraction of sugars, oils and other cellular content from plant cells, such as sugar beets. PEF also can be used to reduce the solid volume (sludge) of wastewater. The effect of PEF can be increased by applying it in combination with other stressing factors such as [27]: 

HHP treatment



the presence of antimicrobial compounds (nisin and organic acids),



increased water activity,



pH and mild heat treatments.


Di Benedetto et al.

PEF is an energy efficient process compared to thermal pasteurization; in fact it would add only $0.03-$0.07/l to final food costs. A commercial-scale PEF system can process between 1,000 and 5,000 litres of liquid foods per hour and this equipment is scalable. Generation of high voltage pulses having sufficient peak power (typically megawatts) is the limitation for processing large quantities of fluid economically. The emergence of solid-state pulsed power systems, which can be arbitrarily sized by combining switch modules in series and parallel, can remove this limitation. PULSED LIGHT IRRADIATION (OR INTENSE LIGHT PULSES ILP) ILP is a new technology based on the use of light pulses, applicable in sterilizing the surface of packaging materials, transparent pharmaceutical products, or other surfaces [28]. Pulsed light may be also used to extend the shelf-life or improve the quality of food. Usually, the packaging material used in aseptic processing is sterilized with hydrogen peroxide, which may leave highly undesirable residues in the food or package [29]; light pulses may be used to reduce or eliminate the need for chemical disinfectants and preservatives. Equipments and Processing Parameters The pulsed light consists of intense flashes of broad-spectrum white light, containing wavelengths from 200 nm in the ultraviolet (UV) to 1000 nm (also to 2600 nm) in the near-infrared region (Fig. 1). The pulsed light distribution is almost similar to that of sunlight, except for the content of UV region under 320 nm, that is an important part of the pulsed light [30]. The material to be treated is exposed to a least 1 pulse of light having an energy density in the range of about 0.01 to 50 J/cm2 at the surface. The material to be sterilized is exposed to at least 1 pulse of light (typically 1 to 20 flashes per s) with a duration range from 1 µs to 0.1 s [31]. For most applications, a few flashes applied in a fraction of a seconds provide a high level of microbial inactivation. Pulsed light (Fig. 6a) is produced using engineering technologies that multiply power many fold. Accumulating electrical energy in an energy storage capacitor over relatively long times (a fraction of a seconds) and releasing this storage energy to do work in a much shorter time (millionths or thousandths of a seconds) magnifies the power applied. The result is a very high power during the duty cycle, with the expenditure of only moderate power consumption [32]. Each pulse has from 20,000 to 90,000 times the intensity of sunlight at sea level. Effect on Microorganisms Light pulses induce photo-chemical or photo-thermal reactions in foods. The UV-rich light causes photochemical changes, while visual and infrared lights cause photo-thermal changes. The mode of action of the pulsed light process is attributed to unique effects of the high peak power and the broad-spectrum of the flash. But UV light has been shown a great importance to inactivate pathogens and indicator organisms [33]. The antimicrobial effects of these wavelengths are primarily mediated through absorption by highly conjugated carbon-to-carbon double-bond systems in proteins and nucleic acids [34]. The damage caused by the broad-spectrum light (200-320 nm) is thought to produce extensive irreversible damage to DNA, proteins, and other macromolecules. In particular, nucleic acids are the primary cellular targets. Inactivation occurs by several mechanisms, including chemical modifications and cleavage of the DNA; the impact of pulsed light on proteins, membranes, and other cellular material probably occurs concurrently with the nucleic acid destruction. For example, the motility of E. coli ceases immediately after exposure to pulsed light. In many studies, loss of motility of protozoan sporozoites was observed after pulsed light treatment of oocysts. As for other lethal physical agents, it is difficult to determine the actual sequence of events due to a possible "domino effect" [35]. ILP Applications Pulsed light is effective for killing both Gram positive and negative bacteria, fungi, spores, virus, oocystes, and the killing effect is higher in a shorter time than with continuous UV treatment [32, 36, 37]. The killing effects of pulsed light are caused by the rich, broad-spectrum UV content, the short duration, and the high peak power of the pulsed light (Fig. 6b) [32]. In fact, the UV region is a very important factor for pulsed light sterilization and the pulsed light contains around 25% in the UV region. It was confirmed that there is no killing effect if a filter, which can remove the UV wavelength region under 320 nm, is used [38]. However, in the case of the pulsed light method, it appears that both the visible and infrared regions work for killing microorganisms.


Energy(peak power)


Continuous light Time of irradiation

pulsed light

Figure 6: Pulsed light experimental apparatus (a); pulsed light and continuous light (b)

Pulsed light provides shelf-life extension and preservation when used with a variety of foods. The lethality of the light pulses is different at different wavelengths. Therefore, the full spectrum or selected wavelength may be used to treat the foods. Intense light pulses (ILP) is a novel decontamination method for food surfaces that could be suitable for disinfecting: minimally processed (MP) vegetables [39- 42]; water [43]; corn meal [44]; strawberry [45]; meat and poultry [37]. For instance, shrimp treated with pulsed light and stored under refrigeration for 7 days remained edible, while untreated shrimp showed extensive microbial degradation and were discolored, foul smelling, thereby not edible [32]. More than 7-log cycles of Aspergillus niger spore inactivation resulted with a minimal number of pulsed light flashes with 1 J/cm2 [31]. A variety of microorganisms including E. coli, Staph. aureus, B. subtilis, and Sacch. cerevisiae have been inactivated by using 1 to 35 pulses of light with an intensity ranging from 1-2 J/cm2. Wavelengths known to produce undesirable products in foods are eliminated by filtering through glass or liquid filters. Future Research Due to failure of light to penetrate opaque and irregular surfaces, there is generally less microbial inactivation with pulsed light, compared to other technologies. Light characteristics (wavelength, intensity, duration and number of the pulses), packaging and food attributes (type, transparency and colour) are considered to be critical process factors. In the case of a fluid food, transparency and depth of the fluid column become critical factors. Despite its minimal effectiveness with opaque foods, pulsed light has been reported to have limited ability to reduce microbial counts (about 1 to 4 log cycles) on eggs, including organisms inoculated onto the surface of eggs and then drawn into egg air pores by a differential temperature [32]. The lethality of the pulsed light increases with increasing light intensity or fluency [35], although formulation of a model for dose-response is not currently possible. Future perspectives in the field of PL are reported in the Fig. 7. Future research

Identification of critical process factors and their effect on microbial inactivation

Suitability of the technology for solid foods and non-clear liquids

Figure 7: Pulsed light future research

Potential formation of unpalatable and toxic byproducts

Resistance of common pathogens

Differences between this technology and that of the more conventional UV (254 nm) light treatment

Mechanisms of microbial inactivation

Understanding of the mechanism and quantification of the benefit attributed to the pulse effect

Di Benedetto et al.


IRRADIATION Irradiation was suggested as a food-processing method in 1897, one year later Roetgens discovered X-rays, and patented in England in 1905. Experiments were conducted in the quartermaster laboratory of the US Army to provide sterilized cans to the US army, resulting in 1962, in the development of the first food irradiation facility; the USDA issued the first food irradiation rules in 1963 to decontaminate wheat and wheat powder [46]. Irradiation involves exposing food to ionizing radiations. In the food sector, irradiation is defined by two processes, radiation pasteurization (radurization), which refers to the inactivation of non-spore-forming bacteria with a low absorbed dose requirement (1-10 kGy) and the sterilization irradiation (radapperdization) to ensure the elimination of Cl. botulinum. For the last one the dose required is higher (between 40 and 50 kGy) than that permitted for commercial food (10 kGy) [47- 49]. IONIZING IRRADIATION Ionizing irradiation occurs when one or more electrons are removed from the electronic orbital of the atom. It can be produced by three different techniques (Table 4), gamma ray processing, high energy electron (called ebeam) and X-ray processing (figures 9.1 and 9.8). [50]

Figure 8: Spectrum light application [5] Table 4: Summary of the depth and efficiency for the three ionised-irradiation technologies used in food processing [48].

a

Gamma ray

X-ray

E-beam

Power source (kW)

≈ 50

25

35

Source energy (MeV)

1.33

5

5-10

Processing speeda(tonnes/h)

12

10

5-10

Penetration depth (cm)

80-100

80-100

8-10

Dose uniformity ratio

≈ 1.7

≈ 1.5

moderate

Dose rate (kGy*/h)

low

high

high

Processing speed to deliver a dose rate of 4 kGy.

*1 Gy = J/kg

Electron Beam Processing It involves irradiation of products using a high-energy cathode ray (also called electron beam or e-beam) accelerator. Electron beam accelerators utilize an on-off technology, with a common design being similar to that of a cathode ray television. Cathode rays are streams of electrons observed in vacuum tubes (Crookes tubes), that are equipped with at least two metal electrodes to which a voltage is applied, i.e. a cathode, or negative electrode, and an anode, or positive electrode. They were discovered by a German scientist. Johann Hittorf, in 1869 and in 1876 named by Eugen Goldstein kathodenstrahlen (cathode rays). In modern tubes, the cathode is assisted by making a thin wire filament and passing an electric current through it; the current heats the filament red hot. The increased random heat motion of the filament atoms assists in knocking electrons out of the atoms at the surface of the filament, into the evacuated space of the tube. This process is called thermionic emission and can reduce the anode to cathode voltage needed to obtain effective currents.



Electron beam processing is used in industry for product modifications: 

cross linking of polymer-based products to improve mechanical, thermal, chemical and other properties;



material degradation often used in recycling;



sterilization of medical and pharmaceutical goods;



food treatment.

For food application electron beams are produced by commercial electron accelerators and therefore can be switch off like all electrical apparatus. They can be directly used for small items such as grains or to remove surface contamination, because they have limited penetration capacity [51]. X-Radiation (Composed of X-Rays) It is a form of electromagnetic radiation. X-rays have a wavelength in the range of 10 to 0.01 nm, corresponding to frequencies in the range 30 petahertz to 30 exahertz (3 × 1016 Hz to 3 × 1019 Hz) and energies in the range 120 eV to 120 keV. They are shorter in wavelength than UV rays. In many languages, X-radiation is called Röntgen radiation. X-rays are primarily used for diagnostic radiography and crystallography, due to the fact that X-rays are a form of ionizing radiation and can be dangerous (Fig. 8). X-rays from about 0.12 to 12 keV are classified as soft X-rays, wherease those included in the range from about 12 to 120 keV as hard X-rays, due to their penetrating abilities. X-rays are produced trough X-ray tube. An X-ray tube is a vacuum tube, including a cathode, which emits electrons into the vacuum, and an anode to collect the electrons, thus establishing a flow of electrical current, through the tube, known as the beam. A high voltage power source, for example 30 to 150 kV, is connected across cathode and anode to accelerate the electrons. The X-ray spectrum depends on the anode material and the accelerating voltage. Electrons from the cathode collide with the anode material, usually tungsten, molybdenum or copper, and accelerate other electrons, ions and nuclei within the anode material. About 1% of the energy generated is emitted/radiated, usually perpendicular to the path of the electron beam, as X-rays. Over time, tungsten will be deposited from the target onto the interior surface of the tube, including the glass surface. As time goes on, the tube becomes unstable even at lower voltages, and must be replaced. In many applications, the current flow (typically in the range 1mA to 1A) is able to be pulsed on for between about 1ms to 1s. Until the late 1980s, X-ray generators were merely high-voltage; in the late 1980s a different method of control was developed, called high speed switching. This approach used the electronics technology of switching power supplies (“aka switch mode power supply”), and allowed for a more accurate control of the X-ray unit, higher quality results, and reduced X-ray exposures. For food treatment the USFDA and the European Commission approved the use of X-ray technology with a maximum energy of 5 MeV; then, the FDA amended the maximum level to reach 7.5 MeV in 2004 [51]. Gamma Rays Originally defined on the basis of wavelength (radiation shorter than λ: 10−11 nm, the break-point, was defined as γ-rays) the distinction between X-rays and gamma rays has changed in recent decades. However, as shorter wavelength continuous spectrum "X-ray" sources such as linear accelerators and longer wavelength "gamma ray" emitters were discovered, the wavelength bands largely overlapped. The two types of radiation are now usually defined by their origin: X-rays are emitted by electrons outside the nucleus, while gamma rays are emitted by the nucleus. The effects of gamma irradiation on the rheological characteristics of guar gum, pectin and sales were investigated [52]. Gamma rays were used for pomegranate juice (from 0 to 10 KGy) carrot, potato and beetroot (3 – 12 KGy) treatment [51]. MECHANISM OF ACTION OF IRRADIATION The molecular bonds in the microbial DNA are the main target of irradiation, but DNA and RNA synthesis, denaturation of enzymes and cell membrane alterations may also be affected. The absorbed dose, measured in

Di Benedetto et al.


grays (Gy); (1 Gy = J/kg = 100 rad), is considered the most important parameter, but the effectiveness of the treatment depends also on the sensitivity of the microorganism, the extrinsic characteristic of the environment (pH, temperature) and the intrinsic characteristics of the food (fat content, salt, additives, etc.). In general, viruses are the most resistant followed by spores and yeasts. Moulds and Gram positive vegetative bacteria are more resistant than Gram negative. Low water activity and low temperature promote resistance while oxygen enhances the irradiation action. Cross-adaptation to other stress as acid has to be considered [46, 48, 53]. Table 5: Irradiation dose for food pasteurization: Current classification

Dose (KGy)

Low doses

< 3.0

Medium doses

3.0 < dose < 7.0

High doses

7.0 < dose < 10.0

Numerous studies have demonstrated that low to medium dose (Table 5) food irradiation, proper packaging and storage are very effective against bacteria (E. coli, Campylobacter, Salmonella, Yersinia, Listeria) as well as against parasite (Taenia solium, T. saginata, Trichinella spiralis) (Table 6). Table 6: Minimum effective dose for rendering some parasites ineffective [54- 56]. Parasites

Minimum dose (kGy)

Toxoplasma gondii

0.5

Fasciola hepatica

0.7

Clonorchis spp

0.15

Angiostrongylus cantonensis

2.0

Cysticercus bovis (Taenia saginata)

0.4

Cysticercus cellulosae (Taenia solium)

0.2-0.6

Trichinella spiralis

0.1-0.3

IRRADIATION DIRECTIVE The technique offers the possibility of processing many foods in great quantities, although it requires a high cost of investment and maintenance. Food irradiation facilities must be designed and constructed in order to ensure the control of the radiological hazard for the personal and the environment; for example reinforced concrete walls must be built around the main source and the treatment area. Additionally, the operational costs are also high and in some countries the same amount of money can be used on quality controls and in good hygiene practice. On the basis of scientific studies, the Food and Agriculture Organization (FAO), the International Atomic Energy Agency (IAEA) and the World Health Organization (WHO) concluded in their report, that any food irradiated up to a maximum dose of 10 kGy is considered to be safe and wholesome [57]. As a consequence of this report a Codex General Standard for Irradiated Foods was adopted in 1983 and a Codex Recommended International Code of Practice for the Operation of Radiation Facilities used for the Treatment of Food was adopted in 1984. In 1997, the FAO, jointly with the WHO and the IAEA, convened a meeting of experts to address issues related to irradiation. It was concluded that food irradiated to any dose appropriate (less than 10 kGy) to achieve the intended technological objective is both safe to consume and nutritionally adequate [58]. The report provides three conclusive assessment: 

food irradiation will not lead to toxicological changes in the composition of food that would have an adverse effect on human health;



the technology will not increase the microbiological risk of the consumer;



food irradiation will not lead to nutrient losses that would have an adverse effect on the nutritional status of individuals.



Irradiation has many application in food safety and it is used to inactive parasites and vegetative form of bacteria from poultry, meat, meat products, fish and other seafood as well as fruit and vegetables. Although a 1999 WHO report concluded on the basis of knowledge derived from over 50 years of research that irradiated foods are safe, European consumers and policy makers remain reticent over the use of the technology. In the USA, the FDA has approved irradiation for a number of commodities: wheat, wheat powder, white potatoes, spices, dehydrated vegetables seasoning, strawberries, pork and poultry. EUROPEAN DIRECTIVE The framework Directive 1999/2/EC [57] concerning foods and food ingredients treated with ionising radiation lays down general and technical aspects for carrying out irradiation (Table 7); it includes the authorised maximum doses, the labelling of irradiated foods and the conditions for authorising food irradiation. The treatment of a specific food item may only be authorised if: 

it presents no health hazard;



it is not used as a substitute for hygiene and health practices or for good manufacturing; or agricultural practice;



there is a reasonable technological need;



it is of benefit to the consumers.

In addition, the Directive lays down that foodstuffs may be only authorised for treatment with ionising radiation if the Scientific Committee on Food (SCF) has expressed a favourable opinion on this particular foodstuff and if the authorised maximum dose does not exceed the dose recommended by the SCF. The list of foodstuffs authorised (Table 8) in the Community for treatment with ionising radiation appears in Directive 1999/3/EC [58] An example of a specification for an irradiated food is that reported for black paper (Table 9). Table 7: Permitted applications for irradiation of foodstuffs (UK and EC Irradiation Legislation) [59] 1 Elimination or reduction of pathogenic organisms causing food poisoning. 2 Reduction, by the retardation or arresting of decay processes and destruction of spoilage organisms. 3 Reduction of waste of food resulting from premature ripening, germination or sprouting. 4 Disinfestations of food from infestation by organisms harmful to plants or plant products. Table 8: Permitted food categories (UK and EC Irradiation Legislation) * Food

Irradiation dose (kGy)

Fruit

2

Vegetables

1

Cereals

1

Bulbs and tubers

0.2

Spices and condiments

10

Fish and shellfish

3

Poultry

7

*Red meat is excluded

Di Benedetto et al.


Table 9: Pre- and Post-irradiation (10 kGy dose) microbiological specification for black pepper [59]. Pre-irradiation

Microbial specification

Total viable count

0) and the inactivation (μ1 convex curves are obtained, while for p

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