Chitosan: Novel Applications in Food Systems 0128216638, 9780128216637

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Chitosan: Novel Applications in Food Systems

Chitosan: Novel Applications in Food Systems

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IOANNIS N. SAVVAIDIS Department of Environmental Health Sciences, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates; Department of Chemistry, Laboratory of Food Chemistry and Food Microbiology, University of Ioannina, Ioannina, Greece

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-821663-7 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Nikki P. Levy Acquisitions Editor: Nina Bandeira Editorial Project Manager: Ali Afzal-Khan Production Project Manager: Greg Harris Cover Designer: R. Vijay Bharath Typeset by MPS Limited, Chennai, India

Dedication I dedicate this book to: My mother Ioanna, my father Nikolaos, and my brother Dimitrios for their continuous aspiration, encouragement, and support while my long stay in Britain and for showing me the way, to follow an academic career. My wife Georgia who taught me what true life is, believed in me, showed me the way to become a better and a stronger, independent person, and encouraged me in my career, bearing patiently during my longterm stays in the Middle East. Finally, limitless thanks and appreciation for her strong will, determination, and patience to growing our little gem “Emmeleia”. Lastly: To my daughter Emmeleia who changed my life completely, showing that life starts any day, never ends, is worth living for, and has so much to offer. Our little gem has been surprising me, challenging me to laugh more, to be more patient, to share more, and to offer endless and unconditional love, as she has always done showing me the way ahead with laughs, enthusiasm, and endless motivation. May God bless us all. Sharjah, UAE

Ioannis N. Savvaidis

Contents List of contributors Preface

1. Application of chitosan in active food packaging

xiii xvii

1

Layal Karam and Angy Mallah 1.1 Introduction 1.2 Active chitosan-based packaging—antimicrobial properties 1.3 Active chitosan-based packaging combined with functional ingredients 1.3.1 Antimicrobial properties 1.3.2 Antioxidant properties 1.3.3 Antimicrobial and antioxidant properties 1.4 Active chitosan-based packaging combined with encapsulated functional ingredients 1.4.1 Metallic nanoparticles encapsulated into chitosan-based films 1.4.2 Plant extracts encapsulated into chitosan-based films 1.4.3 Other compounds encapsulated into chitosan-based films 1.5 Conclusion References

2. Chitosan-based coatings and plant extracts

1 2 4 4 7 10 15 15 22 24 25 25

31

Kataryne Árabe Rimá de Oliveira, Karina Felix Dias Fernandes, Jossana Pereira de Sousa Guedes, Evandro Leite de Souza and Marciane Magnani 2.1 Edible coatings: main aspects and general characteristics 2.1.1 Chitosan-based edible coatings 2.1.2 Plant extracts or essential oils added in coatings 2.2 Effects of coatings on quality parameters and shelf life of food 2.2.1 Antimicrobial effects 2.2.2 Antioxidant effects 2.2.3 Effects on quality parameters in foods (physicochemical, enzymatic activity, and sensory characteristics) 2.3 Future application prospects Acknowledgements References

31 32 34 36 36 39 42 61 62 62

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3. Chitosan nanoparticles as used against food pathogens

69

Daniel Hernandez-Patlan, Bruno Solis-Cruz, Xochitl Hernandez-Velasco and Guillermo Tellez-Isaias Abbreviations 69 3.1 Introduction 69 3.2 Chitosan: generalities 71 3.2.1 Physicochemical properties 71 3.2.2 Extraction methods and characterization 73 3.2.3 Factors that influence its antimicrobial activity 77 3.3 Chitosan-based nanosystems against food pathogens 78 3.3.1 Types of nanosystems 78 3.3.2 Mechanisms of antimicrobial action 84 3.3.3 Factors that affect the antibacterial activity of chitosan nanosystems 85 3.3.4 Antimicrobial activity against food pathogens 86 3.4 Chitosan nanosystems loaded with natural antimicrobials 91 3.4.1 Plant extract/phytobiotics 91 3.4.2 Essential oils 93 3.4.3 Enzymes 96 3.4.4 Bacteriocins 97 3.5 Application of chitosan nanoparticles against pathogens in food systems 103 3.6 Conclusion and future perspectives 103 References 103

4. Chitosan nanoparticles with essential oils in food preservation

115

Layal Karam and Jina Yammine 4.1 Introduction 4.2 Chitosan nanoparticles system for the encapsulation of essential oils: properties, advantages, and formation methods 4.3 Controlled release properties 4.4 Antimicrobial mechanisms of action 4.5 Functional properties of chitosan nanoparticles encapsulating essential oils on food products 4.5.1 Antioxidant activity 4.5.2 Antimicrobial activity—spoilage microorganisms 4.5.3 Sensory characteristics 4.5.4 Physicochemical characteristics 4.6 Conclusions and future perspectives References

115 116 122 125 128 129 132 139 141 143 144

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ix

5. Chitosan as an antimicrobial agent to increase shelf life of foods

155

Gerlane Souza de Lima, Alessandra Silva Araújo, Lúcia Raquel Ramos Berger, Ana Elizabeth Cavalcante Fai, Marcos Antonio Barbosa de Lima, Rodrigo França and Thayza Christina Montenegro Stamford 5.1 Introduction 5.2 Chitosan: emerging and eco-sustainable technology in food preservation 5.2.1 Sources and production 5.2.2 Physicochemical and chitosan derivatives 5.3 Chitosan and derivatives as antimicrobial agents 5.3.1 Antibacterial activity 5.3.2 Antifungal activity: ultrastructural effect of chitosan and chitosan nanoparticles 5.3.3 Chitosan and its derivatives’ application to increase shelf life of foods 5.4 Perspectives References

6. Application of chitosan on seafood safety and quality

155 156 156 159 162 163 163 169 180 180

193

Nikheel Bhojraj Rathod, Nariman El Abed and Fatih Özogul 6.1 Introduction 6.2 Chitosan 6.2.1 Sources 6.2.2 Structure 6.2.3 Extraction 6.3 Health benefits of chitosan 6.4 Bioactivity of chitosan 6.4.1 Antimicrobial properties 6.4.2 Antioxidant properties 6.5 Nanotechnological approaches for application of chitosan 6.5.1 Chitosan for nanoencapsulation 6.5.2 Chitosan-based nanomaterial for packaging 6.5.3 Chitosan-based intelligent packaging 6.6 Impacts of chitosan on seafood safety 6.6.1 Edible coating 6.6.2 Film 6.7 Inclusion of chitosan in combination with other preservation as hurdle concept 6.8 Effect of chitosan on acceptability/sensory quality of seafood 6.9 Conclusion References

193 195 195 197 198 200 202 202 205 207 207 208 210 211 212 214 215 220 221 222

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7. Chitosan and use of pomegranate-based films in foods

235

Hadi Ebrahimnejad, Elham Khalili Sadrabad and Fateme Akrami Mohajeri 7.1 Nutritional and chemical properties of pomegranate 7.2 Functional properties of pomegranate 7.2.1 Bioactivity of pomegranate juice 7.2.2 Bioactivity of pomegranate peel (pericarp) 7.2.3 Bioactivity of pomegranate seed 7.3 Extraction of pomegranate active compounds 7.3.1 Conventional extraction methods 7.3.2 Modern extraction methods 7.4 Chitosan composite films for incorporating pomegranate active compounds 7.5 Encapsulation of pomegranate active compounds in chitosan-based films 7.6 Physicochemical properties of pomegranate-chitosan-based films 7.6.1 Thickness of pomegranate-chitosan-based films

235 236 237 238 239 239 239 240 242 243 244 245

7.6.2 Moisture content of pomegranate-chitosan-based films 7.6.3 Water solubility of pomegranate-chitosan-based films

245 245

7.6.4 Swelling property of pomegranate-chitosan-based films 7.6.5 Water vapor and oxygen permeability properties of pomegranate-chitosan-based films 7.6.6 Optical property of pomegranate-chitosan-based films

246

7.6.7 Mechanical property of pomegranate-chitosan-based films 7.6.8 Thermal property of pomegranate-chitosan-based films 7.6.9 Morphology property of pomegranate-chitosan-based films

248 249 249

7.6.10 Thermogravimetric analysis and differential scanning calorimetry of pomegranate-chitosan-based films

246 247

250

7.7 Functional properties of pomegranate-chitosan-based films 7.7.1 Phenolic content of pomegranate-chitosan-based films

250 250

7.7.2 Antioxidant property of pomegranate-chitosan-based films 7.7.3 Antimicrobial property of pomegranate-chitosan-based films 7.8 Degradation of pomegranate-chitosan-based films

251 251 252

7.9 Application and effects of pomegranate-chitosan-based films on foods 7.9.1 Application of pomegranate-chitosan-based films in fruits and vegetables

253

7.9.2 Application of pomegranate-chitosan-based films in the meat and seafood industry

256

7.10 Conclusion References

253

260 260

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8. Chitosan: a potential antimicrobial agent to enhance microbial safety and shelf life of salad dips and ethnic foods

xi

269

Amin N. Olaimat, Murad Al-Holy, Anas A. Al-Nabulsi and Tareq M. Osaili 8.1 Introduction 8.2 In vitro antimicrobial activity of chitosan against spoilage and pathogenic microorganisms 8.3 Antimicrobial activity of chitosan in fresh produce and salads 8.4 Antimicrobial activity of chitosan in meat and poultry products 8.5 Antimicrobial activity of chitosan in juices 8.6 Antimicrobial activity of chitosan in dips and pastes 8.7 Antimicrobial activity of chitosan in dairy products 8.8 Conclusions References

9. Chitosan and hurdle technologies to extend the shelf life or reassure the safety of food formulations and ready-to-eat/cook preparations/meals

269 270 271 272 272 273 274 276 277

279

Vasiliki I. Giatrakou, Rameez Al-Daour and Ioannis N. Savvaidis 9.1 Introduction 9.2 Chitosan applications to extend the shelf life or reassure the safety of food products, and ready-to-eat/cook preparations/meals 9.3 Applications of chitosan on food formulations, based on bioactive packaging 9.3.1 Utilization of chitosan as a food additive directly incorporated into meat formulations 9.3.2 Application of chitosan as bioactive edible coating or liquid (acid-soluble) additive applied onto food preparations (dipping, spraying, or blending) 9.4 Applications of chitosan and/or bioactive coatings in combination with essential oils on ready-to-eat/cook preparations/meals 9.5 Application of edible chitosan solutions or bioactive chitosan coatings in combination with lysozyme on preserving food quality and safety on ready-to-eat/cook preparations/meals 9.6 Application of chitosan as an active packaging material for controlled release of active compounds on food models and on real food systems Acknowledgments References Index

279 280 284 286

289 298

311

318 323 323 327

List of contributors Fateme Akrami Mohajeri Infectious Diseases Research Center, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Irannces, Yazd, Iran; Department of Food Hygiene and Safety, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Rameez Al-Daour Department of Environmental Health Sciences, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates Murad Al-Holy Department of Clinical Nutrition and Dietetics, Faculty of Applied Medical Sciences, The Hashemite University, Zarqa, Jordan Anas A. Al-Nabulsi Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, Jordan Alessandra Silva Araújo Postgraduate Program in Nutrition, Federal University of Pernambuco, Recife, PE, Brazil Marcos Antonio Barbosa de Lima Department of Biology, Federal Rural University of Pernambuco, Recife, PE, Brazil Lúcia Raquel Ramos Berger Postgraduate Program in Environmental Sciences, Federal University of the Agreste of Pernambuco, Garanhuns, PE, Brazil Gerlane Souza de Lima Postgraduate Program in Nutrition, Federal University of Pernambuco, Recife, PE, Brazil Kataryne Árabe Rimá de Oliveira Laboratory of Food Microbiology, Department of Nutrition, Health Sciences Center, Federal University of Paraíba, João Pessoa, Paraiba, Brazil Jossana Pereira de Sousa Guedes Department of Agroindustrial Management and Technology, Federal University of Paraiba, Bananeiras, Paraiba, Brazil Evandro Leite de Souza Laboratory of Food Microbiology, Department of Nutrition, Health Sciences Center, Federal University of Paraíba, João Pessoa, Paraiba, Brazil Hadi Ebrahimnejad Department of Food Hygiene and Public Health, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran Nariman El Abed Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology (INSAT), University of Carthage, Tunis, Tunisia xiii

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List of contributors

Ana Elizabeth Cavalcante Fai Postgraduate Program in Food and Nutrition, State University of Rio de Janeiro (UERJ), Rio de Janeiro, RJ, Brazil; Basic and Experimental Nutrition, Federal University of the State of Rio de Janeiro (UNIRIO), Rio de Janeiro, RJ, Brazil Karina Felix Dias Fernandes Laboratory of Food Microbiology, Department of Nutrition, Health Sciences Center, Federal University of Paraíba, João Pessoa, Paraiba, Brazil Rodrigo França Department of Restorative Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada Vasiliki I. Giatrakou Hellenic Research and Innovation Center, Athens, Greece Daniel Hernandez-Patlan Laboratory 5: LEDEFAR, Multidisciplinary Research Unit, National Autonomous University of Mexico-Superior Studies Faculty at Cuautitlan, Cuautitlan Izcalli, Mexico; Nanotechnology Engineering Division, Polytechnic University of the Valley of Mexico, Tultitlan, Mexico Xochitl Hernandez-Velasco Department of Avian Medicine and Zootechnics, College of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico, Mexico City, Mexico Layal Karam Human Nutrition Department, College of Health Sciences, QU Health, Qatar University, Doha, Qatar Elham Khalili Sadrabad Infectious Diseases Research Center, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Irannces, Yazd, Iran; Department of Food Hygiene and Safety, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Marciane Magnani Laboratory of Microbial Processes in Foods, Department of Food Engineering, Center of Technology, Federal University of Paraíba, Campus I, João Pessoa, Paraiba, Brazil Angy Mallah Department of Nursing and Health Sciences, Faculty of Nursing and Health Sciences, Notre Dame University-Louaize, Zouk Mosbeh, Lebanon Amin N. Olaimat Department of Clinical Nutrition and Dietetics, Faculty of Applied Medical Sciences, The Hashemite University, Zarqa, Jordan Tareq M. Osaili Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, Jordan; Department of Clinical Nutrition and Dietetics, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates

List of contributors

xv

Fatih Özogul Department of Seafood Processing Technology, Faculty of Fisheries, Cukurova University, Adana, Turkey Nikheel Bhojraj Rathod Department of Post Harvest Management of Meat, Poultry and Fish, Post Graduate Institute of Post-Harvest Management, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Roha, Raigad, Maharashtra, India Ioannis N. Savvaidis Department of Environmental Health Sciences, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates; Department of Chemistry, Laboratory of Food Chemistry and Food Microbiology, University of Ioannina, Ioannina, Greece Bruno Solis-Cruz Laboratory 5: LEDEFAR, Multidisciplinary Research Unit, National Autonomous University of Mexico-Superior Studies Faculty at Cuautitlan, Cuautitlan Izcalli, Mexico; Nanotechnology Engineering Division, Polytechnic University of the Valley of Mexico, Tultitlan, Mexico Thayza Christina Montenegro Stamford Postgraduate Program in Nutrition, Federal University of Pernambuco, Recife, PE, Brazil Guillermo Tellez-Isaias Department of Poultry Science, University of Arkansas, Fayetteville, AR, United States Jina Yammine CNRS, INRAE, Centrale Lille, UMR 8207 – UMET – Materials and Transformations Unit, University of Lille, Lille, France

Preface Chitosan is commercially produced from chitin, recognized as a promising biomaterial for use in the fields of biomedical engineering, food packaging, cosmetics, and agriculture due to its good biodegradability, biological nontoxicity, and film-forming properties. Chitosan possesses antibacterial and antifungal properties and has been extensively studied as a potential natural antimicrobial agent in the pharmaceutical, cosmetic, agricultural, and food industries. Over the years, there has been a rapid increase in research focusing on biodegradable antimicrobial packaging. In this regard, chitosan has been shown to be the optimal candidate to use in making bio-based antimicrobial packaging. Besides its inherent antimicrobial capacity and superior film-forming abilities, chitosan has other desirable attributes, including being natural, biocompatible, and nontoxic. Chitosan can form edible films or coatings on food, the main function of which is to protect the product from external factors. Films made from natural polymers form a good barrier against oxygen and are characterized by high mechanical properties. Chapter 1 deals with one of the most promising applications of chitosan is the preparation of chitosan-based films and coatings, which have emerged as an effective and eco-friendly way to extend the shelf life of perishable foods. Chitosan-based films might be a sustainable alternative to eco-unfriendly synthetic plastics after the incorporation of various components, for example, plant extracts capable of enhancing the antioxidant and/or antimicrobial properties and therefore protective capacity of foods. So far, numerous plant extracts have been studied regarding their effects on techno-functional characteristics of the packaging films intended for food protection. As natural plant extracts possess antimicrobial and antioxidant properties, incorporation into chitosan-based films could contribute to a shelf life extension of perishable foods. Interest in producing films from renewable and biodegradable polymers, such as polysaccharides, has increased in recent years, and this is the main discussion of Chapter 2. The massive and uncontrolled use of food packaging derived from petroleum-based plastics has created a serious environmental problem. Hence, the food packaging industry needs to develop packaging from biodegradable polymers. Among the many raw materials studied in the literature, chitosan is one of the most abundant polysaccharides in the xvii

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nature. Hence, chitosan especially combined with active food packaging has gained attention and offers an alternative to the conventional plastics used in the food industries, as numerous studies conducted so far have shown. Chapters 3 and 4 deal with chitosan nanoparticles as used against food pathogens and in combination with essential oils in food preservation. The consumer demand for preservative-free foods, along with tightened legislation regarding current synthetic or chemical preservatives, has led to increased research into the incorporation of “naturally derived” antimicrobials into food packaging. The recent years have witnessed an astronomical increase in the number of studies focusing on utilizing essential oils. Additionally, essential oils as antimicrobial agents have successfully been incorporated into biodegradable films, which allowed for controlling microbial growth on food surfaces besides prolonging product shelf life and ensuring its quality. It is widely known that chitosan is an advanced biomaterial for antimicrobial packaging to meet the growing needs of safe and biodegradable packaging. The application of natural essential oils as antimicrobial agents effectively controls the growth of spoilage and pathogenic microbes. Thus chitosan edible coatings and films incorporated with essential oils have expanded the general applications of antimicrobial packaging in food products. With the rising demand for fresh and ready-to-eat foods, while at the same time consumer demand and also concern, poses a challenge in the food processing industries to both maintain food quality and reassure safety. To meet such demand, the use of nanoparticles combined with chitosan and/or with added essential oils’ potential antimicrobial packaging has been suggested and developed to control or prevent microbial spoilage or growth of potentially found pathogenic bacteria in foods. Nanomaterials/nanoparticles are currently involved in most human activities such as nutritional, agricultural, industrial, biomedical, and pharmaceutical fields. Nanoencapsulation and nanoconjugation of chitosan with natural antimicrobial agents, including essential oils, can provide many beneficial advantages, in terms of an improvement in food safety, and in the shelf life extension of perishable food products, as essential oils are generally recognized as safe compounds for application in human foods, therefore offering new possibilities for food-related applications. Essential oils (such as eugenol) incorporated in chitosan nanoparticles, as shown in recently conducted studies, exhibit potent bioactivities, which enable their application as antibacterial, antioxidant, antiinflammatory, preservative, and antifungal agents. In the last years research studies have

Preface

xix

highlighted the potential applications of chitosan-essential oils’ composite films or coatings in antimicrobial packaging for food preservation. One of the current challenges faced by the food industry involves the production of natural foods, safer and with extended shelf life. Consumers and public health agencies are concerned about the adverse toxicological effects of using synthetic food additives. Among the critical issues involved in this context are microbial growth, enzymatic browning, and lipid oxidation, among others, in food products. Thus the search for safe, bioactive, and natural additives has attracted the attention of researchers. Recent trends in new concepts of chitosan-based nanoparticles and other forms of chemical modification of this biopolymer with novel properties and applications in the food industry have also been in evidence. Besides the biological potential of chitosan, another aspect that appeals to more attention is the possibility of using waste from the fishing industry precisely through the production of chitosan. This possibility of obtaining a natural additive as a value-added product from an agro-industrial residue appears to be an interesting environmental strategy, aligned with the principles of green chemistry and circular economy. Chapter 5 reviews chitosan and its derivatives’ main sources and techniques for production, focusing on the antimicrobial activity of chitosan, and discusses also diverse forms of adding chitosan in food matrices and how it can promote shelf life extension in these products. Seafood is a rich source of nutrients known to improve consumer well-being based on their nutraceutical value, and they are also highly prone to spoilage by microorganisms and oxidation. Considering the recent advances in chitosan extraction and standardization of process has led to the production of uniform quality. Besides, chitosan production addresses the issue of waste management associated with shrimp processing. Due to their natural origin, nontoxic nature, and ability to inhibit oxidation and microbial growth, chitosan has become popular application for preservation of seafood. Chapter 6 focuses on an overview of chitosan source and extraction, bioactivity and health benefits, as well as novel nanotechnological approaches associated with chitosan and its application in different forms and techniques for preservation and quality retention of seafood. Introducing novel food packaging with plant-based bioactive agents brings the refreshing essence of nature to the food industry. Chitosan film with gel and porous characteristics could trap bioactive compounds of pomegranate and could be a good choice for application in food packaging and alternative to nonrenewable sources. Chapter 7 attempts to give

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an overview of state-of-the-art research studies conducted on chitosan in combination with pomegranate-based films and discusses applications in foods. Chapter 8 attempts to provide recent data and information, although limited to date, on chitosan as an antimicrobial agent to increase the shelf life of salad dips and ethnic foods. As chitosan is a natural antimicrobial agent and as studies have shown, if added at controlled concentrations, it to be a pleasant addition to foods, having a citrus “lemony” zest, it could appeal to consumers seeking natural and home taste like foods, without the presence of chemical preservatives, that are also safe. Ethnic or traditional foods are finding nowadays on global restaurant menu and are challenging continuously, both the well-informed or the less experienced consumer, therefore chitosan added to ethnic and or traditional foods is likely to appeal in new demands as the food is growing worldwide due to globalization and ever-increasing immigration rates. Selected examples, especially conducted on traditional salad dips, of Arabic origin, show the effectiveness of chitosan in preserving, extending the shelf life, and in also assuring the safety of foods, for example, “hummus” is known to be potentially contaminated with Salmonella spp., and when combined as part of a “hurdle” technology, even having a greater efficacy, as a natural antimicrobial agent to be used in potential future applications in food processing. Finally, Chapter 9 summarizes limited data available, on chitosan either added itself into foods, or as part of a “hurdle” technology, in both cases such interventions may be potentially of use and interest to food industries, in designing “natural” food formulations, without the toxicity of chemical preservatives, reassuring, the freshness, shelf life, as well as the safety of foods (ready-to-eat, ready-to-cook, new food formulations). Its use however, as to the present writing, is limited, as further data are needed to establish it, as a safe “natural|” food additive, and also to convince the consumer that its controlled addition may impart a challenging new taste or flavor to existing foods, worth experiencing by the traditional consumer or those seeking a new innovative food formulation. The demand for fresh, ready-to-eat, or semifinished foods is increasing, and the need to maintain food safety and quality further exacerbates the challenges in the supply chain, especially with the globalization of food trade and the use of centralized processing facilities for food distribution. Hence, food products with a prolonged shelf life are needed to reach the largest possible market segments. Also, the potential for spoilage and/or

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pathogenic microorganisms to contaminate food products remains a significant concern, negatively affecting shelf life, and increasing foodborne illness risk. Several traditional food preservation methods that are currently in use can provide some level of preservation but can also negatively impact food quality by reducing its nutritional value. For these reasons, alternative and innovative strategies are required to overcome the challenges and to guarantee food safety and quality, and this hopefully may be done with application of chitosan, as a natural antimicrobial agent, either added itself or in combination with foods. However, as interactions of chemical food constituents are complex, further studies and data will be needed on chitosan activity to be used more in commercial products, and research efforts should be targeted to define the exact mechanism of chitosan’s biological activities. Ioannis N. Savvaidis

CHAPTER 1

Application of chitosan in active food packaging Layal Karam1 and Angy Mallah2 1

Human Nutrition Department, College of Health Sciences, QU Health, Qatar University, Doha, Qatar Department of Nursing and Health Sciences, Faculty of Nursing and Health Sciences, Notre Dame University-Louaize, Zouk Mosbeh, Lebanon

2

1.1 Introduction Food packaging has an essential role in protecting the food from external and environmental factors such as heat, oxidation, and light. Improvements in food packaging studies have created active packaging that aims at extending perishable foods’ shelf life, preventing contamination as well as maintaining, or enhancing the quality and the safety of the food products. As stated by the EU Regulation No 450/2009 (Commission of the European Communities, 2009), “active materials and articles means that are intended to extend the shelf life or to maintain or improve the condition of packaged food; they are designed to deliberately incorporate components that would release or absorb substances into or from the packaged food or the environment surrounding the food.” Moreover, active packaging could be an alternative for the addition of active compounds into foods with the related limited efficiency and interactions with the food matrices (Sharma et al., 2021). Such packaging has the ability to decrease foodborne illness outbreaks as well as food recalls (Karam et al., 2013d; Karam et al., 2016; Vilela et al., 2018). Active packaging has two categories: chemoactive and bioactive. The chemoactive packaging is based on the chemical additives, whereas bioactive packaging is based on the incorporation of natural antimicrobial and antioxidant agents (Karam et al., 2013a; Sharma et al., 2021). Nowadays, researcher’s main focus is being dedicated to the development of bioactive packaging, due to consumers’ concerns toward the chemoactive packaging which has hazardous effects on the health and the environment (Karam et al., 2013b). For example, Domínguez et al. (2018) incorporated butylated hydroxyanisole, which is a synthetic antioxidant into active packaging and reported an enhancement in the quality of the food product. This Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00002-8

© 2023 Elsevier Inc. All rights reserved.

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Chitosan: Novel Applications in Food Systems

improvement was attributed to the butylated hydroxy anisole’s antioxidant characteristic in preventing lipid oxidation. However, it was found that butylated hydroxyanisole may induce problems in the human endocrine system (Pop et al., 2013). The use of natural antioxidants and antimicrobials in food preservation is preferable since the consumers nowadays are moving toward additive free products (Karam et al., 2013c; Ibarra-Sánchez et al., 2020). Hence, researchers have started to look for natural alternatives, where chitosan is taking precedence among all the numerous bio-based materials, due to its antimicrobial and antioxidant characteristics. Chitosan is a linear polysaccharide made of randomly distributed β-(1
4)-linked D-glucosamine and N-acetyl-D-glucosamine and produced by the deacetylation of chitin (Siripatrawan, 2016). Chitosan is also a bio-based eco-friendly material (Bégin & Van Calsteren, 1999) and has a promising future in active packaging as a food preservative material (Siripatrawan, 2016). This chapter focuses on the application of chitosan in active food packaging. Antioxidant and antimicrobial properties of chitosan-based films combined with free or encapsulated antimicrobials and antioxidant agents have been discussed. In addition, this chapter combines the recent advances in the use of chitosan in active food packaging.

1.2 Active chitosan-based packaging—antimicrobial properties Chitosan’s antimicrobial activity is very broad and exhibits inhibition against Gram-positive and Gram-negative bacteria, fungi, as well as yeasts. According to the literature, this activity is explained by the interaction between the amino groups of chitosan and the negatively charged bacterial cell membrane, which increases the permeability of the cell wall leading to the leakage of intracellular components (Siripatrawan, 2016). Many studies have tested the antimicrobials properties of chitosan-based packaging (Table 1.1). For example, it has been reported that the antimicrobial activity of chitosan (CS)—pure poly (vinyl alcohol) (PVA) films was strongly attributed to the antibacterial property of CS since PVA did not display any inhibition zone against tested bacteria (Liu et al., 2018; Narasagoudr et al., 2020a; Tripathi et al., 2009). Pure poly (vinyl alcohol) (PVA) is a relatively harmless and eco-friendly synthetic polymer that has great miscibility and film forming characteristics. PVA can be utilized to immobilize chitosan through the development of hydrogen bonds and

Table 1.1 Recent studies about active chitosan-based packaging with antimicrobial properties. Packaging material

Functional ingredient

Concentration

Design

Target microorganism

Application

References

Poly (vinyl alcohol) (PVA)

Chitosan (CS)

Extruded composite film

Staphylococcus aureus, Escherichia coli

Culture broth

Liu et al. (2018)

Poly (vinyl alcohol)

Chitosan

PVA:CS w/w: 80:20, 75:25, 70:30, 65:35 1% w/w

Casting method

E. coli, S. aureus, Bacillus subtilis

Tripathi et al. (2009)

Poly (vinyl alcohol)

Quaternary ammonium chitosan Chitosan

1%, 2%, 3% w/w

Solution casting method E. coli, S. aureus, Botrytis cinerea

Minimally processed tomato into two forms:(1) as a whole and (2) into two pieces Whole strawberries

1%, 3%, 5% w/w

Melt mixing compression molding

Chitosan oligomers

0.12 g

Multilayer thermocompressing

Plasticized poly (lactic acid)

Thermoplastic corn starch

Aspergillus Potato dextrose agar brasiliensis, mediumSoya casein Penicillium digest lecithin corylophilum, polysorbate broth Fusarium graminearum, S. aureus, E. coli Molds, yeast 3M Petrifilm count plates Fresh whole strawberries, whole moldy ricotta, Mignon breads with cheese flavor

Min et al. (2020) Râp˘a et al. (2016)

Castillo et al. (2017)

4

Chitosan: Novel Applications in Food Systems

thus enhance the mechanical properties of chitosan (Liu et al., 2018). Liu et al. (2018) found that the optimal PVA/CS film was with 75:25 ratio, which demonstrated the highest antibacterial efficacy for inhibiting Staphylococcus aureus (99.16% 6 6.58%) and Escherichia coli (98.8% 6 4.56%) growth. Similarly, Tripathi et al. (2009) tested CS-PVA films on the preservation of minimally processed tomato into two forms: (1) as a whole and (2) into two pieces. The CS-PVA 1 wt.% films demonstrated a higher inhibitory zone capacity against E. coli (1.5 cm) and Bacillus subtilis (1.4 cm) than S. aureus (1.2 cm). In another study, quaternary ammonium chitosan HACC, a chitosan derivative, and PVA coatings were designed and evaluated for their antibacterial and antifogging activities on the storage of strawberries at 25°C for 5 days. Results showed that the increase in HACC content successfully extended the shelf life of strawberries, by retaining their original color, flavor, and freshness (Min et al., 2020) (Table 1.1). Moreover, antifungal and antibacterial activities of plasticized poly (lactic acid) (PLA) films were also enhanced with the incorporation of chitosan (Râp˘a et al., 2016). The biocomposite films containing 1 and 3 wt.% of chitosan showed a significant bacterial count reduction which was higher in E. coli (5.54
5.58 log units, respectively) than in S. aureus (2.7
2.8 log units, respectively) (Table 1.1). According to the literature, PLA film had no capacity to inhibit bacterial growth but is considered as an excellent bio-based and green polymer utilized as a short duration film for the packaging of food (Niu et al., 2018). Moreover, it was concluded that 1 wt.% of chitosan ensured a satisfactory antifungal activity, with no necessary need in increasing its concentration (Râp˘a et al., 2016) (Table 1.1). To summarize, 1 wt.% of chitosan was denoted as the optimum concentration for the antimicrobial properties of the films. Likewise, Castillo et al. (2017) in their study developed biodegradable films by combining chitosan oligomers (CO) and thermoplastic cornstarch (TPS) and tested its antifungal activity on different food products. The films reduced yeasts growth by B58% in strawberries and ricotta, and by 86% in flavored breads (Table 1.1).

1.3 Active chitosan-based packaging combined with functional ingredients 1.3.1 Antimicrobial properties Many studies investigated the abilities of chitosan-based films combined with free antimicrobials for their future use as active food packaging (Table 1.2).

Table 1.2 Recent studies about antimicrobial active chitosan-based packaging combined with free functional ingredients. Packaging material

Functional ingredient

Concentration

Design

Target microorganism

Application

References

Chitosan/poly (vinyl alcohol)

Rutin

0.2%, 0.4%, 0.6% w/w

Escherichia coli, Staphylococcus aureus

Agar plates

Narasagoudr et al. (2020b)

Chitosan, poly (vinyl alcohol)

Boswellic acid

0.2%, 0.4%, 0.6%, 0.8% w/w

S. aureus, E. coli, Candida albicans

Agar plates

Narasagoudr et al. (2020a)

Chitosan-polyvinyl Sodium lactate alcohol/ montmorillonite, polyethylene Chitosan/gelatin Lauroyl arginate ethyl

1%, 3%, 5%, 8%, 10%, 20% w/w

Film forming binary solution blend Solvent casting technique Film forming solution blend Solvent casting technique Coating method

E. coli

E. coli culture broth

Zhang et al. (2017a)

Film forming solution blend Film casting Bilayer films Film forming solution blend Film casting

Listeria monocytogenes, BHIAa plates E. coli, Salmonella typhimurium, Campylobacter jejuni Listeria monocytogenes BHIAa agar E. coli, S. platesBHIBb typhimurium, C. jejuni S. aureus, E. coli Molten agar

0.1% v/v

Chitosan-polyvinyl alcohol

Ethyl lauroyl arginate

Triazole betaine chitosan

Succinyl 3% w/v chitosan sodium salt Grapefruit seed 0.5%, 1.0%, 1.5% extract v/v

Chitosan

a

BHIA: brain
heart infusion agar. BHIB: brain
heart infusion broth.

b

1%, 2.5%, 5%, 10% w/w

Film forming solution blend Film casting Film forming solution blend Film casting

Fungi

Bread slices

Haghighi et al. (2019) Haghighi et al. (2020) Kritchenkov et al. (2020) Tan et al. (2015)

6

Chitosan: Novel Applications in Food Systems

Narasagoudr et al. (2020b) prepared rutin (Rt—known as a plant pigment)-induced CS/PVA bioactive films and reported that the addition of rutin had a strong effect on the antimicrobial capacity of CS/PVA films, which was due to the acidic nature of the hydroxyl groups of Rt. Furthermore, in another study conducted by Narasagoudr et al. (2020a), Boswellic acid (BA) was added to CS/PVA films (CPBA) and reported that CS and BA had a synergistic antibacterial effect on the films. They also reported the increase of BA content and the inhibitory zone efficacy of CPBA active films, highlighting that the CPBA film containing 0.8% of BA showed the maximum inhibitory effects (mm) in Candida albicans (28 6 1), E. coli (26 6 1.5), and S. aureus (26 6 1). In their turn, Zhang et al. (2017a) developed sodium lactate loaded CS-PVA/montmorillonite films and reported an improved inhibition efficacy (%) of the films against E. coli which significantly increased to 70% 6 2.1% with NaL content of 5 wt.% at 37°C for 24 hours (Table 1.2). According to the literature, lauroyl arginate ethyl (LAE) is one of the most effective antimicrobial components among new food additives with significant and fast antimicrobial abilities against foodborne microorganisms when put directly in contact with these pathogens. LAE revealed a high antimicrobial activity, which was attributed to its cationic nature that disrupts the metabolic processes of a bacterial cell and inhibits its growth leading to its death (Haghighi et al., 2019). It was reported that the incorporation of LAE into films containing chitosan and other components demonstrated an enhanced antimicrobial performance and inhibited the growth of Listeria monocytogenes, E. coli, Salmonella typhimurium, and Campylobacter jejuni (Haghighi et al., 2019; Haghighi et al., 2020) (Table 1.2). Moreover, Kritchenkov et al. (2020) mixed triazole betaine chitosan (TBC) with succinyl chitosan sodium salt (SC-Na) and tested its antimicrobial efficacy with different ratios. Results showed that 1:4 TBC: SCNa ratio performed the highest antibacterial activity where the inhibition zones (mm) around S. aureus (36.9 6 0.23) were higher than those of the E. coli (28.2 6 0.37) (Kritchenkov et al., 2020) (Table 1.2). However, few studies are currently available on food applications. Interestingly, Tan et al. (2015) tested the antimicrobial capacity of active chitosan-based film combined with grapefruit seed extract (GFSE) on bread application. It was reported that the films containing 1.5% v/v GFSE in chitosan-based film strongly inhibited fungal growth in bread samples, and fungi were only detected on day 10 (Table 1.2).

Application of chitosan in active food packaging

7

1.3.2 Antioxidant properties Oxidation is an irreversible process that causes deterioration of food products during their processing and storage and alters their nutritional and organoleptic properties. Mainly, foods that are rich in fatty acids are more prone to oxidation particularly known as lipid oxidation. The lipid oxidation process causes discoloration, rancid odor and flavor, development of toxic components, loss of nutrients, and alterations in the texture of the food product. Hence, since it is important to prevent food oxidation, lots of research works have tested the effectiveness of chitosan combined with natural materials in active packaging as an alternative of chemical additives, to ensure the quality of the food products (Sharma et al., 2021). It has been reported that chitosan’s antioxidant property is associated with its scavenging capacity on hydroxyl groups and chelating capacity on ferrous ions. Moreover, this activity is attributed to its efficient chelation that inhibits lipid oxidation by binding to metal ions (Siripatrawan, 2016). Recently, incorporating antioxidant agents from plant extract with active chitosan-based packaging has been examined Agarwal et al. (2021). For example, Agarwal et al. (2021) reported that Larix decidua Mill. Bark incorporated into chitosan-based film exhibited a significant antioxidant activity, which was due to the phenolic components present in bark extract (the total phenolic content reached 894 mg equivalents of gallic acid (GAE)/g dw in chitosan-based film containing 9% of bark extract, while it was 90 mg GAE/ g dw in the control film). Moreover, Bigi et al. (2021) in their study tested the antioxidant activity of nettle leave extract (NLE) and sage leave extract (SLE) blended into chitosan/hydroxypropyl methylcellulose (CS/HPMC) film. They reported that both extracts impressively improved the antioxidant performance of the film; however, CS/HPMC film containing 15% w/w SLE (28.35 μmol TE/g film) exhibited a higher capacity compared to the CS/HPMC film loaded with 15% w/w NLE [17.08 μmol Trolox Equivalent (TE)/g film], which was due to the higher polyphenolic content in SLE (Bigi et al., 2021) (Table 1.3). Similarly, da Rosa Almeida et al. (2022) reported that the antioxidant capacity of chitosan/poly (vinyl alcohol) films increased with the increase in hop (Humulus lupulus L. var. Cascade) extract content in the films (Table 1.3). Similarly, pine needle extract, Ficus carica Linn leave extract, and Aralia continentalis Kitagawa root extract combined with chitosan-based films, or separately, were found to be have a promising potential as antioxidant agents in active food packaging (Kadam et al., 2021;Yilmaz et al., 2022; Wu et al., 2022) (Table 1.3).

Table 1.3 Recent studies about antioxidant properties of active chitosan-based packaging combined with free functional ingredients. Packaging material

Functional ingredient

Concentration

Design

Antioxidant test

Food application

References

Chitosan

Larix decidua Mill. bark

3%, 6%, 9% w/w

Casting method

DPPHa TPCc

NAe

Chitosan/ hydroxypropyl methylcellulose

Nettle leave extract (NLE)Sage leave extract (SLE)

Casting method

TEACd TPCc

NAe

Chitosan/poly (vinyl alcohol)

Hop (Humulus lupulus L. var. Cascade) extracts

7.5%,15% w/w NLE 7.5%,15% w/w SLE 10%, 20%, 40% (v/v)

Agarwal et al. (2021) Bigi et al. (2021)

Casting method

DPPHa ABTSb

NAe

Chitosan

Pine needles (Cedrus deodara) extract

5%, 10%, 20% v/w

DPPHa ABTSb TPCc

NAe

Chitosan

Ficus carica Linn leaves extract

2%, 4%, 6% w/w

Film forming solution blend Casting method Film forming solution blend Casting method

DPPHa TPCc

NAe

da Rosa Almeida et al. (2022) Kadam et al. (2021)

Yilmaz et al. (2022)

Aralia continentalis Kitagawa root extract Hawthorn fruit extract

0.05%, 0.10%, 0.15% v/w 0%, 2%, 4%, 6% w/w

Chitosan

Lycium barbarum fruit extract

0.20, 0.40, 0.60, 0.80, 1.00 g

Chitosan

Mango leaf extract

1%,3%, 5% w/w

Chitosan and glycerol

Citric acid

2% w/w

Chitosan

Chitosan

a

DPPH: 1, 1-Diphenyl-2-picrylhydrazyl. ABTS: 2,20 -Azino-bis (3-ethylbenzothiazo-line6-sulfonic acid). c TPC: total phenolic compounds. d TEAC: Trolox equivalent antioxidant capacity. e TPC: total phenolic compounds. b

Composite film

DPPHa ABTSb

NAe

Wu et al. (2022)

Film forming solution blend Film forming solution blend Film casting Casting method

DPPHa

NAe

Kan et al. (2019)

DPPHa

NAe

Wang et al. (2015)

DPPHa ABTSb TPCc Ferric reducing antioxidant power assay Hydrogen peroxide radical scavenging assay

Crushed cashew nuts

Rambabu et al. (2019)

Green chili

Priyadarshi et al. (2018)

Solvent casting method

10

Chitosan: Novel Applications in Food Systems

Moreover, studies showed that hawthorn fruit extract and Lycium barbarum fruit extract (LFE) added separately to a chitosan-based films significantly enhanced the antioxidant activity of each of the films due to the synergistic antioxidant effect exhibited by each fruit when combined with chitosan-based film (Kan et al.,2019; Wang et al.,2015). For example, it was reported that chitosan films with 6 wt.% of hawthorn fruit extract exhibited a significant DPPH radical scavenging activity that was increased from 33.42% to 84.40% at 5 mg/mL (Kan et al.,2019). Furthermore, it was found that the DPPH scavenging activity of LFE to chitosan, with a 1:1 weight ratio, attained 35.8%, which was 10-fold higher than the chitosan control film (3.7%) (Table 1.3). Due to the promising results and synergistic effect of the combination of chitosan-based films with free antioxidant functional ingredients, various studies tested these films in food applications. For example, Rambabu et al. (2019) investigated the antioxidant properties of chitosan films incorporated with mango leaf extract (MLE) in preservation of cashew nuts (Table 1.3). Cashew nuts were stored for 28 days in the active packaging films and showed a higher oxidation resistance (56%) for the 5% MLE film compared to commercial polyamide/polyethylene film. It was concluded that chitosan and MLE demonstrated a synergistic effect on the antioxidant activity of the films. Moreover, Priyadarshi et al. (2018) reported that chitosan films incorporated with citric acid and glycerol showed a 2.5-fold improvement in antioxidant performance as compared to chitosan films only. In addition, these films preserved the shelf life of green chilies and retained their quality during a period of 7 days at 27°C (Table 1.3).

1.3.3 Antimicrobial and antioxidant properties Various natural extracts, such as apple peel polyphenols (Riaz et al., 2018), Piper betle Linn. leaf extract (Thuong et al., 2019), Chinese chive root extract (Riaz et al., 2020), honeysuckle flower extract (HFE) (Wang et al., 2017), ellagic acid (Vilela et al., 2017), procyanidin (Zhang et al., 2021), amaranth extract (Hu et al., 2020), kombucha tea (Ashrafi et al., 2018), and propolis (Rollini et al., 2017), have been examined to enhance the antioxidant and antimicrobial characteristics of chitosan-based films. Riaz et al. (2018) evaluated the antimicrobial and antioxidant activities of apple peel polyphenols (APP) incorporated into chitosan and reported that both activities were improved with the increase in APP

Application of chitosan in active food packaging

11

concentration. For example, chitosan films’ scores containing 1% APP were 60% and 90% more than the control films’ scores in the DPPH and ABTS assays, respectively. Moreover, these films exhibited the highest inhibitory effect as compared to the other chitosan-based films with lower APP concentration (0.25%, 0.50%, 0.75% w/w) and they had the following different efficiency against the tested bacteria: Bacillus cereus (19.46 6 0.36 mm) . S. aureus (18.11 6 0.36 mm) . E. coli (16.12 6 0.26 mm) . S. typhimurium (14.47 6 0.28 mm) (Table 1.4). Also, it was found that Piper betle Linn. leaf extract (PBLLE) incorporated into chitosan films recorded a significant antimicrobial and antioxidant activity that was proportional to the increase in PBLLE content. Chitosan films containing 3% PBLLE significantly prevented bacterial growth, particularly the growth of S. typhimurium and B. subtilis which were strongly inhibited after the first 6 hours and held for 24 hours even with PBLLE concentration as low as low 1% (Thuong et al., 2019) (Table 1.4). Moreover, Chinese chive root extract (CRE)-chitosan (CS) films with 5%w/w CRE showed improved antioxidant and antimicrobial activities compared to pure CS films (Riaz et al., 2020). The addition of CRE increased its DPPH and ABTS radical scavenging capacity from 6.95% to 47.05% and 11.98% to 57.38%, respectively. Moreover, the CS-CRE films significantly reduced the soybean oil oxidation [the peroxide value of the control sample was 69.23 6 1.38 mEq/kg, while for CS-CRE with 5%w/w CRE it was 31.16 6 1.43 mEq/kg for 28 days of storage (Riaz et al., 2020) (Table 1.4)]. In addition, the CS-CRE films inhibited the growth of B. cereus, S. aureus, E. coli and S. typhimurium, highlighting that this antimicrobial activity was also increased with the increase in CRE concentration. In another study, Wang et al. (2017) developed chitosan and HFE films to be used for active food packaging. Results showed that chitosan films containing 30% HFE induced a significant increase of 95% in DPPH free radical scavenging activity which was twice the score of the pure chitosan film. Furthermore, this film forming solution blend exhibited a wider inhibitory zone against E. coli (127.36 mm2) than the film (36.77 mm2) with 0% HFE (Table 1.4). Furthermore, Vilela et al. (2017) found that chitosan (CH) and ellagic acid (EA) films stopped the growth of both Pseudomonas aeruginosa and S. aureus bacterial strains after 24 hours of incubation and their antioxidant activity was detectable with EA content .0.5 wt.% (it increased from

Table 1.4 Recent studies about antimicrobial and antioxidant properties of active chitosan-based packaging combined with free functional ingredients. Packaging material

Functional ingredient

Concentration

Design

Target microorganism

Antioxidant test

Application

References

Chitosan

Apple peel polyphenols

0.25%, 0.50%, 0.75%, 1.0% w/w

Film forming solution blend Casting method

DPPHa ABTSb

Nutrient broth

Riaz et al. (2018)

Chitosan

Piper betle Linn. leaf extract

1%,2%, 3%v/v

Film forming solution blend

DPPHa

Clonogenic assay

Thuong et al. (2019)

Chitosan

Chinese chive root extract

1%, 3%, 5%w/ w

Film forming solution

DPPHa ABTSb

Agar plates Soybean oil packaging

Riaz et al. (2020)

Chitosan

Honeysuckle flower extract

Film forming solution blend

DPPHa

Nutrient agar

Wang et al. (2017)

Chitosan

Ellagic acid

Chitosan

Extracts of the bark (EB), exudate bark (EEB), and leaves (EL) of Cenostigma nordestinum

0,5%, 10%, 20%, 30% w/w 0.5%, 1.0%, 2.5%, 5.0% w/w 100, 200, and 300 μg/mL of EB, EEB, and EL each

Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium E. coli, Pseudomonas aeruginosa, S. typhimurium, Bacillus subtilis, S. aureus B. cereus, S. aureus, E. coli, S. typhimurium E. coli

Homogenization Casting method

P. aeruginosa, S. aureus

DPPHa

Nutrient broth

Vilela et al. (2017)

Bench casting method

S. typhimurium, Listeria monocytogenes, P. aeruginosa, S. aureus, B. cereus, E. coli

DPPHa

BHIf agar medium

Soares et al. (2022)

Chitosan

Procyanidin

Betalains-rich vegetable, quaternary ammonium chitosan/fish gelatin Chitosan

Amaranth extract

Carboxymethylor microfibrillated cellulose, propolis glycolic extract a

Kombucha tea Chitosan (CS) Propolis glycolic extract

0.05%,0.10%, 0.15%, 0.20%, w/w 0%, 5%,10%, 15% w/w

1%,2%, 3% w/w CS: 77 and 157 kDa

DPPH: 1, 1-Diphenyl- 2-picrylhydrazyl. ABTS: 2,20 -Azino-bis (3- ethylbenzothiazo-line6-sulfonic acid). c TBARS: thiobarbituric acid reactive substances. d PCA: plate count agar. e TPC: total phenolic compounds. f BHI: brain
heart infusion. b

Solution casting method Film forming solution blend

Casting method Film forming solution blend Paper sheets

E. coli, S. aureus, DPPHa Aspergillus niger, Rhizopus E. coli, S. aureus, DPPHa S. typhimurium, L. monocytogenes

E. coli and S. aureus Listeria innocua, S. aureus, E. coli, Pseudomonas putida

TBARSc TPCe

PCAd Cheese cubes (about 8 g) Lysogeny broth agar plate Shrimps

Zhang et al. (2021)

Minced beef samples Tryptic soy broth medium Thinly sliced raw veal meat

Ashrafi et al. (2018) Rollini et al. (2017)

Hu et al. (2020)

14

Chitosan: Novel Applications in Food Systems

0.0% to approximately 28% with the increase in EA concentration from 0.5 to 5.0 wt.%). Moreover, the exposure of the CH/EA films under UV irradiation did not alter their antioxidant and antimicrobial activities. Hence, such films can be used as a food packaging material (Table 1.4). Soares et al. (2022) blended extracts from different parts of the Cenostigma nordestinum plant into chitosan films separately. They reported that the extracts taken from the bark exhibited the highest antioxidant performance compared to those extracted from the leaves and exudate bark. In contrast, the exudate bark demonstrated the most significant antimicrobial activity against all tested strains compared to the extracts of the bark, which exhibited the lowest one (Table 1.4). After viewing the promising antimicrobial and antioxidant properties of active chitosan-based packaging combined with free functional agents in lab culture, various studies tested these films in food applications. For example, when chitosan (CS) was combined with procyanidin (PC), the CS
PC composite films retained cheese main sensory characteristics for 14 days at 5°C, hence proving their active properties and ability to decrease quality loss and nutrition attributes of cheese (Zhang et al., 2021) (Table 1.4). The films’ antioxidant activity increased with the increase in PC content and the highest efficiency was reached at 0.20% w/w. Furthermore, CS
PC films with 0.20% w/w procyanidin exhibited a significant inhibition on the growth of E. coli (15.5 6 0.5 mm) and Aspergillus niger (16.3 6 0.5 mm), a weak one on S. aureus growth (12.3 6 0.4 mm) and did not show any noticeable antifungal activity against Rhizopus (4.5 6 0.2 mm). Furthermore, Hu et al. (2020) in their study prepared a multifunctional food packaging in which betalain-rich vegetable amaranth (Amaranthus tricolor L.) extract (AE) was incorporated into quaternary ammonium chitosan (QC)/fish gelatin (FG) blend films. The antimicrobial activity of the films was more efficient against the Gram-negative bacteria (S. typhimurium and E. coli) than the Gram-positive bacteria (L. monocytogenes and S. aureus). In addition, the antioxidant activity of QCFG-AE films was improved mainly due to the free radical scavenging characteristic of betalains in AE and reached 95.8% with QC-FG-AE films containing 15 wt.% of AE at 5 mg/mL while QC-FG films without AE exhibited the lowest activity (6.8% at 5 mg/mL). Furthermore, since the QC-FG-AE showed sensitivity to volatile ammonia, it was suggested that they could be used to monitor the freshness of shrimp (Table 1.4).

Application of chitosan in active food packaging

15

Moreover, it was reported that chitosan and kombucha tea (KT at 3%) films inhibited the growth of S. aureus, prevented lipid oxidation, retarded the formation of the off odor, and extended the shelf life of minced beef up to 6 days during storage at 4°C (Ashrafi et al., 2018) (Table 1.4). Furthermore, Rollini et al. (2017) created paper sheets by combining chitosan and propolis to achieve antibacterial and antioxidant properties. The study results showed that adding chitosan the paper sheets with propolis exhibited more than ten-fold rise in polyphenols retention capacity. Moreover, the chitosan-propolis active paper achieved a reduction in Listeria innocua counts of around 1 log cycle in 48 hours at 4°C and performed the highest antimicrobial activity against S. aureus on packaged thinly sliced raw veal meat compared to unpackaged samples. This was consistent with the data from the literature stating that the antimicrobial activity of propolis is known to have an effect mostly on Gram-positive bacteria (Mascheroni et al., 2014; Siripatrawan & Vitchayakitti, 2016) (Table 1.4).

1.4 Active chitosan-based packaging combined with encapsulated functional ingredients 1.4.1 Metallic nanoparticles encapsulated into chitosanbased films The combination of polymers and metallic nanoparticles in food packaging has shown promising results toward antimicrobial activity. There are two types of antimicrobials, the first type are the organic antimicrobials such as polymers, organic acids, and enzymes, whereas the second type are the inorganic ones such as metallic nanoparticles including zinc, silver, and titanium, as well as their compounds such as oxides, sulfides, and phosphate (Mesgari et al., 2021). The main advantage of the inorganic antimicrobials is their ability to withstand exposure to high temperature levels as compared to a weak thermal stability for the organic substances. According to literature, metallic nanoparticles such as titanium oxide, silver, zinc, and magnesium oxide exhibit a significant biocidal effect against foodborne pathogens. However, it is recommended to employ them at concentrations below their toxicity threshold level. In addition, using metallic nanocomposite as packaging films presents various important advantages especially their antioxidant as well as antimicrobial properties (Mesgari et al., 2021).Titanium oxide (TiO2) nanoparticles naturally occurs when there is a reaction between titanium and oxygen present in

16

Chitosan: Novel Applications in Food Systems

air. This compound exhibits various properties including high capacity to absorb ultraviolet light and an efficient antimicrobial activity. Furthermore, titanium oxide is used in many areas such as food preservation and active food packaging (Mesgari et al., 2021). TiO2 nanoparticles produces reactive oxygen species (ROS) when exposed to UV light along with the presence of oxygen and water easily found in packaged food applications. These ROS disrupt the cell membrane of the microorganism and lead to its death (Salama & Aziz, 2020; Mesgari et al., 2021). For example, a study has reported that the addition of 1% TiO2 nanocomposites to chitosan-based films enhanced the antimicrobial activity against different bacterial strains and fungi (Siripatrawan & Kaewklin, 2018). Also, Zhang et al. (2017b) reported that chitosan-TiO2 nanopowder film totally inhibited the growth of E. coli, S. aureus, C. albicans, and A. niger in 12 hours and preserved red grapes for 22 days at 37°C. Similar results were documented in another study where green bell pepper was coated with optimized carboxymethyl cellulose and guanidinylated chitosan enriched with (nTiO2) films for 15 days. It was shown that the films inhibited microbial growth and prevented the spoilage of pepper during the storage period (Salama & Aziz, 2020) (Table 1.5). Silver nanoparticle (AgNP) was one of the nanoparticles that gained researcher’s attention because of its excellent antimicrobial property against broad variety of microorganisms. However, they discovered that when AgNPs are directly utilized as antimicrobial substances in food packaging, their leakage might cause toxicity to the human body. Thus it is highly recommended to use a component such as laponite to immobilize silver nanoparticles, in order to lower the risk of leakage (Wu et al., 2018) (Table 1.5). According to the literature, AgNPs, laponite, and chitosan have antimicrobial synergistic effects (Wu et al., 2018). AgNPs can alter the cell’s function and induce its leakage by interacting with sulfur-containing proteins in the cell wall. In addition, this nanoparticle can invade the inside of the cell and cause disruption to its metabolism. Moreover, laponite might be able to immobilize microorganism due to its two-dimensional structure (Wu et al., 2018). For example, chitosan, laponite and AgNPs films showed a strong antimicrobial capacity and were able to maintain the freshness of wrapped litchi for 7 days (Wu et al., 2018) (Table 1.5). Moreover, in another study, the shelf life of red grapes could be extended for up to 18 days when chitosan, gelatin, and 2 mM AgNPs films were used during storage at 37°C (Kumar et al., 2018) (Table 1.5).

Table 1.5 Recent studies about active chitosan-based packaging combined with encapsulated functional ingredients. Packaging material

Functional ingredient

Concentration

Design

Target microorganism

Antioxidant test

Application

References

Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, Aspergillus oryzae, Penicillium roqueforti E. coli, S. aureus, Candida albicans, Aspergillus niger Streptococcus pneumoniae, E. coli, Aspergillus fumigatus

Not applicable

Agar plates

Siripatrawan and Kaewklin (2018)

Not applicable

Red grape

Zhang et al. (2017b)

Not applicable

Agar plates Green bell pepper

Salama and Aziz (2020)

S. aureus, E. coli, A. niger, Penicillium citrinum

Not applicable

Wu et al. (2018)

Molds

Not applicable

Beef extract peptone agar medium, potato dextrose agar Fresh litchi Red grape

E. coli, S. aureus

Not applicable

Microbial scanning electron morphology Fresh white crucian carp (Carassius auratus)

Dai et al. (2022)

Metallic nanoparticles encapsulated into chitosan-based films Chitosan

Titanium dioxide nanoparticles (nTiO2)

0%, 0.25%, 0.5%, 1%, 2% w/w

Ultrasonic homogenization Film forming casting solution

Chitosan

nTiO2

0.05 g

Chitosan biguanidine/ optimized carboxymethyl cellulose Chitosan

nTiO2

1%, 3%, 5%w/w

Ultrasonic homogenization films coated with plastic Ultrasonic homogenization casting technique

Laponite immobilized silver nanoparticles

1%, 2%, 5%, 10% w/wcs

Colloidal silver

1 mM (0.05% w/w), 2 mM (0.1% w/w) 1%, 2.5%, 5% w/wcs

Chitosan, gelatin, polyethylene glycol Chitosan

Covalent organic frameworks and silver nanoparticles

Reactive template grain growth method Casting solvent evaporation technique Composite casting method Emulsification ultrasonication casting method

Kumar et al. (2018)

(Continued)

Table 1.5 (Continued) Packaging material

Functional ingredient

Concentration

Design

Target microorganism

Antioxidant test

Application

References

Fish gelatin/ chitosan

Silver-loaded nTiO2

Composite casting method

E. coli, S. aureus, Botrytis cinerea, molds

Not applicable

Agar plates

Lin et al. (2020)

Chitosan

Zinc oxide nanoparticles (Nano-ZnO) loaded gallic acid Mulberry anthocyanin extract

0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% w/v 30, 50, 70 mg

Solution casting method

Bacillus subtilis, E. coli

DPPHa ABTSb

Nutrient broth, agar Petri-plates

Yadav et al. (2021)

0, 30,60, 90 mg/g

Bionanocomposite casting films

E. coli, S. aureus

DPPHa

LBc medium

Sun et al. (2020)

0%, 0.1%, 0.2%, 0.4% w/v 0.1% ZnO (in ethanol) 2% chitosan (in 0.5% acetic acid) 500, 1000 g/mL

Casting method

E. coli, S. aureus Salmonella enterica, E. coli, S. aureus

Lysogeny broth agar Nutrient broth

Liu et al. (2021)

Film forming solution blend

DPPHa ABTSb Not applicable

Basic catalyzed sol-gel technique Bilayer nanocomposite films Ultrasonic homogenization

Listeria monocytogenes

Not applicable

White cheese

Divsalar et al. (2018)

L. monocytogenes, Shewanella baltic

Not applicable

LBc media

Wang et al. (2020)

Percolation (soaking) method Casting method

E. coli, S. aureus

DPPHa

MHAd Lamb meat

Alizadeh-Sani et al. (2021)

Konjac glucomannan/ chitosan/nanoZnO Chitosan/ZnO nanoparticles Chitosan

Cellulosic paper coated with chitosan-nanoZnO Carboxymethylchitosan

Antioxidant of bamboo leaves Chitosan-nano-ZnO

Nisin

Nano-MgO (magnesium oxide)

0.5%, 1% w/w

Al-Naamani et al. (2016)

Plant extracts encapsulated into chitosan-based films Chitosan nanofiber/ methyl cellulose matrices

Saffron anthocyanins

3% w/v

Chitosan

Eucalyptus globulus essential oil

0.5%, 1.5%, 1.5% v/v

Ultrasonic homogenization

Chitosan nanoparticles

Cinnamon essential oil

0.05%,0.1% w/w

Emulsion-gelation method Ultrasonic homogenization

Chitosan nanoparticles (CS)

Cinnamon essential oil (CE)

Grass carp collagen (GCC)/ Chitosan nanoparticles (CS) Chitosan

Lemon essential oil (LEO)

72 mg (in three sizes 112 nm, 215 nm, 527 nm of CE-NPsj) 0.1%, 0.2%, 0.3%, 0.4% w/w

Turmeric essential oil- magneticsilica nanocomposites

10%, 20%, 30% w/wcs

S. aureus, E. coli, B. cereus, S. entertidis, L. monocytogenes Totalmesophilic aerobic viable count (TMVC) S. aureus Enterobacteriaceae Lactic acid bacteria (LAB) Yeasts and molds

Not applicable

Ionic gelation

Pseudomonas spp., Lactic acid bacteria, Enterobacteriaceae

Oil in water Emulsion and freeze drying

Supercritical carbon dioxide extraction Chemical coprecipitation method Ultrasonic homogenization Casting method

Azadbakht et al. (2018)

Not applicable

Tryptic soy agar Sliced chicken sausages PCAe BPAf supplemented with egg yolk tellurite emulsion VRBGAg MRSh medium YGCi medium 6-Beef patties Chilled pork

Not applicable

POVk TBARSl

Chilled pork

Jiang et al. (2020)

Bacillus cereus

Not applicable

Fish surimi

Surendhiran et al. (2022)

Not applicable

GhaderiGhahfarokhi et al. (2017)

Hu et al. (2015)

(Continued)

Table 1.5 (Continued) Packaging material

Functional ingredient

Concentration

Design

Target microorganism

Antioxidant test

Application

References

Chitosan /poly caprolactone electrospun nanofibers Carboxymethylchitosan

Chlorogenic acid loaded halloysite nanotubes

2%, 4%, 6% w/w

Ultrasonic homogenization

E. coli, S. aureus

DPPHa

LBc culture medium

Zou et al. (2020)

Gliadin/phlorotannin nanoparticles

0%, 3%, 6%, 9%, 12% w/w

E. coli, S. aureus

DPPHa ABTSb

Culture broth

Zhao et al. (2022)

Polylactic acid/ chitosan

Cellulose nanofiber (CNF), rosin (R)

CNF: 0%, 2%, 5%, 8%, 10% w/w RCNF: 0%, 2%, 5%, 8%, 10% w/w

Anti-solvent method Ultrasonic homogenization Casting method Ultrasonic homogenization Casting method Two layer composite film

E. coli, B. subtilis,

Not applicable

LBc agar plates

Niu et al. (2018)

TEMPO-oxidation treatment Ultrasonic homogenization Ionic gelation Ultrasonic homogenization

NA

ABTSb

Not applicable

Hai et al. (2020)

E. coli, S. aureus

Not applicable

Nutrient agar plates

Wu et al. (2019)

Other compounds encapsulated into chitosan-based films Cellulose nanofiber

Chitosan nanofiber

3%,5%,7%,10%, 15%, 20% w/w

Chitosan/sodium tripolyphosphate

ε-Polylysine

0.05 g, 0.1 g

a

DPPH: 1, 1-Diphenyl- 2-picrylhydrazyl. ABTS: 2,20 -Azino-bis (3- ethylbenzothiazo-line6-sulfonic acid). LB: Luria-Bertani. d MHA:Muller
Hinton agar. e PCA: plate count agar. f BPA: Baird Parker Agar. G VRBGA: violet red bile glucose agar. h MRS: Man, Rogosa, and Sharpe. i YGC: yeast extract glucose chloramphenicol. j CE-NPs: chitosan nanoparticles loaded with cinnamon essential oil. k POV: peroxide value. l TBARS: thiobarbituric acid reactive substance. b c

Application of chitosan in active food packaging

21

Moreover, Dai et al. (2022) developed chitosan (CS) film with covalent organic frameworks (COFs) immobilized silver nanoparticles (AgNPs) with various concentration of COFs-AgNPs (1%, 2.5%, 5% w/wcs). According to the study findings, the chitosan-based films with 5% COFsAgNPs showed the best antibacterial activity compared to the other films. Conversely, to the CS pure films, they impressively extended the shelf life of the white crucian carp (Carassius auratus) fish and preserved its quality (12 days at 4°C 1 1°C) (Dai et al., 2022) (Table 1.5). Interestingly, Lin et al. (2020) combined silver to titanium oxide (TiO2) and showed that silver-loaded nanotitania in fish gelatin and chitosan films (0.5% w/v TiO2-Ag) exhibited efficient antibacterial activity against E. coli, S. aureus, and B. cinerea (Table 1.5). Zinc oxide nanoparticles (ZnONPs) demonstrated various functional characteristics including a strong antimicrobial capacity against a wide range of microorganisms, an efficient antioxidant activity and nontoxic nature and stability (Noshirvani et al., 2017; Yadav et al., 2021). Therefore ZnONPs are approved by the Food and Drug Administration of the United States of America and have been extensively considered as a safe substance in food, packaging materials, food additives, water purification, cosmetic, and drug products (Al-Naamani et al., 2016; Noshirvani et al., 2017; Yadav et al., 2021). It was reported that nano-ZnO could be employed as an antimicrobial and antioxidant agent. Zn ions and ROS released from ZnO nanoparticles attack the negatively charged cell membrane of bacteria leading to their death (Yadav et al., 2021). In a study conducted by Yadav et al. (2021), they found that the presence of 70 mg of zinc oxide nanoparticles (ZnO) loaded gallic acid (ZnO@gal) in chitosan films demonstrated a remarkably high scavenging activity toward ABTS assay and DPPH assay (83.43% and 68.51%, respectively) and exhibited the strongest bacteriostatic effect against both B. subtilis and E. coli. compared to the other composite films with lower ZnO@gal content. Moreover, nano-ZnO and mulberry anthocyanin extract (MAE) incorporated into konjac glucomannan (KGM)/chitosan (CS) matrix exhibited inhibitory effect against E. coli and S. aureus, and the films with 90 mg/g MAE recorded the highest antioxidant DPPH radical scavenging activity (71.98%) (Sun et al., 2020). Liu et al. (2021) reported that adding antioxidant of bamboo leaves (AOB) into chitosan/ZnO nanoparticles significantly improved the antioxidant performance of the films. Simultaneously, the synergetic effect of ZnO nanoparticles and AOB improved the antibacterial performance of the films against S. aureus and E. coli (Table 1.5).

22

Chitosan: Novel Applications in Food Systems

Furthermore, in another study, polyethylene (PE) coated with chitosan-ZnO were prepared and their antimicrobial properties were tested (Al-Naamani et al., 2016). It was found that these coatings completely stopped the growth of microorganisms after 24-hour incubation. In addition, Divsalar et al. (2018) reported in their study that the addition of 1000 g/mL nisin to cellulose and chitosan-zinc oxide nanocomposite film sterilized completely L. monocytogenes population of the surface of white cheese stored at 4°C for 14 days (Table 1.5). In addition, another metal nanoparticle known for its high antimicrobial performance and nontoxicity is the magnesium oxide (MgO). MgO nanoparticles produce superoxide, which destroys the bacterial cell membrane. Wang et al. (2020) reported that the addition of 1% nano-MgO greatly improved the antimicrobial activity of carboxymethyl-chitosan (CMCS) films and achieved a 99.99% inhibition rate for both bacteria Shewanella baltica and L. monocytogenes. This activity was proportional with MgO concentration (Wang et al., 2020) (Table 1.5).

1.4.2 Plant extracts encapsulated into chitosan-based films Despite the advantages and properties of metallic nanoparticles, their chemical nature and potential toxicity at high concentrations are undesirable. The use of plant compounds is the top trend to meet increasing consumers demand for natural products. A novel film was developed by immobilizing saffron petal anthocyanins in composite biopolymer films containing chitosan nanofibers and methyl cellulose (Alizadeh-Sani et al., 2021). The films exhibited a stronger antimicrobial activity against S. aureus than against E. coli. It was suggested that both saffron anthocyanins and chitosan nanofibers had antimicrobial characteristics. Particularly when anthocyanins were added to the composite films, their capacity to prevent bacterial development increased, which could be attributed to the anthocyanin’s high content in antimicrobial phenolic groups (AlizadehSani et al., 2021) (Table 1.5). Many studies have incorporated essential oils and their components into chitosan films. When clove essential oil was combined with chitosan, it exhibited a synergistic antimicrobial effect against some microorganisms such as S. aureus and fungi (Sanuja et al., 2014; Adel et al., 2019). Azadbakht et al. (2018) developed chitosan films containing Eucalyptus globulus essential oil (EGO) and tested the film’s antimicrobial activity against L. monocytogenes, which was inoculated on the surface of sliced chicken

Application of chitosan in active food packaging

23

sausages. The films containing 1.5% EGO reduced the growth of L. monocytogenes by 1.01 log (CFU)/mL during storage at 23°C for 3 days (Azadbakht et al., 2018) (Table 1.5). Moreover, it was reported in another study (Ghaderi-Ghahfarokhi et al., 2017) that using chitosan nanoparticles as a carrier of cinnamon essential oil (CEO) in the preservation of beef patties decreased lipid oxidation as well as microbial proliferation and enhanced the stability of the red color of patties, specifically after storing it for 4 days at 4°C (Table 1.5). Similarly, Hu et al. (2015) reported that 527 nm chitosan nanoparticles loaded with cinnamon essential oil (CE-NPs) significantly reduced the microbial growth of chilled pork and retained its redness during 15 days at 4°C. Jiang et al. (2020) reported same results where also GCC/CSLEO films owed their ability to efficiently inhibit microbial growth, prevent lipid oxidation, and retard the deterioration of chilled pork stored at 4°C for 21 days. These antimicrobial activities were attributed to the antibacterial characteristics of CEO and CS (Ghaderi-Ghahfarokhi et al., 2017; Hu et al., 2015). According to studies, the components of CEO attack the phospholipid bilayer of the bacterial cell membrane leading to its destruction (Hu et al., 2015) (Table 1.5). Surendhiran et al. (2022) reported that chitosan (CS)/magnetic nanoparticles (MNPs)/silica (Si)/ turmeric essential oil (TEO) bionanocomposite film slowed and controlled the release of TEO due to the porous nanomatrix used and decreased the B. cereus count from 4.0 to 2.78 log CFU/g in fish surimi during 14 days of wrapping, hence extending the shelf life of fish. Contrary to the CS/TEO film, where TEO was directly mixed into the packaging film, it exhibited a fast-uncontrolled release of TEO from the CS film after 6 days, resulting in bacterial count increase (4.66 log CFU/g after 12 days). Moreover, the inorganic nanocomposites from the CS/MNPs/Si/TEO bionanocomposite film did not penetrate into the food product and only antimicrobial substances were released (Table 1.5). Chlorogenic acid (CGA) is an ester of caffeic and quinic acid, considered as a bioactive substance naturally derived from plants. CGA can be used in packaging to prolong the shelf life of food since it has great antimicrobial and antioxidant characteristics (Zou et al., 2020). For example, chitosan (CS)/polycaprolactone (PCL) electrospun nanofibers combined with CGA loaded halloysite nanotubes (HNTs) as a carrier for CGA were developed (Zou et al., 2020) (Table 1.5). The addition of 6% CGA@HNTs strongly improved the films antioxidant activity, which

24

Chitosan: Novel Applications in Food Systems

reached 51.40% after 24 hours of incubation. Similarly, the antimicrobial activity showed positive association with the ratios of CGA@HNTs in the fibrous mats and was stronger against E. coli than S. aureus (Zou et al., 2020). According to research, carboxymethyl-chitosan (CMCS) film has many functional properties that enable it to be employed in food packaging, but it presents some limitations such as deficiency in antimicrobial and antioxidant properties, which severely narrow its wide opportunities to be used in food application industry (Zhao et al., 2022). However, it was evidenced that the biopolymer matrix’s functional properties can be enhanced if an inorganic or organic nanofiller was incorporated into it (Duan et al., 2021). Hence, Zhao et al. (2022) addressed this issue and incorporated gliadin nanoparticles (GPNPs), which are known for their nontoxicity, biodegradability, biocompatibility, and many more functions, along with phlorotannins into the CMCS films and tested its antimicrobial and antioxidant activity. Results showed that the addition of GPNPs significantly enhanced the antimicrobial and antioxidant activities of the films compared to the CMCS pure films (Table 1.5). Furthermore, Niu et al. (2018) in their study used modified cellulose nanofiber by rosin (RCNF) as a strengthening filler within a PLA matrix and then coated the resulting film with chitosan (CHT). The study results showed that the RCNF/PLA/CHT composite films effectively inhibited the bacterial growth of E. coli (5.81 6 0.27 log CFU) and B. subtilis (5.80 6 0.24 log CF) and recorded a wider inhibition zone against E. coli (10.88/6 mm) than B. subtilis (8.47/6 mm). It was concluded that this antimicrobial activity was attributed to the synergistic effect exhibited by rosin and chitosan (Niu et al., 2018) (Table 1.5).

1.4.3 Other compounds encapsulated into chitosan-based films A simple combination of chitosan nanofiber (ChNF) and cellulose nanofiber (CNF) was formed by Hai et al. (2020). CNF and ChNF are known to have low antioxidant performances; however, their combination increased the antioxidant activity of the CNF-ChNF films. This interesting performance occurred because both CNF and ChNF are polysaccharides; when combined, the reducing ends of the substances might open, hence improving the antioxidant activity. Also, nanofibrillation could boost the hydroxyl groups, which helps in the antioxidant activities (Hai et al., 2020). Moreover, Wu et al. (2019) showed that the antimicrobial capacity of chitosan/ε polylysine (ε-PL) bionanocomposite films against

Application of chitosan in active food packaging

25

E. coli and S. aureus was positively influenced with the increase in ε-PL ratio. This can be explained by the fact that ε-PL can disrupt the physiological functions of microbial cell membrane, and subsequently leading to the bacteria’s death (Wu et al., 2019) (Table 1.5). According to the novel food (Regulation, 1997) Regulation (EC) No 258/97, a food product that contains nano-components, or developed with the use of nanotechnology, may be identified as a novel food and hence needs a safety assessment before being placed on the market (Food safety authority of Ireland, 2010).

1.5 Conclusion Chitosan films have been used in active food packaging to preserve quality, extend shelf life, and enhance safety of food products. They can provide both antimicrobial and antioxidant properties. These functional properties have been enhanced by adding free or encapsulated functional ingredients. Therefore they are being used as an alternative for synthetic additives, synthetic materials, and unrecyclable packaging. They are considered as green, eco-friendly packaging materials and hence can decrease the waste volume and negative impact on the environment.

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231. Sun, J., Jiang, H., Wu, H., Tong, C., Pang, J., & Wu, C. (2020). Multifunctional bionanocomposite films based on konjac glucomannan/chitosan with nano-ZnO and mulberry anthocyanin extract for active food packaging. Food Hydrocolloids, 107, 105942. Surendhiran, D., Roy, V. C., Park, J. S., & Chun, B. S. (2022). Fabrication of chitosanbased food packaging film impregnated with turmeric essential oil (TEO)-loaded magnetic-silica nanocomposites for surimi preservation. International Journal of Biological Macromolecules, 203, 650
660. Tan, Y. M., Lim, S. H., Tay, B. Y., Lee, M. W., & Thian, E. S. (2015). Functional chitosan-based grapefruit seed extract composite films for applications in food packaging technology. Materials Research Bulletin, 69, 142
146. Thuong, N. T., Ngoc Bich, H. T., Thuc, C. H., Quynh, B. T. P., & Minh, L. V. (2019). Preparation and characterization of Piper betle Linn. leaf extract incorporated chitosan films as potential active food packaging materials. ChemistrySelect, 4(27), 8150
8157. Tripathi, S., Mehrotra, G. K., & Dutta, P. K. (2009). Physicochemical and bioactivity of cross-linked chitosan
PVA film for food packaging applications. International Journal of Biological Macromolecules, 45(4), 372
376. Vilela, C., Kurek, M., Hayouka, Z., Röcker, B., Yildirim, S., Antunes, M. D. C., NilsenNygaard, J., Pettersen, M. K., & Freire, C. S. (2018). A concise guide to active agents for active food packaging. Trends in Food Science & Technology, 80, 212
222. Vilela, C., Pinto, R. J., Coelho, J., Domingues, M. R., Daina, S., Sadocco, P., Santos, S. A., & Freire, C. S. (2017). Bioactive chitosan/ellagic acid films with UV-light protection for active food packaging. Food Hydrocolloids, 73, 120
128. Wang, L., Wang, Q., Tong, J., & Zhou, J. (2017). Physicochemical properties of chitosan films incorporated with honeysuckle flower extract for active food packaging. Journal of Food Process Engineering, 40(1), e12305. Wang, Q., Tian, F., Feng, Z., Fan, X., Pan, Z., & Zhou, J. (2015). Antioxidant activity and physicochemical properties of chitosan films incorporated with Lycium barbarum fruit extract for active food packaging. International Journal of Food Science & Technology, 50(2), 458
464. Wang, Y., Cen, C., Chen, J., & Fu, L. (2020). MgO/carboxymethyl chitosan nanocomposite improves thermal stability, waterproof and antibacterial performance for food packaging. Carbohydrate Polymers, 236, 116078.

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Wu, C., Sun, J., Lu, Y., Wu, T., Pang, J., & Hu, Y. (2019). In situ self-assembly chitosan/ε-polylysine bionanocomposite film with enhanced antimicrobial properties for food packaging. International Journal of Biological Macromolecules, 132, 385
392. Wu, S., Li, G., Li, B., & Duan, H. (2022). Chitosan-based antioxidant films incorporated with root extract of Aralia continentalis Kitagawa for active food packaging applications. e-Polymers, 22(1), 125
135. Wu, Z., Huang, X., Li, Y. C., Xiao, H., & Wang, X. (2018). Novel chitosan films with laponite immobilized Ag nanoparticles for active food packaging. Carbohydrate Polymers, 199, 210
218. Yadav, S., Mehrotra, G. K., & Dutta, P. K. (2021). Chitosan based ZnO nanoparticles loaded gallic-acid films for active food packaging. Food Chemistry, 334, 127605. Yilmaz, P., Demirhan, E., & Ozbek, B. (2022). Development of Ficus carica Linn leaves extract incorporated chitosan films for active food packaging materials and investigation of their properties. Food Bioscience, 101542. Zhang, L., Wang, H., Jin, C., Zhang, R., Li, L., Li, X., & Jiang, S. (2017a). Sodium lactate loaded chitosan-polyvinyl alcohol/montmorillonite composite film towards active food packaging. Innovative Food Science & Emerging Technologies, 42, 101
108. Zhang, L., Zhang, Z., Chen, Y., Ma, X., & Xia, M. (2021). Chitosan and procyanidin composite films with high antioxidant activity and pH responsivity for cheese packaging. Food Chemistry, 128013. Zhang, X., Xiao, G., Wang, Y., Zhao, Y., Su, H., & Tan, T. (2017b). Preparation of chitosan-TiO2 composite film with efficient antimicrobial activities under visible light for food packaging applications. Carbohydrate Polymers, 169, 101
107. Zhao, J., Jiang, H., Huang, Q., Xu, J., Duan, M., Yu, S., Zhi, Z., Pang, J., & Wu, C. (2022). Carboxymethyl chitosan incorporated with gliadin/phlorotannin nanoparticles enables the formation of new active packaging films. International Journal of Biological Macromolecules, 203, 40
48. Zou, Y., Zhang, C., Wang, P., Zhang, Y., & Zhang, H. (2020). Electrospun chitosan/ polycaprolactone nanofibers containing chlorogenic acid-loaded halloysite nanotube for active food packaging. Carbohydrate Polymers, 247, 116711.

CHAPTER 2

Chitosan-based coatings and plant extracts Kataryne Árabe Rimá de Oliveira1, Karina Felix Dias Fernandes1, Jossana Pereira de Sousa Guedes2, Evandro Leite de Souza1 and Marciane Magnani3 1

Laboratory of Food Microbiology, Department of Nutrition, Health Sciences Center, Federal University of Paraíba, João Pessoa, Paraiba, Brazil 2 Department of Agroindustrial Management and Technology, Federal University of Paraiba, Bananeiras, Paraiba, Brazil 3 Laboratory of Microbial Processes in Foods, Department of Food Engineering, Center of Technology, Federal University of Paraíba, Campus I, João Pessoa, Paraiba, Brazil

2.1 Edible coatings: main aspects and general characteristics The extension of food shelf-life has become a global public concern with significant economic and public health implications, attracting the attention of the food industry (Liu et al., 2020a). This is related to the fact that the modern consumer is more inclined toward green consumerism, which includes fewer artificial ingredients, ensuring high nutritional value, safety, quality, and extended shelf-life of foods. The evidence on edible films and coatings available shows that they meet the requirements for inclusion in so-called “green” biotechnology and as an alternative to synthetics. However, this type of technology is highly focused on characteristics that must be adequate and specific for the type of food to be protected, and it is a constantly evolving scientific field (Díaz-Montes & Castro-Muñoz, 2021). Edible films and coatings are thin layers of edible materials applied to food products that play an important role in their preservation, distribution, and marketing by accounting for functional properties such as antioxidant and antimicrobial properties that aid in the reduction or inhibition of microbial growth on food surfaces, preventing spoilage, and extending shelf life (Adiletta et al., 2021). In general, edible films are prepared separately and applied to the food surface, whereas coatings are directly applied to the outer surface of the food product as liquids in a variety of ways (spray, immersion, brushing, Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00001-6

© 2023 Elsevier Inc. All rights reserved.

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and dripping) (Fig. 2.1) (Hashemi et al., 2020; Grande-Tovar et al., 2018). Furthermore, edible films are made entirely of food-grade components, whether they are film-forming polymer matrices, solvents in which they are dissolved, or additives incorporated in the films/coatings (Otoni et al., 2017). This chapter will present innovations in the application of chitosan (CH)-based edible coatings incorporated with active ingredients from aromatic plants, highlighting essential oils (EOs), their main biological activities, and impacts on the quality of a variety of foods (plant and animal origin), with a special emphasis on literature published in the last 5 years.

2.1.1 Chitosan-based edible coatings CH is a linear polysaccharide, obtained through partial deacetylation of chitin, the second-most abundant renewable biopolymer in nature, and is majorly composed of 2-amino-2-desoxy-D-glycopyranose and 2-acetamide-2-desoxy-D-glycopyranose interconnected by glycosidic β-1,4 bonds in variable proportions (de Souza et al., 2019). Properties, such as the degree of acetylation, nitrogen composition, molecular size, and polydispersity, are affected by the chitin source. Through acid and alkaline treatments, chitin can be extracted from structural components of arthropod exoskeletons or cell walls of fungi and yeasts. Alkali concentration, incubation time, chitin to alkali ratio,

Figure 2.1 Production and application of edible films and coatings on food surfaces.

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temperature, and atmosphere all play a role in alkaline N formation. CHdeacetylation (Casalini & Giacinti Baschetti, 2022; Adiletta et al., 2021). The degree of deacetylation (removal of an acetyl group) is one of the most significant chemical characteristics of CH. During deacetylation, the acetyl (C2H3O) group is replaced by amino (
NH2) group from the polymer (Kumar et al., 2020). Because the electronegative amino group takes up protons and develops a positive charge, the presence of the amino group in CH provides it with various chemical, physical, and biological properties (Priyadarshi & Rhim, 2020). Furthermore, the degree of deacetylation and molecular weight are important factors in determining the physicochemical properties of the coat-forming solution, such as solubility, appearance, and rheological properties (Díaz-Montes & CastroMuñoz, 2021). At this point, it is characterized as a solution insoluble at neutralalkaline pH, but highly soluble at acidic pH, due to the proportion of protonated amino groups (
NH2) positively charged (Díaz-Montes & Castro-Muñoz, 2021). The CH molecules are typically soluble in pH below of 6 (Priyadarshi & Rhim, 2020). CH can form amorphous and complex 3D structures due to its polycationic nature that allows it to interact electrostatically by hydroxyl groups, or form covalent bonds by
NH2 groups, or other molecules (Díaz-Montes & Castro-Muñoz, 2021). Due to its biocompatibility, biodegradability, nonantigenic, and nontoxicity, CH is becoming a highly recognizable polysaccharide for the active protection and extension of the shelf-life of perishable foods (Casalini & Giacinti Baschetti, 2022; Kumar et al., 2020). CH can extend food shelf-life by regulating gas exchange between food and the environment (selective permeability to gases) and protecting food surface from microbial contamination and discoloration, while also retaining excellent mechanical properties (Priyadarshi & Rhim, 2020; Liu et al., 2020b). In general, three types of CH coating methods have already been investigated: spread coating, spray coating, and dip-coating, resulting in solid, transparent, and flexible components. The dip-coating process, on the other hand, is relatively simple, saves time, and eliminates the need for large equipment, while also being highly effective in preserving food and maintaining its quality (Priyadarshi & Rhim, 2020; Khorshidi et al., 2021).

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Chitosan: Novel Applications in Food Systems

Nonetheless, the CH-based coatings have a significant drawback due to their high permeability to water vapor, which limits their use because adequate moisture transfer control is required for food storage (GrandeTovar et al., 2018). The incorporation of functional compounds in CH is a promising technique in modern barrier technologies for improving their functionality, thereby assisting in the preservation of nutritive and sensory properties as well as the shelf life of food products (Nair et al., 2020). Incorporating unique bioactive compounds into edible coatings can provide functional properties (e.g., antimicrobial activity and antioxidant capacity) with a specific health target, as well as a novel way to maintain food quality during storage (Quintana et al., 2021). Combining CH with EOs can be an effective way to improve the barrier effects of coatings while also minimizing the negative effects of EO sensory impact.

2.1.2 Plant extracts or essential oils added in coatings Much attention has been paid to the biological activities of plant extracts and EOs from plants (herbs, spices), with the goal of developing several novel applications, such as natural coatings for food preservation. EOs has been studied in particular as a food additive in films and coatings to replace synthetic preservatives. The main substances that contribute to these activities are antioxidant and antimicrobial compounds found naturally in some plants, such as phenolic compounds and terpenoids (Aghababaei et al., 2022; Quintana et al., 2021; Sharma et al., 2021). In addition, plant extracts containing phenolic compounds have also been proposed for their application in food products as they provide color, astringency, oxidative stability. 2.1.2.1 Chemical composition of essential oils EOs are hydrophobic volatile liquids derived from aromatic plants, which contain a multitude of bioactive compounds of low-molecular weight (,1000 Da) classified according to their chemical structure including: terpenes (hydrocarbons have several isoprene units), terpenoids (thymol, carvacrol, linalool, linalyl acetate, etc.), and phenylpropanoids (cinnamaldehyde and eugenol) (Amiri et al., 2019; Hashemi et al., 2020; Kumar et al., 2020; Díaz-Montes & Castro-Muñoz, 2021). The qualitative and quantitative differences may influence and increase the biological efficacy of EOs. Additionally, the composition of EOs can vary widely among the plant species, part of the plant, edaphic-climatic conditions, phenological stage of the plant, photoperiod, light intensity, seasonality,

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and extraction method (Grande-Tovar et al., 2018; Casalini & Giacinti Baschetti, 2022; Wang et al., 2022). In general, hydrodistillation produces EOs containing high concentration of antioxidant or antimicrobial volatile compounds (Quintana et al., 2021). However, because this process generally requires long processing times, several techniques have been used as pretreatments of plant material to reduce the time of hydrodistillation and achieve higher efficiency and/ or quality (Chen et al., 2021). Furthermore, Supercritical Fluid Technology has been extensively researched for the extraction of EOs active ingredients and compounds. In comparison to hydrodistillation, this technique allows for the production of high-quality bioactive extracts with a high yield but a lower concentration of volatile compounds (Quintana et al., 2021). EOs are recognized as alternatives for chemical preservatives to protect foods, due to its potential antimicrobial and antioxidative effects and generally recognized as safe (GRAS) by Food and Drug Administration (FDA) (de Souza et al., 2019). Moreover, they are typically nontoxic and do not have any effect on human health if consumed in small quantities (Priyadarshi & Rhim, 2020). However, the direct application of EOs with high doses may have undesirable effects on sensory properties of coated foods (Hashemi et al., 2020). In this context, with a view to maintain the biological activity of EOs and decrease their potential (negative effects) on the sensory properties of food items, EOs have been incorporated in edible coatings. EOs edible coatings are environmentally friendly coatings due to their natural origin. They must, like other food coatings, form thin layers of edible substances that are applied to the food surface and should not be harmful to human health when consumed at the concentrations required to maintain food quality. These coatings can act as a barrier to the exchange of moisture, oxygen, and solid solutes, reducing water loss, respiration, and oxidation reaction rates, and extending shelf-life (Quintana et al., 2021). Furthermore, the addition of plant extracts of a lipid nature influences and possibly improves not only the antimicrobial and antioxidant properties of the final composite, but also its mechanical, barrier, and thermal properties. EOs, for example, can reduce the water vapor permeability of hydrophilic materials and decrease tensile strength while increasing elongation at break. This could be due to the film network’s partial replacement of stronger polymer
polymer interactions with weaker

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Chitosan: Novel Applications in Food Systems

polymer
oil interactions (Casalini & Giacinti Baschetti, 2022). Different aromatic plants have been proposed for combined application in coatings, such as lemongrass, mint, clove, thyme, rosemary, and cinnamon (Contini et al., 2022; Basaglia et al., 2021; Quintana et al., 2021; Soncu et al., 2020).

2.2 Effects of coatings on quality parameters and shelf life of food The main issues affecting food quality and shelf life are microbial deterioration and oxidation processes. The incorporation of antimicrobials and/ or antioxidants in natural active compound coatings has emerged as an alternative to synthetic food additives. Several studies have shown that the use of edible CH coatings combined with plant extracts, such as EOs, reduced, inhibited, or delayed the growth of microorganisms on food surfaces or in food, thereby increasing its post-harvest/post-manufacturing shelf-life (Ewide et al., 2022; Reyna et al., 2022; Langroodi et al., 2021). When applied in CH active coatings, EOs are particularly interesting because they can be released as vapors, sterilizing both the headspace and the food surface (Casalini & Giacinti Baschetti, 2022). Furthermore, combining EOs or plant extracts with edible CH coatings not only improves their antimicrobial and antioxidant properties, but also regulates the water vapor permeability of the hydrophilic coating, improving physical and mechanical properties and providing oxidative protection within the coated product (Khorshidi et al., 2021). The antimicrobial and antioxidant properties of CH and EOs alone and in combination will be described in order to understand and characterize these improvements (attributed to edible coatings).

2.2.1 Antimicrobial effects 2.2.1.1 Antimicrobial effects of chitosan CH has potent antimicrobial activity against pathogenic and spoilage microorganisms, including fungi, which are typically more sensitive to CH than bacteria (Aider, 2010). CH’s antimicrobial activity is influenced by intrinsic, environmental, and microbial factors. When the CH is dispersed, the intrinsic properties can be altered by varying the temperature and exposure time. Furthermore, the degree of deacetylation, concentration, and molecular weight of CH all have an impact on its antimicrobial efficacy. Aside from the type of microorganism, food matrix characteristics

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such as pH, temperature, and the potential development of ionic and/or covalent interactions between the food matrix and CH influence antimicrobial activity (Grande-Tovar et al., 2018; Li & Zhuang, 2020). CH’s intrinsic antimicrobial activity appears to be directed by three distinct mechanisms: ionic surface interaction, penetration of the CH into the nucleus of microorganisms, and the formation of an external barrier inhibiting nutrient uptake (Casalini & Giacinti Baschetti, 2022). However, the most cited and suggested mechanism is due to electrostatic interaction among CH and microorganisms (Adiletta et al., 2021). CH’s antifungal action targets the cell wall, cell membrane, and DNA, resulting in structural changes such as excessive hyphae branching, hyphae size reduction, spore aggregation, swelling of the cell wall, leakage of cytoplasmic constituents, cell lysis and death, and effects on DNA/RNA synthesis (Grande-Tovar et al., 2018). Some studies report that the fungistatic properties of CH are related to the fact that this polysaccharide interferes with the fungal absorption of minerals, especially Ca21, and nutrients, resulting in a delay in spore germination (Elguea-Culebras et al., 2019). Antibacterial mechanisms are complex and rely on electrostatic interactions between CH and bacterial cell wall components (e.g., teichoic and lipoteichoic acids, and lipopolysaccharides), altering rigidity and eventually entering the cell (Díaz-Montes & Castro-Muñoz, 2021). High electrostatic interaction is observed at low pH values between protonated amino (
NH2) groups of CH and anionic carboxyl and phosphate groups of bacterial outer surfaces. These interactions not only change the permeability of the bacterial cell membrane, preventing gas exchange between the interior of the cell and the exterior environment, but also cause cellular dysfunction through membrane rupture and release of intracellular components, eventually leading to cell death (Adiletta et al., 2021; Li & Zhuang, 2020; Mahdavi et al., 2018). 2.2.1.2 Antimicrobial effects of plant extracts or EOs Plant extracts or EOs have antimicrobial activity due to the presence of hydroxyl groups in their constituents, which can damage pathogen cell membranes. This causes the cell constituents to be released and the microorganisms to die. As a result, EOs have a broad antimicrobial spectrum against various pathogenic and spoilage microorganisms (Casalini & Giacinti Baschetti, 2022; Gutiérrez-Pacheco et al., 2020).

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Among its constituents, phenolic compounds can degrade the outer membrane of microorganisms, causing the release of liposaccharides and increasing the cytoplasmic membrane’s permeability to ATP (Adenosine 50 -triphosphate). Withdrawal of ATP causes depletion of cell energy storage and cell death. As a result, higher phenolic compound concentrations may be associated with increased antimicrobial properties (Aghababaei et al., 2022). The chelation of metals by flavonoids also has been considered as a mechanism that prevents the growth of microorganisms, since certain metals (i.e., Fe) are considered essential for cellular growth (Khorshidi et al., 2021). It is worth noting that the antimicrobial activity of the EOs may diminish after dispersion in the polymer, and the effective one will depend on its composition, because different bioactive compounds can act against different microorganisms, and thus some EOs may be more active in certain types of food products. In order to determine the true efficacy of the chosen solution, in vitro and in situ analyses are usually required (Casalini & Giacinti Baschetti, 2022). According to numerous studies, Gram-positive bacteria are more sensitive to antibacterial compounds than Gram-negative bacteria. Gram-positive bacteria have a high sensitivity because they lack a lipopolysaccharide outer membrane, which in Gram-negative bacteria may prevent active compounds from entering the cytoplasmic membrane (Aghababaei et al., 2022). 2.2.1.3 Antimicrobial effects/mechanisms of edible coatings of chitosan with plant extracts Investigations on the effects of coatings formed by CH and EOs or plant extracts can reveal possible additional functionalities and expand the applications of these coatings (de Souza et al., 2019). Generally, the incorporation of EOs into coating can intensifies the antibacterial effects of CH against several spoilage and pathogenic microorganisms (Langroodi et al., 2021). The mechanisms underlying the enhanced antimicrobial effects of CH and EOs in combination have been linked to CH’s ability to alter the permeability of microbial membranes and reduce the synthesis of cell wall components, resulting in a reduced ability of target microorganisms to tolerate the disturbing effects on surface characteristics and microbial cell structure caused by EOs (de Souza et al., 2019). The presence of phenolic compounds in extracts or EO can also increase permeability, and by

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attacking cell membranes and removing cytoplasmic content, they can influence the enzymes in bacterial cell membranes, resulting in cell death (Khorshidi et al., 2021). Several studies have reported antimicrobial effects upon application of edible coatings of CH with plant extracts or EOs on foods (de Oliveira et al., 2019, 2020; Gomes et al., 2020; Macedo et al., 2020; Liu et al., 2020a; Rezaeifar et al., 2020; Yaghoubi et al., 2021). Fresh strawberries are among the most tested fruits for investigating the antimicrobial properties of coating in terms of application to fruits and vegetables. In one recent study, strawberries coated with CH and extracts or EO of rosemary and thyme showed lower fungal decay than the control after 10 days of cold treatment. This activity was linked to the main active constituents of rosemary EOs, namely eucalyptol, thymol, and carvacrol (Quintana et al., 2021). In products of animal origin, it was observed that coating turkey breast meat with CH incorporated with grape seed extract and Origanum vulgare EO was effective against the propagation of different kinds of spoilage microorganisms, including Enterobacteriaceae, Pseudomonas spp., lactic acid bacteria, and yeast-mold during a 20 days of cold storage. Antibacterial effects of O. vulgare EO were related to an increase in cell membrane permeability, what could be caused by components such as thymol and carvacrol. The antimicrobial characteristics of grape seed extract can be triggered via interactions between its active phenolic compounds and sulfhydryl groups of proteins in the bacterial cells (Langroodi et al., 2021).

2.2.2 Antioxidant effects Food oxidation is a deteriorating reaction that results in changes in its chemical, sensory, and nutritional properties. Lipid oxidation is caused by reactive oxygen species (ROS), which increase the formation of lipid oxidation by-products such as malondialdehyde, one of the main decomposition products of polyunsaturated fatty acid hydroperoxides. The thiobarbituric acid reactive substances (TBARS) test, which quantifies malonaldehyde, is widely used to assess the degree and extent of oxidative stability of foods (Contini et al., 2022). Edible coatings containing natural antioxidant compounds can be an alternative to synthetic additives that are widely used in the food industry (Contini et al., 2022). The antioxidant potential of an edible coating is usually determined by its composition. Natural antioxidants can slow the

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Chitosan: Novel Applications in Food Systems

oxidation of proteins, lipids, and other free radicals, which are sensitive food components that cause deterioration. The use of edible coatings can significantly improve the quality, stability, and overall shelf life of food products. Furthermore, adding natural antioxidants may help to preserve or improve the sensory properties of food (Al-Harrasi et al., 2022). 2.2.2.1 Antioxidant effects of chitosan By improving nonenzymatic and enzymatic antioxidant systems, CH coating application delays the changes typically associated with the action of ROS in foods. CH has antioxidant properties due to its ability to scavenge free radicals and chelate metal ions, which can be enhanced by increasing the degree of deacetylation (Anraku et al., 2018; Kumar et al., 2020; Khorshidi et al., 2021). Amino groups in CH can react with free radicals to form more stable macroradicals, which may partially explain the CH elimination effect (Li et al., 2019). Furthermore, such coatings prevent oxygen from penetrating the texture of the foods, resulting in a significant reduction in the rate of lipid oxidation and the subsequent formation of compounds like hydroperoxides (Aghababaei et al., 2022). Endogenous radical-scavenging antioxidants in foods are vitamin E, ascorbic acid (vitamin C), phenolic compounds, including flavonoids, carotenoids, glutathione (GSH), polyphenols, allyl sulfides, curcumin, melatonin, and polyamines; All of which they are involved in the detoxification of ROS, as well as O2 2 and OH scavengers. CH-based coatings act as a barrier film on food surface that preserves the nonenzymatic antioxidant content during storage (Adiletta et al., 2021). Low oxygen permeability in CH-coated foods also inhibits the activity of enzymes involved in bioactive compound oxidative reactions. Although ascorbic acid is an important antioxidant that reduces ROS and is converted to its oxidized form, it is also a cofactor in many enzymes. CH-based coatings control the passage of oxygen from the external environment to the food by inhibiting the activity of ascorbate oxidase, which is responsible for the oxidation of ascorbic acid, thereby reducing losses or increasing the content in foods (Xylia et al., 2021; Zhang et al., 2020) CH coating, alone or in combination with EO, can also significantly increase the activity of superoxide dismutase, an oxygen-scavenging enzyme that plays an important role in maintaining the metabolic balance of ROS (Wang et al., 2022).

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2.2.2.2 Antioxidant effects of plant extracts or essential oils Growing interest in using natural antioxidants such as EOs and plant extracts as alternatives to synthetic antioxidants, particularly by loading them in edible films, has yielded more promising results in terms of improving the shelf-life, stability, and sensory properties of coated foods (Ehsani et al., 2020). The EO components act on the surface of the coated food material to improve its shelf life and to impart the characteristic odor and flavor. However, it is well known that biological activity is strongly influenced by the chemical composition (structure of the compounds) of the various EOs used. The presence or absence of compounds with a specific functional group may have a significant impact on the antioxidant activity in food (Riabov et al., 2020). Phenolic compounds containing polyhydroxyl moieties are usually accountable for overall antioxidant activity. These components have great capability to scavenge and prevent lipid peroxidation (Al-Harrasi et al., 2022). Because phenolic compounds have redox oxidation properties, they act as reducing agents, hydrogen donors, and active oxygen scavengers (Aghababaei et al., 2022). According to research, the double bond of EO constituents increases antioxidant activity, whereas molecules with conjugated double bonds neutralize radicals very quickly (Ðurovi´c et al., 2022). 2.2.2.3 Antioxidant effects of edible coatings of CH with plant extracts or EOs on foods In general, the use of edible coatings and EOs slows down the process of increasing oxidation rates, and increasing the concentration of EOs has a positive effect against the oxidative process (Aghababaei et al., 2022). The degree of lipid oxidation reduction can be attributed to several factors, including the CH coating’s good oxygen barrier properties, which prevent the chain reaction of free radicals from starting in lipid oxidation; the ability to chelate CH cations; singlet-oxygen-quenching activities; and phenolic compounds in EOs, which function as primary antioxidants by donating electrons to stabilize free radicals and interrupt the oxidation chain reaction (Contini et al., 2022). Among the various reactive oxygen species, the chemical activity of the hydroxyl radical _OH is the strongest. Groups in the structure of CH, such as
NH2 and
OH, can eliminate the hydroxyl radical by inhibiting the chain reaction of ROS or can easily react with active hydrogen atoms, forming a more stable compound. The terpenoids present in EOs contain

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double bonds, which can eliminate the hydroxyl radical with _H (unpaired electron) by addition reaction (Li et al., 2019). Several studies have shown that fruit coated with CH and EO maintains the balance of intracellular oxidation metabolism due to its ability to clear cytotoxic compounds via enzymatic and nonenzymatic antioxidants (Abdelgawad et al., 2022; González-Locarno et al., 2020). The ability to reduce fruit browning is highlighted in this context, which is primarily associated with the enzymatic oxidation of phenolic compounds to quinones mediated by polyphenol oxidase (PPO). Coatings inhibit PPO activity due to the low availability of O2 inside the fruit, which is required to initiate browning reactions (Adiletta et al., 2021). In addition to plant-based products, meat and meat products are susceptible to oxidative reactions that reduce their quality while also losing nutrients and safety. TBA is a popular indicator for determining the degree of secondary lipid oxidation in animal products. Cai et al. (2018) suggested that the TBA inhibition of the EOs might be attributed to its antioxidant mechanism, including the prevention of radical chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, and the interaction with the free radicals. The network structure of the CH membrane composed of EOs is more compact than that of CH alone, which reduced the rate of oxygen permeation, thus inhibiting the occurrence of lipid oxidation (Li et al., 2019).

2.2.3 Effects on quality parameters in foods (physicochemical, enzymatic activity, and sensory characteristics) Recently, there has been a significant increase in the number of studies on edible coatings to extend the shelf-life of foods, and among the many methods used, the use of natural-based films and coatings has proven to be a promising area (Casalini & Giacinti Baschetti, 2022; Ewide et al., 2022; Langroodi et al., 2021). Among the main objectives of the application of edible films and coatings, are: The preservation of the natural characteristics of foods and the delay of deterioration, through physical and biological damages and chemical/biochemical reactions, stand out. Considering that food spoilage is influenced by oxygen, water content, temperature, relative humidity, and pH, the general properties (physical, chemical and mechanical) of the films and coating must efficiently control the storage conditions and, consequently, maintain the protective

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43

properties against spoilage and pathogenic microorganisms, increasing the safety and shelf life of foods (Díaz-Montes & Castro-Muñoz, 2021). Because perishable foods are more vulnerable to microbial attack and quality-altering biochemical reactions during storage, edible coatings are primarily used in these products. It is also worth noting that, while using EOs in coating is a good way to improve the quality and safety of foods, sensory evaluation should always be considered when considering its industrial applications. Thus, these and other quality aspects will be presented for various plant (larger field of application) and animal food groups. 2.2.3.1 Fruits and vegetables Fresh fruits and vegetables undergo postharvest changes in weight, soluble solids content (SS), pH, sugar contents, color, and phenolic compounds due to transpiration and respiration processes (Abdelgawad et al., 2022). In the analysis the studies of Table 2.1, it was discovered that adding CH and EOs coatings to fresh fruits and vegetables reduced changes in quality parameters during the maturation process and increased antioxidant activity. As previously stated, coatings have a positive impact on delaying microbial changes. Low weight loss of fruits and vegetables during storage is an indicator of freshness (Yousuf & Srivastava, 2017). Water migration from the fruit to the environment is thought to be the primary cause of fruit weight loss. The incorporation of EOs into the CH formulation results in less weight loss in fruits and vegetables, presumably because CH can form a thin, semipermeable layer around the food, acting as a physical barrier to slow down the rate of respiration, water evaporation, and metabolic activity. Furthermore, due to the inclusion of EO components, the low solubility and hydrophobic nature of the CH-based matrix can contribute to a decrease in water exchange between the food and the environment. These modifications keep the fruits and vegetables in an environment with a controlled atmosphere and low humidity, protecting the fruits against microbial spoilage (Wang et al., 2022; Basaglia et al., 2021; Quintana et al., 2021). Fruit firmness loss can also be attributed to the pectinolytic action (pectin degradation) of spoilage microorganisms. The antimicrobial properties of CH and EOs coatings, as well as their barrier to moisture loss, may play a positive role in maintaining fruit firmness (Abdelgawad et al., 2022).

Table 2.1 Effects of chitosan (CH) coatings and plant extracts or essential oil (EO) on quality parameters (microbiological, physicochemical and sensory) of foods of plant origin. Coating composition CH and tested concentration

Plant extracts or EO and tested concentration

Foods of plant origin

Main effects of coating

References

CH 0.5% (w/v)

Tea tree EO 0.5%, peppermint EO 0.5% (v/v)

Green beans (minimally processed)

Abdelgawad et al. (2022)

CH 0.6% (w/v)

Thyme, citral, methyl anthranilate, and lemongrass EO 1% (v/v)

Bell Pepper (cvs. Khayrat (green), Antonio (red) and Cleopatra (yellow)).

CH 1% (v/v)

Mint EO 0.2% (v/v)

Papaya

All treatments tested significantly increased the shelf life of minimally processed green beans, reducing weight loss while maintaining chlorophyll content, total soluble solids, firmness, ascorbic acid, total phenolic compounds, and total sugars. The combinations were effective in reducing microbial growth during refrigerated storage at 5°C for up to 9 days, as well as in lowering the browning index and increasing antioxidant capacity up to 15 days after harvest. Results demonstrated that complete suppression of mycelial growth and spore germination of Botrytis cinerea was achieved with concentrations of 0.5% and 1.0% of most of the tested essential oils and with CH at a concentration of 6.0 g/L. All treatments, whether used alone or in combination with CH, significantly reduced the incidence and severity of gray mold disease. The most effective treatments were those that combined thyme or citral with CH. In vitro, the applied treatments exhibited an important antifungal activity due to the high inhibition of mycelial growth and conidial germination of Colletotrichum gloeosporioides. In vivo, the lowest incidence and severity of anthracnose was obtained in fruit treated with CH. In addition, the treatments maintained the fruit postharvest quality. CH was effective to inhibit anthracnose development in papaya and the addition of Mint EO did not provide an additional effect.

Ewide et al. (2022)

Reyna et al. (2022)

CH 1% (w/v)

Torreya grandis EO 0.5% and 1.0% (v/v)

Loquat

CH 1% and 2% (w/v)

Cinnamon EO 0.5% and 1% (v/v)

Pineapple (minimally processed)

CH 2% (w/v)

Ruta graveolens EO 0.5%, 1.0%, and 1.5% (v/v)

Pears cv. Packham’s Triumph

CH 1 Torreya grandis EO (1.0%) proved to be the best combination tested in keeping quality attributes and extending the storage period of loquat fruit. This formulation reduced decay index and respiration rate while delaying fresh weight loss and preserving higher levels of total soluble solids and ascorbic acid. This treatment also improved sensory quality, inhibited the increase in polyphenol oxidase and peroxidase activities, and increased superoxide dismutase activity effectively. The coatings used were effective in reducing mold and yeast growth, as well as weight and firmness loss, proving to be effective in extending the shelf life of fruit stored at 5°C for 15 days. The treatment with the highest percentage of CH produced the best results among the treatments tested. The physical-chemical properties of the fruits were examined, revealing that coatings protect against the ripening process. Less ripe fruits have a lower maturity index, decay index, disease damage incidence, and color results. Weight loss and firmness loss were also reduced in coated fruits. Furthermore, after 21 days of storage at 18°C, aerobic mesophilic bacteria and molds were significantly reduced. The sensorial analysis also revealed that all of the formulations were suitable for human consumption and acceptable for the evaluated attributes.

Wang et al. (2022)

Basaglia et al. (2021)

Peralta-Ruiz et al. (2021)

(Continued)

Table 2.1 (Continued) Coating composition CH and tested concentration

Plant extracts or EO and tested concentration

Foods of plant origin

Main effects of coating

References

CH 2%

Thyme and rosemary EO and supercritical extracts 1% and 5%

Strawberries

Quintana et al. (2021)

CH 0.1%, 0.125%, 0.25%, 0.5%, and 1% (w/v)

Majorana EO 1:1000, 1:1500, 1:2000, and 1:2500 (v/v).

Lettuce (minimally processed)

CH 1% (w/v)

Cinnamon EO 0.2% (v/v) and cellulose nanocrystals 0.1% (w/v)

Mango

After 10 days of cold treatment, fruits coated with CH and extracts or EO had lower fungal decay than controls, but EOs had better spoilage preservation than supercritical extracts. Furthermore, in comparison to the fruits coated with extracts, coatings produced with EOs exhibited slightly higher weight loss, but lower pH and soluble solids content, and better preserved the content of phenolic compounds in strawberries. Marjoram essential oil improved the aroma and visual quality of lightly processed lettuce, whereas CH alone had a negative effect on the lettuce color. The combination of treatments increased the phenolic, ascorbic, and carotenoid content, as well as the antioxidant status, improving the nutritional value of the fruit. The activity of enzymes involved in plant tissue browning was reduced by EO treatments (peroxidase activity and polyphenol oxidase). Different treatments demonstrated antimicrobial activity against total viable count, yeast and mold counts, and total viable count of minimally processed lettuce. Composite coatings effectively improved the appearance of mangoes during storage at 25°C for 12 days by reducing yellowing and dark spots and delaying water loss. By regulating the activities of pectin methyl esterase, polygalacturonase, and peroxidase, hardness was maintained and membrane lipid peroxidation was reduced. Furthermore, coating improved nutrient quality by increasing total soluble solids, titratable acid, and ascorbic acid content.

Xylia et al. (2021)

Yu et al. (2021)

CH 5 g/L (w/v)

Mentha piperita and M. 3 villosa Huds EO 0.6
1.2 mL/L (v/v)

Papaya

CH 5 mg/mL (w/v)

Cymbopogon citratus EO 0.6 μL/mL (v/v)

Guava

CH 2% (w/v)

Ruta graveolens EO 0.5%, 1.0%, and 1.5% v/v

Cape gooseberry

CH 0.8% (w/v)

Carnauba Wax 10% and Oregano EO 0.08 g/mL (v/v)

Cucumber

When compared to uncoated papaya, coated papaya had lower firmness, weight loss, total soluble solids, and enzymatic activity, as well as delayed evolution of pulp and peel color during storage (12°C/20 days). Coating formulations had no effect on papaya sensory acceptability and caused delayed maturation without affecting postharvest quality. The coating delayed the ripening during cold storage (15 days, 12°C) resulting in reduced weight and firmness losses, changes in soluble solids, titratable acidity, pH, color, phenolics, and enzymatic activity contributing to improving sensory parameters of the fruit When compared to uncoated fruits, coated fruits lost less weight and had a smaller increase in maturity index. Microbial growth (aerobic mesophylls, molds, and yeasts) was slowed in a concentration-dependent manner. After 12 days of storage (18°C), the coating also preserved the antioxidant properties of the fruit. CH and Oregano EO coatings completely inhibited the in vitro growth of Alternaria alternata and reduced the growth of Salmonella typhimurium, Escherichia coli, mesophilic bacteria, and fungi isolated from decayed cucumbers. The addition of carnauba wax to the formulated CH coatings aided in lowering the water vapor transmission rate. Carnauba wax and essential oil treatments were the most effective in reducing weight loss.

Braga et al. (2020)

de Oliveira et al. (2020)

GonzálezLocarno et al. (2020)

GutiérrezPacheco et al. (2020)

(Continued)

Table 2.1 (Continued) Coating composition CH and tested concentration

Plant extracts or EO and tested concentration

Foods of plant origin

Main effects of coating

References

CH 0.25% and 0.5% (w/v)

Lippia multiflora EO 1% (v/v)

Maize and sorghum seeds

Mohamed et al. (2020)

CH 2% (w/v)

Ruta graveolens EO 0.5%, 1.0% and 1.5% (v/v)

Tomato “Chonto”

CH 2% (w/v)

Ruta graveolens EO 0.5%, 1.0% and 1.5%

Carica papaya

Among all the treatments used to control fungi growth, CH displayed a fungicidal effect against Rhizopus ssp and a fungistatic effect against Aspergillus flavus. Seed coating with CH solution increased the percentage of seed germination and seedling size. An inhibitory effect on fungi growth was also observed when seeds were coated with EO. However, using EO as a coating completely prevented seed germination. Furthermore, using EO and CH emulsion as a coating reduces CH’s antifungal activity, reduces germination percentage, and delays seedling growth. The mature index, weight loss, decay index, and disease damage incidence were significantly different from the control, indicating that the tomato had a preservative effect during storage (4°C/12 days). Furthermore, on the surface of coated tomatoes, the counts of aerobic mesophilic bacteria were significantly reduced, and mold and yeast were completely inhibited, without affecting sensory acceptance. During 12 days at 20°C, coated papaya fruit reduced/ inhibited the growth of Colletotrichum gloeosporioides lesions in a concentration-dependent manner. Furthermore, the emulsions were effective in reducing fruit surface microbiota and delaying papaya ripening without affecting sensory characteristics.

Peralta-Ruiz et al. (2020a, 2020b)

Peralta-Ruiz et al. (2020a, 2020b)

CH 0.5% and 1% (w/v)

Thymus EO 150 and 300 μL/L (v/v)

Grape cv. “Shahroudi”

CH 5 mg/mL (w/v)

Mentha piperita and M. 3 villosa Huds EO 0.3
1.25 μL/mL (v/v)

Papaya

CH 5 g/L (w/v)

Mentha piperita EO 1.25 and 0.6 mL/L (v/v)

Mango cv. Tommy Atkins

CH 1% (w/v)

Thyme EO 500 and 1000 μL/L (v/v)

Persian walnut

The results showed that CH and EO reduced the effect of fungus, decreased decay, respiration rate, and weight loss, and maintained fruit firmness during storage (0
2° C/90 days). Furthermore, sensory analyses revealed beneficial effects in terms of delaying browning and dehydration, as well as maintaining the visual characteristics of the grape without affecting taste or flavors. Development of anthracnose lesions caused by C. gloeosporioides and C. brevisporum isolates was reduced with the application of coatings that contained synergistic and/or additive mixtures of CH and EO, during storage (10 days, 25°C 6 0.5°C). Reduced anthracnose lesion development in papaya coated was comparable to or greater than that caused by a commercial fungicide formulation. During 30 days of cold storage (12°C), coatings formed by synergistic antifungal mixtures of CH and EO delayed senescence and maintained desirable postharvest quality characteristics (retention of pulp firmness, decreased weight loss, and increased acidity, possibly as a result of reduced organic acid degradation). Furthermore, coated mango had lower enzymatic activity, which likely resulted in delayed color development in these fruits. CH and thyme EO coating combined with active packaging had a significant effect on reducing oil oxidation and growth of molds. They also prevented the loss of moisture and a decrease in L value, improving the sensory properties of the samples during storage (120 days/relative humidity 55%/4°C). An increase in the essential oil up to 500 μL/L also improved the functional properties of CH coating.

Ardakani and Mostofi (2019)

Braga et al. (2019)

de Oliveira et al. (2019)

Habashi et al. (2019)

(Continued)

Table 2.1 (Continued) Coating composition CH and tested concentration

Plant extracts or EO and tested concentration

Foods of plant origin

Main effects of coating

References

CH 4 mg/mL (w/v)

Savory and/or tarragon EO 1.25 or 2.5 μL/ mL (v/v)

Kumquats

Hosseini et al. (2019)

CH 0.5% (w/v)

Thymol EO 200 mg/L (w/v)

Peach cv. “Zaferani”

CH 0.5% (w/v)

Thymol EO 200 mg/L (w/v)

Fig cv. “Siah”

CH 2% (w/v)

Ruta graveolens EO 0.5%, 1.0%, and 1.5% (v/v)

Guava

The CH and EOs coatings were effective in reducing weight loss, showed positive effects in maintaining TA and vitamin C content, and retained good sensory acceptability of fruits during storage (7°C/30 days). The preservative effect of CH and thymol was significantly better than the control treatment, with significantly lower weight loss, fungal decay, and TSS. Furthermore, the coated fruits had higher firmness, anthocyanin and carotenoid content, and sensory characteristics than the untreated control, resulting in a longer shelf life (approximately 30 days/6°C). The coated fruits with CH 1 thymol showed significantly lower weight loss, respiration rate, TSS, TSS/TA, and fungal decay incidence than other treatments. Furthermore, the coated fruits exhibited significantly higher firmness, TA, anthocyanin, and sensory characteristics, as well as showed the highest L and C values after 20 days storage at 6°C. The coated guavas exhibited better behavior concerning decay index, weight loss, maturation index, respiration rate, color, firmness, water activity, and microbiological decay as compared to the uncoated guavas. All the fruits coated had greater acceptance and quality than the controls and high percentage of inhibition in the development of anthracnose lesions (caused by Colletotrichum gloeosporioides) for at least 12 days at room temperature.

Rahimi et al. (2019)

Saki et al. (2019)

Tovar et al. (2019)

CH 2% (w/v)

Thymus capitatus EO 0.5%, 1.0%, and 1.5% (v/v)

Strawberries

CH (5 mg/mL) (w/v)

Cymbopogon citratus EO 0.15, 0.3, or 0.6 μL/ mL (v/v)

Guava, mango, and papaya

CH 1% (w/v)

Mentha spicata EO 0.1% and 0.2%

Strawberries

CH 0.4% (w/v) (sprayed)

Cinnamon EO 0.025% and aqueous extract of Roselle calyces (1%)

Strawberries

TSS, total soluble solids; TA, titratable acidity.

Treated fruit exhibited excellent stability with respect to microbial decomposition (aerobic mesophiles, molds, and yeasts), with a positive impact also on the physicalchemical composition (moisture loss, maturation, respiratory rate) and conservation of antioxidant activity (5°C/15 days). Coatings formed by synergistic combinations of CH and EOs decreased anthracnose lesion development in fruits inoculated with any of the tested Colletotrichum species (C. asianum, C. siamense, C. fructicola, C. tropicale, and C. karsti) during storage (25°C/12 days). Overall, the inhibition of anthracnose lesion development in fruit coated was greater than that observed in fruit treated with synthetic fungicides. Treatments resulted in a significant delay in the total viable count, psychrotrophic bacterial, yeasts and molds, weight loss, titratable acidity, and pH, and had also positive effects on water vapor resistance and respiration rate of coated strawberries. In addition, coatings also successfully decreased the population of L. monocytogenes during storage time (4°C/12 days). The formulated coating be considered an effective postharvest technology for the control of Colletotrichum fragariae, during 13 days of storage as a preventive treatment, 10 days as a controlling treatment and 17 days on coated fruit, non-inoculated and stored at 5°C. The weight loss was reduced and the firmness was higher in the fruits treated and stored at 5°C. Under the same conditions, the color and ripeness index as quality parameters were maintained.

Martínez et al. (2018)

Oliveira et al. (2018)

Shahbazi (2018)

VenturaAguilar et al. (2018)

52

Chitosan: Novel Applications in Food Systems

The increase of pH and SS is due to metabolic processes and reactions during postharvest storage, which continue to convert starch and organic acids into sugar (Peralta-Ruiz et al., 2021). Fruit ripening with storage causes an increase in SS, which is caused by the solubilization of complex carbohydrates into simpler structures. The use of CH and EOs coatings is effective in delaying respiratory activity in fruits, resulting in reduced carbohydrate hydrolysis, the preservation of organic acids, a better delay in pH increase, and a reduction in the effect of ripening (Basaglia et al., 2021; Quintana et al., 2021). The barrier property also has a positive impact on SS, reducing the conversion of starch to sugar. Color modification is another distinguishing feature of the ripening process, which is associated with chlorophyll degradation and increased carotenoid content, respectively. Coating CH and EOs reduced respiration rates, which resulted in slower chlorophyll degradation and color pigment production progression. EOs can also protect plant tissues from oxidation and slow the breakdown of chlorophyll pigments, assisting in the preservation of the original colors of fruits and vegetables (Abdelgawad et al., 2022). Enzymatic browning is regarded as one of the most important factors in the marketing and quality of fruits and vegetables (primarily those that have been minimally processed), which may be related to their phenolic compound content. PPO and peroxidase (POD) are two enzymes involved in the oxidative breakdown of phenolic compounds. Coating of CH and EOs reduces O2 permeability, which decreases enzyme activity, reducing the severity of enzymatic browning and oxidative reactions in plant tissues, resulting in higher levels of endogenous phenolic substances found in coated products (Abdelgawad et al., 2022). EOs can participate in enzymatic processes that increase the biosynthesis of phenolic compounds while also decreasing the oxidation process, contributing even more to the increase in phenolic compound levels. The role of EOs is to reduce respiratory enzymes, which results in less sugar loss (Abdelgawad et al., 2022). CH coating, alone or in combination with EOs, can effectively increase superoxide dismutase activity in fruit, improving ROS scavenging potential and postharvest senescence and quality deterioration (Wang et al., 2022). 2.2.3.2 Cereals and oilseeds CH coating containing EOs was also evaluated as an alternative to increase microbial safety and improve the quality parameters of seeds and

Chitosan-based coatings and plant extracts

53

sprouts (Abdelgawad et al., 2022; Habashi et al., 2019). Cereals and pulses, as well as their products, are the foundation of most people’s diets. However, due to unfavorable climatic conditions, poor soil quality, and contamination with a variety of microorganisms, their yields can be consistently low. This type of contamination reduces grain germination, nutritional quality, and output (Lukseviciute & Luksiene, 2020; Mohamed et al., 2020). CH coating with EOs is a potential pest prevention technology because it improves seed quality, germination, and seedling growth, protecting both the seeds and the plant from pests. Mohamed et al. (2020) investigated the use of EOs extracted from Lippia multiflora leaves alone or in combination with CH as coating solutions for maize and sorghum seeds. The use of CH solution as seed coating increased the percentage of seed germination and seedling size, while also inhibiting the growth of contaminating fungi. Another study (Habashi et al., 2019) evaluated the effects of CH coating with thyme EO and different packaging methods on the shelf-life and quality of walnut kernels, and in this study a significant effect on reducing oil oxidation and growth of molds, as also a decreases in L value, loss of moisture and improvement the sensory properties during storage of coated samples and combined with active packaging, were observed. Important to note that the higher L value could be attributed to the antioxidant properties of CH and EOs which inhibited the oxidation/enzymatic browning of phenolic compounds (Habashi et al., 2019). Other works involving grains, seeds and derivatives are summarized in Table 2.1. 2.2.3.3 Meats and meat products The nutritional content and water content of perishable products, such as cheeses and meats (fresh, frozen, and processed), are the most sensitive parameters during storage, as they allow the proliferation of microorganisms and the degradation of the product, primarily through the production of metabolites formed by the reaction of microorganisms with components such as proteins found in cheese, meat, or fish (Díaz-Montes & Castro-Muñoz, 2021). Because of their desirable properties such as preventing gases and moisture loss, reducing oxidative reactions, preserving taste, improving the appearance of the product, preventing color changes and oxymyoglobin formation, preventing liquid loss from lean meat tissue, and maintaining nutritional value, edible coatings can be used to improve the quality (enhance nutritional value) of these products without changing essential ingredients or processing methods (Table 2.2) (Hashemi et al., 2020).

Table 2.2 Effects of chitosan (CH) coatings and plant extracts or essential oil (EO) on quality parameters (microbiological, physicochemical, and sensory) of foods of animal origin. Coating composition CH and tested concentration

Plant extracts or EO and tested concentration

Meats, fish, and derivatives

Main effects of coating

References

CH 1.0%, 1.5%, 2.0% (w/w)

Lemongrass EO 0.5%, 1.5%, 2.5% (v/w)

Chicken patties

Contini et al. (2022)

CH (nondetailed concentration)

Elettaria Cardamomum ethanol extracts and EO (nondetailed concentration)

Chicken drumsticks

CH 2.0% (w/v)

Origanum vulgare EO (1% v/v) and grape seed extract (1% and 2% v/v)

Turkey meat

The higher antioxidant capacity was obtained with 1.0% of CH and 2.5% of EO. Coating applied to hamburgers chicken patties protected the lipidic oxidation (lower TBARS) and maintained the microbiological and pH characteristics. The odor and flavor of EO led to a sensory acceptance index of less than 70%. Peroxide, TVB-N, and pH values for the uncoated sample were higher than those documented for the coated groups at the end during storage (4°C/16 days). The results of the index of TBARS revealed that the sample containing the EO is the best treatment. Microbial analysis revealed that the coating significantly reduces the growth of all bacteria groups tested. It was also discovered that adding EO and extracts can significantly improve the sensory quality of the samples. Results concluded that coating of turkey breast meat with CH, grape seed extract and O. vulgare EO was effective against the propagation of different kinds of spoilage microorganisms including total viable counts (TVC), Enterobacteriaceae, Pseudomonas spp., lactic acid bacteria, and yeast-mold counts. It also postponed lipid oxidation and maintained sensory properties and subsequently prolonged the shelf life of the fresh turkey meat during cold storage (4°C/20 days).

Khorshidi et al. (2021)

Langroodi et al. (2021)

CH 2.0% (w/v)

Berberis vulgaris extract and Mentha pulegium EO (2% and 4% v/v)

Turkey meat

CH 1.0% (w/v)

Artemisia fragrans EO (500, 1000, and 1500 ppm)

Chicken fillets

CH 1.5% (w/v)

Ajwain EO (0.5%, and 1%)

Chicken breast meat

CH (fully deacetylated) 1 and 1.5% (w/v)

Clove EO 0.25% and 0.5%

White shrimp

Shelf life of turkey meat increased after application of CH, Berberis vulgaris extract, and Mentha pulegium EO, with decreasing lipid oxidation and microbial numbers. The combination of Berberis vulgaris extract and Mentha pulegium EO had more detectable inhibitory effects on the microbial count of the turkey samples than any of them applied alone. These treatments revealed also acceptable sensory properties, including texture, odor, taste, color, and total admissibility in the turkey samples. The results showed that the incorporation of Artemisia fragrans EO into CH coating significantly reduced pH, TBARS, and TVBN of chicken fillets during refrigerated storage. The counts of TVC, coliforms, molds, and yeasts were significantly lower, and sensory attributes of coated samples also showed the highest overall acceptability scores than uncoated ones. The results showed that using of Ajwain EO has significant effects on the reduction of all groups of microorganisms (total aerobic mesophilic bacteria, Enterobacteriaceae, total aerobic psychrotrophic bacteria, and Pseudomonas spp.) compared with the control group, and that 1% Ajwain EO treatment was the most effective in microbial groups throughout the storage period (4°C/13 days). CH and clove essential oil (0.25%) edible coating combined with kojic acid (0.25%) effectively lengthened the shelf life of shrimp by inhibiting the increase in total aerobic plate, color change, melanosis, and changes in pH and total volatile basic nitrogen content. Furthermore, these coatings also allowed to shrimp to retain its texture, sensory properties, and moisture during refrigerated storage (4°C/15 days).

Sayadi et al. (2021)

Yaghoubi et al. (2021)

Babolanimogadam et al. (2020)

Liu et al. (2020a, 2020b)

(Continued)

Table 2.2 (Continued) Coating composition CH and tested concentration

Plant extracts or EO and tested concentration

Meats, fish, and derivatives

Main effects of coating

References

CH 2% (w/v)

Lemon verbena EO 0.5% and 1% and ethanol extract 1% and 2%

Rainbow trout

Rezaeifar et al. (2020)

CH 1% (w/w)

Thyme 1% or rosemary EO 1%

Dry-fermented sausages

CH 2% (w/v)

Mentha aquatica L. essence (0.5%, 1%, and 1.5% v/v)

Iranian white cheese

CH 1.5% (w/v)

Santolina chamaecyparissus industrial solid residue extract 1% (w/v)

“Manchego” Cheese

Coating of CH with extract and essential oil of lemon verbena significantly reduced psychrotrophic bacteria, total viable counts, Enterobacteriaceae, and H2S producing bacteria. Lower values of TBARS, peroxide value, TVB-N, and pH were also detected in coated samples. Generally, CH 1 extract 1% 1 EO 0.5% had the best suppress effect on the chemical situation and microbial population in fish meat stored in a refrigerator (4°C/ 16 days). CH, thyme, and rosemary EO treatments inhibited fungal growth on casings while they resulted in lower Gram (1) catalase (1) cocci, Enterobacteriaceae, mold, and yeast counts in sausages. Lower TBARS values were determined for samples coated with EO. CH
EO treatments supported also the protection of characteristic sensory attributes such as acidic and spicy odor and taste during of refrigerated storage (4°C/3 months). The results indicated a remarkable antibacterial activity of the applied active packaging system including CH coating and M. aquatica L. essence against E. coli, S. aureus, and L. monocytogenes and its capability as a natural, healthy, and safe preservative system to improve white cheese shelf-life. By using a CH coating and 1.5% M. aquatica L. essence, after 10 days of storage at 4°C and probably also at the end of the 15th day, E. coli growth was completely stopped. Results showed that the addition of the Santolina chamaecyparissus industrial solid residue extract confers significant antioxidant capacities to the CH coating, increases its antifungal capacities, and enhances some physical properties.

Soncu et al. (2020)

Zavareh and Ardestani (2020)

Elguea-Culebras et al. (2019)

CH 1.5% (w/v)

Citrus EO 1.5%

Pacific mackerel

CH 2% (w/v)

Zataria multiflora EO 0.5% and 1% and propolis extract 1%

Chicken breast meat

CH 0.5% and 2% (w/v)

Clove EO 0.08% and 0.16%

Tambaqui fillets

CH 1.5% (w/v)

Lemon and thyme EO 0.5% and 0.25%

Grass carp fillets

Coating showed significantly greater capacity to eliminate reactive oxygen species (O22 and OH2) and reduce parameters of corruption including physicochemical (drop loss, biogenic amine, and TBARS) and microbiological parameters (total viable count) after 12 days of storage at 23°C. Adding of citrus essential oil has a more significant effect on controlling the deterioration of the quality of Pacific mackerel during superchilled storage than using CH alone. Samples coated with CH 1 propolis extract (1%) 1 Zataria multiflora EO (0.5% and 1%) showed detectably lower microbial count for mesophilic total viable plate counts, lactic acid bacteria, psychotropic bacteria, and Pseudomonas spp. at the last day of storage (16 days). In addition, the results of chemical characteristics (pH, TVB-N, TBARS), revealed that there is a synergistic effect between CH, propolis extract, and Zataria multiflora EO, as they presented less elevation of values and with acceptable sensory characteristics such as color, odor, texture, taste, and total acceptability. CH 1 CEO treatments effectively delayed lipid oxidation, inhibited the growth of culturable psychrotrophic bacteria, and reduced pH and moisture at 120 days (218°C). However, the sensory analysis revealed that panelists preferred CH-treated fillets to CH 1 CEO-treated fillets. CH coating (1.5%) 1 0.5% essential oils treatment maintained higher hardness and retarded the oxidation of lipid and protein. In addition, the most effective treatment was CH (1.5%) 1 lemon EO (0.25%) 1 thyme EO (0.25%), which maintained the color and texture properties, reduced the microorganisms counts, and extended its shelf life to 16 days (2°C).

Li et al. (2019)

Mehdizadeh and Langroodi (2019)

Vieira et al. (2019)

Cai et al. (2018)

(Continued)

Table 2.2 (Continued) Coating composition CH and tested concentration

Plant extracts or EO and tested concentration

Meats, fish, and derivatives

Main effects of coating

References

CH 2% (w/v)

Zataria multiflora EO 1% and hydroalcoholic extract of sumac 2% and 4%

Beef

Langroodi et al. (2018)

CH 1% (w/w)

Thyme 0.2%
1% or rosemary 0.2%
1% EO

Fermented sausages

CH 2% (w/v)

Cumin EO 1%

Turkey breast meat.

The results revealed that Zataria multiflora essential oil and different concentration of the hydro-alcoholic extract of sumac compared to other treatment and control samples were most effective and were able to inhibit the bacterial growth (total viable counts, lactic acid bacteria, Pseudomonas spp., Enterobacteriaceae and yeasts-molds), and apart from the color, they improved the chemical characteristics (significantly lower values of TVB-N, TBARS values and peroxide value) and the sensory quality of meat except flavor factor. Growth of mold on the surface of sausages can be inhibited using CH 1 thyme EO or CH 1 rosemary EO without affecting the interior microbiota. Higher levels of EOs (0.8% 2 1%) were found to be effective against Enterobacteriaceae counts and surface mold growth. Moreover, antifungal treatment did not negatively affect the physicochemical (moisture; aw; pH; titratable acidity), color, and sensory properties during storage. The results of microbial (counts and applied psychrophilic bacteria), physicochemical (pH, TVN-B, and peroxide value) and sensory evaluation analyses indicated that coating on turkey breast fillets may lead to the preservation of qualitative characteristics, improvement of microbial safety (counts decreased) and extension of the shelf life of meat products (4° C/15 days).

TBARS, thiobarbituric acid reactive substances; TVB-N, total volatile base nitrogen.

Soncu et al. (2018)

Taheri et al. (2018)

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Several studies have been conducted on the production of CH-based coatings containing EOs and their application in meat products such as pork, chicken, and red meat. When EOs and their bioactive compounds are incorporated into CH coatings, they can improve the film matrix and increase the shelf-life of meat products (Hashemi et al., 2020). During the processing of meat products the gradual increase in pH values can be attributed to endogenous and microbial enzymes, such as lipase or protease, or the use of amino acids by bacteria (Khorshidi et al., 2021). Application of CH coatings with EOs can reduce pH values in meat, maintain low antioxidant and antibacterial activity. One of the most important indices of chemical parameters, is, Total Volatile Base Nitrogen (TVB-N) and allows to assess the quality and freshness of meat and meat products (Dainty & Mackey, 1992). CH (alone or in combination with EOs) can significantly reduce TVB-N values. This can be attributed to the reduction in the number of spoilage bacteria and their oxidative ability to produce TVB-N from amine compounds (Khorshidi et al., 2021), that usually is responsible for off-odor or offflavor in the spoiled meats or meat products. Lipid peroxidation is another important chemical reaction that occurs during cold storage and has a negative impact on the sensory, functional, and nutritional quality of meats. In general, oxygen and CO2 penetrate edible coatings in very small amounts. As a result, the coatings reduce contact with oxygen and, as a result, the rate of early oxidation and the formation of hydroperoxides. Hydroperoxides are oxidation products that determine the rate of fat oxidation in the early stages and have a significant impact on product acceptability (Khorshidi et al., 2021). In addition, CH coatings increases the activity of some antioxidant enzymes, preventing flesh browning and reducing membrane damage. Malondialdehyde, as measured by TBARS, is the second and most important byproduct of fat oxidation and serves as an indicator of oxidation and rancidity. The TBARS value is commonly used to calculate the rate of fat oxidation and the level of malondialdehyde in meat (Khorshidi et al., 2021). Many researchers reported that CH coating with EOs could effectively prevent lipid oxidation, the microbial degeneration, and consequently reducing values of TBARS (Contini et al., 2022; Yaghoubi et al., 2021). Because of its free radical scavenging property related to nonphenolic terpenoid components of EOs and the strong hydrogen donating capacity of CH, the presence of lemongrass EO in CH-based coatings significantly reduced TBARS in chicken patties (Contini et al., 2022).

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Chitosan: Novel Applications in Food Systems

2.2.3.4 Fish and seafood Fresh fish and seafood, unlike other fresh commodities, are highly perishable and are therefore frequently marketed as frozen or processed products. Deterioration occurs primarily as a result of physicochemical reactions, enzymatic actions, and microbial metabolism, which compromises the sensory and nutritional quality of food and results in economic losses for producers, as well as the presence of foodborne pathogens, which may pose health concerns to consumers (Vieira et al., 2019). The combination of CH and EOs has been studied in recent years in order to extend the shelf-life of fishery products, which are highly perishable due to their high water activity, resulting in microbiological spoilage and quality degradation (Yu et al., 2017). Furthermore, it has been reported that the CH coating incorporated into EOs reduces nucleotidase activity and inhibits ATP decomposition, both of which play an important role as chemical indicators and thus in maintaining the quality of fresh fish/seafood (Cai et al., 2018). In one study, CH coatings combined with clove EO effectively extended the shelf-life of shrimp by inhibiting bacterial growth, color change, melanosis, and pH and TVB-N changes. Furthermore, these coatings allowed the shrimps to keep their texture, sensory properties, and moisture (Liu et al., 2020a). The combination of CH coatings and Clove EOs also demonstrated good promise as a natural preservative, inhibiting chemical (oxidative) and microbial (psychrotrophic bacteria) deterioration of frozen tambaqui fillets for 120 days (Vieira et al., 2019). The use of CH coatings infused with EOs (clove, cinnamon, and lemongrass) was found to help preserve quality and reduce oxidative damage in refrigerated grass carp fillets. In comparison, the CH
clove EO coating had the best effects, inhibiting physicochemical quality deterioration and microbial growth while also maintaining antioxidant enzyme activities during refrigeration storage (Yu et al., 2017). 2.2.3.5 Cheeses Microorganisms (bacteria, molds, and yeasts), photo-oxidation reactions, and excessive surface dehydration in the cheese rind cause significant economic losses in cheese makers, resulting in undesirable flavors and tastes, primarily affecting the cheese surface and causing significant visual alterations (Costa et al., 2018). This can be resolved in an ecological way using different edible and biodegradable active coatings (Elguea-Culebras et al., 2019).

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Edible coatings act as a semipermeable barrier to oxygen, carbon dioxide, and water vapor, allowing for reduced water loss, and control of the ripening rate (Costa et al., 2018). These coatings, in this context, create a modified atmosphere around the merchandise, similar to controlled or modified atmosphere packaging. It is important to note, however, that the requirements for the composition and production of the coatings may vary depending on the type of cheese, as maturation rates, water content, and mechanical stability are all affected by the composition (Costa et al., 2018). According to Cano Embuena et al. (2017), CH coatings containing rosemary and oregano EOs prevented weight loss, reduced lipolytic and proteolytic activities, and improved microbial safety of semihard goat cheeses during ripening. The sensory evaluation also revealed that cheeses doubly coated with CH and oregano EO had the best aroma and flavor attributes. The application of CH coating and essence of Mentha aquatica L. (1.5%), after 10 days of storage at 4°C strongly inhibited Escherichia coli growth in Iranian white cheese (Zavareh & Ardestani, 2020). Also, under the same conditions, significant growth inhibition of 88.54% and 85.9% were obtained for Staphylococcus aureus and Listeria monocytogenes, respectively, which proves that CH coating in combination with an EOs may present a viable alternative to chemical preservatives, to improve both the shelf-life and safety of cheese products (Zavareh & Ardestani, 2020).

2.3 Future application prospects CH coatings with EOs are a strong natural candidate for future replacement of nonbiodegradable plastics and chemical additives. It has been possible to extend the shelf-life of fresh or processed fruits, vegetables, poultry, and meat through the use of edible coatings while maintaining sensory attributes and improving overall quality. However, there are challenges in the use of EOs in food coatings due to their low solubility in water, high volatility, and low sustainability during food processing, which must be thoroughly investigated before any commercial applications in foods are made. Encapsulation of EOs, particularly on the nanoscale, could aid in the resolution of this issue, as well as providing an alternative to reduce the aroma and flavor of coatings and improve consumer acceptance. Encapsulation also improves compound

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Chitosan: Novel Applications in Food Systems

solubility, mask flavors, protects functional compounds from degradation, and regulates active compound release. CH nanoparticles are also emerging as antimicrobial food packaging alternatives, where nanometer-scale control of material properties allows for the exploration of new functionalities. This method is typically used on fruits and vegetables that have been dipped into coating solutions containing the EOs trapped in CH nanoparticles. The incorporation of EOs in nanocomposites also allows for the modification of properties other than antioxidant and antimicrobial activity, such as film transparency or hydrophobicity, while maintaining mechanical strength.

Acknowledgements The authors are grateful the National Council for Scientific and Technological Development (# 402745/2021-3; CNPq-Brazil) and to Coordination of Higher Personnel Improvement (CAPES-Brazil) for a postdoctoral scholarship awarded to K.Á.R. de Oliveira (finance code 001) and an MSc scholarship awarded to K.F.D. Fernandes.

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rosemary essential oils to the volatile composition, microbial profile, and physicochemical and sensory quality of dry-fermented sausages during storage. Meat Science, 166, 108127. Taheri, T., Fazlara, A., Roomiani, L., & Taheri, S. (2018). Effect of chitosan coating enriched with cumin (Cuminum cyminum L.) essential oil on the quality of refrigerated turkey breast meat. Italian Journal of Food Science, 30(3). Tovar, C. D. G., Delgado-Ospina, J., Navia Porras, D. P., Peralta-Ruiz, Y., Cordero, A. P., Castro, J. I., Valencia, M. N. C., Mina, J. H., & Chaves López, C. (2019). Colletotrichum gloesporioides inhibition in situ by chitosan-Ruta graveolens essential oil coatings: effect on microbiological, physicochemical, and organoleptic properties of guava (Psidium guajava L.) during room temperature storage. Biomolecules, 9(9), 399. Ventura-Aguilar, R. I., Bautista-Baños, S., Flores-García, G., & Zavaleta-Avejar, L. (2018). Impact of chitosan based edible coatings functionalized with natural compounds on Colletotrichum fragariae development and the quality of strawberries. Food Chemistry, 262, 142
149. Vieira, B. B., Mafra, J. F., da Rocha Bispo, A. S., Ferreira, M. A., de Lima Silva, F., Rodrigues, A. V. N., & Evangelista-Barreto, N. S. (2019). Combination of chitosan coating and clove essential oil reduces lipid oxidation and microbial growth in frozen stored tambaqui (Colossoma macropomum) fillets. LWT-Food Science and Technology, 116, 108546. Wang, H., Zhang, Z., Dong, Y., & Wang, Y. (2022). Effect of chitosan coating incorporated with Torreya grandis essential oil on the quality and physiological attributes of loquat fruit. Journal of Food Measurement and Characterization, 1
11. Xylia, P., Chrysargyris, A., & Tzortzakis, N. (2021). The combined and single effect of marjoram essential oil, ascorbic acid, and chitosan on fresh-cut lettuce preservation. Foods, 10(3), 575. Yaghoubi, M., Ayaseh, A., Alirezalu, K., Nemati, Z., Pateiro, M., & Lorenzo, J. M. (2021). Effect of chitosan coating incorporated with Artemisia fragrans essential oil on fresh chicken meat during refrigerated storage. Polymers, 13(5), 716. Yousuf, B., & Srivastava, A. K. (2017). A novel approach for quality maintenance and shelf life extension of fresh-cut Kajari melon: effect of treatments with honey and soy protein isolate. LWT-Food Science and Technology, 79, 568
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Yu, D., Xu, Y., Jiang, Q., & Xia, W. (2017). Effects of chitosan coating combined with essential oils on quality and antioxidant enzyme activities of grass carp (Ctenopharyngodon idellus) fillets stored at 4°C. International Journal of Food Science & Technology, 52(2), 404
412. Yu, K., Xu, J., Zhou, L., Zou, L., & Liu, W. (2021). Effect of chitosan coatings with cinnamon essential oil on postharvest quality of mangoes. Foods, 10(12), 3003. Zavareh, S. A. H. T., & Ardestani, F. (2020). Antibacterial effects of chitosan coating containing Mentha aquatica L. essence against Escherichia coli, Staphylococcus aureus and Listeria monocytogenes in Iranian white cheese. International Journal of Dairy Technology, 73(3), 585
593. Zhang, Z., Zhao, P., Zhang, P., Su, L., Jia, H., Wei, X., Fang, J., & Jia, H. (2020). Integrative transcriptomics and metabolomics data exploring the effect of chitosan on postharvest grape resistance to Botrytis cinerea. Postharvest Biology and Technology, 167, 111248.

CHAPTER 3

Chitosan nanoparticles as used against food pathogens Daniel Hernandez-Patlan1,2, Bruno Solis-Cruz1,2, Xochitl Hernandez-Velasco3 and Guillermo Tellez-Isaias4 1

Laboratory 5: LEDEFAR, Multidisciplinary Research Unit, National Autonomous University of MexicoSuperior Studies Faculty at Cuautitlan, Cuautitlan Izcalli, Mexico Nanotechnology Engineering Division, Polytechnic University of the Valley of Mexico, Tultitlan, Mexico 3 Department of Avian Medicine and Zootechnics, College of Veterinary Medicine and Zootechnics, National Autonomous University of Mexico, Mexico City, Mexico 4 Department of Poultry Science, University of Arkansas, Fayetteville, AR, United States 2

Abbreviations CBE CS-NC CS-NP CUR MRS DD DSC EOs FTIR LBL LNP LNPC Lys-CS-NP NCs NP Or-CNPs Th-CNPs NRM TEER

cinnamon bark extract chitosan-coated nanocapsules chitosan nanoparticles curcumin de Man Rogosa Sharpe medium degree of deacetylation differential scanning calorimetry essential oils Fourier transform infrared spectroscopy layer-by-layer lecithin-chitosan nanoparticles lecithin-chitosan nanoparticles containing curcumin lysozyme integrated into chitosan nanoparticles nanocapsules chitosan nanoparticles nanoparticulate systems based on chitosan loaded with EO of Origanum vulgare nanoparticulate systems based on chitosan loaded with EO of Thymus capitatus nuclear magnetic resonance transepithelial electrical resistance

3.1 Introduction Foodborne illness is a significant and substantial burden on public health, as well as economic and social burden throughout the world. In 2015 the World Health Organization (WHO) reported that, each year, 1 in 10 people became ill from food contaminated with microbial or chemical agents, resulting in 2 billion people getting sick and over 1 million deaths worldwide Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00008-9

© 2023 Elsevier Inc. All rights reserved.

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(Havelaar et al., 2015; Kirk et al., 2015). In particular cases such as the United States, foodborne illnesses in 2016 caused 48 million people to get sick, and of these 128,000 were hospitalized and 3000 died (CDC, 2016). Meanwhile, in the European Union, 23 million people are infected with foodborne pathogens and 5000 die from them each year (World Health Organization, 2015). Considering this situation, although the prevention of food contamination with foodborne pathogens through good hygiene and agricultural and manufacturing practices continues to be the main strategy to follow, the use of polymers and biopolymers to develop materials with potential in food applications as a promising and growing alternative could help avoid and reduce such problems (Randazzo et al., 2018). Despite the global polymer market was reported in 2018 to be worth $666.6 billion and was expected a compound annual growth rate of 5.1%, the global polymer market is heavily affected for that of biopolymers. In 2018 the global biopolymer market was estimated at $12 billion and is expected to grow significantly at a compound annual growth rate of 19% between 2019 and 2025 due to their diverse food and nonfood applications and the demand for biodegradable polymers (Baranwal et al., 2022). Biopolymers are simple macromolecules produced by living organisms and are synthesized by processive enzymes that link building blocks such as carbohydrates, proteins, nucleic acids, lipids, amino acids, and peptides (Moradali & Rehm, 2020) or are the organic substances present in natural sources. Currently, three types of biopolymers are recognized based on their monomeric units and structure: polynucleotides (deoxyribonucleic acid and ribonucleic acid), polysaccharides (cellulose and chitosan chitin), and polypeptides (collagen, gelatin, gluten, and whey) (Mohan et al., 2016). It is known that after cellulose, chitin and chitosan are, in fact, the two most predominant natural polymers and considered low cost in terms of production (Kostag & El Seoud, 2021). Due to various attractive properties of chitosan, such as biodegradability, natural origin, abundance, and reactivity, it has applications in areas such as medicine, agriculture, food processing, nutritional enhancement, cosmetics, and water and waste treatment (Ibrahim & El-Zairy, 2015). The biological properties of chitosan and its derivatives include antitumor, antiinflammatory, antioxidant, and antimicrobial activities. However, in the food industry, chitosan has been used mainly as an antimicrobial and antifungal agent in food preservation (Aranaz et al., 2021; Ibrahim & El-Zairy, 2015). Perhaps one of the most important characteristics that chitosan presents is its good film-forming properties, therefore it is highly useful in gels and

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coatings. Unfortunately, chitosan bio-based films have poor mechanical properties and are highly hygroscopic, causing loss of their physical integrity (Torlak & Sert, 2013). However, the addition of nanoparticles to bio-based chitosan films can be a successful approach to improve the physicochemical and mechanical properties of chitosan films. In fact, the use of nanoparticles, in particular chitosan nanoparticles, is often reported to improve the mechanical and barrier properties of other polysaccharide films, composed of pectin or starch (Gomes et al., 2018b). Therefore the objective of this book chapter is to show the applications of chitosanbased nanosystems in the food industry to reduce contamination problems associated with foodborne pathogens.

3.2 Chitosan: generalities Chitosan, a linear polysaccharide that consists of glucosamine and N-acetylglucosamine units, is a deacetylated derivative of chitin derived from the exoskeleton of crustaceans, insects, or fungi that was discovered by Rouget in 1859 after heating chitin in an alkaline medium (Khor, 2001). Several years later, Hoppe Seyer named this material chitosan, although its chemical structure was not clarified until 1950. It is available in a wide range of deacetylation degrees and molecular weights, which are the main factors influencing the nature and quality of the polymer. Chitosan, as a biomaterial, is abundant and cheap and can be easily transformed into various solid and semisolid structures (Szyma´nska & Winnicka, 2015). In this sense, chitosan and its derivatives have attracted attention due to their properties, among which their nontoxicity, biodegradability, biocompatibility and immunopotentiation, antitumor, antibacterial, and antimicrobial activities stand out (Kravanja et al., 2019). Furthermore, it has been reported that chitosan has an influence by decreasing the absorption of cholesterol (Gallaher et al., 2000), interrupting the oxidation process by free radicals (Liu et al., 2017), and acting as an antimicrobial against many bacteria, yeasts, and fungi (Goy et al., 2009). Specifically in terms of the antimicrobial activity of chitosan and its derivatives, it is known that it depends on numerous factors such as degree of deacetylation (DD), molar weight, pH, presence of metallic cations, pKa, and species of microorganisms.

3.2.1 Physicochemical properties It has been reported that the solubility of chitosan depends primarily on its biological origin, followed by molecular weight and DD (Kumari et al., 2017).

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However, chitosan is generally soluble in both inorganic and dilute organic acids with a pH lower than the pKa of chitosan (pKa 5 6.3), forming a nonNewtonian, shear-thinning fluid. At low pH values, the free amine groups are positively charged due to protonation, causing an electrostatic repulsion between the polymer chains, and thus allowing the solvation of the polymer, which translates as an increase in aqueous solubility. In contrast, when the pH is greater than 6, the amine groups of chitosan are deprotonated and the biopolymer loses its charge leading to an insoluble polymer (Szyma´nska & Winnicka, 2015; Yi et al., 2005). Furthermore, chitosan has good mucoadhesive properties due to its ionic character given the presence of free hydroxyl and amino groups in its structure, which allow the polymer to interact with mucin through hydrogen and electrostatic bonds (Szyma´nska & Winnicka, 2015). Table 3.1 confirms the interactions between mucin and low-molecularweight chitosan-coated nanocapsules (CS-NC) at different pHs in an in vitro study. The interactions were evaluated by determining the zeta potential (unpublished data by our research group). It has also been reported that chitosan shows properties that improve the penetration of active components by improving their transport through the intestinal epithelium due to its ability to open tight junctions (Yeh et al., 2011). In this sense, our research group published an article in which curcumin (CUR) was used as a model to evaluate the ability of CS-NC to increase their permeability in an in vitro model of Caco-2 cells (Hernandez-Patlan et al., 2019). The results showed that the permeability of CUR increased 28.6-fold when it was in CS-NC compared to raw CUR (Table 3.2). As explained previously and relating it to CUR, the increase in permeability of CUR was mainly due to the ability of chitosan to temporarily open tight junctions. Therefore transepithelial electrical resistance (TEER) was monitored Table 3.1 Zeta potential of chitosan-coated nanocapsules (CS-NC), mucin and their interaction at different pHs. Component

Zeta potential (mV)

CS-NC Mucin pH 5 1.2 Mucin pH . 7 CS-NC 1 Mucin pH 5 1.2 CS-NC 1 Mucin pH . 7

24.9 1.52 214.6 1.734 211.1

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Table 3.2 Mean apparent permeability and the absorption enhancement ratio of chitosan-coated nanocapsules (CS-NC) and curcumin across Caco-2 cells monolayers after 2 h of incubation. Formulation

PAPP 3 10 6 (cm/s)

R

Curcumin Chitosan-coated nanocapsules

4.96 6 0.36 141.60 6 37.62

28.6

Values are given as the mean 6 SD; n 5 3,  P , .05 significantly different from CUR.

105 100 95

TEER (%)

90 85 80

CTRL

75

CUR

70

CS-NC

65 60

0

200

400

600

800

Time (min)

Figure 3.1 Effect of chitosan-coated nanocapsules (CS-NC, dose per unit area of 32.9 μg/cm2 or 125 mg/mL of polymer), curcumin (CUR, dose per unit area of 32.9 μg/cm2 or 125 μg/mL), and Dulbecco’s Modified Eagle Medium (Control) on the values of transepithelial electrical resistance (TEER) as a function of time. Values are given as mean 6 SD; n 5 3.

during the study (Fig. 3.1) to relate it to the opening of the tight junctions due to the reversible structural reorganization of the junction proteins and a specific redistribution of the F-actin cytoskeleton and the ZO-1 protein (present in the tight junctions), leading to the increase in paracellular transport (Amidi et al., 2010; Pasternak & Miller, 1996; Van der Lubben et al., 2001).

3.2.2 Extraction methods and characterization Chitin belongs to the group of structural polysaccharides, together with cellulose, being the second most abundant polymer in the biosphere.

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Due to its structural nature, it is not only present in the exoskeletons of crustaceans such as shrimp, but also in the exoskeletons of arthropods, in the structures of mollusks, in the cell wall of fungi, protozoa, and bacteria, and in the eggshells of nematodes. However, the main source remains the exoskeleton of crustaceans (Martín-López et al., 2020; Pighinelli et al., 2019). Currently, different extraction methods have been reported, with the chemical method, being the most frequently found in the literature and used for industrial production purposes (Pighinelli et al., 2019). Nevertheless, countries such as the United States, Japan, India, Canada, China, South Korea, Russia, and Norway are trying to change chemical methods of chitin production to more viable methods, since during the chemical methods strong acids and bases are used for its extraction, causing critical points in the process, as well as high production costs due to the materials involved, generation of chemical effluents, and final product with low levels of purity (Flores-Albino et al., 2012; Hajji et al., 2014). Therefore biological processes have become more attractive since they have an affordable production cost, do not generate high-risk effluents (as the chemical processes), and a high-quality final product is obtained (Gortari & Hours, 2013; Jaganathan et al., 2013). Chitin, in addition to having great biotechnological value, generates by-products such as chitosan that also has added value and even more relevant properties. 3.2.2.1 Chemical process of extraction This is currently the most widely used method both in industrial production and in the laboratory (Fig. 3.2). The purpose of this extraction process is to completely remove the organic and mineral content of the raw material. For this, two essential steps are carried out to obtain chitin: deproteinization and demineralization. However, the order of these steps could vary depending on the objective and the chemical components to be used. In addition, a depigmentation and deodorization step can also be carried out during chitin production, if necessary (Mohan et al., 2020; Pighinelli et al., 2019). Before starting the extraction process, the raw material is pretreated to remove impurities and coarse organic residue. In this stage, rinses are carried out with deionized water, and sometimes sodium hypochlorite can be applied. Eventually, the temperature is increased to speed up the cleaning process. Subsequently, a milling process is carried out to improve the interaction with the components of the following stages. These pretreatments

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Pretreatment (Washing, Crashing, and Milling)

Source of Chitin

Demineralization (hydrochloric acid )

Deproteinization (sodium hydroxide )

Depigmentation (potassium permanganate)

CH2OH

CH2OH

Pure Chitin Minerals

O

OH O

H

O

H

Deacetylation

O

OH

NH2

H

Proteins H

NHCOCH3

n

NaOH

n

Chitosan

Chitin

Figure 3.2 Overview of chitin chemical extraction and chitosan production.

are carried out according to the need and condition of the waste used, as well as the species of raw material selected. Deproteinization is the process of eliminating the protein content present in the raw material, for which alkaline solutions of sodium hydroxide (NaOH) or potassium hydroxide (KOH) are used, with the NaOH solution, being the most widely used in industrial production. In addition, for the deproteinization of commercial chitin, temperatures between 25°C and 95°C are usually used, but it is also important to consider that it can cause depolymerization of the material and change some characteristics of chitin, such as viscosity (Pighinelli et al., 2019; Varma & Vasudevan, 2020). In the case of demineralization, this process involves the removal of inorganic matter, such as calcium carbonate and calcium phosphate from the raw material using inorganic acids, such as hydrochloric acid (HCl), nitric acid (HNO3), and sulfuric acid (H2SO4), as well as strong organic acids such as formic acid (HCOOH) and acetic acid (CH3COOH). However, the most widely used acid in the production of commercial chitin is HCl, due to its high efficiency in the removal of the minerals present (Younes & Rinaudo, 2015). Finally, staining is a strong indicator of the presence of impurities in the final product. In this sense, the depigmentation step is carried out using acetone, sodium hypochlorite, hydrogen peroxide, or potassium permanganate (Pighinelli et al., 2019).

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3.2.2.2 Biotechnological process of extraction The biotechnological extraction process of chitin is considered a combination between a chemical and a biological process, in which microorganisms are used. This process has advantages compared to the traditional chemical process, where large amounts of highly reactive chemical inputs are used, which can affect the final quality of the material and the generation of serious chemical effluents in the process (Pighinelli et al., 2019). Furthermore, methods involving biological pathways have been shown to be more assertive, achieving higher chitin purities and with considerably less molecular weight loss than the conventional chemical process (Khanafari et al., 2008). Although chemical processes are still more efficient industrially, the use of biotechnological methods presents a new sustainable vision and a new quality parameter, offering a more suitable biomaterial for its different fields of application. In this case, demineralization can be achieved using microorganisms. When the raw material is deposited together with a microbial culture and a source of sugar, which will provide the necessary nutrients, the production of organic acids is obtained because of this fermentation process (Mao et al., 2013). These acids can react with the minerals, converting them into salts and precipitates. At the end of the process, the salts and precipitates can be eliminated with a simple washing process. It has been reported that lactic acid from the activity of lactic acid bacteria provides better results and greater efficiency in demineralization (Aranday-García et al., 2017). However, the inoculation is carried out according to each species with its respective culture medium, containing the necessary nutrients. These media are basically composed of sugars and fats; amino acids; sources of calcium, iron, and magnesium; among other compounds. For lactic acid bacteria, the most widely used commercial culture media for presenting a balanced composition is de Man Rogosa Sharpe medium. Regarding the elimination of proteins by these biotechnological processes, fermentative processes can be applied, which produce digestive and microbial enzymes that consume organic matter. Hydrolytic enzymes such as proteases are very efficient in deproteinization and can result in the production of hydrolyzed proteins as a by-product of high added value (Pachapur et al., 2016). In fact, proteases enzymes produced by the action of lactic acid bacteria are the most widely used. As in chemical methods, the depigmentation process is important in biotechnological extraction methods. It is known that pigments such as carotenoids are present in crustaceans, which are lipidic in nature; however, they can be removed during fermentation processes and enzymatic

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activity. The main disadvantages of biotechnological processes are: the fermentation time and the high cost of some enzymes. Many times, the biological process is insufficient, so the use of acid and alkaline solutions is required to ensure that the reaction takes place. In contrast to the chemical method, it presents a better deproteinization yield, consequently obtaining a significantly higher quality material due to the low molecular weight loss (Hamed et al., 2016). 3.2.2.3 Characterization The obtained chitin and chitosan should be characterized by using different techniques such as spectral analysis, X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy, differential scanning calorimetry (DSC), and elemental analysis. XRD analysis is used to indicate the crystalline nature of the chitin and chitosan. The FTIR patterns are frequently used to characterize the structure of chitin and chitosan given the bands corresponding to the stretching and vibration of O-H, N-H, and CO bonds. The DD value can be calculated by elemental analysis, potentiometric titration, and FTIR. However, nuclear magnetic resonance (NRM) spectroscopy can be used for the determination of the deacetylation degree of chitin and chitosan and to monitor purification conditions. Furthermore, NRM is commonly used for differentiation of the two isomorphs (Kumari et al., 2015; Younes & Rinaudo, 2015).

3.2.3 Factors that influence its antimicrobial activity Chitosan and its derivatives have a wide field of application in biomedical, food, biotechnological, and pharmaceutical sciences due to their biocompatibility, biodegradability, and safety. Among its biological properties, the antimicrobial and antioxidant activities of chitosan are especially the most important, in the field of food preservation and packaging, providing a suitable alternative to the conventional use of chemical preservatives (being toxic) and due to the good film-forming properties of chitosan (Irastorza et al., 2021). However, it is known that the antimicrobial activity of chitosan depends on both intrinsic and extrinsic factors. While intrinsic factors include the source and derivatives of chitosan, molecular weight, DD, and concentration, mainly, the extrinsic factors are related to pH of the medium, temperature, type, and sensitivity of targeted microorganisms,

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formation of complexes of chitosan with certain materials, and reactive time (Ardean et al., 2021; Hosseinnejad & Jafari, 2016). Each of these factors has a different influence on the antimicrobial activity of chitosan, for example, it has been reported that, in general, the antimicrobial activity of chitosan from a fungal source was lower than that of chitosan obtained from crustacean shells ( Jeihanipour et al., 2007). However, fungal chitosan exhibited better inhibitory effects against Gram-positive bacteria compared to Gram-negative bacteria (Hosseinnejad & Jafari, 2016). Furthermore, factors such as pH significantly affect the antimicrobial activity of chitosan because its solubility is pH dependent and the molecule becomes polycationic when the pH is below its pKa, that is, its antimicrobial effect is reduced as the pH increases (Risti´c et al., 2015). Regarding the molecular weight and DD, these factors are important since it has been shown that low molecular weights of chitosan are desirable to increase antimicrobial activity and the DD is important because it increases the solubility of chitosan, thus improving its properties (Younes et al., 2014). Furthermore, it has been described that chitosan nanoparticles have better antimicrobial properties, due to their small particle size, which gives it a greater surface area and high reactivity, thus enhancing the ionic interaction between their charge and the bacterial surface charge (Romainor et al., 2014).

3.3 Chitosan-based nanosystems against food pathogens As mentioned above, chitosan has shown a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria (Yilmaz Atay, 2019). However, in recent years, nanotechnology has drawn attention in many fields because it has new and promising applications (Chandrasekaran et al., 2020) in food preservation and packaging (Priyadarshi & Rhim, 2020), cosmetics (Chen et al., 2017), pharmacy (Andonegi et al., 2020), and veterinary (Jeon et al., 2016). For example, it has been generally described that chitosan nanosystems have better antimicrobial activity compared to simple chitosan solutions, mainly due to their large surface contact area (Freitas et al., 2022).

3.3.1 Types of nanosystems 3.3.1.1 Nanoparticles Nanosized materials can have improved properties compared to the base material from which they come. In the case of chitosan nanoparticles, it is

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clear that their properties can vary considerably depending on the preparation methods used and the surface modifications that could be made, which can lead to applications in completely different fields (Yanat & Schroën, 2021). The methods used to obtain chitosan nanoparticles involve processes of emulsification, precipitation, ionic or covalent crosslinking, or a combination of these. One of the first methods described for obtaining chitosan nanoparticles was emulsification with cross-linking between the amino group of chitosan and the aldehyde group of a cross-linking agent (Ohya et al., 1994). For this, an aqueous phase consisting of a chitosan solution and an organic phase containing a surfactant, an organic solvent, and a cross-linking agent were used. The formed nanoparticles were separated by centrifugation, several washing steps, and/or vacuum drying or lyophilization. However, since glutaraldehyde was the cross-linking agent of choice, this method was no longer used due to the toxicity of said chemical compound (Yanat & Schroën, 2021). Chitosan nanoparticles can also be obtained from precipitation-based methods with sizes between 600 and 800 nm. This method consists of a phase inversion that includes an emulsification process combined with a precipitation process. Briefly, this method consists of an organic phase and an aqueous phase of chitosan containing a nonionic surfactant that stabilizes the system. Two of the drawbacks of this method are the use of organic solvents and the homogenization process, which is highly energetic. For this reason, there are not many studies considering this technology (El-Shabouri, 2002; Grenha, 2012). The applications of chitosan in nanotechnology have been studied for more than two decades and the ionic gelation method is perhaps one of the most important (Calvo et al., 1997a,b; Hoang et al., 2022). It was described in 1997 (Calvo et al., 1997b) and is based on the ionic cross-linking that occurs between the amino groups of chitosan and the negatively charged groups of a polyanion, such as sodium tripolyphosphate (TPP). Adding an aqueous solution of chitosan to an aqueous solution of TPP under vigorous stirring favors a cross-linking process that leads to the formation of nanoparticles. Unlike the methods described above, this method is simple and free from the use of solvents. Furthermore, the process can be carried out at room temperature and the final size of the nanoparticles can be adjusted by changing the chitosan/TPP ratio (Fan et al., 2012). Finally, chitosan nanoparticles can be coated with other components such as polymers through electrostatic and hydrophobic interactions,

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hydrogen bonds, and/or van der Waals forces, giving them special characteristics (Fan et al., 2012). Furthermore, as a viable alternative, the hydrophobicity of chitosan can be modified by chemical modifications that include acyl-chitosan, chitosan with stearic acid, and PEGylated chitosan since they influence the formation of nanoparticles. These types of nanoparticles are suitable for encapsulating both hydrophilic and lipophilic components (Quiñones et al., 2018; Yanat & Schroën, 2021). Table 3.3 shows a summary of the methods for obtaining chitosan nanoparticles.

Table 3.3 Description of the methods for preparing chitosan nanoparticles (Yanat & Schroën, 2021). Method

Main principle(s)

Advantage(s)

Drawback(s)

Emulsification and crosslinking Reversed micelles

Covalent crosslinking

Simple process steps

Use of harmful chemicals

Covalent crosslinking

Ultrafine nanoparticles (NPs) below 100 nm

Phase inversion precipitation

Precipitation

High encapsulation capacity for specific compounds

Emulsion-droplet coalescence

Precipitation

Time-consuming process Complex application Use of harmful chemicals Requires high shear force Use of harmful chemicals Requires high shear force Use of harmful chemicals

Ionic gelation

Ionic crosslinking

Ionic gelation with radical polymerization

Polymerization and crosslinking

Self-assembly

Electrostatic and/ or hydrophobic interaction

Use of mild chemicals Simple process Ease of adjusting NP size

Highly stable NPs Use of mild chemicals Adjustable procedure

Time-consuming process Complex application Hard to control when carried out a large scale

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3.3.1.2 Nanocapsules Nanocapsules (NCs) are nanometric systems with an inner core and an outer shell. Depending on their composition, NCs have been called lipid NCs if an oily core is stabilized by amphiphilic molecules, and polymeric NCs when the oily core is stabilized by a polymer shell (Fig. 3.3) (Crecente-Campo & Alonso, 2019). Although different strategies to obtain NCs have been described, three methods are the main ones: (1) interfacial deposition method, (2) nanoemulsion template method, and (3) layer-by-layer (LBL) method (Couvreur et al., 2002; Kothamasu et al., 2012; Mora-Huertas et al., 2010). The interfacial deposition method, such as the nanoprecipitation method, was widely used to obtain NCs. This method was first mentioned by Fessi et al. (1989) and consists of an organic phase generally containing oil, an active component, and suitable organic solvents. This phase is then added to an aqueous phase through a fine needle for the formation of NCs. The polymeric shellforming polymer can be dissolved either in the organic phase or in the aqueous phase, depending on the properties of the polymer. Furthermore, it is important to include surfactants to increase the stability of these nanosystems. Eventually, the water suspension of the formed NCs is obtained by eliminating the organic solvent by diffusion or evaporation. The characteristics of the NCs can be influenced mainly by the polymer concentration, the injection method of the organic phase, the volume ratio between the organic phase and the aqueous phase, and the nature of the materials (Deng et al., 2020). As technology progressed, methods for obtaining NCs were developed using high-energy instruments to first preform nanoemulsions (ModarresGheisari et al., 2019; Mustafa & Hussein, 2020). Among the developed

Figure 3.3 General structural composition of nanocapsules. On the left, the shell is formed with a cationic polymer and on the right, with an anionic polymer.

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methods is the diffusion/evaporation of organic solvents and the coacervation of monomers. For both cases, in the preparation of the nanoemulsions, the organic or aqueous phase is emulsified in either the aqueous or organic phase due to the presence of a surfactant, keeping the energy constant, such as sonication or homogenization. During the formation of the nanoemulsions, the surfactants self-assemble at the interface between the organic and inorganic phases to reduce the interfacial tension and thus achieve a stable state. The layer-forming polymeric materials, active substance, oil, and other functional substances can be dissolved or suspended in dispersion phase or continuous phase, depending on the requirements of the formulated NCs (Anton et al., 2008). The main difference between the methods is that the polymer layer is formed and stabilized by physical coacervation or chemical cross-linking. However, emulsion-coacervation methods are mainly used for polyelectrolyte materials or monomers/polymers that have cross-linking functional groups for the formation of NCs due to electrostatic interaction, which means more stable NC systems (Deng et al., 2020). Furthermore, by combining the principle of diffusion/evaporation and emulsion-coacervation, the resulting nanoemulsion can be continuously emulsified in a third phase to form water-in-oil-in-water (W/O/W) and oil-in-water-in oil (O/W/O) type double emulsions, according to the phase sequence. The critical point for the formation of this type of emulsions is to choose the adequate surfactant(s) to provide good stability between the internal and external emulsion interfaces (Ashjari et al., 2012; Bilati et al., 2003). Finally, one of the methods with a promising approach for obtaining multilayer nanocapsular systems is the LBL method. This method allows controlling the targeting and release properties of NCs by modulating the composition and thickness of the polymer shells (Cuomo et al., 2015). The mechanism of NC formation by LBL is generally through electrostatic interactions to achieve sequential deposition of polycations and polyanions on inorganic/organic cores, followed by sacrifice of the template core. Likewise, hydrophobic type interactions can be carried out when the active substance is hydrophobic (Deng et al., 2020). NCs formulated by said LBL can achieve various drug release behaviors in different pH environments (Cook et al., 2013). 3.3.1.3 Nanogels Chitosan nanogels are nanosystems with interesting properties for the administration of active molecules given their nanometric particle size,

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large surface coverage area, rapid response, and easy functionalization. In this sense, chitosan nanogels have reached an outstanding position as nanocarriers due to their biocompatibility, biodegradability, and mucoadhesive nature. Various methods have been reported to prepare chitosan nanogels, being the swelling of the polymeric chains important for association and/or encapsulation of active molecules for their release (Manuel Laza, 2016). According to the characteristics of the materials used in the formation of nanogels, the methods for preparing this type of nanosystems can be divided into two main categories: (1) obtaining from polymer precursors and (2) by heterogeneous polymerization of monomers. The polymer precursors are polymers with amphiphilic character or triblock copolymers that can form nanogels by self-assembly or polymers that have several reactive sites that can be used directly for chemical cross-linking (Fig. 3.4). Likewise, polymers can also be modified with functional groups capable of forming physical or chemical cross-links (An et al., 2011). The preparation of nanogels through the polymerization of monomers includes two steps that take place simultaneously, the polymerization and the formation of nanogels. Compared to the preparation of nanogels using preformed polymers, the synthesis of nanogels through monomer polymerization shows higher efficiency. Physical cross-linking generally occurs between

Figure 3.4 Nanogel synthesis methods: (A) polymer precursor method and (B) heterogeneous monomer polymerization method. Please check the online version to view the color image of the figure.

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polymer precursors with a special nature, while chemical cross-linking can be favored with both polymer precursors and monomers (Li et al., 2021; Zhang et al., 2016).

3.3.2 Mechanisms of antimicrobial action Currently, there are a large number of articles that report the antimicrobial effect of chitosan nanosystems or the synergy exerted by chitosan nanoparticles when used as carriers of metals, antimicrobial drugs, or other active compounds (Perinelli et al., 2018). Although the exact antimicrobial mechanism of action has not been fully elucidated, chitosan nanoparticles are known to have a higher antibacterial effect than simple chitosan solutions. This increase in antimicrobial activity has been attributed to the higher density of positively charged amino groups when chitosan is used as the nanoparticle-forming polymer. In this sense, the higher positive charge density of chitosan nanoparticles promotes a stronger binding to the negatively charged surface of bacteria (Chung et al., 2004), leading to destruction of the cell membrane, leakage of intracellular components, and entry of chitosan into microbial cells (Matica et al., 2019). In the case of Gram-positive bacteria, the positive charges of chitosan interact electrostatically with the negative charges of teichoic acids present in peptidoglycan, leading to a change in membrane permeability that causes an osmotic imbalance and leakage of intracellular substances, resulting in cell death (Yan et al., 2021). It is clear that the most predominant antibacterial activity of chitosan nanosystems is related to electrostatic interactions, which change the permeability of the membrane. However, it has been shown that due to the reduced particle size of chitosan nanosystems, they can enter the bacterial cell and inhibit the replication of the genetic material or protein synthesis, leading to bacterial cell death (Chandrasekaran et al., 2020). The negatively charged phosphate groups on the DNA/RNA react with the positively charged amino groups on the chitosan nanoparticles, thereby inhibiting pathogens (Xing et al., 2009). Likewise, this type of nanosystems is capable of modifying the electron transport chain of bacteria (Birsoy et al., 2015). Another probable mechanism for the antimicrobial activity of chitosan nanosystems is their chelating capacity toward metal ions such as Fe21, Mg21, Ni21, Co21, Cu21, and Zn21, which stimulates the production of toxins, therefore reducing bacterial viability (Ma et al., 2017). It has been

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reported that most of the metal ions (previously described) associated with the cell wall molecules of bacteria are vital for their stability. In Grampositive bacteria, these metal ions bind to teichoic acids found in the bacterial wall, thus minimizing repulsion between adjacent phosphate groups, and achieving stabilization of cell wall structure and integrity (Kern et al., 2010; Wickham et al., 2009). In the case of Gram-negative bacteria, metal ions can minimize the repulsive forces between the negatively charged phosphate groups of lipopolysaccharides, giving bacterial membrane stability (Clifton et al., 2015). The chelation mechanism of chitosan depends on the pH value of the medium, since when it is higher than its pKa value, the unprotonated amino groups of chitosan can donate their lone pair of electrons to the metal ions of the phosphate groups in the lipopolysaccharides and teichoic acids from the surface of the cell membrane to form a metal complex. Thus the positively charged amino groups of chitosan can compete with metal ions for the phosphate groups of lipopolysaccharides and teichoic acids. Therefore such a chelation reaction can lead to instability of the cell surface potential and mutual repulsion between the negatively charged phosphate groups, causing rupture of the bacterial cell membrane (Feng et al., 2021; Liu et al., 2014).

3.3.3 Factors that affect the antibacterial activity of chitosan nanosystems The antibacterial activity of chitosan nanosystems depends on several factors such as the type of bacteria, stage of bacterial growth, pH, zeta potential, degree of acetylation, concentration, and molecular weight (Chandrasekaran et al., 2020). The particle size of chitosan nanosystems is an important parameter that influences its antimicrobial activity and there are several factors that can affect it, such as concentration and molecular weight, mainly (Divya et al., 2017). Likewise, the greater contact surface of these nanosystems is directly related to the increase in antimicrobial activity due to the particle size (O’Callaghan & Kerry, 2016). It is known that the antibacterial activity of both chitosan and chitosan-based nanosystems is highly dependent on molecular weight and degree of acetylation (Chandrasekaran et al., 2020). However, the influence of molecular weight is greater than the degree of acetylation (Goy et al., 2009). Although it has been reported that chitosan nanosystems obtained with low- and medium-molecular-weight chitosan can strongly inhibit bacterial growth (Gomes et al., 2018a), the increase in the

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molecular weight of chitosan can cause an increase in size and decrease in zeta potential, thus reducing its antibacterial activity (Mohammadi et al., 2016). In this sense, a higher zeta potential is related to a decrease in particle size and an increase in ionic interactions with the bacterial membranes. It has been shown that a higher positive charge density in chitosan nanosystems is closely related to the degree of acetylation. Therefore the higher the degree of acetylation, the better the antimicrobial effect, considering the same molecular weight of chitosan (Kong et al., 2008). Regarding the concentration of chitosan in nanosystems, a positive relationship of antimicrobial activity has been seen when an increase is obtained as the number of nanoparticles also increases (Mohammadi et al., 2016; Xing et al., 2008); however, an inconsiderate increase in chitosan concentration is positively correlated with an increase in particle size (Tamara et al., 2018). Chitosan nanosystems have a polycationic nature due to the protonation of their amino group in acid conditions. However, the effect of pH on the antimicrobial activity of chitosan nanosystems depends to a greater extent on the type of bacteria since, in general terms, when the pH increases, its antimicrobial capacity increases (Xing et al., 2008), except for some cases where at low pH levels the antimicrobial activity increases, as do chitosan solutions (Manikandan & Sathiyabama, 2015). Finally, the kinetics of bacterial growth have shown that the susceptibility of bacteria is very different in each of their phases, since the partial charges of the bacterial cell surface are modified as the growth stage changes (Sotelo-Boyás et al., 2017), so that in general, in the mid-exponential phase the antimicrobial activity of the chitosan nanosystems is higher compared to the late exponential phase (Yang et al., 2007).

3.3.4 Antimicrobial activity against food pathogens Foodborne diseases are a major public health problem, especially in people at risk of exclusion and in developing countries. Most foodborne illnesses are related to pathogenic bacteria belonging to the genera Salmonella, Listeria, Escherichia, Clostridium, and Campylobacter. Pathogen contamination of food is known to occur at different stages of food processing, such as harvesting, slaughtering, processing, and distribution (farm to fork) (Zorraquín-Peña et al., 2020). In this sense, the ongoing search for effective antimicrobials for use in the food industry that can be applied both in the processing (conservation) and packaging (safety) stages of food has focused on the use of nanotechnological platforms such as nanoparticles

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and NCs, mainly (Malhotra et al., 2015; Zorraquín-Peña et al., 2020). Therefore the use of chitosan-based nanosystems is perhaps a potential strategy in food processing (food and feed additives) and food safety (packaging) due to its excellent antimicrobial activity already reported (Yilmaz Atay, 2019). 3.3.4.1 Gram-negative bacteria Foodborne pathogens are a major source of morbidity and mortality throughout the world. In most cases, these pathogens are transmitted from contaminated food products, as well as through person-to-person contact. However, a significant proportion of foodborne pathogens are characterized as Gram-negative bacteria (Hernández-Cortez et al., 2017). Salmonella spp. being Gram-negative bacteria, are one of the most important foodborne pathogens that can cause more illness than any other bacteria (Ma et al., 2020; PREVENTION CFORDCAND, 2018). Salmonella is generally transmitted through the consumption of contaminated food of animal origin (mainly eggs, meat, poultry, and milk). As previously described, chitosan and chitosan nanoparticles have antimicrobial activity against pathogens such as Salmonella spp. and Escherichia coli. In an in vitro study (Du et al., 2009), it was shown that chitosan nanoparticles obtained with tripolyphosphate presented better antimicrobial activity against Salmonella compared to chitosan solutions in Muller Hinton broth. In fact, the minimum inhibitory concentrations of chitosan nanoparticles (117 μg/mL) were just over 3 times lower compared to chitosan (468 μg/mL). These chitosan nanoparticles were characterized by presenting an average particle size of 53.99 nm and a positive zeta potential of 151.37 mV, which explains the best antimicrobial activity. However, when these chitosan nanoparticles were loaded with Ag1, Cu21, Zn21, Mn21, or Fe21 metal ions, the antimicrobial activity against Salmonella was enhanced to a minimum inhibitory concentration of 3 μg/mL for Ag1, even with an increase in particle size (90.29 nm), therefore the increase in antimicrobial activity is mainly due to its increase in zeta potential (192.05 mV) (Du et al., 2009). In contrast, the association of Fe21 in the chitosan nanoparticles did not present a potentiating (synergistic) effect since the minimum inhibitory concentration went from 117 to 121 μg/mL (Du et al., 2009). Additionally, another study demonstrated the antimicrobial potential of chitosan nanoparticles during vegetable washing against bacteria such as E. coli and Salmonella typhimurium, two common species of foodborne pathogens often found contaminating fresh vegetables (Paomephan et al., 2018). In the case

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of E. coli, the results of the antimicrobial activity of the chitosan nanoparticles showed that at a size between 300 and 400 nm, the E. coli counts were reduced by around 2 log CFU/mL after 12 hours of exposure, while chitosan nanoparticles with sizes between 500 600 nm and 700 800 nm presented a reduction of 1.73 and 1.50 log CFU/mL, respectively. These findings suggest that the antibacterial activity of these nanosystems is mainly governed by their particle size. Likewise, at polymeric concentrations of 50 200 μg/mL, chitosan nanoparticles exhibited a similar antimicrobial effect at 8 12 h of exposure, but concentrations of 800 μg/mL were significantly better in reducing E. coli counts. Thus the greater susceptibility of S. typhimurium to chitosan nanoparticles could be attributed to the composition of the cell membrane, which makes it more negative and facilitates interactions with the positive charges of chitosan in the nanosystems (Paomephan et al., 2018). 3.3.4.2 Gram-positive bacteria Foodborne microorganisms are the main pathogens affecting food safety and causing human illness worldwide. Both Gram-positive and Gramnegative bacteria have been reported to be capable of producing toxins that cause food poisoning leading to symptoms ranging from gastrointestinal disturbances to paralysis and death (Le Loir et al., 2003). Although Gram-negative bacteria have been reported to account for approximately 69% of cases of bacterial foodborne illness, Gram-positive bacteria such as Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes) are part of the main causes of foodborne diseases and death in the world (Abebe et al., 2020). However, Gram-positive bacterial pathogens also include spore formers such as Bacillus cereus, Clostridium botulinum, and Clostridium perfringens (Wolf-Hall & Nganje, 2017). One of the possible strategies to reduce foodborne pathogens is the development of effective preservation strategies capable of eradicating microbial contamination of food through the use of nanotechnological platforms. In this context, the proliferation inhibition capacity of chitosan nanoparticles with an average size of 361.9 6 31 nm and a zeta potential of 143.8 6 11.25 mV on five different bacteria, including have shown promising results (Barrera-Ruiz et al., 2020). The obtained results indicated that the proliferation of S. aureus decreased in a range of 25% 35% when the concentrations of chitosan (chitosan nanoparticles) were between 150 and 500 μg/mL. This decrease is higher compared to chitosan solutions in bacterial culture medium since the maximum proliferation inhibition capacity of these solutions was around 9% (Barrera-Ruiz et al., 2020). A possible

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explanation for this effect can be centered on the greater interaction of chitosan nanoparticles with teichoic acids, which leads to an increase in membrane permeability, causing its depolarization (Raafat et al., 2008). These results are consistent with others already published where the antimicrobial effect of chitosan nanoparticles against E. coli ATCC 25922, S. aureus ATCC 12222, and Moraxella catarrhalis ATCC 49143 was evaluated (Nguyen & Nguyen, 2022). The results obtained in another study of the antimicrobial activity of chitosan nanoparticles on Gram-positive and Gram-negative bacteria of importance in the food industry are shown in Table 3.4. It is clear that Gram-positive bacteria such as Bacillus cereum (B. cereum) and Bacillus subtilis (B. subtilis) were more susceptible in comparison with Gram-negative when exposed to chitosan nanoparticles, being the particle size again inversely proportional, that is, the smaller the particle size, the higher the antimicrobial efficiency (Sarwar et al., 2014). As for S. aureus, chitosan can cause a permeabilizing effect in B. cereum and B. subtilis, resulting in intracellular potassium leakage (Ke et al., 2021; Mellegård et al., 2011). L. monocytogenes is a species of pathogenic bacteria that are transmitted to food during the harvesting of raw materials, as well as the processing, conditioning (packaging), transportation, and storage of food products. Although infection with L. monocytogenes is relatively rare, it has high mortality rates (20% 30%) compared to other foodborne pathogens (Reda et al., 2016). In a study in which the antibacterial activity of chitosan and nanochitosan (50 100 nm) against L. monocytogenes was evaluated, it was found that although chitosan showed an antimicrobial effect against said bacteria, nanochitosan showed an area of highest inhibition at equal concentrations of 25, 50, 75, and 100 μL/mL, being in addition, its minimum inhibitory concentration 5 times lower (Table 3.5) (El-Zehery et al., 2022). Finally, another important pathogen is C. perfringens, an anaerobic, spore-forming, and foodborne bacterium. Due to its resistance to heat when it is in the form of spores, methods are needed to control it since this pathogen secretes an enterotoxin in the intestine, which changes the permeability of the cell membrane and causes diarrheal syndrome or even death (Chang et al., 2020). Although there are no published studies on the antimicrobial effect of chitosan formulated in nanosystems for the control of anaerobic pathogenic bacteria such as C. perfringens, a study showed that chitosan nanofibers with an average particle size of 87 nm presented a minimum inhibitory concentration of 250 μg/mL when challenged against Clostridium difficile ATCC 700057 (Abadi et al., 2020).

Table 3.4 Minimum inhibitory concentration of the different chitosan nanoparticles on Gram-positive and Gram-negative bacteria by the microtiter broth dilution method (Sarwar et al., 2014). Chitosan molecular weight

70 kDa 375 kDa

Chitosan concentration (mg/mL)

2 4 2 4

Particle size (nm)

196.88 6 3.37 394.79 6 4.03 598.74 6 9.07 872.45 6 7.94

Minimum inhibitory concentration (μg/mL) Gram-positive bacteria

Gram-negative bacteria

Bacillus cereum

Bacillus subtilis

Escherichia coli

Pseudomonas aeruginosa

3.13 6.25 6.25 12.5

1.56 3.13 6.25 6.25

12.5 12.5 25.0 25.0

3.13 3.13 12.5 12.5

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Table 3.5 Antibacterial activity of chitosan and nanochitosan against Listeria monocytogenes (El-Zehery et al., 2022). Antibacterial agents

Listeria monocytogenes (ATCC 19116) Inhibition zone (mm)

Chitosan (µl/mL) 25 50 75 100 Minimum inhibitory concentration (MIC) (μg/mL)

22.0 23.8 28.5 28.6 64.0

Nanochitosan (µl/mL) 25 50 75 100 MIC (μg/mL)

25.0 28.5 30.0 30.0 12.8

3.4 Chitosan nanosystems loaded with natural antimicrobials In order to improve or enhance the antimicrobial activity of chitosan nanosystems, certain natural compounds with biocidal activity can be associated or encapsulated to reduce the growth of bacteria responsible for foodborne diseases (Yilmaz Atay, 2019). Natural antimicrobials include enzymes (Malmiri et al., 2012; Yeon et al., 2019), plant extracts/phytobiotics (Antunes et al., 2021; Hernandez-Patlan et al., 2019), essential oils (EOs) (Granata et al., 2021), organic acids (Jang & Lee, 2008), antioxidants (Quester et al., 2022), and bacteriocins, mainly. Therefore these natural active substances can be used in agriculture, livestock, and food processing as natural products with therapeutic activity or food additives to control foodborne pathogens (Pan et al., 2020).

3.4.1 Plant extract/phytobiotics Plant extracts are widely used as natural pharmaceuticals due to their high availability in nature (e.g., seeds, bark, wood, roots, leaves, flowers, and fruits) and bioactivity, which is attributed to phytochemicals such as simple phenols and phenolic acids, quinones, flavonoids, tannins, coumarins,

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terpenes and terpenoids, alkaloids, lectins, and polypeptides. Among the properties that have been described for these biomolecules is their antioxidant, anticancer, antiinflammatory, and antimicrobial activity (AlSheikh et al., 2020; Antunes et al., 2021). During a study using the microplate microdilution method with concentrations from 0 to 400 μg/mL, the antimicrobial activity between lecithin-chitosan nanoparticles (LNP), nanoparticles of lecithin-chitosan containing curcumin (LNPC), a phytobiotic, and an antibiotic (tetracycline) was compared (Valencia et al., 2021). Although the antimicrobial capacity of curcumin and chitosan has been widely proven, the use of curcumin is limited due to its low aqueous solubility, low permeability, and therefore low bioavailability. However, the results showed that the antimicrobial effect of curcumin was potentiated when it was in NP compared to LNP and the antibiotic tetracycline since the minimum inhibitory concentration was reduced in both Gram-negative and Gram-positive bacteria (Table 3.6) such as Salmonella typhimurium (S. typhimurium, ATCC 14028), Vibrio parahaemolyticus (ATCC 17802), E. coli (ATCC 8739), S. aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), and L. monocytogenes (ATCC 7644) (Valencia et al., 2021).

Table 3.6 Minimum inhibitory concentration of lecithin-chitosan nanoparticles, lecithin-chitosan nanoparticles containing curcumin and a commercial antibiotic (tetracycline) (Valencia et al., 2021). Strains

Salmonella typhimurium Listeria monocytogenes Vibrio parahaemolyticus Staphylococcus aureus Staphylococcus epidermidis Escherichia coli

Minimum inhibitory concentration (μg/mL) Lecithin-chitosan nanoparticles

Lecithin-chitosan nanoparticles containing curcumin

Tetracycline

3.12

1.56

15.62

25.00

1.56

15.62

12.50

3.12

31.25

12.50

1.56

7.81

6.25

3.12

7.81

3.12

0.78

31.25

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S. aureus, Salmonella spp., L. monocytogenes, and E. coli are the main bacterial pathogens that cause foodborne illness and deaths associated with the consumption of contaminated products (Abebe et al., 2020). Therefore the above results are promising because these nanosystems can prevent the growth of these bacteria at concentrations ranging from 1.56 to 3.12 μg/mL. In another publication (Correa-Pacheco et al., 2019) in which films containing ethanolic extracts of propolis in chitosan nanoparticles showed a good antimicrobial effect on L. monocytogenes, S. enteritidis, and E. coli after 24 and 48 hours of exposure. Therefore the use of chitosan nanoparticles with ethanolic extract of propolis in films is a promising alternative for active packaging with antibacterial properties against foodborne pathogens (Correa-Pacheco et al., 2019). Likewise, another published study showed that the association of cinnamon extract present in selenium nanoparticles when conjugated with chitosan nanoparticles significantly improved the growth inhibition of E. coli, S. typhimurium, S. aureus, and L. monocytogenes considering concentrations of the cinnamon extract ranging from 25 to 75 μg/mL (Alghuthaymi et al., 2021). In this sense, the films composed of chitosan nanoparticles with bioactive phytochemicals demonstrated additional activities and applicability for their use in the protection and preservation of food (Alghuthaymi et al., 2021). Finally, in a study similar to the previous one, the antimicrobial activity of a free and nanoencapsulated natural cinnamon bark extract (CBE) was compared in three different chitosan nanosystems on freshly cut lettuce contaminated with L. monocytogenes (Hill et al., 2017). The most effective antimicrobial treatment against L. monocytogenes was the CBE encapsulated in nanoparticles of chitosan-co-poly-N-isopropylacrylamide (chitosan-PNIPAAM) since it presented a significant reduction in the bacterial counts of around 2 log10 CFU/g (P , .05) compared to the other encapsulation systems. Furthermore, it was shown that the most effective antimicrobial concentration was 80 mg/mL for chitosan-PNIPAAM-CBE (Hill et al., 2017). In general, the encapsulation/association of plant extracts in chitosan nanosystems is an important strategy to improve the safety of food products through the reduction of foodborne pathogens.

3.4.2 Essential oils In recent years, natural products have been the focus of multiple research studies because they can be used as both complementary and alternative

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antimicrobial agents (Calo et al., 2015). EOs are natural products derived from plants, which are formed by complex mixtures of biologically active and potential secondary metabolites (Maida et al., 2014). These secondary metabolites are characterized by being volatile and responsible for antibacterial, antifungal, antiinflammatory, antioxidant, anticancer, and antiviral properties (Aumeeruddy-Elalfi et al., 2015; Espina et al., 2011; Hossain et al., 2014; Sharifi-Rad et al., 2015). However, environmental and agronomic conditions are important in the chemical composition of EOs (Mohamed et al., 2014). These types of natural compounds have been used as a food preservative for the control of foodborne pathogens (Perricone et al., 2015). Although EOs have shown excellent antimicrobial activity against different pathogens, their use is limited due to their low aqueous solubility and poor stability under environmental conditions of temperature, humidity, and oxygen. Therefore to resolve these limitations, nanometric-sized chitosan-based formulations encapsulating these EOs emerge as a viable solution, in addition to enhancing their antimicrobial effect in some cases ( Jamil et al., 2016). Recently, an in vitro study evaluated the antimicrobial activity of several EOs and different blends, as well as their effectiveness when associated with nanochitosan on L. monocytogenes and E. coli O157:H7 (El-Zehery et al., 2022). However, the results contrast with those expected since the combination of different EOs and even nanochitosan alone showed a better antimicrobial effect against L. monocytogenes and E. coli O157:H7 compared to nanochitosan combined with EOs (Table 3.7). These results can be attributed to a change in the properties of nanochitosan due to mixing with EOs. In contrast to the previous results, chitosan nanoparticles loaded with clove EO with particle size between 223 and 444 nm showed the highest antimicrobial activity against L. monocytogenes and S. aureus since they presented inhibition halo of 4.80 and 4.78 cm on bacterial cultures, respectively (Hadidi et al., 2020). Thus these types of nanosystems could improve the efficiency of clove EO in food products and in delivery systems for novel applications such as active packaging. Likewise, in another study, the antimicrobial activity of two nanoparticulate systems based on chitosan loaded with EOs of Thymus capitatus (Th-CNPs) and Origanum vulgare (Or-CNPs) was evaluated (Granata et al., 2021). For the case of Th-CNPs, its particle size was around 449, and 407 nm for Or-CNPs. Furthermore, for Th-CNPs and Or-CNPs nanosystems, the Z potential values were 144 6 2 and 146 6 2 mV,

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Table 3.7 Chitosan and chitosan nanoparticles combined with essential oils against Listeria monocytogenes and Escherichia coli (El-Zehery et al., 2022). Treatments

Escherichia coli O157: H7 (ATCC 6933)

Listeria monocytogenes (ATCC 19116)

Inhibition zone (mm)

Chitosan 1 garlic Chitosan 1 thyme Chitosan 1 cinnamon Chitosan 1 clove Chitosan 1 (cinnamon 1 clove) Chitosan 1 (clove 1 thyme) Nanochitosan 1 garlic Nanochitosan 1 thyme Nanochitosan 1 cinnamon Nanochitosan 1 clove Nanochitosan 1 (cinnamon 1 clove) Nanochitosan 1 (clove 1 thyme)

27.6 32 28 30.6 33.5

30 38 32.6 36 40

35

42.5

17 15 12 19 21

23 24 23 28 27.3

25

30

respectively. In this sense, both Or-CNPs and Th-CNPs presented an improved bactericidal activity against S. aureus, E. coli, and L. monocytogenes compared to pure EOs. These nanosystems could represent a viable alternative to food preservatives since they are of interest for maintaining food safety (Table 3.8) (Granata et al., 2021). Continuing to use EOs, one study found that encapsulation of nettle EO in chitosan nanoparticles obtained by a two-stage emulsion-ionic gelation method (average size of 208.3 369.4 nm) had greater antibacterial activity than the free form of nettle EO against S. aureus and E. coli (Bagheri et al., 2021). These results suggest that EO encapsulation in chitosan nanoparticles is a promising candidate for use in food products with novel applications (Bagheri et al., 2021). The use of nanotechnological platforms based on chitosan encapsulating or associating EOs for their application in the food industry is extremely important, such as the fact that the results shown above are not the only ones, but there are others. For example, different EOs have been associated/encapsulated in chitosan-based nanoparticle systems and evaluated for antimicrobial activity on Gram-negative and Gram-positive foodborne

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Table 3.8 Minimum inhibitory concentration of Thymus capitatus (Th), Origanum vulgare (Or), chitosan nanoparticles loaded with Th (Th-CNPs) and Or (Or-CNPs) essential oils, and unloaded chitosan nanoparticles against different pathogens (Granata et al., 2021). Nanosystems

Th-CNPs Th-EO Or-CNPs Or-EO Chitosan nanoparticles

Minimum inhibitory concentration (mg/mL) Staphylococcus aureus ATCC 29213

Escherichia coli ATCC 25922

Listeria monocytogenes ATCC 19118

0.06 2 0.03 4 0.03

0.12 2 0.06 4 0.06

0.03 1 0.03 2 0.03

pathogens. Some of these EOs are lime (Sotelo-Boyás et al., 2017), cinnamon (Hu et al., 2015), Eucalyptus staigeriana (Ribeiro et al., 2013), Limonene (Souza et al., 2014), Cardamom ( Jamil et al., 2016), Schinus molle (Barrera-Ruiz et al., 2020), and Torreya grandis (Wu et al., 2018).

3.4.3 Enzymes Enzymes play an important role in various sectors of the food industry, including dairy, baking, food processing and packaging, animal feed, fruit and vegetable juices, beverages, oil refining, and confectionery (Okpara, 2022). For example, lysozyme, a small monomeric protein, has been used in the preservation of different food products such as raw and processed meat, dairy products, fruits, and vegetables to prolong their shelf life (Tiwari et al., 2009). In this sense, in a published study, lysozyme (Lys) was integrated into chitosan nanoparticles (CS-NP) to improve antibacterial activity against E. coli and B. subtilis. This nanoparticulate system (Lys-CS-NP) was obtained by the ionic gelation technique and presented particle size ranging from 488.8 to 613.5 nm according to the pH of the medium. The results showed that the Lys-CS-NP nanoparticle system improved the antibacterial activity against E. coli and B. subtilis (Table 3.9), suggesting a great potential for its use in the food industry and other applications in the form of direct addition or incorporation into packaging (Wu et al., 2017).

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Table 3.9 Bacterial growth inhibition zone and minimum inhibitory concentration of lysozyme integrated into chitosan nanoparticles (Lys-CS-NP) against Escherichia coli and Bacillus subtilis (Wu et al., 2017). Treatment group

CS-NP Lys-CS-NP

Escherichia coli

Bacillus subtilis

Inhibition zone (mm)

Minimum inhibitory concentration (MIC, mg/mL)

Inhibition zone (mm)

MIC (mg/mL)

10.34 6 1.21 13.11 6 0.48

0.63 0.16

11.70 6 1.51 12.89 6 1.25

0.3125 0.15625

The results of another study are consistent with the previous ones since the integration of lysozyme in chitosan nanoparticles (Lys-CS-NP) by the ionic gelation technique produced nanosystems with particle sizes of 243.1 6 2.1 nm and zeta potential of 22.8 6 0.2 mV. These nanosystems firstly significantly improved the thermal stability of lysozyme and, in addition, exhibited excellent antibacterial properties against P. aeruginosa, Klebsiella pneumoniae, E. coli, and S. aureus (Wang et al., 2020). However, S. aureus was the most sensitive bacterium to Lys-CS-NP (Table 3.10).

3.4.4 Bacteriocins Bacteriocins are peptides produced by bacteria with antimicrobial activity. These molecules have a high efficiency and specificity in the food industry since they can be used as preservatives and food additives. However, they have limitations such as high cost of isolation and purification, narrow spectrum of activity, low stability and solubility, and easy enzymatic degradation. Therefore bacteriocins combined with nanomaterials have emerged as promising strategies to overcome their limitations (Naskar & Kim, 2021). Chitosan has been used with bacteriocins in many studies to obtain materials with synergistic antibacterial activity. For example, it has been reported that chitosan nanoconjugates loaded with bacteriocins have shown synergistic antibacterial activity against L. monocytogenes, with minimum inhibitory concentrations of these nanosystems of 32.5 μg/mL (Namasivayam et al., 2015). Furthermore, release studies revealed that nisin was released from the nanoparticle over a period of 60 days, but its release was pH dependent (Alishahi, 2014). The antibacterial effect of these nanosystems was evaluated by the analysis of cell membrane integrity using a transmission electron microscope, as well as bacterial plate counts. Therefore these nanosystems could be used

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Table 3.10 Minimum inhibitory concentration of chitosan nanoparticles, lysozyme, and lysozyme integration in chitosan nanoparticles against different bacteria (Wang et al., 2020). Treatments

CS-NP Free lysozyme Free lysozyme 1 CS-NP Lys-CS-NP

Minimum inhibitory concentration (mg/mL) Pseudomonas aeruginosa

Klebsiella pneumoniae

Escherichia coli

Staphylococcus aureus

.10 .10 5

10 10 5

10 2.50 0.63

10 1.25 0.31

2.50

1.25

0.63

0.16

Table 3.11 Antimicrobial activity of chitosan, nisin, chitosan nanoparticles, and N-CS nanoparticles against different Gram-positive and Gram-negative bacteria (Lee et al., 2018). Bacterial strain

Staphylococcus aureus Listeria monocytogenes Escherichia coli O157:H7 Salmonella typhimurium a d

Log reduction (CFU/mL) Chitosan (2.5 mg/mL)

Nisin (1 mg/mL)

Chitosan nanoparticles (2.5 mg/mL)

N-CS nanoparticles (1.5 mg/mL)

1.75 6 0.02d

2.73 6 0.01d

2.21 6 0.02d

3.82 6 0.03d

1.58 6 0.01c

2.34 6 0.05c

2.15 6 0.04c

3.61 6 0.05c

1.45 6 0.06b

1.72 6 0.03b

2.03 6 0.03b

3.49 6 0.01b

1.32 6 0.03a

1.56 6 0.03a

1.96 6 0.01a

2.88 6 0.03a

Values within columns with different superscripts differ significantly (P , 0.05).

in food packaging (Alishahi, 2014). Finally, in a current study, nisin-loaded chitosan nanoparticles (N-CS) with an average size of 147.93 6 2.9 nm and a zeta potential of 133.4 mV showed promising results in reducing the counts of Gram-positive bacteria such as S. aureus and L. monocytogenes, as well as Gram-negative bacteria such as E. coli O157:H7 and S. typhimurium in orange juice (Lee et al., 2018). The greatest reduction in bacterial counts occurred for S. aureus (3.82 log CFU/mL) and L. monocytogenes (3.61 log CFU/mL) (Table 3.11). These results suggest that these nanosystems can be used as potent antibacterial agents in food and other related areas (Lee et al., 2018).

Table 3.12 Application of chitosan-based nanosystems in some food systems. Chitosan nanosystem

Physicochemical properties

Challenged pathogen

Effect/purpose

References

Chitosan nanocomposite

Size: 331.1 nm PDI: 0.377 Zeta potential: 34 mV Size: 90 nm diameter

Aspergillus niger Rhizopus stolonifera Botrytis cinerea

Melo et al. (2020)

Chitosan thymol nanoparticles

Size: 293.1 nm PDI: 0.5 Zeta potential: 47.8 mV

B. cinerea

Chitosan nanoparticles loaded with cinnamomum zeylanicum essential oil Chitosan nanoparticles with α-pinene

Size: 97 182 nm

Phytophthora drechsleri

Inhibition of pathogenic fungal growth in strawberries (Fragaria ananassa) Good antifungal activity and delayed color change of strawberries Effective nanosystem to inhibit the mycelial growth of the pathogenic fungus in blueberries and cherry tomatoes Fungal growth completely inhibited in cucumber

Size: 3.9 6 0.5 nm PDI: 0.5 Zeta potential: 13.4 14.9 mV

Alternaria alternata

Inhibition of fungal growth during the cold storage period of bell pepper

Hernández-López et al. (2020)

Chitosan nanofibers

B. cinerea

Sun et al. (2021)

Medina et al. (2019)

Mohammadi et al. (2015)

(Continued)

Table 3.12 (Continued) Chitosan nanosystem

Physicochemical properties

Challenged pathogen

Effect/purpose

References

Chitosan nanoparticles (CS) and chitosan biocomposites loaded with pepper tree essential oil

—

Colletotrichum gloeosporioides

Chávez-Magdaleno et al. (2018)

Nanostructured chitosan functionalized with cinnamon essential oil or trans-cinnamaldehyde

Size: 4.68 nm Zeta potential: 21.94 mV

Fusarium solani

Fungal chitosan nanoparticles

Size: 128.3 nm PDI: 0.903 Zeta potential: 51.4 6 5.77 mV

Chitosan nanoparticles

Size: 26.98 28.82 nm PDI: 0.903 0.341 Zeta potential: 30.1 46.1 mV

Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa Salmonella spp., Escherichia coli S. aureus Salmonella typhimurium

Alternative to preserve avocado fruits from latent infections and control fungal rot, as well as preserving fruit quality Reduction in the severity of F. solani and improvement in the safety and shelf life of cucumbers Inhibitory effect against foodborne pathogenic bacteria in grapes

Increased shelf life of chilled chicken meat and reduction in bacterial load

Elmasry and Dalia (2018)

Chitosan nanoparticles

Size: 10 nm

Improvement in the microbiological quality of fish fingers and increase in shelf life up to 6 months

Abdou et al. (2012)

Coliform bacteria, Psychrophilic bacteria, Proteolytic bacteria, S. aureus, Yeast, and mold

Istúriz-Zapata et al. (2020)

Melo et al. (2018)

Chitosan nanoparticles of cinnamon essential oil and ascorbic acid

Size: 235.6 nm PDI: 0.330 Zeta potential: 25.1 mV

Chitosan nanoparticles loaded with cinnamon essential oil

Size: 527 nm PDI: 0.617 Zeta potential: 25.1 mV

Chitosan-silver nanoparticles

Size: 61.57 and 76.89 nm Zeta potential: 20.0695 to 20.225 mV (at pH 7) Size: 240.6 191.6 nm PDI: 0.09 0.23 Zeta potential: 21.2 35.1 mV Size: 66.4 6 8.9 nm Zeta potential: 231.7 6 2.6 mV

Zein/chitosan nanoparticles loaded with epigallocatechin gallate Nisin-loaded alginatechitosan nanoparticles

S. aureus, total mesophilic aerobic Enterobacteriaceae, Lactic acid bacteria, Yeasts, and molds Lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp.

Application as a novel additive for controlled release and shelf-life extension of beef patties

Ghaderi-Ghahfarokhi et al. (2017)

These nanosystems present excellent antioxidant and antimicrobial properties of chilled pork during 15 days of refrigerated storage Excellent antibacterial activity in minced meat, resulting in promising biological applications in food preservation

Hu et al. (2015)

—

Prevented oxidation of fatty foods

Liang et al. (2017)

L. monocytogenes

Inhibition of microbial growth tested on lean beef stored for 10 and 24 days at 4°C

Zimet et al. (2018)

E. coli, S. typhimurium

Badawy et al. (2019)

(Continued)

Table 3.12 (Continued) Chitosan nanosystem

Physicochemical properties

Challenged pathogen

Effect/purpose

References

Chitosan-myristic acid nanogel associating clove essential oil Polyethylene oxide nanofibers containing nisin-loaded polyγ-glutamic acid/chitosan nanoparticles Chitosan nanoparticle and chitosan-Byrsonima crassifolia nanoparticle Chitosan nanoparticles and chitosan-thyme essential oil nanoparticles

Size: ,100 nm

Salmonella enterica Ser. Enteritidis

Rajaei et al. (2017)

Size: 214.3 402.1 nm PDI: 0.134 0.196 Zeta potential: 35.8 46.1 mV

L. monocytogenes

Size: 351 nm

Pectobacterium carotovorum

Size: 4.5 6 0.6 nm and 6.0 6 0.3 nm

C. gloeosporioides

Nanoparticles composed of chitosan and chitosan with B. crassifolia extract

Size: 420.4 6 192.9 nm and 77.1 6 29.07 nm Zeta potential: 212.3 to 212.9 mV and 240.0 to 243.8 mV

A. alternata

Favorable antimicrobial activity in beef cutlet during chilled storage Satisfactory antibacterial activity against L. monocytogenes on cheese, without impact on the sensory quality Alternative for preserving tomatoes during refrigerated storage Complete inhibition of the growth of the pathogen in vitro and notable reduction of the incidence on avocado cv. Hass Preservation of physicochemical quality and excellent antimicrobial activity

Cui et al. (2017)

Gutiérrez-Molina et al. (2021) Correa-Pacheco et al. (2017)

González-Saucedo et al. (2019)

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103

3.5 Application of chitosan nanoparticles against pathogens in food systems Table 3.12 shows some applications of chitosan nanosystems and their association with some other active components to control and/or inhibit the growth of pathogens in food systems.

3.6 Conclusion and future perspectives In recent decades, interest and attention in the use of chitosan has focused on its antimicrobial properties due to its different areas of application, including the food industry, since it is considered a biodegradable, nontoxic, harmless, and environment-friendly biopolymer. As reviewed in this chapter, the antimicrobial activity of chitosan depends on several factors, such as molecular weight, DD, source of origin, and structural properties of the different bacteria, mainly. However, in recent years, nanotechnology has drawn attention in the food industry because it has new and promising applications in food preservation and packaging. In this sense, it has been reported that chitosan-based nanosystems have a better antimicrobial activity compared to chitosan solutions, mainly due to the smaller particle size and higher zeta potential. Even so, in order to improve or enhance the antimicrobial activity of chitosan-based nanosystems, the association/encapsulation of various natural compounds such as plant extracts/phytobiotics, EOs, organic acids, antioxidants, and bacteriocins with biocidal activity in food has been promising. Therefore in the following years, nanotechnology will play an important role in the food sector, especially to improve food quality through the control of pathogenic agents in food (preservatives) and to incorporate nanomaterials into packaging materials.

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CHAPTER 4

Chitosan nanoparticles with essential oils in food preservation Layal Karam1 and Jina Yammine2 1

Human Nutrition Department, College of Health Sciences, QU Health, Qatar University, Doha, Qatar CNRS, INRAE, Centrale Lille, UMR 8207 UMET Materials and Transformations Unit, University of Lille, Lille, France 2

4.1 Introduction Researchers have been working on the development of innovative strategies to fight microbial contaminations and subsequently improve the quality and extend the shelf life of food products. Among these approaches, natural antimicrobials and particularly chitosan polymers, deacetylated derivatives of chitin, have attracted considerably research interest. Chitosan have shown particular versatile physicochemical properties, potent antimicrobial activities, as well as an ability to be produced in different forms favoring their different applications as in food, medical, pharmaceutical, cosmetics, and textile industries (Chandrasekaran et al., 2020; Keawchaoon & Yoksan, 2011; Ma et al., 2017; Quiñones et al., 2018). Moreover, due to their abundancy in nature, nontoxicity, short-time biodegradability, and low cost, chitosan polymers have been considered excellent candidates for several applications. Particularly, in the food industry, chitosan have received great attention as they are considered as Generally Recognized as Safe in the United States and have been approved to be used as food additives in other countries such as Japan, Finland, and Italy (Bento et al., 2020). Nevertheless, their direct application in their native forms are limited due to their poor water solubility in neutral and alkaline media, as well as their low mechanical and gas barrier properties (Abdollahi et al., 2012; Gyawali & Ibrahim, 2014; Khattak et al., 2019; Ma et al., 2017). Among the different technological approaches proposed to enhance chitosan functionalities, nanoencapsulation has emerged as an effective strategy as it conquers the obstacles of chitosan and enhances their physical as well as their chemical properties (Abdollahi et al., 2012). Nanoencapsulation is the process of incorporating bioactive compounds in a polymeric nanocarrier Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00006-5

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matrix. The matrix could be formed by a single or a combination of compounds such as proteins, polysaccharides, lipids, surfactants, or other polymers. Nanoparticles (NPs), developed using chitosan as a carrier material, have received particular potential for the encapsulation of bioactive compounds such as essential oils (EOs). EOs are natural complex compounds derived from the secondary metabolism of herbal and medicinal plants. They are made of quite different concentrations of volatile active components including terpenes, terpenoids, phenylpropanoids, and aromatic compounds. EOs have shown a broad spectrum of biological activities including antimicrobial (Almeida et al., 2016; García-Salinas et al., 2018; Hu et al., 2019), anticancer (Afoulous et al., 2013), antioxidant (Ahmed et al., 2019; Coté et al., 2017), and antiinflammatory (Avoseh et al., 2015; Coté et al., 2017) functionalities. Despite their various advantages, the use of EOs in food industries is limited due to their hydrophobic nature, high volatility, reactivity with food components, as well as their sensitivity to light, oxygen, pressure, and heat (Amiri et al., 2020). Thus their incorporation into NPs overcomes the different limitations of EOs and promotes their use in several applications. This chapter highlights the recent published data on the different functionalities of chitosan NPs encapsulating EOs in food applications. Moreover, it provides an overview of the formation methods, design, antimicrobial mechanisms of action, and controlled release properties of the developed chitosan NPs incorporating EOs.

4.2 Chitosan nanoparticles system for the encapsulation of essential oils: properties, advantages, and formation methods Chitosan polymers encapsulating EOs have received tremendous interest as antimicrobial agents used in the food sector. Encapsulation may be fundamental to overcome the different obstacles of EOs and allow their protection and stabilization from environmental conditions. In fact, NPs act as barrier agents that provide protection against damage or decomposition of EOs, enhance their bioavailability, and minimize their interactions with food components with a subsequent decrement of unpleasant sensorial impacts (Ahmed & Aljaeid, 2016). As a result of the reduced size and increase in surface-to-volume ratio of NPs, EOs acquire a greater interaction with their target sites and thus enhanced functionalities (Esmaeili &

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Asgari, 2015; Granata et al., 2018; Shah et al., 2013; Shetta et al., 2019). Moreover, NPs exhibit unique advantages such as the controlled and sustained release of EOs leading to long-term effectiveness (Chen et al., 2015; Hosseini et al., 2013; Hu et al., 2018; Jamil et al., 2016; Karimirad et al., 2019; Ngampunwetchakul et al., 2019; Popiolski et al., 2016). Therefore superior applications of chitosan NPs encapsulating EOs have been reported for pest control (Ahmadi et al., 2018; Campos et al., 2018; Ferreira et al., 2019; Rajkumar, Gunasekaran, Dharmaraj, et al., 2020; Rajkumar, Gunasekaran, Paul, et al., 2020), medical applications (Amiri et al., 2020; Ferreira et al., 2019; Julianti Wijayadi & Rusliati Rusli, 2020; Karam et al., 2020; Rajkumar, Gunasekaran, Dharmaraj, et al., 2020; Salehi et al., 2020), food packaging (Correa-Pacheco et al., 2017; de Oliveira et al., 2020; Fernández-Pan et al., 2015; Hadian et al., 2017; Kurek et al., 2013), as well as their use as antioxidant (Amiri et al., 2020; Attallah et al., 2020; Feyzioglu & Tornuk, 2016; Shetta et al., 2019; Su et al., 2020), antibacterial (Alarfaj, 2019; Feyzioglu & Tornuk, 2016; Gadkari et al., 2019; Jamil et al., 2016; Marei et al., 2018; Mohammadi et al., 2020; Ozogul et al., 2020), and antifungal agents (Abeer Ramadan Mohamed et al., 2018; López-Meneses et al., 2018; Yadav et al., 2019). Encapsulated EOs could be either incorporated in food packaging or directly applied on food samples to control spoilage or pathogenic microorganisms. In this chapter, the antimicrobial activity of chitosan NPs encapsulating EOs and applied directly on food products were discussed. For the development, properties and different applications of chitosan NPs encapsulating EOs, several methods have been used. Among the variety of formation methods, ionic gelation (IG) has been widely applied as it is simple, organic solvent free, nontoxic, as well as a controllable technique that avoids the use of high temperatures (Hasheminejad et al., 2019; Karimirad et al., 2019; Mohammadi et al., 2015). Therefore this technique provides the optimal conditions for the applications of chitosan NPs encapsulating EOs in the food sector. IG relies mainly on the formation of inter and intramolecular crosslinkages between oppositely charged polymers and cross-linkers (Hasheminejad et al., 2019). In most of literature studies, the crosslinkages were between chitosan polymers and pentasodium tripolyphosphate (TPP) cross-linker. The protonated amino groups (NH31) of chitosan interact electrostatically with the phosphoric anions (P3O105-) of TPP. Chitosan NPs formed by IG have been reported to successfully load EOs or their active components such as thyme (Barrera-Ruiz et al., 2020;

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Correa-Pacheco et al., 2017; Liu & Liu, 2020), cinnamon (Barrera-Ruiz et al., 2020; Ghaderi-Ghahfarokhi et al., 2017; Hu et al., 2015; IstúrizZapata et al., 2020), savory (Amiri & Morakabati, 2017; Feyzioglu & Tornuk, 2016), oregano (Hosseini et al., 2013), carvacrol (Keawchaoon & Yoksan, 2011), thymol (Liu & Liu, 2020; Medina et al., 2019), and cinnamaldehyde (Gadkari et al., 2019; Istúriz-Zapata et al., 2020). In some studies, IG was followed by freeze drying to induce further reductions in particles’ sizes (Liu & Liu, 2020). Some other techniques were less frequently used for the encapsulation of EOs into chitosan NPs as ultrasonic homogenization (Shokri et al., 2020), emulsion phase inversion (Bento et al., 2020), and solvent displacement method (Correa-Pacheco et al., 2017; Istúriz-Zapata et al., 2020). Ultrasonic homogenization relies on the formation of small particles using a high frequency of ultrasonic waves. This method is highly reproducible at large scale but requires a lot of energy and sophisticated material. The use of emulsion phase inversion method was just to simplify the formation of chitosan NPs and avoid the use of TPP (Bento et al., 2020). This technique relies mainly on an interconversion between two types of simple emulsions: oil-in-water and water-in-oil emulsions and it has an uncontrollable instability (Kumar et al., 2015). In solvent displacement method, an organic solvent diffuses into a surrounding aqueous phase and the encapsulated agent within a carrier material remains into the organic phase. This method is simple but relies on the use of organic solvents which is not preferable in the food industry. After the development of NPs, it is important to determine their different physicochemical characteristics such as their size, size distribution/ polydispersity index (PDI), charge (zeta potential), and encapsulation efficiency (EE) (Table 4.1). These properties could dictate the applications of NPs and may have as well an impact on the efficiency, release mechanism, and stability of the encapsulated agents. The different physicochemical characteristics of the developed NPs are related to the formation techniques and to the carrier materials used. The size of particles plays a major role in the efficiency and release of the encapsulated agents. Smaller particles provide a larger surface area that exposes more of the encapsulated agents to the outer environment. This leads to a faster diffusion and release of the encapsulated agents with subsequent enhanced functionalities (Sahli et al., 2022). The physical stability and distribution of NPs over time is related to their PDI values that represent the homogeneity in the droplets’ size distribution (Shokri et al., 2020). PDI values below 0.3

Table 4.1 Encapsulation methods and physicochemical characteristics of different formulations of chitosan nanoparticles encapsulating essential oils (EOs). EO

Citrus sinensis

Chitosan:EO ratio

Formation method

Physicochemical characteristics

References

Size (nm)

Size distribution

Zeta potential (mV)

Encapsulation Efficiency (%)

54.9 59.2

0.231 0.262

N/Aa

N/A

68 99.5

0.19 0.28

161.72 to 172.5 210.32 to 223.1 122.7 to 140.2

N/A

Ferulago angulata

0.5% (w/v): 0.2 μL/mL 1 g/100 mL:1% 3%

Emulsion phase inversion Ultrasonic homogenization

Savory

1:0 1.25

Ionic gelation (IG)

149.76 210.27

N/A

Cinnamon, thyme, Schinus molle Coriandrum sativum Carum copticum

0.2% (w/v): 125 500 μg/mL

IG

19.7 516.9

0.2 0.74

1:0 1 (w/v)

IG

57 80

N/A

N/A

26.50 75.99

1:0.25% 1.25% (w/w)

IG

30 80

N/A

N/A

7.2 36.2

Savory

0.5% (w/v):1% 1.5% (v:v)

IG

140.25 237.60

N/A

27.54 to 221.12

35.07 40.70

Cinnamaldehyde

1%:2% (w/v)

IG

80 150

N/A

N/A

N/A

32.07 39.93

N/A

Bento et al. (2020) Shokri et al. (2020) Amiri and Morakabati (2017) Barrera-Ruiz et al. (2020) Das et al. (2019) Esmaeili and Asgari (2015) Feyzioglu and Tornuk (2016) Gadkari et al. (2019) (Continued)

Table 4.1 (Continued) EO

Chitosan:EO ratio

Formation method

Physicochemical characteristics

References

Size (nm)

Size distribution

Zeta potential (mV)

Encapsulation Efficiency (%)

Cinnamon

1:0.8

IG

235.6

0.33

125.1

N/A

Clove buds

1:0% 1% (w/v)

IG

223.2 444.5

0.117 0.337

55.8 63.1

Clove

IG

129 148

N/A

Oregano

1 and 1.6:0.25% 1% (w/v) 1:0.1 0.8 (w/w)

IG

40 80

N/A

110.14 to 134.50 131 to 139 N/A

5.45 24.72

Cinnamon

48 105 mg:0 72 mg

IG

112 527

0.258 0.617

N/A

N/A

Cinnamon

Solvent displacement

8

N/A

21.32

N/A

N/A

N/A

22.48

N/A

Cumin

0.05% (w/v):0.1% (v/v) 0.05% (w/v):0.05% (v/v) 1:0.25

IG

52.77

0.181

N/A

17.89

Bitter orange oil

1:0.25 1.25 wt

IG

20 60

0.27 0.42

4.51 15.33

Cyperus articulatus

1:0.25 1.25

IG

119 713

N/A

127.0 to 149.7 N/A

Cinnamaldehyde

31.0 45.8

3.412 40.28

GhaderiGhahfarokhi et al. (2017) Hadidi (2020)

Hasheminejad et al. (2019) Hosseini et al. (2013) Hu et al. (2015) Istúriz-Zapata et al. (2020)

Karimirad et al. (2019) Karimirad et al. (2020) Kavaz et al. (2019)

Carvacrol

1:0.25 1.25

IG

40 80

N/A

125 to 129

14 31

Moringa

1 5 mg/mL:20 mg/mL 1:1

IG

94.3 246.1

0.139 0.432

24.5 41.3

IG followed by freeze drying IG

139.47 123.30 516.9

N/A N/A 0.74

117.2 to 145.1 130.87 132.42 140.2

N/A

Thyme Thymol Schinus molle

0.2% (w/v):0.04 g

80.99 70.72 26.6

1% 1.5% (w/v):1% (v/v) 300 mg:1 mg/mL

IG

34 117

0.32 0.54

IG

293.1

0.5

141 to 147 147.8

Cinnamomum zeylanicum C. zeylanicum

1:0 1

IG

96.93 181.63

N/A

N/A

1.99 16.91

1:0 1

IG

96.93 181.63

N/A

N/A

Heracleum persicum Paulownia tomentosa

1:0.75% (w/v)

IG

40 80

0.38

138.7 to 153.2 N/A

1:1 (w/w)

IG

185

0.67

141.3

40.6

Fennel Thymol

a

N/A: Not applicable.

66.6

N/A

Keawchaoon and Yoksan (2011) Lin et al. (2019) Liu and Liu (2020) LópezMeneses et al. (2018) Maghami et al. (2019) Medina et al. (2019) Mohammadi et al. (2015) Mohammadi et al. (2020) Taheri et al. (2020) Zhang et al. (2019)

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indicate a narrow size distribution that reflects the homogeneity of particles and implies a good relative stability. The magnitude of the charge or electrostatic attraction/repulsion between particles is known as the zeta potential. Zeta potential values of 6 30 mV indicate a good relative stability providing lower attractive forces and aggregation between particles (Bashiri et al., 2020; Moghimi et al., 2018). EE corresponds to the amount of EO successively loaded and enclosed within NPs. It is also related to the type of carrier materials and cross-linkers used, and to the encapsulation method. An increase in particle size provides more space to encapsulate EOs in the wall or core of particles that leads to higher EE percentages (Bashiri et al., 2020). Lower EEs are reported in smaller particles because of the weak absorption of EOs on the surface of NPs, and thus they will be easily removed during centrifugation (Hosseini et al., 2013; López-Meneses et al., 2018). The different physicochemical characteristics of the developed chitosan NPs encapsulating EOs were evaluated in several studies and presented in Table 4.1.

4.3 Controlled release properties Generally, the release of encapsulated agents from NPs occurs by various mechanisms, including diffusion, surface degradation/erosion, and swelling (Esmaeili & Asgari, 2015; Fathi et al., 2012). The dominant mechanisms rely on the diffusion of the incorporated agents out of NPs into the surrounding medium or by the disintegration of the polymeric matrix surrounding the encapsulated agents following a degradation by an enzymatic reaction, an erosion after the dissolution or melting of the carrier materials, or a swelling due to the absorption of great amounts of water (Fathi et al., 2012). The release rate of EOs from chitosan NPs was characterized in most of literature studies by two phases: an initial burst release phase followed by a slower sustained release (Das et al., 2019; Esmaeili & Asgari, 2015; Hosseini et al., 2013; Karimirad et al., 2019). This biphasic release process indicates mainly the time-dependent diffusion of EOs out of chitosan NPs (Das et al., 2021). At the initial stage, the burst release may be related to the rapid diffusion of EO molecules adsorbed on or near the surface of NPs when in contact with a releasing medium. The first stage of fast release might be explained by the difference in Eos’ concentrations between the interior of NPs and the outer releasing medium, in addition to the higher dissolution

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rate of carrier material(s) near the surface of NPs (Esmaeili & Asgari, 2015; Hasheminejad et al., 2019; Hosseini et al., 2013; Kujur et al., 2021; Upadhyay et al., 2021). Moreover, as NPs present a higher surface-tovolume ratio with a subsequent increase in surface area, they allow a faster and more effective release of EO molecules adsorbed on the surface (Hosseini et al., 2013; Kujur et al., 2021). During the second phase, a relatively slower release of EOs occurs until reaching a steady plateau. The releasing medium penetrates into NPs leading to the swelling and degradation of carrier material(s), with an increased surface exposed to the releasing medium that favors the diffusion of EOs out of the swollen polymer (Esmaeili & Asgari, 2015; Karimirad et al., 2019; Keawchaoon & Yoksan, 2011). During this second stage, the reduced concentration gradient of EOs within NPs and the releasing media may be responsible for the slower release and diffusion (Amiri et al., 2020; Hasheminejad et al., 2019). Finally, the release of EOs might be negligible and reach a steady plateau due to the inability of release medium to disintegrate the wall structure of chitosan NPs (Hasheminejad et al., 2019; Kujur et al., 2021). Several studies corroborated this biphasic release profile of EOs from NPs. For example, Hu et al. (2018) spotlighted a fast release of thymol and cinnamaldehyde (26.01% and 38.05%, respectively) from chitosan NPs during the first 7 days, followed by a cumulative release of 26.67% and 38.77%, respectively, after 18 days. Also, Das et al. (2019, 2021) reported an initial release of EOs between 31.27% and 46.96% during the first 8 hours, followed by a reduction in the release rate reaching 12.22% 14.8% after 16 24 hours. Similar findings on the biphasic release profile were suggested by Abreu et al. (2012), where 30% 65% of EOs were rapidly discharged from chitosan NPs during the first 3 hours followed by 35% 74% cumulative release within 24 hours. Also, Upadhyay et al. (2021) reported a 35.74% release of Cananga odorata EO during the first 2 days until reaching 39.53% after 7 days. The release time and profile of EOs from chitosan NPs may vary depending on NPs’ preparation conditions, carrier materials’ compositions, as well as the pH of the releasing medium (Liang et al., 2017; Upadhyay et al., 2021). Most studies reported a higher release of EOs from chitosan NPs at acidic pH compared to their release at neutral and basic pH. This was mainly ascribed to the swelling and partial degradation of chitosan wall material at acidic pH, due to the increased concentrations of H1 that increase the presence and repulsion of free amino groups (NH31) of

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chitosan chains causing an increased surface exposure of NPs to the releasing medium and thus favoring the diffusion of EOs (Amiri et al., 2020; Esmaeili & Asgari, 2015; Hasheminejad et al., 2019; Karimirad et al., 2020; Keawchaoon & Yoksan, 2011). For example, 58.42% 83.47% of Eugenia caryophyllata L. EOs were released from chitosan NPs after 6 24 hours, at pH 3, while 36.30% 68.29% were released at pH 7.4 (Kujur et al., 2021). Also, Hasheminejad et al. (2019) reported a more pronounced release of clove EO at pH 3 as compared to pH 5 after 10 and 56 days of storage. Karimirad, Behnamian, and Dezhsetan (2018) determined a higher release of cumin EO at pH 3 (43.46%) compared to that at neutral pH (24.7%) after 10 days of storage. In another study, during the first 5 hours, the initial release of Carum copticum EO from chitosan NPs was 74.1%, 65.4%, and 73.5% at pH 3, 7.4, and 10, respectively. Even after 24 hours, the release was more pronounced at acidic pH, followed by basic then neutral pH (Esmaeili & Asgari, 2015). The release of EOs was more significant at basic pH compared to neutral pH due to the weakening of ionic interactions between deprotonated free amino groups of chitosan and anionic TPP at basic pH (Esmaeili & Asgari, 2015; Keawchaoon & Yoksan, 2011). Other studies also suggested that the release of EOs at acidic pH was more significant than their release at basic pH (Karimirad et al., 2020; Karimirad, Behnamian, & Dezhsetan, 2018; Kujur et al., 2021). At acidic pH, greater release of EOs was reported as a result of the partial dissolution and swelling of chitosan due to the ionic repulsions of the protonated free amino groups between chitosan chains. Relatively stronger interactions between EOs and chitosan NPs are involved at basic pH as compared to those at acidic pH. Thus the subsequent reduction in NPs surface area exposed to the outer medium at basic pH leads to a slower release of EOs. The distinguished gradual and controlled release properties of chitosan NPs encapsulating EO might be a major reason behind the enhanced and long-term antimicrobial as well as antioxidant activities of EOs (Esmaeili & Asgari, 2015; Hu et al., 2018; Karimirad et al., 2019). In fact, interactions between chitosan polymers and EOs reduce the released amounts of EOs and thus keep relatively high concentrations for the controlled release, ensuring long-term efficiencies (Hasheminejad & Khodaiyan, 2020; Hasheminejad et al., 2019; Yuan et al., 2016). This effect was documented in several studies. For example, encapsulation of E. caryophyllata L. and Coriandrum sativum EOs into chitosan NPs ensured a gradual antifungal effect due to the controlled release of EOs for 148 and 168 hours,

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respectively (Das et al., 2019; Kujur et al., 2021). Also for a similar reason, a mixture of cinnamon, thyme, and ginger EOs loaded into chitosan NPs displayed synergistic and long-lasting (up to 18 days) antimicrobial activities against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis (Hu et al., 2018). Additionally, the remarkable properties of controlled and gradual release of EOs from NPs were also highlighted for the shelf life extension and preservation of food products (Upadhyay et al., 2021). In rice and groundnut seed samples treated with encapsulated EOs, superior inhibitions of fungi and aflatoxin were observed, which delayed the deterioration of seeds for up to 1 year of storage (Das et al., 2019, 2021; Upadhyay et al., 2021). Also, delayed microbial and sensory deteriorations for 15 days were reported in mushroom samples treated with chitosan NPs encapsulating EOs (Karimirad et al., 2019, 2020). The controlled release of ginger EO from chitosan microcapsules delayed senescence, maintained quality, and prolonged the shelf life of jujube fruits for 14 days (Ban et al., 2020). Levels of antioxidant enzymes and phenolic compounds recorded in mushroom samples exposed to chitosan NPs encapsulating EOs were higher as compared to control samples. These higher levels subsequently led to an increased shelf life for 20 days (Karimirad et al., 2018). Beyki et al. (2014) observed a 10 14 days delay in the deterioration of tomato fruits when treated with encapsulated EO compared to a decay from the third day in control samples. Along the enhanced different functionalities of EOs, their controlled release from NPs displays a major another advantage in lowering the amounts of EOs being used in food products. Therefore lower concentrations of EOs provoke negligible sensorial impacts on food products preserving thus their initial qualities (Das et al., 2019, 2021; Hasani et al., 2020).

4.4 Antimicrobial mechanisms of action Within chitosan NPs, chitosan plays a dual role of a carrier material as well as an antimicrobial agent with remarkable effectiveness (Quiñones et al., 2018). Several studies reported an efficient antimicrobial activity of unloaded chitosan NPs (Esmaeili & Asgari, 2015). Thus chitosan NPs encapsulating EOs benefit from the antimicrobial action of both chitosan and the encapsulated EOs. This combination of several antimicrobials together is known as the hurdle technology, and it is expected to enhance

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the antimicrobial activity of both chitosan and encapsulated agent and subsequently contribute to major impacts in food applications. Although the antimicrobial mechanisms of action of chitosan NPs encapsulating EOs are not yet fully inferred, it is suggested that they may act synergistically to enhance the antimicrobial properties of each other (Barrera-Ruiz et al., 2020; Chen et al., 2018; Feyzioglu & Tornuk, 2016; Hu et al., 2018; Zhang et al., 2019). Based on several studies, it can be proposed that microbial membranes are the prime target sites of action of chitosan NPs encapsulating EOs. For chitosan NPs alone, the most widely recognized mechanism of action against microbial cells is through the electrostatic interactions between positively charged amino groups of chitosan polymers at low pH and negatively charged amino and carboxyl groups of microbial cell membranes (Bonferoni et al., 2017; Hafsa et al., 2016; Hu et al., 2015; Khattak et al., 2019; Rozman et al., 2019; Shokri et al., 2020; Verlee et al., 2017). This interaction results in an increased permeability and destabilization of microbial membranes leading to an easier diffusion of EOs into the interior of microorganisms (Alarfaj, 2019; Das et al., 2019; de Azeredo, 2012; Esmaeili & Asgari, 2015; Garcia et al., 2021; Grande-Tovar et al., 2018; Hasheminejad & Khodaiyan, 2020; Hu et al., 2018; Kalagatur et al., 2018; Kavaz et al., 2019; Wang et al., 2015). The facilitated diffusion of EOs originates not only from the destabilization of microbial membranes but also due to the smaller particle size and hydrophilicity of chitosan NPs (Bagheri et al., 2021). In fact, hydrophobic EO molecules have a difficulty to penetrate into the hydrophilic cell walls of microorganisms. Thus their encapsulation into nanoscaled hydrophilic particles promotes their penetration into microbial cell walls (Shetta et al., 2019). Alterations to microbial membranes’ integrity provoke a disruption of the osmotic pressure and enzyme systems, a leakage of essential intracellular constituents responsible for the growth and survival of microorganisms, as well as a damage of cellular functions that subsequently lead to cell death (Barrera-Ruiz et al., 2020; Chen et al., 2018; Correa-Pacheco et al., 2017; Das et al., 2019; Feyzioglu & Tornuk, 2016; Hadidi, 2020; Yang et al., 2021). Specifically for bacterial cells, it has been additionally shown that chitosan NPs with lower molecular weight may enter bacterial cell walls, bind to DNA, and inhibit mRNA together with protein synthesis (Chandrasekaran et al., 2020; Hafsa et al., 2016). Chitosan polysaccharides may also form a polymer membrane on the surface of microbial cells

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preventing nutrients uptake and/or acting as oxygen barriers and consequently inhibiting the growth of bacteria (Hosseinnejad & Jafari, 2016). One more likely mechanism of action is the ability of chitosan to act as a chelating agent toward metal ions associated with microbial cell wall molecules (Chandrasekaran et al., 2020). Chitosan amino groups surround the metal complex, obstruct nutrients and essential metals flow, and lead subsequently to cell death (Hosseinnejad & Jafari, 2016; Kravanja et al., 2019). The synergistic activity of chitosan NPs together with EOs against bacterial cells was reported in several studies. It was observed when loading chitosan with emulsified Pomelo (Citrus grandis Osbeck) peel oil against S. aureus and E. coli. The synergistic activity was probably attributed to chitosan polymers that enhanced the diffusion and contact between pomelo peel oil and bacterial cell walls (Chen et al., 2018). Also, Esmaeili and Asgari (2015) reported an improved penetration of C. copticum EO into S. aureus, Staphylococcus epidermidis, Bacillus cereus, E. coli, Salmonella typhimurium, and Proteus vulgaris outer membranes when loaded into chitosan NPs. Kavaz et al. (2019) determined that an enhanced activity of encapsulated Cyperus articulates rhizome EO due to the higher surface area of NPs promoting EOs penetration and disruption of S. aureus and E. coli cell walls. Additionally, thymol, thyme, and C. zeylanicum EOs were protected from environmental factors after their encapsulation which subsequently improved their antimicrobial activity against S. aureus and E. coli biofilms (Liu & Liu, 2020), as well as against Pseudomonas fluorescens, E. coli, and Erwinia carotovora bacterial cells (Mohammadi et al., 2020). Against fungal cells, the mechanism of action of chitosan polymers involved their diffusion into hyphae and eventual interference with the activity of enzymes responsible for fungal growth (Goy et al., 2009). Chitosan NPs may induce mycelial aggregation and morphological changes characterized by excessive mycelial branching with subsequent cell wall swelling and reduction in hyphae size (Cota-Arriola et al., 2011; Oliveira Junior et al., 2012). This is probably related to the chelating action of chitosan toward calcium ions responsible for hyphae growth and control of signals branching (Cota-Arriola et al., 2011). Moreover, chitosan NPs were shown to be effective in the reduction or inhibition of fungal spore formation and germination, and in partial inhibition of aflatoxin secretion (López-Meneses et al., 2018). Zingiber zerumbet EO encapsulated within chitosan NPs provoked a disruption of fungal cell membranes with subsequent leakage of

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intracellular ions such as K1, Ca21, and Mg21 and thus led to an irregular cell homeostasis with an inhibition of growth of fungi cells (Deepika et al., 2021). Additionally, levels of cellular magnesium oxide (MgO), which plays a major role in aflatoxin secretion, were reduced after treatment with encapsulated EO. This supports the previous findings stating that EOs encapsulated within chitosan NPs interact firmly with fungal cell membranes, affect fungal metabolism, and may probably inhibit the synthesis of aflatoxins (Deepika et al., 2021). Moreover, a significant impairment in intracellular ergosterol synthesis (a major component that maintains the permeability as well as the integrity of fungal cell membranes) was reported after treatment with encapsulated EOs (Chaudhari et al., 2020; Das et al., 2019, 2021; Kujur et al., 2021). Moreover, an irreversible damage of fungal cell membranes with a subsequent leakage of intracellular ions and UV-absorbing materials (nucleic acids and proteins), a disturbance of the osmotic balance, and a functional alteration of different organelles responsible for the secretion of aflatoxin were confirmed (Chaudhari et al., 2020; Das et al., 2019; Kujur et al., 2021). Geranium and lemongrass EOs encapsulated into chitosan particles had also better antifungal activities against Candida albicans, as compared to chitosan powders and to free EOs. This was assigned to their electrostatic interaction with fungal cells causing a destabilization of cell membranes and a subsequent diffusion of EOs (Garcia et al., 2021). Hence, based on the previous studies, it can be concluded that chitosan NPs are effective against both bacteria and fungi and they act synergistically with the encapsulated EOs by disrupting mainly microbial cell walls and allowing an easier diffusion of EOs. Additional studies are further required to investigate the interactions between chitosan NPs and the encapsulated EOs along with the related mechanisms of antimicrobial actions.

4.5 Functional properties of chitosan nanoparticles encapsulating essential oils on food products In food industries, there is a continuous interest in extending the shelf life of food products while using new preservation approaches. Remarkably, chitosan NPs encapsulating EOs have been applied for food preservation and often resulted in promising enhancement in food products’ quality, a reduction in microbial growth with a subsequent decrement in food deterioration, and an extension of products shelf life. In this section, several

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studies that discuss the functional properties and effects of chitosan NPs encapsulating EOs on food products were addressed.

4.5.1 Antioxidant activity Food constituents are susceptible to decomposition and oxidation as they are rich in unsaturated fatty acids, proteins, and water content. The pivotal results of oxidation in food products are the formation of toxic end products, off-flavors, and off-odors. Oxidation may contribute as well to the loss of nutritional values, development of oxidative stress, reduced shelf life, and acceptability and marketability of food products (Amiri et al., 2020; Deepika et al., 2021; Upadhyay et al., 2021). Among the different methods used to assess the antioxidant activity of chitosan NPs encapsulating EOs, DPPH (2,2-diphenyl-1-picrylhydrazil) free radical inhibition assay was the most prevalent technique. DPPH is a stable purple radical at room temperature that turns into a yellow color if its single electron is quenched by an antioxidant compound (Barzegar et al., 2016). Another strategy used to determine the antioxidant activity is by measuring the peroxide values that reflect the process of primary lipid oxidation in food products (Das et al., 2019). Malondialdehyde (MDA) content, a final product of lipid peroxidation, may be also quantified and used to assess the levels of lipid peroxidation (Deepika et al., 2021). Mainly, MDA content is measured after its reaction with thiobarbituric acid (TBA) to produce a red adduct which might determine the antioxidant activity of a compound (Chaudhari et al., 2020). Total phenolic compounds and antioxidant enzyme quantification may be additionally referred to assess the antioxidant activity as they have a considerable impact on the oxidative stability of food products by either restraining the decomposition of hydroperoxides into free radicals or by the inactivation of free radicals (Cetkovic, 2007). A considerable number of studies reported the antioxidant activity of chitosan NPs encapsulating EOs in food matrixes. Das et al. (2019) and Deepika et al. (2021) investigated the levels of MDA in rice and Salvia hispanica seeds after treatment with encapsulated C. sativum and Z. zerumbet L. EOs, respectively. Levels of MDA were the lowest in samples treated with encapsulated EOs [17.7 27.6 and 113.20 142.52 μM/g Fresh Weight (FW), respectively], compared to control samples (148.8 and 381.05 384.09 μM/gFW, respectively). Also, in maize samples, levels of MDA treated with free and encapsulated Origanum majorana L. EO were

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199.2 296.4 and 107.4 153.6 μg MDA/g FW, respectively (Chaudhari et al., 2020). The decrement in MDA content in samples treated with encapsulated EOs may be associated to the protection of EO antioxidant compounds that have a scavenging activity on free radical species that may induce lipid peroxidation (Deepika et al., 2021). In mayonnaise samples stored over a 4-month period, peroxide values for control samples and for those treated with free EO were higher than values recorded for samples treated with different concentrations of encapsulated EO (50.5, 26.68 35.6 and 18.82 28.55 meq peroxide/kg oil, respectively). The superior antioxidant activity of encapsulated EOs could be first attributed to the presence of chitosan polymers that have an antioxidant effect and to the easier penetration of encapsulated EO into the interfacial area that forms between the two phases of mayonnaise water emulsion, affecting thus the oxidation rate of unsaturated fatty acids that takes place mainly in this area. Interestingly, it was also found that peroxide values were declined during the last days of storage for samples treated with NPs, which might be assigned to the controlled release and gradual exposure of EOs to oxidation agents which maintains their functional properties over time. The enhanced antioxidant activity of EOs increased the shelf life of mayonnaise and reduced the production of off-odor and off-flavor compounds (Amiri et al., 2020). Also, in minced beef samples, peroxide values for samples treated with monoterpenes were significantly higher (20.50 28.33 meq O2/kg meat) compared to those treated with monoterpenes encapsulated within chitosan NPs (12.83 18.92 meq O2/ kg meat) (Badawy et al., 2020). Pomegranate arils showed the highest levels (30.58% DPPH) of antioxidant activity when treated with clove EO encapsulated into chitosan NPs after 54 days of storage, as compared to control samples (23.68% DPPH only after 12 days of storage) (Hasheminejad & Khodaiyan, 2020). Additionally, the application of encapsulated EO on pomegranate arils induced a superior activity of antioxidant enzymes with subsequent reduction in the formation and activity of reactive oxygen species and lipoxygenase that are both involved in the destruction of cell membranes. The high antioxidant activity of samples treated with encapsulated EO might be due to the delayed loss of phenolic compounds, flavonoids, or anthocyanins (Hasheminejad & Khodaiyan, 2020). Higher total phenolic content was reported when C. odorata and cumin EOs were used in their encapsulated forms as compared to control or to their free forms in Arachis hypogea L. groundnut seeds (Upadhyay

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et al., 2021) and button mushroom samples (Karimirad et al., 2019). Cumin EO acted as a free radical scavenging and thus diminished the loss of phenolic compounds and delayed the browning of mushroom samples (Karimirad et al., 2019). In the same study, the level of several antioxidant enzymes were maintained after treatment with encapsulated EO compared to free EO and control samples. This clearly reveals the effectiveness of encapsulating EOs in preserving the antioxidant phenolic compounds and enzymes that delay mushrooms’ browning. Also, the lowest activity of polyphenol oxidase, responsible for the oxidation of phenolic compounds, was reported in samples treated with encapsulated EO which might be associated to its gradual release from NPs (Karimirad et al., 2019). In button mushroom samples, the activity of both catalase (CAT) and glutathione reductase (GR) enzymes increased continuously during 15 days of storage after exposure to encapsulated Cuminum cyminum (Karimirad, Behnamian, & Dezhsetan, 2018), Citrus aurantium (Karimirad, Behnamian, Dezhsetan, et al., 2018), and bitter orange EOs (Karimirad et al., 2020). GR and CAT antioxidant enzymes protect cells from oxidative reactions and thus help to enhance or maintain the quality of food products. Moreover, the highest levels of ascorbate peroxidase (APX) and the lowest levels of peroxidase (POD) and polyphenol peroxidase (PPO) enzymes recorded were in samples treated with encapsulated EO (Karimirad et al., 2019, 2020; Karimirad, Behnamian, & Dezhsetan, 2018). Lower levels of POD and PPO enzymes in encapsulated EO treatments were probably due also to the gradual controlled release of EOs from NPs which might retard the oxidation of polyphenolic compounds with a subsequent decrement in the formation of off-colors and offflavors as well as the loss of quality and nutritional values. Accordingly, NPs showed a promising effect through the induction of antioxidant enzymes activities and preserving the quality of Agaricus bisporus fruit (Karimirad et al., 2020; Karimirad, Behnamian, & Dezhsetan, 2018). Therefore as a result of the previous studies, it can be concluded that among the numerous advantages of using NPs, encapsulating EOs improves their antioxidant activity and stability as a result of the protection of phytochemicals and phenolic constituents of EOs from adverse effects as decomposition or evaporation during storage (Badawy et al., 2020; Bagheri et al., 2021; Shetta et al., 2019; Upadhyay et al., 2021). The superior efficacy of encapsulated EOs on oxidation may be also assigned to the reduced size and subsequent increased surface area of NPs that promotes the exposure of EOs to free radicals inhibiting lipid

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peroxidation and thus leading to better antioxidant potencies (Deepika et al., 2021; Upadhyay et al., 2021). Moreover, the controlled and gradual release of EOs from NPs may preserve their functional properties for longer time as they will be gradually exposed to the oxidation agents that contribute to the deterioration of food products’ quality (Amiri et al., 2020; Upadhyay et al., 2021). Thus encapsulated EOs demonstrated their remarkable usefulness in food systems as antioxidant agents that inhibit or delay oxidation and thus improve the shelf life of food products (Chaudhari et al., 2020).

4.5.2 Antimicrobial activity—spoilage microorganisms 4.5.2.1 Yeasts, molds, and mycotoxins Food products are susceptible to postharvest diseases caused by fungal contaminations that lead to significant loss in nutritional values, production yields, and qualities of food products. Moreover, molds are prominent producers of hazardous mycotoxins that may cause contaminations, deleterious food spoilage, and severe health issues. Several studies reported the superior antifungal activity of chitosan NPs encapsulating EOs. This activity was related to several factors such as the type of EOs, concentration used, and food matrix applications. For example, the encapsulation of Satureja khuzestanica EO within chitosan NPs induced a control of Rhizopus stolonifer fungal growth with a subsequent delay in rottenness of tomato samples for 4 days compared to control (Amiri & Morakabati, 2017). Levels of protection displayed against fungal infestation in rice seeds were between 62.43% and 74.12% for samples treated with 2MIC (minimum inhibitory concentration) of free C. sativum or Pimpinella anisum EOs and between 84.17% and 100% for samples treated with 2MIC of encapsulated EOs. Additionally, Cananga odorata EO encapsulated into chitosan NPs exhibited 83.52% and 91.76% protection against fungal contamination at MIC and 2MIC, respectively, in groundnut seeds. While with free EO treatments, levels of protection were 41.75% and 67.03%, respectively (Upadhyay et al., 2021). For both EOs, in their free and nanoencapsulated forms, 100% inhibition of aflatoxin B1 contaminations were recorded, while in control samples aflatoxin levels were between 11.08 and 18.56 μg/kg. Thus EOs in both their free and encapsulated forms had a potent suppression of aflatoxins secretion in rice seeds (Das et al., 2019, 2021). After treatment with free and encapsulated cinnamon EO, yeasts and mold counts in beef patties were reduced by 1.62 1.92 and 2.96 3.16 log cycles, respectively,

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compared to control samples, after 8 days of storage (GhaderiGhahfarokhi et al., 2017). Another advantage of using encapsulated EOs is the possibility of adding lower antimicrobial concentration while achieving higher antifungal activity. Hasheminejad et al. (2019) determined that 1.5 mg/mL clove EO loaded into chitosan NPs inhibited completely Aspergillus niger mycelial growth in pomegranate, while even at concentrations as high as 3 mg/mL, free EO was unable to induce a complete inhibition. Moreover, yeast and mold counts were significantly lower in samples treated with clove EO loaded into chitosan NPs compared to control samples. In another study, chitosan NPs encapsulating clove EO delayed the incidence of fungal decay in ready-to-eat pomegranate arils from 18 to 60 days of storage (Hasheminejad & Khodaiyan, 2020). Upon exposure to chitosan NPs encapsulating C. cyminum EO, yeast and mold contamination in button mushroom samples were inhibited until the fifth day of storage while higher levels were reported in control samples even after 20 days of storage (Karimirad et al., 2019). Moreover, after 5 days of storage, yeast and mold counts in A. bisporus fruit bodies were reduced from 9.19 to 8.56 logs when treated with encapsulated bitter orange oil (Karimirad et al., 2020). MartínezHernández et al. (2017) concluded that after 13 days of storage, yeast and mold counts in carrot samples were reduced by 6.1 logs after exposure to carvacrol loaded into chitosan NPs. Also, maize grains were completely protected for 28 days from fungal growth and hazardous mycotoxin formation after treatment with 700 ppm Cymbopogon martinii EO (Kalagatur et al., 2018). In S. hispanica seeds, nanoencapsulated Z. zerumbet EOs were much more effective than their crude forms against aflatoxin B1 contamination after 1 year of storage. Levels of aflatoxin B1 reported were 36.89 37.24, 4.81 14.95, and 4.42 10.11 μg/kg in control, free, and nanoencapsulated samples, respectively (Deepika et al., 2021). Moreover, C. odorata and O. majorana L. EOs, in both their free and encapsulated forms, ensured a 100% inhibition of aflatoxin B1 contamination in groundnut seeds and maize, respectively, compared to 22.61 26.17 μg/ kg detected in control samples (Chaudhari et al., 2020; Upadhyay et al., 2021). 4.5.2.2 Bacteria High levels of microbial contaminations, exceeding the acceptable upper limits set by international authorizations, may cause serious health concerns as well as costly economical losses for food producers and

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consumers. Moreover, the levels of microbial growth and their subsequent spoilage must be controlled as they are considered key elements for food products’ decay, deterioration, and reduced shelf life. After treatment with different EOs loaded into chitosan NPs, reduced levels of microbial contaminations were reported in several types of food products (Table 4.2). As an example, the encapsulation of cinnamon EO into chitosan NPs had a pronounced impact on levels of total mesophilic aerobic viable counts (TMVC), lactic acid bacteria (LAB), as well as Enterobacteriaceae in beef patties. In fact, TMVC were reduced by 1.19 2 and 2.23 2.86 log cycles after exposure to free and encapsulated EO, respectively. Both treatments showed an efficient reduction with a subsequent extension of the microbiological shelf life of beef patties for 2 4 days. Moreover, LAB counts were kept below 4 log CFU/g up to the eighth day of storage and 3.23 3.86 Enterobacteriaceae log reductions were induced after exposure to encapsulated EO, compared to 1.15 1.79 log reductions when treated with free EO. In button mushroom samples, total aerobic mesophilic counts were inhibited until the 10th and 5th day of storage after treatment with C. cyminum and bitter orange EOs loaded into chitosan NPs, respectively. Moreover, total aerobic psychrophilic bacterial counts reported were lower than in control samples after 20 days of storage (Karimirad et al., 2019, 2020). In fresh pork, the total viable counts were 5.77 and 5.93 log CFU/g, respectively, in samples treated with encapsulated thymol and thyme EO, while in control samples 6.80 log CFU/g were reported at the 12th day of storage. A study conducted by Liu and Liu (2020) indicated a delayed spoilage of pork samples and an extension of their shelf life over 6 days after treatment with encapsulated EO compared to control. Also, in carrot samples, total mesophilic, psychrophilic, Enterobacteriacea, and LAB counts were reduced by 2.3 5.4 logs after treatment with carvacrol loaded into chitosan NPs, with the lowest counts reported at the 13th day of storage as compared to NaOCl, free carvacrol, chitosan powder, and empty chitosan NPs (MartínezHernández et al., 2017). 4.5.2.3 Pathogenic microorganisms Microorganisms, such as Salmonella, E. coli, and S. aureus, are among many common pathogenic bacteria that may compromise the quality and safety of food products. These pathogens are responsible for severe foodborne outbreaks that have induced significant public health concerns worldwide.

Table 4.2 The antimicrobial activity of chitosan nanoparticles encapsulating essential oils (EOs) in food applications. EO

Chitosan:EO ratio

Microorganism

Exposure time

Antimicrobial activity

Food application

References

Savory

1:1

Rhizopus stolonifer

9 days

Delayed rottenness from day 4 to day 8

Tomatoes

Citrus sinensis

0.5% (w/v): 0.2 μL/mL

Escherichia coli O157: H7

30 min

Orange and apple juices

Thyme

44.6% 98.6%: 1.0% 55.0%

Colletotrichum gloeosporioides

6 days

Coriandrum sativum

1.5%:MIC and 2MIC (0.9 and 1.8 μL/mL)

14 different foodborne molds and Aflatoxin B1

180 days

Cinnamon

3.2 mg/mL: 0.05% 0.1%

Staphylococcus aureus Total mesophilic aerobic count

8 days

Around 3 log reductions and lower than 1 log reduction in orange and apple juices, respectively 64% decrease in fungi incidence with 44.6% chitosan: 55% EO proportion The levels of protection against fungal infestation were 66.12% and 84.17% at MIC and 2MIC, respectively, and 100% inhibition of aflatoxin at MIC and 2MIC 3.99 4.59 log reductions 2.23 2.86 log reductions

Amiri and Morakabati (2017) Bento et al. (2020)

Lactic acid bacteria

Clove

0.15:0.15% (w/v)

Enterobacteriaceae Yeasts and molds Yeasts and molds

60 days

Counts were kept below 4 log CFU/g with 0.1% EO 3.23 3.86 log reductions 2.96 3.16 log reductions Delay in fungal decay incidence from day 18 (control) until day 60

Avocado

Rice seeds

CorreaPacheco et al. (2017) Das et al. (2019)

Beef patties

GhaderiGhahfarokhi et al. (2017)

Pomegranate arils

Hasheminejad and Khodaiyan (2020) (Continued)

Table 4.2 (Continued) EO

Chitosan:EO ratio

Microorganism

Exposure time

Antimicrobial activity

Food application

References

Cinnamon

48 105 mg:0 72 mg

15 days

Hu et al. (2015)

0.05% (w/v):0.05% (v/v)

Cucumbers

Istúriz-Zapata et al. (2020)

Cumin

0.5% (w/v):0.05 g

20 days

Agaricus bisporus mushrooms

Karimirad et al. (2019)

Bitter orange oil

1:0.5 wt

Mesophilic bacteria Psychrophilic bacteria Yeasts and molds Mesophilic bacteria Psychrophilic bacteria Yeasts and molds S. aureus

0.82 1.83 log reductions 1.15 2.03 log reductions 0.36 1.98 log reductions 0.1 1.04 log reductions Lower incidence of 48.14% of fungi while it was 62.96% for control 0.87 log reductions 1.53 log reductions

Pork

TransCinnamaldehyde

Total viable counts Pseudomonas Lactic acid bacteria Enterobacteriacea Fusarium solani

20 days

1.05 log reductions 0.68 log reductions 0.67 log reductions

Agaricus bisporus mushrooms

Karimirad et al. (2020)

0.75 log reductions MIC reduced from 20 to 10 mg/ mL compared to free EO Additional 0.87 1.03 log reductions compared to control with an extended shelf life over 6 days

Pork

Liu and Liu (2020)

Prawn shrimp

Liu et al. (2020)

8 days

Thyme, thymol

1:1

Total viable counts

12 days

Clove

1:0.5% (w/v)

Total aerobic counts

15 days

Significant inhibition with around 3 logs lower counts than control

Mesophilic bacteria Psychrotrophic bacteria Pseudomonas Lactic acid bacteria Mesophiles Psychrophiles Enterobacteriaceae Lactic acid bacteria Yeats and molds Total aerobic count

Huso huso fish fillets

Maghami et al. (2019)

Carrot slices

MartínezHernández et al. (2017)

Cucumber

Mohammadi et al. (2015)

7.25 log reductions after 18 days Additional 3 log reductions compared to control at day 15 Not detectable after 11 days 1.78 log reductions at day 13

Cucumber

Mohammadi et al. (2016)

Green beans

Severino et al. (2015)

16 days

4.65 log reductions Around 4 log reductions

Rainbow trouts

Shokri et al. (2020)

16 days

Counts (4.94 log CFU/g) remained below the microbiological threshold (7 log CFU/g) 2.61 log reductions Counts remained below 5 log CFU/g until day 16

Ready to cook pork chops

Zhang et al. (2019)

Fennel combined with modified atmosphere packaging

1% 1.5% (w/v):1% (v/v)

Carvacrol

1% (w/v):2.5 g

Cinnamomum zeylanicum

1.5 g/L of 1:0.25

Zataria multiflora

1:0.25

Total aerobic count Yeasts and molds

21 days

Carvacrol

1:0.05% (w/v)

E. coli Salmonella typhimurium

13 days

Ferulago angulata

1 g/100 mL:3%

Paulownia tomentosa

2% (w/v):1% (v/v)

Total viable counts Total psychrotrophic counts Total viable counts

Lactic acid bacteria Pseudomonas

27 days

13 days

21 days

1.575 log reductions 1.58 log reductions 0.2 log reductions 2.1 log reductions 1.4 log reductions 0.4 log reductions 0.6 log reductions 0.5 log reductions 1.7 log reductions Counts below 5 logs while for control samples they were 11.76 logs

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Few studies addressed the activity of EOs loaded into chitosan NPs on pathogenic microorganisms detected in food products. Bento et al. (2020) studied the effect of encapsulated Citrus sinensis EO combined with mild heat against E. coli O157:H7 Sakai in apple and orange juices. In apple juice, an additional 1.5 log10 cycles inactivation of E. coli O157:H7 Sakai was reported for samples treated with encapsulated EO as compared to EO suspensions. While in orange juice, a similar antimicrobial activity was reported for EO suspension and NPs. This could be explained by the high pectin content of orange juice that interacts with positively charged chitosan polymers at acidic pH, which might limit the antimicrobial activity of encapsulated EO. Ghaderi-Ghahfarokhi et al. (2017) concluded that after 8 days of storage, S. aureus counts in beef patties were reduced by 2.12 2.62 and 3.99 4.59 logs after exposure to different concentrations of free cinnamon and encapsulated EO, respectively. The more pronounced activity of encapsulated EO might be attributed to their controlled release and thus their long-term antimicrobial activity compared to free EO that induces microbial reductions within a limited period. In minced meat stored for 10 days, E. coli levels exceeded the upper acceptable limit of 6 log CFU/g (243 3 104 CFU/g) in control samples, while they were lower, between 40 and 125.83 3 104 CFU/g for samples treated with crude monoterpenes and between 8.33 and 35 3 104 CFU/g for those treated with monoterpenes encapsulated into chitosan NPs (Badawy et al., 2020). NPs cover a larger surface area of meat samples due to their smaller size and thus this favors the antimicrobial activity of EOs. Therefore it can be concluded from the previous studies that the incorporation of EOs into chitosan NPs induces a reduction or inhibition of microbial cells, mycelial growth, fungal infestation, germination, and sporulation, in addition to an inhibition of mycotoxins production. A delayed decay and an increased shelf life of food products, between 4 and 42 days, were observed after treatment with EOs incorporated into chitosan NPs. This emphasizes the importance of the emerging technology of encapsulating EOs into chitosan NPs to reduce microbial contaminations and enhance the shelf life of food products by providing a protection, an enhanced stability, and a more pronounced antimicrobial activity. The superior antimicrobial activity of EOs loaded into chitosan NPs may be attributed to their preservation and protection into the NPs layer that enhances their stability, reduces their volatility and lowers their interaction with food components, and thus increases their potent efficiencies

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(Amiri & Morakabati, 2017; Upadhyay et al., 2021). Moreover, the enhanced activity might be due to the controlled gradual and slower release of EOs from NPs over a long storage period, with the reduced size of NPs to a nanoscale level that provides a larger surface area and promotes an easier penetration of EOs into microbial cell membranes (Hasheminejad & Khodaiyan, 2020; Hasheminejad et al., 2019; Kalagatur et al., 2018; Karimirad et al., 2020; Martínez-Hernández et al., 2017; Upadhyay et al., 2021).

4.5.3 Sensory characteristics Sensory and organoleptic properties such as flavor, texture, firmness, color, and odor are critical aspects that dictate the quality and overall acceptability of food products and thus they must be evaluated and maintained during the storage period. Microbial spoilage and oxidation are the main factors that induce unfavorable sensory attributes in food products. While EOs are effective antimicrobials and antioxidants, their strong flavors and odors can be a major drawback for their direct incorporation into food products. Thus, due to their barrier properties, NPs could be also used to mask these unfavorable characteristics (Chaudhari et al., 2020). Additionally, the reduced concentrations of EOs used in NPs can help to preserve the original sensory attributes avoiding thus any undesirable impact on the appearance, safety, or quality of food products. Several studies discussed the effect of encapsulating EOs on the sensory attributes of food products. For example, encapsulating O. majorana L., E. caryophyllata L. EOs, or carvacrol into chitosan NPs achieved satisfactory scores for the tested sensory parameters (color, texture, odor, overall acceptance, firmness, and/or mouth feel) in maize (Chaudhari et al., 2020; Kujur et al., 2021), carrots (Martínez-Hernández et al., 2017), and rice (Das et al., 2021) samples. Whereas in control samples or those treated with free EO, lower unacceptable scores were reported. Also, the highest scores for sensory attributes in pomegranate arils were recorded after treatment with chitosan NPs encapsulating clove EO after 54 days of storage at 5°C compared to control samples with the lowest scores registered after 12 days of storage (Hasheminejad & Khodaiyan, 2020). In apple juice, NPs of orange EO combined with mild heat displayed higher scores in terms of color and odor in comparison with free EO suspensions, making NPs the optimal option for the preservation of apple juice. While

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in orange juice, both free and encapsulated EOs were accepted by consumers in terms of overall acceptability, color, flavor, and odor (Bento et al., 2020). In untreated beef patties samples or those exposed to free cinnamon EO, unacceptable strong odor, color, and overall acceptability were reported after 4 days of storage, while the same sensorial properties were acceptable until the eighth day of storage in samples treated with encapsulated EO (Ghaderi-Ghahfarokhi et al., 2017). In another study, Ban et al. (2020) addressed a delayed formation of black spots, a maintenance of significantly better sensory properties (appearance, firmness, crunchiness, as well as juiciness) of jujube fruits with a prolonged shelf life of 7 days after exposure to microencapsulated ginger EO. The following color parameters: L [lightness, ranging from black (0) to white (100)], a [ranging from red (160) to green (260)], and b [ranging from yellow (160) to blue (260)] determine any color changes in food samples and are considered critical for consumers’ acceptance (Karimirad et al., 2019). This tristimulus measurement mode is used as it relates with how the human eye perceives colors (Badawy et al., 2020). From these records, Chroma (C ) and hue angle (H ) values can be calculated. C reflects the overall intensity or color saturation, whereas H refers to the specific color appearance parameters and specifically the wavelength of light found along the visible spectrum (Yu et al., 2020). For minced meat samples treated with free monoterpenes, C and H values were 3.82 5.58 and 1.42 2.53, respectively, whereas in samples treated with NPs, C values were significantly higher (4.34 7), while there was no significant difference for H values (1.84 2.33). The higher preservation of red color in meat samples treated with NPs could be attributed to the protective capsules layer(s) that retard(s) the oxidation of myoglobin protein responsible for red coloration in meat samples (Badawy et al., 2020). Also, L values were reduced significantly in control button mushroom samples (75 81.75), while they remained acceptable (84.72) in samples exposed to encapsulated EO. Moreover, control samples were overall unacceptable after 10 days of storage, while they remained acceptable after treatment with both free and encapsulated EO until the 15th day of storage (Karimirad et al., 2019). Mandarin EO encapsulated into chitosan NPs delayed also color changes in pork samples due to the reduced microbial growth (Song et al., 2021). Liu and Liu (2020) reported a discoloration and an appearance of a green color in refrigerated control pork samples due to the accumulation of hydrogen peroxide produced by LAB and their subsequent reaction with nitric

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oxide myoglobin or nitric oxide hemochromogen. Whereas, in samples treated with thymol and thyme EOs encapsulated in chitosan NPs, a suppression of color and appearance changes in pork samples were addressed due to the effective inhibition of LAB. In another study, anthocyanin content, the key element responsible for the red coloration of pomegranate arils, was significantly higher (935.76 mg/L) in samples treated with clove EO loaded into chitosan NPs compared to control samples. NPs could ensure retention and delayed anthocyanin degradation due to the protective layer(s) that lower(s) oxygen supply and subsequently delay(s) the ripening and deterioration of fruits (Hasheminejad & Khodaiyan, 2020). Moreover, the firmness, texture, and weight loss are crucial parameters to determine the quality and predict the shelf life of food products. Firmness of tomato samples increased by applying S. khuzestanica EO loaded within chitosan NPs, while the reverse was reported during storage of control samples. The maintenance of the firmness and texture of tomatoes might be mainly related to the inhibition of fungal growth by chitosan NPs (Amiri & Morakabati, 2017). Karimirad et al. (2019) reported the lowest decrease in firmness in button mushroom samples (10.31%) after 20 days of storage once treated with C. cyminum oil encapsulated into chitosan NPs. However, only after 10 days of storage, the fastest softness (48.46%) occurred in control samples. Chitosan NPs loaded with C. aurantium EO reduced the respiration rate and retarded oxidation of phenolic compounds present in mushroom samples and thus limited the weight loss, the darkening and softening of fruit bodies (compared to the control samples) (Karimirad et al., 2018). It can be concluded that chitosan NPs encapsulating EOs play a vital role in the preservation and maintenance of the principal sensorial characteristics of food products. The reduced oxidation induced by EOs loaded into NPs diminishes oxidative stress as well as the development of rancid tastes, off-odors, toxicity, and deterioration of food products. Also, the superior antimicrobial activities with a higher control of microbial spoilage might preserve remarkably the quality of food products and extend their shelf life. It should be noted that sensorial evaluation of food products supplemented with encapsulated EOs must be exerted before their utilization on a larger industrial scale.

4.5.4 Physicochemical characteristics Various physicochemical parameters such as fat contents, pH, total soluble solids (TSS), and titratable acidity (TA) might be evaluated to determine the quality and safety of food products.

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pH measurement was one of the most evaluated physicochemical parameters as changes in pH are crucial for the shelf life stability of food products. In the reported studies, increased pH values induced microbial spoilage with subsequent deterioration and reduced the shelf life of food. Moreover, the analysis of TSS and TA were commonly practiced in solutions/substances for the determination of total amounts of sugar or acids, respectively, which might predict the quality of food. Several studies determined the physicochemical properties of food products after exposure to EOs loaded into chitosan NPs. For example, in pomegranate arils juice, the lowest levels of TSS and TA determined were in control samples (16 and 0.63, respectively) after 12 days of storage, compared to 17 and 0.83, respectively, reported after 54 days of storage in samples exposed to clove EO encapsulated into chitosan NPs. TSS and TA amounts may be linked to the flavor of fruits; hence, higher amounts will be appreciated and strongly related to fulfill consumers’ acceptance. Moreover, exposure to NPs was the most effective treatment in lowering the increase of pH in juice samples. Higher levels of TSS and TA, as well as the lowest pH changes induced by NPs, may be related to the delayed respiration rate of fruits due to the facilitated interaction of EOs with fruits’ cell membranes affecting thus their metabolic activities. Consequently, NPs encapsulating EOs retarded the senescence and deterioration of fruit products (Hasheminejad & Khodaiyan, 2020). Another study accomplished by Badawy et al. (2020) deduced that pH values reported in minced meat samples were between 5.40 5.76, 5.80 6.06, and 6.09 6.29 for control samples, samples treated with free monoterpenes, and those exposed to NPs encapsulating monoterpenes, respectively. Even though little increase in pH values were reported after exposure to free and encapsulated monoterpenes, minced meat samples were still suitable for consumption as their pH values were between 5.6 and 6.4. Also, in fresh-cut carrot samples, a 0.22 pH unit decrement was reported in samples treated with encapsulated carvacrol after 13 days of storage. Whereas in samples treated with free carvacrol, pH values increased by 2.16 units at the end of the storage period. The highest TA levels of 0.188% were recorded for samples exposed to encapsulated carvacrol, while free carvacrol treatments induced the lowest TA values of 0.010% (Martínez-Hernández et al., 2017). In fresh pork samples, chitosan NPs loaded with thymol or thyme EO inhibited the increase in pH values as they were 6.64 and 6.68, respectively, at the end of the storage period, while in control samples, the pH values were 6.94. NPs extended the

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shelf life of pork samples by inhibiting the growth of spoilage microorganisms that metabolize the basic nitrogen constituents of meat samples responsible for the increase in pH values (Liu & Liu, 2020). GhaderiGhahfarokhi et al. (2017) indicated maintained pH values for 4 8 days in beef patties treated with cinnamon EO loaded into chitosan NPs compared to increased pH values from 5.62 5.65 to 5.84 at the eighth day of storage for control samples. Thus samples treated with encapsulated EO ensured a more preservative effect and an extended shelf life of beef patties as they delayed enzymatic and/or microbial degradation of amino acids that generate volatile bases and deteriorate the quality of patties. In jujube fruits, microencapsulation of ginger EO contributed to maintained soluble solid content and TA values during the storage period which further resulted in preserved quality of fruit products (Ban et al., 2020). Finally, fat content levels reported in minced meat samples treated with NPs encapsulating monoterpenes were in the range of 19.90% -27.46% and of 20.37% 25.90% for those treated with free monoterpenes (Badawy et al., 2020). NPs may retard the decrement in fat content by limiting the activity of lipase or oxidative enzymes. Thus it can be concluded that analyzing the physicochemical properties of food products is crucial for the dictation of their quality and shelf life. More studies are still required to investigate further physicochemical properties of foods after their exposure to EOs encapsulated into chitosan NPs.

4.6 Conclusions and future perspectives During the last years, extensive research has been focused on the development of NPs with natural polymers to reduce microbial contaminations along with their subsequent possible outbreaks and costly economical losses. Chitosan polymers have received particular attention due to their remarkable antimicrobial and antioxidant properties. A highly promising approach in the food industry was their combination with natural EOs. This hurdle technology improved the antimicrobial activity of both active agents and expanded their applications, particularly in the food sector. Safety concerns of chitosan NPs encapsulating EOs should be considered, as at a nanoscale level, particles acquire new properties that allow a greater migration and contact with cell membranes as well as a higher capacity of being absorbed. Available scientific data on the migration of chitosan NPs into food products are still limited but eventually NPs will

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migrate into them. Hence, more studies are required to evaluate the potential toxicity of chitosan NPs when used as preservatives or as packaging materials in food products. Moreover, health and safety risks must be assessed before the application of chitosan NPs encapsulating EOs at an industrial scale in food products. Additionally, more studies are needed for applications in several food products along with better understanding of the exact antimicrobial mechanisms of action of chitosan NPs combined with EOs. Advancements in this field are promising to provide effective natural food preservatives while reducing the antimicrobial resistance challenges associated with the use of high concentrations of antimicrobials.

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Rajkumar, V., Gunasekaran, C., Paul, C. A., & Dharmaraj, J. (2020). Development of encapsulated peppermint essential oil in chitosan nanoparticles: Characterization and biological efficacy against stored-grain pest control. Pesticide Biochemistry and Physiology, 170, 104679. Available from https://doi.org/10.1016/j.pestbp.2020.104679. Rozman, N. A. S., Tong, W. Y., Leong, C. R., Tan, W. N., Hasanolbasori, M. A., & Abdullah, S. Z. (2019). Potential antimicrobial applications of chitosan nanoparticles (ChNP. Journal of Microbiology and Biotechnology, 29(7), 1009 1013. Available from https://doi.org/10.4014/jmb.1904.04065. Sahli, C., Moya, S. E., Lomas, J. S., Gravier-Pelletier, C., Briandet, R., & Hémadi, M. (2022). Recent advances in nanotechnology for eradicating bacterial biofilm. Theranostics, 12(5), 2383 2405. Available from https://doi.org/10.7150/thno.67296. Salehi, F., Behboudi, H., Kavoosi, G., & Ardestani, S. K. (2020). Incorporation of Zataria multiflora essential oil into chitosan biopolymer nanoparticles: A nanoemulsion based delivery system to improve the in-vitro efficacy, stability and anticancer activity of ZEO against breast cancer cells. International Journal of Biological Macromolecules, 143, 382 392. Available from https://doi.org/10.1016/j.ijbiomac.2019.12.058. Severino, R., Ferrari, G., Vu, K. D., Donsi, F., Salmieri, S., & Lacroix, M. (2015). Antimicrobial effects of modified chitosan based coating containing nanoemulsion of essential oils, modified atmosphere packaging and gamma irradiation against Escherichia coli O157:H7 and Salmonella typhimurium on green beans. Food Control, 50, 215 222. Available from https://doi.org/10.1016/j.foodcont.2014.08.029. Shah, B., Davidson, P. M., & Zhong, Q. (2013). Nanodispersed eugenol has improved antimicrobial activity against Escherichia coli O157:H7 and Listeria monocytogenes in bovine milk. International Journal of Food Microbiology, 161(1), 53 59. Available from https://doi.org/10.1016/j.ijfoodmicro.2012.11.020. Shetta, A., Kegere, J., & Mamdouh, W. (2019). Comparative study of encapsulated peppermint and green tea essential oils in chitosan nanoparticles: Encapsulation, thermal stability, in-vitro release, antioxidant and antibacterial activities. International Journal of Biological Macromolecules, 126, 731 742. Available from https://doi.org/10.1016/j. ijbiomac.2018.12.161. Shokri, S., Parastouei, K., Taghdir, M., & Abbaszadeh, S. (2020). Application an edible active coating based on chitosan-Ferulago angulata essential oil nanoemulsion to shelf life extension of Rainbow trout fillets stored at 4°C. International Journal of Biological Macromolecules, 153, 846 854. Available from https://doi.org/10.1016/j.ijbiomac. 2020.03.080. Song, X., Wang, L., Liu, T., Liu, Y., Wu, X., & Liu, L. (2021). Mandarin (Citrus reticulata L.) essential oil incorporated into chitosan nanoparticles: Characterization, anti-biofilm properties and application in pork preservation. International Journal of Biological Macromolecules, 185, 620 628. Available from https://doi.org/10.1016/j.ijbiomac. 2021.06.195. Su, H., Huang, C., Liu, Y., Kong, S., Wang, J., Huang, H., & Zhang, B. (2020). Preparation and characterization of Cinnamomum essential oil chitosan nanocomposites: physical, structural, and antioxidant activities. Processes, 8(7), 834. Available from https://doi.org/10.3390/pr8070834. Taheri, A., Behnamian, M., Dezhsetan, S., & Karimirad, R. (2020). Shelf life ectensions of bell pepper by application of chitosan nanoparticles containing Heracleum persicum fruit essential oil. Postharvest Biology and Technology, 170, 111313. Available from https:// doi.org/10.1016/j.postharvbio.2020.111313. Upadhyay, N., Singh, V. K., Dwivedy, A. K., Chaudhari, A. K., & Dubey, N. K. (2021). Assessment of nanoencapsulated Cananga odorata essential oil in chitosan nanopolymer as a green approach to boost the antifungal, antioxidant and in situ efficacy.

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CHAPTER 5

Chitosan as an antimicrobial agent to increase shelf life of foods Gerlane Souza de Lima1, Alessandra Silva Araújo1, Lúcia Raquel Ramos Berger2, Ana Elizabeth Cavalcante Fai3,4, Marcos Antonio Barbosa de Lima5, Rodrigo França6 and Thayza Christina Montenegro Stamford1 1

Postgraduate Program in Nutrition, Federal University of Pernambuco, Recife, PE, Brazil Postgraduate Program in Environmental Sciences, Federal University of the Agreste of Pernambuco, Garanhuns, PE, Brazil 3 Postgraduate Program in Food and Nutrition, State University of Rio de Janeiro (UERJ), Rio de Janeiro, RJ, Brazil 4 Basic and Experimental Nutrition, Federal University of the State of Rio de Janeiro (UNIRIO), Rio de Janeiro, RJ, Brazil 5 Department of Biology, Federal Rural University of Pernambuco, Recife, PE, Brazil 6 Department of Restorative Dentistry, University of Manitoba, Winnipeg, Manitoba, Canada 2

5.1 Introduction Driven by new techniques developed through research findings and market demand, the food industry is in constant technological innovation, driven by research and market demand. The demand for healthy and safe food for consumption with reduced additives has been increasing in recent years (Batiha et al., 2021). As a result of high consumer demands for fresher, more nutritious, more natural, and additive-free foods, new processing techniques continue to be studied and developed. Thus, during the last few decades, new technological processes have emerged in the food industry. These emerging processes utilize methods such as ohmic heating, high hydrostatic pressure, controlled release packaging, pulsed light, modified atmosphere, ultrasound, ultraviolet radiation, and the use of natural agents such as organic additives (Gómez-López et al., 2021; Khouryieh, 2021; Mesgari et al., 2021). It is important to point out that the food conservation processes are important and aim to avoid occurrences that can deteriorate the food, through the action of biological agents (microorganisms and enzymes) and the occurrence of chemical processes (such as lipid rancidification, color, Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00005-3

© 2023 Elsevier Inc. All rights reserved.

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and flavor compounds) and physical processes (such as agglomeration of powdered products). All these technologies combined with the general principles of good food production practices, from the farm to the table, guarantee the supply of safe food to the consumer and make its logistics viable with an adequate shelf life (Gómez-López et al., 2021; Mota et al., 2021). However, consumers and public health agencies are concerned with the adverse effects of the use of various common food additives in food products and their toxicological safety. This scenario has promoted the research of new natural agents that can be used to complement the traditional food preservation techniques (Batiha et al., 2021). Because of its peculiar physical, chemical, and biological properties, chitosan has been suggested as an option for this emerging demand in the food industry (Mesgari et al., 2021; Oladzadabbasabadi et al., 2022; Teixeira-Costa & Andrade, 2021). This chapter reviews chitosan and its derivatives’ main sources and techniques for production, along with the polymers’ chemical, physical, and biological characteristics. Focusing on the antimicrobial activity of chitosan, the following sections will explain the action mechanisms for their application as antibacterial and antifungal, as well as diverse forms of adding chitosan in food matrices and how it can promote the shelf life extension in these products.

5.2 Chitosan: emerging and eco-sustainable technology in food preservation 5.2.1 Sources and production Chitosan is a natural, amino cationic heteropolymer composed of ß-1,4 D-glucosamine units attached to N-acetylglucosamine residues (Fig. 5.1), which represents a structural component of the fungal cell wall, especially the Mucorales Dumort order and Mucoromycetes Doweld class, and its microbiological production occurs naturally through an enzymatic deacetylation of chitin, with the action of chitin deacetylase enzyme. This biopolymer shows promising characteristics such as biodegradability, biocompatibility, environment-friendly nature, absence of toxicity, antimicrobial activity, and a wide range of potential applications (Berger et al., 2020; Chatterjee et al., 2005). Chitosan can also be obtained by partial deacetylation of chitin present in the exoskeleton of crustaceans, mollusks, annelids, coelenterates,

Chitosan as an antimicrobial agent to increase shelf life of foods

4

H OH 6

O HO H

H

2 NH2

H

1

H

H

H

H OH

H O=

NH H

O

H OH

CH2

OH

H

6 O

H

O

HO

O

HO

H O

O

O 3

H OH

H HO

5 H O

NH2 H

H

NH2 H

H

157

H

5

4 HO

3

1

2 H

O

H NH2

H

Figure 5.1 Schematic representation of the chemical structure of chitosan (
CAS: 901276-4), its three reactive functional groups [amino (2NH2), acetamide group (2NHCOCH2), and primary and secondary hydroxyl groups (2OH) at the carbon in positions 2, 3, and 6 of the glucopyranoside ring].
Chemical Abstracts Service (CAS): CAS Registry Number.

and insects. This thermochemical procedure results in a chitosan molecule with various degrees of deacetylation, and consequently with different properties and applications. Crustacean exoskeletons are considered the traditional and the most common commercial source to obtain chitin and chitosan (Ali et al., 2022). The deacetylation process of chitin extracted from arthropods can involve chemical, enzymatic, fermentative, or bioconversion techniques (Abd El-Hack et al., 2020; Amer et al., 2022; Taser et al., 2021). One important aspect that draws much attention, in relation to obtaining commercial chitosan, is the possibility of using waste from the fishing industry precisely through the production of chitosan. The exoskeleton of crustaceans is one of the residues generated by this industry, being rich in chitin, the most common commercially available raw material to obtain chitosan, as mentioned above (Ali et al., 2022). In this sense, the chitosan is usually obtained by the deacetylation of crustacean chitin through a thermochemical procedure that presents some drawbacks: (1) the raw material (exoskeleton of crustaceans) is considered a source of seasonal and limited supply; (2) the use of high temperatures,

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resulting in high energy costs; (3) this process involves the production of large amounts of concentrated alkaline solution waste that may cause environmental pollution if inadequately discarded; (4) generation of a final product (chitosan) with heterogeneous and variable physicochemical characteristics, difficult to standardize; and (5) the presence of protein residues in the chitosan, which may increase allergic reactions in individuals with shellfish allergy (Berger et al., 2018; El Knidri et al., 2018; Ghormade et al., 2017). Elseways, the extraction of chitin and chitosan produced naturally in the cell wall of fungi can be considered a potential alternative to reduce these limitations. In the fungi cell wall the conversion of chitin to chitosan occurs from its deacetylation performed by the enzyme chitin deacetylase. This is a viable alternative for obtaining chitosan since it is renewable, transposing the barriers of supply and seasonality issues. This process occurs in a controlled manner, under specific cultivation conditions, generating less heterogeneity in physicochemical characteristics such as molecular weight and degree of deacetylation (Berger et al., 2018; Ghormade et al., 2017). Furthermore, the fungal chitosan does not present protein residues that can trigger allergic reactions in individuals, which amplifies its range of applications (Berger et al., 2018; Melo et al., 2020). However, this alternative source of chitosan still remains commercially underexploited. Despite the use of synthetic culture media for fungal growth and chitosan production, a variety of low-cost alternative substrates have been tested with promising results for scaling up fungal chitosan production. In this way, many studies expressed concern and interest to replace the use of synthetic culture media for the fungi growth and chitosan production with alternatives substrates such as vegetable components (e.g., yam bean) and agro-industrial waste (e.g., corn steep liquor and cassava wastewater), which may be considered more inexpensive nutritional sources and environment-friendly (Berger et al., 2016, 2018, 2020; Cardoso et al., 2012; Fai et al., 2011; Silva et al., 2006, 2007; Stamford et al., 2007). This possibility of obtaining a natural additive as a value-added product from an agro-industrial residue appears to be an interesting environmental strategy and is aligned with the principles of green chemistry and circular economy. As the economy grows, more raw materials are needed for production, and waste generation increases. According to Union Nation data, more than 2 billion tons of waste are generated annually. The reason for such volume is based on the current economic model—“extract, produce,

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dispose,” known as linear economy. This would not be much of a problem as long as the economy is smaller than our ecosystem, pointing out that the natural ecosystem is both the source of raw materials and the “sink” of our waste (Clark et al., 2020). The point is that the economy has grown disproportionately, and one must question the effectiveness of the current linear model in which natural resources are used at a rate greater than their regeneration capacity, resulting in an unprecedented scale of waste generation (Brito et al., 2020; MacArthur, 2013). To change this scenario, it is necessary to transition to a system based on the maximum extension of the useful life of products: the circular economy, which is inspired by the mechanisms of natural ecosystems and advocates that waste should be transformed into byproducts or other materials that allow for reuse, recovery, and recycling. Thus the circular economy is a solution that reconciles the sustainable model with the commercial and technological pace of today’s world (MacArthur, 2013). It is urgent to expand the understanding of this new model that favors the care for the environment and remodels the approach with which economic dynamics are perceived. In this logic, sustainability becomes notorious and assumes an elementary role in the global research agenda (Clark et al., 2020). Several sustainability-related approaches are being proposed and the use of waste (such as from the fishing industry or agro-industrial wastes as substrate) to obtain active ingredients, such as chitosan, seems to be an intelligent route that favors the care for the environment and the production of food with more natural additives (Ali et al., 2022; Klai et al., 2021).

5.2.2 Physicochemical and chitosan derivatives In each chitosan’s monomer (C6H11NO4), there are three reactive functional groups (Fig. 5.1): the amino group (C-2), a secondary hydroxyl (C-3), and a primary hydroxyl (C-6). As glucosamine units and N-acetylglucosamine units have different solubility in 1% (v/v) aqueous acetic acid, chitosan and chitin can be distinguished by this property. Since chitin contains more than 40% N-acetylglucosamine, it is insoluble (de Oliveira & de Oliveira Junior, 2020; Li & Zhuang, 2020). Chitosan is soluble in dilute acid solutions (pH , 6.5), but insoluble in water and concentrated organic solvents, such as formic acid, phosphoric acid, propionic acid, succinic acid, lactic acid, malic acid, hydrochloric

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acid, ascorbic acid, and also acetic acid. The last one represents the most frequently used solvent (Bakshi et al., 2020; Hamed et al., 2016; Nascimento et al., 2020; Qiao et al., 2021). At acidic pH, the polymer acts as a polycation due to the protonation of the amino groups (NH31) in the chain. This property promotes the polymer’s positive charge density and reflects its deacetylation degree (Han et al., 2020; Jafarizadeh-Malmiri et al., 2019; Rutz et al., 2017). The more cationic sites are formed by chitosan’s amino group protonation, the more enhanced its solubility. This occurs once the acid solution protonates the molecules’ amino groups, elevating the polarity and the degree of electrostatic repulsion between the positive charges. This results in the disruption of inter- and intramolecular hydrogen bond networks, which breaks chitosan’s crystalline structure and finally dissolves the polymer (Bakshi et al., 2020; Qiao et al., 2021). The dissolution process may be affected by the degree of deacetylation, pH, ionic strength, concentration, polymer molecular weight, and temperature (Bakshi et al., 2020; El Knidri et al., 2018; Qiao et al., 2021). The deacetylation degree and the molecular weight directly influence the chitosan chemical (tensile strength, solubility, surface area, viscosity, conductivity, porosity, and flexibility) and biological properties (adsorption enhancer, biodegradability, antioxidant, bioavailability, and biocompatibility) depending on the process conditions (Yadav et al., 2019). Despite being considered by the Food Drug Administration (FDA) as a food additive “generally recognized as safe” and its wide field of application, chitosan has disadvantages such as its low water solubility and mechanical strength (Romanazzi et al., 2017; Salama et al., 2018). Thus it is necessary to modify its structure chemically or by preparation technologies in order to improve its solubility and biological and chemical properties, further expanding its application (Bakshi et al., 2020; Wang et al., 2020). The amino and hydroxyl side groups in the chitosan chain can facilitate chemical modification reactions in the polymer, resulting in chitosan derivatives. This improves chitosan application since its insolubility and precipitation at pH above 6.5 compromise chitosan use under physiological conditions (Bakshi et al., 2020; Burr et al., 2018; Choi et al., 2016). Chitosan’s chemical modifications can be carried out mainly in the amino group at C-2 and in the primary hydroxyl group at C-6 (Bakshi et al., 2020; Gabriele et al., 2021; Li & Zhuang, 2020).

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On the other hand, the chitosan shows low solubility in water and solutions with neutral to alkaline pH which impairs its application and affects its biological properties (Zhang et al., 2015). Chitosan derivatives can be obtained by chemical modification using techniques such as acylation, alkylation, sulfation, hydroxylation, quaternization, esterification, graft copolymerization, and esterification (Wang et al., 2020; Zhao et al., 2018). Reactions such as these insert new functional groups into the chitosan molecule, breaking its crystalline structure, and resulting in increased solubility (Zhao et al., 2018). In this view, studies have been carried out to test methodologies to chemically modify chitosan (Hafdani & Sadeghinia, 2011), including the replacement of the amino groups in chitosan chain by specific residues which results in chitosan with desired characteristics. For instance, the use of carbohydrates, mainly mono and disaccharides, have been applied to carry out this modification due to its simple molecular structure and its low value. The introduction of carbohydrates can be performed by different chemical reactions, including the Maillard reaction (Gullón et al., 2016). The Maillard reaction involves the condensation reaction between one amino group of amino acids, peptides or proteins, and one carbonyl group of reducing sugars, aldehydes or ketones, being one of the main reactions that occur in processed foods. Thereby, the free amino groups in the chitosan can react in a Maillard reaction. Moreover, some studies suggested the contribution of the products of the Maillard reaction to provide flavors and antioxidant and antimicrobial effects (Bakry et al., 2018; Gullón et al., 2016). Among the water-soluble derivatives of chitosan, chitosan hydrochloride (Fig. 5.2) stands out. It is a chloride salt, positively charged. It’s usually obtained through the enzymatic degradation of chitosan by chitosanases in hydrochloric acid. It is also obtained by dialysis of chitosan solution in acetic acid against sodium chloride and deionized water (Kurozumi et al., 2019; Li et al., 2019). It is known for its nontoxic, biocompatible, biodegradable, and antibacterial properties with common and regular use as a food ingredient in countries such as Japan. This compound enhances the biological benefits of chitosan. As an advantage, it is soluble in neutral and basic media, not requiring acidic solutions to be dissolved (Fortunati et al., 2017; Kurozumi et al., 2019; Wu et al., 2015). Any other way, the polymer properties become more effective when applied in the form of nanoparticles, especially its antimicrobial activity.

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OH

OH

O

O

O HO

O + NH3 CI

+ NH3 CI n

Figure 5.2 Schematic representation of the chemical structure of chitosan hydrochrloride, its three reactive functional groups [amino (2NH31), chrloride group (2Cl22), and primary and secondary hydroxyl groups (2OH)].

The nanoparticles show the change in surface charge, making chitosan positively charged, favoring interaction with the negatively charged cell surface of microorganisms (Hernández-Fernández et al., 2020; JafarizadehMalmiri et al., 2019; Melo et al., 2018). The antimicrobial activity is an important biological property that differentiates chitosan from other polysaccharides and enables its use by the food industry, as it effectively controls spoilage (de Melo Barros et al., 2020; El Knidri et al., 2018; Hamed et al., 2016).

5.3 Chitosan and derivatives as antimicrobial agents Chitosan is considered an effective antimicrobial agent against bacteria, molds, and yeasts. However, fungi are more resistant to the polymer and its derivatives. Concerning bacteria, there is controversy about whether chitosan exhibits better bactericidal activity against Gram-negative or Gram-positive bacteria (Abd El-Hack et al., 2020). The polymer effectiveness is related to the degree of deacetylation, molecular weight, conditions of the matrix where it is applied, pH, the development of interactions between food matrix and the polymer, salinity, solubility, the type of microorganism, and other factors (Abd El-Hack et al., 2020; Alghuthaymi et al., 2020; Leite et al., 2015; Romanazzi et al., 2017). In general, its antimicrobial action increases when the degree of deacetylation is higher and the molecular weight is lower, which may better explain its mechanism of action, since the number of protonated amino groups rises (Hongpattarakere & Riyaphan, 2008).

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5.3.1 Antibacterial activity The mechanism responsible for the antimicrobial action of chitosan is still not fully elucidated. Nevertheless, three represent the most reported. The first one is based on the interaction of the opposite charges between chitosan and the negatively charged microbial cell membrane. It increases membrane permeability and interferes with metabolism, causing bacterial biofilms to rupture and leakage of inner proteins and other components. This sequence of events ultimately leads to cell death. The second mechanism refers to the potential for chitosan penetration into the cytoplasmic content and microbial nuclei by inhibiting the synthesis of mRNA and proteins. The third one is related to the suppression of nutrients essential to cell growth due to the chelating action of chitosan that binds to trace elements, inhibiting the production of toxins and microbial growth (Alghuthaymi et al., 2020; de Oliveira & de Oliveira Junior, 2020; Wang et al., 2020). The antibacterial effect of chitosan on prokaryotic cells is related to the electrostatic interactions between the biopolymer and bacterial cell wall components (e.g., teichoic and lipoteichoic acids, and lipopolysaccharides). These interactions result in the imbalance and damage to the selective permeability of the plasmatic membrane and may cause cell lysis (Díaz-Montes & Castro-Muñoz, 2021). The differences in the cell wall structure of Gram-positive bacteria and Gram-negative bacteria influence the susceptibility of these microorganisms to the antimicrobial effects of chitosan (Fig. 5.3). Gram-positive bacteria have thicker peptidoglycans, while Gram-negative bacteria show an external membrane enriched in lipopolysaccharide (LPS) which have the capacity to attach phosphorylated groups contributing to a more negative charge when compared with Gram-positive. In this way, there is more negative charge in the surface cell of Gram-negative bacteria available to connect with the positive charge of a cationic chitosan in environments with a pH below 6.5 (Ke et al., 2021). Some studies suggest an increased susceptibility to chitosan of Gramnegative bacteria than Gram-positive bacteria (Goy et al., 2016; Hassan, Omer et al., 2018). However, other research works report that Grampositive bacteria are more affected by this polymer (Raafat & Sahl, 2009).

5.3.2 Antifungal activity: ultrastructural effect of chitosan and chitosan nanoparticles In this section, we showed the ultrastructural effects of chitosan and their nanoparticles on relevant postharvest fungi such as Aspergillus niger,

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Figure 5.3 Scheme of the mechanisms of antimicrobial action of high/medium molar weight chitosan and low molar weight chitosan/oligosaccharide in Grampositive (A) and Gram-negative (B) bacteria.

Aspergillus flavus, Colletotrichum gloeosporioides, Colletotrichum siamense, Botrytis cinerea, and Rhizopus stolonifer. The literature has demonstrated the action of natural or modified chitosans from fungi and crustaceans and their nanoparticles on the fungal ultrastructure. Nascimento et al. (2020) reported similar morphological changes with conidia and hyphae of C. gloeosporioides highly wrinkled and withered due to the action of natural chitosan—citric acid at 5 mg/mL. In its turn, Meng et al. (2020) observed that hyphae of Aspergillus ochraceus when exposed to 0.05% and 0.1% chitosan became shriveled, contorted, and shrinkage. On the other hand, Berger et al. (2016) observed the presence of numerous depressions and craters on the hyphae surface of the Fusarium

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oxysporum treated with crustacean chitosan at 3.0 mg/mL. In addition, Berger et al. (2018) also reported that hyphae of the Colletotrichum fructicola treated with fungal chitosan (7500 ppm) showed roughness and depressions on the cell surface. Furthermore, Berger et al. (2020) demonstrated sharp morphological alterations induced by fungal chitosan in concentrations of 4000 µg/mL and 16,000 µg/mL on Scytalidium lignicola and Fusarium solani hyphae, respectively, such as corrugate surfaces with depression, atrophied, irregular and not hyaline branches, flattened, and non-uniform thickness. Chitosan nanoparticles have strong antifungal activity against several phytopathogenic fungi (Abdel-Aliem et al., 2019; Divya et al., 2018; Sathiyabama & Parthasarathy, 2016). Melo et al. (2020) also verified flattened and shrunken hyphae of R. stolonifer submitted to treatment with chitosan nanoparticles. This change suggests loss of cytoplasmic material causing cell death and lysis. The results shown in this work are also corroborated by Dananjaya et al. (2017) in which F. oxysporum mycelium exposed to chitosan nanoparticles (400 µg/mL) was distorted and depressed. Furthermore, it was observed a severe disruption in the hyphae surface, resulting in cell disintegration (Fig. 5.4). Thus the effects of crustacean chitosan on the morphology and cell surface of A. flavus and C. gloeosporioides hyphae can be observed in Fig. 5.5.

Figure 5.4 Scheme of the mechanisms of antimicrobial action of high/medium molar weight chitosan and low molar weight chitosan/oligosaccharide in fungi cell.

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Figure 5.5 Scanning electron microscopy of Aspergillus flavus (A) and Colletotrichum gloeosporioides (B) control hyphae. Hyphae of the A. flavus (C) and C. gloeosporioides (D) exposed to 5 mg/mL of crustaceous chitosan.

The control mycelium of A. flavus (Fig. 5.5A) cultivated in the absence of chitosan exhibits homogeneous hyphae with constant diameter, smooth external surface, and rounded apices. In the control mycelium of C. gloeosporioides (Fig. 5.5B), short, wide, smooth-surfaced hyphae can be observed. On the other hand, A. flavus hyphae treated with 5 mg/mL of crustacean chitosan (Fig. 5.5C) were distorted, with an irregular cell surface and many craters along the entire length of the hyphae. The presence of completely wilted hyphae is also noteworthy. The mycelium of C. gloeosporioides treated with chitosan 5 mg/mL showed hyphae and spores withered and with depressions on the cell surface (Fig. 5.5D). Therefore the serious damages shown on hyphae and spores of A. flavus and C. gloeosporioides reveal the ability of natural crustacean chitosan on inhibiting the fungi growth. The effects of fungal and crustacean chitosan nanoparticles on the morphology of C. siamense, R. stolonifer, A. niger, and B. cinerea are shown in Figs. 5.6 and 5.7. The control mycelium of C. siamense (Figs. 5.6A) and

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Figure 5.6 Scanning electron microscopy of Colletotrichum siamense (A) and Rhizopus stolonifer (B) control hyphae. Hyphae of the C. siamense (C) and R. stolonifer (D) exposed to 0.5 and 4 mg/mL of low-molecular-weight crustacean chitosan nanoparticles, respectively.

R. stolonifer (Figs. 5.6B) cultivated in the absence of chitosan nanoparticles showed a typical structure, and smooth surface, without deformations or damage. In turn, the hyphae subjected to treatment with chitosan nanoparticles showed important morphological and cell surface changes. C. siamense hyphae grown in the presence of chitosan nanoparticles were distorted and wrinkled (Figs. 5.6C), while R. stolonifer hyphae are completely distorted, flattened, and amorphous (Figs. 5.6D). Additionally, changes in the surface ultrastructure of A. niger and B. cinerea hyphae caused by chitosan nanoparticles can be seen in Fig. 5.7. The control hyphae of A. niger (Fig. 5.7A) and B. cinerea (Fig. 5.7B) have a regular and smooth surface. Furthermore, the presence of several conidiophores in the mycelium of A. niger is highlighted. However, A. niger mycelium exposed to 7 mg/mL crustacean chitosan nanoparticles showed completely flattened and amorphous hyphae (Fig. 5.7C). On the other

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Figure 5.7 Scanning electron microscopy of Aspergillus niger (A) and Botrytis cinerea (B) control hyphae. Effects of low-molecular-weight crustacean chitosan nanoparticles (7 mg/mL) on A. niger hyphae (C) and fungal chitosan nanoparticles (0.75 mg/mL) on B. cinerea hyphae (D).

hand, the mycelium of B. cinerea, when subjected to treatment with fungal chitosan nanoparticles, showed flattened, wrinkled hyphae and some cellular rupture points (Fig. 5.7D). Therefore chitosan and its nanoparticles are capable of inhibiting spore germination and mycelial growth, as well as causing heavy morphological and ultrastructural damage to the hyphae. The main ultrastructural changes found in fungi exposed to natural chitosan and its nanoparticles are cells (spores or hyphae) distorted, wide, short, flattened, withered, wrinkled, with depressions and craters, empty, and amorphous. The antifungal activity of chitosan and its derivatives depends on several factors such as the target fungus species and the physicochemical properties of chitosan. The composition of the cell wall and membrane represents an important factor in the sensitivity of filamentous fungi to chitosan

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(Aranda-Martinez et al., 2016; Lopez-Moya et al., 2019; Palma-Guerrero et al., 2010). Furthermore, the degree of deacetylation and the molecular mass are crucial factors in determining chitosan’s mechanism of action (Younes et al., 2014). In a study carried out by Fortunati et al. (2017), chitosan hydrochloride was tested as a film in the postharvest preservation of kiwi fruit and romaine lettuce. It formed a thin film, with great dispersion, and the antimicrobial activity tests, in vitro and in vivo, demonstrated the delay and inhibition of the development of B. cinerea. According to the authors, this chitosan salt has been widely used in the food industry as a preservative to maintain and extend the shelf life of fresh fruits and vegetables. In addition, chitosan is an alternative raw material to produce sustainable biomaterials for different applications, including food packaging (Díaz-Montes & Castro-Muñoz, 2021; Xu et al., 2021). The pandemic of COVID-19 has brought a sense of urgency in the resolution of various environmental problems and has shed a light on the importance of producing safe food, in the nutritional and hygienic-sanitary aspects, and reducing the use of conventional petroleum-based packaging (Barone et al., 2021). Thus the future of the food industry seems to be shaping itself on exactly these pillars of care, and the use of recycled ingredients and materials, like chitosan, is in line with many of these precepts. In essence, the observation formulated by Antoine Laurent Lavoisier in the 18th century, “in nature nothing is created, nothing is lost, everything is transformed” summarizes the goals of this new scenario. Advancing the knowledge of the obtainment and properties of chitosan is the key to broadening and prospecting new applications in the food industry.

5.3.3 Chitosan and its derivatives’ application to increase shelf life of foods The search for fresh food, with quality and extended shelf life, reflects the changes in the profile of consumers’ demand. This creates challenges for the supply chain and food distribution (Flórez et al., 2022; Zhang et al., 2021). This demand has led to the development of food conservation technologies, with emphasis on bioactive packaging that can guarantee an extended shelf life, quality, and microbiological safety of food (Flórez et al., 2022; Zhang et al., 2021). Several traditional conservation technologies have been used for this purpose, such as physical (irradiation, ultraviolet light, and temperature)

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and chemical methods (use of chlorine, bromine, iodine, organic acids, and synthetic fungicides). However, they have disadvantages in sensory and nutritional quality. In addition, some chemical methods can cause health problems for the consumer (Nascimento et al., 2020; Panahirad et al., 2021). An alternative that can overcome the deficiencies mentioned above is the use of eco-friendly, biocompatible, nontoxic, and functional biopolymers. The main application of these bioactive polymers is to obtain edible coatings and films that form a protective barrier around the food. In addition, these packaging systems can also improve sensory characteristics, such as the brightness of the food surface, prevent the outflow of desirable volatile compounds, reduce microbial growth and physiological disturbances, and do not impart odor or flavor to foods. It is important to point out that this conservation method is in accordance with the principles of green chemistry, since it is an edible packaging, which does not generate waste disposal in the environment (Duan et al., 2019; Nor & Ding, 2020). Thus, in addition to protecting, they reduce the harmful impact of plastic packaging disposal on the environment (Cox et al., 2019; Kumar et al., 2020). Edible films are a simple, economical, and efficient type of packaging capable of controlling moisture loss and chemical reaction rates. Therefore they reduce spoilage (inhibition of microorganism growth and microbial contamination) and improve the quality and safety of a wide variety of processed and fresh foods (Kumar et al., 2020). Application of films may follow different techniques such as solution molding, layer by layer, extrusion, and others. Furthermore, during processing, they can incorporate antimicrobial additives, antioxidant substances, flavors, and colorings into the food matrix, which further increases their applications (Kumar et al., 2020; Zhang et al., 2021). Edible coatings are also an innovative preservation method widely used for preserving fruits, vegetables, fish, meats, and derivatives. Its application on the surface of these foods forms a physical barrier that can reduce gas exchange, moisture and solute migration, as well as oxidative reactions (Duan et al., 2019). Moreover, edible coatings or films can be used as a transport vehicle for dyes, nutrients, bioactive compounds, and antimicrobial agents in order to reduce the risk of developing pathogens in the food matrices (Duan et al., 2019; Zhang et al., 2021). Coatings can be applied through various techniques such as immersion, spraying, and fluidized bed. Among these, immersion is the most

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common and has been used to coat fresh products, as it ensures the uniformity of the applied material (Senturk Parreidt et al., 2018). The immersion method consists of immersing the food product in the coating for a specified time, removing it, and draining the excess solution. The system to be used depends on the type of food to be coated, the surface characteristics, and the purpose of the coating (Senturk Parreidt et al., 2018; Suhag et al., 2020). These coatings and films can be made from various types of matrices such as polysaccharides, proteins, and lipids used alone or in combination with each other. Among the natural biopolymers used for the preparation of edible coatings, chitosan and its derivatives are considered the most prominent active principles (Bakshi et al., 2020; Flórez et al., 2022). Chitosan, when prepared as a film for packaging, appears as a transparent film, with good mechanical properties, such as flexibility and breaking strength. The polymeric film acts by forming an effective barrier to gasses (CO2 and O2), having high permeability to water vapor, and antimicrobial action. It can be used in various types of packaging, with the function of improving quality standards, promoting the extension of the shelf life of fresh and processed foods (Aizpurua-Olaizola et al., 2016). Chitosanbased edible coatings and films are excellent carriers of ingredients and functional additives that add flavor, color, vitamins, and antioxidant agents that can increase the nutritional value and functionality of the food matrices (Grande-Tovar et al., 2018; Karthik et al., 2021). Besides that, coatings and films formulated with chitosan or its derivatives can be prepared in various presentations, such as controlled viscosity solutions, gels, microspheres, microcapsules, and nanoparticles (Bakshi et al., 2020; Nair et al., 2020). Coatings and films with chitosan nanoparticles enhance the physicochemical, antimicrobial, and antioxidant properties of this polymer due to increased surface area and charge density (Abu Salha & Gedanken, 2021; Divya et al., 2018). In this form, it has been used as a carrier of bioactive compounds such as organic acids, antimicrobial agents, nutraceuticals, and essential oils (Adiletta et al., 2021; Eratte et al., 2018). Films and coatings using chitosan as an encapsulating material offer controlled release of the incorporated compound, thus maintaining effective doses of the additive throughout the storage period. By carrying compounds sensitive to thermal oxidation or light, chitosan, as an encapsulating agent, promotes increased stability. Thus such polymer properties reinforce its functional properties and those of its derivatives (Alghuthaymi et al., 2020; Fernández-Pan et al., 2015; Yuan et al., 2016).

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Another performance of chitosan, in its various forms, as a food preservative is in increasing the shelf life of beverages. This polymer’s antimicrobial activity has long been proved in different food matrices (Hosseini et al., 2016; Hu et al., 2016; Shah et al., 2016), In addition, chitosan can be easily modified to potentiate its properties, such as increased stability in solution, water solubility, and antimicrobial and antioxidant activities. Chitosan is suitable for a wide range of beverage applications, such as clarification, preservation, encapsulation, and packaging (Khan et al., 2018; Li et al., 2014; Rocha et al., 2017; Uranga et al., 2019). This polymer was applied as a preservative in fruit juices, wine, coconut water, and soy milk. Some uses of chitosan to extend the shelf life of juices involve its direct use. Among other forms of application of the polymer is the association with essential oils, natural compounds that are also recognized for their antimicrobial activity (de Carvalho et al., 2018; Zhong et al., 2020). It is important to highlight that the application of chitosan, like any other preservative in food products, requires vast studies so as not to compromise the product’s quality. Antimicrobial substances may interact with some food components, altering their properties and stability. Besides the food matrices’ peculiarities, chitosan has its own limitations for use, such as its solubility at neutral and alkaline pH values (Abdelhamid & El-Dougdoug, 2020; Ruiz-Rico & Barat, 2021). To circumvent such limitations, the use of water-soluble derivatives of chitosan, as well as chitosan as nanoparticles, is being studied. 5.3.3.1 Meat, fish, and derivative products Beef, sheep, pork, fish, and their derivatives are highly consumed foods around the world and of great economic and nutritional importance (Mauricio et al., 2022; Umaraw et al., 2020). The demand for beef, for example, has increased over the years. In 2018 world consumption was around 346.14 million tons and it is estimated that in 2030 this value will reach 453 million tons (Song et al., 2021). They are important components of the diet due to their composition rich in macro- and micronutrients, minerals, and vitamins (riboflavin, niacin, thiamine, and others from the B complex). They are foods rich in amino acids and essential fatty acids (such as linoleic, oleic, and linolenic acids) and a good source of iron intake, an important mineral for the synthesis of hemoglobin, myoglobin, and essential enzymes. (Song et al., 2021; Umaraw et al., 2020). However, meat products are highly perishable due to their composition and higher water activity, representing a media for microbial growth.

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Even during proper storage conditions, the microflora present can alter its nutritional value, shelf life, and overall quality. The current approach for improving meat product quality is introducing natural substances with antibacterial and antifungal properties that can inhibit pathogens and spoilage microorganisms without causing adverse effects on consumers (Baranenko et al., 2013; Song et al., 2021). The oxidation of lipids and proteins associated with microbial contamination are two relevant aspects of quality deterioration. These factors generate the need for adequate processing and handling to prolong the shelf life of these foods, along with refrigeration (da Silva et al., 2022; Song et al., 2021) Therefore new innovative technologies have been researched to preserve the quality and microbiological safety aspects of meats, such as nonthermal inactivation methods (high hydrostatic pressure) and new packaging systems (active and modified atmospheres) that offer more stable products, combined with renewable resources and cost savings (Baranenko et al., 2013; Dehghani et al., 2018). Among packaging systems, a current approach to improve the quality of meat products is the incorporation of natural substances with antibacterial and antifungal properties that can inhibit pathogens and spoilage microorganisms without causing adverse effects to consumers (Baranenko et al., 2013; Song et al., 2021). There are several studies showing the application of chitosan-based edible coatings or films as a natural antimicrobial agent in fish and meat products such as pork, chicken, beef, and derivatives (Baranenko et al., 2013; da Silva et al., 2022; Mauricio et al., 2022; Sánchez-Ortega et al., 2014). In a study carried out by Alves et al. (2018), the authors reported an increase in the shelf life of refrigerated salmon (Salmo salar), from 4 to 7 days, as a result of the antimicrobial action of the chitosan-based film with chitosan seed extract, grape and carvacrol microcapsules. Regarding the microbiological quality standard, the refrigerated salmon filets have lower values of mesophilic and psychrophilic bacteria and Pseudomonas spp. However, with 7 days of storage, the maximum allowable limit of the values of mesophilic and psychrophilic bacteria was detected. The antimicrobial effect of chitosan films can also be proven through the result of the lower count of psychotropic microorganisms evidenced in restructured chicken products when compared to the control group (Serrano-León et al., 2018). In addition to this result, Mahdavi et al. (2018) also demonstrated a reduction in microbial spoilage in chicken burger samples treated with chitosan edible film enriched with anise

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essential oil (Pimpinella anisum L.). The effectiveness of antimicrobial coatings and films made with chitosan was also evaluated in meat products, in the study conducted by Ashrafi et al. (2018), in which a chitosan-based film containing kombucha tea extract was able to maintain ground beef quality by retarding microbial growth and extending shelf life by 3 days. In seafood, the use of edible coatings can prolong shelf life by retarding oxidation and delaying microbial spoilage, once these products are highly perishable, that is, allowing rapid microbial growth of spoilage and food-borne pathogens. Nevertheless, there are limitations regarding gas permeability and mechanical properties, in comparison to synthetic ones (Dehghani et al., 2018; Sánchez-Ortega et al., 2014). In a research conducted by Martínez et al. (2018), a coating formed by chitosan, alginate, and resveratrol was developed that, when applied to smoked sea bass (Dicentrarchus labrax) filets, inhibited microbiological growth and delayed oxidation. The authors describe that chitosan protected the filets against bacterial growth, showing counts below 101 CFU/g, most of the analysis time during refrigerated vacuum storage. They also report that the microorganism counts showed a point increase in the time of 3 weeks, decreasing in the time of 4 weeks for mesophilic bacteria and anaerobic bacteria, probably due to random contamination. However, for psychrotrophic bacteria the counts remained low throughout the study time (4 weeks). It is important to report that chitosan, even with the oscillation in the count of mesophilic and anaerobic microorganisms, was the material that had the best antimicrobial effect. It is relevant to mention that edible coatings and films correspond to a primary food package. They must be associated with secondary nonedible packaging for proper handling and maintaining sanitary requirements (Dehghani et al., 2018). The type of coatings chosen depends on their biodegradability, biocompatibility, and mechanical properties, but also need to be nontoxic, and nonpolluting. This way, the quality of food is preserved, along with increasing shelf life period and reinforcement of positive environmental actions. 5.3.3.2 Beverages Beverages correspond to any drinkable liquids except water. They are divided into three groups: alcoholic, nonalcoholic, and dairy-based. Another subdivision in the nonalcoholic category is regarding the sugar content, in sugared (fruit juices, fruit nectar, and concentrated fruit juice) and nonsugared (coffee, tea) (Rocha et al., 2017).

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These products are the most common type of functional foods due to their convenient consumption, well adjusting to different lifestyles (Costa et al., 2021; Nazir et al., 2019). Nowadays, one similarity in the market tendency is the search for products that can combine safety, fresh or minimally processed ingredients, high stability, and bioactive components that also promote health benefits (de Carvalho et al., 2018; Nejatian et al., 2022). These requirements challenge beverage industries to develop new products that meet consumers’ needs. Among some strategies that industries pursue to bring healthier and more natural products to the market with a broader shelf life is to invest in natural preservatives combined with improved processing and active packaging (Costa et al., 2021; de Carvalho et al., 2018; Nejatian et al., 2022; Rocha et al., 2017). In fruit juices, products usually with acid pH, the conservation step most applied is heat treatment, for example, pasteurization. Even with the advantages of inactivating enzymes and reducing microbial contamination, there are a few setbacks to this kind of process. When performed at higher temperatures, the products may develop some off-flavors and promote nutritional losses (de Carvalho et al., 2018; Rodríguez-Roque et al., 2015; Soares et al., 2017; Zhao et al., 2020). If done in a mild temperature range (,100°C), the original flavor is preserved but some thermo-resistant fungi may not be inactivated. To resolve this issue, a combination of methods is made, such as mild heat treatment and the addition of preservatives, such as sorbates and benzoates. Albeit their effectiveness, allergy problems have risen from these chemicals’ use. Therefore natural antimicrobial agents have been studied to replace these synthetic preservatives (Ruiz-Rico & Barat, 2021). Among beverages, not only fruit juices have a high susceptibility to microbial growth. Coconut water also carries characteristics that require attention in its preservation, such as its elevated water activity and sugar content, along with a pH of 5.8. A potential solution is the use of essential oils as substitutes for conventional conservatives. An issue with these natural products is their strong flavor, hydrophobicity, and the need for increased doses that can alter the products’ sensory aspects if applied directly to the product (de Carvalho et al., 2018). Therefore adding encapsulated bioactive extracts or essential oils may represent a viable alternative for improving beverages’ shelf life (Costa et al., 2021; Jamali et al., 2021). The role of chitosan in beverage formulations involves their use as wall materials for encapsulating bioactive compounds. These substances, if

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introduced directly into the food matrix, may have their solubility, stability, and bioavailability reduced, once in contact with food components or environmental storage conditions. Another important aspect is that these compounds may require larger doses to express their function in the products, which also affect food organoleptic properties and, finally, consumers’ acceptance. Encapsulation can prevent all the issues mentioned above to the addition of bioactive compounds in foods and promote a controlled release of these ingredients (de Carvalho et al., 2018; Liu et al., 2017; Nejatian et al., 2022). The use of natural antimicrobial substances as a direct component of food has its setbacks, once these substances may interact with the food components, promote instability, alter the products’ properties, or even cause allergic reactions in consumers (Abdelhamid & El-Dougdoug, 2020; Liburdi et al., 2016). So, another approach is their application as an antimicrobial material grafted in regular filters to improve the filtration process. Along with the removal of undesired particles in the product, they eliminate microorganisms without interfering with these products’ properties, since they are immobilized (Cappannella et al., 2016; Ruiz-Rico & Barat, 2021; Zhang et al., 2021). Chitosan has been applied as a covalent immobilizer for lysozyme, an enzyme used in winemaking to control microbial growth, contributing to its removal from the products, avoiding labeling problems, and reducing the risk of allergies. Due to their nontoxic properties, biocompatibility, biodegradability, and antimicrobial activity, chitosan and its derivatives present an excellent potential as food enzyme immobilizers (Liburdi et al., 2016; Wen et al., 2016). In winemaking, oxidation is a significant setback for maintaining or improving the products’ organoleptic characteristics (color, flavors, and taste) and shelf life. The presence of oxygen also reduces the shelf life of wines since it contributes to acetic acid bacteria growth (Valera et al., 2017). An industry strategy is applying sulfur dioxide (SO2) as an antioxidant. In spite of its low cost and simple use, this substance is associated with adverse effects in consumers with hypertension and allergic asthma, which encourages its substitution, producing less processed wines with the maintenance of high quality. In this sense, chitosan may be implemented as an antioxidant because it acts as a chelator for catalytic metals, but it also increases storage time due to its antimicrobial activity (Marín et al., 2019; Valera et al., 2017).

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As seen, chitosan versatility as an active component for beverage preservation is expressed in many ways but is yet to achieve its full potential. This polymer and its derivatives may contribute to decreasing food waste, improving sustainability, and also providing economic benefits. Nevertheless, there is a need for developing more research for application in conditions applicable to the industry level and that can also create products with nutritional and organoleptic characteristics appealing to the market. 5.3.3.3 Fruits and vegetables Foods of vegetal origin have a short postharvest life due to their physiology (such as the high moisture content of 75%95%) and the natural process of senescence (Jafarzadeh et al., 2021; Panahirad et al., 2021). It is estimated that, worldwide, losses of fruits and vegetables reach the range of 20% due to microbiological contaminants (mainly fungi) and the very physiology of this type of food (Blancas-Benitez et al., 2022; FAO, 2018). The deterioration of fruits and vegetables in the postharvest phase leads to nutritional and sensory losses that compromise, in addition to the quality of this type of food, the consumer’s purchase intention (González-Estrada et al., 2021). In order to control microbial contamination and prolong the shelf life of these foods, several postharvest treatments have been used, such as physical and chemical methods. However, they have disadvantages with regard to sensory and nutritional quality and problems related to consumer health, when it comes to chemical methods (Nascimento et al., 2020; Panahirad et al., 2021). In this way, edible coatings can be an alternative technology that addresses the deficiencies in the previously mentioned postharvest treatments, since they perform the barrier function for gasses and moisture transfer, reducing the natural process of deterioration (Muley & Singhal, 2020; Zhang et al., 2021). In addition, they can also improve sensory characteristics such as the brightness of the external surface of the fruit, prevent the outflow of desirable volatile compounds, reduce microbial deterioration, and physiological disturbances, and at the same time be free from toxic effects on the human body (Hassan, Chatha et al., 2018; Nor & Ding, 2020). Several types of matrices can be used to prepare edible coatings, especially chitosan and its water-soluble derivatives (Kurozumi et al., 2019; Zhou et al., 2021). Chitosan-based edible coatings and films are excellent

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ingredient carriers, functional additives that add antimicrobial protection, flavor, color, vitamins, and antioxidant agents that can increase the nutritional value and functionality of the food matrix (Grande-Tovar et al., 2018; Karthik et al., 2021). Many studies have reported the use of edible film coating as a potential technology to be applied on the surface of fruits and vegetables as a thin film layer to maintain the quality and extend the shelf life of these perishable foods (Basumatary et al., 2021; Berger et al., 2018, 2020; Melo et al., 2018, 2020). The polysaccharides, lipids, and proteins can form these coatings, especially the biopolymer chitosan due to its favorable characteristics such as nontoxicity, biodegradability, biocompatibility, antimicrobial effect, and good film-forming properties (Wantat et al., 2022). The chitosan could be applied as fruit coating in a variety of fruit such as blueberry (Vaccinium ashei L.) (Li et al., 2021), pineapple (Ananus comosus) (Basumatary et al., 2021), banana fruit (Wantat et al., 2022), grape (Melo et al., 2018), and strawberry (Melo et al., 2020) to maintain the quality during storage and prolong the shelf life of fruit. The preservative effect, due to antimicrobial action, can be associated with the coating which formed a semipermeable film around the fruit; this thin film could inhibit the growth of pathogens by disturbing the cell membrane of the pathogen causing intracellular leakage and finally cell death. In addition, the chitosan coating can enhance the epidermal structure of fruit and limit the spread of pathogens. Moreover, the coating could assist the cell wall in retaining its integrity against fungal attacks and help in delaying pathogenic infection (Mohamed et al., 2017; Safari et al., 2021). The application of chitosan coating (B/CH) in addition to nanomaterial films such as silicon (B/CH/Nano-SiO2) and titanium (B/CH/NanoTiO2) maintained nutrients and provided the control of microbial growth on fresh blueberry fruits (V. ashei L.) extending its shelf life. The nanomaterial films (B/CH/Nano-TiO2) provided a high inactivation of peroxidase enzyme activities, while the nanomaterial films (B/CH/Nano-SiO2) resulted in the slightest change to acidity and anthocyanin and also minimized the growth of mesophilic aerobic, yeasts, and molds populations on fresh blueberry fruits. These nanofilms form a thin layer, less than 100 nm, over the fruit which can result in the control of dehydration, shelf life limitation, polyphenolics oxidation, transpiration, enzymes, respiration, exposure to oxygen and light, and microbial infection to favor a greater consumer acceptance (Li et al., 2021).

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A previous study evaluated a novel film prepared by chitosan/nano/ SiO2 and chitosan/nano/SiO2/nisin on cantaloupe fruit. The coatings chitosan/nano/SiO2 and chitosan/nano/SiO2/nisin reduced the yeast and mold count by 2.49 and 1.92 log CFU/g, respectively, in cantaloupe fruit’s shelf life on day nine, while only the chitosan coating showed a little effect (below 2.67 log CFU/g) in this same tested condition. Nano/ SiO2 films have been widely used in the packaging industry against the permeation of gasses and these films can prevent bacterial attacks in vivo and in vitro (Sami et al., 2021). These results reinforce the advantages when other substances with biotechnological potential are incorporated into chitosan-based coatings. Chitosan and vanillin (4-hydroxy-3-methoxy benzaldehyde), a phenolic aldehyde organic compound derived from the vanilla bean, were used as edible coatings on tomato fruit (Solanum lycopersicum Mill.) stored at 26° C 6 2°C and 60 6 5 relative humidity to control tomato fruit rot caused by F. oxysporum. The coating composed of 1.5% chitosan 1 15 mM vanillin was able to control disease incidence by 70.84% and severity by 70%. These combinations of coatings were also able to retain phenylalanine ammonia-lyase (PAL), peroxidase activity (POD), and polyphenol oxidase (PPO) enzyme activities as well as prolong the shelf life of tomatoes by up to 15 days (Safari et al., 2021). The combined effect of 1% chitosan plus 400 µL/L thyme oil was evaluated to control anthracnose caused by C. gloeosporioides and improve the shelf life of mango fruit (cv. White Chaunsa). This combined treatment showed more efficiency to control this disease in artificially inoculated mango fruit when compared with the thyme oil (400 µL/L) alone. In addition, the treatment of 1% chitosan plus 400 µL/L thyme oil favored the retention of physicochemical properties such as titratable acidity, total soluble solids, firmness, weight loss, color, and higher scores for sensory perception (Shah et al., 2021). The essential oil from thyme (Thymus vulgaris L.) presents antifungal activity against the fungi that cause postharvest diseases in various fruits, but this effect can diminish rapidly after application due to its high volatility and interaction of essential oil constituents with fruit tissue. In this sense, the combination of thyme oil with edible chitosan can be considered a potential alternative to reduce volatile losses and increase effectiveness (Cháfer et al., 2012). In the general context of the application of chitosan and its derivatives as a multifunctional natural food additive, its potential antibacterial and antifungal properties are pointed out, as well as its antioxidant capacity

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with potential use to control lipid oxidation and prevent enzymatic browning, among other (Novais et al., 2022). Despite its peculiar properties and multifunctionality, there is still a need to better understand the molecular mechanisms of chitosan interaction with microbial cells and other structures, such as nutritional components contained in food matrices. Such an understanding may enable the establishment of more effective commercial technologies that use chitosan for food preservation.

5.4 Perspectives This chapter presented different potential applications of chitosan and its derivatives that have several functionalities due to their biocompatibility, biological activity, biodegradability, reduced toxicity, and mechanical and physical properties. As an antimicrobial agent, this polymer acts by specific mechanisms, not yet fully elucidated. But, as demonstrated throughout the studies presented, it controls microbial decomposition in foods, either in the form of edible coating, film, or additive, besides extending the shelf life of foods. Nevertheless, chitosan’s full utilization is yet to be achieved. There are still many potential sources of its production, especially involving sustainable raw materials and techniques. In food science, chitosan’s use is a great challenge since it requires working with complex matrices. Chitosan represents an alternative to promote a circular economy in food production, the development of safer and more natural products, with less food waste and better food security, besides to represent a reduced use of synthetic plastics and additives promoting healthy food and healthy environment.

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CHAPTER 6

Application of chitosan on seafood safety and quality Nikheel Bhojraj Rathod1, Nariman El Abed2 and Fatih Özogul3 1

Department of Post Harvest Management of Meat, Poultry and Fish, Post Graduate Institute of Post-Harvest Management, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Roha, Raigad, Maharashtra, India 2 Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB), National Institute of Applied Sciences and Technology (INSAT), University of Carthage, Tunis, Tunisia 3 Department of Seafood Processing Technology, Faculty of Fisheries, Cukurova University, Adana, Turkey

6.1 Introduction Chitosan [(1 4)-2-amino-2-deoxy-β-D-glucan] is nontoxic linear biodegradable derivative of chitin (C8H13O5N)n formed by D-glucosamine and N-acetyl-D-glucosamine (Inanli et al., 2020). It is available abundantly in nature after cellulose. Chitosan is obtained by eliminating the acetyl group (deacetylation) from the chitin (Inanli et al., 2020). Commercially, chitosan is produced from seafood processing discards (shrimp and crab shells). The term chitosan was firstly coined by Braconnot for fractions from fungi in 1811. Later, in 1823, material obtained from beetle cuticle was termed as chiton. Chitosan is a derivative of chitin, which was produced by Charles Rouget in 1859 by treating chitin with caustic potash and was found soluble in organic acid (Bakshi et al., 2020). For production of chitosan, crab and shrimp shell wastes are used, which consist of proteins, lipids, chitin, and inorganic salts. They are demineralized (calcium oxide and calcium chloride) using dilute acids (hydrochloric acid) followed by deproteinization using dilute alkali (sodium hydroxide) to form chitin, which is finally deacetylated using sodium or potassium hydroxide at elevated temperatures to form chitosan. The acid employed for demineralization and deproteinization further reduced molecular weight (MW) of the chitosan. Considering the limitations posed by usage of chemicals (hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide, and potassium hydroxide) by reducing the quality of compounds and safety for human applications, several biological agents (proteases, lactic acid, and chitin deacetylase) have been developed high-quality extraction of chitosan, which is safe Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00007-7

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for human applications (Arbia et al., 2013; Dayakar et al., 2021; El Knidri et al., 2018; Oladzadabbasabadi et al., 2022). Chitosan is known for their wide range of biological properties with potential application in medicinal and pharmaceutical fields (Ngo & Kim, 2014). However, properties of chitosan are known to be influenced based upon the source of raw materials, reaction time, and degree of acetylation (DA). The factors are known to influence on the MW of the final compound effecting the functional property of the chitosan (Ngo et al., 2009; Oladzadabbasabadi et al., 2022). The bioactivity of chitosan is mainly related to the MW (lower MW) of chitosan. Antitumor activity and anticancer ability of lower MW chitosan to improve drug delivery and absorption have been reported by several studies (Jeon et al., 2001; Karagozlu & Kim, 2014; Maeda & Kimura, 2004). Similarly, higher antioxidant activity of chitosan with lower MW in salmon was reported by Kim and Thomas (2007). It was suggested that high radical scavenging activity is possessed by chitosan having both lower MW and higher degree of deacetylation (DD) (Park et al., 2004). Due to the ability of chitosan to bind fat, lower serum lipids, and reduce body weight by excreting absorbed fats without getting absorbed in the body, chitosan has gained important applications such as antiobesity (Karadeniz & Kim, 2014; Mhurchu et al., 2004). Chitosan has strong antimicrobial action, across a wide range of microorganisms. The presence of positive charge (NH13) on the chitosan surface reacts with negatively charged cell membrane constituents from microbial cell causing lysis (Andres et al., 2007; Ma et al., 2017; Severino et al., 2015; Tsiligianni et al., 2012). Chitosan modifies cellular permeability causing leakage of cellular components responsible for cell death (Inanli et al., 2020). The antimicrobial activity depends upon charge density, MW, DD as well as pH of medium and temperature (Inanli et al., 2020; Ke et al., 2021). Chitosan acts as binding and chelating agent reducing availability of nutrients and inhibits enzymes hindering/inhibiting the growth of microorganisms (Tsiligianni et al., 2012). Nanotechnology is a novel a multidisciplinary branch of science, dealing with fabrication, manipulation, characterization, and producing nanomaterials (1 100 nm). It has extensively been evaluated for their innovative approach to generate nanomaterials exhibiting unique properties, providing advantages over traditional materials (Cerqueira et al., 2018). Nanomaterial-based approach formulated by using nanotechnology has exhibited improved activity for chitosan, which has been extensively reported in the literature (Alizadeh-Sani et al., 2021; Qin et al., 2019).

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The increased consumer awareness regarding “clean-label foods” has become a momentum. Leading to consumer orientation toward minimally processed and naturally preserved foods has retained nutritional benefits, extended shelf life, and showed no side effects on health (Rathod, Kulawik, et al., 2022; Rathod, Ranveer, Benjakul, et al., 2021). Seafood is regarded as apex of nutritional importance due to the high levels of unsaturated fatty acids, amino acids, vitamins, and minerals (Byrd et al., 2021). The consumption of fish is related to health benefits and positive impacts on human well-being. Due to the benefits, it has been recommended to consume at least 26 pounds of seafood per capita per year as prescribed by United States Department of Agriculture (USDA & HHS, 2020). On the contrary, the seafood is prone to spoilage through different oxidative and microbiological mechanisms causing loss of organoleptic quality (Rathod, Ranveer, Benjakul, et al., 2021) during the storage. This chapter focuses on chitosan, its sources, extraction, bioactivity, and health benefits as well as it covers the application of chitosan in different nanoforms and their ability to impart microbial and oxidative stability to seafood.

6.2 Chitosan 6.2.1 Sources Chitosan is a natural cationic, renewable, and the most abundant biopolymer after the cellulose (Inanli et al., 2020; Kumari & Kishor, 2020; ˇ Kurita, 2006; Simat et al., 2020; Song et al., 2019). Besides, this polysaccharide is characterized by its superb properties, for instance nontoxic, biodegradability, and biocompatibility (Inanli et al., 2020; Kumari & ˇ Kishor, 2020; Simat et al., 2020). The chitosan is considered also a polycationic biopolymer, characterized by its positive charges owing to the existence of an amino group (Biswal & Swain, 2020). Generally, the chitosan is generated by deacetylation of chitin, which is principally obtained from the exoskeletons of mollusks, shellfish, crustaceans, insects, and fungi as shown in Fig. 6.1 (Hamed et al., 2016; Inanli ˇ et al., 2020; Simat et al., 2020; Tayel, 2016). Previous work revealed that the chitin has been extracted from crustacean shells through three processes, including demineralization (DM), deproteination (DP), and decolorization (DC). It was reported that each year approximately 13 million tons of crustaceans worldwide were caught from marine habitats (Harkin et al., 2019). According to the Global Production Statistics in between 1950 and 2014, it was demonstrated that more than 50% of these catches

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Figure 6.1 The main sources of chitosan.

were represented shell lost, which contained a percentage of chitin in the order of 20% 30%, and an amount of calcium carbonate equaled to 30% 40%, as well as a rate of protein in the order of 30% 40% (Cho et al., 1998; Harkin et al., 2019). However, previous works have also proven that the crustacean sources have an economic and environmental merit for the production and preparations of chitosan (Fig. 6.1). Crustacean by-products, for instance, the lobster cephalothorax can be recognized as an appropriate source for the preparation of chitosan on an industrial processing (Kou et al., 2021). Therefore chitosan can be obtained by decolorization (DC) of the aquatic invertebrate or any other source and after the process of deacetylation, which is considered a chemical assay and can occur by several methods. Actually, the techniques applied are either enzymatic or chemical processes. Thus the chitosan can be derived by enzymatic or alkaline process from chitin (Biswal & Swain, 2020; Venkatesan & Kim, 2010). The DD of the biopolymer of chitosan obtained from some biological source was found to be 70% 95% (Annu & Ikram, 2017; Nemtsev et al., 2004; Nwe et al., 2010). The production of chitosan from chitin is carried out by the elimination of the acetyl groups (CH3-CO), which leads the biopolymer soluble in several dilute acids. During the process of deacetylation, the amine (NH) groups are liberated. Hence, this process can provide the biopolyˇ mer of chitosan with cationic characteristics (Simat et al., 2020; Sudatta et al., 2020). The production of the chitosan from chitin is formed through formation of N-deacetylated. The chitosan is characterized by its

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solubility in water and thus it can be applied in diverse research purposes and in several domains, such as food and pharmaceutical applications (Biswal & Swain, 2020). The chitosan can be produced also from the seafood processing, for ˇ instance, the shrimp, crab shells, krill, crayfish, and lobster (Simat et al., 2020). Therefore the crab and shrimp are generally widespread sources mentioned in several literatures as the raw material for the production of chitosan, while other species such as crayfish, lobster, and oyster have also been employed for this purpose (Elieh-Ali-Komi & Hamblin, 2016; Kim, 2010; Kou et al., 2021; Raghvendrakumar, 2017). In this context, the marine sources have attracted a great attention in the production of chitosan (Huq et al., 2022). However, their availability depending on the seasonal variations can affect the extraction process of this biopolymer. Also, the strong mineralization of the exoskeletons of marine sources can make an arduous extraction process (Huq et al., 2022). Besides, terrestrial organisms can be used to extract the chitosan, such as arthropods, honeybee, silkworm, mushroom, and nematodes (Ahmed et al., 2017). Thus the mushrooms, septa, mycelia, and spore of Ascomycetes and Zygomycetes, are considered a superb source of chitosan (Ahmed et al., 2017). In addition, it was reported that the chitosan can be found in Lentinus edodes, which is considered a shitake mushroom belonging to the family of Basidiomycete (Rao et al., 2018). Moreover, earlier works have reported that the chitosan can be isolated from the exoskeleton of insects such as the beetles (Huq et al., 2022). In this regard, the chitosan derived from insects have generally an amount similar to those isolated from crustaceans (Huq et al., 2022; Philibert et al., 2017). Furthermore, the chitosan produced from fungal sources has a lower MW and superior particle size uniformity compared with those obtained from crustaceans and insects (Darwesh et al., 2018). It was reported in previous studies that the chitosan can also be extracted from the microbial biomass (Huq et al., 2022).

6.2.2 Structure Structurally, chitosan is considered a linear polysaccharide, which is composed of β-1, 4-linked polymer of 2-amino-2-deoxy-D-glucose (Bruno et al., 2019; Hamed et al., 2016; Inanli et al., 2020; Song et al., 2019). The chitosan is a biopolymer constituted by copolymer of β-1, 4-linked D-glucosamine with N-acetyl-D-glucosamine (Qin et al., 2019). In fact, the transformation of chitin to chitosan can lead to an increase in the DD,

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which is mostly identified as the glucosamine/N-acetyl-D-glucosamine ratio (Qin & Zhao, 2019). In previous works, when the amount of glucosamine is higher than the proportion of N-acetyl-D-glucosamine, the polysaccharides are known as chitosan; otherwise, the biopolymer is identified as chitin (Qin & Zhao, 2019). However, the chitosan is characterized by the presence of three categories of reactive and functional group, both primary and secondary hydroxyl groups in positions C-2, C-3, and C6, respectively, with an acetamide/anamine. Thus the amino proportions are considered the principal factors, which can influence the variation in their physicochemical properties and structures (Alishahi & Aïder, 2012; Aranaz et al., 2009; Inanli et al., 2020; Qin & Zhao, 2019). Therefore these functional groups can undergo chemical modification with organic or inorganic compounds in order to improve the properties and the functionalities of chitosan (Inanli et al., 2020; Lizardi-Mendoza et al., 2016). The production of chitosan from deacetylation of chitin can be proven by some analysis, such as the Fourier transform infrared spectroscopy. This analysis has revealed the presence of two bands of amide both I and II at 1655 and 1583 cm21, respectively (Ahmed et al., 2017). Hence, the lower intensity of the band II with a greater intensity of the band I demonstrated that there is a good generation of NH2 group with an effective deacetylation (Annu & Ikram, 2017). According to the density of the polymeric chain or the MW of chitosan, this latter can be classified into two types. In fact, there are highdensity chitosan and low-density chitosan (Ahmed et al., 2017). The low MW of chitosan can exhibit several biological activities (Ahmed et al., 2017; Qin & Zhao, 2019). The chitosan has been characterized by its positive charges of the amino groups in glucosamine. This biopolymer is identified as the only cationic polysaccharide that is soluble in water (Qin & Zhao, 2019). In this regard, the chitosan is characterized by its distinctive physicochemical characteristics; for instance, low toxicity, biocompatibility, and biodegradability. Therefore this polymer has an excellent potential to be applied and utilized in various industries (Qin & Zhao, 2019).

6.2.3 Extraction The industrial extraction of chitosan can be carried out by chemical, biological, and also microwave-assisted assays (Abhinaya et al., 2021; Kaur &

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Dhillon, 2015; Oladzadabbasabadi et al., 2022; Shanmuganathan et al., 2019; Sivashankari & Prabaharan, 2017). Chitosan is considered a deacetylated derivative of chitin and it is among the principal biopolymers known in nature (Oladzadabbasabadi et al., 2022). Thus this polysaccharide is generally extracted from crustacean shell waste, mollusks, insects, and fungi (Inanli et al., 2020; Mohan et al., 2020). In this context, the principal extraction technique used to isolate the biopolymer of chitosan included three basic procedures, including demineralization, deproteinization, and deacetylation. The crustacean shells contain not only the chitin, minerals, and proteins, but they are also composed of natural and bioactive pigments, such as carotenoids (Oladzadabbasabadi et al., 2022). Therefore a necessary treatment known by the process of decolorization should be applied by the use of diverse organic and inorganic solvents (hydrogen peroxide, acetone, and sodium hypochlorite), in order to eliminate the undesirable pigments, for example, astaxanthin and β-carotene (Oladzadabbasabadi et al., 2022). The deacetylation process is identified as the functional step to remove the acetyl groups related to chitin and then the replacement of reactive amino groups (Mohan et al., 2020). Actually, the DD can define the amount of the free amino groups and can be useful to distinguish between chitosan and chitin. In fact, the DD is considered an effective parameter for chitosan because it can influence the biological and physicochemical characteristics, such as the capacity to chelate metal ions, biodegradability, electrostatic properties, and the acid base ratio (Hussain et al., 2013; Nessa et al., 2010). Thus the chitin can be transformed to chitosan by the utilization of chemical assays. The extraction of chitosan from insects and crustacean shell using the deacetylation process as a chemical assay was done by the use of alkali-NaOH or acids (Anand et al., 2014; Marei et al., 2016; Mohan et al., 2020; Paulino et al., 2006; Song et al., 2018; Torres-Castillo et al., 2015). However, it has been proven that the use of alkali-NaOH treatment is an effective method compared to the use of acid because such a method is vulnerable to the glycosidic bonds (Hajji et al., 2014). On the other hand, the properties of the chitosan product obtained can be influenced by several factors during the deacetylation process. Thus the processing time and temperature can be defined as the parameters that have the most influence and effect on the MW and DD (Mohan et al., 2020; Rege & Block, 1999). The chemical deacetylation has been known to have some disadvantages, such as the problem of environmental pollution and also the high

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consumption of energy (Raval et al., 2013). Consequently, the enzyme deacetylation has been used as an alternative method to obtain the chitosan from chitin. Thus deacetylation of chitin by enzymatic assay can occur to hydrolyze the bonds of acetamide (Pagnoncelli et al., 2010).

6.3 Health benefits of chitosan Generally, the chitosan and its derivatives are considered as functional compounds due to their chemical and physical properties and therefore these compounds have a great attention for their application in miscellaneous fields, such as medical, pharmaceutical, nutrition, as well as in the food industries (Abd El-Hack et al., 2020; Inanli et al., 2020). Hence, these bioactive compounds can be used as dietary fiber and can prevent the occurrence of several diseases, such as hypertension, diabetes, human tumors, dyslipidemia, and cardiovascular diseases. (Chiu et al., 2017; Inanli et al., 2020; Muanprasat & ˇ Chatsudthipong, 2017; Simat et al., 2020). The chitosan and its derivatives are characterized by their beneficial impacts on human health. Thus the use of chitosan in the food industries as a safe dietary supplement can decrease the level of total cholesterol as well as it has a considerable role in the reduction of the triacylglycerol (TG) both in plasma and liver (Inanli et al., 2020; ˇ Simat et al., 2020). Besides, this functional ingredient is effective in decreasing the level of low-density lipoprotein cholesterol in plasma (Inanli et al., 2020). Therefore it has been revealed that the impacts of chitosan in the decrease of the lipid levels in plasma are due to their capacity to prevent the activity of pancreatic lipase as well as its role in the establishment of an interaction between the bile acids and dietary lipids (Inanli et al., 2020; Muanprasat & ˇ Chatsudthipong, 2017; Ngo et al., 2015; Simat et al., 2020). Thus it can reduce the absorption of intestinal lipids in the gastrointestinal tract ˇ (Muanprasat & Chatsudthipong, 2017; Ngo et al., 2015; Simat et al., 2020). These considerable functions of chitosan make it an effective compound in the prevention of obesity, and, thus, it can lead to weight loss (Inanli et al., ˇ 2020; Simat et al., 2020). Moreover, chitosan and its derivatives can be ˇ applied in the prevention of diseases correlated to obesity (Simat et al., 2020). Earlier works have reported that the chitosan with high MW and high DD can be considered an efficient compound and can provide a great fat-binding capacity compared to the low-MW and low-DD chitosan (Panith et al., ˇ 2016; Simat et al., 2020). Hence, the consumption of chitosan tablets, as a dietary supplement, has been recognized as safe compounds as well as has several beneficial effects for human health.

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The chitosan can make an ionic interaction with several substances due to their superb properties, such as cationic characteristic, biocompatibility, and physicochemical properties, and, thus, it can be used in pharmaceutical forms with local applications (Desbrieres et al., 2019). Thus the chitosan has an excellent role in the therapeutic area and acts as a modulating agent that allows the absorption via diverse mechanisms. Besides, this functional biopolymer is characterized by its biological properties, including hemostatic, antioxidant, antibacterial, and wound healing effects, which makes it and its derivatives as considerable biomedical materials for epithelial regeneration or cutaneous applications (Desbrieres et al., 2019). Moreover, the chitosan is characterized by its antitumor properties. In this context, it has been confirmed that this biopolymer can stimulate the immune system and thus leads to the inhibition of the growth of tumor cells (Aranaz et al., 2009). It has been demonstrated that the chitosan with low MW has a superb inhibitory effect on the development and growth of Ehrlich ascites tumor cells (Inanli et al., 2020). In addition, this functional ingredient can exhibit an inhibitory effect of the tumor-induced neovascularization (Inanli et al., 2020). Furthermore, it has a great role in the inhibition of angiogenesis and leads to the DNA fragmentation and ˇ thus induction of apoptosis (Ngo et al., 2015; Simat et al., 2020). The chitosan can induce an inhibitory ability of MMP-2 melanoma cells due ˇ to the posttranscriptional capacity of this biopolymer on MMP-2 (Simat et al., 2020) as well. However, the inhibitory effect of angiotensin-I-converting enzyme (ACE) is considered a significant signal in the prevention of hypertension (Inanli et al., 2020). Several previous works have proven that the chitosan and its derivatives can exhibit an antihypertensive effect, and, thus, it can reduce the risk of CVD (Inanli et al., 2020; Muanprasat & Chatsudthipong, ˇ 2017; Simat et al., 2020). Hence, the inhibitory activity on ACE is related to the MW and DD of this biopolymer (Panith et al., 2016). The antiinflammatory effect of this functional compound has been investigated both in vitro at a concentration of 20 mg/kg/day and in vivo at different concentrations varied between 10 and 100 mg/kg/day (Muanprasat & Chatsudthipong, 2017). In addition, the chitosan can prevent of the renal diseases and also contribute to the regulation of the functional ability of the immunocompetent cells as well as it induces an improvement of the systemic immune responses (Anraku et al., 2010; ˇ Inanli et al., 2020; Ngo et al., 2015; Simat et al., 2020).

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Moreover, chitosan and its derivatives have been characterized by their potential antidiabetic activity. Several research works have demonstrated that the incorporation of chitosan in the food mixture of both normal mice and neonatal streptozotocin-induced diabetic mice (n-STZ mice) can reduce the amounts of the glucose in blood during 4 weeks (Inanli et al., 2020; Muanprasat & Chatsudthipong, 2017). Thus the chitosan with high MW applied in STZ-induced diabetic rats can exhibit a decrease of their hyperglycemia (Inanli et al., 2020). The antidiabetic effect of this functional compound is due to its ability to activate the proliferation of β-cell as well as to induce an inhibition of the β cells in pancreatic islets (Inanli et al., 2020).

6.4 Bioactivity of chitosan The biopolymers of chitosan have a wide range of biological activities, such as antimicrobial and antioxidant properties. Thus it can be applied in the food industry to improve the quality and the safety of foods as well as their shelf life.

6.4.1 Antimicrobial properties Chitosan is characterized by their considerable antimicrobial capacity (Han et al., 2012; Sarti & Bernkop-Schnürch, 2011). It has been reported that the chitosan extracted from the crab and shrimp shell has a good antimicrobial effect against several microbial species, including Gram-positive and Gramnegative microbes (Arfin, 2017; Chung et al., 2004), molds, yeasts, and various fungi (Arfin, 2017; Friedman & Juneja, 2010). The polycationic property of chitosan makes it a superb functional ingredient that can be used in various fields, such as food industry. The antimicrobial capacity of chitosan is a significant characteristic due to its application for food preservation as well as for the packaging applications. Therefore this biopolymer can contribute to the decrease of the deterioration and spoilage of food products caused by various microorganisms (Biswal & Swain, 2020). The chitosan has a good antimicrobial activity compared to chitin owing to the charge positive on C-2 of glucosamine monomer (Chen et al., 1998). Chitosan presents a higher number of positive charges of amino groups than chitin and, consequently, has higher antimicrobial ˇ capacity (Ahmad et al., 2020; Simat et al., 2020). There are various mechanisms of antimicrobial effect of chitosan against multiple microbes (Kulawik et al., 2019). It has been revealed by

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Mohan et al. (2020) that the possible mechanism for this variability could be due to the interaction between the negative charges of the microbial cellular membranes and the positive charges of the molecule of chitosan. Therefore this type of interaction can induce destruction of the cell walls, liberation of the intracellular compounds, and consequently lead to the death of the microbial cells (Chien et al., 2016; Kulawik et al., 2019; Mohan et al., 2020; Severino et al., 2015). The higher number of amino groups of this biopolymer that are positively charged contributes to its ˇ great antimicrobial activity (Simat et al., 2020). In fact, the existence of 1 the amine groups «NH3 » of glucosamine, which characterized the biopolymer of chitosan, can cause the possibility of such direct interaction with the bacterial cell membranes (Kulawik et al., 2019; Li et al., 2015; Ma et al., 2017). One of the hypotheses suggests that chitosan may prevent the proliferation and the growth of bacteria by competing with fundamental elements or nutrients (Kulawik et al., 2019; Li et al., 2010; Yuan, Chen, et al., 2016). Besides, it has been reported that the chitosan characterized by low MW can pass into the nucleus of microbial cells and then block the mechanism of transcription of RNA from DNA (Inanli et al., 2020; Kumari & Kishor, 2020). In addition, the greater antimicrobial capacity was detected for the chitosan characterized by higher DD. Therefore the mechanism of antimicrobial activity of chitosan depends on different parameters, such as its MW, DD, degree of polymerization, the type and concentration of chitosan used, and the type of target microorganisms (Inanli et al., 2020). Furthermore, another factor of the medium can influence the antimicrobial activity of chitosan, such as the temperature, the coexistence of solutes, and the environmental pH, which can interact with this biopolymer through electrostatic interaction (Inanli et al., 2020). Actually, the antimicrobial effect of chitosan can be increased when the environmental pH value is weak owing to its greater solubility in acid microenvironment (Xing et al., 2016). Chitosan as a functional polymer can exhibit an effective antimicrobial effect against spoilage and foodborne pathogens, such as Staphylococcus aureus and Escherichia coli (Dutta et al., 2012). Nevertheless, this biopolymer is more effective on S. aureus than E. coli due to variability in the cell walls of the two bacterial species. In fact, the S. aureus as a Gram-positive bacterium is characterized by the presence of the peptidoglycan in its cell wall. Thus the peptide polyglycogen layer consists of several pores, which permit the molecule of chitosan to penetrate the cell without difficulty.

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However, the cell wall of E. coli as a Gram-negative bacterium, which is constituted by peptidoglycan and an outer membrane composed of lipoproteins, lipopolysaccharides, and phospholipids, makes the penetration of this biopolymer not easy (Dutta et al., 2012). In contrast, the chitosan extracted from two species of grasshopper, including Oedaleus decorus and Calliptamus barbarous had an excellent antimicrobial activity against Gram-negative and Gram-positive bacteria compared to the several standard antibiotics (Mohan et al., 2020). Hence, the chitosan can exhibit antimicrobial activity against the Gram-negative bacteria, such as Salmonella enteritidis, Vibrio alginolyticus, and Yersinia enterocolitica, and the Gram-positive bacteria, including Listeria monocytogenes, Streptococcus agalactiae, Bacillus subtilis, and Lactococcus garvieae. In fact, the chitosan extracted from both grasshopper species had minimal bactericidal concentrations (MBCs) of 0.16 and 0.32 mg/mL (Kaya et al., 2015; Mohan et al., 2020). Further, both teichoic acid in Gram-positive bacteria and lipopolysaccharides in Gram-negative bacteria have a superb function in the interaction with the biopolymer of chitosan (Raafat et al., 2008; ˇ Simat et al., 2020). The deacetylated chitosan with percentage of 4 extracted from Tenebrio molitor mealworm beetle larvae did not present any antimicrobial activity against E. coli, S. aureus, L. monocytogenes, and B. cereus; while the chitosan with an amount of 8% demonstrated an antimicrobial effect of 1 2 mm of inhibition (Kumari et al., 2017). Also, the combination of silver nanoparticles (NPs) with the chitosan can improve antimicrobial activity of this biopolymer (Wei et al., 2009). In addition, the turmeric-combined chitosan films produced by a crosslinking agent, for instance, the sodium sulfate, can exhibit a good antimicrobial activity against Gram-positive and Gram-negative bacteria (Biswal & Swain, 2020). In this context, several modified chitosan, for instance, hydroxypropyl chitosan (HPCS) and diethoxyphosphorylpolyaminoethyl chitosan (DPECS) can show a great antimicrobial effect identical to the activity of chitosan (Biswal & Swain, 2020). The chitosan NPs can prevent and inhibit the growth of bacteria species, such as Salmonella typhimurium, Salmonella choleraesuis, E. coli, Lactobacillus, and S. aureus (Qi et al., 2004; Risti´c et al., 2015). Moreover, the solution of chitosan can exhibit an excellent antimicrobial activity against Burkholderia seminalis (Lou et al., 2011). Furthermore, earlier works have demonstrated that the chitosan can inhibit the growth of pathogen bacteria, such as Campylobacter spp. (Ganan et al., 2009).

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However, it has also been demonstrated that the antimicrobial capacity of chitosan is more important against molds and yeasts compared to bacteria. In fact, both molds and yeasts are more sensitive to this biopolymer (Tsai et al., 2002).

6.4.2 Antioxidant properties In biological systems, inappropriate generation of reactive oxygen species (ROS), such as hydroxyl radicals (•OH), superoxide anion radicals (O2•-), and hydroxyl radicals (•OH), leads to oxidative stress, and then cellular damage (Inanli et al., 2020; Mohan et al., 2020). In this context, oxidative damage could cause the development of several degenerative diseases, including cancers, neurodegeneration, heart attack, atherosclerosis, inflammation, cataractogenesis, diabetes, retinal damage, and liver injury (Halliwell, 2011; Inanli et al., 2020; ˇ Mohan et al., 2020; Moskovitz et al., 2002; Simat et al., 2020). Besides, the ROS can induce the damage of biomolecules, such as proteins, amine acids, carbohydrates, lipids, lipoproteins, and DNA. Therefore a balance between the ROS content and radical scavenging is necessary to maintain the normal cellular functions and then to prevent the generation of diseases associated with the oxidative damage (Qiu et al., 2022). Several research studies have demonstrated the antioxidants from biological and natural resources (Mohan et al., 2020; Qiu et al., 2022). The biopolymer of chitosan possesses an excellent antioxidant activity through its ROS scavenging capacity (Inanli et al., 2020; LizardiMendoza et al., 2016; Park et al., 2010). The antioxidant activity of the chitosan is extracted from insects, mollusks, shellfish, crustaceans, and fungi (Kaya et al., 2015, 2016; Park et al., 2010; Song et al., 2013; Torres-Castillo et al., 2015; Wu et al., 2013). It has been demonstrated by the previous work of Park et al. (2004) that the antioxidant activity of chitosan depends on its concentration and DD. Besides, the antioxidant activity of this polysaccharide can be correlated also with MW and the source material used for its extraction (Inanli et al., 2020; Olatunde & Benjakul, 2018; Sun et al., 2007). The chitosan extracted from Colorado potato beetle with low MW has been shown to have a greater DPPH radical scavenging activity at an amount of 5 mg/mL; however, the chitosan derived from the larvae of Colorado potato beetle demonstrated a percentage of 33.05 of the scavenging activity (Kaya et al., 2014). Besides, chitosan exhibited a similar effect against the ferric ion reducing test (Kaya et al., 2014). It was proven by Kaya et al. (2014) that the chitosan with higher DA had a good antioxidant activity.

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Moreover, the impacts of MW of chitosan on antioxidant activity have been widely studied (Anraku et al., 2010; Xie et al., 2001). The chitosan with low molecular weight (LMWC) had an excellent antioxidant activity compared to the chitosan with high molecular weight (HMWC) (Anraku et al., 2010). In addition, the LMWC can exhibit a significant antioxidant activity against DPPH radicals (2,2-diphenyl-1picrylhydrazyl) and a good ferrous ion chelating activity compared to both HMWC and medium-molecular-weight chitosan (MMWC) (Chang et al., 2018; Chien et al., 2007). In this context, the high MW of chitosan can induce a weak antioxidant effect against hydroxyl and superoxide radicals. Therefore these findings may be due to the fact that shorter chains are less likely to produce intramolecular hydroxyl bonds, giving a good activation of the amino and hydroxyl groups, which leads to radical scavenging activity (Sun et al., 2007). Moreover, the chitosan with DDA 5 80% and MW 5 307 kDa did not have an antioxidant activity against DPPH radical (Schreiber et al., 2013). However, the biopolymer of chitosan can serve as primary and secondary antioxidants (Olatunde & Benjakul, 2018). In fact, the antioxidant effect of this functional compound can be due to the ability of the interaction of its free amino group with free radical group, as well as the generation of ammonium groups and stable molecular radicals (Yuan, Lv, et al., 2016). Previous work has confirmed that the combination of chitosan-based interpenetrating polymer network hydrogel with gallic acid can exhibit a higher antioxidant activity (Kang et al., 2017). In addition, the combination of chitosan with grafting gallic acid can enhance the antioxidant activity against the DPPH radicals (with percentage of 89.5) as well as exhibit a higher reducing power (Schreiber et al., 2013). In contrast, the investigation of the antioxidant activity of three varieties of chitosan prepared with different MW, such as 5 10, 1 5, and ,1 kDa, has demonstrated that the MMWC (1 5 kDa) can induce an antioxidant capacity against DPPH•, •OH, and •O22 compared to those of higher MW (Je et al., 2004). Moreover, the evaluation of the antioxidant effect of this biopolymer with different MW (28, 67, 223, 444, and 746 kDa) through the DPPH• scavenging activity has demonstrated that the chitosan with lower MW (28 kDa) can exhibit the greater scavenging activity compared to the chitosan with 746 kDa (Cho et al., 2008). Previous study has demonstrated that the evaluation of the antioxidant activity of chitosan with different deacetylation ratio (DR) has shown that the higher DR can exhibit a good

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antioxidant effect due to the presence of amino groups at the position C2 (Chuang et al., 2013; Yen et al., 2008).

6.5 Nanotechnological approaches for application of chitosan Nanotechnology is a novel multidisciplinary division of science, dealing with fabrication, manipulation, characterization, and producing nanomaterials (1 100 nm). It has extensively been evaluated for their innovative approach to generate novel materials at nanoscale exhibiting unique properties, providing advantages over traditional materials (Cerqueira et al., 2018). Nanomaterials increase surface-to-volume ratio providing large space for attachment of biological molecules. Due to the immense bioactivity possessed by the chitosan, they are transformed in several nanoforms improving their bioavailability providing diverse applications. For preparation of chitosan-based nanomaterial, several methods such as ionic gelation, reverse micellar, emulsion-based solvent evaporation, and precipitation methods have been established and reported (Elsaied & Tayel, 2022; Jafari & McClements, 2017).

6.5.1 Chitosan for nanoencapsulation Encapsulation technique is widely adopted to improve quality and impart stability to encapsulated compounds (Ceylan et al., 2020; Chaudhari et al., 2021; Ozogul et al., 2021). Additionally, they are associated with controlled release mechanisms and create barriers against degradation of bioactivity (Chaudhari et al., 2021). Nanoencapsulation of catechin using chitosan for improved stability and activity was evaluated by Kailaku et al. (2014). The NP formed using chitosan (0.2%) exhibited superior stability and no phase separation was observed. Also, it was suggested to estimate the level of encapsulation as lower antioxidant activity, which was possibly attributed to incomplete nanoencapsulation. Chitosan has been evaluated as a potential material for encapsulation of several compounds (Hadidi et al., 2020; Hasani et al., 2018). The bioactive lemon essential oil encapsulated in chitosan and modified starch system was evaluated by Hasani et al. (2018). Higher chitosan ratio in the formulation of nanocapsules ensured slower release efficiency ensuring the stability of essential oil for application in the food and medicine sector. Nanoencapsulation of pomace phenolics (apple and grape) through nanoemulsification using chitosan was reported (Ahmed et al., 2020). The nanocapsules formed encapsulated

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75% of material. In addition, the encapsulation improved the antioxidant activity that increased with duration, suggesting the controlled or delayed release of compounds. The increased antioxidant activity was reported to increase antioxidant activity of juices (apple and pineapple) suggesting as commercial application. Estimation of in vitro drug release evaluation from garlic aqueous extract loaded in a chitosan NP formed by ionic gelation was reported (Gupta et al., 2019). NPs formulated using 0.25% chitosan had the highest encapsulation (75.54%) with acceptable characteristics with highest stability and drug release capacity. Curcumin-based nanoformulations by encapsulation in chitosan exhibited appreciable encapsulation efficiency (66.13%), loading capacity (23.40%), and yield (77.25%), which was reported by Akolade et al. (2017). The release efficiency (after 2 hours) of encapsulated curcumin was superior (71.95%), increasing the release time, reducing loss, and improving inhibition of α-amylase activity. The dietary supplementation (200 mg/kg bw) for 7 days exhibited hyperglycemic effects, suggesting retention of bioactive compounds by encapsulation using chitosan.

6.5.2 Chitosan-based nanomaterial for packaging The biocompatibility, biodegradability, and nontoxic nature of chitosan have gained numerous applications for packaging food products. The issue of food contamination during storage is a major setback for the food industry. Hence, the search for safe antibacterial materials which are compatible, degradable, and eco-friendly for food packaging has increased. Additionally, the antimicrobial and antioxidant activity possessed by chitosan are further improved by the nanoscale alteration and inclusion of natural compounds (Abdollahi et al., 2012; Divya et al., 2018; Qi et al., 2004). The formulation of nanomaterial has exhibited improved preservative ability due to the presence of nitrogen (C-2), higher surface charge density, presence of Schiff bases, and improved membrane permeability (Kulawik et al., 2019). Chitosan in its normal state exhibits good film forming ability with lower mechanical qualities. Hence, formulation and inclusion of chitosan-based nanomaterials such as nanowhiskers, nanofibers, nanocrystals, and nanoribbons, including film forming agents (gelatine, glycerol) have been reported for the mechanical property of films along with water vapor permeability, transparency, tensile strength, film thickness, gas barrier, and adhesion properties attributed to different interaction (Chausali et al., 2022; Fathima et al., 2018;

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Lavorgna et al., 2010; Rathod et al., 2022). Additionally, chitosan nanoformulation handles the problem of solubility in an acidic environment. Coating is regarded as the first line of preservation or packaging of fresh produce. Additionally, incorporating ingredients to enrich other functional characteristics (antioxidant or antimicrobial) has increased. Recently, Homayonpour et al. (2021) reported the preservative property of nanochitosan on sardine fillet. Nanochitosan exhibited significant inhibition of lipid oxidation and microorganisms (total viable count, total plate count, and lactic acid bacteria). Authors suggested the presence of amino groups from chitosan-inhibited lipid oxidation. Chitosan nanofibers based on low watersoluble packaging exhibiting antibacterial activity (S. typhimurium and S. enteritidis) for chicken were successfully demonstrated by Lin et al. (2018). Coating formulated using nanochitosan exhibited higher preservative effect over chitosan-coated silver carp fillets during refrigerated storage (Ramezani et al., 2015). The authors demonstrated the higher antimicrobial activity of nanochitosan by inhibiting total mesophilic and total psychrotrophic counts. Recently, Homayonpour et al. (2021) developed nanochitosan-based film with caraway seed extract in nanoliposomes. Inclusion of caraway seed extract encapsulated in nanoliposomes improves the physical characteristics of the film such as water vapor permeability and flexibility of film. Additionally, the inclusion of seed extract improved the antioxidant and antimicrobial activity of film based on concentration. Combined application of nanochitosan and silver NPs exhibited inhibition of total viable count, S. aureus, and E. coli in caviar during storage (Asl et al., 2021). Chitosan-based nanocomposite and selenium NPs infused edible coating was evaluated for their antimicrobial characters by Alghuthaymi et al. (2021). They reported that packaging material had antibacterial activity against E. coli, S. typhimurium, S. aureus, and L. monocyotgenes. However, the complete lysis of microorganisms was observed after 10 hours. Edible film formation using nanomaterials have been reported by Melo et al. (2020) in earlier study. They showed antifungal capacities of chitosan nanocomposite-based edible coating against phytopathogenic fungi on strawberry. Similarly, silver chitosan (red claw crayfish)-based nanocoating improved the shelf life of fresh-cut melons (Ortiz-Duarte et al., 2019). Recently, Zhao et al. (2022) developed edible film containing chitosan NPs for preservation of red sea bream fillets. The chitosan NP-based film exhibited reduced microbial inhibition (total viable count) and lipid oxidation; besides, the preservation ability could be increased by inclusion of anthocyanidins and cinnamon-perilla essential oil based on pickering nanoemulsions.

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6.5.3 Chitosan-based intelligent packaging Considering the novel trends in packaging apart from acting as a barrier and ensuring preservation, protection, and conservation of food, sensing of contaminants present in foods to ensure safe storage and safety of food for consumers is important (Gokoglu, 2020). Spoilage of foods is due to the growth of several microorganisms (spoilage and pathogenic), which are required to be detected during their growth. They generate several toxic compounds that can be lethal, the gaseous composition of the package is modified, and there is the presence of adulterants, which are the major contaminants that need to be traced (Rathod, Nirmal, et al., 2022; Rathod, Ranveer, Benjakul, et al., 2021). It is a tedious and challenging task to determine the presence of contaminants in food. It is a packaging that communicates with the consumer about the condition of the product packed and its quality/safety (indicators, sensors, and data carriers). They usually consist of barcodes, radio frequency identification tags, time temperature indicators, gas indicators, freshness indicators, and pathogen indicators (Fang et al., 2017). Nanotechnological applications (NPs) have gained importance for their application for sensing based on selective response. The pH has a major role for the indication of spoilage; hence Li et al. (2019), developed a novel intelligent packaging consisting of chitin nanofibers with purple potato extract. Addition of chitosan nanofibers improved the mechanical features of the film (tensile strength, water resistance, and roughness). Besides, combination of chitosan film, chitin nanofibers, and purple potato extracts showed higher antioxidative activity. Several studies have further reported application of methyl cellulose/ chitosan nanofiber in meat (Alizadeh-Sani et al., 2021), black rice extract in chitosan matrix/chitosan nanocrystals, and cellulose acetate nanofibers containing alizarin in fish (Aghaei et al., 2018; Ge et al., 2020), oxidized chitin nanocrystals in konjac glucomannan films (Wu et al., 2020), chitosan, silver NP, and purple corn extract film (Qin et al., 2019) as well as nanoclay from natural and modified montmorillonite (Gutiérrez et al., 2017) for pH sensing. Cheng et al. (2018) developed a gold nanoclusterembedded chitosan nanocapsules and magneto-fluorescent-based nanosensor. The integration of the developed sensor was demonstrated to accurately detect the contamination caused by E. coli O157:H7 in water and milk samples.

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6.6 Impacts of chitosan on seafood safety Seafood is considered nutrient-dense food with a high moisture content, making it extremely prone to spoilage. Recently, the consumer demand for high-quality foods such as minimally processed and preserved using natural preservatives is increasing. Hence, the industrial perspective toward application of natural preservatives has risen. Chitosan a natural biopolymer in addition to preservative action exhibits diverse bioactivity (anticancer, antiinflammatory, hypocholesterolemic, antidiabetic, and ACE activity), which has positive impacts on human health (Hamed et al., 2016; Inanli et al., 2020; Kulawik et al., 2019; Rathod, Ranveer, Benjakul, et al., 2021). The growth of microorganisms and chemical deterioration are the main factors responsible for spoilage of seafood (Rathod, Ranveer, ˇ Benjakul, et al., 2021; Simat et al., 2021). Hence, the requirement of the cold chain to maintain the quality and control delay in proper cold chain facility is associated with growth and proliferation of microorganism in fresh state. The active enzymes present in the fish body begin the deterioration of the seafood by generating several acids by anaerobic respiration (Rathod, Nirmal, et al., 2022). In the case of processed seafood, the role of low temperature (,4°C) and the environment of the package plays a vital role in determining spoilage. Several classes of microorganisms are specific to some packaging or temperature conditions. Hence, the developed modified atmospheric or vacuum packaging plays an essential role in preservation of seafood. Oxygen specifically has a major role in oxidation of fats and proteins further deteriorating the quality (Kedar et al., 2022; ˇ Özogul et al., 2004; Simat et al., 2021). Generally, the alive fish flesh is regarded as sterile, however, upon harvest, the microflora present on the body (gills and intestines) from the environment and processing equipment (cross contamination) invades the fish. Wide range of microorganisms are known to cause spoilage in seafood, prominent amongst them are Gram-negative bacteria such as Acinetobacter, Flavobacterium, Vibrio, Photobacterium, Camplyobacter, Salmonella spp., L. monocytogenes and Gram-positive bacteria such as Aeromonas, Bacillus, Clostridium, Coryneforms, Micrococcus, Photobacterium, Pseudomonas, Staphylococcus, and Shewanella (Authority et al., 2019; Parlapani et al., 2018; Rathod, Nirmal, et al., 2022; Rathod, Phadke, et al., 2021; Rathod, Ranveer, Benjakul, et al., 2021). The microorganisms responsible for spoilage are also associated with several toxins responsible for food poisoning and can be fatal. Considering the ability of spoilage, several limits

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are set for pathogenic microorganisms in place based on region or global regulatory bodies to avoid infections in humans. Additionally, the spoilage microorganisms are also associated with hampering the quality and nutrition. They are known to generate volatile amines (dimethylamine—DMA and trimethylamine—TMA) by reducing trimethylamine-oxide (TMAO). The amines generated are associated with imparting fishy odor, also used as spoilage indicator ( . 15 mg N/100 g and .30 mg N/100 g for fresh and marine water fish). Oxidation is a detrimental process associated with the generation of several compounds (alkanals, alkadianals, ketones, alcohols, and hydrocarbons), imparting offodors and toxicity to humans. Lipid oxidation is usually estimated by thiobarbituric acid reactive substances (TBARS) and peroxide value (PV) (Rathod, Ranveer, Benjakul, et al., 2021; Rathod, Ranveer, Bhagwat, et al., 2021). New technologies or trends are constantly being introduced to preserve the quality of seafood to ensure the availability of safe and nutritious (clean label) for the consumer (Rathod, Kulawik, et al., 2022; Rathod, Nirmal, et al., 2022; Rathod, Phadke, et al., 2021; Rathod, Ranveer, Bhagwat, et al., 2021). Chitosan, based on its compatibility and safe nature, have been extensively used for preservation of seafood by inhibiting the microbial growth and quality retention (Ceylan et al., 2020; Hamed et al., 2016; Inanli et al., 2020; Kulawik et al., 2019).

6.6.1 Edible coating Chitosan (1%) application (coated) on white shrimps (P. vannamei) exhibited a preservative effect (inhibited microorganisms and lipid oxidation) during partial frozen (23°C) storage (Wu, 2014). Significant inhibition of total viable count was observed in samples coated with chitosan, authors suggested synergistic effect of partial frozen storage, chitooligosaccharide, and glutathione treatment. The inhibition of microbial population and lowered pH due to chitosan treatment retarded the evolution of volatile amines. Shelf life extension by 6 days in Nemipterus japonicus fillet treated with 1% chitosan coating with propolis extract was reported (Ebadi et al., 2019). Coating containing chitosan and propolis exhibited lowered mesophilic count and total plate count due to antimicrobial action possessed by positively charged amino group interaction with membrane (negatively charged). Findings corroborated with the results of volatile amines (TVBN), and the positive interaction between propolis extract and chitosan

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retarded the amine formation. Cooked and peeled ready-to-eat shrimps coated with chitosan exhibited microbial safety, when packed under modified atmospheric (40% CO2 and 60% N2) conditions at chilled storage (4°C) (Carrión-Granda et al., 2016). Chitosan-based coating inhibited the microbial growth (total viable count, lactic acid bacteria, Enterobacteriaceae, and psychrotrophic bacteria). Further the inhibition was obtained using chitosan-based emulsion containing essential oil. It was suggested that chitosan-based emulsion improved the prolonged release of essential oil components. Chitosan (2%)-based coating was evaluated for the shelf life extension of Scomberomorus brasiliensis during 180 days stored at 218°C (do Vale et al., 2020). Chitosan coating significantly prolonged the microbial inhibition at frozen storage temperatures and for longer duration. Liu et al. (2021) developed a chitosan coating grafted with phenolic acids (protocatechuic acid and gallic acid) for preservation of sea bass fillets. Chitosan coating alone ( . 1 log CFU/g) significantly improved inhibition of total viable counts over control and further grafting of phenolic compounds ( . 2 log CFU/g) improved the inhibition and retained the total viable counts within the prescribed limits (,7 log CFU/g). Based on microbial limits, the chitosan-coated samples exhibited shelf life extension by more than 4 days (Liu, Lan, et al., 2021). Direct application by spraying or application of chitosan (1%) combined with vacuum packaging was evaluated for preservation of swordfish fillet at refrigerated storage (4°C) (Tsiligianni et al., 2012). Chitosan was found to significantly inhibit the proliferation of total viable count, Pseudomonas spp., H2S-producing bacteria, lactic acid bacteria, and Enterobacteriaceae in stored swordfish samples in comparison to control. The maximum limit for total viable count (7 log cfu/g) was crossed at day 9 for air packed samples and at day 17 for samples packed under vacuum. While reduction in the range of 1.8 2.5 log cfu/g and 1.5 2.3 log cfu/g for Pseudomonas spp. and H2Sproducing bacteria, respectively, was observed in samples coated with chitosan and packed under vacuum. Chitosan exhibited inhibition of evaluated microorganisms and, in combination with vacuum packaging, it improved the durative release of compounds causing inhibition. Superior antimicrobial activity of chitosan (3%) in comparison to thyme essential oil treatment in smoked eel samples was demonstrated (El-Obeid et al., 2018). Chitosan-treated samples reached maximum limits of total plate counts on 49 day (7 log cfu/g) from initial value of 2.85 log cfu/g. Similarly, higher inhibition of Pseudomonas, Shewanella spp., and yeasts and molds was observed.

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6.6.2 Film Fish (Merluccius merluccius and Solea solea) fillets wrapped in chitosan (3%) film packed under air and vacuum were reported by Fernández-Saiz et al. (2013). S. solea fillets packed in films containing chitosan inhibits aerobic mesophiles, Pseudomonas spp., and H2S-producing bacteria for 6 days, with slight growth was reported. Additionally, synergistic effect of vacuum packaging was reported, with increased shelf life by 7 and 9 days comparing to control samples in hake and sole, respectively. The study suggested the effectiveness of chitosan-based antimicrobial film for microbial preservation of hake and sole fillets packed under vacuum (Fernández-Saiz et al., 2013). Chitosan (1%)-based wrapping material for shrimp exhibited inhibition of microorganisms including total viable count, psychrotrophic bacteria, Pseudomonas spp., Pseudomonas fluorescens, Shewanella puterfaciens, Enterobacteriaceae, lactic acid bacteria, and L. monocytogenes (Mohebi & Shahbazi, 2017). Further inclusion of natural extracts (Ziziphora clinopodioides essential oil and pomegranate peel extract) increased the microbial inhibition. Film formed using chitosan and gelatine containing oregano essential oil was evaluated for preservation of grass carp muscle (Wu et al., 2014). The packaging of fish muscle inhibited the bacterial proliferation, thus reducing formation of amines resulted in quality maintaining and shelf life extension. Combination of chitosan treatment (1%) with propolis extract reduced the lipid oxidation (TBARS and FFA) for N. japonicus (Ebadi et al., 2019). The authors suggested inhibition of lipid hydrolysis based on antioxidant activity of chitosan, helping in scavenging of free radicals and reducing its progress. Also significant reduction of PV and volatile amines (TVB-N) in shrimp samples wrapped in chitosan-based film in comparison to control- and gelatin-based film was reported (Mohebi & Shahbazi, 2017). The inhibition of bacterial proliferation (Pseudomonas spp. and psychrotrophic) reduced the generation of lipases and inclusion of plant extracts further reduced the lipid oxidation. Coating of hairtail (Trichiurus haumela) with chitosan-based nanoemulsion containing eugenol retarded the protein denaturation leading to improved water holding capacity of fish muscle (Liu, Shao, et al., 2021). Inhibition of microbial proliferation by chitosan coating has been demonstrated to reduce the decomposition of muscle responsible for evolution of amines (TVB-N and TMA-N) in coated S. brasiliensis fillets extending the quality attributes (do Vale et al., 2020). Besides, coating created a barrier toward exposure to oxygen and

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antioxidant ability of chitosan reduced lipid oxidation during 180-day storage. Chitosan coating alone and enriched with phenolic compounds improved the antioxidant capacity of the coating material for preservation of sea bass fillet (Liu, Lan, et al., 2021). The application of chitosan-based coating with phenolic acids lowered discoloration of sea bass fillet by reducing myoglobin and lipid oxidation along with TVB-N based yellowing. The application of coating retained the textural parameters of the treated sea bass slices due to lowered muscle softening (associated to muscle degradation and protein denaturation) (Liu, Lan, et al., 2021). Production of biogenic amines (cadaverine, putrescine, histamine, and tyramine) was inhibited by the application of chitosan and packaging under vacuum in swordfish steaks (Tsiligianni et al., 2012). In relation to microbial inhibition reported in this study, the relative decrease in amine evolution (TMA-N and TVB-N) was reported. Additionally, chitosan inhibits lipid oxidation due to antioxidant potential and ability was improved in vacuum packaging owing to the elimination of oxygen (Tsiligianni et al., 2012). Treatment with chitosan followed by vacuum packaging in smoked eel samples inhibited oxidative rancidity and retained freshness (TMA-N and TVB-N) as compared to control ad samples treated with thyme essential oil (El-Obeid et al., 2018). Chitosan treatment retained the TVB-N values within the maximum permissible limits (35 mg N/100 g) during 42-day storage (14.9 mg N/100 g) attributed to extension of lag phase and oxygen elimination by vacuum packaging.

6.7 Inclusion of chitosan in combination with other preservation as hurdle concept The demand for fresh, semipreserved, or ready-to-eat foods is increasing, and their security is considered a main existential concern in the world. Therefore the necessity to preserve the quality and the safety of foods is required, and, thus, it is considered a considerable standard of food hygiene. The potential contamination of the food products by the pathogenic and/or spoilage microbes remains a major concern. In fact, the contamination of food products can negatively affect their shelf life and quality and, hence, leads to the increase of the risk of foodborne illness. Previous research works have revealed that the use of traditional food preservation processes can provide some level of conservation; however, these methods have also several unfavorable effects on the quality of food products and lead also to the decrease of their nutritional value (Zhang et al., 2021).

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Consequently, the necessity of innovative and alternative methods is required to prevent and inhibit the contamination of food products and maintain their safety and quality (Appendini & Hotchkiss, 2002). Thus the food industries have increased the investigation about the incorporation of antimicrobials and antioxidant compounds derived from natural sources. The chitosan can be recognized as preservation agent due to its superb properties, such as antioxidant and antimicrobial, and can be considered eco-friendly food preservatives (Falleh et al., 2020; Inanli et al., 2020; ˇ Simat et al., 2020; Zhang et al., 2021). In addition, it has been demonstrated that the combination of chitosan with other preservation compounds, including the essential oils, nisin, etc., can contribute to the improvement of the preservation of food products during extended periods (Zhang et al., 2021) (as shown in Table 6.1). The inclusion of chitosan in combination with other preservation can allow inhibition and prevention of microbial growth on food matrix, prolong their shelf life and security, and ensure their overall quality (Zhang et al., 2021). Several research studies have revealed that the combination of chitosan film with essential oils and their incorporation in food products exhibited an important antimicrobial capacity (Li et al., 2019). In fact, the chitosan film demonstrated its interaction with the essential oils into the food matrix, therefore it can ensure the long durability of antimicrobial activity in the packaging systems (Li et al., 2019; Zhang et al., 2021). The addition of several categories of essential oils into the chitosan films can improve the antimicrobial capacity of this biopolymer against various foodborne pathogens and spoilage microorganisms (Zhang et al., 2021). The combination of chitosan films with the turmeric essential oil can induce an enhancement of the inhibitory capacity on the growth of Aspergillus flavus and the formation of conidial (Li et al., 2019). In this regard, the chitosan packaging films can provide an inhibition of the expression of 16 genes and thus demonstrated a good antiaflatoxigenic capacity of this biopolymer. Besides, the research of Lin et al. (2019) has revealed that the combination of the chitosan nanofiber with chrysanthemum essential oil provides an excellent antimicrobial effect against L. monocytogenes in beef, with a 99.9% inhibition, thus this mixture contributes to the enhancement of the preservation of beef meat. Furthermore, Gómez-Estaca et al. (2010) have investigated the antimicrobial and preservation effects of the mixture of the chitosan-gelatine films with different essential oils against 18 various spoilage and foodborne

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Table 6.1 Overview of some research studies concerning the synergetic effects of the combination of chitosan samples with other preservation agents. Chitosan samples

Other preservation agents

Food matrix

Major findings

References

Chitosan film (2% w/v)

Apple peel polyphenol

Strawberry fruit

Riaz et al. (2021)

Chitosan (0.8% w/v)

Oregano essential oil

Fresh cucumber

Chitosan (1% w/ v)

Bergamot essential oil

Grape

This research study has demonstrated that the combination of chitosan with the apple peel can reduce weight loss and enhance the quality of strawberry fruit. Therefore this combination contributes to the improvement of the preservation effect of this food matrix and extends its postharvest life. The findings of this study have demonstrated that the addition of oregano essential oil to the chitosan films can improve the antimicrobial activity in situ “fresh cucumber.” This study has revealed that coated samples containing the combination of chitosan and bergamot oil can provide an inhibitory activity against Listeria monocytogenes and

GutiérrezPacheco et al. (2020)

SánchezGonzález et al. (2010)

(Continued)

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Table 6.1 (Continued) Chitosan samples

Other preservation agents

Food matrix

Chitosan (0.1% w/v)

Thyme essential oil

Meat pork

Chitosan (2% w/ v)

Oregano and lemon essential oils

Breast meat

Major findings

thus can enhance the antimicrobial activity. Therefore the addition of the bergamot essential oil can improve the antimicrobial ability of the pure chitosan coating. The data of this research work has proven that the mixture of chitosan samples with thyme essential oil has an effective impact on the preservation of meat and pork. Therefore this combination can extend the shelf life of the fresh pork for a long period via the inhibition of the microbial growth. It has been revealed that the chitosanedible film incorporated with the oregano and lemon essential oil in the breast meat can exhibit and antimicrobial activity against

References

Liu and Liu (2020)

Chaleshtori and Chaleshtori (2017)

(Continued)

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Table 6.1 (Continued) Chitosan samples

Chitosan (1.5% w/v)

Other preservation agents

Prunus armeniaca (Apricot) essential oil

Food matrix

Meat beef

Major findings

Shigella dysenteriae, Salmonella typhi, Salmonella typhi, and Streptococcus pyogenes. Besides, this combination can improve the sensory attributes of meat, including its taste and flavor. In addition, it can extend the shelf life and preserve the fresh chicken meat. The incorporation of the mixture of chitosan with the Prunus armeniaca essential oil can exhibit an excellent antimicrobial activity against the strain of L. monocytogenes.

References

Wang et al. (2020)

pathogenic bacteria. The combination of chitosan with clove essential oil has the highest inhibitory effect, followed by rosemary and lavender. In this context, the chitosan film incorporated with clove essential oils can prevent the growth of targeted microbes, such as E. coli and Listeria innocua. Besides, the application of the combination of chitosan with bergamot essential oil can significantly prevent the growth of Penicillium italicum (Sánchez-González et al., 2010). However, the incorporation of food with phenolic compounds can contribute also to the preservation and the maintenance of food safety and quality. Thus the chitosan incorporated with grape seed extract with different concentrations can exhibit an inhibitory effect against several

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spoilage and foodborne pathogens compared to the pure chitosan films (Amankwaah et al., 2020). Moreover, the combination of chitosan with other gums has demonstrated a superb capacity of preservation for foods (Maleki et al., 2022). For instance, the chitosan combined with alginate can induce a preservation effect on food products during storage period (Nualkaekul et al., 2013). Furthermore, the mixture of chitosan with liposomes, which considered as spherical structures, composed of phospholipid groups, can provide an improvement of the preservation effect when they incorporated into food products (Esposto et al., 2021; Jiao et al., 2018; Maleki et al., 2022; Zhou et al., 2018). Besides, the incorporation of the mixture of chitosan with epicatechin gallate, which is a phenolic compound presented in several plants, can exhibit a conservation effect via its important inhibitory activity against the methicillin-resistant S. aureus in pork meat (Guo et al., 2022). In addition, the combination of chitosan with nisin and gallic acid can provide an excellent preservation for pork meat during storage period under modified atmosphere packaging with high oxygen (Cao et al., 2019).

6.8 Effect of chitosan on acceptability/sensory quality of seafood Sensory evaluation is used to measure the actual perception of food quality and acceptability by the evaluator. Different quality parameters such as appearance, color, texture, odor, taste, and overall acceptability are assessed to determine the sensory quality in seafood (Kedar et al., 2022; Pawar et al., 2020). The reduced deterioration of seafood (microbial and oxidative spoilage) by application of chitosan results in maintained sensory quality attributes (Yu et al., 2018). Chitosan (2%)-treated smoked eel fillets were reported to have higher sensorial acceptability (taste and odor) comparing to samples treated with the combination of thyme essential oil and chitosan (El-Obeid et al., 2018). The inclusion of essential oil imparted bitterness, while chitosan alone retained the freshness (taste and odor) of smoked eel samples. The shelf life of chitosan-treated smoked eel packed under vacuum was found to be 49 days in comparison to 35 days for control sample and 42 days for thyme essential oil coated sample. Hence, treatment with chitosan in combination with vacuum packaging can extend the shelf life by more than 14 days. Inclusion of chitosan at 0.5% 2.0% levels in batter formulation for coating fish sticks improved

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the sensorial quality by reducing the frying losses and textural quality (Xavier et al., 2017). Chitosan (1%) treatment inhibited enzymatic and bacterial action leading to acceptable sensory quality for white shrimps (Wu, 2014). Improvement in organoleptic quality of N. japonicus fillets treated by chitosan was found during 12-day storage (Ebadi et al., 2019). The control sample was unacceptable on day 6, while chitosan-coated samples were acceptable for 12 days. Chitosan-based packaging at lower concentration (1%) had no significant impact on the sensory quality of shrimps, whereas inclusion of natural extracts improved the sensory quality (Mohebi & Shahbazi, 2017). Chitosan- and thymol-based emulsion-coated peeled ready-to-eat shrimp retained color, firmness, and odor (Carrión-Granda et al., 2016). Coating inhibited development of putrid or unusual odor during storage. Application of chitosan combined with modified atmospheric packaging was suggested for preserving sensory qualities. Chitosan-based nanoemulsion coating retained the sensory quality of hairtail fish extending shelf life by additional 3 days in comparison to control (Liu, Shao, et al., 2021). Inclusion of eugenol essential oil further extended shelf life by 3 days over chitosan singly and 6 days over control sample. Arancibia et al. (2015) reported the delay in development of melanosis in shrimps by enzymatic action of polyphenols oxidase by chitosan-based coating. Chitosan coating inhibited evolution of off-odor (ammoniacal) development by inhibiting microbial proliferation and evolution of ammoniacal compounds. Application of chitosan-based coating on sea bass fillet retained the sensory quality (color, odor, texture, and overall acceptability) by increased shelf life ( . 10 days) of the coated samples (Liu, Shao, et al., 2021). This is attributed to antimicrobial and antioxidant capacity of chitosan and phenolic compounds in coating.

6.9 Conclusion The chitosan as a biopolymer is characterized by its beneficial and bioactivity properties due to its physicochemical characteristics. Besides, it is not harmful to human health and has several beneficial impacts for the well-being. In fact, this functional compound is recognized as safe and can be used in several sector such as biomedicine, pharmacology, and food. The chitosan has an important role in the food industry and it can be applied for the improvement of the quality and preservation of the food

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products. In addition, this biopolymer is characterized by its potential impact on the extension of shelf life of several food products, particularly the seafood and their products. Moreover, this biomaterial and its derivatives are considered a functional ingredient and can be used for the preservation of seafood products due to their antioxidant and antimicrobial properties and other biological activities. Therefore the chitosan can contribute to an enhancement of the quality of seafood and maintenance of their safety. In fact, this biopolymer and its derivatives applied on seafood products can induce an inhibition of the microbial growth, enhance the sensory attributes, and decrease the lipid oxidation. In addition, the combination of the chitosan with other preservation compounds, such as some gums, essential oils, nisin, phenolic compounds, and other bioactive compounds can improve the quality of seafood products and extend their shelf life more than the use of chitosan alone. Thus these mixtures lead to a better preservation effect on seafood products. Furthermore, nanotechnology processes can be used for the production of chitosan nanosystems and their application on various types of seafood for both safety and quality enhancement. However, more research needs to be done for the application of chitosan in the food industry. In addition, the functionality, nutritional value, and other biological activities of chitosan need to be investigated further.

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CHAPTER 7

Chitosan and use of pomegranate-based films in foods Hadi Ebrahimnejad1, Elham Khalili Sadrabad2,3 and Fateme Akrami Mohajeri2,3 1

Department of Food Hygiene and Public Health, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran Infectious Diseases Research Center, Shahid Sadoughi Hospital, Shahid Sadoughi University of Medical Sciences, Yazd, Irannces, Yazd, Iran 3 Department of Food Hygiene and Safety, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 2

7.1 Nutritional and chemical properties of pomegranate Pomegranate fruit has abundant amounts of nutrients in its various components. Edible and inedible parts of pomegranate fruit contain macro- and micronutrients that can play a valuable role in fulfilling human nutritional needs (Mirdehghan & Rahemi, 2007). About 50% of the weight of pomegranate fruit is allocated to the edible part, that is, fruit juice and seeds (Aviram et al., 2000). Pomegranate juice is obtained from aryl and contains dietary fibers along with several nutritionally valuable components such as polysaccharides, fatty acids, proteins, minerals, and vitamins (Kumar, 2018; Rinaldi et al., 2013). About 85% of pomegranate juice is water. It also contains about 10% sugar with an equal amount of fructose and glucose (Fig. 7.1) (Aviram et al., 2000). Pectin and ascorbic acid are other constituents of pomegranate juice, while its protein content is usually less than 0.5%, including aspartic and glutamic acid as the principal amino acids (Aviram et al., 2000; Rinaldi et al., 2013). High levels of calcium, potassium, sodium, magnesium, phosphorous, zinc, iron, and copper content of edible pomegranate parts are important for human mineral requirements. The ash of pomegranate juice contains about 50% calcium (Aviram et al., 2000; Mirdehghan & Rahemi, 2007). The seed is another edible part of pomegranate fruit, which in addition to pectin, crude fiber, and sugars, also contains estrogenic compounds and tocopherol (Zaouay et al., 2020). There is a noticeable variation in the level of sugars, vitamins, and minerals Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00004-1

© 2023 Elsevier Inc. All rights reserved.

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Figure 7.1 An annotated schematic picture of pomegranate fruit.

reported in pomegranate. The chemical and nutritional composition of pomegranate fruit will vary depending on the pomegranate cultivar, agroclimatic conditions, and the ripening stage of the fruit (Mirdehghan & Rahemi, 2007; Shinde et al., 2020; Çam et al., 2009; Nouri et al., 2020).

7.2 Functional properties of pomegranate Functional foods are foods that have health benefits that go beyond nutritional value. Bioactive or functional compounds usually come from functional food sources such as some fruits (Fernandes et al., 2019). Pomegranate is a rich fruit for these bioactive phytochemicals. Naturally, different pomegranate cultivars have different levels and types of bioactive compounds (Akhavan et al., 2015; Hmid et al., 2017). More than the pomegranate variety, its bioactivity is influenced by the growth climate and region, cultivation conditions, and postharvest fruit storage status (Fernandes et al., 2017). Pomegranate fruit is composed of edible and nonedible compartments. The biological activity of each of these compartments is different. The antioxidant activity of pomegranate is one of its major bioactivities (Amri et al., 2017). Nevertheless, the pomegranate total antioxidant activity does not delineate the exact bioactivity of responsible phytochemicals (Dludla et al., 2018). It is noteworthy that the individual assessment of bioactive compounds is incomplete without considering their synergistic effect.

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Phenolic compounds and fatty acids are the main bioactive compounds in pomegranate. Simple phenols, flavonoids, and hydrolyzable tannins, that is, gallotannins and ellagitannins are notable phenolic compounds found in pomegranate. The hydrolysis of punicalagin, the most abundant pomegranate ellagitannins, produces ellagic acid and punicalin (Sepúlveda et al., 2014; Sun et al., 2017). Gallic acid can be produced through the hydrolysis of gallotannins (Sirven et al., 2019). Furthermore, polyunsaturated fatty acids such as punicic, linoleic, linolenic, and arachidonic acids are the major fatty acids of pomegranate (Melgarejo-Sánchez et al., 2021; Wang et al., 2012).

7.2.1 Bioactivity of pomegranate juice Pomegranate juice is one of the most refreshing and palatable drinks in Asia, North Africa, and the Mediterranean with a taste of sweet, sour, bitter, or astringent that obtains from pomegranate arils. Notably, juicing with pomegranate peel made it more bitter and astringent (Bett-Garber et al., 2014; Wasila et al., 2013). So far, many bioactive compounds have been detected in pomegranate juice. Simple phenolics such as protocatechuic acid, caffeic acid, and ferulic acid have been found in the juice of many pomegranate cultivars (AlMatar et al., 2019; Poyrazo˘glu et al., 2002; Wang et al., 2012). The main phenolic compounds in pomegranate juice are hydrolyzable tannins and their derivatives (Mena et al., 2012). Gallagic acid, punicalagin, punicalin, ellagic acid, gallic acid, and gallagyldilactone (terminalin) are among these natural tannins (Arun & Singh, 2012; Banihani et al., 2014; Kasimsetty et al., 2010; Makino-Wakagi et al., 2012; Poyrazo˘glu et al., 2002; Rojanathammanee et al., 2013; Wang et al., 2012). There are many flavonoids in pomegranate juice, such as myricetin, naringenin, pelargonidin, kaempferol, cyanidin-3-glycoside, rutin, catechin, and quercetin, but luteolin is the most relevant (AlMatar et al., 2019; Poyrazo˘glu et al., 2002; Rocha et al., 2012; Wang et al., 2012). In some studies, punicic acid has been detected as an omega-5 long-chain polyunsaturated fatty acid in pomegranate juice (Banihani et al., 2014; Rocha et al., 2012; Wang et al., 2012). The antioxidant activity of juice is often attributed to ellagitannins, especially punicalagin, which originates from the pomegranate peels (Gil et al., 2000; Tzulker et al., 2007). Studies on pomegranate juice have shown anticancer activity against prostate, breast, and colon cancers (Kasimsetty et al., 2010; Rocha et al., 2012; Wang et al., 2012). This juice has been effective in

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controlling type 2 diabetes, suppressing resistin hormone secretion, and insulin resistance (Banihani et al., 2014; Makino-Wakagi et al., 2012). The antiinflammatory activity of pomegranate juice has mitigated some symptoms of Alzheimer’s disease, such as cognitive impairment or behavioral disturbances (Hartman et al., 2006; Subash et al., 2014, 2015). Finally, cardioprotective and antimicrobial activity of pomegranate juice has been reported in some studies. Pomegranate juice inhibits several Gram-positive and Gram-negative bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Vibrio parahaemolyticus, and Helicobacter pylori (Melgarejo-Sánchez et al., 2021; Pagliarulo et al., 2016).

7.2.2 Bioactivity of pomegranate peel (pericarp) Pomegranate peel is a by-product of the pomegranate juice industry, which contains many functional compounds. Commonly, in literature, the pericarp is referred to as peel and it may comprise up to 50% of the total pomegranate fruit weight (Viuda-Martos et al., 2010). Numerous phenolics such as ferulic acid as well as hydrolyzable tannins and their derivatives such as gallagic acid, punicalagin, punicalin, ellagic acid, and gallic acid are abundantly detected in pomegranate peel and they even may enter the pomegranate juice (Arun & Singh, 2012; Dludla et al., 2018; Fawole et al., 2012; Giamogante et al., 2018; Jalal et al., 2018; Suman & Bhatnagar, 2019; Wang et al., 2014). In addition, there are several flavonoids in pomegranate peel, such as catechin, epicatechin, rutin, cyanidin 3,5-diglucoside, and delphinidin 3,5-diglucoside (Fawole et al., 2012; Gullon et al., 2016). Punicalagin is the most important hydrolyzable tannin in pomegranate peel that causes its antioxidant activity (Gil et al., 2000; Tzulker et al., 2007). However, inhibition of tyrosinase by pomegranate peel is also effective in its antioxidant activity (Fawole et al., 2012). The anticancer role of pomegranate peel, especially against breast and prostate cancer, is mostly due to its high antioxidant activity and DNA repair action (Deng et al., 2017; Melgarejo-Sánchez et al., 2021; Shirode et al., 2015). Antiinflammatory and anticonvulsive effects are other biological activities of pomegranate peel (Olapour & Najafzadeh, 2010). Studies have shown that pomegranate peel extracts inhibit a wide range of microorganisms, such as bacteria, fungi, and viruses (Maroufi et al., 2021; Nair et al., 2018a; Saadat et al., ˇ 2021; Zivkovi´ c et al., 2021).

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7.2.3 Bioactivity of pomegranate seed In biological studies, the pomegranate seed is incorrectly referred to as only the hard pomegranate kernel (Montefusco et al., 2021). Extraction of the pomegranate seed oil is a common practice and its oil content is between % and 20% of the seed weight (Zaouay et al., 2020). Functional compounds such as ellagic acid and punicic acid along with sterols and vitamin E are found in pomegranate seeds (Melgarejo-Sánchez et al., 2021; Rojo-Gutiérrez et al., 2021; Zaouay et al., 2020). Antiinflammatory activity, prevention of growth or metastasis of breast and prostate tumors, and antidiabetic action are some of the main reported biological activities of pomegranate seed (Banihani et al., 2013; Boroushaki et al., 2016; Kim et al., 2002; Wang & Martins-Green, 2014). The impact of pomegranate seed oil in alleviating the symptoms of neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer’s, and Parkinson’s diseases, has also been evaluated in many studies (Gabizon et al., 2018; Mizrahi et al., 2014). The antimicrobial activity of pomegranate seed extract against a range of microorganisms such as bacteria and fungi promises the application of this extract in the food industry (Amri et al., 2020; Okan et al., 2020).

7.3 Extraction of pomegranate active compounds The extraction or sample preparation technique is the first step in research on natural bioactive compounds (Srivastava et al., 2021). There are many methods for the extraction of natural active compounds. The quality and yield of extracted active compounds are related to the method of extraction, the particle size of samples, type of solvents, temperature, extraction ˇ time, and solid:solvent ratio (Zivkovi´ c et al., 2018). The extraction methods could be divided into two categories of conventional and modern extraction methods (Zhang et al., 2018). In the current chapter, only a brief explanation of extraction methods is given in the following sections.

7.3.1 Conventional extraction methods There are different conventional methods for the extraction of pomegranate compounds such as maceration, decoction, percolation, solvent, and soxhlet extraction. Maceration is the conventional method of extraction. The high amount of solvent, energy, and time as well as low extraction efficiency are some disadvantages of maceration (Abbas et al., 2021). The execration yield can be affected by time and temperature and solvent type (Conidi et al., 2017). Through the addition of heat in the maceration

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procedure, the method is named digestion extraction (Srivastava et al., 2021). For bioactive compounds that are heat resistant and water soluble, the decoction method could be applied which is an application of a specific volume of boiled water in samples (Srivastava et al., 2021; Zhang et al., 2018). Percolation is introduced as a conventional method in which continuous solvent replacement is applied to the method (Zhang et al., 2018). Solvent extraction is the application of water in a mixture of organic solvents (Srivastava et al., 2021). Different solvents, including methanol, distilled water, ethanol, hexane, acetone, petroleum ether, ethyl acetate, and chloroform, could be applied in the solvent extraction method (Abbas et al., 2021). According to results of Kumar and Neeraj, extraction of pomegranate peel with methanol showed highest FRAP activity and phenolic content in compared to other solvents, including ethanol, water, hexane, and acetone. Although in study by these researchers, the DPPH and flavonoid contents of pomegranate peel extracted with ethanol reported higher than other solvents (Kumar & Neeraj, 2018). Malviya et al. worked on solvents, including water, methanol, ethanol, ethanol/water (30:70), ethanol/water (50:50), and ethanol/water (70:30) for pomegranate peel extraction. The results revealed that ethanol/water (50:50) had the highest extraction yield and samples extracted with 100% water and ethanol/water (70:30) showed higher phenolic conˇ tent and antioxidant activity (Malviya et al., 2014). Savikin et al. (2018) indicated that among food grade solvents, ethanol could be a good solvent in bioactive compound extraction from pomegranate peel. Soxhlet extraction is a method of continuous reflux and siphoning with a solvent that consumes lesser solution and time in comparison to percolation and maceration (Zhang et al., 2018).

7.3.2 Modern extraction methods Due to problems of conventional methods such as low extraction efficiency, high consumption of solvent, and long extraction time, modern techniques in herbal extraction were provided. There are different modern extraction methods including distillation extraction, microwaveassisted extraction (MAE), vacuum microwave-assisted extraction (VMAAE), ultrasound-assisted extraction, membrane separation, supercritical fluid extraction, pressurized liquid extraction (PLE), pulsed electric field (PEF) extraction, enzyme-assisted extraction, and solid-phase extraction which are briefly explained as follows.

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Distillation extraction is divided into two methods of hydro and steam distillation which are appropriate for the extraction of aromatic or volatile compounds (Srivastava et al., 2021; Zhang et al., 2018). MAE, the application of electromagnetic waves to generate heat, needs less time, less solvent, lower cost, and higher extraction rate compared to the conventional method (Zhang et al., 2018; Zheng et al., 2011). Although the optimum extraction of pomegranate peel with water under MAE condition was related to time, microwave output power, and solvent/pomegranate peel ratio (Zheng et al., 2011). In the study of Turrini et al. (2019), a high content of ellagitannins was detected in microwave drying pomegranate peel comparing traditional oven drying (Turrini et al., 2019). The use of electromagnetic waves with high energy in vacuum condition is known as VMAAE which increases the migration of bioactive material in solid samples to the aqueous phase. Due to the application of low temperature and pressure, the VMAAE could be a selective method for thermosensitive compounds. Along with lower thermal degradation risk, the application of VMAAE facilitates mass transfer and release of bioactive compounds (Skenderidis et al., 2020). In the study of Skenderidis et al. (2020), the total phenolic content of pomegranate peel extracted by VMAAE after 10 minutes reached to 137.97 mgGAE/g. Ultrasound-assisted extraction which is introduced as an environmentfriendly procedure needs shorter time, less solvent, and energy. The rupture of the plant cell wall by prolonged ultrasonic extraction resulted in increased release of polyphenols (Cui et al., 2020; Zhang et al., 2018; ˇ Zivkovi´ c et al., 2018). The research by Turrini et al. (2019) indicated that UAE was more efficient in extraction of pomegranate external peel bioactive compounds (except for ellagitannins) in comparison to traditional maceration and decoction methods. There is a report that methanolic extraction of pomegranate peel assisted with ultrasonic wave had highest ˇ DPPH activity (Cui et al., 2020). According to the results of Zivkovi´ c et al. (2018), the optimum phenolic extraction of pomegranate peel was achieved at 59% ethanol concentration, solid/solvent ratio of 1:44, time of 25 minutes at 80°C. The membrane separation process is a newly developed method that is easy to control and has low operation control, mild temperature and pressure, without need for chemical additives, and less contaminant production. There are different types of membrane separation processes including ultrafiltration, nanofiltration, microfiltration, reverse osmotic, membrane distillation, osmotic distillation, and pervaporation (Conidi et al., 2017).

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Solid-phase extraction is the application of the solid phase to adsorb the unwanted impurities from the pomegranate extract (Surendhiran et al., 2020). Supercritical fluid extraction-carbon dioxide (SC-CO2), widely used, along with good extraction rate and cost efficiency, is safe due to its nonexplosive, nonflammable, and nontoxic material. Ara and Raofie indicated that pomegranate peel essential oil extraction by SCF had higher yield extract in comparison to hydrodistillation method (Ara & Raofie, 2016). PLE is the extraction of bioactive compounds through the combination of solvents with high pressure (from 4 to 20 Mpa) and temperature (García et al., 2021). It was shown that application of pressurized water extraction was effective in recovery of pomegranate peel polyphenols and total phenolic content (Çam & Hı¸sıl, 2010; García et al., 2021). PEF extraction is the destruction of the cell membrane by pulse electric potential (Srivastava et al., 2021; Zhang et al., 2018). The combination of PEF with high-voltage electrical discharge (HVED) could lead to physical and chemical reactions (Lampakis et al., 2021). Application of PEF and HVED resulted in higher yield extraction and polyphenol content of pomegranate peel extraction (Rajha et al., 2019). Enzyme-assisted extraction is the application of hydrolytic enzymes resulting in the denaturation of the cell membrane and facilitating the release of bioactive compounds (Zhang et al., 2018). Mushtaq et al. (2015) showed that combination of enzyme-assisted with supercritical fluid in pomegranate peel extraction resulted in higher yield extract and increase in concentration of phenolic compounds and antioxidant capacity.

7.4 Chitosan composite films for incorporating pomegranate active compounds Chitosan with biodegradable, biofunctional, biological compatibility, and nontoxic characteristics as well as antimicrobial and antioxidant activities could be a good coating for embedding natural and biologically active compositions (Berizi et al., 2018). Moreover, the ability of chitosan to form edible films, gel, and porous scaffolds to trap bioactive compounds makes chitosan a good choice for application in food packaging and alternative to nonrenewable sources (Bertolo et al., 2020; Zheng et al., 2011). There is growing attention to the use of plant extracts especially pomegranate incorporation with chitosan film in the food active packaging industry (Yuan et al., 2016). As mentioned in the previous section, different parts of pomegranate were introduced as important active agents in the chitosan-based film.

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Due to low elasticity, poor mechanical resistance, and low solubility of chitosan in neutral pH, the combination of chitosan and pomegranate extract with other materials, including gelatin (Bertolo et al., 2020), alginate (Nair et al., 2018a,b), gelatin (Saki et al., 2018), zein (Cui et al., 2020), montmorillonite (Qin et al., 2015), pullulan (Kumar et al., 2021b), dialdehyde guar gum hydrogels (Maroufi et al., 2021), Pleurotus eryngii polysaccharide nanofiber (Cai et al., 2021), cinnamon essential oil (Yuan et al., 2016), starch (Lozano-Navarro et al., 2018), active gelatin/cress seed gum based (Soltanzadeh et al., 2022), and tuna skin collagen (Qu et al., 2022), has been investigated.

7.5 Encapsulation of pomegranate active compounds in chitosan-based films The fast release of polyphenols from polymer films into packaged food is a major problem of the direct addition of natural compounds (Cui et al., 2020). Encapsulation of pomegranate extract in chitosan nanoparticles could be an effective way to control the delivery of pomegranate extract in food packaging (Soltanzadeh et al., 2021; Soltanzadeh et al., 2022). Chitosan nanoparticles produced by ionic gelatin methods provide a higher surface-to-volume rate to carry natural compounds (Soltanzadeh et al., 2021). Therefore encapsulation of active compounds was introduced as a reliable method to slow down the release of polyphenols from polymer films into packaging (Cui et al., 2020). On the other hand, the stability of active materials could be improved by nanoencapsulation. Also, a combination of pomegranate peel with chitosan-based nanoparticles could promote their synergistic effects (Cai et al., 2021). There have been some important characteristics, including size distribution and zeta potential which should be considered in nanoencapsulation. The literature provided that in lower pomegranate peel extract concentrations, the size distribution was unimodal and in higher concentrations the size distribution was shifted to bimodal with a high polydispersity (Soltanzadeh et al., 2021). Also, Gull et al. (2021) reported that with an increase in the concentration of pomegranate peel extract, the droplet diameter of nanoemulsions is increased (Gull et al., 2021). The aggregation of nanoparticles was related to the leakage of pomegranate peel extract to the nanoparticle surface. The prevention of aggregation is caused by a high surface charge and strong repellent electrostatic force. The surface charge and stability of nanoparticles could be determined by zeta potential which was lowered in the presence of pomegranate peel extract (Soltanzadeh et al., 2021). The reduction of zeta potential by increase of pomegranate peel polyphenols is

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related to the interaction of amino groups (Cai et al., 2021). Otherwise, Gull et al. (2021) suggested that the addition of pomegranate peel in nanochitosan coating resulted in a heightened electrical charge which was introduced as an important key in the stability of nanoemulsions (Gull et al., 2021). Cai et al. (2021) reported that pomegranate peel polyphenols in the amount of 3 mg/mL had high zeta potential, good encapsulation efficiency, and uniform particle size (Cai et al., 2021). The polydispersity index (PDI) value which is defined as the homogenous distribution of nanoparticles in nanosolutions is set as 0 1 (Cui et al., 2020). The good distribution of nanoparticles is shown in PDI , 0.3 (Cai et al., 2021). In incorporation of encapsulated pomegranate peel extract with chitosan nanoparticles in zein film by ionic gelatin technique, showed a PDI value of 0.287 which indicated the homogenous distribution of nanosolution (Cui et al., 2020). Soltanzadeh et al. (2021) determined that the PDI and particle size were increased by raising pomegranate peel extract concentration. In overall, there were reports that zeta potential and encapsulation efficiency could be negatively affected by pomegranate peel extract addition. Although, the pomegranate peel extract addition positively increased the PDI and particle diameter of chitosan nanoparticles (Soltanzadeh et al., 2021). The lower zeta potential of pomegranate peel extract-chitosan nanoparticles is attributed to lower chitosan amino group accessibility (Potrˇc et al., 2020). Encapsulation efficiency was decreased by higher pomegranate peel extract concentrations which are introduced as a limitation of nanoparticle encapsulation (Soltanzadeh et al., 2021). Electrical conductivity and viscosity are introduced as important agents in the tensile property and morphology of nanofiber in process of electrospinning (Cai et al., 2021). Surendhiran et al. (2020) reported that encapsulation of pomegranate peel extract by electrospun nanofiber could reduce the viscosity by changes in intra- and intermolecular reactions (Surendhiran et al., 2020) and the movement of ions in spinning materials was promoted (Cai et al., 2021). It was shown that the saturation of nanoparticles with pomegranate extract leads to an increase in particle size (Cai et al., 2021).

7.6 Physicochemical properties of pomegranate-chitosanbased films The changes in physicochemical characteristics of pomegranate-chitosanbased films could be related to different hydrophilic and hydrophobic groups and their interaction in the combination of pomegranate peel and chitosan (Zeng et al., 2021). Accordingly, the formation of hydrogen bonds

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between hydroxyl groups of pomegranate peel polyphenols and chitosan molecules is known as the agent of many changes (Cai et al., 2021).

7.6.1 Thickness of pomegranate-chitosan-based films Film permeability which is known as fluid transition could be affected by film thickness and an apparent density. The apparent density increment by the addition of pomegranate extracts due to creating the porous structure in the film enhances the film permeability (Abbas and AbdulRahman, 2020). As reported, by addition of pomegranate peel to chitosan film, the thickness of the film was increased due to higher solid content, the presence of polysaccharides and antioxidants in pomegranate extract, the interaction of chitosan functional groups and phenolic hydroxyl groups of pomegranate extract, as well as homogeneous and uniform distribution (Lozano-Navarro et al., 2017; Min et al., 2021; Pirsa et al., 2020; Soltanzadeh et al., 2022; Zeng et al., 2021). It was shown that incorporation of pomegranate peel which contains high-molecular compounds into chitosan-montmorillonite film elevated the film thickness (Qin et al., 2015). Even though these results differ from Yuan et al. (2016) who reported no significant effects on the film thickness on the concentration of 10 g/L pomegranate peel (Yuan et al., 2016). Probably, different factors including film preparation methods, relative humidity, dish surface, and time of solvent evaporation could be effective in film thickness (Kumar et al., 2021c).

7.6.2 Moisture content of pomegranate-chitosan-based films The total water molecules in the polymeric structure of chitosan film are introduced as moisture content (Gull et al., 2021). Because of the interaction between hydrophilic groups of chitosan and phenolic hydroxyl groups of pomegranate polyphenols, the moisture content of film could decrease by an increase in pomegranate peel concentration (Kumar et al., 2021c; Pirsa et al., 2020; Zeng et al., 2021). Also, the occupation of spaces in chitosan microstructural by pomegranate extract resulted in lowering the moisture content (Gull et al., 2021).

7.6.3 Water solubility of pomegranate-chitosan-based films The integrity of films in an aqueous environment and film biodegradability are determined by water solubility (Gull et al., 2021). The water solubility of the film is related to phenolic content and structural

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characteristics of the matrix (Kumar et al., 2021c). The disruption of the polymer chain by the presence of bioactive materials could lead to changes in film solubility (Gull et al., 2021). The literature indicated that by an increase in the concentration of pomegranate peel extract, the water solubility of the film will reduce (Kumar et al., 2021c). On the contrary in a study of Min et al. (2021), the water stability was increased by higher pomegranate peel extract concentration by breaking the molecular structure and increasing the hydrophilicity of chitosan film. The presence of organic compounds including carboxylic group, carbonyl, amines, and hydroxyls plays an important role in the enhancement of water solubility of chitosan film (Gull et al., 2021).

7.6.4 Swelling property of pomegranate-chitosan-based films The biodegradation and water resistance characteristics of the film could be the result of swelling properties (Kumar et al., 2021c). It was shown that the addition of 1 to 5% of pomegranate peel to chitosan/dialdehyde guar gum hydrogel, showed no effects on swelling ratio, good gel strength, and increase of total phenolic content of hydrogel (Maroufi et al., 2021). On contrary, the results of Kumar et al. (2021c) determined the reduction in swelling properties of chitosan film incorporated by pomegranate peel extract which was related to their cross-linking reaction (Kumar et al., 2021c).

7.6.5 Water vapor and oxygen permeability properties of pomegranate-chitosan-based films The hydrophilic nature of chitosan makes it permeable to water vapor. Thereafter, the addition of hydrophobic compounds could promote the hydrophobic hydrophilic characteristics of chitosan film (Yuan et al., 2016). The barrier moisture property of film known as water vapor permeability (WVP) was enhanced by higher pomegranate peel concentration in chitosan film due to film morphology (Zeng et al., 2021). On the contrary, Yuan et al. (2016), Soltanzadeh et al. (2022), and Pirsa et al. (2020) indicated that the WVP was significantly reduced by the addition of pomegranate peel. The packaging with high WVP capacity could have lower moisture transfer, which could prolong the shelf life of food (Kumar et al., 2021c). Qin et al. (2015) explained that the addition of pomegranate peel in the concentration of 1% 2% into chitosanmontmorillonite film decreased the WVP, otherwise by increasing the

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pomegranate peel to the concentration of 2% resulted in a WVP increase (Qin et al., 2015). The group of factors, including film thickness and uniformity, presence of phenolic compounds, fiber, starch, and pectin in pomegranate peel as well as the formation of agglomerated particles by pomegranate peel in chitosan film, was introduced as effective agents in the increase of WVP (Kumar et al., 2021c; Zeng et al., 2021). To elicit data, the reduction in hydrophilic groups’ availability after interaction of polyphenol compound with chitosan resulted in lowered chitosan affinity to water molecules (Qin et al., 2015). Although, the variation in WVP of pomegranate peel-chitosan film in different literature could be related to the composition of chitosan, method of film preparation, and presence of plasticizer (Yuan et al., 2016). It was illustrated that the incorporation of pomegranate peel in chitosan film lowers the water solubility of film as a result of the lack of hydroxyl groups and polymer amines to react with water as a consequence of the interaction of pomegranate peel phenolic compound and chitosan polysaccharide chain (Abdul-Rahman and Abass, 2021). There is a dearth of research that the addition of 1% 5% w/w of montmorillonite (MMT) in chitosan could improve the WVP and mechanical properties of chitosan film along with the addition of pomegranate peel (Qin et al., 2015). Therefore pomegranate-chitosan-based films could be a good barrier against moisture in food packaging (Zeng et al., 2021). The oxygen permeability is considered an important factor in maintaining the quality of food (Kumar et al., 2021c; Potrˇc et al., 2020). To destroy the action of oxygen, providing a good barrier in packaged food is essential (Potrˇc et al., 2020). The oxygen permeability of chitosan film was promoted by the presence of pomegranate peel (Abdul-Rahman and Abass, 2021). Although, Kumar et al. (2021c) indicated that the addition of pomegranate peel in chitosan film decreased the prevention of oxidation by declining their molecular interactions and enhancing polymer chain mobility (Kumar et al., 2021c).

7.6.6 Optical property of pomegranate-chitosan-based films Since the pomegranate contains pigments such as anthocyanin, the appearance (transparency, opacity, and color) of chitosan film could be affected (Kumar et al., 2021c; Pirsa et al., 2020). The resistance of film against light could be indicated by the transparency or opacity of film (Kumar et al., 2021c). In fact, with an increase in the concentration of pomegranate

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extract, the transparency of film is reduced owing to the presence of polyphenols and changes in pores of film inner structure through hydrolyzable tannin availability (Kumar et al., 2021c; Lozano-Navarro et al., 2017; Pirsa et al., 2020; Yuan et al., 2015, 2016). The higher transparency indicates the lower film opacity (Kumar et al., 2021c; Lozano-Navarro et al., 2017). As mentioned, the film opacity is a crucial issue in the protection of food against visible and ultraviolet light (photooxidation and lipid peroxidation) (Abdul-Rahman and Abass, 2021; Qin et al., 2015). There is some evidence that the phenolic compound of pomegranate peel extract reduces the transmittance of UV light (Zeng et al., 2021). Therefore applications of pomegranate peel extract in chitosan film increase the film opacity and the light stability of foods in the occurrence of oxidative spoilage (Abdul-Rahman and Abass, 2021; Qin et al., 2015; Zeng et al., 2021). The appearance of edible films is related to polyphenols, antioxidants, and anthocyanin pigments presented in pomegranate (Kumar et al., 2021c; Yuan et al., 2016). Therefore, by an increase in pomegranate concentration-incorporated chitosan film, the lightness was decreased (Gull et al., 2021; Kumar et al., 2021c).

7.6.7 Mechanical property of pomegranate-chitosan-based films The mechanical property includes tensile strength (film strength), elongation at break (deformation capacity under pressure), and modulus defined as the ability to protect the integrity of food properties during storage, transportation, and handling (Gull et al., 2021; Zeng et al., 2021). The results of some literatures indicated an increase in tensile strength by pomegranate peel addition due to uniform dispersion of pomegranate peel in chitosan film, the high molecular weight of pomegranate peel, molecular mobility of chitosan chain, formation of hydrogen bonds, and an increase in film thickness (Abdul-Rahman and Abass, 2021; Gull et al., 2021; Kumar et al., 2021c; Pirsa et al., 2020; Qin et al., 2015; Surendhiran et al., 2020; Yuan et al., 2016). The presence of multiple functional groups and the formation of links in the structure of film lead to an increase in tensile strength (Pirsa et al., 2020). Although, Zeng et al. (2021) showed a significant decrease in tensile strength and elongation at break at 9% pomegranate peel concentration. This reduction was attributed to the destruction of compact film structure and formation of H-bands in the chitosan films (Zeng et al., 2021). It was reported that the addition of 10 g/L pomegranate peel extract had insignificant effects

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on tensile strength and elongation at the break of chitosan film (Yuan et al., 2015). Accordingly, it is possible that higher pomegranate peel extract concentration because of the lack of uniform extract dispersion in chitosan film and a reduction in intermolecular interactions would lower tensile strength (Gull et al., 2021). In the study of Qin et al. (2015), pomegranate peel addition had insignificant effects on elongation at break which was attributed to discontinuity of structural development (Qin et al., 2015). Also, there may be a positive relation between elongation at break and moisture content of the film which is reduced by pomegranate peel additions (Gull et al., 2021). By the formation of new interactions, the gel network of PP-chitosan films was weakened. On the other hand, pomegranate peel addition had increased the viscous behavior of film (Bertolo et al., 2020). It was mentioned that mechanical property is related to the flexibility and mobility of film structure and film resistance in face of fracture by the addition of hydrophobic agents (Yuan et al., 2015). The decrease in viscosity of nanochitosan contained pomegranate peel in the research of Gull et al. (2021) was explained by polymeric chain breaks during the ultrasonication process (Gull et al., 2021). In overall, films containing a suitable concentration of pomegranate peel showed good mechanical properties.

7.6.8 Thermal property of pomegranate-chitosan-based films Glass transition temperature defined as the transition temperature of amorphous polymers into a flexible state was decremented in the presence of natural extract in the chitosan-starch film. This reduction is attributed to the instability of antioxidants at high temperatures (Lozano-Navarro et al., 2017). It was shown that by the addition of 1 g/mL of pomegranate peel extracts in chitosan film, the thermal degradation was increased. Therefore enrichment of chitosan film with pomegranate peel extract lessened the thermal stability (Kumar et al., 2021c). As shown by Cai et al. (2021), the increase in temperature resulted in a higher release of pomegranate peel polyphenols in nanofiber (Cai et al., 2021).

7.6.9 Morphology property of pomegranate-chitosan-based films According to FTIR analysis, the application of natural extracts contributed to a wider band at 3500 3200 cm21 (-OH bonds) and shift of the peaks into lower frequencies due to noncovalent reactions of chitosan functional groups with extract (Abbas and Abdul-Rahman, 2020). On the other hand, peak intensification at 1350 1000 cm21 of pomegranate peel

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extract-chitosan films could be representative of high amounts of polyphenol compounds (Gull et al., 2021). Although Soltanzadeh et al. (2022) indicated that application of pomegranate peel extract in chitosan nanoparticles had no changes in IR bands. This fact showed that extract was effectively entrapped in chitosan nanoparticles (Soltanzadeh et al., 2022). Zeng et al. (2021) explained that small dots and corrugation in chitosan and pomegranate peel films are due to the formation of hydrogen bonds between pomegranate peel and chitosan and the presence of insoluble particles. Also, the combination of pomegranate peel and chitosan makes some change in crystalline structure and leads to an increase in amorphous properties of prepared chitosan film (Pirsa et al., 2020; Zeng et al., 2021).

7.6.10 Thermogravimetric analysis and differential scanning calorimetry of pomegranate-chitosan-based films The thermogravimetric analysis and differential scanning calorimetry (DSC) showed weight loss and thermal stability of produced films (Surendhiran et al., 2020). In the research of Abbas and Abdul-Rahman (2020), three weight loss in chitosan-starch film incorporated natural extracts were recorded at 135°C, 135°C 320°C, and 320°C above 400°C, which respectively represent moisture and volatile removal, glycerol and chitosan amino unite decomposition as well as starch plasticization, and -CH2OH group decompositions. In the research of Surendhiran et al. (2020) on active nanofibers encapsulated with pomegranate peel extract in chitosan-polyethylene oxide, thermal degradation at 170°C 240°C (phytochemical decomposition) and 285°C was observed (Surendhiran et al., 2020). Although, Soltanzadeh et al. (2021) manifested that encapsulated pomegranate peel extract-chitosan nanoparticle films had no thermal degradation (Soltanzadeh et al., 2022). Cai et al. (2021) showed that the thermal stability of nanofibers containing pomegranate peel polyphenols was enhanced (Cai et al., 2021). Soltanzadeh et al. (2022) indicated that pomegranate peel extract-chitosan nanoparticle films had an exothermic peak in the DSC thermogram which showed the decomposition (Soltanzadeh et al., 2022).

7.7 Functional properties of pomegranate-chitosan-based films 7.7.1 Phenolic content of pomegranate-chitosan-based films Phenolic compounds, secondary metabolites of plants, have free-radical chelating activity during the oxidation process. As mentioned in the previous section, different parts of pomegranate contained phenolic

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compounds which are known as potential antioxidants. The presence of polyphenolic compounds such as ellagic acid, gallic acid, punicalagin, and catechin in pomegranate extracts resulted in an improvement in the phenolic activity of chitosan film (Kumar et al., 2021c).

7.7.2 Antioxidant property of pomegranate-chitosan-based films The presence of antioxidants in packaging materials could have preventive effects on the deterioration of fatty molecules and shelf life enhancement of packaged foods (Pirsa et al., 2020). The presence of phenolic compounds in plants had a direct relation to antioxidant activity (AbdulRahman and Abass, 2021; Qin et al., 2015). It was reported that the antioxidant activity of chitosan film was improved by the addition of pomegranate extract due to its biological functions (Kumar et al., 2021c). There are some factors known to be important in phenolic content and antioxidant activity variation including pomegranate cultivar, location and season of cultivation, method and time of pomegranate extraction, and type of extraction solvent (Cui et al., 2020; Surendhiran et al., 2020). In some studies, a reduction in total phenolic content of pomegranate-chitosan-based films has been reported as a reason of the interaction of phenolic hydroxyls of pomegranate peel with amine, amide, and hydroxyl groups presented in chitosan and resulted in a decrease in free phenolic hydroxyls (Bertolo et al., 2020).

7.7.3 Antimicrobial property of pomegranate-chitosan-based films Most studies agree that the antimicrobial activity of film could be correlated to antioxidant capacity (Abdul-Rahman and Abass, 2021; Kumar et al., 2021c). The literatures emphasize that the presence of active compounds, as well as sugars and vitamin C in natural extract, improves the antimicrobial activity of chitosan film (Abdul-Rahman and Abass, 2021). Although, the chitosan antimicrobial activity through electrostatic interactions of chitosan and bacteria, changes in nutrients for bacteria, inhibition of enzyme synthesis, loss of bacteria cellular material, chelating ability, acetic acid used in the preparation of chitosan film (lowering the pH) have been proved (Licciardello et al., 2018; Lozano-Navarro et al., 2017). Therefore the antimicrobial activity of films is related to different factors, including properties of matrix film, method of film production, and type of bacteria (Pirsa et al., 2020). Since apparent density will be

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enhanced in the presence of pomegranate extract and these increases lead to moisture reduction and promotion of oxygen barriers which indicated better antimicrobial activity. Furthermore, by these changes, the migration of antioxidant compounds to the cell of the microorganism would be facilitated (Abdul-Rahman and Abass, 2021). The application of natural compounds along with chitosan could promote the antimicrobial activity of film (Lozano-Navarro et al., 2017). The pomegranate peel with a high amount of polyphenols, flavonoids, tannins, and anthocyanins and also the ability to remove nutrients from microorganisms by chelating minerals, carbohydrates, and vitamins could show good antimicrobial activity (Pirsa et al., 2020). In addition, the presence of the bioactive compound in pomegranate (presence of OH groups) could lead to a rupture in the microorganism cell wall by reaction with protein or sulfhydryl groups, decrease in the fluidity of the membrane, and inhibition of the microbial enzyme (Nair et al., 2018a; Zeng et al., 2021). The inhibition activity of pomegranate peel in alginate-chitosan coating against Colletotrichum gloeosporioides was shown (Nair et al., 2018a). Yuan et al. (2015) showed the antimicrobial activity of chitosan film incorporated with 10 g/L pomegranate peel extract against S. aureus (Yuan et al., 2015). The resistance of Gram-negative bacteria in the presence of pomegranate extract in chitosan film was higher than Gram-positive bacteria owing to the multilayer cell wall structure of bacteria (Soltanzadeh et al., 2021; Yuan et al., 2015). Although in a study by Sarhana et al. (2020) chitosan-silver nanoparticles with pomegranate peel showed great antibacterial activity against both Gram-positive and Gram-negative bacteria (Sarhana et al., 2020).

7.8 Degradation of pomegranate-chitosan-based films The thermal degradation without melting at high temperatures is introduced as a problem of chitosan. The low thermal stability of chitosan could be improved by the addition of another compound which resulted from molecular interaction (Surendhiran et al., 2020). Although, anthocyanins are stable at low pH (pH 3 7) by increasing the pH to 7 their degradation happens. Therefore, the color degradation of chitosan film contained pomegranate extract at high pH and under light is probable (Lozano-Navarro et al., 2017). Thus, because antioxidants and sugars present in pomegranate are colored, color changes could be possible during food storage (Abbas and Abdul-Rahman, 2020). These changes in color and antioxidants could be related to pH increases during storage as well as

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environmental conditions including oxygen and light (Lozano-Navarro et al., 2018; Yuan et al., 2015). It was indicated that by adding pomegranate peel to chitosan film, the water solubility was elevated which is related to hydrophilic components of pomegranate peel. By increasing the water solubility of film, its degradability will promote (Zeng et al., 2021). Storage modulus, the resistance to break down the polymer, of films containing pomegranate peel was decreased. There is some evidence that the application of natural compounds in chitosan-based films and less crystalline materials is responsible for the decrease in storage modulus (LozanoNavarro et al., 2017).

7.9 Application and effects of pomegranate-chitosan-based films on foods 7.9.1 Application of pomegranate-chitosan-based films in fruits and vegetables Fruits and vegetables play an important role in a balanced diet that affects the health of the body (Nair et al., 2020). Fruits and vegetables have excellent sources of essential vitamins, minerals, and fiber. Fruits and vegetables also provide a variety of phytochemicals that act as antioxidants, including flavonoids, phytoestrogens, and antiinflammatory agents (Slavin and Lloyd, 2012). Perishability of fruits and vegetables includes ethylene production, increased respiratory rate, expression of cell wall degrading enzymes, insect infestation, postharvest fungal diseases, and other pathological breakdowns postharvest life that lead to the loss of quality and decrease in shelf life (Nair et al., 2020). The lack of proper handling methods and private and public infrastructure has led to an increase in food waste and food loss of these important food commodities, as well as their components (Sagar et al., 2018). For example, Food and Agriculture Organization has reported approximately 1.3 billion metric tons of the edible food produced—one-third of the total—are lost and wasted globally and fruits and vegetables have the highest rates of loss and waste among all types of the food, reaching up to 60% (Sagar et al., 2018). Tayel et al. (2016), fungal chitosan from Mucor rouxii and methanol extract pomegranate peel was developed to evaluate the postharvest pathogens, Penicillium digitatum and Penicillium italicum of citrus fruits. Fruits were dipped in the prepared coating solution containing chitosan/ pomegranate peel extract composite. The appearance of fungal decay was

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monitored after storage for 3, 7, 10, and 14 days. The addition of chitosan to the coating material in combination with pomegranate extract prevented the coated citrus fruits from spoiling with green and blue molds over a 14-day storage period. Most of the active compounds in pomegranate peel extract have been reported to be alkaloids and tannins. It has been reported that the fungistatic or fungicidal activity of chitosan may be enhanced by combination with other antimicrobial agents and treatments (Tayel et al., 2016). Nair et al. (2020) evaluated the effect of 1% chitosan and 2% alginate enriched with 1% pomegranate peel extract on the quality of guavas, respiration rate, ripening index, and instrumental color values for 20 days at 10°C (Nair et al., 2020). In chitosan as well as in alginate incorporation of pomegranate peel extract resulted in further restriction in the increase of ripening index. The respiration rate of the samples in various coating treatments was delayed. The coatings could increase CO2 to significant levels and the quality of the fruit by altering the internal atmosphere. The addition of pomegranate peel extract could also cause a slight change in CO2 production and utilization of O2. Due to the antimicrobial and lipophilic properties of pomegranate peel extract, it enhanced the barrier properties of the coating and restricted gas diffusion. The study showed that 1% pomegranate peel extract in chitosan film could cause a delay in browning and the ripening process in guavas. Because of the action of ascorbic acid oxidase, ascorbic acid is reduced during the storage period and converted ascorbic acid to dehydroascorbic acid and phenol oxidase. Edible coating films have lower oxygen permeability and reduce oxygen levels, so the enzyme activity is reduced thus the oxidative damage of fruits and vegetables is decreased. The most retention of total phenol was determined in chitosan-coated with pomegranate peel extract samples. Edible coating films lead to the accumulation of both the phenolic and ascorbic acid content and thereby the antioxidant activity increases. During the ripening process, flavonoids are either converted to secondary phenolic compounds or attacked by enzymes and depleted. At the end of the storage period, chitosan enriched with pomegranate peel extract and chitosan-coated samples had higher levels of both phenolic and flavonoid content than other samples. The highest antioxidant activity in terms of DPPH and FRAP was obvious in the case of chitosan-extract samples (Nair et al., 2018b). Kumar et al. (2021a) have developed a chitosan-pullulan composite edible coating prepared with pomegranate peel extract on the shelf life of

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mango. The coated fruits reduced the weight loss and maintained total soluble solids (TSS), acidity, pH, and sensory quality (freshness, color, taste, and texture) by treatment. In addition, firmness, phenolic content, and antioxidant activity retained coated mango fruits as compared to the control (Kumar et al., 2021a). To check for potential application of the active packaging, nanochitosan emulsion coatings containing pomegranate peel extract were tested for the shelf life extension of apricot fruit (Gull et al., 2021). Results revealed that apricot fruit coated incorporated with 1% pomegranate peel extract showed significantly reduced decay incidence and weight loss, effectively retained DPPH radical scavenging activity and ascorbic acid, kept titratable acidity, and retained firmness at a higher level than untreated fruit. Nanochitosan coatings containing 1% pomegranate peel extract successfully managed to slow down total psychrophilic bacteria, yeast, and mold count during storage. Findings suggest that chitosan coatings enriched with pomegranate natural peel extract could maintain postharvest quality and extend the shelf life of apricot. Duran et al. (2016) evaluated the efficacy of chitosan coatings as a carrier of pomegranate extract to maintain the quality and extend the shelf life of fresh strawberry (Duran et al., 2016). The use of chitosan and chitosan 1 pomegranate extract film coatings improved the stability of pH, TSS content, and texture of fruits. Chitosan coatings with pomegranate extract were effective against aerobic mesophilic bacteria, yeast, and mold growth on fruits. It has been reported that pomegranate fruit has phenolics, pigments, citric acid, and punicalagin compounds and is effective against mold due to the phenol, tannins, and flavonoid content. Nair et al. (2018a) showed that the usage of chitosan coatings enriched with 1% pomegranate peel extract on capsicum could restrict changes in weight loss, firmness, color, ascorbic acid, and chlorophyll in capsicum while inhibiting the growth of fungal pathogen C. gloeosporioides under cold storage conditions (Nair et al., 2018a). Araújo et al. (2018) have developed the effect of edible cassava starch chitosan coatings incorporated with rosemary pepper essential oil and pomegranate peel extract on the shelf life of tomatoes during storage at 25°C. The analyzed physicochemical properties were not exhibited significantly different between the coated and uncoated tomatoes on day 12 of storage. However, the coatings lowered the weight loss and TSS content, demonstrating their effectiveness in delaying ripening (Araújo et al., 2018).

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Kumar et al. (2021b) reported an improvement in the physicochemical properties of green bell pepper with chitosan-pullulan composite edible coating enriched with pomegranate peel extract during a storage period of 18 days at room and cold temperatures (Kumar et al., 2021b). This packaging resulted in a considerable reduction in browning, maintained TSS, titratable acidity, pH phenolic content, flavonoid content, antioxidant activity, firmness, and sensorial attributes. Due to barrier properties against water loss, reducing synthesis, and control enzymatic activity (PPO/POD) the weight loss of bell peppers during the storage period was retarded. The TSS reduction of the coated bell pepper was due to slowdown synthesis, hydrolysis conversion of starch to simple sugar, metabolic activity, water loss, and hydrolytic enzyme activity, during the storage period. The acidity and pH of bell peppers were maintained during the storage period. It can be due to a reduction of respiration rate, metabolic activity, control enzymatic activity, and anthracnose activation of defense enzymes. They have also reported that chitosan-pullulan composite edible coating has reduced the rate of color. It might be due to the barriers against gases and water and lower respiration rates. Kumar et al. (2021a) demonstrated the use of chitosan-pullulan composite edible coating enriched with pomegranate peel extract to extend the shelf life of tomatoes over 18 days of storage. The authors reported improved postharvest quality such as weight loss, maintained TSS, titratable acidity, color, pH, retained higher phenolic content, flavonoid content, and antioxidant activity and sensory characteristics throughout the storage. Minimized degradation of antioxidant compounds can be due to a decrease in the microbial contamination, activity of polyphenol oxidase, lipid oxidation, peroxidase enzyme, and reduction of the respiration rate throughout storage.

7.9.2 Application of pomegranate-chitosan-based films in the meat and seafood industry Fresh meat and its products are an important protein source in the human diet worldwide. Meat is an ideal medium for microbial degradation and lipid oxidation due to its intrinsic parameters, such as high level of moisture and fat content, availability of nutrients, and optimal pH range (Min et al., 2021). Min et al. (2021) showed that the edible1.5% chitosan film incorporated with 4.0% pomegranate peel extract increased the shelf life of beef from 4 to 7 days (Min et al., 2021). The increase in the concentration of

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pomegranate peel extract in this packaging enhances the film’s thickness, water solubility, and antimicrobial properties against Pseudomonas aeruginosa and S. aureus. Organic compounds have polar functional groups, such as hydroxyl, amines, carbonyl, and carboxylic groups, which can enhance the polarity of water and thus improve the hydrophilicity of the film when incorporated in. When the concentration of pomegranate peel extract was increased, a higher absorption peak in the hydroxyl group, carbonyl group, amide group, and the carboxylic group was seen. Phenolic compounds can inhibit the growth, survival, and proliferation of bacteria by disrupting the bacterial cell membranes. Adhesive binding, protein and cell wall adhesion, enzyme inhibition, and migration of the polyphenol compounds into the cell wall and/or genomic DNA can inhibit cellular functions. Mehdizadeh et al. (2020) reported a prolonged shelf life of beef coated with a chitosan-starch film containing pomegranate peel extract and Thymus kotschyanus essential oil during a storage period of 21 days at 4°C. The results of this study indicated composite films containing chitosanstarch-peel extract (1%) and essential oil (2%) had the highest antilisteria effect and this composite could inhibit lipid oxidation and bacterial counts. During the storage time, microbial spoilage, muscle discoloration, slimy texture, and off-odor were acceptable (Mehdizadeh et al., 2020). Surendhiran et al. (2020) used pomegranate peel extract immobilized electrospun active nanofibers fabricated using chitosan/polyethylene oxide. Increasing the content of chitosan in the blends improves their water-resistance nanofiber mat and it would be more suitable for packaging in the food industry. Although chitosan increase led to a reduction in moisture content, peel extract increased water holding due to its hydrophilicity. Nanofibers composed of chitosan/polyethylene oxide showed weight loss only at above 200°C and have thermal stability up to B400°C. They revealed that for electrospinning nanofibers chitosan was a good alternative due to it being economic, simple, and possible to operate on a commercial scale. Chitosan/polyethylene oxide/peel extract active nanofibers effectively inhibited Escherichia coli O157:H7 growth on beef samples stored at 4°C and 25°C and increased the shelf life (Surendhiran et al., 2020). Abbas and Abdul-Rahman (2020) evaluated the antibacterial activity of chitosan incorporated with pomegranate peel on chicken breast packaging during the 15 days of storage. The findings of the microbiological tests showed that the total count of bacteria, colon bacteria, and

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psychotropic bacteria decreased during the storage period. Sensory characteristics of chicken coated with extracted chitosan membrane are more accepted in general shape, color, smell, and texture than chitosan-coated chicken breast and noncoated chicken breast. The inhibitory action of chitosan against cold-loving bacteria as the main bacteria responsible for spoilage in the refrigerator may be due to the inactivation of polysaccharides of the outer layer of Gram-negative bacteria and antimicrobial activity of pomegranate peel extract to the sensitivity of phospholipids in cell membranes, inhibition microbial enzyme systems, and leakage of cellular components due to increased cell permeability (Abbas and AbdulRahman, 2020). Effect of pomegranate juice dipping and the juice-chitosan coatings incorporated with or without the Zataria multiflora Boiss essential oil was investigated on the shelf life of chicken breast meat during refrigerated storage for 20 days. All total viable counts, Pseudomonas spp., lactic acid bacteria, Enterobacteriaceae, psychotropic bacteria, and yeasts molds during the storage period of the treatments were significantly decreased. Peroxide value, thiobarbituric acid reactive substances, and protein oxidation were significantly lower than control. Sensory evaluation showed that pomegranate juice had a pleasant effect on sensory attributes including taste, color, odor, texture, and overall acceptability in chicken breast meat. This can be correlated to the unique flavor of pomegranate juice. Pomegranate juice can be described as having a sweet and sour taste, musty/earthy notes with fruity odors, and an astringent mouth. It was suggested pomegranate juice can be a replacement for synthetic preservatives and synthetic flavorings in chicken breast meat as well as other kinds of meat products. The coating could reduce phenolic degradation of the juice and maintain nutritional quality so allowing longer storage at refrigerated temperatures (Bazargani-Gilani et al., 2015). In another study, they investigated the influence of aforementioned coatings on chemical and sensory quality of chicken breast meat during frozen storage at 218°C for 6 months. In all treatments total plate counts, peroxide value, thiobarbituric acid value, and carbonyl content were significantly lower than control until 60 days of storage. They revealed that pomegranate juice can be used as a natural antioxidant, natural antimicrobial, flavoring, texturizing, and coloring additive in chicken meat and other meats (Bazargani-Gilani et al., 2016). The application of fungal chitosan coating films incorporated with pomegranate peel extract on Nile tilapia fillets has been studied during

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cooled storage at 4°C for 30 days (Alsaggaf et al., 2017). The sensory properties of odor, texture, color, and overall quality of coated samples showed higher preferences. Maintaining the good microbiological, chemical and sensorial quality, and shelf life extension reported in fish fillets coating with chitosan and chitosan plus pomegranate peel extract. Protein decomposition by microorganisms of fish flesh is due to the TVB-N value increasing; thus the reduced microbial counts in the coated samples could explain the matching TVB-N patterns, during the storage period of 30 days. The addition of pomegranate peel extract to chitosan film could further inhibit protein decomposition by bacteria to its antimicrobial action. Saki et al. (2018) presented the effect of chitosan-gelatin composite and bilayer coating combined with pomegranate peel extract on the quality properties of Belanger’s croaker fillet stored in the refrigerator. The result indicated that this composite retarded the growth of psychrotrophic bacteria, mesophilic bacteria, and Enterobacteriaceae in Belanger’s croaker fillet compared to the control samples. The quality properties of croaker coated with chitosan-gelatin incorporated with peel extract were better than films from chitosan-gelatin coating or peel extract alone during the storage, revealing that there is a synergistic action between chitosangelatin coating and pomegranate peel extract (Saki et al., 2018). Effect of chitosan and locust bean gum combined with pomegranate peel extract on the quality maintenance of white shrimps during cold storage investigated by Licciardello et al. (2018). Findings proved the effective synergy of pomegranate peel extract with chitosan at reducing microbial spoilage, Pseudomonas spp., and the production of volatile bases in shrimps during storage. Chitosan alone and chitosan-peel extract maintained the psychrotrophic microbial load below 7 log CFU/g for 6 days and about 2 log units of Pseudomonas spp. count reduced. Locust bean gum alone did not exhibit an antimicrobial effect, while pomegranate peel extract allowed the reduction of psychrotrophic load by 1 log unit. After 6 days, TVB-N values in shrimps treated with chitosan incorporated with pomegranate peel extract were as low as the levels measured in the noncoated samples after 2 days. Pomegranate peel extract did not exhibit any significant positive effect on the visual quality of shrimps. Yellow-amber hue change was reported in pomegranate peel extract coated samples due to the natural pigments contained in the extract (Licciardello et al., 2018). Berizi, et al. (2018) evaluated the combined effects of chitosan combined with different levels of pomegranate peel extract on the overall quality of rainbow trout during frozen storage for 6 months. The best

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performance in antimicrobial efficacy (psychrophilic, lactic acid bacteria, and mold values) and preventing the oxidation of fats and proteins were in the chitosan combined with the high level (4%) of pomegranate peel extract. But due to its undesirable color, the chitosan incorporated with 2% peel extract was preferred. Textural analysis of fish samples showed that chitosan and peel extract reduced the adverse effects of freezing because chitosan was a barrier to reducing the drip loss and water holding capacity (Berizi et al., 2018).

7.10 Conclusion The application of pomegranate extract in chitosan film, especially in nanoencapsulation form, could improve the film characteristics, including antimicrobial, antioxidant, mechanical, water vapor and oxygen permeability properties, and thermal stability. These pomegranate-chitosan-based films maintain microbiological, chemical, and sensory attributes in food and increase the food shelf life using safe preservatives from natural origins.

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CHAPTER 8

Chitosan: a potential antimicrobial agent to enhance microbial safety and shelf life of salad dips and ethnic foods Amin N. Olaimat1, Murad Al-Holy1, Anas A. Al-Nabulsi2 and Tareq M. Osaili2,3 1

Department of Clinical Nutrition and Dietetics, Faculty of Applied Medical Sciences, The Hashemite University, Zarqa, Jordan Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, Jordan 3 Department of Clinical Nutrition and Dietetics, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates 2

8.1 Introduction Ethnic foods are the food products from countries other than the home market (Marletta et al., 2010). Another definition considers the food is ethnic, it should be culturally and socially accepted by consumers outside the respective its origin (Kwon, 2015). Ethnic foods may include different types of products such as ready-to-eat foods, cooking, table sauces and dips, snacks and accompaniments, meal kits, and seasonings ingredients (Fusco et al., 2015). The consumption of ethnic foods is growing worldwide due to the high immigration rates, increased international travel, multicultural eating habits (culinary tourism), and globalization of food supplies. However, foodborne outbreaks and food recalls associated with ethnic foods are increasing in the recent years (Fusco et al., 2015). Chitosan is a suitable antimicrobial agent that may enhance the microbial safety and quality of fresh and processed foods by extending their shelf life and inhibiting foodborne pathogens due to its unique bioactivity and biodegradability properties (Le Tien et al., 2003). The objectives of this chapter are to review the studies that evaluated the antimicrobial activity of chitosan in ethnic foods and to discuss the factors affecting its inhibitory effect.

Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00003-X

© 2023 Elsevier Inc. All rights reserved.

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8.2 In vitro antimicrobial activity of chitosan against spoilage and pathogenic microorganisms The medium, storage conditions, and structure of chitosan play a major role in its antimicrobial activity. It was found that mild hydrolysis of chitosan using oxidative-reductive degradation, crude papaya latex, and lysozyme improved its antimicrobial activity against spoilage yeasts, including Saccharomycodes ludwigii, Zygosaccharomyces bailii, and Saccharomyces cerevisiae, in malt extract broth supplemented with 2.5 g of glucose at room temperature. However, the extensive degradation of chitosan showed negligible antimicrobial activity. In saline solution containing 0.9% NaCl with a pH value of 6.4, chitosan and hydrolyzed chitosan were not active against spoilage bacteria (Pseudomonas fragi, Bacillus sp.) in nutrient broth at 30°C and yeasts (Cryptococcus albidus) in malt extract broth supplemented with glucose at 25°C by 1 hour. However, native chitosan or hydrolyzed chitosan at a concentration of 0.05% g reduced numbers of Candida sp. by approximately 2 log CFU, while the hydrolysis process enhanced the antimicrobial activity of chitosan against Rhodotorula sp. to reach 1 log CFU/ mL compared to 0.5 log CFU/mL reduction obtained by addition of native chitosan (Rhoades & Roller, 2000). In another study, antimicrobial characteristics of chitosan prepared from red crab chitin that was dominated by 45%, 50%, 55%, and 60% NaOH solutions were tested against food spoilage microorganisms in liquid media. All chitosan types at 100 ppm inhibited the growth of Lactobacillus plantarum up to 20 hours, while at $ 20 ppm, numbers of Serratia liquefaciens in nutrient broth and Lactobacillus fructivorans in De Man, Rogosa and Sharpe (MRS) broth were significantly reduced up to 24 hours. However, chitosan at 70 ppm completely inhibited the growth of Z. bailii in YMPG broth (yeast extract 3 g/L, malt extract 3 g/L, peptone 5 g/L, glucose) up to 24 hours (Oh et al., 2001). Tsai et al. (2000) reported that the minimum inhibitory concentrations (MICs) of a mixture of chitooligosaccharide produced by degradation of shrimp chitosan using cellulose enzyme against Escherichia coli, Shigella dysenteriae, Salmonella Typhimurium, Aeromonas hydrophila, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, Vibrio cholerae, and Vibrio parahaemolyticus in nutrient broth ranged from 5 to 29 ppm which were significantly lower than MICs of the native chitosan which ranged from 20 to 1000 ppm after 2 days at 37°C (28°C for A. hydrophila). Chitosan at 0.1 5.0 g/L completely inhibited the growth of L. fructivorans in MRS

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medium with pH of 6.0 for up to 45 hours at room temperature. In contrast, chitosan at 5.0 g/L was not inhibitory against Salmonella enteritidis in Trypticase soy broth at pH 7.0 (Roller & Covill, 2000). The in vitro antimicrobial activities of high- and low-molecularweight chitosan were evaluated against bacteria (S. aureus, E. coli, L. bulgaricus, and Lactococcus cremoris) and yeasts isolated using broth dilution method. The MICs of chitosan against tested bacteria ranged from 4 to 200 μg/mL. The MICs of low-molecular-weight chitosan against E. coli, S. aureus, L. cremoris, L. bulgaricus, and yeasts were 10, 8, 200, 200 and 1250 μg/mL, respectively, while the corresponding MICs of highmolecular-weight chitosan were 60, 60, 200, 100, and 1500 μg/mL, respectively. E. coli, S. aureus, and yeasts were more susceptible to lowmolecular-weight chitosan than high-molecular-weight chitosan. However, the MICs of chitosan against L. bulgaricus and L. cremoris were more resistant than E. coli and S. aureus (El-Dahma et al., 2017).

8.3 Antimicrobial activity of chitosan in fresh produce and salads In addition to the food type, the antimicrobial activity of chitosan depends on the organic acids used in the preparation of coatings or films. Jovanovi´c et al. (2016) prepared antimicrobial edible chitosan coatings using acetic and lactic acids and investigated their antimicrobial activity against L. monocytogenes on shredded black radish. They found higher activity when bacterial cells were treated with the chitosan coating prepared with acetic acid compared to lactic acid. Chitosan coatings at 1% and 0.5% prepared with 1% (v/v) acetic acid reduced numbers of L. monocytogenes on shredded black radish by 3.1 and 2.6 log CFU/g, respectively, immediately after chitosan addition, while 2.9 and 2.4 log CFU/g reduction were obtained when 1% and 0.5% chitosan coatings prepared with 1% (v/v) lactic acid compared to control. Salad dressing containing highmolecular acid-soluble chitosan at concentrations of 0.375% 1.5% inhibited the growth of microflora of fresh tomato/cucumber salad and vegetable vinaigrette and extended their shelf life during storage temperature of 4°C from 12 to 78 hours (Bugayets et al., 2020). In another study, the sprayed chitosan glutamate on the shrimp that was used to prepare mayonnaise-based shrimp salad at a level of 9 mg/g inactivated the spoilage flora, including total viable count, lactic acid bacteria, and yeasts count by 4 log CFU/g by 28 days of storage at 5°C (Roller & Covill, 2000).

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8.4 Antimicrobial activity of chitosan in meat and poultry products A chitosan coating prepared with 2% chitosan and 0.5% acetic acid solution was able to reduce numbers of S. Typhimurium and E. coli by O157:H7 by 1.7 and 1.1 log CFU/g, respectively, directly after dipping in the emulsion and the numbers remained constant till the end of storage period at 4°C. This treatment did not affect the overall sensory score of chicken fillets (ElKhawas et al., 2020). In another study, Olaimat and Holley (2016) found that 0.2% k-carrageenan/2.0% chitosan-based coatings prepared with 1.5% (v/v) malic acid had stronger antimicrobial activity against L. monocytogenes on vacuum-packed cooked, cured roast chicken with a reduction of 1.5 log CFU/g after 70 days of storage at 4 °C, compared to 0.6 log CFU/g when the coating prepared with 1.5% (v/v) acetic acid and similar results were obtained against aerobic bacteria. However, Olaimat et al. (2014) and Olaimat and Holley (2015) reported that % k-carrageenan/chitosan-based coatings prepared with 1.0% (v/v) acetic acid reduced numbers of Campylobacter jejuni and Salmonella enterica on vacuum-packed chicken breasts by 1.9 and 0.6 log CFU/g at 4°C for 21 days. Shaltout et al. (2019) indicated that treatment of chicken breast fillets with nonchitosan coating (10 mg/mL) completely eliminated Aspergillus flavus after 3 days of storage at 7°C and improved the stability of color, odor and texture of chicken breast fillets for up to 12 days. However, addition of crude onion extract to chitosan coating reduced its antimicrobial activity.

8.5 Antimicrobial activity of chitosan in juices In juices such as apple-elderflower juice , native or degraded chitosan at a concentration of 0.03% g/L suppressed the growth of yeast in the appleelderflower juice (pH 3.3) for up to 13 days at 7°C. While both types of chitosan were able to reduce the counts of total bacteria and lactic acid bacteria by 2 log CFU/mL compared to control at day 4 of storage. After that, however, there were no differences between juices supplemented with chitosan and controls where bacterial numbers grew to reach 7 log CFU/mL by 8 days (Rhoades & Roller, 2000). In another study, Abd and Niamah (2012) reported that addition of 1% chitosan to apple juice reduced numbers of total bacteria by 0.7 log CFU/mL by 30 days of storage at both 4°C and 30°C, while numbers of yeast and mold were reduced by 1.8 and 0.9 log CFU/mL at 4°C or 30°C, respectively, after

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30 days. Furthermore, chitosan enhanced the juice quality by reducing Brix value, enzymatic and nonenzymatic browning and increasing pH values (3.71 4.22). Chitosan also showed a good antimicrobial activity against foodborne pathogens in tropical fruit juices. Furthermore, chitosan at concentrations of 1.5 2.0 mg/mL completely eliminated the initial inoculated numbers of S. Typhimurium (6.7 log CFU/mL) and E. coli O157:H7 (6.4 log CFU/mL) in orange, watermelon, pineapple, and mixed fruit juices during 1 3 days of storage at both 4°C and 28°C. L. monocytogenes (5.8 log CFU/mL) and S. aureus (5.8 log CFU/mL) were also eliminated during 2 6 days of storage at the same storage temperatures (Omogbai & Ikenebomeh, 2019). Chitosan at 0.1 5.0 g/L inhibited the growth of L. fructivorans in apple juice at pH 3.4 for up to 45 hours at 25°C (Roller & Covill, 2000). Sarwar et al. (2014) found that chitosan at 0.5% 1.0% inhibited the total bacterial and fungal counts in orange juice up to 8 days at room temperature and extended its shelf life.

8.6 Antimicrobial activity of chitosan in dips and pastes In hummus (pH 4.2), ,0.1% (w/w) native and degraded chitosan showed no antimicrobial activity against the natural microflora at 7°C. However, native and hydrolyzed chitosan at a concentration of 0.5% reduced the total viable bacteria and the lactic acid bacteria counts by approximately 4 log CFU/g compared to control by 6 days of storage at 7°C and there were no significant differences in the hummus samples treated with native chitosan or hydrolyzed chitosan (Rhoades & Roller, 2000). The antimicrobial activity of chitosan alone or in combination with garlic was evaluated against Salmonella spp., E. coli O157:H7, and L. monocytogenes in hummus dip kept at 4°C for 28 days, 10°C for 21 days, or 25°C for 7 days. Addition of 0.5% and 1.0% and chitosan decreased numbers of tested pathogens by 0.6 2.3 and 1.6 2.9 log CFU/g, respectively. Yet, the addition of 1% garlic did not enhance the antimicrobial activity of chitosan against tested pathogens in hummus (Osaili et al., 2022). The antimicrobial activity of chitosan polysaccharide and chitosan oligosaccharide against L. monocytogenes and S. Typhimurium in oil-in-water emulsion model (20% corn oil, 1.5% Tween 20, 1.5% Trypticase soy broth, 0.58% acetic acid) at 10°C and 25°C for 4 days was evaluated (Zivanovic et al., 2004). It was reported that the addition of 0.1% 0.7% of chitosan polysaccharide significantly enhanced the bactericidal effect of acetic acid in the oil-in-water emulsion where the initial count

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(6.5 7.0 log CFU/g) in the emulsions with 0.58% acetic acid and 0.7% chitosan polysaccharide was reduced to below the detection limits after 24, 48, 72, or 96 hours for L. monocytogenes 310, S. Typhimurium DT104 2576, S. Typhimurium DT104 2486, or L. monocytogenes Scott A, respectively, at 25°C. They also reported that chitosan polysaccharide with a molecular weight of 150 kDa showed stronger bactericidal activity than chitosan oligosaccharide and its antimicrobial activity was reduced at lower temperature (10°C). At this temperature, the reduction of microbial load was delayed for approximately 24 hours compared with 25°C. These results suggest that addition of chitosan polysaccharide would be effective against foodborne pathogens in oil-in-water emulsions including mayonnaise. In another study, addition of chitosan glutamate at a level of 3 g/L to mayonnaise prepared with 0.16% acetic acid reduced numbers of L. fructivorans by 5 log CFU/g and numbers remained below the detection levels for 8 days at 5°C or 25°C. Numbers of Z. bailii were also reduced by about 1 2 log CFU/g at the first day at 25°C and an overall of growth delay of 2 days has been reported. Nonetheless, a regrowth of Z. bailii took place after two days. However, chitosan glutamate was not active against Z. bailii at 5°C. Addition of chitosan also accelerated the inhibition of S. enteritidis after 6 days to reach undetectable levels at 8 days of storage at 5°C and at 2 days at 25°C. However, surprisingly, when using 1.25 lemon juice in the preparation of mayonnaise, the antimicrobial activity of chitosan waned at both storage temperatures against the tested organisms except for S. enteritidis, where approximately 0.5 1 log CFU/g reduction was reported (Roller & Covill, 2000). In another study, chitosan at 100 1000 ppm accelerated death of L. fructivorans and Z. bailii in mayonnaise stored by reducing their numbers by 4 6 log CFU/g after 4 weeks of storage at 25°C (Oh et al., 2001). It has been noticed that chitosan at 0.5% 1.0% extended the shelf life of ginger paste by reducing total bacterial and fungal counts for up to 8 days at room temperature (Sarwar et al., 2014). The total microbial count was not detected in pasteurized palm sap for 3 and 4 days by addition of 0.5% and 1.0% chitosan, respectively. Both concentrations also reduced the mold and yeast count (up to 3 days), lactic acid bacteria (up to 4 days) to undetectable levels in pasteurized palm sap (Naknean et al., 2015).

8.7 Antimicrobial activity of chitosan in dairy products Addition of chitosan to dairy products formulations has been tested for its antimicrobial activity as well as its effect on physiochemical and

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organoleptic properties. Commercially available powdered chitosan or nano-powdered chitosan at concentration levels of 0.3% 0.7% were added to cholesterol-reduced yogurt and the results showed that lactic acid bacteria (LAB) was reduced by ,1.0 log compared to control, which elevated the pH values of treated yogurt from 4.3 to 4.5, compared to 3.9 in control samples after 20 days of storage at 4°C. The color parameters a (redness) and b (yellowness) of yogurt were not significantly affected by the both chitosan types; while the L (lightness) values and overall acceptability of yogurt treated with $ 0.5% nano-powdered chitosan were significantly reduced compared to control (Seo et al., 2009). In another study, Tsai et al. (2000) evaluated the antimicrobial effect of a mixture of chitooligosaccharide produced by degradation of shrimp chitosan using cellulose enzyme. Higher antimicrobial activity of the mixture against E. coli O157, L. monocytogenes, S. Typhimurium, and S. aureus at 4°C than at 37°C was obtained. The reduced effect at high temperature could be possibly due to the interference with milk components at high temperatures the antimicrobially activity of chitosan. Additionally, lowtemperature stress may also change the structure of cell membrane and reduce the growth rate of the bacteria and increase the antimicrobial susceptibility of bacteria to chitooligosaccharide mixture. Furthermore, the mesophilic and psychrotrophic counts in raw milk treated with 0.24% 0.48% (wt/vol) of the oligosaccharide mixture were reduced by $ 3.0 log CFU/g at 4°C after 12 days, and consequently the shelf life of raw milk at 4°C was extended by at least 4 days and this stability was manifested by little changes in pH values. The mixture also inhibited the growth of Salmonella spp. and quickly reduced numbers of Staphylococcus spp. in raw milk. The efficiency of chitosan in inhibiting the growth of spoilage microorganisms in Mozzarella cheese stored at 4°C was evaluated by Altieri et al. (2005) who reported that chitosan at a concentration of 0.075% inhibited the growth of coliforms and Pseudomonas spp., while no antimicrobial effect was detected against Micrococcaceae and LAB. The predicted analysis showed that addition of chitosan prolonged the shelf life of Mozzarella by 0.8 2 days. Furthermore, no significant differences between the sensory properties and pH values of chitosan-treated Mozzarella and chitosan-free samples were reported. However, the addition of chitosan enhanced the texture cheese samples. In general, infant formulae and baby foods are prepared under strict hygienic practices. to preclude the growth of opportunistic pathogens such as Cronobacter sakazakii. This pathogen could be transmitted through infant formula and baby

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foods because of its extreme capability to withstand and survive in low water activity foods. Consequently, this pathogen could pose a potential risk to new born infants. Hence, infant-compatible preventive mesures should be applied to reserict the growth of Cronobacter and other pathogens in reconstituted infant formula and baby foods. When used at concentrations of 1.5 and 2.0%, chitosan resulted in 2 3 log reductions in the C. sakazakii count in infant formula and wheat based infant cereal after 6 h of handling at room temperature (Al-Holy et al., 2014). The antimicrobial activity of chitosan as an edible coating at concentrations of 5, 10, and 15 mg/mL or incorporated into the formulation of Coalho cheese curds at concentrations of 1, 2, and 4 mg/g was evaluated against S. aureus. Coating at 10 and 15 mg/mL reduced numbers of S. aureus by 2.0 and 2.7 log CFU/g after 16 days of storage at 10°C, while when chitosan was added to the mass of cheese at 2 and 4 mg/g, S. aureus numbers were reduced by 0.5 and 1.4 log CFU/g after 16 days at 10°C. It is worth mentioning that these treatments of chitosan did not affect the physicochemical and organoleptic characteristics of the cheese (de Melo Barros et al., 2019). Low-molecular-weight chitosan was added to the milk used in manufacturing of Kariesh cheese to test the inhibitory effect against E. coli, S. aureus, and yeast. Numbers of E. coli, S. aureus, and yeast in Kariesh cheese treated with 0.03% and 0.05% chitosan decreased by 3.7 3.9, 1.3 1.4, and 3.3 3.4 log CFU/g after 10 days of storage at 4°C for the corresponding microorganisms compared with the control (without chitosan). On other hand, addition of chitosan did not affect the growth of LAB in cheese (El-Dahma et al., 2017).

8.8 Conclusions Chitosan is a natural carbohydrate macromolecule prepared with deacetylated chitin which is a major constituent of the crustacean shells. Chitosan is inhibitory against a wide range of foodborne and spoilage microorganisms and it has gained a high consideration to be used as a food preservation agent due to its natural origin and high antimicrobial properties. On other hand, the consumption of ethnic foods has increased worldwide and these foods are becoming popular in most countries. However, ethnic foods have been linked with large numbers of foodborne outbreaks and food recalls. Studies showed that chitosan alone or in combination with other antimicrobials may enhance the microbial safety and extend the shelf life of salad dips and ethnic foods.

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References Abd, A. J., & Niamah, A. K. (2012). Effect of chitosan on apple juice quality. International Journal of Agriculture and Food Science, 2(4), 153 157. Al-Holy, M. A., Sabbah, K., Osailii, T. M., & Qatatsheh, A. (2014). Inactivation of Cronobacter sakazakii in infant formula using chitosan and lactic acid. Journal of Food Processing and Preservation, 39(6), 1229 1234. Available from https://doi.org/10.1080/ 19476337.2020.1772887. Altieri, C., Scrocco, C., Sinigaglia, M., & Del Nobile, M. A. (2005). Use of chitosan to prolong mozzarella cheese shelf life. Journal of Dairy Science, 88(8), 2683 2688. Available from https://doi.org/10.3168/jds.S0022-0302(05)72946-5. Bugayets, N. A., Usatikov, S. V., Lyubimova, L. V., Tereshchenko, I. V., Shantyz, A. K. H., & Miroshnichenko, P. V. (2020). A model for predicting microbiological and organoleptic indicators of salads during storage with the use of chitosan. IOP Conference Series: Earth and Environmental Science, 422012044. Available from https:// doi.org/10.1088/1755-1315/422/1/012044. de Melo Barros, D., de Moura, D. F., Rocha, T. A., Silva Santos, A. E., de Oliveira Silva, M. R., de Oliveira Ferreira, S. A., Bento da Fonte, R. A., & de C. Lima Machado, E. (2019). Coalho cheese with incorporated chitosan and as a coating: Effect on the viability of Staphylococcus aureus and sensory acceptance. Ciências Agrárias, 40, 3477 3492. El-Dahma, M. M., Khattab, A. A., Gouda, E., El-Saadany, K. M., & Ragab, W. A. (2017). The antimicrobial activity of chitosan and its application on kariesh cheese shelf life. Alexandria Science Exchange Journal, 38, 733 745. El-Khawas, K. M., Mashat, B. H., Attala, O. A., & Kassem, G. M. A. (2020). Control of Salmonella and Escherichia coli in chilled chicken fillets using chitosan and lactic acid. CyTA - Journal of Food, 18(1), 445 450. Available from https://doi.org/10.1080/ 19476337.2020.1772887. Fusco, V., Besten, H. M. W. D., Logrieco, A. F., Rodriguez, F. P., Skandamis, P. N., Stessl, B., & Teixeira, P. (2015). Food safety aspects on ethnic foods: Toxicological and microbial risks. Current Opinion in Food Science, 6, 24 32. Jovanovi´c, G. D., Klaus, A. S., & Nikˇsi´c, M. P. (2016). Antimicrobial activity of chitosan coatings and films against Listeria monocytogenes on black radish. Revista Argentina de Microbiologia, 48(2), 128 136. Available from https://doi.org/10.1016/j.ram.2016.02.003. Kwon, D. Y. (2015). What is ethnic food? Journal of Ethnic Foods, 2, 1. Available from https://doi.org/10.1016/j.jef.2015.02.001. Le Tien, C., Lacroix, M., Ispas-Szabo, P., & Mateescu, M. A. (2003). N-acylated chitosan: Hydrophobic matrices for controlled drug release. Journal of Controlled Release, 93, 1 13. Marletta, L., Camilli, E., Turrini, A., Scardella, P., Spada, R., Piombo, L., Khokhar, S., Finglas, P., & Carnovale, E. (2010). The nutritional composition of selected ethnic foods consumed in Italy. Nutrition Bulletin, 35, 350 356. Naknean, P., Jutasukosol, K., & Mankit, T. (2015). Utilization of chitosan as an antimicrobial agent for pasteurized palm sap (Borassus flabellifer Linn.) during storage. Journal of Food Science and Technology, 52(2), 731 741. Available from https://doi.org/ 10.1007/s13197-013-1104-x. Oh, H. I., Kim, Y. J., Chang, E. J., & Kim, J. Y. (2001). Antimicrobial characteristics of chitosans against food spoilage microorganisms in liquid media and mayonnaise. Bioscience, Biotechnology, and Biochemistry, 65(11), 2378 2383. Available from https:// doi.org/10.1271/bbb.65.2378. Olaimat, A. N., Fang, Y., & Holley, R. A. (2014). Inhibition of Campylobacter jejuni on fresh chicken breasts by κ-carrageenan/chitosan-based coatings containing allyl isothiocyanate

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or deodorized oriental mustard extract. International Journal of Food Microbiology, 187, 77 82. Available from https://doi.org/10.1016/j.ijfoodmicro.2014.07.003. Olaimat, A. N., & Holley, R. A. (2015). Control of Salmonella on fresh chicken breasts by κ-carrageenan/chitosan-based coatings containing allyl isothiocyanate or deodorized Oriental mustard extract plus EDTA. Food Microbiology, 48, 83 88. Available from https://doi.org/10.1016/j.fm.2014.11.019. Olaimat, A. N., & Holley, R. A. (2016). Inhibition of Listeria monocytogenes on cooked cured chicken breasts by acidified coating containing allyl isothiocyanate or deodorized Oriental mustard extract. Food Microbiology, 57, 90 95. Available from https://doi. org/10.1016/j.fm.2016.02.001. Omogbai, B. A., & Ikenebomeh, M. J. (2019). Survival of some food-borne pathogenic microorganisms in ozonated tropical fruits juices treated with chitosan. Science World Journal, 14, 52 60. Osaili, T. M., Al-Nabulsi, A. A., Hasan, F., Olaimat, A. N., Taha, S., Ayyash, M., Nazzal, D. S., Savvaidis, I. N., Obaid, R. S., & Holley, R. (2022). Antimicrobial effects of chitosan and garlic against Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in hummus during storage at various temperatures. Journal of Food Science, 87(2), 833 844. Available from https://doi.org/10.1111/1750-3841.16025. Rhoades, J., & Roller, S. (2000). Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods. Applied and Environmental Microbiology, 66(1), 80 86. Available from https://doi.org/10.1128/AEM.66.1.8086.2000. Roller, S., & Covill, N. (2000). The antimicrobial properties of chitosan in mayonnaise and mayonnaise-based shrimp salads. Journal of Food Protection, 63(2), 202 209. Available from https://doi.org/10.4315/0362-028x-63.2.202. Sarwar, M. T., Sidur Rahman, M., Zakir Hossain, M., & Mashiul Alam, M. (2014). Preparation of chitosan and its application on ginger paste and fruit juice as food preservative. Journal of Food and Nutrition Sciences, 2, 243 249. Available from https://doi. org/10.11648/j.jfns.20140206.11. Seo, M. H., Lee, S. Y., Chang, Y. H., & Kwak, H. S. (2009). Physicochemical, microbial, and sensory properties of yogurt supplemented with nanopowdered chitosan during storage. Journal of Dairy Science, 92(12), 5907 5916. Available from https://doi.org/ 10.3168/jds.2009-2520. Shaltout, F. A., El-Diasty, E. M., & Hassan, A. M. A. (2019). Effect of nano-chitosan and onion extract as coating materials on the quality properties of chicken fillet meat during refrigeration. Global Veterinaria, 21(6), 368 372. Tsai, G. J., Wu, Z. Y., & Su, W. H. (2000). Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. Journal of Food Protection, 63(6), 747 752. Available from https://doi. org/10.4315/0362-028x-63.6.747. Zivanovic, S., Basurto, C. C., Chi, S., Davidson, P. M., & Weiss, J. (2004). Molecular weight of chitosan influences antimicrobial activity in oil-in-water emulsions. Journal of Food Protection, 67(5), 952 959. Available from https://doi.org/10.4315/0362028x-67.5.952.

CHAPTER 9

Chitosan and hurdle technologies to extend the shelf life or reassure the safety of food formulations and ready-to-eat/ cook preparations/meals Vasiliki I. Giatrakou1, Rameez Al-Daour2 and Ioannis N. Savvaidis2,3 1

Hellenic Research and Innovation Center, Athens, Greece Department of Environmental Health Sciences, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates 3 Department of Chemistry, Laboratory of Food Chemistry and Food Microbiology, University of Ioannina, Ioannina, Greece 2

9.1 Introduction Chitosan is the name given to a group of substances with varying molecular weights that are created by partially or completely deacetylated chitin and are made of 1,4-linked glucosamine. The deacetylated form of chitin is obtained from crustaceans and/or fungi. Due its hydrophilic, cationic, and biodegradable character, chitosan has been cared for as a biomaterial, medical, pharmaceutical, medication efficiency, textile, agricultural, food additive for preserving, wastewater clarifying, plant pesticide agents, and in wound healing. The most noticeable characteristics of chitosan, a molecule made in a variety of ways, are due to its antibacterial and antioxidant qualities. Chitosan and its derivatives have been the most widely used antibacterial compounds derived from crustaceans for ensuring food safety and extending shelf life. Information about chitosan’s antibacterial activity, its mechanism of action against microbes, and factors influencing its characteristics and use in the food business and for public health have recently started receiving attention (Büyükyörük, 2021). With respect to food businesses, it has been shown that it is currently facing a variety of difficulties related to food safety, public hygiene, and new worldwide regulatory standards. Numerous of these issues relate to the nature of foods and beverages, in terms of chemical contamination, Chitosan: Novel Applications in Food Systems DOI: https://doi.org/10.1016/B978-0-12-821663-7.00009-0

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chemical and physical characteristics (in relation to new or “ameliorated” industrial versions of artisanal products), microbiological contamination, detection of foreign matters, intentional food adulteration, traceability, sustainability, etc. However, due to the obvious connection with nonfood businesses, at least one of these arguments, the contamination and the permitted or prohibited use of antimicrobial agents for food production is not easily understood by food consumers. Actually, the use of antibiotics and related substances in this area is well documented, and there is now a wealth of research available when discussing the associated risks to human health. Speaking of antimicrobials in foods, there are two main aspects to consider: the actual effects on human health and the psychological implications these substances have on consumer behavior (Parisi, 2017). Therefore it is crucial to study the effects of various natural antimicrobials that can add value to food premises.

9.2 Chitosan applications to extend the shelf life or reassure the safety of food products, and ready-to-eat/cook preparations/meals A study conducted on ready-to-eat (RTE) bovine meatballs aimed to assess edible chitosan coating and its impact on shelf life and Listeria monocytogenes management at 5°C. RTE bovine meatballs with and without an edible chitosan covering had the cell injected onto their surface. The samples were kept at 5°C, and the bacterial counts of L. monocytogenes and the aerobic total viable count (TVC) were monitored. On days 0, 1, 7, 14, 21, and 28, the presence of L. monocytogenes, lactic acid bacteria (LAB), and Enterobacteriaceae was determined. At the same time, semitrained panelists assessed the sensory characteristics of RTE bovine meatballs (absence of pathogens). Based on the findings of microbiological analysis, edible chitosan membranes extended the shelf life of the RTE bovine meatballs by 14 days, reducing all of the microbial populations enumerated. Additionally, for the chitosan-coated meatballs, a low population of the (inoculated) L. monocytogenes of around 2 log CFU/g or lower demonstrated that edible chitosan coating inhibited the growth of L. monocytogenes. The sensory data obtained in that study revealed that the panelists were satisfied with the items coated with chitosan, even at day 28, in contrast to the samples without chitosan (control samples), which were rejected at day 14 (Antoniadou et al., 2019).

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Besides RTE bovine meatballs, other animal-based food items were also studied including chicken balls, chicken kebabs, and chicken nuggets, which were examined. (Antoniadou et al., 2019). The samples of lamb kebabs, following a 2 g/100 mL chitosan coating addition, were kept in storage for 14 days. The samples were then analyzed for microbial, sensory, and chemical parameters. Results showed that compared to the uncoated samples, chitosancoated samples had a longer shelf life. With regards to the microbiological indices studied, fecal coliforms were eradicated, while the number of Staphylococcus spp. decreased due to the chitosan coating. Moreover, chitosan being present in all meat products, resulted in a lower lipid oxidation during storage, which was slowed down by coating. There were no significant organoleptic differences between uncoated and chitosan-coated meat products. The effectiveness of chitosan coating, against Escherichia coli, Pseudomonas fluorescens, Bacillus cereus, and Staphylococcus aureus, was also confirmed after enumeration of these species during storage in the (inoculated) meat product packages (Antoniadou et al., 2019). Effective packaging is a critical preservation method, as it can control oxidation of lipids. The shelf life extension of RTE beef products using a chitosan film with thyme essential oil (EO) (0%, 0.5%, 1%, and 2% v/w) was investigated (Quesada et al., 2016). An active packaging system, involving addition of chitosan film with thyme EO (0%, 0.5%, 1%, and 2% v/w), was applied with no direct touch with the meat. The goal of the study was to use a chemotype with a low odor intensity to lessen the effect of thyme EO on the sensory quality of the RTE beef products. During the course of 4 weeks of chilled storage, the pH, color parameters, microbial populations, and sensory characteristics were evaluated. While aerobic mesophilic bacteria, LAB, and enterobacteria were unaffected by the EO in the films, the number of yeast decreased. The presence of EO improved meat color preservation (a value), improving the packaged meat’s sensorial quality (appearance). While packages containing, simply chitosan, showed obvious water droplets, the addition of the chitosan-EO layer minimized water condensation inside the package. Cooked meat was thought to have a pleasant thyme odor, whereas upon increasing the EO concentration made the products’ odor less prominent. The authors concluded that an improved antibacterial treatment would be possible, if the packaging film to present better blended with the EO, to be used as a combination (Quesada et al., 2016). These authors concluded that the use of effective packaging with antimicrobials, such as chitosan may have a positive effect on the preservation of meat products.

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On the other hand, plant-based food products also pose a high risk, if not preserved, especially when combined with another animal-based food product. A study conducted on mayonnaise-based shrimp salads, where researchers looked into the possibility of utilizing chitosan glutamate in mayonnaise and mayonnaise-based shrimp salad, as a natural food preservative (Roller & Covill, 2000). Salmonella enteritidis, Zygosaccharomyces bailii, or Lactobacillus fructivorans were inoculated at 5 6 log CFU/g into the mayonnaise-based shrimp salads and samples were stored at 5°C and 25°C for 8 days. To the mayonnaise-based shrimp salad samples the following were added: chitosan: 3 g/L, acetic acid (0.16% v/w), and lemon juice (1.2% and 2.6% v/w). The results showed that a reduction, by approximately 5 log CFU/g of L. fructivorans, was achieved in the mayonnaise-based shrimp salads, containing chitosan (3 g/L) and acetic acid (0.16% v/w), whereas the number of L. fructivorans stayed below the detection limit throughout the experiment. During the first day of incubation at 25°C, counts of Z. bailii decreased by approximately 1 2 log CFU/g; however, after this period in time, growth was delayed by 2 days. A coating of chitosan (9 mg/g of shrimp) prevented the growth of the spoilage flora in the mayonnaise-based shrimp salads stored at 5°C for up to 4 weeks, reducing the spoilage flora from about 8 log CFU/g in the controls to 4 log CFU/g in the combined treatment (chitosan, acetic acid, and lemon juice). Chitosan, applied to the salad samples at 25°C (temperature abuse), did not prove to be an efficient antimicrobial (preservative agent). The finding of this study supported that chitosan may be effective, as a preservative in particular food applications, when combined with hurdles such as acetic acid, lemon juice, and low-temperature storage (Roller & Covill, 2000). A recent study was conducted on a popular Middle Eastern salad dip product based on tahini (Osaili et al., 2022). Tahini has been gaining an increasing popularity as a RTE salad dip (hummus) among healthy-minded eating consumers and has been also used in plant-related vegetarian salads or formulations, such as felafel. The authors aimed at comparing the antibacterial efficacy against Salmonella spp., L. monocytogenes, and E. coli O157: H7 (inoculated) in a hummus dip of 0.5% and 1% (v/w) chitosan plus 1% (w/w) garlic, with salad samples stored for 7 days. During storage, addition of 0.5% chitosan reduced E. coli O157:H7 and Salmonella spp. and L. monocytogenes. in the hummus samples, by approximately 0.9 2.3 log CFU/g, whereas in general at the higher (1%) concentration chitosan and 1% garlic resulted in reductions for the aforementioned species of up to 2.8 log CFU/g. Addition of 1% of garlic to hummus did not significantly reduce

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their numbers. The most significant reductions in the numbers of Salmonella spp., E. coli O157:H7, and L. monocytogenes were achieved at 1% addition of chitosan and during storage of samples under low-temperature refrigeration (4°C). According to the authors, it is essential to include chitosan, as a potential “natural” antimicrobial agent, in hummus dip, and in view of its potential on reassuring the safety of such product. However, for the commercial application to be beneficial for consumers, careful assessment and evaluation procedures are needed, including the prime material suppliers, as well during the preparation of the salad dip, as recontamination is not excluded, during processing, and these steps must be taken seriously under consideration, as they may raise concerns about the safety of hummus (Osaili et al., 2022). Chitosan films, with or without the use of EOs, offer a cutting-edge, risk-free way to maintain the quality of vegetables, such as fresh shredded cabbage. The use of chitosan-based films combined with natural hurdles, that is, EOs could drastically affect or even lower the prevalence of L. monocytogenes, or of other pathogenic bacteria, that are likely to be encountered in freshly cut produce (shredded or cut as small pieces). RTE salads, where a postprocessing recontamination is more than likely with pathogenic microorganisms, such as Salmonella spp., Listeria, or S. aureus, originating from environmental (contaminated) niches or from food handlers, that may be potential carriers of pathogenic bacteria, may therefore pose serious concerns in public health episodes. A study conducted and aimed at comparing the antibacterial effectiveness of composite chitosan-gelatin films against two strains of Listeria spp. (Jovanovic et al., 2016). Fresh shredded cabbage was (artificially) contaminated) with L. monocytogenes ATCC 19115 and ATCC 19112 species, and the shredded cabbage samples were kept at 4°C and for 7 days. After this period, shredded cabbage samples were examined for the total viable count of Listeria spp., as enumerated on PALCAM agar. Typical L. monocytogenes numbers in the untreated cabbage samples varied from 4.2 to 5.4 log CFU/g and reached values of 7.2 8.6 log CFU/g during storage. Respectively, a decrease of L. monocytogenes ATCC 19115 on shredded cabbage took place after 120 hours in the presence of 0.5% chitosan film. After 144 hours, numbers of L. monocytogenes ATCC 19115 and ATCC 19112 decreased in the presence of 1% chitosan film. The authors reported a strong antibacterial action, as it was seen in all tested chitosan film formulations against the development of both strains of L. monocytogenes. It was concluded that the best results were obtained at a chitosan concentration of 1% (Jovanovic et al., 2016).

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It must be stressed at this point that the use of effective antimicrobials, including “natural” such as chitosan and EOs, can help reduce the burden of foodborne illnesses amongst vulnerable populations, thus saving human lives. Pregnant women and the elderly have the highest rates of listeriosis, which is caused by consuming RTE foods and fresh, shredded, barely processed vegetables. Better preventative measures are advised in order to lower the prevalence of listeriosis among customers.

9.3 Applications of chitosan on food formulations, based on bioactive packaging Consumers, food companies, and governments all want to raise the quality level of food products to a higher extent. The new generation of food products needs to be more appealing, tastier, made with fresher ingredients, but most importantly healthy and safe. Therefore food industry aims to develop products that have a high nutritional density, are based on high-quality and fresh ingredients, and contain natural antioxidants such as spice and herb extracts and less salt and synthetic preservatives. To meet the growing consumer demand for safer and better quality food products with a fresh and natural (green) image, new and novel packaging technologies or materials have been and continue to be developed (Cutter, 2006). Bioactive packaging technologies for extended shelf life for raw or precooked products have become one of the major areas of research in food packaging. Of these active packaging systems, the antimicrobial version is of great importance (Coma, 2008). Antimicrobial active bio-based packaging materials could be developed and used because they can inhibit or kill the microorganisms and thus extend the shelf life of perishable products and enhance the safety of packaged products (Coma, 2008). For example, chitosan-based antimicrobial films have shown potential to be used as packaging material for the quality preservation of a variety of foods, as chitosan is a natural biopolymer that can be formed into fibers, gels, sponges, beads, or even nanoparticles (Dutta et al., 2009). Moreover, it has the ability to dissolve and create film-forming solutions to provide edible protective coating to the food products (dipping, spraying, or blending). Chitosan is an edible and biodegradable polymer derived from chitin, the most abundant natural polymer available, after cellulose (Shahidi et al., 1999). Chitin is the major structural component of the exoskeleton of crustaceans found in marine environment such as lobsters, crabs, shrimp,

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prawn, and krill, as a composite with proteins, lipids, and calcium carbonate. It is also found in abundance in the complex carbohydrate cell wall of fungi and yeasts (Mathur & Narang, 1990). The name “chitin” is derived from the Greek word “chiton,” meaning “coat of mail” (Shahidi et al., 1999). Full (or partial) deacetylation of chitin using hot alkali (concentrated NaOH) or enzymatic hydrolysis produces chitosan. However, commercial chitosan still contains N-acetyl groups. From a chemical point of view, chitosan is a linear polysaccharide composed mainly of β-1 4 2deoxy-2-amino-D-glucopyranose and of β-1,4 2-deoxy-2-acetamidoD-glucopyranose (chitin) residues to a lesser extent (Fernandes et al., 2008). Chitosan is a natural antimicrobial which offers real potential for applications in the food industry, fulfilling the growing consumer demand for foods without chemical preservatives, due to its particular physicochemical properties, short-time biodegradability, biocompatibility with human tissues, antibacterial activity toward spoilage, and pathogenic foodborne microorganisms (Gram-positive and Gram-negative bacteria) (Aider, 2010). It is also effective against several fungi and yeasts and has revealed significant antioxidant properties on muscle foods (Georgantelis, Ambrosiadis, et al., 2007; Georgantelis, Blekas, et al., 2007; Jo et al., 2001; Soultos et al., 2008). The main applications of chitosan on food preservation, based on definitions for bioactive packaging proposed by Coma (2008) could be summarized as follows: 1. Application as a bioactive natural additive, either directly applied onto the food in the absence or presence of other antimicrobial and/or antioxidant agents (spice extracts, tocopherol, sulfites, and nitrites). Chitosan or its modified form (oligomer, glutamate salts, etc.) is used in its initial powder form (insoluble) and incorporated into the core of the foodstuff and, thus, is mainly applied on minced meat mixtures used for preparation of meatballs, patties, sausages, etc. (Darmadji & Izumimoto, 1994; Georgantelis, Ambrosiadis, et al., 2007; Georgantelis, Blekas, et al., 2007; Juneja et al., 2006; Roller et al., 2002; Soultos et al., 2008). 2. Application as a bioactive edible coating or soluble additive (chitosan solutions) directly applied onto the food by dipping, spraying, or blending. This application is based on the fact that chitosan has the ability to be dissolved in aqueous acidic solutions (acetic, lactic, propionic, and citric acid are the most frequently used), creating film-forming solutions that are used as edible coatings or liquid additives for foodstuffs (Beverlya et al., 2008; Chhabra et al., 2006; Kok & Park, 2007;

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Lin & Chao, 2001; Roller & Covill, 2000; Sagoo et al., 2002; Tsai et al., 2006; Waimaleongora-Ek et al., 2008). Nowadays, new promising technologies integrating edible chitosan-based solutions or bioactive coatings in combination with natural antimicrobial substances (EOs, spices, lysozyme, nisin, etc.) have been successfully applied for preservation of fish, meat, poultry or vegetable-based products, as well as on inactivation of foodborne pathogenic bacteria (Giatrakou, 2010; Giatrakou et al., 2010b; Inatsu et al., 2005; Kanatt et al., 2008; Kim et al., 2008; Ojagh et al., 2010; Ponce et al., 2008). Moreover, chitosan solutions in combination with modified atmosphere/vacuum packaging (with or without EOs) have been successfully used for prolonging the shelf life of meat, poultry, dairy products, and fresh homemade pasta (Del Nobile, Cammariello, et al., 2009; Del Nobile, Di Benedetto, et al., 2009; Duan et al., 2010; Giatrakou, 2010; Giatrakou et al., 2010a; Yingyuad et al., 2006). 3. Application as a polymer matrix not directly applied onto the product surface (as described above) but used as carrier for controlled release of other antimicrobials or flavoring substances (EOs or other antimicrobials). In that case edible (or not in some cases) dry chitosan-based films are easily prepared by evaporating from dilute acid conditions using a casting technique (Coma et al., 2002; Fernandez-Saiz et al., 2010; Hosseini et al., 2008; Ouattara et al., 2000; Pranoto et al., 2005; Sanchez-Gonzalez et al., 2010; Zivanovic et al., 2005). The utilization of chitosan as a direct additive (in powder form), an edible coating, or an antimicrobial biopolymer for active packaging of perishable food products has given promising results in the last decade. The potential of “natural” antimicrobials with hurdle technologies has been demonstrated in numerous applications, and some of these are discussed further, in terms of their capacity to extend the shelf life and also to assure the safety and preservation of food products.

9.3.1 Utilization of chitosan as a food additive directly incorporated into meat formulations Direct incorporation of chitosan as a bactericidal agent for protection of meat-based products from microbial deterioration may result in partial inactivation of the active substances by the food constituents and is therefore expected to have only a limited effect on the surface flora (Coma et al., 2002). However, use of chitosan in its initial powder form, without being dissolved in any organic solvent, may offer a good potential for the

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preservation of minced meat products (e.g., patties and meatballs) or meat mixtures used for formulation of emulsion-type or traditional (British or Greek style) sausages, as described in the following section. Soultos et al. (2008) investigated the effect of chitosan powder (MW: 4.9 3 105) added individually or in combination with nitrites (150 ppm) on microbiological, physicochemical, and sensory properties of fresh pork Greek-style sausages stored at 4°C for 28 days. Sausages of this traditional type were made using pork meat and fat, which are chopped and thoroughly mixed with salt and seasonings. The mixture was treated with two levels of chitosan (Dalian Xindie Chitin Co., Dalian, China) in powder form (0.5% and 1% w/w) alone or in combination with nitrites (150 ppm) and finally stuffed into natural casings from the clean intestine of pigs using a filling machine. Chitosan resulted in significant inhibition on microbial growth, while nitrites did not seem to protect sausages from microbial spoilage. Specifically, chitosan at a concentration of 1% w/w in the meat mixture reduced TVCs, Pseudomonas, Enterobacteriaceae, Brochothrix thermosphacta, and LAB by 1.0 1.7 log CFU/g, after 28 days of storage at 4°C. The authors concluded that Gram-positive bacteria were more sensitive than Gram-negative bacteria to the antimicrobial action of chitosan. As regards sensory evaluation, samples containing chitosan-nitrites combination at any level were judged as more acceptable than other treatments. The effect of high-MW chitosan in powder form (1% w/w) on microbial parameters of Greek-style pork sausages was also studied by Georgantelis, Ambrosiadis, et al. (2007), with the exception that the authors combined chitosan (Dalian Xindie Chitin Co., Dalian, China) with rosemary extract and a-tocopherol. The addition of chitosan in the formulation of sausages resulted in significant inhibition of microbial growth during storage at 4°C. In fact, chitosan 1% w/w reduced TVC, Enterobacteriaceae, and yeasts-molds by c. 1.5 1.9 log CFU/g after 20 days of storage at 4°C, as compared to the control. Interestingly, chitosan had a more pronounced effect on Grampositive LAB, as their counts were reduced by c. 2.4 log CFU/g, during the same period, as compared to the control. Counts of Pseudomonas spp. reached the level of 7 log CFU/g on day 9 for control, whereas samples containing chitosan 1% w/w slightly exceeded 6 log CFU/g during the entire storage period. Finally, samples containing the combination of chitosan and rosemary extract (260 mg/kg) exhibited the lowest microbial counts, indicating a possible synergistic effect. Roller et al. (2002) developed a novel preservation system for the chill preservation of British-style pork sausages based on combinations of chitosan

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glutamate (Pronova, Drammen, Norway), carnocin, and low concentrations of sulfite. Sausages were prepared using minced pork and fat. Minced meat was mixed with a mixture of dry ingredients, containing rusk, seasoning, 2% skim milk powder, as well as chitosan glutamate (0.6%) and sodium metabisulphite as additives. Combination of chitosan and low sulfite (170 ppm) proved to be the most effective to reduce TVC, LAB, and yeasts-molds by 3.0 4.0 log CFU/g, especially during the first 7 10 days of storage at 4°C. In conclusion, the authors stated that the combination of chitosan and low sulfite extended nearly three times the shelf life of sausages, based on maximum acceptable limits for total and yeasts counts established for meat (IFST, 1999). Finally, sensory evaluation showed that chitosan-sulfite treatments deteriorated less rapidly and were acceptable for a longer period than other batches by the panelists, based on appearance and odor attributes. Jo et al. (2001) prepared emulsion-type sausages with the addition of a chitosan oligomer (MW: 5000, Shinyoung Chitosan Co, Seoul, Korea). Sausages were prepared using a mixture of ground pork meat and fat, salt, ice water, sugar, nitrites, phosphates, and a spice mix. Chitosan oligomer (in powder form) was incorporated into the meat mixture at a final concentration of 0.2% w/w. Sausages were stuffed with the former mixture into a casing, dried and smoked. Combination of chitosan oligomer with aerobic or vacuum packaging did not affect microbial counts during storage of sausages at 4°C for 21 days. Moreover, the addition of chitosan oligomer in the emulsion-type sausages did not influence flavor, texture, and overall acceptance, according to sensory analysis. Finally, Darmadji and Izumimoto (1994) studied the effect of chitosan (in powder form) on quality attributes of fresh minced beef. Chitosan (Katokichi Co., Japan) was incorporated into the formulation of minced beef in its initial powder form, at final concentrations of 0, 0.2, 0.5, and 1.0% w/w. According to their findings, chitosan at a final concentration of 1.0% w/w reduced TVC, Staphylococci, and coliforms by c. 1 1.4 log CFU/g, as compared to the control, during storage of the product for 10 days at 4°C. Moreover, the addition of chitosan (1.0% w/w) resulted in a significant reduction of Gram-negative bacteria, Micrococci, and Pseudomonas counts by c. 2.0 2.7 log CFU/g at the end of the storage period. Finally, chitosantreated samples resulted in better sensory attributes and maintained redness of the surface of the product in favorable levels. Juneja et al. (2006) were the first to study the effect of chitosan glutamate on controlling spore germination and outgrowth of Clostridium perfringens during storage of cooked ground beef and turkey, in the time

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of abusive cooling from 54.4oC to 7.2°C in predetermined time intervals (hours). Chitosan glutamate (containing 44% glutamic acid, Pronova Biopolymer, Drammen, Norway) was incorporated into ground beef or turkey meat formulations to final concentrations of 0.5% 3.0% w/w. Chitosan-treated and control samples were inoculated with C. perfringens spore cocktail of 3.0 log CFU/g, cooked, and chilled. Results of the study suggest that 3% w/w chitosan reduced C. perfringens spore germination and outgrowth by 4 5 log CFU/g during exponential cooling of beef and turkey in 12, 15, or 18 hours of intervals, but higher chilling time (21 hours) resulted in a lower reduction by c. 2.0 log CFU/g. All above studies suggest that chitosan or chitosan glutamate, both in powder form, can be utilized at concentrations as low as 1.0% w/w, direct additives incorporated into the formulation of food products made of raw ground meat (sausages, patties, meatballs, cocktail salami, etc.), in order to control microbial spoilage and to extend their shelf life during refrigerated storage. An overview of studies testing the antibacterial activity of chitosan (in powder form) directly incorporated into meat products, is given in Table 9.1. Furthermore, the fact that chitosan/low sulfite/spice extracts treatments revealed a possible synergistic effect on delaying microbial spoilage or sensory deterioration of this kind of meat products suggests that the combined application of chitosan with the aforementioned antimicrobial agents allows the use of the latter in lower concentrations than the ones required to achieve the same target if individual additives were used alone. However, chitosan oligomer added in sausages did not prove to be effective in inhibiting microbial deterioration nor in improving sensory characteristics, thus it may not be a promising technology to maintain microbial quality of such meat products. Interestingly, chitosan glutamate at concentrations of 3.0% w/w may reduce the potential risk of spore germination and outgrowth of pathogenic bacteria that remain a major cause of foodborne illness (such as C. perfringens) during improper storage and inadequate cooling practices of cooked ground meat products, in retail food operations.

9.3.2 Application of chitosan as bioactive edible coating or liquid (acid-soluble) additive applied onto food preparations (dipping, spraying, or blending) Chitosan solutions have been investigated as possible means of extending the shelf life and enhancing safety of perishable food products. Till year 2000, little work had been reported on antagonistic properties of chitosan

Table 9.1 Overview of studies testing the antibacterial activity of chitosan (in powder form) directly incorporated into meat products. Food product

Application of chitosan ( 6 other agents)

Concentration applied

Storage temperature/ period/other notes on experimental setup

Bacterial species

Greekstyle pork sausages

Chitosan of high MW 6 nitrites

1% w/w 6 150 ppm

4°C/28 days

Greekstyle pork sausages

Chitosan of high MW 1 rosemary extract

1% w/w 1 260 mg/Kg

Britishstyle pork sausages

Chitosan glutamate 1 sulfites

0.6% w/w 1 170 ppm

Results Reduction of final population (log CFU/g)

Shelf life extension (based on criteria applied)

Natural microflora

1.0 1.7

114 days (threshold limit of TVC 5 7.0 log CFU/g)

4°C/20 days

Natural microflora

1.5 2.4

1More than 10 days (threshold limit of Pseudomonas, yeasts-molds count 5 7.0 log, rancidity level)

4°C, 24 days

Natural microflora

3.0 4.0

1More than 12 days (threshold limit of TVC 5 7.0 log CFU/g, yeastsmolds count 5 6.0 log CFU/g)

Other observations/ comments

References

a) Chitosan-nitrite combination improved sensory. characteristics b) Lipid oxidation lowers in chitosannitrite combination. c) Nitrites did not protect from microbial spoilage. Chitosan-rosemary combination showed the best antimicrobial and antioxidant effect (possible synergistic action). Chitosan-sulfites combination improved sensory characteristics (odor, appearance).

Soultos et al. (2008)

Georgantelis, Ambrosiadis, et al. (2007)

Roller et al. (2002)

Emulsiontype pork sausages

Chitosan oligomer

0.2% w/w

Fresh minced beef

Chitosan (MW not reported)

1% w/w

Cooked ground beef and turkey

Chitosan glutamate

3% w/w

4°C/21 days/ aerobic or vacuum packaging 4°C/10 days

Abusive cooling from 54.4° C 7.2°C after a) 12, 15, and 18 hours b) 21 hours

Natural microflora

No effect

Chitosan reduced lipid oxidation.

Jo et al. (2001)

Natural microflora

1.0 2.7

Chitosan improved sensory attributes, maintained redness, and reduced lipid oxidation and putrefaction.

Darmadji and Izumimoto (1994)

Clostridium perfringens spore cocktail

a) 4.0 5.0 b) 2.0

Not determined

Juneja et al. (2006)

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against pathogenic and/or spoilage microorganisms important in foods; however, over the last 10 years, chitosan has attracted attention of many researchers in order to be exploited as a natural shelf life extender of meat- or fish-based products. Some of the work focused on its possible role in food preservation is presented on the following section. Sagoo et al. (2002) developed a novel preservation system for chilled raw skinless sausages and an unseasoned minced pork mixture, using chitosan glutamate solutions. Antimicrobial solutions were prepared by dissolving chitosan glutamate (Pronova Co, Drammen, Norway) in deionized water. Dipping of skinless sausages in chitosan solution (1.0% w/v) or blending chitosan solution (1.5%, 3.0% w/v) with the formulation of unseasoned minced pork mixture at a final concentration of 0.3% and 0.6% w/w reduced TVC, yeast-molds, as well as LAB populations by up to 3 log CFU/g, after storage for 18 days at 7°C and 4°C, respectively, as compared to control samples. Furthermore, based on the upper acceptability limit of 7.0 log CFU/g established for TVC (IFST, 1999), chitosan dipping resulted in a microbiological shelf life extension of pork sausages by 8 days, as compared to control. In another study, Kok and Park (2007) evaluated the potential of two different chitosan edible coatings to extend the shelf life of a surimi-based product, called “set fish balls,” during refrigerated storage (4°C). Acidsoluble chitosan of low MW (Vanson Halosource, Inc., Redmond, WA) was dissolved in 1% acetic acid solution, to a final concentration of 1% w/ v. Water-soluble chitosan lactate of low MW (Kyowa Technos Co. Ltd., Chiba, Japan) was prepared in water to a final concentration of 0.5% w/v. Fish balls dipped in water-soluble chitosan solution although had (by c. 5.0 log CFU/g) lower aerobic plate counts as compared to the uncoated samples on the third storage day, prolonged storage resulted in a significant loss of antibacterial activity. On the other hand, dipping fish balls in 1% acid-soluble chitosan led to a important antibacterial effect, as TVC remained below 1 log CFU/g during the entire storage period of 21 days, whereas control uncoated samples exceeded 7.0 log CFU/g between 15 and 18 days of storage. The potential of chitosan solutions to prolong shelf life and reduce spoilage microorganisms of meat products was also studied by Lee et al. The authors dissolved chitosan of low MW (30 and 120 kDa) as well as a chitosan oligomer (5 kDa) in lactic acid solution 0.5% (w/v) and water, respectively, in order to achieve final concentration of 0.1% 1.0% w/v. Pork samples were dipped in above solutions for 1 minutes and then

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stored at 10°C for 8 days. Microbiological shelf life of control samples, based on TVC limit of 7.0 log CFU/g was c. 6 days, whereas treatments coated with chitosan solution of low MW (1.0% w/v) had TVC counts lower than 6.0 log CFU/g at the end of the storage period. Interestingly, water-soluble chitosan oligomer (5 kDa) was not effective in reducing TVC and thus prolonging the shelf life of pork samples, whereas chitosan of MW 30 and 120 KDa (1.0% w/v) reduced final TVC counts by up to 1.5 and 2.0 log CFU/g, respectively, as compared to control samples. Use of a natural antimicrobial coating such as chitosan combined with vacuum or modified atmosphere packaging (MAP) may result in foods with better organoleptic and microbiological quality than those of conventional packaged raw or precooked food products. From this point of view, Yingyuad et al. (2006) investigated the combined effect of chitosan coating solutions and vacuum packaging on maintaining the quality of refrigerated grilled pork. Chitosan dipping solutions were prepared by dissolving commercial-grade chitosan (Fisher Scientific, USA) in aqueous acetic acid solution of 1% w/v, to a final chitosan solution concentration of 2.0% and 2.5% w/v. Grilled pork was immersed into above solutions for 1 minute and then vacuum packaged (VP). Chitosan coating (2.0 or 2.5% w/v) in combination with VP reduced TVC by c. 5.0 and 3.0 log CFU/g as compared to air and vacuum uncoated samples, respectively, after 14 days of storage at 2°C. Interestingly, TVC of chitosan-coated samples remained below 4 log CFU/g till the end of the storage period (28 days). TVC of air and vacuum uncoated samples reached 6.85 and 6.3 log CFU/g on 14th and 28th day of storage, respectively, while the quality of the product was unacceptable when TVC exceeded 6.0 log CFU/g. More recently, a study on the combined effects of chitosan and MAP to improve the microbiological quality of a freshly prepared homemade product (amaranth-based fresh pasta) stored under refrigeration was conducted by Del Nobile, Di Benedetto, et al. (2009). Working active solutions were prepared by dissolving chitosan (Danisco, Braband, Denmark) in lactic acid solution (1.38% v/v). These solutions were mixed with the dough of pasta separately, to obtain final concentrations of 2000 and 4000 mg/kg of pasta. The samples were packaged under aerobic or MAP (80:20, 0:100, and 30:70 N2:CO2 combinations) conditions and stored at 4°C. Control treatment (no chitosan added, aerobic packaging) exceeded the legislation threshold limit of mesophilic bacteria for fresh pasta (6 log CFU/g) after c. 20 days of storage, whereas treatments with chitosan

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(2000 and 4000 mg/kg) exceeded aforementioned limit after 47 50 days (4°C). In the three MAP treatments mesophilic counts never exceeded the proposed limit of 6 log CFU/g. Interestingly, the combined application of MAP (80:20, 0:100 N2:CO2) and chitosan resulted in lower mesophilic counts than MAP alone, by c. 1.12 1.55, at the end of the storage period (54th day). Moreover, after the first 30 days of storage, all chitosan-air samples reduced Staphylococcus spp. counts by at least 3.0 log CFU/g, as compared to untreated control samples. The study concluded that MAP and chitosan, irrespective of concentration tested, can act in a synergic mode in controlling the microbial quality loss of fresh pasta during refrigerated storage and thus can be utilized as a valid alternative to more expensive food thermal treatments commonly used to prolong the shelf life of these kind of product. To conclude, all previously described research studies suggest that chitosan-organic acid solutions (mainly lactic or acetic acid) at concentrations of 1.0% 2.5% w/v could be potentially used as effective means of extending the shelf life of various perishable food products, including surimi-based seafood products, fresh homemade pasta, and raw or grilled meat products, during refrigerated storage, without any negative impact on sensorial acceptability. Acid-soluble chitosan applied on food products by dipping or blending proved to significantly reduce TVC by 2 6 log CFU/g, during refrigeration storage (2°C 10°C), and its effectiveness was concentration-dependent. Physical characteristics of chitosan (e.g., molecular weight) seem to be an important factor in controlling its antimicrobial properties on real food products. For example, although watersoluble chitosan glutamate solutions (1.0% 3.0% w/v) managed to reduce TVC, LAB, and yeast-molds populations of raw skinless sausages and minced pork mixture stored under refrigeration by up to 3.0 log CFU/g, other water-soluble chitosan oligomers (0.5% 1.0% w/v) significantly lost their antibacterial properties against spoilage bacteria of seafood or raw pork after 2 4 days of refrigerated storage (4°C). Furthermore, acidsoluble (acetic/lactic acid) chitosan was successfully used in combination with VP or MAP to extend the shelf life of raw and precooked foods and resulted in products with better sensorial and microbiological quality than aerobic packaging, suggesting that chitosan-MAP or VP can act in a synergic mode in controlling microbial and sensorial quality loss. Apart from above studies investigating the ability of chitosan solutions to prolong the shelf life of refrigerated food products, many authors have tried successfully to evaluate its potential to enhance food safety during

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chill storage. The great concern of health-conscious society for better quality and improved safety of food products was the main motive of the following studies to examine the effect of chitosan solutions on several foodborne pathogenic bacteria inoculated into fresh or precooked food products, being stored under refrigeration. L. monocytogenes, S. aureus, and Salmonella enterica are three of a wide range of pathogenic bacteria, implicated in serious foodborne outbreaks caused by the consumption of raw or precooked products (when contaminated with any of these bacteria) and thus research has focused on the potential of chitosan as a natural preservative to inhibit their growth. Refrigerated RTE food products have a great risk of being contaminated and support the growth of L. monocytogenes, a psychotropic and ubiquitous pathogen, presenting a high mortality rate among some highrisk groups, including the newborn, the aged, and people with compromised immune systems. Motivated by the great concern of consumers about the potential harmful effect of synthetic food preservatives traditionally used for preventing pathogen growth, Beverlya et al. (2008) and Albuquerque Bento et al. (2011) evaluated the antimicrobial activity of chitosan solutions against the growth of this specific pathogen, after being inoculated into RTE popular meat products (roast beef, bovine pate) stored at 4°C. Beverlya et al. (2008) evaluated the antimicrobial activity of high- and low-MW chitosan against L. monocytogenes inoculated in RTE roast beef cubes. The low-MW chitosan (Keumco Chemical, Seoul, Korea) and high-MW chitosan (Premix Ingredients, Avaldnes, Norway) were dissolved in acetic or lactic acid solution (1% w/v) to final concentration of 0.5% or 1% w/v. Roast beef cubes, previously inoculated with L. monocytogenes at an initial concentration of 6.65 log CFU/g, were dipped once in above solutions for 30 seconds. The most effective chitosan coating was low-MW chitosan in acetic acid (0.5% or 1% w/v), reducing pathogen counts by c. 2.3 3.3 log CFU/g, as compared to control uncoated samples, after 28 days of storage at 4°C. Albuquerque Bento et al. (2011) aimed at evaluating the efficacy of chitosan in inhibiting L. monocytogenes in bovine meat pâté stored at 4°C. Chitosan originated from Mucor rouxii (fungi isolated from mangrove sediment) and its production was performed by the authors themselves. Obtained chitosan presented molecular weight of 2.60 3 104 g/mol and deacetylation degree of 85%. Preparation of chitosan solution was done by dissolving 50 mg chitosan in 100 mL acetic acid 1% (v/v) and was

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added to bovine pâté samples to achieve a final concentration of 5 mg chitosan/g pâté. The addition of chitosan solution to bovine meat pâté decreased the counts of L. monocytogenes from approximately 7 3 log CFU/g after 6 days of storage at 4°C, whereas in pâté without chitosan added (control) pathogen counts were over 7 log CFU/g already after 2 days of storage. Sensory valuation suggested that addition of chitosan in pâté would be acceptable to consumers, although some negative influence on flavor and taste was found. S. enterica and especially serotype Enteritidis is a well-known pathogenic bacterium being responsible for the most frequently reported zoonotic disease in many countries (WHO, 2002). Although relatively much “in vitro” work has been done on antimicrobial properties of chitosan against S. enterica, very little work (to our knowledge) has focused on its ability to inhibit growth of this pathogen on real food products, stored under low refrigeration temperatures. Results of “in vitro” study of Marques et al. (2008) performed at 10°C, indicated that S. enterica required concentrations of 0.03% 0.05% for an inhibitory or bactericidal effect, respectively, whereas at 20°C MICs were greater than 0.10%. The antimicrobial potential of chitosan solutions sprayed or coated onto fish products (oysters, shrimp-based salad) inoculated with S. enterica was investigated by Chhabra et al. (2006) and Roller and Covill (2000). Chhabra et al. (2006) investigated the fate of Salmonella enterica ser and typhimurium in raw oysters treated with chitosan. Commercial food grade chitosan (T.C. Union Co, Thailand) was dissolved in 0.5% (v/v) HCl (0.6 M) in order to achieve final concentrations of 0.5% 2.0% w/v. Fresh shucked oysters were dipped in chitosan solution for 1 hour at 4°C. Chitosan-treated and control uncoated samples had no significant differences among them as regards S. typhimurium counts, over the 12-day storage period (4°C); however, pathogen counts were reduced from their initial high populations (7.2 log CFU/g) irrespective of treatment. In another study, chitosan glutamate solution (30 g/L, Pronova, Norway) prepared in water was tested against the growth of S. enterica ser. Enteritidis, inoculated onto shrimps (9 mg of chitosan per gram of shrimp), stored at 5° C and 25°C for up to 8 days (Roller & Covill, 2000). Chitosan solution was sprayed over the surface of shrimps (9 mg of chitosan per gram of shrimp). At both temperatures, chitosan did not reveal any antimicrobial effect toward pathogen counts, inoculated in high initial numbers (5 to 7 log CFU/g). Both aforementioned studies indicate that chitosan coating may be ineffective to control Salmonella growth in seafood, especially if

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contamination with this pathogen is at high levels. However, further studies focusing on antimicrobial properties of chitosan in foods contaminated with low levels (1 4 log CFU/g) of this specific pathogen is needed, in order to ensure its potential to control Salmonella growth. S. aureus is a spherical Gram-positive bacterium (coccus), some strains of which are capable of producing a highly heat-stable protein toxin that causes illness in humans. Foods that require considerable handling during preparation and that are kept at slightly elevated temperatures before consumption are frequently involved in staphylococcal food poisoning. Although food handlers are usually the main source of food contamination in staphylococcal food poisoning outbreaks, equipment and environmental surfaces can also be the sources of contamination with S. aureus (FDA, 2009a, 2009b). Various researchers have demonstrated the antimicrobial activity of chitosan against S. aureus in suspension studies, and it was found that chitosan was inhibitory at minimum concentrations of 0.005% 0.20% w/v (Chang et al., 1989; Darmadji & Izumimoto, 1994; Fernandes et al., 2008). However, its antimicrobial potential toward this specific pathogen has only been sparingly investigated in real food systems, with few exceptions. For example, Chhabra et al. (2006) inoculated fresh oysters with S. aureus and observed that dipping in chitosan solution 0.5%, 1.0%, and 2.0% w/v (see previous paragraph for the preparation of chitosan solution) reduced pathogen counts by 3.5, 2.3, and 4.0 log CFU/g, respectively, as compared to untreated samples, after 12 days of storage at 4°C. The antimicrobial effect of chitosan solutions against S. aureus inoculated into semiskimmed milk was studied by Fernandes et al. (2008). In this study chitosan of low, medium, and high MW was dispersed in 1.0% (v/v) acetic acid solution at a final concentration of 0.5% w/v., while COS (Nicechem, Shangai, China) in deionized water was used at a final concentration of 2.5% w/v. Results showed that after an initial positive inhibitory effect of COS, S. aureus viable counts increased afterward, due to possible trapping of their molecules in the milk protein network. However, the highest MW chitosan prevented loss of antibacterial effect against the same pathogen. The results of the aforementioned studies focusing on antibacterial activity of chitosan solutions against Gram-positive pathogenic bacteria, inoculated on real food systems, suggest that chitosan (especially of low MW) could be considered as a possible alternative agent compound for controlling the growth of L. monocytogenes and S. aureus in RTE meat products, fresh seafood, or milk during storage at refrigeration temperatures.

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Antibacterial activity of chitosan against Salmonella spp., although proved to be significant “in vitro,” its effectiveness on real food products seems to be questionable, as acid- or water-soluble chitosan had no influence on the reduction of S. typhimurium or enteritidis during refrigerated storage of seafood (raw oysters and shrimp salad). It is worth mentioning that the majority of studies on the inhibitory effect of chitosan against food-related microorganisms has been carried out in laboratory media, so more research work using involving real food systems needs to be carried out in the future, in order to assess the efficacy of chitosan in inhibiting the survival of pathogens such as L. monocytogenes, S. aureus, or Salmonella spp. An overview of studies testing the antibacterial activity of chitosan in solution form, applied onto food formulations via dipping or spraying onto product surface or via blending with product, is given in Table 9.2. Nowadays, new promising technologies integrating edible chitosanbased solutions or bioactive coatings enriched with natural antimicrobial substances (EOs, lysozyme, nisin, etc.) have been successfully applied for preservation of fish, meat, poultry, or vegetable-based products, as well as on inactivation of foodborne pathogenic bacteria.

9.4 Applications of chitosan and/or bioactive coatings in combination with essential oils on ready-to-eat/cook preparations/meals Recently, several researchers have revealed the beneficial effects of natural antimicrobials such as chitosan and EOs individually and/or in combination when applied on food systems. EOs possess antibacterial, antiviral, antifungal, and antioxidant properties (Burt, 2004; Holley & Patel, 2005). Generally, among EOs, thyme and oregano oil have increasingly attracted interest of researchers as potential “natural” antimicrobials to be used in food industry, because they have revealed strong antibacterial activity against both foodborne pathogens and spoilage organisms due to high percentage of phenolic compounds such as thymol, p-cymene, carvacrol, and γ-terpinene (Burt, 2004; Holley & Patel, 2005). In recent years, highly promising “in vitro” and “in vivo” studies on combined application of active chitosan-based solutions/ bioactive coatings with thyme oil (Giatrakou et al., 2010a, 2010b; Giatrakou, 2010), rosemary, oregano, capsicum and garlic oleoresin (Ponce et al., 2008), allyl isothiocyanates-hop extract (Wasaouro EXT, Inatsu et al., 2005), mint (Kanatt et al., 2008), and cinnamon oil (Duan et al., 2010; Ojagh et al., 2010) have been reported.

Table 9.2 Overview of studies testing the antibacterial activity of chitosan in solution form, applied onto food formulations via dipping or spraying onto product surface or via blending with product. Storage temperature/ period/other notes on experimental setup

Type of chitosan solution

Method of application onto the food product

Bacterial species

a) Raw skinless sausages

a) 7°C/18 days

a) Dipping

b) Minced pork mixture

b) 4°C/18 days

Surimi-based “set fish balls”

4°C/21 days

a) Chitosan glutamate in water (1.0% w/v) b) Chitosan glutamate in water (1.5%, 3.0% w/v) Chitosan of low MW in acetic acid (1.0% w/v)

b) Blending with meat mixture i). 0.3 ii). 0.6% (w/w) Dipping

Grilled pork

2°C/28 days/ vacuum packaging

Food product

Commercialgrade chitosan in acetic acid (2.0, 2.5% w/v)

Dipping

Results Reduction of final population (log CFU/g)

Shelf life extension (based on criteria applied)

a) Natural microflora

a) 1.0 3.0

a) 18 days (threshold limit of TVC 5 7.0 log CFU/g)

b) Natural microflora

b)1.0 3.0

Natural microflora

5.0 7.4

b) i). 1 9 ii). 1 15 days (threshold limit of TVC 5 7.0 log CFU/g) TVC remained too low to determine

Natural microflora

2.5 5.0

1More than 14 days (threshold limit of TVC 5 6.0 log CFU/g)

Other observations/ comments

References

Sagoo et al. (2002)

Chitosan treatment retained TVC below detection limit (,1.0 log CFU/g) throughout 21 days of storage Chitosan-VP combination minimized lipid oxidation and improved sensory characteristics (odor, color, and overall acceptability)

Kok and Park (2007)

Yingyuad et al. (2006)

(Continued)

Table 9.2 (Continued) Food product

Pork

Amaranthbased fresh homemade pasta

Ready-to-eat roast beef cubes

Storage temperature/ period/other notes on experimental setup

Type of chitosan solution

Method of application onto the food product

Bacterial species

10°C/8 days

a) Chitosan of low MW in lactic acid (1.0% w/v)

Dipping

4°C/54 days/ a) Aerobic packaging b) MAP (30 N2/70% CO2) 4°C/28 days

b) Chitosan oligomer in water (1.0% w/v) Chitosan in lactic acid

Blending with pasta dough (2000, 4000 mg/Kg)

Results

Dipping

References

Reduction of final population (log CFU/g)

Shelf life extension (based on criteria applied)

a) Natural microflora

a) 1.5 2.0

Chitosan reduced lipid oxidation and maintained redness

b) Natural microflora

b) No effect

a) 1 More than 2 days (TVC remained below threshold limit of 7.0 log CFU/g during the storage period) b) No effect

a) 127 to 30 days (threshold limit of mesophilic counts 5 6.0 log CFU/g) b) Mesophilic counts never exceeded threshold limit Not determined

Additive antimicrobial effect between MAP and chitosan observed

Del Nobile, Di Benedetto, et al. (2009)

Acetic acid-chitosan is more effective than lactic acid-chitosan coating

Beverlya et al. (2008)

a) Natural microflora

b) Natural microflora a) Chitosan of low MW in acetic acid (0.5, 1.0% w/v)

Other observations/ comments

Listeria monocytogenes

a) 2.3 3.3

Bovine meat pâté

4°C/6 days

Raw oysters

4°C/12 days

b) Chitosan of low MW in lactic acid (0.5, 1.0% w/v) Chitosan of high MW in acetic acid (0.5% w/v) Commercial food grade chitosan in HCl (0.5% 2.0% w/v)

b) 1.0 1.2

Blending with meat formulation (5 mg/g)

L. monocyto- genes

4.0

Not determined

Some negative influence of chitosan on flavor and taste was found

Albuquerque Bento et al. (2011)

Dipping

a) Salmonella typhimurium b) Staphylococcus aureus

a) 2.3 4.1

Not determined

No influence (effect not clear)

Chhabra et al. (2006)

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Giatrakou et al. (2010a) studied the combined application of chitosan solution, thyme oil, and MAP on preservation of a fresh ready-to-cook (RTC) poultry product (chicken-pepper kebab). Stock chitosan solution 2% (w/v) was prepared by dissolving low-MW chitosan (from crab shells) in powder form (Aldrich Co, Athens, Greece) in glacial acetic acid aqueous solution 1% (w/v). The antimicrobials were added to the RTC poultry product, either singly or sequentially; thyme oil was applied using a micropipette (0.2% v/w), whereas chitosan solution was sprayed directly onto the product (1.5% v/w). All samples were stored at 4°C, under MAP (30% CO2/70% N2). MAP-chitosan (M-CH) and MAP-thyme (M-T) treatments significantly affected TVC, LAB, Pseudomonas spp., B. thermosphacta, Enterobacteriaceae, and yeasts-molds, whereas combined application of MAP with chitosan and thyme oil (M-CH-T) had a more pronounced effect, indicating possible additive or synergistic antimicrobial effect between natural agents. For example, M-CH-T treatment reduced aforementioned spoilage microorganisms of the RTC product by 3.0 4.5 log CFU/g, after storage for 14 days at 4°C, as compared to control samples. Moreover, M-CH-T treatment extended the shelf life of the product by 8 9 days, based on microbiological threshold limit for TVC 5 7.0 log CFU/f and sensory analysis (taste attribute) The same authors investigated the combined potential of MAP and chitosan-thyme to inhibit the growth of three inoculated pathogens (L. monocytogenes, S. enterica ser. Montevideo, B. cereus) onto the aforementioned RTC product during refrigerated storage at 4°C (Giatrakou, 2010; unpublished results). According to their findings, thyme oil (M-T treatment) reduced L. monocytogenes counts by 1.0 log CFU/g, as compared to the control (M), while the anti-Listerial activity of chitosan when applied alone (M-CH) decreased progressively over time (Fig. 9.1). However, combined chitosan-thyme treatment (M-CH-T) had a bacteriostatic effect on L. monocytogenes, during storage at 4°C for 8 days. In conclusion, chitosan and thyme oil revealed a possible synergistic effect on inhibiting the growth of this specific pathogen. During storage at 4°C and for a period of 8 days, Salmonella and B. cereus counts were reduced by 0.6 1.0 log CFU/g and 0.5 log CFU/g respectively, in the presence of thyme oil or chitosan, when applied individually onto the product. Combined application of the antimicrobials onto the RTC product (M-CH-T) decreased final population of Salmonella and B. cereus by 1.2 and 0.7 log CFU/g, indicating negligible synergistic action between chitosan and thyme oil toward these species at this specific temperature (Giatrakou, 2010; unpublished results).

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Listeria monocytogenes (4 o C) 6 5,5

Log CFU/g

5 4,5 4 3,5 3 2,5 2 0

1

2

3

4

5

6

7

8

Storage days M

M-CH

M-T

Μ-CH-T

Figure 9.1 Fate of Listeria monocytogenes inoculated on a ready-to-cook poultry product (chicken-pepper kebab) during storage at 4°C under MAP (treatment M), with thyme oil (M-T), with chitosan (M-CH), and with chitosan plus thyme oil (M-CH-T) (Giatrakou, 2010; unpublished results). Please check the online version to view the color image of the figure. MAP, Modified atmosphere packaging.

The combined effect of chitosan solution and thyme oil on inoculated L. monocytogenes was also investigated during storage of the RTC poultry product at 8°C and for a period of 8 days (Giatrakou, 2010; unpublished results). Control treatment (M) was stored under MAP with a gas atmosphere 30% CO2/70% N2. In control samples (M) during the first 6 days of storage at abuse temperature (8°C) L. monocytogenes growth was slow, showing an increase of c. 1.30 log CFU/g from their initial values (Fig. 9.2). However, after the sixth day of storage at 8°C, the pathogen grew faster reaching levels of c. 6.2 log CFU/g (increase of c. 3.0 log CFU/g from the initial value). M-CH treatment decreased pathogen counts by 0.6 0.8 log CFU/g, during the first 4 days of storage. However, from the fourth day and till the end of the storage period (eighth) addition (spraying) of chitosan alone did not decrease L. monocytogenes significantly, as compared to the control. This indicates that antibacterial effect of chitosan against the pathogen significantly weakened over time, probably attributed to the fact that the amine groups of chitosan bind to components of the bacterial cell surface and, therefore, are no longer available to attach to new Listeria cells (Coma et al., 2002). On the contrary, thyme oil (M-T treatment) maintained its

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Chitosan: Novel Applications in Food Systems

Listeria monocytogenes (8 o C)

7

Log CFU/g

6 5 4 3 2 1 0 0

1

2

3

4

5

6

7

8

Storage days M

M-CH

M-T

Μ-CH-T

Figure 9.2 Fate of Listeria monocytogenes inoculated on a ready-to-cook poultry product (chicken-pepper kebab) during storage at 8°C under MAP (treatment M), with thyme oil (M-T), with chitosan (M-CH), and with chitosan plus thyme oil (M-CH-T) (Giatrakou, 2010; unpublished results). Please check the online version to view the color image of the figure. MAP, Modified atmosphere packaging.

antibacterial action against the pathogen to a better degree than chitosan, resulting to a final reduction of c. 1.0 log CFU/g, as compared to control (M). Combined application of chitosan and thyme oil (M-CH-T) reduced pathogen counts by 1.2 log CFU/g, right from the initial day of storage. During the first 4 days of storage, treatments M-CH, M-T, and M-CH-T, revealed no significant changes among them, the latter, however, was the only one to retain Listeria counts below 3.0 log CFU/g until the fifth of storage (8°C) (bacteriostatic effect). Moreover, treatment M-CH-T produced lower counts of the pathogen in the poultry product resulting in c. 1.5 1.6 and 0.7 log cycles as compared to treatments M, M-CH, and M-T, respectively. However, at the end of the storage period, the addition of thyme oil alone or with chitosan (M-T and M-CH-T) reduced viable counts of Listeria by the same magnitude (1.0 log CFU/g). Overall, although the combined application of chitosan and thyme oil (under MAP conditions) reduced Listeria counts by c. 2.0 log CFU/g during storage at 4°C, indicating a possible synergistic effect between two antimicrobials, at 8°C, a different behavior was noted with a reduction in final counts of the pathogen by 1.0 log CFU/g, indicating that the synergistic effect of chitosan and thyme oil alone was less pronounced.

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Chitosan and hurdle technologies to extend the shelf life

Additionally, the combined effect of chitosan and thyme oil on S. enterica ser. Montevideo inoculated onto the RTC poultry product stored at 8°C, for a period of 8 days, was investigated (Fig. 9.3). According to Fig. 9.3, in control (M) samples Salmonella grew fast from an initial level of 3.3 log CFU/g to high final counts of 5.8 log CFU/g, after 8 days of storage at 8°C. Chitosan (M-CH) did not inhibit growth of the pathogen, as Salmonella counts did not differ significantly from the control treatment, during the entire storage period, except from day 4 when a slight difference was observed. Interestingly, thyme oil (M-T) had a more pronounced effect on Salmonella during the first 6 days of storage, reducing pathogen’s counts by c. 1.0 log CFU/g, as compared to the control. However, on final day 8 of storage treatments, M-T and M (control) had almost equal counts, indicating that the antimicrobial effect of thyme oil was progressively weakened. Combination of chitosan and thyme oil (MCH-T) was the most effective treatment in inhibiting Salmonella growth, as compared to all other treatments during the first 6 days of storage at 8°C. Population counts of the pathogen in M-CH-T were by 2.0 log CFU/g lower as compared to M on the sixth day of storage, whereas extended storage resulted in a reduction of a lower magnitude (only by c. 0.7 log

Salmonella enterica (8 oC)

6

Log CFU/g

5 4 3 2 1 0 0

2

4

6

8

Storage days M

Μ-CH

Μ-T

Μ-CH-T

Figure 9.3 Fate of Salmonella enterica inoculated on a ready-to-cook poultry product (chicken-pepper kebab) during storage at 8°C under MAP (treatment M) with thyme oil (M-T), with chitosan (M-CH), and with chitosan plus thyme oil (M-CH-T) (Giatrakou, 2010; unpublished results). Please check the online version to view the color image of the figure. MAP, Modified atmosphere packaging.

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Chitosan: Novel Applications in Food Systems

Table 9.3 Bacillus cereus strains used in the cocktail mixture, inoculated onto a poultry ready-to-cook product during storage under modified atmosphere packaging at 8°C, for a period of 8 days (Giatrakou, 2010). Type strain and reference of source

Description and origin

a

Mesophilic, isolated from a spoiled cheddar cheese Mesophilic, isolated from the air in a cow shed Psychrotrophic, isolated from cooked chilled food Mesophilic, emetic outbreak

ATCC 10987

ATCC 14579 PAL 22 (Z4222, bINRA) PAL 25 (cNCTC 11143) a

ATCC, American Type Culture Collection, Manassas. INRA, Institut National de la Recherche Agronomique, Avignon France. c NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom. b

CFU/g lower than M on day 8). Interestingly, during the first 6 days of storage at 8°C, M-CH-T had significantly lower Salmonella counts than MCH and M-T, indicating that chitosan and thyme oil may potentially exhibit a synergistic or (additive) effect against the pathogen (Giatrakou, 2010; unpublished results). In the same series of experiments, the combined effect of chitosan and thyme oil on a B. cereus cocktail mixture of psychrotrophic and mesophilic strains (Table 9.3), inoculated onto the poultry RTC product, was examined during storage under MAP at 8°C, for a period of 8 days (Fig. 9.4). According to Fig. 9.4, during storage at abuse temperature (8°C), under M treatment (control) B. cereus counts reached c. 4.5 log CFU/g. Treatments with chitosan or thyme oil (M-CH and M-T) did not seem to affect the growth of the pathogen significantly, during the first 5 days of storage (Giatrakou, 2010; unpublished results). However, toward the end of the storage period (days 6 8), these treatments resulted in lower counts of the pathogen (c. 1.0 log CFU/g) as compared to the control (M). Interestingly, the combined application of chitosan and thyme oil (M-CH-T) revealed a more pronounced effect on these species, reducing its viability by almost 2.0 log CFU/g, indicating a possible additive antimicrobial effect between these two antimicrobial agents. In addition, B. cereus counts in treatment M-CH-T remained below 6.0 log CFU/g during the entire storage period, whereas M (control), M-CH, and M-T treatments exceeded this level right from the fifth day. This observation

Chitosan and hurdle technologies to extend the shelf life

307

o

Bacillus cereus (8 C)

9 8 Log CFU/g

7 6 5 4 3 2 0

1

2

3

4

5

6

7

8

Storage days M

M-CH

M-T

Μ-CH-T

Figure 9.4 Fate of Bacillus spp. inoculated on a ready-to-cook poultry product (chicken-pepper kebab) during storage at 8°C under MAP (treatment M) with thyme oil (M-T) with chitosan (M-CH) and with chitosan plus thyme oil (M-CH-T) (Giatrakou, 2010; unpublished results). Please check the online version to view the color image of the figure. MAP, Modified atmosphere packaging.

could lead to the assumption that a combined antimicrobial treatment involving MAP, chitosan, and an EO (e.g., thyme) could be used as a means of an active packaging/antimicrobial technology, for preservation of fresh poultry products, also guaranteeing safety from a likelihood of a Bacillus spp. toxin formation, as it is known that concentrations of 6.0 log CFU/g are required for enterotoxin formation (Beuchat et al., 1997; FDA, 2009a, 2009b; FSAI, 2007; Grant et al., 1993). Such likelihood was not investigated in the aforementioned series of experiments (Giatrakou, 2010; unpublished results). It is worth mentioning that during storage of the poultry product at 8° C, possible synergistic or additive effect between chitosan and thyme oil was more pronounced in the case of Salmonella and B. cereus than for Listeria, whereas at 4°C the opposite effect was observed. Results demonstrate that chitosan loses its antimicrobial action toward Salmonella and Listeria over time; however, combination with thyme oil may be a potential solution for this particular problem, as the combined application of these antimicrobials resulted in a bacteriostatic effect against these bacteria for 8- and 5-to 6 days during storage at 4°C and 8°C, respectively (Giatrakou, 2010; unpublished results).

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With regard to other studies, Inatsu et al. (2005) evaluated the antibacterial effect of chitosan and allyl-isothiocyanate (AIT) against growth of E. coli, S. enteritidis, S. aureus, and L. monocytogenes, inoculated onto Chinese cabbage. These authors prepared a chitosan (Chitosan 10, Wako Pure Chemical, Osaka, Japan) solution in distilled water (5% w/v) and added a proper amount of the former to cups containing Chinese cabbage in order to obtain a final chitosan concentration of 0.1%. Moreover, an emulsion liquid containing AIT and hop extract (AIT-hop, Wasaouro EXT) at a final concentration of 0.2% was added singly, or in combination with the above chitosan solution. According to their findings, combination of chitosan and AIT-hop exhibited a slightly greater bactericidal effect against mesophilic and coliform bacteria compared to the two natural antimicrobials applied individually (c. 2.0 log CFU/g decrease as compared to the control) during storage of the lightly fermented cabbage (10°C, 4 days). Moreover, chitosan alone or with AIT-hop reduced viable counts of E. coli and S. enteritidis by 1.2 and 0.7 log CFU/g, respectively. The combination of chitosan/AIT-hop slightly reduced counts of E. coli when compared to chitosan only, but this was not observed in the case of and S. enteritidis. Finally, the above combination enhanced bactericidal activity toward inoculated L. monocytogenes, with a reduction of c. 1 2 log CFU/g in counts, lower than the singly applied antimicrobials by the end of the storage period. Ponce et al. (2008) evaluated the potential antibacterial and antioxidant benefits of film-forming solutions of chitosan enriched with oleoresins, “in vivo” and “in vitro.” Experiments “in vitro” showed that pure edible coating solution of chitosan, prepared by dissolving 20 g of chitosan in 1% acetic acid and 1% glycerol solution, did not show any significant antimicrobial properties on butternut squash native microflora and L. monocytogenes. Authors attributed the limited anti-Listerial activity of chitosan film-forming solution to the high number of the initial inoculum (c. 106 and 107 CFU/ petri dish) thereby exceeding chitosan inhibition activity. However, enrichment of chitosan film-forming solution with 1% rosemary oleoresin was very effective against L. monocytogenes and the squash native microflora (in vitro). However, the use of chitosan coatings enriched with rosemary and olive oleoresins did not produce a significant antimicrobial effect when applied to butternut squash during “in vivo” study. In another study, Kanatt et al. (2008) developed a novel natural preservative system, consisting of chitosan and mint mixture with antimicrobial and antioxidant properties. Chitosan (Mahatani Chitosan Pvt. Ltd., India) solution was made in 1% acetic acid and mixed with mint extract

Chitosan and hurdle technologies to extend the shelf life

309

solution. Chitosan-mint mixture solution (CM) was incorporated in the meat formulation for the preparation of pork salami, as well as in minced lamb meat, in order to achieve a final concentration of 0.1%. Mint alone did not reveal any antimicrobial action, whereas the antimicrobial action of CM was similar to that of chitosan. CM (0.1%) reduced counts of Gram-negative (E. coli, Pseudomonas, S. typhimurium) and Gram-positive (B. cereus, S. aureus) bacteria, inoculated into the minced lamb meat by 1.0 and 2 3 log CFU/g respectively, after 28 days of storage at 0°C 3° C. Moreover, the same study evaluated the effect of CM on the shelf life of pork cocktail salami. Control salamis spoiled in less than 2 weeks, whereas salamis formulated with CM displayed a shelf life of three weeks (0°C 3°C) without a positive or negative effect on sensory characteristics (color, flavor, taste, and texture). Ojagh et al. (2010) studied the effect of chitosan coating enriched with cinnamon oil on the quality of refrigerated rainbow trout, stored under refrigeration (4°C) for a period of 16 days. Chitosan solution was prepared with 2% w/v chitosan of medium MW (Aldrich Chemical Co.) and glycerol (0.75 mL/g) was added as a plasticizer. Cinnamon oil (1.5%) was dissolved in Tween 80 and then mixed with the above chitosan solution, in order to obtain a final film-forming solution. Fillet samples of rainbow trout were immersed twice in each of the above chitosan coating solutions and were left to drain at 10°C for 5 hours. Final counts of Gram-negative psychrotrophic bacteria were reduced by 1.63 and 1.75 log CFU/g, as compared to the control, in the presence of chitosan and chitosan-cinnamon oil solutions, respectively, after 16 days of refrigerated storage. In addition, the results of the sensory evaluation showed that adding cinnamon oil into chitosan coating enhanced the beneficial effects on color and overall acceptability of raw fish fillets significantly during the final days of storage, whereas chitosan coating, with or without the EO added, did not produce undesirable sensory properties on cooked fish fillet. Duan et al. (2010) investigated the combined effect of chitosancinnamon-krill oil coating and MAP on the storability of cold-stored lingcod (Ophiodon elongates) fillets. Chitosan of low MW (Primex ehf) was dissolved in acetic acid solution 1% with the addition of glycerol 25% (w/w of chitosan) and finally added at a final concentration of 3.0% w/v. Krill oil (20% w/w) was mixed into the chitosan solution with the addition of Tween 80, and cinnamon oil was then mixed into the krill oil-incorporated chitosan combined solution at a concentration of 0 0.1 μL/mL. The solution mixtures were homogenized at 3000 rpm. for 1 minute and used for coating fish fillets

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Chitosan: Novel Applications in Food Systems

right after sample preparation. The lingcod fillets were cut into small pieces, coated using vacuum impregnation procedure to achieve uniform coatings, vacuum or modified atmosphere (MA) (60% CO2 1 40% N2) packaged, and then stored at 2°C for up to 21 days. The combined chitosan coating and vacuum or MA packaging reduced lipid oxidation, chemical spoilage as reflected in TVBN, TVC by 2.2 4.2 log reductions during storage. The addition of cinnamon oil in coating did not provide additional reduction in TVC of chitosan-coated samples, and no difference was observed between samples with vacuum and MA packaging. However, TVC of all chitosancoated samples remained at very low levels (less than 5.0 log CFU/g) during the entire storage period (21 days), while respective counts of control uncoated samples (aerobic packaging) reached c. 8.5 log CFU/g, on 14th storage day. Finally, according to sensorial analysis, consumers preferred the overall quality of chitosan-coated and cooked lingcod samples over the control, based on firm texture and aroma (less fishy). From previously mentioned numerous research studies depicted in this section of the review, there is no doubt that chitosan can be effectively used either, as a food preservative, or as an edible coating material, in order to preserve quality and extend the shelf life of various food products. This potential of chitosan may be enhanced when combined with EOs (e.g., thyme, rosemary, and mint) or allyl-isothiocyanate-hop extract. Based on the results reported on the studies of Duan et al. (2010) and Ojagh et al. (2010), cinnamon seemed to be the less effective among EOs to enhance the antibacterial effectiveness of chitosan coatings against spoilage bacteria but revealed desirable sensorial characteristics when applied on seafood. Combinations of chitosan solutions and EOs have the potential to give food products enhanced microbiological quality, extended refrigerated storage, durability, and improved sensory characteristics (poultry products, fish fillets, and vegetable-based products). Very often, either a possible or a synergistic/additive antimicrobial effect, compatible with the hurdle concept (which incorporates several antimicrobial measures to gain a synergistic antimicrobial net effect) between chitosan and active compounds of EOs, is likely as demonstrated in the aforementioned studies (Giatrakou, 2010; Giatrakou et al., 2010a, 2010b; Inatsu et al., 2005). More importantly, combination of chitosan-EOs may prove effective in controlling growth of certain pathogenic bacteria (L. monocytogenes, Salmonella spp., E. coli, or B. cereus) likely to contaminate or be present on real food products that are stored under aerobic, or VP/MAP conditions at chill or abuse temperatures (Giatrakou, 2010; Kanatt et al., 2008; Inatsu et al., 2005; Ponce et al., 2008).

Chitosan and hurdle technologies to extend the shelf life

311

In some cases, the antibacterial effect of chitosan against pathogenic bacteria may weaken over time, probably due to the fact that the amine groups of chitosan bind to cell debris or surface components of the bacteria and are no longer available to attach to other bacterial surfaces of new cells (Coma et al., 2002). However, combined application of chitosanEOs may be an effective means of resolving this particular problem, maintaining its antibacterial action against pathogens. In the majority of the aforementioned studies, Gram-negative pathogenic (e.g., E. coli, Salmonella spp.) or spoilage bacteria (e.g., Pseudomonas) revealed a greater resistance toward antibacterial action of chitosan than Gram-positive ones, probably due to the outer membrane surrounding their bacterial cells. Thus EOs could enhance the antibacterial activity of chitosan, against Gram-negative bacteria, rendering the outer membrane of these species susceptible to the action of chitosan (Helander et al., 2001).

9.5 Application of edible chitosan solutions or bioactive chitosan coatings in combination with lysozyme on preserving food quality and safety on ready-to-eat/cook preparations/meals During the last decade there has been a great interest within the food industry in using enzymes naturally occurring in foods, such as lysozyme. Lysozyme is one of the most frequently used antimicrobial enzymes and shows antibacterial activity mainly on Gram-positive bacteria. Because of the protective outer membrane surrounding the peptidoglycan layer of Gram-negative bacteria, lysozyme does not show antibacterial activity against these species. However, when lysozyme is combined with other antimicrobial agents such as chitosan, EDTA, or EOs, the antibacterial spectrum may be widened and be effective against Gram-negative bacteria (Del Nobile, Cammariello, et al., 2009; Ntzimani et al., 2010). Kim et al. (2008) developed a novel preservation system, consisting of chitosan and lysozyme for active edible coating of hard-boiled shell-on eggs, as well as on hard-boiled and peeled vacuum-packaged hard-boiled eggs. For preparing chitosan-lysozyme (CL) coating, shrimp derived chitosan (Primex efh, Siglufjordur, Iceland) was dissolved in 1% acetic acid solution, in order to obtain a final 3% chitosan solution. Glycerol 25% (w/w chitosan) was also added. Then, above chitosan solution was mixed with an appropriate amount of a previously prepared (stock) lysozyme

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Chitosan: Novel Applications in Food Systems

solution 10%, in order to achieve a final concentration of 60% (dry weight lysozyme/dry weight chitosan). Finally, chitosan-lysozyme edible coating (CL) was degassed using a vacuum pump. Dried hard-boiled eggs shell-on or peeled eggs were twice coated by immersions in CL solution for 1 minute and 10 seconds, respectively. Inoculated L. monocytogenes was able to grow in hard-boiled shell-on eggs, irrespective of being coated or not. However, CL coating reduced L. monocytogenes counts by 0.8 log CFU/g, as compared to the control, by the end of the 4-week storage period (10°C). On the other hand, CL coating revealed a stronger antimicrobial action against inoculated S. enteritidis, reducing viable numbers by 4.0 log CFU/g compared to the control, during the same storage period. CL coating was also evaluated for controlling the multiplication of spoilage microorganisms of shell-on or peeled (vacuum-packaged) hard-boiled eggs. Total plate counts (TPC) in control hard-boiled eggs exceeded maximum level of TPC (5 log CFU/g for all egg products) after the 4-week storage, whereas shell-on or peeled (vacuum-packaged) eggs, coated with CL, did not reach this value even after 10 or 6 weeks of storage, respectively (reduction by c. 2.3 2.6 log CFU/g). Del Nobile, Cammariello, et al. (2009) combined chitosan with either alginate coating or active coating (lysozyme, ethylenediamine tetra-acetic acid (EDTA), disodium salt) and MAP in order to prolong the shelf life of “Fior di latte” cheese. High-MW chitosan (Sigma-Aldrich, Milan, Italy) was dissolved in lactic acid in order to achieve a final concentration of 1%. Then, a proper amount of the former solution was added into the working milk for cheese making, at a final chitosan concentration of 0.012%. “Fior di latte” cheese was dipped into sodium alginate solution (designed as active coating). Chitosan-active coating-MAP increased the shelf life in comparison to the traditional packaging from 1 to 5 days, due to the synergic effect between the active compounds and the atmospheric conditions in the package headspace (4°C). In fact, chitosan-active coating-MAP treatment, maintained counts of Pseudomonas spp. below threshold limit of 6 log CFU/g over the entire storage period of 8 days and reduced growth of coliforms by c. 2.0 log CFU/g, as compared to the control. An overview of studies testing the combined effect of chitosan solutions/ coatings and EOs/plant extracts or lysozyme on preserving food quality and safety on RTE/RTC preparations/meals, is given in Table 9.4. It needs to be stressed that to date, studies focusing on potential applications of chitosan with lysozyme on real foods are limited, in view of their

Table 9.4 Overview of studies testing the combined effect of chitosan solutions/coatings and essential oils/plant extracts or lysozyme on preserving food quality and safety on ready-to-eat/cook preparations/meals. Food product

Lightly fermented Chinese cabbage

Storage temperature/ period

Type of active packaging technology applied

Bacterial species

10°C/4 days

Chitosan solution (in water) 1 allylisothiocyanatehop extract (AIT-hop, Wasaouro Ext)

Results Reduction of final population (log CFU/g)

Shelf life extension (based on criteria applied)

a) Natural flora

a) 2.0

Not determined

b) Inoculated pathogens i). Escherichia coli ii). Salmonella enteritidis iii). Staphylococcus aureus iv). Listeria monocytogenes

b) i). ii). iii). iv).

1.0 0.7 1.0 1.3 2.5 3.0

Other observations/ comments

References

a) Chitosan and AIT-hop combination exhibited slightly greater bactericidal effect against mesophilic bacteria and coliforms, but not against lactic acid bacteria. b) Synergistic effect between chitosan and AIT-Hop only against L. monocytogenes.

Inatsu et al. (2005)

(Continued)

Table 9.4 (Continued) Storage temperature/ period

Type of active packaging technology applied

Bacterial species

Minced lamb meat

0 3°C/28 days

Chitosan-mint mixture solution

Pork cocktail salami

0°C 3°C/3 weeks

Rainbow trout

4°C/16 days

Food product

Results Reduction of final population (log CFU/g)

Shelf life extension (based on criteria applied)

i). Pseudomonas fluorescens, E. coli, S. typhimurium ii). Bacillus. cereus, S. aureus

i). 1.0 ii). 2.0 3.0

Not determined

Chitosan-mint mixture solution

Natural microflora

.1.0

11 week (based on TVC, oxidative rancidity)

a) Chitosancinnamon oil solution

Natural microflora

a) 1.75

1 More than 6 days (TVC remained below threshold limit of 7.0 log CFU/g during the storage period)

Other observations/ comments

References

Mint extract alone had poor antimicrobial activity, no synergistic effect between chitosan and mint extract observed. Chitosan-mint mixture solution before cooking process minimized oxidative rancidity. Chitosancinnamon oil combination improved sensory characteristics of raw and cooked fish, reduced chemical spoilage.

Kanatt et al. (2008)

Kanatt et al. (2008)

Ojagh et al. (2010)

Lingcod fish fillets

2°C/21 days

Chitosancinnamonkrill oil solution in combina- tion with MAP or VP

Natural microflora

2.2 4.2

1More than 16 days (TVC remained below threshold limit of 7.0 log CFU/g during the storage period)

Ready-to-cook chicken-pepper kebab

4°C/14 days

Chitosan solution 1 thyme oil in combination with MAP

Natural microflora

3.0 4.5

18 to 9 days (threshold limit TVC 5 7.0 log CFU/g, taste attribute)

a) Cinnamon did not provide additional TVC reduction than chitosan only, b) chitosancinnamon combination improved sensory characteristics (less fishy aroma) and c) combination retarded chemical spoilage and lipid oxidation. a) Synergistic or additive antimicrobial effect between chitosan and thyme oil was observed, b) chitosan-thyme oil combination improved sensory characteristics, and c) chitosanthyme oil combination reduced lipid oxidation and maintained redness.

Duan et al. (2010)

Giatrakou et al. (2010a)

(Continued)

Table 9.4 (Continued) Storage temperature/ period

Type of active packaging technology applied

Bacterial species

Ready-to-cook chicken-pepper kebab

4°C/12 days

Chitosan solution 1 thyme oil in combination with aerobic packaging

Ready-to-cook chicken-pepper kebab

a) 4°C/8 days

Chitosan solution 1 thyme oil in combination with MAP

Food product

b) 8°C/8 days

Results Reduction of final population (log CFU/g)

Shelf life extension (based on criteria applied)

Natural microflora

1.0 3.0

13 days (threshold limit TVC 5 7.0 log CFU/g)

Inoculated pathogensa) 4°C: i). L. monocytogenes ii). Salmonella spp. iii). B. cereus b) 8°C: i). L. monocytogenes ii). Salmonella spp. iii). B. cereus

a) 4°C: i). 2.0 ii). 1.2 iii). 0.7

Not determined

b) 8°C: i). 1.0 ii). 0.7 iii). 2.0

Other observations/ comments

References

a) Chitosanthyme oil combination enhanced natural freshness of the product. b) Chitosanthyme oil combination retained acceptable taste-odor attributes over the storage period. a) Chitosan-thyme oil combination exhibited bacteriostatic effect on pathogen growth.

Giatrakou et al. (2010b)

b) Antimicrobial action of chitosan towards Salmonella and Listeria reduced over time at 8°C.

Giatrakou (2010)

Hard-boiled shellon eggs

a) 10°C/4 weeks

ChitosanLysozyme edible coating

b) 10°C/10 weeks

a) Inoculated pathogens: i). L. monocytogenes ii). S. Enteritidis b) Natural microflora

a) i). 0.8 ii). 4.0

a) Not determined

b) 1.5 2.6

b) 16 weeks (Threshold limit TVC 5 5 log CFU/g) 1More than 2 weeks (TVC remained below threshold limit of 5 log CFU/g) 14 days (threshold limit of 6 and 5 log CFU/g for Pseudomonas and coliforms, sensorial parameters)

Hard-boiled peeled eggs

10°C/6 weeks

Chitosanlysozyme edible coating in combination with VP

Natural microflora

2.0 2.3

“Fior di latte” cheese

4°C/8 days

ChitosanalginatelysozymeEDTA, and MAP

Natural microflora

2.0

Kim et al. (2008)

Chitosanlysozyme retarded moisture loss and color changes. Chitosanlysozyme controlled moisture loss and retarded color changes of egg yolk. Synergic effect between active compounds and MAP was observed.

Kim et al. (2008)

Del Nobile, Cammariello, et al. (2009)

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applicability as a natural antimicrobial treatment combination, for either prolonging the shelf life or maintaining the safety of various foods, for example, fresh meat, poultry, or fish/ seafood, including their products.

9.6 Application of chitosan as an active packaging material for controlled release of active compounds on food models and on real food systems Direct surface application of chitosan (via spraying, dipping) or incorporation into food formulations, as described in previous sections, may sometimes challenge some limitations because the active antimicrobial substance could be neutralized, evaporated, or diffused inadequately into the bulk of the food (Pranoto et al., 2005). The use of packaging films based on antimicrobial polymers could prove more efficient, by maintaining high concentrations on food surfaces, where the contamination is prevalent, with a low migration of active substances (Coma et al., 2002; Sanchez-Gonzalez et al., 2010). Dry edible or plastic chitosan-based films are easily prepared by evaporating from dilute acid conditions using a casting technique and possess the ability to retard moisture, oxygen, aromas, and solute transports (Coma et al., 2002; Fernandez-Saiz et al., 2010; Hosseini et al., 2008; Ouattara et al., 2000; Pranoto et al., 2005; SanchezGonzalez et al., 2010; Zivanovic et al., 2005). Moreover, the development of new biodegradable or edible packaging material films has recently been undertaken for environmental aspects (Coma et al., 2002). Although chitosan has intrinsic antimicrobial activity which is effectively expressed in aqueous system, as previously described, antimicrobial properties may become negligible when chitosan is in a form of insoluble films (Ouattara et al., 2000). Incorporation of EOs in chitosan dry films may not only enhance the film’s antimicrobial properties but also reduce water vapor permeability and slow lipid oxidation of the product on which the film is applied (Zivanovic et al., 2005). Its worth mentioning that numerous studies have demonstrated that EOs are more effective in reducing microbial growth, when incorporated into a film or gel applied directly to the product, because of the active substances evaporating or diffusing into the medium (Sanchez-Gonzalez et al., 2010). In addition, the direct application of EOs to foods may pose an adverse effect on the sensory characteristics of the food, therefore incorporation of EOs into edible films may have beneficial applications in food packaging (Hosseini et al., 2008).

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In recent years, highly promising (1) “in vitro” and (2) “in vivo” studies on combined application of chitosan-based dry films and oregano oil (Zivanovic et al., 2005), garlic oil (GO) (Pranoto et al., 2005), thyme oil (Hosseini et al., 2008), clove oil (Hosseini et al., 2008), tea tree oil (TTO) (Sanchez-Gonzalez et al., 2010), or cinnamaldehyde (Ouattara et al., 2000) have been reported. 1. Studies on food models (in vitro): Pranoto et al. (2005) studied the antimicrobial effect of chitosan edible films incorporating GO against several foodborne pathogenic bacteria (E. coli, S. aureus, S. typhimurium, L. monocytogenes, and B. cereus). Edible films were prepared by dissolving shrimp chitosan of high MW in 1% acetic acid solution, in order to achieve a final chitosan concentration of 1% w/v. GO was incorporated into the above chitosan solution and final dry chitosan-GO films were prepared using a casting technique (drying at 40°C). Control chitosan films did not reveal any antimicrobial effect against all tested bacteria, probably due to the immobilization of chitosan molecules within the film. Interestingly, enrichment with GO at concentrations at least 100 μL/g chitosan led to significantly high inhibitory zones for Gram-positive bacteria tested (S. aureus, L. monocytogenes, and B. cereus). L. monocytogenes was the most sensitive species against GO incorporated in the film. Interestingly, chitosan-GO films although reduced bacterial growth of Gram-negative bacteria (E. coli and S. typhimurium) underneath film disks in direct contact with target microorganisms in agar, these did not result in any clearing zone surrounding same bacteria. The results of the study suggested that GO incorporated into chitosan film increased its antimicrobial efficacy without any effect on mechanical and physical properties of the films. A recent study conducted by Sanchez-Gonzalez et al. (2010) analyzed the antimicrobial, mechanical, optical, and barrier properties of chitosan-based film after enrichment with TTO. Films were prepared by dissolving high-MW chitosan (Sigma-Aldrich Quνmica, Madrid, Spain) in acetic acid solution (0.5% w/w) to a final concentration of 1% w/w. TTO was added to the chitosan (CH) solution at concentrations of 0% 2% w/w. Emulsions of CH-TTO were degasified with a vacuum pump and final composite films were obtained by a casting procedure (drying at atmospheric conditions). L. monocytogenes inoculated in TSA plates showed a significant growth from 2 to 8 log CFU/ cm2 after incubation at 10°C, for 7 days. However, pure chitosan films

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(no TTO added) used as coating for TSA plates, retained L. monocytogenes counts at initial levels (2.0 log CFU/g) till the fifth day of storage at 10°C (bacteriostatic effect). Moreover, same film had lower counts of the pathogen by c. 2.0 log CFU/g at the end of the storage period (12 days). The incorporation of 2% w/w TTO in the chitosan film led to a further reduction of pathogen counts by 1.0 1.5 log CFU/g, and no significant effect was observed for lower TTO concentrations (0.5%, 1.0%). A partial loss of antimicrobial activity of chitosan or chitosan-TTO films was observed over time (after the seventh storage day) and the authors attributed this fact to a reduction of volatile compounds concentration (which also contribute to the total antimicrobial activity of EO) during the film drying process and the time required for the microbial experiments. This study concluded that chitosan is a good polymer matrix for entrapping TTO oil which can be used in different applications. Hosseini et al. (2008) evaluated the possible synergistic antibacterial effect of thyme and clove oil incorporated into chitosan-based edible films. Chitosan-based edible film was prepared by dissolving practical grade chitosan from crab shells (Sigma Chemical Co., St. Louis, Mo., USA) in acetic acid solution 1% v/v to a final solution concentration of 2% w/v. Glycerol was mixed into the chitosan solution to a level of 0.50 mL/g, as a plasticizer. Thyme and clove EOs were added to chitosan solution in order to achieve a final concentration of 0.5% 1.5% per film. Final chitosan-based films were prepared by using a casting technique (drying at 25°C). Determination of antimicrobial effect of chitosan-based edible films was done using the agar diffusion method. Chitosan-based control films (no EOs added) did not show any inhibitory zone against several Gram-positive and Gram-negative pathogenic bacteria tested (L. monocytogenes, S. aureus, S. enteritidis, and Pseudomonas aeruginosa), probably due to the immobilization of chitosan molecules within the film, resulting in a limited diffusion ability through the adjacent agar media. However, incorporation of thyme oil at a concentration of 1.0% 1.5% per chitosan film exhibited a clear inhibitory zone against all bacteria tested. Chitosan films with 0.5% clove EO were not inhibitory toward Gram-negative bacteria tested, but increasing concentration at 1.5% enhanced antimicrobial properties. The study indicates that the addition of clove and thyme EO has the potential of application in antimicrobial food packaging, both against Gram-negative and Grampositive bacteria contaminating foods.

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2. Studies on real food systems (in vivo): A study evaluating the feasibility of using antimicrobial films, designed to slowly release bacterial inhibitors, to improve the preservation of vacuum-packaged processed meats during refrigerated storage, was undertaken by Ouattara et al. (2000). Simple chitosan-based antimicrobial films were prepared by dissolving technical grade chitosan from crab shells (Sigma, St. Louis, MO, USA) in acetic or propionic acid solutions (1% v/v) at a final concentration of 2% w/v. In order to obtain translucent chitosan films, the casting technique was conducted (drying at 80°C). Cinnamaldehyde (active component of several EOs) was incorporated into the chitosan solution to reach a final concentration of 1.0% w/w, prior to casting. Antimicrobial chitosanbased dried films were placed on the upper surface of processed meat (bologna, ham, and pastrami). Application of chitosan-based film without cinnamaldehyde added, on vacuum-packaged bologna and pastrami, resulted in a reduction of Enterobacteriaceae counts by c. 3.0 log CFU/g, as compared to control, after storage at 10°C for a period of 11 or 21 days, respectively. Interestingly, incorporation of cinnamaldehyde further decreased Enterobacteriaceae counts by 0.26 0.8 log CFU/g. On the contrary, chitosan-based films did not reveal any significant antimicrobial effect against LAB initially present on bologna and pastrami. Results of the study suggested that although chitosan-based films incorporating cinnamaldehyde could substantially delay the growth of Enterobacteriaceae, successful application on vacuum-packaged processed meat is limited by the fact that the concept was not effective against LAB, being responsible for spoilage of these type refrigerated products. Toward this specific target, later studies focused on using better antimicrobial agents than cinnamaldehyde which were active against a broader range of bacteria and thus improving antimicrobial properties of chitosan dry films and their results are discussed in the following paragraphs. Zivanovic et al. (2005) studied antimicrobial activity of chitosan-based dry films enriched with EOs “in vitro” and on processed meat. Chitosan films were prepared as follows: medium-MW chitosan (Aldrich Chemical Co, Milwaukee, USA.) was dissolved in 1.5% v/v acetic acid at a final concentration of 1.5% w/w. EOs were mixed with Tween 20 and then added to the above solution at various concentrations. Final film-forming chitosan-EOs solution was poured into Petri dishes (10 mg chitosan/cm2) and dried under vacuum (30°C, casting technique). Chitosan films prepared with or without oregano EO were placed between two slices of

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Chitosan: Novel Applications in Food Systems

bologna, previously inoculated with L. monocytogenes and E. coli. Pure chitosan films (no EOs added) reduced L. monocytogenes by 1 3 log, whereas the films with 1% or 2% oregano EO decreased pathogen counts by c. 3.6 4.0 log, after 5 days of storage at 10°C, indicating a possible synergistic or additive effect of two antimicrobial agents. The synergistic effect was more pronounced in the case of E. coli, inoculated on the bologna slices: pure chitosan films did not inhibit E. coli growth, while enrichment with 1% or 2% oregano EO increased the antimicrobial activity, resulting in a significant reduction of E. coli by c. 3 log. These authors concluded that chitosan-oregano oil films have the potential to be utilized for active packaging of processed meat enhancing its safety. Results of the aforementioned studies indicate that chitosan-based composite films have a great potential to improve their antimicrobial properties by incorporating antimicrobial agents such as EOs. Chitosanbased composite films with no EOs added have shown good, limited, or no antimicrobial activity depending on procedure of dry film preparation, type of chitosan used, as well as target microorganism used. Three different types of chitosan have been used for dry antimicrobial film preparation on above type of studies: chitosan of technical (practical) grade chitosan, high MW, and medium MW, whereas low-MW chitosan has not yet been used. “In vitro” studies (Hosseini et al., 2008; Pranoto et al., 2005) reported that dry chitosan-based composite films did not exert any antimicrobial effect against several pathogenic Gram-positive and Gram-negative bacteria (E. coli, S. aureus, S. typhimurium, S. enteritidis, L. monocytogenes, and B. cereus) due to immobilization of chitosan molecules within the films, resulting in limited diffusion ability of active substance through the adjacent medium (Hosseini et al., 2008; Pranoto et al., 2005). However, this was not always the case as recent “in vivo” (processed meat) and “in vitro” studies (agar diffusion method) showed good antimicrobial potential of chitosan-based composite films against L. monocytogenes, (reduction by 1 3 log CFU/g) (Sanchez-Gonzalez et al., 2010; Zivanovic et al., 2005) or Enterobacteriaceae (reduction by 3.0 log CFU/g, Ouattara et al., 2000). Incorporation of EOs (TTO, oregano, thyme, and clove at concentrations of 1% 2.5% w/w or GO 100 μL/g) into chitosan films enhanced inhibitory action against several pathogenic bacteria, as compared to control pure films, but the antimicrobial effect was more pronounced in the case of Gram-positive bacteria, L. monocytogenes and S. aureus (Hosseini et al., 2008; Pranoto et al., 2005; Sanchez-Gonzalez et al., 2010).

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Studies “in vivo” have used dry chitosan films enriched with EOs for active packaging of processed meat such as bologna or pastrami and placing of the film was done, either on the upper surface (Ouattara et al., 2000), or between slices of the meat product (Zivanovic et al., 2005). It is noteworthy that additional research with the prospect of practical application on real foods is required with the view to explore the potential of films (singly or combined with natural antimicrobials) to be utilized as an active packaging solution for preservation of perishable food products, stored under refrigeration. So far, the main issues that need further research are the following: (1) study of the effect of EOs on mechanical and physical properties of the chitosan films, after incorporation; (2) study of the effect of chitosan-EO packaging films on organoleptic properties (odor, taste, and color) of food products; and (3) study of the potential loss of antimicrobial effectiveness of the film due to instable diffusion of the active compounds from the film into the product.

Acknowledgments We thank European Union for financial support of the project “DOUBLE FRESH” (Proposal./Contract no.: PL 023182).

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Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A ACE. See Angiotensin-I-converting enzyme (ACE) 2-acetamide-2-desoxy-D-glycopyranose, 32 Acetic acid (CH3COOH), 75 Acetyl groups (CH3-CO), 196
197 Acid-soluble additive, 289
298 Acid-soluble chitosan, 292, 294 Acinetobacter, 211
212 Active chitosan-based packaging, 2
4 with antimicrobial properties, 3t combined with encapsulated functional ingredients, 15
25, 17t compounds encapsulated into chitosan-based films, 24
25 metallic nanoparticles encapsulated into chitosan-based films, 15
22 plant extracts encapsulated into chitosan-based films, 22
24 combined with functional ingredients, 4
15, 5t antimicrobial properties, 4
6, 10
15, 12t antioxidant properties, 7
15, 8t, 12t Active packaging system, 1
2, 281 Adenosine 50 -triphosphate (ATP), 38 AE. See Amaranthustricolor L. extract (AE) Aerobic mesophilic bacteria, 281 Aeromonas, 211
212 A. hydrophila, 270
271 AIT. See Allyl-isothiocyanate (AIT) Alkali-NaOH treatment, 199 Alkaline treatments, 32
33 Allyl-isothiocyanate (AIT), 308 Alzheimer’s disease, 237
239 Amaranthustricolor L. extract (AE), 14 Amine (NH) groups, 196
197 Amino group, 160
161 2-amino-2-desoxy-D-glycopyranose, 32 Ananus comosus. See Pineapple (Ananus comosus)

Angiotensin-I-converting enzyme (ACE), 201 Anthocyanin, 247
248 Antibacterial activity of chitosan, 163 mechanisms, 37 Antifungal activity chitosan, 163
169 of lower MW chitosan, 194 Antimicrobial(s), 1
2, 284. See also Natural antimicrobials active bio-based packaging materials, 284 activity, 34, 132
139, 162 of chitosan in dairy products, 274
276 of chitosan in dips and pastes, 273
274 of chitosan in fresh produce and salads, 271 of chitosan in juices, 272
273 of chitosan in meat and poultry products, 272 against food pathogens, 86
90 chitosan and derivatives as antimicrobial agents, 162
180 antibacterial activity, 163 antifungal activity, 163
169 chitosan and derivatives’ application to increase shelf life of foods, 169
180 compounds, 34 effects, 36
39 of chitosan, 36
37 mechanisms of edible coatings of chitosan with plant extracts, 38
39 of plant extracts or EOs, 37
38 pomegranate-chitosan-based films, 251
252 properties, 31 active chitosan-based packaging, 4
6, 10
15, 12t 327

328

Index

Antioxidant, 31 activity, 129
132 of chitosan, 205 of pomegranate, 236 effects, 39
42 of chitosan, 40 of edible coatings of CH with plant extracts or EOs on foods, 41
42 of plant extracts or essential oils, 41 properties of active chitosan-based packaging, 7
15, 8t, 12t property of pomegranate-chitosan-based films, 251 Antioxidant of bamboo leaves (AOB), 21 Antitumor activity of lower MW chitosan, 194 AOB. See Antioxidant of bamboo leaves (AOB) APP. See Apple peel polyphenols (APP) Apple peel polyphenols (APP), 10
11 APX. See Ascorbate peroxidase (APX) Aqueous acidic solutions, 285
286 Aromatic plants, 32 Ascomycetes, 197 Ascorbate peroxidase (APX), 130
131 Ascorbic acid, 40, 235
236, 254 oxidase, 254 Aspergillus A. flavus, 163
164, 216, 272 A. niger, 163
164 ATP. See Adenosine 50 -triphosphate (ATP)

B Bacillus sp., 211
212, 270, 306
307 B. cereus, 237
238, 281, 306t B. subtilis, 204 Bacteria, 133
134 Bacteriocins, 97
102 Basidiomycetes, 197 β-1,4
2-deoxy-2-acetamido-Dglucopyranose, 284
285 β-1, 4
2deoxy-2-amino-Dglucopyranose, 284
285 Beverages, 174
177 Bioactive chitosan coatings, application of

in combination with essential oils on ready-to-eat/cook preparations/meals, 298
311 in combination with lysozyme on preserving food quality and safety on ready-to-eat/cook preparations/meals, 311
318 Bioactive edible coating, chitosan as, 289
298 Bioactive packaging, 1
2, 284
298 Bioactive polymers, 170 Bioactivity of chitosan, 194, 202
207 antimicrobial properties, 202
205 antioxidant properties, 205
207 of pomegranate juice, 237
238 of pomegranate peel, 238 of pomegranate seed, 239 Biogenic amines, 215 Biomolecules, damage of, 205 Biotechnological process of extraction, 76
77 Blueberry (Vaccinium ashei L.), 178 Botrytis cinerea, 163
164 British-style pork sausages, 287
288 Brochothrix thermosphacta, 287 Burkholderia seminalis, 204 Butylated hydroxyanisole, 1
2

C Caffeic acid, 237
238 Camplyobacter, 204, 211
212 C. jejuni, 272 Carbohydrates, 161 Carboxymethyl-chitosan (CMCS), 23
24 CAT. See Catalase (CAT) Catalase (CAT), 130
131 Catechin, 237
238, 250
251 CBE. See Cinnamon bark extract (CBE) Cellulose nanofiber (CNF), 24
25 CEO. See Cinnamon essential oil (CEO) Cereals and oilseeds, 52
53 CGA. See Chlorogenic acid (CGA) Cheeses, 60
61 Chemical composition of essential oils, 34
36 Chemical process of extraction, 74
75, 75f

Index

Chemoactive packaging, 1
2 Chinese chive root extract (CRE), 11 Chitin, 32
33, 73
74, 158, 193, 284
285 Chitosan (CH), 1
4, 11, 23, 32, 115, 156
162, 180, 193, 195
200, 259, 269, 279, 284
285, 319
320 antimicrobial activity in dairy products, 274
276 in dips and pastes, 273
274 against food pathogens, 86
90 in fresh produce and salads, 271 in juices, 272
273 in meat and poultry products, 272 antimicrobial effects, 36
37 antioxidant effects, 40 applications CH and/or bioactive coatings in combination with essential oils on ready-to eat/cook preparations/ meals, 298
311 CH as active packaging material for controlled release of active compounds on food models and on real food systems, 318
323 CH as bioactive edible coating or liquid (acid-soluble) additive applied onto food preparations, 289
298 edible CH solutions or bioactive CH coatings in combination with lysozyme on preserving food quality and safety, 311
318 to extend shelf life or reassure safety of food products, and ready-to eat/ cook preparations/meals, 280
284 on food formulations, based on bioactive packaging, 284
298 studies testing antibacterial activity of chitosan, 299t utilization of CH as food additive directly incorporated into meat formulations, 286
289 bioactivity of, 202
207 CH NP-based film, 209 deacetylation, 32
33

329

effect of CH on acceptability/sensory quality of seafood, 220
221 chitosan and derivatives as antimicrobial agents, 162
180 chitosan-based antimicrobial films, 284 chitosan-based coating, 212
213 chitosan-based edible coatings, 32
34, 171 and films, 177
178 chitosan-based films compounds encapsulated into, 24
25 plant extracts encapsulated into, 22
24 chitosan-based intelligent packaging, 210 chitosan-based nanocomposite, 209 chitosan-based nanomaterials, 208
209 packaging, 208
209 chitosan-based nanosystems against food pathogens, 78
90 chitosan-based packaging, 221 chitosan-gelatin films, 283 coatings, 255 in combination with preservation as hurdle concept, inclusion of, 215
220 chitosan samples with preservation agents, 217t composite films for incorporating pomegranate active compounds, 242
243 and derivatives application to increase shelf life of foods, 169
180 edible coatings, 31
36 chitosan-based edible coatings, 32
34 plant extracts or essential oils added in coatings, 34
36 effects of coatings on quality parameters and shelf life of food, 36
61 antimicrobial effects, 36
39 antioxidant effects, 39
42 encapsulation of pomegranate active compounds in chitosan-based films, 243
244 extraction, 198
200 factors affect antibacterial activity of chitosan nanosystems, 85
86 films, 173, 216, 283

330

Index

Chitosan (CH) (Continued) foodborne pathogens, 71
78 functionalities, 115
116 glutamate, 274, 288
289 health benefits of, 200
202 impacts of chitosan on seafood safety, 211
215 mechanisms of antimicrobial action, 84
85 for nanoencapsulation, 207
208 nanosystems, 78
84 nanotechnological approaches for application of, 207
210 perspectives, 180 physicochemical and chitosan derivatives, 159
162 sources, 195
197, 196f and production, 156
159 structure, 197
198 ultrastructural effect of CH and chitosan nanoparticles, 163
169 versatility, 177 in vitro antimicrobial activity of CH against spoilage and pathogenic microorganisms, 270
271 Chitosan nanofiber (ChNF), 24
25, 210 Chitosan nanoparticles (CS-NP), 96, 165, 243
244 application of chitosan nanoparticles against pathogens in food systems, 103 functional properties, 128
143 antioxidant activity, 129
132 physicochemical characteristics, 141
143 sensory characteristics, 139
141 spoilage microorganisms, 132
139 system for encapsulation of essential oils, 116
122, 119t Chitosan nanosystems loaded with natural antimicrobials, 91
102 bacteriocins, 97
102 enzymes, 96
97 essential oils, 93
96 plant extract/phytobiotics, 91
93 Chitosan oligomers (CO), 2
4, 288 Chitosan-coated nanocapsules (CS-NC), 72

effect, 73f mean apparent permeability and absorption enhancement ratio, 73t zeta potential, 72t Chitosan-lysozyme (CL), 311
312 Chitosan-mint mixture solution (CM), 308
309 Chitosan-pullulan composite, 256 Chitosan/hydroxypropyl methylcellulose (CS/HPMC), 7 Chlorogenic acid (CGA), 23
24 Chlorophyll degradation, 52 ChNF. See Chitosan nanofiber (ChNF) Cinnamon bark extract (CBE), 93 Cinnamon essential oil (CEO), 22
23 Circular economy, 158
159 CL. See Chitosan-lysozyme (CL) Clean-label foods, 195 Clostridium, 211
212 C. perfringens, 237
238, 288
289 CM. See Chitosan-mint mixture solution (CM) CMCS. See Carboxymethyl-chitosan (CMCS) CNF. See Cellulose nanofiber (CNF) CO. See Chitosan oligomers (CO) Coating(s), 170
171, 209. See also Edible coating(s) choice of, 174 plant extracts or essential oils added in, 34
36 chemical composition of essential oils, 34
36 Coconut water, 175 COFs. See Covalent organic frameworks (COFs) Colletotrichum C. fructicola, 164
165 C. gloeosporioides, 251
252 C. gloeosporioides, 163
164 C. siamense, 163
164 Colorado potato beetle, 205 Controlled release packaging, 155 properties, 122
125 Conventional extraction methods, 239
240

Index

Coryneforms, 211
212 Covalent organic frameworks (COFs), 21 CRE. See Chinese chive root extract (CRE) Crustacean exoskeletons, 156
157 Cryptococcus albidus, 270 CS-NC. See Chitosan-coated nanocapsules (CS-NC) CS-NP. See Chitosan nanoparticles (CSNP) CS/HPMC. See Chitosan/hydroxypropyl methylcellulose (CS/HPMC) CUR. See Curcumin (CUR) Curcumin (CUR), 72 curcumin-based nanoformulations, 207
208 mean apparent permeability and absorption enhancement ratio, 73t Cyanidin-3-glycoside, 237
238 Cytoplasmic membrane, 38

D D-glucosamine,

193 DA. See Degree of acetylation (DA) Dairy products, antimicrobial activity of chitosan in, 274
276 DC. See Decolorization (DC) DD. See Deacetylation (DD) Deacetylation (DD), 71, 194 process, 199 of chitin, 156
157 Deacetylation ratio (DR), 206
207 Decoction method, 239
240 Decolorization (DC), 195
196 Degree of acetylation (DA), 194 Demineralization (DM), 195
196 Deproteination (DP), 75, 195
196 Derivative products, 172
174 Diethoxyphosphorylpolyaminoethyl chitosan (DPECS), 204 Differential scanning calorimetry (DSC), 77, 250 of pomegranate-chitosan-based films, 250 Dimethylamine (DMA), 212 Dip-coating process, 33

331

2,2-diphenyl-1-picrylhydrazil (DPPH), 129, 206 Distillation extraction, 240
241 DM. See Demineralization (DM) DMA. See Dimethylamine (DMA) DNA synthesis, 37 DP. See Deproteination (DP) DPECS. See Diethoxyphosphorylpolyaminoethyl chitosan (DPECS) DR. See Deacetylation ratio (DR) Dry edible films, 318 DSC. See Differential scanning calorimetry (DSC)

E EA. See Ellagic acid (EA) Edible chitosan solutions, application of, 311
318 Edible coating(s), 31
36, 39
40, 170, 174, 212
213 of CH with plant extracts or EOs on foods, antioxidant effects of, 41
42 of chitosan with plant extracts, mechanisms of, 38
39 chitosan-based edible coatings, 32
34 films, 254 plant extracts or essential oils added in coatings, 34
36 Edible films, 170, 248 and coatings on food surfaces, 32f Edible materials, 31 EDTA. See Ethylenediamine tetra-acetic acid (EDTA) EE. See Encapsulation efficiency (EE) EGO. See Eucalyptus globulus essential oil (EGO) Ehrlich ascites tumor cells, 201 Electrical conductivity and viscosity, 244 Electronegative amino group, 33 Electrostatic interaction, 37 Elemental analysis, 77 Ellagic acid (EA), 11
14, 237
238, 250
251 Emulsion phase inversion, 118 Encapsulation efficiency (EE), 118
122, 244

332

Index

Encapsulation technique, 175
176, 207
208 Endogenous radical-scavenging antioxidants, 40 Enteritidis, 296 Enterobacteria, 281 Enterobacteriaceae, 39, 214, 258, 280, 287, 302 Enzymatic antioxidant systems, 40 Enzymatic browning, 52 Enzymes, 96
97 enzyme-assisted extraction, 240, 242 EOs. See Essential oils (EOs) ε polylysine (ε-PL), 24
25 Equivalents of gallic acid (GAE), 7 Escherichia coli, 61, 203
204, 257, 270
271, 281 Essential oils (EOs), 32, 91, 93
96, 116, 281 antimicrobial effects, 37
38 antimicrobial mechanisms of action, 125
128 antioxidant effects, 41 chemical composition of, 34
36 chitosan nanoparticles system for encapsulation of, 116
122 controlled release properties, 122
125 on ready-to-eat/cook preparations/ meals, applications of chitosan and/or bioactive coatings in combination with, 298
311 Ethnic foods, 269 Ethylenediamine tetra-acetic acid (EDTA), 312 Eucalyptus globulus essential oil (EGO), 22
23 Exoskeletons, 32
33 Extraction method, 34
35 of food shelf
life, 31 of pomegranate active compounds, 239
242 conventional extraction methods, 239
240 modern extraction methods, 240
242

F Fatty acids, 235
237

FDA. See Food and Drug Administration (FDA) Felafel, 282
283 Ferulic acid, 237
239 FG. See Fish gelatin (FG) Films, 171, 214
215 opacity, 248 permeability, 245 “Fior di latte” cheese, 312 Fish (Merluccius merluccius and Solea solea), 214 products, 172
174 and seafood, 60 Fish gelatin (FG), 14 Flavobacterium, 211
212 Fluid transition, 245 Food and Agriculture Organization, 253 Food and Drug Administration (FDA), 35, 160 Food pathogens, antimicrobial activity against, 86
90 Food(s) additives, 156 application and effects of pomegranatechitosan-based films on, 253
260 application of chitosan as active packaging material for controlled release of active compounds on food models and on real food systems, 318
323 application of chitosan as bioactive edible coating or liquid (acid-soluble) additive applied onto food preparations, 289
298 application of edible chitosan solutions or bioactive chitosan coatings in combination with lysozyme on food quality and safety, 311
318 based on bioactive packaging, applications of chitosan on food formulations, 284
298 businesses, 279
280 chitosan and derivatives’ application to increase shelf life of, 169
180 beverages, 174
177 fruits and vegetables, 177
180

Index

meat, fish, and derivative products, 172
174 chitosan applications to extend shelf life or reassure safety of, 280
284 conservation processes, 155
156 effects of coatings on quality parameters and shelf life of, 36
61 antimicrobial effects, 36
39 antioxidant effects, 39
42 effects of coatings on quality parameters in, 42
61 cereals and oilseeds, 52
53 cheeses, 60
61 fish and seafood, 60 fruits and vegetables, 43
52 meats and meat products, 53
59 emerging and eco-sustainable technology in food preservation, 156
162 industry, 31, 155, 169, 284 oxidation, 39 packaging, 1 products, 31, 215
216 utilization of chitosan as food additive directly incorporated into meat formulations, 286
289 studies testing antibacterial activity of chitosan, 290t of vegetal origin, 177 Food pathogens, chitosan nanoparticles against, 70 application in food systems, 103 chitosan-based nanosystems against food pathogens, 78
90 chitosan nanosystems loaded with natural antimicrobials, 91
102 extraction methods and characterization, 73
77 factors that influence antimicrobial activity, 77
78 physicochemical properties, 71
73 Foodborne diseases, 86
87 Foodborne illness, 69
70, 289 Formic acid (HCOOH), 75 Fourier transform infrared spectroscopy (FTIR), 77, 198, 249
250 Fresh produce and salads, antimicrobial activity of chitosan in, 271

333

Fresh Weight (FW), 129
130 Fruit(s), 177
180 application of pomegranate-chitosanbased films in, 253
256 firmness loss, 43 and vegetables, 43
52 effects of CH coatings and plant extracts or EO on quality parameters, 44t, 54t FTIR. See Fourier transform infrared spectroscopy (FTIR) Functional foods, 175, 236 Functional polymer, 203
204 Fungal chitosan coating films, 258
259 Fusarium F. oxysporum, 164
165 F. solani, 164
165

G GAE. See Equivalents of gallic acid (GAE) Gallagic acid, 237
238 Gallagyldilactone, 237
238 Gallic acid, 237
238, 250
251 Generally recognized as safe (GRAS), 35 GFSE. See Grapefruit seed extract (GFSE) Glass transition temperature, 249 Gliadin nanoparticles (GPNPs), 23
24 Glucosamine units, 159 Glutathione reductase (GR), 130
131 Glycosidic β-1,4 bonds, 32 GPNPs. See Gliadin nanoparticles (GPNPs) GR. See Glutathione reductase (GR) Gram-negative bacteria, 87
88, 90t, 251
252 Gram-positive bacteria, 88
90, 90t Grapefruit seed extract (GFSE), 6 GRAS. See Generally recognized as safe (GRAS) Greek-style pork sausages, 287 Green biotechnology, 31 Green chemistry, 158

H Hairtail (Trichiurus haumela), 214
215 Hawthorn fruit extract, 10 HCl. See Hydrochloric acid (HCl) Helicobacter pylori, 237
238

334

Index

HFE. See Honeysuckle flower extract (HFE) High hydrostatic pressure, 155 High molecular weight (HMWC), 206 High-MW chitosan, 287 High-voltage electrical discharge (HVED), 242 HMWC. See High molecular weight (HMWC) Honeysuckle flower extract (HFE), 10 HPCS. See Hydroxypropyl chitosan (HPCS) Hurdle technology, 125
126 HVED. See High-voltage electrical discharge (HVED) Hydrochloric acid (HCl), 75 Hydrodistillation, 35 Hydrophilic materials, 35
36 Hydrophobic volatile liquids, 34
35 Hydroxyl group, 160 Hydroxyl radicals (•OH), 205 Hydroxypropyl chitosan (HPCS), 204

I IG. See Ionic gelation (IG) In vitro antimicrobial activity of chitosan against spoilage and pathogenic microorganisms, 270
271 “In vitro” studies, 322 Interfacial deposition method, 81 Ionic gelation (IG), 117

J Juices, antimicrobial activity of chitosan in, 272
273

K Kaempferol, 237
238 Kombucha tea (KT), 15 KT. See Kombucha tea (KT)

L LAB. See Lactic acid bacteria (LAB) Lactic acid bacteria (LAB), 134, 280
281 Lactobacillus, 204 L. fructivorans, 282 L. plantarum, 270

Lactococcus garvieae, 204 LAE. See Lauroyl arginate ethyl (LAE) Lauroyl arginate ethyl (LAE), 6 Layer-by-layer method (LBL method), 81 LBL method. See Layer-by-layer method (LBL method) Lecithin-chitosan containing curcumin (LNPC), 92 Lecithin-chitosan nanoparticles (LNP), 92 Lentinus edodes, 197 LFE. See Lycium barbarum fruit extract (LFE) Linear economy, 158
159 Linear polysaccharide, 32, 197
198 Lipids, 171 NCs, 81 peroxidation, 59 Lipopolysaccharide (LPS), 37, 163 Liposomes, 220 Lipoteichoic acids, 37 Liquid additive applied onto food preparations, 289
298 Listeria, 283 L. innocua, 216
219 L. monocytogenes, 61, 89, 91t, 204, 237
238, 270
271, 280, 283, 304f LMWC. See Low molecular weight (LMWC) LNP. See Lecithin-chitosan nanoparticles (LNP) LNPC. See Lecithin-chitosan containing curcumin (LNPC) Low molecular weight (LMWC), 206 LPS. See Lipopolysaccharide (LPS) Lycium barbarum fruit extract (LFE), 10 Lys. See Lysozyme (Lys) Lys-CS-NP. See Lysozyme in chitosan nanoparticles (Lys-CS-NP) Lysozyme (Lys), 96, 311
318 Lysozyme in chitosan nanoparticles (LysCS-NP), 97

M M-CH. See MAP-chitosan (M-CH) treatment M-T. See MAP-thyme (M-T) treatment MA. See Modified atmosphere (MA)

Index

Maceration compound, 239
240 MAE. See Microwave-assisted extraction (MAE)Mulberry anthocyanin extract (MAE) Magnesium oxide (MgO), 22, 127
128 Magnetic nanoparticles (MNPs), 23 Maillard reaction, 161 Malondialdehyde (MDA), 59, 129 Mango leaf extract (MLE), 10 MAP. See Modified atmosphere packaging (MAP) MAP-chitosan (M-CH) treatment, 302 MAP-thyme (M-T) treatment, 302 Matrices, 177
178 MBCs. See Minimal bactericidal concentrations (MBCs) MDA. See Malondialdehyde (MDA) Meat products, 172
174 antimicrobial activity of chitosan in, 272 application of pomegranate-chitosanbased films in, 256
260 meats and, 53
59 Meat-based products from microbial deterioration, 286
287 Medium-molecular-weight chitosan (MMWC), 206 Membrane separation process, 240
241 Mentha aquatic L., 61 Merluccius merluccius and Solea solea. See Fish (Merluccius merluccius and Solea solea) Metallic nanoparticles, 15
16 encapsulated into chitosan-based films, 15
22 MgO. See Magnesium oxide (MgO) Microbial enzymes, 59 Microbial spoilage and oxidation, 139 Micrococcus, 211
212 Microorganisms, 36, 211, 239, 270
271 Microwave-assisted extraction (MAE), 240 MICs. See Minimum inhibitory concentrations (MICs) Minerals, 235
236 Minimal bactericidal concentrations (MBCs), 204 Minimum inhibitory concentrations (MICs), 270
271 MLE. See Mango leaf extract (MLE)

335

MMT. See Montmorillonite (MMT) MMWC. See Medium-molecular-weight chitosan (MMWC) MNPs. See Magnetic nanoparticles (MNPs) Metallic nanoparticles (MNPs) Modern extraction methods of pomegranate active compounds, 240
242 Modified atmosphere (MA), 155, 309
310 Modified atmosphere packaging (MAP), 293 Molds, 132
133 Molecular weight (MW), 33, 193
194 Montmorillonite (MMT), 246
247 MS. See Multiple sclerosis (MS) Mucor rouxii, 295
296 Mulberry anthocyanin extract (MAE), 21 Multiple sclerosis (MS), 239 MW. See Molecular weight (MW) Mycotoxins, 132
133 Myricetin, 237
238

N N-acetyl-D-glucosamine, 193, 197
198 N-acetylglucosamine units, 159 n-STZ mice. See Neonatal streptozotocininduced diabetic mice (n-STZ mice) Nano-ZnO, 21 Nanocapsules (NCs), 81
82 structural composition, 81f Nanochitosan, 209 Nanoemulsion template method, 81 Nanoencapsulation, 115
116 chitosan for, 207
208 Nanofibers, 257 Nanogels, 82
84 Nanomaterials, 194, 207 Nanoparticles (NPs), 78
80, 80t, 116, 161
162, 168
169, 243
244 Nanosystems, 78
84 nanogels, 82
84 nanoparticles, 78
80 NCs, 81
82 Nanotechnological approaches for application of chitosan, 207
210 chitosan for nanoencapsulation, 207
208

336

Index

Nanotechnological approaches for application of chitosan (Continued) chitosan-based intelligent packaging, 210 chitosan-based nanomaterial for packaging, 208
209 Nanotechnology, 194, 207 Naringenin, 237
238 Natural antimicrobials, 286, 298 coating, 293 substances, 176 Natural antioxidants, 1
2, 39
40 NCs. See Nanocapsules (NCs) Nemipterus japonicas, 212
213 Neonatal streptozotocin-induced diabetic mice (n-STZ mice), 202 Nettle leave extract (NLE), 7 Nitric acid (HNO3), 75 NLE. See Nettle leave extract (NLE) NPs. See Nanoparticles (NPs) NRM spectroscopy. See Nuclear magnetic resonance spectroscopy (NRM spectroscopy) Nuclear magnetic resonance spectroscopy (NRM spectroscopy), 77

O O/W/O. See Oil-in-water-in oil (O/W/ O) Oedaleus decorus, 204 Ohmic heating, 155 Oil-in-water-in oil (O/W/O), 81
82 Organic acids, 52 Organic compounds, 245
246 Organic solvents, 159
160, 239
240, 286
287 Origanum vulgare, 39 Oxidation, 7, 176 Oxygen, 211 permeability properties of pomegranatechitosan-based films, 246
247

P Packaging, chitosan-based nanomaterial for, 208
209 PAL. See Phenylalanine ammonia-lyase (PAL) Parkinson’s diseases, 239

Pathogenic microorganisms, 38, 134
139 PBLLE. See Piper betle Linn. leaf extract (PBLLE) PC. See Procyanidin (PC) PCL. See Polycaprolactone (PCL) PDI. See Polydispersity index (PDI) PE. See Polyethylene (PE) Pectin, 235
236 PEF extraction. See Pulsed electric field extraction (PEF extraction) Pelargonidin, 237
238 Penicillium P. digitatum, 253
254 P. italicum, 216
219, 253
254 Percolation method, 239
240 Pericarp, 239 Peroxidase (POD), 52, 130
131, 179 Peroxide value (PV), 212 Phenolic compounds, 41, 237 Phenolic content of pomegranate-chitosanbased films, 250
251 Phenylalanine ammonia-lyase (PAL), 179 Phenylpropanoids, 34
35 Photobacterium, 211
212 Physicochemical characteristics, 141
143 Pineapple (Ananus comosus), 178 Piper betle Linn. leaf extract (PBLLE), 11 PLA. See Poly (lactic acid) (PLA) Plant extracts, 34 antimicrobial effects, 37
38 antioxidant effects, 41 encapsulated into chitosan-based films, 22
24 essential oils added in coatings, 34
36 antimicrobial effects of, 37
38 antioxidant effects of, 41 plant extract/phytobiotics, 91
93 Plant-based food products, 282 Plastic chitosan-based films, 318 PLE. See Pressurized liquid extraction (PLE) Pleurotus eryngii, 243 POD. See Peroxidase (POD) Poly (lactic acid) (PLA), 2
4 Poly (vinyl alcohol) (PVA), 2
4 Polycaprolactone (PCL), 23
24

Index

Polydispersity index (PDI), 118
122, 244 Polyethylene (PE), 22 Polymer effectiveness, 162 matrices, 31
32, 286 Polynucleotides, 70 Polypeptides, 70 Polyphenol oxidase (PPO), 42, 130
131, 179 Polyphenols, 243
244 Polysaccharides, 70, 171, 195, 197
198, 235
236 Polyunsaturated fatty acids, 237 Pomegranate, 235
236 application and effects of pomegranatechitosan-based films on foods, 253
260 chitosan composite films for incorporating pomegranate active compounds, 242
243 degradation of pomegranate-chitosanbased films, 252
253 encapsulation of pomegranate active compounds in chitosan-based films, 243
244 extraction of pomegranate active compounds, 239
242 fruit, 235
236 functional properties of, 236
239 bioactivity of pomegranate juice, 237
238 bioactivity of pomegranate peel, 238 bioactivity of pomegranate seed, 239 pomegranate-chitosan-based films, 250
252 juice, 235
236 bioactivity of, 237
238 nutritional and chemical properties of, 235
236 schematic picture of, 236f peel bioactivity of, 238
239 extract-chitosan nanoparticles, 244 physicochemical properties of pomegranate-chitosan-based films, 244
250 Pomegranate-chitosan-based films

337

degradation of, 252
253 on foods, application and effects of, 253
260 application in fruits and vegetables, 253
256 application in meat and seafood industry, 256
260 functional properties of, 250
252 antimicrobial property, 251
252 antioxidant property, 251 phenolic content, 250
251 physicochemical properties of, 244
250 mechanical property, 248
249 moisture content, 245 morphology property, 249
250 optical property, 247
248 swelling property, 246 thermal property, 249 thermogravimetric analysis and differential scanning calorimetry, 250 thickness, 245 water solubility, 245
246 water vapor and oxygen permeability properties, 246
247 Potassium hydroxide (KOH), 75 Potential antioxidants, 250
251 Poultry products, antimicrobial activity of chitosan in, 272 PPO. See Polyphenol oxidase (PPO) Preservation as hurdle concept, inclusion of chitosan in combination with, 215
220 Pressurized liquid extraction (PLE), 240 Procyanidin (PC), 14 Prokaryotic cells, 163 Proteins, 171, 235
236 Protocatechuic acid, 237
238 Protons, 33 Pseudomonas spp., 173, 211
212, 214, 258
259, 274
276, 287, 302, 312 P. aeruginosa, 256
257, 270
271 P. fluorescens, 214, 281 P. fragi, 270 Pulsed electric field extraction (PEF extraction), 240, 242 Pulsed light, 155 Punicalagin, 237
238, 250
251

338

Index

Punicalin, 237
238 PV. See Peroxide value (PV) PVA. See Poly (vinyl alcohol) (PVA)

Q QC. See Quaternary ammonium chitosan (QC) Quality parameters effects of coatings on quality parameters and shelf life of food, 36
61 in foods, effects on, 42
61 Quaternary ammonium chitosan (QC), 14 Quercetin, 237
238

R Reactive oxygen species (ROS), 15
16, 39, 205 Ready-to-eat/cook preparations/meals application of edible chitosan solutions or bioactive chitosan coatings in combination with lysozyme on preserving food quality and safety on, 311
318 applications of chitosan and/or bioactive coatings in combination with essential oils on, 298
311 chitosan applications to extend the shelf life or reassure the safety of, 280
284 Real food systems application of chitosan as active packaging material for controlled release of active compounds on food models and on, 318
323 Renewable biopolymer, 32 Rhizopus stolonifer, 163
164 Ripening process, 52 RNA synthesis, 37 ROS. See Reactive oxygen species (ROS) Rutin, 237
238

S Saccharomyces cerevisiae, 270 Saccharomycodes ludwigii, 270 Sage leave extract (SLE), 7 Salmo salar. See Salmon (Salmo salar) Salmon (Salmo salar), 173

Salmonella spp., 87
88, 211
212, 282
283 S. choleraesuis, 204 S. enterica, 272, 296
297 S. enteritidis, 204, 282 S. typhimurium, 204, 270
271 Scanning electron microscopy, 77 Scomberomorus brasiliensis, 212
213 Scytalidium lignicola, 164
165 Seafood, 195 application of pomegranate-chitosanbased films in seafood industry, 256
260 effect of chitosan on acceptability/ sensory quality of, 220
221 impacts of chitosan on seafood safety, 211
215 edible coating, 212
213 film, 214
215 Selenium NPs, 209 Sensory characteristics, 139
141 Sensory evaluation, 220
221 Serratia liquefaciens, 270 Shelf life, 1 chitosan and derivatives’ application to increase shelf life of foods, 169
180 chitosan applications to extend shelf life or reassure safety of food products, and ready-to-eat/cook preparations/ meals, 280
284 extension, 258
259 pathogens, 269 Shewanella, 211
212 S. puterfaciens, 214 Shigella dysenteriae, 270
271 Silica (Si), 23 Silver nanoparticle (AgNP), 16, 204 SLE. See Sage leave extract (SLE) Sodium hydroxide (NaOH), 75 Solanum lycopersicum Mill. See Tomato fruit (Solanum lycopersicum Mill.) Solid-phase extraction, 240, 242 Soluble solids content (SS), 43 Solvent displacement method, 118 Solvent extraction, 239
240 Soxhlet extraction, 239
240 Spectral analysis, 77

Index

Spoilage microorganisms, 36
37, 274
276 Spoilage of foods, 210 SS. See Soluble solids content (SS) Staining, 75 Staphylococcus aureus, 61, 203
204, 237
238, 270
271, 281 Staphylococcus spp., 211
212, 281, 293
294 Streptococcus agalactiae, 204 Succinyl chitosan sodium salt (SC-Na), 6 Sulfuric acid (H2SO2•), 75 Supercritical fluid extraction, 240 Supercritical fluid extraction-carbon dioxide (SC-CO2), 242 Supercritical Fluid Technology, 35 Superoxide anion radicals (O2•-), 205 Synthetic culture media, 158

T TA. See Titratable acidity (TA) TBA. See Thiobarbituric acid (TBA) TBARS. See Thiobarbituric acid reactive substances (TBARS) TBC. See Triazole betaine chitosan (TBC) TE. See Trolox Equivalent (TE) TEER. See Transepithelial electrical resistance (TEER) Tenebrio molitor, 204 TEO. See Turmeric essential oil (TEO) Terrestrial organisms, 197 TG. See Triacylglycerol (TG) Thermogravimetric analysis of pomegranate-chitosan-based films, 250 Thermoplastic cornstarch (TPS), 2
4 Thiobarbituric acid (TBA), 129 Thiobarbituric acid reactive substances (TBARS), 39, 212 Thyme (Thymus vulgaris L.), 179 Thymus kotschyanus, 257 Thymus vulgaris L. See Thyme (Thymus vulgaris L.) TiO2 nanoparticles. See Titanium oxide nanoparticles (TiO2 nanoparticles) Titanium oxide nanoparticles (TiO2 nanoparticles), 15
16 Titratable acidity (TA), 141 TMA. See Trimethylamine (TMA)

339

TMAO. See Trimethylamine-oxide (TMAO) TMVC. See Total mesophilic aerobic viable counts (TMVC) Tomato fruit (Solanum lycopersicum Mill.), 179 Total mesophilic aerobic viable counts (TMVC), 134 Total plate counts (TPC), 311
312 Total soluble solids (TSS), 141, 254
255 Total viable count (TVC), 280 Total Volatile Base Nitrogen (TVB-N), 59 TPC. See Total plate counts (TPC) TPP. See Tripolyphosphate (TPP) TPS. See Thermoplastic cornstarch (TPS) Transepithelial electrical resistance (TEER), 72
73 Triacylglycerol (TG), 200 Triazole betaine chitosan (TBC), 6 Trichiurus haumela. See Hairtail (Trichiurus haumela) Trimethylamine (TMA), 212 Trimethylamine-oxide (TMAO), 212 Tripolyphosphate (TPP), 79, 117
118 Trolox Equivalent (TE), 7 TSS. See Total soluble solids (TSS) Turmeric essential oil (TEO), 23 Turmeric-combined chitosan films, 204 TVC. See Total viable count (TVC)

U Ultrasonic homogenization, 118 Ultrasound, 155 Ultrasound-assisted extraction, 240
241 Ultrastructural effect of chitosan and chitosan nanoparticles, 163
169 Ultraviolet radiation, 155 United States Department of Agriculture, 195

V Vaccinium ashei L. See Blueberry (Vaccinium ashei L.) Vacuum atmosphere, 309
310 packaging, 293 Vacuum microwave-assisted extraction (VMAAE), 240
241

340

Index

Vacuum packaged (VP), 293 Vegetables, 177
180, 283 application of pomegranate-chitosanbased films in, 253
256 Vibrio, 211
212 V. alginolyticus, 204 V. cholerae, 270
271 V. parahaemolyticus, 237
238, 270
271 Vitamins, 235
236 VMAAE. See Vacuum microwave-assisted extraction (VMAAE) VP. See Vacuum packaged (VP)

W W/O/W. See Water-in-oil-in-water (W/ O/W) Water solubility of pomegranate-chitosanbased films, 245
246 Water vapor permeability (WVP), 246
247 properties of pomegranate-chitosanbased films, 246
247 Water vapour, 34 Water-in-oil-in-water (W/O/W), 81
82 Water-soluble chitosan, 292

Water-soluble derivatives, 161 White shrimps (P. vannamei), 212
213 WHO. See World Health Organization (WHO) World Health Organization (WHO), 69
70 WVP. See Water vapor permeability (WVP)

X X-ray diffraction, 77

Y Yeasts, 132
133 Yersinia enterocolitica, 204

Z Zataria multiflora, 258 Zinc oxide nanoparticles (ZnONPs), 21 Zingiber zerumbet EO, 127
128 ZnONPs. See Zinc oxide nanoparticles (ZnONPs) Zygomycetes, 197 Zygosaccharomyces bailii, 270, 282