Food Safety and Protection [1 ed.] 9781315153414, 9781351649452, 9781351639934, 9781498762885, 9781498762878

Table of contents :

Foodborne Pathogens And Infections. Food-Borne Infectious Diseases. Microbial Risks in Animal Products. Foodborne Pathogens in Fruits - A New Trend or Sporadic Incidence. Influence of Climatic Conditions and Climate Change on the Microbial Safety of Food. Microbial Indicators. Preventing Foodborne Illnesses from Donated or Recovered Foods. Molecular Methods to Characterize Microbial Pathogens. Biosensor in Food Born Pathogen S Detection. Application of Next Generation Sequencing (NGS) for Pathogen Identification. Predictive Microbiology for Safe Foods. Designing Microbiologically Safe Microwaveable Foods: Electromagnetic and Microbial Modeling Approaches. Development and Application of Predictive Models for Food Microbiology. Integration of OMIC Data into Microbiological Risk Assessment. Integrative Aspects in Food Safety Risk Assessment. Practical Examples of Predictive Microbiology Used by the Food Industry. Food Allergens, Contaminants and Toxins. Recent Techniques For Reducing Food Allergenicity To Ensure Food Safety. Risk Assessment Of Unintentional Allergen Cross Contact. Food Safety in Organic Foods. Advances in Food Toxicology. Chemical Safety with Special Reference to Food Additives. Metals Exposure in Foods Nanotechnology and its Implications to Food Safety and Human Health. Preservation of Foods. Prediction of Shelf-Life, and Product’s Microbial Quality Using Smart and Non-Invasive Platforms. Changing the Dogma on Meat Shelf Life. Delivery Systems for Introduction of Natural Antimicrobials into Foods: Need, Formulation, Applications and Current Trends. Metagenomics and Food. Food Packaging. New Trends and Risks of Food Packaging. Modified Atmosphere Packaging Systems, Active and Intelligent, and Antimicrobial Packaging Systems. Novel Nanocomposite Based Packaging. Advanced Food Packaging Systems for Enhancing Product Safety and Quality. Food Safety Laws. Food Laws and Regulations. Recent Developments in Food Fraud Prevention. Current Issues in HACCP Training: Making Systems More Effective. Traceability and Labeling of Foods.

Citation preview

Food Safety and Protection 

Food Safety and Protection 

Edited by

V. Ravishankar Rai and Jamuna A.Bai

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 ©  2018 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper International Standard Book Number-13: 978-1-4987-6287-8 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged, please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice:  Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging‑ in‑ P ublication Data  Names: Rai, V. Ravishankar, editor. | Bai, Jamuna A. (Jamuna Aswathanarayn), editor. Title: Food safety and protection / edited by Ravishankar Rai and Jamuna Bai Aswathanarayan. Description: Boca Raton : CRC Press, [2017] | Includes bibliographical references and index. Identifiers: LCCN 2017012632| ISBN 9781498762878 (hardback : alk. paper) | ISBN 9781315153414 (ebook) | ISBN 9781498762885 (ebook) | ISBN 9781351649452 (ebook) | ISBN 9781351639934 (ebook) Subjects: | MESH: Food Safety | Food Packaging--methods | Foodborne Diseases--prevention & control Classification: LCC RA601.5 | NLM WA 695 | DDC 363.19/2--dc23 LC record available at https://lccn.loc.gov/2017012632 Visit the Taylor & Francis Web site at  http://www.taylorandfrancis.com  and the CRC Press Web site at  http://www.crcpress.com 

Contents Preface.......................................................................................................................ix Editors .......................................................................................................................xi Contributors.......................................................................................................... xiii

Section I  Predictive Microbiology for Safe Foods  1. Semiquantitative and Qualitative Assessment for Determination of Sanitary Risk in Food Service Establishments........ 3 Elke Stedefeldt, Laí s Mariano Zanin, Ana Lúcia de Freitas Saccol, Eduardo Cesar Tondo, Veronica Cortez Ginani, Eneo Alves da Silva Jr., Ana Beatriz Almeida de Oliveira, and Diogo Thimoteo da Cunha 2. Fresh-Cut Apples Spoilage and Predictive Microbial Growth under Modified Atmosphere Packaging .................................................. 29 Predrag Putnik and Danijela Bursać  Kovač ević 

Section II  Food Allergens, Contaminants, and Toxins  3. Analytical Methods for the Detection of Mycotoxins in Milk Samples ........................................................................................................... 49 Myra E. Flores-Flores and Elena Gonzá lez-Peñ as 4. Biotoxins in Seafood..................................................................................... 97 Laura P. Rodrí guez, Juan M. Vieites, and Ana G. Cabado 5. Selection and Characterization of Aptamers for Food Contaminant Monitoring........................................................................... 157 Amina Rhouati, Akhtar Hayat, Gaë lle Catanante, and Jean Louis Marty 6. Allergen Management as a Key Issue in Food Safety ........................ 195 Antó nio Raposo, Esteban Pé rez, Catarina Tinoco de Faria, and Conrado Carrascosa 7. Unintentional Contaminants in Food..................................................... 243 M. Carmen Rubio Armendáriz, Arturo Hardisson de la Torre, Ángel J. Gutié rrez Ferná ndez, Dailos Gonzá lez Weller, Consuelo Revert Gironé s, José  M. Caballero Mesa, and Soraya Paz-Montelongo

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Section III  Preservation of Foods  8. Thermal Inactivation Kinetics of Foodborne Pathogens: An Overview....................................................................................................... 271 Corliss A. O’ Bryan, Nathan A. Jarvis, Philip G. Crandall, and Steven C. Ricke 9. Non-Thermal Preservation Technologies for Meat and Fish Products......................................................................................................... 291 Bruna Leal Rodrigues, Denes Kaic Alves do Rosário, and Carlos Adam Conte-Junior 10. Inactivation of Pathogenic Microorganisms in Foods by High Pressure Processing..................................................................................... 341 Evelyn and Filipa Vinagre Marques da Silva 11. Application of Pulsed Light for the Microbial Decontamination of Foods.......................................................................................................... 379 Marija Zunabovic, Victoria Heinrich, and Henry Jä ger 12. Effect of Commercial Emerging Nonthermal Technologies on Food Products: Microbiological Aspects................................................ 397 Elisabete M. C. Alexandre, Rita S. Iná cio, Ana C. Ribeiro, Álvaro Lemos, Sofia Pereira, Só nia M. Castro, Paula Teixeira, Manuela Pintado, Ana M. P. Gomes, Francisco J. Barba, Mohamed Koubaa, Shahin Roohinejad, and Jorge Saraiva

Section IV  Food Packaging  13. Food Packaging Systems with Antimicrobial Agents......................... 431 Reyhan Irkin 14. Active and Intelligent Food Packaging................................................... 459 Cristina Nerin, Paula Vera, and Elena Canellas

Section V  Food Safety Laws  15. Food Fraud: Detection, Prevention, and Regulations.......................... 495 Jamuna A. Bai and V. Ravishankar Rai 16. Food Safety Regulation and Standards.................................................. 531 Nada Smigic and Ilija Djekic

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17. Food Safety Reforms in the United States: The Food Safety Modernization Act (FSMA)....................................................................... 563 Harmit Singh and Holly M. Greene 18. The HACCP in the Current Food Safety Context................................. 595 Juan Garcí a-Dí ez, Dina Moura, Alexandra Esteves, and Cristina Saraiva 19. Recent Developments in Saffron Fraud Prevention ............................ 651 Anastasia Kyriakoudi and Maria Z. Tsimidou Index......................................................................................................................679

Preface Food safety and protection is a multidisciplinary topic that focuses on the safety, quality, and security aspects of food. Food safety issues involve microbial risks in food products, foodborne infections, and intoxications and food allergenicity. Food protection deals with trends and risks associated with food packaging, advanced food packaging systems for enhancing product safety, the development and application of predictive models for food microbiology, food fraud prevention, and food laws and regulations with the aim to provide safe foods for consumers. The book Food Safety and Protection  covers various aspects of food safety, security, and protection. It discusses the challenges involved in the prevention and control of foodborne illnesses due to microbial spoilage, contamination, and toxins. The book covers new and safe food intervention techniques, predictive food microbiology, and modeling approaches. It deliberates the legal framework, regulatory agencies, and laws and regulations for food protection. The book has five sections dealing with the topics of predictive microbiology for safe foods; food allergens, contaminants, and toxins; preservation of foods; food packaging; and food safety laws. Section  I, on predictive microbiology for safe foods, covers qualitative and quantitative methods for determining the sanitary risk in food services and predictive microbial growth in fresh foods. Section II, on food allergens, contaminants, and toxins, discusses analytical methods for the analysis of mycotoxins in milk, biotoxins in seafood, aptamer designs for food contaminant monitoring, control and management of allergens in foods, and assessment of unintentional contaminants in foods. Section III, on preservation of foods, comprehensively explores developments in food intervention techniques and covers topics such as thermal inactivation kinetics of foodborne  pathogens, nonthermal technologies for the preservation of meat and fish products, high-pressure  processing and ultrasound techniques to inactivate spores in foods, pulsed field techniques, and other emerging nonthermal technologies for food preservation. Section IV, on food packaging, deals with the technological development of antimicrobials, and active, smart, and intelligent packaging for safe foods. Section V, on food safety laws, deals with food fraud— detection, prevention, and regulations; food safety— regulation and standards; the Food Safety Modernization Act; and Hazard Analysis and Critical Control Point. Food Safety and Protection  covers both basic and applied aspects of food safety, hygiene, and protection. Important topics in the multidisciplinary field have been thoroughly and comprehensively covered by international experts in the subjects. The book will be a valued reference for food microbiology

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Preface

students, and researchers, scientists from industries, and food policy makers who are striving to make safe foods available for consumers. I would like to thank all the authors for contributing the chapters and sharing their expertise. Prof. V. Ravishankar Rai  Dr. Jamuna A. Bai 

Editors  V. Ravishankar Rai  earned his MSc and PhD from the University of Mysore, India. Currently, Dr. Rai is working as a professor in the Department of Studies in Microbiology, University of Mysore, India. He was awarded fellowships from the UNESCO Biotechnology Action Council, Paris (1996); the Indo-Israel Cultural Exchange Fellowship (1998); the Biotechnology Overseas Fellowship, government of India (2008); the Indo-Hungarian Exchange Fellowship (2011); and the Indian National Academy Fellowship (2015). Presently, he is the coordinator for the Department of Science and Technology, Promotion of University Research and Scientific Excellence and University Grants Commission innovative programs. Jamuna A. Bai  has earned her MSc and PhD in microbiology from the University of Mysore, India. She is working as a researcher in the University Grants Commission (UGC)-sponsored University with Potential Excellence Project, University of Mysore, India. She has previously worked as an Indian Council of Medical Research (ICMR) senior research fellow on food safety, the role of quorum sensing and biofilms in food-related bacteria, and developing quorum-sensing inhibitors. Her research interests also include the antimicrobial application of functionalized nanomaterials against foodborne pathogens.

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Contributors

Elisabete M.C. Alexandre  Department of Chemistry  University of Aveiro  Aveiro, Portugal  and  Universidade Cató lica Portuguesa  Escola Superior de Biotecnologia  Porto, Portugal  M. Carmen Rubio Armendá riz  Á rea de Toxicologí a Universidad de La Laguna Tenerife, Canary Islands, Spain Jamuna A. Bai  Department of Studies in Microbiology University of Mysore Karnataka, India Francisco J. Barba  Faculty of Pharmacy Universitat de Valè ncia Valè ncia, Spain Ana G. Cabado  Food Safety Division— IDi. ANFACO-CECOPESCA Carretera Colexio Universitario Vigo, Spain Elena Canellas  Departamento de Quí mica Analí tica Universidad de Zaragoza Campus Rio Ebro Zaragoza, Spain

Conrado Carrascosa  Department of Animal Pathology and Production Universidad de Las Palmas de Gran Canaria Arucas, Spain Só nia M. Castro  Department of Chemistry  University of Aveiro  Aveiro, Portugal  and  Escola Superior de Biotecnologia Universidade Cató lica Portuguesa  Porto, Portugal  Gaë lle Catanante  Biocapteurs-AnalysesEnvironnement Université  de Perpignan Via Domitia Perpignan, France Carlos Adam Conte-Junior  Department of Food Technology Universidade Federal Fluminense and Food Science Program Universidade Federal do Rio de Janeiro Rio de Janeiro, Brazil

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Contributors

Philip G. Crandall  Department of Food Science and Center for Food Safety University of Arkansas Fayetteville, Arkansas

Myra E. Flores-Flores  Organic and Pharmaceutical Chemistry Department University of Navarra Pamplona, Navarra, Spain

Diogo Thimoteo da Cunha  Faculdade de Ciê ncias Aplicadas Universidade de Campinas Limeira, Brazil

Ana Lú cia de Freitas Saccol  Curso de Nutriç ã o Centro Universitá rio Franciscano Santa Maria, Brazil

Ilija Djekic  Department of Food Safety and Quality Management University of Belgrade Belgrade, Serbia Alexandra Esteves  Centre of Studies in Animal and Veterinary Science, DCV-ECAV University of Trá s-os-Montes e Alto Douro Vila Real, Portugal

Juan Garcí a-Dí ez  Centre of Studies in Animal and Veterinary Science (CECAV), DCV-ECAV University of Trá s-os-Montes e Alto Douro Vila Real, Portugal Veronica Cortez Ginani  Departamento de Nutriç ã o Universidade de Porto Alegre Porto Alegre, Brazil

Evelyn  Department of Chemical Engineering University of Riau Pekanbaru, Indonesia

Consuelo Revert Gironé s  Á rea de Toxicologí a Universidad de La Laguna Tenerife, Canary Islands, Spain

Catarina Tinoco de Faria  Department of Animal Pathology and Production Universidad de Las Palmas de Gran Canaria Arucas, Spain

Elena Gonzá lez-Peñ as  Organic and Pharmaceutical Chemistry Department University of Navarra Pamplona, Navarra, Spain

Á ngel J. Gutié r rez Ferná ndez  Á rea de Toxicologí a Universidad de La Laguna Tenerife, Canary Islands, Spain

Ana M. P. Gomes  Universidade Católica Portuguesa Escola Superior de Biotecnologia Porto, Portugal

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Contributors

Holly M. Greene  Don B. Huntley College of Agriculture California State Polytechnic University, Pomona Pomona, California Akhtar Hayat  Biocapteurs-AnalysesEnvironnement Université  de Perpignan Via Domitia Perpignan, France and Interdisciplinary Research Centre in Biomedical Materials COMSATS Institute of Information Technology Lahore, Pakistan Victoria Heinrich  Section of Packaging and Resource Management University of Applied Sciences Vienna, Austria Rita S. Iná cio  Department of Chemistry  University of Aveiro  Aveiro, Portugal  and  Universidade Cató lica Portuguesa  Escola Superior de Biotecnologia  Porto, Portugal 

Reyhan Irkin  Department of Food Engineering University of Balikesir Balikesir, Turkey Henry Jä ger  Department of Food Science and Technology University of Natural Resources and Life Sciences Vienna, Austria Nathan A. Jarvis  Department of Food Science and Center for Food Safety University of Arkansas Fayetteville, Arkansas Mohamed Koubaa  Centre de Recherche de Royallieu Université  de Technologie de Compiè gne Compiè gne, France Danijela Bursać  Kovač ević   Faculty of Food Technology and Biotechnology University of Zagreb Zagreb, Croatia Anastasia Kyriakoudi  School of Chemistry Aristotle University of Thessaloniki Thessaloniki, Greece Á lvaro Lemos  Department of Chemistry  University of Aveiro  Aveiro, Portugal 

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Jean Louis Marty  Biocapteurs-Analyses-Environnement Université  de Perpignan Via Domitia Perpignan, France José  M. Caballero Mesa  Á rea de Toxicologí a Universidad de La Laguna Tenerife, Canary Islands, Spain Soraya Paz-Montelongo  Á rea de Toxicologí a Universidad de La Laguna Tenerife, Canary Islands, Spain Dina Moura  Direç ã o de Serviç os de Alimentaç ã o e Veteriná ria da Regiã o Norte Direç ã o Geral de Alimentaç ã o e Veteriná ria Guimarã es, Portugal Cristina Nerin  Departamento de Química Analítica Universidad de Zaragoza Zaragoza, Spain Corliss A. O’ Bryan  Department of Food Science and Center for Food Safety University of Arkansas Fayetteville, Arkansas Ana Beatriz Almeida de Oliveira  Universidade Federal do Rio Grande do Sul Departamento de Nutriç ã o Porto Alegre, Brazil

Contributors

Sofia Pereira  Department of Chemistry  University of Aveiro  Campus Universitá rio de Santiago  Aveiro, Portugal  Esteban Pé rez  Department of Animal Pathology and Production Faculty of Veterinary Universidad de Las Palmas de Gran Canaria Arucas, Spain Manuela Pintado  Escola Superior de Biotecnologia Universidade Cató lica Portuguesa  Porto, Portugal  Predrag Putnik  Faculty of Food Technology and Biotechnology University of Zagreb Zagreb, Croatia V. Ravishankar Rai  Department of Studies in Microbiology University of Mysore Karnataka, India Antó nio Raposo  CBIOS (Research Center for Biosciences and Health Technologies) Universidade Lusó fona de Humanidades e Tecnologias Lisbon, Portugal

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Contributors

Amina Rhouati  Biocapteurs-AnalysesEnvironnement Université  de Perpignan Via Domitia Perpignan, France and Ecole Nationale Supé rieure de Biotechnologie Constantine, Algeria Ana C. Ribeiro  Department of Chemistry  University of Aveiro  Aveiro, Portugal  Steven C. Ricke  Department of Food Science and Center for Food Safety University of Arkansas Fayetteville, Arkansas Bruna Leal Rodrigues  Department of Food Technology Universidade Federal Fluminense Rio de Janeiro, Brazil Laura P. Rodrí guez  Food Safety Division— IDi. ANFACO-CECOPESCA Carretera Colexio Universitario Vigo, Spain Shahin Roohinejad  Division of Food and Nutrition Shiraz University of Medical Sciences Shiraz, Iran Denes Kaic Alves do Rosá rio  Chemistry Institute Universidade Federal do Rio de Janeiro Rio de Janeiro, Brazil

Cristina Saraiva  Centre of Studies in Animal and Veterinary Science DCV-ECAV University of Trá s-os-Montes e Alto Douro Vila Real, Portugal Jorge Saraiva  Department of Chemistry  University of Aveiro  Aveiro, Portugal  Eneo Alves da Silva Jr.  Central de Diagnó sticos Laboratoriais (CDL) Sã o Paulo, Brazil Filipa Vinagre Marques da Silva  Chemical and Materials Engineering Department University of Auckland Auckland, New Zealand Harmit Singh  Don B. Huntley College of Agriculture California State Polytechnic University, Pomona Pomona, California Nada Smigic  Department of Food Safety and Quality Management University of Belgrade Belgrade, Serbia Elke Stedefeldt  Centro de Desenvolvimento do Ensino Superior em Saú de (CEDESS) Universidade Federal de Sã o Paulo Sã o Paulo, Brazil

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Paula Teixeira  Escola Superior de Biotecnologia  Universidade Cató lica Portuguesa  Porto, Portugal  Eduardo Cesar Tondo  Instituto de Ciê ncia e Tecnologia dos Alimentos Universidade Federal do Rio Grande do Sul Porto Alegre, Brazil Arturo Hardisson de la Torre  Á rea de Toxicologí a Universidad de La Laguna Tenerife, Canary Islands, Spain Maria Z. Tsimidou  School of Chemistry Aristotle University of Thessaloniki Thessaloniki, Greece Paula Vera  Departamento de Química Analítica Universidad de Zaragoza Zaragoza, Spain

Contributors

Juan M. Vieites  Food Safety Division— IDi. ANFACO-CECOPESCA Carretera Colexio Universitario Vigo, Spain Dailos Gonzá lez Weller  Á rea de Toxicologí a Universidad de La Laguna Tenerife, Canary Islands, Spain Laí s Mariano Zanin  Programa de Pós-Graduação em Nutriç ã o Universidade Federal de São Paulo São Paulo, Brazil Marija Zunabovic  Department of Food Science and Technology University of Natural Resources and Life Sciences Vienna, Austria

Section I

 Predictive Microbiology for Safe Foods 

1 Semiquantitative and Qualitative Assessment for Determination of Sanitary Risk in Food Service Establishments Elke Stedefeldt, Laí s Mariano Zanin, Ana Lúcia de Freitas Saccol, Eduardo Cesar Tondo, Veronica Cortez Ginani, Eneo Alves da Silva Jr., Ana Beatriz Almeida de Oliveira, and Diogo Thimoteo da Cunha CONTENTS 1.1 Introduction..................................................................................................... 4 1.2 Sanitary Risk in Food Service Setting......................................................... 5 1.3 Risk Assessment.............................................................................................6 1.4 Foodborne Diseases in Brazilian Food Services: Etiological Agents, Food Vehicles, and Contamination Sources.................................7 1.5 Sources of Contamination in Food Services...............................................9 1.6 Causal Factors of FBD.................................................................................. 10 1.7 Use of Inspection Scores to Semiquantitatively Evaluate Food Safety in Food Service Establishments...................................................... 13 1.8 Systematization of Qualitative Method of Risk Assessment.................. 18 1.9 Projects Undertaken in the Context of Sanitary Risk in Food Service Environments.................................................................................. 20 1.9.1 Project 1: Pilot Project Food Service Establishment Categorization................................................................................... 20 1.9.1.1  Project Participants.............................................................. 20 1.9.1.2 Description............................................................................ 20 1.9.2 Project 2: Complementary Technical Assistance Project to the Support Project for the Development of a School Feeding Program in Mozambique: ����������������������������������������������22 1.9.2.1  Project Participants..............................................................22 1.9.2.2 Description............................................................................22 1.10 Final Consideration...................................................................................... 23 References................................................................................................................ 24

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Food Safety and Protection

1.1 Introduction According to the World Health Organization (WHO) (2015), approximately 600 million people become ill after consuming contaminated food every year. Of these victims, an estimated 420,000 die, including 125,000 children under the age of 5 years. Even with the use of advanced food technologies and the investment of substantial financial resources and time, foodborne disease (FBD) is still one of the most important public health problems in the world, as it is a threat to public health and global socioeconomic development. Havelaar et al. (2015) have suggested that the strategies adopted by the food industry to prevent and/or control the survival, proliferation, and contamination of microorganisms in food are inadequate. Even though several FBD outbreaks have been attributed to food processing and production, food service establishments have been associated as well, and in Brazil, they are the public locales where the majority of reported FBD outbreaks have occurred (Brazil 2016). The prevention of microorganism contamination, survival, and proliferation in food is a necessity recognized in all existing regulations and standards and by all responsible professionals and stakeholders. In the United States, for example, the Food Safety Modernization Act (FSMA), established in 2011, consolidated existing laws and changed the focus in food safety promotion from a reactive to a preventive perspective (Grover et al. 2016). The objective of this act was to implement the Hazard Analysis and Riskbased Preventive Controls (HARPC) tool, which focuses on food safety throughout the entire food chain. To support this objective, HARPC was based on other food safety management systems (FSMSs), such as good hygiene practices (GHPs), the Hazard Analysis and Critical Control Point (HACCP), ISO 22000:2005, the Safe Quality Food (SQF) Code (Safe Quality Food Institute), and Global Food Safety Initiative (GFSI) guidelines (Food and Drug Administration 2013). The complexity of these tools requires qualified persons to assess potential hazards in each establishment and identify and implement preventive and control measures by scientifically validated methodologies (Grover et al. 2016). However, it is recognized that the variety among and peculiarities within different food service establishments present a challenge for small businesses to adopt quality systems with the scope described. The difficulty of implementing the Good Handling Practices Program as a prerequisite to HACCP is noteworthy. Research conducted by Grover et al. (2016) indicates that the understanding of tax regulations, implementation costs, deadlines set by law, lack of staff training, and a food safety culture to integrate this preventive approach are important challenges to the implementation of quality systems for small food service establishments, which should not be underestimated.

Semiquantitative and Qualitative Assessment

5

These difficulties may prevent small businesses from adopting quality systems in practice, threatening the consumers’  health. This chapter aims to contribute to the discussion of the application of risk assessment for FBD in food service establishments, using semiquantitative and qualitative methods and considering the main factors associated with these diseases.

1.2  Sanitary Risk in Food Service Setting Established as a global phenomenon, food service has become the main alternative to home food preparation for many people with long commutes or who seek convenience. It is estimated that the food service industry has a global revenue of billions of dollars a year. Food service establishments are facilities that serve meals and snacks for immediate consumption at a particular location, that is, eating out. They include full-service restaurants, fast-food restaurants, catering, cafeterias, food trucks, and other places that prepare, serve, and sell food to the general public for profit. Some, such as hostels, recreational facilities, shopping centers, and retail stores, are located in places that are not dedicated solely to the distribution of meals and snacks (U.S. Department of Agriculture 2015). The food globalization process has had a significant impact on the safety of food sold in food service establishments. This globalization process has focused on production, distribution, and marketing on a large scale and seeks to meet the needs of the expanding global population. An asymmetry of information in the globalization of food, however, can lead to market failures, and standardization may be an impediment to the food sovereignty of a country. Food service is a multifaceted scenario containing complex situations in which the risks are multifactorial and may be associated with some uncertainty and/or ambiguity. Risk analysis in this scenario becomes a challenge but is essential. The use of different ingredients and additives resulting from globalization may potentiate this risk. In the governmental sphere, the Brazilian Health Surveillance Agency uses the concept of sanitary risk to regulate, control, and monitor the production and consumption of products and services related to health. The variety of potential risks indicate the need for a permanent analysis strategy that involves producers, suppliers, food supplier professionals, and the public. The pertinent literature, however, is still scarce, especially considering the magnitude of the associated sanitary risk (Silva and Lana 2014). The sanitary risk relates to the possibility of a health threat incident, and understanding sanitary risks involves a cause– effect examination and demands a careful observation of the conditions and circumstances

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Food Safety and Protection

in which they occurred, as well as of their resulting outcomes. In this sense, contextual and environmental diversity may reflect the intensity of risk (Silva and Lana 2014). As a threat resulting from an activity, service, or substance to the quality of life of a given population, a sanitary risk involves— i n addition to an assessment of objective evidence of health damage from exposure to hazards— t he social, economic, and political environment in which it occurs. The integration of these considerations defines the risk management approach and allows the establishment of effective strategies to combat risk (Agê ncia Nacional de Vigilâ ncia Sanitá ria 2015; Silva and Lana 2014). The management of sanitary risk is complex and broad in nature. It involves a direct and specific association with the regulation field, as well as strong and diverse connections to other related fields. These characteristics result in the need for a management strategy that may be shared, can be integrated daily, and is permanent and participatory (Oliveira 2013). Monitoring and control present a central challenge in the management of the products, services, and processes associated with sanitary risk. It is advisable, therefore, to strengthen interinstitutional cooperation, work in networks, and seek innovations through different initiatives and articulations (Oliveira 2013). Understanding sanitary risk in the food service setting involves predictions of “ health threat,”  “ vulnerability of human health,”  and “ likelihood of harm”  because the characteristics and consequences of risks are not always known, and risk factors are not always identified. It seems clear that the notion of risk as a probability does not always apply to sanitary risks in the food service setting because only the results of the hazards that are already known can be predicted. When dealing with the issue of sanitary risk, this complexity also adds an unknown element to “ sanitary risk”  (Universidade Federal do Ceará  2015). In the food service arena, there is a gap in the approach to sanitary risk. Risk analysis is an iterative and continuous process, consisting of three components: risk management, risk assessment, and risk communication. It can be performed with varying degrees of detail, depending on the risk itself, the purpose of the analysis and information, and available data and resources (OPAS 2008).

1.3  Risk Assessment Risk assessment is the central scientific component of risk analysis. It is the qualitative and/or quantitative characterization of risk and the estimation of the potential for adverse health effects associated with exposure of an individual or a population to a hazard. It was developed primarily to meet the

Semiquantitative and Qualitative Assessment

7

need for information to facilitate decision making aimed at protecting health in a context of scientific uncertainty (OPAS 2008). The risk assessment process consists of four steps: hazard identification, hazard characterization, exposure assessment, and risk characterization. It consists of search, recognition, and description, that is, identification of sources, forms of interaction of these sources, and potential consequences. It can involve historical data, secondary data from scientific publications, expert opinions, and information regarding the needs and procedures of stakeholders (Universidade Federal do Ceará  2015). Risk assessment results may be quantitative, qualitative, or semiquantitative (Manning and Soon 2013). Quantitative risk assessment presents the results numerically, qualitative assessment presents the results in descriptive terms (high, medium, and low), and semiqualitative assessment presents the results through scores or ranking (Manning and Soon 2013; OPAS 2008). According to World Health Organization (2009), “ semi-quantitative risk assessment is a relatively new idea in food safety. Codex Alimentarius Commission and others generally consider just two categories of risk assessment: qualitative and quantitative. Semi-quantitative risk assessment has often been grouped together with qualitative risk assessment, but this understates the important differences between them in their structure and their relative levels of objectivity, transparency, and repeatability.”  Concerning sanitary risk assessment, professionals should rely on prior, empirical, and technical knowledge and norms to identify risks and act on them; strategies based on analysis shall contribute positively to mitigation, prevention, and elimination of risk (Silva and Lana 2014). The impact of these actions may represent a decisive contribution to and fundamental catalyst for the effective management of sanitary risk, and particularly the production of a timely, agile, and powerful sanitary risk communication, which is important and expected by the general population (Oliveira 2013). A very important starting point in this setting is a knowledge of the causative microorganisms, food vehicles, and sources of contamination related to FBD in food service establishments in a given country.

1.4 Foodborne Diseases in Brazilian Food Services: Etiological Agents, Food Vehicles, and Contamination Sources Brazilian FBDs are investigated by the sanitary surveillance services and epidemiological services of municipalities and states. In general, the sanitary surveillance officers of Brazilian municipalities investigate notifiable

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FBD outbreaks, while the state officers give them technical support and are involved, if necessary, in the investigation of complex outbreaks. For the investigation, officers collect food samples, interview involved people, and send the information to the epidemiological surveillance officers, who analyze and interpret the FBD outbreak data. The information is finally sent to the Brazilian National Health Authority, who annually report these data. From January 2000 to July 2016, the Ministry of Health registered 11,051 foodborne outbreaks, with approximately 2.1 million people exposed. Among them, more than 209,000 became sick and 169 people died (Brazil 2015, 2016). Even with very proactive sanitary surveillance services in Brazil, these numbers represent only the “ tip of the iceberg”  concerning FBD because, as occurs in other countries, outbreaks are not always reported, which makes it difficult to know the true incidence of FBD. Considering the foodborne illnesses from 2000 to 2015 that were reported in Brazil, food service establishments were the second most frequently identified FBD location of origin (24.2%), second only to private homes (38.4%), where surveillance services are not allowed to act. The types of food service establishments most frequently reported as the location of origin for these diseases were commercial restaurants, bakeries, and similar establishments (15.5%); schools (8.7%); workplace catering (8.2%); and events (3.6%). The categories “ unknown site”  and “ other establishments”  accounted for 11.3% and 8.3%, respectively. The data from Brazil thus suggest that households are the sites where most FBD outbreaks originate (38.4%) (Brazil 2016), unlike the United States, where restaurants were the places most frequently reported, accounting for 65% of FBD cases (Silva 2016). Although data on FBD outbreaks are underreported, available data may help to indicate differences in eating behaviors that characterize each society, and therefore the priority actions for sanitary risk prevention. In Brazil, the most frequently reported food vehicles of foodborne pathogens were those often prepared in food service establishments, that is, mixed foods (14.1%), eggs and egg products (mainly homemade mayonnaise, 7.8%), and creamy sweets and desserts (2.1%). Water, milk and milk products, red meat and pork meat; poultry were responsible for 6.0%, 2.6%, 2.1%, and 1.4%, respectively. Leafy greens were responsible for only 0.8% of investigated outbreaks. Among the reported Brazilian FBD, Salmonella was the pathogen most frequently identified as the causative agent, accounting for 14.4% of outbreaks, followed by Staphylococcus aureus (7.7%), Escherichia coli (6.5%), and Bacillus cereus (3.1%). Additionally, 58% of the outbreaks did not have an etiological agent identified, highlighting the importance of risk assessment in the sanitary context. An in-depth investigation of the most important Brazilian food pathogens has demonstrated that microorganism strains have been more frequently transmitted and become dominant in some regions. For example, Tondo

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et al. (2015) demonstrated that a specific strain of Salmonella Enteritidis (called SE86) was the major causative agent of salmonellosis in southern Brazil from 1999 to 2013; among reported outbreaks, food service establishments were the most frequently reported location, second only to private homes. The most important food vehicles were homemade mayonnaise made with raw eggs, pastry products, meat, processed meat, chicken, and pork. The majority of these salmonellosis cases occurred during spring, when people may keep food outside of the refrigerator due to the sensation of cold weather in the mornings. The molecular patterns of SE86 were highly similar to those of microorganisms isolated from food vehicles (Oliveira et al. 2007, 2009) and FBD cases (Oliveira et al. 2012) identified in these outbreaks. Further, the SE86 strain was more resistant than other isolated Salmonella species to common sanitizers used in food industries and food services in Brazil (Machado et al. 2010; Tondo et al. 2010). In the same Brazilian state, S. aureus has been identified as the second most frequent etiological agent of foodborne illnesses since the 1990s. Considering the official epidemiological data on S. aureus food poisoning, Lima et al. (2013) reported that S. aureus was identified as the etiological agent of 57 foodborne outbreaks during 2000– 2002. Among these, 42 outbreaks were confirmed by microbiological analyses, and 15 were confirmed by clinical symptoms and/or epidemiological data. Staphylococcal outbreaks were responsible for illness in 5991 people, of whom 1940 (32%) were interviewed by the sanitary surveillance officers. The food vehicles most frequently identified were meat servings (35%), pastries (25%), cheese (23%), pasta (11%), and potato salad with homemade mayonnaise (11%). The majority of the staphylococcal outbreaks occurred inside private homes (33%), followed by food services (28%).

1.5  Sources of Contamination in Food Services Different sources of microbial contamination exist in food service establishments, which may result in cross-contamination of foods and FBD outbreaks. Cleaning cloths and sponges are well-known contamination sources. For example, Bartz and Tondo (2013) investigated the microbial contamination of 35 cleaning cloths collected inside 13 food service establishments in Brazil and observed contamination levels of 6.9, 6.2, and 5.5 log/cm2 for heterotrophic microorganisms, coliforms, and S. aureus, respectively. The same authors demonstrated that boiling the cloths for 15 minutes or washing and disinfecting them using 200 ppm sodium hypochlorite for 15 minutes resulted in a 5-log reduction of bacterial counts. In another study, Bartz et al. (2010) inoculated S. aureus ATCC 25923, E. coli ATCC 25972, and other pathogens that have caused FBDs in Brazilian food service establishments

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(S. Enteritidis and Shigella sonnei) on cleaning cloths and disposable cloths with added organic material and humid conditions. Cloths were incubated at 30° C, and microbial growth was monitored. None of the microorganisms were able to growth on cloths in an initial period of 2 hours; however, the number of pathogenic S. Enteritidis rapidly increased after 3 hours. The authors suggested that disposable or cleaning cloths should not be used for periods longer than 3 hours in food service establishments. Rossi et al. (2012) evaluated the microbiological contamination of 80 kitchen sponges used in Brazilian food service establishments. The results demonstrated that the sponges were contaminated with heterotrophic microorganism counts ranging from 3.4 to 10.4 log CFU/sponge, with an average of 9.1 log CFU/sponge, and fecal coliforms were found in 76.25% of the sponges, with average counts of 8.4 log CFU/sponge. Salmonella and S. aureus were found in 2.5% of samples. After bacterial evaluation, sponges were submitted to boiling for 5 and 10 minutes or to disinfection using 200 ppm sodium hypochlorite for 15 minutes. Both methods significantly reduced bacterial counts, but the boiling method showed a greater reduction than disinfection by sodium hypochlorite. Recently, Kothe et al. (2016) investigated the hygienic conditions of hotdogs collected from street food vendors in Brazil. The results demonstrated that of the 20 hotdog samples, 75% were contaminated with coliforms, 30% were contaminated with fecal coliforms, and 25% had coagulase-positive staphylococci levels above the maximum limit permitted by Brazilian regulations. These results also demonstrated that the majority of vendors defrosted sausages at ambient temperature or employed inadequate cooling. None of the vendors had a thermometer, and several of them used nonpotable water. Other frequent violations were the lack of cross-contamination preventive measures, the lack of time and temperature control, and the use of ingredients of unknown origin.

1.6  Causal Factors of FBD Outbreak investigations, along with laboratory research, are conducted to provide the necessary evidence to understand the occurrence of FBD. Thus, it is possible to identify the four main factors that can cause FBD outbreaks in different parts of the world. They encompass time and temperature control; food contamination by handlers, utensils, and equipment; use of contaminated water and raw ingredients; and indirect contamination (Da Cunha et al. 2016). These causal factors represent situations where FBD can be avoided by risk mediation according to the context of its occurrence. Scientific knowledge addresses theory as it relates to the fact, but this knowledge must be

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translated and integrated into objective actions. Thus, identifying the foods and processes involved in FBD contamination enables the determination and avoidance of the potential dangers, and evaluation of sanitary risk must emphasize the main causal factors through every stage of the food handling process. In a different context, in a study of public schools and daycare centers in Baixada Santista, Brazil, it was observed that other strategic actions were required to guarantee food safety. Scores were used to determine the risk for these strategic actions— a n innovative, effective way to evaluate the food safety practices related to school meal preparation and facilitate the implementation of corrective measures. Among the evaluated establishments, 62% were classified as average sanitary risk, failing to comply with Brazilian food safety legislation. Violations relating to hand hygiene, pest control, food handlers, and food hygiene were most frequently identified. These results reveal the absence of an effective system to monitor and evaluate the sanitary conditions of food service establishments (Da Cunha et al. 2014b). Data on FBD outbreaks in the French Armed Forces called attention to the differences between the hygiene regulations of different countries. In that study, the number of officers affected by FBD that were in their country of origin was compared with the number of those affected during service in other countries. The proportion of cases that occurred overseas was 48.3%. The incidence rate was 2.4 outbreaks/100,000 in France and 26.7/100,000 overseas, with a maximum rate of 39.3/100,000 in Africa. This indicates that places without regulated and monitored sanitary measures may have higher rates of FBD (Mayet et al. 2011). A mistaken idea is that often regular inspections may keep the establishments in strict adherence to guidelines can be directly associated with the prediction of FBD outbreaks and needs clarification. On the other hand, the categorization systems based on sanitary risk and commitment of food service establishments to the process may improve the sanitary conditions of their establishments, as observed by Da Cunha et al. (2016) in practice. Another aspect of sanitary risk in food service establishments, identified by Murphy et al. (2011), is the difference between chain and independent restaurants’  concerns regarding food safety, which is similar in quantity but different in quality. A study conducted in Orange County, Florida, investigated the association of mandatory food safety certification with the inspection results found in chain and independent restaurants; the conclusion was that independent restaurants had more critical violations. A recurrent assertion regarding FBD outbreaks has been the importance of the food handler and the need to empower him or her. Medeiros et al. (2012) observed that the actions of human resources administrations in commercial restaurants affected food safety. This study, conducted in three types of food service establishments in Brazil (buffets by weight, fast-food restaurants, and

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steakhouses or Brazilian barbecues), suggested that investment in the workforce should be a priority in the food sector, as the food handler is often the principal source of food contamination. In addition to the focus on establishments and their food handlers as risks to food safety, food raw materials may also play a role in the transmission of FBD. Concern about fresh products, for example, is ongoing due to the association between numerous FBD outbreaks related to their intake and the increased consumption, large-scale production, and widespread distribution of these foods. Regardless of how and when food contamination happens, its sanitation is compromised if the pathogens are not eliminated by conventional methods (Olaimat and Holley 2012). Alternative methods for pathogen control on fresh produce, such as irradiation, ozone, bacteriophages, and antagonistic bacteria, may not be possible for the majority of food service establishments, even if they participate in a specialized partnership to ensure food safety. As a consequence, the number of FBD outbreaks associated with fresh products has increased rapidly worldwide. Therefore, the importance of purchasing products with assured quality is evident (Olaimat and Holley 2012). In food handling procedures, it is clear, technically, that it is essential to ensure time and temperature control during the preparation of foods as a basic rule to prevent biological hazards and decrease the risk of FBD transmission. Of the outbreaks studied by Silva (2016), 75% were related to microbial growth in food due to either leaving it at temperatures between 10° C and 60° C for longer than 4 hours or using leftovers, 3.6% were due to crosscontamination, and 21.4% of the causes were unknown. In general, FBD outbreaks relate to factors that contribute to the contamination, survival, and proliferation of microorganisms in food. Regarding contamination, the data suggest that of the reported outbreaks, 81.7% were related to contaminated raw food, 55% were due to contaminated food handlers, and 36% were due to contaminated utensils. Regarding contaminant survival, 41.2% of cases were related to food temperatures not reaching 70° C during cooking, and 11.3% to not reaching 70° C during reheating. Regarding contaminant proliferation, 79.2% of cases were related to storing food at temperatures above 10° C, and 83.5% were related to food being exposed to temperatures between 10° C and 60° C for more than 2 hours (Silva 2016). To gain a better understanding of the global state of FBD, the European Food Safety Authority (EFSA) established the Emerging Risks Exchange Network (EREN), composed of organizations that are world leaders in food safety. The EREN is made up of delegates from Member States (MSs) and Norway designated through the advisory forum of EFSA and observers from the European Commission, EU preaccession countries, the Food and Drug Administration of the United States, and the Food and Agricultural Organization of the United Nations (Costa et al. 2016). The exchange of information within the EREN relates to potential emerging problems in the different areas of the food sector, with a particular focus

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on microbiological and chemical hazards. However, discussions on the agenda also include the effect of illegal activity on the food sector, new consumer trends, biotoxins, new technologies and processes, allergens, animal health, environmental pollution, new analytical methods, packaging and technology, and unknown dangers (Costa et al. 2016). The variety of issues contributing to FBD outbreaks require prevention efforts to have different approaches. The manner in which hazards are handled, particularly those related to microorganisms, must be contextualized. The aim must be to address food safety at all stages of food production to produce actions that can objectively prevent microorganism survival, proliferation, and contamination.

1.7 Use of Inspection Scores to Semiquantitatively Evaluate Food Safety in Food Service Establishments The food safety evaluation process of food service establishments must take place in a continuous and strategic way to prevent FBD. Misguided or misunderstood evaluations, however, can lead managers and researchers to misleading conclusions. Quantitative risk assessment is widely used in the food sector and includes the use of the following tools: the HACCP, Quantitative Microbiological Risk Assessment (QMRA), and Fuzzy Risk Assessment Tool (FRAT). In 1995, the World Trade Organization introduced the concept of appropriate level of protection (ALOP) in the Agreement on Sanitary and Phytosanitary Measures (SPS Agreement), which is defined as “ the level of protection deemed appropriate by the Member establishing a sanitary or phytosanitary measure to protect human, animal and plant life or health within its territory”  (World Trade Organization 1995). At a later stage, Food Safety Objectives (FSOs) were introduced to translate the ALOP into a benchmark in the food chain, which is defined as “ the maximum frequency and/ or concentration of a hazard in food at the time of consumption that provides or contributes to the appropriate ALOP”  (International Commission on Microbiological Specifications for Foods 2006). The World Trade Organization Committee on Sanitary and Phytosanitary Measures notes some advantages of the quantitative expressions of risk: “ Quantitative terms, where feasible, to describe the appropriate level of protection can facilitate the identification of arbitrary or unjustified distinctions in levels deemed appropriate in different situations …  use of quantitative terms and/or common units can facilitate comparisons”  (World Health Organization 2009). An ALOP can be expressed in a range of terms, for instance, from broad public health goals to a quantitative expression of the probability of an

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adverse public health consequence or an incidence of disease (Gkogka et al. 2013). The establishment of both ALOP and FSO values is a societal decision and the prerogative of competent authorities (Gkogka et al. 2013). Therefore, quantitative evaluations may facilitate the assessment of FBD in food service establishments, but it remains a challenge due to the presence of uncountable hazards, and the FSOs are the way to go. Many quantitative risk analysis methods are complex and detailed and use mathematical simulations (Manning and Soon 2013). Risk assessments must be cost-effective and able to distinguish the different types of risk (e.g., high, medium, and low) (Health and Safety Executive 2006). One of the most used instruments to evaluate the food safety practices of food service establishments is a checklist. This may be because a checklist is an instrument with reproducibility, cost-effectiveness, and practicality (Stedefeldt et al. 2013). Checklists can easily be used as management and research instruments and have a range of possible applications for evaluation in the food service industry (Da Cunha et al. 2016). Checklists are composed of a list of food safety criteria (or questions) based on legislation (e.g., “ Do food handlers sanitize their hands properly to avoid contamination of food?” ). The evaluator then indicates whether the criterion has been fulfilled or violated, based on his or her observation and experience. In the end, the evaluator calculates the number of violations committed to generate an absolute or relative score. To facilitate decision making, this score should be based on FBD risk. This type of risk analysis, characterized as a semiquantitative risk assessment, establishes a bridge between qualitative and fully quantitative methods (Davidson et al. 2006). Semiquantitative risk assessment is most useful in providing a structured way to rank risks according to their probability, impact, or both together, implying severity, and for ranking risk reduction actions for their effectiveness. A predefined scoring system allows one to map a perceived risk into a category, where there is a logical and explicit hierarchy between categories (World Health Organization 2009). Some researchers have used the semiquantitative method for risk assessment. Lake et al. (2002) studied the risk profile of milk and Mycobacterium bovis in New Zealand; Sumner and Ross (2002) researched seafood safety risk assessment in Australia; and Mataragas et al. (2008), in Europe, studied the risk profiles of pork and poultry meat and risk ratings of various pathogen– product combinations. Most studies research one etiological agent relating the food vehicles and/or contamination sources. Semiquantitative analysis of food service establishments was not found, however, in the research literature. Some studies, such as those performed in Seattle-King County in Washington (Irwin et al. 1989) and Los Angeles County in California (Buchholz et al. 2002), have demonstrated that inspection scores may successfully predict outbreaks of FBD. In contrast, in other studies, foodborne outbreaks were not correlated with inspection scores.

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Food safety inspections are used by health surveillance agencies to measure, manage, and communicate the risk of FBDs within a particular type of food service. Due to the complexity involved in transforming “ situations into scores,”  these agencies present a general compliance score (compliance percentage of all applicable evaluation items) or an overall violation score. These overall scores, though, may not accurately reflect the risk of FBDs, and thus may lead to misinterpretations. The following example illustrates the potential for misinterpretation. A food safety checklist with 100 items was applied in two restaurants. Violations were noted, and the percentage of total violations (violation percentage) was calculated for each establishment. In the first food service establishment (Restaurant 1), the violation percentage was 15% of the listed items, and in the second establishment (Restaurant 2), it was 47% of the total items. One might assume that Restaurant 2 implies a higher risk to consumers because it was cited with more than double the violations of Restaurant 1. However, it is necessary to also analyze the nature of the risks associated with the violations, as shown in Figure  1.1. Although Restaurant 1 had a lower number of violations, they were more highly related to FBD outbreaks (Bryan 1978; ESR 2008; Food and Drug Administration 2000; Todd et al. 2007), mainly because of violations concerning food temperature, hand hygiene, and the cleaningness of utensils and equipment. In this sense, a quantitative assessment attributing weights based on the risk of FBD can generate a metric that is easier to interpret. Inspection scores may be attributed and used in several ways. In many studies, a binary scoring system was used to determine the risk of FBD outbreak. For example, some have assigned one point for each violation (Lockis et al. 2011; Saccol et al. 2013; Veiros et al. 2009; Chapman et al. 2010), allowing the researchers to calculate the violation percentage (or the percentage of correct food preparation procedures). Other studies used a scale to rate Restaurant 1 15% of violations

Restaurant 2 47% of violations

Summary of violations -Inadequate temperature of food storage -Ready-to-eat hot food maintained without controlled temperature -Insufficient frequency of utensil and equipment sanitization -Inadequate hand hygiene

Summary of violations -Physical structure without proper size -Presence of wood boxes in the food storage area -The floor is not made with smooth and durable materials -Presence of exposed water pipes -Presence of exposed light bulbs -Insufficient frequency of sanitization of the waste receptacle -Doors without self-closing mechanism

FIGURE  1.1 Comparison of violations observed in two restaurants.

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the compliance of food safety practices (e.g., full, partial, or nil) but did not assign weights for health or microbiological risks (Baş  et al. 2006a, 2006b; Choudhury et al. 2011). The logic underlying the binary scoring system is that the score will be equal to the overall ratio of compliance to violations. The use of binary scores is not conceptually wrong, but it is limited. Binary scores may be used to assess to what extent the health and food safety legislation is being fulfilled (in a generic way) by food service establishments. However, these scores should not be used to compare establishments, estimate risks, or establish which food service establishments must be prioritized. Arithmetical progression is another widely used technique to establish scores for food safety inspections (Da Cunha et al. 2014b; De Oliveira et al. 2014; Santana et al. 2009; New South Wales 2011). The use of a sequence of scores (i.e., n1, n2, n3, … , nk) (Irwin et al. 1989; Buchholz et al. 2002) is also a common feature of checklists. Mathematical progressions can be used to set scores; however, these scores must have a logical organization with regards to the assessment of the food procedures, hazards, and risks, such as the inspection score systems used in Los Angeles (Los Angeles 2014), New South Wales (New South Wales 2011), and Brazil (Da Cunha et al. 2014b). Table  1.1 presents some examples of the instruments used to assess food safety that utilize risk scores based on the risk of FBD. A panel of experts has been used to set scores based on the risk of foodborne outbreaks in some countries (Da Cunha et al. 2014b; Hoag et al. 2007). In the examples in Table  1.1, the final score has a negative magnitude; that is, the greater the number of points, the greater the number of violations. Simpler solutions can also be used to assess food service establishments. One is to classify the items into categories, as proposed by Da Cunha et al. (2016). In their study, 177 inspection items were classified into five categories of questions: R1, questions that addressed time and temperature aspects; R2, questions that addressed direct contamination; R3, questions that addressed water conditions and raw material; R4, questions that addressed indirect contamination (i.e., structure and buildings); and R5, questions that addressed documentation. The score for each category may be analyzed separately. As another example, Kassa et al. (2010) used binary scores but weighted the scores for critical and noncritical violations. It is important to remember that risk is defined as “ probability × consequence.”  An FBD outbreak affecting three people is less serious than an outbreak affecting thousands of people and much less serious than an outbreak affecting thousands of susceptible people, such as hospital patients. With this in mind, the U.S. Food Code (Food and Drug Administration 2013) has suggested factors that justify increased attention to an establishment: • History of noncompliance with provisions related to foodborne illness risk factors or critical items • Specialized processes conducted

Evaluate and classify food service establishments in general Evaluate and classify food service establishments in New York

Evaluate and classify food service establishments in Los Angeles

Evaluate and classify food service establishments in New South Wales

United States

United States

Australia

United States

Evaluate and classify food service establishments (restaurants and similar)

Objective

Brazil

Country

Risk based with critical and noncritical violations; violation-specific scores range from 2 for lowerrisk/lower-severity violations to 28; each violation has a set of conditions (severity) from I to V; the more severe the violation, the greater the point value for that violation Risk based with minor and major violations; violation-specific scores of 4 for major violations in critical risk factors, 2 for minor violations in critical risk factors; and 1 for violations in low-risk factors Risk based; violation-specific scores ranging from 1 for lower-risk violations to 8 for the highest-risk violations

Binary with critical and noncritical violations

Risk based; violation-specific scores range from 0.02 for lower-risk violations to 139.27 for highest-risk violations

Scores

Total score ranging from 0 (no violation observed) to 120, with four classifications

Total score ranging from 0 (no violation observed) to 100

Total score ranging from 0 (no violation observed) to 1519

Total score ranging from 0.0 (no violation observed) to 2565.95, with five classifications Not presented

Final Score and Classification

Examples of Food Safety Assessment Instruments That Utilize Weights Based on the Risk of Foodborne Disease

TABLE  1.1

Reference

New South Wales 2011

Los Angeles 2014

City of New York 2007

Kassa et al. 2010

Da Cunha et al. 2014

Semiquantitative and Qualitative Assessment 17

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• • • •

Food preparation a day in advance of service Large number of people served History of foodborne illness and/or complaints Highly susceptible population served

1.8  Systematization of Qualitative Method of Risk Assessment According to the Codex Alimentarius Commission (2001), qualitative risk assessment is “ based on data which, while forming an inadequate basis for numerical risk estimations, nonetheless, when conditioned by prior expert knowledge and identification of attendant uncertainties, permits risk ranking or separation into descriptive categories of risk.”  The qualitative method of risk assessment uses techniques based on the construction and integration of knowledge on a topic or an event developed through direct interaction with a team of experts. Some techniques are described below. • Nominal group technique (NGT): This technique is used for the prioritization of items that arise from brainstorming. It is a structured process by which small groups (five to nine experts) make suggestions and then discuss them until they come to a decision. This technique may be used in cases where the intention is to identify and characterize the hazard, assess the exposure and characterize the risk, and select and prioritize all possible solutions (Totikidis 2010). • Delphi: The Delphi technique is used to estimate the probability and impact of future and uncertain events. A group of experts is consulted to help identify risks, assumptions, and their associated premises, and each expert individually presents his or her estimates and premises to a risk manager, who analyzes the data and creates a summary report (Delp et al. 1977). • Scenario analysis: This technique assists in the planning of actions through the study of possible future occurrences in the context of the event (the scenario in this chapter being the food service industry). The analysis involves a consistent vision of the future based on plausible assumptions about relevant events that can influence the occurrence of a hazard. The use of scenarios allows the team of experts to think systematically and strategically about the potential dangers, without the influence of their own biases, opinions, and prejudices (Schwartz 1991).

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• Case study: This technique’ s objective is to analyze an event thoroughly. The aim of a case study would be to perform a detailed examination of a particular situation in the food service environment. Case studies have become the preferred method for risk managers to determine how and why specific events occur when there is little possibility of control over the events studied and when the focus of the case study is on current phenomena that can only be analyzed within their specific context. Observation plays an essential role in the case study. By direct observation, one seeks to understand the appearances, situations, and/or behaviors associated with an event (Yin 2013). Regardless of the technique employed, the results of these qualitative methods are expressed in a descriptive form, indicating, for example, a high, medium, or low risk according to the consensus of the experts involved (Sumner et al. 2004; OPAS 2008). The main difference between qualitative and quantitative assessments is that qualitative assessments bring together a team of experts who are encouraged to dialogue and discuss the risk in depth. These dialogues and discussions can be recorded, transcribed, and interpreted using content analysis. According to Bardin (1993), content analysis “ means a set of communication analysis techniques to obtain, through systematic procedures and description of the objectives of message content, indicators (quantitative or not) that allow the inference of knowledge related to the conditions, production and/ or reception (inferred variables) of these messages.”  The content analysis involves three fundamental phases: preanalysis, material exploration and processing of results using quantitative and/or qualitative techniques, and condensing the results into patterns, trends, or implied relationships. This method of interpretation must go beyond the experts’  words because the risk manager is most interested in reaching the latent content, that is, the meaning that lies beneath the words (Downe-Wamboldt 1992; Joffe and Yardley 2004; Vaismoradi et al. 2013). This analysis may provide a better understanding of an event in the context in which it occurs and of which it is a part, and involves seeking the event’ s meaning, analyzing it in an integrated manner, and considering all relevant points. It also may augment and direct new research and identify actions to be taken during risk management and risk communication. A qualitative risk assessment can help the risk manager to define priorities and formulate public policies (Coleman and Marks 1999). Nestlé  (2003) stated that “ decisions about acceptability involve perceptions, opinions, and values; as well as science, risk is a scientific question…  The acceptability of a given level of risk, however, is a political question to be determined in the political arena.” 

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Davidson et al. (2006) noted that to achieve the objectives of risk assessment, the degree of uncertainty must be recognized and considered in any risk estimates.

1.9 Projects Undertaken in the Context of Sanitary Risk in Food Service Environments The last part of this chapter briefly discusses two projects that developed in a multidisciplinary, multi-institutional, and multisector setting, in which the authors participated, strengthened technical cooperation, and sought innovations through different initiatives and collaborations. 1.9.1  Project 1: Pilot Project Food Service Establishment Categorization 1.9.1.1  Project Participants Diogo Thimoteo da Cunha, Ana Lú cia de Freitas Saccol, Eduardo Cesar Tondo, Ana Beatriz Almeida de Oliveira, Veronica Cortez Ginani, Eneo Alves da Silva Jr., Fabio Montesano, Carolina Vieira Araú jo, Thalita Antony Souza Lima, Angela Karinne Fagundes de Castro, and Elke Stedefeldt. 1.9.1.2 Description Over the past few years, the health departments of cities such as New York, Los Angeles, Toronto, London, Copenhagen, and the state of New South Wales (Australia) have established a method to classify and evaluate restaurants from a food safety perspective; restaurants with better evaluations (that best fulfilled the requirements of the health legislation) received a higher score. The restaurants then received a seal indicating their classification. The aim of this strategy is to encourage healthy competition. Because a potential competitor may have a better score, the owner of the food service establishment may attempt to correct the violations in his establishment to improve his ranking. The result of a greater control of hygienic processes is a reduction in the risk of an outbreak of FBD. The strategy also demonstrates how important risk communication is to the consumer. Based on these positive examples, Agê ncia Nacional de Vigilâ ncia Sanitá ria (Anvisa) managers decided to implement a similar system in Brazil. The 2014 FIFA World Cup in Brazil provided an opportunity to test this strategy. First, Anvisa invited experts in food safety from various universities. They developed an instrument using a semiquantitative risk assessment method. Its creation and evaluation were described in a scientific article in Food Research International, entitled “ Food Safety of Food Services within

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the Destinations of the 2014 FIFA World Cup in Brazil: Development and Reliability Assessment of the Official Evaluation Instrument”  (Da Cunha et al. 2014a). Despite the restaurant categorization strategies being known and used in other countries, the Brazilian article is the first to present, in detail and with scientific evidence, the creation of this type of strategy. After the step of creation and evaluation of the instrument, health assessments by local health surveillance inspectors were performed to categorize the restaurants. The evaluation took place in three stages: (1) self-assessment, in which the property owner was provided the instrument and had the opportunity to make suggestions; (2) diagnostic assessment, in which the health surveillance inspector indicated the observed violations; and (3) final assessment, in which the restaurants received their final classification based on the score received that day. The three-evaluation strategy was established to promote the compliance of the facilities. The facility could then be classified into one of four grades: A (no failure found), B (very little failure or lowrisk failures found), C (few failures found), and “ pending”  (reasonable to a high number of failures or high-risk failures found). After the implementation of the strategy during the 2014 FIFA World Cup, the group of experts convened to assess the effects of the strategy. Overall, 1967 establishments in 27 cities in Brazil were evaluated and categorized. It was observed that sanitary risks were significantly reduced between the evaluations, considering that the lower the score, the lower the sanitary risk. No foodborne outbreak originating from the categorized establishments was reported during the World Cup. Moreover, 2837 people were interviewed (consumers, business owners, inspectors, and health surveillance coordinators) regarding their perceptions of the strategy implementation. The evaluations were positive, and all interviewees indicated that it would be beneficial to transform the strategy into a permanent public policy. The results of the strategy were published in the journal Frontiers in Microbiology (Da Cunha et al. 2016). The responses to the open-ended questions in the 503 interviews with the owners of food service establishments and tax and health surveillance coordinators were interpreted by using thematic content analysis, resulting in a new analysis perspective for food safety in the country and the transparency of food manipulation practices for those involved in both distribution and consuming. The final conclusions of the study indicated that the categorization of restaurants in Brazil has the potential to accomplish the following desirable outcomes: reduce the risk of foodborne outbreaks; motivate the owners of restaurants to ensure the compliance of their establishments with food safety legislation; perform risk communication to the consumer in a simple and concise manner; promote increased acceptance by the public, surveillance, and food sectors involved; and facilitate health inspections, once the instrument has been standardized and focused on controlling factors related to FBD outbreaks. The project proposes recommendations for future work.

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1.9.2 Project 2: Complementary Technical Assistance Project to the Support Project for the Development of a School Feeding Program in Mozambique 1.9.2.1  Project Participants Elke Stedefeldt, Thimoteo Diogo da Cunha, Cristina Gaglianone Murphy, Maria de Fatima Ferreira Menezes, Simone Palma Favaro, and the staff of the Ministry of Education and Human Development from Mozambique. 1.9.2.2 Description Since 2012, the Complementary Technical Assistance Project to the Support Project for the Development of a School Feeding Program in Mozambique, the result of a general agreement on trilateral cooperation among the governments of Mozambique, Brazil, and the United States of America, was developed in Mozambique. The executing institutions are as follows: • Mozambique government: Ministry of Education (MoE), Management of Special Programs (MSP), and Department of Production and Feeding (DPF) • Brazil government: Ministry of Education (MEC), National Fund for Education Development (NFED), and National School Feeding Programme (NSFP) • U.S. government: University of Florida (UF) and Michigan State University (MSU) The project was created to develop activities that strengthen the management of a school feeding program in Mozambique. The project also disseminates knowledge concerning the development of studies and strategies and the operationalization of the information generated by these activities to inform decision making. Among the actions planned in this project, applied research on the topic of good food handling practices was designed in the schools to establish and strengthen recommendations. A risk assessment was developed using a semiquantitative method based on the international standards set by the World Health Organization and the concept of sanitary risk. The Seminar for Good Practices in Food Handling was held in Mozambique, to give context to the results obtained from the survey in the form of discussion of the recommendations developed and the importance of the recommended procedures concerning health and food safety. Two panels were held: (1) the National Food and Nutrition Policy of Brazil and the World Health Organization’ s five keys to food safety and (2) the importance of good food safety practices in the school environment and the results of the research developed in schools in Mozambique. The conclusions and recommendations of the research were discussed with the government representatives, and a consensus was developed through

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participatory methodology. The workshop speeches were interpreted using thematic content analysis, and emergent themes included a profound reflection on the role of school feeding and the country’ s politics, reaching beyond the technical field of nutrition and health. Different cultures can dialogue aiming at the same goal, while still preserving the sovereignty of each country. The project is implementing the recommendations. It was reported that through a dialogue between the Ministries of Health and Education and Human Development, a Department of Nutrition and a health school were established, evidencing a political breakthrough.

1.10  Final Consideration This semiquantitative and qualitative assessment can, in addition to defining the degree of risk, help to expand the possibilities in sanitary risk minimization; draw action plans in the short, medium, and long term; support managers in recognition of uncertainty; and prioritize actions and policy decisions. The integration of the results can be observed in Figure  1.2. From Qualitative assessment

Semiquantitative assessment

c

De

Contamination by food handlers, equipment, and utensils

k

ris

Contaminated water and raw material

Indirect contamination

Time and temperature aspects

Resolution of the unconformities

Political decision

a

ds

se rea

ry

a nit

Priority action

Establishment of action plan (short, medium, and long term)

Recognition of uncertainties

FIGURE  1.2 Integration of the results of semiquantitative and qualitative methods of assessment of sanitary risk in the food service setting.

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these results, risk management decisions can be more assertively made, and strategies can become effective, provided that risk communication is continuous.

References Agê ncia Nacional de Vigilâ ncia Sanitá ria. 2015. Ciclo de Debates em Vigilâ ncia Sanitá ria: desafios e tendê ncias. Textos de Referê ncias. Sã o Paulo: Agê ncia Nacional de Vigilâ ncia Sanitá ria. http://portal.anvisa.gov.br/documents/219201/219401/ Caderno%2BCiclo%2Bde%2BDebates.pdf/06c62703-f8e3– 4424-aa4e5b093e45261e (accessed August 10, 2016). Bardin, L. 1993. L’ analyse de contenu. Paris: Presses Universitaires de France Le Psychologue. Bartz, S., A. C. Ritter, and E. C. Tondo. 2010. Evaluation of bacterial multiplication in cleaning cloths containing different quantities of organic matter. J Infect Dev Ctries 4:566– 571. Bartz, S., and E. C. Tondo. 2013. Evaluation of two recommended disinfection methods for cleaning cloths used in food services of southern Brazil. Braz J Microbiol 44(3):765– 770. Baş , M., A. S. Ersun, and G. Kivanç . 2006a. Implementation of HACCP and prerequisite programs in food businesses in Turkey. Food Control 17(2):118– 126. Baş , M., A. S. Ersun, and G. Kivanç . 2006b. The evaluation of food hygiene knowledge, attitudes, and practices of food handlers in food businesses in Turkey. Food Control 17:317– 322. Brazil, Ministé rio da Saú de, Secretaria de Vigilâ ncia em Saú de— SVS 2013. 2016. Dados Epidemioló gicos— DTA— Perí odo de 2007 a 2016. Brasilia: Ministé rio da Saú de, http://www.saude.gov.br/svs (accessed June 15, 2016). Brazil, Ministé rio da Saú de, Secretaria de Vigilâ ncia em Saú de— SVS 2015. 2015. Dados Epidemioló gicos— DTA— Perí odo de 2000 a 2015. Brasilia: Ministé rio da Saú de, http://www.saude.gov.br/svs (accessed June 15, 2015). Bryan, F. L. 1978. Factors that contribute to outbreaks of foodborne disease. J Food Prot 41(10):816– 827. Buchholz, U., G. Run, J. L. Kool, J. Fielding, and L. Mascola. 2002. A risk-based restaurant inspection system in Los Angeles County. J Food Prot 65(2):367– 372. Chapman, B., T. Eversley, K. Fillion, T. MacLaurin, and D. Powell. 2010. Assessment of food safety practices of food service food handlers (risk assessment data): Testing a communication intervention (evaluation of tools). J Food Prot 73:1101– 1107. Choudhury, M., L. B. Mahanta, J. S. Goswami, and M. D. Mazumder. 2011. Will capacity building training interventions given to street food vendors give us safer food? A cross-sectional study from India. Food Control 22(8):1233– 1239. City of New York, Department of Health and Mental Hygiene, ed. 2007. Inspection scoring system for food service establishments. New York: Department of Health and Mental Hygiene.

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Codex Alimentarius Commission. 2001. Principles and guidelines for the conduct of microbiological risk assessment (CAC/GL 30– 1999). Food hygiene basic texts. Geneva: Codex Alimentarius Commission. Coleman, M. E., and H. M. Marks. 1999. Qualitative and quantitative risk assessment. Food Control 10:289– 297. Costa, M. C., T. Goumperis, W. Anderson, et al. 2016. Risk identification in food safety: Strategy and outcomes of the EFSA emerging risks exchange network (EREN), 2010– 2014. Food Control 73(Pt B):255– 264. Da Cunha, D. T., V. V. De Rosso, and E. Stedefeldt. 2016. Should weights and risk categories be used for inspection scores to evaluate food safety in restaurants? J Food Prot 79(3):501– 506. Da Cunha, D. T., A. B. A. Oliveira, A. L. F. Saccol, et al. 2014a. Food safety of food services within the destinations of the 2014 FIFA World Cup in Brazil: Development and reliability assessment of the official evaluation instrument. Food Res Int 57:95– 103. Da Cunha, D. T., E. Stedefeldt, and V. V. De Rosso. 2014b. The use of sanitary risk scores and classification in food service: An experience in Baixada Santista’ s public schools— Brazil. Brit Food J 116(5):753– 764. Da Cunha, D. T., A. L. F. Saccol, E. C. Tondo, et al. 2006. Inspection score and grading system for food services in Brazil: The results of a food safety strategy to reduce the risk of foodborne diseases during the 2014 FIFA World Cup. Front Microbiol (Online) 7:00–00. Davidson, V. J., J. Ryks, and A. Fazil. 2006. Fuzzy risk assessment tool for microbial hazards in food systems. Fuzzy Set Syst 157:1201– 1210. Delp, P., A. Thesen, J. Motiwalla, and N. Seshardi. 1977. Nominal group technique. In Systems Tools for Project Planning, 5. Bloomington, IN: International Development Institute. De Oliveira, A. B. A., D. T. Da Cunha, E. Stedefeldt, R. Capalonga, E. C. Tondo, and M. R. I. Cardoso. 2014. Hygiene and good practices in school meal services: Organic matter on surfaces, microorganisms and sanitary risks. Food Control 40:120– 126. Downe-Wamboldt, B. 1992. Content analysis: Method, applications, and issues. Health Care Women Int 13(3):313– 321. ESR (Institute of Environmental Science and Research). 2008. Annual summary of outbreaks in New Zealand 2007. Porirua, New Zealand: Population and Environmental Health Group. Food and Drug Administration. 2000. Report of the FDA retail food program database of foodborne illness risk factors, ed. Retail Food Program Steering Committee. Silver Spring, MD: Food and Drug Administration. Food and Drug Administration. 2013. Food code. Silver Spring, MD: Food and Drug Administration. Gkogka, E., M. W. Reij, L. G. M. Gorris, et al. 2013. The application of the Appropriate Level of Protection (ALOP) and Food Safety Objective (FSO) concepts in food safety management, using Listeria monocytogenes in deli meats as a case study. Food Control 29:382– 393. Grover, A. K., S. Chopra, and G. A. Mosher. 2016. Food Safety Modernization Act: A quality management approach to identify and prioritize factors affecting adoption of preventive controls among small food facilities. Food Control 66:241– 249.

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Havelaar, A. H., D. K. Martyn, P. R. Torgerson, et al. 2015. World Health Organization Global estimates and regional comparisons of the burden of foodborne disease in 2010. PLoS Med 12(12):e1001923. http://journals.plos.org/plosmedicine/ article?id=10.1371/journal.pmed.1001923 (accessed June 20, 2016). Health and Safety Executive. 2006. Guidance on risk assessment for offshore installations. HSE Offshore Information Sheet No. 3. Liverpool, UK: Health and Safety Executive. Hoag, M. A., C. Porter, P. P. Uppala, and D. T. Dyjack. 2007. A risk-based food inspection program. J Environ Health 69(7):33– 36. International Commission on Microbiological Specifications for Foods. 2006. A simplified guide to understanding and using food safety objectives and performance objectives. In Ensuring Global Food Safety. North Ryde, NSW: International Commission on Microbiological Specifications for Foods. http://www.icmsf.iit. edu/pdf/FSO% 20Ojectives/GuiaSimplificadoEnglish.pdf (accessed September 3, 2016). Irwin, K., J. Ballard, J. Grendon, and J. Kobayashi. 1989. Results of routine restaurant inspections can predict outbreaks of foodborne illness: The Seattle King County experience. Am J Public Health 79(5):586– 590. Joffe, H., and L. Yardley. 2004. Content and thematica analysis. In Research Methods for Clinical and Health Psychology, (pp 56–68) ed. D. F. Marks and L. Yardley London: Sage. Kassa, H., G. S. Silverman, and K. Baroudi. 2010. Effect of a manager training and certification program on food safety and hygiene in food service operations. Environ Health Insights 4:13– 20. Kothe, C. I., C. H. Schild, E. C. Tondo, and P. S. Malheiros. 2016. Microbiological contamination and evaluation of sanitary conditions of hot dog street vendors in southern Brazil. Food Control 62:346– 350. Lake, R., A. Hudson, and P. Cressey. 2002. Risk profile: Mycobacterium bovis in milk. Christchurch, New Zealand: Institute of Environmental Science and Research. Lima, G. C., M. R. Loiko, L. S. Casarin, and E. C. Tondo. 2013. Assessing the epidemiological data of Staphylococcus aureus food poisoning occurred in the state of Rio Grande do Sul, southern Brazil. Braz J Microbiol 44(3):759– 763. Lockis, V. R., A. G. Cruz, E. H. M. Walter, J. A. F. Faria, D. Granato, and A. S. Sant’ Ana. 2011. Prerequisite programs at schools: Diagnosis and economic evaluation. Foodborne Pathog Dis 8(2):213– 220. Los Angeles. 2014. Reference guide for the food official inspection report, ed. Department of Public Health Environmental Health. Los Angeles: Department of Public Health Environmental Health. Machado, T. R. M., P. S. Malheiros, A. Brandelli, and E. C. Tondo. 2010. Avaliaç ã o da Resistê ncia de Salmonella à  aç ã o de desinfetantes á cido peracé tico, quaterná rio de amô nio e hipoclorito de só dio. Rev Inst Adolfo Lutz 69:475– 481. Manning, L., and J. M. Soon. 2013. Mechanisms for assessing food safety risk. Br Food J 155(3):460– 484. Mayet, A., V. Andreo, G. Bedudourg, et al. 2011. Food-borne outbreak of norovirus infection in a French military parachuting unit. Euro Surveill 16(30):pii: 19930. Mataragas, M., R. N. Skandamis, and E. H. Drosinos. 2008. Risk profiles of pork and poultry meat and risk ratings of various pathogen/product combinations. Int J Food Microbiol 126(1– 2):1– 12.

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Medeiros, C. O., S. B. Cavalli, and R. P. C. Proenca. 2012. Human resources administration processes in commercial restaurants and food safety: The actions of administrators. Int J Hosp Manag 31(3):667– 674. Murphy, K. S., R. B. DiPietro, G. Kock, and J. Lee. 2011. Does mandatory food safety training and certification for restaurant employees improve inspection outcomes? Int J Hosp Manag 30(1):150– 156. Nestlé , M. 2003. Safe Food: Bacteria, Biotechnology and Bioterrorism. London: University of California Press. New South Wales, NSW Food Authority. 2011. Scores on doors: Pilot evaluation report, ed. NSW Food Authority. Newington, NSW: NSW Food Authority. Olaimat, A. N., and R. A. Holley. 2012. Factors influencing the microbial safety of fresh produce: A review. Food Microbiol 32:1– 19. Oliveira, F. A., A. P. Frazzon, A. Brandelli, and E. C. Tondo. 2007. Use of PCRribotyping, RAPD, and antimicrobial resistance for typing of Salmonella Enteritidis involved in food-borne outbreaks in Southern Brazil. J Infect Dev Ctries 1:170– 176. Oliveira, F. A., M. P. Geimba, A. P. Pasqualotto, and E. C. Tondo. 2009. Clonal relationship among Salmonella enterica serovar Enteritidis involved. Food Control 20:606– 610. Oliveira, F. A., A. P. Pasqualotto, W. Padilha, and E. C. Tondo. 2012. Characterization of Salmonella Enteritidis isolated from human samples. Food Res Int 45(2):1000– 1003. Oliveira, N. A. 2013. Gestã o do Risco Sanitá rio: Desafios e Inovaç õ es. Blog Direito Sanitá rio: Saú de e Cidadania. Biblioteca Virtual em Saú de. http://blogs.bvsalud. org/ds/2013/06/14/gestao-do-risco-sanitario-desafios-e-inovacao/ (accessed July 21, 2016). OPAS (Organizaç ã o Pan-Americana da Saú de). 2008. Perspectiva sobre a aná lise de risco na seguranç a dos alimentos. Curso de sensibilizaç ã o. Rio de Janeiro: OPAS. Rossi, E. M., D. Scapin, W. F. Grando, and E. C. Tondo. 2012. Microbiological contamination and disinfection procedures of kitchen sponges used in food services. Food Nutr Sci 3:975– 980. Saccol, A. L. F., A. L. Serafim, L. H. R. Hecktheuer, et al. 2013. Hygiene and sanitary conditions in self-service restaurants in Sã o Paulo, Brazil. Food Control 33(1):301– 305. Santana, N. G., R. C. C. Almeida, J. S. Ferreira, and P. F. Almeida. 2009. Microbiological quality and safety of meals served to children and adoption of good manufacturing practices in public school catering in Brazil. Food Control 20(3):255– 261. Schwartz, P. 1991. The Art of the Long View: Planning for the Future in an Uncertain World. Nova Iorque, Brazil: Doubleday. Silva, A. V. F. G., and F. C. F. Lana. 2014. Meaning the sanitary risk: Modes of action on the risk in health surveillance. Vig Sanit Debate 2(2):17– 26. Silva, E. A., Jr. 2016. Manual de Controle Higiê nico-Sanitá rio em Serviç os de Alimentaç ã o. 7th ed. Sã o Paulo: Editora Varela. Stedefeldt, E., D. T. Da Cunha, E. A. Silva Jr., S. M. Silva, and A. B. A. Oliveira. 2013. Instrumento de avaliaç ã o das Boas Prá t icas em unidades de alimentaç ã o e nutriç ã o escolar: Da concepç ã o à  validaç ã o. Ciê nc Saú de Coletiva 18(4):947– 953. Sumner, J., and T. Ross. 2002. A semi-quantitative seafood safety risk assessment. Int J Food Microbiol 7(1– 2):55– 59.

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Sumner, J., T. Ross, and L. Ababouch. 2004. Application of risk assessment in the fish industry. FAO Fisheries Technical Paper 442:1– 78. Todd, E. C. D., J. D. Greig, C. A. Bartleson, and B. S. Michaels. 2007. Outbreaks where food workers have been implicated in the spread of foodborne disease. Part 3. Factors contributing to outbreaks and description of outbreak categories. J Food Prot 70(9):2199– 2217. Tondo, E. C., T. R. M. Machado, P. S. Malheiros, D. K. Padrã o, A. L. Carvalho, and A. Brandelli. 2010. Adhesion and biocides inactivation of Salmonella on stainless steel and polyethylene. Braz J Microbiol 4:10– 20. Tondo, E. C., A. C. Ritter, and L. S. Sopeñ a. 2015. Involvement in foodborne outbreaks, risk factors and options to control Salmonella enteritidis SE86: An important food pathogen in southern Brazil. In Salmonella, ed. C. B. Hackett, 65– 77. New York: Nova Science Publishers. Totikidis, V. 2010. Applying the nominal group technique (NGT) in community based action research for health promotion and disease prevention. Aust Commun Psychol 22(1):18– 29. Universidade Federal Do Ceará . 2015. Curso Bá sico em Vigilâ ncia Sanitá ria. Agê ncia Nacional de Vigilâ ncia Sanitá ria. Unidade 03: Risco Sanitá rio— Percepç ã o, Avaliaç ã o, Gerenciamento e Comunicaç ã o. Fortaleza: Universidade Federal Do Ceará . https://ares.unasus.gov.br/acervo/bitstream/handle/ARES/3175/ Texto_de_Impressao_Anvisa_Unidade_03.pdf (accessed June 17, 2016). U.S. Department of Agriculture. 2015. Economic Research Service. Food service industry— Market segments. Washington, DC: U.S. Department of Agriculture. http://www.ers.usda.gov/topics/food-markets-prices/food-service-industry/ market-segments.aspx (accessed July 22, 2015). Vaismoradi, M., H. Turunen, and T. Bondas. 2013. Content analysis and thematic analysis: Implications for conducting a qualitative descriptive study. Nurs Health Sci 15(3):398– 405. Veiros, M. B., R. P. C. Proenç a, M. C. T. Santos, L. Kent-Smith, and A. Rocha. 2009. Food safety practices in a Portuguese canteen. Food Control 20:936– 941. World Trade Organization 1995. The WTO agreement on the application of sanitary and phytosanitary measures [SPS Agreement]. Geneva: World Trade Organization. https://www.wto.org/english/tratop_e/sps_e/spsagr_e.htm. (accessed September 3, 2016). World Health Organization. 2009. Risk Characterization of Microbiological Hazards in Food: Guidelines. Microbiological Risk Assessment Series. Geneva: World Health Organization. World Health Organization. 2015. WHO’ s first ever global estimates of foodborne diseases find children under 5 account for almost one third of deaths. Geneva: World Health Organization. http://who.int/mediacentre/news/releases/2015/ foodborne-disease-estimates/en/ (accessed June 16, 2016). Yin, R. K. 2013. Case Study Research: Design and Methods. 5th ed. Thousand Oaks, CA: Sage.

2 Fresh-Cut Apples Spoilage and Predictive Microbial Growth under Modified Atmosphere Packaging Predrag Putnik and Danijela Bursać  Kovač ević  CONTENTS 2.1 Introduction................................................................................................... 29 2.2 Importance and Challenges of Fresh-Cut Apple Production................. 31 2.3 Fresh-Cut Apple Nonmicrobial (Physiological) Spoilage....................... 31 2.4 Fresh-Cut Apple Microbial Spoilage.......................................................... 33 2.5 Microbial Inactivation in Fresh-Cut Apples.............................................34 2.6 Microbial Spoilage and Degradation of Polyphenols in Fresh-Cut Apples ............................................................................................................ 35 2.7 Fresh-Cut Apples in Modified Atmosphere Packaging.......................... 36 2.8 Mathematical Modeling and Modified Atmosphere Packaging .......... 38 2.9 Predicting Microbial Growth in Fresh-Cut Apples Packaged under a Modified Atmosphere................................................................... 39 2.10 Final Remarks................................................................................................ 41 References................................................................................................................ 41

2.1 Introduction The worldwide consumption of fruits and vegetables is constantly increasing, likely due to the promotion of nutrition principles that can be found in various dietary guidelines issued by various governments (USA, European Union [EU], etc.). Such guidelines promote the consumption of fresh produce in order to improve health and decrease diet-related diseases. Freshcut fruits and vegetables are becoming increasingly popular due to their freshness, convenience, and health benefits. Accordingly, the fresh-cut industry is a growing segment of the food industry, with a likelihood of increasing its market share in the future. Apples (Malus domestica ) are commonly consumed fruits, being the second most produced fruit worldwide.

29

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According to the Food and Agriculture Organization (FAO), in 2013 the total worldwide production of apples was 80 million tons. The industrial processing of fresh-cut apples induces plant tissue injury with various degenerative changes that promote microbial and enzymatic activity, respiration, and other negative influences, all with a tendency to shorten the length of storage. The length of storage is one of the most important economic factors for fresh-cut production, during which producers need to guarantee a product’ s safety and quality for the required sales period. Otherwise, apples may lose their market value, as they exhibit microbial appearance and sensory properties not safe and/or appealing to the consumer. Freshcut fruit spoilage has two origins, one internal (e.g., enzymatic and metabolic activity of fruit) and the other external (microbial contamination). Therefore, preventing microbial contamination of fresh-cut products is one of the primary concerns and a major limiting factor in extending the storage of fresh-cut apples. Modified atmosphere packaging (MAP) is one of the most popular approaches to extend the short storage in fresh-cut apples. MAP is based on the concept that selective packaging barriers with different permeances for gases will prevent anaerobic respiration by modifying the atmosphere within the package. Additionally, bacterial growth can be inhibited by altering the volumetric concentration of modified atmosphere gases within the package. Unfortunately, the role of modified atmosphere gases in the respiration process has not yet been clarified, as the respiration process is very complex and influenced by numerous factors. For instance, some of the important factors include temperature, time, concentration of added gases, size of the package, and mass of the packaged produce. The complexity is further multiplied with the addition of the other intricate influences, such as the microbial growth of various bacteria or other microorganisms. Microbial modeling and the development of predictive models for food microbiology are good solutions that are able to give estimates of naturally occurring processes from a large number of parameters. By constructing mathematical equations, predictive modeling may lower manufacturing costs and improve food production. Currently, semifundamental Michaelis– Menten models and fundamental approaches are used for such purposes. Semifundamental modeling is convenient, but it simplifies the naturally occurring process, as it assumes that some variables are constants or their influences are insignificant. Alternatively, fundamental modeling is complex but more flexible, precise, and tailored for each particular set of industrial circumstances. In other words, fundamental modeling can be custom-made for each particular fresh produce and corresponding industrial production. This chapter discusses fresh-cut packaging in a modified atmosphere and the factors that influence the length of apple storage, with the main focus being on the construction of predictive microbial models useful for the prevention of fresh-cut apple spoilage.

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2.2  Importance and Challenges of Fresh-Cut Apple Production Apples (M. domestica ) are popular fruits, with 80 million tons produced in 2013 (FAO 2015). They are the world’ s second most produced fruits, likely due to their reported health benefits, high convenience, and availability (Putnik et al. 2016b; Putnik et al. 2017), along with an increasing number of consumers becoming aware of the nutritional principles from dietary guidelines that promote the consumption of fresh fruits and vegetables (PerezEscamilla and Putnik 2007). Indeed, it is not surprising that fresh-cut apples are regularly found on the market, with production projected to increase in the future (Putnik et al. 2016c). For instance, the U.S. fresh-cut industry recorded $14 billion in sales in 2006 (Nicola et al. 2009), while in the EU the fresh-cut industry has 18% of the food market share (Putnik et al. 2016b). As a result of minimal processing, fresh-cut apples can be stored for shorter periods of time than whole apples (Putnik et al. 2016b). The average length of storage for fresh-cut fruits and vegetables is very brief and ranges from a few days to up to 2 weeks (Anese et al. 2012; Montero-Calderó n and Milagro Cerdas-Araya 2011). Operations such as peeling, coring, and slicing enhance degenerative changes, such as the synthesis of ethylene, phenolic oxidation, and microbial growth (Putnik et al. 2016b). Moreover, with tissue damage in fresh-cut apples comes the induction of enzymatic browning, tissue respiration and softening, the decrease of sensorial and nutritive qualities, and increased microbial spoilage, all with a tendency to shorten the already short length of storage of this type of produce (Putnik et al. 2016c). An increased manifestation of adverse changes in fresh-cut apples can discourage consumers from purchase and will cause loss of market value and economic benefits (Pristijono et al. 2006). The main limiting factor for freshcut production is the length of storage where apples can maintain their original quality and be free from food spoilage (Brecht 1995). The stability of fresh-cut produce is mainly influenced by preservation, processing, and the packaging environment (Rocculi et al. 2012). Accordingly, it is vital for their commercialization to define food engineering conditions for extending the storage of fresh-cut apples while maintaining the highest possible nutritive and microbiological quality (Putnik et al. 2016b; Oms-Oliu and Soliva-Fortuny 2011).

2.3  Fresh-Cut Apple Nonmicrobial (Physiological) Spoilage Fresh-cut apples are considered spoiled and unfit for purchase when their superficial color turns brownish. Hence, superficial color is the main feature that consumers use to evaluate product quality (or food spoilage),

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and it is not surprising that this attribute is the main limiting factor for the length of time that this food can spend on the market (Putnik et al. 2016b). Browning might be caused by the enzymatic or nonenzymatic origin (Corzo-Martinez et al. 2012). Enzymatic browning is created by the phenolic compounds targeted by polyphenol oxidase (PPO) activity, and the nonenzymatic counterpart by Maillard-type reactions (e.g., ascorbic acid browning). The PPO enzymes oxidize polyphenols at pH  =  5 – 7 to quinones that form brown polymer melanins (Corzo-Martinez et al. 2012). The biochemical origins of this type of browning are associated with natural plants’  defense against foreign invaders, where polymerization of melanins is considered an impenetrable barrier among plant tissue and attacking microorganisms (Alzetta 2014). This is considered the main source of browning in fresh-cut apples. Nonenzymatic browning is darkening that might originate from ascorbic acid that is commonly present in apples (Corzo-Martinez et al. 2012; Cropotova et al. 2016). It achieves a maximum rate at pH  =  4 and includes the formation of dehydroascorbic acid, with a tendency to bind amino acids and form brown polymers. Browning prevention is commonly achieved by various agents (Ca-ascorbate, NaCl, citric acid, etc.) or in combination with UV-A or UV-C light and various edible coatings (Chen et al. 2016; Lante et al. 2016; Liu et al. 2016). The use of aqueous solutions of L-arginine also improves the quality of fresh-cut apples by inhibition of browning, and hence extends postharvest life (Wills and Li 2016). Application of innovative technology, such as ultrasound (Jang and Moon 2011; Putnik et al. 2016b), may be used in combination with antibrowning agents to improve their penetration deeper into the apple tissue and to diminish monophenolase enzymatic activity (Jang and Moon 2011). One of the recent studies showed that Cripps Pink and Golden Delicious were the best out of seven studied apple cultivars in terms of resistance to browning. The best antibrowning treatment was Ca-ascorbate, which also showed the best sensory score with or without application of the ultrasound (Putnik et al. 2016a). It was shown that ultrasound may be used in combination with antibrowning agents, likely to improve their access to apple tissue and to diminish monophenolase enzymatic activity (Jang and Moon 2011). This was confirmed in a different study where ultrasound prevented browning only with the combination of ascorbic and citric acid, but not with sole Ca-ascorbate (Putnik et al. 2016b). Superficial browning for industrial production of fresh-cut apples is commonly evaluated by the CIELab color space by assessing color change (∆ Eab  *   ) and Browning index (BI) (Pathare et al. 2013; Rojas-Grau et al. 2006):

∆Eab* =

∆L*2 + ∆a*2 + ∆b*2 (2.1)

where all ∆ L *2 , ∆ a *2 , and ∆ b *2  were calculated in reference to the first day of storage.

Fresh-Cut Apples Spoilage and Predictive Microbial Growth

33



 X − 0.31  BI = 100 ×   (2.2)  0.17 



a * −1.75 × L *  (2.3)  X =  5 . 645 * * 3 . 012 * − × × + L a b  

It is known that consumers base their first purchase of the fresh-cut fruits on visual appearance; their further purchases are made dependent on the sensorial and textural quality of the produce. While sensory characteristics in fresh-cut apples are mainly related to the sugar-to-acids ratio (Beaulieu 2011), another relevant sensory feature to consumers is the fruit’ s texture. In industry, firmness is typically measured by destructive methods with texture analyzers (Beaulieu et al. 2004). Similarly as with taste, loss of firmness of fresh-cut apples during storage will negatively affect the produce’ s market value (Billy et al. 2008).

2.4  Fresh-Cut Apple Microbial Spoilage Microbial food safety is the most important limiting factor for the length of fresh-cut fruit storage, with a direct consequence on public health (Ragaert et al. 2011). Food spoilage is defined as an intolerable number of microbes responsible for the off-flavors in foods (Benner 2014). Tolerance for the presence of spoilage microorganisms in similar foods depends on cultural and legal settings (Putnik et al. 2016c). All fresh-cut produce from the EU should be tested and free from Salmonella  spp., Escherichia coli , and Listeria monocytogenes  (EU Commission 2005). Some EU member states have recommended that fresh-cut products should be tested on Staphylococcus aureus , sulfitereducing clostridia, Enterobacteriaceae (EBac), aerobic mesophilic bacteria (AMB), yeast, and mold (Benussi et al. 2011). EBac and AMB are the main spoilage microorganisms in fresh fruits that are normally present in industrial environments. Mostly, they are not harmful for human health unless their growth reaches a certain amount of colony-forming units (CFU) per gram of product (Benner 2014). The presence of EBac and AMB in industry is commonly evaluated by the ISO standards (ISO 2004, 2013). For instance, the nonlegal limit (M) for spoiled fresh-cut fruits in Croatia is set for EBac to M  ≤   103   CFU/g and for AMB to M  >   105   CFU/g (Putnik et al. 2016c). MAP is a common approach used in the food industry that may inhibit bacterial growth and preserve the quality of fruits over an extended period of time (Caleb et al. 2013). With regard to microbial spoilage predicted from the EBac growth, the longest fresh-cut apple shelf life for the EU market was 9.88 days, and for

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Food Safety and Protection

AMB, 6.47 days. This means that AMB grows almost two times faster than the EBac. This was confirmed by the AMB/EBac maximum growth rates μ max  (EBac)  =  0.25  ±   0.02 log CFU/g*day and μ max  (AMB)  =  0.46  ±   0.02 log CFU/g*day, which were derived from an equation published elsewhere (Putnik et al. 2016d).

2.5  Microbial Inactivation in Fresh-Cut Apples The growth and survival of the microorganisms in fresh-cut produce are affected by the type of microorganism, storage conditions, physical environment, processing, and packaging. Various microbial cultures responsible for afflicting food safety and inducing spoilage are commonly present on the surface of the fresh-cut produce, while their level of contamination is commonly evaluated by the total bacterial count (Qadri et al. 2015). Such contamination may originate from the environment, water, or cross-contamination from industrial equipment (USDA 2014). Containment or complete elimination of microbial contamination in fresh-cut produce may be achieved by various practices, such as good manufacturing practice (GMP), and food safety schemes, such as the Hazard Analysis and Critical Control Point (HACCP) (James and Ngarmsak 2011). These approaches may be embedded in fresh-cut processing, where additional reduction of the total microbial count is achieved by packaging produce in a modified atmosphere (Caleb et al. 2012b). As previously mentioned, the most relevant food safety microorganisms in the production of fresh-cut apples in the EU are Salmonella  spp., E. coli , L. monocytogenes , S. aureus , sulfite-reducing clostridia, EBac, AMB, yeast, and mold (EU Commission 2005). The bacterial counts of these microorganisms can be biocontrolled by their microbial antagonists (Qadri et al. 2015). For instance, the growth of the E. coli  (O157:H7), Salmonella , and Listeria innocua  in fresh-cut apples can be reduced by some strains of the pseudomonads (e.g., L-59– 66 of Pseudomonas syringae ) (Alegre et al. 2013). The growth and survival of L. monocytogenes  or Salmonella enterica  serovar Poona were successfully suppressed by the Gluconobacter asaii  (T1-D1), Candida  sp. (T4-E4), Discosphaerina fagi  (ST1-C9), and Metschnikowia pulcherrima  (T1-E2) in fresh-cut apples (Leverentz et al. 2006). Lactic bacteria were able to suppress the growth of E. coli , L. monocytogenes , Pseudomonas aeruginosa , Salmonella typhimurium , and S. aureus  in fresh-cut Golden Delicious (Trias et al. 2008). Washing with sanitizing solutions is considered the only step to accomplish a reduction in spoilage microorganisms and potential pathogens in fresh-cut produce (Allende et al. 2008; Alegre et al. 2010, 2013). A proper disinfection procedure is considered critical in ensuring the safety of

Fresh-Cut Apples Spoilage and Predictive Microbial Growth

35

fresh-cut fruits; therefore, the number of disinfectants with a potential application to decontaminate fresh-cut produce has increased in recent years (Gil et al. 2009). Another way to control microbial growth in fresh-cut apples is the application of various edible coatings. They are capable of antimicrobial activity and may be engineered with different chemical components, such as organic acids, bacteriocins, esters, polypeptides, plant essential oils, nitrites, and sulfites (Franssen and Krochta 2003). It was shown that edible coatings are effective in the prevention of the microbial growth of different microorganisms, such as psychrotrophics, coliforms, yeast, and molds (Qadri, et al. 2015). Numerous reports showed that various plant extracts and essential oils had growth reduction of various microorganisms (Oms-Oliu et al. 2010). In one example, cinnamon bark extracts incorporated in the antibrowning solutions decreased the growth of E. coli  O157:H7 and L. innocua  (Muthuswamy et al. 2008), and in another, vanillin reduced the growth of the E. coli , P. aeruginosa , Enterobacter aerogenes , Salmonella enterica  subsp. enterica  serovar Newport, Candida albicans , Lactobacillus casei , Penicillium expansum , and Saccharomyces cerevisiae  (Rupasinghe et al. 2006).

2.6 Microbial Spoilage and Degradation of Polyphenols in Fresh-Cut Apples The nutritive value of fruits and their products is commonly evaluated by the content of the polyphenols (Bursać  Kovač ević  et al. 2015b). Polyphenols in fruits have anti-inflammatory, antimicrobial, anticancer, and antiproliferative properties (Putnik et al. 2015a; Forbes-Hernandez et al. 2014). The main polyphenols in the apple are phenolic acids, for example, chlorogenic and coumaric acids, and flavonoids, for example, epicatechin, phloretin, and quercetin (Alzetta 2014). Currently, there is no literature that associates the loss of polyphenols with microbial growth, minimal processing, modified atmosphere, apple respiration, and apple browning. In contrast, it was stated that such data are needed to explain intricate connections between food preservation and MAP soundings (Caleb et al. 2013). Current interests in food processing are focused on the influence of various nonthermal technologies on polyphenolic stability in plants, fruits, and their products (Bursać  Kovač ević  et al. 2015a; Putnik et al. 2015b), but there are fewer investigations focused on the influence of nonthermal processing on polyphenolic stability in fresh-cut produce treated with antibrowning agents and packaged in a modified atmosphere. Ultrasound is one of the advanced technologies commonly used in minimal processing that is able to preserve the natural color, aroma, and nutrient content in apples and in other

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Food Safety and Protection

fruits (Nowacka et al. 2014). Ultrasound exerts pressure on the surface of the apple tissue, removes gas from the intercellular space, and fosters filling of pores with various additives, such as antibrowning agents (Nowacka et al. 2014; Tylewicz et al. 2013). One study examined the influence of a modified atmosphere, apple respiration, and superficial browning on the polyphenolic stability in fresh-cut apples. Identified polyphenols in Cripps Pink and Golden Delicious apple cultivars were coumaric, chlorogenic acid, quercetin, epicatechin, and phloridzin. Cripps Pink had more flavonoids and antioxidant capacity than Golden Delicious. All treatments showed higher antioxidant capacities than the control treatment. During storage, microbial growth was associated only with the quantities of epicatechin, with the possible indication that this compound was oxidized to quercetin with apple. Both antioxidant capacities strongly decreased with an increased log colony-forming units (CFU) per gram of both the AMB and EBac respiration (Putnik et al. 2016e). The other study showed that chlorogenic acid had no influence on bacterial growth, whereas catechol showed antimicrobial activity for Lactobacillus brevis  and Lactobacillus plantarum.  On the contrary, quercetin improved bacterial growth (Alzetta 2014).

2.7  Fresh-Cut Apples in Modified Atmosphere Packaging The major factors responsible for high-quality fresh-cut products are highquality raw material and an efficient cold chain throughout the manufacturing, distribution, and marketing processes (Artes et al. 2007). MAP and refrigerated storage are frequently used to avoid negative physiological effects by reducing respiration rates in fruits, thus extending the shelf life (Li et al. 2011; Montero-Calderon et al. 2008). MAP employs the concepts of packaging produce in a selective barrier that has different permeances for modified atmosphere gases. The most common gases used in MAP are O2 , CO2 , and N2 . Generally, an atmosphere consisting of 2– 5 kPa O2  and 3– 10 kPa CO2  will extend the shelf life in fresh-cut products, mainly through inhibition of surface enzymatic browning and slowing of physiological aging. Also, CO2  is the only gas used in MAP that has a direct and significant antimicrobial effect, hence ensuring the microbial control of fresh-cut products. The CO2  is highly soluble in water and lipids, with a great increase in solubility with a decrease in temperature. Dissolved CO2  forms carbonic acid, and a lower pH reduces the rate of growth for many food spoilage and pathogenic microorganisms by affecting the lag phase, maximum growth rate, and maximum population densities (Devlieghere and Debevere 2000). Aside from temperature, the inhibitory effect of CO2  is also dependent on the microorganism type, growth phase, water activity, and chemical composition of products.

37

Fresh-Cut Apples Spoilage and Predictive Microbial Growth

Selective films in MAP foster selective transport of O2  inside and CO2  outside of the packaging; in other words, a packaging barrier will create an atmosphere rich in CO2  and poor in O2  with impending anaerobic respiration. The two most important parameters for this packaging approach are the permeance of the package film and the respiration rates of the packaged material (Putnik et al. 2016b). The equations required for calculating the relevant parameters for the permeable MAP system are (Putnik et al. 2016c)





RO2 =

RCO2 =

kPO2 x

A



out O2

− yOin2

100 M

V f  dyCO2  kPCO2  − 100  dt  x M Vf



(y

= VTOTAL −

)− V

 dyO2  f   100  dt  (2.4)

A

(y

in CO 2

out − yCO 2

)

100 (2.5)

M Mρ

(2.6)

A = length width

(2.7)

where R  is the respiration rate for a permeable system (cm3 /kg*day*atm), kP /x   =  P  is the permeance of the packaging film (standard temperature and pressure [STP] in cm3 /m2 /day/atm), A  is the package surface area (m2 ), M  is the mass of the packaged apple cultivar (kg), x  is the film thickness (cm), y  is the volumetric concentration of MAP gases (% v/v O2 and CO2 ), M ρ   is the density of the fresh-cut apples, V TOTAL  is the total volume inside the package (cm3 ), and V f  is the free volume inside of the package (cm3 ). Apple respiration is the set of biochemical redox reactions where complex organic species are broken down to simpler units with expenditure of O2  and production of CO2  (Putnik et al. 2016b). By changing the concentrations of modified atmosphere gases, researchers were able to inhibit bacterial growth. However, due to the large number of intricate influences, the clear role of each gas in the fruit respiration process was not fully elucidated (Caleb et al. 2013). Some parameters that interact with microbial growth under modified atmosphere are length and temperature of storage, volumetric concentration of gases, size of package, and mass of produce (Putnik et al. 2016c). MAP applied at refrigeration temperatures efficiently extended the length of storage of fresh-cut apples by reducing their respiration and metabolic rates. Also, it was reported that fresh-cut apples packaged in MAP and treated with antibrowning treatments had an extended length of storage of up to 12 days (Kader 1986; Waghmare et al. 2013).

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Food Safety and Protection

Nevertheless, although MAP and low temperatures may inhibit aerobic spoilage microorganisms, these conditions may facilitate the growth of pathogens (Oliveira et al. 2010). Many research reports were concerned with food safety or health hazards for foods in MAP, particularly with the growth of pathogens able to multiply at low temperatures and under anaerobic conditions. There are seven foodborne pathogenic bacteria associated with fresh produce known to be capable of growth at or below 5° C: Clostridium botulinum  Type E, L. monocytogenes , Yersinia enterocolitica , Vibrio parahaemolyticus , enterotoxigenic E. coli , Bacillus cereus , and Aeromonas hydrophila . Two others grow at temperatures just above 5° C: S. aureus at 6° C (10° C for toxin production) and Salmonella  sp. at 7° C. Thus, it is of great importance that the MAP inhibit the growth of these microorganisms in fresh produce under refrigerated storage. Literature reports showed reduced pathogen counts (E. coli , Salmonella , and L. innocua ) on fresh-cut apples under passive MAP at 5° C for 30 days (Alegre et al. 2010). A study observed that the introduction of the apple cultivars to MAP in an industrial setting successfully prevented browning of both Cripps Pink and Golden Delicious. Browning was successfully prevented if apples were dipped in any tested antibrowning agent. Fresh-cut nonmicrobial spoilage in a modified atmosphere was well predicted from mathematical modeling (Putnik et al. 2016b).

2.8 Mathematical Modeling and Modified Atmosphere Packaging Mathematical modeling in food engineering is the approach used for lowering experimental costs and constructing equations that are capable of approximating the outcomes of natural processes from a large number of relevant predictors (Putnik et al. 2016c). This statistical regression approach is based on empirical data, and it is bound to a particular level of statistical probability. That way, models will have calculated data fitness and accuracy with their predictions in order to gauge their usefulness. Regression modeling is robust and can be applied for different research purposes (Bursać  Kovač ević  et al. 2015a, 2015b; Obranović  et al. 2015; Putnik et al. 2015a, 2016f). There are two main approaches to mathematical modeling for quantifying the roles of O2  and CO2  in the respiration process, namely, semifundamental and fundamental modeling. The first one employs Michaelis– Menten enzyme kinetics and is a simplification of respiration processes (Fagundes et al. 2013; Benitez et al. 2012). The second approach is more complex to use but offers more flexibility, robustness, and preciseness; plus, it is more capable of mimicking natural processes thanks to the use of numerous predictors tailored for each particular packaging circumstance (Putnik et al. 2016c). In

Fresh-Cut Apples Spoilage and Predictive Microbial Growth

39

other words, one equation for predicting microbial growth may be composed of different types of predictors, such as fruit cultivar, volumetric concentration of gases, size of the package, mass of packaged fruits, and type of the packaging film. Created mathematical models can be embedded into computer applications and widely available to all the potential users. A few studies have been conducted on modeling to describe the effects of time and temperature on respiration rate on fresh-cut produce (Waghmare et al. 2013; Caleb et al. 2012a). Respiration rates of fresh-cut produce were successfully modeled using Arrhenius and first-order decay models, which could be useful for selecting suitable packaging film.

2.9 Predicting Microbial Growth in Fresh-Cut Apples Packaged under a Modified Atmosphere In the literature, a limited number of mathematical models have been reported that are useful for fresh-cut apple production; two good examples are Pack-in-MAP®  , which is a commercial web-based software tool (Mahajan et al. 2007), and Anti-browning Apple Calculator— C.A.P.P.A.B.L.E.©  , which is free to use (Pizent et al. 2015). Pack-in-MAP is a compilation of mathematical models on product respiration rates and packaging permeability that are integrated in a user-friendly web-based software tool that helps in designing MAP for fresh and fresh-cut fruits and vegetables. Users of that software may define the type of product, storage conditions, amount of packed product, and size and geometry of the package in order to provide the best storage conditions (Mahajan et al. 2007). However, currently the website seems to be inactive. Anti-browning Apple Calculator— C.A.P.P.A.B.L.E. is the other online software that offers calculation of almost all parameters relevant for fresh-cut apple production. Numerous mathematical equations that are statistically derived from experimental data cover the prediction of browning in apples, whether or not treated with antibrowning agents. For instance, it is possible to detect how much, on average, each of the seven cultivars will naturally sustain browning during storage, or what would be their projected soluble solids content and pH. Similarly, it is possible to calculate the magnitude of browning during storage for two apple cultivars if they are treated with one of the 11 treatments and by inputting initial color (CIELab) parameters. Also, application offers projections for the sensory evaluation for different combinations of cultivars and antibrowning treatments. One of the mathematical models provides calculations for the length of storage in days that two tested cultivars may spend on the market. This equation accounts for the type of cultivar, type of treatment, average color change, pH, soluble solids content, level of initial contamination, and type of spoilage microorganism.

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Food Safety and Protection

Additionally, one of the models estimates the degree of usefulness during storage of the combined application of MAP and antibrowning agents for the packaging of fresh-cut apples with various initial CIELab statuses. Aside from the above-mentioned quality parameters, this online application offers unique predictive models for microbial growth for AMB and Ebac that simultaneously account for the type of cultivar and selection of antibrowning agent, CIELab status, apple respiration, and content of the modified atmosphere. These tools can be helpful in optimizing industrial parameters that will yield the least browning and AMB or Ebac growth of an apple while extending the fresh-cut apple’ s shelf life. In C.A.P.P.A.B.L.E., users can freely choose their own size of packaging, initial volumetric concentration of gases, apple mass inside of the package, and parameters for their own packaging film (e.g., permeance) in order to achieve optimal settings for their packaging systems (Figure  2.1). Additional information about the models is provided in articles published in Journal of Food Safety  and Journal of Food Process Engineering  (Putnik et al. 2016b, 2016c). This way, models provide calculations for very intricate biological processes, together with a great degree of flexibility and a tailor-made approach for the industrial packaging purposes. This is their main advantage, which will likely be accompanied by increased economic benefits and provide quality foods to consumers. On the other hand, models were created on a limited number of apple cultivars, and even though little relevant biological variance

FIGURE 2.1  Example of the Anti-browning Apple Calculator— C.A.P.P.A.B.L.E. computer application with embedded mathematical models.

Fresh-Cut Apples Spoilage and Predictive Microbial Growth

41

is expected among different cultivars, models should still be tested with other samples to confirm or reject their external validity. In conclusion, it was shown that the length of storage, apple respiration rates, and volumetric concentration of packaging gases were the most important predictors for microbial growth. This was expected due to their significance in aerobic respiration (Putnik et al. 2016c).

2.10  Final Remarks Fresh-cut apple production may be challenging, as a shorter shelf life and proneness to fostering microbial growth may pose public health risks and an increased tendency for economic losses. On the other hand, the market’ s demand for these foods will increase in the future, hence leading to increased economic benefits derived from fresh-cut processing. Nonmicrobial freshcut apple spoilage may be decreased with the application of various antibrowning agents and processing technology, while microbial spoilage can be tackled with the application of edible coatings and packaging in a modified atmosphere. Mathematical modeling is a useful statistical approach that may simultaneously account for a large number of packaging factors and help tailor industrial processing for each particular fresh-cut need. Mathematical equations may optimize fresh-cut production by calculating all relevant parameters. That will save production resources and improve economic profitability, and likely help with monitoring microbial spoilage, hence perfecting HACCP procedures. Mathematical models can be incorporated in various computer applications and made conveniently available to a large number of professionals. As a result, safer and quality foods for consumers will be easily engineered at lower costs.

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Leverentz, B., W. S. Conway, W. Janisiewiez, M. Abadias, C. P. Kurtzman, and M. J. Camp. 2006. Biocontrol of the food-borne pathogens Listeria monocytogenes  and Salmonella enterica  serovar Poona on fresh-cut apples with naturally occurring bacterial and yeast antagonists. Applied and Environmental Microbiology  72 (2):1135– 1140. Li, W. L., X. H. Li, Y. J. Sun, Y. Tang, Y. Q. Jiang, and M. Zhang. 2011. Effect of packaging conditions on physiology quality and shelf-life of fresh-cut kiwifruit. Advanced Materials Research  233– 235:1985– 1988. Liu, X. F., J. Y. Ren, Y. X. Zhu, W. Han, H. Y. Xuan, and L. Q. Ge. 2016. The preservation effect of ascorbic acid and calcium chloride modified chitosan coating on fresh-cut apples at room temperature. Colloids and Surfaces A— Physicochemical and Engineering Aspects  502:102– 106. Mahajan, P. V., F. A. R. Oliveira, J. C. Montanez, and J. Frias. 2007. Development of user-friendly software for design of modified atmosphere packaging for fresh and fresh-cut produce. Innovative Food Science and Emerging Technologies  8 (1):84– 92. Montero-Calderó n, M., and M. Milagro Cerdas-Araya. 2011. Fruits and vegetables for the fresh-cut processing industry. In Advances in Fresh-Cut Fruits and Vegetables Processing , ed. O. Martí n-Belloso and R. Soliva-Fortuny, 185– 211. Boca Raton, FL: CRC Press/Taylor & Francis Group. Montero-Calderon, M., M. A. Rojas-Grau, and O. Martin-Belloso. 2008. Effect of packaging conditions on quality and shelf-life of fresh-cut pineapple (Ananas comosus ). Postharvest Biology and Technology  50 (2– 3):182– 189. Muthuswamy, S., H. P. V. Rupasinghe, and G. W. Stratton. 2008. Antimicrobial effect of cinnamon bark extract on Escherichia coli  O157:H7, Listeria innocua  and freshcut apple slices. Journal of Food Safety  28 (4):534– 549. Nicola, S., G. Tibaldi, and E. Fontana. 2009. Fresh-cut produce quality: Implications for a systems approach. In Food Science and Technology , ed. W. J. Florkowski, S. E. Prussia, R. L. Shewfelt, and B. Brueckner, 249. New York: Academic Press. Nowacka, M., U. Tylewicz, L. Laghi, M. Dalla Rosa, and D. Witrowa-Rajchert. 2014. Effect of ultrasound treatment on the water state in kiwifruit during osmotic dehydration. Food Chemistry  144:18– 25. Obranović , M., D. Š kevin, K. Kraljić , M. Pospiš il, S. Neđ eral, M. Blekić , and P. Putnik. 2015. Influence of climate, varieties and production process on tocopherols, plastochromanol-8 and pigments in flaxseed Oil. Food Technology and Biotechnology  53 (4):496– 504. Oliveira, M., J. Usall, C. Solsona, I. Alegre, I. Vinas, and M. Abadias. 2010. Effects of packaging type and storage temperature on the growth of foodborne pathogens on shredded ‘ Romaine’  lettuce. Food Microbiology  27 (3):375– 380. Oms-Oliu, G., M. A. Rojas-Grau, L. A. Gonzalez, P. Varela, R. Soliva-Fortuny, M. I. H. Hernando, I. P. Munuera, S. Fiszman, and O. Martin-Belloso. 2010. Recent approaches using chemical treatments to preserve quality of fresh-cut fruit: A review. Postharvest Biology and Technology  57 (3):139– 148. Oms-Oliu, G., and R. Soliva-Fortuny. 2011. Future trends in fresh-cut fruit and vegetable processing. In Advances in Fresh-Cut Fruits and Vegetables Processing , ed. O. Martí n-Belloso and R. Soliva-Fortuny, 377– 387. Boca Raton, FL: CRC Press/ Taylor & Francis Group.

Fresh-Cut Apples Spoilage and Predictive Microbial Growth

45

Pathare, P. B., U. L. Opara, and F. A. Al-Said. 2013. Colour measurement and analysis in fresh and processed foods: A review. Food and Bioprocess Technology  6 (1):36– 60. Perez-Escamilla, R., and P. Putnik. 2007. The role of acculturation in nutrition, lifestyle, and incidence of type 2 diabetes among Latinos. Journal of Nutrition  137 (4):860– 870. Pizent, G., P. Putnik, D. Bursać Kovačević, and K. Herceg 2015. Anti-browning Apple Calculator – CAPPABLE: http://apple.pbf.hr (IP: 31.147.204.87) In (1.0 ed.). Faculty of Food Technology and Biotechnology, University in Zagreb (accessed May 1st, 2016). Pristijono, P., R. B. H. Wills, and J. B. Golding. 2006. Inhibition of browning on the surface of apple slices by short term exposure to nitric oxide (NO) gas. Postharvest Biology and Technology  42 (3):256– 259. Putnik, P., D. Bursać  Kovač ević , and V. Dragović -Uzelac. 2015a. Optimizing acidity and extraction time for polyphenolic recovery and antioxidant capacity in grape pomace skin extracts with response surface methodology approach. Journal of Food Processing and Preservation  40 (6):1256– 1263. Putnik, P., D. Bursać  Kovač ević , K. Herceg, and B. Levaj. 2016a. Influence of antibrowning solutions, air exposure, and ultrasound on color changes in fresh-cut apples during storage. Journal of Food Processing and Preservation . Submitted for publication. Putnik, P., D. Bursać  Kovač ević , K. Herceg, and B. Levaj. 2016. Influence of cultivar, anti-browning solutions, packaging gasses, and advanced technology on browning in fresh-cut apples during storage. Journal of Food Process Engineering  40, e12400. Putnik, P., D. Bursać  Kovač ević , K. Herceg, and B. Levaj. 2016. Influence of respiration on predictive microbial growth of aerobic mesophilic bacteria and Enterobacteriaceae in fresh-cut apples packaged under modified atmosphere. Journal of Food Safety . 37, e12284. Putnik, P., D. Bursać  Kovač ević , K. Herceg, S., Roohinejad, R. Greiner, A. E. A. Bekhit, and B. Levaj. 2017. Modelling the shelf-life of minimally-processed fresh-cut apples packaged in a modified atmosphere using food quality parameters. Food Control , 81:55–64. Putnik, P., D. Bursać  Kovač ević , K. Herceg, I. Pavkov, Z. Zorić , and B. Levaj. 2016. Effects of modified atmosphere, anti-browning treatments and ultrasound on the polyphenolic stability, antioxidant capacity and microbial growth in freshcut apples. Journal of Food Process Engineering. 10.1111/jfpe.12539. Putnik, P., D. Bursać  Kovač ević  , M. Penic, and V. Dragovic-Uzelac. 2015b. Optimizing microwave-assisted extraction parameters for polyphenols recovery from sage (Salvia officinalis  L.). Annals of Nutrition and Metabolism  67 (Suppl. 1):523– 524. Putnik, P., D. Bursać  Kovač ević , M. Penić , M. Fegeš , and V. Dragović -Uzelac. 2016f. Microwave-assisted extraction (MAE) of Dalmatian sage leafs for the optimal yield of polyphenols: HPLC-DAD identification and quantification. Food Analytical Methods  9 (8):2385– 2394. Putnik, P., S. Roohinejad , R. Greiner , D. Granato , A. E. A. Bekhit , and D. Bursać Kovačević. 2017. Prediction and modelling of microbial growth in minimally processed fresh-cut apples packaged in a modified atmosphere: A review. Food Control  80:411–419.

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Qadri, O. S., B. Yousuf, and A. K. Srivastava. 2015. Fresh-cut fruits and vegetables: Critical factors influencing microbiology and novel approaches to prevent microbial risks— A review. Cogent Food and Agriculture  1 (1121606):1– 11. Ragaert, P., L. Jacxsens, I. Vandekinderen, L. Baert, and F. Devlieghere. 2011. Microbiological and safety aspects of fresh-cut fruits and vegetables. In Advances in Fresh-Cut Fruits and Vegetables Processing , ed. O. Martí n-Belloso and R. Soliva-Fortuny, 53– 75. Boca Raton, FL: CRC Press/Taylor & Francis Group. Rocculi, P., V. Panarese, U. Tylewicz, P. Santagapita, E. Cocci, F. Gó mez Galindo, S. Romani, and M. Dalla Rosa. 2012. The potential role of isothermal calorimetry in studies of the stability of fresh-cut fruits. LWT— Food Science and Technology  49 (2012):320– 323. Rojas-Grau, M. A., A. Sobrino-Lopez, M. S. Tapia, and O. Martin-Belloso. 2006. Browning inhibition in fresh-cut ‘ fuji’  apple slices by natural antibrowning agents. Journal of Food Science  71 (1):S59– S65. Rupasinghe, H. P. V., J. Boulter-Bitzer, T. Ahn, and J. A. Odumeru. 2006. Vanillin inhibits pathogenic and spoilage microorganisms in vitro and aerobic microbial growth in fresh-cut apples. Food Research International  39 (5):575– 580. Trias, R., L. Baneras, E. Badosa, and E. Montesinos. 2008. Bioprotection of Golden Delicious apples and iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria. International Journal of Food Microbiology  123 (1– 2):50– 60. Tylewicz, U., S. Romani, S. Widell, and F. G. Galindo. 2013. Induction of vesicle formation by exposing apple tissue to vacuum impregnation. Food and Bioprocess Technology  6:1099– 1104. USDA (U.S. Department of Agriculture). 2014. Potential for infiltration, survival, and growth of human pathogens within fruits and vegetables. Washington, DC: USDA. http://www.fda.gov/Food/GuidanceRegulation/HACCP/ucm082063. htm (accessed November 6, 2016). Waghmare, R. B., P. V. Mahajan, and U. S. Annapure. 2013. Modelling the effect of time and temperature on respiration rate of selected fresh-cut produce. Postharvest Biology and Technology  80:25– 30. Wills, R. B. H., and Y. X. Li. 2016. Use of arginine to inhibit browning on fresh cut apple and lettuce. Postharvest Biology and Technology  113:66– 68.

Section II

 Food Allergens, Contaminants, and Toxins 

3 Analytical Methods for the Detection of Mycotoxins in Milk Samples Myra E. Flores-Flores and Elena Gonzá lez-Peñ as CONTENTS 3.1 Introduction................................................................................................... 49 3.2 Aflatoxins ...................................................................................................... 52 3.3 Ochratoxins ................................................................................................... 60 3.4 Trichothecenes .............................................................................................. 62 3.5 Fumonisins ................................................................................................... 71 3.6 Cyclopiazonic Acid ...................................................................................... 72 3.7 Ergot Alkaloids ............................................................................................ 73 3.8 Zearalenone and Its Derivatives................................................................. 74 3.9 Multimycotoxin Detection ..........................................................................80 3.10 Conclusions....................................................................................................83 Abbreviations .........................................................................................................83 References ............................................................................................................... 85

3.1 Introduction Food has been proven to be the major source of many toxicants today (Choi et al. 2015). The presence of naturally occurring contaminants that cause severe health effects to humans and animals after chronic exposure at low concentrations is a great concern in terms of food safety. Among these toxic compounds, the presence of mycotoxins is one of the most problematic (Zhang et al. 2014). Mycotoxins are secondary metabolites produced by filamentous fungi that can contaminate raw materials of vegetal origin (cereals and fruits) during their growth in the field or during storage and transport. The major toxin-producing fungal species belong to the genera Aspergillus, Penicillium, and Fusarium, and some of them can produce more than one type of toxin (Zhang et al. 2014). Mycotoxin contamination is hard to eliminate from agricultural crops, foods, and feeds. The mycotoxins are not easily detected by, for instance, a changed organoleptic characteristic in the contaminated products (Binder 2007). The Food and Agriculture Organization of the United Nations (FAO) estimates that approximately 25% of global 49

50

Food Safety and Protection

food production is contaminated by, at least, one mycotoxin (Heussner et al. 2006). In addition, these fungal toxins generally have high resistance to heat, and can appear in a raw material even after the producing fungi have been destroyed. Mycotoxins reach animals and humans through diet, affecting their health and causing mycotoxicosis. In some cases, acute toxic effects have been observed after ingestion of a highly contaminated product, especially in farm animals; however, in terms of human and animal health, the greatest concern is the chronic toxic effects that are generated as a result of continuous long-term exposure to low levels of these toxins. Toxic effects vary due to the different toxicological characteristics of the more than 300 known mycotoxins, including carcinogenicity, genotoxicity, nephrotoxicity, immunotoxicity, and less resistance to infections (Binder 2007). Thus, human exposure to mycotoxins through diet is a significant concern to public health worldwide (Zhang et al. 2014). Among all the different human foods, the study of the presence of mycotoxins in milk is of great interest, due to its key role in children’ s diets and even in many adult diets. The well-documented presence of a broad spectrum of mycotoxins in raw materials and feeding stuffs is a subject of continuous monitoring programs (Zachariasova et al. 2014). It raises the possibility that these toxins reach animals through the diet and are carried over into tissues or biological fluids, such as milk. The number of studies related to the transfer of these compounds to milk (Fink-Gremmels 2008; Flores-Flores et al. 2015), or to the interactions between mycotoxins at absorption and biotransformation level, is very limited (Fink-Gremmels 2008). It is known that rumen flora can transform mycotoxins such as ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), diacetoxyscirpenol (DAS), and T-2 toxin into their metabolites (Kalač  2011; Kiessling et al. 1984). However, this barrier can be altered by animal diseases, changes in the diet, or high mycotoxin contamination in feeding stuffs. For example, the studies of SCOOP (2002) and other authors (Pattono et al. 2011) warn about the possible presence of OTA in milk. In addition, other mycotoxins, such as patulin (PAT), may remain undisturbed by the rumen (Kalač 2011). Aflatoxin M1 (AFM1), the most studied mycotoxin in milk, is produced in the liver by hydroxylation of absorbed aflatoxin B1 (AFB1) (Kalač  2011). Its maximum permissible level in milk has been established at 0.05  µ g/kg in the European Union (EU) (European Commission 2010) and 0.5  µ g/kg in the United States (FDA 2005). In our recent review of the presence of mycotoxins in animal milk (Flores-Flores et al. 2015), it can be observed that approximately 10% of the 22 189 milk samples analyzed for AFM1 contamination worldwide presented concentration levels higher than those established in the EU. Moreover, even when feeding stuffs of ruminants comply with current EU regulations for aflatoxin content, AFM1 can reach milk in levels exceeding the actual permitted maximum level in the EU (Battacone et al. 2009; Han et al. 2013). With regard to other mycotoxins in milk, although few samples have been analyzed worldwide, the presence of fumonisin B1 (FB1), cyclopiazonic acid

Analytical Methods for the Detection of Mycotoxins in Milk Samples

51

(CPA), ZEA, zearalanone (ZAN), α -zearalanol (α -ZAL), α -zearalenol (α -ZEL), deepoxydeoxynivalenol (DOM-1), OTA, fumonisin B2 (FB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), AFB1, aflatoxin B2 (AFB2), and aflatoxin M2 (AFM2) has been encountered (Flores-Flores et al. 2015). In order to increase milk safety, our knowledge in this field must increase. Studies are needed regarding the presence of mycotoxins, their most frequent combinations, and their concentration levels in milk. These studies will help lead to better risk assessment, evaluating compliance with regulatory policies and taking other actions to protect public health (Zhang et al. 2014). The choice of the analytical method, with adequate sensitivity, is of utmost importance in carrying out these studies (Rubert et al. 2012). In addition, due to the presence of fat, proteins, salts, and high water content, milk is a very complex matrix that requires extensive and selective sample cleanup procedures that not only enable the removal of the interference of coextracted compounds, but also preconcentrate the analytes in order to reach the required low detection limits. Figure  3.1 shows the different steps that are usually taken for milk sample preparation before chromatographic analysis of mycotoxins. Some of them are optional (dotted square), depending on the analyzed products and the analytical method. This chapter is devoted to the review of the analytical methods published for mycotoxin detection and quantification in animal milk, including those developed for analysis of a single mycotoxin, as well as those that allow the simultaneous determination of these compounds in milk. Milk sampling Homogenization Hydrolysis of conjugates Extraction Cleanup Derivatization

b-Glucuronidase, aryl sulfatase LLE/SLE (ACN, ACE, MeOH), SPE (C18), QuEChERS Hexane, IAC, charcoal/alumina TMS, HFB, KBr, NDA + KCN

Separation

LC/GC/TLC/CE

Detection

UV/FLD/MS/FID/EC

FIGURE  3.1 General workflow for analysis of mycotoxins in milk by chromatographic-based methods. Dotted square indicates optional steps. Also, the most frequently used reagents or techniques are shown.

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Food Safety and Protection

Different technologies have been applied for this purpose, and they can be classified into two groups: chromatographic and nonchromatographic methods. Among the chromatographic methods, gas chromatography (GC) and especially liquid chromatography (LC), using different detector systems, have been used for the study of the different mycotoxins. Currently, the introduction of LC coupled to mass spectrometry (LC-MS) has improved the performance of the methods, allowing simultaneous detection and quantification of several mycotoxins from different families and with different physicochemical characteristics, with adequate sensitivity, and also allowing structural elucidation of unknown compounds. However, LC-MS requires high-cost equipment and specifically trained staff to operate the equipment and interpret the results. On the other hand, among the nonchromatographic methods, immunoassays are usually used for initial screenings due to their simplicity, low cost, and the fact that it is easy to process a large number of samples. Nonetheless, positive results need to be confirmed (i.e., using chromatographic methods) due to cross-reactivity with related molecules that can give overestimated values (El Khoury and Atoui 2010). The enzyme-linked immunosorbent assay (ELISA) is the most popular format in this category. A wide offer of ELISA-based kits is commercially available for all regulated mycotoxins in different matrixes. Among immunochemical-based methods, lateral flow immunoassay (LFIA), also called immunochromatographic assay or immune-gold colloid (IGC) immunoassay, is relatively new in the field of food safety (Dzantiev et al. 2014), although it is widely used in the field of medical diagnostics (i.e., detection of pregnancy, drug screening, identification of disease biomarkers, etc.). In the case of milk, there are commercially available supplies in both LISA and LFIA formats, although only for AFM1 and OTA detection. Recently, there has been a research trend toward the construction of biosensors for mycotoxin detection, but very few available methods have been published in the case of milk.

3.2 Aflatoxins Aflatoxins are mainly produced by three species of Aspergillus: A. flavus, A. parasiticus, and A. nomius (Prandini et al. 2009), which grow mostly in areas with hot and humid climates (EFSA 2007). They are most commonly known for causing acute or chronic liver disease, but they are also considered immunosuppressive, hepatotoxic, mutagenic, teratogenic, and carcinogenic (Brase et al. 2009). Among aflatoxins, AFB1 is the most commonly found in food, and it is highly toxic under either acute or chronic exposition (Cavaliere

53

Analytical Methods for the Detection of Mycotoxins in Milk Samples

et al. 2006); it has been classified as a human carcinogen (group 1) by the International Agency for Research on Cancer (IARC) (2012). In ruminants, aflatoxins are only partly degraded by the ruminal flora. In the liver, AFB1 is metabolized to AFM1, which may appear in the milk of dairy cows. About 0.3%– 6.2% of AFB1 in animal feed is transferred to milk as AFM1 (Creppy 2002) within 12  h of the ingestion of AFB1-contaminated feed, and no AFM1 levels were found 3 days after the last ingestion of AFB1 (Battacone et al. 2003). AFM1 has been classified as a possible human carcinogen (group 2B) by the IARC (1993). Aflatoxins are the most analyzed mycotoxins in milk. The presence of AFM1 has been especially studied worldwide, and it is the only mycotoxin with a defined maximum limit in milk in the European regulation (FloresFlores et al. 2015). Figure  3.2 shows a survey of the methods published from 2009 until April 2016 for AFM1 detection in milk. ELISA and LC– fluorescence detector (FLD) are the most used techniques and instruments for monitoring purposes; in terms of the development of new methods, LC– mass spectrometry detectors in tandem (MS/MS) is the preferential analytical technique. Currently, biosensors, immunosensors, and immunoassays have been studied and their application to the determination of aflatoxins is in continuous development. Mwanza et al. (2015) evaluated the efficiency of different commercially available rapid kits and techniques (i.e., thin-layer chromatography [TLC], immunochromatographic strips, ELISA, and LC). The study concluded that TLC, LC, and ELISA are sufficiently developed 42

Newly developed methods

34

LS D

ns or

or

os e

ns

Bi

or

se

ns

se

im

m

ta

1

El

ec

tr o

ch e

m

im

pe d

Im

2

ic al

et ric

ap

un

os e

gr ap

m im

ric

2

1

ns or

hy

ry et om

et m di

pe

2

un o

5

at o

LC

Im

Im

m

un

ec

oc hr

tr ofl

uo

rim

-T

U V C-

S

PL

S/ M

Sp

H

PL

C-

M

C-

IS H

5

4

1

H

FL D

2

2D

4

PL

EL

Application of previously developed methods

11

6

A

45 40 35 30 25 20 15 10 5 0

FIGURE  3.2 Publications regarding methodologies for AFM1 determination in milk (2009– April 2016), grouped into newly developed methods and the application of previously developed methods for monitoring purposes.

54

Food Safety and Protection

in this field, and that immunochromatographic strips are not able to detect small quantities of AFM1. Detection limits of commercially available immunochromatographic strips are shown in Table  3.1. Currently, the majority of them achieve limit of detection (LOD) levels sufficient for screenings of AFM1 in milk at the 0.5  µ g/kg level, the maximum limit in some world regions (FAO-WHO 1995; MERCOSUR 2002; FDA 2005), but they are not reliable in the case of the European regulation at 0.05  µ g/kg (European Commission 2010). With regard to the ELISA test, a great variety of kits are commercially available (Table  3.2), but also, new attempts to improve ELISA performance can be found in the literature (Guan et al. 2011a, 2011b; Kanungo et al. 2011; Wang et al. 2011; Vdovenko et al. 2014; Peng et al. 2016). Biosensors based on surface plasmon resonance (Karczmarczyk et al. 2016) and a silicon oxynitride ring resonator (Guider et al. 2015), impedimetric aptasensor (Istamboulié et al. 2016), impedimetric immunosensor (Bacher et al. 2012; Kanungo et al. 2014), electrochemical immunosensor (Neagu et al. 2009; Paniel et al. 2010), immunochromatography combined with gold nanoparticles TABLE  3.1 Commercial Lateral Flow Test Strips for Detection of AFM1 in Milk Manufacturer (Country)

LOD/ Sensitivity

Assay Time

Shenzhen Lvshiyuan Biotechnology Co. (China)

300  ng/L

— 

Qualitative analysis

Charm Sciences Inc. (United States) Neogen Corporation (United States) Bioo Scientific (United States)

350  ng/L

3  min

Qualitative analysis

500  ng/L

5  min

Qualitative analysis

500  ng/L

8  min

Qualitative analysis Designed for field use

Unisensor Diagnostic Engineering (Belgium)

20  ng/L

10  min

Unisensor Diagnostic Engineering (Belgium)

200  ng/L

10  min

Instrumental (quantitative) detection Detection range: 20– 200  ng/L Visual (qualitative) and instrumental (quantitative) detection Detection range: 200– 750  ng/L

Observation

Product Name (Reference) AFM1 Rapid Test Dipsticks (Milk) (Shenzhen Lvshiyuan Biotechnology 2015b) Charm ROSA SLAFM (Charm Sciences Inc. 2016) Reveal for AFM1 (Neogen Corporation 2016a) AuroFlow AFM1 Strip Test Kit (Bioo Scientific 2016a) AflaSensor Quanti 0.05 ppb - Kit 041 (Unisensor Diagnostic Engineering 2016a) AflaSensor Quanti 0.5 ppb - Kit 078 (Unisensor Diagnostic Engineering 2016b)

55

Analytical Methods for the Detection of Mycotoxins in Milk Samples

TABLE  3.2 Commercial ELISA Kits for Detection of AFM1 or OTA in Milk Manufacturer (Country) AFM1 R-Biopharm (Germany)

LOD/Sensitivity

Time (min)

Observation

 8.0 0.6 1.1 2.7 6.0

6.0 6.0 > 7.0

Log Reduction 

O’ Reilly et al. 2000

Patterson et al. 1995

Shigehisa et al. 1991

Hugas et al. 2002

Alpas and Bozoglu 2000

Gervilla et al. 1999

Patterson and Kilpatrick 1998

References 

356 Food Safety and Protection

ATCC 29425 ATCC 25922 CECT 405 (ATCC 10536)

K12 —  — 

Milk Poultry meat Cheese slurry Pork slurry Fresh goat cheese

UHT milk Poultry meat Milk

Liquid whole egg

Skim milk

Food Products 

5.2– 5.4 nr 6.5

nr

6.7

nr

8.0

6.6

pH 

500 400– 500 450– 500

600

345

500

450

550

RT RT RT

RT

20 10 5

15

5

15

50a  50a 

10

15

Time  (min) 

50

50

Average Temperature  (° C) 

Note : RT,  room temperature HPP; nr, not reported; UHT, ultrahigh temperature. a  Initial temperature before compression.

O157:H7

933 931 NCTC 12079

LMM 1020 LMM 1030 LMM 1010 MG 1655 (ATCC 47076) CECT 405 (ATCC 10536) NCTC 12079

O157:H7

O157:H7

— 

K12 K12 K12 K12

Strains 

Pressure  (MPa) 

Inactivation of  Escherichia coli   in Dairy and Poultry Foods by HPTP and HPP Alone 

TABLE  10.6 

8.0 7.5 > 8.0 > 8.0 1.5 3.0 > 6.0 > 6.0 > 8.5

5.5

2.4 2.4 4.7 7.0

Log Reduction 

References 

O’ Reilly et al. 2000 Shigehisa et al. 1991 Capellas et al. 1996

Patterson et al. 1995

Alpas and Bozoglu 2000

Patterson and Kilpatrick 1998

Ponce et al. 1998

Garcí a-Graells et al. 1999

HPP Inactivation of Pathogenic Microorganisms 357

C9490

nr

O157:H7

O157:H7

LMM 1010

NCTC 12079 ATCC 43894

O157:H7 O157:H7

K12

SEA 13B88 ATCC 43895, 932b 

933 933

O157:H7

O157:H7

931

O157:H7

Strains 

3.8 3.5 4.1 3.7 3.8 4.0

6.2 3.0 3.4– 4.5 4.5

Carrot juice Grapefruit juice Orange juice Mango juice

Orange juice Apple juice Tomato juice Orange juice Apple juice Mango juice

3.8 3.8 3.3 3.5 3.7 3.7

3.8

pH 

Apricot juice Orange juice Sour cherry juice Apple juice Apple juice Orange juice

Orange juice

Fruit Juices 

500

250

500

550 550

615

350

345

Pressure  (MPa) 

Inactivation of  Escherichia coli   in Fruit Juices by HPTP and HPP Alone 

TABLE  10.7 

RT

RT

RT

RT RT

RT

2

20

5

5 5

2

5

5

50a 

40a 

Time  (min) 

Average Temperature  (° C) 

> 7.0 > 7.0 > 7.0 5.0 > 7.0 > 5.0

6.4 8.3 > 7.0 > 8.0

> 8.0 > 8.0 > 8.0 > 8.0 > 8.0 0.4 2.2

> 8.0

Log Reduction  References 

(Continued)

Garcia-Graells et al. 1998

Noma et al. 2004

Linton et al. 1999 Hiremath and Ramaswamy 2012 Jordan et al. 2001

Teo et al. 2001

Bayı ndı rlı  et al. 2006

Alpas and Bozoglu 2000

358 Food Safety and Protection

ATCC 11775

ATCC 11775

ATCC 25922

— 

— 

— 

3.8 3.3 4.2 4.1

Cashew apple juice

3.6

3.4

3.5

pH 

Pineapple juice Kiwifruit juice Pear nectar

Mango nectar

Note : RT,  room temperature HPP; nr, not reported. a Initial temperature before compression. b Cocktail of strains.

ATCC 11775

Apple pieces in 24°  Brix GLUCOSE syrup Orange juice

Resistant mutant LMM 1010 Resistant mutant ATCC 11775

— 

— 

K12

Fruit Juices 

Strains 

400

241

300

414

414

600

Pressure  (MPa) 

Inactivation of  Escherichia coli   in Fruit Juices by HPTP and HPP Alone

TABLE  10.7 (CONTINUED)

RT

RT

RT

RT

RT

RT

Average Temperature  (° C) 

3

3

5

1

0.03

10

Time  (min) 

6.5

1.0 4.0 4.0

Guerrero-Beltrá n et al. 2011b Lavinas et al. 2008

Guerrero-Beltrá n et al. 2011a Bermú dez-Aguirre et al. 2011 Buzrul et al. 2008

> 7.0 > 8.0

Vercammen et al. 2012

References 

> 6.0

Log Reduction 

HPP Inactivation of Pathogenic Microorganisms 359

Goat cheese Milk Poultry meat UHT milk Raw milk

Milk

500 375 340

6.5

345

nr nr

6.7

pH 

RT

RT RT

50

Initial Temperature  (° C) 

20

5 15

5

Time  (min) 

> 8.0 > 5.6 0.5 2.0 1.5 2.0

> 8.0

Log Reduction 

References 

Styles et al. 1991

Gallot Lavallee 1998 Patterson et al. 1995

Alpas and Bozoglu 2000

Note : RT,  room temperature HPP (for HPTP, the temperature was the initial one before compression); nr, not reported; UHT, ultrahigh temperature.

Ohio2 nr NCTC 11994 (DSM 15675) Scott A

CA

Food Products 

Pressure  (MPa) 

Inactivation of  Listeria monocytogenes   in Foods by HPTP and HPP Alone 

TABLE  10.8 

360 Food Safety and Protection

Liquid whole egg Strained chicken baby food Pork slurry

SE-4 ATCC 7136

ATCC 14028 775 W

S. enteritidis  S. typhimurium 

S. typhimurium 

a 

Note : RT,  room temperature (20– 25° C); nr, not reported. Initial temperature before compression.

Strained chicken baby food

Milk Milk Liquid whole egg

FDA E 21274 nr

Salmonella enteritidis  Salmonella typhimurium  S. enteritidis 

Salmonella senftenberg 

Food Products 

Strains 

Species 

nr

nr

nr nr

6.7 6.7 8.0

pH 

Inactivation of  Salmonella   in Foods by HPTP and HPP Alone 

TABLE  10.9 

340

400

400 340

345 345 450

Pressure  (MPa) 

RT

RT

50 50 50 RT RT RT

Initial Temperature a   (° C) 

15

10

5 5 15 15 10 15

Time  (min) 

2.5

6.5

> 8.0 > 8.0 > 7.8 5.1 6.0 2.0

Log Reduction 

References 

Metrick et al. 1989

Shigehisa et al. 1991

Bari et al. 2008 Metrick et al. 1989

Alpas and Bozoglu 2000 Alpas and Bozoglu 2000 Ponce et al. 1999

HPP Inactivation of Pathogenic Microorganisms 361

362

Food Safety and Protection

Salmonella  spp. (Tables   10.8 and 10.9). Thus, the minimum processing conditions of 600  MPa and 15 min with an initial temperature of 50° C before compression enabled large reductions of S. aureus , and should actually be used to ensure the HPP inactivation of the most resistant vegetative cells in foods. Vibrio  spp. and other vegetative pathogens, such as C. jejuni , Y. enterocolitica , C. freundii , and A. hydrophila , generally required milder processing conditions (170– 586  MPa, 0– 20 min, and room temperature) to achieve the same viability losses, > 5.0 to 8.0 log (Tables  10.10 and 10.11). Similar inactivation of vegetative pathogens by room temperature HPP between pathogenic strains belonging to the same species has also been observed in most of the past works of many authors compiled. For example, the following ranges of processing conditions were recorded: 400– 500  MPa and 5– 20  min for E. coli  in cheese and pork with > 6.0 to > 8.5 log (Shigehisa et al. 1991; Capellas et al. 1996; O’ Reilly et al. 2000) (Table  10.6), 500– 550  MPa and 2– 5 min for E. coli  O157:H7 in fruit juices with > 5.0 to > 8.0 log (GarciaGraells et al. 1998; Linton et al. 1999; Jordan et al. 2001; Hiremath and Ramaswamy 2012) (Table  10.7), 340– 375  MPa and 15– 20 min for L. monocytogenes  in milk and meat with 0.5– 2.0 log (Styles et al. 1991; Patterson et al. 1995), and 500  MPa and 5 min process resulted in > 5.6 log and 500 MPa and 5 min for L. monocytogenes  in goat cheese with > 5.6 log. (Gallot Lavallee 1998) (Table  10.8); 400– 450  MPa and 15  min for Salmonella  spp. in liquid whole egg with 5.1 to > 7.8 log (Ponce et al. 1999; Bari et al. 2008) (Table  10.9), 586  MPa and 0  min for Vibrio  spp. in oyster with > 5.5 to > 6.5 log (Koo et al. 2006) (Table  10.10), and 375– 400  MPa and 10  min for C. jejuni  and Y. enterocolitica  in meat products with > 6.0– 8.0 log (Shigehisa et al. 1991; Solomon and Hoover 2004) (Table  10.11). However, on the other hand, few researchers observed large resistance differences among S. aureus  in the food products under the same conditions of room temperature HPP, for example, log reductions in the range of 0.6– 6.0 log for S. aureus  after 600  MPa for 6– 20  min (Shigehisa et al. 1991; Patterson et al. 1995; O’ Reilly et al. 2000; Hugas et al. 2002) (Table  10.5), which poses a significant concern of this most resistant vegetative pathogen. Due to a few outbreaks registered in raw unpasteurized acidic fruit juices, E. coli  inactivation by HPP and HPTP was also investigated in this class of beverages, since it might grow in this environment. Some researchers have shown that different fruit juices resulted in large variations in the room temperature HPP resistance of E. coli  O157:H7 at the same processing conditions (Teo et al. 2001; Buzrul et al. 2008). However, Bayı ndı rlı  et al. (2006) worked with a cocktail of resistant E. coli  strains and showed that 350  MPa HPTP with an initial temperature of 40° C for 5  min achieved very high reduction (> 8.0 log) of the pathogenic E. coli  in four fruit juices (Table  10.7). 10.5.2  Kinetic Models Until 2012, modeling studies of vegetative pathogens after HPP in various food products were carried out using first-order kinetics (Table  10.12).

Note : nr, not reported.

Vibrio vulnificus  V. vulnificus  V. vulnificus  Vibrio parahaemolyticus  V. parahaemolyticus  V. parahaemolyticus  V. parahaemolyticus 

Species 

Food Products  Oyster Oyster Homogenized oyster Oyster Homogenized oyster Oyster Clam juice

Strains 

MO-624 MLT 403 nr TX-2103, serotype O3:K6 10 different strains ATCC 43996 T-3765-1

nr nr nr nr nr nr 7.5

pH 

 Inactivation of  Vibrio   in Oysters and Clam Juice by Room Temperature HPP 

TABLE  10.10

586 300 275 586 300 300 170

Pressure  (MPa)  0 2 3 0 3 2 10

Time  (min) 

References  Koo et al. 2006 Ye et al. 2012 Cook 2003 Koo et al. 2006 Cook 2003 Ye et al. 2012 Styles et al. 1991

Log Reduction  > 6.5 > 7.0 > 7.0 > 5.5 > 6.0 7.0 > 5.0

HPP Inactivation of Pathogenic Microorganisms 363

Pork slurry Pork slurry Chicken puree Milk Pork slurry Ground pork Minced beef Ground pork

nr T1 ATCC 35921

nr 9610 nr ATCC 7965

Streptococcus faecalis  Campylobacter jejuni  C. jejuni 

Yersinia enterocolitica  Y. enterocolitica  Citrobacter freundii  Aeromonas hydrophila 

Note : nr, not reported.

Meat Products 

Strain 

Vegetative Cells  nr nr nr nr nr 6.0 5.6– 5.8 6.0

pH  600 400 400 375 400 304 300 253

Pressure  (MPa)  10 10 10 10 10 15 20 15

Time  (min) 

Inactivation of Other Pathogenic Vegetative Cells in Meat Products by Room Temperature HPP

TABLE  10.11 

> 6.0 > 6.0 8.0 8.0 > 6.0 > 7.0 > 6.0 > 6.0

Log Reduction 

Shigehisa et al. 1991 Ellenberg and Hoover 1999 Carlez et al. 1993 Ellenberg and Hoover 1999

Shigehisa et al. 1991 Shigehisa et al. 1991 Solomon and Hoover 2004

References 

364 Food Safety and Protection

HPP Inactivation of Pathogenic Microorganisms

365

However, note that although first-order kinetic parameters were determined by the authors, most charts shown in the publications demonstrated a nonlinear log inactivation behavior (Metrick et al. 1989; Styles et al. 1991; Gervilla et al. 1999; Ponce et al. 1999; O’ Reilly et al. 2000; Koo et al. 2006). The fitting carried out with the conventional first-order linear model simplified the analysis and comparison with the available results from other authors in past literature. S. aureus  was confirmed to have the highest resistance (D 450   =  16.7  min in milk) among other species (Gervilla et al. 1999), whereas V. parahaemolyticus  was shown to be the least resistant vegetative pathogen, with a D 136  value of 5.6  min in clam juice (Styles et al. 1991). Using a secondary model parameter of the first-order kinetics, z P  values of 204 for E. coli  and 359  MPa for S. aureus  indicate low microbial susceptibility to small pressure changes (Table  10.12) (O’ Reilly et al. 2000; Hiremath and Ramaswamy 2012). From this information, the estimated D 600  MPa  values for 5  D of S. aureus  and E. coli  are 28.9 and 0.9  min, respectively.

10.6  Future Perspectives High-pressure-treated foods have been categorized “ novel foods”  in countries in the European Union (EU) and in Canada, due to the capability to retain many of the qualities of the fresh food product, which would otherwise be altered by conventional thermal processing. Standard HPP treatments (400– 600  MPa) can achieve 5.0– 6.0 or more log reductions of most vegetative pathogens since they are susceptible to pressure at ambient temperature. However, a few studies demonstrated higher resistance of a few strains of microbial pathogens in the vegetative form in certain foods. Although > 6.0 log reductions of E. coli  are registered at room temperature HPP, Patterson et al. (1995) could only achieve a low level of inactivation (1.5– 3.0 log) in milk and poultry meat after 600  MPa and 15  min, and Teo et al. (2001) has shown how difficult it is to inactivate a cocktail of E. coli  strains in apple and orange juices. The survivors of E. coli  may grow at low temperature during distribution, posing a human safety concern. Thus, HPP at a temperature of around > 50° C is a possible solution to ensure complete inactivation of E. coli . Furthermore, more research is needed to obtain reliable inactivation models able to predict the nonlinear behavior of vegetative pathogens in HPPtreated foods at different pressure and temperature conditions. HPP alone is not effective when the pasteurization aim is microbial spores, and HPTP at moderate temperatures or higher is required for spore inactivation. In addition to the food matrix being treated, the HPTP treatment conditions (pressure, average temperature, and processing time during the constant pressure phase of the HPP cycle) are important factors affecting spore inactivation. Some species of Clostridium  and

ATCC 29425 Scott A

ATCC 7136

775 W

TX-2103 serotype O3:K6 T-3765– 1

E. coli  K12 Listeria monocytogenes  Salmonella enteritidis  Salmonella typhimurium 

Salmonella senftenberg 

Vibrio parahaemolyticus 

nr 7.5

Clam juice

nr

5.2– 5.4 6.5 8.0 nr

4.5

5.2– 5.4 8.0

6.7

pH 

Cheese slurry UHT milk Liquid whole egg Strained chicken baby food Strained chicken baby food Oyster

Mango juice

Cheese slurry Liquid whole egg

Ovine milk

Food Products 

136

345

340

350 340 400 340

450

400 400

450

Pressure,  P   (MPa) 

5.6

2.0

7.1

19.0 13.2 8.8 7.6

0.72

20.0 14.1

16.7

D  P Value (min) 

nr

nr

nr

nr nr nr nr

204

359 nr

nr

z  P Value  (MPa) 

Styles et al. 1991

Koo et al. 2006

Metrick et al. 1989

Hiremath and Ramaswamy 2012 O’ Reilly et al. 2000 Styles et al. 1991 Ponce et al. 1999 Metrick et al. 1989

O’ Reilly et al. 2000 Ponce et al. 1998

Gervilla et al. 1999

References 

Note : D P   and z  values are the first-order kinetic parameters (Equations  10.1 and 10.2). Initial temperature, ≤ 25° C; nr, not reported; UHT, ultrahigh temperature. Note that although first-order kinetic parameters were determined by the authors, charts demonstrated a log nonlinear inactivation with the time in most of the studies reviewed.

V. parahaemolyticus 

E. coli  O157:H7

S. aureus  Escherichia coli 

CECT 534 (NCTC 4163) ATCC 6538 CECT 405 (ATCC 10536) ATCC 43894

Strain 

Staphylococcus aureus 

Pathogen 

First-Order Parameters 

First-Order Kinetic Parameters for the Inactivation of Pathogenic Vegetative Microorganisms in Food Products after Room Temperature HPP

TABLE  10.12 

366 Food Safety and Protection

HPP Inactivation of Pathogenic Microorganisms

367

Bacillus  are resistant sporeformers of public health concern that can only be inactivated at high pressures and temperatures, not yet achievable by commercial HPP units. The nonlinear trend observed for spore inactivation by HPTP (Evelyn and Silva 2015b, 2016a; Ju et al. 2008) should be followed up due to an increase of microbial spore resistance with processing time. More research is still required to standardize HPTP pasteurization conditions (process criteria, pressure– time– temperature combinations, etc.) in various food products to successfully introduce HPTP in the food industry. As heat has detrimental effects on food quality, an alternative option is the simultaneous or sequential application of HPP and other nonthermal food preservation technologies to enhance the lethal effect of HPP (e.g., irradiation was investigated by Crawford et al. [1996]). Furthermore, the cold storage conditions should be topped up with other hurdles, such as modified atmospheres and the use of preservatives, to inhibit or slow down the growth of resistant sporeformers in HPP pasteurized food products.

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11 Application of Pulsed Light for the Microbial Decontamination of Foods Marija Zunabovic, Victoria Heinrich, and Henry Jä ger CONTENTS 11.1 Introduction................................................................................................. 379 11.2 Pulsed Light................................................................................................. 381 11.2.1 Fundamentals of Pulsed Light...................................................... 381 11.2.1.1 Factor: Treated Matrix..................................................... 381 11.2.1.2 Factor: Microbial Contamination................................... 382 11.2.1.3 Factor: Process Setup....................................................... 383 11.2.2 In-Package Application of Pulsed Light...................................... 386 11.2.3 Antimicrobial Efficacy of Pulsed Light on Different Food Matrixes............................................................................................ 388 11.3 Conclusion................................................................................................... 390 References.............................................................................................................. 390

11.1 Introduction Food spoilage is inevitable. Hence, food business operators (FBOs) take diverse actions to preserve the initial state or desired quality level of foods as long as possible (Prokopov and Tanchev, 2007; Aymerich et al., 2008; Rajkovic et al., 2010). In this context, food processing, and especially food preservation, offers the advantages of increased food safety, continuous food supply during season and off-season, availability of value-added products, and variety in diet. Preservation can be achieved via (1) the delay or inhibition of chemical, microbiological, enzymatic, and nonenzymatic processes (e.g., chilling, freezing, and reduction of pH and aw); (2) the direct inactivation of biological agents and enzymes (e.g., heat pasteurization and sterilization), and (3) the avoidance of (re-)contamination with biological agents in the production process (e.g., hygiene measures and packaging). Further, the preservation technologies can also be classified in microbiological, chemical, mechanical, and physical procedures (Prokopov and Tanchev, 2007).

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The most commonly used physical food preservation method is thermal treatment. Depending on the degree of preservation, it can be distinguished between preservation and sterilization (Prokopov and Tanchev, 2007; Barbosa-Cá novas and Bermú dez-Aguirre, 2011). To achieve the respective level of microbial inactivation, a certain time– temperature regime has to be applied (Prokopov and Tanchev, 2007). Along with the microbial inactivation, however, enzymes and various other food components can be affected. This can have a positive effect in terms of stabilization of the product, but can also have a negative effect in terms of impairment of quality determining constituents or nutrients. In order to prevent the latter, it is indispensable to evaluate the actual need for preservation of each food product and food class treated and to adjust the treatment intensity (Prokopov and Tanchev, 2007; Barbosa-Cá novas and Bermú dez-Aguirre, 2011). Against this background, recent decades have seen a substantial increase in research and development activities aimed at the substitution of these conventionally used and historically proven thermal treatments. The development was induced by the adjustment of FBOs to factors such as changing societal and demographic conditions, consumer trends, expectations, and preferences (Aymerich et al., 2008; Sofos, 2008; Havelaar et al., 2010; Knorr et al., 2011; Weiss et al., 2010). As a result, a steadily increasing proportion and variety of (novel) food products can be found on the market today (Jaroni et al., 2010). The developed alternative technologies thereby range from mild or minimal processing over novel thermal and nonthermal physical food preservation to the combination of different treatments (hurdle technology) (Butz and Tauscher, 2002; Devlieghere et al., 2004; Sun, 2005; Rajkovic et al., 2010; Zhang et al., 2011; Chen et al., 2012; Stratakos and Koidis, 2015). The actual impact on the quality of food, applicability, and overall benefit of these alternatives is, however, dependent on the respective product, contamination, and process setup (Zhang et al., 2011). Further, successful market introduction is dependent on the improvement of shelf life and safety, maintenance of organoleptic and nutritional attributes of the product, freedom from residuals, convenience, economic acceptability, and acceptance from consumers and legislators (Raso et al., 2005; Rajkovic et al., 2010). Focusing on novel physical technologies, novel thermal technologies distinguish themselves from conventional thermal technologies by faster heating rates. Examples of such technologies include ohmic heating, microwave heating, and radiofrequency heating (Sun, 2005; Stratakos and Koidis, 2015). By comparison, novel nonthermal technologies have in common that the treatment is performed at ambient or near-ambient temperature. Examples are high-pressure processing, pulsed electric fields, plasma treatment, ionizing radiation, and pulsed light (PL) (Sun, 2005; Zhang et al., 2011; Stratakos and Koidis, 2015). The following sections aim to provide a concise overview of the underlying principles, the mechanism of microbial inactivation, and the critical

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factors, risks, and limits of the novel nonthermal physical preservation technology PL.

11.2  Pulsed Light 11.2.1  Fundamentals of Pulsed Light PL is a novel nonthermal food preservation technology and is capable of inactivating microorganisms in a rapid, efficient, and residue-free manner. In the food sector, the main application areas are decontamination of gases (e.g., process air), liquids (e.g., beverages) and surfaces of food and food contact materials (e.g., packaging materials) (Dunn et al., 1989, 1995). PL technology comprises the generation of high-power electrical pulses and transformation thereof into short-duration (fractions of a second), high-power pulses of broad spectrum (approximately 180– 1100  nm) electromagnetic radiation (light) via an inert-gas (xenon) flash lamp (Dunn et al., 1989). The basic essentials and schematic layout of a PL device are, for example, provided by Heinrich et al. (2016b). Based on the scientific literature, three main factors affecting the efficacy of a PL treatment were identified. These are the respective (1) type of matrix (e.g., food) treated, (2) the microbial contamination, and (3) the process parameter chosen (Heinrich et al., 2016b). To achieve interpretable, comparable, and reproducible decontamination outcomes, it is of upmost importance to properly record and communicate these factors. Further, it should be aimed at not only a high decontamination rate but also the avoidance of damages to the treated matrix (e.g., changes in color or sensory profile) (Lagunas-Solar and Gó mez-Ló pez, 2006; Gó mez-Ló pez et al., 2007). The following sections discuss the above-listed main factors and their impact on the treatment efficacy of PL from a general point of view. Further on, food category– specific information is given. 11.2.1.1  Factor: Treated Matrix Transparency and opacity, surface characteristics, and composition of the matrix strongly influence the efficacy of a PL treatment. Concerning transparency or opacity, optimal results are achieved when the matrix exhibits a low reflection coefficient and, at the same time, high absorption and transmission coefficients. In other words, aside from transparent liquids or gases, the effect of PL is, in the majority of cases, limited to the surface or upmost layer of a semisolid or solid matrix, according to the matrix’ s capability to absorb and transfer light (Dunn et al., 1989; Palmieri and Cacace, 2005; Gó mez-Ló pez et al., 2007). Further, smooth surfaces can be decontaminated

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more easily as such, exhibiting vast irregularities and light-absorbing matter, since they act as shelter for the microbial contamination and an obstacle for the incident light (Dunn et al., 1995; Gó mez-Ló pez et al., 2005a, 2007; Palmieri and Cacace, 2005; Lagunas-Solar and Gó mez-Ló pez, 2006; Sommers et al., 2009). Last but not least, the matrix should contain as little substances able to competitively absorb light, such as fats or proteins, as possible. Carbohydrates do not show a pronounced light-absorbing effect (Gó mez-Ló pez et al, 2005a; Rajkovic et al., 2010). 11.2.1.2  Factor: Microbial Contamination Microbial contamination impacts the efficacy of a PL treatment in various aspects. These include the respective microorganism and its physiological constitution, population density, and growth parameters (e.g., growth rate and lag time) (Dunn et al., 1989; Augustin et al., 2011; Cudemos et al., 2013). Regarding the respective microorganism, some distinctions in susceptibility can be derived. For example, it seems that due to the differences in cell structure, gram-positive bacteria are more resistant to PL than gramnegative bacteria. Further, mucoid and pigment-forming bacteria seem to be more resistant than their counterparts, fungi seem to be more resistant than bacteria, and bacterial spores tend to be more resistant than their corresponding vegetative cells. Interestingly, the size of bacteria can also have an impact. Namely, the smaller the cell, the faster the heat induced by the electromagnetic radiation can dissipate from the surface and the more resistant the cells are (Rowan et al., 1999; Anderson et al., 2000; Farrell et  al., 2010). Decontamination efficacy can also be impaired by the factors high population density (mutual shading of cells) and stationary growth phase. Consequently, prompt treatment after microbial contamination of the matrix takes place is recommended (Hiramoto, 1984; Anderson et al., 2000; Gó mezLó pez et al., 2005b; Farrell et al., 2010; Rajkovic et al., 2010). Studies related to the viral inactivation on food-related surfaces and in food matrices are scarce. More studies with drinking water and wastewater in this context can be found (Yi et al., 2016; Barrett et al., 2016). The inactivation effect relies on observations in modifications of nucleic acid and capsid (Vimont et al., 2015). The antiviral application of PL in drinking water is promising, as no interference with water hardness (up to 400  mg  L– 1) and conductivity (up to 14.3  mS  cm– 1) was shown (Vimont et al., 2015). Also, varying turbidity showed, for example, 4 log 10 reductions in the infectivity of poliovirus and rotavirus (Vimont et al., 2015). The same authors tested broadband PL against a surrogate of human norovirus on inert, cleaned food contact surfaces and in artificial alginate biofilms. Interestingly, the alginate layer did not have a protective effect against PL. The inactivation of relevant viral groups, such as human noroviruses, was also tested, with its surrogates showing a significantly higher resistance to PL treatments than pathogenic Escherichia coli and Salmonella strains (Huang et al., 2017).

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The inactivation of microorganisms can be explained by photophysical (cell death due to structural damage), photochemical (cell death due to DNA lesions) and photothermal (cell death due to disruption and structural changes) mechanisms (Dunn et al., 1989; Wekhof, 2000; Wekhof and Trompeter, 2001; Takeshita et al., 2003; Farell et al., 2010; Cheigh et al., 2012). Acting parallel or in sequence, these mechanisms make PL superior to conventionally used continuous-wave ultraviolet (CW UV) systems (Dunn et al., 1989). Contrary to the initial expectation, the inactivation curve of PL has been repeatedly shown to be nonlinear. As a consequence, commonly used log-linear mathematical models often cannot be used to accurately describe the inactivation pattern observed (Luksiene et  al., 2007; Uesugi and Muraru, 2009; Farrell et al., 2010; Keklik et al., 2012). In most cases, the authors found a sigmoid-shaped inactivation curve. The specific shape originates from a three-phased inactivation course. First, cells experience a nonlethal injury, which is reflected by the characteristic shoulder of the curve. Second, the surviving fraction rapidly declines because a maximum of cell injury and a minimum of additional energy required to cause high cell death rates are reached. Lastly, a socalled tailing is reached. Here, lack of a homogenous population, multihit phenomena, varying susceptibility of strains, started cell-repair activity, shading of cells by suspended solids or objects, and declined probability of exposure to PL causes decreased cell death rates (MacGregor et al., 1998; McDonald et al., 2000; Yaun et al., 2003; Fine and Gervais, 2004; Gó mezLó pez et al., 2007). In some cases, no shoulder and/or tailing is observed. This can be attributed, on the one hand, to a high initial energy input and, on the other, to a low initial cell population (Otaki et al., 2003; Wang et al., 2005; Farrell et al., 2010). Hence, the Weibull-type mathematical model and the log-linear mathematical model including a shoulder and/or tail phase were found appropriate to describe the inactivation curve of PL in several cases (Geeraerd et al., 2005; Keklik et al., 2005; Bialka et al., 2008a, 2008b; Heinrich et al., 2016a). 11.2.1.3  Factor: Process Setup The chosen process setup has a significant influence on the decontamination efficacy. Factors comprise the spectrum, experimental factors, geometry and setup. In general, the whole spectrum emitted by the PL flash lamp contributes to the lethal effects given above. The high-energy-bearing UV range, and in particular wavelengths below 270  n m, however, are of greatest importance. Consequently, the exclusion of wavelengths in the UV range (e.g., via filter) can have a negative impact on the decontamination efficacy (Dunn et al., 1991; Wekhof, 2000; Takeshita et al., 2003; Wang et al., 2005; Levy et al., 2012). For proper decontamination, the experimental parameters fluence rate, fluence, number of pulses, pulse width, exposure time, frequency, and peak power have to be adjusted to the respective situation

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(Palmieri and Cacace, 2005; BIPM, 2006; Lagunas-Solar and Gó mez-Ló pez, 2006; Gó mez-Ló pez et al., 2007; IUPAC, 2007). Of the parameters listed, the fluence incident on the matrix is often described as the essential measure. Improvements, however, can also be made by use of short-duration, highfrequency pulses (Hiramoto, 1984; Dunn et al., 1989; MacGregor et al., 1998; Anderson et al., 2000; Wekhof, 2000; Panico, 2005; Wang et al., 2005; Gó mezLó pez et al., 2007; Luksiene et al., 2007; Farrell et al., 2010; Levy et al., 2012). Special attention should be paid to the possible overheating of the matrix due to an excessive PL treatment. To avoid this, a sufficient cooling system and cooling period between the pulses, limited treatment duration, and appropriate distance between the flash lamps and matrix should be emphasized (Dunn et al., 1989; Gó mez-Ló pez et al., 2005b). It should be noted that with decreasing distance between the lamps and the treated matrix, the effect of PL on the surface rises. At the same time, however, the frame of high-efficacy treatment narrows down (Gó mez-Ló pez et al., 2005b). In the specific case of globular bodies, multidirectional and uniform illumination of all surfaces can be facilitated by increasing the distance to the light source and the treatment time, but also through relative movement of the body. Another possibility is a conveyor having transparent sections or the installation of reflectors (Lagunas-Solar and Gó mez-Ló pez, 2006). PL can be conducted at various stages in a food processing plant. These are, for example, the decontamination of raw materials and food contact materials used, in-process treatment, avoidance of recontamination, and treatment of the final product prior to or post packaging (Wong, 1998; Lyon et al., 2007; Ferná ndez et al., 2009; Uesugi and Muraru, 2009; Rajkovic et al., 2010). In 1966, the U.S. Food and Drug Administration (FDA) approved PL for food applications in the United States from a technology-oriented approach (21 CFR 179.41). The legal status for food applications in the European Union (EU) is, however, still unclear. A potential approach is food ingredient oriented. Connected with this is Regulation 258/97 (1997), “ Novel Foods and Novel Food Ingredients”  (article 1, item f). Decontamination of food contact materials such as packaging materials is, however, not affected hereby and has been used in the EU and beyond for several years (Dunn et al., 1995; FDA, 1996; EU, 1997). Also, the combination with different thermal and nonthermal decontamination treatments shows great potential. Modified atmosphere packaging (MAP); high hydrostatic pressure (HPP); antimicrobial chemical additives (e.g., nitrates); “ natural”  antimicrobial essential oils, extracts, or bacteriocins (e.g., nisin); and hydrogen peroxide can be applied to improve the decontamination effect synergistically (Heinrich et al. 2016b). Based on the given factors that influence the treatment efficacy, Table  11.1 gives an overview of the main advantages and disadvantages using PL in food applications.

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TABLE  11.1  Overview on Advantages and Limitations in Relation to Main Factors Influencing PL Efficiency Factor

Advantages

Disadvantages

Quality

Applicable in combination with other preservation technologies Decontamination of food (packed/ unpacked) and food contact surfaces

Limited to surface decontamination

Matrix effect

Ecology

Convenience

Effective against pathogenic and spoilage microorganisms 4– 6 times more effective than CW UV Implementation in Hazard Analysis and Critical Control Point systems Resistance formation of bacteria is currently not described Nonthermal surface treatment

Minimally processed technique Safe to apply (industrial safety) Environmentally friendly (lack of residual compounds) Xenon lamps (broad spectrum) Low energy input Short processing times (4– 6  times lower than with CW UV) Integration into existing processes Reduced space requirement In batch or continuous mode Instant action adjustable to product flow Natural cooling of lamps between pulses Light spectrum adjustable by pulse-forming network or filters to the respective situation High UV intensities during a pulse (up to 30% UV)

Product characteristics (e.g,. surface opacity and composition) and contamination degree may reduce effectiveness Limited packaging material can be used Control of postprocess parameters critical for shelf life Photoreactivation possible (SOS response of microorganisms) Colored food products may show undesirable effects Adjustment of processes necessary to avoid product impairment and surface heating Uniformity of treatment is limited Ozone formation —  —  —  Problematic standardization due to geometric configuration and process parameters Only a few suppliers on the market —  —  —  —  — 

—  (Continued)

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TABLE  11.1 (CONTINUED)  Overview on Advantages and Limitations in Relation to Main Factors Influencing PL Efficiency Factor Economic

Advantages Low operation costs High efficiency (40%– 50% conversion of electrical energy into optical) Emerging technology

— 

Acceptance

Good consumer acceptance FDA-approved technology

Disadvantages High investment costs (€ 300,000– € 800,000) Technology for high-value-added products or particular market situations Lamps: Lifetime depends on operating parameters (average lifetime, 6– 12 months; costs (few times higher than for CW UV), about € 700 each due to sophisticated and costly design) Sophisticated and costly driving circuits necessary for long lifetime and high UV output of lamps (10– 100  times more expensive than for CW UV) —  Technology per se not approved in the EU (novel food regulation)

11.2.2  In-Package Application of Pulsed Light PL can be used for in-package application. To obtain satisfactory results, packaging materials have to be selected with regard to transmissibility of light and, in particular, the UV fraction thereof. Conversely, this means that opaque materials and matrixes, such as ink-printed labels or drawings, are not suitable. Only if this condition is met are light incidence from any direction and uniform decontamination of the matrix possible (Dunn et al., 1989; Palmieri and Cacace, 2005; Elmnasser et al., 2007; Han, 2007; Oms-Oli et al., 2010). Of the groups of packaging materials available, only glass and polymeric materials (plastic) qualify for in-package application of PL (Dunn et al., 1989; Eie, 2009). Of the two groups, the importance of glass for the packaging of solid foods is low. Also, depending on the type of glass, transmittance (percentage of light that passes through a sample) for UV light is limited and may therefore reduce the efficacy of the treatment (Eie, 2009). Against this background, plastic packaging materials will be focused on in the following text. Plastics cover a wide range of chemical, mechanical, and physical characteristics. This range is considerably influenced by factors such as the basic structure of the material, processing characteristics, and the crystallinity and incorporation of additives (Kirwan and Strawbrigde, 2003; NOVA Institut, 2013). The last two points exhibit particular influence

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on the transmittance. Amorphous, homogenous polymers, for example, insignificantly absorb light due to hardly any scattering of light, and therefore appear transparent (transmittance greater than 90%). By contrast, crystalline structures and the presence of morphological inhomogeneity cause opacity of the material based on a high scattering power (Hernandez et al., 2000). Further, transmittance is influenced by some of the various end-use additives, which are possibly added for market appeal or functional demands during processing. These are, for example, UV protective agents, dyes, or coatings (Crompton, 2007; Pospí š i l and Neš pů rek, 2008). Against this background, the incident light is partly reflected or absorbed by the packaging material. As a result, even transmissible materials can significantly reduce the light intensity and modify the initial light spectrum (Fellows, 2009). In the context of light transmission, a specific measure below which transmission through a material is negligible (absorbance of 1) is the cutoff wavelength given in nanometers (Hernandez et al., 2000). As mentioned previously, UV light below 270  n m is especially important for the decontamination process (Dunn et al., 1991; Wekhof, 2000; Takeshita et al., 2003; Wang et al., 2005; Levy et al., 2012). This implies that polyolefines (a group comprising polyethylene [PE] and polypropylene [PP]) with a cutoff wavelength below 180  n m seem to be best suited for in-package application of PL. Further, cutoff wavelengths for commonly used plastics are approximately 240  n m for polyvinyl chloride (PVC) and polyamide (PA), 270  n m for polystyrene (PS), 280  n m for polycarbonate (PC), and 310 nm for polyethylene terephthalate (PET). All cutoff wavelengths given are for 10-micron thick films (Carlsson and Wiles, 1986). One should also consider that the given values are reference values that can vary to a great extent with the respective properties and thickness of the material, as well as with the assembly (e.g., multilayer formed by coextrusion or lamination). So far, polyolefines and PA are the main polymers used for in-package application of PL (Heinrich et al., 2015). It is well known that UV light induces reactions and changes in polymers. This can affect the physical, mechanical, and chemical characteristics of a certain material. Therefore, it is recommended, next to the transmissibility of light, to select materials that withstand the intended treatment. Otherwise, loss of packaging integrity, including changes in strength, extensibility, impact strength, and cracking and discoloration, can occur. Further, the mass transfer properties of permeation, migration, and scalping can be altered. Special attention should be paid to the migration of substances from the food contact material to the food, since it is of high interest for consumer protection, has legal relevance, and is becoming increasingly important since it is regulated in EU Regulation 10/2011 (EU, 2004, 2011; Andrady, 2007; Guillard et al., 2010; Castillo et al., 2013). Detailed information on packaging materials and postpackaging application of PL is provided by Heinrich et al. (2015).

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11.2.3  Antimicrobial Efficacy of Pulsed Light on Different Food Matrixes First, the penetration ability of pulsed UV light is seen as a limiting factor for the microbial decontamination of liquid and solid foods due to the complex nature of the matrix. In order to increase the efficacy of the treatment, certain variables in relation to the food properties need to be tested. Fresh-cut produce is mainly eaten raw, and from a microbial safety perspective, food processors are constantly evaluating disinfection procedures carried out through washing steps with different types of sanitizers, such as sodium hypochlorite (Tirpanalan et al., 2011). However, the formation of potential toxic by-products prompted the reduction of dose applications, leading to decreased antimicrobial effects. Agü ero et al. (2016) tested Spinach leaves inoculated with Listeria innocua and E. coli strains with the XeMaticA-2L System (Steribeam Systems GmbH, Germany). Fluences lower than 10 kJ m– 2 showed reductions of 1.85 and 1.72 log CFU g– 1 for L. innocua and E. coli, respectively. The background population (mesophilic, psychrotrophic, and coliforms) present in spinach could also be significantly reduced with PL treatments at 20 and 40 kJ m– 2 (Agü ero et al. 2016). However, the internalization-in-tissue effect of the native microflora is not negligible and may lead to a lower inactivation degree than the inoculated cultures applied in studies. Huang et al. (2017) evaluated the inactivation effect of PL on human norovirus surrogates and outbreak-related bacteria (E. coli O157:H7 and Salmonella enterica serotype Newport H1275) on two types of berries. The results showed the highest sensitivity of E. coli, followed by Salmonella, and finally the virus surrogates. Due to the complex topography of the berries, an increasing fluence of 5.9, 11.4, and 22.5  J  cm– 2 could not enhance the inactivation rate among tested microorganisms, which is mainly due to shadowing effects of the fruits. In addition, a higher inactivation effect could be observed for blueberries than for strawberries. Another study (Kramer et al., 2016) demonstrated the inactivation degree of PL with a reflector composed of three xenon lamps. Bacteria in leafy greens and mung bean sprouts were challenged under simulated washing steps at a distance of 5  cm between the water surface and lamp. The results showed that PL was more effective (2 log for 60  s) than equivalent treatments in electrolyzed water (40  ppm free chlorine) or chlorine dioxide (15  ppm) based on total viable counts (Kramer et al., 2016). Again, the applied energy had less impact on the results than the microbial adhesion with concomitant shadow effects (Kramer et al., 2016). Another approach was demonstrated with so-called repetitive pulsed light (RPL) instead of high fluence rates at one time; periodical exposure with lower PL fluences during storage on cut cantaloupe was applied (Koh et al., 2016). Microbiological results after treatment with two lamps (above and below the samples) using 0.9  J  cm– 2 at every 48  h interval revealed the longest shelf life of 28 days at 4° C  ±   1° C. In terms of microbiological quality parameters, the shelf life could be extended up to 20 days compared with control samples (Koh et al., 2016).

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The disinfection performance of PL on beverages was shown in different studies with varying results related to the light absorption of different liquids tested. The low translucency hampers the penetration of PL (Gó mez-Ló pez et al., 2005). Hwang et al. (2015) demonstrated the inactivation degree of inoculated Pseudomonas aeruginosa at fluences between 0.97 and 29.21  J  cm– 2 in mineral water, isotonic beverage, two types of apple juice, orange juice, grape juice, plum juice, three types of carbonated drink, milk, and coffee beverage without milk. In contrast to the studies on the above-mentioned fruits and vegetables, the bactericidal PL effects are dependent on the total fluence. A comparable reduction of P. aeruginosa by 7 log with 0.97  J  cm– 2 in mineral water could be reached in apple juice, carbonated beverages, and plum juice after treatment with 12.17– 24.35  J  cm– 2 (Hwang et al., 2015). The efficacy of PL treatment in different meat matrices was critically reviewed by Heinrich et al. (2016b). Postprocessing contamination due to handling, slicing, and packaging plays a crucial role for sliced cheese products. Proulx et al. (2015) tested different cheese substrates (cheddar and process cheese) through inoculation of relevant gram-negative and gram-positive bacterial reference strains at fluence levels from 1.02 up to 12.29  J  cm– 2. The inactivation levels reached 3 log reductions for all bacteria at doses below 12  J  cm– 2. Related to the surface topography, cheddar cheese showed a greater number of cavities per unit area, offering more potential for microbial shading (Proulx et al., 2015). Another study, by Innocente et al. (2014), investigated the application of PL as thermal treatment for raw milk with a 3.2 log decrease of total viable counts with fluences of 26.25  J  cm– 2 sufficient for the levels requested for cheese making. The tested parameters showed potential especially for milk types particularly sensitive to heat damage (e.g., goat and donkey). In the RTE meat sector, PL was applied as postprocessing decontamination to inactivate Listeria monocytogenes and Salmonella Typhimurium, known as recontamination agents, and for shelf life prolongation (Ganan et al., 2013; Hierro et al., 2011). Hierro et al. (2011) demonstrated a triple shelf life extension of cooked ham, however, with sensory losses with fluence doses above 2.1  J  cm– 2. The microbial safety of beef and tuna carpaccio could be improved using fluences of 8.4 and 11.9  J  cm– 2 achieving an approximate 1 log reduction of surface-inoculated pathogens, such as L. monocytogenes, E. coli, Salmonella spp., and Vibrio parahaemolyticus (Hierro et al., 2012). Myriad potential applications of PL have been published, but only a few of them have found commercial applications. PL can be used for several types of liquid food, such cold pasteurization of milk and juices and decontamination of fruits, eggs, fish, and meat products. The implementation in manufacturing processes could be verified for beverage filling stations and for in-machine decontamination, such as conveyor belts and knives. Also, PL-treated food contact materials, such as packaging films and cups, are already available on the European market.

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11.3 Conclusion It can be concluded that more research is needed to determine the efficacy of PL treatments in complex food systems, in particular in fluids with limited light transmittance. The low penetration power of PL, in combination with hidden microorganisms, may hamper the inactivation degree. The germicidal effect should be elaborated on at a molecular basis in order to combine the knowledge with tailored technological setups. PL is a promising nonthermal decontamination and preservation technology. However, further studies are needed to demonstrate the scale-up potential for specific food types, such as sliced or cured meat products or leafy greens.

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Farrell, H. P., Garvey, M., Cormican, M., Laffey, J. G., and Rowan, N. J. 2010. Investigation of critical inter-related factors affecting the efficacy of pulsed light for inactivating clinically relevant bacterial pathogens. Journal of Applied Microbiology , 108, 1494– 1508. FDA (Food and Drug Administration). 1996. Irradiation in the production, processing and handling of food. 21 CFR 179.41. Washington DC: Office of the Federal Register, U.S. Government Printing Office. FDA. Fellows, P. J. 2009. Food Processing Technology: Principles and Practice  , chap. 24. Cambridge, UK: Woodhead Publishing. Ferná ndez, M., Manzano, S., de la Hoz, L., Ordó ñ ez, J. A., and Hierro, E. 2009. Pulsed light inactivation of Listeria monocytogenes  through different plastic films. Foodborne Pathogens and Disease , 6 (10), 1265– 1267. Fine, F., and Gervais, P. 2004. Efficiency of pulsed UV light for microbial decontamination of food powders. Journal of Food Protection , 67 (4), 787– 792. Ganan, M., Hierro, E., Hospital, X. F., Barroso, E., and Ferná ndez, M. 2013. Use of pulsed light to increase the safety of ready-to-eat cured meat products. Food Control , 32 (2), 512– 517. Geeraerd, A. H., Valdramidis, V. P., and Van Impe, J. F. 2005. GInaFiT, a freeware tool to assess non-log-linear microbial survivor curves. International Journal of Food Microbiology , 102, 95– 105. Gó mez-Ló pez, V. M., Devlieghere, F., Bonduelle, V., and Debevere, J. 2005a. Intense light pulses decontamination of minimally processed vegetables and their shelf-life. International Journal of Food Microbiology , 103, 79– 89. Gó mez-Ló pez, V. M., Devlieghere, F., Bonduelle, V., and Debevere, J. 2005b. Factors affecting the inactivation of micro-organisms by intense light pulses. Journal of Applied Microbiology , 99, 460– 470. Gó mez-Ló pez, V. M., Ragaert, P., Debevere, J., and Devlieghere, F. 2007. Pulsed light for food decontamination: A review. Trends in Food Science and Technology , 18, 464– 473. Guillard, V., Mauricio-Iglesias, M., and Gontard, N. 2010. Effect of novel food processing methods on packaging: Structure, composition, and migration properties. Critical Reviews in Food Science and Nutrition , 50 (10), 969– 988. Han, J. H. 2007. Packaging for nonthermally processed foods. In Packaging for Nonthermal Processing of Food  , ed. J. H. Han, 3–  16. Ames, IA: Blackwell Publishing Professional. Havelaar, A. H., Brul, S., de Jong, A., de Jonge, R., Zwietering, M. H., and ter Kuile, B. H. 2010. Future challenges to microbial food safety. International Journal of Food Microbiology , 139, S79– S94. Heinrich, V., Zunabovic, M., Bergmair, J., Kneifel, W., and Jä ger, H. 2015. Postpackaging application of pulsed light for microbial decontamination of solid foods: A review. Innovative Food Science and Emerging Technologies , 30, 145– 156. Heinrich, V., Zunabovic, M., Petschnig, A., Mü ller, H., Lassenberger, A., Reimult, E., and Kneifel, W. 2016a. Previous homologous and heterologous stress exposure induces tolerance development to pulsed light in Listeria monocytogenes.  Frontiers in Microbiology , 7, 490. Heinrich, V., Zunabovic, M., Varzakas, T., Bergmair, J., and Kneifel, W. 2016b. Pulsed light treatment of different food types with a special focus on meat: A critical review. Critical Reviews in Food Science and Nutrition , 56 (4), 591– 613.

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Luksiene, Z., Gudelis, V., Buchovec, I., and Raudeliuniene, J. 2007. Advanced highpower pulsed light device to decontaminate food from pathogens: Effects on Salmonella typhimurium  viability in vitro. Journal of Applied Microbiology , 103, 1545– 1552. Lyon, S. A., Fletcher, D. L., and Berrang, M. E. 2007. Germicidal ultraviolet light to lower numbers of Listeria monocytogenes  on broiler breast fillets. Poultry Science , 86, 964– 967. MacGregor, S. J., Rowan, N. J., McIlvaney, L., Anderson, J. G., Fouracre, R. A., and Farish, O. 1998. Light inactivation of food-related pathogenic bacteria using a pulsed power source. Letters in Applied Microbiology , 27, 67– 70. McDonald, K. F., Curry, R. D., Clevenger, T. E., Brazos, B. J., Unklesbay, K., Eisenstark, A., Baker, S., Golden, J., and Morgan, R. 2000. The development of photosensitized pulsed and continuous ultraviolet decontamination techniques for surfaces and solutions. IEEE Transactions on Plasma Science , 28 (1), 89– 96. NOVA Institut. 2013. Market study on bio-based polymers in the world. Capacities, production and applications: Status quo and trends towards 2020. Hü rth, Germany: NOVA Institut. Oms-Oliu, G., Martí n-Belloso, O., and Soliva-Fortuny, R. 2010. Pulsed light treatments for food preservation. A review. Food and Bioprocess Technology , 3, 13– 23. Otaki, M., Okuda, A., Tajima, K., Iwasaki, T., Kinoshita, S., and Ohgaki, S. 2003. Inactivation differences of microorganisms by low pressure UV and pulsed xenon lamps. Water Science and Technology , 47 (3), 185– 190. Palmieri, L., and Cacace, D. 2005. High intensity pulsed light technology. In Emerging Technologies for Food Processing  , ed. D.-W. Sun, 279–  306. London: Elsevier Academic Press. Panico, L. 2005. Instantaneous sterilization with pulsed UV light. In Workshop: Emerging Food Processing Technologies USDA, CSREES , 26– 27. Pullman: Washington State University. Pospí š il, J., and Neš pů rek, S. 2008. Polymer additives. In Plastic Packaging , ed. O. G. Piringer and A. L. Baner, 63– 88. Weinheim, Germany: Wiley-VCH. Prokopov, T., and Tanchev, S. 2007. Methods of food preservation; pp. 3–25. In Food Safety. A Practical and Case Study Approach , ed. A. McElhatton and R. J. Marshall. Berlin: Springer Science + Business Media. Proulx, J., Hsu, L. C., Miller, B. M., Sullivan, G., Paradis, K., and Moraru, C. I. 2015. Pulsed-light inactivation of pathogenic and spoilage bacteria on cheese surface. Journal of Dairy Science , 98 (9), 5890– 5898. Rajkovic, A., Smigic, N., and Devlieghere, F. 2010. Contemporary strategies in combating microbial contamination in food chain. International Journal of Food Microbiology , 141, S29– S42. Raso, J., Pagá n, R., and Condó n, S. 2005. Nonthermal technologies in combination with other preservation factors; pp. 453–475. In Novel Food Processing Technologies , ed. G. V. Barbosa-Canovas, M. S. Tapis, and M. P. Cano. Boca Raton, FL: CRC Press. Rowan, N. J., MacGregor, S. J., Anderson, J. G., Fouracre, R. A., McIllvaney, L., and Farish, O. 1999. Pulsed-light inactivation of food-related microorganisms. Applied and Environmental Microbiology , 65 (3), 1312– 1315. Sofos, J. N. 2008. Challenges to meat safety in the 21st century. Meat Science , 78, 3– 13. Sommers, C. H., Cooke, P. H., Fan, X., and Sites, J. E. 2009. Ultraviolet light (254 nm) inactivation of Listeria monocytogenes  on frankfurters that contain potassium lactate and sodium diacetate. Journal of Food Science , 74 (3), M114– M119.

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Stratakos, A. C., and Koidis, A. 2015. Suitability, efficiency and microbiological safety of novel physical technologies for the processing of ready-to-eat meals, meats and pumpable products. International Journal of Food Science and Technology , 50, 1283– 1302. Sun, D. W. 2005. Emerging Technologies for Food Processing . Amsterdam: Elsevier. Takeshita, K., Shibato, J., Sameshima, T., Fukunaga, S., Isobe, S., Arihara, K., and Itoh,  M. 2003. Damage of yeast cells induced by pulsed light irradiation. International Journal of Food Microbiology , 85, 151– 158. Tirpanalan, Ö ., Zunabovic, M., Domig, K. J., and Kneifel, W. 2011. Mini review: Antimicrobial strategies in the production of fresh-cut lettuce products. In Science against Microbial Pathogens: Communicating Current Research and Technological Advances , ed. A. Mé ndez-Vilas, 176– 188. Vol. 1. Badajoz, Spain: Formatex Research Center. Uesugi, A. R., and Moraru, C. I. 2009. Reduction of Listeria  on ready-to-eat sausages after exposure to a combination of pulsed light and nisin. Journal of Food Protection , 72 (2), 347– 353. Vimont, A., Fliss, I., and Jean, J. 2015. Pulsed light technology: Evaluation of the inhibitory activity against murine norovirus and elucidation of the mechanisms involved. Applied Environmental Microbiology . DOI: 10.1128/AEM.03840– 14. Wang, T., MacGregor, S. J., Anderson, J. G., and Woolsey, G. A. 2005. Pulsed ultraviolet inactivation spectrum of Escherichia coli.  Water Research , 39, 2921– 2925. Weiss, J., Gibis, M., Schuh, V., and Salminen, H. 2010. Advances in ingredient and processing systems for meat and meat products. Meat Science , 86, 196– 213. Wekhof, A. 2000. Disinfection with flash lamps. Journal of Pharmaceutical Science and Technology , 54 (3), 264– 276. Wekhof, A., and Trompeter, F.-J. 2001. Pulsed UV disintegration (PUVD): A new sterilisation mechanism for packaging and broad medical-hospital applications. Presented at the First International Conference on Ultraviolet Technologies, Washington, DC, June 14– 16. Wong, E., Linton, R. H., and Gerrard, D. E. 1998. Reduction of Escherichia coli  and Salmonella senftenberg  on pork skin and pork muscle using ultraviolet light. Food Microbiology , 15, 415– 423. Yaun, B. R., Sumner, S. S., Eifert, J. D., and Marcy, J. E. 2003. Response of Salmonella  and Escherichia coli O157:H7  to UV energy. Journal of Food Protection , 66, 1071– 1073. Yi, J. Y., Lee, N. H., and Chung, M. S. 2016. Inactivation of bacteria and murine norovirus in untreated groundwater using a pilot-scale continuous-flow intense pulsed light (IPL) system. LWT— Food Science and Technology , 66, 108– 113. Zhang, H. Q., Barbosa-Cá novas, G., Balasubramaniam, V. M., Dunne, C. P., Farkas, D. F., and Yuan, J. T. C. 2011. Nonthermal Processing Technologies for Food . Ames, IA: Wiley-Blackwell.

12 Effect of Commercial Emerging Nonthermal Technologies on Food Products: Microbiological Aspects Elisabete M. C. Alexandre, Rita S. Iná cio, Ana C. Ribeiro, Á lvaro Lemos, Sofia Pereira, Só nia M. Castro, Paula Teixeira, Manuela Pintado, Ana M. P. Gomes, Francisco J. Barba, Mohamed Koubaa, Shahin Roohinejad, and Jorge Saraiva

CONTENTS 12.1 Introduction ................................................................................................ 398 12.2 High-Pressure Processing......................................................................... 398 12.2.1 Engineering Principles................................................................... 399 12.2.2 Biological Effects.............................................................................400 12.2.3  Practical Applications  .................................................................... 401 12.2.4 Limitations, Challenges, and Future Trends ............................. 403 12.3 Pulsed Electric Field...................................................................................404 12.3.1 Engineering Principles...................................................................404 12.3.2 Biological Effects.............................................................................405 12.3.3 Practical Applications.................................................................... 406 12.3.4 Limitations, Challenges, and Future Trends.............................. 407 12.4 Atmospheric Cold Plasma......................................................................... 409 12.4.1 Engineering Principles................................................................... 409 12.4.2 Biological Effects............................................................................. 410 12.4.3 Practical Applications.................................................................... 411 12.4.4 Limitations, Challenges, and Future Trends.............................. 414 12.5 Other Commercial Emerging Nonthermal Technologies..................... 414 12.5.1 Osmotic Dehydration .................................................................... 414 12.5.2 Ozone-Based Technology ............................................................. 415 12.5.3 Chlorine Dioxide............................................................................. 416 12.5.4 Ionizing Radiation.......................................................................... 418 12.6 Final Remarks.............................................................................................. 419 Acknowledgments............................................................................................... 420 References ............................................................................................................. 420

397

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12.1 Introduction Consumer demands for high-quality, fresh-tasting, and nutritious foods have generated considerable interest in the development of new food processing techniques. The traditional heat treatments are efficient in microbial inactivation, reducing product decay, and attaining safety targets. However, they have a significant impact on product quality. Some quality parameters, such as texture, color, aroma, flavor, taste, and nutritive value, can be severely affected by temperature. Nonthermal and ecofriendly methodologies, such as high-pressure processing (HPP), pulsed electric field (PEF), atmospheric cold plasma (ACP), osmotic dehydration (OD), ozonation, or the use of chlorine dioxide, have been studied by both industry and academia, in an attempt to meet the challenges of producing safe processed foods with high-quality standards (Balasubramaniam et al. 2015). HPP, PEF, and ACP are among the most commercial technologies being studied, and some processed products can already be found in the market, particularly in the case of HPP, PEF, and ozone application. High pressures in the range of 400– 600  MPa, at room or chilled temperatures, can be useful for food pasteurization of liquid and solid foods, ensuring inactivation of pathogenic and spoilage bacteria, yeasts, molds, viruses, and spores (Balasubramaniam et al. 2015). PEF technology typically involves the application of short-duration pulses (from several nanoseconds to several milliseconds), of high electric field strengths (from 100  V/cm to 80  kV/cm) and a specific energy input in the range of 50– 1000  kJ/kg (Koubaa et al. 2015). This technology is able to inactivate pathogenic microorganisms such as Listeria monocytogenes , Escherichia coli , and Salmonella  Typhimurium, without significant loss of the organoleptic and nutritional properties of food. However, data on the inactivation of some microorganisms, such as Bacillus cereus  and Staphylococcus aureus , by PEF processing are very limited. The application of ACP technology for food processing applications is very recent (Niemira 2012a). However, the strong thermodynamic nonequilibrium nature of this methodology, as well as the low gas temperature, presence of reactive chemical species, and high selectivity of the method, make it very promising for food processing. In this chapter, some of the main engineering principles, biological effects, practical applications and limitations, challenges, and future trends of HPP, PEF, and ACP technologies are reviewed. OD, the use of ozone or chlorine dioxide, and ionizing radiation are briefly discussed.

12.2  High-Pressure Processing The application of HPP started when Hite (1899) verified the effectiveness of pressure to inactivate spoilage bacteria in milk at 680  MPa. In 1990, the first

Effect of Commercial Emerging Nonthermal Technologies on Food Products 399

pressure-treated products (jams and jellies) were introduced in the Japanese market. The next step was the commercialization of pressure-treated guacamole in the United States in 1977. In Europe, sliced cooked ham was the first food processed by HPP sold (Balasubramaniam et al. 2015). At present, HPP is applied at the industrial scale with a pressure ranging between 400 and 600  MPa and a time ranging from a few seconds to several minutes, which is the time required to inactivate microorganisms and enzymes at low temperatures, depending on the food matrix and the type of microorganism to be eliminated (Syed et al. 2016). The main advantage of using HPP is the capacity to expand the shelf life and improve food safety due to the inactivation of pathogenic and spoilage vegetative bacteria, yeasts, and molds (Balasubramaniam et al. 2015). 12.2.1  Engineering Principles HPP has been the most successful alternative technology adopted by the food industry for the pasteurization of foods at refrigeration or ambient temperatures, since a low impact on their functional properties and nutritional values has been verified (Barba et al. 2012, 2015b). The operation of HPP is based on two fundamental physical principles: (1) the isostatic principle and (2) Le Chatelier’ s principle. According to the isostatic principle, the pressure applied is transmitted instantaneously and uniformly throughout the sample, independent of the volume, size, shape, and geometry of the product (Considine et al. 2008). Hence, all parts of foods are exposed to high pressure at the same conditions and at the same time. However, pressurization of liquids or solid foods at room temperature is typically accompanied by an increase in the food temperature, called adiabatic heating, which depends mainly on the food composition. The heat associated with the increase of pressure of high-moisture food materials is close to that of water, 3° C per 100  MPa at 25° C (Balasubramaniam et al. 2015). An example of a schematic diagram for HPP of liquid foods is presented in Figure  12.1. Le Chatelier’ s principle states that reactions that involve a volume change are influenced by pressure, and pressure favors those reactions that result in a decrease in volume (Lou et al. 2015). For pressure application, the packaging material must be flexible because foods decrease in volume under pressure and regain volume during decompression (Lou et al. 2015). Current industrial HPP treatment of solid foods can only be treated in a batch mode (Considine et al. 2008). The product is typically vacuum packaged and placed inside a pressure basket, after being loaded into the pressure vessel. The pressure vessel is then closed. The product pressure is reached through the compression of a pressure-transmitting fluid via the combined action of pumps and intensifiers. During HPP, the product is maintained for the desired time, at the required pressure, and usually at low temperatures. At the end of the treatment, the vessel is depressurized and the product is unloaded. Water is usually used in industrial-scale equipment as the pressure-transmitting fluid (Balasubramaniam et al. 2015).

400

Food Safety and Protection

Feeding

Pressuring

Discharging

Highpressure pump

Water High-pressure vessel Free piston Liquid food Water tank

Treated product Untreated product FIGURE  12.1 Schematic presentation of HPP system of liquid food.

12.2.2  Biological Effects The impact of HPP treatment on microbial inactivation is influenced by the level of pressure, temperature, and treatment time used, as well as by the food matrix characteristics. In fact, the pressure and treatment time are the key factors for microbial inactivation. The levels of pressure used in the food industry only affect noncovalent bonds, such as hydrogen, ionic, and hydrophobic bonds (Lou et al. 2015). Hence, the structures of low-molecular-weight molecules, such as vitamins, peptides, fatty acids, saccharides, and the primary structure of proteins, remain intact because the covalent bonds have very low compressibility at high pressure. On the other hand, the native structure and functionality of macromolecules, such as proteins, enzymes, polysaccharides, and nucleic acids, may change during HPP (Daryaei and Balasubramaniam 2012; Considine et al. 2008). Consequently, the secondary, tertiary, or quaternary structure of proteins is changed, due to rupture alterations in ionic bonds, hydrogen bonds, and hydrophobic and electrostatic interactions that maintain the protein structure (Rendueles et al. 2011). The loss of microorganisms’  viability during HPP is the result of a multiplicity of injuries accumulated in different components of the cell, including cell membranes, nucleoids, ribosomes, proteins, and enzymes (Daryaei and Balasubramaniam 2012; Considine et al. 2008). The biomembrane of the cell is a complex phospholipid bilayer with different protein and lipid

Effect of Commercial Emerging Nonthermal Technologies on Food Products 401

contents. During the compression phase, lipids change their conformation and packing, altering the membrane fluidity. Consequently, the lipid bilayer becomes less permeable to water and protein– lipid interactions are weakened, thus reducing the transmembrane transport (Balasubramaniam et al. 2015). Above 300  MPa, irreversible denaturation of proteins and enzymes was reported, which causes breaking of the cell membrane integrity and the flow of internal substances, leading to bacterial death (Huang et al. 2014a). Below 300  MPa, most of these reactions are reversible. Another effect of HPP is the disruption of ribosome configurations, which leads to a lower protein biosynthesis and inhibition of protein repair. In addition, HPP can also negatively affect the functionality of genetic materials in microorganisms, such as DNA replication and gene transcription, due to condensation of the genetic materials, leading to degradation of the chromosomal DNA (Huang et al. 2014a). 12.2.3   Practical Applications   The efficacy of a high-pressure pasteurization process for microbial inactivation depends on the HPP conditions (pressure level, holding time, treatment temperature, rate of compression, and decompression), microbiota (type and physiological state), and food matrix (pH or acidity and water activity) (Huang et al. 2014a; Syed et al. 2016). In this chapter, the effects of HPP on some of the most important microorganisms (e.g., E. coli , Salmonella  spp. , S. aureus , and L. monocytogenes ) causing food- and waterborne diseases are highlighted. The results summarized in Table  12.1 show the feasibility of using an HPP system to reduce these microorganisms in different food products. In general, an increase of the pressure’ s treatment time and/or an increase of the pressure level causes an increase of the microbial inactivation. An example of this is the inoculation of E. coli  MG1655 in fresh carrot juice that is treated with HPP at different combinations of pressure and holding times (200– 600  MPa for 1– 60  min), having revealed a linear relationship among the inactivation level and treatment times under all the tested pressures (Van Opstal et al. 2005). Regarding the effect of the compression and decompression rates, there is little information available in the literature. The literature reported that the pressure resistance of prokaryotes, grampositive bacteria and cocci exceeds that of eukaryotes, gram-negative bacteria and bacilli, respectively. Moreover, the response can significantly differ among species, and also between strains of the same species (Syed et al. 2016). E. coli  O157:H7 is extremely pressure resistant and has been suggested as the possible indicator of adequate pasteurization of food samples by HPP (USDA 2012). For instance, Tadapaneni et al. (2014) inoculated E. coli  O175:H7 (ENT C9490), S.  Typhimurium (ATCC 14028), and L. monocytogenes  (FRR W2542) in a formulated strawberry-blueberry-based beverage and evaluated the microbiological safety of an HPP-treated (400 and 600  MPa for 1, 5, and 10  min)

Fresh carrot juice Strawberry-blueberry beverage Raw milk cheese Apricot, orange, and cherry juice Dry fermented salami Nuts Apricot, orange, and cherry juice Strawberry-blueberry beverage Dry fermented salami Strawberry-blueberry beverage Dry fermented pork sausage Dry fermented salami Raw cow’ s milk cheese Dry fermented pork sausage Raw cheese Apricot, orange, and cherry juice

Escherichia coli 

Note : nd, not detected (below detection limit).

Staphylococcus aureus 

Listeria monocytogenes 

Salmonella  Typhimurium

Salmonella  Enteritidis

Food Product

Microorganism

5 4 10 5 10 10 10 25– 30

483 and 600 400 and 600 400 483 and 600 500 400 500 250– 450

5 5– 20 25– 30

483 and 600 400 and 600 250– 450 4

10 25– 30

500 250– 450

400 and 600

5– 30 4

Temperature (° C)

200– 600 400 and 600

Pressure (MPa)

2 5– 20

17

1– 5 5

17

1– 5 5 and 10

5 and 10

1– 5 20 5– 20

3 5– 20

1– 60 5 and 10

Time (min)

5.3 (nd) 4.0– 5.7

0.3

1.6– 5.0 (nd) 5.02– 6.34 (nd)

0.6

1.9– 2.4 2.9– 5.8

1.9– 6.0

4.7– 5.8 (nd) 1 4.7– 7.3

5 (nd) 4.9– 6.8

0.5– 8 (nd) 1.9– 5.9

Log Reductions

Rodrí guez et al. (2005) Bayı ndı rlı  et al. (2006)

Jofré  et al. (2009)

Porto-Fett et al. (2010) Rodrí guez et al. (2005)

Jofré  et al. (2009)

Porto-Fett et al. (2010) Tadapaneni et al. (2014)

Tadapaneni et al. (2014)

Porto-Fett et al. (2010) Prakash (2013) Bayı ndı rlı  et al. (2006)

Rodrí guez et al. (2005) Bayı ndı rlı  et al. (2006)

Van Opstal et al. (2005) Tadapaneni et al. (2014)

References

Main Examples of HPP Food Products (Pressure, Temperature, and Time) and Log Reductions Caused in Some of the Most Relevant Food Safety– Related Microorganisms

TABLE  12.1 

402 Food Safety and Protection

Effect of Commercial Emerging Nonthermal Technologies on Food Products 403

sample. E. coli  O157:H7 appeared to be the most pressure-resistant pathogen, and it revealed 2.8 log reductions, while S.  Typhimurium and L. monocytogenes  were inactivated more than 5 log after HPP at 600  MPa for 1  min. In another study, Rodrí guez et al. (2005) reported more than a 5 log reduction of E. coli  O157:H7 inoculated in raw milk cheese after HPP at 500  MPa for 10  min. Recently, Syed et al. (2016) reported that microorganisms in different food matrixes were more resistant to HPP, probably due to the protection provided by food chemical composition (e.g., the presence of fats, proteins, minerals, and sugars). Lower water activity (aw      E. coli   >   L . monocytogenes  and showed that the energy expense needed to inactivate the bacteria was higher than that for yeast. At inlet temperatures higher than 35° C, a synergistic effect between temperature and electric field pulses was found; thus, lower energy for inactivation of microorganisms was needed at higher temperatures. Various juice matrixes caused different degrees of inactivation, mainly determined by pH values. Marsellé s-Fontanet et al. (2009) evaluated the effect of three PEF processing parameters, including electric field strength, pulse frequency, and treatment time, on a mixture of microorganisms typically present in grape juice and wine, including Kloeckera apiculata , S. cerevisiae , Lactobacillus plantarum , L. hilgardii , and Gluconobacter oxydans . The relation between the applied energy to the grape juice and the levels of inactivation of microorganisms was evaluated. The optimal processing

Effect of Commercial Emerging Nonthermal Technologies on Food Products 407

conditions (35  kV/cm field strength, 5  μ s pulse, 303  Hz frequency, and 1000  μ s treatment time) were predicted by polynomial response models and enabled 2.24– 3.94 log cycles of inactivation of microbial populations. Moreover, these authors reported that increasing residence time enhanced the level of microbial inactivation, and the electric field strength and pulse frequency need to be wisely selected for each microorganism. Sharma et al. (2014) studied the inactivation of gram-negative (Pseudomonas aeruginosa  and E. coli ) and grampositive bacteria (S. aureus  and Listeria innocua ) in whole milk using PEF treatment (22– 28  kV/cm for 17– 101  μ s), in combination with controlled preheating (55° C for 24  s) and stepwise intermediate cooling. They concluded that gramnegative bacteria were less resistant to PEF than gram-positive bacteria, and reported microbial inactivation to levels below the detection limit. Bermú dezAguirre et al. (2012) found that the level of fat milk could affect the stability of microorganisms treated under PEF processing. The effectiveness of PEF treatment against B. cereus  spores in skim milk was reported to be higher than that in whole milk. The impact of PEF on some pathogenic strains is summarized in Table  12.2. 12.3.4  Limitations, Challenges, and Future Trends PEF processing for dairy applications continues to be an engineering challenge, because most of the studies have been performed on laboratory-scale equipment, using small volumes, and under batch or laminar flow setup. Upscaling, for the translation of such data to pilot- or commercial-scale production, is frequently difficult because different treatment uniformities, heat conductions, and residence times have to be considered (Buckow et al. 2014). Nevertheless, industrial-scale PEF equipment have been developed and are currently successfully applied for shelf life extension of fruit juices at a capacity up to 8000  L/h in Europe (Irving 2012). Review of the literature shows that the most often used parameters for PEF processing are 20– 40  µ s total treatment time and 30– 35  kV/cm electric field strength (equivalent to 120– 240  kJ/kg specific electrical energy inputs). Under these conditions, at least one intermediate cooling step is required to avoid overheating of the product. Longer treatment times are rarely used at the industrial scale because of the high demand for electrical and cooling energy (Buckow et al. 2014). Although industrial-scale application of PEF processing is possible for improving the safety and quality of food products, further investigations are required, especially for sensorial properties, where the electric field processing may involve minor changes of the product. Compared with the conventional thermal processing methods, the extra costs caused by PEF processing will need to be offset by a premium-priced product. Application of PEF technology at the commercial scale still needs further systematic research and assessment of the safety, quality, and health-promoting aspects of PEF-treated foods to ensure regulatory food safety approval (Buckow et al. 2014).

c 

b 

a 

Number of pulses. Pulse width. Total treatment time.

Staphylococcus aureus 

Salmonella  Typhimurium

Salmonella  Enteriditis

Listeria monocytogenes 

Grape juice Liquid egg Skim milk Whole milk

Skim milk Whole milk Apple juice Liquid egg yolk Liquid egg Orange juice

Apple juice Strawberry juice Liquid egg yolk Strawberry juice Orange juice

Escherichia coli 

Escherichia coli  O157:H7

Food Product 

Microorganism  7.2 18.6 30 18.6 22 20 30 30 35 30 45 22 20 27 40 35 40

E (kV/cm)  —  57.5 105 57.5 22.7 26.9 400 300 393.8 105 10 26.9 57.5 15 5 124 8.9

n  a   2.6 2.6 2.0 2.6 2.6 2.6 1.5 2.0 4 2.0 3.0 2.6 2.6 3.0 3.0 3.7 10.0

τ   (µ s) b   —  150 210 150 59 70 600 600 1575 210 30 59 70 45 15 459 89

t   (µ s) c   —  —  2 —  625 740 170 200 180 2 300 625 740 120 300 250 200

f   (Hz)  57 55 40 55 45 55 25 35 40 40 —  45 55 48.8 —  40 32.5

T  max (° C) 

Most Representative Studies on PEF for Microbial Inactivation in Fluid Food Products 

TABLE  12.2

1.2 3.79 ≈ 5.0 4.71 1.59 2.22 3.0 ≈ 5.0 4.01 ≈ 5.0 4.0 2.05 3.54 3.36 3.0 3.7 5.2

Log 10    Reduction 

Huang et al. (2014b) Gurtler et al. (2010) Monfort et al. (2010) Cregenzá n-Alberti et al. (2014)

McNamee et al. (2010) Zhao et al. (2013) McNamee et al. (2010) Amiali et al. (2004) Monfort et al. (2010) Gurtler et al. (2010)

Ukuku et al. (2010) Gurtler et al. (2011) Amiali et al. (2004) Gurtler et al. (2011) Gurtler et al. (2010)

References 

408 Food Safety and Protection

Effect of Commercial Emerging Nonthermal Technologies on Food Products 409

12.4  Atmospheric Cold Plasma ACP technology is a relatively novel decontamination nonthermal processing method, which is effective against a wide range of pathogenic microorganisms. Therefore, ACP can improve the microbiological safety in conjunction with maintenance of sensory behaviors of the treated foods. The remarkable characteristic features of ACP are its strong thermodynamic nonequilibrium nature, its low gas temperature, the presence of reactive chemical species, and its high selectivity, which provide high potential for use this method in the food industry (Niemira 2012a). However, due to the complexity of ACP, application of this technology is still under development and debate. 12.4.1  Engineering Principles By providing energy, such as by heating, a solid material could change its state to liquid, and then to gas. When further energy is added to gas, the intra-atomic structures break down, yielding plasma, the fourth state of matter. Plasma can be thought as an (partially) ionized gas composed of an assortment of “ light”  (photons and electrons) and “ heavy”  species (ions, atoms, or even molecules in their fundamental or excited states), possessing a net neutral charge (Niemira and Gutsol 2010). To generate plasma, noble gases are often used since lower voltages are required to break down the gas and sustain a discharge, but they are not as reactive and are less expensive than air (Niemira 2012a). Figure  12.3 shows an example of ACP processing of food products. Other gases, that is, O2  or N2 , can also be used to provide the type of reaction needed. The efficiency of the treatment depends High-voltage electrode

Dielectric barriers

Food product Reactive species

Highvoltage source

Plasma

Ground electrode Capacitor Oscilloscope

FIGURE  12.3 Schematic presentation of atmospheric cold plasma processing of food products.

410

Food Safety and Protection

on the nature of the gas used to form the plasma (Kim et al. 2011), and the chemical composition of the feed gas becomes a determining factor in the type of reactions that plasma can initiate (Lieberman and Lichtenberg 2005; Niemira and Gutsol 2010), which often explains the differences in their destructive efficiency (Hury et al. 1998). Depending on the food treated and the processing conditions, effective treatment times can range from 120  s to as little as 3  s (Niemira 2012a). Plasma types can be classified according to several parameters (temperature, thermodynamic equilibrium, pressure, ionization degree, net gas charge, magnetization, frequency, etc.). However, the most used parameter to generate plasma is temperature (Nehra et al. 2008), thus giving two classes: high-temperature (≥ 107   K) and low-temperature (≤ 2  ×   104   K) plasma. Nonthermal plasma (300  =  T  ≤   103   K), also designated nonequilibrium plasma or cold plasma, has two main features that distinguish it from other industrial applied plasma technologies, including the near room temperature at which they operate and the fact that plasma can originate at both atmospheric and reduced pressure, with less power involved than thermal plasma. Until recently, cold plasma treatments were applied under reduced pressure and at a very small scale, with several operational and cost constraints (Lieberman and Lichtenberg 2005). Consequently, research regarding nonthermal plasma has moved forward toward the application of ACP, which has already proven to have advantages over reduced-pressure ACP (Yoon and Ryu 2007). To generate ACP, different principles associated with the energy input from assorted sources have been developed, including corona discharge, dielectric barrier discharges, radiofrequency plasmas, and gliding arc discharge. These mechanisms have been previously described in detail in the literature (Conards and Schmidt 2000; Niemira 2012a). 12.4.2  Biological Effects ACP has already been studied against a wide range of microorganisms, including yeasts and molds, bacteria, spores, and viruses (Kelly-Wintenberg et al. 1998; Ryu et al. 2013; Suhem et al. 2013; San-Cheong et al. 2015; Zimmermann et al. 2011). The effect of plasma can be quite selective, meaning a possible tunable effect between damage to pathogenic organisms, and without injuring the host, or activating some pathways in the microorganisms (Dobrynin et al. 2009). The reaction between the active particles in the plasma and the microbial surrounding environments (water, pH, nutrients, osmotic stability, and temperature) is more interesting to food industry, and in which different levels of lethal effects on microorganisms are generated (Ryu et al. 2013). However, the detailed mechanisms involved in the bactericidal action of the plasma need deeper investigations. Several authors have proposed three main mechanisms associated with the different agents that constitute plasma. Charged particles (electrons and atomic and molecular ions) play an important role in the inactivation process since they can

Effect of Commercial Emerging Nonthermal Technologies on Food Products 411

disrupt the cell membrane by electroporation and formation of cell cracks (Laroussi et al. 2003; Kvam et al. 2012). Excited and reactive species (i.e., O2, NO• , and O3 ) have been reported to be responsible for bacterial reduction due to strong oxidative stresses on the outer membranes of the cells (i.e., lipids and amino acids) (Gaunt et al. 2006). The inactivation of gram-positive bacteria by plasma treatments is believed to be caused by the diffusion of reactive species through the cell membrane, and further reaction with the intracellular compounds (Laroussi et al. 2003). In contrast, the inactivation of gram-negative bacteria is reported to be associated with charge accumulation on the outer surface of the membrane, which overcomes the tensile strength of the membrane, leading to its rupture (Montie et al. 2000). Another mechanism results from ultraviolet (UV) radiation, which erodes the cell membrane and cellular constituents (Lackmann et al. 2013). Finally, the damage of membranes and internal cellular components, such as DNA, can be due to the action of UV photons (Moisan et al. 2002). The relative “ weight”  of each mechanism on the sanitizing processes of a given commodity will also depend on the direct or remote plasma exposure (Laroussi et al. 2003). The type of food and microorganism considered, equipment design, voltage chosen, gas pressure and composition, and distance of the microorganism from the discharge glow are also variables to be analyzed case to case (Afshari and Hosseini 2014; Niemira 2012b). As any other nonthermal technologies, plasma can also affect microorganisms in a sublethal way, in which the metabolic behavior of cells can be significantly altered (Perni et al. 2008a; Rowan et al. 2007) and further compromise the safety of the treated products. 12.4.3  Practical Applications ACP is a novel nonthermal food processing technology that uses energetic, reactive gases to reduce the contaminating microbial load on a wide range of foods, and it could be currently regarded as a potential alternative to chemical (i.e., chlorine treatment) or physical (i.e., HPP and PEFs) methods. The main advantages are the high inactivation efficiency on both pathogens and spoilage organisms on the surface of food products at low temperatures (   B. thermosphacta   >   E. coli . For a food model study, the antimicrobial films effectively reduced the growth of L. monocytogenes  on cooked beef at 4° C (Sung et al. 2014). Cerisuelo et al. (2014) stated in their study that EVOH coatings with carvacrol, citral, marjoram essential oil, or cinnamon bark essential oil on polypropylene (PP) and PET materials are perfectly usable for food packaging applications, and additionally, the incorporation of bentonite nanoclay to their layers was also suggested. Nanotechnology and the incorporation of EOs into edible films have potential in the development of microbiologically safe foods. As an example, pullulan films containing EOs and nanoparticles were tested against foodborne pathogens. The results were indicated that 2% OEO was effective against S. aureus  and S. typhimurium , whereas L. monocytogenes  and E. coli  O157:H7 were not prevented. Recent studies show that pullulan films containing antimicrobial compounds inhibited the pathogens related to vacuum-packaged meat and poultry products stored at 4° C for up to 3 weeks, compared with control films. It can be said that edible films made from pullulan incorporated with EOs or nanoparticles may promote the safety of refrigerated, fresh, or processed meat and poultry products (Kuorwel et al. 2011). In an active film coating application research study, S. aureus , Listeria innocua , Pseudomonas  spp., Salmonella enterica  subsp. enterica , and E. coli  were prevented when exposed to an atmosphere created of 4%– 6% (w/w) of Zataria EO (Akrami et al. 2015). The antimicrobial activities of thymol and carvacrol as major EO compounds in food packaging system applications are shown in Table  13.1.

13.5  Chemically Synthesized Biodegradable Polymers 13.5.1  Poly-L-Lactide-Based Antimicrobial Films It was explained that packaging films incorporating organic acids should have direct conjunction with the food in order to release the active agents to the food surface (Lee et al. 2015). One such aliphatic polyester polymer is polylactic acid (PLA), which is obtained from renewable agricultural sources (corn) following the starch fermentation and condensation of lactic acid. PLA-based nanocomposites are of particular interest for research because of their biodegradation in the environment (Rhim et al. 2013; Ramos et al. 2014). In addition, because PLA is classified as GRAS by the U.S. Food and Drug Administration (FDA) and is approved by the European Commission (Commission Regulation No. 10/2011), it might be applied in contact with food (Ruiz-Cabello et al. 2015).

LDPE/organically modified montmorillonite Polybutylene succinate

LDPE/clay

LDPE/organo-modified montmorillonite LLDPE/organo-modified montmorillonite Polypropylene

Pullulan films

Microencapsulated thymol/ carvacrol in polymer films

Carvacrol/thymol

Carvacrol

Carvacrol

Carvacrol/thymol

Thymol

Carvacrol/thymol

Thymol

Thymol

Polymer Carrier PLA/polytrimethylene carbonate films

Active Component Thymol

Laboratory conditions

Laboratory conditions

Laboratory conditions

Laboratory conditions

Laboratory conditions

Laboratory conditions

Laboratory conditions

Strawberry

Tested Food/Conditions Laboratory conditions

Prolonged and high antimicrobial activity against E. coli  Inhibition effects against E. coli , L. innocua , and Alternaria alternata  Inhibition effects on E.coli  ATCC 8739 Carvacrol and thymol at 8 wt% to PP improved product quality and showed safety aspects (against E. coli  and S. aureus ) in food packaging applications Inhibition effects against B. subtilis  ATCC 6633, S. aureus  ATCC 25923, S. enteritidis  ATCC 13076, and E. coli  ATCC 25922 Inhibition activities on E. coli  O157:H7, S. aureus , L. innocua , S. cerevisiae , and A. niger 

Results Inhibitions of Esherichia coli , Staphylococcus aureus , Listeria  spp., Bacillussubtilis , and Salmonella  spp. Effective inhibition of Botrytis cinerea  on strawberries Inhibition of E. coli  and S. aureus 

Some Food Packaging Applications with Essential Oil Compounds Carvacrol and Thymol

TABLE  13.1  

(Continued)

Guarda et al. 2011

Gniewosz and Synowiec 2011

M. Ramos et al. 2012b

Efrati et al. 2014

Shemesh et al. 2015

Campos-Requena et al. 2015 Petchwattana and Naknaen 2015 Shemesh et al. 2015a

References Wu et al. 2014

Food Packaging Systems with Antimicrobial Agents 443

PET containing carvacrolcoextruded multilater film PP/EVOH 32/PP tray heat-sealed with an active PP/EVOH-29  +  6.5% carvacrol/PP film lid Sodium caseinate plasticized matrixes (transparent active films) Nanoclay bentonite was added to EVOH 29 films

Organoclay Cloisite 30B Gelatin films

Chitosan/CD films

Chitosan-based films

Thymol Carvacrol

Carvacrol

Carvacrol

Carvacrol

Carvacrol

Carvacrol

Polymer Carrier

Active Component

Carvacrol

Laboratory conditions

Chicken fillets

Laboratory conditions Laboratory conditions

Laboratory conditions

Laboratory conditions

Salmon cubes and slices

Fresh salmon fillets

Tested Food/Conditions

Developed material can be used in antimicrobial active food packaging L. innocua  inhibited effectively Films exhibited antimicrobial effects against Pseudomonas aeruginosa  ATCC 9027, E. coli  ATCC 8739, S. aureus  ATCC 6538, and B. subtilis  ATCC 6633 Inhibition effects on lactic acid bacteria, yeast, and fungi Antimicrobial activity for S. enteritidis , B. subtilis , E. coli , and L. innocua 

Inhibitory effects on E. coli  and S. aureus 

Inhibition effects on mesophiles and psychrotrophs Rapid and effective migration of antimicrobial agent to the fish muscle

Results

Some Food Packaging Applications with Essential Oil Compounds Carvacrol and Thymol

TABLE 13.1  (CONTINUED)

Kurek et al. 2013

Higueras et al. 2014

Rodriguez et al. 2014 Kavoosi et al. 2013

Cerisuelo et al. 2012

Arrieta et al. 2014

Cerisuelo et al. 2013

Rollini et al. 2016

References

444 Food Safety and Protection

Food Packaging Systems with Antimicrobial Agents

445

In a study, the effects of natural extract of 2%, 5%, and 6.5% Allium  spp. containing PLA were researched in the packaging of ready-to-eat salad. Although an antioxidant effect was not determined, antimicrobial activity was observed for the film and in the packaged salad. It was reported that yeast and molds were inhibited effectively, but enterobacteria and aerobic bacteria groups were found to be more resistant against the antimicrobial active films (Ruiz-Cabello et al. 2015). In Han et al. (2015), it was stated that 0.5% nisin added to plasticized biodegradable PLA film has the potential to preserve wild edible mushroom (Boletus edulis ) quality and prolong its postharvest life to 18 days stored at 4  ±   1° C. In another study, the packaging mechanism based on PLA was recognized by sol-gel processing and incorporated with natamycin as the active agent. The release of the antifungal determined in food stimulants was also observed at a maximum value of about 0.105  mg/dm2 , a level definitely lower than that permitted by directive for cheese rind. The coating inhibited undesirable mold growth on the surface of commercial semisoft cheese (Lantano et al. 2014).

13.6  Polymer/Clay Nanocomposite-Based Antimicrobial Films Clay minerals, such as montmorillonite, have been extensively used as an agent for drug delivery and in controlled-release mechanisms with good results. Montmorillonite’ s large surface-to-area ratio, cation exchange capacity, and absorption ability make nanoclays optimum drug carrier agents. Several assays to improve antimicrobial agents for food packaging based on clay nanocomposites have applied incorporation into volatile compounds such as antimicrobial materials, for example, carvacrol in nanoclay/EVOH, EOs in alginate/clay nanocomposites, and rosemary EOs in montmorillonite/chitosan bionanocomposites. Among some volatile compounds such as antimicrobial materials in active packaging, carvacrol and thymol contain a broad spectrum of antimicrobial and antifungal effects, and they are currently classified as GRAS agents by the FDA (Campos-Requena et al. 2015). Linear low-density polyethylene (LLDPE) matrix-based active nanocomposite films were obtained with the addition of active nanoclay grains and EO compounds, such as carvacrol, eugenol, and thymol. It was reported that active nanocomposite packaging can keep fresh meat color up to a 4-day storage period, avoiding the surface off-color. Growth of total mesophilic bacteria, lactic acid bacteria, and total yeast and mold counts in the Turkish sliced sucuk samples was controlled by a vacuum packaging system with active nanocomposite films during 30 days of storage (Tornuk et al. 2015).

446

Food Safety and Protection

Nanoclays can be functionalized to obtain antimicrobial functions. Biocidal metals can be incorporated into the clay form as charge-covering ions via ion exchange. Application of inorganic biocides, such as Ag, Cu, Zn, and Mg, can be used with mineral clays as biocide carriers, as previously been determined (Rhim et al. 2013).

13.7 Metal and Metal Oxide NanoparticleBased Antimicrobial Films Many nanostructured metallic particles, such as silver, gold, copper, and zinc, are widely used to obtain nanocomposite packaging materials. Among them, AgNPs have received significant attention in the food packaging area because of their special and wide spectrum of antimicrobial effects against foodborne pathogens. Various antimicrobial bionanocomposites have been demonstrated by incorporating AgNPs into polymeric matrixes, such as agar, chitosan, and cellulose (Kanmani and Rhim 2014a, 2014c). Among metal nanomaterials, nanosilver has been determined to be a potent antimicrobial material. The antimicrobial function of silver is principally assigned to the process of silver ions and metallic AgNPs. It has been stated that silver ions interact with negatively charged biomacromolecular compounds (sulfhydryl or disulfide groups of enzymes) and nucleic acids, leading to structural changes and disruption in bacterial cell membranes and walls that cause the prevention of metabolic conditions, followed by cell death. The bactericidal activity of nanosilver improves by the release of silver ions within bacterial cells (Tavassoli-Kafrani et al. 2016). Nanoparticles with the filler attribute are incorporated in biopolymer films for improving mechanical, water vapor, and thermal barrier properties. Among the nanofillers, ZnONPs have good capacity for nanoscale diffusion and interfacial interactions in protein structures due to their large surface area and high-level surface energy. The incorporation of ZnONP as a particular filler in biopolymers, such as starch-based films, has been reported to cause the development of water vapor and mechanical barrier properties. ZnO has recently been listed as a GRAS agent by the FDA and had earlier indicated high in vitro  antimicrobial effects against foodborne pathogens and spoilage bacteria (Arfat et al. 2014). The antimicrobial mechanism of AgNPs has also been mentioned to be related to membrane destruction due to the free radicals obtained from the surface of the nanoparticles. AgNPs may be stored in the bacterial cytoplasmic membrane, leading to an important increase in permeability and cell death. Recently, CSNPs loaded with several nanoparticles, such as Ag+ , Cu2+ , Zn2+ , and Mn2+ , exhibited a significantly increased antimicrobial activity against E. coli , Salmonella choleraesuis , and S. aureus . Copper ions can inhibit

Food Packaging Systems with Antimicrobial Agents

447

microorganisms and viruses, and copper is vital for life as an agent of metallic enzymes. Copper is regarded as being safe since it is not concentrated by animals, and therefore has few negative effects on higher animals. A polymer-based nanocomposite coating with stabilized copper nanoparticles with antifungal and bacteriostatic properties has previously been suggested for food packaging applications. However, copper is not commonly used in the food packaging area since it is regarded as toxic in contact with food; in addition, it would accelerate the biochemical reaction with foods due to its catalytic effect of oxidation. Metal oxides, such as TiO2 , ZnO, and MgO, can be used for the formulations of antimicrobial packaging films due to their high antimicrobial activity with strong stability compared with organic antimicrobial agents (Rhim et al. 2013). The antimicrobial efficiency of AgNP-containing antimicrobial packaging films is highly affected by several factors, such as the degree of particle agglomeration, the particle size and its distribution, the interaction of silver’ s surface with the base polymer, and the silver content. AgNPs should be highly diffused through the polymer structure without agglomeration. As a result, it is essential to prepare AgNPs with proper measures and determine optimum polymeric materials for the preparation of active antimicrobial packaging films with AgNPs. In addition, the agar/AgNP composite films demonstrated typical antimicrobial effects against both gram-positive and gram-negative pathogenic bacteria (Rhim et al. 2014). In Beigmohammadi et al. (2016), the antimicrobial activities of LDPE packaging films coated with silver, zinc oxide, and copper oxide (CuO) nanoparticles were determined in ultrafiltrated (UF) cheese. Copper decreased the growth rate of E. coli  by more than 99.99%, leading to disruption of the cell walls and changing the bacterial cell contents. The developed active packaging containing 1% w/w CuO nanoparticles in LDPE polymer was processed by melt mixing for packaging UF cheese, and it was confirmed that it can be used to decrease coliform numbers in the cheese without toxicity. In  Sanuja and Umapathy (2015) , nano-ZnO at various concentrations (0.1%, 0.3%, and 0.5%) and neem ( Azadirachta indica  )  EO were incorporated into the chitosan polymer by a solution-cast method to improve the properties of the bionanocomposite film. Antibacterial activity was found to have a high inhibition effect against  E. coli  . Bionanocomposite film applications were carried out for carrot packaging and compared with the commercial film. The antibacterial effect against  E. coli   was determined for all types of films; chitosan/0.5% zinc oxide/neem oil nanocomposite films exhibited high antimicrobial activity when compared with other films.  Montmorillonite modified with quaternary ammonium salt C30B/starch nanocomposite (C30B/ST-NC), AgNP/C30B/starch nanocomposite (AgNP/ C30B/ST-NC), and AgNP/starch nanocomposite (AgNP/ST-NC) films were prepared. Films exhibited inhibition activity against S. aureus , E. coli , and Candida albicans  without significant differences between AgNP concentrations. The migration of compounds from the nanomatrix starch films,

448

Food Safety and Protection

processed by food contact tests, was insignificant and under the limits. Research results showed that the starch films incorporated with C30B and AgNPs have potential to be applied as packaging nanostructured agents. Among all the compositions studied, the AgNP/C30B/ST-NC film with 0.3  mM AgNPs revealed to be the most suitable. (Abreu et al. 2015). AgNPs were incorporated into a hydroxypropyl methylcellulose (HPMC) matrix for processing as food packaging materials. The antibacterial properties of HPMC/AgNP thin films were reported based on a disk diffusion test against E. coli  and S. aureus . The disk diffusion research showed a higher bactericidal effect for nanocomposite films containing 41  nm AgNPs (Moura et al. 2012). Through research, silver nanodots of different sizes (average sizes of 10, 18, and 28  n m by different molecular weights) using a self-assembled polystyrene-b polyethylene oxide (PS-b-PEO) block copolymer were improved. The developed silver nanodot surfaces showed good antimicrobial effect against gram-positive and gram-negative bacteria, resulting in their ability to be used in antimicrobial packaging applications. It was also reported that Pseudomonas fluorescens  (gram-negative bacteria) was more sensitive than S. aureus  (gram-positive bacteria) (Azlin-Hasim et al. 2015). The application of nanotechnology, containing 1– 100  nm particles and indicating novel properties, is a significant novelty in many industrial areas. In the food industry, nanoparticles can be incorporated into food and food contact materials (FCMs), bringing the materials new and developed properties, such as antimicrobial activity, oxygen-scavenging potential, improved thermal stability, biosensing ability, improved moisture and gas barrier, altered color, texture, and improved material strength (Hannon et al. 2016).

13.8 Advantages and Difficulties of Natural Antimicrobial Packaging Systems Synthetic plastic packaging materials are widely used for the packaging of various foods. They can cause serious environmental problems since they cannot easily degrade in the environment after use and they release toxic gases (Sanuja and Umapathy 2015). Environmentally friendly antimicrobial packaging films can increase the shelf life of foods and be used as favorable alternatives to synthetic or petroleum-based packaging materials. Excellent biodegradable packaging agents are obtained from renewable biological resources, generally called biopolymers, with good mechanical and barrier properties. Biopolymers are regarded as potential environmentally friendly materials for use as nonbiodegradable and nonrenewable plastic packaging agents. Biopolymer packaging materials may provide gas and solute barriers by improving quality and prolonging the shelf life of foods. Moreover,

Food Packaging Systems with Antimicrobial Agents

449

biopolymer packaging materials are good vehicles for incorporating various additives, such as antioxidants, antimicrobials, antifungal agents, colors, and other nutrients (Rhim et al. 2013; Kanmani and Rhim 2014b; Sanuja and Umapathy 2015). It was shown that the application of antimicrobial films in food packaging systems could reduce some problems of food processing, such as additive amounts and chemical preservatives, in the food industries (Beigmohammadi et al. 2016). In biopolymers, carrageenans have high effects as a film-forming material. Cooling a hot solution of carrageenan during the film casting and drying process shows the transition of an occasional coil to a double helix, which occurs in the formation of a compact and structured film after dehydration of the solution. In a study, it was concluded that carrageenan can produce a clear film with good mechanical and structural qualities, including high tensile strength (Shojaee-Aliabadi et al. 2013). Alginate is a good alternative for film and coating formation due to its colloidal properties, including strength, thickness, emulsion stability, and gel and film production (Kazemi and Rezaei 2015). Commercialization of carrageenan and alginate biopolymer films is restricted due to their high sensitivity to moisture and their compatibility with other emergent stress factors, such as high pressures, ultrasound, microwave, and gamma radiation (Tavassoli-Kafrani et al. 2016). Another food packaging material is Ag-based nanoparticles, which are applied to the surface of packaging to contribute an antimicrobial property. The large reactive surface of the nanoparticles increases the capacity for AgNPs to oxidize into silver ions (Ag+ ) that come into contact with the food material to allow antimicrobial activity. In a recent study, the migration of AgNPs from an LDPE coating into food materials was examined under accelerated time and temperature conditions. Under microwave conditions, there was an important increase in migration when compared with oven heating; this requires further research, especially where nanopackaging may be applied in microwaveable ready-to-eat foods and food storage containers (Hannon et al. 2016). Today, Ag-based commercial packaging films applied to muscle foods are AgIon®   Life Materials Technology’ s silver-based masterbatch, Irgaguard®   BASF’ s silver-based masterbatch, Surfacine®   Surfacine Development’ s silver-based masterbatch, IonPure®   Solid Spot’ s silver-based masterbatch, d2p®   Symphony Environmental’ s silver-based masterbatch, Bactiblock®   NanoBioMatters’  silver-based masterbatch, and Biomaster®  Linpac Packaging’ s silver-based trays and films (Realini and Marcos 2014). EOs are natural agents classified as GRAS by the FDA, and most of them are obtained from plants. They exhibit substantial antibacterial and antifungal properties obtained from both direct contact and the vapor phase. However, one major disadvantage of EO molecule– based packaging films is the volatile natü re of EOs; therefore, the main application process for their incorporation into polymers is by coating technologies. Then the EOs are

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directly applied into the polymer matrix by high-temperature processing, and antimicrobial activity is achieved. But, this activity is evaluated after film production, and the difficulties considered with controlling and prolonging the activity of these films have not been determined (Shemesh et al. 2015a). PLA-based films have natural, biodegradable, biocompatible, and optimum mechanical and optical properties (Ramos et al. 2014). The growth of antimicrobial PLA films with improved physical and mechanical properties and antimicrobial activity is in question due to the inherent hydrophobicity and brittleness of this polymer. Some of these material limitations can be overcome with the application of current and advanced Technologies, such as nanotechnology (Tawakkal et al. 2014). PLA is produced commercially by four manufacturers in the World: American Polymer Standards Corporation, Mentor, Ohio; NatureWorks, Minnetonka, Minnesota; Purac Biomaterials, Gorinchem, the Netherlands; and Total S.A., Courbevoie, France (Vijayendra and Shamala 2014). In Ruiz-Cabello et al. (2015), research of a commercial product based on Allium  extract (Proallium SO-DMC®  ) was applied by extrusion into PLA to an active food packaging with the aim of extending the shelf life of ready-to-eat salads. Proallium is a condiment based on vegetal extracts that is composed of organosulfur compounds, which are characteristic of the Allium  spp. The result was that the PLA Proallium film exhibited antimicrobial activity for ready-to-eat salads. WPIs have shown potential mechanical features, as well as optimum moisture permeability and good oxygen barrier characteristics, comparable to those displayed by the best synthetic polymer-based films, for example, high-density polyethylene (HDPE), LDPE, EVOH, vinyl alcohol, polyvinylidene chloride (PVDC), polyester, and cellophane. WPI-based films have been confirmed for their excellent biomaterials for application as carriers of such food additives as antioxidants, antimicrobials, flavors, fortifying nutrients, and spices (Ramos et al. 2012a). During the production of composite films, some characteristics can be affected from incorporated materials. For example, composite WPI/clay films have an opaque appearance, which depends on the amount of clay added. The composite films can be slightly less transparent than the neat WPI films. It was found that film properties, such as the surface color, optical, tensile, and water vapor barrier properties, can depend on the clay content of the films (Sothornvit et al. 2010). Organic polymeric agents are the most widely used in food packaging due to their comfort of processing, light weight, variety of design, and low cost. A significant problem with them is their inherent permeability to gases and other small molecules. Various kinds of nanoparticles are applied into the polymers to develop their properties; among them, nanoclay is incorporated into the films to improve their barrier properties to gases. The migration of clay nanoparticles from the two commercialized high-barrier LDPE bags into food products was shown in a study (Echegoyen et al. 2016). It has been

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suggested that avoiding the migration of additives from packing materials to foods should also be researched (Vijayendra and Shamala 2014). Gelatin has excellent film-forming characteristics, but its films are sensitive, and plasticization or solidification is needed for food packaging applications. Gelatin is often applied as an additive in various water-based formulations for contributing antimicrobial or adhesive qualifications to paper packages. Oxygen permeability values of mammalian gelatin films are higher than those for cold-water FG films. Tensile strength, elongation percentage at break, and puncture deformation decreased in gelatin from mammals, warm-water fish, and cold-water fish, respectively. Gelatin is subject to fast biodegradation when the contamination and environmental conditions are sufficient. But disadvantageously, bacterial contamination can be present at different stages of the production, and quality control of gelatin-producing factories indicated that aerobic, thermotropic, and proteolytic endosporeforming bacteria may be present during the production (Coltelli et al. 2016). One of the biopolymers is chitosan, which is obtained from the shells of crab, shrimp, and other shellfish. It is nontoxic and biodegradable and contains antioxidant characteristics, as it is a linear polysaccharide obtained by the deacetylation of chitin (Ninjiarani 2015). Bionanocomposites have potential future prospects, although nowadays the low level of production and high cost restrict them from a wide range of applications (Rhim and Ng 2007). However, chitosan films have a high transfer of water vapor to the food material and are brittle and extremely soluble under dry and wet conditions, which limits their use widely as packaging materials for foods (Gartner et al. 2015). However, further studies are needed to research the potential effects of natural antimicrobial biopolymers in the development of model food products (Kanmani and Rhim 2014c).

13.9 Conclusion Applying antimicrobial-based films for food packaging holds many challenges; however, they provide food safety and reduce spoilage. Antimicrobial packaging can be used with a hurdle technology that, in addition to other processes, such as pulsed light, irradiation, and high pressure, can prevent the risk of pathogen contamination and extend the shelf life of food products. The safety of the packaging materials, if designed for use in food packaging, should be researched thoroughly before being confirmed by regulatory authorities and commercialized. It is important to ensure that antimicrobial-based films are stable to different stress conditions and the environment, so that the spread of application can be increased. Also, keeping the high sensory properties of foods is essential with the use of natural biopolymers. Consumer acceptance and technical feasibility should also be

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considered when the technologies are developed with the use of antimicrobial compounds, in addition to their microbiological, physiological, and chemical effects. Some modeling food studies should also be researched for their biopolymer applications.

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14 Active and Intelligent Food Packaging Cristina Nerin, Paula Vera, and Elena Canellas CONTENTS 14.1 Introduction................................................................................................. 459 14.2 Active Packaging......................................................................................... 460 14.2.1  Antioxidant Packaging................................................................. 462 14.2.2  Oxygen Scavengers....................................................................... 466 14.2.3   Free Radical Scavengers............................................................... 467 14.2.4  Antimicrobial Packaging............................................................. 468 14.2.5   Other Approaches in Active Packaging.................................... 470 14.2.6   Carbon Dioxide Emitters and Scavengers................................. 470 14.2.7  Ethylene Scavenging..................................................................... 471 14.2.8  Moisture Control........................................................................... 471 14.2.9   Flavor or Odor Absorbers and Releasers.................................. 471 14.2.10  Microwaveable Active Food Packaging.................................... 472 14.3 Intelligent Packaging.................................................................................. 472 14.3.1   Indicators and Sensors................................................................. 474 14.3.2  Radiofrequency Identification ................................................... 475 14.3.3   Microwaveable Intelligent Food Packaging.............................. 475 14.4 Nanotechnology, Biomaterials, and Bioactive Compounds Used in Active and Intelligent Packaging......................................................... 476 14.5 Commercially Available Active and Intelligent Packaging.................. 477 14.6 Legislation.................................................................................................... 477 References.............................................................................................................. 482

14.1 Introduction Food packaging has been historically used to contain food, fulfilling the primary functions of containment, protection, and communication. However, these functions have not been sufficient for the food industry for several reasons. Consumers are looking for better-quality, fresh-like, and convenient food products (Ozdemir and Floros 2004), and the food industry is looking

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for an extension of shelf life of packaged food while maintaining the quality. Then the new terms smart  and intelligent  packaging appeared for different types of functional packaging systems. Nowadays, food packaging can be classified into four categories depending on its functionality (Brockgreitens and Abbas 2016): 1. Ergonomic packaging: Packages that improve the transport, use, and storage, for example, the easy-to-open bottles for older adults (Bell et al. 2016). 2. Informative packaging: Packages that enhance the information for consumers about the product’ s manufacturing, composition, and storing conditions. This information can be printed on the package or can be electronically inserted as radiofrequency identification (RFID) tags (Ranky 2006). 3. Active packaging: Where the packaging contains certain compounds that directly interact with the packaged food, affecting its quality and extending its shelf life. These systems are constantly acting on the packaged food without requiring that a change or trigger occur in the food. Some good examples are moisture scavengers and antimicrobial or antioxidant packaging. 4. Responsive or reactive packaging, which is commonly named intelligent or smart packaging: Packaging that gives an informative response as result of a specific trigger or change occurring in the food product or in its headspace or the outside environment. These changes can be foodborne threats, such as the presence of molds, bacteria, and contaminants, or benign factors, such as moisture, pH, or gas levels in the environment or headspace. It is very important to know the difference between active and intelligent packaging. Active packaging works without a specific trigger mechanism. However, intelligent packaging only works when there is a change in the food or headspace, without acting directly on the food (Brockgreitens and Abbas 2016).

14.2  Active Packaging There are different ways found in bibliography to classify active packaging. For example, it can be classified into two categories depending on the effect of the active agent on the food packaging (Brockgreitens and Abbas 2016)

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1. Chemoactive agents: Agents that affect the chemical composition of the food product or headspace inside the package. Examples are moisture control systems, ethylene scavengers, and oxygen or free radicals scavengers. 2. Bioactive agents: Compounds that interact directly with biological molecules such as bacteria and produce changes in the biological process. It is also possible to classify active packaging according to the intended action it has on the packaging, with antioxidant or antimicrobial packaging being the most common one. This classification is justified as a result of the rapid growth of active technologies that have received a great deal of attention in the last decade. However, in most of cases it is difficult to distinguish between them, as both functions occur simultaneously and exert synergistic action on one another. For example, cinnamon essential oil (EO) is simultaneously a potent antimicrobial and a good free radical scavenger, which makes it a good antioxidant. Montero-Prado et al. (2011) demonstrated that cinnamon EO affects the enzymes responsible for oxidation, such as polyphenoloxidase (PPO), and protects natural peaches from natural decay and mold proliferation, acting as an antimicrobial. A classic classification divides active packaging approaches into scavengers and releasers, as Figure  14.1 shows (Lee et al. 2015). For this reason, detailed descriptions of different active packaging approaches are organized by active agent more than by the actions they exercise on the food. Active packaging represents an innovative strategy to incorporate antioxidants or antimicrobials in the material. To remove all synthetic additives from the food while simultaneously extending the shelf life and maintaining the quality of packaged food sounds like a dream. But the advances in technology and the intensive research carried out in the last 15 years make area a close Package Active releasing systems Carbon dioxide Moisture Flavor or odor releasers Antioxidant release Antimicrobial release

FIGURE  14.1  Types of active packaging.

Food

Active agent

Active scavenging systems Oxygen Carbon dioxide Ethylene Moisture Flavor or odor absorbers

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reality. In this chapter, several examples and developments are described. Although not an exhaustive list of everything published, the most important approaches which probably will soon be in the market are mentioned. 14.2.1  Antioxidant Packaging The use of antioxidants in the food industry is historically known. They are organic molecules that act by several mechanisms (Brewer 2011; Becker et al. 2004), inhibiting all reactive oxygen components that produce damage in the metabolic process. But not all antioxidants can be used in food packaging. They must be safe, efficient at very low concentrations, thermally stable, and economically viable; have a long antioxidant capacity; and not modify the organoleptic properties of packaged food (Shahidi 1997). They can be classified into two groups: synthetic and natural antioxidants (Table  14.1). However, specific restrictions are appearing more and more for synthetic ones. For example, tert-butylhydroquinone (TBHQ) is not permitted in Europe, and the use of ascorbyl palmitate is being questioned due to its possible adverse effects on human health (Wojcik et al. 2010; Krishnaiah et al. 2011). Butil Hydroxy toluene (BHT), a well-known antioxidant used in both polymers and food, is not permitted in some countries, as it has toxic effects at high concentrations. For these reasons, the trend is to use natural antioxidants, which have been obtained mainly from plants (Tongnuanchan and Benjakul 2014; Valdes et al. 2015). In principle, there is no restrictive legislation for these substances, nor are they frowned upon by health authorities and consumers. However, they have some disadvantages, such as the contribution of undesirable color, odor, and flavor to food. The main natural antioxidants used for active food packaging are listed in Table  14.1, together with their applications in food and their advantages. The individual substances responsible for the antioxidant properties that are contained in these EOs and extracts from plants are phenolic compounds such as tocopherol, flavonoids, and phenolic acids. The antioxidant performance is controversial. Although many authors insist on the requirement that release antioxidants have efficient active packaging, we believe that in most cases, the release of antioxidants is not necessary. Release means that some compounds are provided inside the packaging to be oxidized before the food, which means that the antioxidant released is sacrificed. Then the oxidation product resulting from this reaction would remain together with the food and likely change the organoleptic properties, as well as influence the food safety. It is true that oxidizable foodstuffs often contain antioxidants to protect them over time, but the purpose of using active (antioxidant) packaging is to reduce or eliminate the added antioxidants and extend the shelf life of food. The main purpose of adding the antioxidants to the packaging material, which will not be eaten, is not met if the antioxidants are released from the packaging. The antioxidant packaging

Oxygen scavengers

Types of Active Packaging Antioxidant release

Technology Used in Food Packaging Synthetic antioxidants • BHT (Nisa et al. 2015; Yehye et al. 2015), butyl hydroxy anisol (BHA) (Kim et al. 2010), propyl gallate, ascorbyl palmitate, TBHQ Natural antioxidants from plant extracts and EOs (Bentayeb et al. 2014) • Tocopherol (vitamin E) (Melo et al. 2016; Amalia et al. 2016) • Ascorbic acid (vitamin C) (Babou et al. 2016) • Phenolic acids (vanilic, galic, and caffeic acids) (AgatonovicKustrin and Morton 2016) • Cinnamic acids (cafeic, ferulic, and p-cumaric acids) • Flavonoids (Heim et al. 2002; Colon and Nerin 2012; Lopez-de-Dicastillo et al. 2012) • Carotenoids, natural chelates, and even amino acids and peptides (Bernald et al. 2010) Enzymatic systems • Glucose and alcohol oxidase (Leyva et al. 2013) Chemical systems • Iron powered oxidation with iron oxide, catechol, ferrous carbonate, iron-sulfur, sulfite salt-copper sulfate, metallic platinum (Lee et al. 2015) • Ascorbic acid oxidation, catalytic conversion of oxygen by platinum catalyst, unsaturated fatty acids such as like oleic or linoleic acid (Ozdemir and Floros 2004) Others • Photosensitive dye oxidation and immobilization of microorganisms in solid holders (Vermeiren et al. 1999)

Types of Active Packaging, Technology Used, and Their Applications

TABLE  14.1 

(Continued)

Vegetables such as tomatoes (Leyva et al. 2013) Fresh fruit such as apples (Di Maio et al. 2015) Strawberry (Kartal et al. 2012) Vine fruit (Tarr and Clingeleffer 2005) Beef and pork (Limbo et al. 2013)

Applications Fresh vegetables such as mushrooms (Wrona et al. 2015) and sweet tomatoes (Lebot et al. 2016) Bulk sunflower oil and mayonnaise (Bholah et al. 2015) Cookies (Aksoylu et al. 2015) Fresh fruit such as peaches (Montero-Prado et al. 2011) Meat (Laranjo et al. 2016), beef (Nisa et al. 2015)

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Technology Used in Food Packaging

Natural phenolic compounds such as hydroquinons and catechins EOs: oregano, cinnamon, rosemary Se nanoparticles (Vera et al. 2016)

Synthetic antimicrobials • Silver zeolites (Shankar et al. 2016) • Polymers (chitosan [Andrei et al. 2016]) • Peptides such as megainins, cecropines, defensins, and lauroyl ethyl arginate (LAE) (Becerril et al. 2013) • Esters and phenolic acid • Metals such as cooper and silver (Kredl et al. 2016) • Chlorine dioxide, sulfur dioxide Natural antimicrobials • Bacteriocins and antibiotics such as nisin (Krivorotova et al. 2016) and natamycin (Colak et al. 2016) • Enzymes such as lactoperoxidase, lysozyme, and lactoferrin (Montiel et al. 2015) • From plant extracts and EOs (Akrami et al. 2015) • Natural phenolic compounds such as hydroquinons and catechins • Terpenes • Fatty acids and steres

Types of Active Packaging

Free radical scavengers

Antimicrobial release

Types of Active Packaging, Technology Used, and Their Applications

TABLE  14.1 (CONTINUED)

(Continued)

Beef (Nerin et al. 2006) Fatty food (Carrizo et al. 2016) Chocolate derivatives, cereals [12] Fruits such as peaches (Montero-Prado et al. 2011) Nuts, walnuts, fried chip potatoes (results not published) Cooked ham (Montiel et al. 2015) Fresh chicken (Sanchez-Ortega et al. 2014) Poultry meat (Ahmed et al. 2016) Bread (Jideani and Vogt 2016) Cheese (Peighambardoust et al. 2016) Fruits such as melon, pine apple (Scollard et al. 2016), and fresh strawberry (Duran et al. 2016) Vegetables such as lettuce and spinach (Poimenidou et al. 2016)

Applications

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Fruits such as strawberry (Aday et al. 2011) Meat such as lamb (Vergara et al. 2009) Fresh pork sausage (Martinez et al. 2006), beef steak (O’ Sullivan et al. 2011) Fruits such as banana (Terry et al. 2007) and kiwifruit (Park et al. 2009) Vegetables such as tomatoes (Taechutrakul et al. 2009) Chips (Liu and Lin 2009) Nuts (Ozdemir and Floros 2004) Spices (Ozdemir and Floros 2004) Biscuits (Rodriguez-Aguilera and Oliveira 2009) Milk power (Vermeiren et al. 1999) Fish Ground coffee Orange and fruit juices (Vermeiren et al. 1999; Biji et al. 2015; Rooney 1995) Popcorn, lasagna, meat pies, bakery goods, chips, hotdogs, pizza, sandwiches (Bohrer 2009; Regier 2014)

Iron powder Calcium, sodium, or potassium hydroxide Silica gel Metal halide (Lee et al. 2015; Ozdemir and Floros 2004) Activated charcoal, silica gel-potassium permanganate (Taechutrakul et al. 2009), zeolite (Lee et al. 2015), kieselguhr, bentonite, Feller’ s earth, silicon dioxide powder, powdered Oya stone, ozone (Ozdemir and Floros 2004) Silica gel, propylene glycol, polyvinyl alcohol, diatomaceous earth (Lee et al. 2015)

Mixture of charcoal and nickel Ferrous salt, citric acid, and ascorbic acid Baking soda Many food flavors (Ozdemir and Floros 2004) Susceptors Shielding Modifiers Moisture-absorbing flexible materials Steam valves

Carbon dioxide scavengers and emitters

Flavor or odor absorbers and releasers

Microwaveable

Moisture scavenging

Ethylene scavenging

Applications

Technology Used in Food Packaging

Types of Active Packaging

Types of Active Packaging, Technology Used, and Their Applications

TABLE  14.1 (CONTINUED)

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has been designed to retard or minimize the natural oxidant food process, and therefore to prolong its shelf life (Realini and Marcos 2014). For this reason, the real active antioxidant packaging should not release any compound, and as was mentioned above, the scavenging action, either of molecular oxygen or free radicals, is more suitable and appropriate for this task. However, there are several approaches that can be mentioned, and all of them can be classified in two different types of antioxidant packaging. The first one modifies the internal atmosphere to gain favorable conditions, and the second one scavenges the free radicals that initiate the oxidation process. In general, any packaging material can be converted into active antioxidant material, although the most common is from polyolefins (polyethylene [PE], polypropylene [PP], polyethylene terephthalate [PET], and polystyrene [PS]), alone or combined with other materials, forming a multilayer film structure (also with other materials, such as aluminum, paper, or cardboard), and more recently, biopolymers (Atares and Chiralt 2016; Valdes et al. 2014). The industry prepares antioxidant materials by several ways: • Incorporation of the antioxidant in the bulk of the polymeric matrix by extrusion technology (Gomez-Estaca et al. 2014). • Incorporation of the antioxidant as a liquid dispersion by coating technologies. This system is widely used, giving rise to many patents and marketed packages (Nerin et al. 2006, 2008; de Dicastillo et al. 2011; Garces et al. 2004). • Incorporation of the antioxidant and an oxygen absorber between different layers in a multilayer material. Although this system is available on the market, it is very expensive technology (Tian et al. 2013). • Incorporation of the antioxidant in multilayer packaging, where the antioxidant agent is placed between inert substrates. For example, there are recent studies that apply selenium nanoparticles as the antioxidant, and they are incorporated in the adhesive used to manufacture the multilayer material (Vera et al. 2016; Nerín et al 2014). The great advantage offered by these active materials is that their antioxidant properties are kept for longer periods. In addition, the number of additives added to the food is reduced, which is beneficial for consumer health and also improves their perception of the packaged food in a positive way. 14.2.2  Oxygen Scavengers The presence of high oxygen levels inside the package produces the oxidation of food constituents and, in many cases, also the proliferation of molds and aerobic bacteria. It causes a change in flavor, color, texture, and nutritive value, producing a loss of quality and reducing the shelf life of the packaged

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food (Realini and Marcos 2014; Kerry et al. 2006). This fact can be minimized by using oxygen scavenger systems, which remove the residual oxygen after packaging, often coming from oxygen dissolved in the food. This active packaging technology is one of the most widely used. It is now more than 10 years old and has become very popular, especially in Asian countries, where almost every packaged sandwich, cake, and so forth, contains an oxygen scavenger. The different systems manufactured are shown in Table  14.1 (Ozdemir and Floros 2004; Lee et al. 2015; Rodriguez-Aguilera and Oliveira 2009). The most commonly used O2  scavenger is based on the oxidation reaction of iron powder (Lee et al. 2015). Traditionally, it was designed in the form of small sachets containing different oxidizing agents inside. But this system is not appropriate for liquid foods and is not good for health, as the sachets can be accidentally broken, producing a risk of ingestion of their contents (Realini and Marcos 2014; Ozdemir and Floros 2004). As an alternative to sachets, another approach is the incorporation of the oxygen scavenger into the packaging structure itself, using cards, sheets, or layers coated on the package’ s inner walls (Biji et al. 2015) or in a multilayer (Ozdemir and Floros 2004; Realini and Marcos 2014). The scavenging action is achieved by rapid diffusion of oxygen from the headspace through the layers to the reactive agent. Of course, the ingredients used in these approaches are not the same as those used in the sachets. In general, the efficiency of this last system is lower than that of the labels or sachets (Day 2008). 14.2.3  Free Radical Scavengers Instead of molecular oxygen, free radicals derived from oxygen can be successfully scavenged, as has been demonstrated in several scientific publications (Carrizo et al. 2014, 2016; Nerin et al. 2008; Pezo et al. 2008; Nerí n 2010; de Dicastillo et al. 2011). As the oxidation reaction is a chemical reaction initiated by free radicals, if they are removed, the oxidation does not take place. Using this principle, antioxidant performance occurs even in the presence of molecular oxygen. Scavenging free radicals, such as oxo, hydroxo, and peroxo, is much easier than scavenging molecular oxygen, as the oxygenderived free radicals are smaller and more reactive and diffuse faster than O2  throughout the polymers. For this task, direct contact with the inner atmosphere is not required if the diffusion of free radicals is demonstrated. The research carried out so far demonstrates that this process occurs through lowdensity polyethylene (LDPE) at up to 90 microns, although 30 or 40 microns increases the efficiency (Vera et al. 2016). This performance opens a new door for antioxidant packaging, which is the incorporation of the scavenger in a multilayer, behind the layer in contact with food. Several approaches using this advantage and either green tea or selenium nanoparticles, both good antioxidants and radical scavengers, have been published (Vera et al. 2016; Colon and Nerin 2012; Carrizo et al. 2014, 2016). Three international patents

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cover these developments (Bosetti et al. 2013; Garces and Nerin 2004; Nerin et al. 2014), and in both cases, the materials are in the market or on the way to the market. The oxidation of lipids is a big problem in the food industry. Unfortunately, big radicals, such as those formed from lipids, cannot cross the polymeric layers. However, lipid radicals are secondary radicals, where the primary ones are always derived from oxygen. This means that these free radical scavengers can also protect lipids from oxidation processes. Vera et al. (2016) demonstrated the extension of shelf life of nuts and walnuts using a multilayer-containing nanoselenium behind the LDPE, and similar results were obtained with chip potatoes (results not published). 14.2.4  Antimicrobial Packaging Antimicrobial packaging acts by reducing, inhibiting, or retarding microbial (yeasts, molds, and bacteria) growth in order to extend the shelf life of packaged food. In contrast to antioxidant packaging, antimicrobials should be released from the packaging, either by direct contact or through the vapor phase, as they should arrive at the cell and kill it or inhibit its growth. A wide variety of antimicrobial agents have been used so far, as shown in Table  14.1, but the list is continuously increasing, as there is not one perfect or ideal agent to be used in any packaging material or foodstuff. The action mechanism of antimicrobial agents can be classified into two types. In the first, the antimicrobial agent migrates to the food. A good example is the migration of volatile compounds that enter into the headspace in a gaseous phase and then come into contact with the food (Mastromatteo et al. 2010), as happens with volatile phenolic compounds of EOs (Kuorwel et al. 2011b; Llana-Ruiz-Cabello et al. 2015). In the second type, the agent acts from the food surface (nonvolatile compounds) in contact with the material (Han 2000), as commercial silver zeolites incorporated into PP, PE, or nylon do (Quintavalla and Vicini 2002). Different approaches have been developed for the application of antimicrobial compounds in packaging: • Incorporation in sachets or pads inside the package, containing the antimicrobial, for example, ethanol. As already mentioned, it acts by absorbing moisture and releasing ethanol vapor to retard mold growth. This approach is mainly used in bakery, cheese, and fish products. Other examples are chloride or sulfur and carbon dioxide agents (Biji et al. 2015; Otoni et al. 2016). • Incorporation of the antimicrobial agents in the bulk of the polymeric matrix by extrusion and melt-blending technology. This technique has great potential, as the antimicrobial agents are homogeneously distributed in the whole material, from where they can be slowly released. Also, this system allows for the addition

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of fungicide or bactericide substances compatible with the food. However, if the antimicrobial agents are volatile, they will be partially volatized during extrusion at high temperature, causing a loss of effectiveness (Tramon 2014). This is the main drawback of extrusion technology. To avoid this inconvenience, new strategies, such as microporous carriers, plasticizers, and encapsulation, have been developed (Kayaci et al. 2013; de Azeredo 2013; Dias et al. 2013). Another alternative is to use an additional inner layer between the film with the antimicrobial substance and the food matrix, to control the antimicrobial release (Bastarrachea et al. 2011). The commercial polymers used are PP, PE, PET, and PE/ethylene vinyl alcohol (EVOH) (Bastarrachea et al. 2011; Raheem 2013; Suppakul et al. 2003), and more recently, research efforts have focused on the use of biopolymer materials (Kuorwel et al. 2011a; Khwaldia et al. 2010; Rhim and Ng 2007) such as PLA (Tawakkal et al. 2014). • Incorporation of the antimicrobial agents by solvent casting methods (Bastarrachea et al. 2011), where the polymer matrix is solubilized in a solvent (water, ethanol, etc.) that contains antimicrobial agents. Then, this complex is cast or sprayed onto a substrate (plastic, paper, or food). After that, the solvent is evaporated at moderate temperature and an active film is obtained. This system is used, for example, for polysaccharides, proteins, and polyols (Van Long et al. 2016). • Incorporation of the antimicrobial agents as liquid dispersion by coating technologies. In this case, the destruction of antimicrobial agents by high temperatures and shearing forces during extrusion is avoided, since the coating is usually applied at nearly room temperature after the packaging film is formed (Chen et al. 2012). An example would be the use of EOs added in active paraffin coating for paper (Gutierrez et al. 2009; Rodriguez-Lafuente et al. 2010). • Incorporation of the antimicrobial compounds immobilized on the polymer either by ionic or covalent link agents producing a slow migration. In this case, the agents used are enzymes, organic acids, and bacteriocins (Krasniewska and Gniewosz 2012). • Polymers that are inherently antimicrobial and are used themselves as films or coatings, for example, macromolecules such as chitosan, used to protect fresh vegetables and fruits from fungal attack. Additionally, it is edible, nontoxic, biodegradable, and biocompatible and has antifungal properties (Aider 2010; Mellinas et al. 2016; Rodriguez-Aguilera and Oliveira 2009; Cha and Chinnan 2004; Krasniewska and Gniewosz 2012) in direct contact applications.

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As above, significant research efforts are being conducted using natural extracts (green tea, grape seed, orange, and lemon seed extract) and EOs, such as thyme, oregano, garlic, and clove, associated with phenolic compounds (carvacrol, eugenol, linalool, and thymol). They are complex mixtures of volatile and nonvolatile compounds from different parts of the plant that have antimicrobial properties. This antimicrobial activity has been widely studied in a great number of microorganisms, demonstrating its effectiveness by direct contact assay (Dias et al. 2013; Becerril et al. 2013) or by the vapor phase (Muriel-Galet et al. 2015; Manso et al. 2015). Another very important and expanding field is the combination of different disciplines, such as nanotechnology, food, and microbiology (Imran et al. 2010), whose objective is the introduction of nanoparticles to provide antimicrobial activity, as well as an enlarged contact area, compared with conventional antimicrobial agents (Mihindukulasuriya and Lim 2014; Echegoyen et al. 2016). Therefore, they have a higher efficiency. An example is the addition of metals such as copper, zinc, and silver in the form of salts and oxides (Espitia et al. 2012) and colloids, such as silver zeolites or elemental nanoparticles (Rhim et al. 2013; Kuorwel et al. 2015). 14.2.5  Other Approaches in Active Packaging In addition to antioxidant or antimicrobial active packaging, other approaches have been proposed with different objectives, although the extension of shelf life while maintaining the quality of packaged food is the constant and main purpose. These approaches can be more specific for the target food, but the technologies used for producing the active packaging are similar to those explained above. The most common ones are described in the next sections. 14.2.6  Carbon Dioxide Emitters and Scavengers The presence of high levels of carbon dioxide retards microbial growth and delays the respiration rates of fresh produce. Therefore, levels between 10% and 80% are usually recommended to extend the shelf life, although the rate depends on the specific food (Kerry et al. 2006). This group can be divided into two types: emitters and scavengers. The first one is used when the package has a high permeability to carbon dioxide; this happens because it is more permeable than oxygen through many plastic films. If this occurs, an emitting system may be necessary to increase the shelf life (Ozdemir and Floros 2004). In contrast, the second type is used for removing the excess CO2  generated by the packaged food. This happens, for example, with flexible packaging materials of roasted coffee beans, which are blown and break as a consequence of the excess carbon dioxide generated during storage (Lee et al. 2015), unless a one-way valve is inserted into the package, allowing the selective release of CO2 , or the packaging material can scavenge the excess CO2  generated.

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14.2.7  Ethylene Scavenging Ethylene is a growth-stimulating hormone that accelerates the respiration of fruits and vegetables, producing fruit ripening, softening, and senescence, and therefore reducing the shelf life of the product packaging (Biji et al. 2015). The different techniques used in this case to absorb ethylene are shown in Table  14.1. The most extensive technology used for ethylene scavenging is potassium permanganate embedded in silica. The system absorbs ethylene, which is oxidized to ethylene glycol, while permanganate reduces to manganese dioxide. The silica can be kept in the package in a sachet, but it is difficult to introduce this agent in a film. In addition, permanganate is a strong oxidant and is not permitted to have contact with food (Ozdemir and Floros 2004). 14.2.8  Moisture Control Excess humidity may have negative results, causing bacterial and mold growth, as well as foggy film formation. This can occur by accumulation of water vapor inside the package due to the low permeability of different materials. In contrast, the lack of humidity is not always beneficial, since the food may dry up (Biji et al. 2015). The moisture scavengers help to control the water excess, as well as to remove water vapor releases by meat products. The different technologies used to control the excess of water accumulation are shown in Table  14.1, as well as the different applications found in the market. Actually, there are several systems already patented and marketed by different companies (Kerry et al. 2006). They consist of a superabsorbent polymer located between two layers of a microporous or nonwoven polymer, or, for example, Dry Fresh resolve (Sirane Ltd.), which consists of drip-absorbing pads placed under meat. Humidity absorbers are well disseminated and used in the food market, as most packaged fresh meat contains an absorbent pad to remove the exudates. 14.2.9  Flavor or Odor Absorbers and Releasers The volatile compounds accumulated inside a package can produce undesirable aromas, flavors, or odors. The formation of these off-flavors and off-odors originate from the oxidation of oils or fats, generating aldehydes, or from the breakdown of fish protein into amines (Vermeiren et al. 2003). This causes significant rejection by consumers. For this reason, absorbers agents are used, as shown in Table  14.1. Although there have been different approaches developed in this area, the European legislation on active and intelligent packaging establishes that the removal of off-odors that can be used as indicators of damaged or rotten food is not permitted. A clear example is the bad odor from fish, which warns the consumer of its bad state. This means that most of these active packaging solutions cannot be used in the European market.

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On the other hand, another important cause of rejection by the consumer is the flavor or odor lost or degradation over time in the package. To avoid this problem, which mainly affects the quality of packaged food, it is necessary to use a high-barrier material or, alternatively, employ an active packaging containing the specific aromas of the particular food. For example, fill the headspace progressively and in a continuous way over the shelf life of the food with volatile compounds in order to obtain the desired flavor. This is used by coffee manufacturers (Ozdemir and Floros 2004). Other systems and applications used are shown in Table  14.1. 14.2.10  Microwaveable Active Food Packaging Microwave cooking is a very convenient way to prepare food, as it is very quick and clean. However, the heat transfer of microwave heating produces nonuniform heat, and temperature is not well distributed in the food. Moreover, some foodstuffs require crisping or browning to be acceptable for consumption, which is not possible with conventional microwave cooking. Microwavable active packaging is designed to solve these cooking disadvantages by using susceptors, shielding, field modification (Regier 2014), moisture-absorbing flexible microwavable packaging materials (Realini et al. 2014), and steam valves (Avery Dennison 2011). Shielding can be applied to achieve differential heating of different portions of the food (Lafferty et al. 2000). Modifiers for microwave heating are systems that alter how the microwaves arrive to the food, thereby resulting in even heating, surface browning, and crisping (Ahvenainen 2003). Microwave susceptors are materials that are able to absorb electromagnetic energy and convert it to heat (Labuza and Meister 1992). They are usually made of metallized film, ceramics, or multilayers containing an aluminum layer in the sandwich mode. Moisture-absorbing flexible microwavable packaging materials are those that absorb the excess grease and water resulting in crispy products (Mondi 2011). Finally, steam valves inserted in the packaging allow the release of vapor once a defined pressure point is reached, resulting in dry food when necessary (Avery Dennison 2011). These active packaging materials are applied to a variety of foods, such as popcorn, lasagna, meat pies, bakery goods, chips, hotdogs, pizza, and sandwiches.

14.3  Intelligent Packaging Intelligent packaging (also described as smart packaging) is, according to EC/450/2009, the material or article that monitors the food packaged conditions or the surrounding environment. It functions are detecting, sensing, tracing, and communicating to give information about the product itself

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(origin, composition, etc.) or the product history (microbial growth, storage conditions, etc.), or to improve external or internal problems of the product to extend its shelf life (Realini and Marcos 2014; Biji et al. 2015) Basically, there are two types of intelligent packaging, indicators and sensors, although both names overlap in meaning. Sensors were initially applied to those devices capable of detecting, locating, quantifying, and transmitting information related to biological or chemistry reactions, with great precision and usually in an electronic way. However, indicators were applied to visual changes provided by the intelligent device, usually a tag, while a sensor was applied to electronic devices that required an automatic or instrumental reading. However, nowadays the term indicator  is less used and sensor  is generally accepted, independently of electronic reading or visual detection. Many authors believe that RFID is also an intelligent packaging. However, this is an advanced label and can only be considered intelligent or smart packaging when there is some active element inserted in the tag that communicates with the packaged food. Some examples are shown in Table  14.2 (Lee et al. 2015). The specific details of each type are described in the next sections. TABLE  14.2  Types of Intelligent Packaging and Their Applications, Advantages, Effectiveness, and Uses in Food Types of Intelligent Packaging Time– temperature indicators

Leak indicators

Freshness indicators

Biosensors

Gas sensors

Chemical sensors

Effectiveness Visual indications of temperature history

Visual indications of a leak Have to be very sensitive (disadvantage) Tracking and controlling of microbial growth or chemical changes Detect and transmit information about biochemical changes Specificity, sensitivity, reliability, portability, and simplicity Detect and quantify gas states

Radiofrequency identification

Detect presence of chemical contaminants Automatic identification and traceability data

Microwaveable intelligent packaging

MDIs Barcode-intelligent microwave

Most Recent Examples Applied to Food Meat (Pennanen et al. 2015; Kim et al. 2013) Fish (Zhang et al. 2016) Fruit (Forney 2007) Meat (Jang and Won 2014) Soap (Smolander et al. 1997) Cod fillets (Dehaut et al. 2016) Meat sausage (Jairath et al. 2015) Cereals (Bougrini et al. 2016) Pepper (Sabela et al. 2016) Meat sausages (Jadan et al. 2016) Wine (Andrei et al. 2016) Mangoes, eggs, and fish (Cui et al. 2016) Tea (Sharma et al. 2016) Cheese (Nakonieczna et al. 2016) Soft drinks (Lin et al. 2016) Milk (Kim et al. 2016) Virgin olive oil and meat (Matindoust et al. 2016) Popcorn, lasagna, meat pies, bakery goods, chips, hotdogs, pizza, sandwiches, etc.

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14.3.1  Indicators and Sensors These indicate the presence of a strange substance at a defined concentration, or if there is a reaction between two or more substances, producing a change. Among them, the following can mentioned: • Time– temperature indicators  (TTIs). These consist of small adhesive labels applied on the external side of the package, which inform the retailer or consumer of the thermal history of the product. They are based on different electrochemical, mechanical, microbiological, or physicochemistry principles, such as enzymatic reactions, compound diffusion, and polymerization processes that are temperature sensitive (Kerry et al. 2006; Biji et al. 2015). Their responses are expressed as color changes or mechanism deformations. There are two basic types in the market: • Critical temperature indicators (CTIs), which change when a temperature exceeds a limit • Critical time– temperature indicators (CTTIs), which change the color when the time and temperature are above the established limits • Leak indicators : These are used to detect O2 or CO2  leaks when modified atmosphere systems are used. They are based on chemical or enzymatic reactions producing a color change. Oxygen indicators are often used to control the proper removal of oxygen absorbers. In the case of CO2  indicators, the response can be altered by the dissolution of CO2  in the food, which usually happens during the first days, and by the production of CO2  by microorganisms, growing during food decay. The best-known system is methylene blue, which has an oxidation reduction reaction (Biji et al. 2015). • Freshness indicators : These indicate microbial growth or chemical compounds generated during food ripening or food aging. There are different market samples that are based on the detection of CO2 , diacetyl, amines, or ammonia, which are related to fish and meat deterioration (Hogan and Kerry 2008). Substances used as indicators of microbial growth, such as n-butyrate, L-lactic acid, D-lactate, and acetic acid, can also be monitored by some indicators (Biji et al. 2015). • Biosensors : These are intelligent systems used to detect, record, and transmit information on biochemical reactions. They are composed of a bioreceptor and a transducer. The former is an organic or biological material, such as a hormone, enzyme, or microbe, that detects the target analytes. The latter transforms this biochemical response into optical, acoustic, or electrochemical response (Biji et al. 2015; Yam et al. 2005). Commercial biosensors are not available on the market,

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although several prototypes have been developed (Nerin et al. 2009). The most recent applications in food are shown in Table  14.2. • Gas sensors : These are used to detect the presence of a gas. There are different types of gas sensors, depending on the specific analyte under study. Examples include oxygen, carbon dioxide, water vapor, ethanol, and amine sensors, and organic conducting polymers or piezoelectric crystal sensors applied to different gases. • Chemical sensors : These detect the presence, composition, or concentration of chemical contaminants, tampering products, or intermediate-chain products by absorption. The most important sensors are composed of nanoparticles, nanotubes, and nanofibers, and their chemical signals are translated in ultraviolet (UV), visible, or infrared (IR) measurements (Biji et al. 2015). 14.3.2  Radiofrequency Identification This is a relatively new technology that uses small chips called tags in order to track, identify, or gather data. These tags contain antennas that allow them to communicate with a reader by radio waves, performing the same function as magnetic codes, but allowing a greater identification distance. RFIDs can be used to store information about humidity, temperature, nutrition, producer, processor, raw materials, operations during the manufacturing process, quality, safety, traceability, and so forth (Biji et al. 2015; Lee et al. 2015), but can only be considered to be intelligent packaging when combined with a real sensor. The most significant drawback is its high implementation and maintenance cost, which can influence the final product price. Several commercial examples try to integrate RFID systems with indicators and sensors able to monitor the quality of food products, for example, an RFID tag with an optical oxygen indicator or pH sensor to test spoilage processes in fish products (Realini and Marcos 2014). Other cases applied to food are shown in Table  14.2. 14.3.3  Microwaveable Intelligent Food Packaging Microwave cooking characteristics have been briefly discussed in Section  14.2.10. One of the disadvantages is the nonuniform heating produced on the foodstuff. As a consequence, hot spots occur throughout the food. To solve this problem, microwave doneness indicators (MDIs) are visual indicators that detect cooler regions that would not have reached acceptable cooking temperatures (Robertson 2006). Therefore, they help to decide when foods are safe to eat. A sophisticated intelligent packaging system for microwave cooking has also been developed. Many times, the cooking instructions on the

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microwaveable packages do not fit for all ovens. This causes bad cooking results. The system proposed by Yam (2000) consists of a barcode in the packaging. The information from the barcode can be scanned and passed on to the microprocessor in the oven. The microprocessor is then be able to control the microwave to ensure perfect cooking with practically zero interaction from the consumer. Although this proposal was published in 2000, there is no currently availability in the market.

14.4 Nanotechnology, Biomaterials, and Bioactive Compounds Used in Active and Intelligent Packaging The use of nanomaterials, biomaterials, or bioactive compounds for active and intelligent packaging is an expansive field that requires a combination of different disciplines, such as nanotechnology, food, and material sciences. Nanotechnology is becoming an important tool for the food industry worldwide, where there are potential applications in active as well as intelligent packaging. In the case of intelligent packaging, there are several practices, such as the use of nanosensors that are sensitive to microbes, toxins, and other contaminants generated during processing and storage chain. Examples are sensors capable of detect Escherichia coli  or salmonella bacteria using nanosized light scattering or silicon/gold nanorod array, respectively (Neethirajan and Jayas 2011). In the case of active packaging, nanotechnology can provide solutions modifying the permeation behavior of layers, increasing barrier properties (mechanical, chemical, and microbial), providing antimicrobial properties, and improving heat resistance properties. An example is the wide use of nanoparticles with antimicrobial properties (silver and ZnO) in matrix coating material or in sachets placed in the package, which can reduce microbial growth in foods such as cheese, sliced meat, and bakery products. Other examples are the use of nanotechnology to produce oxygen scavengers (modifying the nanosized surface), free radical scavengers (SeNP) (Vera et al. 2016), moisture absorber sheets, and ethylene-scavenging bags (using silicate nanoparticules) (Neethirajan and Jayas 2011). On the other hand, society is more and more demanding the use of biopolymer materials because of their sustainability and environmental safety. But, it is known that they have some disadvantages compared with conventional nonbiodegradable materials, such as their poor mechanical and barrier properties. So, research efforts are focused on the use of bionanocomposites (including nanoparticles, nanofibrils, nanorods, and nanotubes) to not only improve their mechanical, thermal, and barrier properties (to gas vapor and water), but also offer functions such as antimicrobial, biosensor, and oxygen scavenger properties (Othman 2014).

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Several applications of bionanocomposite materials are found in the references, such as gelatin films or nanoclay cellulose containing silver nanoparticles, both with antimicrobial capacity. Reactive nanofillers incorporated into biopolymers can act as biosensors with the ability to respond to changes in the environment, such as temperature, humidity, and levels of oxygen. Finally, the use of bioactive compounds where nanotechnology is capable of enhancing the efficiency of functional foods, improving human health, and acting in active and intelligent packaging should be pointed out. Some examples are found in the references, for example, nanocapsules capable of masking the odor of tuna fish oil in the mouth, delaying its breakdown until the stomach, and therefore avoiding an unpleasant taste or odor. However, this approach cannot be used in Europe, as it is forbidden to mask fish odor because it is a good indicator of fish decay. Another example is the enhanced bioavailability of lycopene by the fortification of lycopene nanoparticles with antioxidant capacities in tomato juice, pasta sauce, and jam (Neethirajan and Jayas 2011).

14.5  Commercially Available Active and Intelligent Packaging Nowadays, the commercial use of active and intelligent packaging is limited for several reasons. The first one is that, in most cases, food is in direct contact with the active and intelligent agents, and their substances may migrate into the foods. These intentional and nonintentional migrations must be studied, which is a tedious and time consuming task. Everything incorporated into the food contact materials has to be authorized and cannot affect the consumer’ s health. In addition, active and intelligent materials add cost, which must be justified by the benefit of obtaining a reliable, efficient, and useful system. Besides that, food producers and consumers must accept this new technology. On the other hand, the system must be efficient on a large scale, not only on a laboratory scale. Some examples of commercial active and intelligent packages used for food packaging are shown in Table  14.3 (Kerry et al. 2006).

14.6 Legislation The initial legislation, Regulation (EC) 89/109/EEC, established the basic principles: “ Any material or article, intended to come into direct or indirect contact with food, must be inert, to avoid transfer substances that may endanger human health, cause unacceptable composition or organoleptic

Type of Active or Intelligent Packaging Oxygen scavengers

ATCO®   (EMCO Packaging Systems, United Kingdom) Cryovac®   OS 2000™   (Cryovac Division, Sealed Air Corporation, United States) PureSeal®   (W. R. Grace Co., United States) DarExtend®   (Grace Darex Packaging Technologies, United States) ActiTUF®   (M&G, Italy) ZerO2  (Food Science, Australia)

FreshMax™   and FreshPack (Multisorb Technologies Inc., United States)

Some Commercial Names Ageless®   (Mitsubishi Gas Chemical Co., Japan) Oxyeater®   (Ueno Seiyaku Co., Japan) Freshilizer®   (Toppan Printing Co., Japan) Vitalon®   (Toppan Printing Co., Japan) Seagul®   (Nippon Soda Co., Japan) Sanso-Cut®   (Finetec Co., Japan) ATCO (Standa Industrie, France) Oxyguard®   (Toyo Seikan Kaisha Ltd.) FreshPax®   (Multisorb Technologies Inc., United States)

Commercially Available Active and Intelligent Materials

TABLE  14.3 

Polyester bottles based on iron oxidation Plastic films, bottles, and containers based on photosensitive dye/organic compound.

Bottle crowns based on ascorbate/metallic salts

(Continued)

Plastic trays based on iron oxidation. Packets and strips designed to absorb oxygen inside sealed packaging. Used especially for cooked sliced meat products. Labels designed for adhesion with high-value foods based on iron oxidation. Used especially for cooked sliced meat products. Labels based on iron oxidation. Polymer-based film used to dry and smoke meat products.

Applications Sachets and labels based on iron oxidation. Used especially for meat and poultry products. Sachets based on iron oxidation.

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Moisture control

Ethylene scavengers

Carbon dioxide scavengers

Type of Active or Intelligent Packaging

(Continued)

PE films based on crysburite ceramic. Sorbent packets formed by medical-grade Tyvek®   can be used in food applications.

Based on titanium dioxide catalyst. PE-based films containing activated clay for cut vegetables and fruits.

Sachet, paper, or board based on activated carbon.

Ageless®   (Mitsubishi Gas Chemical Co., Japan) Ethysorb™   (Stay Fresh Ltd.)

NA®   (Johnson Matthey, United Kingdom) Bioka (Bioka Ltd., Finland) Verifrais™   (SARL Codimer, France)

Oxbar®   (CMB Technologies, France)

Neupalon™   (Sekisui Jushi, Japan) Hatofresh™   (Honshu Paper, Japan) Sendo-Mate™   (Mitsubishi Gas Chemical Co., Japan) Bio-Kleen (Kes Irrigations Systems, United States) Everfresh™   and Orega™   (Korea) Profresh™   (Warenhandels GmbH, Australia) Peakfresh™   (Peakfresh Products, Australia) Bio-fresh™   (Grofit Plastic, Israel) BO film™   (Odja Shoji, Japan) Minipax®  

Applications Plastic films, bottles, and containers where the scavenger mechanism is the PET copolymer. Plastic films or bottles based on cobalt-catalyzed polymer oxidation. Labels on platinum group metal catalyst. Sachets based on enzyme. Based on sodium bicarbonate/ascorbate used for fresh meat. Based on ascorbic acid oxidation. Based on potassium permanganate used for fresh fruit.

Shelfplus O2 ®   (Ciba Speciality Chemicals, Switzerland)

Some Commercial Names

Commercially Available Active and Intelligent Materials

TABLE  14.3 (CONTINUED)

Active and Intelligent Food Packaging 479

Some Commercial Names

GOGLIO (Italy) (Bosetti et al. 2013)

Antioxidants: Free radical scavengers

Nor®   Absorbit (Nordenia International AG) Sira-Crisp®   (Sirane Ltd.) SmartPouch®   (VacPac Inc.) LifeLinesFresh-Check

Microwaveable

Time– temperature indicators

BHM™   (EKA Noble and EKA Companies)

Odor absorbers

Microgard™   (Nisin, United States)

ATOX 101 AV, 102 AV, and 104 AV (Artibal, Spain)

Irgaguard®   Emulactiv C-1 and Rycoat F-100 (Repsol, Spain) (Nerin et al. 2005) Artibal

Cryovac®   and Dri-Loc®   (Sealed Air Corporation, United States) Thermarite®   or Peaksorb (Australia) Fresh-R-Pax (Maxwell Chase Technologies, United States) Nisaplin®   (Nisin, United States)

Antioxidants

Antimicrobials

Type of Active or Intelligent Packaging

Commercially Available Active and Intelligent Materials

TABLE  14.3 (CONTINUED)

Applications

(Continued)

Based on polymerization reaction used for different types of ready-made meat and dairy foods.

Concentrated extract used for all types of foods: dairy, culinary, meat, bakery products, and beverages. Film with silver zeolites. Antimicrobial coating for paper and board. Applied to fruits and vegetables. Coating for paper and plastics. Applied to processed food, bakery products, and meat. Coating for paper and plastics. Applied to processed food, bakery products, meat. Multilayer containing a free radical scavenger (green tea) for coffee, chocolate derivatives, cereals, and any kind of food. A film used for all types of foods: dairy, culinary, meat, bakery products, and beverages. Formed by aluminosilicate zeolites to absorb odorous aldehydes for fish products. Moisture-absorbing and flexible microwavable film. Microwave susceptor.

Sorbent packets for meat and poultry products.

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Some Commercial Names

ToxinGuard™   (Toxin Alert, Canada)

Biosensors

Food Sentinel and Food Sentinel System™   (Syra Technologies, United States)

Oxysense®  

Agelles Eye®   (Mitsubishi Gas Chemical Co. Japan) Vitalon®   Samso-Checker FreshTag®   (COX Technologies, United States)

Vitsab®   TTI (COX Technologies, United States)

3M Monitor Mark

Freshness indicators Gas sensors

Gas indicators

Type of Active or Intelligent Packaging

Commercially Available Active and Intelligent Materials

TABLE  14.3 (CONTINUED)

Applications

Labels that react to volatile amines produced during storage used for fish or other seafood products. Fluorescence quenching sensor for measuring the oxygen in headspace, as well as dissolved in liquids. It is used for food and beverages. Immobilizes and protects antibodies in sites on the surface of the polymer film. Capable of detecting the presence of harmful microorganisms through immunological reactions of barcodes.

Based on dye diffusion used for different types of ready-made meat and dairy foods. Based on enzymatic lipase color change used for different types of ready-made meat and dairy foods. Oxygen control labels based on color change.

Active and Intelligent Food Packaging 481

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change in the food.”  This regulation did not include the use of active and intelligent substances. In December 2004, a new Europe regulation (EC 1935/2004) of the European Parliament and Council of October 27, 2004, included, for the first time, active and intelligent materials, repealing Regulation 89/109/EEC. This regulation maintained the basic principles above described, allowing technological innovation in food packaging and accepting some interaction between food and packaging. However, it did not include the list of active substances and their composition. Subsequently, in 2009, Regulation (EC) 450/2009 set the requirements which with active and intelligent materials must comply. Specifically, it details the requirements needed to launch the products into the market. A new feature of this regulation is the creation of a list of authorized substances. This list should contain all substances that can be incorporated in any active system. They must fulfill the basic principles above described, using migration and sensory analysis described in the general materials regulation in contact with food. The regulation also includes the conditions of use for active substances that are currently permitted as food additives. These substances may be used provided they do not exceed their permitted limits. In addition, the systems that are not integrated into the package, for example, sachets, must necessarily include the words “ do not eat”  and the symbol of not edible device. Finally, the companies that manufacture this type of active and intelligent materials must make a declaration of compliance to register the food suitability of these products.

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Section V

 Food Safety Laws 

15 Food Fraud: Detection, Prevention, and Regulations Jamuna A. Bai and V. Ravishankar Rai CONTENTS 15.1 Introduction: Definition of Food Fraud................................................ 496 15.1.1  Types of Food Fraud   ................................................................ 497 15.2 Examples of Food Fraud......................................................................... 499 15.2.1 Meat and Meat Products...........................................................500 15.2.2 Seafood........................................................................................500 15.2.3 Dairy Products........................................................................... 501 15.2.4 Oils............................................................................................... 502 15.2.5 Spices........................................................................................... 502 15.2.6 Honey.......................................................................................... 503 15.2.7 Juice.............................................................................................. 503 15.2.8 Coffee........................................................................................... 503 15.2.9 Organic Vegetables and Fruits.................................................504 15.2.10 Additives.....................................................................................504 15.3 Impact of Food Fraud..............................................................................505 15.3.1 Public Health Impact................................................................. 505 15.3.2 Economic Impact....................................................................... 505 15.4 Detection of Food Fraud ........................................................................505 15.4.1 Genomics....................................................................................505 15.4.1.1 Meat and Meat Products........................................... 506 15.4.1.2 Game Meat..................................................................508 15.4.1.3 Milk and Milk Products........................................... 509 15.4.1.4 Spices........................................................................... 509 15.4.1.5 Seafood........................................................................ 509 15.4.1.6 Plant-Derived Foods.................................................. 510 15.4.2 Metabolomics............................................................................. 511 15.4.2.1 Plant Products............................................................ 512 15.4.2.2 Honey.......................................................................... 514 15.4.2.3 Milk and Milk Products........................................... 514 15.4.2.4 Meat and Meat Products........................................... 515

495

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15.4.3 Proteomics ................................................................................. 516 15.4.3.1 Dairy Products........................................................... 517 15.4.3.2 Meat............................................................................. 517 15.4.3.3 Fish .............................................................................. 518 15.4.3.4 Wine............................................................................. 518 15.5 Prevention of Food Fraud....................................................................... 519 15.5.1 Traceability................................................................................. 519 15.5.2 Authentication............................................................................ 519 15.5.3 Supply Chain Management and Procurement...................... 520 15.6 Regulation of Food Fraud: Laws and Legislation............................... 520 15.6.1 Food Fraud Regulation in the United States.......................... 520 15.6.2 Food Fraud Regulation by the European Commission....... 520 15.6.3 Food Fraud Regulation in the United Kingdom................... 521 15.6.4 Food Fraud Regulation in China............................................. 521 15.6.5 Food Fraud Regulation: Industry Standards and Compliance................................................................................. 521 15.6.5.1  Global Food Safety Initiative................................... 521 15.6.5.2 Safe Supply of Affordable Food Everywhere........ 521 15.6.5.3 U.S. Pharmacopeia..................................................... 522 15.6.5.4 Other Activity............................................................ 522 References ............................................................................................................. 522

15.1  Introduction: Definition of Food Fraud Food fraud  is a collective term used for any deliberate and intentional substitution, tampering, addition, or misrepresentation of food and food ingredients and food packaging, as well as false statements made about the food product with the intention of economic gain (Spink and Moyer 2011; Spink 2013). The Food and Drug Administration (FDA) has defined economically motivated adulteration  (EMA) as the fraudulent, intentional substitution or addition of a substance in a product for either increasing the apparent value of the product or reducing its production cost. It also includes the dilution of products with increased quantities of an already-present substance to the extent that such a dilution poses a known or possible health risk to consumers, as well as the addition or substitution of substances in order to mask dilution (FDA 2009; Spink and Moyer 2011). Food fraud  is also defined as “ t he act of defrauding buyers of food or ingredients for economic gain”  (Johnson 2014). Food fraud includes different forms of EMA, misbranding, food counterfeiting, and even smuggling. Illegal substitution of additives in the raw ingredients, adulterating finished products, tax avoidance smuggling, counterfeiting complete packaged product, mislabeling country of origin, or uplabeling of genetically

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modified as conventional are all various forms of fraud and are economical threats (Moore et al. 2012; Ryan 2016). Food fraud is a part of food protection issue. It needs to be clearly differentiated and understood from other forms of food protection and safety issues. The following provides the definitions of various terms related to food protection (Ellis et al. 2005, 2015; Elliott 2014): Food fraud is a deliberate action of selling foods for financial gain, with the intent of deceiving consumers. Selling foods unfit or harmful for human consumption and mislabeling food with respect to its geographical origin, ingredients, or substitution with lower-value or dangerous contents are the major types of food fraud (Figure 15.1). Contamination involves the unintentional introduction of undesirable physical, chemical, or biological components in food. The unintentional addition of metal or plastic fragments, cleaning reagents and microbes, and their toxins in food products during processing, production, or packaging constitutes food contamination. If these are intentional, it would be food crime, and depending on the intention of deliberate contamination, it would be considered bioterrorism. Food security is the sustainable food production and supply of a secure, sufficient quantity of safe and nutritious food to meet consumer demands. Food authenticity is the correct labeling, or menu section, of a finished food product to be sold to the consumer. Food integrity ensures selling of the quality substance, and nature of food to meet consumer requirements. This chapter provides an overview of the definitions of food fraud, types and examples of food fraud, its detection by various analytical techniques, and the management and regulation of food fraud. 15.1.1   Types of Food Fraud   The Global Food Safety Initiative (GFSI) has defined various types of food fraud (van der Meulen 2015). • Counterfeiting is presenting a product as something different from what it really is. It could be a brand, protected designation, or mark of a religious, quality, or safety profile. • Dilution is increasing the quantity of the product by adding lowvalue substances. It is considered a quality issue, but is a safety issue when harmful substances or substituents are used as diluents. • Substitution is the selling of different species in the food than declared. It is seen in the case of fish and meat products. The use of horsemeat in beef meat products is an example. • Concealment is the nondisclosure of the presence of undesirable aspects in a food product and it reduces the quality of food. The use of harmful colorants in food is an example of concealment.

Counterfeiting

Simulation

FIGURE 15.1 Overview of food fraud— t ypes, detection, and management.

Proteomics

Metabolomics and chemometrics

Genomics

Food fraud detection analytical techniques

Economic impact

Public health risks

Food fraud implications

Theft

Food fraud

Adulteration

Overrun

Tampering

Supply chain management

Traceability

Authentication

Food fraud prevention

Product recall

Legal action

Food fraud response

498 Food Safety and Protection

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499

• Mislabeling is the distortion of the information about the food product provided on the label. Changes in the durability marking on the label is an example of mislabeling. • Unapproved enhancements are the addition of ingredients to food without approval and are related to authorization requirements. An ingredient can be added to the food product only if its required and its usage is approved by regulatory agencies. Similarly, Spink et al. (2016) have also defined the various types of food fraud, some of which are • Adulteration: The addition of a fraudulent substance or an impurity in the finished food product. Example: Addition of melamine to milk and Sudan dyes to saffron. • Tampering: Legitimate product and packaging are changed fraudulently. Example: Changing expiry information of a food product or uplabeling a food product. • Overrun: Legitimate product is manufactured beyond production agreements. Example: Underreporting of manufactured product. • Theft: Stolen legitimate product sold as legitimately procured. Example: Stolen products are mixed and sold with legitimate products. • Diversion: Legitimate products are sold and distributed outside the intended markets. Example: Relief foods for aid are sold in the market. • Simulation: Illegitimate products are designed or imitated to look similar to the legitimate brands of food products. Example: “ Knockoffs”  of popular foods with lower safety and quality. • Counterfeiting: Intellectual property rights (IPR) violation that includes complete replication of all aspects of the product and packaging. Example: Duplication of popular foods without the same food safety assurances.

15.2  Examples of Food Fraud Some food categories that have previously been associated with food fraud are presented in the next sections. These food categories can be assumed to be in high risk of food fraud (Ros 2014).

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15.2.1  Meat and Meat Products Meat and meat products are expensive and adulterated in different ways. The expensive meat parts are substituted with cheaper parts of the animal. For example, skeletal muscle is replaced with offal or collagen tissue and blood. Some of the important factors considered during the purchase of meat are the country of origin, breed of meat, species of meat, rearing condition, and if it is a free-range, pasture-fed, or concentrate-fed animal. By falsely stating the country of origin, or the species of meat, or a breed as a popular meat breed, a higher price is gained by selling such meat. Similarly, vegetable proteins, such as soy protein, are used instead of meat proteins in processed meat products, and plant products are mixed with meat products to increase the volume and weight. Meat weight is also sometimes increased by using water and binding agents (Ballin 2010). In 2008, a company in Sweden had adulterated processed meat products. Lithuanian minced beef was mixed into ­sauteed reindeer meet. The company was found guilty of the fraudulent act, and its largest customer terminated their delivery agreement, as seen in Lycksele Tingsrä tt verdict criminal case B 443-08. In European countries, the adulteration of beef meat with horsemeat garnered media attention as the Horsemeat Scandal. The horsemeat scandal was unraveled in Europe in 2013. Investigation by the Food Safety Authority of Ireland (FSAI) of beef burger products revealed that they had horse and pig DNA. In similar incidences, pork meat containing colorants Azorubine (E122), Sunset Yellow FCF (E110), and Ponceau 4R (E124) was sold as beef tenderloin, and Polish beef with horse meat was sold as Swedish beef tenderloin by a Swedish company (National Food Agency 2013). Consumer and wholesale meat retailers’  complaints led to an investigation by the Swedish Food Agency, revealing the meat food fraud. In Sweden, the presence of horse DNA in Findus lasagna led to the withdrawal of the food product from the market by the Findus Company (FSA 2013). 15.2.2 Seafood Seafood has a high demand, which has led to increased illegal and unreported fishing. The European Union (EU), the largest importer of seafood, imports illegal, unreported, and unregulated (IUU) seafood totaling € 1.1 billion annually. IUU fishing is categorized as seafood fraud. Some of the common examples of seafood fraud are selling cheaper or more abundant fish species as expensive ones, falsely stating their geographical origin, labeling farmed fish as wild or otherwise, and injecting water and binding agents into fish fillets to increase their weight (FSA 2002). During 2010– 2012, 1200 seafood samples were collected and investigated from retail outlets and restaurants in several states in North America. The study showed that 33% of the samples were mislabeled, with the highest percentages of mislabeling of 74% in sushi restaurants, 38% in other restaurants, and 18% in grocery stores

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(Oceana 2013). The species red snapper (Lutjanus campechanus ) was most commonly replaced with other species. For example, on analyzing 120 samples labeled as red snapper, only 7 were red snapper. Similarly, fish sold as tuna were substituted with species such as escolar (Lepidocybium flavobrunneum ). Of 114 samples examined, 67 were actually tuna. Out of 116 cod samples analyzed, 32 were mislabeled as cod (Warner et al. 2013). In Europe and Northern America, 15%– 43% of commercially available seafood are food fraud cases. For example, red snapper is replaced with cheaper and abundant similar species or commonly substituted with tilapia (Galimberti et al. 2013). In Ireland, cod is mostly replaced by cheaper species (Armani et al. 2012). Incidences of mislabeling regarding the production method of fish have been reported, as in the case of Atlantic salmon (Salmo salar ) sold as wild caught and mislabeled in 11 of 54 samples examined from Norway, France, the United Kingdom, and Italy (Thomas et al. 2008). Similarly, the Food Standards Agency (FSA) in the United Kingdom has reported the mislabeling of salmon (S. salar ), sea bass, and sea bream as wild caught (FSA 2007). 15.2.3  Dairy Products Milk and milk products are subject to food fraud in many ways. Diluting milk with water, adding melamine to increase the protein content in milk, and removing constituents such as fat and protein are common types of milk food fraud (FAO 2013). One of the major food fraud incidences concerning milk products was the addition of melamine to raw milk by Chinese milk producers. Melamine, a nonproteinaceous substance, was added to milk to increase the true value of the protein content in milk. Consumption of melamine-adulterated milk in China led to an outbreak of kidney and renal dysfunction among infants. The intake of Sanlu Group infant formula, which contained melamine, led to the outbreak. The Chinese government seized more than 2000 tons of milk powder and recalled 9000 tons of milk powder produced by the Sanlu Group. Twenty-two other infant formula– producing companies in China had melamine in their products. Upon consuming instant formula adulterated with melamine, around 300,000 Chinese infants became sick and 6 infants died. Melamine-containing dairy and bakery products from China had reached 47 different countries by direct import, thirdcountry import, or illegal import (Gossner et al. 2009). Mozzarella cheese, a traditional Italian cheese, is to be labeled with the protected designation of origin (PDO) when produced using milk of water buffalos (Bubalus bubalis ) in the Italian communities of Campania, Latium, and Apulia. The PDO label was assigned by the European Commission (EC) to protect traditional foods. The PDO-labeled mozzarella cheese is sold at a premium price. A fraudulent practice observed in the production of mozzarella cheese is the use of cheaper cow milk in place of buffalo milk by producers to make a profit using a cheaper ingredient. On examining 64 samples from 37 brands of mozzarella cheeses sold by grocery stores, dairy shops, and cheese manufacturers,

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48 samples having the PDO label and 16 lacking it had not stated cow milk as an ingredient, and 80% of the samples contained cow milk. Of the 37 brands examined, only 2 contained buffalo milk in the samples analyzed (Loparelli et al. 2007). Another dairy product that is commonly adulterated is butter. Butter has been adulterated with beef tallow and synthetic chemicals. During 1997– 1999, the Italian financial police conducted on-site inspections and found 16,000 tons of adulterated butter produced from 5,000 tons of beef tallow substances and 400 tons of synthetic chemicals. The adulterated butter was sold in Germany, France, Italy, and Belgium (European Anti-Fraud Office 2009). 15.2.4 Oils Extra virgin olive oil (EVOO) is a product of high value due to its high quality. Adulteration of EVOO by replacement with lower-quality oils, such as refined olive oil or olive pomace, or other oils, such as palm oil, hazelnut oil, soybean oil, or seed oil, is a common fraud (Hodaifa et al. 2012). EVOO from a region known for producing high-quality oil has been replaced with EVOO from another region and sold at a higher price (Frankel 2010). In 1995, a large-scale fraud carried out in the production of olive oil was revealed. Sunflower oil from Turkey was imported to Belgium, Germany, the Netherlands, and Portugal and sold multiple times between different companies before importing to Italy and Spain. Investigations revealed that the oil was actually hazelnut oil, and 20,680 tons of it was used in the production of 103,400 tons of adulterated olive oil (EC 1998). In 2010, the UC Davis Olive Oil Chemistry Laboratory and Australian Oils Research Laboratory found that 70% of the imported EVOO did not meet the sensory standards, and of these, 86% also failed to meet the chemical standard set by the International Olive Council (IOC) and the U.S. Department of Agriculture (USDA). In the case of the ones produced in California, 10% did not meet the sensory standards (Frankel 2010). 15.2.5 Spices Saffron is an expensive spice obtained from the stigmas of the plant crocus (Crocus sativus ). Some of the common frauds practiced with saffron are mislabeling its origin and substituting it with old saffron or stalks of its other parts. Its weight is increased by using honey, syrup, or oil (Alonso et al. 1998). The quality of spices is evaluated by their color. Therefore, chemicals such as lead are used to enhance the color of spices like saffron or turmeric to sell them as a higher-quality product. In 2011, the FDA recalled the sale of turmeric bottles upon discovering elevated levels of lead in the spice (FDA 2011b). Another chemical used to enhance the color of spices such as chili and pepper is the Sudan dye. Sudan IV with Sudan II and Sudan III is carcinogenic in nature, and hence its use in foods is illegal. There were 213 notifications to the Rapid Alert System for Food and Feed (RASFF) in 2005, and 60 notifications in 2006

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regarding the use of Sudan dyes in spices (EC 2007). Pepper powder containing traces of Sudan IV packed in Korea was exported from China to European countries. Using the analytical technique of high-performance liquid chromatography– mass spectrometry (HPLC-MS), Sudan IV was detected in the imported pepper powder and the imported countries were notified through RASFF and the product recalled and destroyed (EC 2012). 15.2.6 Honey The quality of honey is greatly associated with its origin, and thus its prices vary. It is usually adulterated by adding sugars or syrups (Cotte et al. 2003). The fraud practices associated with honey are changing the country or region of its origin and selling cheap, imported honey as locally produced high-quality honey at a higher price. A survey by Fairchild et al. (2003) for the National Honey Board revealed that companies found honey highly and regularly adulterated, and had detected levels of 5%– 43% corn syrup in adulterated honey. The adulterated honey was originating from China and Argentina (Fairchild et al. 2003). A study showed that 35 commercial honey samples from France, Hungary, Spain, and Morocco, upon testing, were found adulterated with three different syrups. Of 18 acacia honey samples tested, 7 were adulterated with added syrup or a honey other than acacia. Of the eight chestnut samples tested, two were adulterated with syrup. Similarly, of nine lavender honeys tested, four had been adulterated with syrup or a different type of honey (Cotte et al. 2003). 15.2.7 Juice Orange juice, a high-value product, is adulterated with different kinds of citrus juices, such as grapefruit, mandarin, tangerine, or lemon. Water, sugar, and citric acid are used to retain the taste of diluted or inferior-quality juice. Pulp wash is also used to adulterate orange juice (Le Gall et al. 2001). Fruit juices such as pomegranate juice have been replaced with pear, apple, cherry, grape, or aronia juice. Cane sugar or corn sugar is used to increase the taste, and anthocyanins from aronia, grape skin, elderberry, black currant, or black carrot are added to enhance color. In a study of 23 commercial pomegranate juices, 14 were found to be adulterated (Zhang et al. 2009). In 2012, the National Consumers League in the United States tested four samples of lemon juices labeled 100% lemon juice concentrate and found all to be adulterated and diluted with water. Citric acid and sugar were used to restore its taste (NCL 2012). 15.2.8 Coffee Coffee, an expensive beverage, is adulterated by using roasted and ground cereals such as barley and corn, seeds, and roots (Oliveira et al. 2009). The

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Association of the Brazilian Coffee Industry found that adulterating coffee affected its quality. One out of six commercial coffee samples were adulterated with corn (Jham et al. 2007). The highly priced arabica (Coffea arabica  L.) had been replaced with the cheaper robusta coffee (Coffea canephora  var. robusta ) and sold for twice the price (International Coffee Organization 2013). 15.2.9  Organic Vegetables and Fruits Conventional crops have been fraudulently sold as organic crops. In 2010, it was found by an Italian inspection agency that false production claims were made by a company that had sold imported conventional crops as organic foods. Crops such as barley, oats, wheat, millet, sorghum, corn, alfalfa, field beans, field pea, linseed, soybeans, rapeseed, sunflower, and potato were falsely sold as organic in Italy, Belgium, France, the Netherlands, and Germany. To prevent traceability, they were sold several times between various companies (European Working Community for Food Inspection and Consumer Protection 2013). 15.2.10 Additives Additives are used in processed foods to improve their texture, color, preservation, and taste. Adulterated additives, such as poor-quality chemicals, have been used in the food industry. In 2011, a large salt scandal was discovered in Iceland. The country’ s leading supplier of salt had sold refined industry-grade salt as food-grade salt for many years (Ministry of Fisheries and Agriculture of Iceland 2012). The unauthorized use of additives, such as sulfites, in seafood to enhance its quality is a food fraud. In 2006, more than 90 notifications regarding sulfites were made to RASFF (EC 2007). Cloudy agents used in food were found to contain plasticizers in Taiwan. Two companies producing cloudy agents had replaced palm oil with bis(2-ethylhexyl)phthalate (DEHP) and diisononyl phthalate (DINP). These cloudy agents containing high amounts of phthalate DEHP were used in preparing capsules for probiotics. The phthalates are usually used as plasticizers in the plastic industry. However, as plasticizers are endocrine disruptors, their usage in food has severe and adverse implications in human health (Li and Ko 2012). The palm oil was replaced with plasticizers due to their low cost and ability to increase the product shelf life. Two companies had sold the adulterated cloudy agents to 8 dealers, who had dispensed the product to 186 food ingredient manufacturers and 229 end-product manufacturers. Taiwan’ s FDA made inspections and recalled 30,000 foodstuffs containing products adulterated with phthalates. Notification was also made of products containing phthalates exported to 22 countries (Li and Ko 2012). Food products such as fruit juices, fruit drinks, jams, and syrups containing phthalates had been exported to the United States (FDA 2011a). Liquid chromatography– tandem mass spectrometry (LC-MS/MS) was used to detect phthalate adulteration in food products (Self and Wu 2012).

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15.3  Impact of Food Fraud 15.3.1  Public Health Impact Although majority of food fraud incidents do not cause public health risk, a few food fraud cases have resulted in high-profile cases of public health risks. One such incidence is the addition of melamine to high-protein feed and milk products to overestimate the protein content in diluted products. In 2007, use of adulterated pet food ingredients in the United States from China caused the deaths of a large number of pet dogs and cats (CRS Report RL34080). Again, consumption of melamine-contaminated baby formula affected 300,000 Chinese children and caused the deaths of 6 infants (Bottemiller 2011). Food fraud by the Peanut Corporation of America caused a Salmonella  outbreak in 2009, resulting in 700 people falling sick and 9 deaths. The company officials had sold and distributed contaminated product (Johnson 2014). Investigations led to the recall of 3912 products containing contaminated peanut butter and peanut paste ingredients manufactured by about 200 companies (CRS Report R40450). 15.3.2  Economic Impact According to the Grocery Manufacturers Association (GMA), food fraud has cost the global food industry $10– $15 billion annually, and affected 10% of all commercially sold food products (GMA 2010; Everstine and Kircher 2013). However, the number of documented food fraud incidents may be a fraction of the true number of incidents (Spink and Fejes 2012). Fraud acts resulting in public health risk can also cause significant financial and public relations consequences to food industries. Food fraud can affect food businesses in terms of lost sales, by 2%– 15% of annual revenues, and may cause bankruptcies in the case of public health consequences (Johnson 2014).

15.4  Detection of Food Fraud In the past two decades, molecular-based technologies have been invaluable tools for the detection of food authenticity, integrity, and safety (Ellis et al. 2016). 15.4.1 Genomics DNA-based techniques use specific DNA sequences or markers for detecting adulterants in food and checking its authenticity, especially the quality and

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origin of ingredients used in food products (Barcaccia et al. 2016). Genomics have been applied for the authentication and traceability of a number of food products (Table 15.1). 15.4.1.1  Meat and Meat Products DNA-based techniques have been used for identifying fraudulent practices in the meat trade, such as mislabeling in the case of meat species specification. In a study in Malaysia, 143 prepackaged beef and poultry meat products purchased from several supermarket chains were analyzed for the presence of common meat species, such as buffalo, cattle, chicken, goat, sheep, duck, and goose, and meats prohibited by Islamic laws, such as cat, dog, monkey, pig, and rat, using species-specific primers. A total of 112 samples were mislabeled where species were falsely declared and the presence of some species was undeclared. Of the 58 samples labeled as beef, buffalo DNA was detected in 40 samples. The presence of undeclared chicken and buffalo DNA was also detected in many beef and chicken products, respectively. The five “ haram” meat sources, however, were not detected in all meat products tested. The majority of the meat samples were legally noncompliant due to substitution and mislabeling of meat products (Chuah et al. 2016). Multiplex polymerase chain reaction (PCR) has been used to identify five meat species forbidden in Islamic foods. Five pairs of species-specific primers targeting mitochondrial ND5, ATPase 6, and cytochrome b genes have been designed to amplify DNA fragments of 172, 163, 141, 129, and 108  bp from cat, dog, pig, monkey, and rat meats, respectively. Species specificity checking in 15 important meat and fish species and 5 plant species showed no cross-species amplification. The single-assay platform can detect 0.01– 0.02  ng of DNA in raw meat and 1% suspected meats in processed foods such as meatball formulation (Ali et al. 2015). Ground meat products sold in the U.S. commercial market were tested for mislabeling by DNA techniques. DNA barcoding of the cytochrome c oxidase I (COI) gene was used to analyze 48 ground meat samples purchased from supermarkets and specialty meat retailers. DNA sequences from meat samples were identified to the species level using the Barcode of Life Database (BOLD). Samples that failed DNA barcoding were tested with real-time (RT) PCR for beef, chicken, lamb, turkey, pork, and horse DNA. Of the 48 samples analyzed, 10 were mislabeled. Nine of the mislabeled samples contained additional meat species based on RT-PCR, and one sample was mislabeled in its entirety. Horsemeat, which is illegal to sell in U.S. markets, was also detected in two of the samples bought from online specialty meat distributors. Mislabeling is due to the intentional mixing of low-cost meat species with high-cost meat products or unintentional mixing during meat processing by cross-contamination (Kane and Hellberg 2016). To identify the presence of undeclared horsemeat in beef products, the UK government Department of the Environment, Food, and Rural Affairs (DEFRA) project developed a RT-PCR method to quantify horse DNA relative to the total amount of mammalian DNA raw meat samples. Single-copy nuclear

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TABLE 15.1 Analytical and Molecular Genomic Techniques Used in Detection of the Food Fraud Detection Techniques  PCR Multiplex PCR DNA barcoding of COI gene RT-PCR DNA barcoding PCR-TTGE PCR-RFLP of COI gene Multiplex TaqMan RT-PCR

PCR targeting mitochondrial 12S rRNA Multilevel PCR D-loop mtDNA DNA barcoding of COI gene

Cyt b and TaqMan probe– based duplex PCR SPED and PCR DNA barcoding DNA barcoding and mini-barcoding PCR of COI gene PCR of COI gene

Barcode and mini-barcoding Barcoding

Nanoengineered DNA encapsulates High-resolution melting analysis DNA barcode trnL Bar-HRM Bar-HRM DNA barcoding DNA barcoding Nano-real-time PCR DNA barcode

Food  Buffalo meat in mixed beef Detection of haram meat Horsemeat in beef Horsemeat in beef Identification of meat species in mixed animal meat Identification of meat sample at the species level 5– 50  pg species-specific identification of chicken, duck, and turkey meat Identification of game meat of red, fallow, and roe deer Unknown meat from wildlife Game meat containing threatened and vulnerable species Murine meat in mutton

References  Chuah et al. 2016 Ali et al. 2015 Kane and Hellberg 2016 Nixon et al. 2015 Colombo et al. 2011

Milk and milk products Chili adulteration in black pepper Seafood

Bloch et al. 2014 Parvathy et al. 2014

Seafood Seafood— identification of European plaice and common sole Fish and shrimp products Seafood— cod, flounder, grouper, tuna, pink cusk eel Extra virgin olive oil

Khaksar et al. 2015 Pappalardo and Ferrito 2015

Fruit juices

Faria et al. 2013

Allergenic tree nut species Legume crops Spice samples Olive oil Edible oil Honey— origin and floral species

Madesis et al. 2013 Ganopoulos et al. 2012 De Mattia et al. 2011 Ganopoulos et al. 2013 He et al. 2013 Valentini et al. 2010; Bruni et al. 2015

Haider et al. 2012 Nq et al. 2014

Fajardo et al. 2007 Parkanyi et al. 2014 Quinto et al. 2016

Fang and Zhang 2016

Chin et al. 2016

Gunther et al. 2016 Carvalho et al. 2015

Puddu et al. 2014

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DNA targets for equine growth hormone receptor and a mammalian or poultry myostatin gene were chosen and assayed. The limit of detection (LOD) was less than 5 horse genome equivalents and was validated for DNA extracted from samples containing raw horsemeat in a raw beef meat background (Nixon et al. 2015). PCR-Temporal temparature gradient gel electrophoresis (PCR-TTGE), along with DNA barcoding, has also been used to detect meat species in mixed animal samples. Identification was performed by matching the “ DNA barcode” zone with the sequences of PCR products obtained from a limited set of “ universal” primers (Colombo et al. 2011). Meat authenticity has also been verified at a cheaper and faster rate by PCR Restriction fragment length polymorphism (RFLP) of part of the COI gene. Raw meat samples of cow, chicken, turkey, sheep, pig, buffalo, camel, and donkey were identified at the species level. The level of COI variation showed that of the seven restriction endonucleases (Hind II, Ava II, Rsa I, Taq I, Hpa II, Tru 1I, and Xba I), only Hpa II was sufficient to generate species-specific restriction profiles for unambiguously distinguishing all targeted species (Haider et al. 2012). A multiplex assay using TaqMan®  RT quantitative PCR (qPCR) for species-specific detection of chicken, duck, and turkey detected low quantities of species-specific DNA from single or multispecies sample mixtures containing 5– 50  pg of starting DNA material (Ng et al. 2014). An improved method involving PCR amplification of the COI gene and detection of species-specific sequences by hybridization led to detection of multiple species, including pork, beef, lamb, horse, cat, dog, and mouse, from a mixed sample in a single assay. Speciesspecific probes and 5  pg of DNA sample was used. A low-cost, high-density DNA-based multidetection test for routine inspection of meat species was developed (Lin et al. 2014). 15.4.1.2  Game Meat Game meat products are often target for fraudulent labeling, as they are highly priced than other meat species (Fajardo et al. 2006). PCR based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene identified meats from red deer (Cervus elaphus ), fallow deer (Dama dama ), and roe deer (Capreolus capreolus ) (Fajardo et al. 2007). Tobe and Linacre (2008) identified 18 common European mammalian species by species-specific multiplex PCR using the mitochondrial cytochrome b gene, which is commonly used in species identification and phylogeny studies. The control region of mtDNA (D-loop) was used for the identification of hair samples of the five hunting game species. The oligonucleotide sequences for multilevel PCR D-loop mtDNA identification of the hunting game species are registered at www.boldsystems.org and can be used for the detection of unknown meat samples from wildlife (Parkanyi et al. 2014). In the United States, 54 samples of whole-cut game meats were analyzed by DNA barcoding of the COI gene using BOLD and GenBank. A total of 18.5% of the samples were potentially mislabeled, and 9.3% of the samples legally contained a near-threatened or

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vulnerable species and were correctly labeled (Quinto et al. 2016). Murine meat has been used as a substitute for mutton in China. The cyt b and TaqMan probe– based duplex RT-PCR was designed for authentication studies. The technique has a LOD lower than 1  pg of DNA per reaction and can detect up to 0.1% murine contamination in meat mixtures (Fang and Zhang 2016). 15.4.1.3  Milk and Milk Products For tracing the products along the food chain, silica particles with encapsulated DNA (SPED) were developed and added to milk at 0.1– 100  ppb. Milk and milk products were thus uniquely labeled with a DNA tag. The DNA tags could be extracted from the food matrixes, identified, and quantified for previously marked products by qPCR with a LOD below 1  ppb of SPED. Approved food additives can be used as DNA carrier (silica  =  E551), and the low-cost technology (i.e., less than US$0.1 per ton of labeling milk with 10  ppb of SPED) can find application in labeling technology for the tracing and authentication of foods (Bloch et al. 2014). 15.4.1.4 Spices The DNA barcoding technique is used to detect species-specific variation in the short region of DNA and is used in the authentication and tracing of agri-food products (Chen et al. 2014). DNA barcoding has been used to detect adulteration in the highly valued black pepper powder. PCR amplification of Piper nigrum  and black pepper powder obtained from the market was performed for three barcoding loci: psbA-trnH, rbcL, and rpoC1. Sequence analysis and a Basic Local Alignment Search Tool (BLAST) search showed chili adulteration in two out of nine market samples. The DNA barcoding technique can be used to detect adulteration at very low levels, such as 0.5% adulteration (Parvathy et al. 2014). 15.4.1.5 Seafood Mislabeling of seafood and processed seafood products is a global problem. DNA barcoding was used to analyze 62 commercially sold seafood raw and processed samples. Full DNA barcoding and mini-barcoding target sequences were obtained and compared using the BOLD and GenBank databases. A total of 81% of the samples were successfully sequenced and analyzed, and of these, 16% were found to be mislabeled (Chin et al. 2016). On targeting mitochondria of the COI region for identifying fish and seafood samples sold in the United States, mislabeling was seen in 28 samples out of the 172 analyzed (Khaksar et al. 2015). In Italy, the frozen seafood market has fraudulent species substitution of fresh and frozen flatfish fillets. COI barcoding has helped in identifying the mislabeling of 35% of European plaice and 41% of common sole. Pleuronectes platessa  was substituted with

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Platichthys flesus , Limanda limanda , and Pangasius hypophthalmus , whereas Solea solea  was substituted with Arnoglossus laterna  (Pappalardo and Ferrito 2015). In Germany, 118 fish and shrimp seafood products from supermarkets and fishmongers were analyzed. Barcodes were successfully generated for 81.4% of the analyzed products. Mini-barcodes were successfully obtained from 43.2% of the failed cases, and samples were identified up to the species level. In seven products, there was a mismatch between labeling and the identified barcodes. In five samples, species did not belong to the genus indicated on the label, and in four products, labeling did not comply with the permitted list (Gunther et al. 2016). The Brazilian Governmental Regulatory Agency (PROCON) confiscated 30 seafood samples of commercial species, including cod, flounder, grouper, tuna, and pink cusk eel, as barcoding revealed that 24% of the samples obtained were mislabeled (Carvalho et al. 2015). 15.4.1.6  Plant-Derived Foods Basmati rice is highly valued due to its fragrance with grain elongation on cooking, giving a characteristic grain shape and integrity. It costs more than twice the price of other ordinary varieties. In the United Kingdom, it currently accounts for about 37% of the dry rice market by value and £ 50 million per year. There are 11 varieties from India and 5 from Pakistan. The FSA revealed that of the 39 samples of Basmati-labeled rice sold in the United Kingdom, more than one sample in six contained high levels of other nonBasmati varieties. In 363 samples examined, 196 samples contained only Basmati rice, and non-Basmati rice was detected in 167 samples. A DNA fingerprinting technique was used to assess the fraud in Basmati trade. Samples were screened to see the characteristic fingerprint of 12 chromosomes, in which chromosome 10 is important for identification (Shears 2008). DNA encapsulates in heat-resistant and inert magnetic particles have been developed. The nanoengineered encapsulates containing DNA can be recovered and analyzed by RT-PCR and Sanger sequencing, and these are stable for 2 years in decalin at room temperature. The magnetic DNA and silica encapsulates can be used for tracing and tagging oils and oil-based products. They require a 1  ppb level of the taggant and allow taggant concentration quantification on a log scale. The low-cost platform developed was able to verify the authenticity of the cosmetic bergamot oil and the food-grade EVOO (Puddu et al. 2014). High-resolution melting analysis was applied to discriminate orange, mango, peach, pear, and pineapple in fruit juices using DNA barcode trnL. The locus trnL proved to be appropriate, as the mean genetic divergence was estimated at 27.7, and all five species were clearly discriminated by the melt curve difference graphs. DNA barcoding can be used to discriminate species in juices in a single-tube analysis (Faria et al. 2013). High-resolution melting analysis using chloroplast barcoding regions (Bar-HRM) has been used to obtain barcoding information for the identification of allergenic tree nut species in processed foods (Madesis et al. 2013).

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Bar-HRM has also been used to analyze legume crops. PDO product “ Fava Santorinis”  adulterated with legumes of Lathyrus  or Vicia  and Pisum  species has been identified by Bar-HRM. It is a highly sensitive tool and can identify as low as 1:100 of non– Fava Santorinis in Fava Santorinis commercial products (Ganopoulos et al. 2012). Similarly, DNA barcoding is used in recognizing commercial spices of the Lamiaceae family. The classical DNA barcoding approach with four candidate barcode regions (rpoB, rbcL, matK, and trnH-psbA) and universal primers were used to differentiate 64 spice samples comprising 6 genera of spice plants: Mentha , Ocimum , Origanum , Salvia , Thymus , and Rosmarinus . The noncoding trnH-psbA intergenic spacer and matK were the best markers, with interspecific genetic distance values ranging between about 0% and 7%, and 0% and 5%, respectively. These two markers distinguished spice species from the closest taxa of all the genera tested except for oregano (De Mattia et al. 2011). Bar-HRM has also been used to detect the adulteration of olive oil with canola oil at a level of 1% (w/w) of canola oil in olive oil. This technique is an accurate, faster, and less expensive method to authenticate vegetable oils, such as olive oil, and to detect mixtures of oils (Ganopoulos et al. 2013). The nano-RT-PCR technique has been used to detect the authenticity of edible oils. The amount of DNA extracted from edible oils and adulterated edible oils is evaluated by RT-PCR with different amplification fragments and nano-RT-PCR. Soybean oil (10  mL) in a background of 40  mL sesame oil was detected by the technique. Gold colloid increased the efficiency and precision of PCR (He et al. 2013). DNA markers have proved to be helpful in the authentication of food even in complex matrixes, such as vegetable oil. DNA markers have been used to authenticate the edible olive oil cultivar identification (Costa et al. 2012). DNA barcoding has been used to identify the plant origins of processed honey. Four multifloral honeys produced in the northern Italian Alps were analyzed using the rbcL and trnH-psbA plastid regions as barcode markers. Thirty-nine plant species were identified in the four honey samples, of which one endemic plant was found in four honey samples, authenticating the geographic identity of the products, and also the DNA of the toxic plant Atropa belladonna  was detected in one sample. Thus, DNA barcoding apart from authentication is also useful in evaluating the safety of honey (Bruni et al. 2015). The DNA barcoding approach combining universal primers and massive parallel pyrosequencing was used to assess plant diversity and the geographical origin of honey. Two commercial honeys, one of a regional origin and the other containing a mix of different honeys, were analyzed in a fast, simple-to-implement, more robust method than the classical methods (Valentini et al. 2010). 15.4.2 Metabolomics The various metabolomic approaches used in the detection and identification of fraud in different types of food products are discussed in the following

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sections. Table 15.2 gives an overview of the various analytical techniques with metabolomic approaches used in food fraud detection. 15.4.2.1  Plant Products Significantly more metabolomic studies related to food authenticity and integrity have been conducted in recent years. Kopi Luwak, an exotic Indonesian coffee, is made from coffee berries that have been eaten by the TABLE 15.2 Metabolomic Techniques Used in Detection of Food Fraud Detection Techniques  GC-MS-based multimarker profiling 1H NMR and chemometrics DRIFTS and chemometrics 1H NMR (qHNMR) LC-HRMS Proton transfer reaction MS FT-MIR FT-IP chemometrics with LC-HRMS High-resolution MAS NMR UPLC-QTOF MS APCI with GC coupled to QTOF MS DART-TOF MS DART-Orbitrap HRMS QTOF MS Colorimetric SERS with gold nanoparticle HILIC-ESI/TOF/MS Multicommuted flow system DART-HRMS

GC-MS and UHPLC-MS 1H NMR LESA MS LC-Orbitrap-HRMS

Food 

References 

Original and fake coffee (Kopi Luwak)

Jumhawan et al. 2013

Adulterant in saffron

Petrakis et al. 2015

Adulterant in saffron Sudan I– IV dyes in saffron Identification of PDO saffron Differentiation of old and new saffron Quality of saffron and mislabeling Fraud in herbs and spices

Petrakis et al. 2017 Petrakis et al. 2017 Rubert et al. 2016 Nenadis et al. 2016 Ordoudi et al. 2014 Black et al. 2016a

Origin of Interdonato lemon Adulteration of fruit juices Differentiation of quality of olive oil

Cicero et al. 2015 Jandric et al. 2014 Sales et al. 2015

Detection of adulteration of hazel nut oil in olive oil Differentiation of Chinese and Japanese star anise Adulteration in honey Melamine in milk

Vaclavik et al. 2009

Melamine in infant formula Adulteration in milk

Inoue et al. 2015 De Souza et al. 2014

Differentiation of milk of farm animal species Detect vegetable oil in dairy products Testing of different grades of beef mince and pork mince Detection of horsemeat in beef Detection of pork, horse, turkey, or chicken meat in beef Detection of process contaminants and toxins in food

Hrbek et al. 2014

Shen et al. 2012 Wu et al. 2017 Lang et al. 2015

Trivedi et al. 2016 Jakes et al. 2015 Montowska et al. 2014 Senyuva et al. 2015

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Asian palm civet (Paradoxurus hermaphroditus ). Gas chromatography– mass spectrometry (GC-MS)– based multimarker profiling differentiated original, fake Kopi Luwak, regular coffee, and coffee blend samples with 50 wt% Kopi Luwak content (Jumhawan et al. 2013). 1H nuclear magnetic resonance (NMR) and chemometrics were used to identify adulterants in saffron. The techniques could identify bulking agents in saffron, that is, Crocus sativus  stamens, safflower, and turmeric. A two-step approach with the application of both orthogonal projections to latent structures– discriminant analysis (OPLS-DA) and Orthogonal-orthogonal Partial Least Square-Discriminant Analysis (O2PLS-DA) models to the 1H NMR data could detect authentic and adulterated saffron. It can be used to assay extensive saffron fraud at a minimum level of 20% (w/w) (Petrakis et al. 2015). Diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS) and chemometric techniques have also been used for testing the adulteration of saffron with six adulterants: C. sativus  stamens, calendula, safflower, turmeric, buddleja, and gardenia. The three-step process can detect and quantify adulteration. It showed 99% correct classification of pure saffron and saffron adulterated at 5%– 20% (w/w) levels and detection limits ranging from 1.0% to 3.1% w/w (Petrakis and Polissiou 2017). An NMR-based approach was used to identify and determine the adulteration of saffron with Sudan I– IV dyes. 1H NMR (qHNMR) was used to detect and quantify Sudan III in varying levels of 0.14– 7.1  g/kg (Petrakis et al. 2017). LC coupled with high-resolution mass spectrometry (HRMS) was used to differentiate saffron cultivated and packaged in Spain, PDO, and saffron packaged in Spain of unknown origin, labeled Spanish saffron. The markers identified by metabolic fingerprinting were glycerophospholipids and their oxidized lipids (Rubert et al. 2016). The nondestructive technique, proton transfer reaction– mass spectrometry (PTR-MS), has been used in quality control of saffron. By monitoring the production of volatile organic compounds (VOCs), fresh saffron and old saffron of poor quality could be easily identified (Nenadis et al. 2016). The Fouriertransform midinfrared (FT-MIR) technique has been applied to assess the quality of saffron, as well as fraud and mislabeling. FT-MIR analysis showed that high-quality samples according to ISO 3632 specifications produced a typical spectrum profile (Ordoudi et al. 2014). The FT-IR screening method coupled to data analysis using chemometrics and a second method using LC-HRMS have been developed to detect fraud in herbs and spices. The twotier testing strategy applied to 78 samples showed adulteration in 24% of all samples tested (Black et al. 2016a). High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) has been used for the metabolic profile of the famous Sicilian lemon known as “ Interdonato Lemon of Messina PGI.”  HR-MAS NMR spectroscopy has been used to analyze and compare the molar concentrations of the main metabolite constituents in the juices of the PGI Interdonato Lemon of Messina and the non-PGI Interdonato Lemon of Turkey. The analytical technique by metabolic fingerprinting can reveal commercial fraud in national and global markets (Cicero et al. 2015).

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Similarly, potential biomarkers for the rapid detection of the adulteration of fruit juices (pineapple, orange, grapefruit, apple, clementine, and pomelo) with cheaper alternatives with the ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS) technique have been developed. The targeted metabolomics method was used to detect adulteration at 1% (Jandric et al. 2014). The novel atmospheric pressure chemical ionization (APCI) source in combination with GC coupled to hybrid QTOF MS has been used for the determination of volatile components of olive oil for the classification of olive oil samples. VOCs in olive oil samples, including extra virgin, virgin, and lampante qualities, have been detected. The analysis used three different steps: a full mass spectral alignment of GC-MS data using MzMine 2.0, a multivariate analysis using Ez-Info, and a statistical model. The technique was validated using blind samples and obtained an accuracy in oil classification of 70% using the official “ panel test”  as reference (Sales et al. 2015). Direct analysis in real time (DART), coupled to a high-resolution time-of-flight mass spectrometer (TOF MS), has been used to authenticate olive oil of varying quality grades by comprehensive profiling of triacylglycerols and polar compounds. Using DART-TOF MS, differentiation among EVOO, olive pomace oil, and olive oil was easily achieved, and the detection of EVOO adulteration with hazelnut oil was also possible. Hazel nut oil at additions of 6% and 15% (v/v) could be easily detected (Vaclavik et al. 2009). Chinese star anise (Illicium verum ) contaminated or adulterated with the toxic Japanese star anise (Illicium anisatum ) or other Illicium  species can be detected with DART ambient ionization coupled to Orbitrap™  -HRMS. The metabolite anisatin signal was > 1000 times larger in Japanese star anise than in Chinese star anise, and thus could be easily determined by DART, and adulteration at 1% (w/w) was measurable (Shen et al. 2012). 15.4.2.2 Honey Apart from common analytical techniques, such as High-performance Anion Exchange Chromatography (HPAEC), GC, and HPLC, used for the detection of syrup adulterants in honey, advanced techniques, including infrared (IR), NMR, and Raman spectroscopy, which enhance the analysis process for larger numbers of samples, have also been used. In recent years, QTOF MS has been used as a metabolomics-based method for detecting complex adulterants in honey (Wu et al. 2017). 15.4.2.3  Milk and Milk Products Nanotechnology-based surface-enhanced Raman spectroscopy (SERS) analyses have been used for the rapid screening and validation of melamine in milk. The colorimetric SERS method uses 30  nm Au nanoparticles to rapidly screen and validate 0.25  ppm melamine in milk in 20  min (Lang et al. 2015).

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Hydrophilic interaction liquid chromatography (HILIC)/TOF/MS and multivariate statistical analysis methods have been used to detect contamination in infant formula. The technique can detect the contamination of melamine and its degradation in infant formula (Inoue et al. 2015). The addition of potassium dichromate, salicylic acid, hydrogen peroxide, and starch in milk is considered adulteration in Brazil. A multicommuted flow system for the sequential screening or determination of dichromate, salicylic acid, hydrogen peroxide, and starch in milk samples developed based on a simple binary “ detect”  or “ no detect”  response could determine analytes quickly, with high performance and easy operation (De Souza et al. 2014). The DART ambient ionization technique coupled with HRMS has been used in the authentication of milk and dairy products. It has been used to discriminate milks obtained from various farm animal species, and milk produced in conventional and organic farming, and to detect vegetable oil in dairy products, such as soft cheese. DART-HRMS distinguished a milk adulteration level of 50% (v/v) and detected the presence of vegetable oils (rapeseed, sunflower, and soybean), added to soft cheese at amounts as low as 1% (w/w) (Hrbek et al. 2014). 15.4.2.4  Meat and Meat Products Different grades of beef mince and pork mince have been tested for adulteration using GC-MS and ultra-high-performance liquid chromatography– mass spectrometry (UHPLC-MS). GC-MS investigates metabolites involved in primary metabolism, and UHPLC-MS uses reversed-phase chromatography to detect lipophilic species. The combined chemometrics and statistical analyses revealed a number of differential metabolites in the two meat types that can be used for meat authentication and labeling (Trivedi et al. 2016). 1H NMR spectroscopy has been used to distinguish beef from horsemeat based on comparison of triglyceride signatures. A simple chloroform-based extraction was used to obtain classic low-field triglyceride spectra in a 10  min acquisition time. Peak integration was sufficient to differentiate samples of fresh beef (76 extractions) and horse (62 extractions). 1H NMR (60  MHz) represents a feasible high-throughput approach for screening raw meat (Jakes et al. 2015). Liquid extraction surface analysis– mass spectrometry (LESA-MS) has been used to analyze five cooked meats— beef, pork, horse, chicken, and turkey— by assessing peptides from samples. The technique could be used to detect 10% (w/w) of pork, horse, and turkey meat and 5% (w/w) of chicken meat in beef. LESA-MS is a faster and simpler tool for meat speciation (Montowska et al. 2014). LC-Orbitrap-HRMS has been increasingly used in food analysis. It has been applied in the analysis of bioactive substances, principally phenolic compounds in foods and various conjugated forms of known mycotoxins. Novel process contaminants were also identified by LC-Orbitrap-HRMS, as well as substances used for food. Untargeted analysis is seen as a major future trend where HRMS plays a significant role (Senyuva et al. 2015). Ambient mass spectrometry (AMS) has been increasingly used in

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the food industry and by regulators globally for detecting food adulteration (Black et al. 2016b). 15.4.3 Proteomics Proteome analysis has been applied to find new marker proteins and peptides to improve the development of assays for detecting adulteration and fraudulent practices (Ortea et al. 2016). The various proteomic approaches used for food fraud detection are discussed in Table 15.3. TABLE 15.3 Proteomic Techniques Used in the Detection of Food Fraud Detection Techniques  Isoelectric focusing MALDI-TOF MS

QTOF MS UHPLC MALDI-TOF MS 2DE LC-MS/MS LC-QTOF MS

MRM-MS Isoelectric focusing Proteome-wide tandem MS Query tandem MS MALDI-TOF Combinatorial peptide ligand libraries Capillary LC-ESI-MS/MS

HRMS

Food  Detection of cow milk in ewe and goat cheese Detection of cow milk in ewe and water buffalo cheese, and in mozzarella from water buffalo Adulterant soy and pea protein in skim milk Adulterants in skim milk Adulteration of fresh milk with powdered milk Identification of animal species in meat Soybean adulteration in milk Differentiation of porcine meat from chicken and beef meat Detection of mixing of beef, pork, horse, and lamb Substitution of high-value fish with low-value fish Substitution of high-value fish with low-value fish Authentication and recognition of fish species Wine authenticity and traceability Detection of fining agents in wine Detection of allergenic proteins used as fining agents in wine Detection of allergenic proteins and egg whites used as fining agents in wine

References  Spoljaric et al. 2013 Cozzolino et al. 2001, 2002

Cordewener et al. 2009 Jablonski et al. 2014 Calvano et al. 2013 Montowska and Pospiech 2012 Leitner et al. 2006 Sarah et al. 2016

Watson et al. 2015 Renon et al. 2005 Barik et al. 2013 Wulff et al. 2013 Chambery et al. 2009 D’ Amato et al. 2010 Monaci et al. 2010

Monaci et al. 2013

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15.4.3.1  Dairy Products Ewe or goat cheese products with a minimum of 1% of cow’ s milk are considered adulterated. The proteomic technique has been used to detect cow’ s milk in ewe and goat cheeses to prevent adulterations and imitations. The method includes the isolation of casein from cheese and isoelectric focusing of γ 2- and γ 3-casein on hydrolysis of β -casein by plasmin. The detection and quantitative determination of γ -casein in cow, ewe, and goat cheese by densitometry can be used to detect and quantify the use of cow’ s milk for ewe and goat cheese production (Spoljaric et al. 2013). The presence of cow’ s milk in either raw ewe or water buffalo milk samples employed in industrial processes and the addition of powdered milk to samples of fresh raw milk have been detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Adulteration is detected by evaluating the protein patterns of whey proteins, α -lactalbumin, and β -lactoglobulin used as molecular markers (Cozzolino et al. 2001). MALDI-TOF MS is also used to differentiate mozzarella made from water buffalo milk and from mixtures of less expensive bovine using whey proteins, α -lactalbumin, and β -lactoglobulins as molecular markers (Cozzolino et al. 2002). A nontargeted protein identification method has been used to test adulterants such as plant proteins in skim milk powder. The LC-MS method using a QTOF MS instrument has been used to identify adulterant protein isolates of soy and pea in skim milk. To identify the plant proteins present in the adulterated skim milk powder, data-dependent LC-MS/MS, in combination with a list of differential peptides, was used (Cordewener et al. 2009). Similarly, using UHPLC with 215  nm detection, chromatographic profiles of skim milk powder and mixtures of it with soy, pea, brown rice, and hydrolyzed wheat protein have been obtained. Adulterations up to 1% and 3% levels could be detected (Jablonski et al. 2014). MALDI-TOF MS has been used to analyze tryptic digests from raw liquid (without heat treatment), commercial liquid, and powdered cow milk. Two-dimensional gel electrophoresis (2DE) was used initially to differentiate liquid and powder milk, and further adulteration with powdered milk was confirmed by MALDI analysis of the in-gel digested proteins. Milk samples adulterated with different percentages of powdered milk and diagnostic peptides were detected at the 1% adulteration level (Calvano et al. 2013). 15.4.3.2 Meat Myosin light chains (MLCs) have been used as markers in the authentication of meat products. MLCs from mixtures of minced meat, frankfurters, and sausages made from cattle, pig, chicken, turkey, duck, and goose were analyzed by 2DE. Species-specific patterns of MLC isoforms were observed in all the mixtures and processed meat products. Species identification of the meat in the samples was possible when the meat content of one species was not lower than 10 (Montowska and Pospiech 2012). Soybean proteins

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are added to processed meat products for economic gain and to improve their functional properties. Chromatographic prefractionation on the protein level by perfusion LC has been used to isolate peaks of interest from extracts of soybean protein isolates and of meat products containing it. By nanoflow LC-MS/MS, different glycinin A subunits could be identified from the peak, discriminating between samples with and without soybean proteins. The protein subunit glycinin G4 subunit A4 can be used as a marker to detect soybean protein adulteration in meat (Leitner et al. 2006). Using LC-QTOF MS, porcine-specific peptide markers were identified to differentiate pork from beef, chevon, and chicken meat. Seven porcine-specific peptides derived from lactate dehydrogenase, creatine kinase, and serum albumin protein were detected. Meat species identification at the peptide level is a robust platform for the authentication of meat products such as halal (Sarah et al. 2016). A rapid multiple reaction monitoring (MRM) mass spectrometric method for the detection and quantitation of the adulteration of meat with undeclared species has been carried out. Corresponding proteins from the different species and corresponding peptides from those proteins (CPCP) were used. The approach allowed the identification of four meat types, beef, pork, horse, and lamb, and could also detect meat mixing at 1% (w/w) (Watson et al. 2015). 15.4.3.3 Fish Isoelectric focusing, a simple and reliable technique, was used to detect fraudulent substitution of cold-smoked fillets of swordfish (Xiphias gladius ) with low-value blue marlin (Makaira mazara ) and blue marlin steaks with Mediterranean spearfish (Tetrapturus belone ). Sarcoplasmic proteins of the fish species have been used to differentiate fish species (Renon et al. 2005). Proteomics has been used to differentially characterize sarcoplasmic peptides of the closely related fish Sperata seenghala  and Sperata aor  (Barik et al. 2013). Proteome-wide MS/MS has been used for species recognition and authentication of fish species. The technique includes protein extraction, digestion, and data analysis. A set of reference spectral libraries is generated for unprocessed muscle tissue from 22 different fish species. Query MS/MS data sets from “ unknown”  fresh muscle tissue samples are searched in the reference libraries. The number of matching spectra can be used to identify the origin of fresh samples. The method can also identify fish species of heavily processed samples (Wulff et al. 2013). 15.4.3.4 Wine MALDI-TOF has been used for profiling peptides from tryptic digests of whole wine proteins. Peptide fingerprints have been obtained for high-quality Campania white wines. Thus, MALDI-TOF can be used to detect wine authenticity and traceability (Chambery et al. 2009).

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Combinatorial peptide ligand libraries (CPLLs) have been used in identifying traces of proteins present in red wines. This technique was used to identify the fining agent used in red wine as bovine casein instead of egg albumin. It had a lower detection limit of 1  μ g/L and could detect 3.8  μ g/L of casein (D’ Amato et al. 2010). Capillary liquid chromatography combined with electrospray ionization– tandem mass spectrometry (CapLC-ESI-MS/ MS) has been used for the detection and identification of casein-derived peptides in fined white wine. The technique is useful in detecting potentially allergenic milk proteins used as fining agents in wine. Peptides arising from α - and β -casein could be detected in white wine fined with casein at 100 and 1000  μ g/mL (Monaci et al. 2010). HRMS has been used to determine various fining agents, such as allergenic milk (casein) and egg white (lysozyme and ovalbumin) proteins in commercial white wines simultaneously, at sub– parts per million levels. The LODs of this technique were in the range of 0.4– 1.1  μ g/mL (Monaci et al. 2013).

15.5  Prevention of Food Fraud Food fraud can be prevented by traceability and authentication of food products and managing the supply chain and procurement process. 15.5.1 Traceability Traceability is to know the origin of product, where it has been, where it is going, and its present location (Spink 2012). Traceability helps to find and monitor the product’ s movement through the supply chain. Traceability can be used to identify when and where a genuine product can be or has been replaced with a fraudulent product. On identifying food fraud, traceability helps in product recall from the marketplace. Some of the tracing techniques are by digitally tracking the product or using readable codes, such as barcode and radiofrequency identification (RFID). Pedigree is a related system that records the history of the product from manufacturing to sale (Dietrich et al. 2006). 15.5.2 Authentication Authentication is to know if a product is original or counterfeit (Spink 2012). The authenticity of a product can be constantly evaluated by continuous or operational authentication. This includes sorting of the product in a warehouse by using numerical identifiers, such as batch, product code, or serial number. The product can also be authenticated on an occasional basis by spot or “ sales,”  by basic monitoring, and sometimes during a specific

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investigation. The consumer or investigator can spot-check to authenticate a product by its numerical identifier (Spink 2009). 15.5.3  Supply Chain Management and Procurement Strict following of supply chain and procurement practices can reduce food fraud incidences. Certain business practices create fraud opportunities in the supply chain. Usually, counterfeit, adulterated, or tampered products enter into sales through the supply chain. During procuring a product, it is essential to verify the source and how it is obtained (Closs and McGarrell 2004; Closs and Mollenkopf 2004; Closs et al. 2008).

15.6  Regulation of Food Fraud: Laws and Legislation 15.6.1  Food Fraud Regulation in the United States The United States passed the Food Safety Modernization Act (FSMA) in January 2011, followed by the publication of the FSMA Preventative Controls (FSMA-PC) rule in September 2015. The FSMA-PC rule contains direction regarding food fraud and EMA (FDA 2009; Spink 2009). The FSMA-PC rule does not limit economically motivated hazards to only adulterant substances in food, but covers any economically motived hazard and addresses prevention of all food safety hazards. There is a section of FSMA that addresses only “ smuggled foods.”  The Food, Drug, and Cosmetic Act of 1938 (FD&C) is in effect and includes violations of the “ Adulterated Foods”  and “ Misbranded Foods”  sections, wherein adulterated foods need not contain an adulterant but can be stolen or spoiled food. The Government Accountability Office (GAO) and Congressional Research Service (CRS) have published two significant U.S. government reports regarding food fraud and emphasized that the FDA would address and prevent all types of food threats. 15.6.2  Food Fraud Regulation by the European Commission The EC has been addressing food fraud within the food integrity focus area after the horsemeat adulteration scandal in 2011 (EP 2013). The EC has clearly defined food fraud and focuses on its prevention (EC 2015). This led to the creation of the Food Fraud Network of government agencies, which share information and intelligence on fraud incidents, and expansion of the EC-wide RASFF food recall system to include adulteration and fraud. The EC has funded the € 12 million Food Integrity Project to protect the European market, with € 9 million for core activities and € 3 million for new research. Although it mainly focuses on food integrity, such as meeting quality and PDO, it also includes food fraud.

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15.6.3  Food Fraud Regulation in the United Kingdom The UK FSA (FSA-UK) works in close collaboration with Ireland (FSA-IRE). The DEFRA has funded Professor Christopher Elliott of Queen’ s University Belfast to review food fraud incident. The Elliott Review guides the UK government to address food crime and food fraud (Elliott 2014). 15.6.4  Food Fraud Regulation in China The Chinese government has a new food safety law that went into effect in October 2015. It expands the Chinese food regulatory system and provides more comprehensive control of the food supply chain, as well as funds research on food safety by the Chinese National Center for Food Safety Risk Assessment (CFSA). The food safety law defines “ traditional”  and “ nontraditional”  food safety, including food defense and food fraud. The CFSA has a broad definition of food fraud and prioritizes human health hazards due to adulterant substances and counterfeit products. 15.6.5  Food Fraud Regulation: Industry Standards and Compliance There are a range of industry or nongovernmental activities relating to food fraud regulation. 15.6.5.1   Global Food Safety Initiative The GFSI is an industry association. It is created to harmonize a food safety management system. The GFSI approves audit schemes, and the scheme owner creates a standard that supports the audit scheme. Food producers follow and implement the standard. A third-party auditor certifies that the company is in compliance with the standard. These result in the producer being “ GFSI certified”  since the GFSI approved the scheme, empowered the scheme owner to set the standards, and approved the auditors. The GFSI has also created a Food Fraud Think Tank (FFTT) to review and make recommendations on the issue. It has come out with a GFSI’ s Food Fraud Position Paper that defined all types of food fraud and focused on its prevention. The second issue proposed is to include a requirement for a food fraud vulnerability assessment and food fraud prevention plan (GFSI 2014). 15.6.5.2  Safe Supply of Affordable Food Everywhere Safe Supply of Affordable Food Everywhere (SSAFE) is an industry group. It is developing a semiquantitative food fraud vulnerability assessment that covers most, but not all, types of food fraud. The gray market production, theft, and diversion are not included, but IPR violations of food counterfeiting are covered by SSAFE (SSAFE 2012).

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15.6.5.3  U.S. Pharmacopeia The U.S. Pharmacopeia (USP) is a standards-setting body that has been followed by countries— including the United States— since the late 1800s. The USP has created the Food Ingredient Intentional Adulteration Expert Panel to address food fraud adulterant substances and to create a food fraud database that includes adulterant substance detection techniques and substances published in scholarly journals. The expert panel has also made recommendations regarding food ingredient adulterant substances, including vulnerability assessments (USP 2009). 15.6.5.4  Other Activity The International Organization for Standardization (ISO) has also conducted activities on food fraud (ISO 2010). The U.S. National Center for Food Protection and Defense (NCFPD), a Department of Homeland Security Center of Excellence, has hosted databases such as the Economically Motivated Adulteration Database (NCFPD 2015).

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16 Food Safety Regulation and Standards Nada Smigic and Ilija Djekic CONTENTS 16.1 Introduction................................................................................................. 531 16.2 Food Safety Issues...................................................................................... 532 16.3 Food Safety Regulation .............................................................................534 16.3.1 Food Safety Regulation at the International Level....................534 16.3.2 Food Safety Regulation in the European Union and the United States����������������������������������������������������������������������������������� 536 16.3.3 Food Safety Regulation in Developing Countries..................... 541 16.3.4 Developed versus Developing Countries ...................................542 16.4 Food Safety Standards ..............................................................................544 16.4.1 Characteristics of Food Safety Standards...................................545 16.4.2 Main Groups of Requirements.....................................................546 16.4.2.1 Prerequisite Programs and Good Practices................. 547 16.4.2.2 Hazard Analysis............................................................... 547 16.4.3 Food Safety Management.............................................................. 549 16.4.4 Effects of Implemented Food Safety Standards......................... 550 16.4.5 Food Safety Audits......................................................................... 551 16.4.5.1 Types of Food Safety Audits........................................... 552 16.5 Final Remarks.............................................................................................. 554 References.............................................................................................................. 554

16.1 Introduction The growing importance of food safety in international trade is influencing the importance of developing and implementing both legal requirements and international standards. Food safety regulations comprise general principles for three main stakeholders— public authorities, food establishments, and consumers. In defining the food safety framework, legislation is distributed via vertical and horizontal approaches. Enforcement of legal requirements by public authorities is overseen through scientific-based risk assessments, official controls and inspections, and foodborne outbreak (crisis or incident) management. On the other side, food establishments throughout the food chain need to 531

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implement requirements laid within product-based and establishment-based food safety requirements. As an outcome, consumers worldwide expect to purchase and consume safe food. International food safety standards are developed for food establishments and are basically implemented on a voluntary basis. Their main features are that they are applicable to all types of food establishments, regardless of size, that are involved in any aspect of food. The majority of food safety standards are generic, although there is a trend in developing tailored standards for specific food industries. In spite of the fact that there are several international organizations that publish various types of food safety standards, they all have three groups of requirements in common: (1) prerequisite programs (PRPs) and good practices, (2) hazard analysis, and (3) food safety management. In order to verify their implementation, three types of audits are present in assuring stakeholders that food safety requirements are implemented. Depending on the role of the organization requesting an audit, the auditee, and the auditors, audits are classified as internal audits (conducted by the organization itself), second-party audits (conducted by the customer or other organization having an interest), and third-party audits (conducted by independent auditing organizations). This chapter gives an overview of these two important pillars in ensuring food safety throughout the food chain.

16.2  Food Safety Issues Today, consumers require high-quality food products in broad assortments throughout the year and for competitive prices. At the same time, they are aware and well informed of all different aspects of food quality and safety. Last but not least, the public also expresses high interest in food-related scandals, such as dioxin crises, “mad cow”  diseases, the Chinese melamine case, various bacterial contaminations of food, toxic chemicals in food, and irradiation and other new technologies (Kä ferstein and Abdussalam 1999). It seems that food safety is receiving more and more attention nowadays, with the consumers’  perception of food safety issues being more negative than before (Bá ná ti 2011). On the other side, the global food supply chain is becoming very complex and internationalized. Food producers are using ingredients from all around the world, transforming the complete food industry into a very complex interconnected system with many different relationships (Trienekens and Zuurbier 2008). Food goes through extensive processes from the farm, via the food producer, to the point of consumption. At each step along the food chain, there is a great chance of food being contaminated with chemical, physical, or microbiological hazards. In addition, there is an increasing

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trend of food ingredients and processed food movement between developing and developed countries, which poses numerous challenges in ensuring the safety of the food (Zach et al. 2012). Thus, rapid globalization has increased the chance of food contamination, and showed some critical gaps in international and national capacity to ensure an adequate level of food safety. Important to note is the fact that food safety issues occurring in developing and developed countries are different. Developed countries often experience food safety problems related to new technologies that are applied, or new materials used in agriculture or food processing. On the other side, the developing countries are still in battle with poor hygiene issues in food production and processing. Both chemical and microbiological contaminations are often seen as on ongoing challenge for food safety. Due to globalization of food supply, food safety problems from developing countries are rapidly become international problems. Despite obvious advances in food science and technology, foodborne illnesses remain an important cause of morbidity and mortality worldwide, but also an important obstacle for socioeconomic development. Foodborne diseases are caused by biological, chemical, and physical hazards that are transmitted though ingested food. Hazards can contaminate food in any step throughout the food chain. Most often, the reported number of foodborne diseases is related to bacterial or viral hazards transmitted via food, as the symptoms of illness are quickly seen and often connected with the cause. On the other side, the burden arising from chemicals and parasites is still unknown. The negative health effects of chemicals may not be observed for years following the exposure (e.g., aflatoxin and liver cancer, lead and cardiovascular disease). In addition, the relevant disease end points related to foodborne chemical hazards may easily arise from many different causes, which create even more complicated estimates of the incidence and mortality (WHO 2015). In recent decades, various scientific studies have been performed in the field of food safety, which were followed by the number of preventive and control measures implemented in the food industry. Nevertheless, epidemiological data from developed countries (the United States, the European Union [EU], Canada, and Australia) indicated that the number of food safety issues and foodborne illnesses remains at a high level (Newell et al. 2010; Havelaar et al. 2010; Mead et al. 1999; EFSA and ECDC 2015). Data from some developing nations do not show the same trend, due to inadequate foodborne illness monitoring and surveillance systems (WHO/FAO 2005; Akhtar et al. 2014). The responsibility for food safety primarily lies on food producers and processors. However, governmental institutions and consumers share some responsibility. Governments should adopt and enforce food safety legislation in the first place, but they also have to interpret inspection data. They are responsible not only for providing adequate medical support for treating foodborne illnesses, but also for gathering and using all epidemiological and foodborne outbreak data (Griffith 2006).

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16.3  Food Safety Regulation The aim of food regulation is to protect the consumer’s health, to increase economic viability, and to harmonize well-being and allow fair trade of foods within and between nations (Aruoma 2006). It is outlined to apply control over all types of food produced, processed, and sold on the market, so that the consumers are guaranteed that the food will not cause any harm within the limits of available scientific knowledge. In most countries, food regulation includes a great number of different laws and ordinances. They set out the government’s requirements to be met by food business operators to ensure that food is safe and of adequate quality. Within the food regulation, different aspects of production, trade, and food handling are included, simultaneously covering food control, food safety, and relevant aspects of food trade. Currently, there are a number of different food safety regulation systems worldwide, some of them well designed and risk based, with a long history of developments and improvements (developed countries), while others are still in the initial phase (developing and/or undeveloped countries). The common platform for food safety regulation lies in the documents adopted and prepared by various international bodies. Nevertheless, the individual member nations may adopt, modify, or make their own food regulations.

16.3.1  Food Safety Regulation at the International Level On the international level, the Food and Agriculture Organization of the United Nations (FAO), the World Health Organization (WHO), and the World Trade Organization (WTO) are major organizations that deal with food safety issues. The impact of WTO on food safety is mainly seen through two agreements: Agreement on Sanitary and Phytosanitary Measures (SPS Agreement) and Agreement on Technical Barrier to Trade (TBT Agreement). Under the SPS Agreement, the WTO sets constraints on member states’  policies relating to food safety, as well as pests and diseases associated with animal and plant health. The definition of sanitary and phytosanitary measures is given in the SPS Agreement as a measure that is applied to protect animal or plant life or health within the territory of the Member from risks arising from the entry, establishment or spread of pests, diseases, disease-carrying organisms or disease-causing organisms; to protect human or animal life or health within the territory of the Member from risks arising from additives, contaminants, toxins or disease-causing organisms in foods, beverages or feedstuffs; to protect human life or health within the territory of the Member from risks arising from diseases carried by animals, plants or products thereof, or from the entry, establishments or spread of pests;

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or to prevent or limit other damage within the territory of the Member from entry, establishments or spread of pests. (WTO 1995)

Within the SPS Agreement, governments have been encouraged to harmonize national measures consistent with international standards, guidelines, and recommendations. In 1962, the FAO and WTO established the Codex Alimentarius Commission to operate as an umbrella organization for policy making regarding different aspects of food on a global level. Codex standards, guides, and recommendation have been cited and recommended in the SPS Agreement, as the most adequate measures for facilitating international food trade. The SPS Agreement adopts codex standards for food additives, veterinary drugs and pesticide residues, contaminants, methods and analysis of sampling, and codes and guidelines of hygiene practices. Therefore, they have been placed as a target against which national food measures and regulations should be enacted and enforced. Where the measures are conformed to international standards (such as codex standards), they are to be considered consistent with the SPS Agreement. The Codex Alimentarius Commission adopted one of the most important guidelines on good hygiene practices in order to prevent, control, and decrease food safety risks (CAC 2003). The SPS Agreement permits individual nation states to take legitimate measures to protect the life and health of consumers given the level of risk that they deem to be “acceptable,”  applying measures that can be justified scientifically and do not unnecessarily hamper food trade. However, they are required to recognize that measures adopted by other countries, although different, can provide equivalent levels of protection. According to the SPS Agreement, WTO members are obliged to apply measures to the extent necessary, and these measures must be scientifically proven and/or outlined in international standards. The EU faced this requirement in the dispute related to hormone-treated beef meat exported from the United States. In 1998, the European Commission prohibited the importation and placement on the market of meat and meat products treated with certain hormones originating from the United States, and as a consequence, the United States complained to the WTO. As there was not enough scientific evidence that hormone-treated meat might present a risk to human health, the United States was found to be right (WHO 1996). Nevertheless, this is still an ongoing issue (Johnson and Hanrahan 2010). The sanitary and phytosanitary measures should not represent a hidden restriction on international trade (WTO 1995). WTO members are also required to recognize sanitary and phytosanitary measures adopted by other countries as being equivalent, if they can demonstrate that measures provide an appropriate level of health protection. Although there are many unclear issues related to the level of equivalence or appropriate level of health, there is no doubt that the SPS Agreement has played an important role in encouraging countries to create national or

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regional regulations in line with international food standards (Neeliah and Goburdhun 2010). Some countries, mainly the developed ones, have seen many benefits, and their food safety legislation is mostly in line with the requirements given in these international agreements. Nevertheless, developing countries are still lagging behind, struggling with a slow implementation process (Das 2008; Neeliah and Goburdhun 2010). As Hooker (1999) indicated in his article, the SPS Agreement has put a heavy burden on developing countries to be in compliance with the international regulations, with the problems related to infrastructure, together with the lack of experts, laboratory resources for testing, and fragmented and uncoordinated structure of the food control system at the national level. 16.3.2 Food Safety Regulation in the European Union and the United States The EU is an economic and political union of 28 member states located in Europe. This great single market is one of the most important food export and import world trade players. The current food legislation that is in place in the EU presents the result of developments and various changes toward the production and marketing of safe food, over a long period of time. In the early nineties, several food safety crises, such as those related to bovine spongiform encephalopathy (BSE), dioxins, high pesticide and antibiotic contents in several foods, and the usage of Sudan Red 1, occurred in the EU. The panic situation related to food safety had driven the European Commission to include food safety among its major priorities and revise the EU food safety system in order to return the loss of consumers’  trust. One of the most important steps in this direction was the publication of a white paper in 2000 (EC 2000), in order to introduce consistency and clarity throughout the food production chain “from the farm to the fork.”  At that time, the white paper presented a new and more risk-based approach to food safety. Consequently, in 2002, the general food law was adapted (EC 2002), while a set of important food safety regulations were adopted in 2004, with a 2-year period for food business operators to comply with the given requirements (EC 2004a, 2004b, 2004c). The package of food safety legislation covered all aspects of food hygiene (often called “the hygiene package” ), food safety, and quality in all stages of the food production chain, from and including primary production and the production of animal feed up to and including the sale or supply of food to the consumer. The European Food Safety Authority (EFSA) was established to effectively control and manage food crises and to protect the health of the public (EC 2002). The EFSA is an independent body with the main goal to perform scientific assessments and evaluations upon the request of the European Commission. Within the general food law, the Rapid Alert System for Food and Feed (RASFF) was established to be a tool for the rapid exchange of information on food safety issues (for both domestic and imported food). EU food legislation, for the first time, introduced

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the precautionary principle, which applies in situations where there is not enough scientific evidence, or when it is inconclusive or uncertain, which might have a negative effect on human health. The Hazard Analysis and Critical Control Point (HACCP)- based food safety system is at the heart of EU food hygiene regulation. It requires food business operators to analyze their food processes regarding the place where hazards may occur and how to deal with them in order to maintain food safety. The implementation of a HACCP system was legally obliged for all food businesses that operate within the EU, except for primary producers (EC 2004a, 2004c). This system was earlier recommended, but not mandated, by the above-mentioned guidelines on good hygiene practices issued by the Codex Alimentarius Commission (CAC 2003). This preventive food safety system does not rely on end-product testing, but focuses on the continuous control and monitoring of hygiene principles related to PRPs and critical control points (CCPs) within the production process. Although many problems have been seen in the process of implementing the HACCP system in EU countries, especially in small and medium food enterprises, the food industry had seen many benefits from its application (Smigic et al. 2012; Tomasevic et al. 2013, 2016). Despite quite stringent regulatory standards that were appointed in the EU, the European Commission and member states are still facing numerous foodborne incidents. In 2008, an outbreak related to dioxin contamination of animal feed occurred in Ireland (Casey and Lawless 2011). This crisis was resolved by the recall of all contaminated products, as a precautionary measure taken by the Irish authorities. Nevertheless, the latter risk assessment indicated that public health risk was of no concern “for this single event”  (Casey and Lawless 2011). The example of the traceability chain breakdown should also be mentioned here, as it was connected with the horsemeat scandal in 2013. Although this issue was mainly related to food fraud, rather than food contamination, the concern was the fact that horsemeat contained traces of the veterinary drug phenylbutazone, which entered the food chain (Bá ná ti 2014). This resulted in great economic loss and demonstrated the importance of proper control at all phases of the food chain. Due to its nature, food safety legislation in the EU (and in other developed countries) is focused mostly on foods of animal origin. Nevertheless, the foodborne outbreak that occurred in 2011 in Germany, caused by Shiga toxin– producing Escherichia coli  O104:H4 in fenugreek sprouts, moved attention to foods of plant origin (Buchholz et al. 2011). It affected consumers in eight countries in Europe and North America, leading to 53 deaths and significant economic losses. Although this outbreak showed fairly good performance of several legislative tools, such as the traceability chain and exchange of information within the RASFF system, ineffective communication between food safety and public health authorities was noted as a concern (McEvoy 2016). In response to this outbreak, an action plan was prepared covering issues such as (1) the risk assessment of pathogens in the food

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of nonanimal origin; (2) microbiological criteria, traceability, and certification related to the production of sprouts; (3) improvement in the dissemination of the RASFF system; and (4) training for the member states conducting the investigation and management of foodborne outbreaks (EC 2011; McEvoy 2016; Bá ná ti 2014). Therefore, the EU, with its food safety control system, has to be prepared for “unexpected”  foodborne issues, related to both food products and food hazards. In order to adequately address food safety issues, the European Commission often asks for scientific evidence from EFSA, to serve as a basis for policy making. Responsive legislation  is the term used by McEvoy (2016), which expresses the way European legislation has been evolving in the last 15 years (Table  16.1). This trend is also expected in the future. The other food safety regulatory system that is worth mentioning is the one established in the United States. After almost 70 years, the United States made a significant change in this area, adopting the Food Safety Modernization Act (FSMA) in 2011 (FDA 2011). The most important change that came with FSMA is the focus shift from reaction to prevention. Two major FSMA rules were issued in 2013, “preventive control for human food”  and “standards for produce safety,”  to be applied for both domestic and imported food (FDA 2015a, 2015c). It is of note that the preventive control approach, which is now emphasized in the FSMA, is not a novelty for the United States food legislation, as the Food Safety and Inspection Service (FSIS) has required preventive control measures to be applied for the production of meat products since 1996 (FSIS 1996). Also, the Food and Drug Administration (FDA) has required control measures for foods such as seafood since 1995 (FDA 1995) and fruit juices since 2001 (FDA 2001). Nevertheless, the adoption of FSMA and the rule on the preventive control for human food now requires the implementation of this preventive approach for all processors and manufacturers of human food, which have to develop a control plan to prevent situations in which their food products could cause foodborne illness (FDA 2015a). This control plan should include the evaluation of hazards, as well as the identification and monitoring of steps or controls to minimize or prevent hazards. Together with this plan, a company has to be prepared for any problems that might arise (action plan). Although it is not named as such, this food safety assurance system is apparently based on the HACCP system, which originated in the United States. Within the new United States food legislation, importance is given to fresh produce (nonanimal products), due to several food safety issues that have been previously experienced (Bowen et al. 2006; Lynch et al. 2009). The FDA rule “standards for produce safety” requires the application of science-based and risk-based minimum standards for the safe growing, harvesting, packing, and holding of fruits and vegetables grown for human consumption (FDA 2015c). Various food safety issues related to imported food (Table  16.1), which participate in great deal in United States food trade, were the major driving force to introduce changes in the food import legislation. FSMA brings some

Developed countries

Foodborne incidents/ imported food

Various food scares (Salmonella  in peanut butter, jalapeñ o peppers from Mexico, etc.) in the United States

Melamine in milk in China

E. coli  O157:H7 outbreak in the United States Salmonella  outbreak in the United States

Mad cow disease in EU

Driving Force for Regulatory Change  Foodborne Dioxin crises in the incidents/ EU domestic food

Consequence  Polychlorinated biphenyls (PCBs) and dioxin-contaminated batch of transformer oil entered the food chain via an animal feed mill in Belgium. This was then fed to broilers and subsequently recycled into pig feed, thus affecting poultry, eggs, pork, and bacon products. As a consequence, a set of new EU food safety legislation was adopted in 2002 and 2004. BSE crisis resulted in loss of consumers’  confidence in the safety of beef meat. This crisis was a “trigger”  to reform the existing European food safety legislation and establish new regulatory institutions across EU. E. coli  O157:H7 outbreak with contaminated hamburgers in restaurants in the western United States was the major driving force to adopt PRPs and the HACCP rule for meat and poultry. The outbreak caused by Salmonella  in peanut butter that occurred in the United States during 2007– 2008 was the first salmonellosis linked to peanut butter. This outbreak, together with others that occurred in the United States, caused a food legislation change in 2011. Melamine-tainted milk caused the death of six infants and serious illness of tens of thousands of infants in China, and consequently, a food safety law was adopted in 2009, with additional media pressure for the effective enforcement of the adopted law. Over the years, a number of different food scares connected with the food imported to the United States resulted in the change of food legislation and adoption of the Food Safety Modernization Act (FSMA). The issue is now more the responsibility of the importer, who has to perform risk-based foreign supplier verification analyses to ensure that imported foods are produced in compliance with HACCP procedures and are not adulterated or misbranded.

Driving Force for Regulatory Change in Developed and Developing Countries

TABLE  16.1

(Continued)

Zach et al. 2012; Paggi et al. 2013; FDA 2011

Broughton and Walker 2010

Sheth et al. 2011

FSIS 1996

Vos 2000; Bá ná ti 2011

References  Bá ná ti 2011; Covaci et al. 2008

Food Safety Regulation and Standards 539

Developing countries

Accession of the market in industrialized countries

West Balkan countries (Serbia, Montenegro, Bosnia and Herzegovina, and Albania) China, countries in Latin America, Asia, and Africa

Listeria monocytogenes  and Cronobacter spp 

New and emerging pathogens

Integration into the regional union, such as the EU

Food safety– related information

Scientific information

Driving Force for Regulatory Change  Melamine in milk incident in the EU

Many developing countries are exporting their often exotic food products to the market of industrialized and developed countries (such as the EU, Japan, and the United States). Due to many food safety issues in the past, a lot of pressure has been put on the developing countries’  governments to adopt adequate food safety legislation, within their food control programs.

Consequence  Due to the presence of melamine in milk, many actions have been taken worldwide. In the EU, after the EFSA gave a scientific opinion on food and feed, the current Regulation (EC) 1881/2006 was amended, giving the maximum level of melamine (mg/kg) for infant formulas and other food. As a need to obtain scientifically based information, to make decisions on a legal basis, the EU introduced EFSA. Many changes in the food safety legislative acts occurred after the EFSA published its scientific opinion, based on the available information published in scientific papers. Scientific knowledge related to, at that time, new pathogens such as L. monocytogenes, and its importance for ready-to-eat food, influenced Regulation (EC) 2073/2005 to introduce this pathogen within microbiological criteria, with which food businesses operators have to be in line. Regulation (EC) 2073/2005 lays down a food safety criterion for Cronobacter  spp. (previously classified as Enterobacter saka zakii ) in dried infant formulas and dried dietary foods for special medical purposes intended for infants below 6 months of age. The political decision of many European countries was to enter the EU. One of the first steps was to harmonize legislation with the European acquis communautaire, and this resulted in many changes in food safety legislation that occurred in these countries.

Driving Force for Regulatory Change in Developed and Developing Countries

TABLE  16.1 (CONTINUED)

Broughton and Walker 2010; Pei et al. 2011; Shukla et al. 2014; Leon and Paz 2014

Glintic 2012; Smigic et al. 2015; Antunovic et al. 2008

EC 2005

EFSA 2010, 2013

References  EFSA 2010; EC 2006

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novelties that will significantly affect the food business operators outside the United States, via American importers (FDA 2015b). This is of ultimate importance for developing economies. To facilitate the process of foreign supplier verification, the FDA has started a plan of deploying its own personnel at United States embassies around the world (Keener et al. 2014). 16.3.3  Food Safety Regulation in Developing Countries Food products originating in developing countries are often present on the global food market, and therefore their control system must be adequately harmonized with at least basic food safety regulation. In many developing countries, especially in Africa and Asia, existing food legislation is outdated, incomplete, and fails to adequately address current and emerging food safety problems, and often the principles of food safety given in the Codex Alimentarius and trade agreements have not been taken into account. Additionally, inefficient enforcement due to the lack of effective food control infrastructure and institutional capacities to ensure compliance is still a bottleneck for the full implementation. Existing food regulation does not provide clear and final responsibility of the main stakeholders involved in food safety, resulting in noncoordinated and overlapped activities. Various pieces of legislation are scattered among many governmental agencies. It is of note that recently, many improvements have been seen in developing countries, which are trying to improve their food control systems, often starting with the adoption and enforcement of food regulation (Chanda et al. 2010; Mwamakamba et al. 2012; Ghaida et al. 2014; Pswarayi et al. 2014; Al-Busaidi and Jukes 2015). Although it is expected that developing countries are doing this to protect the health of their consumers, this is most probably driven by the aspiration to participate in the global food market. In addition, often a driving force for adopting food safety requirements is the presence of multinational food companies in developing countries and the implementation of their internal good hygiene practices and requirements. The most important food importing countries operate on the “principle of equivalence”  for imported food, meaning that exporting countries must demonstrate that their production methods achieve the importing country’s level of sanitary protection and food safety. However, in some cases developing countries consider these requirements to be technical barriers in “fair trade”  since they imply best-available techniques and technologies (Henson and Loader 2001). Therefore, there is still a lot to be done to improve the position of developing countries in the international food market, and to meet the food safety requirements imposed in international agreements (Disdier et al. 2008). Currently, China is one of the most important players in the global food market. Since China’s accession to the WTO in 2001, its food imports and exports have increased, and arguments about food safety have arisen between China and its trading partners, such as the EU and the United

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States. Concern over the Chinese food safety system has increased inside and outside of China. One of the well-known foodborne outbreaks related to Chinese food is the contamination of infant formula with melamine in 2008 (Pei et al. 2011), which affected 300,000 infants and young children, 6 of whom died, in China alone. In addition, there have been many food safety issues related to Chinese aquaculture products (Broughton and Walker 2010). Driven by domestic food safety issues and willingness to participate in the global food market, China enacted a new food law in 2009, that foresees the adoption of the approach of food safety throughout the complete food chain, “from the farm to the fork”, governing the EU regulatory framework. This law is the most important piece of regulation that is used to ensure the safety and quality of food and to protect the health of consumers. It should allow better coordination between national and provincial authorities, which was recognized as a weak point in the previous regulatory system (Jia and Jukes 2013). As in the EU, the primary responsibility for food safety in China lies with food producers. The legal sanctions on food production enterprises and marketing enterprises that violate the rights and interests of consumers are enacted by this law. Still, there are many issues that have to be resolved in coming years, in order to fully implement and capture the positive results of this regulation in the Chinese food sector. As Liu (2014) indicated in his report, there are several issues that the Chinese food sector and government still have to deal with in regard to food safety. They are (1) great production value, (2) the pollution issue, (3) decentralized agricultural production and the traceability of raw materials, (4) the imbalance in rapid food production, and (5) inadequate risk communication for food safety issues. Some developing countries that are geographically closer to the EU, such as the West Balkan countries, have placed integration in the EU to be the national priority. This has been a strong driver for the change and harmonization of food safety regulation with the European acquis communautaire  (Glintic 2012; Smigic et al. 2015; Antunovic et al. 2008), and indirectly with international regulations. The intention of this legal harmonization is to allow subjects in the food chain to perform their activities according to the European regulatory structure. Despite many changes that occurred in West Balkan countries’  regulation, the major obstacles are still seen in the implementation and enforcement of adopted legislative rules (Smigic et al. 2015; Celebicanin 2012). 16.3.4  Developed versus Developing Countries Food safety regulation in developed countries has quite a long history, and is still in the process of construction and development, revision, and improvement. Drivers for this are mainly found in their own reconsideration and reevaluation of food safety issues (Table  16.1). They have better tools to monitor their food systems and provide a fairly consistent and constant supply of safe and wholesome food. On the other side, some developing countries

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High

Leaders

Suppliers

Low

Implementation of food safety regulation

are still battling with an inadequate food supply, which puts food safety in the background. Still, some of them are trying to follow a global food safety trend, due to economic, political, or other reasons. Their food safety regulation is still weak and fragile, and it will defiantly take time to be effective and strong. Figure  16.1 presents an illustration of differences that can be seen between countries regarding the level of food safety regulation adoption and implementation. Four different categories can be distinguished, namely, leaders, suppliers, proactive suppliers, and followers. This classification is made by combining a matrix developed by the Boston Consulting Group (Morrison and Wensley 1991) and food safety culture tool developed by UK Food Standards Agency (FSA 2012). Leaders are countries that strive for food safety improvements in both developing and implementing food safety regulation. Mainly developed countries (e.g., the United Kingdom, Germany, France, the United States, and Australia) can be included in this category. Usually, they work jointly with scientific institutions and introduce new and emerging food safety hazards within legislation based on scientific evidence. They also develop mechanisms for assessing the implementation level of food safety requirements. Proactive suppliers are countries that are already part of a developed market (Croatia, Bulgaria, and Romania in the EU), countries in the candidate status for EU membership (West Balkan countries), and countries trading with developed markets (China, Latin America, Russia, and countries from the Commonwealth of Independent States). Their relatively high level of adoption of food safety regulation is a result of extensive trade with developed markets. It is important to note that their level of implementation is still relatively low, due to the inadequate role of their inspection services.

Proactive suppliers

Followers

High

Low

Adoption of food safety regulation

FIGURE  16.1 Difference in the level of adoption and implementation of food safety regulation in different countries.

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Suppliers are countries with a low level of adopted legal requirements. However, the level of implementation in some sections is high, mainly driven by requirements of big multinational food companies that set a high level of food safety requirements. These food safety standards are incorporated within the food safety culture of these companies and, as such, are a part of their production process wherever they operate worldwide. Also, these countries are often producers of raw food with a low level of processing. Their inspection services express a significant lack of food safety knowledge. Followers are mainly poor, undeveloped countries with a low level of adapted and implemented food safety regulation.

16.4  Food Safety Standards Starting from the late 1990s, the proliferation and evolution of food safety standards was driven predominantly by the development of new regulatory requirements in response to consumers’  concerns about food safety, as well as scientific developments regarding the risks associated with food (Henson and Reardon 2005). Nowadays, the food chain is more complex than ever before, because of demographic, cultural, economic, and technological development (Kleboth et al. 2016). Challenges that initiated introduction of a “management”  concept within food safety are various food industry developments, such as new food technologies and new food products that induced the appearance of unknown hazards and managing risks. In addition, the globalization and development of one global market resulted in an increased need for the management of food safety. As a result, except for the Codex Alimentarius Commission, which defines basic good hygiene requirements, all new developed food safety standards have some management requirements. International trade and travel consequently increased the risk of cross-border transmission and the need for strengthening methods of food control (Van der Spiegel et al. 2005). This initiated the introduction of food safety standards related to specific parts of the food chain, such as logistics, distribution, and retail. It is important to emphasize that food safety standards are mainly intended for food establishments within the food chain and not for the product itself. Requirements of standards specify “what”  should be implemented, but do not specify “how”  the requirements are to be fulfilled. Also, the majority of food safety requirements, such as good hygiene practice, good manufacturing practice, and HACCP are similar to those given in regulations. Although the implementation of food safety standards is on a voluntary basis, various business drivers have the potential to enforce their implementation (Djekic et al. 2011; Clarke 2010). Within the food chain, supply and demand drivers are mainly behind the decision to implement a food safety assurance scheme

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(Tsekouras et al. 2002). As a result, many food retailers and/or multinational food companies require, from their suppliers, full implementation of food safety standards (Van der Spiegel et al. 2005). 16.4.1  Characteristics of Food Safety Standards Basically, there are two types of food safety standards, namely, international and private standards. International standards are developed by international organizations, such as the International Organization for Standardization (ISO), which issued ISO 22000 (ISO 2005). This food safety management standard is applicable to all organizations involved in the food chain and comprises PRPs, HACCP, and management requirements. By the end of 2014, more than 30,500 certificates were granted to food companies worldwide, mostly in Europe and regions of East Asia and the Pacific (ISO 2015b). Another important group of international standards was developed by the Codex Alimentarius Commission, with its fundamental good hygiene practice standard and HACCP principles (CAC 2003). On the other side, starting from the 1990s, many private food safety standards have been developed and published, with the aim to (1) improve supplier consistency, (2) avoid product failures, (3) eliminate multiple audits, and (4) support the consumer– supplier relationship (Trienekens and Zuurbier 2008). An example of private standards developed for primary production is the GlobalG.A.P. standard (GlobalG.A.P. 2016), which comprises 16 separate standards deployed for crop production, fruits and vegetables, and livestock. Private British Retail Consortium (BRC) food safety standards were issued by the BRC and are intended for the food production sector (BRC 2015). The IFS series of standards was first developed by German and French retailers (IFS 2014a), and they have found international implementation in the food production sector. Besides generic standards applicable to all types of food companies, there are some initiatives in developing tailored standards for a specific food sector. An example is the Global Red Meat Standard developed by the Danish Agriculture & Food Council. It prescribes specific requirements for slaughtering, cutting, deboning, and selling red meat and meat products (GRMS 2015). Best aquaculture practice was developed by the Global Aquaculture Alliance and covers standards for finfish, crustacean, and mollusk species (GAA 2015). This type of certification defines the most important elements of responsible aquaculture and provides quantitative guidelines for processing plants, farms, hatcheries, and feed mills. At the end of the food chain, IFS developed a standard for storage, distribution, and transportation, including loading and unloading activities (IFS 2014b). Another dimension that is interesting for consumers is organic products, covering fresh fruits and vegetables, grains, primary products of animal origin, and processed food. The trade in organic food differs from other food commodity networks due to the need for organic certification (EC 2007).

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Food religious standards are also of interest. In the meat industry, there are two slaughtering methods that religions and cultures require around the world, known as the halal and kosher methods, practiced by Muslims and Jews, respectively (Farouk 2013). The halal dietary laws determine which foods are “lawful”  or permitted for Muslims, and kosher dietary laws determine which foods are “fit or proper”  for consumption by Jewish consumers (Regenstein et al. 2003; Regenstein and Regenstein 1991). Religious slaughtering is carried out legally in the EU in licensed slaughterhouses by authorized slaughtermen of the Islamic and/or Jewish faiths (Velarde et al. 2014). Along with the development of standards, their recognition became an obstacle in international trade, due to a great number of available food standards. The modern and global food industry requires universal food safety standards that can be accepted worldwide. As a solution, guidance on recognizing different safety standards along the supply chain was developed by the Global Food Safety Initiative (GFSI 2013). This initiative comprises 400 retailers, manufacturers, service providers, and other stakeholders across 70 countries (GFSI 2015). Table  16.2 presents typical private food safety standards recognized by GFSI throughout the food chain and relevant international standards. 16.4.2  Main Groups of Requirements All food safety standards have requirements regarding PRPs and hazard analysis. PRPs are requirements that need to be fulfilled prior to performing any type of hazard analysis. They present the basic elements of good

TABLE  16.2 Food Safety Standards Recognized by the GFSI within the Food Chain Role in the Food Chain  Primary production

Food processing

Storage and distribution services

GFSI Recognized  SQF Code GlobalG.A.P. CanadaG.A.P. Global Aquaculture Alliance FSSC 22000 Global Red Meat Standard SQF Code IFS Food Standard BRC Global Standard for Food Safety Primus GFS Standard SQF Code BRC Global Standard for Storage and distribution IFS Logistics

Other Standards in Use  ISO 22000:2005

ISO 9001:2008 ISO 22000:2005 HACCP-based food safety system

ISO 9001:2008 ISO 22000:2005

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hygiene practice.* Upon implementation of PRPs, companies need to perform a hazard analysis in order to prevent or decrease food safety risks. In other words, companies have to assess their food safety risks associated with identified hazards. The most recognized hazard analysis in the food industry is performed within the HACCP system. Beside these two, food safety standards have an additional group of requirements, known as food safety management. 16.4.2.1  Prerequisite Programs and Good Practices PRPs present the basic elements and foundation of any risk-based food safety system. These programs are basic conditions and activities that are necessary to maintain a hygienic environment throughout the food chain suitable for the production, handling, and provision of safe end products and safe food for human consumption (ISO 2005). PRPs cover aspects of incoming materials control, product identification and traceability, training of personnel and food safety awareness, and water and energy supply, while good practices are grouped as personal hygiene, pest control, cleaning and sanitation, warehouse and distribution, and layout and premise structure (CAC 2003; Celaya et al. 2007; ISO 2005). Table  16.3 gives a short description of the main requirements outlined in PRPs (ISO 2005; CAC 2003; BRC 2015; IFS 2014a). It is of note that these requirements have also been integrated in food legislative acts worldwide. 16.4.2.2  Hazard Analysis HACCP is a food safety system that has become a preferred method to ensure the production of safe and healthy food. HACCP is the foundation of most food safety standards intended for food processing companies. This approach is based on a detailed assessment and examination of every step in the production process for each food product. The major goal of the HACCP system is to identify the place and time in which food hazards could occur, and also to design effective controls for each identified hazard. It consists of five main steps and seven HACCP principles (WHO 2009). For implementing HACCP, it is necessary to assemble a HACCP team, describe the product (or group of products), identify its intended use, construct flow diagrams, and perform on-site confirmation of all flow diagrams. The HACCP system is based on seven underlying principles: (1) hazard analysis, (2) CCP determination, (3) establishment of critical limits for CCPs, (4) establishment of monitoring procedures for CCPs, (5) establishment of corrective * Depending on the role in the food chain, good practices are known as good agricultural practice (GAP), good veterinarian practice (GVP), good manufacturing practice (GMP), good hygienic practice (GHP), good production practice (GPP), good distribution practice (GDP), and good trading practice (GTP).

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TABLE  16.3 Main PRP Requirements PRP  Layout and premises

Incoming control

Product identification

Personal hygiene

Water supply

Pest control

Cleaning and sanitation

Storage

Transportation

Requirements  All equipment and measuring devices are suitable for the food industry Maintenance of equipment and infrastructure is in place Internal structures and fittings (walls, floors, doors, ceilings, windows, and working surfaces) are built of durable materials and easy to clean Internal design and layout of equipment avoid cross-contamination Lighting fixtures are protected to ensure that food is not contaminated by breakages Quantity and visual control of raw and packaging materials Control of documentation (approvals and/or certificates of conformity) In-house or external testing of sampled raw/packaging materials Traceability backwards (trace all information related to production and suppliers) Traceability forwards (to whom and where final products are sold in case of recall/withdrawal) Instructions regarding the behavior of workers and visitors Workers should wear suitable protective clothing, head coverings, footwear, gloves, and other, whatever is necessary Workers should refrain from coughing, sneezing, or any other activity that could contaminate food Jewelry should not be worn Ongoing training and increasing of food safety knowledge and awareness should be performed on a regular basis Potable water is used for cleaning and sanitation, for workers’  hygiene, or as a food ingredient There is a water sampling plan from all dispensing places All water testing is performed in external/accredited laboratories Contract with an external pest control organization Layout of baits, e.g., the positions of the baits for rodents Insect killers or air curtains that prevent access of flying insects are present Routine checking/observing for the potential presence of any pests Cleaning and sanitation program for all premises Cleaning and sanitation program for equipment and cleaning equipment Routine process hygiene testing (food contact surfaces and hands of workers) Use of adequate and effective chemicals to loosen soil and bacterial film Rotation of goods– first in, first out (FIFO) or first expires, first out (FEFO) Goods are not stored stacked to the walls Goods are placed at least 50  cm from the walls Goods/pallets are raised from the floors Goods with and without allergens are not stored together Hazardous materials and cleaning agents are stored in locked areas Checking hygiene of the transportation vehicle Pest control of transportation vehicles Control of work conditions within the vehicle and/or maintaining the cold chain

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actions, (6) establishment of verification procedures, and (7) establishment of a record system (CAC 2003). Hazard analysis is the first step to establish an effective combination of control measures (Soman and Raman 2016). Hazard analysis is adopted as the first principle of HACCP, and it includes the process of collecting and evaluating information on food hazards that are associated with the specific step in the food production process. Basically, there are three main types of hazards: (1) microbiological hazards, such as pathogens, viruses, yeasts and molds, and parasites; (1) chemical hazards, for example, pesticides, mycotoxins, growth hormones, antibiotics, food additives, and heavy metals; and (3) physical hazards comprising metal parts, stones, soil, wood, and any other foreign particles. In conducting the hazard analysis, one has to take into account the likelihood of a hazard’s occurrence and the severity of its adverse health effects, the qualitative and/or quantitative evaluation of the presence of hazards, the possibility of survival or multiplication of important pathogens, and the possible production or persistence in foods of toxins, chemicals, or physical agents. As a result of hazard analysis, the company should determine those hazards that have or might have significant impacts on food safety. This can be done by using a decision tree and defining CCPs (CAC 2003), or by using matrix that combines hazard occurrence and severity of human health (Soman and Raman 2016). The identified hazards have to be included in the HACCP plan. In the food processing company, hazard analysis is performed according to a flow diagram that covers all steps in the operation for a specific food product. It is necessary to construct as many flow diagrams and perform hazard analyses as needed, depending on the number of products or groups of products. It is of note that hazard analysis is also required within the food safety standards applied in primary production, such as GlobalG.A.P. In this case, hazard analysis is more focused on potential sources of contamination from site location and site history, hygiene, waste pollution, pests, disease and weed carryover, food defense or food fraud, and water supply (GlobalG.A.P. 2016). GlobalG.A.P. also recommends a five-step risk assessment: (1) identifying hazards, (2) deciding who or what might be harmed and how, (3) evaluating the risks and deciding on precautions, (4) recording the work plan and findings and implementing it, and (5) reviewing the assessment and updating if necessary (GlobalG.A.P. 2016). 16.4.3  Food Safety Management The evolution of standards shifted from basic hygiene requirements and hazard analysis to food safety management. The main food safety management requirements cover the process approach; internal audits; corrective and preventive actions; performance monitoring; measuring, reporting, and reviewing against key performance objectives and targets; legal compliance;

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management responsibility; and management review (ISO 2005, 2006; Djekic et al. 2011, 2016). In order to evaluate the effectiveness of a food safety system, companies should develop their own food safety indicators and/or food safety objectives. These indicators or objectives should follow the SMART principle, meaning they should be Specific, Measurable, Achievable, Relevant, and Time-bound. As part of the verification process of the food safety management system, companies should also have an internal audit in place. Very useful management improvement tools are corrective and preventive actions. A corrective action is launched when a problem has occurred and symptoms of the problem provide some data, which can be used in solving it, while preventive action is to eliminate the source of the problem before it appears (Myszewski 2013). Companies with implemented and certified food safety (management) systems often have problems identifying nonconformances and initiating appropriate corrective actions (Djekic et al. 2011). Even if companies have a developed procedure for corrective and preventive actions, root cause analysis and making a clear distinction between corrections and corrective actions are still problems (Djekic et al. 2016). The management reviews provide an opportunity to assess the food safety performance of the organization in meeting the objectives, its food safety policy, and the overall effectiveness. As a part of the review process, analysis of all verification activities (ongoing and periodic) should be included. These activities include review of all testing and inspecting records of products and processes, consumer complaints, external audits and inspections, and emergency situations (ISO 2005, 2014). Innovations in standards development can be overseen in adding new requirements, such as food defense (BRC 2015; IFS 2014a; Kleboth et al. 2016), or new ideas, such as a food safety culture recognized by the BRC. 16.4.4  Effects of Implemented Food Safety Standards Several researchers have highlighted the benefits and difficulties of implemented food safety management standards (Djekic et al. 2016). The main benefit from the implemented food safety management standards is an increase in the safety of food products (Tomasevic et al. 2013; Chen et al. 2015; Mensah and Julien 2011). Many reports have confirmed that consumer confidence is an added value of implemented food safety standards (Karaman et al. 2012; Henson et al. 1999). This is connected with the better reputation and image of the companies within the food chain. It is interesting that food companies recognized food safety as part of quality, and therefore indicated the quality of the product as another benefit (Marthi 2001; Karaman et al. 2012; Vela and Ferná ndez 2003; Mensah and Julien 2011). The attitude of company managers has been identified as one of the main obstacles when implementing food safety requirements (Vela and Ferná ndez

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2003; Herath and Henson 2010; Papademas and Bintsis 2010). In-house capacity remains another constraint, since most companies have confirmed internal problems categorized as “lack of management commitment”  and “lack of knowledge”  (Djekic et al. 2016). Finally, financial assets, associated with infrastructural investments, consulting, and certification services, needed for implementing and maintaining an effective food safety system are recognized as very influential (Karaman et al. 2012; Tomasevic et al. 2013; Macheka et al. 2013; Mensah and Julien 2011). Food companies are faced with the challenge that as the complexity of food safety and quality requirements increases, their organizational knowledge decreases and the time for fulfilling the requirements shortens (Djekic et al. 2013). Analysis of food safety audit findings shows that the main problems are related to PRPs and control of food safety risks in terms of inadequate validations of the control measures (Djekic et al. 2011, 2016). There is a confusion between PRPs and the HACCP plan, their relations, how they should be managed, and understanding which barrier should be handled first due to different understandings between industry personnel, external consultants, and legal authorities (Ramí rez Vela and Martí n Ferná ndez 2003). It is expected that all food safety systems have some type of validation, especially after launching a guideline for validation of control measures (CAC 2008). 16.4.5  Food Safety Audits All types of food safety assessments are activities used to verify that a food producer is following specific guidelines, requirements, or rules (Powell et  al. 2013). An audit is defined as “a systematic, independent and documented process for obtaining audit evidence and evaluating it objectively to determine the extent to which the audit criteria are fulfilled”  (ISO 2011). Audits provide a snapshot limited by audit frequency, auditor competence, and audit scope (Powell et al. 2013). Assessment criteria are requirements used as a reference against which evidence is compared, and these criteria are mainly standards, legal requirements (where they exist), or their combination. Audits may provide audited organizations with a unique opportunity to receive advice, new ideas, and help (Djekic et al. 2011). Audit findings, positive and negative, and statements about the effectiveness of the food safety system can indicate either conformity or nonconformity with audit criteria or opportunities for improvement (ISO 2015c). To ensure adherence to recognized regulations and good manufacturing practices, audits may be supplemented with microbiological and other food safety testing and process inspections by regulatory agencies or industry (Powell et al. 2013). In spite of the fact that assessment of HACCP is under the jurisdiction of inspection services in countries where it is required by food regulation, mistrust occurred regarding the competence of local inspection services

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(Lee and Hathaway 2000; Gagnon et al. 2000; Barnes and Mitchell 2000). Additionally, customers, such as major retail chains and multinational food manufacturers, often require their suppliers to comply with their own private standards for food safety, which may be more stringent than those required by legislation. 16.4.5.1  Types of Food Safety Audits Depending on the role of audit participants, there are three types of audits (Table  16.4) (ISO 2011). First-party audits are conducted by the organization itself, meaning it plans and performs audits using its own resources (trained employees). Internal auditing programs within the food industry are limited, keeping the efforts to a minimum. In the food industry, internal audits of HACCP or similar risk-based assessment programs are not expected beyond the minimum yearly verification (Hepner et al. 2004). If the food safety system is integrated with quality management, the frequency may rise to twice a year (Djekic et al. 2014). Second-party audits are performed when the audit client is the customer or other organization with a specified interest in verifying the effectiveness of a system at the premises of suppliers (auditee). Auditors are either employers of the customers or outsourced to specialized organizations to perform audits on behalf of the customer. Some authors believe that second-party audits are stricter and may identify problems that third-party audits do not (Powell et al. 2013; Djekic et al. 2016). In order to be confident in the safety of food, big food companies also qualify their supplier using second-party auditors (Losito et al. 2011). From a long-term perspective, second-party audits are considered to be an effective tool in directing suppliers toward improvements (Djekic et al. 2016). Third-party audits are also known as certification audits performed by certification bodies. Certification bodies are local and/or global organizations TABLE  16.4 Type of Audit and Audit Participants Type of Audit  Audit Participants

First Party 

Second Party 

Third Party 

Audit client Auditee Audit team (leader and members)

Organization (auditee)

Customer Supplier Working at or subcontracted by customer Customer (outsource company)

Organization (auditee) Organization (auditee) Working at or subcontracted by certification body Certification body

Audit organization

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that provide a variety of auditing services against a large number of standards. When companies decide on certification bodies, basically there are two factors to be included: recognition of the certification body in terms of its accreditation and the competence of auditors (IAF 2011). In order to control the certification process, it is expected that certification bodies are able to demonstrate that they have evaluated risks arising from their certification activities and have adequate (financial) arrangements to cover liabilities arising from certification activities and/or the geographic areas in which they operate (ISO 2015c). As a business opportunity, certification bodies also started providing HACCP certification under various food safety schemes (Djekic et al. 2013). These schemes are either unaccredited with self-made guidelines for auditing HACCP-based food safety systems or in line with accreditation protocols issued by accreditation bodies, such as the Dutch Accreditation Council (RvA 2014). The main reason for using unaccredited schemes is the fact that HACCP is not a food safety management tool. It is considered a food safety control tool applicable only to food processing and does not include any supporting processes, such as maintenance, purchasing, sales, and distribution. The basic requirement for certification bodies to gain accreditation is to have an assessment methodology for certification of management systems (ISO 2015c). As a result, certification bodies provide accredited services for ISO 22000, BRC, and IFS and other food safety management system standards. Food chain stakeholders— producers, customers, and consumers— consider a certificate as proof of an implemented and effective food safety management system. However, some serious concerns have been raised over third-party audits. First, some critics believe that certification is a paper-driven process of limited value for the company performance and is used as a marketing tool (Djekic et al. 2011; Tanner 2000). Also, there is no critical evaluation of potential correlation between (third-party) audit outcomes and foodborne illness outbreaks (Powell et al. 2013). The outbreak of Salmonella typhimurium  linked to the Peanut Corporation of America is cited as an example of a failure in the third-party auditing system. It resulted in a recall of 3900 peanut butter and other peanut-containing products from more than 350 companies. As a result of the foodborne outbreak, 691 people were sickened and 9 died in United States and Canada. The main cause was the lack of competence of both the food safety auditor and food safety inspector (Powell et al. 2013; Sheth et al. 2011). Despite the criticism of the existing performance of audits in the food sector, it is obvious that the audits are now shifting to risk-based auditing (Kleboth et al. 2016; Albersmeier et al. 2009). The latest revisions of ISO management standards with the ultimate introduction of a risk approach confirms the necessity of new audit principles (ISO 2009, 2015a).

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16.5  Final Remarks The major drift in food safety regulation occurred in developed countries with the adoption of the performance-based regulation, in which it is defined what has to be achieved rather than how an outcome must be achieved. This drift is followed with the greater responsibility of food business operators, who should handle food through the development and implementation of hazard analysis. Food safety risk and science-based food safety regulation are a great foundation for the assurance of an adequate level of food safety. They are of great value for food producers to be in line with all requirements. Local inspectors should also shift from checking to advising the application of new and difficult requirements. Also, it is not new that local producers are involved in national surveillance programs to help improve food safety practices. Nevertheless, there is great discrepancy in adopting adequate food safety legislative acts, mainly between developed and developing countries. Along with the legal requirements, voluntarily food safety standards play a very important role in ensuring food safety in the global food market. This is especially seen through the demands of customers, especially major retail chains and big manufacturers. These organizations often require their suppliers to comply with their food safety standards, which may be more stringent than those required by legislation, especially in developing and undeveloped countries. Although the approaches in food safety of both regulations and standards may differ in some aspects, they present two sides of the same coin. Regulation helps in defining certain limits and methods of how to evaluate or test certain food processing or food parameters. Assessments are performed by inspection services that are mostly unannounced. Standards give frameworks for managing food safety issues. These assessments are performed by trained auditors and are mostly announced and planned.

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Johnson, R., and C. E. Hanrahan. 2010. The US-EU beef hormone dispute. CRS Report for Congress. Washington, DC: Congressional Research Service. Kä ferstein, F., and M. Abdussalam. 1999. Food safety in the 21st century. Bulletin of the World Health Organization  77 (4):347– 351. Karaman, A. D., F. Cobanoglu, R. Tunalioglu, and G. Ova. 2012. Barriers and benefits of the implementation of food safety management systems among the Turkish dairy industry: A case study. Food Control  25 (2):732– 739. Keener, L., S. M. Nicholson‐ Keener, and T. Koutchma. 2014. Harmonization of legislation and regulations to achieve food safety: US and Canada perspective. Journal of the Science of Food and Agriculture  94 (10):1947– 1953. Kleboth, J. A., P. A. Luning, and V. Fogliano. 2016. Risk-based integrity audits in the food chain— A framework for complex systems. Trends in Food Science and Technology  56:167– 174. Lee, J. A., and S. C. Hathaway. 2000. New Zealand approaches to HACCP systems. Food Control  11 (5):373– 376. Leon, M. A., and E. Paz. 2014. A perspective of food safety laws in Mexico. Journal of the Science of Food and Agriculture  94 (10):1954–1957. Liu, X. 2014. International perspectives on food safety and regulations— A need for harmonized regulations: Perspectives in China. Journal of the Science of Food and Agriculture  94 (10):1928– 1931. Losito, P., P. Visciano, M. Genualdo, and G. Cardone. 2011. Food supplier qualification by an Italian large-scale-distributor: Auditing system and non-conformances. Food Control  22 (12):2047– 2051. Lynch, M. F., R. V. Tauxe, and C. W. Hedberg. 2009. The growing burden of foodborne outbreaks due to contaminated fresh produce: Risks and opportunities. Epidemiology and Infection  137 (Special Issue 03):307– 315. Macheka, L., F. A. Manditsera, R. T. Ngadze, J. Mubaiwa, and L. K. Nyanga. 2013. Barriers, benefits and motivation factors for the implementation of food safety management system in the food sector in Harare Province, Zimbabwe. Food Control  34 (1):126– 131. Marthi, B. 2001. 6— HACCP implementation: The Indian experience. In Making the Most of HACCP , ed. T. Mayes and S. Mortimore, 81– 97. Cambridge, UK: Woodhead Publishing. McEvoy, J. D. G. 2016. Emerging food safety issues: An EU perspective. Drug Testing and Analysis  8 (5– 6):511– 520. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerging Infectious Diseases  5 (5):607– 625. Mensah, L. D., and D. Julien. 2011. Implementation of food safety management systems in the UK. Food Control  22 (8):1216– 1225. Morrison, A., and R. Wensley. 1991. Boxing up or boxed in? A short history of the Boston Consulting Group share/growth matrix. Journal of Marketing Management  7 (2):105– 129. Mwamakamba, L., P. Mensah, K. Takyiwa, J. Darkwah-Odame, A. Jallow, and F. Maiga. 2012. Developing and maintaining national food safety control systems: Experiences from the WHO African region. African Journal of Food, Agriculture, Nutrition and Development  12 (4):6291– 6304. Myszewski, J. M. 2013. On improvement story by 5 whys. TQM Journal  25 (4):371– 383.

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17 Food Safety Reforms in the United States: The Food Safety Modernization Act (FSMA) Harmit Singh and Holly M. Greene CONTENTS 17.1 Introduction: The History of Food Safety Regulations in the United States................................................................................................ 563 17.2 Events Leading to the Food Safety Modernization Act........................ 568 17.3 Title I: Improving the Capacity to Prevent Food Safety Problems  ....... 569 17.3.1 Produce Safety................................................................................. 574 17.3.2 Food Safety Reforms and Regulations for Functional Foods and Dietary Supplements.................................................. 576 17.3.3 Risk of Nanomaterials in Foods................................................... 577 17.3.4 Food Industry Training................................................................. 577 17.4 Title II: Improving Capacity to Detect and Respond to Food Safety Problems........................................................................................... 581 17.5 Title III: Improving the Safety of Imported Food.................................. 583 17.5.1 Collaboration with Various Government Agencies................... 589 17.6 Title IV: Miscellaneous Provisions........................................................... 591  Disclaimer............................................................................................................. 592 References.............................................................................................................. 592

17.1 Introduction: The History of Food Safety Regulations in the United States On January 4, 2011, President Obama signed into law the Food and Drug Administration (FDA) Food Safety Modernization Act (FSMA), the most comprehensive reform to the U.S. food safety laws in more than 70 years. The signing of this law enabled the U.S. FDA to better protect public health by strengthening the food safety system. Yet, the true establishment of food and drug regulation in the United States has its roots in the late nineteenth century, when state and local governments began to enact food and drug regulations in earnest ( Figure  17.1 ).  The existing governing system for food safety in the United States was formed by two laws passed in 1906: the Federal Meat Inspection Act and 563

1862

19

19

40

76

79 980 981 1 1

19

1950

53

19

57 958 1

19 1960

1920

Federal Import Milk Act

Congress creates Food, Drug, and Insecticide Administration (FDIA) within USDA

Filled Milk Act

23 19

30 19

70 19

FDIA becomes FDA after an agriculture appropriations act

67 19

27 19

19

90 94

19

96

19

97

19

02 20

07 20

11 20

Fair Packaging and Labeling Act Poultry Products Federal Fungicide, Inspection Act Insecticide, and Wholesome Meat Act (amended Rodenticide Act Federal Meat Inspection Act) (FIFRA) FDA transferred to Dept. Egg Products Inspection Act Food Additives of Health, Education, and Amendment Welfare (HEW) pursuant to Environmental Protection Agency Agricultural Reorganization Plan 1 of 1953 established (took over FIFRA) Marketing Act

46 947 1

19

16

19

U.S. Grain Standards Act

Federal Trade Commission Act

14

19

FIGURE  17.1  Selected important dates for food safety in the United States, 1862– 2011. (From Congressional Research Service, The Federal Food Safety System: A Primer, Congressional Research Service, Washington, DC, 2012.)

Toxic Substances Control Act

19

1910 Federal Meat Inspection Act

06 907 1

19

Federal Food and Drugs Act of 1906 (Pure Food and Drug Act)

FDA transferred from USDA to the Federal Security Agency (FSA had been created in 1939)

38

Federal Food, Drug, and Cosmetic Act

Division of Chemistry (precursor to FDA) est.

1900

Division of Chemistry becomes Bureau of Chemistry (precursor to FDA)

01

19

Food Safety and Inspection Dietary Supplement FDA Modernization FDA Amendments Service (FSIS) est. within Health and Education Act of 1997 Act of 2007 USDA in current form Act of 1994 (DSHEA) Animal and Plant Public Health Security FDA Food Safety Infant Formula Health Inspection and Bioterrorism Modernization Act Act of 1980 Nutrition Labeling and Food Quality Protection Service est. (APHIS) Preparedness and Education Act of 1990 (NLEA) Act of 1996 Response Act of 2002 Dept. of Education Organization Act signed into law, HEW becomes Dept. Sanitary Food Federal Tea Tasters of Health and Human Services (HHS) Transportation Act Repeal Act of 1996

19

Tea Importation Act

USDA est.

71

1931

1891

97

18

564 Food Safety and Protection

The Food Safety Modernization Act (FSMA)

565

the Pure Food and Drug Act (PFDA) (Johnson 1982). President Theodore Roosevelt signed the two landmark acts on June 30, 1906, which marked the beginning of the federal efforts to ensure Americans a safe food and drug supply. The signing of these acts occurred after a taxing crusade from the combined efforts of the medical profession, industry, government, and consumers. Prior to 1906, federal regulation of the food and drug industry in the United States was fragmented, and several measures took place between 1820 and 1902 to establish the passing of the 1906 regulation. The path of the 1906 regulation traces back to 1820, with the establishment of the U.S. Pharmacopeia, an authoritative book that catalogs all legally recognized standards for drug substances and dosage forms. Yet, the introduction of the U.S. Pharmacopeia did not alleviate the influx of substandard drugs from Europe. European drug manufacturers were faced with strict government regulations and used the United States, having an absence of drug laws, as a depot for their adulterated products. United States medical professions were unable to prescribe these European drugs with any assurance due to the drugs’ potency instability. In 1848, the U.S. Congress began drug regulation by enacting the Import Drugs Act, which prohibited the importation of any drug that did not meet U.S. Pharmacopeia standards. The Import Drug Act was a comprehensive and ambitious attempt by Congress to solve the problem of imported adulterated drugs (Heath 2004). In the beginning, the statue was strictly enforced, but it lacked public support and the government funding faded. In the mid-1870s, margarine was introduced to the United States and immediately became controversial as an important food adulteration issue. Margarine, then, was made from cattle fatty residue and very often sold as butter, yet the cost to produce it was about half that of butter. Government imposed measures on margarine, to include stamps and proper labeling. By 1886, 27 states had some margarine legislation: 20 regulated labeling and packaging and 7 prohibited its manufacture and sale. Yet, the lack of provisions or resources for enforcement of these state-established regulations put pressure on the federal government to become involved. In 1886, Congress passed the Oleomargarine Bill, which levied a manufacturing tax of 2 cents per pound of margarine and annual licenses fees for manufacturers ($600), wholesalers ($480), and retailers ($48) of margarine. These imposed fees only intensified the selling of margarine as butter to avoid paying for the annual licenses. A shift in state legislatures turned toward regulating the color of margarine, and by the early 1900s, 32 states prohibited the sale of yellowcolored margarine (Dupré  1999). However, with the discovery of hydrogenation in 1909, enforcement of this legislation became even more difficult. The explosion of the price of butter in 1947 generated a great deal of public opinion, and the 1902 Oleomargarine Bill was repealed by the end of 1950. Margarine had finally become a normal food product and was regulated under the Food and Drugs Act. The health of slaughter animals for human consumption has always been associated with meat safety. Prior to the passing of the Meat Inspection Act

566

Food Safety and Protection

of 1891, government inspection of meat did not occur. With a 30% decline of U.S. cattle prices between 1885 and 1890, ways to counter this deterioration became a central issue in the efforts of cattle producers to attain inspection legislation to promote the demand of cattle and meat in the export markets. No evidence of consumer health problems of beef consumption existed, but allegations of slaughtered diseased animals in Chicago packinghouses gave credence to foreign competitors’  accusations that American livestock and meat products were sickening (Libecap 1992). At the same time, claims of trichinosis in American pork brought restrictions on imports in Germany, France, Belgium, and other European countries. The control of Europe prohibiting the importing of American livestock intensified the pretense of the disease issue, and cattle producers lobbied the government to enact federal inspection legislation to aid in rising cattle prices. The 1891 Meat Inspection Act mandated the inspection of all live cattle for export, as well as all live cattle that were to be slaughtered and their meat exported. The law also authorized the inspection of swine and sheep prior to slaughter and interstate shipment. With the passing of this act, for the first time, the federal government was authorized to certify food quality for American consumers. In 1897, the Tea Importation Act was passed, which prohibited the importation of tea into the United States that failed to meet government standards for quality, purity, and fitness for consumption. Adulteration of tea was routine in England, and also with imported tea into the United States. Leaves of plants other than tea leaves would be mixed into the batch and sold as pure tea. In addition, used tea leaves would be sold as new, and consumers purchased certain colored teas that would disguise the inferior quality and the presence of foreign leaves. Sellers of tea leaves would employ methods that increased the weight of the tea leaves, and thus its price. Most of the substances used were relatively safe for human consumption, but some were not. The Tea Importation Act required each lot of imported tea to be sampled at the port of entry to ensure that it met the standard recommended to the secretary of Health and Human Services by the Board of Tea Experts. Tea was the only food or beverage that was sampled upon entry for a comparison with a standard. The Tea Act was repealed by Congress in 1996 (DeWitt 2000). The Biologics Control Act of 1902 was enacted by Congress to ensure the protection of Americans by providing consistently safe biological products. In 1901, there were no mandatory federal manufacturing or product standards for biologics. The death of 13 children in St. Louis in 1901 as a result of receiving tetanus-contaminated diphtheria antitoxin, and other similar incidents, prompted quick action by lawmakers. In 1902, Congress enacted the Biologics Control Act, also known as the virus-toxin law, which gave the government its first control over the processes used for the production of biological products. The first regulations under this act became effective on August 21, 1903, and mandated that producers of vaccines be licensed annually for the manufacture and sale of vaccines, serum, and antitoxins. Manufacturing

The Food Safety Modernization Act (FSMA)

567

facilities were also required to undergo inspections, and licenses could be revoked or suspended when necessary. Production was to be supervised by a qualified scientist. All product labels were required to include the product name, expiration date, address, and license number of the manufacturer. These new controls marked the beginning of a basic change in America’ s federal public health policy and a steadfast commitment to the protection of public health (FDA 2016a). The 1906 PFDA was the first federal law to simultaneously address food, beverages, and drug adulteration, production, distribution, and marketing for import and export (PFDA 1906). Passing of the PFDA replaced all established state standards and developed a collaboration between federal and state authority. On December 5, 1905, President Theodore Roosevelt recommend to the 59th Congress that a law be enacted to regulate interstate commerce in misbranded and adulterated foods, drinks, and drugs. Such a law would protect legitimate manufacture and commerce, and would tend to secure the health and welfare of the consuming public. Traffic in foodstuffs that had been debased or adulterated so as to injure health or deceive purchasers would be forbidden (Roosevelt 1905). The bill was passed in Senate on February 21, 1906, and the house passed a substitute bill 4 months later; Congress produced a compromise bill in only 6 days, and after the signature of President Roosevelt on June 30, 1906, the act went into effect on January 1, 1907. The Meat Inspection Act of 1906 was the beginning of the U.S. federal regulation of the country’ s meat, poultry, and egg supply. In 1906, Upton Sinclair’ s novel The Jungle  was published; it portrayed the harsh working conditions of immigrants in the United States in industrialized cities. However, the American public was more concerned with Sinclair’ s portrayal of the nauseating details regarding the unhealthy practices of Chicago’ s meatpacking district. President Roosevelt deployed Labor Commissioner Charles P. Neill to Chicago to investigate the meatpacking industry, which Neill reported to Roosevelt as being “revolting”  and even worse than the conditions depicted in Sinclair’ s novel. The act of 1906 strengthened requirements for sanitary conditions and established standards for inspecting all meat processing plants that conducted business across state lines (Barkan 1985). The structure of the 1906 PFDA was overhauled in 1938 by the Federal Food, Drug, and Cosmetic Act (FDCA), and that framework still exists today. The FDCA’ s passage was the result of a historical accident in the United States. Elixir Sulfanilamide (the first antimicrobial drug and diethylene glycol used as a diluent) was given to 350 patients during a 4-week period in the fall of 1937. This product was produced by the S. E. Massengill Company of Bristol, Tennessee, a small company that manufactured primarily capsules and tablets. With the demand for a liquid preparation, Massengill’ s chief chemist formulated a raspberry-tasting pink preparation consisting of 10% sulfanilamide, 72% diethylene glycol, 16% water, and small amounts of elixir flavor and raspberry extract. Of the 350 patients given the elixir, there were

568

Food Safety and Protection

105 deaths: 34 children and 71 adults. Following an investigation by the FDA, it was found that these deaths were not due to the sulfanilamide, but rather the diluent used— diethylene glycol. Results from this investigation led to the passage of the 1938 FDCA by the U.S. Congress. This new legislation required toxicity testing before the release of any new drug (Wax 1995). The FDCA focused primarily on ensuring that new drugs be tested for safety before marketing; it also enlarged the authority of the FDA to ensure food safety. Under the FDCA, the FDA was authorized to inspect factories, create identity and quality standards, and establish safety tolerances for unavoidable poisons (Burkett 2012).

17.2  Events Leading to the Food Safety Modernization Act The original FSMA of 2009 was introduced initially as a bill in Congress by Rosa DeLauro, a democrat from Connecticut’ s third congressional district, in 2007. This bill died and was reintroduced in 2009 to establish the Food Safety Administration within the Department of Health and Human Services to protect the public health by preventing foodborne illness, ensuring the safety of food, improving research on contaminants leading to foodborne illness, and improving the security of food from intentional contamination, and other purposes. Further amendments were introduced (by Senators Richard Durbin and Judd Gregg) to the Committee on Health, Education, Labor, and Pensions. Senator Durbin referred to food safety concerns and how the almost 70-year-old food safety acts needed to be revised. He referred specifically to the 2008 incidents of outbreaks in which 1500 people fell sick (21% were hospitalized and 2 died), from 43 different states, the District of Columbia, and Canada, because of Salmonella enterica  serotype Saintpaul. The outbreak led to a governmental investigation that pointed the finger first at tomatoes and then jalapeno peppers in Texas, before settling on serrano peppers in Mexico. In the meantime, more people got sick and the tomato industry lost up to hundreds of millions of dollars. In 2008, peanut butter was tainted with Salmonella , the second case of its kind in 2 years, in which more than 660 people had been sickened, half of them children, and nine people died. More than 2600 products were recalled, a recall that dated back to March 2005 and continued for at least another couple of years, making it one of the biggest food recalls in U.S. history. The U.S. FDA also recognized that about 48 million people (1 in 6 Americans) get sick, 128,000 are hospitalized, and 3,000 die each year from foodborne diseases, according to recent data from the Centers for Disease Control and Prevention (Figure  17.2) (CDC) (2016). This is a significant public health burden that is largely preventable. Finally, the FSMA enables the FDA to better protect public health by strengthening the food safety system. It enables the FDA to focus more on

569

The Food Safety Modernization Act (FSMA)

Attribution of Foodborne Illness and Deaths, 1998–2008 Fish and shellfish

6.10%

Dairy and eggs

20.00%

Meat and poulty

0%

29.00% 46.00%

10%

20%

Deaths

15.00%

22.00%

Produce

Illnesses

6.40%

30%

40%

23.00% 50%

60%

70%

80%

90%

100%

FIGURE  17.2  Contribution of different food categories to estimated domestically acquired illnesses and deaths, 1998– 2008. Chart does not show 5% of illnesses and 2% of deaths attributed to other commodities. In addition, 1% of illnesses and 25% of deaths were not attributed to commodities; these were caused by pathogens not in the outbreak database, mainly Toxoplasma  and Vibrio vulnificus . (From Painter, J. A., Emerg Infect Dis , 19 (3), 407– 415, 2013.)

preventing food safety problems, rather than relying primarily on reacting to problems after they occur. The FSMA is divided into four titles (Table  17.1): Title I is related to the guidelines to improve the facilities to prevent food safety problems and is further divided into 16 subsections. Title II is related to improving the capacity to detect and respond to food safety problems. Title III contains guidelines related to the safety of imported foods. Title IV is related to other miscellaneous provisions.

17.3 Title I: Improving the Capacity to Prevent Food Safety Problems  For the first time, the FDA had a legislative mandate to require comprehensive, prevention-based controls across the food supply to prevent or significantly minimize the likelihood of problems occurring. The five major elements of FSMA are divided into five key areas: preventive controls, inspection and compliance, imported food safety, response, and finally, enhanced partnership. Table  17.2 indicates how the FDA is trying to implement these areas. The FSMA preventive controls for human food rule is final, and compliance dates for some businesses began in September 2016. Preventive measures are now extended in more depth and include produce safety. The Foreign Supplier Verification Program (FSVP) has been established; it requires importers to perform certain risk-based activities to verify that food imported into the United States has been produced in a manner that meets

Title I— ESTABLISHMENT OF THE FOOD SAFETY ADMINISTRATION Sec.   101.   Establishment of the food safety administration. Sec.   102.   Consolidation of food safety functions. Sec.   103.   Additional duties of the administration. Title II— ADMINISTRATION OF FOOD SAFETY PROGRAM Sec.   201.   Administration of national program. Sec.   202.   Registration of food establishments and foreign food establishments. Sec.   203.   Preventive process controls to reduce adulteration of food. Sec.   204.   Performance standards for contaminants in food. Sec.   205.   Inspections of food establishments. Sec.   206.   Food production facilities. Sec.   207.   Federal and state cooperation. Sec.   208.   Imports. Sec.   209.   Resource plan. Sec.   210.   Traceback requirements. Sec.   211.   Accredited laboratories. Title III— RESEARCH AND EDUCATION Sec.   301.   Public health assessment system. Sec.   302.   Public education and advisory system. Sec.   303.   Research. Sec.   304.   Working group on improving foodborne illness surveillance. Sec.   305.   Career-spanning training for food inspectors. Sec.   306.   Food-Borne Illness Health Registry. Sec.   307.   Study on federal resources.

BILL PROPOSED

Comparison of FSM Bill Submitted and the Final FSMA Approved

TABLE  17.1  

(Continued )

ACT S.510 PASSED TITLE I— IMPROVING CAPACITY TO PREVENT FOOD SAFETY PROBLEMS Sec. 101. Inspections of records. Sec. 102. Registration of food facilities. Sec. 103. Hazard analysis and risk-based preventive controls. Sec. 104. Performance standards. Sec. 105. Standards for produce safety. Sec. 106. Protection against intentional adulteration. Sec. 107. Authority to collect fees. Sec. 108. National agriculture and food defense strategy. Sec. 109. Food and Agriculture Coordinating Councils. Sec. 110. Building domestic capacity. Sec. 111. Sanitary transportation of food. Sec. 112. Food allergy and anaphylaxis management. Sec. 113. New dietary ingredients. Sec. 114. Requirement for guidance relating to post harvest processing of raw oysters. Sec. 115. Port shopping. Sec. 116. Alcohol-related facilities. TITLE II— IMPROVING CAPACITY TO DETECT AND RESPOND TO FOOD SAFETY PROBLEMS Sec. 201. Targeting of inspection resources for domestic facilities, foreign facilities, and ports of entry; annual report. Sec. 202. Laboratory accreditation for analyses of foods. Sec. 203. Integrated consortium of laboratory networks.

570 Food Safety and Protection

Title IV— ENFORCEMENT Sec.   401.   Prohibited acts. Sec.   402.   Food detention, seizure, and condemnation. Sec.   403.   Notification and recall. Sec.   404.   Injunction proceedings. Sec.   405.   Civil and criminal penalties. Sec.   406.   Presumption. Sec.   407.   Whistleblower protection. Sec.   408.   Administration and enforcement. Sec.   409.   Citizen civil actions. Title V— IMPLEMENTATION Sec.   501.   Reorganization plan. Sec.   502.   Transitional authorities. Sec.   503.   Savings provisions. Sec.   504.   Conforming amendments. Sec.   505.   Additional technical and conforming amendments. Sec.   506.   Regulations. Sec.   507.   Authorization of appropriations. Sec.   508.   Limitation on authorization of appropriations.

Comparison of FSM Bill Submitted and the Final FSMA Approved

TABLE  17.1 (CONTINUED) Sec. 204. Enhancing tracking and tracing of food and recordkeeping. Sec. 205. Surveillance. Sec. 206. Mandatory recall authority. Sec. 207. Administrative detention of food. Sec. 208. Decontamination and disposal standards and plans. Sec. 209. Improving the training of state, local, territorial, and tribal food safety officials. Sec. 210. Enhancing food safety. Sec. 211. Improving the reportable food registry. TITLE III— IMPROVING THE SAFETY OF IMPORTED FOOD Sec. 301. Foreign supplier verification program. Sec. 302. Voluntary qualified importer program. Sec. 303. Authority to require import certifications for food. Sec. 304. Prior notice of imported food shipments. Sec. 305. Building capacity of foreign governments with respect to food safety. Sec. 306. Inspection of foreign food facilities. Sec. 307. Accreditation of third-party auditors. Sec. 308. Foreign offices of the Food and Drug Administration. Sec. 309. Smuggled food. TITLE IV— MISCELLANEOUS PROVISIONS Sec. 401. Funding for food safety. Sec. 402. Employee protections. Sec. 403. Jurisdiction; authorities. Sec. 404. Compliance with international agreements. Sec. 405. Determination of budgetary effects.

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TABLE  17.2   Major Elements of the FSMA and Their Implementation Guidelines The FSMA Elements Can Be Divided into Five Key Areas 

How the FDA Will Plan to Implement FSMA Elements 

Preventive controls : For the first time, the FDA has a legislative mandate to require comprehensive, prevention-based controls across the food supply to prevent or significantly minimize the likelihood of problems occurring.

Preventive controls for human food : Requires that food facilities have safety plans that set forth how they will identify and minimize hazards. Preventive controls for animal food : Establishes CGMPs and preventive controls for food for animals. Produce safety : Establishes science-based standards for growing, harvesting, packing, and holding produce on domestic and foreign farms.

Imported food safety : The FDA has new tools to ensure that imported foods meet U.S. standards and are safe for consumers. For example, for the first time, importers must verify that their foreign suppliers have adequate preventive controls in place to ensure safety, and the FDA will be able to accredit qualified third-party auditors to certify that foreign food facilities are complying with U.S. food safety standards.

Foreign Supplier Verification Program : Importers will be required to verify that food imported into the United States has been produced in a manner that provides the same level of public health protection as that required of U.S. food producers.

Inspection and compliance : The legislation recognizes that inspection is an important means of holding industry accountable for its responsibility to produce safe food. The FDA is committed to applying its inspection resources in a risk-based manner and adopting innovative inspection approaches.

Third-party certification : Establishes a program for the accreditation of thirdparty auditors to conduct food safety audits and issue certifications of foreign facilities producing food for humans or animals. Sanitary transportation : Requires those who transport food to use sanitary practices to ensure the safety of food. Intentional adulteration : Requires domestic and foreign facilities to address vulnerable processes in their operations to prevent acts intended to cause large-scale public harm. The FDA expects that it will only need to invoke this authority infrequently since the food industry largely honors requests for voluntary recalls. The agency has other new authorities that are also in effect: expanded administrative detention of products that are potentially in violation of the law, and suspension of a food facility’ s registration.

Response : For the first time, the FDA has mandatory recall authority for all food products.

(Continued )

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TABLE  17.2 (CONTINUED) Major Elements of the FSMA and Their Implementation Guidelines The FSMA Elements Can Be Divided into Five Key Areas  Enhanced partnerships  among all food safety agencies— U.S. federal, state, local, territorial, tribal, and foreign.

How the FDA Will Plan to Implement FSMA Elements  The legislation recognizes the importance of strengthening existing collaboration among all food safety agencies— U.S. federal, state, local, territorial, tribal, and foreign— to achieve public health goals. For example, it directs the FDA to improve the training of state, local, territorial, and tribal food safety officials.

applicable U.S. safety standards. A program has been established for the accreditation of third-party auditors as a part of their commitment to provide resources for inspection and compliance. In addition, the FDA has authority to recall all food products; although the FDA expects that the industry will conduct voluntary recalls, it has been given more teeth in this act to detain the product, as well as suspend the facility registration. For the first time, the FDA has a legislative mandate to require comprehensive, science-based preventive controls across the food supply, which include: • Mandatory preventive controls for food facilities: Food facilities are required to implement a written preventive controls plan. This involves (1) evaluating the hazards that could affect food safety; (2) specifying what preventive steps, or controls, will be put in place to significantly minimize or prevent the hazards; (3) specifying how the facility will monitor these controls to ensure that they are working; (4) maintaining routine records of the monitoring; and (5) specifying what actions the facility will take to correct problems that arise. • Mandatory produce safety standards: The FDA must establish science-based, minimum standards for the safe production and harvesting of fruits and vegetables. Those standards must consider naturally occurring hazards, as well as those that may be introduced either unintentionally or intentionally, and must address soil amendments (materials added to the soil, such as compost), hygiene, packaging, temperature controls, animals in the growing area, and water. • Authority to prevent intentional contamination: The FDA must issue regulations to protect against the intentional adulteration of food, including the establishment of science-based mitigation strategies to prepare and protect the food supply chain at specific vulnerable points. Preventive controls are divided into human and animal foods. Hazard analysis, preventive controls, and oversight and management of preventive

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controls using monitoring, corrective actions, and verification are common rules in both animal and human food safety measures. The FDA has finalized the current good manufacturing practices (CGMPs) to be followed for animal food production considering various scenarios in which animal food is produced, such as by-products of animal food production. Processors already implementing human food safety requirements, such as brewers, do not need to implement additional preventive controls or CGMP regulations when supplying a by-product (e.g., wet spent grains, fruit or vegetable peels, and liquid whey) for animal food, except to prevent physical and chemical contamination when holding and distributing the by-product. Examples of physical and chemical contamination include placing trash or cleaning chemicals into the container holding the by-products. The FDA has also identified five ways in which consumers and their pets will be safe: (1) food companies will apply greater controls to help prevent hazards, (2) consumers and their pets will be protected from tainted animal food, (3) eating healthfully and safely will go hand in hand, (4) there will be greater oversight of foods imported from other countries, and (5) consumers will be more confident that their food is safe. The Hazard Analysis and Critical Control Point (HACCP) is based on the critical control points (CCPs) identified by the manufacturers, whereas preventive control under the FSMA includes the CCPs or controls other than CCPs that are appropriate for food safety. The preventive rules require the facilities to control the hazards and take corrective actions to prevent contamination by testing the product and environmental monitoring. The FDA expects companies to more frequently test the products more prone to outbreaks of foodborne illness. Verification of preventive control is also considered very important in this act. The companies should have a hazard analysis and preventive control plan, which should be reanalyzed every 3 years or when the preventive control is found to be insufficient to control the hazard. A qualified individual (either trained or by experience) should be responsible for the development of the preventive control plan. 17.3.1  Produce Safety The FDA has generated several flowcharts to decide whether farms are exempt or subject to produce rules. The preventive controls for human food rule clarified the definition of a farm to cover two types of farm operations: primary production farms and secondary activities farms (Figure  17.3). Agricultural water quality has been given high priority during the FSMA development. Some applications even require total undetectable levels of Escherichia coli , for example, handwashing, water on food contact surfaces, and sprout irrigation. After reviewing scientific literature, the FDA determined that generic E. coli , bacteria found in the intestinal tract of both people and animals, is a consistent indicator of the presence of feces. Identifying fecal contamination is important in assessing the safety of agricultural

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Does your farm grow, harvest, pack, or hold produce? Sections 112.1 and 112.3(c) “Produce” defined in Section 112/3(c)

NO

Your farm is NOT covered by this rule.

YES

Your farm is NOT covered by this rule.

YES

Does your farm on average (in the previous three years) have $25K or less in annual produce sales? Section 112.4(a )

NO

Is your produce one of the commodities that the FDA has identified as rarely consumed raw?

Section 112.2(a)(1) If you grow, harvest, pack, or hold more than one produce commodity, you must ask this question separately for each one to determine whether that particular produce commodity is covered by this rule.

YES

This product is NOT covered by this rule.

NO

Is your produce for personal/on-farm consumption? Section 112.2(a)(2)

YES

This produce is NOT covered by this rule.

NO

Is your produce intended for commercial processing that adequately reduces pathogens (for example, commercial processing with a “kill step”)? Section 112.2(a)(2)

NO

Does your farm on average (in the previous three years) as per Section 112.5, Have