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Food Safety and
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Food Safety and
Protection
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Edited by
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V. Ravishankar Rai and Jamuna A.Bai

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©
CRC Press
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Library of Congress Cataloging‑ in‑ P ublication Data

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Names: Rai, V. Ravishankar, editor. | Bai, Jamuna A. (Jamuna Aswathanarayn),

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editor.
Title: Food safety and protection / edited by Ravishankar Rai and Jamuna Bai
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Aswathanarayan.
Description: Boca Raton : CRC Press, [2017] | Includes bibliographical
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references and index.

Identifiers: LCCN 2017012632| ISBN 9781498762878 (hardback : alk. paper) |
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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

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and the CRC Press Web site at

http://www.crcpress.com
Contents
Preface.......................................................................................................................ix
Editors .......................................................................................................................xi
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Contributors.......................................................................................................... xiii
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Section I Predictive Microbiology for Safe Foods
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1. Semiquantitative and Qualitative Assessment for
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Determination of Sanitary Risk in Food Service Establishments........3
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Elke Stedefeldt, Laí s Mariano Zanin, Ana Lúcia de Freitas Saccol,
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Eduardo Cesar Tondo, Veronica Cortez Ginani, Eneo Alves da Silva Jr.,
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Ana Beatriz Almeida de Oliveira, and Diogo Thimoteo da Cunha op
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2. Fresh-Cut Apples Spoilage and Predictive Microbial Growth

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under Modified Atmosphere Packaging .................................................. 29

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Predrag Putnik and Danijela Bursać Kovač ević

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Section II Food Allergens, Contaminants, and Toxins

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3. Analytical Methods for the Detection of Mycotoxins in Milk

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Samples ........................................................................................................... 49
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Myra E. Flores-Flores and Elena Gonzá lez-Peñ as

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4. Biotoxins in Seafood..................................................................................... 97
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Laura P. Rodrí guez, Juan M. Vieites, and Ana G. Cabado

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5. Selection and Characterization of Aptamers for Food

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Contaminant Monitoring........................................................................... 157

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Amina Rhouati, Akhtar Hayat, Gaë lle Catanante, and Jean Louis Marty
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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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vi Contents
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
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9. Non-Thermal Preservation Technologies for Meat and Fish
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Products......................................................................................................... 291
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Bruna Leal Rodrigues, Denes Kaic Alves do Rosário, and Carlos Adam
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Conte-Junior
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10. Inactivation of Pathogenic Microorganisms in Foods by High
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Pressure Processing..................................................................................... 341
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Evelyn and Filipa Vinagre Marques da Silva
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11. Application of Pulsed Light for the Microbial Decontamination
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of Foods.......................................................................................................... 379
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Marija Zunabovic, Victoria Heinrich, and Henry Jä ger
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12. Effect of Commercial Emerging Nonthermal Technologies on

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Food Products: Microbiological Aspects................................................ 397

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Elisabete M. C. Alexandre, Rita S. Iná cio, Ana C. Ribeiro, Álvaro Lemos,

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Sofia Pereira, Só nia M. Castro, Paula Teixeira, Manuela Pintado, Ana

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M. P. Gomes, Francisco J. Barba, Mohamed Koubaa, Shahin Roohinejad,

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and Jorge Saraiva

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Section IV Food Packaging

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13. Food Packaging Systems with Antimicrobial Agents......................... 431

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Reyhan Irkin
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14. Active and Intelligent Food Packaging................................................... 459

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Cristina Nerin, Paula Vera, and Elena Canellas

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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
Contents vii
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
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19. Recent Developments in Saffron Fraud Prevention ............................ 651
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Anastasia Kyriakoudi and Maria Z. Tsimidou
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Index......................................................................................................................679
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Preface
Food safety and protection is a multidisciplinary topic that focuses on the

safety, quality, and security aspects of food. Food safety issues involve micro-
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bial risks in food products, foodborne infections, and intoxications and food
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allergenicity. Food protection deals with trends and risks associated with
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food packaging, advanced food packaging systems for enhancing product
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safety, the development and application of predictive models for food micro-
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biology, food fraud prevention, and food laws and regulations with the aim
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to provide safe foods for consumers.
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The book Food Safety and Protection covers various aspects of food safety,
security, and protection. It discusses the challenges involved in the prevention
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and control of foodborne illnesses due to microbial spoilage, contamination,
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and toxins. The book covers new and safe food intervention techniques,
predictive food microbiology, and modeling approaches. It deliberates the
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legal framework, regulatory agencies, and laws and regulations for food
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protection.
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The book has five sections dealing with the topics of predictive microbiol-
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ogy for safe foods; food allergens, contaminants, and toxins; preservation of
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foods; food packaging; and food safety laws. Section I, on predictive micro-
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biology for safe foods, covers qualitative and quantitative methods for deter-
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mining the sanitary risk in food services and predictive microbial growth in
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fresh foods. Section II, on food allergens, contaminants, and toxins, discusses
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analytical methods for the analysis of mycotoxins in milk, biotoxins in sea-

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food, aptamer designs for food contaminant monitoring, control and man-
agement of allergens in foods, and assessment of unintentional contaminants
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in foods. Section III, on preservation of foods, comprehensively explores

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developments in food intervention techniques and covers topics such as

thermal inactivation kinetics of foodborne pathogens, nonthermal technolo-
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gies for the preservation of meat and fish products, high-pressure processing
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and ultrasound techniques to inactivate spores in foods, pulsed field tech-

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niques, and other emerging nonthermal technologies for food preservation.

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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
ix
x 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
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Dr. Jamuna A. Bai
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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
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Studies in Microbiology, University of Mysore, India. He was awarded fel-
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lowships from the UNESCO Biotechnology Action Council, Paris (1996);
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the Indo-Israel Cultural Exchange Fellowship (1998); the Biotechnology
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Overseas Fellowship, government of India (2008); the Indo-Hungarian
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Exchange Fellowship (2011); and the Indian National Academy Fellowship
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(2015). Presently, he is the coordinator for the Department of Science and
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Technology, Promotion of University Research and Scientific Excellence and
University Grants Commission innovative programs.
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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
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Grants Commission (UGC)-sponsored University with Potential Excellence

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Project, University of Mysore, India. She has previously worked as an Indian

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Council of Medical Research (ICMR) senior research fellow on food safety,

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the role of quorum sensing and biofilms in food-related bacteria, and devel-
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oping quorum-sensing inhibitors. Her research interests also include the

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antimicrobial application of functionalized nanomaterials against food-

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borne pathogens.
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Contributors
Elisabete M.C. Alexandre Conrado Carrascosa
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Department of Chemistry Department of Animal Pathology
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University of Aveiro and Production
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Aveiro, Portugal Universidad de Las Palmas de Gran
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and Canaria
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Arucas, Spain
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Universidade Cató lica Portuguesa
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Escola Superior de Biotecnologia
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Porto, Portugal Só nia M. Castro
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Department of Chemistry
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M. Carmen Rubio Armendá riz University of Aveiro
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Á rea de Toxicologí a Aveiro, Portugal
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Universidad de La Laguna
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Tenerife, Canary Islands, Spain and

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Jamuna A. Bai Escola Superior de Biotecnologia

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Department of Studies in
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Microbiology Porto, Portugal
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University of Mysore
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Karnataka, India
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Gaë lle Catanante
Biocapteurs-Analyses-
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Francisco J. Barba
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Environnement
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Faculty of Pharmacy Université de Perpignan Via

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Universitat de Valè ncia Domitia

Valè ncia, Spain
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Perpignan, France
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Ana G. Cabado Carlos Adam Conte-Junior

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Food Safety Division— IDi.

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Department of Food Technology

ANFACO-CECOPESCA Universidade Federal Fluminense
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Carretera Colexio Universitario

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Vigo, Spain and

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Food Science Program

Elena Canellas Universidade Federal do Rio de
Departamento de Quí mica Janeiro
Analí tica Rio de Janeiro, Brazil
Universidad de Zaragoza
Campus Rio Ebro
Zaragoza, Spain
xiii
xiv Contributors
Philip G. Crandall Myra E. Flores-Flores

Department of Food Science and Organic and Pharmaceutical
Center for Food Safety Chemistry Department
University of Arkansas University of Navarra
Fayetteville, Arkansas Pamplona, Navarra, Spain
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Diogo Thimoteo da Cunha Ana Lú cia de Freitas Saccol
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Faculdade de Ciê ncias Aplicadas
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Curso de Nutriç ã o
Universidade de Campinas
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Centro Universitá rio Franciscano
Limeira, Brazil
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Santa Maria, Brazil
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Ilija Djekic
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Juan Garcí a-Dí ez
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Department of Food Safety and
Centre of Studies in Animal and
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Quality Management
Veterinary Science (CECAV),
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University of Belgrade
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DCV-ECAV
Belgrade, Serbia
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University of Trá s-os-Montes e Alto
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Douro
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Alexandra Esteves
Vila Real, Portugal
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Centre of Studies in Animal and

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Veterinary Science, DCV-ECAV

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University of Trá s-os-Montes e Alto Veronica Cortez Ginani

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Douro Departamento de Nutriç ã o

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Vila Real, Portugal Universidade de Porto Alegre

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Porto Alegre, Brazil

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Evelyn
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Department of Chemical Consuelo Revert Gironé s

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Engineering Á rea de Toxicologí a

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University of Riau Universidad de La Laguna

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Pekanbaru, Indonesia Tenerife, Canary Islands, Spain

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Catarina Tinoco de Faria

Elena Gonzá lez-Peñ as
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Department of Animal Pathology

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and Production Organic and Pharmaceutical

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Universidad de Las Palmas de Gran Chemistry Department

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Canaria University of Navarra

Pamplona, Navarra, Spain
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Arucas, Spain
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Á ngel J. Gutié r rez Ferná ndez Ana M. P. Gomes

Á rea de Toxicologí a Universidade Católica Portuguesa
Universidad de La Laguna Escola Superior de Biotecnologia
Tenerife, Canary Islands, Spain Porto, Portugal
Contributors xv
Holly M. Greene Reyhan Irkin

Don B. Huntley College of Department of Food Engineering
Agriculture University of Balikesir
California State Polytechnic Balikesir, Turkey
University, Pomona
Pomona, California Henry Jä ger
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Department of Food Science and
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Technology
Akhtar Hayat
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University of Natural Resources and
Biocapteurs-Analyses-
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Life Sciences
Environnement
Vienna, Austria
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Université de Perpignan Via
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Domitia
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Nathan A. Jarvis
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Perpignan, France
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and Center for Food Safety
University of Arkansas
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Interdisciplinary Research Centre in Fayetteville, Arkansas
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Biomedical Materials
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COMSATS Institute of Information Mohamed Koubaa

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Technology Centre de Recherche de Royallieu

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Université de Technologie de
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Lahore, Pakistan
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Compiè gne
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Compiè gne, France
Victoria Heinrich
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Section of Packaging and Resource

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Danijela Bursać Kovač ević

Management
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Faculty of Food Technology and

University of Applied Sciences
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Biotechnology
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Vienna, Austria University of Zagreb

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Zagreb, Croatia
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Rita S. Iná cio
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Anastasia Kyriakoudi
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University of Aveiro
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School of Chemistry
Aveiro, Portugal
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Aristotle University of Thessaloniki

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Thessaloniki, Greece
and
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Á lvaro Lemos
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Universidade Cató lica Portuguesa Department of Chemistry

Escola Superior de Biotecnologia University of Aveiro
Porto, Portugal Aveiro, Portugal
xvi Contributors
Jean Louis Marty Sofia Pereira

Biocapteurs-Analyses-Environnement Department of Chemistry
Université de Perpignan Via Domitia University of Aveiro
Perpignan, France Campus Universitá rio de Santiago
Aveiro, Portugal
José M. Caballero Mesa
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Á rea de Toxicologí a Esteban Pé rez
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Universidad de La Laguna Department of Animal Pathology
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Tenerife, Canary Islands, Spain and Production
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Faculty of Veterinary
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Soraya Paz-Montelongo Universidad de Las Palmas de Gran
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Á rea de Toxicologí a Canaria
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Universidad de La Laguna Arucas, Spain
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Tenerife, Canary Islands, Spain
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Manuela Pintado
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Dina Moura Escola Superior de Biotecnologia
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Direç ã o de Serviç os de Alimentaç ã o op
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e Veteriná ria da Regiã o Norte Porto, Portugal
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Direç ã o Geral de Alimentaç ã o e

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Veteriná ria Predrag Putnik

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Guimarã es, Portugal Faculty of Food Technology and

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Biotechnology
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Cristina Nerin University of Zagreb

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Departamento de Química Analítica Zagreb, Croatia

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Zaragoza, Spain V. Ravishankar Rai

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Department of Studies in
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Corliss A. O’ Bryan Microbiology

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Department of Food Science and University of Mysore

Center for Food Safety Karnataka, India
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University of Arkansas
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Fayetteville, Arkansas Antó nio Raposo

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CBIOS (Research Center for

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Ana Beatriz Almeida de Oliveira Biosciences and Health

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Universidade Federal do Rio Technologies)

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Grande do Sul Universidade Lusó fona de

Departamento de Nutriç ã o Humanidades e Tecnologias
©
Porto Alegre, Brazil Lisbon, Portugal

Contributors xvii
Amina Rhouati Cristina Saraiva

Biocapteurs-Analyses- Centre of Studies in Animal and
Environnement Veterinary Science DCV-ECAV
Université de Perpignan Via University of Trá s-os-Montes e Alto
Domitia Douro
Perpignan, France Vila Real, Portugal
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and
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Ecole Nationale Supé rieure de
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Jorge Saraiva
Biotechnologie
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Constantine, Algeria
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University of Aveiro
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Aveiro, Portugal
Ana C. Ribeiro
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rs
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University of Aveiro Eneo Alves da Silva Jr.
Central de Diagnó sticos
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Aveiro, Portugal
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Laboratoriais (CDL)
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Steven C. Ricke Sã o Paulo, Brazil
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Center for Food Safety Filipa Vinagre Marques da Silva

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University of Arkansas Chemical and Materials

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Fayetteville, Arkansas Engineering Department

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University of Auckland
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Bruna Leal Rodrigues Auckland, New Zealand

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Department of Food Technology

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Universidade Federal Fluminense

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Harmit Singh
Rio de Janeiro, Brazil Don B. Huntley College of
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Agriculture
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Laura P. Rodrí guez California State Polytechnic

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Food Safety Division— IDi.

University, Pomona
ANFACO-CECOPESCA
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Pomona, California
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Carretera Colexio Universitario

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Vigo, Spain
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Nada Smigic
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Shahin Roohinejad Department of Food Safety and

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Division of Food and Nutrition Quality Management

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Shiraz University of Medical University of Belgrade

Sciences Belgrade, Serbia
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Shiraz, Iran
Elke Stedefeldt
Denes Kaic Alves do Rosá rio Centro de Desenvolvimento do
Chemistry Institute Ensino Superior em Saú de
Universidade Federal do Rio de (CEDESS)
Janeiro Universidade Federal de Sã o Paulo
Rio de Janeiro, Brazil Sã o Paulo, Brazil
xviii Contributors
Paula Teixeira Juan M. Vieites

Escola Superior de Biotecnologia Food Safety Division— IDi.
Universidade Cató lica Portuguesa ANFACO-CECOPESCA
Porto, Portugal Carretera Colexio Universitario
Vigo, Spain
Eduardo Cesar Tondo
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Instituto de Ciê ncia e Tecnologia Dailos Gonzá lez Weller
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dos Alimentos Á rea de Toxicologí a
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Universidade Federal do Rio Grande Universidad de La Laguna
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do Sul Tenerife, Canary Islands, Spain
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Porto Alegre, Brazil
na
Laí s Mariano Zanin
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Arturo Hardisson de la Torre Programa de Pós-Graduação em
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Á rea de Toxicologí a Nutriç ã o
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Universidad de La Laguna Universidade Federal de São Paulo
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Tenerife, Canary Islands, Spain São Paulo, Brazil
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Maria Z. Tsimidou Marija Zunabovic
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School of Chemistry Department of Food Science and

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Aristotle University of Thessaloniki Technology

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Thessaloniki, Greece University of Natural Resources and

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Life Sciences
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Paula Vera Vienna, Austria

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Departamento de Química Analítica

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Zaragoza, Spain
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Section I
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for Safe Foods
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Predictive Microbiology
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1
Semiquantitative and Qualitative
Assessment for Determination of Sanitary
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Risk in Food Service Establishments
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Elke Stedefeldt, Laí s Mariano Zanin, Ana Lúcia de Freitas Saccol,
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Eduardo Cesar Tondo, Veronica Cortez Ginani, Eneo Alves da Silva Jr.,
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Ana Beatriz Almeida de Oliveira, and Diogo Thimoteo da Cunha
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CONTENTS op
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1.1 Introduction.....................................................................................................4
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1.2 Sanitary Risk in Food Service Setting.........................................................5

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1.3 Risk Assessment.............................................................................................6

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1.4 Foodborne Diseases in Brazilian Food Services: Etiological

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Agents, Food Vehicles, and Contamination Sources.................................7

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1.5 Sources of Contamination in Food Services...............................................9

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1.6 Causal Factors of FBD.................................................................................. 10

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1.7 Use of Inspection Scores to Semiquantitatively Evaluate Food

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Safety in Food Service Establishments...................................................... 13

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1.8 Systematization of Qualitative Method of Risk Assessment.................. 18

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1.9 Projects Undertaken in the Context of Sanitary Risk in Food

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Service Environments.................................................................................. 20
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1.9.1 Project 1: Pilot Project Food Service Establishment

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Categorization................................................................................... 20
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1.9.1.1 Project Participants.............................................................. 20

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1.9.1.2 Description............................................................................ 20
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1.9.2 Project 2: Complementary Technical Assistance Project

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to the Support Project for the Development of a School

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Feeding Program in Mozambique: ��22

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1.9.2.1 Project Participants..............................................................22

1.9.2.2 Description............................................................................22
1.10 Final Consideration...................................................................................... 23
References................................................................................................................ 24
3
4 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
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under the age of 5 years. Even with the use of advanced food technologies
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and the investment of substantial financial resources and time, foodborne
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disease (FBD) is still one of the most important public health problems in
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the world, as it is a threat to public health and global socioeconomic devel-
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opment. Havelaar et al. (2015) have suggested that the strategies adopted
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by the food industry to prevent and/or control the survival, proliferation,
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and contamination of microorganisms in food are inadequate.
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Even though several FBD outbreaks have been attributed to food process-
ing and production, food service establishments have been associated as
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well, and in Brazil, they are the public locales where the majority of reported
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FBD outbreaks have occurred (Brazil 2016). op
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The prevention of microorganism contamination, survival, and prolifera-
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tion in food is a necessity recognized in all existing regulations and stan-

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dards and by all responsible professionals and stakeholders. In the United

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States, for example, the Food Safety Modernization Act (FSMA), established
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in 2011, consolidated existing laws and changed the focus in food safety pro-
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motion from a reactive to a preventive perspective (Grover et al. 2016).

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The objective of this act was to implement the Hazard Analysis and Risk-
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based Preventive Controls (HARPC) tool, which focuses on food safety

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throughout the entire food chain. To support this objective, HARPC was
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based on other food safety management systems (FSMSs), such as good

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hygiene practices (GHPs), the Hazard Analysis and Critical Control Point
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(HACCP), ISO 22000:2005, the Safe Quality Food (SQF) Code (Safe Quality
Food Institute), and Global Food Safety Initiative (GFSI) guidelines (Food
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and Drug Administration 2013).

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The complexity of these tools requires qualified persons to assess poten-

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tial hazards in each establishment and identify and implement preventive

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and control measures by scientifically validated methodologies (Grover

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et al. 2016).
20
However, it is recognized that the variety among and peculiarities within

different food service establishments present a challenge for small busi-
©
nesses 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 understand-
ing 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.
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1.2 Sanitary Risk in Food Service Setting
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Established as a global phenomenon, food service has become the main
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alternative to home food preparation for many people with long commutes
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or who seek convenience. It is estimated that the food service industry has a
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global revenue of billions of dollars a year.
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Food service establishments are facilities that serve meals and snacks for
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immediate consumption at a particular location, that is, eating out. They
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include full-service restaurants, fast-food restaurants, catering, cafeterias,

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food trucks, and other places that prepare, serve, and sell food to the gen-
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eral public for profit. Some, such as hostels, recreational facilities, shopping
b
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centers, and retail stores, are located in places that are not dedicated solely to
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the distribution of meals and snacks (U.S. Department of Agriculture 2015).

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The food globalization process has had a significant impact on the safety
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of food sold in food service establishments. This globalization process has

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focused on production, distribution, and marketing on a large scale and

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seeks to meet the needs of the expanding global population. An asymmetry

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of information in the globalization of food, however, can lead to market fail-

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ures, and standardization may be an impediment to the food sovereignty of

a country.
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Food service is a multifaceted scenario containing complex situations in

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which the risks are multifactorial and may be associated with some uncer-
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tainty and/or ambiguity. Risk analysis in this scenario becomes a challenge

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but is essential. The use of different ingredients and additives resulting from
18
globalization may potentiate this risk.

20
In the governmental sphere, the Brazilian Health Surveillance Agency

uses the concept of sanitary risk to regulate, control, and monitor the pro-
©
duction 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 pub-
lic. 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
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 dam-
age from exposure to hazards— t he social, economic, and political environ-
y
ment in which it occurs. The integration of these considerations defines
nl
the risk management approach and allows the establishment of effective
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strategies to combat risk (Agê ncia Nacional de Vigilâ ncia Sanitá ria 2015;
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Silva and Lana 2014).
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The management of sanitary risk is complex and broad in nature. It
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involves a direct and specific association with the regulation field, as well as
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strong and diverse connections to other related fields. These characteristics
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result in the need for a management strategy that may be shared, can be inte-
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grated daily, and is permanent and participatory (Oliveira 2013).
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Monitoring and control present a central challenge in the management
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of the products, services, and processes associated with sanitary risk. It is
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advisable, therefore, to strengthen interinstitutional cooperation, work in
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networks, and seek innovations through different initiatives and articula-

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tions (Oliveira 2013).

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Understanding sanitary risk in the food service setting involves predic-

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tions of “ health threat,” “ vulnerability of human health,” and “ likelihood of

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harm” because the characteristics and consequences of risks are not always
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known, and risk factors are not always identified.

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It seems clear that the notion of risk as a probability does not always apply
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to sanitary risks in the food service setting because only the results of the
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hazards that are already known can be predicted. When dealing with the
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issue of sanitary risk, this complexity also adds an unknown element to

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“ sanitary risk” (Universidade Federal do Ceará 2015).

In the food service arena, there is a gap in the approach to sanitary risk.
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Risk analysis is an iterative and continuous process, consisting of three

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components: risk management, risk assessment, and risk communication.

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It can be performed with varying degrees of detail, depending on the risk

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itself, the purpose of the analysis and information, and available data and
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resources (OPAS 2008).

20
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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 indi-
vidual or a population to a hazard. It was developed primarily to meet the
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.
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It can involve historical data, secondary data from scientific publications,
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expert opinions, and information regarding the needs and procedures of
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stakeholders (Universidade Federal do Ceará 2015). Risk assessment results
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may be quantitative, qualitative, or semiquantitative (Manning and Soon
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2013).
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Quantitative risk assessment presents the results numerically, qualitative
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assessment presents the results in descriptive terms (high, medium, and
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low), and semiqualitative assessment presents the results through scores or
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ranking (Manning and Soon 2013; OPAS 2008).
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According to World Health Organization (2009), “ semi-quantitative risk
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assessment is a relatively new idea in food safety. Codex Alimentarius
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Commission and others generally consider just two categories of risk
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assessment: qualitative and quantitative. Semi-quantitative risk assess-

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ment has often been grouped together with qualitative risk assess-
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ment, but this understates the important differences between them in

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their structure and their relative levels of objectivity, transparency, and

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repeatability.”
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Concerning sanitary risk assessment, professionals should rely on prior,

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empirical, and technical knowledge and norms to identify risks and act on
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them; strategies based on analysis shall contribute positively to mitigation,

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prevention, and elimination of risk (Silva and Lana 2014).

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The impact of these actions may represent a decisive contribution to and

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fundamental catalyst for the effective management of sanitary risk, and

particularly the production of a timely, agile, and powerful sanitary risk
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communication, which is important and expected by the general popu-

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lation (Oliveira 2013). A very important starting point in this setting is a

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knowledge of the causative microorganisms, food vehicles, and sources

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of contamination related to FBD in food service establishments in a given

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country.
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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 sani-
tary surveillance officers of Brazilian municipalities investigate notifiable
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 ana-
lyze and interpret the FBD outbreak data. The information is finally sent to the
Brazilian National Health Authority, who annually report these data. From
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January 2000 to July 2016, the Ministry of Health registered 11,051 foodborne
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outbreaks, with approximately 2.1 million people exposed. Among them,
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more than 209,000 became sick and 169 people died (Brazil 2015, 2016). Even
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with very proactive sanitary surveillance services in Brazil, these numbers
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represent only the “ tip of the iceberg” concerning FBD because, as occurs in
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other countries, outbreaks are not always reported, which makes it difficult
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to know the true incidence of FBD.
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Considering the foodborne illnesses from 2000 to 2015 that were reported
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in Brazil, food service establishments were the second most frequently iden-
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tified FBD location of origin (24.2%), second only to private homes (38.4%),
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where surveillance services are not allowed to act. The types of food service
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establishments most frequently reported as the location of origin for these
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diseases were commercial restaurants, bakeries, and similar establishments

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(15.5%); schools (8.7%); workplace catering (8.2%); and events (3.6%). The cat-
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egories “ unknown site” and “ other establishments” accounted for 11.3% and
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8.3%, respectively.
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The data from Brazil thus suggest that households are the sites where
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most FBD outbreaks originate (38.4%) (Brazil 2016), unlike the United States,
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where restaurants were the places most frequently reported, accounting for
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65% of FBD cases (Silva 2016). Although data on FBD outbreaks are under-
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reported, available data may help to indicate differences in eating behaviors

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that characterize each society, and therefore the priority actions for sanitary
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risk prevention.
In Brazil, the most frequently reported food vehicles of foodborne patho-
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gens were those often prepared in food service establishments, that is, mixed
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foods (14.1%), eggs and egg products (mainly homemade mayonnaise, 7.8%),
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and creamy sweets and desserts (2.1%). Water, milk and milk products, red
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meat and pork meat; poultry were responsible for 6.0%, 2.6%, 2.1%, and 1.4%,
18
respectively. Leafy greens were responsible for only 0.8% of investigated

20
outbreaks.
Among the reported Brazilian FBD, Salmonella was the pathogen most fre-
©
quently 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 sani-
tary 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
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 major-
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ity of these salmonellosis cases occurred during spring, when people may
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keep food outside of the refrigerator due to the sensation of cold weather in
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the mornings. The molecular patterns of SE86 were highly similar to those
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of microorganisms isolated from food vehicles (Oliveira et al. 2007, 2009) and
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FBD cases (Oliveira et al. 2012) identified in these outbreaks. Further, the
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SE86 strain was more resistant than other isolated Salmonella species to com-
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mon sanitizers used in food industries and food services in Brazil (Machado
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et al. 2010; Tondo et al. 2010).
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In the same Brazilian state, S. aureus has been identified as the second
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most frequent etiological agent of foodborne illnesses since the 1990s.
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Considering the official epidemiological data on S. aureus food poisoning,
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Lima et al. (2013) reported that S. aureus was identified as the etiological
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agent of 57 foodborne outbreaks during 2000– 2002. Among these, 42 out-

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breaks were confirmed by microbiological analyses, and 15 were confirmed

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by clinical symptoms and/or epidemiological data. Staphylococcal out-

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breaks were responsible for illness in 5991 people, of whom 1940 (32%) were
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interviewed by the sanitary surveillance officers. The food vehicles most

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frequently identified were meat servings (35%), pastries (25%), cheese (23%),
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pasta (11%), and potato salad with homemade mayonnaise (11%). The major-
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ity of the staphylococcal outbreaks occurred inside private homes (33%), fol-
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lowed by food services (28%).

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1.5 Sources of Contamination in Food Services

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Different sources of microbial contamination exist in food service estab-

18
lishments, which may result in cross-contamination of foods and FBD out-

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breaks. Cleaning cloths and sponges are well-known contamination sources.

For example, Bartz and Tondo (2013) investigated the microbial contamina-
©
tion 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 wash-
ing and disinfecting them using 200 ppm sodium hypochlorite for 15 min-
utes 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
(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
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periods longer than 3 hours in food service establishments.
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Rossi et al. (2012) evaluated the microbiological contamination of 80 kitchen
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sponges used in Brazilian food service establishments. The results demon-
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strated that the sponges were contaminated with heterotrophic microorgan-
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ism counts ranging from 3.4 to 10.4 log CFU/sponge, with an average of 9.1
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log CFU/sponge, and fecal coliforms were found in 76.25% of the sponges,
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with average counts of 8.4 log CFU/sponge. Salmonella and S. aureus were
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found in 2.5% of samples. After bacterial evaluation, sponges were submit-
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ted to boiling for 5 and 10 minutes or to disinfection using 200 ppm sodium
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hypochlorite for 15 minutes. Both methods significantly reduced bacterial
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counts, but the boiling method showed a greater reduction than disinfection
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by sodium hypochlorite.
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Recently, Kothe et al. (2016) investigated the hygienic conditions of hotdogs

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collected from street food vendors in Brazil. The results demonstrated that
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of the 20 hotdog samples, 75% were contaminated with coliforms, 30% were
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contaminated with fecal coliforms, and 25% had coagulase-positive staphy-

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lococci levels above the maximum limit permitted by Brazilian regulations.

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These results also demonstrated that the majority of vendors defrosted sau-
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sages at ambient temperature or employed inadequate cooling. None of the

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vendors had a thermometer, and several of them used nonpotable water.

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Other frequent violations were the lack of cross-contamination preventive

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measures, the lack of time and temperature control, and the use of ingredi-
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ents of unknown origin.

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1.6 Causal Factors of FBD

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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 contami-
nated 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 knowl-
edge addresses theory as it relates to the fact, but this knowledge must be
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
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in Baixada Santista, Brazil, it was observed that other strategic actions
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were required to guarantee food safety. Scores were used to determine
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the risk for these strategic actions— a n innovative, effective way to eval-
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uate the food safety practices related to school meal preparation and
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facilitate the implementation of corrective measures. Among the evalu-
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ated establishments, 62% were classified as average sanitary risk, failing
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to comply with Brazilian food safety legislation. Violations relating to
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hand hygiene, pest control, food handlers, and food hygiene were most
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frequently identified.
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These results reveal the absence of an effective system to monitor and eval-
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uate the sanitary conditions of food service establishments (Da Cunha et al.
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2014b). Data on FBD outbreaks in the French Armed Forces called attention
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to the differences between the hygiene regulations of different countries. In

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that study, the number of officers affected by FBD that were in their country
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of origin was compared with the number of those affected during service in
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other countries. The proportion of cases that occurred overseas was 48.3%.
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The incidence rate was 2.4 outbreaks/100,000 in France and 26.7/100,000

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overseas, with a maximum rate of 39.3/100,000 in Africa. This indicates that

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places without regulated and monitored sanitary measures may have higher
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rates of FBD (Mayet et al. 2011).

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A mistaken idea is that often regular inspections may keep the estab-
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lishments in strict adherence to guidelines can be directly associated

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with the prediction of FBD outbreaks and needs clarification. On the

other hand, the categorization systems based on sanitary risk and com-
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mitment of food service establishments to the process may improve the

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sanitary conditions of their establishments, as observed by Da Cunha et

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al. (2016) in practice.

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Another aspect of sanitary risk in food service establishments, identified

18
by Murphy et al. (2011), is the difference between chain and independent

20
restaurants’ concerns regarding food safety, which is similar in quantity but

different in quality. A study conducted in Orange County, Florida, investi-
©
gated the association of mandatory food safety certification with the inspec-
tion 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
steakhouses or Brazilian barbecues), suggested that investment in the work-

force 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
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association between numerous FBD outbreaks related to their intake and the
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increased consumption, large-scale production, and widespread distribution
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of these foods. Regardless of how and when food contamination happens,
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its sanitation is compromised if the pathogens are not eliminated by conven-
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tional methods (Olaimat and Holley 2012).
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Alternative methods for pathogen control on fresh produce, such as irradi-
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ation, ozone, bacteriophages, and antagonistic bacteria, may not be possible
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for the majority of food service establishments, even if they participate in a
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specialized partnership to ensure food safety.
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As a consequence, the number of FBD outbreaks associated with fresh
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products has increased rapidly worldwide. Therefore, the importance of pur-
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chasing products with assured quality is evident (Olaimat and Holley 2012).
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In food handling procedures, it is clear, technically, that it is essential to

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ensure time and temperature control during the preparation of foods as a

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basic rule to prevent biological hazards and decrease the risk of FBD trans-
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mission. Of the outbreaks studied by Silva (2016), 75% were related to micro-
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bial growth in food due to either leaving it at temperatures between 10° C

.C
and 60° C for longer than 4 hours or using leftovers, 3.6% were due to cross-
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contamination, and 21.4% of the causes were unknown.

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In general, FBD outbreaks relate to factors that contribute to the contami-

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nation, survival, and proliferation of microorganisms in food. Regarding

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contamination, the data suggest that of the reported outbreaks, 81.7% were
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related to contaminated raw food, 55% were due to contaminated food han-
dlers, and 36% were due to contaminated utensils. Regarding contaminant
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survival, 41.2% of cases were related to food temperatures not reaching 70° C
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during cooking, and 11.3% to not reaching 70° C during reheating. Regarding
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contaminant proliferation, 79.2% of cases were related to storing food at tem-

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peratures above 10° C, and 83.5% were related to food being exposed to tem-
18
peratures between 10° C and 60° C for more than 2 hours (Silva 2016).
20
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 emerg-
ing problems in the different areas of the food sector, with a particular focus
on microbiological and chemical hazards. However, discussions on the

agenda also include the effect of illegal activity on the food sector, new con-
sumer 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
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efforts to have different approaches. The manner in which hazards are han-
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dled, particularly those related to microorganisms, must be contextualized.
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The aim must be to address food safety at all stages of food production to
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produce actions that can objectively prevent microorganism survival, prolif-
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eration, and contamination.
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1.7 Use of Inspection Scores to Semiquantitatively Evaluate
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Food Safety in Food Service Establishments
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The food safety evaluation process of food service establishments must take
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place in a continuous and strategic way to prevent FBD. Misguided or mis-

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understood evaluations, however, can lead managers and researchers to mis-

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leading conclusions.
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Quantitative risk assessment is widely used in the food sector and includes
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the use of the following tools: the HACCP, Quantitative Microbiological Risk
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Assessment (QMRA), and Fuzzy Risk Assessment Tool (FRAT).

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In 1995, the World Trade Organization introduced the concept of appro-

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priate level of protection (ALOP) in the Agreement on Sanitary and

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Phytosanitary Measures (SPS Agreement), which is defined as “ the level of

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protection deemed appropriate by the Member establishing a sanitary or

phytosanitary measure to protect human, animal and plant life or health
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within its territory” (World Trade Organization 1995). At a later stage, Food
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Safety Objectives (FSOs) were introduced to translate the ALOP into a bench-
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mark in the food chain, which is defined as “ the maximum frequency and/
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or concentration of a hazard in food at the time of consumption that pro-

18
vides or contributes to the appropriate ALOP” (International Commission

20
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 distinc-
tions in levels deemed appropriate in different situations … use of quantita-
tive 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
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
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risk analysis methods are complex and detailed and use mathematical simu-
nl
lations (Manning and Soon 2013).
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Risk assessments must be cost-effective and able to distinguish the differ-
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ent types of risk (e.g., high, medium, and low) (Health and Safety Executive
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2006). One of the most used instruments to evaluate the food safety practices
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of food service establishments is a checklist. This may be because a check-
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list is an instrument with reproducibility, cost-effectiveness, and practicality
Pe
(Stedefeldt et al. 2013). Checklists can easily be used as management and
r
research instruments and have a range of possible applications for evalua-
fo
tion in the food service industry (Da Cunha et al. 2016).
y
op
Checklists are composed of a list of food safety criteria (or questions) based
C
on legislation (e.g., “ Do food handlers sanitize their hands properly to avoid
’s
contamination of food?” ). The evaluator then indicates whether the criterion

or
has been fulfilled or violated, based on his or her observation and experi-
ut
ence. In the end, the evaluator calculates the number of violations committed
b
tri
to generate an absolute or relative score. To facilitate decision making, this

on
score should be based on FBD risk. This type of risk analysis, characterized
.C
as a semiquantitative risk assessment, establishes a bridge between qualita-

C
tive and fully quantitative methods (Davidson et al. 2006).

LL
Semiquantitative risk assessment is most useful in providing a structured

is
way to rank risks according to their probability, impact, or both together,

c
an
implying severity, and for ranking risk reduction actions for their effective-
Fr
ness. A predefined scoring system allows one to map a perceived risk into a
category, where there is a logical and explicit hierarchy between categories
d
an
(World Health Organization 2009).

or
Some researchers have used the semiquantitative method for risk assess-
yl
ment. Lake et al. (2002) studied the risk profile of milk and Mycobacterium
Ta
bovis in New Zealand; Sumner and Ross (2002) researched seafood safety
18
risk assessment in Australia; and Mataragas et al. (2008), in Europe, studied

20
the risk profiles of pork and poultry meat and risk ratings of various patho-
gen– product combinations. Most studies research one etiological agent relat-
©
ing 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 suc-
cessfully predict outbreaks of FBD. In contrast, in other studies, foodborne
outbreaks were not correlated with inspection scores.
Food safety inspections are used by health surveillance agencies to mea-

sure, 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
y
thus may lead to misinterpretations.
nl
The following example illustrates the potential for misinterpretation.
O
A food safety checklist with 100 items was applied in two restaurants.
se
Violations were noted, and the percentage of total violations (violation per-
lU
centage) was calculated for each establishment. In the first food service
na
establishment (Restaurant 1), the violation percentage was 15% of the listed
o
rs
items, and in the second establishment (Restaurant 2), it was 47% of the total
Pe
items. One might assume that Restaurant 2 implies a higher risk to consum-
r
ers because it was cited with more than double the violations of Restaurant
fo
1. However, it is necessary to also analyze the nature of the risks associated
y
with the violations, as shown in Figure 1.1. op
C
Although Restaurant 1 had a lower number of violations, they were more
’s
highly related to FBD outbreaks (Bryan 1978; ESR 2008; Food and Drug
or
Administration 2000; Todd et al. 2007), mainly because of violations con-

ut
cerning food temperature, hand hygiene, and the cleaningness of utensils

b
tri
and equipment. In this sense, a quantitative assessment attributing weights

on
based on the risk of FBD can generate a metric that is easier to interpret.
.C
Inspection scores may be attributed and used in several ways. In many

C
studies, a binary scoring system was used to determine the risk of FBD out-
LL
break. For example, some have assigned one point for each violation (Lockis
is
et al. 2011; Saccol et al. 2013; Veiros et al. 2009; Chapman et al. 2010), allow-
c
an
ing the researchers to calculate the violation percentage (or the percentage
Fr
of correct food preparation procedures). Other studies used a scale to rate

d
an
Restaurant 1 Restaurant 2
or
15% of violations 47% of violations

yl
Ta
Summary of violations Summary of violations

18
-Inadequate temperature of food -Physical structure without proper size

storage
20
-Presence of wood boxes in the food

-Ready-to-eat hot food maintained storage area
©
without controlled temperature -The floor is not made with smooth and
-Insufficient frequency of utensil and durable materials
equipment sanitization -Presence of exposed water pipes
-Inadequate hand hygiene -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.
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 leg-
y
islation is being fulfilled (in a generic way) by food service establishments.
nl
However, these scores should not be used to compare establishments, esti-
O
mate risks, or establish which food service establishments must be prioritized.
se
Arithmetical progression is another widely used technique to establish
lU
scores for food safety inspections (Da Cunha et al. 2014b; De Oliveira et al.
na
2014; Santana et al. 2009; New South Wales 2011). The use of a sequence of
o
rs
scores (i.e., n1, n2, n3, … , nk) (Irwin et al. 1989; Buchholz et al. 2002) is also a
Pe
common feature of checklists. Mathematical progressions can be used to set
r
scores; however, these scores must have a logical organization with regards
fo
to the assessment of the food procedures, hazards, and risks, such as the
y
op
inspection score systems used in Los Angeles (Los Angeles 2014), New South
C
Wales (New South Wales 2011), and Brazil (Da Cunha et al. 2014b).
’s
Table 1.1 presents some examples of the instruments used to assess food
or
safety that utilize risk scores based on the risk of FBD. A panel of experts
ut
has been used to set scores based on the risk of foodborne outbreaks in
b
tri
some countries (Da Cunha et al. 2014b; Hoag et al. 2007). In the examples
on
in Table 1.1, the final score has a negative magnitude; that is, the greater the
.C
number of points, the greater the number of violations.

C
Simpler solutions can also be used to assess food service establishments.

LL
One is to classify the items into categories, as proposed by Da Cunha et al.

is
(2016). In their study, 177 inspection items were classified into five categories
c
an
of questions: R1, questions that addressed time and temperature aspects; R2,
Fr
questions that addressed direct contamination; R3, questions that addressed

water conditions and raw material; R4, questions that addressed indirect con-
d
an
tamination (i.e., structure and buildings); and R5, questions that addressed
or
documentation. The score for each category may be analyzed separately.

yl
As another example, Kassa et al. (2010) used binary scores but weighted the
Ta
scores for critical and noncritical violations.

18
It is important to remember that risk is defined as “ probability × conse-

20
quence.” An FBD outbreak affecting three people is less serious than an out-
break 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 ill-

ness risk factors or critical items
• Specialized processes conducted
©
20
18
Ta
TABLE 1.1
yl
or
Examples of Food Safety Assessment Instruments That Utilize Weights Based on the Risk of Foodborne Disease
an
d Final Score and
Country Objective Fr Scores Classification Reference
Brazil Evaluate and classify food Risk based; violation-specific scores range from 0.02 Total score ranging from 0.0 Da Cunha et al.
an
service establishments for lower-risk violations to 139.27 for highest-risk
cis (no violation observed) to 2014
(restaurants and similar) violations 2565.95, with five
classifications
LL
United Evaluate and classify food
C
Binary with critical and noncritical violations Not presented Kassa et al. 2010
States service establishments in
.C
general on
United Evaluate and classify food Risk based with critical and noncritical violations;
tri Total score ranging from 0 City of New
States service establishments in violation-specific scores range from 2 for lower-
b (no violation observed) to York 2007
Semiquantitative and Qualitative Assessment
New York risk/lower-severity violations to 28; each violation 1519

ut
has a set of conditions (severity) from I to V; the
or
more severe the violation, the greater the point
’s
value for that violation
C
United Evaluate and classify food Risk based with minor and major violations; Total score ranging from 0 Los Angeles
op
States service establishments in violation-specific scores of 4 for major violations in
y violation observed) to 2014
Los Angeles critical risk factors, 2 for minor violations in critical
fo(no
100
risk factors; and 1 for violations in low-risk factors
rP
Australia Evaluate and classify food Risk based; violation-specific scores ranging from 1 Total score New South
er ranging from 0
service establishments in for lower-risk violations to 8 for the highest-risk (no violation Wales 2011
so observed) to
New South Wales violations 120, with four
naclassifications
lU
se
17
O
nl
y
• Food preparation a day in advance of service

• Large number of people served
• History of foodborne illness and/or complaints
• Highly susceptible population served
y
nl
O
se
lU
1.8 Systematization of Qualitative Method of Risk Assessment
ona
According to the Codex Alimentarius Commission (2001), qualitative risk
rs
Pe
assessment is “ based on data which, while forming an inadequate basis for
numerical risk estimations, nonetheless, when conditioned by prior expert
r
fo
knowledge and identification of attendant uncertainties, permits risk rank-
y
ing or separation into descriptive categories of risk.”
op
The qualitative method of risk assessment uses techniques based on the
C
construction and integration of knowledge on a topic or an event developed
’s
or
through direct interaction with a team of experts.

ut
Some techniques are described below.

b
tri
on
• Nominal group technique (NGT): This technique is used for the pri-
.C
oritization of items that arise from brainstorming. It is a structured

process by which small groups (five to nine experts) make sugges-
C
LL
tions and then discuss them until they come to a decision. This tech-
nique may be used in cases where the intention is to identify and
cis
characterize the hazard, assess the exposure and characterize the

an
risk, and select and prioritize all possible solutions (Totikidis 2010).
Fr
• Delphi: The Delphi technique is used to estimate the probability

d
an
and impact of future and uncertain events. A group of experts is

consulted to help identify risks, assumptions, and their associated
or
yl
premises, and each expert individually presents his or her estimates

Ta
and premises to a risk manager, who analyzes the data and creates a
18
summary report (Delp et al. 1977).

20
• 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 indus-
try). 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).
• Case study: This technique’ s objective is to analyze an event thor-

oughly. The aim of a case study would be to perform a detailed
examination of a particular situation in the food service environ-
ment. Case studies have become the preferred method for risk man-
agers to determine how and why specific events occur when there
is little possibility of control over the events studied and when the
y
focus of the case study is on current phenomena that can only be
nl
analyzed within their specific context. Observation plays an essen-
O
tial role in the case study. By direct observation, one seeks to under-
se
stand the appearances, situations, and/or behaviors associated with
lU
an event (Yin 2013).
ona
rs
Regardless of the technique employed, the results of these qualitative
Pe
methods are expressed in a descriptive form, indicating, for example, a high,
r
medium, or low risk according to the consensus of the experts involved
fo
(Sumner et al. 2004; OPAS 2008).
y
op
The main difference between qualitative and quantitative assessments is
C
that qualitative assessments bring together a team of experts who are encour-
’s
aged to dialogue and discuss the risk in depth. These dialogues and discus-
or
sions can be recorded, transcribed, and interpreted using content analysis.

ut
According to Bardin (1993), content analysis “ means a set of communication

b
tri
analysis techniques to obtain, through systematic procedures and descrip-

on
tion of the objectives of message content, indicators (quantitative or not) that

.C
allow the inference of knowledge related to the conditions, production and/

C
or reception (inferred variables) of these messages.”

LL
The content analysis involves three fundamental phases: preanalysis, mate-

is
rial exploration and processing of results using quantitative and/or qualita-

c
an
tive techniques, and condensing the results into patterns, trends, or implied
Fr
relationships. This method of interpretation must go beyond the experts’

words because the risk manager is most interested in reaching the latent
d
an
content, that is, the meaning that lies beneath the words (Downe-Wamboldt
or
1992; Joffe and Yardley 2004; Vaismoradi et al. 2013).

yl
This analysis may provide a better understanding of an event in the

Ta
context in which it occurs and of which it is a part, and involves seeking

18
the event’ s meaning, analyzing it in an integrated manner, and consid-

20
ering 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 percep-
tions, 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.”
Davidson et al. (2006) noted that to achieve the objectives of risk assess-
ment, the degree of uncertainty must be recognized and considered in any
risk estimates.
y
nl
O
1.9 Projects Undertaken in the Context of Sanitary
se
Risk in Food Service Environments
lU
na
The last part of this chapter briefly discusses two projects that developed in
o
a multidisciplinary, multi-institutional, and multisector setting, in which the
rs
authors participated, strengthened technical cooperation, and sought inno-
Pe
vations through different initiatives and collaborations.
r
fo
y
op
1.9.1 Project 1: Pilot Project Food Service Establishment Categorization
C
1.9.1.1 Project Participants
’s
or
Diogo Thimoteo da Cunha, Ana Lú cia de Freitas Saccol, Eduardo Cesar

ut
Tondo, Ana Beatriz Almeida de Oliveira, Veronica Cortez Ginani, Eneo Alves
b
tri
da Silva Jr., Fabio Montesano, Carolina Vieira Araú jo, Thalita Antony Souza
on
Lima, Angela Karinne Fagundes de Castro, and Elke Stedefeldt.

.C
C
LL
1.9.1.2 Description
is
Over the past few years, the health departments of cities such as New York,
c
an
Los Angeles, Toronto, London, Copenhagen, and the state of New South
Fr
Wales (Australia) have established a method to classify and evaluate res-

taurants from a food safety perspective; restaurants with better evaluations
d
an
(that best fulfilled the requirements of the health legislation) received a

or
higher score. The restaurants then received a seal indicating their classifica-
yl
tion. The aim of this strategy is to encourage healthy competition. Because a

Ta
potential competitor may have a better score, the owner of the food service
18
establishment may attempt to correct the violations in his establishment to

20
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 posi-

tive 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
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.
y
After the step of creation and evaluation of the instrument, health assess-
nl
ments by local health surveillance inspectors were performed to categorize
O
the restaurants. The evaluation took place in three stages: (1) self-assessment,
se
in which the property owner was provided the instrument and had the
lU
opportunity to make suggestions; (2) diagnostic assessment, in which the
na
health surveillance inspector indicated the observed violations; and (3) final
o
rs
assessment, in which the restaurants received their final classification based
Pe
on the score received that day. The three-evaluation strategy was established
r
to promote the compliance of the facilities. The facility could then be classi-
fo
fied into one of four grades: A (no failure found), B (very little failure or low-
y
op
risk failures found), C (few failures found), and “ pending” (reasonable to a
C
high number of failures or high-risk failures found).
’s
After the implementation of the strategy during the 2014 FIFA World Cup,
or
the group of experts convened to assess the effects of the strategy. Overall,
ut
1967 establishments in 27 cities in Brazil were evaluated and categorized.

b
tri
It was observed that sanitary risks were significantly reduced between the
on
evaluations, considering that the lower the score, the lower the sanitary risk.
.C
No foodborne outbreak originating from the categorized establishments was

C
reported during the World Cup. Moreover, 2837 people were interviewed
LL
(consumers, business owners, inspectors, and health surveillance coordina-

is
tors) regarding their perceptions of the strategy implementation. The evalua-

c
an
tions were positive, and all interviewees indicated that it would be beneficial
Fr
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
d
an
al. 2016).
or
The responses to the open-ended questions in the 503 interviews with the
yl
owners of food service establishments and tax and health surveillance coordi-
Ta
nators were interpreted by using thematic content analysis, resulting in a new

18
analysis perspective for food safety in the country and the transparency of food
20
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 desir-

able 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, surveil-
lance, 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.
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,
y
Maria de Fatima Ferreira Menezes, Simone Palma Favaro, and the staff of the
nl
Ministry of Education and Human Development from Mozambique.
O
se
1.9.2.2 Description
lU
na
Since 2012, the Complementary Technical Assistance Project to the Support
o
Project for the Development of a School Feeding Program in Mozambique,
rs
the result of a general agreement on trilateral cooperation among the govern-
Pe
ments of Mozambique, Brazil, and the United States of America, was devel-
r
fo
oped in Mozambique. The executing institutions are as follows:
y
op
• Mozambique government: Ministry of Education (MoE),
C
Management of Special Programs (MSP), and Department of
’s
or
Production and Feeding (DPF)

ut
• Brazil government: Ministry of Education (MEC), National Fund

b
tri
for Education Development (NFED), and National School Feeding

on
Programme (NSFP)
.C
• U.S. government: University of Florida (UF) and Michigan State

C
University (MSU)
LL
is
The project was created to develop activities that strengthen the manage-
c
an
ment of a school feeding program in Mozambique. The project also dissemi-

Fr
nates knowledge concerning the development of studies and strategies and

d
the operationalization of the information generated by these activities to

an
inform decision making. Among the actions planned in this project, applied
or
research on the topic of good food handling practices was designed in the
yl
schools to establish and strengthen recommendations.

Ta
A risk assessment was developed using a semiquantitative method based

18
on the international standards set by the World Health Organization and the
20
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
participatory methodology. The workshop speeches were interpreted using

thematic content analysis, and emergent themes included a profound reflec-
tion on the role of school feeding and the country’ s politics, reaching beyond
the technical field of nutrition and health. Different cultures can dialogue aim-
ing at the same goal, while still preserving the sovereignty of each country.
The project is implementing the recommendations. It was reported that
y
through a dialogue between the Ministries of Health and Education and
nl
Human Development, a Department of Nutrition and a health school were
O
established, evidencing a political breakthrough.
se
lU
o na
rs
Pe
1.10 Final Consideration
r
fo
This semiquantitative and qualitative assessment can, in addition to defin-
y
ing the degree of risk, help to expand the possibilities in sanitary risk mini-op
mization; draw action plans in the short, medium, and long term; support
C
managers in recognition of uncertainty; and prioritize actions and policy
’s
or
decisions. The integration of the results can be observed in Figure 1.2. From
b ut
Qualitative
tri
assessment
on
.C
Semiquantitative
C
assessment
LL
c is
an
k
ris
ary
nit
Fr
a Indirect
seds contamination
rea
d
Resolution of the unconformities
Contaminated
c
an
De water and
Political raw material Priority
or
decision Contamination by action

food handlers,
yl
equipment, and utensils

Ta
Time and
temperature
18
aspects
20
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 sani-
tary risk in the food service setting.
these results, risk management decisions can be more assertively made,

and strategies can become effective, provided that risk communication is
continuous.
y
nl
O
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se
lU
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2
Fresh-Cut Apples Spoilage and
Predictive Microbial Growth under
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Modified Atmosphere Packaging
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Predrag Putnik and Danijela Bursać Kovač ević
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CONTENTS
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2.1 Introduction................................................................................................... 29
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2.2 Importance and Challenges of Fresh-Cut Apple Production................. 31
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2.3 Fresh-Cut Apple Nonmicrobial (Physiological) Spoilage....................... 31
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2.4 Fresh-Cut Apple Microbial Spoilage.......................................................... 33

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2.5 Microbial Inactivation in Fresh-Cut Apples.............................................34

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2.6 Microbial Spoilage and Degradation of Polyphenols in Fresh-Cut

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Apples ............................................................................................................ 35
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2.7 Fresh-Cut Apples in Modified Atmosphere Packaging.......................... 36

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2.8 Mathematical Modeling and Modified Atmosphere Packaging .......... 38

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2.9 Predicting Microbial Growth in Fresh-Cut Apples Packaged

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under a Modified Atmosphere................................................................... 39

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2.10 Final Remarks................................................................................................ 41

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References................................................................................................................ 41
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2.1 Introduction
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Ta
The worldwide consumption of fruits and vegetables is constantly increas-

ing, likely due to the promotion of nutrition principles that can be found in
18
various dietary guidelines issued by various governments (USA, European

20
Union [EU], etc.). Such guidelines promote the consumption of fresh pro-
©
duce in order to improve health and decrease diet-related diseases. Fresh-

cut 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 com-
monly consumed fruits, being the second most produced fruit worldwide.
29
According to the Food and Agriculture Organization (FAO), in 2013 the total
worldwide production of apples was 80 million tons. The industrial pro-
cessing of fresh-cut apples induces plant tissue injury with various degen-
erative 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 fac-
y
tors for fresh-cut production, during which producers need to guarantee
nl
a product’ s safety and quality for the required sales period. Otherwise,
O
apples may lose their market value, as they exhibit microbial appearance
se
and sensory properties not safe and/or appealing to the consumer. Fresh-
lU
cut fruit spoilage has two origins, one internal (e.g., enzymatic and meta-
na
bolic activity of fruit) and the other external (microbial contamination).
o
rs
Therefore, preventing microbial contamination of fresh-cut products is one
Pe
of the primary concerns and a major limiting factor in extending the stor-
r
age of fresh-cut apples. Modified atmosphere packaging (MAP) is one of
fo
the most popular approaches to extend the short storage in fresh-cut apples.
y
op
MAP is based on the concept that selective packaging barriers with differ-
C
ent permeances for gases will prevent anaerobic respiration by modifying
’s
the atmosphere within the package. Additionally, bacterial growth can be

or
inhibited by altering the volumetric concentration of modified atmosphere

ut
gases within the package. Unfortunately, the role of modified atmosphere

b
tri
gases in the respiration process has not yet been clarified, as the respiration
on
process is very complex and influenced by numerous factors. For instance,

.C
some of the important factors include temperature, time, concentration of

C
added gases, size of the package, and mass of the packaged produce. The
LL
complexity is further multiplied with the addition of the other intricate

is
influences, such as the microbial growth of various bacteria or other micro-

c
an
organisms. Microbial modeling and the development of predictive models

Fr
for food microbiology are good solutions that are able to give estimates of
naturally occurring processes from a large number of parameters. By con-
d
an
structing mathematical equations, predictive modeling may lower manu-

or
facturing costs and improve food production. Currently, semifundamental

yl
Michaelis– Menten models and fundamental approaches are used for such

Ta
purposes. Semifundamental modeling is convenient, but it simplifies the

18
naturally occurring process, as it assumes that some variables are constants

20
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 indus-
trial 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 pre-
vention of fresh-cut apple spoilage.
Fresh-Cut Apples Spoilage and Predictive Microbial Growth 31
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
y
(Putnik et al. 2016b; Putnik et al. 2017), along with an increasing number of
nl
consumers becoming aware of the nutritional principles from dietary guide-
O
lines that promote the consumption of fresh fruits and vegetables (Perez-
se
Escamilla and Putnik 2007). Indeed, it is not surprising that fresh-cut apples
lU
are regularly found on the market, with production projected to increase
na
in the future (Putnik et al. 2016c). For instance, the U.S. fresh-cut industry
o
rs
recorded $14 billion in sales in 2006 (Nicola et al. 2009), while in the EU the
Pe
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
r
fo
shorter periods of time than whole apples (Putnik et al. 2016b). The average
y
op
length of storage for fresh-cut fruits and vegetables is very brief and ranges
C
from a few days to up to 2 weeks (Anese et al. 2012; Montero-Calderó n and
’s
Milagro Cerdas-Araya 2011). Operations such as peeling, coring, and slicing

or
enhance degenerative changes, such as the synthesis of ethylene, phenolic

ut
oxidation, and microbial growth (Putnik et al. 2016b). Moreover, with tissue
b
tri
damage in fresh-cut apples comes the induction of enzymatic browning, tis-

on
sue respiration and softening, the decrease of sensorial and nutritive quali-
.C
ties, and increased microbial spoilage, all with a tendency to shorten the
C
already short length of storage of this type of produce (Putnik et al. 2016c).
LL
An increased manifestation of adverse changes in fresh-cut apples can dis-

is
courage consumers from purchase and will cause loss of market value and
c
an
economic benefits (Pristijono et al. 2006). The main limiting factor for fresh-
Fr
cut production is the length of storage where apples can maintain their
original quality and be free from food spoilage (Brecht 1995). The stability
d
an
of fresh-cut produce is mainly influenced by preservation, processing, and

or
the packaging environment (Rocculi et al. 2012). Accordingly, it is vital for

yl
their commercialization to define food engineering conditions for extend-

Ta
ing the storage of fresh-cut apples while maintaining the highest possible
18
nutritive and microbiological quality (Putnik et al. 2016b; Oms-Oliu and

20
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 fea-
ture that consumers use to evaluate product quality (or food spoilage),
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 ori-
gin (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
y
acid browning). The PPO enzymes oxidize polyphenols at pH = 5 – 7 to qui-
nl
nones that form brown polymer melanins (Corzo-Martinez et al. 2012). The
O
biochemical origins of this type of browning are associated with natural
se
plants’ defense against foreign invaders, where polymerization of mela-
lU
nins is considered an impenetrable barrier among plant tissue and attack-
na
ing microorganisms (Alzetta 2014). This is considered the main source of
o
rs
browning in fresh-cut apples. Nonenzymatic browning is darkening that
Pe
might originate from ascorbic acid that is commonly present in apples
r
(Corzo-Martinez et al. 2012; Cropotova et al. 2016). It achieves a maximum
fo
rate at pH = 4 and includes the formation of dehydroascorbic acid, with a
y
op
tendency to bind amino acids and form brown polymers. Browning pre-
C
vention is commonly achieved by various agents (Ca-ascorbate, NaCl, citric
’s
acid, etc.) or in combination with UV-A or UV-C light and various edible
or
coatings (Chen et al. 2016; Lante et al. 2016; Liu et al. 2016). The use of aque-
ut
ous solutions of L-arginine also improves the quality of fresh-cut apples

b
tri
by inhibition of browning, and hence extends postharvest life (Wills and

on
Li 2016). Application of innovative technology, such as ultrasound (Jang

.C
and Moon 2011; Putnik et al. 2016b), may be used in combination with anti-
C
browning agents to improve their penetration deeper into the apple tissue
LL
and to diminish monophenolase enzymatic activity (Jang and Moon 2011).

is
One of the recent studies showed that Cripps Pink and Golden Delicious
c
an
were the best out of seven studied apple cultivars in terms of resistance to
Fr
browning. The best antibrowning treatment was Ca-ascorbate, which also

showed the best sensory score with or without application of the ultra-
d
an
sound (Putnik et al. 2016a). It was shown that ultrasound may be used in
or
combination with antibrowning agents, likely to improve their access to

yl
apple tissue and to diminish monophenolase enzymatic activity (Jang and

Ta
Moon 2011). This was confirmed in a different study where ultrasound

18
prevented browning only with the combination of ascorbic and citric acid,
20
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.
 X − 0.31 
BI = 100 ×   (2.2)
 0.17 
 a * −1.75 × L *  (2.3)
X = 
 5 . 645 × L * + a * −3. 012 × b * 
y
nl
O
It is known that consumers base their first purchase of the fresh-cut fruits
se
on visual appearance; their further purchases are made dependent on the
lU
sensorial and textural quality of the produce. While sensory characteristics
na
in fresh-cut apples are mainly related to the sugar-to-acids ratio (Beaulieu
o
2011), another relevant sensory feature to consumers is the fruit’ s texture. In
rs
industry, firmness is typically measured by destructive methods with tex-
Pe
ture analyzers (Beaulieu et al. 2004). Similarly as with taste, loss of firmness
r
fo
of fresh-cut apples during storage will negatively affect the produce’ s market
y
value (Billy et al. 2008). op
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’s
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b
2.4 Fresh-Cut Apple Microbial Spoilage

tri
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Microbial food safety is the most important limiting factor for the length of
.C
fresh-cut fruit storage, with a direct consequence on public health (Ragaert

C
et al. 2011). Food spoilage is defined as an intolerable number of microbes

LL
responsible for the off-flavors in foods (Benner 2014). Tolerance for the pres-
is
ence of spoilage microorganisms in similar foods depends on cultural and

c
an
legal settings (Putnik et al. 2016c). All fresh-cut produce from the EU should
Fr
be tested and free from Salmonella spp., Escherichia coli , and Listeria monocy-
togenes (EU Commission 2005). Some EU member states have recommended
d
an
that fresh-cut products should be tested on Staphylococcus aureus , sulfite-

or
reducing clostridia, Enterobacteriaceae (EBac), aerobic mesophilic bacteria

yl
(AMB), yeast, and mold (Benussi et al. 2011). EBac and AMB are the main
Ta
spoilage microorganisms in fresh fruits that are normally present in indus-

18
trial environments. Mostly, they are not harmful for human health unless
20
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
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).
y
nl
O
se
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2.5 Microbial Inactivation in Fresh-Cut Apples
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The growth and survival of the microorganisms in fresh-cut produce are
o
rs
affected by the type of microorganism, storage conditions, physical environ-
Pe
ment, processing, and packaging. Various microbial cultures responsible for
r
afflicting food safety and inducing spoilage are commonly present on the
fo
surface of the fresh-cut produce, while their level of contamination is com-
y
op
monly evaluated by the total bacterial count (Qadri et al. 2015). Such contam-
C
ination may originate from the environment, water, or cross-contamination
’s
from industrial equipment (USDA 2014). Containment or complete elimina-

or
tion of microbial contamination in fresh-cut produce may be achieved by

ut
various practices, such as good manufacturing practice (GMP), and food

b
tri
safety schemes, such as the Hazard Analysis and Critical Control Point
on
(HACCP) (James and Ngarmsak 2011). These approaches may be embedded

.C
in fresh-cut processing, where additional reduction of the total microbial

C
count is achieved by packaging produce in a modified atmosphere (Caleb et

LL
al. 2012b).
is
As previously mentioned, the most relevant food safety microorganisms

c
an
in the production of fresh-cut apples in the EU are Salmonella spp., E. coli ,

Fr
L. monocytogenes , S. aureus , sulfite-reducing clostridia, EBac, AMB, yeast,

and mold (EU Commission 2005). The bacterial counts of these microor-
d
an
ganisms can be biocontrolled by their microbial antagonists (Qadri et al.

or
2015). For instance, the growth of the E. coli (O157:H7), Salmonella , and
yl
Listeria innocua in fresh-cut apples can be reduced by some strains of the

Ta
pseudomonads (e.g., L-59– 66 of Pseudomonas syringae ) (Alegre et al. 2013).

18
The growth and survival of L. monocytogenes or Salmonella enterica serovar

20
Poona were successfully suppressed by the Gluconobacter asaii (T1-D1),

Candida sp. (T4-E4), Discosphaerina fagi (ST1-C9), and Metschnikowia pulcher-
©
rima (T1-E2) in fresh-cut apples (Leverentz et al. 2006). Lactic bacteria were
able to suppress the growth of E. coli , L. monocytogenes , Pseudomonas aeru-
ginosa , Salmonella typhimurium , and S. aureus in fresh-cut Golden Delicious
(Trias et al. 2008).
Washing with sanitizing solutions is considered the only step to accom-
plish 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 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
y
as organic acids, bacteriocins, esters, polypeptides, plant essential oils,
nl
nitrites, and sulfites (Franssen and Krochta 2003). It was shown that edible
O
coatings are effective in the prevention of the microbial growth of differ-
se
ent microorganisms, such as psychrotrophics, coliforms, yeast, and molds
lU
(Qadri, et al. 2015).
na
Numerous reports showed that various plant extracts and essential oils had
o
rs
growth reduction of various microorganisms (Oms-Oliu et al. 2010). In one
Pe
example, cinnamon bark extracts incorporated in the antibrowning solutions
r
decreased the growth of E. coli O157:H7 and L. innocua (Muthuswamy et al.
fo
2008), and in another, vanillin reduced the growth of the E. coli , P. aeruginosa ,
y
op
Enterobacter aerogenes , Salmonella enterica subsp. enterica serovar Newport,
C
Candida albicans , Lactobacillus casei , Penicillium expansum , and Saccharomyces
’s
cerevisiae (Rupasinghe et al. 2006).

or
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on
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2.6 Microbial Spoilage and Degradation of

C
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Polyphenols in Fresh-Cut Apples

is
The nutritive value of fruits and their products is commonly evaluated by

c
an
the content of the polyphenols (Bursać Kovač ević et al. 2015b). Polyphenols

Fr
in fruits have anti-inflammatory, antimicrobial, anticancer, and antiprolif-

erative properties (Putnik et al. 2015a; Forbes-Hernandez et al. 2014). The
d
an
main polyphenols in the apple are phenolic acids, for example, chlorogenic
or
and coumaric acids, and flavonoids, for example, epicatechin, phloretin, and
yl
quercetin (Alzetta 2014). Currently, there is no literature that associates the

Ta
loss of polyphenols with microbial growth, minimal processing, modified

18
atmosphere, apple respiration, and apple browning. In contrast, it was stated

20
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 vari-

ous 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
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 respi-
ration, and superficial browning on the polyphenolic stability in fresh-cut
y
apples. Identified polyphenols in Cripps Pink and Golden Delicious apple
nl
cultivars were coumaric, chlorogenic acid, quercetin, epicatechin, and phlo-
O
ridzin. Cripps Pink had more flavonoids and antioxidant capacity than
se
Golden Delicious. All treatments showed higher antioxidant capacities than
lU
the control treatment. During storage, microbial growth was associated only
na
with the quantities of epicatechin, with the possible indication that this com-
o
rs
pound was oxidized to quercetin with apple. Both antioxidant capacities
Pe
strongly decreased with an increased log colony-forming units (CFU) per
r
gram of both the AMB and EBac respiration (Putnik et al. 2016e). The other
fo
study showed that chlorogenic acid had no influence on bacterial growth,
y
op
whereas catechol showed antimicrobial activity for Lactobacillus brevis and
C
Lactobacillus plantarum. On the contrary, quercetin improved bacterial growth
’s
(Alzetta 2014).
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ut
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on
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2.7 Fresh-Cut Apples in Modified Atmosphere Packaging

C
LL
The major factors responsible for high-quality fresh-cut products are high-
is
quality raw material and an efficient cold chain throughout the manufac-
c
an
turing, distribution, and marketing processes (Artes et al. 2007). MAP and
Fr
refrigerated storage are frequently used to avoid negative physiological

effects by reducing respiration rates in fruits, thus extending the shelf life (Li
d
an
et al. 2011; Montero-Calderon et al. 2008). MAP employs the concepts of pack-
or
aging produce in a selective barrier that has different permeances for modi-
yl
fied atmosphere gases. The most common gases used in MAP are O2 , CO2 ,
Ta
and N2 . Generally, an atmosphere consisting of 2– 5 kPa O2 and 3– 10 kPa CO2
18
will extend the shelf life in fresh-cut products, mainly through inhibition of
20
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 microor-
ganisms by affecting the lag phase, maximum growth rate, and maximum
population densities (Devlieghere and Debevere 2000). Aside from tempera-
ture, the inhibitory effect of CO2 is also dependent on the microorganism
type, growth phase, water activity, and chemical composition of products.
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 respira-
tion. 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 rel-
y
evant parameters for the permeable MAP system are (Putnik et al. 2016c)
nl
O
se
kPO2 (y out
− yOin2 )− V  dyO2 
lU
O2 f
A  
x 100 100  dt 
na
RO2 =
M
o
(2.4)
rs
Pe
V f  dyCO2  kPCO2 (y in
CO 2 − yCO
out
)
r
2
− A
fo

100  dt  x 100
RCO2 =
y
M op (2.5)
C
’s
M
Vf = VTOTAL −
or
Mρ
ut
(2.6)
b
tri
A = length width
on
(2.7)
.C
C
where R is the respiration rate for a permeable system (cm3 /kg*day*atm),

LL
kP /x = P is the permeance of the packaging film (standard temperature and
is
pressure [STP] in cm3 /m2 /day/atm), A is the package surface area (m2 ), M is

c
an
the mass of the packaged apple cultivar (kg), x is the film thickness (cm), y
Fr
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
d
an
(cm3 ), and V f is the free volume inside of the package (cm3 ).
or
Apple respiration is the set of biochemical redox reactions where com-

yl
plex organic species are broken down to simpler units with expenditure of
Ta
O2 and production of CO2 (Putnik et al. 2016b). By changing the concentra-
18
tions of modified atmosphere gases, researchers were able to inhibit bacte-

20
rial 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, volumet-
ric 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 meta-
bolic 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).
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 con-
ditions. There are seven foodborne pathogenic bacteria associated with fresh
y
produce known to be capable of growth at or below 5° C: Clostridium botuli-
nl
num Type E, L. monocytogenes , Yersinia enterocolitica , Vibrio parahaemolyticus ,
O
enterotoxigenic E. coli , Bacillus cereus , and Aeromonas hydrophila . Two others
se
grow at temperatures just above 5° C: S. aureus at 6° C (10° C for toxin produc-
lU
tion) and Salmonella sp. at 7° C. Thus, it is of great importance that the MAP
na
inhibit the growth of these microorganisms in fresh produce under refrig-
o
rs
erated storage. Literature reports showed reduced pathogen counts (E. coli ,
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Salmonella , and L. innocua ) on fresh-cut apples under passive MAP at 5° C for
r
30 days (Alegre et al. 2010).
fo
A study observed that the introduction of the apple cultivars to MAP in
y
op
an industrial setting successfully prevented browning of both Cripps Pink
C
and Golden Delicious. Browning was successfully prevented if apples were
’s
dipped in any tested antibrowning agent. Fresh-cut nonmicrobial spoilage

or
in a modified atmosphere was well predicted from mathematical modeling

ut
(Putnik et al. 2016b).

b
tri
on
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C
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2.8 Mathematical Modeling and Modified

c is
Atmosphere Packaging
an
Fr
Mathematical modeling in food engineering is the approach used for lower-

ing experimental costs and constructing equations that are capable of approx-
d
an
imating the outcomes of natural processes from a large number of relevant

or
predictors (Putnik et al. 2016c). This statistical regression approach is based

yl
on empirical data, and it is bound to a particular level of statistical prob-

Ta
ability. That way, models will have calculated data fitness and accuracy with
18
their predictions in order to gauge their usefulness. Regression modeling is

20
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 quantify-

ing the roles of O2 and CO2 in the respiration process, namely, semifunda-
mental 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
other words, one equation for predicting microbial growth may be composed
of different types of predictors, such as fruit cultivar, volumetric concentra-
tion of gases, size of the package, mass of packaged fruits, and type of the
packaging film. Created mathematical models can be embedded into com-
puter applications and widely available to all the potential users.
A few studies have been conducted on modeling to describe the effects of
y
time and temperature on respiration rate on fresh-cut produce (Waghmare
nl
et al. 2013; Caleb et al. 2012a). Respiration rates of fresh-cut produce were
O
successfully modeled using Arrhenius and first-order decay models, which
se
could be useful for selecting suitable packaging film.
lU
na
o
rs
Pe
r
2.9 Predicting Microbial Growth in Fresh-Cut Apples
fo
y
Packaged under a Modified Atmosphere op
C
In the literature, a limited number of mathematical models have been
’s
reported that are useful for fresh-cut apple production; two good examples
or
are Pack-in-MAP® , which is a commercial web-based software tool (Mahajan

ut
et al. 2007), and Anti-browning Apple Calculator— C.A.P.P.A.B.L.E.© , which

b
tri
is free to use (Pizent et al. 2015). Pack-in-MAP is a compilation of mathemati-

on
cal models on product respiration rates and packaging permeability that are
.C
integrated in a user-friendly web-based software tool that helps in designing

C
MAP for fresh and fresh-cut fruits and vegetables. Users of that software
LL
may define the type of product, storage conditions, amount of packed prod-
is
uct, and size and geometry of the package in order to provide the best stor-
c
an
age conditions (Mahajan et al. 2007). However, currently the website seems
Fr
to be inactive.
Anti-browning Apple Calculator— C.A.P.P.A.B.L.E. is the other online soft-
d
an
ware that offers calculation of almost all parameters relevant for fresh-cut
or
apple production. Numerous mathematical equations that are statistically

yl
derived from experimental data cover the prediction of browning in apples,

Ta
whether or not treated with antibrowning agents. For instance, it is possible

18
to detect how much, on average, each of the seven cultivars will naturally
20
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 math-
ematical 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.
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 appli-
cation offers unique predictive models for microbial growth for AMB and
Ebac that simultaneously account for the type of cultivar and selection of
y
antibrowning agent, CIELab status, apple respiration, and content of the
nl
modified atmosphere. These tools can be helpful in optimizing industrial
O
parameters that will yield the least browning and AMB or Ebac growth of
se
an apple while extending the fresh-cut apple’ s shelf life. In C.A.P.P.A.B.L.E.,
lU
users can freely choose their own size of packaging, initial volumetric con-
na
centration of gases, apple mass inside of the package, and parameters for
o
rs
their own packaging film (e.g., permeance) in order to achieve optimal set-
Pe
tings for their packaging systems (Figure 2.1). Additional information about
r
the models is provided in articles published in Journal of Food Safety and
fo
Journal of Food Process Engineering (Putnik et al. 2016b, 2016c). This way,
y
op
models provide calculations for very intricate biological processes, together
C
with a great degree of flexibility and a tailor-made approach for the indus-
’s
trial packaging purposes. This is their main advantage, which will likely
or
be accompanied by increased economic benefits and provide quality foods

ut
to consumers. On the other hand, models were created on a limited num-

b
tri
ber of apple cultivars, and even though little relevant biological variance
on
.C
C
LL
c is
an
Fr
d
an
or
yl
Ta
18
20
©
FIGURE 2.1
Example of the Anti-browning Apple Calculator— C.A.P.P.A.B.L.E. computer application with
embedded mathematical models.
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 volumet-
ric 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).
y
nl
O
se
lU
2.10 Final Remarks
ona
Fresh-cut apple production may be challenging, as a shorter shelf life and
rs
proneness to fostering microbial growth may pose public health risks and
Pe
an increased tendency for economic losses. On the other hand, the market’ s
r
fo
demand for these foods will increase in the future, hence leading to increased
y
economic benefits derived from fresh-cut processing. Nonmicrobial fresh-
op
cut apple spoilage may be decreased with the application of various anti-
C
browning agents and processing technology, while microbial spoilage can be
’s
or
tackled with the application of edible coatings and packaging in a modified

ut
atmosphere. Mathematical modeling is a useful statistical approach that may

b
simultaneously account for a large number of packaging factors and help

tri
on
tailor industrial processing for each particular fresh-cut need. Mathematical

.C
equations may optimize fresh-cut production by calculating all relevant

parameters. That will save production resources and improve economic
C
LL
profitability, and likely help with monitoring microbial spoilage, hence per-
fecting HACCP procedures. Mathematical models can be incorporated in
c is
various computer applications and made conveniently available to a large

an
number of professionals. As a result, safer and quality foods for consumers

Fr
will be easily engineered at lower costs.

d
an
or
yl
Ta
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ISO (International Organization for Standardization). 2004. ISO 21528– 2:2004.

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the detection and enumeration of Enterobacteriaceae— Part 2: Colony-count

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method. Geneva: ISO.

an
ISO (International Organization for Standardization). 2013. ISO 4833– 2:2013.

or
Microbiology of the food chain— Horizontal method for the enumeration of

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microorganisms— Part 2: Colony count at 30 degrees C by the surface plating

Ta
technique. Geneva: ISO.

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Section II
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Food Allergens,
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Contaminants, and Toxins
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3
Analytical Methods for the Detection
of Mycotoxins in Milk Samples
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Myra E. Flores-Flores and Elena Gonzá lez-Peñ as
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CONTENTS
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3.1 Introduction................................................................................................... 49
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3.2 Aflatoxins ...................................................................................................... 52
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3.3 Ochratoxins ................................................................................................... 60
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3.4 Trichothecenes .............................................................................................. 62
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3.5 Fumonisins ................................................................................................... 71
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3.6 Cyclopiazonic Acid ...................................................................................... 72

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3.7 Ergot Alkaloids ............................................................................................ 73

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3.8 Zearalenone and Its Derivatives................................................................. 74

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3.9 Multimycotoxin Detection ..........................................................................80

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3.10 Conclusions....................................................................................................83
Abbreviations .........................................................................................................83
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References ............................................................................................................... 85
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3.1 Introduction
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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
or
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severe health effects to humans and animals after chronic exposure at low
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concentrations is a great concern in terms of food safety. Among these toxic

18
compounds, the presence of mycotoxins is one of the most problematic

(Zhang et al. 2014). Mycotoxins are secondary metabolites produced by fila-
20
mentous 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 con-
taminated products (Binder 2007). The Food and Agriculture Organization
of the United Nations (FAO) estimates that approximately 25% of global
49
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, espe-
y
cially in farm animals; however, in terms of human and animal health, the
nl
greatest concern is the chronic toxic effects that are generated as a result of
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continuous long-term exposure to low levels of these toxins. Toxic effects
se
vary due to the different toxicological characteristics of the more than 300
lU
known mycotoxins, including carcinogenicity, genotoxicity, nephrotoxicity,
na
immunotoxicity, and less resistance to infections (Binder 2007). Thus, human
o
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exposure to mycotoxins through diet is a significant concern to public health
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worldwide (Zhang et al. 2014).
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Among all the different human foods, the study of the presence of myco-
fo
toxins in milk is of great interest, due to its key role in children’ s diets and
y
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even in many adult diets. The well-documented presence of a broad spec-
C
trum of mycotoxins in raw materials and feeding stuffs is a subject of contin-
’s
uous monitoring programs (Zachariasova et al. 2014). It raises the possibility

or
that these toxins reach animals through the diet and are carried over into
ut
tissues or biological fluids, such as milk. The number of studies related to

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the transfer of these compounds to milk (Fink-Gremmels 2008; Flores-Flores

on
et al. 2015), or to the interactions between mycotoxins at absorption and bio-

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transformation level, is very limited (Fink-Gremmels 2008).

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It is known that rumen flora can transform mycotoxins such as ochra-

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toxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), diacetoxyscirpe-

is
nol (DAS), and T-2 toxin into their metabolites (Kalač 2011; Kiessling et al.
c
an
1984). However, this barrier can be altered by animal diseases, changes in

Fr
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
d
an
the possible presence of OTA in milk. In addition, other mycotoxins, such as

or
patulin (PAT), may remain undisturbed by the rumen (Kalač 2011). Aflatoxin

yl
M1 (AFM1), the most studied mycotoxin in milk, is produced in the liver by

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hydroxylation of absorbed aflatoxin B1 (AFB1) (Kalač 2011). Its maximum per-

18
missible level in milk has been established at 0.05 µ g/kg in the European
20
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 permit-
ted 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 fre-
y
quent combinations, and their concentration levels in milk. These studies
nl
will help lead to better risk assessment, evaluating compliance with regula-
O
tory policies and taking other actions to protect public health (Zhang et al.
se
2014). The choice of the analytical method, with adequate sensitivity, is of
lU
utmost importance in carrying out these studies (Rubert et al. 2012). In addi-
na
tion, due to the presence of fat, proteins, salts, and high water content, milk is
o
rs
a very complex matrix that requires extensive and selective sample cleanup
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procedures that not only enable the removal of the interference of coex-
r
tracted compounds, but also preconcentrate the analytes in order to reach
fo
the required low detection limits. Figure 3.1 shows the different steps that
y
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are usually taken for milk sample preparation before chromatographic anal-
C
ysis of mycotoxins. Some of them are optional (dotted square), depending on
’s
the analyzed products and the analytical method.

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This chapter is devoted to the review of the analytical methods pub-

ut
lished for mycotoxin detection and quantification in animal milk, includ-

b
tri
ing those developed for analysis of a single mycotoxin, as well as those

on
that allow the simultaneous determination of these compounds in milk.

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Milk sampling
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Homogenization
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Hydrolysis of conjugates b-Glucuronidase, aryl sulfatase

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an
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Extraction LLE/SLE (ACN, ACE, MeOH), SPE (C18), QuEChERS

yl
Ta
Cleanup Hexane, IAC, charcoal/alumina

18
20
Derivatization 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.
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 sys-
tems, have been used for the study of the different mycotoxins. Currently,
the introduction of LC coupled to mass spectrometry (LC-MS) has improved
y
the performance of the methods, allowing simultaneous detection and quan-
nl
tification of several mycotoxins from different families and with different
O
physicochemical characteristics, with adequate sensitivity, and also allowing
se
structural elucidation of unknown compounds. However, LC-MS requires
lU
high-cost equipment and specifically trained staff to operate the equipment
na
and interpret the results.
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On the other hand, among the nonchromatographic methods, immunoas-
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says are usually used for initial screenings due to their simplicity, low cost,
r
and the fact that it is easy to process a large number of samples. Nonetheless,
fo
positive results need to be confirmed (i.e., using chromatographic methods)
y
op
due to cross-reactivity with related molecules that can give overestimated
C
values (El Khoury and Atoui 2010). The enzyme-linked immunosorbent
’s
assay (ELISA) is the most popular format in this category. A wide offer of
or
ELISA-based kits is commercially available for all regulated mycotoxins

ut
in different matrixes. Among immunochemical-based methods, lateral

b
tri
flow immunoassay (LFIA), also called immunochromatographic assay or

on
immune-gold colloid (IGC) immunoassay, is relatively new in the field of

.C
food safety (Dzantiev et al. 2014), although it is widely used in the field of
C
medical diagnostics (i.e., detection of pregnancy, drug screening, identifica-

LL
tion of disease biomarkers, etc.). In the case of milk, there are commercially
is
available supplies in both LISA and LFIA formats, although only for AFM1
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an
and OTA detection.

Fr
Recently, there has been a research trend toward the construction of bio-
sensors for mycotoxin detection, but very few available methods have been
d
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published in the case of milk.

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18
20
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
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
y
levels were found 3 days after the last ingestion of AFB1 (Battacone et al.
nl
2003). AFM1 has been classified as a possible human carcinogen (group 2B)
O
by the IARC (1993).
se
Aflatoxins are the most analyzed mycotoxins in milk. The presence of
lU
AFM1 has been especially studied worldwide, and it is the only mycotoxin
na
with a defined maximum limit in milk in the European regulation (Flores-
o
rs
Flores et al. 2015). Figure 3.2 shows a survey of the methods published from
Pe
2009 until April 2016 for AFM1 detection in milk. ELISA and LC– fluorescence
r
detector (FLD) are the most used techniques and instruments for moni-
fo
toring purposes; in terms of the development of new methods, LC– mass
y
spectrometry detectors in tandem (MS/MS) is the preferential analytical op
C
technique. Currently, biosensors, immunosensors, and immunoassays have
’s
been studied and their application to the determination of aflatoxins is in

or
continuous development. Mwanza et al. (2015) evaluated the efficiency of

ut
different commercially available rapid kits and techniques (i.e., thin-layer

b
tri
chromatography [TLC], immunochromatographic strips, ELISA, and LC).

on
The study concluded that TLC, LC, and ELISA are sufficiently developed
.C
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45 42
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40 Newly developed methods

34
is
35 Application of previously developed methods

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25
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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.
in this field, and that immunochromatographic strips are not able to detect
small quantities of AFM1. Detection limits of commercially available immu-
nochromatographic 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
y
reliable in the case of the European regulation at 0.05 µ g/kg (European
nl
Commission 2010). With regard to the ELISA test, a great variety of kits
O
are commercially available (Table 3.2), but also, new attempts to improve
se
ELISA performance can be found in the literature (Guan et al. 2011a, 2011b;
lU
Kanungo et al. 2011; Wang et al. 2011; Vdovenko et al. 2014; Peng et al. 2016).
na
Biosensors based on surface plasmon resonance (Karczmarczyk et al. 2016)
o
rs
and a silicon oxynitride ring resonator (Guider et al. 2015), impedimetric apta-
Pe
sensor (Istamboulié et al. 2016), impedimetric immunosensor (Bacher et al.
r
2012; Kanungo et al. 2014), electrochemical immunosensor (Neagu et al. 2009;
fo
Paniel et al. 2010), immunochromatography combined with gold nanoparticles
y
op
TABLE 3.1
C
’s
Commercial Lateral Flow Test Strips for Detection of AFM1 in Milk

or
ut
Manufacturer LOD/ Assay Product Name

b
(Country) Sensitivity Time Observation (Reference)

tri
on
Shenzhen 300 ng/L — Qualitative analysis AFM1 Rapid Test

Lvshiyuan Dipsticks (Milk)
.C
Biotechnology (Shenzhen
C
Co. (China) Lvshiyuan

LL
Biotechnology
is
2015b)
c
Charm Sciences 350 ng/L 3 min Qualitative analysis Charm ROSA

an
Inc. (United SLAFM (Charm

Fr
States) Sciences Inc. 2016)

d
Neogen 500 ng/L 5 min Qualitative analysis Reveal for AFM1

an
Corporation (Neogen
or
(United States) Corporation 2016a)

yl
Bioo Scientific 500 ng/L 8 min Qualitative analysis AuroFlow AFM1

Ta
(United States) Designed for field use Strip Test Kit (Bioo
Scientific 2016a)
18
Unisensor 20 ng/L 10 min Instrumental AflaSensor Quanti

20
Diagnostic (quantitative) 0.05 ppb - Kit 041

©
Engineering detection (Unisensor

(Belgium) Detection range: Diagnostic
20– 200 ng/L Engineering 2016a)
Unisensor 200 ng/L 10 min Visual (qualitative) and AflaSensor Quanti
Diagnostic instrumental 0.5 ppb - Kit 078
Engineering (quantitative) (Unisensor
(Belgium) detection Diagnostic
Detection range: Engineering 2016b)
200– 750 ng/L
TABLE 3.2
Commercial ELISA Kits for Detection of AFM1 or OTA in Milk
Manufacturer Time Product Name
(Country) LOD/Sensitivity (min) Observation (Reference)
AFM1
y
R-Biopharm < 125 ng/La 15 Quantitative analysis RIDASCREEN
nl
(Germany) < 125 ng/kgb FAST AFM1
O
(R-Biopharm
se
2016b)
lU
Helica (United 100 ng/L 35 Quantitative analysis Aflatoxin M1 2000
na
States) in Milk (rapid
format) (Helica
o
rs
2016a)
Pe
Neogen 4.3 ng/L 45 Quantitative analysis Veratox for AFM1
Corporation (5– 100 ng/L); (Neogen
r
fo
(United States) cross-reactivity: Corporation
y
AFM2, AFB1, AFB2,
op 2016b)
AFG1, AFG2 (< 1%)
C
EuroClone 2 ng/L 55 Quantitative analysis AFM1 ELISA Kit
’s
(Italy) (2– 100 ng/L) (EuroClone 2014)

or
Tecna (Italy) 5 ng/La 75 Cross-reactivity: AFM2 I’ Screen AFM1

ut
50 ng/kgb (16%), AFB1, AFB2, Milk (Tecna 2016)

b
tri
AFG1, AFG2 (< 0.1%)

on
R-Biopharm 5 ng/La 75 Cross-reactivity: AFM2 RIDASCREEN

.C
(Germany) 5 ng/kgb (< 10%) AFM1

(R-Biopharm
C
2016a)
LL
Immunolab < 10 ng/Lb 140 Cross-reactivity: AFB1 AFM1 ELISA

is
GmbH (10%), AFG1 (5%), (Immunolab

c
an
(Germany) AFB2 (3.5%), AFG2 GmbH 2011)

(2.1%), STC (0.02%)
Fr
Helica (United 5 ng/L 150 Recovery 92%– 100%, Aflatoxin M1 in

d
an
States) precision 2.2%– 4.2%, Milk (high

quantitative analysis sensitivity)
or
(0– 100 ng/L) (Helica 2016b)

yl
Shenzhen 20 ng/L,a — Cross reactivity: AFB1 AFM1 ELISA Test

Ta
Lvshiyuan 100 ng/kgb (30%), AFB2 (5%), Kit (Shenzhen

18
Biotechnology AFG1 (15%), AFG2 Lvshiyuan

20
Co. (China) (7%) Biotechnology

2015a)
©
Sigma-Aldrich 2 ng/L — Recovery 92%– 100%; High-Sensitivity

(United States) precision < 8%; AFM1 ELISA Kit
quantitative analysis (Sigma-Aldrich
(5– 100 ng/L); cross 2016a)
reactivity: AFB1
(100%), AFB2 (77%),
AFG1 (64%), AFG2
(25%)
(Continued)
TABLE 3.2 (CONTINUED)

Commercial ELISA Kits for Detection of AFM1 or OTA in Milk
Manufacturer Time Product Name
(Country) LOD/Sensitivity (min) Observation (Reference)
Sigma-Aldrich — — Sensitivity Rapid AFM1
(United States) 0– 2000 ng/L, ELISA Kit
y
precision < 15%, (Sigma-Aldrich
nl
O
quantitative analysis 2016c)
(100– 2000 ng/L)
se
Romer Labs 18 ng/La — Precision 6.1%– 8.5%; AgraQuant AFM1
lU
(Singapore) 252– 257 ng/kgb recovery 93%– 119%; Sensitive 25/500
na
quantitative analysis (Romer Labs
o
25– 500 ng/L,a 2012b)
rs
270– 5400 ng/Lb;
Pe
cross-reactivity: AFB1
(88%), AFB2 (27%),
r
fo
AFG1 (11.5%), AFG2
y
(4.7%) op
Romer Labs 89 ng/La — Quantitative analysis AgraQuant AFM1
C
(Singapore) 620– 720 ng/kgb 100– 2000 ng/L,a Plus 100/2000
’s
1080– 21600 ng/Lb; (Romer Labs

or
recovery 83%– 97%; 2012a)

ut
precision 6.1%– 8%
b
tri
on
Ochratoxin A
Bioo Scientific 15 ng/L 76 Quantitative analysis MaxSignal
.C
(United States) Ochratoxin A

C
ELISA Test Kit

LL
(Bioo Scientific
is
2016b)
c
Sigma-Aldrich 80 ng/L — Recovery 114% OTA ELISA Kit for

an
(United States) Human and

Fr
Animal Serum
d
and Milk
an
(Sigma-Aldrich
or
2016b)
yl
a Milk.
Ta
b Milk powder.
18
20
(Wang et al. 2011; Zhang et al. 2012; Anfossi et al. 2013; Liu et al. 2016) or immu-
nomagnetic nanobeads (Huang et al. 2014b), and dynamic light scattering
©
(DLS) coupled with gold nanoprobes (Zhang et al. 2013) have been reported.
Some authors use spectrofluorimetry for the determination of aflatoxins in
milk. Ali et al. (2014) use spectrofluorimetry with a Vicam method for sam-
ple preparation consisting of defatting milk by adding NaCl and centrifug-
ing, and then passing the sample through immunoaffinity columns (IACs).
The LOD was 0.02 µ g/kg. Taherimaslak et al. (2014) proposed a magnetically
assisted solid-phase extraction (SPE) method using Fe3O4 nanoparticles. The
authors highlight the high extraction capacity and high efficiency of a low
amount of the magnetic adsorbent due to the high ratio of surface area to
volume and short diffusion route compared with microparticles of the com-
mercially available adsorbents. Moreover, this adsorbent did not change its
analytical performance after reusing it for up to 10 times. The complexation
of AFM1 with β -cyclodextrin enhances its fluorescence emission, allowing a
LOD of 0.052 ng/mL. Amoli-Diva et al. (2015) have developed a low-density-
y
based dispersive liquid– liquid microextraction (DLLME), followed by vortex-
nl
assisted dispersive SPE for the extraction and preconcentration of AFM1 in
O
milk before its quantification by spectrofluorimetry. Five organic solvents (i.e.,
se
ethyl acetate, toluene, 1-heptanol, 1-octanol, and 2-ethylhexanol) were tested,
lU
with 1-heptanol being the one that provided the most suitable extraction. Triton
na
X-100 was used as a signal enhancement agent for AFM1 detection. However,
o
rs
chromatographic methods are the most used ones. Fallah et al. (2010) used the
Pe
two-dimensional (2D) TLC method approved by the Institute of Standard and
r
Industrial Research of Iran for AFM1 determination in milk. Extraction was
fo
made with NaCl and CHCl3, and cleanup was carried out with silica gel col-
y
op
umn chromatography. Separation was performed by 2D-TLC. The first separa-
C
tion was developed with diethylether:methanol (MeOH):H2O (94:4.5:1.5), and
’s
the second one (at 90° direction) with CHCl3:ACE:MeOH (87:10:3). Detection

or
was made by ultraviolet (UV) detector at 364 nm. The AFM1 identity was con-
ut
firmed using trifluoroacetic acid. The achieved LOD was 0.012 µ g/kg.
b
tri
Among chromatographic methods, LC using either fluorescence or mass

on
spectrometer detectors is the main analytical technique in aflatoxin deter-

.C
mination in milk. When an FLD has been used, different sample treat-
C
ments have been described. Herzallah (2009) proposed an extraction with

LL
ACE:H2O (1:1) mixture and diatomaceous earth, followed by a cleanup step

is
with 5%NaCl:hexane (1:1). The aqueous layer was treated with 5% NaCl and
c
an
extracted three times with CHCl3, followed by a second cleanup step with
Fr
SPE– cyanopropyl. Elzupir and Elhussein (2010) applied a two-step sample

treatment proposed by the AOAC (Method 980.21). The first step consisted
d
an
of extraction with CHCl3 and 12% NaCl, and the second extraction was
or
with acetonitrile (ACN):petroleum ether. Scaglioni et al. (2014) used another

yl
AOAC method consisting of an initial extraction with MeOH and celite, fol-
Ta
lowed by partitioning with 4% NaCl and hexane and a second extraction

18
with CHCl3 and anhydrous Na2SO4. Manoochehri et al. (2015) proposed an

20
ultrasound-assisted extraction. First, the fat was removed from the milk by
centrifugation. Next, ACN was added and the sample was sonicated for
©
2 min. Anhydrous MgSO4, NaCl, dehydrated sodium citrate, and disodium

hydrogen citrate were added, and the sample was shaken and centrifuged.
A portion of the supernatant was frozen to impurity precipitation. Primary
secondary amine (PSA) and anhydrous MgSO4 were added to the aqueous
phase; afterwards, the tube was shaken and centrifuged. The supernatant
was evaporated, redissolved in ACN, and injected. Norian et al. (2015) pro-
posed an extraction with MeOH:H2O:hexane (24:6:10) previous to a cleanup
step with IAC.
Dragacci et al. (2001) carried out a collaborative study involving 12

different European countries to evaluate the effectiveness of an IAC cleanup
linked to an LC-FLD method for AFM1 determination in milk in order to
reach enough sensitivity for AFM1 detection at the established European
maximum limit in this matrix. The proposed method consisted of warming
milk to 37° C, centrifuging, and defatting, followed by an IAC cleanup pro-
y
cess. In this collaborative study, reversed-phase (RP) C18 columns were used
nl
and the mobile phases consisted of mixtures of H2O:ACN (75:25 or 67:33),
O
H2O:ACN:MeOH (65:25:10), or H2O:isopropanol:ACN (80:12:8). Detection
se
was carried out using fluorescence detection at 365 n m (excitation) and
lU
435 nm (emission wavelength). LODs of 0.02 ng/mL were achieved. In spite
na
of the wide discrepancy in the laboratory conditions, no particular analyti-
o
rs
cal effects were observed; this was considered proof of the ruggedness of the
Pe
method. Another conclusion of this study was that special attention should
r
be given to new glassware, because it must be soaked in dilute acid for sev-
fo
eral hours before use and then rinsed extensively with water to prevent loss
y
of aflatoxins. op
C
In a comparison study between OASIS® HLB and the expensive IACs,
’s
the results exhibited no significant differences in their cleanup efficiency of

or
AFM1 in milk (Wang et al. 2012).

ut
With regard to LC conditions, C18 is the most used stationary phase.

b
tri
Mobile phases are constituted by binary mixtures of ACN:H2O in gradi-

on
ent (Wang et al. 2012) or isocratic conditions (60:40) (Iha et al. 2013). Also,
.C
ternary mixtures of H2O:MeOH:ACN (6:3:2 or 6:2:2) with HNO3 and KBr

C
for postcolumn derivatization of AFM1 (Norian et al. 2015; Manoochehri

LL
et al. 2015), 1% acetic acid:MeOH:ACN (40:25:35) (Scaglioni et al. 2014), acetic

is
acid:MeOH:ACN (1:65:35) (Elzupir and Elhussein 2010), or H2O:MeOH:ACN

c
an
(57:23:20) (Ruangwises and Ruangwises 2009) have been used. In most of the
Fr
methods, the mobile phase flow is 1 mL/min. The detector conditions set
were emission wavelength of 360 nm or 365 nm and excitation wavelength
d
an
of 418– 460 nm.
or
In the case of using a mass spectrometer detector, Chen et al. (2005)

yl
proposed an extraction with C18 disks, followed by a cleanup step with

Ta
SPE. Speedisk® C18 and Speedisk PolarPlus C18 were tested for extraction.
18
Both disks retained AFM1 well, but Speedisk PolarPlus C18 was discarded
20
because it presented difficulties in the elution of AFM1 when using ACN.

The authors highlight that the method allows analysis of a large sample
©
volume (200 mL) in less time when using a SPE disk in the extraction
step rather than SPE cartridges. In the cleanup, IAC and multifunctional
cleanup columns (MFCs) (Mycosep® 226) are compared. MFC gave worse
recoveries, higher background noise in the MS detector, and higher LOD
than IACs. Campone et al. (2016) reduced the sample preparation proce-
dure with an online SPE. First, they performed a salt-induced liquid– l iq-
uid extraction (LLE) with ACN acidified with formic acid and NaCl. After
centrifugation, a diluted upper phase was injected to SPE coupled online

with the chromatographic analysis. The cartridges ZIC-cHILIC, Atlantis
HILIC Si, OASIS HLB, and BioBasic™ C18 and C8 were evaluated during
online SPE optimization. Finally, the BioBasic C18 cartridge was chosen.
DLLME is a miniaturized LLE technique that allows high throughput,
high recovery, large enrichment factor, and low cost. Campone et al. (2013)
y
proposed a sample preparation procedure as an alternative to the expen-
nl
sive IACs based on the separation of the milk lipid fraction by centrifugation
O
at 4° C, followed by simultaneous precipitation of proteins and extraction
se
of AFM1 (with NaCl and ACN). The ACN phase was extracted by DLLME
lU
(with CHCl3 and H2O). Protein precipitation using acetic acid instead of NaCl
na
was discarded because of a remarkable loss of the analyte due to the ability
o
rs
of caseins to bind AFM1 during coagulation. The final procedure achieves
Pe
a limit of quantification (LOQ) of 2 ng/kg and recoveries in the range of
r
61%– 75%. Huang et al. (2015) proposed an automated hollow-fiber liquid-
fo
phase microextraction. The homemade device was fabricated by combining
y
op
pipette tips (as a needle guide) with hollow fibers. Anti-AFM1 antibody in
C
phosphate-buffered solution (PBS) was used as the extraction phase. The
’s
method is very simple because no special treatment (i.e., protein precipita-

or
tion, filtration, or centrifugation) is required, but the LOQ was 0.21 µ g/kg,
ut
low enough to satisfy the Food and Drug Administration (FDA) and Chinese
b
tri
regulations, but not the European regulation.

on
Turbulent flow chromatography (TFC) is a relatively novel technique

.C
that combines size-exclusion and traditional column chemistry to separate

C
macromolecules from molecules in biological fluids. TFC offers an efficient

LL
removal of interferences from biological fluids in comparison with SPE

is
and LLE. Like online SPE, time and solvent consumption are considerably
c
an
low; moreover, TFC columns are reusable for several hundred injections
Fr
(Couchman 2012). Fan et al. (2015) developed a method based on ultrasound-

assisted extraction, followed by online cleanup using TFC, for the analysis
d
an
of AFB1 and AFM1 in milk. It takes only 9.3 min to perform online cleanup
or
and analysis. Six types of TurboFlow columns with different chemical modi-
yl
fications were tested (C18-XL, C18-P XL, Cyclone, Cyclone-P, Cyclone-MAX,

Ta
and Cyclone-MCX), with C18-XL being the best one. Compared with the SPE
18
offline method, online TFC columns have better precision and higher effi-
20
ciency, but the sample cannot be concentrated enough, achieving an LOQ of

0.1 µ g/kg for AFM1 and AFB1.
©
For LC separation, C18 columns with particle size from 1.7 to 3.5 µ m were
used. MeOH:H2O or ACN:H2O with formic acid (0.01%– 0.2%) or 0.4 mM
ammonium formate or 5 mM ammonium acetate were used as mobile phases
at flows in the range of 0.25– 0.8 mL/min. Chen et al. (2011) tested four col-
umns (BEH C18, BEH HILIC, HSS T3, and BetaBasic™ C18), concluding that
ultra-high-performance liquid chromatography (UHPLC) columns greatly
reduce the chromatographic time and give low instrumental detection limits
compared with high-performance liquid chromatography (HPLC) columns.

The obtained LOD was 0.18 ng/kg for milk and 2.08 ng/kg for milk powder.
These authors also recommend silylation using dimethyldichlorosilane:toluene
(7:93) to reduce the adsorption of AFM1 in the glassware, and destroy AFM1
residues before cleaning and reuse by soaking glassware in 15% sodium
hypochlorite. The most frequently used MS detector is the triple quadrupole
y
with electrospray ionization (ESI). In some cases, the use of a QTrap detector
nl
is reported (Huang et al. 2015; Britzi et al. 2013). Cavaliere et al. (2006) made
O
a comparison and a broad discussion to evaluate the performance of ESI,
se
atmospheric pressure photoionization (APPI), RP, and normal-phase (NP) col-
lU
umns: RP-LC/ESI-MS/MS, NP-LC/APPI-MS/MS, and RP-LC/APPI-MS/MS
na
were compared. NP-LC/APPI-MS/MS gave an adequate LOD (30 ng/L) but
o
rs
did not offer any advantages, and moreover, it uses toluene, a toxic solvent.
Pe
With regard to ion source in RP, APPI achieved a LOD of 6 ng/L, and ESI a
r
LOD of 12 ng/L, both in positive mode. The authors concluded that although
fo
APPI reached a lower LOD value, ESI is preferred because not only it is more
y
op
robust and widespread nowadays but also the achieved LOD is low enough
C
to detect the presence of AFM1 in milk below the maximum permitted level
’s
in the EU. During ESI in positive mode, AFM1 can give [M+H]+ and also
or
[M+Na]+ ions in H2O:MeOH and H2O:ACN solutions. Sodium adduct is, by

ut
far, the most prevalent ion, but the signal for [M+H]+ can be increased by addi-
b
tri
tion of 2 mmol/L ammonium acetate in the mobile phase.

on
.C
C
LL
cis
3.3 Ochratoxins
an
Fr
Ochratoxins are produced by Aspergillus and Penicillium fungi (Monaci and

Palmisano 2004). OTA possesses carcinogenic, nephrotoxic, teratogenic,
d
an
immunotoxic, and possibly neurotoxic properties (EFSA 2006). The IARC

or
(1993) has classified it as possibly carcinogenic to humans (group 2B), and

yl
the EU has recommended a tolerable weekly intake (TWI) of 120 ng/kg body
Ta
weight (EFSA 2006). Ochratoxin B (OTB) is the dechloro analog of OTA and
18
may coexist in naturally contaminated materials; its concentrations are gen-

20
erally low, and it appears to be much less toxic than OTA (EFSA 2006; Mally
et al. 2005).
©
Biotransference of OTA into milk has been demonstrated in animal spe-

cies (Ferrufino-Guardia et al. 2000). In the case of ruminants, the microflora
of the rumen decreases OTA bioavailability through its hydrolysis to ochra-
toxin α (OTα ) (Gonzá lez-Osnaya et al. 2008). Even though only low concen-
trations of OTA are expected in milk, these small amounts may be important
for consumers of large quantities (Gonzá lez-Osnaya et al. 2008), particularly
children, who can reach a total daily intake of OTA greater than the rec-
ommended tolerable daily intake (TDI) of 5 ng/kg body weight/day (SCF
1998). However, the European Commission has not established regulations

for OTA contamination in milk and dairy products (Duarte et al. 2012).
With regard to nonchromatographic methods, Valenta and Goll (1996)
adapted an ELISA-based method to milk; the method was previously devel-
oped for the analysis of OTA in pig serum. Bioo Scientific (2016b) and Sigma-
Aldrich (2016b) have developed commercial ELISA test kits that reach LOD
y
values of 0.015 and 0.08 ng/mL, respectively (see details in Table 3.2).
nl
Chromatographic methods developed for OTA quantification in animal
O
milk are based on TLC or LC using FLD or MS detectors. Shreeve et al. (1979)
se
studied the carryover of OTA and the presence of its metabolite OTα in the
lU
milk of dairy cows using LLE and TLC methods adapted from those applied
na
to the analysis of OTA and OTB in barley. To the best of our knowledge,
o
rs
Breitholtz-Emanuelsson et al. developed the first LC-FLD method for OTA
Pe
determination in rat milk (Breitholtz-Emanuelsson et al. 1993b) and in cow
r
milk (Breitholtz-Emanuelsson et al. 1993a). Other LC-FLD based methods
fo
for cow milk are those published by Valenta and Goll (1996), Skaug (1999),
y
op
Boudra and Morgavi (2006), Bascará n et al. (2007), Gonzá lez-Osnaya et al.
C
(2008), Pattono et al. (2011), and Bulea et al. (2011).
’s
For OTA extraction from milk, CHCl3 (Boudra and Morgavi 2006), MeOH
or
(Gonzá lez-Osnaya et al. 2008), ACN (Pattono et al. 2011), and CHCl3-MeOH

ut
mixtures (Skaug 1999; Valenta and Goll 1996; Breitholtz-Emanuelsson et al.

b
tri
1993a, 1993b) have been used. Usually, a second extraction or a cleanup step
on
is included using hexane (Breitholtz-Emanuelsson et al. 1993b; Pattono et

.C
al. 2011), SPE, or IAC. Breitholtz-Emmanuelson (1993a, 1993b) made the first
C
attempt to apply SPE for OTA extraction from milk in homemade devices,
LL
packing silica gel into Pasteur pipets. After that, Valenta and Goll (1996) and
is
Skaug (1999) tested commercially available silica gel cartridges (LiChrolut®

c
an
and Bond Elut® ). Different commercial IACs have been developed for OTA
Fr
extraction and/or cleanup. Boudra and Morgavi (2006) applied Ochraprep®

columns to OTA extracted from milk. In a comparison study, Bascará n et al.
d
an
(2007) obtained better results when using Ochraprep columns than when
or
using Ochratest® columns. Because prepacked columns are expensive, some

yl
authors have tried to use them more than once applying a reconditioning
Ta
treatment. Boudra and Morgavi (2006) reused them more than three times
18
without any noticeable loss in binding properties when Ochraprep columns

20
were treated with 0.02% (w/v) sodium azide in PBS after being used. Pattono
et al. (2011) recommended washing all glassware with MeOH in order to
©
avoid a loss of OTA by salt formation, precipitation, and/or adsorption to

glassware, as previously reported by Pena et al. (2005) and Remiro et al.
(2010).
In each case, RP chromatography with C18 columns and particle size
of 3 or 5 µ m were used. Some of them used isocratic conditions for chro-
matographic separation using different mixtures of ACN:H2O:CH3COOH
(55:45:0.5, 50:49:1, or 49.5:49.5:1) (Bascará n et al. 2007; Gonzá lez-Osnaya et al.
2008; Pattono et al. 2011) as mobile phases. Other methods used gradient
conditions with 1% CH3COOH:MeOH (Boudra et al. 2007), 10 mM tetrabutyl

ammonium bromide in MeOH:potassium phosphate buffer (51:49 or 50:50)
(Breitholtz-Emanuelsson et al. 1993a, 1993b; Skaug 1999), and ACN:0.008 M
orthophosphoric acid (Valenta and Goll 1996). With regard to the detector,
FLD was usually used with different excitation (330, 333, 334, or 380 nm) and
emission (450, 460, or 464 nm) wavelengths. When OTA, OTB, and OTα were
y
analyzed together, an excitation wavelength at 274 nm and emission wave-
nl
length at 440 nm were set (Boudra and Morgavi 2006). Because ochratoxins
O
are natural contaminants, a confirmation of their presence in the samples
se
is often needed, and there is a broad variety of alternatives that have been
lU
discussed in detail by Li et al. (1998). A simple method consists of the esteri-
na
fication of OTA and its metabolites in the presence of MeOH with boron
o
rs
trifluoride (Breitholtz-Emanuelsson et al. 1993a) or HCl (Li et al. 1998).
Pe
Nowadays, several LC-MS methods for simultaneous determination of
r
ochratoxins (OTA, OTB, and OTα ), together with other mycotoxins in milk,
fo
have been developed, and they are summarized in Table 3.3.
y
op
C
’s
or
ut
b
3.4 Trichothecenes
tri
on
Trichothecenes are the largest group of mycotoxins, produced particularly

.C
by molds belonging to the genus Fusarium, ubiquitous in areas with a moder-

C
ate climate. About 170 trichothecenes have been discovered up to now (Krska
LL
et al. 2001). They are toxic due to an epoxide group in their chemical struc-
is
ture (WHO-IPCS 1990). Trichothecenes are classified into groups according

c
an
to their characteristic functional groups. Type A includes, among others,

Fr
monoacetoxyscirpenol (MAS), DAS, neosolaniol (NEO), and the highly toxic

HT-2 and T-2 toxins. The type B trichothecenes possess a carbonyl functional
d
an
group at the C8 position. The most frequently detected type B trichothe-

or
cenes in food are DON (also known as vomitoxin), 3-acetyldeoxynivalenol

yl
(3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusare-

Ta
non X (FUS-X) (Josephs et al. 2004).

18
Although occasionally nonchromatographic methods, such as liquid

20
scintillation counter, have been used to study the transmission of NIV and
FUS-X to milk (Poapolathep et al. 2004), trichothecenes have usually been
©
analyzed using GC with either flame ionization (Robison et al. 1979), elec-
tron capture (EC) (Prelusky et al. 1984; Swanson et al. 1986), or mass spec-
trometric detection (Robison et al. 1979; Collins and Rosen 1979; Prelusky et
al. 1984, 1987; Seeling et al. 2006). LC using UV (Prelusky et al. 1987; Keese
et al. 2008; Seeling et al. 2006) and MS detectors (Keese et al. 2008) are also
suitable. In terms of gas chromatographic conditions, the first attempts made
included the use of a flame ionization detector (FID) or EC detector and
packed columns (Robison et al. 1979; Swanson et al. 1986). More recently,
©
TABLE 3.3 20
LC-MS/MS Methods for Multimycotoxin Analysis in Milk
18
LC Parameters: Column
Ta
yl Dimensions, Particle Size,
or Column Model, Column
Temperature, Flow Rate,
Injection Volume; Mobile
an
ESI + ESI –
d Sample Treatment Phase; Detector Reference
Fr
AFM1, HT-2, T-2, T-2 triol, DAS, DON, DOM-1, aMethod A (nonbound mycotoxin): 5 mL 150 × 4.6 mm, 5 µ m, Sø rensen
MAS, FB1, FB2 3-ADON, milk.
nc Adjust 2 4
pH = 2 (100 µ L H SO 18%). Phenomenex Luna C18 100A and Elbæ k
15-ADON, OTA, Stand.
is Add 10 mL hexane + 16 mL ACN. and Thermo Electron (2005)
ZEA, α -ZEL, Shake, Lcentrifuge. Reduce 10 mL ACN Hypersil ENV, 25° C, 0.6 mL/
β -ZEL, α -ZAL, phase to L 1C mL (N flow, 60° C). Add 10 mL
2 2
min, 50 µ L. H O:MeOH
β -ZAL 2
H O. Adjust .pH = 8.5 (NaOH). Apply on (0.02% acetic acid), without
OASIS HLB cartridge,
C add 5 mL H O 2 acid for ESI(– ). QqQ.
(wash) and 4 mL MeOH
on (elution). Dry
(60° C), add 500 µ L MeOH
tri 20%, filter
(polytetrafluoroethylene bu[PTFE]).
Method B (total mycotoxin):to 5 mL milk
(pH = 6.6– 7). Add 100 µ L r’s
β -glucuronidase. Stand (37° C, 2 h).
Continue with Method A.
C
o
AFB1, AFB2, AFG1, AFG2, OTA, — 2.5 g sample + 5 mL H O, mix. Add 15 p
2 mL × 2.1 mm, 1.7 µ m, Waters Mol et al.
DON, 3-ADON, 15-O-acetyl-4- ACE (1% HCOOH). Shake, centrifuge, (2008)
y 100BEH-C18, 40° C, 0.4 mL/min,
DON, DAS, HT-2, T-2, FB1, FB2,
f (0.002%
and filter (0.45 µ m). 2
or 5 µ L. H O:MeOH
FB3, ZEA, α -ZEL, β -ZEL, STC, Pformic acid, 1 mM
ergocornine, ergocristine, ammonium
er
ergocryptine, ergonovine,
Analytical Methods for the Detection of Mycotoxins in Milk Samples
so formate). QqQ.
ergotamine na
lU (Continued)
se
63
O
nl
y
©
20 64
TABLE 3.3 (CONTINUED) 18
Ta
yl LC Parameters: Column
Dimensions, Particle Size,
or
an Column Model, Column
d Temperature, Flow Rate,
ESI + ESI – Sample Treatment Phase; Detector Reference
Fr
an
T-2, AFB1, DON — 10 m
ciL milk + 10 mL HCOOH 50 × 2.1 mm, 1.9 µ m, Thermo Filigenzi
(0.1%)s + 12 mL ACN (0.1% HCOOH). Fisher Hypersil Gold AQ, et al. (2011)
Centrifuge. 4
LL Add 6 g MgSO + 1.5 g 35° C, 0.38 mL/min, 20 µ L.
CH COONa
3 C to supernatant. Shake, H O:ACN (0.1% formic acid).
2
centrifuge. Dry . CACN layer (N ). Add 2
40 µ L MeOH + 1o 2
60 µ L H O. Filter
(0.45 µ m, polyvinylidene
nt fluoride
[PVDF]). rib
AFM1, AFG1, AFG2, AFB1, — 2
10 mL milk + 10 mL H O.u Centrifuge. × 2.1 mm, 1.7 µ m, Waters Aguilera-
AFB2, OTA, HT-2, T-2 Luiz
Loan supernatant onto C18 o
t cartridge, add 100BEH-C18, C, 0.35 mL/
5 mL H O + 5 mL hexane (wash)
2 MeOH:H O
2 et al. (2011)
r’s + 5 mL min, 5 µ L. 30°
2
MeOH (elute). Dry (N ), add 1 mLCmobile (5 mM ammonium formate).
phase. op QqQ.
AFM1, AFG1, AFG2, AFB1, — 8 g sample + 32 mL ACN, shake and y 50 × 2.1 mm, 1.7 µ m, Waters Beltrá n et al.
AFB2, OTA 2
centrifuge. Dry supernatant (50° C, N ) fo Acquity BEH-C18, 40° C, (2011)
and dilute until 20 mL with H O. Pass 2

r P0.3 mL/min, 20 µ L.
through AflaOchra HPLC™ , wash (5 mL 2
He O:MeOH (0.1% formic
H O), elute (4 mL MeOH), and dry (50° C,
2 acid,rs0.5 mM ammonium
N ). Redisolve with 1 mL H O.
2 2 acetate). onQqQ.
(Continued)
al
U
se
Food Safety and Protection
O
nl
y
©
20
Ta
or
Fr
an
AFM1, AFM2, AFG1, AFG2, — Enzymolysis:
ci 2 g sample + 100 µ L 150 × 2.1 mm, 3.5 µ m, Thermo Chen et al.
AFB1, AFB2, OTA β -glucuronidase
s 1 mL physiological Hypersil Gold C18, 35° C, (2012)
saline (37°
LLC, 5 h, +water bath). 0.2 mL/min, 10 µ L.
PLE procedure:
C Enzymolyzed ACN:H2 O (5 mm ammonium
sample + 4 g.diatomaceous earth. acetate). QqQ.
C
Extraction with ACN:hexane (50:50),
100° C, 5 min, 1500n
o psi. Centrifuge.
Cleanup: Dry 10 mL ACN 2
tri phase (N ,
b 2
50° C), redissolve with 10u mL H O (pH 7.5
with NaOH). Pass throughtOASIS o HLB
cartridges + 5 mL 20% MeOHr’(wash),s
dry. Elute (4 mL MeOH) and evaporate C
(N , 50° C). Resuspend (ACN:H O) (10:90)
2 2 op
and filter (0.22 µ m, nylon). y
AFM1, AFB1, AFB2, AFG1, ZEA, ZAN 4 mL milk + 150 mg EDTA-Na2 + 40 µ L fo 100 × 2.1 mm, 1.8 µ m, Waters Zhan et al.
AFG2, OTA internal standard (IS) + 5 mL EtOH:ACN r PAcquity HSS-T3, 40° C, (2012)
(1:5). Mix, centrifuge, filter (plug of 0.4e mL/min, 10 µ L.
cotton). To 4.5 mL of clear filtrate add 2
H Or:MeOH formic
4 mL alcohol + 35 mL ACN. Mix, acid, 2.5 ammonium

so mM(0.1%
centrifuge. Dry supernatant (40° C). Add acetate). QqQ.
na
1.25 mL H2 O:EtOH:ACN (6:3:1). Mix, lU
filter (0.22 µ m PTFE). se
(Continued)
65
O
nl
y
©
20 66
Ta
or
Fr
an
NEO, DAS, MAS, HT-2, T-2 NIV, DON, Enzymolysis:
ci 2 g sample + 100 µ L 150 × 2.1 mm, 3.5 µ m, Thermo Chen et al.
DOM-1, FUS-X, β -glucuronidase
s 1 mL physiological Hypersil Gold C18, 35° C, (2013)
3-ADON, saline (37°
LLC, 16 h, +water bath). 0.2 mL/min, 10 µ L.
15-ADON, T-2 Extraction by C ultrasonic bath: ACN:H2 O. QqQ.
triol, T-2 tetraol, Enzymolyzed. sample + 10 mL ACN:H O 2
(90:10). Ultrasonic
ZEA, α -ZEL,
C bath (15 min, 40° C).
β -ZEL, ZAN, Centrifuge. Upper n
o layer + 10 mL hexane,
α -ZAL, β -ZAL mix. Discard hexane phase.
tri
Semiautomated cleanup by
bu SPE (Gilson
Aspec XL4): 10 mL of the extract
to onto
®
Bond Elut Mycotoxin column. r’sDry the
2
eluate (N , 50° C). Resuspend and C filter
(0.22 µ m, nylon). op
AFB1, AFB2, AFG1, AFG2, OTA, ZEA 2
2.5 g sample + 10 mL ACN:H O (80:20) y 50 × 2.1 mm, 1.7 µ m, Waters Beltrá n et al.
FB1, FB2, NIV, DON, FUS-X, (0.1% HCOOH). Shake, filter (paper). fo Acquity UPLC BEH-C18, (2013)
NEO, 3-ADON, 15-ADON, Dilute to 50 mL with H2 O. r P40° C, 0.5 mL/min, 10 µ L.
DAS, T-2 triol, HT-2, T-2 2
He O:MeOH (0.01% formic
0.1 mM ammonium
acid,rs
acetate).
onQqQ.
al (Continued)
U
se
O
nl
y
©
20
Ta
or
Fr
an
AFM1 OTA, ZEA, sample + 8 mL ACN, mix and
2 g c 50 × 2.1 mm, 1.7 µ m, Waters Huang et al.
α -ZEL centrifuge.
is Concentrate to 2 mL (N , 2 UHPLC BEH-C18, 40° C, (2014a)
50° C). Add 2
LL 4 mL H O, adjust pH = 5 2
0.4 mL/min, 5 µ L. H O (0.1%
(PBS). Apply C on OASIS HLB, add 2 mL ammonia):MeOH. QqQ.
H O (wash) and
2
. C4 mL MeOH (elution).
Dry (50° C). Resuspend
46 mycotoxins 12 mycotoxins 15 g sample + 10 mL ACN:H Jia et al.
on and filter (PTFE).
2 × 2.1 mm, 2.6 µ m, Thermo
CH COOH). Mix. Add
3 C-18 aQ, 0.3 mL/ (2014)
tri 6 g O (84:16) (1% 100Accucore
MgSO + 1.45 g CH COONa.
4 3 2
min, 5 µ L. H O:MeOH (0.1%
bu Shake,
t
centrifuge. To 8 mL of upperophase, add formic acid, 4 mM
4
1.2 g MgSO + 108 mg PSA + 4’05 formate).
r s mg C18. ammonium
Mix, centrifuge. To 200 µ L of upper Clayer, Q-Orbitrap.
add 300 µ L MeOH + 500 µ L 8 mM o
ammonium formate buffer. Shake, filtery
p
(0.22 µ m, nylon). fo
295 bacterial and fungal 2 3
5 g sample + 20 mL ACN:H O:CH COOH r150 × 4.6 mm, 5 µ m, Malachová
metabolites in ESI + and – (79:20:1), shake, centrifuge. Add 3.5 mL et al. (2014)
PPhenomenex
e Gemini C18,
ACN:H O:CH COOH (20:79:1) to 3.5 mL
2 3 25° Cr,s1 mL/min, 5 µ L.
supernatant, mix. MeOH:H 2 3

on O: CH COOH
(5 mM ammonium
al acetate),
QTrap. U
(Continued)
se
67
O
nl
y
©
20 68
Ta
or
Fr
an
AFM1, AFB1, AFB2, AFG1, OTα sample + 4 mL MeOH (1% HCOOH),
2 g c 50 × 2 mm, 5 µ m, Varian Tsiplakou et
AFG2, OTA, DAS, HT-2, T-2, mix,iscentrifuge. Freeze upper phase Polaris C18, 0.25 mL/min, al. (2014)
ZEA (12 h). Filter
LL (0.45 µ m, PTFE). 50 µ L. H2 O (0.1% formic acid,
C 5 mM ammonium formate,
.C 0.02% ACN):MeOH (0.1%
formic acid, 5 mM
ammonium formate). QqQ.
on
NIV, DON, DOM-1, FUS-X, — 1 mL milk + 4 mL ACN 150 × 2.1 mm, 2.7 µ m, Supelco Flores-
tri (2% HCOOH).
NEO, 3-ADON, 15-ADON,
b
Shake, centrifuge. Add 60umg C18 Ascentis Express, 45° C, Flores and
DAS, HT-2, T-2 3
t
CH COONa to supernatant.oShake, 0.4 mL/min, 15 µ L. Gonzá lez-
centrifuge. Dry 3.5 mL upper phase H2 O:MeOH (0.1% formic Peñ as
r’s
(65° C). Add 200 µ L mobile phase.C Mix, acid, 5 mM ammonium (2015)
filter (0.45 µ m, PVDF). op formate).
AFM1, OTA, ZEA, AFB1 — 2
5 g milk powder + 25 mL H O, stir, watery 150 × 2.1 mm Thermo Fisher Wang and
bath (40° C, 10 min), filter. Size-exclusion fo Scientific C18, 30° C, 0.2 mL/ Li (2015)
SPE, 8 mL H2 O + 8 mL ACN:H2 O (30:70), rmin. H2 O:ACN. TSQ
0.8 mL/min. quantum MS.
Pe
(Continued)
rs
o na
lU
se
O
nl
y
©
20
Ta
or
Fr
an
AFM1, AFM2, AFB1, AFB2, ZEA, ZAN, sample + 20 mL ACN:ethyl
5 g c 100 × 2.1 mm, 3.5 µ m, Agilent Xie et al.
AFG1, AFG2, OTA, DOM-1, α -ZEL, β -ZEL, acetate:CH 3
is COOH (49.5:49.5:1). Mix, Zorbax SB-C18, 40° C, (2015)
FB1, FB2 α -ZAL, β -ZAL ultrasonic
LLtreatment, centrifuge. Repeat 0.4 mL/min, 10 µ L.
in the precipitate.
C Join both supernatants H2 O:ACN (0.1% formic acid,
and freeze (– .80° C, 20 min). Centrifuge, 5 mM ammonium acetate),
C Add 10 mL MeOH:H O
filter, dry (40° C).o 2 without acid and salts to
(1:9). Apply on OASIS nt HLB, add 5 mL ESI(– ), QqQ.
H O (wash) + 4 mL MeOH:ACN:NH
2
rib 4 OH
(47.5:47.5:5) + 4 mL MeOH:CH 2 2
Dry (N , 40° C). Add 1 mL ACN:H
ut Cl (3:7).
2 2
o O
(10:90) + 3 mL hexane/ACN.rCentrifuge
’s
the lower layer and filter (0.22 µ mC ,
nylon). op
— ZEA, α -ZEL, 500 µ L milk + 20 µ L IS + 1 mL sodium y 100 × 2 mm, 2.8 µ m, Agilent Winkler
β -ZEL, ZAN, acetate buffer + 40 µ L β -glucuronidase. fo Pursuit XRs Ultra C18, 40° C, et al. (2015)
α -ZAL, β -ZAL, Incubation (37° C, 16 h). Add 125 µ L r P0.4 mL/min, 10 µ L.
DON, DOM-1 NaOH 1 M. Mix, centrifuge. Apply 2
He O:ACN:MeOH. QTrap.
supernatant on OASIS HLB, add 1 mL rs
MeOH 5% (wash) and 2 mL MeOH on

(elution). Dry (centrifugal evaporator). al
Add 500 µ L MeOH:H2 O (70:30). Filter U
(PVDF). se
Note: QqQ, triple quadrupole.
69
O
nl
y
Seeling et al. (2006) developed a GC-MS method using an Rtx® -200, 60 m, 0.25

inner diameter, and 0.1 µ m film thickness column (Restek). The carrier gas
was helium at 1.3 mL/min, and the temperature rate was between 115° C and
300° C. With regard to LC methods, Prelusky et al. (1987) and Vudathala et al.
(1994) determined DON and DOM-1 by HPLC-UV with C18 columns and
a mixture of ACN:H2O as the mobile phase. UV was fixed at 220, 234, and
y
254 nm. In addition, for DON and DOM-1 determination, Keese et al. (2008)
nl
used an LC-MS/MS method with a BetaSil™ phenyl/hexyl column (Thermo
O
Electron Corporation) and a mobile phase of 0.13 mM (pH 7.4) ammonium
se
acetate:ACN in gradient conditions.
lU
Milk sample preparation usually starts with an extraction process. LLE
na
with ACN:H2O (Keese et al. 2008) or H2O:MeOH (Seeling et al. 2006) has
o
rs
been used for DON and DOM-1, and CHCl3 (Robison et al. 1979) or ethyl
Pe
acetate (Collins and Rosen 1979) for T-2. Also, solid-supported liquid extrac-
r
tion (SLE) columns have been tested (Swanson et al. 1986; Prelusky et al. 1987;
fo
Vudathala et al. 1994). Trichothecenes have also been extracted from milk for
y
multidetection methods (see Section 3.8). op
C
After extraction, a cleanup step is usually used with petroleum ether (Keese
’s
et al. 2008) or hexane (Collins and Rosen 1979; Robison et al. 1979). Also, SPE
or
using different trademarks of C18 columns has been used (Prelusky et al.
ut
1984; Swanson et al. 1986; Seeling et al. 2006). IACs (Keese et al. 2008) and
b
tri
charcoal/alumina columns (Prelusky et al. 1984, 1987; Vudathala et al. 1994;

on
Keese et al. 2008) have also been used.

.C
Derivatization is widely used to improve trichothecene response in the

C
detection system because of the lack of response of this group of toxins in

LL
the fluorescence and UV detectors. The chosen reagent depends on the pur-
is
pose of the analysis, the toxin concentration, the matrix, and the detection
c
an
system (Koch 2004). Conversion of the hydroxyl groups to trimethylsilyl

Fr
(TMS), trifluoroacetyl, pentafluoropropionyl, or heptafluorobutyryl (HFB)

derivatives of trichothecenes leads to better peak shape and sensitivity (Eke
d
an
and Torkos 2004). For GC, silylation is widely preferred (Robison et al. 1979;
or
Collins and Rosen 1979; Prelusky et al. 1987; Seeling et al. 2006). The selec-
yl
tion of the suitable reagent to form TMS derivatives is of utmost importance.

Ta
While type A trichothecenes react with many available silylating reagents,

18
mixtures containing trimethylsilylimidazole (TMSI) are essential for com-

20
pleting derivatization of type B trichothecenes (Gilbert et al. 1985; Eke and

Torkos 2004; Koch 2004). Also, heptafluorobutyrylimidazole has been used
©
(Prelusky et al. 1984). Swanson et al. (1986) made a comparison between two
derivatizing reagents: TMSI:trimethylchlorosilane (5:1) mixture and hepta-
fluorobutyrylimidazole. HFB ester derivatives of standards gave threefold
greater response factors, but HFB derivatives from milk extracts showed more
complex chromatograms than those of TMS ether derivatives. Moreover, HFB
derivatives obtained from frozen milk samples occasionally presented impu-
rities that coeluted with DON, interfering with its quantitation. In addition,
increased sensitivity was observed when samples were analyzed 18 h after
derivatization. TMS-DON derivatives were stable at least 10 days at room

temperature. No apparent differences have been observed in recoveries or in
the chromatographic profiles of DON and DOM-1 between fresh and frozen
milk. However, frozen milk required homogenization with vigorous shak-
ing at 50° C to prevent the clogging of particulate matter on the top of the
extraction column and, consequently, low recovery. Finally, β -glucuronidase
y
is widely used in DON and DOM-1 analysis to break their glucuronide con-
nl
jugates (Prelusky et al. 1987; Seeling et al. 2006; Keese et al. 2008).
O
se
lU
ona
rs
3.5 Fumonisins
rPe
FB1, synthesized mainly by Fusarium verticillioides and Fusarium proliferatum,
fo
is poorly metabolized in the rumen (Caloni et al. 2000) and could reach
y
op
the milk. The IARC (2002) has classified FB1 as possibly carcinogenic to
C
humans (2B group). A provisional maximum tolerable daily intake (PMTDI)
’s
of 2 µ g/kg body weight/day for FB1, FB2, and fumonisin B3 (FB3), alone or
or
in combination, has been established by the EU (WHO 2011). Fumonisins

ut
do not have a good chromophore in their structures, requiring a chemi-

b
tri
cal modification to become detectable by UV or fluorescence (Krska et al.

on
2007; Ndube et al. 2009). They are not susceptible to GC detection either.
.C
Therefore, MS offers important advances in the study of the presence of

C
these mycotoxins in milk.

LL
There is evidence that some fumonisins are matrix bound, especially to pro-
is
teins and sugars (EFSA 2005); thus, a hydrolysis step with β -glucuronidase is
c
an
recommended before extraction (Scott et al. 1994). For extraction and cleanup
Fr
steps, Maragos and Richard (1994) and Richard et al. (1996) use a MeOH:acetone
(ACE) mixture (50:50), followed by SPE with strong anion exchange columns.
d
an
Scott et al. (1994), Spotti et al. (2001), and Gazzotti et al. (2009) applied milk
or
samples directly over FumoniTest® IACs without previous treatment. SPE C18

yl
cartridges have also been tested for the extraction and purification of amino-
Ta
pentol, a compound produced by the hydrolysis of FB1 in milk (Scott et al.

18
1994).
20
LC-FLD and LC-MS/MS methods have been developed for separation and
detection; C18 columns were used in all of them. Mobile phases for FLD
©
detection were MeOH:H2O:acetic acid (75:24:1) and ACN:H2O:acetic acid

(75:24:1) in gradient conditions (Maragos and Richard 1994); MeOH:NaH2PO4
(0.05 M) (55:45) and ACN:H2O (80:20), also in gradient conditions (Scott et al.
1994); and NaH2PO4 (0.1 M):MeOH (33:67) at pH 6 (Spotti et al. 2001). In each
case, a flow of 1 mL/min was used. FLD excitation wavelengths were set at
250 or 335 nm, and emission was at 440 or 470 nm. Gazzotti et al. (2009) used
ACN:H2O 0.3% formic acid and ACN 0.3% formic acid at 0.3 mL/min for MS
detection.
Before LC-FLD analysis, a derivatization step is needed. Naphthalene-2,3-

dicarboxaldehyde (NDA) with KCN formed a highly fluorescent derivative sta-
ble for more than 24 h (Shephard 1998). The use of NDA allows LODs of 5 ng/
mL for FB1 or FB2 (Maragos and Richard 1994). Other reported derivatization
agents are o-phthaldialdehyde/2-mercaptoethanol and 4-fluoro-7-nitroben-
zofurazan. Scott et al. (1994) reported a LOD of 3 ng/mL for FB1 and FB2
y
when using o-phthaldialdehyde/2-mercaptoethanol derivatization, whereas
nl
a LOD of 7 ng/mL for FB1 was obtained when using 4-fluoro-7-nitrobenzofu-
O
razan. In the case of LC-MS/MS, derivatization is not needed. Gazzotti et al.
se
(2009) proposed an LC-MS/MS method with a LOD value of 0.003 µ g/kg and
lU
an LOQ of 0.1 µ g/kg. It has been observed that milk fat content did not affect
na
FB1 and FB2 recoveries when LC-FLD after strong anion exchange column
o
rs
purification is used (Maragos and Richard 1994), and no losses of FB1 and FB2
Pe
in milk under conditions of freezing, refrigeration, or boiling were detected
r
(Scott et al. 1994). Milk that was near its expiration date yielded an extract
fo
with peaks having retention times close to those of FB1 and FB2 (Maragos
y
op
and Richard 1994) or near aminopentol when stored too long at 4° C (Scott
C
et al. 1994). In addition to sample freshness, other conditions improved the
’s
results, including the use of 4-fluoro-7-nitrobenzofurazan and fresh mobile

or
phase. Moreover, allowing the MeOH-ACE extracts to remain at – 20° C for

ut
several days, the background interference in poor-quality milk is reduced

b
tri
(Maragos and Richard 1994).

on
A commercially available ELISA kit available for screening the presence

.C
of fumonisins in corn has been adapted for analysis of FB1 and FB2 in milk
C
(Maragos and Richard 1994). The method was reproducible without pretreat-
LL
ment of the sample, but substantial amounts of fumonisins must be present

is
for adequate detection with ELISA (LOD: 100– 300 ng FB1/mL).

c
an
Fr
d
an
or
3.6 Cyclopiazonic Acid

yl
Ta
CPA is produced by Aspergillus and Penicillium genera. There is no available

18
information regarding toxicity in humans, although neurotoxicity and pro-

20
tein synthesis inhibition have been described in animals (Losito et al. 2002).
In addition, a synergic toxic effect has been observed when aflatoxins and
©
CPA co-occur (Oliveira et al. 2006).

In order to extract and clean up CPA from milk, Dorner et al. (1994) started
defatting milk with petroleum ether. Next, extraction was carried out with
CHCl3:MeOH (80:20) acidified with HCl. After evaporation, the residue was
dissolved in CHCl3 and mixed with NaOH and saturated NaCl solutions.
The aqueous phase was separated and acidified with HCl and reextracted
with dichloromethane. Prasongsidh et al. (1998) performed extraction and
cleanup with MeOH:2%NaHCO3 (70:30), followed by a defatting step using

hexane. The aqueous phase was reextracted after acidification, using CHCl3,
and finally cleaned by SPE. This procedure was also applied by Oliveira
et al. (2006), with some modifications. Losito et al. (2002) also applied
Prasongsidh’ s procedure, but avoiding the cleanup step with silica columns
in order to achieve a faster sample treatment. Š imů nek et al.’ s (1992) method
y
was based on diffusion of CPA into buffered agar gel inside a petri dish and
nl
successive reextraction from gel by means of diethylether.
O
With regard to analysis, TLC colorimetry, high-performance thin-layer
se
chromatography (HPTLC)-FLD, LC-UV, and LC-MS/MS (ion trap) methods
lU
have been proposed. Š imů nek et al. (1992) performed separation by TLC
na
with a solution of CHCl3:tetrachloromethane:ethyl acetate (7:2:1), visualiza-
o
rs
tion by spraying with p-dimethylamino benzaldehyde solution and HCl,
Pe
and detection by colorimetry with a LOD and LOQ of 0.001 and 1 mg/kg,
r
respectively. Dorner et al. (1994) used HPTLC. Plates were developed in ethyl
fo
acetate:MeOH:NH4OH (85:15:10) and sprayed with 1% p-dimethylamino benz-
y
op
aldehyde in ethanol (EtOH) and ethanolic sulfuric acid. The LOQ achieved was
C
5 ng/g. Prasongsidh et al. (1998) and Oliveira et al. (2006) used LC-UV (279 nm)
’s
with a linear gradient separation using MeOH:H2O (85:15 or 70:30) and 4 mM
or
zinc sulfate. The first study achieved an LOQ of 50 ng/mL and a linear range
ut
from 500 ng/mL to 100 µ g/mL. The second one raised a determination limit of
b
tri
6 ng/mL. Losito et al. (2002) developed an LC-MS/MS (ion trap) method using
on
ACN:ammonium acetate buffer (0.05 M, pH 5) (80:20) as the mobile phase. The

.C
dynamic range was from 5 to 1000 ng/mL, and the LOD and LOQ were 6 and
C
36 ng/mL, respectively. These authors proposed the fragmentation pattern for

LL
CPA on the basis of the ions observed in the MS/MS spectrum.

is
Other proposed methods include that of Roncada et al. (2003) using micel-
c
an
lar electrokinetic capillary chromatography (MEKC) with UV (214 nm)

Fr
detection. The sample treatment is reduced to a centrifugation step to defat

milk. Prasongsidh et al. (1998) made a comparison between LC-UV (279 nm)
d
an
and MEKC-UV (225 nm) for CPA analysis using the same sample treatment
or
for the two methods. The authors concluded that the MEKC-UV method is
yl
significantly more sensitive, although the sample injection volume (8.3 nL)
Ta
was several times lower than that used in the LC method (20 µ L), with the
18
performance of both methods being similar at high concentrations.

20
©
3.7 Ergot Alkaloids

Ergot alkaloids are mainly produced by the fungal genus Claviceps. The
most widespread in Europe, Claviceps purpurea, is known to infect more than
400 plant species with the disease known as ergot (EFSA 2012). Ergotism,
gangrene, convulsions, and vomiting are among the diverse toxic effects
(Capriotti et al. 2012).
Durix et al. (1999) developed an analytical method for ergovaline detec-
tion in milk. Protein precipitation was carried out with ACE, followed by an
extraction step with CHCl3; for cleanup, 100 mg of Ergosil (surface-treated
silica gel) was used. The analysis was performed with LC-FLD for quantifi-
y
cation purposes, and LC-MS/MS was used for identification. LOD and LOQ
nl
were 0.2 and 0.7 ng/mL, respectively. C18 columns and isocratic separation
O
with ACN:ammonium carbonate (2 mM in H2O) (36.5:63.5) or MeOH at a
se
flow rate of 1 mL/min have been used.
lU
Schumann et al. (2009) proposed a method for the analysis of 11 ergot
na
alkaloids in milk. The extraction was performed with a mixture of CH2Cl2,
o
rs
ethylacetate, MeOH, and NH4OH. The cleanup step was carried out using
Pe
Extrelut® columns (SLE); the analysis was carried out by LC-FLD (325 nm
r
excitation and 418 nm emission). A LOD of 5 ng/g was obtained for ergo-
fo
metrinine, ergotamine, ergotaminine, ergocornine, ergocorninine, ergocryp-
y
op
tine, ergocryptinine, ergocristine, ergosine, and ergosinine, and 10 ng/g for
C
ergometrine.
’s
The precursor and product ions used in LC-MS/MS analysis of some ergot
or
alkaloids are shown in Table 3.4.

ut
b
tri
on
.C
C
LL
3.8 Zearalenone and Its Derivatives

is
ZEA is a mycotoxin produced by several Fusarium species that grow in tem-

c
an
perate and warmer climate zones (Mally et al. 2016). ZEA does not degrade at
Fr
high temperatures and remains stable during storage or milling and during
the processing and cooking of food (SCF 2000). ZEA and its metabolites, ZAN,
d
an
α -ZAL, β-zearalanol (β-ZAL), α -ZEL, and β-zearalenol (β-ZEL), are harmful to

or
health, mainly due to their estrogenic activity. These compounds are capable
yl
of binding to the estrogen receptor, and thus are competitive inhibitors for
Ta
the estrogen hormone, which could result, for example, in problems in the
18
mammalian reproductive system (Le Guevel and Pakdel 2001). Moreover,

20
ZEA has also been shown to be hepatotoxic, hematotoxic, immunotoxic, and

genotoxic (Zinedine et al. 2007).
©
For milk sample preparation prior to chromatographic analysis, Palyusik

et al. (1980) performed LLE by means of CHCl3:ACE (1:2) mixture; cleanup
was carried out with hexane. Xia et al. (2009) performed extraction of ZEA
and five derivatives with ACN. During the cleanup step, OASIS MAX car-
tridges (SPE with quaternary amine groups on the surface of the polymeric
sorbent that give both RP and anion exchange functions) were used. The
authors optimized the SPE procedure by using ACN in the washing and
eluting steps, instead of MeOH (as the manufacturer recommend). Mean
TABLE 3.4
Adducts, Molecular Ion, and Product Ions Used in the Analysis of Mycotoxins in
Milk by LC-MS/MS
Adduct Precursor Product

Mycotoxin ESI Type Ion Ions References
y
Aflatoxins
nl
AFB1 + [M+H]+ 313 285, 241, Tsiplakou et al. (2014), Jia
O
269, et al. (2014), Xie et al.
se
128 (2015), Beltrá n et al.
lU
(2011), Malachová et al.
na
(2014), Zhan et al. (2012),
Aguilera-Luiz et al. (2011),
o
rs
Chen et al. (2012), Wang
Pe
and Li (2015)
AFB2 + [M+H]+ 315 287, 259, Tsiplakou et al. (2014), Jia
r
fo
243 et al. (2014), Xie et al.
y
(2015), Beltrá n et al.
op (2011), Malachová et al.
C
(2014), Zhan et al. (2012),
’s

or
Chen et al. (2012)

ut
AFG1 + [M+H]+ 329 243, 311, Jia et al. (2014), Xie et al.
b
200, (2015), Beltrá n et al.

tri
on
215, (2011), Malachová et al.

283 (2014), Zhan et al. (2012),
.C

C
Chen et al. (2012)

LL
AFG2 + [M+H]+ 331 245, 189, Jia et al. (2014), Xie et al.
is
257, (2015), Beltrá n et al.

c

an
217 (2014), Zhan et al. (2012),

Fr

d
Chen et al. (2012)

an
AFM1 + [M+H]+ 329 273, 259, Jia et al. (2014), Xie et al.
or

yl
301 (2014), Zhan et al. (2012),

Ta
Wang and Lie (2015),

Sø rensen and Elbæ k
18
(2005)
20
AFM1 – [M– H] – 327 312 Chen et al. (2005)

©
AFM2 + [M+H]+ 331 273, 285, Xie et al. (2015), Malachová

231, et al. (2014)
259
Ergot Alkaloids
Ergovaline + [M+H]+ 534 223, 268, Durix et al. (1999)
320,
517
(Continued)

Milk by LC-MS/MS
Ergocornine + — 562 268, 223, Malachová et al. (2014),
y
nl
208 Mol et al. (2008)
O
Ergocorninin + — 562 544, 223 Malachová et al. (2014)
se
Ergocristine + — 610 268, 223, Malachová et al. (2014),
lU
592 Mol et al. (2008)
Ergocristinine + — 610 592, 223 Malachová et al. (2014)
na
Ergocryptine + — 576 223, 208 Malachová et al. (2014),
o
rs
Mol et al. (2008)
Pe
Ergocryptinine + — 576 558, 223 Malachová et al. (2014)
Ergometrine + — 326 208, 223 Malachová et al. (2014)
r
fo
Ergometrinine + — 326 208, 223 Malachová et al. (2014)
y
Ergonovine + — 326 op
223 Mol et al. (2008)
C
Ergosinin/ + — 548 223, 208 Malachová et al. (2014)
ergosin
’s
or
Ergotaminine/ + — 582 223, 208 Malachová et al. (2014)

ut
ergotamine
b
Ergotamine + — 582 223 Mol et al. (2008)

tri
on
Ergovalin + — 534 223, 208 Malachová et al. (2014)

.C
Fumonisins
FB1 + [M+H]+ 722 334, 352, Jia et al. (2014), Xie et al.
C
686 (2015), Malachová et al.

LL
(2014), Sø rensen and

is
Elbæ k (2005), Gazzotti

c
an
et al. (2009), Beltrá n et al.

(2013)
Fr
FB2 + [M+H]+ 706 336, 318, Jia et al. (2014), Xie et al.
d
354 (2015), Malachová et al.

an

or
Elbæ k (2005), Gazzotti

yl
et al. (2009)
Ta
FB3 + [M+H]+ 707 336, 319 Jia et al. (2014), Malachová

18
et al. (2014)
20
Ochratoxins
OTA + [M+H]+ 404 221, 358, Tsiplakou et al. (2014), Jia
©
239, et al. (2014), Xie et al.

102, (2015), Beltrá n et al.
386 (2014), Aguilera-Luiz et
al. (2011), Chen et al.
(2012), Wang and Li
(2015)
(Continued)

Milk by LC-MS/MS
OTA – [M– H]– 402 358, 167 Sø rensen and Elbæ k (2005),
y
nl
Huang et al. (2014a)
O
OTB + [M+H]+ 370 205, 103 Jia et al. (2014), Malachová
se
et al. (2014)
lU
OTα – [M– H]– 255 211, 167 Jia et al. (2014), Malachová
et al. (2014)
na
Trichothecenes
o
rs
15-ADON + [M+H]+ 339 137, 261, Malachová et al. (2014),
Pe
297, Beltrá n et al. (2013),
321 Flores-Flores et al. (2015)
r
fo
15-ADON + [M+NH4]+ 356 — Jia et al. (2014)
y
15-ADON – [M– H]– 337 150, 219
op Sø rensen and Elbæ k (2005),
Chen et al. (2013)
C
3-ADON + [M+H]+ 339 231, 213, Jia et al. (2014), Beltrá n
’s
279, et al. (2013), Flores-Flores

or
203 et al. (2015)

ut
3-ADON – [M– H]– 337

b
307, 173 Sø rensen and Elbæ k (2005),

tri
Chen et al. (2013)

on
3-ADON – — 397 59, 307 Malachová et al. (2014)

.C
15-MAS + — 342 265, 247 Sø rensen and Elbæ k (2005),

C
Chen et al. (2013)

LL
4-MAS + [M+NH4]+ 342 157, 105 Malachová et al. (2014)

is
DAS + [M+NH4]+ 384 307, 289, Tsiplakou et al. (2014), Jia

c
247, et al. (2014), Malachová

an
349, et al. (2014), Sø rensen and

Fr
105, Elbæ k (2005), Beltrá n et al.

d
229 (2013), Flores-Flores et al.

an
(2015), Chen et al. (2013)

or
DOM-1 + [M+H]+ 281 241, 215, Jia et al. (2014), Xie et al.
yl
233 (2015)
Ta
DOM-1 – [M– H]– 279 249, 231 Sø rensen and Elbæ k (2005),

Chen et al. (2013), Keese
18
et al. (2008)
20
DOM-1 – — 339 59, 249 Malachová et al. (2014)

©
DON + [M+H]+ 297 249, 231, Jia et al. (2014), Beltrá n

203 et al. (2013)
DON – [M– H]– 295 265, 247, Sø rensen and Elbæ k (2005),
138 Chen et al. (2013), Keese
et al. (2008)
DON – 355 59, 265 Malachová et al. (2014)
FUS-X + [M+H]+ 355 175, 229, Beltrá n et al. (2013)
247
(Continued)

Milk by LC-MS/MS
FUS-X + [M+NH4]+ 372 — Jia et al. (2014)
y
nl
FUS-X – [M– H]– 353 263, 187 Chen et al. (2013)
O
FUS-X – — 413 59, 263 Malachová et al. (2014)
se
HT-2 + [M+H]+ 425 263, 105 Tsiplakou et al. (2014)
lU
HT-2 + [M+NH4]+ 442 215, 323, Jia et al. (2014), Malachová
na
197 Elbæ k (2005), Beltrá n et al.
o
rs
(2013)
Pe
HT-2 + [M+Na]+ 447 345, 285 Malachová et al. (2014),
Chen et al. (2013)
r
fo
NEO + [M+NH4]+ 400 185, 305, Jia et al. (2014), Malachová
y
215,
op et al. (2014), Beltrá n et al.
245 (2013), Chen et al. (2013)
C
NIV + [M+H]+ 313 175, 159, Beltrá n et al. (2013)
’s
91, 177
or
NIV – [M– H]– 311 281, 191 Chen et al. (2013)

ut
b
NIV – – 371 281, 59 Malachová et al. (2014)

tri
NIV – [M+FAc]+ 357 — Jia et al. (2014)

on
T-2 + [M+NH4]+ 484 305, 215, Tsiplakou et al. (2014),

.C
185, Malachová et al. (2014),

C
245 Sø rensen and Elbæ k

LL
(2005), Beltrá n et al. (2013)

is
T-2 + [M+Na]+ 489 387, 245 Chen et al. (2013)

c
T-2 triol + — 383 215, 233 Sø rensen and Elbæ k (2005)

an
T-2 triol + [M+Na]+ 405 303, 273, Beltrá n et al. (2013)

Fr
405
d
T-2 triol + [M+NH4]+ 400 281, 215 Jia et al. (2014), Malachová
an
et al. (2014)
or
T-2 triol – [M– H]– 381 101, 297 Chen et al. (2013)

yl
T-2 tetraol – [M– H]– 297 219, 137 Chen et al. (2013)

Ta
18
Zearalenone and Derivatives

ZAN + [M+H]+ 321 — Jia et al. (2014)
20
ZAN – [M– H]– 319 275, 205, Xie et al. (2015), Zhan et al.
©
107 (2012), Chen et al. (2013),

Xia et al. (2009)
ZEA + [M+H]+ 319 187, 185, Tsiplakou et al. (2014), Jia
275, et al. (2014)
205,
283,
301
(Continued)

Milk by LC-MS/MS
ZEA – [M– H]– 317 131, 175, Xie et al. (2015), Malachová
y
nl
273 et al. (2014), Zhan et al.
O
se
Elbæ k (2005), Beltrá n et al.
(2013), Chen et al. (2013),
lU
Xia et al. (2009)
na
α -ZAL + [M+H]+ 323 — Jia et al. (2014)
o
α -ZAL – [M– H]– 321 277, 259, Xie et al. (2015), Sø rensen
rs
303 and Elbæ k (2005), Chen
Pe
et al. (2013), Xia et al. (2009)
r
α -ZEL
fo
+ [M+H]+ 321 — Jia et al. (2014)
α -ZEL – [M– H]– 319 160, 130, Xie et al. (2015), Malachová
y
op
C
301 Elbæ k (2005), Chen et al.
’s
(2013), Xia et al. (2009)

or
β -ZAL + [M+H]+ 323 — Jia et al. (2014)

ut
β -ZAL – [M– H]– 321 277, 259, Xie et al. (2015), Sø rensen

b
tri
303 and Elbæ k (2005), Chen

on
et al. (2013), Xia et al.

(2009)
.C
β -ZEL + [M+H]+ 321 — Jia et al. (2014)

C
β -ZEL – [M– H]– 319 160, 130, Xie et al. (2015), Malachová

LL
275 et al. (2014), Sø rensen and

is
Elbæ k (2005), Chen et al.

c
(2013), Xia et al. (2009)

an
Fr
Others
d
CIT + [M+H]+ 251 233, 205 Malachová et al. (2014)

an
CIT – [M– H]– 249 — Jia et al. (2014)

or
CPA – [M– H]– 335 180 Losito et al. (2002)

yl
PAT + [M+NH4]+ 172 — Jia et al. (2014)

Ta
PAT – [M– H]– 153 109 Malachová et al. (2014)

18
STC + [M+H]+ 325 310 Jia et al. (2014), Malachová

20
et al. (2014)
©
recoveries ranged between 92.6% and 112.5%. Seeling et al. (2005) applied a
method to milk that had been developed for broiler’ s biological fluids. The
extraction and cleanup were carried out with CHCl3, followed by basifica-
tion with 1 M NaOH and reextraction in acidified toluene. Next, the organic
extract was evaporated and redissolved in ACN and PBS and passed through
a ZEA-specific IAC (Easi-Extract™ Zearalenone). Scott and Lawrence (1988)
used ACN to extract ZEA from the samples. Then they used CH2Cl2 to extract
ZEA from ACN extract and applied the solution onto a SLE column (Chem
Elut™ ). Finally, they performed an SPE by means of a weak anion exchange

column (Bond Elut NH2). Some authors used β -glucuronidase (Hagler et al.
1980; Mirocha et al. 1981; Scott and Lawrence 1988; Seeling et al. 2005) and
aryl sulfatase (Mirocha et al. 1981; Scott and Lawrence 1988) before extrac-
tion to study glucuronide and sulfate conjugates. Scott and Lawrence (1988)
highlighted the need for 16 h of incubation at 37° C to release bound ZEA,
y
α -ZEL, and β -ZEL.
nl
With regard to separation and detection, TLC-UV methods have been used
O
with CHCl3:MeOH (95:5) as the mobile phase (Palyusik et al. 1980; Hagler
se
et al. 1980). But GC and LC with a variety of detectors have preferably been
lU
used. In the case of GC analysis, a prior derivatization step with Tri-Sil BT®
na
was required (Palyusik et al. 1980; Mirocha et al. 1981). For LC-FLD, separa-
o
rs
tion has been performed by MeOH:H2O:ACN (61:35:4) or by ACN:H2O (45:55)
Pe
acidified with phosphoric acid (pH = 2.6); the excitation wavelength was set
r
at 236 or 273 nm and the emission wavelength at 470 or 440 nm (Scott and
fo
Lawrence 1988; Seeling et al. 2005). LC-MS/MS with ESI in the negative mode
y
op
was also used with gradient elution (H2O:ACN); the product ions are listed
C
in Table 3.4 (Xia et al. 2009). The authors reported that there were no signifi-
’s
cant differences in quantitative analysis among milk with 0.5%, 1.5%, or 4%

or
fat content when applying this method.

ut
With regard to immunochemical-based methods for ZEA in milk, Azcona

b
tri
et al. (1990) proposed a preparative cleanup step using monoclonal anti-

on
body– affinity chromatography before analysis of ZEA in milk by ELISA. The

.C
IAC consisted of a 1 mL tuberculin syringe containing 200 µ L of Sepharose®

C
4B previously coupled with the antibody. Then ELISA was carried out to
LL
quantify ZEA in the eluate with a mean recovery of 126%. A biolumines-

is
cent whole-cell biosensor for the detection of ZEA family mycotoxins in milk
c
an
has been developed by Vä limaa et al. (2010) based on a genetically modi-

Fr
fied Saccharomyces cerevisiae strain. The biosensor releases light in the pres-
ence of an estrogenic compound, although the fat content in milk affects the
d
an
response. This method can be used for screening purposes before chemical
or
analysis, due to its short assay time (< 3 h) and the possibility of automatiza-
yl
tion. The LODs obtained for the different ZEA derivatives make the proce-
Ta
dure adequate for detecting ZEA and its derivatives in cow milk.
18
20
©
3.9 Multimycotoxin Detection

Animal feed is prepared using a variety of raw materials. Each of them
can be contaminated with a different type of fungi. Moreover, one type
of fungi is capable of producing mycotoxins that belong to different fami-
lies simultaneously. Thus, animals are always exposed to mixtures rather
than to individual toxins (Heussner et al. 2006), and the co-occurrence of
several mycotoxins in animal tissue and fluids is a likely scenario. In spite

of these facts, the simultaneous presence of mycotoxins in different matrixes
and their involvement in the toxic effects have been scarcely explored, and
further studies are needed (Speijers and Speijers 2004). Due to the proba-
ble co-occurrence of many mycotoxins with very different physicochemical
characteristics, the low levels expected, and the complexity of milk as an
y
analytical matrix, the development of methods for the simultaneous quan-
nl
tification of these compounds in milk is an analytical challenge. However,
O
they will allow the screening of multiclass mycotoxins using a unique same-
se
sample treatment and analytical procedure, resulting in the reduction of
lU
analysis time (Zhang et al. 2014), and in practice, they will allow the surveil-
na
lance of the presence of mycotoxins in milk.
o
rs
Most of the methods developed for analyzing multimycotoxins in milk
Pe
are based on the LC-MS technique because LC with MS detectors, usually
r
using ESI, presents certain advantages over other analytical techniques (i.e.,
fo
universality, selectivity, and sensitivity). In addition, it does not require
y
op
derivatization processes, and in some cases, it may reduce, or even prevent,
C
the pretreatment of the sample. However, some problems are associated with
’s
the use of LC-MS methods for the simultaneous determination of mycotox-

or
ins. For instance, if the full SCAN mode is used and there are multiple ions to
ut
be detected, sensitivity decreases (Tanaka et al. 2006). Alternatively, selected

b
tri
ion monitoring (SIM) in LC-MS or selected reaction monitoring (SRM) in

on
LC-MS/MS can be used. Also, coextracted compounds from milk are able to
.C
provide precursor ions and/or product ions with m/z values close to those
C
of the mycotoxins. Because of this, when other than the SCAN mode is
LL
used, a minimum of three identification points is needed for mycotoxins. In

is
LC-MS/MS, the identification of a precursor ion and two product ions gives
c
an
four identification points. Also, the relative ion intensities of the detected ions
Fr
(in percent) in the sample should correspond to those of the standard solu-
tions (European Commission 2004). Moreover, matrix effects are observed
d
an
for most mycotoxins extracted from milk (Flores-Flores and Gonzá lez-Peñ as

or
2015). Coelution with other compounds from milk could compete with myco-
yl
toxins during the ionization process, changing the intensity of their signals
Ta
obtained in the detector, although the peak shape is not affected (Losito et al.
18
2002). Thus, matrix-matched calibration is of vital importance for a reliable

20
quantification of mycotoxins in milk samples. And last but not least, the high
cost of the equipment, especially in the new generation of high-resolution
©
apparatus, should be taken into account. The characteristics of the multiclass

methods encountered in the literature are summed up in Table 3.3. In addi-
tion, the precursor and product ions used for mycotoxin identification and
quantification in the different methods are displayed in Table 3.4.
With respect to sample treatments for multimycotoxin analysis in milk,
they are very diverse, including simple as well as very complex procedures.
Some of the reported methods have been developed especially for their
application in milk, whereas others have been applied to a broad range of
foodstuffs, including milk. Acidified ACN is the most used organic solvent
for extraction, whereas ACE, MeOH, and ACN:H2O or ACN:ethyl acetate
mixtures have also been used (see details in Table 3.3). Cleanup processes
with hexane and petroleum ether, as well as the use of salts, the quick, easy,
cheap, effective, rugged, and safe (QuEChERS) procedure, or SPE columns,
have been described.
y
Beltrá n et al. (2013), Mol et al. (2008), and Malachová et al. (2014) devel-
nl
oped the simplest proposed methods, applying LLE with acidified mixtures
O
of water and an organic solvent (ACN or ACE). Tsiplakou et al. (2014) pro-
se
posed LLE with acidified MeOH, followed by a freezing step (12 h) in order
lU
to remove fat and coextractives with limited solubility in MeOH. Zhan et al.
na
(2012) proposed an LLE procedure with EtOH and ACN; after centrifugation
o
rs
and filtration, the supernatant was reextracted. The final extract was evapo-
Pe
rated at 40° C, and the residue was dissolved in H2O-EtOH-ACN.
r
Flores-Flores and Gonzá lez-Peñ as (2015) proposed LLE and an additional
fo
step using salt to separate the organic phase from the water associated with
y
op
the milk sample and to promote extraction of mycotoxins into the organic sol-
C
vent. Jia et al. (2014) and Filigenzi et al. (2011) used the QuEChERS approach.
’s
Aguilera-Luiz et al. (2011), Beltrá n et al. (2011), Chen et al. (2012, 2013), Huang
or
et al. (2014a), Sø rensen and Elbæ k (2005), Wang and Li (2015), Winkler et al.
ut
(2015), and Xie et al. (2015) used cartridges for SPE procedures, with the pro-
b
tri
posal by Xie et al. being the most complex process, with an additional freez-
on
ing step. Wang and Li (2015) proposed an automated SPE online with LC-MS/
.C
MS without pretreatment of samples. Huang et al. (2014a) made a compari-

C
son between Mycosep 226 and OASIS HLB cartridges for the analysis of four
LL
mycotoxins (AFM1, OTA, ZEA, and α -ZEL), obtaining better results with the
is
last ones. Different SPE columns for cleaning up mycotoxins extracted from
c
an
milk were considered by Chen et al. (2012, 2013), and finally, Mycosep 226
Fr
Aflazon+, OASIS HLB cartridges, and Bond Elut Mycotoxin® were chosen
and compared. Good results of selectivity and a low matrix effect were found
d
an
when Bond Elut Mycotoxin® cartridges were used for Fusarium toxins (Chen
or
et al. 2013) and when OASIS HLB cartridges were used for six aflatoxins and
yl
OTA (Chen et al. 2012). For aflatoxins and OTA, a comparison was made
Ta
between pressurized liquid extraction (PLE) and commonly shaken extrac-

18
tion using ACN and defatting with hexane in both cases. PLE in terms of
20
recoveries, decision limit (CCα ), detection capability (CCβ ), extraction time,

and volume of solvent used is better than extraction with solvents by shak-
©
ing, but is a big investment (Chen et al. 2012). Also, an optimization (extrac-
tion volume, temperature, and time) of ultrasound-assisted extraction was
carried out for Fusarium toxins (Chen et al. 2013). It was concluded that the
extraction recovery increases with temperature from 20° C to 40° C, but when
temperature is too high, a large fraction of soluble organic matter is coex-
tracted, leading to a higher matrix effect. Aguilera-Luiz et al. (2011) made
an interesting comparison of different approaches: LLE-based, QuEChERS,
and SPE cartridges (C18 and OASIS HLB). They recommended the use of C18
cartridges for the analysis of 6 mycotoxins and an additional 42 pesticides.

Sø rensen and Elbæ k (2005), Winkler et al. (2015), and Chen et al. (2012, 2013)
performed an enzymolysis step with β -glucuronidase before extraction.
y
nl
O
3.10 Conclusions
se
Milk is a highly consumed food, especially for children, a vulnerable group
lU
to toxicants. Among the toxic compounds that can be present in milk, myco-
na
toxins are a problem of concern due to the fact that they are distributed in
o
rs
raw materials and feed worldwide and can ultimately reach milk. The levels
Pe
of mycotoxins in milk are expected to be low, but in order to ensure milk
r
safety, more studies should be carried out regarding their presence, their co-
fo
occurrence, their bioavailability in animals, and their carryover into milk.
y
op
For developing these studies, adequate analytical methods are essential.
C
Nowadays, chromatography, especially LC, using UV, FLD, or MS detectors,
’s
is the most frequently used technique for mycotoxin analysis in milk. Sample
or
treatment is often complex and includes extraction and cleanup steps using
ut
different solvents and techniques. Currently, LC-MS has allowed multimyco-

b
tri
toxin detection in one sample, making surveillance less expensive in terms

on
of sample preparation and output, but increasing the cost due to the equip-
.C
ment and analyst training. In the near future, a new generation of mass spec-
C
trometers will allow simultaneous analysis of a great number of mycotoxins

LL
in a sample, and also, it is expected that new methods will be developed

is
for the analysis of less studied mycotoxins® in milk (e.g., sterigmatocystin

c
an
[STC]), newly discovered mycotoxins®, or masked mycotoxins® (metabolites

Fr
of mycotoxins® produced in vegetables).

d
an
or
yl
Ta
Abbreviations
18
20
15-ADON: 15-Acetyldeoxynivalenol
3-ADON: 3-Acetyldeoxynivalenol
©
α -ZAL: α -Zearalanol
α -ZEL: α -Zearalenol
β-ZAL: -Zearalanol
β-ZEL: -Zearalenol
ACE: Acetone
ACN: Acetonitrile
AFB1: Aflatoxin B1
AFB2: Aflatoxin B2
AFG1: Aflatoxin G1
AFG2: Aflatoxin G2
AFM1: Aflatoxin M1
AFM2: Aflatoxin M2
CIT: Citrinin
CPA: Cyclopiazonic acid
y
DAS: Diacetoxyscirpenol
nl
DLLME: Dispersive liquid–liquid microextraction
O
DLS: Dynamic light scattering
se
DOM-1: Deepoxydeoxynivalenol
lU
DON: Deoxynivalenol
na
EC: Electron capture
o
rs
ELISA: Enzyme-linked immunosorbent assay
Pe
ESI: Electrospray ionization
r
EtOH: Ethanol
fo
FB1: Fumonisin B1
y
FB2: Fumonisin B2 op
C
FB3: Fumonisin B3
’s
FID: Flame ionization detector

or
FLD: Fluorescence detector

ut
FUS-X: Fusarenon X
b
tri
GC: Gas chromatography

on
HFB: Heptafluorobutyryl
.C
IAC: Immunoaffinity column

C
IARC: International Agency for Research on Cancer

LL
IS: Internal standard

is
LC: Liquid chromatography

c
an
LC-MS: Liquid chromatography coupled to mass spectrometry

Fr
LFIA: Lateral flow immunoassay

LLE: Liquid– liquid extraction
d
an
LOD: Limit of detection

or
LOQ: Limit of quantification

yl
MAS: Monoacetoxyscirpenol
Ta
MEKC: Micellar electrokinetic capillary chromatography

18
MeOH: Methanol
20
MFC: Multifunctional cleanup column

MS: Mass spectrometry
©
NDA: Naphthalene-2,3-dicarboxaldehyde
NEO: Neosolaniol
NIV: Nivalenol
OTA: Ochratoxin A
OTB: Ochratoxin B
PAT: Patulin
PBS: Phosphate-buffered solution
PLE: Pressurized liquid extraction

PSA: Primary secondary amine
PTFE: Polytetrafluoroethylene
PVDF: Polyvinylidene fluoride
QuEChERS: Quick, easy, cheap, effective, rugged, and safe
SLE: Solid-supported liquid extraction
y
SPE: Solid-phase extraction
nl
STC: Sterigmatocystin
O
TLC: Thin-layer chromatography
se
TMS: Trimethylsilyl
lU
TMSI: Trimethylsilylimidazole
na
UV: Ultraviolet
o
rs
ZAN: Zearalanone
Pe
ZEA: Zearalenone
r
fo
y
op
C
’s
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4
Biotoxins in Seafood
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Laura P. Rodrí guez, Juan M. Vieites, and Ana G. Cabado
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CONTENTS
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4.1 Introduction ................................................................................................... 98
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4.2 Marine Biotoxins........................................................................................... 99
4.2.1 Okadaic Acid Group Toxins............................................................ 99
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4.2.1.1 Origin and Distribution.................................................... 99
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4.2.1.2 Chemical Structures and Mechanism of Action......... 100
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4.2.1.3 Occurrence and Accumulation in Seafood.................. 101
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4.2.1.4 Episodes of Intoxication.................................................. 101

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4.2.2 Pectenotoxin Group Toxins........................................................... 102

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4.2.2.1 Origin and Distribution.................................................. 102

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4.2.3 Yessotoxin Group Toxins............................................................... 103

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4.2.3.4 Episodes of Intoxication .................................................. 105

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4.2.4 Azaspiracid Group Toxins............................................................. 105

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4.2.5 Saxitoxin Group Toxins.................................................................. 108
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©

4.2.6 Domoic Acid ................................................................................... 111
4.2.7 Ciguatoxin Group Toxins.............................................................. 112
97

4.2.8 Brevetoxins...................................................................................... 114
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4.2.9 Tetrodotoxin.................................................................................... 116
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4.2.9.2 Chemical Structure and Mechanism of Action........... 117
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4.2.10 Palytoxin and Analogs................................................................... 120
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4.2.11 Cyclic Imines................................................................................... 123

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4.3 Methods of Analysis................................................................................... 126

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4.3.1 Lipophilic Toxins............................................................................ 126

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4.3.2 Brevetoxins...................................................................................... 127

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4.3.3 ASP Toxins....................................................................................... 128

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4.3.4 PSP Toxins........................................................................................ 129

4.3.5 Ciguatoxins...................................................................................... 129
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4.3.6 Tetrodotoxin.................................................................................... 130

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4.3.7 Application of the QuEChERS Approach for the Analysis

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of Biotoxins�� 130
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4.4 Risk Assessment for Biotoxins in Seafood.............................................. 131

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4.5 Control Measures for Biotoxin Contamination of Seafood.................. 132

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4.5.1 Legislation ....................................................................................... 132

4.5.2 Managing......................................................................................... 134
©
References ............................................................................................................. 138
4.1 Introduction
Marine toxins consist of a wide range of toxic compounds produced by spe-
cific organisms, mainly microalgae that, under favorable conditions, prolifer-
ate, resulting in a harmful algae bloom (HAB). Once this toxic episode starts,
Biotoxins in Seafood 99
many marine organisms can be contaminated. At present, bivalve mollusks

are the most affected marine invertebrates, but several crustaceans and some
fish can be contaminated as well. These natural events induce enormous
impacts on human health, seafood producers, and associated industries,
such as depuration centers, processing plants, restaurants, and tourism. It is
also very important to mention the economic losses associated with periods
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of when shellfish production areas are closed or when a HAB has started in
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a location and lasts many months, forbidding harvesting or fishing.
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In order to avoid some of the consequences caused by these toxic episodes,
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many countries carry out toxin monitoring programs and have specific legisla-
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tion and methods of analyses. Complementary international institutions partici-
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pate in different tasks in order to evaluate and assess the risk, establish guidelines
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and safe limits linked to marine toxins, and develop methods for the detection
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and quantification of these toxic compounds. Worldwide, many organizations
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are involved in different activities; the following are some of the most rele-
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vant: the Food and Agriculture Organization of the United Nations (FAO), the
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Intergovernmental Oceanographic Commission of UNESCO (IOC), the World
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Health Organization (WHO), the European Food Safety Authority (EFSA), and
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the European Union Reference Laboratory for Marine Biotoxins (EU-RL-MB).

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In addition to the existence of well-studied and legislated toxins, there is

ut
a more recent concern evidenced by the presence of the so-called “ emergent

b
tri
toxins,” such as azaspiracids (AZAs) or cyclic imines (CIs). The occurrence of

on
these new microalgae seems to be due to the increase of seawater tempera-

.C
ture and also to anthropogenic factors that allow the establishment of these
C
new populations in places where they were not detected before, like in the
LL
case of tetrodotoxin (TTX), which has been found in unusual matrixes.

is
In this chapter, an overview related to the occurrence of marine toxins in

c
an
seafood and their origin, structure, and mechanism of action, as well as the
Fr
world distribution of toxic microalgae, is presented. The effects on human

health, depending on the type of toxin, the main regulations, and the meth-
d
an
ods of analysis linked to these toxic substances are also discussed.

or
yl
Ta
18
20
4.2 Marine Biotoxins

©
4.2.1 Okadaic Acid Group Toxins

4.2.1.1 Origin and Distribution
Okadaic acid (OA), isolated for the first time from the marine sponge of the
genus Halichondria (Tachibana et al. 1981) together with its analogs, the
Dinophysis toxins (DTXs), form the OA group toxins, known to cause the diar-
rhetic shellfish poisoning (DSP) syndrome. These toxins, produced by plank-
tonic dinoflagellates, the Dinophysis genus, and the benthic Prorocentrum
genus (Dickey et al. 1990; Yasumoto et al. 1985) were first reported in the
Netherlands in 1961 (Korringa and Roskam 1961). Nowadays, their distribu-
tion is worldwide: Europe (Dahl and Yndestad 1985; Korringa and Roskam
1961; Prassopoulou et al. 2009; Krogh et al. 1985; Lassus et al. 1985; Underdahl
et al. 1985; Vale and Sampayo 1999), Asia (Lee et al. 2011; Wang et al. 2015;
Chen et al. 2013; Yasumoto et al. 1978, 1979), Africa (Elgarch et al. 2008;
y
Pitcher et al. 1993), North and South America (Lembeye et al. 1993; Trainer
nl
and Hardy 2015; Turner and Goya 2015), Australia (Takahashi et al. 2007),
O
and New Zealand (Rhodes and Syhre 1995; Miles et al. 2006).
se
lU
na
4.2.1.2 Chemical Structures and Mechanism of Action
o
rs
OA, a polyether derivative of a C38 fatty acid (Figure 4.1), together with the
Pe
DTXs, is part of one the most relevant groups of phytoplanktonic toxins due
r
to its health effects on human consumers and economical losses (Sassolas
fo
et al. 2013).
y
op
These compounds are specific inhibitors of serine/threonine protein
C
phosphatases (PPs) 1 and 2A, both enzymes involved in the regulation of
’s
many cellular processes (cell death, cell cycle progression, tumor promotion,
or
metabolism, muscle contraction, and exocytosis) (Bialojan and Takai 1988;

ut
Ferná ndez et al. 2002). The inhibition of PP by OA group toxins leads to a

b
tri
rapid increase of phosphorylated proteins in cells, which is responsible for

on
diarrhea and the degenerative changes in the absorptive epithelium of the

.C
small intestine.
C
Some structural modification studies indicated that the carboxylic

LL
group of C-1 is essential for the inhibition of PP activity. Moreover, the

is
hydroxyl groups at C-2, C-7, C-24, and C-27 play important roles in the
c
an
interaction between the toxin and its target, causing a decrease of PP

Fr
inhibition.
DTXs (DTX1 and DTX2) exhibit similar PP binding affinity due to modi-
d
an
fications at C-31 and C-35, assuming the same cyclic conformation as the
or
OA molecule. These results are confirmed by the toxicity and PP2A binding
yl
comparisons between OA and DTX1 or DTX2.

Ta
18
20
CH3
©
O OH
H
O O CH2 H3C
HO
H3C OH O O O
H CH3 O O
HO H3C H
FIGURE 4.1
Chemical structure of OA.
4.2.1.3 Occurrence and Accumulation in Seafood

These lipophilic marine toxins are accumulated by shellfish, mainly bivalve
mollusks (mussels, scallops, clams, etc.), and several fish from Europe,
Japan, and South America (EFSA 2008b; Lawley et al. 2008). Nowadays, DSP
episodes are detected in other countries, such as Canada, Mexico, India,
Thailand, China, and Australia (Wang et al. 2015; Tong et al. 2015).
y
nl
O
se
4.2.1.4 Episodes of Intoxication
lU
The first clinical report of a gastrointestinal disease associated with the
na
consumption of mussels occurred in the Netherlands in 1961 (Korringa and
o
rs
Roskam 1961). Later, in 1970 more than 100 victims suffered severe gastro-
Pe
intestinal disorders after consumption of mussels from Los Lagos (Chile),
r
and the DSP syndrome was described for the first time. This outbreak was
fo
later associated with a bloom of Dinophysis acuta , which was not reported to
y
op
the international community until 1991 (Lembeye et al. 1993). Other gastro-
C
intestinal outbreaks occurred in 1976 and 1977 after eating mussels (Mytilus
’s
edulis ) and scallops (Patinopecten yessoensis ) in northeast Japan. The toxic

or
agent responsible for the intoxication, Dinophysis fortii , was identified 2 years
ut
later by Professor Takeshi Yasumoto, who was among the victims, and the
b
tri
mouse bioassay (MBA) was implemented to quantify this kind of toxicity

on
(Yasumoto et al. 1978, 1979). The main compound responsible for the DSP
.C
outbreak was OA (Murata et al. 1982).

C
During the summer of 1981, an important DSP outbreak was reported from
LL
Galicia (northwest Spain), with more than 5000 victims after eating mussels
is
(Mytilus galloprovincialis ) (Campos et al. 1982), and 3300 people were affected
c
an
in France 2 years later due to the consumption of mussels (M. edulis ) (Alzieu
Fr
et al. 1983). These outbreaks were associated with the presence of Dinophysis
acuminate (Alzieu et al. 1983; Campos et al. 1982; Lassus et al. 1985). The
d
an
following year, in October, a new DSP outbreak occurred in Sweden and

or
Norway, where more than 400 people consumed mussels (Krogh et al. 1985;
yl
Underdahl et al. 1985). The dinoflagellates associated with these events were
Ta
D. acuta and Dinophysis norvegica (Dahl and Yndestad 1985). More DSP cases
18
were detected in London and Portugal between 1997 and 2001 due to the
20
consumption of clams (Donax trunculus ) and razor clams (Solen marginatus )

(Vale and Sampayo 1999; Vale and Sampayo 2002).
©
In 2002, several hundred people were intoxicated after consuming brown

crabs (Cancer pagurus ) in Norway. Together with this outbreak, an unusual
early bloom of D. acuta led to high amounts of DSP toxins in blue mussels
(M. edulis ) in the same area. Analyses of crabs revealed very little free toxin
in the form of OA, maybe due to the accumulation of toxins in crabs after
eating blue mussels (Torgersen et al. 2005).
During 2006– 2007, more DSP episodes occurred in Greece, after eating
mussels from Thermaikos and Saronikos Gulfs (Prassopoulou et al. 2009).
More outbreaks were reported in Canada and China in 2011, where 62 and
57 cases of DSP intoxication were detected, respectively (Chen et al. 2013;
McIntyre et al. 2013; Taylor et al. 2013).
4.2.2 Pectenotoxin Group Toxins
y
nl
O
The pectenotoxins (PTXs) were first isolated from the digestive glands of
se
the Japanese scallop P. yessoensis (Yasumoto et al. 1985), but nowadays PTXs
lU
are found in different bivalve mollusks (mussels, cockles, and scallops)
na
from Australia, Japan, New Zealand, and several European countries (EFSA
o
2009b). Despite the dinoflagellate D. fortii being initially identified as the real
rs
producer of these toxins (Draisci et al. 1996), PTXs were later found in D. acu-
Pe
minata , D. acuta , Dinophysis caudata , Dinophysis rotundata , and D. norvegica
r
fo
(Miles et al. 2004; Ferná ndez et al. 2006; MacKenzie et al. 2005).
y
op
C
’s
or
These lipophilic macrocyclic polyether-lactone toxins are heat-stable poly-

ut
ether macrolide compounds. Their chemical structures include a spiroketal

b
group, three oxolanes, a bicyclic ketal, and a six-membered cyclic hemiketal

tri
on
(Allingham et al. 2007).

.C
PTX2 (Figure 4.2) is suspected to be the main precursor, from which many
PTX analogs are derived through biotransformation during metabolism in
C
LL
bivalves.
An oxidation of PTX2 leading to the formation of PTX1, PTX3, and PTX6
c is
has been suggested by some authors (Draisci et al. 1996; Yasumoto et al.
an
2000). PTX1 and PTX6 are major metabolites found in the Japanese scallop,
Fr
while PTX2 is rapidly metabolized into PTX2 seco acid (PTX2 SA) and its
d
epimer 7-epiPTX2 seco acid (7-epi-PTX2 SA) in mussels and scallops. PTX4
an
and PTX7 were identified as spiroketal isomers of PTX1 and PTX6, namely,
or
yl
Ta
O
18
CH3
H3C
20
O O
©
O O
O
O OH
OH H 3C
O
OH O
H3C O
CH3 CH3
O
FIGURE 4.2
Chemical structure of PTX2.
epi-PTX1 and epi-PTX6, respectively (Sasaki et al. 1998). Later, PTX11 was
reported as a further analog (34Shydroxy-PTX2) (Suzuki et al. 2006).
PTXs are easily destroyed under strong basic conditions and are also labile
under acidic conditions, which transform them into the seco acid forms. In
shellfish, PTX2 SA and 7-epi-PTX2 SA can be metabolized to form corre-
sponding fatty acid esters.
y
Initial studies showed that PTX1 and PTX2 alter F-actin (Zhou et al. 1994;
nl
Spector et al. 1999), and these observations were confirmed by subsequent
O
studies in several cellular types (Ares et al. 2005; Leira et al. 2002).
se
A description of the interaction between PTX2 and actin and the charac-
lU
terization of structure– activity relationships of PTX group toxins have also
na
been obtained (Allingham et al. 2007). The structure– activity studies have
o
rs
shown that the PTX molecule ring is a key determinant of actin binding, so
Pe
that isomerization around the C7 of the PTX molecule and the rupture of the
r
lactone ring would interfere with PTX– actin interactions (Allingham et al.
fo
2007).
y
op
C
’s

or
ut
Different PTX analogs have been found in different bivalve mollusks, such as
b
mussels (Perna canaliculus and M. edulis ) scallops (Pecten novaezelandiae and

tri
P. yessoensis ), and cockles (Cerastoderma edule ), from several regions in the

on
world (Japan, different European countries, and New Zealand, among oth-
.C
ers) (Daiguji et al. 1998; Wilkins et al. 2006; Vale and Sampayo 2002; Suzuki et
C
al. 2001; Pavela-Vrancic et al. 2001).

LL
c is
an

Fr
There are no reports of human illness associated with exposure to PTX

d
group toxins.
an
or
yl
4.2.3 Yessotoxin Group Toxins

Ta

18
20
Yessotoxins (YTXs) were first isolated in Japan in 1986 from the digestive
gland of P. yessoensis , the scallop from which the toxin’ s name is derived
©
(Murata et al. 1987). This group of structurally related polyether toxins is

produced by the dinoflagellates Protoceratium reticulatum (Satake et al. 1997),
Lingulodinium polyedrum (Draisci et al. 1999), and Gonyaulax spinifera (Rhodes
et al. 2006). More recently, YTXs have been reported worldwide in Korea
(Lee et al. 2012), Chile, and New Zealand (Yasumoto and Takizawa 1997).
In Europe, they have been described in mollusks from Norway, Italy, Spain,
and Russia (Arevalo et al. 2006; Ciminiello et al. 1997; Morton et al. 2007;
Vershinin et al. 2006; Lee et al. 1988).

Due to their chemically stable molecular structures, which consist of a ladder-
shaped polycyclic ether skeleton and an unsaturated side chain with one to
three sulfate esters (Figure 4.3), few reports of the chemical conversion of YTXs
are found in the literature. The planer structure was elucidated by nuclear
magnetic resonance (NMR) spectroscopy (Murata et al. 1987), but the relative
y
nl
stereochemistry of YTX was later reported (Satake et al. 1996).
O
Since the YTX molecule has a hydrophobic polyether skeleton, some hemo-
se
lytic activity could be expected; however, it did not induce hemolysis (Ogino
lU
et al. 1997).
na
Different effects in calcium levels were reported in several cellular mod-
o
els after YTX incubation, such as human fresh lymphocytes, HL7702 human
rs
liver cells, the Bel7402 human hepatoma cell line, and primary cultures of rat
Pe
cerebellar neuron (de la Rosa et al. 2001; Pé rez-Gó mez et al. 2006; Pang et al.
r
fo
2011, 2012).
y
The chemical structure of YTX resembles those of brevetoxins (BTXs) and
op
CTXs; however, YTXs did not induce any effect on sodium channels, the tar-
C
gets of the other two groups of toxins, and besides, YTX did not induce any
’s
or
competitive displacement of BTXs from site 5 of the sodium channels (Inoue

ut
et al. 2003). These results demonstrated that the effect of YTX on cytosolic
b
calcium levels is a direct consequence of calcium channel activation, and it is

tri
on
not linked to sodium channels, as happens with BTXs or CTXs.

.C
On the other hand, the interaction between phophodiesterases (PDEs) and

YTX has been studied in a biosensor surface, or by measuring changes in
C
LL
the fluorescence polarization of an enzyme– dye conjugate in the presence of

YTX (Alfonso and Alfonso 2008; Alfonso et al. 2005), which led to the conclu-
cis
sion, after both studies, that YTX binds to cyclic nucleotides PDE1, PDE3, and
an
PDE4 and exonuclease PDE I (Pazos et al. 2006).

Fr
Their mechanism of action is still unclear today, but several investigations

d
have reported that YTXs induce apoptotic changes in the BE(2)-M17 neuro-
an
blastoma cell line and in HeLa cells by caspase activation and the disrup-
or
tion of the E-cadherin– catenin system in epithelial cells (Ronzitti et al. 2004;
yl
Ta
NaO3OS
18
CH2
CH3 CH3
20
HO
O
O
NaO3OS CH2
©
CH2
O
O
O
O O
O CH3
O
O OH
O
CH3 CH3 CH3
FIGURE 4.3
Chemical structure of YTXs.
Leira et al. 2002; Malaguti et al. 2002). Moreover, some effect of YTX in the
protein kinase C (PKC) translocation was shown in primary cortical neurons
(Alonso et al. 2013).
YTXs often coexist with DSP toxins, so initially they were included in the
OA group. DSP toxins are specific inhibitors of Ser/Thr protein phospha-
tases PP1 and PP2A, and YTX also inhibits these enzymes, but the effect and
y
the toxicity are four orders of magnitude lower than those of DSP (Ogino et
nl
al. 1997). Nowadays, YTXs are included in a different group of toxins (FAO/
O
IOC/WHO 2004).
se
lU
na
o
rs
Despite these toxins being described in the digestive glands of scallops,
Pe
YTXs have also been found in other mollusks, such as mussels and oysters,
r
and recently in some gastropods (Lee et al. 2011, 2012; MacKenzie et al. 2002;
fo
Schirone et al. 2013; Samdal et al. 2005).
y
op
C
’s
or
No reports of human intoxication induced by YTXs have been described. In

ut
fact, European Union (EU) legislation changed recently in order to increase

b
tri
the limit of YTXs in shellfish (European Commission 2013).

on
.C
C
4.2.4 Azaspiracid Group Toxins

LL

c is
an
AZAs, responsible for the azaspiracid poisoning (AZP) syndrome, were dis-
Fr
covered in 1995 due to an outbreak of shellfish poisoning in the Netherlands

after consumption of contaminated mussels (M. edulis ) harvested from
d
an
Killary Harbour, Ireland (Satake et al. 1998; McMahon and Silke 1996).
or
These compounds are produced by the dinoflagellate Azadinium spinosum

yl
(Tillmann et al. 2009). More recently, two strains of Azadinium poporum were
Ta
proven to be producers of two new AZA analogs (AZA37 and AZA36) iso-
18
lated from the North Sea and the Korean west coast (Krock et al. 2015).
20
Since then, seafood contamination by AZAs has been reported in different

coastal localizations, including Europe, North and South America, Africa,
©
and Japan (Ueoka et al. 2009; Taleb et al. 2006; Magdalena et al. 2003; Klontz
et al. 2009; James, et al. 2002; Á lvarez et al. 2010).

AZA1, the main compound found in mussels from Ireland, was isolated in
1998 (Satake et al. 1998), but the total structure was later fully elucidated by
synthetic studies (Nicolaou et al. 2006).
Based on the similarities in gastrointestinal symptoms that AZAs have

in common with OA and DTXs, AZAs were originally classified together
with DSP toxins (Twiner et al. 2008). The serine/threonine phosphatases are
known to be inhibited by OA (Cohen 1989). However, the effects of crude
blue mussel extracts containing AZAs demonstrated no indication of PP1
or PP2A inhibition (Flanagan et al. 2001; Twiner et al. 2005), and nowadays
y
AZAs are classified in a separate group.
nl
A cyclic amine (or AZA group), the tri-SPIRO-assembly, and the carboxylic
O
acid group gave rise to the name AZA-SPIR-ACID for these compounds.
se
After the structure elucidation of AZA1 (Figure 4.4), two new analogs,
lU
AZA2 and AZA3, were isolated from mussels collected at Arranmore
na
Island, Ireland, in 1997 and characterized by mass spectrometry (MS) and
o
rs
NMR techniques (Ofuji et al. 1999). These compounds differ in the number
Pe
of methyl groups. AZA3 is lacking the C22 positioned methyl moiety, and
r
AZA2 possesses an additional CH3 at C8 compared with AZA1.
fo
Several investigations had been carried out to discover the target of these
y
op
toxins. The reduction of the AZA cytotoxicity in the presence of a c-Jun
C
N-terminal protein kinase was observed in primary cerebellar granular
’s
cells, but it was not observed in neocortical neurons (Cao et al. 2010). AZAs
or
were tested on several kinases without any indication of inhibition, suggest-

ut
ing that AZAs are not kinase inhibitors (Twiner et al. 2014).
b
tri
AZAs are potent cytotoxic agents causing extensive morphological and

on
cytoskeletal changes to cultured cells (Twiner et al. 2005, 2012a). The effects
.C
of these compounds on actin were assessed, but no direct alteration actin

C
polymerization or depolymerization in vitro was observed, suggesting that

LL
intracellular cytoskeletal rearrangements mediated by AZA toxins are an

is
indirect effect of a toxicological response (Twiner et al. 2014).

c
an
Other investigations have demonstrated that AZAs modify ion flux in dif-
Fr
ferent cell types, and they have been shown to alter intracellular calcium
d
an
H
or
O H
yl
Ta
H
HO O H OH
18
O
H O
CH3
20
O HO
©
H
O H
H
H
NH O
O
O
H
FIGURE 4.4
Chemical structure of AZAs.
flux, proton homeostasis, and membrane hyperpolarization (Alfonso et al.

2006; Roman et al. 2002; Vale et al. 2010).
A recent study demonstrated that AZAs inhibit hERG (human ether-à -go-
go-related gene) channels, a specific type of potassium ion channel (Twiner
et al. 2012b), important in the cardiac action potential. On the other hand,
AZA2 caused an increase in the levels of membrane hERG channels with-
y
out a change in potassium current through the channel. Electrocardiogram
nl
studies in rats showed arrhythmic electrical activity, demonstrating cardio-
O
toxicity (Ferreiro et al. 2014a, 2014b).
se
Despite AZAs having been shown to be cytotoxic, affect cytoskeleton
lU
arrangements, and inhibit potassium channels (hERG), no particular tar-
na
gets have been discovered so far, and the mechanism of action of these com-
o
rs
pounds needs further studies.
rPe
fo
y
op
Mussels (M. edulis ) harvested from Irish coastal waters are the main vectors
C
of AZP in humans (Twiner et al. 2014; EFSA 2008a; Salas et al. 2011). Moreover,
’s
other shellfish species, such as Japanese and flat oysters (Crassostrea gigas and
or
Ostrea edulis ), Chilean mussels (Mytilus chilensis ), razor fish (Ensis siliqua ), and
ut
scallops (Pecten maximus and Argopecten purpuratus ), have also been shown to
b
tri
be contaminated with AZAs (Magdalena et al. 2003; Ló pez-Rivera et al. 2010;

on
Hess 2002). Furthermore, some crustaceans, such as crabs (Cancer pagurus ),

.C
may also accumulate AZAs due to their feeding on mussels, but no human
C
intoxications have been registered due to ingestion of crabs so far (Torgersen

LL
et al. 2008).
c is
an

Fr
Human consumption of AZA-contaminated shellfish can result in severe

d
an
acute symptoms, such as nausea, vomiting, diarrhea, and stomach cramps,

or
similar to DSP toxins (McMahon and Silke 1996). Six human outbreaks have
yl
been confirmed thus far, but due to the similarity of symptoms to those of
Ta
DSP toxins, more undocumented events could occur. Irish mussels and scal-
18
lops were responsible for the intoxications in all cases.

20
The first event occurred in the Netherlands in November 1995, after the
ingestion of mussels harvested from Killary Harbour, Ireland. In this out-
©
break, eight people fell ill with DSP-like symptoms of gastrointestinal ill-
ness (nausea, vomiting, severe diarrhea, and stomach cramps). The absence
of known DSP toxins in the analysis allowed the discovery of the new com-
pound AZA1 (Satake et al. 1998; McMahon and Silke 1996). Two years later,
8 of at least 20 people sought medical attention with gastrointestinal symp-
toms for 2– 5 days due to their consumption of contaminated mussels in
Arranmore Island, Ireland (Ofuji et al. 1999). In September 1998, 10 people
in Italy and 20– 30 in France fell victim to AZP with typical gastrointestinal
symptoms after the ingestion of mussels harvested from Ireland, where

AZAs were found to be present after shellfish analysis (Furey et al. 2010).
Another human poisoning was registered in Warrington, Aylesbury
(United Kingdom), following the consumption of frozen, precooked mussels
harvested from Bantry Bay in Ireland. During this outbreak, 12– 16 people pre-
sented symptoms, including nausea, diarrhea, abdominal pain, and cramps
y
(Ryan et al. 2008). AZAs 1– 3 were detected by liquid chromatography–
nl
tandem mass spectrometry (LC-MS/MS) in the contaminated mussels (Furey
O
et al. 2010).
se
No AZP events occurred until 2008, when many people in France fell ill
lU
due to their ingestion of frozen, precooked mussels from Ireland. The event
na
was categorized by the French government as a “ large outbreak” (European
o
rs
Commission 2008). During the summer of the same year, another AZP event
Pe
occurred in the United States. In this case, two people were intoxicated after
r
the consumption of frozen, precooked mussels from Bantry Bay, Ireland.
fo
Within 5 hours of the meal, each person experienced abdominal heaviness,
y
op
vomiting, and diarrhea for up to 30 hours. Analysis revealed the presence of
C
AZA1– 3 (Klontz et al. 2009). After 2– 3 days, full recovery was reported in all
’s
cases.
or
ut
b
tri
4.2.5 Saxitoxin Group Toxins

on

.C
C
Saxitoxins (STXs), responsible for the paralytic shellfish poisoning (PSP) syn-
LL
drome, are mainly produced by toxic dinoflagellates belonging to the genus

is
Alexandrium (Alexandrium tamarensis , Alexandrium minutum [syn. Alexandrium

c
excavatum ], Alexandrium catenella , Alexandrium fraterculus , Alexandrium fundy-

an
ense , and Alexandrium cohorticula ), Gymnodinium and Pyrodinium have also

Fr
been identified in some cyanobacteria that may occur in fresh and brack-
d
an
ish waters (Hallegraeff 1995; Murray et al. 2011). These genera seem to have
expanded during the last decades; at present, they are distributed practically
or
yl
all over the world (Anderson et al. 2001).

Ta
18

20
Its basic structure is that of a trialkyltetrahydropurine, with positions 2 and

©
8 of the purine ring containing the NH2 groups, which form the two per-
manent guanidinium moieties (Schantz et al. 1975). Variations in functional
groups at four defined positions around the ring define the different divi-
sions that consist of the carbamate toxins, all of which have a carbamoyl
at the R1 position, the N -sulfocarbamoyl toxins, the decarbamoyl toxins,
and the deoxydecarbamoyl toxins. The toxicity of the derivatives varies by
approximately two orders of magnitude, with STX (Figure 4.5) being the
most toxic, followed by neosaxitoxin and gonyautoxins 1 and 3.
H2N O
H
HN N
NH
H2N N NH
y
OH
nl
OH
O
se
FIGURE 4.5
lU
Chemical structure of STX.
na
Since the chemical structure of STX was determined (Schantz et al. 1975),
o
rs
several derivatives have been isolated from toxic dinoflagellates or contami-
Pe
nated shellfish. More than 50 analogs are known today (Wiese et al. 2010).
r
The pharmacological action of PSP toxins is related to the voltage-gated
fo
sodium channel blockade abolishing the propagation of the action poten-
y
op
tial, preventing normal cellular function, and leading to paralysis. The
C
7,8,9-guanidine function has been identified as being involved in the chan-
’s
nel blockade.
or
The mechanism of action of the PSP toxins strongly resembles that of TTX,
ut
assuming that both molecules have the same interaction with the receptor.
b
tri
on
.C

C
Despite mussels and clams being the dominant vectors for PSP toxins, other
LL
organisms, such as gastropods, crustaceans, or certain fish, can produce

is
human intoxications (Table 4.1) (Deeds et al. 2008).

c
an
Fr

d
an
Historical accounts of PSP in humans date back to at least the eighteenth

or
century, when one of Captain George Vancouver’ s crew died and four others
yl
became ill after consuming mussels in Poison Cove, on British Columbia’ s

Ta
central coast (McIntyre et al. 2013).

18
A human intoxication caused by A. catenella occurred in 1927 in the United

20
States, resulting in 106 illnesses and 6 deaths (Wang 2008). Since the 1940s,
cases of PSP have been reported in many countries, including Norway
©
(Langelanda et al. 1984), the United Kingdom (McCollum et al. 1968), Canada
(Tennant et al. 1955), North America (Gessner et al. 1997), Chile (Garcí a et al.
2004), South Africa (Popkiss et al. 1979), Japan, and Indonesia (Kao 1993), in
humans and animals.
In 1942, 2000 dead birds were observed in Washington State, due to an
A. catenella bloom that resulted in six cases of human intoxication. Moreover,
during 1987, 14 humpback whales (Megaptera novaeangliae ) died in the waters
of New England after eating Atlantic mackerel (Scomber scombrus ) containing
TABLE 4.1
Some Nontraditional Vectors of STX to Human Consumers
Nonbivalve Microalgal
Invertebrates Species Source Location Reference
Gastropods Polinices lewisii Alexandrium Canada Quayle (1971)
acatenella
y
nl
Adelomenon ancilla Alexandrium Chile Shumway (1995)
O
catenella
se
Argobuccinum sp.
lU
Nassarius sp. Washington,
United States
na
Neptunea spp. Alaska, United
o
rs
States
Pe
Lunatia heros Alexandrium Massachusetts, Tufts (1979)
tamarense United States
r
fo
Buccinum undatum Quebec, Canada Shumway (1995),
y
op Prakash et al.
(1971)
C
Crepidula fornicata Gulf of Maine, Shumway (1995),
’s
United States White et al.

or
(1993)
ut
Adelomedon brasiliana Argentina Carreto et al.

b
tri
(1996)
on
Rapana venosa Hiroshima Bay, Ito et al. (2004)

.C
Japan
Lambis lambis Pyrodinium Malaysia Ting and Wong
C
LL
bahamense (1989)
Crustaceans Cancer magister Alexandrium Washington, Shumway (1995)
is
catenella United States

c
an
Fabia subquadra
Fr
Pagurus sp.
d
Homarus americans Alexandrium Bay of Gaspe, Desbiens and

an
tamarense Canada Cembella (1995)

or
Portunus pelagicus Pyrodinium Sabah, Malaysia Kan et al. (1986)

yl
bahamense
Ta
Panulirus versicolor
18
Panulirus longipes
20
Fish tissues Scomber japonicus Alexandrium Argentina Montoya et al.

tamarense (1997)
©
Rastrelliger sp. Pyrodinium Brunei Li et al. (1999)

bahamense Darussalam
Sardinella sp.
Sphoeroides nephelus United States Abbott et al.
(2009)
Sphoeroides
testudineus
Sphoeroides splengleri
paralytic shellfish toxins (PSTs). In 1997, at least 117 Mediterranean monk

seals (Monachus monachus ) died along the coast of the Western Sahara, Africa,
due to the presence of A. minutum and Gymnodinium catenatum in the water,
and PSTs were found in all samples tested (Landsberg 2002).
In 2002, fishermen working in the Patagonia Chilean fjords were intoxi-
cated by consumption of the filter-feeder bivalve Aulacomya ater . Two fisher-
y
men died 3– 4 hours after shellfish consumption. High levels of STX were
nl
detected in mussels samples (Garcí a et al. 2004). From 2002 to 2004, 28 puffer
O
fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York
se
were linked to puffer fish (Sphoeroides spp.) harvested from the Indian River
lU
Lagoon (IRL) in Florida. STX and two of its derivatives were detected in sam-
na
ples, leading to the characterization of the food poisoning syndrome as saxi-
o
rs
toxin puffer fish poisoning (SPFP) to distinguish it from the PFP traditionally
Pe
associated with TTX (Landsberg et al. 2006).
r
Symptoms of PSP include paresthesia and numbness, first around the lips
fo
and mouth, and then the face and neck; muscular weakness; sensation of
y
op
lightness and floating; ataxia; motor incoordination; drowsiness; incoher-
C
ence; and progressively decreasing ventilator efficiency, leading to respira-
’s
tory paralysis and death in the most severe cases (Deeds et al. 2008). Around
or
2000 cases of human intoxication are reported every year, with a 15% mortal-
ut
ity rate (Van Dolah 2000).

b
tri
on
.C
4.2.6 Domoic Acid

C

LL
Domoic acid (DA), responsible for the amnesic shellfish poisoning (ASP) syn-
c is
drome, is synthesized by the red alga Chondria armata (Zaman et al. 1997;
an
Takemoto and Daigo 1958) and also from the diatoms Pseudo-nitzschia spp.
Fr
(Kotaki et al. 1999) and Nitzschia (Kotaki et al. 2000). It is distributed mainly
d
in North America (Bill et al. 2006) and western Europe (Blanco et al. 2006),
an
and also in Australia, although several reports have been published in other
or
locations (Lapworth et al. 2001).

yl
Ta
18

20
DA is a cyclic amino acid with three carboxylic acid groups, which are
©
responsible for its water solubility and its relatively high polarity (Quilliam
2001) (Figure 4.6). Different analogs of DA have been reported. DA converts
into epi-DA through long-term storage and degrades and transforms to
epi-DA and iso-DAs through exposure to ultraviolet (UV) light. Moreover,
epimerization is also accelerated by heating. Due to the conjugated double
bond in the aliphatic side chain, DA absorbs UV light and is also the cause of
radical-mediated oxidative metabolism. DA is heat stable, and cooking does
not destroy the toxin (McCarron et al. 2007).
OH
O
O O
HO N
H OH
y
nl
FIGURE 4.6
O
Chemical structure of DA.
se
lU
This neurotoxin acts by blocking some glutamate receptors in the central
na
nervous system, which results in depolarization of neurons (Berman and
o
Murray 1997; Hampson and Manalo 1998).
rs
Pe
r
fo
y
These compounds have been isolated from mollusks such as mussels (M. edu-
op
lis ), razor clams (Siliqua patula ), clams (Mya arenaria ), and scallops. Moreover,
C
DAs have been found in the Dungeness crab Cancer magister and in fish such
’s
or
as anchovies (Engraulis mordax ) and sardines (EFSA 2009a).

ut
b
tri

on
.C
The first documented outbreak of ASP occurred in Canada in 1987, when

4 people died and 100 became ill after consuming contaminated mussels
C
LL
(Bates 1998). Another 21 cases of ASP were reported in Washington State in

1991, after consumption of razor clams. In this case, seven victims required
c is
medical attention, but no deaths were reported (Horner et al. 1997). Moreover,
an
several animals, such as seabirds and marine mammals, have been intoxi-
Fr
cated in Mexico and the United States due to the consumption of anchovies,
d
an
Dungeness crabs, or sardines (EFSA 2009a).

or
yl
4.2.7 Ciguatoxin Group Toxins

Ta

18
20
CTXs, responsible for the ciguatera fish poisoning (CFP) syndrome, are pro-
duced by the benthic dinoflagellate Gambierdiscus spp., mainly associated
©
with tropical and subtropical areas, but recently, CTX group toxins were
identified for the first time in fish from Europe (EFSA 2010a).
Ten Gambierdiscus species were reported in the Atlantic and in the Pacific
region (Litaker et al. 2010), and other new species were found in European
Atlantic waters and in the Mediterranean Sea (Aligizaki et al. 2008a).
Maitotoxins and gambierol, other toxic compounds, are also produced by
species of dinoflagellates of the genus Gambierdiscus , which grow on algae
in tropical waters around the world, accumulate in the body of herbivorous
O HO CH3
CH3
O O
O O
O
HO O O
OH O O CH3
OH
OH
y
nl
O
O
O
se
H3C O
lU
H3C
na
OH
o
rs
Pe
FIGURE 4.7
Chemical structure of the Pacific CTX.
r
fo
fish, and are transmitted through the tropical food chain to carnivorous spe-
y
cies (Caillaud et al. 2010).
op
C
’s
or

utb
CTXs are polyethers with a chemical structure formed by rings 13– 14 fused
tri
by ether linkages (Murata et al. 1989). Various congeners of CTXs have been
on
characterized according to differences in their molecular structure: Pacific

.C
(P-CTX) (Figure 4.7), Caribbean (C-CTX), and Indian (I-CTXs) (Caillaud et al.
C
2010), and more than 30 analogs have been reported worldwide (Silva et al.
LL
2015). These compounds are odorless, tasteless, heat resistant, and lipid sol-
is
uble, so they are not destroyed by cooking processes (Pottier et al. 2002b).
c
an
CTXs bind to nonselective, non-voltage-activated ion channels, causing their

Fr
opening, leading to the increase of intracellular calcium levels to toxic concen-

d
trations. During the acute period in the first 24 hours, gastrointestinal prob-
an
lems appear and cardiovascular complications and neurologic symptoms, like

or
paresthesias, dysesthesias, and hyperesthesias, can occur (Pottier et al. 2002a).

yl
Ta

18
20
The common vectors for these phycotoxins are finfish and some mollusks,
such as the turban snail Lunella cinerea or giant clam Tridacna gigas . Moreover,
©
higher concentrations of CTXs have been detected in top predatory fish, like
certain species of barracuda, snapper, grouper, and jack, that accumulate the
toxins in the flesh, viscera, and liver (FDA 2011).

Worldwide, more than 25,000 persons are affected annually, with the high-
est rates occurring in endemic tropical and subtropical areas. Thirty-three
outbreaks with 103 victims were reported in Japan between 1997 and 2006
due to consumption of several species (Variola louti , Lutjanus bohar , Lutjanus
monostigma , Epinephelus fuscoguttatus , unidentified Lutjanus sp., Plectropomus
areolatus , Oplegnathus punctatus , Epinephelus polyphekadion , Caranxignobilis ,
and moray eel). All these samples were analyzed by LC-MS/MS, and P-CTX-1
was detected (Oshiro et al. 2010).
y
Occasionally, CFP outbreaks occur outside of endemic areas, due to con-
nl
sumption of imported toxic fish. Recent observations suggest the expan-
O
sion of the biogeographical range of Gambierdiscus spp. and ciguatoxic fish
se
(Villareal et al. 2007). One man died 6 days after eating sawtooth barracuda
lU
in Brisbane, Australia, and P-CTX-1, P-CTX-2, and P-CTX-3 were detected in
na
the sample by LC-MS/MS (EFSA 2010a). Another 30 patients were admitted
o
rs
to the hospital in Marseille, France, after the ingestion of the same barracuda
Pe
in Mexico (De Haro et al. 1997).
r
More than 500 people were intoxicated in a single outbreak on the east
fo
coast of Madagascar, and finally, 98 died after consumption of the shark
y
op
Carcharhinus amboinensis . The clinical symptoms led to the conclusion that
C
the poisoning was due to CTX. This was the first occasion with a mortality
’s
rate of 20% (Habermehl et al. 1994).

or
In 2004, the first intoxication related to CFP occurred in Spain linked

ut
to a lesser amberjack (Seriola rivoliana ) captured along the eastern coast

b
tri
of the Canary Islands Archipelago (Perez-Arellano et al. 2005), where

on
Gambierdiscus spp. was also found (Aligizaki et al. 2008b). Finally, C-CTX-1
.C
was confirmed in sample extracts by LC-MS/MS. Two additional outbreaks

C
occurred during 2008 and 2009, and the same implicated fish was confirmed
LL
positive for C-CTX-1 (Boada et al. 2010). This report indicates that carniv-
is
orous fish in temperate waters of the eastern Atlantic could be ciguatoxic,

c
an
reinforcing data suggesting the geographical expansion of CFP endemicity

Fr
worldwide.
d
an
or
4.2.8 Brevetoxins
yl
Ta

18
BTXs, responsible for the neurologic shellfish poisoning (NSP) syndrome,

20
are primarily produced by a dinoflagellate, Karenia brevis (formerly

called Gymnodinium breve and Ptychodiscus brevis ), first identified in the
©
Gulf of Mexico in 1947 (Gunter et al. 1947). However, other algae species
(Chattonella antiqua , Chattonella marina , Fibrocapsa japonica , and Heterosigma
akashiwo ) have also been reported to produce BTX-like toxins (FAO/IOC/
WHO 2004). BTXs have been described in several locations, such as North
America, New Zealand, Australia, Japan, and Scotland (Van Dolah 2000;
FAO/IOC/WHO 2004; Watkins et al. 2008). K. brevi s or K. brevis – l ike
species like K. brevissulcatum have also been reported from Japan, New
Zealand, the West Atlantic, Spain, Portugal, and Greece (Anderson et al.
2001). In particular, Karenia papilionacea was identified as a BTX producer,

and it was found in Scotland (Turner et al. 2015).

BTXs are lipid-soluble cyclic polyether stable compounds. There are two
y
nl
types of BTXs (1 and 2) (Figure 4.8) based on the molecular backbone struc-
O
tures, consisting of 10– 11 transfused rings. Despite several analogs having
se
been identified (BTX1, BTX2, BTX3, BTX-B1, BTX-B2, S-desoxy-BTX-B2, BTX-
lU
B3, BTX-B4, and BTX-B5), BTX1 and BTX2 are considered to be the parent
na
toxins (Baden et al. 2005). These toxins are resistant to heat and steam auto-
o
claving (Poli 1988).
rs
Their mechanism of action is related to binding with high affinity to recep-
Pe
tor site 5 of the voltage-gated sodium channels in cell walls (Baden et al.
r
fo
2005), leading to the activation of these channels with the consequent uncon-
y
trolled Na+ influx into cells and depolarization of neuronal and muscle cell
op
membranes (Watkins et al. 2008).
C
’s
or
ut
b
tri
on
.C
H3C
C
CH3
CH3
LL
O
HO
is
O O
c
H3C O CH2
an
O
O O
O
Fr
O
O O
d
O
an
H
(a)
or
yl
CH3
Ta
18
O O CH3
20
O
©
O CH3
CH3
O
O
O OH
CH3
O H2C O
O
(b) O
H
O
FIGURE 4.8
Chemical structure of (a) BTX1 (A-type backbone) and (b) BTX2 (B-type backbone).

BTXs have been reported in many shellfish species, but the most common
sources of human exposure are through the ingestion of clams, oysters, and
mussels. More recently, these compounds have been retained in the muscle,
gut, and organs of finfish, in much lower concentrations than in mollusks
(Naar et al. 2007). Fish accumulate BTXs by ingestion of viable K. brevis cells
y
nl
and contaminated prey, and by absorption of toxins across gill membranes
O
(Tester et al. 2000).
se
lU
ona
No intoxication outbreaks in humans or occurrences of BTX group toxins in
rs
shellfish or fish have been reported in Europe (EFSA 2010b). However, sev-
Pe
eral intoxications have been reported in the United States and New Zealand,
r
fo
where the largest documented outbreak of NSP occurred between 1992 and
y
1993, due to the ingestion of mussels, cockles, and oysters (Ishida 2004; Ishida
op
et al. 1996).
C
The best documented case in the United States occurred in North Carolina
’s
or
in 1987 due to the ingestion of oysters. In this episode, only one victim was
ut
admitted to the hospital for severe neurological effects (Morris 1991). In 1996,
b
a family was poisoned after their consumption of whelks and clams collected
tri
on
in Sarasota Bay, in Florida. The children were hospitalized with severe neu-
.C
rological symptoms and BTXs were detected in their urine samples (Watkins
et al. 2008).
C
LL
In addition to the human health effects associated with exposure to BTXs,

these compounds can affect animals, such as marine mammals, fish, sea-
c is
birds, or turtles. Large mortality among bottlenose dolphins (Tursiops trun-

an
catus ) and manatees (Trichechus manatus latirostris ) was recorded in different

Fr
red tide events (Bossart et al. 1998; Fire et al. 2007). Some of these deaths
d
were attributed to the ingestion of BTX-contaminated tunicates; however, in

an
several cases animals died due to the inhalation of aerosolized BTX, after
or
the toxin was detected in the nasal and lung tissue of animals (Bossart et al.
yl
Ta
1998). BTXs have been found to persist in seagrass communities long after
the bloom has gone, providing a source for continued intoxication of grazing
18
manatees.
20
©
4.2.9 Tetrodotoxin
TTX was first believed to be present only in puffer fish of the family
Tetraodontidae. However, it has been described in a wide range of phyloge-
netically unrelated organisms, such as newts, algae, frogs, nematodes, star-
fish, crabs, and mollusks. It has also been suggested that TTX is produced
by symbiotic bacteria (Lago et al. 2015). Although TTX is associated with
tropical waters, mainly South Asia and more specifically Japan, it has also
been identified in Mexico and United States (Gessner and McLaughlin 2008).
In Europe, TTX has been reported in migrant puffer fish in Greek waters
and in a gastropod, Charonia lampas lampas , from Portugal (Silva et al. 2012).
The exact origin and pathway for the synthesis and biotransfer of TTX is
not yet fully known. A likely scenario is that synthetic genes for TTX have
y
transferred across species in evolution (Lago et al. 2015). A recent study pro-
nl
posed that the origin of TTX may be due to any of the following combina-
O
tions: exogenous, endogenous, or symbiotic association among the animals
se
acquiring it and the microorganisms that are reported produce it (Jal and
lU
Khora 2015). Recent research suggests that the microalgae Prorocentrum mini-
na
mum could be the producer of TTX found in mussels and clams from Greece
o
rs
(Vlamis et al. 2015).
Pe
r
fo
4.2.9.2 Chemical Structure and Mechanism of Action
y
op
TTX is a heat-stable water-soluble molecule, and it has been described as
C
one of the most toxic compounds among the poisons having low molecular
’s
weight. This toxin has more than 10 analogs, but TTX is the most toxic com-
or
pound among them (Asakawa et al. 2003; Tsuda et al. 1964).

ut
The structure of this compound consists of a single guanidinium moiety

b
tri
attached to a highly oxygenated carbon backbone consisting of a 2,4-dioxa-

on
adamantane structure with five hydroxyl groups (Figure 4.9).

.C
TTX acts by blockage of the sodium channels, reducing the membrane

C
excitability of vital tissues, such as heart myocytes, skeletal muscles, or the

LL
central and peripheral nervous systems. People intoxicated with these com-
is
pounds present symptoms within 30 minutes to 6 hours of ingestion, and

c
an
after 24 hours, victims have usually recovered (Rodrigue et al. 1990; Yang
Fr
et al. 1996). Some of these symptoms are headache, diaphoresis, body numb-
ness, dysarthria, dysphagia, nausea, vomiting, abdominal pain, or lack of
d
an
coordination. In the most severe cases, cardiac arrhythmias, muscle paraly-

or
sis, cranial nerve dysfunction, and even death can occur due to respiratory
yl
failure (William and Shepherd 2001).

Ta
18
20
–
O
©
H
O OH
O OH
NH
NH2+
HO NH
HO
OH
FIGURE 4.9
Chemical structure of TTX.

TTX first believed to be present only in the puffer fish of the family
Tetraodontidae; this compound has since been found in marine, freshwater,
and brackish water organisms (Table 4.2) (Lehman et al. 2004). In marine
puffer fish species, toxicity is generally high in the liver and ovary, whereas in
brackish water and freshwater species, toxicity is higher in the skin (Zaman
y
nl
et al. 1997; Kanoh 1988), suggesting that TTX is present as a defensive sub-
O
stance to protect their eggs or themselves from external enemies.
se
lU
ona
The fugu flesh or musculature is edible and considered a delicacy by many
rs
persons in Japan. Despite careful preparation, fugu remains a common cause
Pe
of fatal food poisoning in that country, accounting for approximately 50
r
fo
deaths annually (Torda et al. 1973).
y
In 1979, a paralytic poisoning occurred due to ingestion of the trumpet
op
shell Charonia sauliae from Shizuoka Prefecture in Japan, and it was con-
C
cluded, from the digestive gland analysis, that the toxin responsible for the
’s
or
incident was TTX (Narita et al. 1981). In 1996, in Nagasaki (Japan) one man
ut
died 1 hour after the ingestion of the puffer fish Takifugu poecilonotus due to
b
respiratory failure (Noguchi and Akaeda 1998). In 1998, eight people were
tri
on
intoxicated after consuming roe of Takifugu oblongus in Taiwan, with five vic-
.C
tims dying (Mahmud et al. 1999). In the same year, four cases, one of which
was fatal, were reported in Nose Be (Madagascar). The application of the
C
LL
MBA suggested poisoning with a TTX as the etiology (Ravaonindrina et al.

2001).
c is
In 2001, an outbreak of TTX poisoning occurred among six Chinese fish-

an
ermen who shared puffer fish in the Taiwan Strait. The symptoms began
Fr
2 hours after ingestion. One of the victims died 1 day after admission as a
d
result of intractable bradycardia (How et al. 2003). Two other incidents of

an
TTX occurred in Taiwan during 2004 and 2005, with seven victims intoxi-
or
cated after consumption of both digestive glands and muscle of the gastro-
yl
Ta
pods Nassarius glans and Nassarius papillosus (Jen et al. 2007; Hwang et al.
2005). Another incident was registered in southeastern Bangladesh in 2008,
18
and two of the nine people affected died after consumption of puffer fish
20
eggs (Islam et al. 2011). In 2010, 81 people were affected due to ingestion of
©
Hapalochlaena fasciata octopus in Taipei, and TTX was identified by LC-MS/

MS in serum and urine from victims (Wu et al. 2014).
Although TTX was regarded as a problem confined to Asian countries, in
recent decades the toxin has threatened the health of consumers all over the
world. Several toxic episodes have been related to TTX. In 1986, in Hawaii,
one man was intoxicated by ingestion of liver of the toxic porcupine fish
Diodon hystrix ; he fully recovered after 6 days (Sims and Ostman 1986). Three
other cases of TTX poisoning occurred in 1996, among chefs in California
TABLE 4.2
Distribution of TTX in Animals and Parts of the Body
Distribution in
Animal Species Animal Body Reference
Flatworms Planocera spp. Whole body Miyazawa et al. (1986)
Ribbonworms Lineus fuscoviridis Miyazawa et al. (1988)
y
nl
Tubulanus punstatus
O
Cephalothrix linearis Ali et al. (1990)
se
Gastropoda Charonia sauliae Digestive gland Narita et al. (1981)
lU
Babylonia japonica Noguchi et al. (1981)
na
Tutufa lissostoma Noguchi et al. (1984)
o
Zeuxis siquijorensis Whole body Narita et al. (1984)
rs
Niotha clathrata Jeon et al. (1984)
Pe
Niotha lineata Hwang et al. (1990)
r
fo
Cynatium echo Digestive gland Narita (1991)
y
Pugilina ternotoma
Cephalopoda Hapalochlaena maculosa
op
Posterior salivary Sheumack and Howden
C
gland (1978)
’s
Yotsu-Yamashita et al.
or
(2007)
ut
Polychaeta Pseudopolamilla occelata Whole body Yasumoto et al. (1989)

b
tri
Xanthidaecrabs Atergatis floridus Noguchi et al. (1983)

on
Zosimus aeneus Yasumura et al. (1986)

.C
Horseshoe crab Carcinoscorpius Eggs Kungsuwan et al. (1987)

C
rotundicauda
LL
Arrowworms Parasagitta spp. Head Thuesen et al. (1988)

is
Flaccisagitta spp.
c
Starfish Astropecten spp. Whole body Maruyama et al. (1984,

an
1985)
Fr
Goby Yongeichthys criniger Skin, viscera, gonad Noguchi and Hashimoto

d
(1973)
an
Newts Taricha spp. Skin, eggs, ovary, Mosher et al. (1965)

or
muscle, blood
yl
Notophthalmus spp. Skin, eggs, ovary Yotsu et al. (1990),

Ta
Yotsu-Yamashita and
18
Mebs (2001)
20
Cynopsis spp. Skin, eggs, ovary, Yasumoto et al. (1988)

muscle, blood
©
Triturus spp. Yotsu-Yamashita et al.

(2007), Yotsu et al.
(1990)
Frogs Atelopus spp. Skin Kim et al. (1975)
Colostethus s p. Daly et al. (1994)
Polypedates sp. Tanu et al. (2001)
Brachycephalus spp. Skin, liver Pires et al. (2002, 2005)
who shared contaminated puffer fish brought from Japan by a coworker as

a prepackaged, ready-to-eat product (CDC 1996). Two other TTX incidents
were reported in the United States in 2007 and 2014 due to the ingestion of
puffer fish belonging to the family Tetraodontidae, purchased in Chicago
and Minneapolis, respectively. In both cases, two victims were registered
and high concentrations of TTX were detected after the chemical analy-
y
sis (Cohen et al. 2009; Cole et al. 2015). On the other hand, in Queensland,
nl
Australia, a kid was bitten by an octopus at a beach, and 10 minutes later he
O
started to present TTX intoxication symptoms (Cavazzoni et al. 2008). Other
se
investigations reported TTX intoxication in dogs in New Zealand due to the
lU
ingestion of the gray side-gilled sea slug Pleurobranchaea maculata (McNabb
na
et al. 2010).
o
rs
Despite all the intoxications produced around the world because of the
Pe
ingestion of fugu, the puffer fish is considered the most delicious in Japan.
r
For this reason, restaurant preparation is strictly controlled by law, and only
fo
qualified chefs are allowed to prepare the fish.
y
op
C
4.2.10 Palytoxin and Analogs

’s
or

ut
b
tri
Palytoxin (PlTX) was first isolated and purified from a Palytoa toxica (Moore
on
and Scheuer 1971), and initially found only in Hawaii and Japan, but cur-
.C
rently it has a worldwide distribution (Deeds and Schwartz 2010). PlTXs

C
have also been detected in other marine zoanthids (soft corals) of the genus
LL
Palythoa (e.g., P. tuberculosa , P. vestitus , P. mamillosa , P. caribaeorum , and P. aff.

is
margaritae ) and benthic dinoflagellates of the genus Ostreopsis (e.g., O. siamen-

c
sis , O. mascarenensis , and O. ovata ) (EFSA 2009c). Blooms of Ostreopsis spp.

an
have also been reported in European countries, such as Spain, France, Italy,
Fr
and Greece (Brissard et al. 2014; Carnicer et al. 2015; Del Favero et al. 2012).
d
an
or

yl
Ta
The chemical structure of PlTX was elucidated in 1981 (Uemura et al. 1981;
18
Moore et al. 1981). This compound is considered one of the most complex
20
and large nonpolymeric natural molecules, containing 129 aliphatic carbon

atoms, 40 secondary hydroxyl groups, 2 diene motifs, a conjugate acryl-
©
amide-enamide system, 3 unsaturated bonds, 2 hydrophobic hydrocarbon

chains, cyclic ether systems, and bicyclic acetals (Figure 4.10). Moreover, its
structure contains 64 chiral centers, and a huge number of conformational
stereoisomers may occur (Moore et al. 1981). Different analogs of PlTX have
been identified. They differ from PlTX as a result of their additional and/or
missing hydroxyl and/or methyl groups, or because of chiralities. Two iso-
mers had been isolated from soft corals: 42-hydroxy-palytoxin (42S-OH-50S-
PlTX) and 42S-OH-50R-PlTX isolated from Palythoa toxica and P. tuberculosa
©
OH
20
O
18
OH
Ta
O
yl O OH OH
H2N
HO OH
or
an OH
d O OH
Biotoxins in Seafood
Fr OH
an
c HO OH
is OH OH
LL OH
C OH
.C
on OH O
HO OH OH
O O R1 OH OH tri
OH
OH
bu
HO N N O to
H H
OH OH OH HO
r’s
HO C OH
O
op
O R2 y
O HO
R3 R4 OH fo OH
OH r
O
OH
Pe
rs
OH OH
R5 OH
onOH
OH
al
U
se
FIGURE 4.10
121
Chemical structure of PlTX. O

nl
y
(Ciminiello et al. 2009, 2014). Other analogs, such as ostreocin-D (Ost-D) and
ovatoxin-a (OVTX-a), have been identified in the dinoflagellates O. siamen-
sis and Ostreopsis cf. ovata in Japan and in the Mediterranean Sea (Ito and
Yasumoto 2009; Ciminiello et al. 2012).
PlTX primarily acts by impairment of Na+ /K+ -ATPase (sodium pump),
converting the pump into a nonspecific open ion channel (Ramos and
y
Vasconcelos 2010). This leads to accumulation of intracellular calcium ions
nl
and cell death. Additionally, the signaling cascade triggered by PlTX leads to
O
actin filament system distortion (Louzao et al. 2008). This compound causes
se
a range of effects in animals and humans, depending on the route of expo-
lU
sure (Tubaro et al. 2011).
ona
rs
rPe
Despite PlTX being the first toxin isolated from the zoanthid P. toxica , it has
fo
also occurred in various other marine organisms living in close association
y
op
with zoanthid colonies, such as sponges, soft corals, gorgonians, mussels,
C
and crustaceans. Moreover, some predators, like polychaete worms, star-
’s
fish, or fish feeding on Palythoa colonies, accumulate high toxin concentra-

or
tions in their organs, where PlTX is stored in its active form (Gleibs and
ut
Mebs 1999).
b
tri
on
.C

C
The cases of PlTX poisoning incidents due to the consumption of contami-

LL
nated seafood are limited, but some human fatalities were involved due to
is
consumption of contaminated crabs in the Philippines, sea urchins in Brazil,

c
an
and fish in Japan, Madagascar, and the United States.

Fr
A fatal case after ingestion of a crab (Demania reynaudii ) occurred in the

Philippines, where a man reported a metallic taste after consuming the crab
d
an
and died 15 hours later (Alcala et al. 1988). In the same period, a case of a
or
man and a woman being poisoned after parrotfish Scarus (Ypsiscarus ) ovi-
yl
frons consumption was described. The symptoms developed were mainly

Ta
dyspnea, myalgia, and convulsions. The man recovered within 1 week, while
18
the woman died of respiratory failure associated with muscular damage,

20
4 days later (Noguchi et al. 1987). Another case of fatal poisoning due to
contaminated tropical sardines (Herklotsichthys quadrimaculatus ) involved a
©
woman who died in Madagascar after fish ingestion. The causative toxin
was identified as PlTX (Onuma et al. 1999). In 2000, 11 people out of 33 that
ate the same food were intoxicated in Japan. Among them, seven were hos-
pitalized after ingestion of the serranid fish (Epinephelus sp.). In this case, all
the patients recovered after more than 1 month (Taniyama et al. 2002).
Other human intoxications associated with the consumption of con-
taminated seafood have been ascribed to PlTXs without any confirmatory
analysis of the involved toxins. These cases have included boxfish (Ostracion
immaculatus ), cowfish (Lactoria diaphana ) (Shinazato et al. 2008), fish of the

Serranidae family, and crabs, such as Daira perlata (Llewellyn 2001).
Recent evidence also suggests that humans are poisoned by PlTX and its
analogs through inhalation and dermal routes (Hamade et al. 2015; Nordt
et al. 2011). The most common signs after inhalation and cutaneous expo-
sures are respiratory distress, bronchoconstriction, mild dyspnea, rhinor-
y
rhea, cough, fever, and a small incidence of dermatitis and conjunctivitis. A
nl
rare case of unilateral PlTX chemical keratitis occurred after a coral directly
O
expressed the toxin into the victim’ s eye (Chaudhry et al. 2016).
se
lU
na
4.2.11 Cyclic Imines
o
rs
rPe
CIs are a group of marine toxins formed by spirolides (SPXs), gymnodimines
fo
(GYMs), pinnatoxins (PnTXs), pteriatoxins (PtTXs), prorocentrolides, and
y
op
spiroprorocentrimine. SPXs are metabolites of dinoflagellates Alexandrium
C
ostenfeldii and Alexandrium peruvianum and are sometimes found in the pres-
’s
ence of other toxins, such as PSP toxins. GYMs are produced by the dino-
or
flagellates Karenia selliformes and were first isolated in oysters from New
ut
Zealand (Seki et al. 1995). PtTXs were first discovered in extracts from the
b
tri
digestive glands of pen shell, Pinna attenuata , in China and Japan (Zheng
on
et al. 1990). PtTXs A, B, and C were isolated from the Okinawan bivalve Pteria
.C
penguin in 2001 (Takada et al. 2001a). Seven PnTXs analogs (PnTXs A– G) have
C
been characterized (Otero et al. 2011). Recently, Vulcanodinium rugosum was

LL
identified as the microalgae that produce PnTx G (Né zan and Chromerat

is
2011). Prorocentrolide A was isolated for the first time from Prorocentrum
c
an
lima (Torigoe et al. 1988), and Prorocentrolide B was first isolated from
Fr
Prorocentrum maculosum in 1996 (Hu et al. 1996b). Finally, spiroprorocentri-

mine was isolated from a laboratory-cultured benthic Prorocentrum species
d
an
of Taiwan (Lu et al. 2001).

or
yl
Ta

18
The SPX group is the largest and best-characterized CI subgroup, with 16

20
SPX analogs (Otero et al. 2011). SPXs A, B, C, D, E, and F, isolated from mus-
sels and scallops from Nova Scotia, Canada, are the six major congeners.
©
The planar structure of SPXs B and D was described as two lipid-soluble

macrocycles containing a spiro-linked tricyclic ether ring system (Hu et
al. 1995). Following further work, the planar structure of SPXs E and F was
also described. These two analogs are thought to be metabolites produced
in shellfish since they have not been detected in phytoplankton samples
from culture and are ketoamines without the characteristic iminium ring.
At first, the CI moiety of this group of toxins seemed to be the spirolide
pharmacore due to the absence of activity in the MBA by intraperitoneal
(i.p.) injection of SPX E or F, where the secondary amine is a reduced prod-

uct of SPX B (Hu et al. 1996a, 1996b). However, the inactivity of SPX H in
the MBA, displaying this function, suggests that the CI moiety is not the
only structural requirement for toxicity (Roach et al. 2009). The differences
in chemical structures between SPXs C and D, which present an additional
methyl substitution on the imine ring, and SPXs A and B, may be significant,
y
as the first two compounds are resistant to oxalic hydrolysis, whereas the
nl
second two are converted to the biologically inactive SPXs E and F when
O
the same reaction is applied (Hu et al. 1996a, 2001). Other SPXs were also
se
identified: 13-desmethyl SPX C, 13,19-didesmethyl SPX C, 20-methyl SPX G,
lU
27-hydroxy-13,19-didesmethyl SPX C, 27-hydroxy-13-desmethyl SPX C, and
na
27-oxo-13,19-didesmethyl SPX C. One of these last isolated, 13-desmethyl SPX
o
rs
C (Figure 4.11a), is the most toxic compound of this group.
Pe
The neurotoxic symptoms observed in mice include hunched appearance,
r
abdominal breathing, respiratory distress, contractions, tremors, and ulti-
fo
mately death (Gill et al. 2003).
y
op
The planar structure of GYM was determined from oysters from the
C
Foveaux Strait, South Island of New Zealand (Seki et al. 1995). GYMs are the
’s
smallest of the CIs. The chemical structure of GYM-A (Figure 4.11b), GYM-B,
or
and GYM-C consisted of a spirocyclic imine ring and a 16-membered mac-

ut
rocycle (Miles et al. 2000, 2003; Stewart et al. 1997). Recently, a new analog,
b
tri
12-methylgymnodimine, was isolated from the dinoflagellate A. peruvianum

on
in the New River, North Carolina. A. peruvianum is the causative organism

.C
of SPXs, and these compounds were also found in samples, suggesting a

C
genetic relationship between SPXs and GYMs (Van Wagoner et al. 2011).
LL
PnTXs are macrocyclic compounds of a 6,7-spiro ring, a 5,6-bicyclo ring,

is
and a 6,5,6-trispiro ketal. The chemical structure of PnTXs (A– G) is closely

c
an
related to that of the SPXs. PnTXs A (Figure 4.11c), B, C, and D were the
Fr
first PnTXs isolated from the viscera of Japanese Pinna muricata (Chou et al.
1996; Uemura et al. 1995; Takada et al. 2001b). Later, PnTXs E, F, and G were
d
an
identified from the digestive gland of Pacific oysters (C. gigas ) from South
or
Australia (Selwood et al. 2010). Most recently, PnTX H was identified from
yl
Vulcanodinium rugosum in China (Selwood et al. 2014).

Ta
SPXs, GYM, and PnTXs target muscular and neuronal nicotinic acetylcho-
18
line receptor (nAChR) subtypes with high affinity (Araoz et al. 2011; Kharrat
20
et al. 2008; Bourne et al. 2010).

The chemical structure of the PtTXs is similar to that of PnTXs, with the
©
cyclohexenyl side chain of the PtTXs ending in a cysteine group. The abso-
lute stereochemistry of PtTXs A (Figure 4.11d), B, and C was confirmed
by total synthesis (Hao et al. 2006). The limited data available show that a
PtTX B/C mix results in 12 times more toxicity than PtTX A (Takada et al.
2001a).
The planar structure of Prorocentrolide A (Figure 4.11e), isolated from
the dinoflagellate Prorocentrum lima , in Japan, was elucidated at the same
time as Prorocentrolide B was isolated from Prorocentrum maculosum .
H3C
CH3
O O
O
N
H3C H N
H3C CH2 H
H3C
y
nl
OH HO
O
O O
O
se
O
lU
H CH3
na
HO
(a) (b)
o
rs
H3C
Pe
H2N
CH3 H 3C CH3
r
fo
HO
S
O
y
N
HOOC
op N
HO
C
H H
CH2 CH2
’s
O
O
or
O O
HO
ut
HO
b
O O O
tri
H3C H3C
O
on
H O O
.C
H3C OH H3C OH
C
H
LL
(c) (d)
is
OH
c
an
OH
HO
Fr
O HO
d
OH
an
N O
N
or
OH
yl
O
Ta
O
HO
18
OH O
20
HO
OH
O O
©
O HO HO3SO
OH
HO OH
(e) (f)
FIGURE 4.11
Chemical structures of CIs. (a) 13-Desmethyl spirolide C; (b) gymnodimine A; (c) pinnatoxin A;
(d) pteriatoxin A; (e) prorocentrolide A; (f) spiroprorocentrimine.
These compounds are described as fast-acting toxins, but no further infor-

mation is available regarding toxicity and no adverse effects are reported in
humans.
The structural elucidation of spiroprorocentrimine (Figure 4.11f), a
polar lipid-soluble macrolide, was conducted using x-ray crystallography
and NMR. No other toxicity data have been published for this compound
y
(Munday 2014).
nl
O
se
lU
SPXs and GYMs have been found in several bivalve mollusks, such as mussels
na
(M. edulis and Mytilus galloprovincialis ), razor clams (Ensis arcuatus ), scallops
o
rs
(Placopecten magellanicus ), oysters (C. gigas ), macha (Mesodesma donacium ), or
Pe
clams (Mulinia edulis , Venerupis pullastra , or Mactra chinensis ) (Á lvarez et al.
r
2010; Hu et al. 1995; Liu et al. 2011; Stirling 2001; Villar Gonzalez et al. 2006).
fo
PnTXs, isolated at first from the bivalves Pinna muricata , Pinna attenu-
y
op
ate, and Pteria penguin , have also been found in mussels (M. edulis ), oysters
C
(C. gigas ), and razor fish (Pinna bicolor ) (Selwood et al. 2010; Rundberget et al.
’s
2011).
or
PtTXs A, B, and C were first reported in the bivalve Pteria penguin and have
ut
not been reported in any other shellfish since. On the other hand, the pres-
b
tri
ence of prorocentrolides or spiroprorocentrimine in shellfish has not been

on
found.
.C
C
LL

is
No human intoxications have been linked to CIs.

c
an
Fr
d
an
or
4.3 Methods of Analysis

yl
Ta
4.3.1 Lipophilic Toxins

18
Regarding methodologies in the EU, the Commission Regulation (EU)

20
No. 15/2011 (European Commission 2011), amending Regulation (EC) No.

©
2074/2005 (European Commission 2005), as regards recognized testing

methods for detecting marine biotoxins in live bivalve mollusks, establishes
the EU-RL LC-MS/MS method as the reference for the detection of lipophilic
toxins, substituting the previous MBA method (Yasumoto et al. 1978) that is
still used in many countries (see Table 4.5).
In the EU, nowadays the LC-MS/MS procedure should be used as matter
of routine, for the purposes of both official controls at any stage of the food
chain and self-checks by food business operators.
The EU-RL-MB reported a detailed protocol, the EU-Harmonised Standard

Operating Procedure for Determination of Lipophilic Marine Biotoxins in
Mollusks, including OA, PTX, AZA, and YTX group toxins, by LC-MS/MS.
Version 5 of this method, published in January 2015, can be applied for the
determination of lipophilic marine biotoxins in different molluscan shellfish
matrixes, both fresh and cooked (EU-RL-MB 2015).
y
The method is based on the extraction of OA, PTX, AZA, and YTX group
nl
toxins with 100% methanol from homogenized tissue. Filtered extracts are
O
directly analyzed by LC-MS/MS in order to investigate the presence of free
se
OA, free DTX1, and free DTX2, PTX1, PTX2, AZA1, AZA2, AZA3, YTX,
lU
homo YTX, 45 OH YTX, and 45 OH homo YTX. To determine the total con-
na
tent of OA group toxins, an alkaline hydrolysis is necessary from metha-
o
rs
nolic extract prior to LC-MS/MS analysis with the aim of converting any
Pe
acylated esters of OA and/or DTXs to the parent OA and/or DTX1 or DTX2
r
toxins. After hydrolysis, extracts are filtered and quantified by LC-MS/MS.
fo
Chromatographic separation is performed by gradient elution, and the chro-
y
op
matographic solvents, conditions, and LC-MS/MS determination are all
C
described in the harmonized protocol.
’s
Since Regulation No. 15/2011 states this method for detecting lipophilic
or
marine biotoxins in live bivalve mollusks, it was necessary to adjust the

ut
protocol for analyzing thermally processed shellfish. Previously, an EFSA

b
tri
panel of experts had recommended considering the effect of processing

on
(cooking, steaming, and autoclaving) when testing shellfish in official con-

.C
trol. Moreover, it was mentioned that since cooking leads to an increase in

C
the level of lipophilic marine biotoxins in shellfish meat, there was a need
LL
for harmonization of sample pretreatment practices (i.e., cooking vs. non-

is
cooking) before the analysis of lipophilic marine biotoxins was carried out.
c
an
In this context, a modification of the protocol was performed for the extrac-
Fr
tion of lipophilic toxins from processed mussels, as described in Annex C.

This takes into account the loss of water during processing due to steaming
d
an
and/or autoclaving. On average, steaming will result in a 30% loss of water

or
and autoclaving 50%. In order to correct for this loss of water and help with
yl
homogenization and extraction, water should be added to the processed

Ta
mussels before testing. This is necessary if the determined toxin concen-

18
tration is to be related to the regulatory limit, which is set for live bivalve
20
mollusks.
©
4.3.2 Brevetoxins
The MBA constitutes the currently accepted method for BTX analyses in the
United States. The procedure was published by the American Public Health
Association (APHA) in 1985, and it is based on diethylether extraction of
shellfish tissue.
After the detection of NSP in New Zealand in 1993, a management strat-
egy to monitor NSP toxins was developed by the Regulatory Authority. The
sample preparation method used was based on acetone extraction of these

lipophilic components, followed by partitioning into dichloromethane. This
procedure was very effective in extracting unknown lipid-soluble toxins from
shellfish containing NSP toxins and presented certain advantages compared
with the APHA protocol (simpler and more suitable for rapid and quanti-
tative separation of organic and aqueous phases of the extract and greater
y
extraction efficiency). However, following the discovery of a novel bioactive
nl
compound (GYM) produced by the dinoflagellate Gymnodinium mikimotoi ,
O
a common species in New Zealand waters during neurotoxic events, the
se
authorities returned to the diethylether extraction procedure of the APHA.
lU
GYM is not extractable by diethylether, but it causes very rapid mouse death
na
when the dichloromethane procedure is used. Since GYM is not considered
o
rs
a risk to human health, the monitoring program now employs diethylether
Pe
extraction as a means of discriminating GYM activity from NSP toxicity
r
(Cembella et al. 1995).
fo
At a time when only the structures of BTX2 and BTX3 were known, a
y
op
competitive radioimmunoassay (RIA) to detect BTX2 and BTX3 with a
C
detectability of 2 nM was developed. A group enzyme-linked immuno-
’s
sorbent assay (ELISA) for ASP, NSP, PSP, and DSP toxins, including YTX,
or
was developed as a screening system for contaminated shellfish samples

ut
(Garthwaite et al. 2001). Thereafter, the suspected samples have to be

b
tri
analyzed by methods approved by international regulatory authorities.

on
Alcohol extraction gave good recovery of all toxin groups. In addition to

.C
MBA, several methods, including chemical analyses, are used at present to

C
detect BTXs (NOAA).

LL
cis
an
4.3.3 ASP Toxins

Fr
European legislation, Commission Regulation (EC) Nº 2074/2005 estab-

d
an
lished in Chapter II, the Amnesic Shellfish Poison (ASP) detection method;
the total content of ASP of edible parts of mollusks (the entire body or
or
yl
any edible part separately) must be detected using high-performance liq-

Ta
uid chromatography (HPLC) or any other recognized method (European

18
Commission 2005). If the results are challenged, the reference method shall
be HPLC. However, in 2007 Commission Regulation (EC) No. 1244/2007
20
of October 24, 2007, amending Regulation (EC) No. 2074/2005 states that
©
for screening purposes, the 2006.02 ASP ELISA method, as published in

the AOAC Journal of June 2006, may also be used to detect the total con-
tent of ASP of edible parts of mollusks (European Commission 2007). In
Asia, different methods are used to detect ASP toxins. Some regions, such
as Indonesia, the Philippines, Singapore, or Thailand, employ the HPLC
method. Nevertheless, in Myanmar or Vietnam, the test kit or LC-MS/MS
could be used (see Table 4.5).
4.3.4 PSP Toxins

Commission Regulation (EC) No. 1664/2006 lays down that the PSP con-
tent of edible parts of mollusks (the whole body or any edible part sepa-
rately) must be detected in accordance with the biological testing method
or any other internationally recognized method (European Commission
2006). The so-called Lawrence method may also be used as an alter-
y
nl
native method for the detection of those toxins as published in AOAC
O
Official Method 2005.06. Nevertheless, if the results are challenged, the
se
reference method shall be the biological method. This regulation men-
lU
tions that it will be reviewed in light of the successful completion of the
na
harmonization of the implementing steps of the Lawrence method by
o
the Community Reference Laboratory for Marine Biotoxins (European
rs
Commission 2006). The MBA was developed more than a half century
Pe
ago and has been refined and standardized by the Association of Official
r
fo
Analytical Chemists (AOAC) to produce a rapid and reasonable accurate
y
measurement of total PSP toxins. This procedure still forms the basis of
op
most shellfish toxicity monitoring programs. Most regulations are set for
C
PSP toxins as a group. At present, biochemical and chemical assays con-
’s
or
stitute good alternatives to the MBA.

ut
A new PSP testing method based on liquid chromatographic postcol-

b
umn oxidation (LC PCOX) was developed in Canada to separate fluid

tri
on
samples at the molecular level (van de Riet et al. 2011). This test replaced
.C
the traditional MBA method that had been used since the 1950s and
allows individual toxic compounds to be identified and measured. The
C
LL
test was developed and validated by the Canadian Food Inspection

Agency’ s (CFIA) Dartmouth Laboratory, in partnership with the National
c is
Research Council Canada’ s Institute of Marine Bioscience. In March 2011,

an
it received approval from AOAC International, the globally recognized

Fr
body for standardization of analytical methods and laboratory activities

d
(CFIA 2011).
an
or
yl
4.3.5 Ciguatoxins
Ta
The high diversity of CTX analogs in complex samples has hindered the
18
development of methods for detection. In addition, CTX standards are

20
scarcely available; only recently has a laboratory produced purified and

©
commercial CTX. Then, this limit constitutes restrictions for the develop-
ment and validation of methods, as well as for evaluating the potential of
toxicity.
The current methods for CTX determination include MBA, bioassays on
animal tissue, in vitro neuroblastoma cell-based assay (CBA), pharmacologi-
cal relative binding affinity (RBA), immunological assay, and physical-chem-
ical analytical methods, such as LC-MS/MS (Caillaud et al. 2010).
4.3.6 Tetrodotoxin
Receptor binding assay, immunological methods (e.g., ELISA), and MBA
have all been used for TTX analysis. MBA is the method most frequently
applied, although chemical assays, such as surface plasma resonance, elec-
trophysiological assays, infrared (IR), NMR, gas chromatography– mass
spectrometry (GC-MS), liquid chromatography with fluorescence detection
y
nl
(LC-FLD), and LC-MS/MS, have been developed for the determination of
O
TTX. At present, the LC-MS/MS methodology is generally considered, for
se
many researchers, the best choice for the detection of TTX and related com-
lU
pounds. Extraction and cleanup methodologies have to be applied to natural
na
samples; in fact, several extraction studies have been conducted to improve
o
the recoveries of TTX (Bane et al. 2014). Many of them include a solid-phase
rs
extraction (SPE) step. Recently, accelerated solvent extraction (ASE) and a
Pe
simple solvent extraction, as well as a UPLC-MS/MS method, were devel-
r
fo
oped and validated in puffer fish and gastropods (Nzoughet et al. 2013).
y
Reverse-phase chromatography was used for many years for the analysis
op
of TTX, but not all analogs of TTX can be separated using this methodol-
C
ogy. A recent review thoroughly compiled the characteristics of MS, matrix
’s
or
effect, quantitation of TTX, and other relevant information related to this

ut
toxin (Bane et al. 2014).

b
tri
on
4.3.7 Application of the QuEChERS Approach

.C
for the Analysis of Biotoxins

C
LL
To analyze lipophilic toxins in shellfish, several LC-MS methods have been

reported, frequently using the extraction of analytes from tissues with
c is
methanol. Then, a cleanup step or SPE with C18 or polymeric cartridges is

an
optional for researchers. Matrix interferences and the complexity of the sam-
Fr
ple, as well as the instrument maintenance and lifetime, are a concern. The
d
quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach was
an
initially developed for pesticides, antibiotics, and mycotoxins, among other

or
contaminants, in different matrixes (Anastassiades et al. 2003; Gonzá lez-

yl
Ta
Curbelo et al. 2014). This strategy comprises the combination of an extrac-
tion using acetonitrile with a high salt content and cleanup using dispersive
18
solid-phase extraction (d-SPE).

20
Recently, a new high-throughput quantitative method for the analyses of

©
lipophilic marine toxins in raw and processed bivalve molluscs, based on

QuEChERS and ultra-high-performance liquid chromatography (UHPLC)-
Q-Orbitrap-high resolution mass spectrometry (HRMS), has been developed
(Rú bies et al. 2015). The extraction of analytes was performed with acetoni-
trile, and then a simple cleanup by d-SPE with C18 was carried out before
extract injection into the chromatographic system. According to these authors,
absolute recoveries for the extraction and cleanup steps were achieved. This
treatment allows the analysis of a large series of samples without special
maintenance of the UHPLC-HRMS system. In order to calibrate the method,

surrogate matrix-matched standards were obtained by spiking blank samples
with standards before extraction. Eprinomectin has shown appropriate behav-
ior to be used as an internal standard for lipophilic toxins (Rú bies et al. 2015).
QuEChERS has also been applied for analyses of PSP toxins in seafood.
In a recent paper, mussel sample treatment, based on QuEChERS, associ-
y
ated with fast protein precipitation, allowed the development of a hydro-
nl
philic interaction liquid chromatography (HILIC)– electrospray ionization
O
(ESI)– MS method for different STX group toxins (Mattarozzi et al. 2016).
se
However, at present, the use of QuEChERS is not widespread in control
lU
laboratories for analyzing marine biotoxins. In our experience, application of
na
QuEChERS, at least to quantify shellfish lipophilic toxins, is not a feasible
o
rs
alternative; this procedure is time-consuming, and the testing for toxins does
Pe
not improve. On the one hand, more cleanup steps using cartridges or salts
r
result in higher costs and longer times of analysis. On the other hand, clean-
fo
ing phases will obviously soil the equipment less, but this has to be evaluated
y
op
by laboratories. A few analyses performed by our group did not reach a rel-
C
evant improvement to justify the costs and the time used in the cleaning step.
’s
It will be necessary to further investigate, with a higher number of samples,

or
if the application of this procedure will help to maintain the equipment’ s life.
ut
b
tri
on
.C
C
LL
4.4 Risk Assessment for Biotoxins in Seafood

is
The risk assessment is divided into four steps: hazard identification, hazard
c
an
characterization, exposure assessment, and risk characterization (Aune 2001).

Fr
In the first step, hazard identification, all the compounds that can result in
harmful effects to human health are identified, usually by epidemiological
d
an
studies. The hazard characterization evaluates the adverse health effects asso-
or
ciated with different agents, comprising the absorption, distribution, metabo-

yl
lism, or excretion of the toxic compounds; the mechanism of action; or the

Ta
target organs, among others. In exposure assessment, the intake of the toxic
18
compounds, together with the concentration of the agent and the pattern of
20
consumption, is necessary, and this information is often lacking or imprecise.

Finally, the risk characterization is based on previous information. This step
©
comprises quantitative estimations, including uncertainties, of the probabil-

ity of harmful effects associated with the exposure to the toxic compounds.
The concept of threshold doses for risk assessment is employed in the case
of nongenotoxic chemicals. On the other hand, from human and experimen-
tal animal data, the no observable adverse effect level (NOAEL) and the low-
est observable adverse effect level (LOAEL) have been established (Table 4.3).
The NOAEL is divided by an uncertainty or safety factor in order to establish
safe levels of exposure to toxic compounds. A safety factor of 100 is applied
when the risk assessment is based on data from the experimental animals,
and a factor of 10 is usually applied in the cases of human data. Due to the
lack of adverse health effects at intake levels close to the estimated LOAELs,
a risk assessment decision has been taken into account, and smaller factors
are usually applied for biotoxins in food.
In Europe, the EFSA has published a series of risk assessments on marine
y
biotoxins in response to a request from the European Commission (EFSA
nl
2009d). It was not possible to establish tolerable daily intakes for all the
O
marine biotoxins, due to the lack of long-term toxicity studies, but acute
se
reference doses (ARfDs) have been established for these compounds
lU
(Table 4.3), such as the amount of toxin that can be ingested during 24
na
hours that would not result in risk to the consumer. Moreover, toxicity
o
rs
equivalency factors (TEFs) were also proposed by EFSA in order to express
Pe
the toxicity of different analogs within a toxin group in terms of the most
r
toxic form (Table 4.3).
fo
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C
’s
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4.5 Control Measures for Biotoxin Contamination of Seafood

b
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4.5.1 Legislation
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.C
In the EU, the limits for PSP, ASP, and lipophilic toxins are laid down in
Regulation (EC) No. 853/2004 in Annex III (European Commission 2004a).
C
LL
Live bivalve mollusks should meet the standards laid down in Chapter V,
including legal limits. Then, for PSP, the limit is 800 µ g/kg STX, and for
cis
ASP it is 20 mg/kg DA, measured in the whole body or any edible part
an
separately.
Fr
This regulation also lays down the maximum levels for lipophilic toxins in
d
bivalve mollusks before they are placed on the market for human consump-
an
tion: for OA, DTXs, and PTXs together, 160 µ g/kg OA, and for AZA, 160 µ g/
or
kg AZA equivalents. Regulation (EU) No. 786/2013 amending Annex III to

yl
Ta
Regulation (EC) No. 853/2004 of the European Parliament and of the Council
as regards the permitted limits of YTX in live bivalve mollusks lays down
18
3.75 mg/kg YTX (European Commission 2013).

20
Current EU legislation does not give quantitative limits for CTXs or state
©
which analogs are relevant. However, the relevant Regulation (EC) No.
854/2004 lays down that checks have to take place to ensure that the follow-
ing fishery products are not placed on the market: fishery products contain-
ing biotoxins such as ciguatera or other toxins dangerous to human health
(European Commission 2004b).
In Japan, the following quarantine levels are established: 0.05 MU/g wet
weight for DSP, that is, 0.2 µ g OA equivalents/g shellfish flesh, and 4.0 MU/g
wet weight for PSP, that is, 80 µ g STX equivalents/100 g shellfish flesh.
TABLE 4.3
ARfDs and TEFs established by the CONTAM Panel for the Regulated Toxins in
Europe
NOAEL/LOAEL
Toxin Group (µ g /kg b.w.) ARfD (µ g /kg b.w.) TEFs
AZA 1.9 (human LOAEL) 0.2 AZA1 eq. AZA1 = 1
y
nl
AZA2 = 1.8
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AZA3 = 1.4
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AZA4 = 0.4
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AZA5 = 0.2
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OA 0.8 (human LOAEL) 0.3 OA eq. OA = 1
o
DTX1 = 1
rs
DTX2 = 0.6
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STX 1.5 (human LOAEL) 0.5 STX eq. STX = 1
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NeoSTX = 1
y
GTX1 = 1
op GTX2 = 0.4
C
GTX3 = 0.6
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GTX4 = 0.7
or
GTX5 = 0.1
but
GTX6 = 0.1
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C2 = 0.1
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C4 = 0.1
.C
dc-STX = 1
C
dc-NeoSTX = 0.4
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dc-GTX2 = 0.2
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GTX3 = 0.4
c
an
11-OH-STX = 0.3
Fr
YTX 5000 (mouse NOAEL) 25 YTX eq. YTX = 1

d
45-OH-YTX = 1
an
1a-homo YTX = 1
or
45-OH-1a-homo YTX = 0.5

yl
PTX 250 (mouse NOAEL) 0.8 PTX2 eq.

Ta
DA 900 (human LOAEL) 30 (DA and epi-DA)

18
Note : eq., equivalents.

20
In Canada, the legal limits are 80 µ g/100 g for PSP toxins, 20 µ g/g for ASP
©
toxins, and 0.2 µ g/g for DSP toxins (OA and/or DTX, singly or in combina-
tion, and PTXs) (CFIA 2014).
In the United States, any detectable level of BTX per 100 g shellfish tissue is
considered potentially unsafe for human consumption. In practice, a residue
toxicity of 20 MU/100 g BTX2 equivalent in the shellfish tissue was adopted,
and it remains the guidance level for prohibition of shellfish harvesting.
Moreover, maximum levels of 0.2 ppm OA and DTX1 in shellfish and 0.01 or
0.1 ppb for P-CTX and C-CTX were established in fish (FDA 2011).
Table 4.4 provides a summary of the limits established for each group of
toxins in different locations.
In Australia, there are four main groups of toxins of concern: PSP, ASP,
DSP, and BTXs, which may accumulate in shellfish tissue and cause illness
in humans. These are named after the poisoning syndrome they cause. The
regulatory limits applied within the Victorian Marine Biotoxin Management
y
Plan meet and in some cases are more conservative than those of the Food
nl
Standards Australia New Zealand (FSANZ) Food Standards Code (FSC)
O
(Victoria State Government 2015). Since July 2012, the following toxins are
se
regulated in Australia: PSP, DSP, BTX, and ASP. On the contrary, YTXs and
lU
AZAs are not regulated in this country; Table 4.5 compiles the analogs of
na
toxins included in each group, the methods of analyses, and the regulatory
o
rs
limit for each group of toxin.
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On the other hand, the Association of Southeast Asian Countries (ASEAN)
r
member countries, in cooperation with the Marine Fisheries Research and
fo
Development (MFRD) and the Japanese Trust Fund II project, has imple-
y
op
mented a program focused on biotoxin monitoring in order to reduce the
C
public health risks associated with the consumption of contaminated sea-
’s
food. The activities undertaken aim to develop methodologies for biotoxin

or
analyses to improve initiatives to monitor and detect these compounds

ut
(Marine Fisheries Research Department 2009). In Table 4.6, methodologies

b
tri
for the determination of biotoxins in the ASEAN countries are compiled.

on
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4.5.2 Managing
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Many countries have developed action levels and control programs for man-
is
aging marine biotoxin threats. Europe and North America have the most
c
an
TABLE 4.4
Fr
Legal Limits of Different Toxins Established in Europe,

d
an
Japan, and North America

or
Region Toxin Limit

yl
EU PSP 800 µ g/kg

Ta
ASP 20 mg/kg
18
OA, DTXs, PTX, AZA 160 µ g/kg

20
YTX 3.75 mg/kg

Japan DSP 0.05 MU/g or 0.2 µ g/g
©
PSP 4 MU/g or 80 µ g/100 g

United States BTX 20 MU/100 g
DSP 0.2 ppm
P-CTX 0.01 ppm
C-CTX 0.1 ppm
Canada PSP 80 µ g/100 g
ASP 20 µ g/g
DSP 0.2 µ g/g
TABLE 4.5
Methods of Analyses and Regulatory Limits of Toxins Established in Australia
Toxins Methods FSC Regulatory Limit
PSP group (STX, GTX1, PSP screening by LC-FLD 0.8 mg/kg (STX eq.)
GTX4, Neo, GTX2, GTX3, (Lawrence Method);* PSP
dc-STX, dc-Neo, dc-GTX2, confirmation by LC-FLD
y
dc-GTX3, C1, C2, C3, C4, AOAC 2005.06 (Lawrence
nl
O
GTX5) Method)*
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DSP group (AZA1, AZA2, LC-MS/MS (McNabb et al. 0.2 mg/kg (OA eq.) (total of
AZA3, total DTX1, free 2005) all DSP toxins), maximum
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DTX1, total DTX2, free limit applied 0.16 mg/kg
na
DTX2, total OA, free OA, (OA eq.)
o
GYM, PTX2, SPX)
rs
ASP: Domoic acid LC-MS/MS (McNabb et al. 20 mg/kg DA eq.
Pe
2005)
r
BTXs (BTX1, BTX2, BTX3) LC-MS/MS 200 MU/kg or 0.8 mg/kg
fo
BTX2 eq.
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Note : eq., equivalents.
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C
* In Australia, a rapid screening test is applied for the analysis of the PSP group. This method
’s
does not separate all the toxins belonging to the PSP group but detects some of them as a
or
group. Individual toxins within this group have different toxicities, but for the purpose of a
ut
rapid assay, all members of the group are assumed to be as toxic as the most toxic member of
b
the group, and the level of toxin in the sample is calculated on that basis.The total toxin detected
tri
is an overestimation of the actual PSP level, so in the event that the “ screen” quantity exceeds
on
FSANZ standards, a second assay (PSP confirmation assay) may be necessary in order to sepa-
.C
rate and analyze the various members of the group (Victoria State Government 2015).
C
LL
TABLE 4.6
c is
Methods of Toxin Analyses in ASEAN Countries

an
Fr
Asian Region Toxin Group Method

d
Indonesia PSP, DSP MBA

an
ASP HPLC
or
Malaysia PSP, TTX GC/MS

yl
Myanmar PSP, DSP, NSP, ASP Test kit, HPLC, LC-MS/MS

Ta
Philippines PSP, DSP, TTX MBA

18
ASP HPLC
20
CTX Test kit

Singapore PSP MBA, test kit
©
ASP HPLC
DSP LC-MS/MS
Thailand PSP MBA, HPLC
DSP, PTX, YTX, AZA MBA, LC-MS/MS
ASP, TTX HPLC
Vietnam PSP MBA, HPLC
DSP MBA and LC-MS/MS or HPLC
ASP LC-MS/MS or HPLC
efficient programs, although in most places, the managing systems estab-

lished in different countries are quite comparable. In nations where control
programs are missing, toxins appear to be marginal and present a low risk
of HABs.
Global standardization should facilitate biotoxin monitoring programs
and harmonization regarding official methods and regulatory action levels
y
in areas where they are needed, enhancing the capacity to protect public
nl
health and guaranteeing international trade. In this context, the FAO and
O
the WHO together established the Codex Alimentarius Commission as an
se
international reference for food standards, including those related to sea-
lU
food biotoxins. There are several codex committees, such as the Commodity
na
Committee on Fish and Fishery Products (CCFFP) applicable to seafood
o
rs
biotoxins. Then, there are current international efforts, notably through the
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Codex Alimentarius Commission, to standardize biotoxin control programs
r
in order to create global equivalency for ensuring seafood safety (FAO 2016).
fo
There are control programs employed by certain North (Canada, Mexico,
y
op
and the United States), Central (Belize, Costa Rica, El Salvador, Guatemala,
C
Honduras, Nicaragua, and Panama), and South (Argentina, Brazil, Chile,
’s
Colombia, Ecuador, Peru, Uruguay, and Venezuela) American countries.

or
In the United States, HABs known to cause PSP, ASP, NSP, DSP, and CFP
ut
have been found in U.S. waters (Anderson et al. 2001). Harvesting clo-
b
tri
sures due to the threat of PSP are common in certain regions of the United
on
States. NSP closures are made when K. brevis cells exceed the established
.C
threshold, and harvesting areas are not reopened until shellfish testing
C
by MBA demonstrates that toxin levels have declined below the action
LL
level (DeGrasse and Martinez-Diaz 2013). There were closures due to

is
a DSP event in 2008, while AZA has not been confirmed to be a haz-
c
an
ard in seafood harvested within U.S. waters. The U.S. Food and Drug
Fr
Administration (FDA) has the authority and responsibility to ensure

food safety, including seafood, with programs that operate according to
d
an
regulations. There is no regulation pertaining specifically to seafood bio-

or
toxin concerns. However, an integral part of the FDA’ s seafood safety

yl
program involves the publication of the Fish and Fishery Products Hazards
Ta
and Controls Guidance (FDA 2011), a critical guide to industry. Chapter

18
6, entitled “ Natural Toxins,” relates to seafood biotoxin risks. PSP, ASP,

20
NSP, DSP, and CFP are covered, and established regulatory action levels
are provided.
©
In order to protect consumers, programs to monitor biotoxin levels and

control the harvesting of toxic shellfish have been established in Canada.
The CFIA is responsible for collecting and analyzing shellfish samples and
making recommendations for the opening and closing of shellfish areas to
Fisheries and Oceans Canada (DFO), which implements and enforces clo-
sures. Each CFIA region must have established sampling sites and frequen-
cies to monitor changes in PSP, ASP, and DSP. The objective of the monitoring
program is to ensure that shellfish areas are closed when toxin levels exceed
the official limit.
DSP and PSP cause serious quality assurance problems for bivalve indus-
tries in Japan. When the toxicity of the bivalves exceeds the quarantine levels
for DSP or for PSP, harvesting ceases. Japan established a monitoring system
in 1979. In addition to the direct monitoring of bivalve toxicity, the occur-
y
rence and abundance of toxic dinoflagellates that cause DSP (Dinophysis spp.)
nl
and PSP (Alexandrium spp. and G. catenatum ) are also monitored. This pro-
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vides an early warning and risk assessment of the chances of toxicity devel-
se
oping in the bivalves. Since 1979, no human poisonings due to contaminated
lU
bivalves distributed on commercial markets have been reported.
na
Strict food safety regulations have been implemented in the EU, requir-
o
rs
ing the development of national marine biotoxin monitoring programs
Pe
(European Commission 2004b). These systems involve the routine monitor-
r
ing of shellfish, ensuring that when toxins are detected, they comply with the
fo
limits prescribed. Similarly, the identification of planktonic species present
y
op
in samples representative of the water column is carried out in order to pro-
C
vide information on the presence of toxin-producing microalgae. The results
’s
of the phytoplankton monitoring are used to provide an early warning of the

or
presence of biotoxin-producing species in shellfish harvesting areas.

ut
It is important to mention that European legislation has different statu-

b
tri
tory instruments for official control of harvesting areas, postharvest control,

on
and end-product control. Implementation of EU legislation is verified by the

.C
EU’ s Food and Veterinary Office (FVO), a branch of DGSanco that inspects
C
the compliance of European monitoring systems with legislation (European

LL
Commission 2016).
is
Contrary to the North American, Australian, and New Zealand systems,

c
an
Europe has, similar to Japan, developed a legal monitoring system with a

Fr
strong emphasis on government involvement in the control of shellfish tox-

ins. Only recently has legislation looked to equilibrate these international
d
an
discrepancies (Hess 2013).

or
Concerning the regulation of the occurrence and monitoring of toxin-

yl
producing phytoplankton species, the current legislation simply states that

Ta
“ classified relaying and production areas must be periodically monitored

18
to check for the presence of toxin-producing plankton in production and

20
relaying waters” (European Commission 2004b). In the EU, preharvest and

postharvest control systems, Hazard Analysis and Critical Control Point
©
(HACCP), and end-product testing differed among member states: the use
of phytoplankton monitoring as alert, the number of monitoring points,
the primary decision-making tool, the methods used, and the number of
samples analyzed, among others (Hess 2013). The FVO inspection reports
outline the major noncompliances in the area of monitoring for potentially
toxic phytoplankton and shellfish toxins and where harmonization has
not been achieved yet. The European Community’ s Rapid Alert System
for Food and Feed (RASFF) allows for an independent assessment of the
functioning of monitoring systems from the consumer’ s point of view.
It is a web-based system allowing for the rapid distribution of informa-
tion pertaining to the noncompliance of food- and feedstuffs (European
Commission 2015).
y
nl
O
se
lU
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5
Selection and Characterization of Aptamers
for Food Contaminant Monitoring
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Amina Rhouati, Akhtar Hayat, Gaë lle Catanante, and Jean Louis Marty
o na
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CONTENTS
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5.1 Introduction................................................................................................. 158
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5.2 Food Contaminants.................................................................................... 159
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5.2.1 Heavy Metals................................................................................... 160
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5.2.1.1 Lead (Pb)............................................................................ 160
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5.2.1.2 Cadmium (Cd).................................................................. 160
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5.2.1.3 Mercury (Hg).................................................................... 160

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5.2.1.4 Arsenic (As)....................................................................... 161

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5.2.2 Pesticides.......................................................................................... 161

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5.2.2.1 Organochlorine Pesticides.............................................. 161

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5.2.2.2 Organophosphorates and Carbamate Pesticides ........ 162

5.2.3 Mycotoxins....................................................................................... 162
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5.2.3.1 Aflatoxins.......................................................................... 162

5.2.3.2 Ochratoxins....................................................................... 163
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5.2.3.3 Fumonisins....................................................................... 163

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5.2.3.4 Trichothecenes.................................................................. 163

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5.2.3.5 Zeralenone......................................................................... 164

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5.2.4 Phenols ............................................................................................ 164

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5.2.5 Pharmaceutical Residues............................................................... 164

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5.3 Aptamers...................................................................................................... 165

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5.3.1 Definition......................................................................................... 165

5.3.2 Aptamer Selection: SELEX Technology....................................... 165
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5.3.3 Aptamer Properties........................................................................ 166

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5.3.3.1 Affinity.............................................................................. 166

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5.3.3.2 Specificity.......................................................................... 167

5.3.4 Structure and Aptamer– Target Interactions.............................. 167
5.3.4.1 Example of a Cadmium Aptamer.................................. 168
5.4 Application of Aptamers in Food Safety................................................. 168
5.4.1 Heavy Metals................................................................................... 168
5.4.2 Pesticides.......................................................................................... 172
5.4.3 Mycotoxins....................................................................................... 174
157
5.4.4 Phenols............................................................................................. 177

5.4.5 Pharmaceutical Residues............................................................... 180
5.5 Food Contaminant Aptasensing: Limitations and Challenges........... 182
5.6 Conclusion and Prospects......................................................................... 183
References ............................................................................................................. 183
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5.1 Introduction
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Foodborne illness may occur after the consumption of food contaminated
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with chemical or biological toxins. Identifying the contamination source is
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thus indispensable to ensure food safety and protect animal and human
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health. For that, the detection and quantification of food contaminants has
y
become critically important in recent years. Traditionally, food contami- op
nant monitoring is based on chromatographic methods coupled to different
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analytical detectors (high-performance liquid chromatography– mass spec-
’s
trometry [HPLC-MS], high-performance liquid chromatography– ultraviolet

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[HPLC-UV], gas chromatography– electron capture detector [GC-ECD], gas

b
chromatography– mass spectrometry [GC-MS], high-performance liquid

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chromatography– fluorenscence [HPLC-FL], etc.) (Ammann 2002; Lehotay

on
et al. 2005; Pittet and Royer 2002; Hirsch et al. 1998). Despite their accuracy,
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these conventional means are expensive and time-consuming and require

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highly trained technicians and pretreatment steps, making them unsuitable

for in situ detection. To overcome these limitations, biochemical methods
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appeared to be a good alternative for their simplicity and low cost.

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Biochemical monitoring of food contaminants, as shown in Figure 5.1, is

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based on the specific binding between the targeted toxic compound, in a

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food sample, and a biological recognition element. This interaction is then

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translated into a measurable signal by using a suitable transducer. Various

or
kinds of ligands have been used in the literature: enzymes, antibodies,

yl
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nucleic acids, molecularly imprinted polymers (MIPs), cells, and microorgan-

isms (Oujji et al. 2013; Hayat et al. 2011; Rhouati et al. 2011; Bakas et al. 2014;
18
Chouteau et al. 2004). The principal methodologies emerging for the rapid
20
diagnosis of food poisoning are immunoassays and aptamer-based assays.

©
The term aptamer represents a class of synthetic oligonucleotides that mimic

antibodies in their binding properties, such as high affinity and specific-
ity, with a better stability and lower costs. Indeed, aptamers are selected in
vitro without the need for animal immunization. Therefore, aptamers have
rivaled antibodies in food control; they have been selected for a wide variety
of food contaminants and used in different conditions and assay formats
(Jayasena 1999). Table 5.1 summarizes the principal differences between
aptamers and antibodies.
Selection and Characterization of Aptamers for Food Contaminants 159
Target molecule
Molecular
Bioreceptor:
recognition
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enzyme, antibody,
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aptamer, …
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Detection
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Optical, electrochemical,
piezoelectric, calorimetric
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FIGURE 5.1
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General principle of biochemical detection methods.
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TABLE 5.1
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Aptamers vs. Antibodies as Biorecognition Elements in Biodetection Approaches
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Aptamers Antibodies
but
In vitro selection In vivo synthesis requiring animal

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immunization
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Stable molecules in pH and temperature Unstable due to their proteic nature

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variations
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Regenerable after denaturation Irreversible denaturation

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Selection conditions can be adapted to Selection restricted by in vivo parameters

obtain the desired properties
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Possibility of attaching functional groups —

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We discuss in this chapter the different selection procedures, struc-

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tures, and applications of aptamers in food safety. A general overview

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of chemical contaminants is exposed in the first part by focusing on

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their occurrence and health effects. Afterwards, we highlight Systematic

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Evolution of Ligands by Exponential Enrichment (SELEX) principles,

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aptamer properties, and their interaction mechanisms with their targets.

18
Finally, the different applications of aptamer-based assays in food analy-

20
ses are reviewed.

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5.2 Food Contaminants

Our food is often the result of a complex chain of actions, including crop
and livestock production, transformation processes, preservation, and
distribution. Many types of residues used during these processes may be

present in the final product. Food contamination can also originate from
naturally occurring or environmental pollutants. These contaminants can
be classified into chemical and biological contaminants (Kantiani et al. 2010).
We focus in this chapter on food contaminants of a chemical nature. This
class of contaminants includes additives, food packaging, environmental
y
pollutants, and chemicals used for animal and plant treatment, in addition
nl
to water disinfection. The main chemical contaminants are heavy metals,
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pesticides, mycotoxins, phenols, and veterinary drug residues.
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5.2.1 Heavy Metals
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Heavy metals are natural or anthropogenic metallic elements characterized
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by a high atomic weight and density. They are released into the environment
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and absorbed by soils and crops, thus contaminating the human food chain.
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Food plants are strong accumulators of heavy metals; after bioaccumulation,
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it is very difficult to remove these toxic elements from living organisms. The
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main threats to ecosystem and human health from heavy metals are associ-
’s
ated with exposure to lead, cadmium, mercury, and arsenic (Jä rup 2003).
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5.2.1.1 Lead (Pb)

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Lead is a widespread pollutant that can be found in air and food. It origi-
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nates from metal smelting and agricultural, industrial, and urban activities.
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Airborne lead is deposited on water and immobilized on soil, where it forms

LL
complexes with the organic matter. It can be consumed via seafood, cereals,
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and vegetables. When the lead concentration in blood exceeds 0.5– 0.8 µ g/
c
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mL, it may cause encephalopathy, anemia, abdominal pains, nephropathy,

Fr
and cardiovascular disorders (Oymak et al. 2009).

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5.2.1.2 Cadmium (Cd)

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Despite its industrial applications, cigarettes and food are the main source of
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cadmium intake. Cd ions are easily absorbed by vegetables, and they are found
18
in animal-based food, mostly distributed in the liver and kidneys. Cd occurs

20
in a variety of foodstuffs, with higher concentrations in rice, wheat, oyster,

mussels, and the kidney cortex of animals (Oymak et al. 2009). This toxic metal
©
can be absorbed via the alimentary tract, penetrates through placenta during
pregnancy, and damages membranes and DNA. It attacks the kidney, liver,
and bones and disturbs the female endocrine system (Peralta-Videa et al. 2009).
5.2.1.3 Mercury (Hg)

For humans, the main contamination of mercury comes from fish and sea-
food. In fish, inorganic Hg is transformed naturally into organic mercury,
such as methylmercury, a very stable compound that accumulates in the

food chain. Phosphate fertilizers used in agricultural processing constitute
another source of mercury contamination. The World Health Organization
(WHO) recommends a maximum intake of methylmercury of 1.6 μ g/kg
per week. The impact of mercury on human health depends on its chemi-
cal form, where organic mercury compounds are the most toxic. They have
y
harmful effects on the nervous, digestive, and immune systems, in addition
nl
to the lungs and kidneys (Ferreira et al. 2015).
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5.2.1.4 Arsenic (As)
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Arsenic is a metalloid, but it is also considered a heavy metal. Exposure to
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arsenic is mainly via intake of food and drinking water. It is found at high
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concentrations in groundwater and surface soil. As has natural sources, and
r
it can be released from smelting and mining activities, agricultural prac-
fo
tices, fabrication and consumption of wood preservatives, and food addi-
y
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tives (Aldrich et al. 2007). The toxic metalloid exists in two chemical forms,
C
where inorganic arsenic is less mobile but more toxic than organic arsenic.
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Both forms can alter metabolic pathways and cause diabetes; cancer of the
or
bladder, lung, and prostate; skin lesions; and encephalopathy. Inorganic

ut
As is particularly accumulated on rice, posing a high health risk for rice-

b
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consuming populations (Zhao et al. 2010). Due to its toxicity and abun-
on
dance, the standard of As in drinking water has been lowered by the U.S.
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Environmental Protection Agency (USEPA) from 50 to 10 g/L (Peralta-Videa

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et al. 2009).
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5.2.2 Pesticides
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Pesticides are chemical substances used in agriculture and public health to

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enhance crop yields by controlling pests and vector-borne diseases. Despite

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their role in the increase of world food production, the use of pesticides
or
may have side effects on the environment, food quality, and human health.
yl
According to the undesired pest they control, pesticides are classified into
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insecticides, herbicides, fungicides, and rodenticides. They can also be cate-

18
gorized by their chemical structure into organochlorine, organophosphorate

20
(OPP), and carbamate pesticides.

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5.2.2.1 Organochlorine Pesticides

Organochlorine pesticides (OCPs) were widely used as insecticides in agri-
culture between 1940 and 1980; after this, their use was restricted around
the world because of their environmental persistence, bioaccumulation and
biomagnification. However, OCPs are still contaminating sediments, air,
water, and foodstuffs. Some OCPs have been classified as persistent organic
pollutants. These toxic compounds have been detected in samples from the
coastal environment, where humans are exposed to OCPs through consum-

ing seafood (Choi et al. 2016). OCPs have harmful effects on humans and
animals; they induce immunotoxicity, reproductive disorder, neurotoxicity,
and carcinogenesis (Jeong et al. 2014).
5.2.2.2 Organophosphorates and Carbamate Pesticides
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OPPs and carbamates are the most used insecticides in the recent decade;
O
they are less persistent and more toxic than OCPs. The most important effects
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of these pesticides are water and soil pollution, leading to the contamination
lU
of vegetables, fruits, milk, food products, seafood, and other living organ-
na
isms (Tahmasbi et al. 2012). Compounds of both classes have the same action
o
on the nervous system: inhibition of cholinesterases (ChEs). They are thus
rs
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considered highly powerful neurotoxic contaminants. Besides their chemi-
cal structure, the main difference between these compounds consists in their
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ability to bind ChEs: OPPs bind irreversibly, while carbamate inhibition is
y
generally reversible (Rhouati et al. 2010). In addition to neurotoxicity, OPPs
op
cause reproductive disorders and oxidative stress, which leads to different
C
cancer types (Tahmasbi et al. 2012). In the literature, some reports have dem-
’s
or
onstrated the effect of carbamates on the immune system; they are impli-
ut
cated in hypersensitivity reactions, some autoimmune diseases, and cancers

b
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(Bini Dhouib et al. 2016).

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5.2.3 Mycotoxins
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Mycotoxins are highly toxic metabolites produced by some species of mold

is
that grow on crops and foods under certain conditions. There are about 100
c
species of toxic molds that produce these mycotoxins, and more than 200
an
types of mycotoxins are known. The most prominent mycotoxins that cause
Fr
health concerns in humans and animals are aflatoxins, ochratoxins, and

d
an
fusarium toxins (Rai et al. 2015).

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5.2.3.1 Aflatoxins
Ta
18
Aflatoxins are difuranocoumarin derivatives produced by filamentous

fungi, mainly Aspergillus flavus and Aspergillus parasiticus . Commodities
20
that have a high potential for contamination with aflatoxins include tree
©
nuts, peanuts, peanut butter, figs, and corn. Around 20 types of aflatox-
ins have been identified and were classified according to their fluores-
cent properties under ultraviolet light (ca. 365 n m) and chromatographic
mobility. However, only aflatoxins B1, B2, G1, and G2 are usually found
in food (Yao et al. 2015). Among those, AFB1 is usually predominant
and is the most toxic and was classified as a Group 1 carcinogen by
the International Agency for Research on Cancer (IARC) in 1988. Two
metabolic products of aflatoxins, including AFM1 and AFM2, were first
isolated from milk of lactating animals fed moldy grains contaminated

with aflatoxins— hence the M designation. AFM1 is relatively stable at
processing treatment and remains present in various dairy products
(Gupta 2011).
5.2.3.2 Ochratoxins
y
nl
O
Ochratoxins are toxic metabolites formed by several Aspergillus and
se
Penicillium genera growing on a wide range of raw food commodi-
lU
ties. Naturally occurring ochratoxins include ochratoxin A (OTA), OTB
na
(dechlorinated OTA), and OTC (ethylated OTA). They are chemically
o
described as 3,4-dihydromethylisocoumarin derivatives linked with an
rs
amide bond to the group of l-β -phenylalanin. OTA is a more prevalent
Pe
and toxic member of the ochratoxin family. It is a very stable compound
r
fo
and can be also contaminate milk or poultry meat from animals fed con-
taminated feed. OTA is a human carcinogen that has also been found to
y
op
cause lesions, as well as teratogenic and neurotoxic effects (Leung et al.
C
2006).
’s
or
ut
b
5.2.3.3 Fumonisins
tri
on
Fumonisins are a group of nonfluorescent mycotoxins produced by various

.C
species of mold, most notably Fusarium verticilloides and Fusarium prolifera-

tum. Approximately 28 different fumonisins have been described in the lit-
C
LL
erature since their discovery in 1988, including the fumonisin A series (FAs),
is
B series (FBs), C series, and P series [8]. Fumonisin is well known to cause
c
two major animal diseases: porcine pulmonary edema and leukoencephalo-

an
malacia in horses (Matsuo et al. 2015). This mycotoxin is also a concern for
Fr
humans, causing esophageal and liver cancers, and may contribute to neural
d
an
tube defects in babies (Missmer et al. 2006).

or
yl
Ta
5.2.3.4 Trichothecenes
18
The trichothecenes constitute a large family of sesquiterpenoid metabo-

20
lites produced by Fusarium culmorum, Fusarium graminearum, Fusarium

sporotrichioides, and Fusarium poae. Theses Fusarium species are widely
©
dispersed, and their toxins reportedly occur in a wide variety of cereals,

grains, and animal feeds. Trichothecenes have been divided in four types
(A– D) according to variations in the functional hydroxyl and acetoxy side
groups. Type A is represented by HT-2 toxin, T-2 toxin, and diacetoxyscir-
penol (DAS), while type B includes deoxynivalenol (DON) and nivalenol
(NIV). However, few of them are toxic to animals and humans, such as T-2
toxin, DON, and NIV, and they are not known to be carcinogenic (Tamura
et al. 2015).
5.2.3.5 Zeralenone
Zearalenone (ZEA) (also known as F-2 toxin) is a nonsteroidal estrogenic
mycotoxin biosynthesized through a polyketide pathway by a variety of
Fusarium fungi, particularly F. culmorum and F. graminearum. It is frequently
implicated in reproductive disorders of farm animals and occasionally in
hyperestrogenic syndromes in humans. There is evidence that ZEA and its
y
nl
metabolites possess estrogenic activity in pigs, cattle, and sheep. The bio-
O
transformation for ZEA in animals involves the formation of two metabo-
se
lites, a-zearalenol (a-ZEL) and b-zearalenol (b-ZEL), and it may coexist with
lU
DON as the same organism. Moreover, ZEA has also been shown to be hepa-
na
totoxic, hematotoxic, immunotoxic, and genotoxic (Zinedine et al. 2007).
o
rs
Pe
5.2.4 Phenols
r
fo
Phenols are chemical pollutants of mostly anthropogenic origin; their tox-
y
icity is related to hydrophobic characteristics and the formation of free
op
radicals. They are introduced into surface water from industrial effluents,
C
and they have harmful effects on human health, such as acute toxicity, his-
’s
or
topathological changes, mutagenicity, and carcinogenicity. Several phenol

ut
derivatives have been identified: nitrophenol, aminophenol, methylphenol,

b
catechol, bisphenol, and so forth (Michał owicz and Duda 2007). In recent

tri
on
years, bisphenols, and bisphenol A (BPA) in particular, have attracted great

.C
interest by the scientific community because of their widespread occurrence

and high toxicity. BPA is used in medical devices (dental sealants), tableware,
C
LL
reusable bottles (baby bottles), and food storage containers. It has been shown
that BPA binds to estrogen receptors and has estrogenic effects in laboratory
cis
studies, thus disrupting the endocrine system. Johanna et al. reported in a

an
detailed review the effects of BPA on human health (Rochester 2013).

Fr
d
an
5.2.5 Pharmaceutical Residues

or
This category of contaminants includes drugs, antibiotics, antiparasitics,

yl
Ta
steroidal hormones, and other residues. They are mostly used to prevent
infections and treat diseases and, in animal production, to increase feed
18
efficiency and promote growth in food-producing animals. Pharmaceutical

20
residues have been detected in environmental water samples and products

©
of animal origin, such as milk and meat (Kantiani et al. 2010). The ingestion
of these toxic compounds via water or food poses a serious health problem to
the human population even at trace levels. For example, antibiotics and ste-
roids cause resistance in natural bacterial populations and endocrine disrup-
tion. Among them, tetracyclines (TETs) and chloramphenicol (Cam) are the
most studied in the food safety field. They are used as antimicrobial agents
or growth promoters applied in human therapy, animal husbandry, aqua
farming, and fruit crop production. TETs damage bones and teeth, causing
gastrointestinal and photosensitive allergies, while Cam causes nephropa-

thy, bone marrow suppression, and gray baby syndrome (Duan et al. 2016).
y
nl
5.3 Aptamers
O
5.3.1 Definition
se
lU
By 1990, a selection procedure was developed to identify, among a large
na
library of synthetic oligonucleotides, specific sequences for a given target.
o
The resulting nucleic acid sequence was called “ aptamer,” derived from the
rs
Latin word aptus, meaning a polymer that “ fits” to its target (Ellington 1990;
Pe
Tuerk and Gold 1990). Aptamers are artificial ligands composed of RNA or
r
fo
single-stranded DNA (ssDNA), selected in vitro according to their ability to
y
bind a target with high affinity and specificity. The selection process begins
op
from a large random pool of nucleic acids to isolate one or a small number of
C
aptamers with particular binding features. The main advantage of this pro-
’s
or
cedure is its applicability for a large variety of targets. Many aptamers have
ut
thus been selected for different food contaminants and used as bioreceptors
b
in bioanalytical methods for food safety.

tri
on
.C
5.3.2 Aptamer Selection: SELEX Technology

C
LL
The process called SELEX is a combination between molecular biology and

combinatorial chemistry. SELEX is a mimic Darwinian procedure that con-
cis
sists of iterative cycles of selection and enzymatic amplification (Mascini 2009).

an
This selective enrichment starts by the screening of a large random library of

Fr
1014 to 1015 individual oligonucleotides adopting different three-dimensional

d
structures. Each one comprises a random sequence of 20– 80 nucleotides,

an
which will constitute the aptamer core, flanked by 5′ and 3′ constant regions
or
providing primer hybridization sites. The molecular diversity or complexity

yl
Ta
of the SELEX library offers a high probability of selecting an aptamer exhibit-

ing strong affinity and specificity for the targeted molecule (Sampson 2003;
18
Luzi et al. 2003). After being prepared by combinatorial chemical synthesis,

20
the random DNA library is incubated with the target under determined
©
SELEX conditions. In the case of RNA aptamer selection, a reverse transcrip-

tion of the DNA library is needed to start the first round of SELEX. After incu-
bation, only candidates able to bind to the molecule of interest are selected,
while the unbound and weakly bound ones are eliminated in the portioning
step. The most commonly used method is based on the immobilization of
the target on a solid support, and the analyte is passed through. Afterwards,
affinity chromatography, magnetic separation, filtration, or centrifugation is
performed for eluting the bound motifs (Mascini 2009). The selected ligands
Library
Cloning and
sequencing
Mol 1 + library
Purification Counterselection
y
nl
SELEX
O
se
lU
Mol 2 + library
na
Amplification Selection
o
rs
Pe
Mol 1: Nontargeted molecule
Mol 2: Targeted molecule
r
fo
y
FIGURE 5.2
General principle of SELEX technology.
op
C
’s
are subsequently amplified by polymerase chain reaction (PCR) and used in

or
the next round of SELEX. In general, 6– 20 iterative rounds of binding, frac-
ut
tioning, and amplification are required to select the aptamer with the stron-
b
tri
gest binding properties to the target. Different characteristics, depending

on
on the three-dimensional structure, could be considered: a fast association

.C
rate, a slow dissociation rate, high affinity to the target, and low affinity to its
C
analogs (James 2006). Usually, the conditions would be made more stringent
LL
during each round, like reducing the target concentration or incubation time
is
to decrease the probability of selecting less specific aptamers. At the end of

c
an
SELEX, the isolated aptamers are cloned and analyzed by sequencing and
Fr
sequence alignment to identify the most specific region to the target. Post-
SELEX modifications are then required to enhance stability or allow aptamer
d
an
immobilization in some analytical assays (Figure 5.2).

or
yl
Ta
5.3.3 Aptamer Properties

18
As mentioned above, the desirable properties of the aptamer are predefined

20
in the selection process by working in different binding conditions. The key

properties of an aptamer are affinity and specificity.
©
5.3.3.1 Affinity
Shape and charge complementarities are the main factors that lead to high
binding affinity. The affinity of an aptamer to its target is determined by
the equilibrium dissociation constant Kd; the affinity is higher when this
constant is low. The latter depends on SELEX conditions and the target
properties, such as functional groups. In addition to charge distribution,
electrostatic and hydrophobic effects dominate the attractive interactions

between the aptamer and its target. Indeed, hydrogen bonds play a key role
in the recognition of nucleic acids. The affinity of aptamers varies widely; in
general, Kd is in the micromolar range for small molecules, while the affinity
of aptamers selected for proteins can achieve nanomolar or picomolar range.
Nieuwlandt et al. (1995) explained the difference of Kd between low and high
y
molecular weight targets by the fact that small molecules are rigid, which
nl
requires a loss of entropy for the aptamer binding. Indeed, the interaction
O
surface is smaller, and thus less functional groups are engaged for the bind-
se
ing interactions (Eaton et al. 1995).
lU
na
5.3.3.2 Specificity
o
rs
Pe
Specificity is the ability of an aptamer to discriminate between two targets
that have common characteristics. Such selectivity is the result of differences
r
fo
in the free energy of interactions and kinetic discriminations. Selection for
y
high-affinity binding automatically leads to highly specific binding because
op
specificity depends on the same factors as affinity (Eaton et al. 1995).
C
The high specificity of aptamers makes them promising receptors in bio-

’s
or
analysis, thus rivaling antibodies. One of the specific aptamers used in food
ut
safety is the OTA aptamer, which binds with 100-fold lower affinity to its
b
tri
dechlorinated analog (OTB) (Cruz-Aguado and Penner 2008). This discrimi-

on
nation is due to the adaptive conformation of aptamers, which acquire the

.C
three-dimensional structure upon binding to the target. The aptamer selec-

tivity can be enhanced by performing counterselection steps. It consists of
C
LL
incubating the oligonucleotide library with the nontargeted molecule, so

is
that sequences exhibiting specificity for this molecule can be eliminated.

c
an
Fr
5.3.4 Structure and Aptamer– Target Interactions

d
an
Aptamers’ function is based on their three-dimensional structure, which

depends on the primary and secondary structures. The replication of the pri-
or
mary structure upon formation of Watson– Crick base pairing and structural

yl
Ta
motives leads, respectively, to the secondary and tertiary structures. The

secondary structure consists of a double helix; it contains a minor groove
18
and major groove, which provide a suitable area for molecular interactions.
20
However, the specificity of these sites remains limited because of the unavail-
©
ability of certain functional groups. Other types of base pairing would bring
additional contributions to the aptamer stability and the specific binding of
the target (Chauveau et al. 2006).
While the stable secondary structure maintains the proper spatial arrange-
ment of the aptamer, the unpaired bases provide specific binding sites for
the target. Many aptamers acquire this stable confirmation only upon bind-
ing to the target. Figure 5.3 shows the possible structural motives in tertiary
structures of the aptamer after the target binding. A detailed review of
G G
G G
G G
G G
Stem–loop Hairpin G-quadruplex
y
nl
FIGURE 5.3
O
Common binding motives of aptamers for their targets.
se
lU
Kulbachinskiy (2007) describes the different structural features of aptamers.
na
For example, G-quadruplexes are four-stranded DNA structures stabilized
o
by coordination cations, for example, K+ and Na+ .
rs
Molecular recognition between an aptamer and its target is based on shape
Pe
complementarities, stacking interactions between aromatic compounds
r
fo
and bases, hydrogen bounds, or electrostatic interactions between charged
y
groups. op
C
’s
5.3.4.1 Example of a Cadmium Aptamer

or
ut
Wu et al. selected a DNA aptamer for cadmium detection. After 11 rounds of

b
selection and counterselection, 12 sequences were identified, among which

tri
on
one aptamer exhibited the highest affinity to Cd. The sequence comprises
30 nucleotides rich in T and G bases. Circular dichroism spectra revealed
.C
that the structural motif of the target binding is stem– loop architecture. The
C
LL
loop may offer sufficient sites for target binding via the coordination bonds
between Cd and the adjacent short fragment rich in T or G, and the stem
c is
maintains the stability of the loop through hydrogen. The dissociation con-
an
stant has been determined as 34.5 nM, and the binding specificity toward
Fr
competitive metal ions was negligible (Wu et al. 2014). The isolated aptamer
d
has been used as a recognition element in the development of a label-free

an
colorimetric method for Cd detection in rice samples using gold nanopar-

or
ticles (AuNPs) (Guo et al. 2014).

yl
Ta
18
20
©
5.4 Application of Aptamers in Food Safety

5.4.1 Heavy Metals
It has been shown that heavy metal cations can bind thymine-thymine (T-T)
mismatches, in a DNA sequence, to promote them to form stable base pairs
(Aragay et al. 2011). Furthermore, certain metal cations play a key role in
inducing DNA folded structures. Smirnov and Shafer (2000) showed that
Pb2+ binds with unusually high affinity to the thrombin binding aptamer
(TBA), inducing a unimolecular folded structure. Based on these findings,

aptamers and DNAzymes have been widely used for heavy metal determi-
nation in the last few years (Table 5.2).
TBA folds into the G-quadruplex and hairpin-like structure upon lead
and mercury binding, respectively. This property has been exploited for
the fluorescent and simultaneous aptasensing of Pb2+ and Hg2+ by probing
y
the aptamer with a fluorophore and a quencher. The selective detection was
nl
based on the change in the DNA strand’ s conformation from the linear to
O
a folded structure upon binding the metal ions (Figure 5.4). These different
se
conformations exhibit different degrees of fluorescence resonance energy
lU
transfer (FRET) between the fluorophore and quencher, allowing the selec-
na
tive detection of Pb2+ and Hg2+ in water and soil samples (C.-W. Liu et al.
o
rs
2009). C.H. Chung et al. (2013) used this method in serum samples by immo-
Pe
bilizing the TBA on AuNPs to stabilize DNA from nuclease degradation.
r
Other nanomaterials have been also used in lead aptasensing, such as quan-
fo
tum dots (QDs), graphene, and carbon nanotubes (CNTBs) (Qian et al. 2015;
y
Zang et al. 2014; Taghdisi et al. 2014). op
C
Besides aptamer conformational conversion, the induced allosteric
’s
G-quadruplex DNAzyme has been explored in the development of aptas-

or
ensing strategies for the selective determination of lead. It is well known

ut
that lead induces a conformational change of K+ -stabilized G-quadruplex

b
tri
DNAzymes and inhibits the peroxidase-like activity. Based on this fact, Li

on
et al. (2010) reported a colorimetric and chemiluminescence sensor for Pb2+

.C
detection in water.
C
Concerning mercury aptasensing, most of the reported strategies are

LL
based on (T)-rich oligonucleotides, because of Hg2+-mediated T-T base

is
pairing, being able to fold ssDNA into duplexes (Tang et al. 2015). As
c
an
an example of fluorescent aptasensors, Chiang et al. described a sensi-

Fr
tive and selective method of Hg2+ detection in aqueous solution using

TOTO-3 (benzothiazolium-4-quinolinium dimer derivative) as a probe
d
an
and polythymine oligonucleotide T33 as a bioreceptor. TOTO-3 is known

or
as a weakly fluorescent dye; its fluorescence increases upon binding to

yl
double-stranded oligonucleotides. In the absence of Hg2+, the fluorescence

Ta
of the probe is weak because the aptamer is found in its flexible structure.
18
Whereas, upon formation of the aptamer-Hg2+ complex through T-Hg2+-T,

20
the aptamer switches to a rigid folded structure, which preferably binds

to TOTO-3, hence increasing the fluorescence (Chiang et al. 2008). In addi-
©
tion to optical detection, electrochemistry has been frequently combined

to T-rich oligonucleotide-based assays in mercury determination. For
example, Liu et al. used an electroactive Fc tag, where the corresponding
redox peak decreases upon the conformational change after Hg2+ binding.
However, probing the aptamer is complicated and increases the prepara-
tion costs (Liu et al. 2009). For that, Gao et al. (2014) described a label-free
aptasensor based on formation of a stable ternary complex between the
aptamer, mercury, and neutral red.
©
TABLE 5.2
20 170
18
Aptamer-Based Assays for Heavy Metal Determination in Food
Ta
LOD
Contaminant Assay Principle Detection Method (nM) Real Sample References
yl
or
2+
Pb 2+
Pb -induced G-quadruplex DNAzyme
an Colorimetric and 32/1 Lake water Li et al. 2010
d chemiluminescence
Pb2+ -induced G-quadruplex aptamer-TMR Fr Fluorescence 1 Lake water D. Zhang et al. 2014
Pb2+ -induced G-quadruplex aptamer-RGO/ an Photoelectrochemistry 0.05 Tap and lake Zang et al. 2014
QDs c water
Pb2+ -induced G-quadruplex aptamer-SWNT 0.42 Tap water, Taghdisi et al. 2014
is Fluorescence
LL serum
2+
Pb -induced G-quadruplex aptamer-QDs Electrochemiluminescence
C 0.0038 River water, Hai et al. 2014
human urine
.C
2+
Pb -induced G-quadruplex aptamer-rGQDs Fluorescence
on 0.6 — Qian et al. 2015
2+
Hg and Pb 2+ 2+
Pb -induced G-quadruplex G33-Tb 3+ Fluorescence tr 10/1 Soil, pond Lin et al. 2011
2+
Hg -induced hairpin-T33/TOTO-3
ib water
2+
Pb -induced G-quadruplex aptamer-AuNPs Fluorescence 121/1283 10– 3 Serum C.H. Chung et al.
ut
2+
Hg -induced hairpin-aptamer-AuNPs 2013
or
’s
Pb2+ -induced G-quadruplex Fluorescence C 5/300 Pond water, C.-W. Liu et al.
FAM-aptamer-DABCYL op soil 2009
Hg2+ -induced y
hairpin-FAM-aptamer-DABCYL fo
Hg2+ Hg2+ -induced hairpin-T33/TOTO-3 Fluorescence 3 r Pond water Chiang et al. 2008
T-Hg2+ -T mismatch/nanogold aggregation Resonance scattering 0.7 Water Jiang et al. 2009
Pe
T-Hg2+ -T mismatch/AuNPs Colorimetric 0.6 rs Tap and lake Li et al. 2009
o water
na
Hg2+ -induced hairpin-tetraphenylethene Fluorescence 244 — Xu et al. 2010
derivative aggregation
lU
T-Hg2+ -T mismatch/TAMRA-aptamer SERS 5 Drinkable Lee and Choo 2011
se
water O
(Continued)
nl
y
©
20
18
Aptamer-Based Assays for Heavy Metal Determination in Food
Ta
LOD
yl
or
2+
Hg -induced hairpin/hydrogel an Fluorescence 10 Lake water Helwa et al. 2012
microparticles d
Hg2+ -induced hairpin/CTAB/Mn-ZnSQDs Fr Room temperature 1.5 Tap and river Xie et al. 2012a
an phosphorescence water
T-Hg2+ -T mismatch/aptamer-FAM-AuNPs c Fluorescence 16 Lake water Tan et al. 2013
T-Hg2+ -T mismatch/aptamer-AO-AuNPs Resonance scattering 30 Tap water Xie et al. 2012b
is
T-Hg2+ -T mismatch/graphene Field effect transistor 0.01 Mussels An et al. 2013
LL
T-Hg2+ -T mismatch/aptamer-Au/Ag
C
SERS 0.01 Drinkable E. Chung et al. 2013
core– shell water
.C
NPs on
T-Hg2+ -T mismatch/aptamer-neutral red Electrochemistry
tri 0.015 River, Gao et al. 2014
b drinking,
and tap
ut
water
or
2+
’s
T-Hg -T mismatch/aptamer-CdTe-CdS Fluorescence polarization C 11 Tap water Jiang et al. 2014
QDs/AgNPs op
T-Hg2+ -T mismatch/aptamer-cellulosic Chemiluminescence 0.01
y River water Liu et al. 2014, 6
paper/phenylene ethylene/nanoporous fo
silver r
T-Hg2+ -T mismatch/FAM-aptamer/ Fluorescence 4.28 Urine S.-H. Chen et al.
Pe
metallothioneins 2015
Selection and Characterization of Aptamers for Food Contaminants
rs
G-quadruplex DNAzyme/T-Hg2+ -T Colorimetric 2.85 nUrine o Tang et al. 2015
mismatch functional chimera aptamer al
Note: TMR, 6-carboxytetramethylrhodamine; SWNT, single-walled carbon nanotube; rGQDs, reduced graphene quantum U dots; RGO/Cds, reduced gra-
3+
phene oxide/Cd quantum dots; Tb , terbium ions; AO, acridine orange; AgNPs, silver nanoparticles; DABCYL, 4-([4-(dimethylamino)phenyl]azo)
se
Mn-doped ZnS quantum
171
benzoic acid; TAMRA, carboxytetramethyl-rhodamine; CTAB/Mn-ZnS QDs, cetyltrimethylammonium bromide-cappedO

dots. nl
y
NaCl
y
nl
O
se
Aptamer AuNPs Pesticide Color change upon
lU
aggregation of AuNPs
na
FIGURE 5.4
o
rs
FRET-based DNAzyme system for lead and mercury detection. (From Liu, C.-W., et al.,
Pe
Analytical Chemistry, 81 (6), 2383– 2387, 2009.)
r
fo
M. Kim et al. selected in vitro a specific aptamer for arsenic by using an
y
op
affinity column, where 10 rounds of selection were carried out. The selected
C
aptamer of 100 nucleotides exhibiting a Kd of 7.05 nM was used for arsenic
’s
removal from groundwater samples (M. Kim et al. 2009). Due to the length of
or
the As aptamer, it is difficult to modify its sequence with a label. Therefore,

ut
the few aptasensors reported for As monitoring were based on aptamer

b
tri
affinity and somecationic polymers or surfactants that induce AuNP aggre-

on
gation. This change in nanoparticle property causes a remarkable change

.C
in color, which can be detected by colorimetry (Wu et al. 2012; Yu 2014). A

C
detailed review discussing the advances in arsenic biosensors is reported in

LL
the literature (Kaur et al. 2015).

cis
an
5.4.2 Pesticides
Fr
Despite its advantages, SELEX technology has been applied for a small num-
d
an
ber of residues. He et al. selected a DNA signaling aptamer for acetamiprid,

or
one of the widely used pesticides. Eighteen rounds of repetitive selection

yl
have been performed; a FAM-labeled library, containing a complementary

Ta
sequence to a biotinylated capture oligonucleotide, was immobilized on

18
streptavidin-coated agarose. The selected candidates were released from

20
the surface after structure switching upon target binding. An aptamer was
identified from 14 selected sequences for its high affinity for acetamiprid
©
with a Kd of 4.98 µ M and a structure characterized by loops (He et al. 2011).
After being selected, this aptamer was used in the colorimetric detection of
acetamiprid in soil samples, by employing AuNPs as the optic probe (Shi
et al. 2013). Weerathunge et al. reported another assay based on the perox-
idase-like nanozyme activity of AuNPs and the acetamiprid aptamer. The
authors showed that the target binding inhibited reversibly the nanozyme
activity, and this inhibition can be detected colorimetrically (Weerathunge
et al. 2014). In a recent report, the acetamiprid aptamer has been modified
with QDs to develop a turn-on aptasensor, while CNTBs have been used
to turn off the fluorescence of the probe (Lin et al. 2016). Electrochemical
aptasensors have been also investigated; Jiang et al. developed an electro-
active nanocomposite (AgNPs anchored on nitrogen-doped graphene). The
sensing strategy is based on the electron transfer inhibition after formation
of an aptamer– target complex on the electrode surface (Jiang et al. 2015).
y
By 2013, a malathion-specific aptamer was selected and conjugated to poly-
nl
mer-AuNP composite microspheres for the sensitive and selective surface-
O
enhanced Raman scattering (SERS) aptasensing (Barahona et al. 2013). A
se
colorimetric aptasensor has been also developed for malathion determina-
lU
tion in water and apples by using AuNP aggregation and cationic polymers
na
(Bala et al. 2016a).
o
rs
In addition to acetamiprid and malathion, four organophosphorous
Pe
pesticides (phorate, profenofos, isocarbophos, and omethoate) have been
r
simultaneously isolated by SELEX technology. For that, the random library
fo
was incubated with the four targets, and after 12 rounds of selection, two
y
op
aptamers were identified for their affinity for the targeted pesticides. The
C
dissociation constants ranged from 0.8 to 2.5 µ M, and the structures were
’s
characterized by stems and loops (Wang et al. 2012). Afterwards, different

or
aptamer-based assays were developed for the detection of organophospho-

ut
rous pesticides in different food matrixes. Bai et al. exploited the ability of
b
tri
ssDNA aptamers to stabilize AuNPs from aggregation to develop a colori-

on
metric assay for the detection of six organophosphorous pesticides in river

.C
water samples (Bai et al. 2015). Other aptasensors based on this principle of
C
AuNP aggregation, shown in Figure 5.5, have been applied for phorate and
LL
omethoate determination (P. Wang et al. 2016; Bala et al. 2016b). Tang et al.
is
(2016) used, for the first time, QDs as signal indicators for the ratiometric
c
an
detection of organophosphorous pesticides by capillary electrophoresis with

Fr
laser-induced fluorescence (CE-LIF). Recently, a fluorescent assay has been

reported for isocarbophos and profenofos detection in water samples. The 5ʹ
d
an
or
yl
Ta
Hg2+
18
20
©
Pb2+
FAM
DABCYL
TBA
FIGURE 5.5
General principle of colorimetric aptasensing using AuNP aggregation.
carboxyfluorescein (FAM)– labeled aptamer was bound to a complementary

3ʹ DABCYL complementary DNA (cDNA) chain; the formed double strand
exhibited a weak fluorescent signal due to the fluorescence energy trans-
fer effect. However, the signal was stronger when the aptamer recognized
its target, thus allowing the determination of the pesticides (Li et al. 2016).
Finally, the high sensitivity of SERS technology has also been exploited for
y
the simultaneous aptasensing of organophosphorous pesticides in apple
nl
juice (Pang et al. 2014).
O
Despite their high toxicity and widespread use, there is still a lack in pesti-
se
cide aptasensing. This may go back to the small size of pesticide residues and
lU
the difficulty of selecting aptamers for such molecules (Table 5.3).
ona
rs
5.4.3 Mycotoxins
rPe
Because of its hazardous effects on animal and human health, OTA is the most
fo
studied mycotoxin in food analysis. After the selection of the OTA aptamer
y
op
by Agado et al., there has been increasing interest in OTA aptasensing (Cruz-
C
Aguado and Penner 2008). We have previously reviewed the different aptamer-
’s
based assays reported in the literature for OTA purification and detection in
or
food (Rhouati et al. 2013). However, more advances have been introduced to
ut
this research field in the last two years. Rivas et al. developed an impedimet-
b
tri
ric label-free aptasensor that combined the advantages of electropolymerized

on
thionine films on a screen-printed carbon electrode (SPCE) and iridium oxide

.C
nanoparticles. The modification of the electrode surface led to high selectiv-

C
ity and reproducibility, showing good applicability in wine samples. The

LL
reported method exhibited the widest linear range and one of the lowest limits
is
of detection (LODs) reported for electrochemical biosensing of OTA (Rivas et

c
an
al. 2015). Moreover, signal generation and amplification techniques are known
Fr
for improving the sensitivity of a biosensor. For example, Xie et al. employed
one of them: loop-mediated isothermal amplification of DNA (LAMP) for the
d
an
ultrasensitive electrochemical aptasensing of OTA in wine. For that, the OTA

or
aptamer was immobilized on the surface by hybridization with a cDNA previ-

yl
ously self-assembled on a AuNP-modified gold electrode. The formation of the

Ta
complex OTA aptamer triggered the amplification; then, the LAMP products,
18
combined with a redox entity, were analyzed by differential pulse voltam-

20
metry (DPV) (Xie et al. 2014). Optical aptasensors have also undergone many
advances by exploring new concepts and strategies for OTA monitoring in
©
food. Recently, we designed in our lab a fluorescent aptaswitch strategy based

on the ability of surface chemistry to quench the response of fluorescent par-
ticles (Figure 5.6). In contrast to most of the quenching aptamer-based assays,
this method allowed fluorescent detection without the need for a fluorophore.
This avoided the loss of signal intensity and decrease of sensitivity that may
be caused by the labeling process. The developed assay has been applied suc-
cessfully for OTA detection in beer samples by using TiO2 nanoparticles as
representative nanomaterial (Sharma et al. 2016).
©
20
18
TABLE 5.3 Ta
Aptamer-Based Assays for Pesticide Determination in Food
yl
Detection LOD
or
Contaminant Assay Principle
an Method (nM) Real Sample References
Acetamiprid AuNP aggregation
d Colorimetric 5 Soil Shi et al. 2013
Peroxidase-like nanozyme Colorimetric 4.49 — Weerathunge et al.
Fr
activity of AuNPs 2014
an
QDs turn on/SWNTs turn off
cis Fluorescence 0.7 River water, Lin et al. 2016
LL cabbage leaves
AgNP-nitrogen-doped Electrochemistry
C 3.3 × 10– 5 Wastewater, Jiang et al. 2015
grapheme nanocomposite .C cucumber,
tomato
Malathion Polymer-AuNP-aptamer SERS 10 × 103 Tap water Barahona et al. 2013
on
microspheres
tri
Cationic polymer/AuNPs Colorimetric 0.06 × 10– 3 Apple Bala et al. 2016a
bu
Phorate AuNP aggregation Colorimetric 0.01 Apple Bala et al. 2016b
to
Phorate, profenofos, QDs-aptamer duplex LI-CE 200, 100, 170, 230 Apple Tang et al. 2016
r’s
isocarbophos, omethoate C
Gold-based nanobeacon Fluorescence 384, 134, 35, 2350 Dried tangerine Dou et al. 2015
op
probe
y peel, running
water
fo
r
5’ FAM-molecular beacon- Fluorescence 19.2, 13.4, 17.2, Pe C. Zhang et al. 2014
DABCYL 3’ 23.4
rs
Isocarbophos, dursban, AuNP aggregation Colorimetric Isocarbophos: River
onwater Bai et al. 2015
phosalone, methamidophos, 346.02
acephate, trichlorfon Others: 2000 ppb
al
U
Note: LI-CE, laser-induced capillary electrophoresis. se
175
O
nl
y
Aptamer-
y
OTA Fluorosphere
nl
stabilized
O
TiO2 NPs
se
lU
FIGURE 5.6
na
Fluorescent determination of OTA based on aptamer impact on fluorescence particles. (From
Sharma, A., et al., RSC Advances, 6 (70), 65579– 65587, 2016.)
o
rs
Pe
By 2012, the aflatoxin B1 aptamer was the first selected and patented
r
by NeoVentures Biotechnology Inc. (Canada) (Le et al. 2010). Afterwards,
fo
Wang’ s group identified specific aptamers for aflatoxins B1 and B2, by
y
op
using magnetic nanoparticles (MNPs) for target immobilization. After 10
C
rounds of selection and amplification, 30 aptamers were isolated. Among
’s
these sequences, the aptamers exhibiting the best affinities for AFB1 and
or
AFB2 were identified with respective Kd values of 11.39 and 9.83 nM. The
ut
selectivity of both aptamers has been demonstrated; the cross-reactivity of

b
tri
AFB1 and AFB2 aptamers to five other toxins did not exceed 15% and 18%,
on
respectively. The selected aptamers have been used in the fluorescent detec-
.C
tion of AFB1 and AFB2 in peanut oil with high sensitivity and good recov-
C
eries (Ma et al. 2014, 2015). On their side, Malhotra et al. identified specific
LL
aptamers for aflatoxins M1 and B1. A total of 11 rounds, including counter-

is
selections, were carried out to select 36 aptamers specific for AFM1 and 5
c
an
for AFB1. Among these sequences, one AFM1 aptamer has been character-
Fr
ized by a unique structure with two overlapping stem loops and the lowest
Kd of 35 nM (Malhotra et al. 2014). These aptamers have been employed as
d
an
recognition elements in different aptamer-based assays for aflatoxin deter-

or
mination in foodstuffs. Nguyen et al. reported a label-free electrochemical

yl
aptasensor to detect AFM1 based on the controlled covalent immobiliza-

Ta
tion of aptamers on carboxy-modified MNPs and a conducting polyaniline

18
interface. It has been demonstrated that the use of polyaniline enhanced the
20
conductivity and sensitivity of the biosensor (Nguyen et al. 2013). Another

covalent attachment has been explored to immobilize the AFB1 aptamer by
©
modifying glassy carbon electrodes with electropolymerized neutral red

and polycarboxylated macrocyclic ligands. Cyclic voltammetry (CV) and
impedimetric measurements have shown the performance of the method
to detect AFB1 in peanuts, cashew nuts, white wine, and soy sauce with
good recoveries (Evtugyn et al. 2014). Recently, we have investigated in our
lab diazonium electrografting for aptamer immobilization to develop label-
free electrochemical aptasensors for AFM1 and AFB1 detection in milk and
alcoholic beverages, respectively (Istamboulié et al. 2016; Yugender Goud
et al. 2016). In addition to electrochemical detection, we have also developed

fluorescent aptasensors for aflatoxin detection. The device was based on
tetramethyl-6-carboxyrhodamine (TAMRA) quenching of the FAM-labeled
AFB1 aptamer. After AFB1 binding, the fluorescence is recovered due to
the release of TAMRA-labeled cDNA (Goud et al. 2016). For a similar prin-
ciple, the ability of graphene oxide for quenching QD fluorescence has been
y
exploited by Lu et al. (2015) to develop a fluorescent aptasensor for AFB1
nl
determination in peanut oil samples. Finally, colorimetric and electroche-
O
miluminescent aptasensors have also been reported for aflatoxin determi-
se
nationin in different food matrixes (Seok et al. 2015; Shim et al. 2014).
lU
The carcinogenic mycotoxin fumonisin B1 has been targeted by different
na
SELEX protocols. First, McKeague et al. reported a SELEX procedure where
o
rs
magnetic beads were used to carry the target (Mag-SELEX). Eighteen rounds
Pe
of increasing stringent conditions were performed to select six candidates.
r
The sequence exhibiting the best affinity displayed low complexity and a
fo
low percent of G (McKeague et al. 2010). Chen et al. selected another aptamer
y
op
for FB1 by using a modified Mag-SELEX without immobilization of the ana-
C
logs in counterselection. After 13 rounds of selection, a high-affinity aptamer
’s
(Kd 62 ± 5 nM) was selected (Chen et al. 2014). In another report, the apamer
or
was thiolated and anchored on AuNPs for the impedimetric detection of FB1
ut
in spiked maize samples (X. Chen et al. 2015). The same research group iso-
b
tri
lated a specific aptamer for ZEA, showing a good applicability in the specific
on
detection of ZEN in real samples based on a magnetic separation and pre-

.C
concentration procedure (Chen et al. 2013).

C
Finally, due to the co-occurrence of mycotoxins in food and feedstuffs,

LL
simultaneous detection of mycotoxins has been reported. Yue et al. designed

is
an aptamer– photonic crystal– encoded suspension array for the fluorescent

c
an
detection of OTA and FB1 in cereals. They immobilized the corresponding

Fr
aptamers on photonic crystals and hybridized them to a fluorescent cDNA.

In the presence of the target, the cDNA was released, resulting in a decrease
d
an
in the microsphere’ s fluorescence intensity. The detection was based on the

or
difference in the fluorescent intensities, while the structure colors or reflec-

yl
tance peak positions of the microspheres confirmed the kind of mycotoxin

Ta
detected (Yue et al. 2014). Double aptasensing of OTA and AFB1 in maize
18
meal has been also reported; the detection was based on SERS labels embed-
20
ded in silver and gold core– shell NPs (Zhao et al. 2015) (Table 5.4).
©
5.4.4 Phenols
Being one of the most significant endocrine disruptors found in food and
beverages, BPA has been widely targeted by aptamer-based assays. Jo and
coworkers selected a DNA aptamer that recognized BPA with a high affin-
ity and specificity. In contrast to the BPA antibody, the isolated aptamer
was specific to BPA without nonspecific binding to it analogs: bisphenol B
and 4,4′ -biphenol. Then, an aptamer-based sol-gel biochip was developed
©
TABLE 5.4
20 178
18
Aptamer-Based Assays for Mycotoxin Determination in Food
Ta
LOD
yl
or
OTA Loop-mediated isothermal amplification
an Electrochemistry 0.3 × 10– 3 Wine Xie et al. 2014
Two-level signal amplification (AuNPs and
d Electrochemistry 0.75 × 10– 3 — Yang et al. 2014
abundant G-rich DNA) Fr
Polythionine and iridium oxide an Electrochemistry 14 × 10– 3 Wine Rivas et al. 2015
NP-modified SPCE cis
Graphene-protected thionine-aptamer Electrochemistry
LL 14 × 10– 3 Wheat Sun et al. 2017
DNase I– based cycling signal amplification C
Diazonium coupling reaction Electrochemistry 0.37 Cocoa beans Mishra et al. 2015
.C
Hyperbranched rolling circle amplification Electrochemiluminescence
on 0.05 × 10– 3 Corn Yang et al. 2015
Biotin aptamer– streptavidin cross-linker Surface plasmon
triresonance 12.38 × 10– 3 Wine, peanut oil Z. Zhu et al. 2015
Dethiobiotin-fiber aptamer-MBs Evanescent wave,ball 3 Oat Wang et al. 2015
Streptavidin-cDNA
utfiber
or
FAM-modified aptamer/SWC nanohorns Fluorescence ’s 17.2 Wine Lv et al. 2016
Displacement assay Fluorescence C 0.005 Beer Hayat et al. 2015
COOH-fluorescent particles (signal- op
generating probe) y
TiO2 quenching of fluorescence particles Fluorescence 1.35 fo Beer Sharma et al. 2016
FAM-aptamer/nanographite aptamer Fluorescence 20
r Wine Wei et al. 2015
hybrid/DNase I amplification
Pe
Peroxidase-like activity of Au@Fe3O4 NPs Colorimetric 0.07 C. Wang et al.
rsCereal
2016
on
AFB1 GCE modified with electropolymerized Electrochemical 0.05 Peanuts, Evtugyn et al.
al cashew
neutral red and polycarboxylated U
nuts, white 2014
macrocyclic ligands
s sauce
wine, soy e
O (Continued)
nl
y
©
20
18
Aptamer-Based Assays for Mycotoxin Determination in Food
Ta
LOD
yl
or
Diazonium coupling reaction an Electrochemical 0.29 Beer, wine Yugender Goud et
d al. 2016
Poly (amidoamine) dendrimers of fourth Fr Electrochemical 0.4 Peanuts Castillo et al. 2015
generation (PAMAM G4) an
AFM1 Diazonium coupling reaction c 3.5 Milk Istamboulié et al.
isElectrochemical 2016
Fe3O4 incorporated polyaniline (Fe3O4/ Electrochemical
LL 0.006 — Nguyen et al.
PANi) film C 2013
AFB1 Fluorescence quenching of GO by QDs Fluorescence 1.4 Peanut oil Lu et al. 2015
.C
FAM-aptamer/TAMRA-cDNA Fluorescence
on 0.608 Beer, wine Goud et al. 2016
Carboxyl-X-rhodamine-aptamer/DNase I Fluorescence tr 1.064 Corn Zhang et al. 2016
amplification
ib
Peroxidase-like DNAzyme activity Colorimetric 0.304 Corn Seok et al. 2015
ut
or
Peroxidase-like DNAzyme activity Chemiluminescence ’s 0.33 Corn Shim et al. 2014
AFB2 AuNP aggregation Colorimetric C 0.08 Luan et al. 2015
FB1 FRET/5’ UCNPs-molecular beacon-AuNPs Fluorescence 0.013
op Maize Wu et al. 2013
3’ y
AuNP-modified GCE Electrochemical 0.002 Maize X. Chen et al. 2015
fo
r
3 4 3 4
Note: Au@Fe O NPs, gold nanoparticle– doped Fe O ; GCE, glassy carbon electrode; GO, graphene oxide.P
e
rs
o na
lU
se
179
O
nl
y
using the identified aptamer for fluorescent detection of BPA in dissolved

water with high sensitivity (Jo et al. 2011). After that, various aptasensing
schemes coupled to different detection strategies were reported (Xu et al.
2015; Ragavan et al. 2013; Kuang et al. 2013). Among them, electrochemi-
cal aptasensors have gained much attention because of their sensitivity,
simplicity, and low cost (Y. Zhu et al. 2015; Zhang et al. 2013). Xue et al.
y
developed the first electrochemical aptasensor for on-site determination
nl
of BPA in drinking water. For that, the BPA aptamer and its cDNA probe
O
were immobilized on the electrode surface and methylene blue was used
se
as redox tags. The principle was based on the competitive recognition of
lU
BPA, which induces the release of cDNA and a decrease in the redox peak
na
current. This method allowed the quantitative detection of BPA in drink-
o
rs
ing water with a LOD as low as 0.284 pg/mL in less than 30 m in (Xue et al.
Pe
2013). However, labeling a biomolecule may change its binding properties,
r
and the yield of the bioconjugation to a label is highly variable. To over-
fo
come these limitations, several label-free aptasensors have been reported
y
op
in the literature for BPA detection in foodstuffs (Zhou et al. 2014; Mei et al.
C
2013).
’s
or
ut
5.4.5 Pharmaceutical Residues

b
tri
on
Most of the developed aptamers and aptamer-based assays for pharmaceu-

.C
ticals are dedicated to antibiotics. Besides food monitoring, these aptam-

ers have other potential applications, such as target drug delivery and the
C
LL
detection of antibiotics in pharmaceutical preparations. The class of TET

has been widely studied for aptamer selection; this class comprises vari-
cis
ous derivatives of the basic TET nucleus, such as oxytetracycline (OTC),

an
doxycycline (DOX), and TET. First, Niazi et al. selected a specific aptamer
Fr
to OTC (Kd 9.61 nM), with a high molecular discrimination over its analogs
d
TET and DOX. For that, the SELEX process was carried out by using tosyl-
an
activated magnetic beads as immobilization support. In total, seven rounds

or
were performed, and the sequences exhibiting affinity for TET and DOX
yl
Ta
were eliminated during counterselection steps. The secondary structure

of the identified 76-mer ssDNA aptamer was rich in stem and loop motifs
18
(Niazi et al. 2008b). The authors extended this work to select an aptamer that
20
binds structurally related TETs and not only OTC. After 12 SELEX rounds
©
using TET, DOX, and OTC as target and countertarget molecules, TET
group– specific aptamers were identified. They exhibited a high affinity,
with Kd varying from 63 to 483 nM (Niazi et al. 2008a). The same research
group modified the OTC aptamer with a thiol function and immobilized
it on a gold interdigitated array (IDA) electrode chip for constructing an
electrochemical OTC aptasensor (Figure 5.7). CV and square-wave voltam-
metry (SWV) measurements showed the high specificity and selectivity of
the biosensor, which can be applied in OTC determination in food samples
Fe(CN)–4
6 Fe(CN)6–3
e–
y
Aptamer
nl
+ OTC
O
se
lU
o na
IDA gold electrode
rs
Pe
FIGURE 5.7
r
fo
Electrochemical detection of OTC based on aptamer-modified IDA gold electrode (From Kim,
y
Y.S., et al., Analytica Chimica Acta, 634 (2), 250– 254, 2009.)
op
C
’s
or
(Y.S. Kim et al. 2009). Later on, they reported an electrochemical aptasensor
ut
for TET detection. The biotinylated aptamer was immobilized on a strepta-

b
tri
vidin-modified screen-printed gold electrode, where CV and SWV analysis

on
showed the high specificity and sensitivity of the biosensor (LOD 10 nM)
.C
(Kim et al. 2010).

C
Cam is a toxicantibiotic that causes adverse health effects; its use is there-
LL
fore forbidden in animal production. However, it is necessary to develop

is
highly sensitive detection methods for Cam monitoring in food. Mehta et al.
c
an
identified, in vitro, the first DNA aptamer that can be used in the aptasens-
Fr
ing of Cam residues in feed and foodstuffs. After eight rounds of selection,
seven candidates were selected; they exhibited a high affinity to Cam with Kd
d
an
values in the micromolar range. The selected clones were G-rich sequences,
or
making them likely to fold into G-quadruplex structures (Mehta et al. 2011).
yl
Later on, the high affinity of the Cam aptamer was combined with mag-
Ta
netic separation for concentration and upconversion nanoparticles (UCNPs)

18
as signal probes for the fluorescent determination of Cam in milk samples.

20
The principle was based on the target-induced conformational change of the

duplex complex of the MNP-conjugated aptamer/UCNP-conjugated cDNA-
©
sequence to the MNP-conjugated aptamer/Cam complex. The release of the

UCNP/cDNA complex resulted in the fluorescence decrease. This method
allowed the concentration and detection of Cam with high sensitivity (LOD
0.01 ng/mL), providing significant improvements in the quality control of
food safety (Wu et al. 2015).
Derbishyre et al. selected RNA aptamers specific to aminoglycosides anti-
biotics; the aptamers recognize; a common streptamine ring. SELEX was
performed by targeting the common structure, and the resulting aptamers
exhibited a high affinity in the nanomolar range. However, unmodified RNA

aptamers cannot be used as recognition elements. For that, 2′ -fluoropyrimi-
dine derivatives, resistant to nucleases, have been used by combination with
AuNP aggregation for the colorimetric aptasensing of aminoglycosides in
the 1– 100 nM range (Derbyshire et al. 2012).
Finally, the SELEX process and AuNP-based colorimetric methods have
y
also been investigated for the determination of other antibiotics in food, such
nl
as kanamycin and ampicillin (Song et al. 2011, 2012; Daprà et al. 2013).
O
se
lU
ona
rs
5.5 Food Contaminant Aptasensing:
Pe
Limitations and Challenges
r
fo
As biosensing strategies are mainly based on the performance of recogni-
y
op
tion elements, the field of aptamers has known many advances. In recent
C
years, many aptamers have been isolated toward a wide variety of food con-
’s
taminants. Given their high affinity and specificity, low cost, easy handling,
or
safety, and reliability, aptamers have been widely used in various sensing
ut
schemes. Despite that, the application of aptamer technology for food safety
b
tri
is still in an early stage. This goes back to certain limitations, such as the poor
on
applicability in situ due to the difference between in vitro experimental con-

.C
ditions and food matrixes. This disadvantage could be overcome by using

C
possible strategies to mimic these conditions during the selection process or

LL
to fix the desired conformation prior to the application. Indeed, SELEX tech-
is
nology is more suitable for targets exhibiting specific features, like positively
c
an
charged groups and aromatic compounds. Therefore, aptamer-based assays

Fr
are rarely commercialized in the food market because of the diversity of con-
taminants and complexity of food matrixes.
d
an
NeoVentures Biotechnology Inc. has selected and commercialized specific

or
aptamers for OTA and AFB1, these two aptamers have been employed for
yl
the development of many aptasensing strategies in the literature. The same

Ta
company has commercialized an aptamer-based diagnostic kit (OTA-Sense

18
system), which consists of an aptamer-based cleanup column and a detection

20
solution. The toxin is first extracted by passing the sample through the OTA-
Sense affinity column, a detection solution containing free aptamer and ter-
©
biumis, which is then used to quantify OTA fluorimetrically. The device has
been applied on wheat and alcoholic beverages. In parallel, Afla-Sense was
developed by using the same system, where an iodine derivatization was
employed to enhance aflatoxin’ s fluorescence. The system was applied for
aflatoxin detection in milk samples.
Finally, in spite of the urgent need of industry to establish simple control
and surveillance measures to ensure food safety, there is still a great lack of
commercially available aptasensing devices.
5.6 Conclusion and Prospects

In conclusion, this chapter provides a detailed description of the differ-
ent categories of molecules that are responsible for the contamination
of food matrixes or food products. In the same context, a relatively new
y
class of assays based on the aptamer as a biorecognition element has been
nl
discussed for the monitoring of different types of food contaminants.
O
Aptamers have emerged as an attractive alternative to the commonly
se
used bioreceptor elements in the design of biosensors in the field of food
lU
contaminant monitoring. Aptamers offer various advantages compared
na
with the other bioreceptor molecules, which include but are not limited
o
rs
to in vitro selection and synthesis of the aptamer strand, stability at room
Pe
temperature for an extended period of time, ease of modification and fic-
tionalization, and a broad spectrum of assay formats. However, despite
r
fo
the above-mentioned advantages, the field of aptamer-based assays is
y
op
still in the development phase compared with immunoassays, which are
C
extensively reported in the literature for food monitoring. This can be the
’s
result of the limited number of aptamers available against various ana-

or
lytes and, subsequently, the lack of command over knowledge of aptamer

ut
immobilization strategies. However, recent years have seen important

b
tri
and rapid advances in the exploration of sequences of new aptamers,

on
along with the integration of new nanomaterials in aptamer-based detec-

.C
tion formats, rapidly improving the existing procedures. Even though

C
substantial progress has been accomplished in the design of aptamer-

LL
based assays, several exciting and improved opportunities still exist to

is
explore the field of aptasensors. For example, the use of a synthetically

c
an
modified nucleotide as a co-component and the integration of the binding

Fr
properties of aptamers with DNAzyme may yield hybrid structures with

superior sensing functions by combining selected binding and catalytic
d
an
properties of aptamers. Overall, the potential of aptamers is immense in

or
food monitoring, and this exciting and challenging area is on the brink
yl
of exponential growth.
Ta
18
20
©
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6
Allergen Management as a
Key Issue in Food Safety
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Antó nio Raposo, Esteban Pé rez, Catarina Tinoco de Faria,
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and Conrado Carrascosa
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CONTENTS
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6.1 Introduction................................................................................................. 196
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6.2 Overview of Food Allergies ..................................................................... 200
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6.2.1 IgE-Mediated Food Allergies........................................................ 201
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6.2.2 Clinical Spectrum of IgE-Mediated Food Allergies.................. 203
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6.2.2.1 Anaphylaxis...................................................................... 203

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6.2.2.2 Acute Urticarial................................................................ 203

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6.2.2.3 Atopic Dermatitis............................................................. 204

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6.2.2.4 Oral Allergy Syndrome................................................... 204

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6.2.3 Mixed IgE- and Non-IgE-Mediated Food Allergies.................. 204

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6.2.4 Characteristics of Patients with Food Allergies......................... 205

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6.2.5 Traceability of Allergenic Foods in the Food Chain Supply.... 206

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6.2.6 Development of Allergen-Free Foods.......................................... 207

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6.3 Overview of Food Intolerances ................................................................ 208

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6.3.1 Gluten “ Intolerance” ...................................................................... 209

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6.3.2 Lactose Intolerance......................................................................... 211

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6.3.3 Fructose Intolerance....................................................................... 212

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6.3.4 Histamine Intolerance.................................................................... 212

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6.4 Food Allergen Risk Management and Communication....................... 213

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6.5 Food Allergy Diagnosis............................................................................. 219

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6.5.1 Food Allergy Prevention............................................................... 221

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6.5.2 Allergen Detection..........................................................................222

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6.5.3 Future Therapies.............................................................................225

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6.6 Conclusion................................................................................................... 226

References.............................................................................................................. 226
195
6.1 Introduction
Current food safety development not only takes into account microbiologi-
cal, physical, and chemical food hazards, but also addresses the problem
of food allergy because it has become a health problem due to the increas-
y
ing prevalence and complexity of modern food and its globalization. In
nl
the last two decades, huge efforts have been made to assess the risk that
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arises from allergenic ingredients in food products for consumers with food
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allergy (Mourano et al., 2014b). Nevertheless, food allergy is an increasingly
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prevalent global health problem in both the industrialized world and in less
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developed countries, where, owing to poor labeling and awareness, a major
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challenge may exist (Gowland and Walker, 2015).
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It is generally believed that food allergy affects 1%– 2% of adults and up
to 8% of children, which equates to 8 million food-allergic individuals in
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the European Union (EU) (Crevel, 2001), and 3%– 5% of adults and 8% of
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children worldwide (Gupta et al., 2011; Sicherer, 2011). For most food-allergic
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individuals, the impact of their allergy affects their quality of life, with a
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small, but significant, number of people who suffer more severe reactions
or
(anaphylaxis and death). Currently, the only way of treating food allergy is
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through elimination diet, where the offending food is avoided (Bruijnzeel-

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Koomen et al., 1995), for example, by hypoallergenic (low-allergen) foods,

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nonallergenic foods, or producing low-allergen foods by genetic modifica-

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tion. Thus, consumers with food allergies rely on food labels to disclose the
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presence of allergenic ingredients.

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However, undeclared allergens can be inadvertently introduced into a

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food via cross-contact during manufacturing. However, allergen removal by

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cleaning shared equipment or processing lines has been identified as one of

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the critical points for effective allergen control (Jackson et al., 2008). For this
reason, cleaning control and use of specific tests for allergens should system-
d
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atically apply after cleaning and disinfection operations. Food handlers play
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a key role in the safety and hygiene of the food consumed by the public.
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As at-risk consumers need to avoid culprit foods as ingredients and from

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cross- contamination with other foods, it is crucial to improve food handlers’

18
allergen management competences when preparing and handling food.

20
There is a general duty of care in the food industry, with obligations set
out in EU legislation to reduce and manage the presence of allergens, along-
©
side other food hazards (Muraro et al., 2014b). For this reason, the EU has
regulated mandatory information for consumers and all the establishments
involved in the food chain through Regulation (EU) No. 1169/2011.
To support consumers with food allergies in avoiding food allergens, EU
food legislation requires the allergenic food components used as ingredi-
ents to be labeled (Anandan and Sheikh, 2005). It also imposes general care
duty in the food industry to reduce, manage, control, and communicate the
presence of allergens, alongside other food hazards. This requires allergenic
Allergen Management as a Key Issue in Food Safety 197
ingredients to be managed rather than completely eliminated from the

food supply (Ward et al., 2010). However, the majority of foods processed
on shared equipment, and so-called allergen cross-contacts, may lead to the
unintended presence of allergens (Muraro et al., 2014b). Yet when process-
ing surfaces are shared with different ingredients or products either with or
without allergens, there is every likelihood of allergen cross-contacts, even
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when they cannot be eliminated by proper cleaning.
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The description of an allergen food profile implies the identification
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of all the potentially allergenic molecules that it contains. This state-
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ment takes for granted the concept that potential allergenic molecules
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represent a finite number of proteins, and the remaining components
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of the proteome of the allergenic source lack the features that cause the
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activation of the immune system, which leads to an allergic reaction
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(Ciardiello et al., 2013).
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To date, the frequency and extent of cross-contact in commercial food
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items are generally unknown. As a result, precautionary allergen label-
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ing, such as “ may contain … ” is frequently used, partly for product
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liability reasons, but also to provide additional consumer safety infor-
’s
mation, even though the application of precautionary labeling may not

or
be evidence based. In addition, major gaps in knowledge on the allergen

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risk management of manufactured food remain to be bridged (Muraro

b
tri
et al., 2014b).
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These are typically classified as food allergies (i.e., reactions that involve
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the immune system) or food intolerances (i.e., reactions that do not involve
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the immune system). Allergen terminology has been published by the World
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Allergy Organization (WAO) and is based on the terminology originally

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proposed by the European Academy of Allergy and Clinical Immunology

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(EAACI).
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A food allergy occurs when an allergen (i.e., a protein in a food, which will
not produce an adverse reaction in the majority of people) sets off a chain of
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reproducible reactions that involve the immune system. Reactions can either
or
be antibody or cell mediated. The former is more frequent and occurs in two
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stages:
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18
1.
Sensitization: Initial contact with an allergen does not evoke an aller-
20
gic reaction, but primes the immune system.

2.
Reaction: Once sensitization has occurred, subsequent exposure
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to that allergen can lead to an allergic reaction; that is, in a sensi-

tized individual, allergenic protein cross-links with the immuno-
globulin E (IgE) antibodies on the surface of mast cells cause the
release of histamine or other substances, such as leukotrienes and
prostaglandins.
Food intolerances do not involve the immune system. Food intolerances

may be categorized as enzymatic (e.g., due to an enzyme deficiency, such as
lactase, required to digest milk sugar lactose) or pharmacological (e.g., due

to amines, such as histamine), or the mechanism may be undefined in some
cases (Sadler et al., 2013).
In reference to food allergen regulation and labeling, a number of coun-
tries and regulatory bodies have recognized the importance of providing
this information by enacting laws, regulations, or standards for food aller-
y
gen labeling of “ priority allergens.” However, different governments and
nl
organizations have taken different approaches to identify these priority
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allergens and to design labeling declaration regulatory frameworks (Gendel,
se
2012). The development of labeling regulations for food allergens has been
lU
complex given the number of foods that are allergens, the range of sensitivi-
na
ties in the allergic population, and the variety of ways that allergenic foods
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and their derivatives are used as ingredients. So after extensively search-
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ing regulatory databases, agency and government websites, literature cita-
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tions, and references in other regulatory documents, it is not surprising that
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Gendel (2012) identified 19 laws, directives, regulations, rules, and ministe-
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rial statements on food allergen labeling. op
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European food law aims to reach a high level of protection of human health
’s
and consumers’ interests. Article 8 of Regulation (EC) No. 178/2002 prohibits

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adulteration of food and any fraudulent, deceptive, or other practices that

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mislead consumers. Likewise, the United States has regulated the Allergens
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and Ingredients of Public Health Concern: Identification, Prevention and

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Control, and Declaration through Labeling.

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Article 14 prohibits unsafe food from being sold, such as food injurious to
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health, including the particular health sensitivities of any specific category

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of consumers (but not exclusively people with food allergy) where food is
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intended for that category of consumers (Gowland and Walker, 2015). Other
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EU legislation (European Directive 2007/68/EC) that amends Directive

Fr
2000/13/EC, the labeling of 13 allergenic foods (or food groups) and derived
products thereof, as specified in Annex IIIa of Directive 2007/68/EC, is man-
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datory when used as ingredients for prepacked foods, regardless of the con-
or
centration of the potentially allergenic ingredient. The 14 allergenic foods (or

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food groups) include the most important foods that cause IgE-mediated and
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non-IgE-mediated allergies, celiac disease, and nonallergic food hypersen-

18
sitivities. The sulfur dioxide and sulfites also listed in this directive cause
20
intolerances. Certain products derived from the foods on the list may be
exempted from labeling requirement if they can be assessed and found to be
©
nonallergenic. For example, wheat-based glucose syrups, including dextrose

or maltose, do not require labeling. Other exceptions are fish gelatin used as
a carrier for vitamins or carotenoids, fully refined soya bean oil, and alco-
holic distillates derived from nuts.
More specifically, Regulation 1169/2011 addresses allergen avoidance risks
relating to composition, labeling, and food safety. The inclusion in prepacked
food of any of 14 major allergens defined by Annex II to Regulation 1169/2011
(replacing Annex IIIa to Directive 2000/13/EC) triggers, with certain limited
exemptions, specific labeling requirements— 14 extended on December 13,

2014, to nonprepacked food, including catering establishments.
Regarding the undeclared allergen trends observed in the U.S. indus-
try, the Food Safety and Inspection Service (FSIS) has recognized a nota-
ble increase in the number of recalls (from 13% in 2008 to 35% in 2012)
that have occurred because of undeclared allergens and ingredients of
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public health concern in products. U.S. regulations only recognize eight
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allergens: wheat, crustacean shellfish (e.g., shrimp, crab, and lobster), eggs,
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fish, peanuts, milk, tree nuts (e.g., almonds, pecans, and walnuts), and soy-
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beans. More than 170 foods have been reported to cause allergic reactions,
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although eight of the most common allergenic foods account for 90% of all
na
food allergic reactions, and are the sources from which many other ingre-
o
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dients are derived (FSIS, 2015).
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The FSIS has found that many of these recalls occurred because of a change
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in product formulation by establishing or making changes in a supplier’ s
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ingredient formulation that was not reflected on the labeling of the finished
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meat or poultry product. If an establishment recalls a product because of
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an undeclared ingredient, it has likely failed to (1) address the chemical
’s
(allergen) food safety hazard in its hazard analyses, (2) support the deci-
or
sions made in hazard analyses, (3) reassess hazard analyses, and (4) effec-
ut
tively implement controls to support the decisions made in hazard analyses

b
tri
(see 9 CFR 417.2(a)(1), 417.5(a)(1), 417.4(a)(3), and 417.3(b), respectively), (FSIS,

on
2015). Establishments are required to declare ingredients on labels if they

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are included in the product formulation (9 CFR 317.2 and 381.118). Allergen-
C
containing products must be properly handled, processed, formulated, and

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stored. If allergens are not declared, then the product is adulterated and
is
misbranded. If an adulterated and misbranded product has already been

c
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shipped to commerce, the FSIS would request its recall.

Fr
Since 2004, the U.S. Food Allergen Labeling and Consumer Protection Act
(FALCPA) requires any products under the jurisdiction of the Food and Drug
d
an
Administration (FDA) to contain a major food allergen to clearly identify the

or
allergen on labels (Public Law 108-282, Title II). The FSIS supports the volun-
yl
tary addition of allergen statements (e.g., “ contains” statements) on meat and

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poultry product labels immediately following the ingredients statement dis-

18
cussed in the FSIS Compliance Assistance: Allergens— Voluntary Labeling

20
Statements. Nevertheless, advisory labels are not regulated by FALCPA, and

the type of tree nut, shellfish, or fish does not need to be disclosed on the
©
advisory label (FSIS, 2015).

All the ingredients used in the formulation of meat, poultry, or egg prod-
ucts must be declared by their common or usual name on the ingredients
statement. Occasionally, a substance may be used in a meat, poultry, or egg
product whose use in that product, which is consistent with the FDA’ s label-
ing definition, would be taken as an incidental additive or processing aid (21
CFR 101.100(a)(3)). If an establishment suspects that a substance is a process-
ing aid or incidental additive in a meat, poultry, or egg product, it should
contact the FSIS to determine its suspicion. The FSIS makes these determina-
tions on a case-by-case basis, as discussed in the FSIS Compliance Guide on
the Determination of Processing Aids (FSIS, 2015). Despite the improvements
made in labeling according to FALCPA, limitations in the law may impose
continued challenges for consumers with a food allergy.
In addition to problems in the food industry, the Food Allergen Act itself
y
fails to include provisions that would protect allergic consumers more effec-
nl
tively (Grossman, 2015). For example, requirements of the act apply only to
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packaged foods regulated by the FDA, so consumers who buy bulk foods
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and foods not in packaged form may not be informed adequately (Grossman,
lU
2015). In addition, the act does not require labeling of allergens in restaurant
na
food, nor does it regulate the use of advisory warnings about the possible
o
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presence of allergens (Derr, 2006). These limitations mean that some con-
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sumers, who are not fully informed, may consume food allergens and risk
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serious allergic reactions (Roses, 2014).
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6.2 Overview of Food Allergies

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Food is essential for life, a major source of pleasure, and often intrinsic to our
on
cultural identity. Most individuals eat three meals a day, plus snacks, and
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typically consume some food at most social gatherings. The average person
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in Westernized societies is likely to eat 2– 3 tons of food in one’ s lifetime.

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Consequently, it is not surprising that food is so frequently implicated in a

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variety of maladies, and that it causes so much distress in individuals who

c
an
believe they are afflicted with food allergy (Sampson, 1999). Some of the
Fr
controversy that surrounds food allergy has stemmed from disparate use of
d
terms. In an attempt to confer uniformity to the nomenclature related to food

an
allergies, the EAACI proposed a mechanistic classification of these disorders

or
(Bruijnzeel-Koomen et al., 1995). Adverse food reactions are defined as any

yl
aberrant reaction after ingesting food or food additive. Adverse food reac-
Ta
tions may be the result of toxic or nontoxic food reactions. Toxic reactions
18
can occur in anyone, provided a sufficient dose is ingested (e.g., histamine

20
in scombroid fish poisoning). Nontoxic reactions depend on individual sus-

ceptibilities and may be the result of immune mechanisms (allergy or hyper-
©
sensitivity) or nonimmune mechanisms (intolerance), or in the majority of

cases, may arise through unknown mechanisms (Ortolani, 1995; Sampson,
1999; Dupont, 2011; Sicherer and Leung, 2011; Vickery et al., 2011; Waserman
and Watson, 2011; Muraro et al., 2014c). According to the National Institute of
Allergy and Infectious Diseases (NIAID)– sponsored guidelines, food allergy
is an “ adverse health effect arising from a specific immune response that
occurs reproducibly on exposure to a given food” (Boyce et al., 2010). This
definition encompasses immune responses that are IgE mediated, non- IgE
mediated, or a combination of both, and is in agreement with other interna-

tional guidelines (Sackeyfio et al., 2011; Urisu et al., 2011). Food allergy is an
increasingly prevalent global health problem in both the industrialized world
(Ring et al., 2001; Nwaru et al., 2014) and less developed countries, where,
owing to poor labeling and awareness, a very significant challenge may exist.
There are well-documented detriments to the quality of life of allergic con-
y
sumers and their families (Avery et al., 2003; King et al., 2009). A systematic
nl
review by RAND Corporation was performed by using prespecified criteria
O
directed toward obtaining articles on epidemiologic aspects of food allergy
se
(Schneider Chafen et al., 2010). It concluded that food allergy affected from
lU
1% to 2% and up to 10% of the population (Chafen et al., 2010).
na
Theoretically, any food that contains protein would be capable of elicit-
o
rs
ing an allergic reaction, although the likelihood of foods provoking aller-
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gic sensitization vastly varies. The Codex Committee on Food Labelling
r
established, after considerable debate, a list of the most common allergenic
fo
foods associated with IgE-mediated reactions on a worldwide basis, which
y
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includes peanuts, soybeans, milk, eggs, fish, crustacea, wheat, and tree nuts.
C
This list was presented to the Codex Alimentarius Commission and was
’s
adopted in 1999 at its 23rd session (FAO and WHO, 2001). These commonly
or
allergenic foods account for more than 90% of all moderate to severe allergic
ut
reactions to foods, although an extensive literature search has revealed that

b
tri
more than 160 foods are associated with sporadic allergic reactions (Hefle
on
et al., 1996). Celery, mustard, sesame, lupine, and molluscan shellfish have
.C
been identified as significant allergens in European countries (Regulation

C
1169/2011), and buckwheat is also a common allergen in Japan (Akiyama

LL
et al., 2011).
is
Allergy to cow’ s milk, egg, wheat, soy, peanut, tree nuts (like walnut,
c
an
almond, hazelnut, cashew, pecan, pistachio, and Brazil nut), fish, and shell-
Fr
fish constitutes the majority of food allergy reactions across different age
groups and regions in Europe (Nwaru et al., 2014). In general, these allergies
d
an
tends to occur in childhood (Patel and Volcheck, 2015).

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Annex II of Regulation 1169/2011, on the provision of food information to

yl
consumers, mentions a list of substances or products that cause allergies or

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intolerances in relatively wide population groups. The list includes cereals

18
that contain gluten, crustaceans, eggs, fish, peanuts, soybeans, milk, nuts, cel-
20
ery, mustard, sesame seeds, sulfur dioxide and sulfite, lupin, and mollusks.
According to the Food and Agriculture Organization (FAO, 1995) of the
©
United Nations, these foods or food groups are widely considered to com-
prise commonly allergenic foods.
6.2.1 IgE-Mediated Food Allergies

The most common type of food allergy is mediated by allergen-specific
IgE antibodies (FAO and WHO, 2001). IgE-mediated reactions are known
as immediate hypersensitivity reactions because symptoms occur within
minutes to a few hours of ingesting the offending food. These type of aller-
gies affect perhaps 10%– 25% of the population in developed countries
(Mekori, 1996), although food allergies represent a small fraction of all aller-
gic diseases. Infants and young children are more commonly affected by
IgE-mediated food allergies than adults; the prevalence among infants under
the age of 3 may be as high as 5%– 8% (Bock, 1987; Sampson, 1990; European
y
Commission, 1998).
nl
In IgE-mediated food allergies, exposure to a specific food and the pro-
O
teins contained therein elicits the development of food allergen– specific IgE
se
antibodies (sIgE). These IgE antibodies attach to the surfaces of mast cells
lU
and basophils, and thus sensitize the individual to react upon subsequent
na
exposure to the specific food. Therefore, to become sensitized, individuals
o
rs
must first be exposed to the food in question. Some food proteins are more
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likely than others to elicit allergic sensitization. Information on levels of
r
exposure to a food that are minimally necessary to illicit allergic sensitiza-
fo
tion in susceptible individuals is extremely limited (FAO and WHO, 2001).
y
op
IgE-mediated food allergies may result in the rapid onset of severe reactions
C
(usually within 2 hours of oral exposure to a given food), which may be
’s
manifested by a variety of signs and symptoms that can involve the gastro-
or
intestinal tract (vomiting and abdominal pain), airways (persistent cough,

ut
hoarse voice, wheeze, stridor, respiratory distress, and nasal congestion),

b
tri
circulatory system (pale and floppy infant or young child, hypotension, or

on
collapse), or skin (urticaria, angioedema, erythema, and pruritus) (Boyce et

.C
al., 2011; Burks et al., 2012). The severity of reactions varies from mild (e.g.,
C
hives) to severe (e.g., anaphylaxis). Subjects can have allergic sensitization

LL
(production of specific IgE antibodies) to food allergens without present-

is
ing clinical symptoms of an allergic reaction on exposure. Thus, sensitiza-

c
an
tion alone does not suffice to define food allergy. An sIgE-mediated food
Fr
allergy requires both the presence of sensitization and the development of

specific signs and symptoms upon exposure to that food (Boyce et al., 2011).
d
an
The severity of allergic reactions varies according to the amount of ingested

or
food, coingestion of other foods, and food preparation (cooked, raw, or pro-
yl
cessed) (Boyce et al., 2011). Severity can also be influenced by patient age,
Ta
and by absorption rapidity, which can be influenced by whether food was

18
eaten on an empty stomach or close to doing exercise. Although reactions

20
after a severe reaction are also likely to be severe (Vander Leek et al., 2000),
mild reactions can also be followed by more severe reactions (Ewan and
©
Clark, 2001).
Food-induced anaphylaxis is a serious allergic reaction with a rapid onset,
and it can even cause death (Sampson et al., 2005). IgE-mediated food-induced
anaphylaxis involves a systemic mediator release from sensitized mast cells
and basophils. In patients with food-dependent, exercise-induced anaphy-
laxis, whether a reaction occurs depends on the amount of time between
food consumption and exercising, usually within 2 hours.
All IgE testing for food allergies must be interpreted in the context of the
patient’ s clinical reactions. Many patients will have positive IgE tests to
foods despite never having a clinical reaction. IgE will also remain positive if
they once had food allergies, but have since developed tolerance (Kurowski
and Boxer, 2008). The most widely used method to assess for food-specific
IgE is the skin prick test (SPT). In such tests, a portion of the commercial food
y
extract in question is pushed into the epidermis with a needle or probe, and
nl
the area is observed for a wheal and flare reaction after 15– 20 minutes. Some
O
allergists believe that fresh extracts of fruits and vegetables have superior
se
sensitivity and specificity, and use them in SPT. Although generalized reac-
lU
tions rarely occur (overall rate of about 0.05%), no deaths have been reported
na
after SPT (Devenney and Fa ̈ ith-Magnusson, 2000). Serum sIgE levels can be
o
rs
measured by immunoassays (ImmunoCAP and Immunlite), which provide
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reliable and reproducible measurements, although results can take hours to
r
days. SPTs are quick and simple to perform. SPT wheal size correlates with
fo
the likelihood of clinical allergy (Knight et al., 2006; Sicherer and Sampson,
y
op
2006), and 95% positive predictive thresholds (wheal size above which there
C
is a > 95% chance of clinical allergy) have been described for common aller-
’s
gens (Sampson, 2001; Roberts and Lack, 2005). However, wheal sizes can vary
or
as a result of age, diurnal variation, body site at which SPT is performed, skin
ut
reactivity, and the SPT device and reagents used. Therefore, 95% positive
b
tri
predictive values (PPVs) established in a specific clinical setting might not be

on
applicable to different populations and settings.

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6.2.2 Clinical Spectrum of IgE-Mediated Food Allergies

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6.2.2.1 Anaphylaxis
an
Fr
Anaphylaxis symptoms occur in multiple organ systems and can include

d
throat swelling, wheezing, rhinorrhea, urticaria, hypotension, and abdomi-

an
nal cramping (Nowak-Wegrzyn and Sampson, 2006). Risk factors for death
or
from anaphylaxis are adolescent or young adult patients; underlying asthma;

yl
allergies to crustaceans, tree nuts, peanuts, or fish; and a delay in or lack of

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administration of epinephrine.
18
20
6.2.2.2 Acute Urticarial

©
Food allergies account for 30% of acute urticaria cases (Legrain et al., 1990).
Patients become symptomatic within minutes to hours of eating the provok-
ing food. As acute urticaria can be one manifestation of anaphylaxis, care
to identify symptoms in other organ systems that would lead to raising the
diagnosis to this more urgent level is warranted. Chronic urticaria is much
less commonly caused by food allergies (3%– 4% of cases) (Kulthanan et al.,
2007).
6.2.2.3 Atopic Dermatitis

About 35% of children with atopic dermatitis have a food allergy, based on
double-blind, placebo-controlled food challenges (Eigenmann et al., 1998).
Skin manifestations improve when suspected foods are removed from the
diet; eggs, milk, and peanuts are more commonly implicated. In breast-fed
infants, the elimination of suspected foods in the mother’ s diet has led to
y
nl
clinical improvement.
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se
6.2.2.4 Oral Allergy Syndrome
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Oral allergy syndrome is the most common food allergy, as it is clinically
o
recognized in up to 10% of patients who have allergic rhinitis or asthma
rs
from grass, weed, or tree pollen (Ma et al., 2003). However, it is believed to
Pe
have a significantly higher prevalence in patients with birch pollen allergy
r
fo
(Ghunaim et al., 2005). Oral allergy syndrome manifestations are brief in
y
duration, limited to the mouth and throat, and are sometimes so mild that
op
the patient may not seek evaluation. Proteins similar to the aeroantigens
C
to which the patient is sensitive are present in apples, carrots, and cherries
’s
or
(birch pollen); kiwi and tomato (grass pollen); and melons (ragweed pollen).
ut
When these foods come into contact with the oropharynx, a local reaction
b
occurs.
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on
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6.2.3 Mixed IgE- and Non-IgE-Mediated Food Allergies

C
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Diagnosing mixed IgE- and non-IgE-mediated food allergies is more chal-

lenging than diagnosing IgE-mediated food allergies. The approach begins
cis
with the clinical history. A clear cause and effect between food ingestion and
an
symptoms might not exist because the symptoms of such food allergies are
Fr
typically chronic rather than immediate. If the clinical history is not defini-
d
tive, diagnosis can usually be made by food elimination, followed by rein-

an
troduction challenge. Food challenges can also be performed to assess when

or
the disease has been outgrown (Sackeyfio et al., 2011). Home introduction
yl
Ta
challenges can be undertaken if the sIgE test result is negative and food pro-
tein– induced enterocolitis syndrome is not suspected. Non-IgE-mediated
18
immunologic reactions (e.g., cell mediated) include food protein– induced

20
enterocolitis, proctocolitis, and enteropathy syndromes. These conditions

©
primarily affect infants or young children who present abdominal com-

plaints, such as vomiting, abdominal cramps, diarrhea, and occasionally
blood in stools, and failure to thrive or poor weight gain. Examples of food
allergy comorbidities with mixed IgE- and non-IgE-mediated causes include
eosinophilic esophagitis and atopic dermatitis (Boyce et al., 2011). Allergic
eosinophilic esophagitis, gastritis, and gastroenteritis are characterized by
infiltration of the esophagus, stomach, and/or intestinal walls with eosino-
phils, basal zone hyperplasia, papillary elongation, the absence of vasculitis,
and peripheral eosinophilia in about 50% of patients. An eosinophilic infil-

trate may involve the mucosal, muscular, and/or serosal layers of the stomach
or small intestine, and clinical symptoms correlate with the extent of eosino-
phil infiltration of the bowel wall (Katz et al., 1977; Moon and Kleinman,
1995). Eosinophilic infiltration of the muscular layer leads to thickening and
rigidity, which provokes obstruction symptoms, whereas infiltration of the
y
serosal area results in ascites that contain eosinophils. Allergic eosinophilic
nl
esophagitis is most frequently seen in infancy and through to adolescence,
O
and presents chronic reflux (gastroesophageal reflux), intermittent emesis,
se
food refusal, abdominal pain, dysphagia, irritability, sleep disturbance, and
lU
failure to respond to conventional reflux medication.
na
Dietary protein enterocolitis syndrome is the disorder most frequently
o
rs
seen in the first months of life when infants first present irritability, pro-
Pe
tracted vomiting, and diarrhea, which frequently results in dehydration
r
(Powell, 1976, 1978). Vomiting generally occurs 1– 3 hours after feeding, and
fo
continued exposure may result in bloody diarrhea, anemia, abdominal dis-
y
op
tention, and failure to thrive. Symptoms are most commonly provoked by
C
cow’ s milk or soy protein– based formulas, but occasionally result from food
’s
proteins passed in maternal breast milk. A similar enterocolitis syndrome

or
has been reported in older infants and children, and is caused by egg, wheat,
ut
rice, oat, peanut, nut, chicken, turkey and fish sensitivity (Sicherer et al., 1998).
b
tri
Hypotension occurs in about 15% of cases after allergen ingestion (Goldman

on
et al., 1963; Sicherer et al., 1998). In adults, shellfish (e.g., shrimp, crab, and
.C
lobster) sensitivity may provoke a similar syndrome with severe nausea,

C
abdominal cramps, and protracted vomiting.

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c is
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6.2.4 Characteristics of Patients with Food Allergies

Fr
Most patients with food allergies have an atopic disorder. However, only 10%
d
of patients with atopic disorders have food allergies (Dreskin, 2006). A family
an
history of food allergy or other atopic disorders increases the risk of developing
or
food allergy. Genetic predisposition, including specific haplotypes, has been

yl
Ta
identified for some common food allergies. Oral allergy syndrome is confined
to patients with allergic rhinitis or asthma. The majority of children outgrow
18
the most common food allergies; those who do not will have persistent aller-
20
gies to the same or different foods. Approximately 70% of children with egg
©
allergy and 85% with milk allergy will outgrow them by the age of 5 (Host
et al., 2002; Ricci et al., 2006). However, about 40%– 60% of these children will
develop asthma and 30%– 55% will develop allergic rhinitis (Host et al., 2002;
Ricci et al., 2006). Risk of persistent allergy to peanut is much greater, with only
20% of children ever developing tolerance (Moneret-Bautrin and Morisset,
2005). Adolescents with persistent allergies and adults with new onsets are
particularly prone to fatal food allergies. Increased risk in adolescents may be
explained by their tendency to eat foods that could contain allergens and to not
carry epinephrine with them (depending on their social situation) (Sampson

et al., 2006). Adults with food allergies usually remain allergic.
6.2.5 Traceability of Allergenic Foods in the Food Chain Supply

Over the last two decades, allergenic foods have become recognized as a
y
food safety hazard. During the same period, knowledge about the biology
nl
and clinical characteristics of food allergy has grown, together with infor-
O
mation that can be used to assess the risk more accurately (Ward et al., 2010).
se
Allergen management has evolved in line with growing knowledge and
lU
increased understanding of the issue. Initially, very little was known about
na
the key determinants of risk; industry’ s approach to date has been based
o
rs
on existing good manufacturing practices (GMPs) by ensuring the segrega-
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tion of allergenic ingredients and the systematic declaration of allergens on
r
labels where mandated. However, much more needs to be done to minimize
fo
risks and provide allergic consumers with consistent risk communication
y
op
and a wide choice of products. Developing knowledge about the relationship
C
between allergen doses and population reactivity, and the tools to translate
’s
this knowledge into practical action to improve the safety and quality of life
or
of allergic consumers, means that it will now be possible to manage food

ut
allergens as effectively as other food safety hazards (Hattersley et al., 2014).

b
tri
Historically, multiple approaches have been considered to assess the risk

on
from low-level residues of allergenic foods that might accidentally be present

.C
in other foods (Taylor et al., 2002; Threshold Working Group, 2008; Madsen
C
et al., 2009; Taylor and Baumert, 2010). Similar approaches could equally be
LL
applied to the consideration of risk to allergic consumers posed by deliber-

is
ate low-level allergenic ingredients. Few regulatory bodies have addressed

c
an
the food allergen risk assessment and risk management issue. However, the
Fr
UK Food Standards Agency (FSA, 2006) has published qualitative guidance

to help food businesses to consider where allergen cross-contact risks could
d
an
arise and how they might be better managed. Faced with uncertainty as to
or
the risk and lack of quantitative guidance, many food manufacturers opt to
yl
use some form of precautionary labeling to alert allergic consumers to the

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possibility of and, consequently, the risks from the inadvertent presence of

18
allergenic food constituents in a variety of phrases (Hattersley et al., 2014).

20
While the intention of such warnings is to help allergic consumers to make

safe food choices, the use of such warnings has become widespread, and it
©
can be difficult to find examples of certain food product groups without them
(FSA, 2002; Sakellariou et al., 2010; Barnett et al., 2011a; Zurzolo et al., 2012).
Faced with the resulting uncertainty, manufacturers often feel obliged to pro-
vide these additional voluntary warnings where they qualitatively determine
that any risk may exist, no matter how low or remote. An evidence-based
approach that leads to the definition and adoption of quantitative allergen
management reference doses would result in consistency and transparency in
risk management decision making, and subsequent consumer and clinician
risk communication. This would, in turn, allow us to clearly discriminate

between foods and their suitability for allergic consumers, and would there-
fore improve food allergy management. However, before this goal can be
reached, a number of data gaps need to be bridged, including the lack of char-
acterization of the allergen hazard (dose distribution curves) (Crevel et al.,
2008; Threshold Working Group, 2008; Madsen et al., 2009) and the relative
y
insensitivity and lack of robustness of some analytical methods (Poms and
nl
Anklam, 2004; Diaz-Amigo and Popping, 2010). Recognition that consistent
O
risk management approaches with agreed quantitative reference doses based
se
on scientifically robust principles will provide optimal consumer risk protec-
lU
tion has grown. In parallel, all stakeholder groups now recognize that a zero
na
risk is unrealistic (Madsen et al., 2010, 2012). Indeed, the EU food safety law
o
rs
explicitly enshrines risk analyses as one of its foundations (European Union,
Pe
2002). These risk management approaches are founded on an understanding
r
that minimizing risk from allergenic foods is a responsibility shared across
fo
stakeholders (patients, clinicians, food manufacturers, retailers, caterers, and
y
op
regulators). Industry’ s current approach to allergen management encom-
C
passes existing GMPs as part of a classic Hazard Analysis and Critical Control
’s
Point (HACCP) approach (Codex Alimentarius Commission, 1997), including

or
traceability through the supply chain, segregation of allergenic ingredients,

ut
and application of “ allergen cleans,” to ensure the production of accurately

b
tri
labeled safe food. A “ visually and physically clean” standard for processing
on
and manufacturing the operations control of allergen cross-contamination,

.C
based on thorough visual inspections of the production line (following clean-

C
ing) and of the final product, has been shown to provide a practical and effec-
LL
tive risk management approach (Jackson et al., 2008), and does away with
is
the need for allergen on-line detection methods. Despite these stringent mea-
c
an
sures, the industry recognizes that it still needs to do more.

Fr
d
an
6.2.6 Development of Allergen-Free Foods

or
Although some studies have offered promising results for the successful
yl
induction of oral tolerance for certain allergens (Clark et al., 2009; Staden
Ta
et al., 2007), a general causative immunotherapy for food allergy is currently

18
not available (Sicherer and Sampson, 2006). Consequently, the treatment of

20
food allergies requires strictly avoiding offending food allergens (De Blok et
al., 2007; Sampson, 2004). Therefore, the reliable labeling of allergenic constit-
©
uents is of paramount importance, which is not yet fully available. Labeling

the most common allergenic foods that are added as ingredients in prepack-
aged foods has improved thanks to regulatory amendments, such as the EU
Labeling Directive 2007/68/EC and the U.S. FALCPA (European Commission,
2007; FDA, 2004). The lack of legal thresholds for adventitious contamination
with allergens, the so-called “ hidden” allergens, is a constant challenge for
consumers with food allergies. For food manufacturers, these hidden aller-
gens represent a problem that is difficult to control. Very little is known about
the extent and frequency of cross-contamination, and producers are chal-

lenged to decide on the use of precautionary labeling. To limit advisory label-
ing to products, if necessary, food manufacturers require sound knowledge
about the frequency and extent of allergen cross-contamination while manu-
facturing ingredients and retail packaged goods. Depending on the applica-
tion of cleaning between product changes and the availability of analytical
y
testing to verify cleaning efficiency, qualified allergen sanitation procedures
nl
may be established and precautionary labeling can thus be avoided.
O
U.S. Agriculture Secretary Tom Vilsack announced on June 24, 2014, a
se
new report on discoveries by the U.S. Department of Agriculture (USDA)
lU
researchers, which have led to new patents and inventions with the potential
na
for commercial application and potential economic growth. The USDA inno-
o
rs
vations in this annual report include USDA-supported research that could
Pe
offer solutions for millions who suffer allergies from peanuts and wheat.
r
Highlighted discoveries from the USDA’ s 2014 Technology Transfer Report
fo
include procedures to remove up to 98% of allergens from peanuts without
y
affecting flavor (USDA, 2015). op
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’s
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6.3 Overview of Food Intolerances

.C
Food intolerances are often confused by the general public with food aller-
C
gies, and vice versa. The difference between them for the scientific commu-
LL
nity is clear, but that is not the case when we focus on food intolerances: the
is
point is that no consensus has been reached in the scientific community as to

c
an
what are and are not intolerances, and whether this term is correct.
Fr
According to the WAO, no agreement on allergen nomenclature intoler-

d
ance exists (Johansson et al., 2003). The nomenclature agreement is based on

an
mechanisms. Hypersensitivity is either nonimmunological or immunologi-

or
cal, that is, allergy. Allergy can be either IgE mediated or due to other mech-
yl
anisms. Despite the fact that allergists do not use intolerance to describe
Ta
allergy or allergic diseases, the word intolerance is used by gastroenterolo-

18
gists and laypersons, which should be discussed in this context. The term
20
intolerance should be better defined and restricted to some nonimmunologi-

cal or nonallergic diseases (Dreborg, 2015).
©
For other authors (Vandenplas, 2015a), intolerance is a term loaded with dif-
ferent meanings and interpretations. Intolerance, or hypersensitivity, relates
to all reactions to foods, while allergy indicates that an immune mechanism
is involved and atopy is the terminology for IgE-mediated reactions.
Adverse reactions to food are divided into toxic reactions and nontoxic
reactions. The occurrence of nontoxic food reactions depends on individual
susceptibility to a certain food. Nontoxic adverse reactions to food are either
immune mediated or non-immune mediated. For non-immune-mediated
reactions, the term food intolerance is recommended, while immune-medi-

ated reactions are referred to as food allergies (Bruijnzeel-Koomen et al.,
1995).
It is possible to classify food intolerances into enzymatic, pharmacological,
and undefined food intolerances. Secondary lactase deficiency affects most
of the adult world population, whereas the majority of other enzyme defi-
y
ciencies are rare inborn errors of metabolism. Pharmacological food intoler-
nl
ance is present in individuals who are abnormally reactive to substances,
O
such as vasoactive amines (like histamine), which are normally present in
se
some foods (Bruijnzeel-Koomen et al., 1995).
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The food intolerance term also includes confirmed adverse reactions to food
na
for which the offending mechanisms are unknown (Bruijnzeel-Koomen
o
rs
et al., 1995).
rPe
fo
6.3.1 Gluten “ Intolerance”
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Gluten is the main structural protein complex of wheat, with equivalent toxic
C
proteins found in other cereals, including rye and barley. The toxic protein
’s
fractions of gluten include gliadins and glutenins, with gliadins containing

or
monomeric proteins and glutenins containing aggregated proteins (Sapone

ut
et al., 2012). Possibly the introduction of gluten-containing grains, which

b
tri
occurred about 10,000 years ago with the advent of agriculture, represented
on
an evolutionary challenge that created conditions for human diseases related

.C
to gluten exposure, the best known of which are mediated by the adaptive
C
immune system: wheat allergy and celiac disease. For both conditions, the
LL
reaction to gluten is mediated by T-cell activation in gastrointestinal mucosa.

is
Gluten-sensitive enteropathy, celiac sprue, and nontropical sprue are terms

c
an
sometimes used to describe celiac disease. The term celiac disease is more
Fr
commonly used and accepted (Anderson and Berrill, 2016).

d
According to the European Food Information Council (EUFIC, 2012) and

an
the Coeliac UK organization (2016), celiac disease is not an intolerance in the

or
strict sense, nor is it a food allergy: it is an autoimmune reaction.

yl
Besides celiac disease and wheat allergy, there are cases of gluten reactions
Ta
in which neither allergic nor autoimmune mechanisms are involved. These

18
are generally defined as gluten sensitivity (Brandtzaeg et al., 1989; Catassi and
20
Fasano, 2008; Anderson et al., 2012). Some individuals who experience dis-
tress when eating gluten-containing products and show improvement when
©
following a gluten-free diet may have gluten sensitivity instead of celiac dis-
ease. Gluten-sensitive patients are unable to tolerate gluten and develop an
adverse reaction when eating gluten that usually, and unlike celiac disease,
does not lead to damage in the small intestine (Sapone et al., 2012).
Celiac disease affects 0.6%– 1.0% of the world’ s population (Fasano et
al., 2003; Hoggan, 2011), with wide regional differences in Europe (e.g., the
prevalence is 0.3% in Germany and 2.4% in Finland) for reasons that remain
unclear (Mustalahti et al., 2010). Celiac disease is also common in developing
countries, particularly in North Africa (Alarida et al., 2011) and the Middle
East (Dalgic et al., 2011).
Celiac disease prevalence increases under at-risk conditions, such as a
family history of celiac disease, autoimmune diseases, immunoglobulin
A (IgA) deficiency, some genetic syndromes (Down, Turner, and William
syndromes), and especially type 1 diabetes and thyroiditis (Sapone et al.,
y
2012).
nl
Serologic screening studies have shown that only a small proportion of
O
cases of celiac disease are clinically recognized (21% according to a recent
se
European study) (Mustalahti et al., 2010). Prevalence is 1.5– 2 times as high
lU
for both women and men. Celiac disease affects all age groups, and its main
na
symptoms are malabsorption, chronic diarrhea, steatorrhea, flatulence, and
o
rs
weight loss or failure to thrive (Motala, 2004).
Pe
The clinical features of celiac disease are protean and reflect its systemic
r
nature. Frequent symptoms and signs also include abdominal distention
fo
(in 40%– 50% of patients). Other manifestations include iron deficiency with
y
op
or without anemia, recurrent abdominal pain, aphthous stomatitis, short
C
stature, high aminotransferase levels, chronic fatigue, and reduced bone
’s
mineral density (Green and Cellier, 2007). Unusual manifestations of celiac

or
disease include dermatitis herpetiformis, a blistering rash with pathogno-

ut
monic cutaneous IgA deposits (Caproni et al., 2009; Salmi et al., 2011).
b
tri
Patients with celiac disease are sensitive to gliadin, the alcohol-soluble

on
portion of gluten found in wheat, oat, rye, and barley. Regarding pathol-
.C
ogy, celiac disease is a dietary protein enteropathy characterized by villous

C
atrophy and inflammatory cell infiltrate. Celiac disease is associated with

LL
the HLA-DQ2 (and DQ8) haplotype. About 90% of patients with celiac dis-
is
ease possess IgA anti-gliadin and antiendomysium antibodies. Endoscopy

c
an
typically reveals profound villous atrophy and extensive cellular infiltrate

Fr
(Motala, 2004).
The clinical spectrum of celiac disease is wide and includes symptomatic
d
an
cases with either classical intestinal (e.g., chronic diarrhea and weight loss)
or
or nonclassical extraintestinal (e.g., anemia, osteoporosis, and neurological

yl
disturbances) features, and silent forms that are occasionally discovered in

Ta
serological screening (Sapone et al., 2012).

18
The pathogenesis of celiac disease involves an external trigger (gluten),

20
changes in intestinal permeability, enzymatically modified gluten, human

leukocyte antigen (HLA) recognition, and innate and adaptive immune
©
responses to gluten peptides that involve self-antigens (e.g., transgluta-

minase), and eventually lead to celiac enteropathy (Jabri and Sollid, 2009;
Schuppan et al., 2009).
Gluten ataxia is a sporadic form of ataxia with positive serologic markers
for gluten sensitization (although the association with celiac disease is still
debated) (Hadjivassiliou et al., 2008). Celiac crisis is a rare life-threatening
syndrome, mostly observed in children, characterized by severe diarrhea,
hypoproteinemia, and metabolic and electrolyte imbalances.
Complications associated with untreated celiac disease include osteopo-

rosis, impaired splenic function, neurologic disorders, infertility or recur-
rent abortion, ulcerative jejunoileitis, and cancer (Fasano and Catassi, 2001).
Enteropathy-associated T-cell lymphoma and adenocarcinoma of the jeju-
num are rare complications of celiac disease (Sharaiha et al., 2012).
y
nl
6.3.2 Lactose Intolerance
O
Lactose is a disaccharide of glucose and galactose. It is abundant in mam-
se
malian milk, sometimes called milk sugar, and is essential for nourishing
lU
newborn infants (Bender, 2009; Mattar et al., 2012).
na
The most common type of carbohydrate maldigestion and malabsorp-
o
rs
tion is caused by intestinal lactase enzyme deficiency. Lactose malabsorp-
Pe
tion or hypolactasia is a common condition caused by poor lactase activity
r
(Vandenplas, 2015b).
fo
In most infants, intestinal lactase activity is maximal during the perinatal
y
op
period. However, after 2– 12 years of age, two distinct groups emerge: a “ lac-
C
tase nonpersistence” group with poor lactase activity (hypolactasia) and a
’s
“ lactase persistence” group of individuals who retain their neonatal level of

or
lactase activity into adulthood (Troelsen, 2005; Rasinperä et al., 2004).

ut
Hypolactasia does not cause any disturbance or discomfort, unless lac-

b
tri
tose-containing food is consumed. Colonic microflora ferment undigested

on
lactose in the intestinal lumen, which leads to the production of short-chain

.C
fatty acids, hydrogen, carbon dioxide, and methane, which are responsible
C
for clinical manifestations (Mattar et al., 2012).

LL
Nonpersistent lactase (natural decline in intestinal lactase that leaves

is
adults minimally able to digest lactose) is poorly associated with so-called

c
an
“ lactose intolerance” (symptoms that result from the ingestion of lactose,

Fr
including flatus, gas, bloating, cramp, diarrhea, and rarely vomiting), and
amounts of lactose up to 12– 24 g on a single occasion are tolerable by almost
d
an
all people, no matter what their lactase status is (Lukito et al., 2015).
or
Reduced lactase activity causes primary lactose maldigestion, a con-

yl
dition that is occasionally asymptomatic. When symptoms are present,

Ta
lactose intolerance is diagnosed. It is important to distinguish between

18
primary hypolactasia and secondary causes of lactose maldigestion,

20
including celiac disease, infectious enteritis, or Crohn’ s disease, which

have distinct pathogenic and therapeutic implications (Rasinperä et al.,
©
2004). Moreover, primary hypolactasia should be distinguished from con-

genital lactase deficiency, a rare autosomal recessive disease with unique
molecular mechanisms that affects infants from birth (Robayo-Torres and
Nichols, 2007).
Lactose intolerance, a concept that emerged in the 1960s, privileges the
European view of health and milk-rich Western diets that have been de
facto universal reference points. The term frames the inability to digest
milk after infancy as a defect— i ntolerance— when, in fact, it is the natural
state of more than two-thirds of the world’ s population, including most

people in Asia. Young children almost universally produce lactase and
can digest lactose in their mother’ s milk. As they mature, most switch off
the lactase gene expression as children are weaned. Only about 35% of
the human population can digest lactose beyond the age of about 7 or 8
(Leonardi et al., 2012).
y
There is evidence that genes play an important role in lactase persistence:
nl
some alleles have arisen in different populations worldwide, where the LCT
O
gene plays an important role, and the variant LCT-13910C> T is completely
se
associated with the lactase persistence phenotype and LCT-22018G> A is
lU
strongly associated. The gene LCT-13910C> T can usually be found in sub-
na
jects of European descent (Mattar et al., 2012).
o
rs
Pe
6.3.3 Fructose Intolerance
r
fo
Fructose is a six-carbon monosaccharide sugar (hexose) that differs from glu-
y
op
cose in that it has a ketone group (at carbon‐ 2) instead of an aldehyde group
C
(at carbon‐ 1). It is also known as fruit sugar or laevulose. It is found as free
’s
sugar in fruits and honey, and as a constituent of disaccharide sucrose. It is

or
1.7 times as sweet as sucrose (Bender, 2009).

ut
The pathophysiology of fructose malabsorption remains unclear, as Ebert

b
tri
and Witt (2016) show in their studies. If there are genetic or epigenetic varia-
on
tions in intestinal fructose transporters, such as GLUT5, GLUT2, or SGLT4,

.C
they need to be elucidated.

C
Since the absorption capacity for fructose is highly individual, it is difficult

LL
to determine the right dose for hydrogen breath tests (HBTs), which are the
is
test normally used to diagnose fructose malabsorption. There is evidence

c
an
that prevalence of fructose malabsorption correlates with age, and is higher

Fr
at younger ages (Hoekstra et al., 1993). There is some agreement that 50 g

of fructose in adults or 2 g/kg fructose in children exceeds the absorption
d
an
capacity of the vast majority (Ebert and Witt, 2016).

or
Whether fructose malabsorption is more common in irritable bowel syn-

yl
drome patients needs to be verified (Ebert and Witt, 2016). Hereditary fruc-
Ta
tose intolerance is caused by catalytic deficiency of aldolase B, a recessively

18
inherited condition that affects homozygotes, to develop hypoglycemic

20
and severe abdominal symptoms after eating foods that contain fructose
and cognate sugars. Continued ingestion of noxious sugars leads to hepatic
©
and renal injury, and also to growth retardation (Ali et al., 1998).
6.3.4 Histamine Intolerance

Biogenic amines are low-molecular-weight organic bases with biological
activity that are formed in foods by either the microbial decarboxylation
of the corresponding amino acids or the transamination of aldehydes and
ketones by amino acid transaminases (Zhai et al., 2012).
Biogenic amines are universal regulators involved in the control of body

homeostasis, and influence all vital body functions. They serve as trans-
mitters for the central and peripheral nervous systems, and are also potent
vasoactive agents. They influence blood supply to organs, act as hormones
(adrenaline and noradrenaline), and influence gastric and intestinal ion
secretion and intestinal motility (histamine and serotonin). Histamine is
y
also a mediator of acute anaphylaxis and vascular permeability, and strongly
nl
influences immune responses (Beaven, 1970; Jutel et al., 2002).
O
Histamine is one of the most important of these amines, is formed by
se
decarboxylation of the amino acid histidine in the body, and is also found in
lU
small amounts in foodstuffs, mainly in fermented products, cheeses, beer,
na
chocolate, sauerkraut, and wines. The excessive release of histamine from
o
rs
mast cells is responsible for many symptoms of allergic reactions. It also
Pe
stimulates gastric acid secretion (Bender, 2009).
r
Approximately 1% of the population has histamine intolerance, and 80%
fo
of these patients are middle-aged (Missbichler, 2004). In healthy persons,
y
op
dietary histamine can be rapidly detoxified by amine oxidases crossing the
C
intestinal epithelial barrier passively, whereas individuals with low amine
’s
oxidase activity are at risk of histamine toxicity. Diamine oxidase (DAO) is

or
the main enzyme for the metabolism of ingested histamine (Maintz and
ut
Novak, 2007; Bodmer et al., 1999).

b
tri
However, upon intake of high loads of biogenic amines with foods, this
on
detoxification system is unable to eliminate biogenic amines sufficiently. A

.C
high histamine level can be considered from 5 mg/g (Rahimi et al., 2012;
C
FDA, 2001).
LL
Sensitive people to histamine present insufficient DAO activity (Bodmer

is
et al., 1999). Besides DAO, monoamine oxidases (MAOs), distributed in dif-

c
an
ferent tissues of the human body, also participate in the physiological inacti-
Fr
vation of biogenic amines (Bodmer et al., 1999).

Histamine has been identified by the FDA as a major chemical hazard of
d
an
seafood products (FDA, 2001). Upon ingestion of food with a high histamine
or
content, such as fish, cheese, meat products, and alcoholic beverages, his-
yl
tamine-intolerant patients have been described to suffer from a variety of

Ta
symptoms, including gastrointestinal discomfort associated with vomiting

18
or diarrhea, urticaria, rhinitis, headache, asthma-like symptoms, and cardio-

20
vascular complaints, such as hypotonia and tachycardia (Maintz and Novak,

2007; Amon et al., 1999).
©
6.4 Food Allergen Risk Management and Communication

Risks that arise from allergenic food can be viewed from the individual and
societal public health perspectives. These two views are not identical; while
regulators and food manufacturers need to be mindful of the perspective of

the allergic individual, their overall management of food allergy risks must
be founded on a public health perspective (Madsen et al., 2010).
Knowledge to characterize the public health risk from allergenic foods is
developing rapidly, as are methodologies to apply this knowledge to quan-
tify risks more accurately (Madsen et al., 2009). A key finding is that the reac-
y
tivity of allergic patients spans at least six orders of magnitude of allergen
nl
doses. One implication is that, in practice, it is often impossible within the
O
constraints that apply to food production for normal consumption to guar-
se
antee that such foods will not provoke reactions in a few allergic individuals.
lU
These findings also reinforce an emerging consensus that, as with other
na
risks in society, a zero risk for food-allergic persons is not a realistic or attain-
o
rs
able option. Scarcity of data and the reluctance to make decisions based on
Pe
available data have led society worldwide to attempt to manage food allergy
r
without setting science-based quantitative management thresholds. One
fo
consequence has been the lack of guidance to risk managers in both the pub-
y
op
lic and food industries, which has led both groups to resort to individual
C
judgments. As these judgments have not been shared, the application of the
’s
resulting risk management measures, such as precautionary labeling, lacks

or
consistency and transparency. This lack of transparency not only creates

ut
uncertainty among food-allergic individuals, but also affects their quality

b
tri
of life. It also reduces public trust in food safety and leads to increased risk
on
taking (Sampson et al., 2006; Greenhawt et al., 2009). Emerging data for, and
.C
new knowledge in, risk assessments offer the chance to develop quantita-
C
tive limits on allergenic constituents present inadvertently in food products

LL
(management thresholds) and to thereby address these concerns. However, a

is
prerequisite to their application is to admit an acceptable level of risk.

c
an
When the question arises as to why food allergy differs from other food
Fr
hazards, it is important to state that toxic chemicals and infectious microor-

ganisms in foods are generally hazardous for the whole population. There
d
an
may be different levels of susceptibility (e.g., those who are ill, the very
or
young, or elderly individuals may be more susceptible), but the overall popu-
yl
lation is at risk. In food allergy, only a minority of the population is at risk,

Ta
and a food that is dangerous for the food-allergic person is often an impor-
18
tant source of nutrients for the rest of the population.

20
Safety risks from toxic chemicals in the modern food and drinking water
supply mostly concern the risk of chronic disease after prolonged expo-
©
sure. For this type of exposure, those who may be affected are “ theoretical”
subjects with an increased probability of developing, for example, cancer.
Manifestations of such effects are also often masked by a multitude of other
influences on a person’ s health. In contrast, food allergens cause immediate
reactions that can be fatal, and those affected are real, identifiable people
with names, faces, and families.
Research has shown that if a risk is perceived to be inequitably distrib-
uted in society and causes damage to identifiable, rather than anonymous,
individuals, it is perceived as less acceptable (WHO, 2001). A perception

that a risk cannot be controlled by those affected and can have severe con-
sequences also reduces its acceptability. Therefore, a risk that arises from
allergenic food is liable to be perceived as less acceptable— at least in allergic
individuals— than the risk from many other food hazards, including those
with acute manifestations, such as microbiological risks.
y
Eliminating all risks to food-allergic individuals from foods for nor-
nl
mal consumption is unrealistic and unachievable, as already briefly
O
discussed— hence the need to identify an acceptable level of risk. An accept-
se
able level of risk might be defined as one at which the probability of an
lU
adverse event is minimized by taking into account the efficacy and feasibil-
na
ity of available risk reduction measures, and the nature and severity of the
o
rs
adverse effect and its impact on society at large.
Pe
To prevent adverse events, people with food allergies must diligently avoid
r
allergenic foods, which depends on food safety controls all along the food
fo
production chain. Food allergen safety begins with producers and grow-
y
op
ers, and continues to include manufacturers, distributors, and transporters,
C
as well as retailers, restaurants, and consumers themselves (Dupois et al.,
’s
2016). Food allergen guidance is provided in the Codex Alimentarius and is

or
embedded in the principles of the HACCP Management System, which offer

ut
a prevention framework for the food industry globally (Codex Alimentarius,

b
tri
2015; Wehr, 1997). In the United States, the FALCPA, passed in 2004, specifies
on
that packaged foods must include labeling for eight of the common allergens
.C
identified by the Codex Alimentarius Committee on Food Labeling: wheat,

C
crustacean shellfish, eggs, fish, peanuts, soy, milk, and tree nuts (Gendel and
LL
Zhu, 2013; Hey and Luedemann, 2001; Kjelkevik et al., 1997; Roses, 2011).
is
For consumers with food allergies, trust in food processing, labeling, and
c
an
handling is essential to manage their potentially life-threatening chronic

Fr
condition.
Food consumed in restaurants or other food service settings accounted for
d
an
approximately one in four food-induced anaphylaxis deaths between 1994

or
and 2006 (Bock et al., 2001, 2007). The role of restaurants in allergy manage-
yl
ment is particularly important since food allergy prevalence is on the rise

Ta
(Branum and Lukacs, 2008) and American families are eating more meals at
18
restaurants and preparing fewer meals at home. In the United States, 49.6%
20
of all food dollars in 2013 were spent away from home (U.S. Department of
Agriculture Economic Research Service, 2014a). Moreover, 20.8% of the food
©
purchased away from home was procured at “ limited-service eating places”

(U.S. Department of Agriculture Economic Research Service, 2014b), where
a customer orders food at a counter and does not receive table service (U.S.
Department of Agriculture Economic Research Service, 2015).
Restaurant employees have critical roles to play in reducing the risk of food
allergy adverse events. Their work requires specialized knowledge based
on HACCP principles, as well as motivation to meet patrons’ needs, self-
efficacy to employ best practices, and resources to execute safe food allergy
management protocols (Choi and Rajagopal, 2013; Muraro et al., 2014b). The
preparation of a food allergy– safe meal begins even before a customer enters
the restaurant, with steps that include careful review, segregation, and stor-
age of ingredients (National Restaurant Association Educational Foundation,
2015b). Once a customer with food allergies enters a restaurant, a food service
worker can employ a number of precautionary steps to mitigate risks, which
y
include having a conversation with the customer to clarify food allergies,
nl
reading food labels, using uncontaminated ingredients, and delivering an
O
allergen-free meal using properly sanitized service ware. Diversion at any
se
point from best practices could have serious consequences for a highly
lU
allergic customer. While most states require some form of food safety train-
na
ing for restaurant workers, only five states and two cities— Massachusetts,
o
rs
Rhode Island, Michigan, New Jersey, Virginia, New York City, and St. Paul
Pe
Minnesota— require additional food allergy training and/or food allergy
r
materials to be displayed in restaurants (Abbot et al., 2007; Food Allergy
fo
Research and Education, 2015; Massachusetts Department of Public Health
y
op
Bureau of Environmental Health/Food Protection Program, 2010; National
C
Restaurant Association Educational Foundation, 2015a). Established train-
’s
ing programs, like the National Restaurant Association’ s ServSafe Allergens

or
Online Course (National Restaurant Association Educational Foundation,

ut
2015b), describe the range of necessary and distinct steps to reduce the risk
b
tri
of food allergy adverse events in restaurants. Food service workers should be

on
prepared to recognize and respond to food allergy adverse events when they
.C
actually occur. In the event of anaphylaxis, prompt administration of epi-

C
nephrine is the preferred and lifesaving emergency response (Kemp et al.,

LL
2008), which should be followed by calling 9-1-1 and transporting the ill per-
is
son to an emergency room (Muraro et al., 2014a; Sampson, 2003).

c
an
Across a broad range of food service environments (e.g., restaurants and

Fr
institutions) and geographical areas (e.g., the United Kingdom, the United
States, Malaysia, and Brazil), several studies have evaluated workers’ food
d
an
allergy knowledge by identifying potential hazards to food-allergic consum-

or
ers (Ahuja and Sicherer, 2007; Ajala et al., 2010; Bailey et al., 2011; Choi and
yl
Rajagopal, 2013; Common et al., 2013; Shafie and Azman, 2015). These stud-
Ta
ies, which were conducted in select populations and convenience samples,

18
have suggested the need for improved training and better adherence to
20
HACCP practices and allergen management among food service workers.

A study conducted in a small town in the United Kingdom reported that
©
food service workers need additional training on food allergy management,

and that proprietors and environmental health officers lack motivation, time,
and money to invest in this issue (Pratten and Towers, 2003, 2004). Low levels
of motivation and concern have also been reported by Ajala et al. (2010), who
studied workers at 12 restaurants in Brazil. A study conducted at a university
cafeteria in the United States revealed specific gaps in food allergy knowl-
edge among food service workers, including challenges in identifying com-
mon food allergens among listed ingredients and lack of knowledge about
appropriate emergency response in the event of a severe food allergy reac-

tion (Choi and Rajagopal, 2013). In Penang, Malaysia, food handlers were
found to have moderate knowledge of safe food allergy management prac-
tices. For example, in that study, only about half of the respondents knew
that eating even a small amount of an allergen could cause a reaction (Shafie
and Azman, 2015). These studies provided important early documentation
y
of potentially serious shortcomings in allergy management in food service
nl
establishments, and indicated the need for additional research that is gener-
O
alizable to other settings.
se
Taking into account the consumers’ perspective, advisory labels are help-
lU
ful if they provide reliable information on allergen contents. However, man-
na
ufacturers widely use them as a “ safety net” to convey a nonspecified risk of
o
rs
possible contamination. An audit by the UK Anaphylaxis Campaign found
Pe
that 69% of cereals and 56% of confectionery items were labeled as contain-
r
ing traces of nuts, despite none listing peanut or tree nuts as an ingredient
fo
(FSA, 2002).
y
op
Allergen labeling causes considerable anxiety for people with allergies
C
and their carers (Cummings et al., 2010; FSA, 2002; Sheth et al., 2010). The
’s
format of labels varies considerably, and it is not uncommon for consumers

or
to miss allergy warnings (FSA, 2002). Use of different expressions on advi-

ut
sory statements is confusing (FSA, 2002; Ng et al., 2011) and may contribute
b
tri
to the increasing trend for consumers to ignore them altogether (Barnett et

on
al., 2011a; Hefle et al., 2007). A UK-based survey found that 60% of parents of
.C
children with nut allergies avoided products labeled “ may contain traces,”
C
but only 40% did so when the statement was more vague, for example,
LL
“ made in a factory that uses nuts” (Noimark et al., 2009). Similar findings
is
have been reported elsewhere (Imamura et al., 2008), which suggests that the
c
an
more ambiguous the warning, the less likely consumers are to heed content.
Fr
However, no correlation has been found between the wording and the risk
of cross-contamination (Crotty and Taylor, 2010; Hefle et al., 2007; Pele et al.,
d
an
2007). Thus widespread use of poorly defined advisory labeling might para-
or
doxically lead to increased risk taking. Do ambiguous labels contribute to

yl
the occurrence of allergic reactions? Published case series suggest that many
Ta
food allergy reactions (including most deaths) happen outside the home after
18
exposure to nonprepackaged foods, like those sold in catering establish-

20
ments, which are currently exempt from allergen labeling legislation (Boden
et al., 2005). However, few well-controlled studies have investigated this. A
©
Canadian study found that labeling remains an important factor in uninten-

tional exposure, with 47% of 651 allergic people attributing their reaction to
a labeling-related issue (Sheth et al., 2010). In 29% of cases, the reaction was
due to not reading the label correctly, while 8.3% were attributed to ignoring
a precautionary statement.
The food industry is increasingly recognizing the role of labels in imple-
menting preventive measures to protect allergic consumers from reactions
through accidentally eating their problem food. However, it is also evident
that key knowledge and skills are essential to support them in undertaking
effective food avoidance. In this context, the indiscriminate use of precau-
tionary labeling has led allergic consumers to lose confidence in this risk
communication tool (Crevel et al., 2014). In addition, the absence of such
labels does not automatically imply that the given food is safe, as precau-
tionary allergen labeling is voluntary. Therefore, appropriate communica-
y
tion strategies are needed (Barnett et al., 2011b), for example, communicating
nl
that reference doses, if available, are associated with a certain risk of reac-
O
tion, and providing guidance according to standards. This, in turn, requires
se
adequately training allergic patients to obtain relevant information on food
lU
products and from food suppliers. Therefore, the key element is close coop-
na
eration and effective communication among patient organizations, food
o
rs
industry representatives, and regulators. Moreover, adequate training of
Pe
individuals who have contact with customers—from helplines, to those in
r
the retailing and catering sectors, is of great importance. This also applies to
fo
those involved in caring for individuals with food allergies in the extended
y
op
community, including personnel in day care centers and nurseries and teach-
C
ers. This is all necessary to increase awareness about food allergies, and to
’s
thus reduce the risk of accidental exposure of food allergens, and to prompt
or
action in the event of such exposure (Muraro et al., 2014a).

ut
Finally, we cannot leave food from genetically modified organisms (GMOs)

b
tri
aside. One generally accepted belief is that genetically modified foods are
on
directly linked with an increase in allergenic reactions, but no agreement on

.C
this point has been reached. Moreover, genetic engineering can be helpful to
C
develop less allergenic foods.

LL
Unlike foods from conventional organisms, whose consumption, from

is
experience, appears safe, in the EU and many other countries, they may
c
an
only be placed on the market when the organisms from which they were
Fr
prepared have passed an authorization procedure with security checks. In

the EU, the marketing of GMO and derived food and feed is controlled by
d
an
Regulation (EC) 1829/2003.

or
In 1996, a task force of the International Food Biotechnology Council (IFBC)

yl
and the Allergy and Immunology Institute of the International Life Sciences
Ta
Institute (ILSI) developed a decision tree approach to assess the potential

18
allergenicity of the plants produced via agricultural biotechnology, where

20
the source of the introduced novel gene or genes is a critical point (Metcalfe
et al., 1996).
©
Some studies have been carried out in the EU to determine differences in

allergenicity between “ natural” food and genetically modified food. This is
the case of the study carried out by Niemann et al. (2016), which determined
a very low impact on the overall assessment of inquiries, as there was only
one case for which there was evidence for increased allergenicity of each
genetically modified plant compared with the conventional baseline. It was a
corn, possessing heat-tolerant alpha-amylase, which was composed of three
genes of different strains of the bacteria of the genus Thermococcales.
Since the introduction of GMOs into the food chain has to be supervised
by different health authorities, there is a risk of increased prevalence in food
allergies, as these GMOs are limited.
y
nl
O
6.5 Food Allergy Diagnosis
se
The evaluation requires a thorough history and physical examination to
lU
consider a broad differential diagnosis, to ascertain possible trigger foods,
na
and to determine a likely general pathophysiological basis, specifically as
o
rs
to whether the food-induced allergic disorder is likely IgE mediated, which
Pe
guides testing (Sicherer and Sampson, 2010). The history should determine
r
the possible causal food or foods, the quantity ingested, the time course of
fo
the reaction, ancillary factors (exercise, aspirin, and alcohol), and the reaction
y
op
consistency (American College of Allergy, Asthma, & Immunology, 2006).
C
The history also focuses on details that might contribute to estimating the
’s
prior probability of an allergic reaction to a specific food. For example, rea-

or
soning dictates that a food ingested infrequently is more likely to be respon-

ut
sible for an acute reaction than one previously tolerated; contamination from
b
tri
a meal by a previously diagnosed allergen should be considered ahead of a

on
less likely explanation, such as development of a new allergy to a previously

.C
tolerated food; major allergens are inherently more likely to be triggers than
C
other foods. To reach a diagnosis, clinicians should consider the epidemio-

LL
logic aspects of the disease (e.g., common triggers and common associations)
is
and the details of the specific history, and then consider appropriate test-
c
an
ing that can be evaluated in the context of these prior probability estimates
Fr
(American College of Allergy, Asthma, & Immunology, 2006).

For IgE-mediated disorders, SPTs provide a rapid means to detect sen-
d
an
sitization (American College of Allergy, Asthma, & Immunology, 2006).

or
Negative SPT responses essentially confirm the absence of IgE-mediated

yl
allergic reactivity (negative predictive accuracy > 90%). However, a posi-

Ta
tive test response does not necessarily prove that food is causal (specific-
18
ity < 100%). Consideration of a clinical history and disease pathophysiology

20
is required to maximize the utility of test results. For example, a positive

SPT response can be considered confirmatory when combined with a recent
©
clear history of a food-induced allergic reaction to tested food. Additionally,

increasing SPT wheal size correlates with an increasing likelihood of clinical
allergy (American College of Allergy, Asthma, & Immunology, 2006; Knight
et al., 2006). Studies have attempted to define wheal sizes above which an
allergy is virtually confirmed based on the test result alone (Hill et al.,
2004; Sporik et al., 2000). However, these studies have been limited to a few
foods in infants and to using specific techniques in only a few populations
(American College of Allergy, Asthma, & Immunology, 2006). One study
with 140 children evaluated peanut allergy; 64 had positive SPT responses,
and 18 reacted during an oral peanut challenge (Pucar et al., 2001). Of the 17
children with an SPT wheal of more than 10 mm, only 8 reacted during the
challenge. Thus, additional studies are needed to continue to define the diag-
nostic accuracy of skin test wheal sizes for different foods, ages, diseases,
and populations; wheal size has not been correlated to severity of outcomes.
y
When evaluating allergy to many fruits and vegetables, commercially pre-
nl
pared extracts are often inadequate because of the lability of the responsible
O
allergen. Therefore, fresh food might be used for testing.
se
Serum immunoassays to determine food-specific IgE antibodies are
lU
another modality to evaluate IgE-mediated food allergy (Hamilton and
na
Franklin, 2004). Increasingly higher concentrations of food-specific IgE lev-
o
rs
els correlate with an increasing likelihood of a clinical reaction, but do not
Pe
generally correlate very well with reaction severity (Boyano-Martinez et al.,
r
2002; Celik-Bilgili et al., 2005; Garcia-Ara et al., 2001; Osterballe and Bindslev-
fo
Jensen, 2003; Perry et al., 2004; Sampson, 2001).
y
op
Different predictive values are being generated from emerging studies,
C
which might represent nuances of diet, age, disease, and challenge protocols
’s
(Osterballe and Bindslev-Jensen, 2003; Celik-Bilgili et al., 2005; Komata et al.,

or
2007). Particular values associated with a high likelihood of clinical allergy

ut
(e.g., > 95%) are often referred to as diagnostic values. Undetectable serum

b
tri
food-specific IgE might be associated with clinical reactions for 10%– 25%

on
(Roberts and Lack, 2005; Sampson, 2001). Consequently, if there is any sus-
.C
picion of possible allergic reactivity, a negative SPT response or a negative

C
physician-supervised food challenge result, or both, is necessary to confirm

LL
the absence of clinical allergy. Nomograms are available where prior prob-
is
abilities can be used along with likelihood ratios (determined from studies
c
an
that have evaluated the diagnostic utility of tests) to predict diagnosis. Yet
Fr
very few studies have provided likelihood ratios, and results vary (American
College of Allergy, Asthma, & Immunology, 2006). A drop in specific IgE
d
an
concentrations is associated with an increasing chance of allergy resolution

or
(Shek et al., 2004).

yl
Despite not being commercially available, determination of specific IgE

Ta
binding epitopes on an allergen might provide increased diagnostic utility

18
(Cerecedo et al., 2008). The specific profiles of bound epitopes might reflect
20
distinctions in binding to areas of an allergen that depend on protein folding

(conformational epitopes), and are a feature of mild or transient allergy ver-
©
sus areas that represent stable linear binding regions, which reflect severe
persistent allergy. Additionally, IgE responses to specific proteins in foods
might account for particular outcomes (Steckelbroeck et al., 2008). For exam-
ple, identification of IgE binding to labile birch pollen– related proteins is
associated with mild reactions, whereas binding to stable lipid transfer
proteins in the same foods is associated with more severe reactions. This
observation forms the basis of an approach termed component-resolved
diagnostics.
Increasingly, more studies are evaluating the utility of the atopy patch
test for disorders in which symptoms are delayed after food ingestion, such
as atopic dermatitis (Mehl et al., 2006), eosinophilic esophagitis (Spergel
et al., 2007), and food protein– i nduced enterocolitis syndrome (Fogg et al.,
2006). The test is run by placing foods under Finn chambers in a manner
akin to testing for contact allergens. Although the atopy patch test is prom-
y
ising, there are currently no standardized reagents, methods of application,
nl
or interpretations, and additional diagnostic information in some studies
O
appears marginal (Mehl et al., 2006; Spergel et al., 2007). Other future diag-
se
nostic modalities might include the basophil activation test (Wanich et al.,
lU
2009). Various tests and procedures (e.g., endoscopy or biopsy and hydro-
na
gen breath tests) might be required to evaluate possible gastrointestinal
o
rs
allergy (Furuta et al., 2007). Unproved or disproved tests, such as pulse
Pe
tests, applied kinesiology (muscle strength tests), cytotoxic tests, electro-
r
dermal tests, and immunoglobulin G (IgG) testing, should not be used
fo
(Bernstein et al., 2008).
y
op
The oral food challenge (OFC) comprises a gradual feeding of a possible
C
allergen under medical supervision to determine tolerance or clinical reac-
’s
tivity. Severe reactions can be elicited; therefore, the procedure is under-

or
taken by properly trained personnel with medications and equipment on

ut
hand to treat anaphylaxis. Feeding is generally stopped when objective or

b
tri
persistent subjective symptoms are elicited (Perry et al., 2004). For chronic
on
disorders in which ingested food currently forms part of the diet, diagnosis
.C
typically includes a period to eliminate the possible trigger food or foods

C
to determine whether symptoms resolve before an OFC. Caution is advised

LL
because acute severe reactions are sometimes noted after the reintroduc-
is
tion of a potential allergen (e.g., positive test result for IgE or suspicion of
c
an
allergy) after prolonged dietary elimination (Flinterman et al., 2006). Open

Fr
or single-blind OFCs are often used to screen for reactions. The double-blind,
placebo-controlled OFC is the gold standard for diagnosing food allergies
d
an
because bias is minimized (Bindslev-Jensen et al., 2004). If the blinded chal-

or
lenge result is negative, it must be confirmed by open supervised feeding

yl
of a typical serving of food in its natural form to rule out a false-negative

Ta
challenge result (approximately 1%– 3%). Several reviews have outlined the

18
procedures involved for OFCs (Bindslev-Jensen et al., 2004; Bock et al., 1988),
20
and a comprehensive clinically oriented guide has been published (Nowak-

Wegrzyn et al., 2009).
©
6.5.1 Food Allergy Prevention

Data on primary prevention of food allergy through dietary means are lim-
ited, although numerous studies with various limitations have addressed
outcomes of atopic disease, such as atopic dermatitis and asthma. Based on
reviews in the available literature, professional organizations (Greer et al.,
2008; Host et al., 2008) have generally concluded that there is insufficient
evidence for reduced atopic disease to recommend maternal avoidance of

allergens during pregnancy or lactation. However, some evidence exists
that allergen avoidance during lactation might be related to reducing atopic
dermatitis. For infants with a family history of atopy, which places them at
increased risk, data primarily support the practice of exclusive breast-feeding
for at least 4 months, compared with feeding intact cow’ s milk formula,
y
to decrease the cumulative incidence of atopic dermatitis and cow’ s milk
nl
allergy in the first 2 years. Similarly, avoidance of solid foods for the first 4– 6
O
months is associated with a reduced risk of atopic dermatitis. For infants not
se
being exclusively breast-fed, whole-protein formula (cow’ s milk or soy), com-
lU
pared with the use of extensively studied or partially hydrolyzed formulas,
na
in the first few months of life appears to be associated with increased risks of
o
rs
atopic dermatitis. After 4– 6 months, there are insufficient studies and data to
Pe
suggest that specific allergen avoidance alters atopy outcomes.
r
fo
y
6.5.2 Allergen Detection op
C
To prevent contamination of the food chain by allergens, detection methods
’s
for allergens in foodstuffs have been developed. The challenge today is the
or
detection and quantification of trace amounts of allergens in miscellaneous

ut
food matrixes, which are able to provoke an allergic reaction, with the sever-
b
tri
ity depending on the allergen and the individual. The quantification of aller-
on
gens in food first aims to guarantee with a high confidence level the absence
.C
of allergens in food for the allergic consumer. In parallel, the quantitative

C
data obtained on patient serum can provide useful information about the
LL
allergenic potential of the food sample and the potential allergic reaction of
is
the patient induced by ingestion of the foodstuff analyzed (Kirsch et al., 2009).
c
an
Among the available immunochemical methods, we first mention the most

Fr
commonly used method in laboratories to detect hidden allergens in food,

the enzyme-linked immunosorbent assay (ELISA). In an ELISA designed to
d
an
screen allergenic proteins in food, antibodies mainly come from the serum
or
of an immunized animal. This serum contains IgG, which is able to bind to

yl
the allergen used to immunize the animal, whereas in tests used for clinical
Ta
diagnostics, the properties of IgE present in human serum are used. The food
18
extract is analyzed in microplate wells. The principle of the quantification is

20
based on the measurement of the enzymatic activity of a second protein-

specific antibody (anti-IgG, e.g., a rabbit anti-human antibody) coupled to
©
an enzyme. This second antibody binds to the allergen– primary antibody

complex. The quantification can also be based on the measurement of the
primary antibody bearing the enzyme label if any secondary antibody is
used, as is the case in the direct ELISA. A reaction with the enzyme sub-
strate produces a colored product, for which the absorption is proportional
(direct, indirect, and sandwich ELISA) or inversely proportional (competi-
tive ELISA) to the quantity of allergen in the food sample. A multiallergen
immunoassay built from the ELISA model has been developed and allows
the simultaneous determination of at least 1 μ g/g protein of each peanut

and tree nut allergen in chocolate, but a limit of quantitation has not been
established yet (Ben Rejeb et al., 2005). ELISA has recently been combined
with inductively coupled plasma mass spectrometry (ICP-MS) to increase
the sensitivity and precision of the detection of a simple ELISA (Careri et
al., 2007). In ELISA-ICP-MS, the secondary antibody is labeled with a stable
y
isotope instead of an enzyme, and can be used for quantification with a mass
nl
spectrometer. As little as 2 μ g of peanut allergens per gram of cereal-based
O
matrix has been detected (Careri et al., 2007).
se
The polymerase chain reaction (PCR), a tool based on nucleic acids, has also
lU
been developed for the indirect analysis of allergenic ingredients in food. It
na
involves targeting a segment of the gene coding for the allergenic protein of
o
rs
interest and amplifying only this deoxyribonucleic acid (DNA) fragment to
Pe
make the protein detectable. This tool is highly specific and sensitive, having
r
a lower limit of detection (LOD) of less than 10 mg/kg for almond, hazelnut,
fo
soy, milk, and peanut (Poms et al., 2004). PCR is also available as PCR coupled
y
op
to ELISA and as real-time PCR. In PCR-ELISA, the detection is gel-free since
C
the amplified DNA fragments are hybridized to a protein probe and detected
’s
by ELISA. In real-time PCR, the detection is gel-free and is performed in real

or
time; amplification of the PCR product results in fluorescence proportional to

ut
the amount of the gene of interest in the food sample. There is the possibility to
b
tri
perform quantification using a unique internal standard to compensate for the

on
variability in DNA extraction and amplification efficiencies (Hirao et al., 2006).

.C
Regarding IgE-based methods, it is important to take into consideration

C
that the IgE level in serum is very low (less than 1/40,000 IgG level) and
LL
a highly sensitive method is prerequisite for accurate diagnosis of allergy

is
(Yunginger et al., 2000). Typically, the conventional in vivo testing method,

c
an
including the SPT with allergens, is known to be sensitive and reliable.

Fr
However, this method has a potential risk of causing adverse reactions, such
as systemic reactions and anaphylactic shocks (Liccardi et al., 2006). In an
d
an
effort to overcome the drawbacks of in vivo tests, in vitro serological analysis

or
of allergen-specific IgE has been developed and employed, along with the in
yl
vivo provocation test for allergy diagnosis, especially when standardized

Ta
allergen extracts are not available (Hamilton and Franklin, 2003; Hamilton
18
and Franklin, 2004).

20
Since the radioallergosorbent test (RAST) (Wide et al., 1967) was reported
in 1967, the original radioisotope assay in RAST has been replaced with the
©
chromogenic enzyme immunoassay or fluorescent enzyme immunoassay

(Fall et al., 2003). Currently, a commercialized CAP system is most widely
used for the analysis of total IgEs and allergen-specific IgEs. Despite the
availability of numerous in vitro methods for diagnosis of allergies, most of
the assays are expensive and time-consuming, requiring large amounts of
reagent and serum (Okochi et al., 1999).
In recent years, the use of magnetic microparticles has gained much atten-
tion in a micro total analysis system (µ TAS) (Deng et al., 2001; Mirowski et
al., 2004; Pamme and Manz, 2004; Pamme, 2006). Due to their easy separation
and conjugation with biomolecules (Safarik and Safarikova, 2002), magnetic
microparticles have been widely used to develop various detection methods
in microfluidic devices as a solid support for reactions (Hayes et al., 2001; Choi
et al., 2002; Fan et al., 1999; Jiang and Harrison, 2000). Meanwhile, magnetic
nanoparticles were employed as a label in a sandwich immunoassay of an
y
analyte (Kriz et al., 1996, 2005; Graham et al., 2004; Enpuku et al., 1999). In this
nl
case, the amount of magnetic nanoparticles that are associated with a target
O
analyte is proportional to the analyte concentration, and the signal generated
se
from the magnetic nanoparticles can be used for quantitative analysis of a tar-
lU
get analyte. For example, the superconducting quantum interference device
na
(SQUID) and giant magnetoresistive (GMR) sensor have been attempted for
o
rs
immunoassays (Enpuku et al., 1999, 2003; Edelstein et al., 2000). The SQUID
Pe
and GMR sensor generally allow a detection of analyte with high sensitivity,
r
but these systems have some drawbacks, such as tedious washing steps, long
fo
analysis time, and cost of instrument. In addition, these methods have a seri-
y
op
ous limitation in multiplexed analysis and improvement of detection limits.
C
Alternatively, Hahn et al. (2007) have demonstrated the magnetopho-
’s
retic immunoassay of mite allergen-specific human IgE in sera under an

or
enhanced magnetic field gradient. The velocity of a magnetic nanoparticle-

ut
conjugated microbead was well correlated with the concentration of human

b
tri
IgE in serum. The developed system exhibited detection limits one order of
on
magnitude lower than those of the conventional test kit (CAP system) for two
.C
types of mite allergens, showing good reliability. In addition, the magneto-

C
phoretic immunoassay system enabled a fast analysis with a smaller amount

LL
of reagents compared with the conventional method, supporting its utility

is
for analysis of disease biomarkers, as well as specific allergens.

c
an
Finally, biosensors are considered an attractive alternative to traditional

Fr
immunoassay methods offering comparable sensitivities and selectivity,

while allowing for on-site detection (Turner, 2013). A number of studies have
d
an
reported the use of biosensor-based quartz crystal microbalance (QCM), sur-

or
face plasmon resonance (SPR), and electrochemical detection for the detec-
yl
tion of milk protein allergens (Rebe Raz et al., 2010; Yman et al., 2006). SPR
Ta
allows for the sensitive, on-line, and label-free detection of milk proteins.
18
Several investigations have been done on the detection of milk allergens

20
using SPR-based sensors. Haasnoot et al. (2004) developed a sensor for the
detection of κ -casein with a detection limit of 0.1% or 100 µ g/mL. Indyk and
©
Filonzi (2005) and Muller-Renaud et al. (2005), designed biosensors against

lactoferrin (LOD 19.9 µ g/mL) and α -s1-casein (LOD 0.87 µ g/mL), respec-
tively (Indyk and Filonzi, 2005; Muller-Renaud et al., 2005). More recently,
researchers have developed SPRi-based affinity tests to diagnose hypersen-
sitivity (Chardin et al., 2014).
6.5.3 Future Therapies

Future therapeutic food allergy options include strategies that target spe-
cific foods and some that block allergic responses and are not food spe-
cific (Li, 2007; Nowak-Wegrzyn and Sicherer, 2008; Sicherer and Sampson,
2009). Of note, immunotherapeutic approaches are now being studied in an
attempt to avoid serious adverse effects that would otherwise be triggered
y
nl
by the injection of native allergens. This has been noted in a study of injec-
O
tion immunotherapy for peanut allergy (Nelson et al., 1997) by changing the
se
administration route or by modifying (engineering) treatment proteins. The
lU
approach that is currently being most investigated is oral immunotherapy
na
(OIT), in which food protein doses are administered in gradually increasing
o
amounts toward a maintenance dose. Jones et al. (2009) enrolled 39 children
rs
with peanut allergy in an open study of OIT. This study did not use initial
Pe
OFCs, but after therapy for 4– 22 months, and initially aimed for 300 mg as
r
fo
a maintenance dose; 27 of the 39 children completed the maintenance phase
y
and tolerated the targeted 3.9 g of open peanut food challenge (18 had no
op
symptoms). The immune parameters followed during the study revealed a
C
decrease in skin test and basophil activation, a drop in peanut-specific IgE
’s
or
levels, and an increase in IgG levels (American College of Allergy, Asthma,

ut
& Immunology, 2006). In a first double-blind trial of milk OIT by Skripak

b
et al. (2008), 19 children (12 completed active treatment and 7 received a pla-
tri
on
cebo) underwent a regime of an initial escalation day (aiming for 50 mg),

.C
8 weekly updosings to a final dose of 500 mg, and maintenance for 3– 4
months. The median dose to elicit a reaction at the baseline was 40 mg,
C
LL
which increased to 5140 mg (range 2540– 8140 mg) in the treated group,
but remained unchanged in the placebo group. OIT is presumed to restore
cis
or induce a tolerant state. However, a distinction must be made between

an
desensitization, in which the allergen is ingested without symptoms during

Fr
treatment, but requires daily ingestion, and tolerance, in which food might
d
be ingested without allergy symptoms despite abstinence periods. Studies

an
to date have indicated that OIT induces desensitization, but it remains

or
unclear whether tolerance is achieved (Buchanan et al., 2007). Staden et al.

yl
Ta
(2007) randomized children to egg or milk OIT (n = 25) or to observation

during dietary elimination (n = 20); after OFCs at about 21 months on ther-
18
apy, the treatment group discontinued daily therapy for 2 months and was
20
rechallenged. Although 64% of the treatment group showed a good or at

©
least partial response to OIT while on treatment, the food challenges per-
formed 2 months off treatment revealed that only 36% continued to have
true tolerance, a percentage that exactly matched the tolerance achieved in
the untreated control subjects. More studies are required to assess safety
(Hofmann et al., 2009), efficacy, and mechanisms.
6.6 Conclusion
Allergic consumers rely on the accessibility, accuracy, and quality of infor-
mation on purchased food. For this reason, all parts in the food chain should
enhance the control risk based on HACCP and other specific measures to
y
avoid allergens in final products according to labels.
nl
It has been well recognized that protecting allergic consumers from unin-
O
tended exposure to allergenic food is a shared responsibility, in which each
se
stakeholder must play its part: industries, EU authorities, consumers, and
lU
food handlers.
na
Finally, we should take into account that while substantial work has been
o
rs
done on detecting and measuring allergens in foods, very few studies on the
Pe
prevalence and levels of undeclared allergens in a wide variety of foods are
available.
r
fo
y
op
C
’s
or
but
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7
Unintentional Contaminants in Food
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M. Carmen Rubio Armendáriz, Arturo Hardisson de la Torre, Á ngel
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J. Gutié r rez Ferná ndez, Dailos Gonzá lez Weller, Consuelo Revert
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Gironé s, José M. Caballero Mesa, and Soraya Paz-Montelongo
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CONTENTS
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7.1 Introduction................................................................................................. 243
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7.2 Environmental Pollutants.......................................................................... 245
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7.2.1 Heavy Metals and Metalloids....................................................... 245
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7.3 Pesticides...................................................................................................... 251
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7.3.1 Dioxins, Furans (PCBs), Brominated Phenols, and

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Brominated Flame Retardants�� 252

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7.3.1.1 Emerging and Novel Brominated Flame Retardants..... 254

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7.3.2 Endocrine Disruptors.....................................................................254

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7.4 Processed Toxics.......................................................................................... 256

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7.4.1 Compounds from the Thermal Oxidation of Fats and

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Mineral Oil Hydrocarbons�� 256

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7.4.2 Maillard Reaction Derivatives...................................................... 257

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7.4.3 Biogenic Amines............................................................................. 258

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7.4.4 Acrylamide...................................................................................... 259

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7.4.5 Polycyclic Aromatic Hydrocarbons and Heterocyclic

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Amines............................................................................................. 260
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7.4.6 N-Nitroso Compounds (Nitrosamines and Nitrosamides)...... 261

References.............................................................................................................. 264
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7.1 Introduction
20
©
A food contaminant is defined as any substance that is not intentionally

added to the food in question, but that is nevertheless found in the food as
production waste products (including treatments to crops and livestock and
in veterinary medical practice), from production, processing, preparation,
treatment, conditioning, packaging, transport, or storage of such food or as a
result of environmental pollution (Reglamento No. 315/93).
243
Unintentional contaminants in food are a group of substances of inorganic

and organic nature whose main physicochemical feature is lipid solubility.
This property gives them a cumulative nature, especially in organs rich in
fat. They are also very persistent in the environment, and their life span in
tissues and organs of living beings is fairly long. Therefore, their elimination
is difficult, and this is what makes them highly toxic in living beings.
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These xenobiotics are also biomagnifiable in biological chains, and particu-
nl
larly in the food chain. Their origin can be accidental, as a result of human
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activities, primarily industrial and agricultural activities, or have a technolog-
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ical origin as a consequence of food processing. Foods also naturally contain
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various toxics that can make them dangerous (natural toxics), but mycotoxins,
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marine biotoxins, and bacterial toxins are not described in this chapter.
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Therefore, the substances shown in Table 7.1 are addressed.
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Persistent organic pollutants (POPs) are found in the environmental
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pollutants group, which is a group that primarily covers polychlorinated
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compounds, such as organochlorine pesticides, polychlorinated biphenyls
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(PCBs), dioxins, and furans. Their properties are toxicologically significant:
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high lipid solubility tends to the bioaccumulation and the biomagnification
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through the trophic chain and can be transported very long distances from
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the emission source without suffering any transformation.

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The large organizations that deal with regulating food safety are the
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World Health Organization (WHO), Food and Agriculture Organization

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of the United Nations (FAO), European Commission (three directorates:

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Agriculture and Rural Development, Health and Consumer Protection, and

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Environment), and European Food Safety Authority (EFSA). The WHO and
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FAO collaborate with the Codex Alimentarius Commission, and the Joint
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FAO/WHO Expert Committee on Food Additives (JECFA) has evaluated the

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safety of food additives since 1956, although nowadays it also evaluates con-
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taminants, natural toxics, and residues of veterinary medicines in food.

The EFSA was created in 2002 by the European Union as an independent
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authority with responsibility for food safety, nutrition, animal health and
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welfare, and plant health protection. The EFSA is the European body in
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TABLE 7.1
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Environmental contaminants Heavy metals and metalloids: Pb, Cd, Hg, and As
Pesticides
©
Dioxins, furans, and brominated phenols

Endocrine disruptors
Processed toxics Maillard reaction derivatives
Fat peroxidation products
PAHs and HCAs
Nitrosamines
Acrylamide
Biogene amines
Unintentional Contaminants in Food 245
charge of assessing risks and providing an independent opinion on issues of

food security and nutrition.
The Food and Drug Administration (FDA) is the agency that controls food
safety in the United States; it was created in 1906 by President Theodore
Roosevelt. In addition, it also controls drugs (for both human and veterinary
use), tobacco, cosmetics, and biological and blood-derived products, as well
y
as devices and equipment used in medicine. One of the six centers the FDA
nl
is divided into is the Center for Food Safety and Applied Nutrition (CFSAN).
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The FDA has close relationships with other national and global agencies, and
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has a global commitment that exceeds the strict North American borders.
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For example, in 2013, an exclusive agreement concerning food safety was
na
signed between its Food Safety Agency (AECOSAN) and Spain.
o
rs
The evaluation of the toxic risk from consuming unintentional contami-
Pe
nants in food is a process (risk modeling) through which researchers, based
r
on the collection of toxic effects posed by chemical substances and of experi-
fo
mental toxicology studies, focus on evaluating this information to determine
y
op
the possible associated risks that would exist from exposure to these sub-
C
stances due to the consumption of food in which they may be present in
’s
unintended ways.
or
In order to perform this risk assessment, it is essential to know the concept

ut
of toxic risk, with this being the probability that a particular hazard may
b
tri
cause deleterious or adverse effects on individuals or population groups,

on
considering “ a danger” to be any chemical, physical, or biological agent

.C
capable of causing damage from exposure to it.

C
Toxic risk assessment from the consumption of foods containing uninten-

LL
tional contaminants consists of the four main phases shown in Table 7.2.
is
Within the risk analysis, in addition to the risk assessment that has been
c
an
discussed in this section, there are two more phases, risk management and
Fr
risk communication, which are performed by the authorities once the results
of the risk assessment studies are known and which involve legislative
d
an
and regulatory decision making, as well as the subsequent communication

or
procedure.
yl
Following on from the previous classification, the first group to look at is

Ta
environmental pollutants.
18
20
©
7.2 Environmental Pollutants

7.2.1 Heavy Metals and Metalloids
Metals such as arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg) are
naturally occurring chemical compounds that can occur as residues in food
because of their presence in the environment, as a result of human activities,
TABLE 7.2
Phases of the Risk Assessment
Danger identification
• Hazard identification includes the collection, organization, and evaluation of all information
concerning the ability of a contaminant to cause one or more adverse effects on human
health. Among the components of hazard identification is evidence of adverse effects in
y
humans, causality, the relevance of the experimental data, and information on toxicokinetics
nl
O
and metabolism, as well as the identification of the most sensitive population range. It
therefore primarily consists of determining the biological, chemical, or physical agents
se
capable of causing adverse health effects and which may be present in food. One of the first
lU
functions could therefore be the comparison of the maximum allowable limits legislated in
na
different foods, thereby checking whether they are suitable for their commercialization and
o
therefore for their incorporation in the commercial food channel.
rs
Danger characterization
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• This is the qualitative and/or quantitative evaluation of the nature of the adverse health
r
effects, relating them to biological, chemical, or physical agents that may be present in
fo
food. In the case of unintended pollutants, parameters have been set that are
y
recommendations for TDI; in some provisional cases (TWI, PTWI, and ADI), it should be
op
kept in mind that these parameters are dependent on body weight and therefore vary for
C
each person. These are defined in general terms as quantities of chemical contaminants
’s
that humans can be exposed to through the ingestion of everyday food, which can also
or
come from environmental pollution, throughout one’ s life without this being a health risk.
ut
Exposure assessment
b
tri
• This is the qualitative and/or quantitative assessment of the likely intake of these
on
contaminants that food may have, as well as exposures from other sources if relevant.
.C
The most widely used model is the estimation of the daily intake of food (EDI) by means
of conducting nutritional surveys. After obtaining the data on estimated food intake, the
C
LL
concentration of a specific contaminant that is ingested by an individual through food

consumption can be determined.
is
Risk characterization
c
an
• This is a qualitative and/or quantitative estimation, including attendant uncertainties, of

Fr
the probability of a known or potential adverse effect occurring and of the severity posed
to the health of a given population based on hazard identification, characterization, and
d
an
exposure assessment.
• When it comes to characterizing the hazard, the aim is basically to integrate information
or
from exposure assessment and data from the dose– response contaminant being studied.
yl
• For example, once the EDI of a given food by the population is known, and knowing the
Ta
concentration of the contaminant problem in food daily intake, the intake of the
18
contaminant would be compared with the values of permissible intakes, whether

provisional or not, to thereby see if those permissible intakes are exceeded— in other
20
words, to know if there is or is not a health risk to the population from eating the food in
©
which the pollutant is present.
or from contamination during food processing and storage. Dietary expo-

sure to these metals occurs by ingesting contaminated food or water, and
may lead to chronic or acute toxic health effects.
Lead is a powerful neurotoxic agent, mainly in children, and it is also
a powerful blood toxic that disrupts the formation of heme. Furthermore,
acute lead poisoning provokes severe abdominal pain (lead colic). Lead
causes the generation of reactive oxygen species (ROS), which results in
critical damage to various biomolecules, such as DNA, enzymes, proteins,

and membrane-based lipids, while it simultaneously impairs the anti-
oxidant defense system (Gagan et al., 2012). The International Agency for
Research on Cancer (IARC) classifies inorganic lead compounds as prob-
ably being carcinogenic to humans (Group 2A) (Kim et al., 2016).
Lead accumulates in the body and most seriously affects the developing
y
central nervous system (CNS) in young children. There is no recommended
nl
tolerable intake level, as there is no evidence of thresholds for a number of
O
critical health effects (EFSA, 2012a).
se
Legislative measures have gradually been introduced to reduce expo-
lU
sure by removing lead from paint, food cans, water pipes, and petrol. The
na
elimination of tetraethyl lead from petrol has dramatically decreased lead
o
rs
levels in food. Food lead levels decreased by about 23% between 2003 and
Pe
2010.
The mean lifetime dietary exposure has been estimated at 0.68 µ g/kg
r
fo
body weight (bw)/day in the European population based on middle bound
y
op
mean lead occurrence. Exposure was highest for toddlers and other chil-
C
dren, with 1.32 and 1.03 µ g/kg bw/day, respectively, while the two infant
’s
surveys ranged between 0.83 and 0.91 µ g/kg bw/day. Adult exposure was
or
estimated at 0.50 µ g/kg bw/day (EFSA, 2012a).

ut
Recent reports in Europe show that contributors to the dietary Pb expo-

b
tri
sure include bread and rolls (8.5%), tea (6.2%), tap water (6.1%), potatoes and
on
potato products (4.9%), fermented milk products (4.2%), and beer and beer-
.C
like beverages (4.1%) (EFSA, 2012a). In Ireland, Pb was detected in 29% of all
C
samples analyzed in the last total diet study (TDS), and alcoholic beverages,
LL
cereals, and vegetables were the major contributors to the total Pb intake in
is
adults (28%, 22%, and 12%, respectively). Children’ s cereals, beverages, and
c
an
vegetables were found to be the major contributors to the total Pb intake

Fr
(37%, 19%, and 22%, respectively) (FSAI, 2016). In Canada, the food groups
contributing most to the Pb dietary intake were also beverages (e.g., beer,
d
an
wine, coffee, tea, and soft drinks), cereal-based foods, and vegetables (Health
or
Canada 2011, 2013).

yl
Cadmium is primarily toxic to the kidney, which can also cause bone
Ta
demineralization. Cd is a powerful endocrine disruptor and a strongly

18
immunosuppressing agent. Cd has been statistically associated with an

20
increased risk of cancer, and although the IARC has categorized Cd as a

Class 1 human carcinogen (IARC, 1993a, 2012), the EPA has classified it as a
©
Group B1 or “ probable” human carcinogen, and the American Conference

of Governmental Industrial Hygienists (ACGIH) considers Cd a suspected
human carcinogen.
Food is the main source of human exposure to Cd in the nonsmoking
population. Cereals and cereal products, vegetables, nuts and pulses, starchy
roots, and potatoes, as well as meat and meat products, contribute the most
to human exposure. High levels have also been found in seaweed, fish and
seafood, food supplements, mushrooms, and chocolate, but as they are
consumed to a lesser extent, they are not major contributors to Cd dietary

exposure.
Rice was found to be contaminated by Cd in the Toyama region of Japan,
causing itai-itai disease, characterized by damage to the bones (osteomala-
cia) mainly in multipara women.
The JECFA established a provisional tolerable monthly intake (PTMI) of Cd
y
of 25 µ g/kg bw, whereas the EFSA set a tolerable weekly intake (TWI) (the
nl
level at which adverse effects are not expected) of 2.5 µ g/kg bw to ensure
O
sufficient protection for all consumers (EFSA, 2012b).
se
Some population groups— vegetarians, children, smokers, and people liv-
lU
ing in highly contaminated areas— can have a higher level of exposure, up
na
to twice the TWI (EFSA, 2009).
o
rs
In an attempt to calculate lifetime Cd dietary exposure in Europe, a middle
Pe
bound overall weekly average was estimated at 2.04 µ g/kg bw and a poten-
tial 95th percentile at 3.66 µ g/kg bw. Individual dietary survey results var-
r
fo
ied between a weekly minimum lower bound average of 1.15 µ g/kg bw to
y
op
a maximum upper bound average of 7.84 µ g/kg bw and a minimum lower
C
bound 95th percentile of 2.01 µ g/kg bw and a maximum upper bound 95th
’s
percentile of 12.1 µ g/kg bw, reflecting different dietary habits and survey
or
methodologies (EFSA, 2012b).

ut
Mercury and its organic derivatives are environmental contaminants that,

b
tri
after entering the food chain, act as potent food toxics. The biotransforma-
on
tion of inorganic mercury discharged into bays is carried out in sediments

.C
by the action of bacteria in marine muds on methylmercury (MeHg) com-

C
pounds that are very soluble and which target the brain. MeHg is particu-
LL
larly toxic to the developing nervous system. Inorganic mercury is less toxic.
is
The IARC has concluded that MeHg compounds are possibly carcinogenic
c
an
to humans (Group 2B) (IARC, 1993b). Mercury is more damaging to the ner-
Fr
vous system in the early stages of human development, as it affects brain

development, as well as the gastrointestinal tract and renal function (Sierra
d
an
and Hardisson, 1991; Myers et al., 2009; Llop et al., 2012; Agamuthu, 2013).
or
Unborn children are the most vulnerable group because MeHg may induce
yl
the production of neurological effects on the fetus, since it is able to cross the
Ta
placental barrier, thereby causing alterations in the normal brain develop-

18
ment of infants and children and, at higher levels, may induce neurological
20
changes in adults and suppression of the immune system (Myers et al., 2009;
Selin, 2010; Agamuthu, 2013; Raimann et al., 2014).
©
The main forms of mercury found in food are MeHg and inorganic mer-
cury, but MeHg is the predominant form of mercury in fish and other sea-
foods. Several studies assume that 100% of the mercury in fish and shellfish
products is present as methylmercury (Park et al., 2014). Fish meat, partic-
ularly tuna, swordfish, cod, whiting, and pike, was identified as the most
important contributors of MeHg exposure in Europe for all age groups, with
the addition of hake for children. As regards MeHg, the beneficial effects
related to long-chain omega-3 fatty acids present in fish may have previously
led to an underestimation of the potential adverse effects of MeHg in fish

(EFSA, 2012c).
Rice is also known to accumulate MeHg, and therefore commercial rice
products sold in Europe have been recently investigated, finding that 30%
of all commercial market rice products exceeded 10% of the provisional tol-
erable weekly intake (PTWI) calculated for toddlers or 13% of products for
y
adults with a rice-based diet. MeHg ranged from 0.11 to 6.45 μ g/kg, with
nl
an average value of 1.91 ± 1.07 μ g/kg. The total Hg ranged from 0.53 to
O
11.1 μ g/kg, with an average of 3.04 ± 2.07 μ g/kg (Brombach et al., 2017).
se
In line with the JECFA, the CONTAM Panel established a TWI for inor-
lU
ganic mercury of 4 µ g/kg bw, expressed as mercury. For methylmercury,
na
new developments in epidemiological studies from the Seychelles Child
o
rs
Developmental Study Nutrition Cohort have indicated that n-3 long-chain
Pe
polyunsaturated fatty acids in fish may counteract the negative effects
r
from methylmercury exposure. Together with the information that benefi-
fo
cial nutrients in fish may have confounded previous adverse outcomes in
y
op
child cohort studies from the Faroe Islands, the panel set a TWI for MeHg of
C
1.3 µ g/kg bw, expressed as mercury (EFSA, 2012c).
’s
The mean dietary exposure across age groups in Europe does not exceed
or
the TWI for MeHg, with the exception of toddlers and other children in some
ut
surveys. The 95th percentile dietary exposure is close to or above the TWI for
b
tri
all age groups. High fish consumers, which might include pregnant women,
on
may exceed the TWI by up to approximately sixfold. The high exposure to

.C
MeHg of pregnant women may result in exposure of the fetus to this toxic
C
metal at a critical period in brain development. Biomonitoring data from

LL
blood and hair indicate that MeHg exposure is generally below the TWI in
is
Europe, but higher levels have also been observed, and therefore exposure to
c
an
MeHg above the TWI is of concern (EFSA, 2012c).

Fr
Death resulting from organic mercury ingestion has been amply docu-
mented following outbreaks of poisoning (Minamata disease) after the con-
d
an
sumption of methylmercury-contaminated fish in Minamata and Niigata

or
(Japan) and after the consumption of grains contaminated with methyl- and
yl
ethylmercury in Iraq.
Ta
Arsenic is a ubiquitous metalloid present at low concentrations in rocks,

18
soil, and natural groundwater. As is a food contaminant that occurs both nat-
20
urally and as a result of human activity. Chronic exposure to As leads to the

development of lung and skin cancer. Natural As pollution is known in the
©
waters from different parts of the world (Argentina, Chile, and Bangladesh)
by cession of arsenic present in the rocks, which has caused various epi-
demics of epidemiologically detailed chronic hydroarsenicism. Inorganic
arsenic is a threat to more than 150 million people living in countries such
as Bangladesh, Cambodia, China, India, Laos, Myanmar, Nepal, Pakistan,
Taiwan, and Vietnam. In addition, endemic areas with dangerously high lev-
els of arsenic in the water have also been reported in Canada, Germany, and
Argentina (Sanz-Gallé n et al., 1993; Singh et al., 2015).
Inorganic arsenic is the more toxic form in which arsenic can appear (As3+,
As5+). As forms that are natural in food, such as arsenocholine, arsenobeta-
ine, and arsenosugars, are not hydrolyzable in the gastrointestinal tract and
not absorbed, and as such, they do not cause acute poisoning.
The inorganic chemical species mentioned above have the same action
mechanism. This mechanism involves the formation of complexes with the
y
sulfhydryl groups of proteins, inactivating enzymes that have thiol groups
nl
in their structure. Arsenic is a carcinogen; inorganic arsenic is classified as a
O
Group 1 carcinogen (Brandon et al., 2014).
se
Foodstuffs are the main source of exposure for the general population.
lU
The main sources of inorganic arsenic intake in Europe are cereal grains
na
and cereal-based products, food for special dietary uses (e.g., algae), bottled
o
rs
water, coffee and beer, rice and rice-based products, fish, and vegetables
Pe
(EFSA, 2014).
The PTWI of total arsenic has been set by the JECFA at 15 µ g of As/kg
r
fo
bw/week, a value that is above intakes established in Europe, although the
y
op
existence of new studies reporting that inorganic arsenic causes lung cancer,
C
urinary tract cancer, and skin cancer, along with a wide range of adverse
’s
effects at exposures lower than those revised by the JECFA, suggests that
or
there is a need to consider reviewing this PTWI value depending on the form
ut
of arsenic.
b
tri
The EFSA has recently updated its analysis of the occurrence of arsenic
on
in food in Europe, and the estimates of long-term (“ chronic” ) dietary expo-

.C
sure to inorganic arsenic were refined and found to be lower than previ-
C
ously reported (EFSA, 2014). The EFSA Comprehensive European Food

LL
Consumption Database was used to estimate chronic dietary exposure to

is
iAs using 28 surveys from 17 European countries. Mean dietary exposure

c
an
among infants, toddlers, and other children ranged from 0.20 to 1.37 μ g/kg
Fr
bw/day, while the 95th percentile dietary exposure estimates ranged from
0.36 to 2.09 μ g/kg bw/day. Mean dietary exposure among the adult popula-
d
an
tion (including adults, the elderly, and the very elderly) ranged from 0.09 to
or
0.38 μ g/kg bw/day, and 95th percentile dietary exposure estimates ranged
yl
from 0.14 to 0.64 μ g/kg bw/day (EFSA, 2014).

Ta
In Europe, for all the age classes except infants and toddlers, the main
18
contributor to dietary exposure to iAs was the food group “ grain-based

20
processed products (non-rice based),” in particular wheat bread and rolls.

Other food groups that were important contributors to iAs exposure were
©
rice, milk and dairy products (the main contributor in infants and toddlers),
and drinking water (EFSA, 2014).
The techniques for detecting these contaminants need a pretreatment
of the sample (digestion). The pretreatment or digestion process of a food
sample to analyze its metal or inorganic contaminants content involves the
destruction of organic matter before the instrumental analysis to avoid inter-
ference with the analytical process. This is achieved with the help of acids
or bases of different strengths, oxidizing agents, or enzymes. The dilution
process before analysis is only necessary in most cases of liquid food. The
most frequent types of digestion are
• Dry digestion (incineration) is based on the complete combustion

(incineration) of the organic components of the sample in electric
furnaces called “ muffles.” After removing other impurities and car-
y
bon particles that may have come from incomplete combustion, the
nl
residue obtained corresponds to the mineral fraction.
O
se
• Wet digestion is the oxidation of organic matter by using differ-
lU
ent strong acids. The most commonly used are mixtures HNO3/
na
H2O2, HNO3/H2SO4/HClO4, and HNO3/HClO4. Wet digestion is
faster than incineration, and higher recovery rates are obtained
o
rs
due to the low volatilization experienced by the metals. Its main
Pe
disadvantages are the long periods of warming, the large amounts
r
of acids required, and the risk of contamination and losses during
fo
mineralization.
y
op
• Digestion by high-pressure microwave ovens. These are closed
C
systems where decomposition takes place under conditions of
’s
or
elevated temperature and pressure. Its advantages are that it

ut
significantly reduces the digestion time (which is completed

b
in 10– 20 m in), so only a few kinds of acid are needed in small

tri
on
amounts (most digestions are performed using only HNO3 and

.C
H2O2), and the risk of loss of volatile elements or air contamina-

tion is eliminated.
C
LL
Once the sample is digested, the most frequently used methods for the
cis
analysis of mineral elements are

an
Fr
• Spectrophotometry
d
an
• Fluorometry
or
• Atomic absorption spectrometry: Determination of mercury by

yl
cold vapor atomic absorption spectrometry and hydride generation

Ta
atomic absorption spectroscopy

18
• Atomic emission spectrometry: Inductively coupled plasma atomic

20
emission spectrometry and inductively coupled plasma mass

©
spectrometry
7.3 Pesticides
This chapter briefly mentions a few ideas about pesticides—an unintentional
group of food contaminants.
Persistent pesticides are organochlorine compounds appearing in the soil,

water, and animal tissues that have been used to combat pests. Organochlorine
pesticides are chlorinated hydrocarbons used extensively from the 1940s
through the 1960s in agriculture and mosquito control. Representative com-
pounds in this group include DDT, methoxychlor, dieldrin, chlordane, toxa-
phene, mirex, kepone, lindane, and benzene hexachloride.
y
Pesticides found in foods have maximum residue limits (MRLs) and some
nl
established acceptable daily intake (ADIs). An MRL is the highest level of
O
a pesticide residue that is legally tolerated in or on food or feed when pes-
se
ticides are applied correctly (good agricultural practice). The amounts of
lU
residues found in food must be safe for consumers and must be as low as
na
possible. The ADI for humans is considered to be a level of intake of a chemi-
o
rs
cal that can be ingested daily over an entire lifetime without any appreciable
Pe
risk to health.
r
Organochlorine compounds produce neurological, immune, and repro-
fo
ductive effects in humans (as they are endocrine disruptors, they are potent
y
op
sterilizers in both men and women), and some even have carcinogenic effects.
C
The control and monitoring of the entire primary production sector is
’s
therefore both necessary and obligatory. Since 1991, pesticide risk assess-
or
ment in Europe has been performed under Council Directive 91/414/EEC,

ut
which requires that a pesticide can be approved if at least one representative

b
tri
use does not pose “ unacceptable” risks to humans and the environment.
on
The implementation of Regulation (EC) No. 1107/2009 further improved

.C
the EU pesticide regulatory framework. Some of the changes that have been
C
introduced, such as impact on biodiversity, hazard-based cutoff criteria,

LL
comparative risk assessment and substitution, and zonal mutual recogni-

is
tion, entailed new challenges to risk assessors and risk managers.

c
an
Moreover, continually changing pesticide usage patterns need to be con-

Fr
sidered when determining analytes of interest for large-scale epidemiology

studies. The Community Duplicate Diet Methodology is a tool for research-
d
an
ers to meet emerging exposure measurement needs, which will lead to more
or
accurate assessments of intake and may enhance decisions for chemical reg-
yl
ulation (Melnyk et al., 2014).

Ta
There are no groups in the human population that are completely unex-
18
posed to pesticides, while most diseases are multicausal to add considerable

20
complexity to public health assessments. Hence, the development of eco-

friendly pesticide alternatives (e.g., EcoSMART) and integrated pest man-
©
agement (IPM) techniques is desirable to reduce the impacts of pesticides

(Kim et al., 2016).
7.3.1 Dioxins, Furans (PCBs), Brominated Phenols,

and Brominated Flame Retardants
Another type of polychlorinated compounds with high toxicological
significance is dioxins and furans, whose sources are industrial and
municipal waste incineration, wood, fire, and so forth. Dioxins are envi-
ronmentally persistent substances that have been associated with human
toxicity. PCBs, used as thermal and electrical insulation, plasticizers in
paints, and so forth, now banned, are highly water-soluble molecules
that have been linked to liver cancer and endocrine disruption. Dioxins
and PCBs are groups of compounds, and some of their congeners may
y
be carcinogens at low levels of exposure over extended periods of time.
nl
A collective intoxication of rice fields occurred in Yusho in Japan (1968),
O
when they were contaminated with industrial waste with PCBs, causing
se
metabolic problems and alterations in the reproductive system.
lU
“ Agent Orange,” a chlorophenoxy compound used by the Americans in
na
the Vietnam War as a defoliant, contained significant amounts of dioxins
o
rs
as impurities. Increased rates of spontaneous abortions and fetal malforma-
Pe
tions and the occurrence of liver cancer and benign tumors in adipose tissue
r
(soft sarcoma tissue) were observed in the Vietnamese population and in the
fo
soldiers.
y
op
Dioxins cause a particular type of chloracne on the skin and are power-
C
ful carcinogens and teratogenic and immunotoxic agents. Their liposolu-
’s
bility within polychlorinated compounds is very high, and therefore fatty

or
foods such as milk, cheese, and meat should be monitored where substantial
ut
amounts of these compounds can accumulate.

b
tri
The Seveso disaster in Italy (1976) highlighted the importance of the indus-
on
trial emissions of these compounds. Likewise, the fraudulent use of indus-

.C
trial fats in the production of dry feed for animal feed surprised Europe, due
C
to the amount of dioxins they contained.

LL
In 1999, fat from a rendering company in Belgium was contaminated with

is
PCBs and dioxin. This product was shipped to animal feed manufacturers
c
an
and introduced into animal feed distributed to poultry, hog, and cattle farms
Fr
in Belgium, France, and the Netherlands, with the majority of the product
going to Belgium. Analysis of chickens and eggs in Belgium revealed ele-
d
an
vated PCB levels and low levels of dioxins (FDA, 1999).

or
In early December 2008, a global recall of Irish pork was initiated as a result
yl
of a subset of the national pork output being contaminated with dioxin.

Ta
It was estimated that approximately 10% of pig meat from the Republic of
18
Ireland was affected by the contamination (Kennedy et al., 2009).

20
The monitoring of dioxins and dioxin-like PCBs (DL-PCB) in food and

feed in Europe has shown that “ meat from eels” and “ fish liver and derived
©
products” contained the highest average contamination levels of both diox-

ins and PCBs. Levels of dioxins and DL-PCBs were above the permitted
maximum levels in 10% of the food samples, respectively (EFSA, 2012d).
Average exposure to dioxins and DL-PCBs was estimated to be between
0.57 and 2.54 pg TEQWHO05/kg bw/day, and the 95th percentile between
1.2 and 9.9 pg TEQWHO05/kg bw/day (EFSA, 2012d).
Fish, meat and dairy products appeared to be the highest-contributing food
groups to dietary exposure. A general decrease in dietary exposure of dioxins
and DL-PCBs was observed between 2002– 2004 and 2008– 2010, estimated to

be between 16.6% and 79.3% for the different population groups (EFSA, 2012d).
Brominated phenols and their derivatives are a complex group of brominated
flame retardants (BFRs), used as reactive as well as additive flame retardants in
a large range of resins and polyester polymers. A limited number of occurrence
data, covering the food group “ fish and other seafood,” has been identified in
y
the literature. Toxicity studies on brominated phenols are scarce and mostly
nl
relate to 2,4,6-tribromophenol (2,4,6-TBP) because it predominates over other
O
brominated phenols (EFSA, 2012e).
se
lU
na
7.3.1.1 Emerging and Novel Brominated Flame Retardants
o
There are at least 17 emerging and 10 novel BFRs, other than
rs
Pe
Polybrominated dipheyl ethers (PBDEs), Polybrominated biphenyls (PBBs),
Hexabromocyclododecanes (HBCDDs), and Tetrabromobisphenol A (TBBPA),
r
fo
and brominated phenols and their derivatives that the EFSA (2012e) has
y
informed about. There are a lack of experimental data on physical-chemical
op
characteristics, stability and reactivity, and current use and production vol-
C
ume of all the emerging and novel BFRs. Due to this very limited informa-
’s
or
tion on occurrence, exposure, and toxicity, a risk characterization has not been
ut
performed, but instead, an attempt has been made to identify those BFRs that
b
tri
could be a potential health concern and should be considered first for future
on
investigations. The following list presents these BFRs and their main toxic char-
.C
acteristics (EFSA, 2012f):

C
LL
• Tris(2,3-dibromopropyl)phosphate and dibromoneopentyl glycol

is
(DBNPG): There is convincing evidence of their genotoxic and carci-

c
nogenic character.
an
Fr
• 1,2-Bis(2,4,6-tribromophenoxy)ethane (BTBPE) and hexabromoben-

zene (HBB): These have been identified as compounds that could
d
an
raise a concern for bioaccumulation based on the limited experimen-

or
tal data on their environmental behavior.

yl
Ta
7.3.2 Endocrine Disruptors

18
20
Endocrine active substances (EASs) are substances that can interact or inter-
fere with normal hormonal action. Endocrine disruptors are EASs that can
©
be shown to cause adverse effects by interacting or interfering with the endo-

crine (hormonal) system
EASs can be naturally occurring (such as phytooestrogens in soya) or
man-made. Several pesticides, environmental pollutants such as dioxins and
PCBs, the food contact material, and bisphenol A (BPA) are examples of EASs
sometimes found in food (European Environment Agency, 2012).
Rates of endocrine diseases and disorders, such as reproductive and devel-
opmental harm in human populations, have changed in line with the growth
of the chemical industry, leading to concerns that these factors may be linked.
For example, the current status of semen quality in the few European coun-
tries where studies have been systematically conducted is very poor: fertility
in approximately 40% of men is impaired.
The EFSA endorses the World Health Organization’ s definition that a
substance must meet three criteria to be considered an endocrine disruptor
y
(EFSA, 2015a):
nl
O
se
1. The presence of an adverse effect
lU
2. The presence of endocrine activity
na
3. A causal relationship between the two
o
rs
Pe
BPA is used in the manufacture of polycarbonate plastics, epoxy res-
ins, and other polymeric materials, as well as for certain paper products.
r
fo
Polycarbonate is used to make food containers, such as returnable beverage
y
bottles, infant feeding (baby) bottles, tableware (plates and mugs), micro-
op
wave ovenware, cookware, and reservoirs for water dispensers, among other
C
nonfood applications. BPA-based epoxyphenolic resins are used as protec-

’s
or
tive linings for food and beverage cans and as a coating on residential drink-
ut
ing water storage tanks.

b
tri
In January 2011, the European Commission adopted Directive 2011/8/EU,

on
prohibiting the use of BPA for the manufacture of polycarbonate infant feed-
.C
ing bottles.
C
LL
• Regulation EU 10/2011 on plastic materials and food contact

is
materials.
c
an
• Directive 2011/8/EU restricted the use of BPA in plastic infant feed-

Fr
ing bottles.
d
an
The use of polycarbonates in the plastics industry has resulted in the

or
assignment of BPA in the packaging of the product. This is especially serious

yl
in baby bottles and has caused controversy in the bottled water industry.
Ta
BPA can migrate in small amounts into food and beverages stored in materi-
18
als containing the substance.

20
Canned food and noncanned meat and meat products are major contribu-
tors to dietary BPA exposure in Europe. Canned food is known to be a dietary
©
source of BPA because of the substance’ s use in the lining of cans (EFSA, 2015a).
BPA is one of a number of chemicals that may have the potential to interact
with hormone systems in the body. It has been known since the 1930s that
BPA can mimic the female sex hormone, estrogen.
According to the EFSA 2015 opinion on BPA, there is no single clearly
defined explanation that substantially completes the scientific understand-
ing of the potential effects of BPA in humans. Therefore, based on the WHO
criteria, it is not possible to conclude that BPA is an endocrine disruptor.
Based on animal studies, high doses of BPA (hundreds of times above the
tolerable daily intake [TDI]) are likely to cause adverse effects in the kid-
ney and liver. BPA is also likely to have effects on the mammary glands of
rodents. How these effects are caused (the “ action mechanism” ) is not clear.
Certain levels of neurological damage, decreased sperm interference oocyte
maturation, thyroid dysfunction, and so forth, have been observed in experi-
y
mental animals (rodents).
nl
Possible effects of BPA on the reproductive, nervous, immune, metabolic,
O
and cardiovascular systems, as well as in the development of cancer, are not
se
considered likely at present, but they could not be excluded (EFSA, 2015).
lU
The lowest dose (“ benchmark dose” ) at which BPA causes a small adverse
na
effect in the kidneys of mice— in this case, a 10% change in the mean rela-
o
rs
tive weight of the organ— would be 8960 µ g/kg bw/day, with the equivalent
Pe
dose for humans being 609 µ g/kg bw/day. An overall uncertainty factor of
150 was applied to the equivalent human dose of 609 µ g/kg bw/day to derive
r
fo
the new TDI of 4 µ g/kg bw/day (EFSA, 2015).
y
op
BPA poses no health risk to consumers in Europe because current exposure
C
to the chemical is too low to cause harm. The EFSA scientific opinion shows
’s
the level of BPA that consumers of all ages are exposed to is well below the
or
estimated level of safe exposure— k nown as the TDI. The EFSA finds that
ut
there is no health concern, as the highest estimates for dietary and aggregate
b
tri
exposure to BPA are three to five times lower than the TDI, depending on the
on
age group. Dietary exposure on its own is more than fivefold below the TDI
.C
for all population groups.

C
The highest estimates for the dietary exposure and for exposure from
LL
a combination of sources (called “ aggregated exposure” ) are three to five

is
times lower than the temporary-TDI of 4 µ g/kg bw/day.

c
an
Dietary exposure is 4– 15 times lower than that previously estimated by the
Fr
EFSA in 2006, depending on the age group considered. This is due to better
data and less conservative assumptions for the exposure calculations. Dietary
d
an
exposure to BPA is highest among infants and toddlers. The highest estimates
or
are four and a half times below the t-TDI. This is explained by their higher
yl
food consumption on a body weight basis. Dietary exposure for bottle-fed

Ta
infants aged 0– 6 months is 50-fold below the t-TDI for the highest estimates.
18
20
©
7.4 Processed Toxics

7.4.1 Compounds from the Thermal Oxidation of
Fats and Mineral Oil Hydrocarbons
Polyunsaturated fats, such as sunflower oils and colza oil, are susceptible to
oxidation by oxygen in the air action and some catalysts, such as certain trace
metals (Fe and Cu) and ultraviolet (UV) radiation. The oxidation of poly-
unsaturated oils, if provided by heat (thermal oxidation), generates many
peroxides that are powerful oxidants in biological systems, generating the
phenomenon of oxidative stress. Peroxides oxidize cell membranes and
cause impaired intracellular calcium homeostasis, which kills cells by the
activation of proteases and phosphatases.
y
Olive oil is more stable to the thermal oxidation process than other oils
nl
because it is monounsaturated.
O
Consumers are dietarily exposed to a range of mineral oil hydrocarbons
se
(MOHs) from many sources, such as food packaging and additives, pro-
lU
cessing aids, and lubricants. Mineral oil saturated hydrocarbons (MOSHs)
na
consist of linear and branched alkanes, and alkyl-substituted cycloalkanes,
o
rs
while mineral oil aromatic hydrocarbons (MOAHs) mainly include alkyl-
Pe
substituted polyaromatic hydrocarbons (EFSA, 2012g). Dietary exposure to
r
MOSHs in Europe has been considered of potential concern. Except for white
fo
oils, exposure to MOAHs is about 20% that of MOSHs. Estimated MOSH
y
op
exposure ranged from 0.03 to 0.3 mg/kg bw/day, with a higher exposure in
C
children (EFSA, 2012g).
’s
Foodborne MOAHs with three or more non- or simply alkylated

or
aromatic rings may be mutagenic and carcinogenic, and therefore of

ut
potential concern. The no-observed-adverse-effect level for the induc-

b
tri
tion of liver microgranulomas by the most potent MOSH, 19 mg/kg bw/

on
day, was used as a reference point for calculating margins of exposure

.C
(MOEs) for background MOSH exposure. MOEs ranged from 59 to 680

C
(EFSA, 2012g).
LL
c is
an
7.4.2 Maillard Reaction Derivatives

Fr
The Maillard reaction occurs during the processing of foods, resulting in the
d
formation of advanced Maillard reaction products (MRPs).

an
Advanced glycation end products (AGEs) are generated in the late stages
or
of the Maillard reaction in foods and biological systems. These products are
yl
Ta
mostly formed by the reactions of reducing sugar or degradation products of

carbohydrates, lipids, and ascorbic acid. AGEs exist in high concentrations in
18
foods, but in relatively low concentrations in most biological systems. Some

20
AGEs have recently been reported to be toxic, and were suggested as being
©
causative factors for various kinds of diseases, especially diabetes and kid-
ney disorder, through their association with AGE receptors (Van Nguyen,
2006).
Melanoidins are compounds formed by the reaction occurring between
amino acids and reducing sugars in high humidity and temperature, in addi-
tion to an alkaline medium. Melanoidin toxicity has been tested in experi-
mental animals and has been demonstrated to be hepatotoxic in character
and allergenic in nature.
7.4.3 Biogenic Amines

Biogenic amines (BAs) are generally produced by microbial decarboxylation
of amino acids in food products. The most significant BAs occurring in foods
are histamine, tyramine, putrescine, cadaverine, tryptamine, 2-phenylethyl-
amine, spermine, spermidine, and agmatine (Gardini et al., 2016).
This process primarily occurs in fermented and cured foods (wine, cheese,
y
nl
fish sauce, and fermented meat), where it is mainly produced by lactic acid
O
bacteria (LAB).
se
Excess biogenic amino acids in fermented and cured foods indicate prod-
lU
uct degradation, possibly due to the presence of excessive decarboxylate
na
bacterial load. In many cases, this degradation results in poor organoleptic
o
qualities in the product. In addition, BAs are heat stable, so that heating food
rs
does not eliminate them.
Pe
BAs are generally considered a food hazard, even though there is no thresh-
r
fo
old for these biomolecules in the European legislation, except for histamine
y
in fishery products. Tyramine and histamine, the most toxic BAs, are often
op
found in high concentrations in certain foods (Linares et al., 2016). When
C
present in high concentrations, they could have toxicological effects, causing
’s
health problems in consumers, especially sensitive persons (Ordó ñ ez et al.,

or
ut
2016).
b
Symptoms of this type of poisoning appear within minutes to several hours

tri
on
after ingestion of food and are characterized by low blood pressure, edema
.C
and facial flushing, headache, diarrhea, and severe respiratory disorders.

Histamine causes a symptomatology known as “ scombroid fish poison-
C
LL
ing,” consisting of flushing of the face, neck, and upper arms; oral numb-
ness and/or burning; metallic taste; headache; itchy rash; heart palpitations;
cis
asthma attacks; hives; gastrointestinal symptoms; and difficulties in swal-

an
lowing. This type of food intoxication is often a result of the consumption

Fr
of scombroid fish (such as tuna, sardines, anchovies, bonito, and mackerel),

d
which are rich in histidine (Gardini et al., 2016).

an
Tyramine toxicity is known as the “ cheese reaction” because it was ini-

or
tially observed following the consumption of cheeses with high levels of

yl
Ta
this BA. Tyramine has a vasoconstrictor effect and causes dietary-induced

migraine, increased cardiac output, nausea, vomiting, respiratory disorders,
18
and high blood glucose. This increase in blood pressure is due to the fact that
20
tyramine can cause heart failure or brain hemorrhage. Besides these effects,
©
tyramine has also been determined to have an effect on the gut microbiota.
The adherence of some enteropathogens, such as Escherichia coli O157:H7,
to intestinal mucosa is increased in the presence of tyramine (Gardini et al.,
2016).
Tyramine has a stronger and more rapid cytotoxic effect than histamine.
Tyramine causes cell necrosis, and histamine induces apoptosis. In order to
prevent health risks, the BA content of foods should be reduced and legal
limits established for tyramine (Linares et al., 2016).
7.4.4 Acrylamide
Acrylamide (AA) is formed when certain foods are prepared at tempera-
tures above 120° C and low moisture, especially in foods containing aspara-
gine and reducing sugars. AA is a contaminant formed in a wide variety
of carbohydrate-containing foods during frying or baking at high tempera-
tures (Kadawathagedara et al., 2016). Bread, crackers, chips, breakfast cere-
y
nl
als, snacks, pizzas, and so forth, whose composition consists of grains and
O
starches that have been subjected to high temperatures when baked, roasted,
se
or fried, show AA levels, but AA has been found at the highest levels in cof-
lU
fee and solid coffee substitutes and fried potato products. In 2016, the FDA
na
issued guidance to help the food industry reduce the amount of AA in cer-
o
tain foods, but these are recommendations and not regulations.
rs
AA is extensively metabolized, mostly by conjugation with glutathione,
Pe
but also by epoxidation to glycidamide (GA). The formation of GA is consid-
r
fo
ered to be the route underlying the genotoxicity and carcinogenicity of AA.
y
Neurotoxicity, adverse effects on male reproduction, developmental toxicity,
op
and carcinogenicity have been identified as possible critical end points for
C
AA toxicity from experimental animal studies (EFSA, 2015b).
’s
or
The IARC classifies AA as a “ probable human carcinogen” based on data

ut
showing it can increase the risk of some types of cancer in lab animals. The
b
evidence in humans was considered to be “ inadequate” at the time of the last

tri
on
IARC review of the subject (1994), and at that time, AA was not known to be
.C
found in foods.
However, although the epidemiological associations have not demon-
C
LL
strated AA to be a human carcinogen, the MOEs indicate a concern for neo-

plastic effects based on animal evidence (EFSA, 2015b). Nevertheless, certain
cis
studies indicate that dietary AA is not related to the risk of most common
an
cancers. A modest association with kidney cancer, and endometrial and

Fr
ovarian cancers, cannot be excluded (Pelucchi et al., 2015). Epidemiologic

d
studies of dietary AA intake have failed to demonstrate an increased risk of

an
cancer. Therefore, continued epidemiologic investigation of AA and cancer

or
risk appears to be a misguided research priority (Lipworth et al., 2012).

yl
Ta
AA reproductive and developmental toxicity in rats and mice has been

studied, observing that it produces a removal pup weight when adminis-
18
tered to pregnant mothers. Furthermore, after administration of AA in

20
drinking water to rats and mice, litter size was lower than that of the control
©
group due to its effects on sperm from males. These observations cannot be
extrapolated to humans, since there is no information, although recent stud-
ies have suggested reduced fetal growth after exposure to high levels of AA
during pregnancy (Kadawathagedara et al., 2016).
In Europe, mean and 95th percentile dietary AA exposures across surveys
and age groups were estimated at 0.4– 1.9 and 0.6– 3.4 µ g/kg bw/day, respec-
tively. The main contributor to total dietary exposure was generally the cat-
egory “ fried potato products (except potato crisps and snacks).” Preferences
in home cooking can have a substantial impact on human dietary AA expo-

sure (EFSA, 2015b).
Included among the most relevant methods to reduce the adverse effects of
dietary AA (Friedman and Levin, 2008) are selecting processing conditions
(pH, temperature, time, processing, and storage atmosphere) that minimize
AA formation and adding food ingredients (acidulants, amino acids, anti-
y
oxidants, nonreducing carbohydrates, chitosan, garlic compounds, protein
nl
hydrolysates, proteins, and metal salts) that have been reported to prevent
O
AA formation.
se
lU
na
7.4.5 Polycyclic Aromatic Hydrocarbons and Heterocyclic Amines
o
rs
These two groups of substances are products of a pyro-organic nature, which
Pe
are formed at temperatures above 300° C.
r
Polycyclic aromatic hydrocarbons (PAHs) are primarily formed by incom-
fo
plete combustion or heat-induced decomposition of organic matter. PAHs
y
op
occur in several foods, such as cereals, vegetable oils, coffee, and home-
C
cooked foods, usually when smoking, heating, and drying processes are
’s
involved, or in seafood from polluted waters. Home cooking, such as grill-

or
ing, roasting, and smoking, particularly charcoal grilled or barbecued foods,

ut
can lead to high concentrations of PAHs (EFSA, 2008).

b
tri
The level of PAHs depends on factors such as distance from heat source,
on
fuel used, level of processing, and cooking times and methods, whereas pro-
.C
cesses like reuse, conching, concentration, crushing, and storage enhance

C
the amount of PAHs in some food items (Singh et al., 2016). In general, PAH
LL
levels increase when the food is barbequed closer to the heat source (Rose
is
et al., 2015).
c
an
Public concern over the toxic effects of PAHs has grown due to the recog-
Fr
nition of their toxicity, carcinogenicity, and teratogenicity (Bansal and Kim,

2015). Some PAHs are highly carcinogenic in laboratory animals and have
d
an
been implicated in breast, lung, and colon cancers in humans (Ramesh et al.,
or
2004).
yl
Based on general PAH exposure levels, there is a low level of concern for
Ta
consumer health in Europe. Benzo[a]pyrene, the only PAH presently regu-

18
lated in food, is not a suitable indicator for the occurrence of PAHs in food,
20
and a total of four or eight PAHs have been proposed as being more suitable
indicators to better protect consumer health (EFSA, 2008).
©
It has been epidemiologically proven that populations consuming diets

rich in these processed foods have a higher incidence of stomach cancer.
Therefore, they are carcinogenic compounds. Likewise, as vegetable oils age,
their PAH contents increase, and the vegetables may be contaminated if they
are grown near roads with heavy traffic whose fumes are rich in PAHs.
Pyrolytic heterocyclic amines (HCAs) form a group of 23 compounds
belonging to two major groups, the carboniles and aminoimidazoazaarenes,
which are potentially carcinogenic and mutagenic compounds. The foods
with pyrolytic HCAs are known to be rich in proteins. Meats cooked at high
temperatures usually contain HCAs known to be mutagenic and carcino-
genic in animals (Sinha, 2002). During the frying of meat and fish, genotoxic
HCAs are formed (Pfau et al., 2001). In addition, when fats are heated, acro-
lein can be produced, which is considered by the IARC as a Group 3 cancer.
The carcinogenic effect of HCAs is possibly accompanied by the pres-
y
ence of ROS, which are also inhibited by antioxidants (Weisburger, 2002).
nl
Therefore, the formation of HCAs during cooking can be decreased by natu-
O
ral and synthetic antioxidants. Advice to the general public about how to
se
reduce the carcinogenic load coming from HCAs would make an important
lU
contribution to cancer prevention (Sugimura et al., 2004).
na
Using information on cooking methods, in addition to food intake, is essen-
o
rs
tial to accurately estimate dietary exposure to HCAs (Keating et al., 1999).
Pe
r
fo
7.4.6 N-Nitroso Compounds (Nitrosamines and Nitrosamides)
y
op
N-Nitroso compounds are potent carcinogens detected in foodstuffs. The
C
importance of dietary nitrosamines in relation to human cancer develop-
’s
ment is, however, uncertain. N-Nitroso compounds have been related to

or
colorectal cancer in humans (Knekt et al., 1999).

ut
Nitrosamines are formed by the reaction of secondary or tertiary

b
tri
amines with a nitrosating agent. In foods, the nitrosating agent is usu-

on
ally nitrous anhydride, formed from nitrite in acidic, aqueous solution.

.C
Ascorbic acid and sulfur dioxide are used to inhibit nitrosamine formation.
C
Nitrosodimethylamine has been shown to be formed in certain foods as a

LL
result of the direct-fire drying process (Scanlan, 1983).

is
The potential associations between the dietary consumption of nitrates,

c
an
nitrites, and nitrosamines and gastric cancer risk have been investigated
Fr
by several studies, but results are inconclusive (Song et al., 2015). The pres-
ence in the diet of L-ascorbic acid blocks the carcinogenic action of N-nitroso
d
an
compounds.
or
Exposure to preformed nitrosamines in food should be minimized by

yl
appropriate technological practices, such as lowering the levels of nitrate and

Ta
nitrite added to foods to the minimum required to achieve the necessary

18
preservative effect and to ensure microbiological safety (EFSA, 2010).

20
The selection of an analysis method for any pollutant depends very much
on the matrix and the expected concentration, which is why it is difficult
©
to decide on a reference method for each one. However, and in general, the
most commonly used methods shown in Table 7.3 could be established.
Table 7.4 presents an overview of various unintentional contaminants and
their most important permissible limits in foods according to some regu-
lations from the European Commission, the Codex Alimentarius, and the
FAO/WHO.
Environmental pollution can affect crops for food production for human
consumption or animal feed intended for food production, as well as surface
TABLE 7.3
Analytical Methods most Frequently Used for the Determination of the Different
Pollutants
Type of Pollutant Most Used Analysis Method
Environmental Pollutants
y
Heavy metals and Pb: Atomic absorption spectrometry with graphite camera and
nl
metalloids: Pb, Cd, Hg, mass spectrometry with inductively coupled plasma.
O
and As Cd: Atomic absorption spectrometry with graphite camera and
se
mass spectrometry with inductively coupled plasma.
lU
Hg: Cold vapor atomic absorption spectrometry.
As: Hydride generation atomic absorption spectrometry.
na
Pesticides Depending on the chemical characteristics of the analyte,
o
rs
high-resolution liquid chromatography or gas chromatography is
Pe
used. There are several types of detectors to quantify these
compounds, such as the flame ionization detector, gas
r
fo
chromatography electronic capture detector, UV detector, and
high-resolution liquid chromatography florescence detector.
y
op
However, the use of conventional detectors, such as UV-visible or
C
electronic capture fluorescence, presents significant uncertainty in
’s
the data they generate.

or
The best detection system (for accuracy and reproducibility) is

ut
coupling a mass detector to a chromatographic separation system.

b
Dioxins, furans, and High-resolution gas chromatography.

tri
brominated phenols High-resolution mass spectrometry.

on
Endocrine disruptors Their analysis depends on the type of substance or disruptor and
.C
its nature, but the most commonly used techniques are gas
C
chromatography coupled to a mass detector or high-resolution

LL
liquid chromatography coupled to a mass detector.

is
Processed Toxics
c
an
Maillard reaction If one focuses on melanoidins as the main product of the Maillard
Fr
derivatives reaction, these are determined by high-resolution liquid

chromatography with an UV detector.
d
an
Fat peroxidation There are a range of methods to assess the degree of oxidation of
products fats and their by-products.
or
There is no test that alone can measure all oxidation reactions at

yl
once, nor is there one capable of measuring all stages of the

Ta
oxidation process.
18
However, in recent years, special attention has been paid to the

development of new techniques that allow a more effective
20
oxidation process control.

©
Solid-phase microextraction (SPME) coupled to gas

chromatography is one of the best alternatives to determine the
by-products of oxidation of a volatile nature (aldehydes, ketones,
and fatty acid esters).
In addition, a significant number of secondary oxidation
compounds have been isolated and identified by SPME combined
with gas chromatography coupled to mass spectrometry.
At present, this technique is a new alternative for an efficient
control of the quality changes associated with lipid oxidation.
(Continued)

Type of Pollutant Most Used Analysis Method
PAHs and HCAs High-resolution liquid chromatography with fluorescence detector
and gas chromatography coupled to mass spectrometry.
Nitrosamines Gas chromatography coupled to different detectors.
The most commonly used detectors are the flame ionization
y
detector, the nitrogen-phosphorus detector or thermionic
nl
detector, the electron capture detector, mass spectrometry, and the
O
thermal energy analyzer.
se
The last is the most widely used and is regarded as the reference
lU
detector for the analysis of N-nitroso compounds in food,
na
especially in terms of volatile nitrosamines.
o
Acrylamide Gas chromatography– mass spectrometry.
rs
Liquid chromatography– mass spectrometry.
Pe
High-performance liquid chromatography– mass spectrometry in
tandem.
r
fo
Biogene amines High-resolution liquid chromatography with fluorescence detector
y
or UV detector. op
C
’s
or
TABLE 7.4
ut
Overview of Various Unintentional Contaminants and Their Permisible Limits in

b
tri
Foods
on
.C
Legislation Chemical Substance Minimum Value Maximum Value

Regulation (EC) Benzopyrene (PAH) 2.0 µ g/kg (oils and 10.0 µ g/kg (bivalve
C
No. 1881/2006 fats) mollusks)

LL
Cd 0.05 mg/kg (fruits 1 mg/kg (some

is
and vegetables) mushrooms)

c
an
Pb 0.02 mg/kg (pure 3 mg/kg (food

Fr
milk) complements)
Hg 0.5 mg/kg (white 1 mg/kg (oily fish)
d
an
fish)
Codex Pesticide residues Highly variable according to food type
or
Alimentarius
yl
Ta
EQT PCDP/FAO/ Amounts of dioxines, 0.2 pg/g (infant 20 pg/g (fish liver
WHO (2006) furanes, and PCBs foods) and cooked
18
similar to dioxines products)

20
©
and ground waters used as sources of drinking water supply, which can also
be used for food production and processing. However, the levels of chemical
contaminants in the environment can be minimized by
• Controlling emissions of pollutants from industry, such as chemical,

mining, metal, and paper, and also from weapons testing industries
• Controlling the emissions caused, both from the production of
energy (including nuclear power plants) and from transportation
• Controlling the disposal and treatment of waste products, both solid

and liquid, from domestic and industrial use
• Controlling the production, sale, use, and disposal of certain toxic
substances that persist in the environment, such as compounds of
lead, cadmium, and mercury
• Ensuring that before introducing new chemicals into the market, espe-
y
nl
cially if they may eventually be released into the environment in signif-
O
icant quantities, they undergo appropriate testing to demonstrate their
se
acceptability from the point of view of health and the environment
lU
• Replacing toxic substances persisting in the environment with
na
more acceptable products from the point of view of health and the
o
environment
rs
Pe
In order to ensure that the levels of chemical contaminants in food are as
r
fo
low as possible and never above the maximum levels considered acceptable
y
or tolerable, as well as to reduce to acceptable levels the risks to consumers
op
from the point of view of health, the following criteria may apply:
C
’s
• Protect the environment and primary production of food

or
ut
• Enhance hygiene measures

b
tri
• Eliminate or control the source of contamination

on
• Reduce contaminant levels

.C
• Identify and separate contaminated food from the food fit for human
C
consumption
LL
is
In addition, if a food is contaminated, this food is to be rejected for food

c
an
use, unless it can be reconditioned and made fit for human consumption. It
Fr
is worth mentioning that removing chemical contaminants in food, as a rule,

is not possible, which is why little can be done to make contaminated food fit
d
an
for human consumption again. That is why eliminating or controlling food

or
contamination at the source is not only more effective at reducing or elimi-

yl
nating the risk of harmful health effects, but also requires fewer resources
Ta
for controlling the food.

18
20
©
References
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Bansal V, Kim KH. 2015. Review of PAH contamination in food products and their
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Brandon EFA, Janssen PJCM, de Wit-Bos L. 2014. Arsenic: Bioaccessibility from sea-
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Brombach CC, Manorut P, Kolambage-Dona PPP, Ezzeldin MF, Chen B, Corns WT,
Feldmann J, Krupp EM. 2017. Methylmercury varies more than one order of
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EFSA (European Food Safety Authority). 2008. Scientific opinion of the Panel on
on polycyclic aromatic hydrocarbons in food. EFSA J 724:1– 114.
EFSA (European Food Safety Authority). 2009. Scientific opinion of the Panel on
y
nl
O
on cadmium in food. EFSA J 980:1– 139.
se
EFSA (European Food Safety Authority). 2010. Panel on Food Additives and Nutrient
lU
Sources Added to Food (ANS): Statement on nitrites in meat products. EFSA J
8(5):1538.
na
EFSA (European Food Safety Authority). 2012a. Question number: EFSA-Q-2012-00563.
o
rs
EFSA J 10(7):2831.
Pe
EFSA (European Food Safety Authority). 2012b. Cadmium dietary exposure in the
European population. EFSA J 10 (1):2551.
r
fo
EFSA (European Food Safety Authority). 2012c. Panel on Contaminants in the Food
y
Chain (CONTAM): Scientific opinion on the risk for public health related to the
op
presence of mercury and methylmercury in food. EFSA J 10(12):2985.
C
EFSA (European Food Safety Authority). 2012d. Update of the monitoring of dioxins
’s
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or
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Section III
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Preservation of Foods
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8
Thermal Inactivation Kinetics of
Foodborne Pathogens: An Overview
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Corliss A. O’ Bryan, Nathan A. Jarvis, Philip G. Crandall,
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and Steven C. Ricke
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CONTENTS
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8.1 Introduction ................................................................................................. 271
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8.2 Introduction to Thermal Processing Parameters .................................. 272
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8.3 Models for Thermal Inactivation Kinetics ............................................. 274
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8.3.1 Primary Models.............................................................................. 274
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8.3.1.1 Log-Linear Model............................................................. 274

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8.3.1.2 Weibull Model.................................................................. 275

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8.3.2 Secondary Models.......................................................................... 276

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8.3.3 Tertiary Models............................................................................... 277

8.4 Factors Influencing Thermal Inactivation Rates of
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Foodborne Pathogens................................................................................. 278
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8.4.1 Intrinsic Factors............................................................................... 278

8.4.1.1 Fat....................................................................................... 278
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8.4.1.2 pH ....................................................................................... 279

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8.4.1.3 Water Activity................................................................... 280

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8.4.1.4 Additives........................................................................... 281

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8.4.2 Extrinsic Factors ............................................................................. 282

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8.5 Conclusions.................................................................................................. 283

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Acknowledgments............................................................................................... 283
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References .............................................................................................................284
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8.1 Introduction
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Thermal processing, the use of heat to reduce the microbial populations in

food, inactivates pathogenic and spoilage microorganisms, resulting in an
increased shelf life; halts unfavorable enzymatic activity that causes spoil-
age; and increases the safety of foods (Bermú dez-Aguirre and Corradini
2012; Silva and Gibbs 2012; Kumar and Sandeep 2014). Thermal processing
is one of the oldest forms of preservation used to ensure the microbial
271
safety of food and is often a critical control point (CCP) in the Hazard
Analysis and Critical Control Point (HACCP) system for food processors.
The CCPs are procedures in a food process where a measure of control
is applied as a means to prevent, eliminate, or reduce to an acceptable
level a specific hazard, such as a microbial pathogen (FDA, 2015). Failing
to ensure that a food product has received an adequate level of process-
y
ing to eliminate foodborne pathogens can be very detrimental to a com-
nl
pany, resulting in millions of dollars lost, loss of brand reputation, and
O
most importantly, the potential for illnesses and even deaths. The Centers
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for Disease Control and Prevention (CDC) reports that about 48 million
lU
Americans contract a foodborne illness annually, 128,000 are hospitalized,
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and 3,000 die (Scallan et al., 2011). Thirty-one known microbial pathogens
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cause 20% of these foodborne illnesses and 44% of the deaths; foodborne
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enteric viruses alone cause 58% of these illnesses (Scallan et al., 2011).
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The top five pathogens associated with acquired foodborne illnesses are
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norovirus, nontyphoidal Salmonella , Clostridium perfringens , Campylobacter
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spp., and Staphylococcus aureus (Scallan et al. 2011). The top five foodborne
C
pathogens that most often result in death are Salmonella , Toxoplasma gon-
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dii , Listeria monocytogenes , norovirus, and Campylobacter spp. (Scallan et al.

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2011). It is noteworthy that although norovirus has a very low death rate
ut
(< 0.1%), it causes 5.5 million illnesses per year, leading to 149 deaths per
b
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year (Scallan et al., 2011).

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In order to prevent over- or underprocessing of food, an effective

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thermal process to inactivate foodborne pathogens must be designed and

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the results verified. This relies on an accurate knowledge of the thermal

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inactivation kinetics of the target microorganism. Inactivation kinetics can

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be influenced by many factors, both internal and external to the organ-

c
an
ism. In this chapter, we first describe the kinetics of heat inactivation in

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general and models used to determine processing parameters, and finish

by discussing the factors that influence the kinetics of heat inactivation of
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microorganisms.
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8.2 Introduction to Thermal Processing Parameters

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Traditionally, the kinetics of thermal destruction of bacteria was assumed

to follow first-order kinetics; that is, the rate of death is expected to be
logarithmic (Moats et al., 1971). Parameters that allow the calculation of
the rate of thermal lethality include the D and z values. The D value is
the amount of time in minutes at a given temperature required to destroy
1 log cycle (90%) of the target microorganisms and is considered to be a
measure of the heat resistance of the target microorganism (Heldman and
Hartel, 1997). A greater (longer) D value at a given temperature indicates
Thermal Inactivation Kinetics of Foodborne Pathogens: An Overview 273
a higher thermal resistance for the target organism. To properly calculate

such values, adequate thermal lethality data are needed initially, usually
in the form of reduction of log colony-forming unit (CFU) plotted over time
at a specific temperature. This set of points can be fitted with a line using
least-square calculations. One underlying assumption is that the bacterial
population is homogenous, and that thermal destruction occurs through
y
a single process or single inactivation of a critical site (Moats et al., 1971).
nl
After numerous inactivation trials at several specific temperatures were
O
conducted, the D values obtained at different temperatures were subse-
se
quently converted to logarithmic values and plotted against temperature;
lU
a straight line is fitted to this plot and the z value calculated. The z value
na
represents the temperature (° C) increase required to achieve a 1 log (90%)
o
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reduction in D values (Heldman and Hartel, 1997). Microorganisms that
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have small z values are highly temperature dependent, whereas those
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with large z values require greater changes in temperature to reduce the
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thermal inactivation time.
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Canned foods provide an anaerobic enough environment for the germina-
C
tion of the spores of Clostridium botulinum , which is the causative organism
’s
for botulism. These bacterial spores can survive most heat treatment pro-
or
cesses, but in modern food production, canned foods are subjected to a time
ut
and temperature process specifically designed to reduce the number of the

b
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most heat-resistant C. botulinum spores by 12 logs at 250°F, also known as a

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12-D process (Heldman and Hartel, 1997). The temperature used in the cal-
.C
culation of most commercial 12-D processes is 250°F, and the D value for C.
C
botulinum at 250°F is 0.21 min.

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The F value for a process is the number of minutes required to kill a

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known population of microorganisms in a given food under specified

c
an
conditions (Heldman and Hartel, 1997). This F value is usually set at 12-D
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values to give a theoretical 12 log cycle reduction of the most heat-resis-

tant species of mesophilic spores in a can of food. For example, if there
d
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were 10,000 spores in a can of food and a 12-D process was given, the ini-
or
tial 10,000 spores (104 spores) would be reduced to a theoretical 10 – 8 living
yl
spores per can or, again in theory, 1 living spore per 108 cans of product (1
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spore per 100 million cans). The actual level of C. botulinum spores in cans
18
is more likely on the level of two or three spores per can (Sevenier et al.,
20
2012), so the 12-D processing provides considerable “ overkill,” as attested

to by the food safety record of the low-acid canned food industry. When
©
F is used without a subscript indicating temperature, 250°F is assumed

and a z value of 18°F is presumed. For instance, the F value for killing
C. botulinum spores is 2.52, which means that the spores are deactivated
when submitted to 2.52 m in of heating time at 250° F. The actual process-
ing time a can of food is given in a retort is always greater than the F
value due to heat penetration requirements, come-up to temperature, and
cooling down to the point the retort can be opened (Heldman and Hartel,
1997).
8.3 Models for Thermal Inactivation Kinetics

8.3.1 Primary Models
Models to describe thermal inactivation of bacteria are divided into three
classes: primary, secondary, and tertiary (Marks, 2008). Primary thermal
y
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inactivation models describe changes in the microbial cell population as a
O
function of time at a defined set of conditions. Secondary models describe
se
the effects that changing conditions, such as pH, water activity (aw ), temper-
lU
ature, or other conditions, have on the parameters of the primary models.
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Tertiary models combine the primary and secondary models in an attempt
o
to make them more applicable to the “ real-world” conditions processors
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experience (Marks, 2008). Table 8.1 contains a list of the main primary mod-
Pe
els available. The best-known and most widely used primary model is the
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log-linear model, followed by the Weibull model. These primary models
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have certain advantages, but also have their limitations (Table 8.2).
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8.3.1.1 Log-Linear Model

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The simplest approach to microbial inactivation kinetics is the log-linear

b
equation, which is based on the assumption that there is a negative and lin-
tri
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ear correlation between microbial cell count and lethal treatment or inactiva-
tion rate, as follows: N = N 0 – kt , where N is the cell count at a given time, N 0
.C
is the initial cell count, t is the independent variable (temperature, time, etc.),
C
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and k is the inactivation rate (Esty and Williams, 1924). The idea of a decimal
is
TABLE 8 .1
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Primary Models Used to Describe the Thermal Inactivation Kinetics of Selected

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Foodborne Pathogens
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Model Pathogen References

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Log-linear Listeria monocytogenes Mackey et al., 1990; Juneja et al.,

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1998; Li et al., 2005

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Escherichia coli O157:H7 Ahmed et al., 1995; Teo et al.,

1996; Blackburn et al., 1997
18
Salmonella Humphrey et al., 1991, 1997;

20
Mañ as et al., 2001, 2003; Li et al.,

©
2005
Staphylococcus aureus Kennedy et al., 2005; Li et al,
2005; Hassani et al., 2006a
Weibull L. monocytogenes Hassani et al., 2006b; Ferná ndez
et al., 2007
E. coli O157:H7 Juneja et al., 2015
Salmonella Li et al., 2014
Modified Gompertz L. monocytogenes Huang, 2009; Miller et al., 2009
Shoulder/tail L. monocytogenes , E. coli O157:H7 Geeraerd et al., 2000
TABLE 8 .2
Overview of the Benefits and Limitations of the Most Used Primary Models
Benefits Limits
Weibull Can fit a wide range of trends Cannot be used for nonthermal
(linear, upward, and downward inactivation
curves) Does not predict decay of
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Only two parameters microorganisms under refrigeration
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Can be used for trends with a tail with a prolonged shoulder
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Shoulder/tail Fits data with shoulder and tail Can only use with homogeneous
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Easy to use populations
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Modified Fits data with shoulder and tail Cannot be used with linear trends
Gompertz Useful for a wide range of May lead to over- or underestimation
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physicochemical data of the death rate
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Does not fit data that has a tail but no
shoulder
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Can only be used with homogenous
populations
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reduction (10-fold) value (D ) was introduced by Ball and Olson (1957), who
’s
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developed the most widely known form of the log-linear equation:

but
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t
log N = log N 0 −
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D
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This model implies that all bacteria have the same heat resistance, and
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therefore does not take into account differences at the cellular level (Cole
is
et al., 1993; Geeraerd et al., 2005). This equation is useful for describing inacti-
c
an
vation kinetics in simple systems, such as in laboratory media or when inac-

Fr
tivating a single strain of bacteria.

d
However, many thermal inactivation experiments do not result in a strict

an
linear pattern (Bermú dez-Aguirre and Corrandini, 2012). Often, the experi-

or
mentally collected data have sharp declines, followed by long tailing, over
yl
an extended period of time, or may have a shoulder (no decrease in cell

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count), followed by a sharp decline, or may have two phases of death. Data
18
in published reports can be categorized as having one or two of the five

20
basic shapes: log-linear, upward concavity, downward concavity, biphasic,

or flat shoulder (Ng et al., 1969; Moats et al., 1971; Bermú dez-Aguirre and
©
Corrandini, 2012). This has led to the development of entirely different mod-
els (other than log-linear) for use in calculating thermal processes.
8.3.1.2 Weibull Model

Often, inactivation curves follow Weibull distributions (Abd et al., 2012;
Bermú dez-Aguirre and Corrandini 2012). The Weibull model produces two
terms, α and β (van Boekel, 2002). α , also called the scale parameter, represents
the time function; β is dimensionless and represents the shape of the curve
(van Boekel, 2002). Some scientists choose to use b and n , respectively, as the
parameter placeholders (Peleg and Cole, 1998; Mattick et al., 2001; Corradini
and Peleg, 2009). The underlying premise of the Weibull model is that every
cell in a microbial population possesses its own unique resistance to the
lethal agent (in this case heat), and that resistance can be expressed as the
y
time of exposure until that cell is no longer viable (Peleg, 2006). The Weibull
nl
function can be expressed as
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N 
log  t  = −bt n
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 N0 
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where b is a nonlinear rate parameter, and n is a shape factor (Peleg and
Cole 1998; Mattick et al., 2001). A concave, upward, semilogarithmic inacti-
r
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vation curve occurs when n < 1, a concave downward curve occurs when
y
n > 1, and when n = 1, the Weibull-based model reduces to the log-linear
op
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model. van Boekel (2002) applied the Weibull model to 55 different sets of
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thermal inactivation data and demonstrated that 39 sets displayed n > 1, 14
or
cases exhibited n < 1, and only one set resulted in n = 1. This is an indication
ut
that the log-linear model is probably too simple for a majority of the thermal
b
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inactivation calculations. The primary drawback to the Weibull model is the

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difficulty of assigning biological significance to its parameters (Diels et al.,

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2007).
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8.3.2 Secondary Models

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Numerous papers discuss the impact of variables, especially temperature, on

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inactivation of target microorganisms. Several reviews summarize the heat

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resistance of various pathogenic organisms as influenced by the food matrix,

an
temperature, pH, and aw , among other parameters (Doyle and Mazzotta 2000;
or
Doyle et al. 2001; Jarvis et al., 2016). We present information later in this chapter
yl
to demonstrate the dependence of survival curves and the resulting D values

Ta
on intrinsic and extrinsic factors of the bacteria, as well as the food matrix.
18
Producing aggregate summaries of D values for many foods or conditions

20
reflects this variability and explains, in part, the wide range of values that can
be obtained at any one given temperature. Thus, a secondary model based on
©
many data points that is able to predict D values over a wide range of physi-
cochemical parameters can be constructed (Marks, 2008). However, the pre-
dicted values are limited to the range of parameters on which the models were
built, and the predicted values are simple mean values with no consideration
of variability around those means. Each model would need to be assessed on
its merits with respect to its application and should only be used within the
ranges for which the model has been validated. See Table 8.3 for some of the
parameters that have been researched for certain foodborne pathogens.
TABLE 8 .3
Secondary Models That Describe the Effects of Various Parameters on Thermal
Inactivation of Foodborne Pathogens
Pathogen Parameters Considered References

E. coli O157:H7 Temperature (54.5° C– 64.5° C), pH Blackburn et al. 1997
(4.2– 9.6), NaCl (0.5%– 8.5%)
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Temperature (55° C– 62.5° C), NaCl Juneja et al., 2015
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(0%– 6.0%), sodium pyrophosphate
se
(4%– 8%), pH (4– 8)
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Listeria monocytogenes Temperature (60° C– 73.9° C), NaCl Juneja et al., 2014
na
(0%– 4.5%), sodium pyrophosphate
(0%– 0.5%), SL (0%– 4.5%)
o
rs
Temperature (55° C– 60° C), pH (5– 7), Chhabra et al. 2002
Pe
Milk fat (0%– 5.0%)
Temperature (55° C– 65° C), pH (4– 8), Juneja and Eblen, 1999
r
fo
NaCl (0%– 6%), sodium
y
pyrophosphate (0%– 0.3%) op
Salmonella spp Temperature (60° C– 71.1° C), Juneja et al., 2013
C
cinnamaldehyde (0%– 1%), carvacrol
’s
(0%– 1%)
or
Fat (1%– 12%), temperature Juneja et al., 2001

ut
(58° C– 65° C)
b
tri
on
.C
8.3.3 Tertiary Models

C
LL
A tertiary model is not really a model, but is the integration of a primary

is
and secondary model with a user-friendly interface (Whiting, 1995). There

c
an
are a few tertiary models available that predict the thermal inactivation
Fr
of foodborne pathogens with various input parameters available on the

Internet. The Pathogen Modelling Program (http://pmp.errc.ars.usda.gov/
d
an
aboutPMP.aspx) has models for several pathogens in culture media and

or
in foods under various environmental conditions. ComBase (http://www.

yl
combase.cc/index.php/en/) provides thermal inactivation models for many

Ta
pathogens. Each of these models states that values predicted by the model
18
are not guaranteed to match values that would be obtained in a particular

20
food system; the models would have to be validated with proposed times
and temperatures for that food system.
©
There are numerous parameters that have an effect on the thermal

inactivation rates of pathogens in foods. For example, low aw increases D
values, which helps to explain why the same organism may have substan-
tially different D values in different foods with differing water activities
(Coroller et al., 2001). In subsequent sections of this chapter, we discuss
the many parameters affecting thermal inactivation of foodborne patho-
gens. We do not present a comprehensive review of the literature, as there
are far too many papers published on the subject for us to review here.
However, we present an overview of the literature to illustrate the manner

in which food components and other factors can impact D values.
y
nl
8.4 Factors Influencing Thermal Inactivation
O
Rates of Foodborne Pathogens
se
lU
Generally speaking, the parameters of food that consistently significantly
na
affect measured D values are intrinsic factors of the foods themselves, such
o
as pH, aw , or food additives. Extrinsic factors, those not pertaining to the
rs
food product, also have an effect, including conditions prior to heat process-
Pe
ing, such as the initial temperature of the food before processing and the
r
fo
growth phase of the bacteria.
y
op
C
8.4.1 Intrinsic Factors
’s
or
8.4.1.1 Fat
ut
Increasing fat content has been implicated in increased thermal resistance of

b
tri
pathogens in food (Juneja et al., 2001; Smith et al., 2001; Murphy et al., 2003;
on
Orta-Ramirez et al., 2005). Ahmed et al. (1995) investigated the effects of fat
.C
level on survival of Escherichia coli O157:H7 in ground beef, pork sausage,

C
chicken, and turkey containing various fat levels. They determined that
LL
higher fat levels in all products resulted in greater D values than lower-fat
is
versions of the same products. However, it was noted that higher fat levels
c
an
were also correlated with lower moisture content (e.g., 7% fat pork sausage
Fr
had 73.3% moisture, while sausage with 30% fat had only 50.1% moisture);
d
thus, it is possible that the entire effect was not caused by the increased fat
an
but by a combination of fat and a decrease in water content of the food. In

or
addition, the survivor curves shown are not log-linear, and the authors did
yl
not fully describe the manner in which the D values were derived. Juneja
Ta
et al. (2001) observed that increasing fat concentrations increased the dura-
18
tion of shoulders, as well as D values, for thermal inactivation curves of

20
Salmonella in chicken and turkey, although there was no statistically signifi-

cant effect on z values. In addition, they noted that D values varied between
©
chicken and turkey. L. monocytogenes in high-fat (30%) ground beef exhibited

higher D values than in low-fat (2%) ground beef (Fain et al., 1991). However,
the link between higher fat content and higher thermal resistance was not
consistent. Kotrola and Conner (1997) did not observe any effect over a range
of fat content (3%– 11%) in minced turkey. Addition of fat to ground beef did
not affect the thermal resistance of L. monocytogenes (Mackey et al. 1990), and
increasing fat concentrations up to 10% in a variety of milk products did not
significantly affect the D value of S. aureus (Kornacki and Marth 1989).
He et al. (2013) compared the thermal inactivation of Salmonella in low-fat

and regular fat formulations of peanut butter and found that higher fat and
lower carbohydrate contents actually reduced the heat resistance. Li et al.
(2014) also observed that there was higher heat resistance of Salmonella in the
low-fat peanut butter and peanut spreads. Kataoka et al. (2014), however, did
not observe a difference in survival of Salmonella in peanut pastes related
y
to varying levels of fat. These inconsistent results may be due to the fat not
nl
being equally distributed in the heated medium. Orta-Ramirez et al. (2005)
O
observed lower heat inactivation rates for an eight-serovar Salmonella cock-
se
tail in whole beef muscle compared with ground beef. They postulated that
lU
this was due to bacteria attached to intramuscular fat in whole muscle being
na
better protected by the fat than when the fat was homogenously spread
o
rs
throughout ground beef, thus diluting it. It has also been speculated that
Pe
the protective effects of fat, when they occur, are due to its inherent lower
r
heat conductivity and lower aw compared with other matrixes (Murphy et al.,
fo
2003).
y
op
C
8.4.1.2 pH
’s
or
Numerous studies have indicated that pH has an influence on the thermal

ut
inactivation of bacteria. Adding different concentrations of acetic acid to poul-

b
tri
try scald water, resulting in different pH values, changed the thermal resis-
on
tance of both Salmonella and Campylobacter . High acetic acid concentrations

.C
(low pH) significantly reduced the thermal resistance of these two pathogens
C
(Okrend et al., 1986). Humphrey and Lanning (1987) used sodium hydroxide
LL
to raise the pH of scald water to 9.0 and determined that the high pH also
is
increased the heat sensitivity of Salmonella and Campylobacter jejuni . Teo et al.
c
an
(1996) determined that a high pH acted synergistically with heat to increase

Fr
the lethality of heat on E. coli O157:H7 and Salmonella enteritidis. Blackburn

d
et al. (1997) likewise observed that both Salmonella and E. coli O157:H7 were
an
most resistant to heat at pH values slightly below neutral (5– 7), and both had
or
lower D values at more acidic or basic pH values. Juneja and Eblen (1999)
yl
observed that low pH increased the heat sensitivity of a four-strain cocktail of

Ta
L. monocytogenes inoculated into beef gravy. Yersinia enterocolitica was reported

18
to have D values in egg whites approximately 10 times less at 55° C than in

20
whole eggs or egg yolks (Favier et al. 2008), presumably because the pH of
whites is higher (pH 9.1) than that of yolks (6.5) or whole eggs (7.4).
©
Stringer et al. (2000) reviewed the thermal inactivation data for E. coli
O157:H7 and concluded that the greatest thermal resistance was present at
a pH slightly below neutral (5.2– 5.9), and a pH below or above this range
unquestionably decreased the thermal resistance. Mañ as et al. (2003) also
demonstrated this effect of pH on D values, but found that the z value was
not affected by pH. The type of acid, organic or mineral acid, may play a role
in changing thermal resistance. Juneja and Novak (2003) used a four-strain
mixture of E. coli O157:H7 to inoculate cook-in-the bag ground beef adjusted
to a pH of 4.5 or 5.5 with either lactic acid or acetic acid. The inoculated
ground beef was subsequently cooked at temperatures ranging from 55° C to
62.5° C. For both acetic and lactic acid, the D values were significantly lower
in beef at pH 4.5 than in beef at pH 5.5, and at the same pH levels, E. coli
O157:H7 was more sensitive to heat when acetic acid was used as the acidu-
lant. Milillo et al. (2011) observed that the effectiveness of a multiple-hurdle
y
treatment combining heat and salts of organic acids against Salmonella
nl
Typhimurium depended in part on the degree of organic acid lipophilicity,
O
which is the ability of the acid to dissolve in fat. In their experiments, sodium
se
acetate was less effective than sodium propionate.
lU
na
o
rs
8.4.1.3 Water Activity
Pe
Most reports on the influence of aw on thermal inactivation kinetics con-
r
fo
clude that thermal tolerance is increased at low aw values. Murrell and Scott
(1966) subjected spores of Clostridium botulinum or Bacillus spp. to heating in
y
op
solutions adjusted to various aw values and reported that the greatest heat
C
resistance in these spores was seen at aw values of approximately 0.2– 0.4,
’s
with D 110 values ranging from 2 to 24 h. Goepfert et al. (1970) determined the
or
ut
thermal resistance of selected Salmonella spp. and E. coli in sucrose, fructose,

b
glycerol, and sorbitol solutions adjusted to a range of aw from 0.75 to 0.99 and
tri
observed that thermal resistance increased as the aw was reduced. Alderton
on
et al. (1980) also observed higher D values for C. botulinum 62A spores when
.C
the aw was in the range of 0.1– 0.5 compared with a higher aw of 0.7– 0.9. In con-
C
trast to spores, it was noted that vegetative cells of S . Typhimurium exhibited
LL
maximum heat resistance at an aw in the range of 0– 0.2 for temperatures

is
from 125° C to 160° C; at higher temperature, thermal resistance decreased

c
an
with increasing aw (Corry, 1973).

Fr
A similar pattern has been observed in food products. Lactobacillus plan-

d
tarum and Saccharomyces cerevisiae were inoculated into wheat flour with an
an
aw value in the range of 0.30– 0.50. When the flour was heated at various
or
temperatures, L. plantarum had the greatest heat resistance at an aw of 0.35,

yl
and S. cerevisiae was most resistant at an aw of 0.40, regardless of temperature

Ta
(Laroche et al., 2005). In skim milk, S. cerevisiae had higher D values in the
18
range of aw 0.30– 0.50, and L. plantarum was most resistant in the range of aw
20
from 0.20 to 0.50, regardless of the heating temperature (Laroche et al., 2005).
©
Villa-Rojas et al. (2013) reported the D value of S . Enteritidis PT30 in almond
flour as 0.42 min at 68° C and 0.95 aw , but it increased to 15.2 min at 70° C
when aw was reduced to 0.60 (Villa-Rojas et al. 2013). Foods with a low aw
may need a higher temperature for a longer time to achieve a 5 log reduction
in pathogen populations, which presents a challenge since prolonged heat-
ing times or higher temperatures will typically adversely affect the quality
of the products (Awuah et al., 2007).
8.4.1.4 Additives
Knight et al. (1999) determined the D and z values for L. monocytogenes Scott
A in liquid whole egg with or without nisin (10 μ g/mL). Nisin significantly
decreased D values at lower temperatures (less than 58° C) in both unsalted
and salted liquid whole egg, but nisin had little effect at higher temperatures
(greater than 60° C). However, when they added nisin 2 h before the heat
y
nl
treatment, D values were significantly reduced at the higher temperatures.
O
Mangalassary et al. (2007) evaluated the effect of nisin and/or lysozyme in
se
combination with in-package pasteurization of a ready-to-eat low-fat turkey
lU
bologna on the inactivation of L. monocytogenes . Bologna was inoculated on
na
the surface with L. monocytogenes and subsequently vacuum packaged and
o
heated at temperatures ranging from 60° C to 65° C. The combination of nisin
rs
and lysozyme (a natural enzyme found in egg white), as well as nisin alone,
Pe
increased the sensitivity of L. monocytogenes to heat at 62.5° C and 65° C but
r
fo
not at 60° C; lysozyme alone did not increase sensitivity to heat.
y
Other combinations have been examined as well. The addition of a
op
mixed solution of acidic calcium sulfate and lactic acid to ground beef
C
decreased the thermal resistance of E. coli O157:H7 (Zhao et al. , 2004). The
’s
or
addition of 1% carvacrol and cinnamaldehyde ground beef reduced the

ut
heat resistance of E. coli O157:H7 approximately three- to sixfold (Juneja

b
and Friedman, 2008). Maks et al. (2010) manufactured bologna with differ-
tri
on
ent levels of sodium lactate (SL), sodium diacetate (SD), or a combination

.C
of both and inoculated them with L. monocytogenes . Inoculated bologna

was dipped in pediocin solution and treated at different temperatures. The
C
LL
formulation with SL or SD (not dipped in pediocin) decreased D values of

L. monocytogenes compared with the formulation without additives. Heat
is
c
and pediocin treatment (2500 and 5000 AU) of inoculated bologna without
an
additives decreased D values, but at higher concentrations (≥ 7500 AU), D

Fr
values increased. Combination treatments increased or decreased D val-

d
ues in relation to the temperature. McMahon et al. (1999) also determined

an
that heat resistance of L. monocytogenes in ground beef decreased with the

or
addition of SL up to 4.8%. Likewise, Schultze et al. (2006) and Gupta (2005)

yl
Ta
observed that increasing SL concentrations increased the heat resistance of

L. monocytogenes in frankfurter slurries and frankfurters. However, other
18
researchers noted that D values for L. monocytogenes increase with the addi-
20
tion of SL. Juneja (2003) found that D values for L. monocytogenes in 75%
©
lean ground beef with SL levels ranging from 1.2% to 4.8% heated at 60° C
increased, indicating that SL was in fact protective to L. monocytogenes .
These experiments illustrate the complexity of the interactions between
additives and temperature, which can affect the thermal inactivation of
foodborne pathogens in food products. Consequently, food processors
must modify their product formulations with care, and then verify that the
old thermal process is still sufficient by testing the new formulation.
8.4.2 Extrinsic Factors

The temperature history of the pathogen prior to thermal treatment can
have an impact on thermal inactivation of the pathogen. The exposure of
organisms to heat induces the production of heat shock proteins, which
are generally believed to protect the organism from heat and other stresses
(Lindquist and Craig, 1988). Mackey and Derrick (1986) demonstrated that
y
nl
S . Typhimurium exhibited increased D values when preincubated at 42° C,
O
45° C, or 48° C prior to thermal treatment at 55° C. In addition, estimated
se
times for a 7-D inactivation increased by 2.6- to 20-fold compared with cells
lU
not preincubated before heat treatment. Wiegand et al. (2009) also observed
na
this phenomenon with E. coli O157:H7 inoculated into ground beef. Arroyo
o
et al. (2012) exposed Cronobacter sakazakii cells to heat (47.5° C, 1 h) and cold
rs
(4° C, 4 h) stress conditions before subjecting them to heat (60° C) treatment.
Pe
The heat shock treatment increased D values by 1.6 times over non-heat-
r
fo
shocked cells. Factors other than heat can also induce the synthesis of heat
y
shock proteins, increasing the thermal resistance of pathogens. Morgan
op
et al. (1986) found that hydrogen peroxide induced heat shock proteins in
C
S . Typhimurium. Thermotolerance has also been induced in L. monocyto-
’s
or
genes , E. coli , and Salmonella by prethermal treatment stress from both star-
ut
vation and acid adaptation (Jenkins et al., 1988; Leyer and Johnson 1993;
b
Lou and Yousef, 1996; Rowe and Kirk, 2000; Arsene et al., 2000; Leenanon
tri
on
and Drake, 2001). Yang et al. (2014) also found that lactic acid– adapted S .
.C
Enteritidis at pH values of 5.3 and 6.3 had higher heat resistance than con-
trol cells. However, Milillo et al. (2011) determined that salts of organic acids
C
LL
(sodium acetate or sodium propionate) repressed numerous genes that pos-

sess functions in heat shock response.
c is
Some types of stress, however, have been shown to lower sensitivity to

an
heat treatments in pathogens. E. coli O157:H7 isolates that had previously

Fr
been stressed by chilling or freezing in ground beef were more heat sensi-
d
tive than those that had not been chilled or frozen (Byrne et al., 2002; Zhao
an
et al., 2004). In contrast, Jackson et al. (1996) demonstrated increased heat

or
resistance of E. coli O157:H7 in ground beef patties that had been frozen
yl
Ta
compared with fresh patties. Juneja et al. (1998) reported no change in the
heat resistance of E. coli O157:H7 in ground beef after freezing. These dis-
18
crepancies in results can likely be explained by differences among these

20
experimental techniques; Byrne et al. (2002) closely modeled the actual

©
production environment of ground beef patties, including freezing or tem-

pering of beef trim and frozen storage of patties prior to cooking. In other
studies, E. coli O157:H7 was added to burger ingredients during burger
formation, and thus was only subjected to one freezing and thawing cycle
applied to formed burgers. Alternate cycles of freezing and thawing are
reported to induce greater cell damage than freezing and frozen storage
alone (Frazier and Westhoff, 1988).
8.5 Conclusions
The level of pathogen inactivation required for any particular food will be
dependent on the levels of pathogen initially present, as well as the thermal
resistance of the pathogen. Thermal resistance varies by genus, species, and
y
strain of bacteria, as well as a host of other factors associated with the food
nl
being treated. In our review of the literature, we found minimal standard-
O
ization across foods and between authorities, making it difficult to directly
se
compare reported thermal lethality values. The concentration of pathogens
lU
within a particular food may differ; for instance, raw chicken is more likely
na
to have Campylobacter present than is raw red meat (Zhao et al., 2001). The
o
rs
same foods from different countries may also differ with respect to the pres-
Pe
ence or concentration of pathogens; for instance, some countries have a high
instance of transovarian transfer of Salmonella from the hen to the shell egg,
r
fo
while other countries do not (Lake et al., 2011).
y
op
Numerous factors influence pathogen D values, and many of these factors
C
can have a considerable effect; for instance, changing the aw from approxi-
’s
mately 0.95 to 0.83 can alter the D value for Salmonella by as much as 140-fold.
or
The pH, fat content, and additives in food, as well as conditions experienced
ut
by the food and pathogen prior to heating, all have been shown to markedly
b
tri
effect the D value measured.

on
We have described the effects of various parameters on the thermal inac-

.C
tivation kinetics of foodborne pathogens. This chapter serves to illustrate

C
that D values vary depending on particular characteristics of a food, and as

LL
a consequence, a set of cooking conditions developed for one food may not
is
necessarily be applicable to another. Caution must also be used when using

c
an
published D values, as inactivation kinetics may vary from the first-order

Fr
rates, which are conventionally assumed to apply, and the presence of shoul-
ders and tails might result in greater thermal tolerance than a consideration
d
an
of the first-order D value would imply. Primary inactivation models show a

or
great deal of variability in D values at any given temperature, but allow for
yl
conservative assessments of thermal inactivation to be made. Secondary and

Ta
tertiary models, however, do not tend to reflect known variability. The safest
18
approach is to have specific thermal inactivation experimentally determined

20
by a competent processing authority.

©
Acknowledgments
Nathan A. Jarvis was supported by a distinguished doctoral fellowship from
the graduate school of the University of Arkansas– Fayetteville.
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9
Nonthermal Preservation Technologies
for Meat and Fish Products
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Bruna Leal Rodrigues, Denes Kaic Alves do Rosário,
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and Carlos Adam Conte-Junior
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CONTENTS
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9.1 Introduction................................................................................................. 291
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9.2 UV Radiation............................................................................................... 295
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9.2.1 UV-C Radiation............................................................................... 295
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9.2.2 Pulsed UV Light.............................................................................. 297
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9.3 Gamma Irradiation..................................................................................... 298

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9.4 Pulsed Electric Fields................................................................................. 301

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9.5 Modified Atmosphere Packaging............................................................. 302

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9.6 High Hydrostatic Pressure........................................................................ 307

9.7 Ultrasound................................................................................................... 310
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9.8 Alternative Chemical Compounds........................................................... 312

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9.8.1 Organic Acids.................................................................................. 313

9.8.2 Essential Oils................................................................................... 314
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9.8.3 Natural Antioxidants..................................................................... 315

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9.8.4 Bacteriocins...................................................................................... 316

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9.8.5 Nanoparticles.................................................................................. 318

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9.9 Conclusion................................................................................................... 320

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References.............................................................................................................. 320
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9.1 Introduction
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Most modern lifestyles include different food patterns and expanded knowl-
edge about the relationship between food and health, leading to an increasing
consumer demand for differentiated and safe products as well as to adapta-
tions from the processed food industry (Siró et al. 2008; Sorenson et al. 2011).
Meat and fish, as well as their derivatives, are among the most important
foods that contribute to the essential nutrition of the population due to their
high-quality content of protein, amino acids, vitamins, and lipids (Biesalski
291
2005; Decker and Park 2010; W. Zhang et al. 2010). However, these charac-
teristics result in high perishability, which leads to microbial growth and
metabolite production favoring the deterioration of the food matrix and
increasing the risks of foodborne illness (Rodrigues et al. 2012; Rodrigues
2013; Hygreeva et al. 2014).
In the United States, the Centers for Disease Control and Prevention (CDC)
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estimates that each year, in general, 48 million people are affected by food-
nl
borne illness, wherein 128,000 are hospitalized and 3,000 die. Each year, one
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in six North Americans get sick by consuming contaminated food or bever-
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ages. Raw foods of animal origin, such as raw meat and poultry, raw eggs,
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unpasteurized milk, and shellfish, are the most susceptible type of food to
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be contaminated (CDC 2015a).
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Several recent outbreaks involving meat products were registered in the
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United States. In 2015, a total of 65 people were infected with Salmonella
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Paratyphi (64 persons) and Salmonella Weltevreden (1 person) by eating
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raw tuna (CDC 2015b). In 2014, 12 people were infected with the toxin-pro-
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ducing Escherichia coli O157:H7 Shiga by eating ground beef (CDC 2014a).
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In addition, in 2014, 188 people were infected with Salmonella serotype I
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4,[5],12:i:-, and 8 with Salmonella Infantis after consuming pork meat (CDC
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2014b). Due to the perishability characteristic and the high incidence of

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these matrixes in causing food outbreaks, the use of preservation via ther-
b
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mal and nonthermal methods has been extensively studied and applied
on
(Lopes et al. 2004; Canto et al. 2015; Rodriguez et al. 2015; Simoes et al. 2015;
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Bottino et al. 2016).

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Thermal processing is the most common preservation method used

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in food; it consists of subjecting the food to high temperatures for a few

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seconds or minutes in order to destroy vegetative microorganisms and

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guarantee its microbiological safety (Zuber et al. 2011). However, thermal

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processing can promote undesired reactions and adverse effects, mainly on

the nutritional and sensorial quality, and for this reason, alternative micro-
d
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bial inactivation methods have been developed (Arnoldi 2001). As an alter-

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native to conventional thermal treatments, the mode of action of a number

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of different nonthermal preservation techniques (Table 9.1), including

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shortwave ultraviolet radiation (UV-C) (Bottino et al. 2016), pulsed UV light

18
(Cheigh et al. 2013), gamma irradiation (Monteiro et al. 2013), pulsed elec-
20
tric field (Bekhit et al. 2016), high hydrostatic pressure (HHP) (Canto et al.
2015), modified atmosphere packaging (MAP) (Rodriguez et al. 2014), ultra-
©
sound (US) (Haughton et al. 2012b), and chemical compounds (Mohan and
Pohlman 2016), has been studied to maintain the initial microbial quality of
the product, as well as its fresh characteristics, ensuring the nutritional and
sensory attributes of the matrix. In addition to presenting the capacity to
ensure safety when applied as an individual treatment, different nonther-
mal hurdles have been verified as having promising potential to decrease
the microbial contamination of foods. A recent trend in food preservation
involves the application of a combination of nonthermal processes and/or
Nonthermal Preservation Technologies for Meat and Fish Products 293
TABLE 9.1
Mode of Action of Different Nonthermal Preservation Techniques for Meat and
Fish Products
Nonthermal
Technologies Mode of Action References
UV-C Disruption of DNA and RNA structures Guerrero-Beltrán and Barbosa-
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radiation and impairment of transcription and Cánovas 2004; Koutchma et al.
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replication of microbial DNA 2009
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Pulsed UV Structural and membrane damage of the Heinrich et al. 2016; Ojha et al.
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light cell and DNA injury 2015
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Pulsed Increase of the transmembrane Heinz et al. 2001; Rajkovic et al.
electric field potential, loss of membrane 2010
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permeability, and disruption of the cell
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membrane
Gamma Damage of chromosomal DNA and Acheson and Steele 2001; Farkas
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irradiation nucleic acids that hinders microbial 2006; Odueke et al. 2016
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growth; alteration of microbial cell
membrane by free radicals produced
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by radiolysis of water
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Modified Bacteriostatic action and decrease of Davies 1997; Sivertsvik et al.

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atmosphere microbial metabolism by CO2 action 2002

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packaging and absence of oxygen; CO2 promotes

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intracellular pH changes and

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modification of microbial protein, as

well as the function and enzyme
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structure, in addition to altering cell

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membrane function, fluidity, and

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permeability
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High Damage of cell membranes, alteration of Considine et al. 2008; Campus

c
hydrostatic permeability and cell transport, loss of 2010; Rendueles et al. 2011
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pressure osmotic state, organelle disruption, and

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inability to maintain pH; modification

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of cell components and cellular

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functions
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Ultrasound Physical, mechanical, or chemical Lamminen et al. 2004; Gogate

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damage in cells; generation of and Kabadi 2009; Gao et al.

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high-temperature zones and high 2014; Sango et al. 2014; São José
pressure that damage microbial cell et al. 2014
18
membrane and create reactive

20
compounds that promote rupture and

fragmentation in the DNA of
©
microorganisms
Organic acids Reduction of microbial internal pH Theron and Lues 2007
(Continued)

Mode of Action of Different Nonthermal Preservation Techniques for Meat and
Fish Products
Nonthermal
Technologies Mode of Action References
Essential oils Inhibition of the cell functional and Fisher and Phillips 2006; Bajpai
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lipophilic properties; rupture of cell et al. 2012; Calo et al. 2015
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membrane by phenolic compounds
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Natural Reduction of free radical derived Wojdyło et al. 2007; Kumar et al.
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antioxidants mainly from decomposition of 2015
hydroperoxides
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Bacteriocins Class I bacteriocins (lantibiotics): Hinder Cotter et al. 2005
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the adequate synthesis of the cell wall
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and promote the formation of pores in
the cell membrane
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Class II bacteriocins (non-lanthionine-
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containing bacteriocins): Cause
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depolarization of cell membrane op
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nonthermal processes and conventional methods (pH, temperature, water

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activity, and redox potential), which promotes a synergism effect by a dif-

b
ferent mode of action at the cellular level (Ye et al. 2012; Pietrasik et al.
tri
2016; Rodrigues et al. 2016). In addition to high microbial reduction counts

on
and extended shelf life, this combination reduces the loss of nutrients and
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preserves the sensorial characteristics of the product (Leistner and Gorris

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1995; Leistner 2000).

Among the aforementioned technologies, HPP and gamma irradiation
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have been a success in terms of commercial application (Ortega-Rivas 2012).

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HPP, even with high-cost products, has been greatly appreciated for its com-
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mercial preservation of different types of foods, such as meat, poultry, and

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seafood, as well as juices, beverages, and vegetables, for more than 15 years,
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due mainly to the higher sensory quality of products (Tonello 2010). Similarly,
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several kinds of foods in different countries have been commercially irradi-

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ated by gamma irradiation. In China, 6 categories and 18 kinds of food are

permitted to be processed by irradiation, and in the United States, irradiated
18
ground beef became the first commercially available product. In the year
20
2000, the United States was leading in providing irradiated prepacked ham-
©
burger patties (Ehlermann 2016). Nevertheless, although most nonthermal

techniques are not yet commercially available, many of them are promis-
ing commercial applications, and schemes for use in the food industry have
already been patented (Ortega-Rivas 2012).
This chapter tackles the most important relevant aspects of different non-
thermal methods of food preservation in meat, fish, and their derivative
products, reporting the significance, benefits, and limiting factors of these
technologies to food quality and safety.
9.2 UV Radiation
9.2.1 UV-C Radiation
UV radiation of short wavelength (UV-C) is a nonthermal technology that
has been applied in food industries to superficial bacterial decontamination
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to improve safety and extend the shelf life of different matrixes as an alter-
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native to conventional thermal technologies, which alter the sensory and
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nutritional properties of products (Guerrero-Beltrán and Barbosa-Cánovas
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2004). This technology is already implemented in water, air, and surface
na
decontamination and has been considered a promising technique for food
o
processing, although its use is still limited in meat products (Koutchma et
rs
al. 2009). UV radiation has numerous advantages over several sanitization
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methods due to its not requiring chemicals or heat, not forming residual
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toxic compounds, and not producing an off-flavor, as well as presenting low
y
cost, simple installation, and maintenance, in addition to being applied to
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ready-to-eat and already packaged products (Bintsis et al. 2000; Chun et al.
C
2009, 2010; Rodrigues et al. 2016). In 2000, the Food and Drug Administration
’s
(FDA) approved the use of UV light in food decontamination as an alterna-

or
ut
tive thermal treatment (FDA 2007).

b
UV light works in a large wavelength range on the electromagnetic spec-

tri
on
trum, comprising from x-rays (100 nm) to visible light (400 nm). However,
only the wavelength band between 200 and 280 nm is considered germicidal,
.C
wherein the maximum effect is obtained at wavelength 253.7 nm (Koutchma

C
LL
et al. 2009). The radiation is generated by low-pressure mercury germicidal

lamps, which effectively inactivate viruses, bacteria, protozoans, yeasts, and
c is
algae (Bintsis et al. 2000), of which fungi and yeasts are among the most
an
resistant microorganisms to UV radiation (Wright et al. 2000; Guerrero-

Fr
Beltrán and Barbosa-Cánovas 2004).

d
The mode of action of this technique consists of the absorption of UV

an
light by microbial nucleic acids (with the strongest light absorbers at 253.7
or
nm), which disrupt DNA and RNA structures, leading to inactivation of

yl
Ta
microorganisms. UV radiation induces the formation of cross-linking bonds

between adjacent pairs of thymine and cytosine of the same microbial DNA
18
band, resulting in impairment of transcription and replication in microbial

20
DNA. This damage causes a decrease in the microbial growth rate, as well as
©
the surface microbial load of the product. Therefore, the efficiency of UV-C
in destroying microorganisms is related to the ability of the nucleic acid in
absorbing UV light, which influences the number of cross-linking bonds in
microbial DNA (Guerrero-Beltrán and Barbosa-Cánovas 2004; Koutchma et
al. 2009). In addition, others factors, such as the microorganism species and
strain, microbial density, type and composition of the matrix, geometric con-
figuration of the reactor, transmitted energy, wavelength, physical arrange-
ments of the UV source, and permeability and topography of the product,
may also influence the microbial inactivation of the technology (Wright

et al. 2000; Sastry et al. 2000; Guerrero-Beltrán and Barbosa-Cánovas 2004;
Woodling and Moraru 2005; Chun et al. 2009; Stoops et al. 2013; Silva et al.
2015). Nevertheless, photoreactivation by enzymatic actions can occur when
injured cells are exposed to wavelengths greater than 330 nm (Liltved and
Landfald 2000). The photolyase enzyme monomerizes thymine and cytosine
y
dimers and repairs damage caused by UV-C radiation. Thus, the radiation
nl
dose applied should be studied to ensure that the food is safe for consump-
O
tion (Stevens et al. 1998).
se
Regarding chemical compounds, in general, unsaturated organic molecules,
lU
conjugated chains, and those that present disulfide bonds absorb strongly at
na
254 nm (Koutchma et al. 2009). Therefore, certain protein and aromatic amino
o
rs
acids, such as phenylalanine, tryptophan, tyrosine, and histidine, are highly
Pe
sensitive and could be degraded by UV-C light. In addition, due to promoting
r
modification in protein structures, UV light can lead to changes in solubil-
fo
ity, heat sensitivity, mechanical characteristics, and digestion by enzymes, as
y
op
well as in the physical properties of proteins, which could result in senso-
C
rial alteration and the production of off-flavors in the product (Spikes 1981).
’s
Furthermore, the absorption of UV light by unsaturated organic molecules

or
promotes the generation of free radical by photochemical reactions, resulting

ut
in the oxidation of vitamins, lipids, and proteins; the degradation of antioxi-

b
tri
dants; changes in texture and color; and the formation of undesirable aromas
on
(Koutchma et al. 2009; Lazaro et al. 2014). Moreover, vitamins (A, B12, D, K, B2,
.C
and E), carotenes, folic acid, tryptophan, ascorbic acid, unsaturated fatty acid,
C
and some food pigments are considered “light-sensitive” nutrients, which

LL
could alter the color of food products (Spikes 1981).

is
Several authors have been studying the use of the technique in meat,
c
an
poultry, and fish products. Chun et al. (2009) verified that UV-C radiation
Fr
(8000 J/m2) reduced the populations of Listeria monocytogenes, Salmonella

Typhimurium, and Campylobacter jejuni by 2.74, 2.02, and 1.72 log CFU/g,
d
an
respectively, on inoculated ready-to-eat sliced ham. In inoculated chicken

or
breasts, count reductions of 1.26, 1.29, and 1.19 log CFU/g of C. jejuni, L. mono-
yl
cytogenes, and Salmonella enterica serovar Typhimurium, respectively, were

Ta
observed when a UV-C dose of 5 kJ/m2 was applied (Chun et al. 2010). In
18
addition, the authors observed that the microbiological population decreases

20
with an increase of UV-C radiation. Lazaro et al. (2014) observed a decrease

in initial bacterial load and an extension of the lag phase and commercial
©
validity in chickens subjected to UV-C radiation (1.95 mW/cm2), indicating

that this technique is an efficient alternative to improving the bacteriologi-
cal quality of chicken meat. Moreover, the same authors verified that UV-C
radiation did not increase lipid oxidation, potentially because the exposure
periods were not sufficient to stimulate oxidation.
In fish, there is still little scientific evidence to support the effectiveness of
this technology. Rodrigues et al. (2016) observed lag-phase formation in meso-
philic and psychrotrophic groups in rainbow trout fillets exposed to UV-C
radiation (106.32 mJ/cm2) and attributed the results to cellular injury caused
by technology, which requires longer microbial adaptation. Furthermore,
UV light caused no significant changes in lipid oxidation values, poten-
tially because the low UV-C dose and exposure time were not able to pro-
mote oxidation. Bottino et al. (2016) verified that a low radiation dose (55.83
mJ/cm2) and high radiation dose (160.97 mJ/cm2) were effective in reducing
y
the growth rate, as well as the number of colonies in the stationary phase of
nl
Enterobacteriaceae, and the mesophilic and psychrophilic bacteria count of
O
natural microbiota of tambacu (Colossoma macropomum × Piaractus mesopota-
se
micus). In addition, the same authors reported that UV-C radiation enhanced
lU
the shelf life of fish fillets by at least 50%. In inoculated bullfrog meat, Silva et
na
al. (2015) studied the effect of UV-C radiation on inactivation of Staphylococcus
o
rs
aureus. In all doses applied (0.65, 1.04, and 1.68 W/cm2) and independent of the
Pe
exposure time, a reduction of 3.0 log CFU/g was observed.
r
fo
y
9.2.2 Pulsed UV Light op
C
Pulsed UV light technology is based on the emission of short (10 –3 to 102 μs)
’s
and high-power UV pulses that cause microbial inactivation (Heinrich et al.

or
2016). This technology was approved by the FDA for application in food dur-
ut
ing production, processing, and handling steps (Palmieri and Cacace 2005;
b
tri
FDA 1996; Heinrich et al. 2016.). The mechanism of action can be defined as
on
photochemical, which leads the cell death due to DNA injury; photothermal,
.C
which causes microbial inactivation through structural damage of the cell

C
(vaporization of water); and photophysical, which promotes cell membrane

LL
injury due to vacuole expansion and protein elution (structural damage)

is
(Ojha et al. 2015; Heinrich et al. 2016).

c
an
The greater bactericidal effect of pulsed UV light is related to higher power

Fr
values at lower pulse durations (Dunn 1996; Heinrich et al. 2016). The main
d
factors that influence the efficiency of pulsed light are the type of matrix,
an
the quantity of microorganisms, and the process parameters, such as light

or
intensity (Heinrich et al. 2016).

yl
Pulsed light is widely studied, and its effectiveness has already been
Ta
proven in meat and fish products. Cheigh et al. (2013) evaluated the effect
18
of several different doses (0.11–1.75 mJ/cm2 per pulse), number of pulses

20
(0–9800 pulses), treatment times (0–1960 s), and total fluences (0–17.2 J/cm2)
in shrimp, salmon, and flat fish fillets. Among conditions, the treatment with
©
1.75 mJ/cm2 per pulse, 3600 pulses, 720 s, and total fluence of 6.3 J/cm2 pro-
duced reductions of 2.2, 1.9, and 1.7 log CFU/g of L. monocytogenes in shrimp,
salmon, and flat fish fillets, respectively, as well as reductions of 2.4, 2.1, and
1.9 log CFU/g of L. monocytogenes, respectively, to each product, when the
conditions of 6900, 1380 s, and total fluence of 12.1 J/cm2 were applied. In
vacuum-packaged salchichón and dry-cured loin, the application of pulsed
UV light in the fluence 11.9 J/cm2 reduced L. monocytogenes counts in 1.81
and 1.61 CFU/cm2, as well as Salmonella Typhimurium in 1.48 and 1.73 CFU/
cm2, respectively. The changes in color, odor, and flavor to salchichón were
not significant when fluences of 0.7, 2.1, 4.2, 8.4, and 11.9 J/cm2 were applied.
In dry-cured loin, the fluences of 8.4 and 11.9 J/cm2 promote color altera-
tion, as well as odor and flavor changes, in comparison with the control sam-
ples, respectively (Ganan et al. 2013). In pork and salmon, the pulsed light
treatments at 3 and 10 J/cm2 (30 pulses) did not induce lipid peroxidation
y
(Nicorescu et al. 2014).
nl
O
se
lU
ona
9.3 Gamma Irradiation
rs
Pe
Gamma irradiation is a nonthermal process of preservation that increases
r
food safety by exposing the matrix to ionizing energy generated by a source
fo
of radioactive material (Li and Farid 2016). Ionizing radiation consists of
y
op
high-energy photon transmission through irradiation sources such as radio-
C
nuclide cobalt-60 and x-rays from machine sources (energies up to 5 MeV),
’s
which possess high penetrating power in food; radionuclide cesio-137, which

or
is allowed by some legislation but is not likely to be used due to difficulties

ut
in managing the isotope; or accelerated electrons (energies up to 10 MeV),

b
tri
which present limited penetration depth in food but can be used in grain
on
on a conveyor or low-density foods, such as ground spices, in addition to

.C
decreasing superficial contamination of prepared meals (CAC 2003; Lee

C
2004). The technique has been used for many years for different purposes,
LL
such as medical diagnosis support and sterilization of medical supplies (Lee

is
2004). However, only in 1921 was there the first indication of using gamma
c
an
irradiation to preserve meat with the inactivation of Trichinella in the pork

Fr
matrix (Diehl 2002). Among them, cobalt-60 is the main ionizing radiation
d
source; it is commonly used in commercial radiation processing. It can be

an
used at a maximum of 10 kGy in food so as not to provide toxicological or

or
nutritional human risk (WHO 1981; Lee 2004).

yl
Gamma irradiation can be classified into radappertization, radicidation,

Ta
and radurization, depending on the doses of irradiation applied (Jay et al.

18
2005; Fellows 2009). Commercial applications in foods generally use the

20
radicidation or radurization process, which consists of the elimination of

spoilage and pathogenic microorganisms, as well as microbial vegetative
©
forms, molds, and yeasts, utilizing doses between 3.0–10.0 kGy and 0.1–2.5
kGy, respectively, extending the shelf life and ensuring the safety of the food
products (Gould 1986; Jay et al. 2005). Due to the high penetration power of
gamma rays, it can be used to treat products even after packaging condi-
tions, preventing recontamination (Farkas 2006).
The direct mechanism of microbial inactivation by gamma irradiation is
mainly due to the damage of chromosomal DNA and nucleic acids, which
make the multiplication of microorganisms impossible, whereas indirect
action involves free radical production, caused by radiolysis of water, which

can alter the cell membrane of microorganisms (Acheson and Steele 2001;
Farkas 2006; Odueke et al. 2016).
The radiation sensitivities of microorganisms depend on many factors,
such as the number of microorganisms present, the strain resistance, the
DNA repair ability to radiation injury, their chemical and physical structure,
y
the amount of radiation applied, the food type, the pH and temperature of
nl
the medium, the moisture content, and the presence or absence of oxygen
O
(Lee 2004; Farkas 2006). Generally, molds and vegetative bacteria present the
se
same radiation sensibility, whereas fungi with melanized hyphae exhibit
lU
a radiation resistance similar to that of bacterial spores (Saleh et al. 1988).
na
Vegetative cells are more sensitive than bacterial spores due to possessing
o
rs
more moisture in their composition (Molins 2001; Fellows 2009). Dutra et
Pe
al. (2016) verified their condition in mortadella, when, after the product was
r
subjected to inoculation (107 spores/g of Clostridium botulinum), irradiation
fo
(10 kGy), and cooking, the count of spores reached 6.28 log CFU/g after 48 h
y
op
of storage (4°C). According to Lewis (1990), a dose of 48 kGy is necessary to
C
achieve a reduction of 12 D of C. botulinum spores. Yeasts and viruses also
’s
show high radiation resistance (Saleh et al. 1988; WHO 1999). The tempera-
or
ture of the medium affects the efficiency of irradiation, wherein low tem-
ut
peratures solidify water content, reducing radiolysis (Molins 2001). The dose
b
tri
is the main factor that interferes with the decontamination of food, wherein
on
higher doses are more effective in microorganism reductions (Kang et al.

.C
2016).
C
The effectiveness of gamma irradiation is already consolidated in the

LL
scientific literature. Since the 1990s, several studies using the individual
is
application of gamma irradiation in the inactivation of pathogens in meat

c
an
have been carried out. Thayer and Boyd (1993) obtained a reduction of 1
Fr
log CFU/g of E. coli O157:H7 in mechanically deboned chicken meat using

a low dose of 0.27 kGy. In ground beef, Clavero et al. (1994) found that the
d
an
dose of 2.5 kGy was sufficient to inactivate 3.1, 8.1, and 10.6 log CFU/g of
or
pool Salmonella serotypes, E. coli O157:H7, and C. jejuni, respectively. Fu et al.

yl
(1995) found in cooked pork chops and cured hams that low doses of 0.75–
Ta
0.9 kGy reduced 2.0 log CFU/g of L. monocytogenes and 1.0–3.0 log CFU/g of
18
Salmonella Typhimurium, as well as doses of 1.8–2.0 kGy, taking the patho-

20
gen counts to undetectable levels.

Decontamination of fresh fish using gamma irradiation was conducted by
©
Huque et al. (2013), who obtained a reduction of up to 5 log CFU/g of natural

microbiota using a dose of 5 kGy. Badr (2012), evaluating the effect of gamma
irradiation on the reduction of L. monocytogenes and Vibrio parahaemolyticus
in cold-smoked salmon, verified a reduction of 6.59 and 6.05 log CFU/g using
doses of 3 and 1 kGy, respectively. In ready-to-eat chicken breasts, irradiation
(48 kGy) was able to reduce 5.30 log CFU/g of aerobic mesophile bacteria to
undetectable levels, even though this dose is above that permitted by law
(Baptista et al. 2014a).
In addition to bacteria, other microorganisms, such as viruses, have been

studied to assess the effectiveness of alternative methods of food preserva-
tion, since they are also highly involved in many foodborne diseases. Kang et
al. (2016) demonstrated the effectiveness of gamma irradiation in the inactiva-
tion of murine norovirus-1 (MNV-1) using doses of 3, 5, 7, and 10 kGy, which
exhibited reductions of 0.59, 0.88, 1.36, and 1.81 log PFU/mL in semidried
y
squid, respectively, requiring a dose greater than 10 kGy to obtain reductions
nl
higher than 2 log. Although up to the present there is a high efficiency in
O
microbial decontamination, irradiation can cause chemical changes in foods,
se
which enables high hydroperoxide production in foods with a high lipid con-
lU
tent, generating off-flavors in the food matrix (Fellows 2009; de Oliveira Silva
na
et al. 2015). This factor was verified by Ayari et al. (2016), who observed an
o
rs
increase in lipid oxidation (thiobarbituric acid reactive substance [TBARS])
Pe
and the concentration of peroxides in ground beef subjected to a dose of 2
r
kGy. However, the lipid oxidation did not differ from control samples when
fo
the gamma irradiation was combined with 1.47% (w/w) cinnamaldehyde,
y
op
an extract bioactive agent of cinnamon, and 0.5% (w/w) ascorbic acid. Kang
C
et al. (2016) also observed an increase of the oxidation index after applying
’s
irradiation doses of 3, 5, 7, and 10 kGy in semidried squid. In addition, the

or
irradiation process promotes a slight breakdown of proteins that contain sul-

ut
fur amino acids, generating undesirable off-flavors. However, Fregonesi et

b
tri
al. (2014) verified that lamb loin cuts (Longissimus dorsi) subjected to 1.5 and
on
3.0 kGy were sensorially acceptable on day 0, after the irradiation process.
.C
Similarly, the irradiation dose of 25 kGy applied in ready-to-eat chicken meat

C
did not affect the acceptability (Feliciano et al. 2014). Furthermore, high-
LL
molecular-weight carbohydrates could also be fragmented in smaller units.

is
However, in general, essential amino acids and fatty acids, minerals, and
c
an
trace elements are preserved under irradiation conditions, whereas some

Fr
vitamins (C and B1) are in part lost (Kilcast 1995).

Chemical metabolites can also be reduced by application of gamma irra-
d
an
diation during storage, due to their effectiveness in decreasing spoilage

or
microorganisms. High irradiation doses (48 kGy) are able to reduce the con-
yl
centration of cadaverine in ready-to-eat chicken breast fillets during stor-

Ta
age (Baptista et al. 2014b). In addition Özogul and Özden (2013) verified that
18
gamma irradiation (2.5 and 5.0 kGy) decreased putrescine, cadaverine, and
20
trimethylamine contents, whereas it increased spermine, agmatine, and

tryptamine concentrations during storage of irradiated sea bream (Sparus
©
aurata). Gamma irradiation was also proved to reduced ochratoxina A by

18.78% in fermented sausage using the dose of 3.07 kGy, as well as decreas-
ing by 25.20% and 33.66% the mycotoxin content in smoked ribs and bacon,
respectively, when the dose of 10 kGy was used (Domijan et al. 2015).
Gamma irradiation has demonstrated efficacy against vegetative cells
when used alone; however, recent studies have demonstrated an improved
effect when it is applied in combination with other preservation methods.
Gamma irradiation (2 kGy) combined with 1.47% cinnamaldehyde (w/w)
provided an apparent reduction of 5.37 log CFU/g of mesophilic bacteria in

ground beef at day 0 of storage, whereas when applied alone, it had a reduc-
tion of only 2.58 log CFU/g of mesophilic bacteria. In addition, when gamma
irradiation was applied in combination with cinnamaldehyde, the meso-
philic and psychrotrophic bacteria count reduction reached undetectable
levels on day 12 of storage, whereas the count reduction of molds and yeasts
y
reached undetectable levels in day 0 of storage (Ayari et al. 2016). Huq et al.
nl
(2015) also demonstrated that L. monocytogenes was more sensitive to gamma
O
irradiation (1.5 kGy) when combined with microencapsulated oregano, cin-
se
namon essential oil, and nisin in cooked ham.
lU
na
o
rs
rPe
fo
9.4 Pulsed Electric Fields
y
op
Electric fields were used for the first time between 1920 and 1930 in the United
C
States in a pasteurization process referred to as electropure (Sitzmann 1995).
’s
It was the first attempt to inactivate microorganisms through electric fields;

or
nevertheless, the capacity to inactivate microorganisms in food was only

ut
demonstrated in 1960 (Vega-Mercado et al. 1997; Jeyamkondan et al. 1999).

b
tri
Pulsed electric fields are based on the generation of electric field pulses of
on
short duration (1–100 µs) and high intensity (10–50 kV/cm) from electrodes,
.C
wherein foods are placed (Vega-Mercado et al. 1997). The application of elec-
C
tric fields results in disruption of the cell membrane and loss of membrane
LL
permeability caused by an increase in the transmembrane potential (Heinz

is
et al. 2001; Rajkovic et al. 2010). The opposite charges formed between cell
c
an
membranes induce an increase of the electric field and generate a compres-

Fr
sion pressure on the membrane. The attraction of opposite charges between

both sides of the cell membrane (animal, vegetable, and microbial) results in
d
an
pore formation (electroporation), which generally occurs when the intensity

or
of electric fields is larger than the critical threshold of transmembrane poten-

yl
tial (Wan et al. 2009; Soliva-Fortuny et al. 2009).

Ta
The efficiency in the inactivation of microorganisms in foods depends on

18
the ability of the matrix to sustain high electric fields and electrical conduc-
20
tivity. Therefore, in liquid foods such as juice, eggs, and milk, the application
of pulsed electric fields to microbial decontamination has been successfully
©
proven (Monfort et al. 2012; Espina et al. 2014; Walter et al. 2016). In meat, low
efficiency is verified mainly to present a high content of lipids and proteins,
which decreases the effectiveness in microbial inactivation. This effect was
confirmed by Haughton et al. (2012a), who verified that application of pulsed
electric fields (3.75 and 15 kV/cm, 10 s, 5 Hz) in raw chicken did not signifi-
cantly reduce the total viable cell count, Enterobacteriaceae, C. jejuni, E. coli,
or Salmonella enteritidis. Bolton et al. (2002) also reported the ineffective action
of this technology in reducing E. coli O157:H7 counts in beef burgers.
In meat products, the pulsed electric fields have been used to improve the
physical and chemical quality. Bekhit et al. (2016) applied a specific pulsed
electric field (10 kV, 90 Hz, 20 μs) in one, two, and three repetition cycles
in hot-boned beef loins (M. longissimus lumborum) and topsides (M. semi-
membranosus) and verified tenderness reduction when three repetition
cycles were performed. However, an increase in proteolysis of troponin T
y
was seen to the largest extent when one repetition cycle was applied. Arroyo
nl
et al. (2015) evaluated the impact of the electric field pulsed effect on turkey
O
breast meat processing under various conditions (100, 200, and 300 pulses;
se
7.5, 10, and 12.5 kV; 10, 55, and 110 Hz, 20 μs), and no significant changes were
lU
observed in weight loss, cook loss, lipid oxidation, texture, and color (raw
na
and cooked) of fresh or frozen samples. The same authors studied sensory
o
rs
changes in turkey breast meat after applying the treatment of 12.5 kV and
Pe
300 pulses, and verified odor and texture changes in relation to the control
r
sample. In cooked lamb meat, no undesirable sensory changes were verified
fo
when they were subjected to an electric field strength of 1–1.4 kV/cm, specific
y
op
energy of 88–109 kJ/kg, frequency of 90 Hz, pulse width of 20 μs, and pulse
C
number of 964 (Ma et al. 2016). Suwandy et al. (2015) applied pulsed electric
’s
field treatments (5 and 10 kV; 20, 50, and 90 Hz) in boned beef longissimus
or
lumborum and semimembranosus and obtained a shear force reduction by

ut
up to 19% for both muscles during storage. Moreover, treatment intensity did
b
tri
not affect the reduction of shear strength in longissimus lumborum, whereas

on
the reduction in semimembranosus was frequency dependent.

.C
C
LL
cis
an
9.5 Modified Atmosphere Packaging

Fr
d
The food packaging contains the product, allows consumers to evaluate it

an
from a hygienic and convenient point of view, serves as a marketing and

or
attractive tool for consumers, and protects the products against deteriorative
yl
factors acting as a barrier between the food and environment (Renerre and
Ta
Labadie 1993; Yam et al. 2005). Actually, packaging not only provides the
18
traditional protection characteristics but also enables different functions for

20
the food packaged (Han 2005).

The permeability of packaging material to atmospheric gases such as oxy-
©
gen and carbon dioxide, as well as to water vapor, is a crucial property that
interferes with the microbiology and shelf life of the product. MAP for meat
and fish products demands a gas barrier packaging for the maintenance of the
gas mixture required during storage time. The gas permeability is variable
with matrix and application. Due to this, the transport of oxygen through a
packaging film must be studied (Jakobsen et al. 2005). In food MAP, the main
polymers used are low-density polyethylene (LDPE), high-density polyethyl-
ene, polypropylene, polytetrafluoroethylene, and polyamide (Han 2005).
MAP is a technique used to increase the shelf life of products through

altering the atmosphere inside the package, which is achieved by removal of
oxygen (O2) or replacement of the atmosphere surrounding the product with
a different gas mixture before sealing in a vapor and gas barrier package
(McMillin et al. 1999; Cutter 2002). The ability to extend shelf life has been
reported since 1920 in apples and 1930 in beef carcasses when high levels of
y
carbon dioxide are used (Davies 1995). Therefore, an increase in consumer
nl
demand for fresh and convenience foods with minimal use of preservatives
O
led to a market expansion of MAP products, which comprise raw and pro-
se
cessed meat, poultry, fish, cheese, vegetables, and others (Masniyom 2011;
lU
Shariat et al. 2013; Jääskeläinen et al. 2016; Wang et al. 2016).
na
Several different MAP conditions are described in the literature, and
o
rs
vacuum packaging (VP) is considered the most common and simple way of
Pe
atmosphere modification (Davies 1995). This technique consists of remov-
r
ing the atmospheric air inside of low-oxygen-permeability packaging, which
fo
is subsequently sealed, leaving residual concentrations (Masniyom 2011).
y
op
The microbial metabolism and respiration of the product result in changes
C
during storage, as well as consumption of the residual oxygen, which is
’s
replaced by carbon dioxide, that appears in higher concentrations (Cutter

or
2002). The absence of atmospheric air in the package results in control of

ut
microbial growth, mainly psychrotrophic aerobic spoilage bacteria, which

b
tri
reduce slime appearance, rancidity, and undesirable discoloration (Salgado

on
et al. 2007). In addition, this condition favors the development of faculta-

.C
tive anaerobic bacteria, such as lactic acid bacteria (LAB), which decrease
C
the growth rate of spoilage organisms and delay deterioration (Genigeorgis

LL
1985). The efficiency of technology in muscle food varies according to the

is
initial microbial load, the types of microorganisms, the storage temperature,

c
an
and the integrity of packaging (Cutter 2002).

Fr
Different from vacuum packaging, MAP consists of removing air by

vacuum and replacing it with a single or different gas mixture with vary-
d
an
ing combinations, mainly oxygen (O2), nitrogen (N2), and/or carbon dioxide
or
(CO2), before sealing the gas-impermeable packaging (Masniyom 2011). In

yl
this condition, the gaseous atmosphere continuously changes during storage

Ta
and no additional manipulation is needed in the surrounding environment

18
of the product (Sivertsvik et al. 2002), which is the opposite of controlled

20
atmosphere packaging (CAP), wherein continuous monitoring is needed to

maintain a stable atmosphere, as well as temperature and humidity, inside
©
the package (Davies 1995; McMillin et al. 1999). As previously discussed, the
gaseous environment is altered in MAP due to the respiration rate of product
and microbial growth, wherein generally oxygen is consumed and carbon
dioxide and water vapor are formed, achieving a stable environment (Brody
1989; Ooraikul 1991).
The main gas responsible for bacteriostatic action in MAP is CO2, which,
in concentrations ranging from 20% to 60%, is responsible for inhibiting a
variety of spoilage organisms, such as Pseudomonas spp., Acinetobacter spp.,
and Moraxella spp., in addition to also having fungistatic properties (Church

and Parsons 1995; Sivertsvik et al. 2002; Malavota et al. 2006). This gas is
solubilized in an aqueous and lipid medium and forms carbonic acid, which
reduces the pH of the product. The pH acidification increases the lag phase
and decreases the growth rate during the log phase, inhibiting microbial
growth (Farber 1991; Devlieghere and Debevere 2000). However, the inhibi-
y
tory effect of CO2 depends on the sensibility of the microorganism and
nl
growth phase, temperature, water activity, and product characteristics. The
O
action of this gas on bacteria culminates in intracellular pH changes; modi-
se
fication of the physicochemical properties of microbial protein, as well as of
lU
the function and enzyme structure; in addition to lead to alterations of the
na
cell membrane function, fluidity, and permeability, delaying the deteriora-
o
rs
tion process (Davies 1997; Sivertsvik et al. 2002). The CO2 solubility in water
Pe
and lipids is potentiated with a decrease of temperature, as well as the con-
r
centration in the food being directly related to the food water and fat content,
fo
in addition to CO2 atmosphere pressure (Ho et al. 1987; Sivertsvik et al. 2002).
y
op
Despite the efficient microbial inactivation action, fish samples subjected to
C
high CO2 MAP conditions tend to present increasing lipid oxidation values.
’s
This behavior occurs due to denaturation of muscle proteins performed by

or
CO2, with consequent liberation of iron, which is a prooxidant component

ut
(Rodrigues et al. 2016). In addition, the compression of food by a gas mix-

b
tri
ture generally results in meat exudation, and the depletion of O2 promotes

on
changes in meat color regarding the decreased conversion of myoglobin to

.C
metmyoglobin (Church and Parsons 1995).

C
The gas mixture depends on matrix characteristics such as the amount of

LL
lipids and myoglobin concentration. The presence of O2 stimulates oxida-

is
tive changes in fat foods, resulting in the production of aldehydes, ketones,

c
an
and alcohols, as well as consists of an important factor in the storage of

Fr
red meats, since O2 complexes with myoglobin (oxymyoglobin), conferring

the bright red color of meat products, which is well accepted by consumers
d
an
(Masniyom 2011). Because of this, O2 gas is generally avoided by MAP of

or
high-fat products, including fish, whereas a proportion of 80% O2 and 20%

yl
CO2 is the most common gas mixture in red meat products (Eilert 2005;
Ta
Masniyom 2011).
18
N2 is an inert and insipid gas that presents low solubility in water

20
and lipids, and is commonly used to replace O2 gas as an alternative to

vacuum packaging or to prevent package collapse due to CO2 absorp-
©
tion by a product (Masniyom 2011). In addition, it decreases the oxidative

rancidity, as well as the rate of microbial growth, especially mesophilic
bacteria (Church and Parsons 1995). However, other gases, such as argon,
xenon, and nitrous oxide (N2O), have been used in MAP conditions, reduc-
ing microbial growth and maintaining the initial quality of the product
(Zhang et al. 2008).
The efficiency of VP and MAP in meat and fish products has been proven
by several studies. Jääskeläinen et al. (2016) investigated the influence of
packaging (vacuum and high-oxygen atmosphere of 80% O2 and 20%

CO2) on the growth of microbial communities and metabolic activities in
chilled beef, wherein they verified that high-oxygen MAP favored hetero-
fermentative species and increased the production of bacterial metabolites,
such as volatile organic compounds, whereas VP favored the growth of
homofermentative species. An off-odor was verified in beef stored under
y
high-oxygen MAP, caused by diacetyl and acetoin formation, in addition
nl
to increasing oxidation of unsaturated fatty acids due to O2 action. The
O
beef conserved in high-oxygen MAP was spoiled more than 10 days ear-
se
lier than beef under vacuum packaging. However, oxygen is usually used
lU
in fresh meat packages to stabilize the color of meat, and when a greater
na
proportion of O2 in red meat products is desired (Eilert 2005; Martínez et
o
rs
al. 2005; Belcher 2006). Wang et al. (2016), studying the changes of lamb
Pe
microbiota under VP and under low-CO2 (20% CO2/80% N2), middle-
r
CO2 (60% CO2/40% N2), and high-CO2 (100% CO2) MAP, verified that the
fo
high-CO2 environment was most effective in inhibiting microbial growth,
y
op
mainly LAB, Pseudomonas spp., and Enterobacteriaceae, delaying changes
C
in the microbial community composition and extending the shelf life by
’s
approximately 7 days compared with VP samples. However, the VP group

or
exhibited a more desirable color than the MAP groups due to metmyoglo-
ut
bin formation, which could be formed at very low O2 concentrations, veri-

b
tri
fied in MAP (>0.3% O2) at the initial stage. In pork and beef minced meat
on
(50%/50%), MAP with a middle-CO2 (20% O2/50% CO2/30% N2) environ-

.C
ment decreased in large proportion the total Enterobacteriaceae count in

C
relation to vacuum packaging and a low-CO2 (20% O2/30% CO2/50% N2)

LL
environment (Djordjevic et al. 2016). Rodrigues et al. (2016) verified that

is
growth rate of mesophilic and psychrotrophic bacteria and the colony-

c
an
forming units in the stationary phase, in addition to the total mesophilic

Fr
count, were reduced. Furthermore, MAP reduced the total production of

ammonia, total volatile basic nitrogen (TVB-N), and putrescine, delaying
d
an
chemical changes. This technology enhances the shelf life of rainbow trout
or
fillets by at least two times. Accordingly, Turan and Kocatepe (2013), study-
yl
ing the shelf life of cultured sea bass packaged in air, vacuum, and MAP
Ta
at different gas percentages (75% CO2/25% N2, 60% CO2/40% N2, and 30%
18
O2/30% CO2/40% N2), verified that the gas proportions of 75% and 60%
20
CO2 were, in general, more effective in total mesophilic and psychrophilic

aerobic bacteria, as well as Pseudomonas spp., count reduction than other
©
packaging conditions, whereas vacuum was the most effective method for
preventing lipid oxidation. Erkan et al. (2007) verified that the production
of chemical compounds was reduced, as well as the growth of microbial
counts in samples of filleted chub mackerel package under vacuum and
MAP (5% O2/70% CO2/25% N2), extending the shelf life by 9 days for air
and vacuum-packaged fish and 12 days for MAP fish.
Active packaging consists of the incorporation of specific compounds
into packaging systems that interact with package material, food, and
the environment, as well as allow other functions to be performed that

promote the extension of commercial validity (Quintavalla and Vicini 2002;
Aymerich et al. 2008). The isolated use of active packaging and the combined
application with other technologies, including MAP, have been extensively
studied to improve the efficacy of techniques (Camo et al. 2008; Shin et al.
2010; Bolumar et al. 2016; Remya et al. 2017). As a branch of active packag-
y
ing, smart packaging recognizes the food properties or environment and
nl
informs the consumer about the conditions of the product, allowing actions
O
to protect it (Kerry et al. 2006). The principal active packaging systems con-
se
sist of oxygen scavengers or absorbers, moisture absorption, carbon dioxide
lU
or ethanol generation, and antimicrobial applications. However, active pack-
na
aging is very important as an antimicrobial tool in food packaging research
o
rs
(Coma 2008).
Pe
Several forms of antimicrobial applications in food have been described.
r
Among them are the direct application of the substance into the package
fo
film; the use of a sachet previously connected with the package that releases
y
op
the compound during the storage period; and through a coating of the pack-
C
aging, in which the matrix acts as a vehicle of substance that can allow the
’s
migration of the compound under food superficially and through the use of
or
bioactive edible coatings that are directly placed into the food (Cooksey 2001;
ut
Aymerich et al. 2008).

b
tri
Remya et al. (2017) prepared an active antimicrobial packaging film from

on
chitosan incorporated with ginger (Zingiber officinale) essential oil. The

.C
authors verified that the addition of essential oil improved the antimicro-
C
bial activity of the chitosan film with no interference in its properties. The
LL
presence of ginger in the chitosan film demonstrated antibacterial property

is
against gram-positive bacteria and a maximum inhibition zone to S. aureus

c
an
and E. coli, wherein the antimicrobial activity was concentration dependent

Fr
for both microorganisms. In fresh lamb steaks, three different conditions of

natural antioxidants (packaging with rosemary active film, packaging with
d
an
an oregano active film, and meat surface sprayed with rosemary extract
or
packaging in a high-oxygen atmosphere) enhanced the oxidative stability of

yl
the products. However, active films with oregano were more efficient than
Ta
those with rosemary, presenting an effect similar to that of the sprayed

18
application of rosemary extract and extending the shelf life of steaks (Camo
20
et al. 2008). Raeisi et al. (2016) studied the effects of shallot (Allium ascaloni-
cum L.) fruit and ajwain (Trachyspermum ammi [L.] Sprague) seed extracts on
©
semifried coated rainbow trout (Oncorhynchus mykiss) fillets and observed

that lipid oxidation and microbiological growth were delayed in treated
samples, and the sensory quality improved. The ajwain seed extract (3%)
presented the best antioxidative and antimicrobial activities, followed by
the shallot fruit extract (3%). Based on the results, the authors recommended
the use of these extracts as natural preservatives for extending the shelf life
of fish products.
9.6 High Hydrostatic Pressure

High Hydrostatic Pressure (HHP), also known as ultra-high-pressure (UHP)
or high-pressure processing (HPP), is a novel nonthermal preservation tech-
nique that consists of subjecting packaged food to general water pressures
y
(from 100 to 1000 MPa) at room temperature, and is compressed in a man-
nl
ner independent of product geometry and size (Aymerich et al. 2008; Guyon
O
et al. 2016). The pressure chamber is loaded, closed, and degassed, and the
se
pressure is isostatically (uniform and instantaneous) transmitted over the
lU
recipient, following Pascal’s law and the principle of Le Chatelier (Hugas
na
et al. 2002). In commercial applications, the pressure level most often used
o
rs
is between 400 and 600 MPa, depending on the product, wherein for each
Pe
100 MPa, an increment of only 3°C is observed, totaling 15°C for a 600 MPa
condition (Aymerich et al. 2008). This processing technology has been used
r
fo
to extend the commercial validity of fresh and processed food products with
y
op
minimal interference to the sensory and nutritional characteristics (Marcos
C
et al. 2013).
’s
Although it was identified as a microbial decontamination technology in

or
1899, only in 1990 was HHP applied industrially in food products in Japan,
ut
and since then, other countries, such as the United States, Italy, Spain,
b
tri
Germany, and Australia, have been using HHP in meat products (Aymerich
on
et al. 2008). HPP processing is considered the best nonthermal process tech-
.C
nique to improve the microbial and technological quality of meat and deriva-
C
tives (Simonin et al. 2012), and it is a potential seafood conservation method

LL
(Yagiz et al. 2007). This technology is used for “cold pasteurization” and
is
commercial sterilization of different products due to its ability to destroy

c
an
spoilage and pathogenic microorganisms and inactivate vegetative microbial

Fr
cells (Rendueles et al. 2011; Guyon et al. 2016). HPP has already been listed by
the U.S. National Advisory Committee on Microbiological Criteria for Foods
d
an
as a nonheating pasteurization technology that can substitute for the pas-

or
teurization method, and the FDA and the U.S. Department of Agriculture
yl
have already approved its use in foods (Barbosa-Cánovas and Juliano 2008).
Ta
However, the resistance of the microorganisms is very variable, depending

18
on the strain barotolerance, the microbial growth stage, and the matrix com-
20
position, as well as the intensity, temperature, and exposure time subjected

to the HPP process (Hugas et al. 2002). The HPP microbial inactivation occurs
©
through a combination of factors that start by damaging cell membranes,

leading to permeability and cell transport alteration, loss of osmotic state,
organelle disruption, and inability to maintain pH (Campus 2010). In addi-
tion, other cell components, such as ribosome, and cellular functions, such as
protein synthesis and enzyme activity, are modified or inhibited (Considine
et al. 2008; Rendueles et al. 2011). Eukaryotic microorganisms are generally
more sensitive to pressure than prokaryotic ones. Pressures ranging from
200 to 400 atm can damage the cell wall, which also seems to be the primary
inactivation factor for yeasts. Regarding prokaryotic microorganisms, gram-
positive bacteria are considered more resistant than gram-negative bacteria
(Hoover et al. 1989), as well as cocci in relation to bacilli due to cell mor-
phology (Huang et al. 2014). In addition, psychrotrophic bacteria are more
sensitive than mesophile bacteria since it seems that heat or pressure treat-
y
ment decreases the ability of growth at low temperature (Garriga et al. 2004).
nl
Furthermore, during the stationary phase, the resistance of microorganisms
O
is higher than during the exponential phase. In the stationary phase, the
se
cell structure and membranes are completely formed and protein synthesis
lU
occurs, which protects the cell against adverse conditions and enhances the
na
stress tolerance (Hill et al. 2002; Patterson 2005). Food matrix composition
o
rs
can also alter the efficacy of the process since it can promote a protective
Pe
effect against pressurization. Meat products that present low water activity
r
and high fat, high protein, and high solute concentrations tend to increase
fo
the barotolerance of microorganisms and decrease the extent of inactivation
y
op
and recovery of damaged cells during storage time (Cheftel and Culioli 1997;
C
Rendueles et al. 2011; Szerman et al. 2011; Canto et al. 2015). In addition, the
’s
degree of inactivation also depends on the level of pressure applied (inten-

or
sity and exposure time) and the temperature during the process. The impair-
ut
ment of cellular processes and structures by HPP occurs mainly the range
b
tri
of 50–300 MPa. In general, the application of 50 MPa leads to inhibition of

on
protein synthesis in microorganisms, 100 MPa causes protein denaturation,

.C
and 200 MPa promotes damage of cell membranes and structures, whereas
C
a pressure of 300 MPa or more causes irreversible denaturation of proteins

LL
and enzymes, as well as cellular membrane rupture, leading to cell death

is
(Abe 2007; Huang et al. 2014). Nevertheless, nucleic acids and DNA struc-
c
an
ture are relatively resistant and stable under commercial pressure (Patterson
Fr
2005). Generally, temperatures above room temperature and a decrease

below room temperature increase the inactivation of microorganisms by the
d
an
HPP process (Butz and Tauscher 2002). The most important hurdle used is
or
the temperature that enhances the microbial vegetative cell inactivation and
yl
allows bacterial spore inactivation. The structure and thickness of bacterial

Ta
spores make them barotolerant, resisting pressure applications above 1000

18
MPa (Cheftel 1995). The inactivation is carried out through the combination
20
of pressurization and high temperatures, close to 100°C. High or moderate

temperatures and pressure cycles consist of technological alternatives in
©
spore inactivation (Huang et al. 2014). The repeated pressure cycles promote
rapid decompression, leading to higher injury and disruption, inactivating
the germinated spores (Nguyen Thi Minh et al. 2010).
The main property of food material packaging for the HPP process is the
flexibility of film to support compression pressure, since the food is gener-
ally packaged prior to high-pressure conditions. At least one side of the pack-
aging should be flexible, and the headspace in the packaging must be low
as possible to guarantee the efficient transmission of pressure and prevent
the damage and rupture of material (Torres and Velazquez 2005; Han 2007).
Currently, flexible packaging, such as polypropylene, polyester tubes, poly-
ethylene, and nylon pouches, is used in HPP applications (Han 2007).
The effectiveness of HPP technology has been reported in different
matrixes on the inactivation of several pathogenic and deterioration micro-
organisms. Garriga et al. (2004), studying the behavior of spoilage and patho-
y
genic microorganisms after application of pressure (600 MPa for 6 min to
nl
31°C) and during chilled storage, verified that HPP is an efficient method for
O
delaying the growth of spoilage microbiota in sliced vacuum-packed cooked
se
ham, dry-cured ham, and marinated beef loin, as well as for controlling risks
lU
associated with Salmonella spp. and L. monocytogenes in sliced marinated
na
meat. In agreement, Bover-Cid et al. (2015) verified that the inactivation of L.
o
rs
monocytogenes and the lethality process in dry-cured ham increases accord-
Pe
ing to the rising of the pressure applied and the water activity, reaching a
r
maximum inactivation of about 7 logs (852 Mpa) in moderate aW (0.92) and
fo
6.8 logs (600 MPa) in high aW (0.96). In rainbow trout, a pressure of 300 MPa
y
op
effectively inactivated the initial microbial count by a 6 log reduction, and
C
in mahi-mahi, by about a 4 log reduction. In addition, microbial growth was
’s
significantly retarded after HPP application (Yagiz et al. 2007). In oyster, HPP
or
was effective for the control of V. parahaemolyticus and V. vulnificus, and the
ut
pressure of 300 MPa for 2 min at 21°C, followed by 5 days of storage in ice or
b
tri
7 days of frozen storage, eliminating V. parahaemolyticus in whole-shell oys-

on
ter and improving the microbial safety (Ye et al. 2013). In ground chicken, a
.C
five-isolate cocktail of Salmonella spp. was inhibited by more than 5 and 8 log
C
CFU/g when pressures of 450 and 550 MPa, respectively, were applied for 10
LL
min, whereas low pressures of 250 and 350 MPa in a single cycle inactivated
is
0.5 and 1.7 log CFU/g, respectively. When multiple cycles were applied (three
c
an
cycles with 5 min/cycle), the reduction counts rose to 1.3 and 3.3 log CFU/g,
Fr
respectively (Sheen et al. 2015). Similar, Kruk et al. (2011) also observed an
effective reduction of Salmonella Typhimurium, E. coli, and L. monocytogenes
d
an
in a chicken breast fillet when the pressures of 450 MPa (4–8 log CFU/g
or
reduction count for 3–14 days) and 600 MPa (6–8 log CFU/g reduction count
yl
for 7–14 days) were applied.

Ta
However, in order to minimize the undesirable changes caused by ultra-

18
high hydrostatic pressures (above 400 MPa), recent studies have proposed
20
the application of low-pressure (100–200 MPa) or moderate-pressure (50–300

MPa) applications, isolated and combined with hurdle technologies with
©
HPP, in meat and meat products (Bajovic et al. 2012). Canto et al. (2012) veri-
fied that especially low-pressure (200 MPa) applications can have positive
effects on the color and texture of alligator meat, wherein the lowest modi-
fication of lightness and redness was observed, as well as decreased hard-
ness. Rodríguez-Calleja et al. (2012) combined HHP (300 MPa) with a liquid
antimicrobial edible coating and MAP and observed that sensory param-
eters, color, and tenderness were maintained during storage of a chicken
breast fillet. In beef (M. pectoralis profundus), a low-pressure level (200 MPa)
promoted minimal alteration in meat quality parameters (lower impact on

color parameters, cooking loss, and lipid oxidation values), whereas increased
pressure levels resulted in color changes, as well as increased cooking loss
and lipid oxidation (McArdle et al. 2010).
In addition to HPP promoting microbial damage, this technology is
associated with altering the structure of high-molecular-weight molecules
y
such as carbohydrates and protein. The secondary, tertiary, and quaternary
nl
structures of proteins are changed by electrostatic and hydrophobic inter-
O
actions, as well as the large hydration of macromolecules, which leads to
se
the denaturation of proteins and disruption of cell structures, resulting in
lU
enzyme inactivation and changes in the structure and texture of products
na
(Butz and Tauscher 2002; Kato et al. 2002; Campus 2010). These character-
o
rs
istics could be used for the development of new products or increment-
Pe
ing the functionality of some ingredients (Hugas et al. 2002; Guyon et al.
r
2016). Moreover, this technique promotes changes in texture parameters,
fo
such as the tenderness, hardness, juiciness, and water holding capacity of
y
op
raw meat, wherein tenderness tends to decrease and hardness increase.
C
However, the changes in texture attributes vary with the temperature and
’s
pressure applied (Guyon et al. 2016). In addition to the texture, the color of
or
raw meat can also be modified since HHP causes globin denaturation. In
ut
general, HHP induces a whitening effect, promoting an increase in light-

b
tri
ness (L*) and a decrease in redness values (a*), which is dependent on the
on
pressure level and time of duration, as well as temperature (Marcos et al.

.C
2010; Bajovic et al. 2012). Due to this, the effect of high pressure in raw
C
meat is more drastic when compared with cooked and cure products, since
LL
the proteins with temperature action are denatured (Bajovic et al. 2012).
is
Furthermore, smaller molecules, such as vitamins, pigments, and flavor

c
an
compounds, as well as chemical changes, are minimal, which guarantees

Fr
the nutritional, chemical, and sensory quality of the products and becomes
a differential in relation to thermal technologies of the food industry (Smelt
d
an
1998; Campus 2010).

or
yl
Ta
18
20
9.7 Ultrasound
©
US consists of sound waves with a frequency that exceed the capacity of

human hearing (>20 kHz) (Knorr et al. 2011). This technology was discov-
ered by Pierre Curie in 1880, and in 1894, Thornycroft and Barnaby noted the
generation of bubbles and cavities in water by vibration of US (Martines et al.
2000). At the beginning of the twentieth century, US application technologies
were developed, focused primarily on surface cleaning procedures, and only
in 1920 did this technique begin to be used in food processing and preserva-
tion (Turantaş et al. 2015). Although there have been many applications and
great developments, US science is still considered a new technology for food

decontamination (São José et al. 2014).
In general, US applications are classified as low energy (low power or low
intensity), which includes frequencies above 100 kHz (Sango et al. 2014),
and high energy (high power or high intensity), which comprises frequen-
cies between 18 and 100 kHz (Knorr et al. 2004; Kentish and Ashokkumar
y
2011), the form most used for meat application (Turantaş et al. 2015). The high
nl
intensity can lead to physical, mechanical, or chemical damage in cells, such
O
as physical disruption and acceleration of certain chemical reactions (Mason
se
2003; Jayasooriya et al. 2004; Brilhante São José and Dantas Vanetti 2012;
lU
Golmohamadi et al. 2013).
na
High-power US has been used for many years for different purposes and
o
rs
has been highlighted in food preservation as a promising technique due to
Pe
its potential to rupture cells and disperse aggregate materials that could
r
inactivate enzymes and microorganisms (Suslick et al. 1999; Knorr et al.
fo
2004; Arzeni et al. 2012). When high-power US is applied, acoustic cavita-
y
op
tion is generated and longitudinal waves are created, causing alternate areas
C
of compression and expansion. In the expansion cycle, an increase in small
’s
bubbles of a liquid medium occurs due to the local pressure reduction of

or
liquid vapor pressure, whereas in the compression phase, the bubble surface
ut
area is reduced. The alternating movements promote implosion of the bub-

b
tri
bles, generating high-temperature zones (up to 5000°C) and high pressure

on
(up to 1000 atm) (Patist and Bates 2008; Mukhopadhyay and Ramaswamy
.C
2012). The high pressure and the implosion lead to microject formation that
C
can damage the cell membrane of microorganisms (Lamminen et al. 2004;

LL
Sango et al. 2014). During the implosion, reactive compounds such as free
is
radicals and hydrogen peroxide can be formed, generating an oxidative

c
an
power (Gao et al. 2014) and causing damage to microbial DNA through rup-
Fr
tures and fragmentation along its length (Gogate and Kabadi 2009; Gao et al.
2014; São José et al. 2014).
d
an
However, the efficacy of US varies with the properties of the medium, the
or
time, and the intensity of treatment, as well as the medium temperature and
yl
pH, which alter the sensibility of microorganisms (Chandrapala et al. 2012).

Ta
In general, gram-positive bacteria are more resistant to the cavitation effect

18
than gram-negative bacteria due to the presence of a strongly adherent and

20
thick peptidoglycan layer (Drakopoulou et al. 2009; Chandrapala et al. 2012).

Although US increases the microbial safety and extends the shelf life of
©
food products, this technology can promote meat softening through rupture
of muscle tissue and acceleration of enzymatic reactions (Jayasooriya et al.
2004; Alarcon-Rojo et al. 2015). Tenderness consists of the most important
parameter that attracts consumers and is effectively promoted by US with-
out causing changes in appearance (Ortega-Rivas 2012). In addition, it main-
tains the initial quality of the matrix regarding nutritional, sensorial, and
functional characteristics, which is different from the thermal process (Cao
et al. 2010; O’Donnell et al. 2010; Bhat et al. 2011).
The effectiveness of US in food preservation is evidenced in different

matrixes. Kordowska-Wiater and Stasiak (2011) observed a decimal reduc-
tion of 2.27 log CFU/cm2 for E. coli and 1.02 log CFU/cm2 for S. enterica on
the skin surface of chicken wings treated with US (40 kHz, 2.5 W/cm2, 20°C,
6 min). In addition, US used individually achieved similar efficiency decon-
tamination in relation to traditional technologies (chlorine compounds and
y
organic acids) currently used at the industrial level. Caraveo et al. (2015),
nl
using US (40 kHz, 11 W/cm2) on bovine semitendinosus muscle, also verified
O
the efficiency of this technology in reducing coliform, mesophilic, and psy-
se
chrophilic bacteria.
lU
Although US is a consolidated technology when applied alone, the combi-
na
nation of this technique with other methods of preservation has improved its
o
rs
antimicrobial effect. The efficacy of US (25 kHz, 200 W, 10.53 min, 74°C) in
Pe
combination with moderate heat (82°C, 16 min) was verified by Cichoski et
r
al. (2015), who observed growth inhibition of lactic acid and psychrotrophic
fo
bacteria and reduction of lipid oxidation in sausages analyzed over 60 days of
y
op
storage. The US allowed the reduction of time and temperature pasteurization.
C
The combination of high-power ultrasound (HPU) and high-pressure
’s
carbon dioxide (HPCD) on cooked ham caused a synergistic effect in the

or
reduction of the natural microbiota in comparison with HPCD used alone.

ut
HPCD + HPU (12 MPa, 45°C, 40 kHz, 10 W) was able to reduce the count of
b
tri
LAB and of molds and yeasts to undetectable levels at 15 and 4 min, respec-
on
tively. To achieve the same efficiency for mesophilic and LAB inactivation, 60
.C
and 45 min of HPCD application (12 MPa, 45°C) were required, respectively
C
(Ferrentino and Spilimbergo 2016). Sara et al. (2014) also studied the combi-
LL
nation of HPCD + HPU (12 MPa, 35°C, 30 kHz, 10 W) and verified the effi-
is
ciency of the inactivation of L. monocytogenes on dry-cured ham, as well as a

c
an
reduction of this pathogen to an undetectable level. In addition, no changes

Fr
were detected for pH, acidity, color, and sensory attributes, compared with
heat treatment. Haughton et al. (2012b), using US and moderate heat (53.1°C,
d
an
20 kW/L), observed a microorganism count reduction of Campylobacter (4.77

or
log CFU/g) and Enterobacteriaceae (3.39 log CFU/g) to undetectable levels in

yl
poultry skin. Moreover, a decimal reduction of 3.36 log CFU/g to total viable
Ta
counts was obtained. In pork, Morild et al. (2011) achieved decimal reduc-
18
tions of 2.0, 2.1, and 2.5 log CFU/cm2 for S. enterica serovar Typhimurium,
20
Yersinia enterocolitica, and E. coli, respectively, after the application of US, heat,
and pressure (30–40 kHz, 130°C, 3.5–5 atm) for 2 s.
©
9.8 Alternative Chemical Compounds

The application of chemicals has been extensively used in the food industry
as a method of preserving the quality and increasing food security, where the
most common compounds used include chlorine, chlorine dioxide, acidified

sodium chloride, and cetylpyridinium chloride (Oyarzabal 2005; Turantaş et
al. 2015). Chlorine was discovered in 1774 (Rutala and Weber 1997), and its
large utilization is related mainly to its oxidative effect, which can inactivate
bacteria, mold, yeast, and viruses. However, it has been proven that chlorine
compounds can form carcinogenic and mutagenic substances, in addition
y
to being highly toxic and not biodegradable (Rutala and Weber 1997; Pérez-
nl
Gregorio et al. 2011).
O
Therefore, there is a tendency to eliminate the use of these compounds
se
in food disinfection, since some European countries have banned their
lU
use in organic food and specific products due to the potential risks to
na
public health (Ölmez and Kretzschmar 2009; Turantaş et al. 2015). In addi-
o
rs
tion, the increase in consumer demand for natural products has stimu-
Pe
lated the substitution of synthetic compounds by substances from natural
r
conserving agents, such as antioxidants, which extend the shelf life of
fo
food products. Based on this context, the use of alternative compounds
y
op
in food preservation is promising. Among the alternative compounds for
C
meat decontamination, organic acids, essential oils, natural antioxidants,
’s
bacteriocins, and nanoparticles have been included in substances, which

or
promote efficient microbial inactivation and delay spoilage and chemical

ut
changes.
b
tri
on
.C
9.8.1 Organic Acids

C
Organic acids are designated by the FDA as generally recognized as safe

LL
(GRAS) for meat products, in which lactic, acetic, propionic, citric, and
is
ascorbic acid are included (Ölmez and Kretzschmar 2009; Mani-López et al.
c
an
2012). These substances are used to prevent food spoilage and increase the
Fr
shelf life, as well as to reduce pathogenic microorganisms that cause food-

borne diseases (Conte-Junior et al. 2010). The mechanism of action is based
d
an
on reducing the internal pH of the microorganisms through the presence

or
of dissociated acid compounds, which are toxic to the cell. In addition, this
yl
acidification distances the optimal intracellular pH range to enzyme activ-

Ta
ity and consequently blocks the protein, as well as DNA/RNA synthesis.

18
Furthermore, there is a high ATP consumption to return to the initial pH,

20
which contributes to the inactivation of microbial cells (Eklund 1985; Ricke

2003; Theron and Lues 2007). In meat decontamination, there are several
©
factors that influence the maximum efficiency of organic acid application,

including the type and amount of organic acid, the tissue type, the bacteria
strain, and the decontamination method used (Smulders 1995; Theron and
Lues 2007).
In order to verify the influence time and type and concentration of organic
acid application, Zaki et al. (2015) evaluated the effectiveness of lactic and
acetic acid at concentrations of 1% and 2%, for 1–3 min in the inactivation
of S. enterica serovar Kentucky in chicken skin immersed in acid solutions.
One percent lactic acid caused 2.05 and 3.36 log CFU/g reductions, while
2% caused 3.24 and 5.01 log CFU/g reductions, at 1 and 3 min, respectively.
One percent acetic acid caused 1.96 and 1.99 log CFU/g reductions, while 2%
caused 2.90 and 2.53 log CFU/g reductions, at 1 and 3 min, respectively. The
authors demonstrated that the time and concentration factors were important
to the efficiency of lactic acid action, while only time was important for acetic
y
acid. In beef trimmings, Mohan and Pohlman (2016) demonstrated a reduc-
nl
tion of approximately 5.0 log CFU/g of E. coli O157:H7 to undetectable levels
O
when applying caprylic acid (30 g/L) solution. Tango et al. (2014) verified
se
that the antimicrobial effect of fumaric acid (0.5%) caused reductions of 2.12,
lU
2.01, 1.81, and 1.60 log CFU/g of L. monocytogenes, E. coli O157:H7, S. enterica
na
serovar Typhimurium, and S. aureus, respectively, in fresh beef immersed in
o
rs
the decontamination solution. In Nile bolti fish immersed in solutions con-
Pe
taining acetic acid (1%) and citric acid (3%) for 5 min, a reduced lipid oxida-
r
tion (TBARS), compared with the control treatment, resulted after 12 days of
fo
storage under refrigeration. Both the acetic and citric acids are effective for
y
op
the preservation of fish (El-Shemy et al. 2015). Similarly, in fresh fish samples,
C
a high reduction in lipid oxidation, as well as in natural microbiota growth,
’s
was observed, compared with control samples, when immersed in a solution

or
containing chitosan incorporated into citric acid (Qiu et al. 2014). In pork pat-
ut
ties, Hwang et al. (2016) found that 0.05% ascorbic acid decreases lipid and
b
tri
pigment oxidation after 12 days of storage. Zhu et al. (2016) evaluated the
on
effect of different concentrations of organic acids (lactic and citric acid) on

.C
the physicochemical and sensory characteristics of drumstick chicken and

C
verified that there were no significant changes in pH, TVB-N, and sensory
LL
acceptance.
c is
an
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9.8.2 Essential Oils

d
Essential oils are aromatic oily liquids obtained from plant source (flowers,
an
buds, seeds, leaves, twigs, bark, herbs, wood, fruits, or roots) by different
or
extraction methods, wherein the steam distillation method is the most com-
yl
Ta
monly used (Burt 2004; Calo et al. 2015). The first official record of obtain-
ing essential oils was by Villanova (1235–1311) (Vergis et al. 2015), and since
18
then, the use of essential oils has demonstrated efficacy in increasing the
20
shelf life of meat products (Karabagias et al. 2011; Jayasena and Jo 2013). An
©
estimated 3000 essential oils are known, and several natural properties have
been reported, such as antibacterial, antiviral, antimycotic, antiparasitic, and
antioxidant, in addition to insecticidal potential (Vergis et al. 2015). The anti-
microbial activity or other biological functions are related to the presence
of bioactive volatile components (Mahmoud and Croteau 2002). However,
although the antimicrobial effect has been studied in several microorgan-
isms, their mechanisms of action are not yet fully understood (Cox et al. 2000;
Calo et al. 2015). In general, the inactivation mechanism can be attributed to
its ability to penetrate the cell and inhibit its functional and lipophilic prop-
erties. In addition, phenolic compounds can rupture the cell membrane and
cause loss of internal contents of the cell (Fisher and Phillips 2006; Bajpai et
al. 2012; Calo et al. 2015). The effectiveness of microorganism inactivation
depends on the type of oil used and the concentration of the target microor-
ganism (Vergis et al. 2015; Ghabraie et al. 2016).
y
Raw chicken fillets immersed in essential oil solutions were investigated
nl
by Khanjari et al. (2013). The authors verified that 1% of oregano essential oil
O
increased the shelf life 6–10 days when compared with the control sample
se
and reduced L. monocytogenes by 1.5 log CFU/g. In addition, when these com-
lU
pounds are combined with N,O-carboxymethyl chitosan, the counts of L.
na
monocytogenes (initial infection of 7 log CFU/g) are reduced to an undetect-
o
rs
able level after 4 days of storage. Furthermore, the effect of oregano essential
Pe
oil and N,O-carboxymethyl chitosan maintained desirable characteristics of
r
taste and odor of raw chicken fillets during storage when compared with the
fo
control. Pesavento et al. (2015) found that the growth of L. monocytogenes and
y
op
S. aureus was restricted in beef meatballs when 1% and 2% of essential oils,
C
respectively, were used. In ready-to-eat meat products, fir essential oil (1%
’s
v/w) and qysoom essential oil (1% v/w) were spread onto the surface matrix.
or
Fir essential oil decreased to 3.09, 5.04, and 6.34 log CFU/g at 0, 7, and 9 days
ut
of storage the initial counts of 3.14, 6.18, and 6.90 log CFU/g of L. monocyto-
b
tri
genes, respectively, and qysoom essential oil (1% v/w) had a similar effect.
on
However, the antilisterial effect was improved when the oils were combined,
.C
reducing to 3.10, 4.11, and 5.53 log CFU/g the counts of this pathogen at 0, 7,
C
and 9 days of storage, respectively (Awaisheh 2013). Similarly, the essential

LL
oil obtained from Satureja horvatii was also reported as an efficient inhibitor
is
agent of the growth of L. monocytogenes when incorporated in pork meat,

c
an
in addition to improving the meat’s color and flavor after 4 days of storage
Fr
(Bukvički et al. 2014).

d
an
9.8.3 Natural Antioxidants

or
yl
Antioxidants are substances that retard the oxidation of easily oxidizable

Ta
biomolecules, such as lipids and proteins that increase the product shelf life
18
(Karre et al. 2013). Synthetic antioxidants, such as butylated hydroxyanisole

20
(BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ),

©
and propyl gallate (PG), have been used in meat products; however, they
possess toxicological potential (Raghavan and Richards 2007; Naveena et
al. 2008; Karre et al. 2013). For this reason, the demand for natural antioxi-
dants has increased. These compounds can be found in different parts of
the plant, such as grains, fruits, nuts, seeds, leaves, roots, barks, and aril.
Most of the natural antioxidants are phenolic compounds, wherein the most
important comprise the tocopherols, flavonoids, and phenolic acids (Kumar
et al. 2015). These substances can be extracted by traditional methods, such
as Soxhlet extraction, including solid–liquid and liquid–liquid extraction.

However, several disadvantages have been described, such as high solvent
consumption, high time extraction, and degradation of thermolabile com-
pounds, which encourage the study and application of new methods, such as
subcritical extraction (Vicente et al. 2013; Veggi et al. 2014; Kumar et al. 2015).
Among foods, some fruits and other vegetables have become excellent
y
sources of natural antioxidants, due to the high content of phenolic com-
nl
pounds (Sebranek and Bacus 2007; Nuñez de Gonzalez et al. 2008). The antiox-
O
idants vary according to their chemical structures, which results in different
se
mechanisms of action. However, the ability of the antioxidant compound to
lU
donate a hydrogen (H+) and reduce the free radicals, present at the beginning
na
of the oxidation reaction or during the decomposition of hydroperoxides, has
o
rs
been described as the major mode of action of these substances (Wojdyło et al.
Pe
2007; Kumar et al. 2015). One of the most important factors that influence the
r
effectiveness of the compound is the number of polymer structures, wherein
fo
the greater the number of OH groups, the greater the antioxidant capacity
y
op
(Ursini et al. 2001). In addition, the concentration of the antioxidant and the
C
composition of the food, especially the lipid content, also influence the effi-
’s
cacy of the compound (Karre et al. 2013; Shi et al. 2014; Kumar et al. 2015).
or
Numerous studies have been conducted to verify the antioxidant potential

ut
of many fruits (plum, grape seed extract, cranberry, pomegranate, and bear-
b
tri
berry) and plants (pine bark extract, rosemary, and oregano) added into meat
on
and poultry products (Lee et al. 2006; Brannan 2008; Nuñez de Gonzalez et al.
.C
2008; Karre et al. 2013; Shi et al. 2014). Gadekar et al. (2014) used sodium ascor-
C
bate (500 ppm) and alpha-tocopherol acetate (10 ppm) added into goat meat
LL
and verified that the color attributes were enhanced with the addition of anti-
is
oxidants, and the amount of free fatty acids were below those in the control.
c
an
In addition, natural antioxidants decreased lipid oxidation during storage and

Fr
improved the sensory attributes of appearance and taste. In silver carp fillets,
Shi et al. (2014) reported that the grape seed (GSE) and clove bud (CBE) extracts,
d
an
when used separately, were effective in retarding lipid oxidation and reducing
or
color stability during storage. However, GSE was more effective in reducing
yl
the lipid oxidation, due to its high concentration of procyanidin (69.06%), the
Ta
antioxidant compound with the most potential. Kim et al. (2013) evaluated the
18
effectiveness of green leafy vegetable extracts in raw beef patties and verified
20
that the microbial count decreased with the addition of the extract, wherein the
higher inhibition effect was achieved with the higher extract concentration. In
©
cooked chicken meat, Sampaio et al. (2012) demonstrated that the antioxidant
effects of oregano, sage, and honey, added as ingredients, reduced lipid oxida-
tion when compared with the control treatment.
9.8.4 Bacteriocins
Bacteriocins can be defined as small peptides produced by bacteria that
present heat stability and high-specificity antimicrobial activity. The use of
bacteriocins produced by LAB is the most common in foods, and nisin is

already regulated by the Joint Food and Agriculture Organization–World
Health Organization Expert Committee (Cotter et al. 2005). LAB bacterio-
cins are also expected to be safe enough to be considered GRAS bacteria.
Furthermore, synthetic bacteriocins can be digested by proteases, having
little or no influence on the intestinal microbiota (Zendo 2013; Woraprayote
y
et al. 2016).
nl
Cotter et al. (2005) suggested different types of bacteriocin classifications,
O
according to their mode of action: Class I bacteriocins (lantibiotics) and
se
Class II bacteriocins (non-lanthionine-containing bacteriocins). In lantibi-
lU
otics (lanthionine-containing antibiotics), the bacteriocin (e.g., mersacidin)
na
can be connected to the main conveyor peptidoglycan subunits, preventing
o
rs
adequate synthesis of the cell wall and leading to cell death. In addition,
Pe
bacteriocin can also act as a docking molecule that generates the formation
r
of pores in the cell membrane, resulting in rapid cell death (e.g., nisin). In
fo
Class II, peptides present an amphiphilic helical structure, which allows
y
op
insertion into the cell membrane, causing depolarization and death. Nisin is
C
the only bacteriocin considered by the FDA as a GRAS compound that can
’s
be applied to meat, poultry, ready-to-eat meat products, and sausage pack-

or
ing (Woraprayote et al. 2016; FDA 2000). Pediocin has been widely studied in
ut
meat and meat products; however, its application has not yet been regulated.
b
tri
The effectiveness of nisin and pediocin depends on several factors, such as

on
the sample preparation method; bacteriocin assay conditions; and pH, tem-
.C
perature, and concentration of NaCl in food, as well as the initial microbial

C
count (Cintas et al. 1998; Martinez et al. 2013).

LL
Bacteriocins can be used in meat and meat products in three main ways,
is
including inoculation of LAB bacteriocin–producing cells, direct application

c
an
of purified or partially purified bacteriocins as a food additive, or in pack-

Fr
aging techniques (Woraprayote et al. 2016). In fermented sausage, Casaburi

et al. (2016) inoculated Lactobacillus curvatus 54M16 bacteriocin producer as
d
an
a starter culture. The number of Enterobacteriaceae reached 3.4 and 2.3 log
or
CFU/g at the end of ripening, in control samples and in sausages, respec-

yl
tively. In charque meat, Biscola et al. (2014) also obtained a reduction of

Ta
pathogenic microorganisms during storage when inoculating an autochtho-

18
nous bacteriocinogenic strain (Lactococcus lactis subsp. lactis 69). Similarly, the
20
bacteriocin sakacin Q produced by Lactobacillus curvatus ACU-1 was used

to control the growth of Listeria innocua inoculated in cooked meat during
©
refrigerated storage. Four different forms were assessed (cell culture, cell-
free supernatant [CFS], and mixture of both forms and freeze-dried recon-
stituted CFS), wherein the use of the freeze-dried reconstituted CFS was the
most effective in controlling the growth of this microorganism, decreasing
by approximately 10 log CFU/cm2 after 4 weeks of storage (Rivas et al. 2014).
Bacteriocins incorporated into packaging have also been reported as an
effective method. Pattanayaiying et al. (2015) investigated the action of nisin
Z with lauric arginate (LAE) incorporated into pullulan film in fresh and
processed meat packaging during refrigerated storage. The result showed

that the antimicrobial activity of the film with nisin Z and LAE was effective
in reducing counts of 6.6, 6.6, 5.7, and 4.9 log CFU/cm2 of pool Salmonella sero-
types, L. monocytogenes, S. aureus, and E. coli O157:H7, respectively, during 2
days of storage.
Regarding sensory changes, in general the presence of bacteriocins or bac-
y
teriocin-producing LAB does not confer sensory changes or any interference
nl
in consumer acceptability of meat products; however, when an alteration
O
is detected, the changes are related to improvements in sensory attributes
se
(Kingcha et al. 2012; Gao et al. 2014; J. Zhang et al. 2010).
lU
ona
9.8.5 Nanoparticles
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Nanotechnology involves the use of compounds and materials comprising
r
size up to 100 nm in one or more dimensions. Its application provides great
fo
opportunities for the development of materials with antimicrobial agents.
y
op
Therefore, interest in nanosize inorganic compounds has been increasing
C
over the past decade (Espitia et al. 2012). Nanosize inorganic compounds
’s
have high antibacterial activity, even at low concentrations, due to their spe-
or
cific chemical and physical properties that create a high ratio of surface to
ut
volume (Sigua et al. 2011; Araújo et al. 2015), and are stable in extreme tem-
b
tri
peratures and pressures (Sawai 2003). Some inorganic compounds are not
on
considered toxic and contain mineral elements essential to the human body
.C
(Roselli et al. 2003; Espitia et al. 2012). Most of the antibacterial inorganic
C
materials are in the form of metal nanoparticles and metal oxide nanopar-
LL
ticles, such as silver (nAg), copper (CuO), and zinc oxide (ZnO) (Bradley et
is
al. 2011; Chaudhry and Castle 2011). In general, nanoparticulate synthesis

c
an
methods can be through precipitation, decomposition, physical vapor, ther-

Fr
mal mechanochemical, and hydrothermal synthesis (Casey 2006; Espitia et

al. 2012; Bernardes et al. 2014).
d
an
The ZnO applications effectively started in 1995, when the antimicrobial

or
activity against some bacterial strains was verified (Sawai 2003). Currently,
yl
ZnO is listed as GRAS by the U.S. FDA and is widely used in the food indus-
Ta
try as a coating of food cans to preserve color and prevent deterioration of

18
meat and fish products (Espitia et al. 2012). The exact mechanism of action of
20
ZnO nanoparticles is still under study. However, it could be attributed to the

release of antimicrobial ions that promote an electrostatic interaction with
©
microorganisms, causing damage in the integrity of the bacterial cell and

formation of reactive oxygen species (Kasemets et al. 2009; Jalal et al. 2010;
Zhang et al. 2017). However, the effectiveness of ZnO depends on the intrin-
sic characteristics of each microorganism, since the response to Zn2+ ions is
different for each strain (Espitia et al. 2012). Contrary to ZnO, nAg ions form
complexes with sulfur, phosphorus, nitrogen, and oxygen, which are pres-
ent in the functional groups of bacterial enzymes, leading to cell membrane
destabilization and disturbances in the metabolism of the microbial cell
(Damm et al. 2007; Rai et al. 2009; Bernardes et al. 2014). Although nanopar-
ticles of silver have been studied in food (Panea et al. 2014; Araújo et al. 2015),
the mechanism of toxicity is poorly explored, limiting it as an application
(Zodrow et al. 2009; Bernardes et al. 2014).
The efficacy of the nanoparticles in foods is highly dependent on the way
in which they are applied, wherein, when incorporated in packaging, pro-
y
longs the antimicrobial effect. Nanoparticles of Ag + ZnO (5%, w/w) incor-
nl
porated into LDPE have antimicrobial activity under aerobic mesophilic
O
bacteria, Enterobacteriaceae, and Lactobacillus present in chicken breast meat.
se
In addition, degradation is reduced, as well as the lipid oxidation, during
lU
storage (Panea et al. 2014). Similarly, pullulan (polysaccharide polymer,
na
C6H10O5) films containing incorporated nAgs promote a reduction of 6.0 and
o
rs
6.5 log CFU/cm2 of S. aureus and L. monocytogenes, respectively, in raw beef
Pe
after 14 days of storage, whereas an increase in microbial counts was veri-
r
fied in control samples (Morsy et al. 2014). In ready-to-eat poultry, Akbar and
fo
Anal (2014) observed that active film containing sodium alginate, glycerol,
y
op
and ZnO nanoparticles in a meat sausage container was able to reduce S.
C
aureus and Salmonella Typhimurium by 4.5 and 4.0 log CFU/g, respectively,
’s
compared with the control, after 6 days of storage. In sheep meat, solutions
or
containing suspended ZnO nanoparticles (6 and 8 nM) were able to retard

ut
the initial growth of L. monocytogenes, E. coli, and S. aureus (Mirhosseini and

b
tri
Arjmand 2014).
on
Overall, no undesirable change has been verified in the physicochemical

.C
and sensory characteristics of meat products conserved by nanoparticles.

C
Suo et al. (2017) found greater retention in pH, total volatile basic nitrogen,
LL
and water holding capacity in pork samples containing ZnO nanoparticle–

is
coated packaging film when compared with the control.

c
an
The application of antimicrobial compounds in conjunction with packag-

Fr
ing has been a great alternative to food preservation since it allows a slow and
continuous diffusion of substances into the matrix, as well as maintains high
d
an
concentrations of them during storage. The forms of interaction between the

or
active substance and packaging material can be by direct incorporation into

yl
the polymer matrix, coating onto the packaging surface, or immobilizing it

Ta
in sachets (Otoni et al. 2016). In addition, packaging materials can be divided

18
into biodegradable and nonbiodegradable. Nonbiodegradable materials

20
are derived from petroleum, which are involved in causing environmental

damage and possibly injuries to public health. Therefore, nonbiodegradable
©
packaging has been replaced by biodegradable materials. Chitosan is a natu-

ral polymer that presents high interaction with antimicrobial compounds
and has great potential applicability in the food industry. However, the high
cost of this compound has limited its commercial use (Sung et al. 2013; Qiu
et al. 2014; Remya et al. 2017). In addition to chitosan, other materials have
been studied, such as the blending of thermoplastic starches (TPSs) with bio-
degradable polyesters such as polycaprolactone (PCL), polylactic acid (PLA),
polyhydroxybutyrate-co-hydroxyvalerate, polybutylene succinate-adipate,
poly(butylene adipate-co-terephthalate), and poly(hydroxyl ester ether)

(Castro-Mayorga et al. 2016; Sung et al. 2013).
y
nl
9.9 Conclusion
O
Nonthermal preservation technologies have been considered an efficient
se
strategy to improve the quality, safety, and extend shelf life of meat, fish,
lU
and derivative products, with minimal interference to the sensorial and
na
nutritional properties of foods. Each emergent technique presents a spe-
o
rs
cific mode of action that promotes punctual damage in the microbial cell
Pe
structure (UV-C, pulsed UV light, pulsed electric field, gamma irradiation,
r
US, and HHP techniques) or, in the case a continuous injuries in cell viabil-
fo
ity, acts throughout the storage period (MAP and chemical compounds).
y
op
Therefore, when applied as a hurdle process, these techniques could decrease
C
the food contamination and delay a microorganism’s growth, conferring
’s
an enhanced effect from both technologies. Moreover, these combinations

or
could be applied in foods already packaged, preventing postcontamination,

ut
in addition to preserving nutritional and sensorial characteristics, which

b
tri
are generally lost by thermal processes. Therefore, a continuous advance

on
in nonthermal technology application is necessary to improve the quality

.C
keeping the intrinsic properties of food, and conferring the possibility of

C
the industry to attend to consumers’ demand of differentiated and safe

LL
products.
c is
an
Fr
d
an
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Inactivation of Pathogenic Microorganisms
in Foods by High Pressure Processing
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Evelyn and Filipa Vinagre Marques da Silva
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CONTENTS
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10.1 Introduction................................................................................................. 341
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10.2 Microbial Pathogens Contaminating Foods............................................342
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10.2.1 Microbial Spores and Vegetative Cells.........................................342
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10.2.2 Pathogenic Sporeformers Relevant for Food Safety..................343
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10.2.3 Vegetative Pathogens Relevant for Food Safety.........................344
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10.3 HPP and HPTP of Foods............................................................................346

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10.3.1 HPP and HPTP Fundamentals and Effect on Microbes ............346

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10.3.2 Mechanism Inactivation of Spores and Vegetative Cells.......... 347

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10.3.3 Kinetic Models for HPP and HPTP Microbial Inactivation...... 347
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10.3.3.1 Primary Models................................................................348

10.3.3.2 Secondary Models............................................................ 349
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10.4 HPTP and HPP Inactivation of Pathogenic Spores in Foods................ 350

10.4.1 Log Reductions................................................................................ 350
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10.4.2 Kinetic Models.................................................................................354

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10.5 HPTP and HPP Inactivation of Vegetative Pathogens in Foods..........354

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10.5.1 Log Reductions................................................................................354

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10.5.2 Kinetic Models................................................................................. 362

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10.6 Future Perspectives..................................................................................... 365

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References.............................................................................................................. 367
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10.1 Introduction
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Foodborne outbreaks continue to be reported presently around the world,

and more than 250 different foodborne diseases have been described (CDC
2015a). Pathogenic bacteria are the main cause of the reported problems,
although viruses and parasites have also been associated with the food-
borne outbreaks. Pathogens are a problem in low-acid foods (pH > 4.6). It is
known that the acidity of high-acid foods (pH < 4.6) inhibits the germina-
tion and growth of microbial spores and the growth of vegetative pathogens.
Therefore, low-acid pasteurized foods are generally stored and distributed
341
under refrigeration (Silva and Gibbs 2009). For pasteurized foods, at least
a 5– 6 log reduction (Betts and Gaze 1992; FDA 2001) in the key pathogen
or spoilage organism is recommended. Thermal processing is the primary
method used by the food industry to achieve this reduction. However, the
heat can alter natural flavors and nutrients in the foods. Researchers and
industry are developing and applying alternative methods for food process-
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ing with less impact on its sensory properties.
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High pressure processing (HPP) has emerged as an attractive pasteuriza-
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tion technology for food preservation. HPP is able to inactivate pathogenic
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and spoilage microorganisms in foods, while retaining their fresh or just
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prepared appearance, organoleptic characteristics, and nutritional quality.
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Guacamole (avocado with spices) is one example of successful HPP-treated
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food. A growing number of foods in the food market are HPP treated. The
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combination of HPP with heat, referred to as HPP-thermal or high-pressure
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thermal processing (HPTP), is possible when more severe process conditions
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are needed. This chapter reviews the updated knowledge dealing with the
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effects of HPP and HPTP on pathogenic microorganisms in foods, includ-
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ing spores and vegetative cells, and kinetic models to describe microbial
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inactivation behavior. Future perspectives of HPP and HPTP foods are also
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discussed.
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10.2 Microbial Pathogens Contaminating Foods

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10.2.1 Microbial Spores and Vegetative Cells

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Microbial cells can exist in different states, vegetative and spores, depend-
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ing on the conditions present. These states of microbes exhibit differences

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in their chemical composition, morphological structure, and physiology

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(Keynan 1969). Vegetative cells are actively growing, metabolizing, and

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dividing. Some microbial species can produce spores, the resting structures,
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which are formed from the vegetative cells. Bacterial spores (endospores, cre-
ated inside the parent vegetative cell) are resistant structures able to survive
18
under environmental stresses such as nutrient deprivation. Under favorable

20
conditions (e.g., water, nutrients, and germinants), the spore breaks its dor-
©
mancy through germination and outgrowth (initiating vegetative growth).

Bacterial spores are well known for their resistance to various agents and
stresses, such as radiation, high temperature, freezing, pressure, desiccation,
extreme pH, and a wide variety of toxic chemicals (Tournas 1994; Setlow
2006; Black et al. 2007; Reineke et al. 2013), and thus are frequently used as
targets in pasteurization and sterilization processes. The resistance of spores
is considered to be due to substantial structural specialization developed
within a mother cell (Moir and Smith 1990).
HPP Inactivation of Pathogenic Microorganisms 343
10.2.2 Pathogenic Sporeformers Relevant for Food Safety

The outgrowth of pathogens in foods can cause contaminations, food spoil-
age, foodborne illnesses, and outbreaks. The main pathogenic sporeform-
ers in foods are Clostridium botulinum , Clostridium perfringens , and Bacillus
cereus. C. botulinum is the most dangerous sporeformer (Carlin et al. 2000a);
it can produce potent and fatal neurotoxins (Brown 2000). C. botulinum
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types A, B, E, and F have been implicated in human foodborne botulism
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(WHO 2016), and incidents from ingestion of the following contaminated
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foods have been reported (Lindströ m et al. 2006): hot-smoked fish (Pace et al.
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1967), canned tuna fish in oil (Mongiardo et al. 1985), canned truffle cream
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or canned asparagus (Therre 1999), pasteurized vegetables in oil (Aureli
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et al. 1999), canned fish (Przybylska 2003), and canned eggplant (Peredkov
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2004). Diagnosis of human botulism is usually from clinical symptoms (gas-
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trointestinal and neurological) (Wells and Wilkins 1996), coupled with the
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detection of toxin in the patient’ s serum and/or feces as a standard method
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(Kautter and Solomon 1977). op
C. perfringens has been identified as the most common cause of food out-
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breaks in ready-to-eat and partially cooked meat and poultry products as
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a result of improper handling and preparation of large quantities of foods

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(Evelyn and Silva 2015a, 2016a; Silva and Gibbs 2009), with type A toxin
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usually being involved in food poisoning (Scallan et al. 2011). In the United
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States, it causes nearly 1 million cases of foodborne illness each year (CDC
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2015b), and it is ranked as an important cause of foodborne illness in the

United Kingdom (Tam et al. 2012) and in several countries (Grass et al. 2013).
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An outbreak associated with vegetables (spinach and fried bean curd dish)
has also been reported with this species (Miwa et al. 1999). Diarrhea, severe
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abdominal cramps, and nausea are the most common symptoms reported
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after 8– 24 h consumption of C. perfringens type A enterotoxin (cpe ) in con-

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taminated foods (Uzal et al. 2014). Based on the symptoms, detection for the
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illness involving C. perfringens (and generally Clostridia species) is usually a

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combination of isolation of Clostridia from feces, blood, or the wound, and

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toxin and serological assays (Berry et al. 1988).

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B. cereus is another sporeforming and pathogenic bacterium commonly

found in meat and in dishes containing meat, resulting in food poisoning,
18
similar in many respects to C. perfringens . Diarrhea and emetic syndromes

20
are two types of diseases caused by B. cereus (Schoeni and Lee Wong 2005).
©
Rice, cereals, and spices are also food commodities often associated with
B. cereus . Some strains have the ability to grow at low temperatures (T < 8° C)
(Dufrenne et al. 1995; Garcí a Armesto and Sutherland 1997; Choma et al.
2000), making them sporeformers frequently isolated from low-acid chilled
foods (Silva et al. 2014; Silva and Gibbs 2010; Carlin et al. 2000b; Dufrenne
et al. 1995). Bacillus licheniformis is another Bacillus species often contaminating
dairy products. Foodborne outbreaks have been registered in cooked meats
and vegetables, raw milk, and commercial baby foods (Salkinoja-Salonen
et al. 1999). Bacillus pumilus human infection is rare, although a few cases of
food poisoning from rice were reported. The symptoms include dizziness,
headache, chills, back pain, stomach cramps, and diarrhea (From et al. 2007).
One of several methods used for the diagnosis of human illness caused by
B. cereus and other Bacillus spp. is the isolation of B. cereus from suspect food
and determining its enterotoxigenicity by serological (diarrheal toxin) or bio-
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logical (diarrheal and emetic) tests (FDA 2014).
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Other than the main pathogenic sporeformers mentioned above, the fol-
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lowing bacteria can also cause diseases in humans after consumption of con-
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taminated foods: Clostridium baratii (infant botulism), Clostridium butyricum
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(infant botulism), Clostridium difficile (diarrhea to fulminant colitis), Bacillus
na
thuringiensis (gastroenteritis), and Bacillus anthracis (gastrointestinal illness
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and diarrhea) (Aureli et al. 1986; Jackson et al. 1995; Barash et al. 2005; Pavic
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et al. 2005; Rupnik and Songer 2010; CDC 2000).
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10.2.3 Vegetative Pathogens Relevant for Food Safety
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Staphylococcus aureus , enterohemorrhagic Escherichia coli O157:H7, Listeria
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monocytogenes , Salmonella spp., and Vibrio spp. are vegetative pathogens

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important to food safety. The toxins of these enteric pathogens (diarrheal

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caused syndrome) can be transmitted via a wide range of foods, causing

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outbreaks of illness following consumption. Food poisoning outbreaks in

on
milk-based products caused by S. aureus enterotoxins have been frequently

.C
reported due to ingestion of foods containing staphylococcal enterotoxins

C
(SEs) (Evenson et al. 1988; Asao et al. 2003; Schmid et al. 2009; Ostyn et al.
LL
2010; Hennekinne et al. 2012). Commonly described symptoms of S. aureus

is
foodborne infection other than diarrhea are nausea, vomiting, abdominal

c
an
cramping, dizziness, and sometimes moderate fever (Hennekinne et al.

Fr
2012). Diagnosis of staphylococcal food poisoning is generally confirmed by

either recovery of S. aureus (≥ 105 /g food) or by the detection of SEs in food
d
an
remnants (Hennekinne et al. 2012).

or
Outbreaks of E. coli O157:H7 have been associated with a wide range of

yl
foods, including beef, vegetables (e.g., lettuce, spinach, and sprouts), raw
Ta
milk (CDC 2014; Armstrong et al. 1996), and very recently, flour (CDC 2016a).
18
The ability of this strain to survive and grow at very acidic conditions and
20
low temperature (Weagant et al. 1994; Conner and Kotrola 1995; Hsin-Yi and
Chou 2001) may explain the occurrence of outbreaks in the following high-
©
acid foods: fruit juices (CDC 1996; Cody et al. 1999), apple cider (Miller and
Kaspar 1994), mayonnaise (Weagant et al. 1994), mustard and ketchup (Tsai
and Ingham 1997), and yogurt (Morgan et al. 1993). Commonly described
symptoms of E. coli O157:H7 food infection include diarrhea and abdomi-
nal cramping, which in some cases progresses to bloody diarrhea (Ibrahim
2015). Human illness is usually diagnosed through laboratory testing of stool
specimens (feces) (CDC 2015c).
Listeria monocytogenes infection (listeriosis) has been a great concern

until now due to its capability to cause severe illness with a high morbid-
ity, hospitalization, and mortality rate in vulnerable populations, such as
pregnant women and the elderly (Henriques and Fraqueza 2015). Flu-like
or gastrointestinal illnesses, miscarriage, stillbirth, septicemia, meningi-
tis, and encephalitis are symptoms detected for listeriosis-infected persons
y
(Gillespie et al. 2010). Disease can be confirmed by isolation of the bacte-
nl
ria from blood, spinal fluid, or amniotic fluid or the placenta (for pregnant
O
women) (CDC 2014). For the years 2014– 2016, multistate outbreaks caused
se
by L. monocytogenes were recorded in the United States, linked to foodstuffs
lU
such as packaged caramel apples and salads, cheese, raw milk, and frozen
na
vegetables (CDC 2016b).
o
rs
Salmonellosis, especially from Salmonella enteritidis and Salmonella
Pe
typhimurium , is the most frequently reported foodborne disease worldwide,
r
and outbreaks have been associated with a diverse range of food vehicles.
fo
Food from animal origins, that is, eggs, meat, poultry, and milk, are the
y
op
main food commodities reported (CDC 1990; Perales and Audicana 1989),
C
although an increasing number of outbreaks was reported in contaminated
’s
green vegetables (Doyle and Erickson 2008; Hanning et al. 2009; WHO 2013).
or
The most common clinical symptoms are diarrhea and abdominal cramps;
ut
however, illness may be accompanied by a fever of 38° C– 39° C (Giannella

b
tri
1996). Stool, blood, urine, and sometimes tissues can be used for the diagno-
on
sis of Salmonella bacteria (Behravesh et al. 2008).

.C
Vibrio parahaemolyticus and Vibrio vulnificus are two major foodborne bac-
C
terial pathogens of great concern in raw foods such as oysters, sushi, and
LL
sashimi, or undercooked seafood, since they are naturally distributed in

is
water (Jay 2000). Large outbreaks of V. parahaemolyticus (O3:K6 serotype)

c
an
occurred during 1997– 1998 in Washington, Texas, and New York, and on

Fr
the West Coast of the United States (Daniels et al. 2000; DePaola et al. 2000),
whereas an outbreak of V. vulnificus was reported occur in coastal states
d
an
from the Gulf of Mexico region (Shapiro et al. 1998). It is estimated that there
or
are 35,000 foodborne infections caused by V. parahaemolyticus in the United

yl
States annually, while V. vulnificus is reported to have the highest mortality

Ta
rate (up to 50%) among other foodborne pathogens (Scallan et al. 2011).
18
Other pathogenic vegetative bacteria, such as Streptococcus faecalis ,

20
Campylobacter jejuni , Yersinia enterocolitica , Citrobacter freundii , and Aeromonas

hydrophila , also pose a health risk due to their association with meat prod-
©
ucts, and outbreaks are sometimes reported (Deming et al. 1987; Hussain
et al. 1988; Tsai and Chen 1996; Gaibani et al. 2013; Grahek-Ogden et al.
2007). Among vegetative pathogens described previously, L. monocytogenes ,
Y. enterocolitica , Salmonella , V. parahaemolyticus , and A. hydrophila are of great
concern because they are able to grow at refrigerated or low temperatures
(D’ Aoust 1991; Penfield et al. 1990), and thus can be a problem in HPP chilled
foods.
10.3 HPP and HPTP of Foods

10.3.1 HPP and HPTP Fundamentals and Effect on Microbes
HPP is a commercial nonthermal food pasteurization technology with less
adverse effects on food quality than conventional thermal processes (Cullen
y
nl
et al. 2012). Industrial HPP typically operates at pressures of 400– 600 MPa
O
to process liquid and solid foods between 5 and 10 min either chilled or at
se
temperatures of ≤ 40° C. According to Le Chatelier’ s principle, hydrostatic
lU
pressure reduces the volume of the pressurized material without chang-
na
ing its shape. Covalent bonds from primary structures of proteins are
o
unaltered by pressure (Mozhaev et al. 1994), making this the central hypoth-
rs
esis behind the preservation of biological activity of functional compounds
Pe
(Balasubramaniam et al. 2015). The main goal of HPP is the nonthermal
r
fo
pasteurization of foods through the inactivation of pathogenic and spoilage
y
vegetative microorganisms, usually by 5 or 6 D. After processing, the foods
op
are usually cold stored and distributed, and the shelf life is influenced by
C
the intensity of the treatments, storage conditions, and other factors, such
’s
as packaging characteristics. The common shelf life of HPP-treated foods is

or
ut
3– 10 times that of untreated HPP foods (Hiperbaric 2013). Fruit juices and
b
smoothies, fruit jams and sauces, yogurt, jelly, guacamole, dips and salsas,
tri
on
ready-to-eat meals, meat and poultry products, and seafood are examples of
commercially available HPP food products worldwide, with manufacturers
.C
located in Japan, the United States, and Europe (Hogan et al. 2005).
C
LL
Room temperature HPP standard treatment does not inactivate most

microbial spores and enzymes. For example, spores of B. subtilis species were
c is
found to survive up to 1200 MPa at ambient temperature (Larson et al. 1918).

an
Thus, as mentioned, HPP foods require a cold chain for distribution. While
Fr
high-acid fruit products (pH < 4.6) do not allow the germination and growth
d
of pathogenic and spoilage sporeformers (Silva and Gibbs 2009; Silva et al.
an
2014), low-acid foods (pH ≥ 4.6), such as meat products, fish, vegetables, milk,
or
and cheeses, pose significant health hazards associated with pathogenic

yl
Ta
sporeforming bacteria and need to be stored and distributed below 7° C. In

addition to cold storage conditions, pressures between 400 and 800 MPa,
18
combined with temperatures higher than 50° C, appear to have potential

20
to reduce spore numbers in foods. However, the resistance of spores varies

©
highly among species, and is also affected by the food matrix. A combination
of HPP with moderate heat or HPTP is inevitably needed to inactivate these
resistant spores, and past works have shown that HPTP pressure and tem-
perature synergistically enhance the spore inactivation (Akhtar et al. 2009;
Silva et al. 2012; Daryaei et al. 2013; Evelyn and Silva 2015b,c, 2016a,b; Evelyn
et al. 2016). Until now, considerable efforts have been made for full HPTP
inactivation of microbial spores, to produce shelf-stable sterilized food prod-
ucts with high quality. However, the HPTP sterilization technology has not
yet been successfully demonstrated; thus, the technology is used industri-

ally for food pasteurization and the production of refrigerated but not yet
shelf-stable foods.
10.3.2 Mechanism Inactivation of Spores and Vegetative Cells
y
Many researchers have thoroughly investigated and reported the mechanism
nl
O
of vegetative and spore cell inactivation by HPP (Mathys 2008; Smelt et al.
se
2001; Lado and Yousef 2002; Patterson 2005; Knorr et al. 2010; Black et al. 2007;
lU
Reineke et al. 2013). Generally, microbial cell death occurs when there are
na
considerable alterations in the cellular structure or physiological functions of
microorganisms after their exposure to pressure (and heat). Regarding veg-
o
rs
etative microorganisms, various HPP inactivation mechanisms suggested
Pe
that result in cell death are the inactivation of essential enzymes (Smelt et al.
r
2001; Ardia 2004), changes in intracellular pH (Smelt et al. 2001), disintegra-
fo
tion of ribosomes in their subunits (Smelt et al. 2001), and rupture of the cell
y
op
membrane due to membrane phase transition and ﬂ uidity changes (Smelt
C
et al. 2001; Ananta et al. 2005), leading to morphological changes in HPP-
’s
treated cells (Patterson 2005).

or
With respect to spores, two-step processes have been widely accepted for
ut
b
the mechanism of spore inactivation, which are mostly based on mechanism

tri
inactivation of Bacillus spores in the buffer: (1) release of dipicolinic acid

on
(DPA) during germination (direct or through activation of nutrient germi-

.C
nant receptors) due to disruption to the spore inner membrane, causing a

C
loss of spore resistance, and (2) subsequent inactivation by pressure and heat
LL
as vegetative cells (Black et al. 2007; Heinz and Knorr 2001; Mathys et al. 2009;
is
Reineke et al. 2013). Spore germination and inactivation pathways depend

c
an
on the pressure– temperature combinations (Reineke et al. 2013; Mathys et

Fr
al. 2009); however, more research is needed to elucidate the mechanisms of

d
the spore inactivation in food products. Various tools, such as flow cytom-
an
etry, fluorescence anisotropy measurement, and electron microscope, have

or
been used to observe cell membrane damage and morphological changes of

yl
spores and vegetative cells (Mathys et al. 2007; Abe 2013; Ananta et al. 2005).
Ta
18
20
10.3.3 Kinetic Models for HPP and HPTP Microbial Inactivation

©
Mathematical models allow many researchers to describe, predict, and

optimize microbial behavior in foods after exposure to certain lethal treat-
ment to ensure the microbiological quality and safety of food products.
According to Whiting (1995), microbial modeling in foods can be classified
as primary, secondary, and tertiary. The use of basic models (the primary
followed by the secondary models) has been frequently reported after HPP
and HPTP treatments in foods, as they are the most straightforward meth-
ods for the users.
10.3.3.1 Primary Models

Primary models aim to describe microorganisms’ response as a function of
time under specific conditions, in which the main goal is to estimate kinetic
parameters such as the inactivation rate. Linear, concave, or sigmoidal trends
are frequently observed in HPP and HPTP treatments based on the shape
of their survival curves. Two common primary models reported to describe
y
nl
microbial log survivors in foods after HPP and HPTP are the simple first-
O
order kinetic (Bigelow) and Weibull models.
se
The simple first-order kinetic model was established to define safe thermal
lU
processes for canned foods, and it has also been successfully applied to other
na
food processes, in which a plot of the logarithm of the surviving fraction
o
against time yields a straight line as a constant intensity of heat or a lethal
rs
factor is applied. With respect to HPP and HPTP, the main kinetic parameter
Pe
of the model is decimal reduction time, or the D P , T value, which is the time
r
fo
in minutes at a certain pressure and/or temperature necessary to reduce the
y
microbial population by 90% (and is calculated from the reciprocal of the
op
slope of Equation 10.1):
C
’s
or
N t
ut
log =− (10.1)
N0 DPT
b
tri
on
where N 0 is the initial or untreated cell population in food (cfu/g or cfu/mL),
.C
and N is the number of survivors after being exposed to HPP or HPTP treat-
C
LL
ment for a specific time t (min).

The Weibull model (Equation 10.2), perhaps the most prominent nonlinear
cis
model used for pressure treatments, is based on the principle of heterogene-

an
ity in the resistance distributed among individual cells within a population

Fr
(vitalistic approach) (Pin and Baranyi 2006).

d
an
N
or
log = −bt n (10.2)

yl
N0
Ta
18
Two parameters obtained from this primary model are b , the scale factor,
20
and n , the survival curve shape factor. b is a rate parameter that is related
to the velocity of the inactivation of the microorganism. n describes the
©
degree of curvilinearity, where n < 1 and n > 1 correspond to concave-

upwards (tailings) and concave-downwards (shoulders) survival curves,
respectively. When n = 1, the Weibull model becomes the simple first-order
kinetics.
The nonlinear models are often more appropriate than first-order kinetics
for HPP microbial inactivation. However, due to their complexity and more
difficult application, they are not commonly used by microbiologists to fit
log microbial survivor data.
10.3.3.2 Secondary Models

The secondary model is an extension of the primary model, in which the
parameters of the primary model (e.g., D value and inactivation rate k ) are
related to the environmental variables/or conditions, such as pressure or
temperature. In the area of inactivation and with respect to first-order linear
kinetics, Bigelow uses the z T value (° C) to model the inactivation rate depen-
y
nl
dence on the applied temperature. The z T value (° C), or the temperature coef-
O
ficient, is the temperature increase for constant pressure, which results in a
se
10-fold decrease in the D value. This is estimated from the negative recipro-
lU
cal of the slope of Equation 10.3:
o na
 D  Tref − T
rs
log  = z (10.3)
Pe
 DTref  T
r
fo
where D Tref is the D value at the reference temperature T ref (can be any refer-
y
ence temperature, ° C), and T is the temperature of the isothermal treatment
op
(° C).
C
Likewise, a similar equation can be used to relate the inactivation D value

’s
or
with the HPTP pressure, for a fixed temperature, or room temperature HPP.
ut
The pressure coefficient, or z P value (MPa), can also be estimated as follows
b
tri
(Equation 10.4):
on
.C
 D  Pref − P
log  = z (10.4)
C
 DTref 
LL
P
is
In the same way, the Weibull model can also relate the inactivation rate
c
an
parameter (b value) to environmental variables, particularly temperature,

Fr
and it can be used to predict the parameter value outside the range of vari-
d
ables tested (Evelyn and Silva 2015a–c, 2016a,b; Evelyn et al. 2016), ideally by
an
validating the secondary model used and understanding the fundamental

or
mechanisms involved.
yl
The polynomial model or response surface model (RSM) is another

Ta
secondary model that has been developed to predict the effect of multiple
18
environmental factors on the inactivation parameters (Ross and Dalgaard

20
2004). Second-order polynomial equations are generally used, involving

©
first-order, second-order (quadratic), and interaction terms, as shown in

Equation 10.5 (Pé rez-Rodrí guez and Valero 2013):
n n n
Y = B0 + ∑B X + ∑B X + ∑B X X + ε (10.5)
i =1
i i
i =1
ii
2
i
j ≠1
ij i j
where Y is the predicted response (microbial log survivors); B 0 , B i ,

B ii , and B ij are the estimated regression coefficients; X i and X j are the
independent variables (environmental factors); and ε is the error term.

Pressure, temperature, and pressure holding time are three main variables
frequently investigated under HPTP treatments. RSM can be graphically
translated; thus, food operators can find the operating conditions that opti-
mize the response. In Sections 10.4 and 10.5, the effects of HPP and HPTP on
the main foodborne sporeforming and vegetative pathogens in foods and
y
their kinetic models are reviewed.
nl
O
se
lU
ona
10.4 HPTP and HPP Inactivation of Pathogenic Spores in Foods
rs
Pe
Overall, spores show great resistance to inactivation requiring the combina-
r
tion of high pressure with thermal processing (HPTP). The genus Clostridium
fo
has the greatest degree of resistance, followed by Bacillus , and then lastly,
y
op
vegetative cells, in which the inactivation is achieved with relatively mild
C
process conditions (see Section 10.5). Simple nonlinear models were used to
’s
describe the spore inactivation after HPTP, whereas the first-order kinetic
or
model was frequently applied for the inactivation of vegetative cells after
ut
HPP or HPTP.
b
tri
on
.C
10.4.1 Log Reductions

C
Tables 10.1 through 10.3 show the spore log reductions obtained for C. botu-
LL
linum , C. perfringens , B. cereus , and other Bacillus spores in food products

is
after high pressure in the range of 345– 827 MPa, combined with tempera-
c
an
tures of 25° C– 86° C. HPTP at 827 MPa and 84° C for 15 min for C. botulinum
Fr
and HPTP at 600 MPa and 75° C for 15 min for C. perfringens were not suffi-
cient to inactivate some strains of these spores (≤ 2 log), indicating high resis-
d
an
tance to the HPTP treatments (Table 10.1). With the exception of C. botulinum
or
strains ATCC 19397, ATCC 25765, and KAP8-B with > 5.5 log after ≥ 600 MPa
yl
and ≥ 80° C for 16 min (Margosch et al. 2004a; Reddy et al. 2006), all others
Ta
reported modest to almost no clostridial spore reductions in foods (0.4– 4.1

18
log) after high pressures at 345– 827 MPa combined with temperatures of

20
60° C– 86° C for 5– 16 min (Kalchayanand et al. 2003; Margosch et al. 2004a;
Reddy et al. 2003, 2006; Evelyn and Silva 2016a).
©
Regarding Bacillus , HPTP in the range of 500– 600 MPa and tempera-

tures of 60° C– 85° C between 1 and 17 min had been used (Tables 10.2 and
10.3). Van Opstal et al. (2004) reported generally similar spore inactivation
(> 5 log) for four B. cereus strains in milk after 500 MPa, 60° C, and 15 min
(Table 10.2). Evelyn and Silva (2015b) obtained 3– 4 log reductions of two
strains of B. cereus spores in skim milk after 600 MPa, 70° C, and 15 min
(Table 10.2). Scurrah et al. (2006) obtained ≈ 4 log for five B. cereus strains also
in skim milk after 600 MPa, 72° C (initial temperature), and 1 min, with the
©
20
TABLE 10.1 18
Inactivation of Clostridium Spores in Foods by HPTP
Ta
Average
Pressure Temperature Time Log
yl
or
Spores Strains Food Products
an pH (MPa) (° C) (min) Reduction References
Clostridium botulinum d
Proteolytic type A 62-A Crabmeat blend nr 827 86 15 2.7 Reddy et al. 2003
Fr
BS-A 3.0
an
Nonproteolytic type B 2B Crabmeat blend
cis 7.2– 7.4 827 84 15 < 1.0 Reddy et al. 2006
17B LL 1.6
KAP9-B C 2.0
KAP8-B > 5.5
.C
nr TMW 2.357 Mashed carrot 5.2 on 600 80a 16 1.2 Margosch et al. 2004a
TMW 2.356 tri 2.6
TMW 2.359 bu 2.6
TMW 2.358 to 4.1
HPP Inactivation of Pathogenic Microorganisms
ATCC 19397 > 5.5

r’s
ATCC 25765 C > 5.5
op
Clostridium perfringens y
NZRM 2621 Beef slurry 6.5 600 75 fo 15 1.5 Evelyn and Silva
(ATCC 12917) r 2016a
NZRM 898 2.0
Pe
(ATCC 14809) rs
1027 Roast beef nr 345 60 5
o na 0.4 Kalchayanand et al.
lU 2003
Note : nr, not reported.
a Initial temperature.
se
351
O
nl
y
©
20 352
18
TABLE 10.2
Ta
Inactivation of Bacillus cereus Spores in Foods by HPTP and HPP Alone
yl
or
an Average
Food d Pressure Temperature Time
Strains Products pH Fr (MPa) (° C) (min) Log Reduction References
ATCC 9818 Cooked rice 6.0 an600 85 4 7.0 Daryaei et al. 2013
NZ 4 (NCTC 8035) Skim milk nr 600
ci 72a 1 4.1 Scurrah et al. 2006
NZ 6
s 4.3
NZ 3/NZ 5/NZ 7 4.4
LL
FRR B2603
C 6.1
.C
NZRM 984 (ATCC 11778) Skim milk nr 600 15 3.0 Evelyn and Silva 2015b
ICMP 12442 (ATCC 9139) 3.5
on70
As 1.1846 Milk buffer 7.0 540 17 6.0 Ju et al. 2008
t71ri
b
LMG 6910 (ATCC 7004) Milk 6.7 500 60 ut 15 5.4 Van Opstal et al. 2004
INRAAV P21S 5.6
or
INRAAV Z4222
’s 5.6
INRAAV TZ415
C 6.6
op
nr Pork slurry nr 600 RT 10
y < 1.0 Shigehisa et al. 1991
NCFB 578 Milk nr 400 RT 15 fo < 0.5 McClements et al. 2001
NCFB 1031 r < 0.5
ATCC 9139 Cheese 5.5 400 RT 15 < 0.5 Lopez-Pedemonte et al.
Pe
2003
rs
o
Note : RT, room temperature HPP; nr, not reported.
a
na
Initial temperature before compression. lU
se
O
nl
y
©
20
18
TABLE 10.3 Ta
Inactivation of Other Bacillus Spores in Foods by HPTP
yl
or
Food Pressure Temperature a Time Log
Species Strains pH (MPa) (° C) (min) Reduction References
an Products
Bacillus licheniformis TMW 2.492 Mashed a
d carrot 5.2 600 80 16 > 7.0 Margosch et al.
2004b
Fr
an
c
B. licheniformis NZ 23/Werribee 260 Skim milkis nr 600 72a 1 1.6 Scurrah et al. 2006
NZ 24/NZ 25 LL 2.0
FRR B2653 C 2.6
(ATCC 9789)
.C
NZ 22 on 2.7
NZ 21(NCTC 6346) tri 3.4
Werribee 229 b 3.6
Werribee 207 4.3
ut
or
Bacillus pumilus NZ 33 Skim milk nr 600 ’s 72a 1 1.8 Scurrah et al. 2006
NZ 32 (NCTC 10327) C 2.9
NZ 27 3.5
op
NZ 31
y 3.7
NZ 29
fo 4.3
r
NZ 28 Pe 4.7
rs
Note : nr, not reported.
o
a Initial temperature before compression.
na
lU
se
353
O
nl
y
exception of FRR B2603 with 6 log (Table 10.2). These authors also found large
differences in the resistance of seven strains of B. licheniformis (1.6– 4.3 log)
and six strains of B. pumilus (1.8– 4.7 log) spores in skim milk after the same
treatment (Table 10.3). Margosch et al. (2004b) reported > 7 log for B. licheni-
formis spores in mashed carrot. These results suggest that species, strain,
and food play significant roles in spore resistance to HPTP; thus, investigat-
y
ing the most resistant spores for each species– strain– food combination is
nl
needed to ensure food safety and quality. HPTP at ≥ 600 MPa and ≥ 85° C for
O
≥ 4 min seems to be needed to achieve ≥ 7 log inactivation of B. cereus spores
se
in cooked rice (Daryaei et al. 2013). HPP treatments (400– 600 MPa) at room
lU
temperatures (≤ 30° C) for 10– 15 min generally showed a negligible effect on
na
B. cereus spores (Table 10.2) (Shigehisa et al. 1991; McClements et al. 2001;
o
rs
Lopez-Pedemonte et al. 2003.).
r Pe
fo
10.4.2 Kinetic Models
y
op
The Clostridium and Bacillus spore inactivation in foods after HPTP was non-
C
linear; thus, the Weibull model was reported (Table 10.4). For 600 MPa pro-
’s
cesses at 70° C– 75° C, the Weibull shape factors (n ) for C. perfringens and B. cereus
or
spores were between 0.39 and 0.74, indicating that the log survival curves have
ut
upward concavity with more pronounced tailings, as processing times increase

b
tri
in some cases (Evelyn and Silva 2015b, 2016a). Response surface methodology
on
was also used to investigate the effects of pressure, temperature, and time on
.C
spore inactivation, and to predict the processing conditions to achieve a desired

C
log reduction of spores. For example, for a 6 log cycle reduction on B. cereus in
LL
milk buffer, HPTP process parameters of at least 540 MPa and 71° C and a hold-
is
ing time of 16.8 min were required, according Ju et al. (2008).

c
an
Fr
d
an
or
10.5 HPTP and HPP Inactivation of

yl
Ta
Vegetative Pathogens in Foods

18
10.5.1 Log Reductions

20
In the studies of foodborne vegetative pathogens, with the exception of

©
Garcí a-Graells et al. (1999) with resistant mutant E. coli strains (LMM 1020,
LMM 1030, and LMM 1010) in skim milk, HPTP (345– 600 MPa, 5– 15 min) at
> 50° C (initial food temperature) generally resulted in large viability losses
(5.5 to > 8.0 log) in the food products (Patterson and Kilpatrick 1998; Gervilla
et al. 1999; Ponce et al. 1998; Alpas and Bozoglu 2000; Bayı ndı rlı et al. 2006)
(Tables 10.5 through 10.9). S. aureus appeared to have the highest resistance to
the treatments among the foodborne vegetative species reported (Table 10.5),
followed by E. coli (Tables 10.5 and 10.6), and then L. monocytogenes and
©
20
18
Ta
yl
TABLE 10.4 or
Modeling the Microbial Spore Inactivation in Foods after HPTP
an
d Average
Food Pressure Temperature Time Model Parameters/
Fr
Products pH Model (MPa) (° C) (min) Equations a References
an
c
Clostridium perfringens is
NZRM 2621 Beef slurry 6.5 Weibull 75 — b = 0.20, n = 0.74 Evelyn and
LL
(ATCC 12917) C600 Silva 2016a
NZRM 898 b = 0.68, n = 0.39
.C
(ATCC 14809) on
Bacillus cereus tri
NZRM 984 Skim milk nr Weibull 600
b — b = 0.55, n = 0.59 Evelyn and
(ATCC 11778) Silva 2015b
ut 70
or
ICMP 12442 ’s b = 0.67, n = 0.57
(ATCC 9139) C
As 1.1846 Milk 7.0 Second-degree 400– 600 60– 80op10– 20 Y = 5.42 + 1.54P + 0.30T Ju et al. 2008
Buffer polynomial y 2 2
+ 0.25t – 0.11P + 0.17T –
(RSM) fo 2
0.23t – 0.23Pt
rP
Note : nr, not reported. er
a
b and n are the Weibull scale and shape factors (Equation 10.2), respectively; Y , P , T , and t are the log reductions
so and pressure, temperature, and hold-
ing time variables of the RSM polynomial equation (Equation 10.5), respectively. na
lU
se
355
O
nl
y
©
20 356
18
Ta
yl
TABLE 10.5
or
Inactivation of Staphylococcus aureus in Foods by HPTP and HPP Alone
an
d
Fr Average
Pressure
an Temperture Time Log
Strains Food Products pH (MPa) c (° C) (min) Reduction References
a
NCTC 10652 Poultry meat nr
i
600 s 50 15 6.0 Patterson and Kilpatrick 1998
(ATCC 13565) UHT milk nr 500 50a 15 6.0
LL
CECT 534 Ovine milk 6.7 500
C 50 15 > 7.0 Gervilla et al. 1999
(NCTC 4163)
.C
485 Milk 6.7 345 a 5 5.5 Alpas and Bozoglu 2000
o50n
765 Milk > 8.0
tri
nr Dry-cured ham nr 600 RT
but 6 0.6 Hugas et al. 2002
Cooked ham or 1.1
Marinated beef ’s 2.7
ATCC 25923 Pork slurry nr 600 RT C10 6.0 Shigehisa et al. 1991
op
NCTC 10652 Milk nr 600 RT 15
y 2.0 Patterson et al. 1995
fo
(ATCC 13565) Poultry meat r 3.0
ATCC 6538 Cheese slurry 5.2– 5.4 600 RT 20 4.5
Pe O’ Reilly et al. 2000
Note : RT, room temperature HPP; nr, not reported; UHT, ultrahigh temperature. rs
a
Initial temperature before compression. o na
lU
se
O
nl
y
©
20
18
TABLE 10.6
Ta
Inactivation of Escherichia coli in Dairy and Poultry Foods by HPTP and HPP Alone
yl
or
an Average
d Pressure Temperature Time Log
Strains Food Products FrpH (MPa) (° C) (min) Reduction References
K12 LMM 1020 Skim milk a6.6n 550 50 15 2.4 Garcí a-Graells et al. 1999
K12 LMM 1030 ci 2.4
K12 LMM 1010
s 4.7
K12 MG 1655 7.0
LL
(ATCC 47076)
C
.C
— CECT 405 Liquid whole egg 8.0 450 50 10 5.5 Ponce et al. 1998
(ATCC 10536)
on
O157:H7 NCTC 12079 UHT milk nr 500 a 15 8.0 Patterson and Kilpatrick 1998
tri 50
Poultry meat 7.5
bu
a
to
O157:H7 933 Milk 6.7 345 50 r 5 > 8.0 Alpas and Bozoglu 2000
931
’s > 8.0
O157:H7 NCTC 12079 Milk nr 600 RT
C 1.5 Patterson et al. 1995
op 15
Poultry meat y 3.0
K12 ATCC 29425 Cheese slurry 5.2– 5.4 500 RT 20fo > 6.0 O’ Reilly et al. 2000
— ATCC 25922 Pork slurry nr 400– 500 RT 10 rP > 6.0 Shigehisa et al. 1991
— CECT 405 Fresh goat cheese 6.5 450– 500 RT 5 er > 8.5 Capellas et al. 1996
(ATCC 10536) so
Note : RT, room temperature HPP; nr, not reported; UHT, ultrahigh temperature.
na
a Initial temperature before compression. lU
se
357
O
nl
y
©
TABLE 10.7
20 358
18
Inactivation of Escherichia coli in Fruit Juices by HPTP and HPP Alone
Ta
Average
yl
Strains Fruit Juices pH (MPa) (° C) (min) Reduction References
or
an
O157:H7 931 Orange juice d 3.8 345 50a 5 > 8.0 Alpas and Bozoglu
2000
933
Fr
an > 8.0
O157:H7 933 Apricot juice c 3.8 350 40a 5 > 8.0 Bayı ndı rlı et al. 2006
Orange juice 3.8
is > 8.0
Sour cherry juice 3.3 > 8.0
LL
Apple juice 3.5
C > 8.0
O157:H7 SEA 13B88 Apple juice 3.7 RT 2 0.4 Teo et al. 2001
. C 615
ATCC 43895, Orange juice 3.7 2.2
on
932b tri
Carrot juice 6.2
b ut 6.4
Grapefruit juice 3.0 or 8.3
O157:H7 NCTC 12079 Orange juice 3.4– 4.5 550 ’s RT 5 > 7.0 Linton et al. 1999
O157:H7 ATCC 43894 Mango juice 4.5 550 C
RT 5 > 8.0 Hiremath and
Ramaswamy 2012
op
O157:H7 C9490 Orange juice 3.8 500 RT
y 5 > 7.0 Jordan et al. 2001
Apple juice 3.5
fo > 7.0
r
Tomato juice 4.1 Pe > 7.0
O157:H7 nr Orange juice 3.7 250 RT 20 rs 5.0 Noma et al. 2004
Apple juice 3.8 o > 7.0
K12 LMM 1010 Mango juice 4.0 500 RT 2 Garcia-Graells et al.
na
> 5.0
1998
lU
(Continued)
se
O
nl
y
©
20
18
Ta
yl
or
TABLE 10.7 (CONTINUED) an
Inactivation of Escherichia coli in Fruit Juices by HPTP and HPP Alone
d
Average
Fr
an
Strains Fruit Juices
cispH (MPa) (° C) (min) Reduction References
Resistant mutant LL
K12 LMM 1010 Apple pieces in 24° Brix 3.5 C RT 10 > 6.0 Vercammen et al. 2012
Resistant mutant GLUCOSE syrup
. C 600
— ATCC 11775 Orange juice 3.4 o414n RT 0.03 > 7.0 Guerrero-Beltrá n et al.
tri 2011a
— ATCC 11775 Mango nectar 3.6 414 b RT 1 > 8.0 Bermú dez-Aguirre
et al. 2011
ut
— ATCC 11775 Pineapple juice 3.8 300 5 1.0 Buzrul et al. 2008
or
’s RT
Kiwifruit juice 3.3 C 4.0
— ATCC 11775 Pear nectar 4.2 241 RTo 3 4.0 Guerrero-Beltrá n et al.
py 2011b
— ATCC 25922 Cashew apple juice 4.1 400 RT fo 3 6.5 Lavinas et al. 2008
Note : RT, room temperature HPP; nr, not reported.
r
a Initial temperature before compression.
Pe
b Cocktail of strains.
rs
o na
lU
se
359
O
nl
y
©
20 360
18
Ta
yl
or
an
d
TABLE 10.8 Fr
Inactivation of Listeria monocytogenes in Foods by HPTP and HPP Alone
an
cis Initial
Food Products pH (MPa) (° C) (min) Reduction References
LL
C
CA Milk 6.7 345 .C 50 5 > 8.0 Alpas and Bozoglu
on 2000
Ohio2 > 8.0
nr Goat cheese nr 500 5 Gallot Lavallee 1998
tri
bRTu > 5.6
NCTC 11994 Milk nr 375 RTt 15 0.5 Patterson et al. 1995
(DSM 15675) Poultry meat
or’s 2.0
Scott A UHT milk 6.5 340 RT C 20 1.5 Styles et al. 1991
Raw milk op 2.0
Note : RT, room temperature HPP (for HPTP, the temperature was the initial one before compression); nr, not reported; UHT, ultrahigh temperature.
y
fo
r Pe
rs
o na
lU
se
O
nl
y
©
20
18
Ta
yl
or
an
d
TABLE 10.9 Fr
Inactivation of Salmonella in Foods by HPTP and HPP Alone
an
c
Initial
is
LL Pressure Temperature a
Time Log
Species Strains Food Products pH C (MPa) (° C) (min) Reduction References
Salmonella enteritidis FDA Milk 6.7 50 5 > 8.0 Alpas and Bozoglu 2000
. C345
Salmonella typhimurium E 21274 Milk 6.7
o
345 n 50 5 > 8.0 Alpas and Bozoglu 2000
S. enteritidis nr Liquid whole egg 8.0 450 tri 50 15 > 7.8 Ponce et al. 1999
15 5.1
bu RT
S. enteritidis SE-4 Liquid whole egg nr 400 10 6.0 Bari et al. 2008
to RT
S. typhimurium ATCC 7136 Strained chicken nr 340

r’RT
s 15 2.0 Metrick et al. 1989
baby food C
S. typhimurium ATCC Pork slurry nr 400 RT 10 6.5 Shigehisa et al. 1991
op
14028 y
Salmonella senftenberg 775 W Strained chicken nr 340 RT
fo15 2.5 Metrick et al. 1989
baby food
rP
Note : RT, room temperature (20– 25° C); nr, not reported.
e
a
rs
Initial temperature before compression. o na
lU
se
361
O
nl
y
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. entero-
colitica , C. freundii , and A. hydrophila , generally required milder processing
y
conditions (170– 586 MPa, 0– 20 min, and room temperature) to achieve the
nl
same viability losses, > 5.0 to 8.0 log (Tables 10.10 and 10.11).
O
Similar inactivation of vegetative pathogens by room temperature HPP
se
between pathogenic strains belonging to the same species has also been
lU
observed in most of the past works of many authors compiled. For example,
na
the following ranges of processing conditions were recorded: 400– 500 MPa
o
rs
and 5– 20 min for E. coli in cheese and pork with > 6.0 to > 8.5 log (Shigehisa
Pe
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 (Garcia-
r
fo
Graells et al. 1998; Linton et al. 1999; Jordan et al. 2001; Hiremath and
y
op
Ramaswamy 2012) (Table 10.7), 340– 375 MPa and 15– 20 min for L. monocy-
C
togenes in milk and meat with 0.5– 2.0 log (Styles et al. 1991; Patterson et al.
’s
1995), and 500 MPa and 5 min process resulted in > 5.6 log and 500 MPa and
or
5 min for L. monocytogenes in goat cheese with > 5.6 log. (Gallot Lavallee 1998)
ut
(Table 10.8); 400– 450 MPa and 15 min for Salmonella spp. in liquid whole egg
b
tri
with 5.1 to > 7.8 log (Ponce et al. 1999; Bari et al. 2008) (Table 10.9), 586 MPa
on
and 0 min for Vibrio spp. in oyster with > 5.5 to > 6.5 log (Koo et al. 2006)
.C
(Table 10.10), and 375– 400 MPa and 10 min for C. jejuni and Y. enterocolitica in
C
meat products with > 6.0– 8.0 log (Shigehisa et al. 1991; Solomon and Hoover
LL
2004) (Table 10.11). However, on the other hand, few researchers observed
is
large resistance differences among S. aureus in the food products under the
c
an
same conditions of room temperature HPP, for example, log reductions in the
Fr
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),
d
an
which poses a significant concern of this most resistant vegetative pathogen.

or
Due to a few outbreaks registered in raw unpasteurized acidic fruit juices,

yl
E. coli inactivation by HPP and HPTP was also investigated in this class of
Ta
beverages, since it might grow in this environment. Some researchers have

18
shown that different fruit juices resulted in large variations in the room tem-
20
perature 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 vari-
ous food products were carried out using first-order kinetics (Table 10.12).
©
20
18
Ta
yl
or
an
d
Fr
TABLE 10.10 an
c
Inactivation of Vibrio in Oysters and Clam Juice by Room Temperature HPP
is
Pressure Time Log
LL
Species Strains Food Products
C pH (MPa) (min) Reduction References
Vibrio vulnificus MO-624
.
Oyster C nr 586 0 > 6.5 Koo et al. 2006
V. vulnificus MLT 403 Oyster nr 300 2 > 7.0 Ye et al. 2012
on
V. vulnificus nr Homogenized oyster tri nr 275 3 > 7.0 Cook 2003
Vibrio parahaemolyticus TX-2103, serotype O3:K6 Oyster
b
nr
ut 586 0 > 5.5 Koo et al. 2006
V. parahaemolyticus 10 different strains Homogenized oyster nr 300 3 > 6.0 Cook 2003
or
V. parahaemolyticus ATCC 43996 Oyster nr ’s 300 2 7.0 Ye et al. 2012
V. parahaemolyticus T-3765-1 Clam juice 7.5
C 170 10 > 5.0 Styles et al. 1991
op
Note : nr, not reported. y
fo
r Pe
rs
o na
lU
se
363
O
nl
y
©
20 364
18
Ta
yl
or
an
d
TABLE 10.11
Fr
an
Inactivation of Other Pathogenic Vegetative Cells in Meat Products by Room Temperature HPP
cis
Pressure Time Log
Vegetative Cells Strain Meat Products (MPa) (min) Reduction References
LL
C pH
Streptococcus faecalis nr Pork slurry . Cnr 600 10 > 6.0 Shigehisa et al. 1991
Campylobacter jejuni T1 Pork slurry nro 400 10 > 6.0 Shigehisa et al. 1991
C. jejuni ATCC 35921 Chicken puree nrn 400 10 8.0 Solomon and Hoover 2004
Milk nr 375 10 8.0
tri
b
Yersinia enterocolitica nr Pork slurry nr 400 10 > 6.0 Shigehisa et al. 1991
ut
Y. enterocolitica 9610 Ground pork 6.0 304 15 Ellenberg and Hoover 1999
or > 7.0
’s
Citrobacter freundii nr Minced beef 5.6– 5.8 300 C 20 > 6.0 Carlez et al. 1993
Aeromonas hydrophila ATCC 7965 Ground pork 6.0 253 op 15 > 6.0 Ellenberg and Hoover 1999
Note : nr, not reported. y
fo
r Pe
rs
o na
lU
se
O
nl
y
However, note that although first-order kinetic parameters were determined

by the authors, most charts shown in the publications demonstrated a non-
linear 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 fit-
ting carried out with the conventional first-order linear model simplified
the analysis and comparison with the available results from other authors
y
in past literature. S. aureus was confirmed to have the highest resistance
nl
(D 450 = 16.7 min in milk) among other species (Gervilla et al. 1999), whereas
O
V. parahaemolyticus was shown to be the least resistant vegetative pathogen,
se
with a D 136 value of 5.6 min in clam juice (Styles et al. 1991).
lU
Using a secondary model parameter of the first-order kinetics, z P values of
na
204 for E. coli and 359 MPa for S. aureus indicate low microbial susceptibility
o
rs
to small pressure changes (Table 10.12) (O’ Reilly et al. 2000; Hiremath and
Pe
Ramaswamy 2012). From this information, the estimated D 600 MPa values for
r
5 D of S. aureus and E. coli are 28.9 and 0.9 min, respectively.
fo
y
op
C
’s
or
ut
10.6 Future Perspectives

b
tri
High-pressure-treated foods have been categorized “ novel foods” in coun-

on
tries in the European Union (EU) and in Canada, due to the capability to
.C
retain many of the qualities of the fresh food product, which would otherwise
C
be altered by conventional thermal processing. Standard HPP treatments

LL
(400– 600 MPa) can achieve 5.0– 6.0 or more log reductions of most vegeta-
is
tive pathogens since they are susceptible to pressure at ambient temperature.

c
an
However, a few studies demonstrated higher resistance of a few strains of

Fr
microbial pathogens in the vegetative form in certain foods. Although > 6.0

log reductions of E. coli are registered at room temperature HPP, Patterson
d
an
et al. (1995) could only achieve a low level of inactivation (1.5– 3.0 log) in
or
milk and poultry meat after 600 MPa and 15 min, and Teo et al. (2001) has
yl
shown how difficult it is to inactivate a cocktail of E. coli strains in apple and

Ta
orange juices. The survivors of E. coli may grow at low temperature during
18
distribution, posing a human safety concern. Thus, HPP at a temperature of

20
around > 50° C is a possible solution to ensure complete inactivation of E. coli .

Furthermore, more research is needed to obtain reliable inactivation mod-
©
els able to predict the nonlinear behavior of vegetative pathogens in HPP-

treated 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 impor-
tant factors affecting spore inactivation. Some species of Clostridium and
©
20 366
TABLE 10.12
18
First-Order Kinetic Parameters for the Inactivation of Pathogenic Vegetative Microorganisms in Food Products after Room
Ta
Temperature HPP
yl
or
an First-Order Parameters
d Pressure, P D P Value z P Value
Pathogen Strain Food Products
Fr pH (MPa) (min) (MPa) References
Staphylococcus aureus CECT 534
a
Ovine milk n 6.7 450 16.7 nr Gervilla et al. 1999
(NCTC 4163) c is
S. aureus ATCC 6538 Cheese slurry LL 5.2– 5.4 400 20.0 359 O’ Reilly et al. 2000
Escherichia coli CECT 405 Liquid whole egg C 8.0 400 14.1 nr Ponce et al. 1998
(ATCC 10536) .C
E. coli O157:H7 ATCC 43894 Mango juice 4.5o 450 0.72 204 Hiremath and
n Ramaswamy 2012
E. coli K12 ATCC 29425 Cheese slurry 5.2– 5.4 350 19.0 nr O’ Reilly et al. 2000
tri
b
Listeria monocytogenes Scott A UHT milk 6.5 ut 340 13.2 nr Styles et al. 1991
Salmonella enteritidis Liquid whole egg 8.0 400 8.8 nr Ponce et al. 1999
or
Salmonella typhimurium ATCC 7136 Strained chicken nr
’s
340 7.6 nr Metrick et al. 1989
baby food
C
Salmonella senftenberg 775 W Strained chicken nr 340 7.1 nr Metrick et al. 1989
op
baby food
y
fo
Vibrio parahaemolyticus TX-2103 Oyster nr 345 r 2.0 nr Koo et al. 2006
serotype O3:K6
Pe
V. parahaemolyticus T-3765– 1 Clam juice 7.5 136 5.6 rs nr Styles et al. 1991
o
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 tem-
na
perature. 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.
lU
se
O
nl
y
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 inactiva-
tion by HPTP (Evelyn and Silva 2015b, 2016a; Ju et al. 2008) should be fol-
lowed up due to an increase of microbial spore resistance with processing
time. More research is still required to standardize HPTP pasteurization
y
conditions (process criteria, pressure– time– temperature combinations,
nl
etc.) in various food products to successfully introduce HPTP in the food
O
industry.
se
As heat has detrimental effects on food quality, an alternative option is
lU
the simultaneous or sequential application of HPP and other nonthermal
na
food preservation technologies to enhance the lethal effect of HPP (e.g.,
o
rs
irradiation was investigated by Crawford et al. [1996]). Furthermore, the
Pe
cold storage conditions should be topped up with other hurdles, such
r
as modified atmospheres and the use of preservatives, to inhibit or slow
fo
down the growth of resistant sporeformers in HPP pasteurized food
y
products. op
C
’s
or
ut
b
tri
on
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Silva, F. V. M., P. A. Gibbs, H. Nunez, S. Almonacid, and R. Simpson. 2014. Thermal
processes: Pasteurization. In Encyclopedia of Food Microbiology , ed. C. A. Batt and
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and D. Knorr, 55– 76. New York: Kluwer Academic/Plenum Publishers.
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www.who.int/mediacentre/factsheets/fs270/en/.
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Microbiology 32:179– 184.
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11
Application of Pulsed Light for the
Microbial Decontamination of Foods
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se
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Marija Zunabovic, Victoria Heinrich, and Henry Jä ger
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CONTENTS
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11.1 Introduction................................................................................................. 379
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11.2 Pulsed Light................................................................................................. 381
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11.2.1 Fundamentals of Pulsed Light...................................................... 381
op
11.2.1.1 Factor: Treated Matrix..................................................... 381
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11.2.1.2 Factor: Microbial Contamination................................... 382
’s
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11.2.1.3 Factor: Process Setup....................................................... 383

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11.2.2 In-Package Application of Pulsed Light...................................... 386

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11.2.3 Antimicrobial Efficacy of Pulsed Light on Different Food

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on
Matrixes............................................................................................ 388
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11.3 Conclusion................................................................................................... 390

References.............................................................................................................. 390
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11.1 Introduction
Fr
d
Food spoilage is inevitable. Hence, food business operators (FBOs) take

an
diverse actions to preserve the initial state or desired quality level of foods as
or
long as possible (Prokopov and Tanchev, 2007; Aymerich et al., 2008; Rajkovic
yl
et al., 2010). In this context, food processing, and especially food preserva-
Ta
tion, offers the advantages of increased food safety, continuous food sup-
18
ply during season and off-season, availability of value-added products, and

20
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 steriliza-
tion), 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).
379
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 inactiva-
y
tion, however, enzymes and various other food components can be affected.
nl
This can have a positive effect in terms of stabilization of the product, but can
O
also have a negative effect in terms of impairment of quality determining
se
constituents or nutrients. In order to prevent the latter, it is indispensable to
lU
evaluate the actual need for preservation of each food product and food class
na
treated and to adjust the treatment intensity (Prokopov and Tanchev, 2007;
o
rs
Barbosa-Cá novas and Bermú dez-Aguirre, 2011).
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Against this background, recent decades have seen a substantial increase
r
in research and development activities aimed at the substitution of these
fo
conventionally used and historically proven thermal treatments. The
y
op
development was induced by the adjustment of FBOs to factors such as
C
changing societal and demographic conditions, consumer trends, expecta-
’s
tions, and preferences (Aymerich et al., 2008; Sofos, 2008; Havelaar et al.,
or
2010; Knorr et al., 2011; Weiss et al., 2010). As a result, a steadily increasing
ut
proportion and variety of (novel) food products can be found on the market
b
tri
today (Jaroni et al., 2010). The developed alternative technologies thereby

on
range from mild or minimal processing over novel thermal and nonther-
.C
mal physical food preservation to the combination of different treatments

C
(hurdle technology) (Butz and Tauscher, 2002; Devlieghere et al., 2004; Sun,
LL
2005; Rajkovic et al., 2010; Zhang et al., 2011; Chen et al., 2012; Stratakos and
is
Koidis, 2015). The actual impact on the quality of food, applicability, and
c
an
overall benefit of these alternatives is, however, dependent on the respec-

Fr
tive product, contamination, and process setup (Zhang et al., 2011). Further,
successful market introduction is dependent on the improvement of shelf
d
an
life and safety, maintenance of organoleptic and nutritional attributes of

or
the product, freedom from residuals, convenience, economic acceptability,

yl
and acceptance from consumers and legislators (Raso et al., 2005; Rajkovic
Ta
et al., 2010).
18
Focusing on novel physical technologies, novel thermal technologies dis-

20
tinguish themselves from conventional thermal technologies by faster heat-

ing 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, ion-
izing 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 under-
lying principles, the mechanism of microbial inactivation, and the critical
Application of Pulsed Light for the Microbial Decontamination of Foods 381
factors, risks, and limits of the novel nonthermal physical preservation tech-
nology PL.
y
nl
11.2 Pulsed Light
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se
11.2.1 Fundamentals of Pulsed Light
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PL is a novel nonthermal food preservation technology and is capable of
na
inactivating microorganisms in a rapid, efficient, and residue-free manner. In
o
rs
the food sector, the main application areas are decontamination of gases (e.g.,
Pe
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
r
fo
comprises the generation of high-power electrical pulses and transforma-
y
op
tion thereof into short-duration (fractions of a second), high-power pulses
of broad spectrum (approximately 180– 1100 nm) electromagnetic radia-
C
’s
tion (light) via an inert-gas (xenon) flash lamp (Dunn et al., 1989). The basic
or
essentials and schematic layout of a PL device are, for example, provided by

ut
Heinrich et al. (2016b).

b
tri
Based on the scientific literature, three main factors affecting the efficacy
on
of a PL treatment were identified. These are the respective (1) type of matrix
.C
(e.g., food) treated, (2) the microbial contamination, and (3) the process param-
C
eter chosen (Heinrich et al., 2016b). To achieve interpretable, comparable, and

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reproducible decontamination outcomes, it is of upmost importance to prop-

is
erly record and communicate these factors. Further, it should be aimed at

c
an
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
Fr
and Gó mez-Ló pez, 2006; Gó mez-Ló pez et al., 2007). The following sections

d
an
discuss the above-listed main factors and their impact on the treatment effi-
cacy of PL from a general point of view. Further on, food category– specific
or
yl
information is given.
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18
11.2.1.1 Factor: Treated Matrix

20
Transparency and opacity, surface characteristics, and composition of the

©
matrix strongly influence the efficacy of a PL treatment. Concerning trans-

parency or opacity, optimal results are achieved when the matrix exhibits a
low reflection coefficient and, at the same time, high absorption and trans-
mission 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
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
y
do not show a pronounced light-absorbing effect (Gó mez-Ló pez et al, 2005a;
nl
Rajkovic et al., 2010).
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se
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11.2.1.2 Factor: Microbial Contamination
na
Microbial contamination impacts the efficacy of a PL treatment in various
o
rs
aspects. These include the respective microorganism and its physiological
Pe
constitution, population density, and growth parameters (e.g., growth rate
r
and lag time) (Dunn et al., 1989; Augustin et al., 2011; Cudemos et al., 2013).
fo
Regarding the respective microorganism, some distinctions in suscepti-
y
op
bility can be derived. For example, it seems that due to the differences in
C
cell structure, gram-positive bacteria are more resistant to PL than gram-
’s
negative bacteria. Further, mucoid and pigment-forming bacteria seem to

or
be more resistant than their counterparts, fungi seem to be more resistant

ut
than bacteria, and bacterial spores tend to be more resistant than their cor-
b
tri
responding vegetative cells. Interestingly, the size of bacteria can also have
on
an impact. Namely, the smaller the cell, the faster the heat induced by the
.C
electromagnetic radiation can dissipate from the surface and the more resis-
C
tant the cells are (Rowan et al., 1999; Anderson et al., 2000; Farrell et al.,
LL
2010). Decontamination efficacy can also be impaired by the factors high

is
population density (mutual shading of cells) and stationary growth phase.

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an
Consequently, prompt treatment after microbial contamination of the matrix

Fr
takes place is recommended (Hiramoto, 1984; Anderson et al., 2000; Gó mez-

d
Ló pez et al., 2005b; Farrell et al., 2010; Rajkovic et al., 2010).

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Studies related to the viral inactivation on food-related surfaces and in

or
food matrices are scarce. More studies with drinking water and wastewater
yl
in this context can be found (Yi et al., 2016; Barrett et al., 2016). The inactiva-
Ta
tion effect relies on observations in modifications of nucleic acid and cap-

18
sid (Vimont et al., 2015). The antiviral application of PL in drinking water is

20
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 infectiv-

ity 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).
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 struc-
tural 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
y
to conventionally used continuous-wave ultraviolet (CW UV) systems
nl
(Dunn et al., 1989). Contrary to the initial expectation, the inactivation
O
curve of PL has been repeatedly shown to be nonlinear. As a consequence,
se
commonly used log-linear mathematical models often cannot be used to
lU
accurately describe the inactivation pattern observed (Luksiene et al.,
na
2007; Uesugi and Muraru, 2009; Farrell et al., 2010; Keklik et al., 2012).
o
rs
In most cases, the authors found a sigmoid-shaped inactivation curve.
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The specific shape originates from a three-phased inactivation course.
r
First, cells experience a nonlethal injury, which is reflected by the char-
fo
acteristic shoulder of the curve. Second, the surviving fraction rapidly
y
op
declines because a maximum of cell injury and a minimum of additional
C
energy required to cause high cell death rates are reached. Lastly, a so-
’s
called tailing is reached. Here, lack of a homogenous population, multihit

or
phenomena, varying susceptibility of strains, started cell-repair activity,

ut
shading of cells by suspended solids or objects, and declined probability

b
tri
of exposure to PL causes decreased cell death rates (MacGregor et al., 1998;

on
McDonald et al., 2000; Yaun et al., 2003; Fine and Gervais, 2004; Gó mez-
.C
Ló pez et al., 2007). In some cases, no shoulder and/or tailing is observed.

C
This can be attributed, on the one hand, to a high initial energy input and,
LL
on the other, to a low initial cell population (Otaki et al., 2003; Wang et
is
al., 2005; Farrell et al., 2010). Hence, the Weibull-type mathematical model
c
an
and the log-linear mathematical model including a shoulder and/or tail

Fr
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,
d
an
2008b; Heinrich et al., 2016a).

or
yl
Ta
11.2.1.3 Factor: Process Setup

18
The chosen process setup has a significant influence on the decontamina-

20
tion efficacy. Factors comprise the spectrum, experimental factors, geome-

try 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 great-
est 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 param-
eters fluence rate, fluence, number of pulses, pulse width, exposure time,
frequency, and peak power have to be adjusted to the respective situation
(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, high-
frequency pulses (Hiramoto, 1984; Dunn et al., 1989; MacGregor et al., 1998;
Anderson et al., 2000; Wekhof, 2000; Panico, 2005; Wang et al., 2005; Gó mez-
y
Ló pez et al., 2007; Luksiene et al., 2007; Farrell et al., 2010; Levy et al., 2012).
nl
Special attention should be paid to the possible overheating of the matrix
O
due to an excessive PL treatment. To avoid this, a sufficient cooling system
se
and cooling period between the pulses, limited treatment duration, and
lU
appropriate distance between the flash lamps and matrix should be empha-
na
sized (Dunn et al., 1989; Gó mez-Ló pez et al., 2005b). It should be noted that
o
rs
with decreasing distance between the lamps and the treated matrix, the
Pe
effect of PL on the surface rises. At the same time, however, the frame of
r
high-efficacy treatment narrows down (Gó mez-Ló pez et al., 2005b). In the
fo
specific case of globular bodies, multidirectional and uniform illumina-
y
op
tion of all surfaces can be facilitated by increasing the distance to the light
C
source and the treatment time, but also through relative movement of the
’s
body. Another possibility is a conveyor having transparent sections or the

or
installation of reflectors (Lagunas-Solar and Gó mez-Ló pez, 2006).

ut
PL can be conducted at various stages in a food processing plant. These

b
tri
are, for example, the decontamination of raw materials and food contact
on
materials used, in-process treatment, avoidance of recontamination, and

.C
treatment of the final product prior to or post packaging (Wong, 1998; Lyon
C
et al., 2007; Ferná ndez et al., 2009; Uesugi and Muraru, 2009; Rajkovic et al.,
LL
2010).
is
In 1966, the U.S. Food and Drug Administration (FDA) approved PL for
c
an
food applications in the United States from a technology-oriented approach

Fr
(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 ori-
d
an
ented. Connected with this is Regulation 258/97 (1997), “ Novel Foods and
or
Novel Food Ingredients” (article 1, item f). Decontamination of food contact

yl
materials such as packaging materials is, however, not affected hereby and
Ta
has been used in the EU and beyond for several years (Dunn et al., 1995;
18
FDA, 1996; EU, 1997).

20
Also, the combination with different thermal and nonthermal decontami-

nation 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 decon-
tamination 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.
TABLE 11.1
Overview on Advantages and Limitations in Relation to Main Factors Influencing
PL Efficiency
Factor Advantages Disadvantages

Quality Applicable in combination with Limited to surface decontamination
other preservation technologies
y
nl
Decontamination of food (packed/ Product characteristics (e.g,. surface
O
unpacked) and food contact opacity and composition) and
se
surfaces contamination degree may reduce
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effectiveness
na
Effective against pathogenic and Limited packaging material can be
spoilage microorganisms used
o
rs
4– 6 times more effective than CW Control of postprocess parameters
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UV critical for shelf life
Implementation in Hazard Photoreactivation possible (SOS
r
fo
Analysis and Critical Control response of microorganisms)
y
Point systems op
Resistance formation of bacteria is Colored food products may show
C
currently not described undesirable effects
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Matrix effect Nonthermal surface treatment Adjustment of processes necessary

or
to avoid product impairment and

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surface heating
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tri
Minimally processed technique Uniformity of treatment is limited

on
Ecology Safe to apply (industrial safety) Ozone formation

.C
Environmentally friendly (lack of —

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residual compounds)
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Xenon lamps (broad spectrum) —

is
Low energy input —

c
Convenience Short processing times (4– 6 times Problematic standardization due to

an
lower than with CW UV) geometric configuration and

Fr
process parameters
d
Integration into existing processes Only a few suppliers on the market

an
Reduced space requirement —

or
In batch or continuous mode —

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Ta
Instant action adjustable to product —

flow
18
Natural cooling of lamps between —

20
pulses
Light spectrum adjustable by —
©
pulse-forming network or filters

to the respective situation
High UV intensities during a pulse —
(up to 30% UV)
(Continued)

Overview on Advantages and Limitations in Relation to Main Factors Influencing
PL Efficiency
Factor Advantages Disadvantages
Economic Low operation costs High investment costs
(€ 300,000– € 800,000)
y
nl
High efficiency (40%– 50% Technology for high-value-added
O
conversion of electrical energy products or particular market
se
into optical) situations
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Emerging technology Lamps: Lifetime depends on
operating parameters (average
na
lifetime, 6– 12 months; costs (few
o
rs
times higher than for CW UV),
about € 700 each due to
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sophisticated and costly design)
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— Sophisticated and costly driving
circuits necessary for long lifetime
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and high UV output of lamps
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(10– 100 times more expensive than
for CW UV)
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Acceptance Good consumer acceptance —

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FDA-approved technology Technology per se not approved in

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the EU (novel food regulation)

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11.2.2 In-Package Application of Pulsed Light

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PL can be used for in-package application. To obtain satisfactory results,

is
packaging materials have to be selected with regard to transmissibility of

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light and, in particular, the UV fraction thereof. Conversely, this means

Fr
that opaque materials and matrixes, such as ink-printed labels or draw-

d
ings, are not suitable. Only if this condition is met are light incidence from
an
any direction and uniform decontamination of the matrix possible (Dunn

or
et al., 1989; Palmieri and Cacace, 2005; Elmnasser et al., 2007; Han, 2007;
yl
Oms-Oli et al., 2010). Of the groups of packaging materials available, only

Ta
glass and polymeric materials (plastic) qualify for in-package application

18
of PL (Dunn et al., 1989; Eie, 2009). Of the two groups, the importance of
20
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 char-
acteristics. This range is considerably influenced by factors such as the
basic structure of the material, processing characteristics, and the crys-
tallinity and incorporation of additives (Kirwan and Strawbrigde, 2003;
NOVA Institut, 2013). The last two points exhibit particular influence
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 con-
trast, crystalline structures and the presence of morphological inhomo-
geneity cause opacity of the material based on a high scattering power
(Hernandez et al., 2000). Further, transmittance is influenced by some
y
of the various end-use additives, which are possibly added for market
nl
appeal or functional demands during processing. These are, for example,
O
UV protective agents, dyes, or coatings (Crompton, 2007; Pospí š i l and
se
Neš pů rek, 2008). Against this background, the incident light is partly
lU
reflected or absorbed by the packaging material. As a result, even trans-
na
missible materials can significantly reduce the light intensity and mod-
o
rs
ify the initial light spectrum (Fellows, 2009).
Pe
In the context of light transmission, a specific measure below which
r
transmission through a material is negligible (absorbance of 1) is the cut-
fo
off wavelength given in nanometers (Hernandez et al., 2000). As mentioned
y
op
previously, UV light below 270 n m is especially important for the decon-
C
tamination process (Dunn et al., 1991; Wekhof, 2000; Takeshita et al., 2003;
’s
Wang et al., 2005; Levy et al., 2012). This implies that polyolefines (a group
or
comprising polyethylene [PE] and polypropylene [PP]) with a cutoff wave-

ut
length below 180 n m seem to be best suited for in-package application of PL.
b
tri
Further, cutoff wavelengths for commonly used plastics are approximately

on
240 n m for polyvinyl chloride (PVC) and polyamide (PA), 270 n m for poly-
.C
styrene (PS), 280 n m for polycarbonate (PC), and 310 nm for polyethylene
C
terephthalate (PET). All cutoff wavelengths given are for 10-micron thick
LL
films (Carlsson and Wiles, 1986). One should also consider that the given
is
values are reference values that can vary to a great extent with the respective
c
an
properties and thickness of the material, as well as with the assembly (e.g.,
Fr
multilayer formed by coextrusion or lamination). So far, polyolefines and

PA are the main polymers used for in-package application of PL (Heinrich
d
an
et al., 2015).
or
It is well known that UV light induces reactions and changes in polymers.

yl
This can affect the physical, mechanical, and chemical characteristics of a

Ta
certain material. Therefore, it is recommended, next to the transmissibility of

18
light, to select materials that withstand the intended treatment. Otherwise,

20
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 protec-
tion, 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 applica-
tion of PL is provided by Heinrich et al. (2015).
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 per-
y
nl
spective, food processors are constantly evaluating disinfection procedures
O
carried out through washing steps with different types of sanitizers, such
se
as sodium hypochlorite (Tirpanalan et al., 2011). However, the formation
lU
of potential toxic by-products prompted the reduction of dose applica-
na
tions, leading to decreased antimicrobial effects. Agü ero et al. (2016) tested
o
Spinach leaves inoculated with Listeria innocua and E. coli strains with the
rs
XeMaticA-2L System (Steribeam Systems GmbH, Germany). Fluences lower
Pe
than 10 kJ m– 2 showed reductions of 1.85 and 1.72 log CFU g– 1 for L. innocua
r
fo
and E. coli, respectively. The background population (mesophilic, psychro-
y
trophic, and coliforms) present in spinach could also be significantly reduced
op
with PL treatments at 20 and 40 kJ m– 2 (Agü ero et al. 2016). However, the
C
internalization-in-tissue effect of the native microflora is not negligible and
’s
or
may lead to a lower inactivation degree than the inoculated cultures applied
ut
in studies. Huang et al. (2017) evaluated the inactivation effect of PL on

b
human norovirus surrogates and outbreak-related bacteria (E. coli O157:H7

tri
on
and Salmonella enterica serotype Newport H1275) on two types of berries.

.C
The results showed the highest sensitivity of E. coli, followed by Salmonella,

and finally the virus surrogates. Due to the complex topography of the ber-
C
LL
ries, 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 shad-
cis
owing effects of the fruits. In addition, a higher inactivation effect could be

an
observed for blueberries than for strawberries. Another study (Kramer et al.,
Fr
2016) demonstrated the inactivation degree of PL with a reflector composed

d
of three xenon lamps. Bacteria in leafy greens and mung bean sprouts were
an
challenged under simulated washing steps at a distance of 5 cm between the

or
water surface and lamp. The results showed that PL was more effective (2
yl
Ta
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
18
et al., 2016). Again, the applied energy had less impact on the results than
20
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).
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,
y
grape juice, plum juice, three types of carbonated drink, milk, and coffee bev-
nl
erage without milk. In contrast to the studies on the above-mentioned fruits
O
and vegetables, the bactericidal PL effects are dependent on the total fluence.
se
A comparable reduction of P. aeruginosa by 7 log with 0.97 J cm– 2 in mineral
lU
water could be reached in apple juice, carbonated beverages, and plum juice
na
after treatment with 12.17– 24.35 J cm– 2 (Hwang et al., 2015).
o
rs
The efficacy of PL treatment in different meat matrices was critically
Pe
reviewed by Heinrich et al. (2016b).
r
Postprocessing contamination due to handling, slicing, and packaging
fo
plays a crucial role for sliced cheese products. Proulx et al. (2015) tested dif-
y
op
ferent cheese substrates (cheddar and process cheese) through inoculation
C
of relevant gram-negative and gram-positive bacterial reference strains at
’s
fluence levels from 1.02 up to 12.29 J cm– 2. The inactivation levels reached
or
3 log reductions for all bacteria at doses below 12 J cm– 2. Related to the sur-
ut
face topography, cheddar cheese showed a greater number of cavities per

b
tri
unit area, offering more potential for microbial shading (Proulx et al., 2015).
on
Another study, by Innocente et al. (2014), investigated the application of PL as

.C
thermal treatment for raw milk with a 3.2 log decrease of total viable counts
C
with fluences of 26.25 J cm– 2 sufficient for the levels requested for cheese
LL
making. The tested parameters showed potential especially for milk types
is
particularly sensitive to heat damage (e.g., goat and donkey).

c
an
In the RTE meat sector, PL was applied as postprocessing decontamination

Fr
to inactivate Listeria monocytogenes and Salmonella Typhimurium, known as

recontamination agents, and for shelf life prolongation (Ganan et al., 2013;
d
an
Hierro et al., 2011). Hierro et al. (2011) demonstrated a triple shelf life exten-
or
sion of cooked ham, however, with sensory losses with fluence doses above
yl
2.1 J cm– 2. The microbial safety of beef and tuna carpaccio could be improved
Ta
using fluences of 8.4 and 11.9 J cm– 2 achieving an approximate 1 log reduction
18
of surface-inoculated pathogens, such as L. monocytogenes, E. coli, Salmonella

20
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 decon-
tamination 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.
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
y
hidden microorganisms, may hamper the inactivation degree. The germi-
nl
cidal effect should be elaborated on at a molecular basis in order to combine
O
the knowledge with tailored technological setups. PL is a promising non-
se
thermal decontamination and preservation technology. However, further
lU
studies are needed to demonstrate the scale-up potential for specific food
na
types, such as sliced or cured meat products or leafy greens.
o
rs
rPe
fo
y
op
C
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bating microbial contamination in food chain. International Journal of Food

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Microbiology , 141, S29– S42.

Raso, J., Pagá n, R., and Condó n, S. 2005. Nonthermal technologies in combination with
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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.
Stratakos, A. C., and Koidis, A. 2015. Suitability, efficiency and microbiological safety
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Sun, D. W. 2005. Emerging Technologies for Food Processing . Amsterdam: Elsevier.
Takeshita, K., Shibato, J., Sameshima, T., Fukunaga, S., Isobe, S., Arihara, K., and
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rs
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b
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20
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IA: Wiley-Blackwell.
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20
18
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12
Effect of Commercial Emerging
Nonthermal Technologies on Food
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Products: Microbiological Aspects
O
se
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o na
Elisabete M. C. Alexandre, Rita S. Iná cio, Ana C. Ribeiro,
rs
Á lvaro Lemos, Sofia Pereira, Só nia M. Castro, Paula Teixeira,
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Manuela Pintado, Ana M. P. Gomes, Francisco J. Barba,
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Mohamed Koubaa, Shahin Roohinejad, and Jorge Saraiva
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CONTENTS
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12.1 Introduction ................................................................................................ 398

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12.2 High-Pressure Processing......................................................................... 398

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12.2.1 Engineering Principles................................................................... 399

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12.2.2 Biological Effects.............................................................................400

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12.2.3 Practical Applications .................................................................... 401

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12.2.4 Limitations, Challenges, and Future Trends ............................. 403

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12.3 Pulsed Electric Field...................................................................................404

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12.3.1 Engineering Principles...................................................................404

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12.3.2 Biological Effects.............................................................................405

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12.3.3 Practical Applications.................................................................... 406

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12.3.4 Limitations, Challenges, and Future Trends.............................. 407

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12.4 Atmospheric Cold Plasma......................................................................... 409

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12.4.1 Engineering Principles................................................................... 409

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12.4.2 Biological Effects............................................................................. 410

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12.4.3 Practical Applications.................................................................... 411

18
12.4.4 Limitations, Challenges, and Future Trends.............................. 414

20
12.5 Other Commercial Emerging Nonthermal Technologies..................... 414

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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
References ............................................................................................................. 420
397
12.1 Introduction
Consumer demands for high-quality, fresh-tasting, and nutritious foods have
generated considerable interest in the development of new food processing tech-
niques. The traditional heat treatments are efficient in microbial inactivation,
y
reducing product decay, and attaining safety targets. However, they have a signifi-
nl
cant impact on product quality. Some quality parameters, such as texture, color,
O
aroma, flavor, taste, and nutritive value, can be severely affected by temperature.
se
Nonthermal and ecofriendly methodologies, such as high-pressure process-
lU
ing (HPP), pulsed electric field (PEF), atmospheric cold plasma (ACP), osmotic
na
dehydration (OD), ozonation, or the use of chlorine dioxide, have been studied
o
rs
by both industry and academia, in an attempt to meet the challenges of pro-
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ducing safe processed foods with high-quality standards (Balasubramaniam
et al. 2015). HPP, PEF, and ACP are among the most commercial technolo-
r
fo
gies being studied, and some processed products can already be found in
y
op
the market, particularly in the case of HPP, PEF, and ozone application. High
C
pressures in the range of 400– 600 MPa, at room or chilled temperatures, can
’s
be useful for food pasteurization of liquid and solid foods, ensuring inactiva-
or
tion of pathogenic and spoilage bacteria, yeasts, molds, viruses, and spores
ut
(Balasubramaniam et al. 2015). PEF technology typically involves the appli-

b
tri
cation of short-duration pulses (from several nanoseconds to several milli-

on
seconds), of high electric field strengths (from 100 V/cm to 80 kV/cm) and a
.C
specific energy input in the range of 50– 1000 kJ/kg (Koubaa et al. 2015). This
C
technology is able to inactivate pathogenic microorganisms such as Listeria

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monocytogenes , Escherichia coli , and Salmonella Typhimurium, without signif-

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icant loss of the organoleptic and nutritional properties of food. However,

c
an
data on the inactivation of some microorganisms, such as Bacillus cereus and

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Staphylococcus aureus , by PEF processing are very limited. The application

of ACP technology for food processing applications is very recent (Niemira
d
an
2012a). However, the strong thermodynamic nonequilibrium nature of this

or
methodology, as well as the low gas temperature, presence of reactive chemi-

yl
cal species, and high selectivity of the method, make it very promising for
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food processing. In this chapter, some of the main engineering principles, bio-
18
logical effects, practical applications and limitations, challenges, and future

20
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 guaca-
mole 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
y
is the time required to inactivate microorganisms and enzymes at low tem-
nl
peratures, depending on the food matrix and the type of microorganism
O
to be eliminated (Syed et al. 2016). The main advantage of using HPP is the
se
capacity to expand the shelf life and improve food safety due to the inac-
lU
tivation of pathogenic and spoilage vegetative bacteria, yeasts, and molds
na
(Balasubramaniam et al. 2015).
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12.2.1 Engineering Principles
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HPP has been the most successful alternative technology adopted by the food
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industry for the pasteurization of foods at refrigeration or ambient tempera-
C
tures, since a low impact on their functional properties and nutritional values
’s
has been verified (Barba et al. 2012, 2015b). The operation of HPP is based
or
on two fundamental physical principles: (1) the isostatic principle and (2) Le
ut
Chatelier’ s principle. According to the isostatic principle, the pressure applied

b
tri
is transmitted instantaneously and uniformly throughout the sample, inde-

on
pendent of the volume, size, shape, and geometry of the product (Considine
.C
et al. 2008). Hence, all parts of foods are exposed to high pressure at the
C
same conditions and at the same time. However, pressurization of liquids

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or solid foods at room temperature is typically accompanied by an increase

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in the food temperature, called adiabatic heating, which depends mainly

c
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on the food composition. The heat associated with the increase of pressure
Fr
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
d
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for HPP of liquid foods is presented in Figure 12.1. Le Chatelier’ s principle

or
states that reactions that involve a volume change are influenced by pressure,
yl
and pressure favors those reactions that result in a decrease in volume (Lou
Ta
et al. 2015). For pressure application, the packaging material must be flexible
18
because foods decrease in volume under pressure and regain volume during
20
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 prod-
uct 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 usu-
ally at low temperatures. At the end of the treatment, the vessel is depressur-
ized and the product is unloaded. Water is usually used in industrial-scale
equipment as the pressure-transmitting fluid (Balasubramaniam et al. 2015).
Feeding Pressuring Discharging
High-
pressure
pump Water
High-pressure
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vessel
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Free piston
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Liquid food
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Water tank
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Treated
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Untreated
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product
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FIGURE 12.1
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Schematic presentation of HPP system of liquid food.

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12.2.2 Biological Effects

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The impact of HPP treatment on microbial inactivation is influenced by the

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level of pressure, temperature, and treatment time used, as well as by the food
c
an
matrix characteristics. In fact, the pressure and treatment time are the key fac-
Fr
tors for microbial inactivation. The levels of pressure used in the food indus-
d
try only affect noncovalent bonds, such as hydrogen, ionic, and hydrophobic
an
bonds (Lou et al. 2015). Hence, the structures of low-molecular-weight mol-

or
ecules, such as vitamins, peptides, fatty acids, saccharides, and the primary
yl
structure of proteins, remain intact because the covalent bonds have very low
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compressibility at high pressure. On the other hand, the native structure and
18
functionality of macromolecules, such as proteins, enzymes, polysaccharides,

20
and nucleic acids, may change during HPP (Daryaei and Balasubramaniam
2012; Considine et al. 2008). Consequently, the secondary, tertiary, or qua-
©
ternary 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 mul-
tiplicity of injuries accumulated in different components of the cell, includ-
ing 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
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 weak-
ened, 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
y
flow of internal substances, leading to bacterial death (Huang et al. 2014a).
nl
Below 300 MPa, most of these reactions are reversible. Another effect of HPP
O
is the disruption of ribosome configurations, which leads to a lower pro-
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tein biosynthesis and inhibition of protein repair. In addition, HPP can also
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negatively affect the functionality of genetic materials in microorganisms,
na
such as DNA replication and gene transcription, due to condensation of the
o
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genetic materials, leading to degradation of the chromosomal DNA (Huang
Pe
et al. 2014a).
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12.2.3 Practical Applications op
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The efficacy of a high-pressure pasteurization process for microbial inacti-
’s
vation depends on the HPP conditions (pressure level, holding time, treat-
or
ment temperature, rate of compression, and decompression), microbiota

ut
(type and physiological state), and food matrix (pH or acidity and water
b
tri
activity) (Huang et al. 2014a; Syed et al. 2016). In this chapter, the effects of
on
HPP on some of the most important microorganisms (e.g., E. coli , Salmonella

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spp. , S. aureus , and L. monocytogenes ) causing food- and waterborne diseases

C
are highlighted. The results summarized in Table 12.1 show the feasibility
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of using an HPP system to reduce these microorganisms in different food

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products.
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In general, an increase of the pressure’ s treatment time and/or an increase

Fr
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
d
an
is treated with HPP at different combinations of pressure and holding times

or
(200– 600 MPa for 1– 60 min), having revealed a linear relationship among
yl
the inactivation level and treatment times under all the tested pressures (Van
Ta
Opstal et al. 2005). Regarding the effect of the compression and decompres-
18
sion rates, there is little information available in the literature.

20
The literature reported that the pressure resistance of prokaryotes, gram-

positive bacteria and cocci exceeds that of eukaryotes, gram-negative bacte-
©
ria 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 micro-
biological safety of an HPP-treated (400 and 600 MPa for 1, 5, and 10 min)
©
TABLE 12.1
20 402
18
Main Examples of HPP Food Products (Pressure, Temperature, and Time) and Log Reductions Caused in Some of the Most Relevant
Food Safety– Related Microorganisms
Ta
Pressure Temperature Time
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Microorganism Food Product an (MPa) (° C) (min) Log Reductions References
Escherichia coli Fresh carrot juice d 200– 600 5– 30 1– 60 0.5– 8 (nd) Van Opstal et al. (2005)
Strawberry-blueberry 400 and 600 4 5 and 10 1.9– 5.9 Tadapaneni et al. (2014)
beverage
Fr
an
Raw milk cheese 500c 10 3 5 (nd) Rodrí guez et al. (2005)
Apricot, orange, and 250– 450 is 25– 30 5– 20 4.9– 6.8 Bayı ndı rlı et al. (2006)
cherry juice LL
Dry fermented salami 483 and 600 C 5 1– 5 4.7– 5.8 (nd) Porto-Fett et al. (2010)
Salmonella Enteritidis Nuts 400 and 600 20 1 Prakash (2013)
. C5– 20
Apricot, orange, and 250– 450 25–
o3n0 5– 20 4.7– 7.3 Bayı ndı rlı et al. (2006)
cherry juice tri
Salmonella Typhimurium Strawberry-blueberry 400 and 600 4 b 1.9– 6.0 Tadapaneni et al. (2014)
beverage
ut 5 and 10
Dry fermented salami 483 and 600 5 1.9– 2.4 Porto-Fett et al. (2010)
or 1– 5
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Listeria monocytogenes Strawberry-blueberry 400 and 600 4 5 andC 10 2.9– 5.8 Tadapaneni et al. (2014)
beverage op
Dry fermented pork 400 10 17 y 0.6 Jofré et al. (2009)
sausage fo
Dry fermented salami 483 and 600 5 1– 5 r1.6– P5.0 (nd) Porto-Fett et al. (2010)
Raw cow’ s milk 500 10 5 5.02– 6e.34 (nd) Rodrí guez et al. (2005)
cheese rs
Staphylococcus aureus Dry fermented pork 400 10 17 0.3 n
o Jofré et al. (2009)
sausage a
Raw cheese 500 10 2 5.3 (nd)
l U Rodrí guez et al. (2005)
Apricot, orange, and 250– 450 25– 30 5– 20 4.0– 5.7
sBayı
e ndı rlı et al. (2006)
cherry juice
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Note : nd, not detected (below detection limit).
nl
y
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. monocyto-
genes 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.
y
Recently, Syed et al. (2016) reported that microorganisms in different
nl
food matrixes were more resistant to HPP, probably due to the protection
O
provided by food chemical composition (e.g., the presence of fats, proteins,
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minerals, and sugars). Lower water activity (aw < 0.9) and/or higher pH of
lU
a food product led to an increase of microbial resistance (Syed et al. 2016).
na
For instance, in nuts treated with HPP at 600 MPa for 20 min, Salmonella
o
rs
counts were reduced to less than 1 log (Prakash 2013). In another study, dry
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fermented pork sausages (aw ≈ 0.93) inoculated with L. monocytogenes and
r
S. aureus were treated by HPP (400 MPa, 10 min) and revealed 0.6 and 0.3 log
fo
reductions, respectively (Jofré et al. 2009). On the other hand, dry fermented
y
op
salami (aw < 0.94) was treated by HPP (483 and 600 MPa for 1– 5 min) and
C
showed a reduction of pathogen numbers of 1.6 to ≥ 5.0 (L. monocytogenes ),
’s
4.7 to ≥ 5.8 (E. coli O157:H7), and 1.9– 2.4 (Salmonella ) log CFU/g (Porto-Fett
or
et al. 2010).
ut
Regarding the effect of pH, Bayı ndı rlı et al. (2006) inoculated different

b
tri
microorganisms (e.g., S. aureus 485, E. coli O157:H7, and S. Enteritidis FDA)
on
into juices (e.g., apricot juice [pH 3.8], orange juice [pH 3.76], and sour cherry
.C
juice [pH 3.30]) and evaluated the effect of HPP treatment (400 and 600 MPa,
C
5 and 10 min) on the microbiological safety of the products. Among the

LL
juices, pressure sensitivity increased more in sour cherry juice (more acidy)
is
than in apricot juice. E. coli was the most resistant to HPP (600 MPa, 1 min),
c
an
showing a 2.8 log reduction, while S. aureus 485 and S. Enteritidis were less
Fr
resistant, showing more than a 5 log reduction. On the other hand, yeasts
and molds have the lowest resistance to HPP, which is usually below the
d
an
detection limit in the range of pressure used for pasteurization (Iná cio et al.
or
2014). Thus, safety evaluation of commercial emerging nonthermal technolo-

yl
gies, such as HPP, on food products depends on reliable estimation of their

Ta
efficacy against pathogenic and spoilage foodborne microorganisms.

18
20
12.2.4 Limitations, Challenges, and Future Trends

©
HPP requires high-pressure equipment, which is expensive and requires

high investment. Intermittent operation and small workload are other limita-
tions that increase the cost of production. Moreover, HPP allows inactivation
of vegetative microorganisms but is not sufficient to substantially destroy
spores (Balasubramaniam et al. 2015). Since 1990, when the first HPP indus-
trial machine was applied, more than 320 machines have been implemented
in industrial operation. This shows exponential growing in the evolution of
this system in the food industry. Compared with other continents, America
and Europe are the main users of this technology. In 2014, more than 500
million kg of food was treated by HPP; 27% corresponded to meat products,
namely, pathogen-free sliced cooked meats, preservative-free deli meats, and
Listeria -free dry-cured products. The vegetable product, juice, and beverage
industries are other food sectors that use HPP, mainly due to the significant
increase in health awareness (Hyperbaric, 2015) and stringent regulations
y
pertaining to the addition of preservatives. In the coming years, further pop-
nl
ularity and use of HPP will be noticed in the food industry, mainly due to
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the reduction of the investment cost with the increased competition between
se
the manufacturers.
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12.3 Pulsed Electric Field
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PEF is a novel nonthermal food preservation technology that is used to erad-
C
icate foodborne pathogens and control spoilage microorganisms in foods,
’s
allowing the making of products with physical, chemical, and organoleptic

or
properties that are similar to those of fresh food products (Barba et al. 2015a).
ut
PEF has a volumetric effect, ensuring fast and homogeneous application of

b
tri
the lethal principle through the treated product. Thus, this technology could
on
be considered a very promising alternative or, at the very least, a complement

.C
to traditional processes for the pasteurization of highly thermal-sensitive

C
foods.
LL
cis

an
Fr
PEF typically involves the application of short-duration pulses (from several

nanoseconds to milliseconds) of high electric field strengths (from 100 V/cm
d
an
to 80 kV/cm) and a specific energy input in the range of 50– 1000 kJ/kg

or
(Koubaa et al. 2015). High-voltage electrical pulses are applied to a set of

yl
electrodes, which then conduct the high-intensity electrical pulses to a prod-

Ta
uct placed between these electrodes (Zimmermann and Benz 1980). Thus,
18
the product experiences a force per unit charge (the electric field), which
20
is responsible for the inactivation of microorganisms by irreversible break-

down of the cell membranes. Figure 12.2 shows a schematic representation
©
of a typical PEF treatment system of a food product.

A PEF system comprises a high-voltage power source, which converts volt-
age from a utility line into high voltage; one or more capacitors that are used
to store the energy and high-voltage switch; a liquid handling system for
preheating and cooling of the product (which are used for pre- and post-
treatments, respectively); a pump, which conveys the product through the
treatment chamber; and monitoring devices (oscilloscope and temperature
sensors) to control and report the conditions (Zhang et al. 1995).
Control
High-voltage
system
pulse generator
T° sensor
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Fluid
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HV electrode
PEF treatment chamber
Pump Cooling coil

direction
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Treated Treatment area
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material Ground electrode T° sensor
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Insulator
Electric field
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HV electrode direction
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y
T° sensor
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Untreated Treated
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product product
’s
or
FIGURE 12.2
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Schematic representation of PEF processing platform of liquid foods. HV, high voltage.
b
tri
on
.C

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The mechanism of PEF processing to inactivate microorganisms is not fully

LL
elucidated, but it is generally accepted that the primary effect on biological

is
cells is related to local structural changes and breakdown of the cell mem-
c
an
brane (Knorr et al. 2008). The dielectric rupture theory considers that the cell
Fr
membrane acts as a capacitor filled with a dielectric medium (Zimmermann

d
1986). However, there is a difference in value of dielectric constant between

an
the cell membrane and cell cytoplasm, which results in a transmembrane

or
potential (TMP). The TMP is generated through the accumulations of free

yl
charges at the inner and outer surface of the cell membrane (Jeyamkondan
Ta
et al. 1999). The application of an external electric field induces ion movement
18
along the field so that they are restrained and accumulated at the membrane,
20
increasing the TMP (Teissié et al. 1985). Opposite-charge ions in and outside
the cell membrane are attracted to each other, causing the compression of
©
the membrane and reducing its thickness. Consequently, when an electrical

field exceeds the critical threshold value of the TMP (typically 0.2– 1.0 V), an
electrical breakdown or pore formation may occur (Teissié et al. 1985).
According to the electroporation theory, when the cell is subjected to a
high electric field, destabilization of proteins and the lipid bilayer of the cell
membrane will occur (Castro et al. 1993). Protein channels present in the
cell membrane are regulated by the TMP. However, when they are exposed
to a high field intensity, they are irreversibly denatured by Joule heating or
electrical modification of functional groups (Tsong 1991). Similarly, when

the cell is under an electric field, the hydrophobic pores of lipid molecules
expand until “ pore inversion” occurs, showing the reorientation of phospho-
lipids in a low-energy configuration, forming hydrophilic pores, and these
phenomena are responsible for electroporation (Glaser et al. 1988).
The applicability of this technology for food pasteurization and its lethal
y
effects on microorganisms depends on PEF processing parameters (equip-
nl
ment design, electrical field strength, specific energy input, pulse width,
O
shape and frequency, and treatment time and temperature), food matrix
se
characteristics (electrical conductivity, pH, and water activity), and target
lU
microorganisms (strains, concentration, cell size or shape, and growth stage)
na
(Wouters et al. 2001). It has been also reported that food composition such
o
rs
as proteins and fat can protect microorganisms against PEF by increasing
Pe
resistance to treatment (Salvia-Trujillo et al. 2011). Thereby, PEF is effective
r
against vegetative bacterial cells, yeast, and molds, while microbial spores
fo
are resistant to this treatment (Mosqueda-Melgar et al. 2008; Grahl and
y
op
Mä rkl 1996). In general, both gram-positive and gram-negative bacteria are
C
more resistant than yeasts (Grahl and Mä rkl 1996), and cells in the expo-
’s
nential growth phase are more sensitive than those in the stationary phase
or
(Pothakamury et al. 1996).

ut
b
tri
on
12.3.3 Practical Applications

.C
Interest in the application of PEF in liquid food processing to produce safe and
C
shelf-stable products has grown over the last decades. PEF technology can be
LL
used to improve the safety and quality of dairy products, such as colostrum,
is
whey, and fruit-dairy drinks, and liquid infant formula. Timmermans et al.
c
an
(2014) investigated the inactivation of Salmonella enterica serotype panama E.

Fr
coli , L. monocytogenes , and Saccharomyces cerevisiae inoculated in fruit juices

with different pH values (e.g., apple juice [pH 3.5], orange juice [pH 3.7], and
d
an
watermelon juice [pH 5.3]), using a continuous-flow PEF system (14 mL/min,
or
20 kV/cm, 2 μ s monopolar pulses, and frequencies of 120– 964 Hz). Under

yl
the same conditions, PEF sensitivity followed the order S. cerevisiae > S. pan-
Ta
ama > E. coli > L. monocytogenes and showed that the energy expense needed
18
to inactivate the bacteria was higher than that for yeast. At inlet tempera-
20
tures 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 oxy-
dans . The relation between the applied energy to the grape juice and the lev-
els of inactivation of microorganisms was evaluated. The optimal processing
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 micro-
bial inactivation, and the electric field strength and pulse frequency need to
be wisely selected for each microorganism. Sharma et al. (2014) studied the
y
inactivation of gram-negative (Pseudomonas aeruginosa and E. coli ) and gram-
nl
positive bacteria (S. aureus and Listeria innocua ) in whole milk using PEF treat-
O
ment (22– 28 kV/cm for 17– 101 μ s), in combination with controlled preheating
se
(55° C for 24 s) and stepwise intermediate cooling. They concluded that gram-
lU
negative bacteria were less resistant to PEF than gram-positive bacteria, and
na
reported microbial inactivation to levels below the detection limit. Bermú dez-
o
rs
Aguirre et al. (2012) found that the level of fat milk could affect the stability
Pe
of microorganisms treated under PEF processing. The effectiveness of PEF
r
treatment against B. cereus spores in skim milk was reported to be higher
fo
than that in whole milk. The impact of PEF on some pathogenic strains is
y
summarized in Table 12.2. op
C
’s
or

ut
PEF processing for dairy applications continues to be an engineering chal-

b
tri
lenge, because most of the studies have been performed on laboratory-scale

on
equipment, using small volumes, and under batch or laminar flow setup.
.C
Upscaling, for the translation of such data to pilot- or commercial-scale

C
production, is frequently difficult because different treatment uniformities,

LL
heat conductions, and residence times have to be considered (Buckow et al.

is
2014). Nevertheless, industrial-scale PEF equipment have been developed

c
an
and are currently successfully applied for shelf life extension of fruit juices
Fr
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
d
an
total treatment time and 30– 35 kV/cm electric field strength (equivalent to
or
120– 240 kJ/kg specific electrical energy inputs). Under these conditions,

yl
at least one intermediate cooling step is required to avoid overheating of

Ta
the product. Longer treatment times are rarely used at the industrial scale
18
because of the high demand for electrical and cooling energy (Buckow et al.
20
2014). Although industrial-scale application of PEF processing is possible

for improving the safety and quality of food products, further investiga-
©
tions 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).
©
TABLE 12.2
20 408
18
Most Representative Studies on PEF for Microbial Inactivation in Fluid Food Products
Ta
E f T max Log 10
a
yl
Microorganism Food Product or (kV/cm) n τ (µ s) b t (µ s) c (Hz) (° C) Reduction References
Escherichia coli Apple juice a n7.2d — 2.6 — — 57 1.2 Ukuku et al. (2010)
Strawberry juice 18.6 57.5 2.6 150 — 55 3.79 Gurtler et al. (2011)
Escherichia coli O157:H7 Liquid egg yolk 30 r
F 105 2.0 210 2 40 ≈ 5.0 Amiali et al. (2004)
Strawberry juice 18.6 2.6 150 — 55 4.71 Gurtler et al. (2011)
an57.5
Orange juice 22
c22.7
is 2.6 59 625 45 1.59 Gurtler et al. (2010)
20 26.9 LL 2.6 70 740 55 2.22
Listeria monocytogenes Skim milk 30 400 C 1.5 600 170 25 3.0 McNamee et al. (2010)
Whole milk 30 300 .C 2.0 600 200 35 ≈ 5.0 Zhao et al. (2013)
Salmonella Enteriditis Apple juice 35 393.8 4 on 1575 180 40 4.01 McNamee et al. (2010)
Liquid egg yolk 30 105 2.0 210
tri 2 40 ≈ 5.0 Amiali et al. (2004)
Salmonella Typhimurium Liquid egg 45 10 3.0 30b 300 — 4.0 Monfort et al. (2010)
Orange juice 22 26.9 2.6 59 625 45 2.05 Gurtler et al. (2010)
ut
or
20 57.5 2.6 70 740
’s 55 3.54
Staphylococcus aureus Grape juice 27 15 3.0 45 120 C 48.8 3.36 Huang et al. (2014b)
Liquid egg 40 5 3.0 15 300 — 3.0 Gurtler et al. (2010)
op
Skim milk 35 124 3.7 459 250 y
40 3.7 Monfort et al. (2010)
Whole milk 40 8.9 10.0 89 200 32.5
fo 5.2 Cregenzá n-Alberti et al. (2014)
r
a Number of pulses.
Pe
b Pulse width. rs
c Total treatment time. o na
lU
se
O
nl
y
12.4 Atmospheric Cold Plasma

ACP technology is a relatively novel decontamination nonthermal processing
method, which is effective against a wide range of pathogenic microorgan-
isms. Therefore, ACP can improve the microbiological safety in conjunction
y
with maintenance of sensory behaviors of the treated foods. The remarkable
nl
characteristic features of ACP are its strong thermodynamic nonequilibrium
O
nature, its low gas temperature, the presence of reactive chemical species,
se
and its high selectivity, which provide high potential for use this method in
lU
the food industry (Niemira 2012a). However, due to the complexity of ACP,
na
application of this technology is still under development and debate.
o
rs
Pe
r
fo
By providing energy, such as by heating, a solid material could change its
y
op
state to liquid, and then to gas. When further energy is added to gas, the
C
intra-atomic structures break down, yielding plasma, the fourth state of
’s
matter. Plasma can be thought as an (partially) ionized gas composed of an

or
assortment of “ light” (photons and electrons) and “ heavy” species (ions,

ut
atoms, or even molecules in their fundamental or excited states), possessing

b
tri
a net neutral charge (Niemira and Gutsol 2010). To generate plasma, noble
on
gases are often used since lower voltages are required to break down the gas
.C
and sustain a discharge, but they are not as reactive and are less expensive
C
than air (Niemira 2012a). Figure 12.3 shows an example of ACP processing
LL
of food products. Other gases, that is, O2 or N2 , can also be used to pro-
is
vide the type of reaction needed. The efficiency of the treatment depends
c
an
Fr
Food product High-voltage electrode

d
an
Dielectric
Tbarriers
r
Reactive
lo
Plasma High-
species
ay
voltage
source
18
Ground electrode
20
©
Capacitor
Oscilloscope
FIGURE 12.3
Schematic presentation of atmospheric cold plasma processing of food products.
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
y
as little as 3 s (Niemira 2012a).
nl
Plasma types can be classified according to several parameters (temper-
O
ature, thermodynamic equilibrium, pressure, ionization degree, net gas
se
charge, magnetization, frequency, etc.). However, the most used param-
lU
eter to generate plasma is temperature (Nehra et al. 2008), thus giving
na
two classes: high-temperature (≥ 107 K) and low-temperature (≤ 2 × 104 K)
o
rs
plasma. Nonthermal plasma (300 = T ≤ 103 K), also designated nonequi-
Pe
librium plasma or cold plasma, has two main features that distinguish it
r
from other industrial applied plasma technologies, including the near room
fo
temperature at which they operate and the fact that plasma can originate
y
op
at both atmospheric and reduced pressure, with less power involved than
C
thermal plasma. Until recently, cold plasma treatments were applied under
’s
reduced pressure and at a very small scale, with several operational and
or
cost constraints (Lieberman and Lichtenberg 2005). Consequently, research

ut
regarding nonthermal plasma has moved forward toward the application of

b
tri
ACP, which has already proven to have advantages over reduced-pressure

on
ACP (Yoon and Ryu 2007). To generate ACP, different principles associated
.C
with the energy input from assorted sources have been developed, including
C
corona discharge, dielectric barrier discharges, radiofrequency plasmas, and

LL
gliding arc discharge. These mechanisms have been previously described in

is
detail in the literature (Conards and Schmidt 2000; Niemira 2012a).

c
an
Fr

d
an
ACP has already been studied against a wide range of microorganisms,

or
including yeasts and molds, bacteria, spores, and viruses (Kelly-Wintenberg

yl
et al. 1998; Ryu et al. 2013; Suhem et al. 2013; San-Cheong et al. 2015;
Ta
Zimmermann et al. 2011). The effect of plasma can be quite selective, mean-
18
ing a possible tunable effect between damage to pathogenic organisms, and

20
without injuring the host, or activating some pathways in the microorgan-

isms (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 bac-
tericidal 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 molec-
ular ions) play an important role in the inactivation process since they can
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-posi-
tive bacteria by plasma treatments is believed to be caused by the diffusion
y
of reactive species through the cell membrane, and further reaction with
nl
the intracellular compounds (Laroussi et al. 2003). In contrast, the inacti-
O
vation of gram-negative bacteria is reported to be associated with charge
se
accumulation on the outer surface of the membrane, which overcomes the
lU
tensile strength of the membrane, leading to its rupture (Montie et al. 2000).
na
Another mechanism results from ultraviolet (UV) radiation, which erodes
o
rs
the cell membrane and cellular constituents (Lackmann et al. 2013). Finally,
Pe
the damage of membranes and internal cellular components, such as DNA,
r
can be due to the action of UV photons (Moisan et al. 2002). The relative
fo
“ weight” of each mechanism on the sanitizing processes of a given com-
y
op
modity will also depend on the direct or remote plasma exposure (Laroussi
C
et al. 2003). The type of food and microorganism considered, equipment
’s
design, voltage chosen, gas pressure and composition, and distance of the
or
microorganism from the discharge glow are also variables to be analyzed

ut
case to case (Afshari and Hosseini 2014; Niemira 2012b). As any other non-
b
tri
thermal technologies, plasma can also affect microorganisms in a sublethal

on
way, in which the metabolic behavior of cells can be significantly altered

.C
(Perni et al. 2008a; Rowan et al. 2007) and further compromise the safety of
C
the treated products.

LL
cis
12.4.3 Practical Applications

an
Fr
ACP is a novel nonthermal food processing technology that uses energetic,

reactive gases to reduce the contaminating microbial load on a wide range
d
an
of foods, and it could be currently regarded as a potential alternative to

or
chemical (i.e., chlorine treatment) or physical (i.e., HPP and PEFs) methods.
yl
The main advantages are the high inactivation efficiency on both pathogens
Ta
and spoilage organisms on the surface of food products at low temperatures

18
(< 55° C– 60° C). Therefore, the thermosensitive products can also be treated,

20
with other features, such as precise generation of plasma that is suitable

for specific intended uses; just-in-time production of the acting agent; low
©
impact on the internal product matrix; and no application of water, sol-

vents, or antiseptic chemicals, with no residues produced (environmentally
friendly). A detailed description of several food products to which ACP has
been applied and the related conditions, as well as the target microorganisms
and the main results obtained, can be found in Table 12.3. Up to the pres-
ent, no food commercial ACP-treated products are available. Even though
this technology holds promise as a nonthermal sterilization treatment, cur-
rent research data are ongoing in order to address fundamental questions,
©
TABLE 12.3
20 412
18
Main Studies Related to ACP Application for Inactivation of Microorganisms in Food Matrixes (Food Product, Plasma Conditions,
and Microorganisms)
Ta
yl
Food Product Microorganism Plasma Conditions Main Effect Reference
or
Mangoes, melons Escherichia coli AC voltage 8– 16 kV at S. cerevisiae was the most resistant Perni et al.
an
Saccharomyces cerevisiae
d 30 kHz, He/O2 mixture microorganism. Cut surfaces (2008a, 2008b)
Pantoea agglomerans Fr markedly reduced the efficacy of
Gluconacetobacterliquefaciens an inactivation.
Listeria monocytogenes Scott A c
Golden Delicious apples Escherichia coli O157:H7
isAC voltage of 15 kV at Bacterial inactivation was shown to be Niemira and
Salmonella Stanley
L Hz, air
60 L a function of flow rate and duration Sites (2008)
C of exposure.
Apples, corn salad, Escherichia coli DSM 1116 Fixed power was observed in all the Baier et al.
. C of 8 W, Ar/ Inactivation
cucumber, tomatoes 2
O mixtureo cases. Extent of log reduction varied (2014)
with the product.
nt
Apples, cantaloupe, Escherichia coli O157:H7 AC voltage 9 kV at Inactivation was observed in all the Critzer et al.
rib
lettuce Salmonella spp. 6 kHz, air L. monocytogenes was the most (2007)
ut
Listeria monocytogenes sensitive microorganism.
or cases.
Lettuce Aeromonas hydrophila Cold oxygen plasma
’s A reduction of 5.0 log incubation Jahid et al.
C(< 15° C). (2014)
o
Pork meat Total aerobic viable counts 2.45 GHz, 1.2 kW, air The pmicrobial was kept under the Frö hling et al.
detection 2
y limitload(10 CFU/g), over 20 (2012)
f
days of storage
or (5° C).
Chicken Listeria innocua 16 kV, 30 kHz, He/O
2 Extent of log P
reduction varied with the Noriega et al.
(skin and muscle) mixture type of chicken surface: skin acts as a (2011)
er
sinactivation.
barrier for plasma o
(Continued)
na
lU
se
O
nl
y
©
20
Main Studies Related to ACP Application for Inactivation of Microorganisms in Food Matrixes (Food Product, Plasma Conditions,
and Microorganisms)
Ta
yl
Food Product Microorganism Plasma Conditions Main Effect Reference
or
Bacon
a
Listeria monocytogenes n Power 125 W, Extent of log reduction varied with the Kim et al. (2011)
Escherichia coli
d 13.56 MHz, He or composition of gas mixture.
Salmonella Typhimurium He/O2 mixture
Fr
Sliced ham, sliced Listeria monocytogenes Power 125 W, 13.6 MHz Extent of log reduction varied with the Song et al.
an
cheese
cis product (2 log for sliced cheese, > 8 (2009)
log for sliced ham).
Prepackaged ready-to- Listeria innocua AC voltage 27 kV, Up to a 2 log reduction of L. innocua Rod et al. (2012)
LL
eat meat ( bresaola)
C
27.8 kHz, Ar/O2 was obtained. No significant effects
mixture (inside of time and intensity were seen.
.C
package) on Multiple treatments increased the
tri reduction.
Egg shells Salmonella Enteritidis 15 kV, 12.7 kHz, airb Reductions were observed for both Ragni et al.
Salmonella Typhimurium strains. Both treatment time and (2010)
ut
relative humidity increased the
or
’s strains’ reduction.
C
Dry almonds Salmonella 549 W, 47 kHz E. coli had the greatest reduction.
op Niemira (2012b)
Escherichia coli O157:H7 y
Cereal bars Aspergillus flavus 20– 40 W, 5– 25 min, Ar Protection under storage conditions
fo Suhem et al.
(25° C, 100% relative humidity).
r (2013)
Pe
rs
o na
lU
se
O
nl
y
including the effect on quality (Grzegorzewski et al. 2011; Lackmann et al.

2013; Suhem et al. 2013).

ACP technology is at a relatively early stage of development, and it still
y
nl
presents some restrictions to certain food applications and further commer-
O
cialization. Both economic aspects and the safety of applied gas for scaling
se
up this technology in the food industry should be addressed to determine
lU
its applicability. According to Niemira and Sites (2008), as the technology
na
moves from lab to commercial scale, it is expected to become increasingly
o
effective not only from an antimicrobial standpoint, but also in terms of
rs
cost. The absolute capital costs will be naturally higher but may be offset by
Pe
improvements in energy efficiency and overall engineering-scale efficiencies
r
fo
(Niemira 2012a). Also, the largely unexplored impacts on the sensory and
nutritional qualities of the treated foods, especially the ones with high lipid
y
op
and vitamin contents, lead to additional issues concerning the food quality
C
and safety (e.g., risk assessment of toxic residues) concerns. Moreover, when
’s
treating prepackaged food products, issues related to packaging material

or
ut
need to be taken into consideration. Combining ACP with other nonthermal

b
processes could be a possible future breakthrough in the food industry. The

tri
plasma composition could be altered through the use of chemical augmenta-

on
tion, as simple as water (Burlica et al. 2010), or even newer, by combination

.C
with essential oils (Matan et al. 2014). Future work is still needed in the appli-
C
cation of ACP to packaged foods, not only to assess the changes in food qual-
LL
ity but also to ascertain the impacts on the packaging material. Packaging,
is
the nature and quantity of substrates, and the temperature and nature of
c
an
microorganisms are highly relevant to the decontamination effect. Because

Fr
this is an emerging technology, regulatory review and approval will also

d
be required. But besides limitations that the application of ACP to the food
an
industry still presents, this is an area of technology that shows promise and
or
is the subject of active research to enhance efficacy.

yl
Ta
18
20
©
12.5 Other Commercial Emerging Nonthermal Technologies

12.5.1 Osmotic Dehydration
OD is a pretreatment that can be used before various processes, such as air
drying, deep fat frying, or freeze drying (Ahmed et al. 2016; Ciurzyń ska
et al. 2016). This method has received special attention by consumers due
to its minimal impact on the nutritional, sensorial, and functional value of
food products (Ahmed et al. 2016; Ciurzyń ska et al. 2016). OD depends on
the moisture diffusion phenomenon from food materials by immersion in

a hypertonic solution. Glucose, corn syrup, sodium chloride, starch concen-
trates, fructose, and sucrose are osmotic agents often selected according to
the intended use of the final product. Briefly, the product is continuously
immersed in solution while water diffuses through the first layer of the cells
to the concentrated solution. The concentration gradient between the solu-
y
tion and the intracellular fluid is the driving force for water removal. Then,
nl
a chemical potential difference of water is developed between the first and
O
second layer of cells. This second layer starts to pump water to the cells of the
se
first layer and shrinks (Ahmed et al. 2016). However, due to the absence of
lU
total selectivity of the cell membrane, some solutes (e.g., organic acids, min-
na
erals, sugars, colorants, and flavors) can also flow to the hypertonic solution.
o
rs
On the other hand, this mechanism is oxygen-free, and there is no need for
Pe
additives to protect against oxidative and enzymatic discoloration (Chandra
r
and Kumari 2015). Besides the type of osmotic agent, OD can be affected
fo
by both osmotic solution (concentration, temperature and time exposure,
y
op
and agitation) and type of food product (geometry and the physicochemical
C
properties of the food materials) (Chandra and Kumari 2015).
’s
This technology has been applied mainly in fruits and vegetables with a
or
notorious reduction of moisture content, up to 50% weight of fresh foods. The

ut
water activity of fruits and vegetables is reduced to 0.94– 0.97, keeping the

b
tri
original quality (Chandra and Kumari 2015). Products treated by OD become

on
more stable during storage due to a lower water activity by solute gain and
.C
water loss. For instance, Castelló et al. (2009) evaluated the effect of OD treat-
C
ment of fresh apples at 30° C using 50° Brix of glucose as the osmotic agent. It
LL
has been shown that mesophilic aerobic counts exceeded 104 CFU/g (micro-
is
biological criterion recommended for fruits and vegetables) after 3 days for
c
an
fresh-cut samples, while for OD-treated samples, these counts exceeded

Fr
104 CFU/g after 6 days. In another study, treatment of papaya with OD in a

sucrose solution (60% and 37° C) for 4.25 h caused 24% of water removal and
d
an
a 4% increment in the dry matter, resulting in a high-quality product, well

or
rated by consumers (Jain et al. 2011). Therefore, OD can be used for partial
yl
concentration of fruits and vegetables by reducing the moisture content, and

Ta
thus the refrigeration load during freezing, saving packaging and distribu-
18
tion costs, which leads to obtaining higher product quality.

20
©
12.5.2 Ozone-Based Technology

Ozone (O3 ) is one of the most powerful oxidizing agents known, with an
oxidation potential of 2.07 mV (only fluorine has a higher oxidation potential
of 3.06 mV). This gas is formed naturally in the stratosphere by sunlight’ s
effect on oxygen molecules (O2 ), but it can also be produced by chemical,
photochemical, thermal, chemonuclear, electrolytic, and electrochemical
procedures (Yousef et al. 2011). The ozone molecule is thermally unstable
and rapidly dissociates into O2 or reacts with other gases. However, O3 is
more soluble in water than O2 , with a half-life in distilled water of 20– 30 min,
before breaking down to oxygen. To increase its concentration in aqueous
solution, it is necessary to have continuous O3 production and low tempera-
tures, since ozone is more stable in the gaseous phase than in aqueous solu-
tions (Alexandre et al. 2016).
Ozone is an exceptionally good sanitizer that has faster kinetics than other
y
widely used chemical solutions, mainly due to its strong oxidation power,
nl
being more effective to eliminate most loads of microorganisms. It is effective
O
against a large spectrum of microorganisms, such as gram-positive bacteria
se
(L. monocytogenes , S. aureus , B. cereus , and Enterococcus faecalis ), gram-negative
lU
bacteria (P. aeruginosa and Yersinia enterocolitica ), yeasts (Candida albicans and
na
Zygosaccharomyces bacilli ), and some molds (Aspergillus niger ) (Restaino et al.
o
rs
1995; Gü zel-Seydim et al. 2004). The germicidal effect of O3 starts on the cell
Pe
surface, with the progressive oxidation of vital cellular components, which
r
results in total or partial destruction of the cell wall and further cell lysis.
fo
Moreover, O3 breaks chromosomes, nitrogen– carbon bonds between sugars
y
op
and bases, DNA hydrogen bonds, and phosphate sugar bonds, which leads
C
to the depolymerization and leakage of the cellular compounds (Alexandre
’s
et al. 2011, 2016). Additionally, O3 does not leave any trace of the residual
or
products on its oxidative reaction, and it is environmentally safe since no

ut
harmful by-products are formed after the ozonation process. In 1997, O3 was
b
tri
declared generally recognized as safe by Food and Drug Administration

on
(FDA), which approved the use of ozone as an antimicrobial agent for the
.C
treatment, storage, and processing of foods in gas and aqueous phases in

C
2001.
LL
The application of O3 started with the disinfection of municipal, indus-

is
trial, and drinking water. Later, the applications were extended to the food
c
an
industry, including food preservation, surface hygiene, food plant equip-

Fr
ment sanitation, water disinfection, and wastewater reutilization. When

dissolved in water, usually at 0.03– 20.0 ppm, O3 has been tested in post-
d
an
harvest treatments of fruits and vegetables (Alexandre et al. 2013), as well

or
as fish products. Figure 12.4 presents an industrial line of fish vitrification

yl
using ozone dissolved in water, which was implemented by SPO3 (Sociedade

Ta
Portuguesa de Ozono, Lda.). Since ozone decomposition is rapid in water,

18
its antimicrobial action may take place only at food surfaces (Aguayo et al.
20
2006). Nevertheless, ozone has greater efficacy when used in the gaseous
form since higher concentrations may be achieved (ca. 20,000 ppm), associ-
©
ated with refrigerated storage and modified atmosphere packaging (Selma

et al. 2008).
12.5.3 Chlorine Dioxide

Chlorine dioxide (ClO2 ) has been used as a disinfectant, and dates back
to the beginning of the twentieth century, when it was first employed for
water disinfection (Tzanavaras et al. 2007). Food applications, and minimally
©
20
18
Ta Ozone Oxygen
yl Water treated generator generator
or GSO 20 Oxygen 5
an
d
Fr
an
c
Dissolving ozone system
is
and separation by
LL
flotation of oxidation
material
C
.C Waste
on
Washing the
f ish before
tri
entering into
but
the freezing or
tunnel ’s
C
op
Fish washing tank
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volume approx. = 1500 L Feeding
fo
r
carpet to the
freezing
Pe
tunnel rs
o
FIGURE 12.4
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Schematic presentation of an industrial line of fish vitrification using ozone dissolved in water. (Courtesy of Sociedade Portuguesa de Ozono, Lda.)
lU
se
O
nl
y
processed products, have been successfully reviewed by Gó mez-Ló pez et al.

(2009), but other purposes have recently been reported in the literature, such
as in seeds and sprouts, meat products, and seafood. ClO2 is a water-soluble
(without reacting with it), aggressive, and unstable gas at concentrations
higher than 10%. The main advantage of ClO2 gas over aqueous sanitizers,
including aqueous solutions of ClO2 , is its higher penetrability (Han et al.
y
2000, 2001), reaching microorganisms often protected by surface irregulari-
nl
ties or even biofilms (Han et al. 2000). It is considered a strong oxidizing
O
agent (oxidation potential 1.50 mV), which can inactivate bacteria, bacte-
se
rial spores, and viruses. ClO2 solutions have shown promising results in
lU
eliminating microorganisms on different fruits and vegetables, along with
na
extending their shelf life (Gó mez-Ló pez et al. 2009), but no complete elimina-
o
rs
tion has been reported. The efficacy of ClO2 gas is often affected by several
Pe
factors, including the amount of the food product relative to the amount of
r
ClO2 applied, the surface integrity of the product, the microorganism loca-
fo
tion, the gas concentration, the treatment time, the relative humidity, and
y
op
the temperature (Han et al. 2000, 2001). The antimicrobial effect of ClO2 is
C
mainly due to its impact on cell membranes; however, this gas seems to alter
’s
the nutrient transport and disrupt the protein synthesis after penetrating
or
into the cells, but mechanisms are not yet entirely understood. Together with
ut
elongation, which might result from inhibition of the cell division, rough-
b
tri
ness and indentations in the surface of B. cereus cells were also reported by
on
Peta et al. (2003).

.C
As a gas, ClO2 has proven to be a tool to extend the shelf life of fresh and
C
fresh-cut products (Gó mez-Ló pez et al. 2009); however, in some cases, it can

LL
induce browning and bleaching. With regard to nutritional quality, it has

is
been known that ClO2 gas reacts with phenolic compounds. Since many phy-
c
an
tochemicals are phenolics, it is possible that ClO2 has an impact on these

Fr
compounds. Moreover, since ClO2 is an oxidant, nutrients such as ascorbic

acid could also be easily affected. As far toxicity is concerned, and since it
d
an
has been mostly used in water, several studies have found no adverse effects
or
in humans living in the United States, where water has been treated with
yl
ClO2 . However, the U.S. Environmental Protection Agency (EPA 2000) has
Ta
considered these studies inconclusive due to methodological problems.

18
Although in the United States fresh processors are legally allowed to use
20
ClO2 (up to 3 mg/L in water) as a disinfectant (FDA 2008), in the European

Union (EU), the application of ClO2 for surface decontamination of fresh
©
products is not permitted. In order to support the use of ClO2 as a food dis-
infectant, extensive research on by-products’ chemistry and their potential
safety is required.
12.5.4 Ionizing Radiation

Ionizing radiation refers to forms of radiant energy that possess suffi-
cient energy to create negatively and positively charged ions in the target
substrate. Gamma radiation, beta radiation (electron beam), and x-rays are
three forms of ionizing radiation approved for food treatment (WHO 1994).
However, the most applied technology in food industry is gamma radiation,
produced by the natural decay of isotopes (60 Co or 137 Cs), since it presents
a high penetrability of the produced photons, allowing irradiation of high
bulk density and volume materials (Koubaa et al. 2016).
y
Food irradiation kills, sterilizes, or renders insects and microorganisms
nl
unable to reproduce by damaging nucleic acid, specifically DNA or RNA
O
(Ward 1991), on the interior of several food products. It has been mainly
se
applied as a phytosanitary treatment (e.g., spices, mangoes, papaya, and
lU
other exotic tropical fruits) to prevent the spread of invasive insects between
na
continents. Another important concept involving the microorganisms’ inac-
o
rs
tivation mechanism is the cells’ sensitivity to heat following irradiation,
Pe
reviewed by Sommers and Fan (2011). Even though increased resistance of
r
microorganisms to ionizing radiation following repeated exposure has been
fo
observed (Parisi and Antoine 1974), research also indicates that ionizing radi-
y
op
ation decreases the virulence (Sommers and Schiestl 2004) and resistance to
C
antibiotics of foodborne pathogens (Niemira and Lonczynski 2006).
’s
Food treatment by irradiation is considered a “ green” technology, as it uti-

or
lizes no chemicals and causes no pollution. However, an innate fear of the

ut
word irradiation creates a negative consumer perception. The safety of irradi-

b
tri
ated foods has been endorsed by the World Health Organization (WHO), the
on
Food and Agriculture Organization of the United Nations (FAO), the U.S.
.C
Department of Agriculture (USDA), Health Canada (HC), the EU, and the
C
FDA. Following the FDA approval for irradiation, consumer attitudes toward
LL
irradiated meat and poultry have changed in the last 20 years (Sommers and
is
Fan 2011), and the global market for irradiated foods has expanded. However,
c
an
in the EU, interest in food irradiation decreased after 1999 due to the enforce-
Fr
ment of an EU regulation in the same year that required strict labeling for
irradiated foods (Commission of the European Communities 1999).
d
an
or
yl
Ta
18
12.6 Final Remarks

20
Two main objectives are usually taken into account by industries before mar-
©
keting a food product: (1) extending the shelf life of foods, which is usually
attained by the use of preservative methods that inhibit microbiological and
biochemical changes, allowing time for distribution, sales, and home storage,
and (2) providing nutritional quality in food products, that is, guaranteeing
the required nutrients for a healthy diet. Heat treatments may be efficient
for the first objective, but they have a significant negative impact on product
quality. The emerging technologies are known to lessen this impact, ensur-
ing product safety. The application of HPP, PEF, ACP, OD, or ozonation and
the use of chlorine dioxide are examples of nonthermal technologies that

have potential applications in the food industry. HPP, PEF, and ozone are
already implemented in the food industry, but only for some products, under
specific conditions and to attain particular objectives. These alternative tech-
nologies should produce microbiologically safe products and increase the
products’ shelf life while reducing the process impact on quality character-
y
istics. Moreover, nonthermal processing does not require high consumption
nl
of energy, and is therefore a more economic and environmentally friendly
O
method.
se
lU
ona
rs
Pe
Acknowledgments
r
fo
This work was supported by national funds from Fundaç ã o para a Ciê ncia
y
op
e Tecnologia— FCT through project UID/Multi/50016/2013 and by FCT/
C
MEC through financial support to the QOPNA research unit (FCT UID/
’s
QUI/00062/2013) and through national funds, and where applicable, was

or
cofinanced by the FEDER, within the PT2020 Partnership Agreement.

ut
b
Authors Elisabete M. C. Alexandre, Rita S. Iná cio, and Só nia M. Castro are

tri
grateful for the financial support of this work by Fundaç ã o para a Ciê ncia e
on
Tecnologia— FCT through fellowship grants SFRH/BPD/95795/2013, SFRH/

.C
BD/96576/2013, and SFRH/BPD/71723/2010, respectively.

C
LL
c is
an
Fr
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Section IV
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Food Packaging
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13
Food Packaging Systems with
Antimicrobial Agents
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Reyhan Irkin
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CONTENTS
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13.1 Introduction................................................................................................. 431
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13.2 Action Mechanisms of Antimicrobial Food Packaging........................ 433
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13.3 Natural Biopolymers..................................................................................433
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13.3.1 Alginate-Based Antimicrobial Films...........................................433
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13.3.2 Gelatin-Based Antimicrobial Films............................................. 435
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13.3.3 Chitosan-Based Antimicrobial Films.......................................... 436

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13.3.4 Carrageenan-Based Antimicrobial Films.................................... 438

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13.3.5 Whey Protein Isolate– Based Films.............................................. 438

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13.4 Essential Oils and Their Compound-Based Films.................................440

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13.5 Chemically Synthesized Biodegradable Polymers.................................442

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13.5.1 Poly-L-Lactide-Based Antimicrobial Films.................................442

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13.6 Polymer/Clay Nanocomposite-Based Antimicrobial Films.................445

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13.7 Metal and Metal Oxide Nanoparticle-Based Antimicrobial Films.....446

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13.8 Advantages and Difficulties of Natural Antimicrobial Packaging

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Systems ........................................................................................................448
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13.9 Conclusion................................................................................................... 451

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References ............................................................................................................. 452
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13.1 Introduction
18
20
Food quality and safety are primary concerns in the food industry.
Biopolymer-based antimicrobial packaging has been considered an emerg-
©
ing technology that has a significant influence on sustaining food qual-

ity and extending the shelf life of packaged foods (Shankar et al. 2016).
Antimicrobial films can be described, as some polymer matrixes may be
loaded with antimicrobial compounds, such as essential oils (EOs), metal
ions, or preservative compounds (Birck et al. 2016). An antibacterial polymer
is a system that covers a polymer matrix and an antimicrobial compound
that inhibits the growth of microorganisms (Urbankova et al. 2015). Today,
consumer concerns are increasing toward biodegradable, edible, renewable
431
films and coatings suitable for food packaging applications, and biopolymer
films are gaining more significance (Tavassoli-Kafrani et al. 2016).
There are several methods for gaining antimicrobial activity in polymeric
films: incorporating antimicrobial agents directly into the polymers, coating
antimicrobial materials onto polymer surfaces, immobilizing antimicrobi-
als by chemical grafting, or using polymers that show intrinsic antimicro-
y
bial properties (e.g., chitosan). Synthetic antimicrobials are generally added
nl
into polymers for packaging, for example, organic or inorganic acids, metals,
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alcohols, ammonium compounds, or amines (Shemesh et al. 2015b).
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The incorporation of antimicrobial agents into films and coatings has been
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shown to act as a stress factor to inhibit pathogen growth and protect the
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spoilage of food. The use of chemical antimicrobial additives, such as sorbic
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acid, potassium sorbate, propionic acid, benzoic acid, and sodium benzoate,
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has limited use in food due to health concerns of consumers. Therefore, con-
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sumers demand the use of natural and healthy additives that are generally
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recognized as safe (GRAS). Antimicrobial agents include organic acids (lac-
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tic, acetic, malic, and citric acids), chitosan, and some plant-derived second-
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ary metabolites, such as EOs. Edible films and coatings with antimicrobial
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specifications can be used as active packaging (Tavassoli-Kafrani et al. 2016).

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Antimicrobial packaging “ reduce[s] the risk of food-borne microbiological

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diseases and also offers other health-related advantages, for example, allow-
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ing better nutritional quality with less severe food treatments and lower
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amounts of additives while providing longer shelf life and wider distribu-
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tion” (Vijayendra and Sharma 2014, pp. 338–357; Singh et al. 2016).
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The use of biopolymer films to prolong shelf life has increased over the
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last two decades because of their environmental advantages compared with

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common synthetic polymeric films. Edible films and coatings made from
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biopolymers are used as barriers for carbon dioxide, oxygen, moisture, and
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lipid transfer; mechanical property modifiers; and food additive carriers in

food systems. Proteins and polysaccharides are the common biopolymers
d
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applied to make edible packaging films (Wu et al. 2015).

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Biodegradable polymer nanocomposites (BPNs) have excessive properties,

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which are important in the prospect of using them as high-performance sub-

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stances over a range of industrial areas. Notably, biopolymers will play a

18
key role in the future alternative to the synthetic polymers because of their
20
biological origin and biodegradable nature properties. Consequently, in

recent years many researchers have focused on biodegradable polymers and
©
modification of their properties. In this regard, use of organic and inorganic

nanomaterials is demonstrated to be a more potent route for the preparation
of BPNs (Bari et al. 2016).
Polysaccharide-based polymers have potential to be used in antimicrobial
packaging mechanisms or can be used together with antimicrobial sub-
stances, including chitosan, carrageenan, cellulose, starch, and alginate. It
was stated that one of the antimicrobial polysaccharide film agents, kefiran,
showed high antimicrobial activity against Streptococcus faecalis KR6 and
Food Packaging Systems with Antimicrobial Agents 433
Fusarium graminearum CZ1, along with complete inhibition of mycelia and

aflatoxin formation by Aspergillus flavus AH3 (Vijayendra and Shamala 2014).
Examples of protein-based materials are whey protein, gelatin, soya protein,
corn zein, and/or their derivatives (Kuorwel et al. 2011).
This chapter focuses on natural biopolymer antimicrobial films associated
with food packaging applications.
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13.2 Action Mechanisms of Antimicrobial Food Packaging
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Antimicrobial activity can be obtained by either indirect contact between the
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product and the antimicrobial package by using volatile releasing systems,
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or direct contact between the antimicrobial package and the food applying
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by nonmigratory antimicrobial systems. Different preservatives, such as sil-
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ver (Ag)-substituted zeolites, salts, organic acids, bacteriocins, and EOs, from
y
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many kinds of plant extracts have been applied to packaging materials to
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obtain antimicrobial activity (Guarda et al. 2011). When a volatile antimicro-
’s
bial compound is incorporated into a package, it is spread mainly by per-

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meation and diffusion onto food surfaces to prevent pathogenic or spoilage

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microorganisms during the shelf life. The amount of antimicrobial agents

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released from the packaging material has an important effect on the antimi-
on
crobial activity and potential use of antimicrobial films in food packaging

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(Kuorwel et al. 2011).

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The use of antimicrobial films can provide advantages compared with

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the addition of preservatives directly to the food, because preservative com-

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pounds are applied to the packages in a way that only optimum levels of
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agents come into contact with the food. Antimicrobial film is an antimicro-
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bial agent distribution mechanism, and only the optimum amount of the
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antimicrobial would be used, and it would not be directly added into the
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food product, and thus the sensory quality of the food would not be nega-
or
tively changed (Irkin and Esmer 2015).

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The mechanisms of action for antimicrobial substances are usually based

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on destroying the cell wall or membrane. In addition, antimicrobial sub-

18
stances work through inhibition of significant enzymes in the microbial cell

20
or destruction of the genetic configuration of the protoplasm (Lee et al. 2015).

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13.3 Natural Biopolymers

13.3.1 Alginate-Based Antimicrobial Films
Among several types of biopolymers, carbohydrate-based polymers are
extensively used to prepare innovative food packaging materials because
of their good film-forming properties. As one such carbohydrate biopoly-

mer, alginate is a good candidate for being investigated in food packaging
applications. Alginate is a naturally occurring pol-anionic polysaccharide
obtained from brown marine algae (Phaeophyceae), and it is commercially
derived from brown seaweed containing giant kelp (Macrocystis pyrifera
and Ascophyllum nodosum ) and several types of Laminaria species, such as
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Laminaria hyperborea , Laminaria digitata , and Laminaria japonica . Alginate is
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composed of a linear block copolymer of 1,4-linked β -d-mannuronic and
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α -l-guluronic residues in varying amounts. Being low cost, easily available,
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biocompatible, and environmentally friendly, it is used in various applica-
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tions in the food and biotechnology industries, for example, as a nontoxic
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food additive, thickening and gelling substance, and colloidal stabilizer
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(Shankar et al. 2016).
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Ternary blend agar/alginate/collagen (A/A/C) hydrogel composite films
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with silver nanoparticles (AgNPs) and grapefruit seed extract (GSE) were
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studied by Wang and Rhim (2015). The results demonstrated that their ter-
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nary blend hydrogel films incorporated with AgNPs or GSE can be used as
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antifogging packaging films for highly respiring fresh agriculture products,
’s
and they can show antimicrobial activity, also. In addition, the GSE- and
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AgNP-incorporated films can be used to prolong the shelf life of food prod-
ut
ucts and obtain quality through preventing postprocessing contamination

b
tri
(Wang and Rhim 2015).

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Shankar et al. (2016) showed that alginate composite films incorporated

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with citrate-reduced AgNPs, silver zeolite, and AgNO3 have high antimi-
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crobial activity against Escherichia coli and Listeria monocytogenes . However,

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the antibacterial activity of AgNPC, AgZ, and AgNO3 was stronger against
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gram-negative bacteria (E. coli ) than gram-positive bacteria (L. monocyto-

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genes ). But on the other hand, laser-ablated AgNPs and metallic silver did
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not exhibit antibacterial activity against both microorganisms.

In another study, single and composite films based on alginate and pectin,
d
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including natamycin as an active material, were prepared, and the release

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behavior in water and diffusion coefficients were evaluated. The single-

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alginate films proved to be more desirable for application in packaging than

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the single-pectin and composite films. The good compatibility of natamycin

18
with alginate, the low diffusion coefficient, and the high physical properties
20
show that this film has great potential to be applied in antimicrobial packag-
ing, such as in cheese packaging to inhibit mold spoilage. However, further
©
research is needed to determine the effectiveness of these films on model

food systems (Bierhalz et al. 2012).
Kapetanakou et al. (2016) researched the efficacy of Na-alginate films
immersed in 40% v/v distillery ethanol and traditional Greek alcoholic bev-
erages (ABs), namely, “ tsipouro,” “ raki,” and “ ouzo,” to prevent L. monocyto-
genes growth on frankfurters and ham slices. However, while pure distillery
ethanol was the most efficient antimicrobial agent during storage, it did not
directly cause reduction of the target microorganism to under levels of enu-

meration after microwave reheating.
In Norajit and Ryu (2011), the preparation of antibacterial alginate films
incorporating extruded white ginseng (EWG) extracts was assayed. The
antimicrobial effect of EWG extract on six selected food pathogenic bac-
teria was tested against the effects of red ginseng (RG) and white ginseng
y
(WG) extracts. Pseudomonas aeruginosa , Bacillus subtilis , and L. monocytogenes
nl
showed the lowest numbers in films incorporating EWG-115; the cell reduc-
O
tions were 9.1, 7.46, and 8.31 log cfu/mL, respectively.
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13.3.2 Gelatin-Based Antimicrobial Films
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Gelatin is a protein obtained by chemical, physical, or biochemical denatur-
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ation and hydrolysis of collagen, which is used extensively in the produc-
r
tion of edible films due to its high film-forming property, low gelling and
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melting point, biodegradability, and abundance (Wu et al. 2015). Gelatin is
y
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a good choice since it corresponds to an equilibrium between human food
C
consumption and valorization of industrial by-products. This biopolymer
’s
is widely used in the food and pharmaceutical industry for its gelling and
or
texturing characteristics (Biscarat et al. 2015). Polymers obtained from gela-

ut
tin have interesting properties due to its low melting point. Films formed
b
tri
using gelatin sources may be desirable to producers due to their edible or

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biodegradable structure, low cost, and full availability. Agents such as EOs,
.C
organic acids, and enzymes can be applied for antimicrobial activity in food
C
packaging. There may be potential to incorporate antimicrobial materials

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into the packaging during the processing stage, but various antimicrobials
is
are sensitive to film process conditions. Packaging materials such as casted

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an
polypropylene (CPP) and polyethylene terephthalate (PET) can be managed

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with antimicrobial agents. It has been stated that beef-derived gelatin con-
taining active antimicrobial substances could potentially be used as a com-
d
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mercial antimicrobial coating for application in conventional plastic-based

or
food packaging or to serve as biodegradable packaging material (Clarke

yl
et al. 2016).
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Proteins are used widely for obtaining biodegradable films because of

18
their relative abundance, film-forming facility, and singularity in terms of

20
the quality of the films obtained, as well as for their high nutritional value
(Kaewprachu et al. 2016).
©
Arfat et al. (2014) studied a fish protein isolate (FPI) and fish skin gela-
tin (FSG) mixture incorporated with 50% and 100% (w/w, protein) basil leaf
essential oil (BEO) without and with the presence of 3% (w/w, protein) zinc
oxide nanoparticles (ZnONPs). As a result, it was reported that FPI/FSG
films produced with BEO and ZnONP could be used as an active food pack-
aging to inhibit the growth of pathogens and extend the shelf life of pack-
aged foods.
It was reported that gelatin/AgNP/clay nanocomposite films can show

strong antibacterial activity against foodborne pathogens. The use of gela-
tin-based nanocomposite films will help to remove bacterial contaminations
and improve the shelf life and quality of food (Kanmani and Rhim 2014a).
Hosseini et al. (2016) studied the development of biobased nanocompos-
ite films from fish gelatin (FG) and chitosan nanoparticles (CSNPs) incorpo-
y
rated with oregano (Origanum vulgare L.) essential oil (OEO). The FG/CSNP
nl
bioactive films showed effective antimicrobial activity against four test food
O
pathogens: Staphylococcus aureus , L. monocytogenes , Salmonella enteritidis , and
se
E. coli . A minimum concentration of 1.2% (w/v) of OEO was necessary to
lU
confirm its antibacterial efficacy.
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Kazemi and Rezaei (2015) researched the effect of gelatin– alginate edible
o
rs
film produced with OEO on spoilage bacteria. The effects of gelatin– alginate
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film containing 1.5% OEO on rainbow trout (Oncorhynchus mykiss ) slices dur-
r
ing refrigerated storage (15 days) were recorded. The edible film enriched
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with OEO showed the highest antimicrobial activity on psychrotrophic bac-
y
op
teria, total viable count (TVC), and Enterobacteriaceae.
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13.3.3 Chitosan-Based Antimicrobial Films

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Among the natural polymers, chitosan is of great interest due to its good
b
tri
antimicrobial activity, and it has the advantages of biocompatibility, bio-

on
degradability, excellent film-forming ability, and nontoxicity (Sunilkumar

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et al. 2012; Ninjiarani 2015). Chemically, chitosan is specified as a copoly-

C
mer containing pyranose cycles of N-acetyl-D-glucosamine (GlcNAc) and

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N-glucosamine (GluN) linked with a glycosidic formation and primarily

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obtained chemically by deacetylation of chitin (alkaline conditions). For these

c
an
reasons, chitosan polymer is applied in several important areas in the food

Fr
packaging, agriculture, biomedical, and cosmetic industries. Furthermore,

due to the potential of good film forming, chitosan can be used in the form of
d
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transparent films or coatings to develop the quality of fresh food and extend
or
its shelf life. Also, chitosan-based films are stable and flexible and require
yl
special attention as an antimicrobial agent for food preservation (Gartner

Ta
et al. 2015; Aljawish et al. 2016).

18
It was stated that biodegradable chitosan-based composite film with the

20
addition of GSE, having high mechanical, physical, and antifungal proper-

ties, is promising for application as a biodegradable biopolymer-based com-
©
posite food packaging to extend the shelf life of the food (Tan et al. 2015).
The antibacterial effect of chitosan-based coatings containing a nano-
emulsion of EOs, gamma irradiation, and modified atmosphere packag-
ing (MAP) systems, alone or in combination, against E. coli O157:H7 and
Salmonella Typhimurium was researched on inoculated green bean sam-
ples. Four different nanoemulsions prepared from carvacrol, mandarin,
bergamot, and lemon EOs were used and compared in terms of minimum
inhibitory concentration (MIC) against the microorganisms. It was con-

firmed that the carvacrol nanoemulsions have strong antimicrobial effects
against two gram negative pathogenic bacteria, E. coli O157:H7 and S.
Typhimurium , than the mandarin, lemon, and bergamot EOs nanoemul-
sions. However, the combination treatment of gamma irradiation, coating,
and MAP resulted in the highest effects against both tested bacteria dur-
y
ing storage, and was also effective in reducing their inoculated population
nl
(Severino et al. 2015).
O
Zhou et al. (2015) showed that chitosan-cross-linked starch has strong
se
activities against E. coli , and they reported a new utilization method of chi-
lU
tosan as an antibacterial agent in foods.
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Generally, food spoilage begins on the food surface; consequently, the
o
rs
antimicrobial efficiency of edible coatings has major importance. He et al.
Pe
(2014) reported that clove oil (CO) and ethylene diamine tetraacetate (E) were
r
added into a chitosan (Ch) coating to develop its antimicrobial activity. The
fo
research demonstrated that Ch + CO and Ch + CO + E coatings could be
y
utilized as active packaging materials for foods.op
C
Chitosan-based composite films with various amounts of GSE (0.5%, 1.0%,
’s
and 1.5% v/v) were produced with a casting technique. The results showed
or
that GSE was distributed homogeneously within all chitosan film matrixes.
ut
Packaging of bread samples with chitosan-based GSE films prevented the

b
tri
growth of fungi compared with control samples. Therefore, it was reported

on
that chitosan-based GSE composite films have the potential to be a useful

.C
agent in food technology (Tan et al. 2015).

C
Cellulose and chitosan composite films are another biodegradable material

LL
with antimicrobial properties, for example, against E. coli and S. aureus . Wu

is
et al. (2016) showed that the composites demonstrated better performance

c
an
than traditional polyethylene packaging material and exhibited good poten-

Fr
tial as food packaging material for sausages. The composite films inhibited
more than 99% bacteria attached to them, and the shelf life of sausages was
d
an
extended at least for 6 days. The prepared composite films also have high
or
transparent and good antimicrobial properties.

yl
Incorporating nisin into chitosan films can improve the antimicrobial

Ta
activity of food packaging. According to Gharsallaoui et al. (2016), films con-

18
taining a nisin concentration above 50,000 IU/g inhibited S. aureus , L. mono-

20
cytogenes , and Bacillus cereus , while retaining good mechanical and optical
characteristics. But, the nisin incorporation did not show antimicrobial activ-
©
ity against B. subtilis .

Low-molecular-weight polyvinyl alcohol (PVOH) can be used to prepare
chitosan/PVOH blends and chitosan/PVOH/montmorillonite nanocom-
posites with the heat pressing method. The antimicrobial activity of chito-
san-based films was tested against the gram-negative bacteria, and it was
shown that chitosan-PVOH films had antibacterial activity against E. coli
(Giannakas et al. 2016).
13.3.4 Carrageenan-Based Antimicrobial Films

Carrageenans are water-soluble hydrocolloids composed of a linear chain of
sulfated galactans and obtained from various species of red seaweed. They
are classified according to the number and placement of a sulfated ester on
3,6-anhydro-d-galactose residues. Carrageenans have high potential as a
film-forming agent (Shojaee-Aliabadi et al. 2013).
y
nl
In Shojaee-Aliabadi et al. (2013), biodegradable composite κ -carrageenan
O
films incorporated with Satureja hortensis essential oil (SEO) and films with
se
SEO effectively inhibited the five microorganisms. Among the tested bacte-
lU
ria, P. aeruginosa indicated the highest resistance, but S. aureus was the most
na
sensitive to SEO-containing films. The results showed that SEO as a natural
o
antibacterial agent can potentially be applied in the packaging of various food
rs
products, especially those that are highly oxidative and microbially sensitive.
Pe
In another food packaging application, carrageenan-based antimicrobial
r
fo
films were obtained with incorporation of GSE at different concentrations
y
into the polymer using a solvent casting system, and their mechanical, physi-
op
cal, and antimicrobial properties were tested. The carrageenan/GSE compos-
C
ite films showed strong antibacterial activity against foodborne pathogens.
’s
or
It was reported that the carrageenan/GSE composite films can be used as an

ut
efficient antimicrobial biodegradable packaging film to extend the shelf life

b
of packaged foods (Kanmani and Rhim 2014b).

tri
on
Alginate and carrageenan can be applied to form film coating for meat and
.C
meat products. The coatings can inhibit microbial contamination, shrinkage,

and surface discoloration by delaying moisture transition (Tavassoli-Kafrani
C
LL
et al. 2016).
Nanocomposite films with chitin nanofibrils (CNFs) and carrageenan by
cis
the solution-casting technique exhibit strong antibacterial activity against

an
a gram-positive foodborne pathogen, L. monocytogenes . Results confirm that

Fr
the CNFs with antimicrobial activity can be used as nanofiller to develop

d
film properties of biopolymer-based packaging films to obtain food safety

an
and prolong the shelf life of foods (Shankar et al. 2015).

or
Antimicrobial bionanocomposite films can be prepared with κ -carrageenan,

yl
Ta
AgNPs, and organically modified clay mineral (Cloisite® 30B). AgNP-

containing nanocomposite films exhibit strong antimicrobial activity against
18
gram-negative bacteria; on the other hand, clay-containing nanocompos-

20
ite films show strong antimicrobial activity against gram-positive bacteria.

©
However, the nanocomposite films with combined application of both nano-

fillers exhibit strong antimicrobial activity against both gram-positive and
gram-negative bacteria (Rhim and Wang 2014).
13.3.5 Whey Protein Isolate– Based Films

Whey is a by-product of the cheese-making process, and it is usually dis-
carded as animal feed or used in sports food, infant formulas, and the
bakery industry. Whey proteins for film productions have been studied in
recent research activities due to their high industrial advantages, mainly as

plastic lacquer and paper coating. Nowadays, edible or biodegradable films
from whey proteins constitute useful means to extend the shelf life of foods
and increase their quality without providing any environmental pollution
(Coltelli et al. 2016).
In a study, antimicrobial films were obtained by incorporating various lev-
y
els of oregano oil (0.5%, 1.0%, and 1.5% w/w in the film processing solution)
nl
into sorbitol-plasticized whey protein isolate (WPI) films. The maximum
O
specific growth numbers of total flora (TVC) and Pseudomonas spp. were
se
significantly decreased with the use of antimicrobial films (1.5% w/w oil in
lU
the film processing solution), while the growth of lactic acid bacteria was
na
entirely inhibited. From the results, it was confirmed that oregano oil con-
o
rs
taining whey protein films extended the shelf life of fresh beef (Zinoviadou
Pe
et al. 2009).
r
In another study, the effectiveness of antimicrobial films against beef’ s
fo
spoilage flora at 5° C storage and the effect of the antimicrobial agents on the
y
op
physical and mechanical properties of the films were tested. Antimicrobial
C
films were processed by incorporating various levels of 3-polylysine (3-PL)
’s
and sodium lactate (NaL) into sorbitol-plasticized WPI films. The antimicro-
or
bial effect of the composite WPI films was reported for fresh-cut beef por-
ut
tions. The maximum specific growth rate of the total flora was significantly
b
tri
decreased with the application of antimicrobial composite films made from

on
0.75% w/w 3-PL in film processing solutions, while the numbers of lactic acid
.C
bacteria were entirely inhibited. Important growth inhibition of the TVCs

C
and Pseudomonas spp. was also observed with the application of films made
LL
with protein solutions (2.0% w/w NaL). Results showed that the effects of
is
the antimicrobial whey protein films prolonged the shelf life of fresh beef
c
an
(Zinoviadou et al. 2010).

Fr
WPI-based films with three types of nanoclays, Cloisite Na+ , Cloisite 30B,
and Cloisite 20A, were processed using a mixture casting method, and their
d
an
physical and antimicrobial properties were researched in order to better

or
realize the effect of nanoclay type on film properties. The WPI/Cloisite 30B
yl
films exhibited an advantageously bacteriostatic effect against gram-positive

Ta
bacteria, L. monocytogenes (Sothornvit et al. 2009).

18
Gadang et al. (2008) studied the inhibition effects of WPI films incorpo-
20
rated with GSE, malic acid (MA), nisin (N), ethylenediamine tetraacetic acid
(EDTA), and their mixture against artificially inoculated L. monocytogenes ,
©
E. coli O157:H7, and S. Typhimurium in a turkey frankfurter system with

surface inoculation (approximately 106 cfu/g) of pathogens. The results dem-
onstrated that the use of a film coating containing nisin, natural extracts, and
organic acids is a means of inhibiting the growth and recontamination of L.
monocytogenes , E. coli O157:H7, and S. typhimurium in ready-to-eat poultry
products.
In another study, the effects of edible films obtained from WPI and glyc-
erol, including the incorporation of lactic acid, propionic acid, natamycin,
and chitooligosaccharides (COSs) (molecular weight 3 kDa) as antimicrobial

agents, were determined. They were evaluated in vitro via agar diffusion and
viable cell counting methods against spoilage microflora generally found on
cheese surfaces. Films combined with lactic acid, propionic acid, or COSs
showed antimicrobial activity against all the microorganisms. COS was
the most effective against gram-negative bacteria; however, lactic acid was
y
the most inhibitory against gram-positive ones. Natamycin was not active
nl
against bacteria, but exhibited the strongest activity against yeasts (O.L.
O
Ramos et al. 2012).
se
The antibacterial activities of albumin– glycerol and whey– glycerol films
lU
were tested in Jones et al. (2015), and bacterial growth was not determined on
na
the bioplastics after 24 h of inoculation. It was concluded that these bioplas-
o
rs
tics can be used in food packaging applications in the future.
rPe
fo
y
op
C
13.4 Essential Oils and Their Compound-Based Films
’s
or
Naturally derived antimicrobial agents can be alternatives for chemically

ut
based antimicrobials. Herbal plant species and spice oils are known to con-
b
tri
tain various organic compounds capable of exhibiting antimicrobial activity

on
(Abdali and Ajji 2015; Irkin et al. 2011). Generally, EOs (also known as volatile
.C
oils) are complex mixtures of volatile agents obtained by living organisms

C
or from plant materials like leaves, buds, seeds, flowers, wood, roots, twigs,
LL
fruits, and barks and processed by physical means (pressing and distillation)
is
from a whole plant or part of plant known as a taxonomic source. The hydro-
c
an
phobicity of EOs allows them to divide the lipid layer of the bacterial cell
Fr
membrane and mitochondrion, and the structures become more permeable.

This condition leads to the outflow of ions and other cell structures, which,
d
an
upon passing a certain limit, leads to lysis and death. The incorporation of
or
EOs into foods for preservation and into food packaging films allows bet-
yl
ter inhibition of microorganisms, as well as provides consumer satisfaction

Ta
from the “ green” earth viewpoint. EOs contribute to food safety from the
18
most important foodborne pathogenic bacteria, such as Listeria , Salmonella ,

20
Staphylococcus , Clostridium botulinum , Aeromonas , Enterobacter , and their tox-

ins (Vergis et al. 2015).
©
In Urbankova et al. (2015), the greatest antimicrobial activity was shown

by composites with linalool 4-allylanisole (immobilized on wood flour),
although good results were reported with molecular sieves and talc.
Antimicrobial agents can be effective through either contact with outside
of food directly or facilitated by the use of volatile compounds of foods indi-
rectly. EOs and their components are highly vaporizing and they can prevent
spoilage growth in terms of vapor phase, which has been verified more effec-
tive than apply in the liquid phase. A study about this subject conducted
by Campos-Requena et al. (2015), combined antimicrobial effect was deter-

mined for the carvacrol (CRV): thymol (TML) mixture against Botrytis cine-
rea compared to a film containing only CRV, when the films were used by
indirect way contact with the strawberries. The IC50 of the EOCs in the film
was decreased from 40.4 mg/g (CRV only) to 13.2 mg/g (CRV:TML 50:50). As
a result the CRV:TML-containing film contributes effective inhibition of B.
y
cinerea but without organoleptic changes in strawberries.
nl
In Alboofetileh et al. (2014), marjoram essential oils exhibited the highest
O
antimicrobial activity, followed by clove and cinnamon, respectively. Next,
se
the antimicrobial films were prepared by incorporating various concentra-
lU
tions of marjoram essential oil, cinnamon, and clove into alginate/clay films.
na
It was reported that the films with EOs incorporated were more inhibitory
o
rs
against gram-positive bacteria (S. aureus and L. monocytogenes ) than gram-
Pe
negative bacteria (E. coli ).
r
Copaiba oil can be another alternative for food packaging. The copaiba
fo
tree is native to tropical areas of Latin America and West Africa. In particu-
y
op
lar, copaiba oil was found to be inhibitory against B. subtilis bacteria. It can
C
be incorporated into paper sheets and plastic films. Results were optimum
’s
concerning antibacterial properties, and without strong smells for biobased

or
agents (Morelli et al. 2015).

ut
In Muriel-Galet et al. (2015), green tea extract (GTE) and OEO were in-
b
tri
corporated into an ethylene vinyl alcohol (EVOH) copolymer. The total

on
antioxidant activity was determined in the food, and the antimicrobial effec-
.C
tiveness of the films was reported in vitro against L. monocytogenes , E. coli ,

C
and Penicillium expansum . The antioxidant property of the films containing

LL
GTE was the most active, and films containing OEO exhibited strong antimi-
is
crobial effects against the microorganisms.

c
an
Cyclodextrins (CDs) are biodegradable cyclic oligosaccharides with a

Fr
hydrophobic inner cavity and a hydrophilic outer surface, which permits the
formation of stable complexes with many organic compounds (Birck et al.
d
an
2016). In a different packaging application study, cellulose sulfate film with

or
antimicrobial characteristics was prepared by incorporating b-cyclodextrin

yl
(b-CD) and mustard essential oil (MO). The addition of MO increased the
Ta
physicochemical properties of the films, and more importantly, films with

18
MO exhibited great antimicrobial effects against E. coli and S. aureus , and

20
lower ones for B. subtilis and Aspergillus niger . The MO amount in NaCS-b-
CD-MO films was 11 mg/g, and it was 7 mg/g in NaCS-MO films processed
©
under the same conditions. The results of the study confirm that MO as a
natural antibacterial material and b-CD as a carrier of slow release can be
applied in edible films or coatings for food packaging for a wide scope of
microbially sensitive products (Chen and Liu 2016).
The incorporation of 0%, 2%, 4%, 6%, and 8% (w/w) Allium sativum essence
oil (AEO) into plastic films was tested against beef-related bacteria L. mono-
cytogenes , E. coli , and Brochothrix thermosphacta . Results demonstrated that
a low-density polyethylene/ethylene vinyl acetate (LDPE/EVA) copolymer
film with 8% AEO significantly decreased the number of bacteria, with the
inhibition level of L. monocytogenes > 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 car-
vacrol, citral, marjoram essential oil, or cinnamon bark essential oil on poly-
y
propylene (PP) and PET materials are perfectly usable for food packaging
nl
applications, and additionally, the incorporation of bentonite nanoclay to
O
their layers was also suggested.
se
Nanotechnology and the incorporation of EOs into edible films have poten-
lU
tial in the development of microbiologically safe foods. As an example, pul-
na
lulan films containing EOs and nanoparticles were tested against foodborne
o
rs
pathogens. The results were indicated that 2% OEO was effective against
Pe
S. aureus and S. typhimurium , whereas L. monocytogenes and E. coli O157:H7
r
were not prevented. Recent studies show that pullulan films containing anti-
fo
microbial compounds inhibited the pathogens related to vacuum-packaged
y
op
meat and poultry products stored at 4° C for up to 3 weeks, compared with
C
control films. It can be said that edible films made from pullulan incorpo-
’s
rated with EOs or nanoparticles may promote the safety of refrigerated,

or
fresh, or processed meat and poultry products (Kuorwel et al. 2011).

ut
In an active film coating application research study, S. aureus , Listeria

b
tri
innocua , Pseudomonas spp., Salmonella enterica subsp. enterica , and E. coli

on
were prevented when exposed to an atmosphere created of 4%– 6% (w/w) of

.C
Zataria EO (Akrami et al. 2015).

C
The antimicrobial activities of thymol and carvacrol as major EO com-

LL
pounds in food packaging system applications are shown in Table 13.1.

c is
an
Fr
d
an
or
13.5 Chemically Synthesized Biodegradable Polymers

yl
Ta
13.5.1 Poly-L-Lactide-Based Antimicrobial Films

18
It was explained that packaging films incorporating organic acids should

20
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).
©
20
18
TABLE 13.1 Ta
Some Food Packaging Applications with Essential Oil Compounds Carvacrol and Thymol
yl
Active Component Polymer Carrier Tested Food/Conditions Results References
or
Thymol PLA/polytrimethylene Laboratory conditions Inhibitions of Esherichia coli , Wu et al. 2014
an
carbonate films d Staphylococcus aureus , Listeria spp.,
Fr Bacillussubtilis , and Salmonella
an spp.
Carvacrol/thymol LDPE/organically modified Strawberry
c Effective inhibition of Botrytis Campos-Requena et al.
montmorillonite
is cinerea on strawberries 2015
Thymol Polybutylene succinate Laboratory conditions Inhibition of E. coli and S. aureus Petchwattana and Naknaen
LL
C 2015
Carvacrol LDPE/clay Laboratory conditions Prolonged and high antimicrobial Shemesh et al. 2015a
.C
on activity against E. coli
Carvacrol LDPE/organo-modified Laboratory conditionstr Inhibition effects against E. coli , Shemesh et al. 2015
montmorillonite L. innocua , and Alternaria alternata
ib
Thymol LLDPE/organo-modified Laboratory conditions Inhibition effects on E.coli ATCC Efrati et al. 2014
ut
montmorillonite 8739
or
’s
Food Packaging Systems with Antimicrobial Agents
Carvacrol/thymol Polypropylene Laboratory conditions Carvacrol and thymol at 8 wt% to

C M. Ramos et al. 2012b
PP improved product quality and
op
showed safety aspects (against
y
E. coli and S. aureus ) in food
fo
packaging applications
r
Thymol Pullulan films Laboratory conditions Inhibition effects against B. subtilis Gniewosz and Synowiec
Pe
ATCC 6633, S. aureus ATCC rs 2011
25923, S. enteritidis ATCC 13076,
o
and E. coli ATCC 25922
na
Carvacrol/thymol Microencapsulated thymol/ Laboratory conditions Inhibition activities on E. coli
l U Guarda et al. 2011
carvacrol in polymer films O157:H7, S. aureus , L. innocua , se
S. cerevisiae , and A. niger
443
O
nl (Continued)
y
©
20 444
18
Ta
Some Food Packaging Applications with Essential Oil Compounds Carvacrol and Thymol
yl
or
Active Component Polymer Carrier an Tested Food/Conditions Results References
Carvacrol PET containing carvacrol- d Fresh salmon fillets Inhibition effects on mesophiles Rollini et al. 2016
coextruded multilater film Fr and psychrotrophs
Carvacrol PP/EVOH 32/PP tray Salmon cubes and slices
an Rapid and effective migration of Cerisuelo et al. 2013
heat-sealed with an active c antimicrobial agent to the fish
PP/EVOH-29 + 6.5% is muscle
carvacrol/PP film lid LL
Carvacrol Sodium caseinate plasticized Laboratory conditions
C Inhibitory effects on E. coli and Arrieta et al. 2014
matrixes (transparent active .C S. aureus
films) on
Carvacrol Nanoclay bentonite was Laboratory conditions Developed material can be used Cerisuelo et al. 2012
added to EVOH 29 films in antimicrobial active food
tri
but packaging
Thymol Organoclay Cloisite 30B Laboratory conditions L. innocua inhibited effectively Rodriguez et al. 2014
or
Carvacrol Gelatin films Laboratory conditions
’s
Films exhibited antimicrobial Kavoosi et al. 2013
C
effects against Pseudomonas
aeruginosa ATCC 9027, E. coli
op
y
ATCC 8739, S. aureus ATCC 6538,
and B. subtilis ATCC 6633
fo
Carvacrol Chitosan/CD films Chicken fillets
r
Inhibition effects on lactic acid
Pe Higueras et al. 2014
bacteria, yeast, and fungi rs
Carvacrol Chitosan-based films Laboratory conditions Antimicrobial activity for o Kurek et al. 2013
S. enteritidis , B. subtilis , E. coli , and
na
L. innocua lU
se
O
nl
y
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
y
active films (Ruiz-Cabello et al. 2015).
nl
In Han et al. (2015), it was stated that 0.5% nisin added to plasticized bio-
O
degradable PLA film has the potential to preserve wild edible mushroom
se
(Boletus edulis ) quality and prolong its postharvest life to 18 days stored at
lU
4 ± 1° C.
na
In another study, the packaging mechanism based on PLA was recog-
o
rs
nized by sol-gel processing and incorporated with natamycin as the active
Pe
agent. The release of the antifungal determined in food stimulants was also
r
observed at a maximum value of about 0.105 mg/dm2 , a level definitely lower
fo
than that permitted by directive for cheese rind. The coating inhibited unde-
y
op
sirable mold growth on the surface of commercial semisoft cheese (Lantano
C
et al. 2014).
’s
or
ut
b
tri
on
13.6 Polymer/Clay Nanocomposite-Based Antimicrobial Films

.C
C
Clay minerals, such as montmorillonite, have been extensively used as an

LL
agent for drug delivery and in controlled-release mechanisms with good

is
results. Montmorillonite’ s large surface-to-area ratio, cation exchange capac-

c
an
ity, and absorption ability make nanoclays optimum drug carrier agents.
Fr
Several assays to improve antimicrobial agents for food packaging based on

clay nanocomposites have applied incorporation into volatile compounds
d
an
such as antimicrobial materials, for example, carvacrol in nanoclay/EVOH,

or
EOs in alginate/clay nanocomposites, and rosemary EOs in montmorillon-

yl
ite/chitosan bionanocomposites. Among some volatile compounds such as

Ta
antimicrobial materials in active packaging, carvacrol and thymol contain

18
a broad spectrum of antimicrobial and antifungal effects, and they are cur-
20
rently classified as GRAS agents by the FDA (Campos-Requena et al. 2015).

Linear low-density polyethylene (LLDPE) matrix-based active nanocom-
©
posite 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 meso-
philic bacteria, lactic acid bacteria, and total yeast and mold counts in the
Turkish sliced sucuk samples was controlled by a vacuum packaging sys-
tem with active nanocomposite films during 30 days of storage (Tornuk
et al. 2015).
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).
y
nl
O
se
lU
13.7 Metal and Metal Oxide Nanoparticle-
na
Based Antimicrobial Films
o
rs
Many nanostructured metallic particles, such as silver, gold, copper, and
Pe
zinc, are widely used to obtain nanocomposite packaging materials. Among
r
them, AgNPs have received significant attention in the food packaging area
fo
because of their special and wide spectrum of antimicrobial effects against
y
op
foodborne pathogens. Various antimicrobial bionanocomposites have been
C
demonstrated by incorporating AgNPs into polymeric matrixes, such as
’s
agar, chitosan, and cellulose (Kanmani and Rhim 2014a, 2014c).

or
Among metal nanomaterials, nanosilver has been determined to be a

ut
potent antimicrobial material. The antimicrobial function of silver is princi-

b
tri
pally assigned to the process of silver ions and metallic AgNPs. It has been
on
stated that silver ions interact with negatively charged biomacromolecular

.C
compounds (sulfhydryl or disulfide groups of enzymes) and nucleic acids,

C
leading to structural changes and disruption in bacterial cell membranes

LL
and walls that cause the prevention of metabolic conditions, followed by cell
is
death. The bactericidal activity of nanosilver improves by the release of silver

c
an
ions within bacterial cells (Tavassoli-Kafrani et al. 2016).

Fr
Nanoparticles with the filler attribute are incorporated in biopolymer

films for improving mechanical, water vapor, and thermal barrier proper-
d
an
ties. Among the nanofillers, ZnONPs have good capacity for nanoscale dif-
or
fusion and interfacial interactions in protein structures due to their large

yl
surface area and high-level surface energy. The incorporation of ZnONP

Ta
as a particular filler in biopolymers, such as starch-based films, has been

18
reported to cause the development of water vapor and mechanical barrier

20
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 cytoplas-
mic 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
microorganisms and viruses, and copper is vital for life as an agent of metal-
lic 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
y
the food packaging area since it is regarded as toxic in contact with food; in
nl
addition, it would accelerate the biochemical reaction with foods due to its
O
catalytic effect of oxidation. Metal oxides, such as TiO2 , ZnO, and MgO, can
se
be used for the formulations of antimicrobial packaging films due to their
lU
high antimicrobial activity with strong stability compared with organic anti-
na
microbial agents (Rhim et al. 2013).
o
rs
The antimicrobial efficiency of AgNP-containing antimicrobial packag-
Pe
ing films is highly affected by several factors, such as the degree of particle
r
agglomeration, the particle size and its distribution, the interaction of sil-
fo
ver’ s surface with the base polymer, and the silver content. AgNPs should be
y
op
highly diffused through the polymer structure without agglomeration. As a
C
result, it is essential to prepare AgNPs with proper measures and determine
’s
optimum polymeric materials for the preparation of active antimicrobial

or
packaging films with AgNPs. In addition, the agar/AgNP composite films

ut
demonstrated typical antimicrobial effects against both gram-positive and

b
tri
gram-negative pathogenic bacteria (Rhim et al. 2014).

on
In Beigmohammadi et al. (2016), the antimicrobial activities of LDPE pack-

.C
aging films coated with silver, zinc oxide, and copper oxide (CuO) nanopar-
C
ticles were determined in ultrafiltrated (UF) cheese. Copper decreased the

LL
growth rate of E. coli by more than 99.99%, leading to disruption of the cell
is
walls and changing the bacterial cell contents. The developed active packag-
c
an
ing containing 1% w/w CuO nanoparticles in LDPE polymer was processed

Fr
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.
d
an
In Sanuja and Umapathy (2015) , nano-ZnO at various concentrations (0.1%,

or
0.3%, and 0.5%) and neem ( Azadirachta indica ) EO were incorporated into the
yl
chitosan polymer by a solution-cast method to improve the properties of

Ta
the bionanocomposite film. Antibacterial activity was found to have a high

18
inhibition effect against E. coli . Bionanocomposite film applications were

20
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 anti-

microbial 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 concen-
trations. The migration of compounds from the nanomatrix starch films,
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)
y
matrix for processing as food packaging materials. The antibacterial proper-
nl
ties of HPMC/AgNP thin films were reported based on a disk diffusion test
O
against E. coli and S. aureus . The disk diffusion research showed a higher
se
bactericidal effect for nanocomposite films containing 41 nm AgNPs (Moura
lU
et al. 2012).
na
Through research, silver nanodots of different sizes (average sizes of
o
rs
10, 18, and 28 n m by different molecular weights) using a self-assembled
Pe
polystyrene-b polyethylene oxide (PS-b-PEO) block copolymer were
r
improved. The developed silver nanodot surfaces showed good antimicro-
fo
bial effect against gram-positive and gram-negative bacteria, resulting in
y
op
their ability to be used in antimicrobial packaging applications. It was also
C
reported that Pseudomonas fluorescens (gram-negative bacteria) was more
’s
sensitive than S. aureus (gram-positive bacteria) (Azlin-Hasim et al. 2015).

or
The application of nanotechnology, containing 1– 100 nm particles and

ut
indicating novel properties, is a significant novelty in many industrial areas.

b
tri
In the food industry, nanoparticles can be incorporated into food and food
on
contact materials (FCMs), bringing the materials new and developed proper-
.C
ties, such as antimicrobial activity, oxygen-scavenging potential, improved

C
thermal stability, biosensing ability, improved moisture and gas barrier,

LL
altered color, texture, and improved material strength (Hannon et al. 2016).
cis
an
Fr
d
an
13.8 Advantages and Difficulties of Natural

or
Antimicrobial Packaging Systems

yl
Ta
Synthetic plastic packaging materials are widely used for the packaging of
18
various foods. They can cause serious environmental problems since they
20
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 barri-
ers by improving quality and prolonging the shelf life of foods. Moreover,
biopolymer packaging materials are good vehicles for incorporating vari-

ous 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
y
amounts and chemical preservatives, in the food industries (Beigmohammadi
nl
et al. 2016).
O
In biopolymers, carrageenans have high effects as a film-forming material.
se
Cooling a hot solution of carrageenan during the film casting and drying
lU
process shows the transition of an occasional coil to a double helix, which
na
occurs in the formation of a compact and structured film after dehydration
o
rs
of the solution. In a study, it was concluded that carrageenan can produce
Pe
a clear film with good mechanical and structural qualities, including high
r
tensile strength (Shojaee-Aliabadi et al. 2013).
fo
Alginate is a good alternative for film and coating formation due to its
y
op
colloidal properties, including strength, thickness, emulsion stability, and
C
gel and film production (Kazemi and Rezaei 2015). Commercialization of
’s
carrageenan and alginate biopolymer films is restricted due to their high

or
sensitivity to moisture and their compatibility with other emergent stress

ut
factors, such as high pressures, ultrasound, microwave, and gamma radia-

b
tri
tion (Tavassoli-Kafrani et al. 2016).

on
Another food packaging material is Ag-based nanoparticles, which are

.C
applied to the surface of packaging to contribute an antimicrobial prop-

C
erty. The large reactive surface of the nanoparticles increases the capacity
LL
for AgNPs to oxidize into silver ions (Ag+ ) that come into contact with the
is
food material to allow antimicrobial activity. In a recent study, the migration

c
an
of AgNPs from an LDPE coating into food materials was examined under
Fr
accelerated time and temperature conditions. Under microwave conditions,

there was an important increase in migration when compared with oven
d
an
heating; this requires further research, especially where nanopackaging may

or
be applied in microwaveable ready-to-eat foods and food storage containers

yl
(Hannon et al. 2016). Today, Ag-based commercial packaging films applied

Ta
to muscle foods are AgIon® Life Materials Technology’ s silver-based mas-

18
terbatch, Irgaguard® BASF’ s silver-based masterbatch, Surfacine® Surfacine

20
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 anti-
fungal 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
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 pro-
longing the activity of these films have not been determined (Shemesh et al.
2015a).
PLA-based films have natural, biodegradable, biocompatible, and opti-
y
mum mechanical and optical properties (Ramos et al. 2014). The growth of
nl
antimicrobial PLA films with improved physical and mechanical properties
O
and antimicrobial activity is in question due to the inherent hydrophobicity
se
and brittleness of this polymer. Some of these material limitations can be
lU
overcome with the application of current and advanced Technologies, such
na
as nanotechnology (Tawakkal et al. 2014). PLA is produced commercially by
o
rs
four manufacturers in the World: American Polymer Standards Corporation,
Pe
Mentor, Ohio; NatureWorks, Minnetonka, Minnesota; Purac Biomaterials,
r
Gorinchem, the Netherlands; and Total S.A., Courbevoie, France (Vijayendra
fo
and Shamala 2014). In Ruiz-Cabello et al. (2015), research of a commercial
y
op
product based on Allium extract (Proallium SO-DMC® ) was applied by extru-
C
sion into PLA to an active food packaging with the aim of extending the shelf
’s
life of ready-to-eat salads. Proallium is a condiment based on vegetal extracts

or
that is composed of organosulfur compounds, which are characteristic of the

ut
Allium spp. The result was that the PLA Proallium film exhibited antimicro-
b
tri
bial activity for ready-to-eat salads.

on
WPIs have shown potential mechanical features, as well as optimum

.C
moisture permeability and good oxygen barrier characteristics, comparable

C
to those displayed by the best synthetic polymer-based films, for example,

LL
high-density polyethylene (HDPE), LDPE, EVOH, vinyl alcohol, polyvinyli-

is
dene chloride (PVDC), polyester, and cellophane. WPI-based films have been
c
an
confirmed for their excellent biomaterials for application as carriers of such

Fr
food additives as antioxidants, antimicrobials, flavors, fortifying nutrients,

and spices (Ramos et al. 2012a).
d
an
During the production of composite films, some characteristics can be

or
affected from incorporated materials. For example, composite WPI/clay films

yl
have an opaque appearance, which depends on the amount of clay added.

Ta
The composite films can be slightly less transparent than the neat WPI films.
18
It was found that film properties, such as the surface color, optical, tensile,
20
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
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 pack-
y
ages. Oxygen permeability values of mammalian gelatin films are higher
nl
than those for cold-water FG films. Tensile strength, elongation percentage
O
at break, and puncture deformation decreased in gelatin from mammals,
se
warm-water fish, and cold-water fish, respectively. Gelatin is subject to fast
lU
biodegradation when the contamination and environmental conditions are
na
sufficient. But disadvantageously, bacterial contamination can be present at
o
rs
different stages of the production, and quality control of gelatin-producing
Pe
factories indicated that aerobic, thermotropic, and proteolytic endospore-
r
forming bacteria may be present during the production (Coltelli et al. 2016).
fo
One of the biopolymers is chitosan, which is obtained from the shells of
y
op
crab, shrimp, and other shellfish. It is nontoxic and biodegradable and con-
C
tains antioxidant characteristics, as it is a linear polysaccharide obtained by
’s
the deacetylation of chitin (Ninjiarani 2015). Bionanocomposites have poten-

or
tial future prospects, although nowadays the low level of production and
ut
high cost restrict them from a wide range of applications (Rhim and Ng 2007).
b
tri
However, chitosan films have a high transfer of water vapor to the food mate-
on
rial and are brittle and extremely soluble under dry and wet conditions, which
.C
limits their use widely as packaging materials for foods (Gartner et al. 2015).
C
However, further studies are needed to research the potential effects of

LL
natural antimicrobial biopolymers in the development of model food prod-

is
ucts (Kanmani and Rhim 2014c).

c
an
Fr
d
an
or
13.9 Conclusion
yl
Ta
Applying antimicrobial-based films for food packaging holds many chal-

18
lenges; however, they provide food safety and reduce spoilage. Antimicrobial
20
packaging can be used with a hurdle technology that, in addition to other

processes, such as pulsed light, irradiation, and high pressure, can pre-
©
vent 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, keep-
ing the high sensory properties of foods is essential with the use of natural
biopolymers. Consumer acceptance and technical feasibility should also be
considered when the technologies are developed with the use of antimi-
crobial compounds, in addition to their microbiological, physiological, and
chemical effects. Some modeling food studies should also be researched for
their biopolymer applications.
y
nl
O
se
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14
Active and Intelligent Food Packaging
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Cristina Nerin, Paula Vera, and Elena Canellas
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CONTENTS
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14.1 Introduction................................................................................................. 459
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14.2 Active Packaging......................................................................................... 460
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14.2.1 Antioxidant Packaging................................................................. 462
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14.2.2 Oxygen Scavengers....................................................................... 466
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14.2.3 Free Radical Scavengers............................................................... 467
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14.2.4 Antimicrobial Packaging............................................................. 468
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14.2.5 Other Approaches in Active Packaging.................................... 470
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14.2.6 Carbon Dioxide Emitters and Scavengers................................. 470

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14.2.7 Ethylene Scavenging..................................................................... 471

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14.2.8 Moisture Control........................................................................... 471

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14.2.9 Flavor or Odor Absorbers and Releasers.................................. 471

14.2.10 Microwaveable Active Food Packaging.................................... 472
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14.3 Intelligent Packaging.................................................................................. 472

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14.3.1 Indicators and Sensors................................................................. 474

14.3.2 Radiofrequency Identification ................................................... 475
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14.3.3 Microwaveable Intelligent Food Packaging.............................. 475

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14.4 Nanotechnology, Biomaterials, and Bioactive Compounds Used

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in Active and Intelligent Packaging......................................................... 476

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14.5 Commercially Available Active and Intelligent Packaging.................. 477

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14.6 Legislation.................................................................................................... 477

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References.............................................................................................................. 482
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14.1 Introduction
Food packaging has been historically used to contain food, fulfilling the pri-
mary functions of containment, protection, and communication. However,
these functions have not been sufficient for the food industry for several rea-
sons. Consumers are looking for better-quality, fresh-like, and convenient
food products (Ozdemir and Floros 2004), and the food industry is looking
459
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 depend-
ing on its functionality (Brockgreitens and Abbas 2016):
y
1. Ergonomic packaging: Packages that improve the transport, use,
nl
and storage, for example, the easy-to-open bottles for older adults
O
(Bell et al. 2016).
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2. Informative packaging: Packages that enhance the information for
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consumers about the product’ s manufacturing, composition, and
storing conditions. This information can be printed on the package
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or can be electronically inserted as radiofrequency identification
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(RFID) tags (Ranky 2006).
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3. Active packaging: Where the packaging contains certain compounds
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that directly interact with the packaged food, affecting its quality
op
and extending its shelf life.
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These systems are constantly acting on the packaged food without

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requiring that a change or trigger occur in the food. Some good

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examples are moisture scavengers and antimicrobial or antioxidant

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packaging.
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4. Responsive or reactive packaging, which is commonly named intel-

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ligent or smart packaging: Packaging that gives an informative

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response as result of a specific trigger or change occurring in the

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food product or in its headspace or the outside environment. These

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changes can be foodborne threats, such as the presence of molds,

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bacteria, and contaminants, or benign factors, such as moisture, pH,

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or gas levels in the environment or headspace.

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It is very important to know the difference between active and intelligent

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packaging. Active packaging works without a specific trigger mechanism.

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However, intelligent packaging only works when there is a change in the

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food or headspace, without acting directly on the food (Brockgreitens and

18
Abbas 2016).
20
©
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)
Active and Intelligent Food Packaging 461
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
y
process.
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O
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It is also possible to classify active packaging according to the intended
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action it has on the packaging, with antioxidant or antimicrobial packaging
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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 atten-
o
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tion in the last decade. However, in most of cases it is difficult to distinguish
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between them, as both functions occur simultaneously and exert synergistic
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action on one another. For example, cinnamon essential oil (EO) is simul-
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taneously a potent antimicrobial and a good free radical scavenger, which
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makes it a good antioxidant. Montero-Prado et al. (2011) demonstrated that
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cinnamon EO affects the enzymes responsible for oxidation, such as poly-
’s
phenoloxidase (PPO), and protects natural peaches from natural decay and
or
mold proliferation, acting as an antimicrobial.

ut
b
A classic classification divides active packaging approaches into scaven-

tri
gers and releasers, as Figure 14.1 shows (Lee et al. 2015).

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For this reason, detailed descriptions of different active packaging

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approaches are organized by active agent more than by the actions they exer-
C
cise on the food.

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Active packaging represents an innovative strategy to incorporate antioxi-

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dants or antimicrobials in the material. To remove all synthetic additives from

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an
the food while simultaneously extending the shelf life and maintaining the
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quality of packaged food sounds like a dream. But the advances in technology
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and the intensive research carried out in the last 15 years make area a close
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Package
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Active releasing Active scavenging

systems systems
20
Carbon dioxide Oxygen

©
Moisture Food Carbon dioxide

Flavor or odor Ethylene
releasers Moisture
Antioxidant release Flavor or odor
Antimicrobial release absorbers
Active agent
FIGURE 14.1
Types of active packaging.
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
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The use of antioxidants in the food industry is historically known. They are
nl
organic molecules that act by several mechanisms (Brewer 2011; Becker et al.
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2004), inhibiting all reactive oxygen components that produce damage in the
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metabolic process. But not all antioxidants can be used in food packaging.
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They must be safe, efficient at very low concentrations, thermally stable, and
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economically viable; have a long antioxidant capacity; and not modify the
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organoleptic properties of packaged food (Shahidi 1997).
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They can be classified into two groups: synthetic and natural antioxidants
r
(Table 14.1). However, specific restrictions are appearing more and more for
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synthetic ones. For example, tert-butylhydroquinone (TBHQ) is not permit-
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ted in Europe, and the use of ascorbyl palmitate is being questioned due to
C
its possible adverse effects on human health (Wojcik et al. 2010; Krishnaiah
’s
et al. 2011). Butil Hydroxy toluene (BHT), a well-known antioxidant used in

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both polymers and food, is not permitted in some countries, as it has toxic
ut
effects at high concentrations.

b
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For these reasons, the trend is to use natural antioxidants, which have been
on
obtained mainly from plants (Tongnuanchan and Benjakul 2014; Valdes et al.
.C
2015). In principle, there is no restrictive legislation for these substances, nor

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are they frowned upon by health authorities and consumers. However, they
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have some disadvantages, such as the contribution of undesirable color, odor,

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and flavor to food.

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The main natural antioxidants used for active food packaging are listed
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in Table 14.1, together with their applications in food and their advantages.
The individual substances responsible for the antioxidant properties that are
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contained in these EOs and extracts from plants are phenolic compounds
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such as tocopherol, flavonoids, and phenolic acids.

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The antioxidant performance is controversial. Although many authors

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insist on the requirement that release antioxidants have efficient active pack-
18
aging, we believe that in most cases, the release of antioxidants is not neces-
20
sary. 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 proper-
ties, 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 anti-
oxidants 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
©
20
TABLE 14.1
18
Types of Active Packaging, Technology Used, and Their Applications
Ta
Types of Active Packaging Technology Used in Food Packaging Applications
yl
or
Antioxidant release Synthetic antioxidants Fresh vegetables such as mushrooms (Wrona et al.
• BHT (Nisa et al. 2015; Yehye et al. 2015), butyl hydroxy 2015) and sweet tomatoes (Lebot et al. 2016)
an
d
anisol (BHA) (Kim et al. 2010), propyl gallate, ascorbyl Bulk sunflower oil and mayonnaise (Bholah et al.
palmitate, TBHQ 2015)
Fr
Natural antioxidants from plant extracts and EOs (Bentayeb Cookies (Aksoylu et al. 2015)
an
et al. 2014) cis Fresh fruit such as peaches (Montero-Prado et al.
• Tocopherol (vitamin E) (Melo et al. 2016; Amalia et al. 2016) 2011)
• Ascorbic acid (vitamin C) (Babou et al. 2016) Meat (Laranjo et al. 2016), beef (Nisa et al. 2015)
LL
• Phenolic acids (vanilic, galic, and caffeic acids) (Agatonovic-
C
Kustrin and Morton 2016)
.C
• Cinnamic acids (cafeic, ferulic, and p-cumaric acids)

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• Flavonoids (Heim et al. 2002; Colon and Nerin 2012;
Lopez-de-Dicastillo et al. 2012)
tri
b
• Carotenoids, natural chelates, and even amino acids and
ut
peptides (Bernald et al. 2010) or
Oxygen scavengers Enzymatic systems Vegetables such as tomatoes (Leyva et al. 2013)
’s
• Glucose and alcohol oxidase (Leyva et al. 2013) C Fresh fruit such as apples (Di Maio et al. 2015)
Chemical systems Strawberry (Kartal et al. 2012)
op
• Iron powered oxidation with iron oxide, catechol, ferrous y Vine fruit (Tarr and Clingeleffer 2005)
carbonate, iron-sulfur, sulfite salt-copper sulfate, metallic Beef and pork (Limbo et al. 2013)
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platinum (Lee et al. 2015) r
• Ascorbic acid oxidation, catalytic conversion of oxygen by
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platinum catalyst, unsaturated fatty acids such as like oleic rs
or linoleic acid (Ozdemir and Floros 2004) o
Others
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• Photosensitive dye oxidation and immobilization of
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microorganisms in solid holders (Vermeiren et al. 1999) se
463
O (Continued)
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20 464
18
TABLE 14.1 (CONTINUED) Ta
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Free radical scavengers Natural phenolic compounds such as hydroquinons and Beef (Nerin et al. 2006)
an
catechins
d Fatty food (Carrizo et al. 2016)
EOs: oregano, cinnamon, rosemary Chocolate derivatives, cereals [12]
Fr
Se nanoparticles (Vera et al. 2016) Fruits such as peaches (Montero-Prado et al. 2011)
an
cis Nuts, walnuts, fried chip potatoes (results not
published)
Antimicrobial release Synthetic antimicrobials Cooked ham (Montiel et al. 2015)
LL
• Silver zeolites (Shankar et al. 2016)
C Fresh chicken (Sanchez-Ortega et al. 2014)
• Polymers (chitosan [Andrei et al. 2016]) Poultry meat (Ahmed et al. 2016)
.C
• Peptides such as megainins, cecropines, defensins, and
on Bread (Jideani and Vogt 2016)
lauroyl ethyl arginate (LAE) (Becerril et al. 2013)
tri Cheese (Peighambardoust et al. 2016)
• Esters and phenolic acid b Fruits such as melon, pine apple (Scollard et al.
• Metals such as cooper and silver (Kredl et al. 2016) 2016), and fresh strawberry (Duran et al. 2016)
ut
• Chlorine dioxide, sulfur dioxide Vegetables such as lettuce and spinach
or
Natural antimicrobials (Poimenidou et al. 2016)
’s
• Bacteriocins and antibiotics such as nisin (Krivorotova et al.C
2016) and natamycin (Colak et al. 2016)
op
• Enzymes such as lactoperoxidase, lysozyme, and lactoferrin y
(Montiel et al. 2015) fo
• From plant extracts and EOs (Akrami et al. 2015)
r
• Natural phenolic compounds such as hydroquinons and
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catechins rs
• Terpenes
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• Fatty acids and steres
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(Continued)
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y
©
20
18
Ta
TABLE 14.1 (CONTINUED) yl
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an
d
Carbon dioxide scavengers Iron powder Fruits such as strawberry (Aday et al. 2011)
and emitters Calcium, sodium, or potassium hydroxide Meat such as lamb (Vergara et al. 2009)
Fr
Silica gel Fresh pork sausage (Martinez et al. 2006), beef
an
c
Metal halide (Lee et al. 2015; Ozdemir and Floros 2004)
is steak (O’ Sullivan et al. 2011)
Ethylene scavenging Activated charcoal, silica gel-potassium permanganate
LL Fruits such as banana (Terry et al. 2007) and
(Taechutrakul et al. 2009), zeolite (Lee et al. 2015), kieselguhr,
C kiwifruit (Park et al. 2009)
bentonite, Feller’ s earth, silicon dioxide powder, powdered
.C Vegetables such as tomatoes (Taechutrakul et al.
Oya stone, ozone (Ozdemir and Floros 2004) 2009)
Moisture scavenging Silica gel, propylene glycol, polyvinyl alcohol, diatomaceous Chips (Liu and Lin 2009)
on
earth (Lee et al. 2015) Nuts (Ozdemir and Floros 2004)
tri
b Spices (Ozdemir and Floros 2004)
Biscuits (Rodriguez-Aguilera and Oliveira 2009)
ut
or
’s Milk power (Vermeiren et al. 1999)
Flavor or odor absorbers Mixture of charcoal and nickel C Fish
and releasers Ferrous salt, citric acid, and ascorbic acid op Ground coffee
Baking soda y Orange and fruit juices (Vermeiren et al. 1999; Biji
Many food flavors (Ozdemir and Floros 2004) et al. 2015; Rooney 1995)
fo
Microwaveable Susceptors Popcorn, lasagna, meat pies, bakery goods, chips,
r
Shielding hotdogs, pizza, sandwiches (Bohrer 2009; Regier
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Modifiers 2014) rs
Moisture-absorbing flexible materials o
Steam valves
na
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se
465
O
nl
y
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 rea-
son, the real active antioxidant packaging should not release any compound,
and as was mentioned above, the scavenging action, either of molecular oxy-
gen or free radicals, is more suitable and appropriate for this task.
However, there are several approaches that can be mentioned, and all of
y
them can be classified in two different types of antioxidant packaging. The
nl
first one modifies the internal atmosphere to gain favorable conditions, and
O
the second one scavenges the free radicals that initiate the oxidation process.
se
In general, any packaging material can be converted into active antioxi-
lU
dant material, although the most common is from polyolefins (polyethylene
na
[PE], polypropylene [PP], polyethylene terephthalate [PET], and polystyrene
o
rs
[PS]), alone or combined with other materials, forming a multilayer film
Pe
structure (also with other materials, such as aluminum, paper, or cardboard),
r
and more recently, biopolymers (Atares and Chiralt 2016; Valdes et al. 2014).
fo
The industry prepares antioxidant materials by several ways:
y
op
C
• Incorporation of the antioxidant in the bulk of the polymeric matrix
’s
by extrusion technology (Gomez-Estaca et al. 2014).

or
• Incorporation of the antioxidant as a liquid dispersion by coating

ut
b
technologies. This system is widely used, giving rise to many pat-

tri
ents and marketed packages (Nerin et al. 2006, 2008; de Dicastillo

on
et al. 2011; Garces et al. 2004).

.C
• Incorporation of the antioxidant and an oxygen absorber between

C
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different layers in a multilayer material. Although this system is

available on the market, it is very expensive technology (Tian et al.
cis
2013).
an
• Incorporation of the antioxidant in multilayer packaging, where the

Fr
antioxidant agent is placed between inert substrates. For example,

d
an
there are recent studies that apply selenium nanoparticles as the

antioxidant, and they are incorporated in the adhesive used to man-
or
yl
ufacture the multilayer material (Vera et al. 2016; Nerín et al 2014).

Ta
18
The great advantage offered by these active materials is that their antioxi-
dant properties are kept for longer periods. In addition, the number of addi-
20
tives 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 oxida-
tion 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
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 oxy-
y
gen scavenger. The different systems manufactured are shown in Table 14.1
nl
(Ozdemir and Floros 2004; Lee et al. 2015; Rodriguez-Aguilera and Oliveira
O
2009).
se
The most commonly used O2 scavenger is based on the oxidation reaction
lU
of iron powder (Lee et al. 2015). Traditionally, it was designed in the form of
na
small sachets containing different oxidizing agents inside. But this system
o
rs
is not appropriate for liquid foods and is not good for health, as the sachets
Pe
can be accidentally broken, producing a risk of ingestion of their contents
r
(Realini and Marcos 2014; Ozdemir and Floros 2004).
fo
As an alternative to sachets, another approach is the incorporation of the
y
op
oxygen scavenger into the packaging structure itself, using cards, sheets, or
C
layers coated on the package’ s inner walls (Biji et al. 2015) or in a multilayer
’s
(Ozdemir and Floros 2004; Realini and Marcos 2014). The scavenging action
or
is achieved by rapid diffusion of oxygen from the headspace through the lay-
ut
ers to the reactive agent. Of course, the ingredients used in these approaches
b
tri
are not the same as those used in the sachets. In general, the efficiency of this
on
last system is lower than that of the labels or sachets (Day 2008).
.C
C
LL
14.2.3 Free Radical Scavengers

is
Instead of molecular oxygen, free radicals derived from oxygen can be suc-
c
an
cessfully scavenged, as has been demonstrated in several scientific publica-

Fr
tions (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 initi-
d
an
ated by free radicals, if they are removed, the oxidation does not take place.
or
Using this principle, antioxidant performance occurs even in the presence

yl
of molecular oxygen. Scavenging free radicals, such as oxo, hydroxo, and

Ta
peroxo, is much easier than scavenging molecular oxygen, as the oxygen-

18
derived free radicals are smaller and more reactive and diffuse faster than O2
20
throughout the polymers. For this task, direct contact with the inner atmo-
sphere is not required if the diffusion of free radicals is demonstrated. The
©
research carried out so far demonstrates that this process occurs through low-
density 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
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
y
radical scavengers can also protect lipids from oxidation processes. Vera et
nl
al. (2016) demonstrated the extension of shelf life of nuts and walnuts using
O
a multilayer-containing nanoselenium behind the LDPE, and similar results
se
were obtained with chip potatoes (results not published).
lU
ona
14.2.4 Antimicrobial Packaging
rs
Pe
Antimicrobial packaging acts by reducing, inhibiting, or retarding microbial
(yeasts, molds, and bacteria) growth in order to extend the shelf life of pack-
r
fo
aged food. In contrast to antioxidant packaging, antimicrobials should be
y
released from the packaging, either by direct contact or through the vapor
op
phase, as they should arrive at the cell and kill it or inhibit its growth.
C
A wide variety of antimicrobial agents have been used so far, as shown

’s
or
in Table 14.1, but the list is continuously increasing, as there is not one per-
ut
fect or ideal agent to be used in any packaging material or foodstuff. The

b
tri
action mechanism of antimicrobial agents can be classified into two types.

on
In the first, the antimicrobial agent migrates to the food. A good example
.C
is the migration of volatile compounds that enter into the headspace in a

C
gaseous phase and then come into contact with the food (Mastromatteo et al.
LL
2010), as happens with volatile phenolic compounds of EOs (Kuorwel et al.

is
2011b; Llana-Ruiz-Cabello et al. 2015). In the second type, the agent acts from
c
the food surface (nonvolatile compounds) in contact with the material (Han
an
2000), as commercial silver zeolites incorporated into PP, PE, or nylon do

Fr
(Quintavalla and Vicini 2002).

d
an
Different approaches have been developed for the application of antimi-

crobial compounds in packaging:
or
yl
Ta
• Incorporation in sachets or pads inside the package, containing the

18
antimicrobial, for example, ethanol. As already mentioned, it acts

by absorbing moisture and releasing ethanol vapor to retard mold
20
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 poly-
meric 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
of fungicide or bactericide substances compatible with the food.

However, if the antimicrobial agents are volatile, they will be par-
tially volatized during extrusion at high temperature, causing a loss
of effectiveness (Tramon 2014). This is the main drawback of extru-
sion technology.
y
To avoid this inconvenience, new strategies, such as microporous carriers,
nl
O
plasticizers, and encapsulation, have been developed (Kayaci et al. 2013; de
se
Azeredo 2013; Dias et al. 2013). Another alternative is to use an additional
lU
inner layer between the film with the antimicrobial substance and the food
matrix, to control the antimicrobial release (Bastarrachea et al. 2011).
na
The commercial polymers used are PP, PE, PET, and PE/ethylene vinyl
o
rs
alcohol (EVOH) (Bastarrachea et al. 2011; Raheem 2013; Suppakul et al. 2003),
Pe
and more recently, research efforts have focused on the use of biopolymer
r
materials (Kuorwel et al. 2011a; Khwaldia et al. 2010; Rhim and Ng 2007) such
fo
as PLA (Tawakkal et al. 2014).
y
op
C
• Incorporation of the antimicrobial agents by solvent casting meth-
’s
ods (Bastarrachea et al. 2011), where the polymer matrix is solubi-

or
lized in a solvent (water, ethanol, etc.) that contains antimicrobial

ut
b
agents. Then, this complex is cast or sprayed onto a substrate (plas-

tri
tic, paper, or food). After that, the solvent is evaporated at moderate

on
temperature and an active film is obtained. This system is used,

.C
for example, for polysaccharides, proteins, and polyols (Van Long

C
et al. 2016).
LL
• Incorporation of the antimicrobial agents as liquid dispersion by

cis
coating technologies. In this case, the destruction of antimicrobial

an
agents by high temperatures and shearing forces during extrusion

Fr
is avoided, since the coating is usually applied at nearly room tem-

d
perature after the packaging film is formed (Chen et al. 2012). An

an
example would be the use of EOs added in active paraffin coating for
or
paper (Gutierrez et al. 2009; Rodriguez-Lafuente et al. 2010).

yl
Ta
• Incorporation of the antimicrobial compounds immobilized on the

polymer either by ionic or covalent link agents producing a slow
18
migration. In this case, the agents used are enzymes, organic acids,
20
and bacteriocins (Krasniewska and Gniewosz 2012).

©
• Polymers that are inherently antimicrobial and are used themselves

as films or coatings, for example, macromolecules such as chito-
san, used to protect fresh vegetables and fruits from fungal attack.
Additionally, it is edible, nontoxic, biodegradable, and biocompat-
ible 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.
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 vol-
atile and nonvolatile compounds from different parts of the plant that have
antimicrobial properties. This antimicrobial activity has been widely stud-
y
ied in a great number of microorganisms, demonstrating its effectiveness by
nl
direct contact assay (Dias et al. 2013; Becerril et al. 2013) or by the vapor phase
O
(Muriel-Galet et al. 2015; Manso et al. 2015).
se
Another very important and expanding field is the combination of differ-
lU
ent disciplines, such as nanotechnology, food, and microbiology (Imran et al.
na
2010), whose objective is the introduction of nanoparticles to provide antimi-
o
rs
crobial activity, as well as an enlarged contact area, compared with conven-
Pe
tional antimicrobial agents (Mihindukulasuriya and Lim 2014; Echegoyen et
r
al. 2016). Therefore, they have a higher efficiency. An example is the addition
fo
of metals such as copper, zinc, and silver in the form of salts and oxides
y
op
(Espitia et al. 2012) and colloids, such as silver zeolites or elemental nanopar-
C
ticles (Rhim et al. 2013; Kuorwel et al. 2015).
’s
or
ut
14.2.5 Other Approaches in Active Packaging

b
tri
In addition to antioxidant or antimicrobial active packaging, other approaches

on
have been proposed with different objectives, although the extension of shelf
.C
life while maintaining the quality of packaged food is the constant and main
C
purpose. These approaches can be more specific for the target food, but the
LL
technologies used for producing the active packaging are similar to those
is
explained above. The most common ones are described in the next sections.
c
an
Fr
14.2.6 Carbon Dioxide Emitters and Scavengers

d
an
The presence of high levels of carbon dioxide retards microbial growth and
or
delays the respiration rates of fresh produce. Therefore, levels between 10%
yl
and 80% are usually recommended to extend the shelf life, although the rate
Ta
depends on the specific food (Kerry et al. 2006). This group can be divided
18
into two types: emitters and scavengers. The first one is used when the pack-
20
age 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 flex-
ible 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.
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.
y
nl
The most extensive technology used for ethylene scavenging is potassium
O
permanganate embedded in silica. The system absorbs ethylene, which is
se
oxidized to ethylene glycol, while permanganate reduces to manganese
lU
dioxide. The silica can be kept in the package in a sachet, but it is difficult to
na
introduce this agent in a film. In addition, permanganate is a strong oxidant
o
and is not permitted to have contact with food (Ozdemir and Floros 2004).
rs
Pe
14.2.8 Moisture Control
r
fo
Excess humidity may have negative results, causing bacterial and mold
y
op
growth, as well as foggy film formation. This can occur by accumulation
C
of water vapor inside the package due to the low permeability of different
’s
materials. In contrast, the lack of humidity is not always beneficial, since the
or
food may dry up (Biji et al. 2015). The moisture scavengers help to control the
ut
b
water excess, as well as to remove water vapor releases by meat products.

tri
The different technologies used to control the excess of water accumulation

on
are shown in Table 14.1, as well as the different applications found in the market.
.C
Actually, there are several systems already patented and marketed by dif-
C
ferent companies (Kerry et al. 2006). They consist of a superabsorbent poly-

LL
mer located between two layers of a microporous or nonwoven polymer, or,

is
for example, Dry Fresh resolve (Sirane Ltd.), which consists of drip-absorb-
c
an
ing pads placed under meat. Humidity absorbers are well disseminated and
Fr
used in the food market, as most packaged fresh meat contains an absorbent
d
pad to remove the exudates.

an
or
14.2.9 Flavor or Odor Absorbers and Releasers

yl
Ta
The volatile compounds accumulated inside a package can produce unde-

18
sirable aromas, flavors, or odors. The formation of these off-flavors and

20
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 differ-
ent 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.
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 neces-
sary to use a high-barrier material or, alternatively, employ an active packag-
ing containing the specific aromas of the particular food. For example, fill
the headspace progressively and in a continuous way over the shelf life of
y
the food with volatile compounds in order to obtain the desired flavor. This
nl
is used by coffee manufacturers (Ozdemir and Floros 2004). Other systems
O
and applications used are shown in Table 14.1.
se
lU
na
14.2.10 Microwaveable Active Food Packaging
o
rs
Microwave cooking is a very convenient way to prepare food, as it is very
Pe
quick and clean. However, the heat transfer of microwave heating pro-
r
duces nonuniform heat, and temperature is not well distributed in the food.
fo
Moreover, some foodstuffs require crisping or browning to be acceptable for
y
op
consumption, which is not possible with conventional microwave cooking.
C
Microwavable active packaging is designed to solve these cooking dis-
’s
advantages by using susceptors, shielding, field modification (Regier 2014),

or
moisture-absorbing flexible microwavable packaging materials (Realini

ut
et al. 2014), and steam valves (Avery Dennison 2011).

b
tri
Shielding can be applied to achieve differential heating of different por-

on
tions of the food (Lafferty et al. 2000). Modifiers for microwave heating are
.C
systems that alter how the microwaves arrive to the food, thereby resulting in
C
even heating, surface browning, and crisping (Ahvenainen 2003). Microwave

LL
susceptors are materials that are able to absorb electromagnetic energy and
is
convert it to heat (Labuza and Meister 1992). They are usually made of met-
c
an
allized film, ceramics, or multilayers containing an aluminum layer in the

Fr
sandwich mode. Moisture-absorbing flexible microwavable packaging mate-

rials are those that absorb the excess grease and water resulting in crispy
d
an
products (Mondi 2011). Finally, steam valves inserted in the packaging allow
or
the release of vapor once a defined pressure point is reached, resulting in dry
yl
food when necessary (Avery Dennison 2011).

Ta
These active packaging materials are applied to a variety of foods, such

18
as popcorn, lasagna, meat pies, bakery goods, chips, hotdogs, pizza, and
20
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 condi-
tions or the surrounding environment. It functions are detecting, sensing,
tracing, and communicating to give information about the product itself
(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 sen-
sors, although both names overlap in meaning. Sensors were initially applied
to those devices capable of detecting, locating, quantifying, and transmitting
y
information related to biological or chemistry reactions, with great precision
nl
and usually in an electronic way. However, indicators were applied to visual
O
changes provided by the intelligent device, usually a tag, while a sensor
se
was applied to electronic devices that required an automatic or instrumen-
lU
tal reading. However, nowadays the term indicator is less used and sensor is
na
generally accepted, independently of electronic reading or visual detection.
o
rs
Many authors believe that RFID is also an intelligent packaging. However,
Pe
this is an advanced label and can only be considered intelligent or smart
r
packaging when there is some active element inserted in the tag that commu-
fo
nicates with the packaged food. Some examples are shown in Table 14.2 (Lee
y
op
et al. 2015). The specific details of each type are described in the next sections.
C
TABLE 14.2
’s
or
Types of Intelligent Packaging and Their Applications, Advantages, Effectiveness,

ut
and Uses in Food

b
tri
Types of Intelligent Most Recent Examples Applied

on
Packaging Effectiveness to Food

.C
Time– temperature Visual indications of Meat (Pennanen et al. 2015; Kim

C
indicators temperature history et al. 2013)

LL
Fish (Zhang et al. 2016)

Fruit (Forney 2007)
is
c
Leak indicators Visual indications of a leak Meat (Jang and Won 2014)
an
Have to be very sensitive Soap (Smolander et al. 1997)

Fr
(disadvantage)
d
Freshness indicators Tracking and controlling of Cod fillets (Dehaut et al. 2016)
an
microbial growth or chemical Meat sausage (Jairath et al. 2015)

changes
or
Biosensors Detect and transmit information Cereals (Bougrini et al. 2016)

yl
Ta
about biochemical changes Pepper (Sabela et al. 2016)

Specificity, sensitivity, reliability, Meat sausages (Jadan et al. 2016)
18
portability, and simplicity Wine (Andrei et al. 2016)

20
Gas sensors Detect and quantify gas states Mangoes, eggs, and fish (Cui
et al. 2016)
©
Tea (Sharma et al. 2016)

Chemical sensors Detect presence of chemical Cheese (Nakonieczna et al. 2016)
contaminants Soft drinks (Lin et al. 2016)
Radiofrequency Automatic identification and Milk (Kim et al. 2016)
identification traceability data Virgin olive oil and meat
(Matindoust et al. 2016)
Microwaveable MDIs Popcorn, lasagna, meat pies,
intelligent packaging Barcode-intelligent microwave bakery goods, chips, hotdogs,
pizza, sandwiches, etc.
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
y
nl
labels applied on the external side of the package, which inform the
O
retailer or consumer of the thermal history of the product. They are
se
based on different electrochemical, mechanical, microbiological,
lU
or physicochemistry principles, such as enzymatic reactions, com-
na
pound diffusion, and polymerization processes that are tempera-
o
ture sensitive (Kerry et al. 2006; Biji et al. 2015). Their responses are
rs
Pe
expressed as color changes or mechanism deformations. There are
two basic types in the market:
r
fo
• Critical temperature indicators (CTIs), which change when a
y
temperature exceeds a limit op
C
• Critical time– temperature indicators (CTTIs), which change the
’s
color when the time and temperature are above the established
or
limits
ut
b
• Leak indicators : These are used to detect O2 or CO2 leaks when modi-
tri
on
fied atmosphere systems are used. They are based on chemical or

.C
enzymatic reactions producing a color change. Oxygen indicators

are often used to control the proper removal of oxygen absorbers.
C
LL
In the case of CO2 indicators, the response can be altered by the dis-
solution of CO2 in the food, which usually happens during the first
c is
days, and by the production of CO2 by microorganisms, growing

an
during food decay. The best-known system is methylene blue, which

Fr
has an oxidation reduction reaction (Biji et al. 2015).

d
an
• Freshness indicators : These indicate microbial growth or chemical

compounds generated during food ripening or food aging. There
or
yl
are different market samples that are based on the detection of CO2 ,
Ta
diacetyl, amines, or ammonia, which are related to fish and meat

18
deterioration (Hogan and Kerry 2008). Substances used as indica-

tors of microbial growth, such as n-butyrate, L-lactic acid, D-lactate,
20
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 biologi-
cal 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,
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
y
piezoelectric crystal sensors applied to different gases.
nl
O
• Chemical sensors : These detect the presence, composition, or concen-
se
tration of chemical contaminants, tampering products, or interme-
lU
diate-chain products by absorption. The most important sensors are
na
composed of nanoparticles, nanotubes, and nanofibers, and their
o
chemical signals are translated in ultraviolet (UV), visible, or infra-
rs
red (IR) measurements (Biji et al. 2015).
r Pe
fo
14.3.2 Radiofrequency Identification
y
op
This is a relatively new technology that uses small chips called tags in order
C
to track, identify, or gather data. These tags contain antennas that allow them
’s
or
to communicate with a reader by radio waves, performing the same function

ut
as magnetic codes, but allowing a greater identification distance. RFIDs can

b
tri
be used to store information about humidity, temperature, nutrition, pro-

on
ducer, processor, raw materials, operations during the manufacturing pro-

.C
cess, quality, safety, traceability, and so forth (Biji et al. 2015; Lee et al. 2015),
C
but can only be considered to be intelligent packaging when combined with

LL
a real sensor.
is
The most significant drawback is its high implementation and mainte-

c
nance cost, which can influence the final product price.

an
Several commercial examples try to integrate RFID systems with indica-

Fr
tors and sensors able to monitor the quality of food products, for example,
d
an
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
or
yl
food are shown in Table 14.2.

Ta
18
14.3.3 Microwaveable Intelligent Food Packaging

20
Microwave cooking characteristics have been briefly discussed in

©
Section 14.2.10. One of the disadvantages is the nonuniform heating pro-

duced 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
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 pack-
aging. 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
y
no currently availability in the market.
nl
O
se
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na
o
14.4 Nanotechnology, Biomaterials, and Bioactive
rs
Compounds Used in Active and Intelligent Packaging
Pe
r
The use of nanomaterials, biomaterials, or bioactive compounds for active
fo
and intelligent packaging is an expansive field that requires a combination
y
op
of different disciplines, such as nanotechnology, food, and material sciences.
C
Nanotechnology is becoming an important tool for the food industry
’s
worldwide, where there are potential applications in active as well as intel-

or
ligent packaging. In the case of intelligent packaging, there are several prac-
ut
tices, such as the use of nanosensors that are sensitive to microbes, toxins,
b
tri
and other contaminants generated during processing and storage chain.

on
Examples are sensors capable of detect Escherichia coli or salmonella bacteria

.C
using nanosized light scattering or silicon/gold nanorod array, respectively

C
(Neethirajan and Jayas 2011).

LL
In the case of active packaging, nanotechnology can provide solutions

is
modifying the permeation behavior of layers, increasing barrier properties

c
an
(mechanical, chemical, and microbial), providing antimicrobial properties,

Fr
and improving heat resistance properties. An example is the wide use of

d
nanoparticles with antimicrobial properties (silver and ZnO) in matrix coat-

an
ing material or in sachets placed in the package, which can reduce microbial
or
growth in foods such as cheese, sliced meat, and bakery products. Other
yl
examples are the use of nanotechnology to produce oxygen scavengers

Ta
(modifying the nanosized surface), free radical scavengers (SeNP) (Vera et al.
18
2016), moisture absorber sheets, and ethylene-scavenging bags (using silicate

20
nanoparticules) (Neethirajan and Jayas 2011).

On the other hand, society is more and more demanding the use of bio-
©
polymer materials because of their sustainability and environmental safety.

But, it is known that they have some disadvantages compared with conven-
tional 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).
Several applications of bionanocomposite materials are found in the refer-

ences, such as gelatin films or nanoclay cellulose containing silver nanopar-
ticles, 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
y
enhancing the efficiency of functional foods, improving human health, and act-
nl
ing in active and intelligent packaging should be pointed out. Some examples
O
are found in the references, for example, nanocapsules capable of masking the
se
odor of tuna fish oil in the mouth, delaying its breakdown until the stomach,
lU
and therefore avoiding an unpleasant taste or odor. However, this approach
na
cannot be used in Europe, as it is forbidden to mask fish odor because it is a
o
rs
good indicator of fish decay. Another example is the enhanced bioavailabil-
Pe
ity of lycopene by the fortification of lycopene nanoparticles with antioxidant
r
capacities in tomato juice, pasta sauce, and jam (Neethirajan and Jayas 2011).
fo
y
op
C
’s
or
ut
14.5 Commercially Available Active and Intelligent Packaging

b
tri
Nowadays, the commercial use of active and intelligent packaging is limited

on
for several reasons. The first one is that, in most cases, food is in direct con-
.C
tact with the active and intelligent agents, and their substances may migrate
C
into the foods. These intentional and nonintentional migrations must be

LL
studied, which is a tedious and time consuming task. Everything incorpo-

is
rated into the food contact materials has to be authorized and cannot affect
c
an
the consumer’ s health.

Fr
In addition, active and intelligent materials add cost, which must be justi-
fied by the benefit of obtaining a reliable, efficient, and useful system. Besides
d
an
that, food producers and consumers must accept this new technology. On
or
the other hand, the system must be efficient on a large scale, not only on a
yl
laboratory scale.
Ta
Some examples of commercial active and intelligent packages used for

18
food packaging are shown in Table 14.3 (Kerry et al. 2006).

20
©
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 indi-
rect contact with food, must be inert, to avoid transfer substances that may
endanger human health, cause unacceptable composition or organoleptic
©
20 478
18
TABLE 14.3
Commercially Available Active and Intelligent Materials
Ta
yl
Type of Active or
or
Intelligent an
Packaging Some Commercial Names
d Applications
Oxygen scavengers Ageless® (Mitsubishi Gas Chemical Co., Japan)
Fr Sachets and labels based on iron oxidation. Used especially
Oxyeater® (Ueno Seiyaku Co., Japan) an for meat and poultry products.
Freshilizer® (Toppan Printing Co., Japan) c Sachets based on iron oxidation.
Vitalon® (Toppan Printing Co., Japan) is
Seagul® (Nippon Soda Co., Japan) LL
Sanso-Cut® (Finetec Co., Japan) C
ATCO (Standa Industrie, France) .C
Oxyguard® (Toyo Seikan Kaisha Ltd.) on Plastic trays based on iron oxidation.
FreshPax® (Multisorb Technologies Inc., United States) Packets and strips designed to absorb oxygen inside sealed
packaging. Used especially for cooked sliced meat
tri
but products.
™
FreshMax and FreshPack (Multisorb Technologies Inc., United
or Labels designed for adhesion with high-value foods based
States) on iron oxidation. Used especially for cooked sliced meat
’s
Cproducts.
®
ATCO (EMCO Packaging Systems, United Kingdom) Labels based on iron oxidation.
op
Cryovac® OS 2000™ (Cryovac Division, Sealed Air Corporation,
y
Polymer-based film used to dry and smoke meat products.
United States)
fo
r
PureSeal® (W. R. Grace Co., United States) Bottle crowns based on ascorbate/metallic salts
Pe
DarExtend® (Grace Darex Packaging Technologies, United States) rs
ActiTUF® (M&G, Italy) Polyester bottles based on iron oxidation
o
ZerO2 (Food Science, Australia) Plastic films, bottles, and containers based on
na
photosensitive dye/organic compound.
lU
se (Continued)
O
nl
y
©
20
18
Ta
yl
Type of Active or or
Intelligent
Packaging Some Commercial Names Applications
an
d
Shelfplus O2 ® (Ciba Speciality Chemicals, Switzerland) Plastic films, bottles, and containers where the scavenger
mechanism is the PET copolymer.
Fr
Oxbar® (CMB Technologies, France) Plastic films or bottles based on cobalt-catalyzed polymer
an
cis oxidation.
NA® (Johnson Matthey, United Kingdom) LL Labels on platinum group metal catalyst.
Bioka (Bioka Ltd., Finland) C Sachets based on enzyme.
Carbon dioxide Verifrais™ (SARL Codimer, France) .C Based on sodium bicarbonate/ascorbate used for fresh
scavengers on meat.
Ageless® (Mitsubishi Gas Chemical Co., Japan) Based on ascorbic acid oxidation.
Ethylene Ethysorb™ (Stay Fresh Ltd.) Based on potassium permanganate used for fresh fruit.
tri
b
scavengers
ut
Neupalon™ (Sekisui Jushi, Japan)
or
Hatofresh™ (Honshu Paper, Japan)
’s Sachet, paper, or board based on activated carbon.
Sendo-Mate™ (Mitsubishi Gas Chemical Co., Japan)
C
Bio-Kleen (Kes Irrigations Systems, United States) Based dioxide catalyst.
op
Everfresh™ and Orega™ (Korea)
yon titanium
PE-basedfofilms containing activated clay for cut vegetables
Profresh™ (Warenhandels GmbH, Australia) and fruits.r
Peakfresh™ (Peakfresh Products, Australia) Pe
Bio-fresh™ (Grofit Plastic, Israel) rs
BO film™ (Odja Shoji, Japan) PE films based on crysburite
on ceramic.
Moisture control Minipax® ®
Sorbent packets formedaby medical-grade Tyvek can be
l
used in food applications.U
(Continued)
se
479
O
nl
y
©
20
480
18
Ta
Type of Active or
Intelligent
yl
or
Cryovac® and Dri-Loc® (Sealed Air Corporation, United States) Sorbent packets for meat and poultry products.
an
d
Thermarite® or Peaksorb (Australia)
Fresh-R-Pax (Maxwell Chase Technologies, United States)
Fr
Antimicrobials Nisaplin® (Nisin, United States) Concentrated extract used for all types of foods: dairy,
an
cis culinary, meat, bakery products, and beverages.
Irgaguard® LL Film with silver zeolites.
Emulactiv C-1 and Rycoat F-100 (Repsol, Spain) (Nerin et al.
C Antimicrobial coating for paper and board. Applied to
2005) .C fruits and vegetables.
Artibal Coating for paper and plastics. Applied to processed food,
bakery products, and meat.
on
Antioxidants ATOX 101 AV, 102 AV, and 104 AV (Artibal, Spain) Coating for paper and plastics. Applied to processed food,
tri
but bakery products, meat.
Antioxidants: Free GOGLIO (Italy) (Bosetti et al. 2013) Multilayer containing a free radical scavenger (green tea)
or
radical scavengers for coffee, chocolate derivatives, cereals, and any kind of
’s
Cfood.
™
Microgard (Nisin, United States) A film used for all types of foods: dairy, culinary, meat,
op
y
bakery products, and beverages.
Odor absorbers BHM™ (EKA Noble and EKA Companies)
fo
Formed by aluminosilicate zeolites to absorb odorous
r
aldehydes for fish products.
®
Pe
Microwaveable Nor Absorbit (Nordenia International AG) Moisture-absorbing and flexible microwavable film.
rs
Sira-Crisp® (Sirane Ltd.) Microwave susceptor. o
SmartPouch® (VacPac Inc.)
na
Time– temperature LifeLinesFresh-Check Based on polymerization reaction used for different types
lU
indicators of ready-made meat and dairy foods.
se
(Continued)
O
nl
y
©
20
18
Ta
yl
or
an
d
Type of Active or
Intelligent
Fr
an
c
3M Monitor Mark is Based on dye diffusion used for different types of
LL ready-made meat and dairy foods.
®
Vitsab TTI (COX Technologies, United States) C Based on enzymatic lipase color change used for different
.C types of ready-made meat and dairy foods.
Gas indicators Agelles Eye® (Mitsubishi Gas Chemical Co. Japan) on Oxygen control labels based on color change.
Vitalon®
Samso-Checker
tri
b
Freshness FreshTag® (COX Technologies, United States) ut Labels that react to volatile amines produced during
indicators storage used for fish or other seafood products.
or
Gas sensors Oxysense®
’s Fluorescence quenching sensor for measuring the oxygen
Cin headspace, as well as dissolved in liquids. It is used for
food and beverages.
op
Biosensors ToxinGuard™ (Toxin Alert, Canada)
y
Immobilizes and protects antibodies in sites on the surface
fo
of the polymer film.
r
Food Sentinel and Food Sentinel System™ (Syra Technologies, Capable of detecting the presence of harmful
Pe
United States) microorganisms through immunological reactions of
rs
barcodes. o na
lU
se
481
O
nl
y
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 main-
tained the basic principles above described, allowing technological innovation
y
in food packaging and accepting some interaction between food and packaging.
nl
However, it did not include the list of active substances and their composition.
O
Subsequently, in 2009, Regulation (EC) 450/2009 set the requirements
se
which with active and intelligent materials must comply. Specifically, it
lU
details the requirements needed to launch the products into the market.
na
A new feature of this regulation is the creation of a list of authorized sub-
o
rs
stances. This list should contain all substances that can be incorporated in
Pe
any active system. They must fulfill the basic principles above described,
r
using migration and sensory analysis described in the general materials
fo
regulation in contact with food.
y
op
The regulation also includes the conditions of use for active substances
C
that are currently permitted as food additives. These substances may be
’s
used provided they do not exceed their permitted limits.

or
In addition, the systems that are not integrated into the package, for exam-
ut
ple, sachets, must necessarily include the words “ do not eat” and the symbol
b
tri
of not edible device.

on
Finally, the companies that manufacture this type of active and intelligent
.C
materials must make a declaration of compliance to register the food suit-

C
ability of these products.

LL
c is
an
Fr
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15
Food Fraud: Detection,
Prevention, and Regulations
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Jamuna A. Bai and V. Ravishankar Rai
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CONTENTS
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15.1 Introduction: Definition of Food Fraud................................................ 496
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15.1.1 Types of Food Fraud ................................................................ 497
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15.2 Examples of Food Fraud......................................................................... 499
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15.2.1 Meat and Meat Products...........................................................500
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15.2.2 Seafood........................................................................................500
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15.2.3 Dairy Products........................................................................... 501

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15.2.4 Oils............................................................................................... 502

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15.2.5 Spices........................................................................................... 502

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15.2.6 Honey.......................................................................................... 503

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15.2.7 Juice.............................................................................................. 503

15.2.8 Coffee........................................................................................... 503
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15.2.9 Organic Vegetables and Fruits.................................................504

15.2.10 Additives.....................................................................................504
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15.3 Impact of Food Fraud..............................................................................505

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15.3.1 Public Health Impact................................................................. 505

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15.3.2 Economic Impact.......................................................................505

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15.4 Detection of Food Fraud ........................................................................505

15.4.1 Genomics....................................................................................505
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15.4.1.1 Meat and Meat Products........................................... 506

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15.4.1.2 Game Meat..................................................................508

15.4.1.3 Milk and Milk Products........................................... 509
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15.4.1.4 Spices........................................................................... 509

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15.4.1.5 Seafood........................................................................ 509

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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
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
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15.5.1 Traceability................................................................................. 519
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15.5.2 Authentication............................................................................ 519
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15.5.3 Supply Chain Management and Procurement...................... 520
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15.6 Regulation of Food Fraud: Laws and Legislation............................... 520
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15.6.1 Food Fraud Regulation in the United States.......................... 520
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15.6.2 Food Fraud Regulation by the European Commission....... 520
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15.6.3 Food Fraud Regulation in the United Kingdom................... 521
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15.6.4 Food Fraud Regulation in China............................................. 521
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15.6.5 Food Fraud Regulation: Industry Standards and
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Compliance................................................................................. 521
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15.6.5.1 Global Food Safety Initiative................................... 521
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15.6.5.2 Safe Supply of Affordable Food Everywhere........ 521
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15.6.5.3 U.S. Pharmacopeia..................................................... 522

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15.6.5.4 Other Activity............................................................ 522

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References ............................................................................................................. 522
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15.1 Introduction: Definition of Food Fraud

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Food fraud is a collective term used for any deliberate and intentional sub-
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stitution, tampering, addition, or misrepresentation of food and food ingre-

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dients and food packaging, as well as false statements made about the food
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product with the intention of economic gain (Spink and Moyer 2011; Spink
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2013).
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The Food and Drug Administration (FDA) has defined economically

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motivated adulteration (EMA) as the fraudulent, intentional substitution

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or addition of a substance in a product for either increasing the appar-

18
ent value of the product or reducing its production cost. It also includes
20
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 sub-

stances 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 ingre-
dients 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 fin-
ished products, tax avoidance smuggling, counterfeiting complete pack-
aged product, mislabeling country of origin, or uplabeling of genetically
Food Fraud: Detection, Prevention, and Regulations 497
modified as conventional are all various forms of fraud and are economi-
cal 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):
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Food fraud is a deliberate action of selling foods for financial gain,
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with the intent of deceiving consumers. Selling foods unfit or harmful for
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human consumption and mislabeling food with respect to its geographi-
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cal origin, ingredients, or substitution with lower-value or dangerous
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contents are the major types of food fraud (Figure 15.1). Contamination
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involves the unintentional introduction of undesirable physical, chemi-
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cal, or biological components in food. The unintentional addition of metal
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or plastic fragments, cleaning reagents and microbes, and their toxins in
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food products during processing, production, or packaging constitutes
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food contamination. If these are intentional, it would be food crime, and
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depending on the intention of deliberate contamination, it would be con-
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sidered bioterrorism. Food security is the sustainable food production
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and supply of a secure, sufficient quantity of safe and nutritious food

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to meet consumer demands. Food authenticity is the correct labeling, or

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menu section, of a finished food product to be sold to the consumer. Food

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integrity ensures selling of the quality substance, and nature of food to

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meet consumer requirements.

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This chapter provides an overview of the definitions of food fraud, types

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and examples of food fraud, its detection by various analytical techniques,

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and the management and regulation of food fraud.

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15.1.1 Types of Food Fraud

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The Global Food Safety Initiative (GFSI) has defined various types of food
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fraud (van der Meulen 2015).

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• Counterfeiting is presenting a product as something different from

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what it really is. It could be a brand, protected designation, or mark

18
of a religious, quality, or safety profile.

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• Dilution is increasing the quantity of the product by adding low-

©
value 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.
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Food fraud implications d Food fraud response
Adulteration
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Public health risks an Legal action
Simulation
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Economic impact LL Product recall
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. Food
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fraud
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Food fraud detection
analytical techniques Food fraud prevention
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Counterfeiting
but Overrun
or Authentication
Genomics ’s
Theft
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Metabolomics and op Traceability
chemometrics y
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Proteomics r management
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FIGURE 15.1
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Overview of food fraud— t ypes, detection, and management.
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• Mislabeling is the distortion of the information about the food prod-

uct 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
y
its usage is approved by regulatory agencies.
nl
O
se
Similarly, Spink et al. (2016) have also defined the various types of food
lU
fraud, some of which are
na
• Adulteration: The addition of a fraudulent substance or an impu-
o
rs
rity in the finished food product. Example: Addition of melamine to
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milk and Sudan dyes to saffron.
r
fo
• Tampering: Legitimate product and packaging are changed fraudu-
y
lently. Example: Changing expiry information of a food product or
op
uplabeling a food product.
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• Overrun: Legitimate product is manufactured beyond production

’s
or
agreements. Example: Underreporting of manufactured product.

ut
• Theft: Stolen legitimate product sold as legitimately procured.

b
tri
Example: Stolen products are mixed and sold with legitimate

on
products.
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• Diversion: Legitimate products are sold and distributed outside

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the intended markets. Example: Relief foods for aid are sold in the
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market.
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• Simulation: Illegitimate products are designed or imitated to

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look similar to the legitimate brands of food products. Example:

Fr
“ Knockoffs” of popular foods with lower safety and quality.

d
an
• Counterfeiting: Intellectual property rights (IPR) violation that

includes complete replication of all aspects of the product and pack-
or
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aging. Example: Duplication of popular foods without the same food

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safety assurances.
18
20
©
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).
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,
y
nl
and if it is a free-range, pasture-fed, or concentrate-fed animal. By falsely
O
stating the country of origin, or the species of meat, or a breed as a popular
se
meat breed, a higher price is gained by selling such meat. Similarly, vegetable
lU
proteins, such as soy protein, are used instead of meat proteins in processed
na
meat products, and plant products are mixed with meat products to increase
o
the volume and weight. Meat weight is also sometimes increased by using
rs
water and binding agents (Ballin 2010). In 2008, a company in Sweden had
Pe
adulterated processed meat products. Lithuanian minced beef was mixed
r
fo
into sauteed reindeer meet. The company was found guilty of the fraudulent
y
act, and its largest customer terminated their delivery agreement, as seen
op
in Lycksele Tingsrä tt verdict criminal case B 443-08. In European countries,
C
the adulteration of beef meat with horsemeat garnered media attention as
’s
or
the Horsemeat Scandal. The horsemeat scandal was unraveled in Europe

ut
in 2013. Investigation by the Food Safety Authority of Ireland (FSAI) of beef

b
burger products revealed that they had horse and pig DNA. In similar inci-
tri
on
dences, pork meat containing colorants Azorubine (E122), Sunset Yellow FCF
.C
(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
C
LL
(National Food Agency 2013). Consumer and wholesale meat retailers’ com-
plaints led to an investigation by the Swedish Food Agency, revealing the
cis
meat food fraud. In Sweden, the presence of horse DNA in Findus lasagna
an
led to the withdrawal of the food product from the market by the Findus
Fr
Company (FSA 2013).

d
an
or
15.2.2 Seafood
yl
Ta
Seafood has a high demand, which has led to increased illegal and unre-
18
ported fishing. The European Union (EU), the largest importer of seafood,
20
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, label-
ing 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 res-
taurants 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
(Oceana 2013). The species red snapper (Lutjanus campechanus ) was most com-
monly 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 ana-
lyzed, 32 were mislabeled as cod (Warner et al. 2013). In Europe and Northern
y
America, 15%– 43% of commercially available seafood are food fraud cases.
nl
For example, red snapper is replaced with cheaper and abundant similar spe-
O
cies or commonly substituted with tilapia (Galimberti et al. 2013). In Ireland,
se
cod is mostly replaced by cheaper species (Armani et al. 2012). Incidences of
lU
mislabeling regarding the production method of fish have been reported, as
na
in the case of Atlantic salmon (Salmo salar ) sold as wild caught and mislabeled
o
rs
in 11 of 54 samples examined from Norway, France, the United Kingdom, and
Pe
Italy (Thomas et al. 2008). Similarly, the Food Standards Agency (FSA) in the
r
United Kingdom has reported the mislabeling of salmon (S. salar ), sea bass,
fo
and sea bream as wild caught (FSA 2007).
y
op
C
15.2.3 Dairy Products
’s
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Milk and milk products are subject to food fraud in many ways. Diluting
ut
milk with water, adding melamine to increase the protein content in milk,
b
tri
and removing constituents such as fat and protein are common types of milk
on
food fraud (FAO 2013). One of the major food fraud incidences concerning
.C
milk products was the addition of melamine to raw milk by Chinese milk
C
producers. Melamine, a nonproteinaceous substance, was added to milk

LL
to increase the true value of the protein content in milk. Consumption of

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melamine-adulterated milk in China led to an outbreak of kidney and renal

c
an
dysfunction among infants. The intake of Sanlu Group infant formula, which
Fr
contained melamine, led to the outbreak. The Chinese government seized

more than 2000 tons of milk powder and recalled 9000 tons of milk powder
d
an
produced by the Sanlu Group. Twenty-two other infant formula– produc-

or
ing companies in China had melamine in their products. Upon consuming

yl
instant formula adulterated with melamine, around 300,000 Chinese infants

Ta
became sick and 6 infants died. Melamine-containing dairy and bakery prod-
18
ucts from China had reached 47 different countries by direct import, third-
20
country 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 fraudu-
lent 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 mozza-
rella cheeses sold by grocery stores, dairy shops, and cheese manufacturers,
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
y
and found 16,000 tons of adulterated butter produced from 5,000 tons of beef
nl
tallow substances and 400 tons of synthetic chemicals. The adulterated but-
O
ter was sold in Germany, France, Italy, and Belgium (European Anti-Fraud
se
Office 2009).
lU
na
o
15.2.4 Oils
rs
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Extra virgin olive oil (EVOO) is a product of high value due to its high quality.
r
Adulteration of EVOO by replacement with lower-quality oils, such as refined
fo
olive oil or olive pomace, or other oils, such as palm oil, hazelnut oil, soybean
y
op
oil, or seed oil, is a common fraud (Hodaifa et al. 2012). EVOO from a region
C
known for producing high-quality oil has been replaced with EVOO from
’s
another region and sold at a higher price (Frankel 2010). In 1995, a large-scale
or
fraud carried out in the production of olive oil was revealed. Sunflower oil from
ut
Turkey was imported to Belgium, Germany, the Netherlands, and Portugal and
b
tri
sold multiple times between different companies before importing to Italy and
on
Spain. Investigations revealed that the oil was actually hazelnut oil, and 20,680
.C
tons of it was used in the production of 103,400 tons of adulterated olive oil (EC
C
1998). In 2010, the UC Davis Olive Oil Chemistry Laboratory and Australian
LL
Oils Research Laboratory found that 70% of the imported EVOO did not meet
is
the sensory standards, and of these, 86% also failed to meet the chemical stan-
c
an
dard set by the International Olive Council (IOC) and the U.S. Department of
Fr
Agriculture (USDA). In the case of the ones produced in California, 10% did
not meet the sensory standards (Frankel 2010).
d
an
or
15.2.5 Spices
yl
Ta
Saffron is an expensive spice obtained from the stigmas of the plant crocus
18
(Crocus sativus ). Some of the common frauds practiced with saffron are misla-
20
beling 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
regarding the use of Sudan dyes in spices (EC 2007). Pepper powder contain-
ing traces of Sudan IV packed in Korea was exported from China to European
countries. Using the analytical technique of high-performance liquid chro-
matography– 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).
y
nl
O
15.2.6 Honey
se
The quality of honey is greatly associated with its origin, and thus its prices
lU
vary. It is usually adulterated by adding sugars or syrups (Cotte et al. 2003).
na
The fraud practices associated with honey are changing the country or
o
rs
region of its origin and selling cheap, imported honey as locally produced
Pe
high-quality honey at a higher price. A survey by Fairchild et al. (2003) for
r
the National Honey Board revealed that companies found honey highly
fo
and regularly adulterated, and had detected levels of 5%– 43% corn syrup
y
op
in adulterated honey. The adulterated honey was originating from China
C
and Argentina (Fairchild et al. 2003). A study showed that 35 commercial
’s
honey samples from France, Hungary, Spain, and Morocco, upon testing,
or
were found adulterated with three different syrups. Of 18 acacia honey sam-
ut
ples tested, 7 were adulterated with added syrup or a honey other than aca-
b
tri
cia. Of the eight chestnut samples tested, two were adulterated with syrup.
on
Similarly, of nine lavender honeys tested, four had been adulterated with
.C
syrup or a different type of honey (Cotte et al. 2003).

C
LL
15.2.7 Juice
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Orange juice, a high-value product, is adulterated with different kinds of

Fr
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.
d
an
Pulp wash is also used to adulterate orange juice (Le Gall et al. 2001). Fruit
or
juices such as pomegranate juice have been replaced with pear, apple, cherry,
yl
grape, or aronia juice. Cane sugar or corn sugar is used to increase the taste,
Ta
and anthocyanins from aronia, grape skin, elderberry, black currant, or black
18
carrot are added to enhance color. In a study of 23 commercial pomegran-

20
ate 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 adul-
terated 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
Association of the Brazilian Coffee Industry found that adulterating coffee

affected its quality. One out of six commercial coffee samples were adulter-
ated 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).
y
nl
15.2.9 Organic Vegetables and Fruits
O
Conventional crops have been fraudulently sold as organic crops. In 2010, it
se
was found by an Italian inspection agency that false production claims were
lU
made by a company that had sold imported conventional crops as organic
na
foods. Crops such as barley, oats, wheat, millet, sorghum, corn, alfalfa,
o
rs
field beans, field pea, linseed, soybeans, rapeseed, sunflower, and potato
Pe
were falsely sold as organic in Italy, Belgium, France, the Netherlands, and
r
Germany. To prevent traceability, they were sold several times between vari-
fo
ous companies (European Working Community for Food Inspection and
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Consumer Protection 2013). op
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15.2.10 Additives
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Additives are used in processed foods to improve their texture, color, preser-
b
tri
vation, and taste. Adulterated additives, such as poor-quality chemicals, have

on
been used in the food industry. In 2011, a large salt scandal was discovered in
.C
Iceland. The country’ s leading supplier of salt had sold refined industry-grade
C
salt as food-grade salt for many years (Ministry of Fisheries and Agriculture of
LL
Iceland 2012). The unauthorized use of additives, such as sulfites, in seafood to

is
enhance its quality is a food fraud. In 2006, more than 90 notifications regard-
c
an
ing sulfites were made to RASFF (EC 2007). Cloudy agents used in food were
Fr
found to contain plasticizers in Taiwan. Two companies producing cloudy

agents had replaced palm oil with bis(2-ethylhexyl)phthalate (DEHP) and
d
an
diisononyl phthalate (DINP). These cloudy agents containing high amounts of

or
phthalate DEHP were used in preparing capsules for probiotics. The phthalates
yl
are usually used as plasticizers in the plastic industry. However, as plasticizers

Ta
are endocrine disruptors, their usage in food has severe and adverse implica-
18
tions in human health (Li and Ko 2012). The palm oil was replaced with plas-
20
ticizers 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 dis-
©
pensed 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 phthal-
ates had been exported to the United States (FDA 2011a). Liquid chromatog-
raphy– tandem mass spectrometry (LC-MS/MS) was used to detect phthalate
adulteration in food products (Self and Wu 2012).
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
y
nl
risks. One such incidence is the addition of melamine to high-protein feed
O
and milk products to overestimate the protein content in diluted prod-
se
ucts. In 2007, use of adulterated pet food ingredients in the United States
lU
from China caused the deaths of a large number of pet dogs and cats (CRS
na
Report RL34080). Again, consumption of melamine-contaminated baby for-
o
mula affected 300,000 Chinese children and caused the deaths of 6 infants
rs
(Bottemiller 2011). Food fraud by the Peanut Corporation of America caused
Pe
a Salmonella outbreak in 2009, resulting in 700 people falling sick and 9
r
fo
deaths. The company officials had sold and distributed contaminated prod-
y
uct (Johnson 2014). Investigations led to the recall of 3912 products contain-
op
ing contaminated peanut butter and peanut paste ingredients manufactured
C
by about 200 companies (CRS Report R40450).
’s
or
ut
b
15.3.2 Economic Impact

tri
on
According to the Grocery Manufacturers Association (GMA), food fraud has

.C
cost the global food industry $10– $15 billion annually, and affected 10% of
C
all commercially sold food products (GMA 2010; Everstine and Kircher 2013).
LL
However, the number of documented food fraud incidents may be a fraction

is
of the true number of incidents (Spink and Fejes 2012). Fraud acts resulting
c
in public health risk can also cause significant financial and public relations
an
consequences to food industries. Food fraud can affect food businesses in

Fr
terms of lost sales, by 2%– 15% of annual revenues, and may cause bankrupt-
d
an
cies in the case of public health consequences (Johnson 2014).

or
yl
Ta
18
20
15.4 Detection of Food Fraud

©
In the past two decades, molecular-based technologies have been invalu-

able 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
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
y
DNA-based techniques have been used for identifying fraudulent practices in
nl
the meat trade, such as mislabeling in the case of meat species specification. In a
O
study in Malaysia, 143 prepackaged beef and poultry meat products purchased
se
from several supermarket chains were analyzed for the presence of common
lU
meat species, such as buffalo, cattle, chicken, goat, sheep, duck, and goose, and
na
meats prohibited by Islamic laws, such as cat, dog, monkey, pig, and rat, using
o
rs
species-specific primers. A total of 112 samples were mislabeled where species
Pe
were falsely declared and the presence of some species was undeclared. Of the
r
58 samples labeled as beef, buffalo DNA was detected in 40 samples. The pres-
fo
ence of undeclared chicken and buffalo DNA was also detected in many beef
y
op
and chicken products, respectively. The five “ haram” meat sources, however,
C
were not detected in all meat products tested. The majority of the meat sam-
’s
ples were legally noncompliant due to substitution and mislabeling of meat

or
products (Chuah et al. 2016). Multiplex polymerase chain reaction (PCR) has
ut
been used to identify five meat species forbidden in Islamic foods. Five pairs
b
tri
of species-specific primers targeting mitochondrial ND5, ATPase 6, and cyto-

on
chrome b genes have been designed to amplify DNA fragments of 172, 163, 141,
.C
129, and 108 bp from cat, dog, pig, monkey, and rat meats, respectively. Species
C
specificity checking in 15 important meat and fish species and 5 plant species
LL
showed no cross-species amplification. The single-assay platform can detect

is
0.01– 0.02 ng of DNA in raw meat and 1% suspected meats in processed foods
c
an
such as meatball formulation (Ali et al. 2015). Ground meat products sold in
Fr
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
d
an
48 ground meat samples purchased from supermarkets and specialty meat

or
retailers. DNA sequences from meat samples were identified to the species
yl
level using the Barcode of Life Database (BOLD). Samples that failed DNA
Ta
barcoding were tested with real-time (RT) PCR for beef, chicken, lamb, turkey,
18
pork, and horse DNA. Of the 48 samples analyzed, 10 were mislabeled. Nine of
20
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 on-
line 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 gov-
ernment 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
TABLE 15.1
Analytical and Molecular Genomic Techniques Used in Detection of the Food Fraud
Detection Techniques Food References
PCR Buffalo meat in mixed beef Chuah et al. 2016
Multiplex PCR Detection of haram meat Ali et al. 2015
DNA barcoding of COI gene Horsemeat in beef Kane and Hellberg 2016
y
nl
RT-PCR Horsemeat in beef Nixon et al. 2015
O
DNA barcoding Identification of meat Colombo et al. 2011
se
PCR-TTGE species in mixed animal
lU
meat
PCR-RFLP of COI gene Identification of meat Haider et al. 2012
na
sample at the species level
o
rs
Multiplex TaqMan RT-PCR 5– 50 pg species-specific Nq et al. 2014
Pe
identification of chicken,
duck, and turkey meat
r
fo
PCR targeting Identification of game meat Fajardo et al. 2007
y
mitochondrial 12S rRNA of red, fallow, and roe deer
Multilevel PCR D-loop Unknown meat from
opParkanyi et al. 2014
C
mtDNA wildlife
’s
DNA barcoding of COI gene Game meat containing Quinto et al. 2016
or
threatened and vulnerable

ut
species
b
tri
Cyt b and TaqMan Murine meat in mutton Fang and Zhang 2016
on
probe– based duplex PCR

.C
SPED and PCR Milk and milk products Bloch et al. 2014
DNA barcoding Chili adulteration in black Parvathy et al. 2014
C
LL
pepper
DNA barcoding and Seafood Chin et al. 2016
is
mini-barcoding
c
an
PCR of COI gene Seafood Khaksar et al. 2015

Fr
PCR of COI gene Seafood— identification of Pappalardo and Ferrito 2015

d
European plaice and

an
common sole
or
Barcode and mini-barcoding Fish and shrimp products Gunther et al. 2016
yl
Barcoding Seafood— cod, flounder, Carvalho et al. 2015

Ta
grouper, tuna, pink cusk

eel
18
Nanoengineered DNA Extra virgin olive oil Puddu et al. 2014

20
encapsulates
©
High-resolution melting Fruit juices Faria et al. 2013

analysis DNA barcode trnL
Bar-HRM Allergenic tree nut species Madesis et al. 2013
Bar-HRM Legume crops Ganopoulos et al. 2012
DNA barcoding Spice samples De Mattia et al. 2011
DNA barcoding Olive oil Ganopoulos et al. 2013
Nano-real-time PCR Edible oil He et al. 2013
DNA barcode Honey— origin and floral Valentini et al. 2010; Bruni et
species al. 2015
DNA targets for equine growth hormone receptor and a mammalian or poul-
try 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
y
species in mixed animal samples. Identification was performed by matching
nl
the “ DNA barcode” zone with the sequences of PCR products obtained from
O
a limited set of “ universal” primers (Colombo et al. 2011). Meat authenticity
se
has also been verified at a cheaper and faster rate by PCR Restriction fragment
lU
length polymorphism (RFLP) of part of the COI gene. Raw meat samples of
na
cow, chicken, turkey, sheep, pig, buffalo, camel, and donkey were identified at
o
rs
the species level. The level of COI variation showed that of the seven restric-
Pe
tion endonucleases (Hind II, Ava II, Rsa I, Taq I, Hpa II, Tru 1I, and Xba I),
r
only Hpa II was sufficient to generate species-specific restriction profiles for
fo
unambiguously distinguishing all targeted species (Haider et al. 2012). A mul-
y
op
tiplex assay using TaqMan® RT quantitative PCR (qPCR) for species-specific
C
detection of chicken, duck, and turkey detected low quantities of species-spe-
’s
cific DNA from single or multispecies sample mixtures containing 5– 50 pg of
or
starting DNA material (Ng et al. 2014). An improved method involving PCR
ut
amplification of the COI gene and detection of species-specific sequences by

b
tri
hybridization led to detection of multiple species, including pork, beef, lamb,

on
horse, cat, dog, and mouse, from a mixed sample in a single assay. Species-
.C
specific probes and 5 pg of DNA sample was used. A low-cost, high-density
C
DNA-based multidetection test for routine inspection of meat species was

LL
developed (Lin et al. 2014).

cis
an
15.4.1.2 Game Meat

Fr
Game meat products are often target for fraudulent labeling, as they are
d
an
highly priced than other meat species (Fajardo et al. 2006). PCR based on
or
oligonucleotide primers targeting the mitochondrial 12S rRNA gene identi-

yl
fied meats from red deer (Cervus elaphus ), fallow deer (Dama dama ), and roe
Ta
deer (Capreolus capreolus ) (Fajardo et al. 2007). Tobe and Linacre (2008) iden-
18
tified 18 common European mammalian species by species-specific multi-

20
plex 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
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).
y
nl
15.4.1.3 Milk and Milk Products
O
For tracing the products along the food chain, silica particles with encapsu-
se
lated DNA (SPED) were developed and added to milk at 0.1– 100 ppb. Milk
lU
and milk products were thus uniquely labeled with a DNA tag. The DNA
na
tags could be extracted from the food matrixes, identified, and quantified
o
rs
for previously marked products by qPCR with a LOD below 1 ppb of SPED.
Pe
Approved food additives can be used as DNA carrier (silica = E551), and
r
the low-cost technology (i.e., less than US$0.1 per ton of labeling milk with
fo
10 ppb of SPED) can find application in labeling technology for the tracing
y
and authentication of foods (Bloch et al. 2014). op
C
’s
or
15.4.1.4 Spices
ut
The DNA barcoding technique is used to detect species-specific variation

b
tri
in the short region of DNA and is used in the authentication and tracing of
on
agri-food products (Chen et al. 2014). DNA barcoding has been used to detect
.C
adulteration in the highly valued black pepper powder. PCR amplification

C
of Piper nigrum and black pepper powder obtained from the market was
LL
performed for three barcoding loci: psbA-trnH, rbcL, and rpoC1. Sequence
is
analysis and a Basic Local Alignment Search Tool (BLAST) search showed
c
an
chili adulteration in two out of nine market samples. The DNA barcoding
Fr
technique can be used to detect adulteration at very low levels, such as 0.5%
adulteration (Parvathy et al. 2014).
d
an
or
15.4.1.5 Seafood
yl
Ta
Mislabeling of seafood and processed seafood products is a global prob-

18
lem. DNA barcoding was used to analyze 62 commercially sold seafood

20
raw and processed samples. Full DNA barcoding and mini-barcoding tar-
get 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 sea-
food 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
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.
y
In seven products, there was a mismatch between labeling and the identified
nl
barcodes. In five samples, species did not belong to the genus indicated on
O
the label, and in four products, labeling did not comply with the permitted
se
list (Gunther et al. 2016). The Brazilian Governmental Regulatory Agency
lU
(PROCON) confiscated 30 seafood samples of commercial species, including
na
cod, flounder, grouper, tuna, and pink cusk eel, as barcoding revealed that
o
rs
24% of the samples obtained were mislabeled (Carvalho et al. 2015).
rPe
fo
15.4.1.6 Plant-Derived Foods
y
op
Basmati rice is highly valued due to its fragrance with grain elongation on
C
cooking, giving a characteristic grain shape and integrity. It costs more than
’s
twice the price of other ordinary varieties. In the United Kingdom, it cur-
or
rently accounts for about 37% of the dry rice market by value and £ 50 mil-
ut
lion per year. There are 11 varieties from India and 5 from Pakistan. The FSA
b
tri
revealed that of the 39 samples of Basmati-labeled rice sold in the United

on
Kingdom, more than one sample in six contained high levels of other non-
.C
Basmati varieties. In 363 samples examined, 196 samples contained only

C
Basmati rice, and non-Basmati rice was detected in 167 samples. A DNA fin-
LL
gerprinting technique was used to assess the fraud in Basmati trade. Samples
is
were screened to see the characteristic fingerprint of 12 chromosomes, in

c
an
which chromosome 10 is important for identification (Shears 2008).

Fr
DNA encapsulates in heat-resistant and inert magnetic particles have been

developed. The nanoengineered encapsulates containing DNA can be recov-
d
an
ered and analyzed by RT-PCR and Sanger sequencing, and these are stable
or
for 2 years in decalin at room temperature. The magnetic DNA and silica
yl
encapsulates can be used for tracing and tagging oils and oil-based prod-
Ta
ucts. They require a 1 ppb level of the taggant and allow taggant concentra-
18
tion quantification on a log scale. The low-cost platform developed was able
20
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 identifica-
tion of allergenic tree nut species in processed foods (Madesis et al. 2013).
Bar-HRM has also been used to analyze legume crops. PDO product “ Fava
Santorinis” adulterated with legumes of Lathyrus or Vicia and Pisum spe-
cies 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 commer-
cial products (Ganopoulos et al. 2012). Similarly, DNA barcoding is used in
recognizing commercial spices of the Lamiaceae family. The classical DNA
y
barcoding approach with four candidate barcode regions (rpoB, rbcL, matK,
nl
and trnH-psbA) and universal primers were used to differentiate 64 spice
O
samples comprising 6 genera of spice plants: Mentha , Ocimum , Origanum ,
se
Salvia , Thymus , and Rosmarinus . The noncoding trnH-psbA intergenic spacer
lU
and matK were the best markers, with interspecific genetic distance values
na
ranging between about 0% and 7%, and 0% and 5%, respectively. These two
o
rs
markers distinguished spice species from the closest taxa of all the genera
Pe
tested except for oregano (De Mattia et al. 2011). Bar-HRM has also been used
r
to detect the adulteration of olive oil with canola oil at a level of 1% (w/w) of
fo
canola oil in olive oil. This technique is an accurate, faster, and less expensive
y
op
method to authenticate vegetable oils, such as olive oil, and to detect mix-
C
tures of oils (Ganopoulos et al. 2013). The nano-RT-PCR technique has been
’s
used to detect the authenticity of edible oils. The amount of DNA extracted
or
from edible oils and adulterated edible oils is evaluated by RT-PCR with dif-
ut
ferent amplification fragments and nano-RT-PCR. Soybean oil (10 mL) in a

b
tri
background of 40 mL sesame oil was detected by the technique. Gold colloid
on
increased the efficiency and precision of PCR (He et al. 2013). DNA mark-
.C
ers have proved to be helpful in the authentication of food even in complex

C
matrixes, such as vegetable oil. DNA markers have been used to authenticate
LL
the edible olive oil cultivar identification (Costa et al. 2012).

is
DNA barcoding has been used to identify the plant origins of processed
c
an
honey. Four multifloral honeys produced in the northern Italian Alps were
Fr
analyzed using the rbcL and trnH-psbA plastid regions as barcode mark-
ers. Thirty-nine plant species were identified in the four honey samples, of
d
an
which one endemic plant was found in four honey samples, authenticating
or
the geographic identity of the products, and also the DNA of the toxic plant
yl
Atropa belladonna was detected in one sample. Thus, DNA barcoding apart
Ta
from authentication is also useful in evaluating the safety of honey (Bruni

18
et al. 2015). The DNA barcoding approach combining universal primers and
20
massive parallel pyrosequencing was used to assess plant diversity and the
geographical origin of honey. Two commercial honeys, one of a regional ori-
©
gin 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
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
y
integrity have been conducted in recent years. Kopi Luwak, an exotic
nl
Indonesian coffee, is made from coffee berries that have been eaten by the
O
se
TABLE 15.2
lU
Metabolomic Techniques Used in Detection of Food Fraud
o na
rs
GC-MS-based multimarker Original and fake coffee (Kopi Luwak) Jumhawan et al. 2013
Pe
profiling
r
fo
1H NMR and Adulterant in saffron Petrakis et al. 2015
chemometrics
y
DRIFTS and chemometrics Adulterant in saffron
op Petrakis et al. 2017
C
1H NMR (qHNMR) Sudan I– IV dyes in saffron Petrakis et al. 2017
’s
LC-HRMS Identification of PDO saffron Rubert et al. 2016

or
Proton transfer reaction MS Differentiation of old and new saffron Nenadis et al. 2016
ut
FT-MIR Quality of saffron and mislabeling Ordoudi et al. 2014

b
tri
FT-IP chemometrics with Fraud in herbs and spices Black et al. 2016a
on
LC-HRMS
.C
High-resolution MAS NMR Origin of Interdonato lemon Cicero et al. 2015

C
UPLC-QTOF MS Adulteration of fruit juices Jandric et al. 2014

LL
APCI with GC coupled to Differentiation of quality of olive oil Sales et al. 2015
QTOF MS
c is
DART-TOF MS Detection of adulteration of hazel nut Vaclavik et al. 2009

an
oil in olive oil

Fr
DART-Orbitrap HRMS Differentiation of Chinese and Shen et al. 2012

d
Japanese star anise

an
QTOF MS Adulteration in honey Wu et al. 2017

or
Colorimetric SERS with Melamine in milk Lang et al. 2015

yl
gold nanoparticle
Ta
HILIC-ESI/TOF/MS Melamine in infant formula Inoue et al. 2015

18
Multicommuted flow Adulteration in milk De Souza et al. 2014

20
system
DART-HRMS Differentiation of milk of farm animal Hrbek et al. 2014
©
species
Detect vegetable oil in dairy products
GC-MS and UHPLC-MS Testing of different grades of beef Trivedi et al. 2016
mince and pork mince
1H NMR Detection of horsemeat in beef Jakes et al. 2015
LESA MS Detection of pork, horse, turkey, or Montowska et al. 2014
chicken meat in beef
LC-Orbitrap-HRMS Detection of process contaminants Senyuva et al. 2015
and toxins in food
Asian palm civet (Paradoxurus hermaphroditus ). Gas chromatography– mass

spectrometry (GC-MS)– based multimarker profiling differentiated origi-
nal, 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
y
stamens, safflower, and turmeric. A two-step approach with the application
nl
of both orthogonal projections to latent structures– discriminant analysis
O
(OPLS-DA) and Orthogonal-orthogonal Partial Least Square-Discriminant
se
Analysis (O2PLS-DA) models to the 1H NMR data could detect authentic
lU
and adulterated saffron. It can be used to assay extensive saffron fraud at
na
a minimum level of 20% (w/w) (Petrakis et al. 2015). Diffuse reflectance
o
rs
infrared Fourier-transform spectroscopy (DRIFTS) and chemometric tech-
Pe
niques have also been used for testing the adulteration of saffron with six
r
adulterants: C. sativus stamens, calendula, safflower, turmeric, buddleja,
fo
and gardenia. The three-step process can detect and quantify adulteration.
y
op
It showed 99% correct classification of pure saffron and saffron adulter-
C
ated at 5%– 20% (w/w) levels and detection limits ranging from 1.0% to 3.1%
’s
w/w (Petrakis and Polissiou 2017). An NMR-based approach was used to

or
identify and determine the adulteration of saffron with Sudan I– IV dyes.
ut
1H NMR (qHNMR) was used to detect and quantify Sudan III in varying
b
tri
levels of 0.14– 7.1 g/kg (Petrakis et al. 2017). LC coupled with high-resolution

on
mass spectrometry (HRMS) was used to differentiate saffron cultivated and

.C
packaged in Spain, PDO, and saffron packaged in Spain of unknown origin,

C
labeled Spanish saffron. The markers identified by metabolic fingerprint-

LL
ing were glycerophospholipids and their oxidized lipids (Rubert et al. 2016).
is
The nondestructive technique, proton transfer reaction– mass spectrometry

c
an
(PTR-MS), has been used in quality control of saffron. By monitoring the pro-
Fr
duction of volatile organic compounds (VOCs), fresh saffron and old saffron
of poor quality could be easily identified (Nenadis et al. 2016). The Fourier-
d
an
transform midinfrared (FT-MIR) technique has been applied to assess the

or
quality of saffron, as well as fraud and mislabeling. FT-MIR analysis showed

yl
that high-quality samples according to ISO 3632 specifications produced a

Ta
typical spectrum profile (Ordoudi et al. 2014). The FT-IR screening method
18
coupled to data analysis using chemometrics and a second method using

20
LC-HRMS have been developed to detect fraud in herbs and spices. The two-
tier 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 meta-
bolic 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).
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 chem-
y
ical ionization (APCI) source in combination with GC coupled to hybrid
nl
QTOF MS has been used for the determination of volatile components of
O
olive oil for the classification of olive oil samples. VOCs in olive oil samples,
se
including extra virgin, virgin, and lampante qualities, have been detected.
lU
The analysis used three different steps: a full mass spectral alignment of
na
GC-MS data using MzMine 2.0, a multivariate analysis using Ez-Info, and
o
rs
a statistical model. The technique was validated using blind samples and
Pe
obtained an accuracy in oil classification of 70% using the official “ panel
r
test” as reference (Sales et al. 2015). Direct analysis in real time (DART),
fo
coupled to a high-resolution time-of-flight mass spectrometer (TOF MS), has
y
op
been used to authenticate olive oil of varying quality grades by comprehen-
C
sive profiling of triacylglycerols and polar compounds. Using DART-TOF
’s
MS, differentiation among EVOO, olive pomace oil, and olive oil was eas-
or
ily achieved, and the detection of EVOO adulteration with hazelnut oil was
ut
also possible. Hazel nut oil at additions of 6% and 15% (v/v) could be easily
b
tri
detected (Vaclavik et al. 2009). Chinese star anise (Illicium verum ) contami-
on
nated or adulterated with the toxic Japanese star anise (Illicium anisatum ) or
.C
other Illicium species can be detected with DART ambient ionization coupled
C
to Orbitrap™ -HRMS. The metabolite anisatin signal was > 1000 times larger

LL
in Japanese star anise than in Chinese star anise, and thus could be easily
is
determined by DART, and adulteration at 1% (w/w) was measurable (Shen

c
an
et al. 2012).
Fr
d
15.4.2.2 Honey
an
or
Apart from common analytical techniques, such as High-performance Anion

yl
Exchange Chromatography (HPAEC), GC, and HPLC, used for the detec-
Ta
tion of syrup adulterants in honey, advanced techniques, including infrared

18
(IR), NMR, and Raman spectroscopy, which enhance the analysis process
20
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) analy-
ses 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).
Hydrophilic interaction liquid chromatography (HILIC)/TOF/MS and multi-

variate 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 consid-
ered adulteration in Brazil. A multicommuted flow system for the sequential
y
screening or determination of dichromate, salicylic acid, hydrogen peroxide,
nl
and starch in milk samples developed based on a simple binary “ detect” or
O
“ no detect” response could determine analytes quickly, with high perfor-
se
mance and easy operation (De Souza et al. 2014). The DART ambient ioniza-
lU
tion technique coupled with HRMS has been used in the authentication of
na
milk and dairy products. It has been used to discriminate milks obtained
o
rs
from various farm animal species, and milk produced in conventional and
Pe
organic farming, and to detect vegetable oil in dairy products, such as soft
r
cheese. DART-HRMS distinguished a milk adulteration level of 50% (v/v) and
fo
detected the presence of vegetable oils (rapeseed, sunflower, and soybean),
y
op
added to soft cheese at amounts as low as 1% (w/w) (Hrbek et al. 2014).
C
’s
or
15.4.2.4 Meat and Meat Products

ut
Different grades of beef mince and pork mince have been tested for adultera-
b
tri
tion using GC-MS and ultra-high-performance liquid chromatography– mass

on
spectrometry (UHPLC-MS). GC-MS investigates metabolites involved in pri-

.C
mary metabolism, and UHPLC-MS uses reversed-phase chromatography to

C
detect lipophilic species. The combined chemometrics and statistical analy-

LL
ses revealed a number of differential metabolites in the two meat types that
is
can be used for meat authentication and labeling (Trivedi et al. 2016). 1H
c
an
NMR spectroscopy has been used to distinguish beef from horsemeat based
Fr
on comparison of triglyceride signatures. A simple chloroform-based extrac-

tion was used to obtain classic low-field triglyceride spectra in a 10 min
d
an
acquisition time. Peak integration was sufficient to differentiate samples of

or
fresh beef (76 extractions) and horse (62 extractions). 1H NMR (60 MHz) rep-
yl
resents a feasible high-throughput approach for screening raw meat (Jakes et

Ta
al. 2015). Liquid extraction surface analysis– mass spectrometry (LESA-MS)

18
has been used to analyze five cooked meats— beef, pork, horse, chicken,
20
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 specia-
tion (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
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 pep-
y
tides to improve the development of assays for detecting adulteration and
nl
fraudulent practices (Ortea et al. 2016). The various proteomic approaches
O
used for food fraud detection are discussed in Table 15.3.
se
lU
TABLE 15.3
na
Proteomic Techniques Used in the Detection of Food Fraud
o
rs
Pe
Isoelectric focusing Detection of cow milk in ewe Spoljaric et al. 2013
r
and goat cheese
fo
MALDI-TOF MS Detection of cow milk in ewe Cozzolino et al. 2001, 2002
y
and water buffalo cheese, op
C
and in mozzarella from
water buffalo
’s
or
QTOF MS Adulterant soy and pea Cordewener et al. 2009

ut
protein in skim milk

b
UHPLC Adulterants in skim milk Jablonski et al. 2014

tri
on
MALDI-TOF MS Adulteration of fresh milk Calvano et al. 2013

with powdered milk
.C
2DE Identification of animal Montowska and Pospiech 2012

C
species in meat
LL
LC-MS/MS Soybean adulteration in milk Leitner et al. 2006

is
LC-QTOF MS Differentiation of porcine Sarah et al. 2016

c
an
meat from chicken and beef

meat
Fr
MRM-MS Detection of mixing of beef, Watson et al. 2015

d
an
pork, horse, and lamb

Isoelectric focusing Substitution of high-value fish Renon et al. 2005
or
with low-value fish

yl
Ta
Proteome-wide tandem Substitution of high-value fish Barik et al. 2013

MS with low-value fish
18
Query tandem MS Authentication and Wulff et al. 2013

20
recognition of fish species

MALDI-TOF Wine authenticity and Chambery et al. 2009
©
traceability
Combinatorial peptide Detection of fining agents in D’ Amato et al. 2010
ligand libraries wine
Capillary LC-ESI-MS/MS Detection of allergenic Monaci et al. 2010
proteins used as fining
agents in wine
HRMS Detection of allergenic Monaci et al. 2013
proteins and egg whites used
as fining agents in wine
15.4.3.1 Dairy Products

Ewe or goat cheese products with a minimum of 1% of cow’ s milk are con-
sidered 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
y
nl
quantitative determination of γ -casein in cow, ewe, and goat cheese by den-
O
sitometry can be used to detect and quantify the use of cow’ s milk for ewe
se
and goat cheese production (Spoljaric et al. 2013). The presence of cow’ s milk
lU
in either raw ewe or water buffalo milk samples employed in industrial pro-
na
cesses and the addition of powdered milk to samples of fresh raw milk have
o
been detected using matrix-assisted laser desorption/ionization time-of-flight
rs
mass spectrometry (MALDI-TOF-MS). Adulteration is detected by evaluat-
Pe
ing the protein patterns of whey proteins, α -lactalbumin, and β -lactoglobulin
r
fo
used as molecular markers (Cozzolino et al. 2001). MALDI-TOF MS is also
y
used to differentiate mozzarella made from water buffalo milk and from
op
mixtures of less expensive bovine using whey proteins, α -lactalbumin, and
C
β -lactoglobulins as molecular markers (Cozzolino et al. 2002). A nontargeted
’s
or
protein identification method has been used to test adulterants such as plant
ut
proteins in skim milk powder. The LC-MS method using a QTOF MS instru-
b
ment has been used to identify adulterant protein isolates of soy and pea in
tri
on
skim milk. To identify the plant proteins present in the adulterated skim milk
.C
powder, data-dependent LC-MS/MS, in combination with a list of differential

peptides, was used (Cordewener et al. 2009). Similarly, using UHPLC with
C
LL
215 nm detection, chromatographic profiles of skim milk powder and mix-
tures of it with soy, pea, brown rice, and hydrolyzed wheat protein have been
cis
obtained. Adulterations up to 1% and 3% levels could be detected (Jablonski

an
et al. 2014). MALDI-TOF MS has been used to analyze tryptic digests from raw
Fr
liquid (without heat treatment), commercial liquid, and powdered cow milk.
d
Two-dimensional gel electrophoresis (2DE) was used initially to differentiate

an
liquid and powder milk, and further adulteration with powdered milk was
or
confirmed by MALDI analysis of the in-gel digested proteins. Milk samples

yl
Ta
adulterated with different percentages of powdered milk and diagnostic pep-

tides were detected at the 1% adulteration level (Calvano et al. 2013).
18
20
©
15.4.3.2 Meat
Myosin light chains (MLCs) have been used as markers in the authentica-
tion 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
are added to processed meat products for economic gain and to improve
their functional properties. Chromatographic prefractionation on the pro-
tein 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 pro-
y
teins. The protein subunit glycinin G4 subunit A4 can be used as a marker
nl
to detect soybean protein adulteration in meat (Leitner et al. 2006). Using
O
LC-QTOF MS, porcine-specific peptide markers were identified to differ-
se
entiate pork from beef, chevon, and chicken meat. Seven porcine-specific
lU
peptides derived from lactate dehydrogenase, creatine kinase, and serum
na
albumin protein were detected. Meat species identification at the peptide
o
rs
level is a robust platform for the authentication of meat products such as
Pe
halal (Sarah et al. 2016). A rapid multiple reaction monitoring (MRM) mass
r
spectrometric method for the detection and quantitation of the adultera-
fo
tion of meat with undeclared species has been carried out. Corresponding
y
op
proteins from the different species and corresponding peptides from those
C
proteins (CPCP) were used. The approach allowed the identification of four
’s
meat types, beef, pork, horse, and lamb, and could also detect meat mixing
or
at 1% (w/w) (Watson et al. 2015).

ut
b
tri
on
15.4.3.3 Fish
.C
Isoelectric focusing, a simple and reliable technique, was used to detect

C
fraudulent substitution of cold-smoked fillets of swordfish (Xiphias gladius )

LL
with low-value blue marlin (Makaira mazara ) and blue marlin steaks with
is
Mediterranean spearfish (Tetrapturus belone ). Sarcoplasmic proteins of the

c
an
fish species have been used to differentiate fish species (Renon et al. 2005).
Fr
Proteomics has been used to differentially characterize sarcoplasmic pep-

tides of the closely related fish Sperata seenghala and Sperata aor (Barik et al.
d
an
2013). Proteome-wide MS/MS has been used for species recognition and
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authentication of fish species. The technique includes protein extraction,

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digestion, and data analysis. A set of reference spectral libraries is generated

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for unprocessed muscle tissue from 22 different fish species. Query MS/MS
18
data sets from “ unknown” fresh muscle tissue samples are searched in the
20
reference libraries. The number of matching spectra can be used to iden-

tify 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-qual-
ity Campania white wines. Thus, MALDI-TOF can be used to detect wine
authenticity and traceability (Chambery et al. 2009).
Combinatorial peptide ligand libraries (CPLLs) have been used in iden-

tifying 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/
y
MS) has been used for the detection and identification of casein-derived
nl
peptides in fined white wine. The technique is useful in detecting poten-
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tially allergenic milk proteins used as fining agents in wine. Peptides arising
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from α - and β -casein could be detected in white wine fined with casein at
lU
100 and 1000 μ g/mL (Monaci et al. 2010). HRMS has been used to determine
na
various fining agents, such as allergenic milk (casein) and egg white (lyso-
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zyme and ovalbumin) proteins in commercial white wines simultaneously,
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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).
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15.5 Prevention of Food Fraud

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Food fraud can be prevented by traceability and authentication of food prod-

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ucts and managing the supply chain and procurement process.

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LL
15.5.1 Traceability
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Traceability is to know the origin of product, where it has been, where it is

c
an
going, and its present location (Spink 2012). Traceability helps to find and
Fr
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
d
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replaced with a fraudulent product. On identifying food fraud, traceability

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helps in product recall from the marketplace. Some of the tracing techniques
yl
are by digitally tracking the product or using readable codes, such as bar-
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code and radiofrequency identification (RFID). Pedigree is a related system

18
that records the history of the product from manufacturing to sale (Dietrich
20
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 continu-
ous 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
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
y
fraud incidences. Certain business practices create fraud opportunities in the
nl
supply chain. Usually, counterfeit, adulterated, or tampered products enter
O
into sales through the supply chain. During procuring a product, it is essen-
se
tial to verify the source and how it is obtained (Closs and McGarrell 2004;
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Closs and Mollenkopf 2004; Closs et al. 2008).
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15.6 Regulation of Food Fraud: Laws and Legislation
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15.6.1 Food Fraud Regulation in the United States
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’s
The United States passed the Food Safety Modernization Act (FSMA) in
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January 2011, followed by the publication of the FSMA Preventative Controls

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(FSMA-PC) rule in September 2015. The FSMA-PC rule contains direction

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regarding food fraud and EMA (FDA 2009; Spink 2009). The FSMA-PC rule
on
does not limit economically motivated hazards to only adulterant substances

.C
in food, but covers any economically motived hazard and addresses preven-
C
tion of all food safety hazards. There is a section of FSMA that addresses only
LL
“ smuggled foods.” The Food, Drug, and Cosmetic Act of 1938 (FD&C) is in
is
effect and includes violations of the “ Adulterated Foods” and “ Misbranded

c
an
Foods” sections, wherein adulterated foods need not contain an adulterant

Fr
but can be stolen or spoiled food. The Government Accountability Office

(GAO) and Congressional Research Service (CRS) have published two sig-
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nificant U.S. government reports regarding food fraud and emphasized that
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the FDA would address and prevent all types of food threats.
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15.6.2 Food Fraud Regulation by the European Commission

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20
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.
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 gov-
ernment to address food crime and food fraud (Elliott 2014).
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15.6.4 Food Fraud Regulation in China
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The Chinese government has a new food safety law that went into effect
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in October 2015. It expands the Chinese food regulatory system and pro-
na
vides more comprehensive control of the food supply chain, as well as funds
o
research on food safety by the Chinese National Center for Food Safety Risk
rs
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Assessment (CFSA). The food safety law defines “ traditional” and “ nontra-
ditional” food safety, including food defense and food fraud. The CFSA has
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a broad definition of food fraud and prioritizes human health hazards due to
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adulterant substances and counterfeit products. op
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15.6.5 Food Fraud Regulation: Industry Standards and Compliance

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There are a range of industry or nongovernmental activities relating to food

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fraud regulation.
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15.6.5.1 Global Food Safety Initiative

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The GFSI is an industry association. It is created to harmonize a food safety

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management system. The GFSI approves audit schemes, and the scheme
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an
owner creates a standard that supports the audit scheme. Food producers
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follow and implement the standard. A third-party auditor certifies that the
company is in compliance with the standard. These result in the producer
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being “ GFSI certified” since the GFSI approved the scheme, empowered the
or
scheme owner to set the standards, and approved the auditors. The GFSI has
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also created a Food Fraud Think Tank (FFTT) to review and make recom-
Ta
mendations on the issue. It has come out with a GFSI’ s Food Fraud Position
18
Paper that defined all types of food fraud and focused on its prevention. The
20
second issue proposed is to include a requirement for a food fraud vulner-

ability 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 counterfeit-
ing are covered by SSAFE (SSAFE 2012).
15.6.5.3 U.S. Pharmacopeia

The U.S. Pharmacopeia (USP) is a standards-setting body that has been fol-
lowed 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 data-
base that includes adulterant substance detection techniques and substances
y
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published in scholarly journals. The expert panel has also made recommen-
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dations regarding food ingredient adulterant substances, including vulner-
se
ability assessments (USP 2009).
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15.6.5.4 Other Activity
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The International Organization for Standardization (ISO) has also con-
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ducted activities on food fraud (ISO 2010). The U.S. National Center for Food
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Protection and Defense (NCFPD), a Department of Homeland Security Center
y
of Excellence, has hosted databases such as the Economically Motivated
op
Adulteration Database (NCFPD 2015).
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©
16
Food Safety Regulation and Standards
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Nada Smigic and Ilija Djekic
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na
CONTENTS
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16.1 Introduction................................................................................................. 531
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16.2 Food Safety Issues...................................................................................... 532
16.3 Food Safety Regulation .............................................................................534
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16.3.1 Food Safety Regulation at the International Level....................534
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16.3.2 Food Safety Regulation in the European Union and the op
United States�� 536
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16.3.3 Food Safety Regulation in Developing Countries..................... 541

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16.3.4 Developed versus Developing Countries ...................................542

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16.4 Food Safety Standards ..............................................................................544

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16.4.1 Characteristics of Food Safety Standards...................................545

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16.4.2 Main Groups of Requirements.....................................................546

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16.4.2.1 Prerequisite Programs and Good Practices................. 547

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16.4.2.2 Hazard Analysis............................................................... 547

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16.4.3 Food Safety Management.............................................................. 549

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16.4.4 Effects of Implemented Food Safety Standards......................... 550

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16.4.5 Food Safety Audits......................................................................... 551

16.4.5.1 Types of Food Safety Audits........................................... 552
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16.5 Final Remarks.............................................................................................. 554

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References.............................................................................................................. 554
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16.1 Introduction
18
20
The growing importance of food safety in international trade is influenc-

ing the importance of developing and implementing both legal requirements
©
and international standards.

Food safety regulations comprise general principles for three main stake-
holders— public authorities, food establishments, and consumers. In defin-
ing the food safety framework, legislation is distributed via vertical and
horizontal approaches. Enforcement of legal requirements by public authori-
ties 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
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,
y
that are involved in any aspect of food. The majority of food safety standards
nl
are generic, although there is a trend in developing tailored standards for
O
specific food industries. In spite of the fact that there are several international
se
organizations that publish various types of food safety standards, they all
lU
have three groups of requirements in common: (1) prerequisite programs
na
(PRPs) and good practices, (2) hazard analysis, and (3) food safety manage-
o
rs
ment. In order to verify their implementation, three types of audits are pres-
Pe
ent in assuring stakeholders that food safety requirements are implemented.
r
Depending on the role of the organization requesting an audit, the auditee,
fo
and the auditors, audits are classified as internal audits (conducted by the
y
op
organization itself), second-party audits (conducted by the customer or other
C
organization having an interest), and third-party audits (conducted by inde-
’s
pendent auditing organizations).

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This chapter gives an overview of these two important pillars in ensuring

ut
food safety throughout the food chain.

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on
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16.2 Food Safety Issues

c is
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Today, consumers require high-quality food products in broad assortments

Fr
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.
d
an
Last but not least, the public also expresses high interest in food-related
or
scandals, such as dioxin crises, “mad cow” diseases, the Chinese melamine
yl
case, various bacterial contaminations of food, toxic chemicals in food, and

Ta
irradiation and other new technologies (Kä ferstein and Abdussalam 1999). It

18
seems that food safety is receiving more and more attention nowadays, with
20
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 com-
plex and internationalized. Food producers are using ingredients from all
around the world, transforming the complete food industry into a very com-
plex 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 chemi-
cal, physical, or microbiological hazards. In addition, there is an increasing
Food Safety Regulation and Standards 533
trend of food ingredients and processed food movement between develop-

ing and developed countries, which poses numerous challenges in ensur-
ing 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.
y
Important to note is the fact that food safety issues occurring in develop-
nl
ing and developed countries are different. Developed countries often expe-
O
rience food safety problems related to new technologies that are applied, or
se
new materials used in agriculture or food processing. On the other side, the
lU
developing countries are still in battle with poor hygiene issues in food pro-
na
duction and processing. Both chemical and microbiological contaminations
o
rs
are often seen as on ongoing challenge for food safety. Due to globalization
Pe
of food supply, food safety problems from developing countries are rapidly
r
become international problems.
fo
Despite obvious advances in food science and technology, foodborne ill-
y
op
nesses remain an important cause of morbidity and mortality worldwide,
C
but also an important obstacle for socioeconomic development. Foodborne
’s
diseases are caused by biological, chemical, and physical hazards that are
or
transmitted though ingested food. Hazards can contaminate food in any step
ut
throughout the food chain. Most often, the reported number of foodborne dis-
b
tri
eases is related to bacterial or viral hazards transmitted via food, as the symp-
on
toms of illness are quickly seen and often connected with the cause. On the
.C
other side, the burden arising from chemicals and parasites is still unknown.
C
The negative health effects of chemicals may not be observed for years fol-
LL
lowing the exposure (e.g., aflatoxin and liver cancer, lead and cardiovascu-
is
lar disease). In addition, the relevant disease end points related to foodborne
c
an
chemical hazards may easily arise from many different causes, which create
Fr
even more complicated estimates of the incidence and mortality (WHO 2015).
In recent decades, various scientific studies have been performed in the
d
an
field of food safety, which were followed by the number of preventive and
or
control measures implemented in the food industry. Nevertheless, epidemio-

yl
logical data from developed countries (the United States, the European Union
Ta
[EU], Canada, and Australia) indicated that the number of food safety issues
18
and foodborne illnesses remains at a high level (Newell et al. 2010; Havelaar
20
et al. 2010; Mead et al. 1999; EFSA and ECDC 2015). Data from some develop-
ing 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 legisla-
tion 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).
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 con-
y
trol over all types of food produced, processed, and sold on the market, so
nl
that the consumers are guaranteed that the food will not cause any harm
O
within the limits of available scientific knowledge. In most countries, food
se
regulation includes a great number of different laws and ordinances. They
lU
set out the government’s requirements to be met by food business operators
na
to ensure that food is safe and of adequate quality. Within the food regula-
o
rs
tion, different aspects of production, trade, and food handling are included,
Pe
simultaneously covering food control, food safety, and relevant aspects of
food trade.
r
fo
Currently, there are a number of different food safety regulation systems
y
op
worldwide, some of them well designed and risk based, with a long history
C
of developments and improvements (developed countries), while others are
’s
still in the initial phase (developing and/or undeveloped countries). The

or
common platform for food safety regulation lies in the documents adopted
ut
and prepared by various international bodies. Nevertheless, the individual

b
tri
member nations may adopt, modify, or make their own food regulations.
on
.C
C
16.3.1 Food Safety Regulation at the International Level

LL
On the international level, the Food and Agriculture Organization of the

c is
United Nations (FAO), the World Health Organization (WHO), and the
an
World Trade Organization (WTO) are major organizations that deal with
Fr
food safety issues. The impact of WTO on food safety is mainly seen through
d
an
two agreements: Agreement on Sanitary and Phytosanitary Measures (SPS

Agreement) and Agreement on Technical Barrier to Trade (TBT Agreement).
or
Under the SPS Agreement, the WTO sets constraints on member states’
yl
Ta
policies relating to food safety, as well as pests and diseases associated with
animal and plant health. The definition of sanitary and phytosanitary mea-
18
sures is given in the SPS Agreement as a

20
©
measure that is applied to protect animal or plant life or health within

the territory of the Member from risks arising from the entry, estab-
lishment 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;
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 harmo-

nize 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
y
nl
making regarding different aspects of food on a global level. Codex stan-
O
dards, guides, and recommendation have been cited and recommended in
se
the SPS Agreement, as the most adequate measures for facilitating inter-
lU
national food trade. The SPS Agreement adopts codex standards for food
na
additives, veterinary drugs and pesticide residues, contaminants, methods
o
rs
and analysis of sampling, and codes and guidelines of hygiene practices.
Pe
Therefore, they have been placed as a target against which national food
measures and regulations should be enacted and enforced. Where the mea-
r
fo
sures are conformed to international standards (such as codex standards),
y
op
they are to be considered consistent with the SPS Agreement. The Codex
Alimentarius Commission adopted one of the most important guidelines on
C
’s
good hygiene practices in order to prevent, control, and decrease food safety
or
risks (CAC 2003).

ut
The SPS Agreement permits individual nation states to take legitimate

b
tri
measures to protect the life and health of consumers given the level of risk
on
that they deem to be “acceptable,” applying measures that can be justified

.C
scientifically and do not unnecessarily hamper food trade. However, they are
C
required to recognize that measures adopted by other countries, although

LL
different, can provide equivalent levels of protection.

is
According to the SPS Agreement, WTO members are obliged to apply

c
an
measures to the extent necessary, and these measures must be scientifi-

cally proven and/or outlined in international standards. The EU faced this
Fr
requirement in the dispute related to hormone-treated beef meat exported

d
an
from the United States. In 1998, the European Commission prohibited the
or
importation and placement on the market of meat and meat products treated
yl
with certain hormones originating from the United States, and as a conse-
Ta
quence, the United States complained to the WTO. As there was not enough
18
scientific evidence that hormone-treated meat might present a risk to human

20
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 hid-

den 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
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, devel-
oping countries are still lagging behind, struggling with a slow implemen-
tation process (Das 2008; Neeliah and Goburdhun 2010). As Hooker (1999)
y
indicated in his article, the SPS Agreement has put a heavy burden on devel-
nl
oping countries to be in compliance with the international regulations, with
O
the problems related to infrastructure, together with the lack of experts, lab-
se
oratory resources for testing, and fragmented and uncoordinated structure
lU
of the food control system at the national level.
na
o
rs
16.3.2 Food Safety Regulation in the European
Pe
Union and the United States
r
fo
The EU is an economic and political union of 28 member states located in
y
op
Europe. This great single market is one of the most important food export
C
and import world trade players. The current food legislation that is in place
’s
in the EU presents the result of developments and various changes toward

or
the production and marketing of safe food, over a long period of time. In
ut
the early nineties, several food safety crises, such as those related to bovine
b
tri
spongiform encephalopathy (BSE), dioxins, high pesticide and antibiotic con-

on
tents in several foods, and the usage of Sudan Red 1, occurred in the EU. The
.C
panic situation related to food safety had driven the European Commission
C
to include food safety among its major priorities and revise the EU food
LL
safety system in order to return the loss of consumers’ trust. One of the most
is
important steps in this direction was the publication of a white paper in 2000
c
an
(EC 2000), in order to introduce consistency and clarity throughout the food
Fr
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,
d
an
in 2002, the general food law was adapted (EC 2002), while a set of impor-
or
tant food safety regulations were adopted in 2004, with a 2-year period for
yl
food business operators to comply with the given requirements (EC 2004a,
Ta
2004b, 2004c). The package of food safety legislation covered all aspects of
18
food hygiene (often called “the hygiene package” ), food safety, and quality
20
in all stages of the food production chain, from and including primary pro-
duction 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 pro-
tect 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 domes-
tic and imported food). EU food legislation, for the first time, introduced
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
y
hazards may occur and how to deal with them in order to maintain food
nl
safety. The implementation of a HACCP system was legally obliged for all
O
food businesses that operate within the EU, except for primary producers
se
(EC 2004a, 2004c). This system was earlier recommended, but not mandated,
lU
by the above-mentioned guidelines on good hygiene practices issued by the
na
Codex Alimentarius Commission (CAC 2003). This preventive food safety
o
rs
system does not rely on end-product testing, but focuses on the continuous
Pe
control and monitoring of hygiene principles related to PRPs and critical con-
r
trol points (CCPs) within the production process. Although many problems
fo
have been seen in the process of implementing the HACCP system in EU
y
op
countries, especially in small and medium food enterprises, the food indus-
C
try had seen many benefits from its application (Smigic et al. 2012; Tomasevic
’s
et al. 2013, 2016).

or
Despite quite stringent regulatory standards that were appointed in the

ut
EU, the European Commission and member states are still facing numerous
b
tri
foodborne incidents. In 2008, an outbreak related to dioxin contamination of

on
animal feed occurred in Ireland (Casey and Lawless 2011). This crisis was
.C
resolved by the recall of all contaminated products, as a precautionary mea-

C
sure taken by the Irish authorities. Nevertheless, the latter risk assessment
LL
indicated that public health risk was of no concern “for this single event”
is
(Casey and Lawless 2011). The example of the traceability chain breakdown
c
an
should also be mentioned here, as it was connected with the horsemeat scan-
Fr
dal in 2013. Although this issue was mainly related to food fraud, rather
than food contamination, the concern was the fact that horsemeat contained
d
an
traces of the veterinary drug phenylbutazone, which entered the food chain
or
(Bá ná ti 2014). This resulted in great economic loss and demonstrated the
yl
importance of proper control at all phases of the food chain.

Ta
Due to its nature, food safety legislation in the EU (and in other devel-
18
oped countries) is focused mostly on foods of animal origin. Nevertheless,

20
the foodborne outbreak that occurred in 2011 in Germany, caused by Shiga

toxin– producing Escherichia coli O104:H4 in fenugreek sprouts, moved atten-
©
tion 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 commu-
nication between food safety and public health authorities was noted as a
concern (McEvoy 2016). In response to this outbreak, an action plan was pre-
pared covering issues such as (1) the risk assessment of pathogens in the food
of nonanimal origin; (2) microbiological criteria, traceability, and certifica-

tion related to the production of sprouts; (3) improvement in the dissemina-
tion 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 prod-
y
ucts and food hazards. In order to adequately address food safety issues, the
nl
European Commission often asks for scientific evidence from EFSA, to serve
O
as a basis for policy making. Responsive legislation is the term used by McEvoy
se
(2016), which expresses the way European legislation has been evolving in
lU
the last 15 years (Table 16.1). This trend is also expected in the future.
na
The other food safety regulatory system that is worth mentioning is the one
o
rs
established in the United States. After almost 70 years, the United States made
Pe
a significant change in this area, adopting the Food Safety Modernization
r
Act (FSMA) in 2011 (FDA 2011). The most important change that came with
fo
FSMA is the focus shift from reaction to prevention. Two major FSMA rules
y
op
were issued in 2013, “preventive control for human food” and “standards for
C
produce safety,” to be applied for both domestic and imported food (FDA
’s
2015a, 2015c).
or
It is of note that the preventive control approach, which is now empha-

ut
sized in the FSMA, is not a novelty for the United States food legislation, as
b
tri
the Food Safety and Inspection Service (FSIS) has required preventive con-
on
trol measures to be applied for the production of meat products since 1996
.C
(FSIS 1996). Also, the Food and Drug Administration (FDA) has required
C
control measures for foods such as seafood since 1995 (FDA 1995) and fruit
LL
juices since 2001 (FDA 2001). Nevertheless, the adoption of FSMA and the
is
rule on the preventive control for human food now requires the implemen-
c
an
tation of this preventive approach for all processors and manufacturers of

Fr
human food, which have to develop a control plan to prevent situations in

which their food products could cause foodborne illness (FDA 2015a). This
d
an
control plan should include the evaluation of hazards, as well as the identi-
or
fication and monitoring of steps or controls to minimize or prevent hazards.

yl
Together with this plan, a company has to be prepared for any problems that
Ta
might arise (action plan). Although it is not named as such, this food safety
18
assurance system is apparently based on the HACCP system, which origi-

20
nated 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 par-
ticipate in great deal in United States food trade, were the major driving
force to introduce changes in the food import legislation. FSMA brings some
©
TABLE 16.1
20
18
Driving Force for Regulatory Change in Developed and Developing Countries
Ta
Driving Force for Regulatory Change
yl Consequence References
Developed Foodborne Dioxin crises in the
or Polychlorinated biphenyls (PCBs) and dioxin-contaminated batch of Bá ná ti 2011;
countries incidents/ EU transformer oil entered the food chain via an animal feed mill in Belgium. Covaci et al.
domestic
an
d This was then fed to broilers and subsequently recycled into pig feed, 2008
food 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.
Fr
Mad cow disease BSE crisis resulted in loss of consumers’ confidence in the safety of beef
an Vos 2000; Bá ná ti
in EU
c
meat. This crisis was a “trigger” to reform the existing European food
is 2011
safety legislation and establish new regulatory institutions across EU.
LL
E. coli O157:H7 E. coli O157:H7 outbreak with contaminated hamburgers in restaurants in
C FSIS 1996
outbreak in the the western United States was the major driving force to adopt PRPs and
United States the HACCP rule for meat and poultry.
.C
Food Safety Regulation and Standards
Salmonella The outbreak caused by Salmonella in peanut butter that occurred in the Sheth et al. 2011
on
outbreak in the United States during 2007– 2008 was the first salmonellosis linked to
tri
United States b
peanut butter. This outbreak, together with others that occurred in the
United States, caused a food legislation change in 2011.
ut
or
Melamine in milk Melamine-tainted milk caused the death of six infants and serious illness
’s Broughton and
in China of tens of thousands of infants in China, and consequently, a food safety
C Walker 2010
law was adopted in 2009, with additional media pressure for the effective
op
enforcement of the adopted law. y
Foodborne Various food scares Over the years, a number of different food scares connected with the food
fo Zach et al. 2012;
incidents/ (Salmonella in imported to the United States resulted in the change of food legislation
r Paggi et al.
imported peanut butter, and adoption of the Food Safety Modernization Act (FSMA). The issue is
Pe 2013; FDA 2011
food jalapeñ o peppers now more the responsibility of the importer, who has to perform
rs
from Mexico, etc.) risk-based foreign supplier verification analyses to ensure that imported
o
in the United foods are produced in compliance with HACCP procedures and are not
na
States adulterated or misbranded. lU
se (Continued)
539
O
nl
y
©
20
540
18
Driving Force for Regulatory Change in Developed and Developing Countries
Ta
Driving Force for Regulatory Change
yl Consequence References
Melamine in milk
or Due to the presence of melamine in milk, many actions have been taken EFSA 2010; EC
incident in the EU worldwide. In the EU, after the EFSA gave a scientific opinion on food 2006
and feed, the current Regulation (EC) 1881/2006 was amended, giving
an
d the maximum level of melamine (mg/kg) for infant formulas and other
food.
Fr
Scientific Food As a need to obtain scientifically based information, to make decisions on EFSA 2010, 2013
an
information safety– related
c
a legal basis, the EU introduced EFSA. Many changes in the food safety
is
information legislative acts occurred after the EFSA published its scientific opinion,
based on the available information published in scientific papers.
LL
New and Listeria
C
Scientific knowledge related to, at that time, new pathogens such as EC 2005
emerging monocytogenes and L. monocytogenes, and its importance for ready-to-eat food, influenced
.C
pathogens Cronobacter spp Regulation (EC) 2073/2005 to introduce this pathogen within
on
microbiological criteria, with which food businesses operators have to be
tri
in line. Regulation (EC) 2073/2005 lays down a food safety criterion for
b
Cronobacter spp. (previously classified as Enterobacter saka zakii ) in dried
ut
infant formulas and dried dietary foods for special medical purposes
or
’s
intended for infants below 6 months of age.
Developing Integration West Balkan
C
The political decision of many European countries was to enter the EU. Glintic 2012;
countries into the countries (Serbia, One of the first steps was to harmonize legislation with the European Smigic et al.
op
regional Montenegro,
y
acquis communautaire, and this resulted in many changes in food safety 2015;
union, such Bosnia and legislation that occurred in these countries.
fo Antunovic
as the EU Herzegovina, and
r et al. 2008
Albania)
Pe
Accession of China, countries in
rs
Many developing countries are exporting their often exotic food products Broughton and
o
the market in Latin America, to the market of industrialized and developed countries (such as the EU,
na Walker 2010;
industrialized Asia, and Africa Japan, and the United States). Due to many food safety issues in the past,
lU Pei et al. 2011;
countries a lot of pressure has been put on the developing countries’ governments Shukla et al.
to adopt adequate food safety legislation, within their food control 2014; Leon and
se
programs. O Paz 2014

nl
y
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 per-
sonnel at United States embassies around the world (Keener et al. 2014).
y
nl
16.3.3 Food Safety Regulation in Developing Countries
O
Food products originating in developing countries are often present on the
se
global food market, and therefore their control system must be adequately
lU
harmonized with at least basic food safety regulation. In many developing
na
countries, especially in Africa and Asia, existing food legislation is out-
o
rs
dated, incomplete, and fails to adequately address current and emerging
Pe
food safety problems, and often the principles of food safety given in the
r
Codex Alimentarius and trade agreements have not been taken into account.
fo
Additionally, inefficient enforcement due to the lack of effective food con-
y
op
trol infrastructure and institutional capacities to ensure compliance is still
C
a bottleneck for the full implementation. Existing food regulation does not
’s
provide clear and final responsibility of the main stakeholders involved in

or
food safety, resulting in noncoordinated and overlapped activities. Various

ut
pieces of legislation are scattered among many governmental agencies.

b
tri
It is of note that recently, many improvements have been seen in develop-

on
ing countries, which are trying to improve their food control systems, often
.C
starting with the adoption and enforcement of food regulation (Chanda et

C
al. 2010; Mwamakamba et al. 2012; Ghaida et al. 2014; Pswarayi et al. 2014;
LL
Al-Busaidi and Jukes 2015). Although it is expected that developing countries

is
are doing this to protect the health of their consumers, this is most probably
c
an
driven by the aspiration to participate in the global food market. In addition,

Fr
often a driving force for adopting food safety requirements is the presence of
multinational food companies in developing countries and the implementa-
d
an
tion of their internal good hygiene practices and requirements.

or
The most important food importing countries operate on the “principle

yl
of equivalence” for imported food, meaning that exporting countries must

Ta
demonstrate that their production methods achieve the importing country’s

18
level of sanitary protection and food safety. However, in some cases devel-
20
oping 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 posi-
tion 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
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).
y
Driven by domestic food safety issues and willingness to participate in the
nl
global food market, China enacted a new food law in 2009, that foresees the
O
adoption of the approach of food safety throughout the complete food chain,
se
“from the farm to the fork”, governing the EU regulatory framework. This
lU
law is the most important piece of regulation that is used to ensure the safety
na
and quality of food and to protect the health of consumers. It should allow
o
rs
better coordination between national and provincial authorities, which was
Pe
recognized as a weak point in the previous regulatory system (Jia and Jukes
r
2013). As in the EU, the primary responsibility for food safety in China lies
fo
with food producers. The legal sanctions on food production enterprises and
y
op
marketing enterprises that violate the rights and interests of consumers are
C
enacted by this law. Still, there are many issues that have to be resolved in
’s
coming years, in order to fully implement and capture the positive results
or
of this regulation in the Chinese food sector. As Liu (2014) indicated in his
ut
report, there are several issues that the Chinese food sector and government
b
tri
still have to deal with in regard to food safety. They are (1) great production
on
value, (2) the pollution issue, (3) decentralized agricultural production and
.C
the traceability of raw materials, (4) the imbalance in rapid food production,
C
and (5) inadequate risk communication for food safety issues.

LL
Some developing countries that are geographically closer to the EU, such
is
as the West Balkan countries, have placed integration in the EU to be the

c
an
national priority. This has been a strong driver for the change and harmo-
Fr
nization of food safety regulation with the European acquis communautaire

(Glintic 2012; Smigic et al. 2015; Antunovic et al. 2008), and indirectly with
d
an
international regulations. The intention of this legal harmonization is to

or
allow subjects in the food chain to perform their activities according to the
yl
European regulatory structure. Despite many changes that occurred in West

Ta
Balkan countries’ regulation, the major obstacles are still seen in the imple-
18
mentation and enforcement of adopted legislative rules (Smigic et al. 2015;

20
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 improve-
ment. Drivers for this are mainly found in their own reconsideration and
reevaluation of food safety issues (Table 16.1). They have better tools to mon-
itor their food systems and provide a fairly consistent and constant supply
of safe and wholesome food. On the other side, some developing countries
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 regula-
tion 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
y
countries regarding the level of food safety regulation adoption and imple-
nl
mentation. Four different categories can be distinguished, namely, leaders,
O
suppliers, proactive suppliers, and followers. This classification is made by
se
combining a matrix developed by the Boston Consulting Group (Morrison
lU
and Wensley 1991) and food safety culture tool developed by UK Food
na
Standards Agency (FSA 2012).
o
rs
Leaders are countries that strive for food safety improvements in both
Pe
developing and implementing food safety regulation. Mainly developed
r
countries (e.g., the United Kingdom, Germany, France, the United States, and
fo
Australia) can be included in this category. Usually, they work jointly with
y
scientific institutions and introduce new and emerging food safety hazards op
C
within legislation based on scientific evidence. They also develop mecha-
’s
nisms for assessing the implementation level of food safety requirements.

or
Proactive suppliers are countries that are already part of a developed mar-
ut
ket (Croatia, Bulgaria, and Romania in the EU), countries in the candidate
b
tri
status for EU membership (West Balkan countries), and countries trading

on
with developed markets (China, Latin America, Russia, and countries from
.C
the Commonwealth of Independent States). Their relatively high level of

C
adoption of food safety regulation is a result of extensive trade with devel-

LL
oped markets. It is important to note that their level of implementation is still

is
relatively low, due to the inadequate role of their inspection services.

c
an
Fr
Implementation of food safety regulation
d
an
High
Leaders Suppliers
or
yl
Ta
18
20
©
Low
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.
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 incorpo-
rated within the food safety culture of these companies and, as such, are a
part of their production process wherever they operate worldwide. Also,
y
these countries are often producers of raw food with a low level of pro-
nl
cessing. Their inspection services express a significant lack of food safety
O
knowledge.
se
Followers are mainly poor, undeveloped countries with a low level of
lU
adapted and implemented food safety regulation.
na
o
rs
Pe
r
fo
16.4 Food Safety Standards
y
op
Starting from the late 1990s, the proliferation and evolution of food safety
C
standards was driven predominantly by the development of new regulatory
’s
requirements in response to consumers’ concerns about food safety, as well

or
as scientific developments regarding the risks associated with food (Henson

ut
and Reardon 2005). Nowadays, the food chain is more complex than ever
b
tri
before, because of demographic, cultural, economic, and technological devel-

on
opment (Kleboth et al. 2016).

.C
Challenges that initiated introduction of a “management” concept within

C
food safety are various food industry developments, such as new food tech-
LL
nologies and new food products that induced the appearance of unknown
is
hazards and managing risks. In addition, the globalization and development

c
an
of one global market resulted in an increased need for the management of

Fr
food safety. As a result, except for the Codex Alimentarius Commission,

which defines basic good hygiene requirements, all new developed food
d
an
safety standards have some management requirements. International trade

or
and travel consequently increased the risk of cross-border transmission and

yl
the need for strengthening methods of food control (Van der Spiegel et al.
Ta
2005). This initiated the introduction of food safety standards related to spe-
18
cific parts of the food chain, such as logistics, distribution, and retail.
20
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 manufactur-
ing 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
(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
y
Basically, there are two types of food safety standards, namely, interna-
nl
tional and private standards. International standards are developed by
O
international organizations, such as the International Organization for
se
Standardization (ISO), which issued ISO 22000 (ISO 2005). This food safety
lU
management standard is applicable to all organizations involved in the food
na
chain and comprises PRPs, HACCP, and management requirements. By the
o
rs
end of 2014, more than 30,500 certificates were granted to food companies
Pe
worldwide, mostly in Europe and regions of East Asia and the Pacific (ISO
r
2015b). Another important group of international standards was developed
fo
by the Codex Alimentarius Commission, with its fundamental good hygiene
y
practice standard and HACCP principles (CAC 2003).op
C
On the other side, starting from the 1990s, many private food safety stan-
’s
dards have been developed and published, with the aim to (1) improve sup-
or
plier consistency, (2) avoid product failures, (3) eliminate multiple audits, and
ut
(4) support the consumer– supplier relationship (Trienekens and Zuurbier

b
tri
2008). An example of private standards developed for primary production is

on
the GlobalG.A.P. standard (GlobalG.A.P. 2016), which comprises 16 separate

.C
standards deployed for crop production, fruits and vegetables, and livestock.
C
Private British Retail Consortium (BRC) food safety standards were issued
LL
by the BRC and are intended for the food production sector (BRC 2015). The
is
IFS series of standards was first developed by German and French retailers
c
an
(IFS 2014a), and they have found international implementation in the food
Fr
production sector.
Besides generic standards applicable to all types of food companies, there
d
an
are some initiatives in developing tailored standards for a specific food sec-
or
tor. An example is the Global Red Meat Standard developed by the Danish
yl
Agriculture & Food Council. It prescribes specific requirements for slaugh-

Ta
tering, cutting, deboning, and selling red meat and meat products (GRMS
18
2015). Best aquaculture practice was developed by the Global Aquaculture

20
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 ori-
gin, and processed food. The trade in organic food differs from other food
commodity networks due to the need for organic certification (EC 2007).
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
y
consumers (Regenstein et al. 2003; Regenstein and Regenstein 1991).
nl
Religious slaughtering is carried out legally in the EU in licensed slaugh-
O
terhouses by authorized slaughtermen of the Islamic and/or Jewish faiths
se
(Velarde et al. 2014).
lU
Along with the development of standards, their recognition became an
na
obstacle in international trade, due to a great number of available food stan-
o
rs
dards. The modern and global food industry requires universal food safety
Pe
standards that can be accepted worldwide. As a solution, guidance on rec-
r
ognizing different safety standards along the supply chain was developed
fo
by the Global Food Safety Initiative (GFSI 2013). This initiative comprises 400
y
op
retailers, manufacturers, service providers, and other stakeholders across 70
C
countries (GFSI 2015). Table 16.2 presents typical private food safety stan-
’s
dards recognized by GFSI throughout the food chain and relevant interna-
or
tional standards.
ut
b
tri
on
16.4.2 Main Groups of Requirements

.C
All food safety standards have requirements regarding PRPs and hazard
C
analysis. PRPs are requirements that need to be fulfilled prior to perform-

LL
ing any type of hazard analysis. They present the basic elements of good
c is
an
Fr
TABLE 16.2
d
Food Safety Standards Recognized by the GFSI within the Food Chain
an
Role in the Food Chain GFSI Recognized Other Standards in Use

or
yl
Primary production SQF Code ISO 22000:2005

Ta
GlobalG.A.P.
CanadaG.A.P.
18
Food processing Global Aquaculture Alliance ISO 9001:2008

20
FSSC 22000 ISO 22000:2005

©
Global Red Meat Standard HACCP-based food safety system

SQF Code
IFS Food Standard
BRC Global Standard for
Food Safety
Primus GFS Standard
Storage and distribution SQF Code ISO 9001:2008
services BRC Global Standard for ISO 22000:2005
Storage and distribution
IFS Logistics
hygiene practice.* Upon implementation of PRPs, companies need to per-

form 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 indus-
try is performed within the HACCP system. Beside these two, food safety
standards have an additional group of requirements, known as food safety
y
management.
nl
O
se
16.4.2.1 Prerequisite Programs and Good Practices
lU
PRPs present the basic elements and foundation of any risk-based food
na
safety system. These programs are basic conditions and activities that are
o
rs
necessary to maintain a hygienic environment throughout the food chain
Pe
suitable for the production, handling, and provision of safe end products
r
and safe food for human consumption (ISO 2005). PRPs cover aspects of
fo
incoming materials control, product identification and traceability, training
y
op
of personnel and food safety awareness, and water and energy supply, while
C
good practices are grouped as personal hygiene, pest control, cleaning and
’s
sanitation, warehouse and distribution, and layout and premise structure

or
(CAC 2003; Celaya et al. 2007; ISO 2005). Table 16.3 gives a short description
ut
of the main requirements outlined in PRPs (ISO 2005; CAC 2003; BRC 2015;
b
tri
IFS 2014a). It is of note that these requirements have also been integrated in
on
food legislative acts worldwide.

.C
C
LL
16.4.2.2 Hazard Analysis

is
HACCP is a food safety system that has become a preferred method to

c
an
ensure the production of safe and healthy food. HACCP is the foundation
Fr
of most food safety standards intended for food processing companies. This
approach is based on a detailed assessment and examination of every step in
d
an
the production process for each food product. The major goal of the HACCP
or
system is to identify the place and time in which food hazards could occur,
yl
and also to design effective controls for each identified hazard.

Ta
It consists of five main steps and seven HACCP principles (WHO 2009). For
18
implementing HACCP, it is necessary to assemble a HACCP team, describe

20
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) estab-
lishment 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).
TABLE 16.3
Main PRP Requirements
PRP Requirements
Layout and All equipment and measuring devices are suitable for the food industry
premises Maintenance of equipment and infrastructure is in place
Internal structures and fittings (walls, floors, doors, ceilings, windows,
y
nl
and working surfaces) are built of durable materials and easy to clean
O
Internal design and layout of equipment avoid cross-contamination
se
Lighting fixtures are protected to ensure that food is not contaminated
by breakages
lU
Incoming control Quantity and visual control of raw and packaging materials
na
Control of documentation (approvals and/or certificates of conformity)
o
In-house or external testing of sampled raw/packaging materials
rs
Product Traceability backwards (trace all information related to production and
Pe
identification suppliers)
r
Traceability forwards (to whom and where final products are sold in
fo
case of recall/withdrawal)
y
Personal hygiene op
Instructions regarding the behavior of workers and visitors
C
Workers should wear suitable protective clothing, head coverings,
footwear, gloves, and other, whatever is necessary
’s
or
Workers should refrain from coughing, sneezing, or any other activity

ut
that could contaminate food

b
Jewelry should not be worn

tri
Ongoing training and increasing of food safety knowledge and

on
awareness should be performed on a regular basis

.C
Water supply Potable water is used for cleaning and sanitation, for workers’ hygiene,
C
or as a food ingredient
LL
There is a water sampling plan from all dispensing places

All water testing is performed in external/accredited laboratories
is
Pest control Contract with an external pest control organization

c
an
Layout of baits, e.g., the positions of the baits for rodents

Fr
Insect killers or air curtains that prevent access of flying insects are
present
d
an
Routine checking/observing for the potential presence of any pests

Cleaning and Cleaning and sanitation program for all premises
or
sanitation Cleaning and sanitation program for equipment and cleaning equipment
yl
Routine process hygiene testing (food contact surfaces and hands of

Ta
workers)
18
Use of adequate and effective chemicals to loosen soil and bacterial film
20
Storage 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
Transportation Checking hygiene of the transportation vehicle
Pest control of transportation vehicles
Control of work conditions within the vehicle and/or maintaining the
cold chain
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
y
step in the food production process. Basically, there are three main types of
nl
hazards: (1) microbiological hazards, such as pathogens, viruses, yeasts and
O
molds, and parasites; (1) chemical hazards, for example, pesticides, myco-
se
toxins, growth hormones, antibiotics, food additives, and heavy metals; and
lU
(3) physical hazards comprising metal parts, stones, soil, wood, and any
na
other foreign particles. In conducting the hazard analysis, one has to take
o
rs
into account the likelihood of a hazard’s occurrence and the severity of its
Pe
adverse health effects, the qualitative and/or quantitative evaluation of the
r
presence of hazards, the possibility of survival or multiplication of impor-
fo
tant pathogens, and the possible production or persistence in foods of toxins,
y
op
chemicals, or physical agents. As a result of hazard analysis, the company
C
should determine those hazards that have or might have significant impacts
’s
on food safety. This can be done by using a decision tree and defining CCPs
or
(CAC 2003), or by using matrix that combines hazard occurrence and sever-
ut
ity of human health (Soman and Raman 2016). The identified hazards have
b
tri
to be included in the HACCP plan.

on
In the food processing company, hazard analysis is performed according

.C
to a flow diagram that covers all steps in the operation for a specific food
C
product. It is necessary to construct as many flow diagrams and perform

LL
hazard analyses as needed, depending on the number of products or groups

is
of products.
c
an
It is of note that hazard analysis is also required within the food safety
Fr
standards applied in primary production, such as GlobalG.A.P. In this case,

hazard analysis is more focused on potential sources of contamination from
d
an
site location and site history, hygiene, waste pollution, pests, disease and
or
weed carryover, food defense or food fraud, and water supply (GlobalG.A.P.
yl
2016). GlobalG.A.P. also recommends a five-step risk assessment: (1) identify-

Ta
ing hazards, (2) deciding who or what might be harmed and how, (3) evaluat-
18
ing the risks and deciding on precautions, (4) recording the work plan and
20
findings and implementing it, and (5) reviewing the assessment and updat-
ing 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 manage-
ment 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;
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 objec-
tives. These indicators or objectives should follow the SMART principle,
meaning they should be Specific, Measurable, Achievable, Relevant, and
y
Time-bound. As part of the verification process of the food safety man-
nl
agement system, companies should also have an internal audit in place.
O
Very useful management improvement tools are corrective and preventive
se
actions. A corrective action is launched when a problem has occurred and
lU
symptoms of the problem provide some data, which can be used in solving
na
it, while preventive action is to eliminate the source of the problem before
o
rs
it appears (Myszewski 2013). Companies with implemented and certified
Pe
food safety (management) systems often have problems identifying non-
r
conformances and initiating appropriate corrective actions (Djekic et al.
fo
2011). Even if companies have a developed procedure for corrective and pre-
y
op
ventive actions, root cause analysis and making a clear distinction between
C
corrections and corrective actions are still problems (Djekic et al. 2016). The
’s
management reviews provide an opportunity to assess the food safety per-

or
formance of the organization in meeting the objectives, its food safety pol-
ut
icy, and the overall effectiveness. As a part of the review process, analysis of
b
tri
all verification activities (ongoing and periodic) should be included. These

on
activities include review of all testing and inspecting records of products

.C
and processes, consumer complaints, external audits and inspections, and

C
emergency situations (ISO 2005, 2014). Innovations in standards develop-

LL
ment can be overseen in adding new requirements, such as food defense

is
(BRC 2015; IFS 2014a; Kleboth et al. 2016), or new ideas, such as a food safety
c
an
culture recognized by the BRC.

Fr
d
16.4.4 Effects of Implemented Food Safety Standards

an
or
Several researchers have highlighted the benefits and difficulties of imple-

yl
mented food safety management standards (Djekic et al. 2016). The main ben-
Ta
efit from the implemented food safety management standards is an increase

18
in the safety of food products (Tomasevic et al. 2013; Chen et al. 2015; Mensah
20
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
2003; Herath and Henson 2010; Papademas and Bintsis 2010). In-house capac-
ity remains another constraint, since most companies have confirmed inter-
nal 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 recog-
y
nized as very influential (Karaman et al. 2012; Tomasevic et al. 2013; Macheka
nl
et al. 2013; Mensah and Julien 2011).
O
Food companies are faced with the challenge that as the complexity of food
se
safety and quality requirements increases, their organizational knowledge
lU
decreases and the time for fulfilling the requirements shortens (Djekic et al.
na
2013). Analysis of food safety audit findings shows that the main problems
o
rs
are related to PRPs and control of food safety risks in terms of inadequate
Pe
validations of the control measures (Djekic et al. 2011, 2016). There is a confu-
r
sion between PRPs and the HACCP plan, their relations, how they should
fo
be managed, and understanding which barrier should be handled first due
y
op
to different understandings between industry personnel, external consul-
C
tants, and legal authorities (Ramí rez Vela and Martí n Ferná ndez 2003). It is
’s
expected that all food safety systems have some type of validation, especially
or
after launching a guideline for validation of control measures (CAC 2008).

ut
b
tri
on
16.4.5 Food Safety Audits

.C
All types of food safety assessments are activities used to verify that a food
C
producer is following specific guidelines, requirements, or rules (Powell

LL
et al. 2013). An audit is defined as “a systematic, independent and docu-

is
mented process for obtaining audit evidence and evaluating it objectively

c
an
to determine the extent to which the audit criteria are fulfilled” (ISO 2011).
Fr
Audits provide a snapshot limited by audit frequency, auditor competence,

and audit scope (Powell et al. 2013).
d
an
Assessment criteria are requirements used as a reference against which

or
evidence is compared, and these criteria are mainly standards, legal require-
yl
ments (where they exist), or their combination. Audits may provide audited
Ta
organizations with a unique opportunity to receive advice, new ideas, and

18
help (Djekic et al. 2011). Audit findings, positive and negative, and statements
20
about the effectiveness of the food safety system can indicate either confor-
mity or nonconformity with audit criteria or opportunities for improvement
©
(ISO 2015c). To ensure adherence to recognized regulations and good manu-

facturing 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
(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 pri-
vate standards for food safety, which may be more stringent than those
required by legislation.
y
nl
16.4.5.1 Types of Food Safety Audits
O
Depending on the role of audit participants, there are three types of audits
se
(Table 16.4) (ISO 2011). First-party audits are conducted by the organization
lU
itself, meaning it plans and performs audits using its own resources (trained
na
employees). Internal auditing programs within the food industry are limited,
o
rs
keeping the efforts to a minimum. In the food industry, internal audits of
Pe
HACCP or similar risk-based assessment programs are not expected beyond
r
the minimum yearly verification (Hepner et al. 2004). If the food safety sys-
fo
tem is integrated with quality management, the frequency may rise to twice
y
a year (Djekic et al. 2014). op
C
Second-party audits are performed when the audit client is the customer or
’s
other organization with a specified interest in verifying the effectiveness of

or
a system at the premises of suppliers (auditee). Auditors are either employ-

ut
ers of the customers or outsourced to specialized organizations to perform

b
tri
audits on behalf of the customer. Some authors believe that second-party

on
audits are stricter and may identify problems that third-party audits do not
.C
(Powell et al. 2013; Djekic et al. 2016). In order to be confident in the safety
C
of food, big food companies also qualify their supplier using second-party
LL
auditors (Losito et al. 2011). From a long-term perspective, second-party

is
audits are considered to be an effective tool in directing suppliers toward

c
an
improvements (Djekic et al. 2016).

Fr
Third-party audits are also known as certification audits performed by cer-

tification bodies. Certification bodies are local and/or global organizations
d
an
TABLE 16.4
or
yl
Type of Audit and Audit Participants

Ta
Type of Audit
18
Audit Participants First Party Second Party Third Party

20
Audit client Organization (auditee) Customer Organization (auditee)

©
Auditee Supplier Organization (auditee)

Audit team (leader Working at or Working at or
and members) subcontracted by subcontracted by
customer certification body
Audit organization Customer Certification body
(outsource
company)
that provide a variety of auditing services against a large number of stan-

dards. 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 con-
trol the certification process, it is expected that certification bodies are able
to demonstrate that they have evaluated risks arising from their certification
y
activities and have adequate (financial) arrangements to cover liabilities aris-
nl
ing from certification activities and/or the geographic areas in which they
O
operate (ISO 2015c).
se
As a business opportunity, certification bodies also started providing
lU
HACCP certification under various food safety schemes (Djekic et al.
na
2013). These schemes are either unaccredited with self-made guidelines for
o
rs
auditing HACCP-based food safety systems or in line with accreditation
Pe
protocols issued by accreditation bodies, such as the Dutch Accreditation
r
Council (RvA 2014). The main reason for using unaccredited schemes is
fo
the fact that HACCP is not a food safety management tool. It is consid-
y
op
ered a food safety control tool applicable only to food processing and does
C
not include any supporting processes, such as maintenance, purchasing,
’s
sales, and distribution. The basic requirement for certification bodies to

or
gain accreditation is to have an assessment methodology for certification

ut
of management systems (ISO 2015c). As a result, certification bodies pro-

b
tri
vide accredited services for ISO 22000, BRC, and IFS and other food safety
on
management system standards. Food chain stakeholders— producers, cus-

.C
tomers, and consumers— consider a certificate as proof of an implemented

C
and effective food safety management system. However, some serious

LL
concerns have been raised over third-party audits. First, some critics
is
believe that certification is a paper-driven process of limited value for the

c
an
company performance and is used as a marketing tool (Djekic et al. 2011;

Fr
Tanner 2000). Also, there is no critical evaluation of potential correlation

between (third-party) audit outcomes and foodborne illness outbreaks
d
an
(Powell et al. 2013). The outbreak of Salmonella typhimurium linked to the

or
Peanut Corporation of America is cited as an example of a failure in the

yl
third-party auditing system. It resulted in a recall of 3900 peanut butter

Ta
and other peanut-containing products from more than 350 companies. As

18
a result of the foodborne outbreak, 691 people were sickened and 9 died in
20
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 man-
agement standards with the ultimate introduction of a risk approach con-
firms the necessity of new audit principles (ISO 2009, 2015a).
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
y
drift is followed with the greater responsibility of food business operators,
nl
who should handle food through the development and implementation of
O
hazard analysis. Food safety risk and science-based food safety regulation
se
are a great foundation for the assurance of an adequate level of food safety.
lU
They are of great value for food producers to be in line with all requirements.
na
Local inspectors should also shift from checking to advising the application
o
rs
of new and difficult requirements. Also, it is not new that local producers
Pe
are involved in national surveillance programs to help improve food safety
practices. Nevertheless, there is great discrepancy in adopting adequate food
r
fo
safety legislative acts, mainly between developed and developing countries.
y
op
Along with the legal requirements, voluntarily food safety standards play
C
a very important role in ensuring food safety in the global food market.
’s
This is especially seen through the demands of customers, especially major

or
retail chains and big manufacturers. These organizations often require their
ut
suppliers to comply with their food safety standards, which may be more
b
tri
stringent than those required by legislation, especially in developing and

on
undeveloped countries.
.C
Although the approaches in food safety of both regulations and stan-

C
dards may differ in some aspects, they present two sides of the same coin.
LL
Regulation helps in defining certain limits and methods of how to evaluate or

is
test certain food processing or food parameters. Assessments are performed

c
an
by inspection services that are mostly unannounced. Standards give frame-

Fr
works for managing food safety issues. These assessments are performed by
trained auditors and are mostly announced and planned.
d
an
or
yl
Ta
18
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17
Food Safety Reforms in the United States:
The Food Safety Modernization Act (FSMA)
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Harmit Singh and Holly M. Greene
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CONTENTS
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17.1 Introduction: The History of Food Safety Regulations in the
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United States................................................................................................ 563
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17.2 Events Leading to the Food Safety Modernization Act........................ 568
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17.3 Title I: Improving the Capacity to Prevent Food Safety Problems ....... 569
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17.3.1 Produce Safety................................................................................. 574
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17.3.2 Food Safety Reforms and Regulations for Functional

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Foods and Dietary Supplements.................................................. 576

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17.3.3 Risk of Nanomaterials in Foods................................................... 577

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17.3.4 Food Industry Training................................................................. 577

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17.4 Title II: Improving Capacity to Detect and Respond to Food

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Safety Problems........................................................................................... 581

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17.5 Title III: Improving the Safety of Imported Food.................................. 583

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17.5.1 Collaboration with Various Government Agencies................... 589

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17.6 Title IV: Miscellaneous Provisions........................................................... 591

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Disclaimer............................................................................................................. 592
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References.............................................................................................................. 592
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17.1 Introduction: The History of Food Safety

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Regulations in the United States

18
20
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
©
97 20 01 06 907 14 16 23 27 30
18 19 19 1 19 19 19 19 19
564
18
1891 1900 Ta 1910 1920
Tea Importation Act Division of Chemistry Federal Meat Filled Milk Act FDIA becomes
becomes Bureau of Inspection Act FDA after an
yl
1862 Chemistry (precursor Federal Trade agriculture
Congress creates Food,
or
USDA est. to FDA) Commission Act appropriations
an Drug, and Insecticide
Federal Food and Drugs U.S. Grain Administration (FDIA) act
Division of Chemistry d
(precursor to FDA) est. Act of 1906 (Pure Food Standards Act within USDA
and Drug Act)
Fr
an6 7 Federal Import Milk Act
38 40 4 4 53 57 958 67 70
19 19 19 c19 19 19 1 19 19
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1931 LL 1950 1960
FDA transferred from C
Federal Fungicide, Poultry Products Fair Packaging and Labeling Act
USDA to the Federal Insecticide, and Inspection Act
Wholesome Meat Act (amended
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Security Agency Rodenticide Act
Federal Meat Inspection Act)
(FSA had been (FIFRA) FDA transferred to Dept.
on
created in 1939) of Health, Education, and
tri Food Additives Egg Products Inspection Act
Federal Food, Drug, Agricultural Welfare (HEW) pursuant to b Amendment Environmental Protection Agency
and Cosmetic Act Marketing Act Reorganization Plan 1 of 1953 ut established (took over FIFRA)
o4 r
71 76 79 980 981 90 9 ’s 96 97 02 07 11
19 19 19 1 1 19 19 19 19 20 20 20
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Toxic Substances Food Safety and Inspection Dietary Supplement FDA Modernization FDA Amendmentsy
Control Act Service (FSIS) est. within Health and Education Act of 1997 Act of 2007 fo
Animal and Plant USDA in current form Act of 1994 (DSHEA) Public Health Security FDA Food Safety r
Health Inspection Infant Formula and Bioterrorism Modernization Act Pe
Service est. (APHIS) Act of 1980 Nutrition Labeling and Food Quality Protection Preparedness and
Dept. of Education Organization Act Education Act of 1990 (NLEA) Act of 1996 Response Act of 2002
rs
signed into law, HEW becomes Dept. Sanitary Food Federal Tea Tasters
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of Health and Human Services (HHS)
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Transportation Act Repeal Act of 1996 lU
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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,
O
Congressional Research Service, Washington, DC, 2012.)
nl
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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 con-
sumers. Prior to 1906, federal regulation of the food and drug industry in
y
the United States was fragmented, and several measures took place between
nl
1820 and 1902 to establish the passing of the 1906 regulation.
O
The path of the 1906 regulation traces back to 1820, with the establishment
se
of the U.S. Pharmacopeia, an authoritative book that catalogs all legally recog-
lU
nized standards for drug substances and dosage forms. Yet, the introduction of
na
the U.S. Pharmacopeia did not alleviate the influx of substandard drugs from
o
rs
Europe. European drug manufacturers were faced with strict government reg-
Pe
ulations and used the United States, having an absence of drug laws, as a depot
r
for their adulterated products. United States medical professions were unable to
fo
prescribe these European drugs with any assurance due to the drugs’ potency
y
op
instability. In 1848, the U.S. Congress began drug regulation by enacting the
C
Import Drugs Act, which prohibited the importation of any drug that did not
’s
meet U.S. Pharmacopeia standards. The Import Drug Act was a comprehensive
or
and ambitious attempt by Congress to solve the problem of imported adulter-

ut
ated drugs (Heath 2004). In the beginning, the statue was strictly enforced, but
b
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it lacked public support and the government funding faded.

on
In the mid-1870s, margarine was introduced to the United States and

.C
immediately became controversial as an important food adulteration issue.

C
Margarine, then, was made from cattle fatty residue and very often sold as
LL
butter, yet the cost to produce it was about half that of butter. Government
is
imposed measures on margarine, to include stamps and proper labeling. By

c
an
1886, 27 states had some margarine legislation: 20 regulated labeling and

Fr
packaging and 7 prohibited its manufacture and sale. Yet, the lack of provi-
sions or resources for enforcement of these state-established regulations put
d
an
pressure on the federal government to become involved. In 1886, Congress

or
passed the Oleomargarine Bill, which levied a manufacturing tax of 2 cents

yl
per pound of margarine and annual licenses fees for manufacturers ($600),
Ta
wholesalers ($480), and retailers ($48) of margarine. These imposed fees only
18
intensified the selling of margarine as butter to avoid paying for the annual
20
licenses. A shift in state legislatures turned toward regulating the color of

margarine, and by the early 1900s, 32 states prohibited the sale of yellow-
©
colored margarine (Dupré 1999). However, with the discovery of hydroge-

nation 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
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
y
credence to foreign competitors’ accusations that American livestock and
nl
meat products were sickening (Libecap 1992). At the same time, claims of
O
trichinosis in American pork brought restrictions on imports in Germany,
se
France, Belgium, and other European countries. The control of Europe pro-
lU
hibiting the importing of American livestock intensified the pretense of the
na
disease issue, and cattle producers lobbied the government to enact federal
o
rs
inspection legislation to aid in rising cattle prices. The 1891 Meat Inspection
Pe
Act mandated the inspection of all live cattle for export, as well as all live
r
cattle that were to be slaughtered and their meat exported. The law also
fo
authorized the inspection of swine and sheep prior to slaughter and inter-
y
op
state shipment. With the passing of this act, for the first time, the federal
C
government was authorized to certify food quality for American consumers.
’s
In 1897, the Tea Importation Act was passed, which prohibited the importa-
or
tion of tea into the United States that failed to meet government standards
ut
for quality, purity, and fitness for consumption. Adulteration of tea was rou-
b
tri
tine in England, and also with imported tea into the United States. Leaves
on
of plants other than tea leaves would be mixed into the batch and sold as
.C
pure tea. In addition, used tea leaves would be sold as new, and consumers
C
purchased certain colored teas that would disguise the inferior quality and
LL
the presence of foreign leaves. Sellers of tea leaves would employ methods
is
that increased the weight of the tea leaves, and thus its price. Most of the
c
an
substances used were relatively safe for human consumption, but some were
Fr
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
d
an
secretary of Health and Human Services by the Board of Tea Experts. Tea
or
was the only food or beverage that was sampled upon entry for a compari-
yl
son with a standard. The Tea Act was repealed by Congress in 1996 (DeWitt
Ta
2000).
18
The Biologics Control Act of 1902 was enacted by Congress to ensure the
20
protection of Americans by providing consistently safe biological products.

In 1901, there were no mandatory federal manufacturing or product stan-
©
dards for biologics. The death of 13 children in St. Louis in 1901 as a result of
receiving tetanus-contaminated diphtheria antitoxin, and other similar inci-
dents, prompted quick action by lawmakers. In 1902, Congress enacted the
Biologics Control Act, also known as the virus-toxin law, which gave the gov-
ernment its first control over the processes used for the production of biolog-
ical 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
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 prod-
uct 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
y
public health (FDA 2016a).
nl
The 1906 PFDA was the first federal law to simultaneously address food,
O
beverages, and drug adulteration, production, distribution, and marketing
se
for import and export (PFDA 1906). Passing of the PFDA replaced all estab-
lU
lished state standards and developed a collaboration between federal and
na
state authority. On December 5, 1905, President Theodore Roosevelt recom-
o
rs
mend to the 59th Congress that a law be enacted to regulate interstate com-
Pe
merce in misbranded and adulterated foods, drinks, and drugs. Such a law
r
would protect legitimate manufacture and commerce, and would tend to
fo
secure the health and welfare of the consuming public. Traffic in foodstuffs
y
op
that had been debased or adulterated so as to injure health or deceive pur-
C
chasers would be forbidden (Roosevelt 1905). The bill was passed in Senate
’s
on February 21, 1906, and the house passed a substitute bill 4 months later;
or
Congress produced a compromise bill in only 6 days, and after the signature
ut
of President Roosevelt on June 30, 1906, the act went into effect on January
b
tri
1, 1907.
on
The Meat Inspection Act of 1906 was the beginning of the U.S. federal regu-
.C
lation of the country’ s meat, poultry, and egg supply. In 1906, Upton Sinclair’ s
C
novel The Jungle was published; it portrayed the harsh working conditions
LL
of immigrants in the United States in industrialized cities. However, the

is
American public was more concerned with Sinclair’ s portrayal of the nau-
c
an
seating details regarding the unhealthy practices of Chicago’ s meatpacking

Fr
district. President Roosevelt deployed Labor Commissioner Charles P. Neill

to Chicago to investigate the meatpacking industry, which Neill reported to
d
an
Roosevelt as being “revolting” and even worse than the conditions depicted
or
in Sinclair’ s novel. The act of 1906 strengthened requirements for sanitary

yl
conditions and established standards for inspecting all meat processing

Ta
plants that conducted business across state lines (Barkan 1985).

18
The structure of the 1906 PFDA was overhauled in 1938 by the Federal
20
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 gly-
col 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
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
y
before marketing; it also enlarged the authority of the FDA to ensure food
nl
safety. Under the FDCA, the FDA was authorized to inspect factories, create
O
identity and quality standards, and establish safety tolerances for unavoid-
se
able poisons (Burkett 2012).
lU
na
o
rs
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r
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17.2 Events Leading to the Food Safety Modernization Act
y
op
The original FSMA of 2009 was introduced initially as a bill in Congress
C
by Rosa DeLauro, a democrat from Connecticut’ s third congressional dis-
’s
trict, in 2007. This bill died and was reintroduced in 2009 to establish the
or
Food Safety Administration within the Department of Health and Human

ut
Services to protect the public health by preventing foodborne illness,

b
tri
ensuring the safety of food, improving research on contaminants leading

on
to foodborne illness, and improving the security of food from intentional

.C
contamination, and other purposes. Further amendments were introduced

C
(by Senators Richard Durbin and Judd Gregg) to the Committee on Health,
LL
Education, Labor, and Pensions. Senator Durbin referred to food safety con-
is
cerns and how the almost 70-year-old food safety acts needed to be revised.
c
an
He referred specifically to the 2008 incidents of outbreaks in which 1500

Fr
people fell sick (21% were hospitalized and 2 died), from 43 different states,
the District of Columbia, and Canada, because of Salmonella enterica serotype
d
an
Saintpaul. The outbreak led to a governmental investigation that pointed the

or
finger first at tomatoes and then jalapeno peppers in Texas, before settling on
yl
serrano peppers in Mexico. In the meantime, more people got sick and the
Ta
tomato industry lost up to hundreds of millions of dollars. In 2008, peanut

18
butter was tainted with Salmonella , the second case of its kind in 2 years, in
20
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, mak-
ing it one of the biggest food recalls in U.S. history. The U.S. FDA also rec-
ognized 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 pre-
ventable. 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
Attribution of Foodborne Illness and Deaths, 1998–2008
Illnesses
Fish and shellfish 6.10% 6.40%
Deaths
Dairy and eggs 20.00% 15.00%
y
Meat and poulty 22.00% 29.00%
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Produce
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46.00% 23.00%
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0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
o na
FIGURE 17.2
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Contribution of different food categories to estimated domestically acquired illnesses and
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deaths, 1998– 2008. Chart does not show 5% of illnesses and 2% of deaths attributed to other
r
commodities. In addition, 1% of illnesses and 25% of deaths were not attributed to commodi-
fo
ties; these were caused by pathogens not in the outbreak database, mainly Toxoplasma and
y
Vibrio vulnificus . (From Painter, J. A., Emerg Infect Dis , 19 (3), 407– 415, 2013.)
op
C
’s
preventing food safety problems, rather than relying primarily on reacting

or
to problems after they occur.

ut
The FSMA is divided into four titles (Table 17.1): Title I is related to the
b
tri
guidelines to improve the facilities to prevent food safety problems and

on
is further divided into 16 subsections. Title II is related to improving the

.C
capacity to detect and respond to food safety problems. Title III contains
C
guidelines related to the safety of imported foods. Title IV is related to other

LL
miscellaneous provisions.
cis
an
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d
an
17.3 Title I: Improving the Capacity to

or
Prevent Food Safety Problems

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Ta
For the first time, the FDA had a legislative mandate to require comprehensive,
18
prevention-based controls across the food supply to prevent or significantly

20
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 partner-

ship. Table 17.2 indicates how the FDA is trying to implement these areas.
The FSMA preventive controls for human food rule is final, and com-
pliance 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
©
20 570
18
TABLE 17.1 Ta
Comparison of FSM Bill Submitted and the Final FSMA Approved
yl
BILL PROPOSED ACT S.510 PASSED
or
an TITLE I— IMPROVING CAPACITY TO PREVENT FOOD SAFETY
Title I— ESTABLISHMENT OF THE FOOD SAFETY ADMINISTRATIONd PROBLEMS
Sec. 101. Establishment of the food safety administration.
Fr Sec. 101. Inspections of records.
Sec. 102. Consolidation of food safety functions. an Sec. 102. Registration of food facilities.
Sec. 103. Additional duties of the administration. c Sec. 103. Hazard analysis and risk-based preventive controls.
Title II— ADMINISTRATION OF FOOD SAFETY PROGRAM is Sec. 104. Performance standards.
Sec. 201. Administration of national program. LL Sec. 105. Standards for produce safety.
Sec. 202. Registration of food establishments and foreign food establishments.
C Sec. 106. Protection against intentional adulteration.
Sec. 203. Preventive process controls to reduce adulteration of food. .C Sec. 107. Authority to collect fees.
Sec. 204. Performance standards for contaminants in food. Sec. 108. National agriculture and food defense strategy.
Sec. 205. Inspections of food establishments. Sec. 109. Food and Agriculture Coordinating Councils.
on
Sec. 206. Food production facilities. Sec. 110. Building domestic capacity.
tri
Sec. 207. Federal and state cooperation. b
Sec. 111. Sanitary transportation of food.
Sec. 208. Imports. Sec. 112. Food allergy and anaphylaxis management.
ut
Sec. 209. Resource plan. Sec. 113. New dietary ingredients.
or
Sec. 210. Traceback requirements.
’s
Sec. 114. Requirement for guidance relating to post harvest
Sec. 211. Accredited laboratories.
C
processing of raw oysters.
Title III— RESEARCH AND EDUCATION Sec. 115. Port shopping.
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Sec. 301. Public health assessment system.
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Sec. 116. Alcohol-related facilities.
Sec. 302. Public education and advisory system.
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TITLE II— IMPROVING CAPACITY TO DETECT AND RESPOND
Sec. 303. Research. TO FOOD SAFETY PROBLEMS
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Sec. 304. Working group on improving foodborne illness surveillance. Sec. 201. Targeting of inspection resources for domestic facilities,
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Sec. 305. Career-spanning training for food inspectors. foreign facilities, and ports of entry; annual report.
rs
Sec. 306. Food-Borne Illness Health Registry.
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Sec. 202. Laboratory accreditation for analyses of foods.
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Sec. 307. Study on federal resources. Sec. 203. Integrated consortium of laboratory networks.
lU
se (Continued )
O
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y
©
20
18
TABLE 17.1 (CONTINUED) Ta
Comparison of FSM Bill Submitted and the Final FSMA Approved
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or
Title IV— ENFORCEMENT Sec. 204. Enhancing tracking and tracing of food and recordkeeping.
Sec. 401. Prohibited acts. Sec. 205. Surveillance.
an
d
Sec. 402. Food detention, seizure, and condemnation. Sec. 206. Mandatory recall authority.
Sec. 403. Notification and recall. Sec. 207. Administrative detention of food.
Fr
Sec. 404. Injunction proceedings. Sec. 208. Decontamination and disposal standards and plans.
an
Sec. 405. Civil and criminal penalties. cis Sec. 209. Improving the training of state, local, territorial, and tribal
Sec. 406. Presumption. food safety officials.
Sec. 407. Whistleblower protection. Sec. 210. Enhancing food safety.
LL
Sec. 408. Administration and enforcement.
C Sec. 211. Improving the reportable food registry.
Sec. 409. Citizen civil actions. TITLE III— IMPROVING THE SAFETY OF IMPORTED FOOD
.C
Title V— IMPLEMENTATION on Sec. 301. Foreign supplier verification program.
Sec. 501. Reorganization plan. Sec. 302. Voluntary qualified importer program.
Sec. 502. Transitional authorities. Sec. 303. Authority to require import certifications for food.
tri
b
The Food Safety Modernization Act (FSMA)
Sec. 503. Savings provisions. Sec. 304. Prior notice of imported food shipments.
ut
Sec. 504. Conforming amendments. Sec. 305. Building capacity of foreign governments with respect to
or
Sec. 505. Additional technical and conforming amendments. food safety.
’s
Sec. 506. Regulations. Sec. 306. Inspection of foreign food facilities.
C
Sec. 507. Authorization of appropriations. Sec. 307. Accreditation of third-party auditors.
op
Sec. 508. Limitation on authorization of appropriations. Sec. 308. Foreign offices of the Food and Drug Administration.
y
Sec. 309. Smuggled food.
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TITLE IV— MISCELLANEOUS PROVISIONS
Sec. 401. Funding for food safety.
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Sec. 402. Employee protections. rs
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Sec. 403. Jurisdiction; authorities.
Sec. 404. Compliance with international agreements.
na
Sec. 405. Determination of budgetary effects.
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se
571
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nl
y
TABLE 17.2
Major Elements of the FSMA and Their Implementation Guidelines
The FSMA Elements Can Be Divided into How the FDA Will Plan to Implement
Five Key Areas FSMA Elements
Preventive controls : For the first time, the FDA Preventive controls for human food : Requires
y
has a legislative mandate to require that food facilities have safety plans that
nl
comprehensive, prevention-based controls set forth how they will identify and
O
across the food supply to prevent or minimize hazards.
se
significantly minimize the likelihood of Preventive controls for animal food :
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problems occurring. Establishes CGMPs and preventive
controls for food for animals.
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Produce safety : Establishes science-based
o
standards for growing, harvesting,
rs
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packing, and holding produce on
domestic and foreign farms.
r
fo
Imported food safety : The FDA has new tools to Foreign Supplier Verification Program :
y
ensure that imported foods meet U.S. Importers will be required to verify that
op
standards and are safe for consumers. For food imported into the United States has
C
example, for the first time, importers must been produced in a manner that provides
’s
verify that their foreign suppliers have the same level of public health protection
or
adequate preventive controls in place to as that required of U.S. food producers.

ut
ensure safety, and the FDA will be able to

b
accredit qualified third-party auditors to

tri
certify that foreign food facilities are

on
complying with U.S. food safety standards.

.C
Inspection and compliance : The legislation Third-party certification : Establishes a

C
recognizes that inspection is an important program for the accreditation of third-

LL
means of holding industry accountable for its party auditors to conduct food safety
is
responsibility to produce safe food. The FDA audits and issue certifications of foreign
c
is committed to applying its inspection facilities producing food for humans or

an
resources in a risk-based manner and adopting animals.

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innovative inspection approaches. Sanitary transportation : Requires those who

d
transport food to use sanitary practices to

an
ensure the safety of food.

or
Intentional adulteration : Requires domestic

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and foreign facilities to address vulnerable

Ta
processes in their operations to prevent

acts intended to cause large-scale public
18
harm.
20
Response : For the first time, the FDA has The FDA expects that it will only need to
mandatory recall authority for all food invoke this authority infrequently since
©
products. 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.
(Continued )

Major Elements of the FSMA and Their Implementation Guidelines
The FSMA Elements Can Be Divided into How the FDA Will Plan to Implement
Five Key Areas FSMA Elements
Enhanced partnerships among all food safety The legislation recognizes the importance
agencies— U.S. federal, state, local, territorial, of strengthening existing collaboration
y
tribal, and foreign. among all food safety agencies— U.S.
nl
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federal, state, local, territorial, tribal, and
foreign— to achieve public health goals.
se
For example, it directs the FDA to
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improve the training of state, local,
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territorial, and tribal food safety officials.
o
rs
applicable U.S. safety standards. A program has been established for the
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accreditation of third-party auditors as a part of their commitment to provide
r
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resources for inspection and compliance. In addition, the FDA has authority
y
to recall all food products; although the FDA expects that the industry will
op
conduct voluntary recalls, it has been given more teeth in this act to detain
C
the product, as well as suspend the facility registration.
’s
For the first time, the FDA has a legislative mandate to require comprehen-
or
ut
sive, science-based preventive controls across the food supply, which include:
b
tri
• Mandatory preventive controls for food facilities: Food facilities

on
are required to implement a written preventive controls plan. This

.C
involves (1) evaluating the hazards that could affect food safety; (2)
C
LL
specifying what preventive steps, or controls, will be put in place to

significantly minimize or prevent the hazards; (3) specifying how the
c is
facility will monitor these controls to ensure that they are working;
an
(4) maintaining routine records of the monitoring; and (5) specifying

Fr
what actions the facility will take to correct problems that arise.
d
an
• Mandatory produce safety standards: The FDA must establish sci-

ence-based, minimum standards for the safe production and har-
or
vesting of fruits and vegetables. Those standards must consider

yl
Ta
naturally occurring hazards, as well as those that may be introduced

either unintentionally or intentionally, and must address soil amend-
18
ments (materials added to the soil, such as compost), hygiene, pack-

20
aging, 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
controls using monitoring, corrective actions, and verification are common

rules in both animal and human food safety measures. The FDA has final-
ized 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
y
not need to implement additional preventive controls or CGMP regulations
nl
when supplying a by-product (e.g., wet spent grains, fruit or vegetable peels,
O
and liquid whey) for animal food, except to prevent physical and chemical
se
contamination when holding and distributing the by-product. Examples
lU
of physical and chemical contamination include placing trash or cleaning
na
chemicals into the container holding the by-products. The FDA has also
o
rs
identified five ways in which consumers and their pets will be safe: (1) food
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companies will apply greater controls to help prevent hazards, (2) consumers
r
and their pets will be protected from tainted animal food, (3) eating health-
fo
fully and safely will go hand in hand, (4) there will be greater oversight of
y
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foods imported from other countries, and (5) consumers will be more confi-
C
dent that their food is safe.
’s
The Hazard Analysis and Critical Control Point (HACCP) is based on the
or
critical control points (CCPs) identified by the manufacturers, whereas pre-

ut
ventive control under the FSMA includes the CCPs or controls other than
b
tri
CCPs that are appropriate for food safety. The preventive rules require
on
the facilities to control the hazards and take corrective actions to prevent
.C
contamination by testing the product and environmental monitoring. The

C
FDA expects companies to more frequently test the products more prone
LL
to outbreaks of foodborne illness. Verification of preventive control is also

is
considered very important in this act. The companies should have a hazard
c
an
analysis and preventive control plan, which should be reanalyzed every 3

Fr
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
d
an
responsible for the development of the preventive control plan.

or
yl
Ta
17.3.1 Produce Safety

18
The FDA has generated several flowcharts to decide whether farms are
20
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 deter-
mined 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
Does your farm grow, harvest, pack, or hold

produce? Your farm is NOT
Sections 112.1 and 112.3(c) NO
“Produce” defined in Section 112/3(c) covered by this rule.
YES
y
Does your farm on average (in the previous
nl
three years) have $25K or less in annual Your farm is NOT
YES
O
produce sales? covered by this rule.
Section 112.4(a )
se
NO
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o na
Is your produce one of the commodities that
rs
the FDA has identified as rarely consumed raw?
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Section 112.2(a)(1)
If you grow, harvest, pack, or hold more than one produce YES This product is NOT
commodity, you must ask this question separately for each one to covered by this rule.
determine whether that particular produce commodity is covered
r
by this rule.
fo
NO
y
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C
Is your produce for personal/on-farm
’s
consumption? This produce is NOT

or
Section 112.2(a)(2)
YES
covered by this rule.
b ut
NO
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on
This produce is ELIGIBLE FOR

.C
Is your produce intended for commercial

EXEMPTION from the rule,
processing that adequately reduces
provided you make certain
C
pathogens (for example, commercial YES

statements in documents
LL
processing with a “kill step”)? accompanying the produce, obtain

Section 112.2(a)(2)
certain written assurances, and keep
is
NO certain documentation, as per

Sections 112.2(b)(2) through (b)(6).
c
an
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Your farm is eligible for a

d
Does your farm on average (in the previous qualified exemption from this rule,
an
three years) as per Section 112.5, which means that you must
Have <$500K annual food sales? comply with certain modified
YES requirements and keep
or
AND
certain documentations, as
A majority of the food (by value) sold directly
yl
per Sections 112.6 and 112.7.

to “qualified end users”?
Ta
Section 112.3(c)
18
NO
20
©
You are covered by this rule.
FIGURE 17.3
Standards for produce safety. The preventive controls for human food rule clarifies the defini-
tion of a farm to cover two types of farm operations: primary production farms and secondary
activities farms. The same definition is used in the produce safety rule (Section 112.3(c)). Shown
are the basic criteria that determine whether an operation that meets the definition of farm is
subject to the produce rule. (Adapted from http://fda.gov. U.S. Food & Drug Administration,
FDA Food Safety Modernization Act, Food/Guidance Regulations)
water. As such contamination increases, so does the likelihood that disease-

causing microorganisms are also present. For untreated water used for this
purpose, the required criteria are a geometric mean of samples of 126 CFU or
less of generic E. coli per 100 mL of water, and a statistical threshold value of
410 CFU or less of generic E. coli in 100 mL of water (CFU is colony-forming
units, a measure of bacteria) (FDA 2016b).
y
Specific directions have been provided to kill the harmful microbes, and
nl
regular testing of water quality should be maintained. If the water does not
O
meet these criteria, corrective actions are required as soon as is practicable,
se
but no later than the following year. Farmers with agricultural water that
lU
does not initially meet the microbial criteria have additional flexibility by
na
which they can meet the criteria and then use the water on their crops. These
o
rs
options include, for example, (1) allowing time for potentially dangerous
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microbes to die off on the field by using a certain time interval between
r
the last irrigation and harvest, but no more than four consecutive days; (2)
fo
allowing time for potentially dangerous microbes to die off between harvest
y
op
and the end of storage, or to be removed during commercial activities, such
C
as washing, within appropriate limits; and (3) treating the water. The FDA is
’s
also in the process of providing guidelines for soil amendments and sprouts,
or
to minimize the risk of contamination. Microbial standards that set limits on

ut
detectable amounts of bacteria (including Listeria monocytogenes , Salmonella

b
tri
spp., fecal coliforms, and E. coli O157:H7) have been established for processes
on
used to treat biological soil amendments, including manure.

.C
C
LL
17.3.2 Food Safety Reforms and Regulations for

Functional Foods and Dietary Supplements
c is
an
The FDA FSMA of 2011 (Pub. L. 111-353) authorizes the FDA to order man-
Fr
datory recalls if there is reasonable probability that an article of food is

adulterated or misbranded, is a major food allergen, or may cause serious
d
an
adverse health consequences or death in humans or animals. The FDA rec-

or
ognizes that improvements are needed in overseeing the safety of dietary

yl
supplements and “functional foods.” Congress has used its legal authority
Ta
and appropriations over the years to direct the Government Accountability

18
Office (GAO) (2000) to examine functional food issues. GAO/RCED-00-156

20
examined the extent to which the FDA, U.S. Department of Agriculture

(USDA), and Federal Trade Commission (FTC) efforts and federal laws
©
ensure the (1) safety of functional foods and dietary supplements and (2)
accuracy of health-related claims on product labels and in advertising. It
concluded that the FDA’ s inability to review safety evidence for functional
foods without a premarket safety notification was a regulatory gap, as well
as its inability to track and analyze instances of adverse effects. Also, the
GAO determined that the distinction between functional foods and dietary
supplements was often difficult for clear and consistent regulatory action. It
recommended that Congress amend the FDCA to require manufacturers of
functional foods to notify the FDA regarding the use of structure– function

claims and to use disclaimers of FDA approval on product labels containing
structure– function claims.
17.3.3 Risk of Nanomaterials in Foods
y
The FDA FSMA does not make a categorical judgment that nanotechnol-
nl
ogy is inherently safe or harmful and intends regulatory approach to be
O
adaptive and flexible and to take into consideration the specific character-
se
istics and effects of nanomaterials in the particular biological context of
lU
each product and its intended use. In June 2011, the FDA issued a draft
na
guidance for industry entitled “Considering Whether an FDA-Regulated
o
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Product Involves the Application of Nanotechnology” to present its think-
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ing on considerations related to nanotechnology (FDA 2016c). In that draft
r
guidance, the FDA indicated that the agency would issue product-specific
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guidance documents in the future, as appropriate. In April 2012, the FDA
y
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issued two product-specific draft guidances for public comment, address-
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ing the use of nanotechnology by the foods and cosmetics industries. Then
’s
in June 2014 and August 2015, the FDA issued final guidance documents
or
for the food industry and other stockholders. In June 2014, after consid-
ut
ering the public comments, the FDA finalized all three guidance docu-
b
tri
ments. Industry remains responsible for ensuring that its products meet all
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applicable legal requirements, including standards for safety— regardless

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of the emerging nature of a technology involved in the manufacturing of

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a product. The FDA encourages industry to consult with the agency early
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in the product development process to address any questions related to

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the safety, effectiveness, or other attributes of products that contain nano-

c
an
materials, or about the regulatory status of such products. The FDA has
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provided other documents for guidance for the industry: “Assessing the
Effects of Significant Manufacturing Process Changes, Including Emerging
d
an
Technologies, on the Safety and Regulatory Status of Food Ingredients

or
and Food Contact Substances, Including Food Ingredients That Are Color
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Additives” (FDA 2016d) and “Guidance for Industry: Use of Nanomaterials

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in Food for Animals” (FDA 2016e).

18
20
17.3.4 Food Industry Training

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While members of the food industry are ultimately responsible for getting
the training they need to comply with the FSMA rules, the FDA recognizes
the importance of its role in facilitating such training. For the agency, this
means joining with public and private partners in state, federal, tribal, and
international governments, industry, and academia in the development
and delivery of training. The produce safety rule and the preventive con-
trols rule both have training components, although they are not the same
for each rule. There will be ample time for farmers and food producers to
TABLE 17.3
Compliance Dates for the FSMA
Compliance Dates for Businesses Are Staggered Over Several Years after Publication of
the Final Rule
Very small businesses (averaging less than $1 January 1, 2016
million per year [adjusted for inflation] in
y
both annual sales of human food and the
nl
O
market value of human food manufactured,
processed, packed, or held without sale): 3
se
years, except for records to support its status
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as a very small business
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Businesses subject to the Pasteurized Milk 3 years (compliance dates extended to allow
o
Ordinance (PMO) time for changes to the PMO safety
rs
standards that incorporate the requirements
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of this preventive controls rule)
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Small businesses (a business with fewer than 2 years
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500 full-time equivalent employees)
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All other businesses 1 year op
Compliance Dates after Publication of the Final Rule for the Requirements of the Supply
C
Chain Program
’s
or
Receiving facility is a small business and its 2 years

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supplier will not be subject to the human

b
preventive controls rule or the produce

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safety rule
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Receiving facility is a small business and its 2 years or 6 months after the supplier is
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supplier will be subject to the human required to comply with the applicable rule,
C
preventive controls rule or the produce whichever is later

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safety rule
Receiving facility is not a small or very small 18 months
c is
business and its supplier will not be subject

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to the human preventive controls rule or the

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produce safety rule

d
Receiving facility is not a small or very small 6 months after the supplier is required to
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business and its supplier will be subject to comply with the applicable rule
the human preventive controls rule or the
or
produce safety rule

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18
come into compliance. Compliance dates for the rules (including the produce
20
safety rule as proposed) are staggered according to the size of the business
(Table 17.3).
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The most important goal that the FDA expects of any training program
is the outcome— t hat it advances knowledge among the food industry to
meet FSMA requirements. There is more than one way to get there, and
there will be a variety of training options and delivery formats, as dis-
cussed below.
The vision of FSMA training began in 2010– 2012 with the creation of
public– private alliances funded primarily by the FDA as a resource for
industry and to facilitate widespread understanding of the new standards to
support compliance. The three FDA-funded alliances (Produce Safety, Food

Safety Preventive Controls, and Sprout Safety) are developing model, stan-
dardized curricula that are intended to meet the needs of, and be used by,
the majority of those affected by the FSMA rules. The agency has entered
into a 5-year cooperative agreement with the National Association of State
Departments of Agriculture (NASDA) that brings together a range of state
y
partners to collaboratively plan implementation of the forthcoming produce
nl
safety rule. In January 2015, the FDA announced that it had joined with
O
USDA’ s National Institute of Food and Agriculture (NIFA) in a collabora-
se
tive partnership to establish the National Food Safety Training, Education,
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Extension, Outreach, and Technical Assistance Program, as mandated in
na
Section 209 of FSMA.
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Recognizing the great diversity among members of the food industry, the
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FDA is building on that investment by funding cooperative agreements that
r
will develop training options for local food production systems and tribal
fo
operations. The FDA is partnering with the USDA’ s NIFA to provide grants
y
op
to fund a national coordination center (NCC) and four regional centers (RCs)
C
to provide training opportunities for owners and operators of farms, small
’s
food processors, and small fruit and vegetable merchant wholesalers. Grants
or
issued through this program will be involved in both key components of

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training— primarily facilitating training delivery and, in certain situations,

b
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facilitating curricula development targeted to specific audiences. This com-

on
petitive grant program, established in January 2015, will provide food safety
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training, education, extension, outreach, and technical assistance. In October

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2015, the FDA awarded the International Food Protection Training Institute,
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located in Battle Creek, Michigan, a grant of up to $600,000 over 3 years to

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establish the NCC, which will lead the coordination of curriculum develop-
c
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ment and delivery to those food businesses covered by the FSMA Section 209
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mandate for implementation of FSMA.

FSMA training will encompass various members of the food industry,
d
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including domestic and foreign food producers and domestic importers. The
or
FDA will work with partners around the world— including the alliances,
yl
regulatory counterparts, and multinational organizations— to promote

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training to the global community of food suppliers. Participants will likely

18
receive documentation of completion for any of the above-mentioned train-

20
ing options. The most long-standing is the Produce Safety Alliance (PSA), a
partnership created between Cornell University, the USDA, and the FDA in
©
2010. The PSA’ s role is to prepare fresh produce growers to meet the regula-
tory requirements included in the final FSMA produce safety rule. These
include:
• A standardized training and education curriculum to assist the

domestic and foreign produce industry, including but not limited
to small and very small farms, as well as regulatory personnel, with
the implementation of the FDA’ s forthcoming produce safety rule
• A train-the-trainer (TTT) course to develop certified trainers and an

interview process for developing certified lead trainers who are quali-
fied to use the curriculum to train farms. The TTT course will include
information on good agricultural practices (GAPs), concepts of adult
learning, and the forthcoming FSMA produce safety standards
• A website at http://www.producesafetyalliance.cornell.edu
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• A network of trainers to support the produce industry and the dis-
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semination of produce safety trainings
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The Food Safety Preventive Controls Alliance (FSPCA), initiated in 2011
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and coordinated by the Illinois Institute of Technology’ s Institute for Food
o
Safety and Health, is developing a standardized training and education pro-
rs
gram and technical information network to help the domestic and foreign
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food industry, including certain mixed-type facilities on farms, comply with
r
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the requirements of the preventive controls rule for human and animal food,
y
as well as the forthcoming rule on the FSVP (Illinois Institute of Technology
op
2016a). This work includes developing
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• Two separate standardized hazard analysis and preventive con-

or
trols training courses and distance education modules— one for the

ut
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human food industry and regulatory personnel and another for the
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animal food industry and regulatory personnel.

on
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• A training curriculum that addresses resources for and preliminary

steps in developing a food safety plan, types of hazards, conduct-
C
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ing a hazard analysis, preventive controls for hazards, monitoring

preventive controls, verification and validation, corrective actions or
cis
corrections, record keeping, and regulatory requirements.

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• Two separate TTT courses for those interested in helping to train food
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facilities— one course for human food and another for animal food.
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• A module on the forthcoming FSVP rule for processors who import

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foods, and a full FSVP course for nonprocessor importers. The alli-
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ance is also encouraging all importers to take the complete preven-

Ta
tive controls training.

18
20
The Sprout Safety Alliance (SSA), initiated in 2012 and coordinated by the
Illinois Institute of Technology’ s Institute for Food Safety and Health, is
©
serving as a network hub and resource for the sprout industry, and federal
and state regulatory agencies. The SSA is developing
• Training materials that will provide techniques to enhance the safe

production of sprouts, and facilitate implementation of relevant
requirements in the forthcoming produce safety rule
• A TTT course for those interested in helping to train farms on safe
sprout production (Illinois Institute of Technology 2016b)
Shinbaum et al. (2016) report that employee training is a very important

aspect of the success of FSMA. One-third of all food recalls recall between
1999 and 2003 were associated with ineffective employee training. Although
the Global Food Safety Initiative (GFSI) has been in place since 2000, and
most companies are compliant with it, GFSI certification and training of
employees has only recently been required. In North America, the top three
y
audit schemes under the GFSI are Safe Quality Food (SQF), British Retail
nl
Consortium (BRC), and Food Safety System Certification 22000 (FSSC 22000).
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se
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ona
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17.4 Title II: Improving Capacity to Detect and
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Respond to Food Safety Problems
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Title II is related to improving capacity to detect and respond to food
y
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safety problems. As discussed above, training various stakeholders is part
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of improving the capacity; however, the FDA has been assigned substan-
’s
tial funds for the new Transforming Food Safety Initiative (Table 17.4). The
or
source of these training funds comes from a cosmetic user fee and a food
utb
contact substance notification user fee.

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The identification and inspection of high-risk facilities will be conducted

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as described under Section 201 of Title II. The secretary, in consultation with
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the secretary of Homeland Security, shall allocate resources to inspect any

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article of food imported into the United States according to the known safety
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risks of the article of food. In each of the 5 years following the 1-year period
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described in clause (i), the secretary shall inspect not fewer than twice the
c
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number of foreign facilities inspected by the secretary during the previ-

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ous year. The secretary of Health and Human Services, the secretary of
d
Commerce, the secretary of Homeland Security, the chairman of the FTC,

an
and the heads of other appropriate agencies may enter into such agreements
or
as may be necessary or appropriate to improve seafood safety. Improvement

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TABLE 17.4
18
U. S. FDA’ s Assigned Funds for the New Initiative of Transforming Food Safety (2016)
20
Category Money Allotted

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Standards setting for food and feed safety $26,687,000

Domestic inspections $4,198,000
Import safety $154,845,000
Integrated food safety system $20,673,000
Foreign inspections $1,418,000
Risk analysis $9,203,000
Science for food safety $8,891,000
Planning and response $1,067,000
in laboratory accreditation for analysis of food is another aspect, and not

later than 2 years after the date of enactment of the FDA FSMA, the secretary
shall establish a program for the testing of food by accredited laboratories.
The secretary shall develop model standards that a laboratory shall meet to
be accredited by a recognized accreditation body for a specified sampling
or analytical testing methodology and included in the registry provided for
y
under paragraph (1). In developing the model standards, the secretary shall
nl
consult existing standards for guidance.
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The FDA directs the Department of Homeland Security to maintain an
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agreement through which relevant laboratory network members and agree
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on common laboratory methods in order to reduce the time required to
na
detect and respond to foodborne illness outbreaks and facilitate the shar-
o
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ing of knowledge and information relating to animal health, agriculture, and
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human health. To enhance tracking and tracing of food and record keeping,
r
the FDA shall establish pilot projects in coordination with the food industry
fo
to explore and evaluate methods to rapidly and effectively identify recipients
y
op
of food to prevent or mitigate a foodborne illness outbreak and to address
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credible threats of serious adverse health consequences or death to humans
’s
or animals as a result of such food being adulterated. The FDA will also
or
establish within the FDA a product tracing system to receive information that
ut
improves the capacity of the secretary to effectively and rapidly track and
b
tri
trace food that is in the United States or offered for import into the United
on
States. Prior to the establishment of such a product tracing system, the secre-
.C
tary shall examine the results of applicable pilot projects and ensure that the
C
activities of such a system are adequately supported by the results of such

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pilot projects. Additional requirements can be set for high-risk foods (HRFs).
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Surveillance of foodborne illness has been suggested to be improved

c
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by working toward automatic electronic searches, for implementation of

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identification practices, including fingerprinting strategies, for foodborne

infectious agents, in order to identify new or rarely documented causes of
d
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foodborne illness and submit standardized information to a centralized

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database. Among other recommendations, an important unique suggestion

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for improving surveillance was establishing a flexible mechanism for rap-

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idly initiating scientific research by academic institutions and integrating

18
foodborne illness surveillance systems and data with other biosurveillance

20
and public health situational awareness capabilities at the federal, state, and
local levels, including by sharing foodborne illness surveillance data with
©
the National Biosurveillance Integration Center (Title II, Section 205).

In terms of response to any food safety issues under Section 206 of Title
II, the mandatory recall authority has given more power to the secretory.
It states if there is a reasonable probability that an article of food (other
than infant formula) is adulterated under Section 402 or misbranded under
Section 403(w), and the use of or exposure to such an article will cause
serious adverse health consequences or death to humans or animals, the
secretary shall provide the responsible party (as defined in Section 417) with
an opportunity to cease distribution and recall such an article.
In regards to keeping a record of all food facilities, the final rule adds cer-
tain new requirements that will improve the food facility registration sys-
tem. All food facility registrations are required to be submitted to the FDA
electronically, although this requirement does not take effect until January
y
4, 2020.
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Registrations are now required to contain the type of activity conducted
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at the facility for each food product category. This was required when the
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final rule became effective on July 14, 2016. The final rule also amends the
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definition of a retail food establishment in a way that expands the number of
na
establishments that are considered retail food establishments, and that are
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therefore not required to register with the FDA as food facilities. However,
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all food establishments, including retail food establishments, continue to
r
have a responsibility to ensure their food is safe.
fo
Title II also gives direction to the FDA to make plans and implement those
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plans for state, local, and tribal governments for assessing, decontaminating,
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and recovering from an agriculture or food emergency. In addition, the FDA
’s
also has additional powers and responsibilities, as indicated in Section 207,

or
“Administrative Detention of Food” ; Section 208, “Decontamination and

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Disposal Standards and Plans” ; Section

209, “Improving the Training
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tri
of State, Local, Territorial, and Tribal Food Safety Officials” ; Section 210,
on
“Enhancing Food Safety” ; and Section 211, “Improving the Reportable Food
.C
Registry.”
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The FSMA requires the FDA to conduct exercises at least annually to eval-
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uate and identify weaknesses in the decontamination and disposal model

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plans and also take into account the priority of such food safety issues accord-
c
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ing to (1) highest-risk biological, chemical, and radiological threat agents; (2)
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agents that could cause the greatest economic devastation to the agriculture
and food system; and (3) agents that are most difficult to clean or remediate.
d
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Apart from training, as discussed in the previous section, the FDA is

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required to identify and provide grants to various agencies to improve infra-

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structure and other needs to enhance food safety. It is also authorized to set
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up centers of excellence headquartered in state health departments that will

18
help tackle foodborne health issues.

20
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17.5 Title III: Improving the Safety of Imported Food

One of the principal driving forces behind the demand for an improved
food safety system is public concern about the safety of imported food,
which now comprises 15% of the U.S. food supply. For some food categories,
more food is imported than produced domestically. For example, 50% of

fresh fruits, 20% of fresh vegetables, and 80% of seafood consumed by
Americans are produced outside of the United States. A plethora of ordi-
nary food ingredients— such as wheat gluten, citric and ascorbic acid, soy
and rice protein, carrageenan, and gum acacia— are primarily sourced
from overseas, often from developing nations. Food is imported from more
y
than 110,000 food manufacturers located in more than 150 countries, many
nl
of which are less developed nations without robust regulatory systems
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in place. A large percentage of the types of foods exported to the United
se
States are considered high risk by food safety experts. Foods make up the
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largest share of FDA-regulated imported product categories, accounting
na
for 59% of reported lines of entry (Figure 17.4). For the first time, importers
o
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have an explicit responsibility to verify that their foreign suppliers have
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adequate preventive controls in place to ensure that the food they produce
r
is safe.
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The FDA regulates $417 billion worth of domestic food and $49 billion
y
op
worth of imported foods. The FDA’ s responsibility in the food area gen-
C
erally covers all domestic and imported food, except meat, poultry, and
’s
processed eggs, which are primarily the responsibility of the USDA Food
or
Safety and Inspection Service (FSIS). This responsibility of the FDA includes
ut
overseeing more than 377,000 domestic and foreign facilities registered

b
tri
with the FDA as a result of the Public Health Security and Bioterrorism
on
Preparedness and Response Act (BT Act) of 2002. More than 1300 full-time
.C
staff years (known as full-time equivalents [FTEs]) are employed with the
C
FDA to conduct field activities, primarily food and feed inspections and
LL
investigational activities.
c is
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Radiological Health, 3%
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Human Foods, 33%

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Devices, 47%
18
20
Animal Feed, 1%
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Housewares and
Food-Related Items, 5%
Drugs and Biologics, 3%
Cosmetics, 8%
FIGURE 17.4
Percentage of imported lines by commodity to the United States for FY 2015. A line is a dis-
tinct product within a shipment. A single shipment may include multiple lines. (Adapted from
http://fda.gov. U.S. Food and Drug Administration, Office of Regulatory Affairs.)
The FSMA gives the FDA unprecedented authority to better ensure that
imported products meet U.S. standards and are safe for U.S. consumers.
New authorities include importer accountability, third-party certification,
certification for HRFs, a voluntary qualified importer program, and author-
ity to deny entry.
In recent years, the FDA has taken significant steps to improve the over-
y
sight of imported food, including implementing a new risk-based analytics
nl
system (Predictive Risk-based Evaluation for Dynamic Import Compliance
O
Targeting [PREDICT]) to improve screening and targeting at the border, and
se
the establishment of foreign offices in China, India, Latin America, Europe,
lU
the Middle East, and Africa. PREDICT uses risk-based data and analytics to
na
help inform entry admissibility decisions, rather than just relying on bor-
o
rs
der examinations. Launched in 2007, the program works by “scoring” food
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entries on the basis of a wide range of risk factors, including inherent risks
r
of the product (such as inherent health risks or risk of the product being
fo
the target of economic adulteration), facility inspections and compliance his-
y
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tory, data anomalies, admissibility history, and intelligence pertaining to the
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manufacturer, foreign locale, or product. Lower-risk lines receive automated
’s
“may proceed” release, while those with higher risk scores are flagged for
or
further review. The FDA is complemented in this effort by collaboration

ut
with the Department of Homeland Security (DHS) Commercial Targeting

b
tri
Analysis Center, allowing access to Customs and Border Protection (CBP)

on
targeting systems that attempt to identify imports that might be problematic

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from a national security standpoint.

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The FSVP mandated by FSMA partners with its foreign counterparts to

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create a global coalition of regulators and strengthen the regulatory capac-

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ity of foreign countries. It leverages public and private third parties to more
c
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effectively verify that modern preventive measures are being taken at the
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tens of thousands of foreign manufacturing facilities producing food for the

American marketplace.
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The law directs the FDA to develop a comprehensive plan to expand the
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capacity of foreign governments and their industries. One component of the

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plan is to address the training of foreign governments and food producers

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on U.S. food safety requirements. The FSMA establishes a program through

18
which qualified third parties can certify that foreign food facilities comply
20
with U.S. food safety standards. This certification may be used to facilitate
the entry of imports. The FDA has the authority to require that high-risk
©
imported foods be accompanied by a credible third-party certification or

other assurance of compliance as a condition of entry into the United States.
A voluntary program must be established for importers that provides for
expedited review and entry of foods from participating importers. Eligibility
is limited to, among other things, importers offering food from certified
facilities. The FDA can refuse entry into the United States of food from a
foreign facility if the FDA is denied access by the facility or the country in
which the facility is located.
Under Section 204(d)(2)(A) of the FSMA, the FDA’ s designation of HRFs

must be based on the following factors (FDA 2016f):
1. The known safety risks of a particular food, including the history

and severity of foodborne illness outbreaks attributed to such a food,
taking into consideration foodborne illness data collected by the CDC
y
2. The likelihood that a particular food has a high potential risk for
nl
microbiological or chemical contamination or would support the
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growth of pathogenic microorganisms due to the nature of the food
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or the processes used to produce such a food
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3. The point in the manufacturing process of the food where contami-
nation is most likely to occur
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4. The likelihood of contamination and steps taken during the manu-
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facturing process to reduce the possibility of contamination
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5. The likelihood that consuming a particular food will result in a
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foodborne illness due to contamination of the food
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6. The likely or known severity, including health and economic
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impacts, of a foodborne illness attributed to a particular food

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The FSMA requires that both microbial and chemical hazards be consid-
b
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ered in HRF designation. The relationship between the criteria in the draft
on
risk model and the factors required by FSMA is shown in Figure 17.5. The
.C
draft risk model includes the following criteria that account for factors (i)
C
through (vi) identified in Section 204(d)(2)(A) of the FSMA, which would be

LL
operationalized based on data and other relevant information. Each factor

is
required by the FSMA would be represented in the model by one or more

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criteria. These criteria are defined as

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• Criterion 1: Frequency of outbreaks and occurrence of illnesses

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• Criterion 2: Severity of illness, taking into account illness duration,

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hospitalization, and mortality

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• Criterion 3: Likelihood of contamination

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• Criterion 4: Growth potential and shelf life

18
• Criterion 5: Manufacturing process contamination probability and

20
intervention
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• Criterion 6: Consumption
• Criterion 7: Economic impact
The classification of foods or categories of food for risk ranking will be

based on the Reportable Food Registry (RFR) commodity definitions (FDA
2016g). The RFR commodity definitions would be appropriate for the risk
ranking because food characteristics, as well as manufacturing processes,
are considered in the definitions (e.g., fresh-cut produce and acidified or
Frequency of outbreaks Likelihood of

and occurrence FSMA contamination
of illnesses (C1) factor (i) (C3)
FSMA
factor (ii)
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Growth potential/shelf life
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(C4)
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FSMA
factor (iii)
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Severity of illness (C2)
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FSMA
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Manufacturing process
factor (iv) contamination/
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intervention (C5)
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FSMA
factor (v)
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Economic impact (C7) op Consumption (C6)
FSMA
factor (vi)
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FIGURE 17.5
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Relationship between criteria in the draft HRF model and factors required by FSMA. (Adapted
b
from http://fda.gov. U.S. Food and Drug Administration, Food/Guidance Regulation, FSMA.
tri
on
low-acid canned foods). Representative foods within each of the RFR catego-
.C
ries would be selected and used in the model (FDA 2016g).

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A 2014 report from the Global Food Traceability Center (GFTC) rating 21
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countries gave the United States a ranking of “vverage” (Charlebois et al.

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2014). With the FSMA, the expectation is to see improved food traceability
c
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capabilities in the United States. Section 204(d)(2)(A) addresses the issue of

tracking and mandates that The secretary shall conduct one or more pilot
Fr
projects in coordination with the processed food sector and one or more such
d
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pilot projects in coordination with processors or distributors of fruits and

or
vegetables that are raw agricultural commodities. The secretary shall ensure
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that the pilot projects reflect the diversity of the food supply and include at
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least three different types of foods that have been the subject of significant
18
outbreaks during the 5-year period preceding the date of enactment of the
20
FSMA, and are selected in order to

©
1. Develop and demonstrate methods for rapid and effective tracking

and tracing of foods in a manner that is practicable for facilities of
varying sizes, including small businesses
2. Develop and demonstrate appropriate technologies, including tech-
nologies existing on the date of FSMA enactment, that enhance the
tracking and tracing of food
3. Inform FDA’s decisions on the exact requirements of traceability, to
help it promulgate implementing regulations
The FDA continues to support the Partnership for Food Protection (PFP)
work groups. In 2012, the FDA and PFP held a 50-state workshop to develop
and implement key deliverables critical to an integrated food safety system.
Both the FSMA and PFP work groups contain federal, state, and local gov-
ernment representatives (FDA 2016h).
Implementing the new authorities and mandates provided by the FSMA
y
will be key to enabling the agency to start to address these challenges, and
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meeting them will extend well beyond the development of regulations
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and guidance. Enforcement will be complicated and resource-intensive.
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Investments will be needed for recruiting and training a cadre of staff to
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audit the FSVP, to oversee the Voluntary Qualified Importer Program and
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the third-party accreditation process, and to develop the information sys-
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tems that will support effective risk-based decision making.
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Anticipating the need for an extended effort to reposition the agency in a
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truly global world, the FDA issued a report on the pathway to global product
fo
safety and quality in 2011. This report, which relates to all FDA-regulated
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products, recognizes that ensuring import safety will involve sustained
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efforts on the part of the agency in four areas:
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1. Partnering with foreign counterparts to form global coalitions of reg-

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ulators: Bilateral and multilateral efforts have been instrumental in

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tri
improving our food safety system, but ultimately, strong collaboration

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and coordination among nations is required to meet the demands of

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a regulatory environment in which product safety and quality know

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no borders. Strengthening the regulatory capacity of other countries

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will be a major focus of the FDA’ s international efforts.

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2. Building a global data information system: A global data infor-

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mation system and network needs to be developed that can allow

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regulators worldwide to regularly and proactively share real-time

d
information and resources. The FDA must work with coalition part-
an
ners to identify critical data elements needed to inform risk models

or
and standardize the reporting of this information to allow for the

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seamless and automated flow of data. Internally, it should work to

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make necessary changes and build new capabilities.

18
3. Becoming an intelligence gathering and data-driven organization:

20
With current resource levels and an ever-growing roster of product

©
manufacturers to monitor, the FDA will need to expand its capa-

bilities in intelligence gathering and use, with an increased focus
on data-driven risk analytics and thoroughly modernized informa-
tion technology capabilities. To enhance its analytics capabilities, the
FDA must work to provide advanced training to current analytics
experts, as well as bring in new employees with significant analyti-
cal talent and experience. To build the necessary support infrastruc-
ture, the FDA must create or identify information technology tools
that will allow experts to quickly access and analyze data across the
various information resources available.
4. Leveraging public and private sector third parties and more effec-
tively allocating FDA resources: The FDA will simply not have the
resources or staff to keep pace with rising imports through its efforts
alone, and thus must make major improvements in its ability to allo-
y
cate its resources based on risk and to leverage the combined efforts
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of government, industry, and public and private sector third parties.
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The FSMA directs the FDA to establish the FSVP and an accred-
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ited third-party inspection program, and these will harness private
na
efforts to help ensure that foreign producers are using effective pre-
ventive controls. In addition, the agency will expand its collaboration
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with foreign governments to rely, where appropriate, on their inspec-
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tion information about the safety of products exported to the United
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States, which will allow the FDA to focus its own resources more effi-
fo
ciently. To make such information most useful, the FDA must expand
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its investment in systems recognition arrangements with foreign
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governments, under which the FDA does a formal assessment of the
’s
foreign food safety system to determine if it offers a level of public

or
health protection comparable to that of the U.S. food safety system.

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If successful, these and other collaborative efforts will result in a global

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food safety system that is both efficiently protective of consumers and facili-
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tates the trade of safe food, providing consumers in the United States, and
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ultimately worldwide, with a wide array of safe and economical food choices.
LL
The appropriation used to inspect facilities registered pursuant to

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Section 415 of the FDCA was approximately $198.5 million. Of this amount,
c
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$145.2 million was used for FDA inspection of domestic facilities and $34.7
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million for FDA inspection of foreign facilities. There were 172,969 active reg-
d
istered domestic food and feed facilities and 285,977 active registered foreign
an
food and feed facilities, for a total of 458,946. During fiscal year (FY) 2011,
or
the FDA’ s Center for Food Safety and Applied Nutrition (CFSAN) identified
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22,325 domestic food firms as high risk per Section 421. Of this inventory,
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11,007 were inspected in FY 2011. In FY 2012, another 8,023 facilities were

18
inspected (or inspection was attempted), totaling 19,030, or 85% of this inven-
20
tory. As of May 2013, the FDA has established a total of 12 foreign posts; the
©
posts have 30 U.S. direct hires (USDHs) and 16 locally employed staff (LES)
and are fully operational (Table 17.5).
17.5.1 Collaboration with Various Government Agencies

The FDA works very closely with the CDC to conduct surveillance; inves-
tigate outbreaks of foodborne illness; develop standards, protocols, and
guidelines; conduct studies and risk assessments; and educate and inform
TABLE 17.5
FDA Foreign Offices
Foreign Post Established USDHs LES Comments
Beijing, China 4 2
Shanghai, China 2 2
Guangzhou, China 2 1
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New Delhi, India 7 2
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Mumbai, India 4 1
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San Jose, Costa Rica 3 2
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Santiago, Chile 1 2
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Mexico City, Mexico 2 2
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Brussels, Belgium 2 0 Staff redeployed in 2012
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London, England 1 0
Parma, Italy 0 0 Staff moved to Brussels in 2012
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Pretoria, South Africa 1 1
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Amman, Jordan 1 1 op
Total 30 16
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Source : FDA, 2013.
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Note : A total of 12 foreign posts with 30 USDHs and 16 LES are fully operational. (Adapted
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from http://fda.gov. U.S. Food & Drug Administration, Food/Guidance Regulation,

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FSMA.)
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the public, consumer groups, public health partners, industry, and other
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stakeholders on food safety. The FDA and CDC also collaborate with the
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USDA FSIS and state health and agriculture departments on these activi-
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ties. The FDA’ s responsibility in the food area generally covers all domestic
is
and imported food, except meat, poultry, and processed eggs, which are pri-
c
an
marily the responsibility of the FSIS. In addition, the FDA cooperates with
Fr
the USDA Agricultural Marketing Service (AMS) on shell egg safety and
d
standards and has regulatory authority over shell eggs and conducts inspec-
an
tions of egg farms. The AMS branch of the USDA is responsible for shell egg
or
surveillance inspections at certain establishments, as mandated by the Egg

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Products Inspection Act (21 USC 1031 et seq.).

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The Foreign Agricultural Service (FAS), through its network of overseas

18
offices and capacity-building efforts, has engaged with foreign officials

20
and the private sector to help keep them up to date on the status of FSMA
implementation, and encourages their participation in the public comment
©
process for the FSMA rules. FDA investigations involve FDA-regulated prod-
ucts distributed via domestic nutrition assistance programs administered by
the Food and Nutrition Service (FNS). Examples of such programs include
the National School Lunch Program and the Emergency Food Assistance
Program. The three branches, FDA, FSIS, and Environmental Protection
Agency (EPA), meet regularly through both the Interagency Strategic
Assessment Team and the Interagency Regulatory Coordinating Group to
coordinate activities on establishing priorities and addressing other issues
related to residues of animal drugs and pesticides in food animals, detect-

ing illegal residues, and taking regulatory action against violators. The
FDA works closely with the U.S. DHS, U.S. CBP regarding the import of
FDA-regulated products. That cooperation reached a new level under the
Bioterrorism Act of 2002, which gave the FDA new authorities to ensure the
safety and security of imported food.
y
Although the FDA is the primary organization responsible for ensuring
nl
that domestic and imported seafood products are safe, sanitary, wholesome,
O
and properly labeled, U.S. the Department of Commerce, National Oceanic
se
and Atmospheric Administration, National Marine Fisheries Service (NMFS)
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conducts, on a fee-for-service basis, a voluntary seafood inspection and
na
grading program that focuses on marketing and quality attributes of U.S.
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fish and shellfish. The FDA provides training and other technical assistance
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to the NMFS. The FDA works with the U.S. Department of Defense (DOD)
r
to enhance information sharing and collaboration, promote the efficient use
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of resources, and build an interagency infrastructure and processes as they
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relate to food at DOD facilities. op
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The U.S. Department of Labor, Occupational Safety and Health
’s
Administration (OSHA) does inspect workplaces where food may be

or
produced, processed, or held. The FDA is working with OSHA to enable

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the agencies to share relevant information obtained during their respec-

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tive inspections of facilities where food is produced, processed, or held.

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OSHA’ s role is to set and enforce standards that will ensure safe working
.C
conditions. The FDA works with the FTC to further the common objective
C
of preventing injury to, and deception of, consumers. Since 1958, the FDA
LL
and FTC have had agreements in place to facilitate information sharing

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(FDA 2016h).
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17.6 Title IV: Miscellaneous Provisions

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Finally, Title IV, with five sections, deals with guidelines related to funding,
18
employee protection, authorities, international agreements, and budgetary

20
effects. This title recommended that FDA increase the recruitment of staff
from 4000 in 2011 to 5000 in 2014. Section 402 is geared to provide protection
©
to employees who report violations.

In summary, the FSMA is designed for the better organization of
records and documentation that can be useful for product tracking and
traceability and importer accountability. It is a long road; the changes are
not going to be easy for anyone with so many moving parts. Funding and
government commitment will be a deciding factor, along with industrial
cooperation. A special challenge seems to be bringing all foreign suppli-
ers on board.
D isclaimer
All the information is intended on the basis of reliable resources but should
not be used to decide on any matter related to food safety. Further interpreta-
tion and application may be very specific, and the FDA and private experts
y
should be contacted for any implementation purposes.
nl
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ona
References
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Barkan, I. D. 1985. Industry invites regulation: The passage of the Pure Food and
Drug Act of 1906. Am J Public Health 75:18– 26.
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Burkett, A. B. 2012. Food safety in the United States: Is the Food Safety Modernization
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Act enough to lead us out of the jungle? Ala L Rev 63:919– 940.
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CDC (Centers for Disease Control and Prevention). 2016. Foodborne outbreak track-
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ing and reporting. Atlanta: CDC. http://www.cdc.gov/foodsafety/fdoss/over-
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view/index.html.
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Charlebois, S., Sterling, B., Haratifar, S., and Naing, S. K. 2014, Comparison of global
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food traceability regulations and requirements. Compr Rev Food Sci Food Saf
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13:1104– 1123.
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DeWitt, P. J. B. 2000. A Brief History of Tea: The Rise and Fall of the Tea Importation Act .
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Cambridge, MA: Harvard Law School.

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Dupré , R. 1999. “If it’ s yellow, it must be butter” : Margarine regulation in North
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America since 1886. J Econ Hist 59:353– 371.

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FDA (Food and Drug Administration). 2016a. The road to the biotech revolu-
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tion— Highlights of 100 years of biologics regulation. Silver Spring, MD: FDA.

an
FDA (Food and Drug Administration). 2016b. FSMA final rule for produce
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safety: How did FDA establish requirements for water quality and test-
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ing of irrigation water? Silver Spring, MD: FDA. http://www.fda.gov/Food/

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GuidanceRegulation/FSMA/ucm472501.htm.
or
FDA (Food and Drug Administration). 2016c. Considering whether an FDA-regulated

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product involves the application of nanotechnology. Silver Spring, MD: FDA.

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http://www.fda.gov/RegulatoryInformation/Guidances/ucm257698.htm.
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FDA (Food and Drug Administration). 2016d. Guidance for industry: Assessing
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the effects of significant manufacturing process changes, including emerg-

ing technologies, on the safety and regulatory status of food ingredients and
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food contact substances, including food ingredients that are color additives.
Silver Spring, MD: FDA. http://www.fda.gov/Food/GuidanceRegulation/
GuidanceDocumentsRegulatoryInformation/ucm300661.htm.
FDA (Food and Drug Administration). 2016e. Guidance for industry: Use of nano-
materials in food for animals. Silver Spring, MD: FDA. http://www.fda.
gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/
GuidanceforIndustry/UCM401508.pdf.
FDA (Food and Drug Administration). 2016f. FDA’ s draft approach for designating
high-risk foods as required by Section 204 of FSMA: February 2104. Silver Spring,
MD: FDA. http://www.fda.gov/downloads/Food/GuidanceRegulation/
FSMA/UCM380212.pdf.
FDA (Food and Drug Administration). 2016g. Reportable Food Summary Report
RFR COMMODITIES Definitions: April 19, 2012. Silver Spring, MD: FDA.
http://www.fda.gov/downloads/Food/FoodSafety/FoodSafetyPrograms/
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RFR/UCM211534.pdf.
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FDA (Food and Drug Administration). 2016h. 2013 annual report on food facilities,
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food imports, and FDA foreign offices. Silver Spring, MD: FDA.
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GAO (Government Accountability Office). 2000. Food safety: Improvements needed
in overseeing the safety of dietary supplements and ‘ functional foods.’ RCED-
na
00-156. Washington, DC: GAO, July.
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rs
Heath, W. J. 2004 America’ s first drug regulation regime: The rise and fall of the
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Import Drug Act of 1848. Food Drug Cosmet Law J 59:169– 200.
Illinois Institute of Technology. 2016a. Food Safety Preventive Controls Alliance.
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Chicago: Illinois Institute of Technology. http://www.iit.edu/ifsh/alliance.
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Illinois Institute of Technology. 2016b. Sprout Safety Alliance. Chicago: Illinois
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Institute of Technology. http://www.iit.edu/ifsh/sprout_safety/.
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Johnson, D. R. 1982. The history of the 1906 Pure Food and Drugs Act and the Meat
’s
Inspection Act. Food Drug Cosmet Law J 37:5– 9.

or
Libecap, G. D. 1992. The rise of the Chicago packers and the origins of meat inspec-
ut
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tion and antitrust. Econ Inquiry 30:242– 262.

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PFDA (Pure Food and Drug Act) of June 30, 1906. 34 stat. 768, ch. 3915. Public Law
on
384.
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Roosevelt, T. 1905. Message to the U.S. Congress. December 5.

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Shinbaum, S., Crandall, P., and O’ Bryan, C. 2016. Evaluating your obligations for
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employee training according to the Food Safety Modernization Act. Food

is
Control 60:12– 17.
c
Wax, P. M. 1995. Elixirs, diluents, and the passage of the 1938 Federal Food, Drug and
an
Cosmetic Act. Ann Intern Med 122:456– 461.

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18
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18
The HACCP in the Current
Food Safety Context
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Juan Garcí a-Dí ez, Dina Moura, Alexandra Esteves, and Cristina Saraiva
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CONTENTS
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18.1 Introduction................................................................................................. 596
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18.2 Hazard Analysis and Critical Control Point........................................... 597
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18.2.1 History of HACCP.......................................................................... 597
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18.2.2 Principles of HACCP: A Practical View....................................... 598
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18.2.2.1 Establish a HACCP Team............................................... 598
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18.2.2.2 Description of the Food Product....................................600

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18.2.2.3 Construct Flow Diagram................................................ 602

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18.2.2.4 In Situ Confirmation of the Flow Diagram.................. 602

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18.2.2.5 Principle 1: Conduct A Hazards Analysis.................... 603

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18.2.2.6 Principles 2– 4: Determine the CCPs, Establish the

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Critical Limits, and Establish a System to Monitor

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Control of the CCP...........................................................608

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18.2.2.7 Principle 5: Establish the Corrective Action to

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Be Taken When Monitoring Indicates That a

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Particular CCP is Not under Control�� 611

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18.2.2.8 Principles 6 and 7: Establish Verification

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Procedures and Establish Documentation and

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Record Keeping��612
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18.3 Evolution of HACCP: Where Are We?..................................................... 612

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18.4 Research on HACCP................................................................................... 613

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18.5 Limitations and Barriers in the Implementation of the HACCP Plan.......618

18
18.6 Compliance of HACCP: Application of Prerequisite Programs

20
and Their Verification and Validation...................................................... 621

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18.6.1 Prerequisite Programs and Differences among Food

Establishments��621
18.6.2 Verification and Validation of HACCP........................................ 627
18.6.2.1 Verification of HACCP.................................................... 627
18.6.2.2 Validation of HACCP...................................................... 627
18.7 Food Safety Training: What Is Really Necessary about HACCP
and Prerequisites? ...................................................................................... 629
595
18.8 HACCP: The New Food Safety Approaches and New Food

Safety Management Systems..................................................................... 635
18.8.1 The 4Cs Approach: Cleaning, Cooking, Cross-
Contamination, and Chilling��635
18.8.2 Hazard Analysis and Risk-Based Preventive Controls............. 636
18.8.3 Application of HACCP in Conjunction with Other Food
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Safety Tools ��638
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18.8.3.1 HACCP and ISO 22000 (FSSC 22000)............................ 638
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18.8.3.2 HACCP and Other Food Standards (BRC
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Standards and IFSs).........................................................640
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References .............................................................................................................640
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18.1 Introduction
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Nowadays, several concepts related to food safety are used, such as food
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security and food quality. Since they present some similar characteristics, it
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is necessary to differentiate each concept.
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Food safety is related to the safety of foodstuffs and includes all the mea-
or
sures applied to guarantee the absence of microbiological, chemical, and

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physical hazards. Thus, food safety includes prerequisite programs (PrPs),

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such as food microbiology control, good manufacturing practices, supplier

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control, cleaning and disinfection (C&D), food contact materials, food label-
.C
ing, food handler training, maintenance, hygienic design, traceability, tem-

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perature control, food handler hygiene, water control, pest control, and food
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transportation (Wallace et al., 2011). Also, it includes the Hazard Analysis

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and Critical Control Point (HACCP) as a worldwide recognized system that

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prevents or reduces the risk of safety hazards in foodstuffs (CAC, 2003).

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Food security is a flexible concept that has been updated in the last 30 years.
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In the 1974 World Food Summit, food security was defined as the “ availability
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at all times of adequate world food supplies of basic foodstuffs to sustain a

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steady expansion of food consumption and to offset fluctuations in produc-

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tion and prices” (UN, 1975). However, the most updated definition defines it
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as “ a situation that exists when all people, at all times, have physical, social
18
and economic access to sufficient, safe and nutritious food that meets their
20
dietary needs and food preferences for an active and healthy life (FAO, 2002).
In summary, food security could be considered a secure supply of food.
©
Food quality can be considered the set of characteristics of a food that meet
the expectations of consumers; it is usually assessed by three characteristics:
sensory value, suitability value, and health value (which could also include
the food safety) (Leitzmann, 1993).
Traditionally, the methods used to guarantee food safety were based on the
basic training of food handlers in the routine inspection of food establish-
ments, as well as the finished foodstuffs by food authorities. However, this
methodology presented several constraints, such as difficulty of application
The HACCP in the Current Food Safety Context 597
in all kinds of food industries and establishments, or the difficulty of food

inspection in loco . Also, if the results of microbiological analysis of the fin-
ished products were incorrect, it was considered that there could be health
risks, and it was necessary to destroy all production.
Today, the safety of foodstuffs is based on preventive measures, with the
HACCP being the cornerstone of food safety. Thus, the HACCP program
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is a systematic preventive system based on principles aimed at identifying
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microbiological, chemical, or physical hazards that are likely to occur at any
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stage of the food chain and implementing measures and controls to prevent
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them from happening.
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In Europe, the European Commission (EC) identified food safety as one
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of its top priorities and established a program of legislative action based on
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the concept “ from farm to table,” as described in the white paper on food
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safety (EU, 2000). Thus, in 2002, the EC set the pillars of food safety with the
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publication of Regulation (EC) 178/2002, laying down the general principles
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and requirements of food regulation, creating the European Food Safety
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Authority (EFSA), and establishing food traceability as mandatory.
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Then, several regulations called the “ food package” (Regulations 852– 854)
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were published by the EC in April 2004, with implementation on January 1,

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2006, with the aim or view to harmonize food safety in the European Union.
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These regulations enforce the implementation of the HACCP principles in

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all food companies, according to the Food and Agriculture Organization of

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the United Nations (FAO), World Health Organization (WHO), and Codex
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Alimentarius Commission (CAC, 2003) recommendations. The advantages

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of their application are related to the efficient use of the resources of an

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enterprise, cost reduction, and enabling food business operators (FOs) to act
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quickly and effectively in the case of foodborne outbreaks, increasing cus-

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tomer confidence and public health.

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18.2 Hazard Analysis and Critical Control Point

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18.2.1 History of HACCP

18
20
HACCP was originally developed in the United States in the early space
programs as a control system for microbiological safety. At that time, most
©
food safety systems and quality control were based on the analysis of the
final product and could not guarantee the safety of foodstuffs. As a conse-
quence, research on a preventive system that would provide a high level of
assurance on the safety of food was necessary. Thus, the HACCP was created.
Pillsbury Company, in conjunction with the National Aeronautics and Space
Administration (NASA) and the U.S. Navy laboratories, was a pioneers in its
development. It was based on an engineering system called failure modes and
effects analysis (FMEA), which analyzes what can go wrong at each stage of an
operation, along with possible causes and the effect they produce (Motarjemi
et al., 1996). After this analysis was put into place, effective control mecha-
nisms were developed to ensure that potential failures do not occur. The
HACCP technique is, in itself, a system of logical and direct control, based on
the prevention of problems, that is, the use of common sense to manage food
safety. Initially, the Food and Drug Administration (FDA) tried to implement
y
the HACCP in the American food industry, but with little success. It was not
nl
until the mid-1980s that various institutions, such as the WHO, International
O
Commission on Microbiological Specifications for Foods (ICMSF), National
se
Academy of Sciences (NAS), and U.S. National Advisory Committee on
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Microbiological Criteria for Foods (NACMCF), boosted its implementation.
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Specifically in 1988, the NACMCF updated the HACCP system, and ICMSF,
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whose main objective is to improve the microbiological safety of foods in
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international trade, promoted its application in the food industries. Twenty
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years later, at the First International Conference on Food Safety, the HACCP
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system acquired importance with the simultaneous publication of the guide-
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lines for its implementation by the NACMCF and the Codex Alimentarius
C
Committee on Food Hygiene (Sperber and Stier, 2009). In 1993, the Codex
’s
Alimentarius Commission adopted these guideline for the implementation

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of the system and annexed them to the Code of General Principles of Food
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Hygiene. These guidelines were subsequently revised in 1997, and the last
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revision to date was carried out in 2003 (CAC, 2003).

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18.2.2 Principles of HACCP: A Practical View

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The HACCP system is a science-based preventive program that uses a sys-

tematic approach to identify specific hazards (biological, chemical, and
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physical) and measures that are necessary to control and prevent the safety
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of foodstuffs. The preventive measures must be described in detail, and the

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person responsible for their implementation should be appropriately trained.

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HACCP involves a careful recording of all details and actions in order to pro-
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vide enough documentation to indicate that the system is operational and all
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potential hazards in food processing are controlled.

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Ta
As defined in the General Principles of Food Hygiene (CAC, 2003), the

implementation of a food safety system consists of the application of the 7
18
principles and 12 steps that must be applied during the development of the
20
HACCP plan, as presented in Table 18.1.

©
18.2.2.1 Establish a HACCP Team

The HACCP team should be multidisciplinary, including experts of repre-
sentative areas, such as engineering, maintenance, food microbiology, food
production, quality control, food policy, and product development. Its main
responsibility is to develop, implement, monitor, and verify that the HACCP
plan removes or reduces the food hazards to ensure the safety of consumers
(Wallace et al., 2012).
TABLE 18.1
Steps for HACCP Implementation
Codex Steps Tasks Required to Develop a HACCP Plan HACCP Steps
1 Establishment of the HACCP team
2 Product description
3 Identification of intended use
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4 Construction of flow diagram
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5 On-site confirmation of flow diagram
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6 Conducting of a hazard analysis 1
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7 Determination of CCPs 2
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8 Establishment of critical limits 3
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9 Establishment of a system to monitor control 4
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of the CCPs
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10 Establishment of corrective actions to be taken 5
when monitoring indicates that a particular
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CCP is out of control
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11 Establishment of verification procedures to
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confirm that the HACCP system is working
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effectively
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12 Establishment of a documentation system 7

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concerning all procedures and records for

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these principles and their application

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HACCP team members should be excellent technicians in their areas of

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expertise, with extensive experience in supervising staff and production

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processes. Although it is recommended that some of the team members

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have previous knowledge or experience in HACCP, this is not fully neces-

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sary, since food safety services can be outsourced or consultants hired to

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improve the process of implementing the HACCP system (usually in small

food establishments) (Yapp and Fairman, 2006). Also, it is not necessary for
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team members to have a thorough knowledge of every detail of the produc-

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tion process of a given product, since the use of support groups within the
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FO can collaborate on solving specific points, as required.

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Once the HACCP team is created, a schedule should be established to ver-

18
ify the achievement of the goals in the process of implementing the HACCP
20
system. In the implementation process, a coordinator should be appointed.

The coordinator of HACCP is the person in charge and should have the
©
knowledge, skills and technical-scientific knowledge in the area of food

safety management to facilitate the development, implementation, and mon-
itoring of the HACCP plan (Surak and Wilson, 2007). He or she should also
have good personal relations with the HACCP team and other workers of the
company to facilitate the handling and coordination, as well establishment
and maintenance, of a pleasant working environment (Azanza and Zamora-
Luna, 2005). The HACCP coordinator is usually the person who knows in
detail the daily activities of the production lines and can maintain fluid
TABLE 18.2
Responsibilities of the HACCP Coordinator
Identify key operators that can serve as internal trainers of operating personnel
Elaborate on lists of instructions and verification
Review and revise, as necessary, operating instructions
Ensure compliance with program prerequisites
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Ensure the implementation and monitoring of corrective actions
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Conduct internal audits
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Initiate and coordinate the resolution of noncompliance situations and problems encountered
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Review records for the HACCP plan
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communication with the engineering staff that provides practical knowl-
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edge on machinery and the work environment with regard to hygiene and
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production design processes. The responsibilities of the HACCP coordinator
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are listed in Table 18.2. Additionally, it is common to incorporate into the
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HACCP team a person from the management department to ensure that deci-
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sions made are consistent with company policies and the team receives all
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the required support of management to successfully implement the HACCP.
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In a small or familiar FO, the development of a HACCP team can be com-

or
promised by the reduced staff. Thus, in this kind of FO, the lack of food
ut
safety technicians implies the hiring of food safety services, which play
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the role of the HACCP coordinator. Also, the FO must define a responsible
on
HACCP, whose function is to inform the external company of all particu-

.C
larities of food production that should be included in the HACCP. Moreover,

C
this person is the connection between the HACCP coordination team (exter-
LL
nal company) and the FO (Losito et al., 2012).

is
The selection of the staff that will be part of the HACCP team is the respon-
c
an
sibility of the HACCP coordinator. The HACCP team composition varies

Fr
from company to company since it should be adapted to the type of FO.

d
However, it is suggested that a representative member from each expertise

an
area be on the HACCP team. In small FOs, the responsibilities, previously

or
indicated, are usually delegated by outsourcing food safety (Alli, 2003).

yl
All the tasks and procedures elaborated by the HACCP team must be
Ta
recorded and should include a list of all persons related to the HACCP plan,
18
indicating who is in charge and responsible, as well providing records of all

20
HACCP team meetings and decisions.

©
18.2.2.2 Description of the Food Product

The description of a food product, including information regarding technologi-
cal and food safety, is usually presented in the technical data sheet (Linton, 2010).
Since some information must be presented in the labeling of the foodstuffs, its
presence on the technical sheet is recommended. Although there is no specific
guideline on how much information the technical sheet should provide, we pres-
ent a brief description of the information that should be included in Table 18.3.
TABLE 18.3
Information Provided in a Foodstuff Technical Sheet
Brand name and description of Commercial brand and a short description of the food
the food product product must be provided.
Information on the FO All information on the food operator, such as name,
address, phone contact, or e-mail, should be provided.
y
nl
Food composition (ingredients All raw products, ingredients, and additives must be
O
and additives) indicated according to the food labeling policy. Also, all
se
ingredients and additives used must be registered for
food use, approved for use in specific foodstuffs, and
lU
defined in the food policy. Thus, all food operators must
na
record the information on the ingredients and additives
o
used, as well as their traceability. Although the quantity of
rs
raw products, ingredients, and/or additives used during
Pe
the food manufacturing is not presented in the technical
sheet, food operators must keep records and be able to
r
fo
demonstrate to the food authority that the quantities used
y
are below the maximum levels defined by law.
op
Nutritional composition Since information on total energy, fat, saturated fat,
C
carbohydrates, sugars, proteins, and salt is compulsory,
’s
technical sheets that present this information are

or
recommended. Since the FO must confirm the accurate

ut
nutritional composition in case of inspection by the

b
tri
official authorities, the existence of laboratory reports is

on
necessary for each foodstuff produced.

.C
Physical, chemical, and Information on aW , pH, or microbiological criteria is

microbiological recommended but not compulsory. Since these criteria are
C
characteristics related to the safety of the foodstuff, food operators must

LL
record all laboratory procedures to demonstrate this

is
information. If microbiological information is included in

c
the technical sheet, the microbiogical criteria should be in

an
accordance with food policy (if applicable). If

Fr
microbiological criteria are not legally defined, the

d
information on the microbiological criteria adopted by the

an
FO should be indicated in the technical sheet.

or
Food processing treatments Information on food processing treatments, such as

yl
pasteurization, freezing, or smoking, can be indicated in

Ta
the technical sheet since they are related and applied to

improving the safety of the food products. It is necessary
18
to indicate all food processing treatments, as well as the

20
conditions in which they are applied (temperature, time,

etc.), which must be defined by law or authorized by the
©
national food authority.

Characteristics of food Information on packaging is also necessary since it is
packaging related to food safety. The information provided should be
related to the type of packaging (vacuum packaging or
modified atmosphere packaging), as well as the packaging
material used. Food operators must guarantee that the
packaging is approved for food contact and must record
its traceability.
(Continued)

Information Provided in a Foodstuff Technical Sheet
Shelflife of the product Since product shelf life is compulsory in food labeling, its
indication on the technical sheet is also recommended.
The determination of the shelf life of a specific foodstuff is
the responsibility of the FO. The food operator must be
able to demonstrate the shelf life of foodstuff to the
y
nl
national food authority.
O
Storage conditions Information on storage is compulsory. Storage conditions
se
must be in accordance with the food policy according to
lU
the food commodities. In case of the absence of legal
requirements for storage, food operators must define the
na
storage condition and also must demonstrate to the
o
rs
official food authority the safety of the foodstuff in the
Pe
specific storage conditions.
Intended use Information on the proper consumption of the food must
r
fo
be provided. In case of those food commodities that have
to be cooked, information on the kind of cooking, such as
y
op
frying, boiling, or oven or microwave heating, is
C
recommended. Also, allergens must be indicated
’s
according to the food policy.

or
Target population of If the foodstuff presents some restrictions for specific

ut
consumers consumer groups (the elderly, infants, pregnant women,

b
or immunosuppressed patients), the warnings must be

tri
indicated in the labeling.

on
.C
18.2.2.3 Construct Flow Diagram

C
LL
The flow diagram is a schematic representation of the phases or steps of food

is
processing. It is very useful for starting the HACCP study, as it allows an

c
an
overview of the process and facilitates the identification of potential sources

Fr
of contamination and the subsequent hazards analysis in each stage of pro-

d
cessing (Varzakas, 2016).

an
A flow diagram should be made by the HACCP team and must include all
or
the steps or stages of food handling and processing. Also, the flow diagram
yl
must represent the raw materials, ingredients, additives, packaging material,

Ta
finished food products, by-products, and residues. A flow diagram should

18
be developed for each product (or product category when they are similar).
20
Thus, in food industries, each product usually has its own flow diagram.
However, for food establishments such as restaurants or pastry shops, which
©
have a great variety of foodstuffs, they are grouped by categories or pro-

cesses, as represented in the Figure 18.1.
18.2.2.4 In Situ Confirmation of the Flow Diagram

All the flow diagrams must be must be confirmed by in situ and in loco
inspection in the food industry or establishment (Figure 18.2). All HACCP
team members must participate in the confirmation of the flow diagram.
Reception*
Storage Cold storage Freezing

storage
Preparation Thawing
y
nl
O
Ready-to-eat Thermal
se
processing
lU
na
Cold
o
maintenance
rs
Heat Cold
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maintenance maintenance
r
fo
Consumption
y
op
C
Dishing
’s
or
ut
b
Washing Residues
tri
on
FIGURE 18.1
.C
Flow diagram example of a restaurant.

C
*Includes raw products and ingredients.

LL
is
Also, all the characteristics observed in situ and in loco must be included. It
c
an
is recommended that in situ confirmation records of the flow diagram pres-

Fr
ent the following information: localization of facilities and equipment; food

processing parameters (temperature, time, etc.); and an indication of the food
d
an
processing and non-food-processing areas, as well as areas of raw products,

or
ingredients, additives, processed foods, finished products, by-products, and

yl
residues. Also, an indication of the critical control point (CPP) is also rec-
Ta
ommended. All flow diagrams must be periodically revised by the HACCP

18
team and all the modifications recorded.

20
©
18.2.2.5 Principle 1: Conduct A Hazards Analysis

Hazard analysis is the first step of a HACCP plan. It consists of the iden-
tification of all the potential hazards (biological, chemical, or physical) in
each food processing step that may harmful to the consumer. Hazard anal-
ysis should be conducted by the HACCP team, and technical expertise is
required to assess all hazards and their severity (Wallace et al., 2014). The
hazard analysis should be realistic, updated, conducted specifically for each
food product, and based on scientific information. Thus, one of the most
1. Milk reception
10
2. Milk cooling
3. Pasteurization
I 4. Rennet addition
8 5. Whey drainage
6. Curd
c 7. Salt addition
9 8. Cooling of cheese
y
nl
d 9. Packaging
O
10. Distribution
b IV
se
II a. Salt/rennet reception
lU
b. Salt/rennet storage
c. Reception of packaging material
na
d. Storage of packaging material
o
rs
4
5 I. Packaging area
Pe
6 a II. Food processing area
7 III. Nonprocessing area
r
fo
V IV. Washing area
3 2 1
y
V.
op Storage room
C
’s
III
or
utb
tri
on
.C
C
FIGURE 18.2
LL
Example of in situ confirmation of flow diagram in the manufacture of traditional fresh cheese.
is
common mistakes is to consider all hazards, mainly microbiological haz-

c
an
ards, found in the literature. Also, compulsory monitoring of several micro-

Fr
biological and chemical hazards in specific food products could be helpful to

achieve this step. To help the readers to conduct a proper hazard identifica-
d
an
tion, a list of microbiological hazards is presented in Table 18.4. In addition,

or
FOs should demonstrate to the official authorities that all the potential haz-
yl
ards have been identified. Moreover, the hazard analysis should consider the
Ta
physical and chemical characteristics of the foodstuff (mainly pH and aW ), as

18
well all the production and manufacturing steps related to the food safety.
20
For example, the 60-day aging rule for unpasteurized milk cheese appears
adequate for producing microbiologically safe products (Brooks et al., 2012);
©
the same is observed in the ripening of traditional meat sausages (Dí ez and
Patarata, 2013). Moreover, it is necessary to consider the target population of
the foodstuffs.
If the manufacture of foodstuffs presents a traditional or novel treatment or
process for controlling foodborne pathogens, the FO should demonstrate its
efficiency. After proper hazard identification, all available information is col-
lected as a framework of reference for subsequent hazard evaluation, which
helps establish its relevance. In other words, it consists of the determination
©
20
18
TABLE 18.4 Ta
Main Chemical and Biological Hazards of Different Foodstuffs
yl
or
Dairy
Foodstuff Sampling Point Hazard (Prevalence %) References
an
Cow raw milk
d
Vending machines Salmonella spp. (2/618) Bianchi et al., 2013
Cow raw milk Vending machines L. monocytogenes (10/618)
Fr
an
Cow raw milk Vending machines c Escherichia coli (1/618)
Cow raw milk Vending machines Campylobacter spp. (9/618)
is
Cheese made from raw milk Retail E. coli (2/41) Brooks et al., 2012
LL
Cheese made from raw milk Retail
C Staphylococcus aureus (3/41)
Cheese Retail S. aureus (42/80) Al-Ashmawy et al., 2016
.C
Ice cream Retail S. aureus (16/40)
on
Yogurt Retail S. aureus (12/40)
tri
Buffalo raw milk Farm and retail
b
Salmonella spp. (8/240)
ut Ahmed and Shimamoto, 2014
The HACCP in the Current Food Safety Context
Cow raw milk Farm and retail Salmonella spp. (4/240)

or
Cheese Retail Salmonella spp. (4/240)
’s
Buffalo raw milk Farm and retail
C
E. coli O157:H7 (16/240)
Cow raw milk Farm and retail E. coli O157:H7 (4/240)
op
Cheese Retail E. coli O157:H7 (9/240)
y
fo
Buffalo raw milk Farm and retail Shigella spp. (27/240) r
Cow raw milk Farm and retail Shigella spp. (9/240)
Pe
Cheese Retail Shigella spp. (20/240)
rs
Raw cow milk Farm S. aureus (162/2650)
o na Jamali et al., 2015
lU (Continued)
se
605
O
nl
y
©
20
606
18
Ta
Raw sheep milk yl Farm S. aureus (86/2650)
or Dairy
Foodstuff anSampling Point Hazard (Prevalence %) References
Cheese from raw milk Retaild S. aureus (31/2650)
Milk (pasteurized/sterilized) Retail F Bacillus cereus (55/55) Kumari and Sarkar, 2014
Milk powder Retail B. cereus (35/52)
ra
Ice cream Retail B. cereus (25/40)
nc
Cheese Retail
is B. cereus (25/33)
Cheese Retail coli (VTEC) (18/23) Reu et al., 2002
LL
Cheese Retail
C E.E. coli (VTEC) (3/83) Caro and Garcí a-Armesto,
2007
.C
Butter Retail Reu et al., 2004
onL. monocytogenes (26/64)
Cheese from cow milk Retail S. aureus Williams and Withers, 2010
tri (4/20)
Meat and Meat Products
bu
Foodstuff Sampling Point Hazard
to (Prevalence %) References
r’s
Raw beef, buffalo, goat, sheep Retail Clostridium difficile Rahimi et al., 2012
Ready-to-eat meat products Retail
C (13/660)
L. monoytogenes (33/628) Wang et al., 2015
o
Meat products Retail S. aureus (29/693) py Crago et al., 2012
Raw chicken Retail Campylobacter jejuni (275/361)
fo Guyard-Nicodè me et al., 2015
Ready-to-eat vacuum and modified Retail L. monocytogenes (22/370) r Kramarenko et al., 2016
atmosphere packaged meat products
Pe
Raw pork Retail Yersinia enterocolitica (20/25) rs Tan et al., 2014
Cooked meat products Retail L. monocytogenes (15/901)
o na Iannetti et al., 2016
Ready-to-eat chicken products Retail Salmonella spp. (4/478) lOsaili
Ready-to-eat beef products Retail Salmonella spp. (1/505)
U et al., 2014
se
(Continued)
O
nl
y
©
20
18
Ta
Ready-to-eat chicken products L. monocytogenes 13/478
yl Retail
Ready-to-eat beef products Retail L. monocytogenes 8/550
or
Fish and Fish Products
an
Foodstuff
d
Sampling Point Hazard (Prevalence %) References
Salted fish Retail Clostridium botulinum (not available) Walton et al., 2014
Fr
an
Raw fish Retail c L. monocytogenes (28/206) Terentjeva et al., 2015
Raw fish Retail is Y. enterocolitica (30/106)
Raw fish Retail Vibrio parahaemolyticus (5/70) Zarei et al., 2015
LL
Raw fish Retail C S. aureus (3/70)
Raw fish Retail Salmonella spp. (2/70)
.C
Fresh shrimp Retail L. monocytogenes (7/60) Rahimi et al., 2012
on
Oyster Retail V. parahaemolyticus (23/38)
tri Yu et al., 2016
Fresh fish Retail
b
Clostridium perfringens (15/50)
ut Herrera et al., 2006
Black tiger prawn Retail Salmonella spp. (2/47) Asai et al., 2008
or
’s
Vegetables
C
Foodstuff Sampling Point Hazard (Prevalence %) References
op
Iceberg lettuce Retail L. monocytogenes (5/24)
y Kovač ević et al., 2013
fo
Leafy vegetables Retail L. monocytogenes (33/11869) r Denis et al., 2016
Raw vegetables Retail V.parahaemolyticus (57/276) Tunung et al., 2010
Pe
Raw salad Retail L. monocytogenes (102/306) rs Ponniah et al., 2010
Vegetables Retail Salmonella spp. (98/1700)
o na Quiroz-Santiago et al., 2009
Minimally processed spinach Food industry E. coli (120/1356) lU Ilic et al., 2008
Minimally processed vegetables Retail Salmonella spp. (4/512) Sant’ Ana et al., 2011
se
Minimally processed vegetables Retail E. coli (14/512)
607
O
nl
y
TABLE 18.5
Risk Factors in Cheese Manufacturing
Geographical area of cheese production Carrascosa et al., 2016
Volume of production
Type of cheese
Handling of cheese
y
nl
Food handler knowledge about food safety
O
Verification of C&D procedures
se
Type of milk (cow, goat, or sheep)
lU
Veterinary management (including udder health) Kousta et al., 2010
na
Milking practices at farm
o
C&D of milk machines at farm Oliver et al., 2005
rs
Use of raw or heat-treated milk
rPe
fo
High Satisfactory Minor Major Critical
y
Likelihood of Medium Satisfactory Minor op Major Major
occurrence Low Satisfactory Minor Minor Minor
C
’s
Negligible Satisfactory Satisfactory Satisfactory Satisfactory

or
Low Medium High

ut
Severity of consequences
b
tri
on
FIGURE 18.3
.C
Bidimensional chart to evaluate the risk of microbiological hazards proposed by the FAO.
(Adapted from FAO 1998. Food Quality and safety systems—a training manual on food
C
LL
hygiene and the hazard analysis and critical control point [HACCP] system. Rome, FAO.)
is
of the probability of a specific hazard occurring and causing serious effects

c
an
on consumers (Table 18.5).

Fr
Risk characterization is defined as an estimation of the probability of

occurrence and the severity of known or potential adverse health effects in
d
an
a population based on hazard identification, hazard characterization, and

or
exposure assessment.
yl
Several approaches have been indicated in the literature, such as FDA-

Ta
iRisk (Chen et al., 2013) and the utilization of spreadsheets (Ross and Sumner,
18
2002). However, an easy approach based on the application of a qualitative

20
bidimensional chart to evaluate the risk of microbiological hazards was pro-

posed by the FAO (1998) and is presented in Figure 18.3.
©
18.2.2.6 Principles 2– 4: Determine the CCPs, Establish the Critical

Limits, and Establish a System to Monitor Control of the CCP
The CCP is the step in which a hazard is eliminated or minimized to accept-
able levels. To determine the CCP, a systematic procedure (decision tree)
(Figure 18.4) is used in each food processing or manufacturing step. It con-
sists of a sequence of questions asked to determine if each step is a CCP
Q1 Do preventive measures exist?
Modify step,
Yes No process, or product
Is control at this step Yes
y
necessary for safety?
nl
O
se
No Not a CCP Stop
lU
na
Is the step specifically designed to eliminate
Q2
o
or reduce the likely occurrence of a hazard to
rs
an acceptable level?
rPe
fo
No Yes
y
op
C
Could contamination with identified
’s
Q3 hazard(s) occur in excess of acceptable

or
level(s) or could these increase to

ut
unacceptable levels?
b
tri
on
Yes No Not a CCP No Stop

.C
C
Will a subsequent step eliminate identified

LL
Q4 hazard(s) or reduce likely occurrence to

is
acceptable level(s)?
c
an
Critical
Fr
Yes No control
d
point
an
or
Not a CCP No Stop

yl
Ta
FIGURE 18.4
18
Decision tree for CCP according to the Codex Alimentarius. Q, question.

20
or not. Also, it is necessary to have preventive measures. However, if there

©
is not a preventive measure that allows control of hazards, a change in the

product or process is necessary to include appropriate preventive measures.
It is also important that the HACCP team understand the difference between
CCPs and control points (CPs) because usually too many CCPs are estab-
lished in order to ensure maximum safety of the products. However, this
undermines the system, causing it to lose credibility and making it difficult
to implement. In contrast, few CCPs can lead to an essential food safety haz-
ard running out of control (Mortimore and Wallace, 2015). When a CCP is
defined in a specific stage of food manufacturing or processing, in addition

to the preventive measures, it is necessary to define the critical limits and
monitoring measures for each type of hazard identified. The aim of monitor-
ing measures is to check whether a CCP is under control in order to detect
in time if there is a deviation from critical limits and to take the necessary
corrective measures immediately. Whenever possible, processes should be
y
corrected when monitoring results indicate a trend toward loss of control at
nl
a CCP, and corrections must be made before the deviation exceeds the criti-
O
cal limit. If a process is not monitored, any possible deviations from critical
se
limits will not be detected, and therefore unsafe food could result.
lU
Due to the serious consequences of a deviation from critical limits, monitoring
na
systems must be effective. Thus, monitoring should be continued, which is pos-
o
rs
sible by using many physical and chemical devices and/or methods (continuous
Pe
measure of time and temperature of heat treatment, continuous pH measure-
r
ment, etc.). In contrast, for other food processing stages, where it is not possible to
fo
continuously monitor a CCP, frequent surveillance that ensures the CCP should
y
op
be established by the HACCP team. To maintain control of the CCP, surveillance
C
systems should provide rapid results to adopt an immediate response to any
’s
deviation from a critical limit related to a process or treatment. It is therefore

or
recommended to use physical and chemical methods instead of microbiological

ut
analysis due to the possibility to apply them in all foodstuffs during processing.
b
tri
For example, if we consider the ultra-high-temperature (UHT) thermal pro-

on
cessing of milk, we can answer the following questions: What is necessary to

.C
monitor? Which is the time/temperature combination that UHT milk should

C
be processed? How are the monitoring measures applied? They are carried
LL
out continuously by probes and computer software. Where are the monitor-
is
ing measures applied? In the milk tank destined for heat treatment. Who is
c
an
responsible for verifying the monitoring measures? The person responsible

Fr
for supervising the process should be identified. What records are needed?
There must be a registration system of temperature and time for each milk
d
an
batch. Since these records are usually automatic, in small FOs, records are
or
frequently made manually. Thus, specific documents should exist to record

yl
all monitoring processes. Although records of the monitoring measures are

Ta
necessary in this step, they are considered part of the last stage of the imple-
18
mentation of the HACCP plan (see Section 18.2.2.8).

20
Erroneous situations can be frequently found in the identification of CCPs.

For example, a common mistake is the consideration of “ bacterial over-
©
growth” as a CCP in a cooling stage. Regarding microbiological hazards,

a cooling stage does not eliminate or reduce the microbiological counts
present in foods because if a food is contaminated with infective counts
of foodborne pathogens, the cooling step will only prevent an increase in
their concentrations, but the infective counts remain in the food. In addition,
an error is observed in the establishment of critical limits and monitoring
measures. Thus, if the biological hazard considered is bacterial overgrowth,
it is necessary to indicate which specific pathogenic bacteria can grow and
then apply monitoring measures to specifically control each kind of bacteria.

Since these monitoring measures would involve the analysis of all foods,
it can be concluded that the CCP bacterial overgrowth in a cooling stage is
just a CP and not a CCP. In contrast, treatments or processes such as thermal
treatments, high hydrostatic pressure, pulsed electric fields, or decontami-
nation by organic acids can be considered as CCPs since they eliminate or
y
reduce pathogenic microorganisms. In this step, if we considered the sur-
nl
vival of specific foodborne pathogens (e.g., Salmonella spp., Listeria monocy-
O
togenes , and Coxiella burnetii ), then the monitoring measures, which include
se
the binomial time intensity, could be defined specifically for each foodborne
lU
pathogen, especially based on scientific reports about inactivation kinetics.
na
As a consequence, the verification measures are not addressed to verify the
o
rs
microbial counts of each food and their reductions (obviously, that is impossible)
Pe
but to check that the time and intensity of the treatment are correctly performed.
r
Regarding chemical hazards, the identification of CPPs is difficult due
fo
to the impossibility of their monitoring. Since some chemicals hazards are
y
op
associated with some processing steps (e.g., acrylamide or biogenic amines),
C
the FO must prove to the food authority that preventive measures are in
’s
place to prevent the formation of these chemical hazards. In a practical way,

or
the FO should analyze the presence of chemical hazards in the foodstuff,

ut
and if results are positive for specific chemical hazards, then the processing
b
tri
conditions (time, temperature, food composition, etc.) should be modified.

on
So, this stage can only be considered as a CP where its control is based on the
.C
application of preventive measures, such as checking processing conditions.

C
Finally, the identification of CCPs related to physical hazards can be eas-

LL
ily carried out by passing all foods through a metal detector, sieve, or filter.
is
Thus, the monitoring measure is correctly accomplished.

c
an
Fr
18.2.2.7 Principle 5: Establish the Corrective Action to Be Taken When

d
Monitoring Indicates That a Particular CCP is Not under Control

an
or
Corrective action is the action to be taken when the results of monitoring of

yl
a CCP indicate a loss of control.

Ta
HACCP is a preventive system, and therefore it is designed to prevent

18
incidents or deviations from critical limits defined in the CCP. The actions
20
taken when a loss of control trend is detected in a stage considered to be a

CCP allow us to adjust the process before a deviation from the critical limits
©
occurs, and return to normal conditions without having affected the product
because it has remained within the tolerance. Despite this, the team must
provide a corrective action plan to carry out during food processing if a
deviation from critical limits in a CCP is found. These corrective measures
should be developed specifically for each CCP and should describe the steps
to ensure, in a quick manner, the correction of the deviation, the removal of
potential food hazards, and that the process is back under control immedi-
ately, and prevent that problem from happening again.
Corrective actions differ according to the foodstuff and processing stage.

Thus, a deviation in thermal processing could be resolved by retreating
foodstuffs, while the addition or presence of a chemical compound implies
the total elimination of an entire batch. It is important to note that all correc-
tive actions must be recorded.
y
nl
18.2.2.8 Principles 6 and 7: Establish Verification Procedures
O
and Establish Documentation and Record Keeping
se
Verification procedures are intended to verify that the HACCP plan is put
lU
in place correctly, as described, effectively reducing or eliminating the haz-
na
ards previously identified. The verification procedures should be specifi-
o
rs
cally defined and recorded and should include the following characteristics:
Pe
(1) what is necessary to verify, and how and where; (2) the frequency of
r
verification procedures; (3) the person responsible for the verification pro-
fo
cedures; and (4) elaboration of a record system of verification procedures.
y
op
Regarding records, they should be as simple and easy to complete as pos-
C
sible (Motarjemi, 2016). They should be adapted according to the type of food
’s
operator, its size, and the number of personnel.

or
Records should also be verified to ensure that all procedures are correctly
ut
recorded, ensuring the safety of foodstuffs (Ryan, 2014). The record system
b
tri
must be stored easily and should be accessible for a period of time deter-
on
mined by the food operator, considering, at a minimum, the technical and

.C
commercial characteristics of foodstuffs. However, storage of information for

C
a minimum period is usually defined in the food law (Regulation 178/2002).

LL
Since all records regarding HACCP must be accessible for food inspectors,
is
they must always be kept up to date and complemented with the necessary
c
an
support documents.
Fr
d
an
or
yl
Ta
18.3 Evolution of HACCP: Where Are We?

18
The HACCP system is the most important and recognized system to ensure
20
food safety from a preventive approach. As previously indicated, its applica-

tion in the European Union is compulsory from January 1, 2006, in all food
©
operators, with the exception of primary production. After 10 years, the

HACCP plans have been adapted to the type of food operator and its size
and needs. Initially, the application of HACCP plans was aimed at, almost
exclusively, reducing or eliminating the microbiological hazards. However,
the identification of chemical hazards in the HACCP has increased in rela-
tion to the potential association of certain chemical substances (added to
foodstuffs as additives or resulting from technological treatments) with the
development of diseases (Asefa et al., 2011; Hwang et al., 2011).
It is possible to observe, with some frequency, HACCP plans with many

CCPs. Although it was thought that this approach guarantees food safety at
maximum levels, today it is known that having many CCPs does not increase
food safety. In addition, excess CCPs imply the application of more monitor-
ing measures than it is impossible to control due to the lack of technical and
human resources. Most of the CCPs considered many years ago (currently
y
considered critical points) are controlled by the application of preventives
nl
measures included in the PrPs.
O
Also, the implementation of HACCP plans is carried out in a linear way
se
independent of the type of FO. This approach is easy to implement in homo-
lU
geneous industrial processes, for example, cheese or biscuit production.
na
However, for food manufacturing processes in which several ingredients,
o
rs
manipulations, and/or different treatments are applied, as observed in res-
Pe
taurants or bakeries, the HACCP plans were updated to a modular approach
r
(Figure 18.1), in which different ingredients and foodstuffs are grouped in
fo
the same processes. Also, the preventive methodology of HACCP has been
y
op
used in other food sectors, such as food packaging industries, primary pro-
C
duction, or even as preventive methodologies in food animal health (Adam
’s
and Brü lisauer, 2010; McAloon et al., 2015).

or
Currently, the evolution of the HACCP and food safety system is associated
ut
with its standardization. Although the HACCP is compulsory for all FOs, it
b
tri
is necessary to elaborate on the verification or supervision process in order

on
to verify it with the same criteria. Thus, in recent years, some standards, as
.C
indicated in Table 18.6, were developed to achieve this objective. The global-
C
ization of food trade implies the need to establish a tool for the global har-
LL
monization of food safety standards that can provide improvements in cost

is
efficiency throughout the supply chain. The Global Food Safety Initiative
c
an
(GFSI) was developed by the food industry, distribution, food safety experts,
Fr
research institutions, and public administrations to promote a harmonized

approach for managing food safety in the industry on a global scale. In other
d
an
words, the objective is to apply food safety methodologies (standards) recog-

or
nized worldwide that are auditable anywhere with the same criteria. Thus, it
yl
is probable that in the near future, the official inspection by food authorities
Ta
will be aimed at controlling the documentation generated by the food safety

18
standards. If the FO meets the prerequisites of the standards, this indicates

20
that food safety is being controlled properly.

©
18.4 Research on HACCP

The HACCP is characterized by its flexibility to adapt to each kind of food
industry or establishment. Due to the diversity and particularities of food
industries and food establishments, the investigation about HACCP has
TABLE 18.6
Standards (Schemes) Recognized by the Global Food Safety Initiative
Food Safety Scheme Sector Applied
PrimusGFS standard Farming of plants
Farming of grains and pulses
Preprocessing handling of plant products
y
Processing of plant perishable products
nl
Processing of animal and plant perishable products (mixed products)
O
Processing of ambient stable products
se
Global Aquaculture Processing of animal perishable products
lU
Alliance/Seafood
na
GLOBALG.A.P. Farming of fish
Farming of plant
o
rs
Pe
Global Red Meat Animal conversion
Standard Processing of animal perishable products
r
fo
y
FSSC 22000 Animal conversion op
C
’s

or

ut
Production of (bio-)chemicals
b
Production of food packaging

tri
on
SQF Farming of animals

Farming of plants
.C
Animal conversion
C
Processing of animal perishable products

LL

c is

an
Provision of storage and distribution services

Fr
Production of food packaging
d
an
CanadaGAP Farming of plants

or
Production of feed
yl
Ta
IFSs Animal conversion

18

20
BRC Global Standards Processing of plant perishable products
©

BRC Institute of Production of food packaging
Packaging (IOP)
Global Standards
IFS Logistics version 2.1 Provision of storage and distribution services
BRC Global Standards Processing of ambient stable products
Provision of storage and distribution services
IFS PACsecure Production of food packaging
Note : SQF, Safe Quality Food; GAP, Global Agriculture Practices.
been addressed to improve its adaptation to guarantee the safety of the

products. Since FOs should demonstrate to the food inspection authorities
aspects such as correct identification of hazards, correct determination of
CCPs, critical limits, and adequate preventive measures, science-based sup-
port for HACCP is necessary.
After the compulsory application of HACCP in FOs, research on HACCP
y
increased. Initially, research on HACCP was aimed at explaining how to cor-
nl
rectly put in place a HACCP plan according to the type of FO. Thus, publi-
O
cations of manuals or guidance by national authorities or food associations
se
were the most common source of information. Although the main objec-
lU
tive of these publications was to help the HACCP teams, the information
na
provided was usually generic or, in some cases, adapted to some food sec-
o
rs
tors. Due to the specificities of each FO, as well as the variety of foodstuffs,
Pe
research regarding HACCP has been addressed to serve as a scientific sup-
r
port for each of the 12 principles defined by the Codex Alimentarius. Some
fo
topics of HACCP research are described below.
y
op
Evaluation and auditing : The evaluation and auditing of a HACCP plan was
C
traditionally based on the manual of HACCP implementation of the Codex
’s
Alimentarius (CAC, 2003). However, new approaches, such as Kohonen’ s arti-

or
ficial neural networks (Trafialek et al., 2015), FMEA (Trafialek and Kolanowski,
ut
2014), multivariate analysis (Kafetzopoulos et al., 2013), and semiquantita-

b
tri
tive studies (Sampers et al., 2012), have been proposed to increase the effec-
on
tiveness of HACCP and improve foodstuff safety. A complex system called

.C
Supply-chain Pedigree Interactive Dynamic Explore (SPIDER), described

C
by Wang et al. (2013), has been proposed as a platform for the public and
LL
governments to explore the information in each part of the food chain with
is
the application of some artificial intelligence techniques, case-based reason-

c
an
ing, rule-based reasoning, fuzzy logic, and neural networks, to be used to

Fr
automatically update and analyze and manage data, mainly regarding risk
analysis and risk management. Regarding the application of FMEA analysis,
d
an
FMEA is a structured approach to discovering potential failures that may

or
exist within the design of a product or process. It improves the operational

yl
performance of the production cycles and reduces their overall risk level
Ta
(Scipioni et al., 2002). This approach has been traditionally used in indus-
18
try, although its application in the food industry has been reported (Scipioni
20
et al., 2002). FMEA has been described in specific food production, such as
confectionery (Ozilgen, 2012; Scipioni et al., 2002), pig farms (Gö dderz et al.,
©
2006), potato chips (Arvanitoyannis et al., 2007), corn curls (Varzakas and
Arvanitoyannis, 2007), and red pepper (Ozilgen et al., 2013), but a new appli-
cation of FMEA that improves the results of food safety audits has been
proposed by Trafialek and Kolanowski (2014). In FMEA analysis applied to
the food industry, the risk of contamination and its presence in hazardous
fractions in the final product is expressed by means of the risk priority num-
ber (RPN), which is defined as RPN = S × O × D , where S is the severity
of a potential risk, O is the probability occurrence, and D is the detection
probability. However, the specific values of S , O , and D must be adapted for
each food industry according to a previously defined methodology. Finally, a
final score resulting from multiplying the values of S , O , and D is obtained,
and corrective actions are suggested for each potential failure mode when
the multiplied values are higher than the critical limit previously defined.
Then, the RPNs, after application of corrective actions, are recalculated to
y
understand the influence of corrective actions on the improvement of the
nl
process (Trafialek and Kolanowski, 2014).
O
New HACCP approaches : New methodologies of HACCP implementa-
se
tion for caterers have been proposed by Taylor and Forte (2008), based on
lU
the application of the Salford model, which consists of the elaboration of a
na
food safety system based on the empirical work at catering establishments.
o
rs
Mataragas et al. (2012) showed that application of specific statistical analy-
Pe
sis of microbiological results of final products as part of the HACCP valida-
r
tion process may improve the microbiological quality of products. Bertolini
fo
et al. (2007) proposed an implementation methodology that combines the
y
op
fault tree analysis (FTA) approach, for the analytical decomposition of the
C
relevant steps in the manufacturing process of a food product, and fuzzy
’s
logic, for quantitative measures of occurrence likelihood, to correctly apply

or
principles 1 and 2 of the HACCP. The SWOT (strengths, weaknesses, oppor-

ut
tunities, and threats) methodology, mainly applied to evaluate the position

b
tri
of an enterprise in the market, has been proposed by Sarter et al. (2010) to

on
evaluate the internal and external factors that could influence the food safety
.C
system. FMEA, a preventive system to identify potential defects in industrial

C
production, was applied in HACCP implementation due to its similarities

LL
with this system (Scipioni et al., 2002).

is
Application of HACCP in specific foodstuffs : To help the food industry and food
c
an
establishments, some research was conducted to support the implementation

Fr
of HACCP of specific foodstuffs by a scientific approach. Thus, implementa-

tion of HACCP plans in ice cream production (Lu et al., 2014), fermented con-
d
an
diments (Oguntoyinbo, 2012), fish processing (Hwang et al., 2011), dry-cured

or
meat products (Asefa et al., 2011), vacuum-packed sauced pork (Wang et al.,
yl
2010), pork paté (Poumeyrol et al., 2010), wine making (Martí nez-Rodrí guez

Ta
and Carrascosa, 2009), bulk tank milk (Vilar et al., 2012), food service (Sun
18
and Ockerman, 2005), cheese (Mauropoulos and Arvanitoyannis, 1999), flour

20
and flour-based products (Arvanitoyannis and Traikou, 2005), butter (Ali

and Fischer, 2005), dry-cured sausages (Conter et al., 2007), and sous-vide
©
meat or pasta (Smith et al., 1990) has been reported.

Improve the microbiological quality of foodstuffs : Investigations of HACCP
also address the improvement in process hygiene by the study of the micro-
biological quality of foodstuffs and the food handlers (Djekic et al., 2016;
Kokkinakis et al., 2011; Domé nech et al., 2013; Soriano et al., 2002).
Investigation of HACCP barriers : The perception of HACCP and its diffi-
culty in application has been discussed in the literature (Toropilova et al.,
2015). Thus, the difficulty of hazard identification in HACCP has also been
reported in the literature (Vela and Ferná ndez, 2003; Wallace et al., 2014).
Since deficiencies in hazard analysis could render the entire HACCP plan
ineffective, Ryu et al. (2013) describe a new methodology to assess the haz-
ards that is based on a model that calculates the risk level for the likelihood
of occurrence of a specific hazard by using background information and
data. Moreover, lack of knowledge on HACCP, lack of PrPs, inadequate phys-
y
ical conditions of the facilities, and lack of motivation were the most common
nl
difficulties observed for putting a HACCP program in place (Baş et al., 2007,
O
Toropilová and Bystrický , 2015).
se
The size of the FO also influences the implementation of the HACCP.
lU
Large FOs have teams dedicated to food safety, while medium and small
na
FOs have some problems, such as a lack of training, a lack of personnel,
o
rs
and maintaining enough records (Taylor, 2001). Adaptation of the HACCP
Pe
to small FOs while avoiding the problem of overdocumenting was studied
r
by Dzwolak (2014). Similar barriers, such as economic cost, time, paper-
fo
work, and unnecessary legal compliance, were referred to in the imple-
y
op
mentation of HACCP on farms (Lowe and Taylor, 2013). Difficulties in the
C
validation process of the HACCP have been investigated by Panisello and
’s
Quantick (2001), who highlighted that technical resources present in the

or
FO are not enough to validate HACCP plans, while the scarce resources
ut
of food authorities are a constraint in the audit and verification of HACCP

b
tri
plans at the national level. Thus, to identify the barriers of HACCP imple-
on
mentation, the utilization of Pareto analysis was indicated (Fotopoulos

.C
et al., 2011).
C
Knowledge : Research on HACCP knowledge and food safety practices has

LL
been studied in restaurant employees (Ko, 2013) and multinational compa-

is
nies (Wallace et al., 2012). Other works reported the difficulty in implement-
c
an
ing HACCP in catering companies due to the lack of well-trained personnel,

Fr
motivation, or financial and economic resources to repair the deficiencies

in the facilities (Garayoa et al., 2011). When the theory of planned behavior
d
an
(TPB) proposed by Ramalho et al. (2015) is applied, it is able to predict the

or
intention to implement a HACCP system in an FO.

yl
Moreover, other aspects with relevant importance in the implementation

Ta
of the HACCP have been studied, such as economic cost, prerequisites, train-
18
ing and knowledge, barriers, and relationship to food standards (ISO 22000,
20
International Food Standards [IFSs], or British Retail Consortium [BRC] stan-

dards) (Escanciano and Santos-Vijande, 2014; Soman and Raman, 2016).
©
Economic cost of HACCP implementation : The economic cost of HACCP

has been reported in the literature, although its benefits are largely demon-
strated today (Cusato et al., 2014), Thus, Lupin et al. (2010) estimated a sav-
ings of about $2 dollars (in preventing failures) for each dollar invested in the
HACCP program.
Research on PrPs : The importance of prerequisites in the implementation of
HACCP was described in the literature (FAO, 1998). However, scarce research
is available on their evaluation by FOs. According to Domé nech et al. (2013),
structure and design, followed by hygiene and cleaning prerequisites, pre-

sented the most frequent nonconformities detected in the majority of the
companies, regardless of size. In addition, this study revealed that noncon-
formities are higher in small FOs than medium FOs.
Application of HACCP in primary production : The application of the HACCP
in primary production has been referred to by some authors, mainly in veg-
y
etable production (Mei Soon et al., 2012; Pardo et al., 2013). However, research
nl
regarding the application of HACCP in other primary sectors, such as rumi-
O
nant production or fish farming aquaculture, has been reported (Garcí a-Dí ez,
se
2012; Kim et al., 2012).
lU
Measuring the HACCP’ s effectiveness : Compulsory implementation of
na
the HACCP and PrPs increases food safety throughout the food chain,
o
rs
although the quantification of its effectiveness is still difficult. Research
Pe
on HACCP is scarce, and the approaches differ. Kafetzopoulos et al.
r
(2013) described a statistical model based on the three main objectives
fo
of the HACCP system: hazards identification, hazards assessment, and
y
op
hazards control. A similar approach based on the degree of achievement
C
of the identification, assessment, and control of foodborne hazards has
’s
been reported by Psomas and Kafetzopoulos (2015) to assess the HACCP

or
effectiveness. Domé nech et al. (2008) proposed a tool for measuring the

ut
effectiveness of the HACCP based on a quantitative risk assessment of the

b
tri
CCPs. In contrast, van der Spiegel et al. (2003) and Cormier et al. (2007)
on
indicated that a full audit of the HACCP system is necessary to assess its
.C
effectiveness.
C
Since the guarantee of the safety of foodstuff is the main objective of the
LL
HACCP, the improvement of some characteristics of the HACCP, such as

is
microbiological quality (as indicated above) and hygiene (Djekic et al., 2016),
c
an
has been indicated for its effective performance.

Fr
It is important to note that appropriate effectiveness of the HACCP can

only be achieved by correct implementation of the PrPs. So, measurement
d
an
of the PrPs should be included. As observed for the HACCP, research on the
or
effectiveness of the PrPs is scarce and only based on audits (Garayoa et al.,
yl
2016, Martins and Rocha, 2014).

Ta
18
20
©
18.5 Limitations and Barriers in the

Implementation of the HACCP Plan
Since its initial development, and after its progressive implementation in
many food businesses, it has been shown that the HACCP system is an effec-
tive preventive tool for routine control of hazards, with greater efficiency
than the old practices based on the inspection (mainly microbiological) of
final products.
TABLE 18.7
Main Difficulties and Barriers Observed in HACCP Implementation
Accessibility to food safety information
Customer perception of food safety/HACCP
Credibility of food inspector authorities
Differences among food establishments
Difficulty in identification and implementation of PrPs
y
nl
Deficiencies in infrastructures and equipment
O
Difficulty in food policy interpretation
se
Difficulty in the hazard analysis
lU
Difficulty of HACCP validation
Difficulty regarding documentation and records
na
Economic cost
o
Food handler hygiene
rs
Lack of human resources
Pe
Lack of knowledge about food safety
r
fo
Although HACCP was made compulsory several years ago, there are sev-
y
op
eral difficulties and barriers in its implementation (Table 18.7) (Jevš nik et al.,
C
2006).
’s
Today, the main barrier in HACCP implementation is associated with the

or
correct application of the hazard analysis step (Vela and Ferná ndez, 2003).
ut
The reason is difficult to explain, although misunderstanding about hazards

b
tri
that should be considered in the HACCP and controlled by specific CCPs

on
and those that should be controlled by the correct application of PrPs (control
.C
points) is a possible explanation (Mortimore and Wallace, 2013).

C
The incorrect design of a food plant or establishment and lack of specific

LL
and/or adequate equipment have also been referred to as barriers (Baş et al.,
is
2007). Thus, the absence of specific rooms for C&D products; the absence
c
an
of separate areas to prevent cross-contamination of ingredients, raw prod-

Fr
ucts, and additives with the final product; and so forth may compromise the
elaboration and on-site verification of the flow diagram, as well as the correct
d
an
application of the PrPs (CAC, 2003).

or
The economic cost of the implementation of a HACCP plan has been indi-
yl
cated (Macheka et al., 2013; Taylor, 2001) as another constraint. Although the
Ta
HACCP should be adapted to the food operator’ s size, food companies usu-
18
ally outsource tasks such as pest control, food and water microbial analysis,
20
and calibration of measuring equipment. Large food companies have their

own teams or departments for food safety and the quality of foodstuffs.
©
However, in small food industries or food establishments, the economic cost

is higher since they should also hire specific technicians to implement the
HACCP system to comply with the food law. Since the HACCP should be
well documented, there should be records for PrPs (C&D, microbial analy-
sis, traceability, temperatures, etc.) and CCPs. These procedures have been
referred to as constraints because they are time-consuming; this is especially
relevant in small food business operations, where human resources are often
scarce (Taylor and Forte, 2008).
Moreover, factors such as lack of training, difficulty in policy interpre-

tation, not enough support by food inspection authorities, or the absence
of guidelines make the implementation of the HACCP difficult, with spe-
cial relevance in small food industries or establishments (Baş et al., 2007;
Ramalho et al., 2015).
The attitudes and behaviors of food handlers or owners of the food
y
industry or establishment also contribute to difficulty in implementing
nl
the HACCP plan. Thus, the absence of economic benefit, an increase in
O
workload, difficulty in understanding, staff turnover, and a lack of food
se
handler motivation have been described (Baş et al., 2007; Ten Eyck et al.,
lU
2006). In addition, differences among the persons in charge of HACCP
na
regarding how records should be implemented and elaborated have been
o
rs
also reported in the literature (Casolani and Del Signore, 2016; Taylor and
Pe
Taylor, 2004).
r
Although the HACCP is an effective tool that is recognized worldwide
fo
to guarantee food safety, there are currently some inherent limitations
y
op
that should be considered before its implementation. First, the HACCP
C
plan, alone, does not guarantee food safety. In other words, its efficiency is
’s
limited. This is because the HACCP is only part of a comprehensive food

or
safety system, together with prerequisites (training, hygiene, traceability,

ut
etc.). Another limitation associated with the HACCP is the hazards analy-
b
tri
sis (principle 1). The lack of information on all potential hazards in each
on
food has limited the successful implementation of HACCP (Panisello and

.C
Quantick, 2001). Although an increase of scientific information on biological

C
hazards has been observed in recent years, more research on foodborne par-
LL
asites, foodborne viruses, and their relationship to the new food techniques
is
and/or processes, mainly in traditional foods, is still necessary (Boelaert

c
an
et al., 2016). Also, the lack of information on critical limits or how to put
Fr
verification measures for these new hazards in place could be considered an

important weakness.
d
an
Moreover, the lack of information on chemical hazards presented in foods

or
due to contamination or synthesis throughout specific food processes repre-

yl
sents an important limitation in the HACCP implementation process. These

Ta
limitations may explain some unexpected food scandals, such as those

18
regarding bovine spongiform encephalopathy (BSE), dioxins, aflatoxins,

20
and melamine (Bá ná ti, 2014; Pei et al., 2011), as well uncommon foodborne
outbreaks, such as listeriosis, with its origin in ice cream, or hepatitis A,
©
from bakery products (Harries et al., 2014; Rietberg et al., 2015). In these
examples, although there were certain risks, these were not included in the
system, so it could not be controlled. Other important limitations of HACCP
are associated with the lack of implementation in the primary production,
as well as the absence of consideration of other public health hazards, such
as radioactivity, food fraud, or improper use of food additives (Xue and
Zhang, 2013).
18.6 Compliance of HACCP: Application of Prerequisite

Programs and Their Verification and Validation
18.6.1 Prerequisite Programs and Differences
among Food Establishments
y
nl
PrPs are described in the General Principles of Food Hygiene (CAC, 2003),
O
as well in the hygiene and food safety policy (Regulation 852/2004), and
se
are considered to be a support network of the HACCP program (Wallace
lU
and Williams, 2001). The main benefit of the application of prerequisites is
na
that they decrease the workload of the HACCP, for example, by reducing the
o
number of CCPs. However, it is necessary to highlight that prerequisites con-
rs
Pe
sider hazards arising from the work environment, including those caused
by cross-contamination. The HACCP plan, on the other hand, considers the
r
fo
specific hazards of the production process.
y
Although the prerequisites are set separately from the HACCP plan, the
op
existence and effectiveness of PrPs should be assessed during the design
C
and implementation of the HACCP plan, and must therefore be documented
’s
or
and verified regularly, together with verification of HACCP as a whole food

ut
safety system.
b
PrPs, such as HACCP, should be adapted to the characteristics of the food

tri
on
industry or food establishment. Although the way they are put in place is
.C
flexible, all of them are mandatory (Regulation 852/2004). Thus, control

and maintenance of equipment and facilities, hygienic design, temperature
C
LL
control, pest control, traceability and recall, microbiological control, good

hygiene and manufacturing practices, C&D, food labeling, food safety train-
cis
ing, and supplier’ s control are considered the most common PrPs (Wallace
an
et al., 2011).
Fr
The information that a prerequisite should include can be variable, but it is

d
an
usually divided into (1) program description and (2) records.

The program description includes the documentation that defines condi-
or
tions, activities, and/or preventive actions that each food facility must com-
yl
Ta
ply with and implement to achieve the objectives defined in the PrP.
The program should include descriptive characteristics of the food facility
18
(such as water pipelines, food areas, entrance of raw ingredients, and barri-
20
ers against pest), as well as the definition of specific activities to be carried

©
out to avoid contamination of foodstuffs. The PrP should also be verified to

ensure the correct application of the PrP. Thus, information on the verifica-
tion or auditing protocol, frequency, and person in charge should be defined.
Finally, it is necessary that the documentation system record the verification
procedures of the PrP.
To increase readers’ knowledge and help food safety professionals, we
present a list of several PrPs and the information that they should include
(Table 18.8) in order to comply with food law. It is important to indicate that
TABLE 18.8
Information of Prerequisite Programs
Prerequisite Program: C&D
Objectives The C&D program consists of a description of the activities carried out in food
facilities to clean and disinfect surfaces, facilities, equipment, or utensils in all
areas.
y
Program Program Description
nl
O
Description of the surface, equipment, or utensil that must be cleaned.
se
Materials used in the C&D process, such as brooms, buckets, mops, and
scrubbers.
lU
Description of all the stages of the C&P. The description of the stages depends
na
on the chemical product used. Thus, the utilization of detergent and
o
disinfectant separately implies five steps (preliminary cleaning, detergent
rs
application, detergent rinsing, disinfectant application, and disinfectant
Pe
rinsing), while the utilization of a mix of detergent and disinfectant implies the
application of three steps (preliminary cleaning, detergent and disinfectant
r
fo
application, and detergent and disinfectant rinsing).
y
The type and/or brand name and concentration of detergents and/or
op
disinfectants must be indicated. Also, the temperature of application and the
C
time of contact must also be defined when applied.
’s
The frequency of all the C&D procedures of all surfaces, equipment, or tools
or
must be defined according to their use and relationship to food safety.

ut
It is important to remark that all the materials used in the C&D must be
b
included in the C&D since they can act as a source of contamination.

tri
on
All C&D products should be approved for the food industry.

It is necessary to indicate the personal protection equipment needed to apply the
.C
C&D products safely.

C
Verification Measures
LL
Visual inspection, microbiological control, or bioluminescence techniques must

is
be defined. Also, the frequency, critical limits, and person in charge must be
c
indicated.
an
Corrective Measures
Fr
Corrective measures should be described in case of deviations (e.g., increase

d
frequency of C&D procedures, training of C&D teams, and utilization of new

an
chemical products for C&D).

or
Records Existence of documentation of each surface, equipment, or tool, which includes

yl
all the information previously described.

Ta
Records of technical sheets of each C&D chemical product.

18
Records of authorization of commercialization of each C&D chemical product

by the health or veterinary national authority.
20
Recording system of the all areas subjected to hygienization.

©
Records of verify measures.
Prerequisite Program: Food Handler Hygiene and Training

Objectives Ensure that food handlers acquire proper knowledge regarding personal
hygiene, food safety, and good manufacturing practices and apply it properly
in their daily work.
(Continued)

This PrP should present all actions to be carried out in loco to achieve hygiene
during food processing. Also, all the training and education programs
(contents) should be described.
y
All the verification procedures, such as visual inspection of food handler
nl
hygiene in loco , visual inspection of dressing, and verification of any food
O
handler presenting symptoms of injury or illness that may affect the food
se
safety or microbiological controls, should be described in the PrP. Also, the
lU
frequency and de critical limits must be defined in the control program.
na
Corrective Measures
o
Corrective measures should be described in case of deviations (e.g., training and
rs
education, and increase microbiological controls).
Pe
Records Update the list of all food handlers of the food establishment.
Task specifications for each process for each food handler.
r
fo
Signed document of hygiene practices that all food handlers must know and
y
comply with. op
Records of all health checkups of all food handlers.
C
Signed document of good manufacturing practices applied in the food
’s
establishment that all food handlers must know and comply with.
or
Records of all training and education programs. They must include the food
ut
handlers’ names, training responsibilities, hours of training, training contents,

b
and records of evaluation.

tri
Records of deviation and corrective measures applied.

on
.C
Prerequisite Program: Maintenance

C
Objectives This prerequisite is aimed at ensuring that all facilities, machinery, and
LL
equipment are correctly inspected and maintained to minimize the probability

of foodstuff contamination by physical, chemical, or biological hazards.
c is

an
This prerequisite should describe all the locations, areas, machines, and
Fr
equipment of the food establishment related to the food processing.

d
an
All the inspections and/or verification of equipment and machines of the food
or
industry should be described. The responsible individual (or company in the

yl
case of outsourcing maintenance services) must be identified.

Ta
A maintenance schedule should be described.

18
Corrective Measures
20
In case of deviations (e.g., machine breakdown), all the corrective measures

should be described.
©
Records Layout of the food establishment, which includes the location and description of
all the food facilities, equipment, and machines.
Records of technical characteristics of all equipment and machines.
Records of all verification procedures.
Records of all corrective actions.
Chronogram of verification and maintenance procedures.
(Continued)

Prerequisite: Microbiological Control

Objectives The objective is to guarantee the microbiological safety of foodstuffs.
The program should describe the type of foodstuffs subjected to microbiological
y
nl
analysis, the parameters analyzed, and the frequency. Also, the microbiological
O
analysis must agree with the microbiological standards defined in food law.
se
lU
A microbiological analysis schedule should be described, including information
na
on microbiological determinations, frequency, and critical limits.
o
Corrective Measures
rs
In case of deviations, corrective measures should be described (e.g., process
Pe
modification and food handler training).
r
Records Schedule of microbiological control.
fo
Information on the microbiological laboratory responsible for the
y
microbiological analysis of foodstuffs. op
Records of all microbiological analysis of foodstuffs.
C
’s
Prerequisite Program: Pest Control

or
Objectives Minimize the contamination of foodstuffs by the presence of pests.

ut

b
tri
Description of physical measures and biological and/or mechanical

on
methodologies used to prevent the occurrence and spread of pests, indicating

.C
the type of pest that being prevented. Indicate the location on a map of the
devices used, if necessary.
C
LL
Preventive Measures
Description of all the procedures aimed at verifying the absence of pests, as well
is
as the correct maintenance of physical, mechanical, and biological barriers.

c
an
Corrective Actions
Fr
Description of all the procedures to be implemented in case of pest

d
contamination.
an
Records Information on and contract of the pest control company.

or
Schedule of pest and barrier verification.

yl
Records (and results) of all inspections and treatment procedures.

Ta
Location map of all mechanical barriers (e.g., insect traps) or biological barriers
(e.g., rat traps). All the rat traps must be numbered.
18
Records of corrective measures.

20
Prerequisite Program: Supplier Control

©
Objectives Guarantee the origin and safety of raw materials, ingredients, additives, and
packaging materials in contact with foodstuffs.
This prerequisite should include all the information on suppliers (name,
address, phone contact, material supplied, food safety standards, etc.).
(Continued)

Specification of the products supplied (technical sheet of each raw product,
ingredient, additive, or packaging material), as well as specification of
transportation, packaging, or microbiological criteria, among others, should be
included.
Preventive Measures
y
nl
Verification procedures of all raw products, ingredients, additives, or packaging
O
materials, when they arrive, should be described.
se
An audit supplier program should also be defined the meet the standards
lU
regarding food safety.
na
Corrective Actions
o
All the corrective measures should be defined (e.g., no acceptance of specific
rs
product by mislabeling).
Pe
Records Updated list of all suppliers.
Record of supplier audits.
r
fo
Records of inspections of raw products, ingredients, additives, or packaging
y
material. op
C
Records of quality and safety (physical, chemical, and microbiological)
’s
agreements of all raw products, ingredients, additives, or packaging material.

or
ut
Prerequisite Program: Temperature Control

b
Objectives This prerequisite is aimed at controlling all the temperatures, including

tri
processing and preservation, at which raw material, intermediate products,

on
and final foodstuffs should be maintained.

.C

C
The prerequisite should describe all the equipment related to thermal or cooling
LL
processes, as well as the temperature limits for each foodstuff according to its
is
step of production. The information on the calibration equipment used in the

c
verification procedures should be included.

an
Fr
The verification, frequency for all the processing temperatures, and critical limits
d
should be described according to the characteristics of the foodstuff.

an
Corrective Measures
or
All the corrective measures (e.g., rejection of frozen foodstuff by twining

yl
associate to equipment breakdown).

Ta
Records Identification, location, and description of all thermal equipment in the food
18
establishment. The technical characteristics of the thermal equipment should be

20
included in the maintenance prerequisite.

Records of all processing temperatures.
©
Records of all corrective measures.
Prerequisite Program: Traceability

Objectives The objective is to track any food, feed, food-producing animal, or substance
that will be used for consumption, through all stages of production, processing,
and distribution.
(Continued)

Previous traceability: Description of identification system of raw materials,
ingredients, or additives and other materials, such as packaging materials and
labels, maintaining correlation with all suppliers.
Internal traceability: The identification of intermediate foodstuffs (prepared or
produced) related to the production data (date in which intermediate/
y
nl
semi-finished foodstuffs are subjected to a specific process or treatment,
O
equipment or facility used, and the amount produced). Also, the input of raw
se
materials, ingredients, and additives used; utilization dates; and amounts used
lU
should be described. The system must guarantee the correlation with all the
raw materials, ingredients, or additives and other materials described in the
na
previous traceability system.
o
Output traceability: Description of system identification of end products
rs
produced. This system must guarantee the correlation with the internal
Pe
traceability, as well with the packaging and labeling material. All end products
r
must be provided by batch number and/or code. This system must also
fo
identify all the companies provided, as well as the type of quantity and
y
delivery date of foodstuffs provided. op
C
The internal audit program of traceability should be described. This program
’s
consists of the selection of a finished foodstuff to verify whether the traceability

or
records are correct and allow identification of the origin of the raw materials,
but
ingredients, additives, and packaging materials.

tri
Corrective Measures
on
In case of loss of traceability of foodstuffs at food facilities, all batches must be

.C
removed until their distribution. In case of food safety problems at the retail
level, a previously described product recall program should be applied.
C
LL
Records Records of all raw materials, ingredients, or additives and other materials, such
as packaging materials and labels.
is
Records of intermediate and/or processed products in the food facilities,

c
an
including all the raw materials, ingredients, or additives used, their quantity,
Fr
and the date of utilization, addition, or application.

Records of all products sold by the food industry identifying the company, type
d
an
of food product, batch number, and quantity supplied.

Records of all verification (audits).
or

yl
Ta
Prerequisite Program: Water Supply

Objectives The water supply control ensures that the water used in the food industry in food
18
and cleaning processes is potable and according to the food and water policy.
20

©
Description of the water used in the food industry.

Description of the water supplier (public of private).
Description of the water pipelines (hot and cold), taps, deposits, etc.
Verification measures must include the microbiological and chemical analyses,
their frequency, and the person responsible for the water analysis (or laboratory
to which it was outsourced) according to the water for human consumption law.
Description of corrective measures in case of deviations.
Corrective Measure
(Continued)

In case of deviations, the corrective measures (e.g., inspection of pipelines and
C&D of water deposits) carried out should be described.
Records Location map of all pipelines, taps, and deposits.
All taps should be identified individually.
Records of all microbiological and chemical analyses and information on the
y
nl
laboratory responsible for the analyses.
O
Records of all corrective measures.
se
In case of the existence of water deposits, the C&D procedures should be
lU
included in the C&D PrP.
na
all the PrPs should define the person responsible for their correct implemen-
o
rs
tation in loco , as well as register all the procedures defined in the PrP. All the
Pe
records (e.g., temperatures, traceability, and C&D) must be dated and signed
r
by the person in charge.
fo
y
op
18.6.2 Verification and Validation of HACCP
C
’s
After implementation of a HACCP plan, its verification and validation are

or
necessary. Although both concepts seem similar, in part because valida-

ut
tion is a component of verification, there are clear differences. Verification

b
tri
includes all methods and/or procedures applied to verify the compliance of

on
the HACCP, while validation indicates that all the procedures described in
.C
the HACCP are effective to guarantee food safety. Since the HACCP is part
C
of a food safety program, as previously described, the PrP must be included

LL
in the verification and validation processes.

c is
an
18.6.2.1 Verification of HACCP

Fr
d
Verification processes should be carried out by the person in charge of the

an
HACCP plan. Some verification measures for PrPs are presented in Table 18.8.
or
In the case of the HACCP program, the verification measures are applied in
yl
those steps where a CCP is defined and should be in accordance with the
Ta
food processing. For example, the verification measures of the thermal treat-
18
ment of milk are usually carried out by observing (and recording) the time
20
and temperature of the process through calibrated measuring devices (ther-

mometers, timers, etc.). In the case of drying meat products, measurement
©
of aW and pH in finished products is the most suitable verification measure.
18.6.2.2 Validation of HACCP

The validation processes should be carried out by elaboration of studies
with scientific support that indicates the efficiency of specific food processes.
Thus, the justification for why a hazard is or is not reasonably likely to occur
can also be viewed as a form of validation (CAC, 2008). Validation procedures
should be conducted by contract laboratories, universities, or equipment or

ingredient manufacturers, or in the food industry itself. Validation proce-
dures include historical knowledge, regulatory documents, experimental
trials, scientific models, operational data, and scientific publications (Scott,
2005) (Table 18.9).
Scientific reports : Utilization of scientific and technical research represents
y
the most important source of information in validation processes.
nl
Experimental trials : This approach consists of designing a methodology to
O
validate a specific process scientifically, for example, the validation of the
se
efficiency of the addition or utilization of a natural agent (e.g., bacteriocins,
lU
essential oils, or organic acid) in a specific food with a specific formulation or
na
composition against selected foodborne pathogens. Although some scientific
o
rs
literature has indicated the efficiency of these natural agents, the particu-
Pe
lar characteristics of the foods (formulation, processing, etc.) imply that the
r
fo
y
TABLE 18.9 op
Examples of Different Validation Procedures
C
’s
Process to Be Type of
or
Validated Methodology Validation Reference

ut
Refrigeration, cutting, Statistical analysis Scientific report Gonzalez-Miret et al.,

b
tri
and packaging of fresh 2001

on
chicken
.C
Thermal processing of Thermal treatment Regulatory Regulation 853/2004

milk for cheese of milk document
C
LL
production
Ripening of cheese Ripening Regulatory Regulation 853/2004
is
made from raw milk document

c
an
Microbiological safety Thermal processing Scientific report Martins and Germano,

Fr
of lasagna 2008
d
Reduction of microbial Application of Scientific report Dormedy et al., 2000

an
counts in beef lactic acid

carcasses
or
Monitoring of fecal On-line monitoring Scientific report Tergney and Bolton,

yl
Ta
contamination system 2006

Shell egg washing Application of Scientific report Khongsak and
18
chlorine water Hourigan, 2002

20
Cooling process for Predictive Scientific report Poumeyrol et al., 2014

©
cooked dishes in microbiology

catering
Elimination of Thermal processing Regulatory Regulation 853/2004
pathogenic of shellfish document
microorganisms of
shellfish
Elimination of Reduction of pH Regulatory FDA, 2009
Staphylococcus aureus in document
fermented meat
products
process must be validated in these conditions. In some cases, nonpathogenic

bacteria (e.g., Listeria innocua instead of L. monocytogenes or Clostridium sporo-
genes instead of Clostridium botulinum ) could be used in experimental valida-
tion processes.
Predictive microbiology : The use of predictive microbiology (Tenenhaus-
Aziza and Ellouze, 2015) in validation processes has arisen in recent years.
y
It consists of the utilization of mathematical models built from experimen-
nl
tal data to predict the microbial growth. There are available some freeware
O
predictive microbiology software, such as predictive modeling programs,
se
ComBase, and seafood spoilage predictors. However, the use of these soft-
lU
ware programs require some previous training. It is also important to
na
highlight that the results of predictive microbial modeling programs are dis-
o
rs
played in specific pH and aW values. As a consequence, it is very important
Pe
to verify whether the predictive modeling program selected fits accordingly
r
with the food process to be validated.
fo
Surveys : Surveys are useful for validating information. Thus, when a food
y
op
industry tries to prove that food labeling (ingredients, storage, preparation,
C
etc.) is clear enough to be understood by consumers, the design of a specific
’s
survey will allow validation of the food labeling.

or
ut
b
tri
on
18.7 Food Safety Training: What Is Really Necessary

.C
about HACCP and Prerequisites?

C
LL
Food safety training is fundamental for any person who works in the food
is
industry, from primary production to the retail level. It is necessary that

c
an
training and formation be adapted to the type of food industry and the per-
Fr
son trained (King, 2012).

Food training is compulsory by law, although some countries have specific
d
an
policies regarding food safety training for specific food sectors. Thus, one
or
of the main constraints of food safety training and education is associated

yl
with the absence of a specific or recognized program by official authorities,

Ta
and also the absence of official evaluation criteria. In addition, training could
18
be conducted by the food company itself, external entities, or entities recog-

20
nized by official authorities.

The elaboration of a training program regarding food safety should include
©
multidisciplinary concepts, such as food microbiology, vegetables, veteri-

nary and foods from animal origin, food engineering, food chemistry, and
food policy. In addition, the importance of proper training and education to
improve food safety has been mentioned, being their lack an important con-
straint in the correct elaboration of the HACCP plan and PrPs (BTSF, 2014).
According to the difficult barrier previously identified, we propose
some topics to be included in the training program (Table 18.10), destined
for the food handlers and technicians responsible for the elaboration and
©
TABLE 18.10
20 630
18
Contents Recommended for Inclusion in a Training and Education Program for Food Handlers and Food Technicians
Ta
yl Food Handlers Technicians
Generic Personal hygiene Cross-contamination
or
concepts an Handwashing
d Clothes
Fr Personal habits
Health status an Diseases related to food safety
c Health condition of food handler and influence on food safety
Good hygiene and Hygienic behaviors on the job
is
manufacturing practices LL Good manufacturing practices
PrP training Temperature control Importance of temperature in
C Elaboration of a temperature control PrP that includes
food conservation • Elaboration of thermal equipment layout
.C
Conservation temperature of on • Identification and classification of foodstuffs by temperature
foodstuffs conservation
Importance of temperate • Implementativon of verification measures of the correct
tri
records
btemperature
ut
• Definition of corrective actions in case of deviations
or
• Correct elaboration of a temperature record system
’s
Cleaning and disinfection Objectives and importance of Elaboration of a layout of all areas, equipment, and tools that need
C
C&D to be subjected to C&D
op
Correct application of C&D Elaboration of hygiene technical sheets for each piece of
y
agents equipment or tool, including procedures, C&D products used,
fo
C&D procedures and frequency r
Importance of C&D records Elaboration of verification measures (visual and microbiological)
Pe
Definition of microbiological critical limits to assess the correct
rs
C&D o
Definition of corrective measures in case of deviations
na
lU
(Continued)
se
O
nl
y
©
TABLE 18.10
20
18
Ta
Traceability Definition of traceability Elaboration of a traceability recording system of all raw products,
or
Importance of traceability in
an ingredients, additives, and packaging material
control of raw products,
d Elaboration of a traceability recording system of intermediate
ingredients, additives, and products (internal traceability)
packaging material Elaboration of a traceability recording system of final products
Fr
Control of intermediate Elaboration a product recall program
an
c
products and final products
is
PrP training Water control Importance of water quality in
LL Elaboration of water supply layout
food processing C Identification and location of all taps or deposits
.C Elaboration of a microbiological, chemical, and physical control
program according to the food policy
Elaboration of corrective measures
on
tri Elaboration of record system of water quality
Training and education Importance of training and
b Elaboration of training programs for food handlers
education on food safety Elaboration of technical sheets for each food handler
ut
or
Elaboration of verification programs to ensure the hygiene of food
’s
handlersC
Pest control Importance of pest control Common pests in the food industry
op
Main foodborne pathogens Elaboration of a layout of barriers presented in the industry or
y
and diseases transmitted by establishment that control the entrance of pests
fo
pests Elaboration of preventive and corrective measures
r
Techniques for pest control Legal documentation of pest control biocides
Pe
Microbiological control Study of the main foodborne Foodborne pathogens of foodstuffs
rs
pathogens Spoilage bacteria of foodstuffso
Elaboration of a microbiological, physical, and chemical program
na
of foodstuffs according the food policy
lU
Definition and implementation of corrective measures
se
(Continued)
631
O
nl
y
©
TABLE 18.10
20 632
18
Food Handlers Technicians
Ta
yl
Maintenance or Importance of correct Elaboration of the layout of equipment, facilities, machinery, and
tools related to food safety
equipment, and machinery Elaboration of preventive maintenance program for all equipment
anmaintenance of facilities,
and
d their relationship to food and machinery
F
safety
ra Equipment calibration
PrP Supplier control Importance
ncof quality of Elaboration of audit program for suppliers
foodstuff supplied
is Elaboration of technical specifications of foodstuffs supplied
HACCP Generic concepts about What is HACCP Land its
training HACCP importance in food
LCsafety
Establish a HACCP team .C Experience in supervising staff and production processes
Describe products
their relationship to food safety
on Physical-chemical characteristics of foodstuffs (pH, aW, etc.) and
additives and processing related to food safety
tri Food
shelf life
buDetermination
Identify intended use Study safety requirements a specific population,
to of the foodof product
with
r’semphasis on those that are weak,ofallergic,
immunosuppressed,
C cancer patients, elderly, or pregnant
Construct a flow
o how to elaborate
Training onp the flow diagrams
diagramand on-site y
confirmation of the flow fo
diagram
rP
HACCP Conduct a hazard analysis To conduct a proper hazard
er analysis, the food team should be
training trained on specific microbiological,
so chemical, and physical
hazards of foodstuffs processed
na in the food industry or
establishment l
Training on searching the scientificUliterature and microbiological
risk analysis is also necessary se
O (Continued)
nl
y
©
TABLE 18.10
20
18
Ta
Determine the CCPs Training on CCPs should indicate that a CPP is the step in which a
or
an hazard is reduced or eliminated; thus, determination of CCPs
d must be related to proper hazard analysis to avoid
misidentification or overidentification
Training should strongly indicate that each CCP identified must
Fr
present critical limits, verification procedures, and corrective
an
cis measures
Establish critical limits LL Determination of critical limits according to food law or by
C scientific literature
Establish a system to .C Manual and automatic monitoring systems in food production
monitor control of the CCP
Establish action to be taken of corrective actions carried out in different deviations
on
when monitoring indicates of critical limits
tri Examples
that a particular CCP is not
bu
under control
to
Establish procedures for Examples
r’s and scientific procedures to validate a HACCP plan
verification to confirm that C
the HACCP system is op
working effectively y
Establish documentation Elaboration of documentation
fo system
concerning all procedures
rP
and records appropriate for er
these principles and their so
application na
lU (Continued)
se
633
O
nl
y
©
TABLE 18.10
20 634
18
Ta
Other training Food transportation Conditions and requirements of food transportation
or
regarding an
food safety d
Food contact materials Food contact material and its
Fr Types of food contact material
relationship to food safety
an Food policy for food contact material
c Chemical hazards of food contact material
Food labeling Importance of labeling in food
is Legal compliance of food labeling
safety LL Nutritional labeling
Food labeling and food fraud
C Determination of food composition
Food policy Generic food safety policy
.C Food policy regarding HACCP, microbiological controls,
on traceability, labeling, etc.
Training for farmers Importance of animal and feed
traceability in food safety
tri
b
Animal diseases and zoonotic ut
diseases or
Correct utilization of ’s
veterinary drugs C
Food safety, biosafety, and op
biosecurity y
Application of good fo
agricultural practices r
Correct utilization of
Pe
phytopharmaceutical rs
products o
Animal well-being
na
lU
se
O
nl
y
implementation of food safety programs. Also, some topics about food safety
for farmers are included.
Regarding HACCP training, it should address all 12 codex points, includ-
ing their characteristics, objectives, and how to put them in place correctly.
Aspects such as correct hazard identification, CCP identification, measure
monitoring, and corrective actions must be thoroughly explained since
y
they constitute the aspects usually incorrectly applied in the HACCP plan
nl
(Mortimore and Wallace, 2001).
O
Training and education regarding PrPs should indicate their importance
se
as the pillars of the HACCP program. In addition, some characteristics tra-
lU
ditionally included in PrPs, such as pest control, temperature control, and
na
C&D, are compulsory by law. Training should be focused on the develop-
o
rs
ment of the program, including the description of preventive and corrective
Pe
measures to guarantee the effectiveness of the PrPs, as well as the elabora-
r
tion of an adequate documentation system that allows the food industry to
fo
demonstrate that the PrPs are correctly put in place.
y
op
Prerequisite training should address temperature control, pest control,
C
C&D, traceability, water control, maintenance, supplier control, microbial
’s
control, training and education, and food handler hygiene. Other topics,
or
such as food contact materials, food transportation, food labeling, and food
ut
policy, should be included in the training program.

b
tri
on
.C
C
LL
18.8 HACCP: The New Food Safety Approaches and

c is
New Food Safety Management Systems

an
Fr
18.8.1 The 4Cs Approach: Cleaning, Cooking,

d
Cross-Contamination, and Chilling

an
In recent years, due to lifestyle changes, the consumption of foods away from
or
home or buying ready-to-eat foods has been associated with an increase of

yl
Ta
food establishments, such as restaurants, cafes, and catering services. In

addition, due to the parents’ work hours in association with the distance
18
from the home, canteen services to children have also increased in recent
20
years.
©
Although the HACCP plan is compulsory in all these food establishments,

as previously mentioned, its implementation is difficult due to the kind of
activity, low number of employees, and scarce economic resources allocated
to food safety (Dzwolak, 2014). Because these establishments use a large vari-
ety of raw foods, ingredients, and/or additives, compliance with the seven
HACCP principles is difficult, particularly with regard to the hazard identi-
fication and determination of CCP.
Thus, to comply with the food policy, application of the 4Cs methodology
(cleaning, cooking, cross-contamination, and chilling) has been proposed
for small kitchens and micro-FOs (FSA, 2015). This methodology includes
the four main ways to prevent foodborne outbreaks, explaining the effective
and preventive measures that should be taken, as described in Table 18.11.
The utilization of this methodology is based on the flexibility described
y
in the food law (Regulation 852/2004), because in certain food businesses, it
nl
is not possible to identify CCPs. As a consequence, the application of good
O
hygienic and processing practices can replace the monitoring of CCPs.
se
However, it is important to mention that elaboration of PrPs is necessary
lU
in these establishments to guarantee food safety and public health. Thus, the
na
cooking and chilling steps should be included in the temperature control
o
rs
prerequisites, while the cleaning and cross-contamination steps should be
Pe
included in the C&D prerequisites. Also, the elaboration of a proper docu-
r
mentation system (e.g., C&D records, temperature records, and traceability)
fo
adapted to the FO size and workload is still compulsory in order to avoid
y
undue burdens. op
C
’s
or
18.8.2 Hazard Analysis and Risk-Based Preventive Controls

ut
As previously described, HACCP is considered an essential tool to guarantee

b
tri
the safety of foodstuffs. However, recent food crises, such as dioxins, aflatox-
on
ins, melanine in Chinese milk, or horsemeat, have caused the questioning of

.C
the food safety plans applied in the food industry.

C
Thus, the Food Safety Modernization Act applied in the United States
LL
indicates that all FOs must implement Hazard Analysis and Risk-Based
is
Preventive Controls (HARPC), a concept similar to HACCP, but with a dif-

c
an
ferent approach (FDA, 2015).

Fr
The HARPC enforces preventive controls in order to identify potential

risks or threats to the food supply and to implement appropriate corrective
d
an
actions proactively to prevent contamination. Thus, HARPC includes plan-

or
ning for potential terrorist acts and/or intentional adulteration and food
yl
fraud. A facility’ s HARPC food defense plan should include additional secu-
Ta
rity, such as visitor access and control.

18
Thus, all FOs (with scarce exceptions, such as very small FOs) must imple-
20
ment a document HARPC plan that includes

©
• Hazard analysis: Identify and evaluate known and reasonably likely

hazards according to the type of food and process. The hazard anal-
ysis must include biological, chemical, physical, and radiological
hazards; natural toxins; pesticides; drug residues; decomposition;
parasites; allergens; and unapproved food and color additives. Also,
naturally occurring hazards or unintentionally introduced hazards
must be included in the hazard analysis.
©
20
TABLE 18.11 18
Main Recommendations of the 4Cs Methodology Ta
Clean yl Cooking
Main Objective Main Objective
or
Stop the spread of harmful bacteria by using good personal hygiene and keeping Food is safely cooked when it reaches a high enough internal temperature to kill
work surfaces, utensils, and hand contact surfaces clean. the harmful bacteria that cause foodborne illness.
an
Tips
d Tips
Always wash work tops before food preparation begins. Bacteria will begin to die when the temperature rises above 60° C.
Fr
Wipe up any spilled food right away. an Hot food must be served above 63° C.
Always sanitize work tops thoroughly after they have been touched by raw c Food should be cooked or reheated to at least 70° C or 75° C, respectively.
meat, including poultry, or raw eggs. is Comminuted meats (e.g., burgers and sausages) should always be cooked, avoiding
Avoid contact of ready-to-eat foodstuffs with food contact surfaces unless it has pink zones on the inside.
been washed thoroughly first. Always preheat the oven before cooking and follow the cooking times according to the
LL
Utilize paper towels to clean up kitchen surfaces. C type and size of the foodstuff.
Rinse fresh fruits and vegetables under running tap water. Also, the application Frozen meat must be thawed all the way through before cooking.
.C
of an approved disinfectant for fruits and vegetables is recommended. Food that is cooked for eating later should be cooled as quickly as possible (ideally
within 90 minutes) and then stored in the refrigerator above raw foods until use.
on
Never reheat cooked food more than once, and make sure to reheat to at least 75° C.
tri
It is recommended to utilize an oven instead of a microwave oven to avoid cold spots
b
inside the food, where bacteria could survive.
ut
Chilling Cross-Contamination
or
’s
Main Objective Main Objective Avoid the transfer of foodborne bacteria from foods— usually raw— to
C
Reduce the temperature (below 5° C) to decrease the growth of foodborne other foods.
pathogens, reducing the risk of food outbreak. Tips
op
Tips
y
Always wash your hands thoroughly after touching raw food.
Cooked foods are covered and ideally labeled with the processing date. Keep raw and ready-to-eat foods separate.
fo
Maintain the FIFO rule: First-in, first-out foods should be stored in the
r
Raw meat or fish should be stored in closed containers to avoid contamination of other
refrigerator correctly (avoid overloading). Cold air needs to circulate around foodstuffs by drip.
Pe
the food. Use different cutting boards, work surfaces, and knives for different raw foods (e.g.,
rs
The refrigerator should be cleaned and disinfected regularly. meat, chicken, fish, fruits, and vegetables) and ready-to-eat foods.
o
Ensure that the refrigerator and freezer are working at temperatures between Apply correct C&D to cutting boards, work surfaces, and knives after use with raw
na
0° C and 5° C and below – 18° C, respectively. food.
Do not leave the door open for extended periods.
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Food to be frozen should be wrapped well to prevent it from drying out. se
Never refreeze defrosted food since it increases the growth of harmful bacteria.
637
O
nl
y
TABLE 18.12
Differences between HACCP and HARPC
In the HARPC program, there is no reference to previous steps of the HACCP (elaboration of a
HACCP team, product description, definition of the intended use, production flow diagram,
and in situ check of the flow diagram). However, HARPC ensures that the analysis is
developed according to the type of food and carried out by a qualified person.
y
The HARPC plan indicates that a radiological hazard must be considered in the hazard
nl
O
analysis. Although radioactivity is not a frequent hazard, it could be presented from natural
sources as water contamination or natural deposits containing radioactive materials, or
se
derived from plant accidents or establishments that handle radioactive materials, as
lU
happened in Fukushima, Japan.
na
The identification of CCP is not indicated in the HARPC, although preventive controls based
o
on risk and science are compulsory. Preventive controls include process control, allergen
rs
control, health checks, training, environmental monitoring, a product recall program, and
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approval of suppliers.
r
While the HACCP plan must be upgraded continuously, the HARPC must be revised every
fo
3 years. Also, records related to the HARPC must be stored for at least 2 years.
y
op
The HACCP is a global standard, while HARPC is still a preventive standard that is only
compulsory in the United States.
C
’s
• Preventive controls: Ensure that the identified hazards that are rea-
or
ut
sonably likely to occur can be minimized or prevented significantly.

b
tri
• Monitoring: Ensure that preventive controls are established as

on
records are generated and carried out.

.C
• Corrective actions: Actions to be taken if there is no control or it is

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ineffective. This would imply a reassessment and modification of

LL
the plan.
is
• Verification: Ensure that the controls are carried out consistently.

c
an
Validation includes the concept that preventive controls are effective

Fr
for the identified hazards.

d
• Records: These include hazard analyses and records of preventive

an
controls, surveillance (monitoring), corrective action, and verifica-

or
tion (including validation).

yl
Ta
Although HARPC is similar to HACCP, HARPC is, in short, a preventive

18
system based on risk mitigation. The most important differences are pre-
20
sented in Table 18.12.

©
18.8.3 Application of HACCP in Conjunction

with Other Food Safety Tools
18.8.3.1 HACCP and ISO 22000 (FSSC 22000)
As previously indicated, there are several food standards, as presented in
Table 18.5. Some of them, such as ISO 22000, the BRC standards, and the
IFSs, present HACCP as the main core, although their approaches and the
food industry in which they are applied vary. ISO 22000, Requirements for
Any Organization in the Food Chain, is the result of an international effort
to harmonize the food safety standards as defined by the International
Organization for Standardization (ISO). ISO 22000 is a food safety manage-
ment system that includes HACCP. However, it also includes other manage-
ment system processes that work together to control food safety throughout
y
the organization. ISO 22000 can be used by all organizations in the food
nl
chain and incorporates the preliminary steps and principles of HACCP
O
(Arvanitoyannis and Varzakas, 2009; Kö k, 2009). Also, it provides an audit-
se
able standard that can be used as part of third-party certification, and
lU
ensures that the process to control food safety is validated, verified, imple-
na
mented, monitored, and managed. The main difference between the two is
o
rs
that HACCP is a system, whereas ISO 22000 is a standard. In addition, ISO
Pe
22000 can and should be used to monitor the success of your HACCP analy-
r
sis (Escanciano and Santos-Vijande, 2014).
fo
ISO 22000 does not have the requirement of a preventive approach, as does
y
op
HACCP. In implementing ISO 22000, the food industry must comply with all
C
the characteristics described in the standard: (1) development of a plan to ensure
’s
food safety; (2) consideration of food safety in the business objectives of the FO;
or
(3) definition of the internal and external food safety communication require-
ut
ments, as well as definition of an adequate emergency response procedure in

b
tri
the case of a crisis; (4) definition of the standard responsible person; (5) defini-
on
tion of proper training programs regarding food safety; (6) elaboration of audit
.C
plans for PrPs; and (7) continuous updating of the food safety manage system.
C
The ISO 22000 certification provides a complete food safety management

LL
system with some benefits, including its applicability to all organizations in

is
the global food supply chain, its recognition as an international standard, its
c
an
compliance with the codex HACCP principles, and its enhancement of the
Fr
communication of hazards with other food operators in the supply chain.

Currently, the standard FSSC 22000 has been implemented in the food
d
an
industry. Although it seems to be a new standard, it can be considered an

or
improved and updated version of ISO 22000. According to the information

yl
available on the official website for FSSC 22000 (www.fssc22000.com), this

Ta
standard is a complete certification scheme for food and feed safety man-
18
agement systems, which are in compliance with the publicly available food
20
safety management systems standard, ISO 22000, and it includes technical

specifications for PrPs, as well other additional scheme requirements. FSSC
©
22000 provides a certification model that can be used in the whole food
supply chain and can cover sectors where such a technical specification for
sector PRPs has been realized. FSSC 22000 follows the food chain category
description as defined in ISO/TS 22003 (2013). But, what are the differences?
ISO 22000 is not recognized by GFSI (see Section 18.3), and its application is
broad in scope. In contrast, FSSC has a more limited scope, including farm-
ing, perishable animal products, food processing, feed production, food
ingredients, and food packaging material manufacturing.
Thus, the implementation of FSSC 22000 instead of ISO 22000 allows FOs
to be recognized by the GFSI. The FSSC 22000 scheme uses ISO 22000 for the
requirements of the management system and also includes requirements for
PrPs.
18.8.3.2 HACCP and Other Food Standards (BRC Standards and IFSs)
y
nl
BRC Global Standards is a scheme developed by the BRC Food Technical
O
Standard to evaluate manufacturers, distributors, and/or retailers to improve
se
the safety and quality of foodstuffs (Havinga, 2006). The IFSs currently com-
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prise eight standards, which have been developed for and by the stakehold-
na
ers involved in all parts of the supply chain. All standards are processes that
o
rs
help users when implementing legal provisions regarding food and/or prod-
Pe
uct safety, and provide uniform guidelines on foodstuffs, product safety, and
r
quality issues (Fulponi, 2006). Both of them set the requirements in terms of
fo
procedures and results in the food safety process. However, they are not
y
op
suited to the whole food chain, as observed in the ISO 22000. These three
C
standards present the HACCP and PrPs as the main tools to guarantee food
’s
safety.
or
Moreover, all of them indicate that all policy regarding food safety must
ut
be fulfilled. However, the main difference among them is that ISO 22000 (or
b
tri
FSSC 22000) is based on results, while BRC standards and IFSs are based on
on
procedures.
.C
C
LL
c is
an
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19
Recent Developments in
Saffron Fraud Prevention
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Anastasia Kyriakoudi and Maria Z. Tsimidou
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CONTENTS
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19.1 Introduction................................................................................................. 651
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19.2 Saffron: The Precious Spice from Crocus sativus Linnaeus ................... 652
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19.3 Chemical Composition of Saffron.............................................................654
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19.3.1 Saffron Secondary Metabolites Related to Its Coloring
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Properties......................................................................................... 655
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19.3.2 Saffron Secondary Metabolites Related to Its Flavor................. 657

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19.4 Saffron Food Uses and Reported Fraudulent Practices......................... 658

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19.5 Recent Developments in Saffron Fraud Prevention............................... 659

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19.5.1 Omic Approaches Developed in the Frame of the COST

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Action FA1101 Saffron-OMICS��661

19.5.1.1 Genomics........................................................................... 662
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19.5.1.2 Proteomics......................................................................... 663
19.5.1.3 Metabolomics...................................................................664
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19.5.2 Other Recent Advances in Saffron Fraud Prevention................ 667

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19.6 Overview...................................................................................................... 671
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Relevant Websites................................................................................................ 672

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References ............................................................................................................. 672
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18
19.1 Introduction
20
Saffron, the dried stigmas of the flower of Crocus sativus Linnaeus, is the most
©
expensive spice in the world, and therefore the target of fraudulent practices.
Many different approaches have been developed for saffron fraud preven-
tion, among which omic ones (genomics, proteomics, and metabolomics)
show great potential. In recent years, many scientists have joined efforts
and expertise to combat saffron fraud by enriching the armory of analyti-
cal methods in a complementary way within the frame of the COST Action
FA1101 “ Saffron-OMICS.” During the same period, other omic approaches
were also reported on the same topic. This chapter covers the major recent
651
analytical achievements, in the light of which existing regulatory measures

for saffron fraud prevention are critically discussed.
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19.2 Saffron: The Precious Spice from
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Crocus sativus Linnaeus
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The genus Crocus (family Iridaceae, subfamily Crocoideae) consists of many
species that occur in the wild and are distributed in central and southern
na
Europe (mainly in the Balkan Peninsula), North Africa, and Asia Minor.
o
rs
Taking into consideration the recent recognition of new species and subspe-
Pe
cies, the latest estimation of their number is circa 150 (Harpke et al., 2013).
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Many crocuses are used as ornamental plants in gardens and parks for their
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colorful flowers. Descriptors for Crocus species have been recently developed
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by Molina et al. (2015) as a useful tool for the investigation of the genetic vari-
C
ability among them, as well as for their conservation.
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The most important representative of the genus cultivated for edible

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purposes is C. sativus L. This perennial plant presents some unique char-

ut
b
acteristics from both the botanical point of view and its composition in sec-
tri
ondary metabolites. In particular, C. sativus L. is a triploid and genetically

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sterile plant that is propagated vegetatively by corms. For this reason, its
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genetic variability is rather limited, with the exception of random muta-

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tions (Ferná ndez et al., 2011). The investigation of the actual genetic vari-
LL
ability of C. sativus on a worldwide scale was the objective of the Genetic

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Resources of Saffron and Allies (Crocus spp.) (CROCUS BANK) project,

c
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which resulted in the creation of the World Saffron and Crocus Collection
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(WSCC) situated in Cuenca (Spain) (http://www.crocusbank.org/). C. sati-

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vus , with 2n = 24 chromosomes, is considered to be an allotriploid hybrid.

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During recent years, efforts have been made with the aid of novel molecu-
or
lar techniques to elucidate how this species was generated. Findings of

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such studies indicate that one of the most probable progenitors of C. sati-
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vus is Crocus cartwrightianus , grown in the wild in many areas of south-

18
ern Greece (e.g., Crete) and the Aegean islands (e.g., Santorini) (Alsayied
20
et al., 2015).
©
C. sativus plant, also known as saffron plant, reaches a height of 10 up

to 25 c m. Its corms constitute subsoil organs of 5– 7 c m in diameter, hav-
ing a characteristic creamy color. Corms are protected by fibrous tunics.
The leaves, usually 5– 11 and deep green in color, are narrow and linear,
with a vein on the outer side, and their length can reach up to 50 c m.
Flowers, which appear before the development of leaves, consist of six
tepals of purple-violet color with darker veins, which are all connected
to a long tube that starts at the top of the ovary. Τ h ree yellow stamens,
Recent Developments in Saffron Fraud Prevention 653
Stigmas
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Stamens
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FIGURE 19.1
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C. sativus L. plant. (From photo gallery of Laboratory of Food Chemistry and Technology,
Aristotle University of Thessaloniki, Thessaloniki, Greece).
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which comprise the male reproductive organs of the plant, emerge from
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the upper part of this tube. The style that originates from the ovary is
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divided in three red branches, the stigmas that all together comprise the
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op
pistil, that is, the female reproductive organ of the plant (ISO 3632-1, 2011;
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Carmona et al., 2006a). The major parts of the C. sativus plant are depicted
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in Figure 19.1.
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Regarding the life cycle of the plant (Figure 19.2), the flowering stage
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takes place once a year for a short period of time (mid-October to mid-
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November). The flowering stage is followed by the vegetative stage, which

on
lasts from December to February. This stage is characterized by the devel-

.C
opment of roots and leaves, as well as the formation of the daughter corms
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that will replace the mother ones, which then shrink. Later on, when the
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c is
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Activation stage Flowering stage

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(September– (mid-October–
d
mid-October) mid-November)
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18
Dormant stage Vegetative stage

20
(June– (December–
©
September) February)
Recapitulation
stage
(March–May)
FIGURE 19.2
Graphical representation of the life cycle of the plant C. sativus L.
temperature begins to rise (March– May), the leaves fall during the recapitu-
lation stage and then in mid-May, the plant enters the dormant stage. This
period of reduced biological activity offers resistance to the plant toward
high temperatures and dryness. However, while nothing appears to happen
above the surface of the soil during the dormant stage, important metabolic
changes occur in the corms related to the composition and content of simple
y
and complex saccharides. Finally, the transition from the dormant to the
nl
active stage takes place around September (Carmona et al., 2006a; Kumar
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et al., 2009).
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From all the vegetative parts of the C. sativus L. plant, the ones with the
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most economic value are the three-branch styles that, after drying, com-
na
prise saffron, the most expensive spice in the world (Melnyk et al., 2010).
o
rs
Saffron is currently produced in Asia (Iran, India, and Afghanistan),
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Europe (Greece, Spain, Italy, and France), and North Africa (Morocco).
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Reported data show that ~90% of the world’ s total annual saffron pro-
fo
duction originates from Iran. Greece is the major producer of saffron
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op
in Europe, whereas Spain is the major exporter (e.g., United Nations
C
Comtrade Database, http://comtrade.un.org, last accessed June 2016).
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Despite the interest, cultivation in the Southern Hemisphere is still in its

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infancy.
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19.3 Chemical Composition of Saffron

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Information about the proximate composition of saffron is limited. The

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maximum limit of moisture content after the drying process that is

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accepted by ISO 3632-1 (2011) for commercial transactions is 12% (w/w) for
saffron in filaments and 10% (w/w) for saffron in powder form. It has also
d
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been reported to contain proteins (~12%, w/w), fats (~5%, w/w), minerals
or
(~5%, w/w), and crude fiber (~5%, w/w) (Sampathu et al., 1984; Rí os et al.,
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1996), but frequent updating of these values for saffron of different origin
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is rather missing. The presence of riboflavin (vitamin B2) and thiamine

18
(vitamin B1) has also been reported in the past by Sampathu et al. (1984)
20
for commercial saffron samples from India (3.4– 5.6 and 0.3– 0.4 µ g/g,
respectively). Higher riboflavin content (5.02 and 13.86 μ g/g) was deter-
©
mined recently by capillary electrophoresis coupled with laser-induced

fluorescence detector by Hashemi and Bedia Erim (2016) in Iranian and
Spanish saffron samples.
Nevertheless, the compounds that are treasured in this spice for their rare
coloring properties and flavor are secondary metabolites, some of which are
present at abundant concentrations. These secondary metabolites, apocarot-
enoids in character, are detailed in the next section in relation to their intrin-
sic properties.
19.3.1 Saffron Secondary Metabolites Related to Its Coloring Properties

The coloring properties of saffron are attributed to a group of water-soluble
apocarotenoids rarely found in nature, the crocins, which are tetraterpenoid
compounds with seven conjugated double bonds. Crocins are sugar esters of
crocetin (8,8′ -diapocarotene-8,8′ -dioic acid) (Figure 19.3) with mono- or oli-
gosaccharides (glucose [g], gentiobiose [G], and neapolitanose [n]). They are
y
nl
called apocarotenoids due to their reduced chain size in comparison with that
O
of usual C40 carotenoids (Carmona et al., 2006a). The first report on the digenti-
se
obiosyl ester of crocetin (trans -4-GG), empirically named in the past as crocin
lU
1 or crocin α , was made by Aschoff (1818). Pfander and Schurtenberger (1982)
na
were the first to separate and identify by high-performance liquid chromatog-
o
raphy– mass spectrometry (HPLC-MS) five crocetin sugar esters in an aque-
rs
ous-ethanolic saffron extract. The identified crocetin esters were trans -4-GG,
Pe
crocetin-(β -D-gentiobiosyl)-(β -D-glycosyl) ester (trans -3-Gg), crocetin-mono-
r
fo
(β -D-gentiobiosyl) ester (trans -2-G), crocetin-(β -D-glycosyl)-ester (trans -2-gg),
y
and crocetin-mono-(β -D-glucosyl) ester (trans -1-g). In 1984, Speranza et al.
op
demonstrated the existence of the cis -4-GG isomer (cis -13 configuration)
C
by subjecting the trans -4-GG isomer to white fluorescence light. Tarantilis
’s
or
but
tri
on
.C
C
LL
c is
an
Fr
d
an
All-trans crocetin 13-cis crocetin

or
yl
Ta
18
20
©
Picrocrocin Safranal
FIGURE 19.3
Three-dimensional chemical structures of the major secondary metabolites present in saffron
(atoms with red stand for oxygen, with dark gray for hydrogen and with light gray for carbon
ones). (From CS ChemDraw Ultra, version 5.0, CambridgeSoftCorporation, Cambridge, MA.
Copyright 1985– 1998.)
et al. (1995) identified by High Performance Liquid Chromatography – Diode

Array Detection-Mass Spectrometry another crocetin ester, [crocetin-tri-
(β -D-glycosyl)-(β -D-gentiobiosyl) ester (trans -5-tG)] in an aqueous methano-
lic saffron extract. Apart from all the above-mentioned crocetin sugar esters,
Carmona et al. (2006b) identified by LC-DAD-MS the presence of another
crocetin ester; namely, the crocetin-(β -D-gentiobiosyl)-(β -D-neapolitanosyl)
y
ester (trans -5-nG) was also found in an aqueous saffron extract.
nl
Despite the many suggestions, only recently has strong evidence for the
O
biosynthesis of the aglycone crocetin and its sugar esters become avail-
se
able. The first step includes the oxidative degradation of zeaxanthin
lU
{C40 H56 O2 , 4-[18-(4-hydroxy-2,6,6-trimethyl-1-cyclohexenyl)-3,7,12,16-tetra-
na
methyl-octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]-3,5,5-trimethyl-cyclohex-3-
o
rs
en-1-ol)} with the aid of the enzyme CCD2, which belongs to the group of
Pe
the carotenoid cleavage dioxygenases (CCDs) (Frusciante et al., 2014). As
r
shown by these authors, using transcriptome analysis, CCD2, expressed
fo
during stigma development, catalyzes the breakage of the 7,8 and 7′ ,8′
y
op
double bonds of zeaxanthin, leading to the formation of crocetin through
C
two intermediates, namely, 3-OH-β -apo-8′ -carotenal and crocetin dialde-
’s
hyde. Glycosylation of crocetin by glucosyl transferases (GTases) results in

or
the formation of crocetin sugar esters. This process is of particular impor-

ut
tance because due to their increased hydrosolubility, crocetin sugar esters

b
tri
are sequestered from chromoplasts, the compartment of their biosynthesis,

on
to vacuoles, where they are stored (Bouvier et al., 2003). This trend seems to
.C
follow the general rule, according to which accumulation of hydrosoluble

C
plant secondary metabolites in those cellular compartments is a prerequisite

LL
to exert their allelochemical roles (e.g., defense mechanisms or communica-

is
tion) (Wink, 2016).

c
an
Total crocetin sugar esters account for the ~30% of the dry weight of
Fr
the spice. Trans -4-GG is the most abundant member. It accounts for more
than 60% of the total crocetin sugar ester concentration, and together with
d
an
the trans -3-Gg, they account for more than 5% (Kyriakoudi et al., 2012).
or
Information about the concentration of total and individual crocetin sugar

yl
esters in saffron is rather limited due to the lack of commercially available

Ta
reference compounds and their high cost. Most of the information reported
18
in the literature is based on the use of synthetic colorants (e.g., 4-nitroaniline)

20
as internal standards (Lozano et al., 1999), the extinction molecular coeffi-

cient (ε ) of individual trans - and cis -crocetin sugar esters (Carmona et al.,
©
2005, 2006b; Caballero-Ortega et al., 2007), or an indirect quantification pro-

cedure to transform chromatographic peak areas to values of crocetin sugar
ester content (Sá nchez et al., 2008). Only very recently has quantification been
reported using laboratory-prepared (Kyriakoudi et al., 2012) or commercial
standards (Garcí a-Rodrí guez et al., 2014).
Apart from their coloring attributes, numerous medicinal properties have
been assigned to crocetin sugar esters and crocetin, which is not naturally
found free, but can be liberated from bound structures after consumption of
saffron or its extracts (Asai et al. 2005). In particular, an increasing number

of studies indicate the action of these apocarotenoids against several types
of cancer, cardiovascular diseases, gastric disorders, Alzheimer disease,
hepatotoxicity, atherosclerosis, mild to moderate depression, and antiradi-
cal properties (Alavizadeh and Hosseinzadeh, 2014; Singla and Bhat, 2011),
as well as against diabetic cataract (Bahmani et al., 2016). Based on all these
y
biological actions assigned to its major apocarotenoids, saffron was named a
nl
functional spice in a recent review article (Kyriakoudi et al., 2015b). However,
O
despite the numerous bioactivity studies of crocetin sugar esters and the par-
se
ent molecule, crocetin, information regarding their bioaccessibility and bio-
lU
availability is rather limited (Asai et al., 2005; Kyriakoudi et al., 2013, 2015a;
na
Lautenschlä ger et al., 2015; Ordoudi et al., 2015a). Such data are of great
o
rs
importance in order to establish oral intake limits or recommendations, since
Pe
only a certain amount of a bioactive ingredient is actually absorbed in the
r
bloodstream and reaches the target issues after ingestion.
fo
Being unsaturated compounds, crocetin sugar esters and crocetin are sus-
y
op
ceptible to degradation. Their stability, a critical factor for their use in food,
C
cosmetics, and pharmaceuticals, has been examined under the influence of
’s
parameters such as temperature, water activity, oxygen, and light (Alonso

or
et al., 1990; Tsimidou and Tsatsaroni, 1993; Tsimidou and Biliaderis, 1997). In
ut
recent decades, there has been a growing interest of the scientific community
b
tri
in the protection of these valuable apocarotenoids through encapsulation.

on
Crocetin sugar esters and crocetin have been so far encapsulated in various
.C
matrixes using techniques such as freeze drying (Selim et al., 2000; Chranioti
C
et al., 2015), spray drying (Zhou et al., 2013; Rajabi et al., 2015), and inclusion
LL
complexation (Kyriakoudi and Tsimidou, 2015).

c is
an
19.3.2 Saffron Secondary Metabolites Related to Its Flavor

Fr
Flavor, described as taste and aroma, is the most important quality charac-
d
an
teristic of saffron after its coloring properties. The characteristic bitter taste
or
of saffron is principally attributed to the colorless monoterpene glucoside

yl
picrocrocin [4-(β -d-glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-car-
Ta
boxaldehyde] (Figure 19.3). However, flavonoids and specifically glycosides of

18
kaempferol (e.g., kaempferol-3-sophoroside-7-glucoside, kaempferol-3,7,4′ -tri-

20
glucoside, and kaempferol-3-sophoroside), although present at low concen-

trations, could also contribute to the overall taste of the spice (Carmona et
©
al., 2006a). Extremely limited is the literature on the bitterness perception of

saffron (Sá nchez et al., 2010; Chrysanthou et al., 2015).
Picrocrocin is the second most abundant constituent of saffron, account-
ing for circa 10% of its dry weight (Kyriakoudi et al., 2012). As mentioned
above, saffron is a unique plant material regarding its composition in sec-
ondary metabolites considering that picrocrocin, together with crocetin
sugar esters, accounts for more than 40% of its dry weight. This value is
much higher than those observed for other plant materials, such as green
and black tea (catechins) (14%– 21% and 8%– 18%, w/w [Anesini et al., 2008])
and sage and rosemary (hydroxycinnamic acids) (~8%, w/w [Rababah et al.,
2011] and ~5%, w/w [Τ avassoli and Djomeh, 2011]). The biosynthetic path-
way of picrocrocin formation is similar to that of crocetin sugar esters. The
oxidative degradation of zeaxanthin with the aid of the CCD2 enzyme also
leads to the formation of 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cy-
y
clohexene (HTCC), which after glycosylation by GTases results in the forma-
nl
tion of picrocrocin.
O
The distinctive aroma of saffron is the result of the presence of many vola-
se
tile organic compounds (VOCs), among which the monoterpene aldehyde
lU
safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde) (Figure 19.3)
na
is the major one (Carmona et al., 2006a). Safranal does not exist in fresh stig-
o
rs
mas but derives from the oxidative or enzymatic degradation of picrocrocin
Pe
that takes place during drying and storage of the spice (Himeno and Sano,
r
1987; Raina et al., 1996). Examination of a large number of saffron samples
fo
has revealed that the major volatile compounds, besides safranal, are the
y
op
isophorone (3,5,5-trimethyl-2-cyclohexene-1-one), the 4-ketoiso-isophorone
C
(2,6,6-trimethyl-2-cyclohexene-1,4-dione), and the HTCC (Maggi et al., 2009).
’s
Saffron samples stored for less than a year after harvest and drying exhibit
or
spicy and floral notes assigned to safranal and isophorone. After prolonged
ut
storage (8– 9 years), these notes degrade while others, like caramel, citrus,
b
tri
and vegetable ones, appear (Maggi et al., 2010).

on
.C
C
LL
c is
19.4 Saffron Food Uses and Reported Fraudulent Practices

an
Fr
Saffron is highly valued in the food industry for exerting color and flavor
to food preparations. It is used as a spice, either in filaments or in powder
d
an
form, in many traditional European dishes, such as the paella Valenciana,

or
the French soup bouillabaisse, and the risotto alla Milanese, as well as
yl
in many Greek bakery products made in the Cyclades and Dodecanese

Ta
islands. Saffron consumption is more common in Iranian and Arab cui-

18
sine. Interestingly, saffron is included in the preparation of special products

20
meant to be consumed over a limited time period. This is the case of “ saf-
fron buns” that are traditionally prepared on St. Lucia Day celebrated during
©
Christmas in Sweden or Labrokouloura over the Easter period in Astypalaia

(Greece). Extracts of saffron are also used in alcoholic and nonalcoholic bev-
erages, such as Benedictine, vermouth, and other bitter drinks, as well as,
more recently, in herbal teas.
Due to its high price (up to € 20,000/kg in the retail market) (http://w3.cost.
eu/fileadmin/domain_files/FA/Action_FA1101/mou/FA1101-e.pdf), saffron
has been subjected to fraudulent practices over the centuries. Such prac-
tices occur more easily when the spice is traded in powder form and not in
filaments. Based on evidence from scientific articles on food fraud, saffron

possesses the fourth position as a target food matrix, after olive oil, milk,
and honey (Moore et al., 2012). Nevertheless, one has to consider that most
of the scientific articles present data for laboratory-made admixtures and not
detection of adulteration in commercial samples. The most frequent fraud-
ulent practices concerning saffron include (1) admixture with old saffron,
y
part of the style or stamens (auto-adulteration); (2) admixture with parts of
nl
other plant materials, such as gardenia dry fruits (Gardenia jasminoides Ellis),
O
safflower (Carthamus tinctorius L.), calendula (Calendula officinalis L.), parts
se
of the flowers of the plant Buddleja officinalis Maxim, curcuma rhizomes
lU
(Curcuma longa L.), or even other Crocus species; (3) addition of synthetic dyes;
na
(4) addition of animal substances (fibers of salted and dried meat); (5) mis-
o
rs
branding or falsification of origin; and (6) impregnation with substances to
Pe
increase weight (e.g., syrups, honey, glycerol, and oils) (US Pharmacopeial
r
Convention, Food Fraud Database, http://www.foodfraud.org/node, last
fo
accessed June 2016). As a consequence, it is important that consumers of all
y
op
ages be acquainted with authentic saffron in order to be able to protect them-
C
selves from buying nongenuine saffron.
’s
or
ut
b
tri
on
19.5 Recent Developments in Saffron Fraud Prevention

.C
C
Food fraud, or better, economically motivated adulteration (EMA), has been

LL
widely encountered in the everyday life of human societies throughout man-

is
kind’ s history. According to the FDA (https://www.fda.gov/Newsevents/

c
an
MeetingsConferencesWorkshops/ucm163619.htm 2009), EMA is defined as

Fr
“ the fraudulent, intentional substitution or addition of a substance in a prod-

uct for the purpose of increasing the apparent value of the product or reduc-
d
an
ing the cost of its production for economic gain.” In the majority of the cases,
or
EMA of a food product does not jeopardize public health since the adulter-
yl
ants are typically used only to replace more expensive ingredients and do
Ta
not intend harm (Everstine et al., 2013). However, in some cases, actual or
18
potential health risks have indeed occurred (e.g., the case of the addition
20
of melamine in milk-based products in 2007, http://www.foodsafetynews.

com). According to data from the Economically Motivated Adulteration
©
(EMA) & Intentional Adulteration (IA) Databases (https://www.foodshield.

org/discover-tools-links/tools/), adulteration is very common in the trade of
spices.
Introducing the word saffron as the only keyword, it is evident that the
interest of the scientific community toward saffron research is continuously
increasing (Figure 19.4) (Scopus, last accessed June 2016). However, as shown
in the figure, the majority of the scientific articles are related to its biological
200
“saffron”
180 “saffron” and “health” and “cancer”
160 “saffron” and “authenticity”

“saffron” and “traceability”
140
Number of publications
y
120
nl
O
100
se
lU
80
na
60
o
rs
40
Pe
20
r
fo
y
0
11 p
20 o
00
01
02
03
04
05
06
07
08
’s 2019
C0
12
13
14
15
16
0
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
Year
or
ut
FIGURE 19.4
b
Trends in the number of publications that contain the words saffron , saffron and health and can-
tri
on
cer , saffron and authenticity , and saffron and traceability in the title, abstract, or keyword fields,
in the period 2000– 2016 (Scopus search carried out in June 2016).
.C
C
actions, whereas publications that contain a combination of the keywords

LL
saffron and authenticity or traceability are limited.

is
The increasing cases of food fraud worldwide prove the inherent weak-
c
nesses in the food chain control and the limitations of authorities to take
an
action a priori (Tsimidou et al., 2016). Based on all the above, it is of out-
Fr
most importance to develop throughput analytical techniques in order to

d
an
combat saffron adulteration and fraud. Saffron is traded under specifica-

tions provided by ISO 3632-1 (2011) using methods described in ISO 3632-2
or
yl
(2010). These trade standards cover mainly quality aspects, whereas meth-
Ta
ods for fraudulent practices are restricted to those for the detection and
quantification of certain synthetic dyes using HPLC. To our knowledge,
18
no legislation covers more aspects for the detection of authenticity as is

20
the case for other spices. However, many scientists working in the area of
©
food authenticity, quality, and safety are paying particular interest in the
prevention of saffron fraud. Many different approaches have been devel-
oped, among which omic ones show great potential. Quoting Raghavachari
(2012), “ ‘ omics ’ refers to the collective technologies used to explore the
roles, relationships, and actions of the various types of molecules that
make up the cells of an organism.” In the last decade, omic approaches
have become an area of major research interest through the development
of high-throughput analytical techniques coupled to chemometrics. Using
90
80
70
Number of publications
60
50
y
40
nl
O
30
se
20
lU
10
na
0
o
2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015
rs
Year
rPe
fo
FIGURE 19.5
Trends in the number of publications that contain the words omics and food in the title, abstract,
y
op
or keyword fields, in the period 2002– 2015 (Scopus search carried out in June 2016).
C
’s
as keywords the combination of omics and food , it is evident that the inter-
or
est of the scientific community is steadily increasing toward this direction

ut
(Figure 19.5). This chapter covers the major recent analytical achievements,
b
tri
taking into account not only the methodology followed but also the valid-
on
ity of samples examined.

.C
C
LL
19.5.1 Omic Approaches Developed in the Frame of

the COST Action FA1101 Saffron-OMICS
cis
an
Since 2011, many scientists have joined efforts and expertise to combat
saffron fraud by enriching the armory of analytical methods in a comple-
Fr
mentary way within the frame of the COST Action FA1101 Saffron-OMICS
d
an
(Omics Technologies for Crop Improvement, Traceability, Determination of

or
Authenticity, Adulteration and Origin in Saffron). (http://w3.cost.eu/filead-

yl
min/domain_files/FA/Action_FA1101/mou/FA1101-e.pdf). In the frame of

Ta
this action, focus was paid to omic approaches. The network of scientists
18
involved devoted time and national sources of funding to collaborate on the

20
genomics, proteomics, and metabolomics of saffron. The outcome of these

efforts was presented in seminars, scientific meetings, and annual confer-
©
ences of the action, at different national and international scientific forums,

and was published in reputable Science Citation Index (SCI) journals. In this
section, only the latter group of publications is cited and discussed. For those
interested in the rest of the outcomes of the network, details are available on
the official webpage of the action.
19.5.1.1 Genomics
According to Zhang et al. (2010), genomics is defined as “ the systematic
study of nucleotide sequences in the genome (i.e. the total DNA) of a cell or
organism.” In the frame of Saffron-OMICS, Alsayied et al. (2015) used the
inter-retroelement amplified polymorphism (IRAP) technique for the deter-
mination of genetic variability of C. sativus L. and related Crocus species. The
y
nl
authors suggested that “ in contrast to the intraspecific variability seen in
O
other Crocus species, C. sativus has minimal genetic variation.”
se
Moreover, Busconi et al. (2015) used the amplified fragment length poly-
lU
morphism (AFLP) and the methyl-sensitive amplified fragment length
na
polymorphism (MS-AFLP) techniques to examine the level of genetic and
o
epigenetic variability inside the species C. sativus L. AFLP is a molecu-
rs
lar marker technique with wide applications, such as population genet-
Pe
ics and linkage mapping (Meudt and Clarke, 2007), while MS-AFPL is
r
fo
a technique that can efficiently monitor variation in the methylation of
y
DNA at several restriction sites (loci) (Schrey et al., 2013). Busconi et al.
op
(2015) suggested that “ epigenetic variations, in contrast to genetic ones,
C
can be influenced by environmental conditions so that samples cultivated
’s
or
in different areas can be characterized by different epigenomes.” In par-

ut
ticular, the authors managed to cluster, in two separate groups, Spanish

b
saffron samples based on their geographical origin, that is, east (Cuenca
tri
on
and Teruel) and west (Toledo and Ciudad Real) Spain, using factorial cor-
.C
respondence analysis (FCA).

Soffritti et al. (2016) used genetic and epigenetic approaches to detect cases
C
LL
of adulteration and auto-adulteration in saffron. In particular, the authors

compared different DNA extraction methods to recover polymerase chain
c is
reaction (PCR)– grade DNA in order to develop DNA markers to identify

an
the presence of adulterants such as B. officinalis , G. jasminoides , C. longa ,

Fr
C. tinctorius , and C. officinalis in saffron. The markers developed based on

d
the sequence of the plastid genes mat K were found to be very selective and
an
allowed the detection and discrimination of the examined plant adulterants

or
from saffron even at low concentrations. Moreover, the authors used the
yl
Ta
AFLP and MS-AFLP techniques to compare the genetic and epigenetic pro-
files of different parts of the C. sativus L. plant, such as stigmas, stamens, and
18
tepals. They concluded that the marker profile of whole stamens (anthers
20
and filaments) differed heavily from that of stigmas and tepals. According to
©
the authors, these findings are of particular importance toward the detection
of cases of auto-adulteration.
Another genomic tool that has gained great attention within the scientific
community for its potential use for specific recognition of plant, animal,
and fungi species is DNA barcoding. Within Saffron-OMICS, DNA mini-
barcodes (i.e., short and standardized regions of the genome), specifically
the ITS and mat K regions, coupled with high-resolution melting (HRM),
were exploited as molecular markers to differentiate C. sativus L. from other
Crocus species, such as Crocus kosaninii , Crocus kotschyanus , Crocus speciosus ,

Crocus olivieri balansae , and C. cartwrightianus (Villa et al., 2016). The authors
suggested that the ITS1 and mat K loci (i.e., specific location of a gene) were
Crocus genus specific, while the ITS2 locus was species specific for C. sativus
and C. cartwrightianus .
y
nl
19.5.1.2 Proteomics
O
According to Chang and Huang (2010), proteomics can be defined as “ the
se
study of all the proteins expressed in a cell, tissue, or biological fluid, i.e.
lU
the ‘ proteome’ , at a given time.” In the frame of the Saffron-OMICS, pro-
na
teomic tools were exploited for the first time to characterize the proteome of
o
rs
saffron and to detect possible frauds (Paredi et al., 2016). In particular, one-
Pe
dimensional (1D) gel electrophoresis was employed to separate the proteins
r
contained in fresh and dried stigmas and styles of the C. sativus plant of
fo
the same geographical origin (i.e., Spanish). The authors suggested that fresh
y
op
stigmas and styles contained more bands than the dried ones. Some of the
C
bands detected in the fresh stigmas and styles were then subjected to peptide
’s
mass fingerprinting (PMF) analysis in order to identify the proteins present

or
(Table 19.1). The second objective of that study was the characterization of
ut
the protein pattern of saffron samples from different geographical origins

b
tri
(Spanish, Italian, Greek, and Iranian) stored for various periods of time after
on
processing using 1D sodium dodecyl sulfate– polyacrylamide gel electropho-

.C
resis (SDS-PAGE). The results showed differences in the number of detected

C
proteins among the examined samples (n = 19, Spanish; n = 16, Italian ones
LL
stored for a few months after processing; n = 19, Greek; and n = 12, Iranian
is
ones stored for a long period). Moreover, the authors used 1D SDS-PAGE to
c
an
compare the protein pattern of saffron with two common plant adulterants,
Fr
that is, the dried petals of C. tinctorius L. and the dried fruits of G. jasminoides
d
an
TABLE 19.1
or
Identity of Proteins Contained in the Sample of Fresh C. sativus L. Stigmas and

yl
Styles.
Ta
Band (kDa) Protein Uniprot Scorea Coverage %

18
99.4 Phosphoenol pyruvate CAPP3_ARATH 83 13

20
carboxylase-3
70 Heat shock cognate 70 kDa HSP7C_PETHY 65 29
©
protein
49.4 Crocetin glucosyl transferase-2 GLT2_CROSA 78 30
42.7 A-1,4-glucan-protein synthase UPTG_PEA 64 22
38.5 Glyceradehyde-3-phosphate G3PC_ORYSJ 85 34
dehydrogenase-2b
Source : Reproduced with permission from Paredi, G., et al., Molecules 21, 167, 2016.
a Mascot score. All values are above the threshold of confidence.
b Identification was further confirmed by matrix-assisted laser desorption/ionization tandem
mass spectrometry (MALDI MS/MS).
Ellis, and found them to differ. The authors suggested that proteome analy-
ses could be exploited for the detection of possible cases of fraud.
19.5.1.3 Metabolomics
Metabolomics, that is, “ the study of global metabolite profiles in a biologi-
y
cal system (organelle, cell, tissue or organism) under specific environmen-
nl
tal conditions,” is one of the newest omics technologies (Benkeblia, 2012).
O
Metabolites are the end products of metabolic reactions and reflect the
se
interaction of the system’ s genome with its environment (Lau et al., 2010).
lU
A metabolomic approach coupled with chemometrics (principle compo-
na
nent analysis [PCA]) was adopted by Ordoudi et al. (2014) for the quality
o
rs
control of traded saffron and, in particular, for the assessment of saffron
Pe
freshness. The authors investigated the changes that occur on the typical
r
Fourier-transform midinfrared (FT-MIR) spectrum of saffron as a result of
fo
storage under conditions that favor oxidative or hydrolytic decomposition
y
op
of bound secondary metabolites, such as the sugar esters of crocetin and
C
picrocrocin. Changes were monitored in the range 4000– 400 cm– 1 . A total
’s
of 52 saffron samples were examined consisting of 17 samples from the

or
Laboratory of Food Chemistry and Technology (LFCT) sample collection

ut
from Saffron Producers Cooperative of Kozani (Greece) (collection years

b
tri
1999, 2000, 2011, and 2012) and 35 samples from the WSCC (Cuenca, Spain)
on
(collection years 2009, 2010, and 2011). The authors suggested that specific
.C
infrared bands associated with the presence of glucose moieties and break-
C
age of glycoside bonds (1028 and 1175– 1157 cm– 1 , respectively) are of the
LL
most importance to monitor the storage effects and detection of deteriora-

is
tion of traded saffron.

c
an
As a continuation of the above contribution, a 1H-nuclear magnetic reso-

Fr
nance (NMR)– based metabolomics approach was adopted by Ordoudi et al.

(2015b) for the detection of saffron quality deterioration in order to overcome
d
an
the limitations of the FT-IR technique regarding the structure elucidation.

or
For the requirements of that study, a total of 98 saffron samples of differ-

yl
ent origins and harvest years, stored under different conditions for various
Ta
periods of time, were examined. In particular, the study involved 51 Greek

18
saffron samples, 21 Spanish and 24 Iranian from the LFCT sample collec-
20
tion (harvest years 1999, 2002, 2004, 2005, 2006, 2008, 2009, 2010, 2011, and
2012), and 2 Italian samples (protected designations of origin [PDOs] of
©
L’ Aquila and Sardenia) (harvest year 2012). The 1H-NMR data of the exam-
ined samples were subjected to PCA and orthogonal projections to latent
structures– discriminant analysis (OPLS-DA), which showed that bound or
free forms of glucose and gentiobiose, as well as fatty acids, can be consid-
ered markers of the quality deterioration of saffron.
A 1H-NMR-based metabolomic approach coupled with chemometrics was
also employed for the detection and identification of four commonly used
plant adulterants in saffron (Petrakis et al., 2015). The latter were stamens
of C. sativus L. (branded as “ safran” ), turmeric (branded as “ like safran” ),

and safflower (branded as “ Turkish saffron” ) that were purchased from
local markets, as well as G. jasminoides fruit extract that was purchased
from a company. The authors observed different signals in the 1H-NMR
spectra that are characteristic for each plant material (Figure 19.6). The use
of the OPLS-DA model resulted in the differentiation of adulterated from
y
pure saffron based on specific secondary metabolites. During a second
nl
step, the O2PLS-DA model allowed the identification of the type of plant
O
adulterant.
se
A combined approach of FT-IR and 1H-NMR metabolomics coupled with
lU
chemometric tools was also employed in order to trace back the “ age” of
na
17 commercial saffron samples of unknown history purchased from open
o
rs
markets and retail shops in major saffron-consuming countries (Qatar,
Pe
9; Israel, 2; Kingdom of Saudi Arabia [KSA], 4; and Italy, 2) (Consonni et
r
al., 2016). In particular, the FT-IR database of authentic fresh (stored for
fo
less than 4 years after processing) and nonfresh (stored for 7– 12 years)
y
op
samples was used to build a PLS-DA, partial least squares— d iscriminant
C
analysis (PLS-DA) model that was employed for the classification of the
’s
examined commercial saffron samples. The authors found that most of

or
them were very close or already had passed the cutoff point of 4 years
ut
after harvest and processing (Figure 19.7) suggested by Ordoudi et al.

b
tri
(2015b). Moreover, data from the 1H-NMR analysis were applied to an

on
OPLS-DA model to evaluate the freshness of the corresponding samples.

.C
The results were in partial agreement with the ones obtained from the
C
FT-IR approach.
LL
All the previous metabolomic approaches took into account mainly the
is
major nonvolatile secondary metabolites, the crocins and picrocrocin, and

c
an
changes that may be encountered due to various processing and storage

Fr
conditions. The volatile fraction was considered in another publication

of the network. In particular, the metabolomic approach using the non-
d
an
destructive proton transfer reaction– mass spectrometry (PTR-MS) tech-

or
nique coupled with chemometrics (PCA) was applied for the first time
yl
for the detection of lower-quality saffron added to fresh saffron (Nenadis

Ta
et al., 2016). PTR-MS that allows quantitative on-line monitoring of volatile

18
organic compounds is a relatively new technique that is gaining popular-

20
ity for food authentication studies (Capuano and Van Ruth, 2012). So far,
PTR-MS has been employed for the differentiation of various grana cheeses
©
(Boscaini et al., 2003), the determination of the geographical origin of olive

oils (Araghipour et al., 2008), and the classification of butter and butter oil
(Van Ruth et al., 2008). In this view, Nenadis et al. (2016) focused on the
volatile fingerprint of saffron that changes upon storage. The authors suc-
ceeded in using only a minute amount of saffron (35 mg) for obtaining the
fingerprint, and they suggested that PTR-MS could be used complementa-
rily with other advanced analytical techniques to address the quality and
authenticity issues of saffron.
Pure saffron
8 7 6 (ppm)
y
nl
O
se
lU
Turmeric
na
o
rs
8 7 6 (ppm)
Pe
(a)
r
fo
y
op
C
’s
Stamens
or
but
8 7 6 (ppm)
tri
(b)
on
.C
C
LL
c is
Safflower
an
Fr
8 7 6 (ppm)
d
(c)
an
or
yl
Ta
18
20
Gardenia
©
8 7 6 (ppm)
(d)
FIGURE 19.6
Selected regions of 1 H-NMR spectra acquired from DMSO-d6 extracts. A squared-top spec-
trum is characteristic of pure saffron. Spectra of pure plant material turmeric, C. sativus sta-
mens, safflower, and G. jasminoides fruit extract) are reported in panels (a)–(d), respectively in
bottom traces, while spiked saffron with a 20% (w/w) concentration of plant adulterants are
reported in panels (a)–(d) in the top traces. (Reproduced with permission from Petrakis, E. A.,
et al., Food Chemistry , 173, 890– 896, 2015.)
22.98
13
16
14
10
9 15
6 1
12 11
7
–17.7 8 30.76
4 3
y
17
nl
2 5
O
Fresh Non-fresh
se
LC2 (45.7%)
lU
na
o
–30.8
rs
LC1 (38.6%)
rPe
fo
FIGURE 19.7
Scatterplot of the PLS-DA model performed considering 52 authentic saffron samples of differ-
y
op
ent geographical origins, and harvest year, and stored under different conditions for different
C
periods (fresh, 0– 4 years of storage; nonfresh, 7– 12 years of storage). LC1 = 38.6%, LC2 = 45.7%,
R2 (Y) = 78%, Q2 = 77%. Blue, green, and white dots represent fresh, nonfresh, and the 17 com-
’s
or
mercial samples of unknown storage history, respectively. (Reproduced with permission from
ut
Consonni, R., et al., Molecules , 21, 286, 2016.)

b
tri
on
19.5.2 Other Recent Advances in Saffron Fraud Prevention

.C
In parallel to the activities of the Saffron-OMICS network of scientists

C
LL
(2011– 2016), other omic approaches have been developed that are summa-
rized in Table 19.2. Regarding genomic approaches, DNA barcoding has
c is
been used by the group of Canini (University of Rome II) (Gismondi et al.,
an
2013) in collaboration with another research group from Spain. In their pre-
Fr
liminary work, they reported the use of DNA barcoding coupled to sequenc-
d
ing as a tool for the C. sativus genetic characterization for future use in cases
an
of saffron adulteration. A similar objective was reported by the Iranian

or
group of Mardi (Nemati et al., 2012) involving polymorphic microsatellite

yl
Ta
markers. Moreover, scientists from two Chinese institutions (Huang et al.,

2015) used this technique to differentiate saffron from other plant materi-
18
als often used as adulterants in their country (e.g., C. tinctorius L., Nelumbo
20
nucifera , Crysanthemum × morifolium , and Zea mays ). The research group of
©
Bruni (Parma University, Italy) developed a method based on sequence-char-

acterized amplified regions (SCARs) to detect plant adulterants (C. tinctorius ,
C. officinalis L., Bixa orellana L., and C. longa L.) in saffron (Marieschi et al., 2012;
Torelli et al., 2014). Three more Chinese groups (Zhao et al., 2016) employed
the loop-mediated isothermal amplification (LAMP) technique in order to
differentiate saffron from common adulterants, such as C. longa , N. nucifera ,
Z. mays , C. officinalis , and C. tinctorius. During the same period, an untar-
geted metabolomic approach using liquid chromatography– (quadrupole
©
TABLE 19.2
20 668
18
Objectives, Employed Techniques, Sample Requirements, and Statistical Treatment of Data Obtained from Methods Developed by
Research Groups outside the Saffron-OMICS Network
Ta
yl
or Data
Statistical
Objective (Reference) Technique Sample Requirements Treatment Main Findings
an
d
Genomic Fr Approaches
Development of SCAR SCAR Leaves from an C. sativus plants originated — SCAR markers offer advantages, such
markers for the detection of markers from bulbs c (Botanical Gardens of Cagliari) as low operating costs and good
seven common bulking cultivated in pots,
is 6 dried commercial interlaboratory replicability. The
agents in saffron (Marieschi saffron samples (stigmas) method allows the detection of low
LL (Morocco, n = 2;
et al., 2012) Spain, n = 2; Afganistan,
C n = 1, Italy, amounts (up to 1%) of each adulterant.
n = 1), seeds of C. tinctorius
. C , Bixa orellana It can be used to screen large saffron
seeds, C. longa rhizomes (collection
on of fresh batches.
plant material, freeze drying, tstorage
– 80° C) rib at
Development of a first set of FIASCO 50 C. sativus samples from differentut — The reported microsatellite markers can
polymorphic microsatellites geographical areas in Iran (Ghayen, or be used for evaluating genetic
in saffron for genetic Gonabad, Birjand, Ferdows, and Istahban) ’s diversity, population structure, genetic
similarity studies (Nemati C conservation, and germplasm
et al., 2012) op management of C. sativus .
Genetic characterization of DNA Corms of the plant C. sativus were obtained y
— The DNA barcoding method was
C. sativus (Gismondi et al., barcoding by producers (Civitaretenga, Italy), Crocus applied to intraspecific discriminant
fo
2013) carpetanus (Spain), Crocus serotinus (Spain),
r analysis (i.e., population studies). It
C. cartwrightianus , Crocus adriaticus , and was proposed to also be used as a tool
Pe
Crocus thomasii (Botanical Gardens of the to certify and trace saffron origin.
rs
University of Rome “ Tor Vergata” )
o na
lU (Continued)
se
O
nl
y
©
20
Ta
Research Groups outside the Saffron-OMICS Networkyl
Data
or
an Statistical
Objective (Reference) Technique d Sample Requirements Treatment Main Findings
Authentication of saffron SCAR 17 commercial saffron samples (ground) — The method had good performance in
Fr
(C. sativus L.) in different markers purchased from shops and supermarkets
an both DNA extraction and amplification
processed retail products (Italy and Spain), 4 samples of dried rice
c stages regardless of the presence of
(Torelli et al., 2014) preparations (ready-made lyophilized
is lipids, sugars, and phenolic
“ Paella valenciana” with fish and compounds. It is a fast, reliable, and
LL
vegetables [n = 1] and ready-made
C low-cost screening method for the
“ Risotto alla minese” [n = 3]), 1 seasoning
.C authentication of saffron.
(declared ingredient list: garlic, salt (25%),
paprika, corn flour, pepper, cloves, and
on
saffron [2.5%]), 1 seasoning (declared
tri
b
ingredient list: turmeric, paprika, garlic,
ut
bay, allspice, coriander, saffron [1%], or
cloves, and pepper), and 1 herbal tea
Recent Developments in Saffron Fraud Prevention
’s
(declared ingredient list: green tea (99%) C
and saffron [1%]) op
Identification of C. sativus DNA 1 authentic saffron sample, 11 sold as —
y A total of 39 DNA sequences from 3
from its adulterants (Huang barcoding “ saffron,” 2 samples of C. tinctorius , 1 fo DNA barcodes (trn H-psb A, rbc L-a, and
et al., 2015) sample of N. nucifera , 1 sample of r ITS2) were generated. trn H-psb A and
Chrysanthemum × morifolium , and 1 sample rbc L-a were capable of distinguishing
Pe
of Z. mays obtained from 12 different rs different accessions. ITS2 could
provinces of China oidentify samples even at the
intraspecific level.
na
(Continued)
lU
se
669
O
nl
y
©
20 670
18
Ta
yl
or
an
TABLE 19.2 (CONTINUED) d
Fr
Research Groups outside the Saffron-OMICS Network
an
cis Data
LL Statistical
Objective (Reference) Technique Sample Requirements
C Treatment Main Findings
Development of a method for LAMP
.
10 saffron samples (Tibet,CXinjiang, and — The LAMP technique is based on a
saffron authentication (Zhao Hubei), 39 plant materialsoas common complex methodology requiring 4– 6
et al., 2016) adulterants (Daucus carota , n n =t 9; C. longa , different primers that are specifically
r
n = 6; N. nucifera , n = 6; Z. mays i,bn = 6; designed to recognize 6– 8 gene
C. officinalis , n = 6; C. tinctorius , n =
ut 6) sequencings. The method developed is
or specific and sensitive.
Metabolomic Approaches
’s
Investigation of the quality LC-QTOF 10 authentic saffron samples (stigmas or
C Authenticity markers were proposed
and authenticity of saffron powder) from Spain and Iran and 10 PLS-DA, (n = 34), but no marker related to
opPCA,
with an untargeted commercial saffron samples (stigmas or y
and geographical origin was found. The
metabolomic strategy powder) from a spice company
f
OPLS-DA
or proposed markers were derivatives of
(Guijarro-Dí ez et al., 2015) Pe kaempferol and geranic acid.
Note : FIASCO, fast isolation by AFLP of sequences containing repeats. rs
on
al
U
se
O
nl
y
time-of-flight) mass spectrometry (LC-[QTOF]MS) and multivariate statisti-

cal analysis (PCA, PLS-DA, and OPLS-DA) was carried out by the research
group of Guijarro-Dí ez et al. (2015). Ten authentic Iranian and Spanish saf-
fron samples and 10 commercial ones were provided by a spice company.
A great number of metabolites were detected as candidates for authentic-
ity and geographical origin by testing mass accuracy against online avail-
y
able databases. A clearance process reduced this number dramatically. Only
nl
authenticity markers were finally proposed (n = 34), and no marker related
O
to geographical origin was found. The proposed markers were derivatives of
se
kaempferol and geranic acid. Their findings need further substantiation, as
lU
the models were built using a confined number of samples.
ona
rs
rPe
fo
y
19.6 Overview op
C
Saffron is a potential target for fraudulent practices all over the world.
’s
Consumers know the name of the spice, but the majority of them ignore how
or
it looks and tastes and can be easily deceived when they buy it in various
ut
spice markets. In recent years, in the frame of Saffron-OMICS many scien-

b
tri
tists joined efforts and exchanged samples and expertise to combat saffron
on
adulteration by enriching the armory of analytical methods in a complemen-

.C
tary way. The results of these efforts justify well those of the COST European
C
framework to “ urge interdisciplinary scientists to jointly develop their

LL
own ideas and new initiatives across all fields of science and technology
is
through trans -European networking of nationally funded research activi-

c
an
ties.” Achievements of other research groups from Europe, China, and Iran
Fr
point to the need for strengthening international collaboration in order to

speed up research against food fraud. Molecular approaches and metabo-
d
an
lomics using spectroscopic methods seem to prevail among the analytical

or
techniques employed.
yl
Ta
18
20
©
Acknowledgments
The European Science Foundation (ESF) is acknowledged for strengthening
the collaboration between the groups involved in the COST Action FA1101
“ Omics Technologies for Crop Improvement, Traceability, Determination
of Authenticity and Origin in Saffron.” This work is part of the continuing
efforts of MZT (chair) to disseminate the outcomes and value of data pro-
duced in the frame of this action. MZT thanks AK (WG4 member, ESR) for
her devotion to the action and multiple tasks undertaken for dissemination
activities of the scientific achievements.
y
Relevant Websites
nl
O
http://www.crocusbank.org
se
http://comtrade.un.org: United Nations Comtrade Database
lU
http://w3.cost.eu/fileadmin/domain_files/FA/Action_FA1101/mou/
na
FA1101-e.pdf: Memorandum of understanding of the COST Action FA1101
o
http://www.foodfraud.org/node: U.S. Pharmacopeial Convention, Food
rs
Pe
Fraud Database
https://www.fda.gov/Newsevents/MeetingsConferencesWorkshops/
r
fo
ucm163619.htm : U.S. Food and Drug Administration
y
http://www.foodsafetynews.com op
https://www.foodshield.org/discover-tools-links/tools/: Economically
C
Motivated Adulteration (EMA) and Intentional Adulteration (IA) Databases

’s
or
ut
b
tri
on
.C
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Index
1,2-Bis(2,4,6-tribromophenoxy)ethane flavor/odor absorbers and releasers,

(BTBPE), 254 471–472
y
nl
1D sodium dodecyl sulfate– free radical scavengers, 467–468
O
polyacrylamide gel legislation, 477, 482
se
electrophoresis (SDS-PAGE), microwaveable, 472
lU
663 moisture control, 471
na
1H-nuclear magnetic resonance (NMR), nanotechnology, biomaterials, and
o
513, 515, 664 bioactive compounds used in,
rs
2D Thin-layer chromatography (TLC), 476–477
Pe
57 other approaches, 470
r
4Cs approach, see Cleaning, cooking, oxygen scavengers, 466–467
fo
cross-contamination, and Acute reference doses (ARfDs), 132
y
chilling (4Cs) approach op
Acute urticarial, 203
5 Carboxyfluorescein (FAM)–labeled Additives, 281, 504
C
aptamer, 174 Adiabatic heating, 399

’s
or
12-D process, 273 ADIs, see Acceptable daily intake (ADIs)

ut
2014 Technology Transfer Report, 208 Adulteration, 499

b
μTAS, see Micro total analysis system Advanced glycation end products
tri
(μTAS) (AGEs), 257

on
AEO, see Allium sativum essence oil

.C
(AEO)
A
C
Aerobic mesophilic bacteria (AMB),

LL
AA, see Acrylamide (AA) 33–34

is
Accelerated solvent extraction (ASE), AF, see Aflatoxins (AF)

c
an
130 Affinity, 166–167

Acceptable daily intake (ADIs), 252 Aflatoxins (AF), 52–60, 162–163
Fr
Acetic acid, 314 AFLP, see Amplified fragment length

d
an
Acetonitrile (ACN), 74, 79, 82 polymorphism (AFLP)

ACGIH, see American Conference of Ag-based nanoparticles, 449
or
Governmental Industrial Agent Orange, 253

yl
Ta
Hygienists (ACGIH) AGEs, see Advanced glycation end

ACN, see Acetonitrile (ACN) products (AGEs)
18
ACP, see Atmospheric cold plasma AgNPs, see Silver nanoparticles (AgNPs)
20
(ACP) Agreement on Sanitary and

©
Acrylamide (AA), 259–260 Phytosanitary Measures (SPS

Active food packaging, 305–306, Agreement), 534, 535–536
460–462 Agreement on Technical Barrier to
antimicrobial, 468–470 Trade (TBT Agreement), 534
antioxidant, 462–466 Agricultural Marketing Service
carbon dioxide emitters and (AMS), 590
scavengers, 470 Alexandrium catenella, 108, 109
commercially available, 477 Alexandrium peruvianum, 124
ethylene scavenging, 471 Alginate, 449
679
680 Index
Alginate-based antimicrobial films, whey protein isolate–based films,

433–435 438–440
Allergic eosinophilic esophagitis, 205 overview, 431–433
Allergy and Immunology Institute, 218 polymer/clay nanocomposite-based
Allium sativum essence oil (AEO), antimicrobial films, 445–446
441–442 Antioxidant packaging, 462–466
AMB, see Aerobic mesophilic bacteria AOAC, see Association of Official
y
nl
(AMB) Analytical Chemists (AOAC)
O
American Conference of AOAC Journal, 128
se
Governmental Industrial APHA, see American Public Health
lU
Hygienists (ACGIH), 247 Association (APHA)
American Public Health Association Apocarotenoids, 655, 657
na
(APHA), 127, 128 APPI, see Atmospheric pressure
o
rs
Amnesic Shellfish Poison (ASP), 128 photoionization (APPI)
Pe
Amplified fragment length Aptamers, 165–182
polymorphism (AFLP), 662 application in food safety, 168
r
fo
AMS, see Agricultural Marketing heavy metals, 168–172
y
Service (AMS) mycotoxins, 174–177
op
Anaphylaxis, 202, 203 pesticides, 172–174
C
Antibacterial alginate films, 435 pharmaceutical residues, 180–182
’s
Antibacterial polymer, 431 phenols, 177–180

or
ut
Antibrowning treatment, 32 definition, 165

b
Antimicrobial agents, 432 food contaminant aptasensing, 182

tri
Antimicrobial bionanocomposite properties, 166–167

on
films, 438 affinity, 166–167

.C
Antimicrobial films, 431, 433 specificity, 167

C
Antimicrobial food packaging, 431–452, SELEX technology, 165–166

LL
468–470 structure and aptamer–target

is
action mechanisms, 433 interactions, 167–168

c
advantages and difficulties, 448–451 example of cadmium aptamer, 168

an
chemically synthesized Aptasensing, food contaminant, 182

Fr
biodegradable polymers, ARfDs, see Acute reference doses

d
442, 445 (ARfDs)
an
poly-L-lactide-based Arsenic contamination (As), 161,

or
antimicrobial films, 442, 445 249–250

yl
essential oils and their compound- ASEAN, see Association of Southeast

Ta
based films, 440–442 Asian Countries (ASEAN)

18
metal and metal oxide nanoparticle- ASE, see Accelerated solvent extraction
20
based antimicrobial films, (ASE)

446–448 ASP, see Amnesic Shellfish Poison (ASP)
©
natural biopolymers, 433–440 Aspergillus flavus, 162

alginate-based antimicrobial Aspergillus parasiticus, 162
films, 433–435 Association of Official Analytical
carrageenan-based antimicrobial Chemists (AOAC), 129
films, 438 Association of Southeast Asian
chitosan-based antimicrobial Countries (ASEAN), 134
films, 436–437 Association of the Brazilian Coffee
gelatin-based antimicrobial films, Industry, 504
435–436 Ataxia, gluten, 210
Index 681
Atmospheric cold plasma (ACP), 409–414 control measures for, 132–138

biological effects, 410–411 legislation, 132–134
engineering principles, 409–410 managing, 134–138
limitations, challenges, and future marine, 99–126
trends, 414 azaspiracid group toxins, 105–108
practical applications, 411–414 brevetoxins, 114–116
Atmospheric pressure photoionization ciguatoxin group toxins, 112–114
y
nl
(APPI), 60 cyclic imines, 123–126
O
Atopic dermatitis, 204 domoic acid, 111–112
se
Audits, food safety, 551–553 okadaic acid group toxins, 99–102
lU
types, 552–553 palytoxin and analogs, 120–123
AuNPs, see Gold nanoparticles (AuNPs) pectenotoxin group toxins,
na
AZA, see Azaspiracid (AZA) group 102–103
o
rs
toxins saxitoxin group toxins, 108–111
Pe
Azadinium poporum, 105 tetrodotoxin, 116–120
Azadinium spinosum, 105 yessotoxin group toxins, 103–105
r
fo
Azaspiracid (AZA) group toxins, methods of analysis, 126–131
y
105–108 ASP toxins, 128
op
chemical structures and mechanism brevetoxins, 127–128
C
of action, 105–107 ciguatoxins, 129
’s
episodes of intoxication, 107–108 lipophilic toxins, 126–127

or
ut
occurrence and accumulation, 107 PSP toxins, 129

b
origin and distribution, 105 QuEChERS approach for, 130–131

tri
Azaspiracid poisoning (AZP) tetrodotoxin, 130

on
syndrome, 105 overview, 98–99

.C
risk assessment for, 131–132

C
Bisphenol A (BPA), 255–256

B
LL
Boston Consulting Group, 543

is
Bacillus cereus, 343, 344 BPA, see Bisphenol A (BPA)

c
Bacillus licheniformis, 343 BPNs, see Biodegradable polymer

an
Bacillus pumilus, 344 nanocomposites (BPNs)

Fr
Bacteriocins, 316–318 Brazilian FBDs, 7–9

d
Barcode DNA High Resolution Melting Brazilian Health Surveillance Agency, 5

an
(Bar-HRM), 510–511 Brazilian National Health Authority, 8

or
BAs, see Biogenic amines (BAs) BRC, see British Retail Consortium
yl
Basmati rice, 510 (BRC)

Ta
Benzothiazolium-4-quinolinium dimer BRC Food Technical Standard, 640

18
derivative, see TOTO-3 BRC Global Standards, 640

20
BFRs, see Brominated flame retardants Brevetoxins (BTXs), 114–116, 127–128

(BFRs) chemical structures and mechanism
©
Bioactive agents, 461 of action, 115

Biodegradable polymer nanocomposites episodes of intoxication, 116
(BPNs), 432 occurrence and accumulation in
Biogenic amines (BAs), 258 seafood, 116
Biologics Control Act, 566 origin and distribution, 114–115
Biopolymer films, 432 British Retail Consortium (BRC), 545,
Biopolymers, 448 640
Biosensors, 474–475 Brominated flame retardants (BFRs),
Biotoxins, 97–138 252–254
682 Index
Brominated phenols, 252–254 CFSA, see Chinese National Center for

BT Act, see Public Health Security and Food Safety Risk Assessment
Bioterrorism Preparedness and (CFSA)
Response Act (BT Act) CFSAN, see Center for Food Safety and
BTBPE, see 1,2-Bis(2,4,6- Applied Nutrition (CFSAN)
tribromophenoxy)ethane CGMPs, see Current good
(BTBPE) manufacturing practices
y
nl
BTXs, see Brevetoxins (BTXs) (CGMPs)
O
Cheese reaction, see Tyramine toxicity
se
Chemical antimicrobial additives, 432
C
lU
Chemically synthesized biodegradable
Cadmium aptamer, 168 polymers, 442, 445
na
Cadmium (Cd) contamination, 160, Chemical sensors, 475
o
rs
247–248 Chemoactive agents, 461
Pe
Campylobacter jejuni, 279 Chinese food safety system, 541–542
Cam, see Chloramphenicol (Cam) Chinese National Center for Food
r
fo
Canadian Food Inspection Agency Safety Risk Assessment
y
(CFIA), 129, 136 op(CFSA), 521
Capillary electrophoresis with Chitin nanofibrils (CNFs), 438
C
laser-induced fluorescence Chitooligosaccharides (COSs), 440
’s
(CE-LIF), 173 Chitosan, 319

or
ut
Capillary liquid chromatography- Chitosan-based antimicrobial films,

b
electrospray ionization- 436–437

tri
tandem mass spectrometry Chitosan nanoparticles (CSNPs), 436

on
(CapLC-ESI-MS/MS), 519 Chloramphenicol (Cam), 164, 165

.C
C.A.P.P.A.B.L.E.©, 39–41 Chlorine, 313

C
Carbohydrate-based polymers, 433 Chlorine dioxide (ClO2 ), 416–418

LL
Carbohydrate biopolymer, 434 Chromatographic methods, 52

is
Carbon dioxide emitters and Ciguatoxin (CTX) group toxins,

c
scavengers, 470 112–114, 129

an
Carrageenan-based antimicrobial chemical structures and mechanism

Fr
films, 438 of action, 113

d
CCP, see Critical control point (CCP) episodes of intoxication, 113–114

an
CDC, see Centers for Disease Control occurrence and accumulation in

or
and Prevention (CDC) seafood, 113

yl
cDNA, see Complementary DNA origin and distribution, 112–113

Ta
(cDNA) CIs, see Cyclic imines (CIs)

18
CDs, see Cyclodextrins (CDs) Class I bacteriocins, 317

20
Celiac disease, 209, 210 Class II bacteriocins, 317

CE-LIF, see Capillary electrophoresis Cleaning, cooking, cross-contamination,
©
with laser-induced and chilling (4Cs) approach,

fluorescence (CE-LIF) 635–636
Center for Food Safety and Applied Clostridium botulinum, 280, 343
Nutrition (CFSAN), 245, 589 Clostridium perfringens, 343
Centers for Disease Control CNFs, see Chitin nanofibrils (CNFs)
and Prevention (CDC), 272, Codex Alimentarius Commission, 7, 136,
292, 568 201, 215, 244, 535, 545, 598
CFIA, see Canadian Food Inspection Codex Committee on Food
Agency (CFIA) Labelling, 201
Index 683
Coeliac UK, 209 Cyclic imines (CIs), 123–126

Coffee, 503–504 chemical structures and mechanism
Cold pasteurization, 307 of action, 123–126
ComBase, 277 episodes of intoxication, 126
Combinatorial peptide ligand libraries occurrence and accumulation in
(CPLLs), 519 seafood, 126
Community Duplicate Diet origin and distribution, 123
y
nl
Methodology, 252 Cyclodextrins (CDs), 441
O
Community Reference Laboratory for Cyclopiazonic acid, 72–73
se
Marine Biotoxins, 126
lU
Complementary DNA (cDNA), 174
D
Complementary Technical Assistance
na
Project, 22–23 Dairy products, 501–502, 517
o
rs
Concealment, 497 Danish Agriculture & Food Council, 545
Pe
Congressional Research Service DAO, see Diamine oxidase (DAO)
(CRS), 520 DART, see Direct analysis in real time
r
fo
CONTAM, see Panel on Contaminants (DART)
y
in the Food Chain (CONTAM) Dartmouth Laboratory, 129
op
Copaiba oil, 441 DA, see Domoic acid (DA)
C
COSs, see Chitooligosaccharides (COSs) DBNPG, see Dibromoneopentyl glycol
’s
COST Action FA1101 Saffron-OMICS, (DBNPG)

or
ut
661–667 DeLauro, Rosa, 568

b
genomics, 662–663 Department of Defense (DOD), 591

tri
metabolomics, 664–667 Department of Health and Human

on
proteomics, 663–664 Services, 568

.C
Counterfeiting, 497, 499 Department of Homeland Security, 582

C
CPLLs, see Combinatorial peptide DFO, see Fisheries and Oceans Canada
LL
ligand libraries (CPLLs) (DFO)

is
Critical control point (CCP), 272, 547, Diamine oxidase (DAO), 213
c
574, 608–612, 613 Diarrhetic shellfish poisoning (DSP)

an
Critical temperature indicators syndrome, 99, 101, 105, 137

Fr
(CTIs), 474 Dibromoneopentyl glycol (DBNPG), 254

d
Critical time–temperature indicators Differential pulse voltammetry (DPV),

an
(CTTIs), 474 174

or
Crocetin, 656 Dilution, food fraud, 497

yl
Crocins, 655 Dinophysis acuta, 101

Ta
Crocus sativus Linnaeus, 652–654 Dinophysis toxins (DTXs), 99, 100

18
CRS, see Congressional Research Service Dioxin-like PCBs (DL-PCBs), 253–254

20
(CRS) Dioxins and furans, 252–254

CSNPs, see Chitosan nanoparticles Direct analysis in real time (DART),
©
(CSNPs) 514, 515
CTIs, see Critical temperature indicators Dispersive liquid–liquid
(CTIs) microextraction (DLLME), 59
CTTIs, see Critical time–temperature DL-PCBs, see Dioxin-like PCBs
indicators (CTTIs) (DL-PCBs)
CTX, see Ciguatoxin (CTX) group toxins DNA barcoding technique, 509, 510, 511
Curie, Pierre, 310 DOD, see Department of Defense
Current good manufacturing practices (DOD)
(CGMPs), 574 Domoic acid (DA), 111–112
684 Index
chemical structures and mechanism Eosinophilic infiltration, 205

of action, 111–112 EPA, see Environmental Protection
episodes of intoxication, 112 Agency (EPA)
occurrence and accumulation, 112 EREN, see Emerging Risks Exchange
origin and distribution, 111 Network (EREN)
DPV, see Differential pulse voltammetry Ergonomic packaging, 460
(DPV) Ergot alkaloids, 73–74
y
nl
Dry digestion, 251 Escherichia coli, 278, 279–280, 282,
O
DSP, see Diarrhetic shellfish poisoning 344, 388
se
(DSP) syndrome ESI, see Electrospray ionization (ESI)
lU
DTXs, see Dinophysis toxins (DTXs) Essential oils, and their compound-
Durbin, Richard, 568 based films, 314–315,
na
440–442, 449
o
rs
Ethylene scavenging, 471
E
Pe
Ethylene vinyl alcohol (EVOH), 441
EAACI, see European Academy EUFIC, see European Food Information
r
fo
of Allergy and Clinical Council (EUFIC)
y
Immunology (EAACI) European Academy of Allergy
op
EASs, see Endocrine active substances and Clinical Immunology
C
(EASs) (EAACI), 197
’s
EBac, see Enterobacteriaceae (EBac) European Commission (EC)

or
ut
EC, see European Commission (EC) Council Directive 91/414/EEC, 252

b
Economically motivated adulteration Directive 2000/13/EC, 198

tri
(EMA), 496, 659 Directive 2007/68/EC, 198

on
EFSA, see European Food Safety Directive 2011/8/EU, 255

.C
Authority (EFSA) Regulation 1169/2011, 198–199, 201

C
Electrospray ionization (ESI), 60 Regulation 89/109/EEC, 482

LL
ELISA, see Enzyme-linked Regulation (EC) 1935/2004, 482

is
immunosorbent assay (ELISA) Regulation (EC) 89/109/EEC, 477

c
Elixir Sulfanilamide, 567 Regulation (EC) No. 1107/2009, 252

an
Elliott, Christopher, 521 Regulation (EC) No. 1244/2007, 128

Fr
EMA, see Economically motivated Regulation (EC) No. 178/2002, 198,

d
adulteration (EMA) 597

an
Emergent toxins, 99 Regulation EU 10/2011, 255

or
Emerging Risks Exchange Network Regulation No. 15/2011, 126, 127

yl
(EREN), 12 Regulation No. 1664/2006, 129

Ta
Endocrine active substances (EASs), 254 Regulation No. 2074/2005, 126, 128
18
Endocrine disruptors, 254–256 Regulation No. 786/2013, 132

20
Enterobacteriaceae (EBac), 33–34 Regulation No. 853/2004, 132

Enterocolitis syndrome, 205 Regulation No. 854/2004, 132
©
Environmental pollutants, 245–251, 261, European Food Information Council

263–264 (EUFIC), 209
heavy metals and metalloids, 245–251 European Food Safety Authority
Environmental Protection Agency (EFSA), 12, 132, 244–245, 250,
(EPA), 247, 418 255, 256, 536
Enzymatic browning, 32 EVOH, see Ethylene vinyl alcohol
Enzyme-linked immunosorbent assay (EVOH)
(ELISA), 53, 54, 61, 80, 128, EVOO, see Extra virgin olive oil (EVOO)
222–223 adulteration
Index 685
EWG, see Extruded white ginseng Fluorescence resonance energy transfer

(EWG) extracts (FRET), 169
Extra virgin olive oil (EVOO) FMEA, see Failure modes and effects
adulteration, 502 analysis (FMEA)
Extruded white ginseng (EWG) FNS, see Food and Nutrition Service
extracts, 435 (FNS)
Food, Drug, and Cosmetic Act of 1938
y
nl
(FD&C), 520
F
O
Food Allergen Act, 200
se
Failure modes and effects analysis Food Allergen Labeling and Consumer
lU
(FMEA), 597, 615 Protection Act (FALCPA), 199,
FALCPA, see Food Allergen Labeling 215
na
and Consumer Protection Act Food allergies
o
rs
(FALCPA) diagnosis, 219–225
Pe
FAO, see Food and Agriculture detection, 222–224
Organization (FAO) future therapies, 225
r
fo
FAS, see Foreign Agricultural Service prevention, 221–222
y
(FAS) overview, 200–208
op
Fat, 278–279 development of allergen-free
C
thermal oxidation of, 256–257 foods, 207–208
’s
Fault tree analysis (FTA), 616 IgE-mediated food allergies,

or
ut
FBD, see Foodborne disease (FBD) 201–204

b
FDA, see Food and Drug Administration mixed IgE- and non-IgE-
tri
(FDA) mediated food allergies,

on
FDCA, see Federal Food, Drug, and 204–205

.C
Cosmetic Act (FDCA) patients characteristics with,

C
FD&C, see Food, Drug, and Cosmetic 205–206

LL
Act of 1938 (FD&C) traceability in food chain supply,

is
Federal Food, Drug, and Cosmetic Act 206–207

c
(FDCA), 567, 568, 576 risk management and

an
Federal Meat Inspection Act, 563 communication, 213–219

Fr
Federal Trade Commission (FTC), 591 Food and Agriculture Organization

d
FFTT, see Food Fraud Think Tank (FAO), 49, 201, 244, 535
an
(FFTT) Food and Drug Administration (FDA),

or
FG, see Fish gelatin (FG) 136, 199, 245, 384, 416, 442, 449,
yl
First-order kinetic model, 348 496, 538, 563, 568–569, 573, 574,
Ta
First-party audits, 552 576–578, 582–583, 585, 588–589,

18
Fish, food fraud, 518 589–591

20
Fish and Fishery Products Hazards and Food and Nutrition Service (FNS), 590
Controls Guidance, 136 Food and Veterinary Office (FVO), 137
©
Fisheries and Oceans Canada (DFO), Food authenticity, 497

136 Foodborne disease (FBD), in
Fish gelatin (FG), 436 food service establishments,
Flavor/odor absorbers and releasers, 3–24, 533
471–472 Brazilian food services, 7–9
FLD, see LC–fluorescence detector (FLD) causal factors, 10–13
Flow diagram, HACCP contamination sources, 9–10
constructing, 602 overview, 4–5
in situ confirmation of, 602–603 projects undertaken, 20–23
686 Index
Complementary Technical pharmaceutical residues, 164–165

Assistance Project, in phenols, 164
Mozambique, 22–23 processed toxics, 256–264
pilot project food service acrylamide, 259–260
establishment categorization, biogenic amines, 258
20–21 Maillard reaction derivatives, 257
qualitative risk assessment, 18–20 N-nitroso compounds, 261–264
y
nl
risk assessment, 6–7 polycyclic aromatic hydrocarbons
O
sanitary risk, 5–6 and heterocyclic amines,
se
use of inspection scores to evaluate 260–261
lU
food safety, 13–18 thermal oxidation of fats and
Foodborne pathogens, 272 mineral oil hydrocarbons,
na
microbial, 342–345 256–257
o
rs
sporeformers for food safety, Food decontamination, pulsed light for,
Pe
343–344 379–390
spores and vegetative cells, 342 antimicrobial efficacy on food
r
fo
vegetative, to food safety, 344–345 matrixes, 388–389
y
thermal inactivation rates of, fundamentals, 381–386
op
278–282 in-package application, 386–387
C
extrinsic factors, 282 overview, 379–381
’s
intrinsic factors, 278–281 Food fraud, 496–522

or
ut
Food contaminants, 159–165 definition, 496–499

b
heavy metals, 160–161 types, 497–499

tri
arsenic (As), 161 detection, 505–519

on
cadmium (Cd), 160 genomics, 505–511

.C
lead (Pb), 160 metabolomics, 511–516

C
mercury (Hg), 160–161 proteomics, 516–519

LL
microbial pathogens, 342–345 examples, 499–504

is
sporeformers, 343–344 additives, 504

c
spores and vegetative cells, 342 coffee, 503–504

an
vegetative pathogens, 344–345 dairy products, 501–502

Fr
mycotoxins, 162 honey, 503

d
aflatoxins, 162–163 juice, 503

an
fumonisins, 163 meat and meat products, 500

or
ochratoxins, 163 oils, 502

yl
trichothecenes, 163 organic vegetables and fruits, 504

Ta
zeralenone, 164 seafood, 500–501

18
pesticides, 161–162, 251–256 spices, 502–503

20
brominated phenols and flame impact, 505–505

retardants, 252–254 economic, 505
©
dioxins and furans, 252–254 public health, 505

endocrine disruptors, 254–256 prevention, 519–520
organochlorine, 161–162 authentication, 519–520
organophosphorates and supply chain management and
carbamate, 162 procurement, 520
phenols traceability, 519
environmental pollutants, 245–251 regulation, 520–522
heavy metals and metalloids, in China, 521
245–251 by European Commission, 520
Index 687
industry standards and regulation, 534–544

compliance, 521–522 developed vs. developing
in United Kingdom, 521 countries, 542–544
in United States, 520 in developing countries, 541–542
saffron uses and, 658–659 in European Union and United
Food Fraud Think Tank (FFTT), 521 States, 536–541
Food globalization process, 5 at international level, 534–536
y
nl
Food handling procedures, 12 standards, 544–553
O
Food industry training, 577–581 audits, 551–553
se
Food integrity, 497, 520 characteristics, 545–546
lU
Food intolerances, 208–213 effects of implemented, 550–551
fructose, 212 main groups of requirements,
na
gluten, 209–211 546–549
o
rs
histamine, 212–213 management, 549–550
Pe
lactose, 211–212 training, 629–635
Food package, 597 Food Safety Administration, 568
r
fo
Food preservation, see Nonthermal Food Safety and Inspection Service
y
preservation technologies; (FSIS), 199, 200, 538, 584, 590
op
Thermal processing Food Safety Authority of Ireland
C
Food quality, 596 (FSAI), 500
’s
Food safety, 11, 13–18, 531–554 Food safety management systems

or
ut
approaches and management (FSMSs), 4

b
systems, 635–640 Food Safety Modernization Act (FSMA),

tri
4Cs approach, 635–636 238, 520, 541, 563, 568–569, 578,

on
hazard analysis and risk-based 585–586, 636

.C
preventive controls, 636–638 Food Safety Preventive Controls

C
and ISO 22000, 638–640 Alliance (FSPCA), 580

LL
and other food standards, 640 Food Safety System Certification 22000
is
HACCP in, 595–640 (FSSC 22000), 638–640

c
approaches and management Food security, 497, 596

an
systems, 635–640 Food Standards Agency (FSA), 206, 521,

Fr
compliance, 621–629 543

d
evolution, 612–613 Food Standards Australia New Zealand

an
history, 597–598 (FSANZ), 134

or
limitations and barriers, 618–620 Foreign Agricultural Service (FAS), 590

yl
overview, 596–597 Foreign Supplier Verification Program

Ta
principles, 598–612 (FSVP), 569, 580, 585

18
research, 613–618 Fourier-transform midinfrared

20
training, 629–635 (FT-MIR) technique, 513

issues, 532–533 Free radical scavengers, 467–468
©
reforms in United States, 563–592 Fresh-cut apples, 29–41

Food Safety Modernization Act degradation of polyphenols, 35–36
(FSMA), 568–569 importance and challenges, 31
history, 563–568 mathematical modeling, 38–39
imported food, 583–591 microbial inactivation, 34–35
improving capacity to detect and microbial spoilage, 33–34
respond, 581–583 in modified atmosphere packaging,
miscellaneous provisions, 591 36–38
preventive controls, 569–581 nonmicrobial spoilage, 31–33
688 Index
overview, 29–30 Game meat, 508–509

Pack-in-MAP® and C.A.P.P.A.B.L.E.©, Gamma irradiation, 298–301
39–41 GAO, see Government Accountability
Freshness indicators, 474 Office (GAO)
FRET, see Fluorescence resonance Gas chromatography–mass
energy transfer (FRET) spectrometry (GC-MS), 70, 515
Fructose intolerance, 212 Gas sensors, 475
y
nl
Fruit sugar, 212 GC-MS, see Gas chromatography–mass
O
FSA, see Food Standards Agency (FSA) spectrometry (GC-MS)
se
FSAI, see Food Safety Authority of Gelatin, 451
lU
Ireland (FSAI) Gelatin-based antimicrobial films,
FSANZ, see Food Standards Australia 435–436
na
New Zealand (FSANZ) Generally recognized as safe (GRAS),
o
rs
FSIS, see Food Safety and Inspection 442, 449
Pe
Service (FSIS) Genetically modified organisms
FSMA, see Food Safety Modernization (GMOs), 218, 219
r
fo
Act (FSMA) Genomics, 505–511
y
FSMA-PC, see FSMA Preventative for food fraud detection, 508–509
op
Controls (FSMA-PC) rule game meat, 508–509
C
FSMA Preventative Controls (FSMA-PC) meat and meat products, 506–508
’s
rule, 520 milk and milk products, 509

or
ut
FSMSs, see Food safety management plant-derived foods, 510–511

b
systems (FSMSs) seafood, 509–510

tri
FSPCA, see Food Safety Preventive spices, 509

on
Controls Alliance (FSPCA) Saffron-OMICS, 662–663

.C
FSSC 22000, see Food Safety System GFSI, see Global Food Safety Initiative
C
Certification 22000 (FSSC 22000) (GFSI)

LL
FSVP, see Foreign Supplier Verification GFTC, see Global Food Traceability
is
Program (FSVP) Center (GFTC)

c
FTA, see Fault tree analysis (FTA) Giant magnetoresistive (GMR), 224
an
FTC, see Federal Trade Commission Global Aquaculture Alliance, 545

Fr
(FTC) Global Food Safety Initiative (GFSI), 497,

d
FT-MIR, see Fourier-transform 521, 546, 581, 613

an
midinfrared (FT-MIR) Global Food Traceability Center (GFTC),

or
technique 587
yl
Fumonisins, 71–72, 163 GlobalG.A.P. standard, 545, 549

Ta
Functional foods, and dietary Global Red Meat Standard, 545

18
supplements, 576–577 Gluten intolerance, 209–211

20
Fusarium culmorum, 164 Glycidamide (GA), 259

Fusarium graminearum, 164 GMA, see Grocery Manufacturers
©
Fusarium proliferatum, 163 Association (GMA)

Fusarium verticilloides, 163 GMOs, see Genetically modified
FVO, see Food and Veterinary Office organisms (GMOs)
(FVO) GMPs, see Good manufacturing
practices (GMPs)
GMR, see Giant magnetoresistive (GMR)
G
Gold nanoparticles (AuNPs), 172, 173
GA, see Glycidamide (GA) Good manufacturing practices (GMPs),
Gambierdiscus spp., 112, 114 206, 207
Index 689
Government Accountability Office constructing flow diagram, 602

(GAO), 520, 576 description of food product,
G-quadruplex DNAzyme, 169 600–602
Grapefruit seed extract (GSE), 434, 437, determination and control of CCP,
438 608–611
GRAS, see Generally recognized as safe establishing corrective action,
(GRAS) 611–612
y
nl
Green tea extract (GTE), 441 establishing team, 598–600
O
Grocery Manufacturers Association in situ confirmation of flow
se
(GMA), 505 diagram, 602–603
lU
GSE, see Grapefruit seed extract (GSE) verification procedures,
GTE, see Green tea extract (GTE) documentation and record
na
Gymnodimines (GYMs), 123, 124, 126 keeping, 612
o
rs
GYMs, see Gymnodimines (GYMs) research on, 613–618
Pe
Hazard Analysis and Risk-based
Preventive Controls (HARPC),
r
H
fo
4, 636–638
y
HAB, see Harmful algae bloom (HAB) HBB, see Hexabromobenzene (HBB)
op
HACCP, see Hazard Analysis and HBTs, see Hydrogen breath tests
C
Critical Control Point (HACCP) (HBTs)
’s
Halal dietary laws, 546 HCAs, see Heterocyclic amines (HCAs)

or
ut
Harmful algae bloom (HAB), 98, 99 Heavy metal, 160–161, 168–172

b
HARPC, see Hazard Analysis and Risk- arsenic (As), 161

tri
based Preventive Controls cadmium (Cd), 160

on
(HARPC) lead (Pb), 160

.C
Hazard Analysis and Critical Control mercury (Hg), 160–161

C
Point (HACCP), 137, 215, 537, and metalloids, 245–251

LL
547, 549, 553, 574, 595–640 Heptafluorobutyryl (HFB), 70

is
compliance, 621–629 hERG, see Human ether-à-go-go-related

c
prerequisite programs, 621–627 gene (hERG)

an
validation, 627–629 Heterocyclic amines (HCAs), 260–261

Fr
verification, 627 Hexabromobenzene (HBB), 254

d
evolution, 612–613 HFB, see Heptafluorobutyryl (HFB)

an
food safety approaches and food HHP, see High hydrostatic pressure
or
safety management systems, (HHP)

yl
635–640 Hidden allergens, 207

Ta
4Cs approach, 635–636 High hydrostatic pressure (HHP),

18
Hazard Analysis and Risk-Based 307–310, 342, 398–404

20
Preventive Controls (HARPC), biological effects, 400–401

636–638 engineering principles, 399–400
©
and ISO 22000, 638–640 and HPTP, 346–365

and other food standards, 640 fundamentals and effect on
food safety training, 629–635 microbes, 346–347
history, 597–598 inactivation, 347–365
limitations and barriers, 618–620 limitations, challenges, and future
overview, 596–597 trends, 403–404
principles, 598–612 practical applications, 401–403
conducting hazards analysis, High-performance liquid
603–608 chromatography (HPLC), 128
690 Index
High Performance Liquid Human ether-à-go-go-related gene

Chromatography-Diode Array (hERG), 107
Detection-Mass Spectrometry Hydrogen breath tests (HBTs), 212
(LC-DAD-MS), 656 Hydroxypropyl methylcellulose
High-performance thin-layer (HPMC), 448
chromatography (HPTLC), 73 Hypolactasia, 211
High-power US (HPU), 311
y
nl
High-pressure carbon dioxide
I
O
(HPCD), 312
se
High pressure processing (HPP), see IACs, see Immunoaffinity columns
lU
High hydrostatic pressure (IACs)
(HHP) IARC, see International Agency for
na
High-pressure thermal processing Research on Cancer (IARC)
o
rs
(HPTP), HPP and, 342, 346–365 ICP-MS, see Inductively coupled plasma
Pe
fundamentals and effect on mass spectrometry (ICP-MS)
microbes, 346–347 IFBC, see International Food
r
fo
inactivation Biotechnology Council (IFBC)
y
kinetic models for, 347–350 IFSs, see International Food Standards
op
of pathogenic spores, 350–354 (IFSs)
C
spores and vegetative cells, 347 IgE, see Immunoglobulin E (IgE)-
’s
of vegetative pathogens, 354–365 mediated food allergies

or
ut
High-resolution magic angle spinning Illegal, unreported, and unregulated

b
nuclear magnetic resonance (IUU) seafood, 500

tri
(HR-MAS NMR), 513 Illinois Institute of Technology, 580

on
High resolution mass spectrometry ILSI, see International Life Sciences

.C
(HRMS), 130, 131, 515 Institute (ILSI)

C
High-risk foods (HRFs), 586 Immunoaffinity columns (IACs), 58, 71

LL
High-temperature plasma, 410 Immunoglobulin E (IgE)-mediated food

is
Histamine, 212–213, 258 allergies, 201–204

c
Honey, adulterants in, 503, 514 clinical spectrum of, 203–204

an
Horsemeat scandal, 500 acute urticarial, 203

Fr
HPCD, see High-pressure carbon anaphylaxis, 203

d
dioxide (HPCD) atopic dermatitis, 204

an
HPLC, see High-performance liquid oral allergy syndrome, 204

or
chromatography (HPLC) description, 201–203

yl
HPMC, see Hydroxypropyl Immunotherapeutic approaches, 225

Ta
methylcellulose (HPMC) Import Drug Act, 565

18
HPTLC, see High-performance thin-layer Imported food safety, 583–591

20
chromatography (HPTLC) Indicators, and sensors, 473, 474–475

HPTP, see High-pressure thermal Inductively coupled plasma mass
©
processing (HPTP) spectrometry (ICP-MS), 223

HPU, see High-power US (HPU) Informative packaging, 460
HRFs, see High-risk foods (HRFs) Institute for Food Safety and Health,
HR-MAS NMR, see High-resolution 580
magic angle spinning nuclear Institute of Marine Bioscience, 129
magnetic resonance (HR-MAS Intelligent packaging, 472–473
NMR) commercially available, 477
HRMS, see High resolution mass indicators and sensors, 474–475
spectrometry (HRMS) legislation, 477, 482
Index 691
microwaveable, 475–476 Lactic acid, 314

nanotechnology, biomaterials, and Lactic acid bacteria (LAB), 317
bioactive compounds used in, Lactobacillus curvatus, 317
476–477 Lactobacillus plantarum, 280
radiofrequency identification, 475 Lactose intolerance, 211–212
International Agency for Research on Laevulose, see Fruit sugar
Cancer (IARC), 53, 60, 162, LAMP, see Loop-mediated isothermal
y
nl
247, 259 amplification (LAMP)
O
International Food Biotechnology Lantibiotics, see Class I bacteriocins
se
Council (IFBC), 218 Lawrence method, 129
lU
International food safety standards, LC-DAD-MS, see High Performance
545 Liquid Chromatography-
na
International Food Standards (IFSs), 640 Diode Array Detection-Mass
o
rs
International Life Sciences Institute Spectrometry (LC-DAD-MS)
Pe
(ILSI), 218 LC-FLD, see Liquid chromatography
International Organization for with fluorescence detection
r
fo
Standardization (ISO), 522, 545 (LC-FLD)
y
Ionizing radiation, 418–419 LC-MS, see Liquid chromatography-
op
ISO, see International Organization for mass spectrometry (LC-MS)
C
Standardization (ISO) LC-MS/MS, see Liquid
’s
ISO 22000, HACCP and, 638–640 chromatography–tandem

or
ut
Isostatic principle, 399 mass spectrometry (LC-MS/

b
IUU, see Illegal, unreported, and MS)

tri
unregulated (IUU) seafood LC-Orbitrap-HRMS, 515

on
LC PCOX, see Liquid chromatographic

.C
postcolumn oxidation (LC

J
C
PCOX)
LL
Japanese Trust Fund II project, 134 LDPE, see Low-density polyethylene

is
Joint FAO/WHO Expert Committee on (LDPE)

c
Food Additives (JECFA), 244, Lead (Pb), 160, 246–247

an
248, 249 Leak indicators, 474

Fr
Journal of Food Process Engineering, 40 Le Chatelier’s principle, 346, 399

d
Journal of Food Safety, 40 LESA-MS, see Liquid extraction surface

an
Juice, adulteration in, 503 analysis–mass spectrometry

or
(LESA-MS)
yl
LFCT, see Laboratory of Food Chemistry

Ta
K
and Technology (LFCT)
18
Kinetic models, for HPP and HPTP Limits of detection (LODs), 174
20
inactivation, 347–350, 354, Linear low-density polyethylene

362–365 (LLDPE), 445
©
primary models, 348 Lipid oxidation, 300

secondary models, 349–350 Lipophilic toxins, 126–127
Kosher dietary laws, 546 Liquid chromatographic postcolumn
oxidation (LC PCOX), 129
Liquid chromatography-mass
L
spectrometry (LC-MS), 81, 517
LAB, see Lactic acid bacteria (LAB) Liquid chromatography–tandem mass
Laboratory of Food Chemistry and spectrometry (LC-MS/MS), 71,
Technology (LFCT), 664 72, 74, 80, 81, 126, 127, 130
692 Index
Liquid chromatography with ciguatoxin group, 112–114

fluorescence detection chemical structures and
(LC-FLD), 57, 58, 61, 71, 72, mechanism of action, 113
74, 80 episodes of intoxication, 113–114
Liquid extraction surface analysis–mass occurrence and accumulation in
spectrometry (LESA-MS), 515 seafood, 113
Liquid–liquid extraction (LLE), 58, 82 origin and distribution, 112–113
y
nl
Listeria innocua, 388 cyclic imines, 123–126
O
Listeria monocytogenes, 278, 281, 282, 345, chemical structures and
se
389 mechanism of action, 123–126
lU
LLDPE, see Linear low-density episodes of intoxication, 126
polyethylene (LLDPE) occurrence and accumulation in
na
LLE, see Liquid–liquid extraction (LLE) seafood, 126
o
rs
LOAEL, see Lowest observable adverse origin and distribution, 123
Pe
effect level (LOAEL) domoic acid, 111–112
LODs, see Limits of detection (LODs) chemical structures and
r
fo
Log-linear model, 274–275 mechanism of action, 111–112
y
Loop-mediated isothermal episodes of intoxication, 112
op
amplification (LAMP), 174, 667 occurrence and accumulation, 112
C
Low-density polyethylene (LDPE), 447 origin and distribution, 111
’s
Lowest observable adverse effect level okadaic acid group, 99–102

or
ut
(LOAEL), 131, 132 chemical structures and

b
Low-temperature plasma, 410 mechanism of action, 100

tri
episodes of intoxication, 101–102

on
occurrence and accumulation, 101

.C
M
origin and distribution, 99–100
C
Maillard reaction products (MRPs), 257 palytoxin and analogs, 120–123

LL
MALDI-TOF-MS, see Matrix- chemical structures and

is
assisted laser desorption/ mechanism of action, 120–122

c
ionization time-of-flight episodes of intoxication, 122–123

an
mass spectrometry occurrence and accumulation in

Fr
(MALDI-TOF-MS) seafood, 122

d
MAP, see Modified atmosphere origin and distribution, 120

an
packaging (MAP) pectenotoxin group, 102–103

or
Margarine, adulteration, 565 chemical structures and

yl
Marine biotoxins, 99–126 mechanism of action, 102–103

Ta
azaspiracid group, 105–108 episodes of intoxication, 103

18
chemical structures and occurrence and accumulation, 103

20
mechanism of action, 105–107 origin and distribution, 102

episodes of intoxication, 107–108 saxitoxin group, 108–111
©
occurrence and accumulation, 107 chemical structures and

origin and distribution, 105 mechanism of action, 108–109
brevetoxins, 114–116 episodes of intoxication, 109–111
chemical structures and occurrence and accumulation, 109
mechanism of action, 115 origin and distribution, 108
episodes of intoxication, 116 tetrodotoxin, 116–120
occurrence and accumulation in chemical structure and
seafood, 116 mechanism of action, 117
origin and distribution, 114–115 episodes of intoxication, 118–120
Index 693
occurrence and accumulation in Microbial contamination, 9–10

seafood, 118 Microbial inactivation, in fresh-cut
origin and distribution, 116–117 apples, 33–35
yessotoxin group, 103–105 Microbial pathogens, in food, 342–345
chemical structures and sporeformers, 343–344
mechanism of action, 104–105 spores and vegetative cells, 342
episodes of intoxication, 105 vegetative pathogens, 344–345
y
nl
occurrence and accumulation, 105 Micro total analysis system (μTAS), 223
O
origin and distribution, 103 Microwaveable active food packaging,
se
Marine Fisheries Research and 472
lU
Development (MFRD), 134 Microwaveable intelligent food
Marjoram essential oils, 441 packaging, 475–476
na
Matrix-assisted laser desorption/ Milk, and milk products, 509, 514–515
o
rs
ionization time-of-flight Mineral oil aromatic hydrocarbons
Pe
mass spectrometry (MALDI- (MOAHs), 256–257
TOF-MS), 517, 518 Mineral oil saturated hydrocarbons
r
fo
Maximum residue limits (MRLs), 252 (MOSHs), 257
y
MBA, see Mouse bioassay (MBA) Mislabeling, 499, 506
op
Meat and meat products, 500, 506–508, Mixed IgE-, and non-IgE-mediated food
C
515–516, 517–518 allergies, 204–205
’s
Meat Inspection Act, 566, 567 MLCs, see Myosin light chains (MLCs)
or
ut
MEKC, see Micellar electrokinetic MNV-1, see Murine norovirus-1 (MNV-1)

b
capillary chromatography MO, see Mustard essential oil (MO)

tri
(MEKC) MOAHs, see Mineral oil aromatic

on
Melamine-adulterated milk, 501 hydrocarbons (MOAHs)

.C
Melanoidin toxicity, 257 Modified atmosphere packaging (MAP),

C
Mercury (Hg) contamination, 30, 302–306

LL
160–161, 248 fresh-cut apples in, 36–38

is
Metabolomics predicting microbial growth,

c
for food fraud detection, 511–516 39–41

an
honey, 514 mathematical modeling and, 38–39

Fr
meat and meat products, 515–516 Moisture scavengers, 471

d
milk and milk products, 514–515 MOSHs, see Mineral oil saturated
an
plant products, 512–514 hydrocarbons (MOSHs)

or
Saffron-OMICS, 664–667 Mouse bioassay (MBA), 127, 130

yl
Metal, and metal oxide nanoparticle- Mozzarella cheese, 501

Ta
based antimicrobial films, MRLs, see Maximum residue limits

18
446–448 (MRLs)
20
Methylmercury (MeHg), 248–249 MRPs, see Maillard reaction products

Methyl-sensitive amplified fragment (MRPs)
©
length polymorphism MS-AFLP, see Methyl-sensitive

(MS-AFLP), 662 amplified fragment length
MFCs, see Multifunctional cleanup polymorphism (MS-AFLP)
columns (MFCs) Multifunctional cleanup columns
MFRD, see Marine Fisheries Research (MFCs), 58
and Development (MFRD) Multimycotoxin detection, 80–83
Micellar electrokinetic capillary Murine norovirus-1 (MNV-1), 300
chromatography (MEKC), 73 Mustard essential oil (MO), 441
Michaelis–Menten models, 30, 38 Mycotoxins, 174–177
694 Index
aflatoxins, 52–60, 162–163 NCC, see National coordination center

cyclopiazonic acid, 72–73 (NCC)
ergot alkaloids, 73–74 Neill, Charles P., 567
fumonisins, 71–72, 163 Neurologic shellfish poisoning (NSP)
multimycotoxin detection, 80–83 syndrome, 114, 127, 128
ochratoxins, 60–62, 163 NIAID, see National Institute of Allergy
overview, 49–52 and Infectious Diseases
y
nl
trichothecenes, 62–71, 163 (NIAID)
O
zearalenone and its derivatives, NIFA, see National Institute of Food and
se
74–80, 164 Agriculture (NIFA)
lU
zeralenone, 164 Nisin, 317
Myosin light chains (MLCs), 517 Nitrosamides, 261–264
na
Nitrosamines, 261–264
o
rs
NMFS, see National Marine Fisheries
N
Pe
Service (NMFS)
Na-alginate films, 434 NMR, see 1H nuclear magnetic
r
fo
NACMCF, see National Advisory resonance (NMR)
y
Committee on Microbiological N-nitroso compounds, 261–264
op
Criteria for Foods (NACMCF) NOAEL, see No observable adverse
C
Nanomaterials risk, in foods, 577 effect level (NOAEL)
’s
Nanoparticles, 318–320 Nonchromatographic methods, 52

or
ut
Nano-RT-PCR technique, 511 Nonenzymatic browning, 32

b
Natamycin, 434 Nonheating pasteurization technology,

tri
National Advisory Committee on 307

on
Microbiological Criteria for Non-lanthionine-containing

.C
Foods (NACMCF), 598 bacteriocins, see Class II

C
National Consumers League, 503 bacteriocins

LL
National coordination center (NCC), 579 Nonmicrobial spoilage, in fresh-cut

is
National Honey Board, 503 apples, 31–33

c
National Institute of Allergy Nonthermal plasma, 410

an
and Infectious Diseases Nonthermal preservation technologies,

Fr
(NIAID), 200 291–320
d
National Institute of Food and alternative chemical compounds,

an
Agriculture (NIFA), 579 312–320

or
National Marine Fisheries Service bacteriocins, 316–318

yl
(NMFS), 591 essential oils, 314–315

Ta
National Research Council Canada, 129 nanoparticles, 318–320

18
Natural antioxidants, 315–316, 462 natural antioxidants, 315–316

20
Natural biopolymers, 433–440 organic acids, 313–314

alginate-based antimicrobial films, commercial emerging, 414–419
©
433–435 chlorine dioxide, 416–418

carrageenan-based antimicrobial ionizing radiation, 418–419
films, 438 osmotic dehydration, 414–415
chitosan-based antimicrobial films, ozone-based technology, 415–416
436–437 gamma irradiation, 298–301
gelatin-based antimicrobial films, high hydrostatic pressure, 307–310
435–436 modified atmosphere packaging,
whey protein isolate–based films, 302–306
438–440 objectives, 419–420
Index 695
overview, 291–294 OPLS-DA, see Orthogonal projections to

pulsed electric fields, 301–302, latent structures–discriminant
404–408 analysis (OPLS-DA)
biological effects, 405–406 OPPs, see Organophosphorates (OPPs)
engineering principles, 404–405 Optical aptasensors, 174
limitations, challenges, and future Oral allergy syndrome, 204
trends, 407 Oral food challenge (OFC), 221
y
nl
practical applications, 406–407 Oral immunotherapy (OIT), 225
O
ultrasound, 310–312 Oregano essential oil (OEO), 436, 441
se
UV radiation, 295–298 Organic acids, 313–314
lU
pulsed UV light, 297–298 Organic polymeric agents, 450
UV-C radiation, 295–297 Organic vegetables, and fruits, 504
na
Nontoxic food reactions, 208 Organochlorine pesticides (OCPs),
o
rs
No observable adverse effect level 161–162, 252
Pe
(NOAEL), 131 Organophosphorates (OPPs), and
NSP, see Neurologic shellfish poisoning carbamate pesticides, 162
r
fo
(NSP) syndrome Orthogonal projections to latent
y
structures–discriminant
op
analysis (OPLS-DA), 666
C
O
OSHA, see Occupational Safety and
’s
OA, see Okadaic acid (OA) group Health Administration (OSHA)

or
ut
toxins Osmotic dehydration (OD), 414–415

b
Obama, Barack, 563 Ostreopsis, 120

tri
Occupational Safety and Health OT, see Ochratoxin (OT)

on
Administration (OSHA), 591 OTA, see Ochratoxin A (OTA)

.C
Ochraprep®, 61 Oxygen scavengers, 466–467

C
Ochratoxin (OT), 60–62, 163 Ozone-based technology, 415–416

LL
Ochratoxin A (OTA), 174

is
OCPs, see Organochlorine pesticides

P
c
(OCPs)
an
OD, see Osmotic dehydration (OD) Pack-in-MAP®, 39–41

Fr
OEO, see Oregano essential oil (OEO) PAHs, see Polycyclic aromatic
d
OFC, see Oral food challenge (OFC) hydrocarbons (PAHs)

an
Oils, adulteration of, 502 Palythoa, 120

or
OIT, see Oral immunotherapy (OIT) Palytoxin (PITX), and analogs, 120–123
yl
Okadaic acid (OA) group toxins, chemical structures and mechanism

Ta
99–102 of action, 120–122

18
chemical structures and mechanism episodes of intoxication, 122–123

20
of action, 100 occurrence and accumulation in

episodes of intoxication, 101–102 seafood, 122
©
occurrence and accumulation, 101 origin and distribution, 120

origin and distribution, 99–100 Panel on Contaminants in the Food
Oleomargarine Bill, 565 Chain (CONTAM), 249
Omics Technologies for Crop Paralytic shellfish poisoning (PSP), 108,
Improvement, Traceability, 129, 137
Determination of Authenticity, Partial least squares—discriminant
Adulteration and Origin in analysis (PLS-DA), 666
Saffron (OMICS), 661–667, Partnership for Food Protection (PFP),
661–667 588
696 Index
Pathogenic spores inactivation, in food, PL, see Pulsed light (PL)

350–354 PLA, see Polylactic acid (PLA)
kinetic models, 354 PLA-based films, 450
log reductions, 350–354 Plant-derived foods, 510–511
Pathogen Modelling Program, 277 Plant products, 512–514
Patinopecten yessoensis, 102, 103 PLS-DA, see Partial least squares—
PCBs, see Polychlorinated biphenyls discriminant analysis
y
nl
(PCBs) (PLS-DA)
O
PCR, see Polymerase chain reaction PMTDI, see Provisional maximum
se
(PCR) tolerable daily intake
lU
PDEs, see Phophodiesterases (PDEs) (PMTDI)
PDO, see Protected designation of origin PnTXs, see Pinnatoxins (PnTXs)
na
(PDO) Polychlorinated biphenyls (PCBs),
o
rs
Peanut Corporation of America, 505, 553 252–254
Pe
Pectenotoxin (PTX) group toxins, Polycyclic aromatic hydrocarbons
102–103 (PAHs), 260, 260–261
r
fo
chemical structures and mechanism Polylactic acid (PLA), 442, 445
y
of action, 102–103 Poly-L-lactide-based antimicrobial
op
episodes of intoxication, 103 films, 442, 445
C
occurrence and accumulation, 103 Polymerase chain reaction (PCR), 166,
’s
origin and distribution, 102 223, 506, 508

or
ut
Pedigree, 519 Polymer/clay nanocomposite-based

b
Pediocin, 317 antimicrobial films, 445–446

tri
PEF, see Pulsed electric fields (PEF) Polynomial model, 349

on
Penicillium, 163 Polyphenols, in fresh-cut apples, 35–36

.C
Persistent organic pollutants (POPs), 244 Polysaccharide-based polymers, 432

C
Pesticides, 161–162, 172–174, 251–256 Polystyrene-b polyethylene oxide (PS-b-

LL
brominated phenols and flame PEO), 448

is
retardants, 252–254 Polyunsaturated fats, 256–257

c
emerging and novel, 254 Polyvinyl alcohol (PVOH), 437

an
dioxins and furans, 252–254 POPs, see Persistent organic pollutants

Fr
endocrine disruptors, 254–256 (POPs)

d
organochlorine, 161–162 Predictive Risk-based Evaluation for

an
organophosphorates and Dynamic Import Compliance

or
carbamate, 162 Targeting (PREDICT), 585

yl
PFDA, see Pure Food and Drug Act Prerequisite programs (PRPs), 532, 546,
Ta
(PFDA) 551, 617–618, 635

18
PFP, see Partnership for Food Protection and differences among food
20
(PFP); Puffer fish poisoning establishments, 621–627

(PFP) and good practices, 547
©
PGI Interdonato Lemon of Messina, 513 Private food safety standards, 545
pH, 279–280 Processed toxics, 256–264
Pharmaceutical residues, 164–165, acrylamide, 259–260
180–182 biogenic amines, 258
Phenols, 164, 177–180 Maillard reaction derivatives, 257
Phophodiesterases (PDEs), 104 N-nitroso compounds, 261–264
Picrocrocin, 657–658 polycyclic aromatic hydrocarbons
Pinnatoxins (PnTXs), 124, 126 and heterocyclic amines,
PITX, see Palytoxin (PITX), and analogs 260–261
Index 697
thermal oxidation of fats and mineral microbial contamination, 382–383

oil hydrocarbons, 256–257 process setup, 383–386
Produce Safety Alliance (PSA), 579 treated matrix, 381–382
Produce safety rule, 574–576 in-package application, 386–387
Prorocentrolide, 123, 124 overview, 379–381
Prorocentrum lima, 124 Pulsed UV light, 297–298
Prorocentrum maculosum, 124 Pure Food and Drug Act (PFDA), 565,
y
nl
Protected designation of origin (PDO), 567
O
501, 502 PVOH, see Polyvinyl alcohol (PVOH)
se
Proteomics
lU
for food fraud detection, 516–519
Q
dairy products, 517
na
fish, 518 QDs, see Quantum dots (QDs)
o
rs
meat, 517–518 Qualitative risk assessment, 18–20
Pe
wine, 518–519 Quantum dots (QDs), 173
Saffron-OMICS, 663–664 Quick, easy, cheap, effective, rugged,
r
fo
Proton transfer reaction–mass and safe (QuEChERS)
y
spectrometry (PTR-MS), 667 approach, 130–131
op
Provisional maximum tolerable daily
C
intake (PMTDI), 71
’s
R
PRPs, see Prerequisite programs (PRPs)
or
ut
PSA, see Produce Safety Alliance (PSA) Radicidation process, 298

b
PS-b-PEO, see Polystyrene-b Radioallergosorbent test (RAST), 223

tri
polyethylene oxide (PS-b-PEO) Radiofrequency identification (RFID),

on
Pseudomonas aeruginosa, 389 473, 475

.C
PSP, see Paralytic shellfish poisoning Radioimmunoassay (RIA), 128

C
(PSP) Radurization process, 298

LL
Pteriatoxins (PtTXs), 126 Rapid Alert System for Food and Feed
is
PTR-MS, see Proton transfer reaction– (RASFF), 137–138, 502, 503, 536,
c
mass spectrometry (PTR-MS) 537–538

an
PtTXs, see Pteriatoxins (PtTXs) RAST, see Radioallergosorbent test

Fr
PTX, see Pectenotoxin (PTX) group (RAST)

d
toxins Regression modeling, 38

an
Public Health Security and Bioterrorism Religious slaughtering, 546

or
Preparedness and Response Repetitive pulsed light (RPL), 388

yl
Act (BT Act), 584 Reportable Food Registry (RFR),

Ta
Puffer fish poisoning (PFP), 111 586–587

18
Pulsed electric fields (PEF), 301–302, Response surface model (RSM), 349
20
404–408 Responsive/reactive packaging, 460

biological effects, 405–406 RFID, see Radiofrequency identification
©
engineering principles, 404–405 (RFID)

limitations, challenges, and future RFR, see Reportable Food Registry (RFR)
trends, 407 RIA, see Radioimmunoassay (RIA)
practical applications, 406–407 Risk
Pulsed light (PL), for food analysis, 6
decontamination, 379–390 assessment, 6–7
antimicrobial efficacy on food for biotoxins in seafood, 131–132
matrixes, 388–389 characterization, 608
fundamentals, 381–386 sanitary, 5–6
698 Index
Roosevelt, Theodore, 245, 565, 567 SELEX, see Systematic Evolution of

RPL, see Repetitive pulsed light (RPL) Ligands by Exponential
RSM, see Response surface model (RSM) Enrichment (SELEX)
S. E. Massengill Company, 567
Semiquantitative risk assessment, 13–18
S
SEO, see Satureja hortensis essential oil
Saccharomyces cerevisiae, 280 (SEO)
y
nl
Safe Supply of Affordable Food Sequence-characterized amplified
O
Everywhere (SSAFE), 521 regions (SCARs), 667
se
Saffron, 651–671 SERS, see Surface-enhanced Raman
lU
chemical composition, 654–658 scattering (SERS); Surface-
coloring properties and secondary enhanced Raman spectroscopy
na
metabolites, 655–657 (SERS)
o
rs
flavor and secondary metabolites, Silver nanoparticles (AgNPs), 434, 446,
Pe
657–658 447, 448
Crocus sativus Linnaeus, 652–654 Sinclair, Upton, 567
r
fo
developments in fraud prevention, Skin prick test (SPT), 203, 219–220
y
659–671 Smart packaging, see Intelligent
op
COST Action FA1101 Saffron- packaging
C
OMICS, 661–667 Solid-phase extraction (SPE) method, 56,
’s
other recent advances, 667–671 57, 82

or
ut
food uses and fraudulent practices, Solvent extraction, 130

b
658–659 SPCE, see Screen-printed carbon

tri
overview, 671 electrode (SPCE)

on
Salmonella enteritidis, 9, 10, 278, 279, 345 SPE, see Solid-phase extraction (SPE)
.C
Salmonella typhimurium, 282, 345, 389 method

C
Sanitary risk, in food service setting, Specificity, 167

LL
5–6 Spectrofluorimetry, 56
is
Sarcoplasmic proteins, 518 Speedisk® C18, 58

c
Satureja hortensis essential oil (SEO), 438 Speedisk PolarPlus C18, 58

an
Saxitoxin (STX) group toxins, 108–111 SPFP, see Saxitoxin puffer fish poisoning
Fr
chemical structures and mechanism (SPFP)

d
of action, 108–109 Spices, adulteration in, 502–503, 509

an
episodes of intoxication, 109–111 SPIDER, see Supply-chain Pedigree

or
occurrence and accumulation, 109 Interactive Dynamic Explore

yl
origin and distribution, 108 (SPIDER)

Ta
Saxitoxin puffer fish poisoning (SPFP), Spirolides (SPXs), 123–124, 126

18
111 Spiroprorocentrimine, 123

20
SCARs, see Sequence-characterized SPR, see Surface plasmon resonance

amplified regions (SCARs) (SPR)
©
Screen-printed carbon electrode (SPCE), Sprout Safety Alliance (SSA), 580

174 SPS Agreement, see Agreement on
SDS-PAGE, see 1D sodium dodecyl Sanitary and Phytosanitary
sulfate–polyacrylamide gel Measures (SPS Agreement)
electrophoresis (SDS-PAGE) SPT, see Skin prick test (SPT)
SE86, see Salmonella enteritidis SPXs, see Spirolides (SPXs)
Seafood, 500–501, 509–510 SQUID, see Superconducting quantum
Second-order polynomial equations, 349 interference device (SQUID)
Second-party audits, 552 SSA, see Sprout Safety Alliance (SSA)
Index 699
SSAFE, see Safe Supply of Affordable TETs, see Tetracyclines (TETs)

Food Everywhere (SSAFE) TFC, see Turbulent flow chromatography
Staphylococcus aureus, 9, 10, 344 (TFC)
Strengths, weaknesses, opportunities, The Jungle, 567
and threats (SWOT), 616 Thermal inactivation
STX, see Saxitoxin (STX) group toxins kinetics models, 274–278
Substitution, food fraud, 497 primary, 274
y
nl
Superconducting quantum interference secondary, 276–277
O
device (SQUID), 224 tertiary, 277–278
se
Supply chain management, and rates of foodborne pathogens,
lU
procurement, 520 278–282
Supply-chain Pedigree Interactive extrinsic factors, 282
na
Dynamic Explore (SPIDER), intrinsic factors, 278–281
o
rs
615 Thermal oxidation, 256–257
Pe
Surface-enhanced Raman scattering Thermal processing, 271–273, 292, 380
(SERS), 173 Third-party audits, 552–553
r
fo
Surface-enhanced Raman spectroscopy Thrombin binding aptamer (TBA),
y
(SERS), 514 op168–169
Surface plasmon resonance (SPR), 224 Time-of-flight mass spectrometer (TOF
C
SWOT, see Strengths, weaknesses, MS), 514
’s
opportunities, and threats Time–temperature indicators (TTIs),

or
ut
(SWOT) 474
b
Synthetic antioxidants, 462 TLC, see 2D Thin-layer chromatography

tri
Systematic Evolution of Ligands by [TLC]

on
Exponential Enrichment TMP, see Transmembrane potential

.C
(SELEX), 159, 165, 165–166, 173 (TMP)

C
TMS, see Trimethylsilyl (TMS)

LL
TMSI, Trimethylsilylimidazole (TMSI)

T
is
TOF MS, see Time-of-flight mass

c
Takeshi Yasumoto, 101 spectrometer (TOF MS)

an
Tampering, 499 Tolerable daily intake (TDI), 256

Fr
TBA, see Thrombin binding aptamer Tolerable weekly intake (TWI), 249
d
(TBA) TOTO-3, 169

an
TBT Agreement, see Agreement on Toxicity equivalency factors (TEFs), 132

or
Technical Barrier to Trade (TBT Toxic risk assessment, 245

yl
Agreement) Train-the-trainer (TTT), 580

Ta
TDI, see Tolerable daily intake (TDI) Transmembrane potential (TMP), 405
18
Tea adulteration, 566 Trichothecenes, 62–71, 163

20
Tea Importation Act, 566 Trimethylsilyl (TMS), 70

TEFs, see Toxicity equivalency factors Trimethylsilylimidazole (TMSI), 70
©
(TEFs) Tris(2,3-dibromopropyl)phosphate, 254

Tetracyclines (TETs), 164 TTIs, see Time–temperature indicators
Tetrodotoxin (TTX), 116–120, 130 (TTIs)
chemical structure and mechanism TTT, see Train-the-trainer (TTT)
of action, 117 TTX, see Tetrodotoxin (TTX)
episodes of intoxication, 118–120 Turbulent flow chromatography (TFC),
occurrence and accumulation in 59
seafood, 118 TWI, see Tolerable weekly intake (TWI)
origin and distribution, 116–117 Tyramine toxicity, 258
700 Index
U Volatile oils, see Essential oils

VP, see Vacuum packaging (VP)
UHPLC, see Ultra-high-performance
liquid chromatography
(UHPLC) W
UHPLC-MS, see Ultra-high-performance
WAO, see World Allergy Organization
liquid chromatography–mass
(WAO)
y
spectrometry (UHPLC-MS)
nl
Water activity, 280
Ultra-high-performance liquid
O
Weibull model, 275–276, 348, 349
chromatography (UHPLC), 59,
se
Wet digestion, 251
130, 131
lU
Whey protein isolate (WPI), 439, 450
Ultra-high-performance liquid
WHO, see World Health Organization
na
chromatography–mass
(WHO)
o
spectrometry (UHPLC-MS),
rs
Wine authenticity, 518–519
515, 517
Pe
World Allergy Organization (WAO),
Ultra-high-pressure (UHP), see High
197, 208
r
fo
Hydrostatic Pressure (HHP)
World Health Organization (WHO), 6,
Ultrasound (US), 310–312
y
136, 161, 244, 255
op
Unapproved enhancements, 499
World Trade Organization (WTO), 534,
C
Unintentional contaminants, see Food
535
’s
contaminants
WPI, see Whey protein isolate (WPI)
or
US, see Ultrasound (US)

ut
WPI–based films, 438–440

U.S. Department of Agriculture (USDA),
b
WTO, see World Trade Organization

tri
208, 579
(WTO)
on
U.S. National Advisory Committee on

.C
Microbiological Criteria for

Foods, 307 X
C
LL
U.S. Pharmacopeia (USP), 522, 565

Xenobiotics, 244
UV radiation of short wavelength (UV-
is
C), 295–297
c
an
Y
Fr
Yersinia enterocolitica, 279

V
d
Yessotoxin (YTX) group toxins, 103–105

an
Vacuum packaging (VP), 303 chemical structures and mechanism

or
Vegetative pathogens inactivation, in of action, 104–105

yl
food, 354–365 episodes of intoxication, 105

Ta
kinetic models, 362–365 occurrence and accumulation, 105

18
log reductions, 354–362 origin and distribution, 103

20
Vibrio parahaemolyticus, 345

Vibrio vulnificus, 345
©
Z
Victorian Marine Biotoxin Management
Plan, 134 ZEA, and its derivatives, 74–80
Vilsack, Tom, 208 Zeralenone (ZEA), 164
Virus-toxin law, see Biologics Control Zinc oxide (ZnO), 318
Act of 1902 Zinc oxide nanoparticles (ZnONPs), 446

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©

V. Ravishankar Rai and Jamuna A.Bai

© 2018 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group, an Informa business

or hereafter invented, including photocopying, microfilming, and recording, or in any information

storage or retrieval system, without written permission from the publishers.

Library of Congress Cataloging‑ in‑ P ublication Data

Names: Rai, V. Ravishankar, editor. | Bai, Jamuna A. (Jamuna Aswathanarayn),

references and index.

ISBN 9781315153414 (ebook) | ISBN 9781498762885 (ebook) | ISBN

9781351649452 (ebook) | ISBN 9781351639934 (ebook)

Visit the Taylor & Francis Web site at

and the CRC Press Web site at

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

Myra E. Flores-Flores and Elena Gonzá lez-Peñ as

Laura P. Rodrí guez, Juan M. Vieites, and Ana G. Cabado

5. Selection and Characterization of Aptamers for Food

Contaminant Monitoring........................................................................... 157

6. Allergen Management as a Key Issue in Food Safety ........................ 195

7. Unintentional Contaminants in Food..................................................... 243

Section III Preservation of Foods

8. Thermal Inactivation Kinetics of Foodborne Pathogens: An

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

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

16. Food Safety Regulation and Standards.................................................. 531

18. The HACCP in the Current Food Safety Context................................. 595

Food safety and protection is a multidisciplinary topic that focuses on the

analytical methods for the analysis of mycotoxins in milk, biotoxins in sea-

in foods. Section III, on preservation of foods, comprehensively explores

developments in food intervention techniques and covers topics such as

and ultrasound techniques to inactivate spores in foods, pulsed field tech-

niques, and other emerging nonthermal technologies for food preservation.

Section IV, on food packaging, deals with the technological development of

Section V, on food safety laws, deals with food fraud— detection, prevention,

Prof. V. Ravishankar Rai

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,

oping quorum-sensing inhibitors. Her research interests also include the

antimicrobial application of functionalized nanomaterials against food-

Elisabete M.C. Alexandre Conrado Carrascosa

Tenerife, Canary Islands, Spain and

Jamuna A. Bai Escola Superior de Biotecnologia

Universidade Cató lica Portuguesa

Faculty of Pharmacy Université de Perpignan Via

Universitat de Valè ncia Domitia

Ana G. Cabado Carlos Adam Conte-Junior

Food Safety Division— IDi.

Department of Food Technology