Trends in Fish Processing Technologies Contemporary Food Engineering

Series Editor Professor Da-Wen Sun, Director Food Refrigeration & Computerized Food Technology National University of Ireland, Dublin (University College Dublin) Dublin, Ireland http://www.ucd.ie/sun/

Trends in Fish Processing Technologies, edited by Daniela Borda, Anca I. Nicolau, and Peter Raspor (2017) Food Biofortification Technologies, edited by Agnieszka Agnieszka (2017) Trends in Fish Processing Technologies, edited by Daniela Borda, Anca I. Nicolau, and Peter Raspor (2017) High Pressure Processing of Fruit and Vegetable Juices, edited by Milan Houška and Filipa Vinagre Marques da Silva (2017) Advances in Meat Processing Technology, Alaa El-Din A. Bekhit (2017) Advances in Postharvest Fruit and Vegetable Technology, edited by Ron B.H. Wills and John Golding (2017) Food Nanotechnology: Principles and Applications, edited by C. Anandharamakrishnan and S. Parthasarathi (2017) Engineering Aspects of Food Emulsification and Homogenization, edited by Marilyn Rayner, Petr Dejmek (2017) Engineering Aspects of Food Biotechnology, edited by Jose A. Teixeira and Antonio A. Vicente (2017) Edible Oils: Extraction, Processing, and Applications, edited by Smain Chemat (2017) Engineering Aspects of Membrane Separation and Application in Food Processing, edited by Robert W. Field, Erika Bekassy-Molnar, Frank Lipnizki, and Gyula Vatai (2017) Advances in Fruit Processing Technologies, edited by Sueli Rodrigues and Fabiano Andre Narciso Fernandes (2016) Thermal Food Processing: New Technologies and Quality Issues, Second Edition, edited by Da-Wen Sun (2016) Enhancing Extraction Processes in the Food Industry, edited by Nikolai Lebovka, Eugene Vorobiev, and Farid Chemat (2016) Engineering Aspects of Cereal and Cereal-Based Products, edited by Raquel de Pinho Ferreira Guine, and Paula Maria dos Reis Correia (2016) Fermentation Processes Engineering in the Food Industry, edited by Carlos Ricardo Soccol, Ashok Pandey, and Christian Larroche (2016) Physical Properties of Foods: Novel Measurement Techniques and Applications, edited by Ignacio Arana (2016) Juice Processing: Quality, Safety and Value-Added Opportunities, edited by Victor Falguera and Albert Ibarz (2016) Modified Atmosphere and Active Packaging Technologies, edited by Ioannis Arvanitoyannis (2016) Advances in Food Extrusion Technology, edited by Medeni Maskan and Aylin Altan (2016) Operations in Food Refrigeration, edited by Rodolfo H. Mascheroni (2016) Emerging Technologies for Food Quality and Food Safety Evaluation, edited by Yong-Jin Cho, Sukwon Kang (2016) Advances in Technologies for Producing Food-relevant Polyphenols, edited by Jose Cuevas Valenzuela, Jose Rodrigo Vergara-Salinas, Jose Ricardo Perez- Correa (2016) Advances in Heat Transfer Unit Operations: Baking and Freezing in Bread Making, edited by Georgina Calderón-Domínguez, Gustavo F. Gutiérrez-López, and Keshavan Niranjan (2016) Innovative Processing Technologies for Foods with Bioactive Compounds, edited by Jorge J. Moreno (2016) Light Scattering Technology for Food Property, Quality and Safety Assessment, edited by Renfu Lu (2016) Edible Food Packaging: Materials and Processing Technologies, edited by Miquel Angelo Parente Ribeiro Cerqueira, Ricardo Nuno Correia Pereira, Oscar Leandro da Silva Ramos, Jose Antonio Couto Teixeira, and Antonio Augusto Vicente (2016) Handbook of Food Processing: Food Preservation, edited by Theodoros Varzakas and Constantina Tzia (2015) Handbook of Food Processing: Food Safety, Quality, and Manufacturing Processes, edited by Theodoros Varzakas and Constantina Tzia (2015) Advances in Postharvest Fruit and Vegetable Technology, edited by Ron B.H. Wills and John Golding (2015) Engineering Aspects of Food Emulsification and Homogenization, edited by Marilyn Rayner and Petr Dejmek (2015) Handbook of Food Processing and Engineering, Volume II: Food Process Engineering, edited by Theodoros Varzakas and Constantina Tzia (2014) Handbook of Food Processing and Engineering, Volume I: Food Engineering Fundamentals, edited by Theodoros Varzakas and Constantina Tzia (2014) Juice Processing: Quality, Safety and Value-Added Opportunities, edited by Víctor Falguera and Albert Ibarz (2014) Engineering Aspects of Food Biotechnology, edited by José A. Teixeira and António A. Vicente (2013) Engineering Aspects of Cereal and Cereal-Based Products, edited by Raquel de Pinho Ferreira Guiné and Paula Maria dos Reis Correia (2013) Fermentation Processes Engineering in the Food Industry, edited by Carlos Ricardo Soccol, Ashok Pandey, and Christian Larroche (2013) Modified Atmosphere and Active Packaging Technologies, edited by Ioannis Arvanitoyannis (2012) Advances in Fruit Processing Technologies, edited by Sueli Rodrigues and Fabiano Andre Narciso Fernandes (2012) Biopolymer Engineering in Food Processing, edited by Vânia Regina Nicoletti Telis (2012) Operations in Food Refrigeration, edited by Rodolfo H. Mascheroni (2012) Thermal Food Processing: New Technologies and Quality Issues, Second Edition, edited by Da-Wen Sun (2012) Physical Properties of Foods: Novel Measurement Techniques and Applications, edited by Ignacio Arana (2012) Handbook of Frozen Food Processing and Packaging, Second Edition, edited by Da-Wen Sun (2011) Advances in Food Extrusion Technology, edited by Medeni Maskan and Aylin Altan (2011) Enhancing Extraction Processes in the Food Industry, edited by Nikolai Lebovka, Eugene Vorobiev, and Farid Chemat (2011) Emerging Technologies for Food Quality and Food Safety Evaluation, edited by Yong-Jin Cho and Sukwon Kang (2011) Food Process Engineering Operations, edited by George D. Saravacos and Zacharias B. Maroulis (2011) Biosensors in Food Processing, Safety, and Quality Control, edited by Mehmet Mutlu (2011) Physicochemical Aspects of Food Engineering and Processing, edited by Sakamon Devahastin (2010) Infrared Heating for Food and Agricultural Processing, edited by Zhongli Pan and Griffiths Gregory Atungulu (2010) Mathematical Modeling of Food Processing, edited by Mohammed M. Farid (2009) Engineering Aspects of Milk and Dairy Products, edited by Jane Sélia dos Reis Coimbra and José A. Teixeira (2009) Innovation in Food Engineering: New Techniques and Products, edited by Maria Laura Passos and Claudio P. Ribeiro (2009) Processing Effects on Safety and Quality of Foods, edited by Enrique Ortega- Rivas (2009) Engineering Aspects of Thermal Food Processing, edited by Ricardo Simpson (2009) Ultraviolet Light in Food Technology: Principles and Applications, Tatiana N. Koutchma, Larry J. Forney, and Carmen I. Moraru (2009) Advances in Deep-Fat Frying of Foods, edited by Serpil Sahin and Servet Gülüm Sumnu (2009) Extracting Bioactive Compounds for Food Products: Theory and Applications, edited by M. Angela A. Meireles (2009) Advances in Food Dehydration, edited by Cristina Ratti (2009) Optimization in Food Engineering, edited by Ferruh Erdoˇgdu (2009) Optical Monitoring of Fresh and Processed Agricultural Crops, edited by Manuela Zude (2009) Food Engineering Aspects of Baking Sweet Goods, edited by Servet Gülüm Sumnu and Serpil Sahin (2008) Computational Fluid Dynamics in Food Processing, edited by Da-Wen Sun (2007) Trends in Fish Processing Technologies

Edited by Daniela Borda Anca Ioana Nicolau Peter Raspor CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742

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Library of Congress Cataloging‑in‑Publication Data

Names: Borda, Daniela, author. | Nicolau, Anca I., author. | Raspor, Peter, author. Title: Trends in fish processing technologies / Daniela Borda, Anca I. Nicolau, Peter Raspor. Description: Boca Raton : Taylor & Francis, 2018. | “A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.” | Includes bibliographical references. Identifiers: LCCN 2017024775| ISBN 9781498729178 (hardback) | ISBN 9781315120461 (e-book) | ISBN 9781498729185 (e-book) | ISBN 9781351646796 (e-book) | ISBN 9781351637275 (e-book) Subjects: LCSH: Fishery processing. | Fishery technology. Classification: LCC SH335 .B65 2018 | DDC 664/.94--dc23 LC record available at https://lccn.loc.gov/2017024775

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com Contents

Series Preface...... ix Preface...... xi Series Editor...... xiii Editors...... xv Contributors...... xvii Introduction...... xix

Chapter 1 Recent Advances in Seafood Technology: An Overview...... 1 Javier Borderías and Helena M. Moreno

Chapter 2 Minimal Heat Processing Applied in Fish Processing...... 27 Jan Thomas Rosnes and Dagbjørn Skipnes

Chapter 3 High-Pressure Processing of Seafood...... 71 Daniela Borda

Chapter 4 Processing of Low-Value Fish, Coproducts, and By-Catch...... 101 Livia Patraşcu and Iuliana Aprodu

Chapter 5 Advances in Surimi Processing...... 121 Ausra Sipailiene

Chapter 6 Reformulation of Preserved Fish Products...... 135 Loreto M. Valenzuela, Allison Leyton, and M. Elena Lienqueo

Chapter 7 New Product Development...... 161 Eirin M. Skjøndal Bar, Sunniva Hoel, and Jørgen Lerfall

Chapter 8 Clean Fish Processing Technologies...... 171 Sanja Vidaček and Rafael Soro

Chapter 9 Innovative Fish Packaging Solutions...... 187 Iulia Bleoanca and Maria Turtoi

vii viii Contents

Chapter 10 Achieving Adequate Protection and Suitable Food Safety Indicators...... 217 Johannes Pucher and Christian Schlechtriem

Chapter 11 Food Safety Management in Fish Processing Units...... 241 Anca Ioana Nicolau and Olafur Oddgeirsson

Chapter 12 Quality and Quality Changes Assessment of Processed Fish...... 255 Mercedes Careche and Isabel Sánchez-Alonso

Chapter 13 Molecular Methods for Assessment of Fish and Fish Product Integrity...... 277 Mette S.R. Fachmann and Jeffrey Hoorfar

Chapter 14 Adulteration and Misbranding of Fish Products...... 291 Marjolein van der Spiegel and Joop van der Roest

Chapter 15 Traceability of Fish Products...... 303 Maitri Thakur

Chapter 16 Fish Trade Regulations...... 313 Isabelle Metaxa

Index...... 323 Series Preface

CONTEMPORARY FOOD ENGINEERING Food engineering is the multidisciplinary field of applied physical sciences combined with the knowledge of product properties. Food engineers provide the technological knowledge transfer essential to the cost-effective production and commercialization of food products and services. In particular, food engineers develop and design processes and equipment to convert raw agricultural materials and ingredients into safe, convenient, and nutritious consumer food products. However, food engineering topics are continuously undergoing changes to meet diverse consumer demands, and the subject is being rapidly developed to reflect market needs. In the development of food engineering, one of the many challenges is to employ modern tools and knowledge, such as computational materials science and nanotechnology, to develop new products and processes. Simultaneously, improving food quality, safety, and security continues to be critical issues in food engineering studies. New packaging materials and techniques are being developed to provide more protection to foods, and novel preservation technologies are emerging to enhance food security and defense. Additionally, process control and automation regularly appear among the top priorities identified in food engineering. Advanced monitoring and control systems are developed to facilitate automation and flexible food manufacturing processes. Furthermore, energy-saving and minimization of environmental problems continue to be important food engineering issues, and significant progress is being made in waste management, efficient utilization of energy, and reduction of effluents and emissions in food production. The Contemporary Food Engineering Series, consisting of edited books, attempts to address some of the recent developments in food engineering. The series covers advances in classical unit operations in engineering applied to food manufacturing as well as topics such as progress in the transport and storage of liquid and solid foods; heating, chilling, and freezing of foods; mass transfer in foods; chemical and biochemical aspects of food engineering and the use of kinetic analysis; dehydration, thermal processing, non-thermal processing, extrusion, liquid food concentration, membrane processes, and applications of membranes in food processing; shelf-life and electronic indicators in inventory management; sustainable technologies in food processing; and packaging, cleaning, and sanitation. These books are aimed at professional food scientists, academics researching food engineering problems, and graduate-level students. The editors of these books are leading engineers and scientists from different parts of the world. All the editors were asked to present their books to address the market’s needs and pinpoint cutting-edge technologies in food engineering. All contributions are written by internationally renowned experts who have both academic

ix x Series Preface and professional credentials. All authors have attempted to provide critical, comprehensive, and readily accessible information on the art and science of a relevant topic in each chapter, with reference lists for further information. Therefore, each book can serve as an essential reference source to students and researchers in universities and research institutions.

Da-Wen Sun Series Editor Preface

Every book has a story, and this is what’s in store for this one….

We have been invited to become editors of a book presenting what is new in fish processing at a time when those interested by the subject could select among many book titles such as Fish Processing Technology (Springer, 1997), Fish Processing: Sustainability and New Opportunities (Wiley-Blackwell, 2010), Seafoods: Chemistry, Processing Technology and Quality (Springer Science & Business Media, 2012), Seafood Processing: Technology, Quality and Safety (Wiley-Blackwell, 2014), Seafood Processing: Adding Value through Quick Freezing, Retortable Packaging and Cook- Chilling (CRC Press, 2005), and even Advances in Fish Processing Technology (Allied Publishers, 2005), to name but a few. The fast rate of science advancement, particularly at molecular level, and the abundance of novelty in fish research made the burden of the job bearable. In addition, the trust of consumers in fish as a healthy and nutritious food resource and its high market demand made fish processing a very dynamic sector, able to take on board many innovative processing and packaging methods. The paradox is that exactly the same dynamism made this important but limited resource vulnerable to food fraud; thus, the need to improve labeling and traceability of fishery products became critical for the fish trade. And there it was, the story slowly started to unfold from the current advancements in fish processing to adulteration, fair trade practices, and the need for added value, clean and sustainable processing in the fish chain. Though in the beginning it seemed difficult even to find an adequate title for our book, later on the title revealed itself as—Trends in Fish Processing Technologies, not only reflecting what is currently new in fish processing but also pointing out where things are heading in this area. Writing a book is not an easy quest; however, the process becomes easier when professionals are merging their efforts. We consider ourselves fortunate as we managed to have around a group of enthusiastic professionals who agreed to write the chapters of the book and enrolled to face the rigor of the written word, text correc- tions, deadlines, copyright permissions, and so on. We are grateful to each and all of them for accepting to partake in this scientific adventure and for their willingness to share their knowledge and experience through the pages of this book. Now, when the book is ready, we are pleased with the result and hope that its readers, either students, professionals from industry, or research, will find it useful and inspiring. By “inspiring,” we mean to find knowledge-based solutions with fore- thought and care or being creative in setting a new way to solve a problem. And, if the readers will find in the book pages new ideas and insights, answers, and more questions able to direct them into either change something or act for improvement, we could say that we have reached our goal.

The Editors

xi http://taylorandfrancis.com Series Editor

Prof. Da-Wen Sun, born in southern China, is a global authority in food engineering research and education; he is a member of the Royal Irish Academy (RIA), which is the highest academic honor in Ireland; he is also a member of Academia Europaea (The Academy of Europe), a fellow of the International Academy of Food Science and Technology, and a Fellow of International Academy of Agricultural and Biosystems Engineering. He is also the founder and editor- in-chief of Food and Bioprocess Technology, one of the most prestigious food science and technology journals; series editor of the Contemporary Food Engineering book series, with already about 50 volumes published; and the founder and president of the International Academy of Agricultural and Biosystems Engineering (iAABE). In addition, he served as the president of the International Commission of Agricultural and Biosystems Engineering (CIGR), the world’s largest organization in the field, in 2013–2014, and is now honorary president of CIGR. He has significantly contributed to the field of food engineering as a researcher, as an academic authority, and as an educator. His main research activities include cooling, drying, and refrigeration processes and systems, quality and safety of food products, bioprocess simulation and optimization, and computer vision/image processing and hyperspectral imaging technologies. His many scholarly works have become standard reference materials for researchers, especially in the areas of computer vision, computational fluid dynamics modeling, vacuum cooling, and related subjects. Results of his work have been published in over 800 papers, including more than 400 peer-reviewed journal-papers (Web of Science h-index = 79, SCOPUS h-index = 83), among them, 33 papers have been selected by Thomson Reuters’s Essential Science Indicators as highly cited papers, ranking him no. 2 in the world in Agricultural Sciences (January 2017). He has also edited 15 authoritative books. According to ESI, based on data derived over a period of 10 years from Web of Science, there are about 4500 scientists who are among the top one percent of the most cited scientists in the category of Agriculture Sciences, and in the last few years, Professor Sun has consistently been ranked among the very top 10 scientists in the world (he was at the 9th position in January 2017) and has been named Highly Cited Researcher in 2015 and 2016 by Thomson Reuters. He received a first class BSc honors and MSc in mechanical engineering, and a PhD in chemical engineering in China before working in various universities in Europe. He became the first Chinese national to be permanently employed in an Irish university when he was appointed college lecturer at the National University of Ireland, Dublin (University College Dublin, UCD), in 1995, and was then progressively promoted in the shortest possible time to senior lecturer, associate professor, and full professor. Dr. Sun is now a professor of food and biosystems engineering and the director of the UCD Food Refrigeration and Computerized Food Technology Research Group.

xiii xiv Series Editor

As a leading educator in food engineering, Professor Sun has trained many PhD students, who have made their own contributions to the industry and academia. He has also frequently delivered lectures on advances in food engineering at academic institutions worldwide, and delivered keynote speeches at international conferences. As a recognized authority in food engineering, he has been conferred adjunct/ visiting/consulting professorships from 10 top universities in China, including Zhejiang University, Shanghai Jiaotong University, Harbin Institute of Technology, China Agricultural University, South China University of Technology, and Jiangnan University. In recognition of his significant contribution to food engineering worldwide and for his outstanding leadership in the field, the International Commission of Agricultural and Biosystems Engineering (CIGR) awarded him the “CIGR Merit Award” in 2000, and again in 2006, the Institution of Mechanical Engineers based in the United Kingdom named him “Food Engineer of the Year 2004.” In 2008, he was awarded the “CIGR Recognition Award” in honor of his distinguished achievements as one of the top 1% among agricultural engineering scientists in the world. In 2007, he was presented with the only “AFST(I) Fellow Award” given in that year by the Association of Food Scientists and Technologists (India), and in 2010, he was presented with the “CIGR Fellow Award”; the title of Fellow is the highest honor at CIGR and is conferred to individuals who have made sustained, outstanding contributions worldwide. In March 2013, he was presented with the “You Bring Charm to the World” Award by Hong Kong-based Phoenix Satellite Television with other award recipients including the 2012 Nobel Laureate in Literature and the Chinese Astronaut Team for Shenzhou IX Spaceship. In July 2013, he received the “Frozen Food Foundation Freezing Research Award” from the International Association for Food Protection (IAFP) for his significant contributions to enhancing the field of food- freezing technologies. This is the first time that this prestigious award was presented to a scientist outside the United States, and in June 2015, he was presented with the “IAEF Lifetime Achievement Award.” This IAEF (International Association of Engineering and Food) award highlights the lifetime contribution of a prominent engineer in the field of food. Editors

Daniela Borda graduated in 1993 from the Faculty of Food Technology; in 1999, she obtained an MSc degree and, in 2005, acquired a PhD degree in Industrial Engineering at the “Dunarea de Jos” University of Galati, Romania (UGAL). She is currently a professor at UGAL, giving lectures in Food Quality and Safety, Dairy Technology, for bachelor and master students. Her main research interests are related with high-pressure processing, dairy science, gas-chromatography, and edible coatings and films. She was an internal and external examiner for several PhD theses. Daniela Borda has served as a reviewer for the Journal of Dairy Science (ADSA), Journal of Food Processing and Preservation (Wiley), Food Chemistry (Elsevier), Food and Chemical Toxicology (Elsevier), and other scientific publications. As food safety expert, Daniela Borda is involved in third-party audits with the Romanian National Accreditation Body–RENAR. Daniela Borda is the director of Romanian Academica Publishing House, with more than 100 scientific books published and over 50 titles in food science. Two of the books coauthored by Daniela Borda and published by Academica Publishing House have received national awards from the “Gh. Ionescu-Şişeşti” Academy of Agricultural and Forestry Sciences in Romania.

Anca Ioana Nicolau graduated as food technologist (1985) and obtained her PhD title in Biotehnology (1999) at the “Dunarea de Jos” University of Galati, Romania. She is currently professor of Food Microbiology, Rapid Methods, and Automation in Microbiology and Hygiene for Food Business Operators at the same institution where she studied and is qualified to coordinate PhD thesis in the domain of industrial engineering, specialization food industry. Her current research is related to the detection of pathogens in food and food processing environments and to the destruction of microorganisms using alternative technologies. She is member of the technological platform Food for Life Romania, where she acts as member of the operational committee and vice president of the working group Quality, Processing and Food Safety. A.I. Nicolau serves as evaluator for European projects and reviewer for several ISI journals (e.g., Frontiers in Microbiology, Food Microbiology, Foods, and Journal of Food Science). She has authored a couple of books in Romanian and many chapters in books edited either in Romanian or in English (Woodhead Publishing, Humana Press, and Springer). She received two academia awards for contributions to technical books in the domain of food industry and food safety: one from the “Gh. Ionescu-Şişeşti” Academy of Agricultural and Forestry Sciences in Romania and the other one from the Romanian Academy.

Peter Raspor is doctor of biotechnological sciences and a professor of industrial microbiology and biotechnology, teaching and researching in Slovenia and Austria. He started as a baker, and later, he finished his education in biochemical engineering with a degree in food science and later activated in biotechnology field. He started the first study program in biotechnology in Slovenia at Ljubljana University and run it from 1994 to 2002, became chair of biotechnology, microbiology, and food safety

xv xvi Editors where he activated until 2013. From 2014 to 2016 he worked at the University of Ljubljana and Institute for Food Nutrition and Health at University of Primorska, where he was member of the managerial team. Under his mentorship, more than 200 students have finished studies in the area of food technology, biotechnology, and microbiology with diploma and 47 at doctoral level. He was the head of the programs of microbiology and biotechnology at University of Ljubljana, Institute for food and health at University of Primorska, and vice dean for research and international cooperation at the Faculty of Health Sciences. He conducted more than two dozen international and national projects in the last 20 years. From 1995 until 2014, he was active with European Cooperation in Science and Technology (COST) and within top commissions of the European Union. He was president of European Federation of Food Science & Technology (EFFoST) from 2007 to 2010 and secretary general of Federation of European Microbiological Societies (FEMS) from 2000 to 2006. He is also involved with other international and national governmental and nongovernmental organizations in Europe. He contributed to European and Global policy documents and declarations in food and microbiology areas. He is a member of many scientific and professional societies and a member of editorial boards or is an editor of highly respected journals in the field. His professional profile is highly respected in the area of food technology and nutrition, industrial microbiology, and biotechnology. He has been awarded with many highest national and international awards. Professionally, he has also been involved as an auditor for ISO 9000 standards and ISO 22000 in terms of HACCP and food safety management issues for more than 20 years in the food and pharmaceutical sector. In the last few years, he has also conducted and chaired a few commissions performing international auditing of quality management systems at prestigious universities on pedagogical and research levels. Contributors

Iuliana Aprodu Jørgen Lerfall Faculty of Food Science and Engineering Norwegian University of Science and Dunarea de Jos University of Galaţi Technology (NTNU) Galaţi, Romania Trondheim, Norway

Iulia Bleoanca Allison Leyton Faculty of Food Science and Department of Chemical Engineering Engineering Center for Biotechnology and Dunarea de Jos University of Galaţi Bioengineering (CeBiB) Galaţi, Romania Universidad de la Frontera Temuco, Chile Daniela Borda Faculty of Food Science and Engineering M. Elena Lienqueo Dunarea de Jos University of Galaţi Department of Chemical Engineering Galaţi, Romania and Biotechnology Center for Biotechnology and Javier Borderías Bioengineering (CeBiB) Institute of Food Science, Technology, Universidad de Chile and Nutrition (ICTAN-CSIC) Santiago, Chile Madrid, Spain Isabelle Metaxa Mercedes Careche Faculty of Food Science and Engineering Institute of Food Science, Technology, Dunarea de Jos University of Galaţi and Nutrition (ICTAN-CSIC) Galaţi, Romania Madrid, Spain Helena M. Moreno Mette S.R. Fachmann Institute of Food Science, Technology, National Food Institute and Nutrition (ICTAN-CSIC) Technical University of Denmark Madrid, Spain Kongens Lyngby, Denmark Anca Ioana Nicolau Sunniva Hoel Faculty of Food Science and Engineering Norwegian University of Science and Dunarea de Jos University of Galaţi Technology (NTNU) Galaţi, Romania Trondheim, Norway Olafur Oddgeirsson Jeffrey Hoorfar Food Control Consultants Ltd. National Food Institute Scotland, United Kingdom Technical University of Denmark Kongens Lyngby, Denmark

xvii xviii Contributors

Livia Patraşcu Eirin M. Skjøndal Bar Faculty of Food Science and Engineering Norwegian University of Science Dunarea de Jos University of Galaţi and Technology (NTNU) Galaţi, Romania Trondheim, Norway

Johannes Pucher Rafael Soro German Federal Institute for Risk AINIA Assessment (BfR) Valencia, Spain Department of Experimental Toxicology and ZEBET Maitri Thakur Berlin, Germany SINTEF Ocean Trondheim, Norway Jan Thomas Rosnes Nofima Research Center Maria Turtoi Stavanger, Norway Dunarea de Jos University of Galaţi Galaţi, Romania Isabel Sánchez-Alonso Institute of Food Science, Technology, Loreto M. Valenzuela and Nutrition (ICTAN-CSIC) Chemical and Bioprocessing Engineering Madrid, Spain Pontificia Universidad Católica de Chile Santiago, Chile Christian Schlechtriem Franhofer Institute for Molecular Joop van der Roest Biology and Applied Ecology (IME) RIKILT Wageningen UR Department of Bioaccumulation and Wageningen, the Netherlands Animal Metabolism Schmallenberg, Germany Marjolein van der Spiegel RIKILT Wageningen UR Ausra Sipailiene Wageningen, the Netherlands Department of Food Science and Technology Sanja Vidaček Kaunas University of Technology Faculty of Food Technology and Kaunas, Lithuania Biotechnology University of Zagreb Dagbjørn Skipnes Zagreb, Croatia Nofima Research Center Stavanger, Norway Introduction

FISH AND TECHNOLOGY FOR THE FUTURE Peter Raspor In 2017, the world’s population is nearly 7.5 billion people, and according to estimates, it could reach 10 billion by 2050. The rising number of earth’s population simultaneously means that more people need to be nourished—a mission difficult to manage, when thinking at several challenges already present and stretching ahead. Scarce of living environment accompanied by continuous population growth, water shortage, climate change, and its consequences are just a few examples of the difficulties the world already has to deal with. Unfortunately, humankind is not always a part of the solution, but mostly a part of the environmental problems through practices such as water contamination via pesticides and other ecologically harmful substances, overfishing that leads to resource depletion, or by deforestation, thus irreversibly affecting natural equilibrium in nature. Moreover, in 2013, the status of the majority of world fishery confirms the previous dismal picture: serious depletions being the norm worldwide, poor management quality, and decline of catch per effort. The performance of stock assessment is challenged by random environmental shifts and by the need to accommodate ecosystem-level effects. Moreover, marine ecosystems and their embedded fisheries are challenged in parallel with climate change by eutrophication, acidification, metabolic disruptors, etc. The global picture of further fish species extinctions, the degradation of ecosystem food webs, and seafood security is indeed alarming. The attempts to remedy the situation need to be urgent, focused, innovative, and global. If in the past, our technological choices were not the most sustainable ones, today, we ought to change it into more responsible ways of producing and processing our food if we want to protect our already vulnerable natural environment. This book discusses clean processing methods, added values to the fish chain, and new processing opportunities as the authors are concerned to responsibly bring the latest novelties to consumers’ plate and enhance their lifestyle and health status with balanced nutrition. Trends in Fish Processing Technologies assesses the state of the art in fish processing throughout 16 chapters, touching on and analyzing some of the recently developed technologies, processes, and research studies with good potential to be in regular production and distribution practice in the near future. Starting with “Recent Advances in Fish Technology: An Overview” by Borderías and Moreno the current practice in processing technologies like heat processing, curing, drying, chilling, freezing, storage, packaging, and thawing is extensively discussed, followed by seafood protein production focused on determination of physicochemical and sensory behavior in proteinaceous products. Quality assessment, divided in eight methodological clusters, is introduced, discussing the advanced methods applied in today’s practice.

xix xx Introduction

“Minimal Heat Processing Applied in Fish Technology” as laid down by Rosnes and Skipnes presents mild heat processing technologies available for reducing the heat load on heat-sensitive fish, different methods for heat treatments before and after packaging, and combined methods. New approaches with combined effects based on microwave, radiofrequency, and pulsed electric field are timely addressed from the perspective of preserving fish nutrients and sensorial properties while reducing the heat load. The integration of these new strategies into practice requires validation for successful processing. It is expected that the market demand of minimal processed fish will continue to increase, so the development of new technologies and new applications for advanced technologies is decisive. Advancement in this area brings new alternatives to conventional processing, able to produce fish products with improved quality and safety attributes. “High-Pressure Processing of Seafood,” addressed by Borda, delivers comprehensive information about HPP effects not only on fish constituents but also on sensorial fish properties. The effect of high-pressure processing (HPP) on fish safety covers extensively the impact on microorganisms, biogenic amines, and allergens. Moreover, high-pressure thermal sterilization, high-pressure freezing, and thawing are introduced. The view into the future is well addressed, with particular emphasis on the importance of gaining the trust of consumers by building a solid reputation of HPP as a clean and stable food technology. The opportunity to synergistically combine HPP with other hurdles by using natural antimicrobials and antioxidants and/ or other minimal processing technologies to improve product safety and quality is a way to overcome the drawbacks associated with intensive processing. A very challenging topic is “Processing of Low-Value Fish, Coproducts, and By-Catch,” addressed by Patraşcu and Aprodu, illustrating trends in processing of low-value fish, coproducts, and by-catch. Most of them are rich sources of valuable nutrients for humans and animals. Selective recovery of proteins from fish materials not intended for human consumption allows for improvements in resource management. In particular, recovery of fish proteins and enzymes shows great added value besides creating a good base for circular economy. There are many “Advances in Surimi Processing,” as illustrated by Sipailiene regarding stabilization of myofibrillar protein obtained from mechanically deboned fish flesh, washed with water and blended with cryoprotectants. Applications of alternative cryoprotectants brought new opportunities to develop products with low salt and low sweetener content. In recent years, the use of the pH shift method proved to be an effective way to get higher-quality proteins with functional properties. Recently, consumer-oriented trends showed a strong demand for new fish products with clean label and improved quality. Valenzuela et al. discuss in “Reformulation of Preserved Fish Products” the reduced-salt-content products and associated safety issues, natural antioxidants, and antimicrobial preservatives. Natural ingredients are a rich source of active components and show an excellent capacity to compensate salt reduction, reduce lipid oxidation, and inhibit bacterial growth. However, there is still room to improve sensorial properties such as color, texture, taste, and flavor of the fish and, consequently, the quality of the end product. However, there are only a few studies that take into account the synergy or antagonistic effects among different ingredients within the food matrix and the overall effect on quality. Industrial Introduction xxi scale validation must be performed that includes consumer validation in terms of food safety, quality perception, and organoleptic approval. All final ingredients must fulfill specific regulations in terms of safety, stability, labeling, and information. “New Product Development” deserves its special place in this book. Speaking about sustainable seafood products side by side with greenhouse gas emissions caused by food production has lately been rightfully receiving increased attention worldwide. Due to limited capacity of the oceans, the seafood industry needs to be sustainable in order to flourish in the future. With increasing attention to illegal and unfair fisheries, sustainable fisheries that are becoming certified and labeled are expected to grow, impact seafood trade, and bring in creative solutions that will support new product development. Vidaček and Soro discuss in the “Clean Fish Processing Technologies” chapter seafood production, processing, and consumption, separately addressing capture and aquaculture in relation to sustainability. The fish processing industry already applies a number of clean techniques that reduce the environmental impact of the industrial activity. The Integrated Pollution Prevention and Control Directive supports implementation of the best available technique to reduce emissions and the impact on the environment. Selected cleaner techniques in the fish processing industry are presented in this chapter. “Innovative Fish Packaging Solutions” are making a real breakthrough in the latest advancement in the packaging industry. Bleoanca and Turtoi present the role of active packaging in fish processing considering the advantages provided by edible films and coatings with essential oils and antimicrobial enzymes, by multifunctional bioblends and nanobiocomposite films. Another interesting area discussed is intelligent packaging of fish that contributes to increased product durability and responds to consumers’ needs. Intelligent packaging is either monitoring the environmental conditions or is using data carriers for a more effective communication between the product and the consumer. The quest for higher food quality and safety is one dominant challenge for food producers, which was often limited by the lack of active and intelligent materials. Now, with so many innovative packaging solutions in the pipeline very soon, there will be new fish products on the market with active or intelligent packaging. Pucher and Schlechtriem show how to achieve an adequate level of protection over a broad range of hazards with potential to have an immediate, interim, or long-term effect on human health, which have been identified in aquatic foods. Consequently, appropriate measures of control to prevent, reduce, or minimize the risks are required to ensure food safety. The measures and indicators used by the regulating authorities to achieve an adequate level of protection for the consumers of fish and other aquatic food products are essential for targeted and efficient risk management. Chapter 11 introduces “Food Safety Management in Fish Processing Units,” discussing the basic structure of food safety management systems (FSMSs). Nicolau and Oddgeirsson assess the available FSMSs and analyze the challenges for fish processing operators, focusing on different aspects such as threats and vulnerabilities, food defense, and food fraud prevention systems and indicating the trends in Hazard Analysis Criterial Control Point and FSMS. These are all viewed as opportunities to continuously improve the overall safety of food products. xxii Introduction

Careche and Sánchez-Alonso present “Quality and Quality Changes Assessment of Processed Fish.” Special attention is given to fish deteriorative changes and indicators to measure changes in properties or composition of fish. They show the need for methodologies to provide objective indicators for quality aspects of fish, applied in all the steps of the processing chain. Presentation of some sensory (Quality Index Method) and instrumental (multisensors or spectroscopic) methods is timely addressed for current needs. Some of them are ready to use for specific situations and others require further development for wider application. Molecular methods for the assessment of fish and fish product integrity are delivered by Fachmann and Hoorfar in “Molecular Methods for Assessment of Fish and Fish Product Integrity.” The application of automated molecular methods for both safety and authenticity assessment of fish and fishery products is discussed, given the great potential for online screening. The use of portable PCR equipment represents a promising tool in detection of fish frauds, but it also can be used for safety and authenticity and traceability assessment. The use of molecular methods in the near future for on-site and real-time testing will have good prospects if validation and standardization protocols will be developed. “Adulteration and Misbranding of Fish Products,” analyzed by van der Spiegel and van der Roest, indicates that traceability in the fish supply chain is of crucial importance to detect fish mislabeling. Incidents of fish substitution have been reported worldwide, despite labeling and traceability regulations. So, the optimization of sample size and frequency together with proper methods, good practices, and management may help in the future to prevent adulteration in aquaculture and fish processing. Future research should focus on collecting data on actual fish substitution and sharing information among governments and the private sector across countries to respond to the incidents reported worldwide. “Traceability of Fish Products,” addressed by Thakur, shows that supply chains of perishable products such as fish require efficient management to be able to maintain product quality. Traceability systems provide an effective tool for managing the fish supply chains and can also be used for cold chain monitoring and for improving the resource efficiency in fish and other food supply chains. To enable supply chain transparency, the ability to track and trace food products should be in place. Automated traceability is based on electronic data capture and exchange. Presentation of current technological trends in developing electronic traceability and temperature monitoring systems for fish products underlines that there is a huge potential to develop applications of traceability systems, including food supply chain management. The last chapter, “Fish Trade Regulations,” written by Metaxa, brings an overview on fisheries product trade and discusses consumption indicators and trade regulations in relation to sustainability, underlying the legislative framework for fish products in Europe. Fish and fish products are part of the human diet and human culture in many countries around the globe. This wholesome food has been consumed by humans since ancient times. Fish has gained a reputation of being a high-quality protein source, rich in omega-3 fatty acids and vitamins such as D and B2 and in calcium and phosphorus and other minerals, such as iron, iodine, magnesium, potassium, and zinc. Introduction xxiii

The technology of catching and processing has been permanently improved and transferred its best solutions to humans, unfortunately at nature’s expense. Even though from a technological standpoint there is currently a broad spectrum of methods and classical technologies that serve reasonably well to the purpose of providing fish products, if there is will to preserve our natural resources for future generations, there is the need not only for incremental improvements on these technologies but also major discoveries supported by inventions to enter into sustainable production systems. Innovative technologies and new methods could bring solutions for solving problems and prevent fatidical scenarios, which will be unavoidable if humankind continues on its current trajectory of unsustainable consumption. This publication tries to fuse cutting-edge research knowledge and present possibilities to integrate it into practice. Hopefully, further development will support a part of this knowledge transfer into food supply nets and will make the lives of producers easier and the lives of consumers more comfortable. Fish should remain a highly valuable resource and a part of a balanced human nutrition if humankind will learn from past mistakes and take the advantage of responsible use of modern technology. http://taylorandfrancis.com Recent Advances 1 in Seafood Technology An Overview

Javier Borderías and Helena M. Moreno

CONTENTS 1.1 Introduction...... 2 1.2 Processing Technologies...... 2 1.2.1 Heat Processing...... 2 1.2.1.1 Thermal-Assisted Processes...... 3 1.2.2 Curing...... 3 1.2.2.1 Salting...... 4 1.2.2.2 Smoking...... 4 1.2.3 Drying...... 5 1.2.4 Chilling...... 6 1.2.5 Freezing...... 7 1.2.5.1 Pressure Shift Freezing...... 8 1.2.5.2 Impingement Freezing...... 8 1.2.5.3 Magnetic Freezing...... 8 1.2.5.4 Hydrofluidization Freezing...... 9 1.2.6 Frozen Storage...... 9 1.2.7 Thawing...... 9 1.2.7.1 High-Power Ultrasound Method...... 10 1.2.7.2 Low-Frequency Acoustic Thawing...... 10 1.2.7.3 Pressure-Assisted Thawing...... 10 1.2.8 Packaging...... 10 1.2.8.1 Active Packaging...... 10 1.2.8.2 Intelligent Packaging...... 11 1.2.8.3 Edible Coatings and Films...... 12 1.2.9 Seafood Proteins...... 12 1.2.9.1 Surimi Gelation...... 12 1.2.9.2 Hydrolysates and Peptides...... 14 1.3 Quality Assessment...... 14 1.3.1 Sensory Methods...... 15 1.3.2 Chemical Methods...... 15 1.3.3 Physical Methods...... 15 1.3.3.1 Image Analysis...... 16

1 2 Trends in Fish Processing Technologies

1.3.3.2 VIS/NIR Spectroscopy...... 16 1.3.3.3 Nuclear Magnetic Resonance...... 16 1.3.4 Microbiological Examination...... 17 1.3.4.1 Electrical Methods...... 17 1.3.4.2 Respiratory Activity...... 17 1.3.4.3 Inmuno-Based Techniques...... 18 1.3.4.4 Molecular Methods...... 18 References...... 18

1.1 INTRODUCTION Worldwide demand for seafood has increased in recent years due to its prodigious nutritional properties. According to the Food and Agriculture Organization of the United Nations (FAO, 2016), in developed countries, processed seafood (frozen, prepared, preserved, and cured) accounts for 92% of total seafood used for food purposes; meanwhile, in developing countries, this amount is only 53%. Ensuring the quality of edible seafood raw material, fresh or processed, is not an easy task, and seafood should therefore be treated with great care from the time of fishing up to the last processing step. Seafood processing methods were initially designed to prolong shelf life and ensure food safety, but nowadays, they also address the creation of new seafood products. Seafood processing applies the most known processing methods and packing technologies to improve quality throughout preparation and storage. At the same time, methods to control all the different aspects of seafood quality need to be optimized, not only in fresh but also in processed seafood. This chapter presents an overview of the latest research in processing technologies and methodologies for preservation of seafood quality.

1.2 PROCESSING TECHNOLOGIES

1.2.1 H eat Processing Heat processing can be subdivided into several groups depending on the temperature regime. The most common method, sterilization, is intended to inactivate all pathogenic bacteria and their spores. The temperature regime is 110°C–135°C for enough time to ensure the inactivation of bacterial spores because the heat resistance of any microorganism will change depending on pH, water activity, or chemical composition, which can also modify the required length of thermal processes (Lewis and Heppell, 2000). Sterilization is regularly applied for canned seafood with high durability, in which it is vital that spores of Clostridium botulinum are destroyed without overprocessing, which results in low quality of the product (Miri et al., 2008). Pasteurization is applied at temperature regime around 70°C–90°C, for a duration of time long enough to inactivate vegetative pathogens such as salmonella; however, some microbial survivors (e.g., sporeformers) might be present in the food after the process. For these reasons, low-acidic (pH < 4.6) pasteurized foods are generally stored, transported, and sold under refrigerated conditions (temperature below 7°C) and with a limited shelf life (Silva and Gibbs, 2012). Combining conventional Recent Advances in Seafood Technology 3 thermal treatments with other different processing methods represents a new trend aimed at overcoming the major drawbacks of the conventional thermal processing associated with Maillard reactions, changes in flavor, and vitamin degradation.

1.2.1.1 Thermal-Assisted Processes One particular type of thermal treatment specific to restaurants that is gaining interest in being industrialized is sous-vide processing, which consists of thermal processing in plastic vacuum pouches at precisely controlled time–temperatures; this improves product shelf life and can enhance the taste and nutritional value of the product (Baldwin, 2012). Products of this kind of minimal processing are a growing segment in developed countries. With conventional heating methods, as used in canned foods, the heat load is higher on the surface than at the center of the product. In these cases, uniform heating can be achieved by rotation of the package during conventional heating (Eisner, 1988) and it can be also combined with ultrasound (Skipnes, 2014). However, in some seafood products, rotation shaking or so will make the seafood fall apart, limiting the use of these methods (Skipnes, 2014). In food processing, ultrasounds commonly operate between 16 and 100 KHz to induce to higher temperatures and pressures in the product (Patist and Bates, 2008). A more recent method in which heat is generated directly in the food rather than transferred to it is ohmic heating; this is an effective solution entailing the application of an electric current (140–150 V and 50–60 Hz) to generate heat throughout the food. Heating with electric fields is more suitable for packaged products with plastic packaging materials (Fryer et al., 1993). Other recent methods, like microwaving, whereby the food is heated inside and outside simultaneously, are not commonly used because of the uneven heating and the high rate of evaporation and the low rate of penetration (Duan et al., 2011). Radiofrequency heating has been also used because it has greater penetrating power, but it can produce uneven heating in nonhomogenous materials, especially mixture of solids and liquids, since heating is produced by depolarization of solvated ions (McKenna et al., 2006; Lyng et al., 2007). However, some researchers have demonstrated lower cooking loss and better texture control in meat products with this system (Lyng et al., 2007).

1.2.2 curing There are various methods of preserving fish by curing, including drying, salting, smoking, marinating, combinations of these methods, and fermentation (FAO, 1983). Curing is an ancient preservation technique. Despite the fact that curing has undergone changes in the course of history, in principle, it remains fundamentally the same. White fish species are traditionally preserved by salting because they contain fewer lipids, while fatty pelagic species are generally preserved by smoking and marinating due to their high fat content. There have been changes in the methodology and/or the equipment used in salting and smoking, but marinating and fermentation processes follow the traditional practices, which are very closely tied to certain geographical regions. 4 Trends in Fish Processing Technologies

1.2.2.1 Salting The salting process depends a lot on the fish species and on local traditions, but it has undergone considerable change in the last few decades. Salted cod (bacalao) is probably the most widely consumed dry-salted product in Europe. For the last century or so, the original dry salting technique has been used (Gallart-Jornet et al., 2007), but lately, new methods of cod salting have been developed that achieved higher production yields, consisting of injection of brine into the muscle or brining followed by dry salting. These new methods accelerate salting, increase automation, achieve better salt distribution, and improve processing yield (Andrés et al., 2005a; Thorarinsdottir et al., 2011). They also improve some sensory characteristics such as texture and appearance (Martínez-Álvarez and Gómez-Guillén, 2005). At the same time, attempts have been made to reduce the proportion of salt to obtain healthier products. To that end, MgCl2, KCl, and CaCl2 have been tested, with negative results, for muscle texture and functional properties (Martínez-Álvarez et al., 2005). To facilitate preparation for the consumer, rehydrated salted cod has recently come on the market, both in frozen and chilled forms. As a result, some special techniques have been applied to accelerate water uptake and salt loss, such as tumbling and vacuum pulses for reducing rehydration time (Bjørkevoll et al., 2004; Andrés et al., 2005b). Lightly salted cod (salt concentration of 2%–3%) is a new product that has been gaining popularity in Southern Europe in recent years. Fish fillets are salted by brine injection or immersion in brine and then frozen. The resulting texture is better, with higher water holding capacity and reduced water drip during storage compared to directly frozen cod (Arason et al., 2014).

1.2.2.2 Smoking Smoking endows fish muscle with flavor and preservative compounds. Smoking was originally a preservation method complemented by heavy salting and/or drying in addition to the smoking process (Alcicek and Atar, 2010); however, smoking nowadays is predominantly applied for adding specific sensory characteristics to these products rather than for preservation purposes (Birkerland et al., 2004). Depending on the temperature and on how the smoke is delivered to the fish muscle, smoking can be defined as hot smoking, cold smoking, liquid smoking, or electrostatic smoking. In the case of cold smoking, which is the method most commonly used in Europe, there are different techniques for smoke generation and/or application of smoke components. These methods are: smoldering of wood by means of an open fire, smoldering of wood using thermostatic plates, smoke generation by friction, and the application of smoke condensates by vaporization or drenching. In 2002, the proportion of these methods used in Europe was approximately smoldering 65%, thermostatic plates 30%, and friction and smoke condensates 5% (Varlet et al., 2007a), although the last one lately has increased significantly. The effects of different processing methods on quality parameters such as texture, color, muscle gaping, liquid loss, and processing yield have been investigated by a large number of authors (Cardinal et al., 2000; Mørkøre et al., 2001; Birkerland and Bjerkeng, 2005). Recent Advances in Seafood Technology 5

Liquid smoking with concentrates is performed by dipping the muscle in a liquid smoking extract produced by dry distillation of wood followed by concentration (smoke condensate). Smoke condensates are generally produced following principles that limit the formation of polycyclic aromatic hydrocarbons (PAHs) and are often further rinsed with water at 15°C to reduce the presence of these components in the condensate (Stolyhwo and Sikorski, 2005; Varlet et al., 2007b). The application of smoke condensates to fish has been investigated by several researchers (Muratore et al., 2007; Varlet et al., 2007b), who have found that the different available smoke flavorings have different effects on taste, usually less salty, depending on the fish species. Moreover, fillets treated with smoke condensates contained less PAHs than traditional smoked fillets did. In electrostatic smoking, the fish is treated with infrared radiation. Muscle is smoked by the creation of a positively charged electrical field while the fish is negatively charged. The electrical field works on the ionized smoke particles to accelerate the smoking process. Electrostatic smoking is fully mechanized, thus entailing savings on labor and production costs. The process renders a higher quality in the final product compared with traditional smoking processes (Arason et al., 2014), although depending on the fish species, the shear force may be lower, as in the case of salmon (Montero et al., 2003). Also in electrostatic smoking, the product tends to have a stronger odor, particularly in herring muscle (Cardinal et al., 2000). Nowadays, the high level of scrutiny from consumers’ organizations determined the European Food Safety Authority (EFSA) to better evaluate the risks of contaminants presence in foods, seafood products being considered one of the highest contributors to PAH presence in the diet. Thus, the efforts for reducing PAH concentration in foods by replacing traditional smoking processes with liquid smoke flavoring is considered a good strategy to reduce the PAH levels in commercially smoked food commodities (EFSA, 2008). Moreover, the trend of reducing the salt intake in the diet, associated with a healthy nutrition and a reduced incidence of coronary diseases, encouraged producers to develop more lightly processed products with a relatively limited shelf life. In this context, it became important to control the possibilities of contamination by combining smoking and salting with preservation methods such as chilling, specific packing methods, of which the most common is vacuum packaging. One potential hazard that may occur in cold smoked products packed under vacuum is the growth of C. botulinum spores, but an appropriate combination of NaCl and low temperature is sufficient to prevent growth of this microorganism during shelf life in vacuum packaging (Dufresne et al., 2000).

1.2.3 Drying Drying means removal of the water content to levels that can slow down the actions of enzymes and microorganisms (Janjai and Bala, 2012). Water removal includes evaporation of water from the surface of fish and transfer of water from internal layers to the surface. The rate of heat flux depends on the air humidity, drying temperature, air velocity, mass, and heat transfer coefficients in interaction with the humidity 6 Trends in Fish Processing Technologies and temperature distribution inside the fish muscle (Gavrila et al., 2008) and retention time. In developing countries, sun drying is the most convenient means of preserving fish products, particularly in places with high levels of solar radiation. However, it presents a major problem in terms of possible infestation by insects (Inmaculate et al., 2012). Moreover, it is difficult to control the drying process in open air, and it requires large areas. Solar drying, on the other hand, minimizes or obviates some of the limitations of open sun drying. The process consists of an enclosed structure that traps heat inside a dryer and makes effective use of it. Thanks to the higher temperature in the dryer, as compared with open air drying, drying time, insect infestation and microbial spoilage can be reduced considerably. Various types of solar dryers have been designed in tropical and subtropical countries (Bala and Debnath, 2012). Solar drying saves energy and time, occupies a smaller area, and improves the quality of the final product (VijayaVenkataRaman et al., 2012). There are two categories of solar dryers: (a) natural convection solar dryer (passive dryer) and (b) forced convection solar dryer (active solar dryer) using a fan. Heat pump dryers have been readily available since the 1970s. These operate more efficiently at lower temperatures than conventional dryers do, which is good for heat-sensitive materials (Hawlader et al., 2006). They are not dependent on weather conditions and are environmentally friendly (Perera and Rahman, 1997). Freeze-drying is an innovative process that involves removing water by sublimation of ice crystals from a frozen material (Ciurzynska and Lenart, 2011). It is the best drying method but also the most expensive, both in terms of equipment and running cost. It is not generally used on fish for practical and economic reasons, but there are some references in the literature to its use on tuna (Rahman et al., 2002), Atlantic mackerel, horse mackerel (Sarkardei and Howell, 2007), and cod (Eikevik et al., 2005). Osmotic dehydration is used as a supplementary treatment in processes such as marinating and smoking. It reduces color loss, promotes volatiles expel, enhances taste, inhibits enzymatic browning, reduces energy cost, and increases shelf life due to the salts and/or sugars used in osmotic solutions (Alakali et al., 2006). Vacuum pulses are applied to the osmotic system to reduce processing time and thus improve the mass transfer rate (Chiralt et al., 1999).

1.2.4 chilling Fish is a highly perishable product urging preservation almost immediately after harvesting. Chilling is the method that produces the least obvious changes; however, storage life is limited depending on fish species, type and season of capture, and of course storage temperature. In 2010, fish marketed live or fresh for human consumption was the most important fish product (40.5%) (Jessen et al., 2014). There are various interrelated physical, microbiological, and chemical reactions that commence just after the fish death and lead to gradual loss of fish quality. Thus, the initial preservation methods applied should be aimed at slowing down the bacterial and enzymatic degradation of fish muscle and reducing the fish quality loss. Traditionally, fresh fish has been preserved either alive or in ice. Ice is the most widespread method of keeping fish fresh, but there are other traditional methods, for instance, placing Recent Advances in Seafood Technology 7 the fish in containers with chilled water, a very common practice for small pelagic fish in European Nordic countries. In Southern Europe, for some years now, small polystyrene containers with iced water have been used to transport small pelagic fish from the coast to major city markets. In one advanced chilling method, ice slurry is used to improve the quality of different aquatic food products. Slurry ice (also known as fluid ice, slush ice, liquid ice or flow ice) is currently viewed as a promising technique for fresh fish storage (Piñeiro et al., 2004). Slurry ice can be defined as a mixture of ice particles and an aqueous solution. This solution is composed of water and other solid components such as salt to reduce its freezing point and achieve temperatures in the negative range, slightly below 0°C, but not so low as to freeze the surface of the product. There are two main advantages to slurry ice: (a) the chilling is faster because it has a higher heat-exchange capacity than flake ice or refrigerated sea water does and (b) there is less physical damage to seafood products from the spherical microscopic crystals characteristic of slurry ice. In some cases, slurry ice can be combined with other additives such as preservatives, antimelanotics, or antioxidants. However, aside from cost considerations, slurry ice has some disadvantages such as clouding of the eyes or some discoloring in crustacean shells (Huidobro et al., 2002). Meanwhile, some authors have reported that slurry ice or flake ice did not slow down the spoilage rate in sea bass, a warm fish species (Martinsdóttir et al., 2002). Studies on other species have reported clear advantages of slurry ice over flake ice in the storage of different species (Losada et al., 2006). In some cases, seafood is stored in what is known as a freeze-chilling state for long distance transportation (Fagan et al., 2003). This consists in partially freezing the fish at temperatures up to −4°C, which prolongs shelf life more than chilling (Adler-Nissen and Zammit, 2011). Another alternative procedure is to apply high isostatic pressure prior to chilling storage. This is a fairly new and important processing method, used mainly to pas- teurize food without altering it as much as when heat is applied. In fish muscle, especially from 200 MPa upward, the muscle becomes whiter and more opaque, and for that reason, it is relatively seldom used on fish. However, be that as it may, Hurtado et al. (2000) and Chevalier et al. (2001) found that pressurizing at 200 MPa on hake and at 180 MPa on turbot significantly prolonged chilled storage life with no obvious changes in the appearance. This technology has been assayed at 400 MPa in oysters, and in that case, the appearance was even better after pressurization, the total plate count was reduced (López-Caballero et al., 2000) and high pressure killed Vibrio parahaemolyticus (Calik et al., 2002). Pressurized oysters are currently marketed in the United States.

1.2.5 Freezing The freezing process aims to decrease bacterial growth and reaction rate of enzymes by converting the water in the fish body into ice crystals at a temperature of −10°C or colder, and the resulting concentration of dissolved solutes in the water that remains will lower the water activity (International Institute of Refrigeration [IIR], 2006). There are four traditional freezing methods for seafood: (a) blast freezing, in which 8 Trends in Fish Processing Technologies a flux of cold air (−40°C) is circulated at high speed over the seafood in a room or over or under a conveyor belt; (b) contact freezing, in which the fish is in direct contact with a cold metal surface; (c) cryogenic freezing, where the seafood is sprayed with liquid nitrogen; and (d) immersion freezing, in which the seafood product is immersed in super-cooled aqueous solutions. New and increasingly sophisticated types of freezing equipment are being developed, but these are only of interest to the fishing industry if the cost is acceptable. The following is an overview of these methods.

1.2.5.1 Pressure Shift Freezing In this method, the water–ice transition is depressed under high isostatic pressure (higher than 100 MPa); the product is then cooled sufficiently (to less than −22°C), and finally, the pressure is released to induce nucleation throughout the product. Pressure release may be either slow, over several minutes (Levy et al., 1999), or fast, lasting only a matter of seconds (Otero et al., 1998). The faster the pressure release, the lower the nucleation temperature and the greater is the resulting degree of supercooling. The higher the pressure and the lower the preexpansion temperature, the more ice is formed, and hence, the shorter is the plateau time for a given cooling temperature (Otero and Sanz, 2003). This method produces very good seafood texture and water binding (Alizadeh et al., 2007), but it is not yet a practical option as the final quality does not outweigh the disadvantages of the high fixed cost.

1.2.5.2 Impingement Freezing This method consists of directing a jet or jets of fluid at the seafood surface at very high velocity (20–30 m s−1). The gas jets break up the static boundary layer of gas that surrounds the seafood products. The resulting medium around the product is turbulent, and consequently, the heat exchange becomes more effective. This method is best suited to products with high ratios of surface area to weight and no thicker than 2 cm, for example, fish fillets. Impingement technology increases the surface heat transfer in air and other freezing systems (Everington, 2001). Experiments with shrimps and fish fillets have been successful in achieving high-quality products evaluated by drip loss and sensory analysis (Jessen et al., 2014). Impingement freezing technology is currently commercially available.

1.2.5.3 Magnetic Freezing Magnetic freezing acts directly on water by orientating, vibrating, and/or spinning molecules to prevent them from clustering during cooling and thus promote supercooling. In this way, magnetic freezing is able to generate very small ice crystals throughout the frozen product; this prevents cell destruction and helps maintain food quality intact after thawing. Otero et al. (2016) have authored an interesting review of magnetic freezing, in which they express some doubts about its mechanisms and efficiency. The brand ABI Co. Ltd. (Japan) commercializes a successful device based on this technology called “CAS (Cells Alive System) freezing”; it uses an oscillatory magnetic field that causes water molecules to spin (as opposed to vibrating as in microwaving) and prevents the formation of ice crystals that can damage the cell Recent Advances in Seafood Technology 9 walls. This method has been used for sardine freezing (ABI Co., Ltd., 2007), in which microscopy pictures showed that the sarcolemma remains intact after freezing.

1.2.5.4 Hydrofluidization Freezing Briefly, hydrofluidization freezing is a combination of immersion freezing and forced liquid fluidization (Peralta, 2009). In this method, an aqueous solution at low temperature is used as a liquid refrigerant. The liquid is pumped upward through orifices or nozzles into a container where the seafood product is loaded, thereby creating agitating jets and turbulent agitation. This produces a fluidized bed of highly turbulent liquid and moving food products, thus achieving high heat transfer coefficients (Peralta, 2009). In these conditions, the critical crystallization zone of water in the muscle is quickly surpassed and very small crystals are formed, preventing damage to cell tissues. The system is especially useful for small fishes or shrimps but not for soft fish fillets, which can be degraded during the process.

1.2.6 Frozen Storage The refrigeration system must be designed with regard to the requirements of the climatic conditions, being adequate to allow sufficient cooling capacity on summer conditions. Temperatures should be low and maintained constant throughout the storage. The majority of cold stores have forced air circulation by means of air coolers. Most of the references from literature recommend a general storage temperature of −30°C (IIR, 2006), but in practice, for other than special fish products (such as tuna for sushi and sashimi), the IIR recommends a storage temperature of −18°C for white fish and −24°C for fatty fish. In retail shops, frozen foods are displayed for sale to the customers in different types of refrigerated open display cabinets (horizontal and vertical display area).

1.2.7 thawing Thawing is the process whereby a frozen product is unfrozen. It involves transferring heat to a frozen product with the purpose of melting the ice that was formed within the muscle during the freezing process (Archer et al., 2008). Thawing is often a longer and more critical process than freezing, as the rate of heat transfer to the muscle decreases when the surface of the food thaws; this happens because the thermal conductivity of the unfrozen food is lower than the one of the frozen food. During seafood thawing, temperatures are higher at the surface, so it is very important not to allow the product surface to become too warm during thawing, as this would accelerate spoilage. Traditional thawing methods include running water, still or ambient air, and forced air. Further new developments in tempering and thawing equipment include different forms of thawing such as steam vacuum, ohmic thawing, dielectric heating, and microwaves. The newest experimental methods include power ultrasound, low-frequency acoustics, and pressure-assisted thawing: 10 Trends in Fish Processing Technologies

1.2.7.1 High-Power Ultrasound Method This method uses a frequency band around 500 KHz and intensities around 0.5 W cm−2. Miles et al. (1999) used this method in cod samples that were thawed to a depth of 7 cm within 2.5 hours.

1.2.7.2 Low-Frequency Acoustic Thawing This method has been used to thaw blocks of Pacific cod 91 mm thick weighing 12.7 kg; these were thawed in water at 18°C and simultaneously exposed to 1500 Hz acoustic energy not exceeding 60 W. In these conditions the time required for conventional thawing was reduced by 71% (Pham, 2014).

1.2.7.3 Pressure-Assisted Thawing Another new experimental method is the high-pressure assisted thawing. High pressures (up to 200 MPa) have the effect of depressing the temperature of ice crystal formation. Murakami et al. (1992) thawed tuna under pressure between 50 and 150 MPa for 30 and 60 minutes. There was a significant reduction in drip loss compared to blocks thawed at atmospheric pressure, but muscle color changed from red to pink. Schubring et al. (2003) reported better quality of fish fillets when thawed under 200 MPa pressure as compared with conventional thawing.

1.2.8 Packaging Fishery products are highly perishable during chilled storage. Degradation is the result of physicochemical, autolytic, and microbiological processes. Modified atmosphere packaging (MAP) is a “classic” technique that prolongs shelf life by providing an atmosphere that retards microbiological growth and attendant deteriorative and chemical processes (Young et al., 1988). MAP generally combines CO2 (40%–60%), O2 (10%–50%), and N2 (the remainder) (Noseda et al., 2012). Another important function of packaging, apart from improving storage duration, is convenience. Portions of seafood can be presented in packaging that is microwaveable, easy to open, reclos- able, etc. Indeed, the packaging itself can be important as a communication interface that informs consumers and often tempts consumer into buying products. MAP is probably the most common method of chilled seafood packaging, but nowadays, it may be considered a conventional methodology. More modern methods of packaging include active packaging, intelligent packaging, and edible coatings and films. There is also a lot of interest currently in sustainable packaging materials made from starch, cellulose, and other renewable bio-based resources that can be reused and recycled.

1.2.8.1 Active Packaging Active food packaging has been defined as a system in which the food product, the package, and the environment interact in a positive way to extent shelf life or to achieve some desirable characteristics that cannot be obtained otherwise (Miltz et al., 1995). Active packaging enhances the protection afforded by general packaging by improving moisture and nutrient retention, deterring oxidation, and inhibiting Recent Advances in Seafood Technology 11 microbial spoilage. The action mechanism of active packaging includes removal of some gases (oxygen, water vapor) from the package head space and emission of desired gases (carbon dioxide, ethanol) to the headspace, and it controls the release of active compounds (antimicrobials, antioxidants, enzymes, flavors, nutraceuticals, etc.) to the headspace (Lee, 2012). Devices used in active packaging include sachets, sheets, blanket liners, adhesive labels, polymer structures, closure liners, coatings, etc., and are located inside the food packages. They can be used for either chilled or frozen foods, although this kind of packaging is more suitable for chilled products.

1.2.8.2 Intelligent Packaging Intelligent packaging is defined as a package containing a sensor that warns consumers that the product is impaired and may begin to undergo harmful changes (Karel, 2000). In general, packaging should provide a good barrier, both to oxygen, to prevent microorganism growth and chemical reactions, and to moisture, to avoid dehydration or freezer burn. However, it is not uncommon for plastic to tear or seals to leak, causing deterioration of the product. There are noninvasive systems that can be used as integrity indicators to warn of oxygen permeation in the packaging. The method requires a colorimetric oxygen indicator that can be assembled using a semiconductor photosensitizer coupled with a redox dye and a sacrificial electron donor in the form of an intelligent ink (Mills and Hazafy, 2008). Other systems use intelligent freshness indicators to track changes in seafood products. The changes that take place in freeze-chilled food products can be categorized as (a) microbial growth and metabolism leading to pH changes; formation of toxic substances; off odors; off flavors; gas or slime formation; and (b) oxidation of lipids and pigments resulting in undesirable rancid flavors, formation of chemicals with adverse biological reactions, or discoloring (Suppakul, 2012). A freshness indicator will monitor the freshness of the seafood product by determining quality based on either metabolites from microbial growth or chemicals from lipid oxidation. Yet another type is time-temperature indicators. These are defined as simple, cost-effective, and user-friendly devices to monitor, record, and cumulatively indicate the overall influence of temperature history on seafood product quality from the manufacturer to the consumer. These are small self-adhesive labels attached to the interior of retail packaging that provide a visual temperature history of the package. Commercial applications identify color changes due to the presence of volatile compounds; this registers as a pH change on the indicator attached to the interior of packaging (Pacquit et al., 2006). The British Standard Institution (1999) divides these time–temperature indicators into the following groups: (a) temperature indicators with ascending functions (thaw or threshold indicators) that measure temperatures in the range −20°C to 30°C; (b) temperature indicators with descending functions that measure temperatures in the range 0 to −6°C; (c) partial indicators, which signal temperature abuse when a temperature threshold is passed; and (d) full indicators, which indicate temperature changes over the full temperature range. Suppakul (2012) gives a very useful overview of different types of temperature indicators. 12 Trends in Fish Processing Technologies

1.2.8.3 Edible Coatings and Films Edible coatings or films are defined as continuous matrices made of edible material consisting of proteins, polysaccharides, and lipids. While edible coatings are applied to or made on seafood, films are independent structures. Their purpose is to prolong shelf life by acting as a barrier to moisture, gas, and solute transmission. Moreover, different ingredients such as antimicrobials, antioxidant flavoring agents, and nutrients can be incorporated in coatings or films to improve safety, stability, and sensory and nutritional properties. The materials used to formulate such coatings and films are biopolymers. Also, small amounts of plasticizers (glycerol) are added to improve flexibility and regulate pH. Coatings and films have been studied, but they are still used very little or not at all by the industry in commercial seafood products. They have been assayed in both chilled and frozen seafood and also in other seafood products like smoked fish (Gómez-Estaca et al., 2007). Fish gelatin-based films containing borage seed extract as a functional component have been used as coverings for horse mackerel patties to protect against lipid oxidation in both chilled and frozen storage (Giménez et al., 2011). Clove essential oil can be added to formulations based on sunflower protein concentrates to prepare edible films for sardine patties, which retard lipid oxidation and growth of total mesophilic microorganisms (Salgado et al., 2013). Also, coating with gelatin and chitosan prevents spoilage by bacteria in cod patties (López- Caballero et al., 2005). Different coating formulas have been assayed on frozen seafood to prevent moisture loss, protein aggregation, and lipid oxidation (Duan and Zhao, 2012).

1.2.9 seafood Proteins Proteins are fundamental food components, both in functional and nutritional terms. On the technological side, they determine physicochemical and sensory behavior in proteinaceous products such as seafood. One of their main functional properties in current food technology is gelation. Moreover, after processing many proteins are converted to hydrolysates and peptides with pronounced bioactive activity.

1.2.9.1 Surimi Gelation Surimi is a myofibrillar protein concentrate produced by mincing, washing, and refining fish. Its most important functional quality is gel-forming ability. A proteinaceous gel made from surimi is an intermediate between solid and liquid, in which strands of protein chains are cross-linked to form a continuous three-dimensional network. This technology is at the base of widely different surimi products. Although surimi is a traditional product in Japan, the technology as we know it today was developed in the 1970s with the introduction of methods for dewatering washed mince and research into new cryoprotectants (Noguchi, 1974). Surimi-based products are processed by grinding thawed surimi with 2%–3% salt to solubilize the proteins. Other ingredients are then added and the product is shaped and heated. In recent years, there have been a number of changes in the technology of production, basically referring to processing, equipment and new ingredients. Recent Advances in Seafood Technology 13

1.2.9.1.1 pH Shift Process This was developed to overcome some of the problems associated with pelagic species (Hulting and Kelleher, 1999). The overall process consists of solubilizing the proteins of muscle tissue with alkali or with acid, followed by centrifugation to remove cellular membranes, connective tissue, and fat. Afterward, the myofibrillar protein solution is adjusted to a pH close to the isoelectric point of the majority of these proteins, which is about 5.3. Kristinsson and Hulting (2003) reported that the pH shift method has some advantages over the traditional method; among them is an improved gel-forming ability. On the other hand, in this process, especially if solubilization takes place at low pH, the hemoglobin molecule is broken and free hemo groups are released, which can lead to rapid oxidation of the lipid fraction of muscle. For that and other practical reasons, the pH shift process is not used by the industry except for the case of Jumbo squid (Dosidicus gigas) protein concentrate production. In this particular case, Sánchez-Alonso et al. (2007) developed a processing method to extract a squid protein concentrate with high gel-forming ability. Various manufacturers are currently processing Jumbo squid on the Peruvian coast using this method, under various different patents.

1.2.9.1.2 Ohmic Heating In this process, a fish paste composed of surimi, salt, and additives is heated by pass- ing an electric current through it. In this way, rapid gelation is achieved and myofibrillar proteins are not subjected to proteolytic enzymes with negative effects. In addition, the fish paste is heated uniformly regardless of size (Okazaki and Kimura, 2012). Other methods in which heat is generated internally in fish pastes are radiofrequency and microwave (Xiangjin et al., 2012).

1.2.9.1.3 High-Pressure Processing One alternative to improve the gel-forming ability of low-quality surimi or low-salt surimi gels is high-pressure processing (HPP). This is a relatively novel technique that has attracted growing interest in the food industry in recent years since it offers a new possibility of modifying functional food ingredients such as proteins and hence expanding the ways in which HPP can be used to modify the textural properties of surimi products. In fact, in recent years, the scientific literature has carried out many studies on HPP treatment influence on fish muscle gelation or surimi making after kneading with salt to induce protein solubilization prior to heat induced gelation (Tan et al., 2010; Moreno et al., 2015). All studies generally recognize that after HPP, the unfolding is reflected in a higher proportion ofα -helix proteins and higher formation of hydrophobic interactions, which play a very important role in the gel networks (Cheftel, 1992; Moreno et al., 2015). Cando et al. (2016) studied the effect of applying HPP to surimi gels to overcome gelation problems with low salt levels for the preparation of low-sodium surimi products with improved physicochemical properties.

1.2.9.1.4 New Ingredients Microbial transglutaminase can help improve gel-forming properties due to its ability to catalyze covalent bonding between the ε-amino group of lysil residues and 14 Trends in Fish Processing Technologies the γ-carboxamide group of glutaminyl residues from adjacent protein molecules, thus improving mechanical properties (Nonaka et al., 1989; Kumazawa et al., 1993). Many manufacturers are using this enzyme to reinforce the strength of surimi. Various additives such as pork plasma, egg white, and others are used as proteinase inhibitors in certain species to prevent enzymatic degradation of surimi gelation during the heating process.

1.2.9.2 Hydrolysates and Peptides The sea is an enormous source for novel compounds, and indeed, the number of novel bioactive substances is growing rapidly. Fish by-products contain valuable lipid and protein fractions as well as other useful compounds. For instance, there has been mas- sive research into the isolation and use of omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are of major commercial interest. More recently, there has been enormous interest in acceleration of hydrolysis with commercial proteases in protein fractions; this offers more possibilities than autolysis for controlling the properties of the product. Bioactive peptides are specific protein fragments that, in addition to providing a good source of nitrogen and amino acids, have numerous potential physiological functions in the human body depending on their structure, hydrophobicity, charge, and microelement binding properties. In fact, some peptides are multifunctional (Cho et al., 2008), exhibiting opioid, immunomodulatory, antibacterial, antithrombotic, anticancer, and antihypertensive activity (Murray and FitzGerald, 2007). The type of bioactive peptides generated from a particular protein depends on two factors: (a) the primary sequence of the protein substrate and (b) the specificity of the enzyme used to produce hydrolysis and generate the peptides (Harnedy and FitzGerald, 2012). Japan has approved several Food for Specified Health Use (FOSHU) products containing fish protein hydrolysates/peptides as functional ingredients. A list of commercially available FOSHU products and other marine- derived proteinaceous components without approved health claims but sold as food supplements in Europe and North America is reported by Harnedy and FitzGerald (2012). So far, most of the biological effects on marine-derived protein hydrolysates and peptides have been observed in vitro or in animal models and the number of human intervention studies is very limited. More controlled human intervention trials need to be performed to demonstrate the efficacy of the alleged bioactivities.

1.3 QUALITY ASSESSMENT “Quality” is a term with a wide range of meanings and various definitions. It was defined by Botta (1995) as “the degree of excellence to which a product meets all of the attributes, characteristics, and features of the product that the buyer and the user of the product and regulatory agencies expect.” The idiomatic expression “top-quality fish” is often applied to expensive species. Processors tend to designate fish as having low quality if it is too small or it is in a poor condition for a specific process or if it results in low yields and profit. Several times, quality is considered as synonym with aesthetic appearance and freshness and it is used to describe the degree of spoilage that the fish has undergone. For health authorities, whose main concern is health hazards, good quality means the absence of harmful agents like Recent Advances in Seafood Technology 15 parasites, chemicals, or pathogenic microorganisms. For wet fish, “freshness” is the most important quality parameter and the freshness period is the time from catch or slaughter until the seafood has lost its initial “freshness characteristics.” The term “shelf life,” on the other hand, simply means the time during which the product is fit for human consumption. The properties considered for wet-fish in ice, to be regarded as fresh, arerigor mortis, sensory volatiles, physical properties, microorganisms, adenosine triphosphate (ATP), proteins, and lipids (Olafsdottir et al., 2004). For other types of processed fish, the quality depends firstly on the raw material and secondly on how it is processed and its storage life. An interesting review on traditional and new instrumental methods to evaluate seafood quality has recently been published by Hassoun and Karoui (2017). There are a number of objective methods for testing freshness and/or determining shelf life:

1.3.1 sensory Methods There are several tests designed specifically for fish: the UE Quality Grading Scheme, the Torry Test, and the “Quality Index Method (QIM).” This last, the QIM, is the most novel one. It is a freshness grading method based on a scheme developed by the Tasmanian Food Research Unit (Bremner, 1985). QIM is based on the specific changes that occur in seafood when it is stored in ice. In the test, the panelists must first consider the characteristic parameters of each fish species, which are described concisely and simply. Demerit points from 0 to 3 are awarded for changes in outer appearance before gutting. In the gutting stage, the color of the blood and fillets and disintegration of the inner ventral part and guts are also evaluated. QIM schemes have been reported for a number of unprocessed species and some frozen and cooked products (Martinsdóttir et al., 2009; QIM Eurofish website: http://www.qim-eurofish.com).

1.3.2 chemical Methods These consist of conventional chemical analysis of muscle to determine the presence of chemicals that correlates with specific sensory attributes. In the case of wet fish, these methods include total volatile basic nitrogen, trimethylamine, hypoxanthine, ammonia content, etc. In the case of frozen seafood, the most common methods are dimethylamine, peroxide value, and thiobarbituric acid. Most of these methods cannot identify deterioration in the early stages. One exception is the K-value; this measures nucleotide degradation, the only chemical method that measures “freshness” and not only “shelf life.” In response to the industry’s need for rapid methods and compact devices, in recent years, researchers have developed kits and various appa- ratuses based on enzymatic reactions or specific sensors that measure all these substances. An interesting review for K-value determination is given by Tejada (2009).

1.3.3 Physical Methods There are a number of physical methods that have been and are currently used to check the quality of fresh and frozen fish. These include color measurement, mechanical properties, Torry meter, pH, etc. However, the methods on which the most recent 16 Trends in Fish Processing Technologies researches have been focusing are based on spectroscopy. Of these, the ones receiving most attention in connection with seafood products are visible/near infrared (VIS/ NIR) spectroscopy, image analysis, and nuclear magnetic resonance (NMR).

1.3.3.1 Image Analysis Image analysis is objective, repeatable, and nondestructive, and its potential has been demonstrated in a number of fish processing operations, including sizing, weighing, grading, recognition, and others. It is now being used and will no doubt be increasingly used in the future to achieve more accurate evaluation of quality in fish products (Dowlati et al., 2012). One of the first instances of its use was to measure connective tissue and fat in salmon fillets using image analysis (Borderías et al., 1999). Although the correlation coefficients registered were low, these authors judged that the method was sensitive enough to detect considerable variations in fat content, which could prove to be useful in industrial applications. More recently, Mathiassen et al. (2011) reported various applications of VIS/NIR imaging spectroscopy, planar and computed tomography, X-ray imaging, and magnetic resonance imaging (MRI) for inspection of fish and fish products. VIS/NIR imaging spectroscopy and MRI are very promising methodologies.

1.3.3.2 VIS/NIR Spectroscopy In VIS/NIR measurement, light is projected onto a sample and then the light emitted by the sample is measured at different wavelengths, ranging from visible to near- infrared. Conventionally, sample measurement using this technique produces a visible or near-infrared spectrum; however, there is a new technique called imaging spectroscopy, which offers spatial in addition to spectral information. In spectroscopy, multivariate analysis is used to relate the spectral information to the target information. VIS/NIR is basically used to assess food constituents. For instance, Sivertsen et al. (2011) used this technique to assess freshness of cod. Then, again, Sivertsen et al. (2011) and Fasolato et al. (2012) applied VIS/NIR image spectroscopy to the detection of freezing in cod and swordfish. This technique also served for accurate and rapid detection of parasites in fish muscle (Sivertsen et al., 2011) and fish muscle rigor mortis, and it has been used to identify the morphological structure of salmo- nids cutlets (Stien et al., 2006). There is a very interesting review by Liu et al. (2013) on the use of NIR in evaluation of fish quality.

1.3.3.3 Nuclear Magnetic Resonance NMR derives information from the fact that atomic nuclei have magnetic properties (Lambert and Mazzola, 2004). It provides detailed information on the structure, dynamics, reaction state, and chemical environment of molecules. NMR methods in food research fall into three groups: high-resolution NMR spectroscopy (HR-NMR), low-field NMR relaxometry (LF-NMR), and MRI. HR-NMR has been used to profile fish products (Aursand et al., 2009) by producing a fingerprint of food material composition. The technique has been used to study marine lipids in fish product samples because the analysis is nondestructive (Aursand et al., 2006). Ye et al. (2012) also used it to analyze the composition of crab paste (amino acids, organic acids, nucleotides, amines, and sugars). Recent Advances in Seafood Technology 17

LF-NMR provides important information about relaxation and diffusion behavior within the fish muscle. It can be used for online quality control and works in the frequency range 2–25 MHz. This technique has been used to analyze the mobility of water and fat in muscles, but it also offers various possible means of linking the behavior of water protons to different technological parameters and fresh seafood quality (Aursand et al., 2009). Moreover, postmortem muscle condition (Bertram and Andersen, 2008), the effects of additives on muscle (Carneiro et al., 2013), and freezing and frozen storage conditions of different muscle species (cod and hake) (Lambelet et al., 1995; Sánchez-Alonso et al., 2014; Sánchez-Valencia et al., 2015) have all been successfully analyzed by LF-NMR. MRI is usually viewed as an extension of LF-NMR and provides additional spatial information on nuclear spins. In MRI, the atoms of target samples are aligned by a powerful magnetic field, then various different frequencies are applied to alter this alignment. These frequencies are used to record a nucleus density image and so construct an image in the scanned area of the object. In the case of seafood, various different protocols have been used to differentiate nuclei (such as 1H and 23Na) in molecules and structures with different chemical environments (Mathiassen et al., 2011). This technique has been used for many purposes: studies on frozen storage of trout (Foucat et al., 2001) and salting and study of changes in muscle texture as a result of water mobility and its interaction with the protein in cod fillets (Veliyulin et al., 2006). Nonetheless, 23Na-MRI can be used to optimize industrial salting processes by monitoring salt distribution; it has also been used for salmon (Aursand et al., 2010) and for “bacalao” (salted cod) (Gallart-Jornet et al., 2007).

1.3.4 M icrobiological Examination Microorganisms are the main cause of spoilage in fresh and lightly processed seafood. Also, food-borne pathogenic bacteria can colonize seafood, causing illness to consumers. Microbiological quality assessment is important to ensure safety and assess quality throughout the chain from catch to consumer’s body. Conventional methods of microbial quality analysis require enrichment, isolation, identification, and enumeration of microorganisms and are laborious and slow. Nowadays, there are various different indirect methods that measure microbial metabolism rather than population counts, among them, electrical methods. Methods based on microscopy and immunological or genetic characteristics are also applied.

1.3.4.1 Electrical Methods Electrical methods measure impedance, conductance, or capacitance changes in liquid growth media, caused by metabolic products released during microorganism growth. A review of these methods as used on seafood products is given by Boziaris and Parlapani (2012).

1.3.4.2 Respiratory Activity Hempel et al. (2011) reported a method of quantifying aerobic microorganisms based on measurement of the increase of respiratory activity in a liquid medium. This experiment was carried out for different fish species (cod, plaice, mackerel, and 18 Trends in Fish Processing Technologies whiting). The method employed fluorescence-based oxygen sensing probes and a fluorescent reader to monitor cell growth via respiration.

1.3.4.3 Inmuno-Based Techniques Methods based on specific antigen-antibody binding (usually enzyme-linked immuno sorbent assay [ELISA]) have been largely used in recent years for detection and identification of food-borne pathogens in seafood (Kumar et al., 2011). These methods have not been used to determine spoilage because their detection limit is low (Boziaris and Parlapani, 2012).

1.3.4.4 Molecular Methods Polymerase chain reaction (PCR)-based methods can identify many microorganisms and viruses that are not detected with the conventional culture-based methods. The 16rRNA gene is the most common approach for isolation and identification. There are various microorganisms in seafood that do not grow on culture media, and so one alternative is direct extraction of DNA. This is followed by PCR amplification of the nucleotide sequence of the rRNA gene and analysis of gene fractions of the different bacteria by various methods, mainly fingerprinting. Macé et al. (2013) used this method to characterize fish spoilage flora. Another procedure, used by Parlapani et al. (2013) to analyze the microbial flora in iced sea bream, involved the isolation of the amplified 16S rRNA gene product by cloning. Various authors have assayed a combination of real-time PCR and ELISA, mainly for quantification of pathogens as in the case of Vibrio species (Canigral et al., 2010). Other methods that have been reported indicate the use of multiple biomarkers to detect Salmonella in seafood (Garrido et al., 2013) and the use of microarray hybridization to detect amplicons generated by PCR from pathogenic bacteria on seafood (González et al., 2004).

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Jan Thomas Rosnes and Dagbjørn Skipnes

CONTENTS 2.1 Introduction...... 28 2.2 Minimal Processing Technologies...... 28 2.2.1 Categories of Minimally Processed Fish Products...... 29 2.2.2 Microbiological Safety Issues Related to Fish Products...... 29 2.3 Examples of Mildly Heat-Treated Fish...... 31 2.3.1 Hot Smoking of Fish Products...... 32 2.3.2 Pasteurization and Sous Vide...... 33 2.4 Strategies to Reduce the Heat Load...... 35 2.4.1 Agitated Heat Processing...... 36 2.4.2 Surface Treatment and Inactivation...... 37 2.5 Validation of Heat Treatment...... 40 2.5.1 Time–Temperature Integrators...... 40 2.5.2 Online Measurement and End-Point Setting of Temperature...... 41 2.6 Combination Effects...... 42 2.6.1 MW and RF Processing of Fish...... 44 2.6.1.1 Implementation of Industrial MW Production...... 44 2.6.1.2 Tempering...... 45 2.6.1.3 Pasteurization...... 45 2.6.1.4 Novel Applications of MW and RF for Fish...... 46 2.6.1.5 Overpressure MW Processing...... 47 2.6.1.6 Subatmospheric Pressure MW Processing...... 49 2.6.1.7 MW Combination Heating...... 49 2.6.1.8 Water Immersed RF Processing...... 49 2.6.1.9 Design and Validation of Thermal Processes...... 50 2.6.1.10 Temperature Measurement...... 50 2.7 PEF Treatment...... 51 2.7.1 Principle of PEF...... 51 2.7.2 Advantages and Application in Foods...... 52

27 28 Trends in Fish Processing Technologies

2.7.3 Challenges of PEF Technology Applied for Food Production...... 53 2.7.4 PEF Used in Fish Products...... 54 2.7.5 PEF and Microbial Inactivation...... 55 2.8 Concluding Remarks...... 56 References...... 57

2.1 INTRODUCTION For consumers, the most important attributes of a food product are its sensory characteristics (Cheng et al. 2015; Nagarajarao 2016). Processing technologies that retain or create desirable sensory and nutritional quality or reduce undesirable changes in food due to processing are therefore a goal of food manufacturers (Pasha et al. 2014). Driving forces for changing consumers’ eating habits into healthier ones are media- and government-sponsored initiatives that support the increase in seafood consumption due to its health benefits. Fish products have attracted considerable attention as a source of protein, vitamins, minerals, and fats (Hibbeln et al. 2007; Tuomiso and Føyland 2008). Manufacturers have, to a large extent, followed the changing nature of the consumer demand, through the development of health-oriented products, with a minimum of processing. An important trend is the development of convenience- orientated products and packaging such as microwaveable formats and individually wrapped products, which is becoming an increasingly central feature of manufacturer’s innovation strategies. Fish is highly perishable and usually spoils faster than other muscle foods; thus, a well-designed process is of vital importance.

2.2 MINIMAL PROCESSING TECHNOLOGIES Efforts have been made to describe foods by distinguishing between different levels of processing, which has led to terms such as “unprocessed,” “minimally processed” foods, or “culinary processed ingredients” (Weaver et al. 2014). The level of a process should be indicated by its intensity and, consequently, by the extent of changes made on raw materials. Many preservation technologies have the potential to extend the shelf life of fish products compared to conventional iced storage in normal atmosphere. However, not all extensions of the shelf life will increase the product lifespan with superior quality for the product. Some of the mild processing technologies will not inactivate autolytic enzymes, and their activity will contribute to spoilage regardless of packaging and storage, leaving temperature as the most critical limiting factor. Minimal processing can roughly be divided into two major strategies: thermal and nonthermal processing. In the range of the traditional thermal processing methods for fish products, e.g., from hot smoking to sterilization, there are different time– temperature combinations resulting in different impacts. Several of the emerging thermal technologies have been examined for application in fish processing, e.g., microwave (MW) processing (Pilavtepe-Celik et al. 2014), ohmic heating (Kumar et al. 2014), and radiofrequency (RF) heating (Archer et al. 2008). Traditionally, pasteurization has been described as a thermal process. In the last decade, however, it has been demonstrated that several nonthermal technologies can obtain the same microbial inactivation as thermal processing. As a Minimal Heat Processing Applied in Fish Processing 29 consequence, pasteurization has been redefined by the National Advisory Committee on Microbiological Criteria for Foods (NACMCF, 2006) as “Any process, treatment, or combination thereof that is applied to food to reduce the most resistant microorganism(s) of public health significance to a level that is not likely to present a public health risk under normal conditions of distribution and storage.” Therefore, this definition considers new alternatives for pasteurization of foods including technologies without heat. Different nonthermal technologies have been under development for many years, and several applications in the industry are implemented. Many technologies are promising for the decontamination of fish products, such as cold plasma (Chiper et al. 2011), ozone (Gelman et al. 2005), pulsed electric field (PEF) (Gudmundsson and Hafsteinsson 2001), and pulsed light (Cheigh et al. 2013; Molina et al. 2014; Ozer and Demirci 2006). Quality optimization for fish products may also be combined with ultrasound (Ghaedian et al. 1998; Sigfusson et al. 2001) and packaging technologies like modified atmosphere (MAP) or active and intelligent packaging (Da-Wen 2005; Nagarajarao 2016). Some nonthermal methods are used in combination with heat treatments (e.g., high-pressure processing [HPP] and heat for inactivation of nonproteolytic Clostridium botulinum) (Lenz and Vogel 2015).

2.2.1 categories of Minimally Processed Fish Products Processed fish products can be separated into major categories, based on the extent of thermal processing or preservation (Hoffman et al. 2003). The categories span from products that are eaten raw without treatments, to mildly processed (e.g., smoked or pasteurized at low temperatures), to fully sterilized products. When minimal processing is used, the fish products are often preserved by hurdle technology by addition of preservatives, salt, smoke, or acidic ingredients to decrease the pH, in order to obtain an extended shelf life with satisfactory safety. The shelf life is normally short, ranging from 7 to about 18 days, for those with the lowest heat treatment. Minimal heat-treated fish products include a diverse group of products, e.g., hot smoked fish, mildly cooked fish, and pasteurized products like sous vide. Depending on the heat treatment and packaging, these products may obtain shelf life of 3 weeks or longer. Traditional thermal treatments are the cornerstones of the food industry, providing required safety profiles and extensions of shelf life. The main aim of minimal and novel processing methods is to decrease negative effects of heat treatment, such as losses of desired organoleptic properties and damage to temperature labile nutrients and vitamins. A huge variety of heat-treated fish products exist in the market, packaged before or after heat treatment. An approach to visualize some important treatments is given in Figure 2.1.

2.2.2 Microbiological Safety Issues Related to Fish Products The microbiological safety of industrially manufactured foods is based on the effective design and implementation of Good Hygienic Practices and Hazard Analysis and Critical Control Points (HACCP) in accordance to applicable guidance and regulations (Anonymous 2005; Food and Drug Administration [FDA] 2011). This chapter is not allocated for a full risk assessment of minimal heat-treated foods but rather 30 Trends in Fish Processing Technologies

Mild heat treatment

Heat treatment before packagingHeat treatment after packaging

Relevant methods: Relevant methods: ohmic heating, frying, water bath, microwaves, steaming, boiling, radiofrequency, heat exchanger retort

MAPHot ﬁll Aseptic ﬁlling Cook/chill Sous vide Sealed trays

FIGURE 2.1 Approaches for minimal heat treatment technologies that can be used for fish products. focuses on key safety issues to prevent pathogenic bacteria to grow and produce toxins. Target organisms for the heat treatments and kinetic inactivation values of the target organisms in food are of uttermost importance. Several pathogens are present in the environment, on raw materials and ingredients, and may contaminate during stages of fish harvesting, processing, or handling. Among the toxin-producing bacteria are psychrotrophic nonproteolytic C. botulinum type B, E, and F and psychrotoler- ant histamine-producing bacteria (photobacteria). The infective microorganisms are Listeria monocytogenes, Vibrio cholerae, Vibrio arahaemolyticus, Vibrio vulnificus, Aeromonas hydrophila, and Plesiomonas shigelloides, while the nonindigenous bacteria are Staphylococcus aureus, C. botulinum proteolytic type A and B, mesophilic histamine-producing bacteria (Morganella morganii), Salmonella spp., Shigella spp., and Escherichia coli (Lunestad and Rosnes 2008). Pathogenic toxin-producing Bacillus cereus is not associated with raw fish materials but may be a risk factor from the ingredients in mixed or minced fish products or in marinades (Feldhusen 2000). Minimally processed food products do primarily rely on refrigerated storage and distribution for preservation, both from microbial and quality standpoints. At low temperature, the major microbiological concern is psychrotrophic pathogens that can grow and mesophilic pathogens that survive under refrigeration and may grow during temperature abuse. The types of microorganisms that are of major concern in minimally processed foods and key time–temperature relationships to inactivate 6 logarithmic units (6D) are listed in Table 2.1. The inactivation of L. monocytogenes has been suggested as a criterion for minimal heat treatment by the European Chilled Food Federation (ECFF 1996). Listeria is regarded as the most heat resistant of the nonsporulating pathogens, and Vibrio species will be more effectively heat inactivated if present (Johnston and Brown 2002; Wong et al. 2002). Pathogenic Vibrio species are common in aquatic habitats of different salinities and are therefore of concern for seafood raw materials. Thermal processes designed for a shelf life of longer than 10 days under chilled conditions require at least a 6 log inactivation (6D) of psychrotrophic nonproteolytic C. botulinum corresponding to 90°C for 10 min (ECFF 2006). Minimal Heat Processing Applied in Fish Processing 31

TABLE 2.1 Target Organisms, Recommended Heat Inactivation Parameters, and Temperature Storage Conditions to Inhibit Surviving Organisms to Grow Recommended Storage Heat Remaining Conditions to Heat Treatment—6D Hazard to be Inhibit Growth Resistance of Target Eliminated or of Surviving Target Organism (min)a Organism Controlled Organisms

b Psychrotropic D90°C = 1.5 90°C/10 min Proteolytic C. <10°C nonproteolytic C. botulinum, type A botulinum type E B. cereus <4°C

B. cereus D100°C = 1–36 100°C for 48 min Proteolytic C. <10°C botulinum, type A

L. monocytogenes D70°C = 0.3 70°C/2 min Proteolytic C. <10°C and other botulinum, type A non-spore- Non proteolytic C. <3°C forming pathogens botulinum, type E B. cereus <4°C a Most heat-resistant species in the target group (ECFF 1996; ECFF 2006). b 10°C—the lowest growth temperature for proteolytic C. botulinum. If food is likely to support B. cereus growth, the limit should be lowered to 4°C, which is the lowest growth threshold for psychrotrophic B. cereus. c 3.0°C—the lowest growth threshold for nonproteolytic C. botulinum (Graham et al. 1997).

Based on risk assessment for L. monocytogenes in ready-to-eat (RTE) foods, the Codex Alimentarius (2009) recommends that the maximum contamination level for L. monocytogenes in food at consumption be less than 100 CFU/g. A Technical Guidance Document from Agence Française de Sécurité Sanitaire des Aliments on shelf life studies for L. monocytogenes in RTE foods has been published (Álvarez- Ordóñez et al. 2015). Such low levels of pathogens in food products has stimulated a development of rapid and alternative high-sensitive detection and quantification techniques as reviewed by Välimaa et al. (2015).

2.3 EXAMPLES OF MILDLY HEAT-TREATED FISH There are many thermal methods available for heat treatments of fish products: static or shaking retorting, MW and RF heating, steaming, cooking, blanching, sterilization, pasteurization, evaporation, extrusion cooking, infrared (IR), ohmic, dehydration, or drying. In the following, some of these methods will be discussed in the light of minimal heat processing. In this context, we have defined minimally processed fish products as a category that has been exposed to a thermal process, typically to a final processing temperature from a low pasteurization at 60°C to a high pasteurization at 95°C for 10 to 30 min, with a water activity >0.85 and pH >4.6. 32 Trends in Fish Processing Technologies

2.3.1 H ot Smoking of Fish Products Smoking is a traditional fish preservation method, and both hot smoking and cold smoking have economic importance for the seafood market (Cardinal et al. 2006). These products are regarded as delicate food items, highly nutritious, containing unsaturated fatty acids, fat-soluble vitamins, essential minerals, as well as proteins consisting of amino acids, some of them being essential for human beings (Erkan 2012). Smoked fish products are usually served as RTE products; thus, they should leave the processing plant with low numbers of pathogens and eventually with preservation hurdles to control growth and activity. For cold smoking of fish at non- lethal temperatures of 25°C–30°C, L. monocytogenes has, for a long time, been a challenge (Lovdal 2015). Microbial risks in hot smoking were reviewed by Sikorski and Kołodziejska (2002), stating that the ultimate core temperature in smoked fish and the time of heating should inactivate the pathogenic bacteria typical for the raw material, as determined in the respective fish and under the applied conditions of salting and smoking. Control of smoke, humidity, temperature, and circulation, as well as of the process time, is necessary to guarantee the required thermal inactivation of the microbiota and the attempted yield of the product; these factors belong to the critical control points in the HACCP system. History, methods, and effects on physical, nutritional, and microbiological properties of different smoking methods are comprehensively reviewed by Arvanitoyannis and Kotsanopoulos (2012) and Lovdal (2015). In the guideline from the US FDA, the internal temperature of hot smoked fish must be maintained at or above 62.8°C throughout the fish for at least 30 min (FDA, 2011, Chapter 13). Additional recommendations for water phase salt (≥3.5%) and nitrate (where permitted use), combined with low storage and distribution temperature, will control surviving spores from nonproteolytic C. botulinum type E/B and F. This heat load will, in theory, reduce L. monocytogenes with 6 log units (6D) as advised by ECFF (1996). Poysky et al. (1997) demonstrated that in salmon steaks inoculated with L. monocytogenes brined to a 2% to 3% salt content in the water phase of the final product, L. monocytogenes survived during hot smoking when the steaks were heated 30 min at 82.8°C measured in the deepest part of the muscle. No survivors were found in samples heated in the same conditions at a temperature higher than 82.8°C, up to 85°C. In a recent study, Shi et al. (2015) pasteurized cold smoked salmon (Oncorhynchus nerka) in laboratory capillary tube experiments. They found that the D-values of L. monocytogenes and Listeria innocua were 0.3 to 14.1 min at 66°C to 58°C, with a z-value of 5.2°C to 6.5°C, which will give the advised 6 log reductions. Hot smoking of fish with mild heat treatments is a typical example where hurdle technology is in active use (Leistner 2000; Singh and Shalini 2016). The combination effect of high sanitary standard of the raw material plus the bactericidal and bacteriostatic effects at different stages of processing and storage reduces chances for growth of microorganisms in the product. Literature from the last decade shows that both shelf life and various quality and safety aspects have been thoroughly investigated to meet the customers’ requirements combined with national legislation (Table 2.2). Minimal Heat Processing Applied in Fish Processing 33

TABLE 2.2 Hot Smoked Fish Species at Different Time–Temperature Combinations and Effects on Quality and Safety Aspects Fish Products Thermal Conditions Postheating examination References Luciobarbus esocinus Smoked at 70°C ± Effects of rosemary (Rosmarinus Coban and fillets (Carp family) 1°C by using a block officinalis) extract on the shelf Ozpolat 2011 of oak life of hot-smoked and vacuum-packed fillets Matrinxa (Brycon 45°C–90°C in 5 h Organoleptic properties, yield, Franco et al. cephalus) and composition 2010 Capelin (Mallotus Heating at 75°C for Lipid and microbial quality Cyprian et al. villosus) and sardine 30 min 2010 (Sardinella gibossa) Rainbow trout 70°C in 4 h Comparison shelf life of MAP Cakli et al. (Onchoryncus mykiss) and vacuum packaging at 2006 3.8°C for 47 days Atlantic Bonito (Sarda 80°C in 1 h (core Quality effects of smoke from Duyar et al. sarda) temp. 76°C) different wood. Stored at 4°C 2008 for 60 days Tilapia (Tilapia mariae) 80°C core temp. in Quality and microbial safety Adeyeye et al. 24 h 2016 Tilapia (Oreochromis 80°C in 45 min Effect of brine concentration on Yanar et al. niloticus) quality and shelf life 2006

2.3.2 P asteurization and Sous Vide Mild pasteurized products have many desirable properties with a minimal use of additives and preservatives. Preservation of sous vide processed fish is achieved by a combination of vacuum packaging in high-barrier flexible bags or pouches and a combination of moderate heat treatment (65°C–95°C), water activity >0.85 and pH >4.5, chilled storage (<8°C), restricted shelf life (21 to 42 days), good raw material quality, and hygienic manufacturing. The products are often named Refrigerated Processed Foods of Extended Durability (REPFED) and may be marketed as RTE or ready to cook (Daelman et al. 2013; Peck 2006; Silva and Gibbs 2010). Both thermal and nonthermal methods may be used to reduce the number of microorganisms in the foods before it leaves the production unit (Rajkovic et al. 2010). The specific challenge regarding REPFED is that the processing temperatures are optimized mainly to kill vegetative bacteria and to retain quality related to the inherent freshness and sensory properties of the food. An additional aim is to reduce as much spores as possible and subsequently control growth of surviving spores (Smelt and Brul 2014). Typically, pasteurized fish products are surimi-based products, soups, and sauces, but also portions of fillets. In the last years, a large market has evolved for ready meals of lunch and dinner meals produced as cook-chill and sous vide. These fish products require refrigerated storage and distribution to maintain food safety. Minimal processing often relies on use of multiple sublethal stresses (or hurdle processes) to achieve a similar level of microbial control as traditionally is 34 Trends in Fish Processing Technologies

TABLE 2.3 Examples of Scientific Studies of Pasteurized Fish Products Based on Sous Vide Technology Thermal Postheating Fish Products Conditions Examination References Salmon (Salmo salar) 80°C for 45 min Stored at 2°C for 0, 5, or Díaz et al. 2011 10 weeks Bonito (Sarda sarda, Bloch, 1793) 70°C in 10 min 4°C for 48 days Mol et al. 2012b 12°C for 27 days (abuse) Whiting (Merlangius merlangus 70°C in 10 min 4°C for 48 days Mol et al. 2012a euxinus, Nordman, 1840) 12°C for 27 days (abuse)

Herring (Etrumeus teres) 70°C in 30 min Loss of vitamin B12 Nishioka et al. 2011 Cobia (Rachycentron canadum) 4 and 12 weeks Shakila et al. 2012 Seabream (Sparus aurata) 60°C Chilled storage at 2°C Espinosa et al. for 48 days 2016 Tambaqui (Colossoma 65°C for Microbiological, Ramos et al. 2016 macropomum) 12.5 min physical, and physicochemical quality parameters Rainbow trout fillets (Oncorhynchus 65°C for Formation of Oz and Seyyar mykiss) 90/150 m heterocyclic aromatic 2016 75°C for amines (HCAs), and the 75/135 m migration level of 85°C for bisphenol-A (BPA) 60/120 m Salmon slices (Salmo salar) 90°C for 15 min Chilled storage at 2 and González-Fandos 90°C for 5 min 10°C for 45 days et al. 2005 65°C for 10 min Rainbow trout (Oncorhyncus 90°C for Chilled storage at 2 and González-Fandos mykiss) 3.3 min 10°C for 45 days et al. 2004 90°C for 1.04 min 70°C for 5.18 min Emperorbream (Lethrinus lethrinus) Cooked in water Chilled storage at 3°C Shakila et al. 2009 fish cakes at 100°C for for 16 weeks 20 min Salmon (Salmo salar) 80°C for 43 min Chilled storage at 2°C Díaz et al. 2009 for 25 days Salmon and trout (From different 90°C for 10 min Chilled storage at 4°C Garcia-Linares et retail stores. Latin names not given) for 45 days al. 2004 (Continued) Minimal Heat Processing Applied in Fish Processing 35

TABLE 2.3 (CONTINUED) Examples of Scientific Studies of Pasteurized Fish Products Based on Sous Vide Technology Thermal Postheating Fish Products Conditions Examination References Albacore tuna (Thunnus alalunga) 90°C for 20 min Products were frozen and Fagan and Gormle Cardinal fish (Epigonus telescopus) reheated in a MW oven 2005 Orange roughy (Hoplostethus to a core temperature of atlanticus) 2 min at 70°C Blue ling (Molva dipterygia) Deepwater redfish (Sebastes mentella) Round nose grenadier (Coryphaenoides rupestris) Greenland halibut (Reinhardtius hippoglossoides)

Source: Rosnes, J.T., Skara, T., Skipnes, D., Food Bioprocess Technol., 4, 833–848, 2011. achieved using a single lethal stress. The quality of fish can be severely reduced if the thermal process is designed for a long shelf life, e.g., >21 days at chilled conditions (Skåra et al. 2002). Designing a thermal process for such a product is challenging as the heat load required for inactivating microorganisms and enzymes may cause undesirable changes such as dry structure and flaking (Ofstad et al. 1996; Skipnes et al. 2008, 2011). Quantification of safety and microbial quality in minimally processed foods is quite complex due to wide biological variability (e.g., in raw material or contaminating organisms) and uncertainty about exactly which factors influence product safety and/or quality (Zwietering 2002). Some bacteria may adapt to sublethal stresses in a manner that can render them less susceptible to additional hurdles. This needs to be taken into account when designing safety or extended shelf life of minimally processed foods (Hill et al. 2002). Concern about the safety of some of these products, and for fishery products in particular, exists, especially when considering potential temperature abuse (Feldhusen 2000). Examples from studies of different vacuum packaged fish products and their thermal processing parameters are presented in Table 2.3.

2.4 STRATEGIES TO REDUCE THE HEAT LOAD Heat transfer rate during a thermal process determines the speed of heating and cooling processes. Thus, knowledge of thermal properties of food becomes important for understanding thermal changes in food during processing. Reducing the thermal load can be done by increasing the heating rate by novel technologies. Another option is to target the specific, potentially contaminated product areas, like the surface area, and avoid heating noncontaminated parts. Agitated heating and MW and 36 Trends in Fish Processing Technologies

RF heating are examples of methods where heat distribution is improved, causing a total lower heat exposure to the product without compromising food safety.

2.4.1 agitated Heat Processing The early scientific developments of retorts operated often with wide microbiological stability margins, due to process control issues and temperature deviations during industrial heating processes. Thus, safety and stability of the process have often been achieved at the expense of food quality degradation caused by overprocessing. Unsteady-state conditions may dominate during heating (or cooling) of foods, causing a change in heat transfer rate over time. When the rate of heat transfer stabilizes, steady-state heat transfer conditions can be achieved. Industrial pasteurization processes often involve unsteady-state heat transfer conditions and the time-temperature history of food products during heating and cooling periods must be measured. Agitating retorts can enhance heat flow rates through forced convection in liquid and semiliquid foods. Through improved heat flow rates, agitated heating can minimize nonuniform temperature zones, overprocessing, and undesirable quality changes. The conventional methods for the agitation of liquid and semiliquid foods are axial rotation and end-over-end (EOE) rotation (Dwivedi and Ramaswamy 2010; Knap and Durance 1998). EOE rotation is an effective means of improving the rate of heating because the air contained by headspace provides mixing of the product, which may reduce the required sterilization time by up to 40% (Eisner 1988). Parameters of great influence on the transfer of heat during agitated sterilization and for agitated pasteurization are (i) rotation speed, (ii) size of headspace, (iii) viscosity of the product, (iv) system geometry, and (v) particle density. In 2006, a new agitating retort with high-frequency reciprocal agitation was developed. This agitating retort model is based on high-frequency longitudinal agitation mechanism, which enables rotation speed higher than 40 rpm (Walden 2008). However, centripetal forces limit the feasible rotation speed for most products to a range between 20 and 40 rpm, with reciprocating agitation now a third alternative. The reciprocating agitation of containers during thermal processing has been patented (Walden and Ferguson 2005), and from 2007, pilot scale autoclaves have been made available under the brand name of Shaka and manufactured under license by a few companies. This new technology opened up an unexplored field of thermally processed products with frequencies up to 320 rpm. Claims from the producers of autoclaves have been that reciprocating agitation allows a huge reduction in processing time, a reduction in required space in the production plant and energy savings compared to static processing. With a Shaka autoclave, Skipnes (2009) found an 18-fold decrease in heating time compared with static processing when using a Bentonite model with viscosity comparable to food products. A wide range of products from low-viscosity fish soup to highly viscous baby foods have been successfully processed in everything from flexible pouches to rigid containers, with remarkable reduction of processing time and less thermal degradation of the products (Skipnes 2009). Ates et al. (2014) treated fish soup in a Shaka autoclave with mild heat treatments at 62°C, 65°C, and 68°C. The soup samples were inoculated with approximately 108 cells/ml L. innocua and the results Minimal Heat Processing Applied in Fish Processing 37 demonstrated that agitating mode could provide equivalent lethality to the model organism L. innocua within significantly shorter heating times compared to static mode. At 62°C, 65°C, and 68°C, L. innocua was not detected after 11.5, 6.8, and 5.5 min processing in agitating mode, while 77, 67, and 52 min processing was needed in static mode. For a similar fish soup, the processing time for inactivation of Bacillus subtilis spores was dramatically reduced by agitating retort treatments. For a 7 log inactivation of B. subtilis spores, 17 min processing in agitating mode was required, compared to 53 min in static mode at 110°C (Ates 2016). Shaka technology is feasible for both mild heat processing and sterilization. For pasteurization at temperatures from approximately 90°C and up, a counter pressure may be desirable for flexible packaging materials and may, in some cases (e.g., easy- peel top film), even be necessary. One disadvantage is that for some fish products, shaking, rotating, etc., will make the fish fall apart and the use is therefore limited. A computational approach with experimental validation for determination of optimal shaking rate for liquid foods has now been published (Erdogdu et al. 2016). In conclusion, the autoclave is well suited for pasteurization compared to other technologies, and the introduction of Shaka technology has improved its competitiveness towards rapid heating technologies for in-pack processed products.

2.4.2 surface Treatment and Inactivation Minimally processed fish products with a core pasteurization value achieved in the thermal center of the product may subsequently be contaminated, e.g., with L. monocytogenes in the production line. Hence, optimization of a surface heat process (pasteurization) is particularly relevant when production involves, e.g., baking or frying of nonpackaged products. Consequently, there is a strong motivation to reduce the microbial loading only on food surfaces instead of a total additional processing. Immediately before the packaging step, a surface heating step may be used to inactivate any recontamination on the surface of the product. Steam treatment may have benefits over water and can enter any cavity large enough to contain a bacterium (Huang and Sites 2012; Morgan et al. 1996). For unprocessed products, hot water is a potential method for reducing pathogenic bacteria on surfaces. A diverse range of food products are subjected to surface decontamination techniques with the main commodities of interest (from a food safety perspective) including meat, seafood, shelled eggs, nuts, vegetables, and fruits (Warriner 2011). Food surface decontamination technologies can be broadly classified as thermal and nonthermal (Figure 2.2). In the current context, the term “surface decontamination” relates technologies that can decontaminate food surfaces without altering the interior of the product. The amount of heat needed to kill 100 bacteria on 1 cm2 is 15 million times less than that required to cook the surface to a depth equal to the length of a bacteria (Morgan et al. 1996). Food surfaces are seldom smooth and microbes can become located in protective sites (e.g., feather follicles on poultry skin, the pores on egg shells, or the muscle lamellae of fish). This is especially relevant, e.g., for a physical nonthermal surface decontamination techniques such as ultraviolet (UV), where shadowing effects protect the microbes from the antimicrobial photons. To be considered successful, a surface decontamination method has to achieve a 5 log reduction of a relevant 38 Trends in Fish Processing Technologies ts .: a cal, e.g gi actic acid - L bacteri - Natural extrac Biolo .: l id e ox er Nontherma Chemical, e.g - P - Ozone - Organic acids .: sm a ound VC ysical, e.g

Ph - U - Ultr as - Cold pla amination oo d surface cont F de d heating ermal radiation Ohmic heating Infrare ermal F) g , R netic g ction ying ve omag Fr heatin ctr Con Contact heatin Ele heating (MW Decontamination technologies that can be used to reduce the microbial surface loading on foods.

FIGURE 2.2 Minimal Heat Processing Applied in Fish Processing 39 pathogen or appropriate surrogate (US FDA 2000). However, due to the need to preserve the sensory characteristics and location of microbes in protective structures (e.g., biofilms, pores, crevices, stomata), the majority of surface decontamination technologies fail to attain a 5 log cfu reduction (Warriner 2011). Yet, it is recognized that even a 2 log cfu reduction reduces the risk of human pathogen carriage and hence deemed acceptable (achievable) by industry and academics. Experiments have shown reductions of 1–2 log units for E. coli K12 and Campylobacter jejuni, applying temperatures of 80°C and 75°C for 20 to 30 s, respectively (Corry et al. 2007). As opposed to liquid media often used to generate models, a structured three- dimensional food product will immobilize bacteria and lead to colony growth (Malakar et al. 2003). Furthermore, it may exert constraints on oxygen availability, mechanical water distribution, and other factors such as availability to organic acids (food preservatives). However, the surface also interacts with the environment, and key parameters such as water content and water activity may change quickly. Finally, there is the issue of channels and microchannels that may harbor reservoirs, which a rapid surface treatment will not inactivate. Efforts have been made to describe the physical parameters and to model the surface treatment, both for dry air (Kondjoyan and Havet 2003; Kondjoyan et al. 2006a, 2006b; Valdramidis et al. 2005) and steam (Kondjoyan and Portanguen 2008; Valdramidis et al. 2008). The experimental data typically show variations way beyond what can be explained by model parameters, thus indicating that the models may be incom- plete with respect to parameters. A considerably improved heat transfer is achieved using steam condensing on the surface. At atmospheric pressure, the steam temperature is 100°C, while the surface treatment temperature will typically be <100°C due to steam being mixed with air on its way from the outlet to the food product surface. James et al. (2000) found a reduction in the total viable counts of contaminated chicken breast portions in the area of log 1.6. Variations were considerable (SD 1 log), and the treatment did not extend the shelf life. Results that are more promising were obtained by Morgan et al. (1996), who introduced a vacuum–steam–vacuum process (VSV process) and achieved a 4 log reduction of L. innocua after only 50 ms of treatment with saturated steam of 138°C. Subsequent studies have revealed an inactivation rate in the range of log 2–4. When applied to catfish samples inoculated with L. innocua, a 3 log reduction was achieved by use of the VSV process (Kozempel et al. 2001). Detailed temperature monitoring on hot dog surfaces during this process led Huang (2005) to hypothesize that a layer of condensate could act as an insulation blanket, which, together with bacteria, was filling the pores and preventing free access of steam, thereby possibly explaining the increased required steam processing time. Skara et al. (2014) used a model fish product to examine steam surface pasteurization as a possible decontamination technology for reducing pathogenic bacteria in different stages of food production. They designed and validated a model system for steam surface pasteurization, assessing different inoculation methods and realistic microbial levels. The response of L. innocua was examined on the model fish product, and the effect of different inoculation levels following treatments with a steam surface pasteurization system was investigated. The variation in the resulting 40 Trends in Fish Processing Technologies inoculation level on the samples was too large (77%) for the contact inoculation procedure to be further considered. In contrast, the variation of a drop inoculation procedure was 17%. Inoculation with high levels showed a rapid 1–2 log decrease after 3–5 s and then no further inactivation beyond 20 s, in agreement with others’ findings (Corry et al. 2007). A low-level inoculation study was performed by analyzing the treated samples using a novel contact plating approach, which can be performed without sample homogenization and dilution (Skara et al. 2014). Using logistic regression, results from this method were used to model the binary responses of Listeria on surfaces with realistic inoculation levels. According to this model, a treatment time of 23 s will result in a 1 log reduction (for P = .1). On the other hand, data from high-level inoculation would indicate a 1 log reduction after 3 s and a 1.5–2 log reduction after 20 s. Including recent works on surface pasteurization, it appears that it is difficult to achieve a 6 log reduction using surface pasteurization. The main effect (1–2 logs) is achieved during the first stages of treatment temperature holding times. Further improvements may be achieved with technological solutions and process/product adaptations, and combinations with growth-inhibiting agents such as lactate/acetate may constitute a useful approach when designing a surface pasteurization process.

2.5 VALIDATION OF HEAT TREATMENT A common requirement for all thermal processes is to prove the safety of the food product through a structured program of thermal process validation. There should be scientific established cooking or pasteurization criteria and a continuous monitoring of the critical process parameters to verify that the process parameters are met (e.g., time and temperature) (FDA 2011, Chapter 16). The core temperature of heat-treated products is usually the most critical point and needs to be measured by insertion of thermocouples. For many processes, with the exception of well-designed and validated retort programs, this procedure has a large degree of uncertainty, and only a few products are checked and a very small volume of the checked products is actually measured. Current practice is often to overcook much of the food to ensure that everything has reached the critical core temperature. For minimally heat-treated foods, a goal is to find strategies that ensure safety in all parts of the product without overheating. If temperature probes cannot be used, alternative approaches to validating microbiological process safety are required. This can be microbiological methods, whereby cells or spores of a nonpathogenic model organism, with similar temperature-induced death kinetics to the target pathogen, are embedded directly into the food or into alginate beads. It can also be simulated trials carried out in a laboratory, where the heat transfer conditions of the process are replicated, or process models that predict, for example, the temperature–time history of the critical food particles as they travel through the heating, holding, and cooling zones of the process.

2.5.1 time–Temperature Integrators A time–temperature integrator (TTI) can be defined as a small measuring device that shows a time–temperature-dependent irreversible change that mimics the change of Minimal Heat Processing Applied in Fish Processing 41 a target attribute when exposed to the same conditions. In practice, a TTI can be an enzyme, such as amylase or peroxidase, that denatures as it is heated in a buffer. If the reaction kinetics of the temperature-induced denaturation match those of the first-order microbial death kinetics, the enzyme can be used as a biochemical marker of a process. TTI has been used both in evaluation of the quality and shelf life of fish in chill chains (Giannakourou et al. 2005; Simpson et al. 2012; Taoukis et al. 1999; Tsironi et al. 2011) and for pasteurization processes (De Cordt et al. 1994; Tucker 1999; Van Loey et al. 1996). Amylase-based TTIs for most commercial pasteurization processes are available, from a few minutes at 70°C up to many minutes at 95°C (Tucker et al. 2002). Several α-amylases from bacterial sources are used for process validation (Grauwet et al. 2010; Mehauden et al. 2008; Tucker 2008; Tucker et al. 2009). Gronqvist et al. (2014) used an α-amylase (BAA70) TTI to validate pasteurization values in fish burgers on a complex frying line with two frying units and a hot air tunnel. Fish burger, being a convenience food prepared out of comminuted fish, spices, and starch, is an important delicacy in fast-food trades and home prepara- tions. They are popular as mild heated lunch or dinner meals or simply eaten without heat treatment as a RTE product. The temperature was measured with thermocouples at different steps in the process and used for validation of a multiphysics com- putation model. The temperature profile calculated from the model was combined with known inactivation kinetics and the residual α-amylase activity was calculated and compared with actual values of the TTIs placed in the core of the burgers. In industrial frying of burgers of minced muscle foods, it is difficult to measure exact accumulated pasteurization values because the heating load is accumulated on a long continuous production line going through several closed heating units. The manufacturer therefore often uses a measured end-point core temperature at the end of the line with a manual handled thermocouple as the only control measure.

2.5.2 online Measurement and End-Point Setting of Temperature It is possible to monitor end-point internal product temperature (EPIPT), a measurement of the temperature of the product, as it exists in the heat process, instead of performing continuous time and temperature monitoring. This approach is suitable if there is a scientific study to validate that the EPIPT selected will provide an appropriate reduction in the numbers of the target pathogen (e.g., 6 log reduction). It is required to conduct a temperature distribution study within the heating system to identify any cold spots. In addition, there may be a need to conduct a heat penetration study that accounts for the slowest heating product under the worst-case heating conditions covered by the study and identify other critical factors of processing and/ or packaging that affect the rate of product heating when scientifically establishing a cooking or pasteurization process (i.e., process validation). This approach might reduce quality loss of the end product and overspending energy. The ideal system should be able to log the temperature in the entire production volume and provide noncontact online temperature measurements for improved control of the cooking process. There are noncontact temperature measurements in use today based on IR measurements and imaging, but these instruments probe the 42 Trends in Fish Processing Technologies surface of the products and not the inner part (Gowen et al. 2010). When the heating process is well defined, it is possible to estimate the core temperature in, e.g., cooked chicken breast by combining noncontact surface temperature with process information like time of heating, heating temperature, etc. (Ibarra et al. 2000). This approach requires detailed quantitative knowledge of the process and how it affects the temperature in the products. Near-IR (NIR) spectroscopy is widely used for rapid determination of food composition. The method is recognized for its rapidity, robustness, and versatility with regard to sampling. It is possible to sample rather large surfaces and volumes, the NIR radiation can penetrate relatively deep into bio-materials, and the measurement principle lends itself to hyperspectral imaging at a macroscopic level. Applications of NIR imaging are already established in the food processing industry for, e.g., quality grading of crabs, salmon fillets, and pork meat (Ellekjaer and Isaksson 1992; O’Farrell et al. 2011; Uddin et al. 2002, 2006; Wold et al. 2006; Segtnan et al. 2009). Tokarskyy et al. (2009) compared different methods for verifying end-point cooking temperatures of fish fillets. Several of the new methods for monitoring processes and product quality in the food processing industry have been based on NIR spectroscopy (Huang et al. 2008). Recently, Wold (2016) showed an online and nondestructive measurement of core temperature in heat treated fish cakes on a belt cooking system by NIR hyperspectral imaging. Two NIR systems (760–1040 nm) were evaluated on heat-treated fish cakes, one point measurement system and one hyperspectral imaging system. Both systems measured several millimeters into the product. Core temperature in the fish cakes (at 10 mm depth) varied between 53°C and 99°C. The point system performed best with a root mean square error of prediction of 2.3°C, while the imaging system was less accurate with an error of 4.5°C. It was demonstrated that temperature changes down to 11–13 mm depth in the fish cakes could be registered by the NIR point system. Such methods are typically fast and noninvasive and well suited for online/inline measurements. However, a major limitation for samples with high water content is the water’s strong absorbance in the NIR region. Spectra of samples with high water content, such as meat and fish, are dominated by the signature from water (Stormo et al. 2012). Thus, when parameters other than the water level are of interest for such samples, efforts to maintain constant water levels are of vital importance. Measures to keep water levels constant may be manageable in lab experiments but not feasible for most of the food processing industry. Stormo et al. (2012) combined visible spectroscopy measurements with multivariate analysis for end-point temperature prediction of surimi samples at low temperatures. The results demonstrated potential for assessing end-point temperatures of surimi by the use of visible spectroscopy and the authors discussed if the basic principle of the method presented could further be transferred to other fish or food products. The temperature range covered was 46.6°C–74.4°C, which enabled validation of a 6 log inactivation of L. monocytogenes.

2.6 COMBINATION EFFECTS In most minimally processed fish products, the thermal process is critical, and a number of factors can affect the thermal process efficiency (e.g., technology, heat Minimal Heat Processing Applied in Fish Processing 43 transfer, etc.). Moreover, additional factors can affect the resistance of microorganisms (e.g., pH, aw), thus facilitating a lighter processing. For processed foods with a potentially small number of surviving organisms, it is imperative that every effort should be made to extend the lag and generation time of potentially present pathogens (Juneja 2006). Hurdle technology is a combination of traditional and innovative preservation techniques in small doses, with the aim of establishing a series of factors that interact in a cumulative or synergetic manner to control the microbial population in food (Leistner 1995). The intelligent selection of hurdles in terms of the number required, the intensity of each, and the sequence of applications to achieve a specified outcome have been proven to have significant potential (Boziaris et al. 2013). The use of hurdles often contributes to a better preservation of a food’s sensory properties after heat treatment. The effectiveness of various preservation methods must be studied through different responses such as microbial growth, sensory analysis, changes in lipids or proteins, and in volatile compounds. For example, Ozer and Demirci (2006) found 1 log reduction of L. monocytogenes on salmon fillets using 60 s treatment of pulsed electric light treatment, but they argued that the increase in temperature during the treatment could be a serious side effect on the bacterial reduction.

In the case of low water activity (aw), this may increase the thermal resistance of L. monocytogenes but limit its growth during subsequent storage. Other factors such as a low pH may increase lethality and reduce subsequent growth, hence the specific regulations for high-acid foods (pH <4.6). The addition of other chemicals can also affect thermal resistance and the growth rate of target bacteria. For example, sodium lactate is increasingly being added to muscle food products and may have an effect on thermal resistance. Whereas Huang and Juneja (2003) found that the addition of sodium lactate (0%–4.5%) had no effect on the thermal resistance of E. coli O157 in ground beef, Juneja (2003) found that sodium lactate (4.8%) increased the heat tolerance of L. monocytogenes in beef. Nevertheless, the addition of sodium diacetate (0.25%) interacted with sodium lactate and rendered L. monocytogenes less resistant to heat. The combination of ultrasound, vanillin, and temperature increased the inactivation rate and decreased the inactivation time of L. innocua (Gastélum et al. 2010). Bacteriolytic enzymes are in the same category. Lysozyme, which is present in a variety of foods, is relatively heat stable, especially under acidic conditions, although it may affect the germination and outgrowth of spores during the storage of sous vide foods (Lindstrom et al. 2003). An important challenge for minimally processed foods is the fact that survivors will have very little competition if they are able to regenerate during the storage period, although this could be counteracted by the addition of competing microbiota. Such organisms should survive processing and potentially have several roles during storage. They could grow during temperature abuse, producing either acid or bacteriocins, or make the product inedible, which would warn the consumer of a hazard (Juneja 2006). This approach has been investigated for cold-smoked salmon by using lactic acid bacteria (Tome et al. 2008, 2009). Among heat-processed products, little information is available for the addition of lactic acid bacteria on seafood (Laursen et al. 2009). 44 Trends in Fish Processing Technologies

2.6.1 M W and RF Processing of Fish Some of the great leaps in innovation have been inventions done by disregarding the marketing reports. The iPad is often used as an example. However, the many small inventions done by industry are based on feedback from the market. This is very much the case when looking at the last decades of electromagnetic heating of foods—development but no revolution. During conventional conduction heating of products, the outer surface will have a much higher heat load than the core of the product. This may be desired but is more often a problem. The MW volumetric heating offers a solution for minimal processing by rapid heating of the core without overcooking the rest of the food. Heat- sensitive foods, e.g., fish, may achieve a quality that is not possible to achieve in other ways. Unfortunately, the process is hard to control and the temperature is difficult to track. For instance, a chef might desire a halibut loin to reach 48°C, while no part should exceed 50°C. Such precise temperature control is a complicated task that was solved by several experiments, numerical modeling, and a dedicated staff (Skipnes and Sone 2014), but in most cases, it would not be possible with conventional heating while MW heating could easily solve the task provided the electromagnetic field was homogenous. MWs are electromagnetic waves with wavelengths from 1 mm to 1 m and corresponding frequencies between 300 MHz and 300 GHz, and two frequencies of MWs have been approved for application to foods at 915 MHz and 2450 MHz. An increasing popularity to MW heating is observed in the food industry for various processing operations like blanching, cooking, drying, pasteurization, baking, and thawing of bulk food products. MW pasteurization has the ability to heat the product 3–5 times faster than the conventional sterilization system, causing food products with better taste and acceptability. Industrial implementation of these technologies relies on a cost-effective production, complexity of operations, and added value to the final products.

2.6.1.1 Implementation of Industrial MW Production Industrial-scale MW cooking is still limited, and even if we include MW tunnels for purposes other than food, these markets are very small compared to the market for domestic MW ovens. The domestic market is estimated to be 70 to 75 million units each year (Osepchuk 2013), which encourages R&D on domestic ovens rather than industrial MW purposes and the total market is growing. This is also reflected in the fact that the number of peer-reviewed papers on MW heating applications has increased steadily. The increase was from 50 in 1990 to over 500 scientific papers in 2013 (WOS core database, Thomson Reuters, December 2014). Another trend is that the manufacturing of both magnetrons and ovens has moved to China and a few other Asian countries. About 16% is still manufactured in Europe, while the production in the United States and Japan is negligible. Advanced domestic MW ovens combined with steam heating, grill function, and hot air circulation is now common in retail stores for household articles. Several MW systems for heating and drying of a number of materials have been industrialized, but still there are few food applications. These applications are tempering, preheating, drying, and some Minimal Heat Processing Applied in Fish Processing 45 few examples of pasteurization. Some of the MW-oven-producing companies offer solutions for all these applications, e.g., the French company Sairem. A Swedish company, MicVac, is offering a complete MW production line including an applica- tor for a valve that will open during pasteurization and let out excessive steam that is generated in the package. The MicVac system is now used in Europe (e.g., Norway and Sweden) and in Asia (e.g., South Korea and Japan) (http://micvac.com/). Another system with some similarities has been launched by the Swiss company Micropast®, with a system originating from a concept called Dream Steam. These few examples document that in-package MW pasteurization has found its niche, even if the applications are still few. The limited industrial use has not stopped the research efforts. Research on industrial MW tunnels or ovens is still carried out and focuses on (i) tempering of frozen foods (e.g., blocks of meat or fish), (ii) pasteurization, and (iii) sterilization. This has led to some novel methods, which follow here.

2.6.1.2 Tempering Tempering is by far the most common MW application in food industry (Higgins 2015). It is not very efficient to let the meat or fish thaw on its own; this leads to a high drip loss, which causes reduced quality and makes further processing difficult. MW systems are not ideally suited to thawing, and they are known for thermal instability (Archer et al. 2008). An example is thawing of large frozen fish blocks, although an MW system below the freezing point (between −5°C and −10°C) is applied to facilitate cutting and further handling of the product. Some parts of the food product can become overheated or even partly cooked, while others remain frozen. There is also the possibility of runaway heating in part-frozen products, where the parts of the food that have thawed will absorb energy preferentially to those that are still frozen. RF thawing systems are also available and can be used for fish products, where the frozen product is placed between two parallel electrodes and alternating RF energy is applied to the electrodes. Compared to MW heating, the temperature rise within the product is relatively uniform, the degree of uniformity being dependent on the size and composition of the product. It is suggested that 5-cm blocks of fish can be thawed in 15 to 45 min. For final temperatures near the thawing point of seafood (typically −1°C), the two frequencies offered by MW processing (2450 and 915 MHz) are no longer adequate as they can create uneven temperature distribu- tions and result in poor quality products. However, RF treatments have more promising attributes for processing seafood. At the lower frequencies of RF, penetration of the RF energy into foods is much greater and enables the temperature of blocks to increase from −20°C to −2 or 0°C. RF systems are available in both batch and continuous formats. Batch RF systems operate from 40 to 350 kg/h, while continuous RF systems can operate from 900 to 3000 kg/h.

2.6.1.3 Pasteurization There are some successful applications for MW pasteurization of liquid foods like milk and juices. Such applications are chosen for their economic competitiveness. 46 Trends in Fish Processing Technologies

With respect to food quality, MW is more interesting for solid foods, which are heated by conduction. All reported commercial MW sterilization and pasteurization systems use 2450-MHz multimode heating cavities (Tang 2015). The products are heated to a desired temperature or to the maximum temperature that can be achieved. Since water is the major constituent of most food products, the boiling point of water is the maximum temperature that can be achieved, i.e., around 100°C at ambient pressure. Some packaging solutions can allow a pressure of 0.1 to 0.2 bar above the ambient pressure; i.e., temperatures up to 105°C may be achieved. One of the main advantages of MW pasteurization is continuous processing, but when balancing the throughput and size of the tunnel, it is evident that the processing times have to be limited and most commonly are less than 5 min. Several bacterial spores may survive at 105°C for 5 min and the products therefore have to be kept under refrigeration during storage. Short treatments usually result in large temperature variations within the product and the heat load achieved at the coldest spot in the product may be much lower than the average for the product. Due to the risk of regrowth of spores, it is essential to cool the products, preferably below 3°C. Blowing cold air over the processed products is the most common way of refrigeration. Compared to the volumetric heating, air cooling is a very slow process and remains the major drawback for continuous MW pasteurization systems. There are alternatives to air cooling of MW-heated foods that are vacuum packed or meals packed in trays are cooling with other gases, water, or ice. Slush ice is, up to now, the most effective cooling media. By addition of salt or antifreeze agents, it is possible to achieve a liquid at −3°C. Baths with stirred slush ice can easily be controlled to avoid surface freezing of the product, which is a challenge in air cooling systems with air at very low temperatures (<−18°C). Some of the advances in minimal processing by MW originate from recent efforts in MW sterilization. Most important is the introduction of counter pressure during MW heating. MW sterilization is an emerging technology that provides faster heat penetration and can significantly reduce the degradation of heat-sensitive nutrients (Resurreccion 2012). It delivers thermal energy to foods under pressure to achieve inactivation of microorganisms. The reduction in processing time improves color, texture, and other sensory attributes of foods while meeting microbial safety requirements. When describing the inactivation kinetics of MW heating, it is recommended to include only thermal effects in the model (US FDA 2000). Several authors recommend that the challenges in obtaining a predictable temperature distribution and history should be overcome before MW sterilization is used for commercial food processing. However, more recently, the US FDA has approved the MW sterilization process for mashed potatoes in trays and pouches with salmon fillet in sauce (Brody 2012). The process involves immersing the packaged food in pressurized hot water and simultaneously heating with MWs at a frequency of 915 MHz, and this technology was developed at Washington State University.

2.6.1.4 Novel Applications of MW and RF for Fish MW heating has been used in different applications related to fish products, like inactivation of microorganisms in fish meals (Bauza-Kaszewska et al. 2014), cooking Minimal Heat Processing Applied in Fish Processing 47

(Baldwin et al. 1971; Sheen et al. 2012), studying changes in fatty acids (Regulska-Iiow and Iiow 2002), and drying of foods (Feng et al. 2012; Vadivambal and Jayas 2007). In MW heating, steam is generated inside the package and there is a risk that such a package may explode. Counter pressure or venting may therefore be necessary to prevent overpressure and package bursting. A number of valves are available that release steam during thermal processing, and during chilling, the condensation of the vapor will generate a “vacuum” in the processed package. Sairem of France and Stalam S.p.A. of Italy offer RF defrosting systems for thawing or tempering seafood, meat, and vegetables. Tempering raises the temperature of frozen blocks of fish products to just below the freezing point to facilitate cutting and further product handling. Their systems operate at a frequency of 27.12 MHz, are built of stainless steel for corrosion resistance, and have polyethylene conveyor belts to facilitate cleaning. They are available with output RF power from 10 kW to 105 kW. Units are operated in parallel for increased production volume and RF systems are designed for continuous processes. RF defrosting systems offer many benefits as compared to conventional systems; however, RF equipment is not widely used by Northwest food processors. Drawbacks of conventional defrosting methods are avoided with RF as heat is generated uniformly, thus producing even drying. The system is compact, fast, and easily controlled, resulting in significant drip loss reductions while minimizing product deterioration due to bacterial growth. The RF process works even for large product blocks and can thaw products inside packaging. Energy savings depend both upon the system being replaced and the product type. Other benefits of RF defrosting include the following:

1. Processing speed and temperature uniformity minimize the risk of product degradation and bacterial growth, even in large blocks. 2. The product can be brought to the correct temperature needed for the next stage of processing. 3. RF equipment requires less floor space than conventional equipment does.

RF drying can be used to dry crackers, cookies, and other snack foods after they have been baked. It can also be used to dry ceramics, synthetic foam, textiles, water- based inks, and adhesives in paper products. RF can also be used to preheat thermo- set plastic molding compounds.

2.6.1.5 Overpressure MW Processing By using a pressurized cavity for heating foods by MW, it becomes possible to go to temperatures above 100°C without boiling and evaporation. MW heating at atmospheric pressure results in rapid evaporation and heat loss from the food that limits the temperature increase and results in drying of the sample. The overpressure MW heating makes it possible to achieve high-temperature short-time pasteurization or even sterilization. An example of research equipment at Nofima, Norway, for this application is shown in Figure 2.3. It has been known for several years that the Belgium company Tops has been using overpressure MW heating for pasteurization of ready meals and some details are now presented by Tang (2015). The Italian 48 Trends in Fish Processing Technologies

FIGURE 2.3 Pilot plant MW heated retort with counter pressure. This programmable unit is equipped with eight fiber optic temperature sensors, pressure indicator, power meter, expansion indicator and direct water cooling. (Courtesy of Nofima.) company Gustosi is another processor of ready meals that started utilizing overpressure MW-heating more recently. Far more attention has been drawn to a group at the Washington State University, who from 1995 on has developed the MW-assisted thermal sterilization (MATS) and MW-assisted pasteurization (MAPS) systems. Professor Juming Tang, who is leading this group, pinpoints three challenges that have been solved (Tang 2016):

1. Stable and uniform heating 2. Cold spot determination in moving food packages 3. Validation of microbial safety Minimal Heat Processing Applied in Fish Processing 49

Despite all the efforts on the MATS development and the tremendous contribution to the science of MW food processing, food products have not come to the retail market yet (2016). The industrial implementation has been limited to WSU’s own commercialization company, 915 Labs. A large number of peer-review articles related to MATS/MAPS have been published on modeling (Birla et al. 2007; Hong et al. 2016; Resurreccion et al. 2013; Wang et al. 2005), dielectric properties (Al Holy et al. 2005; Luan et al. 2015a; Wang et al. 2008; Zhang et al. 2015), and safety and validation (Luechapattanaporn et al. 2004; Tang 2015; Reyes-De-Corcuera et al. 2005).

2.6.1.6 Subatmospheric Pressure MW Processing By reducing the pressure in a MW-heated cavity, it is possible to control the surface temperature as boiling will cool the surface. This was first developed in the 1980s at the Food Refrigeration and Process Engineering Research Center, United Kingdom, and it was shown possibly to dramatically reduce the thawing time compared to conventional thawing methods (James 1984). By using a pressure of 5 kPa, it is possible to avoid temperatures causing protein denaturation and visual changes to the products during thawing. However, the amount of water that has to be evaporated is considerable and the weight loss limits the practical interest to systems where surface water can be sacrificed in a thawing process. For a minimally processed product, subatmospheric MW heating is very interesting as it makes it possible to restrict the temperature at any point in the product. Regarding the previously mentioned problem of heating a halibut loin to 48°C, a pressure of 11 kPa would be ideal to limit the maximum temperature.

2.6.1.7 MW Combination Heating The principles of MW combination heating have been comprehensively reviewed by Datta and Rakesh (2013). Uniformity of combination processes has to be discussed specifically for each combination, like combination of MW and conventional oven heating (Song et al. 2016). IR heating and convection heating by hot air or steam are all in commercial use and sold on the domestic market. The MW combination heating is indeed one of the most promising fields for continued development of new food products because of the ability of both fast volumetric heating and combine a juicy core with a crispy surface. Automated control programs have been the key to make this work in domestic facilities and will also be needed on an industrial scale.

2.6.1.8 Water Immersed RF Processing One of the main advantages of immersing products in water and heating by RF is the combination of maintaining a desired surface temperature by the water while the RF provides rapid volumetric heating. The penetration depth of RF is very high compared to MW and improves temperature uniformity during heating. These advantages have been demonstrated, e.g., for pasteurization of shell eggs (Geveke et al. 2017). Overpressure RF heating in water bath is a combination that opens for RF as a sterilization method. Equipment has been designed with a cooling bath within the same pressurized cavity as the heating, but so far, no one has tried to implement this in an industrial production unit. 50 Trends in Fish Processing Technologies

2.6.1.9 Design and Validation of Thermal Processes Validation of MW heating of food in pouches or ready meals in trays has been widely investigated (Ohlsson and Bengtsson 2001; Zhang 2000). Despite the many attempts to improve temperature distribution in MW ovens, it is still a major challenge to achieve a predictable temperature in all parts of the food to be pasteurized or sterilized. Additionally, the achieved maximum temperature does vary due to composition and geometry. The major problems have been reviewed by Vadivambal and Jayas (2010), who found several studies of specific food materials where non- uniformity caused quality problems either (1) directly, such as drying of surfaces or burning, or (2) indirectly, due to a need for safety margins. Another discrepancy that is ignored in most cases is the atmospheric pressure. As mentioned above, the saturation pressure determines the maximum temperature achieved in moist food. At a low atmospheric pressure of 932 mbar, the boiling point of pure water is 97.7°C. At a high atmospheric pressure of 1011 mbar, the boiling point is 101.1°C. This 3.4°C temperature difference might be important in microbial inactivation. Let’s consider a meal to be pasteurized to 100°C and to be kept there for 1 min for a 6 log inactivation of spores of C. botulinum type E with a decimal reduction time

D90°C = 1.6 min and the temperature change required for 1 log decrease is z = 10.0°C. Using the previous pressures, we would expect D to be 16.3 s at low atmospheric pressure and 7.5 s at high atmospheric pressure. A 1-min processing would result in 3 log inactivation at 97.7°C and 8 log inactivation at 101.1°C, and thus, the atmospheric pressure could make a difference for the product safety. On the other hand, there will usually be a pressure build-up in the package during MW pasteurization and then the achieved pressure in the package will be important, which is more dependent on the packaging than the atmospheric pressure. For systems with an opening valve, it might be important to know the variation in pressure among the valves when they open. Such systems are sometimes validated by using the weight loss during processing for calculating the heat absorbed by the product. The method is fast, inexpensive, and nondestructive, but we have not found any documentation about this method, and research is required. Some topics to be investigated are as follows:

1. May differences in internal geometry of the food packages sometimes result in extraordinary cold spots in such manner that underprocessing may occur even if the total absorbed energy meets the target value? 2. How can the validation method handle variation in composition of food in different packages? 3. Will the validation be affected by the pressure achieved before the valve opens?

These questions should be answered by studies of a range of foods, and one of the major challenges in this work is how to make accurate temperature measurements in packages with valves as an airtight connection is needed. Making a packing gland tight without damaging the fragile fiber optic probes is challenging.

2.6.1.10 Temperature Measurement Thermocouples must be fully grounded if used in electromagnetic systems. Used with insufficient grounding, thermocouples could act as an antenna, spreading Minimal Heat Processing Applied in Fish Processing 51 the radiation outside the cavity, where it was intended to stay. Even when properly grounded, thermocouples may influence the electromagnetic field and is in general not used as a validation tool inside MW or RF systems. In lack of better options, there still have been some attempts to overcome this challenge, e.g., by shielding of thermocouples (Ramaswamy et al. 1998), but with an accuracy of less than 2°C and long response time, the method is not very useful. Wireless temperature loggers are commonly used in pasteurization systems, especially for continuous systems. Such devices contain metal and electronic parts and risk of sparkling, damage to the logger or giving misleading results, and this limits their use. Fully covered in food products, temperature loggers may survive and record temperature. However, concentration of the electromagnetic field at the tip of the temperature probe with misleading high temperatures as a result is likely to occur. Such overheating at the sensor tip has been shown by modeling (Luan et al. 2013, 2015b). The solution that remains for temperature measurement is fiber optic cable systems. Compared to thermocouples, fiber optic probes are slower in responding, are less accurate, are more expensive, and easily break. Properly manufactured fiber optic probes are not expected to change the electromagnetic field in a way that could give misleading results. However, inserting the probe into a food results in a punc- ture that might lead to increased local liquid loss.

2.7 PEF TREATMENT PEF is a nonthermal method of food preservation that uses short pulses of electric- ity for microbial inactivation and causes minimal detrimental effect on food quality attributes. PEF can improve functionality, extractability, and recovery of nutritionally valuable compounds as well as the bioavailability of micronutrients and components in a diverse variety of foods (Barba et al. 2015). The consumer is offered foods of high quality and the technology is considered superior to traditional thermal processing methods. Over the past decades, much research on basic and applied aspects of PEF for the food industry has been carried out, contributing to a better understanding of this innovative food processing technology (Barba et al. 2015; Buckow et al. 2013; Toepfl 2012). From this, a wide variety of applications of PEF has been developed. The lack of reliable and viable industrial equipment limited the exploitation of PEF in the food industry for several years. Recent developments in pulse power generators have permitted the design of appropriate PEF equipment at an industrial scale and it is expected that new ones will be launched in the near future. Still, PEF is mainly used in liquid and semiliquid, pumpable products, emulsions, suspensions, soups, and sauces.

2.7.1 P rinciple of PEF PEF technology consists of an electrical treatment of short time (from several nano- seconds to several milliseconds) with pulse electric field strength ranging from 100–300 V/cm to 20–80 kV/cm (Buckow et al. 2013; Koubaa et al. 2015; Zhao et al. 2014). PEF treatment is conducted at ambient, subambient, or slightly above ambient 52 Trends in Fish Processing Technologies temperature for less than 1 s, in the form of exponentially decaying, square wave, bipolar, or oscillatory pulses and energy loss due to heating of foods is minimized. At high electric fields (>20 kV/cm), it can constitute an alternative to traditional thermal processing to inactivate pathogenic microorganisms and quality related enzymes (Sánchez-Vega et al. 2015). The processing time is calculated by multiply- ing the number of pulse times with effective pulse duration. The process is based on pulsed electrical currents delivered to a product placed between a set of electrodes; the distance between electrodes is termed as the treatment gap of the PEF chamber. After the treatment, the food is packaged aseptically or at strict hygienic conditions and stored under refrigeration. Although the applications of PEF is increasing, it is mainly restricted to food products that can withstand high electric fields, have low electrical conductivity, and do not contain or form bubbles (Mohamed and Eissa 2012). The applied high voltage results in an electric field that causes microbial inactivation. The application of electrical fields to biological cells in a medium (e.g., water) causes build-up of electrical charges at the cell membrane (Schoenbach et al. 1997). Membrane disruption occurs when the induced membrane potential exceeds a critical value of 1 V in many cellular systems, which, for example, corresponds to an external electric field of about 10 kV/cm for E. coli (Castro et al. 1993). Over a long period, several theories have been proposed to explain microbial inactivation by PEF. Among them, the most studied are electrical breakdown and electroporation or disruption of cell membranes (Castro et al. 1993; Sale and Hamilton 1967; Vega- Mercado et al. 1996; Zimmermann 1986).

2.7.2 advantages and Application in Foods Several technical and review articles have been published on the different aspects of application of PEF, including microbial inactivation, plant cell rupture, juice extraction, equipment, and technology aspects to succeed in the food industry (Jeyamkondan et al. 1999; Ho and Mittal 2000; Mosqueda-Melgar et al. 2008; Toepfl et al. 2006, 2007; Zhao et al. 2014). Barba et al. (2015) summarized some main challenges in the food industry in the following points: (i) phenomenon of electroporation, (ii) impact of PEF on different products of biological origin (plant tissues, suspension of cells, by-products, and wastes) and biorefinery applications, and (iii) different modes of PEF-assisted processing (extraction by solvent diffusion or pressing, osmotic dehydration, drying, and freezing) and cold pasteurization of liquid foods (microbial inactivation, bioactive compounds stability, and the potential reduction of food contaminants). Although the main objective of PEF pasteurization is to guarantee food safety, a large proportion of the population of vegetative spoilage microorganisms is also inactivated by the treatment. Therefore, PEF treatments extend the shelf life while better maintaining the original color, flavor, texture, and nutritional value of the unprocessed food (Barsotti et al. 2001; Min et al. 2007; Zhao et al. 2014; Zulueta et al. 2013). PEF has been used in juice processing, to treat microalgae, seaweed, and other aquatic species. It has been used in plant oil extraction, drying enhancement, sugar processing, and meat and fish treatments. PEF treatment was shown to be very Minimal Heat Processing Applied in Fish Processing 53 effective for inactivation of microorganisms, increasing the pressing efficiency and enhancing the juice extraction from food plants and for intensification of the food dehydration and drying (Barbosa-Cánovas et al. 1998, 1999; Estiaghi and Knorr 1999; Vorobiev et al. 2004; Taiwo et al. 2002). Homogeneous liquids with low electrical conductivity provide ideal conditions for continuous treatment with the PEF method. There is a limitation in the particle size of the liquid food in both static and flow treatment modes. The maximum particle size in the liquid must be smaller than the gap of the treatment region in the chamber in order to maintain a proper processing operation. The presence of bubbles may lead to nonuniform treatment as well as operational and safety problems. When the applied electric field exceeds the dielectric strength of the gas bubbles, partial discharges take place inside the bubbles that can volatize the liquid and therefore increase the volume of the bubbles. The bubbles may become big enough to bridge the gap between the two electrodes and may produce a spark. Therefore, air bubbles in the food must be removed, particularly with batch systems.

2.7.3 challenges of PEF Technology Applied for Food Production In a recent survey, Jermann et al. (2015) mapped the trends in novel and emerging food processing technologies around the world. Food professionals from industry, academia, and government identified novel technologies applied either now or with the potential to be commercialized in 5–10 years. The main drivers were higher-quality products (94%), product safety (92%), and shelf life (91%). HPP and MWs were identified as main technologies now and in the next 10 years. PEF was placed after these technologies, indicating a strong belief in commercialization in Europe, while UV and radiation have a better position in North America. The factors in the survey, which limited commercialization, were mainly economic. It was considered that installing a new technology requires a significant investment with an increase in the product price and lack of funding. With increased numbers of commercial PEF production plants, the experiences will most likely be a driver for industrial performance and reliability acceptance. FDA approved the use of PEF in the preservation of liquid eggs more than 20 years ago (Dunn 1996), while the first commercial PEF application for fruit juice preservation was installed in the United States in 2005 (Clark 2006). At present, there are still few but an increasing number of commercial producers of PEF products and retailers that sell the products. The lack of methods to accurately measure treatment delivery limits the validity of conclusions that can be drawn about the effectiveness of particular process conditions. A method to measure treatment delivery would prevent inconsistent results due to variations in PEF systems. In regard to safety of the process, a HACCP system must be approved. Today, every US producer of fruit juice has to prove a 5 log inactivation of the most resistant microorganism of public health significance that is reasonably likely to occur in the juice (FDA 2003). Documentation of achieved performance criterion is difficult because there is no single target pathogen. The pertinent pathogen may vary with the type of juice and the type of treatment used, although typically it would be Salmonella, E. coli O157:H7, or Cryptosporidium 54 Trends in Fish Processing Technologies parvum. Approved validated surrogate organisms that can be used in up-scaled challenge testing of PEF are lacking.

2.7.4 PEF Used in Fish Products Fish is muscle food with dielectric properties that differ from those of liquid foods. The effects of PEFs and a combination of PEF and high pressure were studied on microstructure of salmon, chicken, and lumpfish roes (Gudmundsson and Hafsteinsson 2001). It was shown that PEF treatment with low field strength (less than 2 kV/cm and 20–40 pulses) had considerable effect on the microstructure; i.e., the muscle cells decreased in size and gaping occurred. Further, PEF treatment had greater effect on salmon than chicken samples and roes seemed to tolerate up to 18.6 kV/cm and seven pulses without visible effect. Reduction of microorganisms was still not sufficient at the same time. No direct effect of PEF treatment on protein profile was found by electrophoresis. Thus, it was concluded that the changes in microstructure were associated to permeabilization of the cell membrane and leakage of cell fluid into extra cellular space. This hypothesis was supported by the results of microstructure analysis using cryo scanning electron microscopy in this study (Faridnia et al. 2014). PEF treatment has the potential to increase the water uptake and water holding properties, which makes it an interesting technology in the fish processing industry. This was a focus for Klonowski et al. (2006) using brine injection in fish, combined with PEF. They showed that a PEF treatment (2.0 kV cm−1, 90 pulses) of fish cod samples injected with brine gave a porous structure. In contrast, a study on the effect of PEF treatment (1.1–2.8 kV cm−1, 5–200 Hz, 12.7–226 kJ kg−1) on quality traits of beef semitendinosus muscles suggest that the physiochemical characteristics of beef semitendinosus muscles were not affected immediately after the PEF treatments (O’Dowd et al. 2013). The electro-permeabilization of cell membranes, leading to a drastic increase in mass transfer rates, can be utilized to enhance the drying rates of cellular tissue (Barsotti and Cheftel 1999). An increase in mass transfer rates, resulting in faster water transport to the product surface and therefore reduction of drying time after a pretreatment, will lead to drastic saving of energy and better utilization of production capacities during convective air drying. Taking into account the low energy input required for a PEF treatment of plant or animal tissue (2–20 kJ/kg), it is evident that there is a potential to reduce the total energy input for product drying. The use of PEF for preservation of fish, meat, and other solid food products does not, at present, seem realistic as application of low intensity electric field pulses has a detrimental effect on the microstructure. At the same time, the low field voltage does not effectively reduce the microbial numbers. Fish roes seem to be one of the few solid foods that can withstand PEF treatments of 1200 kV/m and 12 pulses without a visible effect on the microstructure or texture. In the fish industry, a PEF treatment could possibly be used either to dehydrate by-products or to extract some valuable items such as enzymes, fish oils or valuable metabolites. However, these applications need to be thoroughly investigated. Minimal Heat Processing Applied in Fish Processing 55

2.7.5 P EF and Microbial Inactivation The electromechanical instability in the cell membrane provoked by PEF causes the inactivation of vegetative forms of bacteria, yeast, and moulds (Mosqueda- Melgar 2008). While some authors have reported the inactivation of bacterial spores (Marquez et al. 1997) and enzymes (Aguiló-Aguayo et al. 2010; Giner et al. 2000; Ho et al. 1997; Zhao et al. 2007), others have observed that treatments were limited at low temperatures (Grahl and Märkl 1996; Pagán et al. 1998). Spores are more resistant compared to vegetative cells, due to the spore envelope, with a coat and the cortex, that prevent the permeabilization effects of PEF on the spore cytoplasmatic membrane (Pagán et al. 1998). In general, a limited inactivation of bacterial endospores is obtained by PEF if the temperature is lower than 60°C and less than 30 kV/ cm as electric field strength applied (Barbosa-Cánovas et al. 1999; Cserhalmi et al. 2002). However, application of PEFs in combination with thermal energy has been shown to enable spore inactivation with lower heat load compared to only thermal processing. By increasing the temperature and electric field strength, an inactivation of B. subtilis and B. cereus could be achieved (Bermúdez-Aguirre et al. 2012; Siemer et al. 2011, 2014). Thus, for nonthermal use of PEF, other hurdles, such as refrigeration, atmosphere modification, the addition of preservatives, or a combination of these techniques, may be required to preserve the quality and stability of the food during its distribution and storage (Raso and Barbosa-Canovas 2003). To establish the treatment conditions for PEF food pasteurization, the first step is to identify the most PEF-resistant microorganisms that have an impact on public health. This identification depends on the intrinsic PEF resistance of the microorganisms. It may also be influenced by other factors that can affect microbial resistance, such as the treatment medium characteristics or the physiological state of the cells (Aronsson and Rönner 2001; Cebrián et al. 2007). L. monocytogenes is the most PEF-resistant microorganism at neutral pH, but gram-negatives (E. coli, Salmonella spp., Cronobacter sakazakii, C. jejuni) would display a similar or even higher resistance at acidic pH (Cebrián et al. 2016). The factors affecting the resistance of bacterial food-borne pathogens, as well as the magnitude of the effect, vary depending on the technology considered. For example, interspecific and intraspecific differences in microbial resistance to PEF and HPP are much greater than to manosonication (MS) and UV. Similarly, both the pH and aw of the treatment medium highly condition microbial resistance to PEF and HPP but not to MS or UV. Growth phase also drastically affected bacterial HPP resistance. Finally, increasing treatment temperature leads to a significant increase in lethality of the four technologies, which opens the possibility of the development of combined processes including heat. Further work would be required in order to fully elucidate the mechanisms of action of these technologies and to exhaustively characterize the influence of all the factors acting before, during, and after treatment. This would be very useful in the areas of process optimization and combined process design. A smaller organism, e.g., L. innocua, has a higher resistivity to PEF than organisms with higher cell size like E. coli or Bacillus megaterium (Toepfl et al. 2007). Above that, the cell membrane constitution has an important influence on the stability 56 Trends in Fish Processing Technologies of the membrane. The tendency that gram-positive bacteria are more resistant than gram-negative species has frequently been reported (Hulsheger et al. 1981; Vega- Mercado et al. 1996; Wouters et al. 1999). Whereas L. innocua showed a close-to- linear relation between energy input and inactivation rate, for E. coli, a sigmoid curve has been obtained. This curve shape is contrary to earlier studies conducted with parallel electrodes and a field strength in the range of 30–40 kV cm−1 (Heinz et al. 2003). It has been shown that there is no relationship between microbial resistance and the inactivation procedure that is used for microbial destruction. For example, Salmonella senftenberg 775W, which is a very heat-resistant microorganism, is more sensitive to high hydrostatic pressure or PEF than other heat-sensitive Salmonella (Alvarez et al. 2000). Therefore, the target microorganism for PEF pasteurization is not necessarily the same as the target microorganism for thermal pasteurization or other inactivation treatments. The efficacy of PEF against pathogenic microorganisms has generally been based on single-strain studies. There is therefore limited information available in the literature on variations of resistance to PEF among strains of the same microorganism. So far, few studies have demonstrated that the PEF resistance of different strains of bacterial species may vary greatly. Inactivation of nine L. monocytogenes strains by PEF treatment (25 kV/cm, 144 μs) ranged from 0.7 to 3.7 log10 CFU/ml (Lado and Yousef 2003). Inactivation of 15 S. aureus strains by PEF treatment (22 kV/ cm, 200 μs) ranged from 1.2 to 4.0 log10 CFU/ml (Rodriguez-Calleja et al. 2006). Saldana et al. (2009) conducted a complete study on the variability in PEF resistance of different strains of pathogenic microorganisms to identify potential target microorganisms for designing PEF pasteurization processes. The screening study confirmed that the PEF resistance of five strains of L. monocytogenes, S. aureus, E. coli, and Salmonella Typhimurium may vary significantly. As the most resistant strains at low pH are not necessarily the most resistant at neutral pH, the target microorganisms for PEF pasteurization could be expected to be different for foods, depending on their pH (Garcia et al. 2005).

2.8 CONCLUDING REMARKS Minimally processed fish products are requested in the society due to their sensory properties and content of healthy components. It is expected that products with minimal processing will continue to increase, which is a driving force for researchers worldwide to develop new technologies and new applications of existing technologies. The efficiency of the methods is important for an implementation in commercial production, and documentation of quality attributes and food safety is vital. Thermal processing still has a unique position when it comes to the volume of produced minimally processed foods, but new thermal variants are developing and implemented in the industry, like Shaka retorting, MW, and RF heating. Each technology has to be optimized for specific raw materials, and application to fish products has been demonstrated. Overpressure MW heating is one of the promising techniques. New validation methods for electromagnetic heating systems will also Minimal Heat Processing Applied in Fish Processing 57 be important, but most important will be the ability to produce MW systems that can produce homogenous and predictable heating. The development of nonthermal PEF technology has accelerated the last decade, but the use of PEF for preservation of fish is still limited and mostly used for pumpable liquid foods. PEF technology and methods for particulate and muscle foods are increasing, and new applications also for nonpreservation purposes are suggested. Validation is a key factor needed for all new and emerging technologies. International recommendations of a 5 or 6 log reduction of a key target organism should be obtained. With minimal processing, more microorganisms may survive and accepted validation procedures are needed in order to document product safety.

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Daniela Borda

CONTENTS 3.1 Introduction...... 71 3.2 Effect of HPP on Fish Constituents...... 73 3.2.1 Proteins...... 73 3.2.2 Lipids...... 74 3.2.2.1 Antioxidants...... 76 3.3 Effect of HPP on Fish Functions...... 76 3.3.1 Enzymes...... 76 3.4 Effect of HPP on Fish Properties...... 82 3.4.1 Texture...... 82 3.4.2 Color...... 83 3.4.3 Aroma and Taste...... 84 3.5 Effect of HPP on Fish Safety...... 85 3.5.1 Effect of HPP on Microorganisms...... 85 3.5.2 Biogenic Amines...... 91 3.5.3 Allergens...... 92 3.6 Gelation...... 93 3.7 HP Thermal Sterilization...... 93 3.8 HP Freezing and Thawing...... 94 3.9 Concluding Remarks and Future Perspectives...... 95 References...... 95

3.1 INTRODUCTION High-pressure processing (HPP) is for the food industry a relatively young technology that successfully evolved into one of the most engaging alternatives to conventional thermal processing. Only in the last two decades has HPP become economically feasible and led to many industrial applications (Torres and Velazquez 2005; Tintchev et al. 2013). Colloquially named cold pasteurization, up to now, HPP has been widely applied to prolong shelf life of predominantly additive-free foods. At refrigeration, ambient or moderate heating temperature HPP inactivates pathogenic and spoilage microorganisms in foods, with fewer changes in sensorial characteristics and a better retention of micronutrients when compared to other technologies in place (Heinz and Knorr 2005; Torres and Velazquez 2005).

71 72 Trends in Fish Processing Technologies T F/ PA PS ptides pe s unction F me tion Bioactive En zy la Ge ts g essin ty anisms fe oc s rg Sa oo rdle her contaminan ssure pr Hu Biogenic amines Allergens Ot Micr re s e tie e er sh high -p st extur Fi T Ta Aroma Color Prop TS PA e ur ct ru Proteins Lipids St Shelf lif e extension Constituents, properties, function, and safety aspects influencedby together on fish HPP with processingPATS, methods applied.

FIGURE 3.1 pressure-assisted thermal pressure shift sterilization; freezing/pressure-assisted PSF/PAT, thawing. High-Pressure Processing of Seafood 73

One of the earliest high-pressure (HP) applications in the food industry is oyster processing. In this case, the labor-intensive process of manually releasing the adductor muscle from the shell could be replaced by HP. As a result of the HP treatment, oyster shell opening became easy to make, liquid in the oyster is retained, and no meat damage or shell splinters are present. Moreover, HPP also reduces the presence of bacteria such as Vibrio parahaemolyticus and Vibrio vulnificus, occasionally present in natural estuarian microflora of oysters (Cruz-Romero, Kelly, and Kerry 2008). At the present time, almost half of the 200,000 tons of annually HP treated foods are meat, fish, or seafood products (Buckow, Sikes, and Tume 2013). HP treatments affect the main constituents of fish (proteins, lipids, and enzymes), influence its sensorial properties (texture, color, aroma, and taste), and impact product safety (inactivate microorganisms, thus contributing to biogenic amine (BA) reduction and influencing contaminants and allergens presence). The processing applications using HP in seafood are directed toward (1) extending fish refrigerated/ frozen shelf life; (2) pressure-assisted thermal sterilization (PATS); (3) the potential of combining HPP with multihurdle technologies (natural antioxidants and antimicrobials, other minimal processing methods, active packaging, etc.) in order to reduce the intensity of processing and obtain clean label products and/or functionalized ones; (4) texturization induced by pressure (gelation); and (5) HP sterilization and HP freezing/thawing (Figure 3.1).

3.2 EFFECT OF HPP ON FISH CONSTITUENTS

3.2.1 P roteins The primary element of fish muscle is myotome made up of huge numbers of single muscle fibers linked by intramuscular connective tissue named myocommata (Cheng et al. 2014). Three main classes of proteins are present in the fish muscle: (1) myofibrillar proteins, (2) sarcoplasmic proteins, and (3) stroma proteins. Myofibrillar proteins constitute 40%–60% of the total crude protein content of fish and include mainly myosin and actin, but also regulatory proteins such as troponin and tropomyosin (Truong et al. 2015). Sarcoplasmic proteins account for up to 30% of the total muscle proteins and are soluble in water or salt solutions of low ionic strength. Many metabolic enzymes with haem-albumin and myoglobin belong to this class. Sarcoplasmic proteins significantly contribute to the texture, flavor, and color of fish flesh (Truong et al. 2015). The most important stroma protein is collagen. HPP does not influence the primary and secondary structures of proteins; however, the tertiary and quaternary structures of globular proteins have been long known to undergo reversible and irreversible changes under HP (Knorr, Heinz, and Buckow 2006). The bonds that are affected by low to moderate pressures (50–200 MPa) are the ones that stabilize the native structure by various noncovalent forces such as hydrophobic, electrostatic, and van der Waals interactions. Water plays an important role in protein denaturation when forced by pressure into the interior of the protein matrix and favors protein unfolding and disruption of noncovalent bonds. In addition, a reduction of volume is produced by HP according to Le Chatelier’s principle. The stability of the protein largely depends on its compressibility, the void volume of internal 74 Trends in Fish Processing Technologies cavities, and the flexibility to compensate for disruption of noncovalent bonds due to relocation of water molecules (Buckow, Sikes, and Tume 2013). In contrast, pressures of several hundred megapascals can increase the thermostability of proteins, especially when the thermally denatured state occupies a larger volume than the native state (Knorr, Heinz, and Buckow 2006; Buckow, Sikes, and Tume 2013). Early studies on fish muscle, confirmed by later detailed investigations, have shown that reversible depolymerization of F-actin to G-actin takes place at pressures of 100 to 300 MPa. Moreover, in the presence of adenosine triphosphate (ATP), actomyosin dissociates into actin and myosin under pressure. Further, myosin was shown to denaturate faster compared to sarcoplasmatic proteins and actin (Truong et al. 2015). As demonstrated by researchers (Hsu et al. 2007), HP treatments from 100 to 200 MPa applied to actomyosin solutions from tilapia muscle (0.6 M KCl, pH 7) pre- dispose actomyosin to formation of aggregates mainly with hydrogen bonds, while above 200 MPa aggregates were formed due to disulfide bonds. Other studies indicated that carp muscle lost the striation patterns and arrangement of myofribils when different time–pressure regimes were applied, for example, at 150 MPa after 30 min of treatment (Truong et al. 2015). In cod fish, myosin was denatured between 100 and 200 MPa (20 min, 30°C–50°C), whereas actin and sarcoplasmic proteins were denatured at 300 MPa (Buckow, Sikes, and Tume 2013). The larger spatial structure attributed to myofibrillar proteins and higher molecular weight compared to sarcoplasmatic proteins explain an increased susceptibility to denaturation of the former, whereas stroma proteins represented by the collagen network of fish muscles remains almost unaffected by HP. However, the denaturation process largely depends on fish species and processing parameters (Buckow, Sikes, and Tume 2013).

3.2.2 lipids Fish lipids have a unique content of polyunsaturated fatty acids (PUFAs) of the n-3 family, such as eicosapentaenoic acid (C20:5 n-3, EPA) and docosahexaenoic acid (C22:6 n-3, DHA). Numerous health benefits have been associated with high level of PUFAs in diet, including prevention of atherosclerosis, reduction of blood pressure and protection against arrhythmia, improvement of anti-inflammatory response, and development of the brain and eye retina in infants (García-Moreno et al. 2013). There is evidence that HP affects, to some extent, both lipid hydrolysis and lipid oxidation of fish muscle (Buckow, Sikes, and Tume 2013; Truong et al. 2015). Lipid hydrolysis produces an accumulation of free fatty acids (FFAs) that could result in acceleration of lipid oxidation, muscle texture changes, and off- odor development, finally leading to shelf life reduction (Vázquez et al. 2013). The FFA content increases in coho salmon (Ortea et al. 2010) after pressurization at 100–200 MPa, during 10 days of chilled storage and no inhibition in lipases activities was present. An increase in FFA content was also reported after the treatment at relatively low pressures 150–310 MPa in carp fillets (Sequeira-Munoz et al. 2006), turbot (Chevalier, Le Bail, and Ghoul 2001), and cold smoked sardine (Gomez-Estaca et al. 2007) stored for 15 days at 5°C. FFAs have been reported to be produced during the first stage of the chilling process as a result of endogenous enzyme activity (lipase and phospholipases) (Ortea et al. 2010). Furthermore, it High-Pressure Processing of Seafood 75 was hypothesized that HP could accelerate the release of FFA by affecting the electrostatic interactions and hydrogen bonds between myofibrillar proteins and FFA. Other researches showed that applying HP as treatment prior to freezing and frozen storage, a reduction in FFA formation was obtained at a pressure of 300 or 450 MPa, while at 150 MPa, the inhibitory effect was apparent only after 3 months of storage (Vázquez et al. 2013). Nevertheless, both fatty and lean fish species showed significant lipid hydrolysis during frozen storage, this being considered one of the main deteriorative mechanisms that produce shelf life reduction of frozen products. Lipid oxidation is a major deteriorative process in fatty fish species due to high susceptibility to oxidation of the unsaturated fatty acids in fish muscles. It starts postmortem, once the balance between prooxidants and antioxidants in the living tissue is lost in the favor of the former and the autolytic processes release prooxidants, free radicals, and enzymes. Lipid oxidation produces rancidity, color changes, loss in nutritive value, and changes in textural and functional properties when accompanied by simultaneous protein denaturation (Buckow, Sikes, and Tume 2013). Oxidation of unsaturated fatty acids or triglycerides in seafood takes place via free radical formation. HPP enhances lipid oxidation in fish muscle after the treatment and during subsequent chilled storage of cod, carp, and rainbow trout (Sequeira-Munoz et al. 2006; Truong et al. 2015). However, the intensity of oxidation is influenced not only by the pressure–time–temperature regime applied, the fish species, and the type of muscle (white or dark) but also by the antioxidants/ prooxidants present in the system. For example, a partial inhibition of primary oxidation assessed by peroxide formation was observed in coho salmon (Oncorhynchus kisutch) throughout 2 weeks of chilled storage after treatment at 170 and 200 MPa for 30 s (Ortea et al. 2010) and only minor effects were noticed for frozen mackerel (Scomber scombrus) not only after treatment at 150, 300, and 450 MPa with holding times 0, 2.5, and 5 min, subsequently stored at −10°C for 3 months (Vázquez et al. 2013), but also after 150, 175, and 200 MPa, followed by storage at −18°C (Pazos et al. 2015). Changes could merely be attributed to frozen storage time and not to HP treatment. Other researchers reported an increase in the level of primary and secondary lipid oxidation produced by pressure treatments (Bindu et al. 2013; Sequeira-Munoz et al. 2006). Tintchev et al. (2009) used Raman spectroscopy to study the effect of HPP (up to 600 MPa) on astaxanthin and myoglobin in smoked salmon. When pressure is applied, the protein carotenoid complex is thought to partially dissociate, thus releasing astaxanthin, which is then exposed to radicals or to oxidizing species (Tintchev et al. 2009; Kaur et al. 2013). The release of metal ions from heme proteins such as myoglobin also catalyzes the oxidation of PUFAs, EPA, and DHA. Phospholipids from cell membranes also contribute as key substrate in lipids oxidation. Lipases could also play an important role in oxidation and the enzymes display barrostability as they are only inactivated when pressure is combined with moderate or high temperatures. Despite the work that has been done to explain the influence of HP on fish lipids oxidation, the intimate mechanism remains somewhat unclear. Further researches should better clarify which components could tilt the balance toward acceleration of oxidation or toward slowing down the reaction rate. 76 Trends in Fish Processing Technologies

3.2.2.1 Antioxidants In order to slow down the rate of deterioration produced by lipid oxidation, the addition of antioxidants such as butylated hydroxyanisole and butylated hydroxytoluene is approved by EU Regulations EC 1130/2011 with a maximum level of 20 mg/kg single or in combination (expressed on fat) in the preparation and maximum 0.4 mg/kg in the final product (single or in combination). However, in the recent years, there are countless concerns raised by health and consumer organizations against the use of synthesis antioxidants in food. In response to public concern, several studies demonstrate the effectiveness of essential oils and plant polyphenols as natural additives that could represent an alternative to chemically synthetized ones (Hygreeva and Pandey 2016). Nonetheless, up to now, only a few of them have discussed the combined hurdle effects of these antioxidants next to other preservation principles applied to foods and the maximum acceptable concentration in relation with food sensorial properties. Somacal et al. (2015) studied the inhibition of polyunsaturated fatty acids by the use of a natural colorant, annatto, that contains 80% of the car- tonetoid cis-bixin in its composition. Annatto proved to be a powerful antioxidant and reduced the loss of DHA caused by HP processing of herring, from 12% to 7%. Gomez-Estaca et al. (2007) showed the importance of gelatin-based edible films enriched with rosemary, oregano, or chitosan applied to prevent lipid oxidation in pressure-treated cold-smoked sardines (300 MPa, 20°C, 15 min). The researches on the use of natural antioxidants and HP processing are at an early stage, and much attention should be given to the variability in composition of natural antioxidants and compatibility between antioxidants and food matrix.

3.3 EFFECT OF HPP ON FISH FUNCTIONS

3.3.1 enzymes Pressure effects on enzymes have been investigated for many years (Buckow, Sikes, and Tume 2013), and while early studies paid attention particularly to understanding the effect of HPP on individual enzyme inactivation in crude or purified extracts, recent studies are rather oriented toward investigating the effect of pressure regime (time and pressure level) on different food matrixes, correlating the consequences of simultaneous enzymes’ activation, inactivation, resistance, or recovery on food quality (Table 3.1). The proteic nature of enzymes is susceptible to display reactivity upon pressure treatment via the tertiary and quaternary structures, as earlier discussed in this chapter. Accordingly, the active enzyme sites could become partially or entirely covered when the spatial structure changes, depending on the pressure level, come-up rate, treatment time, decompression time, and temperature, and this could inflict on its functionality. Environmental factors such as pH; ionic strength; presence of catalysts, zymogens, and inhibitors; and substrate concentration could also influence the enzyme stability under pressure. Proteases with neutral optimum pH (~6.6) are highly susceptible to pressure compared to acid and alkaline proteases and lipases, which are known for their relative barostability (Truong et al. 2015). HPP affects the integrity of cellular structures High-Pressure Processing of Seafood 77 ( Continued ) Reference Fidalgo et al. 2015 Fidalgo et al. 2014 Effects samples at 450 MPa with 0-min holding time was 1.14-fold with 0-min holding time was samples at 450 MPa After 3 months, the higher than in the control sample. for than in the control samples, except lower was activity with 0-min holding time. the 300 MPa while after 3 months during storage at −10°C, the activity recovery. ~2-fold higher due to activity was samples decreased with 74.7% during 3 months of storage, a 1.43-fold increase the HPP-treated samples exhibited min/20°C. immediately after the treatment at 450 MPa/5 After 1 month of frozen untreated and HPP samples. decreased in samples treated at storage, the lipase activity (0 and 2.5 min), (0, 2.5, and 5 min), 300 MPa 150 MPa (2.5 and 5 min). 450 MPa Compared to frozen after treatments at 450 MPa. of activity though reduced, even was untreated control, the HPP effect statistically significant, particularly at 450 MPa. After 1 month, acid phosphatase activity of pressurized After 1 month, acid phosphatase activity min/20°C, by ~38% at 450 MPa/5 Enzyme is inactivated during storage in untreated control Although the activity between were initially observed No significant differences significant decreases minor but HPP-treated samples showed Enzymes Acid phosphatase Cathepsins B Cathepsin D Lipase Acid phosphatase Conditions Processing and Storage and Storage Processing at 20°C for 0, 2.5, and 5 min, come-up rate 3 MPa/ at −10°C and 5 min, come-up rate decompression time 3 MPa/s, <3 s, up to 3 months of storage at −10°C 150, 300, and 450 MPa; 150, 300, and 450 MPa; up to 3 months of storage for 0, 2.5, 150, 300, 450 MPa; Trachurus ( Trachurus mackerel ) trachurus ( Scomber scombrus ) Atlantic horse Atlantic mackerel TABLE 3.1 Examples StudiesRecent HHP on the of Activity Enzymes of Seafood in Seafood 78 Trends in Fish Processing Technologies ( Continued ) Reference Pazos et al. 2015 Pazos Effects storage, the activity decreased with the HPP intensity. After decreased with the HPP intensity. storage, the activity for 2.5 min produced 1 month, the treatment at 450 MPa After the strongest reduction of enzymatic activity. was 3 months of storage, almost all the initial activity recovered. the At 450 MPa, and 5 min increased the enzyme activity. was An increase in the activity reduced. was activity higher than with end-values noticed for 300 and 450 MPa 3 months of storage. initial control over After during storage. partially recovered was activity higher than initial control were registered 3 months, values 5 min. for the samples treated at 150 MPa, and the HP-treated samples had, at produced by HPP, than enzymatic activities end of storage at −18°C, lower control. Immediately after HP treatment and 1 month of frozen and holding times between 0 Pressure of 150 and 300 MPa Lipase HP treatment produced a reduction of lipase activity. was on acid phosphatase activity An inhibitory effect Enzymes Cathepsin B Cathepsin D Lipase Acid phosphatase Conditions Processing and Storage and Storage Processing at 20°C, 0 min holding time come-up rate 3 MPa/s, decompression time <3 s, frozen immediately after pressurization and stored 9 months at −18°C 125, 150, 175, and 200 MPa; 125, 150, 175, and 200 MPa; ( Scomber scombrus ) Atlantic mackerel Atlantic mackerel TABLE 3.1 (CONTINUED) 3.1 TABLE Examples StudiesRecent HHP on the of Activity Enzymes of Seafood in Seafood High-Pressure Processing of Seafood 79 ( Continued ) Reference 2013 Qiu, Xia, and Jiang Chéret et al. 2007 at 200 MPa; k = at 200 MPa; −1 = −10.43 ± 1.39 mL/ a Effects at 500 MPa; V at 500 MPa; −1 and 175 MPa HPP, inversely correlated with the pressure inversely HPP, and 175 MPa after the observed no inhibition was increase, but level effect During storage, the recovery treatment at 200 MPa. in all samples. obvious was at the end of storage however, higher at 125 MPa; was enzymatic control had lower time, all samples except compared to control. activities a first-order followed Inactivation inactivated. enzyme was kinetic model (k = 0.009 ± 0.16 min led to fish texture of enzymatic activity mol). Inactivation and quality improvement. in muscle compared to went faster Inactivation 300 MPa. extract. purified the control and overrun samples treated at 100 MPa the calpain activity day, samples. Further up to the seventh decreased. 0.0704 ± 1.69 min After freezing, an inhibitory effect was noticed for 125, 150, was After freezing, an inhibitory effect immediately after HP treatment The cathepsin D activity and longer treatments than 10 min, the 300 MPa Above after the HPP treatment at inactivated was Calpain activity increased in the second day of storage Calpain activity Enzymes myofibril-bound serine proteinases Cathepsin B Cathepsin D Extracts of Calpain Conditions Processing and Storage and Storage Processing 0–30 min, come-up rate decompression time 6 MPa/s, <5 s, stored in ice bath (4°C) 5 min, come-up rate decompression rate 3 MPa/s, stored at 4°C up 250 MPa/s, to 7 days 200–500 MPa; at 20°C, for 200–500 MPa; at 10°C, for 100–300 MPa; Hypophthalmichthys ( Hypophthalmichthys molitrix ) ( Dicentrarchus L.) labrax Silver carp Silver Sea bass TABLE 3.1 (CONTINUED) 3.1 TABLE Examples StudiesRecent HHP on the of Activity Enzymes of Seafood in Seafood 80 Trends in Fish Processing Technologies ( Continued ) Reference Teixeira et al. 2013 Teixeira Effects the activity decreased during storage time in all HPP- the activity treated samples and in control. treatment for from that after 100 and 250 MPa different 0–15 min. Pressure holding time and pressure rate did not AU/min/g of 3.7 A minimum activity the activity. affect and 15 min. after 400 MPa registered was for 5–15 min. treatment and 400 MPa of pressure holding time with a contribution values activity only at 400 MPa. treatments. with holding time and pressure level were inactivated they 13 times at 250 (15–30 min) and 450 (5–30) up to values than control. lower Although calpastatin activity seemed unaffected by pressure, seemed unaffected Although calpastatin activity not significantly in the non-treated samples was The activity and 30 min after 250 MPa were observed The highest values lower showed and 400 MPa The treatments at 100 MPa by HPP not significantly affected was Lipase activity however, by HPP at 100 MPa; Calpains were not affected Enzymes muscle and in extract) Calpastatin (in Acid phosphatase Cathepsin B Cathepsin D Lipases Calpains Conditions Processing and Storage and Storage Processing 5, 15, and 30 min, come-up rates 8 and 14 MPa/s 100, 250, and 400 MPa; for 0, 100, 250, and 400 MPa; TABLE 3.1 (CONTINUED) 3.1 TABLE Examples StudiesRecent HHP on the of Activity Enzymes of Seafood in Seafood High-Pressure Processing of Seafood 81 Reference Patterson, and Patterson, Piggott 2005 Lakshmanan, Effects unchanged and decreased only after 300 MPa treatment. unchanged and decreased only after 300 MPa During 18 days of storage at 4°C–5°C, the activity decreased in all samples. inactivated. were largely they 300 MPa, during than cathepsins. Calpains restored their activity storage at 4°C–5°C, after 12 days. Up to 200 MPa, the activity of enzyme remained mostly the activity Up to 200 MPa, by pressure increase, and at Cathepsin B+L were inactivated pressures by lower to a greater extent Calpains were affected Enzymes Cathepsin B Cathepsin B+L Calpains Conditions Processing and Storage and Storage Processing at 9°C, 20 min holding time, come-up rate 3.33 MPa/s, decompression time 2 min 100, 150, 200, and 300 MPa 100, 150, 200, and 300 MPa salmon—pressurized as crude extracts Cold smoked Cold smoked TABLE 3.1 (CONTINUED) 3.1 TABLE Examples StudiesRecent HHP on the of Activity Enzymes of Seafood in Seafood 82 Trends in Fish Processing Technologies such as lysosomes and sarcoplasmatic reticulum and favors the release in cytosol of proteolytic enzymatic systems, such as calpains and cathepsins. The proteolytic activity of proteases affects the myofibrillar proteins and fish texture. Extensive research has been conducted on cathepsin and calpain inactivation by HPP (Table 3.1). These enzymes are likely to be released from their lysosomes after HPP at 100 MPa. Lakshmanan, Patterson, and Piggott (2005) observed that treatment at low pressures between 100 and 300 MPa, at 9°C, after 20 min induced proteolytic activity in fish muscle and that cathepsin B and L activity decreases with the increase in pressure level. However, a reactivation of cathepsin B and L activities was noticed after refrigerated storage. Teixeira et al. (2014) observed that HP determined significant changes in the activities of several enzymes of sea bass muscle. The increases in pressure level and holding time decrease the protein concentration in sarcoplasmatic extracts and the activity of calpains. A maximum activity reduction was obtained at 400 MPa for acid phosphatase, cathepsin D, and calpain. In sea bass treated at 100 MPa, for 5 min, at 10°C, calpains start to lose enzymatic activity due to dissociation of the heterodimeric form, while above 350 MPa in the same conditions, enzymes are completely inactivated. In muscle, the activity of calpain decreases with an increase in HP level (Chéret et al. 2007). Different inactivation profiles of the same class and, sometimes, type of enzyme are observed depending on the HPP parameters, fish species, packaging system, and further storing conditions (refrigerated/frozen, duration, and storage temperature) (Table 3.1) that make very valuable the enzymatic studies conducted for different fish species.

3.4 EFFECT OF HPP ON FISH PROPERTIES

3.4.1 texture Texture represents one of the main features used when one evaluates fish freshness quality. Textural attributes such as hardness, springiness, cohesiveness, gumminess, adhesiveness, and chewiness are employed to assess the perceived fish quality along the fish chain (Cheng et al. 2014; Truong et al. 2015). The biochemical transformations that involve disruption of key myofibrillar proteins, regulated by enzymes, start postmortem and render fish, at the end of the process, a soft texture. This is considered one of the most common texture defects in fresh fish muscle. Many HP-treated fish products displayed a harder texture or higher shear strengths than controls did, for example, blue fish (Ashie, Simpson, and Ramaswamy 1997) treated at 100 MPa for 30 min; cod processed at 200–400 MPa, at 0°C, for 10 min (after cooking) (Matser et al. 2000); vacuum-packed prawn treated at 200 and 400 MPa, and 7°C, for 10 min (Lopez-Caballero et al. 2000); salmon treated at 150 and 200 MPa, at 1°C–5°C for 30 and 60 min (Amanatidou et al. 2000); and black shrimp tiger packed in ethylene vinyl alcohol film treated at 100, 270, and 435 MPa, for 5 min, at 25°C (Kaur et al. 2013). The effect of HP on hardness could be explained by myofibrillar protein denaturation and aggregation (Yagiz et al. 2009). Another possible explanation for the increase in hardness was related to the unfolding of actin and sarcoplasmatic proteins and formations of new hydrogen-bonded networks High-Pressure Processing of Seafood 83

(Truong et al. 2015). It has been suggested that hardening following HP treatment may not be undesirable. In addition, changes in texture of HP-treated fish were reversed by subsequent cooking. The newest methods used to assess fish texture are the nondestructive ones based on image analysis, digital imaging, and spectroscopy techniques for estimation of fish fillet firmness (Quevedo and Aguilera 2010; Cheng et al. 2014). These methods should be employed for better understanding of the textural changes in HP-treated fish. Texture changes of HPP oysters are favorable in the sense that HPP oysters are slightly juicier than the untreated ones (Lopez-Caballero et al. 2000) probably due to an increased moisture content (Murchie et al. 2005). An additional very well-known advantage is related to the increased ability to detach the adductor muscle from shell in shucking the oysters (Yi et al. 2013).

HP treatment applied in modified atmosphere with 50% CO2 and 50% O2 gave an increased softness compared to controls that could be attributed to the increase in solubility of gases in the salmon tissue during prolonged storage (Amanatidou et al. 2000).

3.4.2 color As blunt as human color vision may be compared to fish and amphibians, the subjective visual perception of electromagnetic radiation by an observer is a major deciding factor in buying food. Seafood color is quantified mainly by tristimulus-based Commission Internationale de l’Eclairage system using L* (lightness), a* (redness), and b* (yellowness) parameters. HP impacts fish color, with cooked appearance of fish muscle being one of the most noticed detrimental changes (Truong et al. 2015). Bindu et al. (2013) observed that vacuum-packed Indian white prawn HP treated from 100 to 600 MPa for 5 min at 25°C had significant differences in color compared to controls. The lightness values and yellowness increase with pressure level, while the redness decreases. The increase in L* values was related with denaturation of myofibrillar and sacroplasmatic proteins, while the decrease in a* values was explained by globin denaturation and heme displacement. For almost a fortnight, the color values of stored samples increased and these changes were supposedly related with lipid oxidation of carotenoid pigment, astaxantin. The same behavior was reported by Chéret et al. (2005) in sea bass treated from 100 to 500 MPa (10°C, 5 min) that displayed a white translucent appearance. Chéret et al. (2005) rather attributed these changes to protein denaturation than to changes in pigments, taking into account sea bass muscle white color. Changes in color parameters were reported for pollack, mackerel, tuna, cod, salmon, trout, carp, plaice, and anglerfish treated for 5 min at pressures higher than 150 MPa (Matser et al. 2000). All these changes were explained by protein denaturation between 150 and 200 MPa. The changes in a* and b* values differed as a function of processing conditions and fish species. In carp, L* values increase at 140 MPa and above; however, the a* values also increase with pressure from 100 to 180 MPa and treatment time from 15 to 30 min, and this behavior was related with coagulation of sarcoplasmatic and myofibrillar proteins (Sequeira-Munoz et al. 2006). The same explanation was provided 84 Trends in Fish Processing Technologies by Montiel et al. (2012b) for cold-smoked cod treated from 400 to 600 MPa, 5–10 min, and stored at 5°C for 60 days. This study showed that L* values significantly increase with pressure level applied and with storage time in the first 15 days of storage, causing a brighter and less transparent appearance of muscle. The a* values were reduced by pressurization, while the b* values showed an increase during pressurization; however, no significant differences between control and HP processed samples were noticed after the treatment. The authors concluded that color changes produced by HPP do not induce consumer rejection (Montiel et al. 2012a). In vacuum-packed cold-smoked mahi-mahi, the a* values of fillets increase with pressure level from 200 to 400 MPa, and so did the L* and b* values compared to controls, while in fish packed in slices, there was no significant change of the redness with pressure level during the first 14 days of storage at 5°C (Gomez-Estaca et al. 2007). Red color is very important for consumer acceptability of salmonid muscle and tuna, and its intensity was diminished by HP treatment (Truong et al. 2015). The a* values that indicate red color intensity of salmon declined with the increase in pressure level and treatment duration; however, the unacceptable effect (a* < 13) present in the samples treated above 150 MPa for 10 min was not present in the samples packed in modified atmosphere (50% 2O + 50% CO2). After 14 days of storage at 5°C, all samples packed in modified atmosphere and pressure treated had a* values below threshold, while vacuum-packed samples were at an acceptable level. The changes in redness were attributed to pressure induced denaturation of myofibril and astaxanthin-actinin matrix followed by oxidation of astaxanthin and ferrous myoglobin (Buckow, Sikes, and Tume 2013; Truong et al. 2015). Amanatidou et al. (2000) showed that acceptability changes in fresh salmon color produced by HPP depends not only on pressure level but also on duration and that 60 min treatment at 150 and 200 MPa made fish muscle opaque and unacceptable, with L* values higher than 70. A recent study drew attention to the advantages of hurdle approach by combining HP and the use of a gelatin-lignin film (Ojagh et al. 2011) in order to overcome the negative effects associated with the thermal treatment in ready-to-eat or semi- prepared salmon muscle. The authors highlighted that it is preferable to apply the HP treatment at refrigeration temperatures (300 MPa for 10 min at 5°C), and it is possible to preserve redness coloration and prevent protein denaturation and oxidation without compromising the appearance by covering the fish fillets prior to HP treatments with a gelatin-lignin film.

3.4.3 aroma and Taste In general, HPP is expected to produce a better sensory quality for pressurized fish muscle compared to untreated samples after chilled or frozen storage (Truong et al. 2015). Much of this improved sensory quality is related to the inactivation of enzymes that produce fish alteration. In a study, cold-smoked cod was pressurized and stored; however, no significant differences in appearance, quality, and intensity of the smoky odor were present in HP-treated samples throughout the refrigerated storage (Montiel et al. 2012b). Cold- smoked mahi-mahi was sensorial assessed after HP treatment at 200–400 MPa by High-Pressure Processing of Seafood 85 researchers (Gomez-Estaca et al. 2007) for overall appearance, odor, taste, juiciness, and general acceptability. The results obtained showed that only the HPP-treated samples at 300 MPa achieved 100% general acceptability. Nonetheless, highly favorable scores were awarded by assessors for all the HP-treated samples, while unpressurized smoked fish had lower scores for acceptability, rated as neutral. The effect of the HP treatment (150, 300, and 450 MPa up to 5 min at room temperature) on sensory properties of Atlantic mackerel before freezing was evaluated by Aubourg et al. (2013). Researchers showed that the hardness and chewiness values of HP-treated samples and control samples were similar. However, the most important finding was that the sensorial acceptability of oven-cooked HP-treated samples was better than of controls and similar to that of fresh mackerel, confirming that HPP has a minimal impact on product taste. The taste and flavor of seafood is influenced by the presence of both volatile and nonvolatile components. While volatile components are composed of aromatic hydrocarbons, aliphatic hydrocarbons, esters, aldehydes, ketones, alcohols, and sulphur compounds, the nonvolatiles are mainly represented by nitrogenous compounds such as free amino acids, nucleotides, organic bases, and related compounds (Yue et al. 2016). The presence of undesirable flavors is more closely linked with the presence of lipid oxidation compounds and only slightly with degradation compounds from proteins and carbohydrates (Cruz-Romero, Kelly, and Kerry 2008). The impact of HPP at 260, 500, and 800 MPa applied for 3 to 5 min at 20°C on volatile profile of oysters was investigated (Cruz-Romero, Kelly, and Kerry 2008), and many changes induced by HPP were reported particularly in the profile of sul- fur compounds. Compounds were tentatively identified by proton transfer mass spectrometry. An increase in concentration of dimethyl sulphide, 1-penten-3-one, phenol and 1,2,4-trimethylbenzene compounds, compared to controls, was reported in the HP-treated samples. Squid-specific umami taste is correlated with the presence of nonvolatiles such as free amino acids and nucleotides like 5ʹ-inosine monophosphate, 5ʹ-guanosine monophosphate, and 5ʹ-adenosine monophosphate (Yue et al. 2016). The study of the changes in squid flavor after HP treatment at 200, 400, and 600 MPa for 10 min at 20°C revealed an increase in the most prominent flavor compound (5ʹ-adenosine monophosphate) and an increase in the component responsible for meaty flavor (5ʹ-guanosine monophosphate) after the treatment at 400 and 600 MPa. The increase in flavor contributing ʹ5 -nucleotides was correlated with enzyme inactivation. The free amino acid profile was not significantly altered by the HP treatment; however, significant changes were reported after 10 days of storage. The volatile fingerprint of HP-treated squid showed that concentrations in volatile compounds were higher compared to controls immediately after the treatment; however, differences were attenuated over time (10 days of storage).

3.5 EFFECT OF HPP ON FISH SAFETY

3.5.1 effect of HPP on Microorganisms Bacteriological activity is mainly culpable for fish deterioration occurrence via postcatch slaughtering contamination of fish flesh from skin, gills, gastrointestinal 86 Trends in Fish Processing Technologies tract, or environment, finally leading to fish spoilage. Fish muscle is a media where bacteria could thrive provided the rich protein substrate, pH, endogenous enzymes presence and water activity. Psychrotrophic bacteria commonly responsible for fish flesh alteration belong to the genera Pseudomonas, Moraxella, Acinetobacter, Shewanella, Aeromonas, Flavobacterium, and Vibrionaceae but others such as Bacillus, Micrococcus, Clostridium, Lactobacillus, and Corynebacterium could also contribute to the deleterious processes (Karim et al. 2011a). HP induces many changes in bacteria cells, where cell membranes are thought to be a primary target of pressure (Murchie et al. 2005). HPP effectively reduces the microbial load in fish muscle by producing membrane protein denaturation and inhibition of amino acid uptake, thus providing long shelf life and minimal loss in quality (Wang et al. 2015). Protein denaturation is considered a key target of HP treatments, which may include conformational changes of ribosomes and enzyme inactivation. Interestingly, heat shock proteins including chaperones (DnaK, GrpE, GroES, and GroEL) and proteases involved in the degradation of denatured proteins (ClpB, ClpP, and Lon) are synthesized in cells during exposure to sublethal pressures and in cells recovering from a pressure treatment. These proteins may refold or degrade damaged proteins enhancing the HP resistance of bacterial cells (Gayán, Torres, and Paredes-Sabja 2012). The most sensitive spoilage bacteria to HP are the ubiquitous microorganisms present in fish muscle chilled after 1–2 weeks of storage, namely,Pseudomonas and Shewanella (Truong et al. 2015). HP treatment at 100 MPa and 5°C for 30 min effectively inactivates these bacteria (Amanatidou et al. 2000). It has been reported that, in general, gram-negative bacteria possess higher susceptibility to HP compared to gram-positive bacteria due to the complexity of gram-negative cell membranes, with a notable exception—Escherichia coli O157 (Murchie et al. 2005). Karim et al. (2011b) showed that HPP did not influence the gram-positive cocci (Micrococcus spp. and Staphylococcus spp.) and spore-forming rods (Clostridium spp. and Bacillus spp.) as part of haddock microflora and that microflora did not change significantly during storage of fish in ice at 2°C for 10 days. Bacterial spores are highly resistant to inactivation by HPP; for example, spores of Clostridium botulinum strains can survive extreme treatment conditions (827 MPa for 30 min at 75°C). However, the use of oscillatory HP treatments, where a lower HP favors spores to germinate, allowing their inactivation by a subsequent cycle at a higher HP, has proved successful (Murchie et al. 2005). Inactivation of spores is based on a series of events such as the release of dipicolinic acid (chelate of Ca2+ and pyridine-2,6-dipicolinic acid or Ca-DPA), small acid-soluble spore proteins (SASPs), the hydrolysis of core and cortex, and the decrease of intracellular pH. Spores are sensitized by moderately HP (50–300 MPa), and then it has been postulated that nutrient germinant receptors provoke the release of Ca-DPA. Ca-DPA triggers a cascade of later germination events (hydrolysis of spore cortex by cortex-lytic enzymes, degradation of SASPs, and ATP generation). The resultant germinated spores are sensitive to subsequent heat and pressure treatments and HPs above 500 MPa can rapidly inactivate germinated spores (Ahn and Balasubramaniam 2007; Rendueles et al. 2011). High-Pressure Processing of Seafood 87

Another widespread pathogen that can cause a severe illness is Listeria monocytogenes, which is partially inactivated by HPP up to 250 MPa for 20 min at 9°C in smoked salmon (Lakshmanan and Dalgaard 2004), while in mackerel slurry, L. monocytogenes proved to be more sensitive than E. coli O157:H7 (Ramaswamy, Zaman, and Smith 2008). Listeria innocua was completely inactivated in cold- smoked salmon after 10 s between 700 and 900 MPa (Gudbjornsdottir et al. 2010). An interesting recent study has shown the potential of combining HPP and mild smoking for inactivation of L. monocytogenes and E. coli with more than 6-logs in smoked rainbow trout fillets. After a while, a part of L. monocytogenes cells were reactivated, while E. coli was completely inactivated and no reactivation was possible (Mengden et al. 2015). Another recent study (Du et al. 2016) used as a pressure transmitting medium an acidic electrolyzed water and obtained a synergistic effect of HP and this new pressurizing medium on L. monocytogenes and V. parahaemolyticus inactivation, obtaining log reductions up to 5.71 and 6.08 log10 CFU/g compared to a reduction at 400 MPa after 10 min of 4.31 and 4.74 log10 CFU/g, respectively. Inactivation of Vibrio species associated with raw oysters is also ensured by a combination of HPs (200–250 MPa) and mild temperatures (40°C–45°C) that both reduced V. parahaemolyticus and V. vulnificus to nondetectable levels (Long et al. 2015). Relatively low pressure levels (200–300 MPa) and short time (5–10 min) combinations were effective in inactivation of Anisakis larvae from raw fish dishes (sashimi, carpaccio, cold smoked), marinated fish, and also in hake and finfish or mackerel (Truong et al. 2015). Fish spoilage as a result of microbial activity is often chemically assessed by the presence of off-odors, particularly nonprotein nitrogen compounds, of which the volatile bases ammonia and trimethylamine (TMA) and total volatile based nitrogen (TVB-N) are equally important. In seafood, value of TVB-N below 35 mg/100 g and TMA-N below 15 mg/100 g indicates that fish is outside the spoilage zone (Kaur et al. 2013). In a recent study (Rode and Hovda 2016) on the effect of HPP (200 and 500 MPa, 120 s) on total aerobic bacteria from salmon, cod, and mackerel stored refrigerated at 0.5°C, it was observed that HPP at 500 MPa for cod and mackerel restrained the bacterial flora that did not reach spoilage level after 26 days of storage, opposite to the salmon samples exposed to 500 MPa, which presented spoilage signs after 14 days of refrigerated storage (Murchie et al. 2005). Other recent results discussing HPP influence on refrigerated and frozen seafood shelf life are presented in Table 3.2. Another chemical indicator for fish spoilage is K-value, defined as the ratio of the sum of inosine and hypoxanthine to total concentration of other nucleotides. K-values take into account not only microbial spoilage but also changes in fish tissues that result from autolytic reactions controlled by native enzymes, such as ATP breakdown (Kamalakanth et al. 2011). In yellowfin tuna, K-values decrease with pressure level, indicating an inactivation of Enterobacteriaceae. Predictive microbiology tools, more specifically the reparametrized Gompertz model, were applied by Briones et al. (2010) to assess the microbial growth in salmon and abalone. It was demonstrated that HP treatment at 170–200 MPa for 30 s followed by cold storage increases shelf life to more than 65 days for abalone compared to controls (30 days); 88 Trends in Fish Processing Technologies ( Continued ) Reference 2007 2007 Karim et al. 2011a et al. 2015 Pazos Gomez-Estaca et al. Gomez-Estaca et al. Results pressure treated at 200 MPa/3 min pressure treated at 200 MPa/3 of storage for all the samples (control and pressure The treated) from 0.02 to 0.52 mg N/100 g muscle. TMA on the HP treatment had no significant effect of frozen mackerel. values 15 to 25 mg/100 g after 2 months, while control sample rose from 15 to 28 mg/100 g in the same all samples did not period of storage. However, TVB-N/100 g value. the limit of 35 mg exceed TVB-N The samples in the 21 days of storage at 5°C. than 18 mg/100 g muscle. Only the were lower values TVB-N of 25 mg/100 g in the control sample had a last day of storage. Shelf life extension from 4 to 13 days for the samples Shelf life extension TMA after 6 days of storage TVB-N and Unacceptable slightly increased during 9 months TMA values storage from TVB-N of HP treated samples rose over between the no significant difference There was HP and further stored in ice at 2°C up to 14 days decompression time come-up rate 3 MPa/s, <3 s, frozen immediately after pressurization and stored 9 months at −18°C at 5°C up to 75 days decompression come-up rate 2.5 MPa/s, in nonvacuum time ~2 s, stored packed and conditions: (i) HP treated, (ii) smoked coated with oregano and (iii) smoked, HPP, and HP treated extract 200, 250, 300, MPa at 10°C for 1 and 3 min 200, 250, 300, MPa at 20°C, 0 min holding time, 125–200 MPa at 20°C, 15 min holding time 300 MPa was stored fish cold-smoked Vacuum-packed at 20°C, 15 min holding time, 300 MPa Samples were stored at 5°C for 21 days ) stored on vacuum ) stored on vacuum harengus ice at 2°C packed fillets stored on aeglefinus) ice at 2°C packed vacuum scombru s) frozen after pressurization and stored 9 months at −18°C ( Coryphaena hippurus ) TABLE 3.2 Prolong Seafood to Shelf-Life Examples Treatment Recent HPP of Fish Herring Culpea fillets ( Haddock ( Melanogrammus ( Scomber Atlantic mackerel dolphinfish Cold-smoked ) pilchardus Sardine ( Sardina High-Pressure Processing of Seafood 89 ( Continued ) Reference and Kołodziejska and Kołodziejska 2016 ń czyk Malinowska-Pa Bindu et al. 2013 Results day of storage and at 435 MPa and day of storage and at 435 MPa

accumulated faster in the unpressurized samples accumulated faster stored at −5°C compared with pressurized one and values DMA and FA the ones stored at −20°C. Lower were obtained for HPP samples stored at −5°C compared to control. The the same as for unpressurized samples. was 10 mg N/100 g after TMA did not exceed content of 1 week independently of the storage temperature. of pressurized TMA values After 2 weeks, the than samples at 2°C and 4°C were much lower the threshold values. above control but noticed after HP treatment. In control, 100 MPa the TVB exceed treated samples, and 435 MPa threshold limit after 15, 20, and 25 days of storage, the limit on TMA exceeded In control, respectively. the 15th day of storage, in HPP samples at 100 MPa on the 20th 600 MPa samples after 30 days of storage. 600 MPa Dimethylamine (DMA) and formaldehyde (FA) were (FA) (DMA) and formaldehyde Dimethylamine TMA immediately after pasteurization of The level was TVB-N TMA and A significant reduction of HP holding time, come-up rate 10 MPa/s, and holding time, come-up rate 10 MPa/s, further stored in ice at 2°C up to 30 days 193 MPa, −20°C, 50 min 193 MPa, Samples were stored at 4°C, −5°C and −20°C at 25°C, 5 min 100, 270, 435, and 600 MPa vacuum packed fish packed vacuum indicus ) stored ( Fenneropenaeus on ice vacuum-packed, TABLE 3.2 (CONTINUED) TABLE Prolong Seafood to Shelf-Life Examples Treatment Recent HPP of Fish Cod ( Gadus morhua ) minced Indian white prawn 90 Trends in Fish Processing Technologies Reference Kaur et al. 2013 Results TMA levels after processing; however, values values after processing; however, TMA levels was The threshold value increased during storage. after 10 days of storage in control sample, overpassed while for HPP treated samples at 100 and 270 MPa after 15 days and for the ones exceeded the limit was after 20 days of storage. treated at 435 MPa were where the threshold values for 435 MPa, except higher than permitted only in the 35th day of storage. No significant changes were reported in TVB and No significant changes were reported in TVB values for observed was The same behavior HP holding time, come-up rate 1.67 MPa/s holding time, come-up rate 1.67 MPa/s stored at 2°C up to 35 days 100, 270, and 435 MPa at 25°C, 5 min 100, 270, and 435 MPa mondon ) TABLE 3.2 (CONTINUED) TABLE Prolong Seafood to Shelf-Life Examples Treatment Recent HPP of Fish Black tiger shrimp ( Penaeus High-Pressure Processing of Seafood 91 however, it did not prolong the salmon shelf life but improved its quality during chilled storage. Researchers (Briones-Labarca et al. 2012) emphasized the need to use more tools of predictive modeling in fish spoilage assessment.

3.5.2 biogenic Amines BAs are usually associated with microbial contamination and considered an indicator of food spoilage. BAs are widely distributed in proteinaceous foods and formed in large amounts at the end of proteolysis throughout metabolic processes by decar- boxylation of amino acids. Depending on the type of evaluation, BAs can serve as markers of decomposition processes in fish or can be monitored for their intrinsic toxicity. The complex relationship between microbial loads of food and presence of decarboxylases after the HP treatment correlated with storage temperature can be followed by measuring concentration of BA markers. The evolution of eight BAs, putresceine, cadaverine, spermindine, spermine, histamine, tyramine, tryptamine, and phenyethylamine, was monitored in vacuum-packed trout, HP treated at 300 and 500 MPa, for 10 min, at 20°C and stored at 3.5 and 12°C up to 28 days (Matějková et al. 2013) and in vacuum-packed pike treated in the same conditions as trout and stored up to 70 days (Křížek et al. 2014). The effect of pressurization on the stability of samples was evident both at 300 and 500 MPa for both types of fish. Trout samples treated at 300 MPa did not show signs of decay up to the 28th day of storage, when putresceine level reached a concentration of 16 mg/kg, indicating onset decomposition. A minimal increase in putresceine and cadaverine levels was noticed in the trout samples treated at 500 MPa, regardless of the storage temperature. The first signs of decay in trout samples treated at 500 MPa and kept at 3.5°C were apparent in the 42nd day of storage when putresceine level was 7.4 mg/kg, tyramine 4.9 mg/ kg, spermidine 4.0 mg/kg, spermine 12.7 mg/kg, and the other BAs were below the detection level. Researchers noted that the content of BAs in vacuum-packed trout was reduced as a consequence of HP treatment. Moreover, concentrations of the most toxicologically important amines histamine and tyramine, responsible for allergic-like reactions, did not exceed values of 2 and 13 mg/kg, respectively. Thus, the authors recommended a storage limit for vacuum-packed trout of 5–6 days at 3.5°C with an extension up to 21–28 days for HP-treated samples. Nonetheless, the HP treatment for the samples kept at 12°C was less effective (Matějková et al. 2013). When the same HP treatment was applied to pike by Křížek et al. (2014) during storage at 3.5°C, an increase in cadaverine content was noticed after the seventh day compared to first day and a higher concentration was detected at 12°C. Cadaverine concentration steadily increased throughout storage; however, HP processed samples had lower concentration than controls did. Tyramine formation at 3.5°C in pike samples was suppressed by high pressurization levels; nonetheless, an increase in time was noticed for this BA also. Compared to trout, the results are similar, but histamine concentration was even lower in pike. HP processing could also provide producers with extended shelf life, four times longer when samples of vacuum-pike are treated at 300 MPa and eight times longer at 500 MPa (Křížek et al. 2014). Low concentrations of tryptamine and spermine were identified in HP-treated cold-smoked cod (400 MPa, 10 min), with the highest concentration of 42.92 and 92 Trends in Fish Processing Technologies

2.62 mg/kg, respectively. During storage, the values registered a small variation; however, at the end of refrigeration period in the 60th day of storage, slightly higher values were registered for tryptamine and spermine compared to the beginning of storage in pressurized cold-smoked cod (Montiel et al. 2012b). In general, HPP is considered to actively suppress production of BAs in fish muscle.

3.5.3 allergens Seafood allergy is considered one of the most common food allergies among adults, with a prevalence of shellfish allergies estimated at 0.5%–2.5% of the general adult population, depending on the degree of consumption, by age and geographic region (Woo and Bahna 2011). The allergic reaction is an immune response triggered after allergen ingestion, when immunoglobulin E (IgE) antibodies bind to a specific allergen epitope. The specific epitope can be either linear, having a chain of amino acids along food allergen, or conformational, with a three-dimensional (3-D) folding of the food allergen (Huang et al. 2014). Shellfish allergies are five times more frequent than fish allergies. The major allergen in fish is parvalbumin with 12 kDa molecular weight, while tropomyosin with 36–39 kDa molecular weight is the major allergen in shellfish. HP processing has the capacity to denaturate the tertiary and quaternary structures of proteins where noncovalent interactions between amino acid chains are present. The tertiary structure is the key to allergenicity of proteins, so lately, researchers have explored the potential of HP in reducing food allergenicity (Liu et al. 2012). HP treatment at 300 MPa, 20°C, for 60 min, applied to silver carp did not change the subunit composition or molecular weight (Liu et al. 2012). However, the structure of the allergens was changed above 300 MPa for more than 10 min. In largemouth bass treated from 100 to 400 MPa up to 15 min at 20°C, no changes in allergenicity were noticed at 400 MPa after 15 min of treatment compared to control despite the structural changes, modification of subunits, and molecular weight that were reported (Liu et al. 2012). A very interesting insight of pressure– temperature stability of cod parvalbumin (Somkuti et al. 2012) has demonstrated that an important role in protein denaturation is played by Ca2+ binding to aspartic and glutamic acid residues and that although a pressure level of 500 MPa leads to partially unfolded state at 27°C, the complete unfolding was reached at 890 MPa and 50°C. In fact, neither heat treatment nor combined HP and heat treatment succeeded in reducing the allergenicity of parvalbumin, although irreversible changes occurred presumably due to the capacity of the protein to refold (Somkuti et al. 2012). Better results were obtained for tropomyosin from squid (Jin et al. 2015) treated with HP from 200 to 600 MPa for 20 min at 20°C. Based on in vitro digestibility studies, the authors stated that treatment above 400 MPa improve digestibility and has the potential of alleviating allergenicity of squid. Results of combined HP and thermal treatments on the allergenic potential of shrimp (Long et al. 2015) have shown a reduction with almost 74% of the IgE binding for the samples treated at 55°C and 500 MPa for 10 min compared to boiling. Moreover, tropomyosin treated High-Pressure Processing of Seafood 93 with HP and combined HP and heat was accompanied by reduced histamine levels in serum when proteins were administered to mice. A Chinese patent applies to shrimp meat immersed in 1% papain saline solution and pressurized at 450 MPa, 40°C, for 55 min that lowered the allergenicity of food by papain diffusion into the internal structure of shrimp and succeeded in hydrolyz- ing the allergen protein (Huang et al. 2014).

3.6 GELATION Pressure has the capacity to induce fish protein gelation, a phenomenon observed after pressure release when refolding of proteins establishes new intermolecular interactions that lead to aggregation into a 3-D gel network. Myofibrillar and sacroplasmatic proteins take part in aggregation in HP-treated fish samples as opposed to thermal processes where sarcoplasmatic proteins are washed out (Truong et al. 2015). The main pressure treatment changes responsible for protein gelation include disruption of hydrophobic bonds producing depolymerization of actin and actomyosin, unfolding and solubilization of myofibrillar proteins. Gel formation at temperatures lower than 40°C is catalyzed by an endogenous calcium-dependent transglutaminase (TGase), an enzyme unhindered by pressures between 100 and 300 MPa, that remains active enough to continue working during “setting” phase (Montero et al. 2005). TGase catalyzes a covalent bond formation between the Ɛ-amino group of lysyl residues and the γ-carboxyamide group of glutaminyl residues between adjacent proteins (Uresti et al. 2006). Pressure-induced fish gels can have a better quality, with more elastic and smoother texture compared with heat-induced gelation (Truong et al. 2015), that has many applications in fish texturization. In fact, in recent years, the scientific community has carried on many research studies referring to gelling of fish muscle or surimi under HP after kneading with salt to induce protein solubilization followed by heat-induced gelation (Hsu and Ko 2001; Pérez-Mateos and Montero 2002; Uresti et al. 2004; Moreno et al. 2015), and the subject was extensively reviewed (Truong et al. 2015). An interesting study underlines the possibility of using low-quality flying fish surimi and microbial TGase to make value-added seafood products and describe its physicochemical, rheological, and microstructural properties (Herranz et al. 2013). In order to improve gel quality, several hydrocolloids like carrageenan, alginate, sorbitol, locust bean gum, guar gum, xanthan gum, and sodium carboxymethylcellulose were tested and proved to be effective as gelling agents by reducing the aggregation of myofibrillar proteins and improving the overall gel quality. Nonetheless, the properties of proteins, the gelation method applied, and the processing conditions (pH, salts, hydrocolloids) influence the structure and the mechanical properties of gels.

3.7 HP THERMAL STERILIZATION Albeit HP thermal sterilization has not yet become implemented into practice, the growing interest in high-quality alternatives to retort sterilization makes very attractive the possibility to synergistically combine the effect of elevated temperatures 94 Trends in Fish Processing Technologies

(90°C–121°C) with pressures equal to or above 600 MPa (Sevenich et al. 2013). At this point, the only certified process for sterilization using HP, called pressure- assisted thermal sterilization, is in the United States and was approved to use a regime of 121.1°C and 600 MPa (Sevenich et al. 2013). However, there is much need for research before confirming the advantages of HP thermal sterilization processes over the retort ones. In this regard, an interesting research was presented by Sevenich et al. (2013), who investigated the formation of contaminants such as furan, monochloropropanediols, and their esters in commercially available fish products (tuna in brine, tuna in sunflower oil, sardines) by HP thermal sterilization in comparison with retort sterilization. The results showed that the furan concentration could be reduced by 71% and 97% for the tested temperature– pressure combinations at sterilization conditions where F0 = 7 min. The only concern was related with the formation of monochloropropanediols esters in tuna in sunflower oil, and the suggestion of the authors was to change the recipe of food toward using nonrefined oils (Sevenich et al. 2013). Compared to retort sterilization, the results clearly indicated a reduction of food chemical processing contaminants by HP thermal sterilization.

3.8 HP FREEZING AND THAWING HP freezing at low or subzero temperatures produces phase transitions in fish. Pressure- shift freezing (PSF) generates uniform ice crystals when pressure is released, thus inducing supercooling and instant uniform ice crystal nucleation throughout the sample depth (Cheftel, Vy, and Dumay 2000) if the fish muscle is cooled just above the freezing point. PSF may result in a better preserved texture of some foods, due to the formation of small ice crystals during fast nucleation (Truong et al. 2015). However, ice nucleation and ice crystal growth largely depend on the heat removal rate. PSF studies were conducted on turbot (Chevalier et al. 2000), sea bass (Tironi, LeBail, and De Lamballerie 2007), and Atlantic salmon (Zhu, Le Bail, and Ramaswamy 2003) showing the superiority of texture obtained by PSF compared to conventional freezing. Few studies deal with microbial inactivation through PSF; however, researchers (Picart et al. 2004) have demonstrated that in minced smoked salmon treated at 207 MPa and −21°C followed by further freezing to −25°C at 0.1 MPa, a 1.7 log reduction of L. innocua and 4.6 log reduction of Pseudomonas fluorescens was achieved. Also PSF from 207 MPa and −22°C, followed by further freezing to −25°C at 0.1 MPa, induced 1.4 or 1.9 log cycle reduction of L. innocua, depending on whether pressure was released rapidly (~3 s) or slowly (18 min), respectively (Picart et al. 2005). Despite limitations reported due to protein denaturation and color changes, PSF is still a promising alternative to fish freezing, proving significantly improved cellular protection compared to conventional methods. However, unless solutions for lower- ing the operational costs will be provided, the technology will remain prohibitive (Truong et al. 2015). Pressure-assisted thawing (PAT) is achieved where the phase transition is obtained by heating at constant pressure, while in pressure-induced thawing, the High-Pressure Processing of Seafood 95 phase transition is initiated by a pressure change and continued at constant pressure (Otero and Sanz 2003). PAT applied on fish muscle cod, whiting, redfish, haddock, salmon, and rainbow trout demonstrated that the required phase transition time is reduced by approximately 50% while quality index is similar with the samples thawed at atmospheric pressure. Moreover, the thaw drip is markedly reduced by HP thawing (Schubring et al. 2003). As observed in salmon (Zhu, Le Bail, and Ramaswamy 2003), in cod, redfish, and rainbow trout (Schubring et al. 2003) PAT induced adverse changes such as lightness increase and protein denaturation at 200 MPa; thus, it was suggested that PAT below 150 MPa could result in better quality (Truong et al. 2015). The application of HP freezing/thawing has attracted increased attention from the scientific community; however, more studies are required to optimize both PAT and PSF processing.

3.9 CONCLUDING REMARKS AND FUTURE PERSPECTIVES HPP has transcended the lab and pilot applications area and got onto the production floor where it further has to struggle to gain the trust of consumers by building a solid reputation of a clean and stable food technology. It was demonstrated that, in general, HPP at 300–500 MPa could extend frozen or refrigerated fish shelf life, reduce the microbial load, and improve texture but could have some detrimental effects on color and lipid hydrolysis depending on fish species and processing parameters. The future perspectives of HPP applications in fish are strongly connected with trends in consumers’ preferences that go with the capacity to further reduce fish allergenicity, reduce salts, and obtain products with improved functionality and clean labels. The opportunity to synergistically combine HPP with other hurdles by using natural antimicrobials and antioxidants and/or other minimal processing technologies to improve product safety and quality while being able to counteract the drawbacks associated with singular methods applied by intensive processing deserves further researches in order to develop sustainable solutions for fish processing. Another area of interest in the near future is the application of pressure-assisted processing, such as PATS due to its ability to inactivate microbial spores, and also PAT and pressure shift freezing; however, the industrial equipment market should come with new and feasible solutions for implementing these new technologies.

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Livia Patraşcu and Iuliana Aprodu

CONTENTS 4.1 Fish Protein Recovery...... 103 4.1.1 Fish Protein Concentrates...... 104 4.1.2 Fish Protein Isolates...... 105 4.1.3 Fish Protein Hydrolysates...... 107 4.1.4 Collagen and Gelatin...... 110 4.2 Fish Enzymes...... 114 4.3 Trends in Processing of Low-Value Fish, Coproducts, and By-Catch...... 115 References...... 115

Due to fish and seafood importance in human nutrition, fish catching and processing are economic activities that are performed on a large scale, generating problems such as overexploitation, which has disastrous consequences. In order to reduce fish capture, to avoid stock depletion and the gap between capture fisheries output and increasing world demand for fish and shellfish, better management of the existent catch is required (FAO 2011). In this respect, it can be emphasized that in the 1995–2013 time frame, the European Union (EU) registered a decline of total fishery production of about 37%, or 2.8 million tons of live weight (EUROSTAT 2014). However, on a world scale, fish capture production was reported to be rather constant over the last 10 years, with an average of 91 million tons/year; an increasing tendency was reported for Africa and Asia, and a decreasing one was observed in the United States, Europe, and Oceania (FAO 2012a). Primary processing of fish generates fish processing coproducts and by-products. The use in the common language of the term “waste” to designate fish coproducts and/or by-products is inappropriate because it implies that the material resulting from fish processing cannot be used for any other application and should be disposed of. Both fish coproducts and by-products are generated together with the final product when processing fish. Fish coproducts include products intended for human consumption, such as liver, whereas fish by-products consist of parts that are not meant to be valued as human food, although they retain some valuable components (Penven et al. 2013). The total annual amount of by-products generated by seafood processing through filleting, heading, gutting, skinning, or cutting is difficult to be

101 102 Trends in Fish Processing Technologies assessed; it comprises heads (9%–20%), viscera (12%–18%), trimmings (8%–17%), fins, bones, or cartilage (9%–15%), skin (1%–3%), blood, and eggs (Ramírez 2007; Penven et al. 2013). According to the EU Commission, a valid estimation of total wasted quantities until now could not be accomplished (EUROSTAT 2005). Not even the Food and Agriculture Organization of the United Nations (FAO) could provide accurate data about wasted quantities when reporting world fisheries and aquaculture production (FAO 2012b). However, EFSA (2009) reported rest raw materials (offal) up to 290,000 tons per year arising from the Norwegian aquaculture sector. Scientific literature reported processing wastes ranging from 27% to 60% of the world catch (Kristinsson and Rasco 2000; Bechtel 2003; He et al. 2013). An and Visessanguan (2000) presented some detailed figures on fish wastes for different processing technologies, reported to the total landed weight; for instance, up to 60% of wastes were attributed to the filleting operation, up to 31%–38% of production wastes were ascribed to salmon cannery industry, whereas in the case of shellfish processing, the amount of waste was reported as high as 75%–83%. A significant part is, however, already recovered. Shepherd (2012) estimated that about 25% of fishmeal produced globally uses fish processing waste as an ingredient, while FAO (2013) reported that 90% of the ingredients used in fishmeal produced in Japan come from fish by-products. An additional serious problem is represented by the discarded by-catch originating both from pelagic (shoaling, surface swimming fish) and demersal (bottom swimming fish) fishing. The by-catch is the catch of nontarget fish and ocean wildlife, including both what is brought to port and what is discarded at sea (Keledjian et al. 2014). The discarding of by-catch represents a loss of valuable food and a major threat for the environment and fish biodiversity (FAO 2016). According to FAO estimates in 1994, the region with the highest discard estimate was the Northwest Pacific (Alverson et al. 1994). Hall et al. (2000) gave detailed information on the causes of by-catch and discards, the economic reasons being considered the most important ones. In addition to the lack of space on the ship, the low monetary value of by- catch plays an important role in deciding capture discarding. Moreover, the high perishability of some fish species caught plays a decisive role. Davies et al. (2009) estimated a global quantity of minimum 38.5 million tons of annual by-catch (without counting illegal, unreported, or unregulated fishing), representing 40.4% of the estimated annual global marine catch of 95.2 million tons. Out of these catches, the highest discards were registered for shrimp trawls, with approximately 6 kg of fish for every kilogram of kept shrimp since 2000 (Keledjian et al. 2014). Bottom trawl- ing is known as one of the most deleterious fishing methods, which consists in drag- ging enormous nets weighed down with heavy ballast over the seafloor. Campana et al. (2009) reported only for blue shark catch in the North Atlantic annual discards of 57,000 tons from a total of 84,000 tons captured. Along with the by-catch reducing programs, making it more profitable would contribute to reducing discards of unavoidable by-catch, low-value fish, and by-products. These kinds of programs will end up in higher quantity of fish products on the market (protein derivatives for fish restructured products, lipids, nutraceuticals) and lower amounts of captured fish. Fish muscle proteins recovered from low value fish, by-products, and by-catch can be used as functional ingredients in food systems, such as to make their recovery more Processing of Low-Value Fish, Coproducts, and By-Catch 103 profitable than discarding it (Andrew and Pepperell 1992; Kristinsson and Rasco 2000; Gehring et al. 2011). Clucas (1997) put the large unused by-catch resource on economic difficulties, rather than on technology-related ones. Moreover, Yean (1998) related the problem of unused by-catch to the extreme heterogeneity in terms of composition, bony structure, amount of dark flesh, small size, unattractive appearance and texture, strong flavor, and the possible presence of toxic species. Regarding profitability, Batista (2011) reported that the total cost of fish protein hydrolysate production from sardine by-products is higher with respect to the one of fish meal, whereas Rustad (2003) identified that producing bioactive compounds, such as bioactive peptides, enzymes, or biopolymers, from fish by-products is much more profitable than transforming them into fertilizers. Anyway, the consumer acceptability is a problem to be solved too, because “fish restructured products” are usually perceived as low quality and unhealthy. Another problem for fish coproduct and by-product processing is the lack of appropriate technology. For example, the existent equipment used for evisceration destroys the viscera, making difficult to separate the liver when processing cod (Rustad 2003).

4.1 FISH PROTEIN RECOVERY The high demand for fish proteins exceeds the amounts provided by traditional raw materials and conventional processing technologies employed to deliver value-added fish-based products, thus better use of low value fish, coproducts, and by-catch for producing acceptable products with appropriate functionality is required. Although an attempt for recovering the fish proteins has been made by producing surimi, the success of this technology is limited in the case of processing unconventional raw materials. The main difficulties when processing pelagic species and other fish material rich in dark muscle are related to the presence of high amounts of compounds (oxidatively unstable lipids, heme proteins, etc.), causing gelation, oxidation, and color problems (Kristinsson et al. 2005). The composition of the fish varies with the species, sex, age, health, and nutritional conditions and period of the year (Ghaly et al. 2013). Even the fish species that lack commercial value have equal nutritional quality in terms of proteins to the most preferred ones. The average proximate composition of fish meat is as follows: water, ~75%; proteins, ~18%; and lipids, 4% to 6% (USDA 2016). Proteins from fish muscle can be classified into sarcoplasmic (soluble in water or weak ionic buffers), myofibrillar (soluble in cold neutral salt solutions of rather high ionic strength), and stromal proteins (insoluble connective tissue proteins) (Kristinsson and Rasco 2000). The predominant proteins in fish meat are represented by myofibrillar ones (66%–77%). Over 90% of the myofibrillar proteins can be recovered through different methods, as indicated in Table 4.1. The extraction procedure highly influences the functional properties of the fish proteins, such as gelling, emulsifying capacity and emulsion stability, foaming properties, ability to bind and retain water, oils, and other compounds, etc. In addition to producing fish protein concentrates and isolates, which often display functional or sensory drawbacks, developing different techniques for production of fish protein hydrolysates might be an acceptable way of addressing the problem of 104 Trends in Fish Processing Technologies

TABLE 4.1 Protein Recovery Yield from Low-Value Fish and By-Products, through Different Techniques By-Product/ Protein Method Source Recovery Yield Reference Protein Alcalase, Neutrase, Capelin 51.6%–70.6% Shahidi et al. (1995) hydrolysis Papain (Mallotus villosus) Pescalase 560, – 13%–15% Baca et al. (1991) HT-200, Protease N Isoelectric Various pH domains Not specified 42%–90% Gehring et al. (2011) precipitation and references within Various pH domains Atlantic krill 45%–50% Chen et al. (2009) (Euphausia (d.b.a) superba) Various pH ranges Rainbow trout 77.7%–98% Chen and Jaczynski (Oncorhynchus (d.b.) (2007) mykiss) Alkaline (pH 11.2) Tilapia 71.5% Rawdkuen et al. Acid (pH 3.0) 81.4% (2009) a d.b. = dry basis. underutilization of unconventional raw materials for producing economically valid products. Based on their functional properties, the separated fish proteins can be used as additives for different types of foods. Moreover, various commercial nutraceuticals based on fish proteins, peptides, or amino acids are available mainly in the United Kingdom, United States, and Japan market (Ghaly et al. 2013).

4.1.1 Fish Protein Concentrates FAO defines the fish protein concentrates as powders, with or without odor or flavor, having higher protein concentration with respect to the original fish and total fat content ranging from 0.75% to 3%. According to these criteria and also depending on raw material used for preparation, fish protein concentrates can be classified as follows: type A—characterized by high protein content, low fat content, low water content, colorless, and odorless, and type B—having contents of ~65% protein and ~3% fat and fish odor present (Yean 1998). Fish protein concentrates are among the earliest products developed for human consumption from species of fish of low economic value. In fact, fish protein concentrates used to be considered as a panacea for the world’s food crisis (Pariser 1978). In the 1960s, fish protein concentrate benefited from institutional support from both the US government and the private sector. However, despite being technologically and biologically promising and having considerable interest from the private sector, the so called “fish flour” ultimately failed to meet commercial expectations Processing of Low-Value Fish, Coproducts, and By-Catch 105

(Wintersteen 2012). It was documented that efforts were made from 1930 to 1970, in about 40 countries, to develop commercially viable fish-based protein concentrates for human consumption, but no success in the large-scale production was reported (Pariser 1978, cited by Wintersteen 2012). Despite the nutritional value of fish proteins, given by the well-balanced amino acid composition, the fish protein concentrates are not widely consumed, mainly because of the presence of fish odor. In order to obtain food grade fish protein concentrates, FAO recommends to start processing the raw material (by-catch and by-products) within 12–48 h of landing, while storing it in ice. The technology used for production of type A fish protein concentrates involves chemical extraction of proteins in up to three subsequent steps, using solvents like isopropanol, azeotropic ethylene dichloride, or ethanol, under mild temperature conditions at 20°C–75°C (Kristinsson and Rasco 2000). Although the biological values of the resulting products are high, the protein solubility and the emulsifying properties are very poor, therefore limiting commercial success. In addition to poor functionality, the fish protein concentrates have other drawbacks related to the high production costs and presence of traces of solvent in the final product.

4.1.2 Fish Protein Isolates The main method used for isolating fish proteins consists in applying the principle of pH-dependent solubilization and isoelectric precipitation (isoelectric solubilization/ precipitation processing) under low temperature condition at ~4°C (Tahergorabi et al. 2012). The mechanism standing behind this processing technique for obtaining protein isolates is based on the change of electrostatic charge of proteins at different pH values. Shortly, at a particular concentration of hydrogen ions (H+) in solution, when the proportion between cations and anions is equal, protein solubility is minimum (minimal protein–water interactions are established), while protein–protein interactions are maximum, making it easy to separate them from the solute by precipitation. The pH value at the zero-net electrostatic charge of proteins is known as isoelectric pH (pHi) or isoelectric point (pI). This process is successfully applied in simultaneous protein and fish oil recovery from various raw materials, such as fish by-products and by-catch (Gehring et al. 2011; Matak et al. 2015). The obtained protein isolate has high nutritional quality as well as good functional properties. In general, the protein isolate contains 87%–95% crude protein, 0%–5% lipid, and 2%–6% ash (Matak et al. 2015). As described in detail by Tahergorabi et al. (2012), the processing technology for obtaining fish protein isolates is relatively simple and involves the homogenization of fish mince with a solvent at pH far from isoelectric point, weakening the protein– protein hydrophobic interactions that ensure myofibrillar protein aggregation in fish muscle homogenates. The strong acid (pH 2–3) or alkali (pH 10.5–11.5) pH values determine a net positive or negative charge of proteins, thus causing muscle proteins to disrupt and solubilizing the myofibrilar proteins (Kristinsson et al. 2005; He et al. 2013). In fact, the use of solvent with alkaline pH values ensures obtaining fish protein isolates of better nutritional quality and higher amounts of essential amino 106 Trends in Fish Processing Technologies acids dissimilar to the acidic pH. The phases resulted when suspending the minced fish material into the solvent (about one part fish to five to nine parts of solvent), consisting of oil, protein solution, and insoluble impurities, is additionally separated by centrifugation. In a further step, fish proteins are precipitated by adjusting the pH to its isoelectric point (~5.5), and then are recovered by centrifugation. The fish protein separation procedure is presented in Figure 4.1. The isoelectric solubilization/ precipitation processing ensures high recovery yield of fish proteins, which retain functional properties and nutritional value (Tahergorabi et al. 2012). When recycling processing water resulting in the final centrifugation step, special attention must be paid to the ionic strength. A consequence of the reaction between reagents used for myofibrillar protein solubilization (NaOH) and then for precipitation (HCl) is that high amounts of corresponding salts can be formed, causing the increase of the ionic strength in the resulting water, meant to be recycled. Therefore, the ionic strength increases and the myofibrillar proteins will precipitate at lower pH values than intended. For example, Chen and Jaczynski (2007) reported that when

NaOH 0.5 mol/L Minced ﬁsh material or HCl 0.5 mol/L Homogenization 1 part ﬁsh: 5–9 parts water

Protein solubilization pH reduction to 2.5 or pH increase to 11.5

Centrifugation 10,000 × g

Upper layer Middle layer Bottom layer Fish oil Soluble muscle proteins Residue

Protein aggregation pH adjustment at 5.5

Supernatant– Centrifugation 10,000 × g processing water

Fish protein isolate

FIGURE 4.1 Flow diagram for obtaining fish protein isolate by isoelectric solubilization/ precipitatation (after the methods proposed by Tahergorabi et al. 2012). Processing of Low-Value Fish, Coproducts, and By-Catch 107 the ionic strength increased to 0.2, the pHi of the myofibrillar proteins dropped by ~1 unit, from 5 to 4.

4.1.3 Fish Protein Hydrolysates The process of breaking down the peptide bonds linking the amino acids within proteins, for obtaining peptides of varying sizes, is known as protein hydrolysis. There are three different methods that can be used for protein hydrolysis: chemical, biological/enzymatic, or mixed (chemical and enzymatic) hydrolysis. The final product may be obtained in powder form (freeze-dried or spray dried) or in the form of a moderately viscous liquid at a protein concentration of ~40% when phosphoric acid or other stabilizing additive is added to allow storing the product for a longer period. The protein hydrolysates can be in used in the food industry as functional protein ingredients or nutritional supplements. Chemical hydrolysis is based on the use of either acids or alkali for cleaving the peptide bonds, under high temperature (120°C) and pressure (100 kPa). Although commonly employed due to high yields in shorter time periods, acid hydrolysis has some disadvantages: functional properties (physicochemical and bioactive) are affected, high content of sodium chloride is formed during the neutralization of the digest, and it seems that sensitive amino acids such as tryptophan and methionine are destroyed during acid hydrolysis (Kristinsson and Rasco 2000; Thiansilakul et al. 2007). These particularities of the protein hydrolysates obtained by chemical hydrolysis limit their use for human nutrition, being mostly considered as low-value products and used in other purposes (He et al. 2013). Products obtained via alkali hydrolysis also contain some unwanted compounds such as the highly toxic lysino- alanine, ornithinoalanine, lanthionine, or β-amino alanine (Kinsella 1976). Enzymatic hydrolysis represents another increasingly used method for protein recovery from fish coproducts. It consists of treating a mixture of minced fish:water slurry with different enzymes for periods of time depending on the intended degree of hydrolysis and recovering the obtained peptides by centrifugation (Figure 4.2) or ultrafiltration, which allows separating fractions with specific molecular weights. The processing technique may slightly vary and can be optimized by considering, for instance, different fish:water ratios or proteases of different sources (animal, veg- etal, or of microbial origin) used alone or in subsequent steps in different ratios. The processing technique might include an optional defatting step and different drying processes can be selected. However, enzymatic hydrolysis is a complex process, and the resulting peptide profile depends on some highly important factors, such as specificity of enzyme for substrate, optimum temperature, and pH. Enzymatic hydrolysis can be performed by using fish endogenous enzymes or commercial products. Processes based on autolysis are more difficult to control because of the variation of some critical factors such as enzyme type and amount. The exogenous enzymes allow obtaining higher yields in shorter periods of time and are usually selected for obtaining fish protein hydrolysates meant for human consumption, whereas the hydrolysis assisted by endogenous enzymes is selected for getting feed ingredients. Regardless of the type of enzyme used (endogenous or exogenous), the resulting degree of hydrolysis can vary, with higher ones being favorable from 108 Trends in Fish Processing Technologies

Enzyme Isopropanol Water (distilled) Fish protein coproducts

Mincing

Homogenisation min 1:1 (w/v) or 20% dry basis

Defatting (optional) Fish oil

Washing (twice)

Homogenisation 1:2 (w/v)

Environment adjustment to enzyme optimum conditions pH, temperature

Hydrolysis

Enzyme inactivation t > 85°C, min 15 min

Centrifugation Precipitate ~2000 × g, 10–15 min - Slurry

Supernatant

Freeze drying/ spray drying

Fish protein hydrolysates

FIGURE 4.2 Flow diagram for obtaining fish protein hydrolysates by enzymatic hydrolysis (after the methods proposed by Thiansilakul et al. 2007, Batista 2011, and He et al. 2013). nutritional point of view, although affecting functional properties. The most used enzymes at industrial scale are pepsin and trypsin of animal origin, papain of veg- etal origin, and different microbial endopeptidases. Promising results were obtained when using commercial enzyme products like Alcalase—a serine endopeptidase that consists primarily of subtilisin A, produced through submerged fermentation of a selected strain of Bacillus licheniformis; Protamex—a protease from Bacillus sp.; Neutrase—a bacterial protease produced by a selected strain of Bacillus amyloliquefaciens; and Flavourzyme—a registered trademark of Novozymes Corp., consisting of a fungal protease/peptidase complex produced by submerged fermentation of a Processing of Low-Value Fish, Coproducts, and By-Catch 109 selected strain of Aspergillus oryzae. These enzymes have been tested for hydrolysis of fish proteins under neutral or slightly acidic conditions (Muzaifa et al. 2012). Batista (2011), in a feasibility study on the production of protein hydrolysates from sardine by-products, reported that the price of raw material does not influence significantly the cost of the final product but is highly dependent on the amount of raw material available. The highest benefits of enzymatic hydrolysis consist of avoiding the extreme environmental conditions specific to chemical and physical treatments, minimizing the undesirable reactions that could destroy valuable components in proteins. Moreover, enzyme-assisted hydrolysis was found to improve the functional properties and antioxidant activity of fish proteins (Klompong et al. 2007; Taheri et al. 2013). The inhibition of lipid oxidation by proteins was reported to occur through multiple pathways, including inactivation of reactive oxygen species, scavenging free radicals, chelation of prooxidative transition metals, or reduction of hydroperoxides. The peptides have significant high antioxidant activity due to an increased amount of amino acid residues that can scavenge free radicals and chelate prooxidative metals (Elias et al. 2008). Other advantages of the fish protein hydrolysates obtained through enzymatic procedure are related to very high digestibility and the angiotensin-converting enzyme inhibitory activity (Bougatef et al. 2008). The disadvantages of the enzyme-assisted hydrolysis of fish proteins consist of high costs associated to the exogenous enzyme and large volumes of water used for processing, existence of a bitter taste due to the resulting hydrophobic peptides, and difficulties in controlling the reaction end and obtaining reproducible results because of the variability of the raw materials. The way of reducing the processing costs relies on the use of immobilized exogenous enzymes, which allows their reutilization from one batch to another or even protein hydrolysate production in a continuous process. There are studies in the literature presenting successful applications of enzymatic hydrolysis of fish proteins in research at laboratory scale, and the process was already successfully up-scaled in industry, helping waste reduction and the recovery of valuable nutriments (Kristinsson and Rasco 2000; Ramírez 2007). In this respect, the patented Norwegian Biomega process can be mentioned, which applies continuous hydrolysis to separate different fish components at the end of the processing line. A negative aspect of protein hydrolysates is the bitter flavor associated with the formation of peptides containing bulky hydrophobic groups in their side chains (Benjakul et al. 2014). Proline is an amino acid that contributes mostly to the bitterness of hydrolysates, when present in the center of peptides. Other amino acids with bitter taste are valine, isoleucine, phenylalanine, tryptophan, leucine, and tyrosine (FitzGerald and O’Cuinn 2006). Some efficient methods proposed for debittering the protein hydrolysates are enzymatic hydrolysis of bitter peptides, in which case enzyme specificity and reaction conditions play a major role in obtaining the desired characteristics for the final products, filtration through activated carbon or extraction of bitter peptides with alcohols, peptide cross-linking using transglutaminase, and the plastein reaction (Kristinsson and Rasco 2000; Edens et al. 2005; FitzGerald and O’Cuinn 2006). Defined as the reaction catalyzed by some proteases, such as pepsin or papain, on peptides at specific pH and substrate concentration (generally over 20% protein), the reaction results in a tasteless high-molecular-weight 110 Trends in Fish Processing Technologies protein compound known as plastein. In addition, it was reported that it is possible to generate products with reduced bitter flavor by selecting appropriate enzymes for fish protein hydrolysis. Such an example is Flavourzyme, consisting on a mixture of endopeptidase and exopeptidase enzymes (Kristinsson and Rasco 2000; Thiansilakul et al. 2007).

4.1.4 Collagen and Gelatin Collagens are the most abundant proteins in vertebrates. The aquatic by-products are good alternative sources for mammalian collagen. Fish skin, bones, swim blad- ders, and scales are widely used for collagen production. The skin of eight fish species account for over 95% of the worldwide collagen production: blue shark (Prionace glauca)—43.3%; grenadier (Coryphaenoides rupestres, Macrourus berglax, Macrourus spp.)—12.4%; patagonian grenadier (Macruronus magellanicus)—10.8%; monkfish Lophius( piscatorius, Lophius upsicephalus, Lophius spp.)—11.2%; skate (Raja spp.)—8.8%; argentine hake (Merluccius hubbsi)—4.9%; greenland halibut (Reinhardtius hippoglossoides)—2.8%; and cape hake (Merluccius capensis)—2.6% (Maroto Leal et al. 2011). In nondenaturated form, collagen is widely used in cosmetics, biomedical, and pharmaceutical industries (Huang et al. 2016). Due to the multiple industrial applications, mostly relying on the property of forming insoluble fibers with high tensile strength, collagen is considered one of the most useful biomaterials (Lafarga and Hayes 2014; Schmidt et al. 2016). After denaturation, when turning into gelatin, collagen becomes water-soluble and finds multiple applications as an additive in foods and beverages because of the emulsifying, foaming, texturizing, and binder properties, and also in nonfood industries, such as pharmaceutical, photographic, cosmetic, and packaging industries (Huang et al. 2016). Gelatin is obtained by heating the collagen in acid or alkali solutions, which leads to the cleavage of intramolecular and intermolecular covalent cross-links (Karim and Bhat 2009). According to Ricard-Blum (2011), the collagen superfamily comprises 28 members that share a structural feature, consisting of the existence of a triple helix motif. The percentage of the triple helical content varies for different collagen types; it accounts for about 96% of the collagen I structure and goes down to less than 10% in the case of collagen XII (Ricard-Blum 2011). Different types of collagen are being extracted from diverse animal parts. For instance, collagens II, III, IX, and XI are located in cartilage, collagens I and III in skin, and collagens I and V in cornea, which are organized as fibrils (Ricard-Blum 2011). However, only the first three types are mainly extracted and used, whereas the others are present in very low amounts in animal tissue, being mostly specific to organs (Schrieber and Gareis 2007). The most common type is collagen I from skin, bones, and tendons, followed by collagen II found only in cartilage tissues, while the distribution of type III collagen depends on the animal age: in the skin of young fishes, it is present up to 50%, significantly decreasing with age to approximately 5% (Schrieber and Gareis 2007). As reviewed by Ricard-Blum (2011), collagen members are categorized into subfamilies based on the supramolecular assemblies they form: Processing of Low-Value Fish, Coproducts, and By-Catch 111

– Fibrils (collagens II, XI, and IX or collagens II and III are able to form fibrils in cartilage, collagens I and III in skin, and collagens I and V in cornea) – Beaded filaments (collagen VI) – Anchoring fibrils (collagen VII) – Networks (collagens VIII and X form hexagonal networks, and different isoforms of collagen IV can organize into diverse networks: association of the amino-terminal 7S domains leads to tetramers, whereas the association of two trimeric carboxy-terminal NC1 domains generates hexamers)

Some examples of supramolecular assemblies formed by different collagen types are presented in Figure 4.3. The extraction of food grade collagen from fish can be performed by chemical methods (e.g., isolation with weak organic or inorganic acids or neutral saline solutions) or enzymatic hydrolysis. These methods are batch type and have the disadvantages of being time- and cost-consuming because of the reagents and energy involved. A chemical pretreatment must be normally performed, in mild alkaline and/or acidic environment. This pretreatment allows better cleavage of cross-linked chains, removing noncollagenous proteins, and demineralization and ensures higher processing yields. Concerning the chemical methods used for collagen extraction from fish, organic acids were reported to be more efficient due to their ability of solubilizing the non- cross-linked and some of the interstrand cross-linked collagens (Liu et al. 2015;

Fibrils Anchoring ﬁbrils (collagen VII)

Beaded ﬁlaments (collagen VI) Network (collagen IV)

Hexagonal network (collagens VIII and X)

Triple-helical domain Noncollagenous domain

FIGURE 4.3 Schematic representation of supramolecular assemblies formed by different types of collagens. (From Ricard-Blum, S., Cold Spring Harb. Perspect. Biol., 3, 1–19, 2011.) 112 Trends in Fish Processing Technologies

Schmidt et al. 2016). The process must be performed at low temperatures (4°C) to avoid chain fragmentation (Fruchour and Koenig 1975). In case of by-products rich in calcium-phosphate compounds, for instance, scales (mainly referring to hydroxyapatite [Ca10(PO4)6(OH)2] present in bones up to 70%), a demineralization step must be first performed. For example, pretreatment with 0.2 mol/L ethylenediaminetet- raacetic acid allowed a decalcification ratio over 90%, with minimal protein losses (Wang and Regenstein 2009). Fish bones are a very good source of hydroxyapatite, which can be used as a bone graft material in medical and dental applications (Ghaly et al. 2013). Despite being more expensive, the enzymatic process used to recover food grade collagen from fish by-products gives products with good functional properties. It also generates higher extraction yields and lower waste and the processing time is reduced (Schmidt et al. 2016). Often, the extraction methods are combined, and different collagen fractions are extracted from the same raw material, resulting salt soluble collagen, acid soluble collagen, and enzymatic soluble collagen. All these methods of collagen separation from solution are based on the salting-out effect of proteins—precipitation in solutions with high salt concentrations. The hydrophobic regions of the proteins are normally aggregated together, and the hydrophilic regions are available to interact with the water molecules, in such a way that proteins remain dissolved. In an aqueous environment with a certain ionic strength (depending on protein), the water molecules are no longer able to support the charges of both the ions and the proteins. The result is precipitation of the least soluble solute, such as proteins and large organic molecules. A detailed flow chart of collagen extraction is presented in Figure 4.4. The collagen fractions extracted by the methods presented in Figure 4.4 are mainly type I, due to the presence of the triple helical structure (Wang et al. 2014). When collagen is meant to be used for gelatin preparation, in many cases, the pigmented fish skin is avoided. An additional pretreatment step is required because the pigment compounds are difficult to eliminate from the mixtures, therefore resulting in lower extraction yields (DeVictor et al. 1995). In order to overcome the disadvantages mentioned in the case of the collagen extraction with alkali, acids, or enzymes, Huang et al. (2016) proposed a novel extrusion–hydroextraction process that allows 2–3 times higher extraction yield compared to the conventional methods. This method ensures efficient separation of hydroxyapatite from collagen, which possesses high physicochemical functionalities. Although collagen extraction from fish by-products is easy and safe, particular consideration should be given to the processing temperature because of the low denaturation temperature of the protein, which is considered one of the major drawbacks (Subhan et al. 2015). In addition, in order to obtain high-quality products, manufacturers must pay special attention to appearance (color and particle size), lack of fishy odor and taste, nutritional profile, thermal stability, and shelf life (Maroto Leal et al. 2011). Processing of Low-Value Fish, Coproducts, and By-Catch 113

Fish by-products

Mincing

0.1 N NaOH Alkaline pretreatment - Separation of noncollagenous proteins atment Draining Alkaline solution

Pretre Water Washing (until pH 7.0)

0.1 N HCl Acid pretreatment - Demineralization Draining Acid solution

Water Washing (until pH 7.0) 0.45 M NaCl in 0.05 M Tris-HCl pH 7.5 (1:100 w/v) Salt extraction Centrifugation Collagen solution I

Residue

Water Washing

0.5 M acetic acid (1:10 w/v) Acid extraction

tion Centrifugation Collagen solution II trac

Ex Residue

Water Washing

Pepsin (0.1% w/v) in 0.01 M Enzymatic hydrolysis HCl/or other enzyme Centrifugation Residue

0.7–0.8 M NaCl Collagen solution III in 0.5 M Tris-HCl pH 7.5 Salting-out 2.3 M NaCl - In two steps in 0.5 M Tris-HCl pH 7.5 Centrifugation Supernatant

Collagen 0.5 M acetic acid - A minimum volume Solubilization Acetic acid saline 0.1 M acetic acid Dialysis solution - Desalinization

Puriﬁcation Lyophilization

Collagen powder

FIGURE 4.4 Flowchart for a combined process of collagen isolation from fish by-products (after the methods proposed by Nagai et al. 2008, Vallejos et al. 2014, and Wang et al. 2014). 114 Trends in Fish Processing Technologies

4.2 FISH ENZYMES Another domain where fish by-catch and by-products gained more interest is the enzymes industry. The gastric, intestinal, and hepatopancreas proteolytic enzymes from several fish species have been purified and widely investigated. The most commonly studied enzymes recovered from marine organisms are pepsin, pepsinogen, chymosin, gastricsin, trypsin, chymotrypsin, collagenases, and elastase (Shahidi and Kamil 2001). In addition, the recovery of some enzymes that are fish specific, such as urease, thymidilate kinase, polyphenolases, and trimethylamine oxide demethylase, has been also reported. The fish muscle tissue also contains proteinases, such as cathepsins B, L, and H and alkaline proteinases, located mainly in the lysosomes, sarcoplasm, and the extracellular matrix of the connective tissue neighboring the cells (An and Visessanguan 2000). Among enzymes recovered from fish by-product, hydrolases are the most widely used. These enzymes have various applications in different fields from the food industry to the detergents industry, waste management, or leather production. Regarding the applications in food industry, the digestive enzymes recovered from fish by-products have been tested for obtaining baked products, fermented dairies, vegetable and cereal products, protein hydrolysates, or flavor extracts, and the most promising results were related to the acceleration of the fermentation process for producing fish sauce or matjes herring (Haard 1992; An and Visessanguan 2000). Although it appears that the enzymes recovered from the aquatic environment resemble the structure of similar enzymes obtained from terrestrial organisms, there are some important differences in terms of molecular weight, pH, and temperature condition where optimum enzyme activity occurs, pH and thermal stability, amino- acid composition, inhibition characteristics, or kinetic properties (De Vecchi and Coppers 1996). Due to the particularities of the marine living environment (temperatures usually within 0°C to 5°C), most enzymes recovered from fish are adapted to act at low temperatures. A particular interest was paid during the last decades to the proteolytic enzymes from cold-adapted fish. These enzymes proved to be more active catalysts at relatively low temperatures and low concentrations, compared to similar enzymes from mammals, thermophilic organisms, and plant sources (De Vecchi and Coppers 1996). They are therefore advantageous for some food processing operations where gentle physical and chemical conditions are required, allowing lower thermal energy input, protection of substrates or products from degradation, minimization of side reactions, and prevention of the denaturation of thermally labile compounds associated with raw material (An and Visessanguan 2000). Other examples of unique properties of fish enzymes include low heat stability and ability to catalyze hydrolysis of native proteins (Haard 1992). Pepsin, the major gastric proteinase, is secreted in the stomach as pepsinogen and passes from the zymogen to the active form in an acid environment. Unlike the pepsin of terrestrial animal origins, the corresponding enzymes isolated from cold water fish have lower temperature optimum, lower thermal stability, higher pH optimum, and denaturation temperature approximately 20°C lower, being also very resistant to autolysis at low pH (Haard 1992). As reviewed by Shahidi and Kamil Processing of Low-Value Fish, Coproducts, and By-Catch 115

(2001), pepsin was isolated from the gastric mucosa of various cold- and warm-water marine species and from freshwater fish species, including brown trout and rainbow trout. Crude pepsin preparation can be produced by processing through ultrafiltration, concentration and spray drying the stomach silage. Pepsin of fish origin works optimally at pH 2–4 and temperature of 30°C and can be successfully used as rennet substitute or for gelatin production (Ghaly et al. 2013). The most commonly used proteinases from the intestine of the fish, trypsin, chymotrypsin, and collagenases, which are serine proteases, share similar thermal and catalytic sensitivity characteristics as reported for pepsin. Trypsin has been isolated from several cold- and warm-water fish species, such as sardine, capelin, cunner, Greenland and Atlantic cod, chum salmon, Atlantic salmon, coho salmon, anchovy, palometa, and Atlantic white croaker (Shahidi and Kamil 2001). Although the molecular weight of trypsin varies for different fish species, they appear to share high stability at alkaline pH, unlike the corresponding enzyme from mammals which are mainly stable under acidic pH (An and Visessanguan 2000; Shahidi and Kamil 2001). Trypsin recovered from fish by-products has optimum activity at pH 9 and temperature of 40°C, whereas chymotrypsin, at pH 7–8 and temperature of 50°C (Ghaly et al. 2013). The intestinal collagenases are partially responsible for the degradation of collagen and other extracellular matrix proteins, therefore playing an important role, together with the muscle collagenase, which is zinc metalloproteinases, in the loss of integrity of muscle structure in seafood products. The intestinal collagenases displayed both trypsin- and chymotrypsin-like activities and have optimal activity at temperature of 30°C and pH of 6.5–8.0 (Ghaly et al. 2013). Suganthi and Ramani (2016) proposed a biotechnological process for concomi- tant production of alkaline lipase and protease from fish processing wastes, in a single fermentation step with Streptomyces thermolineatus, revealing that multiple substrate degradation is possible in one pot reaction. The combination of enzymes like lipase and protease was proposed to be used for the treatment of lipid and protein rich industrial wastes and therefore gained increasing interest for environmentally friendly applications.

4.3 TRENDS IN PROCESSING OF LOW-VALUE FISH, COPRODUCTS, AND BY-CATCH Fish by-catch and by-products generated in large amounts when processing raw material into food products are rich sources of valuable nutrients for humans. Selective recovery of proteins from fish materials not intended for human consumption allows improving the resource efficiency and therefore helps create a more circular economy.

REFERENCES Andrew, N. L., and J. G. Pepperell. 1992. The by-catch of shrimp trawl fisheries. Oceanography and Marine Biology. Oceanogr Mar Biol Ann Rev 30:527–565. Alverson, D. L., M. H. Freeberg, J. G. Pope, and S. A. Murawski. 1994. A global assessment of fisheries bycatch and discards. FAO Fisheries Technical Paper. No. 339. Rome: FAO. 116 Trends in Fish Processing Technologies

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Matak, K. E., R. Tahergorabi, and J. Jaczynski. 2015. A review: Protein isolates recovered by isoelectric solubilization/precipitation processing from muscle food by-products as a component of nutraceutical foods. Food Res Int 77:697–703. Muzaifa, M., N. Safriani, and F. Zakaria. 2012. Production of protein hydrolysates from fish by-product prepared by enzymatic hydrolysis. Int J Bioflux Soci 5:36–39. Nagai, T., N. Suzuki, and T. Nagashima. 2008. Collagen from common minke whale (Balaenoptera acutorostrata) unesu. Food Chem 111(2):296–301. Pariser, E. R. 1978. Fish Protein Concentrate, Panacea for Protein Malnutrition. Cambridge, MA, and London: MIT Press. Penven, A., R. Perez-Galvez, and J. P. Bergé. 2013. By-products from fish processing: Focus on French industry. In Utilization of Fish Waste, R. P. Galvez and J. P. Berge (eds.), 1–25. Boca Raton, FL: CRC Press, Taylor & Francis Group. Ramírez, A. 2007. Salmon by-product proteins. FAO Fish Circ, 1027. http://www.fao.org /docrep/010/a1394e/a1394e00.htm (accessed November 16, 2016). Rawdkuen, S., S. Sai-Ut, S. Khamsorn, M. Chaijan, and S. Benjakul. 2009. Biochemical and gelling properties of tilapia surimi and protein recovered using an acid-alkaline process. Food Chem 112(1):112–119. Ricard-Blum, S. 2011. The collagen family. Cold Spring Harb Perspect Biol 3(1):1–19. Rustad, T. 2003. Utilisation of marine by-products. Electronic J Environ Agric Food Chem 2(4):458–463. Schmidt, M. M., R. C. P. Dornelles, R. O. Mello, E. H. Kubota, M. A. Mazutti, A. P. Kempka, and I. M. Demiate. 2016. Collagen extraction process. Int Food Res J 23(3):913–922. Schrieber, R., and H. Gareis. 2007. Gelatine Handbook: Theory and Industrial Practice. Darmstadt, Germany: John Wiley & Sons. Shahidi, F., X. Q. Han, and J. Synowiecki. 1995. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem 53(3):285–293. Shahidi, F., and Y. J. Kamil. 2001. Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci Technol 12(12):435–464. Shepherd, J. 2012. Aquaculture: Are the criticisms justified? Feeding fish to fish. World Agric 3(2):11–18. Subhan, F., M. Ikram, A. Shehzad, and A. Ghafoor. 2015. Marine collagen: An emerging player in biomedical applications. J Food Sci Technol 52(8):4303–4307. Suganthi, S. H., and K. Ramani. 2016. Microbial assisted industrially important multiple enzymes from fish processing waste: Purification, characterization and application for the simultaneous hydrolysis of lipid and protein molecules. RSC Adv 6(96):93602–93620. Tahergorabi, R., S. K. Beamer, K. E. Matak, and J. Jaczynski. 2012. Functional food products made from fish protein isolate recovered with isoelectric solubilization/precipitation. LWT Food Sci Technol 48(1):89–95. Taheri, A., S. A. A. Anvar, H. Ahari, and V. Fogliano. 2013. Comparison of the functional properties of protein hydrolysates from poultry byproducts and rainbow trout (Onchorhynchus mykiss) viscera. Iran J Fish Sci 12(1):154–169. Thiansilakul, Y., S. Benjakul, and F. Shahidi. 2007. Compositions, functional properties and antioxidative activity of protein hydrolysates prepared from round scad (Decapterus maruadsi). Food Chem 103(4):1385–1394. USDA. 2016. National Nutrient Database for Standard Reference Release 28. https://ndb.nal .usda.gov/ndb/search/list (accessed December 2, 2016). Vallejos, N., G. González, E. Troncoso, and R. N. Zúñiga. 2014. Acid and enzyme-aided collagen extraction from the byssus of Chilean mussels (Mytilus chilensis): Effect of process parameters on extraction performance. Food Biophys 9(4):322–331. Wang, Y., and J. M. Regenstein. 2009. Effect of EDTA, HCl, and citric acid on Ca salt removal from Asian (silver) carp scales prior to gelatin extraction. J Food Sci 74(6):426–431. Processing of Low-Value Fish, Coproducts, and By-Catch 119

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Ausra Sipailiene

CONTENTS 5.1 Filleting and Mincing/Deboning...... 122 5.2 Washing...... 122 5.3 Refining...... 123 5.4 Dewatering/Screw Pressing...... 124 5.5 Blending with Cryoprotectants...... 124 5.6 Block Forming...... 126 5.7 Freezing...... 126 5.8 Frozen Storage...... 126 References...... 128

Surimi is stabilized myofibrillar protein obtained from mechanically deboned fish flesh that is washed with water and blended with cryoprotectant (Okada, 1992) and is considered as intermediate product (Agustini et al., 2008). According to literature, surimi originates from Japan (Fatin et al., 2015). The frozen surimi processing technology was developed in the 1960s, when it was discovered that sugar was an effective cryoprotectant for fish protein (Park et al., 2014). The growth of the surimi industry was based on the Alaska pollock or walleye pollock (Theragra chalco gramma) (Park et al., 2014). Due to the limited fish resources, more attention has been paid to dark muscle fish as a potential alternative raw material for processing of surimi (Chaijan et al., 2010). Consequently, various species other than Alaska pollock are commonly used as surimi raw material. They now account for about 50% of total surimi production and include Pacific whiting Merluccius( productus), arrowtooth flounder Atherestes( stomias), blue whiting (Micromesistius poutassou), various types of mackerel (Scomber spp.), menhaden (Brevoortia spp.), bigeye snapper (Priacanthus spp.), threadfin bream Nemipterus( japonicus), lizardfish Saurida( tumbil), croaker (Pennahia, Johnius spp.), tilapia (Oreochromis niloticus) (Tina et al., 2010), hoki (Macruronus novaezelandiae), jack mackerel (Trachurus murphyi), atka mackerel (Pleurogrammus monopterygius), sardine (Sardinia melanostrichus), Northern blue whiting (M. poutassou), Southern blue whiting (Micromesistius aus tralis), Peruvian anchovy (Engraulis ringens), American grondin (Prionotus stepha noprys), bereche (Larimus pacificus), chiri (Peprilus medius), barracuda (Sphyraena spp.), sea eel (Congresoxs spp.), and hairtail (Trichiurus spp.) (Vidal-Giraud and Chateau, 2007; Park et al., 2014).

121 122 Trends in Fish Processing Technologies

However, overfishing of lean species has prompted the surimi industry to process nonfish species such as giant squid Dosidicus( gigas) (Campo-Deaño et al., 2010). On the other hand, the recent development in the aquaculture industry worldwide provides a good ground for freshwater fish as an attractive raw material for surimi processing in the future. Among the aquaculture species, tilapia and silver carp (Hypophthalmichthys molitrix) seem to be the most promising ones (Rohani et al., 1995). However, the gel-forming properties of silver carp muscle proteins depend on the seasons. As reported by Yuan et al. (2005), surimi prepared in cold season (winter and spring) formed gel at 30°C, while surimi prepared in hot season (autumn and summer) required 40°C to induce gelation. Higher gelling temperature for hot season surimi was attributable to the more stable nature of the myofibrillar protein. The quality and characteristic of frozen surimi are affected by various factors such as the fish species used and fish freshness. Benjakul et al. (2005) observed differences in microstructure of surimi gel from different species such as threadfin bream, bigeye snapper, lizardfish, and croaker. Fresh or ice-stored fish are commonly used for surimi production worldwide. According to the literature, surimi gels from frozen fish had more poorly organized network and poorer gel-forming ability compared to those from fresh fish (Benjakul et al., 2005). Besides fish species, differences in habitat temperature, salt solubility, and thermostability of muscle protein, protease and transglutaminase activity also have significant effects on the quality of frozen surimi, especially in terms of its gel-forming ability (Okazaki and Kimura, 2014). Surimi processing is a continuous process involving several operations such as unloading/receiving/holding, sorting, heading, gutting, filleting, mincing/deboning, washing, dewatering, refining, screw pressing, blending with cryoprotectants, block forming, freezing, and frozen storage (Park, 2012).

5.1 FILLETING AND MINCING/DEBONING The heads and viscera are removed before filleting. This step influences the quality and quantity of fish mince, as most of the microorganisms and enzymes are found in viscera and gills. During subsequent processing, the muscle tissues are separated from the skin of fish fillets or frames with deboner with perforated holes of 3 or 5 mm (Kim and Park, 2007).

5.2 WASHING The washing stage of surimi processing is very important since it has a direct influence on its quality. This process is needed not only for removing fat and undesirable materials, such as blood, pigments, and odorous substances, but more importantly, for increasing the concentration of myofibrillar protein in the surimi (Nopianti et al., 2011). The washing process involves mixing the minced meat with cold water. The important parameters of the water-leaching process are temperature, hardness, pH, and salinity of water. The level of chlorination may also exert a bleaching or deodor- izing effect (Park and Lanie, 2000). High concentrations of Ca2+ and Mg2+ in the wash water not only accelerate the denaturation of actomyosin during storage of Advances in Surimi Processing 123 surimi but also decrease the temperature tolerance of proteins during the washing process (Ohshima et al., 1993). Balange and Benjakul (2009) demonstrated that the alkaline-saline washing process for surimi with 0.25% oxidized tannic acid added showed an increase in breaking force and deformation in Mackerel surimi, compared with that of surimi produced using a conventional washing process, thus improving the gel properties. The surimi washing process is, in general, repeated from two to five times. It has been estimated that washing water represents 60% of the total effluent volume, and more than 70% of protein losses occurred during the first washing operation in two-step industrial washing process of surimi manufacture (Jaouen and Quemeneur, 1992). It indicates that all these washing steps require large quantities of freshwater and produce a corresponding volume of wash water rich in organic matter (Martín- Sánchez et al., 2010). In this context, recovering of fish proteins from surimi wash water is important not only to reduce the negative environmental impact or the cost of waste disposal but also to generate potential profits by returning the recovered protein to the process to increase surimi yield (Bourtoom et al., 2009). For instance, 17%–20% soluble proteins are leached during the washing process (An and Visessanguan, 2000). According to Stine et al. (2012), the addition to surimi of 5% recovered protein concentrate from surimi wash water by membrane filtration technology will not adversely affect the storage at −20°C and will give the possibility to obtain a fish protein ingredient from the recovered protein or to add it back to surimi products at a low percentage. On the other hand, most enzymes originating from the muscle are washed out in the waste water. The enzymes identified in surimi wash water were cysteine lyso- zomal cathepsins (cathepsins B and L). It was found that the most predominant protease in surimi wash water is cathepsin L (An and Visessanguan, 2000). Numerous studies have been published on methods of recovering proteins and enzymes from surimi wash water. Methods such as ultrafiltration (Lin et al., 1995), precipitation by shifting the pH and use of organic solvent (Bourtoom et al., 2009), precipitation by shifting the pH and then applying a rapid heat treatment to 60°C (Mireles DeWitt and Morrissey, 2002), membrane filtration (Stine et al., 2012), or combined ohmic heat treatments with ultrafiltration (Huang et al., 1997) can be employed to recover water-soluble proteins from surimi wash water. The method of recovering proteins from surimi wash water should be chosen according to quality, quantity, and end use of the recovered proteins.

5.3 REFINING The refining process removes connective tissues, scales, and pin bones (Park, 2012). There are few studies regarding the effects of connective tissues on surimi quality; however, it is established that connective tissues are generally removed from leached minced fish meat because they lead to brittle surimi gels, whose main protein is thermo-reversible collagen (Okazaki and Kimura, 2014). The refiner is commonly a rotary drum with small size pores of size 1.5–1.7 mm in diameter (Park, 2012). Another important parameter of this process is the running speed of the refiner 124 Trends in Fish Processing Technologies drum. Running the refiner at a slower speed with a smaller screen size results in cleaner surimi with less recovery as compared to running refiner at a faster speed with a larger screen size (Kim and Park, 2007). Temperature control at this stage is very important because an increase in the temperature of the minced fish meat results in denaturation of protein and loss functionality of protein (Okazaki and Kimura, 2014).

5.4 DEWATERING/SCREW PRESSING During the washing step, fish protein picks up water, so the dewatering step is very important in processing of surimi. Excess water in the washed meats is removed mechanically using a screw press. It is ideal to reduce the moisture content to 82%– 85% by controlling the speed of the screw press (Park, 2012). Nowadays, decanter centrifuges are used in many industrial applications for separation of solids from liquid with high solid contents. Its application has been successful in surimi processing technology too. According to Kim and Park (2007), three main factors influence the separation of mince and water in a decanter centrifuge: (I) the design of the decanter, (II) the composition of the liquid and particles to be separated, and (III) process-related aspects (Kim and Park, 2007). Decanter technology has also been successfully used to recover insoluble particles from surimi wash water (Park, 2012). Besides, decanter centrifuges have been adopted to recover large particles from the waste streams in the surimi industry. The use of a decanter centrifuge allows recovering up to 80% insoluble solids from rinsing and dewatering waste water. Surimi produced through a decanter is assigned as recovery-grade surimi, which possesses fairly good color and low impurities, but is usually of lower gel strength (Kim and Park, 2007). To enhance the dewatering efficacy, 0.03% to 0.6% NaCl is incorporated in wash water during the last washing cycle of surimi (Okada and Tomoto, 1986). Also, Lertwittayanon et al. (2013) reported that washing mince with 0.45% NaCl solution containing 20 mM MgCl2 during third washing cycle could improve the dewatering process and gel-forming ability of surimi from yellowtail barracuda. Furthermore, a fine and ordered network could be formed in received surimi.

5.5 BLENDING WITH CRYOPROTECTANTS A mixture of sucrose and sorbitol and polyphosphates is used as cryoprotective additives in surimi production to stabilize fish proteins against denaturation during frozen storage. It is important to maintain a low temperature as much as possible to prevent denaturation of fish meat proteins during this step. A silent cutter equipped with a cooling unit is generally used since the temperature of fish meat tends to increase (Okazaki and Kimura, 2014). Mixture of sucrose and sorbitol (1:1) incorporated in surimi at 8%–9% (w/w) results in an undesirable sweetness in final products. There is interest to identify other cryoprotectants with reduced sweetness for use in surimi (Sych et al., 1991; Liu et al., 2014). Advances in Surimi Processing 125

Recently, many other cryoprotective compounds have been tested that can be used with equal efficiency and have benefits such as reduced sweetness, low tend ency to cause Maillard browning in white gel products, lower price, and reduced caloric value (Sultanbawa and Li-Chan, 1998). Cryoprotectants proper for surimi are well described by Nopianti et al. (2013). Many studies have been carried out investigating low-sweetness sugars, such as lactitol, trehalose, palatinose (isomaltulose), and polydextrose (CAS no. 68424-04-4). Polydextrose is a water-soluble polymer of glucose that provides to foods the bulk and texture of sucrose (Burdock and Flamm, 1999; Nopianti et al., 2013). Trehalose is a disaccharide with low caloric value and sweetness that has only 45% of sucrose sweetness. Besides, it does not cause Maillard browning (Higashiyama and Richards, 2012). Trehalose properties are stable and can protect biological cells under unfavorable conditions (Ren et al., 2001). It was established that trehalose possesses protective effect against thermal inactivation of enzymes and its effec tiveness was correlated with its large hydration volume (MacDonald et al., 2000). Recently, Pan et al. (2010) have declared that trehalose possessed better cryoprotective effect on Grass carp (Ctenopharyngodon idellus) protein than sorbitol/sucrose did. Furthermore, they suggested that trehalose can take the place of sucrose and sorbitol and can be used as a new kind of non sweet cryoprotectant for surimi. Dey and Krushna (2011) tested chitosan that rendered and improved the gel strength of croaker muscle protein without having any adverse effect on acceptability during 6 months of frozen storage. Furthermore, they concluded that chitosan can be effectively used as an alternative cryoprotectant for stabilization of croaker muscle protein during frozen storage. The sodium lactate has no sweetness and appears to have good potential as a cryoprotectant and stabilizer of fish actomyosin. It is used as an emulsifier, flavor enhancer, flavoring agent, humectant, and pH control agent (Zhou et al., 2006). According to the literature, 6% (w/v) sodium lactate and 0.3% sodium tri poly phosphate prevented loss of Ca2+ ATPase activity of actomyosin extracted from croaker muscle more than sucrose/sorbitol, during isothermal storage at (−20 ± 2°C) (Dey and Dora, 2010). MacDonald and Lanier (1994) have reported that 6% (w/v) sodium lactate concentration recovered almost 80% Ca2+ ATPase activity in case of freeze– thaw Tilapia muscle (MacDonald and Lanier, 2006). Carvajala et al. (1999) investigated maltodextrins of different molecular weights (MWs) as cryoprotectants in Alaska pollock surimi. The authors demonstrated that lower-MW maltodextrins possessed cryprotective effect similar to mixtures of sucrose and sorbitol likely via a preferential solute exclusion mechanism. Higher-MW maltodextrins acted as cryoprotectants at lower storage temperatures by a reduced water mobility mechanism (Carvajala et al., 1999). Currently, there is a growing scientific interest in the potential cryoprotective property of protein hydrolysates. According to Cheung et al. (2009), fish protein hydrolysates (FPHs) possessed comparable or better cryoprotective properties than a mixture of sucrose–sorbitol sample. Furthermore, the enhanced cryoprotection exhibited by FPH could also be attributed in part to its composition in free amino acids, especially the high contents of Asp, Glu, Arg, and Lys. 126 Trends in Fish Processing Technologies

Phosphates have been widely accepted as additives in fish and seafood to improve the functional properties of surimi (Chang and Regenstein, 1997). Phosphate is normally added to surimi in combination with sugar or sorbitol (Sultanbawa and Li-Chan, 2001). Julavittayanukul et al. (2006) noted that sodium pyrophosphate exhibited superior gel-strengthening effect, whereas sodium hexametaphosphate showed the adverse effect on surimi gelation. Pyrophosphate has been reported to dissociate protein complexes, leading to improved gel-forming ability (Matsukawa et al., 1996).

5.6 BLOCK FORMING The surimi that has been dewatered and mixed with cryoprotectants is ready for freezing. The product is weighed into blocks of 10 kg each, in polyethylene bags, and placed in freezer pans (Sonu, 1986).

5.7 FREEZING Factors influencing protein denaturation during freezing and frozen storage are influenced by the salt concentration, pH, ionic strength, surface tension, and the physical effects of ice and dehydration (Park, 1994), pretreatment and the degree of the autolytic process before freezing, the freezing rate and temperature, the storage temperature and time, and storage condition stability (Zayas, 1997). As proposed by Moosavi-Nasab et al. (2005), rapid freezing by immersion in liquid air before storage at −20°C is effective in maintaining the secondary structure of proteins during long-term frozen storage.

5.8 FROZEN STORAGE Frozen storage of surimi is very important for the functional properties of the myofibril proteins. Hydrophobic bonds are involved with a formation of intermolecular cross-linkages with same groups nearby, and this process leads to protein aggregation, loss of gelling and water-holding capacity (WHC), and decrease of myosin ATPase activity (Byung-Jin, 2014). One possible opportunity to improve the gelling capacity of frozen surimi is hydrostatic high-pressure (HHP) processing. Moreno et al. (2015) declared that structural changes induced by HHP (40, 125, and 200 MPa) in frozen flying fish (Parexocoetus brachyterus) surimi improved the structural, mechanical, and rheological properties of suwari gels. The results of Cando et al. (2015) demonstrated that application of HHP increased significantly surimi breaking deformation and water-binding capacity. Furthermore, the combination of HHP processing and lysine addition possessed a synergic effect and resulted in an increase of surimi breaking deformation and water-binding capacity, regardless of NaCl concentration. The surimi production growth worldwide increased also the interest for new processing methods able to provide ingredients such as surimi powder obtained by drying and protein concentrates recovered from by-products by pH shift method. Santana et al. (2012) reviewed the technology of surimi powder and discussed its Advances in Surimi Processing 127 potential of application. As the authors have mentioned, surimi powder has good functional properties, such as gelation, WHC, and emulsifying and foaming properties, and the gel-based fish products can be made from surimi powder. Different drying methods such as freeze drying, spray drying, oven drying, solar drying, or mechanical drying can be used for developing surimi powder. The use of the freeze-drying method to produce dried surimi powder has been developed and proposed as mostly suitable for manufacturing of imitation seafood products. The freeze-dried surimi powder from saithe leached mince containing cryoprotectant had superior functional properties and stability than spray-dried sample did (Shaviklo et al., 2010). On the other hand, freeze drying is more expensive in comparison with air drying because of the energy required to maintain the vacuum condition and to keep the temperature low (Ratti, 2008). Spray drying is done at higher temperatures than freeze drying and is the most commonly used method for drying food proteins. Nonetheless, this method is more detrimental to protein quality. Musa et al. (2005) declared that threadfin bream surimi powders obtained after freeze and oven drying can be used as additive in food formulation due to its functional properties. However, oven-dried surimi powder possessed low quality and was affected by the drying conditions. Another potential drying method for producing surimi powder is solar drying. This method is environmentally friendly and less expensive compared to other drying methods. However, as found by Musa et al. (2002), solar-dried surimi powder possessed lower emulsion stability (44%) than oven-dried surimi powder (66%), but there were no differences observed in WHC between solar-dried surimi powder and oven-dried surimi powder. Many proteins, when subjected to freezing and dehydration stress (Shaviklo, 2015), may display problems due to the conformational changes. Consequently, the production of dried surimi powder requires the addition of a cryoprotectant to protect the protein during the drying process (Huda et al., 2001). The tested cryoprotectants as sucrose, sorbitol, polydextrose, isomaltulose, and trehalose played a role in maintaining the functional properties of surimi powder against heat denaturation during the oven drying process at 60°C ± 5°C. Treated surimi powders exhibited higher emulsification, good foaming properties, and better solubility attributes compared to the surimi powder without added cryoprotectant (Huda et al., 2012). Surimi in a freeze-dried state would greatly reduce the product transportation and shipping costs. It also leads to convenience in utilization and eliminates tempering and breaking of blocks before use (Reynolds et al., 2002). The dried surimi powder might be one of the possibilities to reduce expenses of frozen storage in the surimi industry, since in powdered form, surimi is shelf-stable and does not need refrigeration or freezing to be stored (Santana et al., 2012). The protein solubility and molecular-weight of freeze-dried sarcoplasmic proteins from rockfish treated under low and high pH and different NaCl concentrations were explored by Kim et al. (2005). They concluded that the solubility of freeze- dried sarcoplasmic proteins was significantly suppressed at an acidic pH (2.0–4.0) and in the presence of high salt concentration (0.5 M NaCl). Besides, these proteins with improved fracturability properties of surimi positively influenced the texture, but it negatively affected the color of surimi. 128 Trends in Fish Processing Technologies

Acid and alkaline-aided solubilization has been shown to be a potential method for maximal protein recovery from muscle foods (Nopianti et al., 2011). An important feature with the pH shift processes is that the proteins are partly unfolding when muscle proteins are exposed to low and high pH. The subsequent step of this process is an isoelectric precipitation of the proteins by adjusting the pH to their isoelectric point (Kristinsson and Hultin, 2003). The major advantage of this process is that muscle proteins can be recovered through economically feasible processes at a relative high yield and the isolated muscle proteins may have improved functionalities and shelf-life over surimi (Kristinsson and Liang, 2006). The use of the acid and alkali aided processes on tilapia muscle proteins showed that the type of recovered proteins was different. Overall, the acid-treated proteins exhibited poorer gelling ability compared to alkali-treated proteins. At the same time, addition of salt in gels improved gel WHC for acid and alkali treated proteins (Ingadottir and Kristinsson, 2010). As stated by Martín-Sánchez et al. (2009), the extraction yield of proteins from fish is challenging, but successful trials with innovative technologies such as the pH-shift method can be considered steps made into the right direction. Batista et al. (2007) reported that sardine (Sardina pilchar dus) proteins, recovered after acidic or alkaline shift processes, showed poor gelling properties and lower gel strength than conventional surimi did. On the other hand, the global yield of proteins was considerably higher as compared to the traditional surimi process (ca. 28%). Fatin et al. (2015) investigated the efficacy of Japanese scad Decapterus( maruadsi) as raw material for surimi production by the pH-shift method. They found that acid and alkaline shift methods exhibited efficient protein recovery and lipid reduction and resulted in products with better textural properties than conventional surimi from Japanese scad. The surimi production is changing significantly. The availability of new technologies allows the use of fish species that are not suitable for commercial sale and also the higher extraction of fish proteins. The future development of surimi processing should be related with identifying new cryoprotectants (both intrinsic and added) and their synergistic effect. Applications of alternative cryoprotectants and new technologies as HHP or pH-shift method offer new opportunities to develop novel products based on surimi that are with low-salt and low-sweeteners content. The use of the pH shift processes offers several advantages, including producing protein isolates from low-value raw materials, pelagic fish, and by-products. This method is an effective way to get higher-quality protein and improve functional properties of proteins. On the other hand, this technology makes it possible to reduce requirements of water.

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Loreto M. Valenzuela, Allison Leyton, and M. Elena Lienqueo

CONTENTS 6.1 Introduction...... 135 6.2 Reduced Salt Content Products and Associated Safety Issues...... 136 6.3 Natural Antioxidants from Plants as Replacers for Conventional Antioxidants...... 139 6.3.1 Antioxidants from Plants...... 140 6.3.2 Antioxidants from Seaweed...... 140 6.3.2.1 Fucoxanthin...... 140 6.3.2.2 Phlorotannins...... 141 6.3.2.3 Sulfated Polysaccharide...... 141 6.3.3 Application of Natural Antioxidants to Preserve Fish Products...... 141 6.3.3.1 Polyphenols...... 141 6.3.3.2 Algae and Seaweed...... 142 6.4 Antimicrobial Preservatives from Microorganisms and Plants...... 143 6.4.1 Essential Oils...... 143 6.4.2 Plant Extracts...... 144 6.4.3 Chitosan...... 144 6.4.4 Probiotics...... 145 6.5 Concluding Remarks...... 146 Acknowledgments...... 146 References...... 146

6.1 INTRODUCTION Many food products are perishable by nature and require protection from spoilage during their preparation, storage, and distribution to reach final consumers as safe and high-quality products. Because many food products are sold in areas of the world that are far remote from their production sites, the need for extended shelf life for these products has also expanded (Holley and Patel 2005). Although improvement of the technologies applied for food preservation have stimulated international trade, traditional methods such as thermal processing, drying, freezing, or modified atmosphere packaging are still applied and have a large impact in sensory and quality properties of fresh fish and seafood (Singh et al. 2016). As an alternative or in addition to those techniques, fish products can be reformulated by the incorporation

135 136 Trends in Fish Processing Technologies of active ingredients, as additives, coatings or as part of the packaging of the fish product. In this chapter, ingredients used to compensate salt reduction, and natural ingredients used with antioxidant and antibacterial purposes in fish and seafood products, are presented.

6.2 REDUCED SALT CONTENT PRODUCTS AND ASSOCIATED SAFETY ISSUES Sodium chloride (NaCl), commonly known as salt, is the main source of sodium in the human diet (He et al. 2012), which is essential to maintain cellular membrane potentials, maintain blood volume and osmotic pressure, and for nutrient absorption in the small intestine (Kloss et al. 2015). On the other hand, high salt consumption has been recognized as detrimental to health. Its consequences are associated with elevated blood pressure (He and MacGregor 2010; Strazzullo et al. 2009), which is a risk factor in stroke and cardiovascular disease (CVD), as well as in kidney disease, osteoporosis, and stomach cancer (Caron et al. 2016; Cook et al. 2007; He and MacGregor 2010; Strazzullo et al. 2009). In several countries in Europe, over 40% of the population have hypertension, mostly in countries that present a high salt consumption (Brown et al. 2009). The World Health Organization recommends a daily salt intake of 5 g per day, which would reduce 23% of strokes and 17% of CVD (Strazzullo et al. 2009). It is even suggested that a further reduction to 3 g salt per day would improve those numbers even better (He et al. 2013; Kloss et al. 2015). However, sodium intake around the world exceeds the physiological need and recommendations by far. Most adults consume over 9 to 12 g per day (Dötsch-Klerk et al. 2015; Greiff et al. 2015b). In Europe, Czech Republic, Slovenia, Hungary, and Portugal are the countries with the highest reported salt intake (12.3–13.6 g/day) (Kloss et al. 2015).* Although natural food products contain salt, more than 75% of it is received from processed food products (Dötsch-Klerk et al. 2015).† For example, processed salmon and tuna contain up to 10 times more salt than the unprocessed one (Table 6.1).‡ Salt in processed food is used to improve flavor, texture, color, and shelf life (Albarracín et al. 2011; Barat et al. 2013; Martínez-Alvarez and Gómez-Guillén 2006). In particular, it is being used historically to preserve food due to its antimicrobial qualities, derived from its ability to lower water activity in foods, creating an unsuitable environment for bacteria growth (Caron et al. 2016) while improving taste and being low cost (Martínez-Alvarez and Gómez-Guillén 2006). Different salting levels are used for fish preservation, from soft salting that still requires refrigeration of the product,

* European Commission Directorate-General Health and Consumers. Survey on Member States’ Implementation of the EU Salt Reduction Framework. 2013. http://ec.europa.eu/health/nutrition _physical_activity/docs/salt_ report1_en.pdf † World Health Organization Regional Office for Europe, Mapping salt reduction initiatives in the WHO European Region. 2013. http://www.euro.who.int/__data/assets/pdf_file/0009/186462/Mapping-salt -reduction-initiatives-in-the-WHO-European-Region.pdf ‡ World Health Organization. Reducing salt intake in populations: report of a WHO forum and technical meeting, October 5–7, 2006, Paris, France. WHO: Geneva, Switzerland; 2007. www.who.int /dietphysicalactivity/Elliot-brown-2007.pdf Reformulation of Preserved Fish Products 137

TABLE 6.1 Sodium Content of Fish Products Food group Range (mg/100 g) Mean (mg/100 g) Reference Fish and fish products 32–6000 512 Webster et al. 2010 Canned fish 32–6000 501 Tuna 130–950 405 Salmon 47–1170 453 Sardines 57–740 342 Anchovies 500–6000 5607 Other 32–3000 568 Chilled fish 350–1170 789 Frozen fish 185–590 370 Salmon Liem et al. 2011 Raw, steamed 110 Canned 570 Smoked 1880 Tuna Raw 47 Canned in oil, drained 290 Canned in brine, drained 320 to high levels of salting, where usually the fish is dehydrated too (Albarracín et al. 2011; Andrés et al. 2007; Barat et al. 2013). Fish products containing salt include dry-cured fish such as cod, bottarga (dry cured fish roe), or mojama (dry cured tuna loin) (Andrés et al. 2007; Barat et al. 2003, 2013; Thorarinsdottir et al. 2002); sea bream (Chouliara et al. 2004; Goulas and Kontominas 2005); chub mackerel (Goulas and Kontominas 2005); smoked salmon (Gallart-Jornet et al. 2007; Sigurgisladottir et al. 2000); and canned, chilled, and frozen fish (Webster et al. 2010). An assessment done in Australia in 2009 indicates that fish processed products contain between 32 and 6000 mg of sodium/100 g of food, with a mean of 512 mg/100 g (Table 6.1) (Webster et al. 2010). Several countries are making efforts to reduce salt consumption.* Strategies include taxes, labeling, and regulation of nutrition-related health claims. Following these regulations, efforts in food reformulation have followed (Kloss et al. 2015), which represents a challenge for the food industry in terms of reduction of shelf life, food safety, and organoleptic properties (Bidlas and Lambert 2008). NaCl is added to fish products to improve their mechanical properties and, thus, their quality (Munasinghe and Sakai 2003; Nguyen et al. 2011; Ramírez et al. 2002). Nguyen et al. (2011) showed that only reducing NaCl concentration, conformational changes of cod muscle proteins during brine salting occur, due to the protein aggregation. Therefore, when reducing salt content in fish, other ingredients must be

* World Health Organization Regional Office for Europe, Mapping salt reduction initiatives in the WHO European Region. 2013. http://www.euro.who.int/__data/assets/pdf_file/0009/186462/Mapping-salt -reduction-initiatives-in-the-WHO-European-Region.pdf 138 Trends in Fish Processing Technologies used as substitutes to maintain palatability, texture, processing yield, and shelf life (Kilcast and Angus 2007).

Studies have been performed where NaCl is partially replaced with KCl, MgCl2, CaCl2, LiCl, or other salts (Munasinghe and Sakai 2003). However, enzyme activity, protein matrix, fish texture, sensory properties, and water-holding capacity (WHC) of the product are affected with those replacements (Andreetta-Gorelkina et al. 2016; Barat et al. 2013; Lauritzsen et al. 2004; Martínez-Alvarez and Gómez-Guillén 2006, 2013; Reddy and Marth 1990; Rodrigues et al. 2005; Weinbert et al. 1984). Protein stability and solubility depends on the salt present (Baldwin 1996; Zhang and Cremer 2006). Lauritzsen et al. (2004) studied the effect of salt composition and pH in heavily salted cod. They found that salt pH is positively correlated with the muscle pH of the fish but negatively correlated with the protein content, lightness, and sensory firmness of cured fish and that the calcium ions increased lightness and firmness while magnesium ions increased the lightness of the fillet. Rodrigues et al. (2005) compared the effect of the salt composition on the chemical, microbial (total viable counts, total coliforms, enterococci, and staphylococci), and sensory quality of salted cod. They found that although the cod salted in brine containing a mixture of NaCl, MgCl2, and KCl had a slightly higher total viable and staphylococci counts, the presence of Mg2+ and K+ ions improved the color of the product too. Martínez-

Alvarez and Gómez-Guillén (2006) studied the influence of NaCl, KCl, MgCl2, and CaCl2 salts at different pH on the muscle proteins of Atlantic cod. They found that muscle composition depends on the salt and pH used and affect protein composition, extractability, and aggregation. More recently, Martínez-Alvarez and Gómez- Guillén (2013) found that high K+ levels produce high water loss, salt uptake, and hardness of the dry product, while the incorporation of Ca2+ and Mg2+ affects WHC in Atlantic cod. Andreetta-Gorelkina et al. (2016) studied the effect of these salts on the extractability of proteins from fresh and frozen haddock muscle and minces, which is related to its WHC (Richardson and Jones 1987). Although sodium ions produced the highest protein solubility, they showed that at low concentrations of

KCl or MgCl2, solubility is not affected, so partial substitution is possible. They also showed that the protein extractability is lower in frozen tissue compared with fresh. Greiff et al. (2015a) investigated the effect of different concentrations of these cations on raw and cooked haddock mince. They found that reduced salt produces moisture increase and WHC reduction in fish mince. Using KCl had only minor effects, while using MgCl2 provokes major changes, such as lower WHC, cooking loss, and pH. The most recommended salt to replace NaCl is KCl (Aliño et al. 2010; Greiff et al. 2015a). It is being shown that both salts have equivalent antimicrobial properties against different pathogenic bacteria (Bidlas and Lambert 2008; Pelroy et al. 1982). Chayovan et al. (1983a, 1983b) showed that the partial replacement of NaCl with KCl in fish sauce produces acceptable flavor, color, and overall quality on the final product. However, Tahergorabi et al. (2012) and Martínez-Alvarez and Gómez- Guillén (2006) found that when replacing NaCl by KCl to surimi gels, their hardness increases, while its bitter taste is responsible for an off-taste (Desmond 2006). Barat et al. (2013) reviewed the effect of partial replacement sodium by Mg2+ in meat and fish products. They found that magnesium affects the WHC of muscle proteins and their solubility and enzymatic activity, in addition to bitterness and off-flavor. Reformulation of Preserved Fish Products 139

Other strategies of salt reduction in fish include the use of whey, milk-based permeates, plant aqueous extracts, or other compounds. Greiff et al. (2015b) studied how milk minerals (low mineral permeate from cheese production and high mineral permeate from milk) affect the quality of fish pudding when used to reduce salt content. They found that low mineral permeate improves the texture and WHC of puddings without changing their flavor, while the high permeate changes texture, WHC, and salt flavor. Lee (2011) studied 13 extracts from plants, from which there were selected for their salty and umami tastes, allowing the same salty taste with 43% reduced NaCl concentration, but there are no reports of their use in fish or seafood products. Ramírez et al. (2002) added microbial transglutaminase to fish paste of silver carp, obtaining better textural properties in terms of gel strength, cohesiveness, and extracted water.

6.3 NATURAL ANTIOXIDANTS FROM PLANTS AS REPLACERS FOR CONVENTIONAL ANTIOXIDANTS One of the reasons of food spoilage and shelf life expiration is the oxidation of lipids. This process occurs when a hydrogen atom (H•) is abstracted from an unsaturated fatty acid (R:H), forming an alkyl radical (R•); after several chain reactions, an alkyl radical can produce a variety of aldehydes, ketones, alkenes, and alkanes, many of which can lead to the formation of undesirable off-flavors and off-odors (Brewer 2011). Oxidation is also related to changes in color, texture, and decline in nutritive value due to degradation of vitamins (Fennema and Tannenbaum 1996). This whole process is translated in food quality deterioration and product rejection. Seafood is prone to lipid oxidation because of the high concentration in polyunsaturated fatty acids (PUFAs), in particular eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6 n-3). Moreover, the presence of heme pigments (myoglobin and hemoglobin) and trace amounts of metallic ions like iron and copper makes the fish more susceptible to lipid oxidation (Hsieh and Kinsella 1989). To prevent food spoilage and delay shelf life expiration due to oxidation, employment of antioxidants has become a necessity for food products that are sensitive to this type of chemical reaction. Antioxidants act by inhibiting formation of free radicals or by interrupting propagation of the free radicals by one (or more) of several mechanisms: (1) scavenging species that initiate peroxidation, (2) chelating metal ions such that they are unable to generate reactive species or decompose lipid peroxides, (3) quenching •O2−, preventing the formation of peroxides, (4) breaking the autoxidative chain reaction, and/or (5) reducing localized O2 concentrations. Therefore, antioxidants through molecular mechanisms promote preservation of sensory and nutritional properties in food. Antioxidants can be synthetic or natural and can originate from different sources. In the market, there is widespread use of antioxidants of synthetic origin, the following being the most widely used in the industry: hidroxybutylanisol (BHA), butylated hydroxy toluene (BHT), propyl gallate, octylgalate, and tertiary butyl hydro- quinone. However, the use of these compounds has been linked to increased tumoral activity (particularly BHA and BHT) (Kahl and Kappus 1993), resulting in strict regulation of their use in foods; thus, research interest for new sources of natural antioxidants grew. The antioxidants of natural origin are a solution to this problem 140 Trends in Fish Processing Technologies

(Torres et al. 2016). Antioxidants mostly used in the industry are ascorbic acid and derivatives (vitamin C), α-tocopherol and derivatives (vitamin E), polyphenols, and carotenoids. The problem with these natural antioxidants is their lower thermostability compared to the ones with synthetic origin (Said et al. 2002) and lack of versatility to be used in foods with both high lipid content and water.

6.3.1 antioxidants from Plants Plant extracts have been used for thousands of years for several purposes in medi- cine, pharmaceutical, and cosmetic applications as well as in food and beverage flavoring (Bakkali et al. 2008). Nowadays, they are considered as natural preservatives or food additives with strong antimicrobial and antioxidant activities applied by the food industry for raw and processed food preservation (Gyawali and Ibrahim 2014). The antioxidant effects of various plant extracts, as well as individual phenolic compounds, have been evaluated and tested in different food model systems. Plant polyphenolics are the compounds holding more than one phenolic ring and are derived from the secondary metabolism of plants (Parr and Bolwell 2000). Phenolic compounds are present in several foods of plant origin, for example, vegetables, fruits, herbs, spices (Embuscado 2015), coffee, tea (Gyawali and Ibrahim 2014), beer, wine, and chocolate (Hollman and Katan 1999). The major antioxidant plant phenolic compounds can be divided into four general groups: phenolic acids (gallic, protochatechuic, caffeic, tannic, ferulic, hydroxycinnamic, and rosmarinic acids), phenolic diterpenes (carnosol and carnosic acid), flavonoids (quercetin and catechin), and monoterpenes or volatile oils (eugenol, carvacrol, thymol, and menthol) (Shan et al. 2005).

6.3.2 antioxidants from Seaweed Seaweeds are multicellular organisms extensively distributed worldwide in marine environment. Up to date, about 20,000 species are industrially grown and processed and consequently used in the production of hydrocolloids, thickeners, gelling agents, or different products among the food, cosmetic, and pharmaceutical industries (Critchley et al. 1998; Reith et al. 2009; Roesijadi et al. 2010). Seaweeds have been classified according to their pigment constituents into green algae Chorophyta( ), brown algae (Phaeophyta), and red algae (Rhodophyta). They can be also differentiated by their principal polysaccharide composition being alginate, starch, and carrageenan, characteristic to brown, green, and red algae, respectively. Seaweeds are a source of many bioactive compounds such as carotenoids, PUFAs, vitamins, sulfated polysaccharides, and polyphenols, being extensively studied due to their pharmacological and nutraceutical properties (Breton et al. 2011; Glombitza and Pauli 2003; Kim and Himaya 2011; Ortiz et al. 2006; Sánchez-Machado et al. 2004; Singh and Sidana 2013; Zubia et al. 2008).

6.3.2.1 Fucoxanthin Fucoxanthin is one of the major xanthophyll pigments of the carotenoid group present principally in brown seaweed, giving them a brown or olive green Reformulation of Preserved Fish Products 141 color (Kim et al. 2010). The structure of fucoxanthin includes allenic bond and 5,6-monoepoxide (D’Orazio et al. 2012). Previous reports revealed that fucoxanthin isolated from different seaweed species such as Myagropsis myagroides, Undaria pinnatifida, and Sargassum fulvellum exhibited antioxidant effects (Woo et al. 2010). Sachindra et al. (2007) assessed the antioxidant activities of fucoxanthin in vitro, where fucoxanthin exhibited antioxidant activities higher or similar to that of α-tocopherol.

6.3.2.2 Phlorotannins The polyphenols compounds present in brown seaweed, known as phlorotannins, are produced exclusively by these types of macroalgae being biosynthesized via acetate malonate pathway (Heo et al. 2005) and derived from the polymerization of phloroglucinol units, which can constitute up to 15% of the seaweed’s dry weight (Arnold and Targett 2002; Ragan and Glombitza 1986). Phlorotannins are present in algae in soluble form, stored in physodes, and in insoluble form fare, taking part in the structure of the cell walls of algae, forming complexes with proteins and alginic acid (Singh and Sidana 2013). The concentration and molecular size of these compounds vary according to intrinsic factors (reproductive condition, age, and size of the algae), as well as by extrinsic factors (environmental and ecological stimuli) (Glombitza and Pauli 2003; Ortiz et al. 2006; Sánchez-Machado et al. 2004; Singh and Sidana 2013). The molecular size of phlorotannins reported varies between 126 Da (phloroglucinol) and 650 kDa (phlorofucoroeckol) (Glombitza and Pauli 2003), which depend on the types of interlinkage present such as phlorotannins with phenyl linkages, ether linkages or ether and phenyl linkages that are characteristic to different types of phlorotannins (Shibata et al. 2004; Targett and Arnold 1998). Leyton et al. (2016) reported that the extract of Macrocystis pyrifera showed a strong DPPH (1,1-diphenyl-2-pricrylhydrazyl) free radical scavenging activity and identified two types of phlorotannins compounds: phloroeckol and a tetrameric phloroglucinol.

6.3.2.3 Sulfated Polysaccharide Sulfated polysaccharides present in brown algae known as fucoidan are composed mainly of sulfated l-fucose and small residues of galactose, mannose, xylose, glucose, uronic acids, and rhamnose (Cazy 2016). This polysaccharide forms part of the structural cell wall (Deniaud-Bouët et al. 2014), supplying flexibility in the structure, maintaining the ionic balance and protection against osmotic water loss (Hahn et al. 2012; Usov and Zelinsky 2013). Isolates from Laminaria japonica and Turbinaria ornata have antioxidant and radical scavenging activities, which depend on structural variations such as molecular weight, substituted groups and positions, sugar moieties, and glycosidic branching of the polysaccharides (Melo et al. 2004; Yang et al. 2008; Zhang et al. 2008).

6.3.3 application of Natural Antioxidants to Preserve Fish Products 6.3.3.1 Polyphenols The effectiveness of aromatic antioxidants is generally proportional to the number of −OH groups present on the aromatic ring(s) (Brewer 2011). However, there is no consensus on the best antioxidant for each fish product. Some studies have determined 142 Trends in Fish Processing Technologies the antioxidant activity among families of compounds. Among hydroxycinnamic acids with similar structures (caffeic, chlorogenic, o-coumaric, and ferulic acids), caffeic acid was the most effective antioxidant in mince mackerel muscle. Furthermore, Maqsood et al. (2014) reviewed the role of phenolic compounds in retardation of lipid oxidation in different seafood systems. Among these, tannic acid demonstrated the highest efficiency in retarding the lipid oxidation in minced mackerel as evidenced by lower peroxide value and thiobarbituric acid-reactive substance values. Studies that use phenolic acids as antioxidant agents in fish matrices include caffeic acid for mackerel mince, tuna slices, fish muscle, bulk cod liver oils, and fish oil (Iglesias et al. 2009; Maqsood and Benjakul 2010; Medina et al. 2009; Thiansilakul et al. 2013); tannic acid for stripped catfish slices, tuna slices, fish mince, and fish oil (Maqsood and Benjakul 2010; Thiansilakul et al. 2013); ferulic acid for mackerel mince (Maqsood and Benjakul 2010); and hydroxycinnamic acid for minced horse mackerel (Medina et al. 2007). The flavonoids used as antioxidant agents include quercetin, quercetin-3-O- glucoside, myricetin, and morin for fish oil (Huber et al. 2009; Montero et al. 2005; Wanasundara and Shahidi 1998a, 1998b) and catechins for fish oil and mackerel mince and patties (Maqsood and Benjakul 2010; Medina et al. 2007; Sørensen et al. 2008; Tang et al. 2001a, 2001b). Plants or extracts rich in polyphenols with antioxidant effect used to control fish oxidation include green tea extracts for fish oil, red drum Sciaenops( ocellatus), large yellow croaker, crucial carp (Carassius auratus), and black sea bream (Sparus macrocephalus) (Bao et al. 2009; Chan et al. 2011; Feng et al. 2012; He and Shahidi 1997; Li et al. 2012b, 2013; Mustafa 2013; Namal 2013; Shahidi and Alexander 1998; Shahidi and Wanasundara 1992; Wanasundara and Shahidi 1998b); tangerine peel (Citri reticulatae pericarpium) extracts for bream (Megalobrama amblycephala) (He and Xiao 2016); grape extracts for fresh and minced Atlantic mackerel, horse mackerel fillets, and red drum S.( ocellatus) (Li et al. 2013; Pazos et al. 2005a, 2005b; Sánchez-Alonso and Borderías 2008); grape seed extracts for silver carp (Shi et al. 2014); extra virgin olive oil extracts for canned tuna (Medina et al. 1999); rosemary extracts for large yellow croaker and crucial carp (Li et al. 2012a) and fish oil (Namal 2013; Uçak et al. 2011; Wanasundara and Shahidi 2005); clove bud extracts for silver carp (Shi et al. 2014); quince extracts for mackerel fillets (Fattouch et al. 2008); turmeric extracts for rainbow trout (Pezeshk et al. 2011); potato extracts for minced horse mackerel (Farvin et al. 2012); and brown lead seed extracts for minced mackerel (Benjakul et al. 2013). Other polyphenols used to control fish oxidation include barley husk for salmon (Pereira de Abreu et al. 2010), annatto seeds and coriander leaves for white hake fish meatballs (Sancho et al. 2011), 6-gingerol for drum fillets (Mi et al. 2016), and coffee pulp for smoked fish (Adebowale et al. 2012).

6.3.3.2 Algae and Seaweed With respect to algae and seaweed used in fish preservation, Roohinejad et al. (2016) and Gupta and Abu-Ghannam (2011) present a complete review of current strategies, which include the use of ethanolic extract of Alga bifurcaria to control oxidation and Reformulation of Preserved Fish Products 143 bacterial growth in chilled storage of megrim (Miranda et al. 2016) and Graeloipia filicina used to control oxidation in fish oil (Athukorala et al. 2003). From seaweed extracts, cochayuyo, sea lettuce, ulte, and red luche have been used to control oxidation on canned Atlantic salmon (Salmo salar) (Ortiz et al. 2014); cod protein hydrolysate and Fucus vesiculosus ethyl acetate extract for hemoglobin-fortified washed cod mince, iron-containing cod liver oil emulsion (Jónsdóttir et al. 2016), fish cakes (Dellarosa et al. 2015), and washed cod muscle (Wang et al. 2010); eucheuma extract for fish cutlet (Senthil and Mahadevaswamy 2005); nori Porphyra( tenera) and Hijiki (Hijikia fusiformis) for minced tilapia (Ribeiro et al. 2014); and Sargassun kjellmanianurn for fish oil (Yan et al. 1998).

6.4 ANTIMICROBIAL PRESERVATIVES FROM MICROORGANISMS AND PLANTS The microorganisms that commonly cause seafood-borne bacterial illness are Salmonella species, Listeria monocytogenes, Vibrio species, Clostridium botulinum, and Aeromonas hydrophila (Singh et al. 2016). Among all, Salmonella is the main cause of seafood-borne bacterial illness and detention of imports at the border, followed by L. monocytogenes, which is commonly observed in seafood due to its presence as an environmental inhabitant in processing facilities (Norhana et al. 2010). Besides, Vibrio species are present in oysters and the most virulent species are Vibrio vulnificus.Moreover, the toxin produced by C. botulinum, a spore-forming anaerobe, is a big concern because it is heat stable and highly toxic and can grow at temperature exceeding 3.3°C. Finally, A. hydrophila is commonly found in seafood and in fresh-flowing stagnant and brackish water (Singh et al. 2016). An antimicrobial agent is a chemical preservative that can be incorporated into the food or in the packaging material to induce antimicrobial activity. Natural antimicrobials can be derived from plants, animals, algae, and bacteria, and they have been reviewed by Gyawali and Ibrahim (2014) and Singh et al. (2016). They can be applied in different ways, such as dipping and spraying solutions, or they can also be incorporated into edible films or coatings, as carriers (Erkan 2015). Different compounds can also be used combined with other technologies to work synergistically as antimicrobials and antioxidants (Lucera et al. 2012). In this section, some of the natural antimicrobials used in fish and seafood preservation are highlighted.

6.4.1 essential Oils Essential oils (EOs) are aromatic compounds obtained from plants such as flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, and roots (Burt 2004). It is well established that EOs have antimicrobial activity, and they are of hydrophobic nature, so they can disrupt the cell membrane and mitochondria (Burt 2004). Besides, their activity depends on each particular composition, which includes phenolic compounds such as carvacrol, thymol, linalol, limonene, menthol, among others, that disturb the cytoplasmic membrane of bacteria, the proton motive force, the electron flow, the active transport, and producing cell content coagulation (Burt 2004; Singh et al. 2016). In the book by Baser and Buchbauer (2010), EOs have been reviewed in 144 Trends in Fish Processing Technologies terms of their history, sources, methods of extraction, chemistry, activity, and applications, and by Bakkali et al. (2008) in terms of their biological activity. EOs that have been commonly studied to extend shelf life of fish products are derived from thyme, rosemary, bay, marjoram, oregano, basil, garlic, sage, green tea, nettle, flaxseed, black cumin, clove, cinnamon, grapefruit, lemon, and lemon- grass (Patel 2015; Sternisa et al. 2016). Some examples are the use of thyme EO for sea bass fillets (Kostaki et al. 2009); clove EO for silver carp fillet (Jalali et al. 2016), and cod fillets (Gómez-Estaca et al. 2010); oregano EO for rainbow trout (Frangos et al. 2010; Kazemi and Rezaei 2015; Matta et al. 2015; Mexis et al. 2009; Pyrgotou et al. 2010) and grass carp muscle (Burt 2004; Lin et al. 2004; Wu et al. 2015); thyme and laurel EO for blue fish (Erkan et al. 2011); thyme, oregano, lemon, orange, and tangerine EOs for fresh raw catfish fillets (Desai et al. 2012); clove, cumin, and spearmint EOs for turbot fillets (Cai et al. 2015); thyme and rosemary EOs for minced fish (Abdollahzadeh et al. 2014); oregano EO for tarama salad (cod’s roc salad) (Koutsoumanis et al. 1999); and thymol coating for peeled shrimps (Mastromatteo et al. 2010). Moreover, EOs have been used as antioxidant in addition to being an antimicrobial agent in fish matrices. Some examples are cinnamon (Ojagh et al. 2010), oregano (Jouki et al. 2014) or thyme EO for rainbow trout (Jouki et al. 2014; Yang et al. 2015), rosemary EO for silver carp fillet (Abdollahi et al. 2014), thyme and laurel EOs for blue fish (Erkan 2015), thyme EO for sliced fresh Channa argus (Jouki et al. 2014; Yang et al. 2015), lippia alba EO for silver catfish frozen fillets (Veeck et al. 2013), and clove EO for sardine patties (Salgado et al. 2013).

6.4.2 Plant Extracts Although the polyphenols present in plant extracts are mainly known by their antioxidant activity, some of them also present antibacterial properties and have been used in fish preservation. The antimicrobial activity of phenolic extracts has been attributed to cell membrane disruption (Kanatt et al. 2010). Some examples are the use of rosemary plant extracts (Rosmarinus officinalis) for fresh chilled Atlantic salmon (S. salar) (El-Sayed et al. 2015), tangerine peel (Citri reticulatae pericarpium) extracts for bream (M. amblycephala) (He and Xiao 2016), tea polyphenols for black sea bream (S. macrocephalus) (Feng et al. 2012), grape pomace extract for silver carp (Hypophthalmichthys molitrix) fillets (Hasani et al. 2015), green tea polyphenols and rosemary extract in refrigerated large yellow croaker (Li et al. 2012a; Zhu et al. 2015), grape seed extract and tea polyphenols in red drum (S. ocellatus) fillet (Li et al. 2013), coffee pulp in smoked fish (Adebowale et al. 2012), rosemary extract for fish burgers (Uçak et al. 2011), and catechin for Pacific white shrimp (Nirmal and Benjakul 2009).

6.4.3 chitosan Chitosan is a natural polymer that presents antibacterial properties, as reviewed by Kong et al. (2010). Although the exact mechanism is unknown, this activity is related to the electrostatic interaction between the polycationic structure of chitosan and Reformulation of Preserved Fish Products 145 the mainly anionic structure of bacteria cell membrane, altering their permeability (Kong et al. 2010; Rabea et al. 2003; Singh et al. 2016). Chitosan has been used as part of films and coatings (edible or not), with or without other active ingredients, to control bacterial growth and extend the shelf life of fish and seafood products, as reviewed by Singh et al. (2016) and Yuan et al. (2016). For example, it has been used to reduce microbial growth in lingcod fillets (Duan et al. 2010a, 2010b), sea bass (Günlü and Koyun 2013), Atlantic salmon (S. salar) (Soares et al. 2016; Souza et al. 2010; Fernández-Pan et al. 2015), Atlantic cod (Gadus morhua) (Jeon et al. 2002), herring (Clupea harengus) (Jeon et al. 2002), trout fillets (Jasour et al. 2015), rainbow trout (Chamanara et al. 2012; Ojagh et al. 2010), sea bream (Sparus auratus) (Speranza et al. 2013), grass carp (Wu et al. 2014), silver carp (Abdollahi et al. 2014; Fan et al. 2009), large yellow croaker (Pseudosciaena crocea) (Li et al. 2012a), tuna (Fernández-Pan et al. 2015), sardine fish steaks (Fernández-Pan et al. 2015; Mohan et al. 2012; Shakila et al. 2016), C. argus (Yang et al. 2015), Argentinian hake (Merluccius hubbsi) burgers (Schelegueda et al. 2016), fish sausages (Alemán et al. 2016), oysters Crassostrea( gigas) (Cao et al. 2009), and shrimp (Arancibia et al. 2015).

6.4.4 Probiotics Probiotics are live microorganisms that can be beneficial to the host (Pineiro and Stanton 2007). Lactic acid bacteria (LABs) include lactobacilli, bifidobacteria, nonpathogenic Escherichia coli, bacilli, and yeasts such as Saccharomyces boulardii (Dobson et al. 2012). They act in an antagonistic and inhibitory manner against spoilage bacteria, while producing lactic acid that drops the pH, inhibiting microbial growth (Ghanbari et al. 2013). LABs produce bacteriocins, peptides, and proteins with antibacterial activity, in particular effective against gram-positive and spore forming bacteria, as reviewed by Gyawali and Ibrahim (2014), Es and Bhat (2016), Ghanbari et al. (2013), Thomas et al. (2012), and the Food Standards Agency.* This is particularly relevant in fish spoilage from bacteria such as L. monocytogenes, a gram-positive bacterium, which is present in the terrestrial and aquatic environment. Its optimum temperature is 30°C–37°C but it can survive between 1°C and 45°C, which makes it very dangerous if present in raw fish (Thomas et al. 2012). The antimicrobial effect of LABs and their bacteriocins has been demonstrated for catfish fillets (Kim and Hearnsberger 1994; Kim et al. 1995), salmon (Ahmadi et al. 2015; Baños et al. 2016; Nilsson et al. 1999), rainbow trout (Behnam et al. 2015; Nykänen et al. 2000), Indian mackerel fillets (Sudalayandi 2011), smoke rainbow trout (Nykänen et al. 2000), cold smoked salmon (Aasen et al. 2003; Brillet et al. 2005; Duffes et al. 1999; Ghalfi et al. 2006; Leroi et al. 1996; Neetoo et al. 2008; Nilsson et al. 1999, 2004; Perera et al. 2015; Tahiri et al. 2009; Tomé et al. 2007, 2008; Vescovo et al. 2006; Weiss and Hammes 2006; Yamazaki et al. 2003), jumbo squid (Ramírez-Suárez et al. 2015), megrim (Lepidorhombus whiffiagoni) (García- Soto et al. 2015), sardine fillets (Elotmani and Assobhei 2004), turbot (Campos et al.

* Systematic and critical review on the potential use of bacteriophages on foods | Food Standards Agency. 146 Trends in Fish Processing Technologies

2012), fresh plaice (Altieri et al. 2005), mussels (Ahmadi et al. 2015), and shrimp (Einarsson and Lauzon 1995; Fall et al. 2010; Hwanhlem et al. 2015; Karthik et al. 2013; Katla et al. 2002; Matamoros et al. 2009; Shirazinejad et al. 2010). Moreover, both antioxidant and antibacterial activities of LABs had been demonstrated for silver carp (H. molitrix) fillets (Raeisi et al. 2016), tilapia Oreochromis( niloticus) (Ibrahim and Desouky 2009), vacuum-packed rainbow trout (Oncorrynchus mykiss) (Katikou et al. 2007), horse mackerel fillets (Cosansu et al. 2011), and Argentinian hake burgers (Schelegueda et al. 2016).

6.5 CONCLUDING REMARKS Natural ingredients are a rich source of active components and have been studied in order to compensate for salt reduction, reduce lipid oxidation, and inhibit bacterial growth, at least in laboratory or small industrial scale. In this chapter, we did not discuss components’ concentration, but their effectiveness depends on it. A large concentration of a certain ingredient can produce changes in the color, texture, taste, and flavor of the fish, affecting the quality of the final product. Besides, the majority of these studies focus on only one of these three phenomena, missing the fact that many of the components may have multiple activities. In addition, only a few studies study the synergies or antagonistic effects among different ingredients and with the food matrix. Industrial-scale validation must be performed that includes consumer validation in terms of food safety, quality perception, and organoleptic approval. All final ingredients must fulfill specific regulations in terms of safety, stability, labeling, and information to the agencies and the consumer.

ACKNOWLEDGMENTS Daniela Valenzuela, Fernanda Díaz, Katherina Möller, and Paula Llanquileo helped with the literature search and Dr. Wendy Franco reviewed the chapter.

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Eirin M. Skjøndal Bar, Sunniva Hoel, and Jørgen Lerfall

CONTENTS 7.1 Introduction...... 161 7.2 New Product Development...... 161 7.3 Consumer Food Trends...... 163 7.4 Low Salt...... 164 7.5 Sustainable Seafood Products...... 164 7.6 Lightly Processed Seafood...... 165 7.7 Use of Seaweeds and Seaweed Extracts in NPD...... 166 7.8 Concluding Remarks...... 167 References...... 168

7.1 INTRODUCTION This chapter gives a brief introduction to the process of new product development (NPD), touches upon consumer food trends, and gives a short review on current perspective from some of the more promising areas within seafood research that are expected to impact seafood innovation and NPD efforts in the future.

7.2 NEW PRODUCT DEVELOPMENT Most companies are making some sort of changes to their product portfolio or production over time in order to stay in business. NPD is denoted as the transformation of a market opportunity into a consumer product (a physical object) or service (a nonphysical experience; including feeling, customer value, etc.) (Viswanathan and Ulrich 2001) and is seen as a core activity within a company. However, NPD remains a risky business, with a failure rate of 30% (Cooper 1999). Over the last decade, a variety of methods and tools to aid and facilitate the product development processes have been developed and described in literature (Eppinger and Ulrich 2015; Lindahl 2005). The 2010 American Productivity & Quality Center benchmarking study shows that 88% of US businesses employ a Stage-Gate-based system to manage new products, from idea to launch. Companies that adopted the system were found in the study to receive benefits such as improved teamwork, better success rates, earlier detection of failure, and a better launch; in addition, they were also found to have shorter development cycle times (Kahn 2012).

161 162 Trends in Fish Processing Technologies h h unc view age 5 stlaunc re St La Po te 5 Ga age 4 sting and St validation Te te 4 Ga t tion age 3 ec St velopmen De ess dir oc te 3 eneral pr Ga G s age 2 case Build St busines te 2 ening Ga re ea sc y Id te 1 age 1 opin g Ga St Sc Discover Schematic overview of a typical Schematicof overview Stage-Gate model basedCooper on (Stage-Gate (2008) istrademark a Institute). Productof Development

FIGURE 7.1 New Product Development 163

Examples of successful use of the Stage-Gate approach to NPD within the seafood sector can be found in literature (Altintzoglou et al. 2010a; Stewart-Knox and Mitchell 2003). The basic principle of the Stage-Gate model is to function as a conceptual and operational map designed to move new products from the fuzzy front end of idea generation to launch and beyond. The model consists of a combination of steps and gates that are designed to gather information during the stages and pass through gates that function as decision points, in order for the NPD to enter the next stage, shown in Figure 7.1 (Cooper 2008). Underlying the Stage-Gate model are four central success factors (Cooper 1999):

• Cross-functional teams consisting of both technical and marketing representatives; • A holistic process from idea to launch; • Strong market orientation; and • Clear criteria for go/no go at each gate.

Cooper also stressed the fact that the method should be flexible and adaptable and not strictly regarded as one directional but be open to iterations. In addition, he also highlights the importance of scalability and the necessity to overlap stages in order to reduce development time. The Stage-Gate framework can be modified or expanded to fit the need of the company. An adaptation of the model has been successfully deployed in the case of a small-sized seafood enterprise (Howieson et al. 2014) that is classified as a small and medium-sized enterprise (SME) according to the EU definition. In general, NPD has been regarded as a firm-centered activity often undertaken within the company walls; however, recent studies including end customers in the NPD process have shown promising results in order to increase the likelihood of customer acceptance of new fish food products (Banović et al. 2016).

7.3 CONSUMER FOOD TRENDS The modern consumer is well aware of the nutritional value and health benefits linked to intake of seafood, and fish and seafood are widely accepted to be an essential part of a balanced and healthy diet. The world per capita fish consumption has increased steadily since the 1960s, and the highest fish consumption (26.8 kg per capita/year) was reported for industrialized countries (FAO 2016). Nevertheless, the recommended intake of fish is not achieved in all countries, despite fish-promoting campaigns from public health authorities (Carlucci et al. 2015). The consumer purchasing behavior towards fish and seafood products were system- atically reviewed by Carlucci and colleagues (2015). Over the last decade, issues such as convenience, health, ethics, product variety, value for money, sustainability, and safety have become more important. Moreover, the supermarket chains are increasingly acting as key players in setting product requirements (FAO 2016). The focus on convenience has led to the introduction of an assortment of fish products with different degrees of processing, ranging from portion-sized fresh fillets to preprepared fish-based meals. As a result, the customers buy less of what they do not eat, such as skin, bone, and 164 Trends in Fish Processing Technologies head. Fish overprocessing is in general perceived negatively (Altintzoglou et al. 2010b; Debucquet et al. 2012), and consumers seem to appreciate new convenient fish products when the new products are close to the original raw material characteristics (Carlucci et al. 2015). In a French study on oyster-based products, the most processed products were perceived as less natural, healthy, and safe (Debucquet et al. 2012). Following the trend toward more fish processing within the supply chain, the amount of offal and other by-products increases (Hall et al. 2013). Optimization of by-product utilization remains a major challenge and opportunity for the fish processing industry. European customer preference surveys have shown a general preference toward chilled, fresh fish over frozen, canned, smoked, or salted fish (Norwegian Seafood Council 2016). This creates a large market for high-quality, fresh, and lightly processed seafood. Moreover, health and well-being are reported as increasingly influencing drivers of fish consumption (FAO 2016; Norwegian Seafood Council 2016). As a result, more focus has been set on low-salt products, presenting the producers with new challenges to maintain the expected shelf life, food safety, and product quality.

7.4 LOW SALT As people tend to live longer, both the UK and Norwegian health authorities have separately increased their focus on salt reduction in food, due to the known negative health effect of high sodium intake (Greiff et al. 2015; He et al. 2014). Public health and regulatory authorities (FSA 2004; The Norwegian Directorate of Health 2004; WHO 2006) have published advisory guidelines for reduction of salt intakes down to 5 g/day or lower. Still, in most European countries today, the daily salt intake per person is double the maximal recommended intake. Salt is commonly used in fish processing due to its excellent preservative effects, taste enhancement, as well as positive technological effects (Fuentes et al. 2010; Martínez-Alvarez and Gomez-Guillen 2006). For this reason, there is great interest in developing products with reduced salt content without affecting properties related to sensory parameters, texture, yield, and shelf life. Research has shown promising result when using milk- based permeates with favorable combinations of milk minerals and lactose as natural ingredients in for instance meat and fish products as salt replacers (Greiff 2015). As a focus from governmental authorities and increased regulation, in combination with promising research results, more new product innovations from salt-reducing initiatives are expected to hit the supermarket shelves in coming years.

7.5 SUSTAINABLE SEAFOOD PRODUCTS Greenhouse gas emissions caused by food production are rightfully receiving increased attention worldwide. Due to the dire states and capacity limit of the oceans, the seafood industry needs to be sustainable in order to flourish in the future. With increasing attention to illegal and unsustainable fisheries, sustainable fisheries are becoming certified and labeled. To secure a level of trust and to gain competitive advantage between seafood producers, retailers, and customers, there is need for an ongoing growth in both the certification and labeling industry as well as increasing trade with sustainable seafood produce (Kalfagianni and Pattberg 2013; Konefal 2013; New Product Development 165

Ponte 2012). Although at present, most concerns with buying sustainable seafood have been from a business-to-business perspective, it is estimated that sustainable seafood will be trending in the future based on a growing acceptance and demand from consumers (Alfnes 2017).

7.6 LIGHTLY PROCESSED SEAFOOD Lightly processed convenience foods is a growing segment in the European marketplace (Skipnes et al. 2008). The fish-based products are, however, underrepresented due to a number of unsolved problems, which may cause concerns about food safety and quality. Main problems/issues are related to seasonal variations (Mørkøre and Rørvik 2001; Mørkøre et al. 2010; Roth et al. 2005), variations in freshness (Barat et al. 2006), variations in handling between catch and filleting (Mu et al. 2017), and the differences in functional properties depending on the raw material history (fresh versus frozen raw material) (Cardinal et al. 2001; Deng 1977; Sigurgisladottir et al. 2000). The segment of lightly processed products ranges from raw fillets in vacuum or modified atmosphere packages to lightly salted, lightly marinated, and/or lightly pasteurized products. Several technologies including gentle salting (Åsli and Mørkøre 2012; Gallart- Jornet et al. 2007), modified atmosphere packaging (MAP) (Sivertsvik et al. 2002), soluble gas stabilization (SGS) (Rotabakk et al. 2008; Sivertsvik 2000), sous vide cooking (Baldwin 2012), microwave heating (Stephen et al. 2010), and surface pasteurization (Bremer et al. 2002) have been used alone or in different combinations to enhance the quality and safety and or to increase the shelf life of minimally processed seafood products. In the segment of lightly processed seafood, packaging has become an important hurdle because of milder processing technologies and reduced use of additives in processing (Noseda et al. 2014). Vacuum packaging is easily combined with heat processing (Baldwin 2012). On the other side, traditional MAP is often used on fresh fish where the use of carbon dioxide (CO2) inhibits bacterial growth (Sivertsvik et al. 2002). CO2 will penetrate bacterial membranes of the cell, leading to a decrease in intracellular cytoplasmic pH, causing an inhibition of cytoplasmic enzymes and enzyme production but also altering cell membrane functions including nutrient uptake and absorption (Garcia-Gonzalez et al. 2007; Sivertsvik et al. 2002). Traditional MAP is, however, difficult to combine with heating due to the insulat- ing and exponential nature of the presented gasses. To utilize the positive effect of

CO2 (Sivertsvik et al. 2002) in combination with heating, alternative technologies in combination with vacuum must be used. Promising technologies are SGS that allows

CO2 to enter the flesh before heating and the use of CO2 emitters that allows the CO2 to enter the product after heating (CO2 will be released when the cook loss activates the emitter). Documentation of the synergic effect of CO2 and heat is, however, limited to a study on milk, where dispersion of CO2 in the milk before heating was found to increase the thermic inhibition of Bacillus cereus and Pseudomonas fluorescens (Loss and Hotchkiss 2002). In an ongoing joint project between the Norwegian University of Science and Technology, Trondheim, Norway, and Nofima AS, Stavanger, Norway, the combined 166 Trends in Fish Processing Technologies

effect of CO2 and heat on the quality and shelf life of different seafood products was studied (data not published). The industry is continuously searching for easy and economical processing solutions. The best solution for pasteurization is currently autoclaving (Skipnes 2014). Autoclaves provide a counter pressure and a temperature distribution that are much better at low temperatures (<90°C) compared to alternative methods such as steam cabinets, water baths, or traditional microwave ovens. The counter pressure is also important for the heat transfer due to minimization of the dead space between the product and the packaging material that may occur during heating (Skipnes et al. 2002). This is especially important when an SGS step is used prior to the heating process.

7.7 USE OF SEAWEEDS AND SEAWEED EXTRACTS IN NPD The application of seaweeds and seaweed extracts in new food product development was recently reviewed by Roohinejad et al. (2016). Among marine organisms, edible seaweeds and macroalgae are some of the richest sources of natural antioxidants and antimicrobials traditionally consumed by humans as food (Gupta and Abu-Ghannam 2011). Seaweeds used as ingredient in food, or the specific use of extracts or pure compounds raised from seaweeds, are therefore of great interest for the food industry. One example is the use of seaweed powder or extracts against oxidative stress and lipid oxidation in foods. Moreover, the food industry is still the main market for the seaweed hydrocolloids where they are used as texturing agents and stabilizers (Bixler and Porse 2011). Seaweeds have proved to be an excellent source of micronutrients and macronutri- ents (Gupta and Abu-Ghannam 2011). The most important micronutrient compounds found in seaweeds are vitamins (Ferraces-Casais et al. 2012), sterols (Lopes et al. 2011), and minerals (Ferraces-Casais et al. 2012). Seaweeds are moreover known to be one of the best natural sources of iodine (Misurcova et al. 2011). In addition to micronutrients, seaweeds are a rich source of macronutrient compounds, including proteins and amino acids (essential and nonessential amino acids), carbohydrates, fibers, and fats (Marsham et al. 2007; Patarra et al. 2011). Oxidation of lipids is a serious challenge for the food processing industry. Among actions against oxidation in food products, the use of antioxidants is important. Due to the well-documented antioxidant capacity of seaweeds and seaweed extracts, those extracts are promising agents that can be an alternative to replace synthetic antioxidants such as butylated hydroxyl toluene and butylated hydroxyanisole (Roohinejad et al. 2016). This will provide an environmentally friendly and safe source of antioxidants for further use in food products. Seaweeds and seaweed extracts can be used in a variety of food products (Roohinejad et al. 2016), and promising results have been found when added not only to pork meat, frankfurters and breakfast sausages, beef products, chicken products, cereal products, and milk products but to seafood products too. In seafood processing, several applications have been tested to increase the shelf life and to enhance quality of the product. An illustrative example is a study published by Ortiz et al. (2014), which tested the effect of different seaweed extracts, as covering liquids, on the lipid and sensory quality of canned Atlantic salmon (Salmo salar L.). After New Product Development 167

170 days of canned storage at 40°C, all groups except the control were reported to have an acceptable oxidized odor and characteristic flavor scores. The addition of seaweed extracts resulted in reduced secondary peroxidation in the canned salmon and consequently higher polyunsaturated fatty acid and astaxanthin content retention. Moreover, employing different seaweed extracts as covering liquids in canned Atlantic salmon had no significant effect on sensory parameters. In another study by Ribeiro et al. (2014), the possibility of using Nori and Hijiki seaweeds as substitutes of synthetic preservatives to improve the shelf life and quality of minced tilapia was evaluated. The seaweed extracts showed inhibitory influence on total volatile base nitrogen, whereas the sensory panel detected no differences in the rancid aroma, and only minor differences in the color of the products. The treatments of the minced tilapia samples showed no bacterial counts during 180 days of frozen storage and remained within the standards of quality. The application of the seaweed extracts did, however, not affect the chemical composition of the minced tilapia. In another study, the effect of seaweed (Eucheuma) powder on the quality of fish cutlets was tested (Senthil et al. 2005). It was found that the Eucheuma powder could be incorporated up to a 10% level without affecting the sensory properties of the product. It was moreover found that the Eucheuma powder has a high water-holding and swell- ing capacity, which is probably linked to the high amounts of seaweed hydrocolloids. To conclude, seaweeds and seaweed extracts have a large potential in NPD due to their excellent antioxidant and antimicrobial properties. It is moreover important to mention that in spite of all the efforts conducted for the addition of seaweeds to food products, there are still some difficulties and challenges in their commercialization due to their sensory impact on foods. To minimize the impact of the seaweed on the sensory characteristics of the foods and to understand its benefits, more research in the field is necessary.

7.8 CONCLUDING REMARKS Facing the future environmental challenges and a rapidly growing population (FAO 2016), the need for innovation and NPD within the seafood sector becomes evident. The main challenges that lie ahead are how to increase both the amount of raw material utilized for human consumption as well as the bespoke usage of the raw material for optimal nutritional, economic, and environmental gain. In addition, new sources for seafood production will be developed and exploited. In the future, we expect to see more lightly processed fish products in the market, in addition to more heavily processed fractions used as ingredients in order to increase utilization. New commercial products such as seaweeds and seaweed extracts have a large potential in NPD due to their excellent antioxidant and antimicrobial properties. The seaweed products can be combined with several mild processing technologies to obtain synergic effects on product quality and safety. Already, increasing segments of consumers are concerned about personal health and sustainability. The focus on future seafood processing and distribution is expected to be on consumer satisfaction as well as sustainability and quality issues to maintain the freshness, safety, and health benefits associated with seafood consumption, and at the same time to reduce the amount of food loss and waste. 168 Trends in Fish Processing Technologies

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Sanja Vidaček and Rafael Soro

CONTENTS 8.1 Introduction...... 172 8.2 Seafood Production, Processing, and Consumption...... 174 8.2.1 Capture...... 175 8.2.2 Aquaculture...... 175 8.2.3 Fish Processing Chain...... 175 8.2.4 Trade/Transport...... 176 8.3 Sustainability Tools...... 176 8.3.1 Life Cycle Assessment...... 176 8.3.2 Carbon Foot Printing...... 177 8.4 Clean Fish Processing Technologies...... 178 8.4.1 Selected Clean Techniques...... 178 8.4.1.1 Cleaning and Disinfection Procedures...... 178 8.4.1.2 Automatic Cleaning Systems for Small Equipment and Production Devices...... 179 8.4.1.3 Dry Transport...... 180 8.4.1.4 Design Improvement of Discharging Slopes...... 180 8.4.1.5 Good Management of Spraying Devices...... 180 8.4.1.6 Avoiding Scaling if the Fish Is Subsequently Skinned...... 181 8.4.1.7 Using the Filtered Recirculating Scaling Wastewater for Preliminary Fish Rinsing...... 181 8.4.1.8 Thawing with Recirculation, Air Stirring, and Level Control Devices...... 182 8.4.1.9 Hygienic Design of Equipment...... 182 8.4.2 Case Study: Pescanova...... 182 8.4.2.1 Company Profile...... 182 8.4.2.2 Goal and Scope...... 183 8.4.2.3 Methodology...... 183 8.4.2.4 Results...... 184 8.5 Conclusion...... 184 References...... 184

171 172 Trends in Fish Processing Technologies

8.1 INTRODUCTION On the global level, there has been an exceptional interest in the environmental impact associated with food systems, including seafood. This interest was partly due to the analysis of the life cycle environmental impacts related to the consumption in the European Union (EU) within the categories food and drink, healthcare, housing, transportation, clothing, etc. The results showed that food production and utilization cause 20% to 30% of the various environmental impacts of private consumption, which included the full food production and distribution chain “from farm to fork” (Tukker et al. 2006). From the global perspective, it would be interesting to compare the impact of seafood production and utilization with other food categories. However, such comparisons are difficult to achieve, due to differences in goals and scopes, methodologies, and assumptions of different studies as well as differences in production among different types of seafood products. In general, the environmental impact of animal food production is higher than vegetable production, meat and meat products being the most important, followed by dairy products (Tukker et al. 2006). Among the animal food production, beef, lamb, and pork production has more impact on the greenhouse emissions than, for example, salmon aquaculture and fileting; however, broader impact is difficult to assess (Aronsson et al. 2014). In a study on the environmental impact of Danish fisheries, when the seafood supply chain started with capture, the environmental impact of capture, processing, wholesale, transport, retail, and use of wild fish (codfish, flatfish, herring, mackerel, lobster, shrimp and mussels) resulted in that the impact of fisheries equaled the one made by the production of some meat or poultry products (Thrane 2004). Practices that lead to more sustainable fishery products are currently part of one of the emerging research trends that are oriented toward sustainability assessment of every step of the seafood supply chain. There is close relationship between sustainable development and clean production, with, as stated by Hall (2011), the goals of the former being achievable through the activities of the latter. Clean production is defined by the United Nations Environment Programme as the continuous application of an integrated, preventive, environmental strategy practice employed for processes, products, and services to increase overall efficiency and reduce risks to humans and the environment (UNEP 2000). Clean production therefore contributes to sustainability. Assessments of processing and recommended cleaner fish processing options for fish industry are available in a number of reports for different categories, i.e., fish fillets (Thrane 2004; Tomczak-Wandzel et al. 2015); frozen fillets (BREF 2006; UNEP 2000); fish fingers (BREF 2006); canned fish (BREF 2006; Duangpaseuth et al. 2007; Thrane 2004; Tomczak-Wandzel et al. 2015; UNEP 2000); canned shellfish (BREF 2006); frozen crustaceans and mollusks (BREF 2006; Thrane 2004); salted, dried, and smoked fish (Tomczak-Wandzel et al. 2015), pickled herring (Thrane 2004); and ancillary operations, like cleaning (Tomczak-Wandzel et al. 2015; UNEP 2000), steam supplies, or compressed air (UNEP 2000). In many reports, the processing to fish meal is also assessed (Tomczak-Wandzel et al. 2015; UNEP 2000). Clean Fish Processing Technologies 173

Inputs Outputs

Fish (kg) Fish product (kg)

Ancillary P By-products material (kg) r (kg) o c Wastewater Energy e COD, BOD, N, P (kWh) s s Air emission i Water (CO2) (m3) n g Noise Chemicals (kg) Odour

FIGURE 8.1 Consumption and emissions in fish processing industry (in bold are key environmental issues).

A cleaner production assessment is a methodology for identifying areas of inefficient use of resources and poor management of wastes, and for developing cleaner production options (UNEP 2000) by focusing on the environmental aspects and, thus, the impacts of industrial processes. Figure 8.1 presents the required quantitative information in order to assess the environmental challenges in the fish processing industry. Depending on the type of product, input variables in the fish industry are the fish (raw material), ancillary materials (salt, oil, etc.), water (either as such or as ice), energy (for operating automated equipment, freezing, chilling, ice making, and sanitation), and chemicals (for example, antioxidants that are used in freezing, preservatives, cleaning chemicals, and others). Output variables are the product (e.g., frozen fish fillet), by-products or solid waste, wastewater or effluent (with the information on biological oxygen demand, chemical oxygen demand, nitrogen, phosphate), air emissions, noise, and odor. The key environmental issues associated with fish processing operations are the high consumption of water, consumption of energy, and the discharge of a wastewater. Noise, odor, and by-products (solid wastes) may also be concerns for some instal- lations (BREF 2006; Duangpaseuth et al. 2007; Hall 2011; UNEP 2000). However, the processing is just one stage in the entire seafood supply chain. It is well known that fish/seafood is one of the most traded items globally, with a number of different species coming from capture and aquaculture that are processed in a number of different products. Fish processing stage, when compared to the other stages within the fish supply chain, starting from capture to its end use, contributes less to the environment than the other stages (Thrane 2004; Ziegler et al. 2003). Nevertheless, apart from the environmental aspect, cleaner production options offer benefits for the fish processing industry as they reduce operating costs and improve productivity for a company. Cleaner production options may include improving of the housekeeping practices (low cost for the industry), process optimization through the reduction of resources consumption (low to medium costs), hazardous raw material 174 Trends in Fish Processing Technologies substitution with more environmentally friendly materials (medium costs), adapting new technologies, and new product design (higher costs). This chapter covers an overview of global trends in fish (seafood) production and utilization, sustainability tools, and selected cleaner techniques in the fish processing industry.

8.2 SEAFOOD PRODUCTION, PROCESSING, AND CONSUMPTION To evaluate the relative impact of fish processing on the environment in the entire supply chain, data on the seafood production, utilization, and trade should be considered. According to the latest Food and Agriculture Organization of the United Nations (FAO) fisheries report, in 2014, total world fisheries increased in tons of captured and aquaculture species compared to the previous years, with a trend of further growth in the aquaculture sector (FAO 2016). Table 8.1 presents selected data for the world fisheries production for 2006 and 2014 with the aim to compare trends

TABLE 8.1 Selected World Capture and Aquaculture Production and Utilization in 2006 and 2014 (Million Tons) 2006 2014 (Hall 2011) (FAO 2016) Production

Inland Capture 10.1 11.6 Aquaculture 31.6 47.1 Total 41.7 59.0

Marine Capture 81.9 81.5 Aquaculture 20.1 26.7 Total 102.0 108.2 Total capture 92.0 93.4 Total aquaculture 51.7 73.8 Total world fisheries (TWF) 143.6 167.2 Utilization Direct human consumption (DHC) 110 (77% TWF) 146 (86% TWF) Nonfood uses (NFU) 33 (23% TWF) 21 (14% TWF) Live/fresh fish 67 (46% TWF) 67 (46% DHC) Freezing 28 (25% DHC) 44 (29% DHC) Canning 17 (15% DHC) 19 (13% DHC) Curing 12 (11% DHC) 17 (12% DHC) International fish traded in processed form 90% 90% International fish traded in live/fresh whole fish 10% 10% Clean Fish Processing Technologies 175 that will have an impact on the sustainability of the fish processing industry. An extensive description of the sustainability impact of fish production and utilization, from the information provided by FAO fisheries reports for the period from 2002 to 2006, is given in Hall (2011).

8.2.1 capture When one thinks about sustainability, the first association that comes into one’s mind is related to the depletion of the wild fish fund. Capturing of wild fishes is facing a global crisis because the fish stocks are collapsing. A number of factors associated with unsustainable consumption, and production patterns have led to the situation in which 90% of the world’s fisheries are already fully exploited or overfished (FAO 2016). A number of practices and techniques have been implemented with the aim of protection of the global marine wildlife. Some of these include avoiding overfishing, setting up protected areas, educating stakeholders, educating the wider public, etc. Besides direct impacts to targeted fish stocks, fish capture has been associated with substantial by-catch, the disturbance and displacement of benthic communities, and the alteration of trophic dynamics (Pelletier et al. 2007). Recommended cleaner production strategies reported in the literature include technology modifications on boats with the aim of reduction of fuel (diesel use) or reduction of by-catch (Denham et al. 2015). Some proposed measures that lead to the reduction of by-catch may include prohibiting or limiting the use of certain gear types in defined areas or seasons to protect vulnerable life stages. Combining several measures may increase the total effectiveness (FAO 2016).

8.2.2 aquaculture Overfishing of wild species has helped aquaculture to grow tremendously in the last few decades. In 2014, total aquaculture was still less in quantity than wild capture (Table 8.1), but in 2016, aquaculture already provides almost half of all fish for human consumption. However, aquaculture faces many environmental challenges, particularly the intensive cultivation of highly valued shrimp and salmon species. While significant progress has been made over the past decades toward making intensive aquaculture more sustainable, environmental concerns remain due to continued growth of this sector. Overall, environmental impacts from aquaculture are expected to at least double by 2030 (UNEP 2014). The biggest environmental challenge for marine aquaculture is feed production, which is energy consuming. Other potentially problematic practices include breeding, fish growth, and harvest (Denham et al. 2015).

8.2.3 Fish Processing Chain In 2014, more than 86% of world fish production was utilized for direct human consumption, which is an increase when compared to the previous years. Of these products, 46% was in live, fresh, or chilled forms; 12% was utilized in dried, salted, smoked, or other cured forms; 13% in prepared and preserved forms; and 29% in frozen form. Freezing is the main processing method for fish for human consumption. 176 Trends in Fish Processing Technologies

The share of frozen fishery products increased compared to previous years (FAO 2016).

8.2.4 trade/Transport Fish is among the most traded food commodities worldwide, and in 2014, about 78% of seafood products were estimated to be traded internationally. Trade in live, fresh, and chilled fish represented 10% of world fish traded in 2014, similar to the previous years. Fish are increasingly traded as frozen food, 40% of the total quantity in 2014 (FAO 2016). All in all, sustainability implications of trends in world fisheries in 2014 are similar to those in 2007 reported by Hall (2011). An increase in aquaculture implies an increase in energy used for feed production, pollution challenges, and loss of habitat and biodiversity. International trade increases, which implies energy for transport. Frozen fish, as a dominant processed fishery product, needs energy for freezing, storage, and transport. This suggests that aquaculture, transport, and frozen fish supply chain are worth considering in the environmental assessments. There are several options for reducing the energy consumption during the freezing operations, e.g., by reducing the freezing time, controlling the use of heat-generating fans, reducing heat leakage caused by poor insulation of pipes, or by close examination of the freezing process for the specific product type and throughput and sizing of the equipment to achieve good product quality (Hall 2011).

8.3 SUSTAINABILITY TOOLS To apply the cleaner production methods most efficiently within the production and processing, first, the impact of these activities on the environment should be assessed. The impact of food production and processing on the environment can be assessed by a number of different but related tools. The most detailed tool is the life cycle analysis (LCA); however, in food (fish) processing, the most frequently applied tool is carbon foot printing.

8.3.1 life Cycle Assessment LCA is a well-known scientific tool, which identifies all the different life cycle stages that are essential to produce a product. LCA is standardized by the International Organization for Standardization (ISO) in ISO 14040 and 14044 (ISO 2006a, 2006b). It was often referred to as the “cradle-to-grave” approach or from “sea to table” when the product was seafood. However, these linear concepts have been less used and a concept of a circular economy has started being used recently. The circular economy aims to eradicate waste, not just from manufacturing processes, but also systemati- cally, throughout the life cycles and uses of products and their components. LCA is a very comprehensive analysis and has been used for a number of years (Lillywhite 2010). In short, input variables, i.e., uses of biotic and abiotic resources, energy, land, and water during the different life cycle stages, lead to output or emissions of greenhouse gasses (GHGs), nutrient leakage, waste, and wastewater to air, Clean Fish Processing Technologies 177 water, and soil. These emissions to the environment contribute to different environmental impact categories—global warming, acidification, eutrophication, ozone depletion, land use, photochemical smog, eco and human toxicity, and abiotic depletion potential, and biodiversity reduction (Aronsson et al. 2014). These categories are not exclusive and can be made process specific. Of the LCA studies on seafood products, most often employed are climate change, acidification, and eutrophication as impact categories (Pelletier et al. 2007). However, in recent years, the number of the categories has been greater than before, mainly due to the addition of toxicity impact categories and the ozone layer depletion potential category (due to impacts of antifouling paints and refrigerants emissions in fisheries) (Winther et al. 2009; Vazquez-Rowe et al. 2012; Ziegler et al. 2003). Processing, packaging, transport, sale, consumption, and waste management have not been commonly included in life cycle stages in seafood LCAs. This is particularly the case for aquaculture, while studies on fisheries have often included the transport stage as well (Ziegler et al. 2013). LCA has certain limitations. Aronsson et al. (2014) reviewed existing LCA studies to identify the key environmental impacts of different food products in different supply chains and pointed out that there are other aspects that are not adequately included in LCA studies because of lack of consensus methodology and data availability (percentage of waste).

8.3.2 carbon Foot Printing Carbon footprint is a term used, e.g., by British Standard and in ISO-working documents, to describe the amount of GHG emissions of a process to indicate its contribution to climate change (Cederberg 2010). The term “GHG” can be defined as carbon dioxide only or can include other gases, particularly methane (with a GHG effect over 20 times that of carbon dioxide) or as carbon dioxide equivalents (Hall 2011). The carbon footprint of fishing systems is generally dominated by fuel (i.e., energy) use in the fishery; hence, there is not much difference between global warming emissions and energy use. For farmed products, this is not the case, as biological inputs from methane and dinitrous oxide are more important (Winther et al. 2009). A problem with many studies on carbon foot printing of seafood is, as in the case of LCA analysis, that they only consider one product and methodological differences also make it difficult to compare results across studies. However, Ziegler et al. (2013) quantified the carbon footprint of more than 20 Norwegian seafood products, including fresh and frozen, processed and unprocessed cod, haddock, saithe, herring, mackerel, farmed salmon, and farmed blue mussels. There were major differences between seafood products; i.e., the most efficient seafood product was herring shipped frozen in bulk while the least efficient was fresh gutted salmon airfreighted. The authors pointed out that there are many possibilities for considerable improvement within supply chains and in product choices. Fuel use in fishing and feed production in aquaculture are key contributors to GHG emission (Aronsson et al. 2007; Ziegler et al. 2013). Additional key aspects are refrigerants used on fishing vessels, product yield, by-catch, and by-product use. Product form (fresh or frozen) seems to matter only when freezing makes slower transportation possible (Ziegler et al. 2013). Processing and packaging generally 178 Trends in Fish Processing Technologies make small contributions to overall greenhouse emissions (often under 10% of total) except in processing in which emission-intensive materials are used (e.g., metals) or where cooking is involved, etc. (Anonymous 2014).

8.4 CLEAN FISH PROCESSING TECHNOLOGIES The fish industry covers an important amount and variety of food products, from fresh fish to canned products, some heat treated and some commercialized as raw fish. As a consequence, there is also a huge variety of industrial processes, technologies, and equipment. Despite this diversity, a number of common techniques have been identified with the potential of reducing the environmental impact of the industrial activity. The Integrated Pollution Prevention and Control Directive was launched to achieve integrated prevention and control of pollution arising from the industrial activities, leading to a high level of protection of the environment as a whole. The term “best available techniques” (BATs) is defined in Article 2(11) of the Directive as “the most effective and advanced stage in the development of activities and their methods of operation which indicate the practical suitability of particular techniques for providing in principle the basis for emission limit values designed to prevent and, where that is not practicable, generally to reduce emissions and the impact on the environment as a whole.”

8.4.1 selected Clean Techniques The following techniques have been selected for their positive impact in the reduction of environmental impact in the fish industry. Most of them are reported to have more than one environmental benefit. Some of them are considered as BAT (BREF 2006), and some others are added subsequently based in information collected from a number of different sources. The techniques or minimization strategies presented hereinafter are structured in the following sections: operations involved, related environmental aspects, description, and achieved environmental benefits.

8.4.1.1 Cleaning and Disinfection Procedures Operations: cleaning and disinfection of equipment and facilities Related environmental aspects: water consumption, wastewater Description: Cleaning and disinfection operations are critical in the food industry, since they are closely related with the quality and safety of the food product and because it could significantly contribute to resources consumption (water, chemicals, time, energy, etc.). These operations are usually performed manually within the fish sector, since most of the technological steps are performed by the machines that cannot be cleaned by the cleaning-in-place systems. So, good cleaning practices focused on the automation (when possible), rationalization of chemicals and water usage, and training of operators will lead to environmental benefits. Clean Fish Processing Technologies 179

– Cleaning procedures should minimize the use of chemicals, as this will have a positive effect on the amount of rinse water used and the contamination load of the wastewater. Moreover, organohalogen compounds, such as chlorine, commonly used in these operations, are included in the indicative list of the Directive on Industrial Emissions (Directive 2010/75/EU, Annex II) as major pollutants for water emissions. Validation is required in order to ensure that optimized cleaning reaches the expected hygienic levels. The use of oxidizing biocides such as ozone is a good opportunity of improving several environmental indicators (water consumption, wastewater generation). Compared to the use of traditional chemicals, ozone-based techniques provide a number of environmental advantages, mainly because of the absence of chemical residues after their application. – Cleaning operations should be rationally scheduled. When organizing production, cleaning operations should be considered, trying to reduce them to a minimum (reduce product changes, etc.). Reuse of water should be considered (e.g., water from last rinse for the first rinse of the next cleaning cycle in closed systems), but this should always be based on a hygienic validation. – Raw material residues should be removed as soon as possible after processing and materials storage areas should be cleaned frequently. – Automatic dosing systems to prepare the cleaning solutions are recommended. – Operators performing cleaning operations should be trained in proper application of cleaning protocols and in environmental impact of these activities.

Achieved environmental benefits:reduction in water consumption, reduction in volume and contamination load of wastewater generated

8.4.1.2 Automatic Cleaning Systems for Small Equipment and Production Devices Operations: cleaning and disinfection Related environmental aspects: water consumption, wastewater generation, chemical consumption Description: Small equipment and diverse production devices (e.g., plastic boxes, trays, etc.) are usually manually cleaned, either in a dedicated area or near the production line. High or medium pressure washers are commonly used, but their water and chemical consumption is high and the generation of aerosols could be a food safety problem. Cleaning tunnels or cabinets allow a more rational cleaning, with a total control on the main cleaning parameters (time, temperature, pressure, flow, and chemical concentration) and the possibility of recirculation. As a consequence, water and chemical consumption and generation of wastewater are significantly lower. Achieved environmental benefits:reduction in water consumption, reduction in volume and contamination load of wastewater generated 180 Trends in Fish Processing Technologies

8.4.1.3 Dry Transport Operations: filleting, skinning, and other operations generating organic wastes Related environmental aspects: water consumption, wastewater Description: Dry transport of solid wastes from different processing operations represents a significant environmental advantage in terms of water consumption and wastewater generation. As an example, in a filleting machine, a channel with an appropriate slope (e.g., 20%) mounted under the gutting wheel may collect viscera, fatty belly strips, and the water from the wheel. Viscera strips and water slide on from the channel to a fine meshed synthetic cloth positioned on a conveyor belt, through which the water is drained. The viscera are transported to a container. Additionally, vacuum suction for removing and transporting skin (from the skinning drum) or fat and viscera (from skinning and cutting) would also contribute to a reduction in water consumption (compared to the traditional method based on water removal and transport). Achieved environmental benefits:reduction in water consumption, reduction in volume and contamination load of wastewater generated

8.4.1.4 Design Improvement of Discharging Slopes Operations: solid waste removal from gutting, deheading, filleting, and skinning Related environmental aspects: water consumption, wastewater generation Description: Prolonged contact of organic wastes with water in the process lines leads to a significant increase of the contaminant load of wastewater in fish processing plants. Wastes are often emptied through slopes without the necessary angle, toward a collecting bin. Water used to keep the slope clean and to facilitate the wastes movement often ends up in the same container, together with the solid waste. In this process, water dissolves part of the organic matter, which, together with small parts of waste, will end up in the sewage. Discharging slopes may be improved to deviate water before falling into the solid waste containers. An appropriate slope angle will minimize the need of water to keep the slope clean and facilitate transport. Another option is to install a perforated tray (mesh size of 1 mm as an orientation) at the beginning of the slope so that water can fall to a collecting channel while solid wastes continue towards the container. Achieved environmental benefits:reduction in volume and contamination load of wastewater generated.

8.4.1.5 Good Management of Spraying Devices Operations: maintenance Related environmental aspects: water consumption, wastewater generation Description: Water sprayers are commonly used in the fish industry with the main purpose of washing or removing small parts (e.g., peeled skin). It is reported that in fish skinning and cutting, reducing both the number and size of spray nozzles can lead to water savings of about 75%. In fish filleting, a reported reduction of 60% to 75% Clean Fish Processing Technologies 181 in water consumption is obtained by using the following means, which combine a planned use of nozzles and other techniques:

• Removing unnecessary water nozzles • Using water nozzles instead of water pipes for washing the product • Using low flow nozzles, adjusting the needed pressure to the specific need • Proper orienteering of nozzles • Using mechanical devices instead of water nozzles to move the fish from the tail cut • Replacing the nozzles for washing the driving wheels on the filleting part with mechanical scrapers • Replacing existing nozzles by nozzles with a lower water consumption • Using pulsating water nozzles • Replacing the waste drain by mesh conveyors and closing the nozzles in the waste drain. The waste will be separated from the process water directly near the filleting machine, resulting in shorter contact time and less entrain- ment of, e.g., fat. • Using presence-activated sensors to control when the nozzles operate

Achieved environmental benefits:reduction in water consumption, reduction in volume and contamination load of wastewater generated

8.4.1.6 Avoiding Scaling if the Fish Is Subsequently Skinned Operations: scaling Related environmental aspects: water consumption, wastewater generation Description: Scaling equipment typically consists of a perforated rotating drum onto which water is introduced to flush scales away. So, an important water consumption (an average of 10–15 m3/t of raw material) and wastewater generation are involved in this operation. Depending on the final use of the product, scaling can be avoided if the fish is subsequently skinned. So, this would only be an option when at the time of processing raw fish, it’s already known that the final product will be skinned product. Achieved environmental benefits:reduction in water consumption, reduction in volume and contamination load of wastewater generated

8.4.1.7 Using the Filtered Recirculating Scaling Wastewater for Preliminary Fish Rinsing Operations: scaling Related environmental aspects: water consumption, wastewater generation Description: Using the filtered recirculated scaling wastewater for preliminary fish rinsing minimizes overall water consumption. A proper management of the recirculation water is essential from the food safety point of view. Disinfection techniques such as ultraviolet treatment may contribute to guarantee the hygienic quality of the recirculated water. 182 Trends in Fish Processing Technologies

Achieved environmental benefits:reduction in water consumption, i.e., water savings when comparing to the use of fresh water could reach 70% (BREF 2006); reduction in volume and contamination load of wastewater generated

8.4.1.8 Thawing with Recirculation, Air Stirring, and Level Control Devices Operations: thawing Related environmental aspects: water consumption, wastewater generation Description: Processing of frozen fish is common; thus, thawing is widely applied in these sectors. The traditional thawing of fish takes place under running water. In this case, the unpacked fish are completely immersed in pools with water. Recirculation of water and stirring by mechanical devices or by blowing air will improve the effectiveness of the thawing process. The amount of water fed into the tank should be automatically controlled by level switches, so that no water overflow occurs. Achieved environmental benefits:reduction in water consumption (according to BREF document, a water consumption decrease from 5 to 2 m3/t of raw fish is reported, compared to running water), reduction in volume and contamination load of wastewater generated

8.4.1.9 Hygienic Design of Equipment Operations: all Related environmental aspects: water consumption, wastewater generation, energy consumption Description: The main objective behind hygienic design is to reduce/eliminate the risk of equipment becoming a source of product contamination, but this will lead most of the times to economic and environmental benefits since easy cleanable equipment will surely need less water, energy, and chemicals to reach the required hygienic level. The EU funded project Ecodhybat (LIFE12 ENV/ES/001070) is currently trying to demonstrate and quantify the relationship hygiene–environment. Its main objective is to demonstrate that the hygienic design of equipment is a cost-effective preventive approach to reduce the consumption of water, energy, and chemicals and to reduce wastewater and CO2 emissions during sanitation. So far, promising results have been obtained in the test conducted in the fish industry, with savings of more than 30% in water consumption for cleaning some of the equipment tested (hygienically vs. unhygienically designed equipment).

8.4.2 case Study: Pescanova 8.4.2.1 Company Profile The Nueva Pescanova Group, to which the Centro Industrial Porriño belongs, is a Spanish multinational company leader in the frozen foods sector, engaged in Clean Fish Processing Technologies 183 fishing, aquaculture, processing, and marketing of seafood products, both fresh and deep-frozen. Incorporated in 1960, Pescanova is present in many parts of the world with sub- sidiary companies located in 26 countries and employing more than 12,000 people. Over the years, Pescanova products have become a benchmark, promoting healthy eating habits by providing food products that meet consumers’ needs in terms of quality, convenience, and nutrition. Pescanova is committed to the preservation of aquatic ecosystems and marine environment by implementing sustainable development strategies, streamlining its fishing and farming activities always bearing in mind the protection of the environment.

8.4.2.2 Goal and Scope The aim of this internal project was to achieve a significant reduction in water consumption during cleaning operations.

8.4.2.3 Methodology The project was structured in three phases:

a. Analysis of the current cleaning procedures and operations More than 40 improvement measures were identified (lack of working procedures for some operations, inadequate sequence of operations, intensive water rinsing, lack of awareness about the cost of water, inadequate nozzles in some cases, etc.). b. Design of action plan The improvement plan was built upon four main premises: proper waste collection, improved solid wastes withdrawal, accessibility to machinery, waste minimization. c. Implementation and staff training A set of improvement measures were implemented. In the following list, some of them are presented: • Complete opening of equipment during emptying processes • Proper organization and tidiness of ancillary equipment and dismount- able parts (by applying 5S methodology—sort, set in order, shine, stan- dardize, and sustain) • Improvement of equipment protection devices • Hygienic design and hygienic installation of equipment • Waste disposal in dry conditions • Minimization of water hosing down of floors

Apart from these measures related with the cleaning procedures, a number of actions were taken to reduce the water consumption: Koopens refrigeration system (hydraulic system refrigeration unit was replaced), accumulator in the blancher (a modification in the system led to a considerable water consumption reduction), water recovery system in the chilling tunnels, etc. 184 Trends in Fish Processing Technologies

8.4.2.4 Results Once the action plan had been implemented, a reduction of 13% in water consumption was found in cleaning operations and a 5% reduction was reported as a consequence of improvements in other operations (refrigeration, equipment modifications, etc.).

8.5 CONCLUSION The fish processing industry may use a number of clean techniques that reduce the environmental impact of the industrial activity. However, fish processing is one step in the entire supply chain. Therefore, to ensure the greatest reduction in environmental impact, evaluation of the entire supply chain management system is recommended.

REFERENCES Aronsson, A.K.S., Landquist, B., Esturo, A., Olafsdottir, G., Ramos, S., Pardo, G., Nielsen, T., Viera, G., Larsen, E., Bogason, S., Ingólfsdóttir, G.M., Yngvadóttir, E. 2014. The applicability of LCA to evaluate the key environmental challenges in food supply chains. Proceedings of the 9th International Conference on Life Cycle Assessment in the Agri-Food Sector. 8–10 October 2014, San Fransico, USA. R. Schenck and D. Huizenga (eds.), 55–63. ACLCA, Vashon, WA, USA. Anonymous. 2014. The Seafish Guide to Greenhouse Gas Emissions in Seafood. Seafish, UK. BREF. 2006. Reference Documents on Best Available Techniques in the Food, Drink and Milk Industries. European Commission, Seville, Spain. Cederberg, C. 2010. Improving nutrient management in agriculture to reduce eutrophication, acidification and climate change. In Assessment of the Environmental Impacts of Food Production, U. Sonesson, J. Berlin, and F. Ziegler (eds.), 3–15. Cambridge: Woodhead Publishing Limited. Denham, F.D., Howieson, J.R., Solah, V.A. et al. 2015. Environmental supply chain management in the seafood industry: Past, present and future approaches. Journal of Cleaner Production 90: 82–90. Duangpaseuth, S., Das, Q., Chotchamlong, N. et al. 2007. Seafood Processing. Asian Institute of Technology, School of Environment, Resource & Development, Pathum Thani, Thailand. EC Directive 2010/75/EU on industrial emissions (Integrated Pollution Prevention and Control, IPPC). FAO (Food and Agriculture Organization of the United Nations). 2016. The State of World Fisheries and Aquaculture (Rome) 2016, FAO, Rome, Italy. Hall, G.M. 2011. Sustainability impacts of fish-processing operations. In Fish Processing— Sustainability and New Opportunities, G.M. Hall (ed.), 112–137. Oxford: Blackwell Publishing Ltd. ISO (International Organization for Standardization). 2006a. ISO 14040:2006(E). Environmental Management—Life Cycle Assessment—Principles and Framework. Geneva, Switzerland: ISO. ISO (International Organization for Standardization). 2006b. ISO 14044:2006(E). Environmental Management—Life cycle Assessment—Requirements and Guidelines. Geneva, Switzerland: ISO. Lillywhite, R. 2010. Footprinting methods for assessment of the environmental impacts of food production and processing. In Assessment of the Environmental Impacts of Food Production, U. Sonesson, J. Berlin, and F. Ziegler (eds.), 255–271. Cambridge: Woodhead Publishing Limited. Clean Fish Processing Technologies 185

Pelletier, N.L., Ayer, N.W., Tyedmers, P.H. et al. 2007. Impact categories for life cycle assessment research of seafood production systems: Review and prospectus. International Journal of Life Cycle Assessment 12 (6): 414–421. Thrane, M. 2004. Environmental impacts from Danish fish products. PhD dissertation, Aalborg University. Tomczak-Wandzel, R., Vik, E.A., and Wandzel, T. 2015. BAT in Fish Processing Industry— Nordic Perspective. Nordic Council of Ministers, Copenhagen, Denmark. Tukker, A., Huppes, G., Guinée, J. et al. 2006. Environmental Impact of Products (EIPRO). Main report IPTS/ESTO project. Technical Report EUR 22284 EN. European Commission Joint Research Centre (DG JRC), Seville, Spain. UNEP. 2000. Cleaner Production Assessment in Fish Processing. Paris, France: Division of Technology, Industry and Economics, United Nations Environment Programme (UNEP). UNEP. 2014. The 2014 Year Book—Emerging Issues in Our Global Environment. Paris, France: Division of Technology, Industry and Economics, United Nations Environment Programme (UNEP). Vazquez-Rowe, I., Hospido, A., Moreira, M.T. et al. 2012. Best practices in life cycle assessment implementation in fisheries. Improving and broadening environmental assessment for seafood production systems. Trends in Food Science and Technology 28: 116–131. Winther, U., Ziegler, F., Skontorp Hognes, E. et al. 2009. Carbon Footprint and Energy Use of Norwegian Seafood Products. SINTEF Fisheries and Aquaculture report: SFH80 A096068. Trondheim, Norway: SINTEF Fisheries and Aquaculture. Ziegler, F., Nilsson, P., Mattsson, B. et al. 2003. Life cycle assessment of frozen cod fillets including fishery-specific environmental impacts. International Journal of Life Cycle Assessment 8 (1): 39–47. Ziegler, F., Winther, U., Hognes, E.S., Emanuelsson, A., Sund, V., Ellingsen, H. 2013. The carbon footprint of Norwegian seafood products on the global seafood market. Journal of Industrial Ecology 17 (1): 103–116. http://taylorandfrancis.com Innovative Fish 9 Packaging Solutions

Iulia Bleoanca and Maria Turtoi

CONTENTS 9.1 Introduction...... 187 9.2 AP of Fish...... 189 9.2.1 EFs and Coatings for Fishery Products as AP...... 189 9.2.1.1 EFs and Coatings with EOs...... 193 9.2.1.2 EFs and Coatings with Antimicrobial Enzymes...... 196 9.2.2 Multifunctional Bioblends for Fish Packaging...... 197 9.2.3 Nanobiocomposite Films Used for Fish Packaging...... 198 9.3 IP of Fish...... 198 9.3.1 IP Monitoring Environmental Conditions...... 199 9.3.1.1 Time–Temperature Indicators...... 199 9.3.1.2 Seal and Leak Indicators...... 200 9.3.1.3 RH Sensors...... 201 9.3.2 IP Monitoring Quality Attributes...... 201 9.3.2.1 Fish Freshness Sensors and Indicators...... 202 9.3.2.2 Biosensors...... 202 9.3.3 Data Carriers...... 202 9.3.3.1 Barcode Labels...... 202 9.3.3.2 Radio-Frequency Identification...... 206 9.4 Concluding Remarks and Future Perspectives...... 207 References...... 207

9.1 INTRODUCTION In the last decades, trends in food packaging have evolved toward the development of a new generation of packaging system with improved technological functionality and eco-effectivity, able to decrease the environmental burden through waste prevention while providing industrial process optimization, for closed-loop recyclability. The beginning of the current millennium brought to life the concept of smart packaging (SP), which is based on two synergistic packaging solutions: active packaging (AP), able to act upon the changes in the product or its environment, and intelligent packaging (IP), responsible for monitoring changes in the product or in its environment (EC450 2009).

187 188 Trends in Fish Processing Technologies

The current growing interest in these new packaging systems is demonstrated by the $15.7 billion global market for AP and IP in 2015 and the estimation of a value of $32.7 billion to be reached by 2022 (MRC 2016). AP complements the protection function of the traditional, passive packaging. This innovative packaging system relies on the positive interaction between the product, packaging, and the environment, mediated by a component that enables the release or absorption of substances into/from the packaged food or the environment surrounding the food product (Vanderroost et al. 2014). Thus, this revolutionary concept actively involves packaging in preserving food quality and safety. The remarkable developments during the last 10 years in the field of food AP focused on preventive strategies to mitigate the deleterious effects upon food quality and safety, including nutrient loss, color change, microbial growth, and off-odors, and created a more mature market when compared to IP. This novel alternative to the conventional use of preservatives or modified atmosphere packaging (MAP) technology is based on either absorbing or releasing systems such as moisture or odor absorbers, oxygen scavengers, carbon dioxide scavengers or emitters, preservative releasers (edible films [EFs] and coatings), and ethanol emitters (Kuorwel et al. 2015). IP, a valuable extension of the traditional packaging communication function, has the ability to monitor and communicate to the consumer the changes that appear in the product or its environment during products’ life cycle (EC450/2009 2009). Thus, IP can use sensors or indicators that either monitor the environmental conditions influencing the quality attributes (time–temperature indicators [TTIs], gas leakage indicators), monitor quality attributes and indicator compounds of the product itself (biosensors, freshness sensors and indicators, microbial growth indicators), or use data carriers for a more effective communication between the product and the consumer (radio-frequency identification [RFID] tags, antitheft devices) (Heising et al. 2014). Not only consumers could benefit from IP systems, but also food producers who use them as a valuable traceability solution. From a global perspective, IP can improve the supply chain performance, contributing to the global reduction of food waste. The deliberate interaction between components of active or IP and the food or its environment challenges the evaluation of food safety in terms of substance migration from the packaging to the food. In this context, EU Regulation Nos. EC1935 (2004) and EC450 (2009) set up special requirements for active and intelligent food contact materials (FCMs), as well as their labeling, traceability, and food manufacturing practices. Authorization for new active materials and articles use is granted by the EU Commission (DG SANCO) based on a safety assessment made by the European Reference Laboratory for Food Contact Materials, from the European Food Safety Authority. The risk assessment evaluation focuses on the migration of active and intelligent substances, migration of their degradation and/or reaction products, and their toxicological properties. The approval for the new active and intelligent systems will be entered in the Regulation and is not general, but only for the petitioner. In the case of non-edible FCM from active and intelligent materials and articles, covered by EU Regulation EC10 (2011), additional labeling requirements are Innovative Fish Packaging Solutions 189

FIGURE 9.1 Labeling symbol “DO NOT EAT” for active and intelligent materials (EC450 2009). indicated by EC450 (2009): clear indication with the words “DO NOT EAT” and always where technically possible, with the symbol reproduced in Figure 9.1. SP is an exciting new trend that offers the perspective to combine IP and AP in a closed-loop packaging system where the IP component can monitor the changes in the product or its environment, while the integrated AP system is able to react appro- priately upon those changes in favor of food product quality and shelf life extension. In order to make this system fully functional, the development of new technologies is required (Vanderroost et al. 2014). According to market reports (Marketsandmarkets 2015), the SP segment is currently estimated at $39.7 billion, growing at a compound annual growth rate of 4.8% from 2014 to 2020. The ongoing food packaging innovation activities consider the current trend toward a circular economy with zero waste, with packaging waste prevention being a top priority. In this regard, the development of a sustainable packaging should be performed considering the modern design concept of cradle-to-cradle (C2C) instead of the cradle-to-grave (C2G) one (Vanderroost et al. 2014). While the C2G concept considers a one-way flow out of materials from the factory, depleting at a very high rate the material and fossil resources, the C2C redesigns the materials flow in a sustainable manner. For food packaging, the C2C concept would mean resource reuse for as many as possible cycles or recycling in a closed-loop process with zero loss in material, rather than struggling to eliminate waste afterwards. This chapter provides an overview of the trending active and IP advances applied to fish and seafood industry, considering sustainable packaging design: low-environmental-impact AP, EFs and coatings, and IP to fish industry monitoring environmental conditions (TTIs, seal and leak indicators, relative humidity [RH] sensors), quality attributes (freshness sensors and indicators, biosensors), as well as data carriers (barcodes, RFID). The growing interest in innovative packaging solutions that promote higher food quality and safety, as well as convenience for both consumers and food producers, boosted the development of AP and IP (Figure 9.2) as a valuable extension of the basic functions of the traditional, passive packaging (containment, convenience, protection, and communication).

9.2 AP OF FISH

9.2.1 eFs and Coatings for Fishery Products as AP Traditional food packaging, standing for 50% of the global packaging industry (FAO 2014), could be oriented in the future toward environmentally friendly packaging solutions, an upcoming novel technology being EFs and coatings use. A considerable 190 Trends in Fish Processing Technologies

O2 scavengers CO2 emitters Active Moisture absorbers Containment Protection packaging Preservatives releasers (edible ﬁlms and coatings, nanobiocomposites ﬁlms)

TTI, gas leakage indicators Intelligent Convenience Communication Freshness indicators, biosensors packaging RFID tags

Traditional Smart packaging packaging

FIGURE 9.2 Active and intelligent packaging as complements to the traditional packaging functions. amount of research was performed in the last decade for developing new bio-based polymers and applications onto food (Heising et al. 2014; Valencia-Chamorro et al. 2014; Vanderroost et al. 2014), which led to the design of green primary packaging, associated with multiple environmental benefits (reduced anticipated pollution and generation of waste material, improvement of material recovery), maintaining at the same time packaging ability to extend shelf life and enhance fresh food products quality. EFs are preformed continuous layers of biopolymer matrix suitable for consumption, with thickness ranging from 0.050 to 0.250 mm (Jooyandeh 2011), based on GRAS ingredients (proteins, lipids, polysaccharides, or composite materials— combination of lipids with hydrocolloids), which, once formed, can be placed on or between food components (Falguera et al. 2011; Rossman 2009). After an EF is applied, it remains on the product during storage and it will be disintegrated during cooking or mastication process (Falguera et al. 2011). Edible coatings (ECs) are a particular form of EFs, formed from thinner layers than in the case of EF, the film forming solution being applied as a coating directly on a food product usually by immersion, spraying, or brushing. Consequently, there are two main differences between films and coatings: one is their thickness and the second is the fact that EFs are first molded into stand- alone sheets and further transformed into pouches, wraps, capsules, bags, or casings (Ramos et al. 2012a), while ECs are part of the final product, without the possibility of being disposed of separately from the food. Their edibility, biocompatibility, aesthetic appearance, nontoxicity, nonpolluting nature and low cost recommend EF and EC to be considered for food preservation (Vásconez et al. 2009). EFs and ECs are typically biopolymer or lipid based, with a solvent and additives, which either improve the basic function of the packaging or improve the quality and safety of the packed food product (Figure 9.3). For improving polysaccharide- or protein-based EF flexibility, nonvolatile plasticizers are typically added to hydrocolloid film forming solution: polyols (propylene glycol, glycerol, sorbitol, polyethylene glycol), oligosaccharides (sucrose), or water (Han 2014). Innovative Fish Packaging Solutions 191 , 5, , 5, d od r fo , , g ts ed , colo vors idan , stability an , fla ox ts Improvin ality Anti nutraceuticals agen s antimicrobials safety of pack the qu ditive , in g Ad ts ts di fy s, s, ticizer king agen as s slin emulsifier Pl reinforcemen os of the material basic functionality cr Improving/mo onent the comp gs s in at ethanol r, te olvent S Wa s, e ee fr , s, ed s Edible films and co sucros s, ipid triglyceride , shellac resin s, acetylac ers xe tty acid fa est Wa monoglyceride s , scin, onent sL , ca Main oteins ey comp rotein ybean pr So sunflower protein, keratin, fish proteins gelatin, wh wheat gluten, corn zein, , olymers sP s, s, ums g s, Biop inale er s , alg fib chitosan in s, olysaccharide starches Cellulose derivative P Components edible films of C. Musso, Y. P., L. Ortiz, Di Giorgio, and and A, coatings.Mauri, Salgado,(From Curr Opin Food Sci pect

FIGURE 9.3 86–92, 2015.) 192 Trends in Fish Processing Technologies

Considering that mass transfer is involved in most cases of food quality loss due to either moisture, oxygen, undesirable odor absorption, or flavor loss, packaging permeability is an important parameter to consider, which can greatly influence the rate of food deterioration. Thus, EF and common plastic materials are classified (Table 9.1) according to their oxygen and water vapor permeability as inferior, marginal, good, and superior. Both EF and EC create a modified overhead atmosphere (Ramos et al. 2012a) for the food product, altering the gas transfer (O2, CO2) and acting as a migration barrier for moisture and volatile compounds. The release of active compound from EF and EC is performed through a reservoir system-like mechanism (Del Nobile and Conte 2013), following a zero-order kinetic, exhibiting a constant release rate. The release process takes place in four steps: (1) diffusion of the active agent within the EF and EC reservoir; (2) dissolution or partitioning of the active agent between the EF and EC reservoir carrier fluid and the barrier; (3) diffusion through the barrier and partitioning between the barrier and the elution medium (i.e., the surrounding food); and (4) transport away from the barrier surface (EF and EC) into the food; the rate- limiting step in the release of the active ingredient is diffusion through the EF and EC polymeric barrier (Del Nobile and Conte 2013). Novel controlled-release systems EF and EC can be functionalized by incorporating active substances with specific migration rates, like antimicrobial, antioxidant, coloring, or flavoring agents, probiotics, or nutraceuticals (vitamins, minerals). The development of EF and EC for food products gained a considerable interest in the last decade, demonstrated by the growing number of published research articles (Falguera et al. 2011; Han 2014; Vanderroost et al. 2014) and the total annual rev- enue for food EF and EC exceeding $100 million in 2009 (Pavlath and Orts 2009). Edible packaging was successfully applied to RTE foods, such as fruit and vegetable cuts (Bierhals, Chiumarelli, and Hubinger 2011; Muriel-Galet et al. 2012), to bakery products (Silveira et al. 2007; Soukoulis et al. 2014), cheese (Di Pierro et al.

TABLE 9.1 Permeability Characteristics of EF and Common Plastics Classification Parameter Inferior Marginal Good Superior Oxygen permeability >1000 1000–100 100–10 <10 (cm3 ∙μm∙m−2∙d−1∙KPa−1) LDPE HDPE Cellophane PVDC Starch: Gly BW WPI: Gly SPI: Gly Water vapor permeability >10 10–1 1–0.1 <0.1 (g∙mm∙m−2∙d−1∙KPa−1) WPI: Sor WPI: BW: Gly Shellac HPMC: BW: PEG Pea protein: Gly Cellophane LDPE, HDPE

Source: Han, J., Edible films and coatings: a review, in Innovations in Food Packaging, 2nd ed., Han, J. (ed.), Elsevier Ltd., 2014, 213–255. Note: LDPE, low-density polyethylene; Gly, glycerol; HDPE, high-density polyethylene; BW, beeswax; WPI, whey protein isolate; PVDC, polyvinylidene chloride; SPI, soy protein isolate; Sor, sorbitol; HPMC, hydroxypropyl methylcellulose; PEG, polyethylene glycol. Innovative Fish Packaging Solutions 193

2011; Guldas et al. 2010; Pires et al. 2008; Ramos et al. 2012b), sliced meat products (Emiroglu et al. 2010; Fernández-Pan, Mendoza, and Maté 2013), and fish fillets (Jouki et al. 2014; Kilincceker, Dogan, and Kucukoner 2009; Kim and Min 2012). Consumers’ mounting demand for high-quality fresh fishery products has intensi- fied researches on reducing their extreme perishability, which is mainly caused by the presence of increased concentrations of free amino acids and volatile nitrogenous bases in fish compared to other types of meat. A successful strategy for shelf life extension and enhancement of sensorial properties of fishery products is the use of EF and EC, which provide attractive advantages, such as the ability to retard lipid oxidation (Ojagh et al. 2010a; Bonilla et al. 2012; Han 2014), to slow down proteolysis, to prevent superficial dehydration and decrease oil absorption during frying of fishery products (Kilincceker, Dogan, and Kucukoner 2009). Examples of recent advances on EF and EC application to fishery products are presented in Table 9.2. To meet consumer’s choice for natural preservatives, food industry researchers turned toward natural antimicrobial compounds (essential oils [EOs], enzymes) to be used in EF and EC preparation.

9.2.1.1 EFs and Coatings with EOs EOs have been employed with promising results in the preservation of a wide variety of food products, due to their antimicrobial and antioxidant properties, which are directly correlated to the presence of bioactive and aroma volatile compounds (alcohols, acids, esters, epoxides, aldehydes, ketones, amines, and sulfides) (Calo et al. 2015). Gómez-Estaca et al. (2010) functionalized a gelatine chitosan (CH)-based EF with clove EO and tested its antibacterial effect against Pseudomonas fluorescens, Shewanella putrefaciens, Photobacterium phosphoreum, Listeria innocua, Escherichia coli, and Lactobacillus acidophilus. The gelatin CH-based EF (gelatin 6.00%, CH 2.00%, sorbitol 0.15 g·g−1 gelatin plus CH, clove EO 0.75 ml·g−1 biopolymer, soya lecithin 0.125 g·g−1 gelatin) was used for wrapping cod fillets, which were vacuum packed and stored at 2°C for 11 days. The clove-based EF delayed or even prevented both the growth of pathogenic and spoilage bacteria: after 12 days of storage, total viable bacteria (TVB) reached around 4 log CFU·g−1 in film-wrapped cod −1 samples compared to almost 9 log CFU·g in the control samples; H2S-producing microorganisms were completely inhibited in film-wrapped cod samples after 3 days of storage and the state was maintained throughout the entire storage period, while in the untreated samples, they had multiplied, reaching 8 log CFU·g−1 at the end of the 12th day of storage. Pseudomonas spp. doubled its number in the first 3 days of storage in the control sample and maintained throughout the rest of the storage period its initial cell count at around 7 log CFU·g−1. In the cod wrapped with film, a decrease in Pseudomonas CFUs was noticed after the first 3 days of storage, while after 12 days of storage, the concentration value was of about 2.2 log CFU·g−1, below the initial value. Lactic acid bacteria (LAB) counts reached levels of around 6 logs counts at the end of storage. Counts for Enterobacteriaceae were found to be high in untreated samples despite the storage temperature, while in the cod wrapped in film, their growth was continuously inhibited from the beginning of the experiment, with a complete inhibition being recorded after 9 days of storage. The antimicrobial 194 Trends in Fish Processing Technologies ( Continued ) (2014) (2015) Reference and Montero and Javadian and Javadian López de Lacey, López de Lacey, Kim et al. (2012) López-Caballero, Heydari, Bavandi, Bavandi, Heydari, Jouki et al. (2014) ) and TVB-N −1 ; DPPH assay CFU·g −1 10 0.04 mg·mL 50 Indicative Effect Indicative fish) maintained below the limits of acceptability fish) maintained below −1 S-producing microorganisms (~5.00 log S-producing microorganisms 2 ). −1 0.60 mg·mL were two times lower compared to uncoated samples); significant reduction of times lower were two changes during 21 days of storage; preferred for fish glossiness and smell volatile compared to uncoated samples. compared to untreated samples. (~23 mg TVB-N·100g refrigeration storage (4°C ± 1°C) compared to the control. and lactic acid bacteria counts during Pseudomonas spp., Enterobacteriaceae 18 days refrigerated storage (4°C). TVC, H in fish. EC: retarded lipid oxidation (after seven days of storage at 20°C TBARS values values TBARS days of storage at 20°C EC: retarded lipid oxidation (after seven EF: antioxidant properties (ABTS assay: IC < .05) lower TVB-N content and lipid oxidation during 16 days of Significantly ( p < .05) lower extension by 11 days. Significant reduction of Significant microbial shelf life Retarded lipid oxidation. Extended shelf-life of at least a week and increase the beneficial lactic acid bacteria Edible Film or Coating Edible Film EF and EC with xanthan 5.00% (w/w) (QSM), glycerol 35% (w/w) EO (2.00% QSM weight, thyme v/v), 0.1 and 0.2% (w/v) Tween 80 1.00% w/v horsemint EO 80 (0.25 g/g of Tween (HEO), HEO) 2.00% glucose, green tea probiotic bacteria: extract; spp. Lactobacillus paracasei LAFTI-L26 and paracasei Bifidobacterium animalis spp. lactis LAFTI-B94 Defatted mustard meal composite Defatted EF 1.00% quince seed mucilage EF 1.50% (w/v) sodium alginate, 1.00% glycerol, EF 1.50% agar, Oncorhynchus ( Oncorhynchus mykiss ) fillets Aristichthys nobilis ) ( Aristichthys fillets capensis ) fillets TABLE 9.2 Examples EF and of EC Applied Fishery to Products Product Fishery Smoked salmon Smoked Rainbow trout Rainbow Bighead carp ( Merluccius Hake Innovative Fish Packaging Solutions 195 (2009) (2009) (2007) (2010b) Reference Ojagh et al. Sathivel et al. Sathivel Vásconez et al. Vásconez Cao, Xue, and Liu ). ). −1 −1 CFU·g 10 in control samples −1 in control samples after −1 CFU·g 10 CFU·g 10 Indicative Effect Indicative (highest acceptable level 25.00 mg N·100 g (highest acceptable level after 16 days of refrigerated storage, while in control −1 −1 compared to the 7.88 log compared to 8.43 log −1 −1 CFU·g CFU·g 10 10 16 days of storage. after 12 days of storage (maximal recommended limit 7.00 log , and native lactic acid bacteria from , and native , Micrococcus , Moraxella Vibrionaceae oysters. aerobic mesophilic and psychrophilic cell counts, acceptable weight loss). yield. Significantly ( p < 0.05) higher thaw reached 42.93 mg N·100 g PTC 6.68 log TVB-N 14.23 mg N·100 g Extended shelf life at 5°C from 8–9 days to 14–15 due inhibition of Extended global quality during refrigerated storage to six days at 2°C (reduced during eight months storage at −35°C. down of oxygen slowed Diffusion Shelf life extended from 12 to 16 days for refrigerated (4°C) coated samples. Shelf life extended TVC <6.00 log chitosan used −1 Edible Film or Coating Edible Film for 10 min immersion chitosan, 0.64% (w/w) glycerol used for 3 min. fish immersion dipping frozen fillets at 4°C for 30 s acid, 0.75% glycerol, 0.20% 80, 1.50% cinnamon oil Tween EC 5.00 g·L EC solution made of 1% (w/w) EC 1.00% w/w chitosan used for EC 2.00% chitosan, 1.00% acetic thiobarbituric acid reactive substances; PTC, psychrotrophic bacteria count. thiobarbituric acid reactive

-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid; DPPH, 1,1-diphenyl-2-picrylhydrazyl; acid; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ABTS, 2,2 ′ -azino-bis-3-ethylbenzthiazoline-6-sulphonic TVC, total viable count; base nitrogen; TVB-N, total volatile TBARS, gigas ) Oncorhynchus ( Oncorhynchus ) skinless gorbuscha fillets Oncorhynchus ( Oncorhynchus mykiss ) fillets Crassostrea Oyster ( Crassostrea TABLE 9.2 (CONTINUED) 9.2 TABLE Examples EF and of EC Applied Fishery to Products Product Fishery Salmon Pink salmon Note: Rainbow trout Rainbow 196 Trends in Fish Processing Technologies effect corresponded with the delay in TVB production registered during fish storage by the cod-film lot. Therefore, it was concluded that EF with clove EO could ensure an extended shelf-life for chill-stored fish. In order to inhibit the growth of pathogenic bacteria on salmon, Song, Shin, and Song (2012) prepared a film made of barley bran protein (3%), gelatin (3%), sorbitol (1%), and grapefruit seed extract (1%). After 15 days of storage at 4°C, populations of E. coli O157:H7 and Listeria monocytogenes inoculated on salmon packaged with the film decreased by 0.53 and 0.50 log CFU·g−1, respectively, compared to the control. At the same time, the EF had a positive impact on the lipid oxidation rate by decreasing the peroxide value with 23.00% and that of thiobarbituric acid by 23.40%. Promising results were reported for the cold-smoked sardine fillets (Gómez- Estaca et al. 2007) covered with gelatin (4% w/v) EF functionalized with oregano, rosemary, or CH and high pressure treated (300 MPa/20°C/15 min.). The combination of high pressure with EF yielded the best results for oxidation prevention and microbial growth inhibition in the sardine fillets, which indicates the possible use of EF as part of hurdle technologies applied for food products with extended shelf life. An important source of proteins not enough valorized yet for EF and EC production is represented by the by-products of the fish and seafood industry. Although several research studies (Gomez-Estaca, Montero, and Gomez-Guillen 2014; Kim and Min 2012; Leerahawong et al. 2011; Pires et al. 2013) present successful preparation of EFs based on muscle proteins extracted either from fish or squid mantles, the number of reports on edible packaging tested directly on fish is limited at the present time. For example, Teixeira et al. (2014) assessed the antimicrobial activity of an EF made of proteins recovered from cape hake (Merluccius capensis) by-products (4 mg hake protein·cm−2 film) and functionalized with clove, garlic, and oregano EOs (1 μL·cm−2 film). The incorporation of EOs significantly increased the DPPH radical-scavenging activity of fish protein EFs, the highest antioxidant activity being registered by clove and garlic EFs (72% of inhibition). All EFs inhibited L. innocua, while garlic and oregano EFs inhibited Brochothrix thermosphacta and L. monocytogenes, whereas garlic and clove EFs inhibited S. putrefaciens.

9.2.1.2 EFs and Coatings with Antimicrobial Enzymes Another way of extending food products shelf life is to apply antimicrobial enzymes in EF and EC. Jasour et al. (2015) used for rainbow trout fillets an EC with chitosan (1.5% (w/v), lactoperoxidase system (LPOS) (5% v/v), and glycerol as plasticizer (0.75 mL∙g−1). Fish samples individually coated by immersing in CH solution for 1 min (2:1 ratio of coating solution to fish) were further stored under refrigeration conditions at 4°C ± 1°C for 16 days. In the early days of storage, a significant reduction, especially in H2S-producing bacteria (around two log cycles), was observed in CH and LPOS coated samples, followed by a gradual increase as the number of storage days raised. Samples of CH and LPOS group had significantly lower number of S. putrefaciens, P. fluorescens, psychrotrophic, and mesophilic bacteria than the CH and control group had during the storage period (4–16 days, p < .05). The total counts of bacteria in the control and CH group increased progressively with storage time, Innovative Fish Packaging Solutions 197 while control and CH samples reached the values of 6–7 log CFU∙g−1 in psychrotrophic and mesophilic bacteria on the 12th and 16th days of storage, respectively. The value was regarded as over the acceptability limit based on sensory scores, while CH and LPOS samples did not reach the same counts of cells during the whole 16 days of storage. TVB-N measurement, one of the most widely used indices of seafood quality, directly related with the growth of microorganisms, had the lowest (p < .05) value (22.07 mg N∙100 g−1 fish muscle) in CH and LPOS samples during the entire period of refrigeration. The TVB-N value of coated samples did not exceed the upper acceptability limit for spoilage initiation in cold water fish (30–35 mg N∙100 g−1) compared to the control samples, which surpassed this limit within 12–16 days of storage. This result indicates that the synergistic effect of CH and LPOS in the presented EC could ensure for chill-stored trout fillets an extended shelf life by at least 4 days as compared to the control samples. The antimicrobial effect of LPOS against L. monocytogenes was previously demonstrated by Min, Harris, and Krochta (2005). LPOS was incorporated into a whey protein isolate film (WPIF) and coating (WPIC), both applied to smoked salmon. The effect of this antimicrobial edible packaging was monitored during storage at 4°C and 10°C for up to 35 days. WPIF incorporating 29 mg LPOS per gram of film (dry basis) inhibited 4.20 log CFU·cm−2 of L. monocytogenes inoculated on agar media. WPIC prepared with LPOS at 0.7% (w/w) initially reduced >3 and 1 log CFU·g−1 of L. monocytogenes and total aerobic microorganisms, respectively, in smoked salmon. The WPIC incorporating LPOS prevented L. monocytogenes growth in smoked salmon at 4°C for 35 days and at 10°C for 14 days, as well as yeasts and molds. Thus, LPOS-WPIC shows greater potential than WPIF of inhibiting microorganisms present on RTE fish with tensile, barrier, and color properties identical to those without LPOS.

9.2.2 M ultifunctional Bioblends for Fish Packaging A novel concept in food packaging is represented by multifunctional bioblends, resulting from the use of conventional polymeric films as vehicles for transport of active substances (antioxidants, antimicrobials), thus improving their stability, efficiency, and molecule release into food products (Cerqueira et al. 2014). Employment of such innovative packaging, made of low-density polyethylene (LDPE) film with natural tocopherols (5.0% NUTRABIOL-T90), already available on the market, applied to frozen salmon muscle induced a reduction with 40% in lipid oxidation during 21 days of 4°C storage (Barbosa-Pereira et al. 2013). Another research group reported a retarded oxidation of polyunsaturated fatty acid in blue shark muscle slices packed in LDPE film with natural antioxidant derived from barley husks during storage of the frozen samples for 12 months (Pereira De Abreu et al. 2011). The antimicrobial effect of a CH-coated plastic film incorporating 9 mg·cm−2 sodium lactate was tested on cold-smoked salmon. For refrigerated samples a complete inhibition of L. monocytogenes growth was achieved for 6 weeks, while the accelerated shelf life tests performed at room temperature (~20°C) indicated a complete inhibition of L. monocytogenes for 10 days (Ye, Neetoo, and Chen 2008). 198 Trends in Fish Processing Technologies

9.2.3 nanobiocomposite Films Used for Fish Packaging Another promising development in the food packaging industry is represented by nanobiocomposite films, obtained by combination of conventional polymers with nanometer sized particles. An example of nanotechnology application to food packaging industry is represented by the inclusion of CH tripolyphosphate (TPP) nanoparticles (NPs) (4% CH, 4% TPP) in a coating used for fish fingers, stored at −18°C for 6 months (Abdou, Osheba, and Sorour 2012). Results indicated reduced total bacteria counts in the nanocoated sample (2.87 log CFU·g−1 compared to 5.27 log CFU·g−1 in the uncoated samples), psychrophilic bacteria of 2.52 log CFU·g−1 compared to 3.34 log CFU·g−1 in the control samples and coliform bacteria of 1.88 log CFU·g−1 in the CH nanocoated samples compared to 2.96 log CFU·g−1 in the uncoated samples. Besides the improved antimicrobial effect, this research indicated that CH NP also decreased oil absorption during frying. CH NP coated whiteleg shrimps (Litopenaeus vannamei, formerly Penaeus vannamei), stored for 10 days at 4°C exhibited significant (p < .05) decrease in TVB-N content, TBA amount, and TVC, as well as the higher hardness and springiness compared to uncoated samples (Wang et al. 2014). Other researches indicated zinc oxide NP as effective antimicrobial substances (Espitia et al. 2012); thus, they were used for bionanocomposite packaging materials obtained through its incorporation into glycerol plasticized pea starch (Ma et al. 2009). Other novel nanobiocomposite films prepared from CH, potato starch, glyc- erin, cyclophosphamide, and 2%–10% Fe3O4 NP were tested against E. coli and Staphylococcus aureus (Shariatinia and Mahbubeh 2015). The tested nanobiocomposite films showed greater antibacterial activity againstS. aureus than E. coli, exhibiting inhibition zones ranging from 13.94 ± 0.48 to 14.69 ± 0.44 mm and 11.89 ± 0.50 to 12.76 ± 0.47 mm, respectively. However, despite the high effectiveness of nanocomposite packaging in controlling food spoilage and pathogen microorganisms, there is a strong need for more in-depth studies and risk assessment of NP migration from the packaging into the food matrix in order to be able to assess long-term toxicological effects of nanopack- aging use. Even though the commercial use of EF and EC containing active ingredients is still reduced in the present, EF and EC could become in the near future a valuable complement to the conventional packaging systems, offering substantial advantages while increasing food product quality and safety. While studies of EF and EC applied to real food matrices will prove the effectiveness of the developed edible packaging, the industrial use of these materials depends on the economic and environmental impact, the regulatory aspects, and last but not least on the consumer acceptability.

9.3 IP OF FISH The uniqueness of IP consists in its ability to carry out intelligent functions such as detecting, sensing, recording, and tracing and to communicate information about certain aspects of a food. It always accompanies the food throughout the supply Innovative Fish Packaging Solutions 199 chain cycle, senses the environment on both sides of the package, and communi- cates the changes to consumers (Yam, Takhistov, and Miltz 2005). These actions facilitate decision making to improve food quality, enhance safety, extend shelf life, ensure more convenience, provide information, and warn when a safety problem arises (Robertson 2013; Yam, Takhistov, and Miltz 2005). The intelligent function is achieved by indicators, sensors, and/or devices introduced in the headspace of the package, attached to the lid or as label outside the package, incorporated into, or printed onto the food packaging material (Heising et al. 2014; Yam, Takhistov, and Miltz 2005). All the devices positioned inside the package have to meet the status of FCM according to EU regulation EC1935/2004 (2004).

9.3.1 iP Monitoring Environmental Conditions IP used for monitoring environmental conditions consists of indicators and sensors able to watch carefully conditions that influence the changes of quality attributes of the food (Heising et al. 2014). Indicators inform about a change that happened in a food or its environment, for example, temperature or pH through visual changes such as color changes (Realini and Marcos 2014; Yam, Takhistov, and Miltz 2005), while sensors monitor the food moisture and internal package RH (Pursula et al. 2013). The quality of food in the supply chain can be predicted with mathematical models obtained from the information on the food conditions. To be reliable, the predictions need a known and constant initial quality of the food measured immediately after packaging at the manufacturer (Heising et al. 2014). Devices with application to fish and fish product packaging are noninvasive TTIs, gas leakage indicators (integrity and oxygen indicators), and RH sensors.

9.3.1.1 Time–Temperature Indicators TTIs are small tags or labels attached to/introduced in the package (Lee and Mijanur Rahman 2013), which provide visual information about temperature history during distribution and storage of chilled or frozen foods. Thus, TTIs inform whether a threshold temperature has been exceeded over time or estimate the minimum length of time a product has spent above the threshold temperature (Biji et al. 2015; EC450/2009 2009). TTIs are able to show an easily measurable change of the temperature of a product during time. Usually, this is a visible and irreversible mechanical deformation, color development, or color movement based on mechanical, chemical, electrochemical, enzymatic, or microbiological reactions (Kerry, O’Grady, and Hogan 2006). Therefore, currently available TTIs are diffusion-, polymer-, photochromic-, enzymatic-, and microbial-based systems (Lee and Mijanur Rahman 2013). TTIs are extensively used in the fish processing industry for monitoring the traceability, the quality, and the shelf life of fish in the chilled and frozen chain. Meanwhile, researchers are concerned to improve and/or develop new TTIs to allow a realistic control of fish cold chain and predict the fish shelf life based on mathematical models. The most used in the fish processing industry are enzymatic-, photochromic-, and polymer-based TTIs. Thus, Giannakourou et al. (2005) used 200 Trends in Fish Processing Technologies an enzymatic TTI based on lipase (VITSAB AB, Malmö, Sweden) to monitor the gilthead sea bream quality in the chill chain and found a good correlation of TTI with end of shelf life at 107 CFU·g−1 Pseudomonas spp. after 109 h at 2°C. A photochromic TTI (OnVu, Ciba Specialty Chemicals & Freshpoint, SW), UV activated, has been used to monitor the quality of MAP gilthead sea bream fillets in the temperature range of 0°C to 15°C. It showed a good correlation with the end of shelf life at 6 log LAB level and allowed the validation of the model developed for MAP fish quality (Tsironi et al. 2011). Fresh turbot was monitored with a polymer-TTI (Fresh Check, TempTime Corporation, USA) under fluctuating temperature (Nuin et al. 2008). A good correlation of TTI with the sensory rejection time was found for 105–106 CFU·g−1 bacterial load at 0°C, 5°C, 10°C, and 15°C. Other authors compared two different TTIs, enzymatic (Check Point), and photochromic TTI (OnVu), using them for different fish and storage conditions. Giannoglou et al. (2014) monitored the quality and shelf life of frozen Blueshark slices and arrow squid and concluded that selecting an optimum TTI, a realistic control of fish cold chain could be obtained. Tsironi et al. (2008) applied a Safety Monitoring and Assurance System on the shelf life optimization of chilled tuna fish, vacuum packed, and observed a good correlation of both TTIs with the end of shelf life at 6.5 log LAB level (at 4°C). They also established and validated a kinetic model for growth of spoilage bacteria on vacuum packed tuna slices in the temperature range of 0°C to 15°C.

9.3.1.2 Seal and Leak Indicators Package integrity is essential to maintain the quality and safety of fish in vacuum packaging (VP) and MAP. In VP, a softened film with low oxygen permeability is placed over the product and vacuum applied. Sometimes, the film may be heat- molded over the product. In the case of delicate products such as smoked pickled fish and soft seafood, vacuum skin packaging is used (Slattery 2010). MAP represents packaging of food in an atmosphere consisting of a mixture of gases with a composition different of the air. This atmosphere is continuously modified as a result of respiration of the packaged food, selective permeability of the packaging material, and the presence of “atmosphere modifiers” (Berk 2013; Cooksey 2014; Velu et al. 2013). VP and MAP provide several mutual benefits: barrier against air and moisture, protection from dust, and tamper protection. VP alone ensures protection against dehydration and compressed packaging for fragmented fish. However, VP causes product compression followed by drip release, and it strongly depends on the initial quality of the fish and on a suitable temperature control throughout storage (Manju et al. 2007). MAP provides a better mechanical protection of fish than VP. MAP is also more effective than VP in preventing histamine production during storage and in ensuring a longer shelf life of fish (Gunsen, Ozcan, and Aydin 2011). Some of the most recent applications of MAP to fish and seafood are presented by Speranza et al. (2009), Bono et al. (2016), Calliauw et al. (2016), and Rodrigues et al. (2016). The active component in MAP is CO2. It is easily soluble in water, acts as a microbial inhibitor, and promotes the growth of Photobacterium phosphorum and LAB. Because CO2 is absorbed by the fish over time, the walls of gas-tight semirigid packs cave in when high levels of CO2 are used. Therefore, safe and high- quality MAP fish needs the control of gas mixtures and tight temperature (Slattery Innovative Fish Packaging Solutions 201

2010). Gas mixes used for nonfatty fish are mostly 30% 2O , 40% CO2, 30% N2, or 40% CO2, and 60% N2 for smoked and fatty fish (Robertson 2006). Other solution is to use MAP combined with superchilling to obtain an extended shelf life. Wang et al. (2008) used MAP (50% CO2/45% N2/5% O2) and −0.9°C storage temperature, thus obtaining a shelf life of fresh cod loins with 1.5 times higher than of MAP at 1.5°C. The gas composition within the package in MAP can easily change due to interaction of fish with its environment. For such situations, gas indicators offer a noninvasive alternative to traditional destructive techniques to determine the integrity of a package of MAP, for example, leaking seals (Vanderroost et al. 2014). Gas indicators have to be in direct contact with the gaseous atmosphere from inside the package and also FCM, typically signaling the presence or absence of O2 and/or CO2, whether there is a gas leakage in the package, or verifying the efficiency of an oxygen scav- enger. They usually provide qualitative or semiquantitative information about altered gas concentrations (CO2, O2, water vapor, etc.) through visual colorimetric changes (Vu and Won 2013). The simplest integrity indicators are time indicators that provide information about how long a package has been opened, the presence of oxygen, indicating that the package has a leak or has been tampered. Colorimetric oxygen indicators used to detect oxygen in MAP are created as an intelligent ink system by using a redox dye (Dox), a sacrificial electron donor (SED), a semiconductor photosensitizer (SC), and a coating polymer (Mills and McGrady 2008; Vu and Won 2013). In this system, the SC absorbs UV-light in an activation step and creates electron-hole pairs. The photogenerated holes react rapidly with the

SED and the electrons react with the Dox (Mills and Hazafy 2009; Mills and Lawrie 2011; Mills and McGrady 2008; Ollis, Mills, and Lawrie 2016).

9.3.1.3 RH Sensors Monitoring of fish moisture and/or internal RH of package may help in making decisions in order to control water activity for reducing microbial growth on fish, prevent condensation from fresh fish, remove melting water from frozen fish or ice used for ice-chilled fish, and keep the rate of lipid oxidation low (Codex Alimentarius 2012). These are mainly made by AP using moisture absorbers. However, the literature mentions a combination of RFIDs with RH sensors such as the passive high- frequency and ultrahigh-frequency RFID transponder developed by Pursula et al. (2013) for RH and temperature monitoring in packaged fish.

9.3.2 iP Monitoring Quality Attributes Fish is predisposed to large variations in quality attributes due to various species, feeding or environmental conditions, and changes in the product during storage and can also carry various spoilage or even pathogenic microorganisms. Therefore, the maintenance of quality is of utmost importance in processing and commercialization of fish and fish products. Thus, quality indicator compounds are monitored by measuring of quality-related compounds formed in the product (freshness sensors and indicators) or microorganisms in/on the product (biosensors) (Heising et al. 2014; Mohebi and Marquez 2015). 202 Trends in Fish Processing Technologies

9.3.2.1 Fish Freshness Sensors and Indicators The quality of fish is directly influenced by freshness so that there is an increased requirement for on-site measurement of this quality attribute during any stage of fish distribution (Itoh et al. 2012). The freshness of the fish is usually evaluated using sensory attributes such as pupil color, eye position, flesh elasticity, muscle contraction, gills color, and skin odor (Chun, Kim, and Shin 2014). It may express in certain metabolic products, gaseous or dissolved in the product (van Veen and van Dongen 2010), as a result of microbial degradation. The number of microorganisms on the skin and gill surfaces of the fish increases progressively after death, spreading within various tissues (Hamada-Sato et al. 2005). They are known as specific spoilage organisms and are usually consisting of Pseudomonas spp. (Boulares, Mejri, and Hassouna 2011), Shewanella spp., and genera of the Enterobacteriaceae family (Skandamis and Nychas 2012). The products of microbial degradation are volatile compounds such as trimethylamine

(TMA), dimethylamine, and ammonia (NH3), which are known as total volatile basic nitrogen (TVB-N) and used as specific marker for fish (Bhadra et al. 2015a; Bhadra, Thomson, and Bridges 2015b; Heising et al. 2012; Kuswandi et al. 2012; Pacquit et al. 2006, 2007). Another indicator of fish freshness is adenosine-5'-triphsophate (ATP), which is present in the muscle and begins to degrade to uric acid after the death of the fish (Barat et al. 2008; Itoh et al. 2012). Table 9.3 presents several research results related to freshness indicators and sensors for fish and fish product packaging.

9.3.2.2 Biosensors Biosensors are mostly applied to pathogen detection and safety of packaged food, even though they have also been used to indicate the freshness of fish and meat (Vanderroost et al. 2014). They can be placed inside the food package or integrated into the packaging material, although there are also handheld or desktop biosensors (LaGier, Fell, and Goodwin 2007; Yam, Takhistov, and Miltz 2005). When used in IP, biosensors have the disadvantage of not being able to continuously monitor the quality of the food (Heising et al. 2014). Selective identification is based on fluorescence, magnetic devices, conductance, or bioluminescence, although enzyme sensors and immune sensors also exist (Mahalik and Nambiar 2010). Among fish pathogens detected in most of the recent researches are bacteria such as Aeromonas hydrophyla (Jafari et al. 2015; Ligaj et al. 2014; Tichoniuk et al. 2010), Aphanomyces invadans (Kuan et al. 2013), and viruses such as fish nervous necrosis virus (Su et al. 2015; Toubanaki, Margaroni, and Karagouni 2015). Nanosensors, comprising of NPs, which attach to pathogens or other contaminants (Kaittanis, Santra, and Perez 2010), are applied for fish pathogen detection (Jafari et al. 2015; Kuan et al. 2013; Toubanaki, Margaroni, and Karagouni 2015). Table 9.4 presents several recent examples of biosensors used for pathogen detection in fish.

9.3.3 D ata Carriers 9.3.3.1 Barcode Labels A barcode contains information about the product to which it is attached or printed and allows tracking the location of a product at any point in the supply chain. Innovative Fish Packaging Solutions 203 ( Continued ) (2012) References Kuswandi et al. Kuswandi and Dekker (2015) and Dekker Pacquit et al. (2007) Pacquit Pacquit et al. (2006) Pacquit Heising et al. (2012) Heising, van Boekel, Boekel, Heising, van

−1 −1

5 CFU·g 7 CFU·g 7 was was −1 to 10 4 CFU·g 7 (10 h) to 3.9 × 10 −1 CFU·g 5 Analysis Results Analysis after 26 h (at room temperature, 19°C–21.5°C— −1 (threshold of bacteria spoilage). −1 CFU·g 8 Correlation of the Sensor Response with the Biochemical of the Sensor Response with Biochemical Correlation from the sensor signal based on TMA. from the sensor signal based on reached. (end of shelf life) after about 18 h and stabilized at around 10 (24 h) at room temperature (25°C). 14 h, at 25°C. headspace of the package, could be nondestructively monitored headspace of the package, could be nondestructively in the aqueous phase. CFU·g in fish spoilage trial). after 18 h at room temperature. of 10 when the specific level TVB-N level) Mathematical model to predict the freshness of the packed fish Mathematical model to predict the freshness of packed increased from 85% to 100% of TVC at around Pseudomonas increased from 85% to 100% of film changed from green to blue at 5 × 10 Color of the PANI Changes in the ammonia content, released from fish counts rose from 65% to 100% of the TVC counts Pseudomonas counts rose from 65% to 100% of the to green (due changes in The sensor color changes from yellow TVC increased from 2.9 × 10 Total viable counts (TVC) increased from ca. 10 Total TMA TMA TVB-N TVB-N TVB-N TVB-N Marker Specific Ammonia fillets Product grenadier Fish/Fish Fish/Fish Cod fillets Milkfish— Packed cod Packed cod, cardinal, Fillets of fresh Fillets of fresh and roundnose and main bone without innards cod and whiting -ISE) and a reference electrode -ISE) and a reference electrode + 4 + 4 NH NH (PANI) (Ag/ AgCl) ( (Ag/ AgCl) polyethylene terephthalate (PET) coated polyethylene with a typical sensor solution (cellulose bromocresol green as acetate as binder, dye, an ammonium salt, and pH-sensitive a plasticizer) and placed in sandwich (PTFE) between a polytetrafluoroethylene permeable membrane and a clear gas cover adhesive protective ( Colorimetric sensor containing polyaniline TABLE 9.3 Summary Freshness to Research of Results and Related Indicators Sensors Fish for Indicator or Sensor Freshness Ammonium ion-selective electrode Ammonium ion-selective Colorimetric sensor: discs of optically clear Ammonium ion-selective electrode Ammonium ion-selective 204 Trends in Fish Processing Technologies References Shin (2014) Bhadra et al. Thomson, and Chun, Kim, and Bridges (2015b) (2015a); Bhadra, Barat et al. (2008) ) −1

−1 CFU·g 7 CFU·g 5 at 30 h, room −1 during initial 9 h then −1 CFU·g 8 during the initial 16 h then rose . a −1 CFU·g in 4 days at 4°C, reaching 5 7 Analysis Results Analysis to 10 CFU·g 5 4 evolution

at 27 h. −1 at the end of trial (7 days for minced meat). −1 at 88 h. −1 CFU·g 9 CFU·g 9 CFU·g 8 – 10 8 Correlation of the Sensor Response with the Biochemical of the Sensor Response with Biochemical Correlation during the initial 20 h, then to 10 temperature (19°C–21°C). became stable after 48 h. spoilage of fish. 10 in both trials: at 15 h (24°C) and 48 (4°C). to 10 rose to 10 fish fillets, which is in good correlation with changes as pH, Ino, and Hx and IMP, counts rapidly increased to 10 Pseudomonas fragi The color of freshness indicator began to change at 15–30 h and The color of freshness indicator began The pH increased from 6.2 to ca. 6.5 in 4 days due rapid TVC increased from 10 Trial at 24°C: Trial increased from 10 TVC slowly TVC to near 100% after 32 h. Pseudomonas count rose from 78% of of fish (10 Sensor distinctly identifies the rejection level count rose from 75% of TVC to near 100% after 12 h. Pseudomonas count rose from 75% of at 4°C: Trial TVC increased from 10 Redox potential of Au and Ag electrodes changed after 4 days for Au and Redox potential of TMA TVB-N TVB-N Marker Specific compounds ATP-related ATP-related fillets bream Product Mackerel Fish/Fish Fish/Fish Fillets and Tilapia fish Tilapia gilthead sea minced meat of IMP, inosine 5 ′ -monophosphate; Ino, inosine; Hx, hypoxanthine. IMP, pH-sensitive dye bromocresol green pH-sensitive (BCG) metal oxide and silver/silver chloride metal oxide and silver/silver (Ag/ AgCl) electrodes and calomel reference electrode

Polymer matrix solution containing a TABLE 9.3 (CONTINUED) 9.3 TABLE Summary Freshness to Research of Results and Related Indicators Sensors Fish for Indicator or Sensor Freshness a Hydrogel-coated pH-electrode: mixed Hydrogel-coated pH-electrode: mixed Gold (Au) and silver (Ag) potentiometric Gold (Au) and silver Innovative Fish Packaging Solutions 205

TABLE 9.4 Summary of Research Results Related to Biosensors Used in Fish Processing Industry Linear Range (LR), Detected Detection Limit (DL), Biosensor Type Analyte and Response Time (RT) References Amperometric biosensor with Xanthine (X) and LR: Cubukcu, gold nanoparticles (Au-np) hypoxanthine 5.0 × 10−7–1.0 × 10−5 M (X) Timur, and and xanthine oxidase (XOD) (Hx) in canned 5.0 × 10−6–1.5 × 10−4 M (Hx) Anik (2007) tuna fish DL: 5 μM (X), 80 μM (Hx) Amperometric Streptococcus LR: 101–107 CFU∙mL−1 Vasquez et al. immunobiosensor agalactiae DL: 10 CFU∙mL−1 (2017) pathogen RT: 15–60, optimum: 30 min Amperometric nanobiosensor Hx as freshness LR: 3.0 × 10−8–2.8 × 10−5 M Zhang et al. indicator DL: 10 nM (2010) Electrochemical ATP sensor ATP as freshness LR: 0.0–200.0 μM (standard Itoh et al. indicator solution); 0.0–7.0 μmol·g−1 (2012) (jack mackerel) DL: 8.9 μM RT: 0–80 s Electrochemical DNA Aeromonas LR: 1.0–10 μM DNA Tichoniuk hybridization sensor hydrophila DL: 2.5 μg·cm−3 et al. (2010) pathogen RT: 15 min Electrochemical DNA sensor Aphanomyces Step-by-step hybridization: Kuan et al. based on Au-np invadans LR: 5.0 fM to 5.0 pM (2013) pathogen DL: 5.0 fM Premix hybridization: LR: 0.05 fM to 1.0 pM DL: 0.5 fM Lateral flow paper biosensor Fish nervous LR: 135 pg–1 ng/reaction Toubanaki, with Au-np electrode necrosis virus DL: 270 pg of total RNA Margaroni, (NNV) or RT: 20 min and Karagouni Nodavirus (2015) Surface-enhanced fluorescence Microcystin-LR LR: 0.02–16.00 ng∙mL−1 Li et al. (2014) immunobiosensor toxin DL: 0.007 ng∙mL−1 Electrochemical biosensor: Xanthine (X) as LR: 1.0–400.0 μM Reza et al. Tin oxide quantum dots fish freshness RT: 10 s (2013)

(SnO2 QDs ~1/5 nm) deposited indicator onto indium-tin-oxide glass

The information can be read by an optical barcode scanner that sends the information to a system, where it is stored and processed (Lee and Mijanur Rahman 2013). This system has become the most widespread form of data carrier because it is very simple and has a low price. Product coding systems and automatic identification of codes have been developed to overcome diversity and abundance of products on the market. The Universal 206 Trends in Fish Processing Technologies

Product Code (UPC) has been designed in 1973 as a linear barcode with 12 digits to be used throughout North America (United States and Canada). In Europe, the European Article Numbering (EAN) Association has introduced EAN-13 barcode in 1977. Later on, the cooperation between Universal Code Council and EAN International led to the launch of the global data synchronization network in 2004 and the Global Standards (GS1) in 2005 with the new universal barcode Global Trade Item Number (GTIN). GTIN describes a family of GS1 (EAN·UPC) global data structures that employ 14 digits and can be encoded into various types of data carriers (GS1 2016). Currently, GTIN is used exclusively within barcodes, but it could also be used in other data carriers such as RFID (Overbosch and Blanchard 2014). Although barcodes are used on a large scale, their major disadvantage is that they provide very limited information about the product and leave no room for other information (Lee and Mijanur Rahman 2013). To overcome this situation, advanced barcoding systems are being introduced, either one-dimensional barcodes such as GS1 DataBar (formerly Reduced Space Symbology) or two-dimensional barcodes such as GS1 DataMatrix (similar to Portable Data File, PDF417) and GS1 QR code (Quick Response) (GS1 2016). Lately, a rapidly emerging global initiative that involves the use of arbitrary DNA sequence to characterize species has been developed (Maralit et al. 2013). This is called DNA barcoding and it is used as a reliable method for food traceability (Galimberti et al. 2013) and authenticity (Nicole et al. 2012). The US Food and Drug Administration proposed the DNA barcoding for the authentication of fish-based commercial products (FDA 2015). Thus, DNA barcode sequences were generated for fish contained in the Regulatory Fish Encyclopedia, an FDA web-based resource. Additionally, the Fish Barcode of Life initiative (FISH-BOL) is an international research focused on a standardized reference library of DNA barcodes for all fish species derived from voucher specimens with authoritative taxonomic identifications (IBOL 2017). During the last few years, a huge number of researches generated DNA barcoding data for fish species identification (Armani et al. 2015; Brandao et al. 2016; Carvalho et al. 2015; Gunther, Raupach, and Knebelsberger 2017), detection of mislabeled seafood products (Chin et al. 2016; Lamendin, Miller, and Ward 2015; Maralit et al. 2013), or for achieving better monitoring, conservation, and management of certain fish species (Zhang et al. 2017).

9.3.3.2 Radio-Frequency Identification Although RFID systems have been available for many years, their application in packaging has only begun in recent years (Kerry, O’Grady, and Hogan 2006; Siro 2012). The packaging industry uses passive RFID tags because they are much cheaper than the active tags (Kuswandi et al. 2011). RFID technology offers important benefits to the food supply chain, improving traceability and facilitating automation (Peppa and Moschuris 2013). In addition, RFID has a long reading range, being detected at distances of more than 100 m, the signal is very strong and can work under extreme temperatures and various pressures (Abad et al. 2009; Mohebi and Marquez 2015). The main disadvantages of RFID are the high cost of tags used in individual primary packages (Roberts 2006), the difficulty of reading information from tags on food containing a high amount Innovative Fish Packaging Solutions 207 of moisture that absorb the signals or metals that reflect them (Kumar et al. 2009), and the disclosure of consumer information such as buying habits (Lee and Mijanur Rahman 2013). The use of RFID systems in the fish supply chain is largely described in literature. Thus, Abad et al. (2009) reported the results regarding a demonstration of RFID use along an intercontinental fresh fish logistic chain, Martínez-Olmos et al. (2013) presented an RFID tag with an optical oxygen indicator that is perfectly suitable for use in MAP where the oxygen concentration is reduced below 2%, and Smits et al. (2012) estimated the freshness of cod fish using an RFID connected with sensors able to measure temperature, humidity, and the presence of volatile amine compounds. RFID systems are used for fish traceability (Smits et al. 2012; Trebar, Lotric, and Fonda 2015; Zhang et al. 2009), cold chain monitoring (Jedermann, Ruiz-Garcia, and Lang 2009; Trebar et al. 2013), shelf life prediction, and quality monitoring (Hafliðason et al. 2012; Tingman, Jian, and Xiaoshuan 2010).

9.4 CONCLUDING REMARKS AND FUTURE PERSPECTIVES The quest for higher food quality and safety is one of the biggest challenges for food producers worldwide. Innovative packaging solutions developed in the last decade, AP and IP, are perceived as more than an integral part of the food system and have the potential of becoming in the near future an everyday packaging commodity. Despite the numerous advantages of innovative fish packaging solutions, a considerable gap in market applications exists. Even though a cost–quality analysis of AP and IP use indicates a positive result, the cost–benefit analysis is negative (Dainelli et al. 2008) for the time being. As consequence, the main limiting factors for development of active and intelligent materials are cost and consumer acceptance related (Ghaani et al. 2016). Another important aspect to consider is the limited effectiveness of active and intelligent materials in real food packaging applications compared to their in vitro high efficiency. A future challenge for the active and IP industry is to develop sustainable long- lasting devices instead of the one-use SP devices.

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Johannes Pucher and Christian Schlechtriem

CONTENTS 10.1 Production Systems of Aquatic Food...... 218 10.1.1 Fish and Other Aquatic Products as Food...... 218 10.1.1.1 Capture Fisheries...... 218 10.1.1.2 Aquaculture...... 219 10.2 Objectives and Regulations to Ensure Food Safety...... 220 10.3 Assessment and Management of Food Risks from Aquatic Products...... 223 10.3.1 Environmental Contaminants...... 223 10.3.1.1 Origin, Pathways, and Exposure of Environmental Contaminants...... 223 10.3.1.2 Regulatory Definition of Safety of Environmental Contaminants...... 224 10.3.1.3 Measures to Ensure an Adequate Level of Protection from Environmental Contaminants...... 225 10.3.2 Feed Contaminants...... 225 10.3.2.1 Origin, Pathways, and Exposure of Feed Contaminants....225 10.3.2.2 Regulatory Definition of Safety of Feed Contaminants.....226 10.3.2.3 Measures to Ensure an Adequate Level of Protection from Feed Contaminants...... 227 10.3.3 Algae Toxins within the Natural Food Chain...... 228 10.3.3.1 Origin, Pathways, and Exposure of Algae Toxins...... 228 10.3.3.2 Regulatory Definition of Safety of Algae Toxins...... 228 10.3.3.3 Food Safety Indicators and Measures to Ensure an Adequate Level of Protection from Algae Toxins...... 229 10.3.4 Microbiological Contamination...... 230 10.3.4.1 Origin, Pathways, and Exposure of Microbiological Contamination...... 230 10.3.4.2 Regulatory Definition of Safety of Microbiological Contamination...... 232 10.3.4.3 Food Safety Indicators and Measures to Ensure an Adequate Level of Protection from Microbiological Contamination...... 232

217 218 Trends in Fish Processing Technologies

10.3.5 Parasites...... 234 10.3.5.1 Origin, Pathways, Contamination, and Hazards of Parasites...... 234 10.3.5.2 Regulatory Definition of Safety in Relation to Parasites....234 10.3.5.3 Food Safety Indicators and Measures to Ensure an Adequate Level of Protection against Parasites...... 235 10.4 Conclusion...... 235 References...... 235

10.1 PRODUCTION SYSTEMS OF AQUATIC FOOD

10.1.1 Fish and Other Aquatic Products as Food Fish and other aquatic food products are an important source for well-digestible, high quality proteins and lipids, which are fundamental to a well-balanced, healthy diet (Food and Agriculture Organization [FAO] 2014a). Fish are seen as healthy food due to their profile and content of essential amino acids, polyunsaturated fatty acids (e.g., eicosapentaenoic acid and decosahexaenoic acid), vitamins, and minerals. Globally, total fish demand is increasing due to population growth, rising per capita consumption, and better access to global and local markets (FAO 2014a). Since the 1990s, the growing demand for aquatic food products cannot be satisfied by capture fisheries alone and caused a steady increase of aquaculture production of on average 8.8% annually. This makes aquaculture the fastest growing agricultural sector globally, especially in Asia (FAO 2014a). In 2012, the global fish production accounted for 158.0 million tons, while capture fishery produced 91.3 million tons and aquaculture production accounted for 66.6 million tons (data from 2012; FAO 2014a). Aside from the supply of fish to markets, the quality and safety of the food products is a prerequisite for trading. It is important to understand the globally established production systems and processing procedures to assess the potential risks for food safety and to maintain an adequate level of protection for consumers.

10.1.1.1 Capture Fisheries In general, capture fishery supplies the markets with organisms of higher trophic levels like piscivorous or carnivorous fish species (pollock, tuna, cod, hake, drums, croakers, snappers, groupers, flatfish, breams, basses, etc.), mollusc species (squid, cuttlefish, octopus), and crustaceans (shrimp, lobsters, crabs) (Neori and Nobre 2012; Tacon et al. 2010). Freshwater fisheries, with only 13% of the global capture production, play a minor role in fishery-based food production compared to marine fisheries production, which amounts to 57% of total fishery production. The remaining part, representing 21.7 million tons of capture fishery, is used to produce fishmeal and fish oil for the feed industry, while these feed resources are dominantly used in feeds for aquaculture (Shepherd and Jackson 2013). The diversity of species and fishing grounds causes a high variation in quality and safety of food products. Potential risks are the bioaccumulation of pollutants or contamination with harmful parasites, marine toxins, bacteria, or viruses (Table 10.1). Achieving Adequate Protection and Suitable Food Safety Indicators 219

TABLE 10.1 Food Safety Hazards, Their Origin, the Production System, and Potentially Affected Aquatic Food Organisms Potential Food Production Potential Affected Safety Hazards Hazard Origins Systems Aquatic Organisms Heavy metals Polluted water, natural food Fishery, aquaculture High trophic organisms web, sediments PCBs Polluted water, natural food Fishery, aquaculture High trophic organisms web, sediments Marine toxins Algae Fishery, aquaculture Filter feeding and herbivorous organisms (e.g., bivalves), to less extent high trophic organisms Parasites Infested natural food webs Fishery, extensive Fish, especially marine aquaculture fish Pesticides Water, natural food web, Inland fisheries and All plant-based feed resources aquaculture Veterinary substances Inadequate application Intensive aquaculture Fish and crustaceans Bacterial contamination Water (esp. for bivalves), Fishery, aquaculture All feed, processing, storage Fungal contamination Feed, processing, storage Fishery, aquaculture All Viral contamination Water (esp. for bivalves), Fisheries Bivalves and sediments crustaceans Histamine Fishery, aquaculture All

10.1.1.2 Aquaculture In contrast to fisheries, aquaculture is defined as having “…some sort of intervention in the rearing process to enhance production, such as regular stocking, feeding, protection from predators…” (FAO 1997, p. 6). The FAO divides aquaculture production into about 520 single species or groups of species (excluding plants and mammals) cultured in marine, brackish, or freshwater. The species differ greatly in their nutritional requirements and feeding behavior (planktivorous, herbivorous, detrivorous, omnivo- rous to piscivorous, and carnivorous) and are raised under various culture conditions. In general, aquaculture is classified into extensive, semi-intensive, and intensive aquaculture. Under extensive aquaculture, fish from lower trophic levels are grown solely on natural feed resources without substantial inputs of external feeds and fertilizer. In such an aquaculture production, similar risks for food safety as in capture fisheries are present (Table 10.1). In semi-intensive aquaculture, fish are grown on a combination of external supplemental feed and natural feed resources supported by fertilizer inputs. These systems often use feed and fertilizer resources of lower nutritional quality, which enhance specific feed and fertilizer originating hazards for food safety. 220 Trends in Fish Processing Technologies

In intensive aquaculture, fish are grown solely on external feed inputs, which are formulated to supply the cultured fish with all their required nutrients and energy. Intensive aquaculture enables the highest control over the culture conditions including water quality, feed utilization, hygiene condition, and health management of the cultured species. It also enables the highest stocking densities through the use of technical equipment (pumps, filters, disinfection units, heaters/coolers, feeders…) to meet the needs of the cultured species. Under intensive aquaculture, potential hazards are insufficient quality of feeds and water as well as inadequate utilization of veterinary medicines and production technologies. While extensive and semi-intensive aquaculture productions serve mainly local and regional markets, intensive aquaculture provides highly traded goods for international markets. According to FAO (2014a), 37% of the seafood produced globally was exported in 2012, which implies a strong imbalance in geographical supply and demand in seafood. In 2012, 49% of the import value of developed countries originated from developing countries (FAO 2014a). Consequently, seafood products consumed in industrialized countries are often produced in countries with lower standards of production, processing, and trading. Often, seafood is produced in one country, processed in another, and the products are finally exported to markets in further countries. This makes harmonized and internationally accepted standards and regulations for production, processing, and trading of aquatic foods essential to ensure an adequate level of protection of the consumer. Fish and other aquatic food products are traded fresh or preserved at different stages of processing. The trading of fresh products necessitates highly controlled cold chain management and fast transportation. Preservation technologies for aquatic food products include freezing, chilling, heating, frying, boiling, drying, salting, smoking, pickling, fermenting, and canning. Highly processed fish products include convenient food products like fish salads or fish crackers as well as functional foods or food supplemental products like cod liver oil or ingredients extracted from algae. These different food products can present potential hazards for consumers such as contamination of the products with undesired substances or toxins from the abiotic environment or from feeds and inadequate veterinary treatments, microbiological contamination with bacteria or viruses, infection with parasites, and degradation due to unsuitable hygiene managements, processing, packaging, storage, or cold chain management.

10.2 OBJECTIVES AND REGULATIONS TO ENSURE FOOD SAFETY To protect consumers from potential safety hazards of fish and other aquatic food products, a variety of regulations and standards are established at international, multinational, national, and regional levels (Figure 10.1). Some of them follow the three-step approach of scientific risk assessment, risk management, and risk communication. Hazard is defined as a biological, chemical, or physical agent in, or condition of, food with the potential to cause an adverse health effect. Risk is defined as a function of the probability of an adverse health effect and the severity of that effect, consequential to hazards in food (CAC 1999). Achieving Adequate Protection and Suitable Food Safety Indicators 221

Industry standards International and multinational regulatory – GAP institutions and consortia: – GHP – Codex Alimentarius – GMP – FAO – Traceability – WHO – Quality and safety monitoring – WTO – HACCP – OIE – IPPC – OECD – EU

Private sector certiﬁcations: National regulations: – Safety standards – Food and feed safety regulations – Water regulations – Sustainability standards – Veterinary and health regulations

FIGURE 10.1 Overview of international and national regulations, certificates, and standards for food and feed safety. EU, European Union; FAO, Food and Agriculture Organization; GAP, Good Agricultural Practice; GHP, Good Hygienic Practice; GMP, Good Manufacturing Practice; HACCP, Hazard Analysis Criterial Control Point; IPPC, International Plant Protection Convention; OECD, Organization for Economic Co-Operation and Development; OIE, World Organization for Animal Health; WHO, World Health Organization; WTO, World Trade Organization.

Food safety regulations impose the food and feed industry to control and ensure the safety and the traceability of their products along their production chains including production, processing, packaging, storage, transportation, and distribution to the consumers. In the European Union (EU), the food industry is responsible for the safety of their products along the production chain (EC 2004a) and the precautionary principle prohibits the placing of unsafe food on the market (EC 2002b). Further regulations lay down specific hygiene rules for unprocessed and processed food products of animal origin (EC 2004b). The European Commission demands that the industry constantly monitor the production and processing of seafood and feeds for quality and safety by applying procedures based on the Hazard Analysis Criterial Control Point (HACCP) approach (see Chapter 11) (EC 2004a), together with preliminary programs such as: good hygiene practices, good manufacturing practices, good handling practices, and others. HACCP is a science-based approach to ensure food quality and safety by identifying specific hazards and control measures in order to prevent, eliminate or reduce hazards to acceptable levels along the production chain. Apart from the industry-based quality and safety systems for foods and feeds, governmental laboratory analyses, audits, and inspections also monitor the food and feed products along the production chains (EC 2004c). There is a wide range of analytical, microbiological, and microscopic/macroscopic methods in use that are used as indicators for food safety (Table 10.2). While several unaccredited methods and tests might be applied by the industries as indictors for safety along the production chain, finally, accredited methods must be used to confirm the safety of products. International standards for quality and safety support the control of internationally traded food. In 1963, the FAO and the World Health Organization (WHO) established the Codex Alimentarius to develop harmonized international food standards, 222 Trends in Fish Processing Technologies Season of Production Level Species/Trophic Species/Trophic of Feed of Feed Commodities Contamination Environmental Environmental Contamination Sensory Texture and Texture Tests Quick Quick Observation Macroscopic Macroscopic Microscopic/ Assays Microbial. Microbial. Analytics Chemical Dark: clear indication; grey: limited indication; white: no indication. Dark: clear indication; grey: contamination TABLE 10.2 Food Safety Indicators Heavy metals Heavy PCBs Pesticides Marine toxins Parasites Microbiological Histamine Note: Achieving Adequate Protection and Suitable Food Safety Indicators 223 guidelines, and codes that help to protect consumer health and promote fair practices in global food trade. Codex Alimentarius standards, guidelines, codes, and recommendations cover a broad range of aspects including food labeling, food hygiene, food safety risk assessment, sampling and analysis, inspection and certification procedures, as well as animal food production. The Code of Practice for Fish and Fishery Products is especially important for foods from fish and other aquatic biomass. Additionally, the Codex Alimentarius sets maximum residue levels (MRLs) for pesticides in foods and feeds and sets conditions for the use of food/feed additives and veterinary drugs in foods. All these documents are written for all stakeholders in the food sector from production, processing, trade, regulatory and safety authorities, and consumers. Member states of the World Trade Organization follow the Codex Alimentarius standards under the Agreement on Sanitary and Phytosanitary Measures (SPS Agreement). In many states, the Codex standards serve as a basis for national legislation, while many states apply stricter food safety measures than those set by Codex Alimentarius (e.g., the EU and United States). Scientifically based recommendations on animal health and veterinary medical use in animals for food production are internationally set by the World Organization for Animal Health (OIE). Additionally to the standards, guidelines, and codes, national/European food and feed safety surveillance and monitoring systems have been established to ensure food safety and quality. Samples with concentrations exceeding the regulatory thresholds are published in the Rapid Alert System for Food and Feed (RASFF), which is an information exchange tool for national control authorities within the EU and is accessible by all stakeholders. In the case of a serious health risk from foods or feeds, measures such as withdrawal or recalling food or feed products from the market or blocking certain trade channels in order to protect consumers’ health may be taken. Risk analyses are constantly in progress adopting regulatory measures and governmental monitoring systems to react on changes in the food/feed supply as well as emerging risks, to minimize the potential risks for consumers. In the EU, this task is fulfilled by the European Food Safety Authority (EFSA), which provides scientific advices on food and feed safety issues and emerging risks to the European Commission, the European Parliament, and EU Member States. On national level, similar institutions are established in each European member state and in many countries around the globe (e.g., Australia, Canada, Japan, and United States).

10.3 ASSESSMENT AND MANAGEMENT OF FOOD RISKS FROM AQUATIC PRODUCTS

10.3.1 environmental Contaminants 10.3.1.1 Origin, Pathways, and Exposure of Environmental Contaminants Chemical pollution is one of the main sources of impact affecting aquatic ecosystems. Almost all anthropogenic pollutants are transported via rivers into lakes and seas. Contamination of the aquatic environment can come from many different sources such as industrial and municipal discharges, agricultural practices or storm water runoff. Also, the direct input of harmful substances into the water from ships and drilling rigs, by dumping of sewage sludge, or the discharge of dredge material as well as 224 Trends in Fish Processing Technologies atmospheric input of contaminants from gases, aerosols, or airborne particles may lead to significant contamination of the aquatic (marine) environment (FAO 2014b). Environmental pollutants that give rise to considerable concern are heavy metals (e.g., organo-tins, mercury, cadmium, lead) and industry chemicals (e.g., perfluori- nated alkylated substances; polychlorinated biphenyl, PCB; polybrominated diphenyl ethers). Also, polycyclic aromatic hydrocarbons, which are natural constituents of mineral oil, dioxins from combustion processes, pesticides from agricultural use, and other chemicals like fragrances, cyanides, and radioactive substances, need to be mentioned in this context (FAO 2014b; Hastein et al. 2006). Marine aquaculture may also have a significant impact on environmental pollution, especially concerning the use of biocides, antibiotics, and further veterinary drugs. The safety of human food can directly be affected when pollutants dissolved in the water or bound to sediments enter the aquatic food chain and accumulate in wild fish and shellfish or farmed organisms (FAO 2004, 2014b). Bioaccumulation is the process by which chemicals are taken up by an organism either directly from exposure to contaminated water (bioconcentration) or by consumption of feed containing the chemical (biomagnification). Generally, bioaccumulation increases with increasing hydrophobicity of chemicals, which tend to accumulate in fatty tissues like skin, organs, and filet. Fish can also accumulate heavy metals and their organometallic derivatives from feed, water, and sediments. Mercury, for instance, is a naturally occurring element, which is released into the environment from both natural resources and industrial pollution. In water, inorganic mercury is converted by bacteria into methylmercury, which shows an increased potential to accumulate in fatty tissues in fish compared to the inorganic element. The presence of chemical contaminants in fish and shellfish is highly influenced by the geographic locations of the fishing grounds. Fish from coastal areas with a high input of waste water or effluents from industries are often more contaminated than fish from the open oceans. However, contamination also depends on the fish species. Species at a higher trophic level (e.g., swordfish, tuna) may accumulate larger amounts of contaminants (e.g., methylmercury) through eating other fish compared to lower trophic organisms. Furthermore, older and larger fish often show higher levels of persistent chemicals which bioaccumulate over time (FAO 2014b).

10.3.1.2 Regulatory Definition of Safety of Environmental Contaminants The consumption of fish and shellfish has several health benefits. However, bioaccumulation of environmental contaminants may lead to high levels of toxic substances such as heavy metals and dioxins in aquatic food (Costa 2007; FAO 2011; Verbeke et al. 2005). The complete absence of a chemical hazard (zero tolerance) is often desired by the public seeking maximum safety of the products consumed. However, there can be no zero risk (total absence) for most contaminants especially of natural contaminants such as mercury in fish and seafood. Different food safety measures are thus defined for environmental contaminants to ensure an adequate level of protection of consumers. Tolerable levels of hazardous substances in foods have been defined for a wide range of substances following a scientific risk analysis. Provisional tolerable weekly intake values have been established by the food safety authorities (e.g., EFSA, United States Enviromental Protection Agency, FAO/WHO) describing Achieving Adequate Protection and Suitable Food Safety Indicators 225 the amount per unit body weight of potentially harmful substances or contaminants in food that can be ingested over a lifetime without risk of adverse health effects. Additional intake guidelines (e.g., for pregnant, young, or older people) allow consumers to enjoy the benefits of fish or shellfish within safe limits of intake (e.g., FAO 2001; JECFA 2004, 2011).

10.3.1.3 Measures to Ensure an Adequate Level of Protection from Environmental Contaminants An efficient way to ensure an adequate level of protection is to obtain raw materials from areas with low or insignificant environmental contamination. The monitoring of environmental pollution and food safety control for specific harvesting zones on a sufficiently frequent basis is required to provide confidence that chemical hazards are constantly below acceptable limits. Governmental bans of products from specific capture zones are an effective measure to avoid trading of fishery products which are likely to be contaminated (FAO 2004, 2014b). Environmental contamination in products from aquaculture production is highly dependent on the method of production, management practices, and environmental conditions (e.g., water source). While in intensive aquaculture, environmental contaminants are mainly bioaccumulated from the water or the feed, in extensive aquaculture systems, pollutants may be transferred from water and natural food resources. Consequently, information on the aquaculture production system may provide an indication of possible contamination of aquaculture products, which should be regularly confirmed as part of monitoring activities (FAO 2004, 2014b). The development of faster detection methods of environmental contaminants are under development and will finally need to be validated against the analytical reference methods. An overview and discussion of analytical methods for contaminants in aquaculture products is provided by Justino et al. (2016).

10.3.2 F eed Contaminants 10.3.2.1 Origin, Pathways, and Exposure of Feed Contaminants Feed contamination can result in hazards in food of animal origin, posing a threat to human health (Amlund et al. 2012; CAC 2009). Potential hazards associated with feed are caused by mycotoxins, infectious agents, veterinary drugs, as well as agricultural and other chemicals (FAO 1998). Mycotoxins are produced by naturally occurring fungi commonly known as molds. One of the most predominant examples is the contamination with aflatoxins, which are produced by molds such as Aspergillus flavus. Aflatoxins are of major concern because of their carcinogenicity. Plant ingredients have a higher potential than animal ingredients for contamination with aflatoxins. However, fishmeal and complete feeds can also be contaminated (Royes and Yanong 2002). Bacterial contamination of feed ingredients or diets with potential pathogens can be caused by both plant- and animal-derived feed resources and is relevant across the whole aquaculture sector (FAO 2009). Salmonellae, for instance, are widely distributed in nature. Contaminated feed has shown to be a major vector for transmission of salmonella to aquaculture sites and processing plants (FAO 1998, 2009). 226 Trends in Fish Processing Technologies

Fat-soluble environmental pollutants such as dioxins and dioxin-like PCBs are ubiquitous in the marine ecosystem and are readily biomagnified along the natural food chain. Fish oils, extracted from marine pelagic fish species and used in high- energy fish feeds, are considered to be the main source of lipophilic organochlorines in farmed fish such as salmon (Berntssen 2005; Jacobs et al. 2002; WHO 1999). However, a large variation exists in the background levels of persistent organic pollutions (POPs) found in fish oil depending on the geographic origin of capture, harvest season, and fish species (Amlund et al. 2012). The most common dietary contaminants have been the organochlorine pesticides dichloro-diphenyl-trichloroethane, dichloro-diphenyl-dichloroethylene, dieldrin, endrin, and industrial chemicals such as PCB, phthalate esters, and hexachlorobenzene. Most, but not all, residues are in the finished diets due to the presence of the contaminated marine ingredients fish oils and/or meal (FAO 1980; Justino et al. 2016). During the last years, marine ingredients have been partially or fully replaced by plant-derived feed ingredients. Most plant ingredients have lower levels of POPs than marine ingredients, and the use of plant ingredients may thus lower the load of POPs in aquafeeds (Amlund et al. 2012). In contrast, plant-based feed ingredients may be contaminated with agricultural chemicals. With an increasing amount of plant material in aquaculture diets, there is an increasing risk for pesticide residues in aquaculture diets and their transfer into aquaculture products. Pesticides may accumulate in fish and potentially lead to significant residue levels in edible fish commodities. Heavy metals originating from natural or anthropogenic sources are pollutants of major concern in the aquatic environment. Fishmeal may contain high levels of heavy metals such as cadmium, lead, and mercury (Amlund et al. 2012) and thus lead to increased levels of heavy metals in feeds. However, contamination of aquaculture diets with heavy metals may also occur by using mineral mixtures of essential elements as feed supplements which are contaminated with such elements. Veterinary drugs are intentionally included into feeds for treating infectious diseases. In some cases, during production of medicated fish feed, cross-contamination might occur to feeds, leading to contamination of nonmedicated diets (Amlund et al. 2012; FAO 1980). Examples of hazards in fish products that can be linked to feed have created concern at national and international levels and require a strong regulatory framework to ensure feed safety.

10.3.2.2 Regulatory Definition of Safety of Feed Contaminants Safe feed is an essential element to reduce and prevent undesired substances entering the food chain (FAO 2010). Regulations addressing contamination of animal feeds are most comprehensive in Europe and North America. Codex Alimentarius provides a general standard for contaminants and toxins in feeds (CAC 2009). In addition to that, the European legislation provides further regulations ensuring that feedstuffs do not cause any danger to human or animal health. Maximum levels in any feed (e.g., maximum levels for POP in feed fish oil and fishmeal) are defined under directive 2002/32/EC (EC 2002a) for undesirable substances in animal feed. Requirements for feed hygiene are laid down in Regulation (EC) No. 183/2005 (EC 2005a). For licensed drugs and pesticides, MRLs are prescribed to ensure food Achieving Adequate Protection and Suitable Food Safety Indicators 227 safety (e.g., EC regulation 37/2010, EC 2010; CAC 2009). Residues of pesticides in food and feed are covered by Regulation (EC) No. 396/2005 (EC 2005b) providing MRLs of pesticides in or on food and feed of animal origin. The European pesticide regulation (Regulation [EC] No. 1107/2009; EC 2009) requires fish metabolism and fish feeding studies (EC 2013; Schlechtriem et al. 2016a), where residues in fish feed exceed 0.1 mg kg−1 of the total diet (dry weight basis) to enable the setting of appropriate MRLs in fish commodities (Schlechtriem et al. 2016b).

10.3.2.3 Measures to Ensure an Adequate Level of Protection from Feed Contaminants Contaminant levels in feed shall be as low as reasonably achievable through best practice such as Good Agricultural Practice and Good Manufacturing Practice following an appropriate risk assessment. Feeds used in aquaculture should comply with the code of practice on good animal feeding (CAC 2004; FAO 1998). Where foodborne hazards may originate in feed, the feed contamination should be adequately controlled through quality assurance programs including analytical measurements. Measures to prevent and reduce contamination in feeds are available. On one hand, POP contamination in farmed fish can be reduced by replacing fishmeal and fish oil in the feed by plant-derived material or by using marine proteins and oils that are known to be low in POPs (Hardy and Lee 2010). On the other hand, technologies for decontamination of marine feed ingredients exist and might be applied if economically feasible (Oterhals 2011). Also, to alter the levels of arsenic and heavy metals in aquafeeds, the use of plant-derived feed ingredients is a possibility (Amlund et al. 2012). However, new contaminants (e.g., pesticides) may be introduced by these measures and need to be adequately addressed. More knowledge is required about how much and which pesticides are found in the feed ingredients, how much is transferred to the edible part of the fish, and what effect this has on product quality and safety as well as potential dose effects and exposure to the consumer. Veterinary drugs or medicated feeds should be used according to manufacturer instructions, with particular attention to withdrawal periods to eliminate the risk of chemotherapeutic contamination of farmed fish products (Hardy and Lee 2010). Veterinary drugs for use in aquaculture should comply with national and international guidelines such as code of practice for control of the use of veterinary drugs (CAC 1993b) and the regulatory program for control of veterinary drug residues in food (CAC 1993a, 2003). Cross-contamination of nonmedicated feed with veterinary drugs during feed production must be avoided. The safest procedures would involve having a separate production line for medicated feed (FAO 1980). To avoid spoilage and fungal and bacterial contamination of feeds, transportation and storage of feeds under appropriate good practices should be adhered to. Aquafeed ingredients should be regularly tested for mycotoxins such as aflatoxins. The chemical analysis of aflatoxins, e.g., as part of the governmental monitoring of feed ingredients, includes extensive extraction processes, followed by thin-layer chromatography or high-pressure liquid chromatography (Royes and Yanong 2002). However, the industry is seeking faster and cheaper methods for early detection of mycotoxin contamination along the feed production chain. Simple on-farm 228 Trends in Fish Processing Technologies inspection should be done regularly to identify changes in feed quality as early as possible. The use of black light (UV-A), which causes a bright greenish/yellow fluorescence if A. flavus is present, can help to gain early indications of feed contamination. Alternatively, commercial test kits are available for detecting concentrations of aflatoxins in feeds (Royes and Yanong 2002). If feed is contaminated with aflatoxins, toxin in-activator or mycotoxin adsorption agents may help to control mold development and accumulation of toxins (Amlund et al. 2012; FAO 2008; Royes and Yanong 2002). However, an absolute assessment of the level of contamination can be obtained only by using the accredited analytical methods. Generally, the traceability of all feed ingredients ensures the identification of origin, use, and time of production/application at any stage of the feed and food chain. This allows the identification of the origin of the contamination and eliminates the related risk as part of the industry-internal quality/safety procedures as well as the regulatory approaches. If the appropriate actions to prevent or reduce contamination of feed are applied, the risks to human health associated with feed contamination are relatively low in comparison to other food safety hazards along the food production/processing chain (FAO 1998).

10.3.3 algae Toxins within the Natural Food Chain 10.3.3.1 Origin, Pathways, and Exposure of Algae Toxins There are several natural algae toxins that may accumulate in aquatic organisms and pose a threat for humans. Especially lipophilic toxins from marine algae pose a hazard in food products from fisheries and aquaculture. These toxins are produced either by algae directly or the algae produce precursors of toxins which are metabolized along the food chain to toxic compounds. Certain environmental conditions (e.g., temperature, pH, salinity, availability of nitrogen and phosphorus) trigger the synthesis of toxins in algae, and consequently, the content of toxins in the algae cells may differ. Temporal blooms of potentially toxic algae may occur regionally in response to environmental conditions (reviewed by Tirado et al. 2010). Filter feeding organisms, like mussels and oysters, are especially at risk to be contaminated with marine algae toxins as they directly feed on algae or particulate matter and may accumulate especially lipophilic toxins (García-Altares et al. 2016). Fish and other aquatic organisms may either be contaminated by feeding on poison- ous algae directly or the toxins may biomagnify along the natural food web. There are several groups of marine algae toxins that pose potential food hazards for humans and may cause different poisoning effects. About 80 algae species, mainly diatoms and dinoflagellates, are known to potentially produce toxins, which may cause paralytic, diarrhetic, neurotoxic, or azaspiracid shellfish poisoning as well as the ciguatera fish poisoning in humans (Tirado et al. 2010; Vlamis and Katikou 2014).

10.3.3.2 Regulatory Definition of Safety of Algae Toxins Due to the threat to human health from algae toxins, many countries (including EU member states) ratified regulations on the maximum permitted levels (MPLs) of algae toxins in food products. The European Commission decided on MPLs of Achieving Adequate Protection and Suitable Food Safety Indicators 229 marine biotoxins in whole body or edible parts of fresh bivalves of 800 μg per kilogram for PSP, 20 mg of domoic acid per kilogram for paralytic shellfish poison, 20 mg of domoic; 160 μg of okadaic acid equivalents per kilogram for okadaic acid, dinophysis toxins, and pectenotoxins together; 1 mg of yessotoxin equivalents per kilogram; and 160 μg of azaspiracid equivalents per kilogram (EC 2004b). Safety management of ciguatera toxins is currently discussed while the potential analytical methods (cytotoxicity assays, receptor binding assays, enzyme linked immunosorbant assays [ELISA] and liquid chromatography coupled to mass spectrometry [LC–MS]) are not yet validated due to limited availability of reference material or limited sensitivity (reviewed by Friedemann 2016).

10.3.3.3 Food Safety Indicators and Measures to Ensure an Adequate Level of Protection from Algae Toxins The contamination of seafood by marine toxins does not change the sensory and visual character of the product and makes it difficult for producers and consumers to determine whether seafood products are contaminated or not. Furthermore, most of the algal toxins are heat stable and are consequently not inactivated by heat treatments (Vidaček 2014). A set of regulatory and industry-based safety measures are installed to reduce the risk of serious seafood poisoning by combining intensive monitoring of phytoplankton species composition in the harvesting areas with the use of chemical analyses as well as bioassays of the seafood products along the production chain. The use of monitoring programs of phytoplankton in combination with environmental data sets (temperature, nutrient availability, pH, salinity, intraspecific/ interspecific interactions) helps to understand the temporal and regional occurrence of toxic algae blooms (Ferrante et al. 2013). Increased abundance of toxic algae necessitates more intensive monitoring and may lead to a temporary ban of harvests at the affected production sites. Based on coastal management plans and former results of the implemented monitoring systems, coastal areas and regions of increased risks might be identified to improve future monitoring systems. Validated LC–MS techniques are commonly used as methods for the detection of lipophilic biotoxins in the flesh of seafood (EC 2011). As these techniques are expensive and require a certain infrastructure and skilled staff, the industry searches for faster, cheaper, and easier applicable methods for the detection of biotoxins in environmental samples and in the final seafood product. Currently, for single toxins, there are ELISA assays, protein phosphatase inhibition assays, and lateral flow immuno assays available that are debated for future supplemental use (Johnson et al. 2016). Solid phase adsorbing toxin tracking devices have been suggested as an early-warning tool to detect lipophilic toxins in production areas for bivalves (MacKenzie et al. 2004). But the use of these devices without coupling with validated analytical techniques may lead to an overestimation of toxin levels in shellfish as critically discussed by Pizarro et al. (2013). Therefore, a validated LC–MS method is the method of choice for the detection of lipophilic biotoxins for official controls at any stage of the food chain and should be used as a reference method during internal checks by food business operators. 230 Trends in Fish Processing Technologies

10.3.4 Microbiological Contamination 10.3.4.1 Origin, Pathways, and Exposure of Microbiological Contamination Microbiological contamination is one of the main hazards in foods from fish and other aquatic organisms for human consumption. These contaminations might occur during the production and growing phase of the organisms or during the processing, transportation, and storage. Several foodborne illnesses from consuming seafood are caused by bacterial, fungal, or viral contamination along the production chain. These illnesses might be caused either by the infection with the pathogens themselves or by toxins formed by bacteria or fungi before consumption or in the intestinal tract. Seafood products can be classified into risk categories according to their stage of preprocessing (Huss et al. 2000b). For the consumer, filter feeding organisms (e.g., bivalves) that are consumed raw pose an especially high risk due to potential microbiological contamination during their production. Filter feeding organisms feed on small plankton and particles but might also extract bacteria and viruses from the water and accumulate them in their filtering and digestion system. These seafood species are normally cultured in coastal, nutrient-rich waters with a certain plankton abundance that significantly supports their growth. These areas, like estuaries, are more likely to be contaminated with sewage and polluted run-off water from land. Of highest concern are enteric bacterial pathogens (e.g., Escherichia coli, Salmonella spp.), viral pathogens (norovirus, viruses causing hepatitis), or naturally occurring bacterial pathogens (Vibrio spp.). Cooking of fresh or frozen crustaceans and fish prior to consumption can lower the risk associated to microbial contamination (Huss et al. 2000b). The lowest risk is associated with the consumption of lightly preserved fish products (salted, marinated, fermented, cold-smoked, and graved fish), semipreserved fish (like caviar), mildly heat-processed (pasteurized, hot-smoked), and heat-processed (sterilized, sealed packaging) (Huss et al. 2000b). Further, seafood-borne pathogenic bacteria can be divided into three groups according to their occurrence and origin: (1) bacteria ubiquitously present in the environment, (2) bacteria that are naturally present in aquatic habitats, and (3) bacteria with a reservoir in animals or humans but being present in environments due to unsuitable sanitary/hygiene conditions (Huss et al. 2004). Bacteria occurring in the (aquatic) environment, which represent highest risk for consumers, are Vibrio spp., Clostridium botulinum, Clostridium perfringens, Listeria monocytogenes, Plesiomonas shigelloides, Aeromonas spp., and Bacillus spp. (e.g., reviewed by Gauthier 2015). The genus Vibrio, which consists of anaerobic bacteria of which several species are foodborne pathogens, is frequently isolated from fish, bivalves, crustacean, and cephalopods used as food for humans. There are three species of major concern (Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae) that may negatively affect human health after consumption of raw or undercooked contaminated seafood (e.g., reviewed by Martinez-Urtaza et al. 2010). V. parahaemolyticus infection can cause symptoms like diarrhea, headache, vomiting, nausea, abdominal cramps, and low fever (Su and Liu 2007). V. vulnificus poses a lethal hazard for persons with underlying predisposing illness like liver disease, diabetes mellitus, Achieving Adequate Protection and Suitable Food Safety Indicators 231 and immunodeficiency. V. cholerae (strains O1 and O139) may be present in aquatic systems with lower sanitary standards and water regulations and can cause strong diarrhea, vomiting, and intense dehydration with even lethal outcome without immediate and adequate treatment. C. botulinum is an anaerobic bacterium that can be found ubiquitously in aquatic and terrestrial environments and forms heat-resistant endospores. Under anaerobic, low-acid conditions, C. botulinum can produce different forms of the heat-labile botulinum neurotoxin (Vidaček 2014). If only mildly heat treated, pasteurized or understerilized, seafood may still contain spores which under certain conditions may lead to botulinum toxin contamination. L. monocytogenes is a ubiquitous bacterium causing both invasive and non-invasive infections (Huss et al. 2000a). It resists freezing, drying, and heat and can cause symptoms like septicemia, meningitis, encephalitis, or intrauterine and cervical infections in pregnant women. Especially immunosuppressed humans are at risk. As well as naturally occurring pathogens, bacteria with a reservoir in animals or humans can pose a risk for consumers. For the safety of aquatic products, the pathogens of concern are Salmonella spp., Shigella spp., E. coli, Campylobacter jejuni, and Staphylococcus aureus. Salmonella spp. are facultative anaerobic, nonsporulating bacteria that are globally one of the main pathogens of foodborne diseases. Contamination of seafood occurs within the production (fishery or aquaculture) or during processing and trading (e.g., reviewed by Amagliani et al. 2012). Symptoms of an infection include nausea, vomiting, cramping abdominal pain, fever, headache, and diarrhea. E. coli are coliform bacteria and are widely used as an indicator for fecal contamination (Newell et al. 2010). The pathogenic strains are most often associated with foodborne infections in human and cause intestinal infection, urinary infections, meningitis, and other extraintestinal infections. S. aureus are non-spore-forming, facultative anaerobic bacteria that can produce harmful and heat-resistant enterotoxins. Contamination of aquatic products most often occurs during processing and can be prevented most effectively by hygiene programs along the processing chain (Vidaček 2014). Antibiotic compounds might be used as therapeutic or prophylactic agents in the production of seafood products. An emerging risk is the development of resistance genes that code the resistance against antibiotic compounds (Newell et al. 2010). These resistance genes can be present on mobile genetic elements and can be transferred to other pathogenic bacteria (Liebert et al. 1999). This might create pathogenic bacteria being resistant toward therapeutic treatments in humans and leading to an increased risk with respect to foodborne hazards caused by bacterial contamination. Contamination with viruses can also present a serious hazard to consumers. The virus groups noroviruses, hepatitis A, and rotaviruses are of highest concern regarding the safety of seafood in general and especially of filter feeding mollusks that are consumed raw. Not only the presence of pathogenic viruses, microbes, or their toxins are a hazard to consumers, but also metabolites (e.g., biogenic amines like histamine) formed by decomposition processes in fish products. Histamine is a metabolite of the amino acid histidine, which is present in high amounts in fish species of the 232 Trends in Fish Processing Technologies families Scombridae, Clupeidae, Engraulidae, Coryfenidae, Pomatomidae, and Scombresosidae (Sumner et al. 2004). In food products from these fish species, maximum histamine levels are set by the EC (2007) and special requirements for processing of such products (effective cold chain management) are defined.

10.3.4.2 Regulatory Definition of Safety of Microbiological Contamination Microbiological standards for food in the EU are established by Regulation (EC) No. 2073/2005 (EC 2005c) and its amendment Regulation (EC) No. 1441/2007 (EC 2007) by setting limits for pathogens and defining suitable reference methods. In these regulations, microbiological criteria are set for the safety of foodstuff, including aquatic products. Sampling schemes and specific limits of contamination are set for E. coli in shelled and shucked products of cooked crustaceans and molluscan shellfish; for coagulase-positive staphylococci in shelled and shucked products of cooked crustaceans and molluscan shellfish; for Salmonella in cooked crustacean and molluscan shellfish, as well as in live bivalve mollusks, echinoderms, tunicates, and gastropods; and for L. monocytogenes in foods depending on their ability to support growth. In addition, on a national level, recommendations from different organizations and industry associations exist for specific pathogens. Foods that do not meet these standards may lead to sorting, reprocessing, rejection, or destruction of the products. Regulation (EC) No. 2073/2005 (EC 2005c) and its amendment Regulation (EC) No. 1441/2007 (EC 2007) set limits for histamine in fishery products of fish families (Scombridae, Clupeidae, Engraulidae, Coryfenidae, Pomatomidae, and Scombresosidae) that are particularly rich in histidine.

10.3.4.3 Food Safety Indicators and Measures to Ensure an Adequate Level of Protection from Microbiological Contamination The best way for ensuring the microbiological quality and safety of seafood is to prevent the contamination or reduce the bacterial growth. This can be achieved by establishing suitable environmental conditions during production in combination with hygienic standards further along the processing chain, including harvest/ capture, processing, storage, and trading as well as food preparation for human consumption. This may include methods that act against the bacterial growth like chilling, freezing, heating, reduction of oxygen, washing with potable water, drying, salting, and changes in pH or by adding suitable preservatives. Staff must be trained in handling the products and cross-contaminations must be avoided along the production chain. Such may occur at any point postharvest but are often associated with contamination at the production plant or during food preparation. Similarly, as histamine cannot be eliminated if present, minimizing histamine levels in seafood products must be achieved by destroying bacteria by heat treatment after harvest or by effective cold chain management from farm to fork. For fishing grounds and harvesting areas for bivalve mollusks, the most effective measure is the implementation of adequate regulations for open waters, waste waters, and industrial effluents, as well as hygiene and sanitary measures for agriculture, animal production, and other potential sources for contaminating surface waters. European coastal areas and regions for the production of bivalves are classified according to the presence of fecal contamination using E. coli as indicator Achieving Adequate Protection and Suitable Food Safety Indicators 233

(EC 2004c). This classification according to the potential fecal contamination is not necessarily a suitable indicator for other pathogens. If bivalves are moderately contaminated, an effective method of reducing the number of bacterial pathogens is to keep them for a suitable time in clean water under controlled conditions, which is called depuration. For aquaculture, the quality of water, feeds, and stocking organisms as well as the hygienic status of the production area and facility with its filtration, nitrification, and disinfection units is essential. This is of importance in extensive aquacultures as well as in intensive flow-through systems and recirculation systems. After capture or harvest (fishery or aquaculture), the organisms must quickly be processed (eviscerating, heading, gutting, etc.) under hygienic conditions using potable water for washing and immediately be cooled/freezed. Heat treatments of products must be applied at effective time–temperature regimes. To protect the products from spoilage along the production chain, suitable cold chain management and packaging are essential. Similarly important is the ability to trace the products along the production chain, including all steps from the producers of raw materials, feeds, processors, suppliers of ingredients and package materials, and retailers (McKean 2001). For trading in the EU, food business operators must fulfill these requirements following Regulation (EC) No. 853/2004 (EC 2004b). Additionally to the industry-based hygiene standards and quality and safety systems for foods and feeds (e.g., HACCP), governmental inspections are carried out by accredited laboratories for the most important foodborne bacteria, their toxins, and metabolites, including Enterobacter spp., L. monocytogenes, Salmonella spp., C. botulinum, and staphylococcal enterotoxins by using validated reference methods (EC 2007). However, such detection methods are time consuming, require a level of skilled staff, and are consequently expensive. Furthermore, not all methods applied are sensitive enough to detect small numbers of pathogenic bacteria in the presence of large numbers of nonpathogenic bacteria or to detect the ability to synthesize toxins. Alternative methods may be used by food business operators if coupled with validated reference methods. Sensitive and pathogen-specific methods as alternatives to the conventional methods range from membrane filtration, automated electrical techniques, immunological assays to polymerase chain reaction (PCR), and real- time PCR methods (Amagliani et al. 2012; Kumar et al. 2008; Martinez et al. 2005). However, these alternative methods also have their disadvantages. The PCR methods for instance might be sensitive for the detection of a toxin forming gene, but a positive detection does not necessarily mean that the toxin is present in the product. Therefore, suitable combinations of alternative and conventional methods must be used to ensure product safety. Currently, antibiotic resistance is one of the major concerns in various sectors ranging from agriculture, food production, animal husbandry, and medical treatments. The prudent use of antimicrobial agents in all sectors is a key aspect of the prevention and control of antimicrobial resistance (Newell et al. 2010). The control of temperature and hygienic conditions along the entire product chain from harvest to the consumer is of highest importance to minimize the microbiological contamination and potential product decomposition. 234 Trends in Fish Processing Technologies

10.3.5 Parasites 10.3.5.1 Origin, Pathways, Contamination, and Hazards of Parasites Parasites especially in marine fish and bivalves can be hazardous for the consumers. Parasites from infested seafood might migrate to human organs or they can release substances into seafood that may induce allergy-like reactions in susceptible consumers after eating contaminated seafood (Prester 2016). Most of the human infections are associated with the consumption of raw, undercooked, or inadequately preserved seafood. Especially in Asia, infestations with zoonotic parasites are frequent due to specific aquatic food consumption habits and lower sanitary standards for open waters (Lima dos Santos and Howgate 2011; Phan et al. 2010). Nearly all natural marine organisms are potentially infected by parasitic nematodes, trematodes, cestodes, or protozoa, of which a few species are harmful to humans (Butt et al. 2004). In freshwater, trematodes are using a wide range of fish and some crustaceans as second intermediate host (Lima dos Santos and Howgate 2011). In humans, the infection with trematodes may cause fever, diarrhea, anemia, colic, cachexia, and undernourishment. Cestodes use a wide range of wild and farmed freshwater and marine fish as intermediate hosts (Lima dos Santos and Howgate 2011) and are present especially in cold water habitats (Vidaček 2014). Cestodes can infect consumers after consumption of undercooked or raw fish and cause cysts. Of special concern for food safety is the parasitic nematode Anisakis spp., which is responsible for the most common parasitic infection caused by seafood consumption (Butt et al. 2004). Anisakis spp. can infest crustaceans, fish, and squids while the level of infestation depends on age, size, and lipid content of the host species as well as the region of harvest (Konishi and Sakurai 2002; Strømnes and Andersen 2003). Infection of humans may cause diarrhea, abdominal pain, and enteritis. In sensitized consumers, ingestion of seafood infested with Anisakis larvae, regardless of the pretreatment (Tejada et al. 2014), can cause a disease known as anisakiasis, with symptoms similar to true seafood allergy (Prester 2016), causing acute abdominal pain, epigastric distress, linked with nausea, diarrhea, and vomiting. In the RASFF from the European Commission (EC 2016), particularly anchovies, hake, and mackerel are shown to be susceptible to infestation. Commonly, Anisakis spp. infests the viscera and musculature of fish (Mattiucci and Nascetti 2008), but migration into the flesh may also occur postmortem (Cipriani et al. 2016; Šimat et al. 2015; Smith 1984). In some fish species, e.g., Alaska salmon, postmortem migration was not recorded (Karl et al. 2011). However, also the allergenic substances released by Anisaki larvae were reported to migrate within the fish and pose a threat to consumers (Tejada et al. 2014).

10.3.5.2 Regulatory Definition of Safety in Relation to Parasites Parasites in fish should be inactivated or removed during processing to avoid health hazards. Products with more than 20 nematode larvae per kilogram are not suitable for human consumption, and the nematode larvae in the product should not be alive. In the EU, fishery products to be consumed raw or almost raw must be frozen at temperatures below −20°C in all parts of the product for at least 24 hours Achieving Adequate Protection and Suitable Food Safety Indicators 235 to inactivate the parasites. Cold-smoked fishery products from herring, mackerel, sprat, and (wild) Atlantic and Pacific salmon or marinated and/or salted fishery products must be frozen for at least 24 hours (EC 2004b). Heat treatment at temperatures above 60°C is sufficient to destroy parasites like nematode larvae in fish products. Documentation of such pretreatments must accompany the product along the production chain. If epidemiological data for specific harvest areas indicate that no health hazard from parasites exists, the responsible authority may waive the requirement for freeze treatment of the products (EC 2004b).

10.3.5.3 Food Safety Indicators and Measures to Ensure an Adequate Level of Protection against Parasites The level of infestation of parasites in captured organisms can only be decreased by choosing fishing grounds with less infested fish populations and by harvesting certain species that are known to be less infested (Vidaček 2014). Infestation of parasites in organisms from aquaculture might also occur in systems that are linked to natural waters (e.g., pond aquaculture, net cages). Direct processing (e.g., chilling, degutting) of the fish directly after capture might reduce the migration of parasites and the release of allergenic substances into the flesh (Smith 1984; Tejada et al. 2014). Inspections on a light table (candling) can be used to visualize and remove parasites by cutting out heavily infested parts. If parasites cannot be removed, freeze or heat treatments are suitable methods to destroy/inactivate the parasites in the flesh (Tejada et al. 2014).

10.4 CONCLUSION A broad range of hazards, of different types and origins, has been identified in aquatic foods that may have an immediate, interim, or long-term effect on human health. Appropriate measures of control to prevent, minimize, or reduce the risks are required to ensure food safety. Food safety indicators allow the identification of potential hazards at different stages of the production chain and are essential for targeted and efficient risk management. The development of faster, more reliable, flexible, and cost-effective indicators is required to further improve the implementation and enforcement of food safety standards and to secure an adequate level of protection. But the use of such indicators needs to be validated and suitably combined with the accredited analytical methods for the respective parameters.

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Anca Ioana Nicolau and Olafur Oddgeirsson

CONTENTS 11.1 Food Safety Management Systems—Definition, Structure, and Challenges for Fish Processing Operators...... 241 11.2 Food Defense and Food Fraud Prevention Systems...... 245 11.3 Trends in HACCP and FSMS...... 246 11.4 Conclusions...... 252 References...... 252 Internet Sites...... 254

11.1 FOOD SAFETY MANAGEMENT SYSTEMS— DEFINITION, STRUCTURE, AND CHALLENGES FOR FISH PROCESSING OPERATORS The way food safety is managed by the food industry in recent years has changed considerably at the world level. Changes are fuelled by consumer expectations regarding what they eat, increasing regulatory requirements, and efforts to fulfill the safety objectives stated by food companies. The result is that anyone who is running a food business is legally required to have an approved written food safety management system (FSMS) in place to ensure food is safe for consumption. In fish processing units, food safety is managed in a similar way as in any other branch of the food industry, so similar challenges have to be faced. An FSMS is defined by the International Organization for Standardization (ISO)/ TS 22003:2013 as a set of interrelated or interacting elements to establish policy and objectives (ISO, 2013). The elements are used to direct and control an organization with regard to food safety, complementing the food safety system foreseen by the European Commission, created as a holistic system of prevention, prepared- ness, and own-check activities to manage food safety and hygiene in a food business (European Commission—DG Health and Food Safety, 2016). The key elements of any FSMS are Prerequisite Programs (PRPs), Hazard Analysis Criterial Control Point (HACCP)-based procedures, and basic management elements, as well as top management elements and communication. The elements of an FSMS are illustrated in Figure 11.1. Similar to other food industries, the preliminary and operational programs in fish processing have different names around the world, in some countries being referred to as prerequisite programs, while in others being known as legal parameters,

241 242 Trends in Fish Processing Technologies call P, ures MOM, EH ed oc raceability and re n pr oli cy C p RP ny HA Communicatio Compa sT training GHP and GMP P, P: PR OPR call P, P CC ures ed MOM, EH oc raceability and re y pr and VA olic CP p AC ny Communication Compa PT sT CC HA training P, GHP and GMP Elements a food safety of Food management business (FSMS). system operators can choose to implement coupled HACCP with TACCP P:

OPR PR FIGURE 11.1 and VACCP or HARPC,and Good according Good Practices; VACCP Manufacturing Hygiene with. to GMP, the to comply GHP, legislation they have Practices; Vulnerability Threats Hazard Analysis Critical Analysis Control AnalysisHACCP, Critical Critical Control TACCP, Control Points; VACCP, Points; HARPC,Points; Hazard Analysis manufacturing Controls; MOM, and Preventive Risk-Based operations enterprise management; ERP, resource planning. Food Safety Management in Fish Processing Units 243 autocontrols, and good practices, which include Good Hygiene Practices and Good Manufacturing Practices, or Sanitation Standard Operating Procedures (Oddgeirsson, 2012). No matter the name, they include construction and layout of buildings and work- space, installation and equipment, maintenance, utilities (water, air, ice, energy), waste management, measures to prevent cross-contamination, pest management, cleaning and sanitizing, personnel hygiene and facilities, temperature control, management of purchased materials, trainings, warehousing, final product information and consumer awareness. ISO 22000/2005 introduced allergen control as a required PRP too. HACCP-based procedures include Hazard Analysis Critical Control Points (HACCP), a global system, the evolution of which proved to be effective and efficient in management of food safety, or Hazard Analysis and Risk-Based Preventive Controls (HARPC), an updated US standard incorporated into the Food Safety and Modernization Act (FSMA) (Maloy, 2015). As HRPC includes planning for potential terrorist acts and/or intentional adulteration and food frauds, the companies that implemented an HACCP system should introduce a complementary Threats Analysis Critical Control Points (TACCP) and Vulnerability Analysis Critical Control Points (VACCP) systems to counterbalance the defense and fraud part incorporated by HARPC. The HACCP system, which celebrates this year 58 years since its presentation to the public, evolved over the years and proved to be the most effective system to ensure food safety (Surak, 2009). As food safety is still evolving, the same happens with HACCP. We can talk today about traditional HACCP, which focuses on preventing or completely eliminating a hazard, and risk-based HACCP, which implies that a certain level of risk, not zero, is acceptable (Overbosch, 2013), taking into consideration whether the exposure to a hazard has a meaningful impact on public health (Buchanan, 2011). The HARPC system has a similar concept (is risk based) and goal (control of significant hazards) as HACCP but works with a qualified individual or a team of qualified individuals, analyzes radiological hazards and intentional hazards too, and has a different approach regarding the way hazards are evaluated. Under HACCP, hazard analysis is firstly conducted, then is followed by a qualitative risk assessment of the hazards to determine their significance. This means that the risk is determined taking into account the preventive measures already existing at the examined step, so the result is fewer hazards and a lower risk. Under HARPC, the risk assessment is done assuming that there are no preventive measures in place, so it ends up with many more significant hazards requiring a preventive control (Howlett, 2016). In other words, in an HARPC system, a Food Safety Plan is set, which includes a Preventive Control Plan, where basic controls are promoted to the same status as Critical Control Points (CCPs), which means HARPC requires more documentation for non-CCPs than HACCP does actually, the same amount of work is dedicated for Preventive Controls as for CCPs in terms of monitoring, verification, setting corrective actions and preventive actions (CAPAs), and reviewing. The basic management elements as manufacturing operations management (MOM) and the instant traceability and recall management and enterprise resource planning (ERP) are some of the important management elements of an FSMS worth mentioning. MOM refers to control of plant floor activities, including production 244 Trends in Fish Processing Technologies tracking and scheduling, scaling and rejection tracking, overall equipment effectiveness reporting, and analyses in order to avoid costly breakdowns. It is moreover used to develop a knowledge base of technical experience, respond quickly to maintenance problems and maximize productivity, and use supervisory control and data acquisi- tion to assist in managing inventory by enabling processors to easily track consumed ingredients and intermediate inventory in real time. To processors, MOM is a key to achieve overall efficiency. Traceability is an important element in an FSMS as companies must track every ingredient from receipt to finished product delivery, and at least one-up and one-back in the supply chain. This capability is often referred to as e-pedigree or product genealogy usage (Farooq et al., 2016). The goal of the Recall Management program is to protect consumers from the possible event of a product safety failure by removing all suspect products from the distribution channels in the least amount of time, once a product withdrawal or recall is initiated. ERP systems have traditionally included only finance and accounting, but today’s ERP solutions also include advanced costing, customer resource management, human resources, maintenance, and purchasing. In order to develop a successful system, top management must be committed to establish, implement, and maintain the FSMS and has to start the process by preparing an organizational strategy and generating food safety policies and food safety objectives. Adequate resources have to be provided to implement, maintain, and improve the FSMS. As part of this process, a food safety team leader and a multidisciplinary food safety team should be appointed by top management to develop the FSMS. Communication, either internal or external, is an essential element in developing, implementing, and maintaining a functioning FSMS (Stier, 2014). In fish processing units, where exports represent an important part of the activities developed by companies, external communication necessitates special attention due to challenges emerging from different cultural confrontations. Implementation of a safety management system has an associated cost, and in fish processing industry, as in any other food business, a successful such system is considered the one able to meet customer requirements and government regulations at a manageable cost. Despite having implemented FSMS, fish processing plants are still facing food safety performance challenges worldwide, which include food borne outbreaks, recalls, and the associated economic losses (Adawo, 2013). Many fish companies need improvements of their FSMS and reduction of context riskiness to assure good food safety performances, but the direction of these improvements is different in developed countries than in developing or underdeveloped countries. While fish processing companies from developed countries had the opportunity to voluntarily and gradually introduce HACCP into their food safety programs and then move to industry-driven programs, with regulatory and control agencies being in charge of certifying the food facilities and conducting on-site verification of proper HACCP implementation (Ryder, Karunasagar, and Ababouch, 2014), fish processing companies from developing and underdeveloped countries are confronted with a different situation. Very few of them, except exporting and large companies, have implemented HACCP and ISO 22000:2005, the others being Food Safety Management in Fish Processing Units 245 now in a position to try the HACCP system, implemented and in force. A study revealed that in Tanzanian fish processing units, the FSMS activity levels could be improved through automation of production processes (such as filleting, packaging, and sanitation), strict raw material control, proper follow-up of CCP analysis, developing specific sanitation procedures and company-specific sampling design and measuring plans, independent validation of preventive measures, and establishing comprehensive documentation and record-keeping systems (Kussaga et al., 2014a, 2014b). In another study, it was demonstrated that the majority (67%) of Kenyan fish- exporting companies operated at moderate- to high-risk context, but with an average performance in control and assurance activities and to improve their FSMS, it was suggested to recruit high-skilled technological staff and use scientific information sources, historical results, and own experimental trials in their preventive, intervention, and monitoring systems (Onjong, Wangoh, and Njage, 2014). However, such intervention measures for improvements could be taken in phases, starting with less expensive ones (such as sanitation procedures) that can be implemented in the short term, to more expensive interventions (setting up assurance activities) to be adopted in the long-term. These measures are essential for fish processing companies to move toward more effective FSMS.

11.2 FOOD DEFENSE AND FOOD FRAUD PREVENTION SYSTEMS According to Oceana’s report on fish and seafood, 58% of the samples identified as substitute species in the analysis performed at global level in 2014 carried a species- specific hazard, which could not be adequately screened or mitigated due to the mislabeling and put consumers at risk. These hazards included parasites, environmental chemicals, aquaculture drugs, and other natural toxins as histamine, ciguatera, tetrodotoxin, saxitoxin, and gempylotoxin (http://usa.oceana.org/sites/default /files/global_fraud_report_final_low-res.pdf). Unfortunately, mislabeling is not the single type of fraud associated to fish and seafoods, and this category of food is not the single one being fraudulent, although it is considered one of the most fraudulent foods. The safety problems generated by food frauds have resulted in authorities taking measures for preventing them. Considering HACCP a successful tool in relation to food safety, two other preventive systems were developed to cover both economically motivated adulteration (EMA) and malicious tampering (sabotage, extortion or ter- rorism) of food. The two new systems were named TACCP and VACCP, similarly to HACCP, although CCPs are not involved in any of them. Even if the names’ similarity indicates an association, it has generated confusion when food business operators (FBOs) proceeded to implement the new systems. At present, the methodology for TACCP is described by Publicly Available Specification (PAS) 96:2014 (2014), which was developed by the British Standard Institution. PAS guides food businesses managers through approaches and procedures to improve the resilience of supply chains to fraud or other forms of attack on food and drinks, but a more detailed guide on this subject is expected to be issued by the Global Food Safety Initiative (GFSI) at the end of 2016. 246 Trends in Fish Processing Technologies

Meanwhile, the specialists from Techni-K Consulting, a company from the United Kingdom, are proposing to unify the actions taken under the umbrella of TACCP and VACCP systems, to name them Threat & Vulnerability Assessment, which is more adequate, and to replace CCPs with Vulnerable Threat Points (Marsh, 2015). The methodology they established is presented in a book entitled Assessing Threat Vulnerability for Food Defence (https://techni-k.co.uk/shop/books/assessing-threat -vulnerability-for-food-defence/), which received the appreciation of Chris Elliot, the leader of the UK government’s team for independent review of food systems. In the United States, planning for preventing potential terrorist acts and/or intentional adulteration and food fraud is included in the HARPC system (Maloy, 2015). No matter which methodology is applied, it is important to perform regularly the assessment of threats and vulnerabilities as they are changing over time. In order to help professionals with these assessments, specific tools have been created. For example, in Europe, SSAFE and its partners (RIKILT–Institute of Food Research from Wageningen University and Research, Free University of Amsterdam, and Pricewaterhouse Cooper) developed a free online tool available in three formats (Excel, online, and mobile app) to guide users through a decision tree and assessment questionnaire to determine fraud opportunities, motivators, and gaps in existing controls. The tool, which will be available in 11 languages by the spring of 2017, produces a report that the users can include with their FSMS documentation (http:// www.ssafe-food.org/our-projects). In the United States, the Grocery Manufacturers Association and Battelle produced EMAlert, a subscription-based online tool to assess vulnerability from EMAs (https://www.battelle.org/battelle-newsroom/news -details/the-grocery-manufacturers-association-(gma)-and-battelle-launch-tool-to -fight-food-fraud), and the Food and Drug Administration (FDA) developed a user- friendly free food defense software tool, Food Defense Plan Builder, to assist FBOs with developing personalized food defense plans for their facilities. To stay informed on historical and emerging threats to the supply chain, individuals conducting vulnerability assessments have at their disposal the US Pharmacopeial Convention’s food fraud database.

11.3 TRENDS IN HACCP AND FSMS Amanda Evans (2015), a food safety consultant and compliance specialist from Australia, identified five trends in relation to HACCP starting with 2015 and presented them on the HACCP Mentor site, in which she is the facilitator. These five trends are focusing on supply chain, using prevention models rather than reaction, implementing mobile technology, using social media, and complying with customer requirements (https://haccpmentor.com/haccp/top-5-haccp-trends-2015/). Meanwhile, Lori Carlson (2016) identified eight food industry trends fueled by FSMA and presented them in Food Safety Tech, an industry-specific eMagazine, and a Conference series serving the global food industry. These trends and some more identified by the authors of this chapter are presented in the following. Focusing on supply chain is in trends because hazards associated with raw materials and ingredients can be controlled only if their origin and their journey to the processing unit are known. For example, when fish comes from aquaculture, regulations Food Safety Management in Fish Processing Units 247 for unapproved drugs (e.g., antibiotics) and regulations on approved aquaculture drug must be known. It is common practice for many FBOs to require HACCP plans for their suppliers. Thus, with an HACCP-based system in place at the food processing establishment, there is a good possibility that a problematic raw material or ingredient will not leave the supplier’s dock, farm, or warehouse. The specialists from EtQ Inc. advise their clients to use Supplier Management tools such as the following:

• Detailed supplier ratings, to benchmark and rank suppliers. • Secure supplier portals, to allow suppliers to manage corrective action requests assigned to them. • Customized inspection rules, to audit suppliers based on their performance history (Tracy, 2016).

HACCP receiving legislative support was and continues to be a trend. Along the years, the use of HACCP passed from voluntary implementation driven almost by the marketplace to compulsory implementation driven by regulations. It is recognized that regulations enacted by the European Union (EU) and the United States have increased the pace and set the trend for many other countries, especially the major commercial partners of the EU and the United States (http://www.fao.org /docrep/008/y5924e/y5924e0b.htm). Now, governments around the world are renewing legislative frameworks to be in line with international agreements (e.g., Codex Alimentarius) and standards (e.g., ISO 22000). An example is the FSMA, in force since 2011 in the United States. Based on this act, FDA has, for the first time, a legislative mandate to require comprehensive, science-based preventive controls across the food supply, like the ones required by HACCP, but under the frame of a new system named Hazard Analysis and Risk-Based Preventive Controls. Another example is Safe Food for Canadians Act, in force since 2012 in Canada, which consolidates the authorities of the Fish Inspection Act, the Canada Agricultural Products Act, the Meat Inspection Act, and the food provisions of the Consumer Packaging and Labelling Act. Canada, even if in 2008 it was considered a country having one of the best safety systems in the world, based on factors such as rates of food-borne illness, inspections, education programs, use of agricultural chemicals and strategies on bioterrorism, risk management, and food recalls (http://www.cbc.ca/news /canada-s-food-safety-rated-among-world-s-best-1.888680), decided to renew its legislation to be aligned to new achievements regarding food safety. A package of regulations dedicated to Food Safety and Standards has also been in force in India since 2011 (http://www.fssai.gov.in/home/fss-legislation/fss-regulations.html), while China’s new laws, which officially took effect from October 2015, are widely considered the toughest food safety laws ever. Some of the changes include central- izing supervision of food production and trading activities under the China Drug & Food Administration, greater focus on risk prevention, stricter food ingredient and processing regulations, and tougher penalties for violations (https://foodindustry .asia/how-regulation-and-technology-are-driving-food-safety-in-asia). Similar things are happening in the Asian and African countries that both have fish processing industries more developed than other food industries. More than that, even international agreements and standards are renewing. For example, ISO 22000, which 248 Trends in Fish Processing Technologies was published in 2005, is now under revision. It is common practice for an ISO standard to be evaluated every 5 years to determine whether a revision is necessary or not. Following the years when the standard was considered still relevant and useful to food businesses operators, in 2014, it came out that a revision is necessary. According to the declaration of the ISO, currently (January 2017), the standard is at the committee draft stage, and the revised version will be available for publication in October–November 2018 (http://www.iso.org/iso/iso22000_revision). The availability of risk assessment tools has increased. The new vision on designing a HACCP plan created a demand for user-friendly tools to help FBOs in analyzing and updating their management systems. Risk assessment tools currently available to the food industry are diverse in functionality and vary in scope and cost. For example, chemical and microbiological hazards in foods through process models, which quantify risk across scenarios and predict the effectiveness of control strategies, can be assessed using the free online tool FDA-iRISK 2.0, available from FDA. Then, for supply chain management and food safety auditing, the Safefood 360° software can be used, which is commercially available from Safefood 360, Inc. (Manhattan, New York), and EtQ, from EtQ Inc. (Farmingdale, New York). Applying modern methods for monitoring operational prerequisite programs (OPRPs) is also a trend taking into consideration that in an HARPC system, such points need the same attention as a CCP. Some examples include the following:

• Pest e-monitoring using a device installed on top of the bait station that instantly sends a signal to a facility device to alert that pest activity is occurring. • Hand washing and sanitizing monitoring systems. • Smart ID badges used for zone movement monitoring, hand sanitizing monitoring by location or interval, and bathroom/hand wash monitoring. • Adenosine triphosphate (ATP) bioluminescence cleanliness monitoring. Most of the luminometers used for cleanliness monitoring based on ATP bioluminescence are able to report and analyze the data immediately after the measuring process. Thus, safety managers can compare how things are evolving in the same facility or in different facilities and take appropriate measures to keep contamination under control.

Applying rapid methods for pathogen detection is another trend as well due to the fact that they can be used in developing a HACCP system and for verifying that the system is working. The interest in rapid methods is shown equally by small food companies to develop food safety programs and by larger companies to reduce bottom-line costs while ensuring the safety and wholesomeness of their product lines. Researchers are still developing such methods in order to gain improvements in terms of rapidity, sensitivity, specificity, and suitability for in situ analysis and distinction of the viable cells (Law et al., 2014; Zhao et al., 2014). Communication via color-coding is another trend based on the fact that this type of communication goes beyond language. Besides being an important part of food safety programs by preventing cross-contamination due to pathogens, allergens, and foreign bodies, color-coding enhances the level of traceability and aids organizational Food Safety Management in Fish Processing Units 249 efforts too. So, usually used to separate zones based on risk, distinguish between food contact and non-food-contact surfaces, and distinguish between cleaning and material- handling tools from different areas, color-coding can be used to a diversity of items used in food processing environments (cutting boards, hair nets, footwear, clothing, gloves, mats, bins). To know which color to assign to an object, a color-coding program should exist in a written form and included into the HACCP program. Authorities responsible for food safety are in favor of color-coding because it is a method that can easily be documented and followed by employees (Vogel, 2015). Meanwhile, they consider color-coding as a supplementary preventive measure to protect food. Implementation of mobile technology is another trend. Although behind other industries, the food industry is starting to see the benefits of using mobile technology to increase productivity by converting paper compliance forms into electronic mobile applications. A significant impact on implementation of mobile technology had Bluetooth Smart technology (https://www.bluetooth.org). This version remarked by its power efficiency (allows portable devices to work for longer periods with smaller batteries). Mobile technology is useful for keeping records and setting up in-house online training programs. For example, the °FreshTemp company is able to create HACCP-compliant logbooks and detailed reports from the wireless temperature data collected by Bluetooth devices (http://www.freshtemp.com/). Record keeping using mobile technology has already become a business opportunity. Any FBO can implement mobile technology due to affordable associated costs. Using social media is also a trend both for consumers and for food safety authorities. Consumers are using social media because they find it easier and quicker to express an opinion via Facebook or Twitter than directly complaining to the authorities or to the FBOs. It is estimated that this trend will increase with customer dissatisfac- tion regarding certain foods. On the other hand, food safety authorities are choosing to disseminate important information on food quickly and directly to social media users. Institutions such as Centers for Disease Control and Prevention and FDA in the United States, European Food Safety Authority in Italy, Food Standards Agency in the United Kingdom, and the Centre for Food Safety of the Food and Environmental Hygiene Department in Hong Kong use Facebook to provide the public a wide range of information including food alerts, food safety reports, food safety tips, results of theme studies, and publications issued by them, considering that Facebook pages facilitate better communication and interaction with the public. According to Wu (2015), Facebook could be also an efficient tool of risk communication related to food safety. Using cloud technology is a solution to replace paper-based recording systems, to eliminate redundancy of data, and to allow instant access to data and systems from anywhere in the world, while providing total coverage of the organization. Cloud computing is a synonym for distributed computing over a network and means the ability to run a program on many connected computers at the same time (Corkery et al., 2013). A cloud-based ERP system should operate a dynamic database-driven electronic HACCP program and produce traceability records instantly or distrib- ute very fast notifications for CAPA. When cloud computing is chosen, a monthly subscription fee has to be paid, but costs are lower than those for traditional information technology management, which includes system upgrades, database management, antivirus programs, system integration, and data archiving and retrieval. 250 Trends in Fish Processing Technologies

The solutions using cloud computing are better known as software as a service, infrastructure as a service, and platform as a service. Specialists consider that cloud computing gives manufacturers a significant competitive advantage in a low-margin, highly competitive marketplace (Nessen and Cowan, 2012). An example of cloud- based technology combined with mobile technology is the Estabman system (www .estabman.com), which allows users to create their own tailor-made template to carry out self-assessment, trend analysis, and presentation of results. Trainings based on e-learning proved to be a very good solution to cover the training needs for those who are working in the food industry. Many studies have concluded that the lack of understanding of HACCP and other FSMS processes is one of the key barriers to the implementation of an effective and sustainable FSMS. This demonstrates that the workforce responsible for implementing HACCP-based systems have to be adequately trained. It was also demonstrated that to increase knowledge retention and job performance, it is necessary to have shorter and more frequent targeted trainings (Carlson, 2016). In these circumstances, e-learning trainings are able to provide targeted contents with shorter duration in an interactive format. Depending upon the training provider (dedicated training service providers as well as universities, trade associations, regulatory agencies, scheme owners, certification bodies), online trainings may be distributed through a learning management system to provide additional training tools, assess training effectiveness, and manage the training activities and competencies of all participants (Carlson, 2016). Food safety managers have to be highly qualified persons as they are the on-site food safety and regulatory authorities at the facilities. They must have an advanced degree in food science, food engineering, or related fields; direct working experience in a plant; and expertise in food safety matters. Besides these qualifications and expertise, it is not uncommon for employers to ask for more from a food safety manager. Usually, excellent written and verbal communication skills, demonstrated ability to work cross-functionally, innovative thinking to create novel solutions, and persuasion skills to lead/influence others, including third parties in the supply chain, are the supplementary characteristics that are added to the profile of a food safety manager. Recently, verifying some profiles for food safety managers, we were sur- prised to find that Six Sigma* credentials were asked for a Vice President of Quality and Food Safety and Black Belt† was preferred. High qualification is also needed for any PCQI; the new position appeared in an FSMS based on HARPC. Such a person needs the technical knowledge of the food being made and processes in use to evaluate risk and identify the means to control them and to assume responsibility for the implementation of various provisions of an FSMS: preparation of the food safety plan, validation of the preventive controls, review of records, and r-analysis of the food safety plan. Another trend refers to updating of food safety systems based on self-assessments. This trend gives FBOs the opportunity to reconsider hazards and vulnerabilities

* Six Sigma is a method that provides organizations tools to improve the capability of their business processes. This increase in performance and decrease in process variation lead to defect reduction and improvement in profits, employee morale, and quality of products or services. Six Sigma is considered the gold standard for process and quality management of process outputs in organizations. † Leads problem-solving projects. Trains and coaches project teams. Food Safety Management in Fish Processing Units 251 and upgrade food safety controls along with the management system. Several self- assessment tools are available. We mention here the ones developed in the frame of the FP6 PathogenCombat project, which are differentiated in diagnostic tools (FSMS Diagnostic Instrument and Microbial Assessment Scheme), selection tools (Microbial Assessment Scheme selection tool and Quality Assurance Grid), and improvement tools (Roadmaps for improvements, Protocol Validation, and Verification of FSMS). These tools, which have been transformed in an FSMS support application, are freely available via the Internet by accessing the project site (www.pathogencombat.com) (Jacxsens et al., 2011). Self-assessments can lead to significant improvements in food safety management, product safety and quality, and even operational efficiency. There is also an increasing trend toward major customers requiring third-party certification as part of food business trading terms. Complying with this requirement may have a serious financial impact on the food business because multiple food safety and HACCP certification standards need first implementation and then third-party auditing, if not audited directly by the customer. Several private standards and initiatives came into operation, such as the British Retail Consortium standard, the International Food Standard, GLOBAL G.A.P., Aquaculture Standard, Best Aquaculture Practices, GFSI, as well as the ISO 22000 standard on food safety and the newest one, the Food Safety System Certification Standard/Food Safety 22000. Although standards are not a legal requirement and it was expected to have enforced such standards in small and medium enterprises focusing on maximizing opportunities to continuously improve the overall safety of the organizations delivering products to the supermarkets or being involved in exports, it happened that FBOs decided to become certified against different food safety and quality standards as key to maintaining and improving reputation and legal liabilities, as well as against additional standards such as labor, environmental, and animal welfare standards as strategies for customer loyalty and market shares (Fulponi, 2006). More than that, major food retailers now demand their suppliers to adhere to GFSI, as well as other food safety standards such as ISO 22000, and demonstrate that they have incorporated safety and quality initiatives into their food production processes. No matter which certification scheme is chosen, they are similar enough that the development and implementation will be pretty much the same. This is why it is said that it is easier for a company to implement a new standard if it is successfully managed than to implement another one. On the other hand, the decision to seek global versus national and/or industry- specific certification is dependent on where/with whom a company is doing business and what standards are expected or required. A global certification may eliminate the need for duplicate audits by different certification bodies. Certification is needed not only for food safety systems as a whole but also for equipment, materials (e.g., cleaning and disinfection materials, food industry work wear), and services (e.g., trainings). When such an item is HACCP certified, it means that it satisfies the HACCP standards. Products that demonstrate food safety excellence in design, materials, and manufacture may carry the HACCP certification mark, confirming their “fitness for purpose” in food processes governed by HACCP programs. The same applies to production equipment, such as packaging machines and filleting machines, which are of particular interest in fish processing establishments because they are complicated and have many rough parts, which are difficult 252 Trends in Fish Processing Technologies to clean and disinfect. Similar to other food industries, in fish processing units, it is recommended to have equipment designed and constructed based on hygiene principles. The European Hygienic Design Group, National Sanitation Foundation International, 3A Sanitary Standards Inc., or other organizations are setting the general principles for enhanced cleanability of equipment, thus decreasing the risk of biological, physical, and chemical contamination. On the market, there is a large choice of hygienic equipment, much of which has been certified by one of the previously mentioned organizations, so fish processors can select the most suitable ones to construct processing lines that meet the hygienic requirements and to set an appropriate food safety management plan in their units.

11.4 CONCLUSIONS • FSMS focuses on maximizing opportunities to continuously improve the overall safety of food products. • To be successful in implementation of FSMS in their companies, FBOs have to rely on modern technologies and very well-trained personnel. • To assess how efficient an FSMS is, third-party certification is needed.

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INTERNET SITES http://usa.oceana.org/sites/default/files/global_fraud_report_final_low-res.pdf https://techni-k.co.uk/shop/books/assessing-threat-vulnerability-for-food-defence/ http://www.ssafe-food.org/our-projects https://www.battelle.org/battelle-newsroom/news-details/the-grocery-manufacturers-association -(gma)-and-battelle-launch-tool-to-fight-food-fraud) https://haccpmentor.com/haccp/top-5-haccp-trends-2015/ http://www.cbc.ca/news/canada-s-food-safety-rated-among-world-s-best-1.888680 https://foodindustry.asia/how-regulation-and-technology-are-driving-food-safety-in-asia http://www.iso.org/iso/iso22000_revision http://www.freshtemp.com/ Quality and Quality 12 Changes Assessment of Processed Fish

Mercedes Careche and Isabel Sánchez-Alonso

CONTENTS 12.1 Introduction...... 255 12.2 Fish Deteriorative Changes...... 257 12.3 Indicators to Measure Changes in Properties or Composition of Fish...... 258 12.3.1 Quality Index Method...... 259 12.3.2 Multisensor Mimicking Human Senses: Artificial Quality Index....262 12.3.3 Electronic and Vibrational Spectroscopy...... 264 12.3.3.1 Time–Temperature Estimation by FTIR of the Lipid Fraction in Frozen Hake...... 265 12.3.4 Low-Field 1H Nuclear Magnetic Resonance Relaxometry...... 267 12.3.4.1 Time–Temperature Modeling in Frozen Hake...... 267 12.3.5 Dielectric Spectroscopy...... 268 12.4 Opportunities and Challenges...... 269 12.5 Future Trends and Expectations...... 270 12.6 Conclusion...... 270 Acknowledgments...... 270 References...... 270

12.1 INTRODUCTION Quality is a complex concept with many dimensions and context dependent so that it is relative to person, place and time. It can be defined as “the requirements necessary to satisfy the needs and expectations of the consumer” and can be divided into 13 different consumer requirements, which in turn can be grouped into those that are considering the product as food or as an object of trade (Peri 2006). The first group comprises safety, commodity, nutritional, and sensory requirements and the latter constitute a way of interaction between products and consumers since consumers can perceive them. The set of these four requirements constitutes the core of the quality of the product as such. On the other hand, psychological requirements concerning the product context (i.e., origin, tradition, etc.) and the system of values (ethical) are becoming increasingly important immaterial requirements of quality, and both psychological and product requirements are those that regard the product as a food. The second group, in which the product is considered as an object of trade, encompasses

255 256 Trends in Fish Processing Technologies guarantee (certification, traceability), product/packaging system (functional and aesthetic requirements, information, and convenience), as well as product/market system, which covers dimensions such as availability and price (Peri 2006). The concepts of food quality have also been classified as (a) product oriented, where quality can be measured as a set of physical, chemical, microbiological characteristics, properties, or performances, (b) process oriented, related to the stability of the product-oriented quality in terms of a prespecified level, and both of them are considered objective quality, or (c) user oriented, the subjective perception of either an intermediate or end user in the food chain (Grunert 1995). Within the requirements related to the product as food, some of them are considered as immaterial since they primarily satisfy psychological, cultural or ethical needs whereas others are measurable and can be verified and certified. These are the ones which constitute the core of the food technologists’ activity. Bremner (2000) pointed to the gap existing between the overall quality concept and the particular characteristics or performances that are actually measured and, thus, the need for workable definitions of quality with well-defined limits, so that parameters and measurements could be derived from them. He proposed an approach that links them by adding specific, concrete levels with descending order of abstraction, so that from the overall quality (QUALITYC), which in itself is not measurable, the concept would be linked to standard descriptive phrases such as the ones used in the ISO definition (QUALITYG). The missing link would be filled by inserting a set of definitions closely related to the material, product, circumstances, or uses (QUALITYD1….Di), in terms of a number of properties that can be quantified, and these would embrace each one of the dimensions of the quality concept. For a given QUALITY Di, criteria and specifications related to a set of inherent characteristics or performances need to be set up by mutual agreement among all the interested parties and, at the bottom of the hierarchical ladder, these could be determined by prescribed test methods, so that the values obtained could be compared against these agreed criteria and specifications. The need for estimating product- and process-oriented quality of fish in a variety of scenarios is well acknowledged and these include, for example, compliance with regulatory specifications, agreements, or standards; avoidance of counterfeit and fraud; the modification or introduction of new product, processes, or species by the manufacturing industry; improvement of the communication in the seafood processing chain by the use of agreed test methods; as well as for linking subjective quality as perceived by the consumers into objective product characteristics and performances. The inherent properties of seafood products can be affected by a number of factors, in many cases interrelated. These factors are linked to the fish living conditions, type of rearing or harvest system, processing and packaging, storage and distribution, and preparation for consumption, thus affecting the whole seafood processing chain. Sensory requirements play a key role on the quality of fish as food and most of the indicators relate to this parameter directly or indirectly. Moreover, one of the characteristics of seafood is their high perishability, so that time and temperature of storage become critical variables, since they can condition the maintenance of the products’ inherent sensory properties. Quality and Quality Changes Assessment of Processed Fish 257

The eating acceptability of fish during storage does not usually follow a linear pattern. For example, in chilled fish, it suffers a rapid decline during the first hours or days, followed by a plateau and later by a fast decrease until the fish is rejected by the sensory panel (Bremner et al. 1987; Hyldig and Nielsen 1998). However, wholesalers, processors, or retailers would need to have the product early enough so that there is time to process, pack, or store it before selling, and consumers would also need some time to store or cook the fish product well before it is unacceptable for consumption. In this context, there is a need for methodologies to provide objective indicators related to the loss of attributes and in particular for those affected by time and/ or temperatures of storage (Jorgensen et al. 2003) in all the steps in the processing chain (Green-Petersen et al. 2012). These methodologies could be used to meet QUALITY Di as one of the definitions needed to construct the several dimensions of QUALITYC for a given processed seafood product.

12.2 FISH DETERIORATIVE CHANGES The muscle of fish just after death is totally relaxed and the soft elastic texture (pre- rigor) usually persists for some hours. Then the muscle contracts and becomes hard and rigid entering in rigor mortis. Rigor lasts for some hours and its resolution makes the muscle less rigid but no longer as elastic as before. The process of tenderization, where cytoskeletal links to sarcomere and sarcolemma as well as the extracellular matrix structure are degraded, starts to occur soon after the death of the fish. When oxygen supply ceases due to lack of blood circulation, the glycogen is metabolized anaerobically, leading to accumulation of lactic acid in muscle, resulting in a pH drop. Mitochondria and sarcoplasmic reticulum deterioration due to pH fall and osmotic pressure changes results in the release of calcium ions in the cytosol. Eventually, adenosine triphosphate (ATP) levels cannot be maintained and contraction occurs. ATP degradation products are also accumulated in the muscle. The biochemical changes taking place additionally favor initiation of lipid oxidation. Moreover, trimethylamine oxide (TMAO) is changed to trimethylamine by endogenous enzymes and later by bacteria, the activity of microorganisms being the main limiting factor of fresh fish shelf-life. The early postmortem events and subsequent storage at chilling temperatures lead to an array of new chemical compounds and changes affecting taste, smell, colour and texture of the fish, which depends on species and are affected by a number of factors including size and physiological condition of the fish, as well as slaughtering and postmortem handling and processing (Olafsdottir et al. 1997; Delbarre-Ladrat et al. 2006; Cheng et al. 2015). When fish is to be preserved for long periods of time, frozen storage is the most effective method to prolong shelf life since microbial growth is halted, and the rate of many physical and chemical reactions is decreased. This results in fewer changes in the characteristics of the fish muscle as compared to other preserving methods. Also in this case, several factors including species, season, fishing and slaughtering methods, postmortem handling, freezing and frozen storage conditions, as well as the interactions of all these factors may eventually affect the fish flesh structure, composition, and functionality, all of which are directly linked to the acceptability by the consumer and may exert a detrimental effect on their commercial value. 258 Trends in Fish Processing Technologies

In lean species, changes in frozen stored fish are mainly due to the loss of texture attributes, which result in hard, dry, and fibrous products. In fatty fish, the most important factor affecting frozen storage stability is the development of rancidity, and semifatty species are the most stable toward frozen storage deterioration. Texture changes have been attributed to denaturation and aggregation of myofibrillar proteins, resulting additionally in loss of their extractability in salt solutions or loss of water holding capacity of the muscle (e.g., Schubring 2004; Herrero et al. 2005; Burgaard and Jørgensen 2010). Denaturation and/or aggregation of myofibrillar proteins can occur due to dehydration by sublimation of ice crystals, concentration of solutes in the tissue as a result of freezing of the water in the fish muscle, the formation in some species of compounds such as formaldehyde (FA), from TMAO by demethylation into dimethylamine and FA, or the interaction of proteins with products from lipid hydrolysis or oxidation (Shenouda 1980; Haard 1992; Sikorski and Kolakowska 1994; Jaczynski et al. 2012). Protein secondary and tertiary structure modifications occur during frozen storage (Careche et al. 1999; Herrero et al. 2004); also the morphology of muscle fibers is altered, and reduction of space between myofibrils (Howgate 1979) and interfila- ment distance occurs (García et al. 1999). All these may be accompanied by further alterations in the protein and water interactions and in the distribution and mobility of water (Herrero et al. 2005). Lipid degradation by oxidation and hydrolysis during frozen storage, result in the production of a number of substances which negatively affect flavor, odor, color, texture, and the nutritional value of fish muscle. Oxidation occurs by an autocata- lytic process through a series of chain reactions in which hydroperoxides and subsequently secondary oxidation products such as aldehydes, ketones, alcohols, or carboxylic acids are formed. Hydrolysis of triglycerides and phospholipids release free fatty acids (FFA), which can contribute to toughening (Shenouda 1980; Haard 1992; Sánchez-Alonso et al. 2012a).

12.3 INDICATORS TO MEASURE CHANGES IN PROPERTIES OR COMPOSITION OF FISH Many of the changes described earlier can be measured by standardized methodologies. They include sensory, physical, chemical, biochemical, microbiological, and techno functional methods and are designed to meet a wide scope of needs of the different actors in the food processing chain. The methods can measure the overall characteristics of the fish as food (e.g., sensory acceptability), some particular characteristics or performances (e.g., brightness of the skin, smell, rigor), or the product components (e.g., chemical, microbial, biological, physical objects), structural organization, or specific properties (e.g., physical, technofunctional, nutritional). The outputs of the different methodologies need to be calibrated directly against agreed criteria for a given definition of quality (e.g., sensory analysis, microbial counts), through intermediate indicators calibrated previously with the former (e.g., the use of a chemical compound known to be directly related with postmortem deterioration, for the calibration of a spectroscopic output), or with some external Quality and Quality Changes Assessment of Processed Fish 259 parameters (e.g., time or temperature), and they can be used alone or in combination with other methodologies. Relevant examples of indicators will be addressed in this section with emphasis on those related to storage time and temperature.

12.3.1 quality Index Method The Tasmanian Food Research Unit originally developed a demerit point scoring system with the aim of providing a sensory test that gave a linear response with time of storage and with the further requirement that it should be able to integrate time– temperature effects (Bremner 1985; Bremner et al. 1987) so that it could be applied to products stored at different or fluctuating temperatures. The demerit point scoring system was first developed for chilled storage, where the main cause of deterioration is bacterial growth, but the authors discussed that the principles should be the same in other conditions (i.e., frozen storage), where there may be different major factors causing the deterioration of seafood (Bremner et al. 1987). These authors (Bremner et al. 1987) revised several empirical relationships that relate the reaction rates with temperature for the main processes involved in the deterioration of seafood, including Arrhenius equation and Bělehrádek functions, and observed that at least microbial spoilage and enzyme activity (i.e., nucleotide breakdown) obeyed Bělehrádek power functions with an exponent of 2 and that the biological zero (i.e., the hypothetical temperature where the reaction rate is zero) of these reactions was similar. Since changes in ice-stored fish that are perceived by the senses of sight, smell, and touch can be mostly associated to microbiological and enzymatic deteriorative changes, they were used to develop a sensory score system and shown to follow the same deterioration rate as the previously mentioned microbial and enzymic processes. The score system summed up the demerit points or defects for each attribute on the assumption that if the individual components of the sensory score followed the same temperature function, the system as a whole would also have the same temperature dependency and they found that this was the case in their system (Bremner et al. 1987). The performance of the method was initially proven in several tropical species (Bremner 1985), and soon afterward, the score system was further developed by some European laboratories and adopted the name of “Quality Index Method” (QIM) (Larsen et al. 1992). The QIM is thus a sensory method designed so that the sum of demerit points or defects given to each of the attributes (QI) correlates linearly with storage time. The system is based on demerit points since assessors can more easily judge defects rather than degree of perfection. At start, the number of demerit points is 0 for each attribute and the higher the number of demerit points, the more defects the product has. The score is constructed so that there is a sufficient number of attributes but with no high scores for any single attribute (a minimum of 0 to a maximum of 3 sensory points) so that no special weight is given to any of them as compared to the rest. Therefore, the system does not reject a sample based on only one criterion; all of the attributes must be judged and minor differences in judgments in any parameter do not over-influence the total score. 260 Trends in Fish Processing Technologies

The parameters for the QIM scores are selected among well-defined characteristic changes of outer appearance (e.g., brightness, iridescence of the skin, surface mucus, cloudiness and shape of the eyes, colour of the gills, discoloration of the belly cavity), smell (e.g., gills), and touch (e.g., rigor, softness or lack of elasticity of the muscle). Attributes need to be chosen for each species and condition and, in general, proven to be relevant to the various groups operating in the fisheries sector (Larsen 1998; Jorgensen et al. 2003). The linearity with storage time implies that the equivalent of the number of days at 0°C the fish have been stored can be estimated. If there is an agreement at which score the product should no longer be consumed (e.g., when the cooked fish is rejected by the sensory panel or when a given microbial load has reached its maximum allowable value), the remaining shelf life can be calculated, within the limits of the uncertainty in the estimation. Determination of the absolute storage time is known to be influenced by the initial conditions, including intrinsic, environmental, catching, handling, and processing factors before storage (Hyldig et al. 2011), and thus, the estimation of the shelf life, by QIM or by any other method, will be affected by these initial conditions. The advantages of the tool were early discussed in many fora and new score sheets disseminated (Hyldig and Nielsen 1998; Larsen 1998; Luten and Martinsdottir 1998; Martinsdottir 1998; Martinsdottir et al. 2003). It is an easy-to-use system and no deep knowledge of the species is necessary to learn how to assess the different attributes (Bremner et al. 1987). The method has become very popular and practical at the marketplace in some countries (Luten and Martinsdottir 1998; Martinsdottir et al. 2003). It is also used in research laboratories, so that, at present, developments of more than 50 score sheets for different species (finfish, cephalopods, crustacea) or fish products (e.g., whole, gutted fish, fillets) have been published in refereed journals (Hyldig et al. 2011; Bernardi et al. 2013 and references therein). Schemes for preservation methods other than storage in ice have been developed, such as the development of QIM for maatjes herring packed in air and modified atmospheres (Lyhs and Schelvis-Smit 2005) or for the ice storage of thawed Greenland halibut (López-García et al. 2014). The conditions for developing and validating new QIM schemes are fully described in Hyldig et al. (2011). QIM has been introduced as an indicator to evaluate the effect of several processing treatments. Some examples are the study of storage in water and ice of anchovies and sardines (Careche et al. 2002; García and Careche 2002) and the effect of washing in gilthead seabream (Huidobro et al. 2000; López-Caballero et al. 2002). However, there are very few publications on the adaptation of the QIM score sheet for frozen fish (Warm et al. 1998; Herrero et al. 2003). A QIM score sheet consisting of nine parameters with a total of 17 sensory points for headed and gutted hake (M. capensis and M. paradoxus) stored frozen was designed by Herrero et al. (2003). Part of the score sheet, consisting of 13 sensory points (shape of the fish, surface appearance in the ventral area, dehydration, firmness, elasticity, water loss, odor) provided information about quality in terms of apparent storage time at −20°C and the second part consisted of up to four sensory points for commercially important quality parameters (remains of guts in the belly, catch handling signs) not directly related to storage time (Table 12.1). Quality and Quality Changes Assessment of Processed Fish 261

TABLE 12.1 QIM Score Sheet for Frozen Headed and Gutted Hake Parameter Attributes Sensory Points Shape of fish Normal round shape as freshly caught hake 0 Mechanically damaged during freezing. Deformed 1 Surface appearance (ventral area) Silver bright. Bright, shining. No bleaching 0 Slight bleaching 1 Dull and very bleached. Freeze dried 2 Odor Fresh marine, seaweed, or neutral, fruity 0 Slightly sour, metallic or cold stored 1 Strong sour and metallic, or cold stored 2 Dehydration/roughness on head Between 0% to 10% 0 cut area Between 10% to 50% 1 More than 50% 2 Firmness Very soft 0 Soft 1 Firm 2 Very firm 3 Elasticity Marked by pressure 0 Not marked by pressure 1 Water loss None with slight pressure 0 Some with slight pressure 1 Much with slight pressure 2 Partial score Remains of guts in the belly None 0 Few 1 Many 2 Catch handling (bruises or marks) None 0 Few 1 Many 2 Total score

Source: Herrero, A. M., Huidobro, A. and Careche, M.: Development of a quality index method for fro zen hake (M-capensis and M-paradoxus). J Food Sci. 2003. Vol. 68. Pp. 1086–1092. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission. License number 4118240950496.

When developing new score sheets, the effect of temperature on the deterioration rate has seldom been checked, and in most papers, if not all, the initial assumptions about the similarity of the temperature relation of the sensory attributes with the main deteriorative changes have been adopted. However, there was a nonlin- ear decrease with time in the QI values of temperature-abused refrigerated Arctic charr (Salvelinus alpinus) (Cyprian et al. 2008), which suggests that the QIM score should be reevaluated for each condition. As regards the QIM score sheet of frozen and thawed hake, and although it was developed at a fixed temperature of storage 262 Trends in Fish Processing Technologies

(i.e., −20°C), it can be calculated from data published by Kent et al. (2005), who used this score for calibration purposes, that the rate of increase of the QIM score followed an Arrhenius type of behavior, and therefore, it could have a time–temperature integration function. Versions for consumers have been developed (C-QIM) (Larsen et al. 2003), and a QIM-Eurofish alliance (www.qim-eurofish.com) has contributed to disseminate a reference manual with several QIM schemes in 11 languages. Several QIM tables are also available on the Internet (Archer 2010) and the QIM of thawed fish stored at refrigerated temperatures for eight species has been collected and recently published in a reference manual in Spanish (Romero et al. 2014). User-friendly applications for smartphones are available for some species so that the fisheries sector and the consumer can have in a matter of seconds the remaining shelf life of a number of species. Thus, the number of applications is increasing, and QIM has also been used for calibration of several indicators measured by instrumental devices, as will be described in the following section.

12.3.2 M ultisensor Mimicking Human Senses: Artificial Quality Index Sensory evaluation of fish using well-defined schemes such as QIM can indeed give reliable information of quality in terms of storage time at a given temperature. Although it is the method of choice in many situations, there can be some restrictions to its application. For example, the use of sensory panels can be expensive, time consuming, or not available in a particular part of the fish processing chain. Although QIM is an objective sensory method suitable for many applications, there are some circumstances where instrumental methods are preferred. Many approaches have been explored that measure physical, chemical, enzymatic, microbiological, and biological parameters related with some properties of fish as food. These include equipment, which can, in some way, be considered as candidates to replace human senses such as image analyzers, colorimeters, electronic noses, or texture analyzers. Artificial senses are widely applied in food analysis for process monitoring and determining the quality and authenticity of foods (Sliwinska et al. 2014). Likewise, instrumental texture analysis has been extensively used for evaluation of fish quality (Barroso et al. 1998a, 1998b; Sánchez-Alonso et al. 2010; Cheng et al. 2013). Many different instrumental measurements render information about fish freshness or quality; however, it has been considered that by themselves, they are insufficient to provide an unambiguous answer to freshness or quality (Olafsdottir et al. 1997). The fish sector, although acknowledging the need for instrumental measurements, does not consider individual attributes (i.e., colorimeters, texture analyzers, or electronic noses) as important, in agreement with the fact that complex concepts such as freshness or quality cannot be quantified by a single attribute measurement (Jorgensen et al. 2003). One of the approaches in assessing the quality of fish has been to develop a multisensor by selection of complementary rapid physical techniques that are adapted to measure the quality of fish in terms of days in ice. This concept was developed and tried in cod stored in ice (Olafsdottir et al. 2004). Outputs from the instrumental devices (i.e., texturometer, colormeter, electronic nose) were calibrated to provide Quality and Quality Changes Assessment of Processed Fish 263 an estimation of the corresponding human sense so that their estimation could be treated the same way as QIM attributes. Thus, data collected from each instrument were analyzed separately and a partial least squares model was calculated to predict those QIM attributes relevant to each device, followed by their integration into an artificial quality index (AQI). A score could thus be built that behaved like the QIM with respect to storage time in ice (Figure 12.1), and consequently, it was proposed that it be called Artificial Quality Index (Di Natale 2003). The AQI considered a subset of attributes from QIM that retained most of the classification properties of the whole QIM, and in the case of cod, the AQI was shown to be as accurate and precise as the QIM (Olafsdottir et al. 2004), since it described the freshness of this species with the uncertainty of the predicted storage time being less than 0.5 days. This concept was applied to build a fish freshness indicator of sardine during refrigerated storage (Macagnano et al. 2005). The benefits of this approach include that the skills of the sensory panel could be transferred to the multisensor system. Moreover, the measurements are rapid and potentially cheaper than a sensory panel, and the monitoring could be done online or at places non accessible to the panel (Di Natale 2003). The similarity of the time and temperature relationships of this AQI with the main deteriorative processes in fish is implied through the calibration with individual QIM parameters. However, it should ideally be checked as proposed by Bremner et al. (1987). The method should be extended for more species and conditions; it needs some improvements to be faster and easier to operate before it is accepted by companies. Nevertheless, the introduction of instruments mimicking human senses seems

QIM sensory attributes Instrumental techniques

Appearance Calibration Color Skin Firmness Calibration Texture analyzer Eyes Cornea Form Color of pupil Gills Color Calibration Electronic nose Smell Mucus Blood Alternative techniques: Color Σ VIS spectroscopy Fillets Image analysis Color Electrical testers

Σ= QIM score Artiﬁcial quality score Σ = AQI

FIGURE 12.1 Artificial quality index. Order Detail ID: 70534901. (Adapted from Olafsdottir et al., 15:86–93, 2004, Trends Food Sci Technol by PERGAMON. Reproduced with permission of PERGAMON in the format Book via Copyright Clearance Center.) 264 Trends in Fish Processing Technologies a promising approach to obtain an estimation comparable to the judgment of trained panels (Di Natale 2003; Olafsdottir et al. 2004; Sliwinska et al. 2014).

12.3.3 electronic and Vibrational Spectroscopy Another approach for instrumentally monitoring the characteristics or performances relevant to the quality of fish is the use of electronic (ultraviolet or visible) or vibrational (infrared [IR] or Raman) spectroscopy. The growing interest in spectroscopic techniques in food applications is due to their potential of being highly specific, nondestructive, noninvasive, cost-effective, and fast in addition to allowing the simultaneous determination of several parameters, while traditional methods are usually labor-intensive and/or destructive. Many applications have been developed for fish and fish products during the last years, using visible IR (VIR), near-IR (NIR), mid- IR, Raman, and spectral imaging coupled with multivariate analysis. These spectroscopic methods have been mainly calibrated with intermediate attributes related to the deterioration of fish, i.e., physical chemical methods measuring specific compounds by classical analytical techniques or by technofunctional properties, such as water-holding capacity, as well as with some microbiological indicators. There are fewer instances where the output is calibrated with the corresponding human sense (e.g., to measure colour, odor, texture) or by using storage time (e.g., for shelf life evaluation). Spectroscopic methods have been applied also to differentiate among known classes of fish (e.g., according to origin, species, fresh vs. frozen, salt content). Thus, methods have covered studies on composition analyses of fish and fish products, changes in protein and water structure, lipid profiling and hydrolysis, lipid oxidation, presence of defects such as bruising, monitoring changes of the fish as affected by process variables (e.g., salting, smoking, atmospheres, freeze drying), for parasite detection, for measurements of contaminants, microbial spoilage, differentiation of fish species and by products, designation of origin, differentiation between fresh and frozen thawed fish, for establishing the storage time, as well as for prediction of colour, flavor, or instrumental texture, and recent reviews have been published on the applications of these techniques to foods (Li-Chan et al. 2010; Cozzolino and Murray 2012; Cheng et al. 2013; Liu et al. 2013; Cheng and Sun 2014; Cozzolino 2015; Hassoun and Karoui 2017; He et al. 2015; Qu et al. 2015). In relation to storage time in ice, VIS/NIR spectroscopy has been successfully used for cod and salmon, and good correlations of prediction and errors of estimation were found for both species (Nilsen et al. 2002). Freshness assessment of cod (Gadus morhua) and Atlantic salmon (Salmo salar L.) by VIS/NIR spectroscopy was found to be determined on individual fillets using a handheld interactance probe and with an imaging spectrometer for doing online analysis at industrial speed of one fillet per second (Sivertsen et al. 2011; Kimiya et al. 2013). The authors indicated that the spectral changes could be explained mainly by the oxidation of heme proteins. For salmon fillets, the prediction results were comparable to the QIM with trained panelists. VIS has also been calibrated with QIM scores in cod with a relatively narrow band in the mid visible spectral range (Nilsen and Esaiassen 2005). The storage time of frozen Quality and Quality Changes Assessment of Processed Fish 265 hake products has been studied by visible spectroscopy (Heia et al. 2003) and by Fourier transform mid-IR (FTIR) of the lipid fraction (Sánchez-Alonso et al. 2012a).

12.3.3.1 Time–Temperature Estimation by FTIR of the Lipid Fraction in Frozen Hake Spectroscopic methods offer the possibility of having not only analytical but also structural information so that the compound or groups of compounds or properties which are being measured can be in many instances characterized. For example, mid-IR can provide information on polar groups such as C=O, N-H, and O-H with strong IR stretching vibrations and also on nonpolar groups such as C=C, C-C, and S-S, albeit with less intense bands, and it has been used as a tool to monitor changes in lipid hydrolysis and oxidation. Since FFAs formed by hydrolysis of triglycerides and phospholipids, and lipid oxidation products have been reported to have an effect on the technofunctional and textural characteristics of fish muscle during frozen storage as well as detrimental effects on flavor and color changes, FTIR spectroscopy was used to investigate the lipid deterioration process in hake muscle frozen stored at a high temperature (i.e., −10°C), where frozen storage deterioration was achieved in a relatively short time (Sánchez-Alonso et al. 2012a). Among the structural and compositional features observed by FTIR, the changes in the carbonyl stretching bands from esters and carboxylic acids and the disap- − pearance of the bands from the asymmetric stretching PO2 group showed strong time dependence at this temperature, which suggested phospholipid hydrolysis as a contributor to the toughening of hake upon storage. Since FTIR spectroscopy could provide markers for modeling the time and temperature history of frozen fillets (Sánchez-Alonso et al. 2012a), the lipids extracted from hake fillets frozen stored at different temperatures for up to 150 weeks were analyzed in a second set of experiments. Three spectral regions known to carry specific physical chemical information on the changes in fish lipids were studied separately, corresponding to ν(C-H) vibrations (3100–2750 cm−1), ν(C=O) vibra- −1 − tions from ester and carboxylic groups (1800–1670 cm ), and νas ()PO2 phospholipids vibrations (1330–1127 cm−1) (Sánchez-Alonso et al. 2013; Careche et al. 2015) (Figure 12.2). Principal component (PC) scores of the spectroscopic output of these three regions were used as the response variables for modeling the time–temperature relationships. Three Arrhenius models were obtained, one for each region, and the best one resulted from the one corresponding to the phosphorous moiety of the phospho- 2 lipids with Radj = 09. 3 (Figure 12.3). Whenever a method is calibrated with time and temperature, it is highly desirable that the temperature dependence would follow the same rate as the deterioration of the fish, in the same principles described earlier for the initial construction of the QIM scales (see Section 12.3.1). In this study, the rate of change of the three regions of the FTIR spectra was similar to that found for shear resistance (Sánchez-Valencia et al. 2014), which was reported to be closely related to lipid hydrolysis (Herrero et al. 2005; Sánchez-Alonso et al. 2012a). 266 Trends in Fish Processing Technologies

0.20 v(CH) methylene and methyl groups Fingerprint region Ester linkage C = O stretch 2923 106 2 174 2 0.16 Phospholipid Lipid choline – vas(PO2) 108 8 CNC stretch 720 115 4 123 4 0.12 2853 ance 969 sorb

Ab 0.08 146 5 2954 821 926 137 7 FFAs 0.04 3012 C = O stretch 165 6

0.00 3500 3000 2500 2000 15001000 Wavenumber (cm–1)

FIGURE 12.2 Infrared spectrum (3500–650 cm−1 region) of lipids extracted from hake muscle.

3.0

2.5

ed 2.0 rv

1.5 d and obse 1.0 dicte Pre 0.5

0.0

020406080100 120140 160 Weeks

FIGURE 12.3 Observed (open symbols) and predicted (closed symbols) values for PC2 of the 1330–1127 cm−1 region of FTIR spectra as a function storage time for hake fillets stored at −10°C (square), −20°C (triangle), −30°C (circle), and −80°C (diamond). Quality and Quality Changes Assessment of Processed Fish 267

The modeling of these markers provides useful information of the time and temperature relationships that may help fish processors to establish more precise definition of quality specifications, to classify the product into different categories, or to define premium quality products. Also, following a similar experimental approach for other species or conditions, a more product-specific shelf life could be agreed upon among the fishery sector. The models proposed here could also be applied for inspection and authentication purposes. For example, they could be used to monitor the temperature exposure of fish lots along distribution and they could serve as an authentication tool when fraud or mislabeling occurs. Their actual implementation by the fish industry, however, would require validation with new batches of fish.

12.3.4 low-Field 1H Nuclear Magnetic Resonance Relaxometry Low-field 1H nuclear magnetic resonance relaxometry (LF NMR) can provide valuable information about relaxation and diffusion behavior and can be used for noninvasive determination of water and fat contents. The assessment of proton relaxation behavior can be either longitudinal (spin–lattice, or T1) relaxation or transversal (spin–spin, or T2) relaxation. There are more studies on fish using the T2 transverse relaxation times, in part because they are faster to measure than T1. The applications of LF NMR relaxometry in fishery products have increased during the last years, and it can be considered as an important analytical method to evaluate raw material characteristics, including fat and water content, water content and distribution, seasonal variation in water distribution, or water holding capacity, as well as the effect of postmortem events such as preslaughter handling stress, rigor mortis development, or different processing and storage factors (salting, desalting, dry salting, rehydration, smoking, superchilling, pressure treatment, freezing rates, and time and temperature of storage) on product characteristics (Erikson et al. 2012; Kirtil and Oztop 2016). In model systems with proteins and other biopolymers, as well as in muscle, it has been shown that the transverse relaxation times (T2) can provide information about the physical–chemical nature and spatial organization of muscle components (Belton 2011; Erikson et al. 2012).

12.3.4.1 Time–Temperature Modeling in Frozen Hake Using an approach similar to that of FTIR spectroscopic analysis of the lipid fraction of hake muscle, the potential of LF NMR relaxometry to produce indicators to estimate the quality and shelf life of frozen hake (Merluccius merluccius L.) muscle as affected by storage temperature and time was explored. Previous analysis on hake muscle stored at a fixed temperature (i.e., −10°C) showed that T2 relaxation times provided information (Sánchez-Alonso et al. 2012b), which was correlated with storage time and other parameters such as water-holding capacity and shear resistance. LF NMR has also provided useful information on changes observed in the muscle as affected by freezing rate and their interactions with final freezing temperature and storage time (Sánchez-Alonso et al. 2014). Based on the fact that the results of that study suggested that the output of LF NMR might be useful for kinetic modeling, LF NMR T2 transverse relaxation time measurements were performed on fillets stored at −10°C, −20°C, and −30°C for up to 150 weeks (Sánchez-Valencia et al. 268 Trends in Fish Processing Technologies

2015). The PC scores from the LF NMR distribution of T2 relaxation times followed an Arrhenius-type pattern, and using the PC scores of the 12–400 ms range, corresponding to both T21 and T22 regions, resulted in the best model. The temperature dependency of the rate of reactions was similar to that previously found for shear resistance and for the hydrolysis of phospholipids (Sánchez-Valencia et al. 2014; Careche et al. 2015, Section 12.3.3.1), well-known markers related to the sensory quality in this species. Thus, results from those studies suggest the potential of these methodologies to evaluate the frozen storage time and temperature in hake as well as to provide an indication of the expected quality of the fillet. The method would share the advantages of FTIR of the lipid fraction and in addition provides faster sample preparation and it is less time-consuming since measurements are directly done on pieces of fillets.

12.3.5 Dielectric Spectroscopy The deteriorative biochemical and microbiological processes that take place during storage affect the dielectric properties of the material in the microwave frequency region. The shape of the spectrum as a function of frequency depends not only on the dielectric properties of liquid water but also on the effects of other constituents on the water (e.g., rotational hindering and ionic conduction effects) (Kent et al. 2002). With this methodology, the complex dielectric properties of the fish as a function of frequency were studied and subsequent transformation of these spectra using the method of PC analysis was performed (e.g., measurements of added water). A simplification for the method consisting of performing measurements in the time domain (time domain reflectrometry [TDR]), rather than in the frequency domain, has led to the construction of a handheld dielectric instrument and, together with multivariate data processing, has been used to predict the storage time or the QIM score of cod stored in ice, producing reasonable estimates of this last factor (Kent et al. 2004a). In hake muscle, results of the prediction of days in ice by using TDR rendered slightly lower coefficients of calibration than for cod. However, the classical measurement of the nucleotide breakdown relationship, the K value, gave more consistent values regarding changes in fish freshness, with a highly linear response with time (Tejada et al. 2007). Dielectric spectroscopy has also been used for the evaluation of frozen fish so that, by PC regression, good predictions of time and temperature of storage were obtained for cod fillets stored in the range of −10°C to −30°C. Sensory variables also showed good relationship with the dielectric method (Kent et al. 2004b). In frozen hake stored at different freezing temperatures, a good relationship of time of storage was obtained as well as with sensory analysis of cooked samples. Nevertheless, results were highly dependent on the batch so that they should be classified first into a given category before predicting storage time or sensory analysis (Kent et al. 2005). The origin for the modifications of the signal has not been studied in detail for this system. Nevertheless, the results shown here suggest that dielectric spectroscopy is a promising technique for evaluation of some important process and storage parameters, which share much of the advantages of other spectroscopies mentioned in this chapter. Quality and Quality Changes Assessment of Processed Fish 269

12.4 OPPORTUNITIES AND CHALLENGES The needs and expectations of the consumers have to be translated into specific product characteristics or features for increasing or maintaining perceived quality, or for developing new products, modifying formulations or processing steps, which in turn are aimed to get benefits (nutritional, sensorial, economic, etc.) from seafood purchase and consumption. On the other hand, the complex nature of fish trade and the high variety of seafood, with different production methods, many species, processing, storage conditions, etc., give rise to a broad range of marketed products, but they also are a potential source of problems that need to be addressed by the seafood processing chain and regulatory authorities. Therefore, in all these scenarios, objective product characteristics or performances need to be assessed at some points and constitute an opportunity for the applications of new assessment methods. In fact, the fisheries sector itself has expressed their interest for new methodology and instru- mentation to measure the quality of fish (Jorgensen et al. 2003). Nowadays, there are solutions for problems that can be applied for specific situations. Some of them are cost-effective and others are even available at zero cost (e.g., QIM tables on the internet or applications in smartphones). In cases where the technology developed for applications on fish is ready to use, the uptake of the existing methods is not always as general as it might be expected. In those situations, stimulation of campaigns by authorities for the use of specific methods (e.g., QIM) should be pursued. Other methods need further development for a broad use; they may be still expensive or not ready to being sufficiently versatile, easy to operate or perform, or too lengthy. Many of the procedures described in the literature are in the proof of concept phase and would need to be developed more thoroughly and validated. Studies where the temperature dependence of the changes along time is monitored by either sensory or instrumental methods should be further developed, together with kinetic modeling or chemometrics, so that the methods have time–temperature integration function. Many of the methodologies (i.e., spectroscopic) could be equally adequate for similar purposes (e.g., estimating changes due to time and temperature of storage), but it is difficult to compare them due to the differences in the experimental designs among the published works. The problem of the initial conditions of the fish affecting the estimation of some parameters (e.g., storage time) calls for a more thorough understanding on their effect on the chemical, physical, biochemical, and microbiological changes and mechanisms. This will lead to the identification of new markers and, above all, relate all the existing ones so that more precise models can be constructed. Despite the fact that direct measurements of objective quality characteristics are very important, and sometimes mandatory, and that the need for new developments is generally acknowledged, the different actors across the fish chain should see the benefits of the new approaches as compared to their in-house evaluating ones. According to Peri (2006), it is very difficult to rank the requirements of quality in terms of importance, and in this context, in addition to the perceived technical problems of the developed methodologies as such, it is possible that the assessment of objective quality by new procedures is envisioned as one small part of the many other dimensions inherent to the quality concept. The uptake of these technologies 270 Trends in Fish Processing Technologies needs to be integrated with the rest of the components and actors of quality and adapted to wider contexts.

12.5 FUTURE TRENDS AND EXPECTATIONS It is expected that new technologies will be developed in engineering, information technology, and communications that will have an impact on the development of methods for objective quality assessment. The societal demands as well as the needs from industry will lead to tailor-made applications for specific problems. On the other hand, use of instruments applicable not only for fish but also to a wide variety of food commodities may be particularly important for the development of instruments at a reasonable cost. More user-friendly applications for smartphones (e.g., IR sensor) or multisensors, able to provide a more comprehensive and integrated view of all the objective quality dimensions, may be among the future developments. Nondestructive and noninvasive, fast methods with high analytical and, if possible, structural power would greatly benefit not only the industry but also the scientific community. Such methods would allow generating better models, which would account for the effect of the initial characteristics of the fish on the changes during processing and storage, which in turn would lead to a new generation of more robust methodologies as well as a deeper knowledge of the underlying mechanisms of deterioration of fish.

12.6 CONCLUSION There is a need for methodologies to provide objective indicators related to some aspects of the quality of fish of use in all the steps in the processing chain. In this chapter, some sensory (QIM) and instrumental (multisensors or spectroscopic) methods were described. Some of them are ready to use for specific situations and others require further development for a wider application.

ACKNOWLEDGMENTS Thanks are due to Professor Iciar Martinez for review of this manuscript. This work has been financed by the Spanish project ANIRISK (AGL2015-68248-C2) MINECO/FEDER.

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Mette S.R. Fachmann and Jeffrey Hoorfar

CONTENTS 13.1 Introduction...... 277 13.2 Methods in Safety Assessment of Fishery Products...... 281 13.3 Molecular Analysis of Quality Attributes in Fish...... 283 13.4 Molecular Tools for Testing of Authenticity of Fish...... 285 13.5 Future Perspectives...... 286 References...... 286

13.1 INTRODUCTION Integrity as applied in the context of food chains covers not only the safety and quality of food products but also their authenticity and traceability. It is important that consumers can trust that the food product is safe and has not been adulterated. This is especially true for seafood, where products are traded globally and fraud occurs regularly. In recent years, consumption of seafood has increased. This increase of consumption, related to the perception of health benefits, has, however, made seafood a leading cause of food poisoning (BCC Research 2014). In a recent report by Centers for Disease Control and Prevention (CDC), fish was the most commonly implicated food category, causing 21% of the recorded outbreaks in United States in 2014 (CDC 2016). Consumer demand for fish and seafood means that the food safety testing market value is expected to increase by 5.2% each year (estimated for 2014– 2019) and testing costs for fish/seafood are estimated to increase to $986 million by 2019 (BCC Research 2014). Seafood-related food poisoning can be caused by toxic residues or fish-borne pathogens. The latter include bacterial pathogens (listed in Table 13.1), viruses, and parasitical agents. Bacterial pathogens are the most important hazards, as 53% of foodborne outbreaks in 2014 with confirmed single etiologic agent were caused by bacteria (CDC 2016) and testing costs for bacterial pathogens account for around 89% of total testing market value (BCC Research 2014). Although fish-borne pathogens are a major concern, spoilage microorganisms (listed in Table 13.2) are of great importance. Spoilage microorganisms are responsible for the production of biogenic amines, e.g., scombroid toxin (histamine), that in 2014 was associated with 16 fish-borne outbreaks in the United States (CDC 2016). Other metabolites produced by spoilage microorganisms include volatile

277 278 Trends in Fish Processing Technologies Reference and Papadopoulou 2014; and Papadopoulou et al. 2010; Iwamoto et al. 2015 Novoslavskij 2010; Zhong et al. 2016 and Berthe 2013; Ryder, Ababouch 2014 Iddya, and 2010; Postollec et al. 2011; et al. 2010 Velusamy et al. 2015; Novoslavskij et al. 2010 Velusamy Kim, Labbe, and Ryu 2000; Postollec et al. 2011; Ryder, Ababouch 2014 Iddya, and Postollec et al. 2011; Ryder, Ababouch 2014 Iddya, and Arvanitoyannis, Kotsanopoulos, Kotsanopoulos, Arvanitoyannis, et al. Austin 2010; Iwamoto Grim 2013; Haenen, Evans, et al. 2010; Mayr Iwamoto Lambertz et al. 2000; et al. 2010; 2011; Iwamoto FDA et al. 2015; Novoslavskij Test Method Test of bacteria biosensor real-time PCR or biosensor or biosensor PCR for detection ELISA for toxin or PCR, LAMP, PCR Enrichment + PCR Enrichment + qPCR Enrichment + qPCR Products fermented, or salted fish products originating from the Baltic Sea seafood raw seafood and MAP raw products seafood raw products products Vacuum-packed, Vacuum-packed, or Improperly cooked or Improperly cooked or Improperly cooked food Vacuum-packed and handled fish Cooked RTE fish and Smoked Hazard storage and toxin production disease gastrointestinal disease gastrointestinal storage, leading to disease gastrointestinal improperly handled food storage leading to disease gastrointestinal illness or invasive Growth during cold Growth infection and Wound infection and Wound Gastrointestinal disease during cold Growth produced in Toxin during cold Growth microbiota microbiota fish (freshwater) microbiota contamination contamination Reservoir/Source Indigenous fish Indigenous fish Indigenous Fecal contamination Fecal contamination Processing Processing MAP, modified atmosphere packing; RTE, ready-to-eat. modified atmosphere packing; MAP, and ( hydrophila) Plesiomonas shigelloides spp. Salmonella , Shigella and E. coli , and Bacillus cereus Clostridium perfringens TABLE 13.1 Examples Important of Pathogens Bacterial Fish in and Fishery Products Pathogen Clostridium botulinum spp. Vibrio spp. Aeromonas Campylobacter , spp. Yersinia , Staphylococcus aureus Listeria monocytogenes Note: Molecular Methods for Assessment of Fish and Fish Product Integrity 279 ( Continued ) Reference Podeur et al. 2015; Ryder, Iddya, Podeur et al. 2015; Ryder, and Ababouch 2014 2000; Mamlouk et al. 2012; Ababouch 2014 Iddya, and Ryder, Macé et al. 2013; Gram and Huss, 2002; Jørgensen, Dalgaard 2000 and Dalgaard Berthe 2013; Ryder, Iddya, and Berthe 2013; Ryder, Ababouch 2014 Cheng and Sun 2015 Iddya, and Ababouch 2014 Iddya, and 2008; Mamlouk et al. 2012 Björnsdóttir-Butler et al. 2010; Björnsdóttir-Butler Cheng and Sun 2015; Dalgaard Cheng and Sun 2015; Dalgaard Björnsdóttir-Butler et al. 2010; Björnsdóttir-Butler Grim 2013; Haenen, Evans, and Grim 2013; Haenen, Evans, Ryder, Iddya, and Ababouch 2014; Iddya, and Ryder, Gram and Dalgaard 2002; Ryder, 2002; Ryder, Gram and Dalgaard Lyhs 2009; Mamlouk et al. 2012 Lyhs Dalgaard 2000; Lyhs and Björkroth and Lyhs 2000; Dalgaard Cheng and Sun 2015 a Test Method Test hybridization spectroscopy hybridization hyperspectral imaging hyperspectral hyperspectral imaging hyperspectral qPCR or colony lift qPCR or colony qPCR, FTIR qPCR or colony lift qPCR or colony PCR FTIR spectroscopy, FTIR spectroscopy, PCR qPCR qPCR FTIR spectroscopy, FTIR spectroscopy, Products (e.g., tuna and mackerel) cooked shrimp in MAP cooked and MAP products fermented fish semipreserved marinated, semipreserved and fermented fish vacuum or MAP vacuum High histidine fish species Cold-smoked fish and Cold-smoked Fresh and cold-smoked fish Fresh and cold-smoked Fresh fish Fresh chilled fish Fresh chilled fish Lightly salted and Cold-smoked, lightly salted,Cold-smoked, Processed products in Metabolites Major Spoilage hydrogen sulfide hydrogen other biogenic amines sulfide sulphur compounds sulfide tyramine, acetoin, diacetyl, and sulphur compounds sulphur compounds Histamine Acetoin, diacetyl, and TMA, histamine, and TMA and hydrogen TMA and hydrogen Ammonia, esters, and TMA and hydrogen TMA and hydrogen N/A Acetic acid, ammonia, Biogenic amines and environmental environmental contamination contamination fish microbiota microbiota microbiota fish microbiota microbiota microbiota Reservoir/Source Fecal/ Process Indigenous marine Indigenous fish Indigenous fish Indigenous marine Indigenous fish Indigenous fish N/A b bacteria spp. and Proteus thermospacta spp. Lactic acid spp., Morganella Raoultella spp., Brochothrix Brochothrix Photobacterium spp. Aeromonas Pseudomonas spp. spp. Shewanella TABLE 13.2 Examples Important of Organisms Spoilage Microbial Fish and of Fishery Products Spoilage Organism Lactococcus Lactobacillus spp. 280 Trends in Fish Processing Technologies Reference 2000; Podeur et al. 2015; Ryder, 2000; Podeur et al. 2015; Ryder, Ababouch 2014 Iddya, and Jørgensen, Huss, and Dalgaard Jørgensen, Iddya, and 2000; Ryder, Ababouch 2014 2000 Mamlouk et al. 2012 Björnsdóttir-Butler et al. 2010 Björnsdóttir-Butler Jørgensen, Huss, and Dalgaard Jørgensen, Banerjee and Bhunia 2010; Cheng and Sun 2015; Dalgaard Dalgaard 2000; Lyhs 2009 2000; Lyhs Dalgaard Dalgaard 2000; Lyhs 2009 2000; Lyhs Dalgaard Dalgaard 2000; Lyhs 2009; 2000; Lyhs Dalgaard a Test Method Test hybridization hyperspectral imaging hyperspectral PCR or colony lift PCR or colony PCR Biosensor FTIR spectroscopy, FTIR spectroscopy, N/A N/A qPCR Products in vacuum products incl. fish roe fermented fish and cooked shrimp in MAP and cooked Cold-smoked fish stored Cold-smoked Cold-smoked fish Cold-smoked Cold-smoked fish Cold-smoked Brined, gravad, and smoked Brined, gravad, Gravad fish Gravad Fresh, lightly salted and Hot smoked and gravad fish and gravad Hot smoked Metabolites Major Spoilage compounds tyramine, acetoin, diacetyl, and sulphur compounds Histamine Histamine and cadaverine TMA and cadaverine TMA, histamine, sulphur Acetic acid, ammonia, Diacetyl, tyramine Tyramine microbiota contamination Reservoir/Source N/A N/A Indigenous fish Fecal/environmental Fecal/environmental N/A N/A N/A Dominant spoilage organism and spoilage metabolites depend on products and storage conditions. FTIR, Fourier transformed infrared; MAP, modified atmosphere transformed infrared; MAP, and spoilage metabolites depend on products storage conditions. FTIR, Fourier Dominant spoilage organism TMA, trimethylamine. packing; N/A, not available; 16S rDNA-based methods are often used for analysis of entire microbial communities. 16S rDNA-based of pathogenic bacteria, e.g., Listeria . to suppress growth This group of bacteria has been shown liquefaciens Note: spp. Enterobacter Hafnia alvei Serratia Serratia Enterobacteriacea Leuconostoc spp. Enterococcus Carnobacterium TABLE 13.2 (CONTINUED) 13.2 TABLE Examples Important of Organisms Spoilage Microbial Fish and of Fishery Products Spoilage Organism a b Molecular Methods for Assessment of Fish and Fish Product Integrity 281 compounds, e.g., trimethylamine, which result in off-odors and flavors that decrease the quality of fish products. Spoilage metabolites, as well as other quality attributes like color and moisture level, can be determined by molecular techniques. Another important issue, which relates to both health and quality of fish and fishery products, is the authenticity of the fish, both in origin and species, as fraud can have adverse effects for both consumers and fish stocks. Fortunately, fraud with fish products can be monitored by molecular analysis. This chapter highlights some of the molecular techniques and genes involved in safety, quality, and authenticity of fish and fishery products.

13.2 METHODS IN SAFETY ASSESSMENT OF FISHERY PRODUCTS The general trend in food safety is a shift from time-consuming culture-based methods to rapid molecular methods. For bacterial pathogens, culture coupled with molecular detection ensures that mostly viable pathogens are detected. For pathogens that are difficult to culture and for viral pathogens, molecular methods are key in assessing the safety of the fishery products. The detection limits of these methods can, however, be below the contamination level; thus, some form of concentrating sample preparation may be necessary. An example thereof is immunomagnetic separation (IMS). It is a separation method based on capture with antibody coated magnetic beads. While IMS is not at detection method by itself, it can be used for sample preparation for molecular detection to replace time-consuming selective enrichment (Corry et al. 2007). Other concentrating sample preparation methods include physiological, physical, or biochemical techniques, and the choice of sample preparation method depends on the detection method. The molecular detection methods can be roughly divided in two categories: immuno based and nucleic acid based. Immuno/antibody-based detection methods include the following:

• Enzyme-linked immunosorbent assay (ELISA) • Lateral flow assay (LFA) • Protein microarray • Biosensors

ELISA and other immunoassays rely on the specific binding between antigen and antibody. This binding can be coupled with a chromogenic catalyst or even a cellular cascade, resulting in detection. Immunoassays can be used not only to screen for parasitic pathogens and harmful residues (e.g., histamine, cyanotoxins, brevetoxins, and dioxin) but also for indirect detection of Vibrio cholera and other toxin-producing bacteria (Arvanitoyannis, Kotsanopoulos, and Papadopoulou 2014; Banerjee and Bhunia 2010). LFA is a form of immunochromatographic dipstick that can be used for similar purposes as ELISA and interpreted easily. It can be in a format similar to a home pregnancy test and is well suited for point-of-care diagnostics. It does, however, require a high amount of target and is therefore less suited for direct detection of pathogens found in low numbers (Corry et al. 2007; Dwivedi and Jaykus 2011). The specificity of antigen-antibody binding has also been the basis of a biosensing protein microarray designed for specific detection of bacterial pathogens such 282 Trends in Fish Processing Technologies as Salmonella and Escherichia coli (Bouguelia et al. 2013). With this chip-based microarray, surface plasmon resonance imaging allowed for real-time monitoring of specific bacterial growth. Other biosensors include antibody coated bioluminescent mammalian B-cells (Banerjee and Bhunia 2010; Velusamy et al. 2010). These are based on the interaction between the B-cells and the analyte and are able to distinguish pathogenic from nonpathogenic and active from inactive toxin, thus giving a more accurate estimation of the risk associated with the agents (Banerjee and Bhunia 2010). The immuno-based assays can be used to rapidly screen for the presence of pathogens/toxins, and some can be automated for high throughput. They are, however, less suitable for quantification of pathogens and nonspecific reactions can cause false positives (Bohaychuk et al. 2005). Nucleic-acid-based methods on the other hand are less prone to false-positives and some are available for quantification, as described next. Nucleic-acid-based detection methods include the following:

• Polymerase chain reaction (PCR), real-time, reverse transcriptase (for RNA- virus detection) • Digital PCR • Loop-mediated isothermal amplification (LAMP) • Lab-on-a-chip (microfluidic chips) • DNA microarrays • DNA fingerprinting and sequencing (16S-rDNA-based methods)

PCR is a favored molecular detection technique due to its high specificity and versatility. When coupled with fluorescent dyes, the detection can be real-time, and with the improvement of thermo-cyclers, the time of detection can be less than an hour (Huggett, O’Grady, and Bustin 2015). Through use of specific fluorescent probes, real-time PCR allows for detection of multiple targets simultaneously. Real-time PCR also allows for quantification, either by comparison to a reference gene (relative quantification) or a calibration curve (absolute quantification) (Pfaffl 2004). Another form of PCR that can be used for quantification is digital PCR (Huggett, O’Grady, and Bustin 2015). Digital PCR, also referred to as digital droplet PCR (ddPCR), involves partitioning of the sample into nanoliter droplets and has been shown to have higher detection rates than real-time PCR (Doi et al. 2015). The technique can be adapted for microfluidic chips as highlighted in a recent review (Zhang and Jiang 2016) and have been used for detection of norovirus in oysters (Polo et al. 2016). A major challenge with ddPCR is in obtaining the droplets, and thus, the technique has not yet been implemented for routine diagnostics (Zhang and Jiang 2016). LAMP and other isothermal amplification techniques are attractive for point-of- care diagnostics as they, in contrast to PCR, do not require large and expensive thermos-cyclers and results can be inspected by simple visual comparison. A specific LAMP coupled with propidium monoazide allowed for detection of as little as 14 copies/g of only viable Vibrio parahaemolyticus without interference from different seafood matrices (Zhong et al. 2016). Microfluidic chips can be used for both immunoassays and different nucleic acid assays. They are available for a wide range of pathogens found in fish products Molecular Methods for Assessment of Fish and Fish Product Integrity 283

(Safavieh et al. 2014). Often, both the sample preparation and detection can be integrated on the chip (Hellberg and Morrissey 2011). Although the cost of the chips will most likely become lower in the future, they are still costly and problems still exist with sensitivity due to the small amount of sample that can be loaded (Safavieh et al. 2014). DNA microarrays are chips spotted with oligonucleotide probes that allow monitoring of several DNA sequences simultaneously. This technique can either be used to screen for multiple pathogens in one assay or for typing of specific pathogens (Byrne et al. 2015). The hybridization principle from DNA microarrays has also been used to screen for histamine producing bacteria through coupling with colony lift (Björnsdóttir-Butler et al. 2010). For rapid identification and classification of bacteria by 16S rDNA, several strategies have been used and DNA microarrays are one. The most popular technique for studying seafood microbiota is currently 16S rDNA sequencing of bacterial isolates (Parlapani, Kormas, and Boziaris 2015). This technique can be coupled with a dena- turing gradient gel electrophoresis, which by itself gives a DNA fingerprint and is often used for studying dynamics in bacterial communities (Svanevik and Lunestad 2011).

13.3 MOLECULAR ANALYSIS OF QUALITY ATTRIBUTES IN FISH While traditionally assessed by sensory evaluation, sensory quality attributes like odor, texture, and appearance (color, size, shape) can be assessed by methods based on machinery/computer vision systems, electric noses, and electric tongues (Cheng et al. 2015). Specific molecular quality assessment methods include the following:

• Hyperspectral imaging • Electronic nose (EN) and tongue • Colorimetric spoilage sensor array • Nuclear magnetic resonance (NMR) • Chromatographic methods • Matrix-assisted laser desorption-ionization–time of flight mass spectrometry (MALDI–TOF MS)

Hyperspectral imaging is a rapid nondestructive method with potential for online screening of multiple factors. It can be used to detect spoilage (Cheng et al. 2016) and texture in relation to freshness (Khoshtaghaza et al. 2016) as well as freshness authentication (Sivertsen, Kimiya, and Heia 2011; Sone et al. 2012) and estimation of bacterial loads (Cheng and Sun 2015). Automatic parasite screening is also possible (Sivertsen et al. 2012). The method relies on imaging and spectroscopy coupled with multivariate analysis to provide spectral and spatial information (Sivertsen, Kimiya, and Heia 2011; Sone et al. 2012). Hyperspectral imaging is essentially an artificial eye that builds on the visual ins pection of fish. Sight is, however, not the only sense that has been artificially re-created, as also ENs and tongues have been described. ENs are gas sensors that detect volatile compounds produced by microbes or changes in food due to oxidation, and thus, 284 Trends in Fish Processing Technologies they can be used to monitor spoilage. The gas sensors are often based on metal oxide semiconductors (MOSs). The volatile gasses adsorb to the MOSs and causes changes of conductivity that can be measured. A recent study by Han et al. (2014) described an EN based on nine MOS gas sensors for detection of volatile compounds in fish. Gas sensors can, however, also be based on simple colorimetric reaction with indicative dyes as described by Morsy et al. (2016). The described colorimetric sensor array is based on reaction of volatile spoilage compounds with different indicative dyes and is noninvasive, user friendly, and low cost. It was suggested as a way to monitor the quality and safety of fish products in their packages (Morsy et al. 2016). Electronic tongues (ET) are electrochemical sensors that measure ionic composition changes or production of nonvolatile compounds in a liquid phase or on the sample itself (Di Natale and Ólafsdóttir 2009). ET can be used in combination with EN to assess fish freshness in a nondestructive manner (Han et al. 2014). NMR spectroscopy can be used to assess the fatty acid quality in whole fish, and to monitor qualitative and quantitative changes in fishery by-products (Cai et al. 2016; Shumilina et al. 2016). Since the fatty acid profile of fish muscle is species specific, the lipid analysis can also be used to discriminate farmed species from wild ones and to distinguish between fake and real salmon (Cai et al. 2016; Gribbestad, Aursand, and Martinez 2005). NMR can also be used to detect water pollutants in fish and fishery products (Wang et al. 2016). The advantages with NMR is that the method is high throughput, little sample preparation is needed and the results can be obtained directly and noninvasively (Cai et al. 2016). Different chromatographic techniques have been described for the analysis of fish quality, e.g., gas chromatography, ion pair chromatography, thin layer chromatography (TLC), and high-performance liquid chromatography (HPLC). Often, they are used to detect unwanted chemicals (e.g., dioxins and antibiotics) and heavy metals (e.g., mercury and arsenic), but they can also be used to detect spoilage metabolites. Liquid chromatography methods separate nonvolatile analytes. HPLC, which is based on analyte affinity for a mobile phase, is the most widely used method for detection of biogenic amines and it can be coupled with different forms of detectors, e.g., ultraviolet, fluorescent, and mass spectrometry (Evangelista et al. 2016). TLC is a technique that separates nonvolatiles through the adsorption to a sheet coated with a relevant matrix, e.g., glass with silica gel (Arvanitoyannis, Kotsanopoulos, and Papadopoulou 2014). The technique can be used for semiquantitative measurement of, e.g., biogenic amines. A recent study favored TLC over HPLC in histamine screening of fresh fish due to the inexpensive and simple setup (Arulkumar et al. 2016). MALDI–TOF MS can be used for several purposes. As mass spectrums are species and genus specific, this technique can be used to type both pathogenic and spoilage bacteria (Böhme et al. 2013). The technique is especially good for anaerobic, fastidious, and slow-growing bacteria where traditional identification methods are time consuming, complicated, and expensive, e.g., Clostridium spp. (Biswas and Rolain 2013). Furthermore, the ability to discriminate between species has also been used for authentication purposes to detect species substitutions (Siciliano, D’Esposito, and Mazzeo 2016). Molecular Methods for Assessment of Fish and Fish Product Integrity 285

13.4 MOLECULAR TOOLS FOR TESTING OF AUTHENTICITY OF FISH Species identification and tracking of geographical origin are important for several reasons. Tracking of allergens and bioaccumulative organic contaminants (e.g., dioxins and polychlorinated biphenyls) (Arvanitoyannis, Kotsanopoulos, and Papadopoulou 2014; Ryder, Iddya, and Ababouch 2014), some of which can be detected by the previously mentioned techniques for measuring quality attributes, are important especially for the consumers. However, mislabeling not only deceives consumers; it could also adversely affect estimates of stock size if it influences the reporting of catch data that are used in fisheries management (Ryder, Iddya, and Ababouch 2014). Thus, authentication of fish products is of great importance. As in food safety testing, the trend in authentication is to use DNA-based techniques. This is because DNA is less sensitive to degradation than proteins are, only small amounts of sample is needed for analysis, and DNA-based methods are more easily replicated and standardized (Martinsohn 2011). Examples of DNA-based methods used in authentication include the following:

• PCR with universal primers or species specific (and probes) • PCR coupled with restriction fragment length polymorphism (RFLP) or single strand conformation polymorphism analysis (SSCP) • PCR sequencing (DNA barcoding) and NGS • DNA microarrays

PCR can be utilized in a number of ways to authenticate fishery products. PCR with universal primers, which are often based on mitochondrial genes, can establish whether or not a specific product contains fish. However, for identification of the fish species, further characterization is needed. This could be done by RFLP, SSCP, or sequencing (Abdullah and Rehbein 2016). Although RFLP and SSCP are less informative than sequencing, these methods are also faster, simpler, and less expensive and can also reveal the number of species present in a sample (Rehbein 2009). Primers for species specific PCR are also available for most fish species, but it requires at least some knowledge on which type of fish is expected and careful primer and probe design as some genera can be hard to differentiate (Hellberg and Morrissey 2011). On the other hand, species-specific real-time PCR may be used to determine the ratio of a particular fish species in a mix (Luekasemsuk, Panvisavas, and Chaturongakul 2015). PCR sequencing relies on amplification of a specific region that is found in all fish species but can be used to differentiate between them. Mitochondrial genes (cytochrome b and cytochrome c oxidase subunit I) are good for differentiation between closely related species and there have been generated great amounts of data for these genes in databases like Genbank, FISH-BOL, and FishTrace (Abdullah and Rehbein 2016; Hellberg and Morrissey 2011), which greatly improves the traceability of fish products. Mitochondrial genes are also used in PCR-RFLP and DNA microarrays 286 Trends in Fish Processing Technologies

(Hellberg and Morrissey 2011). They are, however, not suited for quantitation of tissue or differentiation between hybrids and maternal species (Rehbein 2009). Other genes used in species identification in fishery products are introns and exons of protein coding genes, ribosomal RNA genes, internal transcribed spacer of ribosomal genes, and microsatellites (Rehbein 2009). Microsatellites (also called short-tandem repeats) are mainly used for characterization of populations but can also be used for species identification (Behrmann et al. 2015). With regard to authentication, microsatellites can be used to trace the origin of both farmed and wild fish (Yue et al. 2012). A specific case study on mislabeling of cod used microsatellites to distinguish between the Eastern Baltic cod and cod from the North Sea and North Atlantic, where the former is of inferior quality (Nielsen 2012).

13.5 FUTURE PERSPECTIVES DNA-based methods are increasingly preferred in both safety and authenticity assessment of fish and fishery products. These methods are, however, currently associated with expert laboratories as analysis requires both advanced equipment and skilled users. In the future, we may see a shift from laboratory testing to on-site and even real-time testing. Portable PCR equipment is already available and is a promising tool for use in the detection of fish fraud, but it may also be possible to combine aspects of quality, safety, and authenticity. This was exemplified in a recent study where both the species and freshness of fish were assessed using an EN (Güney and Atasoy 2015). When it comes to evaluation of quality, safety, and authenticity, traceability is important, but so are validation and standardization of methods. Many methods are published but not validated. Validation of automated, real-time and on-site methods is essential to obtain faster release of low risk fish products in acceptable sensory quality, and to more easily detect fish fraud.

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Marjolein van der Spiegel and Joop van der Roest

CONTENTS 14.1 Traceability in the Fish Supply Chain...... 291 14.2 Fish Mislabeling...... 292 14.3 Species and Origin Identification...... 293 14.4 Locations of Fish Substitution in the Flatfish Supply Chain...... 296 14.5 Sampling to Monitor Fish Substitution throughout the Flatfish Supply Chain...... 297 14.6 Conclusion and Future Perspective...... 299 Acknowledgment...... 299 References...... 299

14.1 TRACEABILITY IN THE FISH SUPPLY CHAIN Fish are among the most internationally traded food commodities. However, due to the globalization of markets, illegal species substitution is becoming an important concern (Hellberg and Morrissey 2011, Martinsohn et al. 2011, Toldrá et al. 2013), which may have economical, health, and environmental consequences. Tracking and surveillance throughout the food supply chain is important to demonstrate that fish products derive from sustainably exploited stocks and to assure consumers that a product has been produced according to defined environmental standards (Brécard et al. 2009, Martinsohn et al. 2011) and fulfills the declared authenticity. Currently, traceability in the fish sector is mainly assured by documentation and labeling of products, as obliged by European Union (EU) legislation (see Table 14.1). Furthermore, several organizations assess fish species on sustainability via audits, like the Marine Stewardship Counsel (MSC), or inform via a traffic light system about catch area and method, fish species, and production method (wild/farmed), like the Dutch VISwijzer (Good-Fish-Foundation 2016), Seafood Choices of the United States (GreenBiz 2010), and the Southern African WWF-SASSI (SASSI 2014).

291 292 Trends in Fish Processing Technologies

TABLE 14.1 EU Legislation on Traceability in Fish Supply Chains EU Legislation Requirements Regulation (EC) Fisheries and aquaculture products on the market in the community shall be No. 1224/2009 adequately labeled to ensure traceability. Regulation (EC) All food destined for human consumption must be traceable through all stages No. 178/2002 of production, processing, and distribution. Regulation (EC) Appropriate species traceability and labeling of fishery and seafood products, No. 2065/2001 with a particular care for the scientific name of the species. • Fresh fishery and aquaculture products offered for retail sale must be labeled with an approved commercial designation, the production method used, and the area in which the fish were caught. • EU Member States are responsible for publishing a list of the common names that are acceptable for each species of commercially available fishery product in their territory. Directive 2000/13/EC Seafood must not be sold under a name that could mislead the purchaser as to its identity Regulation (EC) • To prevent, deter, and eliminate illegal, unreported, and unregulated No. 1005/2008 fishing. • Includes a commitment to improve product traceability. • Fishery products can only be imported into the EU when accompanied by a catch certificate. • The certification scheme aims to ensure product traceability at all stages of production, from catch to processing, and transport to marketing, and to be a tool to enhance compliance with conservation and management rules.

14.2 FISH MISLABELING Although the traceability of fish products is regulated, processed fish products are sometimes intentionally or mistakenly mislabeled (Marko et al. 2004, Sanjuan and Comesaña 2002). Commercial frauds can be related to taxonomic problems. For example, a common vernacular name assigned to a specific species can correspond to different taxa, like palombo that can be sold in Italy both as Mustelus mustelus and Mustelus asterias. Furthermore, the same species in different regions can be identified with different vernacular names (Galimberti et al. 2013). For instance, Mugil cephalus has a variety of common names, like striped mullet, black mullet, and sea mullet. Changes in taxonomy also result in mislabeling. The common dab was first named Pleuronectes limanda, but it has also been adjusted to other genera, including Liopsetta, and it is now known as Limanda limanda. Therefore, common dab is sometimes incorrectly called plaice due to the former species name Pleuronectus. To avoid frauds and mislabeling, Galimberti et al. (2013) suggests that the vernacular name should be written together with the correct scientific name and the reference to the DNA barcode sequence. Intentionally mislabeling is often due to substitution of cheaper species with more expensive, popular species (Rasmussen and Morrissey 2009, Sanjuan and Comesaña 2002) or commercial species with endangered or vulnerable species (Filonzi et al. 2010, Hellberg and Morrissey 2011). Fish substitution Adulteration and Misbranding of Fish Products 293 can cause health problems through consumption of species from contaminated areas (Van Leeuwen et al. 2009) or different standards for pathogens, heavy metals, or sanitary controls of farming sites (Filonzi et al. 2010, Hellberg and Morrissey 2011). Mislabeling of fish species occurs at a global scale (Galimberti et al. 2013, Marko et al. 2004, Miller et al. 2012). For example, Oceana found that 33% (401 out of 1215) of seafood samples from retail outlets in the United States were nationwide mislabeled (Warner et al. 2013), whereas Marko et al. (2004) even reported that between 60% and 94% of fish sold as red snapper in the United States did not comply with their label. Filonzi et al. (2010) found that 32% (22 out of 69) of analyzed samples in Italy revealed an incorrect fish species declaration (Filonzi et al. 2010). In Ireland, 25% (39 out of 156) cod and haddock products in supermarkets, fishmongers’ shops, and take-away restaurants were mislabeled (Miller and Mariani 2010). Espiñeira et al. (2008) found that 43% (13 out of 30) of flatfish samples were incorrectly labeled. The number of mistakes was higher in processed samples (69%) than in whole fish (31%). The species with the highest rate of mislabeling was the European plaice (Pleuronectes platessa) (Espiñeira et al. 2008). European plaice is the most commercially important flatfish in European waters, especially at the frozen fillets market (Caldelli et al. 2014). Espiñeira et al. (2008) found that 17% of the European plaice labels were incorrect and that the species present in the commercial product concerned flounder Platichthys( flesus) in the case of whole fish and American plaice and flathead flounderHippoglossoides ( spp.) in the case of frozen fillets. These results correspond with another study that showed that 37% (10 out of 27) of plaice samples turned out not to be P. platessa as declared on the label due to substitution by making packages with fillets all different from plaice and partial substitutions with packages with mixed fillet of plaice and other species (Berrini et al. 2009). Several studies reported that European plaice (P. platessa) is quite frequently substituted with other flatfish with less economical or nutritional values (Caldelli et al. 2014, Sotelo et al. 2001, Tepedino et al. 2001), for example, with yellowfin sole (Limanda aspera), dab (Limanda limanda), (Northern) rocksole (Lepidopsetta polyxystra, Lepidopsetta bilineata), and flounder P.( flesus).

14.3 SPECIES AND ORIGIN IDENTIFICATION The frequent substitution of flatfish shows the need for identification of the species. This identification of flatfish is of interest to consumers as well as the fish industry that buys the raw fish for use in the production of convenience products (Sotelo et al. 2001). Fish species identification is traditionally based on external morphological features, like body shape and pattern of colors (Van Emmerik 2007). When these external morphological features have been removed and the products have been processed (frozen, breaded, filleted), species identification is complicated without alternative identification methods (Caldelli et al. 2014, Hellberg and Morrissey 2011, Tognoli et al. 2011, Toldrá et al. 2013, Wong and Hanner 2008). Therefore, analytical methods for species identification and authenticity testing are necessary (Filonzi et al. 2010). The identification can be focused on genetic or compositional features of fish, or a combination of both, to determine the species, geographical origin, and/or production method. 294 Trends in Fish Processing Technologies

With respect to the identification of European plaice from other flatfishes, a -num ber of analytical methods have been used for species and origin identification of (see Table 14.2). These methods include spectroscopic techniques, enzyme-linked immunosorbent assays (ELISAs), molecular methods, and gas chromatography (GC). To date, DNA barcoding seems the most promising approach to solve taxonomic attri- bution in foiling food mystification (Caldelli et al. 2014, Filonzi et al. 2010, Griffiths et al. 2014, Kochzius et al. 2010). In addition to these methods, analysis methods for identification of fatty acid profiles (spectrometric methods like GC-MS, NMR spectrometry), volatile compounds profiles (PTR-MS), peptide profiles (LC-MS/MS), and isotope ratios (IRMS), may also be useful. However, these have not been applied to European plaice. Emerging methods are real-time polymerase chain reaction, microarray technology, and Next Generation Sequencing (NGS) (Hellberg and Morrissey 2011, Kochzius et al. 2010). The application of NGS in flatfish genomics research has just started (Cerdà and Manchado 2013). The rapid progress of DNA analysis technologies will initiate the development of new traceability tools. New sequencing technologies have declined in costs, whereas the speed and quality of analysis have increased. These methods are on lab-scale; currently, no cost- effective handheld analytical device for fish traceability is available (Martinsohn et al. 2011).

TABLE 14.2 Overview of Analysis Methods Used in Other Studies to Differentiate European Plaice (Pleuronectes platessa) from Other Flatfishes Type of Analysis Method Analysis Method Reference Spectroscopic MALDI-TOF Mass Spectrometry Mazzeo et al. 2008 techniques ELISA Indirect ELISA Céspedes et al. 1999a Molecular methods Isoelectric focusing (IEF) Bossier and Cooreman 2000, Tepedino et al. 2001 Polymerase Chain Reaction (PCR) Espiñeira et al. 2008, Tognoli et al. 2011 PCR-Restriction Fragment Length Caldelli et al. 2014, Céspedes et al. Polymorphism (PCR−RFLP) 1998a, 1998b, Dooley et al. 2005, Hold et al. 2001, Kijewska et al. 2009, Sanjuan and Comesaña 2002, Sotelo et al. 2001 PCR–Single Strand Conformational Céspedes et al. 1999b, Rehbein and Polymorphism (PCR–SSCP) Schiefenhövel 2012 DNA barcoding Filonzi et al. 2010, Kochzius et al. 2010 Pyrosequencing De Battisti et al. 2013 Real-time PCR Hird et al. 2012 DNA microarray Kochzius et al. 2010 Chromatography GC-FID Sirot et al. 2008 Adulteration and Misbranding of Fish Products 295

Each analysis method has specific advantages and disadvantages (see Table 14.3). The suitability of the analysis method depends on the aim of the sampling strategy, e.g., identification or verification of a species, proving origin (geographical area), or production process (farmed/wild). The sampling strategy could be used for periodic checks, selection of new suppliers, testing suspected batches or a particular supply chain, or verification as part of a quality label. Furthermore, the analysis method should be selected on cost-effectiveness, sensitivity, and the ability to analyze processed and/or mixed samples. The laboratory performing the analyses should be accredited for the methods offered.

TABLE 14.3 Advantages and Disadvantage of Analysis Methods to Identify Fish Species and Origin (Gigliarelli et al. 2013, Hellberg and Morrissey 2011, Sotelo et al. 2001, Teletchea 2009, Tognoli et al. 2011) Analysis Method Advantage Disadvantage Protein-based methods Generally reliable for testing fresh, Impractical in heavily processed or frozen, or lightly processed dried foods seafoods ELISA Use with heat-treated seafood Does not work well with closely products related species and requires development of species-specific antibodies IEF Refrigerated or frozen seafood Not for food products subjected to products thermal treatment, requires the use of authentic species protein extracts, which have to be analyzed together with the unknown samples DNA-based methods Compared to protein-based methods: Sample processing time and costs, higher information content, greater equipment and start-up costs, resistance to degradation, increased reproducibility, reliability, range of specificity and sensitivity, and target species, and ability to recover presence in all cell types and identify DNA from processed products, complex food matrices, and mixed-species samples PCR sequencing High reproducibility Costly and time-consuming, cannot be used for mixed-species samples PCR–RFLP High reproducibility, less costly than Vulnerable to errors from PCR sequencing, mixed-species intraspecies variation, provide not samples the high level of information acquired with PCR sequencing PCR-SSCP Do not require prior knowledge of Reduced reproducibility compared DNA sequence information with other methods 296 Trends in Fish Processing Technologies

14.4 LOCATIONS OF FISH SUBSTITUTION IN THE FLATFISH SUPPLY CHAIN Fish substitution can occur at any point along the fish supply chain (Everstine et al. 2013), from ocean to fork. The Dutch flatfish production chain consists of fisheries, fish auctions, fish processors, and customers, including fish producers, retailers, restaurants and catering (see Figure 14.1). Flatfish species like European plaice are caught in the North Sea. The fish is mostly sold through the fish auction to fish- fil leters and fish processors. Other internationally caught flatfish species are supplied indirectly via the fish auction or directly to the fish filleter or fish processor. Fish processors manufacture fish products through processes such as filleting, glazing, freezing, frying, and packaging. Most products are exported to fish producers, retailers, restaurants/catering in Scandinavia, Italy, United Kingdom, France, Austria, and Germany. Areas of highest risk in the supply chain should be addressed to decide on a sampling strategy. Fish substitutions may occur not only on the fishing vessel or at the dock because species that are morphologically similar are caught together but also at retailers due to mislabeling (Marko et al. 2004). However, at the fishing vessel and fish auction, flatfish species are still visually recognizable, and therefore, substitution can be corrected during sorting of the fishes. Jacquet and Pauly (2008) concluded that mislabeling most often occurs at the distribu- tor or the point of retail sale to increase profits and that the people responsible for catching or harvesting the fish do not typically benefit from the extra profit

Fish producer

Wholesaler Auctioneer

Fisherman Fish processor

Retailer Fish ﬁlleter/ﬁsh processor

Caterer, restaurant

FIGURE 14.1 Actors of the flatfish production chain. Adulteration and Misbranding of Fish Products 297 earned. In general, the critical points for substitution in the Dutch flatfish production chain include the following:

• Entrance inspection of processed or frozen fillets delivered by suppliers at fish processors, fish producers, wholesalers, and retailers; • Skinning of fishes at fish processors; • Relocation of products in production areas where more than one species are simultaneously processed; • Further processing at fish producers; • Repackaging and reuse of products after failures at fish processors, fish producers, wholesalers, and retailers; and • Labeling of fish products and documentation in the entire flatfish production chain.

14.5 SAMPLING TO MONITOR FISH SUBSTITUTION THROUGHOUT THE FLATFISH SUPPLY CHAIN The development of protocols for assurance and control of fish safety is currently a priority (Tognoli et al. 2011). To protect fish production from mistaken or intentional substitution, a sampling protocol for species and geographic origin assignment in the fish supply chain is required. All actors in the flatfish production chain have to monitor fish substitution throughout the flatfish supply chain to ensure that their raw materials, half fabricates, and/or processed products contain the labeled species and to test suspected incoming materials and/or products processed by customers. Samples have to be taken for investigating compliance of suspected batches, testing of new suppliers, monitoring as a routine control activity, and for surveillance as verification as part of a quality label. Each of these strategies has its own requirements for sampling:

1. Testing compliance of suspected batches and new suppliers Sometimes, incoming fish products or products processed by customers or competitors are suspected of noncompliance with their labels or documents. When fish products are suspected of noncompliance, the batch concerned can be sampled randomly to investigate the authenticity compared to the label or specification. In case of positive results, the supplier has to be informed and possibly a new test should be done as a second opinion. New suppliers have no history of compliance, and therefore, they should be monitored during a specific period. If no fish substitution is found, the frequency may be reduced. 2. Routine monitoring Routine monitoring can be integrated in the production system of an organization. The advantage is that the producer can be completely certain that products comply with the specifications. Currently, applied routine monitoring consists of visual inspections and using mass balances as required by MSC. For routine monitoring during fish production, the risk level of fish substitution in the flatfish production chain determines the 298 Trends in Fish Processing Technologies

frequency of sampling. Basically, every batch that enters the processor’s premises has to be sampled. However, in case of a supplier’s history of no fish substitution, it can be decided to only verify the batches periodically, e.g., once a month. However, after detecting adulteration, the sampling frequency has to be increased again. Higher risks require more frequent sampling, and lower risks, less frequent sampling. 3. Verification of authenticity or sustainability Assurance of authenticity or sustainability via a quality label or a quality assurance system may be used for guaranteeing nonsubstitution of fish species or origin. This strategy fits within the Global Food Safety Initiative (GFSI) food fraud vulnerability requirements to integrate fraud in GFSI acknowledged food safety management systems (e.g., British Retail Consortium, International Featured Standard–Food). These requirements include a documented food fraud vulnerability assessment and control plan. For ensuring authenticity or sustainability, actors in the flatfish production chain have to identify and document the potential locations along the supply chain where fish substitution may occur and what measures are taken to prevent and control substitution. All processes and activities have to be traceable in the entire chain. The documentation and implementation of such an assurance system have to be assessed by an independent auditor at least once a year. If this audit shows that a company does not comply with one or more aspects of the system, an extra inspection will be required. As part of the assurance, the authenticity of the fish products has to be randomly monitored and verified by independent laboratory analysis to demonstrate the compliance of the products. In case the results show noncompliance, corrective measures have to be taken, like informing the customer and, if necessary, a recall. Subsequently, the causes of substitution have to be investigated, and the sampling frequency has to be increased. Possibly, the inspection and analysis results can be combined with those of the MSC chain of custody standard for seafood traceability or a GFSI acknowledged food safety management system.

The Codex Alimentarius Commission (2004) provides general guidelines on sampling (CAC/GL 50-2004) to consider the sample size that represents the variability in one batch (FAO 2004). A variable sampling plan with unknown standard deviation can be used for sampling in the flatfish production chain, since the expected fish substitution is still unknown as periodic checks are commonly not performed due to the related costs. Based on this sampling plan, five fishes per 500 kg should be taken to randomly sample noncompliant fish species in a certain batch. In some cases, the optimal sample size and frequency may be not feasible in the flatfish production chain due to the costs of the analyses, although the cost-effectiveness of analyses may change in future due to new developments. Screening of a pooled sample can be considered if the selected analysis method allows this. After identification of positive samples, an identification step should be done afterward. The sample size for authentication identification of flatfishes is dependent on the ability of the analytical method to distinguish one fish species from another or one Adulteration and Misbranding of Fish Products 299 location of origin from another. The required sample size has to be larger for a method with a lower ability to distinguish between groups.

14.6 CONCLUSION AND FUTURE PERSPECTIVE Incidents of fish substitution have been reported worldwide, despite labeling and traceability regulations. The substitution of flatfish species is currently mainly controlled by documents and labeling and less frequently by using analytical methods in case of suspected batches. An appropriate and recognized sampling plan of substitution of species and origin throughout the flatfish production chain is needed in order to ensure the correct labeling of products, to protect endangered fish species, and to obtain confidence in product authenticity. The design of a sampling plan (location, sample size, frequency) depends on the purpose of the sampling strategy, including periodic checks as a routine control activity, selection of a new supplier, testing suspected batches or a particular supply chain, or verification as part of a quality label. In some cases, the optimal sample size and frequency may be not feasible due to the costs of the analyses, although these costs may decrease in the future due to new developments. These insights could also be applied in other sectors of aquaculture and fish processing. Future research should focus on collecting data on actual fish substitution in order to determine an optimal sample size, developing new analysis methods to identify species and origin in mixed samples, and combining the sampling plan with audits for a quality label, like the traceability certification by MSC, in order to save costs. Information sharing among government and the private sector across countries will be required for a faster worldwide response to incidents.

ACKNOWLEDGMENT The authors acknowledge the Ministry of Economic Affairs (EZ) of the Netherlands and the European Fisheries Fund for financial support of this study.

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Maitri Thakur

CONTENTS 15.1 Food Supply Chains...... 303 15.2 Traceability in Fish Supply Chains...... 303 15.3 Standardization of Information Exchange...... 304 15.4 Technologies for Information Exchange...... 304 15.5 Applications of Traceability...... 308 15.5.1 Cold Chain Monitoring...... 308 15.5.1.1 Temperature Sensors...... 308 15.5.1.2 Web-Based Monitoring...... 309 15.5.1.3 EPCIS Temperature Events...... 309 15.5.2 Food Supply Chain Management...... 310 15.5.3 Resource Efficiency...... 311 15.6 Conclusion and Future Perspectives...... 311 References...... 311

15.1 FOOD SUPPLY CHAINS Food supply chain management is a complicated task because of the intrinsic focus on product quality (Luning and Marcelis, 2006). Transportation and logistics are integral parts of food supply chains and the transportation segment has the greatest risk associated with it due to multiparty involvement, mishandling of information, and the length of the segment. These complexities can lead to spoilage, losses, and changes in the shelf life of food. Food safety and quality issues generally occur due to incorrect processing and handling of food products (Gustavsson et al., 2011). Monitoring the flow of products, their quality, and the process parameters throughout production and linking them to each transition state throughout the supply chain are effective ways of implementing and ensuring product safety and traceability.

15.2 TRACEABILITY IN FISH SUPPLY CHAINS One of the cornerstones of the General Food Law is also the requirement that food should be traceable. The terms “tracking” and “tracing” are commonly used to describe traceability. Tracking (forward) is the ability to follow the downstream path of a particular trade unit in the supply chain, while tracing (backward) is the ability to

303 304 Trends in Fish Processing Technologies identify the origin of the products used in a particular trade unit. Thus, tracking is a top–down approach and tracing is a bottom–up approach. Both tracking and tracing play a very important role in the overall supply chain traceability. According to Van Dorp (2002), tracking and tracing provide the visibility to the supply chain as these create a historical record by means of recorded identifications. A good traceability system should have the capability of performing both functions efficiently. Tracking can be done manually though paper-based record keeping systems, but adopting new technologies such as radio-frequency identification (RFID) for information exchange opens new opportunities and benefits more than one link in the food chain. Fish is a highly perishable product and requires efficient handling, logistics, and supply chain management procedures. Fish is essentially handled as a bulk product until it arrives at the production facility from the fishing vessels. Figure 15.1 shows a typical supply chain for mackerel in Norway including the flow of material and information. The production process at the producer/packer link in the chain is shown in detail. Traceability in fish supply chains is important for various reasons, including documentation of origin, food safety, regulatory compliance, etc. In addition to documentation of origin and chain of custody, it is important to maintain an efficient cold chain monitoring system to maintain the quality and safety of the fish products.

15.3 STANDARDIZATION OF INFORMATION EXCHANGE Globalization combined with the ever-increasing complexity of food supply chain networks has led to an increase in the significance of efficient systems for information exchange between food businesses. The effectiveness of information exchange is influenced by the lack of internal traceability systems and standardized way of information exchange. The absence of internal systems makes it impossible to con- nect the information related to incoming products to that of the outgoing products in any organization. Individual companies have made great progress in proprietary technologies for automated data capture and electronic data coding. However, the benefit of these is lost when the data element transmission is required for use outside the originating company as it is effective only when there is an identical software system at the receiving end (Donnelly et al., 2008). Studies have shown that there is currently no standardized way of formatting information for exchange in traceability systems. Research suggested that structured data lists, vocabularies, and ontology will be appropriate tools in achieving effective universal data exchange (Donnelly et al., 2009a, 2009b; Dreyer et al., 2004; TRACE 2, 2008). According to Folinas et al. (2006), standards must describe how information can be constructed, sent, and received and also how the data elements in the information should be identified, measured, interpreted, and stored. The traceability information needs to be captured in a precise, effective and electronic manner (FSA, 2002; Storøy et al., 2013).

15.4 TECHNOLOGIES FOR INFORMATION EXCHANGE Automated traceability is based on electronic data capture and exchange. Electronic data capture can be optical or radio-wave systems, for example, barcodes and Traceability of Fish Products 305 Customer customer Shipment to storag e Container Importer Processo r Cold storag e Transporter Refrigeration packer exporter Producer/ Packing Norwegian Grading Sales organization Pumping Flow of goods Flow of information Typical mackerel supply chain mackerel supply andTypical production process. Vessel Receiving

ﬁshing vessel FIGURE 15.1 306 Trends in Fish Processing Technologies

RFID technology. The interest in these systems for traceability has been increasing recently. RFID tags essentially contain Electronic Product Codes (EPCs) generation 2 (EPCglobal, 2015). Most of the research in this field presents traceability solutions where only the product packaging is tracked through the supply chains but fails to address the internal traceability issues linked to the production events within a food facility. Regattieri et al. (2007) presented the application of an RFID system integrated with alphanumeric code to trace Parmigiano Reggiano cheese through the complete supply chain. Shanahan et al. (2009) proposed the use of RFID for the identification of individual cattle and biometric identifiers for verification of cattle identity. They also proposed a data structure for RFID tags and a middleware software to convert animal identification data to the EPC data structure. EPC provides a method for unique identification of all items in a supply chain. The use of EPC also makes it possible to register internal and external events electronically that are related to the movement of tagged items. The standard for using RFID is based on EPC global standard—Electronic Product Code Information Services (EPCIS). EPCIS is an EPC global standard designed to enable EPC-related data sharing within and across enterprises (EPCIS Standard, 2016). Automated traceability systems have existed in various countries for several years, but EPCIS makes the data capture and exchange electronic, thus making EPCIS an applicable standard. There are two kinds of EPCIS data, event data and master data. Event data are created in the process of carrying out business processes and are captured through the EPCIS Capture Interface and made available for query through the EPCIS Query Interfaces. Master data are additional data that provide the necessary context for interpreting the event data. They are available for query through the EPCIS Query Control Interface. The EPCIS events cover normal logistic and stock control processes by the use of the Event classes: ObjectEvent, AggregationEvent, QuantityEvent, and TransactionEvent. The basic chain traceability requirements with respect to managing and recording transactions between different business actors are directly covered by EPCIS Events. EPCIS has promising properties related to food supply chain traceability (Thakur et al., 2011). The use of EPCIS and RFID is limited to tracking the product packages between stakeholders, but the additional food product transformations (or transitions) that include process and quality parameters are not covered under the basic EPCIS specification (EPCIS Standard). Before EPCIS can be implemented as a tool for food traceability information exchange, it is crucial to identify the specific transitions that take place internally at a food business operator in order to identify the critical points where temperature and other product information must be monitored. Figure 15.2 shows the states and events in the frozen mackerel production process. The numbers represent different events in the frozen mackerel production processes that show the transition of the raw material from one state to the next. Various products and product and quality data can be recorded and linked to each event and thus carried to the next state. Thakur and Forås (2015) presented an EPCIS-based real-time and online temperature monitoring system that could provide better control over the supply chain and food quality. Traceability of Fish Products 307 21 27 26 25 Get pallets Stor e 24 Shipping Transit out To external 23 28 22 17 Palleting Palleting grade B Palleting grade A Palleting grade C Unpacking 20 29 19 18 Pallet equipment ready Get frozen produc t 16 Refrigerating 12 15 Get boxes Packin g Packing grade A Packing grade B Packing grade C 11 14 13 9 10 t Packing machine ready Sortin g 8 7 Manual check Weight control Distribution to bel 6 5 4 3 Pump ready Vessel empty Product receiving 2 1 Transit in From external States in and frozen events mackerel production process. Forås, (From E., Hurburgh, Thakur, C.R., Sørensen, Bjørnson, M., C., F.O.,

., 103, 417–433, 2011.) 417–433, 103, ., FIGURE 15.2 Food EngJ. 308 Trends in Fish Processing Technologies

15.5 APPLICATIONS OF TRACEABILITY Among all drivers of traceability, food safety is the most important. However, there are several other applications of traceability that are of importance. In this section, three applications of traceability, including cold chain monitoring, food supply chain management, and resource efficiency, are discussed.

15.5.1 cold Chain Monitoring Temperature monitoring is the most critical requirement in the management of a cold product chain like a fish supply chain. Consumers expect that the food they buy in retail stores must be of good quality and adequate shelf life (Smith and Sparks, 2004). Fish needs to be stored and transported in a temperature-controlled environment to maintain quality. There are a number of difficulties in managing temperature- controlled food supply chain networks such as the short shelf life, which puts additional requirements on speed and reliability of logistics systems and requires specialized transportation and storage equipment. Furthermore, modern chains dis- tribute multiple types of products—often with multiple temperature regimes. This means that a “one size fits all” approach to temperature control may not be optimal for all types of products (Van der Vorst et al., 2007). Food degradation is related to environmental conditions (such as temperature and humidity), intrinsic properties (such as microbial contamination, and composition), and the time that food products are exposed to these conditions (Van der Vorst et al., 2007). Several studies have been conducted on food quality decay modeling and time–temperature impact of the quality throughout the distribution (Aung and Chang, 2014; Kim et al., 2015; Shi et al., 2010). The key focus in cold chain monitoring is to maintain the product quality and safety while maintaining the required product temperature during handling and processing.

15.5.1.1 Temperature Sensors EPCglobal Ultra-High-Frequency Class 1 Generation 2 semiactive RFID tags with integrated temperature sensors are shown in Figure 15.3. These sensors can be used

FIGURE 15.3 RFID-based temperature sensors with external and internal sensors, respectively. Traceability of Fish Products 309

FIGURE 15.4 Active RFID temperature sensor (left) and near-field communication temperature sensor (right). for both ambient temperature monitoring as well as the product temperature. For example, during transportation, the RFID tags with internal temperature sensors can be used to monitor the ambient temperature in the truck while the tags with external temperature probe can be used to measure the product core and surface temperature as well as the temperature inside the boxes containing the fish products. Figure 15.4 shows an active RFID temperature sensor and a near-field communication (NFC) temperature sensor. The NFC temperature sensor is the size of a credit card, does not require any special readers, and can be read using a mobile application.

15.5.1.2 Web-Based Monitoring Web-based monitoring systems can be used for real-time temperature monitoring during the transportation of fish products. The RFID tags with integrated temperature sensors can communicate with the main unit installed in the truck and transmit the data to an EPCIS-based system, which is accessible through a web interface. The temperature logs and the geo-location of the truck can be communicated through a Global Positioning System. Using the EPCIS framework, the temperature data are linked to a specific event with the parameters shown in Table 15.1.

15.5.1.3 EPCIS Temperature Events An EPCIS event is created every time temperature is read (based on the prede- termined frequency, e.g., temperature readings every 10 min) and linked to each uniquely identified tag. An example of an EPCIS-temperature event is shown in Table 15.1. The eventid refers to a unique temperature record and the EPC code identifies the RFID tag. Temp1 and Temp2 refer to the temperature measurements made by the internal and external temperature sensors, respectively. The current EPCIS standard requires modification to incorporate the temperature data in an efficient manner. So, the business step in the EPCIS vocabulary can be defined as temptracking and disposition as in_transit for logging of the temperature data in a fish supply 310 Trends in Fish Processing Technologies

TABLE 15.1 EPCIS—Temperature Event Structure Eventid 26234 Epc urn:epc:id:giai:0000000.121 eventTime 23. nov 2011 12:21 readPoint urn:hrafn:readpoint:licenseplate:SINTEF bizLocation urn:hrafn:readpoint:licenseplate:SINTEF BizStep urn:hrafn:temperature:bizstep:temptracking disposition urn:epcglobal:cbv:disposition:in_transit Temp1 0,3 Temp2 2,3 chain. An example of the XML code generated for an Object Event where temperature is recorded is shown in the following.

2011-11-23T12:21 urn:epc:id:giai:0000000.121 OBSERVE urn:hrafn:temperature:bizstep:temptracking urn:epcglobal:cbv:disposition:in_transit urn:hrafn:readpoint:licenseplate:SINTEF urn:hrafn:readpoint:licenseplate:SINTEF 0.3

15.5.2 F ood Supply Chain Management Several recent studies have argued that traceability systems can be used to improve supply chain performance. Bosona and Gebresenbet (2013) propose that food traceability should be considered as an important and integral part of logistic management in contemporary food supply chains. Ringsberg and Mirzabeiki (2014) studied Traceability of Fish Products 311 the effects of implementation of an EPCIS-based traceability system on the logistics operations in a cod supply chain. The study found that implementation of an EPCIS- based traceability system would potentially reduce the time required for capture and transfer of information between different processes as well as actors in the supply chain. According to Dabbene et al. (2014), together, new technologies for automatic identification and sensing such as RFID sensors and new computational and simulation models and of mechanical systems for the segregation of lots can pave the way for solutions for a higher level of supply chain control.

15.5.3 resource Efficiency Decision making in logistics and transportation planning of fresh food is characterized by product quality deterioration during transport in addition to the inherent properties of all supply chains in general. The amount of food wasted due to quality deterioration is particularly important. Aramyan et al. (2007) suggested that waste elimination is one of the performance indicators of food traceability systems, and Regattieri et al. (2007) suggested that RFID and traceability systems result in improved management of perishable items, and continuous monitoring of item routing reduces waste and improves customer service levels. Using real-time temperature data from a traceability system can provide control over the quality and food safety of food products, thus reducing food waste and improving resource efficiency (Thakur and Forås, 2015).

15.6 CONCLUSION AND FUTURE PERSPECTIVES Supply chains of perishable products such as fish require efficient management to maintain their quality. Traceability systems provide an effective tool for managing the fish supply chains and can also be used for cold chain monitoring and improving the resource efficiency in fish and other food supply chains. Future research should focus on dynamic shelf life estimation based on temperature data connected to individual items gathered through RFID-based temperature sensors. Supply chain wide implementation of traceability and information exchange systems still does not exist on most fish supply chains and should be a topic of further research.

REFERENCES Aramyan, L.H., Lansink, A.G.J.M., Van der Vorst, J.G.A.J., van Kooten, O., 2007. Performance measurement in agri-food supply chains: A case study. Supply Chain Management: An International Journal, 12 (4), pp. 304–315. Aung, M.M., Chang, Y.S., 2014. Temperature management for the quality assurance of a perishable food supply chain. Food Control, 40, pp. 198–207. Bosona, T., Gebresenbet, G., 2013. Food traceability as an integral part of logistics management in food and agricultural supply chain. Food Control, 33 (1), pp. 32–48. Dabbene, F., Gaya, P., Cristina, T., 2014. Traceability issues in food supply chain management: A review. Biosystems Engineering, 120, pp. 65–80. Donnelly, K.A.-M., Karlsen, K.M., Olsen, P., van der Roest, J., 2008. Creating standardized data lists for traceability—A study of honey processing. International Journal of Metadata, Semantics and Ontologies, 3 (4), pp. 283–291. 312 Trends in Fish Processing Technologies

Donnelly, K.A.-M., van der Roest, J., Höskuldsson, S.T., Olsen, P., Karlsen, K.M., 2009a. Improving information exchange in the chicken processing sector using standardised data lists. Communications in Computer and Information Science, 46, pp. 312–321. Donnelly, K.A.-M., Karlsen, K.M., Olsen, P., 2009b. The importance of transformations for traceability—A case study of lamb and lamb products. Meat Science, 83, pp. 68–73. Dreyer, C., Wahl, R., Storøy, J., Forås E., Olsen, P., 2004. Traceability standards and supply chain relationships. Proceedings of the 16th Annual Conference for Nordic Researchers in Logistics, NOFOMA 2004, Challenging Boundaries with Logistics, Linköping, Sweden, pp. 155–170. EPCglobal, 2015. The GS1 EPCglobal Architecture Framework Version 1.7. EPCIS Standard, 2016. EPC information Services Version 1.2. Folinas, D., Manikas, I., Manos, B., 2006. Traceability data management for food chains. British Food Journal, 108 (8), pp. 622–633. FSA, 2002. Traceability in the Food Chain—A Preliminary Study. Food Standards Agency. Available: https://www.food.gov.uk/. Gustavsson, J., Cederberg, C., Sonesson, U., Otterdijk, R., Meybeck, A., 2011. Global food losses and food waste. Food and Agriculture Organization of the United Nations. Available: http://www.fao.org/docrep/014/mb060e/mb060e00.pdf. Kim, W.R., Aung, M.M., Chang, Y.S., Makatsoris, C., 2015. Freshness Gauge based cold storage management: A method for adjusting temperature and humidity levels for food quality. Food Control, 47, pp. 510–519. Luning, P.A., Marcelis, W.J., 2006. A techno-managerial approach in food quality management research. Trends in Food Science and Technology, 17, pp. 378–385. Regattieri, A., Gamberi, M., Manzini, R., 2007. Traceability of food products: General framework and experimental evidence. Journal of Food Engineering, 81, pp. 347–356. Ringsberg, H.A., Mirzabeiki, V., 2014. Effects on logistic operations from RFID- and EPCIS- enabled traceability. British Food Journal, 116 (1), pp. 104–124. Shanahan, C., Kernan, B., Ayalew, G., McDonnell, K., Butler, F., Ward, S., 2009. A framework for beef traceability from farm to slaughter using global standards: An Irish perspective. Computers and Electronics in Agriculture, 66 (1), pp. 62–69. Shi, J., Zhang, J., Qu, X., 2010. Optimizing distribution strategy for perishable foods using RFiD and sensor technologies. Journal of Business & Industrial Marketing, 25 (8), pp. 596–606. Smith, D., Sparks, L., 2004. Temperature controlled supply chains. In: Food Supply Chain Management (eds. M.A. Bourlakis and P.W.H. Weightman), Blackwell Publishing Ltd, Oxford, UK. doi: 10.1002/9780470995556.ch12. Storøy, J., Thakur, M., Olsen, P., 2013. The Tracefood framework—Principles and guidelines for implementing traceability in food value chains. Journal of Food Engineering, 115 (1), pp. 41–48. Thakur, M., Sørensen, C., Bjørnson, F.O., Forås, E., Hurburgh, C.R., 2011. Managing food traceability information using EPCIS framework. Journal of Food Engineering, 103 (4), pp. 417–433. Thakur, M., Forås, E., 2015. EPCIS based online temperature monitoring and traceability in a cold meat chain. Computers and Electronics in Agriculture, 117, pp. 22–30. TRACE 2, 2008. Annex I–TRACE—Tracing Food Commodities in Europe ‘Description of Work’, FP6-2003-FOOD-2-A Proposal No. 006942, Sixth Framework Programme. Van der Vorst, J.G.A.J., Van Kooten, O., Marcelis, W., Luning, P., Beulens, A.J.M., 2007. Quality controlled logistics in food supply chain networks: Integrated decision-making on quality and logistics to meet advanced customer demands. In: Proceedings of the 14th International EurOMA Conference, Ankara, Turkey. Van Dorp, K., 2002. Tracking and tracing: A structure for development and contemporary practices. Logistics Information Management, 15 (1), pp. 24–33. 16 Fish Trade Regulations Isabelle Metaxa

CONTENTS 16.1 Overview on Fisheries Products Trade...... 313 16.2 Current EU Legislation on Labeling of Fishery and Aquaculture Products.... 315 16.3 Environmental Issues of the Fisheries Products...... 319 Bibliography...... 321

16.1 OVERVIEW ON FISHERIES PRODUCTS TRADE Fisheries (wild capture and aquaculture) are important sources of food, nutrition, income, and livelihood for hundreds of millions of people around the world. Fisheries are of immense scale and economic importance, which also means they have a significant environmental impact that must be managed effectively to ensure sustainability (Sumaila and Bellmann, 2016). According to the United Nations Food and Agriculture Organization (FAO), marine and inland fisheries and aquaculture (excluding aquatic plants) supplied the world with about 167.2 million tons in 2014. Global total capture fishery production in 2014 was 93.4 million tons, of which 81.5 million tons were from marine waters and 11.9 million tons were from inland waters. Production of aquatic animals from aquaculture in 2014 amounted to 73.8 million tons, with an estimated first-sale value of US$160.2 billion. When aquatic plants are included, world aquaculture production reached 101.1 million tons in 2014, representing 52% of total fisheries production (195.7 million tons). The FAO State of World Fisheries and Aquaculture report 2016 states that about 87% (more than 146 million tons) of estimated world fish production was used for direct human consumption. The remaining, 21 million tons, was destined for nonfood products, of which 76% was converted to fishmeal and fish oil in 2014, the rest being largely utilized for a variety of purposes including as raw material for direct feeding in aquaculture. Increasingly, the utilization of by-products is becoming an important industry by itself, with a growing focus on handling in a controlled, safe and hygienic way, thereby also reducing waste. World per capita fish supply reached a new record high of 20 kg in 2014, thanks to vigorous growth in aquaculture, which now provides half of all fish for human consumption, and to a slight improvement in the state of certain fish stocks due to improved fisheries management (FAO, 2016). The per capita consumption in the European Union (EU) is only marginally higher than the world average at 23 kg. The EU covers its domestic consumption mostly through imports, the majority of which are either frozen or prepared products. Shrimps, tuna, white fish, and fishmeal are the most imported products. Norway (mainly salmon and cod) and China (processing country for white fish) are the main EU suppliers (EUROFISH, 2016a). Tuna was the most consumed

313 314 Trends in Fish Processing Technologies product in the EU, with a per capita consumption of 2.6 kg in 2014, followed by cod, whose per capita consumption increased 22% from 2012 to 2014 (European Market Observatory for Fishery and Aquaculture Products [EUMOFA], 2016). Fish and fishery products are among the most traded food commodities in the world. World trade in fish and fishery products has grown significantly also in value terms, with exports rising from US$8 billion in 1976 to US$148 billion in 2014, at an annual growth rate of 8.0% in nominal terms and 4.6% in real terms (FAO, 2016). China is now the world’s largest producer (more than 60% of world aquaculture production), consumer, importer, and exporter of seafood products. In the EU, the main markets for Chinese products are Germany, Spain, the Netherlands, and the UK. China has contributed over a half of all Alaska Pollack to these markets, and a quarter of the whitefish fillets, which are used extensively by EU secondary processors. On average, in recent years, fish fillets have accounted for some 64% of imports to EU countries, and crustaceans and molluscs, a further 20% (Seafish, 2015). The fisheries and aquaculture sector in the EU is very significant in terms of fisheries production. With landings of more than 6,100,000 tons per annum, the Union of 27 ranked fifth in the world, after China, Indonesia, India, Peru, and Vietnam, in 2013 in terms of magnitude of fisheries (80% from catches and 20% from aquaculture) production, representing 3.2% of the world total production (European Commission, 2016). In relative terms, though, the European fishing sector represents a very low percentage of the overall European economy: its contribution to the European Gross Domestic Product is only around 0.1% compared to 0.4% in Japan, 0.7% in Norway, close to 1% in Korea, and up to 10% in Iceland. In the United States, the percentage goes down to 0.02% (Lado, 2016). Since April 2013, the European Commission has been running and managing the EUMOFA, developed to facilitate structural planning and decision making for economic operators, nongovernmental organizations, and policy makers. The Observatory is an EU-wide interactive web tool offering up-to-date data on volume, value, and price of fisheries and aquaculture products throughout the supply chain, from when they land to port to when they are displayed on supermarket shelves. The Observatory is updated daily with production information from the local to the EU-wide level and includes data on imports, exports, and consumption trends, data mainly provided by authorities in Member States (plus Norway and Iceland) and European institutions. It is expected to support market transparency and stability and let producers identify new market opportunities and optimize value of production. This should in turn help moving away from production strategies based solely on volume and thus contribute to social, economic, and environmental sustainability. According to EUMOFA, in terms of value, the EU is the largest trader of fishery and aquaculture products in the world. EU consumers spent EUR 54 billion for buying fisheries and aquaculture products in 2015, reaching the highest amount ever recorded. Per capita fish consumption per year increased to 25.5 kg in 2015. EU covers its domestic consumption mostly through imports, the majority of which are either frozen or prepared products. Cod, shrimps, and salmon are the most imported products. In 2015, the EU registered a trade flow amounting to EUR 49.3 billion and 13.8 million tonnes. Fish alone represents almost 20% of the overall EUR 120 billion worth of food products imported by the EU. The EU is by far the largest single Fish Trade Regulations 315 market for fish imports, valued at EUR 22.3 billion in 2014 (intra-EU trade is excluded). Exports from the EU registered EUR 4.5 billion, being mainly determined by exports of tuna from Spain and fishmeal from Denmark. The global trade patterns are obviously influenced by major trade agreements, such as the EU and Canada signing the Comprehensive Economic and Trade Agreement (CETA) on October 30, 2016. The European Parliament voted in favor of CETA on February 15, 2017. CETA will be fully implemented once the parliaments in all Member States ratify the deal according to their respective domestic constitutional requirements (European Commission, Trade policy, 2017). Once applied, it will eliminate virtually all tariffs in imports between the two economies, harmonize and reduce trade regulations and related structural barriers, and provide a mechanism to resolve disputes concerning trade, investment, and other economic matters. Regarding fish and seafood, CETA will eliminate tariffs that on most products are already low (many under 5% ad valorem). However, some important items, such as lobsters from Canada and sardines from the EU, currently face significant import tariffs. When these tariffs are phased down, prices of such products are expected to fall, leading to increased demand and consumption (EUROFISH, 2016b). According to the annual statistical yearbook on commercial fisheries, Fisheries of the United States (US) 2015, released by the US government, the imports of edible fishery products totaled $18.8 billion. Fresh and frozen products make up a majority of US imports and include tropical shrimp, salmon, and tuna, among a wide array of other products. The leading sources of US imports are Canada, China, India, Thailand, Indonesia, Vietnam, and Chile (75.1% of the total). The EU (mainly the United Kingdom, Spain, and Germany) supplied 2.5% of total US imports. US exports of edible fishery products totaled $3.1 billion, down by 7.7% from 2014. As with imports, fresh and frozen products make up the bulk of US exports. Important products include fresh and frozen salmon, surimi, live and frozen lobsters, canned salmon, and roe, among many other fishery products. The single largest market for US exports is the EU (mainly the Netherlands, Germany, the UK, and France), accounting for 21.4% of total US exports. Other leading export markets are Canada, China, Japan, and India.

16.2 CURRENT EU LEGISLATION ON LABELING OF FISHERY AND AQUACULTURE PRODUCTS The valuable consumer information on fish and seafood products provided through label will guarantee the consumers their right to information and will help them to make informed choices. It will guarantee market transparency and protect consumers against mislabelling. Consumers, processing, and retail sectors increasingly share these concerns and require guarantees that the fish they consume and sell originates from well-managed and sustainable fisheries. With better labeling, the consumer will be able to choose fish and fish products from sustainable fishing and responsible aquaculture. The general food law (Regulation (EC) No. 178/20021) lays down guiding principles and establishes common definitions. Furthermore, the Regulation puts the overall responsibility for producing safe food on the food business operator. It requires 316 Trends in Fish Processing Technologies the food business operator to have a system in place enabling them to identify the immediate supplier(s) and immediate customer(s) of their products to ensure traceability. Other issues that are covered within this Regulation are the principles of risk analysis, the precautionary principle, and withdrawal of food from the market by the food business operator if safety is at stake. The traceability system as an element of food safety is strictly connected with the hygiene requirements. The common hygiene and traceability legislation for fish and bivalve molluscs is contained in the hygiene legislation package. The hygiene legislation package, adopted in April 2004 by the European Parliament and the Council, became applicable on January 1, 2006. This legislation aims to create a single, transparent hygiene policy applicable to all food and all food business operators, together with effective instruments to manage food safety and potential future food crises, throughout the food chain. The package consists of the following key acts:

– Regulation (EC) No. 852/2004 on the hygiene of foodstuffs. This regulation lays down general hygiene requirements to be respected by food businesses and applies to all stages of food and feed chain including primary production. The Regulation requires all food business operators to put in place, implement, and maintain a permanent procedure based on Hazard Analysis and Critical Control Point (HACCP) principles with the exception of those involved in primary production. Guides to good practice for hygiene and for the application of the HACCP principles developed by the food business sectors themselves, either at national or at community level, should help businesses to implement HACCP-based procedures tailored to the characteristics of their production. In addition, the Regulation requires food businesses to be registered with the competent authority, this being a simple procedure whereby the competent authority is informed about the address of the establishment and the activity carried out. – Regulation (EC) No. 853/2004 laying down specific hygiene rules for food of animal origin. Establishments handling products of animal origin, including those involved in the production of fisheries products, can operate only if a competent authority has approved them. Approval procedures involve an on-site visit by the competent authority to verify if the establishment fulfils all the requirements concerning infrastructure, equipment, and hygiene. The exceptions are the establishment carrying out only primary production, transport operations, the storage of products not requiring temperature-controlled storage conditions or most retail operations. – Regulation (EC) No. 854/2004 laying down specific rules for the organization of official controls on products of animal origin intended for human consumption, including obligations placed on competent authorities and food business operators. It also lays down specific rules for each sector of production for food of animal origin, including bivalve mollusc and fisheries products. The regulation also includes procedural requirements for the import of products of animal origin from third countries. – Regulation (EC) No. 882/2004 on Official Feed and Food Controls provides general rules about the performance of official controls to verify Fish Trade Regulations 317

compliance with EU rules related to food safety assurance and communication. It covers the entire range of activities covered by feed and food law, including animal health and animal welfare. The Regulation provides for a set of general rules applicable to the official controls of all feed and food at any stage of production, processing, and distribution, whether produced within the EU or exported to or imported from third countries. In addition to these rules, there are other specific control measures that are important to maintain a high level of protection and therefore must be kept in place. This is, for example, the case for the specific veterinary control rules on imports of animals and food of animal origin or for the specific controls rules for organic products. – Council Directive 2002/99/EC5, which deals with animal health issues, lays down the animal health rules governing the production, processing, distribution, and introduction of products of animal origin for human consumption. In an Annex to the Council Directive, the diseases relevant to trade in products of animal origin and for which control measures have been introduced under community legislation are listed. – Directive 2004/41/EC, which repeals the old legislation, a total of 17 Directives concerning food hygiene and health conditions for the production and placing on the market of certain products of animal origin intended for human consumption.

In response to the increasing concern about food composition from consumers and industry, since December 2014, the rules on quantity labeling of prepackaged foods have changed. Fish and seafood products are also subject to comply with rules regarding the mandatory quantity labeling requirements of Regulation (EU) No. 1169/2011 of the European Parliament and of the Council on the provision of food information to consumers (FIC). The legislation came into effect on December 13, 2011, went into application on December 13, 2014, and became mandatory as of December 13, 2016. It affects all food manufacturers producing or selling within the territories of the EU member states. The EU has established Regulation 1379/2013 on the common organization of the markets (CMO) in fishery and aquaculture products. This regulation includes standards for the proper labeling of fishery products and mandatory information regarding labeling of fishery products to be marketed in the EU. One significant difference between the two regulations is that the CMO includes non-prepacked seafood, whereas the FIC does not. Council Regulation (EC) No. 1224/2009 establishes the minimum labeling and information requirements available to consumer at retail stage. The mandatory information on fish label as required by the European legislation is as follows:

– Commercial designation and the scientific name of the species: Nowadays, the consumers demand clear and reliable information about the species they eat. Therefore, both the commercial and scientific names used in the product labeling must be those on the list drawn up and published by each EU country. 318 Trends in Fish Processing Technologies

The commercial designation and the scientific name can either correspond fully with the name of the food (FIC Regulation) or partly, as would be the case where additional particulars must be added to the name. If an EU country wants to change any of the commercial names on the list, it must notify the Commission of this and the Commission must inform the other EU countries. – Production method: Production method (“…caught…” or “…caught in freshwater…” or “…farmed…”), correct identification and labeling of fish as farmed or wild, is relevant for the protection of wild and endangered stocks. – The area where the product was caught or farmed: Catch area for wild fish/country and body of water for wild freshwater fish/farming country for farmed fish: the location where the fish is caught or farmed is also important since some areas are considered clean, and others, polluted. The FAO designates global fishing areas by numeric code. These in turn are often subdivided into subareas and then, further, to divisions. For fishery products caught in the Northeast Atlantic (FAO Fishing Area 27) and the Mediterranean and Black Sea (FAO Fishing Area 37), the name of the subarea or division is to be provided instead. In addition, this should include the name of this zone expressed in terms understandable to the consumer or a map or pictogram showing that zone. In case of aquaculture products, the Member State or the third country in which the product reached more than half of the rearing period must be mentioned, or in case of shellfish, where it underwent a final rearing or cultivation stage of at least 6 months. – Category of fishing gear used for capture: Any of the seven gear type categories listed in the CMO regulation must appear on the label: seines, trawls, gillnets and similar nets, surrounding net and lift nets, hooks and lines, dredges, and pots and traps. – Whether the product has been defrosted: This information is not necessary if fishery and aquaculture products are ingredients present in the final product or have been previously frozen for health safety purposes or have been defrosted before smoking, salting, cooking, pickling, drying, or a combination of these processes or are foods for which freezing is a technologically necessary step of the production process. – “Best before” and “use by” dates: The date of minimum durability must be preceded by the words “best before” when the date includes an indication of the day (or “best before end…” in other cases), and accompanied by either the date itself or a reference to where the date appears on the label and if necessary followed by a description of the storage conditions that must be observed if the product is to keep for the specified period. All prepacked products that are not highly perishable must display the “best before” date. The “use by” date is for foods that are highly perishable and therefore likely to pose an immediate danger to health after a short period (e.g., ready-to-eat fish). The “best before” date is just an indication of the date before which the product is of best quality. For all non-prepacked products, products prepacked for direct sale or on sales premises at the consumer’s request, EU Fish Trade Regulations 319

countries can decide whether to adopt national rules stipulating that the “best before” or the “use by” date should be displayed. – Frozen unprocessed fishery products must carry the date of freezing or the date of first freezing, preceded by the words “frozen on” and accompanied by the date itself or a reference to where the date is given on the label; the date must consist of the day, the month, and the year, in that order and in uncoded form. – Ingredient declaration: The lists of ingredients for the prepacked products must include each ingredient used to make the product in order of predominance by weight, expressed as a percentage. – Allergen declaration: Allergens must be highlighted in the ingredients list; this includes substances produced or derived from allergens or used in processing the food. Where no list of ingredients exists, the presence of allergens must be indicated as follows: “Contains…” – Storage conditions and usage instructions: Storage conditions must be included if necessary to maintain the quality of the products. Examples include “once opened, keep refrigerated and consume within 3 days” and “once defrosted do not refreeze.”

In addition to the mandatory information required, the following information can be provided if it is clear, unambiguous, and verifiable and does not mislead the consumer: date of catch or harvest; date of landing; information on the port of which the product was landing; more detailed information on fishing gears; flag state of the vessel; information on environmental, ethical, or social nature; information on production techniques and practices; nutritional content of the product; and quick response code. Specific EU labeling requirements apply only to canned sardines and tuna.

16.3 ENVIRONMENTAL ISSUES OF THE FISHERIES PRODUCTS The growing awareness of the ecological crisis and the increased level of trade in fish products have led to a proliferation of legal and institutional responses (Young, 2011). In the fisheries area, there is a series of environmental measures introduced in various countries, which in one way or another act as trade barriers to attain a sustainable utilization of fish resources. Consumers have begun to demand environmentally friendly seafood products. Certification is an integral and indispensable part of any ecolabeling scheme. In respect to fisheries ecolabeling schemes, it provides assurance to buyers and consumers that a certain fish or fishery product comes from a fishery that conforms to the established standard for sustainable fisheries (Albert, 2010). Certification of fisheries provides a means of addressing both fishery-related objectives toward, and consumer interest in, an ecologically sustainable harvest (Potts and Haward, 2007). Documentation and traceability are important for environmental labeling. Producers may choose to adopt specific requirements that permit them to label their products as environmentally friendly or produced in respect of certain social values. Examples of such labeling include “organic production” labels, “fair trade” labels, 320 Trends in Fish Processing Technologies

“dolphin-safe tuna” labels, or other ecolabels. An ecolabel is a tag or label placed on a product that certifies that the production was environmentally friendly. The label provides information at the point of sale that links the product to the production process. Ecolabeling of fish products has been in use since the beginning of 1990s and started with the “dolphin safe” label. Several species of tuna socialize with dolphins, the dolphins swimming alongside them and protecting them (just with their presence) from the sharks. The capture of dolphins in the nets, especially pure seines (large nets that are drawn shut by a rope passed through rings), was not incidental: fishing vessels in search of tuna would seek out dolphin pods, which were easier to find, and then encircle the pods to capture both the dolphins and tuna at once (Schiffman, 2011). The by-catch of dolphins in the Eastern Tropical Pacific Ocean specifically was the highest because fishermen used to intentionally capture both tuna and dolphins and then attempt to release the dolphins from the net. However, release wasn’t always successful and the stress alone often caused serious damage: large numbers of dolphins were injured and killed in the netting process. Concern over the by-catch of the dolphin in tuna purse-seine fisheries (the fishery that underpins the canned tuna industry) led to US government requirements that imported tuna be caught in a way that minimized this by-catch. The introduction of this requirement led to Mexico taking the United States to a World Trade Organization (WTO) dispute panel, claiming that the US requirements were discriminatory and thus outside the scope of WTO rules (Potts and Haward, 2007). Nowadays, Marine Stewardship Council (MSC) labeling probably has the greatest influence. MSC is an independent nonprofit organization established by the World Wide Fund for Nature and Unilever. There are three principles of the MSC Fisheries Standard that have to be met all together to be certified as a sustainable fishery (www.msc.org). As an independent organization, the MSC has established a program of assessment, product certification, and promotion of sustainable fisheries. Ensuring sustainable fish stocks principles considers that fishing activity must be at a level that ensures it can continue indefinitely. Minimizing environmental impact principles considers that the fishing operations must be managed to maintain the structure, productivity, function, and diversity of the ecosystems they’re working in. Effective fisheries management principles consider that if fisheries are poorly managed, environmental impacts go unchecked. Unsustainable fishing practices put seafood resources and fishing livelihoods at risk. Several types of certification based on an environmental approach exists. Other schemes (for example, KRAV in Sweden and Naturland in Germany) provide certification of organic aquaculture and relatively small-scale sustainability certification schemes for marine wild capture fisheries (MRAG, 2009). To be MSC certified, fisheries must comply with relevant laws and have a management system in place that is responsive to changing circumstances. This international organization certifies fish stocks worldwide. In addition, there have been calls for a ban on the consumption of some fish species to prevent overfishing. The Ecolabel is the European brand for certification and services, which was launched in 1992 by the European Commission in light of developing a Europe-wide voluntary environmental labeling scheme that consumers could trust. The ecolabel is already displayed on many aquatic products in over 100 countries around the world. Fish Trade Regulations 321

It is a type of certification assuring consumers that the product has been produced according to a given set of environmental standards. These address issues such as the sustainability of the resources used and the environmental impact of the production method. The underlying idea is that if consumers are properly informed, their choices could possibly stimulate the production and consumption of environmentally friendly products. Consumers could thus influence the behavior of producers and policy makers. Most MSC product is sold in the United States and in specific EU markets (the Netherlands, France, Sweden, United Kingdom, and Germany), although an increasing number of licenses have also been sold in China. In terms of fisheries, sustainability typically refers to the sustainability of stocks. To paraphrase one of the best-known definitions of sustainable development, that was elucidated by the Brundtland Commission in its report from 1987, it is referring to the exploitation of fish stocks to meet the needs of current generations without compromising the ability of future generations to meet their own needs. A clear corporate commitment to sustainability is one of the keys to ensuring that food quality and safety are based on sustainable principles (EUROFISH, 2014). According to the European strategy for food safety “from farm to fork” (European Commission, 2009b), the quality and safety of animal feed stuffs is a major crossroad, encompassing the environment from which feed ingredients are derived, farming practices, animal health, and food safety. As succinctly presented, fisheries products trade will more and more depend on the interrelation between offer, demand, and regulation stemming from human health and consumer perspective and major environmental issues related to the sustainability of the fisheries industry and its impacts on the ecosystems. Fisheries products trade between current major blocks in itself may be a challenge in the coming years if the current patterns of globalization are impacted by the growing levels of protection- isms. Instead of harmonizing trade and environmental regulations, the latter could be used, on top of tariff barriers, to create additional obstacles to fisheries products trade. Failing to find the right balance at the appropriate level, future generations would risk losing healthy proteins provided by nature: it is therefore essential that public policy interacts as efficiently as possible with fisheries industries and consumers, locally and globally.

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EUMOFA. 2016. The EU Fish Market. Brussels, Belgium: European Commission. EUROFISH. 2016a. EUMOFA data provides snapshot of EU seafood sector. Eurofish Magazine, Issue 3, 2016 (May/June), 16. EUROFISH. 2016b. New EU–Canada trade agreement seeks to reduce consumer prices and boost trade. Eurofish Magazine, Issue 6, 2016 (November/December), 14. European Commission. 2009a. Green Paper—Reform of the Common Fisheries Policy, COM (2009) 163 final. European Commission. 2009b. White Paper on Food Safety. Brussels, COM (1999) 719 final. European Commission. 2014. A Pocket Guide to the EU’s New Fish and Aquaculture Consumer Labels. Luxembourg, Publications Office of the European Union. European Commission. 2016. La politique commune de la peche en ciffre. Publication Office of the European Union. Food and Agriculture Organization. 2005. Causes of Detentions and Rejections in International Fish Trade: FAO Fisheries Technical Paper, No 473, Food and Agriculture Organization, Rome. Food and Agriculture Organization. 2016. The State of World Fisheries and Aquaculture 2016 (SOFIA). Food and Agriculture Organization, Rome. Frid, C. L., Paramor, O. A. 2012. Feeding the world: What role for fisheries? International Council for the Exploration of the Sea Journal of Marine Science, Volume 69, Issue 2, 145–150. IUCN. 2009. Guide for the Sustainable Development of Mediterranean Aquaculture 3. Aquaculture Responsible Practices and Certification. Gland, Switzerland and Malaga, Spain: IUCN. Lado, E. P. 2016. The Common Fisheries Policy: The Quest for Sustainability. Brussels, Oxford: Wiley Blackwell. Lelieveld, H. L., Holah, J., Napper, D. 2013. Hygiene in Food Processing: Principles and Practice. Woodhead Publishing, Cambridge, UK. Luten, J., Jacobsen, C., Bekaert, K., Sæbø, A., Oehlenschläger, J. 2006. Quality, Safety and Processing of Wild and Farmed Fish. Wageningen, the Netherlands: Wageningen Academic Publishers. MRAG. 2009. Review of Fish Sustainability Information Schemes. Final Report. London, UK: Marine Resources Assessment Group. Pauly, Z., Daniel, D. 2017. Comments on FAOs State of World Fisheries and Aquaculture (SOFIA 2016). Marine Policy, Volume 77, 176–181. Potts, T., Haward, M. 2007. International trade. Eco-labeling and sustainable fisheries— Recent issues, concepts and practices. Environment, Development and Sustainability, Volume 9, 91–106. Schiffman, H. S. 2011. Green Issues and Debates: An A-to-Z Guide. Sage Publication, London. Seafish. 2015. Seafish Ethics Profile—China. UK: Seafish. Sumaila, U. R., Bellmann, C. 2016. Fishing for the future: An overview of challenges and opportunities. Marine Policy, 173–180. Tidwell, J. H., Allan, G. L. 2001. Fish as food: Aquaculture’s contribution. European Molecular Biology Organization, Volume 2, Issue 11, pages 958–963, November 2001 Toldrá, F. 2009. Safety of Meat and Processed Meat. Springer, New York. Vidacek, S., Janci, T. 2016. Safety of fish products, in Regulating Safety of Traditional and Ethnic Foods, Prakash, V., Martin-Belluso, O., Keener, L., Astley, S. B., Braun, S., McMahon, H., Lelieveld, H. (eds.), Academic Press, San Diego (CA). 79–97. Waisundara,V., Jayawardena, N., Watawana, M. 2016. Safety of fermented fish products, in Regulating Safety of Traditional and Ethnic Foods. Prakash, V., Martin-Belluso, O., Keener, L., Astley, S. B., Braun, S., McMahon, H., Lelieveld, H. (eds.), Academic Press, San Diego (CA). 149–168. Young, M. A. 2011. Trading Fish, Saving Fish: The Interaction between Regimes in International Law, Cambridge University Press, UK. Index

A phlorotannins, 141 from plants, 140 Acinetobacter, 86 polyphenols, 141–142 Active packaging (AP), 10, 187, 189–198 from seaweed, 140–141 EFs and coatings with antimicrobial sulfated polysaccharide, 141 enzymes, 196–197 AP, see Active packaging (AP) EFs and coatings with EOs, 193–196 AQI, see Artificial quality index (AQI) EFs and coatings for fishery products, 189–196 Aquaculture, 219–220 multifunctional bioblends for fish packaging, 197 Aristichthys nobilis, 194 nanobiocomposite films used for fish Artificial quality index (AQI), 262–264 packaging, 198 Aspergillus flavus, 225, 228 Adenosine triphosphate (ATP), 74, 202, 248 Aspergillus oryzae, 109 Adulteration and misbranding of fish products, Assessment of fish, see Molecular methods for 291–301 assessment of fish and fish product fish mislabeling, 292–293 integrity future perspective, 299 Atherestes stomias, 121 locations of fish substitution in flatfish supply ATP, see Adenosine triphosphate (ATP) chain, 296–297 Authenticity of fish, molecular tools for testing, sampling to monitor fish substitution throughout 285–286 flatfish supply chain, 297–299 species and origin identification, 293–295 traceability in the fish supply chain, 291 B Aeromonas, 86 Bacillus, 86 Aeromonas hydrophila, 30, 143 Bacillus amyloliquefaciens, 108 Aeromonas spp., 230, 278 Bacillus cereus, 30, 55, 165, 278 Aflatoxins, 225 Bacillus licheniformis, 108 Agitated heat processing, 36–37 Bacillus megaterium, 55 Alga bifurcaria, 142 Bacillus spp., 230 Algae toxins, 228–229 Bacillus subtilis, 37, 55 food safety indicators and measures to ensure Bacteriolytic enzymes, 43 adequate level of protection from, 229 Barcode labels, 202–206 origin, pathways, and exposure of, 228 Best available techniques (BATs), 178 regulatory definition of safety of, 228–229 BHA, see Hidroxybutylanisol (BHA) Allergens, 92–93 BHT, see Butylated hydroxy toluene (BHT) Anisakis spp., 234 Bioaccumulation, definition of, 224 Antimicrobial preservatives from Biogenic amines, 91–92 microorganisms and plants, 143–146 Biosensors, 202 chitosan, 144–145 Blacklight, 228 essential oils, 143–144 Brevoortia spp., 121 lactic acid bacteria, 145 Brochothrix thermosphacta, 196, 279 plant extracts, 144 Brycon cephalus, 33 probiotics, 145–146 Butylated hydroxy toluene (BHT), 139 Antioxidants, 139–143 By-catch, see Low-value fish, coproducts, and activities, 141 by-catch (processing of) algae and seaweed, 142–143 application of natural antioxidants to preserve C fish products, 141–143 effects of HPP on, 76 Campylobacter jejuni, 39, 55, 231 fucoxanthin, 140–141 CAPAs, see Corrective actions and preventive natural, 139 actions (CAPAs)

323 324 Index

Capture fisheries, 218 Consumer food trends, 163–164 Carassius auratus, 142 Coproducts, see Low-value fish, coproducts, and Carbon foot printing, 177–178 by-catch (processing of) Carnobacterium, 280 Corrective actions and preventive actions Centers for Disease Control and Prevention (CAPAs), 243 (CDC), 277 Coryfenidae, 232 CETA, see Comprehensive Economic and Trade Corynebacterium, 86 Agreement (CETA) Coryphaena hippurus, 88 Channa argus, 144 Coryphaenoides rupestris, 35, 110 Chemical hydrolysis, 107 Crassostrea gigas, 145, 195 Chilling, 6–7 Critical Control Points (CCPs), 243 Chitosan, 144–145, 193 Cronobacter sakazakii, 55 Chorophyta, 140 Cryoprotectants, 124–126 Citri reticulatae pericarpium, 142, 144 Cryptosporidium parvum, 53–54 Clean fish processing technologies, 171–185 Ctenopharyngodon idellus, 125 aquaculture, 175 Culpea harengus, 88 automatic cleaning systems for small Curing, 3–5 equipment, 179 avoiding scaling, 181 D capture, 175 carbon foot printing, 177–178 Data carriers, 202–207 case study, 182–184 Depuration, 233 cleaning and disinfection procedures, Dicentrarchus labrax L., 79 178–179 Dielectric spectroscopy, 268 design improvement of discharging slopes, Digital droplet PCR (ddPCR), 282 180 Dosidicus gigas, 13, 122 dry transport, 180 Drying, 5–6 filtered recirculating scaling wastewater, use of, 181–182 E fish processing chain, 175–176 hygienic design of equipment, 182 Economically motivated adulteration (EMA), life cycle assessment, 176–177 245 seafood production, processing, and Edible coatings (ECs), 12, 190 consumption, 174–176 Edible films (EFs), 12, 188 spraying devices, good management of, E-learning, 250 180–181 Electronic Product Codes (EPCs), 306 sustainability tools, 176–178 Electronic tongues (ET), 284 thawing, 182 ELISA, see Enzyme-linked immunosorbent trade/transport, 176 assay (ELISA) Clostridium, 86 EMA, see Economically motivated adulteration Clostridium botulinum, 2, 5, 29, 30, 32, 50, 86, (EMA) 143, 230, 231, 233, 278 End-over-end (EOE) rotation, 36 Clostridium perfringens, 230, 278 End-point internal product temperature (EPIPT), 41 Cloud technology, 249 Engraulidae, 232 Clupea harengus, 145 Engraulis ringens, 121 Clupeidae, 232 Enterobacteriaceae, 87, 193, 202 Codex Alimentarius, 221, 223 Enterobacter spp., 233, 280 Cold chain monitoring, 308–310 Enterococcus, 280 EPCIS temperature events, 309–310 Enterprise resource planning (ERP), 243 temperature sensors, 308–309 Environmental contaminants, 223–225 web-based monitoring, 309 bioaccumulation, definition of, 224 Cold pasteurization, 71; see also High-pressure environmental pollutants, 224 processing (HPP) of seafood measures to ensure adequate level of Colossoma macropomum, 34 protection from, 225 Comprehensive Economic and Trade Agreement origin, pathways, and exposure of, 223–224 (CETA), 315 regulatory definition of safety of, 224–225 Congresoxs spp., 121 Environmental pollutants, 224 Index 325

Enzymatic hydrolysis, 107 Food safety management in fish processing units, Enzyme-linked immunosorbent assay (ELISA), 241–254 18, 281, 294 cloud technology, 249 Enzymes, 76, 114 color-coding, communication via, 248–249 EOE rotation, see End-over-end (EOE) rotation definition, structure, and challenges for fish EOs, see Essential oils (EOs) processing operators, 241–245 EPCs, see Electronic Product Codes (EPCs) economically motivated adulteration, 245 Epigonus telescopus, 35 e-learning, 250 EPIPT, see End-point internal product food defense and food fraud prevention temperature (EPIPT) systems, 245–246 ERP, see Enterprise resource planning (ERP) mobile technology, 249 Escherichia coli, 30, 55, 87, 145, 193, 198, 230, operational prerequisite programs, 248 231 risk assessment tools, 248 Escherichia coli K12, 39 social media, 249 Escherichia coli O157:H7, 53, 196 trends in HACCP and FSMS, 246–252 Essential oils (EOs), 143, 193 Food Safety and Modernization Act (FSMA), 243 ET, see Electronic tongues (ET) Fourier transform mid-IR (FTIR) spectroscopy, 265 Etrumeus teres, 34 FPHs, see Fish protein hydrolysates (FPHs) Euphausia superba, 104 Free fatty acids (FFAs), 74, 258 European Article Numbering (EAN) Freezing, 7–9 Association, 206 Frozen storage, 9 FSMA, see Food Safety and Modernization Act F (FSMA) FTIR spectroscopy, see Fourier transform mid-IR FAO, see Food and Agriculture Organization of (FTIR) spectroscopy the United Nations (FAO) Fucoxanthin, 140–141 FBOs, see Food business operators (FBOs) Fucus vesiculosus, 143 FCMs, see Food contact materials (FCMs) Feed contaminants, 225–228 G aflatoxins, 225 measures to ensure an adequate level of Gadus morhua, 89, 145 protection from, 227–228 Gas chromatography (GC), 294 origin, pathways, and exposure of, 225–226 General Food Law, 303, 315 regulatory definition of safety of, 226–227 GHGs, see Greenhouse gasses (GHGs) Fenneropenaeus indicus, 89 Global Food Safety Initiative (GFSI), 245 FFAs, see Free fatty acids (FFAs) Global Trade Item Number (GTIN), 206 FIC, see Food information to consumers (FIC) Graeloipia filicina, 143 “Fish flour,” 104 Greenhouse gasses (GHGs), 176 Fish packaging solutions, see Packaging solutions Green primary packaging, 190 (innovative) Fish product integrity, see Molecular methods H for assessment of fish and fish product integrity Hafnia alvei, 280 Fish protein Hazard Analysis Criterial Control Point hydrolysates (FPHs), 125 (HACCP), 221, 246, 316 recovery, see Protein recovery Hazard Analysis and Risk-Based Preventive Fish trade regulations, see Trade regulations Controls (HARPC), 243 Flatfish supply chain, 296–299 Heat processing, 2–3 Flavobacterium, 86 HHP processing, see Hydrostatic high-pressure Food and Agriculture Organization of the United (HHP) processing Nations (FAO), 2, 318 Hidroxybutylanisol (BHA), 139 Food business operators (FBOs), 245 High-performance liquid chromatography Food contact materials (FCMs), 188 (HPLC), 284 Food and Drug Administration (FDA), 246 High-power ultrasound thawing, 10 Food information to consumers (FIC), 317 High-pressure processing (HPP) of seafood, 71–100 Food safety indicators, see Protection and food allergens, 92–93 safety indicators, achievement of antioxidants, 76 326 Index

aroma and taste, 84–85 IP, see Intelligent packaging (IP) biogenic amines, 91–92 Isoelectric pH (pHi), 105 cold pasteurization, 71 color, 83–84 J enzymes, 76–82 fish constituents, 73–76 Johnius spp., 121 fish functions, 76–82 fish properties, 82–85 K fish safety, 85–93 future perspectives, 95 K-value, 87 gelation, 93 HP freezing and thawing, 94–95 L HP thermal sterilization, 93–94 lipids, 74–76 Lactic acid bacteria (LABs), 145, 200 microorganisms, 85–91 Lactobacillus, 86 pressure-assisted thawing, 94 Lactobacillus acidophilus, 193 pressure-assisted thermal sterilization, 94 Lactoperoxidase system (LPOS), 196 pressure-shift freezing, 94 Laminaria japonica, 141 proteins, 73–74 Larimus pacificus, 121 texture, 82–83 Lateral flow assay (LFA), 281 Hijikia fusiformis, 143 LCA, see Life cycle analysis (LCA) Hippoglossoides spp., 293 Lepidopsetta bilineata, 293 Hoplostethus atlanticus, 35 Lepidopsetta polyxystra, 293 Hot smoking of fish products, 32 Lepidorhombus whiffiagoni, 145 HPLC, see High-performance liquid Lethrinus lethrinus, 34 chromatography (HPLC) Leuconostoc spp., 280 HPP, see High-pressure processing (HPP) of LFA, see Lateral flow assay (LFA) seafood LF NMR, see Low-field nuclear magnetic Hydrofluidization freezing, 9 resonance relaxometry (LF NMR) Hydrolysates and peptides, 14 Life cycle analysis (LCA), 176 Hydrostatic high-pressure (HHP) processing, Limanda aspera, 293 126 Limanda limanda, 292, 293 Hypophthalmichthys molitrix, 79, 122, 144, 146 Liopsetta, 292 Lipid(s) I effects of HPP on, 74–76 hydrolysis, 74 Image analysis, 16 oxidation, 75 Immunomagnetic separation (IMS), 281 Listeria innocua, 32, 37, 39, 87, 94, 193, 196 Impingement freezing, 8 Listeria monocytogenes, 30, 31, 32, 43, 55, 56, Information exchange, 304–307 87, 143, 145, 196, 197, 230, 233, 278 Integrated Pollution Prevention and Control Litopenaeus vannamei, 198 Directive, 178 Lophius piscatorius, 110 Intelligent packaging (IP), 11, 187, 198–207 Lophius spp., 110 barcode labels, 202–206 Lophius upsicephalus, 110 biosensors, 202 Low-field nuclear magnetic resonance data carriers, 202–207 relaxometry (LF NMR), 267 fish freshness sensors and indicators, 202 Low-frequency acoustic thawing, 10 monitoring environmental conditions, Low-value fish, coproducts, and by-catch 199–201 (processing of), 101–119 monitoring quality attributes, 201–202 amino acids with bitter taste, 109 radio-frequency identification, 206–207 collagen superfamily, 110 RH sensors, 201 enzymes, 114–115 seal and leak indicators, 200–201 “fish flour,” 104 time–temperature indicators, 199–200 isoelectric pH, 105 vacuum packaging, 200 protein hydrolysis, 107 International Organization for Standardization protein recovery, 103–113 (ISO), 176, 256 trends, 115 Index 327

LPOS, see Lactoperoxidase system (LPOS) pasteurization and sous vide, 33–35 Luciobarbus esocinus, 33 PEF treatment, 51–56 Lysozyme, 43 strategies to reduce heat load, 35–40 surface treatment and inactivation, 37–40 M technologies, 28–31 time–temperature integrators, 40–41 Macrocystis pyrifera, 141 validation of heat treatment, 40–42 Macrourus berglax, 110 Misbranding of fish products, see Adulteration Macrourus spp., 110 and misbranding of fish products Macruronus magellanicus, 110 Mobile technology, 249 Macruronus novaezelandiae, 121 Modified atmosphere packaging (MAP), 165, Magnetic freezing, 8–9 188 Mallotus villosus, 33, 104 Molecular methods for assessment of fish and fish Maltodextrins, 125 product integrity, 277–290 Manufacturing operations management (MOM), authenticity of fish, molecular tools for 243 testing, 285–286 MAP, see Modified atmosphere packaging (MAP) electronic tongues, 284 MAPS system, see MW-assisted pasteurization future perspectives, 286 (MAPS) system immunomagnetic separation, 281 Marine Stewardship Counsel (MSC), 291, 320 metal oxide semiconductors, 284 MATS system, see MW-assisted thermal methods in safety assessment of fishery sterilization (MATS) system products, 281–283 Maximum permitted levels (MPLs), 228 quality attributes in fish, molecular analysis Maximum residue levels (MRLs), 223 of, 283–284 Megalobrama amblycephala, 142 Molecular weights (MWs), 125 Melanogrammus aeglefinus, 88 Molva dipterygia, 35 Merlangius merlangus euxinus, 34 MOM, see Manufacturing operations Merluccius capensis, 110, 194, 196, 260 management (MOM) Merluccius hubbsi, 110, 145 Moraxella, 86 Merluccius merluccius, 267 Morganella morganii, 30 Merluccius productus, 121 Morganella spp., 279 Metal oxide semiconductors (MOSs), 284 MOSs, see Metal oxide semiconductors (MOSs) Microbiological contamination, 230–233 MPLs, see Maximum permitted levels (MPLs) depuration, 233 MRLs, see Maximum residue levels (MRLs) food safety indicators and measures to ensure MSC, see Marine Stewardship Counsel (MSC) adequate level of protection from, Mugil cephalus, 292 232–233 Mustelus asterias, 292 origin, pathways, and exposure, 230–232 Mustelus mustelus, 292 regulatory definition of safety of, 232 MW-assisted pasteurization (MAPS) system, 48 Micrococcus, 86 MW-assisted thermal sterilization (MATS) Micrococcus spp., 86 system, 48 Micromesistius australis, 121 MW and RF processing of fish, 44–51 Micromesistius poutassou, 121 design and validation of thermal processes, Microorganisms, effect of HPP on, 85–91 50 Microwave (MW) processing, 28; see also MW implementation of industrial MW production, and RF processing of fish 44–45 Minimal heat processing, 27–69 MW combination heating, 49 agitated heat processing, 36–37 novel applications of MW and RF for fish, categories of minimally processed fish 46–47 products, 29 overpressure mw processing, 47–49 combination effects, 42–51 pasteurization, 45–46 end-point setting of temperature, 41–42 subatmospheric pressure MW processing, examples of mildly heat-treated fish, 31–35 49 hot smoking of fish products, 32 temperature measurement, 50–51 microbiological safety issues related to fish tempering, 45 products, 29–31 water immersed RF processing, 49 MW and RF processing of fish, 44–51 Myagropsis myagroides, 141 328 Index

N PEF treatment, 51–56 advantages and application in foods, 52–53 Nanobiocomposite films, 198 challenges of PEF technology applied for Nanoparticles (NPs), 198 food production, 53–54 Near-IR (NIR), 264 PEF and microbial inactivation, 55–56 Nemipterus japonicus, 121 PEF used in fish products, 54 New product development (NPD), 161–170 principle of PEF, 51–52 consumer food trends, 163–164 Penaeus mondon, 90 lightly processed seafood, 165–166 Penaeus vannamei, 198 low salt, 164 Pennahia, 121 modified atmosphere packaging, 165 Peprilus medius, 121 soluble gas stabilization, 165 Persistent organic pollutions (POPs), 226 sustainable seafood products, 164–165 Phaeophyta, 140 use of seaweeds and seaweed extracts in, Phlorotannins, 141 166–167 Phosphates, 126 Next Generation Sequencing (NGS), 294 Photobacterium phosphoreum, 193, 200 NPD, see New product development (NPD) Photobacterium spp., 279 NPs, see Nanoparticles (NPs) Plants Nuclear magnetic resonance (NMR), 16–17 antimicrobial preservatives from, 143–146 antioxidants from, 140 O Platichthys flesus, 293 Plesiomonas shigelloides, 30, 230, 278 Oncorhynchus gorbuscha, 195 Pleurogrammus monopterygius, 121 Oncorhynchus kisutch, 75 Pleuronectes limanda, 292 Oncorhynchus mykiss, 33, 34, 104, 194, 195 Pleuronectes platessa, 293 Oncorhynchus nerka, 32 Polymerase chain reaction (PCR), 18, 233, 282 Operational prerequisite programs (OPRPs), Polyphenols, 141–142 248 Polyunsaturated fatty acids (PUFAs), 74, 139 Oreochromis niloticus, 33, 121 Pomatomidae, 232 POPs, see Persistent organic pollutions (POPs) P Porphyra tenera, 143 Preserved fish products, reformulation of, Packaging, 10–12 135–160 Packaging solutions (innovative), 187–215 antimicrobial preservatives from active packaging, 187, 189–198 microorganisms and plants, 143–146 edible coatings, 190 chitosan, 144–145 food contact materials, 188 essential oils, 143–144 future perspectives, 207 lactic acid bacteria, 145 green primary packaging, 190 natural antioxidants from plants as replacers intelligent packaging, 187, 198–207 for conventional antioxidants, 139–143 modified atmosphere packaging, 188 probiotics, 145–146 smart packaging, 187 reduced salt content products, 136–139 vacuum packaging, 200 Pressure-assisted thawing (PAT), 10, 94 Parasites, 234–235 Pressure-assisted thermal sterilization, 94 food safety indicators and measures to ensure Pressure-shift freezing (PSF), 8, 94 adequate level of protection against, Priacanthus spp., 121 235 Prionace glauca, 110 origin, pathways, contamination, and hazards Prionotus stephanoprys, 121 of, 234 Probiotics, 145–146 regulatory definition of safety in relation to, Processing technologies, 2–14 234–235 chilling, 6–7 Parexocoetus brachyterus, 126 curing, 3–5 PAS, see Publicly Available Specification (PAS) drying, 5–6 Pasteurization, sous vide and, 33–35 freezing, 7–9 PAT, see Pressure-assisted thawing (PAT) frozen storage, 9 PCR, see Polymerase chain reaction (PCR) heat processing, 2–3 PEF, see Pulsed electric field (PEF) packaging, 10–12 Index 329

seafood proteins, 12–14 Quality Index Method, 259–262 thawing, 9–10 time–temperature estimation by FTIR of lipid Protection and food safety indicators, fraction, 265–267 achievement of, 217–240 algae toxins, 228–229 R aquaculture, 219–220 assessment and management of food risks Rachycentron canadum, 34 from aquatic products, 223–235 Radiofrequency (RF) heating, 28 capture fisheries, 218 Radio-frequency identification (RFID), 188, depuration, 233 206–207, 304 environmental contaminants, 223–225 Raja spp., 110 feed contaminants, 225–228 Raman spectroscopy, 264 hazard, definition of, 220 Raoultella spp., 279 microbiological contamination, 230–233 Ready-to-eat (RTE) foods, 31, 192 objectives and regulations to ensure food Recent advances in seafood technology, 1–25 safety, 220–223 chilling, 6–7 parasites, 234–235 curing, 3–5 production systems of aquatic food, drying, 5–6 218–220 freezing, 7–9 risk, definition of, 220 frozen storage, 9 Protein recovery, 103–113 heat processing, 2–3 chemical hydrolysis, 107 microbiological examination, 17–18 collagen and gelatin, 110–113 packaging, 10–12 concentrates, 104–105 processing technologies, 2–14 enzymatic hydrolysis, 107 quality assessment, 14–18 hydrolysates, 107–110 seafood proteins, 12–14 isolates, 105–107 thawing, 9–10 Proteins, effects of HPP on, 73–74 Reformulation of preserved fish products, Proteus spp., 279 see Preserved fish products, Pseudomonas, 86 reformulation of Pseudomonas fluorescens, 94, 165, 193 Reinhardtius hippoglossoides, 35, 110 Pseudomonas spp., 202, 279 Restriction fragment length polymorphism Pseudosciaena crocea, 145 (RFLP), 285 PSF, see Pressure-shift freezing (PSF) RF heating, see Radiofrequency (RF) heating Publicly Available Specification (PAS), 245 RFID tags, see Radio-frequency identification PUFAs, see Polyunsaturated fatty acids (RFID) (PUFAs) RF processing of fish, see MW and RF Pulsed electric field (PEF), 29 processing of fish Rhodophyta, 140 Q Rosmarinus officinalis, 144 RTE foods, see Ready-to-eat (RTE) foods Quality Index Method (QIM), 259 Quality and quality changes assessment of S processed fish, 255–275 demerit point scoring system, 259 Saccharomyces boulardii, 145 dielectric spectroscopy, 268 Safety management, see Food safety management electronic and vibrational spectroscopy, in fish processing units 264–267 Salmonella, 18, 53 fish deteriorative changes, 257–258 Salmonella senftenberg 775W, 56 future trends and expectations, 270 Salmonella spp., 30, 55, 230, 233 indicators to measure changes in properties or Salmo salar, 34, 143, 264 composition of fish, 258–268 Salt content products, 136–139 low-field nuclear magnetic resonance Salt reduction in food, 164 relaxometry, 267–268 Salvelinus alpinus, 261 multisensor mimicking human senses Sarda sarda, 33, 34 (artificial quality index), 262–264 Sardina pilchardus, 88 opportunities and challenges, 269–270 Sardinella gibossa, 33 330 Index

Sardinia melanostrichus, 121 Thunnus alalunga, 35 Sargassum fulvellum, 141 Tilapia mariae, 33 Sargassun kjellmanianurn, 143 Time domain reflectrometry (TDR), 268 SASPs, see Small acid-soluble spore proteins Time–temperature integrator (TTI), 40, 188 (SASPs) TLC, see Thin layer chromatography (TLC) Saurida tumbil, 121 TMA, see Trimethylamine (TMA) Sciaenops ocellatus, 142 Total viable bacteria (TVB), 193 Scomber scombrus, 75, 77, 78, 88 Total volatile based nitrogen (TVB-N), 87, 197, Scomber spp., 121 202 Scombresosidae, 232 TPP, see Tripolyphosphate (TPP) Scombridae, 232 Traceability of fish products, 303–312 Seafood proteins, 12–14 applications of traceability, 308–311 Seaweed, antioxidants from, 140–141 cold chain monitoring, 308–310 Sebastes mentella, 35 food supply chain management, 310–311 Serratia liquefaciens, 280 future perspectives, 311 SGS, see Soluble gas stabilization (SGS) resource efficiency, 311 Shewanella, 86 standardization of information exchange, 304 Shewanella putrefaciens, 193, 196 technologies for information exchange, Shewanella spp., 202, 279 304–307 Shigella spp., 30, 278 traceability in fish supply chains, 303–304 Single strand conformation polymorphism Trachurus murphyi, 121 analysis (SSCP), 285 Trachurus trachurus, 77 Six Sigma, 250 Trade regulations, 313–322 Small acid-soluble spore proteins (SASPs), 86 environmental issues of fisheries products, Smart packaging (SP), 187 319–321 Social media, 249 EU legislation on labeling of fishery and Soluble gas stabilization (SGS), 165 aquaculture products, 315–319 Sous vide, pasteurization and, 33–35 fisheries products trade, overview on, SP, see Smart packaging (SP) 313–315 Sparus aurata, 34, 145 Trichiurus spp., 121 Sparus macrocephalus, 142 Trimethylamine (TMA), 87, 202 Sphyraena spp., 121 Tripolyphosphate (TPP), 198 SSCP, see Single strand conformation Trypsin, 115 polymorphism analysis (SSCP) TTI, see Time–temperature integrator (TTI) Staphylococcus aureus, 30, 56, 198, 231 Turbinaria ornata, 141 Staphylococcus spp., 86 TVB, see Total viable bacteria (TVB) Sulfated polysaccharide, 141 TVB-N, see Total volatile based nitrogen Surimi gelation, 12–14 (TVB-N) Surimi processing, advances in, 121–133 blending with cryoprotectants, 124–126 U block forming, 126 dewatering/screw pressing, 124 Undaria pinnatifida, 141 filleting and mincing/deboning, 122 United Nations Environment Programme, 172 freezing, 126 Universal Product Code (UPC), 205–206 frozen storage, 126–128 refining, 123–124 V washing, 122–123 Vacuum packaging (VP), 200 T Vibrio, 143 Vibrio arahaemolyticus, 30 TDR, see Time domain reflectrometry (TDR) Vibrio cholera, 30, 230, 281 Thawing, 9–10 Vibrionaceae, 86 Theragra chalcogramma, 121 Vibrio parahaemolyticus, 73, 87, 230, 282 Thermal-assisted processes, 3 Vibrio spp., 230, 278 Thin layer chromatography (TLC), 284 Vibrio vulnificus, 30, 73, 87, 143, 230 Threats Analysis Critical Control Points Visible IR (VIR), 264 (TACCP), 243 VIS/NIR spectroscopy, 16 Index 331

VP, see Vacuum packaging (VP) Whey protein isolate coating (WPIC), 197 Vulnerability Analysis Critical Control Points Whey protein isolate film (WPIF), 197 (VACCP), 243 World Health Organization (WHO), 221 World Trade Organization (WTO), 320 W Y Water-holding capacity (WHC), 126, 138 Web-based monitoring, 309 Yersinia spp., 278