Improving Seafood Products for the Consumer

WPNL0206 Improving seafood products for the consumer WPNL0206 Related titles: Safety and quality issues in fish pro...

Author: Torger Borresen

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WPNL0206

Improving seafood products for the consumer

WPNL0206

Related titles: Safety and quality issues in fish processing (ISBN 978-1-85573-552-1) The processing and supply of fish products is a huge global business. Like other sectors of the food industry it depends on providing products which are both safe and which meet consumers' increasingly demanding requirements for quality. With its distinguished editor and international team of contributors, Safety and quality issues in fish processing addresses these two central questions. Maximising the value of marine by-products (ISBN 978-1-84569-013-7) Over-exploitation and declining fish stocks are creating an urgent need for better utilisation of seafood by-products. Currently a large proportion of the catch is wasted, even though marine by-products contain valuable protein and lipid fractions, vitamins, minerals and other bioactive compounds beneficial to human health. This collection covers marine by-product characterisation, recovery, processing techniques, food and non-food applications: essential information for those involved in seafood by-product valorisation. Lawrie's meat science Seventh edition (ISBN 978-1-84569-159-2) Lawrie's meat science has established itself as a standard work for both students and professionals in the meat industry. Its basic theme remains the central importance of biochemistry in understanding the production, storage, processing and eating quality of meat. At a time when so much controversy surrounds meat production and nutrition, Lawrie's meat science provides a clear guide which takes the reader from the growth and development of meat animals, through the conversion of muscle to meat, to the point of consumption. The seventh edition includes details of significant advances in meat science which have taken place in the last eight years, especially in areas of eating quality of meat and meat biochemistry. Details of these books and a complete list of Woodhead titles can be obtained by: · visiting our web site at www.woodheadpublishing.com · contacting Customer Services (email: [email protected]; fax: +44 (0) 1223 893694; tel.: +44 (0) 1223 891358 ext. 130; address: Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, England)

WPNL0206

Improving seafood products for the consumer Edited by Torger Bùrresen

WPNL0206

WPNL0206

Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, England www.woodheadpublishing.com Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2008, Woodhead Publishing Limited and CRC Press LLC ß 2008, Woodhead Publishing Limited The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing Limited ISBN 978-1-84569-019-9 (book) Woodhead Publishing Limited ISBN 978-1-84569-458-6 (e-book) CRC Press ISBN 978-1-4200-7434-5 CRC Press order number: WP7434 The publishers' policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elementary chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Project managed by Macfarlane Book Production Services, Dunstable, Bedfordshire, England (e-mail: [email protected]) Typeset by Godiva Publishing Services Limited, Coventry, West Midlands, England Printed by TJ International Limited, Padstow, Cornwall, England Cover photograph: Frank Gregersen, Nofima

WPNL0206

Contents

Contributor contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xv

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xxv

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Bùrresen, Technical University of Denmark, Denmark 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Structure of the book . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Part I

1 3 8 9

Consumers and seafood

2

Introduction to Part I: consumers and seafood . . . . . . . . . . . . . . . . . K. Brunsù, University of Aarhus, Denmark 2.1 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

1

Consumer attitudes and seafood consumption in Europe . . . . . . K. Brunsù, K. B Hansen and J. Scholderer, University of Aarhus, Denmark, P. Honkanen and S.O. Olsen, Nofima, Norway and W. Verbeke, Ghent University, Belgium 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Consumer motives and barriers to seafood consumption . . . . . 3.3 Overview of cross-cultural investigation of consumption patterns and attitudes towards fish . . . . . . . . . . . . . . . . . . . . . . . . . . .

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16 17 20

vi

Contents 3.4 3.5 3.6 3.7 3.8

European consumption patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Attitudes and preferences across Europe ± differences and similarities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Convenience and fish consumption . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

Improved eating quality of seafood: the link between sensory characteristics, consumer liking and attitudes . . . . . . . . . . . . . . . . . . E. MartinsdoÂttir and K. SveinsdoÂttir, MatõÂs, Iceland, D. Green-Petersen and G. Hyldig, Technical University of Denmark, Denmark and R. Schelvis, Wageningen University and Research Centre, The Netherlands 4.1 Introduction: why is the eating quality important for the industry and for the consumer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Methods for evaluation of sensory quality of seafood . . . . . . . . 4.3 Sensory characteristics of cod and salmon . . . . . . . . . . . . . . . . . . . 4.4 Consumer liking of different seafood products related to sensory characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Consumer segmentation across different European countries, related to attitudes and product preferences . . . . . . . . . . . . . . . . . . 4.6 The Seafood Sensory Quality Model . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 4.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

Evaluating consumer information needs in the purchase of seafood products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. Verbeke and Z. Pieniak, Ghent University, Belgium, K. Brunsù and J. Scholderer, University of Aarhus, Denmark and S.O. Olsen, Nofima, Norway 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Empirical findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 5.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

Consumer evaluation of tailor-made seafood products . . . . . . . . . S.O. Olsen and K. Toften, Nofima, Norway, D. Calvo Dopico and A. Tudoran, University of CorunÄa, Spain, and A. Kole, Wageningen University and Research Centre, The Netherlands 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 The product in its environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Intention and behavioural indicators . . . . . . . . . . . . . . . . . . . . . . . . .

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40 42 45 50 53 55 57 58 58 63

63 65 69 80 82 83 85

85 87 93

Contents 6.4 6.5 6.6 6.7 Part II

Perceived quality and satisfaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results from a project about consumer evaluation of a Norwegian fish burger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 95 98 102 104

Health benefits of seafood

7 Introduction to Part II: health benefits of seafood . . . . . . . . . . . . . . G. Schaafsma, HAN University, The Netherlands 7.1 Developments in nutrition science . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Nutritional role of seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Protective effects of fish consumption in relation to gastrointestinal health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Lund, Institute of Food Research, United Kingdom and E. Kampman, Wageningen University and Research Centre, The Netherlands 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Colorectal cancer (CRC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Inflammatory bowel disease and fish consumption . . . . . . . . . . . 8.4 Fish consumption and other gastrointestinal tract cancers . . . . 8.5 Possible importance of other nutritional aspects of fish consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6 The FISHGASTRO study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9 Fish consumption and the health of children and young adults I. Thorsdottir and A. Ramel, University of Iceland, Iceland 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Effect of fish consumption on obesity . . . . . . . . . . . . . . . . . . . . . . . 9.3 Effect of fish consumption on blood lipids . . . . . . . . . . . . . . . . . . 9.4 Effect of fish consumption on maternal and child health . . . . 9.5 N-3 fatty acids and postpartum depression . . . . . . . . . . . . . . . . . . . 9.6 Bone health and n-3 fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7 Communicating nutritional effects of fish to young adults and children and developing functional fish products for improved health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.8 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.9 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 9.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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viii 10

Contents

Fish, omega-3 fatty acids and heart disease . . . . . . . . . . . . . . . . . . . . . I.A. Brouwer, Free University Amsterdam, The Netherlands 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Fish, omega-3 fatty acids and heart disease . . . . . . . . . . . . . . . . . . 10.3 Omega-3 fatty acids from fish and cardiac arrhythmias . . . . . . 10.4 Possible mechanisms of effects of omega-3 fatty acids on the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5 Conversion and metabolism of omega-3 fatty acids in the human body . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.6 Future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 10.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Part III 11

12

13

165 165 165 170 174 175 176 176 176

Ensuring seafood safety

Introduction to Part III: ensuring seafood safety . . . . . . . . . . . . . . . B. DoreÂ, Marine Institute, Ireland 11.1 Risks associated with seafood consumption . . . . . . . . . . . . . . . . . . 11.2 Relative incidence of microbiological illness associated with seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Control of risks associated with seafood and legal requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Contribution of SEAFOODplus to seafood safety . . . . . . . . . . . . 11.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detecting virus contamination in seafood . . . . . . . . . . . . . . . . . . . . . . . A. Bosch and R. M. PintoÂ, University of Barcelona, Spain, D.H. Lees, Centre for Environment, Fisheries and Aquaculture Science, United Kingdom, C.-H. von Bonsdorff, University of Helsinki, Finland, L. Croci and D. De Medici, Instituto Superiore di SanitaÁ, Italy and F. S. Le Guyader, Ifremer, France 12.1 Introduction: viruses and shellfish contamination . . . . . . . . . . . . 12.2 Methods for detecting viruses in shellfish . . . . . . . . . . . . . . . . . . . . 12.3 Potential emerging virus problems . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reducing microbial risk associated with shellfish in European countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Pommepuy, F.S. Le Guyader and J.C. Le Saux, Ifremer, France, F. Guilfoyle and B. DoreÂ, Marine Institute, Ireland, S. Kershaw, D. Lees, J.A. Lowther and O.C. Morgan, Centre for Environment, Fisheries and Aquaculture Science, United Kingdom, J.L. Romalde,

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194 198 201 204 205 212

Contents Universidad de Santiago de Compostela, Spain and D. Furones and A. Roque, Institute of Agro-Food Research and Technology, Spain 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 REDRISK project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3 Potential strategies to limit microbial contamination of shellfish and tools to implement them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Bacterial pathogens in seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R.J. Lee and R.E. Rangdale, Centre for Environment, Fisheries and Aquaculture Science, United Kingdom, L. Croci, Istituto Superiore di SanitaÁ, Italy and D. Hervio-Heath and S. Lozach, Ifremer, France 14.1 General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Principal bacterial pathogens associated with seafood . . . . . . . . 14.3 Sources of bacterial pathogens in seafoods . . . . . . . . . . . . . . . . . . 14.4 Control of bacterial contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5 Seafood-associated bacterial illness . . . . . . . . . . . . . . . . . . . . . . . . . . 14.6 Conventional methods for the detection, enumeration and identification of bacterial pathogens . . . . . . . . . . . . . . . . . . . . . . . . . 14.7 Molecular methods for the detection, enumeration and identification of bacterial pathogens . . . . . . . . . . . . . . . . . . . . . . . . . 14.8 Molecular approaches to microbial typing . . . . . . . . . . . . . . . . . . . 14.9 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.10 General discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.11 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 14.12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Histamine and biogenic amines: formation and importance in seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. Dalgaard and J. Emborg, Technical University of Denmark, Denmark and A. Kjùlby, N.D. Sùrensen and N.Z. Ballin, Danish Veterinary and Food Administration, Denmark 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Histamine fish poisoning (HFP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3 Legislation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4 Formation of histamine and other biogenic amines in seafoods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5 Determination of histamine and biogenic amines in seafood . 15.6 Management of histamine formation and histamine fish poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.7 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 15.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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212 217 225 237 239 241 247

247 248 254 255 258 262 267 271 278 281 281 282 292

292 293 306 307 314 315 315 316 316

x

Contents

Part IV 16

17

18

Seafood from source to consumer products

Introduction to Part IV: seafood from source to consumer product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. B. Luten, Nofima, Norway 16.1 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Developing functional seafood products . . . . . . . . . . . . . . . . . . . . . . . . . M. Careche, Consejo Superior De Investigaciones Cientificas, Spain, J. B. Luten, Nofima, Norway, A. Kole and R. Schelvis, Wageningen University and Research Centre, The Netherlands, F. Saura-Calixto, Consejo Superior De Investigaciones Cientificas, Spain, O. E. Scholten, Wageningen University and Research Centre, The Netherlands, M. E. Diaz-Rubio, Consejo Superior De Investigaciones Cientificas, Spain, M. A. J. Toonen and E. Schram, Wageningen University and Research Centre, The Netherlands, A. J. Borderias, I. SaÂnchez-Alonso, P. Carmona and I. SaÂnchez-Gonzalez, Consejo Superior De Investigaciones Cientificas, Spain, T. R. Gormley, Ashtown Food Research Centre (Teagasc), Ireland, J. OehlenschlaÈger and S. Mierke-Klemeyer, Federal Research Centre for Nutrition and Food, Germany, E. O. Elvevoll, University of Tromsù, Norway, M. Leonor Nunes and N. Bandarra, IPIMAR, Portugal, I. Stoknes, Mùre Research, Norway and E.H. Larsen, Technical University of Denmark, Denmark 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Consumer studies with respect to the development of new functional seafood products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Novel ingredients for incorporation into functional restructured/ fillet-based seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.4 Development of functional restructured seafood products. Addition of dietary fibre and antioxidant dietary fibre to minced fish muscle and surimi gel-based products . . . . . . . . . . . . . . . . . . . 17.5 Aquaculture production of functional seafood . . . . . . . . . . . . . . . . 17.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 17.8 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

327 330 331

331 333 337 343 349 354 356 356 356

Mild processing techniques and development of functional marine protein and peptide ingredients . . . . . . . . . . . . . . . . . . . . . . . . . 363 G. Thorkelsson, S. Sigurgisladottir, M. Geirsdottir and R. JoÂhannsson, Matis, Iceland, F. GueÂrard and A. Chabeaud, UniversiteÂ de Bretagne Occidentale, France, P. Bourseau and L. Vandanjon, UniversiteÂ de Bretagne Sud, France, P. Jaouen and M. Chaplain-Derouiniot, UniversiteÂ de Nantes, France, M. Fouchereau-Peron, O. MartinezAlvarez and Y. Le Gal, MuseÂe National d'Histoire Naturelle, France,

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Contents R. Ravallec-Ple, ProBioGEM, France, L. Picot, UniversiteÂ de La Rochelle, France, J.P. Berge, Ifremer, France, C. Delannoy, Copalis, France, G. Jakobsen and I. Johansson, Marinova, Denmark and I. Batista and C. Pires, Ipimar, Portugal 18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2 Improved yield in traditional fish processing . . . . . . . . . . . . . . . . 18.3 Processing of marine proteins and peptides . . . . . . . . . . . . . . . . . . 18.4 Bioactive properties of fish protein hydrolysates and peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5 Functional properties of dried marine proteins and peptides . 18.6 Market for functional marine proteins and peptide products . 18.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 18.9 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Hurdle technology to ensure the safety of seafood products . . . F. Leroi and J.J. Jofftaud, Ifremer, France, J.C. Arboleya, F. Amarita, Z. Cruz, E. Izurieta, A. Lasagabaster, I. MartõÂnez de MaranÄoÂn, I. Miranda, M. Nuin and I. Olabarrieta, AZTI-Tecnalia, Spain, H.L. Lauzon, Matis, Iceland, G. Lorentzen and I. Bjùrkevoll, Nofima, Norway, R. Olsen, University of Tromsù, Norway, M.F. Pilet, H. PreÂvost, X. Dousset and S. Matamoros, ENITIAA, France and T. Skjerdal, National Veterinary Institute, Norway 19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.2 Salt hurdle in seafood processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3 Biopreservation of lightly preserved seafood products . . . . . . . 19.4 Antimicrobial compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.5 Antimicrobial packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.6 Pulsed light as a novel decontamination technology . . . . . . . . . 19.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.8 Source of further information and advice . . . . . . . . . . . . . . . . . . . . 19.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 Preventing lipid oxidation in seafood . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Jacobsen, Technical University of Denmark, Denmark, I. Undeland, Chalmers University of Technology, Sweden, I. Storrù, SINTEF Fisheries and Aquaculture, Norway, T. Rustad, Norwegian University of Science and Technology, Norway, N. Hedges, Unilever, United Kingdom and I. Medina, Consejo Superior De Investigaciones Cientificas, Spain 20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2 Processes leading to lipid and protein oxidation in seafood . . 20.3 Common analytical methods to evaluate oxidation . . . . . . . . . . .

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363 364 368 372 380 384 386 387 388 388 399

399 400 405 411 413 415 418 419 420 426

426 427 433

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Contents 20.4 20.5 20.6 20.7 20.8 20.9 20.10 20.11 20.12 20.13

Part V

Introduction to model systems for use in seafood oxidation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetics and modelling of lipid oxidation in liposomes and emulsions using the oxygen uptake rate . . . . . . . . . . . . . . . . . . . . . . Effect of emulsifiers and antioxidants on lipid oxidation in oil-in-water emulsion model systems . . . . . . . . . . . . . . . . . . . . . . . . Washed fish mince as model systems . . . . . . . . . . . . . . . . . . . . . . . . Natural antioxidants in fish products . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of further information and advice . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

434 435 439 442 446 450 451 452 453 453

Seafood from aquaculture

21

Introduction to Part V: seafood from aquaculture ± added value possibilities and potential impacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 B. DamsgaÊrd, Nofima, Norway

22

The biological basis of variability in the texture of fish flesh . . I.A. Johnston, University of St Andrews, Scotland 22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 Muscle texture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3 Organisation, structure and biochemistry of fish myotomes . . 22.4 Cellular and molecular mechanisms of muscle growth . . . . . . . 22.5 Relationship between muscle structural traits and texture . . . . 22.6 Proteolytic enzymes and post-mortem softening of the flesh . 22.7 Environmental influences on muscle structural traits . . . . . . . . . 22.8 Heritability of muscle structural traits . . . . . . . . . . . . . . . . . . . . . . . . 22.9 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.10 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.11 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 Fish welfare and ethical qualities in aquaculture . . . . . . . . . . . . . . . B. DamsgaÊrd, Nofima, Norway 23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 Terms and definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.3 Fish farmers and consumers seeking a common destiny . . . . . 23.4 Welfare during the production cycle . . . . . . . . . . . . . . . . . . . . . . . . . 23.5 Welfare during slaughter of farmed fish . . . . . . . . . . . . . . . . . . . . . 23.6 Monitoring ethical qualities in farmed fish . . . . . . . . . . . . . . . . . . . 23.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 23.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contents Part VI 24

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Seafood traceability to regain consumer confidence

Introduction to Part VI: traceability in a changing world . . . . . E. P. Larsen, Technical University of Denmark, Denmark

513

25 Improving traceability in seafood production . . . . . . . . . . . . . . . . . . . J. Storùy, G. Senneset and E. ForaÊs, SINTEF Fisheries and Aquaculture, Norway, P. Olsen and K.M. Karlsen, Nofima, Norway and M. Frederiksen, Technical University of Denmark, Denmark 25.1 The METHODS project: introduction . . . . . . . . . . . . . . . . . . . . . . . . 25.2 The vocabulary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.3 Good Traceability Practice manual . . . . . . . . . . . . . . . . . . . . . . . . . . 25.4 The SEAFOODplus map service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.5 The IMPLEM project: introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 25.6 Data capture technology ± Radio frequency identification data (RFID) tags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.7 Choice of technology/equipment: introduction . . . . . . . . . . . . . . . 25.8 Testing radio frequency temperature loggers at Fjord Seafood Herùy, Norway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.9 Fish chain process studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.10 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

516

529 530 537

26

539

Validation of traceability in the seafood production chain . . . . . B. PeÂrez-Villarreal, F. AmaÂrita, C. Bald, M.A. Pardo and I. Sagardia, AZTI-Tecnalia, Spain 26.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.2 Principles for the validation of traceability . . . . . . . . . . . . . . . . . . . 26.3 Establishment of indicators for the validation of a traceability system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.4 Specific tools for validation of the most important traceable data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.5 Methods for validation of traceability to ensure safety . . . . . . . 26.6 Validation of traceability for quality concerns . . . . . . . . . . . . . . . 26.7 Validation methodologies to prevent fraud . . . . . . . . . . . . . . . . . . . 26.8 Validation of data management and information flow . . . . . . . 26.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.10 References and bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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539 540 542 545 545 550 554 559 559 560 567

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Contributor contact details

(* = main contact)

Preface and Chapter 1 Professor Torger Bùrresen Technical University of Denmark National Institute of Aquatic Resources Department of Seafood Research Building 221, Sùltofts Plads DK-2800 Kgs. Lyngby Denmark E-mail: [email protected]

Chapter 2 Professor K. Brunsù MAPP, Aarhus School of Business University of Aarhus Haslegaardsvej 10 DK-8210 Aarhus V Denmark E-mail: [email protected]

Chapter 3 Professor K. Brunsù,* K.B. Hansen and Professor J. Scholderer MAPP, Aarhus School of Business University of Aarhus Haslegaardsvej 10 DK-8210 Aarhus V Denmark E-mail: [email protected] Dr P. Honkanen Nofima PO Box 6122 NO-9291 Tromsù Norway E-mail: [email protected] Professor S.O. Olsen Department of Social Science and Marketing Norwegian College of Fishery Science University of Tromsù N-9037 Tromsù

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Contributors

Chapter 5

Norway E-mail: [email protected]

Professor W. Verbeke* and Dr Z. Pieniak Department of Agricultural Economics Ghent University Coupure links 653 B-9000 Gent Belgium E-mail: [email protected]

Professor W. Verbeke Department of Agricultural Economics Ghent University Coupure links 653 B-9000 Gent Belgium E-mail: [email protected]

Chapter 4 Ms E. MartinsdoÂttir* and Ms K. SveinsdoÂttir MatõÂs Skulagata 4 IS-101 ReykjavõÂk Iceland E-mail: [email protected] Ms D. Green-Petersen and Dr G. Hyldig Technical University of Denmark National Institute of Aquatic Resources (DTU Aqua) Building 221, Sùltofts Plads DK-2800 Kgs. Lyngby Denmark E-mail: [email protected] Ms R. Schelvis Wageningen IMARES (Institute for Marine Resources & Ecosystem Studies) P.O. Box 68 1970 AB IJmuiden The Netherlands E-mail: [email protected]

Professor K. Brunsù and Professor J. Scholderer MAPP, Aarhus School of Business University of Aarhus Haslegaardsvej 10 DK-8210 Aarhus V Denmark E-mail: [email protected] Professor S.O. Olsen Department of Social Science and Marketing Norwegian College of Fishery Science University of Tromsù N-9037 Tromsù Norway E-mail: [email protected]

Chapter 6 Professor S.O. Olsen* Nofima and Department of Social Science and Marketing Norwegian College of Fishery Science University of Tromsù N-9037 Tromsù Norway E-mail: [email protected]

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Contributors

Chapter 8

Dr K. Toften Nofima Market PO Box 6122 NO-9291 Tromsù Norway E-mail: [email protected]

Dr E. Lund* Gastrointestinal Biology and Health Institute of Food Research Norwich NR4 7UA UK E-mail: [email protected]

Dr D. Calvo Dopico and Ms A. Tudoran Department of Economic Analysis and Business Administration Faculty of Economics University of CorunÄa Campus ElvinÄa s/n 15071 A CorunÄa Spain E-mail: [email protected]

Professor E. Kampman Wageningen University and Research Centre Division of Human Nutrition 6703 HD Wageningen Netherlands E-mail: [email protected]

Mr A. Kole The Centre for Innovative Consumer Studies, The Institute for Marine Resources and Ecosystems Studies Wageningen University and Research Centre PO Box 16 6700 AA Wageningen The Netherlands

Chapter 7 Professor Gertjan Schaafsma HAN University P.O. Box 6960 6503 GL Nijmegen The Netherlands E-mail: [email protected]

xvii

Chapter 9 Professor Inga Thorsdottir* and Dr Alfons Ramel Unit for Nutrition Research Landspitali University Hospital and Department of Food Science and Human Nutrition University of Iceland Eiriksgata 29 101 Reykjavik Iceland E-mail: [email protected] [email protected]

Chapter 10 Dr Ingeborg A. Brouwer Institute of Health Sciences Free University Amsterdam De Boelelaan 1085 1081 HV Amsterdam The Netherlands E-mail: [email protected]

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Contributors

Chapter 11

Chapter 13

Mr Bill DoreÂ Marine Institute Rinville Oranmore Galway Ireland E-mail: [email protected]

Dr M. Pommepuy* and J.C. Le Saux French Research Institute for the Exploitation of the Sea (Ifremer) Centre de Brest 29280 Plouzane France E-mail: [email protected]

Chapter 12

Dr F.S. Le Guyader French Research Institute for the Exploitation of the Sea (Ifremer) Nantes France E-mail: [email protected]

Dr A. Bosch* and Dr R.M. PintoÂ University of Barcelona Barcelona Spain E-mail: [email protected] Dr D. Lees Centre for Environment, Fisheries and Aquaculture Science (CEFAS) Weymouth Dorset DT4 8UB UK E-mail: [email protected] Professor C.-H. von Bonsdorff University of Helsinki Helsinki Finland E-mail: carl-henrik.vonbonsdorff@ helsinki.fi Dr L. Croci and Dr D. De Medici Istituto Superiore di SanitaÁ Viale Regina Elena 299 00161 Rome Italy E-mail: [email protected] [email protected] Dr F.S. Le Guyader French Research Institute for the Exploitation of the Sea (Ifremer) Nantes France E-mail: [email protected]

Mr S. Kershaw, Dr D. Lees, J.A. Lowther and O.C. Morgan Centre for Environment, Fisheries and Aquaculture Science (CEFAS) Weymouth Dorset DT4 8UB UK E-mail: [email protected] Dr J.L. Romalde and M.L. VilarinÄo Universidad de Santiago de Compostela Campus Sur s/n 15782 Santiago de Compostela Spain E-mail: [email protected] Dr D. Furones and Dr A. Roque The Institute of Agro-Food Research and Technology (IRTA) 43540 Sant Carles de la Rapita Tarragona Spain E-mail: [email protected] Mr F. Guilfoyle and Mr B. DoreÂ Marine Institute Rinville Oranmore

WPNL0206

Contributors Galway Ireland E-mail: [email protected]

Chapter 14 Dr R.J. Lee* and Dr R.E. Rangdale Centre for Environment, Fisheries and Aquaculture Science (CEFAS) Barrack Road The Nothe Weymouth Dorset DT4 7TF UK E-mail: [email protected] [email protected] Dr L. Croci Istituto Superiore di SanitaÁ Viale Regina Elena 299 00161 Rome Italy E-mail: [email protected] Dr D. Hervio-Heath and S. Lozach French Research Institute for the Exploitation of the Sea (Ifremer) Centre de Brest PO Box 70 29280 Plouzane France E-mail: [email protected]

Chapter 15 Dr P. Dalgaard* and Dr J. Emborg Technical University of Denmark National Institute of Aquatic Resources Department of Seafood Research Building 221, Sùltofts Plads DK-2800 Kgs. Lyngby Denmark E-mail: [email protected]

xix

Ms A. Kjùlby, N.D. Sùrensen and N.Z. Ballin Danish Veterinary and Food Administration Region East Sùndervang 4 4100 Ringsted Denmark

Chapter 16 Dr J.B. Luten Nofima PO Box 6122 NO-9291 Tromsù Norway E-mail: [email protected]

Chapter 17 Dr M. Careche,* Professor F. SauraCalixto, Dr M.E. DõÂaz-Rubio, Professor A.J. BorderõÂas, Dr I. SaÂnchez-Alonso and Dr I. SaÂnchez-GonzaÂlez Instituto del FrõÂo (CSIC) JoseÂ Antonio Novais 10 28040 Madrid Spain E-mail: [email protected] Dr J.B. Luten Nofima PO Box 6122 NO-9291 Tromsù Norway E-mail: [email protected] Ms R. Schelvis and Mr E. Schram Wageningen IMARES (Institute for Marine Resources & Ecosystem Studies) P.O. Box 68 1970 AB IJmuiden

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Contributors

The Netherlands E-mail: [email protected] [email protected] Mr A. Kole The Centre for Innovative Consumer Studies, The Institute for Marine Resources and Ecosystems Studies Wageningen University and Research Centre PO Box 16 6700 AA Wageningen The Netherlands E-mail: [email protected] Dr O.E. Scholten, M.A.J. Toonen Plant Research International (PRI) Wageningen University and Research Centre PO Box 16 6700 AA Wageningen The Netherlands E-mail: [email protected] Dr P. Carmona Institute of Structure of Matter (CSIC) Serrano 121 28006 Madrid Spain E-mail: [email protected] Dr T.R. Gormley Ashtown Food Research Centre (Teagasc) Dublin 15 Ireland E-mail: [email protected] Professor J. OehlenschlaÈger and Dr S. Mierke-Klemeyer Federal Research Centre for Nutrition and Food

Department of Seafood Research Palmaille 9 D-22767 Hamburg Germany E-mail: [email protected] Professor E. Elvevoll Norwegian College of Fishery Science Institute for Marine Biotechnology (IMAB) University of Tromsù Tromsù Norway E-mail: [email protected] Professor M. Leonor Nunes and Dr N. Bandarra National Institute of Biological Resources INRB/IPIMAR Research Unit of Marine and Aquaculture Fish Products Upgrading Avenida de Brasilia 1449-006 Lisboa Portugal E-mail: [email protected] Dr I. Stoknes Mùre Research PO Box 5075 N-6021 Aalesund Norway Professor E.H. Larsen The National Food Institute Technical University of Denmark 2860 Sùborg Denmark E-mail: [email protected]

WPNL0206

Contributors

Chapter 18

Station de Biologie Marine 29900 Concarneau France E-mail: [email protected]

Mr G. Thorkelsson,* Dr S. Sigurgisladottir, Dr R. JoÂhannsson and Ms M. Geirsdottir Matis Skulagata 4 IS 101 Reykjavik Iceland E-mail: [email protected] Dr F. GueÂrard and Ms A. Chabeaud UniversiteÂ de Bretagne Occidentale PoÃle universitaire Pierre-Jakez Helias 18 avenue de la Plage des Gueux 29018 Quimper cedex France E-mail: [email protected] Professor P. Bourseau and Dr L. Vandanjon Laboratoire Polymeres et Procedes Universite de Bretagne Sud Rue de Saint Maude BP 92116 56321 Lorient Cedex France E-mail: [email protected] Professor P. Jaouen and M. Chaplain-Derouiniot ISOMer, Cnt Rech & Trans Tech Lab Genie Procedes UniversiteÂ de Nantes Blvd Univ BP 406 F-44602 St Nazaire France E-mail: [email protected] Dr M. Fouchereau-Peron, O. Martinez-Alvarez and Professor Y. Le Gal UMR 5178 BOME

xxi

Dr R. Ravallec-PleÂ ProBioGEM Polytech' Lille BP 179 F-59653 Villeneuve Dascq France E-mail: [email protected] Dr L. Picot UFR Sci Fondamentales & Sci Ingn FRE 2766 CNRS, Lab Biotechnol & Chim Bioorgan UniversiteÂ de La Rochelle Batiment Marie Curie F-17042 La Rochelle France E-mail: [email protected] Dr J.P. Berge French Research Institute for the Exploitation of the Sea (Ifremer) BP211105 F-44311 Nantes 03 France E-mail: [email protected] Mr C. Delannoy Copalis B.P.239 62203 Boulogne sur Mer Cedex France E-mail: [email protected] Ms G. Jakobsen and Ms I. Johansson Marinova Adelvej 11 Hoejmark DK-6940 Lem St. Denmark E-mail: [email protected]

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Contributors Norway E-mail: [email protected]

Mr I. Batista and Dr C. Pires National Institute of Biological Resources INRB/IPIMAR Research Unit of Marine and Aquaculture Fish Products Upgrading Avenida de Brasilia 1449-006 Lisboa Portugal E-mail: [email protected]

Dr M.F. Pilet, Professor H. PreÂvost, X. Dousset and S. Matamoros UMR INRA 1014 SECALIM ENITIAA Rue de la geÂraudieÁre 44322 Nantes Cedex 03 France E-mail: [email protected]

Chapter 19 Dr F. Leroi* and Dr J.J. Joffraud French Research Institute for Exploitation of the Sea (Ifremer) Rue de l'Ile d'Yeu BP 21105 44311 Nantes Cedex 03 France E-mail: [email protected] Dr T. Skjerdal National Veterinary Institute P.O. Box 750 Sentrum N-0033 Oslo Norway E-mail: [email protected] Ms G. Lorentzen and Mr I. Bjùrkevoll Nofima Muninbakken 9-13 Postbox 6122 NO-9291 Tromsù Norway E-mail: [email protected] [email protected] Professor R.L. Olsen Norwegian College of Fishery Science University of Tromsù N-9037 Tromsù

Dr F. Amarita, J.C. Arboleya, Z. Cruz, E. Izurieta, A. Lasagabaster, I. MartõÂnez de MaranÄoÂn, I. Miranda, M. Nuin and I. Olabarrieta AZTI-Tecnalia Food Reseach Division Txatxarramendi Ugartea z/g. 48395 Sukarrieta Spain E-mail: [email protected] Ms H.L. Lauzon MatõÂs Skulagata 4 IS-101 Reykjavik Iceland E-mail: [email protected]

Chapter 20 Dr C. Jacobsen* Technical University of Denmark National Institute of Aquatic Resources Department of Seafood Research Building 221, Sùltofts Plads DK-2800 Kgs. Lyngby Denmark E-mail: [email protected]

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Contributors Dr I. Undeland Food Science, Chalmers University of Technology SE-412 96 GoÈteborg Sweden E-mail: [email protected] Dr I. Storrù SINTEF Fisheries and Aquaculture N-7465 Trondheim Norway E-mail: [email protected] Professor T. Rustad Department of Biotechnology Norwegian University of Science and Technology (NTNU) NO-7491 Trondheim Norway E-mail: [email protected] Dr N. Hedges Unilever UK E-mail: [email protected]

xxiii

Chapter 22 Professor Ian A. Johnston Gatty Marine Laboratory School of Biology University of St Andrews St Andrews Fife KY16 8LB Scotland E-mail: [email protected]

Chapter 24 Mr Erling P. Larsen Technical University of Denmark National Institute of Aquatic Resources Department of Seafood Research Building 221, Sùltofts Plads DK-2800 Kgs. Lyngby Denmark E-mail: [email protected]

Chapter 25

Dr I. Medina IIM/CSIC Eduardo Gabello 6 36208 Vigo Spain E-mail: [email protected]

Mr J. Storùy,* Mr Gunnar Senneset and Mr Eskil ForaÊs SINTEF Fisheries and Aquaculture N-7465 Trondheim Norway E-mail: [email protected]

Chapters 21 and 23 Dr Bùrge DamsgaÊrd Nofima PO Box 6122 NO-9291 Tromsù Norway E-mail: [email protected]

Mr Petter Olsen and Ms Kine Mari Karlsen Nofima PO Box 6122 NO-9291 Tromsù Norway E-mail: [email protected]

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Contributors

Dr Marco Frederiksen Technical University of Denmark National Institute of Aquatic Resources Department of Seafood Research Building 221, Sùltofts Plads DK-2800 Kgs. Lyngby Denmark E-mail: [email protected]

Chapter 26 Dr B. PeÂrez-Villarreal,* Dr F. AmaÂrita, Dr C. Bald, Dr M.A. Pardo and I. Sagardia AZTI-Tecnalia Food Research Division Isla de Txatxarramendi 48395 Sukarrieta (Bizkaia) Spain E-mail: [email protected]

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Preface

The background to this book is the Integrated Research Project SEAFOODplus, which started officially on 1 January 2004, supported by funding from the European Union following an application from a consortium of more than 70 partners, including universities, research institutes and private companies. The research plan covered a 412-year period and comprised all the topics presented in this book. The authors finished their chapters by Autumn 2007, and so the final results from the projects have not been included in the present edition. However, this research should be considered as a contribution within a continuum of ongoing development, and the presentations given in the book should be viewed in this context. I should like to express my gratitude to all the authors for their contributions to the book and their great commitment to the research carried out as part of SEAFOODplus. The consortium was built up over a period of about two years before the funding application to the European Commission was submitted. In total, more than 200 researchers have been engaged in the research work, supported by 14.4 million euros from the EU, and a total budget of 26 million euros with the addition of the contributions from participating member institutions and companies. One important aspect of this project has been the integration of research from different fields, covering human nutrition, consumer studies, seafood safety, seafood quality, production systems, aquaculture and traceability. The research environments within each of these fields in Europe were scattered and uncoordinated, so it was a considerable challenge to pull together the best expertise and make new collaborative connections between the research groups. However, the results have been very rewarding. When researchers were brought together with other researchers with whom they had not had regular contact, new

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Preface

inspiration emerged. The extension of traditional seafood technological research to also include human nutrition and consumer studies has resulted in a considerable step forward within seafood research ± hence the term SEAFOODplus. When creating the consortium, we were encouraged to be ambitious and break new ground within seafood research. It gives me great pleasure to thank Mr Liam Breslin, then Head of the Unit for Food Safety within EC DG Research, for his constant inspiration. It was later a pleasure to have Mr Ciaran Mangan as the project's Scientific Officer, and I would like to take this opportunity to thank him for providing excellent contact with the EC throughout the project period. The first demonstration of a successful integration of European seafood research is now evident with the publication of this book, in which the research topics are presented collectively. Thanks are due to the many people who have made this possible: firstly the research coordinators and project leaders within SEAFOODplus, who have contributed so successfully to the book as authors, among whom Dr Joop Luten deserves special thanks for his very dedicated work to manage the research area of SEAFOODplus. It is further my pleasure to thank the members of the Industry, Training and Dissemination (ITD) team: Dr Lucay Han-Ching, Dr Maria Leonor Nunes, Dr BegonÄa Perez-Villarreal, Dr SjoÈfn Sigurgisladottir, Prof. JoÈrg OehlenschlaÈger and Dr Mercedes Careche. Above all, my thanks go to the staff at the SEAFOODplus secretariat for their very skilled work: Secretariat Manager Mrs Jette Donovan Jensen and Financial Manager Mr Jim Codd. Last but not least, thanks go to all the SEAFOODplus consortium members and their families for allowing work to be carried out outside regular working hours, in spare time and during holiday periods. Thank you for your commitment! Professor Torger Bùrresen Coordinator SEAFOODplus Copenhagen

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1 Introduction T. Bùrresen, Technical University of Denmark, Denmark

1.1

Introduction

While most consumers perceive seafood as healthy and nutritious, there is a lack of a thorough understanding, enabling comparison across Europe, of aspects that determine the levels of seafood consumption, such as consumers' motives, barriers, quality perception and information requirements. It is well known that a diet containing seafood, and in particular omega 3 fatty acids, will reduce the occurrence of cardiovascular diseases, but less is known about the other health benefits of a seafood diet. Some consumers are concerned about seafood safety, as some products may contain contaminants that lead to illness or components which could possibly lead to long-term negative effects on health. It is therefore important to reduce these obstacles so that consumers can obtain all the benefits of seafood consumption. The objective of this book is to provide an overview of the most recent research into understanding the consumers' attitudes toward seafood, the essential factors relating to seafood nutrition, safety and eating quality, as well as the opportunities for new supplies presented by aquaculture. The opportunity to compile this overview was very timely as the lead authors were the coordinators and project leaders of the Integrated Research Project SEAFOODplus. The overall approach to the research for this project is illustrated in Fig. 1.1, in which the seafood production chain is shown. A fork-to-farm consideration was applied, starting with the factors of importance for consumers' health and wellbeing, and working backwards in the chain, seeking the best raw materials and processing conditions. This leads to seafood products with optimal features with regard to nutrition, safety and eating quality.

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Improving seafood products for the consumer

Fig. 1.1 The overall approach for research within SEAFOODplus. Percentages for amounts of fish originating from capture fisheries and from aquaculture are given by FAO statistics 2006 (FAO, 2007) and are likely to shift to higher percentages originating from aquaculture in the future.

The book is divided into parts and chapters reflecting this approach, starting with studies of how to gain a better understanding of consumer perceptions of seafood and how messages about seafood nutrition and safety can best be communicated to them. Seafood consumption is balanced between positive health messages and frequently observed statements about the potential risks from environmental contaminants, particularly in oily fish. This is unfortunate, as oily fish are also the species that contain most of the health-positive omega 3 fatty acids. It should be emphasised that the negative statements concern potential risks, as most of the experimental background for recommending reduced seafood intake results from animal studies, often with exaggerated doses of the different components. Obviously, intervention studies with contaminants cannot be performed on human beings, and until now few epidemiological studies have allowed firm conclusions to be drawn. However, some evidence is gathering to show that consumers who have ingested large amounts of seafood with high contaminant levels are actually performing better in health tests than comparable groups with a lower seafood intake, in spite of the accumulated higher doses of contaminants ingested. It thus seems that a seafood diet counteracts the potential negative effects, which would be real if only the contaminants themselves were ingested. A study by Mozzafarian and Rimms (2006) leads to the simple conclusion that the benefits of fish intake exceed the potential risks. The message that seafood may contain harmful substances is continuously broadcast by the media and seems to come across to the public in a simplified form, resulting in the information being understood as `fish is harmful'. Michael T. Morrissey at Oregon State University expresses his concerns about the situation in an editorial called `Misinformation' (Morrissey, 2003), in which he warns that over-complex messages transmitted by, e.g. medical doctors, are interpreted as `the consumer should not eat seafood'.

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Introduction

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In the SEAFOODplus safety projects, the focus was on the real safety risks which may occur as a consequence of contamination by viruses or bacteria. If the incidence of direct illnesses caused by viral or bacterial infections or by the toxins produced by bacteria could be eliminated, consumers would be safer when having a seafood meal. Combined with a production chain with full traceability, this would give consumers the basis for the most credible seafood products.

1.2

Structure of the book

Throughout the book, reference is made to project work carried out in SEAFOODplus, reflecting the different projects, in most cases giving only the acronym of the specific project in question. In order to give a better picture of all the projects of SEAFOODplus and to explain what lies behind each acronym, the following overview of projects and objectives is included, and it is indicated in which parts of the book the different projects are being presented. Part I: Consumers and seafood The fact that seafood consumption seems to be dropping in spite of general knowledge that seafood is healthy is being addressed in baseline studies and research, revealing consumer motives and barriers to seafood consumption across Europe. The lack of knowledge about consumers' preferential behaviour, demands for information, and the impact and effectiveness of health, safety and ethical messages relating to seafood, is also addressed. Finally, there is discussion leading towards a better understanding of how the sensory-quality attributes of seafood are perceived by consumers. Project CONSUMERSURVEY: Seafood Consumption ± Explaining attitudes, preferences and eating habits across consumer segments in Europe The objective was to develop an integrated approach towards explaining seafood consumption covering two areas: consumers' choice of food and consumers' choice of seafood. Important levels of analysis covered by this project include motives and barriers to seafood consumption, cross-cultural variations in Europe, attitudes and preferences in relation to seafood, and last but not least, how these aspects are linked to lifestyles, perceived health and well-being from a consumer's point of view. Project SEAFOODSENSE: Improved seafood sensory quality for the consumer The objective was to develop and apply consumer-oriented Seafood Sensory Quality Models that will enable the seafood industry to improve the eating quality of seafood available to consumers, encourage increased seafood consumption, and in so doing, contribute to improved consumer health.

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Improving seafood products for the consumer

Project SEA-INFOCOM: Seafood Information and Communication ± Assessment of consumers' needs for seafood information and the development of effective seafood communication The objective was to assess consumers' needs for information about seafood and develop effective communication about seafood, relating to traceability, health, safety and ethical issues. Project CONSUMEREVALUATE: Consumer evaluation and willingness to buy convenience and tailor-made seafood products The objective was to explore and explain consumers' preferences, evaluation and buying behaviour relating to convenience and tailor-made seafood products. Part II: The health benefits of seafood The relevance of seafood in the diet to diminish the increased incidence of nutrition-related chronic diseases (cardiovascular, cancer and inflammatory) was addressed in SEAFOODplus by performing dietary intervention and epidemiological studies in areas where seafood may contribute to reducing or preventing the development of such diseases. Another focus area was the health of young populations, to treat obesity, prevent the development of osteoporosis and specifically focus on the high rate of postpartum depression observed in women giving birth. Project FISHGASTRO: Gastro-intestinal health with special emphasis on the reduction of the risk of colon cancer and inflammatory bowel disease The objective was to clarify to what extent fish consumption improves the health of the gastrointestinal tract, what aspects of fish are important in this respect, and what are the mechanisms of protection. Project YOUNG: Health of young European families and fish consumption The objective was to increase knowledge about the nutritional effects of fish constituents, to promote health and prevent diseases in young European families. Project METAHEART: Metabolism of n-3 fatty acids and heart disease The objectives were to provide proof about the major protective effect of seafood against the risk of heart disease; to unravel its underlying mechanism; to study the potential of different dietary n-3 fatty acids; and to determine how the conversion and metabolism of n-3 fatty acids in the human body are controlled and how they can be modulated by other dietary factors. Furthermore, this project investigated whether and how dietary n-3 fatty acids can prevent cardiac arrhythmia and related heart disease risk, and which specific n-3 fatty acids in seafood and other foods may be responsible for this effect.

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Part III: Ensuring seafood safety Among the clearest food safety risks connected with seafood consumption are diseases caused by viral shellfish poisoning and the continuing occurrence of histamine poisoning in Europe. These topics were selected as relevant research areas, as well as studies of how contamination with the pathogenic bacteria, such as Vibrio species, can be controlled. Project REFHEPA: Development of standard reference methods for Hepatitis A virus and Norovirus in bivalve molluscan shellfish The objective was to develop sensitive, quantitative and standardised ISO (International Standards Organisation) polymerase chain reaction (PCR)-based methods for the detection of Hepatitis A virus and Norovirus in bivalve molluscan shellfish. Project REDRISK: Reduction of risk in shellfish harvesting areas The objectives were to identify pollution sources and the conditions responsible for microbial contamination in shellfisheries, and to determine their impact on viral contamination in shellfisheries. This will provide a framework for the development of a preventative strategy to reduce the virus risk associated with shellfish, by using a risk management approach in shellfish harvesting areas, based on HACCP principles. A preventative strategy of this kind will reduce the virus risk associated with the consumption of bivalve molluscan shellfish for the European consumer. Project SEABAC: Enhanced assessment of bacterial-associated contamination The objective was to develop standardised techniques to detect and characterise pathogenic Vibrios. This will facilitate future assessment of the health risks posed to European consumers by these organisms. Project BIOCOM: Biogenic amines in seafood ± assessment and management of consumer exposure The objective was to provide data that will reduce European consumers' intake of biogenic amines from seafood and reduce the incidence of histamine fish poisoning (HFP). Part IV: Seafood from source to consumer product In order to retain the intrinsic qualities of seafood, it is necessary to consider the whole production chain. Special attention is needed to obtain tailor-made products with satisfying eating characteristics such as taste and texture. Emphasis was placed on the prevention of contamination with pathogens during the production of perishable seafood convenience products. Another challenge was to exploit the health-promoting compounds contained in fractions that today are considered as by-products.

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Improving seafood products for the consumer

Project CONSUMERPRODUCTS: Consumer-driven development of innovative tailor-made seafood products, with functional components of plant or marine origin, to improve the health of consumers The objectives were to develop innovative, functional seafood products from both capture (under-utilised) and farmed fish, containing health-promoting compounds aimed at the improvement of intestinal health and lipid metabolism, as well as the potential prevention of cancer. Project PROPEPHEALTH: High-added value functional seafood products for human health from seafood by-products by innovative mild processing The objectives were to screen, map and recover `new' health-beneficial compounds from seafood by-products by advanced mild refining processes; to develop `new' bioactive (functional) seafood ingredients; and to use these novel ingredients, either directly in the food industry, or in the CONSUMERPRODUCTS project for the development of new, functional seafood products, accepted by the target consumers. Project HURDLETECH: Hurdle technology, including minimal processing, to ensure the quality and safety of convenience seafood The objective was to ensure the safety and quality of convenience seafood products. Project LIPIDTEXT: Preventing seafood lipid oxidation and texture softening to maintain healthy components and quality of seafood The objectives were to secure and maintain the high sensory quality (colour, flavour, texture parameters) and nutritional value (high level of anti-oxidants, n3 lipids, and low levels of potentially toxic oxidation products) of seafood products, including fresh and frozen fish fillets, fish-based products and fish oilenriched systems. Part V: Seafood from aquaculture Intensive production of seafood from aquaculture presents both opportunities and potential environmental impacts. In order to meet consumer needs and expectations of healthy, high-quality seafood, the full potential of farmed fish has to be developed for a diversity of species reared in sustainable and environmentally friendly systems. Quality and ethical factors were addressed in studies investigating genomic and physiological traits, as well as husbandry practices and slaughtering methods for a wide range of European farmed fish, including freshwater species. Project BIOQUAL: Physiology and genetics of seafood quality traits The objectives were to establish novel endocrinological, physiological and genetic tools in order to identify quality traits in finfish aquaculture; to apply these to fish-fed novel diets; to lay the foundations for the establishment of high-

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Introduction

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throughput protein-array technology to assess muscle quality and produce gene microarrays, to aid in broodstock selection based on quality traits. Project ETHIQUAL: Ethical quality traits in farmed fish ± the role of husbandry practices and aquaculture production systems The objective was to examine how husbandry practice, aquaculture systems and pre-slaughter conditions contribute to the flesh quality and ethical quality of finfish seafood. Part VI: Seafood traceability to ensure consumer confidence In today's production systems valuable information is lost, leading to products for which documented authenticity cannot be provided. It is thus a challenge to implement validated traceability systems, agreed and accepted by all the players in the production chain. Project METHODS: Methodology The objectives were to define the vocabulary for the `shall elements' in the existing Tracefish standard so that it can be easily used in the whole fishery industry in practice; to add and define new elements of information from the results of other research areas in the SEAFOODplus project; and to develop a Good Traceability Practice (GTP) guideline `manual'. Project IMPLEM: Implementation The objectives were to study current information flow in case chains; to specify what changes are needed in each link in order to ensure that traceability is in place; to test, evaluate and make suggestions for the improvement of advanced technology for global batch identification and data catch; and to integrate data captured with advanced technology into traceability software with functionality for data storage and transmission. Project VALID: Validation The objectives were to validate the traceability systems developed and implemented in different fish production chains across Europe; and to validate the traceability data coming from the chains by testing different tools, such as authenticity methods and other specially adapted tools and methods for the validation process. Although the research undertaken in each of the projects is described in the specific chapters, there was considerable integration between the different projects. This is highlighted throughout the book and collaboration between the researchers in the various disciplines is explained.

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1.3

Improving seafood products for the consumer

Future trends

Based on the results emerging from SEAFOODplus, new avenues for research can be indicated and new directions given for the further development of the seafood sector. As already mentioned, media misinformation to the general public makes it difficult for consumers to take full benefit of seafood diets. Observations made in the consumer studies show that consumers do not place very much trust in media information, and it leads to confusion. Medical doctors, however, come at the top of the list of information sources that consumers do trust. If, for example, a negative message about consuming seafood originating from the media is channelled through a medical doctor, the effect is transformed into something that the consumer trusts. A new strategy could thus be formulated, targeting medical doctors to inform consumers about the health-beneficial effects of seafood diets. Evidence has been gathered in SEAFOODplus showing that it is not only omega 3 fatty acids that have positive health effects; other components, including the proteins, also have positive effects. In intervention studies with hypocaloric diets for weight reduction, it has been shown that such diets containing lean or oily fish lead to greater weight loss than a control diet, and the effect is more pronounced in men than in women. These are observational studies and the mechanisms behind the effects should be studied further to understand how seafood can be better used in the fight to reduce overweight and obesity. Other components, such as selenium and taurine, are also known to be present in high amounts in fish, but vary among fish species and tissues. A new concept has been investigated in SEAFOODplus, in which farmed fish is being used as a carrier for important trace elements. It has been shown that selenium can be enriched in plants from soil, and these can be included in the fish feed to produce farmed fish containing consistently high levels of selenium. In future, further work should be carried out to demonstrate the health effects and validity of such a concept to deliver necessary nutrients through seafood diets. Another promising development concerns functional seafood. Functional food is an area that is consistently expanding, and for seafood, new combinations have been tried in SEAFOODplus, showing that it is possible to add, e.g. plant fibre by-product fractions containing natural antioxidants, to restructured fish products, controlling oxidation and adding fibre components to the product. It is thus possible to utilise nutritionally valuable components from by-products. The by-product fractions from fish processing also contain many valuable components, and the screening for effects, e.g. within the pharmaceutical sector, as done in SEAFOODplus, is only the start of a trend that will expand considerably in the future. Some of the studies in the human nutrition area of SEAFOODplus have shown that depression in women after giving birth may be considerably reduced if they are given a seafood diet through pregnancy. This is an example of how seafood may impact on human psychology. Other studies have also revealed that

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Introduction

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seafood may have a pronounced effect on the development of brain functions. It is believed that further studies in this area will be very rewarding, particularly relating to very early development and through childhood.

1.4

References

(2007) The state of world fisheries and aquaculture 2006, FAO Fisheries and Aquaculture Department, Rome, 2007. MORRISSEY, M.T. (2003) Misinformation, J. Aquatic Food Prod. Technol. 12(2) 1±2. MOZZAFARIAN, D. and RIMMS, E.B. (2006), Fish Intake, Contaminants, and Human Health ± Evaluating the Risks and the Benefits, JAMA 296, 1885±1899. FAO

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Part I Consumers and seafood

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2 Introduction to Part I: consumers and seafood K. Brunsù, University of Aarhus, Denmark

In general motive and value fulfilment are major antecedents for consumer food decision making as well as being very important for consumers' seafood choices. Four general motives or values for food and seafood choices have earlier been distinguished; they are health, taste, convenience and processrelated characteristics (Brunsù and Grunert, 2007), and the achievement of desired consequences, such as the expected health benefits achieved by eating specific foods, is an important driver for consumers' food and seafood choices. Earlier studies have revealed that many consumers consider fish and seafood as healthy, nutritious and tasty, and, as mentioned, health and taste are major drivers motivating consumers' food choices. Nonetheless, a number of European countries have experienced a decline in the overall consumption of fish. It has furthermore been established that young consumers especially consume less seafood compared to older generations, and that there are major differences in consumption levels across Europe. In order to improve the understanding of consumer attitudes, preferences and seafood choices, a number of new studies have been initiated, and the chapters in this section will provide new scientific insights related to consumers' seafood and fish choices in several respects. Chapter 3 `Consumer attitudes and seafood consumption in Europe' presents a consumer-oriented approach for explaining the variations in consumption levels across countries applying methodologies leading to comparable and valid results. New findings related to consumer attitudes and seafood consumption in Europe, e.g. motives and barriers to seafood consumption, are presented along with results on consumption patterns and how to understand differences in consumption levels based on attitudes and preferences. Furthermore, the particular

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Improving seafood products for the consumer

issue of convenience will be investigated, since seafood consumption to some degree seems to depend on the perception of seafood convenience as well as on how consumers perceive barriers in relation to the purchase and preparation of seafood. Chapter 4 `Improved eating quality of seafood: the link between sensory characteristics, consumer liking and attitudes' especially focuses on the important aspect of taste as perceived by consumers in various European countries. No earlier studies on a European level have managed to use real seafood samples in a cross-cultural setting to investigate the relationship between quality and taste evaluations as performed by experts and the experienced eating quality by consumers; new interesting findings in this respect will be presented and discussed. Furthermore, a sensory quality model for `translating' consumers' perception of eating quality to sensory characteristics perceived by key decision makers in the total seafood production chain will be introduced and discussed in terms of points of quality decision making, methods and measures used in the chain. Chapter 5 `Evaluating consumer information needs in the purchase of seafood products' focuses on consumers' use of and trust in information sources related to seafood, and here a number of important aspects of consumers' seafood choices are discussed. Very few studies have been made regarding the impact of health, safety and ethical information on consumer decision making in the case of seafood products. Consumer interest in different information cues, labelling and traceability is an important topic, since often consumer decision making and utility maximisation are disturbed by imperfect information or because consumers lack knowledge about how to use information cues. Different aspects will be discussed, especially how consumers perceive traceability and ethical issues related to seafood consumption and production (a production-related characteristic) will be treated, as well as how consumers differ in their use and trust in information across countries. Chapter 6 `Consumer evaluation of tailor-made seafood products' specifically deals with how to contribute to a deeper understanding of consumers' preferences and willingness to buy tailor-made seafood products. The chapter establishes and discusses a conceptual, theoretical and methodological platform for designing and measuring consumer evaluation and preferential behaviour related to new and tailor-made seafood products. Among other things the chapter discusses how different testing conditions/contexts influence the evaluation and motivation to buy products and the effect of time pressure. Also real tailor-made seafood products targeted at specific consumer segments are tested both in-home and out of home, and new insights into how consumers balance various health benefits, convenience benefits and taste benefits are presented and discussed. The chapters in the present part of the book describes the research confined to the projects dealing with consumer studies within SEAFOODplus, but as the research has been highly integrated, it will be evident in the individual chapters how research has been performed in concert with studies presented in the other

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Introduction to Part I: consumers and seafood

15

parts of the book as well. The closest contact has been to project issues reported in Part IV of the book.

2.1

References and GRUNERT, K. G. (2007). Consumer attitude measures and food product development. In H. MacFie (Ed.), Consumer-led food product development, pp. 197±222. Cambridge: Woodhead Publishing Ltd.

BRUNSé, K.

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3 Consumer attitudes and seafood consumption in Europe K. Brunsù, K. B. Hansen and J. Scholderer, University of Aarhus, Denmark, P. Honkanen and S. O. Olsen, Nofima, Norway and W. Verbeke, Ghent University, Belgium

3.1

Introduction

Research on consumer attitudes towards seafood and how consumer preferences can be used to explain cross-cultural differences is a fairly new research area in Europe. Comparisons of seafood consumption across European countries have revealed considerable differences in consumption levels, in spite of the fact that most consumers seem to perceive seafood as healthy and nutritious, and that health has proven to be a major driver motivating the consumption of food in general (Brunsù, 2003). Some countries have even experienced a decline in the consumption of fish. From a European health policy perspective, knowledge on what determines the consumption levels across Europe from a cross-cultural consumer perspective will be crucial for future attempts to change or increase seafood consumption. As a consequence, one of the main objectives of the CONSUMERSURVEY project in SEAFOODplus has been to explain the crosscultural differences and to investigate the contradiction in consumer knowledge and behaviour. Several new CONSUMERSURVEY studies have therefore been conducted to understand and explain the differences in seafood consumption levels in Europe by means of motives and barriers, attitudes, preferences and eating habits across consumer segments. Various scientific methodologies have been applied, e.g. in-depth qualitative investigations of motives and barriers to seafood consumption as well as quantitative studies of representative population groups in several countries. In this chapter we start by introducing earlier findings from the research field to define our point of departure, and next we

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Consumer attitudes and seafood consumption in Europe

17

will present results from the qualitative and quantitative studies focusing on four important aspects: 1. Results on motives and barriers revealed in group interviews. 2. Results on cross-cultural consumption patterns revealed in the consumer survey. 3. Similarities and differences in attitudes and preferences across Europe. 4. Convenience and consumer segments.

3.2

Consumer motives and barriers to seafood consumption

Fish is an important part of a healthy diet (Adams and Standridge, 2006; Mozaffarian and Rimm, 2006). It is an important source of a number of nutrients, particularly protein, retinol, vitamin D, vitamin E, iodine, selenium and the essential long-chain polyunsaturated fatty acids (PUFA), i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Welch et al., 2002). It is recommended that fish and seafood products take a prominent position in the human diet due to their beneficial effect on chronic degenerative diseases. The consumption of fish may protect against cancers (Caygill et al., 1996; Fernandez et al., 1999) and cardiovascular diseases (Nestel, 2000). Therefore, health authorities and the food industry have a joint interest in increasing the consumption of fish. Despite some negative news about the potential adverse health impact of contamination in wild or farmed fish (KrisEtherton et al., 2002; 2003), this food group maintains a healthy image among nutrition and food scientists, governments as well as consumers (Brunsù, 2003; Gross, 2003; Pieniak et al., 2004). Still, despite the healthy image of seafood, there are considerable consumption variations across Europe, making research with a focus on improved understanding of underlying drivers of consumption highly relevant. Earlier studies investigating motives for consumers' food choice in general and seafood choices in particular have shown that four general motives seems to be important to most consumers when choosing food including seafood: health benefits (here freshness seems to be a prominent aspect), taste, convenience and process characteristics (Brunsù et al., 2002; Grunert, 2005; Nielsen et al., 1997). Common motive fulfilments when consuming seafood are keeping the family healthy, preparing a meal for the whole family, social enjoyment and pleasure. But consumers also perceive barriers to consuming fresh fish. Fish is perceived as time-consuming to buy and to prepare, and some consumers have an aversion to the bones in fresh fish (Nielsen et al., 1997). The findings are consistent with results of other studies on fish with regard to the central attributes or evaluation criteria used by the consumers when buying or not buying fish (Marshall 1988; Olsen and Kristoffersen 1999; Verbeke and Vackier, 2005). A study conducted in the United Kingdom revealed the same trend: that boneless, filleted fish, with guaranteed freshness and more knowledge about cooking methods, would be an important incentive for respondents to increase their purchase of different kinds

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Improving seafood products for the consumer

of fish (Baird et al., 1987). Many of the aspects mentioned above are related to the level of experience, which seems to be a strong determinant of future consumption levels of fish (Myrland et al., 2000), together with consumer habits. A further investigation into why and how level of consumption and experience have this impact on food and seafood choice and consumption requires new in-depth research, with a particular focus on the difference between heavy and light consumers, in order to understand what may facilitate or hamper a future increase in seafood consumption in Europe. As a consequence, qualitative in-depth studies were conducted in two European countries ± Spain and Belgium ± with the aim of identifying motives and barriers to fish consumption. The choice of these two countries was deliberate because of their difference in seafood consumption levels. According to FASonline (2002) Spain has one of the highest consumption levels of fish in the world; approximately 40 kg/capita/year, whereas Belgium is among the countries with the lowest consumption of fish in Europe; approximately 10 kg/ capita/year (Brunsù, 2003). These countries served as a case to compare consumer preferences in a heavy and a light seafood user environment, and in-depth focus group discussions were carried out with consumers in each country. Owing to very different consumption levels in the two countries, the consumption levels of heavy and light users were specified differently in Spain and Belgium. A heavy user in Spain consumes fish 4±5 times a week, while a heavy user in Belgium consumes fish once a week or more. A Spanish light user consumes fish once or twice a week while the Belgian light user consumes fish once a month and some even more rarely. All in all six focus groups were carried out, three in each country, one with heavy users and two with light users. All respondents recruited for the focus group discussions were responsible for shopping and cooking. All groups were mixed as regards age in order to have both old and young consumers in each group. A common interview guide was developed to ensure consistency across groups and countries. In both countries professional research agencies were employed for the focus group interviews, e.g. agencies conducted the recruitment of participants by phone based on agreed recruitment criteria, secured adaptation and translation of the interview guides, and undertook professional facilitation of the group interviews. In Spain the group discussions were carried out in two different cities (Madrid and Bilbao) in order to explore possible regional differences between coastal and inland areas in Spain. In Belgium all groups were carried out in Ghent. The group interviews lasted between 150 and 180 minutes, they were videotaped and transcribed for subsequent analysis. 3.2.1 Major motives and barriers to seafood consumption In both countries, the most important motives for fish consumption are health and taste. Regardless of species, fish is perceived as healthy and important for human well-being. Fish is also thought of as essential in a balanced diet;

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Consumer attitudes and seafood consumption in Europe

19

however, consumers find it difficult to explain why fish is actually healthy. Except for the fact that fish is easy to digest, is recommended by doctors and has a low content of fat and cholesterol, hardly any of the consumers could further clarify what makes fish a healthy food product. When prompted, a number of participants, however, were able to mention omega-3 polyunsaturated fatty acids (PUFA) as the most likely reason for fish being healthy (Brunsù et al., forthcoming). Regardless of consumption level, the most often mentioned barrier to higher fish consumption is price, and in all focus groups price was mentioned as a reason for not increasing the consumption of fish. Consumers also brought up that there are no cheap fish species or fish meal solutions compared to, for instance, meat that can be bought at various price levels. One Belgian consumer did, however, mention that fish farming had lowered the prices for some species, such as, for example, salmon and lobster. Fish is perceived as light and easy to digest, which some perceive as an advantage, while others find it a problem that fish is not as substantial as meat. Some consumers therefore feel a need for consuming (and buying) more fish to feel satisfied, which adds to the perception of fish as expensive compared to other foods. In both countries, smell is considered a negative characteristic of fish. For instance, some consumers stated that after cooking a fish meal, not only does it leave an unpleasant odour in the kitchen but in the whole house. Another mentioned the presence of children, who are generally not considered to fancy fish, i.e. the composition of a household ± i.e. number and age of children ± may also influence the frequency of fish meals. The focus group participants explained children's dislike of fish with taste, smell and bones. Bones, however, are primarily seen as a problem in Belgium, while Spanish consumers do not think of bones a barrier to fish consumption (Brunsù et al., forthcoming). This result reflects the different levels of experience with fish: whole, fresh fish where consumers have to remove the bones are more popular in Spain, whereas Belgian consumers prefer filleted, fresh fish, pre-packed fresh fish and deepfrozen fish, which are all easy to prepare. Time is another barrier, which is, however, only relevant to Spanish consumers and only to some of the consumer groups. In particular, light users are concerned about time and tend to perceive the cooking of fish as more difficult and time consuming than heavy users who generally like to cook and who cook fish very often. The elderly heavy users generally spend much time in the kitchen and they cook using sophisticated recipes. Young heavy users also tend to like cooking; however, most of them work and as a consequence have less time for cooking. In contrast to heavy users, light users do not fancy cooking much, and prefer easy recipes. Lack of time is an important factor for not liking to cook, and in Belgium, for instance, light users claimed that the `consumption of fish must be ``planned'' in detail' (Brunsù et al., forthcoming). A busy lifestyle will limit the time left for cooking, even when enjoying cooking, and this might be particularly detrimental to fish consumption.

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Improving seafood products for the consumer

Overall, consumers perceive all fish species as healthy, wild fish is preferred to farmed, and national fish is considered to be of higher quality than foreign fish. In addition, most consumers prefer fresh fish to frozen, which is highly consistent with findings from several other studies (Marshall, 1988; Nielsen et al., 1997; Olsen and Kristoffersen, 1999; Peavey et al., 1994). Light users in Belgium, however, like frozen fish fillets because they are easy and fast to prepare and in addition do not smell. Convenience seems to be quite a new concept in Spain. On the one hand young women especially would like to save some time and find some easy solutions. On the other hand there is a very strong food tradition in Spain. It is part of the culture to spend time cooking and eating and the fastfood idea still does not appeal to most of the Spanish respondents. In Belgium convenience is a well-known and important concept. In general the respondents do not find shopping for and cooking of fish very convenient ± fish requires careful and cooled treatment and must be consumed soon after purchase, if fresh. Also, fish shops are few and far between, and some respondents even had to drive to another town to purchase fish. Especially the Belgian heavy users mentioned convenience solutions and fast cooking as one of the important purchase criteria for choosing fish, while light users found it much more inconvenient and claimed that the purchase and preparation of fresh fish, especially, had to be planned well in advance. As can be seen from the results, there are both similarities and differences between the two countries investigated. It is interesting that even though we are dealing with countries with very different consumption levels, we find the same attitudinal motives and barriers to eating fish. But when it comes to perceptions of fish preparation and convenience, there is quite a difference between the Belgian and the Spanish consumer. We find that the more experienced consumers in Spain are more skilled in choosing and cooking fish and less aware of convenience solutions compared to the less experienced consumers in Belgium.

3.3 Overview of cross-cultural investigation of consumption patterns and attitudes towards fish In order to validate the findings from the focus group discussions, representative consumer surveys were conducted in five European countries. A comprehensive questionnaire was developed covering various aspects of consumer behaviour in relation to fish consumption taking into account the motives, barriers and preferences identified in the focus group discussions. The Theory of Planned Behaviour (Ajzen, 1991) was applied as the theoretical framework, and questions about behaviour, intention, attitudinal constructs and beliefs were included. In addition, a number of other relevant constructs were included in the questionnaire, e.g. involvement and convenience as well as health concerns.

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Consumer attitudes and seafood consumption in Europe

21

Data were collected by randomly selected representative household samples from Denmark (N 1110), Poland (N 1015), Belgium (N 852), Spain (N 1000) and the Netherlands (N 809), resulting in a total of N 4786 respondents. The fieldwork and the questionnaire pre-test were handled by local market research agencies. Interviews were conducted in various ways in the five countries: in Poland and Spain, the interviews took place face-to-face in participants' homes. In Denmark and Belgium, data were collected by mail surveys, with response rates of 79% in Denmark and 53% in Belgium. In the Netherlands, consumers were asked to participate by means of a web survey. In all countries a quota sampling procedure was applied with age and region as main control factors. The person mainly responsible for food shopping and cooking was selected as the respondent from each household, and as a result 77% of the respondents in the total sample were females. Except for the proportion of men and women, samples are representative for each country in terms of basic sociodemographics such as age, education, town size and region.

3.4

European consumption patterns

Owing to cultural differences, seafood consumption differs across Europe with respect to quantity, type of fish and species. According to our results, European consumers eat fish 1.49 times a week on average, which is less than the recommended level of twice a week. This figure includes both consumption at home and away from home. As can be seen in Table 3.1, the overall consumption frequency differs significantly between countries; while the Spanish consumers eat fish 2.6 times a week on average, the Dutch consumers eat it less than once a week. Thus, Spain is the top fish eating country followed by Denmark, where consumers eat fish 1.41 times a week. In Belgium, the Netherlands and Poland it is less common to eat fish and here the average consumption of fish is about once a week. In general, consumers primarily eat fish at home, thus on average 81% of all fish meals are consumed at home. Table 3.1

Average* frequency of fish consumption

Belgium Denmark The Netherlands Poland Spain Total

N

Mean*

851 1096 809 1012 999 4767

1.10 1.41 0.95 1.20 2.60

F

Sig

222.698

0.000

* Times per week (based on the sum of fish eaten at home and outside home)

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Home

Out

0.88 1.12 0.69 1.05 2.12 1.20

0.22 0.31 0.26 0.15 0.49 0.29

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Improving seafood products for the consumer

Table 3.2

Consumption of different product types: shares of total consumption (%) BE

DK

NL

PL

SP

Typical in:

Whole fresh fish 13.3 Filleted fresh fish 24.3 Raw fresh fish 6.6 Pre-packed fresh fish 13.3 Deep-frozen fish 17.3 Ready to eat meals with fish 5.8 Canned fish 13.3 Fish in glass (marinated) 5.8 Total 100.0

9.8 11.6 3.7 7.3 8.5 2.7 29.0 27.1 100.0

8.8 19.3 11.8 13.6 17.1 8.8 14.9 6.6 100.0

9.9 12.5 5.7 5.2 15.4 9.4 22.7 18.8 100.0

28.5 15.7 4.8 6.9 13.2 6.4 20.6 3.6 100.0

SP BE, NL NL BE, NL BE, NL, PL NL, PL DK, PL DK, PL

BE = Belgium, DK = Denmark, NL = Netherlands, PL = Poland, SP = Spain.

3.4.1 Types of fish In order to investigate how the intake of fish differs in relation to the types of fish consumed in the five countries, respondents were asked to state the consumption frequency of eight typical European types of fish. Table 3.2 shows that whole fresh fish, for instance, is commonly consumed in Spain, where it makes up 28.5% of the types of fish examined. The preference for whole fresh fish may be due to the fact that Spanish consumers eat much fish and therefore have more experience in the handling of fish. In countries with a relatively low fish consumption, consumers prefer more convenient types of fish than in Spain. In Belgium and the Netherlands, for example, consumers prefer filleted fresh fish, pre-packed fresh fish and deepfrozen fish, which are all products that are easier to prepare than whole fresh fish. In Denmark and Poland, canned and marinated fish are the most commonly consumed fish products. Deep-frozen fish is much more common in Poland than in Denmark. Results also show that ready-to-eat meals are most common in the Netherlands and Poland. 3.4.2 Fish species To investigate the consumption of various fish species, consumers were asked to state the consumption frequency of 11 different species (see Table 3.3). Across the five countries tuna is the most commonly consumed species, while the consumption of eel and plaice is low in most countries. In Belgium, cod and salmon are the most common species, while Danish consumers prefer herring that count for 21.9% of the total consumption. Another 18.6% of the fish consumed in Denmark is tuna. Of the fish consumed by Danish consumers 10.4% are plaice, which is quite a lot compared to other countries. In the Netherlands, tuna is the most popular species, followed by salmon, cod and herring. Eel counts for 6.6% of the Dutch fish consumption, which is relatively high compared to other European countries. In Poland, herring is nearly as popular as in Denmark, while mackerel counts for 18.9% of the consumption.

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Consumer attitudes and seafood consumption in Europe Table 3.3

23

Consumption of fish species: shares of total consumption per country

Cod Salmon Sole Trout Tuna Plaice Hake Mackerel Eel Herring Alaska Pollock Total

BE

DK

NL

PL

SP

18.3 16.2 9.6 5.6 15.7 6.1 2.0 6.1 2.5 6.6 11.2 100.0

6.1 10.8 1.8 4.7 18.6 10.4 1.1 16.5 2.5 21.9 5.0 100.0

13.3 15.0 7.1 8.0 15.9 1.3 2.7 9.7 6.6 12.8 8.0 100.0

8.8 4.7 1.5 5.0 12.1 1.8 10.0 18.9 2.1 20.4 15.0 100.0

8.5 9.3 13.9 7.4 24.3 2.0 23.1 6.0 1.4 2.2 1.8 100.0

Typical in: BE, NL BE, NL SP NL, SP SP DK SP DK, PL NL DK, PL PL, BE

BE = Belgium, DK = Denmark, NL = Netherlands, PL = Poland, SP = Spain.

Poland has the highest share of Alaska Pollock. Tuna and hake make up nearly half of the Spanish fish consumption. The share of hake is much lower in other European countries (1.1% to 10.0%). In general, we must conclude that the choice of species varies significantly between countries in Europe. 3.4.3 Shopping Based on focus group statements, we know that shopping for fish can be described as often semi-impulsive, i.e. consumers tend to decide in advance that they want to buy fish but do not plan on the exact type of fish. Since the buying situation is semi-impulsive, consumers may choose to buy meat instead and the decision is often influenced by, for instance, price, freshness, supply and appearance of the fish. According to the focus group discussions consumers prefer buying fish at traditional fish shops, which are associated with confidence, quality and freshness and where the salesman is seen as a good and trustworthy adviser. However, as can be seen from Table 3.4, supermarkets are the most common place to shop for fish across Europe (2.69 times per week) followed by fishmongers (2.59 times per week). The result may be because some consumers, particularly young ones, find it difficult to take the time to go to the fishmongers, and as consequence they shop for fish at supermarkets. Supermarkets are generally perceived as more convenient than speciality shops but the level of expertise and advice is considered lower here than at the fishmongers. In Belgium, Denmark and the Netherlands consumers primarily shop for fish at the supermarkets, while in Spain and Poland fishmongers are the most common shopping place. In all countries fishmongers and supermarkets are the two most popular places to shop for fish.

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Improving seafood products for the consumer

Table 3.4

Average* shopping frequencies at different outlets

Fishmonger Supermarket Marketplace Fisherman Own catch Total

BE

DK

NL

PL

SP

F

Sig.

Total

0.21 0.50 0.14 0.08 0.03 0.96

0.26 0.46 0.08 0.09 0.06 0.96

0.34 0.41 0.28 0.04 0.03 1.11

0.62 0.41 0.16 0.10 0.09 1.38

1.16 0.91 0.63 0.09 0.05 2.84

260.44 80.62 132.58 2.94 5.80

0.000 0.000 0.000 0.000 0.000

2.59 2.69 1.29 0.41 0.27

* Times per week BE = Belgium, DK = Denmark, NL = Netherlands, PL = Poland, SP = Spain.

As expected, we can conclude that there are major differences in the consumption of fish across the five countries in relation to consumption levels, types of fish and shopping patterns. In the following section we investigate attitudes and preferences across countries in order to gain insight into and to explain the differences in the consumption of fish.

3.5 Attitudes and preferences across Europe ± differences and similarities According to the Theory of Planned Behaviour, attitudes are determined by underlying salient beliefs (Ajzen, 1991). The relationship between attitudes and beliefs has its origin in Fishbein's summative models of attitudes (Fishbein and Ajzen, 1975). It assumes that a person may process large numbers of beliefs about a particular behaviour, but at any one time only a limited number of these are likely to be salient. It is the salient beliefs that are assumed to determine a person's attitude. In the marketing literature, these salient beliefs are defined and assessed as quality attributes (Peter et al., 1999). In order to develop a pool of relevant salient quality attributes, especially in relation to fish, we used the results from focus group discussions as well as results from earlier research on fish for inspiration. The attribute items were formulated using both positive and negative framing and were measured on a seven-point agree-disagree Likert scale (1 = totally disagree and 7 = totally agree) to reveal salient beliefs. We also intentionally included two items to measure negative affect (includes the word `unpleasant') since Olsen (2001) and Verbeke and Vacker (2005) have shown that negative affect can be negatively associated with motivation (involvement or intention) to consume seafood. The measured salient quality attributes can be seen in Table 3.5. 3.5.1 Overall attitudes and preferences Our results confirm the image of fish as a safe, healthy and nutritious food product. Consumers in this study also consider fish as delicious and tasty, while

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Consumer attitudes and seafood consumption in Europe Table 3.5

Eating Eating Eating Eating

fisk fish fish fish

25

Cross-cultural attitudes and preferences

is is is is

healthy nutritious safe risky

BE Mean

DK Mean

NL Mean

PL Mean

SP F-value p-value Mean

6.10 5.74 4.84 2.87

6.38 6.30 5.01 3.11

5.99 5.62 5.06 2.97

6.45 6.17 5.74 2.77

6.25 6.22 5.45 2.51

25.71 62.26 66.06 19.11

0.000 0.000 0.000 0.000

5.93 4.94

5.97 5.83

5.41 4.40

6.33 5.83

5.87 4.78

52.16 179.84

0.000 0.000

Fish has a good taste Eating fish is delicate Fish has an unpleasant smell The bones in fish are unpleasant

3.69

3.70

4.15

4.28

4.22

21.95

0.000

5.65

5.17

5.56

5.62

5.33

13.07

0.000

Eating fish is ethically correct Eating fish is trendy Eating fish is boring

4.77 3.70 2.38

4.60 4.15 2.31

4.61 3.77 2.68

5.11 4.64 2.52

4.95 3.54 2.99

20.30 79.95 28.00

0.000 0.000 0.000

5.71

5.32

5.32

5.82

5.28

28.72

0.000

4.46

4.72

4.70

4.74

4.10

28.93

0.000

Eating fish is expensive Fish give you value for money

BE = Belgium, DK = Denmark, NL = Netherlands, PL = Poland, SP = Spain.

bones are thought of as unpleasant, i.e. the results confirm that bones are a barrier to fish consumption, as discussed in the focus groups. Consumers' perception of the odour of fish is neutral (average = 4.01), so in this respect the results are not fully consistent with the indications from the focus groups, where bones and odour were mentioned as major barriers to fish consumption. Earlier studies have shown that fish is generally perceived as expensive, and in addition consumers have stated that they would eat more fish if it were less expensive (Baird et al., 1987; Nielsen et al., 1997). Our results confirm that fish is perceived as expensive, but at the same time the value for money is relatively high, indicating that the relationship between price and quality is considered to be relatively fair (average = 4.54). Particularly in Belgium and Poland the price is thought of as very high, indicating one of the reasons for the low consumption of fish in these two countries. Across countries, results show that health is an important motive for eating fish, since consumers in all five countries agree that fish is both healthy and nutritious. Eating fish is also considered relatively safe as opposed to unsafe but Polish and Spanish consumers think of fish as being much safer than the other European consumers. The European consumers generally perceive fish as delicious and tasty but Dutch consumers are less positive towards fish than other nationalities. As regards fish odour, the average evaluation is around 4, i.e. the smell is considered neither pleasant nor unpleasant. Consumers across the five countries agree that bones are unpleasant.

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Improving seafood products for the consumer

Eating fish is in general perceived neither as trendy nor boring. In Poland, however, it appears that fish is considered trendier than in other cultures and this may be related to the fact that the price of fish is considered very high, i.e. in Poland fish is thought of as a luxury product. As can be seen from the analysis, nationality certainly has an impact on consumers' opinions about fish. 3.5.2 Light versus heavy users Earlier findings have shown that the distinction between consumers with high and low consumption of fish is highly relevant when investigating consumers' attitudes and preferences towards fish (Juhl and Poulsen, 2000). In the following, the total sample of consumers is divided into three groups depending on their in-home frequency of eating fish. The total sample (excluding consumers who never eat fish) was categorised as follows: `seldom eat fish/light users' consume fish at home once a month or less, `regularly eat fish/medium users' consume fish at home from 2±3 times a month to once a week and `often eat fish/heavy users' consume fish at home twice a week or more. When dividing consumers into these three categories, we find that: · 23.6% eat fish at home only once a month or less · 47.8% eat fish between 2±3 times a month and once a week, and · 28.6% eat fish at least twice a week, as recommended. Four of the attitude statements are closely related to health and for each of these there were significant differences between the three groups (p 0:000). The table of multiple comparisons (Table 3.6) shows that consumers who seldom eat fish, perceive it as less healthy and less nutritious than those who eat it more frequently, and light users, especially, consider fish less safe/more hazardous. When comparing consumers who eat fish regularly and consumers who eat it often, the results follow the same pattern, i.e. heavy users are in every sense more positive towards fish than medium users. Consumers generally agree that fish is rather delicious, but light users in particular think of fish as less tasty than other consumer groups. The smell of fish was perceived as neither pleasant nor unpleasant, while bones were considered unpleasant by all consumer groups. The study shows that the more positive consumers' attitudes are towards the sensorial aspects of fish, the more fish they consume. There are significant differences between the three groups with respect to the four statements about taste, smell and bones (p 0:000) as can be seen in Table 3.6. However, when comparing the three groups' multiple comparisons, only some of the mean differences are significant at the 0.05 level. With respect to `Eating fish is delicate' and `Fish has an unpleasant smell' no significant differences can be found between medium and heavy users. The tendency, however, follows the traditional pattern, i.e. medium users are less positive than heavy users, as expected.

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Consumer attitudes and seafood consumption in Europe Table 3.6

27

Attitude statements Seldom eat fish Mean

Regularly eat fish Mean

Often eat fish Mean

6.01 5.73 4.87 3.15

6.32 6.08 5.29 2.81

6.45 6.33 5.56 2.61

52.653 80.656 74.793 33.883

0.000 0.000 0.000 0.000

Fish has a good taste Eating fish is delicate Fish has an unpleasant smell The bones in fish are unpleasant

5.49 4.92 4.31 5.76

6.10 5.32 3.92 5.47

6.29 5.44 3.77 5.13

137.164 36.652 24.938 36.270

0.000 0.000 0.000 0.000

Eating fish is ethically correct Eating fish is trendy Eating fish is boring

4.58 3.92 2.86

4.87 4.10 2.48

5.02 3.90 2.38

24.723 8.081 30.212

0.000 0.000 0.000

Eating fish is expensive Fish give you value for money

5.57 4.16

5.56 4.71

5.38 4.71

6.754 48.056

0.001 0.000

4.45

3.93

3.36

105.713

0.000

4.42

3.93

3.32

113.474

0.000

3.35

2.92

2.69

41.300

0.000

3.65 4.91

3.33 4.96

2.96 5.29

47.515 14.918

0.000 0.000

3.86

3.43

3.22

25.464

0.000

3.79

3.46

3.06

58.454

0.000

3.12

3.92

4.58

220.039

0.000

Eating Eating Eating Eating

fish fish fish fish

is is is is

healthy nutritious safe risky

Problem to evaluate the quality of fish Not confident with regard to evaluate if fish is fresh and safe I feel lost when having to choose fish I never know if I make the right choice of fish It is a problem for me to clean fish It is a problem for me to prepare fish To prepare fish for dinner is very time-consuming I have a lot of knowledge of how to prepare fish for dinner

F-value p-value

Two attitude statements are related to price and value for money, and as it appears from Table 3.6 that light and medium users fully agree that fish is expensive, i.e. in this respect there is no significant mean difference between these two groups. On the other hand, we can conclude that among light and medium users the price of fish is considered higher than among heavy users. The perceived value for money differs significantly between the three groups (p 0:000). As expected, light users consider the value of fish lower than others, whereas medium users have the same perception of value for money as heavy users. The analysis also included items about perceived problems in relation to buying, cleaning and preparing fish, and the results show that even though consumers experience some problems in this respect they are not totally lost when it comes to handling fish. The results more or less follow the same

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Improving seafood products for the consumer

pattern as above, i.e. the more often consumers eat fish, the less the perceived problems. When it comes to cleaning of fish, the results are, however, not as expected, since heavy users perceive this task as more complicated compared to light and medium users. Further analysis, however, revealed that heavy users of fish eat whole fresh fish much more often than other consumers, and since this type of fish requires cleaning, these consumers think of the cleaning as more problematic than other consumers. In other words, heavy fish users are not considered to be less talented in cleaning of fish, but are the ones who actually clean fish now and then, and therefore also realise whether it is complicated or not. Based on the analysis above we can conclude that there are significant differences in attitudes and preferences among light, medium and heavy users across Europe, and this emphasises the need for developing and promoting fish targeted especially at light users. Seen in this light the topic of convenience becomes very important, since lack of knowledge, skills, abilities and time to prepare home meals influence consumers' food attitudes and choices towards more convenience food (Gofton, 1995). From a consumer point of view convenience is more than just ease of purchase or quick consumption. Convenience means the saving of time, physical or mental energy at one or more stages of the overall meal process: planning and shopping, storage and preparation of products, consumption, and the cleaning up and disposal of leftovers (Gofton, 1995). Since light users of fish especially experience these problems when purchasing and consuming fish, we focus on how convenience is related to fish attitudes and consumption in the last part of the chapter.

3.6

Convenience and fish consumption

The role of convenience in explaining food attitudes, food choices and consumption has been explored in several recent studies (Candel, 2001; Jaeger and Meiselman, 2004; Mahon et al., 2006; Scholderer and Grunert, 2005). In a study of food consumption habits in the UK, Gofton and Marshall (1992) found that consumers regarded fish as inconvenient because of a perceived need to invest large amounts of time and effort at different stages of the provisioning process, and because fish meals were perceived to require unfamiliar vegetable side dishes. However, they also found that some aspects of the `inconvenience' of fish were related to taste preferences and habits. In the analysis of convenience and fish consumption, it is important to distinguish between convenience orientation (Candel, 2001) and perceived product convenience (Darian and Cohen, 1995; Lockie et al., 2002; Steptoe et al., 1995). Whilst the former refers to an aspect of the consumer, the latter refers to a property of the food, i.e. how consumers evaluate convenience attributes associated with a specific product, product category, or meal solution (Olsen et al., 2007). Results from the investigation of cross-cultural differences of convenience orientation and perceived convenience of fish will be presented

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Consumer attitudes and seafood consumption in Europe

29

below as well as the relationships between convenience orientation, attitudes, and fish consumption in five European countries: Denmark, Poland, Spain, Belgium and the Netherlands. Four items from Candel's scale measuring convenience orientation in meal preparation (CONVOR) were used with the purpose of assessing convenience orientation. In accordance with the theoretical outline above (Gofton, 1995), the items were modified slightly, also referring to planning and buying (in addition to preparing or cooking) to cover more stages of the consumption process that might be associated with meal convenience: · `I prefer meals that are easy to plan, buy (provide), prepare and cook' · `The less physical effort (work, energy) I need to buy and prepare a meal, the better' · `I prefer meals that are quick to plan, buy (provide), prepare and cook' and · `I prefer meals that can be prepared and cooked quickly'. Perceived product inconvenience was measured by three items: · `Preparing fish for dinner is very time-consuming' · `It is difficult to plan, provide, prepare and cook fish for a meal (dinner)' and · `It takes a lot of time to plan, provide, prepare and cook fish as a meal (dinner)'. These three items refer to time and ease/difficulty, the main dimensions previously used for assessing perceived product convenience (Lockie et al., 2002; McEnally and Brown, 1998; Steptoe et al., 1995). As above, two of the items were formulated in such a way that they referred to convenience at different stages in the consumption process. All items were measured on a seven-point Likert scale (1 = totally disagree and 7 = totally agree). The tests (post-hoc multiple comparisons ± Turkey HSD) indicated that convenience orientation was highest in Poland, followed by Spain, while Denmark and the Netherlands showed the lowest convenience orientation (see Table 3.7). The difference between Denmark and the Netherlands was not significant (p 0:42). Perceptions of fish as an inconvenient product largely followed the same pattern as convenience orientation, except for the figures from the Netherlands, where consumers actually reported the highest perceived inconvenience. Consumers in Poland and Spain reported a similar level of perceived inconvenience as consumers in the Netherlands, while consumers in Table 3.7

Comparison of mean scores of main constructs amongst the countries

Constructs

DE

PL

BE

NL

SP

F-value

p-value

Convenience Product inconvenience

4.30 2.85

5.13 3.40

4.53 2.96

4.35 3.51

4.73 3.28

49.8 29.2

0.000 0.000

Note: High score value indicates higher convenience orientation and more agreement about fish as an inconvenient product. BE = Belgium, DK = Denmark, NL = Netherlands, PL = Poland, SP = Spain.

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Denmark and Belgium perceived fish as least inconvenient. The differences between Belgium and Denmark (p 0:59), the Netherlands and Spain (p 0:60), and Poland and Spain (p 0:46) were not significant. To the extent that evaluations of a product's convenience aspects (perceived product convenience) are generalised into overall evaluations of the same product (attitude toward the product), a consumer's attitude toward the product should mediate the relationship between perceived product convenience and consumption frequency (Eagly and Chaiken, 1993; Fishbein and Ajzen, 1975). Since we found a significant positive relationship between convenience orientation and perceived inconvenience of fish across the five countries, the results suggest that convenience orientation can be crucial for fish consumption (Olsen et al., 2007). The link between convenience orientation and the (more specific) perceived inconvenience of fish is important because it suggests that more general, value-like constructs might be the basis from which more specific beliefs and attitudes are formed. In that respect, the importance of convenience orientation should be taken into consideration even though it is only indirectly related to consumption. 3.6.1 Convenience orientation and segments As explained above, Olsen et al. (2007) found a significant positive relationship between convenience orientation and perceived inconvenience of fish across the five countries. The results indicate that convenience orientation has an indirect influence on the fish consumption among European consumers, and to further investigate convenience orientation among European consumers we conducted a pan-European cluster analysis to look for convenience segments. The aim of the segmentation was to identify possible groups of consumers with different levels of convenience orientations in relation to food and cooking. Among other things we aimed at investigating: · · · ·

Which consumers like convenience? Which consumers are pro ready meals? How do demographics influence convenience orientation? To what extent is convenience orientation related to the consumption of fish?

In order to carry out the convenience segmentation, we used a number of items all covering different aspects of convenience in general. Thus we deliberately excluded all items mentioning aspects of `fish' to make sure that the segmentation was based on factors relating to general convenience orientation only. Later, when profiling the convenience segments, items relating to fish will be included in the analysis. All in all the segmentation was based on ten items about `convenience orientation' and six items about consumers' `perceived obligation to serve convenience food'. All items were measured on a sevenpoint scale (1 = totally disagree and 7 = totally agree). First a factor analysis was conducted to extract factor scores for the segmentation. We chose this procedure with the aim of improving the interpretation

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and significance of clusters. By grouping the 16 items into a smaller number of factors, the segments' convenience orientation becomes clearer and more distinct. The factor analysis (principal components, varimax rotation) resulted in four factors. The ten items covering convenience orientation were divided into two factors, one concerning `Easy Cooking' and one concerning `Real Ready Meals'. The `Easy Cooking' factor consists of seven statements related to aspects of time, effort, planning and thinking when preparing a meal: `I prefer meals that are easy to plan, buy (provide), prepare and cook', `The less physical effort (work, energy) I need to buy and prepare a meal, the better', `The less thinking I need to plan, buy and prepare a meal, the better', `I prefer meals that are quick to plan, buy (provide), prepare and cook', `I want to spend as little time as possible on planning and buying of what to have for meals', `I prefer meals that can be prepared and cooked quickly' and `It is a waste of time to spend a long time in cleaning up after meals'. The `Real Ready Meals' factor includes one item about buying and using ready meals and two about the consumer's attitude towards ready meals: `I often use/buy ready meals', `Eating ready meals gives me a good feeling' and `Ready meals are good value for money'. The six items about perceived obligation to serve convenience food were divided into two factors labelled `Moral Obligation' and `Compensation'. `Moral Obligation' describes the feeling that may result from serving family, friends and children convenience food. The factor consists of four statements: `I feel bad serving convenience food for my family', `I think it is alright to serve convenience foods for my friends', `Serving convenience foods for my children makes me feel like a bad person' and `Before using convenience foods, I always think of what my friends would do'. After eating convenience food some consumers may have a guilty conscience so that they feel a need to make amends. This aspect is covered by a `Compensation' factor which consists of two statements: `I always try to make amends if I have been eating convenience foods' and `I always try to make amends if I have been serving convenience foods'. Based on the derived factor scores, a K-means cluster analysis (SPSS 14.0) was conducted to establish convenience-based segments, where consumers with similar patterns of convenience orientation were grouped together: four distinct convenience segments could be identified. Analysis of variance was then used to study differences in the demographic variables between segments (age, number of persons in the household, gender, age, working hours). In addition, analysis of variance was used to analyse differences in consumers' health orientation, interest in food and attitudes towards fish. Table 3.8 shows the major differences with respect to the segments' convenience orientation, and below we describe and compare the four segments. The Convenience segment (29%) The Convenience segment has a much more positive attitude towards consuming ready meals compared to the other three segments, and these consumers also buy

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Table 3.8

Convenience variables by segments (numbers indicate segment means) Independence segment

I feel bad serving convenience food for my family I think it is alright to serve convenience foods for my friends I always try to make amends if I have been eating convenience foods I always try to make amends if I have been serving convenience foods Serving convenience foods for my children makes me feel like a bad person Before using convenience foods, I always think of what my friends would do I prefer meals that are easy to plan, buy, prepare and cook The less physical effort (work, energy) I need to buy and prepare a meal, the better The less thinking I need to plan, buy and prepare a meal, the better I prefer meals that are quick to plan, buy, prepare and cook I want to spend as little time as possible on planning and buying of what to have for meals I prefer meals that can be prepared and cooked quickly It is a waste of time to spend a long time in cleaning up after meals I often buy and use ready meals Eating bought ready meals give me a good feeling Bought ready meals are good value for money

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Convenience segment

Critical segment

Traditional segment

2.5 5.5 4.1 3.8 1.9 1.6 5.4

3.3 4.2 3.9 3.7 3.0 2.8 5.2

5.2 2.4 4.8 4.2 4.6 2.8 6.1

5.0 2.6 4.5 4.0 4.3 2.8 3.7

4.5 4.2 4.8

4.8 4.9 5.1

5.4 5.4 5.7

2.5 2.5 2.7

4.3 4.6 3.9 2.0 1.9 2.1

4.9 5.0 5.0 4.4 4.3 4.4

5.3 5.5 5.0 1.9 1.7 2.2

2.5 2.6 2.9 1.5 1.6 2.2

Consumer attitudes and seafood consumption in Europe

33

and eat ready meals more often than other consumers. While most consumers in this study in general feel bad about eating ready meals, the Convenience consumers think that eating ready meals gives them a good feeling. Compared to the other segments, the Convenience consumers are also much more positive towards the question of whether ready meals are good value for money. This segment consists of young consumers: the average age (39.5 years) is significantly lower compared to the other three segments. The households also tend to be smaller in the Convenience segment. However, the difference is only significant when compared to the Independence segment (see below), which has the highest average number of persons per household. Our total sample consists of 22.7% men, but in the Convenience segment the proportion of men is 25.7%, i.e. men are rather over-represented in the Convenience segment. The Independence segment (22%) The Independence segment appears to have a rather mixed attitude towards convenience. On the one hand, these consumers think that it is all right to serve their friends, family and children convenience food. On the other hand, the Independent consumers only rarely buy and eat convenience food themselves. One reason may be that they do not feel that ready made meals are good value for money or are too expensive. Still this segment is rather keen on easy cooking. When they do buy convenience food, the Independence consumers do not consider what other people think or expect, they feel confident enough to make their own decisions. The Critical segment (23%) The Critical segment seems to be busy, but nonetheless suspicious towards ready meals; they are against ready meals but still pro easy cooking. They do not like to serve their family, children or friends convenience food. The Critical segment thus has an anti-ready meal attitude, but likes other ways of easy and fast cooking, they want to cook their own meals, but do not want to spend too much time doing it. The average age in the Critical segment is 43.5 years, which is the second highest in the sample, only Traditional consumers are older on average, and 28.7% of the Critical consumers in this survey live in a household with four persons, which is much more than the sample average (23.0%) ± a typical family segment. In other words, due to their felt obligations Critical consumers feel bad about serving and eating convenience food, but still have a need for fast and easy solutions for their own cooking. The Traditional segment (26%) In the Traditional segment, consumers are not at all concerned about the time aspect of cooking, on the contrary, these consumers like spending time cooking. One reason may be these consumers' age, which is significantly higher than in the other segments. There is also an under-representation of consumers who work full time and an over-representation of persons who do not work at all. The average age of the Traditional segment is 45.2 years. With respect to moral

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obligations the Traditional consumers are very similar to the Critical consumers. This is also the case when looking at their need to make amends if they have eaten or served convenience food and like the Critical consumers, the Traditional consumers only rarely buy and use ready meals. Even though the Critical and the Traditional consumers have the same negative attitude towards ready meals, the Traditional consumers like to spend time in the kitchen while the Critical consumers want quick and easy cooking. 3.6.2 Differences across segments in relation to attitudes towards fish and health orientation The description of segments has so far been based on their general convenience orientation, and below, differences across segments as regards attitudes towards fish and health will be explored. To investigate the four segments' fish-related behaviours and opinions, we analysed consumers' statements about their attitudes towards fish. As can be seen from Table 3.9 the Convenience segment differs significantly from the other groups of consumers. For example, they do not appreciate fish as much as other European consumers. However, it is very important to note that the Convenience consumers actually do like fish (average for `Fish has a good taste' = 5.6). The Critical consumers appreciate fish more than Convenience consumers, but less than other consumers. However, consumers in the Critical segment still have the lowest consumption of fish in our sample. One reason may be fish bones, which the Critical consumers consider a major problem because they find it difficult to remove all the bones. The consumers in the Critical (family) segment are therefore significantly more interested in buying de-boned fish than other consumer groups. When looking at the Traditional segment, results show that these consumers are significantly less concerned about bones than the others. Even though there are differences in consumers' opinion about fish bones, it is important to note that according to our results most European consumers find it difficult to remove fish bones (4.9 in average), and therefore they also prefer filleted fish (5.3 in average). This may be significant when considering new product developments of fish. Some consumers think that compared to other kinds of meals, fish is more expensive. Compared to Critical and Independent consumers Traditional consumers consider fish to be good value for money. Fish is considered most expensive by the Critical consumers. This may be another reason why these consumers do not eat as much fish. The Independent segment is the least price sensitive with respect to fish. Consumers have different levels of knowledge about fish that can influence their opinion about this food product. Below, we compare the four consumer groups to detect whether there are any differences in the level of knowledge across segments. The average evaluations of the questions show that, in general, consumers do not consider themselves knowledgeable about fish. When looking at knowledge compared to an average person, we can conclude that Traditional consumers assess their own knowledge of fish as being better than Independent

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Table 3.9

Segment profiles Independence segment

Convenience segment

Critical segment

Traditional segment

Total

18.4b

17.9a

18.8b

18.8b

18.4

15.298

0.000

Health orientation (mean) Consumption of fish (times per week) At home Out Taste (7-point scale) Fish has a good taste I appreciate fish very much Intention to eat fish (7-point scale) Plan to eat fish Expect to eat fish Desire to eat fish Fish bones (7-point scale) I find it difficult to remove all bones out of fish I prefer to buy fish without bones Price (7-point scale) Fish give you value for money To buy fish for dinner is expensive Knowledge about fish Compared to an average person, I know a lot about fish My friends consider me as an expert on fish I have a lot of knowledge of how to prepare fish for dinner I have a lot of knowledge how to evaluate the quality of fish Age (mean) Household size (number of persons) Gender (%) Male Female Own working hours (%) Full time Part time Not working

F-value p-value

1.3b 0.3a

1.2b,c 0.3a

1.1a,c 0.3a

1.3b 0.3a

1.2 0.3

3.683 0.874

0.012 0.454

6.0b 5.1b,c

5.6a 4.7a

5.9b 4.9b

6.0b 5.2c

5.9 5.0

18.534 16.020

0.000 0.000

2.6a 2.7a 3.6a

2.8a,c 2.8a,c 3.3a,b

2.7a,d 2.7a,d 3.3a

2.9b,c,d 3.0b,c,d 3.6a,b

2.7 2.8 3.4

3.452 2.743 3.859

0.014 0.042 0.009

4.9b 5.2b

5.0b 5.2b

5.3c 5.8c

4.6a 4.9a

4.9 5.3

25.618 45.401

0.000 0.000

4.4a 4.4a

4.5a,c 4.6b,c

4.4a 5.1c

4.6b,c 4.6d,e

4.5 4.7

3.455 30.052

0.016 0.000

3.4a 2.6a 3.9b 3.7b 42.8b 3.0b

3.6a,c,d 3.0b,d 3.7a 3.7b 39.5a 2.7a,c

3.2a,c 2.4a 3.5a 3.3a 43.5b 2.9b,c,d

3.7b,d 3.0c,d 4.2c 4.0c 45.2c 2.8a,d

3.5 2.8 3.9 3.7 42.8 2.7

20.7 79.3

25.7 74.3

18.2 81.8

24.8 75.2

22.7 77.3

17.568 25.991 31.140 21.850 43.491 6.422 7.096

0.000 0.000 0.000 0.000 0.000 0.000 0.000

59.1 14.4 26.5

54.3 17.5 28.2

49.1 18.5 32.3

46.1 19.2 34.6

52.0 17.5 30.4

10.946

0.000

The a, b, c and d indicate significantly different means at the 0.05 level using Bonferroni Post Hoc

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and Critical consumers. Furthermore, consumers in the Convenience segment tend to think they know more about fish than Critical consumers. None of the consumer groups think that their friends consider them to be experts on fish. However, Traditional consumers rate themselves higher than the consumers in the Independent and the Critical segments. Traditional consumers also rate their own abilities higher than other consumers when it comes to cooking fish for dinner. Critical consumers, in contrast, feel less confident when cooking a fish meal than other consumer groups. When asked if they know how to evaluate the quality of fish, the pattern is the same. Traditional consumers feel more confident than others, while consumers in the Critical segment feel less confident than others. Overall we can conclude that Traditional consumers think they know more about fish than others, while Critical consumers think they know less about fish than others. The results indicate that the level of knowledge influences the amount of fish consumed and vice versa. Finally, the level of health orientation was analysed to find out if some segments were more interested in health than others. In order to measure overall health orientation a new health variable was computed as a sum score of three statements: `Health means a lot to me', `I care a lot about health' and `Health is very important to me'. Our results show that consumers in the Critical and the Traditional segments are most concerned about general health aspects, and the Convenience segment is significantly less concerned about health aspects compared to the other consumers.

3.7

Conclusion and future challenges

In this chapter, we have presented several new results about European consumers' attitudes towards fish, and have elaborated further on their convenience orientation. The focus group discussions in Belgium and Spain revealed that consumers perceive all fish species as healthy, wild fish is preferred to farmed fish, and national fish is considered to be of higher quality than foreign fish. In addition, most consumers prefer fresh fish to frozen, which is highly consistent with findings from several other studies (Marshall, 1988; Nielsen et al., 1997; Olsen and Kristoffersen, 1999; Peavey et al., 1994). Light users in Belgium, however, like frozen fish fillets because they are easy and fast to prepare and in addition do not smell. Thus we found both similarities and differences even though the selected countries represent very different consumption levels. The representative surveys conducted in Denmark, Poland, Belgium, Spain and the Netherlands confirmed most of the findings from the focus group discussions, and both similarities and differences between countries could be found. Earlier findings on differences between more and less experienced fish consumers (Nielsen et al., 1997) could also be confirmed and extended by the cross-cultural analysis across all samples that revealed more differences when splitting the samples based on consumption levels. Thus, we can conclude that there are significant differences in attitudes and preferences among light,

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medium and heavy users across Europe, and this emphasises the need for developing and promoting fish targeted especially at light users, and to focus more on the concept of convenience. Finally, new results based on the convenience concept showed that consumers in general perceive fish as inconvenient, and a cross-cultural segmentation analysis taking point of departure in consumers' convenience orientation resulted in four pan-European segments that have very different demands and needs in relation to convenience ± some segments are really into convenience solutions while other segments are not. This result indicates a clear need to develop convenient products, to educate consumers about where to buy and how to prepare fish in convenient ways, and change some consumers' beliefs and attitudes about fish being an inconvenient product. Also, the analysis shows that all countries have some consumers that perceive fish as convenient, probably because of their knowledge of and experience with the product (Gofton, 1995), and thus a future challenge for the fishing industry is to know precisely which consumer segments to approach and also how to meet and fulfil the needs and wants of the selected segment(s) by new targeted product developments.

3.8

References

and STANDRIDGE, J. B. (2006). What should we eat? Evidence from observational studies. Southern Medical Journal, 99(7), 744±748. AJZEN, I. (1991). The theory of planned behavior. Organizational Behavior and Human Decision Processes, 50, 179±211. BAIRD, P. D., BENNETT, R. and HAMILTON, M. (1987), 'The consumer Acceptability of Some Underutilised Fish Species', in: Thomsen, D. M. H. (Ed.), Food Acceptability, Elsevier Applied Science, London, pp. 431±442. BRUNSé, K. (2003). Consumer research on fish in Europe. In J. Luten, J. Oehlenschlager and G. Olafsdottir (Eds.), Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Wageningen: Wageningen Academic Publishers, pp. 335±344. BRUNSé, K., FJORD, T. A. and GRUNERT, K. G. (2002). Consumers' food choice and quality perception. MAPP working paper 77, Aarhus School of Business. BRUNSé, K., VERBEKE, W., OLSEN, S. O. and JEPPESEN, L. F. (forthcoming). Motives, barriers and quality evaluation in fish consumption decisions: A comparison between heavy and light users in Spain and Belgium. British Food Journal. CANDEL, M. J. J. M. (2001). Consumers' convenience orientation towards meal preparation: Conceptualization and measurement. Appetite, 36(1), 15±28. CAYGILL, C. P. J., CHARLETT, A. and HILL, M. J. (1996). Fat, fish, fish oil and cancer. British Journal of Cancer, 74(1), 159±164. DARIAN, J. and COHEN, J. (1995). Segmenting by consumer time shortage. Journal of Consumer Marketing, 12 (1), 32±44. EAGLY, A. H. and CHAIKEN, S. (1993). The Psychology of Attitudes. Fort Worth, TX: Harcourt Brace Jovanovich. FASONLINE (2002). Fishery Products Market News. www.fas.usda.gov FERNANDEZ, E., CHATENOUD, L., LA VECCHIA, C., NEGRI, E. and FRANCESCHI, S. (1999). Fish ADAMS, S. M.

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consumption and cancer risk. American Journal of Clinical Nutrition, 70(1), 85± 90. FISHBEIN, M. and AJZEN, I. (1975). Belief, Attitude, Intention and Behaviour: An Introduction to Theory and Research. Reading, Mass.: Addison-Wesley. GOFTON, L. (1995). Dollar rich and time poor? Some problems in interpreting changing food habits. British Food Journal, 97(10), 11±16. GOFTON, L. and MARSHALL, D. W. (1992). Fish: a Marketing problem. Bradford: Horton Publishing. GROSS, T. (2003). Consumer attitudes towards health and food safety. In J. Luten, J. Oehlenschlager and G. Olafsdottir (Eds.), Quality of Fish from Catch to Consumer: Labelling, Monitoring and Traceability. Wageningen: Wageningen Academic Publishers, pp. 401±411. GRUNERT, K. G. (2005). Food quality and safety: consumer perception and demand. European Review of Agricultural Economics, 32 (3) 369±391. JAEGER, S. R. and MEISELMAN, H. L. (2004). Perceptions of meal convenience: the case of athome evening meals. Appetite, 42, 317±325. JUHL, H. J. and POULSEN, C. S. (2000). Antecedents and effects of consumer involvement in fish as a product group. Appetite, 34, 261±267. KRIS-ETHERTON, P. M., HARRIS, W. S., APPEL, L. J. and COMM, N. (2002). Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation, 106(21), 2747±2757. KRIS-ETHERTON, P. M., HARRIS, W. S., APPEL, L. J. and COMM, A. N. (2003). Omega-3 fatty acids and cardiovascular disease ± New recommendations from the American Heart Association. Arteriosclerosis Thrombosis and Vascular Biology, 23(2), 151±152. LOCKIE, S., LYONS, K., LAWRENCE, G. and MUMMERY, K. (2002). Eating green: motivation behind organic food consumption in Australia. Sociologia Ruralis, 42 (1), 23±40. MCENALLY, M. R. and BROWN, L. G. (1998). Do perceived time pressure, life cycle stage and dimorphic characteristics affect the demand for convenience? European Advances in Consumer Research, 3, 155±161. MAHON, D., COWAN, C. and MCCARTHY, M. (2006). The role of attitudes, subjective norms, perceived control and habit in the consumption of ready meals and takeaways in Great Britain. Food Quality and Preference, 17 (6), 474±481. MARSHALL, D. (1988). Behavioural variables influening the consumption of fish and fish products. In: D. M. H. Thomson (Ed.), Food Acceptability. Essex: Elsevier, pp. 219±231. MOZAFFARIAN, D. and RIMM, E. B. (2006). Fish intake, contaminants, and human health ± Evaluating the risks and the benefits. JAMA ± Journal of the American Medical Association, 296(15), 1885±1899. MYRLAND, é., TRONDSEN, T., JOHNSTON, R. S. and LUND, E. (2000). Determinants of seafood consumption in Norway: lifestyle, revealed preferences, and barriers to consumption. Food Quality and Preference, 11(3), 169±188. NESTEL, P. J. (2000). Fish oil and cardiovascular disease: lipids and arterial function. American Journal of Clinical Nutrition, 71(1), 228S±231S. NIELSEN, N. A., SéRENSEN, E. and GRUNERT, K. G. (1997). Consumer motives for buying fresh or frozen plaice: A means end chain approach. In J. B. Luten, T. Bùrresen and J. OehlenschlaÈger (Eds.), Seafood from Producer to Consumer: Integrated Approach to Quality. Amsterdam: Elsevier, pp. 31±43. OLSEN, S. O. (2001). Consumer involvement in seafood as family meals in Norway: an application of the expectancy-value approach. Appetite, 36(2), 173±186.

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and KRISTOFFERSEN, E. M. (1999), Sjùmat i norske husholdninger. Norwegian Institute for Fisheries and Aquaculture, Tromsù, Rapport 19. OLSEN, S. O., SCHOLDERER, J., BRUNSé, K. and VERBEKE, W. (2007). Exploring the relationship between convenience and fish consumption: a cross-cultural study. Appetite, 49, 84±91. PEAVEY, S., WORK, T. and RILEY, J. (1994). Consumer attitudes towards fresh and frozen fish. Journal of Aquatic Food Product Technology, 3 (2), 71±87. PETER, J. P., OLSON, J. C. and GRUNERT, K. G. (1999). Consumer Behaviour and Marketing Strategy, European edition. Maidenhead: McGraw-Hill. PIENIAK, Z., VERBEKE, W., FRUENSGAARD, L., BRUNSé, K. and OLSEN, S. O. (2004). Determinants of fish consumption: Role and importance of information. Polish Journal of Human Nutrition and Metabolism, 31 (Suppl. 2), 409±414. SCHOLDERER, J. and GRUNERT, K. G. (2005). Consumers, food and convenience: The long way from resource constraints to actual consumption patterns. Journal of Economic Psychology, 26, 105±128. STEPTOE, A., POLLARD, T. M. and WARDLE, J. (1995). Development of a measure of the motives underlying the selection of food: the food choice questionnaire. Appetite, 25, 267±284. VERBEKE, W. and VACKIER, I. (2005). Individual determinants of fish consumption: application of the theory of planned behaviour. Appetite, 44, 67±82. WELCH, A. A., LUND, E., AMIANO, P., DORRONSORO, M., BRUSTAD, M., KUMLE, M., et al. (2002). Variability of fish consumption within the 10 European countries participating in the European Investigation into Cancer and Nutrition (EPIC) study. Public Health Nutrition, 5(6B), 1273±1285. OLSEN, S. O.

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4 Improved eating quality of seafood: the link between sensory characteristics, consumer liking and attitudes E. MartinsdoÂttir and K. SveinsdoÂttir, MatõÂs, Iceland, D. Green-Petersen and G. Hyldig, Technical University of Denmark and R. Schelvis, Wageningen University and Research Centre, The Netherlands

4.1 Introduction: why is the eating quality important for the industry and for the consumer? Fish consumption varies greatly across Europe. Welch et al. (2002) provided an overview of the fish consumption in 10 European countries. The highest total fish consumption was in Spain, but the lowest in Germany and the Netherlands. However, generally more fish was consumed in Northern compared to Southern Europe. The proportion of fat fish species of the total consumption was relatively higher in coastal areas of Northern Europe, such as in Denmark and Sweden and in Germany compared to countries in central and Southern Europe. More variety of fish species was consumed in Southern than Northern Europe. Fish consumption was generally higher in areas with greater costal access. Further, cod and salmon were among the most frequently consumed species in many European countries. Cod was the most commonly consumed species in Norway, France, Greece, Sweden, UK, Italy, the third most common in Spain and the Netherlands, the fourth in Denmark, but the seventh in Germany. Salmon was the second most commonly consumed species in Norway, France and the Netherlands, the third most common in Denmark, Sweden and Germany, the sixth most common in Italy but the tenth in Spain. Cod is an example of a lean fish species but salmon is a fat fish species and are very different in sensory characteristics and nutritional values. Both species are caught wild and farmed.

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Improved eating quality of seafood

41

In the near future most of the increase in fish production is expected to come from aquaculture (FAO, 2006). The worldwide production of fresh seafood has increased gradually since 1994, from approximately 30 000 000 T to 50 000 000 T per year in 2002, while the production of frozen fish has remained around 25 000 000 T per year (Vannuccini, 2004). However, production of frozen fish products is expected to increase in the near future (Agriculture and Agri-Food Canada, 2005). Seafood products are very perishable products and their sensory characteristics depend on various factors, such as packaging methods, storage methods and storage time. It is important to consider how the product is presented to consumers. In some areas, such as Northern Europe, fillets are the most common product, while in Southern Mediterranean countries consumers usually buy whole fish. The effects of freezing on quality of fish are well documented. Frozen/thawed cod products are generally characterised by lower eating quality compared to fresh, e.g., due to decreased freshness, drier and tougher texture (MagnuÂsson and MartinsdoÂttir, 1995; MartinsdoÂttir and MagnuÂsson, 2001). Storage life of fresh fish and fresh fish product is relatively short and in order to meet consumer demands for fresh products, food products packed in modified atmosphere (MA) have increased their market share, with the advantage of extended shelf life. The use of modified atmosphere packaging (MAP) has been found to increase the keeping quality of fish products (see, e.g., Sivertsvik et al., 2002). Consumers are sensitive to the different sensory aspects of fish products caused by storage time, storage and packaging methods (e.g., SveinsdoÂttir et al., 2003a; SveinsdoÂttir et al., 2008; Green-Petersen et al., 2008). The changing composition of the market for seafood products raises questions about consumer preferences for the different products on the market. Eating quality preference decisions are ultimately made during consumption. Eating quality will vary from one species of seafood to another, and then again due to choice of storage, handling, packaging, transportation, etc., made at each point in the chain from seafood catch, or slaughter, to consumption. Consumers in different countries may have different experiences with seafood, related to traditions, availability and frequency of consumption, that will determine individual preferences. Key decision makers determine quality at each stage in the seafood handling chain. However, the basis of their quality decision may not relate well with that of another quality decision maker later in the chain, or ultimately with that of the consumer, who is the final judge of eating quality. Therefore, it is important to relate eating quality to the evaluations of sensory quality carried out by key persons in the seafood handling chain, and from descriptive sensory evaluations carried out in parallel by trained panels. Eating quality has to be communicated from the consumer back through the quality chain to catch. This chapter provides an overview over the sensory methods used for evaluation of seafood and the sensory characteristics of cod and salmon products. Few studies have been published on consumer likings in relation to sensory characteristics of seafood. Results from the SEAFOODplus project

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Improving seafood products for the consumer

added considerably to this knowledge and are discussed here. Further, within the project, consumers were segmented across different European countries, related to attitudes and specific preferences of seafood products and the results discussed in relation to previous findings. In addition, sensory quality models relating consumers' perceptions of eating quality with sensory characteristics perceived by key quality decision makers at each point in the fishery production chain, from catch or slaughter, through handling and distribution to final consumption are described from the SEAFOODplus project. Further, some future trends are mentioned and guidelines on the applications of above items for the fish industry and consumer are given.

4.2

Methods for evaluation of sensory quality of seafood

Several methods may be applied to evaluate the sensory quality of seafood depending on the objective. The objective could be to use the results of the evaluation in quality and process control, product development, shelf life studies or consumer preference research. Sensory evaluation of seafood products is mostly used in quality control, but also in product development and optimisation. The Quality Index Method (QIM) may be used to estimate the freshness quality of whole/raw seafood species and has been developed for various species, such as cod (Larsen et al., 1992) and farmed Atlantic salmon (Salmo salar) (SveinsdoÂttir et al., 2003b). The QIM-Eurofish Foundation (www.qimeurofish.com) provides a detailed list of references concerning QIM, and the method has been described in detail in the literature (MartinsdoÂttir et al., 2003; Hyldig et al., 2007; MartinsdoÂttir et al., 2008). For sensory evaluation of fish fillets, it is common to cook the fillets and evaluate their odour and flavour. The Torry scale was the first detailed scheme developed for evaluating the freshness of fish (Shewan et al., 1953). The Torry scheme has been used in the fish industry in Europe and by British retailers of fish. Recently QIM-schemes have been developed for thawed, raw cod fillets, thawed cooked cod fillets (Gadus morhua) (Warm et al., 1998) and fresh raw cod fillets (Gadus morhua) (Bonilla et al., 2007). In order to obtain a complete sensory description of a product, which includes detailed descriptions of all aspects regarding appearance, odour, flavour and texture, methods such as Quantitative Descriptive Analysis (QDA) (Stone and Sidel, 1985; Lawless and Heymann, 1999; Meilgaard et al., 1999) are used. With the QDA, all detectable aspects of a product are described and listed by a trained panel, generally consisting of 6±12 panellists. The list is then used to evaluate the products and the panellists quantify the sensory aspects of the product using an unstructured scale. A wide range of sensory descriptors has been used to describe the sensory quality of seafood. Hyldig (2007) and Hyldig and Nielsen (2007) provide a detailed overview of terms and definitions which have been used. Sensory descriptors specific for cod and salmon products are listed in Tables 4.1 and 4.2.

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Table 4.1 Sensory vocabulary for cooked samples of different cod (Gadus morhua) products (Sveinsdottir et al., 2008) Sensory attribute Odour sweet boiled milk boiled potatoes butter vanilla meaty frozen storage table cloth TMA sour sulphur putrid

Description of attribute sweet odour boiled milk, fruity/mushy odour odour of boiled potatoes butter odour, popcorn vanilla odour, sawdust, timber meaty odour, reminds of boiled meat reminds of odour found in refrigerator and/or freezing compartment reminds of a table cloth (damp cloth to clean kitchen table, left for 36 h) TMA odour, reminds of dried salted fish, amine sour odour, spoilage sour, acetic acid sulphur, matchstick putrid odour

Appearance light/dark colour

left end: light, white colour; right end: dark, yellowish, brownish, grey homogenous/ left end: homogenous, even colour; right end: discoloured, heterogeneous heterogeneous, stains white precipitation white precipitation in the broth or on the fish

Flavour salt sweet metallic sour taste butter meaty frozen storage pungent TMA putrid Texture flakiness firm/soft dry/juicy tough/tender mushy meaty clammy rubbery

salt taste sweet flavour metallic flavour sour taste, spoilage sour butter flavour, popcorn meaty flavour, reminds of boiled meat, meat sour, farmed fish reminds of food which has soaked in refrigerator/freezing odour pungent flavour, bitter TMA flavour, reminds of dried salted fish, amine Putrid flavour the fish portion slides into flakes when pressed with the fork left end: firm; right end: soft. Evaluate how firm or soft the fish is during the first bite left end: dry; right end: juicy. Evaluated after chewing several times: dry ± pulls juice from the mouth left end: tough; right end: tender. Evaluated after chewing several times mushy texture meaty texture, meaty mouth feel clammy texture, tannin rubbery texture, chewing gum

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Table 4.2 Sensory vocabulary for cooked samples of different salmon (Salmo salar, Oncohynchus keta, Oncohynchus kisutch) products (Green-Petersen et al., 2006, 2008) Sensory attribute

Description of attribute

Odour Seaweed Sourish Sweet Rancid Sour

Fresh seaweed, fresh sea smell Acidic, Fresh citric acid Sweet Rancid fish, paint, varnish Sour dishcloth/sour sock

Appearance Discoloured Colour

Brown or yellow spots, dark areas Salmon colour

Flavour Fresh fish oil Sweet Sourish Cooked potatoes Mushroom Rancid Salt

Fresh oil, fresh green hazelnut Sweet, hot milk Acidic, fresh citric acid Cooked peel potatoes Mushroom flavour Rancid fish, paint, varnish Salt

Texture Juicy Firm Oily

The samples ability to hold water after 2±3 chews Force required to compress the sample between the molars Amount of fat coating in the mouth surfaces

For objective sensory evaluation, panellists or inspectors need to be selected and trained according to standards (ISO 8586-1, 1994; Meilgaard et al., 1999). In MartinsdoÂttir et al. (2001), selection and training of panellists specific for evaluation of seafood is described. A panel leader supervises the training, manages the samples and maintains the skills and motivation of the panel. Analysis and interpretation of the sensory data requires understanding of the methods and is a part of the sensory evaluation. Facilities to carry out sensory work have been widely described in the literature and standards (ISO 8589, 1988; Meilgaard et al., 1999; MartinsdoÂttir et al., 2001). For consumer tests, people representing the consuming population are chosen. Acceptability of seafood, the degree of liking and disliking, are usually estimated using a scalar method and the most common ones are seven- or ninepoint structured hedonic scale (dislike extremely = 1, like extremely = 7 or 9). Validity of data generated using this method can be influenced by factors such as unequal size category intervals in the scale, the tendency of consumers to avoid extreme values on the scale and to score close to the midpoint. The hedonic scales have been widely studied and have been shown to be useful in the hedonic assessment of food. The samples are served to the consumer in random order and the consumers are asked to indicate their hedonic response to the

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sample on the scale. The results are affected by the test location and the most used locations are laboratory test, central location test and home use test. The choice of test depends on various factors and has advantages and disadvantages (Meilgaard et al., 1999).

4.3

Sensory characteristics of cod and salmon

The sensory characteristics of different species are very different, whether raw (MartinsdoÂttir, et al., 2001) or cooked (Cardello, et al., 1982; Prell and Sawyer, 1988; Chambers and Robel, 1993). This chapter describes the sensory characteristics of different cod and salmon products. 4.3.1 Cod products Shelf life of whole fresh cod stored in ice is short and has been reported to be up to 15 days (MartinsdoÂttir, et al., 2001). Sensory characteristics of whole raw cod as evaluated with the Quality Index Method (Larsen et al., 1992) may be used to estimate the storage time of whole cod kept in ice. At the beginning of storage time, the skin is bright and iridescent, the eyes clear, convex and black (Fig. 4.1), and the gills bright red with clear mucus and fresh seaweed or metallic odour. At the end of shelf life, the skin has become dull and discoloured, the eyes milky, sunken and the pupil has become gray, while the gills have become brownish and discoloured, with milky, dark and opaque mucus with yeast, or very sour odour. Similarly, the sensory characteristics of raw cod fillets can be used to estimate the storage time with the Quality Index Method (Bonilla et al., 2007). At the beginning of storage time, the skin is iridescent with thin and transparent mucus, the flesh is firm, transparent and whitish with fresh or neutral odour. At the end of shelf life the skin has become dull with clotted, thick and

Fig. 4.1

Appearance of the eyes of newly caught cod (Gadus morhua).

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yellowish mucus, and the flesh has become soft, milky and yellowish, with sour or acetic odour. Various terms may be used to describe the sensory quality of cooked cod products. The vocabulary described in Table 4.1 was developed by a trained sensory panel to describe the sensory characteristics of different cod (Gadus morhua) products (SveinsdoÂttir et al., 2008), such as wild and farmed, fresh or frozen/thawed at various stages of storage. At the beginning of storage time, lean fish are generally described with sweet, boiled milk odours and watery, metallic and meaty flavours when cooked (Shewan et al., 1953). Fresh, wild cod has a sweet odour and sweet and metallic flavour when cooked (Bonilla et al., 2007; SveinsdoÂttir et al., 2008) and very juicy, soft (SveinsdoÂttir et al., 2003a) and tender texture (SveinsdoÂttir et al., 2008). As storage time progresses, odour and flavour characteristics of freshness become less evident, and when the cod approaches end of shelf life, the cod is more described by table cloth, sour and TMA (trimethylamine) odour, sour and TMA flavour, dark and discoloured appearance (SveinsdoÂttir et al., 2008). At the end of shelf life, TMA, table cloth, rotten, sour and sulphur odour together with TMA, sour, rotten and pungent flavours have become dominating (Bonilla et al., 2007). The shelf life of fresh cod fillets stored at 0±1 ëC has been reported from 8 days (Bonilla et al., 2007) to 10±12 days (MagnuÂsson and MartinsdoÂttir, 1995). Wild cod after short storage (3 days at 0±1 ëC) in modified atmosphere (MA) has generally similar sensory characteristics to fresh wild cod of the same storage time and temperature, but the texture of MA-packed cod is not as soft (SveinsdoÂttir et al., 2008) and tender (SveinsdoÂttir et al., 2003a). However, the MA-packed cod keeps the freshness characteristic odours and flavours considerably longer (Wang et al., 2007). In addition, cod after extended storage (10 days at 0±1 ëC) in modified atmosphere was less described by storage characteristics such as TMA and sour odour and TMA flavour than fresh cod after the same storage time at the same temperature. However, the texture of the MA-packed cod was less soft, tender and mushy, but more meaty, clammy and rubbery. The use of modified atmosphere packaging (MAP) has been found to increase the keeping quality of fish products (see, e.g., Sivertsvik et al., 2002) and shelf life has been reported up to 20 days for cod fillets packed in MA (Dalgaard et al., 1993) and more than 24 days in combination with super chilling (Wang et al., 2007). Newly frozen wild cod has similar appearance, odour and flavour characteristics to fresh, new wild cod when cooked within two or three days after thawing (MagnuÂsson and MartinsdoÂttir, 1995; MartinsdoÂttir and MagnuÂsson, 2001; SveinsdoÂttir et al., 2008). Nielsen and Jessen (2007) reported that the sensory quality of newly frozen cod stored at stable temperatures at ÿ30 ëC or lower has comparable taste as fresh cod. However, the texture is less juicy, tender (SveinsdoÂttir et al., 2003a, 2008) and soft, but more meaty, clammy and rubbery (SveinsdoÂttir et al., 2008). The longer the cod has been stored frozen, the faster the freshness characteristics fade after thawing (MartinsdoÂttir and

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MagnuÂsson, 2001). After three months in frozen storage at ÿ30 ëC or below, cold storage flavours become evident and characteristic flavour fades (Nielsen and Jessen, 2007). According to SveinsdoÂttir et al. (2008), cod stored five months frozen has less sweet odour, but more odour of boiled potatoes, in addition to hints of table cloth odour and TMA flavour. However, the texture was more soft, juicy and tender compared to newly frozen cod which is similar as observed by MartinsdoÂttir and MagnuÂsson (2001) after similar storage time. Cod fillets kept at ÿ25 ëC for 12 months received very low freshness scores (MagnuÂsson and MartinsdoÂttir, 1995) and the texture was very dry and tough (MartinsdoÂttir and MagnuÂsson, 2001). This is in agreement with Nielsen and Jessen (2007), who stated that frozen cod stored at low and stable temperature may have a shelf life up to 12 months. However, fish processing companies usually regard the storage time of fresh frozen cod fillets as one to two years, and most commonly 18±24 months at ÿ18 ëC or below. The sensory characteristics of fresh farmed cod are very different from fresh wild cod. The main difference is with regard to texture, as the farmed cod has meaty texture, more rubbery, and clammy texture, and is less soft, juicy and tender. The appearance of the farmed cod is also very different as it has a very white and even colour and a very high degree of white precipitation on the cooked sample. The odour of farmed cod is mainly characterised by meat odour and flavour, more sweet odour and flavour compared to wild cod (SveinsdoÂttir, et al., 2008). Similar results were reported by Luten et al. (2002), who found that farmed cod received higher scores for white and dull appearance, cod taste and fibrousnesses, but lower scores for juiciness. 4.3.2 Salmon products Shelf life of whole fresh farmed salmon (Salmo salar) stored in ice has been reported up to 20 or 21 days (SveinsdoÂttir et al., 2002, 2003b). Sensory characteristics of whole raw farmed salmon as evaluated with the Quality Index Method (SveinsdoÂttir et al., 2002) may be used to estimate the storage time of whole salmon kept in ice. At the beginning of storage time, the skin is pearlshiny with clear mucus, the eyes clear, convex and black, and the gills bright red with clear mucus (Fig. 4.2) and fresh, seaweed odour. At the end of shelf life, the skin has become yellowish (mainly near the abdomen) with yellow and clotted mucus, the eyes mat, gray and sunken, while the gills have become brownish or gray, with brown and clotted mucus and sour, mouldy or rotten odour. The vocabulary described in Table 4.2 was developed by a trained sensory panel to describe the sensory characteristics of different salmon (Salmo salar, Oncohynchus keta, Oncohynchus kisutch) products, such as wild and farmed, fresh or frozen/thawed at various stages of storage time (Green-Petersen et al., 2006, 2008). Farmer et al. (2000) in addition provides a vocabulary that was used to evaluate wild and farmed, fresh and frozen salmon (Salmo salar). At the beginning of storage time, fresh, fatty fish are generally described with butter, margarine or fatty odour and meaty, shellfish, slightly bitter or metallic

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Fig. 4.2 Appearance of the gills of newly slaughtered salmon (Salmo salar).

flavour when cooked (Shewan et al., 1953). However, at the beginning of storage time, fresh farmed salmon (Salmo salar) has been described with characteristic seaweed and oily odour and characteristic salmon, metallic, sweet and oily flavour (SveinsdoÂttir et al., 2002), similar to what was reported by SveinsdoÂttir et al. (2003b) in addition to cucumber odour, juicy and firm texture. The sensory characteristics do not change much the first two weeks of storage (Green-Petersen et al., 2006, 2008). However, at the end of shelf life, sour, rancid and musty odour, sour and rancid flavour and discolouration become evident (SveinsdoÂttir et al., 2002). Similar characteristics were reported by SveinsdoÂttir et al. (2003b), in addition to amine odour and flavour. Furthermore, it has been reported that the texture becomes less firm during storage in ice (SveinsdoÂttir et al., 2002, 2003b; Andersen et al., 1995). Farmer et al. (2000) demonstrated that texture of farmed salmon (Salmo salar) becomes less moist, light and tender by freezing at ÿ24 ëC, with less juicy and moist appearance, and oily flavour. Though, only minor changes were observed with time of frozen storage up to eight months. However, sensory attributes related to spoilage, such as cold storage/frozen storage, rancid and sour odours and flavours were not evaluated. Waagbù et al. (1993) reported less juiciness of frozen salmon compared to fresh salmon. Further, Waagbù et al. (1993) and Refsgaard et al. (1998) found a significant reduction in juiciness with time in frozen storage. Other studies of frozen salmon indicated that four months was the maximum shelf life for frozen salmon fillets stored at ÿ26 ëC (PoÂrisson and BragadoÂttir, 1992). Whole frozen salmon can be stored longer, Refsgaard et al. (1998) reported only minor sensory changes in whole salmon during eight months of storage at ÿ30 ëC. However, PoÂrisson and BragadoÂttir (1992) found that eight months was the maximum shelf life of whole frozen salmon at ÿ26 ëC as thereafter changes occurred in colour and flavour. Green-Petersen et al. (2006) compared the sensory characteristics of farmed Atlantic salmon (Salmo salar) stored whole in ice (seven and 16 days), frozen whole (six months) and frozen fillets (one and six months). Samples stored in ice for seven or 16 days and frozen as fillets for one month were characterised by sea/seaweed odour, juicy and oily texture, fresh fish oil and sweet flavour. However, after six months in frozen storage as

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whole and as fillets, the samples were described with firm texture, rancid flavour and discoloured appearance. An alternative to storage in ice and freezing is modified atmosphere (MA) packaging. Emborg et al. (2002) found that the shelf life of salmon (Salmo salar) fillets packed in MA was between 14 and 21 days. Studies on whole Atlantic salmon (Salmo salar) packed in MA compared to storage in ice (Sivertsvik et al., 1999a,b) indicated that the sensory quality of cooked MApacked salmon was equal or better compared to salmon stored in ice, such as less off-odour, off-flavour, rancid odour and flavour. However, they reported that the storage in MA had an undesirable effect on the appearance of whole raw salmon as the gills became grey sooner and the eyes lost clearness sooner compared to ice stored salmon. Fletcher et al. (2002) studied the spoilage of King salmon (Oncorhynchus tshawytscha) fillets stored in different atmospheres. The shelf life of the salmon stored in a 40:60 carbon dioxide and nitrogen mix was longer compared to storage in ambient air. Altogether 34 sensory attributes including sour and bitter flavour, moistness and chewiness were evaluated by a trained panel. Brown et al. (1980) studied the effect of MA storage compared to storage in ambient air of Silver salmon (Oncorhynchus kisutch), and found that storage in modified atmosphere reduced the development of strong aromas. GreenPetersen et al. (2006) studied products bought on the Danish market and found that sensory profile of Atlantic salmon (Salmo salar) stored in MA for seven days was marked with rancid and sour odour. However, this did not occur in Salmo salar stored in MA for five days nor in samples stored in ice for seven and 16 days, whereas no clear sensory differences were found between the samples. During recent decades, the production of farmed salmon has increased and thus the share of farmed salmon on the market. Several researchers have aimed at studying differences between farmed and wild caught salmon, and how eating quality is influenced by different farming conditions. Rasmussen (2001) published a review on how the quality of salmonids is affected by different farming conditions. Einen and Thomassen (1998) reported that fresh flavour was significantly reduced in Atlantic salmon (Salmo salar) when the fish was starved for 86 days compared to 30 days, and stress before stunning has been found to reduce the firmness of cooked fillets (Sigholt et al., 1997). River and sea-caught salmon have different sensory characteristics, and even more different than wild and farmed salmon (Salmo salar) (Farmer et al., 2000). River-caught salmon had more stagnant, earthy odour and flavour (which was related to the water quality) compared to sea-caught salmon, while salmon-like and oily odour and flavour, moist, light and tender texture were more characteristic for the farmed salmon. Farmer et al. (1995) also found that river-caught salmon had enhanced earthy flavour compared with sea-caught salmon, but did not find any significant difference between the flavour and odour of wild and farmed salmon. Green-Petersen et al. (2006) compared sensory characteristics of Salmo salar, Oncohynchus keta and Oncohynchus kisutch and showed that Oncohynchus keta was not particular different from Salmo salar, but Oncohynchus kisutch was different from Salmo salar. Oncohynchus kisutch

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was characterised by firm texture, rancid flavour and discoloured appearance, but a low intensity of juicy and oily texture, sea/seaweed odour, sweet, fresh fish oil and mushroom flavour. However, Green-Petersen et al. (2008) found sensory differences between all three species (Salmo salar, Oncohynchus keta and Oncohynchus kisutch). Samples of Oncohynchu keta and Oncohynchus kisutch had a more sour and rancid odour, discoloured appearance and a firm texture, combined with low intensity of sea/seaweed odour, fresh fish oil flavour, oily and juicy texture, though Oncohynchus keta had much lower intensity of these descriptors. Furthermore, Oncohynchus keta had high intensity of salt and rancid flavour, discoloured appearance, cooked potatoes and mushroom flavour, but low intensity of salmon colour, sweet odour and flavour and sourish odour and flavour. However, in both studies the samples from the different species were not treated in the same way (primary differences in frozen storage time), consequently influencing the sensory characteristics.

4.4 Consumer liking of different seafood products related to sensory characteristics Various terms used by consumers to describe cooked samples of several fish species are introduced by Sawyer et al. (1988). Flavour was cited as the main reason for liking or disliking, though there were some indications that texture was relatively more important for those who disliked fish. Further, Sawyer et al. (1988) compared evaluation by an untrained consumer panel to a trained sensory panel of 13 attributes for 18 common Atlantic fish species (cooked), which correlated significantly for most of the attributes. However, consumers usually find it difficult to explain in detail why they prefer one product to another, and the results may be difficult to interpret. Descriptive sensory analysis carried out by trained sensory panels provides accurate and detailed description of the products under study. The consumer acceptance or preference may then be related to the sensory characteristics of products by preference mapping (Greenhoff and MacFie, 1994; McEwan, 1996), and has been used to study acceptability of various food products. This section describes how different sensory characteristics of cod and salmon influence consumer liking. 4.4.1 Consumer preference of cod Few studies comparing consumer acceptability and sensory properties of different cod products have been published. SveinsdoÂttir et al. (2003a) studied acceptability of Icelandic consumers and the sensory quality of fresh, thawed and MA-packed cod fillets of different storage time. Consumers preferred thawed and MA-packed fillets to unpacked fresh fillets. The thawed and MApacked fillets were determined to be more dry and tough when compared to fresh fillets, according to a trained sensory panel. In addition, the consumers found differences between fresh and stored cod fillets (stored two and 10 days).

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The consumers preferred the more fresh fish, which was more described by fresh sweet odour and flavour, juicy and tender texture, while the cod stored 10 days was more described by sour and frozen storage odour and flavour and less juicy and tender texture. Luten et al. (2002) studied preferences of Dutch consumers and the sensory quality of wild and farmed cod. The farmed cod was slightly more appreciated by consumers. Compared to wild cod, the farmed cod received higher scores for white and dull appearance, cod taste and fibrousness, but lower scores for juiciness when evaluated by a trained sensory panel. SveinsdoÂttir et al. (2008) studied the liking of different cod products (Gadus morhua) (cooked) among consumers in Iceland (n 112), Denmark (n 107), Ireland (n 109) and the Netherlands (n 50) in a central location test carried out simultaneously in the four countries. At the same time a trained sensory panel evaluated the products with quantitative descriptive analysis. The products were of fresh farmed cod fillets (stored at 0±1 ëC for three and six days), fresh wild cod fillets (stored at 0±1 ëC for three and 10 days), MA-packed wild cod fillets (stored at 0±1 ëC for three and 10 days) and frozen fillets of wild cod (stored at ÿ24 ëC for nine days and five months). Icelandic and Irish consumers had higher liking for the cod products compared to Dutch and Danish, presumably due to different fish consumption patterns within the countries. Overall, frozen cod products, either after long or short frozen storage, were most preferred, but fresh cod after long storage was least preferred, but the preferences were different by countries. Irish consumers liked cod after extended frozen storage (5 months at ÿ24 ëC), while Icelandic and Dutch consumers liked cod after short frozen storage (9 days at ÿ24 ëC). These liking differences were somewhat in sequence with consumption traditions and consumption of different fish products within the countries. The consumers were segmented to analyse if groups of consumers with similar preferences existed. The cluster analysis identified five distinct clusters of consumers that were viewed with preference mapping. The clusters were different with regard to age, and marginal significance was observed for test location/country. The farmed cod particularly appealed to consumers in the first cluster, which could be explained by the meaty texture, flavour and odour, and light colour of farmed cod. Consumers in the second and third cluster were positively influenced by similar sensory characteristics, such as sweet and metallic flavour, and the absence of sensory attributes present in cod products after extended storage time, such as table cloth odour, TMA odour and flavour. Though the second and third cluster were influenced by similar sensory characteristics, they were very different with regard to liking scores. The second cluster discriminated much more between the products, while the third cluster generally scored all products low. The fourth cluster included consumers who had the highest preference for wild cod after extended frozen storage (five months at ÿ24 ëC). Generally, their preferences were accredited to attributes such as dark colour, frozen storage, table cloth and TMA odour, which are more characteristic for cod products after extended storage time. On the other hand, attributes characteristic for very fresh and farmed cod appeared not to appeal to

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this cluster. The fifth cluster included consumers who had high preferences for all the cod products indiscriminately. 4.4.2 Consumer preference of salmon Only a few studies have been made on consumer preference of salmon products. Sylvia et al. (1995) studied the acceptability of 189 consumers in Oregon (USA) of three types of fresh cooked salmon; farmed Atlantic salmon (Salmo salar), wild and farmed Chinook salmon (Oncorhynchus tschawytscha). The overall enjoyment of wild Chinook salmon was significantly higher than of the two other types of salmon. No differences were found in overall enjoyment between the two types of farmed salmon. In addition to hedonic assessment of the salmon, the consumers evaluated eleven sensory attributes related to flavour, texture and colour. Wild Chinook was found to have a more delicate/fresh flavour than both farmed Chinook and farmed Atlantic salmon, but the farmed Chinook and farmed Atlantic salmon were not different with regard to this attribute. Green-Petersen et al. (2008) studied consumer liking of different cooked salmon products. The products were farmed Atlantic salmon (Salmo salar) stored in ice (eight and 15 days), frozen (six weeks and five months) and MApacked (six and eight days), Oncohynchus kisutch and Oncohynchus keta stored frozen for eight and nine months, respectively. Altogether 381 consumers completed the test, which was performed in Iceland (n 121), Denmark (n 102), Ireland (n 109) and the Netherlands (n 49). Overall, the Salmo salar products received the highest average liking scores followed by Oncohynchus kisutch and Oncohynchus keta. No liking differences were found between the Salmo salar products, but a significant effect of storage time was found. Extended storage of Salmo salar products in ice (15 days), frozen (five months) and MA-packed (eight days) resulted in lower liking compared to salmon stored for a short time in ice (eight days), frozen (six weeks) and MA-packed (six days). Some liking differences were observed between the countries, as the Icelandic consumers had higher liking for Salmo salar stored frozen for five months compared to the other countries. However, the consumers in Denmark had higher preferences for Oncohynchus kisutch and Oncohynchus keta, which were stored frozen for eight and nine months. The connection between the consumer liking and the objective sensory description was studied using external preference mapping. Oncohynchus keta received in general lowest liking scores which can be explained by a high intensity of sour and rancid odour, rancid flavour, salt taste, discoloured appearance and firm texture, combined with a low intensity of sea/seaweed odour, fresh fish oil flavour, oily and juicy texture. Oncohynchus kisutch was generally scored second lowest for overall liking, which can also be explained by a high intensity of sour and rancid odour, discoloured appearance and firm texture, together with a low intensity of sea/seaweed odour, fresh fish oil flavour, oily and juicy texture. However, the profile was not as extreme as for Oncohynchus keta. The frozen storage period of Oncohynchus kisutch and

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Oncohynchus keta was considerably longer than of the Salmo salar and therefore, it was not clear if the overall liking differences and differences in sensory profiles were caused by differences between species. After extended storage of Salmo salar, the trend was towards a high intensity of sour odour, rancid odour and flavour, but a lower intensity of fresh fish oil flavour and oily texture.

4.5 Consumer segmentation across different European countries, related to attitudes and product preferences Consumers in different countries experience seafood differently, related to traditions, availability and frequency of consumption influencing individual preferences. Honkanen et al. (2005) studied fish consumption in five European countries, and the average fish consumption frequency in Spain was 2.6 times a week, or two to three times more frequent compared to mid-European countries such as Belgium (1.1 times per week) and the Netherlands (1.0 times per week) in 2004. The average fish consumption frequency in Northern European countries is rather high, 1.4 times per week in Denmark (Honkanen et al., 2005) and in 2002, close to 90% of Icelandic consumers consumed fish once per week or more often (SteingrõÂmsdoÂttir et al., 2003) and Brunsù (2003) reported a high fish consumption in Iceland compared to central Europe. Demographic differences have been demonstrated for liking of various food products, such as coffee (Heidema and de Jong, 1997), meat (Prescott et al., 2001) and chocolate (Januszewska and Viaene, 2001). Frequency of consumption influences attitudes towards foods and vice versa. Consumers from countries with high pork consumption have been shown to be more positive towards pork quality counter to consumers in countries with lower pork consumption (Bryhni et al., 2002). Furthermore, higher liking of lamb meat were shown among consumers in countries with high traditional lamb meat consumption (SanÄudo et al., 2007). SeÂmeÂnou et al. (2007) were able to show to some degree a link between the preferences of consumers from different countries and sensory characteristics of smoked salmon within their country. SveinsdoÂttir et al. (2008) showed that consumer preferences for different cod products were somewhat related to differences in fish consumption of four European countries. SveinsdoÂttir et al. (2006) segmented consumers across four European countries (Iceland, Denmark, Ireland and the Netherlands, approximately 120 fish consumers were recruited in each country) based on general attitudes towards food, fish and health, fish consumption motives and barriers in order to describe their fish consumption behaviour and predict seafood preferences. The products were cod and salmon of wild and farmed origin, fresh, MA-packed, frozen/thawed, and of short and extended storage time. Three consumer clusters were identified; the fish lovers (n 242), the low health and food involved (n 44), and the inconvenience group (n 175).

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The fish lovers consumed fish, fresh cod and salmon more frequently and purchased fish more often at the fishmonger (speciality shop) compared to the other two clusters. Overall, the fish lovers had high preferences for the fish products, and the highest preferences for fresh farmed cod after short storage time and wild cod stored frozen for a short time. For those consumers, sensory characteristics of newly caught fish seemed to be the most attractive, presumably due to higher consumption and familiarity of fresh fish. The low health and food involved and the inconvenience cluster scored high on inconvenience and insecurity towards fish purchase. These two clusters generally consumed fish less frequently and purchased fish less often compared to fish lovers. However, the inconvenience group consumed fish more often out of home and tended to purchase more pre-packed fish and consumed ready to eat chilled and frozen cod products most frequently. This indicated that these consumers tended to purchase more convenient seafood. The low health and food involved consumers had generally low preferences for the cod and salmon products. The liking differences of the clusters indicated that general positive attitudes towards fish resulted in higher overall liking. The clusters did not differ by country, which indicated that within each of the countries groups of similar seafood consumers exist. However, in the same study, SveinsdoÂttir et al. (2008) showed that the consumption of cod products and the places of purchase were different between the countries. The attitudes were also different. Icelanders were the most convinced that fish is healthy and had easy access to fish. Easy access to purchase fish might encourage fish consumption, as convenience has been found to have positive correlation to fish consumption frequency (Olsen et al., 2007). Icelandic consumers did not find fish expensive, as opposed to Danes, but price has been found to be one of the main barriers for fish consumption (Verbeke and Vackier, 2005). Further, in SveinsdoÂttir et al. (2008) it was shown that easier access, high frequency of purchase in fishmonger shops in Iceland and tradition of high fish consumption indicated a higher liking of sensory attributes characterising very fresh cod. However, the Irish consumers consumed frozen cod products more frequently and have a tradition of consuming much more frozen fish compared to fresh fish (National Statistics, 2001), which may have been reflected in their preferences for cod characterised by frozen storage attributes. The results of SveinsdoÂttir et al. (2008) showed that young consumers were considerably less health and food involved in comparison to older consumers. In addition, the younger consumers were more insecure regarding fish purchase, found fish preparation problematic and scored low on the fish liking factor. Further, Verbeke and Vackier (2005) showed that young adults had more negative attitudes related to fish (bones and price), but bought their own food less frequently than the older, and scored lower on experience with seafood, such as purchase, preparation and knowledge about seafood. Olsen (2003) also found positive correlation between age and attitudes, dealing with health concern and perceived convenience of seafood. High health and food involvement

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(Olsen, 2003) and convenience (Olsen et al., 2007) can positively influence fish consumption, but fish consumption is considerably lower among young adults (SteingrõÂmsdoÂttir et al., 2003; Verbeke and Vackier, 2005; Olsen, 2003). Easy access of convenient seafood products, clear and visible information on preparation and seafood handling for young consumers could contribute to an increase in their fish consumption. Sensory liking is the strongest determinant for fish consumption intention (Verbeke and Vackier, 2005). However, sensory liking of seafood appears somewhat to be subject to traditions within each country. Nevertheless, similar segments with regard to attitudes and product preferences exist within the countries.

4.6

The Seafood Sensory Quality Model

The fish chain can be defined from the catch of wild fish or slaughtering of farmed fish to consumption. The fish chain contains many links depending on fish species and product type. The fish farm, vessels, fish processors, transporters and retailers can, for example, be parts of the chain. In each link the fish is exposed to different factors such as temperature, handling and packaging. All these factors have an influence on the sensory quality and the sensory characteristics of the product. Besides, the raw material has a key influence on the product quality and sensory characteristics. Furthermore, it is possible to use sensory methods in all of the steps in the production chain to obtain information on the products sensory quality (Hyldig et al., 2007). Owing to the importance of the sensory quality, a Seafood Sensory Quality Model will enable the seafood industry to improve the eating quality of seafood available to consumers, and thereby encourage increased seafood consumption, and by doing so contribute to improved consumer health. To develop and establish Seafood Sensory Quality Models that relate consumers' perceptions of eating quality with sensory characteristics perceived by key quality decision makers at each point in the fishery production chain (from catch or slaughter, through handling and distribution to final consumption), information is needed from each stage of the fish chain. A schematic figure of the Seafood Sensory Quality Model is shown in Fig. 4.3. The methods used for sensory evaluation of seafood are described in Section 4.2, and the sensory characteristics of seafood products are described in Section 4.3. To understand consumer perceptions of eating quality, investigations of consumer preferences, in relation to objective sensory data, have been described in Section 4.4. Two different seafood chains (cod and salmon) have been studied in SEAFOODplus regarding the use of sensory evaluation and the application of the results (Schelvis et al., 2005). In four European countries, Ireland, Iceland, Denmark and The Netherlands, 8±17 companies throughout the fish production chain were selected to obtain knowledge about their sensory quality evaluation procedures and the descriptive

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Fig. 4.3

A schematic figure of the Seafood Sensory Quality Model.

terms they use. The fishery chain is represented by consumers, retail, storage/ distribution, fish processor, wholesaler, auction and fishing vessel/fish farmer. Selection of the companies aimed at providing useful information, not necessarily fully representative for the whole fishery sector. For this questionnaire, key persons were defined as persons within a company who have knowledge of quality assurance and assessment methods in their company. The objective of the questionnaire was to provide answers to the following questions: which points in the chain are in place for quality decision making? Are sensory methods used (for quality determination) in the fishery chain? Which methods are used? What do they measure in their opinion and why do they measure this? The results showed that there was a large variation in the way the information on sensory quality was structured and documented in each of the individual companies throughout the European fishery production chain. Almost all companies assessed the products by appearance and described general quality criteria, often related to freshness or other product specifications, but not in a systematic way. For companies using specific methods (EU scheme, QIM and Torry), it is relatively easy to describe norms and tolerances to evaluate the sensory quality on attributes. However, information on results of the sensory evaluation is not always communicated between companies in the chain.

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When the results were compared with the Seafood Sensory Quality Model, 60±100% of the vessels, farms and auctions used sensory tests of the incoming raw material (Descriptive test 0). Between 95 and 100% of the wholesalers, processors and exporters used sensory test (Descriptive test 2) and they used sensory tests at the raw material, in the processing and the final products. All the storage companies used sensory tests (Descriptive test 3) of the incoming material, but only 50% in the processing and 75% of the final goods. Most (90%) of the retailers used sensory tests (Descriptive test 5) of the incoming raw material and around 70% of the final goods. In the descriptive test used for quality control of raw material, mostly appearance and odour were evaluated, but for quality evaluation of the final products, taste was also included. Information obtained by the companies through sensory testing was rarely well documented and often not traceable. Further data collection on sensory data in the chain from consumer to catch is needed as an input to be able to finalise the Sensory Quality Model. Data collected with consumers and with key quality decision makers, will be related to develop consumer oriented Seafood Sensory Quality Models for each fish species studied within SEAFOODplus. These models will statistically link consumers' perceptions of eating quality, based on both intrinsic and extrinsic factors, with sensory characteristics perceived and measured by key decision makers in the fish handling chain. The Quality Index Method (QIM) is an important and very useful method within the seafood handling chain. It has been shown in various researches that there is a strong correlation between the Quality Index and the sensory attributes of cooked samples. In many articles on development of Quality Index Methods, a comparison has been made of sensory evaluation of whole raw fish and sensory evaluation of cooked samples, and an overview of these references is provided on www.qimeurofish.com. The freshness or storage time of the whole fish evaluated by QIM will be reflected in the sensory quality of the fillets or the final products. Therefore QIM will be one of the key methods used within the Sensory Quality Model.

4.7

Future trends

In the future, environmental factors such as functional products, hurdle technology, and aquaculture will be in focus. In addition, consideration will be given to key quality issues such as the ultimate effect of various environmental factors on the perceived eating quality. There will be possibilities of controlling sensory quality during seafood handling or processing and the potential for new product developments with unknown effects on sensory quality. The focus within the processing and distribution of seafood should be on quality issues and to maintain the freshness of the products as needed for the consumers to receive high quality product. Use of sensory methods to evaluate the quality of the products at each stage in the chain from catch to consumer gives unique information, which is useful for product and quality management.

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The seafood producer should know the consumer attitudes, motives and barriers for fish consumption to be able to choose market strategies. Although consumers liking and attitudes differ somewhat between countries, different segments of consumers can be found within each country and are comparable to segments found in other countries. Young consumers seem to be insecure regarding fish preparation and the food habits of young people are changing. Consumption of food products ready to cook or ready to eat is increasing. Decreasing fish consumption and attitudes of young consumers can have various negative impacts on future marketing and sales of seafood products. Education on the benefits of seafood consumption and preparation of seafood dishes is recommended. Seafood should be a part of a healthy diet of the consumers.

4.8

Sources of further information and advice

Further information on the role and descriptions of sensory evaluation in the fishery chain from catch/slaughter to consumer can be found in following books web-sites or books: http://www.qim-eurofish.com/ NOLLET, L.M.L. (ed.). (2007). Handbook of Meat, Poultry and Seafood Quality. Blackwell Publishing, Iowa, USA. Â LAFSDOÂTTIR, G. (eds.). (2003). Quality of Fish from LUTEN, J.B., OEHLENSCHLaÈGER, J. and O Catch to Consumer, Labelling, Monitoring and Traceability Wageningen Academic Publishers, The Netherlands. BREMNER, H.A. (ed.). (2002). Safety and Quality Issues in the Fish Processing Woodhead Publishing Ltd., Cambridge, England. Â TTIR, E., SVEINSDO Â TTIR, K., LUTEN, J., SCHELVIS-SMITH, R. and HYLDIG, G. (2001). MARTINSDO Sensory Evaluation of Fish Freshness. Reference Manual for the Fish Sector. QIM-Eurofish.

4.9

References

(2005). Fish and Seafood Sector Profile, The Netherlands, March 2003. Canadian Embassy in the Hague, Netherlands. Available: http://www.ats.agr.gc.ca/europe/3911_e.htm. Accessed 28.03.2007. Ê , A.M.B. (1995). Texture properties of farmed ANDERSEN, U.B., THOMASSEN, M.S. and RéRA Atlantic salmon (Salmo salar). Influence of storage time on ice and smelt age. In: Andersen U.B. Measurements of texture quality in farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) (III) Doctor Scientiarum Thesis. Ê S, Norway ISSN 0802-3220, ISBN 1995: 26, Agricultural University of Norway. A 82-575-0265-01-26. pp. 1±26. Â TTIR, K. and MARTINSDO Â TTIR, E. (2007). Development of Quality BONILLA, A.C., SVEINSDO Index Method (QIM) scheme for fresh cod (Gadus morhua) fillets and application in shelf life study. Food Control, 18 (4), 352±358. BROWN, W.D., ALBRIGHT M., WATTS, D.A., HEYER, B., SPRUCE, B. and PRINCE R.J. (1980). Modified atmosphere storage of rockfish (Sebastes miniatus) and silver salmon (Oncorhynchus kisutch). Journal of Food Science, 45, 93±96. AGRICULTURE AND AGRI-FOOD CANADA

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(2003). Consumer research of fish in Europe. In J.B. Luten, J. OehlenschlaÈger Â lafsdoÂttir, Quality of fish from catch to consumer (pp 335±344). and G. O Wageningen: Wageningen Academic Publishers.

BRUNSé, K.

BRYHNI, E. A., BYRNE, D. V., RéDBOTTEN, M., CLAUDI-MAGNUSSEN, C., AGERHEM, H.,

JOHANSSON, M., LEA, P. and MARTENS, M. (2002). Consumer perceptions of pork in Denmark, Norway and Sweden. Food Quality and Preference, 13, 257±266. CARDELLO, A.V., SAWYER, F.M, MALLER, O. and DIGMAN, L. (1982). Sensory Evaluation of the Texture and Appearance of 17 Species of North Atlantic Fish. Journal of Food Science, 47, 1818±1823. CHAMBERS, E. and ROBEL, A. (1993). Sensory Characteristics of Selected Species of Freshwater Fish in Retail Distribution. Journal of Food Science, 58 (3), 508±512. DALGAARD, P., GRAM, L. and HUSS, H.H. (1993). Spoilage and shelf-life of cod fillets packed in vacuum or modified atmospheres. International Journal of Food Microbiology, 19, 283±294. EINEN, O. and THOMASSEN, M.S. (1998). Starvation prior to slaughter in Atlantic salmon (Salmo salar) II. White muscle composition and evaluation of freshness, texture and colour characteristics in raw and cooked fillets. Aquaculture, 169, 37±53. EMBORG, J., LAURSEN, B.G., RATHJEN, T. and DALGAARD P. (2002). Microbial spoilage and formation of biogenic amines in fresh and thawed modified atmosphere-packed salmon (Salmo salar) at 2 ëC. Journal of Applied Microbiology, 92, 790±799. FAO (2006) Projection of World Fishery Production in 2010. FAO fisheries. Available: http://www.fao.org/fi/highligh/2010.asp. Accessed 14.03.2006. FARMER, L.J., MCCONNELL, J.M., HAGAN, T.D.J. and HARPER, D.B. (1995). Flavour and offflavour in farmed and wild Atlantic salmon from locations around Northern Ireland. Water Science Technology, 31, 259±264. FARMER, L.J., MCCONNELL, J.M. and KILPATRICK, D.J. (2000). Sensory characteristics of farmed and wild Atlantic salmon. Aquaculture, 187, 105±125. FLETCHER, G.C., SUMMERS, G., CORRIGAN, V., CUMARASAMY, S. and DUFOUR, J.P. (2002). Spoilage of king salmon (Oncorhynchus tshawytcha) fillets stored under different atmospheres. Journal of Food Science, 67 (6), 2362±2374. GREENHOFF, K. and MACFIE, H.J.H. (1994) Preference mapping in practice. In: H.J.H. MacFie and D.M.H. Thomson, Measurements of Food Preferences (pp 137±166). London: Blackies Academic and Professional. GREEN-PETERSEN, D.M.B., NIELSEN, J. and HYLDIG, G. (2006). Sensory profiles of the most common salmon products on the Danish market. Journal of Sensory Studies, 21, 415±427. Â TTIR, K., SCHELVIS, R. and MARTINSDO Â TTIR, E. GREEN-PETERSEN, D.M.B., HYLDIG, G., SVEINSDO (2008). Consumer preference and description of salmon in association with sensory characteristics. Journal of Aquatic Food Product Technology (submitted). HEIDEMA, J. and DE JONG, S. (1997). Consumer preferences of coffees in relation to sensory parameters as studied by analysis of covariance. Food Quality and Preference, 9(3), 115±118. HONKANEN, P., OLSEN, S. O., BRUNSé, K., VERBEKE, W., SCHOLDERER, J., FRUENSGAARD, L. and PIENIAK, Z. (2005). Deliverable 5: Report on cross-cultural eating habits and segments. Project 2.1 CONSUMERSURVEY. Integrated Project FOOD-CT-2004506359. HYLDIG, G. (2007). Sensory Profiling of Fish, Fish Products, and Shellfish. Nollet, L.M.L. (ed.). Handbook of Meat, Poultry and Seafood Quality (pp. 511±528). Blackwell Publishing, Iowa, USA.

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and NIELSEN, D. (2007). Texture of Fish, Fish Products, and Shellfish. Nollet, L.M.L. (ed.). Handbook of Meat, Poultry and Seafood Quality (pp. 549±561). Blackwell Publishing, Iowa, USA. HYLDIG, G., LARSEN, E. and GREEN-PETERSEN, D.M.B. (2007). Fish and sensory analysis in the fish chain. Nollet, L.M.L. (ed.). Handbook of Meat, Poultry and Seafood Quality (pp. 499±510). Blackwell Publishing, Iowa, USA. ISO 8586-1 (1994). Sensory analysis ± General guidance for selection, training and monitoring of assessors ± Part 1: Selected assessors. ISO 8589 (1988). Sensory analysis ± General guidance for the design of test rooms. JANUSZEWSKA, R. and VIAENE, J. (2001). Sensory segments in preference for plain chocolate across Belgium and Poland. Food Quality and Preference, 12, 97±107. LARSEN, E., HELDBO, J., JESPERSEN, C.M. and NIELSEN, J. (1992). Development of a method for quality assessment of fish for human consumption based on sensory evaluation. In: Huss, H.H., Jakobsen, M. and Liston, J. (eds.) Quality Assurance in the Fish Industry (pp. 351±358). Elsevier Science Publishing, Amsterdam. LAWLESS, H.T. and HEYMANN H. (1999). Sensory Evaluation of Food. Principles and Practices. An Aspen Publication, Aspen Publishers, Inc., Gaithersburg, Maryland. È G, M. and AKSE, L. (2002) LUTEN, J., KOLE, A., SHELVIS, R., VELDMAN, M., HEIDE, M., CARLEHO Evaluation of wild cod versus wild caught, farmed raised cod from Norway by Dutch consumers. ékonomisk Fiskeriforskning, 12, 44±60. Â TTIR, E. (1995). Storage quality of fresh and frozen-thawed Â SSON, H. and MARTINSDO MAGNU fish in ice. Journal of Food Science, 60 (2), 273±278. Â TTIR, E. and MAGNUÂSSON, H. (2001). Keeping quality of sea-frozen thawed cod MARTINSDO fillets on ice. Journal of Food Science, 66 (9), 1402±1408. Â TTIR, E., SVEINSDO Â TTIR, K., LUTEN, J., SCHELVIS-SMITH, R. and HYLDIG, G. (2001). MARTINSDO Sensory Evaluation of Fish Freshness. Reference Manual for the Fish Sector. QIM-Eurofish. Â TTIR, E., LUTEN, J.B., SCHELVIS-SMIT, A.A.M. and HYLDIG, G. (2003). Developments MARTINSDO Â lafsdoÂttir, G. of QIM ± past and future. In: Luten, J.B., OehlenschlaÈger, J. and O (eds.) Quality of Fish from Catch to Consumer, Labelling, Monitoring and Traceability (pp. 265±272). Wageningen Academic Publishers, The Netherlands. Â TTIR, E., SCHELVIS, R., HYLDIG, G. and SVEINSDO Â TTIR, K. (2008). Sensory MARTINSDO evaluation of seafood ± Methods. In: Rehbein, H. and OehlenschlaÈger, J. (eds.) Fishery Products: Quality, Safety and Authenticity. Blackwell Publishing, Oxford. MCEWAN, J.A. (1996). Preference mapping for product optimization. In Nñs, T. and Risvik, E. (eds.) Multivariate Analysis of Data in Sensory Science (pp. 71±102). London: Elsevier Science B.V. MEILGAARD, G., CIVILLE, V. and CARR, B.T. (1999). Sensory Evaluation Techniques, 3rd. edn, New York, CRC Press. NATIONAL STATISTICS (2001). National Food Survey Northern Ireland 2000. Annual Report on Food Expenditure, Consumption and Nutrient Intakes. Department of Agriculture and Rural Development. Economics and Statistics Division. Available: http://www.dardni.gov.uk/nfs2000.pdf. Accessed 21.03.2007. NIELSEN, J. and JESSEN, F. (2007). Quality of Frozen Fish. In Nollet, L.M.L. (ed.). Handbook of Meat, Poultry and Seafood Quality (pp. 577±586). Blackwell Publishing, Iowa, USA. OLSEN, S.O. (2003). Understanding the relationship between age and seafood consumption: the mediating role of attitude, health involvement and convenience. Food Quality and Preference, 14 (3), 199±209. HYLDIG, G.

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and VERBEKE, W. (2007). Exploring the relationship between convenience and fish consumption: a cross-cultural study. Appetite, 49 (1), 84±91. Â RISSON, S. and BRAGADOÂTTIR, M. (1992). Geymsluìol a Â frystum laxi. Rit Rf 34. PO RannsoÂknastofnun fiskiônaôarins, Reykjavik, Iceland (in Icelandic). (Shelf life of frozen salmon, IFL report no. 34). Available: http://www.matis.is/media/utgafa/ matra/Rit_Rf_34.pdf (accessed April 2007). PRELL, P.A. and SAWYER, F.M. (1988). Flavor profiles of 17 species of North Atlantic fish. Journal of Food Science, 53 (4), 1036±1042. PRESCOTT, J., YOUNG, O. and O'NEILL, L. (2001). The impact of variations in flavour compounds on meat acceptability: a comparison of Japanese and New Zealand consumers. Food Quality and Preference, 12, 257±264. RASMUSSEN, R.S. (2001). Quality of farmed salmonids with emphasis on proximate composition yield and sensory characteristics. Aquaculture Research, 32, 767±786. REFSGAARD, H.H.F., BROCKHOFF, P.B. and JENSEN, B. (1998). Sensory and chemical changes in farmed Atlantic salmon (Salmo salar) during frozen storage. Journal of Agricultural and Food Chemistry, 46 (9), 3473±3479. OLSEN, S.O., SCHOLDERER, J., BRUNSé, K.

Â N, R., THORKELSSON, G., VALDIMARSDO Â TTIR, T., Ä UDO, C., ALFONSO, M., SAN JULIA SAN

ZYGOYIANNIS, D., STAMATARIS, C., PIASENTIER, E., MILLS, C., BERGE, P., DRANSFIELD,

and FISHER A.V. (2007) Regional variation in the hedonic evaluation of lamb meat from diverse production systems by consumers in six European countries. Meat Science, 77 (4), 610±621. SAWYER, F.M., CARDELLO, A.V. and PRELL, P.A. (1988). Consumer evaluation of the sensory properties of fish. Journal of Food Science, 53 (1), 12±24. E., NUTE, G.R., ENSER M.

Â TTIR, K., VAN RUTH, S., SCHELVIS, R., VELDMAN, M., HYLDIG, G., GREEN-PETERSEN, D., SVEINSDO Â TTIR, E. (2005). European fish industry chain rarely uses FAYOUX, S. and MARTINSDO

specified sensory methods to describe sensory quality in a systematic way. RIVO report C017/05 July 2005. SEÂMEÂNOU, M, COURCOUX, P., CARDINAL, M., NICOD, H. and OUISSE, A (2007). Preference study using a latent class approach. Analysis of European preferences for smoked salmon. Food Quality and Preference, 18 (5), 720±728. SHEWAN, J.M., MACINTOSH, R.G., TUCKER, C.G. and EHRENBERG, A.S.C (1953). The Development of a Numerical Scoring System for the Sensory Assessment of the Spoilage of Wet White Fish Stored in Ice. Journal of Science and Food Agriculture, 4 (June) 283±298. SIGHOLT, T., ERIKSON, U., RUSTAD, T., JOHANSEN, S., NORDTVEDT, T.S. and SELAND, A. (1997). Handling stress and storage temperature affect meat quality of farmed-raised Atlantic salmon (Salmo salar). Journal of Food Science, 62, 898±905. SIVERTSVIK, M., NORDTVEDT, T.S., AUNE, E.J. and ROSNES J.T. (1999a). Storage quality of superchilled and modified atmosphere packaged whole salmon. In Proceedings from 20th International Congress of Refrigeration, Sydney Australia, 19±24 Sept. Vol IV. pp. 2488±2495. SIVERTSVIK, M., ROSNES, J.T, VORRE, A., RANDELL, K., AHVENAINEN, R. and BERGSLIEN, H. (1999b). Quality of whole gutted salmon in various bulk packages. Journal of Food Quality, 22, 387±401. SIVERTSVIK, M., JEKSRUD, W.K. and ROSNES, J.T. (2002). A review of modified atmosphere packaging of fish and fishery products ± significance of microbial growth, activities and safety. International Journal of Food Science and Technology, 37 (2), 107±127.

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and OÂLAFSDoÂTTIR, S. A. (2003). Hvaô borôa ÂIslendingar? KoÈnnun aÂ matarñôi ÂIslendinga 2002, Helstu niôurstoÈôur (in Icelandic). (The Diet of Icelanders, Dietary Survey of The Icelandic Nutrition Council 2002. Main findings) LôheilsustoÈô. Available: http://lydheilsustodvefur.eplica.is/media/ manneldi/rannsoknir/skyrsla.pdf. Accessed 10.04.2006. STONE, H. and SIDEL, J.L. (1985). Sensory Evaluation Practices. Orlando, FL: Academic Press. Â TTIR, K., MARTINSDO Â TTIR, E., HYLDIG, G., JéRGENSEN, B. and KRISTBERGSSON, K. SVEINSDO (2002). Application of quality index method (QIM) scheme in shelf-life study of farmed Atlantic salmon (Salmo salar). Journal of Food Science, 67, 1570±1579. Â TTIR, K., THORKELSDO Â TTIR, A Â . and MARTINSDO Â TTIR, E. (2003a). Consumer survey: SVEINSDO cod fillets packed in air and modified atmosphere (MAP). Proceedings of the TAFT 2003 conference, 10±14 June 2003 Reykjavik, Iceland. The Icelandic Fisheries Laboratories, ISBN 9979-74-005-1. Â TTIR, K., HYLDIG, G., MARTINSDOÂTTIR, E., JéRGENSEN, B. and KRISTBERGSSON, K. SVEINSDO (2003b). Quality Index Method (QIM) scheme developed for farmed Atlantic salmon (Salmo salar). Food Quality and Preference, 14, 237±245. Â TTIR, K., MARTINSDO Â TTIR, E., GREEN-PETERSEN, D., HYLDIG, G. and SCHELVIS R. SVEINSDO (2006). Deliverable 8: Report on development of the Preference Map models. Focus on the consumer test in March/April 2005, data analysis. Project 2.2 SEAFOODSENSE. Integrated Project FOOD-CT-2004-506359. Â TTIR, K., MARTINSDO Â TTIR, E., GREEN-PETERSEN, D., HYLDIG, G., SCHELVIS, R. and SVEINSDO DELAHUNTY, C. (2008). Sensory characteristics of different cod products related to consumer preferences and attitudes. Food Quality and Preferences (submitted). SYLVIA, G., MORRISSEY, M.T., GRAHAM, T. and GARCIA, S. (1995). Organoleptic qualities of farmed and wild salmon. Journal of Aquatic Food Product Technology, 4 (1), 51±64. VANNUCCINI, S. (2004). Overview of fish production, utilization, consumption and trade: based on 2002 data. FAO, Fishery Information, Data and Statistics Unit. Rome, Italy. Available: http://www.fao.org/fi/Prodn.asp. Accessed 16.03.2006. VERBEKE, W. and VACKIER I. (2005). Individual determinants of fish consumption: application of the theory of planned behaviour. Appetite, 44 (1), 67±82. WAAGBé, R., SANDNES, K., TORRISSEN, O.J., SANDVIN, A. and LIE, é. (1993). Chemical and sensory evaluation of fillets from Atlantic salmon (Salmo salar) fed three levels of N-3 polyunsaturated fatty acids at two levels of vitamin E. Food Chemistry, 46 (4), 361±366. Â TTIR, K., MAGNUÂSSON, H. and MARTINSDO Â TTIR, E. (2007). Combined WANG, T., SVEINSDO application of modified atmosphere packaging and superchilled storage to extend the shelf-Life of fresh cod (Gadus morhua) loins. Journal of Food Science. Available online from 28 November 2007. WARM, K., BOKNáS, N. and NIELSEN, J. (1998). Development of Quality Index Methods for Evaluation of Frozen Cod (Gadus morhua) and Cod Fillets. Journal of Aquatic Food Product Technology, 7 (1), 45±59. Â TTIR, L., üORGEIRSDOÂTTIR, H. STEINGRõÂMSDO

WELCH, A.A., LUND, E., AMIANO, P., DORRONSORO, M., BRUSTAD, M., KUMLE, M., RODRIGUEZ, M., LASHERAS, C., JANZON, L., JANSSON, J., LUBEN, R., SPENCER, E.A., OVERVAD, K., TJONNELAND, A., CLAVEL-CHAPELON, F., LINSEISEN, J., KLIPSTEIN-GROBUSCH, K., BENETOU, V., ZAVITSANOS, X., TUMINO, R., GALASSO, R., BUENO-DE-MESQUITA, H.B., OCKE,

and SLIMANI, N. (2002). Variability of fish consumption within the 10 European countries participating in the European Investigation into Cancer and Nutrition (EPIC) study. Public Health Nutrition, 5(6B), 1273±1285. M.C., CHARRONDIERE, U.R.

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5 Evaluating consumer information needs in the purchase of seafood products W. Verbeke and Z. Pieniak, Ghent University, Belgium, K. Brunsù and J. Scholderer, University of Aarhus, Denmark and S.O. Olsen, Nofima, Norway

5.1

Introduction

Consumers' cognitive mechanisms and their perception of product attributes may be markedly affected by information (Caporale and Monteleone, 2004). Consumers seem to want information to help them achieve a balanced diet, and eventually also to avoid certain allergens or ingredients that have proven not to agree with them. Furthermore, contemporary consumers seem to want to know the origin and environmental, ethical and technological conditions under which their food was produced, processed, stored and distributed. The food industry has been responding to these demands through the establishment of quality management systems, quality labelling and branding schemes, and with the establishment of traceability, with the latter being commissioned and regulated to a large extent by European and national governments. By communicating and sending a message to consumers, such as an advertisement, public health recommendations, or labelling information, not only will consumers be informed, but also their attitudes, intentions or behaviour can be influenced. At least, this would be the strategic policy or commercial objective of providing consumer information. However, providing (more) information to consumers does not necessarily mean better informed consumers, as has already been understood with respect to non-food products (Dranove et al., 2003; de Garidel-Thoron, 2005). Information is likely to be effective in terms of altering attitude, intention or food choice behaviour, only when it addresses specific information needs and can be processed and used adequately

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by its target audience. Risks of information overload and potential adverse effects resulting from consumers' indifference when confronted with too much information have been recognised (Verbeke, 2005). Increasing the amount of information, for example on the product label, may overload the label or package, and make a given and desired amount of information harder to extract, or simply cause individuals without time, ability or willingness to process information to ignore it (SalauÈn and Flores, 2001). Hence, insights in and a better understanding of consumers' needs for information are required before these needs can be addressed effectively and efficiently. Thus far, only a few studies concerning consumer information needs, the role of different information sources and labelling, have concentrated specifically on seafood. Kaabia et al. (2001) reported that increased information available to consumers about the relationship between diet and health, had a positive but relatively small impact on fish and poultry consumption, at the expense of red meat consumption. Scholderer and Grunert (2001, 2003) investigated a generic advertising campaign that was launched in 1996 in Denmark and that aimed at stimulating fresh fish consumption in line with public health recommendations. A series of television advertisements together with supplementary point-of-sales materials and the introduction of modified atmosphere-packed fresh fish fillets in the supermarkets resulted in an increase of both the intention to buy fresh fish and reported fish purchase. Also relatively little research is available about the type of information consumers seek and use on product labels. Most of the work in this area has focused on the fresh meat product category (Wandel, 1997; Bernues et al., 2003; Verbeke and Ward, 2006) and on how consumers use food labels in terms of attention paid to particular pieces of information (Capps, 1992; Abbott, 1997). None of these studies focused specifically on consumers' use of seafood labelling information. Consumer interest in information, labelling and traceability related to seafood has been covered specifically within the SEAFOODplus project 2.3 SEAINFOCOM. The rationale for this scope was that, in many of today's food markets, consumer decision-making and utility maximisation are hampered because information is imperfect, incomplete, inaccessible, asymmetrically distributed, non-standardised or costly to collect. The potential market failure resulting from decision-making under uncertainty is that food choice is not fully in line with actual preferences, which ultimately restricts consumer well-being. This potential failure holds specifically in situations where product differentiation is relatively low and mainly based on so-called credence characteristics, like healthiness, safety, sustainability or ethical characteristics. Needless to say that these areas are highly and increasingly relevant in the case of seafood, though they remained largely un-investigated so far. This chapter is organised as follows. First, materials and methods used for collecting primary consumer data with respect to the use, trust and interest in information are presented. Second, findings from exploratory focus group discussions performed in Spain and Belgium during May 2004 are summarised. Third, quantitative descriptive findings are presented based on the pan-European

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Structure of the findings presented in this chapter.

SEAFOODplus consumer survey performed in Belgium, Denmark, The Netherlands, Poland and Spain. The empirical findings presented in this book chapter are partly based on and extended from Pieniak et al. (2007a, 2007b). Figure 5.1 provides an overview of the different components of the quantitative study as covered in this chapter. First, consumers' use of and trust in information sources related to seafood are used to segment the seafood market. Next, the resulting segments are profiled both in terms of socio-demographics, fish consumption behaviour, interest in seafood information cues and traceability, which can form the basis for targeted information provision. Finally, the segments are compared with respect to health perceptions, risk and safety perceptions, and perceptions relating to ethical issues. Whereas the implementation and challenges facing seafood traceability is extensively dealt with in Part VI of this book, the current chapter reports findings with respect to traceability from the consumer perspective, more specifically consumers' interest in seafood traceability and information that can be backed up by traceability systems.

5.2

Materials and methods

5.2.1 Data collection Exploratory study First, qualitative exploratory research has been performed in May 2004 through six focus group discussions in two European countries: three in Belgium and three in Spain. Focus groups are an established way of obtaining deeper insights into beliefs and subjective meaning structures of consumers. With the objective to investigate consumers' interest in information related to fish in both a heavy user and a light user country, it was decided to segment the participants according to their fish consumption level. Spain has the second highest fish intake in the world, with a consumption level of 40 kg/capita/year, while Belgium is among

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the countries with the lowest consumption of fish in Europe with an intake of 10 kg/capita/year. These countries were therefore evident choices for this exploratory research that aimed at gathering consumer opinions as diverse as possible. It was chosen to have one heavy user group and two light user groups in both Spain and Belgium in order to obtain an in-depth knowledge of the barriers that prevent consumers from eating fish1 (an insight which we expected to get primarily from the light users). Owing to the very different consumption levels in the two countries, the definition of heavy users and light users varied considerably. It was assumed that a heavy user in Spain consumes fish four to five times a week, while a heavy user in Belgium consumes fish at least once a week. A Spanish light user consumes fish only once or twice a week while the Belgian light user consumes fish less than once a week. In both Belgium and Spain professional marketing research agencies assisted in conducting the focus group discussions. Participants were recruited from the local areas (Madrid and Bilbao in Spain; Ghent in Belgium) by telephone. The aim was to recruit 8 to 10 participants for each of the six focus groups. Consumers were only admitted for participation if they were women, responsible for purchasing and preparing fish in their own household. Both young and older consumers were recruited, provided that they fulfilled the screening criteria. In total, 48 women participated in this study, i.e. 22 in Belgium and 26 in Spain. An interview guide used for structuring the group discussions was initially developed by the research team, then translated to the languages of the respective countries and strictly adhered to during the discussion sessions. All question items were presented in an open-ended format in order to obtain as much information as possible, and to stimulate interaction among participants. All sessions lasted between 150 and 180 minutes, were facilitated by a professional moderator and were attended live in a neighbouring room by the researchers. Additionally, the sessions were videotaped and transcribed literally for subsequent analyses. Only questions related to information about fish will be presented within this chapter. Consumer survey After gaining preliminary insights into consumers' use of information and information needs about fish, a quantitative cross-sectional consumer survey was carried out in November±December 2004 in five European countries: Belgium, Denmark, The Netherlands, Poland and Spain. Age and region have been reported previously as important determinants of fish consumption (Myrland et al., 2000; Trondsen et al., 2003; Verbeke and Vackier, 2005). Therefore, a quota sampling procedure with age and region as quota control variables was used. A total sample of 4,786 consumers (n 800±1,100 respondents per country) was obtained (for additional methodological issues about the 1. This study focused on finfish consumption only, i.e. including fresh, deep-frozen, canned and smoked finfish, though excluding shellfish and other seafood like algae.

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pan-European SEAFOODplus consumer survey, see Chapters 2 and 3). Samples are representative within each country for age and region. All respondents were responsible for food purchasing within their household. The gender, age, income, education and country distribution within the total sample is presented in Table 5.1. A questionnaire was developed in English and pre-tested through pilot studies in all languages (Dutch, French, Danish, Polish and Spanish). The questionnaire measured a wide variety of constructs including behaviour, attitude, beliefs, perceptions, knowledge with respect to fish, and use of and interest in informaTable 5.1

Socio-demographic and behavioural profile of the clusters Consumer segments

Total Sceptics Enthusiasts Confidents sample Size (% sample)

24.0

41.4

34.6

Age (mean) Age (classes) < 25 (%) 25±55 (%) > 55 (%)

43.4

42.7

41.3

42.7

11.3 67.7 21.0

10.3 71.1 18.6

10.6 74.0 15.4

10.2 70.9 18.9

Gender Male Female

28.1 71.9

21.1 78.9

24.0 76.0

23.7 76.3

Income Lower Middle Upper

28.4 44.5 27.1

23.8 50.4 25.8

25.1 48.1 26.8

25.7 47.8 26.5

Nationality Belgian Danish Dutch Polish Spanish

12.6 11.4 11.8 50.2 14.0

15.8 18.5 16.2 20.2 29.3

17.7 29.7 26.9 5.5 20.2

17.8 23.2 16.9 21.2 20.9

p-value

Pearson 2 /F-value

100 <0.001 0.005

9.04 14.95

<0.001

17.57

0.029

10.80

<0.001

826.76

Intention to eat fish* 3.82a

4.61b

3.71a

4.13 (2.09)

< 0.001

88.972

Fish consumption** at home

1.02a

1.43b

1.06a

< 0.001

54.120

out of home

0.23a

0.36b

0.25a

< 0.001

17.224

total

1.25a

1.79b

1.31a

1.20 (1.22) 0.29 (0.64) 1.48 (1.50)

< 0.001

60.859

The a, b indicate significantly different means using Tukey HSD Post Hoc * 7-point scale (1 = very unlikely; 7 = very likely) ** number of times per week

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tion cues. Respondents were either personally contacted at home or recruited online by the market research agencies involved in the fieldwork. Upon their agreement to participate, participants were asked to self-administer and return the questionnaire. 5.2.2 Measures Use of and trust in information sources Respondents were asked about their use of different information sources for obtaining information about fish. A 7-point Likert scale ranging from `never' (1) to `very often' (7) was used. Potential information sources included were: family and friends, food and fish industry, consumer organisation, government, scientists, fisherman/fish farmers, supermarkets, fish monger, doctor and dietician (Rosati and Saba, 2004; and focus group results, see Section 5.3.1 for more details). These sources were considered as those most frequently involved in communicating benefits, potential risks and other information about seafood. Additionally, typical mass media information sources were included in the questionnaire: newspaper, television, advertising, public health recommendations and radio. Again, those media were considered as the most involved in giving information with respect to seafood. Furthermore, trust in information sources was investigated using a single item measure (Rosati and Saba, 2004). Respondents were asked to rate each of the above-mentioned information sources and mass media on the question `To what extent do you trust information about fish from the following sources' on 7-point Likert scales ranging from `completely distrust' (1) to `completely trust' (7). Use of and interest in information cues Next, respondents were asked to report how often they use seven information cues that (can) appear either on the package or on the supermarket shelf or on the product label of fish. These were: `fish species/name'; `price'; `weight'; `expiry date'; `nutritional composition'; `brand name'; and `capture area'. Use of these cues was measured on a 7-point Likert scale ranging from `never' to `always'. These seven items were chosen based on the fact that most of these are mandatory either for fresh food in general (price, weight, expiry date) or for fish in particular (species, capture area). During the exploratory study, consumers effectively reported to believe that these cues are or should be mandatory on fish labels. Finally, consumers were asked about their interest in emerging information cues. Nine possible information cues were selected, based also mainly on the results of the focus group discussions and regulatory developments with respect to food and fish labelling. The additional information cues were: `method of preparation'; `wild/farmed'; `health benefits'; `recipes'; `safety guarantee'; `quality mark'; `batch number for product identification'; `environmentally friendly'; `fish welfare'. Moreover, four information cues with specific

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relevance for farmed fish were included: `country of origin'; `feed used during farming'; `fed with genetically modified feed'; and `colorants used'. Respondents had to indicate to what extent they are interested in each of the included potential information cues. A 7-point Likert scale ranging from `not interested at all' (1) to `very interested' (7) was used. 5.2.3 Analyses procedures First, the transcripts from the focus group discussions were analysed by coding responses and examining the discussions' content for common themes according to content analysis procedures (Miles and Huberman, 1994; Morgan and Krueger, 1997). Second, the quantitative data from the consumer survey were analysed using the statistical software SPSS version 12.0. Descriptive analysis was performed in order to get the general view on consumers' use of and interest in information sources and cues. Next, a cluster analysis was performed. Finally, cluster profiles were determined using chi-square cross-tabulation and ANOVA with post-hoc Tukey HSD comparison of mean scores.

5.3

Empirical findings

5.3.1 Exploratory study Use of information sources Personal sources were found to be the most important source of information about fish. Both in Spain and in Belgium the salesperson (fishmonger or fisherman) is believed to be a source of confidence and plays an important role as an adviser and opinion leader. Belgian consumers pointed to the practical dimension of the information received through this face-to-face contact with the salesperson: `The salesman tells me how to prepare the fish; what fish combines best with other fish; what vegetables and sauces I need to prepare and finally what wine I should serve with the fish I prepare.' (Belgium). Additionally, and particularly in Belgium, friends, relatives or acquaintances were seen as one of the most important information sources. During all three focus group discussions with Belgian consumers, many participants pointed at one particular shop after being advised to shop there by friends and relatives; some participants even informed other participants who were not yet aware of this shop. Spanish respondents had not recently noticed any mass media information regarding fish. By the time the research was carried out, no public health or generic advertising campaigns with regard to fish consumption were running in Spain. In general, the fish intake in Spain is already on a high level; therefore campaigns to promote fish consumption are probably not common. Nevertheless, Spanish respondents believed that mass media information related to seafood is needed to some extent. In Belgium, although a campaign promoting fish consumption was running during the period of data collection, none of the respondents spontaneously referred to it. In general, there was a feeling among

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participants that not much advertising about fish exists and that little information about fish is available. Advertising was not perceived by heavy users as a source of information about fish: `Advertising is not information; if you want to know and learn something about fish, you have to search yourself for information' (Spain). Moreover, `If some stories or information is published, then is tends to be mainly negative. Good news is no longer shown ± you have to really search for it' (Belgium). Participants were convinced that there were many more scandals with regard to meat, and they believed that as a result, more effort is made to advertise meat, so as to counterbalance the negative press. One participant believed that there is no need for more information about fish. She stated that there is already too much information, even about fish: `People are too much manipulated and our choices are too heavily influenced by the information we receive. When more information about fish was provided, its primary aim was to influence consumers to eat more fish, rather than to inform people in an objective manner' (Belgium). The heavy users' group stated that appropriate advertisement (understood as public health campaigns) could be successful in promoting fish consumption towards children, especially when reference people or opinion leaders would be involved: `Famous people, for instance sportsmen or royals, should give the good example; it should be shown that these people also eat fish, which may have a favourable impact on both mothers' and children's motivations towards serving or eating fish' (Spain). Use of potential and existing information cues on-label The focus group discussions also explored consumer awareness, interest and use of information cues on fish labels. The most important on-label information cues for Spanish consumers were: name of the fish, expiry date, and price. In Belgium, expiry date as an indication of freshness and shelf life, price, cooking recommendations, nutritional information and eventual harmful substances content emerged as the most important information cues. Furthermore, in both Spanish light user groups the `date of death' (catch date) was stated as an important information cue, but to a lesser extent as compared with the previously mentioned information cues. Other issues mentioned by the Spanish consumers were: animal welfare, nutritional information, production date, information related to the quality controls of fish and cooking recommendations. All these issues were, however, considered dispensable and the last three as `nice to have' only in the ideal situation. Additional issues mentioned by the Belgian respondents with regard to fish labelling were: production and packaging date; weight and name of the fish. Moreover, Belgian heavy users indicated that they would like to receive information about the origin of fish, meaning both capture area and information whether the fish is wild or farmed: `For me, it is important to know the origin of fish because then I have an idea of the cleanliness of the water where the fish was caught. For me, this is an indication for eventual contamination and safety risks' (Belgium).

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Furthermore, because of an increasing role of traceability as an information system also for fish (Bùrresen et al., 2003), consumers' interest in traceability as an additional on-label information cue was investigated. None of the Spanish respondents, neither the light users nor the heavy users, recognised the word `traceability'. However, when the concept was explained, it was quite well understood. In Belgium both the word and the concept were understood, which is most likely the result of the debates and initiatives about traceability in the meat chain. However, the awareness of this specific concept was very low. One Belgian participant stated that traceability may have the opposite effect: `It is better not to know anything about the fish; if you know where it comes from, you may not want to eat it any more' (Belgium). In general, there was no perceived need for comprehensive information about fishing methods and processing. Both heavy and light users agreed that reading all information from the label is very time consuming, difficult to understand and ultimately only loosely related to the intrinsic fish quality. Additionally, Spanish respondents did not show any interest in this information: `They can write whatever they want on the label. I am not going to read it anyway' (Spain); `I really do not care very much whether this fish comes from the north or from the south; I don't care since this tells nothing about the quality' (Spain). On the other hand, Belgian participants displayed somewhat more interest in information, thus they were keener on extrinsic attributes like information that may help them to evaluate fish quality. In this case, information cues perform the function of a heuristic or easy decision rule that help the less experienced Belgian consumers to evaluate fish quality and form quality expectations. Spanish respondents felt more confident with evaluating fish quality, because of their experience, and hence they may be less dependent on using extrinsic information cues to evaluate fish quality and form quality expectations. Trust in information sources Most of the respondents, particularly light users, did not trust labels and their claimed need for information was low: `Can information and labels after all be trusted?' (Belgium); `Is it true what is written on the packages or on the labels?' (Spain). Moreover, Belgian consumers expressed serious doubts whether traceability is feasible and can be trusted in the particular case of wild fish. In the case of farmed fish perceived feasibility was higher: `Fish swim around all over the ocean how to control this and how to provide a trustworthy guarantee about the origin or history of wild fish?' (Belgium). On the other hand, consumers trusted that the fish has been inspected by an independent or government control organisation before reaching the retail stage. This may explain why traceability and a detailed level of information were not considered extremely important, at least not directly, to consumers. The respondents simply did not think that quality control and follow-up is a consumer's task or responsibility. They seem to expect government and public institutions to take care of quality and they trust that these institutions perform the jobs they are assigned to. Another explanation could relate to the predominantly healthy

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image of fish ± consumers are already convinced and do not need more information with regard to health benefits or the nutritional value of fish. Many respondents, both in Spain and in Belgium, expressed that they would rather not know too many details. This way, it seems that people start to worry when they are informed and, therefore, there seems to be a potential risk of raising unnecessary concerns or building up a crisis by suddenly providing more information than consumers are used and expecting to receive: `at the time the fish reach the stores it has all been controlled; or at least, it should have been decently controlled . . . if we could not even trust this, where would this all end up?' (Spain); `In Europe there are so many controls that the food must be good; there are so many safety norms, regulations, as well as regulatory and controlling bodies that we do not really have to think that we get rubbish on our plates' (Belgium). 5.3.2 Consumer survey Use of and interest in information sources and cues The survey participants, i.e. fish consumers from Spain, Belgium, The Netherlands, Denmark and Poland, displayed the highest level of trust in personal information sources about fish. These include among others doctors, dieticians, fishmonger and family or friends. Trust levels were significantly lower for mass media or commercial information sources like retailers and industry advertisements, although on average they were not alarmingly low. This was also reflected in consumers' use of information sources (Fig. 5.2), with commercial sources receiving the highest use level, in particular the fishmonger and supermarkets. Combining trust and use levels yields the following picture:

Fig. 5.2

Mean scores and standard deviation for the use of information sources about fish (n 4; 786).

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Fig. 5.3 Mean scores and standard deviation for the use of mandatory information cues (n 4; 786).

doctors, dieticians, scientists and consumer organisations received relatively high trust scores, but failed to be frequently used. On the contrary, retail or industry advertisements about fish, mainly communicated through television, were rather frequently used but not well trusted. Expiry date, price, species name and weight were the most used information cues on seafood labels, packages or shelves (Fig. 5.3). Consumers were most familiar with these cues and they felt able to derive clear quality expectations from the information these cues convey. Other cues like capture area, brand, nutritional composition or date of capture were far less used. The likely reasons are consumers' lack of familiarity with this information, and lack of trust in these cues that signal typical credence attributes, i.e. attributes that consumers can hardly verify themselves, even upon purchase or during consumption of the product. European consumers claimed a high interest in additional seafood information. The strongest interest was displayed for a safety guarantee and a quality mark for seafood (Fig. 5.4). Whereas consumers showed little interest in a batch identification number ± how could they ever interpret or use this direct indication of traceability? ± their interest in information cues that logically can result from traceability (namely a safety or quality guarantee) was extremely strong. Consumer interest in information from traceability was determined by several factors. Interest in information from traceability was higher among consumers who have a high level of trust in fish information. It was also stronger among consumers who find ethical issues (i.e., preservation of natural fish stocks and fish welfare) more important, and among consumers who perceive more health and safety risks from consuming fish.

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Fig. 5.4

Mean scores and standard deviation for use of potential information cues (n 4; 786).

Use of and trust in information sources-based segmentation Consumers' mean use of and trust in information sources' scores have subsequently been used for cluster analysis, with the aim to obtain use of and trust in information-based segments of fish consumers (see Pieniak et al., 2007b for methodological details). A three-cluster solution emerged as optimal from the cluster analysis. The profile of each cluster in terms of size, sociodemographic characteristics and behaviour is presented in Table 5.1. Cluster 1 is the smallest segment, including 24.0% of the sample; this cluster is the oldest consumer group (43.4 years of age) with relatively fewer of the middle-aged respondents and more of the older ones. It consists of relatively more men to women, relatively more lower income and fewer middle income respondents; more skilled consumers; more Polish respondents and less Belgian, Danish, Dutch and Spanish. Members of this group, together with individuals belonging to cluster 3, report the lowest scores on intention to eat fish and the lowest fish consumption level (total, at home and out of home). Respondents from this segment display low use of mass media and independent information sources and moderate to rather low use of health-related information sources, such as doctor, dietician and public health recommendations. However, their trust level is the lowest among the three groups. They seem to be very distrustful and insecure about information sources in general. Individuals belonging to this group do not trust and do not use any particular information source more in comparison with the other two clusters. Therefore, they may be called `Sceptics'. Cluster 2 is the biggest and accounts for 41.4% of the sample. This segment includes relatively more women to men; fewer of the respondents with lower income level and more of those with middle income level; and more Spanish

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consumers and fewer Dutch, Danish and Polish ones. Members of this group display the highest fish consumption level and scored the highest on the intention to eat fish. Respondents from this group are the least differentiated in terms of use of and trust in information sources. They scored the highest on the use of all information sources about fish. Simply, they are very involved in information search related to fish consumption. Additionally, they display the highest trust in almost all information sources (except in government); they may be called `Enthusiasts'. Cluster 3 accounts for 34.6% of the sample. Gender distribution of this fish consumer segment is very similar to the one of the total sample. Individuals belonging to this group are the youngest (41.3 years of age) with relatively more of the middle-aged respondents and fewer of the older ones. This segment includes relatively more Belgian, Dutch and Danish consumers and fewer Polish and Spanish ones. Members of this group display together with Sceptics the lowest intention to eat fish and fish consumption level. Individuals belonging to this segment did not actively search for information about fish (themselves). They were rather `passive' in information search about fish (the lowest scores on use of almost all information sources). However, they held trust in independent information sources, such as government, scientists and consumer organisations (score on the use of government is the highest among the three clusters). Therefore, individuals belonging to this consumer group might be called `Confidents', or those who `rely on the system'. Use of information cues between segments Comparison of the use of information cues about fish between segments revealed significant differences (Table 5.2). Enthusiasts (cluster 2) report the highest usage of all mandatory information cues and the highest interest in emerging information cues related to first of all health and safety issues of fish, then to convenience, fish sustainability, farming process and finally origin of fish. They are simply interested in any information related to fish. Sceptic respondents (cluster 1) report high usage, together with Enthusiasts, of price and expiry date and moderate usage of weight, nutritional composition, brand name and capture area. Additionally, they scored the lowest on usage of fish species name. Members of this group display the lowest interest in all potential information cues. Finally, respondents belonging to the third cluster, Confidents, report the lowest usage for most of the mandatory information cues (except for fish species name), and the lowest interest in almost all newly emerging information cues (except moderate interest in information cues related to fish sustainability). Interest in traceability issues between the segments Confident respondents report a moderate score on the traceability item `It is important for me to have direct access to as much information as possible about the fish' but the lowest scores on the other three traceability items included, as well as on the direct interest in traceability construct (Table 5.3). Apparently, their confidence or their passiveness is translated into a lower interest in direct

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Table 5.2 Mean scores on use of and interest in fish information cues between segments (n 4,786) Consumer segments

F-value p-value

Sceptics

Enthusiasts

Confidents

5.63a 5.92b 5.50b 6.29b 3.88b 3.83b 2.94b

5.95b 5.96b 5.71c 6.38b 4.77c 4.49c 3.90c

5.51b 5.58a 5.15a 6.03a 3.49a 3.44a 2.68a

31.85 27.51 45.92 23.84 199.95 130.85 186.46

< < < < < < <

0.001 0.001 0.001 0.001 0.001 0.001 0.001

5.49c 5.67b 5.04c 4.82b 5.03b

5.05b 5.13a 4.52b 4.19a 4.32a

83.25 82.34 72.12 109.25 78.82

< < < < <

0.001 0.001 0.001 0.001 0.001

Use of information cues Fish species name Price Weight Expiry date Nutritional composition Brand name Capture area

Interest in information cues related to . . . Convenience 4.79a Health/safety 5.09a Fish sustainability 4.33a Origin 4.09a Farming process 4.34a

The a, b, c indicate significantly different means using Tukey HSD Post Hoc on a 7-point scale

Table 5.3

Mean scores on traceability items between segments (n 4,786) Consumer segments Sceptics

If there was a computer in the shop that could supply me with more information about the fish, I would use it I'm willing to pay more for a fish that has better documentation It is important for me to have direct access to as much information as possible about the fish I prefer the retailer to keep the information about the fish and make it available to me on request Direct interest in traceability

F-value p-value

Enthusiasts

Confidents

3.66a

4.65b

3.79a

118.02

< 0.001

2.89a

3.76b

3.02a

105.22

< 0.001

3.70a

4.70c

3.92b

128.54

< 0.001

4.90a 3.93a

5.33b 4.68b

5.03a 4.04a

23.82 160.52

< 0.001 < 0.001

The a, b, c indicate significantly different means using Tukey HSD Post Hoc on a 7-point scale

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traceability information. On the other hand, respondents belonging to the second segment (Enthusiasts) scored the highest on all items related to traceability and on direct interest in traceability. Undoubtedly, they are most strongly interested in direct traceability information about seafood. Finally, Sceptical respondents scored the lowest on all items with respect to interest in traceability as well as for the direct interest in traceability. Furthermore, the data obtained from the consumer survey allowed analysis of associations between health, safety and ethical perceptions of seafood on the one hand, and consumer interest and use of information on the other hand. The results are presented below. Information and seafood health perception Comparison of the health-related beliefs about seafood between the information segments did not reveal significant differences for the beliefs that eating fish is healthy and nutritious and for the health involvement construct (Table 5.4). In general, people hold a strong belief that eating fish is very healthy and nutritious (mean scores above 6.0 on a 7-point Likert scale), and this belief hardly differs between consumer segments. Furthermore, all fish consumers are found to be very much involved with their health (mean scores about 6.20). On the other hand, significant differences between segments were found for two constructs, namely satisfaction with life and interest in healthy eating. Enthusiasts (cluster 2) together with Confidents (cluster 3) are more satisfied with their life, whereas Sceptics scored the lowest on this construct, indicating that they are the least satisfied with their life. Finally, Sceptic and Enthusiastic respondents are the most interested in healthy eating, whilst Confidents are the least interested in healthy eating. To sum up, Enthusiasts are both the most satisfied with their life and most interested in healthy eating. Information and seafood risk perception Next, consumers' attitudes towards seafood safety and risk perception with respect to potential food poisoning from eating fish are compared between the segments and the results are presented in Table 5.5. In general, consumers Table 5.4 Mean scores of consumers' attitudes towards seafood health and health constructs between segments (n 4,786) Consumer segments

Eating fish is healthy Eating fish is nutritious Satisfaction with life Healthy eating Health involvement

F-value p-value

Sceptics

Enthusiasts

Confidents

6.22 6.00 4.47a 5.95b 6.19

6.26 6.05 4.91b 5.90b 6.20

6.28 6.02 4.98b 5.63a 6.18

0.80 0.60 56.98 35.30 0.12

ns ns < 0.001 < 0.001 ns

The a, b, c indicate significantly different means using Tukey HSD Post Hoc on a 7-point scale

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Table 5.5 Mean scores of consumers' attitudes towards seafood safety and perceived risk between segments (n 4,786) Consumer segments Sceptics

Enthusiasts

Confidents

5.33b 5.20b 5.21

5.12a 5.07a 5.18

8.97 3.61 1.52

< 0.001 0.027 ns

2.47a

2.75b

2.34a

26.88

< 0.001

2.32b

2.53c

2.15a

24.37

< 0.001

2.27b

2.52c

2.13a

26.50

< 0.001

2.40b

2.78b

2.38a

30.86

< 0.001

2.43a

2.71b

2.34a

23.57

< 0.001

a

b

a

35.98

< 0.001

Eating fish is safe 5.29b Fresh wild fish-unsafe/safe 5.10a,b Fresh farmed fish-unsafe/safe 5.11 I do not want to eat fish too often because I am afraid of food poisoning from chemical contamination I do not want to eat fish too often because I am afraid of food poisoning from bacterial contamination I am very concerned about the possibility of getting ill from eating fish Fish is more risky to eat with respect to food poisoning from chemical contamination than other kinds of food Fish is more risky to eat with respect to food poisoning from bacterial contamination than other kinds of food Risk perception

F-value p-value

2.38

2.66

2.26

The a, b, c indicate significantly different means using Tukey HSD Post Hoc on a 7-point scale

consider fish mainly as a safe product (mean scores above 5 on a 7-point Likert scale). Enthusiasts (cluster 2), together with Sceptics (cluster 1) report the highest scores on attitudes towards seafood safety, meaning that they perceive fish in general as the safest from the three consumer groups. With regard to the safety perception of fresh wild fish, Enthusiasts scored the highest, followed by Sceptics. Confidents indicated the lowest belief in fish safety in general, and in fresh wild fish safety in particular, although their mean scores on the safety perception items is still substantially above the neutral point of the scale. Additionally, consumers perceive fresh farmed fish as safe. However, no differences between the three groups are found. Enthusiast respondents scored the highest on all five risk perception items. Sceptic respondents scored moderately on two items: `I do not want to eat fish too often because I am afraid of food poisoning from bacterial contamination' and `I am very concerned about the possibility of getting ill from eating fish' and

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together with the Enthusiasts the highest on the item `Fish is more risky to eat with respect to food poisoning from chemical contamination than other kinds of food'. Finally, Confident consumers scored the lowest on all of the items. With regard to the overall risk perception construct, Enthusiasts perceived fish consumption as the most risky; whereas Sceptics and Confidents reported the lowest risk perception related to fish consumption. However, fish is perceived as a safe product to consume by respondents from the three consumer segments. On average, two thirds of the respondents are not afraid of food poisoning from eating fish. Additionally, it should be noticed that specifying a particular origin of food poisoning from eating fish (i.e., through microbiological versus chemical contamination), does not really change consumers' safety or risk perceptions. Respondents seem not make any distinction between risk related to either chemical or bacterial contamination. Nevertheless, the mean scores for fear of food poisoning from chemical contamination are somewhat higher than for poisoning from bacterial contamination, although this difference is not statistically different. Information and ethical issues related to seafood Finally, consumers' attitudes towards ethical issues related to seafood consumption and production are compared between segments (Table 5.6). The results show that Enthusiast respondents (cluster 3) report the highest scores on attitudes towards ethical issues, meaning that they perceive eating fish (in general as well as fresh wild) as the most ethically correct from all consumer Table 5.6 Mean scores of consumers' attitudes towards ethical issues, ethical concern about fish and perceived consumer effectiveness between segments (n 4,786) Consumer segments Sceptics Eating fish is ethically correct 4.77b Fresh wild fish ± unethical-ethical 5.01a Fresh farmed fish ± unethical-ethical 4.88b Fish can't feel pain 2.93a Fish ethics 5.52a,b Through my choice of fish species, I can really contribute to save the fish-stock from over-fishing 4.30a If I choose farmed fish I can really contribute to save the fish-stock from over fishing 4.39a

F-value p-value

Enthusiasts

Confidents

5.00c

4.62a

25.47

< 0.001

5.24b

5.03c

10.74

< 0.001

4.95b 3.13b 5.60b

4.73a 3.00a,b 5.41a

8.51 4.83 8.06

< 0.001 < 0.001 < 0.001

4.65b

4.16a

38.20

< 0.001

4.68b

4.41a

16.59

< 0.001

The a, b, c indicate significantly different means using Tukey HSD Post Hoc on a 7-point scale

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groups. Additionally, they scored the highest on the item `fish cannot feel pain', meaning that they agree the most with this statement. Moreover, they attach more importance to fish ethics than individuals gathered in the other two segments. Sceptic consumers (cluster 1) show moderate scores on attitudes towards ethical issues related to fish in general, and the most positive (together with Enthusiasts) perception of fresh farmed fish as ethically correct. Furthermore, members of this group most disagree that fish cannot feel pain. Consumers belonging to cluster 3 (Confidents) have the lowest perception of fish (in general and when the origin of fish is given) as ethically correct. Finally, perceived consumer effectiveness has been compared between consumer segments. Consumers with the highest perceived effectiveness, i.e. the belief that their personal behaviour can make a difference, belong mainly to the second consumer segment. Thus, Enthusiast respondents hold stronger belief that they can contribute through their own personal choice and behaviour to save the fish-stock from depletion. On the contrary, consumers who scored lower on the perceived effectiveness items, meaning that they have less strong belief that through their choice of fish species or by choosing a farmed fish, they can contribute to save the fish-stock from over-fishing, belong mainly to the first and third consumer segment, thus Sceptics and Confidents.

5.4

Conclusions

The results from this study indicate that consumers (will) use those information cues and information sources they are most familiar with, because these cues allow them to make quality expectations that most likely will be confirmed by experienced product performance, i.e. upon tasting and enjoying seafood products. The conclusion from this study is that consumers show a strong interest in information cues that need some kind of traceability as a back up in order to be credible or trustworthy. More than one third of the European seafood consumers show a very strong interest (scoring 6 or more on a 7-point scale) in a safety guarantee and a quality mark for fish (information cues related to health/ safety), which is exactly what a good working traceability system can warrant to consumers. However, a strong interest in direct indications of traceability, such as capture area or a batch identification number is reserved to only a niche representing less than 15% of the market. Furthermore, our findings with respect to fish confirm that traceability in the strict sense of a reference code or identification number, in the absence of easily interpretable quality verification, has little apparent value to consumers. The primary role of traceability is within the chain, with considerable potential though to guarantee safety and quality to the fish consumer as end user. Three distinct market segments were identified based on consumers' use of and trust in fish information sources: Sceptic (24.0%), Enthusiast (41.4%) and Confident (34.6%). Those consumer segments differed significantly with respect to use of and interest in information cues on fish labels, fish consumption

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behaviour, socio-demographic and attitudinal profile, which yields opportunities for targeted information provision efforts. The major contribution of the activities with the SEAFOODplus project 2.3 SEA-INFOCOM pertains to the quantification of consumers' interests in seafood information and the identification of distinct information-based seafood market segments. Further studies, e.g. using experimental designs for information impact testing, are recommended in order to develop and test whether consumers belonging to these different segments effectively respond differently to targeted seafood information provision. Interestingly, this study reveals there is no group of consumers who report very low trust levels, but at the same time high use levels of information sources related to seafood. This finding indicates a minimum level of trust might be required before information sources are critically examined and (reported as) used. In the extreme situation of very low trust, consumers are unlikely to examine information sources in any way; that is neither critically nor uncritically. This underscores the importance of traceability and related information provision: only when backed up by a trustworthy and watertight traceability system can information sources, messages and labelling cues rightfully be trusted by consumers. Sceptical distrusting but involved consumers do not seem to exist, at least not with respect to seafood information sources. Whether this conclusion holds similarly to other seafood categories deserves further attention in future studies, for example with respect to shellfish and processed seafood products where consumer perceptions may be shaped by slightly different expectations and beliefs, e.g. relating to convenience or microbiological safety, which may entail different information needs and interests. The research activities within the SEA-INFOCOM project of SEAFOODplus have provided us with insights on European consumers' information needs and use, and their interest in traceability information in relation to seafood. During the course of the project, a number of additional seafood-and-informationrelated issues or trends have emerged, which deserve further attention in the future. First, despite the fact that public health authorities have been recommending to eat seafood twice a week for its proven health benefits, it has become clear that the share of consumers meeting this recommendation remains quite low, particularly so in countries with a weak seafood consumption tradition. Questions rise about finding more effective ways for conveying this message to consumers and causing behavioural change to happen. Further research focusing on alternative communication and information provision strategies is recommended in order to provide public health authorities and the industry with concrete guidelines for improving the effectiveness and efficiency of their communication efforts. A second trend relates to the regulatory and marketing evolutions in the field of nutrition and health claims. A better understanding of consumers' reactions to health claims in general, and claims on seafood and products derived from seafood in particular is required if the seafood industry wants to take full advantage of its current pole position in the market of food products that may

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become entitled to carry nutrition and health claims in the European Union. This research should deal with the full range from consumers' attention to, liking and usage of nutrition labelling information and claims, until the potential impact of behavioural change induced by health claims on health and well-being, Last but not least, given the globalisation of seafood production and trade and the increasingly prominent role occupied by aquaculture in the supply of seafood, it can expected that European authorities as well as consumers will remain quite vigilant to the quality, safety and healthiness of the seafood products on their plate. This will inevitably lead to heightened and newly emerging information needs at the consumer level, e.g. relating to the origin of seafood species. Also, the European citizen and consumer are likely to express ever stronger expectations that public and private stakeholders in the seafood chain take up their responsibility for providing them with safe and healthy seafood products that are produced with respect to environmental, ethical and sustainability conditions. Newly emerging concerns and information needs will have to monitored continuously. Without doubt, watertight and reliable traceability systems as the key to realising this consumer (re)assurance of seafood quality, safety, healthiness and wholesomeness, will remain crucial.

5.5

Sources of further information and advice

Readers are referred to a series of journal papers that have been published since the start of the SEAFOODplus integrated project in 2004. A first category of papers provide additional background related to the role and importance of information in consumer decision-making and food choice, either in general, or related to fish and seafood in particular: and OLSEN, S.O. (2004). Determinants of fish consumption: Role and importance of information. Polish Journal of Human Nutrition and Metabolism, 31 (S2), 409±414. PIENIAK, Z., VERBEKE, W., BRUNSé, K. and OLSEN, S.O. (2006). Consumer knowledge and interest in information about fish. In Luten, J., Jacobsen, C., Bekaert, K., Súùbo, A. and Oehlenschlager, J. (eds). Seafood Research from Fish to Dish: quality, safety and processing of wild and farmed fish. Wageningen: Wageningen Academic Publishers, 229±240. VERBEKE, W. (2005). Agriculture and the food industry in the information age. European Review of Agricultural Economics, 32 (3), 347±368. VERBEKE, W., FREWER, L.J., SCHOLDERER, J. and DE BRABANDER, H.F. (2007). Why consumers behave as they do with respect to food safety and risk information. Analytica Chimica Acta, 586, 2±7. PIENIAK, Z., VERBEKE, W., FRUENSGARD, L., BRUNSé, K.

The second category of readings informs more generally on consumers' attitudes and beliefs in relation to seafood consumption. This work was realised in close collaboration with project 2.1 CONSUMERSURVEY. Other related papers can be found in Chapter 2.

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and VACKIER, I. (2005). Individual determinants of fish consumption: application of the theory of planned behaviour. Appetite, 44 (1), 67±82. VERBEKE, W., SIOEN, I., PIENIAK, Z., VAN CAMP, J. and DE HENAUW, S. (2005). Consumer perception versus scientific evidence about health benefits and safety risks from fish consumption. Public Health Nutrition, 8, 422±429. VERBEKE, W., VERMEIR, I. and BRUNSé, K. (2007). Consumer evaluation of fish quality as basis for fish market segmentation. Food Quality and Preference, 18, 651±661. VERBEKE, W., SIOEN, I., BRUNSé, K., DE HENAUW, S. and VAN CAMP, J. (2007). Consumer perception versus scientific evidence of farmed and wild fish: exploratory insights in Belgium. Aquaculture International, 15, 121±136. VERBEKE, W.

The third category includes papers that provide the full methodological, analytical and empirical details of the findings that have been summarised in this book chapter: and OLSEN, S.O. (2007). Consumer interest in fish information, traceability and labelling: exploratory insights. Journal of International Food and Agribusiness Marketing, 19 (2/3), 117±141. PIENIAK, Z., VERBEKE, W., SCHOLDERER, J., BRUNSé, K. and OLSEN, S.O. (2007). European consumers' use of and trust in information sources about fish. Food Quality and Preference, 18, 1050±1063. PIENIAK, Z., VERBEKE, W., BRUNSé, K.

5.6

References

(1997). Food and nutrition information: a study of sources, uses, and understanding. British Food Journal, 99, 43±49. BERNUES, A., OLAIZOLA, A. and CORCORAN, K. (2003). Labelling information demanded by European consumers and relationships with purchasing motives, quality and safety of meat. Meat Science, 65 (3), 1095±1106. BùRRESEN, T., FREDERIKSEN, M. and LARSEN, E. (2003). Traceability from catch to consumer in Denmark. Quality of Fish from Catch to Consumer: Labelling, Monitoring and Â lafsdoÂttir. Wageningen, Traceability J. Luten, J. OehlenschlaÈger and G. O Wageningen Academic Publishers, pp. 93±100. CAPORALE, G. and MONTELEONE, E. (2004). Influence of information about manufacturing process on beer acceptability. Food Quality and Preference, 15 (3), 271±278. CAPPS, O. (1992). Consumer response to changes in food labeling: Discussion. American Journal of Agricultural Economics, 74, 1215±1216. DE GARIDEL-THORON, T. (2005). Welfare-improving asymmetric information in dynamic insurance markets. Journal of Political Economy, 113 (1), 121±150. DRANOVE, D., KESSLER, D., MCCLELLAN, M. and SATTERTHWAITE, M. (2003). Is more information better? The effects of `Report cards' on health care providers. Journal of Political Economy, 111 (3), 555±588. KAABIA, M. B., ANGULO, A. M. and GIL, J. M. (2001). Health information and the demand for meat in Spain. European Review of Agricultural Economics, 28 (4), 499±517. MILES, M. B. and HUBERMAN, A. M. (1994). Qualitative Data Analysis. Thousand Oaks, CA: Sage. MORGAN, D. L. and KRUEGER, R. A. (1997). The Focus Group Kit. Thousand Oaks, CA: Sage. MYRLAND, O., TRONDSEN, T., JOHNSTON, R. S. and LUND, E. (2000). Determinants of seafood ABBOTT, R.

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consumption in Norway: lifestyle, revealed preferences, and barriers to consumption. Food Quality and Preference, 11 (3), 169±188. PIENIAK, Z., VERBEKE, W., BRUNSé, K. and OLSEN, S.O. (2007a). Consumer interest in fish information, traceability and labelling: exploratory insights. Journal of International Food and Agribusiness Marketing, 19 (2/3), 117±141. PIENIAK, Z., VERBEKE, W., SCHOLDERER, J., BRUNSé, K. and OLSEN, S.O. (2007b). European consumers' use of and trust in information sources about fish. Food Quality and Preference, 18, 1050±1063. ROSATI, S. and SABA, A. (2004). The perception of risks associated with food-related hazards and the perceived reliability of sources of information. International Journal of Food Science and Technology, 39 (5), 491±500. È N, Y. and FLORES, K. (2001). Information quality: meeting the needs of the SALAU consumer. International Journal of Information Management, 21, 21±37. SCHOLDERER, J. and GRUNERT, K. (2001). Does generic advertising work? A systematic evaluation of the Danish campaign for fresh fish. Aquaculture Economics and Management, 5, 253±271. SCHOLDERER, J. and GRUNERT, K. (2003). Promoting seafood consumption: an evaluation of the Danish campaign for fresh fish. Quality of fish from catch to consumer: labelling, monitoring and traceability. J. Luten, J. Oehlenschlager and G. Olafsdottir. Wageningen, Wageningen Academic Publishers, pp. 367±374. TRONDSEN, T., SCHOLDERER, J., LUND, E. and EGGEN, A. E. (2003). Perceived barriers to consumption of fish among Norwegian women. Appetite, 41 (3), 301±314. VERBEKE, W. (2005). Agriculture and the food industry in the information age. European Review of Agricultural Economics, 32 (3), 347±368. VERBEKE, W. and VACKIER, I. (2005). Individual determinants of fish consumption: application of the theory of planned behaviour. Appetite, 44 (1), 67±82. VERBEKE, W. and WARD, R. W. (2006). Consumer interest in information cues denoting quality, traceability and origin: An application of ordered probit models to beef labels. Food Quality and Preference, 17 (6), 453±467. WANDEL, M. (1997). Food labelling from a consumer perspective. British Food Journal, 99 (6), 212±220.

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6 Consumer evaluation of tailor-made seafood products S. O. Olsen and K. Toften, Nofima, Norway, D. Calvo Dopico and A. Tudoran, University of CorunÄa, Spain and A. Kole, Wageningen University and Research Centre, The Netherlands

6.1

Introduction

Product evaluation is of vital importance in product and marketing development of food products. Food scientists use trained people to evaluate the products' preferences and differences in physical aspects in controlled environments or laboratories. People are perceived as neutral evaluators, using their sensory receptors to perform an `objective' evaluation of specific product attributes, or as a general evaluation. Marketing scholars are interested in studying the evaluation among real and potential buyers or consumers in more realistic buying and consumer settings, and the evaluative outcome is defined in individual and subjective terms. Several models have been developed over recent years in order to integrate aspects of the food or product (flavour, texture, odour) with characteristics of the individual (perceptions, preferences, attitudes, knowledge) or characteristics of the environments (situation, social, culture, season) (see Shepherd, 1989; Steenkamp, 1993). In general, most comprehensive models are essentially theoretical frameworks. Empirical models tested in real settings are more restricted and focus on few and important aspects of products, individuals and/or the environment (Shepherd and Sparks, 1994). Intention to buy is suggested to be one way to estimate a potential demand for new products (Lilien and Kotler, 1983). Very few marketing or consumer scholars have used willingness to pay (WTP) as a theoretical and empirical

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construct in explaining consumer attitudes and motivation, or as a predictor of product evaluation (Cameron and James, 1987). The most frequently used construct in performing an evaluation of products or services over the past twenty years has been perceived quality (Zeithaml, 1988) and/or consumer/ customer satisfaction (Oliver, 1997). These constructs have been used to evaluate the performance of single attributes as well as a general evaluation of products and services, and as an important antecedent to behaviour intention or loyalty (e.g., Olsen, 2002). The background for the present chapter is the SEAFOODplus project CONSUMEREVALUATE, where the main objective is to explore and explain consumers' preferences, evaluation and buying behaviour related to convenience and tailor-made seafood products. We would like to highlight some of the results obtained in the project and to consider special seafood aspects in the perspective of a more general food context. In particular we want to discuss how different aspects of the food or the meal `in the context of real food' (Meiselman, 1992) might influence perceived quality, consumer satisfaction or attitudes as a general evaluation of a given product or meal. Some focus will be on a discussion of how different testing conditions/contexts influence the evaluation and motivation to buy products (de Graaf et al., 2005; Hersleth et al., 2005; Weber et al., 2004). These aspects deal with what Meiselman (1992) terms `real dining situations' in his suggestion for performing real product testing among real consumers. It also deals with the general aspects of how situation and context affect product evaluation and choice (Belk, 1975; Meiselman, 1996). In addition to knowing how well or poorly a given product is evaluated among trained or untrained, new or established, segmented or global consumers, business and marketing managers are also interested in explanations of why consumers buy or do not buy their products. For these reasons, product evaluation is also integrated into more extensive theoretical frameworks that include individual and motivational variables and external and social aspects in addition to aspects of the product. Individual aspects include food-specific motives and attitudes such as variety seeking, food neophobia, restrained eating, personality, values, attitudes, emotions, norms, perceived risk, knowledge, skills, age and other demographic variables (Grunert, 2002; Shepherd, 1989; Steenkamp, 1993). Our research (Olsen et al., 2005) includes convenience orientation (Gofton, 1995), social norms and moral obligation (Olsen et al., 2007; Raats et al., 1995), ethical concerns (Honkanen and Olsen, 2007; Honkanen et al., 2005; Lindeman and VaÈaÈnaÈnen, 2000), health concern and perceived health (Steptoe et al., 1995), innovativeness (Goldsmith and Hofacker, 1991) food neophobia (Pliner and Hobden, 1992), and subjective knowledge and skills (Brucks, 1985; Gofton, 1995) to name some of the important constructs. Several of these aspects (e.g., health/nutrition, convenience, ethical concerns) are discussed as both an aspect of the individual as well as an attribute or belief associated with the product under evaluation. The general theoretical framework used in our research of how and why consumers evaluate different tailor-made seafood products (Olsen et al., 2005) is

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Fig. 6.1

87

A conceptual model for studying consumer evaluation and willingness to buy tailor-made seafood products.

presented in Fig. 6.1. Our main contribution in the area of testing new products is increased focus on the use of `real and realistic consumers' and a deeper understanding of individual and environmental differences on the evaluative outcome. In the following, we will discuss some of the different constructs in the model and how they are manipulated or measured in experiments and field conditions. Some preliminary results are presented, but some of our field experiments are yet to be performed or are in the early stage of data analyses. For this reason we will not go into a deep discussion of our results in this chapter and will mostly leave out a discussion of the importance of how and when individual variables (values, general attitudes, involvement and resources) and environments (location, situation and time) influence the evaluation and intention to behave. Our main hypotheses are briefly discussed in the concluding part of this chapter.

6.2

The product in its environment

In our experiments we manipulate differences in the physical attributes of the product (including meal/form), information about the product and production methods, as well as the context or environments in which the experiments will be done. In the following discussion, we will focus on how testing environments might influence consumers' evaluations of food products. 6.2.1 The product attributes and informational cues Within the product development and marketing literature, a product is defined as the object of the exchange process and includes several product levels (e.g., Baker and Hart, 1998). This implies that several decisions are needed to frame what kind of product should be evaluated in food product testing. Within marketing and consumer theory, a product is perceived and evaluated by a

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collection of different product attributes. Some of these attributes are related to physical quality or feature, design, packaging or brand name. Some marketing textbooks (e.g., Kotler and Armstrong, 1999) include warranty and other services as a part of the product concept. Most product development usually deals with testing of bites or sips of products, rarely with multi-component meals or longer-term patterns of eating (Meiselman, 1996). Testing a food product in a real consuming situation often includes aspects of a meal as a unit of evaluation. Different aspects of a meal that are possible to evaluate could include preparation methods, combinations of ingredients in a meal, the meal situation and social interactions. Meiselman (1996) suggests that one should integrate sensory research into a defined contextual or situational setting in order to improve our understanding of foodrelated behaviour, including our ability to predict buying and other real-life behaviour. Therefore, he proposes that context research begin on the level of the whole food and the whole sensory response. Sensory research on the more analytical level is not properly part of the food context. To conclude, what is a real product is different for different people depending on the situation. Consumers evaluate and buy ingredients and raw materials as well as complete meals depending on the purpose and situation, and product tests should be planned accordingly. This includes specifying the object under evaluation, deciding on a suitable form of the product and deciding on the use of instructions, number of attributes and products or variants to test. Our research (e.g., Dopico et al., 2007b; Honkanen and Olsen, 2007; Olsen, 2006a; 2006b; Olsen et al., 2007; Tudoran et al., 2006) attempts to combine sensory experiments in laboratory and field settings as well as field experiments as close to a real food or meal situation as possible. Attributes to be manipulated are: · physical content of core products (e.g., different fibres in surimi products) · production methods (e.g., ethical versus unethical production methods) · form/meal (e.g., degree of convenience as time and effort used to prepare a meal). Influences from cues and information Consumers use information cues and expectations to infer their sensory evaluation and perceived quality of food products (Grunert, 1997; Steenkamp, 1990). Product and process information is one of the extrinsic factors that have been shown to affect consumer choice. The marketing literature has focused on the effect of brand, price, store and country-of-origin as some of the main informational cues (Verlegh and Steenkamp, 1999). Sensory claims, labelling, product and process information (e.g., Kihlberg et al., 2005) have proved to influence product evaluation, willingness to pay (Lange et al., 2002) and other intentional and behavioural variables in food science studies (see, e.g., Mialon et al., 2002 for a recent review). A relevant study for our experiments related to tailor-made seafood products is a study by Mialon et al. (2002) on how dietary fibre information influences

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product evaluation in a cross-cultural setting. Also, studies discussing interactions between product information and individual consumer characteristics are important to follow-up in our experiments (Pelchat and Pliner, 1995). For example, Tuorila et al. (1998) found that the acceptance of new products is affected by information and different consumer characteristics such as food neophobia. As described in Olsen et al. (2005), we designed our experiments in a manner that makes it possible to test the effects of relevant informational cues related to the physical attributes that are being manipulated. In a study of farmed cod in Spain, we used three different labels on the products; one with basic information (Farmed cod), a second with general ethical information (`This cod has been farmed in order to meet all environmental and ethical standards'), and the third one with more specific ethical information. In a study of enriched seafood in Spain we (Careche et al., 2006) gave the respondent different kinds of information, including product content (types of fibre) and different levels of health benefit information. 6.2.2 Test situations Research on the relationship between product evaluation and actual behaviour often provides low correspondence (e.g., Cardello et al., 2000), and contextual variables have been suggested to have a great impact on this relationship (e.g., Meiselman, 1994; Rozin and Tuorila, 1993). Therefore, it is important to include realism in the product testing process. Realism in product testing refers to the extent to which the product test can portray the purchase, use or consumption of the product in such as way as to match its purchase, use or consumption in a natural or uncontrived situation (Baker and Hart, 1998) Despite Meiselman's (1992) suggestion and call for `real food to be tested by real people in real dining situations', few studies are conducted by food scientists and marketing scholars who follow his advice (de Graaf et al., 2005; Hersleth et al., 2005). This is unfortunate, given that consumers' food preferences are influenced by the eating situation (Bell and Meiselman, 1995), and the conditions for testing the food are often far different from the conditions/situations in which food is typically eaten (Cardello and Schutz, 1996). Most hedonic food testing is conducted in laboratories, at central locations or at the location of the product's intended use, such as in-home. Different types of test locations exhibit different degrees of control over the experiment, where a laboratory setting is expected to provide the best control of the testing and an in-home test is expected to provide the least control (Hersleth et al., 2005; Pound and Duizer, 2000). The challenge for the researcher is thus to balance the need for control of the experiment and the desire for a realistic situation of consumption. Pros and cons of different test locations Different locations have different advantages and disadvantages, and the actual research is thus of vital importance with respect to the selection of test sites.

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When planning a product test, the researcher has to balance the need for control of the experiment with the desired realism of the test. Apparently, laboratories imply the best-controlled test environment (e.g., Hersleth et al., 2005; Pound and Duizer, 2000). In this setting, the environmental and stimuli variables and social interactions can be controlled, thereby reducing systematic and random error due to various influences (de Graaf et al., 2005). This enables focusing on specific aspects of a product, such as sensory characteristics (Hersleth et al., 2005). Laboratory testing can further represent the advantages of low costs (Pal et al., 1995) due to the small number of subjects and amount of product samples. Also, since these experiments are often of a limited nature with simultaneous testing of the subjects, the data feedback can be rapid (Pal et al., 1995). The disadvantages of laboratory testing are, however, several. First, this type of test location represents far different conditions or situations for eating (Meiselman, 1992; Schutz, 1988; 1994), questioning the generalisability of the test results. Second, the subjects have only a limited product exposure (Pal et al., 1995), since the test product is often just a mouthful and the time available for testing of each product is short. This limited product exposure might jeopardise the validity of the test. Third, experience has shown that certain consumer groups can be difficult to attract to laboratory testing (Pound and Duizer, 2000). The testing might take place at times of the day when mostly retired people, unemployed and students are available. This might be acceptable if these groups are the targeted consumer groups, but not if the study attempts to be representative for a larger population or, say, `working women'. Also, if the testing involves several test sessions, this strengthens the problem of recruiting consumers who might be interested, willing and capable, but not able to participate. Fourth, laboratory testing in terms of preference and acceptance measures has shown to be a poor predictor of consumption (Cardello et al., 2000; Kozlowska et al., 2003). Testing at central locations, such as shopping malls, retail outlets and trade shows, includes the possible advantages of capturing a large number of subjects for the testing (Pal et al., 1995) without being restricted by, for example, a limited number of testing booths. It is also possible to partly control environmental and stimulus variables and social interaction (Hersleth et al., 2005). Moreover, the extensive use of students or own-company employees (Pal et al., 1995) for testing purposes can easily be avoided. Finally, this testing situation also represents a more realistic setting than laboratory testing (Hersleth et al., 2005). Combined, this form of product testing provides higher external validity than laboratory testing. The potential disadvantages are, however, represented by, for example, less control of the experiment than for laboratory tests (Pal et al., 1995), raising concerns of possible `irrelevant' variables influencing the test results. Also, the setting does not encourage too lengthy or distasteful tasks, and the instructions to the subjects can only be of limited nature (Pal et al., 1995). In-home testing, on the other hand, represents the least-controlled environment. This implies that the researcher cannot control the environmental and stimuli variables and social interactions. The great advantage of this setting is

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that the products are tested under actual use situations, providing a realistic setting for the test (Pal et al., 1995). Testing under more realistic situations, i.e. in-home environments, has proven to have better predicting value than laboratory testing (e.g., Kozlowska et al., 2003). Another advantage of in-home testing is the possibility of including an all-family opinion of the product (Pal et al., 1995), representing a realistic real-life situation. In addition, respondents tend to be more relaxed in in-home testing, allowing the respondents to eat as much as they wish (McEwan, 1997). Further, by using in-home testing, there is a possibility for learning processes to take place (Kozlowska et al., 2003), thereby increasing interest in the test product. The possible disadvantages of this test location are largely a result of this lack of controlled test environment. First, there will be variability in preparation of the food, the time of day for the eating and any combinations with other foods (Hersleth et al., 2005; Meilgaard et al., 1991) when consumers are to consume the test product. This implies that it can be difficult to assess the testing procedure and validity of the data (Hersleth et al., 2005). Also, the potential for misuse is greater than for alternative testing locations (Stone and Sidel, 1992; Hersleth et al., 2005). In addition, this test can be expensive and time-consuming to organise (Pal et al., 1995). When looking into the various advantages and disadvantages of these three major test locations, it appears that the locations are suitable for different situations or purposes. Another issue that is also part of the testing environment is the possibility to test products that are prepared by the individual consumer and for (served to) the individual consumer. According to self-serving bias or other theories discussing consumers as co-producers, respondents may perceive the value of the meal depending on their own participation in the food preparation process (Bendapudi and Leone, 2003; Hanefors and Mossberg, 2003; Toffler, 1980) as well as their individual preferences of meal solutions. This may affect the test situation and the physical aspects of the product that researchers want to inspect in a field experiment. We (see Olsen et al., 2005) will investigate this issue in our further research, and also test new seafood products in different test locations to explore advantages and disadvantages with different test locations. Since one of our main research issues deals with convenience orientation and convenience products, in the following we will discuss this issue in relation to one in-home study of farmed cod among Spanish households. 6.2.3 Time and convenience Numerous attempts have been made to define convenience (Berry et al., 2002; Yale and Venkatesh, 1986) derived a definition of convenience from a series of focus groups. They isolated two major dimensions of convenience: time and energy/effort. Studies indicate that people differ in their temporal orientation, including perceived time scarcity, the degree to which they value time and their sensitivity to time-related issues (see Berry et al., 2002 for a recent review). Consumers' energy expenditures, or efforts, can be subdivided into physical,

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cognitive and emotional effort (Berry et al., 2002). Out of these facets, most research in marketing, psychology, economics and decision theory has focused on cognitive or mental effort. The convenience construct is also related to different stages or activities of buying, using and disposing of a product or service (Berry et al., 2002; Yale and Venkatesh, 1986). Based on the discussion of saving time and effort in different stages of the food consumption process, Candel (2001) suggests a domainspecific definition of food convenience orientation as `the degree to which a consumer is inclined to save time and money in regard to meal preparation'. He argues that the preparation stage seems to be the most time- and energyconsuming process. As outlined in Olsen et al. (2005), we have in our research used Candel's (2001) food convenience scale as a general framework, but include items with some focus on planning, disposal and cognitive effort (both in planning and preparation). We also make a distinction between convenience orientation and product convenience. While the former describes some aspects of the individual's value, attitude or lifestyle/behaviour, the latter describes some aspects of the product investigated: how consumers evaluate convenience attributes associated with a specified product or meal. In experiments, it is possible to manipulate different facets of convenience. This can be done by manipulating the form of the product (e.g., a whole fish versus a fillet), preparation method (fried in a pan versus baked in an oven), information on the package (how to do it the easy way versus doing it your way) and test-location (at home versus in a restaurant). We believe that the most appropriate way to manipulate or control the degree of convenience in a product is to use `in-home' tests. Such tests will make it possible to test aspects of preparing, eating and disposing of a meal. In our in-home test of farmed cod in Spain we (e.g., Honkanen and Olsen, 2007) manipulated convenience by randomly providing the respondents with two different situations. The first situation was manipulated with the following suggestions for how the cod should be prepared: Imagine yourself in a situation when you are under time pressure. For example, you have less than 20 minutes to prepare a dinner for yourself and your family. Please choose a dinner which you find suitable for such a situation. The other situation was framed in a similar way, but with no time options and they were not under time pressure. In both cases a meal solution with different time uses were suggested. The results of the manipulated information resulted in significant differences in time used to prepare the dinner. About 40% of the respondents under time pressure used less than 15 minutes and two out of three used less than 20 minutes. On the other hand, consumers with no time pressure (almost two out of three) used more than 20 minutes, and nearly 50% used more than 30 minutes. The frequency of time used for the two manipulated time situations are presented in Fig. 6.2.

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Fig. 6.2 The effect of manipulating time pressure in an in-home study of farmed cod in Spain.

6.3

Intention and behavioural indicators

6.3.1 Willingness to pay Willingness to pay (WTP) has been defined as the maximum amount of money a customer is willing to spend for a product or service (Cameron and James, 1987; Krishna, 1991). It is a measure of the value a person assigns to a consumption or usage experience in monetary units. Economists refer to WTP as the reservation price (Monroe, 1990). Despite the importance of price-related issues in studies of product evaluation of consumer satisfaction, few studies have studied the link between consumer satisfaction and price tolerance or willingness to pay (Anderson, 1996; Homburg et al., 2005). A few studies have tested willingness to pay in relation to product development and market pre-tests (Cameron and James, 1987; Mitchell and Carson, 1989). Studies of willingness to pay have also been conducted in the food area (Loureiro et al., 2002; Moon and Balasubramanian, 2003). Several approaches can be used to elicit consumers' willingness to pay for products or services including contingent valuation, experimental auctions, conjoint analysis and hedonic price methods (Lee and Hatcher, 2001). The choice of willingness to pay measurement methods will influence the estimates of consumers' valuations, and the different methods can have different advantages and disadvantages depending on the purpose of the study, aspects of the product or services (object) estimated, availability of resources to conduct a study, and aspects of the individual investigated (see Lee and Hatcher, 2001 for a discussion). In general, contingent valuation has its strength in that it collects data directly from consumers and is less expensive and simpler than

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Table 6.1 Factor loadings and Cronbach's Alpha for the willingness to pay scale in three different experiments

What do you expect this product costs in a typical store? What is the highest price you are willing to pay? What would you suggest is an `expensive' price per kilo for this product? What would you suggest is a `fair price' per kilo for this product? What would you suggest is an `inexpensive' price per kilo for this product? Cronbach Alpha

Fish burger Norway

Fish burger Spain

Cod Spain

0.84 0.88

0.90 0.89

0.89 0.90

0.80

0.84

0.82

0.91

0.95

0.94

0.88 0.89

0.93 0.93

0.82 0.90

Extraction Method: Principal Component Analysis; Rotation Method: Varimax with Kaiser Normalisation.

experiments. The major weakness of contingent valuation is overestimation of consumers' willingness to pay. However, this depends on familiarity with the product and how the questions are constructed: a hypothetical product is difficult to evaluate and consumers with no previous experience of `real products' will have more problems evaluating products than consumers with high knowledge and product experience (Schoormans et al., 1995). As a common procedure in our studies of willingness to pay, we first gave the respondents an indicator of the price range for similar products (e.g., 85 NOK per kilo) in order to reduce non-responses and provide a `realistic' anchor for evaluation in an open-ended question (Simonson and Drolet, 2004). We then used multiple questions to assess willingness to pay for this product in a typical store. In Table 6.1 we have presented the factor score and reliability (Cronbach's Alpha) for three different studies; fish burger in Norway and Spain (Dopico et al., 2006; Olsen et al., 2006; Tudoran et al., 2006), and ethical farmed cod in Spain. 6.3.2 Intention to consume and potential loyalty Intention is most often defined as indications of how hard people are willing to try, how much effort they are planning to exert, in order to perform the behaviour (Ajzen, 1991). Intention is assumed to capture the motivational factors that influence human behaviour within several models in social psychology (Eagly and Chaiken, 1993; Sheeran and Orbell, 1999) and consumer psychology (e.g., Bagozzi and Warshaw, 1990). It is also used as the most appropriate indicator of customer loyalty (Fornell, 1992), and in a few studies it is used as a mediator between satisfaction and repurchase behaviour/loyalty (Mittal and Kamakura, 2001). The most widely agreed-upon definition of loyalty is `behavioural response expressed over time by some decision-making unit with respect to one or more

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alternatives' (Dick and Basu, 1994; Jacoby and Chestnut, 1978). Oliver (1997, p. 392) defines loyalty as `a deeply held commitment to rebuy or repatronise a preferred product or service in the future'. Several authors tend to consider loyalty on a multi-dimensional basis, by adding an attitudinal (cognitive and/or affective components), motivational or conative (intention or commitment to consume) component (e.g., Macintosh and Lockshin, 1997; Oliver, 1999) to a behavioural loyalty concept. In our studies of different new seafood (e.g., Olsen et al., 2007) products we limited our definition of loyalty to attitudinal and intentional aspects because the products are new and in most circumstances are not available on the market. Definitions and measurements are based on earlier research on intended loyalty (Chaudhuri and Hoolbrook, 2001), likelihood of retention and recommending or complaining behaviour (Bloemer and de Ruyter, 1998; Johnson et al., 2001). Several meta-analyses and individual studies suggest that there is a positive relationship between intention and future behaviour (Armitage and Conner, 2001), intention and past behaviour (Ouellette and Wood, 1998), and intention and loyalty (Olsen, 2004). Based on these recent theories of intention, several authors make theoretical distinctions among terms like will (estimate), expect (estimate), should (norm), wish/intend (goal) and determined or want (desire) in their assessment of intention (Chapman, 2001; Conner and Sparks, 1996). However, we are aware of different types of intentions (plan/intention, expectation/likelihood, or wants/ wish/will) associated with different types of strength to other variables (e.g., Fishbein and Stasson, 1990). It has also been suggested that scale correspondence between action and intention (e.g., frequency estimates) might be used to improve the predictive ability of intention (Courneya, 1994), compared to traditional probability estimates (e.g., `unlikely/likely'). We have in some of our studies tested both a `frequency estimate' of intention and a probability estimate in order to investigate if they have discriminant validity and predictive validity. The items used for intention are plan, expect and want to eat (e.g., the fish burger in the future). Some preliminary results from our fish burger test of Norwegian parents (Olsen, 2006a; Olsen et al., 2006) prove high correlation (about 0.70) between the two intentions scales. A confirmatory factor analysis suggested low discriminant validity between the two different formats. The correlation between the two intention scales and attitudinal loyalty was about the same (0.50), so our preliminary results prove the same predictive validity for both scales.

6.4

Perceived quality and satisfaction

Our research (e.g., Dopico et al., 2007a; 2007b; Honkanen and Olsen, 2007; Olsen et al., 2007; Tudoran et al., 2006) integrates several general constructs in the definition and assessment of product evaluation; evaluation of sensory attributes, perceived quality, customer satisfaction and general attitudes towards a seafood product. Our approach is based on attitude theory and research (Eagly

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and Chaiken, 1993). It is possible to anticipate that people form their beliefs based on sensory and other quality experiences with the product, and that the sum of belief or attribute evaluation forms the general evaluation like satisfaction, general attitude and expectation (Olsen, 2002). Within our framework, it is possible to vary our use of theoretical constructs and assessments from different theoretical fields within food science, marketing and product development. 6.4.1 Sensory evaluation and perceived quality of product attributes/ quality beliefs Sensory evaluation is defined as the scientific discipline used to evoke, measure, analyse and interpret reactions to characteristics of food as perceived through the senses of sight, smell, taste, touch and possibly hearing. Food products possess physiochemical characteristics that make them unique and incomparable. Specific characteristics play a crucial role in product evaluation. People proceed to a sensory assessment by using one or more of the five senses to judge, or form an opinion about, some aspects of the food product. Taste is often the most important attribute in sensory and product evaluation of food products (AcebroÂn and Dopico, 2000; Roininen et al., 1999), including seafood product (Nielsen et al., 1997; Honkanen et al., 2005). Perceived quality has been conceptualised, operationalised and applied in various manners and at different levels including excellence, value, conformance to specifications or requirements, fitness for use, loss avoidance and meeting and/ or exceeding customers' expectations (see Reeves and Bednar, 1994 for a discussion). While the early literature used conformance-to-specifications and fitness for use definitions of quality (Juran, 1974), the marketing and service literature have contributed with their focus on defining quality from a customer's point of view by introducing terms such as perceived quality, subjective quality, quality cues and quality expectations (Zeithaml, 1988). Quality, in the utilitarian sense, has been assessed through the product's ability to serve its intended purpose or perform its proper function (Sweeney and Soutar, 2001), or its ability to provide satisfaction (Monroe and Krishnan, 1985, p. 212). We use an attitudinal approach (Olsen et al., 2005) to assess perceived quality, including sensory evaluation (Moskowitz, 1995). In the theory of planned behaviour, attitudes are held to be determined by underlying salient beliefs (Ajzen, 1991). This part of the model, the relationship between attitudes and beliefs, has its origin in Fishbein's summative models of attitudes (Fishbein and Ajzen, 1975). It assumes that a person may process a large number of beliefs about a particular behaviour, but at any one time, only some of these are likely to be salient. The salient beliefs are assumed to determine a person's attitude. Within the marketing literature, these salient beliefs are defined and assessed as quality beliefs/attributes (Peter et al., 1999). It also fits well with several definitions of product quality as a process evaluation of relevant quality attributes formed from both physical product characteristics and cues in the environment (Grunert et al., 1996; Steenkamp, 1990).

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A proper understanding of which beliefs or attributes are more important for overall liking or satisfaction is helpful for establishing meaningful criteria for product development and optimalisation. Salient and important attributes change between product, persons, situations, and over time (Holm and Kildevang, 1996). When formulating a successful product, a food company must perform consumer studies to determine which attributes and products are liked or disliked by consumers. These tests indicate which attributes and perhaps attribute levels a product should exhibit in order to be successful in the market. In our research (e.g., Dopico et al., 2007a; 2007b; Honkanen and Olsen, 2007; Tudoran et al., 2006), we integrate sensory quality (sensory beliefs) with a marketing and consumer approach to quality by including different salient and important beliefs/attributes besides sensory attributes. Which attributes to include will, of course, depend on what is important for the consumer, but also what is important to investigate in the different studies. The most salient quality attributes in our research deal with sensory aspects (taste, texture, appearance, odour, and nutrition), convenience, health, moral and ethical issues. 6.4.2 Attitude and satisfaction In more formal terms, an attitude is defined as a psychological tendency that is expressed by evaluating a particular entity or object with some degree of favour or disfavour (Eagly and Chaiken, 1993). Most attitude theories keep to a onecomponent view of attitude and suggest that attitudes consist of evaluative or affective responses to attitude objects, and that affective responses are based upon cognition and are used synonymously with evaluation itself (Fishbein and Ajzen, 1975). We define attitude as an association in memory between a given object (e.g., a food product) and a given summary evaluation of the object (Fazio, 1995). Attitude and evaluative responses in attitude research are defined by their valence and extremity. In our questionnaires, we have decided to use items covering several facets of the attitude constructs without trying to make any particular effort to investigate the possible different dimensions. Because satisfaction is one of the most-used constructs to define and assess a general evaluation of products and services (Oliver, 1997), we intend to include satisfaction measures as one facet of our general evaluation construct. Satisfaction has been defined and measured in different ways over the years (Oliver, 1997). While earlier studies defined satisfaction as a transaction-specific product episode, recent studies argue to define satisfaction as a customer's overall experiences to date ± as cumulative satisfaction, like attitude (Johnson and Fornell, 1991; Olsen, 2002). An important advantage of the cumulative satisfaction construct over a more transaction-specific view is that it is better able to predict subsequent behaviours and economic performance (Johnson et al., 2001). In our research (Olsen et al., 2005) we define individual satisfaction as a consumer's personal overall evaluation of satisfaction and pleasure with a given product. Even though our experiments will cover situations defined as a

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transaction-specific evaluation, they will be influenced by previous experiences with similar products and expectations towards the product. Making a formal distinction between cumulative satisfaction and transaction-specific satisfaction will not be necessary in our case. The items we use to measure satisfaction are unsatisfied/satisfied, unpleasant/pleasant. In addition it is possible to add one or more of the attitude scale such as like/dislike, terrible/delightful, dull/exiting, negative/positive, bad/good, etc.

6.5 Results from a project about consumer evaluation of a Norwegian fish burger In this section, we present some of the findings we obtained from a part of our project `Consumer tests of convenience tailor-made seafood products', developed within the SEAFOODplus research programme. The main objective of the project was to explore consumer evaluation, satisfaction and buying intention for a convenient seafood product (a fish burger) targeting to satisfy Norwegian children or adolescents. The testing has mainly been concentrating on young consumers (Norwegian pupils and Spanish students), but has been supplied by studies of the Norwegian pupils' parents. It took place in the period of January/February of 2006 in Norway and April of 2006 in Spain. A total of 296 Norwegian pupils and 349 Spanish students randomly allocated to an inhome test group and a school canteen test group, participated in this study. Further, 149 parents of the pupils participated and conducted the fish burger test in-home. 6.5.1 Differences in evaluations across groups, countries and test locations Looking into cross-country and inter-group comparisons of product evaluation, our findings (Dopico et al., 2007b; Olsen, 2006a; Olsen et al., 2007) showed that, in general, the respondents from Norway perceived the fish burger as a meal and were more satisfied with the product compared to Spanish respondents. On average terms parents scored highest on global evaluation (average score of 5.42 on a seven-point scale), followed by the Norwegian pupils and the Spanish students in third place (see Table 6.2). With regard to the perceived quality, health and youth appeals, considerable differences were found between countries and groups. The Norwegian respondents evaluated the taste and healthy aspect of the product significantly better compared to Spanish respondents. The Norwegian parents again scored significantly higher than any other group, except for the health aspects where, on average, they registered similar results to the Norwegian pupils. Furthermore, parents were more likely to find the product appealing to youth than were young people themselves (see Table 6.2). Concerning the intention to eat the fish burger in the near future, on average the respondents adopted a neutral position in Norway (average scores around 4)

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Table 6.2 Differences in evaluation of a new fish burger product in three different consumer segments (n 894) Country/Group

Global evaluation Unsatisfactory (1) ± Satisfactory (7) Sensory quality/attribute evaluation Bad (1) ± Good taste (7) Unhealthy (1) ± Healthy (7) Not appealing to youth (1) ± Appealing (7) Intention: Disagree (1) ± Agree (7) I want to eat this product in the next three weeks Loyalty: Disagree (1) ± Agree (7) I will recommend this product to others

F-value p-value

Norway Pupils

Norway Parents

Spain Students

4.69b

5.42c

4.08a

40.145

< 0.001

5.32b 6.08b

5.89c 6.21b

4.06a 4.41a

96.884 204.164

< 0.001 < 0.001

4.43b

5.20c

4.05a

26.968

< 0.001

3.90b

4.30c

2.42a

78.323

< 0.001

5.15b

5.77c

4.01a

72.477

< 0.001

a, b, c indicate significantly different means using Duncan Post Hoc on a 7-point scale semantic differential scale (1±7) and a Likert scale (disagree±agree).

and showed to be quite sceptical towards this issue in Spain (average score around 2.5). Moreover, the results also revealed that about half of the Norwegian parents are inclined to buy the product while three out of four Spanish students would definitively not. Significant differences were found between countries and groups with respect to the loyalty towards the product. The Norwegian respondents were much more willing to recommend the product (average scores of 5.15 for pupils and 5.77 for parents) versus Spanish respondents who were not particularly motivated to do this and thus showed a neutral position concerning this issue (average score of 4.01). Our results suggest that even though this product received very high evaluations among the target group (Norwegian pupils), it did not satisfy the taste of the Spanish students. However, elderly consumers in Norway liked the product best ± and this gives the company new opportunities in positioning this product against new consumer groups. With reference to our previous discussion (Olsen et al., 2005), we also tested the differences in evaluation between canteen and in fish burgers prepared and eaten at home. Some of the results are presented in Table 6.3. These findings suggest that the test location had no significant effect on the evaluation of the fish burgers in either country. The tendency between the countries differs. While the burgers got the highest scores at the in-home test in Norway, the Spanish students had a tendency to evaluate the burger in the canteen a bit higher than in the in-home test. Some earlier studies confirm that testing conditions may have no effect on product evaluation (e.g., Hersleth et al., 2005), but this may differ between products and individuals (Boutrolle et al., 2005; de Graaf et al., 2005).

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Table 6.3 Differences in evaluation for the fish burger served in two different test locations (mean values and p-values) Norwegian pupils

Spanish students

Canteen In-home Sign (p) Canteen In-home Sign (p) Global evaluation Unsatisfactory (1) ± Satisfactory (7) Messy (1) ± Neat to eat (7) Quality/attribute evaluation Bad taste (1) ± Good taste (7) Unhealthy (1) ± Healthy (7) Non appealing to youth (1) ± Appealing (7) Intention: I want to eat this product in the next three weeks: Disagree (1) ± Agree (7) Loyalty: I will recommend this product to others: Disagree (1) ± Agree (7)

4.62 3.84

4.81 3.94

0.65 (ns) 0.67 (ns)

4.11 4.21

4.05 3.95

0.73 (ns) 0.09 (ns)

5.22 6.07

5.49 6.11

0.19 (ns) 0.76 (ns)

4.11 4.64

4.02 4.18

0.59 (ns) 0.00

4.36

4.64

0.72 (ns)

4.19

3.91

0.10 (ns)

3.91

3.89

0.58 (ns)

2.45

2.39

0.71 (ns)

5.05

5.32

0.06 (ns)

4.00

4.02

0.92 (ns)

ns = not significant, p > 0.05

The interaction between evaluation and individual characteristics will be investigated in future studies of our data. 6.5.2 The relationship between quality, satisfaction and behavioural intention Furthermore, we explored the interrelationships between quality beliefs and satisfaction with the product, and their influence on intention to consume the product in the future. Our research integrated both, sensory quality evaluations (taste, texture and appearance) of the product and quality beliefs about product healthiness and convenience issues. The analysis was based on data collected only from young consumers. The findings suggest that perceived sensory quality is the main antecedent of satisfaction with the product, and satisfaction plays a mediating role between sensory quality and behavioural intention (Fig. 6.3). Comparatively, quality beliefs about product healthiness and convenience have no significant influence on young consumer satisfaction with the product. Even though many young respondents may understand and value the health or convenience benefits that new seafood products may offer, it cannot determine their perception of product efficacy, and therefore, their satisfaction, unless the product is enjoyable in sensory aspects. However, the analysis brings evidence that the perception of health- and convenience-related benefits in the fish burger could be good reasons to purchase or consume the product in the future. Indeed, as Fig. 6.3

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Fig. 6.3 The relationship between quality, satisfaction and intention to consume.

shows, health and convenience exert significant and positive effects on behavioural intention. Overall, the study allows us to infer how young consumers weight the sensory attributes against perceived healthiness and convenience related benefits when arriving at overall preferences for a new product. Sensory appeal, health and convenience are important factors in the intention to consume the product, but only sensory quality plays a decisive role in satisfaction with the product. Variation in perceived health benefits is certainly not the most important factor in forming global satisfaction of a seafood product; most consumers totally agree that seafood products are healthy even though they like the taste and other attributes or not. This supports the argument that a multidimensional approach to motives governing seafood choice is appropriate. Finally, we concluded that if taste took precedence over health, then strategic marketing communications emphasising healthy seafood products that were also tasty, might be of greater value than messages emphasising health alone. 6.5.3 Using the theory of planned behaviour in new product testing Several models have been developed over recent years in order to understand consumers' preferences, choice or behavioural aspects (see Shepherd and Sparks, 1994). One of the most used recent theories for studying food attitudes and behaviour is Ajzen's (1991) Theory of Planned Behaviour (TPB). This theory is acknowledged for its parsimony and ability to generalise across situations, behaviour, objects, individuals and cultural settings (Armitage and Conner, 2001). The authors are not aware of any study which has used this theory to explain intention to consume a new product. Using this theory in a cross-cultural study of food products is an extension to the very few crosscultural studies of this theory. The results of a multi-group analysis in LISREL indicated no group differences in the structure of the TPB in explaining intention to consume the

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fish burger (Olsen et al., 2007). Attitudes, social norms and perceived behavioural control explained 46% in the variance of intentions across groups. Perceived behavioural control is more important than attitudes and norms in explaining intention to consume the fish burger across cultures (Spain and Norway) and across generations (parents and their children). This indicates that this theory can be used not only in explaining intention to consume common food products, but also in the area of new-product testing. It also supports a possible stability in the model structure across groups even though the evaluation of attitudes, norms and perceived control varies in their mean-values in the different groups.

6.6

Conclusions

Integrating the different concepts presented in Fig. 6.1 (see Olsen et al., 2005 for a discussion), the suggested relationships among the variables can be expressed by the following overall hypotheses. These relationships guide the experiments that are being performed to develop new or improve on existing seafood products. H1: The form of the meal, different test locations and other aspects of the contextual environment, and informational cues influence individual evaluations. As was pointed out earlier, several intrinsic and extrinsic cues are likely to affect and determine the different types of evaluations consumers can make. Since there are different types of product evaluations, in development and improvement of products it is important to know what types and levels of information as provided (by the product) affect what type of evaluation most in order to leave the best overall impression. Since the evaluations were defined as `beliefs', the most direct relationship can be expected to be between product attributes and evaluations at the same level: intrinsic sensory attributes (e.g., texture, flavour, colour) cue directly sensory experiences; extrinsic information (e.g., health claim information, but also price, brand label, package) cue related intrinsic and extrinsic quality beliefs; situational (location), contextual (meal, occasion) and extrinsic product factors (e.g., price, convenience) cue extrinsic experiences (i.e., `convenient product'). Systematic variation of these factors is required in experimentation to understand the full scope of product evaluation and satisfaction. Subsequently, three factors are essential in explaining the relationship between cues and the resulting evaluations according to our model. First, interactions between product and situational information are likely to occur during evaluation (Meiselman, 1994). E.g. the perceived level of healthiness is likely to be affected by the perceived level of convenience of the product. The perceived level of convenience depends on the consumption situation; etcetera. Systematic variation of situations, convenience levels and health information in

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experimentation may explain product evaluations better. Second, interactions between evaluations themselves and the formation of overall evaluations need to be accounted for (Fishbein and Ajzen, 1975). The perceived level of healthiness of a product is likely to interact with perceived flavour, according to one's individual associations. Clever analyses should reveal such co-variations that can be very dominant. Third, due to these interactions, but also due to their own nature, relationships between cues and perceptions are often not linear, but can be quadratic or even higher order. This is especially true for global evaluations, since they can be assumed to result from a weighed sum of lower level evaluation outcomes. Moreover, appropriateness judgments and expectation (dis-)confirmation can affect (global) evaluations in a non-linear fashion. H2: There is a positive relationship between the different individual evaluations (sensory, perceived quality, attitude/satisfaction) and different behavioural indicators (willingness to pay, intention to consume, buy and recommend). According to planned behaviour theory (Ajzen, 1991), positive attitudes lead to positive intentions to use the product (again). In the case of planned behaviour, intentions are needed first to actually express the behaviour. It need not be rational behaviour, as in cognitive theory `action tendencies' are used to explain the relationship between emotions and behaviour as well. However, measuring behavioural intentions and intended behaviour depends on a positive experience and acceptation as opposed to rejection. Positive global evaluations increase the probability of a product to be reconsidered for consumption and/or purchase (e.g., Olsen et al., 2007). From the results it can be seen that consumers feel that their behavioural intent should be more affected by their satisfaction based upon sensory characteristics, than by health considerations. The differences between intention types show that different considerations can affect behavioural intentions: a low tendency for repeated consumption doesn't mean that the fish burger product is not recommendable to others (Tables 6.2 and 6.3). Of course, in real life there are many more factors that determine actual behaviour, thereby reducing the predictive value of intention measurements to actual behaviour. We can conclude that the current model and more controlled experiments are especially suited to study the evaluation of products, in interaction with their environment. More spontaneous behaviour observing experiments are needed to predict behaviour, especially when it comes to buying behaviour. H3: Individual variables such as convenience orientation, health concern, social norms, moral/ethical concern, knowledge, innovativeness and food neophobia moderate the relationships proposed in H1 and H2, as well as have a direct influence on evaluation and behavioural indicators. Individual differences are background variable to all of these processes (Olsen et al., 2005). They come in different varieties: states or moods that might

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induce particular cravings, thus either moderating (emotional) responses in general, or in interaction with product or situational cues. For example, a high fat product can seem more appealing when very hungry as compared to a satiated state. Longer-term tendencies and preferences often were formed over experiences and can be either more physiological (preference for sour) or more cognitive (preference for healthy food). Cognitive predispositions often depend on (subjective) knowledge or beliefs. For example, the belief that seafood is healthy may lead to a positive predisposition towards anything with seafood. The belief that deep-frying is unhealthy may cause a negative predisposition towards anything deep fried. Again, these predispositions can affect either the evaluation or behavioural intention directly, or the interaction between product characteristics and individual evaluations. This transfer of attitudinal affect is often associative and therefore subjective and not necessary logic or deductive. The belief that fish need space for their welfare is not true in the case of catfish, but awareness of the packed conditions of farmed catfish may lead to negative catfish product evaluations. Subjective knowledge and beliefs are formed and available through associative memory. Attitudinal believes do involve sensory predispositions (e.g., Dopico et al., 2007b), but also more complex ones as preferences for or avoidance of new products, moral attitudes, preference for convenience (interacting with situations), etcetera. For the current purpose, probably most interesting is the time frame: longterm dispositions can help us to segment different consumer groups that might need a different approach in order to elicit the most positive of evaluations in response to new seafood products. On the other hand, short-term or unstable dispositions offer possibilities to add to a more positive product experience. For example, filling in a lack of knowledge about catfish natural habits might improve the image of the production conditions and subsequently product evaluations. Since perceptual effects of intrinsic and extrinsic cues and their interactions are not external factors, but internal dispositions of the human information processing system, untrained consumers will be unable to assess the different evaluations independently. Therefore in modern product development and improvement, elaborating on these cues is supposed to be an integral part of product design and testing. For this matter, our experiments focus on these interactions (Olsen et al., 2005).

6.7

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KOZLOWSKA, K. JERUSZKA, M., MATUSZEWSKA, I., ROSZKOWSKI, W., BARYLKO-PIKIELNA, N.

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Part II Health benefits of seafood

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7 Introduction to Part II: health benefits of seafood G. Schaafsma, HAN University, The Netherlands

7.1

Developments in nutrition science

The health benefits of seafood consumption can be viewed against the background of developments in nutrition science, as described by Schaafsma and Kok (2005) and summarized here briefly. Since the beginning of the 20th century nutrition science developed rapidly and in the first period of this science (until about 1970), all indispensable nutrients, that we know now, were discovered and their biochemical role in the body was elucidated. Deficiencies of one or more of these nutrients are known to cause the classical deficiency diseases, like beri beri (vitamin B1), rickets (vitamin D), scurvy (vitamin C) and anaemia (iron). Research in that period resulted in setting up so-called Recommended Daily Intakes (RDIs) of most nutrients for various groups of the population. These RDIs are very helpful for nutrition education, for the planning of diets, for the evaluation of the adequacy of food intake and for the assessment of the nutritional value of single foods. Although new research may lead to further refinement and substantiation of the RDIs, it is not expected that new indispensable nutrients will be discovered. Whereas in Western societies classical nutrient deficiencies have become scarce, they unfortunately still occur on a large scale in food-deprived Third World countries. After this so-called period of classical nutrition, scientists and health care authorities became aware that, particularly in affluent Western societies, adequate nutrition is more than just the prevention of classical nutrient deficiencies. Unbalanced diets appeared to be involved in the occurrence of chronic diseases, such as cardiovascular disease, diabetes type II, osteoporosis and cancer. Since then, nutrition science became involved in finding the optimal (balanced) diet.

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Dietary guidelines, aiming to reduce the risk of chronic diseases, were disseminated. This is a continuing process and it is expected that future research, including that of SEAFOODplus, will generate new knowledge in this field. Until about 1990, the main focus of nutrition science in Western countries was on the adequacy of total diets, rather than on health aspects of individual foods. Whereas nutrition scientists continue their efforts to improve and substantiate further the dietary guidelines, since about 1990, under the influence of Asian countries (e.g. Japan), the concept of functional foods came up in Europe. These foods can be considered as foods, which are designed especially to exert a health benefit. They are marketed with nutrition claims and health claims.

7.2

Nutritional role of seafood

For many population groups, seafood is a nutritionally significant part of the diet, but between groups and between individuals within groups, seafood consumption may show a wide variation. From a nutritional point of view and in conformity to the text above, seafood can be considered in three ways: (1) source of indispensable nutrients, (2) food that lowers the risk of chronic diseases, and (3) food that serves as a basis for the design of functional foods. 7.2.1 Seafood, an excellent source of indispensable nutrients It is well known that seafood (lean and fatty fish, shell fish) is an excellent source of a large variety of nutrients. These include high-quality protein, vitamins (especially vitamins A and D in fatty fish, and B vitamins), minerals and trace elements (especially iodine and selenium) and long chain polyunsaturated omega-3 fatty acids. Because of its high nutrient content, seafood consumption can make a significant contribution to cover the needs of indispensable nutrients. Its high content of a large variety of nutrients compared to its energetic value, means that seafood perfectly fits into low-energy diets, designed for weight management. Challenging new information is reported in Chapter 9 in this book where new aspects of diets for weight management have been found when including fish or fish oils in the diets. 7.2.2 Seafood as a food that lowers the risk of chronic diseases Research in various population groups on the relationship between food consumption and the occurrence of chronic diseases (so-called epidemiological nutrition research) has demonstrated protective effects of fish consumption against cardiovascular disease, colon cancer and the metabolic syndrome. The metabolic syndrome is a condition which includes elevated blood pressure, elevated blood lipids (especially triglycerides), and raised blood glucose levels and body fat content. The metabolic syndrome is associated with a strongly

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increased risk of cardiovascular disease and diabetes type II. A disadvantage of epidemiological research is that this type of research cannot assess whether these beneficial relationships are causal. Theoretically, they could be spurious, because of confounding factors related to life style and not directly to fish consumption. Therefore it remains necessary to perform controlled intervention trials in humans in which the effects of fish consumption on the risk of the chronic diseases mentioned above are directly investigated. And this is exactly the aim of the nutrition research within SEAFOODplus. In the following chapters, written by Dr Liz Lund, Prof. Inga Thorsdottir and Dr Ingeborg Brouwer, studies are described in three areas. A specific focus in these respective chapters is on the beneficial effects of fatty acids from fish as well as on fish protein. 7.2.3 Seafood as a basis for the design of functional foods It is beyond the scope of this introduction to address in detail this particular issue. For a description of the concept of functional foods, the reader is referred to an overview written by Schaafsma and Kok (2005) and in Part IV of this book examples are given of how functional foods and/or functional food ingredients, with seafood as a basis, can be made by novel food processing and aquaculture techniques, which is also covered in Part V of this book.

7.3

References and KOK FJ (2005), Nutritional aspects of food innovations: a focus on functional foods. In: Jongen WMF and Meulenberg MTG (ed), Innovation in Agrifood Systems. Wageningen Academic Publishers, pp 207±220.

SCHAAFSMA G

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8 Protective effects of fish consumption in relation to gastrointestinal health E. Lund, Institute of Food Research, United Kingdom and E. Kampman, Wageningen University and Research Centre, The Netherlands

8.1

Introduction

Fish consumption is well recognized by the general public to be beneficial to health, but this association is probably more generally linked to prevention of heart disease and arthritis. However, there is an accumulating body of evidence to suggest that fish consumption may well be protective in relation to diseases of the gastrointestinal tract, particularly the colon and rectum (Geelen et al., 2007) and possibly other regions of the intestinal tract. As with heart disease and arthritis, most of the beneficial effects of consuming fish have been linked to the high n-3 content of some fish and this is the area that has been studied most systematically. The gastrointestinal tract is subject to a range of diseases both acute, such as food poisoning, and chronic, such as a number of cancers, gastric and duodenal ulcers and inflammatory bowel disease. There is no evidence to suggest that fish consumption is protective in relation to food poisoning and indeed shellfish may be a source of food-borne pathogens which, although potentially dangerous to the individual do not contribute significantly to the overall disease burden at a population level. In contrast there is evidence that fish or at least fish oils may be protective in a number of chronic diseases. Limited data suggest that PUFAs in general might be protective in relation to duodenal and gastric ulcers (Manjari and Das, 2000), more evidence exists in relation to inflammatory bowel disease although this is not compelling (MacLean et al., 2005), but probably the most important effects are seen in relation to cancers. Colorectal cancer (cancer of the large bowel) is one of the most significant causes of morbidity and mortality in most Westernised

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countries and is increasing in incidence in many developing countries. For this reason the main focus of this chapter relates to colorectal cancer.

8.2

Colorectal cancer (CRC)

8.2.1 Background Colorectal cancer is the third most common cancer across the world (Parkin et al., 2005) with about 2 million new cases worldwide recorded in 2002. There is a particularly high incidence in most European countries, in North America, Australia and New Zealand (Fig. 8.1) and in recent years in countries such as Japan, where a largely Westernised lifestyle is now established. Similarly, Japanese moving to America have greatly increased incidence of colorectal cancer (Key et al., 2002) and African Americans have over 60-fold increased risk of colorectal cancer compared to those living in Africa (O'Keefe et al., 2007). These type of data suggest that lifestyle is an important contributory factor in the risk of developing this cancer with diet (Doll and Peto, 1981), low physical activity and obesity (Giacosa et al., 1999) considered to be the major contributory factors. There are two major genetic causes of colorectal cancer; familial adenomatous polyposis (FAP) in which the APC tumour suppressor gene is non-functional (Nakamura et al., 1992) and hereditary non-polyposis colorectal cancer (HNPCC) which encompasses a group of at least five genes associated with DNA mismatch repair (Lynch and de la Chapelle, 1999). However, in combination these two syndromes only account for around 5% of

Fig. 8.1

Age-standardised incidence rates, by sex, colorectal cancer, world regions, 2002 estimates (adapted from IARC GLOBOCAN 2003).

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all cases of colorectal cancer, supporting the theory that environmental factors are key; although there are now known to be other genetic polymorphisms that may predispose someone to developing a cancer in the presence or absence of a particular environmental factor, for example polymorphisms in the vitamin D receptor affect response to a number of dietary factors (Murtaugh et al., 2006). Colorectal cancer is very much a disease of old age with incidence increasing in both men and women from the age of 50. The chance of cancer developing and the rate at which an initial defect leads to a tumour may well be modifiable by alterations in lifestyle both earlier in life and from this point onward. Tumour development is believed to occur over a period of decades and involves the accumulation of a series of genetic defects during progression towards an increasingly neoplastic state (Vogelstein et al., 1988). Even before any mutations have occurred, the mucosal tissue lining the colon may become more susceptible to DNA damage as a result of increased epithelial cell proliferation (Mills et al., 2001), modification of gene expression by direct signalling or as a consequence of DNA methylation (Malfoy, 2000), or because removal of damaged cells by apoptotic cell death is compromised (Kerr et al., 1972, Martin et al., 2002). Dietary constituents such as polyunsaturated fatty acids are known to be able to modify such factors in either a positive (Latham et al., 1998) or negative manner (Rao et al., 2001). Traditionally much of the research in relation to the underlying mechanisms as to how diet may influence cancer risk has focussed on negative aspects of food, such as the presence of carcinogens in the diet, with protective factors such as fibre being seen as a means to ameliorate any toxic effects. In this context secondary bile acids have received a great deal of attention with fibre acting to dilute any toxins. A high consumption of processed and red meat is a known risk factor for this disease (Bingham et al., 2002) and originally this was considered to be due to the presence of high levels of heterocyclic amines, particularly PhIP, in cooked or burnt meats, but this has been questioned on the basis that the patterns of mutation found in the DNA of cancer patients is not typical of heterocyclic amine (HCA) exposure in animal experiments and that high protein foods such as fish and white meat, which may contain relatively high levels of HCAs (Busquets et al., 2004), are not associated with increased cancer risk. Currently, the formation of nitroso-compounds in the colon from high protein diets containing significant levels of the haem moiety provides the most likely explanation for the increased incidence of colorectal cancer in high meat eaters (Bingham et al., 2002). The complexity in interpreting the effects of a mixed diet is well illustrated by the evidence from the EPIC cohort in which increased colorectal cancer risk associated with high meat consumption is to a large extent counteracted by the consumption of fibre (OR1.09 for high fibre-high meat eaters but 1.5 for low fibre-high meat eaters) and fish (OR1.12 for high fish-high meat eaters but 1.63 for low fish-high meat eaters; Norat et al., 2005). Fibre and fish consumption are not the only dietary factors seen as protective, indeed the protective effects of vegetables have received extensive attention especially in relation to the identification of more specific anticancer properties (Bingham, 1996, Johnson, 2004). Similarly

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more mechanistic studies in relation to n-3 fatty acids such as those found in oilrich fish have also been the subject of extensive research (Lund, 2006). 8.2.2 Observational studies in relation to fish consumption and colorectal cancer While observational studies do not provide unequivocal evidence of cause and effect, rather reflecting an association between an environmental factor and disease risk, they can provide useful indications of factors that might be relevant to the issue being studied. In contrast to the effects of meat consumption, such observational studies suggest that fish intake is generally associated with no increased risk of colorectal cancer or possibly a decreased risk. There have been in total 26 well-conducted case-control studies covering 35 separately defined populations in which fish consumption has been considered in relation to risk of bowel cancer. Of these, three have shown a significantly increased risk (Amaral et al., 2002, Chiu et al., 2003, Zaridze and Filipchenko, 1991) and five have shown a significantly decreased risk (Fernandez et al., 1999, Franceschi et al., 1997, Haenszel et al., 1973, Kune et al., 1987, Yang et al., 2003, Yeh et al., 2003) amongst those eating most fish. Case-control studies investigating the role of diet in disease risk are known to potentially suffer bias in that people's recall of what they were eating twenty years previously is poor and tends to reflect more recent diet, which itself may be modified in response to symptoms of disease, or overt disease. If we therefore consider cohort studies in which diet is recorded in a relatively young population and participants are then followed up to see what diseases they succumb to over subsequent decades, these should better reflect the diet at a time prior to disease development. In a recent systematic review MacLean et al. collated data from nine studies involving seven different cohorts in which CRC risk and n-3 fatty acid intake was assessed and concluded that there was no significant association between n3 fatty acid intake and disease risk (MacLean et al., 2006). They also reported on fish intake and CRC and again found no evidence to support any benefit of a high intake of fish. These conclusions were based on a relatively small selection of the relevant studies. More recently, Geelen et al. (2007) have published a detailed meta-analysis of 14 prospective cohort studies with data on fish consumption in which they conclude that fish consumption, and possibly n-3 fatty acid intake, inhibits colorectal carcinogenesis particularly if fish consumption is high and that each extra portion of fish per week corresponds to a 4% reduction in cancer risk. From these 14 studies, two describe the data for men and women separately giving 16 different populations. No studies included show a significantly increased risk and two report significantly protective effects (Kato et al., 1997, Norat et al., 2005) of fish consumption. Many of these studies suffer from small numbers and a generally low consumption of fish such that even the highest quartile or quintile of consumption included people only eating one portion of fish a week so that the variation in intake is not large enough to detect any difference in risk.

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Fig. 8.2 Risk of colorectal cancer in Europeans in relation to frequency of fish consumption. Based on data from Norat et al. 2005 in which a portion of fish is assumed to weigh in the region of 120g. * shows significant reduction after adjustment for dietary assessment methodology and confounding factors such as meat intake.

In 2005 The European Prospective Investigation into Cancer and Nutrition (EPIC) published a paper showing that people eating on average 80 g fish/day or more had a significantly reduced risk of getting cancer of the large bowel compared to those consuming less than 10 g/d (Fig. 8.2). This study has the advantage of size and a wide range of fish intake within the population. However, even this study, in common with all other observational studies, has little information on the type of fish consumed and, for example, how it has been cooked or whether it is high in n-3 fatty acids. The dietary information on which these studies were based was obtained using a questionnaire approach including only 3±5 questions on fish intake. This lack of data reflects low awareness of the potentially beneficial effects of fish in the diet at the time of setting up most cohort studies of reasonable duration and which are now identifying significant numbers of disease cases. The method of preparation of the fish in the EPIC study was reported (Rohrmann et al., 2002), but without specific reference to salted and fermented fish, and in the analysis of fish intake in relation to colorectal cancer risk for this study this was not taken into account (Norat et al., 2005). This is important as other studies have suggested that salted and fermented fish may actually increase cancer risk in both the stomach and colon (Ahn, 1997, Chen et al., 1992, Phukan et al., 2006, Yang et al., 2003); illustrating the limitations of many observational studies. The accuracy of dietary assessment in relation to fish intake and in particular n-3 fatty acid intake is further undermined by the quality of information available in dietary databases on polyunsaturated fatty acid content in many foods and by the great variability of content depending on the source of the fish. Also, not all studies appear to have recorded the intake of fish oil capsules as an

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additional variable. Furthermore, the extent to which circulating levels of fatty acids reflect dietary intake is uncertain. Welch et al. (2006) have shown that intake of n-3 fatty acids assessed using either food frequency questionnaires or diaries only explain 25% of the variation in plasma phopholipid n-3 PUFAs. Whether this is because there are other homeostatic processes in place to maintain the functionality of the membrane from which these phospholipids are composed or whether the tools for dietary assessment are inadequate it is hard to tell; possibly a combination of factors explains this discrepancy. 8.2.3 Nutrient-gene interactions in relation to fish intake A further complication in interpreting observational studies is that not everyone in a population will respond in the same way to a particular nutrient. This might be because of other factors in their diet and lifestyle or most interestingly because of the background genotype of the individual. For example, some people handle toxins and drugs in a different manner because of small variations in the genetic sequence of genes such as the GSTs (Loktionov et al., 2001). These single nucleotide polymorphisms (SNPs) exist for many genes involved in nutrient metabolism and DNA repair. Siezen et al. (2005, 2006) have investigated the role of SNPs in genes associated with fatty acid metabolism and signalling (see below), and fish consumption. These analyses show that people with particular SNPs in the COX-2 gene and PPAR have reduced risk of colorectal adenoma and colorectal cancer. Moreover, fish consumption appeared to be protective in these studies, with the exception of those people carrying a minor SNP (c.-789C>T) in the PPAR gene in which the protective effects of fish consumption in relation to adenoma formation found in the majority of cases was reversed in this sub-group (<10% of the population). No significant interaction between fish consumption with SNPs in the genes involved in the arachidonic pathway and colorectal cancer was observed in the later study investigating actual tumour formation (Siezen et al., 2006). 8.2.4 Intervention studies and colorectal cancer To undertake a long-term intervention study to investigate the impact of fish consumption on colorectal cancer incidence would be completely impractical. In fact no such studies have ever even been undertaken using fish oil capsules, a potentially simpler type of study but which is still limited by the long period of time over which colorectal cancer develops. There is limited information in relation to cancer risk available from studies looking at fish oil intake and cardiovascular disease end-points but even the largest of these trials, the GISSIPrevenzione trial, only reports total cancer incidence (GISSI, 1999). Often the interventions have also been too short-term in relation to tumour development. For example, the GISSI trial involved a 3.5 y fish oil intervention, which is very short in relation to the timescale of cancer development, and so is unlikely to have a significant effect on overt tumour development if the main effect relates to prevention rather than cure.

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An alternative approach has been to measure end-points that, based on our understanding of the development of colorectal cancer, are likely to be indicative of increased risk. Such biomarkers of risk are not well developed in the area of cancer research. However, there are a few available. For example, increased levels of cell proliferation in the epithelial tissue lining the large bowel has been considered to indicate increased risk (Mills et al., 2001) and more recently increased apoptotic cell death has been even shown to be a marker of reduced risk (Chang et al., 1997, Martin et al., 2002). The presence of early signs of tissue dysplasia such as adenomas is also considered to be a marker of increased risk as removal of such adenomas reduces tumour incidence (Lefton et al., 1996), but only a small percentage of these features actually develop into tumours. Currently, these end-points are the best available indicators of risk, although they are recognized to be poor in comparison to those used in the study of cardiovascular disease. These methods all require examination of the colon and usually collection of biopsy samples and are therefore not straightforward to implement in the context of large-scale intervention trials. However, such human intervention trials have shown a protective effect of n-3 supplementation on crypt cell proliferation (Anti et al., 1992, Bartoli et al., 1993) and apoptosis (Cheng et al., 2003). These results in humans are replicated in a diverse range of animal models using chemical carcinogens (Reddy and Sugie, 1988, Chang et al., 1997), the APCmin mouse model (Paulsen et al., 1997) and implantation of tumour cells (Calder et al., 1998). Such studies also provide evidence of the relative protective effects of different fatty acids such that EPA and DHA provide most protection, compared to a sunflower or safflower oil or even an olive oil diet. The data on -linolenic acid and linseed oil are inconsistent with some studies suggesting a small protective effect on tumour development while others suggest an increased risk relative to sunflower oil, although not to the extent that conjugated linoleic acid or linolenic acid have been reported to do so (Wahle et al., 2004). We have previously shown in the rat DMH model that fish oil (n-3 diet) suppresses cell proliferation and increased apoptosis relative to corn oil fed (n-6) animals when fed after exposure to the carcinogen (Latham et al., 1999). This suggests a post initiation protective effect of n-3 consumption, compared to n-6, that is maintained for up to 18 weeks (Chang et al., 1997) when a reduction in ACF has been reported. These observations are consistent with epidemiological data provided by Oh et al. (2005) that there maybe a post initiation protective effect of fish consumption. That is, they suggest that fish consumption does not particularly protect against any initial damage leading to the formation of the precancerous adenomas, but may prevent the development of adenomas to tumours, which is consistent with the pro-apoptotic effects of n-3 fatty acids seen in animal studies. 8.2.5 Mechanisms underlying the effects of fatty acids on colorectal cancer risk The mechanisms by which n-3 fatty acids reduce cell proliferation and increase apoptosis are not well understood and are likely to be multifaceted. Redox

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modification would appear to be an important aspect in relation to induction of apoptosis. Rats given BSO in their drinking water, to reduce intracellular glutathione levels, have increased levels of apoptosis, an effect that is exacerbated by the replacement of n-6 fatty acids in the diet with n-3 fatty acids (Latham et al., 2001). In cell culture, pre-treatment with lipid soluble anti-oxidants prevents apoptosis and loss of mitochondrial membrane potential (Ng et al., 2005) supporting the hypothesis that the oxidizability of long chain, highly unsaturated PUFAs (LC-PUFAs) is important in relation to their protective role. Classically, induction of apoptosis has been considered to be regulated post-transcriptionally with pro-caspases always present, ready to be activated in response to either intrinsic or extrinsic signalling. But, it is entirely feasible that the sensitivity of a cell to a pro-apoptotic signal may be modified by altering not only the structure of cellular membranes, including mitochondrial membranes, but also by changing gene expression of proteases and receptors involved in apoptosis. Potential mechanisms relating to dietary fatty acids include modification of Fas-L expression and/or activity on the cell surface, changes in mitochondrial potential, down regulation of anti-apoptotic molecules such as bcl-2 or up regulation of proapoptotic molecules (Cheng et al., 2003). Control of gene expression in relation to apoptosis, cell proliferation and differentiation by fatty acids may be exerted through binding to PPAR receptors. Both PPAR and PPAR are expressed in the colon, are rarely mutated during tumour progression and control genes associated with cell proliferation, apoptosis and differentiation (Murtaugh et al., 2005). PPAR activation has been shown to induce apoptosis and reduce adenoma number in the colon, possibly by down regulating Cox-2 expression (Yang and Frucht, 2001). However, in the APCmin mouse some synthetic ligands have been reported to increase tumour progression but others are protective (Niho et al., 2003). As natural ligands, n-3 fatty acids are protective in this model (Paulsen et al., 1997) yet n-3 and n-6 fatty acids show similar affinity for PPAR . However, n-3 fatty acids do have a higher affinity for the RxR which combines with PPAR to form the active heterodimer (Fan et al., 2003), potentially explaining the differential effects of n-3 and n-6 fatty acids. Alternatively, oxidized lipids increase PPAR controlled luciferase expression more than the unoxidized forms (Bull et al., 2003). The highly unsaturated LCPUFAs with 5 or 6 double bonds, which are found in fish oil, will oxidize more readily than for example linolenic acid with only two double bonds, so may therefore bind more strongly to PPARs and have an exacerbated effect. Normally PPAR is increasingly expressed as cells move up the crypt axis and onto the luminal surface but in some tumours and in the APCmin mouse PPAR is over expressed throughout the length of the crypt as a result of -catenin over expression (Jansson et al., 2005). The APC protein is also reported to control PPAR expression through the -catenin ± Tcf-4 pathway such that wild type APC promotes degradation of -catenin and down regulation of PPAR (Gupta et al., 2000) and, although luciferase reporter assays suggest that PUFAs are agonists for PPAR /RxR in the colon (Fan et al., 2003), research based on gel shift assays suggest that DHA, and to a lesser extent by EPA, inhibits PPAR and PPAR

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binding to the response element (Lee and Hwang, 2002). The target genes for PPAR are those associated with increased cell proliferation, such as c-myc and cyclin D1 and suppression of apoptosis. Thus PUFAs may act by both reducing the proliferative effects of PPAR over expression and increasing the pro-apoptotic effects of PPAR but interpretation of the literature remains equivocal. A diverse range of alternative mechanisms to explain the protective effect of n-3 fatty acids in relation to colorectal cancer have been proposed and comprehensively reviewed in two recent articles (Roynette et al., 2004, Larsson et al., 2004). These include a number of mechanisms involving extrinsic influences on the colonocyte genotype and phenotype from both the colonic lumen and the circulation as well as local intercellular signalling molecules including NO. For example, in the lumen n-3 fatty acids may change the bacterial conversion of bile acids to less genotoxic secondary bile acids and reduce the production polyamines. The hyperproliferative effects of circulating levels of insulin like growth factor II (IGF-II) are counteracted by the ability of n-3 PUFAs to induce the binding protein IGFBP-6 and the effects of epidermal growth factor (EGF) are counteracted by chronic exposure to lipid peroxides, which down regulate ornithine decarboxylase activity. It has also been demonstrated using reporter assays that DHA down regulates EGF mediated AP-1 (jun-fos) transcription factor controlled gene expression which would be predicted to reduce cell transformation and tumour promotion (Liu et al., 2001). The presence of chronic inflammation in the colon is associated with an increased risk of colorectal cancer (Rhodes and Campbell, 2002) and it may well be that the well known anti-inflammatory effects of fish oils, particularly EPA, act by modifying this aspect of intestinal health. EPA acts as a substrate for the cyclooxygenase and lipoxygenase catalysed formation of eicosanoids and related oxidized metabolites, competing with arachidonic acid and generally forming less inflammatory mediators. Many studies have shown reduced PGE2 (prostaglandin E2) production in response to consumption of n-3 fatty acids in both colorectal tumour cell lines and in tissue. In the latter case we cannot be sure whether the effect relates to PGE formation by the epithelial cells or that of associated leukocytes. However, there is considerable support for the concept that PGE2 is a tumour promoter as treatment with Cox-2 inhibitors reduces tumour number in several systems (Tapiero et al., 2002). Furthermore there is evidence to suggest that arachidonic acid derived eicosanoids are involved in tumour promotion via PPARs (Gupta et al., 2000), and, for example, 15d-PGJ2, can inhibit PPAR induced cell death (Shimada et al., 2002). The potential role of n-3 fatty acids as anti-inflammatories in the prevention of colorectal cancer has been highlighted in a recent review by Chapkin et al. (2007).

8.3

Inflammatory bowel disease and fish consumption

Inflammatory bowel disease (IBD), as well as being a debilitating condition in itself, is also associated with an increased risk of colorectal cancer (Rhodes and

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Campbell, 2002). There are two distinct forms of IBD, ulcerative colitis (UC) and Crohn's disease (CD). These both show distinct geographical variation, being most common in North America and North-western Europe (Loftus, 2004) and in one study were associated with consumption of a highly refined, high carbohydrate diet (Sakamoto et al., 2005). Few studies have considered the relationship between fish consumption and IBD in epidemiological terms and those that have reported on this provide conflicting evidence. Sakamoto et al. (2005) report fish consumption to be positively associated with CD in a casecontrol study while Magee et al. (2005) suggest fish consumption is beneficial in patients with ulcerative colitis. A recent review by MacLean et al. (2005) of intervention studies conducted using fish oil supplements in both ulcerative colitis and Crohn's disease reported that the available data was insufficient to conclude any benefit in relation to a number of end-points, although reduction in the requirements for corticosteroid treatment was consistently reported across three studies. Studies in animal models, in which other aspects of diet are better controlled and which the disease is usually of only one modality, are more supportive of a protective effect of fish oil (Camuesco et al., 2005). For example, colonic lymphocytes from mice fed fish oil rather than corn oil have a lower proliferative response to a number of pro-inflammatory cytokines (Kuratko, 2000). Furthermore, it has recently been reported that colitisassociated tumorigenesis is suppressed in mice with endogenously high levels of n-3 fatty acids (Nowak et al., 2007).

8.4

Fish consumption and other gastrointestinal tract cancers

Although the colon and rectum are the major sites of gastrointestinal cancers, gastric and oesophageal cancers are also relatively common. The incidence of oesophageal cancer has increased rapidly in recent years, particularly in the United Kingdom, suggesting an environmental aspect to its aetiology. Oesophageal adenocarcinoma is associated with obesity and gastric reflux of acid/bile. No particular dietary factor has been linked to the increase in disease but the few case-control studies in existence suggest that consumption of fish does appear to be protective (Fernandez et al., 1999, Chen et al., 2002) and a small case-control study from Italy suggests a protective effect of fish consumption in relation to adenocarcinoma of the small intestine (Negri et al., 1999). Whether the effects in relation to oesophageal cancer are linked to the n-3 fatty acids in the fish is questionable as in our recent intervention study with eicosapentaenoic acid in patients with the pre-neoplastic condition Barrett's oesophagus, we showed no significant effects after six months' treatment (Mehta et al., 2006) and in an animal model of this disease no effect on tumour number has been observed (Lee et al., 2005). It may be that the previously reported impact of fish on controlling appetite and thus obesity may be an important factor in this disease (Borzoei et al., 2006).

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8.5 Possible importance of other nutritional aspects of fish consumption Combined results from both observational and laboratory-based studies suggest that fish oils may be the key protective factor related to fish consumption and gastrointestinal health. The epidemiology does not, however, effectively assess the types of fish eaten and this may be important as the n-3 content of the fish may not be the only important component in relation to risk of disease development. For example, fish is also known to be a good source of bioavailable selenium (Fox et al., 2004) and oily fish is also the major source of vitamin D when exposure to sunlight is limited. Both these factors have been shown to be protective in relation to colorectal cancer (Donaldson, 2004) and enhancement of levels within seafood may improve the functionality of such products. There is another important question in this context. That is to ask how other aspects of diet might interact with the effects of fish. For example, from the EPIC study we might consider the interactions with fibre and meat content of the diet. Also epidemiological studies suggest that vegetable consumption is important in relation to colorectal cancer risk and more specific mechanistic studies suggest that cruciferous vegetable such as cabbage, Brussel sprouts and broccoli may be particularly important in this respect (Lund, 2003, Lynn et al., 2006). Indeed we know that the most bioactive ingredient found solely in this group of vegetables can reduce cell number of colorectal cancer cells by different mechanisms to those mediated via fish oils (Smith et al., 2005) and thus one might speculate that there may be potential to develop seafood-based products containing both components which could have additional or even synergistic effects. Such products would be predicted to provide wider benefit than just those associated with gastrointestinal health, as these foods have also been shown to be potentially protective in relation to other cancers, particularly prostate (Chan et al., 2005).

8.6

The FISHGASTRO study

Until now there have been no systematic human intervention studies with fish to look at the effect of increased intake on aspects of gastrointestinal health. Although observational studies can provide us with evidence to suggest fish might be protective, they cannot provide proof of an effect. Also by undertaking an intervention study the researcher can begin to look at what particular aspects of fish consumption might be important and begin to address specific mechanisms within the context of a physiological dose. We therefore decided to undertake such a study within the SEAFOODplus project to address these issues, focused on colorectal health. The FISHGASTRO intervention involved feeding volunteers with lean fish (cod) and oil-rich fish (salmon) and comparing changes in a range of end-points with those in people just given the standard general nutritional advice given in the country in which the volunteer is resident. For this study it was planned that 270 patients due to be examined by

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colonoscopy would be recruited from local hospitals in The Netherlands and United Kingdom; to date 240 volunteers have been recruited. The volunteers were taken from three major patient groups: those with no visible disease at colonoscopy, people who had previously had polyps removed and those with the inflammatory bowel disease, ulcerative colitis. They were then randomly assigned to one of three six-month interventions: control (no additional fish provided), cod (300 g/week) or salmon (300 g/week). All participants have been given standard dietary advice given in the UK or Netherlands. Between eight and ten biopsies were collected at the start and end of the trial and dietary intake assessed by food frequency questionnaires and 7-day food diaries at the start and end of the study. Blood and faecal samples were also collected. The main end-point of the study is the number of apoptotic cells per crypt, a known risk marker for colorectal tumour which is inversely related to risk. Crypt length, cell proliferation and distribution of mitoses within the crypts are also being assessed as known risk markers used in previous fish oil intervention trials. Biopsies are also being used to develop novel markers of tumour risk based on gene expression analysis using a combination of micro-array analysis and real-time RT-PCR of a selected panel of genes selected from the initial array based transcriptomic assessment. One approach to assessing the affect of diet on a specific tissue is to measure DNA damage by the `COMET' assay (McKelveyMartin et al., 1993). This method has previously been successfully applied to cells isolated from rectal tissue (Pool-Zobel et al., 2004) and therefore biopsies from a sub-set of patients in the study have been analyzed by this method to see if fish consumption can protect against colorectal damage. As collection of biopsy tissue is very invasive, faecal samples are also being collected for the development of markers of risk which we expect to be considerably more accurate than the currently available faecal occult blood tests. For example, addition of the water phase from faecal samples to cultured colorectal cells followed by an assessment of DNA damage using the COMET assay has been shown to predict tumour outcome in animal studies (Klinder et al., 2004). The faecal water samples from the FISHGASTRO study are therefore being examined by this group as an important part of the assessment of effect of fish consumption on risk. Blood samples have been collected for the purposes of assessing potential confounding factors such as vitamin D status and selenium intake as well as to measure plasma fatty acids and levels of circulating inflammatory signalling molecules. We are hopeful that such approaches, in combination with other studies, will allow the development of more accurate predictions of risk and which factors are amenable to dietary manipulation. Results from this highly novel multinational parallel intervention study, using a real food, should become available from 2008 onward. Longer-term, more detailed investigation into the impact of fish consumption on actual disease outcomes would be desirable but, as previously alluded to, the costs of undertaking intervention studies of sufficient scale in terms of numbers of participants over the required timescale of probably 10±20 years would be prohibitively expensive and in reality such assessments will only be achievable

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using improved methodologies of dietary assessment in large cohort studies similar to the EPIC study. Should future data continue to support a beneficial role for seafood in the diet in respect to gastrointestinal health then the exact form that would provide most benefit would need to be elucidated. For example, how important are the n-3 fatty acids, is the amino-acid content of the protein important and would the production of selenium enriched fish in aquaculture be beneficial?

8.7

Summary

The concept that fish consumption may play a positive role in relation to gastrointestinal health and cancer prevention has received increasing attention in recent years. Although the epidemiological data is currently equivocal, more recent studies have provided more convincing evidence of benefit. Observational studies have been, and to a lesser extent still are, limited by the quality of data in relation to: · the lack of variance in the amount of fish consumed within a particular study population · questionnaire design and thus limitations in discriminating between different types of fish and whether preserved or fresh, etc. · limitations in accuracy in relation to dietary recall especially in case-control studies considering diet of more than one decade · the impact of genetic polymorphisms on response to fish consumption. The health benefits of eating fish are usually attributed to the unusual fatty acid content but fish have also been shown to be a good, well-absorbed source, of selenium (Fox et al., 2004). There is some evidence that fish consumption is associated with reduced adiposity and oil-rich fish are also an important source of vitamin D for people with inadequate exposure to sunlight. All these factors are likely to be protective in relation to prevention of cancers of the gastrointestinal tract. Human, animal and in vitro studies support the hypothesis that n3 fatty acids may be protective in relation to colorectal cancer and a range of potential mechanisms of action have been proposed. For example, by increasing the ratio of n-3 to n-6 fatty acids in tissues, fish may increase apoptosis and reduce hyper-proliferation of epithelial cells. Inflammation has the opposite effect on the colonocytes and fish oil may counteract this directly, as has been reported in cell culture, or by modifying the behaviour of the lymphocytes within the mucosal tissue. Fish oil has been shown to modify cellular responses by: acting as a ligand for transcription factors and changing gene transcription, changing the redox state of the cell and so potentially changing the behaviour of a wide range of enzymes, and thirdly by altering membrane fluidity due to its unusual physical properties. Members of the FISHGASTRO team from the UK, Netherlands and Germany are currently undertaking a very novel human intervention study,

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combined with a number of in-vitro tests, to investigate whether adding moderate amounts of fish to the human diet can reproduce the effects previously reported for fish oil supplements in relation to health of the large bowel and whether potential benefit is solely associated with the consumption of oil-rich fish such as salmon. As part of this study we are assessing the impact of fish in the diet on circulating inflammatory markers and on selenium status. Such information may lead to the development of new methods of aquaculture to produce fish providing greater health benefits or the formulation of potentially beneficial fish products.

8.8

Acknowledgements

The authors would like to acknowledge the following contributions made toward the FISHGASTRO study: funding from SEAFOODplus and The Food Standards Agency (UK), salmon was provided by Marine Harvest and cod by Pescanova. We would like to thank our numerous clinical collaborators in both The Netherlands and UK and all our scientific collaborators at The Institute of Food Research, Norwich, UK; Wageningen University and Research Centre, The Netherlands and The Friedrich Schiller University of Jena, Germany. In particular we would like to mention the excellent input of Anouk Geelen, Nina Habermann, Linda Harvey, Gosia Majsak-Newman, Beatrice Pool-Zobel, and Gerda Pot.

8.9

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and GALCERAN, M. T. (2004) Occurrence of heterocyclic amines in several home-cooked meat dishes of the Spanish diet. J Chromatogr B Analyt Technol Biomed Life Sci, 802, 79±86. CALDER, P. C., DAVIS, J., YAQOOB, P., PALA, H., THIES, F. and NEWSHOLME, E. A. (1998) Dietary fish oil suppresses human colon tumour growth in athymic mice. Clin Sci (Colch), 94, 303±11. BUSQUETS, R., BORDAS, M., TORIBIO, F., PUIGNOU, L.

CAMUESCO, D., GALVEZ, J., NIETO, A., COMALADA, M., RODRIGUEZ-CABEZAS, M. E., CONCHA, A., XAUS, J. and ZARZUELO, A. (2005) Dietary olive oil supplemented with fish oil, rich in EPA and DHA (n-3) polyunsaturated fatty acids, attenuates colonic inflammation in rats with DSS-induced colitis. J Nutr, 135, 687±94. CHAN, J. M., GANN, P. H. and GIOVANNUCCI, E. L. (2005) Role of diet in prostate cancer development and progression. J Clin Oncol, 23, 8152±60. CHANG, W. C., CHAPKIN, R. S. and LUPTON, J. R. (1997) Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis, 18, 721±30. CHAPKIN, R. S., DAVIDSON, L. A., LY, L., WEEKS, B. R., LUPTON, J. R. and MCMURRAY, D. N. (2007) Immunomodulatory Effects of (n-3) Fatty Acids: Putative Link to Inflammation and Colon Cancer. J Nutr, 137, 200S±204S. CHEN, C. S., PIGNATELLI, B., MALAVEILLE, C., BOUVIER, G., SHUKER, D., HAUTEFEUILLE, A.,

and BARTSCH, H. (1992) Levels of direct-acting mutagens, total Nnitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China. Mutat Res, 265, 211±21.

ZHANG, R. F.

CHEN, H., WARD, M. H., GRAUBARD, B. I., HEINEMAN, E. F., MARKIN, R. M., POTISCHMAN, N. A.,

and TUCKER, K. L. (2002) Dietary patterns and adenocarcinoma of the esophagus and distal stomach. Am J Clin Nutr, 75, 137±44.

RUSSELL, R. M., WEISENBURGER, D. D.

CHENG, J., OGAWA, K., KURIKI, K., YOKOYAMA, Y., KAMIYA, T., SENO, K., OKUYAMA, H., WANG, J., LUO, C., FUJII, T., ICHIKAWA, H., SHIRAI, T. and TOKUDOME, S. (2003) Increased intake of n-3 polyunsaturated fatty acids elevates the level of apoptosis in the normal sigmoid colon of patients polypectomized for adenomas/tumors. Cancer Lett, 193, 17±24. CHIU, B. C., JI, B. T., DAI, Q., GRIDLEY, G., MCLAUGHLIN, J. K., GAO, Y. T., FRAUMENI, J. F., JR. and CHOW, W. H. (2003) Dietary factors and risk of colon cancer in Shanghai, China. Cancer Epidemiol Biomarkers Prev, 12, 201±8. DOLL, R. and PETO, R. (1981) The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst, 66, 1191±308. DONALDSON, M. S. (2004) Nutrition and cancer: a review of the evidence for an anti-cancer diet. Nutr J, 3, 19. FAN, Y. Y., SPENCER, T. E., WANG, N., MOYER, M. P. and CHAPKIN, R. S. (2003) Chemopreventive n-3 fatty acids activate RXRalpha in colonocytes. Carcinogenesis, 24, 1541±8. FERNANDEZ, E., CHATENOUD, L., LA VECCHIA, C., NEGRI, E. and FRANCESCHI, S. (1999) Fish consumption and cancer risk. Am J Clin Nutr, 70, 85±90.

FOX, T. E., VAN DEN HEUVEL, E. G., ATHERTON, C. A., DAINTY, J. R., LEWIS, D. J., LANGFORD, N. J., CREWS, H. M., LUTEN, J. B., LORENTZEN, M., SIELING, F. W., VAN AKEN-SCHNEYDER, P.,

and FAIRWEATHER-TAIT, S. J. (2004) Bioavailability of selenium from fish, yeast and selenate: a comparative study in humans using stable isotopes. Eur J Clin Nutr, 58, 343±9.

HOEK, M., KOTTERMAN, M. J., VAN DAEL, P.

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and DECARLI, A. (1997) Food groups and risk of colorectal cancer in Italy. Int J Cancer, 72, 56±61.

NANNI, O.

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GEELEN, A., SCHOUTEN, M., KAMPHUIS, C., STAM, B., BUREMA, J., RENKEMA, J., BAKKER, E.-J.,

and KAMPMAN, E. (2007) Fish consumption, n-3 fatty acids and colorectal cancer: a meta-analysis of prospective cohort studies. Am J Epidem, 166, 1116±25. GIACOSA, A., FRANCESCHI, S., LA VECCHIA, C., FAVERO, A. and ANDREATTA, R. (1999) Energy intake, overweight, physical exercise and colorectal cancer risk. Eur J Cancer Prev, 8 Suppl 1, S53±60. GISSI (1999) Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto miocardico. Lancet, 354, 447±55. GUPTA, R. A., TAN, J., KRAUSE, W. F., GERACI, M. W., WILLSON, T. M., DEY, S. K. and DUBOIS, R. N. (2000) Prostacyclin-mediated activation of peroxisome proliferator-activated receptor delta in colorectal cancer. Proc Natl Acad Sci USA, 97, 13275±80. HAENSZEL, W., BERG, J. W., SEGI, M., KURIHARA, M. and LOCKE, F. B. (1973) Large-bowel cancer in Hawaiian Japanese. J Natl Cancer Inst, 51, 1765±79. JANSSON, E. A., ARE, A., GREICIUS, G., KUO, I. C., KELLY, D., ARULAMPALAM, V. and PETTERSSON, S. (2005) The Wnt/beta-catenin signaling pathway targets PPARgamma activity in colon cancer cells. Proc Natl Acad Sci USA, 102, 1460±5. JOHNSON, I. T. (2004) New approaches to the role of diet in the prevention of cancers of the alimentary tract. Mutat Res, 551, 9±28. KATO, I., AKHMEDKHANOV, A., KOENIG, K., TONIOLO, P. G., SHORE, R. E. and RIBOLI, E. (1997) Prospective study of diet and female colorectal cancer: the New York University Women's Health Study. Nutr Cancer, 28, 276±81. KERR, J. F. R., WYLLIE, A. H. and CURRIE, A.R. (1972) Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer, 26, 239±57. KEY, T. J., ALLEN, N. E., SPENCER, E. A. and TRAVIS, R. C. (2002) The effect of diet on risk of cancer. Lancet, 360, 861±8. KLINDER, A., FORSTER, A., CADERNI, G., FEMIA, A. P. and POOL-ZOBEL, B. L. (2004) Fecal water genotoxicity is predictive of tumor-preventive activities by inulin-like oligofructoses, probiotics (Lactobacillus rhamnosus and Bifidobacterium lactis), and their synbiotic combination. Nutr Cancer, 49, 144±55. KUNE, S., KUNE, G. A. and WATSON, L. F. (1987) Case-control study of dietary etiological factors: the Melbourne Colorectal Cancer Study. Nutr Cancer, 9, 21±42. KURATKO, C. N. (2000) Proliferation of colonic lymphocytes in response to inflammatory cytokines is lower in mice fed fish oil than in mice fed corn oil. CANCER LETTERS USA Texas Tech Univ, Hlth Sci Ctr, Dept Pathol, Lubbock, TX 79430 USA, 148, 27±32. LARSSON, S. C., KUMLIN, M., INGELMAN-SUNDBERG, M. and WOLK, A. (2004) Dietary longchain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms. Am J Clin Nutr, 79, 935±45. LATHAM, P., LUND, E. K. and JOHNSON, I. T. (1998) Modulation of colonocyte proliferation and apoptosis by dietary fish oil in experimental colorectal carcinogenesis. Biochem Soc Trans, 26, S158. LATHAM, P., LUND, E. K. and JOHNSON, I. T. (1999) Dietary n-3 PUFA increases the apoptotic response to 1,2-dimethylhydrazine, reduces mitosis and suppresses the induction of carcinogenesis in the rat colon. Carcinogenesis, 20, 645±50. LATHAM, P., LUND, E. K., BROWN, J. C. and JOHNSON, I. T. (2001) Effects of cellular redox balance on induction of apoptosis by eicosapentaenoic acid in HT29 colorectal VAN'T VEER, P.

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LEE, G., MEHTA, S., SAMS, V., SEERS, J., SMITH, T., BELSHAW, N., DOLEMAN, J., HINDMARSH, A.,

JOHNSON, I., RHODES, M. and LUND, E. (2005) Impact of aspirin and fish oil on development of esophageal adenocarcinoma in a rat surgical model assessed microscopically and by gene expression, Cpg island methylation and western blot Gastroenterology, 128 (4), A22±A23. LEE, J. Y. and HWANG, D. H. (2002) Docosahexaenoic acid suppresses the activity of peroxisome proliferator-activated receptors in a colon tumor cell line. Biochem Biophys Res Commun, 298, 667±74. LEFTON, H. B., PILCHMAN, J. and HARMATZ, A. (1996) Colon cancer screening and the evaluation and follow-up of colonic polyps. Prim Care, 23, 515±23. LIU, G., BIBUS, D. M., BODE, A. M., MA, W. Y., HOLMAN, R. T. and DONG, Z. (2001) Omega 3 but not omega 6 fatty acids inhibit AP-1 activity and cell transformation in JB6 cells. Proc Natl Acad Sci USA, 98, 7510±15. LOFTUS, E. V., JR. (2004) Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences. Gastroenterology, 126, 1504±17. LOKTIONOV, A., WATSON, M. A., GUNTER, M., STEBBINGS, W. S., SPEAKMAN, C. T. and BINGHAM, S. A. (2001) Glutathione-S-transferase gene polymorphisms in colorectal cancer patients: interaction between GSTM1 and GSTM3 allele variants as a riskmodulating factor. Carcinogenesis, 22, 1053±60. LUND, E. (2003) Non-nutritive bioactive constituents of plants: dietary sources and health benefits of glucosinolates. Int J Vitam Nutr Res, 73, 135±43. LUND, E. (2006) Dietary fatty acids and colon cancer. Scand J Food Nutrition, 50, 39±44. LYNCH, H. T. and DE LA CHAPELLE, A. (1999) Genetic susceptibility to non-polyposis colorectal cancer. J Med Genet, 36, 801±18. LYNN, A., COLLINS, A., FULLER, Z., HILLMAN, K. and RATCLIFFE, B. (2006) Cruciferous vegetables and colo-rectal cancer. Proc Nutr Soc, 65, 135±44. MACLEAN, C. H., MOJICA, W. A., NEWBERRY, S. J., PENCHARZ, J., GARLAND, R. H., TU, W., HILTON,

and MORTON, S. C. (2005) Systematic review of the effects of n-3 fatty acids in inflammatory bowel disease. Am J Clin Nutr, 82, 611±19. L. G., GRALNEK, I. M., RHODES, S., KHANNA, P.

MACLEAN, C. H., NEWBERRY, S. J., MOJICA, W. A., KHANNA, P., ISSA, A. M., SUTTORP, M. J., LIM, Y.

and MORTON, S. C. (2006) Effects of omega3 fatty acids on cancer risk: a systematic review. Jama, 295, 403±15. MAGEE, E. A., EDMOND, L. M., TASKER, S. M., KONG, S. C., CURNO, R. and CUMMINGS, J. H. (2005) Associations between diet and disease activity in ulcerative colitis patients using a novel method of data analysis. Nutr J, 4, 7. MALFOY, B. (2000) The revival of DNA methylation. J Cell Sci, 113, 3887±8. MANJARI, V. and DAS, U. N. (2000) Effect of polyunsaturated fatty acids on dexamethasoneinduced gastric mucosal damage. Prostaglandins Leukot Essent Fatty Acids, 62, 85±96. W., TRAINA, S. B., HILTON, L., GARLAND, R.

MARTIN, C., CONNELLY, A., KEKU, T. O., MOUNTCASTLE, S. B., GALANKO, J., WOOSLEY, J. T.,

and SANDLER, R. S. (2002) Nonsteroidal anti-inflammatory drugs, apoptosis, and colorectal adenomas. Gastroenterology, 123, 1770±7. MCKELVEY-MARTIN, V. J., GREEN, M. H., SCHMEZER, P., POOL-ZOBEL, B. L., DE MEO, M. P. and COLLINS, A. (1993) The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res, 288, 47±63. MEHTA, S., BODDY, A., COOK, J., LUND, E., SAMS, V., JOHNSON, I. and RHODES, M. (2006) A randomised study of the effects of n-3 fatty acid supplementation on COX-2 SCHLIEBE, B., LUND, P. K.

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expression in Barrett's epithelium. Gastroenterology, 130 (Supp 2), A180±181. and GUNN, A. (2001) Colonic crypt cell proliferation state assessed by whole crypt microdissection in sporadic neoplasia and familial adenomatous polyposis. Gut, 48, 41±6.

MILLS, S. J., MATHERS, J. C., CHAPMAN, P. D., BURN, J.

MURTAUGH, M. A., MA, K. N., CAAN, B. J., SWEENEY, C., WOLFF, R., SAMOWITZ, W. S., POTTER, J. D.

and SLATTERY, M. L. (2005) Interactions of peroxisome proliferator-activated receptor gamma and diet in etiology of colorectal cancer. Cancer Epidemiol Biomarkers Prev, 14, 1224±9. MURTAUGH, M. A., SWEENEY, C., MA, K. N., POTTER, J. D., CAAN, B. J., WOLFF, R. K. and SLATTERY, M. L. (2006) Vitamin D receptor gene polymorphisms, dietary promotion of insulin resistance, and colon and rectal cancer. Nutr Cancer, 55, 35±43. NAKAMURA , Y., NISHISHO, I., KINZLER, K., VOGELSTEIN, B., MIYOSHI, Y., MIKI, Y., ANDO, H. and HORII, A. (1992) Mutations of the APC (Adenomatous Polyposis Coli) Gene in FAP (Familial Polyposis Coli) Patients and in Sporadic Colorectal Tumors,. J Exp Med, 168, 141±7. NEGRI, E., BOSETTI, C., LA VECCHIA, C., FIORETTI, F., CONTI, E. and FRANCESCHI, S. (1999) Risk factors for adenocarcinoma of the small intestine. Int J Cancer, 82, 171±4. NG, Y., BARHOUMI, R., TJALKENS, R. B., FAN, Y. Y., KOLAR, S., WANG, N., LUPTON, J. R. and CHAPKIN, R. S. (2005) The role of docosahexaenoic acid in mediating mitochondrial membrane lipid oxidation and apoptosis in colonocytes. Carcinogenesis, 26, 1914± 21. NIHO, N., TAKAHASHI, M., KITAMURA, T., SHOJI, Y., ITOH, M., NODA, T., SUGIMURA, T. and WAKABAYASHI, K. (2003) Concomitant suppression of hyperlipidemia and intestinal polyp formation in Apc-deficient mice by peroxisome proliferator-activated receptor ligands. Cancer Res, 63, 6090±5. NORAT, T., BINGHAM, S., FERRARI, P., SLIMANI, N., JENAB, M., MAZUIR, M., OVERVAD, K., OLSEN, A., TJONNELAND, A., CLAVEL, F., BOUTRON-RUAULT, M. C., KESSE, E., BOEING, H., BERGMANN, M. M., NIETERS, A., LINSEISEN, J., TRICHOPOULOU, A., TRICHOPOULOS, D., TOUNTAS, Y., BERRINO, F., PALLI, D., PANICO, S., TUMINO, R., VINEIS, P., BUENO-DEMESQUITA, H. B., PEETERS, P. H., ENGESET, D., LUND, E., SKEIE, G., ARDANAZ, E., GONZALEZ, C., NAVARRO, C., QUIROS, J. R., SANCHEZ, M. J., BERGLUND, G., MATTISSON, I., HALLMANS, G., PALMQVIST, R., DAY, N. E., KHAW, K. T., KEY, T. J., SAN JOAQUIN, M.,

and RIBOLI, E. (2005) Meat, fish, and colorectal cancer risk: the European Prospective Investigation into cancer and nutrition. J Natl Cancer Inst, 97, 906±16. NOWAK, J., WEYLANDT, K. H., HABBEL, P., WANG, J., DIGNASS, A., GLICKMAN, J. N. and KANG, J. X. (2007) Colitis-associated colon tumorigenesis is suppressed in transgenic mice rich in endogenous n-3 fatty acids. Carcinogenesis, 28, 1991±5. O'KEEFE S, J., CHUNG, D., MAHMOUD, N., SEPULVEDA, A. R., MANAFE, M., ARCH, J., ADADA, H. and VAN DER MERWE, T. (2007) Why do African Americans get more colon cancer than native Africans? J Nutr, 137, 175S±82S. OH, K., WILLETT, W. C., FUCHS, C. S. and GIOVANNUCCI, E. (2005) Dietary marine n-3 fatty acids in relation to risk of distal colorectal adenoma in women. Cancer Epidemiol Biomarkers Prev, 14, 835±41. PARKIN, D. M., WHELAN, S., FERLAY, J. and STORM, H. (2005) Cancer Incidence in Five Continents, Vol. I to VIII IARC CancerBase No. 7, Lyon. PAULSEN, J. E., ELVSAAS, I. K., STEFFENSEN, I. L. and ALEXANDER, J. (1997) A fish oil derived concentrate enriched in eicosapentaenoic and docosahexaenoic acid as ethyl ester suppresses the formation and growth of intestinal polyps in the Min mouse. HEMON, B., SARACCI, R., KAAKS, R.

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and MAHANTA, J. (2006) Dietary habits and stomach cancer in Mizoram, India. J Gastroenterol, 41, 418±24. POOL-ZOBEL, B. L., DORNACHER, I., LAMBERTZ, R., KNOLL, M. and SEITZ, H. K. (2004) Genetic damage and repair in human rectal cells for biomonitoring: sex differences, effects of alcohol exposure, and susceptibilities in comparison to peripheral blood lymphocytes. Mutat Res, 551, 127±34. RAO, C. V., HIROSE, Y., INDRANIE, C. and REDDY, B. S. (2001) Modulation of experimental colon tumorigenesis by types and amounts of dietary fatty acids. Cancer Res, 61, 1927±33. REDDY, B. S. and SUGIE, S. (1988) Effect of different levels of omega-3 and omega-6 fatty acids on azoxymethane-induced colon carcinogenesis in F344 rats. Cancer Res, 48, 6642±7. RHODES, J. M. and CAMPBELL, B. J. (2002) Inflammation and colorectal cancer: IBDassociated and sporadic cancer compared. Trends Mol Med, 8, 10±16. PHUKAN, R. K., NARAIN, K., ZOMAWIA, E., HAZARIKA, N. C.

ROHRMANN, S., LINSEISEN, J., BECKER, N., NORAT, T., SINHA, R., SKEIE, G., LUND, E., MARTINEZ, C., BARRICARTE, A., MATTISSON, I., BERGLUND, G., WELCH, A., DAVEY, G., OVERVAD, K., TJONNELAND, A., CLAVEL-CHAPELON, F., KESSE, E., LOTZE, G., KLIPSTEIN-GROBUSCH, K., VASILOPOULOU, E., POLYCHRONOPOULOS, E., PALA, V., CELENTANO, E., BUENO-DE-

and SLIMANI, N. (2002) Cooking of meat and fish in Europe±results from the European Prospective Investigation into Cancer and Nutrition (EPIC). Eur J Clin Nutr, 56, 1216±30. ROYNETTE, C. E., CALDER, P. C., DUPERTUIS, Y. M. and PICHARD, C. (2004) n-3 polyunsaturated fatty acids and colon cancer prevention. Clin Nutr, 23, 139±51. MESQUITA, H. B., PEETERS, P. H., RIBOLI, E.

SAKAMOTO, N., KONO, S., WAKAI, K., FUKUDA, Y., SATOMI, M., SHIMOYAMA, T., INABA, Y., MIYAKE, Y., SASAKI, S., OKAMOTO, K., KOBASHI, G., WASHIO, M., YOKOYAMA, T., DATE, C.

and TANAKA, H. (2005) Dietary risk factors for inflammatory bowel disease: a multicenter case-control study in Japan. Inflamm Bowel Dis, 11, 154±63. SHIMADA, T., KOJIMA, K., YOSHIURA, K., HIRAISHI, H. and TERANO, A. (2002) Characteristics of the peroxisome proliferator activated receptor gamma (PPARgamma) ligand induced apoptosis in colon cancer cells. Gut, 50, 658±64. SIEZEN, C. L., VAN LEEUWEN, A. I., KRAM, N. R., LUKEN, M. E., VAN KRANEN, H. J. and KAMPMAN, E. (2005) Colorectal adenoma risk is modified by the interplay between polymorphisms in arachidonic acid pathway genes and fish consumption. Carcinogenesis, 26, 449±57. SIEZEN, C. L., TIJHUIS, M. J., KRAM, N. R., VAN SOEST, E. M., DE JONG, D. J., FODDE, R., VAN

KRANEN, H. J. and KAMPMAN, E. (2006) Protective effect of nonsteroidal antiinflammatory drugs on colorectal adenomas is modified by a polymorphism in peroxisome proliferator-activated receptor delta. Pharmacogenet Genomics, 16, 43±50. SMITH, T. K., LUND, E. K., CLARKE, R. G., BENNETT, R. N. and JOHNSON, I. T. (2005) Effects of Brussels sprout juice on the cell cycle and adhesion of human colorectal carcinoma cells (HT29) in vitro. J Agric Food Chem, 53, 3895±901. TAPIERO, H., BA, G. N., COUVREUR, P. and TEW, K. D. (2002) Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies. Biomed Pharmacother, 56, 215±22. VOGELSTEIN, B., FEARON, E. R., HAMILTON, S. R., KERN, S. E., PREISINGER, A. C., LEPPERT, M., NAKAMURA, Y., WHITE, R., SMITS, A. M. and BOS, J. L. (1988) Genetic alterations during colorectal-tumor development. N Engl J Med, 319, 525±32.

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and ROTONDO, D. (2004) Conjugated linoleic acids: are they beneficial or detrimental to health? Prog Lipid Res, 43, 553±87. WELCH, A. A., BINGHAM, S. A., IVE, J., FRIESEN, M. D., WAREHAM, N. J., RIBOLI, E. and KHAW, K. T. (2006) Dietary fish intake and plasma phospholipid n-3 polyunsaturated fatty acid concentrations in men and women in the European Prospective Investigation into Cancer-Norfolk United Kingdom cohort. Am J Clin Nutr, 84, 1330±9. YANG, C. X., TAKEZAKI, T., HIROSE, K., INOUE, M., HUANG, X. E. and TAJIMA, K. (2003) Fish consumption and colorectal cancer: a case-reference study in Japan. Eur J Cancer Prev, 12, 109±15. YANG, W. L. and FRUCHT, H. (2001) Activation of the PPAR pathway induces apoptosis and COX-2 inhibition in HT-29 human colon cancer cells. Carcinogenesis, 22, 1379± 83. YEH, C. C., HSIEH, L. L., TANG, R., CHANG-CHIEH, C. R. and SUNG, F. C. (2003) Risk factors for colorectal cancer in Taiwan: a hospital-based case-control study. J Formos Med Assoc, 102, 305±12. ZARIDZE, D. G. and FILIPCHENKO, V. V. (1991) [The role of nutrition in the etiology of cancer of the large intestine]. Vopr Onkol, 37, 152±8. WAHLE, K. W., HEYS, S. D.

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9 Fish consumption and the health of children and young adults I. Thorsdottir and A. Ramel, University of Iceland, Iceland

9.1

Introduction

A strategic objective of the SEAFOODplus Integrated Program, supported through the 6th framework program of the EU, is to reduce health problems and to increase well-being among European consumers by applying the benefits obtained through consumption of health-promoting and safe seafood products of high eating quality. Health of young European families and fish consumption (YOUNG) is one of the 20 projects of SEAFOODplus and one of the three nutrition projects. The idea of YOUNG is on the one hand related to suggested beneficial physiological effects of seafood, some of which have to be investigated more thoroughly before used in advice to people, and on the other hand on the public health concern about increasing poor health of young families in Europe, as all over the world, related to the increasing prevalence of overweight and obesity at younger age than earlier seen. Seafood is rich in long chain n-3 fatty acids which are known for their effects in the prevention of diseases associated with overweight and obesity. Recent results from YOUNG indicate that seafood might also themselves directly be important in the battle against overweight and obesity. SEAFOODplus YOUNG Intervention is a large, carefully controlled dietary intervention trial in Iceland, Ireland and Spain, investigating young overweight and obese adults, a high risk group for metabolic complications, and their possibilities for control of body weight, blood lipids and better health, when eating healthy weight loss diets varying in seafoods or fish oil supplement. SEAFOODplus YOUNG is the first intervention trial assessing the effects of lean fish, fatty fish and fish oil versus a control diet without any seafood. The

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originality in the design of SEAFOODplus YOUNG is that no other study in humans has ever tried to distinguish between consumption of lean and fatty fish, or to compare intake of lean fish versus meat, during weight loss. Countries throughout the world experienced a marked increase in the prevalence of overweight and obese children and young adults from the 1980s to 1990s and evidence suggests that this upward trend has continued in the 21st century.1±13 The high prevalence of overweight and obesity in children and young adults could be explained by any of the factors that influence energy intake, through diet, or energy expenditure, through physical activity. A change in dietary patterns in recent decades, including an increased consumption of soft drinks and candy and a decreased consumption of fruits and vegetables in many countries, and probably also the reduction of the intake of n-3 fatty acids have been associated with the increase in childhood and young adult obesity.14±16 Other constituents of seafood might also influence weight control. An early study also showed that taurine, an amino acid found in seafood, decreases body weight in hyperglycemic obese mice,14 and ingestion of 3 g taurine per day for seven weeks has been found to be associated with weight loss in humans. Obesity has become a serious worldwide healthcare problem becoming increasingly prevalent among young adults and children.1,2 It is therefore of great importance to help young overweight adults to lose weight and adopt appropriate eating habits and physical activity. It is important for their own health and also their children's as lifestyle factors are at least partly learned from parents. In recent years it has become clear that seafood lipids can be effective in the prevention of chronic nutrition-related diseases, e.g. cardiovascular and inflammatory diseases. The major causes of premature morbidity and mortality in Europe are cardiovascular disease (approx. 40%) and cancer (approx. 25%). Epidemiological studies provide convincing evidence that seafood consumption is related to improved health and reduced risk of chronic diseases. It is generally assumed that the most pronounced effect is due to the n-3 fatty acids, even though not certain. Pollutants possibly following seafood are many times more likely to be found in or with lipids than with other nutrients. Therefore there is an increasing interest in studies exploring intake of varying amounts of marine lipids to define lower and upper limits and the optimal intake. A beneficial action of fish protein, i.e. cod protein, on insulin sensitivity has been observed in experimental animals, but effects in humans have not been investigated.17 It is possible that fish protein and other seafood constituents are responsible for other health effects of seafood. Age-related loss of bone mass and bone fragility are major risk factors for osteoporosis, leading to an increased risk of fractures representing a significant public health problem. Experimental, epidemiological and clinical evidence suggests that n-3 fatty acids may have beneficial effects on bone metabolism in animals and man.18±20 There is some evidence of a negative association between fish intake or n-3 fatty acids and general depression.21 Symptoms and characteristics of postpartum depression are much like those for general depression. Seen in this light, an association between postpartum depression and fish intake

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seems plausible. Postpartum depression afflicts 5±20% of childbearing women and can have serious consequences for the mother and the infant. A recent crossnational ecological analysis indicated that occurrences of postpartum depression correlate inversely with seafood consumption and content of DHA in breast milk across populations.22 However, the contention needs to be tested more rigorously in more powerful epidemiological designs among pregnant women. In this chapter we describe the current status of knowledge on effects of seafood on health. The focus is on health of YOUNG families as studied in the project SEAFOODplus Integrated Program, i.e. overweight and obesity, blood lipids, pregnancy outcomes and pregnancy complications, bone health and postpartum depression. In the last part of the chapter we discuss how to communicate nutritional effects of fish to young adults, the development of functional fish products to improve health, and decribe future trends for fishand health-related issues.

9.2

Effect of fish consumption on obesity

Obesity is a serious and worldwide problem related to premature death and diseases, e.g. type 2 diabetes, hypertension and atherosclerosis. Inclusion of fish in a weight-loss diet has been shown to have positive effects on several healthrelated variables23,24 which could be due to n-3 fatty acids or other seafood constituents such as fish proteins, as has been reported in animal studies.25±27 Beneficial effects of fish consumption in relation to cardiovascular health have been thoroughly described, and mainly attributed to n-3 fatty acids.28±31 Studies in rodents have demonstrated that marine n-3 fatty acids enriched diet decreases adipose growth and increase beta-oxidation.32,33 Additionally, taurine, an amino acid abundant in fish protein, has been suggested to decrease body weight.34 The effects of seafood, n-3 fatty acids and fish protein on weight control in overweight and obese humans were studied for the first time in a large intervention trial in the project SEAFOODplus YOUNG. In this controlled, randomized intervention study an energy-restricted diet for eight weeks containing either lean fish, fatty fish or fish oil resulted in significantly more weight loss than an isocaloric diet without seafood.35 This is the first time seafood is shown to increase weight loss compared to an isocaloric non-seafood diet. The effect of lean fish as part of an energy-restricted diet is of specific interest as up to now the main hypothesis on the health benefits of fish consumption has been contingent on the effect of n-3 fatty acids. The study indicates that consuming a diet relatively low in n-3 fatty acids, but high in quality fish proteins gives similar results as a diet rich in n-3 dietary fatty acids and quality fish proteins. SEAFOODplus YOUNG contributes to a better understanding of seafood as a whole and indicates that n-3 fatty acids are not the only bioactive compounds of clinical relevance in seafood.35 If confirmed, this might be important public health information, especially since some populations consume more lean fish than fish rich in n-3 fatty acids.34,36 The intake during

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the intervention was verified by a validated specific food frequency questionnaire on seafood intake,37,38 additional to a three-day food registration at the beginning and end of the intervention period. The validation study was done on 56 subjects not involved in the intervention and ahead of the intervention study.37 The compliance study showed that at baseline the intake of seafood in Iceland was much lower among the overweight and obese young adults than in the general population.38 Importantly the study of compliance also showed that these young adults could easily comply with a high seafood diet. The significance of this study is that the beneficial effects of fish consumption on body weight could be demonstrated in a randomized, controlled intervention trial. An important novel finding of SEAFOODplus YOUNG are the effects of cod on weight loss. The comparison of a diet including cod with a diet including a non-fish protein source (meat), but with the same amount of protein, further underlines the novel findings. Additionally, the results from fish oil supplement and fatty fish (salmon, which includes both fish protein and fish fat) increase the knowledge in that field substantially. Seafood-derived n-3 fatty acids have been shown to decrease the growth of the adipose cell, probably through stimulated beta-oxidation.32,33,39 Recently, this finding was supported by a study on mice showing that the anti-adipogenic effect of eicosapentaenoic and docoxahexaenoic acids may involve a switch in adipocytes that includes increase in beta-oxidation and upregulation of mitochondrial biogenesis.40 Furthermore, a recent review by Madsen et al. also describes that n-3 fatty acids may affect adipocyte differentiation.41 N-3 polyunsaturated fatty acids decrease adipose tissue mass and the development of obesity in rodents by targeting a set of key regulatory transcription factors involved in adipogenesis and lipid homeostasis.41 The same set of factors are targeted by n-6 polyunsaturated fatty acids, but their effect seems to be dependent on feeding status and hormonal background and n-6 therefore react either as anti- or proadipogenic agents. 41 However, Garaulet et al. have recently shown, for the first time in humans, that n-3 and n-6 fatty acids are related to a reduced adipocyte size depending on tissue localization.42 A specific effect of seafood n-3 fatty acids may be responsible for an additional reduction in body weight during a weight loss diet, but this cannot explain the similarly effective lean fish diet, which had only slightly higher amounts of total n-3 than a control diet without seafood.35 Fish proteins may be the candidate for this effect. Inclusion of fish in a weight loss diet has been shown to have positive effects on several health-related variables in a study of 63 subjects,24,43 but to our knowledge the effects of seafood to increase weight loss in humans has not been seen before. It is indicated that there are components of fish, e.g. particular combinations of amino acids that may be beneficial to human health beyond the n-3 fatty acids or other bioactive constituents. A plausible candidate is taurine, an amino acid abundant in fish protein. An early study showed that taurine decreases body weight in hyperglycemic obese mice,34 and ingestion of 3 g taurine per day for seven weeks has been found to be associated with weight loss

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in humans.44 Additionally, a recent study found dietary taurine supplementation to prevent obesity in mice by increased resting energy expenditure.45 Further studies are needed to examine possible mechanisms. In addition, closer investigation of the effects of portion size and more frequent fish consumption as part of a varied and balanced diet is required. It can be assumed that the results obtained from SEAFOODplus YOUNG apply to other populations, considering that the difference in weight loss between diet groups was similar in all three participating countries. It remains a significant priority to help overweight individuals to lose weight. How the weight loss is brought about can make a difference to both total kg lost and to reduction of cardiovascular risk factors. The subjects in the recent study were 20±40 years old, which means that some are parents or are likely to have children in the near future.35 Parental influence on children's dietary habits is well known,46,47 and if fish consumption proves to be effective in assisting weight reduction and improving weight maintenance in humans, the 20±40 year age group is a key target group for dietary interventions aimed to increase fish intake. It can be concluded, that in young, overweight men, the inclusion of either lean or fatty fish, or fish oil as part of a hypoenergetic diet resulted in ~1 kg more weight loss after four weeks, than did a similar diet without seafood or supplement of marine origin. The addition of seafood to a nutritionally balanced energy-deficient diet may boost weight loss. In general, energy balance is the key to weight control and weight loss maintenance, and greater energy expenditure than energy intake is needed to achieve weight loss. However, dietary components from fish can assist in weight loss when necessary to prevent obesity complications and can facilitate body weight control in obesity management. In future bioactive fish components and their exact mechanisms leading to improved weight loss will be pinpointed facilitating well defined assistance in obesity therapy.

9.3

Effect of fish consumption on blood lipids

Different blood lipids have various effects on atherosclerosis and heart disease. The type of dietary fat and other nutrients can affect the concentrations of circulating blood lipids and thus influence development and progress of disease. The effects of seafood, n-3 fatty acids and fish protein on blood lipids in overweight and obese humans were studied for the first time in a large intervention trial in the project SEAFOODplus YOUNG. Countries with high intake of fish have been shown to have lower rates of coronary heart disease (CHD) compared to other countries. During the past 20 years several studies have demonstrated the beneficial effects of fish consumption.28,29,48 These findings are supported by two recent meta-analyses suggesting that fish intake may be an important component for the prevention of CHD.49,50 There are many properties in fish that may protect against CHD,51 n-3

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fatty acids being the most frequently studied. There is increasing concern that the ratio of the n-6/n-3 fatty acids in the diet in some European populations is too high, in the region of 12:1, where a desirable level is estimated to be nearer to 6:1, and by some scientists' suggestions even lower. By consuming more seafood this imbalance can be corrected. N-3 and n-6 fatty acids are precursors of eicosanoids (Fig. 9.1), hormone-like agents which affect inflammation, coagulation, vasodilation and body temperature, where n-3 and n-6 derived eicosanoids often have opposing effects. N-3 fatty acids have been shown to protect against arrhythmia, reduce platelet aggregation and thereby have an antithrombotic effect, and also to lower triglycerides in the blood.52±55 However, the benefits of n-3 fatty acids on cardiovascular health are still a debate.56 The triglyceride lowering effects of n-3 fatty acids are well documented.57 Hypertriglyceridemia is an important risk factor for coronary heart disease,58,59 and n3 fatty acids contribute to lower plasma triglyceride levels by inhibiting the synthesis of very-low-density lipoprotein cholesterol and decreasing hepatic triglycerides.57 Inclusion of fish in a weight loss diet and its effects have only been reported in one randomized controlled study,23,24 in which the beneficial effects of fish have primarily been attributed to the presence of n-3 fatty acids. The role of

Fig. 9.1

From omega-6 and omega-3 fatty acids to eicosanoids.

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other seafood constituents, such as fish proteins, has been studied in animals,29,48,60,61 but to a very limited degree in humans.62 The specific effects of seafood consumption on blood lipids during weight loss in young European overweight individuals have been investigated by the SEAFOOplus YOUNG project, comparing iso-caloric weight loss diets containing either lean fish, fatty fish or supplemental fish oil capsules, to a diet without seafood but including supplemental placebo capsules.63 Inclusion of fish or fish oil to a weight loss diet among young overweight or obese individuals resulted in greater reduction in triglyceride concentration and lowering of total cholesterol when compared with a group receiving diet without food of marine origin. However, the reduction of total cholesterol was partly due to reduction in HDL cholesterol in the group receiving relatively low amounts of n3 fatty acids.63 The hypotriglyceridemic effects of n-3 fatty acids apparent in that study, where the greatest reduction in triglyceride concentration was seen in the group receiving the largest amount of n-3 fatty acids and is therefore in accordance with the extensive literature on the triglyceride lowering effects of n-3 fatty acids.63 The novelty and significance of this SEAFOODplus YOUNG project is that the study suggests that consuming lean fish three times per week could lower triglyceride concentration to a similar degree as 1.5 g per day of n-3 fatty acids. The different components of fish contributing to cardiovascular health have been assessed in the rat. Cod protein has been shown to lower hepatic triglyceride concentration and triglyceride secretion rate in the rat when compared with casein, although without significantly affecting plasma triglyceride concentrations.64 In the SEAFOODplus YOUNG project proteins and lipids exerted main effects on several lipid variables but did not interact, indicating that their actions were independent of one another.64 Two to four grams of longchain n-3 fatty acids per day have been recommended for patients needing triglycerid lowering.65 High quality fish proteins could contribute to triglyceride lowering and it cannot be excluded that some of the triglyceride lowering effects of fish seen in previous studies could be attributable to the fish proteins, not only the n-3 fatty acids. The significance of SEAFOODplus YOUNG is related amongst others to the positive health effects of lean fish consumption, which are for the first time to be demonstrated in a large-scale human intervention trial. This is of main interest for the public, health professionals and the fish industry. Positive health effects of lean fish consumption are highly relevant for many populations and areas, e.g. the Baltic Sea area, where fatty fish has been found to be contaminated.66,67 Lean fish consumption may also be especially relevant in understanding how, e.g., Icelanders have protected their health, i.e., low rates of cardiovascular disease and type 2 diabetes ± where the lean fish certainly makes a difference (cod and haddock have been the main species consumed by the population). A recent systematic review on the effects of n-3 fatty acids on serum markers of cardiovascular disease risk show that the net effect on total cholesterol, LDL, and HDL cholesterol levels are small.68 In previous studies, fish proteins have

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been found to decrease plasma total cholesterol concentration in comparison with casein.25,27 Hypocholesterolemic effect of fish protein in rats64 may be caused by a decrease in liver cholesterol output into the circulation due to a stimulation of cholesterol to bile acid conversion and to increased excretion of cholesterol and its metabolites into feces,69 or reduced activity of Acyl-CoA: cholesterol acyltransferace (ACAT).70 This suggests that fish proteins might have cardioprotective effect and be involved in the regulation of plasma cholesterol. In a study by Wergedahl et al.70 the proportion of HDL cholesterol was increased in association with fish protein intake. Fish proteins have also been found to increase HDL in rabbits.71 In a recent systematic review of the literature it was suggested that fish oil may be most effective at raising (or stabilizing) HDL levels in people whose HDL levels would otherwise decrease with time.68 A favorable effect of intake of lean fish diet on HDL2 cholesterol has been seen,72 suggested to be attributed to the presence of small quantities of n-3 fatty acids in lean fish.73 In a 1994 study by Mori et al. 73 one daily fish meal (fatty fish) reversed the fall in HDL in comparison to a diet without fish. The fat content of the diets used in the present study was around 30% of total energy intake. Fall in HDL cholesterol is usual with a low-fat diet, around or less than 30E%.74,75 It seems therefore that n-3 fatty acids in combination with high quality fish proteins could be the best means to decrease cardiovascular risk during weight loss using diet relatively low in fat content. It seems that both high quality fish proteins and n-3 fatty acids experience the greatest overall beneficial effects with respect to blood lipid profile during weight loss. Perhaps it is rather unrealistic to advise consumption of fat fish, e.g. salmon, three times per week, but it is suggested that a variety of fish species, in combination with consumption of fish oil, should be consumed at least three times per week during weight loss. What the SEAFOODplus YOUNG taught us is that we should be careful when trying to substract fish constituents from their natural environment as our results suggest, supported by animal studies,64 that fish protein, in combination with fish oil, may contribute to the beneficial effects of fish consumption on the lipid profile, and perhaps better than one of the constituents alone. Future reseach must not focus on n-3 fatty acids alone, but will determine which combinations of bioactive fish components is most effective in improving blood lipid profile during weight loss.

9.4

Effect of fish consumption on maternal and child health

9.4.1 Effect of fish consumption on pregnancy and birth outcome According to the Barker hypothesis, several serious diseases in later life including coronary heart disease, hypertension and type 2 diabetes mellitus originate from impaired intrauterine growth and development, and may be the consequences of fetal programming.76 Intake of fish as well as fish liver oil supplements in pregnancy, have been positively related to babies' birth size,

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both in epidemiological and intervention studies.77±79 Birth weight of term infants in Iceland is high (3779 469 g) compared to other countries.80,81 It is possible that a higher mean birth weight in some populations, e.g. Iceland or Faroe Islands, than in the genetically related neighboring populations of the Nordic countries, might be related to higher fish consumption82,83 and/or to the large percentage of the population that take fish liver oil as a daily supplement. The association between smaller size at birth and adult metabolic disturbances such as glucose intolerance, hypertension and coronary artery disease84±87 gives this hypothesis an important dimension. This may be especially relevant because the prevalence of type 2 diabetes, systolic blood pressure and coronary heart disease are lower in Iceland than in the neighboring countries of genetically related populations.88±91 However, in contrast to earlier publications, a high intake of marine fats has recently been related to lower birth weight and length in the Faroe Islands.92 In an Icelandic cohort study, frequency of fish consumption was found to be positively related to both length and head circumference at birth (Table 9.1). Women with the lowest fish consumption had children of smaller size at birth than women consuming higher amounts, which is in accordance with earlier studies.77,78 However, length and head circumference at birth was lowest in the group consuming the highest amount of fish liver oil in pregnancy (Table 9.2). Olsen and co-workers found that the length of the newborn increased with the frequency of seafood dinner meals in pregnancy but only up to a consumption level of about three meals per week, or in another study, 15 g per day.77,78 This is in accordance with other research showing that consumption of >20 g of fish per Table 9.1 Mean birth size of infants born to women according to monthly frequency of consumption of fish as a main meal, both before and after adjustment,y Reykjavik, Iceland, 1998 Monthly consumption < 4 times 4±6 times > 6 times 12.8% 50.4% 36.8% Birth weight (g) Unadjusted Adjusted Birth length (cm) Unadjusted Adjusted Head circumference (cm) Unadjusted Adjusted Ponderal index (kg/m3) Unadjusted Adjusted

P-value

3,750 3,725

3,790 3,780

3,795 3,810

18 50

0.595 0.098

51.9 51.8

52.1 52.1

52.3 52.3

0.18 0.35

0.216 0.007*

35.7 35.6

36.0 36.0

36.0 36.1

0.13 0.24

0.162 0.005*

26.6 26.6

26.7 26.7

26.5 26.5

ÿ0.099 ÿ0.043

0.519 0.34

* p < 0:05. y Adjusted for weight gain in pregnancy, maternal height, parity, smoking, infant's gender, gestational length, and fish liver oil supplementation.

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Table 9.2 Mean birth size of infants born to women according to fish liver oil intake group,y both before and after adjustment,z Reykjavik, Iceland, 1998 1 55.7% Birth weight (g) Unadjusted 3,800 Adjusted 3,805 Birth length (cm) Unadjusted 52.2 Adjusted 52.3 Head circumference (cm) Unadjusted 36.1 Adjusted 36.1 Ponderal index (kg/m3) Unadjusted 26.6 Adjusted 26.6

Intake groups 2 3 14.6% 16.1%

4 13.6%

P-value

3,825 3,795

3,815 3,800

3,680 3,695

ÿ9 ÿ8

0.086 0.184

52.2 52.1

52.2 52.2

51.7 51.8

ÿ0.05 ÿ0.04

0.017* 0.036*

36.1 36.0

36.0 35.9

35.4 35.5

ÿ0.05 ÿ0.04

0.001* 0.003*

26.7 26.8

26.6 26.6

26.6 26.6

0.02 0.02

0.497 0.598

* p < 0:05. y Group 1: no intake; group 2: 0.1±0.7 g; group 3: 0.71±8.7 g; group 4: >8.7 g. z Adjusted for weight gain in pregnancy, maternal height, parity, smoking, infant's gender, gestational length, and fish consumption.

day does not result in an additional increase in birth size. Increase in birth size with fish consumption might be due to a higher intake of long chain omega-3 fatty acids,78 but might also be due to the eventual higher protein consumption or even protein composition.79 Recent studies suggest bioactivity of fish proteins, which might be of importance.25,27,79 As no relationship was found between fish consumption and weight gain in pregnancy, the higher birth weight of babies born to fish-consuming mothers, seems very unlikely to be due to a higher energy intake of fish consumers.80 Not all studies agree on the effect of seafood consumption on pregnancy and birth outcome. Most of the women taking in fish liver oil in pregnancy in this study had been supplementing before pregnancy, while in the intervention studies fish liver oil supplements to women of habitual low intake were commenced during pregnancy,93,94 which might partly explain the results. It is also possible that a large majority of the women in a fishing community are already consuming the minimum amount necessary of fish or fish products for the beneficial effect on gestational length and therefore this relationship is not found. It seems that supplementation with fish liver oil has a positive effect on birth size up to a certain point, among nations with low intake,78 but might have a negative effect on birth size at very high intake levels. The important contribution of our Icelandic cohort study is that it reflects a very wide range of intake levels, being able to detect effects of higher consumption of seafood or fish oil on birth outcome which can not be detected in other cohorts with low to medium intake. Whether fish oil itself or accompanying nutrients or possible pollutants

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are responsibe for this growth limiting effect, remains to be elucidated. Overall, the supplementation with fish oil during pregnancy should be regarded as beneficial, considering frequent suboptimal intakes of n-3 fatty acids and vitamin D in young women. 9.4.2 Effect of fish consumption on pregnancy and delivery complications Hypertensive disorders are the leading cause of morbidity and mortality in pregnancy. Furthermore, there is a tendency to more ischaemic heart disease deaths in women who have suffered hypertensive disorders in pregnancy. Thus, prevention of elevated blood pressure during pregnancy is not only a matter of prenatal health care but also important for women's health in general. Hypertensive disorders in pregnancy, including gestational hypertension and preeclampsia, are among the most common complications associated with pregnancy, affecting 5±10% of all pregnancies worldwide.95±99 Studies on fish liver oil focusing on intake of n-3 fatty acids, suggest a moderate beneficial effect on blood pressure in nonpregnant hypertensive as well as normotensive subjects. Imbalance between n-3 fatty acids (low) and n-6 fatty acids (high, particularly arachidonic acid) in erythrocytes has been suggested to lead to an increased risk of preeclampsia.100,101 An intervention study on pregnant women concluded that 2.7 g/day of marine n-3 fatty acids provided in the third trimester (from week 30) of normal pregnancy showed no effect on blood pressure.102 However, this study was rather short term and does not account for possible effects of fish oil consumption earlier in pregnancy and of long-term supplementation. Fishing communities with high consumption of both fish and intake of fish liver oil give an important opportunity to study whether these dietary factors are related to pregnancy and delivery complications. In two Icelandic cohorts an increased risk of gestational hypertension has been related to very low and very high intake of fish liver oil each day ( 1 tablespoon).99,103 Odds ratio for hypertensive disorders (P 0:008) and gestational hypertension (P 0:035) suggested a u-shaped curve with the odds ratio being lowest in the second and third quartiles. The lowest figure for frequency of hypertensive disorders was in the groups consuming between 0.1 and 0.9 g of n-3 fatty acids from fish liver oil per day. Because fish liver oil is also a good source of vitamin A, women taking in the highest amount ( 1 tbsp/day) throughout their pregnancy had a vitamin A intake from fish liver oil three times higher than the recommendation. A study on serum antioxidant vitamins and blood pressure in the United States found that serum vitamin A was significantly associated with higher odds of hypertension.104 It is possible that beneficial effects of the n-3 fatty acids on hypertension vanish in pregnant women, if the vitamin A intake exceeds a certain point. Pre-eclampsia it is a rapidly progressive condition characterized by high blood pressure and the presence of protein in the urine. Swelling, sudden weight gain, headaches and changes in vision are important symptoms. Oxidative stress, i.e. an imbalance between maternal prooxidants and antioxidants, is thought to

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be involved in the pathogenesis of pre-eclampsia.105,106 Therefore, maternal intake of polyunsaturated fatty acids is of interest. It has been suggested that pregnancy itself may be a stimulus for lipid peroxidation.107 Some studies focusing on the intake of n-3 LCPUFA suggest a reduced risk of pre-eclampsia,108,109 whereas others have found no association with preeclampsia,110,111 and still others an increased risk.107,112 In an Icelandic cohort study99 high maternal consumption of fish liver oil in early pregnancy was related to the development of pre-eclampsia. Further analysis of the data indicates that large amounts of n-3 LCPUFA, rather than retinol and vitamin D, nutrients accompanying fish liver oil in high dosage, are associated with the adverse effect. Owing to the observational design of the study, the relationship between fish liver oil and pre-eclampsia could also result from some related lifestyle factor. Taking into consideration only randomized controlled trials, a recent meta-analysis did not find evidence that n-3 LC-PUFA supplementation influences the rate of pre-eclampsia.113 Available information on a possible effect of fish consumption or n-3 fatty acids on Caesarean section is rare. A positive association was seen between fish consumption and Caesarean sections in an Icelandic cohort.103 It is hard to explain why there are more Caesarean deliveries in the highest fish consumption and fish frequency groups. Fish consumption, which has been seen to increase duration of gestation,114 might also increase the duration of labor resulting in more women ending up in an emergency caesarean delivery. Also, the largest babies (birth length, head circumference and birth weight indicated) are found in the groups with the higher frequency of fish consumption,115 which creates a plausible explanation for an increase in Caesarean delivery, as it is known that more difficulties are often involved when delivering a large baby than a smaller one.116 It can be said that fish oil intake during pregnancy is related to a lower frequency of hypertensive disorders, as long as intake does not exceed the recommended dose. Frequent fish consumption might increase the risk for Caesarean section, although this has to be confirmed by additional studies. Maternal consumption of n-3 LCPUFA in adequate amounts is important for growth and development of the fetus, but future research has to determine the exact amount and proportion in which various LCPUFAs need to be provided for optimal health of the mother and fetus during the perinatal period. Current knowledge suggests that the optimal dose lies between 0.1 and 0.9 g of n-3 LCPUFA from fish per day to achieve the health benefits.

9.5

N-3 fatty acids and postpartum depression

The prevalence of clinical depression and of depressive symptoms increases during the first weeks and months after birth. Research in Western countries has shown that between 5 and 20% of women suffer from more frequent depressive symptoms some period after birth. Apart from the adverse consequences they

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can have for women themselves, there is a negative impact on the relationship between mother and child and on the child's development.117±123 Several lines of evidence indicate that a negative association exists between n-3 fatty acids and depression. This relationship is observed in both observational and experimental research.21 Both n-6 and n-3 fatty acids are required for the normal function and structure of the mammalian nervous system.124 Maternal concentrations of EPA and DHA decrease during pregnancy, particularly in the third trimester,125±127 if there is no nutritional replenishment, and DHA concentrations may take up to 1 year to normalize.128 Symptoms and characteristics of postpartum depression are much like those for general depression. Seen in light of the fairly convincing evidence of an association between fish intake/n-3 fatty acids and general depression, an association between postpartum depression and fish intake seems plausible. Cohort studies have examined the relationship between n-3 fatty acids status and postpartum depression. In a study by Otto et al.,129 postpartum depression, measured retrospectively using the Edinburgh Postnatal Depression Scale (EPDS) 32 weeks after delivery, was associated with slower DHA normalization as indicated by the increase in the ratio of DHA to n-6 docosapentaenoic acid (DPA). Improvement in DHA status according to this index was significantly greater for 88 non-depressed versus 24 participants with depressive symptoms after multivariate adjustment. Concentrations of EPA, ALA and total n-3 fatty acids were lower, and concentrations of LA, AA and total n-6 fatty acids were higher among participants with depressive symptoms versus non-depressed at time of delivery and 32 weeks postpartum. In another cohort study,130 lower concentrations of DHA and total n-3 fatty acids and a higher ratio of n-6 to n-3 fatty acids were observed among ten participants with postpartum depression as compared to 38 non-depressed controls; each difference was statistically significant. In this study, blood samples were taken shortly after delivery, and postpartum depression was diagnosed retrospectively 24 to 40 weeks after delivery. Failure to control for potential confounders is a limitation in this study. An association between depressive symptoms and plasma DHA at 6 months postpartum was also determined in a prospective cohort of women.131 Women were classified with symptoms of depression if they has a score of 12 or above on the EPDS. The results indicated that a 1% increase in plasma DHA was associated with a 59% reduction in reporting of depressive symptoms. These associations need to be interpreted with caution because plasma DHA was positively influenced by maternal education and negatively influenced by maternal smoking. In an analysis of data from the Danish National Birth Registry,132 which is part of the SEAFOODplus YOUNG project, the question whether fish intake is associated with a lower rate of postpartum depression was examined in a prospective observational design. In this very large cohort investigating 19,470 mothers, fish consumption was negatively associated with depressive symptoms in a bivariate analysis with odds ratios between 0.65 and 0.72 of low to high fish

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intake in comparison to zero fish intake. However, in the multivariate analysis correcting for potential confounders like social status, smoking and previous mental illness, the odds ratios were between 0.81 and 0.87, but no longer significant. Viewing these results it should be considered that self reports on postpartum depression have to be interpreted carefully. In the case of the Danish National Birth Registry a re-analysis of the data will be performed including the diagnosis of depression done by a medical doctor. Inclusion of depression diagnosed by a doctor and the size of the cohort will provide results of highest quality. The results from SEAFOODplus YOUNG project will significantly contribute to the scientific discussion whether seafood consumption can help to prevent postpartum depression. A postpartum randomized controlled intervention trial,133 using a low dose of DHA (0.2 g/day), failed to observe an association between supplementation and outcome. Only a low percentage of the participants were moderately depressed, which may explain the non-association. The experimental study above and others134 mentioned above have not shown promising results. However, the results of these trials should not be taken as definite answers, because doses, treatment timing and investigated patient groups are debatable. In future intervention studies it is important to ensure that the investigated women are provided with a sufficient amount of EPA or DHA to compensate the transfer to the fetus during pregnancy, and the continued transfer to the child via breast feeding. Overall, it seems that fish oil or fish consumption can help to decrease postpartum depression, considering that most cohort studies agree on a negative association between n-3 fatty acids and postpartum depression. Future randomized intervention studies have to confirm these findings where women are provided with a sufficient amount of n-3 fatty acids to compensate losses during pregnancy and breastfeeding.

9.6

Bone health and n-3 fatty acids

Life expectancy has increased considerably over the last century in the Western countries. Age-related loss of bone mass and bone fragility are major risk factors for osteoporosis, leading to an increased risk of fractures, representing a significant public health problem. Therefore, nutritional strategies and lifestyle changes that prevent age-related osteoporosis and improve the quality of life for the elderly population are urgently needed.135 The change in fat intake during the last decades has led to an elevated ratio of n-6/n-3 fatty acids during the 20th century. Evidence suggests that the high intake of n-6 with an inadequate amount of n-3 fatty acids in the diet contributes to the development of certain chronic diseases, including those of the skeletal system.136 The importance of essential fatty acids in relation to calcium homeostasis was first reported many decades ago. Early studies showed that essential fatty acid

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deficiency was associated with loss of normal synthesis of bone connective tissue matrix, loss of normal cartilage, and bone demineralisation.135,137 Today, various epidemiological, clinical and experimental evidence suggests that different fatty acids may have different effects on calcium metabolism in animals and man.138 Dietary fat may influence bone metabolism by altering eicosanoid biosynthesis,139±141 which regulate both bone formation and bone resorption.142 The effect of n-3 fatty acids on bone health was examined in animal models. Iwami-Morimoto et al.143 compared fish oil or corn oil for 6 weeks and found out that dietary fish oil reduced osteoclastic activity and subsequent bone resorption. Bones from rats given n-3 fatty acids with a low calcium diet were as strong as those from rats on a normal diet in the bone rupture test and were stronger than those from rats given a low calcium diet.144 In another animal study n-3 fatty acids resulted in higher bone mineral density (BMD) compared to n-6 fatty acids.19 In a recent cohort study the association between the ratio of dietary n-6 to n-3 fatty acids and BMD in men and women aged 45±90 y was investigated. In ageand multiple-adjusted linear regression analyses there was a significant inverse association between the ratio of dietary linoleic acid to alpha-linolenic acid and BMD at the hip in men, women not using hormone therapy and women using hormone therapy. An increasing ratio of total dietary n-6 to n-3 fatty acids was also significantly and independently associated with lower BMD at the hip in all women and at the spine in women not using hormone therapy.18 In a longitudinal study the relationship between PUFA and bone metabolism in renal transplant patients was investigated using 22 recipients of a first renal allograft at baseline and after a mean 24.4 month follow-up. A multivariate regression analysis showed that BMD improvements at follow up were positively related to plasma phospholipid n-3 fatty acids, and negatively to plasma phospholipid arachidonic acid.145 So far only few intervention studies have been performed on the effects of PUFA on humans for osteoporosis prevention and treatment. These involved a total of 190 women using mixtures of evening primrose oil (rich in n-6 GLA) together with n-3 rich fish oil. Results have been rather contradictory. Kruger et al. performed a randomized placebo controlled study in 65 postmenopausal women with low bone mass.146 Women were fed 6 g per day of a mixture of evening primrose oil and fish oil. These two oils provided linoleic acid (60%), -linolenic acid (8%), eicosapentaenoic acid (4%) and docosahexaenoic acid (3%). After 18 months, women on the active treatment maintained bone mass at the lumbar spine while women on placebo lost about 3%. Women on the active treatment had 1.3% increase at the femoral neck while women on placebo had a 2.1% decrease.20 Vanpapendorp and colleagues146 supplemented the diet of 40 osteoporotic patients with evening primrose and fish oil or olive oil (placebo) for 16 weeks. Patients supplemented with the PUFA rich oils showed an improvement in calcium absorption and a stimulation of osteoblastic activity indicated by a rise in osteocalcin and procollagen both markers of bone formation.

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Bassey et al.147 failed to show any effect in total bone mineral density in 43 premenopausal women and 42 postmenopausal women randomized to either a mixture containing 4 g of primrose oil, 1 g of calcium and 440 mg of marine fish oil per day or placebo for 12 months. No significant difference was observed either in markers of bone turnover, which can be explained by the low dose of fish oil used in this study. The specific effects of seafood consumption on bone turnover during weight loss in young European overweight individuals has been investigated by a controlled randomized intervention study in course of the SEAFOOplus YOUNG project, comparing iso-caloric weight loss diets containing either lean fish, fatty fish or supplemental fish oil capsules, with a diet without seafood but including supplemental placebo capsules. According to a preliminary analysis inclusion of fish or fish oil did not significantly affect bone formation and resorption measured as serum osteocalcin, serum bone specific alkaline phosphatase, serum crosslaps and urinary N-telopeptides of Type I collagen. Weight loss is believed to increase bone mobilization and loss in these individuals;148,149 however, whether these effects of weight loss on bone can be generalized to younger adults is unclear. This study is one of the first studies to observe increased bone resorption with moderate weight loss (5.8%) over a relatively short period of time (8 weeks) in young adults aged 20±40 years, and contributes to our understanding that during weight loss moderate intake of seafood does not prevent from bone resorption. Optimizing bone development in the young and reducing bone resorption to maintain bone mass and restore skeletal integrity in the older are still the best means to control the disease. The best prevention of osteoporosis is to build strong bones early in life by consuming a well-balanced diet (with regards to vitamin D, calcium, probably n-6 and n-3 fatty acids, and phytochemicals) and to follow a routine exercise program pre- and postmenopause. Future intervention studies providing higher doses of fish oil or seafood for a prolonged period of time will be necessary to further elucidate the association between n-3 fatty acids or seafood and bone health seen in prospective cohort studies.

9.7 Communicating nutritional effects of fish to young adults and children and developing functional fish products for improved health 9.7.1 Communicating nutritional effects of fish to young adults and children Child and young adulthood represents a window of opportunity to prepare for a healthy adult life. During this time, nutritional problems originating earlier in life can potentially be corrected, in addition to addressing current ones. It is also a timely period to shape and consolidate healthy eating and lifestyle behaviors, thereby preventing or delaying the onset of nutrition-related chronic diseases in adulthood. Young adults are in the process of establishing responsibility for their

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own health-related behaviors, including diet. It is therefore an appropriate time for health promotion programmes based on documented relationships between behavior in this age group, obesity, cardiovascular and other chronic disease risk factors. Many adolescents are in school, which provides an effective and efficient opportunity for reaching large portions of the population. According to a large body of dietary survey data, it appears that some dietary patterns are consistently observed among adolescents, and put them at risk of unhealthy eating: the consequence of snacking, usually on energy-dense but otherwise nutrient-poor items; meal skipping; irregular eating patterns; and a wide use of fast food for meals and snacks. Television and magazines probably have more influence than any other form of mass media on adolescents' eating habits.150 Fish is a part of a healthy diet and therefore it is officially recommended to have fish at least twice a week as a major part of a meal. This is recommended additionally to other seafood consumption, i.e. as part of salads and sandwich meals. Seafood consumption has decreased in countries where it has traditionally been high, both in northern and southern parts of Europe. This trend, which seem especially strong among young adults of lower social status, will affect health negatively. It is therefore of high importance to prevent diminishing intake as well as to stimulate increased intake of fish to promote health.150 As for any other age group, interventions using an integrated approach in order to reach adolescents are required. The most effective and sustainable health programmes reportedly offer a variety of services, including counselling, family-life education, as well as physical examinations, and treatment of diagnosed conditions, e.g. obesity. Comprehensive programmes directed at multiplerisk behaviors are more likely to be successful than those targeting single specific behaviors, as concluded from studies on adolescents' risk behaviors in general or related to health. There is mounting evidence in developed countries that programmes targeting young adults are not effective when they are too short, single focused, too late, and when they stress negative behaviors to avoid, rather than promoting positive behaviors, whereas others, school-based or community-based, most probably had positive outcomes because of the holistic approach. The holistic or integrated approach does not mean that one given project should attempt to do it all, but rather, that programmes addressing different needs and providing different skills and knowledge are forming networks that enable them to meet the multiple needs of youth in a flexible and efficient manner.150 Nutrition promotion, as an integral part of health promotion, should involve the promotion of healthy eating, physical activity and other components of healthy lifestyle. Promotional activities are to be conducted through the media, and for interpersonal communication, through schools, health facilities, communities and even work-sites; For promoting healthful nutrition practices in adolescents, the challenge is to develop interventions that succeed in increasing motivation, while decreasing barriers to eating a healthful diet. Interventions have to be culturally appropriate. Adolescents of low social classes should be focused as a priority group, because it has been reported that they often had less

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adequate eating patterns and were more overweight. Food-based dietary guidelines are a useful tool in this regard for promoting nutrition among the population.150 9.7.2 Development of functional fish products for improved health Scientific and technological developments in the field of food have led to a marked shift in the way consumers deal with food and health. There is a growing awareness that the dietary source and form of food may affect the overall health of the consumer. The role of food as an agent for improving health has initiated the development of new classes of food ± functional foods. Nutrients and other bioactive substances isolated from fish as well fish in itself can be used as ingredients for functional foods. There is good evidence (Fig. 9.2) that regular seafood consumption reduces cardiovascular mortality. Many observational studies have shown an inverse relationship between higher seafood consumption and coronary heart disease. On the basis of mainly epidemiological research there are strong indications that regular seafood consumption could help to reduce diseases with an inflammatory component, e.g. diabetes type 2 and osteoporosis. The role of dietary seafood consumption as a component of a weight loss diet in overweight individuals has been reported in the SEAFOODplus YOUNG project and

Fig. 9.2

Effects of fish constituents on health factors in young families.

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seafood consumption may play a significant role in weight management, and help in the prevention of obesity among young adults.35 These beneficial effects should be verified and the underlying mechanism elucidated, before health claims for functionality could be attached to seafood consumption. Seafood is an important focus for research and development as well as industrial activity in the Nordic countries. A Nordic integrated multidisciplinary network has been created to strengthen the marine based food industry in the development of marine functional foods or marine functional ingredients. The Nordic Network for Marine Functional Food (MARIFUNC) will target the following main specific areas: health effects and claims for marine food or ingredients in marine foods, consumers' attitude to marine functional foods, possibilities for developing innovative new marine functional food products, needs, ideas and strategy for marine functional foods from small/medium enterprises and industrial partners. This pro-active platform will share the strategic intent and common goals for marine functional foods through for discussion and communication between industrial stakeholders and scientists from various disciplines and act as an initiator and catalyst for strategic activities.

9.8

Future trends

It has become clear that seafood lipids can be effective in the prevention of chronic nutrition-related diseases, e.g. cardiovascular and inflammatory diseases. Although the positive health effects of fish and/or fish oil are convincing for some diseases, e.g. heart disease,151 health effects for other diseases have to be confirmed in large prospective cohort studies as well as in randomized controlled trials. The beneficial health effects of seafood have mainly been related to n-3 fatty acids, but a fully exploration of different effects of seafood lipids versus seafood protein has not been done. The n-3 fatty acids from seafood have positive effects on human health, but over a certain level or in high doses they seem to be detrimental to some important measures of health. This is a fact which is true for most nutrients, and the upper level for long n-3 fatty acids intake has to be officially defined. Mechanisms of protection of fish or seafood constituents have to be elucidated, gene-nutrient interactions have to be explored and dose finding studies have to be conducted in order to provide the best possible nutritional prevention and therapy. Because fish and seafood are susceptible to bacterial spoilage, and can contain unwanted pollutants, an effort must be made during production, transport and handling in order to guarantee first class quality and food safety. In order to increase intake of health-relevant fish and seafood products in concerned population groups, authorities and educational institutions have to increase awareness and knowledge on the health benefits of regular fish and seafood consumption.

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Sources of further information and advice

In this section a short list of useful internet links is provided. These homepages can give valuable and interesting information on seafood and health: · · · · · ·

http://www.seafoodplus.org/ http://www.lydheilsustod.is/stodflokkar/english http://www.who.int/en/ http://en.fiskforsk.norut.no/fiskeriforskning/nyheter http://www.gissi.org/EngIntro/T_Intro_ENG.php www.americanheart.org

9.10 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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RK. Fish protein hydrolysate reduces plasma total cholesterol, increases the proportion of HDL cholesterol, and lowers acyl-CoA:cholesterol acyltransferase activity in liver of Zucker rats. J Nutr 2004; 134: 1320±1327. 71. BERGERON N, DESHAIES Y, JACQUES H. Dietary fish protein modulates high density lipoprotein cholesterol and lipoprotein lipase activity in rabbits. J Nutr 1992; 122: 1731±1737. 72. BEAUCHESNE-RONDEAU E, GASCON A, BERGERON J, JACQUES H. Plasma lipids and lipoproteins in hypercholesterolemic men fed a lipid-lowering diet containing lean beef, lean fish, or poultry. Am J Clin Nutr 2003; 77: 587±593. 73. MORI TA, VANDONGEN R, BEILIN LJ, BURKE V, MORRIS J, RITCHIE J. Effects of varying dietary fat, fish, and fish oils on blood lipids in a randomized controlled trial in men at risk of heart disease. Am J Clin Nutr 1994; 59: 1060±1068. 74. PETERSEN M, TAYLOR MA, SARIS WH, VERDICH C, TOUBRO S, MACDONALD I, ROSSNER S, STICH V, GUY-GRAND B, LANGIN D, MARTINEZ JA, PEDERSEN O, HOLST C, SORENSEN TI,

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Relationship between dietary intake of cod liver oil in early pregnancy and birthweight. BJOG 2005; 112(4): 424±429. MEEUWISSE G, OLAUSSON PO. Increasing birthweight in the Nordic countries; a growing proportion of neonates weigh over four kg. Swed Med J 1998; 95: 5488± 5492. OLSEN SF, JOENSEN HD. High liveborn birth weights in the Faroes: a comparison between birth weight in the Faroes and in Denmark. J Epidemiol Community Health 1985; 39: 27±32. BIRGISDOTTIR BE, GUNNARSDOTTIR I, THORSDOTTIR I, et al. Size at birth and glucose intolerance in a relatively genetically homogenous, high birth weight population. Am J Clin Nutr 2002; 76: 399±403. GUNNARSDOTTIR I, BIRGISDOTTIR BE, BENEDIKTSSON R, et al. Size at birth and hypertension in a genetically homogenous high birth weight population. J Hypertens 2002; 20: 623±628. BARKER DJP. Mother, babies and health in later life. Edinburgh: Churchill Livingstone, 1998. GUNNARSDOTTIR I, BIRGISDOTTIR BE, THORSDOTTIR I, et al. Size at birth and coronary artery disease in a population of high birth weight. Am J Clin Nutr 2002; 76: 1290± 1294. VILBERGSSON S, SIGURDSSON G, SIGVALDASON H, et al. Prevalence and incidence of NIDDM in Iceland: evidence for stable incidence among males and females 1967± 1991 ± the Reykjavik Study. Diabet Med 1997; 14: 491±498. CHAMBLESS L, KEIL U, DOBSON A, et al. Population versus clinical view of case fatality from acute coronary heart disease: results from the WHO MONICA Project 1985±1990. Multinational MONItoring of Trends and Determinants in Cardiovascular Disease. Circulation 1997; 96: 3849±3859. WOLF HK, TUOMILEHTO J, KUULASMAA K, et al. Blood pressure levels in the 41 populations of the WHO MONICA Project. J Hum Hypertens 1997; 11: 733±742. Ecological analysis of the association between mortality and major risk factors of cardiovascular disease. The World Health Organization MONICA Project. Int J Epidemiol 1994; 23: 505±516. GRANDJEAN P, BJERVE KS, WEIHE P, et al. Birthweight in a fishing community: significance of essential fatty acids and marine food contaminants. Int J Epidemiol 2001; 30: 1272±1278. HELLAND IB, SAUGSTAD OD, SMITH L, et al. Similar effects on infants of n-3 and n-6 fatty acids supplementation to pregnant and lactating women. Pediatrics 2001; 108: E82. OLSEN SF, SORENSEN JD, SECHER NJ, et al. Randomised controlled trial of effect of fish-oil supplementation on pregnancy duration. Lancet 1992; 339: 1003±1007. MACKAY AP, BERG CJ, ATRASH HK. Pregnancy-related mortality from preeclampsia and eclampsia. Obstet Gynecol 2001; 97: 533±538. THADANI R, STAMPFER MJ, HUNTER DJ, MANSON JE, SOLOMON CG, CURHAN GC. High body mass index and hypercholesteremia: risk of hypertensive disorders of pregnancy. Obstet Gynecol 1999; 94: 543±550. SKULADOTTIR GV, STEINGRIMSDOTTIR L.

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MEHER S, DULEY L. Interventions for preventing pre-eclampsia and its consequences: generic protocol. The Cochrane Database of Systematic Reviews. 2005. È. ARNARDOTTIR GA, GEIRSSON RT, ARNGRIMSSON R, JONSDOTTIR LS, OLAFSSON O

Cardiovascular death in women who had hypertension in pregnancy: a casecontrol study. BJOG 2005; 112: 286±292. OLAFSDOTTIR AS, SKULADOTTIR GV, THORSDOTTIR I, HAUKSSON A, THORGEIRSDOTTIR

Relationship between high consumption of marine fatty acids in early pregnancy and hypertensive disorders in pregnancy. BJOG 2006; 113(3): 301±309. OGBURN PL JR, WILLIAMS PP, JOHNSON SB, HOLMAN RT. Serum arachidonic acid levels in normal and preeclamptic pregnancies. Am J Obstet Gynecol 1984; 148(1): 5±9. WILLIAMS MA, ZINGHEIM RW, KING IB, ZEBELMAN AM. Omega-3 fatty acids in maternal erythrocytes and risk of preeclampsia. Epidemiology 1995; 6(3): 232±237. SALVIG JD, OLSEN SF, SECHER NJ. Effects of fish oil supplementation in late pregnancy on blood pressure: a randomised controlled trial. Br J Obstet Gynaecol 1996; 103(6): 529±533. BIRGISDOTTIR BE, GEIRSSON RT, THORSDOTTIR I. Association between consumption of fish and intake of fish liver oil and pregnancy delivery complications among women of normal weight before pregnancy. Submitted 2006. CHEN J, HE J, HAMM L, BATUMAN V, WHELTON PK. Serum antioxidant vitamins and blood pressure in the United States population. Hypertension 2002; 40(6): 810±816. MORETTI M, PHILLIPS M, ABOUZEID A, CATANEO RN, GREENBERG J. Increased breath markers of oxidative stress in normal pregnancy and in preeclampsia. Am J Obstet Gynecol 2004; 190: 1184±1190. SCHOLL TO, LESKIW M, CHEN X, SIMS M, STEIN TP. Oxidative stress, diet, and the etiology of preeclampsia. Am J Clin Nutr 2005; 81: 1390±1396. HUBEL CA. Oxidative stress in the pathogenesis of preeclampsia. Proc Soc Exp Biol Med 1999; 222: 222±235. OLSEN SF, SECHER NJ. A possible preventive effect of low-dose fish oil on early delivery and pre-eclampsia: indications from a 50-year-old controlled trial. Br J Nutr 1990; 64: 599±609. DYERBERG J, BANG HO. Pre-eclampsia and prostaglandins. Lancet 1985; 325: 1267± 1268. OLSEN SF, SECHER NJ, TABOR A, WEBER T, WALKER JJ, GLUUD C. Randomised clinical trials of fish oil supplements in high risk pregnancies. BJOG 2000; 107: 382±395. BULSTRA-RAMAKERS MT, HUISJES HJ, VISSER GH. The effects of 3 g eicosapentaenoic acid daily on recurrence of intrauterine growth retardation and pregnancy induced hypertension. Br J Obstet Gynaecol 1995; 102: 123±126. CLAUSEN T, SLOTT M, SOLVOLL K, DREVON CA, VOLLSET SE, HENRIKSEN T. High intake of energy, sucrose, and polyunsaturated fatty acids is associated with increased risk of preeclampsia. Am J Obstet Gynecol 2001; 185: 451±458. SZAJEWSKA H, HORVATH A, KOLETZKO B. Effect of n-3 long-chain polyunsaturated fatty acid supplementation of women with low-risk pregnancies on pregnancy outcomes and growth measures at birth: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2006; 83: 1337±1344. Review. OLSEN SF, SECHER NJ. Low consumption of seafood in early pregnancy as a risk factor for preterm delivery: prospective cohort study. BMJ 2002; 324(7335): 447. H, STEINGRIMSDOTTIR L.

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Improving seafood products for the consumer Association of fish and fish liver oil intake in pregnancy with infant size at birth among women of normal weight before pregnancy in a fishing community. Am J Epidemiol 2004; 160(5): 460±465. MERCHANT KM, VILLAR J, KESTLER E. Maternal height and newborn size relative to risk of intrapartum caesarean delivery and perinatal distress. BJOG 2001; 108(7): 689±696. ARMSTRONG KL, O'DONNEL H, MCCALLUM R, DADDS M. Childhood sleep problems: association with prenatal factors and maternal distress/depression. J Paediatr Child Health 1998; 34: 263±266. BECK CT. Predictors of postpartum depression: an update. Nursing Resarch 2001; 50: 275±285. MORGAN M, MATTHEY S, BARNETT B, RICHARDSON C. A group programme for postnatally distressed women and their partners. J Adv Nursing 1997; 26: 913± 920. THOME M. Predictors of postpartum depressive symptoms in Icelandic women. Archives of Womens Mental Health 2000; 3: 7±14. WATT S, SWORD W, KREUGER P, SHEEHAN D. A cross-sectional study of early identification of postpartum depression: Implications for primary care providers from The Ontario Mother and Infant Survey. BMC Family Practice 2002; 3: 5. THORSDOTTIR I, BIRGISDOTTIR BE, HALLDORSDOTTIR S, GEIRSSON RT.

WEBSTER J, PRITCHARD MA, LINNANE JW, ROBERTS JA, HINSON JK, STARRENBURG SE.

Postnatal depression: use of health services and satisfaction with health-care providers. J Qual Clin Practice 2001; 21: 144±148. 123. THOME M, ALDER EM, RAMEL A. A population-based study of exclusive breastfeeding in Icelandic women: is there a relationship with depressive symptoms and parenting stress? Int J Nurs Stud 2006; 43(1): 11±20. 124. HALLAHAN B, GARLAND MR. Essential fatty acids and their role in the treatment of impulsivity disorders. Prostaglandins Leukot Essent Fatty Acids 2004; 71(4): 211± 216. 125. HORNSTRA G. Essential fatty acids in mothers and their neonates. Am J Clin Nutr (2000); 71: 1262S±1269S. 126. AL MDM, VAN HOUWELINGEN AC, KESTER AD, HASAART TH, DE JONG AE, HORNSTRA G. Maternal essential fatty acid patterns during normal pregnancy and their relationship to the neonatal essential fatty acid status. Br J Nutr 1995; 74: 55± 68. 127. AL MDM, VAN HOUWELINGEN AC, HORNSTRA G. Relation between birth order and the maternal and neonatal docosahexaenoic acid status. Eur J Clin Nutr 1997; 51: 548± 553. 128. VAN HOUWELINGEN AC, HAM EC, HORNSTRA G. The female docosahexaenoic acid status related to the number of completed pregnancies. Lipids 1999; 34: S229. 129. OTTO SJ, DE GROOT RH, G. HORNSTRA G. Increased risk of postpartum depressive symptoms is associated with slower normalization after pregnancy of the functional docosahexaenoic acid status. Prostaglandins Leukot Essent Fat Acids 2003; 69: 237±243. 130. DE VRIESE R, CHRISTOPHE AB, MAES M. Lowered serum n-3 polyunsaturated fatty acid (PUFA) levels predict the occurrence of postpartum depression: further evidence that lowered n-PUFAs are related to major depression. Life Sci 2003; 73: 3181± 3187.

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Docosahexaenoic acid and post-partum depression ± is there a link? Asia Pac J Clin Nutr 2003; 12 Suppl: S37. STROM M. Maternal nutrition and risk of post partum depression. Masters thesis 2005. Faculty of Health Sciences, Institute of Public Health, University of Copenhagen. LLORENTE AM, JENSEN CL, VOIGT RG, FRALEY JK, BERRETTA MC, HEIRD WC. Effect of maternal docosahexaenoic acid supplementation on postpartum depression and information processing. Am J Obstet Gynecol 2003; 188: 1348±1353. MARANGELL LB, MARTINEZ JM, ZBOYAN HA, CHONG H, PURYEAR LJ. Omega-3 fatty acids for the prevention of postpartum depression: negative data from a preliminary, open-label pilot study. Depress Anxiety 2004; 19(1): 20±23. ALBERTAZZI P, COUPLAND K. Polyunsaturated fatty acids. Is there a role in postmenopausal osteoporosis prevention? Maturitas 2002; 42(1): 13±22. SIMOPOULOS AP. Evolutionary aspects of omega-3 fatty acids in the food supply. Prostaglandins Leukot Essent Fatty Acids 1999; 60: 421±429. BORLAND VG, JACKSON CM. Effects of a fat free diet on the structure of the kidney in rats. Arch Pathol 1931; 11: 687±708. BAGGIO B. Fatty acids, calcium and bone metabolism. J Nephrol 2002; 15(6): 601± 604. Review. WATKINS BA, SHEN CL, ALLEN KG, SEIFERT MF. Dietary (n-3) and (n-6) polyunsaturates and acetylsalicylic acid alter ex vivo PGE2 biosynthesis, tissue IGF-I levels, and bone morphometry in chicks. J Bone Miner Res 1996; 11: 1321±1332. WATKINS BA, SHEN CL, MCMURTRY JP, XU H, BAIN SD, ALLEN KG, SEIFERT MF. Dietary lipids modulate bone prostaglandin E2 production, insulin-like growth factor-I concentration and formation rate in chicks. J Nutr 1997; 127: 1084±1091. LI Y, SEIFERT MF, NEY DM, GRAHN M, GRANT AL, ALLEN KG, WATKINS BA. Dietary conjugated linoleic acids alter serum IGF-I and IGF binding protein concentrations and reduce bone formation in rats fed (n-6) or (n-3) fatty acids. J Bone Miner Res 1999; 14: 1153±1162. MARKS SC, JR., MILLER SC. Prostaglandins and the skeleton: The legacy and challenges of two decades of research. Endocr J 1993; 1: 337±344. IWAMI-MORIMOTO Y, YAMAGUCHI K, TANNE K. Influence of dietary n-3 polyunsaturated fatty acid on experimental tooth movement in rats. Angle Orthod 1999; 69: 365±371. SAKAGUCHI K, MORITA I, MUROTA S. Eicosapentaenoic acid inhibits bone loss due to ovariectomy in rats. Prostaglandins Leukot Essent Fatty Acids 1994; 50: 81± 84. MAKRIDES M, CROWTHER CA, GIBSON RA, GIBSON RS, SKEAFF CM.

BAGGIO B, BUDAKOVIC A, FERRARO A, CHECCHETTO S, PRIANTE G, MUSACCHIO E,

MANZATO E, ZANINOTTO M, MARESCA MC. Relationship between plasma phospholipid polyunsaturated fatty acid composition and bone disease in renal transplantation. Transplantation 2005; 80(9): 1349±1352. 146. VANPAPENDORP DH, COETZER H, KRUGER MG. Biochemical profile of osteoporotic patients on essential fatty-acids supplementation. Nutr Res 1995; 15: 325±334. 147. BASSEY EJ, LETTLEWOOD JE, ROTHWELL MC, PYE DW. Lack of effects of supplementation with essential fatty acids on bone mineral density in healthy pre and post menopausal women: two randomized controlled trial of EfacalÕ v calcium alone. Br J Nutr 2000; 83: 629±635.

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Intentional and unintentional weight loss increase bone loss and hip fracture risk in older women. J Am Geriatr Soc 2003; 51: 1740±1747 149. MACDONALD HM, NEW SA, CAMPBELL MK, REID DM. Influence of weight and weight change on bone loss in perimenopausal and early postmenopausal Scottish women. Osteoporosis Int 2005; 16: 163±71. 150. WORLD HEALTH ORGANIZATION. Nutrition in adolescence: issues and challenges for the health sector: issues in adolescent health and development, 2005. 151. BRESLOW JL. n-3 Fatty acids and cardiovascular disease. Am J Clin Nutr 2006; 83(6 Suppl): 1477S±1482S. Review. OF OSTEOPOROTIC FRACTURES RESEARCH GROUP.

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10 Fish, omega-3 fatty acids and heart disease I. A. Brouwer, Free University Amsterdam, The Netherlands

10.1

Introduction

The METAHEART project of the SEAFOODplus programme intended to learn more about the heart health effects of seafood and its fatty acids. As heart disease is the number one cause of death in the Western world, an increase in knowledge on this subject could be of great benefit to the consumer. This chapter discusses the relationship between intake of fish, omega-3 fatty acids and heart disease. It starts with a description of omega-3 fatty acids in our diet and then continues with the relationship between fish, omega-3 fatty acids and heart disease. The next part reviews more specifically the association between omega-3 fatty acids and cardiac arrhythmia. Human studies in which the various forms of cardiac arrhythmia are studied will be explained. Animal and in vitro studies are briefly mentioned to provide more information on the possible underlying mechanisms. The SEAFOODplus programme has contributed and is still contributing new scientific knowledge to all of these topics. This chapter will not only deal with direct effects of omega-3 fatty acids from fish, but also with possible effects of the vegetable oil alpha-linolenic acid. Both direct effects of alpha-linolenic acid and the conversion to the longer chain omega-3 fatty acids are discussed. Finally, possibilities for future research and sources of further information and advice are mentioned.

10.2

Fish, omega-3 fatty acids and heart disease

10.2.1 Omega-3 fatty acids Omega-3 fatty acids, otherwise named n-3 fatty acids, are polyunsaturated fatty acids which are characterized by the first double bond at the third position

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counted from the methyl end of the molecule. They are essential fatty acids. This implies that the body needs them but cannot synthesize them by itself. Thus, it is essential for humans to have a sufficient intake from food or other sources. There are two main groups of omega-3 fatty acids; alpha-linolenic acid and the very-long chain omega-3 fatty acids. Alpha-linolenic acid (C18:3 n-3) is mainly found in vegetable oil and nuts and has 18 carbon molecules and three double bonds. The very long-chain omega-3 fatty acids are mainly found in fish and other marine products and have at least 20 carbon atoms and five or more double bonds. The main omega-3 fatty acids from fish and fish products are eicosapentaenoic acid (EPA; C20:5 n-3) and docosahexaenoic acid (DHA; C22:6 n3). Fatty fish is the main source of EPA and DHA. Fatty fish is fish with a relative high amount of oil. Lean, white fish also contains omega-3 fatty acids, but because this fish contains less oil it also contains less omega-3 fatty acids. Although fatty fish are the richest sources of omega-3 fatty acids, one should keep in mind that the actual amount of omega-3 fatty acids not only varies from species to species, but also within species.1 10.2.2 Observational studies Observational studies are studies in which behaviour of people is observed in the free living situation without actually intervening in their choices. So, in these studies, behaviour of people, for example their dietary choices, is studied and measured, but subjects are not asked to change anything. In the 1970s, researchers from Denmark observed that Inuits from Greenland had a low prevalence of cardiovascular disease. They suggested that intake of fish and omega-3 fatty acids from fish could be related to cardiovascular disease. These remarkable observations formed the basis of the hypothesis that intake of fish fatty acids could prevent cardiovascular disease.2,3 Since then many observational studies have been performed investigating the relationship between intake of fish or fish fatty acids and cardiovascular disease. The Zutphen study in 1985 was the first prospective cohort study which showed an association between intake of fish and mortality due to coronary heart disease. Middle-aged men who consumed fish once or twice per week had a 50% lower risk of dying of coronary heart disease than men who consumed no fish.4 This study was followed by many other observational studies that showed that a higher intake of fish or fish fatty acids was associated with a lower risk of cardiovascular disease.5±10 However, there are also studies that showed no relationship or even a slightly increased risk of cardiovascular disease in those subjects with a higher fish intake.11±14 The higher rates of fatal coronary heart disease in those subjects with a high fish intake in the studies performed in Finland may be explained by adverse effects of high mercury concentrations in fresh water fish in Finland.14 Whelton et al.15 and He et al.16 have performed so-called meta-analyses. In these meta-analyses they have taken all available observational studies together and they have quantitatively calculated the overall risks. The meta-analyses of Whelton et al.15 and He et al.16 both suggested that people who ate at least once

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fish meal per week had approximately 15% lower risk of dying of coronary heart disease than people who ate a fish meal less than once per month. Some studies have investigated the relationship between omega-3 fatty acids in blood and the risk of fatal heart disease. In the Seattle area Siscovick et al. (1995) performed a population-based case-control study comparing cases with a primary cardiac arrest with non-cases. The study showed a strong inverse association between both intake of fish and levels of omega-3 fatty acids in red cell membranes and the risk of a primary cardiac arrest.17,18 As depicted in Fig. 10.1, a level of omega-3 fatty acids of 5.0% of total fatty acids (the mean of the third quartile) was associated with a 70% lower risk of a primary cardiac arrest compared with a red blood cell membrane of 3.3% (mean of the lowest quartile).18 The Physicians' Health Study, a large cohort study, reported the relationship of fish consumption and omega-3 fatty acid levels in the blood with the incidence of sudden cardiac death.8,19 Fish consumption of at least once per week was associated a relative risk of 0.48 (95% confidence interval, 0.24 to 0.96) compared to no fish consumption. The relative risk (RR) is the ratio of the risk of disease or death among the exposed versus the risk among the unexposed. Thus, in this case, men who reported to eat fish at least once per week had a 52% lower risk of sudden death compared to men who reported not to eat any fish.8 The same group also reported a strong adverse association between blood levels of omega-3 fatty acids and the risk of sudden death. Men with blood levels of omega-3 fatty acids in the fourth quartile of the distribution had a relative risk of 0.19 (95% CI 0.05±0.71) compared to men with levels in the lowest quartile.19 There was no association between intake and fish and risk of non-fatal coronary heart disease in the Physicians' Health Study.11 The Cardiovascular Health Study is a population-based cohort study of elderly people aged 65 years and above. In this study higher levels of omega-3 fatty acids in plasma phospholipids were associated with a lower risk of fatal coronary heart disease, but not with a lower risk of non-fatal myocardial infarction.20 Another publication from the same Cardiovascular Health Study

Fig. 10.1 Odds ratio for risk of a primary cardiac arrest for quartiles of omega-3 fatty acid concentration in red cell membranes (based on Siscovick et al.18).

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suggested that the beneficial effects of fish consumption on heart disease may depend on the type of fish consumed. Consumption of tuna or other broiled or baked fish two times per week or more was associated with lower risk of fatal ischemic heart disease compared with a consumption of less than once per month. However, consumption of fried fish or fish sandwiches was not associated with a lower risk, but even with trends towards higher risks.21 In the same study population, consumption of tuna or other broiled or baked fish was associated with a lower heart rate, lower systemic vascular resistance and a greater stroke volume compared to non-consumption. A lower heart rate, lower systemic vascular resistance and a greater stroke volume all suggest a better functioning heart in those subjects with a higher consumption of tuna or other broiled or baked fish. In contrast, consumption of fried fish or fish sandwiches was associated with structural abnormalities of the heart.22 The contradictory effects of the tuna and other broiled or baked fish versus the fried fish and fish sandwiches might be caused by the low content of omega-3 fatty acids in the fried fish and fish sandwiches or the unfavourable effects of frying in the wrong, unhealthy fatty acids. The tuna or other broiled or baked fish provided around 270 mg/d of the very long-chain omega-3 fatty acids, whereas the fried fish and fish sandwiches provided only around 65 mg/d. Furthermore, higher intake of tuna or other broiled or baked fish was associated with higher omega-3 levels in the plasma phospholipids, but higher intake of fried fish and fish sandwiches (fish burgers) was not associated omega-3 levels in the blood.21 Thus, the studies that investigated the association between blood levels of omega-3 fatty acids and fatal heart disease support the idea that the omega-3 fatty acids in the blood are the component responsible for the cardio-protective effect. In summary, the observational studies suggest that a high intake of fish compared to a lower intake of fish is associated with a lower risk of fatal coronary heart disease, but not with non-fatal heart disease. However, all observational studies have the disadvantage that other lifestyle factors may affect the associations. So, people who eat fish may live healthier lives than people who do not eat fish, and people who eat tuna or other broiled or baked fish may live healthier lives than people who eat fried fish or fish burgers. 10.2.3 Intervention studies/trials In intervention studies or trials researchers actively intervene in the habits of the people by either advising them or actually supplementing them. Preferably the `treatment' is compared with a placebo treatment which is indistinguishable from the real supplement. Three trials have been published showing direct effects of omega-3 fatty acids from fish on fatal cardiovascular disease.23±25 In the Diet and Reinfarction trial (DART), patients who previously suffered from a myocardial infarction received advice to increase their fish intake to at least two fish meals per week or they did not receive this advice. Those patients who received the fish advice had a 29% lower mortality rate after two years of intervention compared to the patients who did not receive the advice.25

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However, a post-trial follow-up showed no beneficial effects on survival in the long term. The reduction in mortality over the first two years was even followed by a higher risk over the following three years.26 The problem with this followup study26 is that it is unclear whether patients in either of the two groups changed their behaviour after the study ended. It is not unlikely that patients changed their fish intake after they learned the results of trial. The GISSIPrevenzione trial showed a clear protective effect. Patients with a recent myocardial infarction randomly received supplements which contained either n3 PUFA from fish (900 mg/d), or vitamin E, or both, or they received no treatment. After 3.5 years of follow-up the study showed that patients who received fish oil had 10±15% lower combined risk of mortality, non-fatal myocardial infarction and stroke.23 In contrast, the second DART trial of Burr et al.24 in 3114 patients with stable angina without a myocardial infarction showed no beneficial effect of n-3 PUFA. In this trial, advice to eat fatty fish did not reduce mortality, and intake of fish oil supplements was even associated with a higher risk of cardiac and sudden death.24 Compliance to advice was only shown for a sub-sample of patients. Unfortunately, both participants and providers were not masked, which implies that the intake of fish oil may have modified the patients' or the physicians' behaviour towards intake of medication or diet and lifestyle. Furthermore, recruitment for DART-2 was interrupted for a year because of funding problems.27 Thus, there are some methodological concerns about this trial. Overall, the trials suggest that intake of omega-3 fatty acids prevents fatal cardiovascular disease in patients with a prior myocardial infarction. 10.2.4 Systematic reviews In 2006 two systematic reviews were published. In a systematic review all available evidence is taken together and reviewed in a systematic way. The systematic review of Hooper et al.28 concluded on the basis of 48 randomized clinical trials and 41 cohort studies that long chain and shorter chain omega-3 fats did not have a clear effect on total mortality, combined cardiovascular events, or cancer. However, the authors have received criticism because of their approach. They pooled the results of alpha-linolenic acid with omega-3 fatty acids from fish, while epidemiological evidence of protective effects of alphalinolenic acid is not very convincing and there is no evidence from randomized controlled trials. Furthermore, fatal and non-fatal cardiovascular events were taken together in the Hooper review29 although several earlier meta-analyses have shown a protective effect of fish intake on stroke and on fatal coronary heart disease.15,30,31 Hooper et al.28 made clear that their overall conclusion on omega-3 fatty acids from fish was quite heavily influenced by the DART 2 study of Burr et al.24 Of course the results of the DART-2 trial should not be ignored. However, because of the methodological concerns it is useful to also report the results without DART-2. Removal of DART 2 from the meta-analysis resulted in a relative risk of death was 0.83 (95% confidence interval 0.75±0.91), which

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implies a protective effect for long chain omega-3 fatty acids.28 Another review, by Wang et al.32 was based on 14 randomized clinical trials, 25 prospective cohort studies and 7 case-control studies. These authors came to the conclusion that increased consumption of omega-3 fatty acids from fish or fish oil supplements, but not of alpha-linolenic acid, reduces the rates of all-cause mortality, cardiac and sudden death, and possibly stroke. They also concluded that the evidence for the benefits of fish oil is stronger in secondary than in primary prevention settings and that adverse effects appear to be minor.

10.3

Omega-3 fatty acids from fish and cardiac arrhythmias

10.3.1 Ventricular tachycardia and ventricular fibrillation The beneficial effects of omega-3 fatty acids are thus mainly seen on fatal cardiovascular disease. More specifically, the most pronounced effects of omega-3 fatty acids are shown on sudden death.8,18,19,23 Therefore, omega-3 fatty acids may prevent sudden death by prohibiting cardiac arrhythmia. Sudden death from cardiac causes is the main cause of all deaths from cardiovascular causes.33 Most of the acute sudden deaths are caused by ventricular tachyarrhythmia.34 Ventricular tachycardia and ventricular fibrillation are lifethreatening ventricular tachyarrhythmias. Ventricular tachyarrhymias are arrhythmias that occur in the ventricles ± the chambers ± of the heart. These arrhythmias are more dangerous that those that occur in the atria of the heart. Three trials have used patients with implantable cardioverter defibrillators (ICDs) to test whether omega-3 fatty acids from fish oil can prevent ventricular tachycardia and ventricular fibrillation.35±37 An ICD device detects arrhythmic events and can treat these events by delivering smaller or larger electric shocks. The disturbances in the cardiac rhythm and the given treatment are stored in the memory chip of the ICD. Patients receive an ICD because they are at high risk of having ± recurrent ± life-threatening cardiac arrhythmia (Fig. 10.2).

Fig. 10.2 Implantable cardioverter defibrillator (ICD) detects (D) arrhythmia and provides a shock (S) to restore normal rhythm (reproduced from Brouwer et al.39).

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The first trial of Raitt et al.35 from Portland included 200 patients with an ICD who had recently experienced a ventricular tachycardia or ventricular fibrillation. These patients received daily either fish oil containing 1.8 grams of fish oil containing 76% omega-3 fatty acids or 1.8 grams of olive oil (placebo). The patients were followed for a maximum of two years. The number and type of arrhythmias were monitored during the entire intervention period. This trial did not show a reduction in life-threatening cardiac arrhythmia. After the intervention period, 66% of the patients in the fish oil group versus 60% of patients in the placebo group had experienced at least one ventricular tachyarrhythmia (p 0:19). Patients with a ventricular tachycardia (n 133) as index arrhythmia before their ICD implantation were even significantly worse of if they were allocated to the fish oil treatment.35 The second trial, named FAAT, from Leaf et al.36 from Boston included 402 patients and followed these for 12 months. Patients randomly received either 2.4 grams of fish oil or olive oil per day (placebo). The FAAT trial showed a borderline significant (p 0:057) protective effect of fish oil.36 The third trial, the SOFA trial was from our group.37 This was the largest trial until now and was integrated in the so-called METAHEART project of the ongoing SEAFOODplus project. It has been performed in 26 cardiology clinics in 8 countries in Europe. In total 546 patients received at random either 2 grams of fish oil or 2 grams of oleic acid rich sunflower oil (placebo). The fish oil contained approximately 900 mg of long chain omega-3 fatty acids. Survival without life-threatening ventricular arrhythmias was 70% in the fish oil group versus 67% in the placebo group (p 0:33, log-rank).37 Taken together, the three trials do not show a strong protective effect of fish oil on life-threatening ventricular arrhythmia.38 In addition to these trials a small pilot study and a cross-sectional analysis have been performed. To assess the immediate effects of fish oil on the induction of sustained ventricular arrhythmia the investigators did electrophysiological testing in ten patients with an ICD. Tachycardia was induced in seven out of ten patients without infusion of omega-3 fatty acids. After infusion of omega-3 fatty acids such tachycardia could not be induced in five out of seven patients. Even though the patient group was small and though there was no placebo group the study suggests that infusion of omega-3 fatty acids has immediate protective effects on inducible sustained tachycardia in patients with an ICD.39 The crosssectional analysis of Christensen et al.40 showed that ICD patients with a high concentration of omega-3 fatty acids in their serum had less chance of having ventricular tachycardia or ventricular fibrillation than ICD patients with a high concentration of omega-3 fatty acids in their serum.40 The major problem with this study is the cross-sectional design. This makes it exclude the possibility to say whether there is a causal relationship between fish fatty acids and tachycardia. Furthermore, the study was small and the samples were taken after the follow-up period, which excludes patients that died of arrhythmia of the analysis. Thus, the current evidence suggests that omega-3 fatty acids from fish may prevent life-threatening arrhythmia in some patients, but may be harmful in others.

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10.3.2 Atrial fibrillation The most common forms of sustained arrhythmias are not the ventricular tachyarrhythmias, but atrial fibrillation. The prevalence of atrial fibrillation increases with age, which makes it particularly a problem among the elderly.41 The prognosis of atrial fibrillation is related to the underlying disease. The prognosis is very good if the atrial fibrillation is idiopathic, which implies that the fibrillation seems to occur without other underlying disease. However, the prognosis is much poorer when it is due to ischaemic cardiomyopathy.42 Three cohort studies have investigated the association between intake of omega-3 fatty acids and the incidence of atrial fibrillation. The Cardiovascular Health Study showed that consumption of tuna or other broiled or baked fish of 1 to 4 times per week was associated with a 28% lower risk of atrial fibrillation than consumption of less than once per month.43 In the same study among the elderly, intake of fried fish and fish burgers was not associated with a lower incidence of atrial fibrillation. The authors suggest that the higher content of omega-3 fatty acids in tuna and other broiled or baked fish compared to fried fish and fish burgers explains the association with a protective effect.43 In contrast, in the Danish Diet, Cancer and Health study consumption of omega-3 fatty acids from fish was not associated with the risk of atrial fibrillation. The Danish authors divided fish consumption in quintiles of intake. In the lowest quintile intake of omega-3 fatty acids from fish was 0.18 grams per day versus 1.38 grams per day in the highest quintile of intake. The hazard ratios for atrial fibrillation did not significantly differ between the quintiles. Thus, fish consumption was not associated with reduction in atrial fibrillation.44 This finding is in line with what we found in the Rotterdam Study.45 In this study 312 subjects had developed atrial fibrillation after a mean follow-up of 6.4 ( 1.6) years. The study showed no association between intake of EPA and DHA and the incidence of atrial fibrillation.45 An Italian study investigated the effect of fish oil supplementation on the development of atrial fibrillation after coronary bypass graft surgery (CABG). They randomized 160 CABG patients over either receiving 2 grams of omega-3 fatty acids daily from at least 5 days before the surgery until the day of discharge from the hospital, or no treatment. So, unfortunately the study was not blind. In the untreated group 27 patients (33%) developed atrial fibrillation after surgery versus only 12 of the patients (15.2%) treated with omega-3 fatty acids. Thus, intake of fish oil supplements before surgery might prevent postoperative atrial fibrillation.46 The three cross-sectional studies do not show a consistent association between intake of fish fatty acids and spontaneous atrial fibrillation. However, the study from Calo et al.46 suggests that fish oil might help for the prevention of post-operative atrial fibrillation. 10.3.3 Premature ventricular complexes Premature ventricular complexes (PVCs) are a form of arrhythmia that is quite common and in itself innocent (Fig. 10.3). Almost everyone has one or two

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Fig. 10.3 A = Normal rhythm. B = Premature ventricular complex (PVC) (reproduced from Brouwer et al.39).

PVCs per day, but some people have many more (up to thousands per 24 hours). Although PVCs are innocent in themselves, they may trigger more serious arrhythmias, such as ventricular tachycardia or ventricular fibrillation. PVCs occur in subjects with and without underlying heart disease.47 Frequent PVCs are a risk factor for sudden death and mortality in patients who previously had a myocardial infarction or patients with a decreased ejection fraction.48±50 Ejection fraction is a measurement for the output of the heart. Thus, patients with a diminished heart function who suffer from frequent PVCs have a higher risk of sudden death or mortality than similar patients who do not suffer from PVCs. Middle-aged men who had no earlier signs of cardiovascular disease, but in whom frequent PVCs occurred during exercise had a higher risk of cardiovascular death than men who did not have these PVCs during exercise.51 Hardarson et al.52 were the first to test in a small study whether supplementation with omega-3 fatty acids, in this case cod liver oil, would prevent PVCs. In that study cod liver oil did not affect PVCs.52 Sellmayer et al. included 68 patients with at least 2000 spontaneous PVCs per 24 hours. They considered a reduction in PVCs of more than 70% clinically relevant. After the supplementation period, 44% of the patients in the fish oil group showed such a reduction in PVCs, whereas only 15% of the patients in the placebo group showed a similar reduction (p < 0:01).53 As pre-study to gain information that could be used in the SEAFOODplus project we did a study with 84 patients who suffered from at least 1440 PVCs per 24 hours (on average at least one per minute). Patients randomly received daily either 1.5 grams omega-3 fatty acids from fish oil or placebo oil. PVCs were registered by two 24-hour Holter recordings at the start of the intervention period and two after 14 weeks of intervention. The average number of PVCs decreased 6% more in the fish oil group than in the placebo group. This small difference was not significant.54 This is in line with two small studies which also showed no significant effects of omega-3 fatty acids on the number of PVCs.55,56 In contrast, in a study with 33 patients with on average 200 PVCs per 24 hours, six months of intervention with

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1 gram of fish oil per day resulted in a significant reduction in PVCs.57 If all currently available studies are taken together it is questionable whether intake of omega-3 fatty acids can reduce PVCs.

10.4 Possible mechanisms of effects of omega-3 fatty acids on the heart Several animal and in vitro models have been used to gain more insight into the possible mechanism underlying anti-arrhythmic properties of omega-3 fatty acids. Billman et al.58,59 used a dog model in which a main coronary artery could be occluded. The dogs were trained to run on a treadmill and at the end of the exercise the artery was occluded. Dogs that developed ventricular arrhythmia after the occlusion of the artery were included in the study. Intravenous infusion of fatty acids from fish oil led to less arrhythmia in these dogs after the same exercise programme.58,59 In rats and marmoset monkeys electrophysiological stimulation was used to trigger ventricular fibrillation. A diet rich in fish oil prevented these induced arrhythmias in the rats and marmoset monkeys.60±62 An important disadvantage of these studies is that pro-arrhythmic behaviour can never be detected because only animals that show reproducible arrhythmia are included in the studies. In the framework of the SEAFOODplus project Coronel et al.63 showed in a pig study that a diet rich in omega-3 fatty acids led to more life-threatening arrhythmias than a control diet. The study suggested that intake of omega-3 fatty acids reduced excitability and did not prevent, but rather caused arrhythmia during induced ischemia. Charnock suggested that eicosanoids may prevent ventricular fibrillation during myocardial ischaemia and reperfusion. It is known that omega-3 fatty acids influence the production of several eicosanoids. These eicosanoids may make the heart less vulnerable to arrhythmias and in this way prevent ventricular fibrillation.64 Another option is that omega-3 fatty acids cause increased membrane fluidity65 through incorporation in the membrane phospholipids of the cardiomyocytes.66 Although it is known that feeding omega-3 fatty acids leads to increased levels in the membranes it is unknown whether this phenomenon indeed affects electrophysiology in the whole heart in vivo. Cardiomyocytes from animals can retain their own rhythm when isolated and cultured. Omega-3 fatty acids have been shown to change the conductance of the membrane of these cardiomyocytes.67 This can be expected to affect the arrhythmogenic property of the cells and thereby of the whole heart. In vitro studies have generated several possible mechanisms of action. Ion channels are very important for good conductance of the sodium, calcium and potassium currents through the heart cell membranes. This is essential for a regular heart rhythm. Acutely administered omega-3 fatty acids reduce the sodium channels.68±70 However, incorporated omega-3 fatty acids do not seem to do so.71,72 Both acutely administered and incorporated omega-3 fatty acids reduce the so-called L-type calcium current. This current contributes to the duration of

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the ventricular action potential and is responsible for the plateau. Acute administration of omega-3 fatty acids lowers the plateau.73,74 Incorporation of omega-3 fatty acids in the sarcolemma leads not only to a lower plateau of the action potential, but also inhibits `reopening' of the calcium channel at plateau potential. This may have pro-arrhythmic as well as anti-arrhythmic properties.71 Verkerk et al. suggest that this may explain why omega-3 fatty acids seem protective against arrhythmias in patients with healed infarction, but not in patients with acute ischemia (angina pectoris). Thus, whether dietary fish oil is anti-arrhythmic or pro-arrhythmic may depend on the underlying disease.71 Thus, animal and in vitro experiments suggest mechanisms by which omega3 fatty acids may affect arrhythmia, but the exact mechanism is still unclear. Furthermore, the effect of fish oil may depend highly on the underlying pathology. Within the currently ongoing SEAFOODplus project we are doing further animal and in vitro studies to investigate this dependence on the underlying pathology.

10.5 Conversion and metabolism of omega-3 fatty acids in the human body One question that remains is whether alpha-linolenic acid from vegetable sources can have similar effects to the very long-chain omega-3 fatty acids from fish. If alpha-linolenic acid by itself could prevent heart disease, it could be an alternative for EPA and DHA in that respect. However, knowledge on the direct effects of alpha-linolenic acid on heart disease is very limited. A limited number of cohort studies have been performed. Our meta-analysis including these cohort studies suggested that alpha-linolenic acid may protect against heart disease. However, it was based on only a few studies.75 In addition, three earlier trials have been published on this subject. However, in the Lyon Diet Heart trial the intervention with alpha-linolenic acid was part of a complete dietary advice, which makes it impossible to single out alpha-linolenic acid as the effective factor.76 The results of the two other trials cannot be believed because the main author Singh has been accused of fraud.77,78 Thus, it is too early to make any statements about alpha-linolenic acid being protective against heart disease or not. Alpha-linolenic acid is the mother compound of the longer chain fatty acids. Enzymes, named desaturases and elongases are used to convert the precursor alpha-linolenic acid in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The desaturase makes it possible to add an extra double bond to the molecule; the elongase is used to lengthen the carbon atom chain with two carbon atoms. Omega-3 fatty acids use the same desaturases and elongases as omega-6 fatty acids. Alpha-linolenic acid is the precursor of the omega-3 series, whereas linoleic acid (C18:2 n-6) is the precursor of the omega-6 series. This implies that there is competition between omega-3 and omega-6 fatty acids with respect to the use of desaturases and elongases. The absolute amounts of the

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consumed alpha-linolenic acid and linoleic acid do affect the conversion more than the ratio between the omega-3 and omega-6 fatty acids.79,80 The conversion of linoleic acid to arachidonic acid (C20:5 n-6) goes easier than the conversion of alpha-linolenic acid to DHA. Only approximately 7% of the alpha-linolenic acid consumed with the diet actually reaches the phospholipid fraction of the plasma. Almost all absorbed alpha-linolenic acid is converted to EPA, but less than 1% is via docosapentaenoic acid (C22:5 n-3) converted to DHA. Thus, this last step in the conversion is the rate limiting one.81 Therefore it can be stated that alpha-linolenic acid cannot be used to substitute DHA in the diet. Humans need fish, fish products or other sources of DHA to ensure a sufficient intake of DHA.

10.6

Future research

Future research should focus on which groups of healthy subjects and patients will benefit from omega-3 fatty acids and which groups will not. Furthermore, knowledge on effects of alpha-linolenic acid is still very limited. It would therefore be very useful to learn more about health effects of this fatty acid.

10.7

Sources of further information and advice

For further information I refer to the overview of Mozaffarian et al. This overview again makes clear that the benefits of consuming fish, fish products and fish oil are greater than the risks.82 Therefore, the advice to the general population should be to consume fish twice a week of which at least one time should be fatty fish.

10.8 1. 2. 3. 4. 5. 6.

References

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Improving seafood products for the consumer Dietary fish oil prevents ventricular fibrillation following coronary artery occlusion and reperfusion. Am Heart J 1988; 116(3): 709±17. MCLENNAN PL, BRIDLE TM, ABEYWARDENA MY, CHARNOCK JS. Comparative efficacy of n-3 and n-6 polyunsaturated fatty acids in modulating ventricular fibrillation threshold in marmoset monkeys. Am J Clin Nutr 1993; 58(5): 666±9. CORONEL R, WILMS-SCHOPMAN FJ, DEN RUIJTER HM, et al. Dietary n-3 fatty acids promote arrhythmias during acute regional myocardial ischemia in isolated pig hearts. Cardiovasc Res 2007; 73: 386±94. CHARNOCK JS. Omega-3 polyunsaturated fatty acids and ventricular fibrillation: the possible involvement of eicosanoids. Prostaglandins Leukotrienes and Essential Fatty Acids 1999; 61(4): 243±7. LEIFERT WR, MCMURCHIE EJ, SAINT DA. Inhibition of cardiac sodium currents in adult rat myocytes by n-3 polyunsaturated fatty acids. J Physiol Lond 1999; 520 Pt 3: 671±9. MCLENNAN PL. Myocardial membrane fatty acids and the antiarrhythmic actions of dietary fish oil in animal models. Lipids 2001; 36 Suppl. S: S111±S14. LEAF A, KANG JX, XIAO YF, BILLMAN GE. n-3 fatty acids in the prevention of cardiac arrhythmias. Lipids 1999; 34 Suppl: S187±S9. KANG JX, LEAF A. Evidence that free polyunsaturated fatty acids modify Na+ channels by directly binding to the channel proteins. Proc Natl Acad Sci USA 1996; 93(8): 3542±6. XIAO YF, KANG JX, MORGAN JP, LEAF A. Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes. Proc Natl Acad Sci USA 1995; 92(24): 11000±4. XIAO YF, WRIGHT SN, WANG GK, MORGAN JP, LEAF A. Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the alpha-subunit of the human cardiac Na+ channel. Proc Natl Acad Sci USA 1998; 95(5): 2680±5. VERKERK AO, VAN GINNEKEN AC, BERECKI G, et al. Incorporated sarcolemmal fish oil fatty acids shorten pig ventricular action potentials. Cardiovasc Res 2006; 70(3): 509±20. LEIFERT WR, JAHANGIRI A, SAINT DA, MCMURCHIE EJ. Effects of dietary n-3 fatty acids on contractility, Na(+) and K(+) currents in a rat cardiomyocyte model of arrhythmia. J Nutr Biochem 2000; 11(7±8): 382±92. XIAO YF, GOMEZ AM, MORGAN JP, LEDERER WJ, LEAF A. Suppression of voltage-gated Ltype Ca2+ currents by polyunsaturated fatty acids in adult and neonatal rat ventricular myocytes. Proc Natl Acad Sci USA 1997; 94(8): 4182±7. FERRIER GR, REDONDO I, ZHU J, MURPHY MG. Differential effects of docosahexaenoic acid on contractions and L-type Ca2+ current in adult cardiac myocytes. Cardiovasc Res 2002; 54(3): 601±10. BROUWER IA, KATAN MB, ZOCK PL. Dietary alpha-linolenic acid is associated with reduced risk of fatal coronary heart disease, but increased prostate cancer risk: a meta-analysis. J Nutr 2004; 134(4): 919±22. DE-LORGERIL M, RENAUD S, MAMELLE N, et al. Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease. Lancet 1994; 343(8911): 1454±9. WHITE C. Suspected research fraud: difficulties of getting at the truth. BMJ 2005; 331(7511): 281±8. HORTON R. Expression of concern: Indo-Mediterranean Diet Heart Study. Lancet 2005; 366(9483): 354±6. MCLENNAN PL, ABEYWARDENA MY, CHARNOCK JS.

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WIJENDRAN V, HAYES KC. Dietary n-6 and n-3 fatty acid balance and cardiovascular health. Annu Rev Nutr 2004; 24: 597±615. 80. GOYENS PL, SPILKER ME, ZOCK PL, KATAN MB, MENSINK RP. Conversion of alphalinolenic acid in humans is influenced by the absolute amounts of alpha-linolenic acid and linoleic acid in the diet and not by their ratio. Am J Clin Nutr 2006; 84(1): 44±53. 81. GOYENS PL, SPILKER ME, ZOCK PL, KATAN MB, MENSINK RP. Compartmental modeling to quantify alpha-linolenic acid conversion after longer term intake of multiple tracer boluses. J Lipid Res 2005; 46(7): 1474±83. 82. MOZAFFARIAN D, RIMM EB. Fish intake, contaminants, and human health: evaluating the risks and the benefits. JAMA 2006; 296(15): 1885±99.

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Part III Ensuring seafood safety

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11 Introduction to Part III: ensuring seafood safety B. DoreÂ, Marine Institute, Ireland

11.1

Risks associated with seafood consumption

Seafood is generally recognised as a nutritious and healthy source of food. Despite this, large and relatively frequent outbreaks of food poisoning associated with seafood consumption have been recorded. A wide range of risks have been associated with seafood including chemical contamination, biotoxins and allergic reactions. However one of the most clearly identified acute risks to consumers results from microbiological agents. In particular, even a cursory review of the literature identifies human enteric viruses found in sewage contaminated bivalve molluscs, pathogenic bacteria (e.g. Vibrio species) and the formation of biogenic amines (histamine poisoning) caused through bacterial activity in particular fishery products as among the most significant illnesses associated with seafood consumption. 11.1.1 Microbiological risks Microbiological risks associated with the consumption of seafood can occur either as a direct result of contamination of the aquatic environment with pathogens through human waste disposal or from naturally occurring microorganisms which can be pathogenic to humans. Human faecal pollution in the marine environment resulting in the microbiological contamination of bivalve molluscan shellfish such as oysters, mussels and clams has long been recognised as a significant threat to human health. As long ago as the late 1800s, the consumption of raw oysters (then a food for the masses) was associated with Typhoid fever caused by the bacterial pathogen Salmonella Typhi. The first link

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to viral illness associated with sewage contaminated shellfish was made relatively more recently (Roos, 1956). Despite this, viral illness particularly infectious hepatitis caused by hepatitis A virus (HAV) and gastroenteritis caused by noroviruses (NoV), is generally recognised as one of the most significant risks to consumers of seafood (Lees, 2000). In fact it is worth noting that the largest recorded outbreak of food-borne illness ever recorded was associated with the consumption of clams in Shanghai in 1988, when almost 300,000 people contracted hepatitis A (Halliday et al., 1991). The ability of bivalve shellfish to concentrate microbiological contaminants through their filter feeding activity, and a widespread consumer preference to eat shellfish products raw or only lightly cooked are fundamental reasons for the high level of illness associated with their consumption (Lees, 2000). Viral infections are not generally associated with the consumption of fish except occasionally when contamination of the product may occur at the point of sale and post processing due to poor hygienic practice. Despite the extensive implementation of sanitary controls to prevent this problem worldwide, outbreaks of viral illness associated with bivalve shellfish consumption continue to occur throughout the world. Bacterial illness associated with the consumption of seafood also occurs (Gillespie et al., 2001; Olsen et al., 2000). This may be as the result of contamination of the marine environment by human and potentially animal waste, pathogenic bacteria occurring naturally in the aquatic environment or as a result of the growth of pathogenic bacteria post harvest of the seafood product. Illness may be caused by ingestion of toxins pre-formed in the seafood by bacteria during growth (intoxication) or ingestion of sufficient number of viable bacteria, (minimum infectious dose), to initiate bacterial growth and cause illness (infection). Therefore ingestion of viable bacteria is not required in order for illness to occur. Toxins are often thermostable and can withstand extensive processing without a reduction in toxicity. Principal among the bacteria causing intoxications associated with seafood consumption are Clostridium botulinum, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Bacteria that have commonly been associated with causing infections following seafood consumption include Listeria monocytogenes, Salmonella sp., Shigella sp., Vibrio parahaemolyticus and Vibrio vulnificus. Salmonella sp., Shigella sp. and Vibrio cholera are associated human faecal waste whereas Vibrio vulnificus and Vibrio parahaemolyticus are naturally occurring pathogens of marine environments. The role these bacterial pathogens play in seafood borne outbreaks of illness are reviewed in detail in Chapter 14. A further highly significant hazard associated indirectly with bacterial growth in seafood is the formation of biogenic amines. Biogenic amines are produced by decarboxylation of amino acids present in fish proteins as a result of the metabolic activity of bacteria commonly found in seafood products. Biogenic amines can provoke a severe and rapid allergic reaction when ingested but often symptoms are mild and illness may not be recorded. Histamine produced from histidine is the biogenic amine most often associated with intoxication and has resulted in the reaction being termed histamine fish poisoning (HFP). In turn

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HFP is often called scombroid fish poisoning because of the frequent association with scombroid fish such as tuna (Thunnus spp.) and mackerel (Scomber spp.) although non-scombroid fish have also been implicated in outbreaks. Once formed biogenic amines are thermostable and are capable of withstanding temperatures in excess of those associated with cooking. HFP has usually been associated with temperature abuse at some point in the supply chain which allows bacterial growth (Lehane and Olley, 2000). However, despite strict legislative controls in Europe, and elsewhere throughout the world, and the stringent use of good hygienic practice, outbreaks of HFP continue to occur. More recent research has demonstrated that biogenic amine production may be increasingly linked to the growth of psychrotolerant bacteria (Emborg and Dalgaard, 2006; Kanki et al., 2007) and the recent discovery of a new species of bacteria Morganella psychrotolerans (Emborg et al., 2006) capable of inducing biogenic amine formation at low temperatures questions the effectiveness of current health controls to protect public health. 11.1.2 Further acute risks associated with seafood consumption Most parasites present in fish, and in particular helminths (worms), reside in the visceral organs and intestinal tract and are thus usually discarded during processing and are generally of little public health concern (Olsen, 1987). In addition, controls within Europe require that fish products that will be consumed raw must be frozen prior to sale and this appears to be effective in destroying the parasites of concern. Therefore in a European setting, parasitic infections associated with fish consumption can occur but are rare and in general cause only mild disease. However, in some parts of the world, notably Southeast Asia, parasitic infections can be prevalent because of the cultural consumption patterns. Marine biotoxins produced by naturally occurring phytoplankton are widely distributed in the environment and contaminated seafood can represent a significant risk to consumers (Ahmed, 1991). In particular, bio-accumulation of biotoxins by bivalve shellfish represents the most significant risk for European consumers. The most important shellfish associated toxic syndromes caused by biotoxins are Paralytic Shellfish Poisoning (PSP) initiated by saxitoxin, Azaspiracid Poisoning, Amnesic Shellfish Poisoning initiated by domoic acid, Diarrhetic Shellfish Poisoning (DSP) initiated by okadaic acid and pectenotoxins and Neurotoxic Shellfish Poisoning initiated by brevotoxins. Toxic events associated with PSP are rapid in onset, attack the nervous system and can cause death due to paralysis of the respiratory system in severe cases. Toxicity associated with other intoxications vary in the range of symptoms and severity. Despite the widespread distribution of biotoxins in the marine environment and potential severity of the syndromes caused, intoxications are a relatively rare event in Europe. Data from the UK over a seven-year period between 1992 and 1999 recorded only one outbreak of DSP associated with shellfish consumption (Gillespie et al., 2001). The application of extensive biotoxin monitoring

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programmes and regulatory programmes in place in Europe appear to be relatively successful in controlling the public health risk. A further risk associated with the presence of biotoxins in seafood is ciguatera poisoning. Ciguatera poisoning is a significant intoxication associated with the consumption of tropical and subtropical fish (Isbister and Kiernan, 2005). Fish become contaminated by accumulating several species of naturally occurring dinoflagellates (algae). Intoxications of ciguatera in humans usually involve a combination of gastrointestinal, neurological, and cardiovascular disorders. In Europe ciguatera poisoning is rare and associated with imported fish.

11.2 Relative incidence of microbiological illness associated with seafood The true incidence of illness associated with the consumption of seafood, as for all foods, is of course unknown. The extent and quality of national data collection on food-borne outbreaks is variable from country to county and disease statistics presented can only be treated as guide to trends and the relative extent of disease burden. Despite this, a review of available data reveals that the incidence of disease varies considerably from country to country depending on consumption patterns. Two major factors that clearly influence the incidence of illness are the level of seafood consumption and cultural considerations such as the willingness and desirability of consuming raw and lightly cooked seafood products. For example, reports have indicated that seafood outbreaks in the USA constituted just 11% of the total food-borne outbreaks compared with over 70% in Japan which consumes a much larger amount of seafood and has tradition of raw consumption (Eyles, 1986). The most complete data on food-borne outbreaks of illness exist in the UK and USA. In the USA during the period between 1993 and 1997 shellfish and fish represented 1.7% (number 47) and 5.1% (number 140) of all identified food-borne outbreaks respectively (CSPI, 2001; Olsen et al., 2000). Although fish consumption was responsible for a higher number of outbreaks than shellfish, the number of cases of illness associated with shellfish outbreaks (1,868) were considerably higher compared with those associated with the fish outbreaks (696). This possibly reflects the highly infectious nature of the viral illness often associated with bivalve shellfish consumption. Further data over an eight-year period from 1990 to 1998 from the USA identified that HFP was responsible for 50% of outbreaks and almost 50% of cases of disease associated with fish consumption (CSPI, 2001). During the same period V. parahaemolyticus was identified as the cause of 27% of outbreaks of food poisoning associated with molluscan shellfish. This was closely followed by norovirus which was responsible for 23% of seafood associated outbreaks. However, it is significant that from these outbreaks NoVs were responsible for 66% of cases of illness associated with shellfish. In the UK between 1992 and 1999, 1,425 outbreaks of infectious intestinal disease from all foodstuffs were recorded. Seafood was confirmed as being

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responsible for 148 of these outbreaks (Gillespie et al., 2001). Of the seafood outbreaks scombrotoxin was identified as responsible for 47 outbreaks which were all related to fish consumption. Viruses were responsible for 26 outbreaks which were generally related to consumption of bivalve molluscs. A variety of bacterial infections and intoxications made up the majority of the remaining identified outbreaks. However, it is worth noting that in 52 of the seafood outbreaks the aetiological agent was not accounted for. Thirty-one of these outbreaks were associated with molluscs and it likely that these were viral in nature but that a lack of sensitive methods for detecting viruses in shellfish during this period failed to identify the causative agent. While data may be less clear in other European countries further studies would also support the general view that HFP associated with fish consumption and viral infection associated with mollusc consumption are the principal health risks associated with seafood within Europe. Risks of bacterial infections or intoxications appear to be significantly lower than the threat of HFP or viral infection. However, an increasing reliance on imported products may increase the risk of bacterial infection associated with seafood consumption. In particular the risk of Vibrio sp. infection associated with imports of seafood from warmer climates should not be underestimated.

11.3 Control of risks associated with seafood and legal requirements As may be expected with such well-defined and recognised public health risks a comprehensive and wide ranging suite of control measures and regulations, aimed at reducing these risks, is in place throughout the world. In Europe a new suite of food hygiene regulations strictly controlling the placing on the market of all foods, including seafood, came into effect on 1 January 2006. These lay down procedures to be followed by primary producers, processors and competent authorities in Member States. The three principal regulations of importance are laid out below: 1. Hygiene 1. Regulation EC No. 852/2004 on the hygiene of foodstuffs. 2. Hygiene 2. Regulation EC No 853/2004 Laying down specific hygiene rules for food of animal origin. 3. Hygiene 3. Regulation EC No 854/2004 Laying down specific rules for the organisation of official control on products of animal origin intended for human consumption. The Hygiene 1 regulations sets down the general requirements for food operators and establishes that the principle responsibility for food safety lies with the food business operator (FBO). The regulation introduces the requirements for application of hazard analysis critical control point (HACCP) principles. However this does not currently apply to primary production. Hygiene 2 gives the requirements for foods of animal origin for industry. The specific rules for

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the production of live bivalve molluscs and fishery products are given in these regulations. Hygiene 3 concerns the organisation and application of official controls for products of animal origin by competent authorities in Member States. In addition EU Regulation 2073/2005 sets out the microbiological criteria which food stuffs, including seafood, must meet. Failure to comply with these standards implies the food is unfit for human consumption and may not be placed on the market. Standards in this regulation include acceptable limits on levels of histamine in fish susceptible to biogenic formation and causing HFP. In addition to the implementation of regulation, the adoption of quality control programmes such as HACCP plans, good hygiene practice and good manufacturing practice are commonly employed in the seafood industry and form the cornerstone of efforts to produce safe products for the consumer (Huss. et al., 2004). Despite the high level of regulation and quality programmes in use in the seafood industry, problems associated with microbiological contamination of seafood continue to occur. In particular the relatively high incidence of HFP and viral infection associated with bivalve shellfish consumption indicates that there is a systemic and underlying failure of the controls applied to these hazards rather than the application of poor handling and management procedures during production and processing.

11.4

Contribution of SEAFOODplus to seafood safety

When developing the work programme for the seafood safety pillar within SEAFOODplus an early decision was made to focus on the principal risks associated with seafood consumption. Therefore three areas were identified for which projects in SEAFOODplus should concentrate on. These were the risks associated with viruses in shellfish, HFP poisoning associated with fish and finally bacterial risks associated with seafood in general. Four projects were formulated to cover these areas, two to cover aspects of viruses in shellfish and one on each of the two other areas. In the area of viruses in shellfish the establishment of reliable robust standardised methods for the detection of HAV and NoV were recognised as a priority. In Europe there is a growing desire to introduce virus standards into legislation to control the virus risk. However, the lack of standardised methods has prevented this from occurring. Therefore a project was initiated which aims to develop procedures for detecting HAV and NoV in molluscs. The methods developed will fulfil the requirements of the International Standards Organisation (ISO) for standard methods. The project is targeting molecular procedures and in particular real-time polymerase chain reaction (PCR) technology to develop these methods. A further project in the area of viruses in shellfish is aimed at developing risk-based management procedures in shellfish harvest areas to reduce the virus risk associated with shellfish. A third project in the seafood safety pillar addresses the issue of developing molecular-based methods for detecting bacteria in seafood. The

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projects focus on the developing procedures for the detection Vibrio sp. and in particular procedures that are capable of distinguishing between pathogenic and non-pathogenic strains of Vibrio parahaemolyticus. Recognising the significance of HFP as a major cause of food poisoning the final project in the seafood safety pillar is aimed at assessing and managing consumer exposure with particular focus on the role of psychrotolerant bacteria in the formation of biogenic amines. The following four chapters describe in greater detail the three major areas of risk associated with seafood consumption identified in the SEAFOODplus project. They explain the research undertaken to address these issues and highlight the major achievements in each area.

11.5

Future trends

There is no doubt that the progress made in understanding and combating food poisoning risks associated with seafood in recent years and the work presented in the following pages will have a positive impact on the number of incidences of illness associated with seafood consumption in Europe. An understanding of the causes leading to illness associated with seafood, such as identification of the role of psychrotolerant bacteria in HFP and conditions leading to virus contamination, will identify critical control points which can be managed to prevent illness. Improved procedures for detecting pathogens in seafood will also have a positive effect, allowing better control of the risk. Despite this, there are new challenges on the horizon for risk managers in Europe. Globalisation of trade and an increasing reliance on imported seafood into the European Union present particular risks. Developing appropriate procedures, which ensure the equivalent safety of products entering Europe, presents a considerable scientific and political challenge in the context of world trade agreements. Maintaining a high level of consumer protection in the light of potentially increased threats to food safety, for example increased risk of ciguatera poisoning with increased import of tropical fish, should remain a high priority for risk managers. This places a significant responsibility on the scientific community to develop appropriate tools for product monitoring and to provide the scientific knowledge to put the results from such monitoring into context. Further future threats to public health through seafood consumption which require careful management are likely to occur as a result of global warming. Global warming is now an accepted fact and the recent figures indicate that the earth's temperatures may rise by as much 6.4 ëC by the year 2100 (IPCC, 2007). This has potential implications for the incidence of food poisoning in general and for incidents associated with shellfish consumption in particular. Already increasing seawater temperatures have been linked to an outbreak of gastroenteritis caused by V. parahaemolyticus associated with the consumption of Alaskan raw oysters in waters previously considered too cold to support the growth of this organism (McLaughlin et al., 2005). Increasing temperatures also

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pose difficulties for controlling product temperature at all stages of the supply chain. Temperature control is critical in preventing the proliferation of directly pathogenic bacteria and bacteria responsible for the production of biogenic amines in fish. Global warming is also linked to increasing storms events. Storm events are in turn linked to the overflow of untreated sewage into aquatic environments where sewage treatment plant infrastructure cannot cope with surges in influent volumes. Such events can lead to viral contamination of shellfisheries and subsequent outbreaks associated with shellfish consumption. Increased contamination of aquatic environments may also lead to infiltration of water supplies used in processing of seafood products, causing contamination of the product. This may be a particular problem in developing countries, where both sewage and water treatment processes may be less developed. Clearly the threats to public health from globalisation and global warming require careful consideration and developing a robust understanding of the processes involved in seafood-borne illnesses and developing tools to monitor for pathogens and toxins will be at the forefront of developing controls to combat these problems.

11.6

References

(1991). Naturally occurring seafood toxins. Journal of Toxicology Toxin Reviews 10, 263±287. CSPI (2001). Outbreak Alert. Closing the Gaps in our Federal Food-safety Net. Washington DC: Centre for Science in the Public Interest. EMBORG, J. and DALGAARD, P. (2006). Formation of histamine and biogenic amines in cold-smoked tuna: An investigation of psychrotolerant bacteria from samples implicated in cases of histamine fish poisoning. Journal of Food Protection 69, 897±906. EMBORG, J., DALGAARD, P. and AHRENS, P. (2006). Morganella psychrotolerans sp. nov., a histamine-producing bacterium isolated from various seafoods. International Journal of Systematic and Evolutionary Microbiology 56, 2473±2479. EYLES, M. J. (1986). Microbiological hazards associated with fishery products. CSIRO Food Research Q 46, 8±16. GILLESPIE, I. A., ADAK, G. K., O'BRIEN, S. J., BRETT, M. M. and BOLTON, F. J. (2001). General outbreaks of infectious intestinal disease associated with fish and shellfish, England and Wales, 1992±1999. Communicable disease and public health 4, 117± 123. HALLIDAY, M., KANG, L. Y., ZHOU, T. K., HU, M. D., PAN, Q., FU, T. Y., HUANG, Y. S. and HU, S. L. (1991). An epidemic of hepatitis A attributable to the ingestion of raw clams in Shanghai, China. Journal of Infectious Diseases 164, 852±859. HUSS., H. H., ABABOUCH, L. and GRAM, L. (2004). Assessment and management of seafood safety and quality. FAO Fisheries Technical Paper 444. Food and Agriculture Organization of the United Nations, Rome, 1±227. IPCC (2007). Climate Change 2007: The Physical Basis ± Summary for Policy Makers. Geneva: Intergovernmental Panel on Climate Change. ISBISTER, G. and KIERNAN, M. (2005). Neurotoxic marine poisoning. The Lancet Neurology 4, 219±228. AHMED, F. E.

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and BABA, E. (2007). Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury. Applied and Environmental Microbiology 73, 1467±1473. LEES, D. N. (2000). Viruses and bivalve shellfish. International Journal of Food Microbiology 59, 81±116. LEHANE, L. and OLLEY, J. (2000). Histamine fish poisoning revisited. International Journal of Food Microbiology 58, 1±37. MCLAUGHLIN, J., DEPAOLO, A., BOPP, C., et al. (2005). Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. New England Journal of Medicine 353, 1463±1470. OLSEN, R. E. (1987). Marine fish parasites of public health importance. In Seafood Quality Determination, pp. 339±355. Edited by K. D. E. and J. Liston. Netherlands: Elsevier Science. OLSEN, S. J., MACKINNON, L. C., GOULDING, J. S., BEAN, N. H. and SLUTSKER, L. (2000). Surveillance for foodborne-disease outbreaks ± United States, 1993±1997. Morbidity and Mortality Weekly Report 49, 1±59. ROOS, B. (1956). Hepatitis epidemic conveyed by oysters. Svenska Lakartidningen 53, 989±1003. KANKI, M., YODA, T., TSUKAMOTO, T.

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12 Detecting virus contamination in seafood A. Bosch and R. M. PintoÂ, University of Barcelona, Spain, D. H. Lees, Centre for Environment, Fisheries and Aquaculture Science, United Kingdom, C.-H. von Bonsdorff, University of Helsinki, Finland, L. Croci and D. De Medici, Instituto Superiore di Sanita, Italy and F. S. Le Guyader, Ifremer, France

12.1

Introduction: viruses and shellfish contamination

Viruses are obligate intracellular parasites depending on living cells for replication and usually infecting only a restricted range of hosts. Thus viruses cannot multiply in dead or processed food and therefore are not responsible for food spoilage. Human viruses may contaminate seafood but since the commercially exploited species (fish and shellfish) are widely divergent from humans, there is no evidence that they can act as a replication vector. Viral problems are thus limited to the role of seafood in passive transfer of viruses to humans. The viruses most adapted and likely to be carried in this way are those transmitted by the fecal-oral route. These include viral agents causing gastrointestinal disease in humans but also agents such as hepatitis A virus and polio virus which although being transmitted by the fecal-oral route, and often having a growth phase in the gut, exhibit their classical clinical symptoms elsewhere in the body. Such viruses can contaminate seafood at source through fecal pollution of the aquatic environment, or through poor hygiene during seafood processing (Fig. 12.1). Many viruses transmitted by the fecal-oral route are widely prevalent in the community and infected individuals can shed many millions of virus particles in their feces. Consequently viruses of many types occur in large numbers in municipal sewage and may also occur in other sources of human fecal contamination (Bosch, 1998). Sewage treatment processes are generally only partially effective at virus removal (depending on the treatment level) and may also be bypassed during periods of heavy rain or during emergencies (see Chapter 13). Other sources of fecal pollution, for example

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Possible sources of contamination for shellfish growing in coastal area (source: Ifremer, www.ifremer.fr/envlit/).

septic tank overflows or boat discharges, may also contribute to marine contamination. Following marine discharge from such sources, viruses are capable of survival for long periods (Callahan et al., 1995; Gantzer et al., 1998; Nasser, 1994). Thus, seafood harvested from coastal locations is vulnerable to contamination with enteric viruses of potential health significance for man (Fig. 12.1). However, of the many harvested seafood species, only the filter-feeding bivalve molluscan shellfish (bivalve molluscs) have consistently proven to be an effective vehicle for the transmission of human viral diseases (Lees, 2000). Bivalve molluscs are a type of shellfish that have two shell halves which hinge together. Species commonly commercially exploited in Europe include the native or flat oyster (Ostrea edulis), pacific oyster (Crassostrea gigas), common blue mussel (Mytilus edulis) and Mediterranean blue mussel (Mytilus galloprovincialis), cockles (Cerastoderma edule), king scallops (Pecten maximus) and queen scallops (Chlamys opercularis), and various clams including the native clam or palourde (Tapes descussatus), the hard shell clam (Mercenaria mercenaria), the manila clam (Tapes philippinarum), and the razor shell clam (Ensis spp.). The bivalve molluscs are an effective vehicle for transmission of enteric disease agents for several reasons. A principal factor is that these animals obtain their food by filtering small particles from their surrounding water. In the process of filter-feeding, bivalve molluscs may also concentrate and retain human pathogens derived from sewage contamination of growing waters. Many bivalve molluscs are harvested from sheltered in-shore coastal locations, such as river estuaries, which are often also susceptible to fecal

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pollution (see Chapter 13). Important additional compounding factors are that a number of species of bivalve mollusc are traditionally consumed raw, or only lightly cooked, and are also consumed whole, i.e. including the viscera, which contains the bulk of contaminants. Disease incidents are most commonly associated with species consumed raw (or lightly cooked) and whole, such as oysters and clams and infrequently, or not at all, with species that are well cooked and where the viscera is not consumed, such as scallops. Disease incidents associated with bivalve molluscs have been extensively reported and have been reviewed by several authors (Richards, 1985; Rippey, 1994; Jaykus et al., 1994, Lees, 2000). The potential of bivalve molluscs to transmit enteric pathogens acquired through sewage pollution of growing areas first became recognised in the late 19th and early 20th century with numerous outbreaks of typhoid fever in several European countries, the US and elsewhere (Allen, 1899). Since this time there has been increasing recognition of the importance of human enteric viruses as the predominant aetiological agent in human illness incidents associated with consumption of bivalve molluscs. It is now well recognised that the most common illness associated with bivalve mollusc consumption is gastroenteritis caused by Norovirus (NoV). Other gastro enteric viruses, such as astroviruses and parvoviruses, have also occasionally been implicated in shellfish-related outbreaks, although their true epidemiological significance is not clear. NoV causes a relatively `mild' gastroenteritis, often including nausea, diarrhoea, vomiting, fever and abdominal pain. The incubation period is 1 to 4 days with a duration of about 2 days and generally followed by complete recovery. NoV has previously been known as Norwalk-like virus or as small round structured virus (SRSV). Human NoV cannot be propagated using cell culture (Duizer et al., 2004) therefore characterisation and classification has been achieved largely using molecular techniques. It is now known that the Norovirus genus belongs to the Caliciviridae family and comprise a genetically diverse group of viruses which can be separated beneath this level into genogroups, clusters or genotypes, and individual strains (Zheng et al., 2006; Hansman et al., 2006). NoVs infecting humans group into genogroup one (up to 8 clusters), and genogroup two (up to 17 clusters) (Zheng et al., 2006). Genetically related animal NoV strains have also been described (Oliver et al., 2006); however, currently there is no evidence that they are capable of directly infecting man. The genetic diversity of NoV strains presents a difficult challenge for the design of molecular diagnostics capable of detecting all strains of health significance. It is now generally accepted that NoV is one of the most common causes of infectious intestinal disease in both outbreaks and in the community (Evans et al., 1998; Tompkins et al., 1999). Infections occur in all age groups including older children and adults. NoV is highly transmissible and often becomes noticeable through epidemic spread of diarrhoea and vomiting in closed communities such as hospitals, cruise and military ships and old people's homes. NoV appears to be prevalent throughout the world. Infected individuals shed large amounts of virus in their

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faeces thus domestic sewage and other polluted waters can normally be expected to be heavily contaminated with this virus (Lodder and Husman 2005), with the obvious risks for impacted bivalve molluscs. There are numerous reports in the scientific literature documenting the occurrence of NoV gastro enteric illness outbreaks associated with consumption of bivalve mollusc. These have been reviewed by several authors (Jaykus et al.,1994; Lees, 2000) and continue to occur (Doyle et al., 2004). The need for measures to more adequately protect the consumer against viral infection are widely noted in such outbreak reports. US FDA risk assessments estimate cases of NoV mediated gastroenteritis related to seafood consumption at some 100,000 per year (Williams and Zorn, 1997). In addition to these direct health consequences, bivalve molluscs may also present a potent vector for emergence of recombinant NoV strains of enhanced virulence following contamination with multiple strains from human (and potentially animal) sources. Mixed human infections following consumption of bivalve molluscs with multiple contaminating NoV strains has been commonly reported (Gallimore et al., 2005; Prato et al., 2004; Kageyama et al., 2004). The other fecal-oral transmitted virus of major significance in bivalve molluscs related outbreaks is hepatitis A virus (HAV). HAV is a positive-strand RNA virus classified in its own genus of Hepatovirus within the Picornaviridae. There is only a single major serotype of HAV with three human antigenic variants and a number of genotypes identified by sequence analysis (CostaMattioli et al., 2003). Compared to other enteric viruses HAV has an extended incubation period of about 4 weeks (range 2 to 6 weeks) and causes a serious debilitating disease progressing from a non-specific illness with fever, headache, nausea and malaise to vomiting, diarrhoea, abdominal pain and jaundice. HAV is self-limiting and rarely causes death but patients may be incapacitated for several months. Age has an important bearing on the severity of the infection with young children frequently experiencing only mild illness whereas overt hepatitis develops in the majority of infected adults. Recovery is complete and leads to long-term immunity from reinfection. HAV is a common endemic infection in developing countries with most children being seropositive by six years of age. However improving sanitary conditions in developed countries have lead to declining prevalence and resulted in large sectors of the population being susceptible to infection. HAV can be readily demonstrated in stools by molecular techniques (Yotsuyanagi et al., 1996) and has also been demonstrated in sewage effluents and polluted receiving waters (Tsai et al., 1994). Thus bivalve molluscs have frequently been implicated as food vehicles in outbreaks of hepatitis A (Klontz and Rippey 1991; Conaty et al., 2000; Bosch et al., 2001). With the advent of molecular diagnostic methods in more recent years the polymerase chain reaction (PCR) has been used to study the contamination of molluscan shellfish with NoV and HAV at the low concentrations found in field samples. Various studies have shown rather high rates of viral contamination of commercially produced bivalve shellfish placed on the market in a number of different countries (Costantini et al., 2006; Cheng et al., 2005; Chironna et al., 2002; Formiga-Cruz et al., 2002; Nishida et al., 2003; Boxman et al., 2006)

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illustrating the potential health hazards and the urgent need for diagnostic methods capable of identifying the risk and better protecting the consumer. The REFHEPA project within the SEAFOODplus consortium is aimed at the development of standardised procedures for the detection of HAV and NoV in bivalve molluscan shellfish to the point at which they could be used successfully in a routine diagnostic context to increase the safety of shellfish delivered to the consumer. The methodologies developed in the REFHEPA project have been successfully applied in another project, REDRISK, to detect viruses in naturally contaminated shellfish from France and Spain.

12.2

Methods for detecting viruses in shellfish

Viruses are present in shellfish in very low numbers; however, in sufficient amounts to pose a health risk (Bosch et al., 1994; SaÂnchez et al., 2002, Le Guyader et al., 2003, 2006a). This low contamination makes necessary the development of highly sensitive viral extraction methods ensuring the virus recovery from shellfish tissues. The hypothesis made in the 1980s, that viruses are concentrated in digestive diverticulum tissues (Metcalf et al., 1980), represented a major step for the progress of the extraction methodologies. This hypothesis was later confirmed by detection of HAV using an in situ system in oysters artificially contaminated following virus bioaccumulation (Romalde et al., 1994) as well as through the tissue-specific quantification of infectious enteric adenoviruses and rotaviruses in mussels previously contaminated by bioaccumulation of such viruses (Abad et al., 1997a). Additionally, a very interesting result has recently been described: the occurrence of a specific binding of Norwalk virus to oyster digestive tissues through the interaction with a N-acetylgalactosamine-containing receptor (Le Guyader et al., 2006b). Analysis of digestive tissues provides several advantages, including increased sensitivity, decreased processing time and decreased interference with RT-PCR (Atmar et al., 1995). Focusing the analysis of shellfish on the digestive tissues, where many of the viruses are concentrated, enhances assay performance by eliminating tissues (i.e. adductor muscle) that are rich in inhibitors but contain relatively little virus (Abad et al., 1997a). As a matter of fact, the digestive tissues represent about one tenth of the total animal weight for oysters and mussels. Except for small species, such as clams or cockles, because dissection may be technically difficult, most of recent methods are based on dissected tissues and thus will be discussed here. Methods cited in the literature are diverse, complex, poorly standardised and restricted to a few specialist laboratories. It seems obvious that quality control and quality assurance issues must be solved, as well as simplification and automatation, of molecular procedures before they could be adopted by routine monitoring laboratories. All these latter issues have been addressed in the REFHEPA project of SEAFOODplus. Extraction of enteric viruses from shellfish is based on several steps: virus elution from shellfish tissues, recovery of viral particles, and then virus

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concentration. The weight analysed generally ranges from 1.5 to 2 g of digestives tissues. Some recent methods propose larger weights for the first step but thereafter analysing only part of them (Boxman et al., 2006). Viruses are eluted from shellfish digestive tissues using various buffers (i.e. chloroformbutanol or glycine) before being concentrated either by polyethylene glycol precipitation or ultracentrifugation (Atmar et al., 1995; Nishida et al., 2003; Myrmel et al. 2004). Those approaches used in the analysis of whole shellfish meat, such as acidic adsorption prior to virus elution, are not applied to dissected tissues (Shieh et al., 1999; Mullendore et al., 2001). When working on dissected tissues, and applying molecular techniques, direct lysis of virus particles can also be used. For example, proteinase K, or Trizol and lysis of shellfish tissues using Zirconia beads and a denaturing buffer have all been used for virus and/or nucleic acid elution (Lodder-Verschoor et al., 2005; Jothikumar et al., 2005). A disadvantage of this direct approach is that a lower quantity of shellfish tissue is analysed in the RT-PCR assay. Since the most relevant shellfish-borne viral pathogens, enteric hepatitis viruses A and E and noroviruses, are non-culturable RNA viruses, RT-PCR and now real-time RT-PCR are the methods of choice to set up sensitive protocol for their detection. The methods used for nucleic acid extraction are dependent on those used for virus elution and concentration. Most methods are based on guanidium extraction either using the methods described by Boom et al. (1990) or using a kit, based on similar chemistry (QIAamp or RNeasy kit by QiagenÕ) (Shieh et al., 1999; Loisy et al., 2000; Schwab et al., 2000). Capsid lysis by proteinase K and then purification of nucleic acid using phenol-chloroform and CTAB precipitation is a more labour-intensive but was one of the first successful methods described (Atmar et al., 1993). One of the goals of the extraction methods is to remove inhibitors of the RT and PCR reactions sufficiently to allow detection of viral nucleic acids. Polysaccharides present in shellfish tissue are at least one substance that can inhibit the PCR reaction (Atmar et al., 1993). Several reported methods eliminate inhibitors to varying degrees, although no systematic evaluation of the efficiency of inhibitor removal has been performed, and only a few of them have been applied on naturally contaminated shellfish. Inhibitor elimination is difficult to evaluate and depending on the time of the year and shellfish life, different compounds may be present (Di Giralimo et al., 1977; Burkhardt and Calci, 2000). Internal control standards have been used to detect the presence of significant sample inhibition, and the amount of sample inhibition has varied depending upon the shellfish tissue being analysed (Atmar et al., 1995; Schwab et al., 1998; Le Guyader et al., 2000). Dilution of the extracted sample is the approach often used to overcome the inhibitor problem, leading to a smaller quantity of shellfish tissues being analysed. For most methods in the literature, the weight of digestive tissues analysed in each RT reaction varies between 0.01 g and 2.5 g. The method analysing the smallest shellfish tissue weight (0.01 g) is based on direct lysis of virus without a concentration step (Jothikumar et al., 2005), while the method analysing the largest tissue weight (2.5 g) is based upon direct extraction of all nucleic acids

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followed by purification of nucleic acid using a poly A capture (Goswami et al., 2002). It is important to promote methods allowing the biggest tissue analysis as it helps to improve the detection sensitivity. Beside the inhibitor problem, molecular analysis of viruses in shellfish samples includes other frequent difficulties such as low virus concentrations, and sequence variation. As the extraction-concentration procedure is not virus specific, the nucleic acid of several viruses can be extracted at the same time. RT-PCR must be performed under stringent conditions and confirmed by hybridisation. The first important step for sensitivity and specificity is the synthesis of the complementary DNA (cDNA) by reverse transcription (RT). Most assays utilise a virus-specific primer in the RT reaction (Atmar et al., 1995; Le Guyader et al., 2000; Kingsley et al., 2002; Formiga-Cruz et al., 2002; SaÂnchez et al., 2002; de Medici et al., 2004; Myrmel et al., 2004; Boxman et al., 2006) but random hexamers are also used in some assays (Chung et al., 1996; Green et al., 1998; Cheng et al., 2005). PCR amplification is usually performed for at least 40 cycles; some methods use nested PCR formats with fewer than 40 cycles in the first amplification reaction. Probe hybridisation is then performed as a confirmation step and enhances both assay sensitivity and specificity (Atmar et al., 1995; Chung et al., 1996; Shieh et al., 1999; Le Guyader et al., 2000; SaÂnchez et al., 2002; Costantini et al., 2006). Sometimes it is necessary to analyse the amplified sequence in order to characterise the viral strains, and virus-specific amplicons must be sequenced to obtain additional information about the virus(es) present in the sample. This is particularly important for NoV detection, due to its wide strain diversity. However, sequence analysis is hampered by the scarce product sometimes obtained after PCR amplification from shellfish tissues. One of the limitations in developing RT-PCR assays for the detection of NoV has been the selection of proper primer and probe combinations that allow the detection of most or all strains of concern. The high genetic diversity of NoV makes it necessary to use broadly reactive primers. Despite several improvements in the methodology, up to now no single primer set is able to amplify all strains (Atmar and Estes, 2001; Vinje et al., 2003). In the absence of such a universal primer set, multiple sets increase the chance to detect a greater number of strains, and the homology of the primers with the NoV strain is important in terms of sensitivity (Le Guyader et al., 1996a, 2000, 2003, 2006a). No single assay stands out as the best by all criteria, such as evaluation of sensitivity, detection limit and assay format, not even for the stool analysis being clearly more difficult in the case of shellfish samples with such very low contamination (Atmar and Estes, 2001; Vinje et al., 2003). For example, in three outbreak reports, primer sets targeting different regions of the NoV genome were needed to be able to amplify the strain both in clinical or environmental samples (Shieh et al., 2000; Le Guyader et al., 1996b, 2003, 2006a). For HAV, primer selection is easier since the degree of variation, particularly in the 50 non-coding region, is significantly lower (SaÂnchez et al., 2004; Costafreda et al., 2006). However, when genotyping is required other regions

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must be analysed such as the VP1X2A junction (Robertson et al., 1992; SaÂnchez et al., 2004) or even larger fragments. However, the low virus concentration very often hampers the amplification of such large fragments. Real-time RT-PCR assays, in which the RT, PCR and hybridisation assays are combined in a single well, are being developed and used successfully to detect enteric viruses in shellfish (Nishida et al., 2003; Loisy et al., 2005; Jothikumar et al., 2005; Costafreda et al., 2006). This technology takes advantage of not merely detecting but also quantifying the viruses present in the sample. However, for this last purpose it is necessary not only to develop but also to standardise the methodologies by including several controls at those most critical steps, let's say the nucleic acids extraction and the RT reaction (Costafreda et al., 2006). The efficiency of the virus nucleic acids extraction must be controlled by means of a model virus while the efficiency of the RTPCR reaction must be traced by means of a RNA molecule amplifiable and detectable with the same combination of primers and probes as those used for the actual virus. When these two reagents are added at known concentrations their recovery can be measured. Costafreda and colleagues (2006) proposed the use of the Mengo virus to evaluate the nucleic acid extraction efficiency, in general for any enteric virus and in particular for HAV, while the RNA molecule should be specific for each assay. The use of such an internal RNA control for the evaluation of the molecular reactions inhibition has been extensively used even in qualitative assays (Schwab et al., 1998; Le Guyader et al., 2003). Regarding other viral pathogens, such as rotaviruses and astroviruses, an interesting alternative exists based on their capability of replication in some tissue culture systems, such as the CaCo-2 cells, which represents a universal in vivo amplification system for the enteric viruses (PintoÂ et al., 1994) combined with either molecular (Abad et al., 1997b; PintoÂ et al., 1994, 1996, 1999) or immunological (Abad et al., 1998; Bosch et al., 2004) detection methods. Other cell culture molecular integrated systems have been proposed for enteroviruses (Reynolds et al., 1996). Interestingly, these combinations allow the quantification of infectious viruses (Abad et al., 1997a). However, although these techniques have been satisfactory evaluated and used in water samples, their application in shellfish is not common due to the infrequent shellfishborn viral outbreaks other than enteric hepatitis and norovirus gastroenteritis. In summary, the quantitative assays open a new view in terms of analysis of the sanitary risks associated to the consumption of virus contaminated shellfish.

12.3

Potential emerging virus problems

The gut as a `factory' of viruses is strongly selective. On one side, the alimentary tract with strong salivary enzymatic activity together with large pH shifts followed by bile acids and pancreatic enzymes aiming at breaking down foodstuff into its smallest components sets harsh conditions for a virus to survive. Most of the enteric viruses are small, non-enveloped RNA-viruses

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possessing an icosahedral capsid. However, an increasing number of enveloped viruses have also emerged that are capable of surviving the enteric route. The abundance of viruses in the gut offers exceptionally favorable conditions for genetic modifications of enteric viruses. Not only mutations, but recombinations and reassortments may facilitate the appearance of new variants of the already recognised viruses. An example of this is the recent appearance of a new variant of the well-known GII.4 type NoV. Within weeks it was able to spread via a variety of epidemiological routes throughout the world causing extensive outbreaks. This pandemic also demonstrates how fast an enteric pathogen may spread. It emphasises the threat posed by a pathogen of high medical impact, too. There is only a semantic difference between a virus called `new' and just a genetically modified old one. For practical reasons, a virus is `new' when the population immunity is missing totally or to a considerable part. An example would be the above mentioned GII.4 new variant NoV. Owing to their error prone polymerase, viruses that possess a ssRNA genome are constantly modified by mutations and may lead to strains or variants of high virulence. An example of this was the poliovirus type 3 that caused an outbreak in a vaccinated population in Finland (Hovi et al., 1986). Human rotaviruses, having a segmented genome, can undergo genetic changes through interchange of RNA segments, i.e. give rise to reassortants. This has been demonstrated clearly among group A rotaviruses (Maunula and von Bonsdorff, 2002). Waterborne outbreaks caused by group A rotaviruses have been detected (Villena et al., 2003; Divizia et al., 2004) but it is not known to which extent that virus possibly was modified. Rotaviruses of group B cause extensive outbreaks among adults, which appear to be restricted almost exclusively to China (Hung et al., 1984). Also in China new unclassified rotaviruses have emerged causing outbreaks that are still only poorly defined (Yang et al., 2004). Rotaviruses of group C have been involved in cases of gastroenteritis throughout the world, both in sporadic cases and in outbreaks (Jiang et al., 1996; Brown et al., 1989). However, in general, the rotavirus C infections seem to be rare (Abid et al., 2007). Group C viruses are also found in animals, preferentially in pigs. The porcine strains are, however, not identical to the human ones. For both rotaviruses belonging to groups B and C there is the potential that they may undergo changes that could increase their pathogenicity. Some zoonotical agents have caught a lot of attention due to their potential to spread emerging infections. One of these agents is the severe acute respiratory syndrome (SARS, Peiris et al., 2005). The causative virus belongs to the family Coronaviridae and is able to overcome the harsh alimentary tract conditions and is excreted in stools. However, whether this observation indicates an effective infection route for SARS remains to be determined, and thus a risk for seafood safety seems presently rather remote. Another group of emerging viruses that has evoked a lot of attention are the highly pathogenic avian influenza viruses (HPAI), preferentially the ones classified as H5N1 and H7N3, reviewed by Horimoto and co-workers (2005).

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These viruses have evolved from viruses of low pathogenicity by a mutation in the cleavage site of the hemagglutinin protein. This site is affected both by the local protein and the carbohydrate moieties (Stieneke-Gruber et al., 1992; Kawaoka and Webster, 1988). The trypsin specific cleavage is changed to a less specific protease cleavage (Li et al., 2004; Glaser et al., 2005). In seabird colonies, among which all known influenza A viruses reside, they seem to cause very little harm. Such pathogenic strains emerge occasionally, as is the case at the time of the writing of this chapter. In birds the viral infection is enteric, i.e. the virus is secreted in the chloaca. Especially waterfowl, such as ducks and other dabblers, that reside and excrete the virus in shallow waters are of importance (Markwell and Shortridge, 1982). The inactivation of the viruses in water is rather slow lasting from weeks to months, depending on the conditions (Stallknecht et al., 1990). Thus the viruses in water pose an infection risk for humans, too. The HPAI viruses show a varying pathogenecity among bird species. In general they cause mass death among cultured fowl like chicken, geese and turkey. Among wild bird species the pathogenicity varies. The reason for additional concern is the fact that they may infect humans and that the infections concur with a high mortality, up to 50%. The infections in man are, however, rare due to the receptor distribution in the respiratory pathway. It appears, that the `right' sialic acid construction is found only in alveolar cells, not in nasopharynx (2,3- vs 2,6-sialic acid bond) (Matrosovich et al., 2004). Thus only directly inhaled viruses that reach the susceptible cells will lead to an infection. Although an infected duck may excrete as much as 1,010 infectious doses per day, and the virus is able to survive in contaminated waters as long as 4 days at 22 ëC and 30 days at a 0 ëC, the risk to acquire the infection through bathing in contaminated waters has been estimated to be negligible (WHO, 2006). Like HAV, hepatitis E virus (HEV) replicates in the gut epithelium. It evolves, however, into a systemic infection mostly affecting the liver. The disease is similar to that of HAV-infection with the exception of its devastating effect on pregnant women. Up to 20±30% of them succumb as a consequence of the infection (Khuroo, 1980). It is still unclear what causes this high mortality. Large waterborne outbreaks caused by HEV have been reported originally from India, but the virus circulates widely in tropical and subtropical areas. HEV underwent several classification steps before it was placed into its present own family Hepeviridae (Reyes et al., 1990; Tam et al., 1991). Most of the human hepeviruses belong to one serogroup, although a genome-based division into four genotypes has been defined (Schlauder and Mushahwar, 2001). Apart from the human HEVs, they have also been broadly found among animals, most commonly among swine (Meng et al., 1997; Tei et al., 2003). The swine HEV appear in three clusters, in two of these, human cases have been identified. The swine farms when contaminated provide a rich source of HEV with direct close contact to man but will also enter the circulation via water. The detection of porcine HEV in cases of human disease (van der Pool et al., 2001; Meng et al., 2002; Tamada et al., 2004; Li et al., 2006) indicates that this threat is real.

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Nipah virus is a newly recognised zoonotic virus. The virus was 'discovered' in 1999 (Chua et al., 2000). It has caused disease in animals and in humans, through contact with infectious animals. The virus is named after the location where it was first detected in Malaysia. Nipah is closely related to another newly recognised zoonotic virus called Hendra virus, named after the town where it first appeared in Australia. Both Nipah and Hendra are members of the virus family Paramyxoviridae (Eaton, 2001). Although members of this group of viruses have only caused a few focal outbreaks, the biologic property of these viruses to infect a wide range of hosts and to produce a disease causing significant mortality in humans has made this emerging viral infection a public heath concern. In symptomatic cases, the onset is usually with `influenza-like' symptoms, with high fever and muscle pains (myalgia). The disease may progress to inflammation of the brain (encephalitis) with drowsiness, disorientation, convulsions and coma. Fifty percent of clinically apparent cases die. It is unlikely that Nipah virus is easily transmitted to man, although previous outbreak reports suggest that Nipah virus is transmitted from animals to humans more readily than Hendra virus. Pigs were the apparent source of infection among most human cases in the Malaysian outbreak of Nipah, but other sources, such as infected dogs and cats, cannot be excluded. Human-to-human transmission of Nipah virus has not been reported. The low stability of the paramyxovirus virions makes the shellfishborne transmission of Nipah virus an unrealistic possibility. Advances in the detection tools for the `classic' enteric virus pathogens (rotavirus, astrovirus, adenovirus and norovirus) also evidenced the occurrence of a variety of other agents such as Aichi virus, belonging to genus Kobuvirus within the Picornavirus family, and picobirnavirus in the Birnaviridae family. Their apparent rather limited circulation or low pathogenicity for man may be just temporary. With the increasing spread and efficiency by which especially food- and waterborne viruses are propagated all over the world, one can foresee the emergence of some of them as pathogens with more serious impact on the disease burden. Another important issue in the emergence and re-emergence of viruses is their potential implication in bioterrorism. Apart from the airborne route of infection, the most damaging spread of a pathogen is achieved if (drinking) water can be contaminated. This also applies for possible contamination of molluscs. For this purpose, viruses normally not transmitted through water or food may be employed, smallpox being an obvious candidate. However the potential of poliovirus as a bioterrorism weapon in a future immunologically naõÈve population if poliomyelitis is finally eradicated should not be underestimated.

12.4

Conclusions

One key element in reducing foodborne spread of viruses is the implementation of surveillance, controls on the products before the commercialisation and awareness. Additionally, consumer-information campaigns must be

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strengthened, including the promotion of suitable procedures of food preparation and consumption. The REFHEPA project (SEAFOODplus) has produced methods for the detection of HAV and NoV in bivalve molluscan shellfish to the point at which they could be included in regulatory standards for viruses in molluscan bivalves which would greatly increase the safety of these products for public consumption.

12.5

References

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S, BOSCH A

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(2006), Review of latest available evidence on risks to human health through potential transmission of avian influenza (H5N1) through water and sewage. http:// www.who.int/water_sanitation_health/emerging/avianflu/en/index.html. WILLIAMS R A and ZORN D J (1997), `Hazard analysis and critical control point systems applied to public health risks: the example of seafood', Rev Sci Tech Off Int Epiz, 16, 349±358. YANG H, MAKEYEV E V, KANG Z, JI S, BAMFORD D and VAN DIJK A A (2004) `Cloning and sequence analysis of dsRNA segments 5, 6 and 7 of novel non-group A, B, C adult rotavirus that caused an outbreak of gastroenteritis in China', Virus Res 106, 15± 26. YOTSUYANAGI H, KOIKE L, YASUDA K, MORIYA K, SHINTANI Y, FUJIE H, KUROKAWA K and IINO S (1996), `Prolonged fecal excretion of hepatitis A virus in adult patients with hepatitis A as determined by polymerase chain reaction', Hepatology, 24, 10±13. ZHENG D P, ANDO T, FANKHAUSER R L, BEARD R S, GLASS R I and MONROE S S (2006), `Norovirus classification and proposed strain nomenclature', Virology, 346, 312± 323. WHO

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13.1

Introduction

Bivalve molluscan shellfish such as oysters, mussels and cockles feed by filtering large volumes of seawater. During this process shellfish can accumulate human pathogenic bacteria and viruses when grown in sewage-contaminated waters. Such contaminated molluscs may present a significant health risk when consumed raw or lightly cooked. Numerous outbreaks of illness associated with the consumption of such shellfish have been recorded throughout the world (Lees, 2000; Butt et al., 2004). Within Europe, legislative controls exist aimed at preventing shellfish outbreaks. These primarily rely on the use of bacteriological monitoring programmes to determine the sanitary quality of shellfish harvesting areas. Levels of E. coli are used to categorise harvesting areas and prescribe levels of treatment required before they can be sold to consumers (Table 13.1). The current controls have been effective at reducing the risk of bacteriological illness to minimal levels. However, outbreaks of viral illness associated with the consumption of shellfish continue to occur within Europe (Lees, 2000; Sanchez et al., 2002; Le Guyader et al., 2003, 2006a; Prato et al., 2004; Gallimore et al., 2005; Shieh et al., 2007). The major illnesses associated with sewage-contaminated shellfish are gastroenteritis caused by norovirus (NoV)

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Table 13.1 European regulatory limits for shellfish harvesting areas based on E. coli monitoring and prescribed treatment options Category

Microbiological standard

Acceptable treatment

Class A

<230 E. coli per 100 g flesh and intravalvular fluid.

May go direct for human consumption.

Class B

<4,600 E. coli per 100 g flesh and intravalvular fluid.

Purification in depuration tanks. Heat treatment by an approved process. Relayed to meet class A requirements.

Class C

<46,000 E. coli per 100 g flesh and intravalvular fluid.

Relayed for a long period to meet category A requirements. Heat treatment by an approved process.

Prohibited

>46,000 E. coli per 100 g flesh and intravalvular fluid

Harvesting prohibited.

and infectious hepatitis caused by hepatitis A virus (HAV). When we focus on data obtained from shellfish collected from producing areas or from the market, and showing no bacterial contamination as defined by current regulations, noroviruses were detected from 6% to 37% of the time (Henshilwood et al., 1998; Le Guyader et al., 2000; Formiga-Cruz et al., 2002; Nishida et al., 2003; Cheng et al., 2005; Costantini et al., 2006). Given the failure of the current arrangements to fully protect public health there is a clear need to develop better approaches to controlling this problem. Long term the most effective way forward is developing further programmes aimed at reducing the degree of sewage contamination of shellfisheries at source (Pommepuy et al., 2005). European legislation exists, aimed at reducing levels of contamination in shellfisheries. However, progress has been relatively slow in this area. Given the high level of investment required in wastewater treatment plant infrastructure to achieve good water quality in at risk harvesting areas, a significant risk to consumers is likely to remain for the foreseeable future. In addition, even where improvements to sewage treatment have improved the quality of shellfish harvesting areas, there is still a risk of contamination by untreated sewage to shellfisheries through overflows as a result of process failures or during extreme rainfall events (Miossec et al., 1998). An alternative approach is to develop risk-based management procedures for shellfisheries. However, to develop this approach it is necessary to identify the pollution sources and conditions responsible for microbial contamination in shellfisheries and in particular to determine their impact on viral contamination. If it is possible to predict when viral contamination occurs in a shellfishery, it may then be possible to target intervention measures for public health protection at that time. Such measures could include temporary closure of the area or implementing increased treatment measures. The aim of the REDRISK project was to identify environmental conditions responsible for virus contamination in shellfisheries and to determine whether it may be possible to identify monitoring strategies that

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allow real-time intervention action. This information would provide the basis for a framework to allow the development of a preventative strategy for reducing the virus risk associated with shellfish by using a risk management approach. 13.1.1 Risk associated with contamination of shellfish with faecal material In recent years the epidemiological data shows that human enteric viruses are the most common cause of illness associated with bivalve mollusc consumption (Sanchez et al., 2002; Butt et al., 2004; Boxman et al., 2006; Le Guyader et al., 1996, 2003, 2006a; Shieh et al., 2007). Viruses transmitted by the faecal-oral route are widely prevalent in the community and infected individuals can shed millions of virus particles in their faeces (Chan et al., 2006). Consequently viruses, of many types, occur in large numbers in sewage and in other sources of aquatic pollution potentially contaminated with human faeces. Sewage treatment processes, if present, are only partially effective at virus removal (Sorber, 1983; Rose et al., 1996; Le Cann et al., 2004). Following introduction into the environment, viruses can survive for long periods either in the water column or by attaching to particulate matter and accumulating in sediments (Callahan et al., 1995; Gantzer et al., 1998; Nasser, 1994). Thus filter-feeding bivalve molluscs are vulnerable to contamination and numerous human health incidents have been reported (Butt et al., 2004). Various studies have shown significant rates of viral contamination of commercially produced bivalve shellfish placed on the market in a number of different counties (Costantini et al., 2006; Cheng et al., 2005; Chironna et al., 2002; Formiga-Cruz et al., 2002) potentially indicating a serious ongoing contamination problem. Although numerous enteric viruses can be found in the marine environment, the viral illness most commonly associated with contaminated bivalve mollusc consumption is gastroenteritis caused by norovirus. Noroviruses cause a relatively `mild' gastroenteritis, often including nausea, diarrhoea, vomiting, fever and abdominal pain. The incubation period is 1 to 4 days with a duration of about 2 days and generally followed by complete recovery. It is now generally accepted that Norovirus is one of the most common causes of infectious intestinal disease in both outbreaks and in the community (Food Standards Agency, 2000). Thus sewage can normally be expected to be heavily contaminated with this virus and, indeed, this has been found to be the case (Lodder and de Roda Husman, 2005). The other virus of major significance in shellfish-related outbreaks is hepatitis A virus. Hepatitis A virus has an extended incubation period of about 4 weeks (range 2 to 6 weeks) and causes a serious debilitating disease progressing from a non-specific illness with fever, headache, nausea and malaise to vomiting, diarrhoea, abdominal pain and jaundice. Hepatitis A is self-limiting and rarely causes death but patients may be incapacitated for several months. Whilst human enteric viruses (norovirus and hepatitis A) are generally the aetiological agents responsible for shellfish associated outbreaks animal strains of norovirus have been identified and the potential exists for mixed infection

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from bivalve molluscs contaminated in the environment with both human and animal faeces leading to the emergence of virulent recombinant strains (Lopman et al., 2002, 2003, 2004a; Costantini et al., 2006; Symes et al., 2007). Whilst this is considered to be a low risk by some, this possibility exists and needs to be considered (Sugieda et al., 2002; Oliver et al., 2003, 2007). Although most disease reports are viral in nature, occasional reports of bacterial illness (generally salmonellosis) associated with bivalve molluscs continue to occur. Salmonella spp may be acquired from both human and animal sources. It should also be noted that in many outbreaks the definitive aetiological agent, and cause of contamination, is not established. Thus whilst the greatest risk of shellfishassociated contamination is clearly from human faecal contamination, risks from animal faecal contamination cannot be discounted. 13.1.2 Sources of microbial pollution Contamination of bivalve molluscs with viruses, bacteria and other agents occurs principally because these animals obtain their food by filtering small particles, such as algae, from their surrounding water. Many of the commercial bivalve molluscs species (mussels, oysters, clams, cockles) grow in inshore estuaries or similar shallow or drying areas where nutrient levels are high and the waters are sheltered. Unfortunately such shallow, in-shore, growing waters are also frequently contaminated with human sewage, land run-off, and other sources of microbiological contaminants. In the process of filter-feeding, bivalve shellfish may also concentrate and retain human pathogens derived from such contamination. The hazards posed by bioaccumulation of harmful microorganisms are compounded by the traditional consumption of certain bivalve molluscs species raw, or only lightly cooked, and by the consumption of the whole animal including the viscera. Examination of the literature relating to illness associated with bivalve mollusc consumption shows that, where a polluting event is identified, contamination of the harvest area with human sewage is almost always responsible for the health incident. The most common route for human faecal contamination of the bivalve molluscs harvest area is through discharges of the municipal sewerage system generally caused by heavy rainfall. Such overflows generally release untreated sewage into the aquatic environment. Untreated sewage is likely to be heavily loaded with enteric viruses and thus poses the highest risk of all potential contamination sources (Fig. 13.1). A number of outbreaks have been linked to rainfall associated spills from sewerage systems (Miossec et al., 1998). In Australia a large oyster-associated gastroenteritis outbreak involving some 2000 persons occurred during the summer of 1978 (Murphy et al., 1979). This outbreak was linked to sewage contamination of the oyster harvesting area near Sydney caused by heavy rainfall. In 1982 in the USA 103 outbreaks involving more than 1000 persons were associated with clam or oyster consumption in New York State (Morse et al., 1986). Norovirus was implicated as the predominant aetiological agent.

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Fig. 13.1

Primary stage of shellfish contamination.

Shellfish originated from several north-eastern states with faecal contamination caused by runoff due to heavy spring rains the most likely cause. In the USA a number of gastroenteritis outbreaks have also been attributed to overboard dumping of faeces from boats by infected individuals. In 1993 a large multistate outbreak of gastroenteritis occurred in the US in Louisiana (Kohn et al., 1995), Maryland, North Carolina, Mississippi, Texas and Pennsylvania (Dowell et al., 1995). The outbreak was linked to oysters harvested in a small area in Louisiana over a 4-day period in November 1993. Twenty-five separate clusters of norovirus illness were identified with the authors calculating that as many as 186,000 people may have become ill (Dowell et al., 1995). The oysters were harvested by boat from a remote bed thought to be free of any sewage pollution. However the investigation showed oyster harvesters routinely disposed of faeces overboard and furthermore that one harvester, with a high level of antibodies to Norwalk virus, had experienced gastroenteritis just prior to the responsible harvesting period. The long incubation period of hepatitis A complicates linkage of this agent to particular food poisoning incidents. However, linkage is still possible during large incidents. In Shanghai, China in 1988 almost 300,000 HAV cases were traced to the consumption of clams harvested from a sewage-polluted area (Halliday et al., 1991; Tang et al., 1991). Other incidents were also reported in Australia and in Europe (Conaty et al., 2000; Bosch et al., 2001). A sizeable HAV episode in the USA during 1973 was linked to Louisiana oysters following flooding of the harvesting areas from the Mississippi causing sewage contamination. Hepatitis A virus was suspected to have been retained in shellfish for at

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least 6 weeks following the contamination event. At the time of harvesting oysters were fully compliant with the US sanitation programme standard (Mackowiak et al., 1976). Many other HAV outbreaks linked to bivalve shellfish have been reported but the initiating faecal contamination event is generally difficult to ascribe due to the protracted incubation period for this disease. In summary, where a cause is ascribed, most contamination incidents are linked to failures or bypassing treatment processes, often due to heavy rainfall. The problem is generally that heavy rain causes the storage capacity of the sewage treatment works to be exceeded. In combined sewer and rainfall systems this leads to storm spills. Such discharges will release untreated effluent heavily contaminated with micro-organisms. This may be particularly true during the `first flush'. Other causes are flooding of harvest areas with contaminated river water and overboard disposal of faeces from infected individuals. It is interesting to note that although the antiviral efficiency of sewage treatment can be questioned, fully treated effluents are not usually associated with bivalve mollusc disease incidents. A major problem with detection and control of contamination incidents is that viruses can persist for weeks to months in the marine environment (Callahan et al., 1995; Gantzer et al., 1998; Nasser, 1994) which is appreciably longer than for bacterial indicators (Solic and Krstulovic, 1992). Also, viral particles may persist for months in shellfish tissues either via ionic binding or specific attachment (Burkhardt and Calci, 2000; Loisy et al., 2005; Le Guyader et al., 2006b). Monitoring using faecal bacterial indicators will tend to significantly underestimate the duration of the viral contamination risk. Many outbreak reports have noted the problem with control of viral risk using bacterial indicators and the urgent need for measures to reduce risks from viral contamination.

13.2

REDRISK project

Given the slow progress in developing pollution reduction programmes and the significant challenges in moving this forward in the future it has to be accepted that sewage contamination of shellfisheries will occur into the foreseeable future. With this in mind, the risk of viral illness associated with shellfish consumption will continue. Therefore there is a need to introduce better risk management arrangements in shellfisheries. The major aim of the REDRISK project was to identify pollution sources and conditions responsible for viral contamination in shellfisheries. It was hoped that this information could be used to determine environmental monitoring procedures that would allow real-time identification of viral contamination in shellfisheries and replace existing flawed microbiological monitoring programmes. This would be considered to be a first step towards developing a framework to allow the development of a preventative strategy for reducing the virus risk associated with shellfish. To achieve this, the project was broken down into two phases. Firstly sanitary surveys were conducted in selected sites in four European countries to identify pollution sources

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and environmental conditions responsible for microbial contamination. On the basis of the sanitary surveys sampling programmes were developed using semiquantitative PCR procedures to determine the impact of the identified microbial contamination sources and environmental conditions on the viral contamination of shellfish in the area. Monitoring of shellfish for viruses and of environmental conditions was conducted concurrently over a year-long period. 13.2.1 Site selection In the REDRISK project, selection of appropriate sites was determined by accessing data from national shellfish monitoring programmes and supplementary data available from appropriate regulatory and environmental agencies. The selected harvesting areas were classified as class A or B under European hygiene regulations. By conducting the project in a range of harvesting areas throughout Europe it was hoped to ensure that a range of geographically diverse conditions and pollution types would be investigated (Fig. 13.2). The study sites were chosen from review of literature and of existing available bacterial and environmental data, descriptions of point and non-point sources, catchment characteristics and hydrodynamics within the harvesting area.

Fig. 13.2 Location of the selected shellfish harvesting areas (REDRISK).

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13.2.2 Spanish sites Owing to the different characteristics of the Atlantic and Mediterranean coasts in Spain, two sites, one in Galicia and one in Catalonia, were selected for study. The Galician site was located in a RõÂa representing approximately 14% of the Galician mussel production (247 000 tons in 2003). This is an estuary which can be divided into three zones. The innermost zone is characteristic of a typical estuary, and is influenced by tides and a large river with a mean annual discharge of 13 m3/s. The central zone, where the sampling point used in the study is located, is influenced by both continental and oceanic inputs. It contains 172 floating mussel rafts in two areas classified as category B and a beach area classified as category C where clams (Ruditapes decussates and R. phylippinarum) and cockles (Cerastoderma edule) are harvested. The RõÂa is densely populated and there is a large city with a population of approximately of 500 000 persons with a significant number of sewage treatment plants impacting the area. In Catalonia, a site situated in an estuarine bay at the delta of the largest Spanish river (catchment of 88 935 km2) was selected for study (B classified). The bay covers a total area of 5600 ha with a mean depth of 3.13 m (maximum depth of 6.5 m). The bay is a moderately eutrophic estuary, with an average water residence time of approx. 10 days and the salinity range depends mainly on the discontinuous fresh water inputs from numerous irrigation canals along the bay. The delta (total surface of 32 059 ha) consists of rice fields (80%) and lagoons (20%), with an urban area (1200 ha) of 43 847 people in total. Bivalve culture revolves around mussels (Mytilus galloprovincialis) and Pacific oyster (Crassostrea gigas) grown in suspended systems and clams (Ruditapes decussatus and R. semidecussatus) grown in bottom systems. Shellfish production in the delta is around 3000 Mt per year, and this activity supports 90 mussel producing facilities, along with the corresponding depuration plants and bivalve retailing centres. 13.2.3 United Kingdom sites One of the UK sites is a wide, shallow estuary, with mud flats at low tide fringed by salt marsh on the upper shores, with shingle, shell banks and offshore islands as a feature of the tidal flats. The estuary is 28.5 km long and it is a well-mixed system, with salt water present through most of the estuary. At high water the estuary within covers an area of approximately 30 km2. It has an average depth of approximately 4 m and a maximum depth of 13.6 m at chart datum, lowest astronomical tide (LAT). The estuary is characterised by a large low-lying catchment area of approximately 1200 km2 drained by two main rivers which flow into the head of the estuary. The first of these has a sub-catchment area of 337 km2 and a mean river flow of 1.38 m3/s. The second has a catchment area of 534 km2 and a mean river flow of 1.93 m3/s. This river flows 72.6 km to the tidal limit. There are several large towns within the catchment and the total population in the area is over 340 000. Average population density within the catchment is

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283 persons/km2. The nearest town to the fishery population c. 20 000 is within 6 km of the shellfish beds. There are over 1400 regulated pipeline discharges within the catchment, including 62 continuous discharges from sewage treatment works. The catchment is an important agricultural area, predominantly supporting arable farming, but also some livestock. The 2005 farm census recorded over 45 000 animals (average 375 km2). There are three marinas in the wider estuary plus 12 sailing clubs. Overall it is estimated that there are over 3000 moorings and berthing facilities in the wider estuary as a whole. The whole of the estuary and approaches to the estuary were designated as a bivalve mollusc production area. One creek in the upper estuary was chosen as the focus for the study. This area supports an important Pacific oysters (C. gigas) fishery. The water quality falls short of that necessary to support a class `A' under the hygiene legislation due to intermittent peaks in E. coli contamination and consequently is designated as a B area. 13.2.4 French site The French site is an estuary located on the western French Atlantic coast. The total catchment area is 113 km2 and the distance between the source of the rivers and the estuary mouth is about 87 km. The main river represents 43% of the subcatchment area. Five smaller rivers also flow into the estuary. Approximately 6562 people live in the catchment with a mean density close to 58 inhabitants/ km2 (188 inhabitants/km2 near the sea). Less than 40% of this population live in small villages. The largest town with 1772 inhabitants is located upstream of the estuary and is served by a sewage treatment plant (STP), built in 1985 composed of three nested ponds. The sewage network collects around 245 m3dÿ1. Only 155 m3dÿ1 are of urban origin and the remaining volume is due to rainfall water input to the network. Previous studies suggest that the estuary is significantly affected by the river inputs and suggest that they make up 85% of the E. coli fluxes in the estuary. The STP is believed to represent fluxes which are approximately 100 times lower than those of the river inputs. The area is subjected to intensive agriculture. Livestock is made up of cattle (9443), pigs (60 177) and poultry (434 254). There are 169 farms which cover 62% of the land surface of the catchment. Within the estuary shellfish, oysters (Crassostrea gigas) and clams (Ruditapes decussatus) are produced. The area is classified as category B for oysters and C for clams. 13.2.5 Irish site The selected site was a large (c. 26 000 ha), westerly-facing bay on the west coast around 12 km wide and 16 km long, classified A and B depending on the shellfish location. The shellfish industry in the bay is mostly composed of bag and trestle growing of pacific oysters (Crassostrea gigas) and long line mussel (Mytilus edulis) production. The licensed aquaculture activity covers approximatly 180 ha of the inner bay. A small but significant wild native oyster

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(Ostrea edulis) fishery also exists within the bay. The average production and value per annum is: native oysters, 12 tonnes valued at ''45 000, Pacific oysters, 700 tonnes valued at ''960 000 and rope mussels, 400 tonnes valued at ''220 000. The population on the coast surrounding the bay is approximately 11 000 with a further 3500 people living within catchments of the river entering the bay. The major pollution source identified as impacting the shellfishery was an area of the bay selected for study. This is a modern wastewater treatment facility serving a population of 6000 people with secondary treatment comprising filtration, aeration and settlement but no disinfection and a large stormwater storage capacity. From the sanitary survey it was assessed that other pollution sources were likely to have a negligible effect on viral contamination. Three sampling points were selected and monitored at locations at increasing distances up to 4.5 km from the sewage treatment works. 13.2.6 Results The REDRISK project represents the first systematic investigation of shellfish harvesting areas using relative quantitative procedures, i.e. real-time PCR methods, to determine the factors responsible for contamination of shellfish with human pathogenic viruses. The project was the first in Europe to use quantitative PCR procedures for viral detection. This testing strategy was developed in the REFHEPA project within SEAFOODplus (see Chapter 12). The aim of the work was to establish whether it may be possible to identify environmental conditions responsible for increased viral contamination in shellfish. Subsequently could this information be used to establish real-time environmental monitoring to predict viral occurrence? The results from the project indicate that environmental conditions responsible for viral contamination could be identified on a number of occasions; however, the conditions responsible and the point at which they became significant varied considerably from site to site. Therefore while the potential exists to develop site-specific real-time environmental monitoring programmes to predict viral contamination, it must be recognised that these will require in depth characterisation of the harvesting area in question and will be resource intensive. Such programmes could be built on a common framework approach and potential strategies for controlling viral risks in shellfish, drawing on the experience of the REDRISK project are presented here. Determination of the major factors leading to viral contamination: REDRISK site experience The REDRISK sites offer a broad range of growing conditions which one can meet on the European littoral: ria, bay and estuary were selected, climate and tidal conditions varying from one country to another, as well as the shellfish species grown (Table 13.2). The site characteristics are presented in Table 13.3. A sanitary survey was carried out for each selected harvesting area. This included a review of existing available bacterial and environmental data and a description of point and non-

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Table 13.2

Characterisation of shellfish areas

Site

Shellfish production (T)

RõÂa de Vigo MoanÄa, Spain Alfacs Bay, Spain

34 580 1 280

Blackwater Estuary, UK Daoulas Estuary, France

>100 200

Clew Bay, Ireland

1 110

Shellfish species

Class

Mytilus galloprovincialis, Ruditapes spp; Cerastoderma edule Mytilus galloprovincialis, Crassostrea gigas, Ruditapes spp. Crassostrea gigas Crassostrea gigas, Mytilus edulis, Ruditapes decussatus Crassostrea gigas, Ostrea edulis

B/C B A/B B A/B

point sources, catchment characteristics and hydrodynamics within the harvesting area. Targeted collection of additional data was then undertaken and the survey was conducted taking account of best practice from within Europe. Conditions and parameters linked to continuous contamination or intermittent pollution events were investigated further to establish their significance as a cause of shellfish harvesting area deterioration. Comparison of the results obtained in the different sites Table 13.4 synthesises data obtained on sites. When located in the vicinity of sewage input, the presence of norovirus was found in shellfish for a great percentage of the year. These results indicated that the proximity does not allow the dilution of viral contamination, even if they are excreted at low level (Figs 13.2 and 13.3). When dilution occurred, seasonal contamination was observed (Area 2) and or was rarely present (Area 3) because of favourable hydrodynamic conditions (high currents, low residential time of particles in the Bay). The possibility for a contaminated plume to reach shellfish beds depending on the distance and currents was previously reported (Pommepuy et al., 2004; Fiandrino et al., 2003; Riou, 2007). Factors leading to viral contamination Among the monitored parameters to assess the links between environmental factors and viral occurrence in shellfish, rainfall, river flow, epidemic in population and sewage overflow were found to be the more relevant parameters (Table 13.5). Conclusion The relation between virus occurrence and other parameters is site-dependent and no single parameter was found to explain viral contamination over all the sites, except the distance of the shellfish beds to the river or sewage outfall. Some factors were more relevant in the northern sites than the southern ones.

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Table 13.3 Main characteristics of REDRISK sites Site

Length or surface

Hydrodynamic characteristics

Max. tidal range

Salinity and Temp. range

Human population Density/km2

WTP (Distance from shellfish beds)

33 km

Well-mixed estuary

3.5 m

31±37ù 10±20 ëC

500 000 >400/km2

WTP* (<1 km)

Alfacs Bay, Spain

5,600 ha

Stratified waters

0.2 m

16±38ù 7±31 ëC

43 900 nd

WTP (<1 km)

Blackwater Estuary, UK

28.5 km

Well-mixed estuary

5.3 m

14±34ù 14.4±34 ëC

340 000 283/km2

WTP (6 km)

Daoulas Estuary, France

7.5 km

Partially mixed estuary

5.5 m

12±35ù 5.5±23.3 ëC

6 500 58/km2

Ponds (2 km)

Clew Bay, Ireland**

16 km

Stratified/wellmixed bay

5m

10±35ù nd

6 600 nd

Sec. WTP (0.3±4.5 km)

RõÂa de Vigo MoanÄa, Spain

* WTP wastewater treatment plant; ** 3 sample points.

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Table 13.4

Occurrence of norovirus in shellfish during the REDRISK survey Area 1 Area 2 Heavy Moderate contaminated sewage impacted area area (close to sewage discharge)

Area 3 Low contaminated area

Norovirus occurrence

High

Low

Very low Accidental?

Comments

Weak seasonal variation

Seasonal variation

Rare contamination in winter

The differences between the regions could also be attributed to other parameters, such as the water temperature acting on viral survival or the tidal effect on plume dilution. On the Mediterranean coast, the principal parameters ± rainfall and epidemics in the population ± were not found to be relevant to explain the contamination, but climate features could be the reason for the lack of relationship (dry climate with short, strong rainfall in summer). Another explanation could lie in the fact that the survey was too short (12 months), probably insufficient to cover different climatic or epidemic conditions. The same observation is also valid in the UK where the REDRISK study period was characterised by a drought condition. The most significant parameter obtained and available on all the sites studied is the proximity of shellfish beds to viral sources (river, urban outfall, sewageoverflow point, etc.). All the critical points located in the vicinity of the shellfish

Fig. 13.3 Recorded number of gastroenteritis cases for 100,000 inhabitants, in France (Brittany), from January 1992 to October 2006. www.sentiweb-hebdo.

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225

Relevant factors and viral contamination Spanish sites

UK site

Irish site

French site

Rainfall effect

ÿ

(winter)

ÿ

River flow impact

ÿ

(and salinity)

Distance input to beds Sewage overflow impact

nd

ÿ (drought conditions) ÿ (drought conditions) nd

Season or epidemic in population

nd

Probable (peak in March)

overflow

Rainfall and epidemic occurrence

nd

ÿ (drought conditions) Seasonal trends

WWTP/river (winter, spring)

Relation virus/other parameters

Sampling site and mollusc species

Probable Proximity outfall, season WTP discharge

Rainfall and epidemic

Nd: no data

beds are a potential source of viruses and were clearly possible causes of contamination.

13.3 Potential strategies to limit microbial contamination of shellfish and tools to implement them 13.3.1 General approaches to risk management identified from the REDRISK project Hazard identification While it is recognised that contamination with human viruses is not the only microbiological risk associated with shellfish consumption, it is clearly the most significant and as such when conducting sanitary surveys particular attention should be focussed on human sewage contamination sources. It is well established that viral contamination of shellfish occurs at source. Unlike other foods there is no proof that other routes such as infected food-handlers, could be the origin of the contamination (Koopmans and Duizer, 2003). Most of the time, sewage and in particular, untreated sewage, is the origin of viral presence in shellfish. Results obtained during the REDRISK project clearly demonstrated the presence of norovirus in treated wastewater when norovirus is epidemic in the population (Table 13.6).

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Improving seafood products for the consumer Table 13.6 Results obtained in raw sewage effluent (France) during gastroenteritis in local population (November 2005 to April 2006) Norovirus (RNAcopies/m)

Genotype I

Genotype II

<30 30< to <104 >104 Total

9 3 3 15 samples

13 1 1 15 samples

It is often difficult to gather information on sewage inputs into the marine environment. However, a fundamental element of understanding the risks associated with a shellfishery is to establish the extent of sewage contamination entering a harvesting area. In particular establishing when, and how often, sewage overflows are triggered is important, as these are known to have a significant effect on viral contamination. The REDRISK project results indicated that viral contamination of oysters 4.5 km away from a sewage treatment plant was believed to be associated with sewage overflows at that STW following high rainfall. Identification of high risk periods High risk periods for viral contamination of shellfish and subsequent illness in consumers occur during the presence of elevated levels of pathogens in sewage effluents. This is linked to epidemics in the population. In general data from national monitoring programmes in France and UK indicates the number of community cases of gastroenteritis (GE) is related to season. In winter it was demonstrated that viruses, and in particular noroviruses, are the agents most often responsible for GE cases in the population (Lopman et al., 2003; Atmar and Estes, 2006; Svraka et al., 2007). Seasonally the number can typically vary from 200 per 100 000 population during the summer season, to 800 and more during the winter (Lopman et al., 2004b). However, this may vary from region to region, and in particular different patterns may be observed between Mediterranean countries and North European countries. Little data is available on the seasonal distribution of GE cases in Mediterranean countries. Some years appear to be more favourable to winter epidemics of norovirus in the population. For example, during the last major outbreak of norovirus GE in France, 6 million people became ill over a 6-week period (January 2001 to February 2001). Considering an attack rate equivalent to 3% of the population, and a viral concentration in faeces of about 30 million particles/patient, the viral flux from 15 000 inhabitants could be estimated at 600 000 viruses/per minute. During these periods the risk is undoubtedly further compounded when shellfish are contaminated with untreated sewage, maybe as a result of overflows during high rainfall periods. Identification of the probability of occurrence of viruses in the environment during REDRISK period Identification of virus occurrence (outbreaks) in the population is possible from national monitoring programmes. Epidemiological and clinical networks giving

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epidemiological data are now widespread in Europe and allow the possibility to get this information rapidly (Lopman et al., 2004a, 2004b; Ando et al., 1995; Blanton et al., 2006). However, the level of data acquisition and availability varies between countries and may limit the use of this as a tool in some countries. This is an area of risk management that should be developed and further co-ordination of a European-wide network of data collection should be further encouraged. In France the sentinelle network managed by the French Health Ministry (www.sentiweb-hebdo) gave comprehensive data on the number of gastroenteritis (GE) cases in the western part of the France where the site study was conducted. During the REDRISK study, input of virus mainly occurred during the end of the winter period (from early February to the end of March). However it was apparent that the epidemic winter events were less important during the study period compared with those observed during the 2001±2002 period (Fig. 13.3). Where direct epidemiological data of GE outbreaks is not available, an approach for identifying risk periods has been proposed as a surrogate to monitor sales of anti-diarrhoeic medicines in local pharmacies. At the Irish site, pharmacy sales were recorded (Fig. 13.4). The results indicated a seasonal variation consumption of anti-diarrhoeic medicines peaking during the winter months consistent with the expected high-risk periods for GE cases. July also showed a peak in sales; however, this is considered as a result of sales of medicine for travellers going abroad on holiday. The input of viruses consistent with the monitoring results found in wastewater (Table 13.6) was therefore demonstrated through simple indirect means during the REDRISK. This demonstrates the potential of these procedures for epidemic occurrence in the community leading to periods of high risk for viral contamination. The effects of rainfall on river flows were also studied during the same period. For example, the average monthly river flow totals for the UK site are presented for the period June 2005 to June 2006 and compared to the average period January 1991 to June 2005 in the UK site (Fig. 13.5). As would be expected, the most significant river flows are generally recorded from October

Fig. 13.4

Anti-diarrhoeic pharmacy sales, Irish site.

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Fig. 13.5 Average river flows of the Rushes lock.

to April, with peaks usually occurring in December and January. During the period of the REDISK project significantly lower than normal river flows, due to low rainfall, were observed compared with the average flow (1991±2005). Similar, but less significant observations, were reported in other countries involved in the project. In Ireland it was possible to monitor rainfall and identify overflows of sewage into the shellfisheries. However, this was only possible after the event. Systems to allow early warning of sewage overflows by authorities responsible for STPs could be developed to allow intervention procedures to be introduced to control the risks in shellfisheries. Monitoring of sewage treatment plants observed demonstrated where overflows of raw sewage were observed as a result of high rainfall events there was often a significant increase in the viral load in shellfish. Combining identified environmental conditions such as rainfall and the epidemic status in the community allows a relative risk rating to be determined (Table 13.7). Generally the low rainfall levels observed in some sites during the REDRISK project were not representative of an average period and may have represented a lower than normal risk. Nevertheless `risk months' could be defined as occurring when both river flow and epidemic events were at maximum, i.e. during the period from January to March. Considering Table 13.7, the second column (R) Table 13.7 Rainfall

Probability of occurrence of the presence of viruses in the environment

Epidemic

Low

R R R R

Low Normal Important Large outbreak low;

probable;

Normal

high.

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Storm event

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seems to correspond to the conditions prevailing during the period of the REDRISK study. 13.3.2 Proposed approach to risk analysis-risk matrix approach Risk analysis and risk assessment An assessment of identified risks was defined by the probability of damage and the level of damage involved. The risk approach adopted in REDRISK was a portfolio method (a general instrument of business management) enabling the probability of the risk occurring to be defined. The overall risk can be considered to be a combination of the probability of occurrence (Table 13.8) and the likely impact (Table 13.9). The following factors would be considered to describe the impact: the sewage treatment, the distance of shellfish to source, the area hydrology and hydrography and the seafood species harvested. To describe the probability of occurrence, the following parameters would be considered: seasonal variations in human and animal populations and seasonal harvesting of shellfish. A REDRISK matrix was built to summarise the critical factors of each site and to underline the major critical points of the harvesting areas. The matrix makes it possible to identify the management priorities selected according to the viral health risk. At that time we considered that the main viral contamination was due to faecal human discharges. Nevertheless, individual sources (farms or villages) in the catchment could lead to faecal or viral contamination, thus the matrix includes agricultural activities. Even though some information exists concerning the presence of norovirus in cattle faeces, the major sources are those commonly reported. Depending on the site, different strategies to reduce the risk could be proposed. For example, sewage load reduction or elimination, land management (e.g., farming practices) or coastal management (e.g., zoning of activities). The management strategy also concerns shellfish beds and strategies could be Table 13.8 Probability of occurrence Probability of occurrence Low Medium High Table 13.9

Weight 1 2 3

Allocation of impact (modified from Wagner and Strube, 2005)

Impact

Health effect

Minimum impact Moderate impact Maximum impact

Minimal human health effects Temporary impairment of health Serious illness

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proposed, including post-harvesting treatment (e.g., depuration or heat treatment), closure or relocation or relaying of shellfish. Some examples of the risk matrix applications are presented from REDRISK sites (Tables 13.10, 13.11 and 13.12). Potential risk management strategies could be defined and solutions applied, depending on the level of contamination. The following REDRISK matrix applications refer to the areas presented in Table 13.4 and classified into three categories: heavy, limited and rarely contaminated sites. Area 1, with a high and permanent viral contamination (Table 13.10) This is a C class area, where the contamination originated from outfalls located near the shellfish beds: wastewater, the quality of which is probably very poor, is directly discharged into the bay less than 500 m from the shellfish beds. Along with possible failures in the sewage network leading to raw sewage input, this can lead to the presence of viruses in shellfish regardless of the season, because of the poor dilution of sewage in the sea. Management strategy must be addressed in order to reduce the sources of contamination. However, owing to the hydrodynamics of the area and the distance from the outfall, a more precise study is needed to evaluate the costbenefit of investing in the wastewater treatment plant or repairing the network. At present, shellfish areas are closed for much of the year and relocating or relaying the shellfish beds could be the better solution to manage the risk. Table 13.10 Area 1, high viral contamination: scoring matrix of health risk associated with microbiological impact Source

Potential risk occurrence probability

Impact Management priorities

Sewage discharges

3

3

+++

Sewerage overflows

3

3

+++

Septic tanks Sewage sludge/ irrigation Boats Wildlife Domesticated animals Animal manure Shellfish management

0

0

/

0 1 0 0 0

/ + / / /

0 1 1 0 0 strategy

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Sewage attenuation (biological and disinfection treatment) Significant modification and repairs of sewage network / / Limitation / / / Closure of shellfish area Relocation or relaying of shellfish

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Area 2 with seasonally limited viral occurrences (Table 13.11) The second example corresponds to an area that was approximately 3.5 km away from the closest waste water treatment plant and where viral contamination occurred at times during the winter period. This is a B class zone under EU regulations. The presence of insufficiently treated sewage, discharged near the shellfish beds, could explain why enteric viruses are sometimes observed. In addition the potential for nearby septic tanks to cause contamination was also identified and regular assessment of their efficiency could also identify risks. The management strategy could assess whether the current regulations of agriculture activities are applied, including the elimination of human sewage and animal manure on the catchment. Specific wastewater improvements could be undertaken, such as additional treatment (biological treatment, filtration, UV) or tidal phasing of discharge to reduce the flow of faecal material into the harvesting area. Additional shellfish management procedures could be introduced into the area such as extended depuration or re-laying during the winter period. Table 13.11 Area 2, seasonal limited viral contamination: scoring matrix of health risk associated with microbiological impact Source

Potential risk occurrence probability

Impact Management priorities

Sewage discharges Sewerage overflows

1 1

2 1

++ +

Septic tanks

2

3

+++

Sewage sludge/ irrigation Boats Wildlife Domesticated animals

0 0 1 2

0 0 1 1

/ / / +

Animal manure

3

2

++

Shellfish management strategy

Management strategies

Sewage attenuation Reduced spill frequency/ telemetry alarms Setting up watertight septic tanks / / / Land management, e.g. farming practices Post-harvesting treatment, e.g. depuration or relaying

Area 3, limited contamination (Table 13.12) In example 3 (Area 3, A classified), viral contamination was observed in shellfish very rarely at low levels. The low level contamination was associated with high-risk periods coinciding with sewage overflows associated with high rainfall events and periods of high prevalence of norovirus in the community. At this site

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Table 13.12 Area 3, limited contamination: scoring matrix of health risk associated with microbiological impact Source

Potential risk occurrence probability

Impact Management priorities

Management strategies

Sewage discharges Sewerage overflows Septic tanks Sewage sludge/ irrigation Boats Wildlife Domesticated animals

1 1 1 0

1 2 1 0

+ ++ + /

Sewage elimination Sewage network repairs / /

1 1 1

1 1 1

+ / +

Animal manure

1

1

+

/ / Land management, e.g. farming practices

Shellfish management strategy

Survey and warning system

it was considered possible to identify a relatively low number of occasions where virus contamination occurred during high-risk periods. This was particularly evident during periods of sewage spills as a result of high rainfall. The management strategy could first assess whether current regulations of agriculture activities are applied, including the elimination of human sewage and animal manure on the catchment. Specific actions could also be set up, such as secondary or tertiary treatment (biological treatment, filtration, UV) to reduce the faecal charges. In this area, shellfish management involves post-harvesting treatment, for example shellfish depuration. The ability to identify risk periods through early warning schemes linked to epidemiological surveys could allow the opportunity to take additional shellfish treatment procedures or even suspend harvesting to remove the risk. Waste management options would likely focus on additional measures to ensure sewage spills are minimised. 13.3.3 Management strategy Reducing levels of contamination at source: site-specific strategies and speciesspecific strategies The risk of shellfish contamination by pathogenic human viruses is linked to the presence of these viruses circulating in the community, viral shedding rates from the community and the potential for virus-contaminated sewage and sewage sludge to impact on shellfish growing waters. Opportunities to reduce viral pollution levels in contaminated sewage sources firstly require identification of the sources that have the potential to affect the shellfish production areas. These can include both point source inputs such as

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discharges from sewage treatment plants and storm or emergency overflows from the sewerage system or diffuse sources such as runoff from spreading of sewage sludge on land or discharges from boats. Sanitary surveys for all new harvesting areas are now a requirement under EC Regulation No 854/2004 (Anon., 2004b). Under this Regulation there is a requirement for member state competent authorities to make an inventory of the sources of pollution of human and animal origin, likely to be a source of contamination for the production area and examine the quantities of organic pollutants that are released during the different periods of the year (Anon., 2006c). Traditional sewage treatment such as primary treatment (settlement of solids) followed by secondary (biological) treatment will typically reduce the overall microbiological load by a 2 log10 reduction from in faecal indicator bacteria across the process, but geometric mean levels of faecal coliforms in the final effluent level will still be in the range 1:7 4:5 105 cfu/100 ml (Kay et. al., unpublished) and this level of treatment will not eliminate pathogenic bacteria and viruses in wastewater effluent. Tertiary treatment systems are often used to effect further reductions in microbiological numbers of effluent discharging to sensitive waters, e.g. bathing and shellfish waters, and little information exists on viral removal (Rose et al., 1996). To date these disinfection techniques have generally been applied to target reduction of bacterial faecal indicator organisms to achieve compliance with current receiving water standards, e.g. such as the guideline level for faecal coliforms in shellfish flesh under the Shellfish Waters Directive 79/923/EEC (Anon., 1979) (now replaced by the codified Shellfish Waters Directive, 2006/113/EC Anon., 2006c) and the coliform and Faecal streptococci parameters identified under the EC Bathing Water Directive (70/ 169/EEC Anon., 1976). Under typical operating conditions of transmittance >45% for suspended solids concentrations of <30 mg/l and a received dose >32 mJ/cm2 ultraviolet (UV) disinfection typically achieves a 3 log10 reduction in faecal coliform bacteria through the process (Conlan, and Wade, 2003). Relatively few data are available on virus reduction through sewage treatment stages, and treatment efficacy is often difficult to measure if the target organism is only present at relatively low levels to start with. Longer-term retention lagoons or constructed wetlands offer some scope for further reduction in final effluent viral numbers (ranging from 1 to 3 log, Ifremer unpublished data). In a study of a 6-year old, sub-surface flow (SSF) wetland, Vidales et al. (2003), achieved results, suggesting that after a 5Ý-day retention period a 99% reduction in viruses was achieved. Recent data demonstrated that effectiveness in removing viruses or phages can vary dramatically over time by a factor of 3 or 4 log, ranging from low values <1 log to 5±6 log viral reduction (Le Cann et al., 2004; van der Berg et al., 2005) therefore coastal discharges constantly release human viruses into the marine environment. However, recent work (Kato et al., 2005) in applying a combined photo catalyst (TiO2)/UV disinfection system to disinfect wastewater appeared to decompose norovirus to the extent that they were not detectable by genetic analysis. But no methods are currently available for easily confirming the infectivity of noroviruses, it is therefore not possible to measure

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the effectiveness of existing non-barrier sewage treatment systems including UV disinfection at the present time (Pommepuy and Le Guyader, 1998). Micro-filtration `membrane' barrier treatment processes can achieve up to 6 log reduction in facal indicator bacteria and offer the opportunity to directly removal viruses at source. Experimental studies of poliovirus transmission through 0.2 m microfiltration (MF) and 30 kD cut-off ultrafiltration (UF) membranes. It was found that UF membranes give complete rejection of virus and that MF membranes gave significant removals under appropriate conditions (Madaeni et al., 1995). To conclude, disposal of sewage sludge from wastewater treatment has increased in Europe due to the ending of sewage sludge disposal at sea in the 1980s and the requirements for minimum levels of treatment under the Urban Waste Water Treatment Directive (Anon., 1991). However, effective regulation and measures are necessary to protect sensitive shellfish waters. Management improvements include the location of sludge storage sites and their containment and catchment sensitive farming approaches in relation to spreading of sewage sludges including exclusion and buffer zones and treatment such as sludge pasteurisation. It is assumed that long residence time treatment lagoons, microfiltration and optimised UV plants currently offer the best options for significant reductions in viruses in continuous discharges (Le Guyader, pers. com.). As quantitative methods for enumeration of viruses improve, surveillance of the virus template directly in sewage may offer an appropriate contribution to risk assessment in the future that will enable targeting of improvement measures and/ or appropriate management actions to be undertaken. Concerning the role of non-point sources from agriculture on viral contamination, relatively little work has been undertaken on the potential for animals to act as vectors for the transmission of human viral pathogens to shellfish waters and this area therefore requires further work. However, good management practices that can be adopted in relation to storage and application of sewage sludge on farms apply equally to farm animal wastes. Contamination monitoring in real time: REDRISK experience To avoid shellfish contamination during the risk period, necessary control points have to be established. The basis of an early warning system was set up during the project and tested in a coastal area (Fig. 13.6). The concept of the system takes into account the different parameters that could have an impact on viral contamination. These parameters are gathered in a database and when trigger values are observed, the system alerts users. On the experimental REDRISK site (Baie de Daoulas, France), we selected the following parameters: rainfall, river flow, salinity and E. coli concentrations and tested the relationships between them. The results demonstrated that salinity could be an available proxy to assess the entropic input and thus the contamination in term of E. coli (Fig. 13.7). Recent development of such models based on a simple relationship between observed rainfall and pathogen concentrations, or other systems based on

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Fig. 13.6 Basis of an early warning system implementation.

complex models of the prevalent mixing and transport processes are already used for bathing waters risk management (EAP, 1999). These first results are promising but need further investigation to take into account the relationship of uptake of contaminants in shellfish and the overlaying waters. The work in the French site indicated that models could be produced which would potentially

Fig. 13.7

Model of the effect on rainfall on river flow in Daoulas estuary. (....... observed river flow values) (Ð calculated river flow values)

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allow the use of early warning in estuarine production sites in Europe. Such a monitoring network has already been proposed for activities in estuaries (Butler et al., 2001; Le Saux et al., 2006). Trigger values Virus quantification is still under development (REPHEPA Chapter 12) and quantitative data on food implicated in outbreaks is still very rare. Using realtime RT-PCR, each oyster implicated in an international outbreak was estimated to be contaminated by about one hundred copies of NoV genome (Le Guyader et al., 2006b). In a previous outbreak, using MPN RT-PCR, about the same amount of virus per oyster was found (Le Guyader et al., 2003). In using the same methodology for virus extraction and RNA amplification, about 102 or 104 genome copies/oyster were detected in naturally contaminated oysters in Japan (Nishida et al., 2003, 2007). Post treatment It is obvious to say that the most effective and reliable approach to control viral contamination of shellfish is to harvest them from areas with good water quality preventing sewage pollution of these zones (Lees, 2000; Papafragkou et al., 2006). However, several factors, such as the increase of population in coastal areas, recreational activities or the high investment needed for efficient sewage treatment processes, make it difficult to get and to maintain high standards of water quality in certain shellfish harvesting areas. Therefore, for these cases control of contamination through mollusc processing procedures is necessary. There are two different forms of commercial processing available for reducing microbial contamination of shellfish, both being included in the EU regulations. Contamination may be reduced by extending the natural filterfeeding processes in clean seawater to purge out microbial contaminants (depuration and re-laying) or by heat treatment. Regulation (EC) 853/2004, Annex III, Section VII (Anon., 2004a), specifies the purification and re-laying requirements for food business operators in order to place live bivalve molluscs in the market. Depuration or controlled purification, is the process of reducing the microbial load in live shellfish by placing them in a controlled water environment. Purification periods are not stipulated in the legislation but are generally for 2±3 days. It is usually carried out in tanks provided with a supply of clean and disinfected (by ultraviolet light, chlorine or ozonation) seawater under specific operating conditions (Sobsey and Jaykus, 1991). Nevertheless viral depuration studies suggested that viral elimination is longer than for E. coli due to viral binding or attachment to digestive tissues (Burkhardt and Calci, 2000; Le Guyader et al., 2006b). Re-laying consists in the transfer of shellfish from B or C areas, to ad hoc approved waters. Although re-laying is cheaper than depuration, it requires extended periods (at least two months). Additionally, such special areas have to be approved by the authority, since their management has to be very robust regarding handling, traceability, geographical borders, etc. According to the Regulation (EC) 853/2004, Annex III, Section VII,

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Chapter II (Anon., 2004a) live bivalve molluscs collected from B areas can be either depurated or re-layed, whereas, those coming from C areas can not be depurated, re-laying being the only option. Bivalve molluscs from class C areas must be re-layed for a long period (Regulation 853/2004 specifies at least two months unless a risk analysis justifies a lesser period) prior to being placed on the market. Moreover, before entering the market for human consumption and regardless of the production area of origin (A, B or C), all live bivalve molluscs must meet the same microbiological and biotoxin criteria, as well as the organoleptic characteristics associated with freshness and viability. For faecal contamination the requirement of Regulation (EC) 2073/2005 is that live bivalve mollucs sold contain less than 230 E. coli per 100g of shellfish flesh (Anon., 2005). Regarding heat treatment, the same Regulation (EC) No 853/2004 (Anon., 2004a) specifies that live bivalve molluscs from such areas that have not been submitted for purification or re-laying may be sent to a processing establishment, where they must undergo treatment to eliminate pathogenic micro-organisms. The permitted treatment methods are: (a) sterilisation in hermetically sealed containers; and (b) heat treatments. The heat treatments included are (i) immersion in boiling water for the period required to raise the internal temperature of the mollusc flesh to not less than 90 ëC and the maintenance of this minimum temperature for a period of not less than 90 s; (ii) cooking for three to five minutes in an enclosed space where the temperature is between 120 and 160 ëC and the pressure is between 2 and 5 kg/cm2, followed by shelling and freezing of the flesh to a core temperature of ÿ20 ëC; and (iii) steaming under pressure in an enclosed space satisfying the requirements relating to cooking time and the internal temperature of the mollusc flesh mentioned under (i). In all cases, a validated methodology must be used, and procedures based on the HACCP principles must be in place to verify the uniform distribution of heat. It is interesting to point out that, in some cases (i.e., bivalve species and/or size), heat treatment may lead to undesirable changes of the organoleptic characteristics such as toughening of meat texture, which could result in a rejection by the consumer. Other approaches suggested for sanitisation of bivalve molluscs are ionising radiation and high hydrostatic pressure processing (HPP) (Papafragkou et al., 2006; Kingsley, 2002; Calci et al., 2005). Whereas HPP seems to be a promising alternative interesting for the shellfish industry since it has previously been shown to eliminate Vibrio species in oysters, while maintaining the organoleptic properties of the raw shellfish meat (Berlin et al., 1999). Ionising radiation has also been tested to inactivate viruses in shellfish but the results obtained and its high costs, make it a non-viable procedure. Neither of these two methods are considered in the new EU legislation.

13.4

Future trends

The shellfish industry is directly dependent on the quality of the production waters, both to support shellfish life and to ensure healthy products for the

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consumer. In the EU, regulations to monitor and achieve the adequate conditions for shellfish waters are under parallel (environmental and food safety) legislation (Anon., 2006d). At present, most shellfish monitoring programmes in the EU countries are still based on the rather static criteria established by the `vertical' legislation developed, on the one hand, to set the quality requirements for shellfish waters and stipulate the monitoring needed for certain key environmental and biota parameters (Directive 79/923/EEC, Anon., 1979) and, on the other hand, the EU Directive 91/492/EEC (Anon., 1991), which states the conditions for placing live bivalves on the market. Both Directives have a great deal of overlap regarding the criteria for biota parameters. To comply with such Directives, EU countries have implemented monitoring programmes for indicators of water quality and toxic phytoplankton in production areas, as well as for marine biotoxins and microbiological and chemical contamination in the shellfish themselves. The current horizontal EU legislation regulating all water bodies and all food commodities is established by the Water Framework Directive (WFD, Anon., 2000, 2006b) and by the new EU food safety regulations (Anon., 2004a,b,c). These laws were also designed to supersede the current legislation regarding shellfish (Directive 79/923/EEC and Directive 91/492/EEC, Anon., 1979; Anon., 1991). The new legislation is very complex. In general terms, its implementation requires compilation and interpretation tools for a wide variety of data series including catchment microbial dynamics (Kay et al., 2007). The WFD is heavily `ecosystem' based and, in the case of food legislation (Anon., 2004a,b; Anon., 2006a,b), integrated work strategies (e.g., HACCP) and risk-based approaches for the whole food chain have been introduced as requirements. In the case of live molluscs, criteria for official controls are developed in Annex II of Regulation (EC) 854/2004 (Anon., 2004a), which requires classification of production areas and their monitoring, as previously stipulated in Directive 91/492 (Anon., 1991). The broad requirement is that all production areas should be monitored according to their E. coli content into zones of either class A (<230 E. coli per 100 g shellfish flesh), class B (<4600 E. coli per 100 g shellfish flesh) or class C (<46,000 E. coli per 100 g shellfish flesh). The level of pollution determines the subsequent processing required prior to placing the product on the market. Products from class A zones may be directly marketed, class B products must be purified by treatment in an approved purification plant or relay centre or must be heat treated by an approved method, class C products must be purified by relaying for an extended period or must be heat treated by an approved method. Products exceeding class C level of contamination cannot be placed on the market. An important requirement of EU legislation is that official control monitoring plans should be performed according to scientific principles. Microbiological points should be representative of the pollution contamination conditions and sampling programmes should be conducted on a systematic basis. Regulation 854/2004 (Anon., 2004b) requires a scientific approach to selection

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of monitoring points based on a through assessment of the polluting influences. Regulation 854/2004 point 6 requires that: If the competent authority decides in principle to classify a production or re-laying area, it must: (a) make an inventory of the sources of pollution of human or animal origin likely to be a source of contamination for the production area; (b) examine the quantities of organic pollutants which are released during the different periods of the year, according to the seasonal variations of both human and animal populations in the catchment area, rainfall readings, waste-water treatment, etc.; (c) determine the characteristics of the circulation of pollutants by virtue of current patterns, bathymetry and the tidal cycle in the production area; and (d) establish a sampling programme of bivalve molluscs in the production area which is based on the examination of established data, and with a number of samples, a geographical distribution of the sampling points and a sampling frequency which must ensure that the results of the analysis are as representative as possible for the area considered. The process of gathering this data and making an assessment is commonly described as a `sanitary survey'. In general, implementation of these requirements is complex and requires acquisition and analysis of complex datasets. Therefore, overall, the new legislation evolves from a rather `close' approach, where criteria are fixed for both spatial and time scales, to a more dynamic, deductive prevention strategy, where shellfish production areas are considered by their complexity and inherent variations. However, the regulation does not provide the methodology to set up and run monitoring plans and deal with data treatment. This would need a great deal of co-ordination among competent authorities and official laboratories as well as collaborative work with R&D institutions in order to develop appropriate harmonisation tools for use in the EU territories. However, an EU working group chaired by the European Community Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs has recently considered these issues. A guide to good practice for microbiological monitoring has been developed to assist Competent Authorities and scientific institutes responsible for implementing these requirements (downloadable from www.crlcefas.org).

13.5

Conclusion

Although seafood can generally be regarded as a wholesome safe and nutritious food, it may occasionally pose consumer risks. Regulations are currently based on routinely monitoring shellfish for faecal bacteria to determine their sanitary

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quality. However, viral contamination of bivalve molluscs is currently recognised as one of the major causes of illness associated with seafood. Outbreaks of infectious hepatitis and gastroenteritis caused by the hepatitis A virus and norovirus are frequently documented. The REDRISK study was the first application in Europe of a viral genome quantification-based method to be carried out in the environment, and more particularly, on shellfish contamination in farming areas. Real time PCR methods for detection of NoV (gI and gII) were found to be very sensitive, robust and reproducible. The application of these methods showed the presence of NoV in different locations. However, the presence of levels of NoV in shellfish does not indicate the risk of norovirus infection and as it stands interpretation must be carefully done in term of sanitary risk. Further studies are required to establish the link between NoV levels observed in shellfish and the health risks for consumers. To answer the question `How can the risk be reduced?' The REDRISK study highlighted the following points: · Investigation for risk management requires basic knowledge about the sources (occurrence and level of viruses) and dilution mechanisms of the area where shellfish are grown. The widespread application of sanitary surveys would provide this information. · The REDRISK selected sites were found to be most significantly influenced by identified point-source contaminated input. Sewage treatment plants close to the shellfish farming areas are often the main source of viral contamination and the survey of overflow incidents is of the greatest concern for managing the risk. However, owing to the size of the flow the river inputs must be taken into account, to appreciate the `episodic' flux of faecal material during storm events which is one of the key risk periods for shellfish non-compliance. On many occasions the dilution was not sufficient to reduce the contamination of the plume. In areas close to faecal input (less than 1 km) NoV was regularly found. When located far from the sources, shellfish were submitted to intermittent contamination · The introduction of risk management procedures is appropriate in areas subject to intermittent contamination. Sanitary surveys can provide an initial assessment of the likely risk of NoV contamination during `risk periods'. In most of the countries `risk periods' can be defined as the conjunction of rainfall events while a winter viral epidemic is occurring in the population. Recent shellfish outbreaks were clearly linked with such events leading to overflowing of raw water or significant contaminated water. · To limit shellfish contamination, the most desirable and effective option is to reduce the viral input. Appropriate EU Directives have recently emerged and are identifying pressures and impact and to the design of a programme of measures on catchment (Anon., 2006b). This may be done by limiting the urban input in the watershed. The villages and dwellings there must be equipped with small individual treatment tanks to comply with the regula-

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tions. Another solution would be to relocate shellfish aquaculture away from the contamination sources. For example, towards the outer estuary where the dilution is greater. · Rainfall clearly has a major effect on contamination in shellfisheries. This may be because of increased river flows and the discharge of untreated raw sewage. On all sites, except in Spain, we demonstrated that these events could be predicted one or two days before the contamination. An early warning system based on site-specific criteria including salinity variations, rainfall and overflow telemetry/alarm systems could be proposed to advise the administration and shellfish farmers about possible shellfish contamination.

13.6

References

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study to assess human enteric viruses in shellfish', Appl Environ Microbiol, 66, 3241±3248. LE GUYADER F S, NEILL F H, DUBOIS E, BON F, LOISY F, KOHLI E, POMMEPUY M and ATMAR RL (2003), `A semi-quantitative approach to estimate Norwalk-like virus contamination of oysters implicated in an outbreak', Int J Food Microbiol, 87, 107±112. LE GUYADER F S, BON F, DEMEDICI D, PARNAUDEAU S, BERTOME A, et al. (2006a), `Detection of noroviruses in an international gastroenteritis outbreak linked to oyster consumption', J Clin Microbiol, 44, 3878±3882. LE GUYADER F S, LOISY F, ATMAR R L, HUTSON A M, ESTES M K, RUVOEN-CLOUET N, POMMEPUY

and LE PENDU J (2006b), `Norwalk virus specific binding to oyster digestive tissues', Emerg Inf Dis, 12, 931±936. LE SAUX J C, DEROLEZ V, BREST G, LE GUYADER F and POMMEPUY M (2006), `Elaboration of a strategy to limit shellfish viral contamination', in Molluscan Shellfish Safety, Proceeding of the 5th International Conference on Molluscan Safety, Galway, ed. Marine Institute, Ir. 342±349. LODDER W J and DE RODA HUSMAN A-M (2005), `Presence of noroviruses and other enteric viruses in sewage and surface waters in the Netherlands', Environ Microbiol, 71, 1453±1461. LOISY F, ATMAR R L, LE SAUX J-C, COHEN J, CAPRAIS M-P, POMMEPUY M and LE GUYADER S F (2005), `Use of Rotavirus virus like particles as surrogates to evaluate virus persistence in shellfish', Appl Environ Microbiol, 71, 6049±6053 LOPMAN, B A, BROWN D W and KOOPMANS M (2002), `Human caliciviruses in Europe', J Clin Virology, 24, 137±160. LOPMAN B A, REACHER M H, VAN DUIJNHOVEN Y, HANON F-X, BROWN D and KOOPMANS M. (2003), `Viral gastroenteritis outbreaks in Europe, 1995±2000', Emerg Infect Dis, 9, 90±96. LOPMAN B, H VENNEMA, E KOHLI, P POTHIER, A SANCHEZ, et al (2004a), `Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant', Lancet, 363, 682±688. M

LOPMAN, B A, REACHER M H, VIPOND I B, HILL D, PERRY C, HALLADAY T, BROWN D W, EDMUNDS

W J and SARANGI J (2004b), `Epidemiology and cost of nosocomial astroenteritis, Avon, England, 2002±2003', Emerg Infect Diseases, 10, 1827±1834. MACKOWIAK P A, CARAWAY C T and PORTNOY B L (1976), `Oyster-associated hepatitis: lesions from the Louisiana experience', Am J Epidemiol, 103, 181±191 MADAENI S S, et al (1995), `Virus removal from water and wastewater using membranes', J Membrane Sci, 102, 65±75 MIOSSEC L, LE GUYADER F, HAUGARREAU L, COMPS M A and POMMEPUY M (1998), `Possible relation between a winter epidemic of acute gastroenteritis in France and viral contamination of shellfish', J Shellfish Res, 17 (5), 1661±1664. MORSE D L, GUZEWICH J J, HANRAHAN J P, STRICOF R, SHAYEGANI M, DEIBEL R, GRABAU J C,

and BLACKLOW N R (1986), `Widespread outbreaks of clam- and oyster-associated gastroenteritis: Role of Norwalk virus', N Engl J Med, 314, 678±681. MURPHY A M, GROHMANN G S, CHRISTOPHER R J, LOPEZ W A, DAVEY G R and MILLSOM R H (1979), `An Australia-wide outbreak of gastroenteritis from oysters caused by Norwalk virus', Med J Aust, 2, 329±333. NASSER A M (1994), `Prevalence and fate of hepatitis A virus in water', Crit Rev Environ Sci Technol, 24, 281±323. NISHIDA T, KIMURA H, SAITOH M, SHINOHARA M, KATO M, et al. (2003), `Detection, NOWAK N A, HERRMANN, J E, CUKOR G

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quantitation, and phylogenic analysis of noroviruses in Japanese oysters', Appl Environ Microbiol, 69, 5782±5786. NISHIDA T, NISHIO O, KATO M, CHUMA T, KATO H, IWATA H and KIMURA H (2007), `Genotyping and quantitation of norovirus in oyters from two distinct sea areas in Japan', Microbiol Immunol, 51, 177±184. OLIVER S L, DASTJERDI A M, WONG S, EL-ATTAE L, GALLIMORE C, BROWN D W G, GREEN J and BRIDGER J C (2003), `Molecular characterization of bovine enteric caliciviruses: a distinct third genogroup of noroviruses (Norwalk-like viruses) unlikely to be risk to humans', J Virol, 77, 2789±2798. OLIVER S L, ASOBAYIRE E, CHARPILIENNE A, COHEN J and BRIDGER J C (2007), `Complete genomic characterization and antigenic relatedness of genogroup III, genotypes 2 bovine noroviruses', Arch Virol, 152, 257±272. PAPAFRAGKOU E, D'SOUZA D H and JAYKUS L-A (2006), `Food-borne viruses: prevention and control', in: Viruses in foods (S M Goyal, ed.), pp. 289±330. Springer Science + Business Media, LLC, New York. POMMEPUY M and LE GUYADER F (1998), `Molecular approaches to measuring microbial marine pollution', Current Opinion Biotech, 9, 292±299. POMMEPUY M, DUMAS F, CAPRAIS M P, CAMUS P, LE MENNEC C, PARNAUDEAU S, HAUGARREAU

and LE GUYADER F (2004), `Sewage impact on shellfish microbial contamination', Wat Sci Tech, 50, 117±124. POMMEPUY M, HERVIO-HEATH D, CAPRAIS M P, GOURMELON M, LE SAUX J C and LE GUYADER F (2005), `Fecal contamination in coastal area: an engineering approach', in Oceans and Health: Pathogens in the marine environment, (S Belkin, ed.) Kluwer Academic/Plenum Publishers, USA, 331±360. PRATO R, LOPALCO P, CHIRONNA M, BARBUTI G, GERMINARIO C and QUARTO M (2004), `Noro gastroenteritis general outbreak associated with raw shellfish consumption in South Italy', BMC Infect Dis, 21, 37. RIOU P, LE SAUX J C, DUMAS F, CAPRAIS M-P, LE GUYADER S F and POMMEPUY M (2007), `Microbial impact of small tributaries on water and shellfish quality in shallow coastal areas', Wat Res, 41, 2774±2786. ROSE J B, DICKSON L J, FARRHA S R and CARNAHAN R P (1996), `Removal of pathogenic and indicator microorganisms by full-scale water reclamation facility', Wat Res, 30, 2785±2797. Â NCHEZ G, PINTO Â R M, VANACLOCHA H and BOSCH A (2002), `Molecular characterization of SA hepatitis A virus isolates from a transcontinental shellfish-borne outbreak', J Clin Microbiol, 40, 4148±4155. L, SARRETTE B, VILAGENES P, POTHIER P, KOHLI E

SHIEH Y C, KHUDYAKOV Y E, GANOVA-RAEVA L M, KHAMBATY F M, WOODS J W, VEAZEY J E,

and FIORE AE (2007), `Molecular confirmation of oysters as the vector for hepatitis A in a 2005 multistate outbreak', Virol 70, 145±150 SOBSEY M D and JAYKUS L-A (1991), `Human enteric viruses and depuration of bivalve molluscs', in: Molluscan shellfish depuration (Otwell W S, Rodrick G E, Martin R E, eds), pp. 71±114. CRC Press, Boca Raton, FL. SOLIC M and KRSTULOVIC N (1992), `Separate and combined effects of solar radiation, temperature, salinity, and pH on the survival of faecal coliforms in seawater', Mar Pollut Bull, 24, 411±416. SORBER C A (1983), `Removal of viruses from wastewater and effluent by treatment processes', in: Vir pollut environ, G. Berg (ed.). CRC Press, Boca Raton, FL, pp. 39±52. MOTES M L, GLATZER M B, BIALEK S R

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and NAKAJIMA S (2002), `Viruses detected in the caecum contents of healthy pigs representing a new genetic cluster in genogroup II of the genus ``Norwalk like viruses'''. Virus Research, 87, 165±172. SVRAKA S, DUIZER E, VENNEMA H, DE BRUIN E, VAN DER VEER B, DORRESTEIJN B and KOOPMANS M (2007), `Etiological role of viruses in outbreaks of acute gastroenteritis in The Netherlands from 1994 through 2005', J Clin Microbiol, 45, 1389±1394. SYMES S J, GUNESEKERE I C, MARSHALL J A and WRIGHT P J (2007), `Norovirus mixed infection in an oyster-associated outbreak: an opportunity for recombination', Arch virol, on line. TANG Y W, WANG J X, XU Z Y, GUO Y F, QIAN W H and XU J X (1991), `A serologically confirmed case-control study of a large outbreak of hepatitis A in China associated with consumption of clams', Epidemiol. Infect, 107, 651±658. VAN DER BERG H, LODDER W, VAN DER POEL W, VENNEMPA H and DE RODA HUSMAN A M (2005), `Genetic diversity of noroviruses in raw and treated sewage water', Res Microbiol, 156, 532±540. VIDALES J A, GERBA C P and KARPISCAK M M (2003), `Virus removal from wastewater in multispecies subsurface-flow constructed wetland', Wat Envir Research, 75, 238± 245. WAGNER M and STRUBE I (2005), `Risk management in waste water treatment', Wat Sci Technol, 52, 53±61. SUGIEDA M

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14 Bacterial pathogens in seafood R. J. Lee and R. E. Rangdale, Centre for Environment, Fisheries and Aquaculture Science, UK, L. Croci, Istituto Superiore di SanitaÁ, Italy and D. Hervio-Heath and S. Lozach, Ifremer, France

14.1

General introduction

A number of bacterial illnesses may arise from the consumption of seafood that has either been contaminated at source or which becomes contaminated during the processing and retail chain. Such illnesses may arise from infection with the bacteria themselves or by the ingestion of toxins formed in the foodstuff prior to consumption. This division is actually too simplistic: for example, the toxins of Clostridium botulinum and Staphylococcus aureus are preformed in food during bacterial growth, the toxin of Clostridium perfringens is usually only formed when the bacteria sporulate in the intestinal tract while the toxin of Vibrio cholerae O1 (and O139) is produced when the bacteria multiply in the intestinal tract. One of the most common illnesses associated with seafood is scombrotoxin, due to the production of histamine, and possibly related compounds, by certain types of Gram-negative bacteria growing on histidine rich fish tissue. This subject is explored in more detail in Chapter 15 in this book and therefore it will not be considered in depth here. This chapter will consider the range of bacteria that may cause illness in humans following seafood consumption, the incidence of such infections that are reported in certain countries for which the data is available, and will review conventional and molecular techniques for the detection, identification and typing of the bacteria. There is increasing interest in the vibrios as agents of bacterial illness associated with the consumption of bivalve molluscan shellfish and other types of seafood and particular attention will be paid to this group of bacteria.

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14.2

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Principal bacterial pathogens associated with seafood

The principal bacterial hazards associated with seafood are shown in Table 14.1. In general, the bacterial hazards associated with seafood consumption, apart from the specific case of the marine vibrios pathogenic for humans, do not differ significantly from those associated with other foods. Some types of seafood, such as bivalve molluscs eaten raw (principally oysters), other raw seafood, or fermented seafoods, pose particular problems. Invasive listeriosis is a severe disease mainly associated with a specific risk group of people (Crum, 2002). It causes meningitis, encephalitis, bacteremia, and febrile gastroenteritis. Most disease occurs in immunosuppressed individuals. The organism occurs widely in the environment. Although there is a large concentration on testing of seafood products for Listeria monocytogenes, and some other Listeria spp., the level of risk associated with this organism from seafood consumption is probably small ± the US risk assessment on L. monocytogenes only identified four seafood associated outbreaks worldwide in the period 1970±2000, one associated with raw seafood, one with smoked seafood and two with smoked mussels (FDA, 2003). Table 14.2 gives some of the principal clinical features of the illnesses caused by these organisms, together with the principal sources. The latter is discussed further in Section 14.3. 14.2.1 Vibrios involved in food poisoning Vibrios are Gram-negative bacteria that are primarily associated with estuarine and coastal marine environments. A number of species have been associated with intestinal or extra-intestinal infections in humans (see Table 14.3). All vibrios have an absolute requirement for Na+ for growth although some, such as V. cholerae only require trace amounts. The surface and intestinal microbial flora of harvested fish and shellfish, particularly those from brackish, estuarine and coastal environments, will tend to contain high numbers of members of the genus Vibrio. Only a small proportion of the vibrios present will belong to species that may be pathogenic in humans and, of these, only a small proportion of the isolates of such species may possess the pathogenicity traits that enable them to colonise and cause disease in the human body. Table 14.1

Bacterial hazards associated with seafood

Type of illness

Causative agent

Infections

Salmonella spp., Shigella spp., Campylobacter spp., Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, other Vibrio spp., Listeria monocytogenes, E. coli O157

Intoxications

Clostridium botulinum, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus

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Table 14.2 Clinical features and principal sources of seafood-associated bacterial illness Micro-organism Infections Listeria monocytogenes

Salmonella Typhi and Paratyphi

Incubation period

Duration

Principal signs and symptoms

Principal source of contamination of seafood

3 to 90 days, with the median incubation period being three weeks

±

Fever, muscle aches, nausea or diarrhoea Central nervous system infection ± headache, stiff neck, confusion, loss of balance, convulsions

Marine environment or crosscontamination ± usually during processing

Typhi: 1±3 weeks

Typhi: up to 4 weeks

Malaise, headache, fever, cough, nausea, vomiting, constipation, abdominal pain, chills, rose spots, bloody stools

Human faeces/sewage

Paratyphi: 1±10 days

Paratyphi:

Other source: 7±28 days, mean 14 days Other Salmonella

6±72 hours, mean 18±36 hours

4±7 days

Abdominal pain, diarrhoea, chills, fever, nausea, vomiting, malaise

Human faeces/sewage or animal/bird faeces/slurry

Campylobacter

2±7 days

3±6 days

Diarrhoea, (often bloody), severe abdominal pain, fever anorexia, malaise, headache, vomiting

Animal/bird faeces/slurry

Shigella

24±72 hours

5±7 days

Abdominal pain, diarrhoea, bloody and mucoid stools, fever

Human faeces/sewage

Vibrio parahaemolyticus

2±48 hours, mean 12 hours

2±14 days (average 2.5)

Abdominal pain, diarrhoea, nausea, vomiting, fever, chills, headache

Marine environment

Vibrio vulnificus

16 hours mean <24 hours

2±3 days

Malaise, chills, fever, prostration, cutaneous lesions, fatalities occur

Marine environment

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Table 14.2 Continued Micro-organism

Incubation period

Duration

Principal signs and symptoms

Principal source of contamination of seafood

Vibrio cholerae

1±5 days, usually 2±3 days

2±5 days

Profuse, watery diarrhoea (rice-water stools), vomiting, abdominal pain, dehydration

O1/O139: human faeces/sewage Other serotypes: marine environment

Other Vibrio spp.

2±3 days

Watery diarrhoea (varies from loose stools to cholera-like diarrhoea)

Marine environment

Intoxications Bacillus cereus ± diarrhoeic

6±15 hours

Watery diarrhoea, abdominal cramps, and pain

Environment

Early signs of intoxication consist of marked lassitude, weakness and vertigo, usually followed by double vision and progressive difficulty in speaking and swallowing. Difficulty in breathing, weakness of other muscles, abdominal distention, and constipation may also be common symptoms ± paralysis and death may occur

Type F Cl. botulinum spores occur widely in the marine environment

24 hours

Clostridium botulinum

18±36 hours

Clostridium perfringens

8±22 hours

<24 hours

Abdominal cramps and diarrhoea

Human faeces/sewage or animal/bird faeces/slurry and the general environment

Staphylococcus aureus

4±6 hours

<24 hours

Nausea, vomiting and abdominal cramps

Faecal or skin contamination of the marine or processing environment Food handlers

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Bacterial pathogens in seafood Table 14.3

Vibrio spp associated with foodborne illness

Vibrio-species 1. V. V. 2. V. 3. V. 4. V. 7. V. 8. V. 9. V. 10. V.

251

cholerae O1/O139 cholerae non O1/ non O139 parahaemolyticus vulnificus fluvialis furnissii hollisae mimicus metschnikovii

GI tract ++ ++ ++ + ++ (+) ++ ++ (+)

Primary septicaemia

Bacteraemia

(+)

(+) (+) +

++ (+) (+)

Source: Modified from European Commission (2001) (which used material from West, 1989 and Oliver and Kaper, 1997)

Doubling time of vibrios under ideal conditions may be as short as 10 minutes or less, but usually much longer in seafoods at ambient temperature. Little growth of pathogenic vibrios usually occurs in seafood stored below 10 ëC. A one-log reduction of pathogenic vibrios in seafoods takes between 1 and 10 minutes at 50 ëC, depending on species and strain. Vibrios die out gradually at refrigeration temperatures and numbers are markedly reduced by freezing and thawing. Marine vibrios naturally contaminating bivalve molluscs have been shown to be less easily removed by depuration than are faecal bacterial indicators such as E. coli (Rodrick and Schneider, 1991). Such processing methods may therefore not provide the necessary level of public health protection if significant levels of pathogenic vibrios are present in harvested product. The infectious dose of human pathogenic marine vibrios may be relatively high (Sanyal and Sen 1974) and therefore the presence of low numbers in final product may therefore not be of significance. It is also known that strains of the potentially pathogenic species vary in their pathogenicity. Only a small proportion of the vibrios present will belong to species that may be pathogenic in humans and, of these, only a small proportion of the isolates of such species may possess the pathogenicity traits that enable them to colonise and cause disease in the human body. These factors enhance the difficulty of isolating potentially pathogenic Vibrio spp from seafoods and also in fully determining the potential significance of those that are detected. Further information may be found in the US risk assessment on V. parahaemolyticus (FDA, 2005), the European Commission report on V. parahaemolyticus and Vibrio vulnificus in seafood (European Commission, 2001) and the FAO/WHO risk assessments on V. vulnificus (FAO/WHO, 2005a) and V. cholerae (FAO/WHO, 2005b).

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Vibrio cholerae Epidemic and pandemic cholera is associated with toxigenic strains of Vibrio cholerae O1 and O139. The toxin is produced in the intestine during multiplication and causes marked loss of fluid into the intestine, resulting in extremely watery diarrhoea which leads to extreme dehydration and, if not treated, to death. Contamination of seafood is usually either by faecal contamination of the marine environment or by contact with faecally contaminated fresh water during preparation of the food. Vibrio cholerae serotypes other than O1 and O139 may be pathogenic. Some may be able to produce cholera toxin but lack the ability to produce epidemics and pandemics. Although V. cholerae O1 and O139 infections are potentially severe, and the public health consequences of epidemics may be grave, infections due to other V. cholerae serotypes are much more commonly associated with seafood and, in general, cause a much higher level of morbidity and mortality from this source. Vibrio cholerae O1 outbreaks have also been reported in association with raw shellfish consumption (Baine et al., 1974) or raw seafood or cooked crab (Weber et al., 1994). Whereas contamination with other vibrios arises from bacteria naturally present in the marine environment, that with V. cholerae, in particular serovar O1, may be associated with faecal contamination. V. cholerae non-O1 serotypes have also been reported from the marine environment in the UK, France and Italy (Bashford et al., 1979; Dumontet et al., 2000; Hervio-Heath et al., 2002). Vibrio parahaemolyticus Vibrio parahaemolyticus food-poisoning associated with seafood consumption has been identified in many countries (Blake et al., 1980). Food-poisoning with this organism is most commonly reported from countries with both a high ambient temperature and where seafood is consumed raw. Seafoods which may be associated with human infection due to V. parahaemolyticus are bivalve molluscs, finfish and crustacea, particularly those served raw or superficially cooked and which have been exposed to temperature abuse (see below). In Europe, the main type of seafood eaten raw or lightly cooked are species of bivalve molluscan shellfish and thus arguably bivalve molluscs, such as oysters and mussels, constitute a risk to the European consumer. V. parahaemolyticus can be commonly isolated from estuarine environments that form suitable habitats for the growth of a variety of shellfish species and it has been shown to be present in the marine environment of several European countries, including the UK (Ayres and Barrow, 1978), France (Hervio-Heath et al., 2002), Spain (Lozano-LeoÂn et al., 2003), Italy (Cavallo and Stabili, 2004) and Greece (Davies et al., 2001). There is a well-recognised correlation between pathogenicity of V. parahaemolyticus isolates and their ability to form Thermostable Direct Haemolysin (TDH) or TDH-Related Haemolysin (TRH) (Takeda, 1983; Honda and Iida, 1993). However, the presence of V. parahaemolyticus in the absence of thermostable haemolysins may not represent a pathogenic risk as the proportion of strains isolated from environment possessing the ability to

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produce these putative pathogenicity principles is reportedly low (Nishibushi and Kaper, 1995). For example, in the United States, only 0.2±3.2% of environmental isolates of V. parahaemolyticus isolates contain the tdh gene which is associated with the potential to cause gastroenteritis in humans, with the proportion varying by geographical region (FDA, 2001). Currently in Europe the prevalence and distribution of TDH and TRH producing V. parahaemolyticus in the marine environment or indigenously produced seafoods is not known. Vibrio vulnificus Vibrio vulnificus, is associated with significant mortality in persons with underlying predisposing illness who consume raw shellfish or who are subject to contamination of open wounds by seawater. These predisposing conditions include liver disease, diabetes mellitus and immunodeficiency (including that caused by AIDS). Illness caused by consumption of raw oysters usually manifests as a primary septicaemia, where the systemic illness is not preceded by any apparent localised infection ± i.e. there may not be any gastrointestinal symptoms. The mortality rate is approximately 50%. Reports of infection with this organism have originated mainly from the United States but there have also been isolates in other parts of the world (Hlady, 1997; Chuang et al., 1992). In Europe, V. vulnificus has been reported as having been isolated from mussels in Denmark (Hùi et al., 1998) and France (Hervio-Heath et al., 2002) and from cutaneous infections in Scandinavia and the Low Countries (Melhus et al., 1995; Veenstra et al., 1993). Other Vibrio spp. · Vibrio mimicus is a bacterium that resembles Vibrio cholerae in many characteristics, including its ability to grow in low concentrations of sodium and its antigenic structure. It has been implicated in individual cases and outbreaks of gastroenteritis and some strains can also produce cholera toxin. · Vibrio fluvialis is an inhabitant of estuarine and coastal waters and has been identified as causing individual cases and outbreaks of gastroenteritis. As with many other vibrios, infection is usually associated with the consumption of raw or undercooked seafood, especially raw oysters. · Vibrio furnissii is closely related to Vibrio fluvialis both genetically and phenotypically. However, it has been isolated much more rarely from faecal samples of patients with gastroenteritis and so there is some doubt as to its significance as a seafood-associated pathogen. · Vibrio hollisae is a marine vibrio that has been reported to cause gastroenteritis following consumption of raw shellfish. The significance of this vibrio is difficult to estimate because of its failure to grow on conventional vibrio media (Morris et al., 1982).

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14.3

Improving seafood products for the consumer

Sources of bacterial pathogens in seafoods

14.3.1 Contamination at source Contamination of seafood with bacterial pathogens at source (i.e., in the sea) primarily arises from two different origins. The first with bacteria that occur naturally in the marine environment which, when consumed in seafood in large enough numbers, will cause illness in humans. This primarily relates to the vibrios. Some species of the genus Aeromonas are considered to some to possibly cause gastroenteritis in humans and these may also be present naturally in the marine or, more especially, the estuarine environment. Spores of type F Clostridium botulinum are found widely in marine sediments and the intestinal tract of fish and shellfish and, if seafood is stored under conditions (principally in the absence of oxygen) that allows the spores to germinate and the bacteria to multiply, toxin may be formed in the seafood and then cause botulism in humans when the food is consumed. The other source of contamination is from faecal contamination of the marine environment. This may arise from contamination with human faeces from sewage discharges, boats and ships or, in some countries, direct defaecation into the marine environment or rivers and streams flowing into it. Some bacteria may also arise from the contamination of the marine environment by animal faeces. Again, this may arise from discharges from animal keeplots or slurry pits, or direct defaecation into the marine environment or rivers or streams flowing into it. Some bacteria, such as Salmonella Typhi and Vibrio cholerae, are principally associated with human faecal contamination while non-typhi Salmonella and Campylobacter, may arise from either source. Seafood harvested from such environments may constitute a risk if: · the bacteria are initially present in sufficient numbers that they exceed the infectious dose · or the seafood is stored under conditions that allow a lower initial concentration to multiply to the point where the bacteria exceed the infectious dose · and, if any control measures are applied, they are not sufficient to reduce to, or maintain the bacterial concentration, at a low enough level. Similar criteria apply to the production of toxin by toxin-forming bacteria. In general, contamination at source is a greater problem with respect to bivalve molluscs than other seafoods due to their filter-feeding activities which tend to concentrate pathogens within the gut of the bivalves. Following harvest, these tend to be eaten raw or lightly cooked, thus potentially allowing the survival of any pathogens present in the bivalves at harvest. The issue of temperature control following harvesting is particularly important with respect to the risk of illness from pathogenic vibrios. In the USA, it has been shown that the cooling time and subsequent time and temperature of transport and storage are significant risk factors with respect to V. parahaemolyticus infection from oysters (FDA, 2005). In Europe, it is not the norm for bivalve molluscs to be refrigerated between harvest and either depuration or dispatch.

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14.3.2 Cross-contamination Cross-contamination may take place in the processing environment or during food preparation prior to consumption. Such contamination may be from the environment to the food, from a contaminated food batch or item to a previously uncontaminated one, or from an infected food handler to one of more items of food. Some pathogenic bacteria can become established in the processing environment and, if not eradicated, can be a long-term source of contamination of batches. This applies particularly to L. monocytogenes in fishery product establishments (Miettinen, 2006). Once this organism becomes established in a factory it can be difficult to remove by normal cleaning procedures. As noted above, live bivalve molluscs, being contained in their shells, are generally less prone to cross-contamination than other seafoods. However, if several batches of bivalves from different areas are immersed in the same seawater tank(s), during either wet storage or depuration, then crosscontamination may occur from one batch to another. Contamination may also arise if the seawater itself is not of sufficient initial quality, or is not otherwise properly treated prior to use. Salmonella spp. has been shown to be viable after excretion in oyster faeces (Rowse and Fleet, 1982). A potential route of contamination of cooked crustacea and bivalve molluscs is the use of untreated seawater for cooling the shellfish following the cooking process (European Commission, 2001). This procedure is particularly prone to take place when cooking is undertaken on board boats.

14.4

Control of bacterial contamination

The pursuit of a high level of protection of human life is the fundamental objective of Regulation (EC) No 178/2002, which prohibits the placing on the market of unsafe food and provides a uniform basis for the use of the precautionary principle. To this aim Regulation (EC) No. 852/2004 lays down general rules for food business operators, which have the primary responsibility for food safety, that must be assured throughout the food chain, starting with primary production, by means of the implementation of procedures based on HACCP principles, together with the application of good hygiene practice (GHP). Besides this, the Regulation (EC) No. 853/2004 lays down specific hygiene rules, necessary for certain foodstuffs, that shall be applied to unprocessed and processed products of animal origin. Then on the basis of the Regulation (EC) No. 854/2004, laying down specific rules for official controls on foodstuff animal origin, the Competent Authority of the Member States must carry out inspections to control the observance of the quality criteria of the products to assure the health of the consumer. Microbiological standards are established by the Regulation (EC) No. 2073/2005, foods not meeting standards are in noncompliance and in this case the regulatory control actions may lead to sorting, reprocessing, rejection or destruction of

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product, and/or further investigation to determine the appropriate actions to be taken. Microbiological limits should be appropriate for the food and be applicable at one or more points in the food chain. Limits for specific hazards should be compatible with any FSOs (Food Safety Objective) that may have been established. In particular for the microbial contamination of the seafood it must be considered that the dominant population of bacteria on finfish and shellfish is mainly composed of saprophytic species, however the level of contamination of living fish with bacteria of public health significance can vary greatly among the localities. Molluscan shellfish are normally considered separately from the other seafood because of their different physiologies, modes of life, feeding and handling/processing requirements after harvest. 14.4.1 Harvesting, handling and transport Fresh fishery products Finfish are caught in areas more or less remote from the processing plants. After capture, to ensure microbiological quality and safety, they must be protected from spoilage during the transport to the processing plant, that can vary from a few hours to some weeks. Beneficial effects on storage life can be obtained reducing the bacteria number or preventing the flesh contamination. Finfish may be eviscerated on board the vessel and, as the contamination of fish from handling and storage procedures can affect subsequent storage (Ward and Baj, 1988), the operations such as heading and gutting must be carried out hygienically and as quickly as possible. The products must be washed thoroughly with potable water or, on board vessels, clean water immediately after these operations. Containers used for the dispatch or storage of unpackaged prepared fresh fishery products stored under ice must ensure that melt water does not remain in contact with the products. On board vessels and after landing the products must be transported in cooled water until arrival at the first establishment. Food business operators must ensure all these requirements according to the Regulation (EC) No. 853/2004. Live bivalve molluscs Live bivalve molluscs must be harvested only from areas that the Competent Authority has classified as being of class A, B, or C in accordance with Regulation (EC) No. 854/2004. Classification is based on levels of E. coli in bivalves in the harvesting area as an indicator of the risk of the presence of pathogens associated with faecal contamination. Recommendations for such monitoring have been produced by a European Working Group (CRL, 2007). Bivalve molluscs collected from class B or C production areas may be placed on the market for human consumption, only after a purification treatment performed respectively in a purification centre or after relaying over a long period in accordance with Regulation (EC) No. 853/2004. Purification was originally

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developed for the removal of S. Typhi from bivalve molluscs: it has been reported that 0.1% of the original concentration of salmonella remains in clams after 24 hours purification (Timoney and Abston, 1984). Oysters naturally contaminated with pathogenic bacteria were found to retain a significant proportion (44%) after two days purification and therefore the process is not effective in removing all types of bacterial pathogens (Jones et al., 1995). Longterm relaying is intended to remove viral contamination. However, relaying in high salinity seawater (>30 parts per thousand) has been suggested as a way of removing pathogenic vibrios from bivalves (Motes and DePaola, 1996). During all production processes, food operators must use harvesting techniques and further handling that do not cause additional contamination or damage to the animals, adequately protecting them from crushing, abrasion or vibration and not exposing them to extreme temperatures or re-immersing them in water that could cause additional contamination. After harvesting or treatment food business operators storing and transporting live bivalve molluscs must ensure that they are kept at a temperature that does not adversely affect food safety or their viability. The products must not be reimmersed in, or spayed with, water after they have been packaged for retail sale and left the dispatch centre. 14.4.2 HACCP plans Food operators have the responsibility to apply, along the food chain, a series of control measures that contribute to providing safe products. These measures, applied at different steps of the production, aim to eliminate, prevent or reduce the hazard to an acceptable level, according to the GHP and HACCP programmes. The GHP programme, based on the application of the basic hygienic practices, also includes other activities such as raw material selection, lot identification, product information, labelling, consumer education, handling/storage instructions and so on. Its application provides the foundation for HACCP systems. 14.4.3 Failure of control measures With fishery products, there are two principal ways that failure may occur in such a way as to affect the bacteriological quality of the product. The first is a failure to maintain adequate temperature control which may allow proliferation of bacteria in the product. These bacteria may be pathogens, in which case illness may ensue if the resulting concentrations exceed those needed to initiate infection. If the bacteria are non-pathogens, the failure of temperature control may result in spoilage. The failure in temperature control may be at any point along the chain after harvest. However, in order to increase the risk of illness, the failure must be after the stage at which pathogens were first present in the product. The exact point at which this occurs is not always evident. The second way is by crosscontamination as outlined in Section 14.3.2. Such cross-contamination may occur

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at any point post-harvest but is often associated with contamination at the production plant or during food preparation. There is obviously the possibility of both events occurring: cross-contamination introducing a pathogen to the product and inadequate temperature control exacerbating the risk by allowing multiplication of the pathogen. The toxin of Clostridium botulinum is formed when the organism multiplies under the right conditions. Two principal food processes are associated with botulism. One is canning when insufficient heating during the canning process leaves spores which can then grow in the anaerobic conditions in the can. The other is fermentation of fish products, especially when whole or inadequately cleaned fish are fermented for prolonged periods of time to form fish products or sauces. Salmonella infection primarily occurs due to the consumption of undercooked food contaminated at the time of harvest or by cross-contamination after cooking. The risk of infection is increased by the storage of cooked food at ambient temperature prior to consumption. The assessment of risk associated with bivalve mollusc harvesting areas based on E. coli monitoring depends partly on the design and implementation of the monitoring programme (Younger et al., 2003). There is no relationship between faecal indicators and the presence of pathogenic vibrios, not even Vibrio cholerae (European Commission, 2001), and therefore classification does not reflect the risk from these pathogens. In general, the European legislative controls on commercially produced bivalve molluscs have addressed the problems of bacteria associated with faecal contamination that can arise from consumption of this type of seafood. However, this does not address bivalves gathered by the public for their own consumption. Also, unfortunately, bivalves for commercial sale may not fully comply with the legislative requirements, either by intent or by ignorance. An example of this is an outbreak of Salmonella in the UK which was due to cockles which had been harvested from an unclassified area and had been inadequately cooked in a domestic environment before sale (Greenwood et al., 1998).

14.5

Seafood-associated bacterial illness

The level of epidemiological reporting of seafood-associated illness varies markedly between different countries. Reporting of internationally notifiable illnesses, such as cholera and typhoid, may be expected to be more consistent, but even this can be subject to political interference as countries may not wish to be identified as having such a disease. This means that global, and even regional, illness statistics related to seafood consumption are not reliable. Even within individual countries, several factors affect the level of reporting and complicates the assessment of outbreak data (O'Brien et al., 2002). This also means that the statistics from individual countries will not be comparable. However, comparison of data from countries with relatively good reporting systems will give

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an impression of the problems and the relative risk from the consumption of different seafoods. Epidemiological data usually relates to the reporting of outbreaks and not individual infections. An outbreak consist of two or more linked cases of the same illness. Individual infections may be reported if the illness is formally notifiable, or if they cause mortality (e.g., primary septicaemia due to V. vulnificus). Scientific reviews of illness may be confined to individual foods or types of micro-organisms. These may contain information not available from the epidemiological data. The fullest reviews of seafood-associated outbreaks are available for the United States and England and Wales although some information is available for other countries. Most of these reviews were last undertaken for the 1990s and have not been systematically assessed more recently. These reviews will be examined and compared here. Other data on the occurrence of illness related to vibrios will be separately examined in more depth. Over the period 1992±1999, there were 1425 outbreaks in England and Wales associated with fish and shellfish and 181 of these (13%) were due to seafood consumption (Gillespie et al., 2001). The seafood type and identified cause are shown in Table 14.4. This shows that the predominant illness associated with fish consumption was scombrotoxin and that associated with bivalve mollusc consumption was viral gastroenteritis. Bacterial infections associated with the consumption of both fish and crustacea in temperate developed countries appear to be those associated with foods in general and none that is specific to the origin and processing of the foods. This is not the case with bivalves, which, in Europe, are largely either sold as live in-shell animals (except for cockles which are sold as commercially Table 14.4 Seafood type and identified cause of seafood-associated outbreaks in Enlgand and Wales 1992±1999 Cause

Scombrotoxin Salmonella Campylobacter Cl. perfringens B. cereus Staph aureus Norovirus Astrovirus DSP Unknown Total

Seafood type Fish

Bivalves

Crustacea

Not defined

Mixed

Total

47 7 1 1 1 0 0 0 0 12 69

0 1 0 0 0 0 19 2 1 31 54

0 4 1 1 0 1 3 0 0 7 17

0 2 0 1 0 0 2 0 0 1 6

0 0 1 0 0 0 0 0 0 1 2

47 14 3 3 1 1 24 2 1 52 148

Source: Gillespie et al. (2001)

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Table 14.5 US outbreaks from 1990 to 1998 associated with the consumption of fisha Agent

Scombroid Ciguatera Botulism Salmonella Haff disease S. aureus E. coli O157 V. cholerae C. perfringens Norwalk Tetrodotoxin `Chemical' Total

Outbreaks

Cases

Number

%

131 98 14 11 2 1 1 1 1 1 1 1 263

50 37 5 4 1 <1 <1 <1 <1 <1 <1 <1 100

Number 759 394 43 305 6 2 3 26 25 37 3 58 1661

% 47 24 3 18 <1 <1 <1 2 2 2 <1 4 100

a Only those outbreaks for which an aetiological agent had been identified. Source: Huss et al. (2004).

heat-treated products) and which may, especially in the case of oysters, be consumed raw. This would suggest that the small numbers of bacterial outbreaks associated with fish and crustacea are due to post-harvest contamination, the live in-shell bivalves being naturally protected from this. In the US, between 1993 and 1997, there were 2751 reported food-associated outbreaks and 47 (1.7%) were identified as due to shellfish consumption and 140 (5.1%) due to shellfish consumption (Huss et al., 2004). Further details are available for US outbreaks between 1990 and 1998 and the seafood types and identified causes are shown in Table 14.5 (fish) and Table 14.6 (bivalve shellfish). A worldwide review of foodborne disease was published in 1997 (Todd, 1997). This included some consideration of seafood-related disease. In France, from 1990 to 1992 inclusive, 60 (8.4%) of the 717 reported outbreaks were associated with the consumption of seafood: Salmonella (21 outbreaks); S. aureus (4); other (35); no seafood-related outbreaks were attributed to C. perfringens. The same review showed that seafood was incriminated in 31.8% of outbreaks in the Republic of Korea and 21.7% of outbreaks in Japan. Sumner and Ross (2002) reported that 32 seafood-related outbreaks involving 2158 people occurred in Australia in the period 1990±2000 and that 6 outbreaks involving 159 cases were due to bacterial pathogens. A review of bivalve mollusc-associated disease in the US from 1984±1993 showed that, of the 2548 reported cases of illness, 546 were due to vibrios as were 95 of the 96 reported deaths (Wittman and Flick, 1995). The other bacterial causes were Salmonella (8 cases), Shigella (76 cases) and Campylobacter (12 cases).

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US molluscan shellfish-associated outbreaks 1990±1998a

Agent

V. parahaemolyticus Norwalk/virus PSP/toxin Salmonella Scombroid Ciguatera Shigella Campylobacter V. vulnificus V. alginolyticus C. perfringens Giardia Total

Outbreaks

Cases

Number

%

18 15 16 6 2 3 2 2 1 1 1 1 66

27 23 20 9 3 5 3 3 <1 <1 <1 <1 100

Number 733 2175 92 183 4 5 17 6 2 4 57 3 3281

% 22 66 3 6 <1 <1 <1 <1 <1 <1 2 <1 100

a Outbreaks for which an aetiological agent had been identified. Source: Huss et al. (2004).

In 2006, there were 154 isolates of vibrios from laboratory confirmed illness by the CDC's FoodNet Surveillance network in 10 US States. Of the 147 for which a species identification was given, 94 (64%) were V. parahaemolyticus, and 18 (12%) were V. vulnificus (CDC, 2007). It was noted that this represented the highest level of vibrio infection reported by the network since it started and that most cases were due to raw seafood, principally oysters. Todd (1997) reported that the proportion of foodborne outbreaks due to Vibrio spp. in a number of countries were: Australia (3%), Japan (47.3%), Korea (37.6%), Taiwan (55.3%). No outbreaks caused by vibrios were reported from 16 European countries. While most of the reported vibrio-associated outbreaks were due to V. parahaemolyticus, a number of outbreaks due to V. cholerae O1 were also reported, including one in Japan related to imported Korean clams. The National Institute of Infectious Diseases in Japan reported that there were 496 outbreaks of V. parahaemolyticus food poisoning during the period 1996± 1998 (102 in 1996, 160 in 1997, and 234 in 1998). Two outbreaks involved more than 500 cases each. One of these outbreaks was associated with boiled red queen crabs and serotype O3:K6 V. parahaemolyticus. The other large outbreak was associated with catered meals and multiple serotypes of V. parahaemolyticus; the nature of the meals was not stated. Two large outbreaks of V. parahaemolyticus occurred in Chile in 2004 and 2005, affecting approximately 1500 people in 2004 and approximately 3600 people in 2005 (Fuenzalida et al., 2006). The outbreaks were due to the O3:K6 pandemic clone which had been first seen in south-east Asia in 1996. Before 2004, V. parahaemolyticus infections had been rare in Chile.

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14.6 Conventional methods for the detection, enumeration and identification of bacterial pathogens 14.6.1 Introduction Conventional methods for detection of foodborne pathogenic micro-organisms are dependent on the growth of micro-organisms, to the extent of being able either to form visible colonies in solid media, or to produce a detectable chemical change in liquid media. In seafood, as in other food, pathogenic bacteria are often present in relatively low numbers, outnumbered by spoilage micro-organisms and possibly damaged by processing operations. Then the qualitative or quantitative detection is a complex procedure involving the different stages: resuscitation or pre-enrichment, enrichment and selective plating, followed by subsequent identification through morphological, biochemical and/or immunochemical tests. Resuscitation may involve preliminary incubation in nonselective media or, alternatively the addiction of protective compound such as pyruvate or egg yolk to solid selective media. Enrichment is applied where pathogenic micro-organisms are present only in small number, particularly if large numbers of spoilage micro-organisms are present. Although in many cases enrichment media are used in conjunction with selective plating media, they may be used to enumerate specific bacteria or group of bacteria using the Most Probable Number (MPN) technique. In such cases it is necessary to incorporate a diagnostic reaction to distinguish the micro-organism being enumerated from micro-organisms (i.e. `sulphide' reaction in media for clostridia). However such differentiation must be regarded as presumptive and confirmatory tests (biochemical and/or immunochemical tests) are required. Within Europe, reference methods are specified for the detection and/or enumeration of bacteria for which legislative standards are defined (European Communities, 2005). It is assumed that the reference methods will be use for official control purposes. Alternative methods may be used for testing undertaken by food business operators if they are validated against the reference method. In particular, proprietary methods have to be certified by a third party in accordance with the protocol set out in EN/ISO standard 16140 or another internationally accepted similar protocol. The European Commission, via the European standardisation organization, CEN, has commissioned the validation of a number of methods for which the available information was deemed to be insufficient. These approaches should help to ensure that the results obtained by different laboratories are equivalent. Many methods traditionally used for pathogens in foods have been qualititative, using enrichment (non-selective and/or selective) followed by plating on a differential selective medium. A standard weight of 25 g of food has customarily been used in such tests. However, the different pathogens vary widely in their infectious dose and therefore results obtained using this approach do not relate to the risk posed by the pathogen/food combination. This drawback has been recognised for listeria in the EU microbiological criteria for foods where

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different standards are specified depending on whether the foods are able to support the growth of listeria and also whether they are intended to be eaten by particular at risk groups such as infants. This approach has not, however, been taken for all of the pathogens covered by the Regulation. Classical methods are characterised by a good sensitivity, specificity and are able to determine only the viable cells, but they present some disadvantages. Firstly, they tend to be labour-intensive and time-consuming. Secondly, they do not always allow the detection, or more especially the enumeration, of relatively small numbers of bacterial cells of pathogenic species in the presence of large numbers of cells of non-pathogens. Thirdly, they do not necessarily allow the reliable detection of strains of potential pathogens that possess pathogenic principles, e.g. the ability to form toxins, especially in the presence of nonpathogenic forms of the same species. Therefore, many research efforts have been devoted to the development of rapid and sensitive alternative methods (Vaneechoutte and Van Eldere, 1997; Croci et al., 2004; Panicker et al., 2004). Application of DNA and RNA probes, immunochemical methods and detection of bioluminescence are some of the tools currently used (Hill, 1996; Meer and Park, 1995; Jaykus, 2003; Nelson, et al., 2007). In particular the Polymerase Chain Reaction (PCR) technique allows improvements in detection and characterisation of bacteria, using species-specific DNA regions and specific traits of pathogenicity. In addition to present several advantages over conventional methods with respect to detection limit, speed and potential for automation (Vaneechoutte and Van Eldere, 1997). 14.6.2 Conventional methods for specific pathogens Conventional methods for the detection of some seafood-associated pathogens, with particular attention to pathogenic vibrios, are reported. Salmonella spp The genus Salmonella, belonging to the Enterobacteriaceae family, includes Gram-negative bacteria which have both a respiratory and fermentative metabolism of carbohydrates. The cultural methods for the isolation and detection of salmonella in food are well established. Regulation (EC) No. 2073/2005 for microbiological criteria in foodstuff reports that the salmonella must be absent in 25 g and the analysis must be performed on five sampling units by means of the EN/ISO 6579 reference method. This method currently involves nonselective pre-enrichment in buffered peptone water followed by selective enrichment in Rappaport Vassiliadis Soya Broth and Muller-Kauffmann tetrathionate/ novobiocin broth followed by selective plating on xylose lysine deoxycholate agar (XLD) and one other selective agar of the laboratory's choice. Any suspect salmonella strains that are isolated are confirmed by biochemical and serological test methods. The ISO method has not been fully validated for S. Typhi and S. Paratyphi and so, if these species are of specific interest, it is presently advisable to

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subculture the buffered peptone water to selenite broth as an additional selective enrichment, followed by plating on Bismuth Sulphite agar. Clostridia The genus Clostridium currently contains more than 80 species diverse in terms of metabolic activity, nutritional requirements and DNA relatedness. It comprises Gram-positive endospore-forming bacteria which are obligate anaerobes. Two species Cl. botulinum and Cl. perfringens are primarily involved in food poisoning but also Cl. baratii and Cl. butyricum have been involved in botulism cases. The EU microbiological criteria regulation does not contain any stipulations relating to clostridia. The method of CDC (Botulism in the United States, 1899±1996. Handbook for epidemiologists, clinicians and laboratory workers. (1998) CDC, Atlanta, GA, USA) is suggested with some modifications for the detection botulism neurotoxin producing clostridia. In particular for the detection of Cl butyricum type E spores and Cl. baratii Type F, the heat treatment of the sample should be carried out at 70 ëC and in the isolation of the toxic strains also the lipase negative colonies should be considered. Listeria The genus Listeria comprises Gram-positive, coccoid to rod-shaped, bacteria with a characteristic tumbling motility at room temperature. Between the species currently recognized only L. monocytogenes is consistently associated with human diseases and two other hemolytic species, L.ivanovii and L. seeligeri have been implicated in rare occasions. L. monocytogenes is a well-known cause of abortion, encephalitis and septicaemia in man and animals. The micro-organism can cause both invasive and non-invasive infections. L. monocytogenes is ubiquitous bacterium occurring in both terrestrial and aquatic habitat and has been isolated from fish and fishery products from different part of the world. For foods, other than those that may be fed to infants and for special medical purposes, the EU microbiological criteria regulation specifies two different limits and associated reference methods. For foods that can support the growth of L. monocytogenes there is a requirement for absence in 25 g in five sampling units at the processing plant, with the reference method specified as EN/ISO 11290-1. For those same foods on the market, and for foods which do not support the growth of L. monocytogenes, the limit is 100 colony forming units (cfu) per gram in five sampling units. The latter group of foods is deemed to include live bivalve molluscs. For these, the reference method is EN/ISO 11290-2. The presence/absence method EN/ISO 11290-1 consists of an initial enrichment step in Half-Fraser broth followed by a second enrichment in Fraser broth. Both broths are cultured on Agar Listeria according to Ottaviani and Agosti (ALOA) and a second medium of the laboratory's choice. The enumeration method EN/ISO 11290-2 consists of a plate count on ALOA following brief resuscitation in either buffered peptone water or Half-Fraser base. Confirmatory tests are carried out on presumptive colonies from both media.

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Staphylococcus aureus The genus Staphylococcus contains a number of species which have been implicated as causative agents of disease in man and animals. Staphylococcus aureus is a major cause of food poisoning in man as well as of a range extraintestinal infections. Staphyloccocci are gram-positive rods. S. aureus isolates pathogenic for humans usually coagulate human and rabbit plasma (the coagulase test). Regulation (EC) No. 2073/2005 for microbiological criteria in foodstuff states that for process hygiene of shelled and shucked products of cooked crustacea and mussels, the Staphylococcus aureus number must not exceed 1000 cfu/g in two of five sampling units and in the remaining must not exceed 1000 cfu/g. The reference method for this is EN/ISO 6888-1 or 2. The first of these is a plate count on Baird-Parker (BP) agar and the second is a pour plate in BP-RPF (Rabbit plasma + bovin fibrinogen) Agar. Colonies on BP Agar require confirmation by the coagulase test while BP-RPF agar gives this confirmatory result in situ. Vibrios Bacteria of the genus Vibrio are ubiquitous in marine and estuarine aquatic ecosystems. Among more than 20 Vibrio species known to be associated with human disease, V. cholerae, V. parahaemolyticus and V. vulnificus are most important. To date, the detection of pathogenic vibrios in seafood products is mostly performed by conventional cultural methods, including the new technical specifications ISO TS 28172 parts 1 and 2, followed by biochemical identification of the isolated strains. Part 1 is for V. parahaemolyticus and V. cholerae while part 2 is for other pathogenic vibrios. Both methods consist of an initial enrichment in Alkaline Salt Peptone Water (ASPW) followed by a second enrichment in the same broth, with the cultures from both being plated on Thiosulphate Citrate Bile Salt Sucrose Agar (TCBS) and one other agar. The difference between the two methods comes at the enrichment stages. For fresh products, both the first and second enrichments are undertaken at 41.5 ëC in the part 1 method while they are undertaken at 37 ëC in the part 2 method. For frozen products, the initial enrichment in part 1 is performed at 37 ëC in order to allow better recovery of damaged cells. Following isolation on the solid media, the presumptive colonies are identified biochemically (up to five colonies per plate). If they belong to a pathogenic species, they may then be submitted to a reference laboratory for the detection of pathogenic principles. Cultural methods for pathogenic vibrios, including these ISO methods, suffer greatly from the drawbacks referred to earlier: the pathogenic vibrio species are often vastly outnumbered by non-pathogenic species and the strains of potentially pathogenic species possessing pathogenicity traits are usually vastly outnumbered by the non-pathogenic forms. This means that the probability of identifying a colony of a potential pathogen showing the pathogenicity traits is very low. Enumeration of potentially pathogenic vibrios in seafoods is usually performed by one of two methods. The first is to perform plate counts directly on

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TCBS or one of the other selective isolation media. However, the recovery efficiency of these agars is usually only approximately 50% and may be even lower for stressed cells. The other method is to perform the initial enrichment in a 3 3 Most Probable Number format and then follow the rest of the isolation procedure for each initial broth. One problem with this is that the procedure results in a large number of presumptive colonies requiring confirmation. Another is that void MPN combinations are often obtained, with a lower than expected number of positives at the first dilution, presumably due to overgrowth by non-target bacteria. Some laboratories have used a semi-quantitative method with a single primary broth at each dilution, but the uncertainty associated with this method is very large. For identification of isolated strains the miniaturised biochemical tests API 20E (commonly used for the identification of clinical isolates) and API 20NE, supplemented with NaCl, are widely used (Croci et al., 2001; O'Hara et al., 2003; Truu et al., 1999). Despite different studies reporting the inadequacy of miniaturised tests for the identification of some isolates of the Vibrionaceae family (Colodner et al., 2004; Dalsgaard et al., 1996a; Israil et al., 2003b; Martinez-Urtaza et al., 2006), some authors pointed out the ability of the API 20NE system to provide a higher number of correct identifications than API 20E or other systems, particularly on the analysis of environmental isolates (Israil et al., 2003a; Toti et al., 1996). Another biochemical key for the identification of Vibrio spp. has also been proposed (Alsina and Blanch, 1994a,b) and, after optimisation of the conditions (temperature, time of incubation and media composition) by other authors (Ottaviani et al., 2003), good identifications have been obtained on a large number of reference and environmental Vibrio strains. Recently other systems have been proposed as Biolog GN system, based the bacteria identification on the exchange of electrons generated during respiration, leading subsequently to tetrazolium-based colour changes, That seems to provide results on environmental strains in agreement with API 20NE (Truu et al., 1999). A comparison between some commercially available systems, Crystal E/NF panel, Rapid Neg ID, Vitek GNI+, ID-GNB cards and API 20E, showed that only Vitek cards and API20E presented an accuracy of 90% on V. parahaemolyticus identification. However, a large proportion of misidentifications, with non-pathogens being identified as pathogens and vice versa, have been shown. A study for the evaluation of biochemical identification methods, API 20E and API 20NE and Alsina's by means of an intra and inter-laboratory test in order to determine the accuracy and concordance of each method was performed at the beginning of the SEAFOODplus project. The Alsina's scheme resulted as the most efficient identification for Vibrio, but required a larger amount of work for the laboratory (including the preparation of the culture media and their quality control). The API systems, despite the advantages brought by their quick and easy execution, showed a lower efficiency in the identification of V. parahaemolyticus. However the authors concluded that in order to avoid false positive results the confirmation of the results by means of a reliable molecular method it is necessary (Croci et al., 2007).

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14.7 Molecular methods for the detection, enumeration and identification of bacterial pathogens Detection of pathogenic bacteria in seafood is essential to ensure safe products for consumers and sustainable fish and shellfish growing activities. Molecular diagnostic methods have evolved significantly in the last few years and some are established as useful and reliable to allow the rapid detection and identification of pathogens. Molecular detection, identification and enumeration of Vibrio spp. are largely based on PCR-amplification following purification of nucleic acids from the samples. Although less sensitive and more time consuming, DNA or oligonucleotide probe-based hybridisation methods have been proposed for Vibrio spp. in food. In this section, the different methods commonly applied to pathogenic bacteria are discussed with particular emphasis on enteropathogenic vibrios ± scope of the SEABAC project. 14.7.1 Detection and identification methods A number of PCR-based assays have been utilised to amplify Vibrio spp. from food, blood, faecal and environmental samples. While largely used for the detection of pathogens in the clinical diagnosis of microbial infections and also for the detection of these organisms in food and environmental samples, they were first developed to confirm the identification of isolated strains in place of biochemical tests. Polymerase chain reaction (PCR) assays for identification Several genes have been utilised as targets in confirmatory assays for Vibrio spp. Venkateswaran et al. (1998) recommended the gyrB gene for differentiation of V. parahaemolyticus and V. alginolyticus although subsequently Kim et al. (1999) identified a number of cross-reacting V. alginolyticus strains. The outer membrane regulator toxR gene has been reported to effectively distinguish between V. parahaemolyticus, V. vulnificus and V. alginolyticus (Lin et al., 1993; Kim et al., 1999). Multiplex PCR including a thermostable haemolysin gene (tlh) and pathogenicity markers (tdh and trh) for confirmation of the presence of pathogenic strains of V. parahaemolyticus has also been used (Bej et al., 1999). However, evaluation of this approach did not include vibrio strains of the European origin and latterly Robert-Pillot et al. (2002) have shown that tlh could not differentiate between V. alginolyticus and V. parahaemolyticus isolated in France. A highly conserved region termed pR72H has also been proposed as an identification tool for V. parahaemolyticus (Lee et al., 1995; Wu and Lee, 1998). Most V. vulnificus identification assays target the haemolysin gene (vvh), although recently the efficacy of variable regions of the 16S rRNA gene have been reported (Kim and Jeong, 2001). Most PCR methods for V. cholerae O1/ O139 have concentrated on detection of virulence cassette genes (i.e. ctxAB, zot, ace) (Kobayashi et al., 1990) and serotype specific determinants (rfb region)

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(Hoshino et al., 1998). In non-O1/O139 V. cholerae that do not produce enterotoxins, genes encoding NAG-specific heat-stable toxin (st), thermostable direct haemolysins, shigella-like toxin and classical haemolysin have been variously identified (Lyon, 2001). Within the SEABAC project evaluation of multiple PCR targets for V. parahaemolyticus confirmation showed use of the toxR gene generated data that most closely concurred with biochemically confirmed V. parahaemolyticus derived from European seafoods. It was suggested that this approach could be used to complement or replace traditional biochemical confirmatory tests for V. parahaemolyticus (Croci et al., 2007). Polymerase chain reaction (PCR) assays for detection Several conventional PCR assays have been used successfully for the detection of V. parahaemolyticus by employing species-specific PCR primers designed on the nucleotide sequences of several targeted genes. Amongst others, the tlh gene, the gyrB gene, the toxR gene, as well as the pR72H cloned fragment have been targeted as markers for all V. parahaemolyticus strains (Taniguchi et al., 1986; Venkateswaran et al., 1998; Kim et al., 1999; Lee et al., 1995). The genes encoding major virulence determinants have been used to characterise and distinguish potentially enteropathogenic from all V. parahaemolyticus strains. Tada et al. (1992) reported primers for the detection of tdh and trh genes encoding for the production of two thermostable haemolysins associated with disease, Thermostable Direct Haemolysin (TDH) and TDH-related Haemolysin (TRH), respectively. An open reading frame (ORF8) in a lysogenic filamentous phage was selected for specific detection of V. parahaemolyticus strains belonging to the pandemic clone, O3:K6 (Nasu et al., 2000). Until recently, the pR72H, the toxR, the tdh and the trh PCR assays have been the most widely used in epidemiological investigations, surveillance and shellfish purification studies in Asia, America and Europe (Cordova et al., 2002; Croci et al., 2002; Hervio-Heath et al., 2002; Alam et al., 2003; Martinez-Urtaza et al., 2004a; Robert-Pillot et al., 2004). Multiplex PCR assays for simultaneous detection of total and enterotoxigenic V. parahaemolyticus (Bej et al., 1999) or of V. parahaemolyticus and other Vibrio spp. (Tarr et al., 2006) or marine bacteria (Gonzales et al., 2004) have been developed. They have been tested for the identification of isolated V. parahaemolyticus strains or for the detection of these bacteria in seeded oyster tissue homogenates but they were rarely used for field investigations because they lack sensitivity. Lee et al. (2003) reported that the combination of a multiplex PCR with a colorimetric microwell plate sandwich hybridisation assay improved the sensitivity and permitted verification of the amplified DNA. Reverse transcription-PCR (RT-PCR), which targets short-lived mRNA molecules, has become an increasingly used molecular method for assessing the viability of bacteria, culturable (VC) or nonculturable (VBNC). The latter state is a survival strategy adopted by bacteria who are exposed to a hostile environment and are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. Vibrio parahaemolyticus

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RT PCR assays, targeting transcripts of the rpoS and the tdh2 genes, has been described to detect cDNA from VBNC cells for extended periods of time (Coutard et al., 2005). DNA microarrays and biosensors Next generation assays under development include biosensors and DNA microarrays that potentially have the capability for near real-time and on-line monitoring for multiple pathogens in foods or environmental samples. Several studies have demonstrated the applicability of DNA arrays to environmental microbial detection (Gushin et al., 1997). Combined or not with multiplex PCR assays, these oligonucleotide arrays have been employed to detect and discriminate V. parahaemolyticus from other human pathogenic vibrios or other foodborne pathogenic bacteria (Panicker et al., 2004; Vora et al., 2005; Jin et al., 2005; Eom et al., 2006). 14.7.2 Enumeration methods Colony hybridisation In recent years, the use of DNA probes to identify colonies growing on nonselective or semi-selective agar media have been proposed for detection and enumeration of V. parahaemolyticus and V. vulnificus. Alkaline phosphatase (AP)-labelled probes (available commercially) or digoxigenin (dig)-labelled amplicon probes can be used to identify the presence of V. parahaemolyticus and strains harbouring the tdh gene, and detecting V. vulnificus. Although it is generally considered that PCR methods are more sensitive and less time consuming than either DNA or oligonucleotide probe-based hybridisation methods, a number have been proposed for detection, biotyping and confirmation of the identification of Vibrio spp. in food. McCarthy et al. (1999) evaluated an alkaline phosphatase- and a digoxigenin- (DIG) labelled tlh probe for V. parahaemolyticus specificity. An AP-labelled tdh probe and two DIG-labelled tdh and trh probes for the numeration of enterotoxigenic V. parahaemolyticus were also described. These methods have been widely used for the enumeration of both V. parahaemolyticus and V. vulnificus in regulatory laboratories and surveillance studies across the United States (DePaola et al., 2000; Cook et al., 2002; FDA, 2004) but in a limited number of studies in Europe. Their applicability for the detection of V. vulnificus and V. parahaemolyticus has been verified within the SEAFOODplus project using a large number of strains of different origins (ATCC, NCT, clinical, environmental). The method for the V. vulnificus detection using vvh probe demonstrated good performance. On the contrary the method for the V. parahaemolyticus detection using tlh probe generated some false negative results for strains isolated from Adriatic Sea, Africa, imported fisheries. The development of alternative probe using toxR gene has been planned. By means of the alignment of the sequences of all strains available and on the basis of Blast (NCBI) a probe was designed and provided good results, performing well in the range 80±90 ëC. The experimental condi-

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tions (hybridisation temperature, buffer composition, etc.) have been optimised. Experiments performed under repeatability conditions using reference strains and recently collected environmental isolates have yielded good results. Specificity has been confirmed against a large panel of non-parahaemolyticus Vibrio spp. and non-Vibrio spp. Most probable number (MPN) ± polymerase chain reaction DNA hybridisation and PCR methods combined with an enrichment in an MPN format have been used extensively to enumerate bacterial pathogens. A PCR and a multiplex PCR method, targeting the pR72H fragment, the toxR gene and the tlh, tdh and trh genes, respectively, were combined with a MPN technique to enumerate the total and enterpathogenic V. parahaemolyticus in mussels, seawater and marine organic material (Croci et al., 2002; Alam et al., 2002). This combination proved to be more sensitive and specific than the conventional culture-based MPN method. This was used in a recent study to evaluate the levels of total and tdh positive V. parahaemolyticus in retailed seafood in Japan (Miwa et al., 2006). Real-time polymerase chain reaction While conventional PCR-based methods are specific and sensitive and provide good alternative assays to conventional culture for the detection of V. cholerae, V. vulnificus and total and/or enteropathogenic V. parahaemolyticus in seafood, they are not useful for enumeration. Real-time PCR combines the specificity of conventional PCR with the quantitative measurement of fluorescence for determining the presence of specific types of nucleic acids in environmental samples. These offer the potential for a more rapid and quantitative analysis for the detection, and enumeration, of pathogenic bacteria. SYBR Green or the use of TaqManÕ probes are the more frequently selected for the numeration of Vibrio spp. Many authors reported the application of realtime PCR for quantitative detection of Vibrio spp. in seafood and seawater (Panicker et al., 2004; Blackstone et al., 2007, Takahashi et al., 2005a, Ward and Bej, 2006). However, detection of these bacteria was possible only after enrichment of tissue homogenates or water. To date, only one study refers to detection of V. parahaemolyticus directly from naturally contaminated seafood (Cai et al., 2006), the number of detectable cells varying from 4:3 103 to 4:7 105 1per ml. These results indicate that PCR amplification can be inhibited by components from the different food matrices and that these methods still lack sensitivity for direct testing. It is for this reason that this methodology has been selected for optimisation and standardisation within the scope of the SEAFOODplus programme particularly for the estimation and detection of total and enteropathogenic V. parahaemolyticus in shellfish. Research performed with the SEAFOODplus (SEABAC) project has enabled progress in sample preparation (concentration of micro-organisms) and nucleic acid extraction and the optimisation of real-time PCR for the detection of the bacteria in shellfish. The transfer of the methodology in inter-laboratory trials will in the

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future enable elaboration of a standard real-time PCR method for V. parahaemolyticus. 14.7.3 Conclusion A drawback to these methods: a positive result in PCR or real-time PCR, for example with a probe or primers specific for a toxin gene, only indicates that the bacteria carrying this gene are present in the sample and thus, have the potential to be toxigenic. However, it does not indicate if the gene is expressed or the toxin produced. Furthermore, if more rapid than culture, these methods still lack sufficient sensitivity and specificity for direct testing. Indeed with few exceptions, almost all assays used to detect specific pathogens in foods require some growth in an enrichment medium before analysis. However, considering the increasing importance of Vibrio spp., the molecular approach is a reliable alternative procedure for routine microbial screening and monitoring of clinical, food and environment samples and offers interesting perspectives for epidemiological, phylogenetic and environmental studies.

14.8

Molecular approaches to microbial typing

14.8.1 Introduction Molecular typing methods involve the examination of chromosomal or extrachromosomal DNA or RNA. Based on the technical approach, four different categories are usually recognised: plasmid analysis; hybridisation; nucleic acid amplification-based methods using PCR with or without sequencing and macrorestriction pattern (MRP) analysis by the use of infrequent cutting enzymes and pulsed-field gel electrophoresis (PFGE). To varying extents intra-species characterisation of pathogenic seafood borne bacteria has been achieved using the majority of these approaches with various degrees of discriminatory success. The basis for all molecular typing methods is that all pathogenic agents within an infectious chain are clonally related. Thus successful typing systems effectively determine markers that can optimise discrimination between epidemiological related and unrelated isolates of the pathogens of interest (Streulens, 1998). In food microbiology genotyping methods are most commonly employed in outbreak investigations. However, they have also been used in confirmation or delineation of the patterns of transmission, testing hypotheses on the origin and vehicles of transmission, and in the monitoring of reservoirs of clinically significant bacterial strains. Typing has also in some cases contributed to epidemiological surveillance and evaluation of control measures, by assessment of the temporal prevalence and distribution of epidemic clones. In this section, typing strategies commonly applied to seafood borne bacteria are discussed in brief with more extensive consideration given to the application of PFGE and RAPD techniques to Vibrio spp. within the context of the SEAFOODplus research programme.

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14.8.2 Plasmid analysis Plasmids are autonomously replicating extrachromosomal DNA found within bacteria. Frequently, related strains contain the same number of plasmids with the same molecular weight and similar phenotypes. There are two major applications ± typing of a specific plasmid profile and plasmid fingerprinting. Plasmid profiling is based upon the number and molecular weight of plasmids whereas, in plasmid fingerprinting, plasmids are digested with restriction enzymes before analysis. Both approaches have been used in a number of studies to sub-type a range of bacterial pathogens isolated from seafood or seafood products (Kumao et al., 2002, Teophilo et al., 2002, Chen et al., 2003, MartinezUrtaza et al., 2004a). Whilst plasmid analysis has been shown to be useful in the characterisation of specific bacterial strains, e.g. exotoxin producing E. coli from fish and crustacaea (Teophilo et al., 2002), many pathogens can spontaneously acquire or lose plasmids and this limits there use in epidemiological investigations. 14.8.3 Hybridisation approaches Ribotyping The most commonly utilised hybridisation approach is known as ribotyping. Ribotyping utilises variations in the ribosomal genes and their adjacent sequences. These genes are usually highly conserved and vary in number and position. Chromosomal DNA is isolated, restricted and the resulting DNA fragments carrying the rRNA genes are separated by electrophoresis. Detection is via hybridisation with labelled E. coli 16S, 23S or 5S probes (Grimont and Grimont, 1991). Over the last decade automated ribotyping systems have been introduced (Bruce, 1996). Automation has a number of benefits including increased standardisation, speed, reproducibility and repeatability and a decrease in nontypeable isolates (Suihko et al., 2002). Ribotying has been used extensively in sub-typing of pathogenic bacteria derived from seafoods. In one notable study, Hùi et al. (1997) characterised Danish and US isolates of V. vulnificus using ribotyping and RAPD PCR. Ribotyping enabled the identification of the first reported isolation of V. vulnificus biotype 2. The authors concluded that ribotyping was useful for biotyping whereas RAPD PCR was unable to correlate isolates with sources or to differentiate biotypes. In general, it has been reported that ribotyping produces fewer patterns and clusters requiring less complex interpretation than many other sub-typing methods, and that ribotypes show a good relationship between geographical distribution and genotype (Tamplin et al., 1996). 14.8.4 Nucleic acid amplification-based typing methods by using polymerase chain reaction A number of PCR based typing approaches have been applied to characterisation of seafood borne bacteria. Most commonly these have included repetitive element PCR (Rep PCR) and randomly amplified polymorphic DNA (RAPD).

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Repetitive element polymerase chain reaction (Rep PCR) Rep (PCR) uses PCR amplification of the DNA between adjacent repetitive extragenic elements to obtain strain specific DNA fingerprints (Versalovic et al., 1991). Three main rep-PCR methods (ERIC-PCR, REP-PCR, and BOX-PCR) are usable for bacterial typing. Each method uses one or several oligonucleotides homologous to a defined sequence that is present in multiple copies on the bacterial chromosome. In a study on the relatedness of outbreak related strains of V. parahaemolyticus Marshall et al. (1999) concluded that ERIC PCR exhibited good discrimination and could be used to evaluate genetic and epidemiological relationships. Wong and Lin (2001) reported on the design and evaluation three rapid PCR typing methods, including ERIC PCR, in addition to ribosomal gene spacer sequence (RS), and repetitive extragenic palindromic sequence. In this study, in which the efficacy of each method was compared to PFGE patterns, typing patterns and clustering analysis differentiated between 15 and 27 patterns amongst 40 V. parahaemolyticus strains presenting a wide range of PFGE profiles. High discrimination indices were recorded for all methods however, assay reproducibility was identified as a potential drawback when using ERICPCR. Khan et al. (2002) used ERIC PCR followed by conventional PCR amplification of a unique 327 bp fragment in newly emerged pathogenic strains of O3:K6 in a Texan outbreak However, only a limited number isolates were tested (18) in this study, and the reproducibility and reliability of this approach has been questioned by others (Myers et al., 2003). Random amplified polymorphic DNA (RAPD) Randomly amplified polymorphic DNA (RAPD) is a PCR-based method utilising short random primers to rapidly detect genomic polymorphisms under low-stringency conditions. RAPD analysis has been is widely used for differentiating between seafood-borne bacterial isolates and relies upon small quantities of genomic DNA, which can be of particular benefit when typing slow growing or fastidious bacteria. RAPD PCR has frequently been used to sub-type isolates of V. vulnificus as it been identified that other methods such as PFGE are not suitable due to the high level of intra-specific diversity of isolates from clinical and environmental sources (Buchreiser et al., 1995; Tamplin et al., 1996) and the sensitive discriminatory capability of PFGE. Warner and Oliver (1998) developed a RAPD PCR protocol for the detection V. vulnificus. The authors reported clear differentiation between clinical and environmental isolates and unique banding patterns indicating that V. vulnificus comprised a highly heterogeneous group. In a more recent study Lin et al. (2003) used RAPD PCR to analyse the temporal and spatial intra-specific diversity of V. vulnificus strains isolated from Galveston Bay water and oysters. Cluster analysis of RAPD PCR profiles confirmed a high level of intra-specific diversity among the strains with no correlation between the variation and sampling site or source of isolation. However, a major shift in population structure was observed with rising water

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temperatures. The authors concluded that RAPD PCR enabled sensitive monitoring of temperature sensitive genetic population changes. Ripabelli et al. (2003) utilised RAPD PCR in molecular characterisation studies on V. vulnificus isolated from Mussels in Italy. The results demonstrated extreme intra-specific variability of V. vulnificus. Arias et al. (1998) analysed RAPD PCR profiles of V. vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, plus seawater and shellfish isolates from the western Mediterranean coast, including reference strains) comparing the results to ribotyping. Both ribotyping and RAPD PCR indicated a high level of homogeneity of diseased-eel isolates, in contrast to the genetic heterogeneity of Mediterranean isolates. It has been suggested that RAPD PCR is superior for rapid typing in the routine analysis (Ripebelli et al., 2003). Research carried out within SEAFOODplus has described the first molecular characterisation of Vibrio spp. isolated in the EU using RAPD. These results have demonstrated that RAPD is a relatively quick, reproducible and easy method for subtyping bacterial organisms. RAPD has been shown to differentiate between different Vibrio species isolated from the environment as well as enabling identification of intra-species variation in V. parahaemolyticus (Fig. 14.1). Macrorestriction pattern analysis and pulsed field gel electrophoresis Pulsed field gel electrophoresis (PFGE) is a technique that enables analysis of entire chromosomal DNA previously digested with a restriction endonuclease to yield large DNA fragments (Towner and Cockayne, 1993). Over recent years PFGE has emerged as the `gold standard' in typing methodology (Maslow and Muligan, 1996; Streulens, 1998). It is the current method of choice for typing nosocomial and community acquired pathogens (Tenover et al., 1997). In comparison with other typing methods it shows a high level of discriminatory ability and most systematic studies of typing performance have included comparisons with PFGE. PFGE has been reported to have the most discriminatory power with respect to the characterisation many seafood borne bacterial pathogens including V. cholerae (Cameron et al., 1994; Dalsgaard et al., 1996b; Kam et al., 2003), L. monocytogenes (Johansson et al., 1999), Clostridium spp. (Hyytia et al., 1999), and Salmonella spp. (Martinez-Urtaza et al., 2004b). In South East Asia and North America where amongst seafood-borne bacterial pathogens Vibrio spp. are considered to be predominant, PFGE has been used extensively (Bag et al., 1999; Chowdury et al., 2000; Lu et al., 2001). It has been reported that PFGE has provided adequate discrimination and is widely considered as a superior cf alternate typing procedures. In a Tawainese study on the heterogeneity of V. parahaemolyticus strains recovered from seafood and the environment, Wong et al. (1999) demonstrated a high level of genetic diversity in V. parahaemolyticus isolates. Likewise in Europe, MartinezUrtaza et al. (2004a) demonstrated a considerable degree of heterogeneity amongst isolates from Spanish coastal waters and seafood. Extensive heterogeneity has been confirmed amongst non TDH/non-TRH strains collected from

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Fig. 14.1 Clustering of RAPD patterns of V. parahaemolyticus and associated banding pattern. Intraspecies RAPD banding similarities are shown as percentage similarities.

European fish and shellfish as part of the SEAFOODplus programme (Fig. 14.2). PFGE has also been used in the analysis of clinical isolates from Spain and strains from Galician oysters. PFGE clearly distinguished Spanish clinical isolates from the Asian and North American pandemic clones. Recently research carried out within SEAFOODplus has identified clonal relationships between Spanish clinical strains and strains originating from a patients with enteritis in the UK (Fig. 14.3). One major drawback of PFGE is the lack of standardisation of PFGE protocols and resultant difficulties in inter-study comparison. Most authors have recognised that to promote effective bacterial and epidemiological tracking and disease control, internationally standardised typing procedures and an international PFGE fingerprint database should be established. It is for these reasons that this methodology has been selected for development and standardisation within the scope of the SEAFOODplus programme particularly for application to Vibrio spp. Research forming part of the SEAFOODplus programme has enabled the elaboration of a standard PFGE method for V. parahaemolyticus using two restriction enzymes (NotI and SfiI). In inter-laboratory comparisons this method has demonstrated excellent resolution and reproducibility. Adoption

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Fig. 14.2 Heterogeneity amongst non-clinically significant V. parahaemolyticus isolated from fish and shellfish in Europe determined using the SEAFOODplus developed standardised PGFE methodology. WPNL0206

Fig. 14.3 Clonal relationships between Spanish and UK clinical isolates of V. parahaemolyticus observed using SEAFOODplus developed standardised PGFE methodology.

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of standardised PFGE protocols will in the future enable data generated in single and multi-laboratory studies to be reliably compared.

14.9

Future trends

14.9.1 Emerging bacterial risks In terms of bacteriological food safety emerging disease risks can be described as the first linkage between a particular bacteria or bacterial strain and disease, those that cause a sudden increase in frequency or severity of illness particularly in non-endemic regions, or when a recognised pathogen reappears as a significant cause of disease in the community. Numerous and diverse bacterial species have been isolated from seafood in the absence of definitive aetiologic connection to fish or shellfish related outbreaks. Although many such organisms pose significant health risks for immune-compromised individuals or other susceptible groups, several, such as Pseudomonas spp., Aeromonas spp., and Pleisomonas spp. commonly form part of the natural flora of seafood. Whereas many of these organisms have usually been considered harmless or related to bacterial spoilage, the emerging pathogen Stenotrophomonas maltophilia, formerly known as Pseudomonas maltophilia, has received attention in recent years due its putative role in a number of clinical syndromes such as cystic fibrosis, bacteremia, endocarditis, skin and soft tissue infection, ocular infection, and urinary infection (Ben-Gigirey et al., 2000, 2002). Over the last decade DNA probe techniques have become available to enable isolation and differentiation of S. maltophilia from commensal non-pathogenic species in high value commercial seafood products such as albacore tuna. Increasingly, application of molecular methods in food microbiology will make possible the detection, identification and differentiation of emerging bacterial risks associated with seafoods. Although not usually related to faecal contamination of water, a number of studies have indicated an inverse association between the concentration of bacteria in the marine environment and faecal indicator levels (Kaper et al., 1979; Tamplin et al., 1982). Bacterial counts tend to be highest in warm summer months, particularly when water temperatures exceed 15±20 ëC (Baross and Liston, 1970) and this has led to a concern that a rise in global seawater temperatures may result in increased consumer risk from seafood-associated bacterial pathogens. International trade in wild caught seafood and an increase in aquaculture seafood products has facilitated the introduction of new and existing pathogens into novel geographic areas, as well as seafood and human communities. For example, Salmonella Agona, which has been increasingly responsible for outbreaks of salmonellosis in recent years in Europe, was first introduced with imported Peruvian fish meal and has subsequently been reported in many other food products, including aquacultured seafood (Flemming et al., 2006). Thus the international trade in seafood and fisheries products can signify an increased risk of exposure to geographically exotic pathogens. Similiarly,

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trade in aquaculture commodities from world regions where licensed or illegal antimicrobial prophylaxis is practised can encourage the spread of antibiotic resistance within and often between bacterial species. Finally, it is worthy of note that of the four bacteria identified in the United States as of major concern in food-borne disease namely E. coli O157:H7, Salmonella enteriditis, Listeria monocytogenes and Campylobacter jejuni only S. enteriditis was an established food-borne disease agents 20 years ago (Martinez et al., 2005), highlighting the relatively rapid appearance of bacterial food borne illness. 14.9.2 Application of new methods Over the last 20 years numerous new non-classical methods in food microbiology have become available. Many of these have utilised techniques in molecular biology for the detection of particular bacterial species, specific pathogenicity genes or enterotoxins using PCR amplification and gene probes. Whilst there is currently no universal method that permits identification of all pathogenic bacteria, a number of bacteria associated with fish and fisheries products have been successfully detected including L. monocytogenes; Campylobacter coli, C. jejuni, Campylobacter spp.; Clostridium botulinum, enterotoxigenic strains of E. coli, Vibrio spp., Salmonella spp. and Shigella flexineri (Martinez et al., 2005). The advent of real-time quantitative PCR has signalled a move towards the development of methods for the direct quantitation of a number of target organisms in seafood matrices (Cai et al., 2006; Campbell and Wright, 2003; Takahashi et al., 2005b). However, to date most studies utilising this technology have recommended the use of enrichment culture prior to PCR amplification to concentrate bacterial cells and to eliminate the presence of inhibitors. Thus, so far most published methods enable presence/absence determination but not enumeration of pathogens from the seafood matrix. At present within the EU no molecular-based method is included in the list of analytical reference methods stipulated for use in official control testing of fish or fisheries products. The unsuitability of some existing cultural methods for seafood-borne pathogens has been acknowledged previously (Feng, 1998) and it is noted in the EU microbiological criteria for foodstuffs that there is a need for development of reliable methods for certain microbial hazards, e.g. V. parahaemolyticus (Anon, 2005). Thus it is probable that following appropriate standardisation and validation, molecular techniques for detection and/or enumeration of bacterial pathogens will be incorporated into the European legislative framework. Currently through the European Committee for Standardisation (CEN) several molecular standard methods are being elaborated for use in food microbiology, e.g. Clostidium botulinum neurotoxins by PCR, Yersinia enterocolitica by PCR and V. parahaemolyticus by nucleic acid hybridisation. The latter methodology for enumeration of potentially pathogenic V. parahaemolyticus in seafood having been developed during work carried out within the SEAFOODplus work programme.

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14.9.3 Emerging treatment technologies For many years food scientists have sought alternative procedures that can preserve shelf life and reduce public health risks without compromising the taste and texture of products of highly perishable fish and fisheries products (West, 1989). Certain seafoods, such as some species of crab, smoked fish, caviar and some imitation crab based surimi products, have been traditionally pasteurised to reduce risks associated with Clostridium botulinum (type E and nonproteolytic B and F) Listeria monocytogenes and Vibrio vulnificus. In several countries guidelines for specific foodstuffs or reductions in target bacterial species have been adopted as part of HACCP procedures (ECFF, 1999; Gould, 1999; FDA, 2006). However, pasteurisation involves a mild or moderate heat treatment and is therefore not appropriate for a number of fresh seafood or fishery products usually intended for consumption with no further processing. Additionally, it has been suggested that pasteurisation followed by chilling does not remove all public health risks (Pace et al., 1988). And, that it may even facilitate the emergence in importance of members of the genera Bacillus and Listeria, that can survive pasteurisation and subsequently proliferate under refrigeration conditions (West, 1989). A number of workers have reported that fish and seafood can be effectively decontaminated through irradiation, increasingly referred to as `cold pasteurisation'. Relatively low doses (1.0±2.0 kGy) have been shown to eliminate V. parahaemolyticus and V. vulnificus from a range of matrices (Matches and Liston, 1971; Molins et al., 2001) without affecting the quality of fresh raw products such as live oyster and clams (Molins et al., 2001). In the EU the use of irradiation to prolong shelf life and prevent food-borne diseases by reducing the number of viable micro-organisms in meat, poultry and seafood is foreseen under EC Directive 1999/2/EC (Anon., 1999). In 2005, irradiation facilities were approved in 10 Member States and whereas a number of countries listed fish and fisheries products amongst the irradiated products (Anon., 2007) in practice, the use of this technique is limited in Europe compared with elsewhere. More recently, high pressure (HP) processing has been investigated as an alternative method for food preservation that allows microbial inactivation whilst maintaining sensory and nutritional properties (Murchie et al., 2005). The use of HP as a means of elimination of pathogenic bacteria associated with shellfish (oysters) has become of increasing interest in recent years, particularly in the United States. This is largely due to the susceptibility of human pathogenic Vibrio spp. to relatively low HP treatments (circa 300MPa) (Voisin, 2001, 2002; Calik et al., 2002). In addition, HP treatment has been reported to enhance the appearance of oysters compared with untreated products (Johnston et al., 2003) and to cause detachment of the abductor muscle assisting in opening of the shell (He et al., 2002). The advantages of HP over alternative processing methods have been demonstrated for other seafood products especially in Japan, where it is used increasingly in the production of surimi and kamaboko (Ashie et al., 1996). Despite the reported advantages of HP for the decontamination of fresh raw fish and fisheries products to date its use outside the US and Japan are strictly limited.

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General discussion

The proportion of outbreaks due to seafood varies markedly around the world, from around 10% to 30% of all foodborne outbreaks, and appears to be highest in south-east Asia although little information is available for Africa. The differences are undoubtedly related to variations in the proportion that seafood makes up of the diet; the nature in which seafood is usually eaten (i.e., whether a significant proportion is raw or partially cooked); the local ambient temperature; local level of public health. The proportion will also depend on whether vibrios are a significant cause of illness in a country although this is in itself is related to a number of other factors, as well as the occurrence of pathogenic species and forms in the environment. International travel and trade is also an important factor ± a significant proportion of vibrio infections in Europe are due to persons infected abroad and returning during the incubation period or while still ill. Global change may lead to an increase in some types of seafood-associated illness. However, for some high-risk seafoods, such as oysters, new processing technologies may offer the potential for providing a safer product, as long as the result is acceptable to the consumers, particularly those that are used to eating raw products. Conventional methods of detection, enumeration, identification and characterisation have been successfully used for some bacterial pathogens in seafoods but less successfully for others, such as vibrios, where they are unable to adequately distinguish pathogenic forms from non-pathogens. Better methods may provide additional information necessary for undertaking quanitative risk assessments for most pathogens. Newer molecular methods have the potential to overcome some of the drawbacks and thus contribute to seafood safety. In this respect, the molecular methods that have been investigated within the SEAFOODplus project are contributing to the establishment of better standard methods within the aegis of the International Standardisation Organisation.

14.11

Sources of further information and advice

The following websites provide information relating to the microbiological safety of seafood. · Community Reference Laboratory for monitoring bacteriological and viral contamination of bivalve molluscs: http://crlcefas.org · European Commission ± Food Safety: http://ec.europa.eu/food/index_en.htm · FAO Fisheries and Aquaculture Department: http://www.fao.org/fi/ · FAO Agriculture and Consumer Protection Department: http://www.fao.org/ ag/agn/ · UC Davis Seafood Network Information Centre: http://seafood.ucdavis.edu/ · WHO Food Safety: http://www.who.int/foodsafety/en/ · National Food Safety/Standards Agencies will provide information specific to individual countries.

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14.12

References

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and RUBY E G (2007), A novel lux operon in the cryptically bioluminescent fish pathogen Vibrio salmonicida is associated with virulence, Appl Environ Microbiol, 73, 1825±1833. NISHIBUCHI M and KAPER J B (1995), Thermostable direct haemolysin gene of Vibrio parahaemolyticus: a virulence gene acquired by a marine bacterium, Infect Immun, 63, 2093±2099. O'BRIEN S J, ELSON R, GILLESPIE I A, ADAK G K and COWDEN J M (2002), Surveillance of foodborne outbreaks of infectious intestinal disease in England and Wales 1992± 1999: contributing to evidence-based food policy?, Public Health, 116 (2), 75±80. O'HARA C M, SOWERS E G, BOPP C A, DUDA S B and STROCKBINE N A (2003), Accuracy of six commercially available systems for identification of members of the family Vibrionaceae, J Clin Microbiol, 41, 5654±5659. OLIVER J D and KAPER J B (1997), `Vibrio species', in Doyle M P, Beuchat L R. and Montville T J (Eds), Food Microbiology ± Fundamentals and Frontiers, Washington DC, ASM Press, 228±264. OTTAVIANI D, MASINI L and BACCHIOCCHI S (2003), A biochemical protocol for the isolation and identification of current species of Vibrio in seafood, J Appl Microbiol, 95, 1277±1284. PACE J, WU C Y and CHAI T (1998), Bacterial flora in pasteurised oysters after refrigerated storage, J Food Sci, 53, 325±327 (348). PANICKER G, CALL D R, KRUG M J and BEJ A K (2004), Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays, Appl Environ Microbiol, 70 (12), 7436±7444. RIPABELLI G, SAMMARCO M L, MCLAUCHLIN J and FANELLI I (2003), Molecular characterisation and antimicrobial resistance of Vibrio vulnificus and Vibrio alginolyticus isolated from mussels (Mytilus galloprovincialis), System Appl Microbiol, 26, 119± 126. ROBERT-PILLOT A, GUENOLE A and FOURNIER J M (2002), Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA-DNA hybridisation, FEMS Microbiol Lett, 215, 1±6. ROBERT-PILLOT A, GUEÂNOLEÂ A, LESNE J, DELESMONT R, FOURNIER J M and QUILICI M L (2004), Occurrence of the tdh and trh genes in Vibrio parahaemolyticus isolates from waters and raw shellfish collected in two French coastal areas and from seafood imported into France, J Food Microbiol, 91, 319±325. RODRICK G E and SCHNEIDER K R (1991), Vibrios in depuration. In Molluscan Shellfish Depuration, Otwell W S, Rodrick G E and Martin R E (Eds), Boca Raton, FL, CRC Press, 115±125. ROWSE A J and FLEET G H (1982), Viability and release of Salmonella charity and Escherichia coli from oyster feces, Appl Environ Microbiol, 44, 544±548. SANYAL S C and SEN P C (1974), Human volunteer study on the pathogenicity of Vibrio parahaemolyticus. In International Symposium on Vibrio parahaemolyticus, Fujino T, Sakaguchi G, Sakazaki R and Takeda Y (Eds), Tokyo: Saikon Publishing Co., 227±230. STREULENS M J (1998), Molecular epidemiologic typing systems of bacterial pathogens: current issues and perspectives, Mem Inst Oswaldo Cruz Rio de Janeiro, 93, 581±585. NELSON E J, TUNSJé H S, FIDOPIASTIS P M, SéRUM H

È RNSDO Â TTIR B, TORKELSSON G, BREDHOLT S, SUIHKO M-L, SALO S, NICLASEN O, GUDBJO È BERG A-M and GUSTAVSSON P (2002), Characterization of Listeria SJO

monocytogenes isolates from the meat, poultry and seafood industries by automated ribotyping, Int J Food Microbiol, 72, 137±146.

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and TAKEDA Y (1992), Detection of thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus by polymerase chain reaction, Mol Cell Probes, 6, 477±487. TAKAHASHI J, HARA-KUDO J, MIYASAKA J, KUMAGAI S and KONUMA H (2005a), Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus, J Microbiol Meth, 61 (1), 77±85. TAKAHASHI J, IWABE Y, KONUMA H and HARA-KUDO J (2005b), Development of a quantitative real-time PCR method for estimation of the total number of Vibrio parahaemolyticus in contaminated shellfish and seawater, J Food Protect, 68 (5), 1083±1088. TAKEDA Y (1983), Thermostable direct hemolysin of Vibrio parahaemolyticus, Pharmac Ther, 19, 123±146. TAMPLIN M L, RODRICK G E, BLAKE J and CUBA T (1982), Isolation and characterization of Vibrio vulnificus from two Florida estuaries, Appl Environ Microbiol, 44, 1466± 1470. TAMPLIN M L, JACKSON J K, BUCHRIESER C, MURPHREE R L, PORTIER K M, GANGAR V, MILLER

and KASPAR C W (1996), Pulsed-field gel electrophoresis and ribotype profiles of clinical and environmental Vibrio vulnificus isolates, Appl Environ Microbiol, 62, 3572±3580. TANIGUCHI H, HIRANO H, KUBOMURA S, HIGASHI K and MIZUGUCHI Y (1986), Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin from Vibrio parahaemolyticus, Microb Pathog,1, 425±432. TARR C L, PATEL J S, PUHR N D, SOWERS E, BOPP C A and STROCKBINE N A (2006), Identificaton of Vibrio isolates using a multiplex PCR assay and rpoB sequence determination, J Clin Microbiol, 45, 134±140. LG

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(1997), How to select and interpret molecular typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists, Infect Cont Hosp Ep, 18, 426± 439. TEOPHILO G N, VIERIRA D F, RODRIGUES D P and MENEZES F G (2002), Escherichia coli isolated from seafood: toxicity and plasmid profiles, Int Microbiol, 5, 11±14. TIMONEY J F and ABSTON A (1984), Accumulation and elimination of Escherichia coli and Salmonella typhimurium by hard clams in an in vitro system, Appl Environ Microbiol, 47, 986±988. TODD E C D (1997), Epidemiology of foodborne diseases: a worldwide review, Rapp trimest statist sanit mond, 50, 30±50. TOTI L, CROCI L, SERRATORE P, STACCHINI A, MILANDRI S and COZZI L (1996), Bacteria isolated from seawater and mussels: identification and toxin production, Lett Appl Microbiol, 32, 57±61. TOWNER K J and COCKAYNE A (1993), Molecular Methods for Microbial Identification and Typing, London: Chapman & Hall. TRUU J, TALPSEP E, HEINARU E, STOTTMEISTER U, WAND H and HEINARU A (1999), Comparison of API 20NE and Biolog GN identification systems assessed by techniques of multivariate analyses, J Microbiol Meth, 36, 193±201. VANEECHOUTTE M and VAN ELDERE J (1997), The possibilities and limitations of nucleic SOCIETY FOR HEALTHCARE EPIDEMIOLOGY OF AMERICA

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acid amplification technology in diagnostic microbiology, J Med Microbiol, 46, 188±194. VEENSTRA J, RIETRA P J G M, GOUDSWAARD J, KAAN J A, VAN KEULEN P H J and STOUTENBEEK C P (1993), Extra-intestinale infecties door Vibrio spp. (in Dutch), Ned Tijdschr Geneeskd, 137, 654±657. VENKATESWARAN K, DOHMOTO N and HARAYAMA S (1998) Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp, Appl Environ Microbiol, 64, 681±687. VERSALOVIC J, KOEUTH T and LUPSKI J R (1991), Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes, Nucleic Acids Res, 19, 6823±6831. VOISIN E (2001), United States Patent 6427435. Process elimination of bacteria in shellfish and of shucking shellfish, Issued 17 April 2001. VOISIN E (2002) United States Patent 6426103. Process elimination of bacteria in shellfish and of shucking shellfish, Issued 30 July 2002. VORA G J, MEADOR C E, BIRD M M, BOPP C A, ANDREADIS J D and STENGER D A (2005), Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp, Proc Natl Acad Sci USA, 102, 19109±19114. WARD D R and BAJ N J (1988), Factors affecting the microbiological quality of seafoods, Food Technol, 42, 85±89. WARD L N and BEJ A K (2006), Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes, Appl Environ Microbiol, 72, 2031±2042. WARNER J M and OLIVER J D (1998), Randomly amplified polymorphic DNA analysis of starved and viable but nonculturable Vibrio vulnificus cells, Appl Environ Microbiol, 64, 3025±3028. WEBER J T, MINTZ E D, CAIZARES R, SEMIGLIA A, GOMEZ I, SEMPERTEGUI R, et al. (1994), Epidemic cholera in Ecuador: multidrug resistance and transmission by water and seafood, Epidemiol Infect, 112, 1±11. WEST P A (1989), Human pathogens and public health indicator organisms in shellfish, in Austin B and Austin D A (Eds), Methods for the microbiological examination of fish and shellfish, Chichester, Ellis Horwood Ltd, 273±308. WITTMAN R J and FLICK G J (1995), Microbial contamination of shellfish: prevalence, risk to human health, and control strategies, Ann Rev Publ Health, 16, 123±140. WONG H and LIN C (2001), Evaluation of typing of Vibrio parahaemolyticus by three PCR methods using specific PCR, J Clin Microbiol, 39, 4233±4240. WONG H C, HO C Y, KUO L P, WANG T K, LEE C L and SHIH D Y (1999), Ribotyping of V. parahaemolyticus isolates obtained from food poisoning outbreaks in Taiwan, Microbiol Immunol, 43, 631±636. WU M S and LEE C Y (1998), Sequence analysis of the flanking regions of Vibrio parahaemolyticus R72H fragment, 98th General Meeting of the American Society for Microbiology, Atlanta, GA. YOUNGER A D, LEE R J and LEES DN (2003), Microbiological monitoring of bivalve mollusc harvesting areas in England and Wales ± rationale and approach, in Villalba, A, Reguera B, Romalde J L, Beiras R (Eds), Molluscan Shellfish Safety, Santiago de Compostela, Spain, ConsellerõÂa de Pesca e Asuntos MarõÂtimos da Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, 265±277.

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15 Histamine and biogenic amines: formation and importance in seafood P. Dalgaard and J. Emborg, Technical University of Denmark, Denmark and A. Kjùlby, N.D. Sùrensen and N.Z. Ballin, Danish Veterinary and Food Administration, Denmark

15.1

Introduction

Histamine fish poisoning (HFP) is an intoxication that can be caused by consumption of many different types of marine finfish. It is a common characteristic of these seafoods that, at some stage between catch and consumption, specific bacteria have been able to grow to high concentrations and to form histamine and other biogenic amines in the products. HFP is common and occurs world wide. Thus, control of the factors that allow seafood to cause HFP deserves to be improved. To illustrate this point it is sufficient to mention that HFP during the 1990s caused 32% of all seafoodborne incidents of human disease in England and Wales. And that the situation is similar in the USA where HFP caused 38% of all seafood-borne human disease outbreaks. These outbreaks (i.e., incidents where two or more people became ill after consuming the same food) accounted for 18% of all the people that became ill due to consumption of seafood. HFP have been responsible for 8% of all the food-borne disease outbreaks and 1.2% of all the people that became ill due to food consumption in the USA (McLauchlin et al., 2006; Dewall et al., 2006). Increased seafood consumption is desirable in many parts of the world. Some developing regions need seafood as a source of protein and nutrients, whereas in developed regions particular culinary properties and health-promoting effects contribute to the demand for seafood. Interests in the potential of seafood to reduce the risk of cardiovascular and coronary heart diseases, cancer, diabetes, hypotension as well as neuropsychiatric and autoimmune disorderes has been considerable (Narayan et al., 2006). In fact, several national authorities

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recommend increased seafood consumption. Recommendations are, for example, two portions of fish per week in the UK (Anon., 2004), 200±300 g of fish per week in Denmark (Andersen et al., 2003) and two 85 g servings per week in the USA (Anon., 2006). To meet these recommendations a positive perception for seafood by consumers is required and it is imperative to reduce the occurrence of seafoodborne human disease. HFP and viruses together with salmonellas account for most incidents and cases of human seafood-borne diseases in UK and USA (CSPI, 2005; Hughes et al., 2007). This situation is probably similar for many countries in Europe although outbreak statistics are incomplete as discussed below in Section 15.2.1. Consequently, efforts to reduce HFP deserve a high priority (i) to prevent seafood-borne disease, (ii) to reduce economic losses in the seafood sector and (iii) to contribute to increased seafood consumption with potential corresponding health benefits. The present chapter reflects the priority given to HFP within the EU integrated programme SEAFOODplus. The text results from activities within the BIOCOM project (Biogenic amines in seafoods ± assessment and management of consumer exposure, January 2004 to June 2007). The overall objective of BIOCOM has been to reduce the occurrence of HFP. Firstly, incidents of HFP were studied with focus on chemical and microbiological characteristics of the implicated seafoods including of course their concentration of histamine and other biogenic amines. Secondly, bacteria responsible for biogenic amine formation were identified. Finally, product characteristics and storage condition that reduce histamine formation were identified. The project focused on psychrotolerant bacteria with potential to form toxic concentrations of histamine and other biogenic amines in seafood at storage temperatures as low as 0 ëC to 5 ëC.

15.2

Histamine fish poisoning (HFP)

Between 1940 and 1960, several studies of HFP, including some larger outbreaks in France and Japan, were reported. These early studies found the responsible toxin(s) to be formed during storage of the implicated seafoods. The toxic products were not necessarily unacceptable with respect to sensory properties such as flavour, texture or colour. The pH, total volatile nitrogen or trimethylamine concentrations of the implicated products could be increased but this was not always observed. It was also established that HFP symptoms (described in Section 15.2.3 below) were due to histamine and possibly other compounds produced by bacteria in seafood with a high initial concentration of free histidine. In Japan, histamine formation in mackerel was shown to be rapid and extensive at storage temperatures of 17±23 ëC but slower and less pronounced both at 6±7 ëC and at 35 ëC. The bacterium Morganella morganii (initially named Achromobacter histamineum) was pointed out as primarily responsible for histamine formation. Although, information about HFP was relatively limited, early studies suggested ways to prevent the formation of

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histamine by using good hygiene onboard fishing vessels and chilling throughout distribution of relevant finfish (van Veen and Latuasan, 1950; Kawabata et al., 1955b; Sapin-Jaloustre and Sapin-Jaloustre, 1957; Kimata, 1961). Later, HFP and formation of histamine in different seafood by various histamine producing bacteria have been extensively studied. The information has been used to determine critical limits for histamine in seafood, maximum temperatures of storage, good manufacturing practices (GMP) and safety management relying on HACCP. Specifically, it has been shown that histamine, when formed in seafood, is relatively stable and not inactivated by freezing or heating such as normal cooking, hot smoking or even canning (Arnold and Brown, 1978; Taylor, 1986; Lehane and Olley, 2000; Flick et al., 2001; FDA/ CFSAN, 2001; Kim et al., 2003). It needs to be noted, however, that despite the considerable information available, it has not been possible to reduce the occurrence of HFP either in Europe or the USA. Thus, HFP remains one of most common seafood-borne diseases as explained above. Therefore, it seems that the available information about HFP and histamine formation in seafood could be incomplete or perhaps not used appropriately for management of this safety hazard. Clearly, a combination of these two possibilities cannot be excluded as an explanation for the high occurrence of HFP. The BIOCOM project has studied all outbreaks of HFP that occurred during 2004±2006 in Denmark. To provide an up-to-date status on information about HFP our results are presented in this section together with information that we have collected from the literature and from other countries. 15.2.1 Histamine fish poisoning: incidents and cases Outbreaks of HFP usually involve fewer than 10 people (Hughes et al., 2007) but due to its high frequency this seafood-borne disease is quantitatively important as already mentioned and as documented below (Table 15.1). The incubation time for HFP is short and people often develop symptoms while they are still eating. Nevertheless, the occurrence of HFP is underreported (Taylor and Lyons, 1984; Wu et al., 1997) and this is mainly because: · Many countries do not collect data on incidents of HFP. The BIOCOM project has contacted authorities in various countries. Just within the EU, we have been informed that this is the situation for Austria, Germany, Italy, Poland, Portugal and Spain. · Symptoms can be mild and of short duration so that a physician may not be contacted. · HFP symptoms can be incorrectly identified and recorded, e.g. as food allergy. · Some statistics exclusively include cases reported as part of outbreaks. However, single HFP cases are common. As an example, for Hawaii, 691 people were reported ill with HFP between 1990 and 2003 but outbreaks only accounted for 526 of these with the remaining 165 people (24% of the total number of patients) representing single cases (CSPI, 2005; Kaneko et al., 2004).

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Table 15.1 Overview of incidents and cases of histamine fish poisoning in various countries. To facilitate comparisons, data are sorted according to the mean annual rate (cases/year/million people) Country

Year

Incidents or outbreaks

Cases

Cases/year/ million people

Taiwan

1990±2003 1986±2005 2001±2005 1951±1954 1970±1980 1994±2005 1987±2005 1983±1994 1998±2005 1986±2001

111 64 11 14 42 68 123 26 15 8

526 489 62 1215 4122 1523 2635 73 89 535

31 4.9 3.1 3.7 3.2 1.1 2.5 1.3 2.1 1.5

UK

1976±2004

515

1300

0.8

Norway Switzerland South Africa

1999±2000 1966±1991 1992 2004 1995±2003 1973±1987 1990±2003 1993±2000 1975±1995 1999±2005 1995±2004

4 76 10 3 12 202 341 5 39 4 ?

6 111 22 21 77 1216 1651 22 109 15 124

0.7 0.7 0.5 0.4 0.4 0.4 0.4 0.3 0.2 0.2 0.2

Hawaii, USA Denmark New Zealand Japan France Finland

Australia USA Sweden Canada Netherlands Philippines a

References CSPI (2005) DVFA (2006)a, Emborg et al. (2006a,b) ESR (2006) Kawabata et al. (1955a) Taylor (1986) Kanki (2006)b WHO (2000, 2003), INVS (2006)c Maijala et al. (1996) WHO (2003), NFSAE (2006)d Kow-Tong and Malison (1987); Hwang et al. (1995, 1997); Su et al. (2000); Wu and Chen (2003); Tsai et al. (2005) Bartholomew et al. (1987); Scoging (1998); McLauchlin et al. (2006) WHO (2003) Marie et al. (1992) MuÈller et al. (1992) Auerswald et al. (2006) Bell (2002); Dalton et al. (2004); Ashbolt (2004) Bean and Griffin (1990) CSPI (2005) WHO (2000, 2003) Todd (1997) NIPHE (2006)e Azanza (2006)

Danish Veterinary and Food Administration (DVFA). Homepage and personal communication from Annette Perge. Personal communication by Masashi Kanki of data from Ministry of Health, Labour and Welfare in Japan. c Institut de Veille Sanitaire (INVS), personal communication from Gilles Delmas. d National Food Safety Authority Evira, personal communication from Taina Niskanen. e National Institute for Public Health and the Environment, personal communication from Yvonne van Duynhoven. b

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Fig. 15.1 Reported occurrence of HFP in different countries as a function of seafood consumption (data from Table 15.1). Seafood consumption is expressed as kg live weight of fish/year/person (Laurenti, 2004) corresponding to approximately two times the amount of fish flesh consumed. For Hawaii the seafood consumption data was obtained from Pan (1998).

The BIOCOM project has collected information about the occurrence of HFP world wide (Table 15.1). This effort was carried out because the last overview of this type was more than 20 years old (Taylor, 1986). We have used the mean annual rate of HFP (cases/year/million people) to facilitate comparison of data between regions of different population size and for different recording periods (Table 15.1). The particularly high occurrence of HFP in Hawaii (Table 15.1, Fig. 15.1) is mainly caused by consumption of yellowfin tuna (44%), mahi-mahi (24%), bigeye scad (10%) and marlin (9%) (Sasaki, 2001). Seafood consumption in Hawaii is about three times higher than the average for the USA but the occurrence of HFP is about 75 times higher. Thus, the total amount of seafood consumed in Hawaii cannot alone explain the significant problem with HFP. As shown in Fig. 15.1 there is no clear relation between the total amounts of seafood consumed in different countries and the reported occurrence of HFP. One example is Norway where seafood consumption is high but the reported occurrence of HFP is very low (Fig. 15.1). Another example is Denmark where the seafood consumption is moderate (Fig. 15.1) and the relatively high occurrence of HFP (Table 15.1) is caused primarily by consumption of tunabased products (fresh, frozen, canned or cold-smoked), fresh garfish and escolar (fresh, frozen or smoked) (Emborg et al., 2006b).

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The precision of data collection must be kept in mind when HFP occurrence is compared between countries but within a given country this factor is likely to be less important. In Japan the occurrence of HFP has decreased after 1980 (Table 15.1). This observation is noteworthy as we have found no other countries where the occurrence of HFP seems to decrease consistently over a period of time (Table 15.1). The decreased occurrence of HFP in Japan has been attributed to improved chilling of fish and to a reduced consumption of dried fish products (Taylor and Lyons, 1984; Masashi Kanki, pers. comm.). In fact, it has been shown recently that a high proportion of semi-dried fish products in Tokyo still have critically high concentrations of histamine (Kan et al., 2005). The reported occurrence of HFP (Table 15.1) clearly documents the need to improve controls and to reduce this seafood-borne disease. 15.2.2 Histamine fish poisoning: implicated products and their characteristics Many species of marine finfish have caused HFP (Table 15.2) and they belong to various families. Previously, scombroid fish from the families Scombridae and Scomberesocidae primarily tuna, mackerel and sauries were believed to cause HFP. Therefore, the disease was called scombroid fish poisoning. However, this name is misleading as several other fish species have caused numerous HFP incidents, particularly, mahi-mahi (Coryphana hippurus), escolar (Lepidocybium flavobrunneum), sardines (sardine spp.), kahawai (Arripis trutta), garfish (Belone belone), marlin (Istiophoridae), swordfish (Xiphiidae) and these are not scombroid fish. Interestingly, no freshwater fish seem to cause HFP. It has long been known that HFP is caused by consumption of meat from dark-fleshed finfish and that the flesh of the implicated species contains a high concentration of free histidine. BIOCOM has confirmed the high concentration of free histidine in seafood that cause HFP (Emborg and Dalgaard, 2007). More than 2000 mg of free histidine/kg is typical but above 10 000 mg of free histidine/kg is usual for several species of tuna (Thunnus, Euthynnus and Katuswonus). Also, flesh of fish that cause HFP tends to have a relatively low pH of about 6. A notable exception is salmon that has been reported to cause typical HFP symptoms although its flesh contains as little as 130±1090 mg of free histidine/ kg. Concentrations of histidine correspond to the low histamine concentrations of less than 10±170 mg of histamine/kg in the implicated salmon products (Bartholomew et al., 1987; Gessner et al., 1996; Emborg and Dalgaard, 2007). However, for these HFP patients histamine or histamine degradation products have not been measured in blood or urine and it cannot be excluded that another toxin(s) may have caused the observed symptoms. The BIOCOM project has collected data from 142 HFP incidents including the 16 incidents from Denmark that we have studied in 2004, 2005 and 2006. As shown in Table 15.2 the large majority of incidents and cases (90%) were caused by seafood with a histamine concentration of more than 500 mg/kg. In

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Table 15.2 Overview of outbreaks (n 142) and cases (n 1998) of histamine fish poisoning as a function of the concentration of histamine in different seafoods (Emborg and Dalgaard 2007) Histamine (mg/kg)

Outbreaks

Cases

Seafood or fish species

Number

%

Number

%

>5000

14

10

98

5

1000±5000

66

47

937

47

500±1000

26

18

772

39

<500

36

25

191

10

Escolar, kahawai, kingfish, marlin, saury, tuna, yellowfin tuna Amberjack, anchovies, bluefish, cape yellowtail, castor oil fish/ escolar, kahawai, mackerel, mahimahi, marlin, pilchard, red tuna, sailfish, sardines, swordfish, tuna Anchovies, garfish, kahawai, mahimahi, mackerel, marlin, sardines, tuna Anchovies, bonito, escolar, mackerel, mahi-mahi, pilchard, red tuna, sardines, skipjack, salmon, tuna

agreement with this a study of 148 HFP incidents in England and Wales (1987± 1996) found 91% of the implicated fish to contain above 200 mg of histamine/kg whereas 79% contained above 1000 mg histamine/kg (Scoging, 1998). Table 15.2 shows that 191 people (10%) became ill due to seafood with less than 500 mg of histamine/kg. It is interesting, that for 80% of these people the histamine analysis of the seafood were conducted on a sample from the same batch of fish but not on a `plate-sample' of the fish actually implicated in the HFP incident (Table 15.2). Such samples are analysed, e.g. when absolutely no left-overs are available to be studied. This is important to point out because the concentration of histamine can vary considerable from fish-to-fish within a batch and even between different portions of a single fish. An example of this is shown in Fig. 15.2 for tuna. Similar results have been obtained with other fishes (Kawabata et al., 1955b; Lerke et al., 1978; Ijomah et al., 1991; BeÂdry et al., 2000). But variability in histamine concentrations in seafood is not always observed (Middlebrooks et al., 1988; LoÂpez-Sabater et al., 1996). Consequently, determination of histamine in samples other than the `plate-sample', that actually caused HFP, can be quite uninformative. In fact, it cannot be excluded that in some situations where seafood with very low concentrations of histamine have been reported to cause HFP, the seafood-sample actually consumed contained a much higher concentration of histamine. For seafood implicated in incidents of HFP, information about concentrations of biogenic amine other than histamine is limited. We have collected literature data from 14 incidents. This information has been considerably expanded by the BIOCOM project where concentrations of nine biogenic amines were deter-

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Fig. 15.2

299

Example of within-fish variation in histamine concentrations (Frank et al., 1981).

mined for seafoods implicated in 16 incidents of HFP (Emborg et al., 2006b). The concentrations of other biogenic amines were typically much lower than the concentration of histamine (Fig. 15.3). Although a high concentration of cadaverine in bluefish (740 mg/kg) and of tryptamine in sailfish (1850±2080 mg/kg) have been observed (Etkind et al., 1987; Hwang et al., 1995). Interestingly, the concentration of histamine in various products (tuna, escolar, marlin, mackerel, mahi-mahi, swordfish and salmon) was proportional to the concentration of cadaverine as well as to the sum of the concentrations of putrescine, cadaverine, tryptamine, spermine, spermidine, tyramine and -phenethylamine. No clear difference was observed between data from the literature and from the BIOCOM project (Fig. 15.3). As discussed later, biogenic amines in seafood may potentiate the oral toxicity of histamine. It is therefore both interesting and of practical importance that concentrations of biogenic amines in seafood that cause HFP seem proportional to the concentration of histamine. In fact, this information can reduce costs of seafood inspection and facilitate exposure assessment studies. Bacteria responsible for histamine formation in seafood that actually caused HFP have been identified but in a very limited number of studies (Table 15.3). BIOCOM has identified the bacteria responsible for five incidents of HFP and these were due to fresh tuna, cold-smoked tuna and tuna heated in a flexible packaging film (Table 15.3; Emborg and Dalgaard, 2006; Emborg et al., 2006b). Thus the BIOCOM project has contributed importantly to our present understanding of the importance of psychrotolerant bacteria for histamine formation in seafood (Table 15.3). In fact, we have identified Morganella psychrotolerans as a new psychrotolerant and strongly histamine-producing species within the genus Morganella (Emborg et al., 2006a). Today it is clear that both mesophilic bacteria (Morganella morganii, Hafnia alvei and Raoultella planticola) and psychrotolerant bacteria (Morganella psychrotolerans and Photobacterium phosphoreum) can produce toxic concentrations of histamine and other biogenic amines in seafood and thereby cause HFP (Table 15.3). Prior to 2004 it was a general opinion that HFP was caused exclusively by the activity of mesophilic histamine-producing bacteria (Kim et al., 2004).

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Fig. 15.3 Relation between concentrations of histamine and cadaverine (a) and between concentrations of histamine and of the sum of the concentrations of putrescine, cadaverine, tryptamine, spermine, spermidine, tyramine and -phenethylamine (b). Data from the literature (solid symbols) and data from the BIOCOM project (open symbols) (Emborg and Dalgaard, 2007).

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Table 15.3 Incidents of histamine fish poisoning where the bacteria responsible for histamine formation have been identified Implicated seafood Mesophilic bacteria Fresh tuna Fresh tuna Fresh tuna Fresh tuna Tuna heated in flexible film

Bacterium

Reference

Morganella morganii Hafnia alvei Morganella morganii Raoultella planticola

Kawabata et al. (1956) Havelka (1967) Sakabe (1973) Lerke et al. (1978)

Morganella morganii

Emborg et al. (2006)

Psychrotolerant bacteria Dried sardines Photobacterium phosphoreum Tuna in chilli-sauce Morganella psychrotolerans and/or Photobacterium phosphoreum Cold-smoked tuna Photobacterium phosphoreum Cold-smoked tuna Morganella psychrotolerans Fresh tuna Photobacterium phosphoreum

Kanki et al. (2004) Emborg et al. (2005) Emborg and Dalgaard (2006) Emborg and Dalgaard (2006) Emborg et al. (2006)

M. psychrotolerans and P. phosphoreum can grow at 0 ëC (i.e., they are psychrotolerant) but not at 37 ëC and their temperature optimum is 20±30 ëC. Interestingly, this corresponds to early studies from Japan where histamine formation in mackerel was faster and more pronounced at 17±23 ëC compared to both 6±7 ëC and 35 ëC (Kimata, 1961). In contrast, when histamine is produced primarily by mesophilic bacteria then the formation at 35 ëC is much faster than at 17±23 ëC (Frank et al., 1981, 1983). Information about the bacteria that produce histamine in seafood is important. Firstly, to reduce histamine formation it is essential to inhibit growth of the specific bacteria that actually produce this compound. Secondly, microbiological methods for seafood inspection must target the bacteria of importance and therefore the characteristics of these bacteria need to be known. Psychrotolerant bacteria in seafood can be detected by using a medium with 1% NaCl and incubation at 15 ëC (Dalgaard, 2000; NMKL no.184 2006). In contrast, P. phosphoreum cannot be detected (i) on microbiological media without NaCl (e.g., standard Plate Count Agar), (ii) on media incubated at 37 ëC, (iii) with pour plating as it is killed by the 45 ëC warm and melted agar used for this technique. Nivens agar (Niven et al., 1981), frequently used for enumeration of histamine producing bacteria, relies on pour plating and incubation of plates at 37 ëC. Consequently, this method will never detect neither P. phosphoreum nor M. psychrotolerans in seafood. With thousands of reported HFP incidents and cases it is surprising that the histamine producing bacteria have been identified for as few as ten of the implicated products (Tables 15.1 and 15.3). Clearly, heat treatment during canning, hot smoking or during cooking prior to consumption will inactivate bacteria and thereby make their isolation impossible. P. phosphoreum is also

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inactivated by normal frozen storage of seafood (Emborg et al., 2002). Nevertheless, it seems little priority has been given to this area and this is unfortunate. In fact, the BIOCOM project and other recent studies (Kanki et al., 2004) suggests that histamine formation by psychrotolerant bacteria causes an important part of the reported HFP incidents (Table 15.3). This information is new and highly important for the ways in which histamine formation in seafood can be controlled. Once isolated from seafoods the bacteria with potential to produce histamine and biogenic amines can be pointed out by culturing of isolates in appropriate media. Fish infusion broths or other complex media with added free amino acids have been used (Taylor, 1986; Dalgaard et al., 2006). It is imperative to evaluate formation of biogenic amines by bacterial isolates at appropriate temperatures. Thus, incubation above 25 ëC is not relevant to evaluate histamine formation by bacteria from chilled seafood as these may be heat labile and unable to produce histamine or even to grow at such temperatures. Nevertheless, incubation at above 30 ëC has frequently been used (Taylor et al., 1978, 1979; Omura et al., 1978; Taylor and Speckhard, 1983; Gingerich et al., 2001; Kim et al., 2001a, 2001b). These high incubation temperatures may be another reason why psychrotolerant histamine producing bacteria were not until 2004±2005 found responsible for incidents of HFP (Table 15.3). The BIOCOM project used incubation of cultures at 10 ëC to evaluate their formation of histamine and other biogenic amines (Emborg and Dalgaard, 2006). To identify the bacteria responsible for histamine and biogenic amines formation in a product, the dominating microflora was first isolated and identified. Biogenic amine formation by isolates representing the dominating groups of bacteria was then evaluated in broth. Table 15.4 shows an example where histamine and biogenic amine formation in cold-smoked tuna was explained by the activity of P. phosphoreum whereas other bacteria from the dominating microflora of the implicated product formed very different profiles of biogenic amines. Table 15.4 Profile of selected biogenic amines in cold-smoked tuna from incidence of HFP and profiles produced by different bacteria isolated from the implicated seafood. Modified from Emborg and Dalgaard (2006) Biogenic amines (mg/kg or mg/l) Histamine Putrescine Cadaverine Tyramine -phenylethylamine Cold-smoked tuna that caused HFP

4548

20

212

150

54

Photobacterium phosphoreum Brochothrix thermosphacta Lactic acid bacteria Pseudomonas fluorescens

3777 <5 <5 <5

6 9 9 78

107 <5 <5 <5

290 <5 23±275 <5

60 <5 <5 <5

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Related to incidents of HFP it is usually attempted, but often not possible, to evaluated the storage conditions of the implicated products. Such retrospective analyses indicate that fish exposed to extended storage at 10±44 ëC (lack of chilling or long-time smoking) can cause HFP (Hughes et al., 1977; Russell and Maretic, 1986; Gessner et al., 1996; Maher et al., 2000). In fact, it is frequently and categorically stated that HFP is due to time/temperature abuse of certain species of fish (FDA/CFSAN, 2001; Kim et al., 2004). Results from the BIOCOM project support that time/temperature abuse is a very important risk factor for HFP. Nevertheless, we have also clearly shown that other factors are significant to control HFP and must be taken into account. Firstly, BIOCOM showed that HFP can be caused by psychrotolerant bacteria (Table 15.3) and that they can produce toxic concentrations of histamine at temperatures as low as 2 ëC (Emborg et al., 2005). Consequently, histamine formation during extended storage of fish at low temperature must not be disregarded. Secondly, BIOCOM found three of the 16 studied HFP incidents to be caused by smoked tuna with very little salt (0.5±2.2% NaCl in the water phase of the products). The temperature history of these products was not elucidated. However, the low salt concentrations allow histamine to be formed at the intended storage temperature of less than 5 ëC. Finally, BIOCOM found one of the 16 studied HFP incidents was due to tuna heated in a flexible packaging film. This packaging was inappropriately sealed which resulted in a post heating contamination of Morganella morganii and subsequent histamine formation and HFP (Table 15.3). To reduce the occurrence of HFP it is important to manage all these important risk factors as further discussed in Section 15.6. 15.2.3 Histamine fish poisoning: symptoms and toxicology HFP symptoms and their duration are important, e.g. to appropriately recognize the disease. The BIOCOM project has recorded symptoms for 15 HFP incidents in Denmark with a total of 56 patients. This information has been compared with data from 125 incidents in Finland, UK and Japan as well as with symptoms for 515 individual patients from Australia, France, South Africa, Japan, Switzerland, USA and Taiwan (Table 15.5). Major HFP symptoms are cutaneous (rash/ urticaria), neurological (flushing, headache) and gastrointestinal (diarrhoea, vomiting). Other symptoms include oral tingling, irregular or increased heart beat, i.e. palpitations, abdominal cramps and localized edema. Each patient typically experience several but rarely all of these symptoms. The symptoms are often mild but can be severe and in rare situations even life-threatening, particularly for individuals with asthma or heart disease. The sensitivity of consumers to HFP differs markedly and it is unusual that everybody eating a seafood becomes ill even when it contains 1000±5000 mg of histamine/kg or more. After eating a toxic product symptoms begin rapidly within two minutes to three hours and last from a few hours up to one day. The physiological effects of histamine include dilatation of the peripheral blood vessels and capillaries, increase in capillary permeability, contraction of intestinal smooth muscles as

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Table 15.5 Symptoms of histamine fish poisoning recorded for incidents (with one or several people involved) and for cases (individual patients) Symptoms

Incidentsa (n 140)

Casesb (n 515)

Flushing Rash Headache Diarrhoea Vomiting

40±79% 18±73% 20±71% 29±53% 27±50%

NRc±100% NR±100% 4±100% NR±90% NR±53%

a

Arnold and Brown (1978); Bartholomew et al. (1987); Maijala et al. (1996); Emborg et al. (2006) Kawabata et al. (1955a); Merson et al. (1974); Russell and Maretic (1986); Kow-Tong and Malison (1987); Smart (1992); MuÈller et al. (1992); Marie et al. (1992); Wu et al. (1997); Boutin et al. (1998); Wu and Chen (2003); Leask et al. (2004) c Not reported (NR) b

well as increased heart rate and increased force of heart contraction. These effects of histamine explains the symptoms of HFP (Taylor, 1986; Lehane and Olley, 2000; Parsons and Ganellin, 2006; GloÂria, 2006). Histamine in small amounts is not toxic for humans as it is metabolized (detoxified) prior to reaching the blood circulation. The enzymes histamine-Nmethyltransferase (HMT), monoamine oxidase (MAO) and diamine oxidase (DAO or histaminase) transform histamine to less toxic metabolites that are excreted in urine and faeces. The enzymes are found primarily in the small intestine and liver of humans (Taylor, 1986; GloÂria, 2006). However, if the normal histamine metabolism is reduced or very large amounts of histamine are consumed, then the concentration of histamine in the blood increases, resulting in the symptoms described above. Symptoms can be resolved by antihistaminic drugs (antihistamines) including chlorpheniramine, diphenhydramine, hydroxyzine, promethazine and cimetidine. These drugs block the binding of histamine to specific receptor in different tissues and thereby its effect and HFP symptoms (Parsons and Ganellin, 2006; GloÂria, 2006). The large majority of HFP incidents is caused by seafood with more than 500 mg/kg of histamine and with a total concentration of other biogenic amines above 50 mg/kg (Table 15.2, Fig. 15.3). A meal size of 100 g seafood then corresponds to an intake of above 50 mg of histamine (with 100±500 mg being most common) together with more than 5 mg of other biogenic amines and with higher amounts (10±50 mg) being typical (Table 15.2, Fig. 15.3). Related to a HFP outbreak caused by escolar it has been shown that persons consuming less than 113±215 mg of histamine experienced fever symptoms and of shorter duration than persons consuming more of the fish and thereby higher amounts of histamine (Feldman et al., 2005). In some challenge studies with human volunteers, 67.5 to 300 mg of histamine administered in water, grapefruit juice or fish resulted in none or mild symptoms only. But it has also been found that 180 mg histamine resulted in severe headache and flushing (Motil and Scrimshaw, 1979; van Gelderen et al., 1992). Thus, available data from challenge studies with

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human volunteers suggest that pure histamine cannot always explain the toxicity of histamine-containing seafood. This apparently low toxicity of pure histamine may, to some extent, be explained by (a) sensitivity of the few volunteers used in most studies and (b) the relatively low amounts of histamine (<100±500 mg) evaluated. In addition, two different hypotheses to explain the apparently low toxicity of histamine have been extensively discussed in the scientific literature. To explain how seafood with low concentrations of histamine can cause HFP, it was proposed that seafood may contain a mast cell degranulator (or activator), and that this compound could be cis-urocanic acid. The degranulator should cause a release of histamine from mast cells in the human intestinal tissue, and HFP would then be due to indigenous histamine. In contrast, histamine in seafood should not necessarily contribute to HFP symptoms according to this hypothesis (Arnold and Brown, 1978; Taylor, 1986; Clifford et al., 1991; Ijomah et al., 1991; Lehane and Olley, 2000). The hypothesis is consistent with the fact that antihistamine therapy eliminates symptoms of HFP. Nevertheless, experimental evidence to support the hypothesis is very limited. When mast cells are degranulated they release histamine together with tryptase and prostaglandin D2. However, significant concentrations of these compounds or their degradation products were not detected in serum or urine from patients with HFP (Morrow et al., 1991; Sanchez-Guerrero et al., 1997). Thus, for these incidents the observed symptoms and the increased concentration of histamine in serum and urine resulted from histamine in seafood rather than histamine from mast cells. With respect to cis-urocanic acid as a specific mast cell degranulator it remains to be determined if the concentrations found in seafood (or even higher concentrations) have any effect on histamine release from human mast cells in the gastrointestinal tract. Mackie and FernaÂndez-Salguero (1977) found less than 50 mg of urocanic acid/kg during storage of mackerel at 0, 10 and 23 ëC but data for other seafoods are lacking. The other hypothesis is older and suggests the oral toxicity of histamine in seafood can be potentiated by different compounds including other biogenic amines (Kawabata et al., 1955b; Taylor, 1986). Numerous experiments with laboratory animals (cats, dogs, guinea pigs, pigs, rabbits and rats) have demonstrated that various compounds can inhibit the normal histamine metabolizing enzymes (DAO, HMT and MAO) and thereby increase the oral toxicity of histamine. With laboratory animals this has been observed for agmatine, cadaverine, -phenylethylamine, putrescine, trimethylamine, tyramine, combinations of these compounds and for ethanol (Taylor and Lieber, 1979; Lyons et al., 1983; Hui and Taylor, 1985; Satter and Lorenz, 1990). However, extrapolation of the quantitative effects from such data to humans may not be possible as distribution of the key histamine metabolizing enzymes in tissues differ between species (Buffoni, 1966; Taylor, 1986; Satter and Lorenz, 1990; Lehane and Olley, 2000). Data from human volunteer studies are limited but tuberculosis patients taking the drug isoniazid, which is known to inhibit DAO and HMT have increased sensitivity to histamine in seafood (Uragoda and

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Kottegoda, 1977; Miki et al., 2005). This supports the hypothesis that compounds which inhibit histamine degradation increase the oral toxicity of histamine. However, van Gelderen et al. (1992) found 22 mg of cadaverine and 18 mg of putrescine unable to potentiate the oral toxicity 88±90 mg of histamine for eight volunteers. Newer studies are lacking and this is somewhat surprising. Thus available information suggests that HFP is caused primarily by histamine in seafood. Consequently, to reduce HFP, efforts to reduce histamine formation in seafood should be the main objective.

15.3

Legislation

Existing regulations on critical limits for histamine in seafood and on storage temperatures is summarized here. The information is used later to discuss management of histamine formation and of HFP. 15.3.1 Critical limits for concentrations of histamine in seafood EU regulation (EC 2073/2005) includes limits for critical concentrations of histamine in two groups of fishery products: (i) `fishery products from fish species associated with a high amount of histidine' and (ii) `fishery products which have undergone enzyme maturation treatment in brine, manufactured from fish species associated with a high amount of histidine'. Of nine samples the average (m) should be below 100 mg/kg for (i) or below 200 mg/kg for (ii). Two of the nine samples are allowed to have a histamine concentration higher than `m' but the maximum allowable concentration in any sample (M) is 200 mg/kg for (i) or 400 mg/kg for (ii). The EU regulation does not include critical limits for other biogenic amines (EU, 2005). The EU regulation particularly refer to fish species of the families Scombridae (bonitos, kingfish, mackerels, seerfish, tunas and wahoo), Clupeidae (herrings, shads, sardines and manhadens), Engraulidae (anchovies), Coryphaenidae (dolphinfishes including mahi-mahi), Pomatomidae (bluefishes) and Scombresosidae (sauries). It is surprising that Belonidae (garfish), Gempylidae (escolar and oilfish), Istiophoridae (marlin and sailfish) and Xiphiidae (swordfish) are not mentioned as these fishes have caused many incidents of HFP (Table 15.2). For USA the defect action level (m) is 50 mg of histamine/kg, whereas the toxicity level is indicated to be 500 mg of histamine/kg. Eighteen fish per lot should be analyzed individually or composited into, e.g. 6 units of fish each, but then the critical limit is reduced accordingly from 50 mg/kg to 17 mg/kg. The legislation apply to many fish species including escolar, oilfish, and marlins but not garfish, sailfish and swordfish (www.cfsan.fda.gov/~comm/haccp4c1.html accessed October 2004) (FDA/CFSAN, 2001).

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15.3.2 Storage temperature EU regulation (EC 853/2004) requires `Fresh fishery products, thawed unprocessed fishery products, and cooked and chilled products from crustaceans and molluscs, must be maintained at a temperature approaching that of melting ice' (EU, 2004). With respect to practical seafood inspection this is interpreted as temperatures between 0 ëC and +2 ëC. This also applies for fishing vessels but the time to reach this temperature is not specified. Lightly preserved seafood with less than 6% salt and pH above 5, e.g. smoked and gravad products, should be kept at 5 ëC or less. Regulations in the USA are more detailed but allow fresh fish to be kept at higher temperatures. Thus, it is recommended for fresh fish (not previously frozen) that `fish should be placed in ice or in refrigerated seawater or brine at 4.4 ëC or less within 12 hours of death, or placed in refrigerated seawater or brine at 10 ëC or less within 9 hours of death'. In addition `Fish exposed to air or water temperatures above 28.3 ëC or large tuna (i.e. above 9.1 kg) that are eviscerated before on-board chilling, should be placed in ice (including packing the belly cavity of large tuna with ice) or in refrigerated seawater or brine at 4.4 ëC or less within 6 hours'. `Large tuna that are not eviscerated before onboard chilling should be chilled to an internal temperature of 10 ëC or less within 6 hours of death.' Furthermore fresh fish `should not be exposed to temperatures above 4.4 ëC for more than 4 hours, cumulatively, if any portion of that time is at temperatures above 21 ëC; or the fish should not be exposed to ambient temperatures above 4.4 ëC for more 8 hours, cumulatively, as long as no portion of that time is at temperatures above 21 ëC after chilling on board the harvest vessel'. Finally, it is mentioned that further chilling towards the freezing point is desirable to safe-guard against longer-term, low-temperature development of histamine (FDA/CFSAN, 2001).

15.4 Formation of histamine and other biogenic amines in seafoods Toxic concentrations of histamine in seafood are produced by bacteria and high concentrations of strongly histamine-producing bacteria are required. Therefore, it is possible to control histamine formation by limiting the contamination and by reducing the growth of these bacteria. 15.4.1 Histamine producing bacteria and hygiene Numerous bacteria from seafood have the ability to produce small amounts of histamine (Taylor et al., 1978; Kim et al., 2003). In contrast relatively few species are strong, or so called prolific, histamine-producers able to form more than 1000 mg of histamine/kg medium. As previously pointed out, these bacteria include both mesophilic and psychrotolerant species (Table 15.3). Mesophiles do not produce toxic concentrations of histamine below 7±10 ëC. These bacteria

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include the Gram-negative Morganella morganii, Hafnia alvei, Raoultella (Klebsiella) planticola, R. (K.) ornithinolytica, K. oxytoca, Citrobacter braaakii, C. freundii, Enterobacter aerogenes, Proteus vulgaris, Pr. mirabilis, P. damselae subsp. damselae (previously C-group bacteria or P. histaminum), Serratia fonticola and the Gram-positive Staphylococcus epidermis and Tetragenococcus muriaticus (Taylor et al., 1978; LoÂpez-Sabater et al., 1994; Kimura et al., 2001; Kim et al., 2001b, 2003; Kanki et al., 2002; Takahashi et al., 2003). Morganella psychrotolerans and Photobacterium phosphoreum are strongly histamine-producing psychrotolerant bacteria (Takahashi et al., 2003; Kanki et al., 2004; Emborg et al., 2005, 2006a; Emborg and Dalgaard, 2006; Dalgaard et al., 2006). Morganella is unique as all isolates from both species of this genus seems to be strong histamine-producers (Taylor et al., 1978; Takahashi et al., 2003; Emborg et al., 2006a). In contrast, the other species include negative or weakly histamine-producing isolates and these variants of the species can occur more frequently in seafood than the strongly histamineproducing isolates. The intestinal content of many fish contain P. phosphoreum in high concentrations, often 106±108 cfu/g. Other strongly histamine-producing bacteria including M. morganii, R. (K.) planticola and P. damselae subsp. damselae are also found in fish intestines but less it known about their typical concentrations (Omura et al., 1978; Taylor et al., 1979; Yoguchi et al., 1990; Dalgaard, 2006). Gills of different fish can also contain various strongly histamine-producing bacteria (Omura et al., 1978; Kim et al., 2001b). A high concentration of strongly histamine-producing bacteria in the intestinal content of fish corresponds to the observation that histamine formation often is most pronounced in fish flesh close to the abdominal cavity (Fig. 15.2). Consequently, efficient gutting and cleaning and removal of gills is recommended (Frank et al., 1981) but not possible in practice for smaller fish. Quantitative information about the occurrence of histamine-producing bacteria in different natural habitats, on fishing vessels, on fishing gear and in seafood processing environments is limited. Also, routes for contamination of seafood with histamine-producing bacteria are incompletely understood (Kim et al., 2001b; Staruszkiewicz et al., 2004). Clearly, prevention of contamination has not been sufficient to reduce histamine formation in seafood. Increased knowledge about routes of contamination may improve this situation. However, at present it seems more promising to reduce growth of the strongly histamineproducing bacteria in products of the relevant marine finfish. 15.4.2 Time and temperature To describe the effect of time and temperature on histamine formation in seafood the BIOCOM project has collected data from the literature (Fig. 15.4). As discussed above, HFP is almost exclusively caused by seafood with more than 500 mg of histamine/kg. This concentration of histamine can be formed at ÿ1 ëC to 5 ëC but the formation is substantially faster at higher storage

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Fig. 15.4 Effect of temperature on histamine formation in seafood. Data from 124 storage trials with naturally contaminated seafood have been compiled by the BIOCOM project. Modified from Emborg and Dalgaard (2007).

temperatures (Fig. 15.4). In fact, after processing 500 mg of histamine/kg can be formed in naturally contaminated seafood within a few hours at above 16 ëC, after about two days at 11 ëC to 15 ëC, after about three days at 6 ëC to 11 ëC and after as little as four but up to more then 25 days at ÿ1 ëC to 5 ëC (Fig. 15.4). Variability in histamine formation as shown in Fig. 15.4 most likely results from different storage temperatures, different degrees of contamination with strongly histamine-producing bacteria and different characteristics e.g. pH of the various fish. According to Fig. 15.4 high concentration of histamine in seafood and thereby HFP can be explained by exposure of the implicated products to high temperature during one or more relatively short periods of time between catch and consumption. Nevertheless, Fig. 15.4 clearly shows that toxic concentrations of histamine (above 500 mg/kg) also can be formed in naturally contaminated seafood during storage at temperature in agreement with existing regulation (2 ëC for EU and 4.4 ëC for USA). Prediction of histamine formation in seafood, as a function of time and temperature, can be used to avoid storage conditions that result in toxic products. Based on data similar to those included in Fig. 15.4, Frank et al. (1983) suggested a simple and entirely empirical mathematical model for the formation of histamine in Skipjack tuna between 70 ëF and 100 ëF (21.1±37.8 ëC) (Eqn. 15.1):

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15:1

where Time(h) is the storage time in hours and Temp is the storage temperature in ëF. The BIOCOM project found this simple model to be reasonably accurate for Skipjack tuna but histamine formation in other species was typically predicted with unacceptable precision (Table 15.6). By using the same approach as above Frank and Yoshinaga (1987) developed a model for histamine formation in Skipjack tuna between 30 ëF and 60 ëF (ÿ1.1 ëC to 15.6 ëC). The BIOCOM project also evaluated this model. Unfortunately we found the predicted histamine formation to be much lower than observed for various fish species (results not shown). Histamine in seafood is formed by specific groups of bacteria. It therefore seems logical to develop mathematical models that at the same time predict growth of these bacteria and their histamine formation. The BIOCOM project has developed such a model for growth and histamine formation by M. psychrotolerans. This model relies on the expanded logistic growth curve and a constant yield factor to relate growth and histamine formation (Fig. 15.5). The predicted histamine formation at 4.4 ëC is much faster than at 2.0 ëC and this clearly illustrates the importance of efficient chilling. The model can predict the effect of hygiene (Fig. 15.5b) as well as the effect of various time/temperature scenarios. The Food and Drug Administration in the USA indicates the safe shelf-life of fish can be as short as 5 to 7 days at 4.4 ëC due to histamine Table 15.6 Comparisons of histamine concentrations in storage trials with seafood and predictions by the model of Frank et al. (1983) Fish species

Temp. (ëC)

Time (h)

Skipjack Skipjack Skipjack Skipjack Skipjack Skipjack Skipjack Skipjack Skipjack Skipjack Mackerel

26.7 29.4 32.2 35.0 37.8 22.0 22.0 21.1 21.1 31.0 23.0

Mackerel Mackerel Sardines Sardines Anchovy

25.0 25.0 25.0 24.0 32±38

a

Histamine (mg/kg) Predicted

Observed

24 24 24 24 24 48 72 36 49 10 36

329 744 1654 3519 7201 2035 14600 368 1645 17 709

1020 1140 2480 3690 6430 3000 3500 270 1600 332 50

48 48 24 24 12

5570 5570 192 192 1750

5080 59 2350 3844 121

References

Frank et al. (1981)a Frank et al. (1981)a Frank et al. (1981)a Frank et al. (1981)a Frank et al. (1981)a Silva et al. (1998) Silva et al. (1998) Rossi et al. (2002) Rossi et al. (2002) Staruszkiewicz et al. (2004) FernaÂndez-Salguero and Mackie (1979) Okuzumi et al. (1984) Ritchie and Mackie (1979) Ababouch et al. (1991) Ababouch et al. (1996) Brillantes et al. (2002)

Histamine concentrations were measured in the anterior part of the fish.

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Fig. 15.5 Predicted growth and histamine formation by Morganella psychrotolerans. A) Growth and histamine formation at 2.0 ëC (dashed lines) and 4.4 ëC (dotted lines). B) Growth and histamine formation at 2.0 ëC with an initial concentration of the bacteria of 10 cfu/g (dashed lines) and 1000 cfu/g (solid lines).

formation (FDA/CFSAN, 2001). In fact, this corresponds to the predicted time for production of 500 mg of histamine/kg in fish that initially contains 1000 M. psychrotolerans/g. We believe predictions of the type shown in Fig. 15.5 are valuable to determine critical combinations of storage time and temperature. Such mathematical models can therefore contribute to reduce HFP. Clearly,

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development of models for other important histamine-producing bacteria, including M. morganii and P. phosphoreum, is important to improve the usefulness of the approach for management of HFP in practice. 15.4.3 Storage atmosphere, salt and pH Storage of seafood below 2 ëC or 4.4 ëC, as requested by EU and USA regulations, prevents histamine formation by mesophilic bacteria and reduces the rate of histamine formation by psychrotolerant bacteria. However, psychrotolerant bacteria can produce toxic concentrations of histamine in chilled seafood at 2 ëC to 4.4 ëC (Table 15.7, Fig. 15.5). Consequently, it is important to evaluate how storage atmosphere (packaging) and preservation with NaCl, pH adjustment or other treatments can be used to control histamine formation. This is specifically relevant for vacuum-packed, dried or smoked fish, including escolar, kahawai, mackerel and tuna, that has caused numerous incidents of HFP (Table 15.2). Modified atmosphere packaging (MAP) is used increasingly by the seafood sector and its effect on histamine formation has been studied by the BIOCOM project. As shown in Table 15.7 vacuum-packing and MAP with gas mixtures containing carbon dioxide (CO2) and nitrogen (N2) have no important inhibiting effect on histamine formation in fresh seafood. In contrast, MAP with gas mixtures containing CO2 and oxygen (O2) has provided most promising results with chilled tuna. In fact, this gas mixture prevented the formation of histamine in toxic concentrations above 500 mg/kg (Table 15.7). A pronounced inhibiting effect of oxygen on histamine formation has also been observed for the Grampositive halophilic lactic acid bacterium Tetragenococcus muriaticus (Kimura et al., 2001). High concentrations of NaCl inhibit growth of bacteria and thereby histamine formation in seafood. Clearly, the use of NaCl to control histamine formation requires firstly that histamine is not formed in the fish raw material prior to the addition of salt. Secondly, the addition of NaCl must be sufficient to reduce growth of the relevant histamine forming bacteria. With mackerel at 20 ëC, 1± 2% NaCl slightly stimulated histamine formation, whereas toxic histamine formation was delayed from one to two days by 3% NaCl and from one to four days by 4% NaCl (Yamanaka et al., 1985). For vacuum-packed cold-smoked tuna at 5 ëC, histamine formation by P. phosphoreum was prevented by 4.4% 0.8% water phase salt (WPS). M. psychrotolerans was slightly more NaCl resistant, and to prevent its histamine formation more than 5% WPS and a declared shelf-life at 5 ëC of 3±4 weeks or less has been recommended (Emborg and Dalgaard, 2006). High concentrations of histamine and other biogenic amines have been detected in fish sauce, fermented fish and salted-ripened fish. The profile of the biogenic amines in these products differs markedly from that of fresh fish (Fig. 15.3). In fact, the concentration of the sum of other biogenic amines is similar to or higher than the concentration of histamine (Emborg and Dalgaard, 2007).

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Table 15.7 Effect of atmosphere (air, vacuum and modified atmosphere packaging) on histamine formation in seafood Fish

Time and temp.

Histamine (mg/kg)

Effect of vacuum and MAP with CO2 and N2: Tuna Morganella morganii Air Tuna Morganella morganii Vacuum Garfish Natural microflora Air Garfish Natural microflora 40% CO2, 60% N2 Tuna Morganella morganii Air Tuna Morganella morganii 60% CO2, 40% N2 Mackerel Natural microflora Air Mackerel Natural microflora 80% CO2

15 d, 10 ëC 15 d, 10 ëC 12 d, 5 ëC 12 d, 5 ëC 14 d, 10 ëC 14 d, 10 ëC 2 d, 20 ëC 2 d, 20 ëC

3000 3500 1270 1610 6577 4560 3380 1420

Wei et al. (1990) Wei et al. (1990) Dalgaard et al. (2006) Dalgaard et al. (2006) Oka et al. (1993) Oka et al. (1993) Watts and Brown (1982) Watts and Brown (1982)

Effect of MAP with CO2 and O2: Tuna Natural microflora Tuna Natural microflora Tuna M. psychrotoleransa Tuna M. psychrotoleransa Tuna P. phosphoreumb Tuna P. phosphoreumb

19 19 13 13 14 14

140 35 3000 230 3500 0

LoÂpez-GaÂlvez LoÂpez-GaÂlvez Emborg et al. Emborg et al. Emborg et al. Emborg et al.

a b

Natural microflora or inoculation of product

Atmosphere

Air 40% CO2, 60% O2 Vacuum 40% CO2, 60% O2 Vacuum 40% CO2, 60% O2

Morganella (M). Photobacterium (P).

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2 ëC 2 ëC 2 ëC 2 ëC 2 ëC 2 ëC

References

et al. (1995) et al. (1995) (2005) (2005) (2005) (2005)

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Despite high concentrations of both histamine and other biogenic amines these products have rarely been reported to cause HFP (Murray et al., 1982). This is probably because they are intensely flavoured and consumed in small amounts per meal (Brillantes, 1999). Fish sauce and salted-ripened fish can be prepared with high concentrations of NaCl (~10%±~40%). They are often not chilled during storage where the concentratrion of histamine can increase. It remains unclear if this histamine formation is exclusively due to activity of histidine decarboxylase produced by bacteria prior to the mixing of fish and NaCl. Alternatively, halotolerant Gram-positive bacteria including Staphylococcus epidermis and Tetragenococcus muriaticus may produce histamine but the quantitative importance of their activity in highly salted seafood is not clear. Histamine formation in frigate tuna (Auxis thazard) was faster and more pronounced at pH 6.2 as compared to both pH 5.6 and pH 6.7 (Kimata and Kawai, 1953). In agreement with this it has been shown that P. phosphoreum produce about twice as much histamine at pH 6.1 compared to pH 6.5 although growth is practically identical at the two conditions (Dalgaard et al., 2006). Thus, lowering of pH in seafood to about 6 may contribute to increased histamine formation.

15.5 Determination of histamine and biogenic amines in seafood As already mentioned in Section 15.1 early studies found HFP to be caused by seafood that appeared normal with respect to sensory properties such as flavour, texture and colour. Later, determination of histamine in seafood by sensory evaluation has been a much debated topic. If it is assumed consumers do not intentionally eat spoiled seafood then the thousands of reported HFP cases suggests many people are unable to taste or smell even high concentrations of histamine in seafood (Table 15.1, Table 15.2). In agreement with this some laboratory studies found toxic concentration of histamine (above 500 mg/kg) in seafood that was sensorily acceptable (Fletcher et al., 1995; Hwang et al., 1995; LoÂpez-Sabater et al., 1996). On the other hand, concentrations of histamine and biogenic amines have been suggested for different indices of sensory seafood quality (Mietz and Karmas, 1977, 1978; Veciana-NogueÂs et al., 1997; Jùrgensen et al., 2000). In line with this, sensory evaluation by experts is actually used in Denmark as a screening method for histamine inspection of canned and cooked tuna. Positives and suspected samples are subsequently analyzed by HPLC. EU regulation (EC 2073/2005) indicate a HPLC method (Malle et al., 1996) for determination of histamine in seafood whereas a less instrument demanding fluometric method (AOAC Official Method 977.13) is used in several other countries including the USA. In addition, many histamine test kits are available and several perform well when compared to AOAC 977.13 (Rogers and Staruszkiewicz, 2000; Emborg and Dalgaard, 2007).

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15.6 Management of histamine formation and histamine fish poisoning Despite decades of (i) research, (ii) efforts by the seafood sector and (iii) efforts by national and international authorities HFP remains common indicating that histamine formation is not appropriately managed. Our evaluation of HFP in the present chapter suggests that the established critical limits for histamine in seafood are appropriate. The limits of 100 mg/kg (EU) or 50 mg/kg (USA) are lower than the toxic concentration and this is needed due to variability of histamine concentrations in seafood. The BIOCOM project has not found evidence to support establishment of critical limits for other biogenic amines in seafood. Firstly, it remains unclear if they potentiate the oral toxicity of histamine for humans (Section 15.3.2). Secondly, the concentration of histamine in HFP causing seafoods seems proportional to the concentration of other biogenic amines (Fig. 15.3). Therefore, critical limits for histamine may remain sufficient even if new data document a potentiating effect of other biogenic amines. It is well accepted that adequate refrigeration is the key to control histamine formation and thereby HFP. The BIOCOM project has documented the importance of the psychrotolerant bacteria with respect to HFP and with respect to histamine formation in seafood at low temperature (Table 15.3, Fig. 15.4). These data suggest fresh fish stored aerobically, vacuum-packed or in MAP with gas mixtures of CO2 and N2 should be kept at 0 ëC to prevent the formation of toxic concentration of histamine. At higher storage temperatures, such at 2 ëC and 4.4 ëC, a declared shelf-life of products must be used in addition to temperature control to prevent critical histamine formation. To help establish a realistic declared shelf-life depending on temperature variation during chilled distributions, BIOCOM suggest validated predictive models can be useful and further development of such models is needed. Chilled lightly preserved seafood (including salted and smoked products) has longer shelf-life than fresh fish and they are typically distributed at 5 ëC. Several of these products are important for HFP. The BIOCOM project suggests salt concentration, storage temperature and declared shelf-life can be used in combination to prevent critical histamine formation in lightly preserved seafood. For packed and heated seafood, contamination after processing is the key issue. BIOCOM agree with the suggestion of McLauchlin et al. (2006) that focus should be on adequate refrigeration after opening of heated packed or canned seafood.

15.7

Concluding remarks

The recent discovery of psychrotolerant bacteria, as major producers of histamine in seafood, provides new options to reduce HFP. To evaluate and exploit these options further studies are needed as indicated below:

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· Seafood implicated in incidents of HFP in different countries should be studied to evaluate the relative importance of mesophilic and psychrotolerant bacteria for histamine formation. · These studies should also evaluate the correlation between concentrations of histamine and other biogenic amines. If results from BIOCOM are confirmed then seafood inspection using HPLC analyses can be simplified and costs reduced. · To control histamine formation in chilled lean tuna MAP with CO2 and O2 is promising and this type of packaging deserves further study with different seafoods and storage temperatures. · To predict and manage histamine formation mathematical models particularly for P. phosphoreum (psychrotolerant) and M. morganii (mesophilic) deserve further development. · To prevent contamination of seafood with strongly histamine-producing bacteria more quantitative data on their occurrence in marine and seafood processing environments are required. This information is particularly needed for the newly identified species M. psychrotolerans. · To evaluate the oral toxicity of histamine, challenge studies with human volunteers are needed. Specifically, effects of histamine in combination with different biogenic amines deserve evaluation. It should be studied how these combinations influence symptoms and concentrations of histamine, histamine metabolites, tryptase and prostaglandin D2 metabolites in plasma and/or urine of volunteers. The results will help determine if potentiators of histamine toxicity and/or endogenous histamine released from mast cells can explain HFP when caused by seafood including products with low concentrations of histamine.

15.8

Sources of further information and advice

SeafoodNIC: Seafood Network Information Center Compendium of Fish and Fishery Product Processes, Hazards, and Controls. Chapter 27: Scombrotoxin (Histamine) http://seafood.ucdavis.edu/haccp/compendium/Chapt27.htm (Accessed on 14 January 2007).

15.9

References

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histamine is the causative toxin of scombroid-fish poisoning. New England Journal of Medicine 324, 716±720. MOTIL, K.J. and SCRIMSHAW, N.S. (1979) The role of exogenous histamine in scombroid poisoning. Toxicology Letters 3, 219±223. MuÈLLER, G.J., LAMPRECHT, J.H., BARNES, J.M., DEVILLIERS, R.V.P., HONETH, B.R. and HOFFMAN, B.A. (1992) Scombroid poisoning. Case series of 10 incidents involving 22 patients. South African Medical Journal 81, 427±430. MURRAY, C.K., HOBBS, G. and GILBERT, R.J. (1982) Scombrotoxin and scombrotoxin-like poisoning from canned fish. Journal of Hygiene 88, 215±220. NARAYAN, B., MIYASHITA, K. and HOSAKAWA, M. (2006) Physiological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ± A review. Food Reviews International 22, 291±307. NIVEN, C.F., JEFFREY, M.B. and CORLETT, D.A. JR. (1981) Differential plating medium for quantitative detection of histamine-producing bacteria. Applied and Environmental Microbiology 41 (1), 321±322. NMKL NO.184 (2006) Aerobic count and specific spoilage organisms in fish and fish products, pp. 1±6. Nordisk MetodikkomiteÂ for Nñringsmidler/Nordic Committee on Food Analysis. OKA, S., FUKUNAGA, K., ITO, H. and TAKAMA, K. (1993) Growth of histamine producing bacteria in fish-fillets under modified atmospheres. Bulletin of the Japanese Society of Scientific Fisheries 44, 46±54. OKUZUMI, M., YAMANAKA, H., KUBOZUKA, T., OZAKI, H. and MATSUBARA, K. (1984) Changes in numbers of histamine-forming bacteria on/in common mackerel stored at various temperatures. Bulletin of the Japanese Society of Scientific Fisheries 50, 653±657. OMURA, Y., PRICE, R.J. and OLCOTT, H.S. (1978) Histamine-forming bacteria isolated from spoiled skipjack tuna and jack mackerel. Journal of Food Science 43, 1779±1781. PAN, M. (1998) Multilevel and Multiobjective Programming Model for Hawaii Fisheries Management. University of Hawaii, Dept. of Agriculture and Resource Economics. PARSONS, M. and GANELLIN, C.R. (2006) Histamine and its receptors. Brtish Journal of Pharmacology 147, S127±S135. RITCHIE, A.H. and MACKIE, I.M. (1979) The formation of diamines and polyamines during storage of mackerel (Scomber scombrus). Torry Jubilee Publication, 489±494. ROGERS, P.L. and STARUSZKIEWICZ, W.F. (2000) Histamine Test Kit Comparison. Journal of Aquatic Food Product Technology 9, 5±17. ROSSI, S., LEE, C., ELLIS, P.C. and PIVARNIK, L.F. (2002) Biogenic amines formation in Bigeye tuna steaks and whole Skipjack tuna. Journal of Food Science 67, 2056±2060 RUSSELL, F.E. and MARETIC, Z. (1986) Scombroid poisoning ± mini review with casehistories. Toxicon 24, 967±973. SAKABE, Y. (1973) Studies on allergylike food poisoning 1. Histamine production by Proteus morganii. Journal of Nara Medical Association 24, 248±256. SANCHEZ-GUERRERO, I.M., VIDAL, J.B. and ESCUDERO, A.I. (1997) Scombroid fish poisoning: A potentially life-threatening allergic-like reaction. Journal of Allergy and Clinical Immunology 100, 433±434. SAPIN-JALOUSTRE, H. and SAPIN-JALOUSTRE, J. (1957) Une toxi-infection alimentaire peu connue: L'intoxication histamique par le thon. Le Concours Medical 79, 2705± 2708. SASAKI, D.M. (2001) Scombroid fish poisoning. A review. Communicable Disease Report November/December, 5±6.

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and LORENZ, W. (1990) Intestinal diamine oxidases and enteral-induced histaminosis: studies on three prognostic variables in an epidemiological model. Journal of Neural Transmission [Supplementum] 32, 291±314. SCOGING, A. (1998) Scombrotoxic (histamine) fish poisoning in the United Kingdom: 1987 to 1996. Communicable Disease and Public Health 1, 204±205. SILVA, C.C.G., DA PONTE, D.J.B. and ENES DAPKEVICIUS, M.L.N. (1998) Storage temperature effect on histamine formation in Big Eye tuna and Skipjack. Journal of Food Science 63, 644±647. SMART, D.R. (1992) Scombroid poisoning. The Medical Journal of Australia 157, 748± 751. STARUSZKIEWICZ, W.F., BARNETT, J.D., ROGERS, P.L., BRENNER, R.A., WONG, L.L. and COOK, J. (2004) Effect of on-board and dockside handling on the formation of biogenic amines in Mahimahi (Coryphaena hippurus), Skipjack tuna (Katsuwonus pelamis), and Yellowfin tuna (Thunnus albacares). Journal of Food Protection 67, 134±141. SU, S.C., CHOU, S.S., CHANG, P.C. and HWANG, D.F. (2000) Determination of biogenic amines in fish implicated in food poisoning by micellar electrokinetic capillary chromatography. Journal of Chromatography B: Biomedical Sciences and Applications 749, 163±169. TAKAHASHI, H., KIMURA, B., YOSHIKAWA, M. and FUJII, T. (2003) Cloning and sequencing of the histidine decarboxylase genes of gram-negative, histamine-producing bacteria and their application in detection and identification of these organism in fish. Applied and Environmental Microbiology 69, 2568±2579. TAYLOR, S., GUTHERTZ, L.S., LEATHERWOOD, M., TILLMAN, F. and LIEBER, E.R. (1978) Histamine production by food-borne bacterial species. Journal of Food Safety 1, 173± 187. TAYLOR, S.L. (1986) Histamine food poisoning: toxicological and clinical aspects. CRC Critical Reviews in Toxicology 17, 91±128. TAYLOR, S.L. and LIEBER, E.R. (1979) In vitro inhibition of rat intestinal histaminemetabolozing enzymes. Food and Cosmetics Toxicology 17, 237±240. TAYLOR, S.L. and LYONS, D.E. (1984) Toxicology of Scombroid Poisoning. In Seafood toxins. ed. Ragelis, E.P. pp. 417±430. Washington, DC: American Chemical Society. TAYLOR, S.L. and SPECKHARD, M.W. (1983) Isolation of histamine-producing bacteria from frozen tuna. Marine Fisheries Review, National Oceanic and atmospheric Administration 45, 4±6. TAYLOR, S.L., GUTHERTZ, L.S., LEATHERWOOD, M. and LIEBER, E.R. (1979) Histamine production by Klebsiella pneumoniae and an incident of scombroid fish poisoning. Applied and Environmental Microbiology 37, 274±278. TODD, E.C.D. (1997) Seafood-associated diseases and control in Canada. Revue Scientific et Technique Office International des Epizooties 16, 661±672. TSAI, Y.H., KUNG, H.F., LEE, T.M., CHEN, H.C., CHOU, S.S., WEI, C.I. and HWANG, D.F. (2005) Determination of histamine in canned mackerel implicated in a food borne poisoning. Food Control 16, 579±585. URAGODA, C.G. and KOTTEGODA, S.R. (1977) Adverse reactions to isoniazid on ingestion of fish with a high histamine content. Tubercle 58, 83±89. VAN GELDEREN, C.E.M., SAVELKOUL, T.J.F., VAN GINKEL, L.A. and VAN DOKKUM, W. (1992) The effects of histamine administered in fish samples to healthy volunteers. Clinical Toxicology 30, 585±596. VAN VEEN, A.G. and LATUASAN, H.E. (1950) Fish poisoning caused by histamine in Indonesia. Documenta Neerlandica et Indonesica de morbis tropicis 18±20. SATTER, J.

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and VIDAL-CAROU, M.C. (1997) Biogenic amines as hygienic quality indicators of tuna. Relationships with microbial counts, ATPrelated compounds, volatile amines, and organoleptic changes. Journal of Agricultural and Food Chemistry 45, 2036±2041. WATTS, D.A. and BROWN, D. (1982) Histamine formation in abusively stored Pacific mackerel: Effect of CO2-modified atmosphere. Journal of Food Science 47, 1386± 1387. WEI, C.I., CHEN, C.-M., KOBURGER, J.A., OTWELL, W.S. and MARSHALL, M.R. (1990) Bacterial growth and histamine production on vacuum packed tuna. Journal of Food Science 55, 59±63. WHO (2000) WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe. WHO Europe. WHO (2003) WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe. WHO Europe. WU, M.L., YANG, C.C., YANG, G.Y., GER, J. and DENG, J.F. (1997) Scombroid fish poisoning: An overlooked marine food poisoning. Veterinary and Human Toxicology 39, 236± 241. WU, S.F. and CHEN, W. (2003) An outbreak of scombroid fish poisoning in a kindergarten. Acto Peadiatrica Taiwanica 44, 297±299. YAMANAKA, H., ITAGAKI, K., SHIOMI, K., KIKUCHI, T. and OKUZUMI, M. (1985) Influence of the concentration of sodium chloride on the formation of histamine in the meat of mackerel. Journal of the Tokyo University of Fisheries 72, 51±56. YOGUCHI, R., OKUZUMI, M. and FUJII, T. (1990) Seasonal variation in number of halophilic histamine-forming bacteria on marine fish. Nippon Suisan Gakkaishi 56, 1473± 1479. VECIANA-NOGUEÂS, M.T., MARINEÂ-FONT, A.

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Part IV Seafood from source to consumer products

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16 Introduction to Part IV: seafood from source to consumer product J. B. Luten, Nofima, Norway

The strategic objective of the SEAFOODplus project is to reduce health problems and to increase well-being among European consumers by applying the benefits obtained through consumption of health promoting and safe seafood products of high eating quality. In order to properly address this strategic objective a consumer oriented (fork-to-fish) approach is being considered in a total chain context. While embracing the total seafood chain production, one of the strategic areas `Seafood from source to consumer products' (Fig. 16.1), which will be described in the chapters in this part of the book, the development of a number of consumer driven tailor-made, functional seafood products and processes to improve health and to ensure nutritional quality and safety in a concept of full utilisation of raw materials from aquaculture production and from traditional fisheries will also be presented. As availability of marine resources is becoming limited, a rise in total quantity of by-products, resulting from increasing aquaculture production is observed, emphasising the necessity of full utilisation of seafood by-products. Consumers are concerned about the decline in marine resources. Fishmeal, fish sauce and fish silage are traditional products in the by-products area. However, there are promising opportunities in upgrading by-products into high value-added marine ingredients with beneficial health properties. Of course there is a long way to go from the starting material, being a by-product to a final product being an ingredient with proven bioactivity. Nevertheless, at the present time mild processing techniques, such as, for example, pH shift method, appropriate enzymatic hydrolysis, ultra- and nanofiltration, offer a wide scope of opportunities for upgrading seafood byproducts into valuable ingredients for use in food or as `nutraceuticals'. Protein isolates made from seafood by-products could be re-used in seafood products improving sensory and water binding properties. Screening the

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Fig. 16.1

Seafood from source to consumer products in SEAFOODplus.

bioactivity of fractionised fish protein hydrolysates with in-vitro methods will be a first step to map potential `new' health beneficial components from seafood by-products. The experience in the production of fish hydrolysates in small and medium scale industry combined with scientific expertise in the area of separation technology of bioactive compounds are key factors for success in this context. The next step after the mapping of bioactive substances is further chromatographic isolation, purification and identification of the bioactive peptides and testing the bioactive compounds in in-vivo animal studies. All essential elements of such studies are covered in Chapter 18. There is an increase in consumers' need for convenience seafood products such as lightly preserved products and ready to eat meals. However, assurance of safety and keeping quality at a high level are important issues for these types of products because they are frequently contaminated or could contain nongrowing pathogens that start growing after transformation to a ready to use product and subsequent chilled storage. Application of preservative factors, hurdles, inhibiting the growth or causing the death of the micro-organisms will improve the microbial safety and stability of seafood. Examples of classical hurdles in seafood products, as detailed in Chapter 19, are salt, smoke, acids and fermentation. More recent in food production is the use of `ingredients' such as organic acids, bacteriocins, chitosan, etc., as well as the application of advanced decontamination techniques such as microwave, inductive heating, high pressure, pulsed light, etc. Combining various hurdles in order to establish additive antimicrobial effects and/or synergy is a new concept with promising opportunities to improve safety without compromising the sensory quality. This hurdle technology might be of particular importance for convenience seafood products based upon traditional WPNL0206

Introduction to Part IV: seafood from source to consumer product 329 products such as rehydrated salt-cured or dried cod, or for lightly preserved seafood products such as cold smoked salmon. Cross contamination with microorganisms, including pathogens, during the production of these lightly preserved seafood products is a risk factor. Prevention of the growth of micro-organisms will contribute to the safety of the final products and also to postpone spoilage. Seafood is well known for its health beneficial effects of the long chain n-3 polyunsaturated fatty acids (PUFA). Several recent reviews reach the conclusions that fish consumption decrease the risk for coronary vascular diseases (CVD) and CVD-mortality in primary prevention. Even low consumption (1±3 times/month) of fish reduces the relative risk of CVD-mortality by about 11± 17% compared to no fish consumption (He et al., 2004). Increasing fish consumption will further decrease the risk of CVD-mortality. The unsaturated nature of n-3 fatty acids makes them highly susceptible to lipid oxidation, which can lead to flavour and colour deterioration and loss of endogenous antioxidants. Oxidation will have a negative impact on consumer acceptance of seafood products. Oxidation may also involve the seafood proteins affecting their solubility, decrease gel elasticity, and affect water distribution in muscle foods, with an overall negative influence on the texture of the fish. Therefore, understanding the mechanisms and kinetics behind lipid and protein oxidation will ensure high sensory quality of fresh and frozen fish. Owing to the complexity of the various interactions, it is essential to generate knowledge about the oxidation process starting in relative simple oil-in-water model systems to finally minced fish models as described in Chapter 20. The seafood consumption among the young generation is low as explained in Parts I and II of this book, despite the awareness of the beneficial health effects. Taking away obstacles for low seafood consumption by developing tailor-made seafood products in a consumer-driven approach should be a key issue. There is a fast growing consumer market in the area of functional foods. The term functional food has been linked with definitions that vary from simple statements to more complex scientific ones. In 1999, a European Community Concerted Action on Functional Foods Science in Europe (FUFOSE) tightened the definition of functional foods. The Concerted Action came to the conclusion that food can be regarded as functional if it is satisfactorily demonstrated to affect one or more target functions in the body beyond adequate nutritional effects, in a way that is relevant either to an improved state of health and well-being and/or the reduction of risk of disease. Dietary fibres, phytosterols, carotenoids, natural antioxidants, PUFA or folic acid are widely used nowadays as functional ingredients in the design of functional foods. New functional components like antioxidant dietary fibres, taurine and anti-carcinogenic selenium compounds are emerging new ones as detailed in Chapter 17. The development of a new range of functional seafood products beyond the existing intrinsic nutritional value of seafood could provide target groups of consumers with such products. It is well established that seafood itself has a lot of functional characteristics. It contains very nutritious proteins, lipids and trace elements. But seafood lacks insoluble and non-digestible elements that are WPNL0206

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necessary for a normal function of the intestine. These dietary fibres with antioxidant properties may further solve the problem of lipid oxidation. Selenium is an essential element and it forms a part of at least eleven selenoproteins in the human body. Seafood, relatively rich in selenium, is an important source for the intake by consumers. The estimated dietary intake of selenium in Europe varies from country to country (24±110 g/day) (Scientific Committee on Food, 2000). It is argued that the current intake of selenium does not meet the socalled Population Reference Intake of 55 g Se/day in some European countries. Dietary modulation of farmed fish with selenium and in particular with anticarcinogenic Se-methyl-selenocysteine originally present in garlic and broccoli gives the opportunity to develop functional seafood with specific compounds at a natural level or if needed at enriched level reached in a natural way. The development of new food products is a risky activity. This is best exemplified by the high percentage of failure in new product development process. The level of failure is 30% of all products launched in the market and up to 75±80% of all new products ideas. One of the important critical success factors in new product development is dedication to consumer-voice. Although new technological developments are important for the production of attractive products the technology-driven development as such will not be the answer to the needs of the consumer. Therefore a consumer-driven approach involving the needs and demands of consumers should be involved. Besides taste, smell and texture many other factors play a role in consumer appreciation. These factors are related to the way consumers perceive the role of food in their life, their attitudes towards food and their food-related behaviour such as buying, preparing, and eating the food. These food-related `lifestyles' in combination with underlying needs of the consumers can be used as the basis for further development of new seafood product with appropriate properties taking into account the technological possibilities. The R&D area in SEAFOODplus `Seafood from source to consumer product', as reflected in the following chapters in this book, has focused on the upgrading by-products into high added-value marine ingredients with beneficial health properties, the hurdle technology as tool for assuring the safety and quality of convenience seafood products, the understanding of the mechanisms and kinetics behind lipid and protein oxidation of fresh and frozen fish and the development of tailor made functional seafood products in a consumer-driven approach. As a consumer-driven approach has been used, extensive integration with research reported in Part I of this book has been achieved.

16.1

References

(2004) Accumulated evidence on fish consumption and coronary heart disease mortality: a meta-analysis of cohort studies, Circulation 109: 2705±11. SCIENTIFIC COMMITTEE FOR FOOD, COMMISSION OF THE EUROPEAN COMMUNITIES (2000) Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Selenium, SCF/CS/NUT/UPPLEV/25 Final, pp. 1±18. HE K, SONG Y, DAVIGLUS M L, LIU K, VAN HORN L, DYER A R, GREENLAND P

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17 Developing functional seafood products M. Careche, Consejo Superior De Investigaciones Cientificas, Spain, J. B. Luten, Nofima, Norway, A. Kole and R. Schelvis, Wageningen University and Research Centre, The Netherlands, F. Saura-Calixto, Consejo Superior De Investigaciones Cientificas, Spain, O. E. Scholten, Wageningen University and Research Centre, The Netherlands, M. E. Diaz-Rubio, Consejo Superior De Investigaciones Cientificas, Spain, M. A. J. Toonen and E. Schram, Wageningen University and Research Centre, The Netherlands, A. J. Borderias, I. SaÂnchez-Alonso, P. Carmona and I. SaÂnchezGonzalez, Consejo Superior De Investigaciones Cientificas, Spain, T. R. Gormley, Ashtown Food Research Centre (Teagasc), Ireland, J. OehlenschlaÈger and S. Mierke-Klemeyer, Federal Research Centre for Nutrition and Food, Germany, E. O. Elvevoll, University of Tromsù, Norway, M. Leonor Nunes and N. Bandarra, IPIMAR, Portugal, I. Stoknes, Mùre Research, Norway and E.H. Larsen, Technical University of Denmark, Denmark

17.1

Introduction

European policymakers and scientists acknowledge that an increased consumption of seafood can contribute to improving the health of European citizens. One approach could be to offer new seafood products that overcome existing barriers for their consumption or increase the motivation to eat seafood. There are two trends that might have a direct impact on the development of seafood products. One of them is the increasing consumer interest in optimising their health through food, which is the basis of the exceptional development of the functional food market. The other is the growth in the consumption of convenience foods and ready-meals made from fish products that is taking place in wealthy countries (SOFIA 2004). Changes in the family structure and roles, higher consumer purchasing power, less time for preparation in the kitchen, developments

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in food science and technology, and the need for brand differentiation of the different food industries are among some of the underlying factors for this trend (SOFIA 2000). One could, for example, use capture fish for making functional, convenient seafood products. Seafood matrices such as fish fillets, pieces of fillets, minced fish, or surimi can be regarded as excellent vehicles for the incorporation of functional ingredients. However, there is an increasing number of over-exploited or fully exploited species, thus restricting those that can be used, and also necessitating a more efficient utilisation of fish and fishery products (Sakaguchi 2004). The other alternative is aquaculture production, now experiencing a very fast growth (SOFIA 2004), which provides an excellent opportunity for producing tailor-made raw materials for functional seafood products because many of the factors that determine the composition of the edible parts of fish can be controlled. However, there are only a few examples of aquaculture production of tailor-made farmed fish. Both approaches are, in principle, complementary since some of the functional ingredients can be successfully added via dietary modulation, whereas others can only be included in restructured seafood. There is clearly potential for increasing the variety of new products, which could be seen as an opportunity for the seafood industry that is looking for new markets. As a result, the development of new ingredients and food products is the current challenge for the food industry and researchers. Some functional ingredients such as dietary fibres (DF), phytosterols, carotenoids, natural antioxidants, n-3, n-6 fatty acids or folic acid are widely used in the design of functional foods and new ones are emerging. This is the case for antioxidant DF (Saura-Calixto 1998), selenium (Larsen et al. 2006) and taurine (Gormley 2006a). At present, seafood is used as a source of the components used to manufacture functional foods, such as fish oils or protein powders from by-products, although the actual production of functional seafood is limited compared to other foods. The requirements for customising food products to have the required functional and sensory characteristics include: obtaining a stable environment for the bioactive ingredient, gaining knowledge of the interactions between the ingredient and other ingredients in the vehicle matrix, maximising and maintaining the bioactive ingredients' health benefit, and achieving the desired sensory/organoleptic properties in the final product (IFT Expert Report 2005). These technological developments have to be translated into attractive products; but technology-driven development as such may not be enough for the market. Despite considerable promotional expense and effort to explain, for example, the health benefits to consumers, many food products face problems when put on the market, or even fail. Apparently they do not meet any perceived need well enough to make consumers actually buy these products. In addition, the demands from various consumer segments must be integrated with the new technological developments. Taking the above considerations into account, the objective of the work carried out in the CONSUMERPRODUCTS project was to develop functional seafood convenience products with the characteristics required by target consumer groups.

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For that reason, consumer studies supported the development of the seafood products throughout the project. This development looked at two different scenarios: (a) restructured muscle or fillets from captured species to which taurine, DF or antioxidant DF were added, and (b) aquaculture fish where selenium was incorporated via dietary modulation. Preparation of the non-commercial ingredients used, such as antioxidant DF and selenium-enriched garlic, was required prior to the development of the actual seafood products and will also be described. To complete the integrated development path from consumer input to production of products then back to the consumer again, the products were tested during industrial as well as normal household processing. The possible changes or losses of the health-promoting components present in or introduced to the seafood products, such as polyunsaturated fatty acids, selenium, taurine, or other lowmolecular weight compounds, were monitored during processing and storage in order to achieve the optimum retention of functional compounds.

17.2 Consumer studies with respect to the development of new functional seafood products The first goal in product development should be to search for market niches where consumers' needs are not met (van Kleef et al. 2005a). These studies aim to provide market-related guidelines for functional product development. Food has to taste good, but there are many more factors that play a role in final consumer satisfaction. These factors relate to the way consumers perceive the role of (sea)food in their daily lives and involve their attitudes towards food and their food-related behaviour: buying, preparing, and eating the food. Together these factors express themselves in people's food-related `lifestyles' (Brunsù and Grunert, 1995; Scholderer et al. 2004). For consumer groups with different food-related, or even more specifically, seafood-related lifestyles, different products will be needed if they are to be successful. Thus, a series of studies was planned to cover the following stages of consumer-driven product development. Firstly, sample results from a number of Dutch consumers were grouped according to their food-related lifestyles using the data from a paper-and-pencil survey and cluster analysis. Secondly, the underlying needs of the different consumer groups were identified and translated into more concrete product properties. This was done by means of group discussions and interviews with consumers. It also involved food technologists, since current technological developments in the project were taken into account. Thirdly, since product characteristics are never evaluated in isolation by consumers, combinations of properties were tested within more complete product concepts by means of a conjoint analysis study. For example, healthy ingredients can be positive factor, but in a snack product that is fulfilling a `need' to enjoy oneself, they might get a negative `responsible' connotation. The information derived from these studies was integrated with the technology developments. This integration led to several product concepts for different consumer

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segments, giving rise to new designs. After all, final consumer acceptance to a large extent depends on design factors (e.g. Bech et al. 1997). 17.2.1 Consumer attitudes In 2004, a large survey was performed among 973 members of the home-based consumer panel TasteNet, run by several institutes for applied research in The Netherlands. The questionnaire contained a broad range of questions on foodrelated attitudes and behaviour including ones on Food-Related Lifestyle (FRL) (Brunsù and Grunert 1995). The questionnaire contained sections on the following subtopics: shopping behaviour (e.g., importance of product information, enjoyment of shopping, price consciousness); special interests (e.g., health, novelty, organic products); meal preparation attitudes (e.g., involvement with cooking, convenience, interest in new ways of cooking); desired consequences (e.g., social relationships, self-fulfilment). The survey results were used to identify different consumer types so that it would then be possible to deduce the needs and specific attitudes of each of the consumer groups towards food, and fish products in particular. Six groups were identified that more or less confirmed earlier findings for food-related lifestyle segmentation throughout Europe: Involved consumers, Hedonistic consumers, Naturalistic consumers, Social consumers, Conservative consumers, Uninvolved consumers. From these groups, two (`Conservative' and `Involved') were selected as target groups for further stages in seafood product development, with a focus on `healthy' and `convenient' products. 17.2.2 Specification of consumer needs The two groups were involved in two sessions of focused group discussions. The primary goal was to translate their general food-related attitudes, in particular towards convenience and health, into more concrete product attributes that could be used interactively with technological developments for new seafood products. Since the project's technological developments are focused on the increase of functional ingredients, especially enhanced levels of selenium in farmed fish through dietary modulation and the addition of DF to restructured seafood products, the second focus group session centred around these topics. The `Conservative' or practical consumers form one of the largest segments (20%); they do not consume a lot of fish and appear family-oriented. `Convenience' for them means products that are easy and fast to prepare, and appeal to the whole household. `Healthy' to them primarily means meeting the required daily intake of nutrients for each member of the household. They are not very interested in innovative products. The `Involved' consumers are much more interested in new products and are much more quality-oriented. They like to experiment and to spend time in the kitchen with high-quality ingredients. To them, convenience means that it becomes easier for them to perform more complex food processing, something they are willing to do, which the Practical

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people may not be. Healthy is interpreted in a `good for you' sense and is related to fresh, quality products, rather than being interpreted in terms of nutritional value. The focus of convenience for the first group would be on reducing time and effort, for the second group on adding quality. `Naturalness' appeared to be a central theme with respect to seafood products for both segments. A perceived lack of naturalness makes the conservative group especially wary of functional ingredients, such as DF or extra selenium. This has led to the suggestion that in the context of seafood, using seaweed as a natural source for antioxidant DF will lead to better consumer acceptance compared to other non-marine sources. The interest in functional ingredients seems to be partly dependent on the consumers' health-consciousness in general, and the extent to which they value health as an aspect of seafood products. On average, the involved consumers seem to value vitamins, minerals, DF, and healthiness of their seafood products slightly more highly than do the more conservative consumers. Both groups find these functional ingredients most appropriate for their weekly meals. 17.2.3 Conceptual product testing So far the results point toward differences between desired product properties such as health, convenience, and naturalness, and an interest in particular functional ingredients individually. However, these aspects never appear separately when being applied in products. The next stage in developing consumer-driven product concepts is combining these properties to study how consumers perceive their interaction in complex products. A concept test was performed with 63 combinations of nine different seafood products with seven different functional ingredient claims (no claim, DF added, antioxidant DF added, n-3 and n-6 fatty acids added, naturally enhanced levels of n-3 and n-6 fatty acids, selenium added, naturally enhanced levels of selenium). The nine products were selected to be different with respect to convenience (three levels: high, medium, low) and naturalness (also three levels: high, medium, low). `Convenience' was determined according to a matrix assuming that overall convenience consists of the (expected) mental effort, physical effort, and time spent on consecutive stages of product handling (e.g. buying, preparation, eating, cleaning up, etc.) (Scholderer and Grunert 2005). `Naturalness' was defined so that the least `natural' product would be the product with the highest level of processing and where no original fish parts would be visible. Thus a full factorial design of 3 3 7 mini-concepts was set up, each mini-concept consisting of a description of the seafood product together with a very brief description of the health function the functional ingredient might serve (Durgee et al. 1998; van Kleef et al. 2005b). The 63 concepts were judged by 283 consumers (that had previously also filled in the food-related lifestyle questionnaire) for their `willingness to try' and for `perceived convenience' of the product. Conjoint analysis revealed two to three different consumer groups having a particular preference for added

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Fig. 17.1 Consumer segments with similar patterns of preference for the functional ingredient claims. Health 1 = no claim, Health 2 = `digestive fibre added', Health 3 = `Antioxidant fibre added', Health 4 = `Selenium added', Health 5 = `Naturally enhanced levels of Selenium', Health 6 = `Omega-3 and 6 fatty acids added', Health 7 = `Naturally enhanced levels of Omega-3 and 6 fatty acids'.

selenium and DF, but not for n-3 and n-6 fatty acids, or naturally high levels of selenium (see Fig. 17.1). These groups did not match the conservative or involved food-related lifestyle segments that were chosen earlier. The result that consumers showing a particular interest in functional ingredients prefer the addition of selenium instead of naturally enhanced levels or extra n-3 fatty acids seems counterintuitive. One explanation might be that seafood is considered to be high in n-3 fatty acids, whereas it is not clear to consumers what other sources

Fig. 17.2 Interaction between `naturalness' of the products and perceived `Convenience' level. Dependent variable is `willingness to try' the product.

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of selenium might be available in their diet. The preference for its addition over naturally enhanced levels might come from the assumption that `addition' is a more controlled way of supplementation. The test also revealed that for the most natural product category (least processed, i.e. raw fish varieties), increasing convenience leads to an increased willingness to try the product (see Fig. 17.2). In the least natural (i.e. most processed) product category, increased convenience leads to less appreciation of the products. These effects were similar for both the involved and the conservative consumers. One of the most interesting conclusions is that, apparently, an increase in convenience, which usually involves processing, is considered positive, despite a reduction in naturalness, for the most natural products. For the least natural products, enhanced convenience is not positive. Furthermore, there is definitely a substantial group of consumers that are positive about functional seafood products, although their feelings about their willingness to buy such products seem rather complex. This calls for further research.

17.3 Novel ingredients for incorporation into functional restructured/fillet-based seafood The production of non-commercial ingredients, and product development began at the same time as the consumer studies which, during the project, might reorient some parts of the product development. Results from the previous section show that in the right context, balanced between convenience and preserving the natural character of the product, the addition of functional ingredients to seafood products could be a success. This supported the initial choices for technological development. Four possible ingredients were selected: DF, taurine, antioxidant DF, and selenium. DF and antioxidant DF could only be incorporated into restructured products. Taurine was injected into fish fillets, whereas organoselenium compounds were added to fish feed and used in aquaculture production. The role of DF in nutrition and health is now well established (Anderson et al. 1990; Jenkins et al. 1995; Hill and FernaÂndez 1990). Knowledge of the beneficial effects of high DF diets on gastrointestinal health, the prevention of cardiovascular disease and some types of cancer has led to the development of a large and profitable market for DF-rich products. In fact, among the different functional ingredients used in food products, DF is the most common, representing over 50% of the total number of functional ingredients on the market. The World Health Organisation and other scientific organisations recommend an increase of at least 50% in the consumption of DF in European countries. Fibres from cereals like bran and betaglucans, soya, fruit fibre, galactommanans, and psyllium are most commonly used. The clinical use of taurine in relation to cardiovascular health has been demonstrated by a number of researchers (Azuma et al. 1992; Franconi et al. 1995; Hayes et al. 1989). It has been shown that taurine modifies endothelial dysfunction in young smokers and restores normal flow-mediated dilation in the

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brachial artery (Fennessy et al. 2003). Beneficial effects of dietary taurine on those at risk of cardiovascular diseases have been observed in both animal and human studies (Militante and Lombardini 2002; Yamori et al. 2001, 2004). Both dietary fibre and taurine are available commercially. The other ingredients used, such as antioxidant dietary fibres and organo selenium material, were produced for this project and their properties and compositional characteristics will be described in the next subsections. 17.3.1 Antioxidant dietary fibre (DF) DF was first defined as non-starch polysaccharides and lignin which are resistant to hydrolysis by the human digestive enzymes (Trowell 1976). On the basis of this definition, the analytical methodology to determine DF in foods was developed and a large number of clinical and epidemiological studies were performed. However, knowledge has increased since the first definition of DF and the general tendency among nutritionists nowadays is to extend the concept of DF to include other compounds of proven resistance to the action of digestive enzymes, such as resistant starch, indigestible protein and certain polyphenols. The main sources of DF are those derived from cereals, legumes, vegetables, fruits, and from marine origins (seaweed). Some fibres, from fruit for example, generally have better nutritional qualities than others, such as those derived from legumes or cereals, because of the presence of significant amounts of some associated bioactive compounds. Most DFs available on the market do not exhibit any antioxidant capacity (Larrauri et al. 1997). Non-commercial DFs obtained from tropical fruits such as mangoes, pineapples, limes and grapes or from seaweed, such as fucus vesiculosus, have shown a remarkably high antioxidant capacity, which can be explained by the presence in the fibres of significant amounts of phenolic compounds. Antioxidant activities of 280 and 120 mol eq Trolox/g were found in white grape and Fucus seaweed fibres respectively (ABTS method). On the basis of this, a new type of natural product, named antioxidant DF, has been defined (Saura-Calixto 1998), combining in a single material the physiological effects of both DF and antioxidants. To clarify the concept of antioxidant DF, it was proposed that a vegetable material should fulfil the following requirements to be considered as antioxidant DF (Saura-Calixto 1998): · DF content should be higher than 50%. · One gramme of antioxidant DF should have the capacity for inhibiting lipid oxidation equivalent to, at least, 200 mg of vitamin E, and a free radical scavenging capacity equivalent to, at least, 50 mg of vitamin E. · The antioxidant capacity must be an intrinsic property derived from natural constituents of the material, not by added antioxidants nor by constituents released by previous chemical or enzymatic treatments. The antioxidant DFs produced initially were from different grape sources coming from waste fractions after wine production. Grape antioxidant DF used

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Composition of antioxidant dietary fibre from grape

Components

% dry matter

Total dietary fibre Total phenolics Protein Lipids Ash

76.27 6.28 7.31 5.50 24.48

339

1.33 0.32 0.21 0.06 0.30

in the present research fulfils these requirements (see Tables 17.1 and 17.2). Moreover, it has been shown that the utilisation of antioxidant DF from grapes as a source of antioxidant DF lowers serum total cholesterol and LDL cholesterol concentrations in hypercholesterolemic rats (MartõÂn-CarroÂn et al. 2000). Believing that consumer studies suggested a higher acceptance of antioxidant DF from marine origin in seafood products, this ingredient was then prepared from fucus vesiculosus. Seaweeds are rich in polysaccharides, minerals, vitamins, sterols, and phenolic compounds (Truus et al. 2004) such as the phlorotannins in fucus vesiculosus. A high content of polysaccharides in the algal cell wall contributes to their high DF content. Different types and amounts of DF exist depending on the seaweed phyla. In brown seaweeds (phaeophyta, as fucus vesiculosus), soluble fibres are alginates, in laminarans and in fucus vesiculosus, fucans (fucoidan) are the main polysaccharides found. Fucoidan (fucan sulfates) are unique polysaccharides not occurring in any other seaweed or land plant other than fucus vesiculosus. Several biological activities have been attributed to the fucoidans: anticoagulant, antithrombotic, anti-inflammatory, antitumoral, contraceptive and antiviral (Durig et al. 2003). They have been described as inhibitors of the replication of several enveloped viruses such as the herpes simplex virus and human cytomegalovirus. Tables 17.3 and 17.4 show that these DFs also fulfil the compositional requirements for being considered as antioxidant DFs.

Table 17.2

Antioxidant activity of antioxidant dietary fibre from grape

Method FRAPa ABTSb ORACc DPPH (EC 50%)d

372.91 34.81 Trolox mol Eq/g dry matter 284.32 24.27 Trolox mol Eq/g dry matter 418.06 26.58 Trolox mol Eq/g dry matter 1.75 0.06 g dry matter/g DPPH

a

Ferric reducing antioxidant power method (Pulido et al. 2000) ABTS radical cation decoloration assay (Re et al. 1999) c Oxygen radical absorption capacity (ORAC) method (Cao et al. 1993) d DPPH method (Brand-Williams et al. 1995). The parameter EC 50% reflects the depletion of DPPH* free radical to 50% b

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Composition of antioxidant dietary fibre from fucus vesiculosus

Components Total dietary fibre Total phenolics Total carotenoids Protein Lipids Ash Ca Mg Zn Na K Cu Fe Mn

Table 17.4

61.22 1.09% dry matter 5.367 0.001% dry matter 0.052 0.004% dry matter 9.62 0.11% dry matter 2.77 0.09% dry matter 24.48 0.03% dry matter 934.58 12 mg/100 g dry matter 668.53 4 mg/100 g dry matter 3.62 0.04 mg/100 g dry matter 2101.55 68 mg/100 g dry matter 3170.13 100 mg/100 g dry matter 0.34 0.005 mg/100g dry matter 9.24 0.57 mg/100 g dry matter 11.89 0.25 mg/100 g dry matter

Antioxidant activity of antioxidant dietary fibre from fucus vesiculosus

Method FRAPa ABTSb ORACc DPPH (EC 50%)d

71.62 2.23 Trolox mol Eq/g dry matter 119.66 2.14 Trolox mol Eq/g dry matter 565.88 31.84 Trolox mol Eq/g dry matter 3,51 0.10 g dry matter/g DPPH

a

Ferric reducing antioxidant power method (Pulido et al. 2000) ABTS radical cation decoloration assay (Re et al. 1999) Oxygen radical absorption capacity (ORAC) method (Cao et al. 1993) d DPPH method (Brand-Williams et al. 1995). The parameter EC 50% reflects the depletion of DPPH* free radical to 50% b c

17.3.2 Beneficial health effects of selenium and selenium in garlic and other crops Selenium is an essential micro-nutrient and occurs predominantly in free or protein bound form in humans and animals. Concentrations of selenium in crops are highly correlated to the selenate concentration in soils, which differs between regions. As a result, selenium concentrations in humans differ greatly. Its deficiency results in disease, whereas high amounts are toxic. Dietary selenium is essential for human health (Rayman 2000; Birringer et al. 2002) and several studies report the anti-carcinogenic effects of dietary selenium in humans at levels above the dietary requirement (Ip 1998). Animal studies with Se-enriched garlic or yeast have shown that it has a protective effect against cancer (Ip et al. 2000). Comparable results have been obtained with Se-enriched broccoli (Finley 2003). Results of studies on the relationship between Se-uptake

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and colon cancer were not always unambiguous (Duffield-Lillico et al. 2004). Negative correlations were found between Se-concentrations in blood and the risk of colon cancer (Jacobs et al. 2004). Results of a large intervention study carried out over a period of 10 years, with volunteers who received either a placebo or 200 g Se per day, demonstrated a decrease in the occurrence of and mortality by prostate cancer (70%), lung cancer (50%) and colon cancer (50%) (Clark et al. 1996; Finley 2003). Currently, another large intervention study is underway (Lippman et al. 2005; Duffield-Lillico et al. 2004). Cancer prevention is not directly associated with either seleno-protein production or with the selenium-concentration in tissues (Ip et al. 2000). The availability of certain components, such as selenate, Se-methyl-selenocysteine and glutamyl-Se-methylselenocysteine, that can easily be taken up by the stomach and gut is of great importance. Although not all selenium compounds are stable, it is known that intestinal Se uptake from fish is not influenced by cooking (Fox et al. 2004). Selenium is present in all kind of foods and supplements but fish, cereals, mushrooms and yeast, in particular, are rich in selenium. Determination of total selenium is relatively easy, but estimating the content of various compounds is very complex (Larsen et al. 2006). In Se-enriched garlic, most selenium is present in the form of gamma-glutamyl-Se-methylselenocysteine (Ip et al. 2000; Larsen et al. 2006). In other studies Se-methyl-selenocysteine was the predominant form found in garlic (Cai et al. 1995; Bird et al. 1997). In Se-enriched yeast, selenium was mainly present in the form of selenomethionine. A comprehensive review about selenium speciation from food source to metabolites has recently been published by Dumont et al. (2006). The total amount of selenium in crops either for direct consumption or for use as ingredients in other products can be increased by fertilising the soil with selenate. The latter approach was used in this project. Se-enriched garlic from different accessions was produced. No problems were encountered on a larger scale production of Se-enriched garlic to obtain Se-enriched garlic powder for fish feed experiments. It was shown that the selenium in the garlic was present mostly in the form of glutamyl Se- methyl selenocysteine (Larsen et al. 2006). The enriched garlic powders were incorporated into the aquaculture feed for farming experiments with African catfish (Section 17.5). In the first experiment it was demonstrated that fertilisation with selenate strongly increased the selenium content in garlic (Larsen et al. 2006). Parallel analysis of the sample extracts produced by cation exchange and reversed-phase HPLC with ICP-MS detection showed that -glutamyl-Se-methyl-selenocysteine amounted to 2/3, whereas methylselenocysteine, selenomethionine and selenate each amounted to a few per cent of the total selenium in all garlic samples. In a second experiment, different garlic accessions were included and large differences in Se accumulation between a genebank accession and a commercially available cultivar were observed (see Table 17.5).

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Table 17.5 Quantitative results for selenium species in lyophilised garlic samples originating from two garlic accessions: (1) a genebank accession, (2) cultivar `American Early' using cation or anion exchange HPLC±ICP±MS Selenium species concentration/g Se gÿ1 dry mass (protease extraction)* Garlic

1 2

Total Se

1394 585

-GLUMeSeCys

MeSeCys

SeMet

Selenite

Selenate

g/g

% of total

g/g

% of total

g/g

% of total

g/g

% of total

g/g

% of total

883 357

69 69

55 13

4 3

66 32

5 6

5.2 2.3

0.4 0.4

147.8 52.1

11.5 10.1

* -GLU-Se-MeSeCys = -glutamyl-Se-methyl-selenocysteine, MeSeCys = methylselenocysteine, SeMet = selenomethionine.

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17.4 Development of functional restructured seafood products. Addition of dietary fibre and antioxidant dietary fibre to minced fish muscle and surimi gel-based products Restructured seafood products are made from minced and/or chopped muscle with or without additional ingredients, to which a new appearance and texture are given. Seafood products based on surimi, a concentrate of stabilised myofibrillar protein made from fish muscle, have the advantage that they can resemble other value-added seafood products, with a positive image for the consumer. The reason for restructuring fish muscle has usually been the use of alternative resources either from under-exploited seafood species or from wastes of more noble ones. The fact that the muscle is broken at different degrees of integrity and then reshaped gives an excellent opportunity for including some functional ingredients in the matrix that otherwise would not be possible. This is the case for dietary or antioxidant dietary fibres. Most of the DFs used in seafood products are soluble and include carrageenan, alginates, garrofin, guar, xanthan, or pectins (Park 2005). These ingredients are selected for their technological properties, such as a high water binding, emulsifying, thickening or gel-forming ability. On the other hand, there is very limited experience in using insoluble DFs in seafood products (Yoon and Lee 1990; Ang and Miller 1991), and none on the use of antioxidant dietary fibres. Thus the technological characteristics of formulations made with these ingredients, and their interactions with the seafood matrix, were explored in this research. 17.4.1 Technological uses of dietary fibre in seafood products Two types of DF with different origins and characteristics were mainly used: wheat DF (VitacelÕ) and grape DF concentrates. Following recommendations from the results of the consumer studies, the effect of seaweed antioxidant dietary fibre was incorporated. Wheat dietary fibre Together with its physiological benefits, wheat dietary fibre, which is insoluble and composed mainly of cellulose and hemicellulose, has been reported to have advantageous technological properties such as high water and fat binding capacity (Guillon et al. 2000) and it can be considered as a good ingredient for achieving high yields and reduced costs for the industry. Different formulations with wheat DF have been studied: minced muscle from lean (hake), semi-fatty (horse mackerel) and fatty (salmon) species, surimi gels made from Alaska pollack and giant squid surimi gels, which could potentially be caught in large numbers. These matrices covered the use of different types of restructured seafood (surimi gels vs non-gelled products), underutilised species (horse mackerel, giant squid), the by-products from regular processing of high-value species (minced hake), as well as fish muscle and surimi obtained commercially (salmon, Alaska pollack). Some of these formulations have been

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Fig. 17.3

Schematic presentation of the development of functional restructured based seafood products with (anti-oxidant) dietary fibres.

included into product concepts (minced fillet and shredded surimi products) and produced in large amounts for further consumer tests (see Fig. 17.3). For surimi gels, the study with wheat DF (3 and 6%) was performed at constant moisture. No differences in colour were observed among the samples. Some of the technological properties, such as expressible moisture during centrifugation and hardness were lower than the controls (SaÂnchez-Alonso et al. 2006a, 2007a), and dependent on the DF concentration. Sensory analysis showed that panellists could distinguish between gels that contained DF and those that did not, but in all cases they were considered as acceptable formulations. In the case of surimi, it would be advantageous to modify the texture to some degree to reduce the rubbery feel of gel seafood products. For minced fish muscle, up to 6% of the fish muscle was replaced by wheat DF, also in conditions of constant moisture. Virtually no effect on appearance was observed. The main technological advantage of this type of DF is its water holding capacity. It is possible to add more water to restructured products, which will be more efficiently bound, even after cooking. The minced products with added DF were not significantly different in terms of shear strength when tested using the Kramer press cell. Likewise, the use of maximum force under compression at 25% did not uncover any significant differences among the formulations. Sensory analysis showed differences in the samples with and without added DF. The samples with 3% of DF were all accepted, while the samples with 6% DF added were rated worse (SaÂnchez-Alonso et al. 2007c), primarily

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due to a sensation of dryness. This could be overcome by combining this fibre with other ingredients such as carrageenan. Grape antioxidant dietary fibre Grape DF concentrates produced from various white or red grape by-products (skin, seeds or pomace) were introduced as a source of antioxidant DF into restructured products made from horse mackerel (Trachurus trachurus) (SaÂnchez-Alonso and BorderõÂas 2006; SaÂnchez-Alonso et al. 2006b, 2007b, 2007d). Antioxidant DF concentrates from either white or red grapes added to minced fish muscle in proportions of 2 or 4%, significantly increased the water retained during frozen storage after a physical stress, in direct proportion to the amount of added fibre. Also, the addition of these DFs reduced the thawing drip after frozen storage and significantly increased the cooking yield due to the reduction of drip losses during heating. The addition of grape DF concentrates significantly affects the textural characteristics of the samples, in which hardness and cohesiveness decreased with increasing amounts of DF. A major advantage of these DFs, both from white and red grapes, is that they possess notable antioxidant properties and are able to significantly inhibit the development of lipid oxidation in frozen minced fish muscle stored at ÿ20 ëC for up to 180 days. Sensory analysis indicated that samples of horse mackerel minced muscle with 2% of grape DF added were very acceptable, but when 4% of grape DF was added, samples were less acceptable due to changes in texture although no changes in taste were evident. In order to obtain final products with characteristics close to the initial control, these formulations have been optimised in terms of the addition of ingredients such as carrageenans that help achieve the desired characteristics. Seaweed antioxidant dietary fibre According to the consumer studies, the idea of introducing DF of marine origin into functional seafood can be of interest to some market niches, and the different possibilities for its incorporation are currently being explored. Previous work with seaweed antioxidant DF extracted from fucus vesiculosus seaweed showed that this ingredient gave the products a dark green colour and strong flavour. The challenge for the technologists is to make this fibre blander in terms of taste and colour. 17.4.2 Structural changes in surimi gels and minced muscle with added dietary fibre A general description of the protein-protein and water-protein interactions involved during the gelling process for surimi has been established in the literature, which to some extent has outlined the order of interaction of the proteins (Lanier et al. 2005). However, there is still some uncertainty about the precise mechanism responsible for gel formation, in part due to less work having been done on analysing in situ the structural changes that occur in the surimi

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proteins, and on the structure and mobility of water during the gelation process. Therefore the structural and rheological changes of proteins and water during surimi gelation were studied by Raman spectroscopy and stress relaxation tests, and a method for H2O/D2O exchange was developed to study the structural organisation of water in surimi products (SaÂnchez-GonzaÂlez et al. 2006, 2008). This method could also be used for the analysis of formulations with ingredients that, as expected for DF, can modify the water-protein interactions and/or the three-dimensional structure of gel networks. Structure changes with wheat dietary fibre An initial set of experiments with functional ingredients, previously checked for their technological properties as described in Section 17.4.1, was performed with a wheat DF (VitacelÕ). The first question to be answered was whether there were any structural changes in the seafood gel matrix due to the addition of fibre and, if so, at what level those changes took place. Light microscopy showed that wheat DF caused some discontinuity of the surimi matrix (SaÂnchez-Alonso et al. 2006). The rheological parameters from using the stress relaxation test showed distinct features depending on whether the DF was substituting for water or protein in the formulation. When protein was substituted by fibre at a constant moisture, which is the condition typically used for a new product, the observed changes in the rheological parameters were interpreted as a mixture of: (a) a lower protein density due to less protein concentration, (b) less homogeneity of the threedimensional network, and (c) the effect of the DF itself reinforcing the matrix and increasing its water holding ability (SaÂnchez-GonzaÂlez et al. 2005a). The wheat DF, in turn, could cause some structural alterations and local dehydration of the proteins. The study of the effect of the wheat DF on the protein and water, monitored by Raman spectroscopy (SaÂnchez-GonzaÂlez et al. 2008), showed that the wheat DF actually provokes an increase in -sheet proportion, which was explained in terms of a higher aggregation of the proteins, in going from the surimi paste to the final gel. There was a marked increase in the C-H stretching band of the surimi paste in the presence of wheat DF, which can be interpreted in terms of a lower hydrophobic interaction. Since the latter plays an important role in the formation of the gel protein network, these results would partly account for an impaired network due to the presence of wheat DF. Moreover, analysis of the fibre suggested that water transfer from protein to WDF occurs in surimi gels (SaÂnchez-GonzaÂlez et al. 2008), with more bound water in the formulations with WDF. This was confirmed in a separate series of H2O/D2O exchange experiments. The effect of wheat DF on the structure of minced fish products was studied by rheology and Raman spectroscopy. The rheological parameters, using the stress relaxation test, also showed different patterns depending on whether the DF was a substitute for water or protein. Generally speaking, an increase was observed in the `solidity' of the system and in the elastic and viscous elements when water was being substituted by fibre, and slightly lower values for these parameters were observed when fish muscle was being substituted by the fibre.

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Raman spectroscopy showed an increase in beta sheet proportion of the proteins in cooked mince samples as compared to the controls containing no fibre, thus suggesting a similar effect of the DF in minced products. Structure changes with grape antioxidant dietary fibre The experiments described above were considered necessary for the analysis of antioxidant DF concentrates with a more complex composition. Changes in secondary protein structures of the gels containing red and white grape fibres were analysed by Raman spectroscopy, and results showed that the formulations with grape fibre did not have significantly different proportions of secondary structures, as compared to the controls containing no fibre. The effects of adding grape antioxidant DF concentrates to minced fish muscle on the rheological and structural parameters indicate that grape antioxidant DF did not affect the secondary protein structure in either frozen/thawed or cooked mince (SaÂnchezGonzaÂlez et al. 2005b, 2005c). This may be a consequence of the presence of lignin in the antioxidant dietary fibre, which surrounds the cellulose fibres (Besombes and Mazeau 2005). The effect of the aromatic groups of the lignin on the hydration of cellulose and its relation to the secondary structure of the proteins are currently being investigated. 17.4.3 Addition of taurine to fish muscle: health component from seafoods Fish is a good taurine source (Kim et al. 1999). This was confirmed by analysis of the taurine content in samples from four species purchased in supermarkets. The average taurine content in the muscle tissue was 1.26 g/kg for plaice, 0.93 g/ kg for cod, 0.69 g/kg for mackerel and 0.53 g/kg for farmed salmon. Values for spot samples of a number of other species were albacore tuna 1.55 g/kg, ray wing 1.28 g/kg, wild salmon 0.53 g/kg, siki shark 0.44 g/kg, whiting 0.35 g/kg, Greenland halibut 0.28 g/kg, roundnose grenadier 0.06 g/kg and Baird's smoothhead 0.05 g/kg. Tests with plaice fillets indicated that chilled storage, with and without modified atmosphere packaging, for up to 10 days did not influence taurine content. This finding was unexpected as a loss of taurine during storage was anticipated (Gormley et al. 2006). Taurine was added to tuna portions using a vacuum tumbling procedure (taurine/phosphate solution) with a target taurine content in the fish flesh of circa 1% on a fresh weight basis. This level was achieved in the fish thus creating a taurine-rich functional fish (Gormley 2006a, 2006b). Sensory tests indicated that the taurine/phosphate solution did not affect the sensory properties of the fish. When the taurine-enriched tuna was processed by freezing, chilling, freeze-chilling and sous vide cooking, there was a good retention of the added taurine. Cooking tests on the frozen samples indicated that grilling resulted in the highest level of taurine retention followed by microwaving and steaming; the latter having a leaching effect on the taurine (Gormley et al. 2006). A brine injector was also used to achieve a taurine content of circa 1% in the flesh of farmed salmon sides. However, there was a wide variation in taurine

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content for different parts of each side and there was no pattern in the data, e.g. no head to tail or similar effect. These findings are of significance to both processors and consumers. The former have the potential to produce taurine-rich seafood products, while attractive products can be provided to the latter, who are becoming increasingly aware of functional foods. 17.4.4 Underutilised fish species as raw materials for new products During the last few years, there has been an increasing interest in exploring underutilised fish species that can replace or supplement the available commercially exploited species. The innovative seafood products made from captured fish in this project are mostly being developed using underutilised species (horse mackerel, giant squid surimi) or by-products from the processing of more noble ones (minced hake). A selection of further underutilised fish species was made as possible raw materials for further product development. It is important that the new species can compete with the traditional species with regard to both sensory properties and nutritional values. The protein content and water holding capacity of the proteins are important factors, as a high protein content and proteins with a good water-holding capacity often improve the texture of the fish flesh. Silver smelt/greater argentine (Argentina silus), northern wolf fish (Anarhichas denticulatus) and polar cod (Boregadus saida) were chosen as species of interest for further investigation with regard to raw material properties and chemical composition, and some physical attributes were analysed. The qualities of silver smelt have been tested by three European research centres (Stoknes et al. 2006). The proximate composition, water holding capacity, cook loss and colour of the fish flesh were examined. Silver smelt had high protein (18.7%) and low water content (79.1%). It is a lean species with a fat content around 1%. The water-holding capacity of frozen and thawed fish flesh (78.8%) was found to be very good compared to the reference species cod (Gadus morhua) (66.3%). The sensory scores were high for steamed fillets, fishcakes and breaded nuggets made from both these species. Silver smelt steamed fillets received 9 preferences compared to 3 for steamed cod in a 12person paired comparison taste panel. In the case of fishcakes, those from silver smelt were ranked 1st (rank sum 20; low values best) in comparison with cod (2nd; rank sum 31), white pollock (3rd; rank sum 36), ling (4th; rank sum 41) and a commercial fish cake (5th; rank sum 52) by a 12-taster panel who were asked to rank the samples from 1st to 5th for preference. In the case of breaded nuggets, silver smelt samples received 13 preferences compared to 2 for a commercially produced nugget (fish species not stated on the pack) in a 15person paired comparison taste panel. The inorganic contaminants were also analysed and small amounts of the heavy metals cadmium and lead were found (0.75 and 15.8 g/kg respectively). Considering the raw material properties, silver smelt seems to be suitable as a high-quality raw material for the consumer-driven development of new tailor-made seafood products.

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Aquaculture production of functional seafood

Many factors that determine the composition of the edible portions of fish can be controlled by aquaculture production. These include species, sex, degree of sexual maturation, size and diet (Heidmann Soccol and Oetterer 2003). Current aquaculture production as a basis for functional seafoods is limited to marine algae in Japan (Murata and Nakazoe 2001), and salmon, which are functional seafood by nature because of their high levels polyunsaturated fatty acids. Polyunsaturated fatty acids are probably the most investigated functional components from animal origins. Seafood is further used as a source of functional components for use in manufacturing functional foods. Examples are fish oils (Heidmann Soccol and Oetterer 2003) and protein powders from herring byproducts (Sathivel et al. 2004). But farmed fish, which might be regarded as a functional food for its elevated levels of functional components other than polyunsaturated fatty acids, is not at present commercially available. Clearly the large potential for the aquaculture production of raw material for functional seafood is underutilised. 17.5.1 Tailor-made African catfish enriched in selenium As a first step, the aim was to investigate the possibilities for tailor-made farming of fish with enriched levels of functional selenium and create a vehicle for the incorporation of organic selenium compounds originating from garlic (see Fig. 17.4). African catfish (Clarias gariepinus) was used as a model species. Seleniumenriched garlic was included in their diet as the source of functional selenium. The high selenium levels in enriched garlic make direct human consumption of the garlic impossible. A vehicle is required to incorporate the organo-selenium in human diets. Incorporation of selenium-rich garlic in aquaculture feeds and

Fig. 17.4 Schematic presentation of the development and testing (sensorial, speciation, nutrient losses) of Se-enrichment of African catfish with feed containing Se-enriched garlic.

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subsequent production of selenium-rich farmed fish through dietary modulation is a potential solution. In the first farming trial, a dose response relation and the retention of the organic selenium compounds in the fish were established. In addition, growth performance was assessed in relation to the garlic and selenium content of the diet. It was found that the selenium level in the fish fillet increased linearly with the level in the feeds. The highest level of selenium reached in the fillet was 0.85 mg/kg during a feeding period of 6 weeks with an Se-enriched (8.5 mg Se/ kg feed) garlic-containing feed while the control treatments yielded selenium levels of 0.24 mg/kg. Neither dietary garlic nor high dietary selenium levels affected fish performance negatively in terms of feed intake, feed conversion rate and specific growth rate. Speciation of selenium in the fish fillet revealed that Se-methylselenocysteine, to which anti-carcinogenic properties are attributed (Ip 1998), was present in the fillet (Schram et al. accepted for publication). Once it was shown that African catfish can actually be enriched with organoselenium compounds, use of the so-called finishing diet was considered. In this diet the enriched feed is not provided to the fish during the entire production period but for a short period prior to harvest. In a second farming trial, the required length of the feeding period to reach our target selenium concentration in the fillet was studied. It was found that a selenium concentration of 0.7 mg/kg can be reached in the fillet after 10 days of feeding using a diet with a selenium level of 11.7 mg/kg. The inclusion of garlic in our experimental fish diets raised questions regarding the taste of the final product. A preliminary trial revealed that garlic does affect taste. In the taste evaluation of the final product, the depuration period should, however, be considered. Depuration consists of depriving fish from feed and transferring them to clean water for typically 1 to 4 days. This procedure is commonly applied to fish farmed in recirculation systems to eliminate off-flavour-causing compounds that accumulate in the fish during production, prior to harvest and slaughter. During this period the fish empty their stomachs and lose up to 5% of their body weight. As depuration may greatly affect the selenium level in the fillet as well as the taste caused by dietary garlic, a third farming trial was set up to investigate these aspects. At present, the results of this part of our work are not available. 17.5.2 Changes in the nutritional functional components of African catfish during household preparation The thorough investigation of a farmed African catfish was performed with respect to the retention/losses of important nutritional components such as selenium, taurine and polyunsaturated fatty acids (n-3) during storage and household preparations (see Fig. 17.4). The Se-enriched African catfish fillets are not usually eaten raw but consumed in prepared meals, where heat treatment might cause loss of the nutritional components with beneficial health effects. In

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addition, the fish have usually been stored for a certain period of time prior to consumption. There are, as we already know, both positive and negative effects from storage and, in particular, from thermal processing of food, and the severity of the changes depends upon the treatment applied. Thermal degradation of components, browning reactions and loss of water-soluble compounds are some detrimental effects of heat treatment. When preparing fish and meat, sarcoplasmic proteins, peptides, amino acids and other water-soluble compounds are lost with drip, and heat treatment generally amplifies these losses (Elvevoll and ésterud 2003). Previous studies indicate up to 70% loss of taurine and other LMW substances during the processing or preparing of seafood (Roe and Weston 1965; Larsen et al. 2007). In addition to the beneficial effects of dietary taurine on the incidence of cardiovascular disease (CVD) observed in both animal and human studies (Militante and Lombardini 2002; Chen et al. 2004; Oudit et al. 2004; Yamori et al. 1987, 2001, 2004; Elvevoll et al. 2008), as explained in Section 17.3, it has been documented that peptides obtained from the digestion of seafood inhibit the angiotensin I-converting enzyme (ACE). Many studies on animals, and clinical and epidemiological research have pointed to the importance of (n-3) polyunsaturated fatty acids (n-3 PUFA) in the prevention of coronary heart disease, the decrease of the incidence of breast cancer, rheumatoid arthritis, multiple sclerosis, psoriasis and inflammation, and on the development of the brain and retina. Marine lipids are the main source of this (n-3) PUFA; within the most important fatty acids are EPA [eicosapentaenoic acid, 20:5 (n-3)] and DHA [docosahexaenoic acid, 22:6 n-3)]. Due to the importance of these components for nutrition and health, a general daily dietary intake of 500 mg for healthy adults and 1000 mg per day for patients with coronary artery disease is recommended (Kris-Etherthon et al. 2001). Taking this into account, it is important to evaluate the (n-3) PUFA level of functional seafood products obtained via dietary modulation and the effect of typical culinary treatments on the retention of such constituents. Therefore the losses of nutritional compounds like selenium, taurine, and ACE inhibitors, as well as n-3 polyunsaturated fatty acids, during household preparation were evaluated in African catfish. The preparation techniques chosen were: baking fillets in aluminium foil (180 ëC, 27 min in an oven with circulating air), deep frying (160 ëC, 4 min) in hydrogenated vegetable oil, and poaching in a cooking pouch (90 ëC, 10 min). Selenium For the evaluation of possible selenium losses, the prepared freeze-dried samples were analysed for selenium content by a graphite furnace AAS operating with Zeeman-background correction. The selenium concentration of the prepared samples was compared to the selenium concentration in the raw fillets, which was initially approx. 1 mg/kg fillet calculated on a wet-weight basis. During the cooking procedure, the African catfish fillets lost about 30% of the initial weight of the fillets as cooking loss and about 10% of the original

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selenium content was lost simultaneously. During baking in aluminium foil, the fillets lost about 21% of their initial weight together with about 15±20% of their selenium concentration. During the deep-frying procedure, the fat uptake was estimated to be about 4.4% of the weight of the prepared samples. The liquid loss during deep frying was more than 35% of the initial weight. In contrast to the other household preparation techniques, the concentration of selenium seemed to remain quite stable. So with respect to the selenium content, deep frying has to be recommended because fish with the highest selenium concentration should be consumed (Mierke-Klemeyer et al. 2007). Loss of free amino acids and angiotensin I-converting enzyme (ACE) inhibitors Household preparation and storage of fish (African catfish) raw material generally lead to losses of water-soluble compounds such as free amino acids (taurine, alanine and glycine). Such losses may also be regarded as markers for losses of similar `free-floating' low molecular weight substances. The loss of taurine as a result of household preparation varied from 30±50% depending on the treatment: simmering, deep frying or baking (see Fig. 17.5). The optimal preparation method for maintaining taurine concentration was found to be baking, followed by simmering or cooking by boiling in water, with deep frying causing the highest loss of taurine. The retention of glycine was higher than taurine and alanine, and the retention of alanine was, in general, better than the retention of taurine. The literature on losses of free amino acids from seafood and meat prepared with different heat treatments generally agrees with these results (Larsen et al. 2007). The storage of the raw material also leads to significant losses in free amino acids. The impact of household preparation techniques on the measured activities of ACE inhibitors was not found to be significant. Fatty acids No significant effect (p > 0:05) was registered for fatty acid profiles, (n-3) PUFA or EPA and DHA contents in African catfish. With regard to the influence of culinary treatments, steaming, baking and deep-frying (sunflower oil) did not promote significant decreases in either EPA or DHA levels. Nevertheless, the fatty acid profiles of deep-fried catfish were different from those of steamed and baked fish due to the vegetable oil absorption and the water leaching out. This fact is reflected in the thrombogenecity index (14:0+16:0+18:0)/((0.5*MUFA)+(0.5* n-6 PUFA)+(3* n-3 PUFA)+(n-3 PUFA/n-6 PUFA)) since the highest level (0.94) was found for the deep-fried product, whereas the values were 0.36 and 0.32 respectively for steamed and baked products. Following the ISSFAL (2004) recommendations for health, it is necessary to consume around 150 g of catfish (boiled, baked or fried), the usual serving portion recommended by nutritionists, to obtain the recommended levels of EPA and DHA. Results of the retention and losses of nutritionally important components during household preparation show that no general recommendation

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Fig. 17.5 Mean SD losses of taurine, glycine and alanine in mg/g dry matter after three different heat treatments: simmering, baking and frying. The diet of the African catfish was enriched with high, medium, and low selenium.

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for a particular preparation method can be given for all three of the components investigated. However, baking in aluminium foil was preferable for the retention of fatty acids and taurine, while most selenium was retained during deep frying.

17.6

Future trends

Translating the results from consumer studies to the specific functional ingredients and product `carriers' that are central in this project, it can be concluded that we have been able to define consumer segments that could be interested in DF and/or selenium as a functional ingredient. However, the acceptance of these functional ingredients depends on intrinsic factors such as the perceived naturalness and contextual factors such as the convenience of the product. Future studies will focus on the relevance of other intrinsic and contextual factors, such as the (marine) source of DF in seafood, whether the product will be used for daily meals or as snack food, as well as the introduction of other functional ingredients such as taurine. After a thorough characterisation of the antioxidant dietary fibres of grapes and fucus seaweed, it can be concluded that these new ingredients have all the properties to be classified as antioxidant dietary fibre and from the nutritional/ functional composition, they are suitable for use as ingredients in restructured seafood products. To use them in a wider range of matrices, methods to make these antioxidant DFs blander in taste and colour without compromising their antioxidant DF properties will need to be investigated. No problems were encountered in a larger scale production of Se-enriched garlic to obtain Se-enriched garlic powder for additional fish feed experiments. However, a disadvantage of high concentrations of Se in soil is the toxic effect on plants and the inability of plants to survive. Different garlic accessions vary in their potential uptake of selenium and production of glutamyl-Se-methylselenocystein (Scholten et al. unpublished). This implies that lower amounts of Se-fertilisation are needed for those accessions that result in a high uptake. Arbuscular mycorrhizal fungi easily associate with garlic roots and further increase the uptake of Se from the soil (Larsen et al. 2006). Breeding for garlic accessions with improved Se uptake would be an interesting option as well. Unfortunately, breeding is hampered due to the fact that seed production of garlic is restricted. However, breeding broccoli for improved Se-uptake could be an interesting option. In Brassica oleracea, a genetic component has been discovered for the accumulation of Se (Kopsell and Randle 2001). The development of formulations with dietary fibre has been successfully performed for some seafood products. Nevertheless, there are several challenges remaining for the use of some of the ingredients such as seaweed fucus. The studies on the structural changes of the main components and their interactions, successfully studied for wheat DF, need to be finalised for more complex DFs with a significant percentage of associated bioactive compounds. Also, the study of household preparation and storage will also need to be

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completed for the restructured fish products developed, in the same way as for aquacultured fish. Some of the innovative formulations studied could be used directly in preexisting products, for example the surimi-based formulations and functional shellfish analogues, or the taurine-enriched tuna portions. Prior to their inclusion in these products, consumer tests should be carried out. A positive outcome of a consumer test would be considered as a big step forward, since it would give a higher chance of success for the use of this project's results by companies or SMEs. The design of new products, with multidisciplinary teams composed of product developers, cooks, consumer scientists, and industry is a step towards industrial application of some of the results obtained by this project. Preparation of restructured fish products containing dietary fibre may be a good way to offer consumers new functional fish products with nutritional claims. The use of restructured fish with specific health claims such as `contains omega-3', `high protein', `source of dietary fibre' and `contains natural antioxidants' appears to be feasible on the basis of the work performed in this project. The recently approved Regulation of the EU on nutrition and health claims includes dietary fibre among the list of nutrients and specifies the health claims that can be made for this food constituent. Dietary fibres from vegetable or marine origin tested in this project present suitable technological and nutritional properties as required by the EU regulation. Antioxidant dietary fibres tested in SEAFOODplus may, in addition, use the nutritional claim `contains natural antioxidants'. As far as health claims are concerned, grape antioxidant dietary fibres used as dietary supplements have shown significant bioavailability of their natural antioxidants and potential health properties related to gastrointestinal and cardiovascular health in recent biological and animal experiments and in clinical studies (MartõÂn-CarroÂn et al. 1999, 2000; SaÂnchez-Moreno et al. 2000; GonÄi et al. 2005; PeÂrez-Jimenez 2007). However, to establish health claims for fish products enriched with antioxidant fibres, further biological and clinical studies specific to this type of food product are needed. Biological and health effects derived from antioxidants should be the focus of further research, including not just free radical scavenging activity but also the effects on gene expression. Although antioxidant properties are important, the bioavailability of many dietary compounds is generally too low to have any substantial direct effect on reactive radical species. Recent work has shown that such dietary components, even at very low concentrations, may have a far greater impact than previously appreciated on the regulation of gene expression, profoundly affecting metabolism. Mechanisms of cellular detoxification, proliferation, differentiation, survival and death are particularly relevant targets for protection against cardiovascular disease (CVD) and cancer (Fearon and Vogelstein 1990; Beckman and Ames 1998; Manna et al. 2000; Sumi et al. 2001). The studies carried out demonstrate that enrichment of farmed fish via dietary modulation is very feasible. A limited number of farming trials has shown some of the technical and practical aspects of enriching a certain fish species with a

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specific functional component. Results so far indicate that Se-methylselenocysteine was recovered in the fillet, to which anti-carcinogenic properties are attributed. The anti-carcinogenic activity of Se-enriched fish in cell cultures or in mice studies needs to be addressed to also prove the presence of these properties of this compound in the fish matrix. Clearly the enrichment of African catfish with selenium is just one example of the large potential of aquaculture production of functional seafood.

17.7

Sources of further information and advice

This chapter can only cover some highlights of the research currently being performed in CONSUMERPRODUCTS. For more comprehensive information, the reader is directed to the scientific publications or conference proceedings where parts of this work have been disseminated. More information can be found in: · www.seafoodplus.org · Popular articles in EUROFISH MAGAZINE 2004±2006: http:// www.seafoodplus.org/Popular_articles.327.0.html · Details about CONSUMERPRODUCTS subproject: http:// www.seafoodplus.org/Project_4_4_CONSUMERPR.59.0.html · Underutilised fish species: http://www.teagasc.ie/research/reports/ foodprocessing/5097/eopr-5097.htm · Facts about selenium: http://dietary-supplements.info.nih.gov/factsheets/ selenium.asp · Health effects of polyunsaturated fatty acids (n-3): Mozaffarian D. and Rimm E.B. 2006. Fish intake, contaminants, and human health ± Evaluating the risks and the benefits. JAMA 296 (15) 1885±1899. · Information about taurine: http://www.mgwater.com/taurine.shtml

17.8

Acknowledgement

Thanks are due to Oddvar Dahl for the design of the figures.

17.9

References

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BANDARRA, N.M.; PARREIRA, R.; ANDRADE, A.M.; NUNES, M.L.; SCHRAM, E. J.

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and LOMBARDINI, J.B. (2002). Treatment of hypertension with oral taurine: experimental and clinical studies. Amino Acids 23, 381±393. MURATA, M. and NAKAZOE, J. (2001). Production and use of marine algae in Japan. JARQ 35(4), 281±290. OUDIT, G.Y.; TRIVIERI, M.G.; KHAPER, N.; HUSAIN, T.; WILSON, G.J.; LIU, P.; SOLE, M.J. and BACKX, P.H. (2004). Taurine supplementation reduces oxidative stress and improves cardiovascular function in an iron-overload murine model. Circulation 109, 1877±1885. PARK, J.W. (2005). Ingredient technology for surimi and surimi seafood. Chapter 13. In Park JW Editor. Surimi and Surimi Seafood. 2nd edition. Food Science and Technology 142. A Series of Monographs. CRC Press, Taylor and Francis Group. Boca Raton FL, pp. 649±707. PEREZ-JIMENEZ, J. (2007). Effect of grape antioxidant dietary fibre on antioxidant status and cardiovascular risk factors in humans. In Metodologia para la evaluacioÂn de ingredients funcionales antioxidants. PhD report. Facultad de Ciencias. Universidad AutoÂnoma de Madrid. Madrid, Spain. PULIDO, R.; BRAVO, L. and SAURA-CALIXTO, F. (2000). Antioxidant Activity of dietary Polyphenols as determined by a modified ferric Reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry 48, 3396±3402. RAYMAN, M.P. (2000). The importance of selenium to human health. The Lancet 356, 233± 241. RE, R.; PELLEGRINI, N.; PROTEGGENTE, A.; PANNALA, A.; YANG, M. and RICE-EVANS, C. (1999). Antioxidant Activity applying an improvement ABTS radical cation decoloration assay. Free Radical Biology and Medicine 26, 1231±1237. ROE, D.A. and WESTON, M.O. (1965). Potential significance of free taurine in diet. Nature 205, 287±288. SAKAGUCHI M. (Ed.) (2004). More efficient utilization of fish and fisheries products. Developments in Food Science 42. Prooceedings of the International Symposium on the occasion of the 70th anniversary of the Japanese Society of Fisheries Science, held in Kyoto, Japan, 7±10 October 2001. Elsevier Ltd. Oxford. Â NCHEZ-ALONSO, I. and BORDERIÂAS, A.J. (2006). Technological effect of red grape SA antioxidant dietary fibre added to minced fish muscle. International Journal of Food Science and Technology (on line doi: 10.1111/j.136- 2621.2007.01554x). Â NCHEZ-ALONSO, I.; HAJIÂ-MALEKI, R. and BORDERIÂAS, A.J. (2006a). Effect of wheat fibre in SA frozen stored fish muscle gel. Eur. Food Res. Technol. 223, 571±576. Â NCHEZ-ALONSO, I.; JIMEÂNEZ-ESCRIG, A.; SAURA-CALIXTO, F. and BORDERIÂAS, A.J. (2006b). SA Antioxidant protection of white grape pomace on restructured fish products during frozen storage. LWT-Food Science International (on line, doi:10.1016/ j.lwt.2007202002). Â NCHEZ-ALONSO, I.; SOLAS, M. and BORDERIÂAS, A.J. (2007a). Technological implications of SA addition of wheat dietary fibre to giant squid (Dosidicus gigas) surimi gels. J Food Engineering 81, 404±411. Â NCHEZ-ALONSO, I.; SOLAS, M.T. and BORDERIÂAS, A.J. (2007b). Physical study of minced SA fish muscle with a white-grape by-product added as ingredient. Journal of Food Science 72(2), 94±101. Â NCHEZ-ALONSO, I.; HAJIÂ-MALEKI, R. and BORDERIÂAS, A.J. (2007c). Wheat fiber as a SA functional ingredient in restructured fish products. Food Chemistry 100, 1037±1043. Â NCHEZ-ALONSO, I.; JIMEÂNEZ-ESCRIG, F.; SAURA-CALIXTO, F. and BORDERIÂAS, A.J. (2007d). SA Effect of grape antioxidant dietary fibre on the prevention on lipid oxidation in minced fish: Evaluation by different methodologies. Food Chemistry 101, 372±378. MILITANTE, J.D.

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and CARECHE M. (2005a). Estudio de la adicioÂn de fibra dieteÂtica de trigo en las propiedades reoloÂgicas y estructurales de geles de surimi, NutricioÂn Hospitalaria 20, S1, 134. Â NCHEZ-GONZA Â LEZ I.; CARMONA, P. and CARECHE, M. (2005b). Study of grape dietary fiber SA addition on the rheological, colorimetric and structural properties of minced horse mackerel, Intradfood. Conference. EFFoST-Innovations in traditional foods, 2005, 25±28 October 2005. Abstract. Â NCHEZ-GONZA Â LEZ, I.; CARMONA, P. and CARECHE M. (2005c). Study of Protein and Water SA Structure in Alaska Pollack Surimi Gels in the Presence of Wheat Dietary Fiber. 11th European Conference on the Spectroscopy of Biological Molecules, Aschaffenburg, Germany, 2005, 3±8 September, Poster. Book of Abstracts of the 11th ECSBM, UniversitaÈt Frankfurt am Main, pp 138PC-2. Â NCHEZ-GONZA Â LEZ I.; CARMONA, P.; MORENO, P. and CARECHE, M. (2006). Estudio de los SA cambios en la estructura y disposicioÂn del agua mediante espectroscopõÂa FTRaman en el proceso de gelificacioÂn de surimi, CYTALIA Conference, 2006, March 2006. Â NCHEZ-GONZA Â LEZ, I.; CARMONA P. SA

Â NCHEZ-GONZA Â LEZ I.; CARMONA, P.; MORENO, P.; BORDERIÂAS, J.; SA Â NCHEZ-ALONSO, I.; SA RODRIÂGUEZ-CASADO, A. and CARECHE, M. (2008). Protein and water structural

changes in Alaska Pollock surimi during gelation as revealed by H/D exchange and Raman spectroscopy. Food Chemistry 106, 56±64. Â NCHEZ-GONZA Â LEZ, I.; RODRIÂGUEZ-CASADO A., CARMONA, P. and CARECHE M. (2008). SA Raman spectroscopic study of surimi gelation by addition of wheat dietary fibre. Food Chemistry, accepted for publication. Â NCHEZ-MORENO C., JIMEÂNEZ-ESCRIG A. and SAURA-CALIXTO F., (2000). Study of lowSA density lipoprotein oxidizability indexes to measure the antioxidant activity of dietary polyphenols. Nutr Res 20, 941±953. SATHIVEL, S.; BECHTEL, P.J.; BABBITT, J.; PRINYAWIWATKUL, W.; NEGULESCU, I.I. and REPPOND, K.D. (2004). Properties of protein powders from arrowtooth flounder (Atheresthes stomias) and herring (Clupea harengus) byproducts. J. Agric. Food Chem. 52, 5040±5046. SAURA-CALIXTO, F. (1998). Antioxidant Dietary Fibre Product: A new concept and a potential food ingredient. Journal of Agricultural and Food Chemistry 48, 4303± 4306. SCHOLDERER, J. and GRUNERT, K.G. (2005). Consumers, food and convenience: the long way from resource constraints to actual consumption patterns. Journal of Economic Psychology 26(1), 105±128. SCHOLDERER, J.; BRUNSé, K.; BREDAHL, L. and GRUNERT, K.G., (2004). Cross-cultural validity of food-related lifestyles instrument (FRL) within Western Europe. Appetite 42, 197±211. SCHRAM, E.; PEDRERO, Z.; CAMARA, C.;. VAN DER HEUL, J.W. and LUTEN J.B. Enrichment of African catfish with functional selenium originating from garlic (accepted for publication). SOFIA (2000). The State of World Fisheries and Aquaculture, 2000. FAO document. http://www.fao.org Accessed November 2006. SOFIA (2004). The State of World Fisheries and Aquaculture, 2004. FAO document. http://www.fao.org Accessed November 2006. È GER, J. and GORMLEY, R. (2006). Quality evaluation of silver STOKNES, I.S.; OEHLENSCHLA smelt (Argentina silus) and its suitability for seafood products: Compilation of results from three European research centres. In: Seafood research from fish to dish

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(J.B. Luten, C. Jacobsen, K. Bekaert, A. SaeboÈ, J. OehlenschlaÈger, eds.). Wageningen Academic Publishers, pp. 439±456. SUMI, S-I., TSUNEYOSHI, T., MATSUO, H. and YOSHIMATSU, T. (2001). Isolation and characterisation of the genes up-regulated in isolated neurones by aged garlic extract. Journal of Nutrition 131, 1096S±1100S. TROWELL, H. (1976). Definition of dietary fiber and hypotheses that it is a protective factor in certain diseases. American Journal of Clinical Nutrition 29 (4), 417±427. TRUUS, K.; VAHER, M.; KOEL, M.; MAHAR A. and TAURE I. (2004). Analysis of bioactive ingredients in the brown alga fucus vesiculosus by capillary electrophoresis and neutron activation analysis. Anal. Bioanal. Chem. 379, 849±852. VAN KLEEF. E.; VAN TRIJP, H.C.M. and LUNING, P. (2005a). Consumer research in the early stages of new product development: a critical review of methods and techniques. Food Quality and Preference 16, 181±201. VAN KLEEF, E.; VAN TRIJP, H.C.M. and LUNING, P. (2005b). Functional Foods: Health claimfood product compatibility and the impact of health framing on consumer evaluation. Appetite 44, 299±308. YAMORI, Y.; NARA, Y.; KIHARA, M.; MANO, M. and HORIE, R. (1987). Urinary Amino Acids as Biological Markers for Dietary Protein Intake. In: Yamori Y., Lenfant C. (eds.): Prevention of Cardiovascular Diseases: An Approach To Active Long Life, Elsevier, pp. 99±104. YAMORI, Y.; LIU, L.; IKEDA, K.; MIURA, A.; MIZUSHIMA, S.; MIKI, T. and NARA, Y. (2001). Distribution of twenty-four hour urinary taurine excretion and association with ischemic heart disease mortality in 24 populations of 16 countries: results from the WHO-CARDIAC study. Hypertension Research ± Clinical & Experimental 24, 453±457. YAMORI, Y.; MURAKAMI, S.; IKEDA, K. and NARA, Y. (2004). Fish and Life style related disease prevention: Experimental and epidemiological evidence for antiatherogenic potential of taurine. Clin Exp Pharmacol Physiol. 31, Suppl 2, S20±23. YOON, K.S. and LEE, C.M. (1990). Effect of powdered cellulose on the texture and freezethaw stability of surimi-based shellfish analog products. Journal of Food Science 55, 87±90.

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18 Mild processing techniques and development of functional marine protein and peptide ingredients G. Thorkelsson, S. Sigurgisladottir, M. Geirsdottir and R. JoÂhannsson, Matis, Iceland, F. GueÂrard and A. Chabeaud, UniversiteÂ de Bretagne Occidentale, France, P. Bourseau and L. Vandanjon, UniversiteÂ de Bretagne Sud, France, P. Jaouen and M. Chaplain-Derouiniot, UniversiteÂ de Nantes, France, M. Fouchereau-Peron, O. Martinez-Alvarez and Y. Le Gal, MuseÂe National d'Histoire Naturelle, France, R. Ravallec-PleÂ, ProBioGEM, France, L. Picot, UniversiteÂ de La Rochelle, France, J.P. Berge, Ifremer, France, C. Delannoy, Copalis, France, G. Jakobsen and I. Johansson, Marinova, Denmark and I. Batista and C. Pires, Ipimar, Portugal

18.1

Introduction

The raw materials that come from traditional fisheries and aquaculture can be regarded as great and valuable sources of protein for both animal and human nutrition. Fish meal, fish sauce, surimi and fish silage are traditional proteinbased products. Over 6 million tonnes of fishmeal are produced worldwide each year from about 25±30 million tonnes of industrial fish (FIN, 2007). Demand is increasing with the growth in aquaculture and the price has been rising (Klickhardt, 2006). Every year, 2±3 million tons of wild fish are processed worldwide to produce about 750 000 tonnes of surimi, the figures having doubled over the last 10 years (GRP, 2007). The quantity of fish sauce produced each year is about 400 000 tonnes (Dissaraphong et al., 2006). Fish silage is used almost entirely for feed. Norway is the major fish silage producer ± producing

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about 140 000 tonnes per year, mainly from aquaculture by-products (Rustad, 2003). It may be possible to increase the value of marine by-products and underutilised fish by using mild processing techniques to convert them into protein and peptide ingredients, both to improve the yield of traditional seafood and to be used as nutraceuticals or in functional foods. Mild techniques such as pH shift, fermentation, enzymatic hydrolysis, filtration, centrifugation, and spray and freeze drying can be used for the processing and production of value-added products. The utilisation of protein sources from fisheries and aquaculture can be divided into two categories: · Improved yield and utilisation of proteins in traditional fish processing. · Marine proteins and peptides with functional and bioactive properties. This chapter is an overview of the latest developments in the use of mild processing techniques for the production and use of functional marine protein and peptide ingredients. It is also written in association with the PROPEPHEALTH project within SEAFOODplus (www.seafoodplus.org). The aims of PROPEPHEALTH are to screen, map and recover `new' (health) beneficial compounds from seafood by-products by advanced mild refining processes, to develop 'new' bioactive (functional) seafood ingredients, and to use the ingredients for the development of new functional seafood products, accepted by the target consumers. The project is divided into three parts. The first part looks at the use of pH-shift methods to produce protein isolates from by-products and pelagic fish, and the insertion of the protein isolates into fish fillets and ready-to-eat seafood products. The second part looks at fish protein hydrolysates and bioactive peptides from an industrial point of view. Commercially produced fish protein hydrolysates (FPH) from three companies were screened in vitro for different biological activities. The FPHs with the most promising activities were subjected to ultrafiltration and nanofiltration in order to optimise the amounts of the active components. Chromatographic separation was carried out on a few of the FPHs in order to isolate and identify the active peptides. In the last part of the project, the in vivo activity of two selected products will be tested.

18.2

Improved yield in traditional fish processing

About 76% of the global fish supply was used for human consumption in 2002 (FAO, 2004), of which about 40 million tonnes were used for manufacturing products for direct human consumption. But up to 50±70% of the fish may end up as co-products as the yield from filleting operations is 30±50% (Arason, 2002; Kristinsson et al., 2006; Mackie, 1982). About 6 million tonnes of trimmings and co-products from fish processing are turned into fish meal (FIN, 2007) and the rest is used in fish silage or as a fertiliser or discarded. Enormous economic, nutritional and environmental gains can be achieved by increasing

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the yield of raw material from the fish filleting operation and in the production of ready-to-eat seafood products. The meat, poultry and fish industries in the USA and other parts of the world are adding up to 12% brine to modify both fresh retail and further processed products. This is done to improve quality, firmness and juiciness and to meet increasing consumer demand for convenience food items while at the same time increasing yield. The brine is made up of water, salt, phosphates, and sometimes other functional or flavour ingredients such as sodium lactate, polysaccharide gums, fish proteins and protein hydrolysates, hydrolysed whey and soy proteins, and modified starches (Xiong, 2005). Innovative technologies for improving the yield from fish filleting are using raw materials from co-products such as cut-offs and backbones of the same species. This applies both to the SuspenTecÕ system (Christensen, 2006; www.suspentec.com) and acid and alkali extraction (pH-shift) of fish proteins (Batista, 1999; Hultin and Kelleher, 1999; Hultin et al., 2004; Nishioka and Shimizu, 1983). Chemical processing methods for protein recovery have been extensively reviewed in two recently published books (Hultin et al., 2005 and Shahidi, 2006). The SuspenTecÕ process is an automated method of reducing fish trimmings to micron-sized particles at low temperatures (ÿ4±6 ëC) and incorporating them into traditional brines to create homogeneous suspensions. The controlled temperature ensures efficient protein binding and dispersal of the suspensions into the whole-muscle product (Christensen, 2006). The pH-shift protein isolate can be added to fresh seafood of the same species by needle injection into fillets, static soaking, or vacuum tumbling. Another interesting technique is NutraPureÕ protein processing, a technology to reduce fat in deep-fried fish products (Kelleher and Williamson, 2007; www.proteusindustries.com). Acid and alkali extracted fish protein isolates have a GRAS (Generally Regarded as Safe) status in the United States (FDA, 2004). The pH shift methods are an alternative to surimi production and are more suitable than the surimi process for complex raw materials like whole fish and co-products. The process is shown in Fig. 18.1. It involves solubilising muscle proteins by subjecting diluted, finely homogenised fish meat to either a very low pH (~2.5±3) or a very high pH (~10.8±11.2) at low temperatures. Solids such as bones, scales, neutral fat and disrupted cellular lipid membranes are then removed by centrifugation and the soluble protein is precipitated by adjusting the pH to the isoelectric point of the myofibrillar proteins to give a protein isolate (Kristinsson et al., 2006). This method gives a higher yield of proteins than surimi processing. Sarcoplasmic proteins, which are washed away in surimi processing, are nearly all recovered in the pH-shift process (Choi and Park, 2002; Kristinsson and Demir, 2003; Kristinsson et al., 2005; Undeland et al., 2002). Microorganisms are also partly removed during the first centrifugation step (Hultin and Kelleher, 1999). The protein isolate was found to have a lower number of aerobic bacteria and longer bacterial shelf life than surimi from catfish (Kristinsson et al., 2005).

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Fig. 18.1 Schematic representation of the acid and alkaline processes used in the production of fish protein isolates. The process involves solubilising muscle proteins at low or high pH, separating them from undesirable muscle components via centrifugation and recovery of the proteins of interest by isoelectric precipitation. The final protein isolate can then be used directly, or stabilised with cryoprotectants and frozen until used. (Adapted from Kelleher and Hultin, 2000.)

The protein isolate from the pH processes is claimed to have a substantial absence of both neutral and membrane lipids and their removal is expected to greatly improve the oxidative stability of the product. The alkali-assisted process gives a more oxidative stable isolate than the acid-assisted process and one that is sometimes more stable than surimi (Kristinsson and Demir, 2003; Petty and Kristinsson, 2004). Haemoglobin, a principal catalyst of oxidation in fish muscle, is highly pro-oxidative at low pH but very stable at high pH (Kristinsson and Hultin, 2004). Also, more heme proteins are removed compared to using the surimi process, yielding a whiter product which is more stable with regards to oxidation if processed with high pH (Kristinsson, 2002).The acid process leads to denaturation and co-precipitation of heme proteins, giving a less stable and darker product (Kristinsson and Hultin, 2004; Kristinsson et al., 2005; Choi and Park, 2002). There are conflicting results on the influence of high and low pH on the gelforming and water-binding properties of the isolated fish protein. They depend on species, condition of the raw material and processing conditions. The alkali-

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assisted process is claimed to produce gels that are superior to those from both the acid-assisted process and the surimi process (Davenport and Kristinsson, 2004; Kristinsson and Ingadottir, 2006 and Yongsawatdigul and Park, 2001). The lower strength of the acid-process gels was explained by proteolysis that can decrease gelation and water-binding properties. This has not, however, been demonstrated in cod (Hultin and Kelleher, 1999; Kristinsson and Hultin, 2003) and catfish (Kristinsson et al., 2005). Sarcoplasmic proteins are removed from surimi because they are believed to interfere with gelation (Park et al.,1997; Shimizu et al., 1992). Then again, other studies have shown that the presence of sarcoplasmic protein has either no effect or a positive effect on gel strength (Hultin and Kelleher, 1999; Kristinsson and Crynen, 2003; Kristinsson and Liang, 2006; Ko and Hwang, 1995; Morioka and Shimizu, 1990). Separations in the pH shift process are usually performed by centrifugation with relatively expensive equipment that requires qualified personnel. Decanter centrifuges are normally used, which require relatively large amounts of material to achieve steady-state running conditions. Most small- and mediumsized fish processors do not have sufficient quantities of fish material for a processing solution using decanter centrifuges. It has been shown that replacing the first centrifugation with sieving improves the yield of protein isolate but reduces the quality of the produced gel. Replacing the second centrifugation with filtration has no influence on the yield or the quality of the protein isolate (Nolsoe et al., 2007). Applying the pH shift process for production has been tried on an industrial scale in Iceland in the PROPEPHEALTH project where a pilot plant with a continuous process was set up. There were difficulties in removing fat during protein isolation of herring and there was extensive lipid oxidation during processing. Trials to reduce the oxidation were unsuccessful (Geirsdottir, 2006). But on a laboratory scale, high-speed centrifugation (10 000 g) reduced initial TBARS levels by about 50% and process-induced oxidation was prevented by adding reducing agents (0.2% erythobate) and metal chelator (0.2% EDTA or sodium tripolyphosphate) during either the prewashing step or homogenisation. To achieve good stability during frozen storage, ETDA or sodium tripolyphosphate had to be added during the homogenisation step (Undeland et al., 2005). The injection of brine containing fish protein isolates or homogenised muscle has been found to increase the weight gain in cod and haddock fillets by 5±20% and also increase cooking yield. There are indications that fish protein isolates give a higher cooking yield and microbiologically more stable products than those injected with fish mince (Thorarinsdottir et al., 2005; Valsdottir et al., 2006a, 2006b). The use of fish protein isolates to reduce fat in deep-fried fish products has also been tested on a laboratory scale. The effects of the frying time and the addition of cod protein isolate (5±20%) to fish blocks on the fat uptake in deep-fried battered and breaded cod and saithe were evaluated by tumbling and coating 1 10 10 cm pieces with 2±6% isolate solution,. The fat uptake increased more than 50%, that is from 8% to 12%, by increasing the frying time

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from 60 to 180 seconds, but the addition of protein isolate to the fish block and having a pre-batter containing protein isolate did not change the fat content of the finished product in the set-up and conditions used in the tests (Einarsdottir et al., 2007). The alkali-aided process produced better gels than the acid-aided process (Batista et al., 2006) but they were not as good as those obtained from fish muscle. The proteins recovered after alkaline solubilisation of unwashed mince showed the best textural properties but the gels obtained were of medium quality according to the results of a folding test score. The protein isolates were used as ingredients in the preparation of frankfurter-type sausages (Pires et al., 2007a, 2007b).

18.3

Processing of marine proteins and peptides

18.3.1 Enzymatic hydrolysis Proteases are used industrially to produce fish protein hydrolysates. The hydrolysates can be processed further by separating them into fractions using centrifugation, sieving, ultrafiltration and nanofiltration and then concentrating them by evaporation and drying. The products can be used as ingredients in fish and other food formulations, as seafood flavours or as ingredients for functional foods, nutraceuticals and cosmetics. The most recent reviews are by Kristinsson (2006) and GueÂrard (2006b). The enzymes are active under mild pH conditions and temperatures. The type of enzymes applied depend on the intended final product. The production of a wide range of protein ingredients from by-products and underutilised fish using controlled conditions has been documented. Preliminary steps in the production of fish protein hydrolysates are the selection, storage and handling of the raw material, depending both on the needs and demands of end-users as well as on the needs and wishes of the raw material producers. The raw material is either co-products or underutilised fish. The coproducts come from both fisheries and aquaculture, which need the co-products to be collected, disposed of and/or utilised in the most cost-effective manner. Cost limits the choice of raw material to underutilised fish and co-products such as viscera, backbones, skins, cut-offs, saw dust, washing waters and cooking juices. The quality of the raw material at the processing site will determine the manufacturing possibilities. Good handling is extremely important. Prevention and control of microbial and chemical contamination is important when producing fish proteins and peptides from co-products. Regulation (EC) No 178/2002 lays down general food safety requirements, according to which food must not be placed on the market if it is unsafe. It also applies to production of fish proteins and peptides. Regulation (EC) No 2073/ 2005 lays down microbiological criteria for foodstuffs and is a guide on the acceptability of foodstuffs and their manufacturing, handling and distribution processes. They are an integral part of the implementation of HACCP-based procedures and other hygiene control measures in the companies.

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Fish protein supply is very diverse compared to other protein sources. This diversity creates opportunities but can also create problems. Besides diversity of species, there is diversity in fishing vessels, production and processing sites, and operations. This can create problems in collecting by-products and in the quality and condition of the raw material so that it becomes unfit for processing into high-value products. A stable and sustainable supply is necessary to start up businesses in fish proteins and peptides. The condition of the wild fish stocks and the seasonality of the catches must be considered and can cause problems when planning a large-scale production of proteins and peptides. Many of the stocks are declining while others are in good condition and even increasing. The first stages in the production of fish protein hydrolysates are mincing, homogenisation and mixing with enough water to ensure easy access for the enzymes (Slizyte et al., 2005). The next step is the actual hydrolysis, which includes setting the slurry to the right temperature, adjusting the pH and adding the enzymes. The choice of enzymes depends not only on the end-product but also on cost. Mixing fish viscera into the slurry is the least expensive method; the fish enzymes are active at very low temperatures, which minimises microbiological and quality problems, but variations in enzyme level and activity as well as lower yields can cause problems. Commercial enzymes, usually a combination of endopeptidases and exopeptidases, are used in controlled hydrolysis (GueÂrard, 2006b), producing FPHs with very different properties. Limited hydrolysis requires specific enzymes but a mixture of enzymes is used in more extensive hydrolysis. Several studies have been documented using enzyme preparations from Novo Nordisk: Alcalase, Flavourzyme, Protamex, Neutrase and Kojizyme (GueÂrard, 2006b), but others such as Newlase from Amano, fungal protease type II from Sigma (GueÂrard, 2006b) and Corolase PN-L and 7089 have also been tested with success (Kristinsson, 2006). 18.3.2 Membrane filtration Ultrafiltration (UF) using membranes with different molecular weight cut-offs (MWCO) is used in the production of ingredients with different biological and physicochemical properties from protein hydrolysates. Peptides can be separated from non-hydrolysed proteins and proteolytic enzymes using UF membranes with a high MWCO, approximately 20 kDa or above. The peptides in the permeate can then be fractionated according to their molecular weight (MW) with UF membranes of intermediate MWCO, approximately 4±8 kDa. Peptide solutions can also be concentrated with nanofiltration (NF) membranes of low MWCO, approximately 200±300 Da, which will retain almost all the peptides excepted the smallest ones (amino-acids, di- or tri-peptides) (Vandanjon et al., 2007). Ultrafiltration and nanofiltration can also be used in diafiltration for refining solutions (e.g., desalination, partial deodorisation with a nanofiltration membrane) (Simon et al., 2002; Vandanjon et al., 2005). A high concentration of pepsin can be obtained by ultrafiltration of the aqueous phase from cod stomach silage preserved with formic acid, and a

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concentrate of trypsin-like enzymes can be obtained by ultrafiltration of fish sauce produced by salt fermentation of cod intestines. The permeate from the ultrafiltration contains the greatest amount of proteinous material (peptides and amino acids), and has a palatable taste similar to traditional fish sauce (Gildberg, 1992). Cod frame protein hydrolysate can be separated into fractions with different physicochemical and bioactive properties. Permeate from a 10 kDa MWCO shows high antioxidative activity, while permeate from a 3 kDa membrane has excellent ACE inhibitory activity. A fraction between 10 and 30 kDa shows excellent emulsion properties and whippability (Jeon et al., 1999). The antioxidative properties of an ultrafiltrated fraction of yellowfin sole frame protein hydrolysate have been studied and the active peptide from the fraction showing the highest activity was isolated and identified, using chromatographic methods, to be a 13 kDa peptide molecule with 10 amino acids (Jun et al., 2004). Ultrafiltration was used to fractionate Alaska pollack frame protein hydrolysate to concentrate and separate peptides with antioxidative properties. The fraction with the smallest MWCO had the highest activity. It was further purified using consecutive chromatographic methods. The sequence of the purified peptide was Leu-Pro-His-Ser-Gly-Tyr (Je et al., 2005). Enzymatic hydrolysis is usually a batch operation. It has been criticised for being costly as the enzymes cannot be reused and the process is labour intensive with a low yield and inconsistent quality. A UF membrane reactor for the hydrolysis of proteins has been applied to overcome these problems. Several studies have concluded that the production of protein hydrolysates using a continuous membrane reactor results in higher productivity and more uniform products compared to using batch-type reactors (Chiang et al., 2006). Figure 18.2 shows the features of a continuous reactor. 18.3.3 Fermentation There are challenges and opportunities in studying, adapting and changing traditional foods made by methods like fermentation. Fish sauce and dry-cured fermented meat products are good examples. Fermentation is a traditional food processing and food preservation method. Many traditional food products are fermented. Starter cultures of lactic acid bacteria and other microorganisms are used in the production of fermented beverages, and dairy, meat and vegetable products. Fermented products have an extended shelf life, and distinct flavour profiles and textures. The preservation effect is due to the production of lactic acid and other organic acids, which reduce the pH and inhibit the growth of pathogenic and spoilage organisms. Fermentation is also used to produce functional dairy products with ACE inhibitory peptides that may exert an antihypertensive effect (Chen et al., 2007; Gobbetti et al., 2000; Nakamura et al., 1995; Yamamoto et al.,1999). Lactic acid bacteria have not been used commercially to any great extent in the production of seafood for human consumption. The fermentation of surimi with lactic acid bacteria to obtain a product with Angiotensin I-converting

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Fig. 18.2 Schematic diagram of continuous spiral-wound membrane system. Protein solution is prepared by stirring and heating, then passed through a 100-mesh sieve to remove large particles. The reaction vessel is filled with the desired volume of the protein filtrate and the desired temperature maintained in the tank. Then the enzyme is added to the protein solution. Inlet pressure and flow rate can be controlled and adjusted. The reaction mixture is pumped to a spiral-wound membrane where the large particles, such as intact proteins or enzymes that cannot penetrate the pores of ultrafiltration membrane, are recycled to the reaction vessel. The permeate, containing particles small enough to penetrate the membrane are collected and processed further by further filtration, evaporation or drying. (From Chiang et.al. 2006. By permission from Elsevier Limited.)

enzyme (ACE) inhibitory activity that can help improve surimi fish sausage has been reported (Shan et al., 2007). Hydrolysed and fermented minced mackerel have been shown to have antioxidative activity (Yin et al., 2005). In Norway, traditional northern-European dry-cured meat sausage technology has been used to develop a new kind of fermented, smoked and dried fish product resembling a firm and sliceable dry-cured meat sausage but with a high content of polyunsaturated (PUFA) omega-3 fatty acids (Nordvi et al., 2007). It is made from salmon, saithe, and fish oil with whey protein-based ingredients and fermented with Lactobacillus sakei. The fish oil is microencapsulated simultaneously with the microparticulation of the protein. This product can be in liquid, powder or emulsion form and is suitable for the enrichment of a variety of food items and beverages or it may be consumed directly (Bakkene et al., 2007). Recent results from research on dry-cured ham indicate that dipeptides with a strong ACE inhibitory activity can be produced during meat ageing (Sentandreu and Toldra, 2007). A traditional way of preserving fish in South-East Asia is to process them to make a fermented sauce. Fish sauce is mainly produced from anchovies, mackerel and herring. The heavily salted fish, with two to three parts water, are

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fermented in closed tanks at 30±40 ëC for 6±12 months or longer. The fermentation process normally continues for a long time to ensure solubilisation as well as flavour and colour development (Dissaraphong et al., 2006). Biochemically, fish sauce is a salt-soluble protein in the form of amino acids and peptides. It is developed microbiologically with halophilic bacteria which produce proteases that, together with proteases from the fish muscle and viscera, hydrolyse the fish and are principally responsible for its flavour and aroma (Lopetcharat et al., 2001; Gildberg and Thongthai, 2001; Fukami et al., 2004). There is great interest in producing low-salt fish sauce. A high salt content inhibits the action of the enzymes in the fish, making the processing time very long, and the high quantities of salt in the product make marketing it as a health product very difficult. The processing time can be shortened by adding fish viscera or proteases (Kim et al., 1997; Morioka et al., 1999) or reducing the salt concentration to under 20% (Gildberg and Thongthai, 2001; Morioka et al., 1999). It has also been documented that high-pressure treatment of 60 MPa at 50 ëC for 48 hours can be used to produce an autolysate such as fish sauce without adding any salt (Okasaki et al., 2003). Fish sauce can also contain bioactive peptides. A peptide with antioxidative properties has been isolated from fermented blue mussel sauce (Jung et al., 2005) and ACE inhibitory dipeptides have been isolated from fermented anchovy sauce, which also stimulated insulin secretion by cultured insulinoma cells (Ichimura et al., 2003). Fish sauce is mostly produced in South East Asia but its successful production from Arctic species of pelagic fish like capelin has been reported (Gildberg 2001; Hjalmarsson et al., 2007).

18.4 Bioactive properties of fish protein hydrolysates and peptides Various fish protein hydrolysates and peptides have been shown to have different types of in vitro bioactivities such as antioxidative, immunomodulatory, antihypertensive, anticancer and antithrombotic activities (Kim and Mendis, 2006). The peptides are usually 2±14 amino acids long and usually have hydrophobic amino acid residues in addition to proline, lysine or arginine groups. Bioactive peptides are also resistant to the action of digestion peptidases (Kitt and Weiler, 2003; Seki et al., 1996; Yamamoto et al., 2003). Tables 18.1 and 18.2 show published examples of FPHs with their biological effects, the raw material they are produced from, the enzymes used and the amino acid sequence of the active peptides. Most of the publications are on their hypotensive or angiotensinconverting enzyme inhibition (Anti-ACE) effects, and their antioxidative effects. 18.4.1 Angiotensin I-converting enzyme (ACE) inhibition Inhibitory peptides that have an antihypertensive effect have been isolated from enzymatic digests of various food proteins. Many of them show in vitro ACE inhibitory activity and also in vivo activity in spontaneously hypertensive rats (SHR) but few of them have been tested on humans (Vercruysse et al., 2005). To

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Table 18.1 Examples of bioactive protein hydrolysates/peptides with ACE inhibitory/hypotensive effects derived from fish and crustaceans Raw material

Process

Peptide sequence (Single letter code)*

References

Katsuobushi Dried bonito bowels

Thermolysin

LKPNM; GYPHK; IRPVQ

Sardine

Bacillus licheniformis Alkaline protease

VY

Skipjack tuna

Pepsin

Cod frames

Crude proteinase from tuna Pyloric caeca Alcalase, Pronase E, collagenase

VAWKL; WSKVVL SSKVPP; CWLPVY

Yokoyama et al., 1992; Fujii et al., 1993; Matsumura et al., 1993 Karaki et al., 1993; Fujita et al., 1995; Fujita and Yoshikawa, 1999 Yoshikawa et al., 2000; Kouna et al., 2005 Sugiyama et al., 1991; Matsui et al., 1993; Matsufuji et al., 1994; Kawasaki et al., 2000; Matsui et al., 2002; Matsumoto et al., 2004 Astwan et al., 1995

Alaska pollack skin Sardine Cod heads Shrimp Chum salmon Pearl oyster Alaska pollack frame Heshiko fermented mackerel Sea bream scales Tuna broth Shrimp Kamaboko Yellowfin sole Arabesque greenling surimi

Thermolysin Alkaline protease Pepsin Fermentation Alkaline protease Orientase Protease from Bacillus sp.98011 Gastrointestinal proteases Protein proteases Chymotrypsin Lactobacillus delbrueckii

GPL

Jeon et al., 1999 Byun and Kim, 2001 Bordenave et al., 2002

FY; AW; VW; GW FGASTRGA GY; VY; GF; VIY FCVLRP; IFVPAF; KPPETV MIFPGAGGPEL IAW; YNR

Ono et al., 2003 Katano et al., 2003 Je et al., 2004 Itou and Akahane, 2004 Fahmi et al., 2004 Hwang and Ko, 2004 Hai-Lu et al., 2006 Nagai et al., 2006 Jung et al., 2006 Shan et al., 2007

* A ± Alanine, C ± Cysteine, D ± Aspartic Acid, E ± Glutamic Acid, F ± Phenylalanine (Phe), G ± Glycine, H ± Histidine, I ± Isoleucine, K ± Lysine, L ± Leucine, M ± Methionine, N ± Asparagine, P ± Proline, Q ± Glutamine, R ± Arginine, S ± Serine, T ± Threonine, V ± Valine, W ± Tryptophan, Y ± Tyrosine

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Table 18.2 Examples of bioactive protein hydrolysates/peptides with biological effects derived from fish and crustaceans Biological activity

Raw material

Process

Antioxidative

Cod frames

Crude proteinase from tuna pyloric caeca Proteinase XXIII Asp.oryzea Pepsin and mackerel crude enzyme

Seven antiox. peptides RPDFDLEPPY

Fermentation

FGHPY

Pepsin, trypsin and -chymotrypsin

NADFGLNGLEGLA NGLEGLK FDSGPAGVL NGPLQAGQPGER GPLGPL LPHSGY

Tuna cooking juice Yellowfin sole frame Fish Fermented sauce of Blue mussels Giant squid muscle

Peptide sequence (single letter code)*

Giant squid skin

Trypsin

Hoki skin gelatin Alaska pollack frame Shrimp Kamaboko Mussels

Trypsin Mackerel crude enzyme Protease from Bacillus sp.98011 Gastrointestinal proteases In vitro gastrointestinal digestion system Protein proteases Pepsin Flavourzyme Alcalase, Flavourzyme

Tuna backbone Round scad Yellow stripe trevally

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References Jeon et al., 1999 Jao and Ko, 2002 Jun et al., 2004 Guerard et al., 2003 Jung et al., 2005 Rajapakse et al., 2005 Mendis et al., 2005b

Mendis et al., 2005a Je et al., 2005 Hai-Lu et al., 2006 Nagai et al., 2006, 2007 LVGDEQAVYAVCVY Jung et al., 2007

VKAGFAWTANQQLS Je et al., 2007 Thiansilakul et al., 2007a,b Klompong et al., 2007

Immunomodulation/ immunostimulation

Cod stomach White fish

Fermentation

Gildberg et al., 1996 Duarte et al., 2006

Satiety/Growth/ Secretion of digestive enzymes

Sardine Cod muscle

Alcalase Alcalase

Ravallec-Ple et al., 2001 Ravallec-Ple and Wormhoudt, 2003

Calcitonin gene related peptide

Cod muscle Shrimp heads sardine Portugese dogfish

Alcalase Alcalase Alcalase Copalis

Fouchereau-Peron et al., 1999

Shrimp heads

Rousseau et al., 2001 Martinez-Alvarez et al., 2007

* A ± Alanine, C ± Cysteine, D ± Aspartic Acid, E ± Glutamic Acid, F ± Phenylalanine (Phe), G ± Glycine, H ± Histidine, I ± Isoleucine, K ± Lysine, L ± Leucine, M ± Methionine, N ± Asparagine, P ± Proline, Q ± Glutamine, R ± Arginine, S ± Serine, T ± Threonine, V ± Valine, W ± Tryptophan, Y ± Tyrosine

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exert an antihypertensive effect after oral ingestion, ACE inhibitory peptides have to reach the cardiovascular system in an active form. Therefore they need to remain active during digestion by human proteases and be transported through the intestinal wall into the bloodstream. The bioavailability of some ACE inhibitory peptides has been studied. For example, (hydroxy)proline-containing peptides are generally resistant to degradation by digestive enzymes. Peptides can be absorbed intact through the intestine by paracellular and transcellular routes, but the potency of the bioactivity after absorption is inversely correlated to chain length (Vermeirssen et al., 2004). Peptides from chicken muscle and ovalbumin failed to show antihypertensive activity in SHR (Fujita et al., 2000). The authors classified the peptides into three groups: (1) an inhibitor type that is not affected after preincubation with ACE; (2) substrate-type peptides that are hydrolysed by ACE to give peptides with weaker activity; and (3) pro-drug-type peptides that are converted to true inhibitors by ACE or gastrointestinal proteases. Peptides belonging to the 1st and the 3rd groups exert antihypertensive activities in SHR even after oral administration. Dried bonito bowel (`Katsuobushi') is a seasoning used in Japan, made from thin slices of boiled, dried bonito. Thermolysin hydrolysate of bonito bowels has shown in vitro ACE inhibitory activities (Yokoyama et al., 1992) that are increased 16-fold by treating it with ultrafiltration and chromatography (Fujii et al., 1993). The hydrolysate reduced systolic blood pressure in SHR and humans (Fujita et al., 1995). The active peptides were isolated and the effects confirmed in an in vivo test on SHR (Karaki et al., 1993). The LKPNM peptide, which was isolated from the thermolysin digest of dried bonito, is a pro-drug ACE-inhibitor whose activities were increased 8-fold by ACE itself and which showed a prolonged effect after oral administration (Fujita and Yoshikawa, 1999; Yoshikawa et al., 2000). Recent results suggest that the antihypertensive mechanism of the effect induced by dried bonito peptides involves direct action on vascular smooth muscle in addition to ACE-inhibitory activity (Kouna et al., 2005). Sardine protein hydrolysate produced by Bacillus licheniformis alkaline protease showed inhibitory effects in both in vitro and in vivo tests with SHR before and after in vitro digestion. When FPH was orally administered at a dosage of 2.0 g protein/kg to SHR, blood pressure was reduced and the reduction continued for 6 hours after administration. A diet containing FPH as the sole protein source had a stronger hypotensive effect and a higher survival ratio in stroke-prone SHR than a commercial diet (Sugiyama et al., 1991; Matsui et al., 1993). The most active peptide is a dipeptide VY that has a significant antihypertensive effect on mildly hypertensive people via angiotensin-I-converting enzyme inhibition, as well as on SHR (Kawasaki et al., 2000). Other raw materials that have been converted into hydrolysates and active peptides with ACE-inhibitory effects include Alaska pollack frames (Je et al., 2004) and skin (Byun and Kim, 2001); cod frames (Jeon et al., 1999) and heads (Bordenave et al., 2002); skipjack tuna (Astwan et al., 1995) and tuna broth (Hwang and Ko, 2004); shrimp (Bordenave et al., 2002 and Hai-Lu et al., 2006);

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Table 18.3 Examples of ACE inhibitory/hypotensive effects of commercial fish protein hydrolysates from the PROPEPHEALTH project compared with reference sample Sample

ACE IC50 (g. mLÿ1)*

Captopril Blue whiting 1 Blue whiting 2 Cod Plaice Saithe Salmon Portuguese dogfish

4.78 10ÿ3 50 1350 75 4 200 220 260

* IC50 corresponds to the hydrolysate concentration (g.mLÿ1) inhibiting 50% of ACE activity.

salmon and chum salmon (Bordenave et al., 2002 and Ono et al., 2003); fermented sauce of pearl oysters (Katano et al., 2003); fermented mackerel (Itou and Akahane, 2004); sea bream scales (Fahmi et al., 2004), yellowfin sole (Jung et al.,2006); and finally fermented surimi (Shan et al., 2007) and hydrolysed kamaboko (Nagai et al., 2006). Commercial samples of FPH from the companies Copalis (France), Marinova (Denmark) and Primex (Iceland), partners in the PROPEPHEALTH project, were screened for ACE inhibititory effects. In vitro ACE inhibitory activities of some of the fish hydrolysates in PROPEPHEALTH are summarised in Table 18.3. Most of them exhibited a moderate ACE inhibitory activity with IC50 ranging from 4 to 1350 g.mLÿ1 of hydrolysate. The highest activity was measured for plaice hydrolysates (IC50 = 4 g.mLÿ1), which was still 836-fold less active than captopril, the most potent synthetic inhibitor of ACE. These results are in agreement with previous data indicating that fish protein hydrolysates constitute a good source of peptides, exerting moderate activity on ACE. LKPNM, the pro-drug peptide isolated from hydrolysed dried bonito, had IC50 = 2.4 g.mLÿ1 that increased 8-fold in activity when hydrolysed by ACE (Fujita and Yoshikawa, 1999). 18.4.2 Antioxidant properties There is a growing interest in finding safe and natural antioxidants that enhance the body's antioxidant defences through dietary supplementation, and inhibit lipid oxidation in foods. Under normal conditions, reactive oxidative species (ROS) are effectively eliminated by the antioxidant defence system, such as antioxidant enzymes and nonenzymatic factors. However, under pathological conditions, the balance between the generation and the elimination of ROS is broken and biomacromolecules, including DNA, are damaged by ROS-mediated oxidative stress. Lipid peroxidation in foods affects the nutritive value and may cause disease following the consumption of products that could potentially cause a toxic reaction (Je et al., 2007; Jung et al.,, 2007).

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A list of published experiments on antioxidative activities of FPHs and peptides can be seen in Table 18.2. An ultrafiltrated fraction of 10±30 kDa with a high antioxidant activity was isolated from cod frames hydrolysed with crude protease extracted from tuna pyloric caeca (Jeon et al., 1999). Antioxidative peptide was isolated from tuna cooking juice hydrolysed by Protease XXIII, from Aspergillus oryzae (Jao and Ko, 2002). The antioxidant activity of fish enzyme hydrolysates could be improved 20±30% by reacting them with glucose (GueÂrard and Sumaya-Martinez, 2003). An antioxidative peptide of 10 amino acids was isolated from yellowfin sole frame proteins (Jun et al., 2004). The same group of scientists also tested Alaska pollack frames hydrolysed with a mackerel intestine crude enzyme. They isolated a six amino acid peptide after fractionation with ultrafiltration and further purification using chromatographic methods (Je et al., 2005). The most active peptide from tuna backbones had 14 amino acid residues. It significantly inhibited lipid peroxidation in a linoleic acid emulsion system and also quenched free radicals (DPPH, hydroxyl and superoxide) in a dose-dependent manner (Je et al., 2007). Antioxidant peptides were also isolated from giant squid muscle and skin (Mendis et al., 2005b; Rajapakse et al., 2005). A seven amino acid peptide was isolated from hoki skin gelatine after hydrolysis with trypsin (Mendis et al., 2005a). Another group has recently published results on the antioxidative properties of round scad and yellow stripe trevally. The results revealed that the antioxidative activity of protein hydrolysates from yellow stripe trevally meat was determined by the degree of hydrolysis and the enzyme type used (Klompong et al., 2007; Thiansilakul et al., 2007a, 2007b). Antioxidative peptides can also be found in processed seafood. An antioxidative peptide with five amino acids was isolated from blue mussel sauce after 6 and 12 months fermentation (Jung et al., 2005). Kamaboko, a surimibased ready-to-eat product, also showed higher oxidative activities after hydrolysis using three gastrointestinal proteases and protein proteases (Nagai et al., 2006). These results were confirmed on walleye pollack hydrolysates. The authors conclude that Kamaboko products, with high amounts of essential amino acids, are of benefit not only for health food diets but also to patients with various diseases such as cancer, cardiovascular diseases and diabetes (Nagai et al., 2007). Antioxidant and free-radical scavenging activities were demonstrated in many FPHs in the PROPEPHEALTH project of SEAFOODplus (Chabeaud et al., 2006a). More active fractions of saithe and shrimp processing-waste hydrolysates could be isolated using ultrafiltration and nanofiltration (Chabeaud et al., 2006b; GueÂrard et al., 2007). Table 18.4 summarises the results of the antioxidant assays in the PROPEPHEALTH project (GueÂrard, 2006a). 18.4.3 Immunostimulation There is increasing interest in identifying new immunomodulators to enhance non-specific host defence mechanisms for the improvement of stress-induced

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Table 18.4 Antioxidative properties of commercial fish protein hydrolysates from the PROPEPHEALTH project compared with reference samples Samples

FPH from PROPEPHEALTH Ascorbic acid BHA Trolox EDTA

-carotene IC50

DPPH scavenging assay IC50

Chelating activity IC50

Reducing power at OD = 0.5 (mg/mL)

0.17±1.8

10±36

0.3±7.7

4.1±18.5 0.043 0.057 0.084

0.0049

0.056

0.06

From GueÂrard 2006a

immunosupression and general well-being and as a way to reduce treatment costs. Immunostimulants are used in aquaculture, enhancing the resistance of cultured fish to disease and stress. Acid peptide fractions from Atlantic cod stomach hydrolysate stimulated superoxide production in Atlantic salmon macrophages (Gildberg et al.,1996) and supplementing the feed of juvenile fish with cod milt cationic proteins improved their resistance to this bacterial infection (Pedersen et al., 2004). SeacureÕ is a protein supplement derived from the fermentation of fish protein using a proprietary yeast strain. Its effects on the mucosal immune response in an in vivo model were evaluated and the conclusion was that the product is an immunomodulating food with a demonstrated capacity to enhance non-specific host defence mechanisms (Duarte et al., 2006). 18.4.4 Secretagogue and calciotropic activities Gastrin and cholecystokinin (CCK) are small intestinal hormones belonging to the secretagogue family. Cholecystokinin is a peptide hormone that reduces gastic acid secretion and stimulates the intestinal digestion and absorption of fat and protein, and controls satiety/appetite. CCK mediates satiety by acting on the CCK receptors (CCK1 and CCK2) distributed widely throughout the central nervous system. Administering CCK to humans causes nausea and anxiety and weakly decreases the desire to eat (Fink et al.,1998). The calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide derived from the calcitonin gene with widespread effects such as in the heart, blood vessels, pituitary, thyroid, lung and gastrointestinal tract, where it decreases food intake by expressing gastric acid secretion, and with a wide array of biological effects including neuromodulation, vasodilatation, cardiac contractility and bone growth (Wimalawansa, 1996). The presence of a biologically related CGRP molecule in sardine hydrolysates has been reported (Fouchereau-Peron et al.,1999). CCK-like peptides and a calcitonin gene-related peptide (CGRP)

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respectively were detected in Alcalase(R) hydrolysates of cooked sardine wastes (Ravallec-PleÂ et al., 2001, Rousseau et al., 2001) and measured in cod muscle and shrimp head extracts and alcalase hydrolysates (Fouchereau-Peron et al., 1999, Ravallec-PleÂ and Wormhoudt, 2003). The FPHs in the PROPEPHEALTH project were screened for gastrin/ cholecystokinin (CCK) and calcitonin gene-related peptide-like molecules. The highest activities were detected in Portuguese dogfish or siki hydrolysates but activities were also found in cod hydrolysates. Ultrafiltration had no effect on the yield of gastrin/CCK-like molecules but improved the yield of CGRP in a siki protein hydrolysate. Molecular exclusion chromatography was used to purify and isolate a 1.5 kDa fraction that could stimulate adenylate cyclase activity (Martinez-Alvarez et al., 2007). 18.4.5 Other properties Diazepam-like effects of cod protein hydrolysate on stress responsiveness in rats have been reported (Bernet et al., 2000) and the authors concluded that these effects of the hydrolysate (Gabolysat PC60) agree with the anxiolytic properties of this nutritional supplement, previously reported in both rats and humans. High-fat feeding led to severe whole body and skeletal muscle insulin resistance in rats fed with casein or soy protein, but feeding them cod protein fully prevented the development of insulin resistance. It was demonstrated that feeding cod protein prevents obesity-induced muscle insulin resistance in highfat-fed obese rats, at least in part through the direct action of amino acids on insulin-stimulated glucose uptake in skeletal muscle cells (Lavigne et al., 2001; Tremblay et al., 2003). The antiproliferative activity of FPH from the companies Copalis (France), Marinova (Denmark) and Primex (Iceland) was measured in the PROPEPHEALTH project of SEAFOODplus on two human breast-cancer cell lines grown in vitro. Samples of blue whiting, cod, plaice and salmon hydrolysates were identified as significant growth inhibitors on the two cancer cell lines (Picot et al., 2006).

18.5 Functional properties of dried marine proteins and peptides Food proteins, especially soy and dairy proteins but also fish proteins are used in many food applications because of their good functional properties, i.e. their water-binding capacity, oil-holding capacity, viscosity, foaming properties, emulsifying properties, gelation and solubility. Functional properties are influenced by the protein source, and production and environmental parameters. Production parameters are isolation, precipitation, drying or dehydration, concentration and modification (enzymatic, alkaline, acid hydrolysis, chemical), and environmental parameters include temperature, pH and ionic strength. Each protein has a different isoelectric point based on its source and thus the

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functionality of proteins differs when measured at different pH values (Kinsella, 1976). Enzymatic hydrolysis changes the functional properties of fish proteins. Heating is used to stop enzyme reaction and in the drying of FPH. It may have a deleterious effect on the functional properties and on nutritional and quality factors because of the denaturation of proteins (Kinsella, 1976). Spray drying is considered a gentle form of drying and gives a product where some retention of functional properties can be observed. Drum drying can, on the other hand, cause losses in solubility. 18.5.1 Water-holding capacity Good water-holding capacity in fish is important as it affects both the economic and sensory attributes of the products. There is about 80% water in lean fish muscle and most of it is held within the myofibrils, mainly by capillary forces. The water in fish muscle is divided into bound water, immobilised water and free water. Bound water is less than 10% of the total and forms layers of water molecules around the myofibrillar proteins. This water has reduced mobility and is very resistant to freezing and evaporation by heat. Immobilised water is held within the structure of the muscle but is not bound to proteins. Factors that can influence the retention of immobilised water include manipulation of the net charge on the myofibrillar proteins and the structure of the muscle cell and its components, as well as the amount of extracellular space within the muscle itself. Free water flows from fish during pressing or centrifugation (Kolczak et al., 2007) and can be lost from fish muscle during handling, transportation, storage and processing. The goal of many fish processors is to maintain as much water as possible. Shrinkage during cooking causes water to be expelled out of the fish by pressure and causes the great water losses that occur with heating, which depend on the method of heating. Too much shrinkage influences the quality and sensory properties of the cooked fish. Maintaining low cooking loss is therefore the goal of both the processors as well as the buyers and users of the fish. Factors that affect the water-binding capacity of proteins are, for example, protein concentration, pH, ionic strength, temperature, other food components such as polysaccharides, lipids and salts, and rate and length of heat treatment (Zayjas, 1997). Very little has been documented about the use of dried hydrolysed fish protein in injected or tumbled products. It has been reported that dried cod protein hydrolysates have better water-holding capacity than soy proteins but that the soy protein results in lower drip and higher cooking yield (Thorarinsdottir et al., 2004). 18.5.2 Solubility Intact fish myofibrillar proteins are quite insoluble in water over a wide pH range. Smaller peptides produced by hydrolysis have more of the hydrophilic

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polar amino acid side groups exposed and can bind more readily to water than the intact protein can (Kristinsson and Rasco, 2000). High solubility over a wide range of pH is important for many food applications as it influences other functional properties, such as emulsifying and foaming. Good solubility for fish protein hydrolysates over a wide range of pH that increased with the degree of hydrolysis has been reported several times (Gbogouri et al., 2004; Geirsdottir et al., 2007; Klompong et al., 2007; Sathivel and Bechtel, 2006; Sathvivel et al., 2005; Shahidi et al., 1995; Thiansilakul et al., 2007a). 18.5.3 Emulsifying properties There is a growing trend within the food industry to replace synthetic emulsifiers with more natural ones. Proteins can be used as emulsifiers in foods because of their ability to facilitate formation, improve stability, and produce desirable physicochemical properties in oil-in-water emulsions. The protein emulsifiers have the advantage of protecting polyunsaturated lipids from iron-catalysed oxidation (Hu et al., 2003). At pH values below their isoelectric point, proteins form positively charged interfacial membranes around oil droplets that electrostatically repel any Fe2+ and Fe3+ ions present in the aqueous phase. Thus, iron is prevented from catalysing oxidation of the polyunsaturated lipids contained within the droplets. The emulsifying properties of many fish proteins and fish protein hydrolysates have been reported. Interfacial activities (emulsion activity index, emulsion stability index, foaming capacity, foam stability) increase with limited hydrolysis but increased hydrolysis decreases them (Thiansilakul et al., 2007b; Klompong et al., 2007; Sathivel and Bechtel, 2006; Sathvivel et al., 2005; Gbogouri et al., 2004; Shahidi et al., 1995). Good solubility, emulsifying activity, and cooking stability have been reported for purified fish collagen (Kim and Park, 2005). Initial heating of the raw material before hydrolysis decreased the emulsifying properties of FPH (Slizyte et al., 2005). FPHs often show poorer emulsifying properties than dairy and soy proteins. The emulsifying capacity and stability were found to be lower in hydrolysed herring and herring by-products than in egg and soy proteins (Sathivel et al., 2003) and fish gelatin had a much lower oil-in-water emulsification capacity than beta-lactoglobulin (Suhr et al., 2006). The superior emulsifying properties of sodium caseinate, the susceptibility of whey protein emulsions to increasing flocculation on storage, and the coalescence of fish gelatin emulsions following centrifugation have been demonstrated (Dickinson and Lopez, 2001). Freezedried salmon protein hydrolysates had comparable solubility to that of egg albumin and their water-holding capacity was better than that of egg albumin or soy protein but their emulsifying properties were similar or lower (Kristinsson and Rasco, 2002). However, a soluble fraction of ca.12 kDa of sardine sarcoplasmic proteins heated to over 60 ëC showed excellent emulsifying activity (Kawai et al., 1995). There are surprisingly few reports on the emulsifying properties of purified

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fractions or components of FPH. Two protein fractions extracted from cod were able to form and stabilise oil-in-water emulsions (PeÂtursson et al., 2004). A fraction between 10 and 30 kDa of cod frame protein hydrolysates showed excellent emulsion properties and whippability (Jeon et al., 1999). Moderate foaming and emulsifying capacities and stabilities have been reported in some of the commercial FPH tested in the PROPEPHEALTH project (Geirsdottir et al., 2005). 18.5.4 Sensory challenges The flavour of protein hydrolysates depends on the raw material, the kind of protease applied and the hydrolytic conditions. A major problem with most fish protein hydrolysates is their bitter flavour (Kristinsson and Rasco, 2002). The bitterness is normally caused by a number of medium-sized peptides with hydrophobic amino acid residues. Principally, this problem may be solved either by performing mild hydrolysis to reduce the production of medium-sized peptides or by running extensive hydrolysis to digest the troublesome peptides to free amino acids. Although mild hydrolysis may improve both flavour and nutritional properties, it will normally reduce the yield significantly. The latter is a big problem if maximal utilisation of the raw material is a major concern. Extensive digestion is probably more convenient although it may reduce nutritional quality. Producers of commercial enzymes claim that the problem of bitterness may be solved by applying certain enzyme products with specific `non-bitter' properties. However, this does not always hold true (Gildberg et al., 2002). Lipid oxidation is a problem in dried fish proteins and fish protein hydrolysates. Kristinsson and Rasco (2002) suggested producing powder from lean fish by enzymatic hydrolysis and spray drying since the problems of lipid oxidation can be reduced by mild processing conditions. Lipid oxidation is influenced by storage time and temperature. The prospects for using fish powder as a food ingredient depend on whether it is possible to stabilise the residual lipid by suitable processing techniques. Measures must be taken to prevent oxidation during all processing steps. Oxidation problems could be controlled by washing the lipids and heme proteins from the raw material, by adding antioxidant before hydrolysis and by selecting enzymes that do not operate at low pH and high temperatures (Kristinsson, 2006). There are very few publications on the oxidative stability of dried fish powders. Bragadottir et al. (2007) reported advanced lipid oxidation in fresh spray-dried enzyme-hydrolysed saithe powder. They recommended ways of optimising processing parameters including reducing access to prooxidants and oxygen, preserving endogenous antioxidants in the raw material by mild processing techniques and using added antioxidants. The drying of pH-shift processed protein could even increase the problem (Geirsdottir, 2006).

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18.6 Market for functional marine proteins and peptide products The market for extracted, hydrolysed, isolated and dried functional protein and peptide products is not very large compared with that of dairy and plant proteins and peptides. The traditional and most significant market is in seafood flavours and extracts for several small companies with products in liquid form, as pastes, dried flakes or powders. The markets for them are primarily in Europe and Asia. The annual production of collagen/gelatine is about 300 thousand tonnes but fish gelatine accounts for only 1±2% of the production. It is sold for both its physical and bioactive properties. The companies Marinova and Copalis in the PROPEPHEALTH project are on this market and are focussing their attention more towards the functional food and food supplement market. It is the fastest growing part of the food market. In Japan it grew from 3.8 billion dollars in 1990 to 17 billion dollars in 2006. FOSHU (Food for Specified Health Use) accounts for 30% of that market. There are many products containing proteins and peptides, which are mainly soy and dairy based. The number of collagen-based products is growing very quickly and the number of collagen health foods and supplement products is remarkable (Functional Foods Japan, 2006). In Europe and the United States they are mostly sold either as food supplements or to the cosmetic industry. Fish collagen peptides have a variety of functions, the most representative ones being improving skin quality and preventing increases in blood pressure. In particular, they have been found to improve skin dryness and roughness, and are therefore already being used widely in health and beauty applications. There are far fewer fish protein and peptide products with approved health claims in comparison to similar soy and dairy products, with none in Europe and North America. They are mostly sold as food supplements and Table 18.5 shows examples of some of them. Only two products, both from Japan, have been approved by the authorities. They have achieved FOSHU status and both are claimed to reduce blood pressure. One is the Katsuobushi oligopeptide made by hydrolysis of dried bonito with the enzyme thermolysin. It is marketed as PEPTIDE ACE 3000 in Japan and was the first dietary supplement that was FOSHU approved. Its sales in 2005 were worth 3.5 million dollars (Functional Foods Japan, 2006). It is also marketed in the United States as VasotensinÕ and PeptACETM and in Canada as LevenormTM. The other is the sardine peptide SP100N, a hydrolysed extract from sardine muscle that as well as other products is sold as a drink, LAPIS SUPPORT, in Japan and achieved sales worth 1.5 million dollars in 2005. SEACUREÕ, a white fish protein hydrolysate concentrate, has been on the market since 1994 in the United States. It is claimed to support the cells in your gastrointestinal tract and regulate bowel functions. NutripeptinÕ is a peptide powder for reducing blood sugar, made from codfish from France/ Norway. It can be added to several different types of food such as bread, chocolate, ice cream, hamburgers, and beverages.

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Table 18.5 Examples of commercially available functional foods or food ingredients carrying bioactive peptides Product name

Manufacturer

Hydrolysed dried bonito bowels Peptide ACE 3000 Nippon Supplements, Japan

Type of food

Health claims

References

Nutraceutical Soup mix Nutraceutical Nutraceutical Nutraceutical

Lowers blood pressure

www.nippon-sapuri.com/english/

Lowers blood pressure Lowers blood pressure Lowers blood pressure

www.metagenics.com http://us.naturalfactors.com/ http://www.onc.ca/

VasotensinÕ PeptACETM LevenormTM

Meatgenics, USA Natural factors, USA Ocean Nutrition, Canada

Peptides from sardines Lapis Support Peptidea

Tokiwa Yakuhin, Japan Abyss Ingredients

Functional drink

Lowers blood pressure Relaxing

http://www.tokiwayakuhin.jp/

Collagen peptides Bifidus & Collagen EnocerideÕ

Kagome Japan Laboratories LeStum

Yogurt drink Nutraceutical

Beautifies the skin

http://www.kagome.co.jp/ http://www.labo-lestum.com/

Hydrolysed whitefish Seacure

Proper Nutrition, USA

Nutraceutical

http://www.propernutrition.com/

Protizen AntiStress 24

Copalis, France Forte Parma, France

Nutraceutical Nutraceutical

Improves gastrointestinal health Relaxing Relaxing

Fortidium Nutripeptin

Nutraceutical

Food ingredient

Reduces oxidative stress Lowers glycaemic index

Maripep

Marinova, Denmark

Food ingredient

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http://www.copalis.fr/ http://www.fortepharma.com/fr/ index.html http://www.biothalassol.com/ www.nutrimarine.com; www.copaplis.fr

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Future trends

This chapter is an overview of the latest developments in the use of mild processing techniques for the production and use of functional marine protein and peptide ingredients. The pH-shift methods show great promise for the better utilisation and upgrading of by-products and underutilised species (Kristinsson et al., 2006). Applying protein isolates as water binders in injected and tumbled products will result in greater additional economic, nutritional and environmental values by increasing the yield of raw materials in the fish filleting operation and the production of ready-to-eat seafood products. There would be an even greater economic advantage if pH-shift methods could be used to produce high-quality isolates from raw material that today is unfit for traditional processing. There are oxidation and technological problems that must be solved and much more research and development are needed into applying the isolates as commercial food ingredients or food products. Fish protein hydrolysates cannot compete on price, size and quality with plant and dairy proteins in the functional ingredient market. Plant and dairy ingredients will continue to be a part of formulating ready-to-eat convenience seafood products and the marine ingredients will be used for culinary and nutritional reasons and for their special bioactive properties. But we still have a long way to go. More research is needed into process optimisation and how to scale up the hydrolysis, separation and concentration processes, and how to solve problems with lipid oxidation. It takes a long time to go from molecules to megatonnes. There are many small companies like Marinova and Copalis but no large-scale factories for manufacturing specific marine proteins and peptides as there are for fish meal and for protein ingredients from other sources. Ten years ago it was concluded in a review article that biotechnology within the fish industry was still in its extreme infancy compared with other areas of food production and processing. The main reasons were said to be the small number and diversity of utilised species and processing methods of the industry compared with agricultural production, and it was forecast that the biotechnological fish processing industry would have a long gestation period (Vilhelmsson 1997). Since then the production and utilisation of FPH have come a long way, as has been described in this chapter. Future priorities for research into healthy, safe and nutritious seafood, based on results from SEAFOODplus, were presented at the 2nd Joint Trans-Atlantic Fisheries Technology Conference in QueÂbec City 2006 (Bùrresen, 2006). One of the priorities was to develop lean, nutrious, tasty and convenient seafood products to control weight and reduce obesity in Western populations. But there are other needs that must also be addressed. Some of them have been mentioned in this chapter and in some cases there are already products on the market to meet those needs. More supplements from FPH can be developed to reduce high blood pressure but they will face heavy competition from other protein sources. The antioxidant properties of FPH can be employed in supplements and food products to enhance the antioxidant defences of the body against oxidative stress. They can

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also be used as immunomodulators to enhance non-specific host defence mechanisms. Specific protein products can even be made to control food intake in the fight against obesity and there are also products on the market and future possibilities for developing FPH to lower the glycaemic index. The market for such products made from fish proteins is not big but it will grow and there are also opportunities for adapting traditional food processes like fermentation to enhance the bioactive properties of FPH and to use them in products that consumers already know. Low-salt fish sauce and fish flavours with tailor-made bioactive properties are likely the future. Bioactive properties and functional seafood bring us straight to the complicated situation that exists for the scientific documentation and official acceptance of health claims. Sufficient scientific evidence must be produced if companies are to produce and sell products with health claims. It means providing evidence that the active ingredient is present in the quantity and the form needed to perform a specific function, providing evidence from human studies, using a valid scientific method, of the effect of the food or food component and, finally, evaluating and excluding any risk that the consumption of the products could pose to public health, including allergic potential. Getting a health claim accepted requires much research and legal work. Private companies, universities and other research organisations can work together on special hydrolysates or peptides but the cost might be too high for small companies, so a global collaboration may be needed in the interests of fisheries, fish processing industries and consumers worldwide.

18.8

Sources of further information and advice

The following books are recommended for further information as they are of special relevance to fish proteins and peptides and the upgrading of co-products and underutilised species. 2004 M. Sakagushi (ed.), More Efficient Utilization of Fish and Fisheries Products. Elsevier Science Publishing Company, London, England, 464 pp. 2005 J.W. Park (ed.), Surimi and Surimi Seafood, 2nd edn. CRC Press, Boca Raton, FL, USA, 960 pp. 2006 Y. Le Gal and R. Ulber (eds.), Marine Biotechnology I. Advances in Biochemical Engineering/Biotechnology. Vol. 96. Springer Verlag, Berlin, Germany, 288 pp. 2006 F. Shahidi (ed.), Maximizing the Value of Marine By-Products. Woodhead Publishing Limited, Cambridge, England. 560 pp. The following websites are suggested: · http://www.functionalfoodnet.eu/ is a network and an information centre for companies and contains many results from functional food research funded by the EU Commission under its framework programmes.

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· http://www.nordicinnovation.net/ is the home page of the Nordic Innovation Centre. It supports five Nordic projects on functional foods with a common goal to support the Nordic food industry in becoming more innovative and competitive in the functional food market, targeting health claims for marine functional foods, consumer acceptance of marine functional foods, innovative consumer-driven marine functional food products and ingredients development, networking, cooperation and international positioning. · http://www.marifunc.org/ is one of the Nordic projects and it focuses on the use of fish, nutrients and other bioactive substances isolated from fish as ingredients in functional foods.

18.9

Acknowledgements

The contribution of all participants in PROPEPHEALTH in SEAFOODplus is gratefully acknowledged. Also the financial support from the Nordic Functional Food programme of the Nordic Innovation Centre, the Fund for Added Value of Seafood and the Technology Development Fund in Iceland is also gratefully acknowledged.

18.10

References

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functional properties of its enzymatic hydrolysates', Journal of Food Agriculture & Environment, 5 (2), 76±81. NAKAMURA Y, YAMAMOTO N, SAKAI K, OKUBO A, YAKASAKI S and TAKANO T (1995), `Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk', J Dairy Sci, 78 (4), 777±783. NISHIOKA F and SHIMIZU Y (1983), 'Recovery of proteins from washings of minced fish by pH-shifting method', Nippon Suisan Gakkashi, 49 (4), 795±800. NOLSOE H, IMER R and HULTIN H O (2007), `Study of how phase separation by filtration instead of centrifugation affects protein yield and gel quality during an alkaline solubilisation process ± different surimi-processing methods', International Journal of Food Science & Technology, 42 (2), 139±147. NORDVI B, EGELANDSDAL B, LANDSRUD O, OFSTAD R and SLINDE E (2007). `Development of a novel, fermented and dried saithe and salmon product', Innovative Food Science and Emerging Technologies,8 (2), 163±171. OKASAKI T, SHIGETA Y, AOYAMA Y and MAMBA K (2003), `Autolysis of unsalted fish protein under pressurization', Fisheries Science 69 (6), 1257±1263. ONO S, HOSOKAWA M, MIYAHITA K and TAKAHASHI K (2003), `Isolation of peptides with Angiotensin I-converting enzyme inhibitory effect derived from hydrolysate of upstream chum salmon muscle', J Food Sci, 68 (5), 1611±1614. PARK J W, LIN T M and YONGSAWATATDIGUL J (1997), `New developments in manufacturing of surimi and surimi seafood', Food Rev Int, 13 (4), 577±610. PEDERSEN G M, GILDBERG A and OLSEN R (2004), `Effects of including cationic proteins from cod milt in the feed to Atlantic cod (Gadus morhua) fry during a challenge trial with Vibrio anguillarum', Aquaculture, 233 (1±4), 31±43. PETTY H T and KRISTINSSON H G (2004), `Impact of antioxidants on lipid oxidation during acid and alkali processing of Spanish mackerel', IFT Annual Meeting, Las Vegas, NV, Abstract 49B-7. PETURSSON S, DECKER E A and MCCLEMENTS D J (2004), `Stabilization of oil in water emulsions by cod protein extracts', J Agric Food Chem, 52 (12), 3996±4001. PICOT L, BORDENAVE S, DIDELOT S, FRUITIER-ARNAUDIN I, SANNIER F, THORKELSSON G, BERGEÂ J P, GUEÂRARD F, CHABEAUD A and PIOT J M (2006), `Antiproliferative activity of fish

hydrolysates on human breast cancer cell lines', Process Biochemistry, 41 (5), 1217±1222. PIRES C, GODINHO V, BATISTA I and RIBEIRO A T (2007a), `Preparation of Frankfurt fish sausages with proteins recovered from Cape hake by-products', in Actas do 8ë. Encontro de QuõÂmica dos Alimentos;Alimentos Tradicionais, Alimentos SaudaÂveis e Rastreabilidade, Instituto PoliteÂcnico de Beja, Escola Superior AgraÂria de Beja, Sociedade Portuguesa de QuõÂmica. Beja, MarcËo, 99±103. PIRES C, BATISTA I, FRADINHO P and COSTA S (2007b), `Utilization of alkaline-recovered proteins from Cape hake by-products in the preparation of Frankfurter-type sausages', in Book of abstracts of the 37th WEFTA annual meeting, Ipimar, Lisbon. RAJAPAKSE N, MENDIS E, BYUN H G and KIM S K (2005), `Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems', Journal of Nutritional Biochemistry, 16 (9), 562±569. RAVALLEC-PLEÂ R and VAN WORMHOUDT A (2003), `Secretagogue activities in cod (Gadus morhua) and shrimp (Penaeus aztecus) extracts and alcalase hydrolysates determined in AR4-2J pancreatic tumour cells', Comp Biochem Physiol B, 134, 669± 679.

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RAVALLEC-PLEÂ R, CHARLOT C, PIRES C, BRAGA V, BATISTA I, VAN WORMHOUDT A, LE GAL Y

and FOUCHEREAU-PERON M (2001), `The presence of bioactive peptides in hydrolysates prepared from processing wastes of sardine (Sardina pilchardus)', J Sci Food Agric, 81 (11), 1120±1125. ROUSSEAU M, BATISTA I, LE GAL Y and FOUCHEREAU-PERON M (2001), `Purification of a functional antagonist for calcitonin gene related peptide action from sardine hydrolysates', Electronic Journal of Biotechnology, 4 (1), 1±8. RUSTAD T (2003), `Utilisation of marine by-products', Elec J Env Agric and Food Chem, 2, 458±463. SATHIVEL S and BECHTEL P J (2006), `Properties of soluble protein powders from Alaska pollock (Theragra chalcogramma)', Int J Food Sci Technol, 41 (5), 520±529. SATHIVEL S, BECHTEL P J, BABBIT B, SMILEY S, CRAPO C, REPPOND K D and PRINYAWIWATKUL (2003), `Biochemical and functional properties of herring (Clupea harengus), byproduct hydrolysates', J Food Sci, 68 (7), 2196±2200. SATHIVEL S, SMILEY S, PRINYAWIWATKUL W and BECHTEL P J (2005), `Functional and nutritional properties of red salmon (Oncorhynchus nerka) enzymatic hydrolysates', J Food Sci, 70 (6), C401±C408. SEKI E, OSAJIMA K, MATSHUFUJI H, MATHUI T and OSAJIMA Y (1996), `Resistance to gastrointestinal proteases of the short chain peptides having reductive effect in blood pressure', Journal of the Japanese Society of Food Science and Technology ± Nippon Shokukin Kagaku Kogaku Kaishi, 43 (5), 520±525. SENTANDREU M A and TOLDRA F (2007), `Oligopeptides hydrolysed by muscle dipeptidyl peptidases can generate angiotensin-I-converting enzyme inhibitory dipeptides', European Food Research and Technology, 224 (6), 785±790. SHAHIDI F (2006), Maximising the value of marine by-products, Cambridge, Woodhead. SHAHIDI F, HAN X-Q and SYNOWIECKI J (1995), `Production and Characteristics of protein hydrolysates from capelin (Mallotus villosus)', Food Chemistry, 53 (3), 3285± 3293. SHAN J, OGAWA Y, WATABE T, MORIMOTO R, OOTA S, SEIKI M and MIYAMOTO T (2007), `Angiotensin I-converting enzyme inhibitory activity of fermented surimi by lactic acid bacteria', Journal of the Japanese Society of Food Sciene and Technology ± Nippon Shokukin Kagaku Kogaku Kaishi, 54, 160±166. SHIMIZU Y, TOYOHARA H and LANIER T (1992), `Surimi production from fatty and dark fleshed fish species', in Lanier and Lee, Surimi Technology, New York, Marcel Dekker, 181±207. SIMON A, VANDANJON L, LEVESQUE G and BOURSEAU P (2002), `Concentration and desalination of fish gelatine by ultrafiltration and continuous diafiltration processes', Desalination, 144 (1), 313±318. SLIZYTE R, DAUKSAS E, FALCH E, STORRé I and RUSTAD T (2005), `Yield and composition of different fractions obtained after enzymatic hydrolysis of cod (Gadus morhua) byproducts', Process Biochemistry, 40 (3±4), 1415±1424. SUGIYAMA K, TAKADA K, EGAWA M, YAMAMOTO I, ONZUKA H and OBA K (1991), `Hypotensive effect of fish-protein hydrolysate', Nippon Nogeikagaku Kaishi ± Journal of the Japan Society for Bioscience Biotechnology and Agrochemistry, 65 (1), 35±43. SUHR J, DECKER E A and MCCLEMENTS D J (2006), `Properties and stability of oil-in-water emulsions stabilized by fish gelatin', Food Hydrocolloids, 20 (5), 596±606. THIANSILAKUL Y, BENJAKUL S and SHAHIDI F (2007a), `Antioxidative activity of protein hydrolysate from round scad muscle using Alcalase and Flavourzyme', J Food Biochem, 31 (2), 266±287.

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and YOSHIKAWA M (1992), `Peptide inhibitors for angiotensin Iconverting enzyme from thermolysin digest of dried bonito', Biosci Biotechnol Biochem, 56 (10), 1541±1545. YONSAWATDIGUL J and PARK J W (2001), `Gelation characteristics of alkaline and acid solubilisation of fish muscle', IFT Annual Meeting. New Orleans, LA, Abstract 100-1. YOKOYAMA K, CHIBA H

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and TAKAHATA K (2000), `Bioactive peptides derived from food proteins preventing lifestyle-related diseases', Biofactors, 12 (1±4), 143±146 ZAYJAS J F (1997), Solubility of Proteins, Functionality of Proteins in Food. Berlin Heidelberg, Springer Verlag, Berlin, Germany, 6±67.

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19 Hurdle technology to ensure the safety of seafood products F. Leroi and J. J. Jofftaud, Ifremer, France, J. C. Arboleya, F. Amarita, Z. Cruz, E. Izurieta, A. Lasagabaster, I. MartõÂnez de MaranÄoÂn, I. Miranda, M. Nuin and I. Olabarrieta, AZTI-Tecnalia, Spain, H. L. Lauzon, Matis, Iceland, G. Lorentzen and I. Bjùrkevoll, Nofima, Norway, R. Olsen, University of Tromsù, Norway, M. F. Pilet, H. PreÂvost, X. Dousset and S. Matamoros, ENITIAA, France and T. Skjerdal, National Veterinary Institute, Norway

19.1

Introduction

The microbial safety and stability of most food are based on an application of preservative factors called hurdles. Each hurdle implies putting microorganisms in a hostile environment, which inhibits their growth or causes their death (Leistner, 2000). Some of those hurdles have been empirically used for years to stabilise meat, fish, milk and vegetables. This sometimes leads to completely different product with its own new taste characteristics. Examples of hurdles in marine products are salt (salted cod, klipfish), smoke (cold or hot smoked salmon, herring), acids (marinated products, pickles), temperature (high or low), fermentative microorganisms (traditional Asian sauces) and more recently redox potential (vacuum-packed products). Those preservative factors have been studied for years, but a large amount of potential hurdles for food have already been described, including organic acids, bacteriocins, chitosan, nitrate, lactoperoxidase, essential oil, modified atmosphere packaging, etc., as well as novel decontamination technologies such as microwave and radio frequency, ohmic and inductive heating, high pressure, pulsed electric field, high voltage arc discharge, pulsed light, oscillation magnetic field, ultraviolet light, ultrasound, X-ray, electrolyse NaCl water, ozone, etc. (Kim et al., 1999; Weber, 2000; Mahmoud et

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al., 2006). Hurdles that have a positive effect by inhibiting microorganisms may have a negative one on other parameters such as nutritional properties or sensory quality, depending on their intensity. As an example, salt content in food must be high enough to inhibit pathogens and spoilage microorganisms, but not so high to impair taste. In order to lower the preservative level, the hurdle technology concept has been developed (Leistner, 1985), consisting of using combined hurdles to establish an additive antimicrobial effect, and even sometimes a synergetic one, thus improving the safety and the sensory quality of food. For fish products manufactured in industrialised countries, the hurdle technology has been identified of the most interest for two groups of products: · Convenience products based on traditional products, like rehydrated saltcured or dried fish. The raw material is a preserved semi-finished (PSFP) product but as the preservative is removed during processing, surviving pathogens in the raw material may recover. Minimising the survival of pathogens in the PSFP is therefore, beside the hygienic process conditions, necessary to ensure product safety. · Lightly preserved fish products (LPFP) which are uncooked or mildly cooked products, with low level of preservatives (NaCl < 6% WP, pH >5), such as cold-smoked salmon (CSS), carpaccio, slightly cooked shrimp. LPFP are usually produced from fresh seafood and further processing involves one or a few additional steps that increase risk of cross contamination. The treatments are usually not sufficient to destroy pathogens, and, as several of these products are eaten raw, minimising the presence and preventing growth of pathogens is essential for food safety. Some microorganisms that do not represent a health risk for consumer may sometimes be responsible for organoleptic damages such as off-odours and taste, pasty texture, visual defaults, etc. Preventing the growth of those spoilage microorganisms is therefore also a challenge. This chapter focuses on five potential hurdles that might contribute to ensuring the microbial safety and quality of those two groups of convenience products: a traditional hurdle (salt), three innovative hurdles (bioprotective microorganisms, chitosan and bioactive packaging) and a novel decontamination technology (pulsed light). Some examples of application that have been developed within the framework of the HURDLETECH project from the SEAFOODplus Integrated Project will be specifically addressed.

19.2

Salt hurdle in seafood processing

Preservation methods like salt-curing and drying have been used for centuries to obtain fully preserved products and access to good, safe and nutritious food in all seasons and areas where the availability of fresh food is limited. Salt-cured cod, the precursor to klipfish, and known as the traditional product bacalao in Spain and bachalau in Portugal, has had this position for centuries, but today salt-cured

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cod is popular due to its sensory properties rather than lack of availability of other foods. However, the consumption decreases, and one important reason is the time consuming preparation. The salt-cured cod must be rehydrated (soaked in water) for at least 24 hours for most dishes in order to lower the salt concentration from approx. 20% to 2.5±3.0% before the meal can be prepared. Commercially soaked products have been developed in order to meet the demands for convenient products with the traditional taste of salt-cured cod. Salt-cured and dried fish products are generally regarded as safe, even though they are produced in relatively open houses with limited possibilities to regulate temperature and maintain good hygienic conditions. It is considered that saltcuring is an effective barrier against bacteria. However, rehydrated salt-cured cod spoils rapidly, and it is found that this is due to growth of Psychrobacter spp. These bacteria are present on the skin of fresh fish, survive in a nongrowing mode during salt-curing, but recover and grow during and after rehydration (Bjùrkevoll et al., 2003). A number of other bacteria have also been found to survive the salt-curing step (Vilhelmson, 1997; Skjerdal et al., 2002; Barat et al., 2006). Listeria spp. and Staphylococcus spp. are occasionally found in salt-cured cod products (Pedro et al., 2004), but it has not been clear whether these bacteria survive in the fish if introduced to the fish prior to salt-curing, or only when they are introduced directly to the salt-cured cod shortly before the sample is taken. Commercially rehydrated salt-cured cod is stored for some days from rehydration to consumption, indicating that surviving pathogenic bacteria may get the opportunity to grow before the consumer eats the product. Another element is that the salt-curing and rehydration processes are usually carried out in different countries. For risk management in a farm-to-fork perspective, it is therefore essential to know whether the salt-curing step eliminates the pathogenic bacteria or not. The objectives for our studies have been to investigate how salt-curing influence the survival of growth of pathogenic bacteria, primarily Listeria spp., that are introduced at different steps in the production process of salt-cured cod. Salt-cured cod products contain 15±21% salt, and the salt-curing period lasts for approximately three weeks. 19.2.1 Survival and growth of Listeria spp. during salt-curing and rehydration/soaking The survival of Listeria spp. and Staphylococcus spp. after exposure to high salt concentrations was performed in a semi-quantitative study in order to investigate whether it is likely that these bacteria survive salt-curing. The surrogate pathogen bacteria Listeria innocua and Staphylococcus xylosus were used as indicators for the pathogenic bacteria Listeria monocytogenes and Staphylococcus aureus, respectively. Those bacteria were inoculated in levels from log10 5 to log10 9 CFU mlÿ1 in fish juice supplemented with NaCl in the range 0±21% and stored for up to three weeks at 4 ëC. The fish juice was prepared by the method of Dalgaard (1995) from wild-caught, newly killed cod (Gadus morhua). The results are shown in Table 19.1. Li. innocua survived at all inoculation and stress

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levels for at least 21 days of incubation at 4 ëC. Similar experiments with Li. innocua and six Li. monocytogenes strains and inoculation levels of 2.5 log10 CFU ml-1 showed that most strains survived for at least 60 days in 21% NaCl (results not shown). Surviving S. xylosus was also detected after 21 days at all stress conditions when the highest inoculation level was used, but not for lower inoculation levels (Table 19.1). Thus, both Staphylococcus and Listeria are able to survive during exposure to high salt-concentrations. The survival of Li. innocua in cod during salt-curing and rehydration was further investigated in a quantitative study by inoculating newly caught cod with 1 to 6 log10 CFU g±1 prior to salt-curing. The obtained results from eight experiments are shown in Table 19.2. After salt-curing and rehydration, Li. innocua was present in the inoculated fish samples in levels less than 1 log10 CFU g±1 lower than the corresponding inoculation level. The growth of Li. innocua during storage of the rehydrated fish samples at 4 and 8 ëC were also analysed. When the fish was stored at 8 ëC, growth of Li. innocua was observed in most experiments within five days, and in all experiments after ten days. In fish stored at 4 ëC, on the other hand, growth was not observed in any of the experiments after five days, but in three of the experiments after ten days (experiments 2±4). The increase in Li. innocua levels between five and ten days in experiment 2±4 were within 1 0.3 log10 CFU gÿ1 in fish stored at 4 ëC. In experiment 1, there might have been a similar but undetected growth, as the inoculation level was below the detection level in the quantitative analysis. Li. monocytogenes and Li. innocua showed similar results (experiments 6±8). In conclusion, Listeria spp. are able to survive in cod during salt-curing, and after some time to recover and grow after rehydration. The lag phase indicates that Listeria introduced to the fish prior to salt-curing has limited impact on the food safety risk in rehydrated salt-cured cod unless it is stored at unsafe temperature. Table 19.1 Survival of L. innocua and S. xylosus during exposure to salted fish juice, incubated at 4 ëC from 3 to 21 days. Detection of colony forming bacteria are presented as present or ÿ absent, for inoculation levels 109/107/105 CFU mlÿ1, respectively Days

NaCl (%) 5

9

12

15

18

21

Li. innocua 3 21

// //

// //

// //

// //

// //

// //

St. xylosus 3 7 10 13 17 21

// //ÿ //ÿ // //ÿ //

// //ÿ // //ÿ //ÿ //ÿ

// //ÿ // //ÿ //ÿ /ÿ/

// //ÿ // //ÿ /ÿ/ /ÿ/ÿ

// //ÿ // /ÿ/ÿ /ÿ/ÿ /ÿ/ÿ

// /ÿ/ /ÿ/ÿ /ÿ/ÿ /ÿ/ÿ /ÿ/ÿ

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Table 19.2 Levels of L. innocua CCUG 15531 T (experiments 1±4, 7) and L. monocytogenes no 4006 (experiment 6) in cod prior to salt-curing, after rehydration and during storage at 4 and 8 ëC. Experiment 5 and 8 are uninoculated controls Experi- Inoculated ment bacteria

1 2 3 4 5 6 7 8

Listeria level (log10 CFU gÿ1 fish) Prior to After salt-curing rehydration

Li. innocua Li. innocua Li. innocua Li. innocua none Li. monocytogenes Li. innocua none

1.0* 2.7 4.5 6.3 nd na na nd

nd 1.7 4.0 5.6 nd 4.3 5.4 nd

During storage at 4 ëC, after 5/10 days

During storage at 8 ëC, after 5/10 days

nd/nd 1.6/2.8 3.7/4.5 5.3/7.2 nd/nd 4.6** 5.6** nd**

nd/5.0 3.0/6.7 4.4/8.0 7.9/8.6 nd na na na

* estimated, below detection level ** after 8 days of storage na: not analysed nd: not detected, below 1.7 log10 CFU gÿ1 fish

19.2.2 The impact of contamination point of Listeria spp. The rehydration process of salt-cured cod products is a source of contamination, as usually done in non-aseptic conditions and as nutrients released from the fish give favourable conditions for bacteria (Skjerdal et al., 2002). The growth kinetics of Li. innocua introduced to the fish from the rehydration water was therefore investigated and compared to that for Li. innocua introduced prior to salt-curing. The results obtained with fish inoculated with 10±500 CFU g±1 of Li. innocua are showed in Fig. 19.1. In fish inoculated during rehydration, the observed lag phases of Li. innocua were relatively short: two to four days at 4 ëC and two days at 8 ëC. In fish that was inoculated prior to salt-curing, on the other

Fig. 19.1 Growth of Listeria innocua in rehydrated salt-cured cod. The fish was contaminated either prior to salt-curing or during rehydration.

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Table 19.3 Days until the Listeria innocua level in rehydrated salt-cured cod during storage reached 50 and 100 CFU/gÿ1, respectively, of rehydrated salt-cured cod. The fish was inoculated with app 10 CFU/gÿ1 fish either prior to salt-curing or during rehydration Storage of rehydrated salt-cured cod at 8 ëC (days) Treatment

Listeria inoculated prior to salt-curing

Listeria inoculated during rehydration

<50 CFU/gÿ1 >100 CFU/gÿ1 <50 CFU/gÿ1 >100 CFU/gÿ1 Air packed Vacuum packed Sodium benzoate, air packed Potassium sorbate, air packed Sodium sulphite, air packed Un-inoculated controls

2 2 4±7 2 7 >10

2±3 2±3 7±8 3±4 8±9 >10

0 0 4 2 4 >10

1±2 1±2 6±7 3±4 5±6 >10

hand, the lag periods were approximately seven and two to four days when the fish was stored at 4 and 8 ëC, respectively. As in earlier experiments, the growth rate of Li. innocua was significantly higher at 8 than at 4 ëC. From a practical point of view, the results illustrate that Listeria introduced to the fish during rehydration has the potential to grow to higher numbers, i.e. to reach the infective dose earlier than Listeria introduced to the fish prior to salt-curing, and thereby represents a higher food safety risk. 19.2.3 Effect of preservatives on shelf life and Listeria innocua growth of rehydrated salt-cured cod Vacuum packing and some preservatives are found to inhibit growth of Psychrobacter spp. and thereby prolong the sensory shelf life of rehydrated saltcured cod (Skjerdal et al., 2002; FernaÂndez-Segovia et al., 2003 and 2006; Magnusson et al., 2006). In the present project, the effect of these treatments on Li. innocua growth in rehydrated salt-cured cod was investigated. The results obtained with fish inoculated with app. 1 log10 CFU gÿ1 and stored at 8 ëC are shown in Table 19.3. The Li. innocua growth was delayed by sodium benzoate and sodium sulphite, but not by vacuum packing. The shelf life of rehydrated salt-cured cod was estimated based on Psychrobacter content and sensory analysis (results not shown). The sensory shelf life of preserved salt-cured cod was, in case of vacuum packed, sodium benzoate and sodium sulphate treated samples, longer than the time period required for a 100-fold doubling of Listeria in the fish, indicating that the fish may become unsafe to eat raw or undercooked for vulnerable consumers before the fish is sensory spoiled. 19.2.4 Concluding remarks Salt-curing of cod is not an effective barrier against Listeria spp., but leads to a longer lag-phase for the Listeria spp. growth after rehydration. The lag-phase

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becomes shorter and the growth rate faster when the fish is stored at unsafe temperature. Listeria spp. introduced to the fish during rehydration, i.e. bacteria that have not been through the salt-curing process, have a shorter lag-phase. Some of the treatments that extend the sensory shelf life of rehydrated salt-cured cod inhibit the growth of Listeria but to a lesser extent. They may therefore lower the food safety of the products because the products may become unsafe before they are sensorily spoiled. These aspects should be considered in risk management of commercially rehydrated salt-cured cod products.

19.3

Biopreservation of lightly preserved seafood products

Biopreservation is a technology used to extend the shelf life and/or control the growth of pathogenic flora of refrigerated products by the inoculation of bacteria selected for their inhibition properties towards undesirable bacteria. In nonfermented food like LPFP, these bacteria should not modify the organoleptic and health qualities of the product. Lactic acid bacteria (LAB) are usually chosen for these applications as they produce a wide range of inhibitory compounds such as organic acids, hydrogen peroxide, diacetyl and bacteriocins. In addition, they are associated to fermented products and thus have the GRAS (generally recognised as safe) status granted by the US-FDA (US Food and Drug Administration) and for some of them the QPS (qualified presumption of safety) status given by the European Food Safety Authority (www.efsa.europa.eu/). LAB also benefit from an healthy image associated with dairy products (Rodgers, 2001). 19.3.1 Use of lactic acid bacteria to control pathogenic flora in lightly preserved fish products LPFP are highly perishable products. The major risk associated with LPFP is the pathogenic bacteria Li. monocytogenes responsible of listeriosis a foodborne disease generally associated with a high mortality rate (20±40%). Li. monocytogenes has frequently been isolated from LPFP products like CSS (Jorgensen and Huss, 1998; Hoffman et al., 2003; Nakamura et al., 2004; Miettinen and Wirtanen, 2005). The contamination comes from raw fish or can occur during the process (Huss et al., 2000). Li. monocytogenes is of special concern to the CSS industry because it is not destroyed by the different stages of processing (Ribeiro Neunlist et al., 2005) and is able to grow at low temperature in the presence of high NaCl concentration and in anaerobic conditions (Cornu et al., 2006). It has been shown that the bacterial flora of CSS is dominated by LAB like Carnobacterium maltaromaticum (previously named as Cb. piscicola) and Lactobacillus spp. at the end of storage (Leroi et al., 1998). For that reason, protective cultures are usually selected among these bacteria. Moreover, many LAB from the genus Carnobacterium and Lactobacillus are able to produce bacteriocins active against Li. monocytogenes (Drider et al., 2006). Several

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strains of Cb. maltaromicum have been successfully tested to prevent the growth of Li. monocytogenes in CSS for up to 30 days at chilled temperature (Nilsson et al., 1999; Katla et al., 2001; Duffes et al., 1999; Yamazaki et al., 2003). Other species like Lb. sakei, Lb. casei or Lb. plantarum have also been used to limit Li. innocua development in this product (Vescovo et al., 2006). In most of these studies, inhibition can be attributed to the production of bacteriocins with antilisterial activity (for a review, see Drider et al., 2006). However, a strain of Cb. maltaromicum exhibits an anti-listerial activity due to nutrient competition (Nilsson et al., 2005). In the HURDLETECH project, three anti-listerial strains selected from a previous work, Cb. maltaromaticum V1 and SF668, and Cb. divergens V41 were tested. These strains produce one or two bacteriocins that have been totally or partially characterised (Bhugaloo-Vial et al., 1996; MeÂtivier et al., 1998). Their inhibition activity has been shown with the agar diffusion method on Petri dishes towards 57 strains of Li. monocytogenes representative of the smoked-salmon industry (Brillet et al., 2004). To confirm these observations in the product, each Carnobacterium strain was tested in coculture with a set of 5 strains of Li. monocytogenes at respective levels of 105 and 102 CFU gÿ1 in sterile CSS during vacuum storage for nine days at 4 ëC and 19 days at 8 ëC (with a temperature break of 2 h at 20 ëC after 19 days). The growth of Li. monocytogenes strains alone reached 105 to 106 CFU gÿ1 at the end of the storage. It was maintained respectively below 50 and 100 CFU gÿ1 with Cb. divergens V41 and Cb. maltaromaticum V1 whereas a reduction of 1 to 1.5 log CFU gÿ1 was observed with Cb. maltaromaticum SF668 (Fig. 19.2). The inhibition activity of Cb. divergens V41 in CSS was clearly attributed to the divercin V41 production (Richard et al., 2003), although the bacteriocin could not be detected in the product, as a bacteriocin negative mutant of Cb. divergens V41 did not inhibit growth of Li. monocytogenes. 19.3.2 Use of lactic acid bacteria to control spoilage flora in lightly preserved fish products The efficacy of LAB to control spoilage flora in food products and particularly in fish products is not well documented. Leroi et al. (1996) significantly increased the shelf-life of smoked-salmon slices by inoculating them with strains of Carnobacterium spp., but results varied depending on the batch treated (Leroi et al., 1996). Only a slight extension of the smoked-salmon shelf-life was obtained with Cb. maltaromaticum (Paludan-Muller et al., 1998). No sensory improvement was found in cooked shrimps and no inhibition of the specific Gram-positive spoilage bacteria Brochothrix thermosphacta was observed (Laursen et al., 2005). However, recently, Altieri et al. (2005) succeeded in inhibiting Pseudomonas spp. and P. phosphoreum in vacuum-packed fresh plaice fillets at low temperatures by using a Bifidobacterium bifidum starter. A French patent was also developed for the biopreservation of cooked shrimps using the strain of Lactococcus lactis (Daniel and Lorre, 2003). It is used in

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Fig. 19.2 Growth of Listeria monocytogenes (mix of 5 strains) and protective Carnobacterium strains co-inoculated in cold smoked salmon stored under vacuum for 9 days at 4 ëC and 19 days at 8 ëC (with a break of 2 h at 20 ëC after 19 days). Mean of three experiments.

France on cooked peeled shrimps stored under modified atmosphere to extend the shelf-life of the products (Meyer, 2005). However no information concerning its mechanisms of inhibition and its effect on the quality of the product is available. The strain Cb. divergens V41, which was selected in the HURDLETECH project for its inhibition activity towards Li. monocytogenes in CSS, was inoculated on commercial CSS slices from four different producers to evaluate its impact on the natural flora. The results showed that when the natural microflora was initially weak (two batches < 20 CFU gÿ1), Cb. divergens V41 quickly reached 107ÿ8 CFU gÿ1 and a slight inhibition of endogenous Enterobacteriaceae, lactobacilli and yeasts was observed (Fig. 19.3). On the contrary, when the natural microflora was initially high (2 batches > 104ÿ5 CFU gÿ1), no effect on the microflora was detected (data not shown) (Brillet et al., 2005). Considering these results, this strain could not be used to prevent the growth of spoilage flora in CSS, but its interest on other LPFP should be tested as the spoilage flora is highly variable among the different products. The collection of protective cultures available in the HURDLETECH project was widened by new LAB strains recently isolated from various marine products. These strains were selected on their capacity to inhibit spoiling and pathogenic Gram-positive and Gram-negative marine bacteria. In order to obtain LAB strains competitive with psychrotrophic spoilage, the isolation was performed at 8 ëC and the strains growing at temperature up to 30 ëC were eliminated. The screening led to the selection of 52 psychrotrophic strains that were clustered on seven groups, on the basis of their inhibition spectrum and

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Fig. 19.3 Growth of Enterobacteriaceae, lactobacilli and yeasts and moulds in presence or absence of Carnobacterium divergens V41 in commercial cold smoked salmon stored under vacuum for 9 days at 4 ëC and 19 days at 8 ëC (with a break of 2 h at 20 ëC after 19 days). Straight line: control; dashed line: inoculated with C. divergens V41. Mean of three experiments.

identification at genus level. One strain per group was lastly selected and identified by sequencing of the 16S rRNA gene as Leuconostoc gelidum (3 strains), Lactococcus sp. (2 strains), Lactobacillus fuchuensis (1 strain) and Carnobacterium alterfunditum (1 strain). These seven strains were used for an application in cooked tropical shrimps where their ability to grow in the product and to control the spoilage was evaluated. Each LAB strain was inoculated at a level of 105 CFU g±1 on two batches of peeled shrimps (different wild or farmed species). The shrimps were cooked, inoculated and stored at 8 ëC for 28 days under vacuum packaging. For each trial, a non-inoculated sample was used as control. After seven and 28 days of storage, samples were analysed for sensorial quality (seven trained judges for odour descriptors and spoiling level) and for microbiological quality. For sensory evaluation, a quality index (QI) was calculated, based on the percentage of judges considering the product as non-spoiled, lightly spoiled and strongly spoiled. A QI up to 2 corresponds to a spoiled product (rejected by most of the trained panel). Figure 19.4 shows (batch 1), that after seven days, the control was considered as non-spoiled. The samples inoculated with Le. gelidum and Lc. sp. strains were the closest to the control. On the contrary, samples inoculated with Lb. fuchuensis and Cb. alterfunditum were considered as lightly or strongly spoiled. After 28 days, the control was considered as strongly spoiled whereas the samples inoculated with Le. gelidum EU2247 and EU2262 kept their fresh initial sensory quality. Those two strains as well as the two Lc. sp. also delayed the spoilage of shrimp in batch 2.

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Fig. 19.4 Evolution of the Quality index of peeled frozen shrimps inoculated with seven different strains of bioprotective lactic acid bacteria (105 UFC/g), after 7 days and 28 days of storage under vacuum at 8 ëC. Control: non inoculated sample. Le. gelidum EU2213, 2247, 2262; Lactococcus sp. EU2229, 2241; Lb. fuchuensis EU2255; Cb. alterfunditum EU 2257.

Lactic acid flora counts confirmed that the seven inoculated LAB strains were at the expected level, and were able to grow during the storage (data not shown). Total mesophilic flora, total psychrotrophic flora and enterobacteria increased in the control and in the inoculated samples, without showing any correlation with the sensory parameters. To conclude, two Le. gelidum strains greatly extended the shelf-life of both batches of shrimps, two Lc. sp. strains had a moderate effect, two were spoilers (Lb. fuchuensis and Cb. alterfunditum) and the last one (Le. gelidum) showed highly variable results depending on the batch considered. 19.3.3 Additional selection properties of lactic acid bacteria (LAB) used in biopreservation of lightly preserved fish products Besides their ability to prevent the growth of pathogenic or spoiling flora, other properties of the protective culture must be characterised for an application in food. First, the protective cultures should not have spoilage activities and even induce noteworthy organoleptic changes in the product. LAB from the genus Carnobacterium have often been selected for the biopreservation of CSS because they are usually described as non-spoiling organisms (Paludan-Muller et al., 1998; Nilsson et al., 1999; Brillet et al., 2005). In contrast, some species of Lactobacillus are responsible for specific spoiling activities (Stohr et al., 2001). However, the effect of the protective strains on the organoleptic qualities of the products is rarely investigated in biopreservation studies. In the HURDLETECH project, experiments were performed to evaluate the organoleptic changes caused by the inoculation of the protective Carnobacterium strains in CSS. These tests were investigated with the three strains of

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Carnobacterium on sensory properties and physicochemical parameters in sterile CSS stored in the conditions previously described. The results showed that after three weeks of storage, none of the three strains acidified the product, produced total volatile basic nitrogen nor caused sensory spoilage. The same experiments were performed on four commercial CSS batches inoculated and stored in the same conditions and results confirmed that Cb. divergens V41 did not induce major organoleptic or physicochemical changes in the product. For the seven LAB recently selected, the results presented previously showed that Lb. fuchuensis and Cb. alterfunditum strains are not retained for biopreservation of shrimps as they caused a notable spoilage after seven days of storage at 8 ëC. For the other strains, results are variable depending on the trial tested, but strains EU2247 and EU2262 (Le. gelidum) were the best candidates for a food application. 19.3.4 Regulation concerning the use of bioprotective culture in lightly preserved fish products The application of LAB for the biopreservation of seafood products is slightly different from the traditional use of lactic starters in fermented products. However, at this time, there is no regulation in Europe concerning the application of already known positive flora in food products except the directive 94/40/EC (European Commission, 1994) that is applied to microorganisms coming in the food chain from animal feeding (probiotic LAB). The regulation `novel foods' (Regulation 258/97/EC, European Parliament and Council, 1997) is suitable for genetically modified microorganisms, but does not include the use of already characterised LAB in the food chain (Wessels et al., 2004). The European Commission has written a working paper (European Commission, 2003) that proposes a decision tree for the determination of the QPS status. Some of the main conditions are the taxonomic information available on the strain, the exclusion of pathogenic potential and production of undesirable metabolites, and evidence of the absence of acquired antibiotic resistance. In order to forecast the emerging European regulation concerning the safety assessment of the LAB strains for the biopreservation of seafood products, some safety properties were investigated within the HURDLETECH project, like the production of biogenic amines, and the antibioresistance. The production of histamine has not been detected after culture in histidine containing culture media for any of the strains tested. These results have been confirmed during the storage of inoculated smoked salmon for the three inhibiting Carnobacteria (Brillet et al., 2005) and the Le. gelidum and Lactococcus sp. strains. The antibiotic resistance observed at this time on the strains are usually described on these genus as non transmissible, some results are still in acquisition. 19.3.5 Conclusion Results in the HURDLETECH project have shown that biopreservation is a very promising additional hurdle to ensure quality and safety of convenient seafood

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products. When the target is clearly identified (Li. monocytogenes for instance), the strains selected in the project are quite effective whatever the seafood product tested and we now have to bring the biopreservative technology to a stage where it can become available for the industry. Concerning the spoilage, this technology has to be tailor-made for each industry as spoiling microorganisms vary within plants, depending on the hygienic conditions. At the moment, no strain is performing to inhibit spoiling and pathogens at the same time. More work is needed to use mixed LAB cultures to master both quality and safety.

19.4

Antimicrobial compounds

Emergence of psychrotrophic food-borne pathogens has been a main concern in either ready to cook or to eat processed products. Based on this fact, reevaluation of food preservation methods is unavoidable matter. Therefore, the introduction of new or improved methods that comply with some current needs as chilled products with low levels of artificial preservatives is essential. In this context, the food industry and food research have driven towards the use of `natural' ingredients, i.e. naturally produced preservatives (biopreservatives). Biopreservation often implies the use of LAB, their metabolic products or both to improve safety and quality of foods that are not generally considered fermented (Montville and Winkowski, 1997). The use of other antimicrobial compounds of plant, animal or microbial origin is also considered in biopreservation (Ray, 1992). Typical examples of these compounds are lactoperoxidase (milk), lysozyme (egg white, figs), saponins and flavonoids (herbs and spices) as well as chitosan (shrimp shells). Previously in this chapter, biopreservation and different applications have been described and therefore, at this point, the use of chitosan as a biopreservative will be addressed. Chitosan, a natural polymer derived from crustacean shells after deacetylation of chitin, has been considered a potential novel food preservative due to its biodegradability, non-toxicity (Coma et al., 2002) and capacity to inhibit the growth of several bacteria and fungi in vitro (Roller and Covill, 1999). The mechanism of the antimicrobial activity of chitosan involves extensive cell surface alterations, changing membrane permeability with loss of barrier function (Helander et al., 2001). As a chelating agent, chitosan has the ability to selectively bind trace metals, which prevents production of toxins and microbial growth (Cuero et al., 1991). Regarding regulatory issues, chitosan is considered as a GRAS product in USA and in some countries, such as Korea and Japan, it has been incorporated into food products as a functional ingredient. However, chitosan is not currently regulated in Europe for food applications, although it has been used in the food industry as a safe and natural fat digestion and trapped lipid compound (Coma et al., 2002). The inhibitory action of chitosan has been reported widely in the scientific literature, mainly on the basis of in vitro trials against individual micro-

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organisms (Gemma and Du, 1996; Genta et al., 1997; Cruz et al., 2006). However, the evidence in the literature regarding antimicrobial activity is contradictory. Reported minimum inhibitory concentrations for same species vary several orders of magnitude (Roller, 2002). These variations were suggested to be due to different types of chitosan (e.g. degree of acetylation, chain length and concentration used), the testing conditions (e.g. pH, temperature, medium) and target organism (Roller, 2003). Therefore, the success of chitosan's application will be a result of an appropriate parameter selection. Due to its antimicrobial properties, chitosan has been proposed as a novel food preservative (Chen et al., 1998; Rhoades and Roller, 2000; Shahidi et al., 1999; Tsai et al., 2000). However, only few investigations have been carried out in seafood products. Chitosan has been applied to lightly-salted and dried horse mackerel (Ahn and Lee, 1992), fresh fish fillets (Skonberg, 2000), shrimps (Simpson et al., 1997), salmon (Sathivel, 2005; Tsai et al., 2002), oysters (Chen et al., 1998), cod and herring (Jeon et al., 2002). It can be foreseen that the application of chitosan in food matrixes could lead to decreased antimicrobial activity compared to in vitro tests due to interactions with different compounds, such as proteins and fats (Rhoades and Roller, 2000). Neutralisation of antimicrobial properties has also been reported for other natural compounds, lysozyme, bacteriocins such as sakacin K (Leroy and De Vuyst, 1999) and curvacin (Verluyten et al., 2002). This phenomenon has been confirmed for LPFP within the HURDLETECH project of the SEAFOODplus IP where the evaluation of the antimicrobial activity of several chitosan formulations was conducted. In a micro-well assay, some chitosan preparations (C4 and C8: low and high viscosity dissolved in acid) were found to be inhibitory to several pathogenic and fish spoilage bacteria, but generally influenced by pH and temperature when tested (0.02% w/v) in a model liquid system at 8 and 15 ëC (Fig. 19.5). Interestingly, growth of the protective culture Cb. divergens V41 was not inhibited in presence of chitosan, but to the contrary it was apparently stimulated under some conditions (Fig. 19.5). Antimicrobial effectiveness of chitosan coatings on real products was not as promising as the in vitro results suggested. Chitosan concentrations ranging from 0.002 to 0.2% (w/ v) showed a drop of 5 log CFU mlÿ1 on Li. innocua counts in CSS juice while only a reduction of 1 log CFU cm±2 was induced in CSS coated with chitosan (2% w/v) and maintained for five days. When the same coating was applied on surimi products the cell inactivation was greater, reaching a 4 log-drop in Li. innocua counts which was maintained during the 20 days of storage. Therefore, the ability of chitosan to reduce Li. innocua counts and to inhibit microbial growth does not only vary from in vitro tests to studies performed in seafood products, but is influenced by the type of food matrix. Extensive work is required for a better understanding of the chitosan antimicrobial efficacy and before a commercial exploitation of chitosan as a novel preservative can occur. The studies should also be focused on the possible changes in the organoleptic and textural properties of the real products.

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Fig. 19.5 Bacterial development (% maximum growth) in cold-smoked salmon juice (control) at pH 6.2 and 7 over a time period at 8 and 15 ëC, in presence of acidified chitosan preparations (C4 or C8) or acid control (acidified solvent with no added chitosan). Results shown are an average of two measurements.

19.5

Antimicrobial packaging

Active packaging is one of the innovative food packaging concepts that have been introduced as a response to market trends and the continuous changes in current consumer preferences towards mildly preserved, fresh, tasty and convenient foods with a prolonged shelf-life. Active packaging can be defined as `a type of packaging that changes the condition of the packaging to extend shelf-life or improve safety or sensory properties while maintaining the quality of the food'.

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Packaging regulations require compounds in contact with food to be on approved lists of compounds. Traditionally, a well functioning food packaging should be more or less inert. The overall migration limit of substances from packaging into the food was set at a maximum of 60 mg per kg of food. This may be said to be inconsistent with the objective of active packaging that releases substances in order to extend shelf-life or improve quality. Therefore a new approach of packaging regulations was required. The approval in 2004 of the EU Framework Regulation 1935/2004 on materials and articles intended to come into contact with food, triggered the serious research in this area in Europe. In this regulation the active packaging concept was defined for the first time, among other basic definitions. This is only a starting point, legislation about active packaging materials is still in the elaboration process. The publication of the new legislation in active packaging (2007/2008) will enhance the competitiveness of the European food industry, especially with the USA, Australia and Japan. 19.5.1 Antimicrobial packaging description The term antimicrobial packaging covers any packaging technique used to control microbial growth in a food product. This concept includes mostly packaging materials (where the antimicrobial material is incorporated in the surface of the plastic films) and edible films or food coatings that contain antimicrobial activity. The antimicrobial efficiency can be given by preservatives that are released slowly from the packaging materials to the food surface or by preservatives that are firmly fixed and do not migrate into the food products. Both techniques are assumed to control growth of undesirable microorganisms if there is a good and intensive contact between the food product and the packaging material. In antimicrobial films and coatings, either the functional groups that have antimicrobial activity (e.g. bacteriocins) are added and immobilised on the surface of a polymer film which is in contact with the food surface, or the antimicrobial activity comes from the polymer itself used as filmforming entity or coating (e.g. chitosan). 19.5.2 Chitosan as a potential polymer for antimicrobial packaging As explained earlier, chitosan is a high-molecular weight cationic polysaccharide that exhibits antibacterial and antifungal activity. The advantage of using this polymer as part of an active packaging, apart of these characteristics, is its good film-forming properties. Several studies have been made about the film-forming ability of chitosan (Butler et al., 1996). Unfortunately chitosan films are brittle (Suyatma et al., 2005) so there is a need of adding a plasticiser which will increase the free volume in the matrix. This affects the film ductility and handling properties positively but has a negative effect on barrier properties, thus a compromise between mechanical properties and barrier properties must be found (Olabarrieta, 2005). Chitosan forms tough, long-lasting, flexible,

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transparent films which resemble plastic films. Chitosan film-forming and physicochemical properties will depend on the chitosan production process (Nadarajah et al., 2006) film-casting solvent (Caner et al., 1998), degree of deacetylation (Hwang et al., 2003), molecular weight (Mw) (Park et al., 2002; Hwang et al., 2003), drying conditions (Srinivasa et al., 2004) and plasticiser used (Caner et al., 1998). Chitosan films appear to be a promising prospect for edible films. In addition to its antimicrobial properties, due to its good gas barrier properties, chitosan coating can be expected to modify the internal atmosphere as well as decrease the transpiration losses. Therefore, the use of chitosan coating and films in food packaging applications could result in a delay in ripening and control of decay. Within the HURDELTECH project, chitosan films were successfully produced. Glycerol and polyethyleneglycol (PEG) were used in order to make the films flexible. The mechanical tests showed a great improvement in film elasticity for films with glycerol, whereas the improvement for PEG films was not as extensive. The addition of plasticisers to the initial chitosan formulations did not affect the antimicrobial activity of the chitosan film-forming solution (data not shown). However, their films were very hydrophilic and sensitive to humidity. It is a challenge to develop chitosan-based antimicrobial films that are less sensitive to humidity. Further research should be directed towards maintaining the oxygen barrier and antimicrobial properties of chitosan films while improving water-vapour barriers and mechanical properties. To conclude, chitosan shows potential to be used as part of an active packaging together with, e.g., synthetic plastic films. The biopolymer film could be incorporated into the polymer matrix or absorbed onto the film surface by spraying, dipping or coating after a surface modification to improve adhesion between the different materials. Research has shown that chitosan-based coatings and films could help to obtain less perishable food products. However, as regards seafood product applications, very few references have been found with chitosan/plastic active systems. Thus, further research is needed in this area.

19.6

Pulsed light as a novel decontamination technology

19.6.1 Pulsed light technology description Pulsed light technology is a novel non-thermal decontamination process which consists of a successive repetition of high power pulses of broadband emission light. The emitted light spectrum includes wavelengths from 200 to 1000 nm with a considerable amount of light in the short-wave UV spectrum (Wekhof, 2000). For the emission of a single light pulse, the electric power is stored in an energy storage capacitor and later released quickly to a Xenon lamp (Wekhof, 2000). Then, this lamp emits short duration (Lasagabaster and MartõÂnez de MaranÄoÂn, 2006) and high intensity light flashes that are transmitted to the surface of the products (Fig. 19.6). Even though the peak power of each pulse is

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Fig. 19.6

Pulsed light process.

high due to its short duration, the total pulse energy is relatively low. Therefore, since the average power requirement for a pulsed light treatment is moderate, it can be considered economical (Dunn et al., 1995). Pulsed light technology could be applied as an alternative method to traditional thermal and chemical treatments to improve the safety and increase the shelf-life of foodstuffs. However, since penetration capability of the light is poor, pulsed light technology could be limited to reduce microbial contamination of the surface of solid products (e.g., seafood products), clear liquids, processing devices (e.g., seafood processing chain) or packaging materials. Moreover, packed products could also be decontaminated whenever pulsed light is optimally transmitted through the packaging materials (Dunn et al., 1997). The US-FDA (FDA, 2003) approved the use of pulsed light technology `for production, processing and handling of foods' up to light doses of 12 J.cmÿ2. 19.6.2 Impact of pulsed light on survival and growth of spoilage and pathogenic bacteria Pulsed light process has been shown to be effective in inactivating a wide range of microorganisms (vegetative bacteria, moulds, bacterial, fungal spores, etc.) involved in food products spoilage (Arrowood et al., 1996; GoÂmez-LoÂpez et al., 2005; Lasagabaster and MartõÂnez de MaranÄoÂn, 2006; MartõÂnez de MaranÄoÂn and Gartzia, 2002; Roberts and Hope, 2003). The specific mechanism by which pulsed light causes microbial inactivation still remains unclear. Different hypotheses have been proposed in the literature which could be due to the characteristics of the pulsed light devices like the peak power of each pulse, the kind of flashlamp and so on. The main inactivating effect of pulsed light could be attributed to UV inducing DNA-damage, such as formation of single strand breaks and pyrimidine and thymine dimers (Wang et al., 2005). Furthermore, Takeshita et al. (2003) showed DNA damage and structural changes, such as cell membrane damage, when Saccharomyces cerevisiae was treated by pulsed light technology. These authors hypothesised that the high content in UV wavelengths of pulsed light could play an important role not only in DNA damages but also in cell structure modifications. Otherwise, Wekhof (2000) reported that pulsed light induced microbial inactivation

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could be attributed to cell disintegration after an instantaneous overheating of cellular constituents due to very high pulsed light doses. However, Rowan et al. (1999) and Krishnamurthy et al. (2004) found only minimal heating after pulsed light treatments inducing high levels of microbial inactivation, concluding that this high efficacy would be due to the effect of UV and not to a rise in temperature. The impact of pulsed light to inactivate microorganisms isolated from fish products was studied (Lasagabaster and MartõÂnez de MaranÄoÂn, 2006) within the HURDLETECH project of SEAFOODplus IP. Results showed that a short treatment time (325 s) at relatively low dose induced high inactivation (> 7 log10 CFU mlÿ1 or cmÿ2) of Li. innocua inoculated in liquids and on the surface of agar petri dishes, with no significant increase in sample temperature confirming previous results (see above). Pulsed light efficacy depended on the light dose received by microorganisms, which was modified by some process factors such as pulse energy, number of pulses, etc. Li. innocua and Li. monocytogenes were the most pulsed light resistant bacteria among the different seafood spoiling and pathogenic strains studied. Therefore Li. innocua could be considered as a surrogate for Li. monocytogenes and as a reference microorganism for pulsed light treatment optimisation in seafood products. The impact of some physicochemical factors on the effectiveness of pulsed light treatment was also determined for model media. Results showed that Li. innocua inactivation did not depend either on process temperature or NaCl concentration (up to 5%). Moreover, cell concentration inoculated on solid models did not affect the pulsed light efficacy to inactivate Li. innocua. However, this effectiveness would slightly depend on the physiological state of cells. 19.6.3 Application to seafood products Pulsed light technology has also been shown to be effective in inactivating Li. innocua from the surface of fish products such as CSS (Lasagabaster and MartõÂnez de MaranÄoÂn, 2006). Although microbial inactivation was less pronounced in inoculated seafood products (e.g., CSS, desalted cod) than in food models, the work performed within the HURDLETECH project pointed out that pulsed light processing could improve the safety (Listeria hazard) of seafood products. Moreover, pulsed light processing would increase the shelf life of CSS (by, at this stage of the project, taking into account only microbiological criteria). Although more studies are needed, pulsed light technology appears as an efficient non-thermal decontamination process that could be applied to improve the safety and increase the shelf-life of food products, in particular seafood products. Since the time required to inactivate microorganisms is very short, this technology could be successfully implemented in high-speed processing lines for the food industry.

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19.7

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Future trends

The convenience food trend is strong and is expected to continue through the coming years. The term convenient includes both the easy-to-use aspect, which is assumed to be most important for the consumers, and long shelf-life, which is important for the producers, distributors and shops to avoid loss. Results presented in this chapter show that traditional preservation processes and more recently developed preservation methods may contribute to increased safety and quality of convenient seafood products. In some cases, they may also introduce new food safety risks or unusual behaviour of the product that must be carefully taken into consideration (production of toxic metabolites, favourable conditions for unexpected pathogens, products that become unsafe before they are spoiled, etc.). For that reason, results obtained in model media or with artificially inoculated seafood matrix (challenge tests) must be validated in real products, and all safety and quality aspects must be checked carefully. Combining different hurdles seems to be a very promising way to increase the antimicrobial effects. However, attention must be paid to the fact that bacteria submitted to a high stress (salt, acid, temperature, starvation, etc.) may synthesise stress shock proteins that make them more tolerant to other stresses. This appears mainly when the preservative level is quite elevated. On the other hand, bacteria that are simultaneously submitted to various stresses require more energy to synthesise several shock proteins and become metabolically exhausted (Leistner et al., 2000). Therefore, lowering the level of each hurdle, which is also very interesting to maintain acceptable sensory characteristics, and applying them all together may be more efficient than using a single preservative at high concentration. In some cases, not only a cumulative effect between hurdles but also a synergistic one has been observed. This is achieved if the hurdles hit different targets at the same time such as cell membrane, DNA, ribosome, proteins, enzyme system, intracellular pH etc. (Leistner, 1995; MaoÁas and PagaÂn, 2005). For that reason, elucidating the inhibitory mechanism of hurdles is of great importance to anticipate their adequate combination to get the most efficient effect. In the future, it will be interesting to test different combinations of the preservative factors described in this chapter. Some of the studied hurdles are more efficient at spoiling microflora and other pathogenic bacteria, therefore combining them could simultaneously enhance quality and safety of food. Potential synergistic effects may be anticipated as the inhibiting mechanism varies within the tested hurdles. Enhancement of bioprotective bacteria growth has already been observed in the presence of chitosan constituting a promising indicator of synergetic antimicrobial effects. All the results presented in this chapter, if completed by the suggested work, could lead to a very efficient system for ensuring both quality and safety of seafood convenient products.

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Source of further information and advice

Some sources on further information concerning: Hurdle technology

(2005), `Update on hurdle technology approaches to food preservation'. Antimicrobiol Food Third Ed, 143, 621±631.

LEISTNER L, GOULD G W

Biopreservation

(2001), `Preserving non-fermented refrigerated foods with microbial cultures ± a review'. Trends Food Science Technol, 12, 276±284.

RODGERS S

RICHARD C, LEROI F, BRILLET A, RACHMAN C, CONNIL N, DRIDER D, PILET M F, ONNO B, DOUSSET X, PREVOST H (2004), `Control development of Listeria monocytogenes in smoked salmon: interest of the biopreservation by lactic bacteria'. Lait, 84, 1±2, 135±144. DROSINOS E H, MATARAGAS M, METAXOPOULOS J (2005), `Biopreservation: a new direction towards food safety'. New Dev Food Policy Control Res, 31±64. EFSA, Qualified presumption of safety of micro-organisms in food: www.efsa.europa.eu.

Chitosan

(2002), `Edible antimicrobial films based on chitosan matrix'. J Food Sci, 67, 1162±1168.

COMA V, MARTIAL-GROS A, GARREAU S, COPINET A, SALIN F

Pulsed light

(1999), `Pulsed-light inactivation of food-related microorganisms'. Appl Environ Microbiol, 65(3), 1312±1315.

ROWAN N J, MACGREGOR S J, ANDERSON J G, FOURACRE R A, MCILVANEY L, FARISH O.

Results from the HURDLETECH project

(2006), `Antmicrobial effect of chitosan on micro-organisms isolated from fishery product', in Luten J B, Jacobsen C, Bekaert K, Sñbù A, Oehlenschlager J, Seafood research from fish to dish: Quality, safety and processing of wild and farmed fish. Wageningen, Wageningen Academic Publishers, 387±393. Â N I. (2006), `Inactivation of microorganisms ÄO LASAGABASTER A, MARTIÂNEZ DE MARAN isolated from fishery products by pulsed light', in Luten J B, Jacobsen C, Bekaert K, Sñbù A, Oehlenschlager J, Seafood research from fish to dish: Quality, safety and processing of wild and farmed fish. Wageningen, Wageningen Academic Publishers, 381±386. MATAMOROS S, PILET M F, PREVOST H, LEROI F (2006), `Selection of psychotrophic bacteria active against spoilage and pathogenic micro-organisms relevant for seafood products', in Luten J B, Jacobsen C, Bekaert K, Sñbù A, Oehlenschlager J, Seafood research from fish to dish: Quality, safety and processing of wild and farmed fish. Wageningen, Wageningen Academic Publishers, 395±402. PILET M F, BRILLET A, MATAMOROS S, BLANCHET-CHEVROLLIER C, LEROI F, PREÂVOST H (2006), `Selection of non-tyramine producing Carnobacterium strains for the biopreservation of cold smoked salmon', in Luten J B, Jacobsen C, Bekaert K, Sñbù A, Oehlenschlager J, Seafood research from fish to dish: Quality, safety and processing of wild and farmed fish. Wageningen, Academic Publishers, 403±410. Â N I, AMARITA F ÄO CRUZ Z, LAUZON H L, ARBOLEYA J C, NUIN M, MARTIÂNEZ DE MARAN

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MEÂTIVIER A, PILET M F, DOUSSET X, SOROKINE O, ANGLADE P, ZAGOREC M, PIARD J C, MARION

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antimicrobial-producing lactic acid cultures in vacuum-packed cold smoked salmon'. Food Microbiol, 23, 689±693. VILHELMSSON O, HAFSTEINSSON H, KRISTJAÂNSSON J K (1997), `Extremely halotolerant bacteria characteristic of fully cured and dried cod'. Int J Food Microbiol, 36, 163± 170. WANG T, MACGREGOR S J, ANDERSON J G, WOOLSEY G A (2005), `Pulsed ultra-violet inactivation spectrum of Escherichia coli'. Water Res, 39, 2921±2925. WEBER D E (2000), `Kinetics of microbial inactivation for alternative food processing technologies'. J Food Sci Special supplement, 65, 1±107. WEKHOF A (2000), `Disinfection with flash lamps'. J Pharm Sci and Technol, 54, 264± 276. WESSELS S, AXELSSON L, HANSEN E B, DE VUYST L, LAULUND S, LAHTEENMAKI L, LINDGREN S,

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20 Preventing lipid oxidation in seafood C. Jacobsen, Technical University of Denmark, Denmark, I. Undeland, Chalmers University of Technology, Sweden, I. Storrù, SINTEF Fisheries and Aquaculture, Norway, T. Rustad, Norwegian University of Science and Technology, Norway, N. Hedges, Unilever, United Kingdom and I. Medina, Consejo Superior De Investigaciones Cientificus, Spain

20.1

Introduction

Long chain (LC) n-3 polyunsaturated fatty acids (PUFA) from fish and marine animals have a number of beneficial health effects (Boissonneault, 2000; Narayan et al., 2006). This is one of the reasons why intake of fish is encouraged (Simopolous, 2002). Thus, even low consumption (1±3 times/month) of fish reduces the relative risk of cardiovascular heart disease mortality by about 11± 17% compared to no fish consumption (Konig et al., 2005). Owing to the unsaturated nature of n-3 fatty acids they are highly susceptible to lipid oxidation, which can lead to flavour and colour deterioration and loss of endogenous antioxidants. In the case of severe lipid oxidation, the content of omega-3 fatty acids may even decrease. Oxidative deterioration of fish may not only affect the lipids as the proteins are also susceptible to oxidation. Protein oxidation has been shown, for example, to affect protein solubility, decrease gel elasticity, and affect water distribution in muscle foods, which in turn may affect the texture of the fish (Badii and Howell, 2002; Ooizumi and Xiong, 2004; Rowe et al., 2004; Bertram et al., 2007). It is therefore important to prevent lipid and protein oxidation to maintain sensory and nutritional quality of seafood. The aim of the SEAFOODplus project LIPIDTEXT has been to understand the mechanisms and kinetics of the processes leading to rancidity and texture changes in different types of seafood products. An important aim has also been to evaluate the efficacy of phenolic antioxidants and to explain the mechanism behind their possible antioxidative effects. To obtain this goal the project has

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performed experiments in different model systems ranging from relatively simple oil-in-water emulsion and liposome systems via washed fish muscle to the more complex fish mince model. In this chapter, the theoretical background behind the work carried out in LIPIDTEXT will be summarized together with some of the major findings from the project.

20.2 Processes leading to lipid and protein oxidation in seafood 20.2.1 The lipid oxidation reaction in brief As mentioned above, the fact that seafood lipids are highly unsaturated is a disadvantage from an oxidation stability point of view. The multiple double bonds in the LC n-3 PUFA, EPA (20:5) and DHA (22:6), give rise to numerous 1,4-cispentadiene systems that are easily attacked by radicals/initiators. As shown in Fig. 20.1, the resulting lipid radicals, L., quickly react with oxygen, yielding peroxy radicals, LOO.. During the propagation phase, LOO. attacks intact fatty acids forming odourless and tasteless primary oxidation products, lipid hydroperoxides (LOOH). Low molecular weight (LMW) and heme-bound transition metals quickly break down LOOH to an array of new radicals, .OH, LO. and LOO., which can re-initiate oxidation reactions. LO. can also be cleaved in a -scission reaction into various secondary products like aldehydes, ketones and alcohols. When originating from LC n-3 PUFA, such oxidation products have extremely low odour thresholds; e.g. down to 0.001 g/kg for 1,5-octadien-3-one, which makes

Fig. 20.1 Schematic illustration of the lipid autoxidation process including different techniques which can be used to follow its progress. LH = fatty acid, X. = initiator, L. = alkyl radical, LO. = alkoxy radical, LOO. = peroxy radical, LOOH = hydroperoxide, AH = antioxidant, ESR = electron spin resonance, PV = peroxide value, CL = chemiluminescence, A234,268,400-420 = absorbance at 234, 268 or 400±420 nm, GC-MS = gas chromatography with mass spectroscopy detection, TBARS = thiobarbituric acid reactive substances test. Adapted after Undeland, I. Lipid oxidation in fish ± causes, changes and measurements. In Methods to determine the freshness of fish in research and industry, pp. 241±257, International Institute of Refrigeration (IIR), Paris, France, 1998.

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oxidation a much larger sensory problem in seafoods than it is in more saturated systems such as meat. Some of the secondary products can also react further, e.g. with free amino groups of proteins, yielding tertiary products such as Schiff's bases. These products can polymerize into yellow-brownish pigments (Pokorny et al., 1974). Proteins can also interact with oxygen or lipid radicals, which can result in protein-cross-linking (S-S bridges) or protein-lipid complexes; both reducing, e.g. water holding capacity and solubility of proteins (Liu and Wang, 2005). 20.2.2 Oxidation in fish The reactants and catalysts of lipid oxidation The LC n-3 PUFA of fish are located in phospholipids (PL) and triacylglycerols (TG). The PL give structure and fluidity to membranes and are found at relatively constant levels ~0.5±1% (w/w) (Ackman and Ratnayake, 1992). TGs are found both in adipose tissues and integrated into muscle tissue, and their levels vary widely with species and environmental conditions. From an oxidation point of view, it is important to highlight that PL are more unsaturated than TG, and also that the surface area they expose to the aqueous phase is ~50± 100 times greater than the oil droplet surface areas on a weight basis (Hultin, 1994). Most pro-oxidants are located in the aqueous phase, and therefore the location of PL in the membrane is crucial for lipid-pro-oxidant interactions. Although rapid oxidation in the muscle of fatty fish species has traditionally been attributed to its high total lipid content, i.e., TG-level (Aubourg et al., 1999), recent research has shown that the type and level of pro-oxidants in such fish appear to be of greater importance. Strong rancid odour developed in a reduced lipid (~0.1%) washed cod muscle system fortified with whole trout blood (Richards and Hultin, 2001). The rate and extent of rancidity was not increased by the presence of >6 times more membrane lipids. A crucial step in oxidation initiation is the conversion of ground state oxygen, 3 O2, to active oxygen species (the superoxide anion radical, O2.ÿ, hydroperoxyl radical, HO2., hydrogen peroxide, H2O2, and the extremely reactive hydroxyl radicals, .OH). In contrast to 3O2, these forms can directly react with fatty acids. Both LMW- and heme-bound Fe are involved in formation of active oxygen species in post mortem muscle, e.g. through the Fenton-Haber-Weiss reaction. It should be stressed that the most reactive radicals, like .OH, seems to be the least selective (Buettner, 1993; Davies, 2005). .OH is produced in the aqueous phase of the cell, and it is believed that this radical reacts, e.g., with proteins before it reaches the hydrophobic interior of the membranes (Hultin, 1994; Davies, 2005). Other radicals, such as protein radicals, with lower redox potentials then mediate the oxidation process into the lipid phase. This is one driving force behind the interest in LIPIDTEXT to study how interactions between proteins and lipids lead to oxidation. A second type of 3O2 activation that can take place in fish is the production of singlet oxygen, 1O2. This is a species which can directly add to a C-C double bond and thereby initiate oxidation. The production of 1O2 may occur following

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exposure to light of photosensitizers such as porphyrins, and riboflavins in the presence of 3O2. In fish, a third type of oxidation initiation can also occur via lipoxygenases, which are iron containing enzymes being situated in the cell cytosol or microsomal fraction (Harris and Tall, 1989). The enzyme catalyses the insertion of one molecule of 3O2 into an unsaturated fatty acid containing a 1,4-cispentadiene group (Belitz and Grosch, 1987). Lipoxygenases have been localized in both skin (Mohri et al., 1999), gills (Liu and Sun Pan, 2004) and muscle (Stodolnik et al., 2005) of various fish. Lipoxygenase-derived volatiles may be important both for the fresh and spoiled odour of fish (Hsieh and Kinsella, 1989; Lindsay, 1990). Also another enzyme, myeloperoxidase, which was isolated from trout leukocytes, can initiate lipid oxidation in the presence of hydrogen peroxide and halides such as bromide and iodide (Kanner and Kinsella, 1983). This way of initiation might be critical during processing of fish, when the contact between air and blood is increased. Heme-proteins as pro-oxidants Both myoglobin (Mb) (Baron and Andersen, 2002) and hemoglobin (Hb) appear to control the onset of oxidation via several mechanisms. Hb is a tetrameric molecule with allosteric O2-binding properties while Mb is a monomer without allosterism. The ratio between Mb and Hb is roughly equal in dark muscle while Hb dominate in light fish muscle (O'Brien et al., 1992). In general the ratio between Mb and Hb is lower in fish than in beef (Livingston and Brown, 1981; Matsuura and Hashimoto, 1954). This is one reason why the work in LIPIDTEXT has been more focused on Hb as a pro-oxidant. The natural pH-drop that rapidly takes place in fish muscle post mortem, particularly in pelagic species (e.g., from ~7 to ~6.4 in herring), activates Hb as a pro-oxidant by different mechanisms as shown in Fig. 20.2 (Undeland et al., 2004). This feature may, together with the abundance of Hb in these fishes, be the key behind the susceptibility of pelagic dark muscle species to lipid oxidation. The reduced pH will induce Hb deoxygenation. Deoxy-Hb has been suggested to act as a strong pro-oxidant in itself (Richards et al., 2002a), but particularly, it is more susceptible than oxy-Hb to formation of the highly catalytic met-Hb (i.e. Hb-Fe3+) (Livingston and Brown, 1981). Hb dissociation, which can occur at 0.15 M Hb (Manning et al., 1996) is a second feature increasing the susceptibility for Met-Hb-formation. Mbs become autoxidized via some mechanism not linked to oxygen-affinity (Richards et al., 2005). The highly pro-oxidative role of met-Hb and Mb is linked to their ability to generate different free radicals. Among these are oxygen radicals (O2.ÿ , HO2., H2O2 .OH), out of which H2O2 can react with met-Hb or met-Mb to form a hypervalent ferryl-Hb (Fe4+=O) radical capable of initiating lipid oxidation (Kanner and Harel, 1985). Lipid radicals such as LOO. and LO. then also emerge from the breakdown of pre-formed lipid hydroperoxides by deoxy-Hb, met Hb/ met-Mb or heme/hemin (Ryter and Tyrrell, 2000). Recent studies (Richards et al., 2005; Grunwald and Richards, 2006a,b) have revealed that heme/hemin-

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Fig. 20.2 Example of a figure showing the many routes by which Hb in combination with low pH can accelerate oxidation of muscle. Adapted after Undeland, I. Hemoglobin catalyzed oxidation of washed cod muscle phospholipids ± effect from pH and hemoglobin source, Lecture given at the 1st TAFT-meeting, Reykjavik, Iceand, 11±14 June 2003.

release may actually be the most critical peroxide breaking species. The release occurs as the binding of heme/hemin to the proximate histidine gets weakened (Everse and Hsia, 1997). The low polarity of the free hemin is expected to aid dissolving it into the hydrophobic interior of membranes. Several comparisons have been made regarding pro-oxidation characteristics of Hb and Mb; but with contradicting results. One of the few comparisons made in a fish system (washed cod mince) showed that trout Hb was more prooxidative than trout Mb (Richards et al., 2005). However, when comparing the ferryl species of Hb and Mb, the latter was formed more quickly (Kanner and Harel, 1985), and has also been ascribed to be the most active pro-oxidant (Everse and Hsia, 1997). LMW-Fe versus heme-bound Fe as pro-oxidants When a critical level of peroxides is formed, low molecular weight Fe (LMWFe) can also be released from hemin and theoretically act as an initiator of lipid oxidation (Puppo and Halliwell, 1988). Fe is well suited to catalyze redoxreactions as it has a number of different oxidation states which enables it to transfer electrons. Fe is usually chelated in the tissue, e.g. with ADP, ATP, amino acids (e.g. histidine) or ascorbate (Hultin, 1994). Fe contamination during processing, and liberation from the Fe-storage protein ferritin are other sources of LMW-Fe in muscle. Storage induced release of LMW iron (1.4±1.6 fold) has been reported in mackerel and sardine (Decker and Hultin, 1990; Decker and Welch, 1990; Chaijan et al., 2005). In simpler lipid systems such as bulk oil,

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liposomes and emulsions, Fe has a strong role as catalyst (see Section 20.2.3 below). However, from several studies in fish muscle systems, there is a clear lack of evidence that endogenous levels of LMW-Fe contribute significantly as an oxidation catalyst. Moreover, several studies seem to suggest that heme is a stronger pro-oxidant than LMW-Fe (Chiu et al., 1996; Undeland et al., 2002). Recent results on the comparison of prooxidative effects of LMW-Cu and LMW-Fe seem to suggest that while LMV-Fe induces lipid oxidation and the formation of a yellow colour in fish muscle, Cu may promote oxidation of protein (Thanonkaew et al., 2006). 20.2.3 Lipid oxidation in emulsified foods Due to the health benefits of n-3 PUFA, there is an increasing interest from the food industry to incorporate these lipids into food products such as milk, yoghurt, mayonnaise and salad dressing. These foods are examples of oil-inwater emulsions, where oil droplets are dispersed in the continuous aqueous phase. In water-in-oil emulsions, such as margarine and butter, the opposite is the case, i.e. water droplets are dispersed in the continuous oil phase. The two phases are separated by an interface comprised of amphiphilic compounds (emulsifiers). Due to the complexity of emulsions, their oxidation mechanisms may be very different from those in bulk oils. Hence, to successfully apply n-3 PUFA in such foods it is important to understand the oxidation mechanisms in the food system in question. Lipid oxidation can be prevented by the addition of antioxidants. The efficacy of the antioxidants is highly dependent on the composition of the food systems. Some of the most important factors affecting the oxidation rate and the efficacy of antioxidants in food emulsions are summarized in the following with particular focus on the factors that were further investigated in LIPIDTEXT and which will be summarized in Section 20.6. Metal catalysis Several food ingredients including refined oils contain trace levels of metal ions. Therefore, metal-catalyzed decomposition of pre-existing lipid hydroperoxides is probably the most important initiator of lipid oxidation in many emulsified foods. As previously mentioned, the decomposition of lipid hydroperoxides not only generates free radicals, which may initiate further oxidation reactions, but may also lead to the formation of secondary volatile oxidation compounds, which may lead to unpleasant off-flavour formation. Transition metals are also capable of directly breaking down unsaturated lipids (LH) into alkyl radicals (L.), but this reaction occurs slowly and is therefore not believed to be important in promoting lipid oxidation (Reische et al., 1998). Emulsifiers (proteins and surfactants) Emulsifiers can generally be categorized in two groups. The first type is macromolecules such as proteins, while the second type is small amphipilic

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molecules such as phospholipids, free fatty acids, mono- and diacylglycerols and synthetic surfactants. Among the macromolecules, proteins are used both to facilitate the formation of oil or water droplets and to enhance the stability of food emulsions. Proteins are absorbed to the oil droplet surface during the homogenization/emulsification process. The absorbed proteins will lower the surface tension and prevent coalescence of droplets by forming protective membranes around the droplets. They may also provide the emulsion droplets with a positive or negative electrical charge at pH values below or above their pI, respectively. The electrical charge of the droplet surface (oil±water interface) is often measured as the Zeta potential. Since all droplets will either have a negative or a positive charge, the droplets repel each other whereby coalescence is prevented and the physical stability of the emulsion increases. The electrical charge of the interfacial layer around the oil droplet may also significantly influence lipid oxidation in food emulsions containing trace metals (Mei et al., 1997, 1999). If the charge of the interface is negative, metal ions will be attracted to the interface and promote oxidation. This is because lipid oxidation is generally believed to be initiated at the oil-water interface where water soluble metal ions can interact with the more lipid soluble lipid hydroperoxides. In contrast, if the charge of the interface is positive, metal ions will be repelled from the interface and thereby their ability to promote lipid oxidation will be low. The electrical charge of the interface depends on the type of emulsifier used and on the pH in the emulsion. Other factors than the charge of the emulsion droplets also seem to influence the effect of proteins on the oxidative stability. Hence, the ability of different proteins to influence the thickness or packing of the emulsion droplet interface most likely also influences lipid oxidation of emulsions (Coupland and McClements, 1996). Moreover, the amino acid composition of proteins, which may affect their antioxidative activity can also affect the oxidation rate of the emulsion (Hu et al., 2003; Tong et al., 2000). Recently, it was shown that different homogenization conditions changed the protein composition at the oilwater interface in fish oil enriched milk and that this in turn affected lipid oxidation (Sùrensen et al., 2007). Physical structure of the emulsion A large interfacial area is created during emulsification. The total interfacial area depends on the size distribution of the oil droplets and can be calculated by As = 6/D32, where D32 is the mean surface diameter. The increased interfacial area increases the potential contact area between the oil droplet and trace metals in the continuous, aqueous phase. Contradicting literature is available about the relationship between droplet size and lipid oxidation rates. In fish oil enriched mayonnaise, lipid oxidation thus increased with decreasing droplet size (Jacobsen et al., 2000), whereas the opposite was the case in fish oil enriched milk (Let et al. 2007; Sùrensen et al., 2007). In the case of milk, the decreased droplet size seemed to be less important in comparison with the change in the protein composition that occurred concomitantly. Hence, these results indicate

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that the composition of the food system determines whether the droplet size has a large or small impact on lipid oxidation. pH As already discussed, pH plays a very important role in lipid oxidation. This is due to the fact that pH affects reactivity, solubility, partitioning and interactions of several of the reactive compounds that participate in oxidation and antioxidant reactions. Metal ions are generally more soluble at low pH than at high pH (Frankel, 2005). In accordance with this, lipid oxidation is generally slowest at high pH values. In fish oil-enriched mayonnaise, decreasing pH from 6.0 to 3.8 was found to increase lipid oxidation (Jacobsen et al., 1999, 2001). Similar results were observed in fish oil-in-water emulsions (10% oil) with different emulsifiers (Haahr and Jacobsen, 2008). However, the opposite effect of pH was observed in salmon oil-in-water model emulsions (5% oil), where lipid oxidation was greater and more rapid at pH 7.0 than at pH 3.0 (Mancuso et al., 1999). Hence, it seems that in complex food emulsions pH may affect lipid oxidation in different directions depending on a number of different factors such as the effect of pH on the emulsifier charge and composition.

20.3

Common analytical methods to evaluate oxidation

The most common analyses to use when studying muscle lipid oxidation are shown in Fig. 20.1. A number of different methods are available to determine lipid oxidation in food systems, but no single method alone can give a complete and satisfactory description of the oxidative status (Frankel, 1998; Falch, 2005). The most common methods used to determine primary oxidation products are peroxide value and conjugated dienes. Because peroxides are labile compounds, these methods have to be combined with determination of secondary oxidation products. Thiobarbituric acid reactive substances (TBARS) and anisidine value are commonly used to determine content of aldehydes, which are secondary oxidation products. Totox value is a combination of peroxide value and anisidine value and is a commonly used oxidation parameter. The Oil stability index, the Rancimat test and Oxidograph are based on accelerated oxidation (Falch, 2005). New techniques for evaluating lipid oxidation include free radical assessment using ESR spectroscopy and determination of primary and secondary oxidation products using chromatographic techniques such as GC-MS and LCMS. Yellow pigments or co-oxidation of Hb (i.e., redness loss) can be followed with a colorimeter. In addition, oxygen, fatty acid or antioxidant consumptions can be followed, e.g. with a Clark electrode, GC and high performance liquid chromatography (HPLC), respectively. In this chapter we will show examples from data obtained with a range of the above-mentioned methods and particular attention will be given to the possibilities for using the measurement of oxygen uptake rate for modelling lipid oxidation reactions. For further details of the

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oxidation processes and analytical determinations on the reaction compounds, the reader should refer to the numerous reviews on the lipid oxidation process in fish (Kamal-Edin and Yanishileva, 2002; Frankel, 2005).

20.4 Introduction to model systems for use in seafood oxidation studies Lipid oxidation in multicomponent foods is an interfacial phenomenon where the rate of oxygen diffusion and its interactions with unsaturated lipids, metal initiators, radical generators and terminators in different compartments are crucial (Frankel, 2005). Many different model systems have been used to simulate foods or biological samples in research on lipid oxidation. Although the use of model systems is important, it can be misleading because many of the model systems oversimplify the interfacial interactions of the components. Thus, model system development is a delicate compromise between reality and simplicity. Both oxidation substrate, the surrounding matrix and storage conditions should resemble `the real world' as much as possible. Yet, the system should be simple enough to be able to draw mechanistic conclusions. Within marine research, examples of models that have been used to study the oxidation process in fish tissue are bulk fish oils, emulsions (e.g., from linoleic acid), isolated bilayer structures (fish microsomes, marine PL-liposomes) and fish mince (washed and unwashed) (Fig. 20.3). Below, details of the preparation and use of liposomes, emulsions and washed fish minces will be given. It should be stressed that results obtained with both these and other model systems must be interpreted carefully, and conclusions made should, if possible, subsequently be confirmed in real food systems. 20.4.1 Liposomes and emulsions as model systems Liposomes are microscopic structures of one or more concentric lipid bilayers enclosing an equal number of aqueous compartments. Many phospholipids form

Fig. 20.3 Examples of model systems commonly used in fish muscle oxidation research. All except the bulk oil system have been used within SEAFOODplus. Adapted after Undeland, I. The use of a marine liposome based model system to study the mechanism of water soluble, fish-derived antioxidants, Lecture given at the 23rd Nordic Lipidforum Symposium, Reykjavik, Iceland, 1±4 June 2005.

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liposomes spontaneously in water (Chatterjee and Agarwal, 1988). The advantage of using liposomes in the study of lipid oxidation is that liposomes allow easy manipulation of lipid composition, pH, temperature, contents of various agents including those involved in biological oxygen defence mechanisms such as vitamin E, etc. Liposomes can be easily used to study the effect of metals on oxidation and also the oxidative interaction of haemoglobin and cell membrane. The effect of different initiating systems has also been studied using liposomes. In addition, several researchers have used liposomes to study the effect of different antioxidants (Chatterjee and Agarwal, 1988). Another model system, which is often used, is simple oil-in-water (o/w) emulsions, which consist of buffer emulsified in oil using surfactants such as Tween, SDS, Brij or more complex emulsifiers such as whey protein, sodium caseinate as already described above. These simple model emulsions are not very good models for fish muscle, but are often used to mimic more complex food emulsions such as dressing, milk etc. Model emulsions have often been used to study the effect of pro- and antioxidants including the partitioning of antioxidants between the different phases in multiphase systems.

20.5 Kinetics and modelling of lipid oxidation in liposomes and emulsions using the oxygen uptake rate 20.5.1 Introduction to the principles behind the modelling of lipid oxidation ± approach used by LIPIDTEXT As previously described the two most important reactants in lipid oxidation are the unsaturated fatty acid and oxygen. The reaction products from the oxidation are numerous and there are many possibilities for measuring lipid oxidation as already shown. But due to the low sensory threshold value for secondary oxidation products, measuring lipid oxidation is still a challenge. The sensory threshold level for lipid oxidation products from n-3 fatty acids from the marine environment (0.001±0.01 g/g) is lower than the threshold level from n-6 fatty acids (0.1±2 g/g) (Frankel, 1998; KulaÊs et al., 2003). This low threshold level and the fact that most of the reaction products from lipid oxidation are more or less stable intermediate products, makes quantification of lipid oxidation difficult. Another complicating factor is that the breakdown pathways of the lipid oxidation products can vary depending upon the physical and chemical conditions under which the oxidation takes place. Change in pathways makes it difficult to rely on one or a few oxidation products to quantify lipid oxidation. It is therefore interesting to try to use the reaction substrates as indicators for oxidation. Decrease in the concentration of fatty acids could be a possible measure. With a sensory threshold level of around 0.1 g/g of oxidation products, we will have to detect approx 1 g/g of reduced fatty acid concentration. This sensitivity of the fatty acid analysis is hard to reach. The other substrate, oxygen, can be measured as long as the lipids are dispersed in a water phase. Sensitivity in measuring oxygen concentration is in

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the range of 1±5 M, and close to the sensory threshold level for products formed from lipid oxidation (approx. 1 M). The concentration of oxygen in water at room temperature is around 230 M, enough to create rancidity. Concentration of dissolved oxygen can be easily measured by a polarographic electrode, such as the Clark electrode. One big advantage is that measurements can be done continuously and that systems for measuring lipid oxidation without mass transfer limitation can be constructed. Moreover, there is no need for sample preparation when consumption of dissolved oxygen is used for assessing lipid oxidation. Direct measurement and easy quantification of the rate of oxygen incorporated in lipids makes this technique excellent for kinetic studies of lipid oxidation. The system can be constructed in several ways, either with the use of liposomes or emulsions. In liposomes made mainly from phospholipids, systems comparable to biological membranes can be studied. In addition, oxidation stability of liposomes intended for drug deliveries can be investigated. Measuring oxygen uptake in emulsion systems gives important information of oxidation rates in food-like emulsions. Different pro-oxidants can be characterized with respect to their catalytic activity for lipid oxidation by this system. In addition, the effect of both fat and water soluble antioxidants can be studied and antioxidant strategies can be developed using this model system. The effect of different salts and changing pH, even during the oxidation reaction, can also be investigated. The consumption of oxygen as part of the lipid oxidation reaction can be measured both in gas phase as reduction in partial pressure and in water phase as reduction of dissolved oxygen. The reduction in oxygen gas partial pressure is usually measured by decrease in oxygen pressure in an oxygen bomb at elevated pressure (5 atm) and temperature (up to 200 ëC) (MikroLab, Aarhus, Denmark). Another system used is the Wahrburg apparatus (Brimberg and Kamal-Eldin 2003; Braughler et al., 1986) where the reduction of the partial pressure of oxygen in gas phase is measured at atmospheric pressure and temperatures close to room temperature. The most common method is however by using a Clark (polarographic) electrode to measure oxygen dissolved in water (Genot et al., 1999, 1994; Tampo and Yonaha, 1996; Wang et al., 1994; Niki et al., 1984, 1985; Niki, 1991; Fukuzawa, 1992, 1988; Braughler et al., 1986). Roginsky and Lissi (2005) used oxygen uptake as a method to determine antioxidant activity. 20.5.2 Mass balance PV, Tbars, CD, mass spec ± results from LIPIDTEXT In the kinetic studies performed in LIPIDTEXT, liposomes were made as described by Mozuraityte et al. (2006a). Marine phospholipids (98% pure) were as standard procedure sonicated in a 5 mM MES (2-morpholinethanoesulfonic acid, Sigma) buffer, pH 5.5 with an ultrasonic disintegrator. Lipid oxidation was measured as the consumption of dissolved oxygen by the liposomes in a closed, stirred, water jacketed cell. The concentration of dissolved oxygen was measured continuously by a polarographic oxygen electrode.

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Fig. 20.4 Concentration of dissolved oxygen in liposome solution after addition of Fe2+ or Fe3+. Each addition increased iron concentration by 7.5 M. The phospholipid concentration in the liposome solution was 6 mg/mL.

When Fe2+ was added to liposomes, the oxidation was divided in two phases: a fast initial phase and a slower linear phase of oxygen uptake (Fig. 20.4). In the fast oxidation phase, lipid peroxides interacted with Fe2+ resulting in formation of lipid radical and Fe3+ and a fast increase in lipid peroxide and TBARS values (Fig. 20.5). Addition of Fe3+ induced only the linear phase of oxygen uptake. In the linear phase, an equilibrium is established between Fe2+ and Fe3+. Peroxides reduce Fe3+ to Fe2+ at a slower rate than the reverse reaction resulting in formation of peroxy radicals. The fast initial drop in oxygen was proportional to the added Fe2+ concentration. (Mozuraityte et al., 2008). At iron concentrations

Fig. 20.5 Changes in dissolved oxygen concentration [O2], peroxide [PV] and TBARS as a function of time. Phospholipid concentration in liposomes was 9 mg/ml, and 7.5 M of Fe2+ was used to catalyze oxidation.

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between 0 and 15 M Fe2+, the constant oxidation rate was proportional to the added amount of iron. The oxidation rate as a function of pH was bell shaped with a maximum at pH 4± 5. The lipid oxidation rate increased with increasing temperature, following Arrhenius kinetics. The activation energy varied between 60 and 86 kJ/mol and varied between different phospholipid preparations, but was not dependent upon the lipid or the iron concentration. The oxidation rate did not depend on the dissolved oxygen concentration in the range between 100% saturation and down to the detection limit around 2±5% of saturation. The cations (Na+, K+, Ca2+, Mg2+) did not influence the rate of oxidation in the tested range of ionic strength (I) (0± 0.14 M). Among the tested anions, sulphates and nitrates did not change oxygen uptake rate significantly, but chlorides (KCl, NaCl, CaCl2) reduced the oxidation rate by approximately 55% and dihydrogen phosphate (pH = 5.5) by 86%, when ionic strength was 0.14 M (Mozuraityte, 2006b). To reach 50% inhibition of the oxidation rate, dihydrogenphosphate was approximately 500 times more effective than chlorides. The effect of Cl± and H2PO4± was additive, but not synergistic indicating that the ions work through two different mechanisms. Addition of salts and changes in pH affected the Zeta potential (i.e. surface charge) of liposomes. By increasing chloride concentration, an apparent linear relationship between Zeta potential and oxygen uptake rate was observed, where higher Zeta potential resulted in lower oxygen uptake rate. However, when phosphates were added, no relationship between oxygen uptake rate and Zeta potential was observed. When changing the pH of the liposome solution, there was a relationship between oxygen uptake rate and Zeta potential, but pH also has an effect on autooxidation of iron which could be important for the oxidation rate of phospholipids. Thus, the effects of pH and Zeta potential are confounded and it is therefore difficult to interpret the effect of Zeta potential. When changing one component at a time some prediction of the oxygen uptake rate can be done based on Zeta potential. But the relationship between oxygen uptake rate, Zeta potential and salt concentration is complex due to the fact that cations influence the Zeta potential, but only Cl- and H2PO4ÿ modified the oxygen uptake rate. Therefore, the absolute values of the Zeta potential alone cannot be used for prediction of oxygen uptake rates of PUFA rich phospholipids. 20.5.3 Modelling of lipid oxidation Mathematical models can be classified in different ways. Models can be based on understanding phenomena in physics and chemistry, so-called first principle based models, or models can be made directly from experimental results without any a priori knowledge of underlying principles or mechanisms. These models are called data driven models. In LIPIDTEXT project both models have been used to model lipid oxidation on data from oxygen uptake measurements. The models used compared an empirical polynomial approach to one based on more detailed chemical and physical models, which is the traditional

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approach taken for chemical systems. In this way, an assessment is made of the advantages and disadvantages of the experimental design approach as opposed to benchmark methods in the area of chemical reaction optimization. An empirical approach is used for the predictions obtained from the theoretical model for the purpose of getting information on which factors that are responsible for possible discrepancies. The effect of five variables (iron-, lipid-, and sodium chloride concentration, pH and temperature) on the rate of lipid oxidation was investigated using experimental design and response surface modelling. All the design variables, all of the two-factor interactions and one quadratic effect were found to be significant by the response surface model. In addition to the empirical response surface model, the traditional approach based on more detailed chemical knowledge was used for fitting and for making predictions. The traditional approach consisted of both theoretically and empirically determined parts. When comparing the predictions, the response surface model showed a slightly lower average prediction error than the mechanistic model. Lipid oxidation can be modelled by these two mathematical models, showing that oxidation of fatty acids in simple systems follows the rules of chemical reactions. Moreover this LIPIDTEXT work shows that prediction of the rate of lipid oxidation is possible and that predicting shelf life due to development of rancidity can be possible (Hùy et al., 2007).

20.6 Effect of emulsifiers and antioxidants on lipid oxidation in oil-in-water emulsion model systems Many of the previously performed studies on oil-in-water emulsions have used non-food surfactants such as SDS and Brij as emulsifiers (Cho et al., 2002; Stockmann et al., 2000). Moreover, only few investigations on the effect of the interactions between emulsifiers and antioxidants have been carried out. In the LIPIDTEXT project, the effect of food emulsifiers was investigated and interactions between emulsifiers and selected polyphenolic antioxidants were also investigated. 20.6.1 pH and emulsifiers ± results from LIPIDTEXT The following emulsifiers (Tween 80, Citrem, Lecithin, sodium caseinate) were evaluated with respect to their effect on lipid oxidation in fish oil-in-water emulsions at pH 3 and 7 (Haahr and Jacobsen, 2008). The emulsions had similar droplet sizes (D32 ranging from 0.2±0.5 m). It was found that the rate of oxidation decreased in the following order: Tween80 > Citrem > Lecithin > sodium caseinate and that oxidation was more pronounced at pH 3 compared to pH 7. The `antioxidative' effect of Citrem, Lecithin and sodium caseinate is illustrated in Table 20.1, which shows how much oxidation was reduced in emulsions with these emulsifiers compared to an emulsion with Tween as

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Table 20.1 Percentage inhibition of formation of selected volatiles in emulsions with Citrem, Lecithin or sodium caseinate as emulsifiers compared to emulsions with Tween as emulsifier at pH 3 and 7 in the presence of Fe after 12 days of storage at room temperature 1-Penten- Pentanal 1-Penten- 2-Pentenal Hexanal 3-one 3-ol

Heptanal 2-Heptenal Octanal

2,4Nonanal Heptadienal

2Nonenal

pH 3 Citrem Lecithin Sodium caseinate

51 82 88

11 44 93

37 88 78

73 90 94

63 63 96

83 96 96

49 ÿ25 96

51 90 98

74 90 80

18 91 89

90 86 94

pH 7 Citrem Lecithin Sodium caseinate

66 95 96

83 98 102

81 86 87

69 100 97

77 91 99

90 98 98

35 72 94

55 97 100

87 89 86

34 81 83

97 102 96

Adapted after Hahhr and Jacobsen (2008).

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emulsifier. As previously mentioned, emulsifiers may affect the oxidative stability via their surface charge, as positively charged droplets may repel positively charged metal ions and thereby decrease oxidation. However, in this case the surface charge of the droplets could only partly explain the results. Thus, the sodium caseinate emulsion had as the only emulsion a positive droplet charge at pH 3 and this could explain its antioxidative effect at this pH. However, at pH 7 the droplet charge was more negative for sodium caseinate emulsions than for Tween emulsions. Moreover, at both pH values Citrem emulsions had more negative droplet charges than the Tween emulsions and it was still a better `antioxidant'. The ability of sodium caseinate to reduce oxidation was suggested to be due to its metal chelating and free radical properties (Haahr and Jacobsen, 2008). The antioxidative effects of Citrem and Lecithin were also suggested to be due to their ability to chelate metal ions. These data clearly demonstrate that emulsifiers should be carefully chosen when new food emulsions with fish oil are developed. 20.6.2 Antioxidants in food emulsions The antioxidant efficacy in multiphase systems depends on many factors. The partitioning of the antioxidant into different phases of the systems appears to be one of the most important factors. This is due to the so-called polar paradox theory described first by Porter (1993) and later supported by (Frankel et al., 1994; Huang et al., 1996). According to this paradox, polar antioxidants such as ascorbic acid and Trolox will have a better antioxidative effect in non-polar media like bulk oil than their more non-polar counterparts ascorbyl palmitate and tocopherol, respectively. This is probably due to the fact that polar antioxidants are located at the air-oil interface where oxidation is taking place. In contrast, tocopherol and ascorbyl palmitate have a better antioxidative effect in more polar systems like emulsions, because they are located in the oil phase where oxidation propagates. 20.6.3 Interactions between antioxidants, emulsifiers and pH ± results from LIPIDTEXT Since pro-oxidants, antioxidants, pH as well as emulsifiers may affect lipid oxidation, it is important to study how interactions between antioxidants and emulsifiers at different pH will affect oxidation in emulsions in the presence of pro-oxidants such as iron. Preferably, this should be done by the use of carefully designed experiments using statistical tools. In LIPIDTEXT, the effect of different antioxidants (Caffeic acid, Rutin, Narengenin, Coumaric acid) were evaluated in emulsions at pH 3 and 6 with and without iron ions where either Citrem or Tween were used as emulsifiers. The choice of these naturally occurring phenolic compounds is further elaborated upon in Section 20.8. Lipid oxidation was evaluated by PV, volatiles and electron spin resonance (Sùrensen et al., 2008). The results clearly showed that when iron was present the pH was

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crucial for the progress of lipid oxidation. At pH 3 the phenolic compound may reduce Fe3+ to Fe2+, which increased lipid oxidation at this pH. Moreover, among the tested phenols caffeic acid had the most significant effects on the lipid oxidation progress. Even though PV and ESR measurements showed some antioxidative effect of caffeic acid it seemed that caffeic acid had prooxidative effects regardless of pH, emulsifier and iron as the formation of most volatiles generally increased. The other evaluated phenols were prooxidative at pH 3 in Citrem-stabilized emulsions and had no significant effect at pH 6 in Citrem or Tween-stabilized emulsions based on the development of volatiles. Importantly, the data showed that different analytical methods used to evaluate lipid oxidation showed different effects of the same antioxidant.

20.7

Washed fish mince as model systems

In recent years, the use of washed fish minces has emerged as a particularly useful tool in fish muscle research. This is because washed fish mince provides a matrix that has the structure of muscle, i.e., with intact myofibrillar proteins and membranes, but is virtually free of endogenous triacylglycerols, pro- and antioxidants. Controlled physiological levels of any of these groups of compounds can then be added back and studied in relation to lipid oxidation under various conditions of pH, moisture, etc. By adding an antibacterial agent, the `window' during which oxidation can be studied during ice storage is extended. To date, most trials with washed fish mince as a model have been done using cod light muscle as this species naturally is very low in both lipids and catalysts (Undeland et al., 2002, 2003, 2004; Grunwald and Richards, 2006a,b; Richards and Li, 2004; Li et al., 2005; Sannaveerappa et al., 2007; Richards and Hultin, 2001; Richards et al., 2002a, 2005). However, a few studies exists where washed minces from other species are used, including mackerel (Kelleher et al., 1992), horse mackerel (Eymard et al., 2005) and herring (Undeland et al., 1998b). Some of them are parts of storage stability tests of traditionally made surimi. 20.7.1 Hb as a pro-oxidant in washed fish mince Endogenous levels of fish Hbs (5.8±20 M), usually in the form of hemolysates, have almost exclusively been used to start oxidation in washed cod mince (Undeland et al., 2002, 2003, 2004; Grunwald and Richards, 2006a,b; Richards and Li, 2004; Li et al., 2005; Sannaveerappa et al., 2007; Richards and Hultin, 2001; Richards et al., 2002a, 2005). When running trials at the slightly acidic pHvalues (6.2±6.5), commonly found in post mortem fish, oxidation is then usually measurable within 1±5 days of ice storage. A few studies also exists where sperm whale Mbs have been added to washed cod (Grunwald and Richards, 2006a,b) or to washed mackerel (Oshima et al., 1988). A reason for focusing on heme proteins has been the wish to elucidate the role of deoxygenation, autoxidation, hemin-loss and Fe-loss in lipid oxidation. Other tasks have been to compare Hb with Mb and

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also different forms of Hbs (e.g., anodic/cathodic and Hb from different fish species). Some results that have emerged include that variants of human Hb that have higher ability to form subunits stimulate washed cod lipid oxidation more efficiently than those with lower subunit formation ability. This study, together with a series of studies on mutant sperm whale Mbs point at the rate of hemerelease as the most important factor predicting the onset of lipid oxidation in washed cod mince; even higher than the autoxidation rate (Grunwald and Richards, 2006a,b; Richards et al., 2005). Washed cod has also been used to evaluate whether LMW-Fe has a role equal to Hb in catalyzing lipid oxidation in fish. When 15 M Fe2+-ADP-NADPH was compared with 12 and 24 M heme-bound Fe in washed cod with/without added oil, it was seen that LMW-Fe did not give rise to any oxidation, which was in contrast to the Hb (Undeland et al., 2002). Further, Richards and Li (2004) found that during 2.5 days ice storage of Hb-fortified washed cod mince at, 7% of the Hbassociated Fe was released from Hb, but still, EDTA had no effect on oxidation. This also suggested a minor role of LMW-Fe. Thirdly, it was found that a sperm whale Mb variant sensitive to heme-degradation (L29F/H64Q) was a weaker prooxidant than wild type Mb not having this feature (Richards et al., 2005). Thus, releasing the Fe from the porphyrin ring decreased the pro-oxidant capacity. Regarding results on species differences in Hb-activity, a recent study (Undeland et al., 2004) showed that four different Hbs were ranked as follows regarding the ability to oxidize washed cod mince; pollock, mackerel, menhaden, flounder. All Hbs had higher catalytic activities at pH 6 than at pH 7.2, which corresponded with higher formation of deoxy- and met-Hb. It was hypothesized that fish adapted to colder temperatures have more unstable Hb, which could in part refer to the heme-globin linkage (Grunwald and Richards, 2006a,b). Since Hb has been a major catalyst used in washed fish mince models, the opportunity has also been given to follow loss of redness as a useful additional way of monitoring oxidation. Conversion of the red oxy-Hb to the greyish-brown met-Hb correlates very strongly to lipid oxidation development (Wetterskog and Undeland, 2004). Other oxidation parameters commonly followed in the washed cod mince systems are rancid odour (sensory analysis), peroxide value (PV), TBARS and yellowness (increased b*-value). Together they give a comprehensive picture of the oxidation progress. An effort has also been made to monitor the volatile oxidation products responsible for the rancid dour in washed cod mince fortified with cod and char Hb (JoÂnsdoÂttir et al., 2007; Olafsdottir et al., 2006). Volatiles contributing to rancidity in these models were primarily hexanal (odour threshold, OT = 4.5 ppb), cis-4-heptenal (OT = 0.04 ppb), 1octen-3-ol (OT = 10 ppb), and 2,4-heptadienal (OT = 10 ppb). 20.7.2 Results from LIPIDTEXT In LIPIDTEXT, the washed fish muscle systems have been used to study a range of factors (Larsson et al., 2007); (i) whether model systems can be made from

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other species than cod (salmon, herring, horse mackerel), (ii) the kinetics by which different products of Hb-mediated lipid oxidation form (peroxides, TBARS, met-Hb, volatiles) (iii) role of added neutral oil in Hb-mediated oxidation in washed cod mince, (iv) role of moisture content and (v) role of Hbmediated lipid oxidation for protein insolubilization. The idea behind comparing different species in making washed minces is that washed cod mince certainly cannot mimic oxidation reactions of all fish species, and that the closer to reality one is working, the more reliable data will be obtained. It was obvious from the comprehensive compositional analyses that were made of both the crude and washed minces that there are individual characteristics of all these fish species when it comes to, for example, fatty acid pattern, lipid class distribution, antioxidant and pro-oxidant content. Most of these compositional differences were evened out after washing the different minces, but some small differences remained which will be discussed below. During ice storage, Hb immediately induced rancid odour in the herring model (no lag phase), followed by the cod (0.8 day lag phase) and then the salmon model (1 day lag phase) (Table 20.2). The same order of rates was also found for PV. However, while maximum rancidity scores were about the same in all three systems, higher maximum PV-levels were reached in the salmon model, followed by herring and then cod. The latter order was the same as that found regarding the residual lipid levels in the models; salmon model (3.4± 3.6%, w/w) > herring model (1±3%) > cod model (0.6±0.7%) (Table 20.2). It is therefore believed that the higher lipid substrate level simply yielded higher levels of peroxides. That both primary (PV) and secondary (rancid odour) oxidation products increased more or less simultaneously contradicts the classic sequential development of primary and secondary products which is often suggested from bulk oil studies (Gardner, 1983). That the measured oxidation products levelled out, and sometimes even declined after a few days indicates fast reactions of secondary products, e.g. with proteins of the models. The latter was confirmed by the slight increases in yellowness (Schiff's bases polymerization) in the cod and herring models in the same time span as odour and PV Table 20.2 Rancid odour development together with compositional information (fat, PUFA, -tocopherol, Fe, Cu and Zn) for model systems made from cod, herring and salmon caught in the spring and fall of 2004. w/w = wet weight, PUFA = polyunsaturated fatty acids Model

Cod Salmon Herring

Days Total until fat rancid content odour (% w/w) detected 0.8 1.0 0.0

0.6±0.7 3.4±3.6 1±3

PUFA (mg/g model)

tocopherol (g/g model)

2.0±2.1 10.6±12.7 2.9±9.3

2.2±3.5 1.9±2.2 0.25±0.9

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Total Cu (g/g model)

Total Zn (g/g model)

0.3±0.4 0.07±0.01 2.0 0.3±0.7 0.1±0.2 1.3±1.8 0.7±1.1 0.4 2.4±2.5

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changed. Salmon model behaved somewhat differently in this aspect in that yellowness rather decreased, probably due to carotenoid bleaching resulting from co-oxidation of lipids. The small differences in the onset of Hb-mediated oxidation (lag phase length) between models from the three species were probably not linked to the total lipid or n-3 PUFA levels (Table 20.2), and also not to the relative amount of n-3 PUFA in the fat (cod > herring ~ salmon). Rather, it could be linked to residual levels of vitamin E in the washed models (cod > salmon > herring) (Table 20.2). Visual inspection further revealed a much higher level of carotenoids in the salmon model compared to in the other two models. Both vitamin E and carotenoids therefore probably contributed to the longer oxidation lag phase seen in washed salmon mince. The oxidation differences could also be linked to the small residues of trace elements in the three models (Table 20.2). Total Fe and Zn ranked the models as herring > cod > salmon, and Cu: herring > salmon > cod. The metals could be responsible for the pre-formed peroxides present in the washed minces, which, upon addition of the trout Hb, quickly break down to free radicals. From PV-analyses of the models prior to Hbaddition, it was seen that the herring models indeed had higher PVs than the other ones. Upon adding Hb to the herring model, the PV immediately increased by 33%, while in the other models, a short storage on ice was needed to increase their PVs. When no Hb was added, the cod and salmon models stayed completely stable, while there was a tiny development of rancid odour in the herring model. This showed that the residual metals per se, without extra Hb, could not induce any significant oxidation. In later trials with washed horse mackerel light muscle, it was found that this model behaved almost exactly like herring light muscle model, both with/without Hb. It was found that the washed fish models could also be used to study lipid oxidation during storage at ÿ18ëC. Cod and herring models obtained elevated rancid odour scores, PVs and b*-values after 4±9 weeks. In general, oxidation was somewhat more linear during frozen storage than during ice storage, where the classic sigmoid behaviour was seen. In order to increase the complexity of the washed cod mince model, Hbmediated oxidation kinetics were evaluated in the presence or absence of 10% non-refined cold pressed herring oil. It was found that the extent of rancid odour was not altered by the addition of 10% herring oil. However, both the rate and maximum PVs were slightly higher in high-lipid samples. It thus appears as the total amount of secondary oxidation products released to the head space or aqueous phase is unaffected by the total fat content, although there is a higher amount of pre-cursor lipid and hydroperoxide content. Increasing the moisture of washed cod mince from the normal 84±85% up to 90% shortened the lag phase with 1 day, despite the fact that the ratio between oxidizable substrate (membranes and proteins) and Hb was lower. A reason might be the increased mobility of the Hb, creating better access to the lipid/ peroxide substrate (Fenema 1996). A scoop within the LIPIDTEXT project has been to link lipid oxidation to

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protein changes. Using washed cod and herring mince models, protein salt solubility was therefore followed along with Hb-mediated lipid oxidation during ice and frozen storage. The general conclusion from these trials have been that the initial salt solubility of the proteins of washed fish mince systems was fairly low (40±60%) since the sarcoplasmic proteins were removed, and since the models have been through a fairly rough treatment, often with double freezing. This limits the window where one can measure further changes in salt solubility due to Hb/lipid oxidation. However, despite a low starting solubility (54% and 43% in cod and herring models), the solubility during 16 weeks at ÿ18ëC was lowered by another 40%. It was interesting to note, though, that these solubility losses were recorded both with and without added Hb, although lipid oxidation only proceeded with Hb. It thus appears as if the freezing process per se causes more harm to the proteins than the Hb. During ice storage, on the other hand, the solubility decreased by 36% in the Hb-fortified sample, while only a slight decrease was observed in the control. Solubility trials with -mercaptoethanol + salt as well as SDS indicated that the insolubilzation recorded during both ice and frozen storage was not due to formation of disulphide bonds, but that electrostatic interactions were involved. Based on the results from the ice storage, it remains to be proven whether there is a direct cross reaction between, e.g. lipid radicals with proteins and vice versa, or if the two reactions just occur in parallel. In conclusion, the washed fish mince models are highly flexible models that work very well for studying the kinetics of Hb-mediated lipid oxidation, and how this reaction can be prevented. It is possible to also make models from dark muscle fish and from salmonoids, which allows more valuable conclusions to be drawn if there is an interest in these species. One has to keep in mind that both the pH and moisture content of the models are highly controlling factors, with lower pH and higher moisture both speeding up the reaction. If the wish is to study changes in protein solubility, it is worthwhile considering not freezing the models before preparing the oxidation samples, and also not after taking out samples during storage trials. The latter will lower the initial salt solubility and thus, minimize the `window' where possible further changes can be monitored. Finally, it is strongly suggested that hypotheses on anti- or pro-oxidants developed from washed fish mince studies are confirmed in unwashed muscle before making recommendations about how to minimize oxidation, e.g. to industry.

20.8

Natural antioxidants in fish products

20.8.1 Introduction to antioxidant effects in fish products Inhibition of lipid oxidation is critical for increasing the shelf-life of fish species during storage and processing, and for maintaining its sensory and nutritional values. Oxidative stability is controlled by the balance among pro-oxidants and antioxidants in live fish but the post-mortem reactions change this balance

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(Hultin, 1994). Therefore, the dynamism of fish tissues complicates the procedures aimed to reduce or minimize lipid oxidation. Fish rancidity cannot be totally avoided, but some procedures can minimize the rate of lipid oxidation. The use of antioxidants as food additives has increased as an effective methodology for inhibiting lipid oxidation and its deleterious consequences (Madrid and Cenzano, 2000), but the antioxidant efficacy in fish muscle is difficult to predict. As already mentioned in Section 20.2 the antioxidants can show different efficacies depending on the type of fat or food, the processing or manipulation (Frankel, 1998). Usually, oxidative rancidity is controlled by the use of synthetic phenolics as butyl-hydroxy-anisol (BHA) and butyl-hydroxytoluene (BHT). Now, the safety of synthetic antioxidants has been discussed (Yu et al., 2000) and the current legislation and the restrictions and preferences of consumers limit their use in foodstuffs. The capacity of some natural compounds to effectively scavenge free radicals proved to be useful in preventing oxidation of many lipid systems (Shahidi and Naczk, 1995). Vegetable extracts as those resulting from tea (Ishihara et al., 2000; Tang et al., 2001) rosemary (Vareltzis et al., 1997), olive oil (Medina et al., 1999), ginger extracts (Fagbenro and Jauncey, 1994) or grapes seeds (Pazos et al., 2005a) composed by flavonoids, polyphenols, terpenoids, etc., have inhibited successfully rancidity of seafood products such as fish patties, fermented fish, canned fish, emulsified fish, etc. Other natural extracts as those obtained from materials such as fish light muscle have been also utilized in fish systems (Gunnarsson et al., 2006). In order to establish the mechanisms involved in the antioxidant activities, some of the isolated compounds like catechins and their gallate esters (He and Shahidi, 1997), procyanidins (Pazos et al., 2006a), hydroxytyrosol (Pazos et al., 2006a), flavonoids (Ramanathan and Das, 1992), carnosic acid and their derivative by-products (Medina et al., 2003), or the derivatives of elenoic acid (Medina et al., 2003) were tested. The activity found was related with the molecular structures and polarity. Some of these components have been recently demonstrated to inhibit the enzymatic oxidative activity of lipoxygenase in fish muscle (Banerjee, 2006). Tocopherol isomers, particularly alpha-tocopherol, have also been used to reduce oxidation in fish oils (Nawar, 1996). They can also be supplemented through the diet normally in form of tocopherol acetates (Yan et al., 2006). Ascorbic acid is usually supplemented with tocopherol, but its use must be carefully optimized since it can be a promoter of lipid oxidation (Lauritzsen and Olsen, 2004). The activity of these natural compounds is greatly influenced by the lipid substrate (Frankel, 1998). Some antioxidants are very effective in fish oils, but not in fish muscle or in fish oil emulsified products. The polarity and the incorporation into the sensitive oxidative sites of fish muscle are factors which largely affect the antioxidant activity of phenolics on fish lipids (Raghavan and Hultin, 2005). Molecular features as the extensive hydroxylation and polymerization and properties as the ability for chelating iron or the capacity for donating electrons are indicated as possible mechanisms for explaining its efficiency (He and Shahidi, 1997; Pazos et al., 2006b).

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Several of these phenolic compounds have lately attracted much attention in relation to their physiological role as antimutagenic and antitumorigenic agents (Hong et al., 2001). Thus, the seafood products supplemented with natural antioxidants can offer the combined action of fish bioactive compounds such as n-3 PUFA and natural polyphenols. The group of hydroxycinnamic acids is particularly attractive for being employed as natural antioxidants in seafood. They are widely distributed in plants and vegetables and can be obtained at low cost. They have also shown a high antioxidant activity in different in-vitro assays and in different lipid systems (Rice-Evans et al., 1996; Marinova et al., 2006). 20.8.2 Effect of natural antioxidants in fish mince ± results from LIPIDTEXT The LIPIDTEXT project has demonstrated that caffeic, chlorogenic acid, ocoumaric acid and ferulic acid have a high potency for inhibiting rancidity in fish minced muscle. Minced fish muscle provides an excellent matrix for making controlled test aimed to study the antioxidant activity of several compounds. It comprises all muscle components. By mincing, its external surface is high and the oxidation occurs fast. Additionally, it is possible to get a homogeneous fish sample with similar levels of PUFA, iron, haemoglobin, and other components involved in the oxidative reactions. These facts allow controlled and non-extensive experiments as those needed to check the activity of antioxidants. It is particularly useful for chilled experiments supplemented with antibacterial agents and for frozen tests. This system has been used in different works focused on the antioxidant activity of phenolics (Vareltzis et al., 1997; Medina et al., 1999; Ishihara et al., 2000; Tang et al., 2001) and the results obtained could be confirmed in whole fish fillets (Pazos et al., 2006a). In LIPIDTEXT, the above cited hydroxycinnamic acids were supplemented in minced horse mackerel and minced salmon muscle and its antioxidant activity was tested during chilling and frozen storage. Caffeic acid showed the highest effectiveness for retarding lipid oxidation of chilled horse mackerel muscle followed by ferulic, chlorogenic and o-coumaric acids (Table 20.3). Such order of effectiveness was corroborated in minced salmon muscle. The antioxidant inhibition achieved by the supplementation of 100 mg/kg of caffeic acid was Table 20.3 Formation of TBARS (Thiobarbituric Reactive Substances) of chilled minced horse mackerel muscle supplemented with 100 mg/kg of phenolic antioxidants (mmol MDA/Kg muscle) Days 5 7

Control

Caffeic

Chlorogenic

o-coumaric

Ferulic

BHT

Propyl gallate

4.17 6.24

0.04 0.08

0.08 0.31

1.21 2.79

0.09 0.22

0.10 0.52

0.03 0.08

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comparable to that shown by propyl gallate and higher than that achieved by BHT. In accordance with the results obtained in chilled fish, cinnamic acids inhibited oxidation in frozen horse mackerel. Studies performed at two experimental temperatures, ÿ10ëC and ÿ18ëC demonstrated a significant efficiency of caffeic acid for inhibiting the formation of off-flavours and oxidation byproducts in frozen horse mackerel and frozen salmon. The order of antioxidant efficiency obtained in frozen horse mackerel stored at ÿ10ëC and ÿ18ëC was the same as that observed in chilled horse mackerel. The capacity of phenolic acids for donating electrons showed a high correlation with their ability to retard lipid oxidation in fish muscle. In contrast, their ability for chelating metals or their polarity was not correlated with their inhibiting activities. In spite of the effect of hydroxycinnamic acids on inhibiting lipid oxidation of fish minced muscle, they did not show any effect on inhibiting the change of protein solubility. Control samples of minced horse mackerel and salmon muscles and those supplemented with phenolic acids showed similar pattern of decrease in protein solubility during frozen storage. Thus, it seems that the inhibition of lipid oxidation by the addition of phenolic antioxidants has no effect on the change in muscle proteins related with the loss of solubility. 20.8.3 Effect of exogenous antioxidants on the oxidative reductor system During early post-mortem stage, fish tissues show reduced capacity for activating free iron (LMW iron) present in its ferric form and usually associated to metabolites (Kanner, 1994). As previously discussed, Hb activation is also considered a major catalyzer of lipid oxidation in fish muscle of several fish species (Richards et al., 2002b). On this basis, inhibition of oxidation promoted by Hb and non-Hb iron in fish muscle is difficult. Some phenolic compounds have inhibited Hb autooxidation during the post-mortem storage (Gorelik and Kanner, 2001). Chlorogenic acid can deactivate haemoproteins through a bond between the phenolic group and the protein (Carlsen et al., 2000). Pazos et al. (2006b) have found a relationship between chelating capacity of the exogenous phenolic and the inhibition of oxidation promoted by Hbs in fish microsomes. A possible loss of iron from the porfyrinic structure was suggested. In post-mortem stages, endogenous antioxidants of fish muscle are sequentially consumed for inhibiting lipid oxidation or they can be lost during processes as washing or filleting. Their loss provokes a rapid increase of the rate of oxidation by concluding the induction period. Recent studies have described that alpha-tocopherol is the last defence of fish muscle against oxidation (Pazos et al., 2005b). Similar works have demonstrated the loss of alpha-tocopherol during storage and processing of fish oils (Frankel et al., 2002). The supplementation of hydroxycinnamic acids to minced horse mackerel retarded the loss of glutathione and alpha-tocopherol during chilling and frozen storage in the studies performed in LIPIDTEXT. Again, caffeic acid showed the highest activity for inhibiting the loss of endogenous antioxidants followed by ferulic acid and then o-coumaric acid. The retardation achieved by the use of phenolic

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acids was correlated with the increment of the induction periods of peroxide and TBARS formation detected in fish muscle. Such inhibition is attributed to the prevention action of the hydroxycinnamic acids, but also a synergistic or protection effect. Caffeic acid was demonstrated to be active for regeneration of alpha-tocopherol from its tocoferoxyl radical in Electronic Spin Resonance Spectroscopy experiments. Data suggested that the addition of 100 ppm of caffeic acid could regenerate endogenous alpha-tocopherol from its oxidized forms resulting in an antioxidant synergy consistent with the reduction of lipid oxidation observed in fish muscle supplemented with phenolic acids. Recent works have also shown alpha-tocopherol regeneration in micelles and LDL due to catechins and other flavonoids (Zhu et al., 2000; Zhou et al., 2005).

20.9

Conclusions

On the basis of the above review of the theoretical background for and the results obtained in LIPIDTEXT the following main conclusions can be drawn: Previous studies on fish lipid oxidation clearly show that hemoglobin (Hb) appears to be a major pro-oxidant in fish; especially in combination with pH < 6.5; which activates the pro-oxidative properties of Hb. Likewise, iron also seems to play an important role for lipid oxidation reactions in many food emulsion systems enriched with fish oil. Model systems in fish muscle oxidation research are a delicate compromise between reality and simplicity. Among the model systems commonly used today are lipid bilayers, lipid emulsions, washed fish mince and whole fish mince. Liposomes are a good model system for studying lipid oxidation. Lipid oxidation can be studied by measurement of oxygen uptake, which is a sensitive and rapid method. The oxygen uptake rate is directly proportional to the concentration of low molecular weight iron (Fe2+ and Fe3+). The equilibrium between Fe2+ and Fe3+ is important for lipid oxidation rate and the rate limiting ion is Fe3+. The cations (Na+, K+, Ca2+, Mg2+) did not influence the rate of oxidation. Sulphates and nitrates did not change oxygen uptake rate significantly, but chlorides (KCl, NaCl, CaCl2) reduced the oxidation rate by approximately 55% and dihydrogen phosphate (pH=5.5) by 86%. To reach 50% inhibition of the oxidation rate, dihydrogenphosphate was approximately 500 times more effective than chlorides. The effect of Cl- and H2PO4ÿ was additive indicating that the ions work through two different mechanisms. Within certain limits it is possible to predict lipid oxidation rates in liposomes and simple oil-in-water emulsions. Further research on more complex systems is necessary. In emulsions, our results showed a clear effect of the emulsifier type on lipid oxidation and it is thus possible to control oxidation by selecting the right emulsifier. This approach should also be investigated in more complex food emulsions. Washed fish minces from several species (herring, horse mackerel, salmon and cod) proved to be highly useful in studying Hb-mediated fish lipid oxidation

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during ice storage. Important aspects for concluding this were that control samples without added Hb stayed stable and that oxidation with Hb started within 1±2 days on ice. When comparing the different models, oxidation developed fastest in washed herring and horse mackerel minces, followed by cod and then salmon. This was explained by the fact that the salmon model had most antioxidants, while the herring model had most pre-formed hydroperoxides and trace elements. In general, oxidation in the washed models increased by reduced pH and increased moisture content. Adding 10% extra cold pressed herring oil to washed cod mince (original fat content 0.6±0.7%) did not affect rancid odour development or intensity, but raised the maximum peroxide values reached. Together with previous data this strengthens that the total lipid content plays a minor role for the rate of rancidity development when strong pro-oxidants are present. When following protein solubility changes in the washed fish mince models, it was seen that this parameter went down by 40% both with/without Hb during 16 wk storage at ÿ18 ëC. However, during ice storage, only proteins in the Hb-containing samples lost solubility (by 36%). Hydrophobic interactions between proteins appeared to be responsible for solubility losses. In fish mince, our results suggested that natural cinnamic acids can be successful additives to seafood products by improving their stability toward oxidation, and their nutritional and flavour quality. The effectiveness of inhibiting oxidation in fish muscle seems to be highly dependent on the intrinsic redox capacity of the antioxidant and the protection of the endogenous reductor system. The research in LIPIDTEXT showed that the formulation of fish products rich in n-3 PUFA in combination with the antioxidant properties attributed to natural phenolics could result in stable and nutritional foods. However, it was also found that cinnamic acids were not efficient antioxidants in fish oil enriched emulsions. Moreover, in emulsions antioxidants and emulsifiers were found to interact in a complex way and the interaction was depending on pH and the presence of pro-oxidants and this influenced the efficacy of the antioxidants. Such interactions may be even more complex in real food emulsions and needs further investigation as results obtained in one model system cannot be interpolated to other food systems.

20.10

Future trends

The research in LIPIDTEXT has increased our understanding of the lipid oxidation mechanisms and their kinetics in fish products. A few indications on the link between lipid oxidation and protein changes have also emerged. However, more research is still needed on the following topics in order to be able to develop efficient strategies to prevent both lipid oxidation and related protein changes: · Kinetic models on the factors affecting lipid oxidation rates should be expanded to include complex systems such as fish muscle and different fish oil enriched food systems in order to be able to predict how fast oxidation develops under different conditions in different food matrices containing fish lipids.

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· Interactions between antioxidants, emulsifiers and other ingredients should also be evaluated in more complex food systems to be able to explain how such interactions affect oxidation and to be able to predict antioxidant efficacies to a greater extent. An important research topic will be to obtain more knowledge about the reactions taking place at the interfaces in both emulsified systems and in fish muscle systems. · It is important to realise that changes in texture and odour are not separate events, but that they often go hand in hand and are due to oxidative reactions. Therefore, it is necessary to obtain a better understanding of protein and lipid interactions during storage in complex food systems containing both lipids and proteins. Unfortunately, the methods currently available to study protein oxidation are not sensitive and specific enough to get a complete picture of the protein oxidation kinetics. A high priority is therefore to develop better methods to assess protein oxidation. Once having this tool, more detailed studies on the possibility for lipid and protein oxidation reactants to interfere with one another should be conducted.

20.11

Sources of further information and advice

Euro Fed Lipid Varrentrappstr. 40±42 D-60486 Frankfurt/Main Germany Phone: +49 69 7917 345 Fax +49 69 7917 564 www.eurofedlipid.org American Oil Chemists Society 2710 S. Boulder, Urbana IL 61802-6996 USA Phone: +1-217-359-2344 Fax: +1-217-351-8091 www.aocs.org Nordic Lipidforum Secretary General Professor Sigmundur Gudbjarnason Department of Biochemistry, Laeknagardur University of Iceland IS-101 REYKJAVIK, ICELAND Phone: +354-525 4797 Fax: +354-525 4886 E-mail: :[email protected] http://www.lipidforum.org www.lipidforum.org

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Cyber lipids: http://www.cyberlipid.org/cyberlip/home0001.htm The International Lecithin and Phospholipid Society Website: http://www.ilps.org/ilps_main.htm Lansbury Research Site, literature database: http://lansbury.bwh.harvard.edu/literature.htm

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Acknowledgements

The authors wish to thank the following co-workers in LIPIDTEXT for their contributions to the results obtained in the LIPIDTEXT project: K. Larsson, A. Almgren, J. M. Gallardo, M. J. GonzaÂlez, S. Lois, M. Pazos, L. Berner, A.-D. M. Sùrensen, A.-M. Haahr and R. Mozuraityte.

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Part V Seafood from aquaculture

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21 Introduction to Part V: seafood from aquaculture ± added value possibilities and potential impacts B. DamsgaÊrd, Nofima, Norway

Seafood products are sourced from either traditional fisheries or aquaculture. Most consumers have little knowledge on whether a seafood product is fished or farmed, and would not be able to taste the difference. However, the added value possibilities of the product and its potential negative impact can differ markedly between each source. Various aspects of product quality and the number of health-promoting factors in the products depend to a large extent on the starting point of the value chain, in addition to handling at slaughter. The potential negative impacts of fisheries or aquaculture, such as those regarding social, economic and ecological sustainability, are comparable only to a minor extent. Overexploitation of scarce wild resources is one of the most important threats for sustainable fisheries, and aquacultural food production can be regarded as a necessary progression in sourcing seafood, if fish consumption is to be maintained at current levels. Any further increase in fish consumption may depend on the availability of healthy, high quality seafood products. Aquaculture may become an increasingly important source of a wide range of seafood products, including both functional food and tailor-made added value products and more traditional styles of seafood. The industry can deliver year round product quality and composition, potentially increasing the market penetration of healthy seafood. Compared to the capture-based seafood industry, aquaculture has the potential to be more consumer driven, as the industry can speedily adapt to market demands. Currently, consumers have several concerns about aquaculturally produced seafood and these concerns have to be addressed. Some consumers might

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believe that farmed fish have poorer taste and texture compared with wild fish, and express fears about product contamination from polluted water sources or contaminated fish feed. Consumers might also be concerned about the sustainability of marine feed sources, including the use of marine proteins and lipids in fish feed. As the aquaculture industry grows, it is becoming increasingly necessary to develop alternative feed sources as marine resources decline. Aquacultural production may also have adverse environmental effects, both directly by disease and organic pollution from the farms, and indirectly such as escapees' interaction with wild fish populations. In addition, during recent years consumers have expressed ethical concerns over intensive production and slaughter. Such consumer concerns, whether based on actual or perceived negative impacts, may undermine the developmental potential of the aquaculture industry. Aquaculture is to a large extent a knowledge-based industry. Cultivation of any animal depends on managing biological processes throughout its lifespan. Basic knowledge of fish physiology and behaviour is crucial in order to optimise production possibilities, and to address the potential negative impact of aquaculture. In addition, as European farming is mostly intensive, industrial technologies have to develop in parallel with biological knowledge. SEAFOODplus focuses on the production potential of aquaculture, and also the challenge of finding a compromise between intensive rearing and consumer demands for ethically and sustainable produced seafood. The aquaculture component of SEAFOODplus has focused on dietary modulation, fish physiology, genetics, fish welfare and pre-slaughter conditions, incorporating data from both traditional farmed species and emerging species. The following two chapters about aquaculture focus on muscle texture in farmed fish, including postmortem softening and heritability of muscle structural traits (Chapter 22) and fish welfare issues during production, including the effects of water quality in intensive rearing and the effects of pre-slaughter treatments (Chapter 23).

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22 The biological basis of variability in the texture of fish flesh I. A. Johnston, University of St Andrews, Scotland

22.1

Introduction

Firmness is a critical factor in determining the acceptability of raw fish products (Veland and Torrissen, 1999). Soft flesh leads to reduced consumer acceptability (Ando, 1999) and also causes problems when fillets are sliced by machine during industrial processing (Michie, 2001). An associated problem but not always related to soft flesh is fillet gaping (Mùrkùre and Rùrvik, 2001). Gaping involves the post-mortem rupture of the connective tissue matrix between muscle fibres that detract from the appearance of the product and impede secondary processing. The secondary processing of Atlantic salmon involves filleting, curing, smoking, and preparation of the final consumer product. Monetary loss during secondary processing mainly arises due to variations in colour, bloodspotting, gaping, lacing and soft flesh (Michie, 2001). It has been estimated that soft flesh and gaping combined represent around 40% of the causes of downgrading losses during secondary processing within the industry in Scotland (Michie, 2001). In the future, the opportunities for genetic selection, control over rearing conditions and slaughter provided by fish farming may enable flesh texture to be optimised for particular markets and seafood products. For this vision to be realised a greater understanding of the cellular, biochemical and genetic factors influencing muscle texture is required. This chapter summarises current knowledge on the biological basis of variation in the texture of fish flesh and describes some of the new research emerging from the SEAFOODplus programme.

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Muscle texture

Fillet texture can be measured using trained taste panels or a variety of instrumental methods (reviewed in Hyldig and Nielsen, 2001). The advantage of taste panels is that the results are more likely to be directly related to the eating experience of consumers. Sensory evaluation of texture is often subdivided into perceptions of mouth-feel, chewiness, juiciness, dryness, and firmness. The disadvantages of taste panels are that they are highly skilled, require sophisticated statistical knowledge to interpret and are labour intensive and therefore very expensive. The design of taste panels requires replicate samples to be tasted by multiple panellists and as a consequence only small numbers of fish can be handled, and the data obtained at different times and by different laboratories cannot be combined. Instrumental texture analysis when performed under standardised conditions may provide more precision and repeatability relative to taste panels (Veland and Torrisen, 1999) and is relatively high throughput making it is suitable for large numbers of fish. With appropriate cross-validation, measurements from different laboratories can be directly compared. Various instrumental techniques to measure texture have been developed involving puncture, compression, shear and tensile techniques (Casas et al., 2006). There is no consensus about the most appropriate instrumental texture method for fish muscle. Furthermore few studies have directly compared instrumental texture measurements with results from trained taste panels using the same samples. Therefore the extent to which instrumental methods reflect the eating experience or behaviour of the product during mechanical processing is largely unknown. Texture varies between pre-rigor, rigor and post-rigor states and therefore the timing of measurements must be carefully controlled, particularly for instrumental methods. Post-mortem softening of fish flesh occurs after storage on ice for several days (Ando et al., 1991; Hatae et al., 1985; Sigurgisladottir et al., 2001). In general, long-term storage of fish flesh, even at sub-zero temperatures, results in an increase in firmness eventually reaching the point of consumer unacceptability (Love, 1988). The texture of fish flesh is known to vary with slaughter method and the degree of struggling prior to death which produces associated metabolic changes in the muscle, including a drop in pH (Kiessling et al., 2004). After controlling for extrinsic factors influencing flesh texture including husbandry conditions, slaughter method, rigor, and storage time, there are still significant differences found between individual fish due to variation in the intrinsic biological properties of the muscle (Fauconneau et al., 1995; Bugeon et al., 2003). The biological factors contributing to texture are different for raw, smoked and cooked fish products. In raw and smoked fish, muscle fibres and associated proteins, the connective tissue, lipid, pH and water content can all contribute to the texture. However, after fish flesh is cooked, the connective tissue matrix no longer significantly contributes to texture, which contrasts markedly with the situation found in beef, mutton and pork. Different smoking procedures also have a significant influence on muscle texture (Birkeland et al., 2004). Post-

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The biological basis of variability in the texture of fish flesh 467 mortem softening of the flesh is also related to intrinsic differences in the content of muscle proteolytic enzymes and their inhibitors (Lund and Nielsen, 2001). Factors influencing muscle texture have been most extensively studied in salmonids, particularly Atlantic salmon. In this species, texture decreases with increasing body size (Johnston et al., 2006) and increases in a rostral to caudal direction along the trunk (Casas et al., 2006), in both cases reflecting variation in muscle structural components. Texture varies with season often in association with sexual maturation (Red sea bream, Pagus major: Touhata et al., 1998; Atlantic halibut: Hagen et al., 2007). Since the early days of salmon farming, factors such as genetic selection, improved diets, the use of accelerated smolts and treatments for sea lice have reduced the time to produce marketable salmon of 4±5 kg from 4.5 years to a little over 2 years. Johnston and co-workers tested the hypothesis that fast growth rate increases the incidence of soft flesh and gaping (Johnston et al., 2007). To increase the robustness of hypothesis testing, two different strains of Atlantic salmon were studied in Scotland and northern Norway farmed under very different environmental conditions. Individual growth rates were estimated using the thermal growth coefficient (TGC) (Cho, 1992). Although TGC values at the high latitude site were amongst the highest recorded for Atlantic salmon in the literature, the hypothesis was still rejected and it was concluded that fast growth per se does not produce soft texture or an increased incidence of gaping (Fig. 22.1).

Fig. 22.1 Fish growth rate and muscle texture. There was no significant relationship between thermal growth coefficient (TGC) and the work required (WD) in mJ to shear a standardised slab of flesh for Atlantic salmon grown at a site in northern Norway and fed either satiation or restricted ration levels and harvested in September (open circles) and November (closed circles) respectively. Thermal growth coefficient (TGC) was calculated according to the formula: TGC = [(W20.333 ÿ W10.333) (degree days)ÿ1 1000], where W1 and W2 were the initial and final body weights for each period respectively. Degree day values are the sum of the ëC values for each day of the growth trial. Modified from Johnston et al. (2007).

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22.3 Organisation, structure and biochemistry of fish myotomes The fillet is made up of segmentally arranged structures called myotomes or myomeres, the shape of which varies along the length of the body. In three dimensions, the myomeres constitute a series of overlapping cones that are bounded by connective tissue sheets or myocommata called myosepta. Typically, a transverse steak through the fillet will cut through several myotomes at different levels. Each myotome contains a lateral superficial strip of dark muscle primarily composed of slow contracting fibre types that are used for sustained swimming activity (Johnston et al., 1977). The muscle is dark due to high concentrations of myoglobin and the rich blood supply associated with an aerobic tissue type. The bulk of the myotome is composed of white muscle that is pigmented in salmonids due to carotenoid pigments absorbed from the diet. White muscle is composed of fast contracting fibres that primarily rely on anaerobic metabolic pathways and are recruited during burst swimming (Johnston et al., 1977). White or fast muscle fibres comprise the bulk of the edible portion of the fillet, 80±95% depending on species. The contractile proteins are packed into filaments that are organised into organelles called myofibrils with each muscle fibre containing several hundred or thousand myofibrils. The individual muscle fibres have a complex orientation and only the superficial fast and slow fibres run parallel to the longitudinal axis of the fish. The majority of fast fibres trace a helical pattern between adjacent myotomes. The orientation of the muscle fibre trajectories also changes along the trunk as does the average fibre diameter which is reduced in the more caudal myotomes. The muscle fibres insert into collagenous sockets in the myocommatal sheets (Fig. 22.2A) via short tendons. Longer more specialised tendons are found towards the tail, and these are particularly well developed in tuna fish where they direct muscle force directly to the tail fin rays. The extracellular matrix (ECM) in muscle has a complex organisation and is composed of collagen, noncollagenous glycoproteins and proteoglycans. Most of the collagen is located in the myocommata separating the individual myotomes. In addition, a complex network of collagen surrounds bundles of muscle fibres (perimysium) and individual muscle fibres (endomysium). The major collagen present in muscle is collagen type I, with collagen V a less abundant component (Sato et al., 1989a; Eckhoff et al., 1998). The ratio of type-V to type-I collagen is higher in the endomysium than in the myocommata fraction (Sato et al., 1989b). Collagen in the lateral tendons of tuna is largely type-I with every third amino acid residue being glycine with proline and hydroxyproline contributing a further 22% of residues (Gemballa and Vogel, 2002). The unusually high hydroxyproline content of collagen is often used as the basis for determining collagen concentration. In rainbow trout, rapid post-mortem softening was found to be associated with the solubilisation of collagen-V whereas there was no change in collagen-I concentration (Sato et al., 1991). Each collagen molecule comprises three polypeptide chains that contain at least one domain of repeating Gly-X-Y sequences per chain which are wound

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Fig. 22.2 Scanning electron micrographs (SEM) of myotomes in Atlantic salmon. (A) A post-rigor sample. The arrows show two examples of the connective tissue sockets into which the muscle fibres tendons insert at the myosepta. (B) Structure of the cold-smoked salmon product. The arrowheads show spaces between the muscle fibres due to shrinkage during the salting and smoking process. Note the large accumulation of adipocytes containing fat at the position of the myosepta (arrows). From Li et al. (2005).

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together in a tight triple helix. Many of the X and Y positions are occupied by the relatively uncommon amino acid residues: proline (X) and hydroxyproline (Y) which fit perfectly inside the helix. For collagen types I and V which form fibrils, the signal peptide is first removed, and then certain proline and lysine residues are hydroxylated, followed by the glycosylation of some hydroxylysine and asparagine residues (Myllyharju and Kivirikko, 2004). Three C-terminal propeptides associate to form a nucleus for assembly of the triple helix, which propagates from the C-terminus to the N-terminus. Procollagen molecules are then transported through Golgi stacks, aggregate laterally, then the N and C propeptides are cleaved to facilitate further aggregation, which leads to the selfassembly of fibrils. The mechanical rigidity of collagen is provided by post-translational modifications of the nascent polypeptides. The enzyme lysyl oxidase (LOX) is the only protein coding gene required to initiate collagen crosslink formation involving the formation of deaminlysyl residues There are two categories of collagen crosslink, immature or reducible and mature and irreducible. The crosslinking process starts with the oxidation of the e-NH2 group in certain lysine/hydroxylysine residues to produce reducible intermediates including dihydroxylsinonorleucine, hydroxylsinorleucine, lysinonorleucine, deoxypyridinoline, and pentosidine (Saito et al., 1997). Mature crosslinks, including hydroxylysylpridinoline (HP) and lysylpryridinoline are derived from the aldol condensation of two ketoamine crosslinks. Extracting salmon muscle with 0.1 M NaOH results in an alkaline-insoluble fraction which contains 100% of the mature collagen crosslinks (Li et al., 2005). The alkaline-soluble fraction, on the other hand, is thought to contain the nascent collagen polypeptides and collagen molecules containing reducible crosslinks (Li et al., 2005). In the flesh of Atlantic salmon less than 1% of the collagen molecules are linked by mature crosslinks (Li et al., 2005). This is in marked contrast to the situation in beef and pork skeletal muscle where the majority of collagen molecules contain mature crosslinks (McCormick, 1999). It is likely that there are many more immature reducible crosslinks in fish muscle which help to stabilise the collagen network and these may be gradually converted to mature crosslinks with increasing age. The lipid content of the muscle affects the colour as well as the flavour of fresh and smoked salmon (Robb et al., 2002). Lipid is stored in specialised cells called adipocytes which occur interspersed between the muscle fibres, but are most abundant at the myosepta (Fig. 22.2B). Excessive lipid storage at the myosepta produces a prominent white banding pattern giving the fillet an unsightly appearance whereas high fat deposition in the abdominal area of the belly wall increases dress-out losses during processing.

22.4

Cellular and molecular mechanisms of muscle growth

The muscle fibres themselves are a multi-nucleated syncitium. Since muscle is a differentiated tissue its growth is dependent on a population of myogenic

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The biological basis of variability in the texture of fish flesh 471 progenitor cells (MPCs) that retain the ability to divide. MPCs are thought to be derived from an embryonic structure equivalent to the amniote dermomyotome (Stellabotte et al., 2007; Hollway et al., 2007), which persists in larval, juvenile and adult stages providing a source of proliferative cells required for growth. These myogenic progenitor cells (MPCs) migrate from the external muscle layer to become resident in the myotomal muscle. Once activated and committed to differentiation they have one of two fates: either fusing together to form a multinucleated myotube or being absorbed into established muscle fibres as they expand in diameter. Paired box transcription factor 7 (Pax7) is thought to be important for the maintenance of MPCs and has been used as a marker for these cells in mouse (Seale and Rudnicki, 2000) and zebrafish (Hollway et al., 2007). In SEAFOODplus the complete coding sequence of the Pax-7 gene was determined in Atlantic salmon. RNA was isolated from alevin and adult stages and 10 splice variants (alternatively transcribed mRNAs involving insertions or deletions) of the gene were identified (Gotensparre et al., 2006). Evidence was obtained for two paralogues of the gene (see Section 22.8) based on the length and sequence of intron 3 (218 and 248 bp respectively) and associated insertions. In situ hybridisation with cRNA probes confirmed that Pax7 was expressed in the mononuclear myogenic progenitor cells (Gottenspare et al., 2006). The myogenic regulatory factors (MRFs) are a conserved family of four MyoD proteins (myf5, myoD, myogenin and MRF4) which are required for the specification of cells to a myogenic lineage and muscle differentiation. The MRFs are potent transcription factors that activate muscle-specific genes, due to two domains conserved in each family member: the basic region and helix-loophelix domain (Weintraub et al., 1991). Gene `knockout' studies in mice have shown that MRF genes show partial redundancy in vivo but have evolved a unique expression pattern and specialist function in initiating or maintaining myogenesis (Rudnicki et al., 1993; Hasty et al., 1993). The lineage leading to modern teleosts has undergone whole genome duplication relative to the common ancestor to the tetrapods (Jaillon et al., 2004). A salmonid-specific genome duplication is also thought to have occurred 10±25 million years ago (Allendorf and Thorgaard, 1984). Approximately 50% of the duplicated genes have subsequently been lost from the genome and are represented by a single paralogue (Bailey et al., 1978). In SEAFOODplus we have obtained the intron-exon structures of all known Atlantic salmon myoD family member genes (Fig. 22.3). Myogenin and MRF4 appeared to be present as a single copy in the genome whereas three paralogues of myoD were identified (Macqueen and Johnston, 2006; Maqueen et al., 2007). Atlantic salmon are usually harvested as young adults at 4 to 5 kg body weight sometime after the final fibre number has been reached. A cross-section through the trunk in fish of harvest size reveals a wide spectrum of diameters with only the smallest size classes (0±15 m) absent. The mosaic pattern of fibre diameters observed reflects the successive formation of myotubes on the surface of muscle fibres. The number of muscle fibres found per unit cross-sectional area is the fibre density, and this is a particularly important parameter in relation

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Fig. 22.3 The structure of all known myoD family member genes in Atlantic salmon: the master transcription factors controlling muscle development and growth. Each gene is represented by three exons (black boxes) and two introns (lines). The known sizes of exons and introns are shown. Introns with a double line are of unknown size (but in each case greater than 1 kb). All the intron-exon boundaries are experimentally supported. From Macqueen et al. (2007).

to flesh texture (Johnston et al., 2000a). Muscle fibre density in Atlantic salmon declines significantly once new muscle fibres are no longer being added (Johnston et al., 2000a). For adult Atlantic salmon sexual maturation occurs after the end of fibre recruitment in fast muscle and neither sex nor maturation affects fibre number. In contrast, for Atlantic halibut (Hippoglossus hippoglossus L.) sexual maturation occurs before muscle fibre production has stopped and is associated with a reduction of growth which reduces the value of male fish in farming. In view of the importance of the composition of muscle fibre diameters for texture in SEAFOODplus we investigated muscle growth in male and female halibut from a commercial site over a full twelve month period (Hagen et al., 2006). We found that the number of fibres per myotomal cross-

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The biological basis of variability in the texture of fish flesh 473 section (at 0.55 FL) was 24.5% higher in female than male fish prior to sexual maturation. Muscle fibre production in immature females slowed in the winter as day-length and water temperature decreased, but resumed in the spring/early summer. In contrast, maturation in male fish was associated with a complete cessation in growth and a temporary halt in the production and expansion of muscle fibres. Subsequent studies of wild and farmed fish up to 85 kg body mass have shown that fast muscle fibres continued to be produced until ~50 cm forklength (FL) in males and ~80 cm Lf in females, reflecting differences in their ultimate body size (female Atlantic halibut can reach 300 kg whereas males rarely exceed 50 kg). The final fibre number was also higher in female than in male fish, equivalent to 1 600 000 and 880 000 per trunk cross-section at 0.55 FL respectively (Hagen et al., 2008). Individual fish have a maximum fibre diameter that is determined by diffusional constraints and is strongly influenced by temperature and massspecific metabolic rate. As body mass increases the mass-specific metabolic rate declines with body mass±0.25, resulting in a relaxation of diffusional constraints and an increase in the maximum permissible diameter (Dmax) (Johnston et al., 2003a). In Atlantic salmon, Dmax reaches a limiting value of about 200 m at 2 kg body mass (Johnston et al., 2003a). In a range of teleost species, myotube production in fast muscle continues until around 40% of the ultimate body length (Weatherly et al., 1988). In Atlantic salmon, the number of fast muscle fibres in a cross-section of the trunk at the level of the first dorsal fin ray increases from around 7000 in yolk-sac alevins to 50±80 000 at the end of freshwater life, reaching a final fibre number of 500±900 000 around 6 to 12 months after transfer to seawater (Johnston et al., 2000a, 2003a). The duration of fast muscle fibre recruitment in Atlantic salmon varies between strains, but has usually stopped by 1.2 to 2.5 kg body weight (Johnston et al., 2000a). Once fibre recruitment has finished all subsequent growth involves the hypertrophy of the muscle fibres with diameters less than Dmax that were formed earlier during ontogeny.

22.5 Relationship between muscle structural traits and texture The genetic and phenotypic determinants of fibre number/density and their relation to flesh texture have been most studied in Atlantic salmon. A positive correlation was found between muscle fibre density and fillet firmness in 3±4 kg Atlantic salmon as assessed by trained taste panels, which could explain up to 34% of the total variation in texture (Johnston et al., 2000b). Similarly, significant negative correlations were found between the average muscle fibre diameter (which is inversely related to fibre density) and estimates of firmness between various fish species determined by trained taste panels (Hurling et al., 1996). In contrast, using instrumental texture analysers, correlations between muscle fibre density and flesh texture for fish of a similar body size are either

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absent or only apparent in certain seasons (Hagen et al., 2007; Atlantic halibut), or at the population (Periago et al., 2005; sea bass Dicentrarchus labrax) or family level (Johnston et al., 2004a; Atlantic salmon), probably reflecting differences in the nature of what is being measured by the two approaches. The extracellular connective tissue matrix and particularly the collagen proteins are another class of structural components that have a major influence on muscle texture. The total content of soluble collagen in myotomal muscle (moles gÿ1 dry mass) was found to range from 6±31 in different Atlantic salmon populations (Li et al., 2005; Johnston et al., 2006) and was around 12 in farmed Atlantic halibut during the summer months (Hagen et al., 2007). For both these species there was no significant relationship between the soluble collagen content in the muscle and texture as measured using an instrumental shear test. Recently a high performance liquid chromatography method has been developed for measuring the low concentration of mature pyridinoline (PYD) crosslinks in fish muscle (Li et al., 2005). Average values of PYD concentration (pmole g dry massÿ1) in fast muscle ranged from 400±500 in Atlantic salmon of 3±5 kg (Johnston et al., 2006) compared to around 1500 in Atlantic halibut of ~2 kg (Hagen et al., 2007). Thus assuming one collagen molecules contains ~200 HYP molecules and one PYD is capable of connecting three collagen polypeptides then ~3% of the total collagen has mature crosslinks in halibut muscle which is three times higher than in Atlantic salmon, contributing to the firmer texture of halibut flesh (Hagen et al., 2007). Fillets from a wild Atlantic salmon population with high firmness had a high concentration of alkaline insoluble collagen, but similar PYD concentrations indicating other crosslink species including immature crosslinks may contribute to the observed differences in texture (Johnston et al., 2006). In Atlantic salmon, positive correlations have been found between PYD concentration and fillet firmness measured with an instrumental shear test, explaining 25 and 16% of the variation in texture for the fresh and smoked product respectively (Li et al., 2005). PYD was relatively resistant to the smoking process only showing a 11.7% decrease relative to the fresh flesh measured 3d post-rigor (Li et al., 2005). Muscle fibre density, collagen crosslink and other structural variables that influence texture can be expected to be correlated with each other. To address this issue in SEAFOODplus we used multiple linear regression analysis with backward exclusion of independent variables to investigate the relative contribution of structural and biochemical parameters (pH, muscle fibre density, alkaline-insoluble collagen, alkaline-soluble collagen, PYD and water content) to flesh texture (Hagen et al., 2007). Texture was assessed as the work (mJ) required to shear standardised slabs of myotomal muscle. The most important contribution to fillet firmness in this species was PYD crosslinks, explaining 64% of the total variation (Fig. 22.4A). The influence of muscle fibre density on texture was significant for male but not female fish (combined samples over a 12-month period), explaining 19% of the variation (Fig. 22.4B), and correlations were higher still (r2 0:42) in the late spring indicating seasonal effects (Hagen

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Fig. 22.4 (A) The relationship between hydroxylysyl pyridinoline concentration and the texture of Atlantic halibut flesh measured with an instrumental shear method (n 98, r2 0:64, P < 0:001). Fish were sampled at different times of the year. A first order linear regressions was fitted to the data, shear work = 0.04 (PYD) + 5.361. (B) Regression analysis of fibre density (FD) and texture in males (pooled data, n 44, r2 0:19, P < 0:01). A first order linear regressions was fitted to the data, shear work = 0.014 (FD) + 6.356. Symbols indicate fish sampled as follows: 24.05.04 (l), 20.08.04 (n), 26.11.04 (), 18.02.05 (t), 05.05.05 (l).

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Fig. 22.5 The relationship between flesh lipid content (%) and fillet firmness (work done to shear a standardised slab of muscle (WD), mJ) for (A) a strain of Atlantic salmon farmed in Scotland and (B) a different strain of Atlantic salmon farmed in northern Norway. Open and closed indicate fish harvested at different times of the year. No significant correlation between the variables was found in (A) whilst for (B) there was a significant inverse relationship and a first order regression fitted to the data had the following equation: Fillet firmness ÿ16:3 507:0 WD; R2 0:25, F1,55=18.6; P < 0:001. From Johnston et al. (2007).

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The biological basis of variability in the texture of fish flesh 477 et al., 2007). In contrast, post-rigor pH was not found to be a good predictor of texture. It was concluded that mature collagen crosslinks were a major, and muscle fibre density a minor factor for explaining intrinsic variation in flesh texture in this species. From a practical perspective these results suggest that the optimal season for harvesting Atlantic halibut is in the autumn or early winter when both nutritional state and texture are good. Thus both muscle fibre density and collagen crosslinks affect muscle texture in the raw and smoked product, although their relative contributions vary between species and with growing conditions, e.g. muscle fibre density makes a greater, and PYD crosslinks a lesser contribution, to muscle texture in salmon than halibut. Another structural component which may affect flesh texture is the number of adipocytes containing stored fat. Both genetic and environmental factors are likely to influence the relationship between muscle lipid content and texture because correlations between these variables are not always found. For example in Atlantic salmon, muscle lipid values were found to be correlated with texture assessed using a shear method in some populations but not others (Fig. 22.5). A significant inverse relationship between fat content and fillet firmness was also found in farmed rainbow trout, measured as resistance to compression (Mùrkùre et al., 2002).

22.6 Proteolytic enzymes and post-mortem softening of the flesh Net protein accretion during growth is a function of the balance between protein synthesis and protein degradation. Protein degradation in muscle is thought to involve three main proteolytic systems: (1) the ATP-dependent ubiquitinproteosome complex, (2) a complex of calcium activated cysteine proteinases (calpains) and (3) lysosomal enzymes including cathepsins (Mommsen, 2004). It is thought that the highly complex proteasome pathway is much less important for protein degradation in teleosts than in mammals (Mommsen, 2004). Indeed, the ubiquitin-proteosome pathway was not up-regulated in spawning-induced muscle proteolysis in the rainbow trout which is associated with deterioration in flesh quality (Salem et al., 2006). There is an extensive literature in mammals concerning the involvement of the calpain/calpastatin system in meat tenderisation during post-mortem storage (Ilian et al., 2004a; Sentandreu et al., 2002). Calpains are calcium-dependent cysteine proteinases. The calpain/calpastatin ratio is a good predictor of the ultimate tenderness of beef (Ouali and Talmant, 1990). In mammals, the calpain/calpastatin system is thought to be more important than cathepsins for the meat tenderisation process, although numerous other, often poorly characterised peptidases, undoubtedly play a role (Sentandreu et al., 2002). The ubiquitous - and m-calpains catalyse the limited proteolysis of cytoskeletal and membrane proteins and are regulated by Ca2+ concentration and the specific protein inhibitor calpastatin (Goll et al., 2003). -calpain (calpain 1) is

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active at M calcium concentration and m-calpain (calpain 2) is active at mM calcium concentrations. In the well characterised proteins from mammals each calpain has a common 30 kDa regulatory subunit and a unique 80 kDa catalytic subunit. The catalytic subunit has four domains: Domain I (autolytic activation), Domain II (cysteine catalytic site), Domain III (switch domain) and Domain IV (calmodulin-like calcium binding domain). In skeletal muscle, the calpain/ calpastatin system has numerous physiological roles including protein turnover and growth, cell cycle progression and myoblast fusion (Goll et al., 2003). A large number of tissue specific calpains including Calpain 3 (p94) (Jones et al., 1999) and Calpain 10 (Ma et al., 2001) have been identified that are expressed in a fibre type-specific manner. Calpain 3 mRNA transcripts are more abundant in muscle than those of the Calpains 1 and 2. The purified Calpain 3 protein is unstable on isolation and in vivo it is thought to be stabilised by interaction with titin (Sorimachi et al., 2000), a giant 3.7 Mda cytoskeletal protein that spans the muscle half sarcomere from M to Z line. Calpain 3 (Ilian et al., 2004a) and Calpain 10 (Ilian et al., 2004b) are also involved in the tenderisation of sheep meat through limited proteolysis of specific muscle structural proteins such as titin and nebulin. In mammals, the calpain inhibitor calpastatin (CAST) has four homologous C-terminal inhibitory domains (I-IV) downstream of a noninhibitory leader domain (L) and an N-terminal XL sequence and it also has several isoforms that are also expressed in a fibre type-specific fashion. The calpain/calpastatin system in fish has been much less studied. Calpain 1 and 2 were partially purified from the Chinook salmon (Oncorhynchus tshawytscha) (Geesink et al., 2000) and rainbow trout (Saito et al, 2007) and calpain 2 has been isolated from carp (Sakamoto et al., 1985), tilapia (Wang et al., 1993) and sea bass (Ladrat et al., 2002). Recently, full-length cDNAs have been obtained for Calpain 1 and 2 from the rainbow trout and these sequences show around 65% identity with the mouse orthologues (Salem et al., 2005). Starvation for 35 d in the rainbow trout resulted in the up-regulation of mRNA transcripts for Calpain 1 (2.2-fold), calpain 2 (6.0-fold) and calpastatin (1.6-fold) (Salem et al., 2005). These results indicate that season of harvest and preslaughter starvation period are likely to affect the calpain/calpastatin system and hence flesh texture and storage characteristics. Whereas tenderisation is a positive attribute in red meat, in fish, softness represents a loss of quality and hence economic value. Verrex-Bagnis et al., (2002) used Western Blotting to show that Calpain 2 released -actinin and desmin following in vitro degradation of myofibrils. Calpains were shown to degrade troponin T and -actinin in sea bass (Delbarre-Ladrat et al., 2004). The carboxyterminal region of dystrophin, a cytoskeletal actin binding protein, was highly sensitive to degradation by Calpain 2 (Bonnal et al., 2001). Several studies have shown that during the pre-rigor period, cytoskeletal proteins are affected by the first proteolytic events. These cleavages disrupt connections between myofibrils and the extracellular matrix, induce segmentation of myofibrillar cores, and modify the rheological properties of the tissue (Bonnal et al., 2001), presumably decreasing the firmness of the flesh. Dystrophin release

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22.7

Environmental influences on muscle structural traits

Salmon of the same genetic stock reared with identical husbandry practices on the same farm site in successive years can show major differences in muscle fibre density due to uncontrolled environmental influences. In the data presented in Fig. 22.6 the final fibre number was the same between year classes but muscle fibre density differed due to a lower average fibre diameter in fish harvested in 2004 than 2005, presumably reflecting different patterns of hypertrophic growth (Vieira et al., 2007).

Fig. 22.6 Environmental influences on muscle growth in farmed Atlantic salmon (Salmo salar L.). The figure shows the relationship between fork length (FL) (cm) and muscle fibre density (FD) (number fibres per mm2 muscle cross-sectional area) for female (circles) and male (triangles) fish sampled in 2004 (open symbols) and 2005 (closed symbols). All fish were from a population of the Fanad-Mowi strain and had been farmed on the same site using the same husbandry routine and diet. First order linear regressions were fitted to the combined male and female data in 2004 (solid line) and 2005 (dashed line) using a least squares method and the following regression equations obtained: For 2004; FD = 427.8 ÿ 3.55 (FL); R2 0:46; F1,140 = 119.9; P < 0:0001. For 2005; FD = 243.9 ÿ 1.78 (FL); R2 0:24; F1,68 = 21.1; P < 0:0001. From Vieira et al. (2007).

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Other experiments have shown that the final fibre number can be modified by about 20% by varying egg incubation temperature by just a few degrees centigrade in the hatchery (Johnston et al., 2003a). The resulting effects on fibre density were sufficient to influence muscle texture, with salmon reared at cooler ambient temperatures at the early stages having a firmer flesh than salmon reared in heated water at 8 ëC (I. A. Johnston, unpublished results). In a recent study, partially supported by SEAFOODplus, salmon eggs were incubated at a range of temperatures and it was established that in order to obtain the highest final fibre number, and density, in adult salmon the embryos should be incubated at 5 ëC (D. Macqueen, D. Robb and I.A. Johnston, unpublished results). Furthermore these effects of egg incubation temperature on adult fibre number were shown to occur prior to the `eyed stage' around half way through embryonic development. Fish reared at 5 ëC to the `eyed stage' were smaller as smolts, but grew faster in seawater eventually catching-up with the 8 and 10 ëC groups. The reason hatchery temperature can have persistent effects on adult fibre number is probably related to the formation of the embryonic external cell layer which is thought to be the source of myogenic precursor cells for postembryonic muscle growth (Hollway et al., 2007). In the future it may be possible to optimise the final fibre number in adult salmon by cooling the eggs for a much more limited period in the hatchery (possibly around the time the embryonic external cell layer is formed). In Atlantic salmon, we have shown that the expression of some (myf5 and MRF4), but not all (myoD, myogenin), of the myogenic regulatory factors regulating muscle development varied with respect to developmental stage (Macqueen et al., 2007). Morpholino knock-down experiments of myoD and myf5 in the zebrafish resulted in an increase in the number of Pax3/7 expressing external cells on the lateral surface of the somite (Hammond et al., 2007). Thus the heterochronies in MRF expression in Atlantic salmon observed in the SEAFOODplus experiments provide a potential mechanisms that could explain some of the changes in muscle phenotype that occur with development temperature (Johnston, 2006), including changes in final fibre number. Photoperiod is another powerful factor that can influence the final fibre number and hence muscle fibre density at certain stages of the life-cycle in Atlantic salmon. Previously, we found that using artificial lights in 1-sea winter salmon at the time the natural photoperiod was decreasing rapidly resulted in much higher levels of fibre recruitment than under ambient conditions (Johnston et al., 2003b). Light treatment at this stage in the life-history resulted in a final fibre number that was 20% greater than in fish reared under ambient photoperiod and a correspondingly higher muscle fibre density at slaughter (Johnston et al., 2003b). In contrast, the photoperiod manipulation techniques used to produce accelerated smolts had no lasting impact on muscle fibre number or density in adult salmon (Vieira et al., 2005). The short-days and low temperatures (<6 ëC) associated with winter result in a cessation of feeding activity in farmed halibut, resulting in a breakdown of fast muscle protein and an associated increase in water content (Hagen et al., 2006,

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The biological basis of variability in the texture of fish flesh 481 2007). On a dry tissue mass basis this was found to be accompanied by a significant decrease in alkaline-soluble and to a lesser extent alkaline-insoluble collagen, but no significant change in PYD concentration (see Table 1 in Hagen et al., 2007). During the winter the alkaline-insoluble collagen content fraction is enriched in PYD as water content increased leading to a firmer texture of the flesh (Hagen et al., 2007).

22.8

Heritability of muscle structural traits

In Atlantic salmon, heritabilities have been estimated for carcass traits (Gjerde and Gjerdrem, 1984) and traits related to body composition including abdominal fat deposition and muscle fat content (Rye and Gjerde, 1996; Quillet et al., 2007). Muscle fibre number and density (FD) are known to vary within and between families, and between different populations or strains (Johnston et al., 2000b). Evidence for very rapid genetic selection of muscle fibre number has been found for dwarf populations of Arctic charr (Johnston et al., 2004b). In a study of 28 families of Atlantic salmon (Fanad-Mowi strain), average final fibre number per family at the level of the adipose fin increased by 34% from the highest to lowest ranked family, from 3.48 105 to 4.65 105 (Vieira et al., 2007). Vieira et al. (2007) estimated the heritability of the final muscle fibre number and FD in 215 Atlantic salmon from 28 full-sib families based on a multi-trait genetic model with sex nested within year. Muscle fibre number and densities showed medium heritabilities of 0.33. Values for the heritability were within the range reported for mammals, where the majority of estimates are from 0.2 to 0.5 (Rehfeldt et al., 1999). There was a strong and significant negative genetic correlation between fat content and both fibre number (ÿ0.85) and fibre density (ÿ0.76) (Vieira et al., 2007) which suggests that selection against fish with high breeding values for flesh lipid would also result in an increase in muscle fibre density. This strong genetic correlation suggests that the two traits are being influenced by some of the same genes. Indeed, myogenic cells and adipocytes are thought to be derived from a common pleuripotent stem cell population (Wada et al., 2002). The much weaker phenotypic correlation found between muscle fibre density and flesh lipid indicates that environment is not influencing these traits in the same manner. The estimation of fibre number by morphometric techniques is too laborious to be practical for use in selective breeding programs. With partial support from SEAFOODplus we explored the relationship between the density of myogenic precursor cells, identified with a specific antibody to Pax7, and muscle fibre density. Although a significant correlation was obtained between these variables it was not sufficiently strong to form the basis of a quick and indirect method of estimating muscle fibre density (Vieira et al., 2007). In contrast, fat content can be measured non-invasively and with high throughput by NIR techniques (Solberg et al., 2003), and selecting for low fat should also result in a higher muscle fibre density (Vieira et al., 2007).

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Future trends

A number of single genes that have been identified with potentially large phenotypic effects on muscle texture including lysyl oxidase (LOX) (Consuegra and Johnston, 2006), calpastatin (CAST) (I. A. Johnston, unpublished results) and myostatin, a negative regulator of muscle fibre hyperplasia (McPherron and

Fig. 22.7 Polymorphism of the lysyl oxidase gene in salmonids. (A). Alignment of LOX Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) amino acid sequences. Dots indicate identity and gaps introduced to maximise alignment are indicated by dashes. Underlined codons indicate the LOX domain. Bases in bold correspond to the functional domains and the `copper-talon' is shown in italics. Positive selected sites identified by at least two methods are indicated by asterisks. Nucleotide sequences were deposited in the GenBank under accession numbers DQ167812DQ167851 and EF514520-EF514532. (B) Frequency of alleles of the lysyl oxidase gene identified in three Atlantic salmon populations with different life histories and growth rates: the landlocked Bleke salmon, a wild anadromous population from the River North Esk, Scotland and a farmed Mowi derived strain. From Consuegra S and Johnston I A (2008).

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The biological basis of variability in the texture of fish flesh 483 Lee, 1997). For example, in pigs, allelic variation in the CAST gene was shown to be correlated with variation in meat quality parameters including raw firmness score, and the tenderness, juiciness and chewiness of the cooked meat (Ciobanu et al., 2004). In SEAFOODplus we have continued our work to identify genes that inhibit myotube formation and potentially control the final fibre number in fish. Previously four candidate genes were identified that were strongly and specifically up-regulated in the fast muscle of the model teleost Takifugu rubripes concomitant with the cessation of muscle fibre recruitment (Fernandes et al., 2005). In SEAFOODplus we cloned the complete coding sequence and a partial 50 untranslated region of the Atlantic salmon orthologue of one of these candidates called cee, a hitherto uncharacterised gene that is apparently required for normal growth and development (Fernandes et al., 2008). Comparative genomic analyses indicate that cee arose some 1.6±1.8 billion years ago and is found as a single copy in the genomes of most species. The Cee protein has no functional motifs or known domains, yet is highly conserved, particularly among vertebrates. In developing salmon embryos Cee was expressed concomitantly with pax7 and myoD family members in the external cell layer and myotomal compartment respectively (Fernandes et al., 2008). Research to elucidate the function of Cee and to identified other genes with putative roles in controlling muscle fibre number and hence muscle fibre density in Atlantic salmon are continuing. The existence of multiple paralogues and the presence of a large number of splice variants complicate the task of identifying genetic polymorphisms that influence muscle texture in teleosts. The genetics of LOX, the gene involved in collagen crosslink formation, has been studied in Atlantic salmon within SEAFOODplus. In Atlantic salmon, up to four alleles are expressed per individual (Consuegra and Johnston, 2006, 2008) indicating all duplicated paralogues have been retained. The LOX gene is highly polymorphic and a farmed population was found to have fewer LOX alleles than a wild anadromous and a land-locked population (Fig. 22.7A, B). For the future, studies on the effect of particular alleles of LOX and CAST on muscle structural components and texture, such as those being conducted within SEAFOODplus, are important for identifying candidate genes that might one day be used in marker assisted selection to produce farmed fish with superior eating and processing qualities.

22.10

Acknowledgements

The author is grateful to support from the Integrated Project SEAFOODplus granted by the European Union under contract No. 506359. I am also grateful to Dr Vera Vieira of the Fish Muscle Research Group, St Andrews for her patience in proof reading the draft manuscript.

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22.11

References

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The biological basis of variability in the texture of fish flesh 489 Atlantic salmon (Salmo salar)', Aquaculture, 246, 197±208. and JOHNSTON I A (2007), `Heritability of fibre number and size parameters and their genetic relationship to flesh quality traits in Atlantic salmon (Salmo salar L.)', Aquaculture, doi: 10.1016/j.aquaculture.2007.08.028. WADA M R, INAGAWA-OGASHUIWA M, SHIMIZU S, YASUMOTO S and HASHIMOTO N (2002), `Generation of different fates for multipotent stem cells', Development, 129, 2987± 2995. WANG J H, MA W C, SU J C and JIANG S T (1993), `Comparison of the properties of m-calpain from tilapia and grass shrimp muscles', J Agricult Food Chem 41 (9) 1379±1384. WEATHERLEY A H, GILL H S and LOBO A A (1988), `Recruitment and maximum diameter of axial muscle fibres in teleost and their relation to somatic growth and ultimate size', J Fish Biol, 33, 851±859. VIEIRA V L A, NORRIS A

WEINTRAUB H, DAVIS R, TAPSCOTT S, THAYER M, KRAUSE M, BENEZRA R, BLACKWELL T K,

and HOLLENBERG S (1991), `The myoD gene family: nodal point during specification of the muscle cell lineage', Science, 251, 761±766.

TURNER D, RUPP R

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23 Fish welfare and ethical qualities in aquaculture B. DamsgaÊrd, Nofima, Norway

23.1

Introduction

Human food consumption in Europe has changed drastically in the last century. Our grandparents' generation was mostly concerned about the amount of food, our parents' generation to a larger extent about food quality, while our generation has a complex relationship with food. During the last decades, an increasing focus on human health and lifestyle has changed our expectation of food products. These changes might be looked upon as a development from a nutrient regime to a sound food regime and to food choices based more on feelings. In a society with enough food resources, people are generally more concerned about potential problems and benefits of the different food components, including how our food species have been treated during production. These ethical and animal welfare issues include both how the animals are treated during their lifetime, including the fulfilment of basic needs, and questions about how the animals are treated before and during slaughter. 23.1.1 The relationship between humans and other animals The moral status of non-human species is not a new discussion, but a very old philosophical question that suddenly took on a new charm of novelty. The way we understand animals is clearly related to how we understand ourselves as humans. According to the Greek philosopher Aristotle in the 4th century BC, non-human species were without reason (`logos') and belief (`doxa'), and they were thus far below humans because of their alleged irrationality. Despite extensive behavioural descriptions of several animal species, Aristotle stated

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that animals were created and unchanged. This interpretation was the guiding principle not only for how humans understood animals, but also for how they treated them. The view had a strong influence on Western Christianity, represented, for example, by the view of Augustine in the early 5th century AD, and the view of the French philosopher ReneÂ Descartes in the 17th century who argued that animals had no soul or conscious mind, and therefore could not think or feel pain. This view of Aristotle existed for two thousand years, until JeanBatiste Lamarck, John Stuart Mill and others developed ethology as a scientific field during the 18th and early 19th centuries. This issue was based upon the need to `understand' animals, and especially to describe individual differences in behavioural traits. The term `animal welfare' was used by the British philosopher Jeremy Bentham in the early 19th century, who argued that animals should be treated well because they could suffer. After the scientific paradigm shift caused by Charles Darwin's evolutionary theories in the late 19th century, many biologists started to show an increasing interest in animal behaviour. It soon became clear that animals indeed had a complex range of reasonable behaviours. During the early 20th century, Ivan Pavlov focused principally on individual instincts as the driving forces for behaviour, while Konrad Lorenz and Niko Tinbergen developed animal behaviour in the direction of understanding behaviour in an integrative way, studying animal behaviour in their natural environments. The debate has now taken several directions, and one of them is the development of the use of evolutionary methods in behavioural ecology in order to describe how the behaviour of an individual animal can be understood with both proximate models of regulatory mechanisms and ultimate fitness models in an evolutionary time scale. This historical development is based on the question of what are the differences between humans and other animals. The most distinct difference is perhaps human consciousness or self-awareness, which some authors have termed a human `I-am-ness'. Such consciousness may be defined as a sense of the `I' and how this `I' relates to the environment. The term `sentience' usually refers to the ability to respond to and perceive external stimuli, but the relationship between sentience and consciousness is still poorly understood. Non-human species may have a reasonable behaviour and they may communicate, but many people will still say they lack the linguistic syntax to discuss who they are. With such moral background, most humans feel we have the moral right to culture, kill and eat other animals. The discussion of sentience and consciousness in animals, including fish, may change the future moral status of some animal species. The question of moral status is general, but the discussion of animal welfare is mainly limited to farming conditions, where animals are held under our control. Humans have reared animals from prehistoric times, and the possibility that we have to show compassion and care for non-human species may be an important part of our social instinct. One might say that humans become hosts for other species in non-symmetric relationships. At the same time, the cruelty to farmed animals was one of the main starting points for the increasing debate

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over animal welfare in modern times. Ruth Harrison's book, `Animal Machines' (1964), resulted in a report to the British Parliament on animal welfare in agriculture, known as the Brambell Committee, in 1965. The report stated that welfare should be fulfilled with the so-called `five freedoms', including freedom from hunger and thirst, freedom from discomfort, freedom from pain, injury or disease, freedom to express normal behaviour, and freedom from fear and distress. 23.1.2 Why such concern about fish? Fish have been reared for a long time in Europe, but only during the last decade has the focus on fish welfare emerged and it may significantly change the way we treat and produce fish for food. There are several reasons for this increasing interest. The moral status of fish seems to be increasing. Apparently, such status cannot be explained merely as an animal's evolutionary position, what is often referred to as `lower' and `higher' species. Teleost fish have an unclear position in such classification and might be considered the boundary between higher and lower animals. In biological classification, however, such separation is misleading as the evolution is developing into several lines, and fish cannot be regarded as a simpler form of higher animals, but as a highly successful and diverse group of animals. New knowledge about fish biology has demonstrated that functions in fish are not so different from other animal groups. Many tend to believe that the negative focus on farmed fish must be a consequence of an increasing urban lifestyle far removed from food production. This view may be understood as that fish farmers always know what is best for the animals, while consumers have a romantic and unrealistic view about the food they eat. The general picture, however, is much more complex, and includes elements of changes towards more intensive fish farming, combined with megatrends in lifestyles and political changes in Europe. Many people, urban or not, are generally more concerned about the fish they eat. The quality aspects of seafood now extend from taste, risk factors and health effects to aspects of sustainability, husbandry methods and slaughtering, the so-called ethical qualities. There are not necessarily links between the muscle quality and the ethical quality of a food product, since our food choices are based largely on feelings. An egg from a free-range hen does not necessarily have to taste better or be healthier than an industrially produced egg, but the consumer feels better because our biological needs for food have less negative effects on other animals or the environment. There are several reasons why this trend developed later for fish than other species. Compared with terrestrial farm animals, many farmed fish have a relatively short evolutionary history of farming. Most of the other terrestrial farmed species developed thousands of years ago, while large-scale salmon farming in Europe has a history of far less than a hundred years. Biologically, the number of terrestrial farm animals is quite low, and very few new candidates

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have emerged since the start of agriculture. Fish farming in Europe is currently based largely on fish species such as Atlantic salmon, rainbow trout, sea bass, sea bream, carp species, eel and turbot, and we may expect emerging species such as Atlantic cod, halibut and Arctic charr. The present farmed fish species have mostly been evaluated for high growth rate and market price and not to a large extent whether a fish species is acceptable for welfare reasons. The scientific background for the knowledge of fish is generally based on wildlife biology, developing into experimental studies of fish biology. Such studies have focused on optimisation of growth and solving of practical problems for the industry, and despite the large number of salmon studies, for example, there has been little focus on behavioural needs and fish welfare until the last decade. Thus, the knowledge of fish as a farm species is far less than for other animal production, especially concerning how we understand the behaviour of farmed fish. The method used is also challenging. Behavioural studies are based on how we as humans interpret what we observe. One of the methodological problems with the development of fish welfare is that fish expressions are generally very different from human expressions, and behavioural differences may be overseen or wrongly interpreted.

23.2

Terms and definitions

Only a decade ago, farmers and scientists seldom used the terms fish welfare and ethical aquaculture, while these questions are now basic issues in the development of European fish farming. The terms, however, are poorly understood and defined, and aquaculture stakeholders use them differently. 23.2.1 How do we understand and assess fish welfare? Animal welfare as a scientific term is not regarded as a research field on its own, but rather as a cluster of scientific areas based in several research fields. There are definitions of animal welfare based on biological functions, subjective perception or species-dependent behavioural needs (Duncan and Fraser, 1997). In biological terms, animal welfare is regarded as a coping mechanism, defined, for example, as the `individual's subjective experience of its mental and physical state as regards its attempt to cope with its environment' (Anon., 2005), which is developed from a definition by Broom (1986). The definition indicates that welfare is a property of the individual animal, and that it deals with this individual's subjective experience of its state as the balance between positive and negative perceptions (Braastad et al., 2006). The term `mental state' thus includes emotional and cognitive elements that are affected by stimuli and memories of previous experiences with similar stimuli. The term `physical state' includes physiological states that potentially influence mental states, and `environment' includes the physical environment (rearing conditions, water quality), social interactions and all other biotic factors such as pathogens, parasites and predators, in addition to interactions with humans.

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Coping with physiological, immunological and behavioural functions means that an animal is functioning well biologically with a low stress level, adequate growth and reproduction and good health, which in sum might be termed `a good quality of life' (Broom, 1986, 1991). According to Broom's definition, homeostasis is the ultimate goal, and coping comprises the biological mechanisms an animal utilises to handle a significant threat to its stability or homeostasis and to regain control. One of the five freedoms in the Brambell Committee document was the right to express normal behaviour. It is easy to think that what is `normal' for an animal must also be the best for its welfare. This is not always true for farmed animals. For example, aggression and fighting are normal behaviours in nature, but are probably not behavioural needs. They are also unwanted behaviours in culture. Welfare is not a question of imitation natural habitats, and most fish welfare scientists address welfare issues as a coping strategy. Being in a tank or sea cages is clearly not natural for a fish, but welfare issues are not about making the system as natural as possible, but rather to understand how the fish is coping with this unnatural situation. A farmed fish has good welfare if it can cope with its farming conditions, including factors such as the water environment (e.g., temperature), physiological needs (e.g., food), social intraspecific interactions (e.g., aggression), and interactions with humans (e.g., handling). Behavioural models in fish welfare are often based on a cost-benefit analysis. For example, a fish will be aggressive when the benefit of performing the behaviour (e.g., more food) outweighs than the cost of the behaviour (e.g., more energy utilisation and risk of wounds). Such models are based on the assumption that a fish may make a `decision'. A behavioural decision does not have to be a cognitive choice, but is simply a way to say that an individual fish has a number of alternatives, and these in some way have a consequence for survival and fitness. Some of these decisions are frequent, such as should the fish feed or not or should it fight or not, while others, often called life-history strategies, happen only once or very few times. Habitat shifts such as seaward migration and mating are examples of evolutionarily important decisions, including a long preadaptive period before and a short vulnerable period during smoltification and reproduction, respectively. The other definitions of welfare go beyond the question of coping, and address issues such as the animal's subjective experience or the possibility for an animal to utilise its full behaviour repertoire for which it is genetically preadapted. According to the definition of an individual's subjective experience, a good quality of life requires that there be no mental suffering (Duncan, 1993), such as negative emotions related to stress, fear and pain. According to the definition by Spruijt et al. (2001), animal welfare is `the balance between positive (reward, satisfaction) and negative (stress) experiences or states'. Dissatisfaction, or even frustration, is the result of an inability to achieve this homeostasis. Another alternative definition of animal welfare is based more on the natural life and suggests that farmed animals should live in an environment as close to their natural habitat as possible (Kiley-Worthington, 1989). This

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principle is often used as a platform for organic farming, including organic aquaculture. The use of the term `ethical' provides a wider definition than animal welfare, and goes beyond biological regulation and coping. Handling of fish before slaughter may thus be regarded as a welfare issue, while the killing itself is not necessarily a regulatory welfare question, but might still be an important ethical issue. On a higher level, animal welfare and ethical questions are parts of what is termed `sustainable aquaculture'. Together with environmental sustainability, welfare forms the biological platform of fish sustainability, being parallel to economic and social sustainability of aquaculture. The term `Welfare Indicator' usually refers to a parameter that may be used to measure welfare. An assessment may be based upon a list of needs of a farmed species, for example measuring the non-fulfilment of these needs, or be based upon the deviation from normality. The assessment can be deduced by how far an individual animal has deviated from what is normal for animals in that environment. Normality is not necessarily that is natural for the fish, and an assessment of such deviation must be based upon base line studies covering the complexity between the individuals and their rearing environment. Any welfare indicator can be directly linked to the biology of single individuals, to a population of fish (e.g., all fish in a tank or a seacage) or indirectly to the whole fish farm. The term `Operational Welfare Indicator' (OWI) usually refers to a parameter that may be used in practical farming. Welfare indicators may be measured by the farmers or by the authorities, for example as a part of a fish health assessment. If each indicator is estimated within a defined range, e.g. from 0 to 10, a `Welfare Index' may sum up the result from several indicators, weighting the indicators in comparison to each other. A `Welfare Assessment System' (WAS) is an aggregation of welfare indicators, including a total evaluation of the farm. The term welfare indicators is, however, used in multiple ways by various aquaculture stakeholders. 23.2.2 Wild versus farmed fish Farming of fish is based on biological materials either caught in natural waters or bred in culture. Most farmed species are domesticated throughout a number of generations, often in breeding programmes aiming to increase growth or minimise production problems. Behavioural traits are both genetically and environmentally determined. Farmed fish often have similar behaviour as in nature, but domestication may increase or decrease the expression of the behaviour. Some species have a higher behavioural plasticity and are thus better able to cope with a changing environment during domestication. There are several important differences between nature and a fish farm. In the sea, space is not limited, but the habitats may still be small and fish density variable. In culture, space is limited and the density is more constant and higher. In nature, the environment and food qualities and quantities are highly spatial and temporally variable compared with less variation in a farm. During short

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periods in nature, food might be in excess, but limited food resources and competition for food is much more pronounced in nature. Nature is `cruel', and most fish in natural habitats have to fight for food in situations that might look like lifelong suffering with hunger and pain. Fish in nature also have more predators and parasites compared with cultured fish, and the natural survival rate is much lower than in fish farms. On the other hand, farmed fish have no possibilities to escape from adverse situations and thus may be less able to cope with stressors. The consequences of chronic stressors may be physiological stress effects and reduced immune function, increasing disease susceptibility and heath-related problems. However, it is irrelevant to ask what is best or worst of nature and culture because according to legislation, we are responsible for the welfare of the fish as soon as we bring them into culture, and also because the definition we use for welfare is based on how the fish cope with culture, not on how they would have lived in a natural environment.

23.3

Fish farmers and consumers seeking a common destiny

Several large national and pan-European NGOs work actively with welfare questions related to fish, and they play important roles in the development of legislation and regulation of fish farming. The interest in fish welfare and ethical considerations of aquaculture varies amongst European countries, and these differences apparently do not depend on the volume of fish production or fish consumption, but rather a combined effect of several driving forces, including cultural views in different countries. The aquaculture industry and farming organisations have generally shown divergent views on welfare issues. Many fish farmers expect national and international welfare regulations to increase production costs, for example with a reduction in maximum fish density. Producers also fear that regulations in their own country may reduce European competitiveness compared with countries without such regulations, selling cheap and less ethically produced fish into the European markets. Some farmers also think that fish farming is already ethically sound and does not really need national and international regulations, simply based on the fact that farmers would not do anything that would harm their own animals. At the same time, production intensity has increased in the farming industry. In Norway today, compared with 20 years ago, a smolt farmer often produces three to four times more fish per year, and despite the fact that the average fish today is much bigger than it was 20 years ago, the farmer uses only approximately one-tenth of the running freshwater. The main reason for this change is simply better technology for oxygen regulation in the water, and a more effective industrial intensive production. On the other hand, many producers also believe that welfare improvements and expenses may be repaid in terms of higher production rate, less variation in size, fewer health problems and better fish quality. The ethical issues of fish farming are now high on the agenda in most fish farming organisations.

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European consumers, retailers and consumer organisations are increasingly aware of ethical issues of fish welfare in general. In some countries, farmed fish is an important protein source, while in Europe, the demand for a particular fish product is regarded more as a luxury product with an elastic demand. The more a product costs and the richer the consumers are, the more likely it is that they will buy something else if a product is regarded as unhealthy or unsound. The importance of ethical questions in consumer behaviour studies has been addressed during recent years, probably as a part of a general megatrend in the way consumers perceive food (Fig. 23.1). Generally, the interest in how and where our food is produced seems to be increasing, but there is great variation amongst countries and amongst segments of the market within a country. Consumers' concerns about aquaculture included a range of factors, including a divergent taste and texture of farmed fish compared with wild fish, fear of contamination from feed, depletion of natural marine resources used in fish food, adverse environmental impact associated with pollution and interaction with wild stocks, and ethical concerns over intensive production and slaughter. Most consumers do not distinguish amongst these issues, but probably respond negatively to farmed fish for a variety of reasons. For example, the relationship between the sustainability of wild-caught and farmed fish is complex. Some consumer groups presumably do not distinguish between wild and farmed fish. Environmentally oriented consumers are often negative towards wild-caught fish because of depletion of the natural fish population, and even see the possibilities that farming of Atlantic cod, for example, will decrease the fishing pressure on wild cod stocks. In all European countries, there are consumer groups who prefer certified organic products, such as `ecological salmon'. The factors determining such ecological products are mainly based on sustainability and environmental

Fig. 23.1 Food megatrends during the last 50 years. Each line represents the relative importance of fish food quality traits, moving towards healthy and safe seafood, produced in an ethically responsible way.

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effects, and only to a minor extent on direct welfare issues. Organic fish produced without the use of health treatment on outbreaks of diseases may even have poorer welfare than non-organic fish. The eco-labelling is still an important driving force for the development of alternative production regimes for farmed fish, and may in the future include fish welfare to a greater extent. Most European consumers have relatively good knowledge about the positive effects of eating fish and other seafood products. Too much information about welfare aspects may have negative consequences for the consumption of fish. Consumer behaviour may be both irrational and inconsistent, and low price is probably more important than any other factor in determining fish preference. However, consumers do not necessarily directly determine the minimum ethical level of fish production. Large retail organisations probably have the knowledge necessary to set production standards, and may thus strongly affect consumer behaviour. Legislation and policy-making around fish farming in general consist of a large variety of international regulations and recommendations, in addition to national laws and farming regulations. Over the last decade, an increasing amount of this legislation includes welfare issues, and the number of such issues is expected to rise in the future. England was the first country with welfare legislation in 1822, followed by Norway in 1935, while China has the most recent law enacted in 2005. Norway aims to be a leading country in the development of animal welfare, especially regarding fish production. A White Paper on animal husbandry and welfare (Anon., 2003) states that animals, including fish, have an `intrinsic value' and should be treated with respect to the species' natural needs. Few other European countries have welfare acts that clearly include fish farming, but welfare-related aspects may be nationally regulated by other legislation. The EU strategy for sustainable aquaculture development focuses on the availability to consumers of products that are healthy, safe and of good quality, as well as promoting high animal health and pan-European welfare standards. Several large animal protection groups have included fish, and especially farmed fish, in their areas of interests. These groups focus mainly on a small number of welfare-related problems, such as pain and fish density. The questions are not necessarily the most important welfare issues, but they are easy to understand and may thus attract public attention.

23.4

Welfare during the production cycle

Welfare issues cover the entire lifespan from egg to brood stock or slaughter, and each factor is size- and life-history dependent and varies between species and systems. The husbandry and water quality requirements during intensive rearing of juvenile fish are good examples of the complexity in fish welfare issues.

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23.4.1 Intensive production systems and rearing technology European farmed fish are reared in a large range of production systems, from extensive carp farming with low energy input to highly intensive industrial production of Atlantic salmon or turbot, with fully controlled water recirculation. In extensive systems, the farmer has no possibility to control the water quality and few means to avoid suboptimal levels of, for example, oxygen concentration. Intensive industrial farming, on the other hand, includes high fish density produced with less water, high energy feed and fast growth, short generation time and season-independent production. In these systems, the fish farmer may easily monitor and change water quality traits, but the systems face the challenge of finding a balance between what is economically optimal for the farmer and the limits for acceptable fish welfare. The present knowledge about lethal levels of different water quality parameters is not adequate to evaluate the welfare consequences of suboptimal rearing conditions. Each fish species has developed a set of minimums and maximums for tolerance for each environmental factor, and the ranges may be wide or narrow, developing the species into opportunistic generalists or specialists. The plasticity of the opportunistic generalists is the key to the evolutionary success of the fish, and enables individual fish to cope with a changing environment, including large changes in water temperature and other water quality traits. Compared with warm-blooded terrestrial animals, fish are poikilothermic and have few possibilities to regulate their body temperature. They have developed a life strategy aiming to meet a changing and unpredictable environment. Relatively minor factors during early life stages may have severe effects in later stages. Complex causal relationships exist amongst water quality requirements, stress, growth and immune function, and chronic factors may have longterm effects on both coping strategies and on general quality traits. The physiological stress responses are similar between fish and mammals. The primary stress responses include release of stress-related hormones (e.g., adrenalin, cortisol), leading to a secondary stress responses that stimulate oxygen uptake, mobilisation of energy substrates, and finally reallocate energy away from growth and reproduction. The stress response is mainly adaptive, but chronic exposure to stressors will be maladaptive when the adaptive capacity is exceeded. Farmed fish that are exposed to suboptimal rearing conditions over time will experience irreversible consequences for welfare, health and mortality, including maladaptive stress responses such as behavioural changes, reduced feed intake and growth, and decreased immune function. Several experiments of the effects of chronic exposure to intensive rearing conditions in juvenile Atlantic salmon demonstrate a clear relationship between the degree of intensive rearing and the negative effects afterward on growth and susceptibility to diseases after seawater transfer (Toften et al, unpubl., Fig. 23.2). Most people tend to think that fish density is the most critical parameter in fish production, and maximum density is often suggested as a key issue to maximise fish welfare. Several studies, in both freshwater tanks and sea cages, have revealed that density per se is probably neither the most critical factor nor

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Fig. 23.2 Relative growth (a) and relative mortality due to IPN virus susceptibility (b) as a function of the degree of intensive rearing in juvenile Atlantic salmon. Each circle represent an experiment with different water quality treatments, and each point represent an average of the experiment groups estimated as a percentage of the control groups (dashed line). All treatments are within the normal range of salmon farming, and the treatments within each experiment range from optimal (left side) to suboptimal (right side). Toften et al., unpubl.

the best indicator of welfare. On the contrary, many species reared in low density may increase the aggression amongst the fish when the fish start to scramble for food beyond a certain fish density. In addition, fish do not usually distribute equally in a tank or a cage, and actual density may be much higher then average density. At high density, there might be greater variation in water quality, but as long as the water quality and water current are adequate, the density itself affects the fish only to a minor extent. Independent of the density, a reduction in the oxygen concentration or an increase in the carbon dioxide concentration, however, may seriously affect the welfare of the fish.

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23.4.2 The effects of high carbon dioxide on fish welfare Some carbon dioxide (CO2) is present in all types of fresh or sea water, but the large increase in CO2 commonly measured in fish farms is mainly due to the metabolites from the fish's respiration. The carbon dioxide reacts with the water and creates carbonic acid (H2CO3), whereas the HCO3 fraction leads to a reduction in water pH and will thus change all pH-dependent responses in the fish. With high fish density or low water flow, the CO2 concentration may build up in the farming unit, leading to hypercapnea (high CO2), which starts a stress response and is known to affect gill functions. The Atlantic salmon responds to elevated CO2 levels with decreasing feed intake and growth (Toften et al., 2006), an increase in general stress parameters like cortisol, leading to higher post-smolt mortality (Fivelstad et al., 2003), and impaired immune functions and increasing susceptibility to diseases after transfer to sea water (Toften et al., 2006). A SEAFOODplus study on the effects of hypercapnea in juvenile Atlantic cod revealed that intensive rearing of cod might have similar effects as reported for Atlantic salmon (Toften et al., unpubl.). In the study, cod weighing approximately 100 grams were reared in high fish densities (60 kg m±3) with either a high specific water flow (SWF; 0.84 litre water kg fish±1 min±1) or low SWF (0.17 litre water kg fish±1 min±1). The two groups were compared with a control group reared at low density (30 kg m±3) and high SWF (0.84 litre water kg fish±1 min±1). The oxygen concentration was held constant at 85%, while the low water flow resulted in an increase in CO2 and a decrease in pH in the water, leading to an increase in the partial pressure of CO2 and HCO3 in the blood plasma of the cod. The most intensive group (with high density and low SWF) had a significantly lower growth rate compared with the two other groups (Fig. 23.3). The fish in this group also developed nephrocalcinosis due to

Fig. 23.3 Specific growth rate of juvenile Atlantic cod reared in groups of increasing intensity. The control groups had low fish densities (30 kg mÿ3) and high specific water flow (SWF; 0.84 litre water kg fishÿ1 minÿ1). The treatment groups had high fish densities (60 kg mÿ3) and high SWF or low SWF (0.17 litre water kg fish±1 minÿ1). Toften et al., unpubl.

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calcium secretion in the kidney, also an indication that intensive rearing challenges the physiological coping mechanisms in cod and affects the welfare of the fish.

23.5

Welfare during slaughter of farmed fish

The slaughter period includes the effects of pre-slaughter treatment such as transportation, handling and stunning, in addition to the killing of the fish. Most fish species in Europe are starved for a period before slaughter. During starvation, the fish will lose biomass as they mobilise energy stores such as lipids, but after a number of days, the biomass reduction will slow down as the fish become hypometabolic and downregulate their metabolisms. Depending on the production system, the fish is then caught, stunned and killed or transported by some means to a slaughter site. This last period in the fish's production cycle is probably the time when various welfare issues most strongly affect general muscle quality traits. In addition, the killing of an animal is easily understood by the consumers as either `cruel' or `humane', and information about slaughter methods may thus significantly affect consumer behaviour and human fish consumption. The slaughter of fish is largely linked with the debate over pain and suffering in fish. Most people have problems distinguishing between the terms `pain' (or nociception) and `pain perception'. The question whether `the fish feels pain' is partly irrelevant as there is no scientific doubt that the fish can feel the pain, but it is still an open question to what extent it perceives pain similarly to humans. Fish have nerve ends dedicated to pain nociception, and have a nociceptive neuronal pathway similar to mammals to communicate nociception from the body to the brain. Some scientists argue that fish and other animal groups cannot perceive pain because they lack a neocortex, the most important area for pain perception in mammals (Rose, 2002). On the contrary, scientists argue that other brain structures may also have the same functions as the neocortex (Braithwaite and Huntingford, 2004), and there are clear indications that fish have numerous pain receptors and show long-term behavioural indicators when exposed to pain stressors (Sneddon et al., 2003). Suffering is an even more complex question than pain perception, focusing more on the animal's consciousness and cognitive skills. The debate over pain and suffering will probably continue, and the way we understand these questions may have a large impact on how we handle and slaughter farmed fish. 23.5.1 Stunning of fish Stunning is applied to render a fish unconscious and insensible until death, without avoidable excitement, pain or suffering (Van de Vis et al., 2003). Adding carbon dioxide (CO2) to a fish tank has been a common way of stunning Atlantic salmon, for example. This leads to a drop in blood pH and a disruption

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in the central nervous system, followed by immobility. The method leads to asphyxia under stressful conditions and is no longer recommended as a stunning protocol. Cooling of fish prior to or during stunning has also been used to calm the fish and improve muscle quality. Cooling slows down the behavioural responses of the fish, but does not result in loss of consciousness and sensibility without avoidable excitement for fish species such as Atlantic salmon, rainbow trout, sea bream, sea bass, eel and African catfish. Even though the fish moves more slowly, cooling before death will probably only prolong the time period of poor welfare before killing. For example, African catfish during live chilling had an increase in heart rate from 70 to 300 beats per minute in a conscious animal (Lambooij et al., 2006), indicating a poor welfare status. One of the other alternatives is electrical stunning (Van de Vis et al., 2003), which will probably be commonly used in European fish farming in the future. The scientific criterion for electrical stunning or any other new methods must be an immediate loss of consciousness and sensibility to pain stimulus. Behavioural measures are insufficient for assessment of loss of consciousness and sensibility. Sensibility is required, since unconscious fish should not be aroused during gutting, for instance. Because of exhaustion or paralysis, a conscious fish may not be able to show spontaneous behaviour and responses to administered stimuli. In contrast to warm-blooded animals, the spinal cord in fish controls a major part of the coordinated movements. Unconscious fish can be classed as conscious and sensible. Therefore, the use of EEG recordings, as well as evoked responses on the EEG, is recommended for an unequivocal assessment of the level of brain function in animals. The ECG can be used to establish whether heart failure occurs or whether a slow stunning method may cause stress to the animals. For an instantaneous electrical stun, sufficient current should be passed through the brain of the fish. The applied voltage across the head of the animal or electrodes in water only is not a sound criterion for guaranteeing immediate loss of consciousness and sensibility. For example, when sea bass were exposed to 50 V with electrode plates at 50 cm distances in seawater (conductivity 52 mS cm±1), the animals were immediately stunned. When the animals were exposed to the same electric field (1.0 V cm±1) in freshwater (conductivity 1 mS cm±1), the animals were not stunned (Lambooij and Van de Vis, unpubl.). Several studies have demonstrated that fish may not be killed by the electricity. This could be because permanent heart failure cannot be induced by electricity. In order to prevent recovery of a stunned fish, a killing method has to be applied. The use of electricity, on the other hand, may lead to fractures and haemorrhages, which can be due to strong muscle stimulations, but carcass damage may have several causes and a further optimisation is needed to minimise or prevent damages. As an alternative stunning method in carp, electrical stunning has been tested in SEAFOODplus (Van de Vis et al., 2006). Common carp reared in ponds are usually slaughtered after 30 minutes' asphyxia in air followed by a manually applied blow to the head. Asphyxia is used to exhaust the carp before killing it. It is known that the asphyxia period is stressful for the fish, and a manually

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applied blow is often inaccurate and not hard enough to result in immediate loss of consciousness. In the SEAFOODplus experiment in Poland, carp from the pond were drained and crowded into a canal, where the fish farmer caught the fish with a hand net. The fish were stunned by applying electricity and immediately chilled on flake ice or a slurry of ice and water. The effectiveness of electrical stunning was monitored by EEG registration, and the muscle quality changes were measured by analysis of colour and muscle pH. The EEG recordings revealed that carp lost consciousness immediately using a current density of at least 0.14 A dm±2 with 50 Hz AC for one second in water with conductivity 0.2 mS cm±1. The fish would not recover from stunning after five seconds of electricity combined with chilling. The experiment demonstrated that carp stunned with electricity had a significantly higher pH value in the fillets during a seven-day period after slaughter (Fig. 23.4). The results conclude that electrical stunning in carp leads to a more humane slaughter and may improve muscle quality. For comparison, carp have also been stunned mechanically using an instrumental blow to the head. EEG recordings revealed the appearance of theta, delta waves and spikes, which were proceeded by no brain activity on the EEG providing evidence of unconsciousness and insensibility. In 18 of 20 fish, an iso-electric line was observed after an average of 16 12 seconds. However, two carp responded to pain stimuli at 0.5 minutes and one carp also at 3 minutes post stunning, demonstrating there is no certainty for instantaneous loss of consciousness and sensibility (Lambooij et al., 2007). The percussive gun was characterised by using a high-speed camera (5000 frames second±1), and the head was X-rayed to show the damage to the scull as a result of the blow. The displacement of the nylon cylinder in the percussion pistol, which delivered the blow on head, reached a maximal velocity of 19.13 0.76 m second±1. The resulting kinetic energy at maximal velocity was 10.99 0.88 J.

Fig. 23.4 pH in the fillets of common carp slaughter by electrical stunning followed by chilling compared with control stunning (asphyxia followed by a blow to the head). Van de Vis et al. (2006).

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Monitoring ethical qualities in farmed fish

In order to improve the welfare of farmed fish, some measurements of welfare are needed to move towards defining protocols and standards of fish husbandry. A set of rapid, inexpensive and non-invasive screening methods may be used as welfare indicators. The most obvious indicators are related to feed intake, growth, health, injuries, damages or stress, while indicators of motivational and emotional states are much more difficult to identify and validate. Measurements and monitoring of fish welfare will probably have to include sets of integrated measures, perhaps combinations of broad indicators affected by multiple stressors, and more specific physiological and behavioural indicators. In most experimental fish welfare studies, one or several physiological samples are analysed to measure the level of biological coping, such as measurements of blood plasma cortisol to indicate the general stress level. However, it is difficult to obtain blood samples fast enough without stressing the fish and such measurements will only give a glimpse of the situation. There is no single physiological parameter showing sensitive and linear responses to all factors that could cause potential distress to the fish. The development of online, non-invasive methods enables continuous measurements of fish welfare in free-swimming fish. This approach requires knowledge of the reference levels of the measured welfare parameters and that the relationship amongst measured response and relevant types of acute and chronic distress are established in a series of experimental validation studies. For experimental validation, this may be done before, during and after exposing the fish to potential stressful episodes. 23.6.1 Breathing patterns as a welfare indicator Several laboratory studies suggest that measurements of breathing patterns in fish are a promising indicator of a wide range of important welfare factors. Ultimately, fish breathe to supply oxygen and remove waste. If the animal is stressed, oxygen requirements increase and this is reflected in the breathing pattern. Likewise, the gills also represent the major arena of interaction between the fish organism and its external medium. If environmental quality changes, the breathing pattern may also change in order to maintain functional homeostasis. The fish's breathing pattern results from integrated processing in its central nervous system and is generated through nervous innervation of the different breathing muscles. The physiological and affective statuses of the fish are thus continuously processed and incorporated into a breathing pattern that reflects the status and needs of the organism as a whole relative to the changing qualities of its external medium. As such, breathing pattern is known to be significantly affected by a variety of factors such as hypoxia (low O2), hypercapnea (high CO2), changes in water pH or metabolite levels, toxic or subtoxic levels of metabolites, anaemia and diseases, as well as during the general stress response and factors that presumably also include psychological responses such as shelter, fear and pain. For example, the breathing pattern in Atlantic cod changed as a

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Fig. 23.5 Breathing patterns in Atlantic cod in a swim tunnel, measured with the SmartTag. The audio frequency represents the pressure (in cm H2O) in the buccal cavity. Fish from the control group (a) with high oxygen and low CO2 concentration with a breathing frequency on 38 minÿ1 and an amplitude of 0.8 cm H2O, from the partial hypoxia/hypercapnea group (b) with 65% oxygen and 13 mg lÿ1 CO2 with a breathing frequency on 42 minÿ1 and an amplitude of 1.7 cm H2O, and from the hypoxia/ hypercapnea group (c) with 45% oxygen and 35 mg lÿ1 CO2 with a breathing frequency on 31 minÿ1 and an amplitude of 1.1 cm H2O. Aas-Hansen et al., unpubl.

response to both low oxygen and high CO2 in a swim tunnel experiment, affecting both the frequency of breathing and the amplitude of each breath (AasHansen et al., unpubl., Fig. 23.5). The use of breathing pattern as a welfare indicator has been tested in SEAFOODplus, including the development of a `SmartTag' technology (AasHansen and DamsgaÊrd, 2006) together with the R&D company THELMA (www.thelma.no). The transmitter contains a differential pressure sensor that measures the water pressure inside the buccal (mouth) cavity of the fish relative to the surrounding water pressure. The output from the transmitter is a continuous frequency-modulated acoustic signal in the ultrasound range (50±120 kHz). The acoustic signal from each tag is picked up by a hydrophone, amplified, and displayed and stored on a computer. The relationship between the frequencymodulated acoustic signal and actual water pressure is constant for each tag and determined through simple calibration. The online screenshot thus shows the

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Fig. 23.6 Breathing pattern in Atlantic cod in a sea cage, before, during and after a twominute disturbance (a) measured with the SmartTag. The audio frequency represents the pressure (in cm H2O) in the buccal cavity. Typical breathing patterns before and after the stress period are enlarged in (b) and (c). Aas-Hansen et al., unpubl.

actual changes in water pressure inside the mouth cavity (i.e., the breathing pattern), with additional online calculation of the breathing frequency and changes in breath amplitude during inspiration and expiration. The present tag weight is six grams in water with an estimated battery life of 25 days. The tag has been tested in various situations during the production cycle, including shortterm handling stress and changes in water quality traits such as a decrease in O2 concentration or an increase in CO2 level (Fig. 23.5). These factors affected both breathing frequency and amplitude, indicating that the breathing pattern may be a suitable candidate as a welfare indicator. The tags have been tested in full scale farming, for example to measure the effects of short term handling in sea cages (Aas-Hansen et al., Fig. 23.6).

23.7

Future trends

The further development of welfare issues in Europe depends on a compromise amongst various driving forces. The interest of ethical questions is expected to rise further in the future and the variation amongst European countries is expected to decrease. The increasing focus on animal welfare will strongly

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affect our everyday lives, our food preferences and how we treat animals. The consumers and the market have and will always have a strong impact on fish farming and are expected to be the most important driving force for fish welfare. Consumers and food producers are equally dependent on each other's trust. Even small changes in food confidence may have large market consequences. European farmers are in a unique position to be able to market a fish that is not only tasteful, safe and healthy to eat, but also a traceable seafood product that is produced in a way that accommodates the welfare of the animals. Finding a compromise between the current trend towards intensive rearing of fish and the increasing demand for healthy, high-quality seafood produced in an ethical husbandry system will be the main challenge of sustainability. Given the development of questions regarding welfare issues during the last decade, the future of fish welfare may take several directions. The first scenario is a further development of legislation and regulation of fish farming, both nationally and within the EU, leading step-by-step to a change in the way fish is produced. The consumers will be increasingly aware of ethical issues and will include these questions in a general quality discussion, separating the market in segments of consumers who are price- or quality-oriented. Traceability may become an important tool to communicate ethical qualities of fish products. On the other hand, if farmers experience an increasing focus on the reduction of production costs in order to meet a price pressure, we might expect few positive effects of an increased ethical development. In such case, welfare will be more of a problem than a possibility for the farmers. Another and much more drastic scenario may develop into an ethical disaster for fish farming. Consumer awareness of fish welfare may increase together with general food awareness, but the fish farmers may not be able to meet these questions by scientifically based knowledge and improvements in the production. In such case, we might expect incidents in the farming industry that will hit the headlines and be actively used by anti-aquaculture groups to establish a picture of farmed fish as unsound and unethical, ultimately leading to a drastic decrease in the consumption of farmed fish and fish in general.

23.8

Sources of further information and advice

More information about European fish farming can be found on the website of the European Aquaculture Society, EAS (http://www.easonline.org), and the Federation of European Aquaculture Producers, FEAP (http://www.feap.info). The World Organisation for Animal Health (OIE) has focused on animal welfare during recent years (http://www.oie.int). The European Food Safety Authorities, EFSA, publish expert opinions on welfare of European fish species, including a report on welfare aspects of animal stunning and killing methods, and forthcoming reports on the welfare of various European farmed fish species (http://www.efsa.europa.eu). Several EU projects have developed websites with information about fish welfare, including SEAFOODplus, project ETHIQUAL,

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`Ethical quality traits in farmed fish' (http://www.seafoodplus.org), WEALTH, `Welfare and health in sustainable aquaculture' (http://wealth.imr.no), FASTFISH, `On farm assessment of stress level in farmed fish' (http://fastfish.imr.no), BENEFISH, `Evaluation and modelling of benefits and costs of fish welfare interventions in European aquaculture' (http://www.benefish.eu), CONSENSUS, `Sustainable aquaculture in Europe' (http://www.euraquaculture.info) and the COST Action 827, `Welfare of Fish in Europe Aquaculture' (http:// www.fishwelfare.com). Other scientific Internet pages on fish welfare include a website developed by Stirling University, UK (http://www.fishwelfare.net), and the report from FSBI, 2002. `Fish Welfare. Briefing Paper 2', the Fisheries Society of the British Isles, Granta Information System, Cambridge, UK (http:// www.le.ac.uk/biology/fsbi/welfare.pdf). More information on the SmartTag method can be found on THELMA's website (http://www.thelma.no). A strategic programme on fish welfare in Norway is presented in the report by DamsgaÊrd et al., 2006, `Welfare in Farmed Fish', which can be downloaded from the website of Fiskeriforskning, NO (http://www.fiskeriforskning.no).

23.9

References

(2003). Om dyrehold og dyrevelferd [On farming and animal welfare]. White Paper no. 12 (2002±2003). The Agriculture Department, Norway (in Norwegian). ANON. (2005). Forskningsbehov innen dyrevelferd i Norge [Research needs in animal welfare in Norway]. Report from the steering committee for `Animal welfare ± research and knowledge gaps'. The Research Council of Norway (in Norwegian). AAS-HANSEN, é. and DAMSGAÊRD, B. (2006). Non-invasive methods for assessment of fish welfare. In: Welfare in Farmed Fish. DamsgaÊrd, B., Juell, J.E., and Braastad, B.O. (eds.). Report 5/2006 Fiskeriforskning, Tromsù, Norway, pp. 27±29. Ê RD B. and JUELL, J.-E. (2006). Animal welfare ± A new concept in BRAASTAD, B.O, DAMSGA aquaculture and fisheries. In: Welfare in Farmed Fish. DamsgaÊrd, B., Juell, J.E., and Braastad, B.O. (eds.). Report 5/2006 Fiskeriforskning, Tromsù, Norway, pp. 5±13. BRAITHWAITE, V.A. and HUNTINGFORD, F.A. (2004). Fish and welfare: do fish have the capacity for pain perception and suffering? Animal Welfare, 13 Suppl.: 87±92. BROOM, D.M. (1986). Indicators of poor welfare. British Veterinary Journal, 142: 524±526. BROOM, D.M. (1991). Animal welfare: concepts and measurement. Journal of Animal Science, 69: 4167±4175. DUNCAN, I.J.H. (1993). Welfare is to do with what animals feel. Journal of Agricultural and Environmental Ethics, 6, Suppl. 2: 8±14. DUNCAN, I.J.H. and FRASER, D. (1997). Understanding animal welfare. In: Animal Welfare. Appleby, M.C. and Hughes, B.O. (eds.). CAB International, Wallingford, pp. 19± 31. Ê SGA Ê RD, T., BáVERFJORD, G., RASMUSSEN, T., VINDHEIM, T. and FIVELSTAD, S., OLSEN, A., A STEFANSSON, S.O. (2003). Long-term sub-lethal effects of carbon dioxide on Atlantic salmon smolts: ion regulation, haematology, element composition, nephrocalcinosis and growth parameters. Aquaculture, 215: 301±319. ANON.

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(1964). Animal machines: the new factory farming industry. Vincent Stuart Publisher, London. KILEY-WORTHINGTON, M. (1989). Ecological, environmental and ethically sound environments for animals: Toward symbiosis. Journal of Agricultural and Environmental Ethics, 2: 323±347. LAMBOOIJ, E., KLOOSTERBOER, R.J., GERRITZEN, M.A. and VAN DE VIS, H. (2006). Asssesment of electrical stunning of farmed African catfish (Clarias gariepinus.) and chilling in ice water for loss of consciousness and sensibility. Aquaculture, 254: 388±395. LAMBOOIJE, E., PILARCZYK, M., BIABWAS, H., VAN DEN BOOGART, J.G.M. and VAN DE VIS, J.W. (2007). Electrical and percussive stunning of the common carp (Cyprinus carpio L.): Neurological and behavioural assessment. Aquaculture Engineering, 37: 171± 179. ROSE, J.D. (2002). The neurobehavioral nature of fishes and the question of awareness and pain. Reviews in Fishery Science, 10: 1±38. SNEDDON, L.U., BRAITHWAITE, V.A. and GENTLE, M.J. (2003). Do fishes have nociceptors? Evidence for the evolution of a vertebrate sensory system. Proceedings of the Royal Society. B. Biological Sciences, 270: 1115±1121. SPRUIJT, B.M., VAN DEN BOS, R. and PIJLMAN, T.A. (2001). A concept of welfare based on reward evaluating mechanisms in the brain: anticipatory behaviour as an indicator for the state of reward systems. Applied Animal Behaviour Science, 72: 145±171. Ê RD, B. and ARNESEN, A.M. (2006). TOFTEN, H., JOHANSEN, L.-H., SOMMER, A.-I., DAMSGA Optimising intensive rearing conditions to secure fish welfare and health. In: Welfare in Farmed Fish. DamsgaÊrd, B., Juell, J.E., and Braastad, B.O. (eds.). Report 5/2006 Fiskeriforskning, Tromsù, Norway, pp. 83±89. HARRISON, R.

È GER, J., LAMBOOIJ, E., MU È NKNER, W., VAN DE VIS, H., KESTIN, S.C., ROBB, D.F.H., OEHLENSCHLA È NKNER, W., KLOOSTERBOER, R.J., TEJADA, M., HUIDOBRO, A., TEJADA, KUHLMANN, H., MU

and NESVADBA, P. (2003). Is humane slaughter of fish possible for industry? Aquaculture Research, 34: 211±220. VAN DE VIS, H., BIALOWAS, H., PILARCZYK, M., MACHIELS, M., REIMERT, H., VELDMAN, M. and LAMBOOIJ, B. (2006). Comparison of commercial and experimental slaughter of farmed carp (Cyprinus carpio) with respect to development of rigor mortis and flesh quality. In: Seafood research from fish to dish. Luten, J.B., Jacobsen, C., Bekaert, K., Sñbù, A. and OehlenschlaÈger, J. (eds.). Wageningen Academic Publisher, Wageningen, The Netherlands, pp. 201±210. M., OTTERAÊ, H., ROTH, B., SéRENSEN, N.K., ASKE., L., BYRNE, H.

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Part VI Seafood traceability to regain consumer confidence

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24 Introduction to Part VI: traceability in a changing world E. P. Larsen, Technical University of Denmark, Denmark

Traceability has become a `hot issue' in the food sector in the last decade. There is no single reason for the increase in interest. Recent food scares and also issues which had an impact worldwide, such as the 11 September attacks on the US, have played a role. One major food scare, the BSE (Bovine Spongiform Encephalopathy) crisis in the 1990s led to the identification of individual animals. The events of 9/11 prompted the US to release of a set of new laws demanding full traceability of imported goods by requesting Country of Origin Labelling (COOL) to protect its population. The use of GMO (genetically modified organisms) in the food sector has also increased demand for full traceability, not only of food items, but also of food ingredients and the feed that has been used in animal production. Organic food producers now have to justify that no GMO products have been used in the production chain. The seafood area has been spared a major food safety scare until now. There have been several incidents that could easily have developed into a crisis. Dioxins were found in wild-caught salmon and herring from the Baltic Sea, for example, and malachite green was found in too great concentrations in imported farmed fish products from East Asia, resulting in restrictions on imports to both the US and Europe. In both of these cases, it has not been proven that low concentrations of these substances have deleterious effects on human health, but in the turmoil of an intensive media storm this can be forgotten. One episode illustrates this effect and its effect on exports. In 1986 a herring fillet with three live nematodes was pictured on German television while the presenter explained that the fillet was from Denmark. This resulted in a drop of 30% in export value of Danish herring fillets. It took three years before the export value was back to the original level. It rather quickly transpired that the herring fillet was not even from Denmark, but the first impression counts in crises like this.

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During the first years of this decade, the EU General Food Law was prepared and its requirements gradually implemented. The traceability part of the Food Law became mandatory as part of EC Regulation 178/2002 from 1 January 2005. This regulation sets out the principles of food safety and states who is responsible for ensuring that the requirements of food safety law are met. It also covers the implementation of food traceability, which is essential to food safety and a responsibility of partners along the entire food chain. In §18 the text says that `food and feed business operators shall be able to identify any person from whom they have been supplied with a food, a feed, a food-producing animal, or any substance to be, or expected to be, incorporated into a food or feed'. In the next section it states that: `Food and feed business operators shall have in place systems and procedures to identify the other business to which their products have been supplied'. This can of course be done by using paper and pen and by having large batch sizes, but if available technologies such as on/at line sensors or data carriers were used, this would speed up product registration and open up possibilities for the use of the data in the management of the food production chains. The data captured by traceability systems could then be used for other purposes such as process rationalisation, process optimisation, and marketing (the information about the origins of the product could be used to promote it). From the start of SEAFOODplus in 2004, the aim of the traceability pillar has been to look into possible ways of developing suitable systems which can help the seafood sector deal with these issues. For traceability systems to be more efficient and to facilitate communication between the links in the chain it is essential to have common standards and guidelines and everyone must understand the words that are being used. The IT language should also be understandable by everybody in the chain. These are the key elements of the traceability part of SEAFOODplus. The title Validated Traceability Systems has been chosen to describe the activities of the traceability part. This was selected because it signals co-operation between the links in the chain from the primary producer to the end-user; traceability is not just a control tool. This is described in more detail in Chapter 26. The objective of Validated Traceability Systems is to create the basis for the development of validated traceability systems in the seafood sector. Several chains in the seafood sector are studied in the project, all of which have been chosen because they illustrate as many aspects of the complexity of the seafood area as possible. The following cases were selected: Aquaculture · Salmonides ± Atlantic salmon from Norway to Spain and on to the final consumer. · Other aquaculture species ± Wild caught tuna, kept in cages until they have reached market size. Wild caught fish · Pelagic fish species, both European and imported ± Tuna and small fatty fish species, anchovies and herring, deepwater prawn.

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· Demersal fish species, both European and imported fish species ± Merlucidae ± the hake family. The chains were also selected because they cover different seafood production chains: aquaculture, distant water wild caught frozen fish and wild caught fresh fish. A chain with imported fish species was selected for two reasons. First, because traceability systems for chains beginning in an arrivals terminal need to take into account not only the legal and regulatory issues involved with importing seafood, but also the traceability requirements of the next link in the chain. Second, because the chain allows authenticity tools and quality methods, which have already been developed, to be tested. The traceability area has been split into three projects, which are interconnected to a high degree. The three projects are METHODS, IMPLEM and VALID. The results of the two first projects are described in Chapter 25, and the latter in Chapter 26. The most important result of the METHODS project has been the development of a Good Traceability Practice Manual ± GTP. A more distinct list of information elements that can be used in communicating between the links in the chains has been developed. This has been named the Vocabulary. Collaboration with other projects under the EU 6th Framework Programme, such as IP TRACE and TraceCore, is already creating a very useful platform for both developing and disseminating the results achieved. The development of a new agreed vocabulary for the bivalve sector will be finished in 2008. The vocabulary will allow the exchange of information elements in a unanimous way between the different links in bivalve production chains. In IMPLEM, the continuing testing of Radio Frequency Identification (RFID) tags has resulted in recommendations for the fish sector when introducing or facing the introduction of this technology. The recommendations for the different fish chains analysed and mapped for full or partial traceability implementation can be found on the SEAFOODplus homepage. In VALID the guides for validation of the most important traceable data have been the major outcome. These include a DNA database and reference materials (plasmidic standards) as validation tools for the authentication of fish species. Validation of the traceability data of a chain in which fish products are imported to the EU has been another major achievement. The seafood area is complex, and one of the most frequently used excuses for not having a functional traceability system or even sufficient information on the fish product sold to the consumer, is that the product passes through many links, even changing ownership several times. The result of this is that data is lost due to the lack of cooperation between the different businesses in the chain. Theses issues have to be overcome and the results from RTD 6 `Seafood traceability to ensure consumer confidence' will be a major step towards the achievement of full traceability in the fish sector, by the introduction of the concept of validated traceability systems: Validated traceability systems are traceability systems that can be `trusted': internally in a company and externally by all customers and authorities.

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25 Improving traceability in seafood production J. Storùy, G. Senneset and E. ForaÊs, SINTEF Fisheries and Aquaculture, Norway, P. Olsen and K.M. Karlsen, Nofima, Norway and M. Frederiksen, Technical University of Denmark, Denmark

As described in the introduction to this part of the book, the SEAFOODplus traceability research was conducted under three different projects. This chapter describes the developments and achievements of the METHODS and IMPLEM projects within the general context of traceability.

25.1

The METHODS project: introduction

The METHODS project addressed the fundamental issues of traceability, the basic format of the information elements to be transferred in electronic traceability software systems (the `vocabulary') and the development of a Good Traceability Practice (GTP) manual to be used by seafood companies complying with traceability requirements. A map service that enables software systems to convert nautical positions to easily understandable graphical maps has also been developed. The maps can be used for end consumers. The vocabulary is a set of files that software developers can use when they develop traceability software systems. The files describe the names of selected information elements translated into different languages and their format. Different software systems can use the vocabulary to exchange information without any risk of human error in the translation process and without any additional costs. The GTP manual is still in progress and will be finished mid-2008. The first parts of the manual are presented in this chapter. The manual advises the seafood

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industry on good practice and the basic considerations when analyzing production and developing ways of improving traceability through the production processes. The GTP manual will enable the seafood industry to make better choices when deciding the size of lots, data formats and data transfer because it increases awareness of the consequences of their decisions. Such consequences might include limiting the economic risks in case of a recall, and the ability to optimize production both internally and externally with partners through standardized data format and transfer. The possibility of utilizing selected data elements to inform end customers is an added benefit that could make companies more competitive in high-value markets.

25.2

The vocabulary

The vocabulary developed in METHODS is only to be used by software developers for traceability software. The vocabulary is `help files' to be used in connection with the technical TraceFish standard. These files have been translated to XML files and are available on www.tracefish.org. The original files made in Excel are available at www.seafoodplus.org. The vocabulary consists of 21 files where selected parameters are translated into the main EU languages. In the files, the parameters have been standardized and translated into as many languages as possible given the language abilities available in the working group. All parameters are always presented in English. The technical TraceFish standard is now multilingual and more selfexplanatory. All the standardized FAO 10462 fish species have been added to the Species type file. The standardized EU size groups have been added to make the standard more useful in practice.

25.3

Good Traceability Practice manual

25.3.1 Introduction Recommendations for Good Traceability Practice (GTP) in the seafood sector will be available mid-2008. The purpose of developing GTP is to support the implementation of traceability. The recommendations for good traceability practice are now being tested in several pilot projects. The starting point for the GTP guidelines for the fish sector was the development of three standards for voluntary recording and exchange of traceability information in the seafood chain at the beginning of this decade (www.tracefish.org). The standards relate to: (a) farmed fish, (b) captured fish and (c) technical standards. For further details see the European Standardization Body CEN (www.cenorm.be/cenorm/index.htm). The information in the farmed and captured fish standards is categorized in `shall', `should' and `may'. `Shall' are information elements necessary to identify and trace the movement of the products as they move through the supply chain.

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Fig. 25.1

Components of the Good Traceability Practice Guidelines.

`Should' are information elements required by law, or crucial parameters relating to food safety, labelling or quality. The `may' category includes optional data elements possibly relevant for internal or external reporting. The technical TraceFish standard defines the way information is transferred using the Internet and XML as language, but the original version had some deficiencies. For instance, the fish species was defined at an overall level, but there was no unique definition of the fish name. So one of the aims of this project was to enable the technical standard to use a vocabulary where the different information pieces were predefined and already translated into many of the European languages. The guidelines were developed as a co-operation between the two EU Integrated Research projects TRACE and SEAFOODplus, and are discussed in other research fora where traceability is the main topic. The document will be continuously revised based on experiences in these projects. The newest version of GTP will always be available at the website www.tracefood.org. Our aim is that these guidelines should become THE global guide to food chain traceability, and constitute a major part of the TraceFood framework. To illustrate how GTP is constructed, Fig. 25.1 shows the different components. 25.3.2 Defining traceable units The concept of traceable units is a principal concept in chain traceability in general. A trade unit (TU) is defined as `any item upon which there is a need to retrieve predefined information and that may be priced, or ordered, or invoiced

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at any point in any supply chain'. In practice, it often refers to the smallest traceable unit that is exchanged between two parties in the supply chain. A crate of fish or crate of meat is often a trade unit (www.tracefood.org). A logistic unit (LU) is defined as `an item of any composition established for transport and/or storage that needs to be managed through the supply chain'. In practice, it is made up of one or more separate trade units. In some cases, the trade unit and the logistic unit are the same. A logistic unit is often a pallet of fish/meat crates being distributed from a producer to a receiver. 25.3.3 Unique identification of traceable units The trade units must be uniquely identified. A minimum of additional information needs to be linked to a trade unit throughout its lifetime, and these data should be accessible via the unique identification number. The size of the smallest traceable unit varies across and within different industries. In the fish farming business, a bucket of roe, the entire contents of a well boat or the contents of a fish crate are typical trade units. In the meat sector, a crate of meat is a typical trade unit. Identifying the logistic unit Typical logistic units in the fishing industry might be a pallet or a 40-foot container of fish crates. GS1 provides a globally unique data element for the identification of a logistic unit, called the Serial Shipping Container Code (SSCC). TraceFood requires that the IDs of the separate trade units within the logistic unit are linked to the logistic unit identifier, in practice to the SSCC. The SSCC number structure is (00) 235467985462312345, where 00 is the Application Identifier and the following figure is a unique, 18-digit number. Identifying the trade unit The GS1 128 symbology does not have a single data element for the unique identification of a trade unit (i.e., a particular fish crate). However, the symbology provides a Global Trade Item Number (GTIN), which identifies a variant of a trade unit (i.e., a crate of 20 kg fresh Superior Atlantic salmon, 4±5 kg each fish). To uniquely identify the particular crate, one has to add one or more predefined data elements. In the TraceFood standard, this identifier is called GTIN+, where the + indicates that additional information is needed for this purpose. To make up the GTIN+, the GTIN (AI 01) must be combined either with a batch number (AI 10) and a serial number (AI 21), or with the date and time of production (AI 8008). GS1 defines the batch number as an internal number for a production batch. It is common practice to allocate this number to all produced units with similar properties (i.e., origin/farm area, time of arrival, supplier, etc.) and/or produced within a certain time period (i.e., hour, shift, day, week, etc.). Since a group of trade units is commonly given the same batch number, unique identification for each separate trade unit demands further specification. An appropriate solution

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Fig. 25.2 ID numbering, from NSF ± CWA 1459 (TraceFish standard).

is to allocate a serial number to each trade unit (i.e., a meat crate). See below and Fig. 25.2. For example, a GTIN+ with the batch and serial number might look as follows: (01)07038010000065(10)123456(21)1234567890 The alternative is to make up a unique identification for a trade unit by combining the GTIN and date and time of production (AI 8008). For example, (01)07038010000065(8008)040915125603 The figures for AI(8008) have a structured format, representing year/month/ time/minute/second. In some cases a logistic unit and a trade unit will be of equal size (i.e. the entire contents of a cargo boat carrying grain). 25.3.4 Keeping track of transformations To be able to trace both backwards to find origin and forwards to find all related units it is vital to record all transformations (splits and joins) a trade unit is subject to. The four steps below specify how to keep track of transformations. 1. Define the trade unit according to industry. 2. Record the IDs of received trade units (raw materials and/or ingredients). ± If the trade unit has a unique ID, record it. ± If the trade unit does not have a unique ID, allocate one to it. 3. Record the ID of the trade units that enter the production process, and give all trade units produced a unique ID.

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In practice, the ID of trade units that goes into the production process will at that stage be linked to a production batch and therefore a batch number. Every trade unit produced must be allocated a unique number (GTIN+). In this way the ID of trade units received will be linked to the ID of trade units produced. This practice ensures forward traceability internally. Where possible and relevant, it is also recommended to record the fraction (%) and/or the net weight of each trade unit that enters production. 4. Record the ID of all trade units dispatched. Fulfilling the requirements in steps 2±4 provides both a link between received and dispatched trade units, via the production process (internal traceability), and a link to the previous and subsequent operators (chain traceability). Mapping these relations provides transformation information. Figure 25.3 shows trade unit relationships within a business. For instance, trade unit 21 is composed of all of trade unit 11 and some of trade unit 12. Both fractions (%) and net weight are indicated. In this figure, the production step is removed, and only the relationship between received and dispatched trade units is shown. 25.3.5 Additional data recording (i.e. food safety data) Traceability is defined as `The ability to trace the history, application or location of an entity by means of recorded identifications' (ISO 8402). Hence, information related to history (i.e., temperature records, production process information), application (property-related information such as weight, species, fat percentage, etc.) and location (distribution route) should be recorded and linked to a traceable unit. Figure 25.4 shows how traceability-related information can be split into different categories. Within each of these categories, there are one or more data element(s) to be recorded in the forms presented in the sectorspecific standards. 25.3.6 Traceability friendly production Traceability oriented implementation processes include re-engineering of data recording, and material flow and production processes. There are two main ways of dealing with traceability, either to document the existing material flow, or to modify the material flow based on traceability friendly criteria and document the new processes. Designing optimal, cost-effective production processes and purchase policies has long been a major element of improvements in the food industry. Modern food factories are streamlined, with specialized production of narrowly defined products (i.e., cutlets, fillets, etc.). Where the risk of contamination is low, implementing a traceability friendly production regime may not improve profits. However, if there is a substantial risk of contamination, handling and production procedures can be modified to simplify traceability, as this may help to isolate and recall contaminated items efficiently, hence reducing direct costs.

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Fig. 25.4 Different information categories within the traceability concept.

When considering re-engineering existing production practices to improve traceability, the following corrective actions should be considered: · Reduce the size of inputs (raw materials and ingredients). This is especially important for long shelf-life ingredients, such as salt, spices, flour, etc. Contamination from such ingredients can have far-reaching effects, because they may have been used in a large number of products over a long period of time. · Reduce the size of production batches. In principle, operators are free to define the size of their production batches. But in a food scare case, large batch sizes could mean the withdrawal of an enormous quantity of products. If production is split into small batches, for instance one day's production, any withdrawal will affect only a limited amount of product. · Keep the mixing of raw materials from different origins to a minimum. Contamination is often not detected before a trade unit has reached the shops. If the probable cause of the contamination is the raw materials, it can be hard to tell which raw material is the source. If these raw materials also have been used in other production lines, any recall will probably include products made of `clean' raw materials as well as those containing the contaminated raw materials. Due to this, mixing raw materials of different origins in a production batch should be avoided. Decisions taken regarding instituting traceability friendly production should be based on risk analysis and cost/benefit calculations.

25.4

The SEAFOODplus map service

SEAFOODplus mapservice.xls is a map service that translates a nautical position into a user-friendly map, based on the nautical positions defined in the TraceFish technical standard (www.tracefish.org). The service is available online on a GIS-server free of charge, and the file includes instructions. The user sends a position to the server and an overview and a more specific map (two bitmap files) are returned to an email address defined by the user. The position is converted to a highlighted square on both maps, exact enough for the producer/

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retailer and end user, but not so precise that, for instance, a fisherman reveals the exact fishing position to his competitors. The maps can be used by retail shops and home delivery services to show catch areas to the end consumer. The service is provided from a DIFRES online server. Software engineers can incorporate the map service into other software packages. Guidelines are given at the SEAFOODplus website (www.seafoodplus.org).

25.5

The IMPLEM project: introduction

The work conducted in IMPLEM focused mainly on: · Developing a method for food chain process mapping, by systematic analysis of material and information flow. · Applying this method in selected case chains, identifying information loss related to traceability, and suggesting improvements. · Testing and evaluating new technology for automatic capture of traceabilityrelated data throughout the food chain; e.g. identifiers and temperature data. The aim of conducting such process mappings is to identify current practices and to suggest possible improvements. It is particularly important to observe where critical traceability information is lost and to correct this.

25.6 Data capture technology ± radio frequency identification data (RFID) tags The aim of using radio frequency identification data (RFID) tags on packaging and carriers is to improve several functions, including: · · · ·

accurate identification of cargo units; speed of data capture; cargo sorting; and recording of historical data throughout the food chain (e.g., food safetyrelated information such as temperature).

Supermarkets Wal-Mart, Metro, Sainsbury's and Marks & Spencer have conducted their own RFID projects and have shown great interest in implementing this new technology in their supply chains. The main focus of this activity is the testing of equipment for automatic identification and data capture (AIDC) using state-of-the-art RF technology. One major goal is to evaluate the technical and economic advantages and disadvantages in the distribution chain for farmed fish compared to the use of bar codes and traditional temperature loggers. The testing includes transfer of data between equipment for data capture and information systems. The SEAFOODplus data capture technology activity consisted of four main parts:

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Initial survey of available technology. Establish functional specifications. Conduct workshops as an arena for sharing experience and discussion. Test RFID tags in realistic environments.

25.6.1 Status and trends Today, the dominant technology used to identify logistic and trade units is the bar code. The use of GS1 (previously EAN-UCC) barcode standards is recommended by the Tracefish standard and by major producers, wholesalers and retailers (for example, in the ECR D-A-CH, ECR France and ECR Spain 2004). However, GS1, government bodies, and major companies in manufacturing and retail are looking at RFID technology to replace or supplement the use of bar codes. The major reasons for looking into this technology are: · · · · · ·

Several RFID tags can be read at the same time. Line of sight is not required for reading. The tags are less exposed to mechanical damage than barcode labels. The tags are smaller than barcode labels. Possible to store more information, and also add/update information. Possible to combine identification with environmental data (e.g., RFID tags with temperature sensors). However, there are some obstacles:

· Costs for tags and readers (expected to decrease with increasing volumes). · Work on international standards is still in progress. · Physical constraints related to reading distance, type of goods (metal, liquids), interference, etc. The technology has been on the market for several years, and products are developed with functionality for a wide range of applications. Naturally, increasing functionality corresponds to a higher price per tag (see Fig. 25.5). The current focus for consumer goods supply chains is on the use of passive tags in the HF and UHF frequency range. The concept of item-level tagging using low cost, read-only tags with a fixed Electronic Product Code (EPC) was developed by the Massachusetts Institute of Technology's Auto-ID Center. Further developments based on the proposals from Auto-ID are now coordinated by EPCGlobal, established by GS1. In supply chain applications, it is common for many tags, in the hundreds, to be in the same area, so anti-collision functionality is important. Tags with writable memory are more expensive, but can be more flexible: · The need for online database access is reduced, because more information can be stored in the tag. · Information can be added to the tag at selected stages in the distribution chain.

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Fig. 25.5 RFID tag categories, functionality and price.

Active tags (battery powered) also have some important applications, especially where higher read ranges are required (for example, tracking of containers in large yards) or where tags are combined with sensors (temperature, pressure, etc.). 25.6.2 International standards A more detailed summary of technology issues and status of relevant standards can be seen in Table 25.1, which shows the topics addressed in the ISO standards most relevant to the use of RFID tags in the distribution chain for fish. In parallel with the work in ISO, considerable effort is being made to develop the technology and standards for simple RFID tags used in the supply chains for consumer goods. This work is driven by large retail chains, their suppliers and leading electronics equipment manufacturers. ECPGlobal was established by GS1 to coordinate this work. An increasing number of specifications are published by EPCGlobal. Some of the most important are: · EPC Tag Data Standard (v 1.3 at time of writing). · Class 1 Generation 2UHF Air Interface Protocol Standard (v 1.0.9 at time of writing). · Low Level Reader Protocol (LLRP) Standard (v. 1.0.1 at time of writing). · Application Level Events (ALE) Standard (v 1.0 at time of writing). The EPC Tag Data Standard gives directions for implementing GS1 barcode standard data elements on read/write RFID tags (for example GTIN ± Global Trade Item Number, SSCC ± Serial Shipping Container Code, etc.).

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Overview of the most relevant ISO standards

ISO Standards

Description

ISO-IEC 15693.1±3

The standards are developed for the use of vicinity cards, but the technology has also been used extensively for logistics applications. The standard defines physical properties, radio communication requirements, anti-collision handling and data transfer protocols.

ISO-IEC 15961 ISO-IEC 15962 ISO-IEC 15963

The standards respectively describe the protocols for transfer and storage of data on RFID tags used for identification of physical items: · The instruction set and the data syntax for exchange of information between an application and the RFID tag. · Data storage formats in the RFID tag memory. · Numbering system for tags with permanent unique ID. The standards are independent of the radio communication interfaces described in the ISO-IEC 18000 series.

ISO-IEC 18000.1 ISO-IEC 18000.2-n

The standards describe the communication interfaces between RFID tag and RF interrogator (reader). 18000.1 describes the functionality and parameters to be defined for each of the frequencies to be used for RFID tags. The standards 18000.2-n describe how the functionality should be implemented for the different frequency ranges. Frequency ranges currently in process: · 18000.2: 135 kHz and below · 18000.3: 13.56 MHz · 18000.4: 2.45 GHz · 18000.5: 5.8 GHz (currently withdrawn) · 18000.6: 860±930 MHz · 18000.7: 433 MHz

The need for common global standards is evident, and EPCGlobal and the ISO SC31 WG4 committees are coordinating standardization efforts. Another challenge for worldwide use of the technology is the use of common radio frequencies and signal strengths. For example, in some parts of the world the UHF frequency range is reserved for other purposes. Work is also in progress to resolve these issues.

25.7

Choice of technology/equipment: introduction

Ideally, several types of equipment should be tested. This would allow comparisons between different solutions for technology and data communication interfaces, as well as measuring the performance of different types of equipment/technology. This should be done incrementally, using a limited number of products for the initial testing to make sure that the technology is suitable for the demanding environments in the fish industry.

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When selecting suppliers for participation, several criteria should be considered. Among these are: · Available RFID technology (for example frequency range). · Relevant experience (including from other industries). · General background in the AIDC industry (including barcode equipment, traditional temperature loggers). · Participation in standardization work and policy on conformation to standards. It is of course essential that the suppliers are willing to commit resources (equipment, personnel) to the project. 25.7.1 Identification of trade/logistic units Barcode technology The GS1 barcode standards will be the basis for comparison with the use of RFID technology. Barcode technology is well proven, and the GS1 standards are well known. The emphasis on barcodes in this project will be more on whether the potential for improving traceability is realized, not technical tests. The use of 2D barcodes is quite limited in supply chain operations compared to traditional barcodes, and will not be considered in this project. Radio frequency identification data technology International standardization efforts and product developments are currently focused on the UHF (860±960 MHz) and HF (13.56 MHz) frequency ranges. Different products have different characteristics, which make them more or less suitable for the demanding environments in the distribution chain for fish. Because of this, it is important to test equipment for both these frequency areas, and ideally also products from several suppliers. When starting RFID technology projects three to four years ago (Seafood Plus, SPINK, IFSAT), we soon discovered that the RFID UHF tag technology was less mature and applicable than expected. In most cases we had problems reading the tag ID in the fish food chain environment. This was due to the especially challenging conditions, including ice, water, and organic materials with high absorption rates for UHF radio signals. However, over the last couple of years, a lot of effort has been put into the development of both international standards and the technology. The ISO 18000.6C (EPC Gen 2) was approved in 2006, and the current tags and readers show very promising results, with read rates approaching 100%. This is due to efforts in several areas, e.g. tag antenna design, non-collision protocols, readers, etc. It is expected that the EPC Gen 2 tags will continue to improve, and with increased focus on developing tags for use in harsh environments, including tags for moulding into plastic crates, pallets and tubs. This means that the EPC Gen 2 will be the preferred standard in the fish value chain. ISO 15963 (HF) tags will still be used for special company internal applications.

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25.8 Testing radio frequency temperature loggers at Fjord Seafood Herùy, Norway The loggers can in theory be identified and the data transferred to a computer using radio frequency (RF) readers. This will be an advantage over current systems, because loggers placed inside cases can be read or monitored at critical points in the distribution chain without opening the pallets or cases. The loggers were placed inside cases with salmon on ice and two different RFID temperature loggers were tested: · Elpro Hamster R loggers, with reader/antenna from Elpro, 868 MHz (www.elpro.com). · KSW Microtec TempSens loggers, with reader/antenna from Scemtec, 13.56 MHz (www.ksw-microtec.de). 25.8.1 Functionality to be tested The basis for the tests was the Tracefish standards, where both temperature check and temperature record are recommended information elements. The time used for data transfer is an important parameter, and the tests included both the readout of current temperature (a single value with a timestamp) and the complete log. 25.8.2 Test procedures The test was done using a pallet of fresh salmon on ice in cases of expanded polyester, with dimensions 800 400 200 mm (L W H). The pallet had nine layers of three cases each, i.e. a total of 27 cases. The net weight of salmon in each case was about 20 kg, and there was about 3 kg of ice in each case. Tag location on pallet and orientation relative to reader antenna Tags were placed at different positions in the box and in the pallet as shown in Fig. 25.6. In an operational environment, it is expected that the current cost levels of loggers will allow for a maximum of one logger on each pallet. Thus, all the tests were done with only one logger on the pallet. Distance from logger to reader antenna The two types of loggers used for these tests have quite different characteristics. The Elpro logger operates on frequency 868 MHz (UHF) and is active. The theoretical maximum distance is stated as 100 metres. The KSW Microtec logger operates on frequency 13.56 MHz (HF) and is passive. The theoretical maximum distance is around 1 metre. 25.8.3 Results Apart from the frequencies, there was one other major technical distinction between the loggers; the Elpro logger used active communication, whereas the

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Fig. 25.6

Tag locations used for testing.

KSW logger was semi-passive. This means that the Elpro logger used internal battery power for communication with the reader, while the KSW logger depended on power generated from the radio waves emitted from the reader. This naturally influenced the reading distances obtained. The ID of the Elpro logger was readable at all box /pallet positions shown in Fig. 25.6. The temperature log was only readable from stationary pallets up to a distance of 20 metres. The KSW TempSens reader had no contact with the tag when the pallet was moving. The logger could only be read when the pallet was stationary in front of the antenna, at a distance of about 20±25 cm. The results are shown in Fig. 25.7. The results suggest that the Elpro logger can be used in real life conditions for monitoring temperatures inside a case of salmon. The reading range of the semi-passive KSW TempSens was too short to be used in an industrial environment.

25.9

Fish chain process studies

The aim of conducting process mappings is to identify current practices and suggest improvements. It is of special interest to notice where critical traceability information is lost, and correct this.

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Test results from testing the Elpro tag at all locations.

25.9.1 Method The objective of process mapping is to analyze material and information flows, and in particular to identify systematic information loss. The overall steps for process mapping are outlined in Fig. 25.8. Companies in a pelagic supply chain in Denmark, a tuna supply chain in Spain and a farmed salmon supply chain in Norway were chosen to be pilot companies for SEAFOODplus. These companies were visited in 2004 and 2005, and the process mapping study was carried out. A walk-through of each company was followed by detailed interviews of the staff. The first step in process mapping was to identify the end product. The method `Analysis of traceability in food supply chains ± Standard method' was used (Olsen, 2008). This method was developed for exactly this type of analysis. The principle and sequence of events is illustrated in Fig. 25.9. When performing process studies to document material and information flow, each of the nine steps can be converted into a recording form to be used in the mapping. Note that steps 2, 4, 6 and 8 deal with the transformation information ± the documentation of what happens exactly at the point and time when the product moves from one context to the next. Steps 1, 3, 5, 7, and 9 deal with durations ±

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Fig. 25.8

Overview of the steps in the process mapping.

what happens or what is the state during transportation, pre-processing, production and packaging of the product. Figure 25.9 shows how to map one product, starting with a form or table where information about transportation to the next link is recorded. As the process mapping moves against the material flow, it is likely that multiple tables or forms will be needed. In particular this is true when moving from mapping the process parameters (step 5) to the application of raw materials and ingredients (step 6). If only one product, process and transportation route is documented, there will be only one set of questions to ask (one form or table) in steps 1, 2, 3, 4, and 5. If multiple raw materials or ingredients are used, then each of these will be documented on a separate form 6, and each separate form 6 will then have to be traced through steps 7, 8 and 9. 25.9.2 Process mapping of farmed Norwegian salmon Material flow and identification A salmon supply chain from breeding to production of salmon fillets in Norway was studied (Fig. 25.10), including production of vitamins and pigment colour and production of salmon feed. The first step in process mapping of this type is to identify the end product. The product chosen to map was salmon fillets.

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Fig. 25.9 Overview of the method in process mapping to analyze the material flow and the information flow.

Fig. 25.10 Overview of the salmon supply chain in Norway. The grey links in the supply chain have been analyzed by using the analysis schemes.

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Producer of vitamins and pigment colour The producer of vitamins and pigment colour was a supplier of vitamins to the producer of salmon feed. The vitamins were based on chemical products. All the steps, including the natural gas supply, were internal in the company. The internal traceability of vitamin production was not evaluated in this study, which instead focused on chain traceability out from the producer of vitamins and pigment colour. The transformation information in the Enterprise Resource Planning system (ERP) between producerID, production batchID and CustomerID, indicated that the producer of vitamins and pigment colour could trace each batch of an article to a defined number of customers. A barcode labelling and reading system was implemented and running. The system was based on EAN 128 code, which is the preferred system for global unique identification. Producer of salmon feed The producer of salmon feed received raw materials from more than 100 different suppliers. The sizes of the received batches varied between a few kilos in a single box of vitamins to several tons in a bulk cargo of fishmeal. The study focused on the methods and systems for receiving raw materials from the producers of vitamins and pigment colour. Traceability between the producer of vitamins and pigment colour and producer of salmon feed was based on manual recordings of identifications and additional traceability information. The identifications used were only partly based on an internationally standardized system. Breeder The breeder produced salmon roe and delivered it to the juvenile salmon producer. This link was not analyzed in this study. Juvenile salmon producer The juvenile salmon producer received salmon roe. Feed, water and oxygen were added to grow the salmon into juveniles, and temperature and light were controlled to optimize the growing conditions. During the production, the original fish groups were split up. The identification of trade units was unique both for reception and dispatch of fish groups. Input factors such as feed were not recorded with unique trade or logistic unit IDs. Traceability of feed was therefore only possible at the level of feed type. For the salmon itself, developing from roe to juvenile, the information loss was not significant. Salmon from one origin/generation was kept separate from other salmon at all stages, from roe to juvenile. The roe could be distributed across numerous cylinders, and the juveniles in many tanks, but the splitting, mixing and joining that happened did not cause significant information loss, as the fish was uniform. There is a concern, however, that relevant information pertaining to the feed could be lost unnecessarily ± if a recall based on feed batch ID occurred, for the juvenile salmon producer to show `No fault' might be problematic.

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Smolt producer The smolt producer received juvenile salmon. Feed and water were added to grow the salmon from juveniles to smolt (ready for salt water), temperature and light were controlled to optimize the growing conditions, and the fish were vaccinated against disease. Salmon smolt were delivered to fish farms either in September/October of the same year as received (0 yearlings) or in April/May the following year (1 yearlings). Traceability of the fish trade units was considered to be good. During the production, the original fish groups were split but not mixed. The ID of trade units were unique both for reception and dispatch of fish groups. The input factor feed was not recorded with unique trade and logistic unit IDs at reception. At consumption, feed name and batch ID were linked to the actual fish groups. Traceability of feed was therefore possible at feed batch level per fish group. The software was however not capable of reporting these references. Because of this, the traceability was not electronic. The input factor vaccine was recorded with unique trade and logistic unit IDs. Traceability of vaccine was possible at the trade and logistic unit level per fish group. Fish farms The fish farms received smolt. Feed was added to grow the salmon from smolt to 4±6 kg salmon. Temperature and light were controlled to optimize the growing conditions, and the fish were chemically treated against lice. Salmon smolt were received at fish farms either in September/October or in April/May. It took 10± 18 months to grow the salmon from smolt to 4±6 kg. Traceability of the fish trade units was considered to be good. During the production, original fish groups were split but not mixed. The ID of trade units was unique both for reception of smolt and dispatch of salmon for harvesting. These IDs were internal, proprietary and were not used as a link by the live fish transporters. Input factors such as feed were not recorded with unique trade or logistic unit IDs. Traceability of feed was therefore only possible for feed type per fish group. Well boat Well boats transported live salmon from the fish farms to the first processor. This link was not analyzed in this study. First processor Live salmon was received from well boats and placed in waiting cages. The salmon in each cage were assigned a production batch ID and processed one cage at a time. Salmon from the cages were pumped one cage at a time to a cooling tank. The production lines were emptied between batches to make sure that batches were not mixed. To keep the salmon calm, CO2 was added to the cooling tank. From the cooling tank, the salmon were pumped to a station for `throat cutting', and then on to a bleeding tank. The salmon were then sent through a grader for sorting by size, and sent to the appropriate gutting line.

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The salmon in each waiting cage were treated as one separate batch. When a new batch was started, the production plant information system assigned a batch ID. The operator chose a supplier (fish farm) from a list, and could also enter the fish farm cage number. The batch number assigned to the salmon in each waiting cage was kept through the production plant, and was printed on both box labels and pallet labels. Transport The transport company transported salmon from the first processor to the second processor. Customer orders were loaded at the second processor according to a freight manifest, printed from the plant IT system. This was a standardized document with basic information about the transport, and each manifest had a unique consignment number. This number was also printed on the document as a barcode. The processing plant printed the customer order number on the freight manifest as a reference between the transport and the customer order. The document was signed by sender, transporter and receiver, each party kept a copy. For each transport order, the transport company assigned a transport order number. This was used as the internal reference in the transport company for tracing the transport. As an external reference, the transport company normally linked the second processor's customer order number to each transport order number. The invoice number was also linked to the transport order number. One transport (transport order) consisted of one or several trips, identified by a unique trip number. The trip numbers were linked to the transport order number. The information stored for each trip was origin and destination, date/time of start and arrival, truck registration number, etc. For international transports, the transport company issued an international freight manifest. In addition to the name of the receiver, the reference to the second processor's customer order on this document was the transport company transport order number. When the transport company stored salmon on the terminals, the transport company kept track of pallets/boxes by assigning a physical area for each client. The location of individual pallets/boxes was not managed by a warehouse management system (WMS). When a sale was made, the transport company received an order with a picking list with reference to individual boxes. Second processor Fresh salmon were received from the first processor in 20 kg styrofoam boxes on pallets. The transport was made by refrigerated trucks. The outgoing products could vary between a few kilos to several tons of smoked salmon in 10 kg styrofoam boxes. The level of external traceability was poor, as traceability links were missed at both ends of the internal chain ± the reception of raw materials and the shipment of outgoing products. When raw materials were received, the box or pallet labels were not scanned. Although some data from the box labels were entered manually onto paper forms, nothing that could be used as unique backward links (to the slaughtering plant or to the transporter) was entered.

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The situation was similar at shipment. Production lot numbers were stamped on the boxes using ink-stampers, but the numbers were not globally, nor even internally, unique. The boxes received printed labels (Marel system) as well, but the labels only identified customer and transporter at a generic level (name only), and did not include the production lot number (as this was stamped on the box). Conclusions and recommendations In general, traceability was good along the production chain. Internal traceability was acceptable at all the links. The methods and systems of chain traceability were, however, more insecure and incomplete. Based on the findings, general recommendations for reengineering each link in the salmon supply chain were made. These guidelines were presented as a separate report (ForaÊs et al., 2006). The Norwegian salmon farming industry saw substantial production processing reengineering during the period from 2002 to 2004. This reengineering has led to an improved granularity of traceability. But there are still multiple challenges to optimal chain traceability. Further efforts should focus on implementing globally unique IDs and an improved solution for live fish carriers.

25.10

Bibliography

(2003), `Traceability of fishery products ± Specification of the information to be recorded in farmed fish distribution chains', Tracefish Workshop Agreement (CWA). TM EAN-UCC (2002),'Global Tag GTAG Technical Specification Draft V0.2', August 2002. EAN-UCC (2002), `Traceability of fish guidelines', November 7, 2002. ECR D-A-CH, ECR FRANCE and ECR SPAIN (2004), `Using traceability in the supply chain to meet consumer safety expectations', ECR (Efficient Consumer Response) Europe. EPCGLOBAL (2007) Documents available at www.epcglobalinc.org FINKENZELLER K (2003), RFID Handbook Fundamentals and Applications in Contactless Smart Cards and Identification, Second Edition. Ê S E, STORéY J and PETTER O (2004), `Kjedesporbarhet innen fiskeri og FORA havbruksnñringen (STF80 A044068)', SINTEF Fisheries and Aquaculture. Ê S E, FREMME K and SENNESET G (2006), `TELOP Trace (Project report March 2006)', FORA Internal project report, TraceTracker. HARTMANN C S (2002), `A Global SAW ID Tag with Large Data Capacity', in Proceedings of 2002 IEEE Ultrasonics Symposium, Munich, Germany, October 2002. HARTMANN C S, BROWN P and BELLAMY J (2004), `Design of Global SAW RFID Tag Devices', Second International Symposium on Acoustic Wave Devices For Future Mobile Communication Systems, Chiba University, Japan, March 2004. ISO 8402 (1994), `Quality Management and Quality Assurance ± Vocabulary', International Organization for Standardization, 1 April 1994. KARLSEN K M, OLSEN P and STORéY J (2006), `TraceFish basert innfùring av sporbarhet i CEN

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norsk fiskerinñring', Fiskeriforskning report 11/2006, ISBN 978-82-7251-585-9. (2008), `Analysis of traceability in food supply chains ± Standard Method'. Journal of Supply Chain Management (submitted). Ê S E and OLSEN P (2006), Recommendations for Good Traceability Practice STORéY J, FORA in the food industry, Version 1.0 2007.04.20, Report SFH80 A074016, ISBN 97882-14-04161-3. OLSEN P

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26 Validation of traceability in the seafood production chain B. PeÂrez-Villarreal, F. AmaÂrita, C. Bald, M. A. Pardo and I. Sagardia, AZTI-Tecnalia, Spain

26.1

Introduction

Ensuring traceability throughout the whole fishery chain is a great challenge for all the agents involved. Team work and collaboration is nowadays necessary as each link of the fish value chain must contribute its own traceability plan and facilitate at the same time adequate and agile information exchange between chain partners so that other links can fulfil their specific traceability requirements. The importance of ensuring traceability is even more evident if it is understood that traceability is not only an important tool in incidents affecting safety, but also plays a role in problems affecting quality and is in addition a method for guaranteeing the reliability of commercial transactions, in other words, preventing fraud. That is why a traceability system, beyond its fundamental requirement of protecting consumers, will also help to maintain the image and prestige of the companies. All the information that accompanies a fish product through the whole chain, until it reaches the consumer, acts as its curriculum vitae. It should not only contain the mandatory and necessary data to identify the fish product, but also enough information to reconstruct its history at any point along the production and commercialization chain. It should be noted that identification and traceability are concepts with different natures and objectives. While traceability is a tool intended for tracking purposes that may be useful in ensuring, in first instance, food safety, the purpose of labelling is to provide the consumer with the information necessary for making purchasing decisions. A good traceability system must first be efficient. In case of an incident, it has to be able to provide all the registered information about a particular product in

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an adequate time for corrective and preventive measures to be established promptly. General rules state not only that food companies must ensure that their products satisfy the requirements of food law but, furthermore, that they shall also verify that these requirements are met (EC Regulation No 178/2002 Art. 17). This means that food companies have to be able to demonstrate that their traceability system is working with efficacy and that all the information it provides is truthful and error-free. In other words, food companies must show that their system is reliable. The way they can accomplish this task is closely linked to other mandatory requirements laid down by specific legislation like the regulation that states the general principles of HACCP based quality assurance systems (EC Regulation No 852/2004 Art. 5). Every food company should therefore define, document and put into practice a protocol for the verification of its traceability plan. They should measure systematically and periodically not only its efficiency but also its reliability. Checking for reliability entails verifying through different methods that the information contained in the `curriculum vitae' of a product is true and serves its purpose. The procedures defined with this objective are known as validation methods. In the first part of this chapter the basic principles for validation of traceability are explained. A methodology for validation of traceable information through the definition of suitable indicators of reliability is also proposed and some examples are given. Finally, this chapter presents an overview of the available and most suitable methodologies or tools for validation of traceable information related with seafood safety, quality, fraud and data management, which aim to serve as a starting point for anyone who is searching for an approach to validate traceability throughout the fishery chain.

26.2

Principles for the validation of traceability

Despite existing legislation, there is no general provision that defines exactly how a traceability system should be designed or implemented in food companies. Food companies have to be able to demonstrate, though, that the implemented system is functioning as it has been defined and also that it serves its final purpose. In other words, they have, to verify that the system is not only efficient but also effective. For the purposes of definition, conformity in the context of validation of traceability means that the records demonstrate that the activities are carried out according the established traceability procedures and the control parameters are under the established limits. Compliance means that the system meets the regulatory requirements. Standardization may play a critical role, not only for control but also for the purposes of information interchange. A number of initiatives have been undertaken to propose different voluntary standards of reference for traceability implementation in the food industry resulting in the publication of sector

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specific guides (Derrick and Dillon, 2004) as well as generic and sector specific Good Traceability Practices and Standards of language for interchanging electronically traceable information (TracecoreÕ XML, Tracefood framework). A food company must verify that its traceability system conforms with the established standards. In order for the control tasks to be considered effective, they should, at least, cover the following aspects: · A study and evaluation of the documents corresponding to: ± Definition of the lots and of the records that allow the product to be traced, including the retention period of those records. ± Documented results from the verification of the traceability system, including the reaction speed. · Verification through auditing of the system that it complies with the standards established. · Execution of a simulation tracing exercise within the company. · Validation of the reliability of the traced information. The competencies of the official control authorities have been defined in EC Regulation No 882/2004. They must verify that there exists documented evidence of all the necessary aspects and that the design of the traceability system established by the company as well as its implementation comply with the objectives pursued through the regulations. However, there are no given specific rules or guidelines for how companies should accomplish this. The way to achieve the above is through the establishment of validation procedures. Control authorities should verify that there is documentary evidence of the implementation of validation procedures. It is advisable that this evidence includes the following: · General description of the validation procedure. · Details of person/s who carries/carry out the validation of the system, not only internal staff, but also, if applicable, external company staff. · Periodicity of the tasks. · Records of the results obtained in each validation (including the reaction time in delivering the information). · Records of incidents (direct and indirect), correcting measures and verification of their efficiency. It is important to differentiate verification from validation procedures. By verifying traceability, the food company states if all traceable information is available, if `they are doing things right', as previously stated in the established standard. By validating, the food company goes a step further and checks for the efficacy of all this information, in other words, they check `if they are doing the right things'. Through validation the food operator evaluates whether the traceable information meets the requirements of the regulations and protects the interests and expectations of the consumers providing enough information to facilitate their choice (Regulation EC No 178/2002, Art. 8).

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Validation provides evidence to support the traceability plan. With this purpose, it will demonstrate that the traceable data registered and obtained through the control tasks are relevant for answering the questions posed about the safety, quality or truthfulness of the product and that the level of uncertainty of the conclusion is known and sufficiently small for the intended purpose. To achieve these objectives, validation has to be targeted to ensure that: · The definition of relevant traceable information is supported by scientific and technological knowledge. · The reliability of the information provided by the records of the traceability system is being validated by suitable testing methods. The demonstration of the reliability of the traceable data is not sufficient, though, for validating traceability. When an incident that may affect the safety of the consumer occurs, the system has to guarantee the correct response in an adequate timescale. A minimum level of information must be available for the competent authorities upon immediate request for product recall purposes. Therefore, it is also necessary to ensure the adequate and reliable flow of information throughout the chain.

26.3 Establishment of indicators for the validation of a traceability system When implementing traceability in the seafood chain, an important step is to identify the records in which the information about the product is registered. The way to demonstrate traceability relating to a specific product is through the verification of the information contained in these records. Therefore, for verification and validation purposes, it is necessary, first to define the information that should be registered and checked as a verification parameter. Once the parameters have been defined, the aim of the verification tasks will be to ensure that the traceability system works, that the records are completed, that the good practices of traceability are respected and that there are no information gaps or lack of information. The indicators of efficiency are the records that demonstrate that these parameters are being controlled. The purpose of validation is to ensure that the information given by the records is reliable. The system shall define the suitable indicators that will tell the food operator and the competent authorities that the control parameters are really within the control limits with a known acceptable uncertainty. In order to define a map of indicators for verification and validation purposes, a useful way may be to classify the information with respect to the objective to be accomplished. This means to identify control parameters and their indicators of efficiency and reliability in relation to safety and quality assurance, fraud prevention and for correct data management vs. information flow. For each parameter, suitable indicators of efficiency and reliability should be defined (Fig. 26.1) as in the example given in Table 26.1. These indicators of reliability

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Fig. 26.1

Map of verification parameters and their allocation by means of the criteria or aspect they target. For each parameter indicators of efficiency and reliability shall be defined.

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Table 26.1 Indicators of efficiency and reliability for verification parameters related to safety of fishery products Verification parameter

Efficiency indicator

Reliability indicator

Species fraud

Catch area/ Certificate of origin

What

How

Species identification

Morphological characterization

Records of product identification Origin fraud

Catch area/ Certificate of origin Records of product identification

Molecular techniques (DNA/proteins) Geographical Genetic analysis origin/production method Analysis of the protein/enzyme profile in certain organisms Analysis of oil and grease components (lipid profile and structure) Analysis of stable isotopes Analysis of trace elements

Product presentation Records of product (over-glazing, identification frozen-thawed vs fresh) a

Product presentation

Physical analyses (NMRa) Physiological analyses Enzymatic analyses

Nuclear Magnetic Resonance

should be evaluated or measured with the adequate methodologies based on official methods of reference where defined. If alternative methods are used (e.g., rapid routine tests, online methods, kits, sensors), they should be validated or contrasted with reference analytical methods. There are no reference guides that define or propose the adequate verification parameters and their indicators. To elaborate and propose a practical guide of reference for verification and validation of traceability was therefore one of the aims of VALID, a subproject of SEAFOODplus. The guide is available online at http://www.azti.es/valid/. It maps indicators of efficiency and reliability of traceable information within each link of the fishery chain. The links considered are those defined in CEN 14660 standard (CEN 2003b) for the captured fish chain: fishing vessels, vessel landing businesses, auction markets, processors, transporters, stores, traders, wholesalers, retailers and caterers. The guide also gives references to the validated official methods and other methods proposed

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for testing each indicator, chosen because of their reliability and convenience for routine control in the chain.

26.4 Specific tools for validation of the most important traceable data When choosing the adequate methodology for testing purposes, some aspects have to be taken into account: · Specificity for the parameter to be measured and selectivity to avoid interference. · Accuracy and precision. · Repeatability and reproducibility. · Robustness. This means the ability to give the same result when the operating conditions change. The continuous advances in food technology and other convergent technologies, the diversification of products and the increasing complexity of the fish chain, have motivated the development of new methodologies. These have been implemented where official methods are not available or where official methods can be made more rapid, specific and/or easy to use. The validation of these new methods should be considered to be of primary importance. Methods of validation can be divided into the same categories as the traceable data that those methodologies or tools are intended to evaluate; i.e. they can be divided into the following categories: · Methods to ensure the safety of the product. · Methods to ensure that the expected quality is provided to customers and consumers. · Methods to prevent fraud in the seafood chain. · Methods to ensure correct data management and information flow.

26.5

Methods for validation of traceability to ensure safety

Traceability system records should give valuable information about the status of the control parameters defined in every link of the chain in order to recover information about the history of a product or a batch when a problem related with safety is detected or even when it is suspected to represent a threat to consumer health. Seafood safety hazards are essentially physical, chemical or biological. Biological risks may be caused by naturally occurring organisms but physical and chemical risks are mainly derived from human activity. The methodologies described below focus on the former. Biological risks arise from the activity of micro-organisms, the side-products of their metabolism or the toxins they may

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produce and from some associated organisms like parasites. Therefore, the most suitable indicators of reliability are the presence of: · · · ·

Histamine and biogenic amines. Biotoxins. Pathogens (bacteria and viruses). Parasites.

26.5.1 Histamine and biogenic amines Histamine (or scombrotoxin), putrescine, cadaverine, tyramine and agmatine are biogenic amines produced from the decarboxylation of histidine, ornithine, lysine tyrosine and arginine respectively by decarboxylase enzymes from some bacteria present in spoiling fish. Histamine fish poisoning (HFP) is a mild foodborne disease, but is important in relation to food safety and international trade. Issues such as the incidence of HFP among consumers, the fish species affected, toxicological aspects, current legislation, the importance of the different groups of bacteria, specially psychrotolerant species, the influence of process conditions in the formation of histamine, etc., are treated in detail in Chapter 15. Methods for detection of histamine UE regulation 2073/2005 establishes HPLC as the reference method. Fluorimetric techniques have been developed for an accurate measurement of histamine and since these new techniques have evolved, new validation procedures were tested. The chromatographic techniques, such as gas chromatography (GC), high performance liquid chromatography (HPLC) (German reference method 1999), high performance thin layer chromatography (HPTLC), as well as capillary electrophoresis now allow the simultaneous analysis of histamine and other biogenic amines in fish and fishery products (Jeya Shakila et al., 2001, Male and Luong, 2001). For quality control purposes, it is advisable to use a rapid method for screening, even if it is semi-quantitative. Some immuno-enzymatic kits have been commercialized (Rogers and Starustzkiewicz, 2000) and other techniques like colorimetric and thin layer chromatography (TLC) may be employed (Shalaby, 1999). 26.5.2 Biotoxins in seafood There are five different syndromes known to be caused in humans through the consumption of contaminated seafood products. Apart from ciguatera fish poisoning (CFP), which occurs after the ingestion of tropical and subtropical reef species (e.g. grouper, moray, barracuda, mackerel and tiger fish), vector organisms for intoxications are filtering species, mainly bivalve molluscs. Marine biotoxins are relatively heat stable, are not destroyed by either cooking or freezing and do not alter the taste or odour of the food. The toxins causing these syndromes, the micro-organisms that produce them and their geographical distribution are summarized in Table 26.2.

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Table 26.2 Main syndromes caused by marine biotoxins, micro-organisms responsible of their production and their geographical distribution (adapted from AZTI, 2002, internal report on marine biotoxins) Syndrome

Toxins

Responsible micro-organism

Geographic distribution

DSP, diarrhoetic shellfish poisoning

Okadaic acid Dinofisistoxins Pectenotoxins Yessotoxins

Dinophysis spp.a Prorocentrum spp.a

Japan and Europe

Azaspiracids

Gonyaulax polyedraa Protoceratium reticulatuma (Unknown)

PSP, paralytic shellfish poisoning

Saxitoxin Neosaxitoxin Goniautoxins

Alexandrium minutua A. tamarensea Gymnodinium catenatuma Pyrodinium bahamensea

North-east USA, Asia, South America and Europe

ASP, amnesic shellfish poisoning

Domoic acid

Pseudo-nitzschiab

Canada, North-east USA and Northern Sea

NSP, neurotoxic shellfish poisoning

Brevetoxins

Gymnodinium brevea

Florida, Mexico and New Zealand

CFP, ciguatera fish poisoning

Ciguatoxins Maitotoxins

Gambierdiscus toxicusc

Tropical and subtropical waters

a

Dynoflagellates.

b

Diatoms. c Macro algues

EC regulation related to marine biotoxins The Council Directive 91/492/EEC states the maximum permitted levels (MPL) of domoic acid (DA) and PSP toxins. The Commission Decision 2002/226/EC derogates the former and states additional MPL under restricted conditions specifically for Pecten maximus and Pecten jacobaeus species. The limits for Paralytic shellfish poison (PSP), Amnesic shellfish poison (ASP) and lipophilic toxins of the diarrhoetic shellfish poisoning (DSP) complex (okadaic acid and dinophysistoxins), yessotoxins, pectenotoxins and azaspiracids are laid down in Regulation (EC) No 853/2004. Routine methods for quality control of biotoxins For DA, protocols such as receptor binding assays, immunoassays and thin layer chromatography can be used for routine monitoring. The liquid chromatography-mass spectrometry method allows confirmation of the presence of DA isomers.

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The AOAC standardized mouse bioassay (MBA) is widely used as a routine monitoring method in most countries (Botana, 2000) but its lack of specificity and the ethical issues, relating to the use of live animals for experimentation, mean that the use of this method is being questioned in some countries. Commission Decision 2002/225/EC states that techniques such as receptor binding assay (in vitro bioassay), immunoassays (ELISA-based commercial test kits) and functional assays like the phosphatase inhibition assay and postcolumn-derivatization liquid chromatography can be used as alternative methods for routine monitoring. Other methods such as high-performance capillary electrophoresis (HPCE) have been studied as an alternative to HPLC widely used as for the analysis of yessotoxins (de la Iglesia et al., 2007). The `pre-column oxidation method' described by Lawrence et al., (2004), a method which is much simpler in terms of instrumentation, is approved as the European norm by CEN and by AOAC as the official method of analysis (CEN 2002). It was documented by the working group on biotoxins of the European Committee for Standardization (CEN) and approved as European Norm EN 14526 (CEN, 2004). Reference validation methods for analysis of biotoxins The European Commission Regulation only stipulates that for DA the reference method shall be an instrumental method, HPLC. Liquid chromatography linked to UV detection is currently the preferred analytical technique for the determination of DA in shellfish. The Quillian method, a LC-UV detection method variant, is recommended by Codex Alimentarius as reference method. This method has been approved as European Norm EN 14176 (CEN, 2003a) and is recommended by the Community Reference Laboratory on Marine Biotoxins (CRLMB). In the EU, according to Commission Regulation (EC) No 2074/2005, MBA remains the reference method only when the results of the analyses performed show discrepancies between the different methods or if the results are challenged. In addition to biological testing methods, alternative detection methods, such as chemical methods and in vitro assays, are allowed if it is demonstrated that the performance of the chosen methods is at least as effective as the biological method and that their implementation provides an equivalent level of public health protection. At the moment, certified standards for some toxins are commercially available from National Research Council Halifax, Canada. Reference material for calibration is distributed by the Centre for Food Safety and Applied Nutrition, FDA, USA and by the Joint Research Centre ± Institute for Reference Materials and Measurements (JRC/IRMM), Geel, Belgium. 26.5.3 Methods for microbiological safety assessment of fishery products Fish bacterial flora is composed mainly of psychrotrophic bacteria, microorganisms that grow well at refrigerating temperatures. The most frequently

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found food-borne pathogens are Vibrio spp., Clostridium botulinum and Listeria monocytogenes. All other pathogenic micro-organisms have their origin in human activity, Salmonella spp. being the most important. The range of seafood associated pathogens, the illnesses they cause, their incidence, some ecological aspects, references to published regulations and a review of the conventional and new molecular biology based methods for pathogen detection, identification and typing are treated in detail in Chapter 14. Parameters for microbiological control of seafood Analysis of coliforms is used as an indicator of the sanitary conditions during food processing. Faecal streptococci analysis may be employed as a good indicator of hygiene and appropriate handling of frozen food. In the same way Staphylococcus aureus can be used as an index of inappropriate handling, as it is a well-defined inhabitant of human skin, hair and nose. Clostridia presence is an indicator of bad processing conditions thus, evidences a deficient system of control of time versus temperature in food processing. For E. coli and coliform bacterial counts, the Most Probable NumberConfirmed test for coliforms described by the US Food and Drug Administration, specified in ISO 16649-3 and in Commission Regulation (EC) No 2074/ 2005 Annex VIII, may be used as reference and validation method (Feng et al., 1998). Some validated methods available are: `Enumeration of Escherichia coli in raw molluscs by the multiple tube technique' (Health Protection Agency, 2004a) or `Direct Enumeration of E.coli' (Health Protection Agency, 2004b), both from the National Standard Method of the Health Protection Agency (HPA), UK. At present, several Salmonella and Shigella rapid detection methods are available. Listeria monocytogenes may be analyzed by one of the several methods offered by culture media producers, as RAPID'L.Mono from Bio-Rad In both cases, the methods described by the ISO standards or USFDA are those of choice for validation purposes (see Chapter 14). Bivalves are a special case because of their special food intake method: they are filtering organisms that concentrate bacteria, viruses and/or particles present in their environment. After harvesting it is good practice to depurate bivalves in running water for several days before they entering the commercial chain, depending on the quality of water they were grown in. Once again analysis of coliforms is mandatory, analysis of Escherichia coli, Salmonella spp. And Shigella spp. must be done, too, due to the risk of faecal contamination. Bivalves that live under low oxygen conditions might be analyzed for Clostridium botulinum because of the risk of the presence of botulinic toxin in the substrate. Since it is an emerging pathogen, analysis of Listeria monocytogenes might also be performed. Vibrio spp. has become an increasingly serious problem in seafood derived from warm waters and the discrimination of the forms that cause illness from those non pathogenic presents a special challenge for the new techniques (see Chapter 14).

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26.5.4 Viruses Hepatitis A virus (HAV) is the leading cause of acute viral hepatitis throughout the world and noroviruses (NoV) are the most commonly identified cause of outbreaks and sporadic cases of acute gastroenteritis (see Chapter 13). Molluscs, as filtering organisms are likely to act as vectors of transmission. Routine analysis of microbial indicators of faecal contamination is not a reliable indicator of viral contamination as indicators of faecal contamination are not representative of viral contamination levels. A dramatic improvement in diagnostic virology comes from the emergence of real-time-PCR (RT-PCR). The objective of project REFHEPA within SEAFOODplus Integrated Programme has been the development of standardized RT-PCR assays for the detection of NAH and NoV in molluscan bivalves. Quality control methods such as standards and internal controls have been implemented enabling its inclusion in regulatory standards (see Chapter 12). 26.5.5 Parasites Some parasites, such as as Anisakis spp., a nematode with worldwide distribution, that infects consumers of raw or under-cooked fish, have recently been a cause of public concern. Regulations (EC) 853/2004 and 854/2004 set out the requirements governing parasite checks during handling of fishery products on shore and on board vessels. The only reference procedure for detection is visual inspection. This means `nondestructive examination of fish or fishery products with or without optical means of magnifying and under good light conditions for human vision, including, if necessary, candling' (detailed rules given in Regulation EC 2074/2005). There is a lack of fast and reliable techniques for identification of parasites in fish products, but the development of molecular biology methods is very promising for the near future. A DNA-based method (RT-PCR) has recently been developed and reported on by LoÂpez and Pardo (2006) for detection of Anisakis simplex and its allergens, and assayed with success in different highly processed fish products.

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Validation of traceability for quality concerns

Taking into account the biochemical-biological origin of the spoilage process, control measures should focus on controlling the three main factors that accelerate spoilage: · Physical damage (bruises, tears, squeezing) that makes tissues more accessible and vulnerable to the enzymes and bacteria present. Physical damage may begin from the moment of the capture. · Temperature is known to determine the speed of all chemical reactions and the activity of micro-organisms. · The degree of contamination.

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A seafood product is considered `fresh' when its global properties are near those corresponding to living fish. `Freshness' is a complex concept that clusters a number of attributes: a combination of sensory, biochemical, physical and microbiological parameters. Therefore, validation methodologies should be designed to measure and to evaluate the efficacy of the control measures implemented by measuring the degree of seafood spoilage through: · Sensory evaluation of freshness. · Analysis of biochemical indices of spoilage. · Microbiological analysis of specific spoilage micro-organisms (SSO). 26.6.1 Sensory evaluation of freshness During the last 50 years many schemes have been developed for sensory analysis of raw and cooked fish. The first modern and detailed method was developed in the Torry Research Station (Shewan et al., 1953). The method proposed involved observation and description of the changes in each sensory property over time. The changes are then arranged to form scales to which numerical scores are assigned. Freshness scales can be related to storage time and linear regressions can be constructed for prediction of days in ice or freshness scores. The most frequently used method for evaluating the quality of fresh fish in the European trade and inspection is that of Regulation 2406/96 of 26 November 1996 laying down common marketing standards for certain fishery products. This method establishes three degrees of freshness: E, A and B, which correspond to the different stages of spoilage. E (Extra) is the highest possible quality, while below B is the level where fish is considered unfit for human consumption. A multilingual guide to EU freshness grades for fishery products is available (Howgate et al., 1992). However, this method is to some extent limited as it does not take into account the important differences between species. Other evaluation methods have been developed for the sensory evaluation of seafood. The Quality Index Method (QIM) was originally developed by the Tasmanian Food Research unit (Bremner, 1985) and introduced in Europe 15 years ago (Larsen et al., 1992). Changes in each of the important sensory parameters are quantified using demerit scales of 0±1, 0±2 or 0±3 points. The sum of the scores for all parameters gives an overall sensory score named the Quality Index. QIM schemes have been developed for several species: Andrade et al., 1997; Huidobro et al., 2000; Martinsdottir et al., 2001; Baixas-Nogueras et al., 2003; Herrero et al., 2003; Barbosa. and Vaz-Pires, 2004; Vaz-Pires. and Seixas, 2006; È zogul et al., 2006; Cardenas et al., 2007; Pons-SaÂnchez-Cascado et al., 2006; O PeÂrez-Villarreal et al., 2007. Additionally, different organizations in Europe have developed guides and manuals including photos that illustrate the changes in freshness that occur in the fish species most important around their fishing area: BIM in Ireland, OFIMER

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in France, AZTI in Spain and QIM Eurofish. The guide developed by AZTITechalia, is available at www.azti.es and presents a number of QIM and Torry's schemes for several species of commercial interest in the south of Europe. Further development of sensory schemes are needed, especially for fish species processed in different conditions, such as filleted products and those further preserved in modified atmosphere packaging, vacuum, freezing, slurry ice, etc. The use of sensory analysis to evaluate how process conditions affect fish quality all along the fishery chain and the cross linking of this information with the consumer's perceptions and preferences has been proposed (see Chapter 4). The correlation between QI and other classical and novel analytical techniques for fish quality assessment is also of increasing interest (OlafsdoÂttir et al., 2004). 26.6.2 Volatile amines Volatile amines are the characteristic molecules responsible for the fishy odour and flavour present in fish several days after they have been caught and they are commonly used as criteria for assessing fish quality. By `volatile amines' are understood largely three molecules: ammonia, dimethylamine (DMA) and trimethylamine (TMA). DMA and TMA result from the degradation of trimethylamine oxide (TMAO), a fish typical molecule which has an important role in osmoregulation, DMA is mostly produced by endogenous enzymes and TMA by bacterial enzymes. Ammonia is present in freshly caught fish. During chilled storage it is formed by endogenous and bacterial enzymes; it is a poor indicator of fish freshness and cannot be considered an effective marker of fish spoilage. However, it is considered a good indicator of quality in cephalopods. DMA is present at very low concentration in freshly caught fish, about 0.2 mg/100 g. It is formed from TMAO, under TMAO-ase action, an endogenous enzyme which is mainly present in gadoids. Total Volatile Basic Nitrogen (TVB-N) or Total Volatile Bases (TVB) represents the sum of ammonia, DMA, TMA and other basic volatile nitrogenous compounds under the analysis conditions. In freshly caught fish its content is generally higher than 10 mg/100 g and does not exceed 15 mg/100 g except for in pelagic fish. DMA-N can be considered as a marker for fish freshness of some white fish species containing TMAO-ase enzyme and it can be used to monitor the quality of frozen-stored gadoid fish. In gadoids, it accompanies the formation of formaldehyde, which causes denaturation of muscle proteins, making DMA a good indicator of frozen fish quality. TVB-N is an indicator of spoilage of some fish species such as red fish, flat fish, gadoids, hake and Atlantic salmon. TMAN is an indicator of spoilage due to specific bacteria activity. It does not reflect the earlier stages of spoilage, and its level increases rapidly once bacterial growth after lag phase has begun (PeÂrez-Villarreal and Howgate, 1987; PeÂrezVillarreal and Pozo, 1990).

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When using volatile amines as indicators of fish spoilage, it must be taken into account that volatile amine production is linked to the initial concentration of TMAO in the muscle which depends on the species, season and fishing ground (Oehlenschlaeger, 1997). Also processing conditions and preservation methods may have a significant influence on their presence, and results have to be interpreted carefully. It is known, for example, that volatile amines are watersoluble compounds that may be washed away when processing of seafood involves washing steps. Their concentration also depends on the size of fish and the type of ice in which it is being kept. Proper filleting of fish also prevents volatile amine formation to some extent as it eliminates bacterial load coming from those parts of fish where the flora is present like viscera and skin. EU regulation for levels of volatile amines The Commission Regulation (EC) No 2074/2005 specifies in its Annex II the species categories for which TVB-N limit values are fixed and the reference method of analysis to be used in the event of dispute. Analytical reference methods for volatile amine determination The analytical methods may be classified as specific methods, which provide results for a single substance, and multi-parameter methods, which allow simultaneous determination of DMA, TMA and TVB. For TVB-N, Commission Regulation (EC) No 2074/2005 in its Annex II specifies the reference analytical method to be used. Although accurate, this method is tedious and time consuming. Regulation also proposes three methods that may be used for routine analysis: the micro-diffusion method described by Conway and Byrne (1933), the direct distillation method (Antonacopoulos, 1968) and the distillation of an extract deproteinized by trichloroacetic acid proposed as reference method by the Codex Alimentarius Committee on Fish and Fishery Products in 1968 (FAO/WHO, 1968; Malle and Tao, 1987). For TMA-N determination the current reference method AOAC (1995) is based on the reaction of TMA with picric acid to form a coloured complex. Methods for routine assessment of volatile amines To date many analytical methods have been developed for quantitative measurements of TVB-N and TMA-N concentrations. Among all these methods, steam distillation and the colorimetric method of Dyer (1945) are the most known and widely used procedures for TVB-N and TMA-N determination. Some novel analytical methods have been evaluated for TMA and TVB such as flow injection/gas diffusion (FIGD) (Baixas-Nogueras et al., 2001; Dhaouadi et al., 2007). For TVB, the AOAC Official method 999-01, `Volatile bases in fish, ammonia ion selective electrode method', which is more rapid and easier to perform than the reference method, could be compared to the EU official procedure to facilitate quality control in the chain (AOAC, 2002). For TMA-N, specific sensors and probes recently developed (Pacquit et al., 2006) and others being developed in European projects (Barranco, 2007), must

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be validated to assess the possibility of their use as quality control tools for the fish industry. 26.6.3 Micro-organisms responsible for spoilage The bacterial flora present in a fish product is dependent on the environment rather than the fish species. Species from warmer environments have a more abundant flora than those from colder and cleaner waters. Most raw seafood spoilage micro-organisms are are of a limited number of genera, mostly Gram-negative psychrotrophic ones. Acinetobacter spp., Flavobacterium spp., Moraxella spp. Photobacterium phosphoreum, Pseudomonas spp. or Shewanella putrefaciens are some of them. The development of offodours and off-flavours derived from bacterial metabolism is evidence of microbial spoilage. The off-odours and off-flavours produced are mainly several biogenic and volatile amines, as previously described, and/or hydrogen sulphide. These compounds contribute to the development of the fishy odour characteristic of spoiled seafood. Other compounds such as aldehydes, ketones or even organic acids can help the development of the spoilage characteristics. Legislation in European countries requests microbiological analysis of mesophilic aerobes (Commission Decision 1993/51/EEC; Commission recommendation 2001/337/EC), but since natural seafood flora is usually composed of psychrotrophic bacteria and since seafood is commercially refrigerated, mesophilic aerobic plate counts (MAPC) are not considered as good indicators of the micro-flora present. A specific spoilage micro-organism plate count (SSMPC) can be a better indicator of food quality. Depending on the nature of the bacteria under analysis, these micro-organisms might be evaluated by growth at 20 ëC for up to four days (for bacteria of cold water seafood) or growth at 30 ëC for up to two days (for bacteria of warm water seafood). Raw and lightly preserved seafood is often distributed under vacuum or in modified atmosphere packaging. Under such conditions growth of the common seafood flora is usually slowed down and growth of more adapted microorganisms takes place. Thus lactic acid bacteria (LAB) and related microorganisms can colonize seafood. Although faecal streptococci may be associated with faecal contamination their presence in food is not always due to contamination with waste water. These micro-organisms are frost-resistant and are often associated with inappropriate handling. Faecal streptococci analysis may be employed as a good indicator for hygiene in frozen food.

26.7

Validation methodologies to prevent fraud

The main objective for the labelling of fishery products is to facilitate consumer choice by providing sufficient and relevant information about the product. From

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the point of view of traceability, all this information should accompany the product and be available at any point in the production and commercialization chain. Providing information that the consumer considers relevant is key. Essentially information should be displayed that makes consumers confident that the product will satisfy their expectations. This becomes more evident when the information provided not only affects consumers' choice but also determines the price they are willing to pay. EU regulations try to respond to these needs by laying down rules that will guarantee provision of information for consumers. With this aim, Commission Regulation (EC) No 2065/2001 states in Article 8 about traceability and control that the information required concerning the commercial designation, the production method and the catch area shall be available at each stage of marketing of the species concerned. This information together with the scientific name of the species concerned shall be provided by means of the labelling or packaging of the product, or by means of a commercial document accompanying the goods, including the invoice. Food operators and control authorities must put into practice control measures to ensure that the information is provided and it is not misleading. There are no provisions, though, about how to implement adequate measures and the methods for verifying the reliability of the traceable data. 26.7.1 Methods for authentication of fish species For many years, methodologies to guarantee fish identification were strongly linked to the morphological characteristics of the whole fish according to certain taxonomic keys. These taxonomic keys are included in several species catalogues compiled by Food and Agriculture Organization of the United Nations (FAO). However, when the external morphological characteristics of fish are removed, there is no reliable and objective methodology based on morphological characteristics. To deal with this problem several molecular techniques have been developed. These techniques are based on the analysis of biomolecules (molecular markers) that are unique to the species. The analysis of a specific genetic characteristic (polymorphism) present within a molecular marker is therefore a viable method that can be used to draw distinctions between various species of fish. Protein analysis techniques are based on the physicochemical differences in size, net charge and aminoacidic composition. These differences can reveal several protein polymorphisms which are useful for fish identification. The way to analyze these polymorphisms is through a great variety of biochemical techniques including electrophoretic, chromatographic and immunological methodologies (Rehbein et al., 1995; Mackie, 1996; Chen et al., 1992; Scobbie and Mackie, 1988; Sotelo et al., 2003; Asensio, 2003). Protein analysis has been

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extensively used in fresh, frozen and minimum processed seafood products. When the fish is processed (i.e., canned products), though, protein integrity and structure is strongly modified and it is no longer possible to use protein analysis for identification purposes. DNA, however, is much more thermo-stable and informative. Therefore, it has recently been revealed to be the ideal marker for species identification in seafood products. Basically, DNA methodologies can be separated into two groups: methods not requiring a previous knowledge of the sequence (Scheider et al., 1997; Rehbein et al., 1999; Congiu et al., 2001) and methods which use sequence information (Bartlett and Davidson, 1991; Quinteiro et al., 1998; Terol et al., 2002; Pardo and PeÂrez-Villarreal, 2004). Both approaches require an analysis of the amplified marker by sequencing, electrophoresis and other techniques. At the present time the most robust DNA methodology for fish species identification is direct sequencing or Forensically Informative Nucleotide Sequencing (FINS) of a DNA fragment amplified by PCR (PCR-FINS). Nowadays efforts are also focused on obviating this analysis step and creating a one-step protocol. This would simplify the detection system and could be used as a rapid tool in the fishing industry and government quality control laboratories (Trotta et al., 2005; LoÂpez and Pardo, 2005). To enforce the standardization of DNA methodologies AZTI-Tecnalia has proposed a validation model based on the use of FINS as validation methodology with a fish database including reference DNA sequences available at http://www.azti.es/DNA_database/ and also the interchange of plasmidic standards as reference material for inter-laboratory trials (Pardo, 2007). 26.7.2 Identification of geographical origin Regulation (EC) No 2065/2001 states that in the case of products caught at sea, the label must refer one of the areas mentioned in the Regulation's Annex, which correspond to the classification areas of the FAO (FAO, 2000). In the case of farmed products, the label needs to refer to the Member State or third country in which the product undergoes its final stage of development. However, as with other food products, a more precise definition of the geographical origin is necessary. In the case of seafood products, the catching area must be defined. Consumers' perception of some harvesting regions may play a role in their buying decisions for environmental reasons. Therefore EU regulation also states that operators may indicate a more precise catching area. There are no validated methodologies for identification of the geographical origin of fish, and validation methods are still at a research stage. Different approaches have been taken: · The differentiation between fish stocks by molecular techniques using microsatellites (Pardo and Estonba, 2006) or by 1H and 13C NMR spectroscopic studies of lipid extracts including fatty acid composition, lipid classes and the positional distribution of mono-, di- and polyunsaturated fatty acids in the triacyl-glycerides (Aursand et al., 2007).

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· Through the effect of environmental conditions on fish protein expression, on the ratios of the stable isotopes of oxygen (16O/18O) and hydrogen (2H/1H) or through differences in the isotope distributions of trace elements (Campana et al., 2000; Jonsdottir et al., 2006). The two main techniques used to determine the isotope ratios of natural products are isotope ratio mass spectrometry (IRMS) and site-specific natural isotope fractionation studied by nuclear magnetic resonance (SNIF-NMR) (MartõÂnez et al., 2003).

26.7.3 Production method There are several reasons why it is necessary to discriminate farmed from wild fish. The need for information and some ethical concerns are the main drivers. First, domestication of the major species in European aquaculture happened relatively recently in comparison to the domestication of terrestrial farmed animals. Second, aquaculture regulations and procedures were adopted without the necessary basis of knowledge in marine biology, oceanography and ecology, resulting in sub-optimal productions systems, which compromised animal welfare (Torrissen, 2007). In addition, fisheries authorities may be interested in protecting some endangered wild stocks from overexploitation, and therefore ensuring that no specimens from these wild stocks may be found in the market. The reference to the production method in accordance with Article 4(1) (b) of Council Regulation (EC) No 104/2000, must consist of one of the following expressions, according to whether the product in question was caught, at sea or in freshwater, or resulted from aquaculture: `. . . caught . . .' or `. . . caught in freshwater . . .' or `. . . farmed . . .' or `. . . cultivated . . .'. A review of the analytical approaches to verify the production method of fish has been made by MartõÂnez (2006). Morphological, genetic, lipid, protein and carotenoids analyses are discussed. The author concludes that reliable analytical methods and databases with genetic sequences, lipid and protein composition data of authentic samples still need to be developed for relevant species. The fatty acid profile of fish muscle is known to be species-specific and to reflect the fatty acid composition of the feed. Vegetable oils have been introduced into fish feed in order to protect endangered stocks which are used for fish oil and meal production as well as for safety reasons (dioxin concentration throughout the trophic chain). Therefore, analyses of lipids may be used to discriminate farmed from wild species. There are several methods suitable for this purpose: gas chromatography (GC), nuclear magnetic resonance (NMR), and near infrared spectroscopy (NIR). 1H and 13C NMR spectroscopic studies have been also employed to discriminate the production method of fish and their origin (Masoum et al., 2007) as well as for authentication of fish oils (Aursand et al., 2007). Magnetic Resonance (MR) spectroscopy is a multi-component detection technique that allows biological tissue to be studied non-invasively and non-destructively both in vivo and in vitro (Gribbestad et al., 2005). Individual tagging of fish would be an optimal, trustworthy method, as the technology is advanced, and reliable tag systems are available, but it entails

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extra costs that would only be justified if fish from particular farms or regions were to achieve a higher market price. 26.7.4 Product presentation and processing conditions Some processing conditions can have an important influence on the quality and/ or safety of fishery products. Salted and dried fish produced in the traditional way are safer products, as a lower water activity is reached, their shelf life is longer and they perform better in traditional recipes, therefore are preferred by consumers (AZTI, 2001). More modern methods, though, such as freezing-thawing of fish and salting by immersion or injection of brine are advantageous in industrial production (in terms of yield, water retention and production time). There are no validated methods to determine which drying and salting process has been used. Today, proton nuclear magnetic resonance (1H NMR) is widely used to measure the physical properties of foods, such as the waterholding capacity (WHC) of fish muscle (Lakshmanan et al., 2007). 23Na-NMR has been sparsely used for the monitoring of salt distribution in food, but both methods seem to be promising for the determination of the processing conditions, as they can be used to determine the water and salt distribution respectively in fish muscle tissue (MartõÂnez et al., 2003). Frozen-thawed fish is usually drier due to liquid loss and its lower waterholding capacity. It is also poorer in bioactive components of nutritional interest due to liquid losses during its preparation. DMA analysis by GC or HPLC methods may be used to discriminate a frozen product but only in case of DMA forming species like gadoids as previously described. Because of this, the suitability of 1H NMR spectroscopy as a multiparameter analytical method for determining the presence of DMA and of bioactive compounds like taurine, betaine, anserine, creatine, and trimethylamine oxide (TMAO), indicators of fish quality and processing parameters, has been investigated (MartõÂnez et al., 2005), The determination of the heating temperature that the product may have undergone is also important from the point of view of safety, especially when the product is a ready to eat meal and, thus, intended to be consumed without further cooking. There are several techniques that have been proposed for the determination of the heating temperature based mainly through measurement of the degree of denaturation of muscle proteins: coagulation tests, SDS- PAGE, differential scanning calorimetry (DSC), but, these methods are still at research stage and have not yet been validated. Many processing related factors may influence the degree of denaturation of proteins as measured by DSC: salting, drying, freezing, thermal treatment and deterioration processes (Jensen et al., 2003; Thorarinsdottir et al., 2002; Schubring, 2006). Therefore, the results of any methodology employed have to be carefully interpreted taking into account the whole history of the product.

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559

Validation of data management and information flow

The goal for validation of data management is to ensure that there are no information gaps in the transmission of data between links in the commercialization chain, so that a product can be traced backwards and forwards throughout the whole fishery chain, from the origin of its raw materials until it reaches the consumer. The only way to test this ability is by simulating recall of fish products. There are no standard accepted methods for conducting such an exercise. A description of the methods is beyond the objectives of this chapter but there are some published experiences that may be consulted (Karlsen and Senneset, 2006).

26.9

Conclusions

Despite existing legislation, there is no general provision that defines exactly how traceability systems should be implemented, but food companies have to be able to demonstrate that the system put in place is efficient and effective. Validation procedures for established traceability systems have been recommended in this chapter. Control parameters, together with a map of indicators of efficiency and reliability in relation to safety and quality assurance, fraud prevention and data management and information flow, have been defined in a practical guide of reference available on line in the form of a web page specifically constructed for this purpose. There are multiple traceable data important for the fish sector and, for each one, diverse methodologies have been described for measuring them. In the majority of cases, there are not standards that allow easy or simple comparison of data between interested parties, and not every methodology is suitable for each link in the fish chain. Official methods do exist for measurement of some parameters and these have been indicated, although the majority of them are time consuming and quite slow to produce results that could be used to make decisions along the fish chain. Some specific sensors and probes have recently appeared, but in general there is a lack of validation, and reference methods are needed before they can be considered for adoption as rapid quality control tools for the fish industry. As the development of new, faster and simpler methods continues, identity, safety and quality parameters will be more frequently controlled, contributing to a safer and more reliable fish distribution chain. The establishment of a validated system for traceability management including the set-up of standards for the analysis of relevant traceability parameters and inspection procedures in each link of the fish chain is of primary importance in these circumstances. Special attention must be paid to the latest advances in the molecular biology techniques applied to traceability. In this sense, the project VALID of SEAFOODplus has paid special attention to species identification in seafood products. It has proposed a validation model for the standardization of DNA methodologies based on the use of FINS as validation methodology, constructed

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products. Food Chem, 103 (3), 1049±1053. (1945). Amines in fish muscle. I. Colorimetric determination of trimethylamine as the picrate salt. J Fish Res Board of Canada, 6, 351±358. EMBORG J, LAURSEN B.G., DALGAARD P. (2005). Significant histamine formation in tuna (Thunnus albacares) at 2 ëC ± effect of vacuum- and modified atmospherepackaging on psychrotolerant bacteria. Int J Food Microbiol, 101 (3), 263±279. FAO/WHO (1968). Method for the determination of total volatile basic nitrogen (TVB) in fish muscle. Presented to Codex Committee on Fish and Fishery Products, 3rd session, Bergen, 7±11 October 1968, as Codex Fish 1/7, 9. FAO (2000). FAO Yearbook of Fishery Statistics 1998, Vol. 86/1. ISBN 9250044240. FDA (FOOD AND DRUG ADMINISTRATION) (1982). Defect action levels for histamine in tuna; availability of guide. Fed Reg, 470, 4048740487. FENG P., WEAGANT S.D., GRANT M.A. (1998). Enumeration of Escherichia coli and the Coliform Bacteria. Ch.4. In US Food and Drug Administration (USFDA) Bacteriological Analytical Manual, 8th edn (revision A), http://www.cfsan.fda.gov/~ebam/ bam-4.html. (CD-ROM version). R.L. Merker (Ed.). AOAC International, Gaithersburg, MD. GERMAN REFERENCE METHOD (1999). Analysis of foods. Determination of biogenic amines in fish and fish products. HPLC reference method. Amtliche-Sammlungvon-Untersuchungsverfahren-nach-Paragraph-35-LMBG, L 10.00-5, 3 p.005, 263± 279. 1 GRIBBESTAD I.S., AURSAND M., MARTINEZ I. (2005). High-resolution H magnetic resonance spectroscopy of whole fish, fillets and extracts of farmed Atlantic salmon (Salmo salar) for quality assessment and compositional analyses. Aquaculture, 250, 445± 457. Guidance Document. Implementation of certain provisions of Regulation (EC) No 852/ 2004 on the hygiene of foodstuffs. European Commission Health and Consumer Protection Directorate-General. Brussels, 21 December 2005. Guidance on the implementation of articles 11, 12, 16, 17, 18, 19 and 20 of regulation (EC) No 178/2002 on general food law. Conclusions of the Standing Committee on the Food Chain and Animal Health. December 2004. HEALTH PROTECTION AGENCY (2004a). Enumeration of Escherichia coli in raw molluscs by the multiple tube technique. National Standard Method F 16 Issue 4. http:// www.hpa-standardmethods.org.uk/pdf_sops.asp. HEALTH PROTECTION AGENCY (2004b). Direct enumeration of Escherichia coli. National Standard Method F 20 Issue 1. http://www.hpa-standardmethods.org.uk/ pdf_sops.asp. HERRERO A.M., HUIDOBRO A., CARECHE M. (2003). Development of a quality index method for frozen hake (M. Capensis and M. Paradoxus). J Food Sci, 68 (3), 1086±1092. HOWGATE, P., JOHNSTON A. AND WHITTLE A.D.J. (1992). Multilingual Guide to EC Freshness Grades for Fishery Products, Torry Research Station, Food Safety Directorate, Ministry of Agriculture, Fisheries and Food, Aberdeen, Scotland. HUIDOBRO A., PASTOR A., TEJADA M. (2000). Quality Index Method Developed for Raw Gilthead Seabream (Sparus aurata). J Food Sci, 65 (7), 1202±1205. JENSEN K.N., JORGENSEN B.M., NIELSEN J. (2003). Low-temperature transitions in cod and tuna determined by differential scanning calorimetry. Lebensmittel-Wissenschaft und-Technologie ± Food Science and Technology, 36 (3): 369±374. JEYA SHAKILA R., VASUNDHARA T.S., KUMUDAVALLY K.V. (2001). A comparison of the TLCdensitometry and HPLC method for the determination of biogenic amines in fish DYER W.J.

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Improving seafood products for the consumer

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a free database including reference DNA sequences and developed specific plasmidic standards as reference materials.

26.10

References and bibliography

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WPNL0206

Index

ACE inhibitors 351, 352, 370±1, 372±7 acid extraction 365±8 active oxygen species 428 active packaging 413±15 active RFID tags 526 adipocytes 469, 470, 477, 481 advertising 64, 69±70 Aeromonas spp. 254 African catfish, selenium-enriched 349±54 changes in nutritional functional components during household preparation 350±4 Aichi virus 204 alanine 352, 353 algae, marine 349 alkali extraction 365±8 alpha-linolenic acid 166, 175±6 alpha-tocopherol 447, 449±50 Alsina biochemical identification scheme 266 amines biogenic see biogenic amines; histamine volatile 552±4 amino acids, free 352, 353 ammonia 552±4 amnesic shellfish poisoning (ASP) 187, 547 analytical methods biotoxins 548

evaluation of oxidation 427, 433±5 histamine 314, 546 volatile amines 553±4 angiotensin I-converting enzyme (ACE) inhibitors 351, 352, 370±1, 372±7 animal studies 174±5 animal welfare 490±2, 493, 494±5, 507±8 Anisakis spp. 550 anisidine value 433 antibiotic resistance 410 antihistamines 304 antimicrobial compounds 411±13 antimicrobial packaging 413±15 antioxidant dietary fibres 337, 338±40, 354, 355 functional product development 343±7 antioxidants antioxidant properties of fish protein hydrolysates and peptides 374, 377±8, 379 effect on lipid oxidation in oil-in-water emulsion model systems 439±42 natural 8, 446±50 AntiStress 24 385 API 20E test 266 API 20NE test 266 apoptosis 122±4 appearance cod 43, 45±7 salmon 44, 47±50 appetite control 379±80

WPNL0206

568

Index

aquaculture 6±7, 41, 332, 463±4 distinguishing between wild and farmed fish 557±8 fish welfare and ethical qualities 490±510 common interests of fish farmers and consumers 496±8 future trends 507±8 monitoring ethical qualities 505±7 welfare during production cycle 498±502 welfare during slaughter 502±4 wild vs farmed fish 495±6 muscle texture see texture process mapping of farmed Norwegian salmon 532±7 production of functional seafood 349±54, 355±6 validation of traceability 557±8 Aristotle 490±1 arrhythmias, cardiac 170±4 arthritis 116 ascorbic acid 447 asphyxia 503±4 astroviruses 196, 201 Atlantic cod 501±2, 505±7 Atlantic halibut 474±7 Atlantic salmon 467, 469, 471±2, 473, 474±7, 479, 480, 481, 482 atrial fibrillation 172 attitudes see consumer attitudes Australia 215, 260, 261 authentication of fish species 555±6 automated ribotyping systems 272 avian influenza viruses 202±3 Bacillus cereus 250 bacteraemia 251 bacteria 3 histamine producing 299±303, 307±8 microbiological safety assessment 548±9 pathogenic see bacterial pathogens spoilage see spoilage bacteria bacterial pathogens 5, 116, 186, 190±1, 247±91 application of new methods 279 control of bacterial contamination 255±8 detection, enumeration and identification 262±71 conventional methods 262±6, 281 molecular methods 267±71, 279, 281 emerging bacterial risks 278±9

emerging treatment technologies 280 future trends 278±80 impact of pulsed light technology 416±17 LAB and control of in LPFPs 405±6, 407 molecular typing methods 271±8 principal bacterial pathogens associated with seafood 248±53 safety assessment methods 548±9 seafood-associated bacterial illness 248, 249±50, 251, 258±61 sources 254±5 baked fish 168 bar codes 525, 528 Barker hypothesis 143±4 barriers to consumption of seafood 17±20 batch number 519±20 behavioural indicators 93±5, 100±1, 103 behavioural models 494 Belgium 53±5 consumer attitudes and seafood consumption 18±20, 21±30 consumer information needs 65±6, 67, 69±80 beliefs, salient 24, 25, 96±7 Bentham, Jeremy 491 beta-phenylethylamine 302 BIOCOM project 5, 293, 294, 296, 297, 298±9, 302, 303, 315 biogenic amines 5, 186±7, 191, 292±324, 410 determination in seafood 314 formation in seafoods 307±14 validation of traceability 546 see also histamine; histamine fish poisoning (HFP) Biolog GN system 266 biopreservation 411 chitosan 411±15 lightly preserved seafood products 405±11 biopsies 127 BIOQUAL project 6±7 biosensors 269 biotechnology 386 bioterrorism 204 biotoxins 187±8, 546±8 EU regulation 547, 548 reference validation methods for analysis 548 routine methods for quality control 547±8 birth size 143±6

WPNL0206

Index bitterness problem 383 bivalve molluscs 5, 258, 549 contamination 195±6, 197, 212, 214, 254 cross-contamination 255 harvesting, handling and transport 256±7 processing and viral contamination control 236±7 sources of contamination 215±17 see also shellfish; shellfish harvesting areas blood lipids 140±3, 153 blood samples 127 bone mineral density (BMD) 150 bones barrier to fish consumption 17, 19, 24±5, 26, 34 bone health 137, 149±51, 153 bonito bowel, dried 376, 384, 385 botulism 258 bound water 381 Brambell Committee report 492, 494 breathing patterns 505±7 broiled fish 168 BSE (Bovine Spongiform Encephalopathy) crisis 513 butyl-hydroxy-anisol (BHA) 447 butyl-hydroxy-toluene (BHT) 447, 448±9 by-products 8 cadaverine 299, 300, 302 Caesarean sections 147 caffeic acid 448±50 calciotropic activities 375, 379±80 calcitonin gene-related peptide (CGRP) 375, 379±80 calcium ion channel 174±5 calpain/calpastatin system 477±9 calpastatin (CAST) gene 482±3 Campylobacter spp. 249 jejuni 279 cancer 116±17, 137 antiproliferative activity of FPH 380 colorectal 117±24, 128 other gastrointestinal tract cancers 125 selenium's protective effects 340±1 canteens, testing in 99±100 captured fish (TraceFish) standard 517±18 carbon dioxide 312, 313 high and fish welfare 501±2 stunning 502±3 cardiac arrhythmias 170±4

569

cardiomyocytes 174±5 cardiovascular disease 137, 140±3, 153, 166 see also heart disease Cardiovascular Health Study 167±8, 172 Carnobacterium spp. 405±11 divergens V41 406, 407, 408, 412, 413 maltaromaticum 405±6 carotenoids 445 carp 503±4 case-control studies 119 catalysts, oxidation 428±9, 431 catfish, African 349±54 cathepsins 477 cee gene 483 cell proliferation 122±4 central location testing 90 centrifugation 367 chemical reaction modelling 438±9 children and young people 4, 19, 136±64 bone health 137, 149±51, 153 communicating nutritional effects of fish 151±3 development of functional fish products 153±4 fish burger 98±102 fish consumption and blood lipids 140±3 future trends 154 obesity 137, 138±40, 153±4 postpartum depression 137±8, 147±9, 153 pregnancy and birth outcome 143±6 pregnancy and delivery complications 146±7 Chile 261 China 216 chitosan 411±13 antimicrobial packaging 414±15 chlorogenic acid 448±9 cholecystokinin (CCK) 379±80 cholera 252 cholesterol 142±3 chromatography 433, 546 chronic diseases 114±15, 116 ciguatera fish poisoning (CFP) 188, 547 cinnamic acids 448±50, 451 cis-urocanic acid 305 Citrem 439±41 clams 195 Clark electrode 436 Clostridium spp. 549 botulinum 250, 254, 258 detection methods 264

WPNL0206

570

Index

perfringens 250 cluster analysis 51±2, 67, 74±5 cockles 195 cod 40, 126±7 Atlantic cod 501±2, 505±7 polar cod 348 salt-cured 400±5 sensory evaluation 43, 53±4 consumer preferences 50±2 sensory characteristics 45±7 washed fish mince and lipid oxidation 444±6 and weight loss 139 cohort studies 119 cold-smoked salmon (CSS) 405±6, 407±8, 409±10, 417 coliforms 549 collagen 468±70 crosslinks 470, 474±7 peptides 384, 385 colony hybridization 269±70 colorectal cancer (CRC) 117±24, 128 intervention studies 121±2 mechanisms underlying the effects of fatty acids on risk of 122±4 nutrient±gene interactions 121 observational studies 119±21 COMET assay 127 commercial enzymes 369 communication 151±3 compensation 31±6 compliance 540 conceptual product testing 335±7 Confident consumer segment 67, 75±80 conformity 540 conjugated dienes 433 consciousness 491 Conservative consumer segment 334±5 consumer attitudes 3, 13±14, 16±39 convenience and fish consumption 28±36 differences across segments 34±6 cross-cultural investigation 20±1 and development of functional seafood products 334 European consumption patterns 21±4 attitudes and preferences across Europe 24±8 future challenges 36±7 motives for and barriers to seafood consumption 17±20 perceived quality and satisfaction 96±8 segmentation and 53±5

consumer-driven product development 330 consumer evaluation 4, 14, 85±110 intention to consume and potential loyalty 94±5 Norwegian fish burger 98±102 perceived quality and satisfaction 95±8 product attributes and informational cues 87±9 test situations 89±91 time and convenience 91±3 willingness to pay 93±4 consumer information needs 4, 14, 63±84 analysis procedures 69 consumer survey 66±8, 72±80 data collection 65±8 exploratory study 65±6, 69±72 measures 68±9 consumer needs, specification of 334±5 consumer preferences cross-cultural investigation 24±8 segmentation and 53±5 and sensory characteristics 50±3 see also consumer attitudes consumer surveys consumer attitudes and seafood consumption 20±30 information needs 66±8, 72±80 CONSUMEREVALUATE project 4, 86 CONSUMERPRODUCTS project 6, 331±2 consumers 3±4, 13±15 concerns about aquaculture 496±8 consumer studies and development of functional products 333±7 segmentation see segmentation of consumers see also consumer attitudes; consumer evaluation; consumer information needs; sensory quality CONSUMERSURVEY project 3, 16 consumption of seafood 13±14, 16±39 convenience and 17, 28±36, 37 cross-cultural investigation 20±1 European consumption patterns 21±8, 40, 65±6 frequency of consumption 21 light vs heavy users 26±8, 65±6 motives for and barriers to 17±20 contingent valuation 93±4 continuous membrane reactor 370, 371 convenience 20, 418 consumer evaluation and 91±3 quality, satisfaction and intention 100±1

WPNL0206

Index functional seafood product development 335±7 and seafood consumption 17, 28±36, 37 convenience foods 331±2, 400 convenience orientation 28±36, 92, 103±4 and segments 30±4 Convenience consumer segment 31±3, 34±6 conventional bacteria detection/ enumeration methods 262±6, 281 cooling 503 Copalis 384, 385, 386 coping 494 copper 431 co-products 368 coronary bypass graft surgery (CABG) 172 coronary heart disease (CHD) 140±1, 166±7, 329 o-coumaric acid 448±9 critical limits for histamine 306, 315 Critical consumer segment 32, 33, 34±6 Crohn's disease (CD) 125 cross-contamination 255, 257±8 cross-cultural attitudes 20±37 crosslinks, collagen 470, 474±7 cruciferous vegetables 126 customer satisfaction see satisfaction Danish Diet, Cancer and Health study 172 dark (slow) muscle 468 Darwin, Charles 491 data capture technology see radio frequency identification data (RFID) tags data driven models 438 data management 543, 545, 559 decanter centrifuges 367 deep-fried fish products 367±8 delivery complications 146±7 Denmark 53±5, 295, 296 consumer attitudes and seafood consumption 21±30 consumer information needs 67, 72±80 herring fillet scare 513 depression 153 postpartum 137±8, 147±9, 153 depuration 236±7, 256±7, 350 desaturases 175±6 diamine oxidase (DAO) 304 diarrhoeic shellfish poisoning (DSP) 187, 547

571

Diet and Reinfarction Trial (DART) 168±9 DART±2 169 dietary fibre (DF) 118, 337, 343±7, 354±5 antioxidant DF 337, 338±40, 343±7, 354, 355 structural changes in surimi gels and minced fish muscle 345±7 technological uses 343±5 digestive tissues 198±9 dimethylamine (DMA) 552±4 dioxins 513 direct lysis of viruses 199 disinfection systems 233±4 distance from viral sources 222, 224, 225 DNA damage and pulsed light technology 416 species authentication 556 DNA microarrays 269 docosahexaenoic acid (DHA) 122, 124, 166, 175±6, 352 dried bonito bowel (Katsuobusi) 376, 384, 385 dried proteins and peptides 380±3 drum drying 381 drying 381 validation of traceability 558 dystrophin 478±9 early warning system 234±6, 241 ecological products 497±8 efficiency indicators 542±4 egg incubation temperature 480 eicosanoids 141, 174 eicosapentaenoic acid (EPA) 122, 124, 166, 175±6, 352 electrical stunning 503±4 elongases 175±6 Elpro temperature logger 529±30, 531 emerging disease risks 201±4, 278±9 emulsifiers 431±2 effect on lipid oxidation in oil-in-water emulsion model systems 439±42 emulsifying properties of dried proteins and peptides 382±3 emulsions lipid oxidation in 431±3, 440 model systems 434±5, 450 kinetics and modelling of lipid oxidation in 435±9 oil-in-water 435, 439±42 physical structure 432±3

WPNL0206

572

Index

end-point measures 122 endomysium 468 energy expenditure/effort 91±2 England 259±60 see also United Kingdom Enoceride 385 enrichment 262, 265 Enthusiast consumer segment 67, 74±80 environmental influences on muscle structural traits 479±81 enzymatic hydrolysis 368±9, 370 EPC Gen 2 tags 526, 528 EPCGlobal 525, 526±7 epidemics in population 222, 224, 225±6, 228±9 epidemiological data 226±7 bacterial illness 258±61 epidermal growth factor (EGF) 124 Escherichia coli (E. coli) 212, 213, 238, 256, 258, 549 O157:H7 279 ethical issues 7, 103±4, 490±510 information segments and 79±80 monitoring ethical qualities 505±7 reasons for concern about fish 492±3 relationship between humans and other animals 490±2 see also welfare ETHIQUAL project 7 Europe 3, 13±14, 16±39 consumer segmentation related to attitudes and preferences 53±5 consumption patterns 21±4, 40, 65±6 attitudes and preferences across Europe 24±8 convenience 28±36 European Prospective Investigation into Cancer and Nutrition (EPIC) study 120 European Union (EU) Concerted Action on Functional Foods Science (FUFOSE) 329 methods for detection of bacteria 262±3, 279 regulation 368 bacterial contamination 255±7 biopreservation of LPFPs 410 dietary fibre 355 food hygiene regulations 189±90 freshness grades 551 General Food law 514 histamine 306, 307 marine biotoxins 547, 548 microbial contamination of shellfish

212, 213, 233, 236±7, 238±9 packaging 414 volatile amines 553 Water Framework Directive 238 sustainable aquaculture 498 evening primrose oil 150±1 FAAT trial 171 factor analysis 30±1 faecal contamination bacterial pathogens 254 viruses and shellfish 194±5, 214±17 see also sewage contamination faecal samples 127 faecal streptococci analysis 554 familial adenomatous polyposis (FAP) 117 farmed fish see aquaculture farmed fish (TraceFish) standard 517±18 fatty acids and colorectal cancer 120±1 mechanisms underlying effects of fatty acids 122±4 losses in household preparation of African catfish 352±4 measurement of lipid oxidation 435 omega-3 fatty acids see omega-3 fatty acids omega-6 fatty acids 123, 141, 175±6 profile of fish muscle and validation of traceability 557 fermentation/fermented products 312±14, 370±2 ferulic acid 448±9 fibre see dietary fibre fibrillation atrial 172 ventricular 170±1 fillet gaping 465 finfish harvesting, handling and transport 256 see also under individual types of fish finishing diet 350 first principle based models 438 fish burgers 98±102, 168 fish density 499±500 fish farms see aquaculture fish growth rate 467 fish liver oil supplements 144, 145±6 fish meal 363, 364 fish mince, washed 434, 442±6, 450±1 fish muscle see muscle, fish fish protein hydrolysates (FPHs) 327±8, 364, 368±72, 386±7

WPNL0206

Index bioactive properties 372±80 functional properties of dried FPHs 380±3, 386±7 fish protein isolates 327±8, 365±8, 386 fish sandwiches (fish burgers) 98±102, 168 fish sauce 312±14, 363, 370, 371±2 fish silage 363±4, 364 fish welfare see welfare FISHGASTRO project 4, 126±9 fishmongers 23, 24 `five freedoms' 492, 494 fixed code passive RFID tags 525, 526 Fjord Seafood Herùy, Norway 529±30, 531 flavour/taste cod 43, 45±7 dried fish proteins and peptides 383 motive for seafood consumption 17, 18±19 salmon 44, 47±50 selenium-enriched garlic 350 see also sensory quality focus groups consumer attitudes and seafood consumption 18±20 consumer information needs 65±6, 69±72 food hygiene regulations 189±90 food megatrends 497 food neophobia 103±4 food preparation 17, 19, 20 changes in nutritional components of selenium-enriched African catfish 350±4 convenience 31±6 test situations 91 food safety objectives (FSOs) 256 food scares 513 foodborne illness/outbreaks bacterial pathogens 248, 249±50, 251, 258±61 HFP 292, 293, 294±7 relative incidence 188±9 viruses in shellfish 186, 188, 189, 212 epidemics in population 222, 224, 225±6, 228±9 identification of probability 226±9 Forensically Informative Nucleotide Sequencing (FINS) 556 Fortidium 385 France 260 gastroenteritis cases 224, 226 REDRISK project site 218, 220, 221±5

573

sentinelle network 227 fraud prevention 543, 545, 554±8 free amino acids 352, 353 free water 381 freshness, sensory evaluation of 551±2 fried fish 168 frozen fish 558 cod 46±7 salmon 48±9 fucoidans (fucan sulphates) 339 fucus vesiculosus antioxidant DF 338, 339, 340, 345 functional products 6, 8, 114, 115, 329±30, 331±62 aquaculture production 349±54, 355±6 consumer studies and development of 333±7 development of for improved health 153±4 development of restructured seafood products 343±8 future trends 354±6 marine proteins and peptide products 384±5, 386±7 mild processing see mild processing novel ingredients for incorporation into restructured/fillet-based seafood 337±42 gaping 465 garlic, selenium-enriched 340, 341, 342, 349±50, 354 gastrin 379±80 gastrointestinal health 4, 116±35 colorectal cancer 117±24, 128 FISHGASTRO study 126±8 inflammatory bowel disease 124±5 other gastrointestinal tract cancers 125 other nutritional aspects of fish consumption 126 gels formation 345±7 pH-shift processes and 366±7, 368 genes genetic modifications of viruses 202 and muscle texture 471, 472, 481, 482±3 nutrient-gene interactions 121 genetically modified organisms (GMOs) 513 geographical origin, identification of 556±7 GHP programmes 257 GII.4 norovirus 202

WPNL0206

574

Index

GISSI-Prevenzione trial 121, 169 Global Trade Item Number (GTIN) 519±20 global warming 191±2, 278 globalization 191, 278±9 glycerol 415 glycine 352, 353 Good Traceability Practice (GTP) manual 516±23 additional data recording 521, 523 defining traceable units 518±19 keeping track of transformations 520±1, 522 traceability friendly production 521±3 unique identification of traceable units 519±20 grape antioxidant dietary fibre 338±9, 345, 347 HACCP systems 257, 280 haem proteins 366 as pro-oxidants 429±30 haemoglobin 366, 429±30, 450 as a pro-oxidant in washed fish mince 442±3, 444±5 halibut, Atlantic 474±7 handling 256±7 harvesting 256±7 see also shellfish harvesting areas Hawaii 295, 296 hazard identification 225±6 HDL cholesterol 142±3 health 1, 2, 4, 17, 103±4, 113±15 children and young people see children and young people consumer information needs and segments 77, 81±2 consumer needs and functional seafood development 334±5 convenience segments and attitudes towards health orientation 34±6 development of functional fish products for improved health 153±4 gastrointestinal see gastrointestinal health heart disease see heart disease motive for seafood consumption 17, 18±19, 25 quality, satisfaction and intention 100±1 health claims 81±2, 387 health promotion programmes 151±3 heart disease 116, 153

coronary heart disease 140±1, 166±7, 329 omega-3 fatty acids and 4, 165±81 cardiac arrhythmias 170±4 future research 176 intervention studies/trials 168±9 observational studies 166±8 possible mechanisms of effects 174±5 systematic reviews 169±70 heat treatment 237 detection of temperature used 558 heavy users 26±8 hedonic scales 44±5 hemin 429±30 Hendra virus 204 hepatitis A virus (HAV) 5, 186, 197±8, 212, 214, 216±17, 550 detection 198, 200±1 hepatitis E virus (HEV) 203 hereditary non-polyposis colorectal cancer (HNPCC) 117 heritability 481 herring Danish herring fillet scare 513 washed fish mince and lipid oxidation 444±6 heterocyclic amines (HCAs) 118 high pressure processing 237, 280 high risk periods 226, 240 highly pathogenic avian influenza (HPAI) viruses 202±3 histamine 5, 186±7, 191, 247, 292±324 critical limits for concentrations in seafood 306, 315 formation in seafoods 307±14 histamine producing bacteria 299±303, 307±8 management of formation 315 methods of detection 314, 546 validation of traceability 546 within-fish variations in concentration 298, 299 histamine fish poisoning (HFP) 5, 186±7, 292±306, 315±16, 546 implicated products and their characteristics 297±303 incidents and cases 292, 293, 294±7 management of 315 symptoms and toxicology 303±6 histamine-N-methyltransferase (HMT) 304 histidine 297 homeostasis 494

WPNL0206

Index hurdle technology 6, 328±9, 399±425 antimicrobial compounds 411±13 antimicrobial packaging 413±15 biopreservation of lightly preserved seafood products 405±11 future trends 418 pulsed light as a decontamination technology 415±17 salt hurdle 400±5 HURDLETECH project 6, 400, 409±10, 412, 415, 417 hybridization 200, 201 colony hybridization as enumeration method 269±70 ribotyping 272 hydroxycinnamic acids 448±50, 451 hydroxyl radical 428 hygiene regulations 189±90 hypercapnea 501±2 hypertensive disorders 146±7, 153 Iceland 53±5 immobilised water 381 immunostimulation 375, 378±9 implantable cardioverter defibrillators (ICDs) 170±1 IMPLEM project 7, 515, 524±37 choice of technology/equipment 527±8 fish chain process studies 530±7 RFID tags 524±30 testing radio frequency temperature loggers 529±30, 531 in-home testing 90±1 fish burger 99±100 in vitro experiments 174±5 Independence consumer segment 32, 33, 34±6 indicators behavioural 93±5, 100±1, 103 for validation of traceability 542±5 welfare indicators 495, 505±7 individual differences 103±4 infections bacterial 186, 248, 249±50 viral 186 inflammation, in the colon 124 inflammatory bowel disease (IBD) 124±5 information categories and traceability 521, 523 consumer information needs see consumer information needs flow and validation of traceability 543, 545, 559

575

information cues consumer evaluation 87±9, 102±3 interest in 68±9, 70±1, 72±4 use of 68, 70±1, 72±4 use between segments 75, 76 information sources 2 trust in 8, 68, 71±4 segmentation by 74±5 use of 68, 69±70, 72±4 segmentation by 74±5 inhibitor elimination 199 innovativeness 103±4 inputs, reducing size of 523 instrumental texture measurement techniques 466 insulin like growth factor II (IGF-II) 124 insulin resistance 380 integrated approach to interventions 152 intensive production systems and rearing technology 499±500 intention to consume 94±5, 103 quality, satisfaction and 100±1 interfacial area 432 intervention studies colorectal cancer 121±2 FISHGASTRO study 126±8 omega-3 fatty acids and heart disease 168±9 intoxications 186, 248, 250 Involved consumer segment 334±5 ion channels 174±5 ionizing radiation 237, 280 Ireland pharmacy sales 227 REDRISK project site 218, 220±5 iron 429±30 low molecular weight (LWF-Fe) 430±1, 443 irradiation 237, 280 irreducible (mature) crosslinks 470 ISO standards for RFID tags 526±7 isotope ratio mass spectrometry (IRMS) 557 Japan 188, 260, 261 functional foods market 384 HFP 295, 297 juvenile salmon producer 534 Kamaboko 378 Katsuobushi (dried bonito bowel) 376, 384, 385

WPNL0206

576

Index

knowledge 103±4 about fish and convenience segments 34±6 Korea, Republic of 260, 261 KSW TempSens logger 529±30 laboratory testing 90 lactic acid bacteria (LAB) 370±1, 405±11 additional selection properties in biopreservation of LPFPs 409±10 control of bacteria in LPFPs pathogenic bacteria 405±6, 407 spoilage bacteria 406±9 Lactobacillus spp. 405±11 Lapis Support 384, 385 Lecithin 439±41 Levenorm 384, 385 lifestyle 117 light users 26±8 lightly preserved fish products (LPFPs) 400 biopreservation 405±11 lignin 347 limited viral contamination 231±2 linoleic acid 175±6 lipid hydroperoxides 427 lipid oxidation 6, 329, 383, 426±60 analytical methods to evaluate 427, 433±5 in emulsified foods 431±3, 440 in fish 428±31 future trends 451±2 model systems 434±46, 450±1 effect of emulsifiers and antioxidants in oil-in-water emulsion model systems 439±42 kinetics and modelling 435±9 liposomes and emulsions 434±5 washed fish mince 435, 442±6, 450±1 natural antioxidants in fish products 446±50 reactants and catalysts 428±9 reaction in brief 427±8 LIPIDTEXT project 6, 426±7, 428, 429 modelling of lipid oxidation 435±42, 443±6 natural antioxidants 448±9 liposomes 434±5, 450 kinetics and modelling of lipid oxidation in 435±9 lipoxygenases 429 Listeria spp. detection methods 264

innocua 401±4, 417 monocytogenes 248, 249, 255, 279, 405±6, 413, 549 detection 264 salt hurdle 401±5 impact of contamination point 403±4 survival and growth during saltcuring and rehydration/soaking 401±3 listeriosis 248 logistic units (LUs) 519, 520 identifying 519, 528 long residence time treatment lagoons 233±4 Lorenz, Konrad 491 Louisiana norovirus outbreak 216 low molecular weight iron (LMW-Fe) 430±1, 443 low-salt fish sauce 372 loyalty 94±5 Lyon Diet Heart trial 175 lysyl oxidase (LOX) 470, 482±3 macrorestriction pattern analysis 274±8 malachite green 513 mandatory information cues 73 map service 523±4 marine algae 349 Marinova 384, 385, 386 Maripep 385 mast cell degranulator (activator) 305 maternal health 143±9, 153 postpartum depression 137±8, 147±9, 153 pregnancy and birth outcome 143±6 pregnancy and delivery complications 146±7 mathematical models of histamine formation 309±12, 316 maximum muscle fibre diameter 473 meat 118 mechanical stunning 504 medical doctors 8 membrane filtration 234, 369±70, 371 membrane fluidity 174 mesophilic aerobic plate counts (MAPC) 554 mesophilic bacteria 299, 301, 307±8 metabolic syndrome 114±15 METAHEART project 4, 165, 171 metal catalysis 431 METHODS project 7, 515, 516±24 GTP manual 516±23 SEAFOODplus map service 523±4

WPNL0206

Index vocabulary 516, 517 microbiological monitoring 234±6, 238±9 microbiological risks 185±7 bacterial pathogens see bacterial pathogens biogenic amines see biogenic amines histamine see histamine; histamine fish poisoning (HFP) relative incidence of microbiological illness 188±9 shellfish harvesting areas see shellfish harvesting areas viruses see viruses microfiltration 234 microsatellites 556 mild processing techniques 6, 363±98 bioactive properties of fish protein hydrolysates and peptides 372±80 functional properties of dried marine proteins and peptides 380±3, 386±7 future trends 386±7 improved yield in traditional fish processing 364±8 market for functional marine proteins and peptide products 384±5 processing of marine proteins and peptides 368±72 minced fish muscle 343±8, 355 miniaturised biochemical tests 266 model systems 434±46, 450±1 kinetics and modelling of lipid oxidation in liposomes and emulsions using oxygen uptake rate 435±9 liposomes and emulsions as 434±5 oil-in-water emulsions and effect of emulsifiers and antioxidants 439±42 washed fish mince 434, 442±6, 450±1 modified atmosphere packaging (MAP) 312, 313, 315, 316 cod 46 salmon 49 molecular methods 267±78, 279, 281 detection and identification 267±9 enumeration 269±71 typing methods 271±8 molecular weight cut±offs (MWCO) 369 monitoring biotoxins 547±8 contamination of shellfish harvesting areas 234±6, 238±9 ethical qualities in farmed fish 505±7

577

national monitoring programmes 226±7 monoamine oxidase 304 moral obligation 31±6 Morganella spp. morganii 293 psychrotolerans 299, 301, 308 model for growth and histamine formation 310±11 most probable number polymerase chain reaction (MPN-PCR) 270 motives for seafood consumption 17±20 multiplex PCR assays 268 muscle, fish functional product development 343±8, 355 mechanisms of growth 470±3 structural traits environmental influences 479±81 heritability 481 and texture 473±7 texture see texture muscle fibre density 471±7 heritability 481 muscle fibre diameter, maximum 473 muscle fibre number 473 environmental influences 479±80 heritability 481 mussels 195 myeloperoxidase 429 myoD family member genes 471, 472 myofibrils 468 myogenic progenitor cells (MPCs) 470±1, 481 myogenic regulatory factors (MRFs) 471 myoglobin 429±30 myostatin 482±3 myotomes (myomeres) 468±70 national monitoring programmes 226±7 natural antioxidants 8, 446±50 naturalness 335±7 nematodes 513 Netherlands, The 53±5 consumer attitudes and seafood consumption 21±30 consumer information needs 67, 72±80 neurotoxic shellfish poisoning (NSP) 187, 547 new product testing 101±2 `new' viruses 202 Nipah virus 204 nitrogen 312, 313 nitroso-compounds 118

WPNL0206

578

Index

Nordic Network for Marine Functional Food (MARIFUNC) 154 normal behaviour 494 noroviruses (NoV) 5, 186, 196±8, 212±13, 214±15, 240 detection 198, 200 GII.4 type 202 REDRISK project 222, 224, 225±6, 240 sources of shellfish contamination 215, 216 validation of traceability 550 northern wolf fish 348 Norway 295, 296, 498 fish burger 98±102 process mapping of salmon supply chain 532±7 nuclear magnetic resonance (NMR) 556, 557, 558 nucleic acid amplification-based typing methods 272±8 NutraPure protein processing 365 nutrients indispensable 113, 114 nutrient±gene interactions 121 Nutripeptin 384, 385 nutrition 113±15 changes in nutritional components of African catfish during household preparation 350±4 developments in nutrition science 113±14 fish consumption and gastrointestinal health 121, 126 promotion of nutritional effects of fish to children and young adults 151±3 role of seafood 114±15 nutrition claims 81±2 obesity 137, 138±40, 153±4 observational studies colorectal cancer 119±21 omega-3 fatty acids and heart disease 166±8 oesophageal cancer 125 oil-in-water emulsions 431 model systems 435 effect of emulsifiers and antioxidants on lipid oxidation 439±42 omega-3 fatty acids 1, 137, 141, 154, 165±6 and blood lipids 141±3 and bone health 149±51

colorectal cancer 120±1, 122±4, 128 conversion and metabolism in the body 175±6 in the diet 165±6 and heart disease see heart disease lipid oxidation see lipid oxidation and postpartum depression 147±9 pregnancy and delivery complications 146±7 omega-6 fatty acids 123, 141, 175±6 operational welfare indicators (OWIs) 495 organic production 494±5, 498 organoleptic changes 409±10 osteoporosis 149±51 oxidation see lipid oxidation; protein oxidation oxidative reductor system 449±50 oxygen 312, 313 singlet 428±9 uptake rate 435±9 oysters 185, 195 packaging antimicrobial 413±15 modified atmosphere packaging 46, 49, 312, 313, 315, 316 vacuum packaging 312, 313, 404 pain (nociception) 502 pain perception 502 paired box transcription factor 7 (Pax7) 471 panels, sensory evaluation 44, 466 paralytic shellfish poisoning (PSP) 187, 547 parasites 187, 550 parents 98±102 parvoviruses 196 passive RFID tags 525, 526 pasteurization 280 Pavlov, Ivan 491 PeptACE 384, 385 Peptide ACE 3000 384, 385 Peptidea 385 peptides 363±98 bioactive properties 372±80 functional properties 380±3 future trends 386±7 market for functional peptide products 384±5 processing 368±72 perceived product convenience/ inconvenience 28±30 perceived quality 95±7

WPNL0206

Index perimysium 468 permanent viral contamination 230 peroxide value (PV) 433, 436±8 peroxy radicals 427 pH formation of histamine 312±14 lipid oxidation 429 interactions between antioxidants, emulsifiers and pH 441±2 pH and emulsifiers 433, 439±41 pH-shift processes 365±8, 386 pharmacy sales 227 phenolic antioxidants 447±50 phospholipids 428 Photobacterium phosphoreum 299, 301, 302, 308 photoperiod 480±1 Physicians' Health Study 167 picobirnavirus 204 pigment colour producer 534 plasmid analysis 272 plasmid fingerprinting 272 plasmid profiling 272 plasticisers 414±15 Poland 21±30, 67, 72±80 polar cod 348 polar paradox theory 441 poliovirus 204 type 3 202 pollution sources of microbial pollution 215±17 REDRISK project 217±25 see also faecal contamination; sewage contamination polyethyleneglycol (PEG) 415 polymerase chain reaction (PCR) 197, 221, 263 bacterial pathogens 267±9 assays for detection 268±9 assays for identification 267±8 MNP-PCR 270 molecular typing 272±8 PCR-FINS 556 real-time PCR 270±1, 279, 550 real-time RT-PCR 199, 201 Rep PCR 273 RT-PCR 199, 200±1, 268±9 post-mortem softening of flesh 477±9 postpartum depression 137±8, 147±9, 153 PPAR receptors 123±4 pR72H PCR assay 268 pre-eclampsia 146±7 preferences see consumer preferences pregnancy 143±9, 153

579

and birth outcome 143±6 and delivery complications 146±7 postpartum depression 137±8, 147±9, 153 premature ventricular complexes (PVCs) 172±4 preservatives 404 preserved semi-finished products (PSFPs) 400 price 19, 25, 27 primary cardiac arrest 167 primer sets 200 process mappings 524, 530±7 farmed Norwegian salmon 532±7 method 531±2, 533 processing conditions and validation of traceability 558 improved yield in traditional processing 364±8 mild see mild processing technologies and bacterial pathogens 280 product attributes 87±9 sensory evaluation and perceived quality 96±7 product convenience 92 perceived 28±30 see also convenience product development 6, 329±30 functional foods see functional products product evaluation see consumer evaluation product type and heart disease 168 and seafood consumption in Europe 22, 41 production batch size 523 production chains 1, 2, 5±6, 327±30, 514±15 aquaculture see aquaculture process mappings 524, 530±7 Seafood Sensory Quality Model 55±7 traceability see traceability; validation of traceability production cycle, welfare during 498±502 production intensity 496, 499±500 production method aquaculture see aquaculture validation of traceability 557±8 PROPEPHEALTH project 6, 364, 377, 378, 379 propyl gallate 448±9

WPNL0206

580

Index

prostaglandin E2 (PGE2) 124 protein analysis techniques 555±6 protein hydrolysates see fish protein hydrolysates protein isolates 327±8, 365±8, 386 protein oxidation 426±60 analytical methods 427, 433±5 future trends 451±2 model systems 434±46 natural antioxidants in fish products 446±50 processes leading to 427±33, 440 proteins 363±98 bioactive properties of fish protein hydrolysates 372±80 emulsifiers 431±2 functional properties of dried marine proteins 380±3 future trends 386±7 market for functional marine proteins 384±5 processing 368±72 protein-protein interactions 345±7 protein-water interactions 345±7 salt solubility and lipid oxidation 445±6 proteolytic enzymes 477±9 Protizen 385 proximity to viral sources 222, 224, 225 Pseudomonas spp. 413 Psychrobacter spp. 401 psychrotolerant bacteria 299, 301, 302, 308 pulsed field gel electrophoresis (PFGE) 274±8 pulsed light technology 415±17 application to seafood products 417 impact on survival and growth of bacteria 416±17 purification 236±7, 256±7 putrescine 302 pyridinoline 474±7 quality 82 perceived quality and satisfaction 95±8 satisfaction, intention and 100±1 sensory see sensory quality texture see texture validation of traceability 543, 545, 550±4 quality control programmes 190 Quality Index Method (QIM) 42, 57, 551 Quantitative Descriptive Analysis (QDA) 42±4

radio frequency identification data (RFID) tags 524±30 choice of technology/equipment 527±8 international standards 526±7 status and trends 525±6 testing temperature loggers 529±30, 531 rainfall 215±16, 217, 222, 224, 225, 241 randomly amplified polymorphic DNA (RAPD) 273±4, 275 raw materials, minimal mixing of 523 reactive oxidative species (ROS) 377 ready meals 31±6, 331±2 real-time PCR 270±1, 279, 550 real-time RT-PCR 199, 201 realism, in test situations 88, 89±91 recommended daily intakes (RDIs) 113 REDRISK project 5, 198, 213±14, 217±25, 240±1 approaches to risk management identified from 225±9 comparison of results from different sites 222, 224 factors leading to viral contamination 221±5 French site 218, 220, 221±5 Irish site 218, 220±5 site characteristics 221±2, 223 site selection 218 Spanish sites 218, 219, 221±5 UK sites 218, 219±20, 221±5 reducible (immature) crosslinks 470 reference methods bacteria 262 biotoxins 548 volatile amines 553 REFHEPA project 5, 198, 205, 221, 550 refrigeration 315 regular users 26±8 regulation 189±90 aquaculture 508 and welfare 496, 498 biopreservation 410, 411 EU see European Union (EU) histamine 306±7 packaging 414 shellfish harvesting areas 212, 213 rehydration 401±4 re-laying 236±7, 257 reliability indicators 542±4 repetitive element PCR (Rep PCR) 273 response surface modelling 439 restructured fish products 337±48, 355 development of functional restructured products 343±8

WPNL0206

Index novel ingredients 337±42 resuscitation 262 reverse transcription PCR (RT-PCR) 199, 200±1, 268±9 real-time RT-PCR 199, 201 ribotyping 272 risk associated with faecal contamination of shellfish 214±15 associated with seafood consumption 185±8 control of risks 189±90 emerging bacterial risks 278±9 information segments and seafood risk perception 77±9, 82 potential 2 see also safety risk analysis-risk matrix approach 229±32 risk assessment 229±32 risk management 225±37, 240±1 approaches identified from REDRISK project 225±9 strategies 232±7 risk matrix 229±32 risk periods 226, 240 river flow 222, 225 effect of rainfall 227±9 RNA 201 rotaviruses 201, 202 safety 5, 185±93 bacterial pathogens see bacterial pathogens biogenic amines see biogenic amines; histamine contribution of SEAFOODplus 190±1 control of risks 189±90 future trends 191±2 information segments and perception of 77±9, 82 relative incidence of microbiological illness 188±9 risks associated with seafood consumption 185±8 shellfish harvesting areas see shellfish harvesting areas validation of traceability 543, 544, 545±50 viruses see viruses salient beliefs 24, 25, 96±7 salmon 40, 126±7, 349 Atlantic salmon 467, 469, 471±2, 473, 474±7, 479, 480, 481, 482

581

HFP 297 process mapping for farmed Norwegian salmon 532±7 sensory evaluation 44, 53±4 consumer preferences 52±3 sensory characteristics 47±50 washed fish mince and lipid oxidation 444±5 salmon breeder 534 salmon feed producer 534 Salmonella spp. 215, 249, 258, 549 Agona 278 cold-smoked salmon 405±6, 407±8, 409±10, 417 detection methods 263±4 enteriditis 279 Paratyphi 249 Typhi 249 salt formation of histamine 312±14 hurdle in seafood processing 400±5 validation of traceability and salting 558 salt-cured cod 400±5 sanitary surveys 239, 240 sarcoplasmic proteins 365, 367 satiety, control of 379±80 satisfaction attitude and 97±8 quality, intention and 100±1 scallops 195 Sceptic consumer segment 67, 74±80 scombroid fish poisoning see histamine fish poisoning (HFP) SEA-INFOCOM project 4, 64 SEABAC project 5, 267 Seacure 379, 384, 385 Seafood Sensory Quality Model 55±7 SEAFOODplus 1, 2, 3±9, 136, 464, 483 contribution to seafood safety 190±1 map service 523±4 molecular typing of bacterial pathogens 274, 275±8 projects 3±7 SEAFOODSENSE project 3 seasonal limited viral contamination 231 seaweed antioxidant dietary fibre 338, 339, 340, 345 secondary oxidation projects 427±8, 433 secretagogue activities 375, 379±80 segmentation of consumers consumer information needs and 67, 74±81

WPNL0206

582

Index

interest in traceability issues between the segments 75±7 use of informational cues between segments 75, 76 use of and trust in information sources-based segmentation 74±5 convenience segmentation 30±4 differences across segments in relation to attitudes to fish and health orientation 34±6 related to attitudes and product preferences 53±5 selective plating 262 selenate 341, 342 selenium 8, 126, 128, 330 functional product development 337, 340±2 losses during household preparation of African catfish 351±2 selenium-enriched African catfish 349±54 selenium-enriched garlic 340, 341, 342, 349±50, 354 sensory descriptors 42±4 sensory quality 3, 14, 40±62 cod 43, 45±7, 50±2, 53±4 consumer preferences related to sensory characteristics 50±3 consumer segmentation across Europe 53±5 dried proteins and peptides 383 evaluation of freshness and validation of traceability 551±2 evaluation and texture 466 future trends 57±8 importance for industry and consumer 40±2 methods for evaluation 42±5 perceived quality and satisfaction 95±8 salmon 44, 47±50, 52±4 satisfaction, intention and 100±1 Seafood Sensory Quality Model 55±7 sensory characteristics of fish 45±50 sentience 491 sentinelle network 227 septicaemia 251, 253 serial number 520 Serial Shipping Container Code (SSCC) 519 severe acute respiratory syndrome (SARS) 202 sewage contamination 185±6, 194±5, 214±17, 226 due to sewage overflow 192, 222, 225

reduction at source 213, 232±4 see also faecal contamination sewage treatment systems 233±4 shelf-life 404 shellfish 5, 185±6, 188, 190 biotoxins 187±8, 546±8 bivalve molluscs see bivalve molluscs viruses 190, 194±211 methods for detection in shellfish 198±201 potential emerging virus problems 201±4 shellfish contamination 194±8 shellfish harvesting areas 5, 212±46 European regulation 212, 213, 233, 236±7, 238±9 future trends 237±9 REDRISK project 5, 198, 213±14, 217±25, 240±1 risk associated with faecal contamination 214±15 sources of microbial pollution 215±17 strategies to limit microbial contamination 225±37 general approaches to risk management 225±9 management strategy 232±7 risk analysis-risk matrix approach 229±32 Shigella spp. 249, 549 shopping 23±4 shrimps 408±9, 410 silver smelt 348 single nucleotide polymorphisms (SNPs) 121 singlet oxygen 428±9 site-specific natural isotope fractionation studied by NMR (SNIF-NMR) 557 site-specific strategies 232±4 Skipjack tuna 309±10 slaughter 502±4 smart cards 526 SmartTag 506±7 smell/odour 19, 25, 26 cod 43, 45±7 salmon 44, 47±50 smoked, dried, fermented fish product 371 smolt producer 535 social norms 103±4 sodium benzoate 404 sodium caseinate 439±41 sodium sulphite 404

WPNL0206

Index soft flesh problem 465 softening, post±mortem 477±9 solubility of dried proteins and peptides 381±2 Spain 53 consumer attitudes and seafood consumption 18±20, 21±30 consumer information needs 65±6, 67, 69±80 fish burger product 98±102 REDRISK project sites 218, 219, 221±5 species consumption in Europe 22±3 and HFP 297, 298 methods for authentication of 555±6 species-specific strategies 232±4 underutilised 348, 368 specific spoilage micro-organism plate count (SSMPC) 554 spoilage bacteria control using lactic acid bacteria 406±9 impact of pulsed light 416±17 validation of traceability 554 spray drying 381 standards RFID tags 526±7 validation of traceability 540±1 Staphylococcus spp. 401 aureus 250, 265, 549 xylosus 401±2 starvation 502 Stenotrophomonas maltophilia 278 storage atmosphere 312±14 storage temperature see temperature storm spills 192, 217 stress 380, 496 responses 499 stress shock protein synthesis 418 structural changes 345±7 stunning 502±4 suffering 502 supermarkets 23, 24 surfactants 431±2 surimi 363, 365 functional products based on 343±8, 355 SuspenTec process 365 sustainable aquaculture 495 synergistic effects 418 systematic reviews 169±70 tachycardia, ventricular 170±1 taste see flavour/taste

583

taste panels 44, 466 taurine 8, 137, 337±8 addition to fish muscle 347±8 loss in household preparation of African catfish 351, 352, 353 and obesity 139±40 TDH PCR assay 268 TDH-related haemolysin 252±3, 268 technical TraceFish standard 517±18 temperature control 192, 254 failure in 257±8 egg incubation temperature 480 histamine regulation and storage temperature 307, 315 time, temperature and formation of histamine 308±12 temperature loggers 529±30, 531 tendons 468 tertiary oxidation products 428 test locations 88, 89±91, 102±3 evaluation of fish burger 98±100 texture 6±7, 465±89 cellular and molecular mechanisms of muscle growth 470±3 cod 43, 45±7 environmental influences on muscle structural traits 479±81 factors influencing 466±7 future trends 482±3 heritability of muscle structural traits 481 measurement 466 myotomes 468±70 proteolytic enzymes and post-mortem softening of flesh 477±9 relationship to muscle structural traits 473±7 salmon 44, 47±50 theory of planned behaviour (TPB) 20, 24, 96 using in new product testing 101±2 thermostable direct haemolysin (TDH) 252±3 TDH PCR assay 268 thiobarbituric acid reactive substances (TBARS) 433, 436±8, 448±9 time barrier to seafood consumption 17, 19 consumer evaluation 91±3, 104 formation of histamine and 308±12 Tinbergen, Niko 491 tocopherol isomers 447

WPNL0206

584

Index

Torry scale 42, 551 total volatile bases (TVB) 552 total volatile basic nitrogen (TVB-N) 552, 553 totox value 433 toxR PCR assay 267±8 trace elements 8, 445 traceability 7, 508, 513±15, 516±38 consumer information needs 63, 65, 71, 73, 80 interest in traceability issues between segments 75±7 fish chain process studies 530±7 GTP manual 516±23 IMPLEM project 7, 515, 524±37 map service 523±4 METHODS project 7, 515, 516±24 RFID tags 524±30, 531 validation see validation of traceability traceability friendly production 521±3 traceable units 518±20 defining 518±19 keeping track of transformations 520±1, 522 unique identification of 519±20 trade 278±9 trade units (TUs) 518±19 identifying 519±20, 528 keeping track of transformations 520±1, 522 Traditional consumer segment 32, 33±6 transformations, keeping track of 520±1, 522 transport control of bacterial contamination 256±7 process mapping for farmed Norwegian salmon 536±7 TRH PCR assay 268 triacylglycerols (TGs) 428 trimethylamine (TMA) 552±4 trimethylamine oxide (TMAO) 552, 553 trust in information sources 8, 68, 71±4 segmentation by 74±5 tryptamine 299, 300 tuna 168 histamine and biogenic amines 302, 303, 309±10 typing, molecular 271±8 tyramine 302 ubiquitin-proteosome complex 477 ulcerative colitis (UC) 125 ultrafiltration 369±70, 371 ultraviolet (UV) disinfection systems

233±4 underutilised fish species 348, 368 United Kingdom (UK) REDRISK project sites 218, 219±20, 221±5 seafood-associated illness 188±9, 259±60 United States of America (USA) country of origin labelling (COOL) 513 HFP 295, 296 regulation of histamine 306, 307 seafood-associated illness 188, 215±17, 260±1 vacuum packaging 312, 313, 404 VALID project 7, 515, 544±5, 559±60 validated traceability systems 514±15 validation of traceability 7, 515, 539±66 data management and information flow 543, 545, 559 ensuring safety 543, 544, 545±50 establishment of indicators for 542±5 prevention of fraud 543, 545, 554±8 principles for 540±2 quality concerns 543, 545, 550±4 specific tools 545 Vasotensin 384, 385 vegetables 126 ventricular fibrillation 170±1 ventricular tachycardia 170±1 verification 541 Vibrio spp. 5, 191, 248±53, 549 cholerae 250, 251, 252 detection methods conventional 265±6 molecular 267±71 fluvialis 251, 253 foodborne outbreaks 260±1 furnissii 251, 253 hollisae 251, 253 mimicus 251, 253 parahaemolyticus 191, 249, 251, 252±3, 261 vulnificus 249, 251, 253 viruses 3, 5, 185±6, 190, 194±211 contamination of shellfish harvesting areas see shellfish harvesting areas elution and concentration 198±9 identification of probability of occurrence of viruses in the environment 226±9 methods for detection in shellfish 198±201

WPNL0206

Index potential emerging virus problems 201±4 and shellfish contamination 194±8 validation of traceability 550 see also hepatitis A virus (HAV); noroviruses (NoV) viscera 369 vitamins A 146 D 126, 128 E 444, 445 producer of for aquaculture 534 vocabulary sensory evaluation 42, 43, 44 traceability 516, 517 volatile amines 552±4 Wahrburg apparatus 436 Wales 259±60 see also United Kingdom (UK) washed fish mince 434, 442±6, 450±1 water-holding capacity 381 water-in-oil emulsions 431 water-protein interactions 345±7 weight reduction 8 welfare 7, 490±510 animal welfare 490±2, 493, 494±5, 507±8

585

common interests of fish farmers and consumers 496±8 during the production cycle 498±502 during slaughter 502±4 future trends 507±8 monitoring ethical qualities 505±7 reasons for concern about fish 492±3 understanding and assessing fish welfare 493±5 wild vs farmed fish 495±6 welfare assessment system (WAS) 495 welfare indicators 495, 505 breathing patterns as 505±7 well boats 535 wheat dietary fibre 343±5, 346±7 white (fast) muscle 468 wild fish captured fish standard 517±18 validation of traceability 557±8 welfare vs farmed fish 495±6 willingness to pay (WTP) 93±4 wolf fish, northern 348 yield, technologies to improve 364±8 YOUNG project 4, 136±64 Zeta potential 432, 438

WPNL0206