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]

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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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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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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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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


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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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 Ìmproved 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 Èvaluating 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

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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

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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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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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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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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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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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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

22


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


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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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 ùnpleasant') 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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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


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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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 òften 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 Èating 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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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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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 ìnconvenience' 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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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: · Ì 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' · Ì prefer meals that are quick to plan, buy (provide), prepare and cook' and · Ì 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' · Ìt is difficult to plan, provide, prepare and cook fish for a meal (dinner)' and · Ìt 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 Èasy Cooking' and one concerning `Real Ready Meals'. The Èasy Cooking' factor consists of seven statements related to aspects of time, effort, planning and thinking when preparing a meal: Ì 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', Ì prefer meals that are quick to plan, buy (provide), prepare and cook', Ì want to spend as little time as possible on planning and buying of what to have for meals', Ì prefer meals that can be prepared and cooked quickly' and Ìt 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: Ì often use/buy ready meals', Èating 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: Ì feel bad serving convenience food for my family', Ì 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: Ì always try to make amends if I have been eating convenience foods' and Ì 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


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', Ì 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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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 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 (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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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

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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


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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Cardiac benefits of fish consumption may depend on the type of fish meal consumed ± The Cardiovascular Health Study. Circulation 2003; 107(10): 1372±7. 22. MOZAFFARIAN D, PRINEAS RJ, STEIN PK, SISCOVICK DS. Dietary fish and n-3 fatty acid intake and cardiac electrocardiographic parameters in humans. J Am Coll Cardiol 2006; 48(3): 478±84. 23. INVESTIGATORS G-P. 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 1999; 354(9177): 447±55. 24. BURR ML, ASHFIELD WP, DUNSTAN FDJ, et al. Lack of benefit of dietary advice to men with angina: results of a controlled trial. Eur J Clin Nutr 2003; 57(2): 193±200.

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Improving seafood products for the consumer et al. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: diet and reinfarction trial (DART). Lancet 1989; 2(8666): 757±61. NESS AR, HUGHES J, ELWOOD PC, WHITLEY E, SMITH GD, BURR ML. The long-term effect of dietary advice in men with coronary disease: follow-up of the Diet and Reinfarction trial (DART). Eur J Clin Nutr 2002; 56(6): 512±18. BURR ML, DUNSTAN FD, GEORGE CH. Is fish oil good or bad for heart disease? Two trials with apparently conflicting results. J Membr Biol 2005; 206(2): 155±63. HOOPER L, THOMPSON RL, HARRISON RA, et al. Risks and benefits of omega 3 fats for mortality, cardiovascular disease, and cancer: systematic review. BMJ 2006; 332(7544): 752±60. GELEIJNSE JM, BROUWER IA, FESKENS EJ. Risks and benefits of omega 3 fats: health benefits of omega 3 fats are in doubt. BMJ 2006; 332(7546): 915; discussion 916. BUCHER HC, HENGSTLER P, SCHINDLER C, MEIER G. N-3 polyunsaturated fatty acids in coronary heart disease: a meta-analysis of randomized controlled trials. Am J Med 2002; 112(4): 298±304. HE K, SONG Y, DAVIGLUS ML, et al. Accumulated evidence on fish consumption and coronary heart disease mortality: a meta-analysis of cohort studies. Circulation 2004; 109(22): 2705±11. WANG C, HARRIS WS, CHUNG M, et al. n-3 Fatty acids from fish or fish-oil supplements, but not alpha-linolenic acid, benefit cardiovascular disease outcomes in primary- and secondary-prevention studies: a systematic review. Am J Clin Nutr 2006; 84(1): 5± 17. ZIPES DP, WELLENS HJ. Sudden cardiac death. Circulation 1998; 98(21): 2334±51. HUIKURI HV, CASTELLANOS A, MYERBURG RJ. Sudden death due to cardiac arrhythmias. N Engl J Med 2001; 345(20): 1473±82. RAITT MH, CONNOR WE, MORRIS C, et al. Fish oil supplementation and risk of ventricular tachycardia and ventricular fibrillation in patients with implantable defibrillators: a randomized controlled trial. JAMA 2005; 293(23): 2884±91. LEAF A, ALBERT CM, JOSEPHSON M, et al. Prevention of fatal arrhythmias in high-risk subjects by fish oil n-3 fatty acid intake. Circulation 2005; 112(18): 2762±8. BROUWER IA, ZOCK PL, CAMM AJ, et al. Effect of fish oil on ventricular tachyarrhythmia and death in patients with implantable cardioverter defibrillators: the Study on Omega-3 Fatty Acids and Ventricular Arrhythmia (SOFA) randomized trial. JAMA 2006; 295(22): 2613±19. BROUWER IA, GEELEN A, KATAN MB. n-3 Fatty acids, cardiac arrhythmia and fatal coronary heart disease. Prog Lipid Res 2006; 45(4): 357±67. SCHREPF R, LIMMERT T, CLAUS WP, THEISEN K, SELLMAYER A. Immediate effects of n-3 fatty acid infusion on the induction of sustained ventricular tachycardia. Lancet 2004; 363(9419): 1441±2. CHRISTENSEN JH, RIAHI S, SCHMIDT EB, et al. n-3 Fatty acids and ventricular arrhythmias in patients with ischaemic heart disease and implantable cardioverter defibrillators. Europace 2005; 7(4): 338±44. KANNEL WB, WOLF PA, BENJAMIN EJ, LEVY D. Prevalence, incidence, prognosis, and predisposing conditions for atrial fibrillation: population-based estimates. Am J Cardiol 1998; 82(8A): 2N±9N. MORRIS DH. Methodologic challenges in designing clinical studies to measure differences in the bioequivalence of n-3 fatty acids. Mol Cell Biochem 2003; 246(1± 2): 83±90. BURR ML, FEHILY AM, GILBERT JF,

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et al. Fish intake and risk of incident atrial fibrillation. Circulation 2004; 110(4): 368±73. FROST L, VESTERGAARD P. n-3 Fatty acids consumed from fish and risk of atrial fibrillation or flutter: the Danish Diet, Cancer, and Health Study. Am J Clin Nutr 2005; 81(1): 50±4. BROUWER IA, HEERINGA J, GELEIJNSE JM, ZOCK PL, WITTEMAN JC. Intake of very longchain n-3 fatty acids from fish and incidence of atrial fibrillation. The Rotterdam Study. Am Heart J 2006; 151(4): 857±62. CALO L, BIANCONI L, COLIVICCHI F, et al. N-3 Fatty acids for the prevention of atrial fibrillation after coronary artery bypass surgery: a randomized, controlled trial. J Am Coll Cardiol 2005; 45(10): 1723±8. JIMENEZ RA, MYERBURG RJ. Sudden cardiac death. Magnitude of the problem, substrate/trigger interaction, and populations at high risk. Cardiol Clin 1993; 11(1): 1±9. Risk stratification and survival after myocardial infarction. The Multicenter Postinfarction Research Group. N Engl J Med 1983; 309(6): 331±6. MAGGIONI AP, ZUANETTI G, FRANZOSI MG, et al. Prevalence and prognostic significance of ventricular arrhythmias after acute myocardial infarction in the fibrinolytic era. GISSI-2 results. Circulation 1993; 87(2): 312±22. STATTERS DJ, MALIK M, REDWOOD S, HNATKOVA K, STAUNTON A, CAMM AJ. Use of ventricular premature complexes for risk stratification after acute myocardial infarction in the thrombolytic era. Am J Cardiol 1996; 77(2): 133±8. JOUVEN X, ZUREIK M, DESNOS M, COURBON D, DUCIMETIERE P. Long-term outcome in asymptomatic men with exercise-induced premature ventricular depolarizations. N Engl J Med 2000; 343(12): 826±33. HARDARSON T, KRISTINSSON A, SKULADOTTIR G, ASVALDSDOTTIR H, SNORRASON SP. Cod liver oil does not reduce ventricular extrasystoles after myocardial infarction. J Intern Med 1989; 226(1): 33±7. SELLMAYER A, WITZGALL H, LORENZ RL, WEBER PC. Effects of dietary fish oil on ventricular premature complexes. Am J Cardiol 1995; 76(12): 974±7. GEELEN A, BROUWER IA, SCHOUTEN EG, MAAN AC, KATAN MB, ZOCK PL. Effects of n-3 fatty acids from fish on premature ventricular complexes and heart rate in humans. Am J Clin Nutr 2005; 81(2): 416±20. CHRISTENSEN JH, GUSTENHOFF P, EJLERSEN E, et al. N-3 Fatty acids and ventricular extrasystoles in patients with ventricular tachyarrhythmias. Nutrition Research 1995; 15(1): 1±8. CHRISTENSEN JH, GUSTENHOFF P, KORUP E, et al. N-3 fatty acids and ventricular arrhythmias in post-myocardial infarction patients with a low ejection fraction. In: Kristensen SD, Schmidt EB, De Caterina R, Endres S, eds. N-3 fatty acids: prevention and treatment in vascular disease. London: Springer Verlag; 1995: 107±14. SINGER P, WIRTH M. Can n-3 PUFA reduce cardiac arrhythmias? Results of a clinical trial. Prostaglandins Leukot Essent Fatty Acids 2004; 71(3): 153±9. BILLMAN GE, KANG JX, LEAF A. Prevention of sudden cardiac death by dietary pure omega-3 polyunsaturated fatty acids in dogs. Circulation 1999; 99(18): 2452±7. BILLMAN GE, KANG JX, LEAF A. Prevention of ischemia-induced cardiac sudden death by n-3 polyunsaturated fatty acids in dogs. Lipids 1997; 32(11): 1161±8. MCLENNAN PL. Relative effects of dietary saturated, monounsaturated, and polyunsaturated fatty acids on cardiac arrhythmias in rats. Am J Clin Nutr 1993; 57(2): 207±12. MOZAFFARIAN D, PSATY BM, RIMM EB,

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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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Fig. 12.1

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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 ìnfluenza-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

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and LEMON S M (1992), `Genetic relatedness of hepatitis A virus strains recovered from different geographical regions', J Gen Virol, 73, 1365±1377. ROMALDE J L, ESTES M K, SZUCS G, ATMAR R L, WOODLEY C M and METCALF T G (1994), Ìn situ detection of hepatitis A virus in cell cultures and shellfish tissues', App Environ Microbiol, 60, 1921±1926. MARGOLIS H S, ISOMURA S, ITO K, ISHIZU T, MORITSUGU Y

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and BOSCH A (2002), `Molecular characterization of hepatitis A virus isolates from a transcontinental shellfish-borne outbreak' J Clin Microbiol, 40, 4148±4155. Â NCHEZ G, VILLENA C, BOSCH A and PINTO Â R M (2004), `Molecular detection and typing of SA hepatitis A virus', in Spencer J F T and Ragout de Spencer A L, Methods in Molecular Biology vol 268: Public Health Microbiology: Methods and Protocols, Totowa, NJ, Humana Press, 103±114. SCHLAUDER G G and MUSHAHWAR I K (2001), `Genetic heterogeneity of hepatitis E virus', J Med Virol, 65, 282±292. SCHWAB K J, NEILL F H, ESTES M K and ATMAR R L (1998), Ìmprovements in the RT-PCR detection of enteric viruses in environmental samples', Wat Sci Tech, 38, 83±86. Â NCHEZ G, PINTO Â R M, VANACLOCHA H SA

SCHWAB K J, NEILL F H, FANKHAUSER R L, DANIELS N A, MONROE S S, BERGMIRE-SWEAT D A, ESTES M K and ATMAR R L (2000), `Development of methods to detect ``NorwalkLike Viruses'' (NLVs) and Hepatitis A virus in delicatessen foods: application to a food-Borne NLV outbreak', Appl Environ Microbiol, 66, 213±218. SHIEH Y S C, CALCI K R and BARIC R S (1999), À method to detect low levels of enteric viruses in contaminated oysters', Appl Environ Microbiol, 65, 4709±4714. SHIEH Y S C, MONROE S S, FANKHAUSER R L, LANGLOIS G W, BURKHARDT W and BARIC R S (2000), `Detection of Norwalk-like virus in shellfish implicated in illness', J Inf Dis, 181, 360±366. STALLKNECHT D E, SHANE S M, KEARNEY M T and ZWANK P J (1990), `Persistence of avian influenza viruses in water', Avian Dis, 34, 406±411. STIENEKE-GRUBER A, VEY M, ANGLIKER H et al. (1992), Ìnfluenza virus hemagglutinin with multiple cleavage sites is activated by furin, a subtilisin-like endoprotease', EMBO J, 11, 2407±2414. TAM A W, SMITH M M, GUERRA M E et al. (1991), `Hepatitis E virus (HEV): molecular cloniong and sequencing of the full-length viral genome', Virology, 185, 120±131. TAMADA Y, YANO K, YATSUHASHI H, INOUE O, MAWATARI F and ISHIBASHI H. (2004), `Consumption of wild boar linked to cases of hepatitis', Eur J Hepatol, 40, 869±870. TEI S, KITAJIMA N, TAKAHASHI K and MISHIRO S. (2003), `Zoonotic transmission of hepatitis E virus from deer to human beings', Lancet, 362, 371±373.

TOMPKINS D S, HUDSON M J, SMITH H R, EGLIN R P, WHEELER J G, BRETT M M, OWEN R J,

and COOK P E (1999), À study of infectious intestinal disease in England: microbiological findings in cases and controls', Comm Dis Pub Health, 2, 108±113. Y L, TRAN B, SANGERMANO L R and PALMER C J (1994), `Detection of poliovirus, hepatitis A virus, and rotavirus from sewage and ocean water by triplex reverse transcriptase PCR', Appl Environ Microbiol 60, 2400±2407. DER POOL W H, VERSHOOR F, VAN DER HEIDE R et al. (2001), `Hepatitis E virus sequences in swine related to sequences in humans', Emerg Infect Dis, 7, 970±976. BRAZIER J S, CUMBERLAND P, KING V

TSAI

VAN

Â R M, GUIX S, DONIA D, BUONOMO E, PALOMBI L, CENKO F, BINO VILLENA C, GABRIELLI R, PINTO

and DIVIZIA M (2003), À large infantile gastroenteritis outbreak in Albania caused by multiple emerging rotavirus genotypes', Epidemiol Inf, 131, 1105±1110.

S, BOSCH A

VINJE J, VENNEMA H, MAUNULA L, VON BONSDORFF C-H, HOEHNE M, SHREIER E, RICHARDS A,

GREEN J, BROWN D, BEARD S, MONROE S, DE BRUIN E, SVENSSON L and KOOPMANS M P G (2003), Ìnternational collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of Noroviruses', J Clin Microbiol, 41, 1423± 1433.

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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

45% for suspended solids concentrations of 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 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

100 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 à 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


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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419

Source of further information and advice

Some sources on further information concerning: Hurdle technology

(2005), Ùpdate 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), Èdible 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), Àntmicrobial 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), Ìnactivation 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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19.9


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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 àntioxidative' 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 àntioxidant'. 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


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

20.12

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.

20.13

References

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GUNNARSSON G, UNDELAND I, SANNAVEERAPPA T, SANDBERG A S, LINDGARD A, MATTSSON-

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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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22.2


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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The biological basis of variability in the texture of fish flesh 479 which can be detected using specific antibodies has proved to be a very useful marker of the early events of proteolysis during post-mortem storage of the flesh (Bonnal et al., 2001).

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 èyed stage' around half way through embryonic development. Fish reared at 5 ëC to the èyed 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 (

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