Improving the safety and quality of eggs and egg products
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Related titles: Improving the safety and quality of eggs and egg products: Volume 1 (ISBN 978-1-84569-754-9) Eggs are a convenient and economical food of high nutritional value, yet are also a significant source of foodborne disesase. The two volumes of this important collection review essential research and best practice in the production and processing of eggs with particular emphasis on their safety, nutritional and sensory quality. Opening chapters in Volume 1 set the scene with information on egg production and consumption worldwide. Following chapters introduce essential topics in egg chemistry and provide and overview of egg quality parameters. Chapters in parts III and IV then focus on egg production systems and their influences on egg safety and quality. The final section of the volume reviews the nutritional quality of eggs and their use as food ingredients. Food safety control in the poultry industry (ISBN 978-1-85573-954-3) Consumers’ expectations about the safety of products such as poultry meat and eggs have never been higher. The need to improve food safety has led to renewed attention on controlling contamination at all stages of the supply chain from ‘farm to fork’. This collection reviews the latest research and best practice in ensuring the safety of poultry meat and eggs, both on the farm and in subsequent processing operations. Microbiological analysis of red meat, poultry and eggs (ISBN 978-1-84569-059-5) Microbiological analysis has long been used to monitor the microbial quality and safety of red meat, poultry and eggs, whether in relation to guidelines, product specifications or legally enforceable standards. These food products are, or have been, major global causes of foodborne human disease and are also susceptible to microbial growth and spoilage. Therefore monitoring their safety and quality remains a concern. With the recent development of more preventative, risk-based approaches to food safety control, microbiological testing of foods now has a more significant role to play in food safety management. With chapters written by international experts, this collection reviews the keys issues in this dynamic area of food microbiology. Details of these books and a complete list of Woodhead’s titles can be obtained by: ∑ visiting our web site at www.woodheadpublishing.com ∑ contacting Customer Services (e-mail:
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Woodhead Publishing Series in Food Science, Technology and Nutrition: Number 214
Improving the safety and quality of eggs and egg products Volume 2: Egg safety and nutritional quality Edited by Filip Van Immerseel, Yves Nys and Maureen Bain
Oxford
Cambridge
Philadelphia
New Delhi
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Published by Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge CB22 3HJ, UK www.woodheadpublishing.com Woodhead Publishing, 1518 Walnut Street, Suite 1100, Philadelphia, PA 19102-3406, USA Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India www.woodheadpublishingindia.com First published 2011, Woodhead Publishing Limited © Woodhead Publishing Limited, 2011; Chapter 5 © Crown copyright, 2011 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 Control Number: 2011934931 ISBN 978-0-85709-072-0 (print) ISBN 978-0-85709-392-9 (online) ISSN 2042-8049 Woodhead Publishing Series in Food Science, Technology and Nutrition (print) ISSN 2042-8057 Woodhead Publishing Series in Food Science, Technology and Nutrition (online) The publisher’s 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 elemental chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Replika Press Pvt Ltd, India Printed by TJI Digital, Padstow, Cornwall, UK
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Contents
Contributor contact details..................................................................
xi
Woodhead Publishing Series in Food Science, Technology and Nutrition............................................................................................... xvii Preface................................................................................................. xxxi Part I Microbial and chemical contamination of eggs................
1
1 Microbiology and safety of table eggs ..................................... M. T. Musgrove, United States Department of Agriculture, USA 1.1 Introduction....................................................................... 1.2 Washing table eggs........................................................... 1.3 Table egg facility sanitation.............................................. 1.4 Regulations........................................................................ 1.5 Microbiology of table eggs............................................... 1.6 Bringing eggs and foodborne disease into perspective..... 1.7 Acknowledgements........................................................... 1.8 References and further reading.........................................
3
2 Foodborne disease associated with eggs: microbial hazards and Salmonella Enteritidis risk assessment............................. M. Chemaly and G. Salvat, Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses), France 2.1 Introduction....................................................................... 2.2 Hazard identification......................................................... 2.3 Quantitative risk assessment: Salmonella Enteritidis in eggshells............................................................................ 2.4 Conclusion......................................................................... 2.5 References and further reading.........................................
3 6 13 14 15 22 25 25 34
34 35 38 41 41
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vi Contents 3 Internal contamination of eggs by Salmonella Enteritidis..... R. Raspoet, I. Gantois, R. Devloo, F. Pasmans, F. Haesebrouck, R. Ducatelle and F. Van Immerseel, Ghent University, Belgium 3.1 Salmonella Enteritidis and eggs: a close connection........ 3.2 Eggshell surface contamination........................................ 3.3 Eggshell penetration.......................................................... 3.4 Contamination of the egg during development in the reproductive tract.............................................................. 3.5 Salmonella Enteritidis virulence factors involved in chicken reproductive tract colonization............................ 3.6 References.........................................................................
46
4 Chemical residues and contaminants in eggs.......................... C. Jondreville, A. Fournier and C. Feidt, Institut National de la Recherche Agronomique (INRA), Nancy Université, France, A. Travel, Institut Technique de l’Aviculture (ITAVI), France and B. Roudaut, Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses), France 4.1 Introduction....................................................................... 4.2 Chemical contaminants in animal-derived foodstuffs: origins and regulatory context.......................................... 4.3 Modes of transfer into the egg.......................................... 4.4 Monitoring strategies......................................................... 4.5 Origin of non-conformities and preventing risk during rearing............................................................................... 4.6 Conclusion......................................................................... 4.7 References.........................................................................
62
74 76 77
Part II Salmonella control in laying hens .....................................
81
46 47 47 49 51 57
62 63 66 71
5 Detection and monitoring of Salmonella in laying hen flocks . ......................................................................................... 83 R. Davies and J. J. Carrique-Mas, Veterinary Laboratories Agency, UK 5.1 Introduction....................................................................... 83 5.2 What to sample................................................................. 84 5.3 Recommended sampling regime of laying houses............ 91 5.4 Serology............................................................................ 94 5.5 Methods used for the 2004/2005 baseline survey and Salmonella control programmes in flocks of laying hens in the European Union...................................................... 95 5.6 Factors affecting the detection of Salmonella infection... 96 5.7 Significance of under-detection......................................... 99 5.8 References......................................................................... 100
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Contents vii 6 Epidemiology of Salmonella infections in laying hens with special emphasis on the influence of the housing system........ J. Dewulf, S. Van Hoorebeke and F. Van Immerseel, Ghent University, Belgium 6.1 Introduction....................................................................... 6.2 Effect of the housing system on Salmonella prevalence.. 6.3 Factors related to housing systems and Salmonella prevalence.......................................................................... 6.4 Presence of Salmonella serotypes other than S. Enteritidis in outdoor production systems.................... 6.5 Conclusions....................................................................... 6.6 Acknowledgement............................................................. 6.7 References......................................................................... 7 Pre-harvest measures to control Salmonella in laying hens... R. K. Gast, United States Department of Agriculture, Agricultural Research Service, USA 7.1 Introduction....................................................................... 7.2 Vaccination........................................................................ 7.3 Genetic selection for naturally occurring resistance......... 7.4 Gastrointestinal colonization control................................ 7.5 Future trends..................................................................... 7.6 Sources of further information and advice....................... 7.7 References......................................................................... 8 Management and sanitation procedures to control Salmonella in laying hen flocks................................................. R. Ducatelle and F. Van Immerseel, Ghent University, Belgium 8.1 Introduction....................................................................... 8.2 Management procedures to prevent introduction of Salmonella on the farm or to suppress the infection pressure from the environment......................................... 8.3 Sanitation and decontamination........................................ 8.4 Future trends: management and sanitation procedures for a Salmonella-free production chain............................. 8.5 Sources of further information and advice....................... 8.6 References......................................................................... 9 Egg decontamination by washing............................................. W. Messens, Institute for Agricultural and Fisheries Research (ILVO), Belgium, J. Gittins, ADAS, UK, S. Leleu, Institute for Agricultural and Fisheries Research (ILVO), Belgium and N. Sparks, SAC, UK 9.1 Introduction....................................................................... 9.2 Historical and commercial perspective............................. 9.3 The egg washing process..................................................
107 107 108 111 114 115 115 115 120 120 121 127 129 134 135 136 146 146 148 153 157 158 158 163
163 164 166
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viii Contents 9.4
9.5 9.6 9.7 9.8
Factors that influence the microbiological quality of washed eggs...................................................................... Post-washing treatments.................................................... Benefits and problems associated with egg washing........ Conclusions....................................................................... References.........................................................................
10 Alternative egg decontamination techniques to washing........ A. Berardinelli, C. Cevoli, A. Fabbri, M. E. Guerzoni, G. Manfreda, F. Pasquali, L. Ragni and L. Vannini, University of Bologna, Italy 10.1 Introduction....................................................................... 10.2 Washing methods currently used in industry.................... 10.3 Hot air pasteurization........................................................ 10.4 Microwave pasteurization................................................. 10.5 Gas plasma........................................................................ 10.6 Pulsed light........................................................................ 10.7 Conclusions and future trends........................................... 10.8 References.........................................................................
169 173 175 176 177 181
181 182 182 186 188 192 194 194
Part III Eggs in nutrition and other applications........................... 199 11 The nutritional quality of eggs.................................................. I. Seuss-Baum, University of Applied Sciences Fulda, Germany and F. Nau and C. Guérin-Dubiard, AGROCAMPUS Ouest, France 11.1 Reputation of the egg........................................................ 11.2 Nutritional evaluation of eggs: composition . .................. 11.3 Nutritional evaluation of eggs: macronutrients ............... 11.4 Nutritional evaluation of eggs: micronutrients ................ 11.5 Improving the nutritional quality of eggs......................... 11.6 Conclusions....................................................................... 11.7 Sources of further information and advice (food tables).. 11.8 References......................................................................... 12
Eggs, dietary cholesterol and disease: facts and folklore....... B. A. Griffin, University of Surrey, UK 12.1 Egg nutrition: facts and folklore....................................... 12.2 Serum cholesterol and dietary cholesterol as risk factors for coronary heart disease................................................. 12.3 Impact of cholesterol perception on egg consumption..... 12.4 Evidence from egg-feeding studies in humans................. 12.5 The relative effects of dietary saturated fat and dietary cholesterol on serum cholesterol....................................... 12.6 Current consensus and recommendations ........................ 12.7 Conclusion......................................................................... 12.8 References.........................................................................
201
201 203 217 222 226 228 228 229 237 237 239 243 243 245 246 250 250
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Contents ix 13
Egg allergy.................................................................................. Y. Mine and M. Yang, University of Guelph, Canada 13.1 Introduction....................................................................... 13.2 Egg allergy: an overview ................................................. 13.3 Egg proteins ..................................................................... 13.4 Egg yolk allergenicity....................................................... 13.5 Effects of processing on the allergenicity of egg proteins.............................................................................. 13.6 Conclusion and future trends............................................ 13.7 References......................................................................... 14
Modifying egg lipids for human health.................................... F. Sirri and A. Meluzzi, University of Bologna, Italy 14.1 Introduction....................................................................... 14.2 Egg lipid fractions............................................................. 14.3 Fatty acids metabolism in laying hens.............................. 14.4 Effects of hen’s diet on lipid components........................ 14.5 Sensory characteristics of enriched eggs.......................... 14.6 Conclusion......................................................................... 14.7 References.........................................................................
15 Egg enrichment with vitamins and trace minerals................. A. Schiavone, University of Torino, Italy and A. C. Barroeta, Universitat Autònoma de Barcelona, Spain 15.1 Introduction....................................................................... 15.2 Egg enrichment with vitamins.......................................... 15.3 Water-soluble vitamin enrichment . ................................. 15.4 Egg enrichment in trace minerals..................................... 15.5 References......................................................................... 16 Bioactive fractions of eggs for human and animal health...... M. Anton, Institut National de la Recherche Agronomique (INRA), France and F. Nau and C. Guérin-Dubiard, AGROCAMPUS Ouest, France 16.1 Introduction....................................................................... 16.2 Egg fractions..................................................................... 16.3 Antibody applications....................................................... 16.4 Bioactive properties of eggs.............................................. 16.5 Cryoprotective activity...................................................... 16.6 Conclusions....................................................................... 16.7 References.........................................................................
254 254 255 259 263 264 266 266 272 272 274 276 279 283 284 285 289 289 290 301 304 314 321
321 322 327 329 336 339 340
17 Using egg IgY antibodies for health, diagnostic and other industrial applications................................................................ 346 J. Kovacs-Nolan and Y. Mine, University of Guelph, Canada 17.1 Introduction....................................................................... 346 17.2 Overview of the avian immune system and IgY biosynthesis....................................................................... 347 © Woodhead Publishing Limited, 2011
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x Contents
17.3 17.4 17.5 17.6 17.7 17.8
Production and purification of IgY................................... Advantages of eggs as an alternative antibody source..... Applications of IgY........................................................... Immunotherapeutic applications of IgY............................ Conclusion and future trends............................................ References.........................................................................
18 Strategic planning for the development of the egg nutraceutical industry................................................................ V. Guyonnet, Burnbrae Farms Ltd., Canada 18.1 Introduction....................................................................... 18.2 Egg nutraceutical business – strengths, weaknesses, opportunities and threats (SWOT) analysis...................... 18.3 Strategic goals for the egg nutraceutical business............ 18.4 Action plan for the egg nutraceutical business................. 18.5 Conclusion......................................................................... 18.6 References.........................................................................
348 350 352 354 361 361 374 374 375 387 389 392 392
Index..................................................................................................... 400
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Contributor contact details
(* = main contact) Editors F. Van Immerseel Department of Pathology, Bacteriology and Avian Diseases Faculty of Veterinary Medicine Ghent University Salisburylaan 133 B-9820 Merelbeke Belgium E-mail:
[email protected]
Y. Nys Institut National de la Recherche Agronomique INRA UR83 Recherches Avicoles F-37380 Nouzilly France E-mail:
[email protected]
M. Bain Institute of Biodiversity, Animal Health and Comparative Medicine College of Medical, Veterinary and Life Sciences University of Glasgow Garscube Estate Bearsden Road Glasgow G61 1QH UK E-mail:
[email protected]
Chapter 1 M. T. Musgrove Egg Safety and Quality Research Unit Agricultural Research Service United States Department of Agriculture Richard B. Russell Agricultural Research Center 950 College Station Road Athens, GA 30604 USA E-mail:
[email protected]
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xii Contributor contact details Chapter 2 M. Chemaly* and G. Salvat Research Unit Quality and Hygiene of Poultry and Pork Products Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses) BP 53 22440 Ploufragan France E-mail:
[email protected]
Chapter 3 R. Raspoet, I. Gantois, R. Devloo, F. Pasmans, F. Haesebrouck, R. Ducatelle and F. Van Immerseel* Research Group Veterinary Public Health and Zoonoses Department of Pathology, Bacteriology and Avian Diseases Faculty of Veterinary Medicine Ghent University Salisburylaan 133 B-9820 Merelbeke Belgium E-mail:
[email protected]
Chapter 4 C. Jondreville*, C. Feidt and A. Fournier Institut National de la Recherche Agronomique (INRA) Nancy Université Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires (ENSAIA) Unité de recherches Animal et Fonctionnalités des Produits Animaux (UR AFPA) 2, avenue de la Forêt de Haye 54500 Vandoeuvre-lès-Nancy France E-mail: catherine.jondreville@ensaia. inpl-nancy.fr;
[email protected];
[email protected]
A. Travel Institut Technique de l’Aviculture (ITAVI) Unité de Recherches Avicoles Centre de Tours 37380 Nouzilly France E-mail:
[email protected]
B. Roudaut Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses) Laboratoire de Fougères La Haute Marche 35133 Javené France E-mail:
[email protected]
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Contributor contact details xiii Chapter 5 R. Davies* and J. J. Carrique-Mas Department of Food and Environmental Safety Veterinary Laboratories Agency – Weybridge Woodham Lane New Haw Addlestone KT15 3NB UK
Chapter 7 Richard K. Gast United States Department of Agriculture Agricultural Research Service Egg Safety and Quality Research Unit 950 College Station Road Athens, GA 30605 USA
E-mail:
[email protected]
E-mail:
[email protected]
Chapter 6 J. Dewulf* and S. Van Hoorebeke Veterinary Epidemiology Unit Department of Reproduction Obstetrics and Herd Health Faculty of Veterinary Medicine Ghent University Merelbeke Belgium
Chapter 8 R. Ducatelle* and F. Van Immerseel Research Group Veterinary Public Health and Zoonoses Department of Pathology Bacteriology and Avian Medicine Faculty of Veterinary Medicine Ghent University Salisburylaan 133 B9820 Merelbeke Belgium
E-mail:
[email protected]
F. Van Immerseel Research Group Veterinary Public Health and Zoonoses Department of Pathology, Bacteriology and Avian Diseases Faculty of Veterinary Medicine Ghent University Salisburylaan 133 B-9820 Merelbeke Belgium
E-mail:
[email protected]
Chapter 9 W. Messens* Biological Hazards (BIOHAZ) Unit European Food Safety Authority (EFSA) Largo N. Palli 5/A I-43121 Parma Italy E-mail:
[email protected]
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xiv Contributor contact details J. Gittins ADAS Woodthorne Wergs Road Wolverhampton WV6 8QT UK
G. Manfreda and F. Pasquali Department of Food Science University of Bologna Via del Florio 2 – 40064 Ozzano dell’Emilia Italy
E-mail:
[email protected]
E-mail:
[email protected];
[email protected]
S. Leleu Institute for Agricultural and Fisheries Research (ILVO) Animal Sciences Unit Scheldeweg 68 B-9090 Melle Belgium E-mail:
[email protected]
N. Sparks Avian Science Research Centre SAC Ayr KA6 5HW UK E-mail:
[email protected]
Chapter 10 A. Berardinelli*, C. Cevoli, A. Fabbri and L. Ragni Agricultural Economics and Engineering Department University of Bologna Piazza G. Goidanich 60 – 47023 Cesena (FC) Italy
M. E. Guerzoni and L. Vannini Department of Food Science University of Bologna Viale Fanin 46 – 40127 Bologna Italy E-mail:
[email protected];
[email protected]
Chapter 11 I. Seuss-Baum* University of Applied Sciences Fulda Department of Food Technology Marquardstr, 35 36039 Fulda Germany E-mail:
[email protected]
F. Nau and C. Guérin-Dubiard AGROCAMPUS Ouest INRA UMR1253 Science et technologie du lait et de l‘oeuf France
E-mail:
[email protected];
[email protected];
[email protected];
[email protected].
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Contributor contact details xv Chapter 12 B. A. Griffin Faculty of Health and Medical Sciences University of Surrey Guildford GU2 7XH UK E-mail:
[email protected]
Chapter 13 Y. Mine* and M. Yang Department of Food Science University of Guelph Guelph, ON N1G 2W1 Canada E-mail:
[email protected]
Chapter 14 F. Sirri* and A. Meluzzi Department of Food Science University of Bologna Via del Florio 2 40064 Ozzano dell’Emilia Italy E-mail:
[email protected];
[email protected]
Chapter 15 A. Schiavone Department of Animal Production Epidemiology and Ecology University of Torino Via Leonardo da Vinci 44 10095 Grugliasco Torino Italy
A. C. Barroeta* Animal Nutrition Welfare Service (SNiBA) Department of Animal and Food Science Universitat Autònoma de Barcelona Edifici V – Campus de la UAB 08193 Barcelona Spain E-mail:
[email protected]
Chapter 16 M. Anton* UR1268 Biopolymères Interactions Assemblages Equipe Interfaces et Systèmes Dispersés INRA F-44316 Nantes cedex 3 France E-mail:
[email protected]
F. Nau and C. Guérin-Dubiard UMR 1253 INRA AGROCAMPUS OUEST Science et Technologie du Lait et de l’Œuf 65 rue de Saint-Brieuc CS 84215 35042 Rennes cedex France
E-mail:
[email protected]
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xvi Contributor contact details Chapter 17 J. Kovacs-Nolan and Y. Mine* Department of Food Science University of Guelph Guelph, ON N1G 2W1 Canada
Chapter 18 V. Guyonnet Burnbrae Farms Limited 3356 County Road 27 Lyn, ON K0E 1M0 Canada
E-mail:
[email protected]
E-mail:
[email protected]
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Woodhead Publishing Series in Food Science, Technology and Nutrition
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xx Woodhead Publishing Series in Food Science, Technology and Nutrition 71 Safety and quality issues in fish processing Edited by H. A. Bremner 72 Minimal processing technologies in the food industries Edited by T. Ohlsson and N. Bengtsson 73 Fruit and vegetable processing: improving quality Edited by W. Jongen 74 The nutrition handbook for food processors Edited by C. J. K. Henry and C. Chapman 75 Colour in food: improving quality Edited by D. MacDougall 76 Meat processing: improving quality Edited by J. P. Kerry, J. F. Kerry and D. A. Ledward 77 Microbiological risk assessment in food processing Edited by M. Brown and M. Stringer 78 Performance functional foods Edited by D. Watson 79 Functional dairy products Volume 1 Edited by T. Mattila-Sandholm and M. Saarela 80 Taints and off-flavours in foods Edited by B. Baigrie 81 Yeasts in food Edited by T. Boekhout and V. Robert 82 Phytochemical functional foods Edited by I. T. Johnson and G. Williamson 83 Novel food packaging techniques Edited by R. Ahvenainen 84 Detecting pathogens in food Edited by T. A. McMeekin 85 Natural antimicrobials for the minimal processing of foods Edited by S. Roller 86 Texture in food Volume 1: semi-solid foods Edited by B. M. McKenna 87 Dairy processing: improving quality Edited by G. Smit 88 Hygiene in food processing: principles and practice Edited by H. L. M. Lelieveld, M. A. Mostert, B. White and J. Holah 89 Rapid and on-line instrumentation for food quality assurance Edited by I. Tothill 90 Sausage manufacture: principles and practice E. Essien 91 Environmentally-friendly food processing Edited by B. Mattsson and U. Sonesson 92 Bread making: improving quality Edited by S. P. Cauvain 93 Food preservation techniques Edited by P. Zeuthen and L. BøghSørensen 94 Food authenticity and traceability Edited by M. Lees 95 Analytical methods for food additives R. Wood, L. Foster, A. Damant and P. Key 96 Handbook of herbs and spices Volume 2 Edited by K. V. Peter 97 Texture in food Volume 2: solid foods Edited by D. Kilcast 98 Proteins in food processing Edited by R. Yada 99 Detecting foreign bodies in food Edited by M. Edwards 100 Understanding and measuring the shelf-life of food Edited by R. Steele © Woodhead Publishing Limited, 2011
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Woodhead Publishing Series in Food Science, Technology and Nutrition xxi 101 Poultry meat processing and quality Edited by G. Mead 102 Functional foods, ageing and degenerative disease Edited by C. Remacle and B. Reusens 103 Mycotoxins in food: detection and control Edited by N. Magan and M. Olsen 104 Improving the thermal processing of foods Edited by P. Richardson 105 Pesticide, veterinary and other residues in food Edited by D. Watson 106 Starch in food: structure, functions and applications Edited by A-C. Eliasson 107 Functional foods, cardiovascular disease and diabetes Edited by A. Arnoldi 108 Brewing: science and practice D. E. Briggs, P. A. Brookes, R. Stevens and C. A. Boulton 109 Using cereal science and technology for the benefit of consumers: proceedings of the 12th International ICC Cereal and Bread Congress, 24–26th May, 2004, Harrogate, UK Edited by S. P. Cauvain, L. S. Young and S. Salmon 110 Improving the safety of fresh meat Edited by J. Sofos 111 Understanding pathogen behaviour in food: virulence, stress response and resistance Edited by M. Griffiths 112 The microwave processing of foods Edited by H. Schubert and M. Regier 113 Food safety control in the poultry industry Edited by G. Mead 114 Improving the safety of fresh fruit and vegetables Edited by W. Jongen 115 Food, diet and obesity Edited by D. Mela 116 Handbook of hygiene control in the food industry Edited by H. L. M. Lelieveld, M. A. Mostert and J. Holah 117 Detecting allergens in food Edited by S. Koppelman and S. Hefle 118 Improving the fat content of foods Edited by C. Williams and J. Buttriss 119 Improving traceability in food processing and distribution Edited by I. Smith and A. Furness 120 Flavour in food Edited by A. Voilley and P. Etievant 121 The Chorleywood bread process S. P. Cauvain and L. S. Young 122 Food spoilage microorganisms Edited by C. de W. Blackburn 123 Emerging foodborne pathogens Edited by Y. Motarjemi and M. Adams 124 Benders’ dictionary of nutrition and food technology Eighth edition D. A. Bender 125 Optimising sweet taste in foods Edited by W. J. Spillane 126 Brewing: new technologies Edited by C. Bamforth 127 Handbook of herbs and spices Volume 3 Edited by K. V. Peter
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xxii Woodhead Publishing Series in Food Science, Technology and Nutrition 128 Lawrie’s meat science Seventh edition R. A. Lawrie in collaboration with D. A. Ledward 129 Modifying lipids for use in food Edited by F. Gunstone 130 Meat products handbook: practical science and technology G. Feiner 131 Food consumption and disease risk: consumer–pathogen interactions Edited by M. Potter 132 Acrylamide and other hazardous compounds in heat-treated foods Edited by K. Skog and J. Alexander 133 Managing allergens in food Edited by C. Mills, H. Wichers and K. Hoffman-Sommergruber 134 Microbiological analysis of red meat, poultry and eggs Edited by G. Mead 135 Maximising the value of marine by-products Edited by F. Shahidi 136 Chemical migration and food contact materials Edited by K. Barnes, R. Sinclair and D. Watson 137 Understanding consumers of food products Edited by L. Frewer and H. van Trijp 138 Reducing salt in foods: practical strategies Edited by D. Kilcast and F. Angus 139 Modelling microorganisms in food Edited by S. Brul, S. Van Gerwen and M. Zwietering 140 Tamime and Robinson’s Yoghurt: science and technology Third edition A. Y. Tamime and R. K. Robinson 141 Handbook of waste management and co-product recovery in food processing Volume 1 Edited by K. W. Waldron 142 Improving the flavour of cheese Edited by B. Weimer 143 Novel food ingredients for weight control Edited by C. J. K. Henry 144 Consumer-led food product development Edited by H. MacFie 145 Functional dairy products Volume 2 Edited by M. Saarela 146 Modifying flavour in food Edited by A. J. Taylor and J. Hort 147 Cheese problems solved Edited by P. L. H. McSweeney 148 Handbook of organic food safety and quality Edited by J. Cooper, C. Leifert and U. Niggli 149 Understanding and controlling the microstructure of complex foods Edited by D. J. McClements 150 Novel enzyme technology for food applications Edited by R. Rastall 151 Food preservation by pulsed electric fields: from research to application Edited by H. L. M. Lelieveld and S. W. H. de Haan 152 Technology of functional cereal products Edited by B. R. Hamaker 153 Case studies in food product development Edited by M. Earle and R. Earle
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Woodhead Publishing Series in Food Science, Technology and Nutrition xxiii 154 Delivery and controlled release of bioactives in foods and nutraceuticals Edited by N. Garti 155 Fruit and vegetable flavour: recent advances and future prospects Edited by B. Brückner and S. G. Wyllie 156 Food fortification and supplementation: technological, safety and regulatory aspects Edited by P. Berry Ottaway 157 Improving the health-promoting properties of fruit and vegetable products Edited by F. A. Tomás-Barberán and M. I. Gil 158 Improving seafood products for the consumer Edited by T. Børresen 159 In-pack processed foods: improving quality Edited by P. Richardson 160 Handbook of water and energy management in food processing Edited by J. Klemeš, R. Smith and J-K. Kim 161 Environmentally compatible food packaging Edited by E. Chiellini 162 Improving farmed fish quality and safety Edited by Ø. Lie 163 Carbohydrate-active enzymes Edited by K-H. Park 164 Chilled foods: a comprehensive guide Third edition Edited by M. Brown 165 Food for the ageing population Edited by M. M. Raats, C. P. G. M. de Groot and W. A. Van Staveren 166 Improving the sensory and nutritional quality of fresh meat Edited by J. P. Kerry and D. A. Ledward 167 Shellfish safety and quality Edited by S. E. Shumway and G. E. Rodrick 168 Functional and speciality beverage technology Edited by P. Paquin 169 Functional foods: principles and technology M. Guo 170 Endocrine-disrupting chemicals in food Edited by I. Shaw 171 Meals in science and practice: interdisciplinary research and business applications Edited by H. L. Meiselman 172 Food constituents and oral health: current status and future prospects Edited by M. Wilson 173 Handbook of hydrocolloids Second edition Edited by G. O. Phillips and P. A. Williams 174 Food processing technology: principles and practice Third edition P. J. Fellows 175 Science and technology of enrobed and filled chocolate, confectionery and bakery products Edited by G. Talbot 176 Foodborne pathogens: hazards, risk analysis and control Second edition Edited by C. de W. Blackburn and P. J. McClure 177 Designing functional foods: measuring and controlling food structure breakdown and absorption Edited by D. J. McClements and E. A. Decker
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xxiv Woodhead Publishing Series in Food Science, Technology and Nutrition 178 New technologies in aquaculture: improving production efficiency, quality and environmental management Edited by G. Burnell and G. Allan 179 More baking problems solved S. P. Cauvain and L. S. Young 180 Soft drink and fruit juice problems solved P. Ashurst and R. Hargitt 181 Biofilms in the food and beverage industries Edited by P. M. Fratamico, B. A. Annous and N. W. Gunther 182 Dairy-derived ingredients: food and neutraceutical uses Edited by M. Corredig 183 Handbook of waste management and co-product recovery in food processing Volume 2 Edited by K. W. Waldron 184 Innovations in food labelling Edited by J. Albert 185 Delivering performance in food supply chains Edited by C. Mena and G. Stevens 186 Chemical deterioration and physical instability of food and beverages Edited by L. H. Skibsted, J. Risbo and M. L. Andersen 187 Managing wine quality Volume 1: viticulture and wine quality Edited by A. G. Reynolds 188 Improving the safety and quality of milk Volume 1: milk production and processing Edited by M. Griffiths 189 Improving the safety and quality of milk Volume 2: improving quality in milk products Edited by M. Griffiths 190 Cereal grains: assessing and managing quality Edited by C. Wrigley and I. Batey 191 Sensory analysis for food and beverage quality control: a practical guide Edited by D. Kilcast 192 Managing wine quality Volume 2: oenology and wine quality Edited by A. G. Reynolds 193 Winemaking problems solved Edited by C. E. Butzke 194 Environmental assessment and management in the food industry Edited by U. Sonesson, J. Berlin and F. Ziegler 195 Consumer-driven innovation in food and personal care products Edited by S. R. Jaeger and H. MacFie 196 Tracing pathogens in the food chain Edited by S. Brul, P. M. Fratamico and T. A. McMeekin 197 Case studies in novel food processing technologies: innovations in processing, packaging, and predictive modelling Edited by C. J. Doona, K. Kustin and F. E. Feeherry 198 Freeze-drying of pharmaceutical and food products T-C. Hua, B-L. Liu and H. Zhang 199 Oxidation in foods and beverages and antioxidant applications Volume 1: understanding mechanisms of oxidation and antioxidant activity Edited by E. A. Decker, R. J. Elias and D. J. McClements
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Woodhead Publishing Series in Food Science, Technology and Nutrition xxv 200 Oxidation in foods and beverages and antioxidant applications Volume 2: management in different industry sectors Edited by E. A. Decker, R. J. Elias and D. J. McClements 201 Protective cultures, antimicrobial metabolites and bacteriophages for food and beverage biopreservation Edited by C. Lacroix 202 Separation, extraction and concentration processes in the food, beverage and nutraceutical industries Edited by S. S. H. Rizvi 203 Determining mycotoxins and mycotoxigenic fungi in food and feed Edited by S. De Saeger 204 Developing children’s food products Edited by D. Kilcast and F. Angus 205 Functional foods: concept to product Second edition Edited by M. Saarela 206 Postharvest biology and technology of tropical and subtropical fruits Volume 1: Fundamental issues Edited by E. M. Yahia 207 Postharvest biology and technology of tropical and subtropical fruits Volume 2: Açai to citrus Edited by E. M. Yahia 208 Postharvest biology and technology of tropical and subtropical fruits Volume 3: Cocona to mango Edited by E. M. Yahia 209 Postharvest biology and technology of tropical and subtropical fruits Volume 4: Mangosteen to white sapote Edited by E. M. Yahia 210 Food and beverage stability and shelf life Edited by D. Kilcast and P. Subramaniam 211 Processed meats: improving safety, nutrition and quality Edited by J. P. Kerry and J. F. Kerry 212 Food chain integrity: a holistic approach to food traceability, safety, quality and authenticity Edited by J. Hoorfar, K. Jordan, F. Butler and R. Prugger 213 Improving the safety and quality of eggs and egg products Volume 1 Edited by Y. Nys, M. Bain and F. Van Immerseel 214 Improving the safety and quality of eggs and egg products Volume 2 Edited by F. Van Immerseel, Y. Nys and M. Bain 215 Feed and fodder contamination: effects on livestock and food safety Edited by J. Fink-Gremmels 216 Hygiene in the design, construction and renovation of food processing factories Edited by H. L. M. Lelieveld and J. Holah 217 Manley’s technology of biscuits, crackers and cookies Fourth edition Edited by D. Manley 218 Nanotechnology in the food, beverage and nutraceutical industries Edited by Q. Huang 219 Rice quality: A guide to rice properties and analysis K. R. Bhattacharya 220 Advances in meat, poultry and seafood packaging Edited by J. P. Kerry 221 Reducing saturated fats in foods Edited by G. Talbot
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xxvi Woodhead Publishing Series in Food Science, Technology and Nutrition 222 Handbook of food proteins Edited by G. O. Phillips and P. A. Williams 223 Lifetime nutritional influences on cognition, behaviour and psychiatric illness Edited by D. Benton 224 Food machinery for the production of cereal foods, snack foods and confectionery Ling-Min Cheng
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Hy-D Layer 115x190:Layout 1
01.07.2011
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Seite 1
strength to succeed
Strong Shells = Increased Returns Hy•D delivers… • Healthier, stronger hens • Increased rates of lay • Superior persistency of lay • Thicker, more robust shells • Reduced carcass condemnations • Reduced proportion of broken eggs Hy•D® is proven to greatly improve bone and shell formation leading to more profitable flocks DSM Nutritional Products Ltd PO Box 2676, CH-4002 Basel, Switzerland www.dsmnutritionalproducts.com
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Preface
Eggs are a cheap source of high-quality bio-available proteins, vitamins and unsaturated fatty acids. Enriching eggs with n-3 polyunsaturated fatty acids (PUFAs), minerals or vitamins by manipulating the hen’s diet can even increase the nutritional quality of eggs, and create ‘functional eggs’. Because of the high unsaturated fat content relative to the saturated fat content and the lack of ‘trans’ fat, the link between egg consumption and an increase in serum cholesterol and coronary heart disease is now considered more a myth than a fact. Eggs are a source of bioactive fractions that can be used in medical, pharmaceutical, biotechnological and nutraceutical applications. In Part III of this book, eight chapters give overviews on the nutritional quality of eggs and how this can be increased using enrichment or modification of egg nutrients. In addition, an update is given on facts and folklore related to eggs and cholesterol, and on the use of bioactive egg fractions and IgY for non-food applications. Eggs can thus be considered as healthy food. On the other hand, egg allergy is one of the most prevalent food hypersensitivities and eggs can contain a set of chemical contaminants, including veterinary drugs, dioxins and polychlorobiphenyls. Even more, the eggshell and the egg content can be contaminated by a variety of bacterial species. The most well known, Salmonella Enteritidis, has caused a worldwide pandemic and is still not completely under control despite many efforts from authorities, the poultry industry, researchers and the pharmaceutical and feed industry. In Part I of this book, general overviews on the chemical and bacterial contaminants of eggs are provided, including an update on the mechanisms by which the major public health threat related to eggs, i.e. the bacterium Salmonella Enteritidis, contaminates eggs. In Part II of this book detailed overviews are given on the monitoring, detection and control methods that are used to combat Salmonella Enteritidis. Both pre- and post-harvest control methods are thoroughly discussed. The editors of this book have tried to compile a book that is meaningful and helpful for the egg and egg-related industry, the authorities and the © Woodhead Publishing Limited, 2011
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xxxii Preface research community. The contributions of experts in the respective fields were greatly appreciated and it is only because of the help of these experts and the critical remarks of numerous referees that this book could be produced. We hope that the book improves knowledge of the readers and is stimulating researchers to further gain novel information on the fascinating world of the egg and its interaction with the environment, resulting in both a lower health risk for the consumers and novel applications for the egg-related industry. Filip Van Immerseel Yves Nys Maureen Bain
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1 Microbiology and safety of table eggs M. T. Musgrove, United States Department of Agriculture, USA
Abstract: This chapter describes the microbiology of table eggs, effects of processing, regulatory influences, relative risk of egg-borne disease, and the role of retail and consumer practices in outbreaks. Effects of washing, refrigeration, and facility sanitation in US commercial facilities will be described and their contribution to shelf-life and food safety will be discussed. Current regulations, recent changes, and the influence of Safe and Quality Foods (SQF), a voluntary program required by some retailers, are described. A general discussion of table egg microbiology, pathogens, and emerging pathogens is followed by a description of sampling methods. Finally, the relative risk of egg-borne illness in the US and the contribution by retail and consumer practices will be discussed. Key words: table eggs, table egg microbiology, table egg sanitation, table egg processing, table egg safety.
1.1 Introduction Eggs, a nutritious and inexpensive food, are an important part of human diets worldwide (McNamara, 2003). Modern operations allow for the washing and packaging of thousands of eggs an hour (Klippen, 1990). Since large-scale operations became prevalent in the 1970s, there have been many modifications to the process (Moats, 1978; Hutchison et al., 2003). Understanding how shell egg microbial populations are affected by processing (washing, grading, packing) is important to ensuring product quality and safety.
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4 Improving the safety and quality of eggs and egg products 1.1.1 United states table egg industry In the early 1900s, 90% of US commercial eggs were produced on multipurpose farms by 100–300 birds that roamed freely, fed with waste grain, insects and forage (Bell 1995, 2002). After World War II, farms became modernized, and flock sizes were increased to take greatest advantage of efficient systems (Bell, 2002). Multiple-tier cages became common and came equipped with automated transport belts for gathering eggs. Currently, more than 80% of US eggs are gathered by this method (Fig. 1.1 ). Mechanical feed and watering devices are present in 90% of layer houses. Temperature, humidity, feed intake, water consumption, and all other mechanical operations are electronically monitored. These conditions allow egg companies to employ only 15 persons per million hens. Prior to environmentally controlled housing, eggs were only produced in the spring and summer months. After eggs had been separated according to cleanliness and size, washed and clean eggs were stored until needed for retail. Today, eggs are now transported to retail outlets almost as soon as they are packaged (Stadelman, 1995; Bell, 2002; Zeidler, 2002). Modern equipment for washing, candling, sizing, and packaging can handle over 180,000 eggs per hour. Photographs and a diagram of this type of operation are depicted in Fig. 1.1. As late as the 1950s, it took egg producers most of their time to clean, size, and pack eggs. Production was ~1.4 cases/person hour (1 case = 360 eggs). Eggs that are transported to processing and packaging rooms by conveyor from hens housed in buildings attached to the processing facility are known as ‘in-line’ eggs. Eggs that are transported from remote housing are known as ‘off-line’ eggs. Conveyor systems, mass candling, automatic check (crack) detection, and electronic egg scales with computer controls have allowed for the transformation in production capacity (Zeidler, 2002; Curtis, 2002; Curtis et al., 2004) (Fig. 1.2).
Belt
Hens
Belt
Hens
Processing plant
Fig. 1.1 Inline shell egg layer house and plant layout.
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Microbiology and safety of table eggs 5 Rewash belt Oiler Scales
Checkcandle
Washer 2
Washer 1
Accumulator
Dryer Packer lane
Transfer
Packer lane Case belt
Sorter
Packer lane
Off-line belt
Packer lane Packer lane Packer lane
Fig. 1.2 Schematic of shell egg processing facility.
Per capita egg consumption has also undergone a great deal of change. Highest per capita egg consumption was in 1945 at 402 while the nadir occurred in 1991 at 233.9 eggs. Health concerns associated with egg-related outbreaks of salmonellosis caused by Salmonella Enteritidis and a desire to reduce cholesterol intake are regarded as the principal reasons for the decline. Currently, most nutritionists and medical doctors recommend daily egg consumption. In 2009, per capita egg consumption was 247.7 (American Egg Board, 2010). As late as the 1960s, many eggs were obtained directly from farms or home delivered by milk companies (Bell, 1995). Today, most eggs are sold in supermarkets. Once size (pee wee to jumbo), color (brown or white egg tables), and quality (AA, A, B, or ungraded) were the only egg choices consumers could make. Now, specialty eggs comprise ~ 5% of the market. Examples include organic, vegetarian fed, free range, cage free, or fertile. Some egg types boast higher contents of nutrients such as vitamin E or omega-3 polyunsaturated fatty acids while one type claims 25% less cholesterol than traditional eggs. Even generic eggs provide a number of essential fatty acids, vitamins, and minerals. Human milk is the only food source with a higher biological protein value for people (Anderson, 2003). In 2009, 57.8% of eggs went to retail, 30.8% were further processed, 8.5% went to foodservice use, and 3.0% of eggs were exported. Top foreign markets for table eggs were Canada, Hong Kong, and the European Union, while Canada and Japan were the top importers of egg products. Egg quality allows the US to out-compete competitors such as China, even though they are able to produce eggs more cheaply.
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6 Improving the safety and quality of eggs and egg products
1.2 Washing table eggs In the United States, Canada, and Japan, shell eggs are washed and graded prior to being packaged for retail (Zeidler, 2002). Though washing eggs was once disallowed in the US, it is now required for plants that participate in the Agricultural Marketing Service (AMS) voluntary grading program (USDA, 2003). Washing eggs with water colder than the egg, heavily contaminated with bacteria, containing large amounts of soluble iron, or in machines whose surfaces are contaminated with large numbers of microorganisms are factors determined to increase chances of bacterial cross-contamination during egg washing (Moats, 1978; Zeidler, 2002). Such conditions are addressed by AMS guidelines (see www.ams.usda.gov) and when attention is given to these factors, modern commercial shell egg washing operations result in improved microbiological egg quality (Baker and Bruce, 1995). 1.2.1 Effects of washing Initially, table eggs in the US were not washed but were sanded to remove stains. In the 1940s, at the height of per capita consumption, a non-automated system involving submersion of eggs into water was common. Water conditions were not monitored closely so some eggs were washed with dirty, colder water. After eggs were separated according to cleanliness and size, washed and clean eggs were stored until needed for retail (Stadelman, 1995; Bell, 2002; Zeidler, 2002). After the USDA published Market Research Report Number 757, submersion of eggs was no long advised. Since that time, research has been conducted to verify and further improve the efficacy of egg washing. Kinner and Moats (1981) inoculated simulated wash water with bacteria previously isolated from shell eggs. Temperature, pH, and detergent affected the survivability of pure cultures. When wash water pH was > 10, Escherichia coli, Salmonella, Citrobacter, Enterobacter, Proteus, Klebsiella, Alcaligenes, Flavobacterium, and Pseudomonas; Escherichia coli Pseudomonas were almost instantly destroyed. Staphylococcus aureus was adversely affected by detergent though protected by 1% egg solids. Streptococcus faecalis was the most resistant of the organisms tested, surviving for over 2.5 h. Catalano and Knabel (1994) analyzed the effects of pH and rapid chilling on S. Enteritidis destruction during simulated commercial egg processing. Eggs were immersed in inoculated fecal slurry before being washed at pH 9 or 11 in 37.7 °C wash water followed by rapid or slow chilling. Wash water pH significantly affected shell surface survival of Salmonella. Significant cross-contamination was observed between inoculated eggs and control eggs at wash-water pH 9 (75.0%) but was decreased at pH 11 (8.3%), based on shell surface counts. Slow chilling increased S. Enteritidis survivability regardless of wash-water pH. At pH 9, S. Enteritidis penetration into egg contents increased.
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Microbiology and safety of table eggs 7 Leclair et al. (1994) describe a model for inactivation of Listeria monocytogenes and S. Typhimurium in simulated wash water. Temperature, egg solids, pH, and chlorine were the treatments used to generate the data used in the models. Higher temperature and lower egg solid negatively affected survivability of both organisms. S. Typhimurium survivability decreased significantly affected at higher wash water pH and chlorine levels. Egg solids reduced the survivability of L. monocytogenes but promoted S. Typhimurium survivability. Linear equations calculated for each organism were used to estimate washing conditions that would reduce the time for a 4-log reduction in viable counts to a period of less than 30 minutes. When higher egg solids are present, pH must be increased from 10.5 to 10.8 and temperature from 42 to 47.4 °C. Whiting et al. (2000) described a stochastic model for estimating S. Enteritidis growth during shell egg collection, processing, storage, and transportation. Equations for internal egg temperature, vitelline membrane integrity, and S. Enteritidis growth rate were included. Monte Carlo simulations were used to determine that S. Enteritidis were unlikely to grow during an average 4.5 day progression from oviposition through transportation. However, parameter fluctuations in this model indicate that ambient air temperature was a key factor. These authors conclude that ensuring refrigeration during transport and cooling eggs as quickly as possible were likely to increase egg safety. Srikaeo and Hourigan (2002) published a report on the use of statistical process control to enhance validation of critical control points during shell egg washing. Control measures analyzed were pH of wash water (11–13), wash water temperature (32–44 °C), rinse water temperature (41–49 °C), and chlorine level (100–200 ppm). This model was generated based on literature recommendations for the parameters included. However, pH levels used were higher than typically observed in many shell egg wash water samples and the model does not take into account the effects of egg solids or other organic materials that are always observed in modern operations. Moats (1980) surveyed commercial shell egg washing facilities in Maryland and Pennsylvania. Washed and unwashed eggs, wash water, and equipment surface swabs were collected. An aerobic plating method was employed to enumerate microorganisms and selected isolates were identified to the genus level. Aerococcus, Streptococcus faecalis, Propionibacterium, and Lactobacillus populations were reduced by washing. Escherichia coli were reduced by 67%. Greater numbers of actinomycetes were found on equipment surfaces than were found on unwashed eggs. Alcaligenes and Moraxella were the most frequently recovered Gram-negative bacteria from washed or unwashed eggs but 71% of microorganisms recovered from unwashed eggs were Gram-positive cocci. Moats (1981) collected additional samples from the plants visited in 1980, including wash water, brushes, egg conveyors, washed eggs, and unwashed eggs. A sanitizing rinse was added to operations on sampling
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8 Improving the safety and quality of eggs and egg products days. Chlorinated spray lowered bacterial counts only on conveyor samples though thorough rinsing equipment at the end of daily operations appeared to reduce bacterial counts on equipment surfaces and in wash water. Bacterial counts from washed eggs correlated significantly with equipment surface and wash water counts but not with unwashed eggs. Wash water counts correlated with counts from equipment surfaces but not from unwashed eggs. Moats concluded that the major source of bacteria in the wash water was the equipment rather than the eggs and that the sanitizing rinse was of no benefit. However, it seems likely that the bacteria on the equipment originated from the eggs. This work underscores the value of daily plant sanitation. Sodium hypochlorite works best in a pH range of 6–7 and most egg wash water pH values are greater than 9. This may have contributed to the limited effectiveness of the chlorinated sanitizer on equipment surfaces, particularly if there was wash water present at the time of application. In a study published in 1979, Moats visited commercial facilities in Maryland and Pennsylvania that used different combinations of washing compounds and sanitizing or water rinses. Counts in plants using sanitizer rinses were very low (<50 cells/shell), significantly lower than one plant using an unsupplemented water rinse. However, when sanitizer rinse was temporarily cut off in plants that employed this type of rinse, counts did not change. Moats concluded that a lack of significant change in eggshell bacterial numbers indicated that sanitizer rinse was at most an indirect effect. Moats further concluded that due to low correlation between wash water and eggshell data, cross-contamination was minimized by commercial shell egg washing conditions. Catalano and Knabel (1994) assayed eggs and wash water samples from processing plants in southeastern Pennsylvania for aerobic microorganisms and Salmonella. A greater number of microorganisms survived the washing process at relatively low wash water temperature (32.2–35°C) and pH values (9–10). Salmonella from serogroup D1 were recovered from wash water and eggs that operated under those water parameters. When plants operated with higher wash water ranges for pH (11–1.5) and temperature (37.7–42.3 °C), no Salmonella were detected and wash water counts were lower. High detergent concentration and reduced egg solids also contributed to the destruction of Salmonella. Jones et al. (1995) collected egg samples from various stages of production from layer houses and processing samples from the adjoining packing plant. Samples collected from the layer house environment (flush water, ventilation fan swabs, egg belt and egg collector swabs) tested 72.0% Salmonella positive. Eggshell rinses prior to processing were 7.8% (7/90) Salmonella positive while post-processing rinses were only 1.1% (1/90) positive. None of the 180 egg content samples tested positive for the organism. As was the case with the work of Catalano and Knabel (1994), Salmonella was detected on shell rinses from an egg collected at a time when the wash water pH was at the lowest measure (10.19). Salmonella serotypes recovered from eggshells prior
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Microbiology and safety of table eggs 9 to processing were S. Heidelberg and S. Montevideo. Production serotypes were identified as S. Agona, S. Typhimurium, S. Infantis, S. Derby, S. Heidelberg, S. California, S. Montevideo, S. Mbandaka, and untypeable. Murase et al. (2001) surveyed two layer houses and the attached in-line processing facility at a single farm. Samples from the production environment as well as from pre- and post-washed eggs, and the processing environment, were collected over a five-year period. Pulsed-field gel electrophoresis (PFGE) patterns from Salmonella serotypes collected in the processing plant drains matched serotype patterns consistently recovered from one of the layer houses. Other serotype PFGE patterns (S. Cerro, S. Mbandaka, and S. Montevideo) from production and processing samples also matched. These authors concluded that a single Salmonella clone colonized the production facilities and that egg belts are likely the means by which Salmonella spread from house to house and to the processing facility. Davies and Breslin (2003) investigated Salmonella contamination at production facilities and farm egg-packing plants. Swabs from the floor, grading tables, conveyor belts or roller, and candlers were often Salmonellapositive (23.1–30.8%). Even after disinfection, contamination ranged from 5.0–12.6%. Sterilized eggs passed through facilities showed a contamination rate of 0.3%. These results indicate that cross-contamination may contribute significantly to the external contamination of shell eggs. De Reu et al. (2003) compared aerobic microbial shell populations on eggs collected from production through retail from cage and organic production systems. Their results indicated that air quality affected shell counts regardless of production system. However, because eggs were collected in Belgium and eggs are not washed in the European Union, it is difficult to make comparisons with eggs processed and packaged in the US. Knape et al. (2002) compared aerobic microbial populations on shell surfaces of eggs from four in-line (IL) and four off-line (OL) commercial processing plants. Eggs were collected from four different sites: accumulator or transfer belt, after washing but before application of sanitizer rinse, immediately after sanitizer rinse, and just before packaging. Five different collection times were observed, spaced equally over the processing day. Wash water temperature, pH, and aerobic microbial populations were also monitored. Though counts from eggs prior to processing (accumulator or transfer belt) were only 0.3 log cfu/ml between IL and OL, by the time eggs were ready to be packaged, OL eggs were 1.4 log cfu/ml greater than IL egg counts. Knape et al. (2002) concluded that OL eggs were more difficult to clean because organic material would have had longer to adhere to shells and therefore be more difficult to remove. OL eggs are stored at 12.8–15.6 °C for as long as a week before being processed whereas IL eggs are processed within 24 h of oviposition (Curtis, 2002). As a result, OL internal egg temperatures average 16.7–20 °C while IL internal egg temperatures typically average 31.1–35.6 °C. Average wash water temperatures for all eight plants and all five sampling times were approximately 34 °C. If the difference between
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10 Improving the safety and quality of eggs and egg products the wash water temperature and the internal egg temperature is greater than ~7 °C, thermal checks and cracks increase (Curtis et al., 2004). Also, OL eggs are transported from a layer house to the processing facility and during this process eggs may be jostled causing minute shell damage that can expand once the eggs encountered warm wash water. In 2005, Musgrove et al. (2005a) conducted a study to gather in-depth information on how microbiological populations associated with shell egg surfaces fluctuate throughout US commercial table egg processing. Five different shell egg surface populations (aerobic, yeasts/molds, Enterobacteriaceae, Escherichia coli, and Salmonella) were monitored at 12 points along the processing line (accumulator, pre-wash rinse, washer one, washer two, sanitizer, dryer, oiler, scales, two packer head lanes, re-wash entrance, re-wash exit). Three commercial facilities were each visited three times allowing for the sampling of 990 eggs and subsequently analyzed by 5220 microbiological samples. Though variations existed in levels of microorganisms recovered from plant to plant, the patterns of fluctuations for each population were similar at each plant (Table 1.1). On average, aerobes, yeasts/molds, Enterobacteriaceae, and E. coli prevalence were reduced by 30%, 20%, 50% and 30%, respectively, by end of processing. Log10 cfu/ml rinse on eggs collected from packer head lanes were decreased by 3.3, 1.3, 1.3, and 0.5, respectively, when compared to rinses from eggs collected at the accumulator. Salmonella was recovered from 0 to 48% of pooled samples in the three repetitions. Average pH of wash water for the entire study was 10.2. On the day that 48% of the eggs were contaminated with Salmonella, the pH Table 1.1 Mean populations of microorganisms recovered from the surface of commercial shell eggs collected at 12 sites on processing lines in three commercial plants Site A Accumulator B Pre-wash C Wash # 1 D Wash # 2 E Sanitizer F Dryer G Oiler H Scales/Check I Re-wash enter J Re-wash exit K Pack # 1 L Pack # 2
Population (log10 cfu/ml) Aerobes a
4.61 3.34c 2.35d 1.90e 2.36d 1.16g 1.22g 1.24g 3.77b 4.03b 1.57f 1.21g
Yeasts/molds ab
1.76 0.78cd 0.55d 0.51d 0.68cd 0.65cd 0.91c 0.71cd 1.69b 2.01a 0.76cd 0.68cd
Enterobacteriaceae a
1.32 0.58c 0.10d 0.12d 0.17d 0.11d 0.02d 0.07d 1.03b 1.05b 0.04d 0.02d
E. coli 0.56a 0.19b 0.04c 0.05c 0.11bc 0.05c 0.00c 0.01c 0.22b 0.11bc 0.04c 0.05c
a,b,c,d,e,f,,g
Values in the same column that are not followed by the same letter are significantly different (P ≤ 0.001) for yeasts/molds, Enterobacteriaceae, and E. coli; (P ≤ 0.0001) for aerobes.
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Microbiology and safety of table eggs 11 was only 9.1, there were mechanical malfunctions that increased the rate of broken eggs, and the well water source was contaminated with Salmonella. Salmonella serotypes recovered included Typhimurium, Heidelberg, and Kentucky. More Salmonella was recovered from pre-processed than inprocess or ready to pack eggs. If the correct conditions are observed, current commercial practices decrease microbial contamination of egg shell surfaces and improve quality and safety. 1.2.2 Eggshell sampling methodology A variety of methods have been developed for the recovery of microorganisms from eggshells. Determination of bacterial numbers on eggshells has been accomplished using surface rinses (Penniston and Hedrick, 1947; Gillespie et al., 1950, Conner et al., 1953; Forsythe et al., 1953; Gentry, 1970; Gentry and Quarles, 1972; Gunaratne and Spencer, 1973; Moats, 1979; Berrang et al., 1991, shaking crushed shells with glass beads (Haines, 1938), blending egg shells and membranes (Brant and Starr, 1962; Board et al., 1963; March, 1969; Winter et al., 1955), as well as surface swabbing and blending (Penniston and Hedrick, 1947). In 1970, Gentry described a very simple procedure in which an individual egg is placed in 10 ml of a sterile, isotonic buffer in a plastic bag and massaged by hand for 1 min before soaking in the buffer for an additional 5 min. Many shell rinse methods are a variation on Gentry’s method. Berrang et al. (1991) described a method in which individual eggs were aseptically cracked, contents discarded, and eggshells were placed into a bag with diluent where they were hand massage for a minute prior to sampling. Gunaratne and Spencer (1973) recovered more Pseudomonas from inoculated eggs by blending than surface rinsing. However, Penniston and Hedrick (1947) found that rinsing and blending methods were equivalent in their ability to recover bacteria from artificially dirtied but washed eggs. Once eggs were chemically sanitized using chlorine, counts were 3–6 times higher from blended than rinsed samples. Moats (1980, 1981) concluded that whether greater numbers are recovered by surface rinsing/swabbing or by blending the entire shell depends on whether bacteria reside on the surface or embedded within the pores or membranes of the shell. It has been reported (Musgrove et al., 2011) that when evaluating broiler hatching egg disinfectants, the method of inoculation and the method of microbial recovery greatly affect the interpretation of chemical efficacy. A lenient test of efficacy would result when a method of inoculation less likely to result in sub-surface contamination (droplet) is combined with a shell rinse method. Inoculation by immersion and temperature differential and sampling by homogenization of shells and membranes would provide the most rigorous test of sanitizer efficacy. Rinsing is also used for the recovery of microorganisms from poultry carcasses. Lillard (1988) has reported that subsequent rinses (as many as 40
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12 Improving the safety and quality of eggs and egg products were performed) of poultry carcasses respectively yield bacterial numbers equal to the initial carcass rinse. However, data from multiple rinsing of egg shells do not display the same pattern unless the eggs are extremely dirty. Musgrove et al. (2003) rinsed eggs up to eight times, plating after the 1st, 2nd, 4th, and 8th rinses to enumerate aerobes and Enterobacteriaceae. Even the 8th rinse for very dirty eggs recovered the same rate and with similar numbers as the 1st rinse. Unwashed eggs gave lower population levels on subsequent rinses, but the second rinse gave comparable recovery rates to the initial rinse. Washed eggs were different even on the second rinse. This work suggests that a significant portion of shell surface populations will be removed with the initial wash or rinsing of the egg. Moats (1979) sampled washed and unwashed eggs from commercial shell egg processing plants. He reported that though a great deal of variability was noted, there were generally much lower numbers recovered from washed eggs, particularly those sampled by a surface rinse method. Moats also compared a whole egg surface rinse technique to blending of the shell and membranes. He concluded that rinsing was more important because the surface bacteria that were recovered would be more likely to contaminate the egg contents when the shell was broken. Surface rinse methods are easily and rapidly performed. However, surface rinse or swab methods may not be adequate for microbial recovery of washed or previously rinsed eggs. In consideration of the literature, it seems that the more effective the detergent or sanitizer, the more likely it is that a shell homogenization method may be required to recover surviving microorganisms. Choosing the appropriate method is an important consideration when evaluating the efficacy of washing or sanitizing steps in the processing chain. In 2005, Musgrove et al. (2005c) compared shell rinse and shell crush methods for recovery of naturally occurring bacteria from table eggs collected at commercial shell egg processing facilities. Shell rinse (SR) techniques are easy to perform and widely used. An alternative sampling method involves crushing and rubbing the shell (CR). In order to determine the most appropriate method for shell eggs, 358 shell eggs were collected from a commercial egg processor and sampled by SR and CR techniques. Total aerobic microorganisms and Enterobacteriaceae were enumerated on plate count and violet red bile glucose agar plates, respectively. Unwashed (PREP), in-process (INPR), and clean eggs (POST) were evaluated in the study. Aerobic microorganism prevalence for eggshells sampled was similar for both methods (~100%) but log cfu/ml were higher from SR than CR samples (3.2 and 2.2, respectively). Average Enterobacteriaceae recovery was similar for both methods (45% SR v. 40% CR) when all eggs were considered together. This population was detected more often by SR when PREP eggs were sampled (90% SR v. 56% CR), equally by SR and CR for INPR eggs (30% SR v. 29.3% CR) but more often by CR for POST eggs (10% SR v. 36% CR). SR was easier to perform and recovered larger numbers of aerobic organisms, particularly for PREP eggs. However, CR was
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Microbiology and safety of table eggs 13 more efficient for recovery of Enterobacteriaceae from POST eggs. Stage of shell egg processing may be an important consideration when choosing egg sampling methodology. In 2008, Kawasaki et al. sampled Salmonella inoculated eggs using a shell swab method or the crush method developed by Musgrove et al. (2005c). The crush method recovered 2 log cfu/ml sample more than the swab method. When the shell was rinsed out using warm diluents, another log cfu/ml was recovered. Methods which allow for a thorough sampling of shells and membranes are more likely to recover Salmonella, particularly if the eggs have been washed. Musgrove et al. (2005c) determined that the crush method recovered more naturally occurring Salmonella than the shell rinse method.
1.3 Table egg facility sanitation If processing and packing are executed with contaminated equipment or in an unhygienic environment, then the benefits of processing may be negated due to recontamination. In 2002, a survey of egg-contact and non-contact surfaces was conducted in 11 commercial egg processing facilities (Jones et al., 2003; Musgrove et al., 2004a). Samples were collected before and after facility disinfection. Aerobic microorganisms and Enterobacteriaceae were enumerated. These microbial populations are indicators of the level of hygiene; higher counts mean poorer conditions (Kornacki and Johnson, 2001). Neither population decreased in numbers after sanitation procedures were performed and the most contaminated surfaces were identified. More in-depth studies have focused on packer head brushes, transfer cups, and nest run cart shelves (Musgrove and Jones, 2006; Jones and Musgrove, 2007; Musgrove et al., 2008b). Musgrove and Jones (2006) sampled eggs and packer head brushes samples from two processing plants. Samples were enumerated for aerobic microorganisms and Enterobacteriaceae and enriched for Salmonella, Campylobacter, and Listeria (Table 1.2). The second plant had Table 1.2 Prevalence (% positive) of Enterobacteriaceae of packer head brushes and table eggs collected from two commercial processing facilities Sample
Plant 1
Plant 2
Unwashed eggs Ungraded, washed eggs1 Packer head brushes Packaged eggs2
53.3 0.0 40.0 3.3
30 6.7 85.7 41.7
Based on chi square analysis, differences between plants were significant (P < 0.0001). 1 Eggs were collected as they left the washers and had not come into contact with packer head brushes. 2 Packaged eggs passed through the packer head brushes.
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14 Improving the safety and quality of eggs and egg products consistently higher prevalence of Enterobacteriaceae and numbers of aerobic microorganisms on packer head brushes. While very low average counts were determined for eggs from either facility (0–0.4 log cfu Enterobacteraiceae/ ml), a significantly higher prevalence for this population was observed from samples collected at the second facility. Salmonella, Campylobacter, and Listeria were recovered at least once from packer head brushes at this facility. None of the pathogens was recovered from egg samples. These data demonstrate the importance of facility sanitation in maintaining the benefits of washing. Previous studies have surveyed processing facilities for Salmonella contamination (Poppe et al., 1992; Jones et al., 1995; Murase et al., 2001; Davies and Breslin, 2003). The only one of the studies conducted in the US included only six types of surfaces from a single facility. Equipment and environmental samples were analyzed for numbers of indicator microorganisms and for Salmonella contamination from a single facility (Musgrove et al., 2008a). If reservoirs of contamination are not determined and addressed, multi-species biofilms may form. A biofilm is a sessile bacterial community characterized by cells that are attached to a surface, polysaccharide interface, or each other (Kumar and Anand, 1998). Microorganisms that are incorporated into a biofilm are 100¥ more resistant to sanitizers and heat (Allison, 1993). Hazard analysis critical control point (HACCP) failures are often attributed to a lack of focus on key areas of contamination during sanitation (Northcutt and Smith, 2010). Keeping equipment and facilities clean is crucial to producing safe, high quality food. It is estimated that 25% of outbreaks are due to recontamination most often due to poor facility hygiene, crosscontamination, or unsanitary equipment.
1.4 Regulations In 1946, the us Agricultural Marketing Act was passed (USDA, 2003). This law gave the Secretary of Agriculture the authority to set standards for agricultural products and on a voluntary basis on the part of producers, to grade and certify conformity of agricultural products to set standards. Inspections are performed by the Agricultural Marketing Service personnel (USDA, 2000). Eggs are judged on external qualities, internal qualities, and weight. This program is voluntary but companies who participate pay for grading and inspection services. Plants that are not under USDA inspection are governed by state laws which are equal to or more stringent than AMS guidelines (Johnson, 2002). While attention to quality may impact food safety positively, shifting focus recalls a rethinking of practices that have been acceptable in previous years. Eventually, egg processors will be under HACCP-based regulations. Many retailers now require shell egg producers to obtain Safe Quality Food (SQF) certification. This program was launched © Woodhead Publishing Limited, 2011
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Microbiology and safety of table eggs 15 in 1994 and is administered by the Food Marketing Institute. The goal of SQF is to improve food safety, increase consumer confidence, and improve relations between government and the food production and retail industry. Many retailers now require that egg producers be SQF certified. SQF is a HACCP program relying on management of critical control points, record keeping, and prerequisites of Good Manufacturing Practices (GMPs) and Sanitation Standard Operating Procedures (SSOPs). Definitive critical control points based on scientific information are required for an effective HACCP or SQF program (Sperber, 2004). Though designed for enhancing quality there are parameters inherent in the procedure described in 7 CFR part 56 that also affect egg safety. Only approved cleaning compounds may be used in the wash water. Wash water must be changed every 4 h or more often if it becomes unsanitary or at least at the end of the shift. Wash water must be potable and soluble iron content should not exceed 2 parts per million. Washed eggs should be spray-rinsed with water at a temperature equal to or warmer than the temperature of the wash water and containing an approved sanitizer of 50–200 ppm of available chlorine or its equivalent. Alternatives may be approved by the National Supervisor. In July 2009, the US food and Drug Administration (FDA) published Prevention of Salmonella Enteritidis (SE) in shell eggs during production, storage, and transportation (21 CFR parts 16 and 118) (FDA, 2009). This will require producers to enforce and keep records of biosecurity, rodent/ pest control, refrigerate eggs at off-line farms at 36 h after oviposition, clean and disinfect between flocks and to monitor pullets for SE. Additionally, each house will be tested by drag swab when any group of hens in the house reach 40–45 weeks of age or 4–6 weeks after molting. If the test is positive then eggs may be diverted to a breaker or other use that includes a process with a 5-log kill for Salmonella. After interventions, eggs from the flock must be demonstrated to be Salmonella Enteritidis free. Negative status is determined by sampling 4000 egg contents over an 8-week period. Producers are responsible for testing.
1.5 Microbiology of table eggs It is estimated that 90% of eggs are microbiologically sterile at oviposition (Board, 1966). Potential sources of contamination include dust, nesting material, and feces (Board, 1966; Mayes and Takeballi, 1983; Board and Tranter, 1995). However, three potential routes of infection have been described. Trans-ovarian infection occurs while the ovum or yolk is still connected to the ovary. Oviduct contamination is an infection from the vitelline membrane or albumen as the eggs pass through any portion of the oviduct. Trans-shell contamination is when bacteria pass from the outer to the inner surface of the shell (Bruce and Drysdale, 1994). © Woodhead Publishing Limited, 2011
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16 Improving the safety and quality of eggs and egg products Physical resistance to bacterial contamination is provided by the cuticle, shell, inner shell membrane, and the outer shell membrane (Mayes and Takeballi, 1983; Solomon, 1991). Sometimes referred to as bloom or shell accessory material, the cuticle is a 0.01 mm thick protein-like substance that coats the outside of the shell. It provides protection in two ways. First, by adding to shell thickness, it increases shell strength. Secondly and most importantly, it prevents flow of water, bacteria, or other materials through the shell pores. Each shell is perforated by numerous pores (Fig. 1.3). There are 7000–17,000 pores, many of which are located around the equator or the blunt end of the shell. Pore diameter ranges from 9 to 35 mm. These openings are wider at the top than the bottom. Some are malformed but many pores run from the outer surface to the shell membrane. The integrity of these layers is affected by organic material, egg handling (easily damaged immediately after oviposition), length of storage (shrinks, dries, and flakes with time), storage temperature (increases with temperature), fumigation (chemical damage), and cleaning processes (physical and chemical damage). If the cuticle is removed, bacteria gain access to the egg’s interior much more easily, though this is the most expendable of the barriers to trans-shell contamination (Board, 1966; Mayes and Takeballi, 1983; Solomon, 1991; Baker and Bruce, 1995). Shell thickness, influenced by nutrition, environment, management practices, and genetics, has a significant effect on the ability of bacteria to pass through the shell (Solomon, 1991). This ability has been demonstrated by correlation of bacterial penetration and poor shell quality (Haines and Moran, 1940). In particular, eggs with cracked shells are invaded more often and with greater numbers of bacteria, including pathogens (Todd, 1996; Ricke et al., 2001). There are two shell membranes that are held closely together except at the blunt end of the egg where the air cell is located (Romanoff and Romanoff, 1949; Solomon, 1991). An inner membrane lies over the albumen and the outer membrane is attached to the calciferous shell. Three layers of random fibers make up the outer membrane; two indistinct layers form the inner membrane. Most researchers estimate the outer membrane to be double the thickness of the inner membrane with a combined thickness of approximately 80 mm. These membranes are thought to serve as a bacterial filter (Garibaldi and Stokes, 1958; Kraft et al., 1958). Bacterial penetration is thought to be most effectively prevented by the inner shell membrane, shell, and outer shell membrane. Other researchers believe that the two shell membranes together form the most effective barrier (Board, 1966; Mayes and Takeballi, 1983; Solomon, 1991; Board and Tranter, 1995). When the cuticle, shell, and shell membranes are unable to prevent bacterial penetration, albumen contamination will occur (Lock et al., 1992). However, there are numerous factors that limit the ability of microorganisms to survive and grow in this material (Mayes and Takeballi, 1983; Board and Tranter, 1995). Firstly, the viscous nature of albumen proteins, particularly when
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Microbiology and safety of table eggs 17
(a)
(b)
Fig. 1.3 Scanning electron micrograph of (a) an eggshell showing pores and (b) a cross-section of an eggshell shell, pores, and membranes.
eggs are freshly laid, makes microbial motility difficult. Viscosity coupled with a lack of available water and nutrients make albumen inhospitable to bacterial growth. However, the main impediment to bacterial growth or survival in the albumen is chemical. There are several important naturally occurring antimicrobial compounds within the albumen. Ovotransferrin and
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18 Improving the safety and quality of eggs and egg products conalbumen chelate metal ions, particularly iron. Ovomucoid inhibits trypsin. Lysozyme causes hydrolysis of b-1,4-glycosidic bonds in peptidoglycans. Ovoinhibitor inactivates several proteases, ovoflavoprotein chelates riboflavin and avidin binds biotin. In addition, albumen pH is in the alkaline range. Immediately after oviposition, pH ranges from 7.6 to 7.9 but a gradual increase is observed during storage. As carbon dioxide is lost to the environment, pH increases to greater than 9, beyond the tolerance of most microorganisms. Lysozyme, conalbumen, and pH are considered to be the most important of the antimicrobial factors naturally occurring in albumen (Mayes and Takeballi, 1983). Quality and safety may be impacted by trans-ovarian, oviducal, and trans-shell contamination (Mayes and Takeballi, 1983). Of these three, transovarian has been considered the least important. Harry (1963) killed and skinned hens before transferring them to a clean necropsy room. Oviducts, ovaries, ova, and eggs from the oviduct were removed and examined for the presence of various bacterial species. All the ovaries were found to be contaminated with bacteria. Most of the bacteria recovered were determined to be Gram-positive micrococci and lactobacilli though coliform and Pasteurella haemolytica bacteria were also infrequently encountered. Of the 28 ova that were examined, 64.3% were found to be contaminated by enrichment but only 26.3% were positive by direct plating. For this reason, it was concluded that bacteria were present in fairly low numbers. Another reason contamination prior to laying has been discounted as a major source of infection is that 90% or more of eggs are sterile at oviposition. However, for some bacterial species and serotypes, trans-ovarian and oviducal contamination may be very important (Barnhart et al., 1991; Gast et al., 1992; Baumler et al., 2000; Ricke et al., 2001). For most serotypes of Salmonella, trans-shell contamination is probably the most important route of egg contamination. In the case of S. Enteritidis, this does not appear to be the case. S. Enteritidis is recovered from egg contents but not from shells or from hen fecal samples. Mawer et al. (1989) analyzed 360 eggs from a small free-range flock that had been implicated in a salmonellosis outbreak. He recovered S. Enteritidis from egg contents but from none of the eggshells. In another study involving an outbreak-implicated flock, Humphrey et al. (1989) found 194 intact eggs contaminated with S. enteritidis but no fecal samples from the flock that laid them. Even when birds are orally challenged with large numbers of organisms, they produce few contaminated eggs (Gast and Beard, 1992). This is not the case with other serotypes (Humphrey et al., 1991). Other researchers have recovered S. Enteritidis from the reproductive tract but not from the gastrointestinal tract (Lister, 1988; Bygrave and Gallagher, 1989). Though most eggs are microbiologically sterile at the time of lay, opportunities for contamination abound the instant they leave the oviduct (Board and Tranter, 1995). Eggs are likely to have their first exposure to bacteria as they pass through the cloaca. Egg temperatures are around 42 °C,
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Microbiology and safety of table eggs 19 generally warmer than ambient air. Eggs contract as they cool, creating a negative pressure that can pull material into the pores. As a result, eggs are potentially contaminated by any surface with which they come into contact. Several factors can affect the extent of trans-shell contamination that occurs. Positive correlations exist between shell or environmental moisture, ambient storage temperature, condition of the cuticle, and shell damage (Board et al., 1994; Todd, 1996; Ricke et al., 2001). Eggshell bacterial numbers fluctuate widely, from zero to hundreds or even millions (Mayes and Takeballi, 1983; Board and Tranter, 1995). The average number of bacteria per shell is considered to be 100,000 for unwashed or untreated eggs (Board, 1966). Heavily soiled eggs are likely to harbor millions of bacteria while lightly soiled and visibly clean egg bacterial numbers are difficult to predict (Board, 1966; Ricke et al., 2001). This natural variability requires that a great number of eggs need to be collected and sampled to allow adequate statistical data analysis. A recent study indicated that for non-graded eggs (including visibly dirty) approximately 40 eggs were required statistically but that for graded eggs 20 often sufficed (De Reu et al., 2003). Sources of bacterial shell contaminants can include caging material, nesting materials, water, hands, broken eggs, blood, insects, and transport belting, though dust, soil, and feces are probably the most important (Board and Tranter, 1995; Ricke et al., 2001; Davies and Breslin, 2003). There have been several general surveys of the types of bacteria than be recovered from shell eggs prior to washing and packing. In order of prevalence, Micrococcus, Staphylococcus, Arthrobacter, Bacillus, Pseudomonas, Acinetobacter, Alcaligenes, Flavobacterium, Cytophaga, Escherichia, Enterobacter, Streptococcus, Sarcina, Aeromonas, Proteus, and Serratia have found to be present on eggshells (Ayres and Taylor; 1956; Board et al., 1963; Board and Tranter, 1995). In addition, Kurthia, Propionibacterium, Microbacterium, Lactobacillus, Moraxella, Acetobacter, and yeasts have been detected on washed and unwashed eggs (Moats, 1980). Jones et al. (2004) stored washed and unwashed eggs and plated shell rinses onto violet red bile glucose agar plates for each of six weeks. In a follow-up study, Musgrove et al. (2004b) randomly selected presumptive Enterobacteriaceae isolates and identified them using biochemical tests. Genera identified from eggs that had not been washed included Escherichia coli, Enterobacter cloacae, Enterobacter sakazakii, Enterobacter spp., Citrobacter youngae, Klebsiella pneumoniae, Klebsiella spp., Serratia odorifera, Serratia spp., Kluyvera spp., Providencia rettgeri, Providencia spp., Pantoea spp., Rahnella aquatilis, Salmonella spp., Yersinia spp., Flavimonas oryzihabitans, and Xanthomonas maltophilia. Enterobacter amnigenus and Salmonella arizonae were the only identified isolates from washed eggs, recovered from shell rinses at week 5 of storage. In a larger study published in 2005, Musgrove (2005a) and others isolated Enterobacteriaceae from eggs collected at three commercial facilities. Biochemical tests were used to make identifications. Escherichia coli and Enterobacter spp. were
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20 Improving the safety and quality of eggs and egg products isolated from each of the nine plant visits. Other genera isolated from at least one of the three plants included Cedecea, Citrobacter, Erwinia, Hafnia, Klebsiella, Kluyvera, Leclercia, Morganella, Proteus, Providencia, Rahnella, Salmonella, and Serratia. Non-Enterobacteriaceae isolated and identified included Aeromonas, Chryseomonas, Listonella, Pseudomonas, Sphingobacterium, Vibrio, and Xanthomonas. Lower numbers and prevalence were noted for washed eggs when compared with unwashed eggs. Some bacteria are associated more often with spoilage of eggs, particularly during prolonged storage (Haines, 1938; Forsythe et al., 1953; Ayres and Taylor, 1956; Florian and Trussell, 1956; Board et al., 1963; Board, 1966; Mountney and Day, 1970; Moats, 1979, 1980, 1981; Plusquellec, 1995). Recognized spoilage flora include Pseudomonas fluorescens, Pseudomonas putida, Alcaligenes, Proteus, Escherichia, Serratia, Xanthomonas maltophilia, Aeromonas, Hafnia, Citrobacter, Enterobacter, Cytophaga, Achromobacter, Micrococcus, and Pseudomonas aeruginosa. Based on such findings, Gramnegative bacteria are considered to be the primary culprits in terms of egg spoilage. A number of rots have been described that render eggs unfit for human consumption. Bacteria with relatively simple nutritional requirements and those that can survive and grow at refrigerated temperatures are also favored. Some species of Pseudomonas can break down cuticular proteins, allowing for growth of yeasts and molds (Board et al., 1979). Yeasts and molds, able to withstand harsher environmental stresses, can grow on or in eggs and cause spoilage (Beuchat and Cousin, 2001; Ricke et al., 2001). Particularly when eggs are packed while wet or stored at high relative humidity, molds can be an important quality concern (USDA, 2000). Egg solids readily pick up off odors, including those caused by mold growth. When storage conditions are particularly humid, mycelial growth can cover eggs in what are known in the industry as ‘whiskers.’ In 2008, Musgrove (2008b) and others identified 380 yeasts collected from washed and unwashed eggs stored under refrigeration. Candida famata and Candida lusitaniae were the most common species identified. Other genera included Cryptococcus, Hansenula, Hyphopichhia, Metschnikowia, Rhodotorula, Sporobolomyces, and Torulaspora. 1.5.1 Pathogens Though current regulations and guidelines are geared to address quality concerns, there are pathogenic species of bacteria that are of concern to the egg industry and regulatory agencies (Sharar, 2004). Several serotypes of Salmonella have been implicated in egg-borne salmonellosis (Humphrey, 1999; Ricke et al., 2001). Campylobacter jejuni has been implicated in at least one outbreak of egg-borne disease (Finch and Blake, 1985). Other organisms, Listeria monocytogenes, Staphylococcus and Yersinia enterocolitica, are capable of growing in eggs or surviving shell egg and shell egg product processing, or even cooking (Ricke et al., 2001). Salmonella is the most
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Microbiology and safety of table eggs 21 important pathogen associated with shell eggs and egg products (Fazil et al., 2000; Andrews et al., 2001; Ricke et al., 2001). Prior to the passage of the Egg Products Inspection Act in 1970, many different serotypes were associated with foodborne illnesses (Gast et al., 1992). Mandatory USDA inspection, removal of cracked or dirty eggs, and pasteurization standards reduced egg-borne illness associated with Salmonella (Baker and Bruce, 1995). However, since the 1980s, human salmonellosis caused by S. Enteritidis (SE) has increased dramatically (Humphrey, 1999). In 1990, the number of outbreaks in the US peaked but has remained at 45–50 per year for the past several years (Centers for Disease Control, 2003). When outbreaks and sporadic cases are investigated, undercooked and raw eggs are the most common sources of SE infection. The Centers for Disease Control uses the Salmonella Enteritidis Outbreak Surveillance System to decipher foods associated with illness caused by this serotype. Each year since the system has been in place, from 55 to 80% of all SE outbreaks have been somehow linked to shell eggs. Of the 309 confirmed SE outbreaks that occurred from 1990 to 2001, 241 (78.0%) were associated in some way with shell eggs. Salmonellosis caused by S. Enteritidis has also been associated with such foods as juices, salsa, meat, sprouts, fruit, and salads. Most outbreaks, including those considered to be egg-associated, are also linked to an interruption in the cold chain during transport or storage, improper handling, crosscontamination with other foods, or inadequate cooking. A large proportion of SE-illnesses (90%) and deaths (67%) occur in institutional settings where such breakdowns in hygiene or kill steps affect large numbers of people. There were 14,319 cases of illness during this timespan, though 72.7% of them occurred from 1990 to 1995. This overall decrease in SE incidence from 1996 to 2001 may be attributed in part to implementation of a number of preventative measures and education programs. United Egg Producers (2004) initiated a ‘5-star’ Total Quality Assurance Food Safety Program designed to support the efforts of egg producers, processors, and marketers to establish programs that address food safety concerns. This program’s key points include cleaning and disinfection of poultry houses, rodent and pest elimination, proper egg washing, biosecurity, and refrigeration at 7.2 °C from the point of packing through delivery. Poultry improvement plans have been implemented in many states. One of the benefits these programs are designed to produce is a reduction of S. Enteritidis (and other human or avian pathogens) from the environment of layer houses and breeder farms (Henzler et al., 1998). There is considerable data that indicate that factors such as rodent populations and manure levels increase the likelihood that flocks or their environment will be positive for S. Enteritidis. Cleaning and disinfecting hen houses between flocks, following biosecurity measures, monitoring hen mortality, and using SE-free chicks and pullets are also key in reducing a flock’s risk of SE colonization (Sheehan, 2002). A comparison of flocks in Pennsylvania (90% of flocks are in the Pennsylvania Egg Quality Assurance Program) from 1990 to 1998 show a
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22 Improving the safety and quality of eggs and egg products dramatic reduction in the percentage of flocks with SE positive environment (48 v. 10) and percent flocks with SE positive egg (26 v. 2) (Eckroade, 2000). In terms of outbreaks of foodborne disease associated with eggs, clearly Salmonella Enteritidis is the microorganism most often implicated. In terms of sporadic cases of foodborne illness involving eggs, other bacteria may be involved. Campylobacter spp. is another important foodborne pathogen in developed countries that has been isolated from laying hens and eggs. Campylobacter is often recovered from poultry intestinal tracts, feces, and carcasses. Listeria spp., which can grow at refrigerated temperatures, have been isolated from eggs and processing plants in the US and in Canada. Listeria spp. was found to be a frequent contaminant on transfer suction cups. A procedure for analysis using electrolyzed water was developed that did not have a caustic effect on the cups themselves (Jones and Musgrove, 2007). Listeria spp., including Listeria monocytogenes, has been recovered from 2 of 42 samples of raw liquid egg collected from a commercial facility (Foegeding and Leasor 1990). Yersinia enterocolitica, a member of the family Enterobacteriaceae (includes Salmonella, E. coli), is also psychrotrophic. Yersinia entercolitica can penetrate egg shells and membranes. Excess iron (20 ppm) encourages growth and when stored at 10 °C, the organism was able to grow. Aeromonads are ubiquitous in many foods and are thought to play a role in foodborne disease (Isonhood and Drake, 2002). In 2005, Musgrove et al. (2005a) reported that 4.6% of eggs collected from three commercial facilities were contaminated with Aeromonas spp. or Vibrio spp. These organisms are able to survive and grow slowly at refrigerated temperatures (Isonhood and Drake, 2002). Any pathogen associated with eggs that has the ability to grow at refrigerated temperatures is potentially a cause of sporadic egg-borne disease (Ricke et al., 2001). Bacillus cereus has been recovered from egg mélange, raw liquid egg products, pasteurized whole liquid eggs, and egg-based bakery ingredients. Musgrove and Jones (2006) recovered bacilli, including Bacillus cereus from 28% of retail shell eggs. Staphylococcus aureus has been isolated from dirty eggs stored without washing and disinfection as well as from liquid contents. S. aureus has been associated with hard-boiled eggs in Canada and Sweden. Musgrove and Jones found 20% of retail eggs were contaminated Staphylococcus spp. Aeromonads are ubiquitous in many foods and are thought to play a role in foodborne disease (Isonhood and Drake, 2002).
1.6 Bringing eggs and foodborne disease into perspective Food safety is an important concern worldwide (Holmes, 2003). It is estimated that there are 6.5–33 million cases of foodborne illness in the United States annually (Buzby et al., 1996). While most of these cases are mild in nature, © Woodhead Publishing Limited, 2011
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Microbiology and safety of table eggs 23 it is estimated that up to 9000 deaths are attributable to foodborne illness (Mead et al., 1999). Fungi, viruses, protozoa, and bacteria are included in the list of >40 microbial foodborne pathogens that cause these illnesses (Reid and Harris, 1999). Quite often foods of animal origin are implicated as contributing to foodborne illness caused by bacteria though there are often other factors that influence their occurrence. Salmonella is one of the most frequent causes of bacterial foodborne illness (Andrews et al., 2001; Bell and Kyriakides, 2002). Usually, the food itself is blamed for disease regardless of how it is handled during transit or preparation. Several factors may contribute to salmonellosis: preparation of food too far in advance, storage of food at ambient temperatures, inadequate cooling or re-heating, contamination of processed foods, undercooking, cross-contamination, consumption of raw food, incorrect handling while foods are warmed, infected food handlers, improper handling of leftovers, or preparation of excessive amounts of food (catering, institutionally) (Bell and Kyriakides, 2002). 1.6.1 Role of consumer and retailer More work has been conducted on the farm than the fork side of the food chain. In terms of sporadic illness, it is thought that if consumers would handle food properly, 85% of foodborne illnesses would be avoided annually. In a case control study of sporadic cases of SE infection in the UK, takeout chicken was the second highest risk factor for infection after dishes containing raw egg (Cowden et al., 1989). This would indicate that poultry meat is also a source of SE. Yet, many consumers, even when they know the importance of washing hands after touching raw meat, fail to do so (Redmond and Griffith, 2003). Most Salmonella infections involve only a single person at a given time and are referred to as sporadic cases. In fact, it is estimated that 95% of foodborne illnesses occur sporadically (as opposed to outbreaks). Outbreaks are reported more often and are more easily investigated. Most foodborne illnesses occur 12–72 hours after the food is consumed, so rarely is any of the suspect food left to be analyzed. Questioning of those affected provides the only clues. In this way, a food will be implicated circumstantially, and information gathered may or may not include information on how food was stored, handled, or prepared. Certainly these details are often omitted when an outbreak is reported. Table 1.3 lists food types and their relative levels of risk for transmission of salmonellosis to humans. Note that only raw eggs are listed. Eggs have gained an undeserved reputation as being an unsafe food but mishandling and inadequate cooking often contribute to egg-related salmonellosis. According to the CDC, in outbreaks where the place in which the food was eaten was known, food service establishments and group feeding situations are the most common sites where foodborne illness is acquired. It is likely that improper handling or cooking contributed to these outbreaks.
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24 Improving the safety and quality of eggs and egg products Table 1.3 Levels of concern for exposure to food borne salmonellosis for various foods1 Risk level
Food
Highest
Raw milk ripened soft cheeses, sprouts
High
Raw eggs, salami, dry cured ham, chocolate, infant dried milk powder, raw poultry, cooked poultry, unpasteurized fruit juice
Medium
Prepared salads, sushi, pasteurized milk ripened cheese, Brie, cooked meat, pasteurized milk hard cheese, raw red meats
Low
Raw fish and shellfish, cod, plaice, mussels
Lowest
Chub paté, products cooked in pack
Source: Bell and Kyriakides (2002).
In 2006, two outbreaks of salmonellosis were linked to egg consumption. A high end restaurant prepared a sauce with raw eggs and kept it under a heat lamp. Over 100 people were sickened and S. Heidelberg was recovered from the sauce and raw eggs. In another case, over 50 patients at a psychiatric hospital became ill after nest run eggs were broken to prepare an egg dish. In order to prevent the edges from drying, the dish was not completely cooked. While the primary function of the kitchen is the location where food is prepared, it may also serve as a study, laundry, work room, and as housing for pets. As well as foods, people, water, pets, insects, rodents, and even the air can be a source of bacteria in the home, including the kitchen. However, when an outbreak is reported, seldom is more than the commodity and number of people mentioned – though many other factors may have contributed to the disease occurrence (cross-contamination, improper storage temperatures, inadequate cooking, etc.) (Redmond and Griffith, 2003). Historically, the consumer has remained the least studied link in the food chain. Most information concerning consumers has been collected anecdotally. However, in recent years, more work has been published in this area. In April 2003, the World Health Organization and the Food and Agriculture Organization of the United Nations announced a ‘Food Chain Approach’ to food safety. Similar in nature to ‘Farm to Fork’ approaches used in developed countries worldwide, the FAO-WHO launched initiative emphasized the need for consumer education. Information regarding appropriate handling and preparation is readily available from university extension departments, in retail advertisements, and even on product packaging. Recent studies describe how and why consumers behave as they do in terms of food safety.
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Microbiology and safety of table eggs 25
1.7 Acknowledgements Chapters from Commercial Chicken Meat and Egg Production, Egg Science and Technology, and Microbiology of the Avian Egg were extremely helpful in preparing this chapter. Review papers on egg washing by W. A. Moats and M. L. Hutchison were also very helpful.
1.8 References and further reading allison d. g.
1993. Biofilm associated exopolysaccharides. Microbiology, 12:
16–19. 2010. U.S. Egg Industry Fact Sheet. http://www.aeb.org. Date accessed: Mar. 5, 2010. anderson, k. e. 2003. The nutritious egg. Pages 26–34 in Proceedings of the National Egg Quality School, North Carolina State University, Raleigh, NC. andrews, w. h., r. s. flowers, j. silliker, and j. s. bailey. 2001. Salmonella. Pages 357–380 in Compendium of Methods for the Microbiological Examination of Foods, 4th ed., F. P. Downes and K. Ito (eds.), American Public Health Assoc., Washington, DC. ayres, j. c. and b. taylor, 1956. Effect of temperature on microbial proliferation in shell eggs. Appl. Microbiol. 4: 355–359. bailey, j. s., j. e. thomson, and n. a. cox, 1987. Contamination of poultry during processing, Pages 193–211 in The Microbiology of Poultry Meat Products, F. E. Cunningham and N. A. Cox (eds), Academic Press, New York. bailey, j. s., n. j., stern, p. j., fedorka-cray, n. a., cox, d. r., cosby, s. r. ladely, and m. t. musgrove, 2001. ‘Survey of Salmonella contamination in commercial broiler facilities’, J. Food Prot. 64: 1001–1008. baker, r. c. and c. bruce, 1995. Effects of processing on the microbiology of eggs. Pages 153–181, in Microbiology of the Avian Egg, R. G. Board and R. Fuller (eds.), Chapman and Hall, London. barnhart, h. m., d. w. dreesen, r. bastien, and o. c. pancorbo, 1991. Prevalence of Salmonella enteritidis and other serovars in ovaries of layer hens at time of slaughter. J. Food Prot. 54: 488–491. baumler, a. j., b. m. hargis, and r. m. tsolis. 2000. Tracing the origins of Salmonella outbreaks. Science 287: 50–53. bell, c. and a. kyriakides, 2002. Salmonella: A Practical Approach to the Organism and its Control in Foods. Blackwell Science, Cornwall, England. bell, d. d. 1995. Forces that have helped shape the U.S. egg industry: the last 100 years. Poult. Tribune 1: 30–43. bell, d. d. 2002. Introduction to the U.S. table-egg industry. Pages 945–964 in Commercial Chicken Meat and Egg Production, 5th ed., D. D. Bell and W. D. Weaver, Jr. (eds.), Kluwer Academic, Norwall, MA. berrang, m. e., n. a. cox, j. s. bailey, and l. c. blankenship, 1991. Methods for inoculation and recovery of Salmonella from chicken eggs. Poult. Sci. 70: 2267–2270. american egg board
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26 Improving the safety and quality of eggs and egg products beuchat, l. r.
and m. a. cousin. 2001. Yeasts and molds. Pages 209–216 in Compendium of Methods for the Microbiological Examination of Foods, 4th ed., F. P. Downes and K. Ito (eds.), American Public Health Assoc., Washington, DC. board, r. g. 1966. Review article: the course of microbial infection of the hen’s egg. J. Appl. Bact. 219: 319–341. board, r. g. and h. s. tranter, 1995. The microbiology of eggs. Pages 81–103 in Egg Science and Technology, 4th ed., W. J. Stadelman and O. J. Cotterill (eds.), Food Products Press, New York. board, r. g., j. c. ayres, a. a. kraft, and r. h. forsythe, 1963. The microbiological contamination of egg shells and egg packing materials. Poult. Sci. 43: 584– 595. board, r. g., s. loseby, and v. r. miles, 1979. A note on microbial growth on hen egg shells. Br. Poult. Sci. 20: 413–420. board, r., c. clay, j. lock, and j. dolman, 1994. The egg: a compartmentalised, aseptically packed food. In Microbiology of the Avian Egg, R. Board and R. Fuller (eds), Chapman and Hall, London. brant, a. w. and p. b. starr, 1962. Some physical factors related to egg spoilage. Poult. Sci. 41: 1468–1473. bruce, j. and e. m. drysdale, 1994. Trans-shell transmission. Pages 63–91 in Microbiology of the Avian Egg, R. G. Board and R. Fuller (eds.), Chapman & Hall, London. buzby, j. c., t. roberts, c. t. j. lin, and j. m. macdonald, 1996. Bacterial Foodborne Disease: Medical Costs & Productivity Losses. Agricultural Economic Report Number 741. An Economic Research Service Report. USDA. bygrave, a. c. and j. gallagher. 1989. Transmission of Salmonella enteritidis in poultry. Vet. Rec. 124: 333. catalano, c. r. and s. j. knabel, 1994. Destruction of Salmonella enteritidis by high pH and rapid chilling during simulated commercial egg processing. J. Food Prot. 57: 592–595. centers for disease control 2003. Outbreaks of Salmonella serotype Enteritidis infection associated with eating shell eggs – United States, 1999–2001. Mortal. Morbid.Week. Report 51: 1149–1152. conner, j. w., s. e. snyder, and h. orr, 1953. The influence of washing and oiling on grade and bacterial content of eggs stored for a nine month period. Poult. Sci. 23: 227–235. cowden, j. m., d. lynch, c. a. joseph, m. o’mahoney, s. l. mawer, b. rowe, and c. l. r. bartlett, 1989. Case-control study of infections with Salmonella Enteritidis phage type 4 in England. Br. Med. J. 299: 771–773. cox, n. a., n. j. stern, k. l. hiett, and m. e. berrang, 1999. Transmission of Campylobacter from breeders to commercial broiler chickens. Page 67 in Proceedings of the International workshop on Campylobacter, Helicobacter, and related organisms, Copenhagen, Denmark (abstract). cox, n. a., n. j. stern, k. l. hiett, r. j. buhr, s. e. craven, j. s. bailey, j. e. wilson, m. e. berrang, m. t. musgrove, and g. r. siragusa, 2000. Eggborne transmission of Campylobacter jejuni from the breeder hen to her progeny and the presence of
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Microbiology and safety of table eggs 27 other human foodborne pathogens in the breeder hen’s reproductive system. Pages 1–5 in Proceedings of Campylobacter Control in Poultry. Arhus, Denmark. cox, n. a., j. s. bailey, l. j. richardson, r. j. buhr, k. l. hiett, g. r. siragusa, d. e. cosby, j. l. wilson, and d. v. bourassa, 2004. Detection of Campylobacter and Salmonella in the mature and immature reproductive follicles of late life broiler breeder hens. Poultry Sci. 83: (Suppl):1 (abstract). curtis, m. and e. johnston, 1998. Chlorine disinfection. Available at : http://www. ce.vt.edu/environ2/wtprimer/chlorine/chlorine.html. Accessed Jan. 2004. curtis, p. a. 2002. Clean eggs, our most important merchandising tool. Pages 56–61 in Proceedings of the National Egg Quality School, North Carolina State University, Raleigh, NC. curtis, p. a., k. e. anderson, and f. t. jones, 2004. Designing a HACCP plan for shell egg processing plants. http://www.ces.ncsu.edu/depts/foodsci/ext/pubs/ haccp2.html. Accessed Jan. 2004. davies, r. h. and m. breslin, 2003. Investigation of Salmonella contamination and disinfection in farm egg-packing plants. J. Appl. Microbiol. 94: 191–196. de reu, k., k. frijspeerdt, m. heyndrickx, m. uytendaele, and l. herman, 2003. Bacterial eggshell contamination in the egg production chain and in different housing systems. Pages 180–185 in Proceedings of the XVIth European Symposium on the Quality of Eggs and Egg Products, Cotes d’Amor, France. diamond systems , 2010. Products. http://www.diamondsystem.com/english/. Accessed Jan. 2010. eckroade, r. j. 2000. Shell eggs and egg products – safety issues: Egg quality assurance programs. Pages 51–60 in Shell Egg and egg products – safety issues, Institute of Food Technologists, Atlanta, GA. fazil, a. m., r. a. morales, a. m. lammerding, a. s. vicari, and f. kasuga, 2000. hazard identification and hazard characterization of Salmonella in broilers and eggs, Food and Agriculture Organization of the United Nations and World Health Organization, Rome, Italy. fda , 2007. Bacteriological Analytical Manual. Chapter 5. Salmonella. http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/ BacteriologicalAnalyticalManualBAM/ucm070149.htm. Accessed May, 2010. fda, 2009. Prevention of Salmonella Enteritidis in shell eggs during production, storage, and transportation. 21 CFR parts 16 and 118. finch, m. j. and p. a. blake, 1985. Foodborne outbreaks of campylobacteriosis: the United States experience, 1980–1982. Am. J. Epidemiol. 122: 262–268. fletcher, d. l. and d. p. smith, 1990. A comparison of seven microbiological sampling procedures for hard-cooked eggs. Poult. Sci. 69: 1428–1432. florian, m. l. e. and p. c. trussell. 1956. Bacterial spoilage of shell eggs. IV. Identification of spoilage organisms. Food Technol. 11: 56–60. foegeding , p . and s . leasor , 1990. Heat resistance and growth of Listeria monocytogenes in liquid whole egg. J. Food Prot. 53: 9–14. folkes, l. k., l. p. candeias, and p. wardman. 1995. Kinetics and mechanisms of hypochrolrous acid reactions. Archives of Biochem. Biophysics 323: 120– 126.
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28 Improving the safety and quality of eggs and egg products and inspection service, 1999. All about shell eggs. http://www.fsis. usda.gov/OA/pubs/shelleggs.htm. Accessed March 2003. foodsafety.gov, 2004. Gateway to government food safety information. http:// www.foodsafety.gov/ Accessed March 2003. forsythe, r. h., j. c. ayres, and j. l. radlo. 1953. Factors affecting the microbiological populations of shell eggs. Food Technol. 7: 49–56. garibaldi, j. a. and j. l. stokes. 1958. Protective role of shell membranes in bacterial spoilage of eggs. Food Res. 23: 283–290. gast, r. k. and c. w. beard, 1992. Detection and enumeration of Salmonella enteritidis from internal organs of experimentally infected hens. Av. Dis. 34: 991–993. gast, r. k., n. a. cox, and j. s. bailey, 1992. Salmonellae in eggs. Pages 49–67 in Food Poisoning, Tu, A. T. (ed), Marcel Dekker, New York, NY. gentry, r. f. 1970. Measurement and control of egg shell contamination. U.S. Agricultural Research Service, ARS–91 101: 28–32 gentry, r. f. and c. l. quarles, 1972. The measurement of bacterial contamination of egg shells. Poult. Sci. 51: 930–933. gillespie, j. m., m. r. j. salton, and w. j. scott, 1950. Studies on the preservation of shell eggs V. The use of chemical disinfectants in cleaning machines. Austr. J. Appl. Sci. 1: 531–538. gunaratne, j. w. b. and j. v. spencer, 1973. A study of methods to enumerate the microbial flora of the avian egg shell. J. Milk Food Technol. 36: 101–102. haines, r. b. 1938. Observations on the bacterial flora of the hen’s egg, with a description of a new species of Proteus and Pseudomonas causing rots in eggs. J. Hyg. Camb. 38: 338–355. haines, r. b. and t. moran, 1940. Porosity of, and bacterial invasion through, the shell of the hen’s egg. J. Hygiene 40: 453–561. haque, a. k. m., j. m. vandepopuliere, and o. j. cotterill. 1989. Microbial counts and Salmonella in shell eggs from seven retail outlets. Poult. Sci. 68: 62 (abstract). harry, e. g. 1963. Some observations on the bacterial content of the ovary and the oviduct of the fowl. Br. Poult. Sci. 4: 63–70. henzler, d., d. kradel, and w. sischo, 1998. Management and environmental risk factors for Salmonella enteritidis contamination of eggs. American Journal of Veterinary Research 59: 824–829. holmes, s. 2003. From farm to table: a global approach to food safety. Food and Agriculture Organization of the United Nations. www.fao.org/english/newsroom/ news/2003. Accessed March 2003. humphrey, t. j. 1999. Contamination of eggs and poultry meat with Salmonella enterica serovar Enteritidis. Pages 183–192 in Salmonella enterica serovar Enteritidis in Humans and Animals: Eidemiology, Pathogenesis, and Control. A. M. Saeed, R. K. Gast, M. E. Potter, P. G. Wall (eds), Iowa State University Press, Ames, IA. humphrey, t. j., a. baskerville, s. mawer, b. rowe, and s. hopper, 1989. Salmonella enteritidis phage type 4 from the contents of intact eggs: a study involving naturally infected hens. Epidemiol. Infect. 103: 415–423. food safety
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and b. rowe, 1991. Numbers of Salmonella enteritidis in the contents of naturally contaminated hens’ eggs. Epidemiol. Infect. 106: 489–496. hutchison, m. l., j. gittins, a. walker, a. moore, c. burton, and n. sparks, 2003. Washing table eggs: a review of the scientific and engineering issues. World’s Poult. Sci. J. 59: 233–248. International Food Information Council, 1999. Worth the risk – putting activities into perspective. http:/www.ific.org/foodinight/1999/nd/worthriskfi699.cfm. Accessed March 2003. isonhood, j. and m. drake, 2002. Aeromonas species in foods. J. Food Prot. 65: 575–582. johnson, h. 2002. What is covered by the Egg Products Inspection Act (EPIA) and what is covered by the voluntary grading program. Pages 140–146 in Proceedings of the National Egg Quality School. North Carolina State University, Raleigh, NC. jones, d. r. and m. t. musgrove, 2007. Assessment of microbial contaminants present on vacuum loaders in shell egg processing facilities. J. Food Safety 28: 346– 354. jones , d . r ., j . k . northcutt , m . t . musgrove , p . a . curtis , k . e . anderson , n. a. cox, and d. l. fletcher, 2003. Survey of shell egg processing plant sanitation programs: effects on egg contact surfaces. J. Food Prot. 66: 1486– 1489. jones, d. r., m. t. musgrove, and j. k. northcutt, 2004. Variations in external and internal microbial populations in shell eggs during extended storage. J. Food Prot. 67: 2657–2660. jones, f. t., d. v. rives, and j. b. carey, 1995. Salmonella contamination in commercial eggs and an egg production facility. Poult. Sci. 74: 753–757. kawasaki t. m., m. musgrove, m. murata, n. tominaga, and s. kawamoto, 2008. ‘Comparative study of shell swab and shell crush methods for the recovery of Salmonella from shell eggs, J. Food Safety, 28: 482–498. kinner, j. a. and w. a. moats, 1981. Effect of temperature, pH, and detergent on survival of bacteria associated with shell eggs. Poult. Sci. 60: 761–767. klippen, k. 1990. Egg production and processing. Dairy, Food and Environment. San. 10: 266–267. knape, k. d., c. chavez, r. p. burgess, c. d. coufal, and j. b. carey, 2002. Comparison of eggshell surface microbial populations for in-line and off-line commercial egg processing facilities. Poult. Sci. 81: 695–698. kornacki, j. l. and j. l. johnson, 2001. Enterobacteriaceae, Coliforms, and Escherichia coli as quality and safety indicators. Pages 69–82 in Compendium of Methods for the Microbiological Examination of Foods, 4th ed, F. P. Downes and K. Ito (eds), American Public Health Assoc. Washington, DC. kraft, a. a., l. e. elliott, and a. w. brant, 1958. The shell membrane as a barrier to bacterial penetration of eggs. Poult. Sci. 37: 238–240. kumar, c. g. and s. k. anand, 1998. Significance of microbial biofilms in the food industry. Intl. J. Food Microbiol. 42: 9–27.
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30 Improving the safety and quality of eggs and egg products leclair, k., h. heggart, m. oggel, f. m. bartlett,
and r. c. mckellar, 1994. Modeling the inactivation of Listeria monocytogenes and Salmonella typhimurium in simulated egg wash water. Food Microbiol. 11: 345–353. lillard , h . s . 1988. Comparison of sampling methods and implications for bacterial decontamination of poultry carcasses by rinsing. J. Food Prot. 51: 405–408. lister, s. a. 1988. Salmonella enteritidis infection in broilers and broiler breeders. Vet. Rec. 123: 350. lock, j. l., j. dolman, and r. g. board, 1992. Observations on the mode of bacterial infection of hens’ eggs. FEMS Microbiol. Letters 100: 71–74. march, b. e. 1969. Bacterial infection of washed and unwashed eggs with respect to salmonellae. Appl. Microbiol. 17: 98–101. mawer, s. l., g. e. spain, and b. rowe, 1989. Salmonella enteritidis phage type 4 and hens’ eggs. Lancet 1: 280–281. mayes, f. j. and m. a. takeballi. 1983. Microbial contamination of the hen’s egg: a review. J. Food Prot. 46: 1092–1098. mcnamara, d. j. 2003. Being positive about eggs. Pages 230–245 in Proceedings of the National Egg Quality School, North Carolina State Univeristy, Raleigh, NC. mead, p. s. , l. slutsker, v. dietz, l. f. mccaig, j. s. bresee, c. shapiro, p. m. griffin, and r. v. tauxe, 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5: 607–625. moats, w. a. 1978. Egg washing – a review. J. Food Prot. 41: 919–925. moats, w. a. 1979. The effect of washing eggs under commercial conditions on bacterial loads on egg shells. Poult. Sci. 58: 1228–1233. moats, w. a. 1980. Classification of bacteria from commercial egg washers and washed and unwashed eggs. Appl. Environ. Microbiol. 40: 710–714. moats, w. a. 1981. Factors affecting bacterial loads on shells of commercially washed eggs. Poult. Sci. 60: 2084–2090. mountney, g. j. and c. day, 1970. Microbiological contamination of water used for washing eggs. Poult. Sci. 49: 146–149. murase, t., k. senjyu, t. maeda, m. tanaka, h. sakae, y. matsumoto, y. kaneda, t. ito, and k. otsuki, 2001. Monitoring of chicken houses and an attached eggprocessing facility in a laying farm for Salmonella contamination between 1994 and 1998. J. Food Prot. 64: 1912–1916. musgrove, m.t., and d.r. jones (2006). Microbiological contamination of packer head brushes in US commercial shell egg processing plants. XII European Poultry Conference Proceedings, Verona, Italy. musgrove, m. t., d. r. jones, j. k. northcutt, and m. a. harrison, 2003. Multiple rinses of eggshells for recovery of aerobes and Enterobacteriaceae. Poult. Sci. 82:54 (abstract). musgrove, m. t., d. r. jones, j. k. northcutt, p. a. curtis, k. e. anderson, n. a. cox, and d. l. fletcher. 2004a. Survey of shell egg processing plant sanitation programs: effects on non egg contact surfaces. J. Food Prot. 67: 2801– 2804.
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Microbiology and safety of table eggs 31 musgrove, m. t., d. r. jones, j. k. northcutt, n. a. cox,
and m. a. harrison. 2004b. Identification of Enterobacteriaceae from washed and unwashed commercial shell eggs. J. Food Prot. 68: 2819–2822. musgrove, m. t., d. r. jones, j. k. northcutt, m. a. harrison, and n. a. cox, 2005a ‘Identification of Enterobacteriaceae and related organisms from rinses of eggs collected during processing in commercial shell egg processing plants in the Southeastern United States. Poultry Sci, 87: 1211–1218. musgrove, m. t., d. r. jones, j. k. northcutt, m. a. harrison, and n. a. cox, 2005b. Impact of commercial processing on the microbiology of eggs. J Food Prot, 68: 2367–2375. musgrove, m. t., d. r. jones, j. k. northcutt, m. a. harrison, and n. a. cox, 2005c. Shell rinse and shell crush methods for the recovery of aerobic microorganisms and Enterobacteriaceae from table eggs. J. Food Prot. 68: 2144–2148. musgrove, m. t., m. e. berrang, and k. a. liljebjelke, 2008a. Attached bacterial cell contamination of shell egg processing facilities. Pages 66–67 in Proceedings of the 37th United States-Japan Cooperative Program in Natural Resources. musgrove, m. t., d. r. jones, j. d. shaw, m. sheppard, and m. a. harrison. 2008b. Enterobacteriaceae and related organisms isolated from nest run cart shelves in commercial shell egg processing facilities. Poultry Sci. 88: 2113– 2117. musgrove, m. t., n. a. cox, m. e. berrang, r. j. buhr, j. s. bailey, and j. m. mauldin, 2011. Effect of inoculation and application methods on the performance of chemicals used to disinfect Salmonella contaminated eggs. J. Appl. Poult. Res. (in press). northcutt, j. and d. smith, 2010. Microbiological and chemical analyses of ice collected from a commercial poultry processing establishment. Poult. Sci. 89: 145–149. penniston, v. and l. r. hedrick, 1947. The reduction of bacterial count in egg pulp by use of germicides in washing dirty eggs. Food Technol. 1: 240–244. plusquellec, a. 1995. Eggs and egg products. Pages 431–435 in Microbiological Control for Foods and Agricultural Products, C. M. Bourgeois, J. Y. Leveau, D. Y. C. Fung (eds), VCH Publishers, Inc. New York, NY. poppe, c., r. johnson, c. forberg, and r. irwin, 1992. Salmonella enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment. Can. J. Vet. Res. 56: 226–232. redmond, e. c. and c. j. griffith, 2003. Consumer food handling in the home: a review of food safety studies. J. Food Prot. 66: 130–161. reid, d. s. and l. j. harris, 1999. Microorganisms and microbial toxins. Pages 9–21 in Impact of Processing on Food Safety, L. S. Jackson, M. G. Knize, and J. N. Morgan (eds), Kluwer Academic, New York, NY. reynells, r. d. 2004. Egg Products Inspection – the USDA Food Safety and Inspection service has issued a Final Rule, which took effect on January 12.
[email protected]. ricke, s. c., s. g. birkhold, and r. k. gast, 2001. Eggs and egg products. Pages 473–479 in Compendium of Methods for the Microbiological Examination of
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32 Improving the safety and quality of eggs and egg products Foods, 4th ed., F. P. Downes and K. Ito (eds), American Public Health Assoc. Washington, DC. romanoff, a. l. and a. j. romanoff, 1949. The Avian Egg., Wiley, New York, NY. schutze, g. e., h. a. fawcett, m. j. lewno, e. l. flick, and r. s. kirby, 2002. Prevalence of Salmonella enteritidis in poultry shell eggs in Arkansas. www. sma.org/smj/96sept8.htm. Accessed Jan. 2004. sharar, a. k. 2004. Policy needs for eggs and egg products. Oral presentation at the FSIS-ARS annual meeting, Shepardstown, WV. sheehan, j. f. 2002. The federal egg safety action plan. Pages 218–232, in Proceedings of the National Egg Quality School, North Carolina State University, Raleigh, NC. solomon, s. e. 1991. Egg and Eggshell Quality, Wolfe Publishing Ltd, Aylesbury, UK. sperber, w. h. 2004. HACCP does not work from Farm to Table. Food Control 16: 511–515. srikaeo, k. and j. a. hourigan, 2002. The use of statistical process control (SPC) to enhance the validation of critical control points (CCPs) in shell egg washing. Food Control 13: 263–273. stadelman, w. j. 1995. The egg industry. Pages 1–8 in Egg Science and Technology, 4th ed., W. J. Stadelman and O. J. Cotterill (eds.), Food Products Press, New York. stadelman, w. j., v. m. olson, g. a. shemwell, and s. pasch, 1988. Shell egg production and processing. Pages 40–51 in Egg and Poultry Meat Processing, Weinheim Publishing, New York, NY. todd, e. c. d. 1996. Risk assessment of use of cracked eggs in Canada. Intl. J. Food Microbiol. 30: 125–143. united egg producers. 2004. Five Star Food Safety Program. http://unitedegg.org/ html/safety/fivestar.html. Accessed March 2003. united states department of health and human services public health service. 1998. Principles of Epidemiology, 2nd ed., Centers for Disease Control, Atlanta, GA. usda. 1996. Pathogen reduction; hazard analysis and critical control point (HACCP) systems, Final rule. 9 CFR Part 304, Washington, DC. usda. 1998. Salmonella Enteritidis risk assessment: shell eggs and egg products, final report. Food Safety and Inspection Service, Washington, DC. usda. 2000. Egg-grading manual. USDA, Washington, DC. usda. 2003. Regulations governing the inspection of eggs (Egg Products Inspection Act). 7 CFR Part 57, Washington, DC. whiting, r. c. and r. l. buchanan, 1997. Predictive Modeling. Pages 728–739 in Food Microbiology Fundamentals and Frontiers, M. P. Doyle, L. R. Beuchat, and T. J. Montville (eds), ASM Press, Washington, DC. whiting, r. c., a. hogue, w. d. schlosser, e. d. ebel, r. a. morales, a. baker, and r. m. mcdowell, 2000. A quantitative process model for Salmonella Enteritidis in shell eggs. J. Food Sci. 65: 864–869.
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Microbiology and safety of table eggs 33 winter, a. r., b. burkhart, p. clements,
and l. mcdonald. 1955. Cleaning eggs with detergents and detergent sanitizers. Ohio Agr. Exp. Sta. Bull. 726. zeidler , g . 2002. Processing and packaging shell eggs. Pages 1129–1162 in Commercial Chicken Meat and Egg Production, 5th ed.
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2 Foodborne disease associated with eggs: microbial hazards and Salmonella enteritidis risk assessment M. Chemaly and G. Salvat, Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses) France
Abstract: Eggs and egg products can be contaminated by a variety of pathogens at any stage of the production chain. Although Salmonella Enteritidis is by far the major risk associated with egg consumption, other hazards may occur during egg production and processing. The purpose of this chapter is to describe potential hazards associated with egg production and egg products and review existing risk assessment models related to Salmonella infection. Key words: pathogens, eggs, egg products, risk assessment.
2.1 Introduction Food poisoning is an illness resulting from contaminated food consumption; although it is commonly a mild illness, it can sometimes be deadly. Food poisoning falls into two main categories: food infection and food intoxication. Food infections are caused by the presence of bacteria or other microbes in the food, which lead to an infection after consumption. Food intoxications occur when ingesting toxins contained within the food such as bacterial exotoxins. Typical symptoms of food poisoning include nausea, vomiting, abdominal cramping, and diarrhoea, the onset of which can start rapidly, within 48 hours after consuming the contaminated food or drink. Until the mid-1980s, eggs and egg products were considered one of the safest foods, because egg content was considered to be sterile at the time of lay (Board, 1994). The emergence of Salmonella Enteritidis in laying hens in
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Foodborne disease associated with eggs 35 the late 1980s made shell eggs one of the most common sources of human salmonellosis worldwide (Greig and Ravel, 2009). Because of the ability of the bacteria to transmit vertically, infections in laying hen flocks have been responsible for numerous foodborne disease cases and outbreaks associated with raw or undercooked egg consumption worldwide. Despite the fact that Salmonella Enteritidis represents by far the most important risk associated with egg consumption, other hazards may occur during egg production and transformation that must also be taken into consideration. The purpose of this chapter is to describe the potential risks associated with egg production and egg product manufacturing and consumption, to review risk analyses associated with these hazards, and to suggest ways to control these risks at the various stages of the production process.
2.2 Hazard identification Epidemiological investigations reveal that poultry meat and eggs are major sources of foodborne diseases affecting humans (Luber, 2009). Salmonellosis and campylobacteriosis are the most frequently reported foodborne illnesses worldwide. In European countries, a total of 131,468 and 190,566 confirmed cases of human salmonellosis and campylobacteriosis respectively were reported in 2008 (EFSA, 2010a). Although eggs and egg products are often incriminated in outbreaks caused by S. Enteritidis (Greig and Ravel, 2009), they are susceptible to hosting other pathogens as well, such as Campylobacter and Listeria monocytogenes. In this section, we will discuss the contamination of fresh eggs and egg products by these different pathogens. 2.2.1 At the farm Zoonotic agents such as Salmonella and Campylobacter are commonly found in the intestines of a broad range of domestic and wild animals; as a result, they represent potential sources of infections in a wide variety of foodstuffs. Transmission to humans often occurs when these pathogens are introduced in the food in which they then multiply (for example, due to inadequate storage temperatures, inadequate cooking or other reasons). These organisms may also be transmitted by direct contact with the infected animals or humans or in faecally contaminated environments. A European baseline study conducted in 2004 in all Member States estimated the prevalence of Salmonella spp. in laying hen flocks in European countries to vary between 0 and 79.5% (EFSA, 2006). The five most frequently isolated serovars were Salmonella Enteritidis, Infantis, Typhimurium, Mbandaka and Livingstone. The observed prevalence of S. Enteritidis in the EU was found to be 18.4%. Epidemiological studies conducted in laying hen farms determined that several risk factors such as flock size, housing and farming
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36 Improving the safety and quality of eggs and egg products characteristics, season and poultry age (Garber et al., 2003; Castellan et al., 2004; Mollenhorst et al., 2005; Huneau-Salaün et al., 2009) could be linked to the prevalence of the pathogen. In France, prevalence was noted to be significantly higher in cage flocks than in on-floor flocks (Huneau-Salaün et al., 2009). The risk of Salmonella contamination in cage flocks increased with flock size and the passing of delivery trucks by poultry-house entrances. In floor-housed flocks, contamination risks increased with multistage management (presence of hens of different ages on the farm) and if a previous flock on the farm had been contaminated by S. Enteritidis. Although Campylobacter contamination is more often associated with poultry meat and broiler flocks where its prevalence can reach 100% (EFSA, 2010b), this pathogen can also be found in laying hen farms (Doyle, 1984; Shane et al., 1986; Rasschaert et al., 2007; Sulonen et al., 2007; Schwaiger et al., 2008; Cox et al., 2009). The prevalence of Campylobacter in laying hen farms varies among studies and countries between 35% (Rasschaert et al., 2007) and 84% (Sulonen et al., 2007). Campylobacter is frequently isolated from caeca but is able to colonize other organs and tissues such as the ovarian follicles, the reproductive tract, the spleen and the liver-gallbladder (Cox et al., 2009). Although the ability of Campylobacter to colonize reproductive tracts has been demonstrated, there is no evidence for vertical transmission of the pathogen (Sahin et al., 2003; Callicott et al., 2006; Fonseca et al., 2006). Listeria monocytogenes is not frequently studied in poultry flocks, possibly because poultry products are rarely involved in cases of listeriosis. Nevertheless, an investigation carried out in poultry flocks in France revealed that L. monocytogenes was present in 30% of caged hen flocks (Chemaly et al., 2008a). The most frequently detected serotype was the 1/2a (84%), which is the second major cause of human listeriosis after the serogroup 4 (Goulet et al., 2004). It is responsible for most of the human cases, especially in single origin outbreaks (Goulet et al., 2004). L. monocytogenes may present a risk to egg and egg product consumers because of its ability to transfer to eggshells and occasionally to egg products (Protais et al., 2007; Rivoal et al., 2010). 2.2.2 Eggshells Owing to the ability of S. Enteritidis to colonize laying hen ovarian tissue and reproductive tracts as well as its presence within the contents of intact shell eggs, the pathogen has been presented as an important virulence factor, making it a potential source of pandemics (Gantois et al., 2009). However, the vertical transmission of S. Enteritidis to the egg yolk is considered to be only a marginal occurrence (Poppe et al., 1992; Henzler et al., 1998) and epidemiological data gathered after an outbreak showed that only 6 out of 355 eggs (1.7%) presented yolks positive for S. Enteritidis (Fravalo et al., 2006). Moreover, eggshells can also become contaminated either from
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Foodborne disease associated with eggs 37 uterine infections (shell gland) or environmental conditions due to bacteria shed by infected animals. In a recent investigation conducted on eggs and eggshells collected from Salmonella-positive flocks, it was discovered that while 39% of the contaminated flocks had positive eggshells only 1.05% of the egg shells tested positive for Salmonella. Therefore, eggs can remain Salmonella-free even when the flock is contaminated (Chemaly et al., 2009). A multivariate analysis conducted on a data set collected from 30 Salmonella-positive flocks reveals that environmental conditions are potential factors that can influence eggshell contamination: flocks with high holding capacity (>30,000 laying hens), with high laying rate (>96%), where delivery trucks pass by the air entrances of the poultry house, having more than five environmental samples positive for Salmonella, and and those with farmers working in other animal production are more likely to have contaminated eggshells by Salmonella than flocks presenting the opposite characteristics. Eggshells collected from pathogen-free flocks can also become contaminated during packing. It has been shown that egg-packing plants present a contributory factor to the external contamination of eggshells (Davies and Breslin, 2003; Jones and Musgrove, 2008). Eggshell contamination by Campylobacter and L. monocytogenes has not been widely investigated. Some studies have reported that the presence of Campylobacter on eggshells remains scarce despite flock infection (Doyle, 1984; Shane et al., 1986; Jones and Musgrove, 2007; Schwaiger et al., 2008; Widdicombe et al., 2009). Shane et al. (1986) demonstrated that the pathogen C. jejuni cannot survive more than 16 hours in a desiccated environment. A study performed in Trinidad found that 1.1% of sampled table eggs were contaminated by Campylobacter while all samples tested negative for Listeria (Adesiyun et al., 2005) and a study performed in Italy showed no egg contamination by L. monocytogenes (Busani et al., 2005). 2.2.3 Egg products Both the microbiological quality of egg contents and eggshells can influence the quality of egg products. It has been demonstrated that Salmonella on eggshells can migrate to egg content under normal storage and moisture conditions (Humphrey, 1994; De Reu et al., 2005) and therefore become a potential source of contamination in egg dishes or other food items due to cross-contamination during handling. Greig and Ravel (2009) analysed international outbreak data for source attribution and found that S. Enteritidis was the most frequent Salmonella serotype associated with outbreaks and eggs were among the most frequently reported food categories affected. Recently, a prevalence study involving three main egg-breaking plants in France revealed that raw egg products could present Salmonella contamination levels as high as 90%. Pasteurization was shown to significantly decrease the percentage of positive samples down to 3.9% (Rivoal et al., 2009).
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38 Improving the safety and quality of eggs and egg products During egg processing, the control of factors such as salt and sugar concentrations, pH and temperature in whole egg products can offer a potential means of restricting S. Enteritidis contamination in egg products (Chemaly et al., 2006, 2008b, 2010). Egg product processing may involve sweet or salty preparations intended for the food industry. In the case of sweet egg products, changes in temperatures (ranging from 1 to 25 °C) and sugar concentrations (ranging from 0 to 50%) can significantly affect (p = 0.00) the growth rate of S. Enteritidis. Inhibition improves by decreasing temperatures and increasing sugar concentrations. Interactions between temperature (from 1 to 25°C), sugar concentration (from 0 to 42%) and pH (from 5.0 to 9.0) significantly inhibit growth of S. Enteritidis (Chemaly et al., 2008b). In the case of salty egg products, changes in temperature and salt concentrations (from 0 to 24%), individually or as a combination, can significantly affect (p = 0.00) the growth rate of S. Enteritidis. Inhibition improves by decreasing temperatures and increasing salt concentrations (Chemaly et al., 2010). Based on a quantitative risk assessment study, the estimated annual cases of human salmonellosis caused by pasteurized whole eggs without salt or sugar, 10% salt and 10% sugar, were found to be 1763, 407 and 0 respectively (Latimer et al., 2008). Very few studies have reported the occurrence of Campylobacter and L. monocytogenes in egg products (Izat and Gardner, 1988; Busani et al., 2005; Protais et al., 2007; Ohkochi et al., 2009; Rivoal et al., 2010; Sato and Sashihara, 2010). The reported incidence of Campylobacter and L. monocytogenes varied among the countries and the studies. In France, the reported prevalence of Campylobacter and L. monocytogenes in raw egg product samples was 4 and 17% respectively (Protais et al., 2007; Rivoal et al., 2010); in Japan, Campylobacter was isolated in 27.9% of raw liquid whole eggs (Sato and Sashihara, 2010) while L. monocytogenes was detected in 8 and 55% of the samples depending on the egg-breaking facility (Ohkochi et al. 2009). The authors found that pasteurization totally eliminates Campylobacter contamination, confirming that the pathogen reacts to heat. Meanwhile, L. monocytogenes could still be detected after pasteurization (Protais et al., 2007; Ohkochi et al., 2009; Sato and Sashihara, 2010) and L. monocytogenes contamination appears to be seasonal with higher levels detected during the summer and winter months with an apparent dominance of the serovar 1/2a (Rivoal et al., 2010).
2.3 Quantitative risk assessment: Salmonella Enteritidis in eggshells Since the first publication by Whiting and Buchanan (1997), numerous quantitative risk assessment studies on the presence of Salmonella Enteritidis in shells and liquid eggs have been published both in scientific literature
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Foodborne disease associated with eggs 39 and by national and international consortia. The first model published at a national level described the five basic modules that must be taken into consideration to perform a reliable risk assessment of egg infection (i.e. internally contaminated eggs) (FSIS, 1998). These five modules are: ∑
the egg production module, which estimates the number of eggs produced internally contaminated with Salmonella Enteritidis; ∑ the eggshell module, the egg product module, and the preparation and consumption module, which estimate the increase or decrease in numbers of Salmonella Enteritidis organisms in the eggs or egg products as they pass through the various stages of storage, transportation, processing and preparation; ∑ the public health module that calculates the incidence of illnesses and their consequence on human and public health. Following the initial publication of risk assessment models, many other models have since been published, some of which apply the Bayesian statistical techniques to model egg contamination and exposure risks for the consumer while also taking into account different means of product treatment. Probable efficiencies of the various intervention strategies scenarios have also been modelled. The most important models published at the national or international level were from Canada (Paoli, 2001; Food Directorate, 2008), WHO and FAO (2002), Finland (EELA, 2006), and the most recent one published by EFSA (2010c). Other publications dealing more with the methodology of quantitative risk assessments used table egg contamination by Salmonella Enteritidis as an example to support the fitness of their mathematical models (Ebel and Schlosser, 2000; Whiting et al., 2000; Hope et al. 2002; Almonacid et al., 2002; Hald et al., 2004; Mokhtari et al., 2006; Kelly et al., 2009; Evers and Chardon, 2010). The Quantitative Microbial Risk Assessment (QMRA) provided by the WHO/FAO (2002) and EFSA (2010c) will be further discussed. Among the major risk factors evaluated in the WHO/FAO risk assessment study (2002), within-flock prevalence was considered one of the most important factors proportionate to the risk to humans. The risk of illness per serving in the case of eggs originating from a flock with 10% within-flock prevalence increased 100-fold versus eggs originating from a flock with only 0.1% within-flock prevalence. Flock prevalence itself was also considered a major risk factor by the QMRA. For instance, by increasing flock prevalence from 0.1 to 5% the risk of illness per egg serving increased 40-fold and by increasing it from 0.1 to 25% the risk of illness increased 200-fold. Thus, intervention strategies at the farm level to reduce S. Enteritidis flock prevalence and within-flock prevalence are a major concern (WHO/FAO, 2002). All the intervention strategies such as regular sampling and monitoring, culling of positive breeder flock pullets and laying flocks, biosecurity measures, vaccination with killed and/or live vaccines, pasteurization of eggs from
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40 Improving the safety and quality of eggs and egg products positive flocks, are detailed in another chapter of this book and are to be taken into consideration when implementing an S. Enteritidis eradication program. Conversely, initial quantities of Salmonella in egg content at the time of lay are not considered critical in terms of risk to consumers and the amount of S. Enteritidis in freshly laid eggs is estimated to be as low as 1 to 100 cfu/egg. When considering the quantities of S. Enteritidis per serving, major risks were attributed to the multiplication of the pathogen during the transportation, storage and preparation stages. The authors consider that while reducing egg shelf-life to less than 14 days will not have any effect on the contamination risk per serving, reducing shelf-life to less than 7 days would decrease the risk by 60%. Keeping retail storage temperatures at less than 7.7 °C would have a similar effect on risks per serving. Thus, a shorter shelf-life and chilling of the eggs could be considered efficient intervention strategies (WHO/FAO, 2002). EFSA also investigated the efficiency of egg chilling as a strategy to control Salmonella Enteritidis infections in humans (EFSA, 2009). It was concluded that, despite the fact that egg chilling prevented the growth of Salmonella Enteritidis in egg content, a risk/benefit analysis needs to be performed vs. the ‘problems associated with this measure, including those resulting from an inability to maintain the cold chain and the consequential water condensation on the egg surface which facilitates growth and penetration of microorganisms into the egg’ (EFSA, 2009). Additionally, EFSA indicated that rapid cooling may cause eggshells to crack due to temperature gradients and this may in turn further facilitate microbial migration in the shells (EFSA, 2009). The final scientific report from EFSA on quantitative risk assessments of Salmonella Enteritidis in shell eggs in Europe (EFSA, 2010c) provided the EU member states with a model to estimate, based on a two-stage Bayesian model, the number of eggs contaminated (internally and externally) with Salmonella Enteritidis. The first stage of the model estimated the average flock prevalence over a laying period in a national production system and the second one estimated the proportion of contaminated eggs issued from an infected flock. Combined stages estimated the number of infected eggs per million, taking the example of two member states. The first member state chosen had approximately 3500 flocks, sampled at 25, 40, 55 and 70 weeks and a number of flocks infected at each sampling time of 4, 8, 5 and 7 respectively. The average prevalence of this first member state was 0.69%. The second member state chosen had approximately 325 flocks, sampled at 24, 39, and 54 weeks and a number of flocks infected at each sampling time of 2, 1 and 7 respectively. The average prevalence of this second member state was 2.15%. Two models of within-flock prevalence from pooled results and individual results issued from studies undertaken by the Veterinary Laboratories Agency (VLA, UK) were compared and combined in this study. The results from the combined model estimate the average contamination at 9.5 contaminated eggs per million for the first member
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Foodborne disease associated with eggs 41 state and 56.8 contaminated eggs per million for the second one (EFSA, 2010a, 2010c). In the study carried out by FSIS, the estimated number of contaminated eggs was close to 50 per million (FSIS, 1998), but only internal contamination was considered. A scientific opinion by EFSA (EFSA, 2010d) regarding a quantitative estimation of the public health impact of setting a new target for the reduction of Salmonella in laying eggs suggests a linear relationship between flock prevalence of S. Enteritidis and the number of infected eggs to be laid but highlights the uncertainty due to lack of data on the number of contaminated eggs produced by an infected flock. It has been concluded that further research is required in order to evaluate within-flock prevalence and the average number of contaminated eggs produced by an S. Enteritidis-positive flock (EFSA, 2010d). In spite of this, the model provided by the EFSA scientific opinion (EFSA, 2010c) will be a convenient tool for the EU member states to provide their own risk assessment, well fitted to the production conditions in their country. All of these models are very useful both to estimate the average number of foodborne diseases attributed to egg consumption and to measure the year-by-year progress of the eradication policy implemented in the EU member states.
2.4 Conclusion Eggs and egg products are prone to microbiological contamination at any stage of the production chain. Salmonella remains the dominant pathogen in public health risks. Controlling this pathogen at the farm level is essential to limit the consequences at subsequent stages, and producers and manufacturers must carry out good sanitary practices to produce safe eggs and egg products. Although eggshells are exposed to bacteria colonizing the intestines of laying hens, shell egg content contamination remains a very rare event. Eggs can thus still be considered a relatively safe food product. Considerable efforts made by egg producers and authorities to control Salmonella Enteritidis and Typhimurium in egg production have a measurable impact on public health.
2.5 References and further reading adesiyun a., offiah n., seepersadsingh n., rodrigo s., lashley v., musai l., georges k.
2005. Microbial health risk posed by table eggs in Trinidad. Epidemiology and Infection. 133, 1049–1056. almonacid s., gutierrez j., jaques a., simpson r. 2002. Salmonella Enteritidis risk assessment: a kinetic analysis. Journal of Food Science. 67, 1115–1120. board r.g. 1994. Microbiology of the avian egg. Chapman and hall, London. 196. busani l., cigliano a., taioli e., caligiuri v., chiavacci l., di bella c., battisti a., duranti a., gianfranceschi m., nardella m.c., ricci a., rolesu s., tamba m., marabelli r., caprioli
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42 Improving the safety and quality of eggs and egg products a. 2005. Prevalence of Salmonella enterica and Listeria monocytogenes contamination in foods of animal origin in Italy. Journal of Food protection. 68, 1729–1733. callicott k.a., friethriksdóttir v., reiersen j., lowman r., bisaillon j.r., gunnarsson e., berndtson e., hiett k.l., needleman d.s., stern n.j. 2006. Lack of evidence for vertical transmission of Campylobacter spp. in chickens. Applied and Environmental Microbiology. 72(9), 5794–5798. castellan d.m., kinde h., kass p.h., cutler g., breitmeyer r.e., bell d.d., ernst r.a., kerr d., little h.e., willoughby d., riemann h., ardans a., snowdon j.a., kuney d.r. 2004. Descriptive study of California egg layer premises and analysis of risk factor for Salmonella enterica serotype enteritidis as characterized by manure drag swabs. Avian Disease. 48, 550–561. chemaly m., hervé g., protais j., huchet v., fravalo p. 2006. The behaviour of Salmonella Enteritidis in fresh and frozen egg products. International Symposium Salmonella and Salmonellosis. pp 457–458. chemaly m., toquin m.t., le nôtre y., fravalo p. 2008a. Prevalence of Listeria monocytogenes in poultry production in France. Journal of Food Protection, 71(10), 1996–2000. chemaly m., g. hervé v. huchet j. protais p. fravalo 2008b. Modelling the behaviour of Salmonella Enteritidis in pasteurised whole egg products. 4th annual meeting MedVetNet, p. 24. chemaly m., huneau-salaun a., labbé a., houdayer c., le marec f., ligouy l., fravalo p. 2009. Isolation of Salmonella enterica in laying hen flocks and assessment of eggshell contamination in France. Journal of Food Protection. 72, 2071–2077. chemaly m., hervé g., huchet v., protais j., fravalo p. 2010. Behaviour of Salmonella Enteritidis in pasteurised whole egg products based on a modelling approach. International Symposium Salmonella and Salmonellosis. June. cox n.a., richardson l.j., buhr r.j., fedorka-cray p.j. 2009. Campylobacter species occurrence within internal organs and tissues of commercial caged leghorn laying hens. Poultry Science. 88, 2449–2456. davies r.h., breslin m. 2003. Investigation of Salmonella contamination and disinfection in farm egg-packing plants. Journal of Applied Microbiology. 94, 191–196. de reu k., grijspeerdt k., messens w., heyndricks m., uyttendaele m., debevere j., herman l. 2005. Egshell factors influencing eggshell penetration and whole egg contamination by different bacteria, including Salmonella Enteritidis. International Journal of Food Microbiology. 112, 253–260. doyle m.p. 1984. Association of Campylobacter jejuni with laying hens and eggs. Applied and Environmental Microbiology. 47, 533–536. ebel e., schlosser w. 2000. Estimating the annual fraction of eggs contaminated with Salmonella Enteritidis in the United States. International Journal of Food Microbiology. 61, 51–62. eela, Salmonella in Egg Production in Finland – a Quantitative Risk Assessment, Eelan Julkaisu, EELA report, April 2006. efsa, 2006. Analysis of the baseline study on the prevalence of Salmonella in laying hen flocks of Gallus gallus. EFSA Journal. 81, 1–71. efsa, 2008. Scientific opinion of the panel on biological hazards on a request of the European Commission on a quantitative assessment on Salmonella in meat. EFSA Journal, 625, 5–32. efsa, 2009. Special measures to reduce the risk for consumers through Salmonella in table eggs – e.g. cooling of table eggs (Question No EFSA-Q-2007-198) Adopted on 22 January 2009. EFSA Journal. 957, 1–29 efsa, 2010a. The Community Summary Report on Trends and Sources of Zoonoses and Zoonotic Agents and foodborne outbreaks in the EU in 2008. EFSA Journal. 8(1), 1496. efsa, 2010b. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU,
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Foodborne disease associated with eggs 43 2008, Part A: Campylobacter and Salmonella prevalence estimates. EFSA Journal. 8(2), 1503. efsa, 2010c. Quantitative risk assessment of Salmonella Enteritidis in shell eggs in Europe. EFSA Journal. 8(4), 1588. efsa, 2010d. Scientific opinion on a quantitative estimation of the public health impact of setting a new target for the reduction of Salmonella in laying hens. EFSA Journal. 8(4), 1546. eu, 2003a. Regulation (EC) No 2160/2003 of the European Parliament and of the Council of 17 November 2003 on the control of salmonella and other specified foodborne zoonotic agents. Official Journal of the European Union. 12/12/2003. eu, 2003b. Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents, amending Council decision 90/424/EEC and repealing Council Directive 92/117/EEC. Official Journal of the European Union. 12/12/2003. evers e., chardon j. 2010. A swift quantitative microbiological risk assessment (sQMRA) tool. Food Control. 21, 319–330. fda, 2009. Prevention of Salmonella Enteritidis in shell eggs during production, storage, and transportation; final rule. fonseca b.b., soncini r.a., gimaraes a.r., rossi d.a. 2006. Campylobacter sp. in eggs from cloacal swab positive breeder hens. Brazilian Journal of Microbiology. 37, 573–575. food directorate, 2008. Risk Assessment of Salmonella Enteritidis in Canadian Shell Eggs. fravalo p., kerouanton a., bily l., hervé g., brisabois a., salvat g. 2006. Detection and characterisation of Salmonella Enteritidis in eggs. International Symposium Salmonella and Salmonellosis. 397–399. fsis, 1998. Salmonella Enteritidis risk assessment. Final report. gantois i., ducatelle r., pasmans f., haesebrouck f., gast r., humphrey t.j., van immerseel f. 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Review. 33(4), 718–738. garber l., smeltzer m., fedorka-cray p. ladely s., ferris k. 2003. Salmonella enterica serotype Enteritidis in table egg layer house environments and in mice in US layer houses and associated risk factors. Avian Disease. 47, 134–142. goulet v., c. jacquet p. martin v. vaillant e. laurent h. de valk. 2004. Surveillance de la listériose humaine en France, 2001, Bulletin Epidémiologique Hebdomadaire (B.E.H.) 9, 33–35. greig j.d., ravel a. 2009. Analysis of foodborne outbreak data reported internationally for source attribution. International Journal of Food Microbiology. 130, 77–87. hald t., vose d., wegener h.c., koupeev t. 2004. A Bayesian approach to quantify the contribution of animal-food sources to human salmonellosis. Risk Analysis. 24, 255–269. henzler d., kradel d., sischo w.m. 1998. Management and environmental risk factors for Salmonella Enteritidis contamination of eggs. American Journal of Veterinanry Research (RJVR). 59, 824–829. hope b.k., baker a.r., edel e.d., hogue a.t., schlosser w.d., whiting r., mcdowell r.m., morales r.a. 2002. Risk Analysis. 22, 203–218. humphrey t.j. 1994. Contamination of egg shells and contents with Salmonella Enteritidis: a review. International Journal of Food Microbiology. 21, 31–40. huneau-salaün a., chemaly m., le bouquin s., lalande f., petetin i., rouxel s., michel v., fravalo p., rose n. 2009. Risk factors for Salmonella enterica subsp. enterica contamination in 519 French laying hen flocks at the end of the laying period. Preventive Veterinary Medicine. 89, 51–58. izat a.l., gardner f.a. 1988. Incidence of Campylobacter in processed egg products. Poultry Science. 67, 1431–1435.
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44 Improving the safety and quality of eggs and egg products jones d.r., musgrove m.t.
2007. Pathogen prevalence and microbial levels associated with restricted shell eggs. Journal of Food Protection. 70, 2004–2007. jones d.r., musgrove m.t. 2008. Assessment of microbial contaminants present on vacuum loaders in shell egg processing facilities. Journal of Food Safety. 28, 346–354. kelly l., murchie l., xia b., whyte p., madden r. 2009. Probabilistic model for contamination of egg dishes with Salmonella spp. made from shell eggs produced on the island of Ireland. International Journal of Food Microbiology. 135, 187–192. latimer h.k., marks h.m., coleman m.e., schlosser w.d., golden n.j., ebel e.d., kause j., schroeder c.m. 2008. Evaluating the effectiveness of pasteurization for reducing human illnesses from Salmonella spp. In egg products: Results of a quantitative risk assessment. Foodborne Pathogens and Disease. 5, 59–68. luber p. 2009. Cross-contamination versus undercooking of poultry meat or eggs – which risk need to be managed first? International Journal of Food Microbiology. 134, 21–28. mokhtari a., moore c., yang h., jaykus l.a., morales r., cates s., cowen p. 2006. Consumerphase Salmonella enterica serovar Enteritidis risk assessment for egg-containing food products. Risk Analysis. 26, 753–768. mollenhorst h., van woudenbergh c.j., bokkers e.g.m., de boer i.j.m. 2005. Risk factors for Salmonella enteritidis infections in laying hens. Poultry Science. 84, 1308–1313. ohkochi m., nakazawa m., sashihara n.. 2009. Detection of Listeria monocytogenes in commercially broken unpasteurized liquid egg in Japan. Journal of Food Protection. 72, 178–181. paoli g. 2001. Risk Assessment of Salmonella Enteritidis in Canadian Shell Eggs Model Version 4 2001. Prepared by Greg Paoli Decisionalysis Risk Consultants, Inc. poirier e., watier l., espie e., weill f. x., de valk h., desenclos j.c. 2008. Evaluation of the impact on human salmonellosis of control measures targeted to Salmonella Enteritidis and Typhimurium in poultry breeding using time-series analysis and intervention models in France. Epidemiology and Infection. 136, 1217–1224. poppe c., johnson r.p., forberg c.m., irwin r.j. 1992. Salmonella Enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment. Canadian Journal of Veterinary Research. 56, 226–232. protais j., queguiner s., boscher e., chidaine b., ermel g., gerault p., salvat g., federighi m., jugiau f. 2007. Campylobacter sp. et Listeria monocytogenes dans l’oeuf entier liquide. Journées de la Recherche Avicole. 7, 532–535. rasschaert g., houf k., van hende j., de zutter l. 2007. Investigation of the current colonization with Campylobacter and Salmonella in poultry flocks and assessment of the sampling site for status determination at slaughter. Veterinary Microbiology. 123, 104–109. rivoal k., s. p rotais j., quéguiner s., boscher e., chidaine b., rose v., gautier m., salvat g. 2009. Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of Salmonella serotype Enteritidis, Typhimurium and Infantis isolates obtained from whole liquid eggs. International Journal of Food Microbiology. 129, 180–186. rivoal k., queguiner s., boscher e., bougeard s., ermel g., salvat g., federighi m., jugiau f., protais j. 2010. Detection of Listeria monocytogenes in raw and pasteurized liquid whole eggs and characterization by PFGE. International Journal of Food Microbiology. 138, 56–62. sahin o., kobalka p., zhang q. 2003. Detection and survival of Campylobacter in chicken eggs. Journal of Applied Microbiology. 95, 1070–1079. sato m., sashihara n. 2010. Occurrence of Campylobacter in commercially broken liquid eggs in Japan. Journal of Food Protection. 73, 412–417. schwaiger k., schmied e.m.v., bauer j. 2008. Comparative analysis of antibiotic resistance characteristics of Gram-negative bacteria isolated from laying hens and eggs in conventional and organic keeping systems in Bavaria, Germany. Zoonoses and Public Health. 55, 331–341. © Woodhead Publishing Limited, 2011
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Foodborne disease associated with eggs 45 shane s.m., gifford d.h., yogasundram k.
1986. Campylobacter jejuni contamination of eggs. Veterinary Research Communications. 10, 487–492. sulonen j., kärenlampi r., holma u., hänninen m.l. 2007. Campylobacter in Finnish organic laying hens in autumn 2003 and spring 2004. Poultry Science. 86, 1223–1228. whiting r.c., buchanan r.l. 1997. Development of a quantitative risk assessment model for Salmonella enteritidis in pasteurized liquid eggs. International Journal of Food Microbiology. 36, 111–125. whiting r.c., hogue a., schlosser w.d., ebel e.d., morales r.a., baker a., mcdowell r.m. 2000. A quantitative process model for Salmonella Enteritidis in shell eggs. Journal of Food Science. 65, 864–869. who/fao, 2002. Risk assessments of Salmonella in eggs and broiler chickens. widdicombe j.p., rycroft a.n., gregory n.g. 2009. Hazards with cracked eggs and their relationship to egg shell strength. Journal of the Science of Food and Agriculture. 89, 201–205.
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3 Internal contamination of eggs by Salmonella Enteritidis R. Raspoet, I. Gantois, R. Devloo, F. Pasmans, F. Haesebrouck, R. Ducatelle, and F. Van Immerseel, Ghent University, Belgium
Abstract: Egg contamination by Salmonella Enteritidis is one of the most important causes of foodborne gastroenteritis in humans throughout the world. This chapter critically overviews the possible routes of internal egg contamination by S. Enteritidis, ranging from penetration through the shell to incorporation into the forming egg. Survival of Salmonella in the forming egg and survival and multiplication strategies in eggs post-lay are also discussed. Salmonella Enteritidis seems to harbour a specific battery of virulence factors, enabling it to efficiently contaminate laying hen eggs and to survive in the hostile egg white compartment. Key words: Salmonella Enteritidis, egg contamination, reproductive tract colonization.
3.1 Salmonella Enteritidis and eggs: a close connection Salmonella is an important source of foodborne disease in humans throughout the world. An important change in the epidemiology of Salmonella occurred since the mid-1980s, when Salmonella Enteritidis (S. Enteritidis) became a major contaminant of eggs and egg products. In 2007, the overall European Union (EU) prevalence of Salmonella in table eggs was 0.8%. S. Enteritidis was by far the most frequently isolated serovar while other serovars were only found in a limited number (EFSA, 2009). Although egg contamination by S. Enteritidis is a sporadic event and although, in an infected flock, only a limited number of eggs become contaminated, eggs and egg products are the major source of foodborne salmonellosis. In the United States (US), 80% of the human S. Enteritidis outbreaks between 1985 and 1999 could © Woodhead Publishing Limited, 2011
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Internal contamination of eggs by Salmonella Enteritidis 47 be traced back to eggs (Patrick et al., 2004). In addition, the most recent study of the European Surveillance System (2007) showed that 64.5% of the 155,540 confirmed salmonellosis cases were caused by S. Enteritidis and that eggs and egg products were the predominant food vehicle in these outbreaks (EFSA, 2009). On top of this, S. Enteritidis remains one of the most frequently isolated serotypes in chickens. In the EU, 59.9% (890) of the 1486 Salmonella positive laying hen flocks were infected with S. Enteritidis in 2007. This means that, although other serovars are found in laying hen holdings, Enteritidis is by far the most recovered serovar from eggs (EFSA, 2007). These data suggest that S. Enteritidis harbours some intrinsic properties, leading to a unique interaction with the chicken reproductive tract and/or egg components. Eggs can be contaminated by S. Enteritidis on the outer shell or inside the egg. Passage of the egg through the contaminated cloaca or contamination with environmentally present Salmonella bacteria can cause outer shell contamination. Internal egg contamination can be caused by shell penetration through cracks (horizontal transmission) (Messens et al., 2005; De Reu et al., 2006) or by colonization of the reproductive tract and thus incorporation into the forming egg (vertical transmission) (Keller et al., 1995; Miyamoto et al., 1997; Okamura et al., 2001a).
3.2 Eggshell surface contamination After oviposition, the surface of the egg can become contaminated with Salmonella Enteritidis or any other serotype that is present in the environment. Survival of S. Enteritidis on the eggshell surface is supposed to be facilitated by low relative humidity and low temperature, due to a slower bacterial metabolism (Messens et al., 2005). Also, the presence of organic material on the shell surface facilitates the survival of Salmonella as it provides all the necessary nutrients for growth and protects the bacteria from environmental stressors (Schoeni et al., 1995). Consequently, removal of faecal contamination could theoretically reduce the number of infections caused by S. Enteritidis positive eggs. Nevertheless, extensive examination of eggs for cracks, and washing and disinfection of the eggshell have not eliminated egg contamination with S. Enteritidis in the US. This suggests that internal egg contamination is more important than shell surface contamination with Salmonella Enteritidis (Braden, 2006). The major part of this chapter will therefore focus on internal egg contamination.
3.3 Eggshell penetration In order to contaminate the content of an intact egg by eggshell penetration, Salmonella bacteria contaminating the outer eggshell have to pass through © Woodhead Publishing Limited, 2011
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48 Improving the safety and quality of eggs and egg products three mechanical (physical) defence barriers. First in line is the cuticle. This outermost layer is a waxy membrane formed prior to oviposition and covering the eggshell pores (Rzedzichi and Stepien-Pysniak, 2009). From the outside to the inside, the cuticle covers the crystalline eggshell itself and the shell membranes. The shell membranes are built up in three layers, i.e. the outer and inner membranes and the limiting membrane. The former are located directly under the shell and consist of a randomly oriented fibre network creating a biological filter system to exclude potential contaminants. The limiting membrane on the other hand consists of homogeneous material that avoids leakage of the albumen and prevents microbial invasion (Wong-Liong et al., 1997). In spite of these physical defences different studies have shown that many bacteria, including Salmonella Enteritidis, are able to cross these lines of defence (Sauter and Peterson, 1969; Mayes and Takeballi, 1983; Jones et al., 2002; De Reu et al., 2006). Many aspects of the eggshell defence, reviewed by Messens et al. (2005), seem to determine the risk of eggshell penetration. Although results between different studies are conflicting, these aspects include cuticle deposition, the numbers of pores and cracks in the shell and environmental parameters such as temperature and humidity. The eggshell seems to be more prone to bacterial penetration immediately after lay (Sparks and Board, 1985; Padron, 1990; Miyamoto et al., 1998). During this period the cuticle is still immature and some pores in the shell are open. Another reason for this phenomenon is that a temperature difference is created between the freshly laid egg (warm) and the ambient air temperature (cool) generating a negative pressure pulling the bacteria through the pores and into the egg (Board, 1966; Bruce and Drusdale, 1994). Consequently, cooling of eggs shortly after lay reduces bacterial penetration, as the temperature difference between the egg and the environment is decreased (Miyamoto et al., 1998). This is, however, not easy to perform in practice. In a study conducted by De Reu et al. (2006), agar filled eggs were used to investigate the eggshell penetration potential of seven unrelated bacterial strains (Staphylococcus warneri, Acinetobacter baumanii, Alcaligenes sp., Serratia marcesens, Carnobacterium sp., Pseudomonas sp. and S. Enteritidis). S. Enteritidis was less frequently isolated from the agar (43%) than Pseudomonas sp. (60%) and Alcaligenes sp. (58%), but more frequently than Staphylococcus warneri, Acinetobacter baumanii, Serratia marcesens, Carnobacterium sp., indicating that Gram-negative, motile, non-clustering bacteria penetrate the shell more easily. When this study was performed with whole eggs instead of agar filled ones, S. Enteritidis was most frequently isolated from the internal egg contents compared to all other bacterial strains. Moreover, in a study by Humphrey et al. (1991b), different Salmonella serotypes could be isolated from eggs obtained from hens naturally infected with Salmonella, but only the serotype Enteritidis was found in internal egg contents. This would mean that, although many bacteria have the ability to penetrate eggs, S. Enteritidis has mechanisms to survive in the internal egg contents. As a matter of fact, once the bacteria have penetrated the shell membranes, a wide
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Internal contamination of eggs by Salmonella Enteritidis 49 range of chemical antimicrobial defence mechanisms, such as lysozyme and ovotransferrin, await them in the albumen. Lysozyme cleaves the b-(1,4) linkage between N-acetylneuramine and N-acetylglucosamine in the cell wall of Gram-positive bacteria (Board, 1969), while ovotransferrin functions as an iron chelator, thus depriving the bacteria of iron and making it impossible for them to grow (Garibaldi, 1960). These data point out that eggshell penetration on itself is not a specific trait used by the serotype Enteritidis to contaminate eggs, since also other serovars and even unrelated bacteria seem to be capable of egg penetration. Nevertheless, this trait may be used for internal egg contamination by S. Enteritidis after outer shell contamination, taken into account that different bacterial species and even different Salmonella serovars behave differently inside the egg once they have passed the eggshell membranes. Indeed, survival and multiplication strategies inside the eggs can be different between bacterial strains, including Salmonella serotypes. This issue is discussed further in this chapter.
3.4 Contamination of the egg during development in the reproductive tract Although eggs can be contaminated by penetration of the eggshell, it is clear that Salmonella can also contaminate eggs after colonization of the ovary or oviduct and thus become incorporated in the forming egg. Several studies support the idea that contamination of the forming egg may be more important than shell penetration: first of all, no correlation could be found between faecal carriage and Salmonella contamination of the egg content in experimentally infected hens (Gast and Beard, 1990a; Humphrey et al., 1991a). Second, even in the absence of intestinal contamination, isolation of S. Enteritidis from the reproductive tissue has been described (Lister, 1988). Depending on the site of reproductive tract colonization, Salmonella can be incorporated in the egg at different locations. Once maturated in the ovary, follicles containing the egg yolk will ovulate and be captured by the infundibulum of the oviduct. Subsequently the egg passes the magnum and isthmus of the oviduct, were respectively the egg white and eggshell membranes are deposited. Next the egg reaches the uterus were the shell is formed and after which oviposition through the vagina takes place. Consequently, infection of the ovary will result in yolk colonization, while contamination of the albumen will occur during passage of the egg through the oviduct. Salmonella bacteria infecting the oviduct could thus be incorporated in either the albumen or the eggshell membranes, depending on the site of colonization in the upper oviduct (magnum, or isthmus respectively). S. Enteritidis has been found in both yolk and albumen of eggs laid by infected hens. However, there is disagreement as to on whether the albumen or yolk
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50 Improving the safety and quality of eggs and egg products is the principal site of contamination. In naturally infected hens, it has been demonstrated that the albumen is the major contamination site, but owing to the low numbers of contaminated eggs after natural infection and the fact that it is labour-intensive to bacteriologically analyse separated egg fractions, this statement has not been confirmed in further studies (Humphrey et al., 1991b). In experimental infection trials, most authors report the albumen (Gast and Beard, 1990b; Keller et al., 1995; Miyamoto et al., 1997), but others report the yolk (Bichler et al., 1996; Gast and Holt, 2000a; Gast et al., 2002) as the most frequently contaminated egg fraction. 3.4.1 Colonization of the vagina Contamination of the vagina by S. Enteritidis can be the consequence of an ascending infection from the cloaca (Keller et al., 1995). Several studies investigated the role of the vagina in the production of S. Enteritidis contaminated eggs. Intravaginal inoculation of laying hens with S. Enteritidis resulted in a high frequency of positive eggs without reduction of egg production. Even more, the number of contaminated eggs was higher after infection with S. Entertidis compared with other serovars (Miyamoto et al., 1997; Okamura et al., 2001b). On top of that, S. Enteritidis shows a higher association with vaginal explants and a higher invasion of the vaginal epithelium compared with other serovars (Mizumoto et al., 2005). Egg contamination after vaginal colonization may occur by penetration through the eggshell or by incorporation into the forming egg. Incorporation into the forming egg could be the result of an ascending infection from the vagina to the upper oviduct, but this is rarely seen in intravaginally inoculated hens, and is thus thought to be, if at all possible, a marginal event (Miyamoto et al., 1997). Passage of the fully formed egg through the contaminated vagina or cloaca would result in eggshell surface contamination (Barrow and Lovell, 1991; Keller et al., 1995). 3.4.2 Colonization of the upper oviduct Colonization of the upper oviduct by S. Enteritidis and consequent incorporation in the albumen of the forming eggs has been reported in several studies (Gast and Beard, 1990a; Hoop and Pospischil, 1993; Keller et al., 1995). This colonization can in theory be the result of an ascending infection from the cloaca or vagina, a descending infection from the ovary and/or a systemic spread of Salmonella, resulting in oviduct tissue colonization. Salmonella bacteria have been found on the mucosal surface and within epithelial cells of the isthmus and magnum in both naturally and experimentally infected hens (Hoop and Pospischil, 1993; De Buck et al., 2004). In a study carried out by De Buck et al. (2004) it was shown that the tubular gland cells of the isthmus are a site of colonization. Contamination of the eggshell membranes, pointing to isthmus contamination, has been reported to occur frequently
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Internal contamination of eggs by Salmonella Enteritidis 51 (Humphrey et al., 1991b). In some studies it is even reported to be the most infected site of contaminated eggs (Miyamoto et al., 1997; Okamura et al., 2001a,b). It is thus possible that the bacteria are persistently carried within the epithelial cells of the isthmus and are released from the isthmus cells into the oviduct lumen, resulting in contamination of forming eggs in the eggshell membrane. Also egg white contamination after colonization of the magnum is a possible route. Since most serotypes are able to spread systemically and since most serotypes can invade and multiply in oviduct cells in vitro (Gantois et al., 2008b), oviduct colonization is most likely not a unique characteristic of the serotype Enteritidis. 3.4.3 Colonization of the ovary Colonization of the ovary is most likely the result of systemic spread of Salmonella. Following experimental oral inoculation of laying hens with S. Enteritidis, bacteria were isolated from the tissue layers surrounding the yolk in pre-ovulatory follicles (Thiagarajan et al., 1994). These findings indicate that Salmonella can interact with the cellular components of the pre-ovulatory follicle. Indeed, S. Enteritidis has been shown to interact with the granulosa cells and to invade and multiply in these cells, as in all other cell types (Thiagarajan et al., 1994, 1996a). Blood-borne organisms may be deposited near the basement membrane of the theca cells as many blood vessels terminate near the basement membrane (Thiagarajan et al., 1994). In another report most of the ovarian infections after intravenous inoculation with S. Enteritidis were found to be confined to the interstitial tissue and not to the yolk contained in the large follicles (Barrow and Lovell, 1991). It is also shown that Salmonella can invade ovarian follicles in vitro, depending on the maturity of the follicles (Howard et al., 2005). Whether yolk contamination is really an important aspect of egg contamination still needs to be seen, because multiplication of Salmonella in the nutrient-rich follicles would most likely lead to degeneration of the follicles and thus inhibit ovulation. Although, some studies found that S. Enteritidis colonized the ovaries and pre-ovulatory follicles more heavily than five other serotypes (Okumara et al., 2001a,b), ovarian colonization is not believed to be a trait that is specific to the serotype S. Enteritidis to contaminate eggs. Indeed, systemic spread is a common trait of most, if not all, Salmonella enterica serotypes, suggesting that all these serotypes can colonize the ovary, independent on their epidemiological association with egg infections.
3.5 Salmonella Enteritidis virulence factors involved in chicken reproductive tract colonization Salmonella contains numerous virulence genes which are located on Salmonella Pathogenicity Islands (SPIs) (Hensel, 2004). Most well-known and maybe also © Woodhead Publishing Limited, 2011
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52 Improving the safety and quality of eggs and egg products most important are SPI-1 and SPI-2, involved in invasion in epithelial cells (SPI-1) and survival in different cell types, including macrophages (SPI-2) (Galan and Curtiss, 1989; Shea et al., 1996; Hensel et al., 1998). Genes involved in intestinal colonization (such as SPI-1 genes) and systemic spread (such as SPI-2 genes) will definitely be essential for oviduct colonization and egg contamination after oral uptake because of their general function in virulence. For example, Bohez et al. (2008) showed a decrease in oviduct colonization of a SPI-2 mutant after oral and intravenous inoculation of laying hens compared with the wild type strain. SPI-1, SPI-2 and other SPIs virulence genes that are contained by multiple, if not all, serotypes, are general virulence attributes that can also be involved in some specific interaction with the oviduct tissue, but they cannot explain why the serotype Enteritidis would be more capable of colonizing the oviduct or contaminate eggs. A genome-wide screen of Salmonella Enteritidis was performed by Gantois et al. (2008a) in order to identify genes that are specifically expressed during infection of the chicken oviduct. Most of the genes identified in this study are involved in the biosynthesis of amino acids (asnS, folC, lysS, glyA, leuS, valS, serS), nucleic acids (tmk, nkdD, dnaX, rpoZ and purA) or in the carbohydrate and energy metabolism (GpmA and AceA). Another group of genes is involved in cell membrane and cell wall integrity (TatA, HflK, peg/yohN and murA) which are essential for the survival of S. Enteritidis in egg albumen. Also two stress-related genes (UspAB and yrfI), one motility gene (flgG), two virulence plasmid genes (repB, repA) and two genes with unknown function were identified. Additionally, one regulatory gene (lrp), which is a positive regulator of type 1 fimbriae expression through direct interaction with fimZ, was identified. Fimbriae are proteinaceous surface appendages mediating bacterial interaction with host cell. De Buck et al. (2003) already suggested that type 1 fimbriae are capable of binding immobilized isthmus glandular secretions. In addition, the receptor for these fimbriae is also present within the tubular gland cells of the isthmus, suggesting that S. Enteritidis binds to the isthmus secretions inside the cell, which may result in an efficient translocation, and thus incorporation, in the forming egg. However, no difference could be found in reproductive tract colonization and egg contamination between S. Enteritidis strains with distinct fimbrial expression patterns (Thiagarajan et al., 1996b). 3.5.1 Survival in forming eggs and damage control mechanisms of Salmonella Enteritidis Depending on the reproductive tract colonization site, bacteria can be incorporated in different compartments of the forming egg. Colonization of the ovary will result in contamination of the yolk. However, deformed and degenerated ovarian follicles have been found in chickens experimentally infected with S. Enteritidis (Kinde et al., 2000). These abnormalities are probably caused by rapid multiplication of S. Enteritidis in the nutrient-rich
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Internal contamination of eggs by Salmonella Enteritidis 53 yolk and result in a drop of egg production (Barrow and Lovell, 1991). Gast and Holt (2001) demonstrated that the vitelline membrane surrounding the yolk is more frequently contaminated than the yolk itself. Penetration across this membrane has been investigated and seems to be dependent of temperature. Frequent penetration occurred within 24 h after inoculation at 25–30 °C, but infrequently at 15 or 42 °C (Gast and Holt, 2001; Gast et al., 2005, 2007; Guan et al., 2006) The latter indicates that penetration through the vitelline membrane is unlikely to occur during oviposition, as the passage through the oviduct takes about 26 hours at a temperature of 42 °C. Consequently, yolk content is most likely contaminated after lay, underlining the importance of a rapid refrigeration of laid eggs. Interestingly, differences can be found in the number of contaminated forming eggs and contaminated laid eggs. In a study by Barrow and Lovell (1991), no samples from the contents of laid eggs were positive, although a number of eggs removed from the oviduct were infected. This discrepancy between the isolation rate of Salmonella between forming and laid eggs was confirmed by Keller et al. (1995), who showed that freshly laid eggs were positive in a limited number of eggs (below 1%), while developing eggs in the oviduct were very frequently positive (30%) after oral infection. Incorporation of Salmonella in the forming egg could thus lead to killing of the bacteria, and thus production of non-contaminated eggs. The ovum spends about 26 hours in the oviduct, of which 21 hours in the uterus, where the shell is deposited. It is proposed that the antibacterial properties of the albumen control Salmonella contamination at this site, where the temperature is 42 °C. The temperature of 42 °C seems to be a limiting factor for survival in the egg albumen. This is demonstrated in a study by Guan et al. (2006) where none of the 32 strains tested, survived in albumen after an incubation of 96 hours at 42 °C, while all of the strains survived for 120 hours at 37 °C. Indeed, during their stay in the albumen, the bacteria encounter multiple antimicrobial components like ovotransferrin and lysozyme. Ovotransferrin chelates iron that is necessary for bacterial replication (Garibaldi, 1960). Lysozyme is a muramidase that hydrolyses the b-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of bacteria (Board, 1969). In Gram-negative bacteria this layer is protected by the outer membrane. It is possible that other proteins in the egg white, such as proteins containing bactericidal/permeability-increasing (BPI) domains, enhance the permeability of the outer membrane to increase access of lysozyme to the peptidoglycan layer. These BPI-domains are usually involved in protein binding and neutralizing lipopolysaccharides (LPS) (Elsbach and weiss, 1998). In 2004, a genome-wide screen identified a single b-defensin cluster encoding 13 different b-defensins in the chicken (Xiao et al., 2004). Eleven of these are expressed in all segments of the oviduct and five of them showed an increased expression in the vagina in response to LPS treatment (Mageed et al., 2008). It is thought that b-defensins bind to the bacterial membrane
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54 Improving the safety and quality of eggs and egg products as a consequence of electrostatic interaction between the negatively charged LPS and the positively charged defensins. This interaction will ultimately result in loss of membrane integrity (Sugiarto and Yu, 2004). In addition to proteins that target the bacterial membrane, DNA damaging proteins have also been found in egg white (Lu et al., 2003). It is clear that all of the above mentioned factors must be overcome by S. Enteritidis, in order to survive in the forming egg. During evolution, bacteria have developed a wide variety of defence mechanisms against these antimicrobial factors, such as lipid A modification strategies (Guo et al., 1997) and the production of lysozyme inhibitors (Callewaert et al., 2008). A transposonbank approach identifying genes involved in egg albumen resistance at 37 °C showed that egg albumen susceptible mutants frequently had insertions in genes encoding proteins involved in the structure and function of the cell wall (Clavijo et al., 2006). These results are in line with an in vivo expression technology (IVET) study in which genes involved in cell membrane and cell integrity were identified as being highly expressed in the chicken reproductive tract (Gantois et al., 2008a). In addition, a gain of function study approach testing for albumen resistence showed that YafD, a putative DNA repair enzyme and XthA, exonuclease III, were important for survival in egg albumen (Lu et al., 2003). Next to the antimicrobial components in egg white targeting the bacterial cell membrane and DNA, many antimicrobial components present in egg white target LPS. This implies that LPS, and more specific the structure of the O-antigen, which is a determinant of serotype specificity, plays a major role in the survival of S. Enteritidis in forming eggs. The high body temperature of the hen may result in structural changes of LPS, followed by an enhanced binding of lysozyme to LPS resulting in an enhanced reversible inhibition of lysozyme (Ohno and Morrison, 1989a,b). The role of LPS in egg white survival was also observed in an IVET-study in eggs, in which the rfbH gene was found to be up-regulated in eggs. This gene belongs to the rfb operon that encodes approximately 20 genes involved in O-antigen synthesis. A mutation in this gene made S. Enteritidis more susceptible for inhibiting factors in albumen. The authors propose that an rfbH mutation changes the LPS composition as the phage P22 susceptibility pattern was altered (Gantois et al., 2009). Increased survival in the forming egg could be a major reason why the serotype Enteritidis is found more in laid eggs than other serotypes of Salmonella. For that reason, some research groups have compared the ability of different Salmonella strains to survive in egg white. For example, Gantois et al. (2008b) compared the egg white survival potential of five different Salmonella serotypes by incubating them for 24 hours at 42 °C. This study indicated that, although S. Enteritidis had a significant higher survival than S. Virchow and S. Hader, no significant difference could be found between S. Enteritidis and S. Heidelberg. Moreover S. Enteritidis was less capable than S. Typhimurium to survive in egg white. In contrast, inoculation of laying hens with three different Enteritidis and Typhimurium strains showed that both
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Internal contamination of eggs by Salmonella Enteritidis 55 serotypes were equally effective in contaminating the forming eggs, but that only Enteritidis could be found in eggs after oviposition (Keller et al., 1997). Additionally, Clavijo et al. (2006) showed that survival in egg albumen at 37 °C was higher for the serotype Enteritidis compared with Typhimurium and E. coli. In the studies mentioned above, only few strains belonging to few serotypes were used to make a thorough comparison. Therefore a largescale egg survival study using 90 different Salmonella strains belonging to 25 serotypes, were compared regarding egg white survival at 42 °C (authors, unpublished data). This study clearly showed that S. Enteritidis was by far more capable of surviving in egg white compared to the other serovars. 3.5.2 Behaviour of Salmonella Enteritidis in laid eggs As already mentioned earlier, eggs can be contaminated by penetration of Salmonella through the eggshell after oviposition, or by incorporation into the forming egg after oviduct or ovarian colonization, and thus incorporation will be in the yolk, albumen or eggshell membranes depending on the site of reproductive tract colonization. It has been shown after experimental and natural inoculations that mostly the albumen and not the yolk contents are contaminated (Gast and Beard, 1990b; Humphrey et al., 1991b). Gast and Beard (1990b) showed that after experimental inoculation with S. Enteritidis whole yolks and albumen, but not yolk contents was contaminated, suggesting deposition of S. Enteritidis in the albumen or the vitelline membrane. Furthermore, there is a delay in multiplication of Salmonella in contaminated eggs, pointing to albumen and not yolk contamination, as the latter is a very rich medium supporting fast growth (Humphrey et al., 1991b; Humphrey and Whitehead, 1993). Indeed, when inoculated in the yolk or on the vitelline membrane, Salmonella multiplies to huge levels at ambient temperature within a few days, in contrast to inoculations in the albumen (Takase et al., 1999; Gast and Holt 2000b; Guan et al., 2006). When eggs of less than 28 days of storage were inoculated with 500 S. Enteritidis cfu in the albumen and incubated at 20 °C for 5 days, the increase in the number of S. Enteritidis was negligible, but storage for 42 days before inoculation led to multiplication to 106 cfu within 5 days at 20 °C (Humphrey and Whitehead, 1993). In a large-scale study of Humphrey et al. (1991b), 32 of the 5700 eggs from 15 naturally infected flocks were positive. Of these, 3 contained more than 100 bacteria in the contents, and 2 more than 1000. All of these eggs were stored for more than 21 days at room temperature, while all eggs that were stored for less than 21 days contained less than 20 cfu/egg. It is hypothesized that older eggs have a partial loss of vitelline membrane integrity, resulting in leakage of nutrients into the albumen. This could trigger chemotaxis of Salmonella towards and penetration of the vitelline membrane, and subsequently invasion of the yolk. In the yolk, extensive multiplication will occur as the yolk contains all molecules that are necessary for survival and growth (Cogan et al., 2004).
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56 Improving the safety and quality of eggs and egg products While flagella are probably necessary for migration to the vitelline membrane, curli fimbriae are thought to be required for penetration through this membrane. Indeed, non-flagellate host-specific serotypes Pullorum and Gallinarum and aflaggelate S. Enteritidis strains (fliC mutants) do not grow in eggs. The reason is that FliC mutants of S. Enteritidis are not able to migrate from albumen to the yolk (Cogan et al., 2004). Curli fimbriae on the other hand, are proposed as essential for attachment to the vitelline membrane in order to facilitate yolk invasion and bacterial multiplication (Lock and Board 1992; Cogan et al., 2004). 3.5.3 What makes the serotype Enteritidis more efficient at contaminating eggs Although more than 2500 different Salmonella serotypes exist, S. Enteritidis is the predominant serovar associated with laying hens and more specifically with the reproductive tract and/or the hen’s eggs. Although the exact mechanism for this tropism is still largely unknown, many hypotheses have been put forward. It could be that S. Enteritidis acquired a unique set of genes which made it possible for this serotype to colonize the hen’s reproductive tract and/or survive in the forming egg more easily than other serotypes. It has been shown by Thomson et al. (2008) that S. Enteritidis harbours clusters of genes, called regions of difference (RODs), that are not present in S. Typhimurium. Although the role of these RODs in S. Enteritidis pathology is currently not described, it could be that these islands have fundamental roles in the colonization of the chicken reproductive tract or the survival in the forming egg. Additionally, it is possible that small genetic changes in gene sequence are responsible for the reproductive tract tropism and/or the ability to survive in the egg albumen. It has already been shown that mutations can have an important influence on the gene function. For example, the genome sequence of S. Enteritidis and of S. Gallinarum are highly similar, though mutations in 5 of 50 motility genes of S. Gallinarum made the difference between a motile S. Enteritidis strain and a non-motile S. Gallinarum strain (Thomson et al., 2008). It is thus possible that mutations in genes exist that make the serotype S. Enteritis more capable of colonizing the chicken reproductive tract and the egg. This hypothesis is supported by a study of Guard-Bouldin in which single nucleotide polymorphisms (SNPs) were identified between an egg contaminating and a non-egg contaminating S. Enteritidis strain (Guard-Bouldin, 2006). Ultimately, differences in gene expression profile could lead to an enhanced interaction of S. Enteritidis with the laying hen, as environmental factors may influence the expression of virulence genes. Although the human pandemic of Salmonella Enteritidis seems to be gradually coming to an end, unravelling the molecular mechanisms of the
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Internal contamination of eggs by Salmonella Enteritidis 57 tropism of Salmonella Enteritidis for the laying hen oviduct and eggs is still of importance. Firstly, it will yield information that can be used to develop specific control methods to prevent Salmonella Enteritidis from contaminating eggs. Secondly, it can yield valuable information on markers that can be used to predict whether certain emerging serotypes have the potential to cause an egg-related pandemic. Using the new molecular methodologies developed in the last decade one can considerally increase the knowledge on how S. Enteritidis used its genetic potential in order to develop an efficient way of establishing itself in an unusual ecological niche, being the egg. Such research should not come to a standstill because the human pandemic is ending, but it should be maintained in order to gain information that can be used to prevent similar problems, with or without other serotypes, in the future.
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58 Improving the safety and quality of eggs and egg products (2006). ‘Eggshell factors influencing eggshell penetration and whole egg contamination by different bacteria, including Salmonella Enteritidis’, Int J Food Microbiol, 112, 253–260. efsa (2007), ‘Report of the task force on zoonoses data collection on the analysis of the baseline study on the prevalence of Salmonella in holdings of laying hen flocks of Gallus gallus’, EFSA J, 97, 1–84. efsa (2009), ‘The community summary report on trends and sources of zoonoses and zoonotic agents in the European Union in 2007’, EFSA J, 223, 21–108. elsbach p and weiss j (1998),’ Role of the bactericidal/permeability-increasing protein in host defence’, Curr Opin Immunol, 10(1), 45–49. galan j e and curtiss r (1989), ‘Cloning and molecular characterization of genes whose products allow Salmonella Typhimurium to penetrate tissue culture cells’, Proc Natl Acad Sci USA, 86(16), 6383–6387. gantois i, pasmans f, haesebrouck f, ducatelle r and van immerseel f (2008a), ‘Salmonella enterica serovar Enteritidis genes induced during oviduct colonization and egg contamination in laying hens’, Appl Environ Microbiol, 74(21), 6616–6622. gantois i, eeckhaut v, pasmans f, haesebrouck f, ducatelle r and van immerseel f (2008b), ‘A comparative study on the pathogenesis of contamination by different serotypes of Salmonella’, Avian Pathol, 37(4), 399–406. gantois i, ducatelle r, pasmans f, haesebrouck f and van immerseel f (2009), ‘The Salmonella Enteritidis lipopolysaccharide biosynthesis gene rfbH is required for survival in egg albumen’, Zoonoses Public Health, 56(3), 145–149. garibaldi j a (1960), ‘Factors in egg white which control growth of bacteria’, Food Res, 25, 337–344. gast r k and beard c w (1990a), ‘Isolation of Salmonella Enteritidis from internal organs of experimentally infected hens’, Avian Dis, 34(4), 991–993. gast r k and beard c w (1990b), ‘Production of Salmonella Enteritidis-contaminated eggs by experimentally infected eggs’, Avian Dis, 34, 438–446. gast r k and holt p s (2000a), ‘Deposition of phage type 4 and 13a Salmonella Enteritidis strains in the yolk and albumen of eggs laid by experimentally infected hens’, Avian Dis, 44(3), 706–710. gast r k and holt p s (2000b), ‘Influence of the level and location of contamination on the multiplication of Salmonella Enteritidis at different storage temperatures in experimentally inoculated eggs’, Poult Sci, 79, 559–563. gast r k and holt p s (2001), ‘Assessing the frequency and consequences of Salmonella Enteritidis deposition of the egg yolk membrane’, Poult Sci, 80, 997–1002. gast r k, guard-petter j and holt p s (2002), ‘Characteristics of Salmonella Enteritidis contamination in eggs after oral, aerosol, and intravenous inoculation of laying hens’, Avian Dis, 46(3), 629–635. gast r k, holt p s and murase t (2005), ‘Penetration of Salmonella Enteritidis and Salmonella Heidelberg into egg yolks in an in vitro contamination model’, Poult Sci, 84(4), 621–625. gast r k, guraya r, guard-petter j and holt p s (2007), ‘In vitro penetration of egg yolks by Salmonella Enteritidis and Salmonella Heidelberg strains during thirty-six-hour ambient temperature storage’, Poult Sci, 86(7), 1431–1435. guan j, grenier c and brooks b w (2006), ‘In vitro study of Salmonella Enteritidis and Salmonella Typhimurium definitive type 104: survival in egg albumen and penetration through the vitelline membrane’, Poult Sci, 85(9), 1678–1681. guard-bouldin j (2006), ‘Comparative genome sequencing of Salmonella Enteritidis isolates that vary in virulence characteristics’, Proceedings of the 110th US Animal health Association, 97–101. guo l, lim k b, gunn j s, bainbridge b, darveau r p, hackett m and miller s (1997), ‘Regulation of lipid A modifications by Salmonella Typhimurium virulence genes phoP-phoQ’, Science, 276(5310), 250–253. herman l
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Internal contamination of eggs by Salmonella Enteritidis 59 (2004), ‘Evolution of Pathogenicity islands of Salmonella enterica’, Int J Med Microbiol, 294(2–3), 95–102. hensel m, shea j e, nikolaus t, banks g, vazquez-torres a, gleeson c, fang f c and holden d w (1998), ‘Genes encoding putative effector proteins of the type III secretion system of Salmonella Pathogenicity Island 2 are required for bacterial virulence and proliferation in macrophages’, Mol Microbiol, 30(1), 163–174. hoop r k and pospischil a (1993), ‘Bacteriological, serological, histological and immunohistochemical findings in laying hens with naturally acquired Salmonella Entertidis phage type 4 infection’, Vet Rec, 133(16), 391–393. howard z r, moore r w, zabala-diaz i b, landers k l, byrd j a, kubena l f, nisbet d j, birkhold s g and ricke s c (2005), ‘Ovarian laying hen follicular maturation and in vitro internalization’, Vet Microbiol, 108, 95–100. humphrey t j and whitehead a (1993), ‘Egg age and the growth of Salmonella Enteritidis PT4 in egg contents’, Epidemiol Inf, 111(2), 209–219. humphrey t j, chart h, baskerville a and rowe b (1991a), ‘The influence of age on the response of SPF hens to infection with Salmonella Enteritidis PT4’, Epidemiol Infect, 106(1), 33–43. humphrey t j, whitehead a, gawler a h l, henley a and rowe b (1991b), ‘Numbers of Salmonella Enteritidis in the contents of naturally contaminated hens’ eggs’, Epidemiol Inf, 106(3), 489–496. jones d r, anderson k e, curtis p a and jones f t (2002), ‘Microbial contamination in inoculated shell eggs: I. effects of layer strain and hen age’, Poult Sci, 81(5), 715–720. keller l h, benson c e, krotec, k and eckroade r j (1995), ‘Salmonella Enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens’, Infect Immun, 63, 2443–2449. keller l h, schifferli d m, benson c e, aslam l s and eckroade r j (1997), ‘Invasion of chicken reproductive tissues and forming eggs is not unique to Salmonella Enteritidis’, Avian Dis, 41, 535–539. kinde h, shivaprasad h l, daft b m, read d h, ardans a, breitmeyer r, rajashekara g, nagaraja k v and gardner i a (2000), ‘Pathologic and bacteriologic findings in 27week old commercial laying hens experimentally infected with Salmonella Enteritidis , phage type 4’, Avian Dis, 44(2), 239–248. lister s a (1988), ‘Salmonella Enteritidis infection in broilers and broiler breeders’, Vet Rec, 123(13), 350. lock j l and board r g (1992), ‘Persistence of contamination of hens’ egg albumen in vitro with Salmonella serotypes’, Epidemiol Infect, 108(3), 389–396. lu s, killoran p b and riley l w (2003), ‘Association of Salmonella enterica serovar Enteritidis yafD with resistance to chicken egg albumen’, Infect Immun, 71, 6734– 6741. mageed a m, isobe n and yoshimura y (2008), ‘Expression of avian beta-defensins in the oviduct and effects of lipopolysaccharides on their expression in the vagina of hens’, Poult Sci, 87(5), 979–984. mayes f j and takeballi m a (1983), ‘Microbial contamination of the hen’s egg: a review’, J Food Prot, 46, 1092–1098. messens w, grijspeerdt k and herman l (2005), ‘Eggshell penetration by Salmonella: a review’, World’s Poult Sci J, 61, 71–85. miyamoto t, baba e, tanaka t, sasai k, fukata t and arakawa a (1997), ‘Salmonella Enteritidis contamination of eggs from hens inoculated by vaginal, cloacal and intravenous routes’, Avian Dis, 41, 296–303. miyamoto t, horie t, baba e, sasai k, fukata t and arakawa a (1998), ‘Salmonella penetration through the eggshell associated with freshness of laid eggs and refrigeration’, J Food Prot, 61(3), 350–353. mizumoto n, sasai k, tani h and baba e (2005), ‘Specific adhesion and invasion of hensel m
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60 Improving the safety and quality of eggs and egg products Salmonella Enteritidis in the vagina of laying hens’, Vet Microbiol, 111(1–2), 99–105. ohno n and morrison d c (1989a), ‘Effects of lipopolysaccharides chemotype structure on binding and inactivation of hen egg lysozyme’, Eur J Biochem, 186(3), 621–627. ohno n and morrison d c (1989b), ‘Lipopolysaccharide interaction with lysozyme. Binding of lipopolysaccharide to lysozyme and inhibition of lysozyme enzymatic activity’, J Biol Chem, 264(8), 4434–4441. okamura m, kamijima y, miyamoto t, tani h, sasai k and baba e (2001a), ‘Differences among six Salmonella serovars in abilities to colonize reproductive organs and to contaminate eggs in laying hens’, Avian Dis, 45, 61–69. okamura m, miyamoto t, kamijima y, tani h, sasai k and baba e. (2001b), ‘Differences in abilities to colonize reproductive organs and to contaminate eggs in intravaginally inoculated hens and in vitro adherences to vaginal explants between Salmonella Enteritidis and other serovars’, Avian Dis, 45, 962–971. padron m (1990), ‘Salmonella Typhimurium penetration through the eggshell of hatching eggs’, Avian Dis, 34(2), 463–465. patrick m e, adcock p m, gomez t m, altekruse s f, holland b h, tauxe v and swerdlow d L (2004), ‘Salmonella Enteritidis infections, United States, 1985–1999’, Emerg Inf Dis, 10(1), 1–7. rzedzichi j and stepien-pysniak d (2009), ‘Antimicrobial defence mechanisms of chicken eggs and possibilities for their use in protecting human and animal health’, Annales, 64(2), 1–8. sauter e a and petersen c f (1969), ‘The effect of eggshell quality on penetration by Pseudomonas fluorescens’, Poult Sci, 45, 825–829. schoeni j l, glass k a, mcdermott j l and wong a c (1995), ‘Growth and penetration of Salmonella Enteritidis, Salmonella Heidelberg and Salmonella Typhimurium in eggs’, Int J Food Microbiol, 24(3), 385–396. shea j e, hensel m, gleeson c and holden d w (1996), ‘Identification of a virulence locus encoding a second type III secretion system in Salmonella Typhimurium’, Proc Natl Acad Sci USA, 93(6), 2593–2597. sparks n h c and board r g (1985), ‘Microbial penetration of recently developed oviposited shell of hen’s eggs’, Aust Vet J, 62, 169–170. sugiarto h and yu p l (2004), ‘Avian antimicrobial peptides: the defense role of betadefensins’, Biochem Biophys Res Commun, 323(3), 721–727. takase k, nakayama t, kawai t and fujikawa h (1999), ‘Growth of Salmonella Typhimurium and Salmonella Enteritidis in egg yolks from highly immunized hens’, J Vet Med Sci, 61, 959–960. thiagarajan d, saeed a m and asem e k (1994), ‘Mechanism of transovarian transmission of Salmonella Enteritidis in laying hens’, Poult Sci, 73(1), 89–98. thiagarajan d, saeed m, turek j and asem e (1996a), ‘In vitro attachment and invasion of chicken ovarian granulosa cells by Salmonella Enteritidis phage type 8’, Infect Immun, 64, 5015–5021. thiagarajan d, thacker h l and saeed a m (1996b), ‘Experimental infection of laying hens with Salmonella Enteritidis strains that express different types of fimbriae’, Poult Sci, 75(11), 1365–1372. thomson n r, clayton d j, windhorst d, vernikos g, davidson s, churcher c, qual m a, stevens m, jones m a, watson m, barron a, layton a, pickard d, kingsley r a, bignell a, clark l, harris b, ormond d, abdellah z, brooks k, cherevach i, chillingworth t, woodward j, norberczak h, lord a, arrowsmith c, jagels k, moule s, mungall k, sanders m, whitehead s, chabalgoity j a, maskell d, humphrey t, roberts m, barrow p a, dougan g and parkhill j (2008), ‘Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways’, Genome Res, 18(10), 1624–1637. wong-liong hw, frank j f and bialey s (1997), ‘Visualization of eggshell membranes and
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Internal contamination of eggs by Salmonella Enteritidis 61 their interaction with Salmonella Enteritidis using confocal scanning laser microscopy’, J Food Prot, 60(9), 1022–1028. xiao y, hughes a l, ando j, matsuda y, cheng j f, skinner-noble d and zhang g (2004), ‘A genome-wide screen identifies a single beta-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins’, BMC Genomics, 5(1), 56.
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4 Chemical residues and contaminants in eggs C. Jondreville, A. Fournier and C. Feidt, Institut National de la Recherche Agronomique (INRA), Nancy Université, France; A. Travel, Institut Technique de l’Aviculture (ITAVI), France, and B. Roudaut, Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (Anses), France
Abstract: This chapter takes stock of the knowledge of the organic chemical contaminants or residues that may be present in hen eggs. The contaminants originate from veterinary drugs or feed additives, from pesticides used for cereal production and from persistent pollutants of the environment. For veterinary drugs and additives, official surveys reveal little about concentrations over the maximum residue limits. However, concentrations of dioxins, furans and polychlorobiphenyls in eggs from hens reared outdoors exceeding the maximum concentrations allowed were recorded. The worst cases arose in home-produced eggs, probably as a result of practices that stimulate ingestion of environmental matrices, especially soil, by hens. Key words: layers, chemical contaminants, veterinary drug, risk, egg.
4.1 Introduction During rearing, animals can come into contact with chemical substances which are likely to be transferred to animal-derived foodstuffs. Such substances, which can be found in animal products, in addition to their degradation products and metabolites, pose a potential danger to consumer health, and could be the source of technological problems in processing. These chemical compounds can be intentionally administered to the animals (veterinary drugs, feed additives), or unintentionally given through their feed, as is the case when pesticides are used in the production of raw materials. Animals can also be in contact with residual contaminants in the environment, such
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Chemical residues and contaminants in eggs 63 as organochlorine pesticides and other organic compounds unrelated to agriculture. Yet consumers are demanding healthy products, free of any chemical contamination that could endanger their health. This demand is largely passed on by the sanitary authorities and through regulations, and it is the responsibility of each farmer to ensure that the products they are putting on the market comply with regulatory residue levels. This chapter focuses on the chemical compounds which laying hens can be exposed to either intentionally or inadvertently during their rearing, explaining the origins of these compounds and the regulation that oversees safe product supply. The chapter will go on to look into the scientific principles which provide the basis of this regulation. These scientific studies allow us to evaluate the risk of transfer of chemical compounds into the egg, to understand the modes of this transfer and to highlight its variation factors, whether they are tied to the molecular characteristics or to animal performance. The results of regulatory and non-regulatory surveys are then presented and the sources of non-conformity are analysed.
4.2 Chemical contaminants in animal-derived foodstuffs: origins and regulatory context Regulations differ substantially depending on the type of compound the animal is exposed to. Some are authorized, but their use is strictly regulated. However, others, such as certain persistent organic pollutants (POPs), can be found residually or accidentally in the animal rearing environment or in animal feed. 4.2.1 Regulated substances Among the regulated molecules are phyto-pharmaceutical substances used in the production of raw materials intended for animal feed. These molecules are not intentionally added to animal feed. Veterinary drugs and animal feed additives, however, are allowed and used intentionally, but adhere to strict dosage schedules, so as to guarantee their safety and effectiveness. All these substances are evaluated in terms of risk before being allowed to go on the market. The concept of maximum residue limit (MRL) A concept was laid down for these substances: foodstuffs cannot contain any residue at a level susceptible of presenting a risk to the consumer’s health. A ‘residue’ is considered as any pharmacologically active substance found in animal-derived foodstuffs (meat, offal/giblets, milk, eggs and honey) coming from animals exposed to a preparation. It can mean not only the active principle(s) of the administered preparation and excipients, but also
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64 Improving the safety and quality of eggs and egg products the degradation products and the metabolites of these active ingredients. This is the case for nitrofurans (banned substances), whose testing deals with the research of protein-linked metabolites. Evaluating the toxic potency of a residue allows us to determine the no observable effect level (NOEL) in animals and to extrapolate the acceptable daily intake (ADI), for humans taking into account safety factors. Having determined the evolution of the residues through studies on metabolism, it is then possible to define the residue marker and calculate the maximum residue limits (MRL) which will keep ADI from being exceeded. For veterinary drugs, this sort of risk management is based on the withdrawal time specific to each veterinary drug so as to avoid having concentrations of residues over the established MRL for active substances. Setting maximum residue limits Directive 2001/82/EC instituted a European Community code pertaining to veterinary drugs that could be authorized in food-producing animals. Regulation (EC) no. 470/2009, which replaced Council Regulation (EEC) no. 2377/90, and has been in effect since 6 May 2009, sets up the abovementioned procedure for setting MRLs in foodstuffs of animal origin. A listing of pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin is given in Commission Regulation (EU) No. 37/2010. This evaluation was carried out by the veterinary division of the European Medicines Agency (EMA). Veterinary drugs that cannot be assigned an MRL are banned in all member states of the European Union: these include chloramphenicol, nitrofurans and nitroimidazoles. Several classes of veterinary drugs are allowed for poultry: antibiotics, antiparasitics, anthelmintics and coccidiostats. However, the number of available molecules is restricted for laying hens due to their continuous egg production and the risk of residue transfer to the eggs. The treatments are mainly administered orally in their drinking water or food for 5–7 days, except when using external anti-parasitics, which is done by spraying the surfaces around the animals. At the European level, nine MRLs have been established for antibiotic residues in eggs (Table 4.1) belonging to the classes of tetracyclines, polypeptides, macrolides, aminoglycosides and pleuromutilines. Sulfamide, penicillin and quinolone are not allowed for laying hens in production. MRLs were also established for three anti-parasitics and one coccidiostat. One coccidiostat can also be administered orally without an MRL: amprolium. MRLs generally pertain to the mother molecule. Withdrawal times are then determined for each veterinary field in such a way that the MRLs are not exceeded in a given tissue. Respecting withdrawal times is particularly tricky for laying hens insofar as the producer has to take the egg production off the market during treatment and, in certain cases, for several days after treatment. The molecules that have received an MRL for eggs usually do not go over this MRL during treatments recommended by the veterinarian.
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Chemical residues and contaminants in eggs 65 Table 4.1 Maximum residue limits (MRL) for veterinary drugs in eggs Class Molecule Marker residue Antibiotics
Chlortetracycline Oxytetracycline Tetracycline Colistin Lincomycin Erythromycin Tylosin Neomycin Tiamulin
MRL (mg/kg)
Species
Chlortetracycline and 4-epimer 200 Oxytetracycline and 4-epimer 200 Tetracycline and 4-epimer 200 Colistin 300 Lincomycin 50 Erythromycin A 150 Tylosin 200 Neomycin B 500 Tiamulin 1000
Fowls Fowls Fowls Fowls Fowls Fowls Fowls Fowls Hens
Anthelmintics Flubendazole Piperazine
Flubendazole Piperazine
Fowls Hens
External antiparasitics
Phoxim
Phoxim
Coccidiostats Lasalocid
Lasalocid
400 2000 60
Hens
150
Fowls
The prohibition of the use of growth promoting substances such as hormones or ß-agonists is established with Council Directives No. 96/22/EC and 2003/74/ EC. Since 1 January 2006 according to Regulation (EC) No. 1831/2003 the use of antibiotic growth promoting substances as additives for use in animal nutrition is forbidden. However, coccidiostats and histomonostats, antibiotics intended to kill or inhibit protozoa, are still authorised for use as feed additives in accordance with Regulation (EC) No. 1831/2003. The evaluation of feed additives is carried out by the European Food Safety Authority (EFSA), in accordance to Regulation (EC) No. 1831/2003. Some of these coccidiostatic additives are only authorized for pullets bred for laying eggs only until the age of 12–16 weeks: halofuginone, diclazuril, lasalocid, salinomycin and monensin. Pesticide residue in animal-derived foodstuffs is governed by Council Directive 86/363/EEC, and the MRLs are set in regulation (EC) no. 396/2005. They are established as the duo ‘active principle/foodstuff’. Studies in transfer are necessary when phytosanitary treatments lead to an excess of 0.1 mg/kg of plants used in the feed of milk-producing cows or egg-producing hens, or when studies in metabolism indicate a risk of bioaccumulation. This is the case for lipophilic pesticides, which show a log Kow (the partition coefficient for octanol/water) higher than 3. 4.2.2 Unintentional contaminants Contaminants such as POPs can be found in an animal’s environment or feed. POPs are organic substances that combine properties that make them
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66 Improving the safety and quality of eggs and egg products toxic, with a potential for bioaccumulation, stability in different environments and an ability to survive long-range travel, notably by air. These molecules are a concern for international regulators: they have drawn up a list of 12 substances for the Stockholm Convention, which was signed in May 2001. Figuring among these molecules are pesticides (DDT, endrin, aldrin, dieldrin, hexachlorobenzene, etc.), industrial products like polychlorinated biphenyls (PCBs), as well as substances involuntarily produced during an incomplete combustion process of organic materials, such as dioxins (PCDDs) and furans (PCDFs), which mainly come from the incineration of waste, and polycyclic aromatic hydrocarbons (PAHs), which mainly come from combustion in urban areas and ground transportation. Nine additional chemical products, pesticides (lindane, chlordecone, etc.) and brominated flame retardants (certain polybrominated diphenyl ethers, PBDE) have recently been added to this list. European regulators have set maximal limits in foodstuffs of some of these contaminants (except pesticides, Commission Regulation (EC) No. 1881/2006). They have imposed a maximum of 3 pg OMS-TEQ PCDD/F/g of fat content in eggs and egg products. These values are doubled when dioxin-like PCBs (DL PCBs) are included. Discussions are underway to set the maximum legal limit at 50 ng/g fat for non-dioxin-like PCBs (NDL PCBs). Maximum regulatory values are 0.01 mg lindane, 0.05 mg DDT and 0.02 mg aldrin + dieldrin per kg of eggs. However, these maximums in eggs have not been established for PAHs or in animal-derived foodstuffs for PBDEs. Limiting the levels of unwanted substances in raw materials and complete feed used in animal feeding (Directive 2002/32/EC) helps in preventing risks tied to contamination of animal products by substances such as POPs and heavy metals. However, these contaminants can be taken directly from the environment by animals via the ingestion of soil, plants, pedofauna or residue from building materials.
4.3 Modes of transfer into the egg Knowing the transfer modes and the kinetics inside the animal of the compounds to which it is exposed is fundamental for evaluating the risk of these compounds being present in eggs. The main means of transmission is oral, including that for veterinary drugs. These drugs are usually intended to act systemically, except for certain molecules that act directly on the gastro-intestinal tract (coccidiostats) and/or those which are poorly absorbed (colistin). 4.3.1 General outline of transfer in animals Pharmacokinetics or toxicokinetics, which refers to the absorption, distribution, metabolism and excretion (ADME) of chemical compounds (Renwick, 2001), © Woodhead Publishing Limited, 2011
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Chemical residues and contaminants in eggs 67 allows the fate of a compound in an organism to be described and quantified. For veterinary drugs, pharmacokinetics makes it possible, among other things, to determine the withdrawal time required to avoid having residues from undesirable substances in tissues used for consumption. After absorption in the intestines, the molecules move to the liver. The first site of metabolization is the enterocyte in the duodenum (Brugère, 1992), but most of the transformations (Anadon et al., 1993) take place in the liver (reactions in phase I: oxidation, reduction, hydrolysis; and in phase II: conjugation). These reactions are a means of defence for the organism, which leads to the transformation of part of the xenobiotic ingested in one form that can be eliminated, such as through urine or bile. After having reached the bloodstream, the molecules are distributed among different tissues and eliminated. The egg is one of the excretion routes for drugs and xenobiotics in laying hens. Numerous factors linked to the characteristics of the ingested molecule, such as its lipophilicity, its polar character, its capacity to bind with proteins and its metabolic sensitivity are decisive elements in its rates and transfer kinetics into the egg. Furthermore, certain factors relative to the animal may constitute important modulation factors in the transfer of xenobiotics into the egg. 4.3.2 Distribution of contaminants and drugs in the egg’s components Lipophilic molecules such as chloramphenicol (Samouris et al., 1998) and POPs, are drawn to the yolk, which is rich in lipids. However, weak acids (sulfamides, quinolones), which have an extra cellular distribution in organisms, are concentrated in the egg white (Roudaut, 1997), a polar environment made up of 88% water. The distribution of antibiotics between the two components of the egg is quite variable, ranging from 23% to 95% for the egg white. Table 4.2 compares ratios of concentrations between the white and yolk for different classes of veterinary drugs. 4.3.3 Transfer kinetics of contaminants and drugs After ingestion, residues appear rapidly in the white (24 hours after first being administered), and later in the yolk (24 to 48 hours after first being administered). Indeed, the moment xenobiotics or drugs are first ingested, the yolk of an egg laid the following day has already gone through its growth phase, and as such, is not exposed to the contaminant; on the other hand, the white, formed in the final 24 hours prior to being laid, risks containing residues. For highly metabolized or poorly stored molecules, as is the case for most veterinary drugs, the flow of residues into the yolk is proportional to the yolk deposits (Donoghue et al., 1997). This type of model can also be applied to contaminants that are lipophilic but very sensitive to metabolization, such as benzo[a]pyrene (Fig. 4.1). Taking into account the
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68 Improving the safety and quality of eggs and egg products Table 4.2 Distribution of veterinary drug residues in egg (from Kan and Petz, 2000) Molecules
pKa
Sulfonamides 5.5–7.5 Quinolones 6.2–6.3 Tetracyclines 3.3–8.3 Macrolides 7.1–8.7 Aminoglycosides >10 Chloramphenicol neutral Nitrofurans 7.2–9.3 Nitroimidazoles Ionophoric anticoccidials Nicarbazin (DNC) Flubendazole
Ratio white egg/yolk 2–4 0.4–9.6 0.2–3 0.3–1.4 0.05 0.3 0.5–0.8 0.8 0.04–1.25 0.007 0.14
development and the depositing of the yolk by accretion, which takes about 8 days, the plateau is only reached after about 10 days. Decontaminating eggs is also a process that does not exceed the time it takes for the yolk to develop, with usually rapid elimination kinetics. On the other hand, for molecules that accumulate significantly, like certain contaminants that are not very sensitive to metabolization (e.g. lindane presented in Fig. 4.1) and lipophilic drugs (chloramphenicol), the flows of yolk and contaminants are not directly proportional. The time needed to reach a state of equilibrium, as well as the time needed to find the basal contamination level of the egg after discontinuing exposure, are even longer (Arnold and Somogyi, 1986; MacLachlan, 2008). The models used to predict the transfer of these types of compounds are more complex and bring into play the flows that increase or decrease stores of this compound in the body adipose tissue (Van Eijkeren et al., 2006). For example, McLachlan et al. (2008) compared several pesticides. These authors calculated that the plateau is reached after 10 days for molecules sensitive to metabolization, whereas it is only reached after 25 days for lindane. For poorly metabolized lipophilic molecules, the carry-over rates and kinetics depend on performance factors, essentially the laying rate and the corporal composition of the animals. For example, diflubenzuron displayed a higher concentration in eggs at equilibrium and a longer half-life with White Leghorns than with hens producing brown eggs such as the brown Warren (Opdycke and Menzer, 1984; MacLachlan, 2008). This difference is attributed to a larger fatty peripheral compartment found in the latter breed. Moreover, in the case of chronic exposure to a persistent lipophilic contaminant, the decrease in laying performance, which limits its excretion, favours its accumulation in body fat. Figure 4.2 simulates the effect of interrupting laying on the PCB concentration in the eggs in hens subjected to chronic exposure to PCBs.
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Chemical residues and contaminants in eggs 69 60
3.0 B[a]P Yolk
40
2.0
30
1.5
20
1.0
10
0.5
0
0.0 0
2
4
6 Day (a)
8
10
12
25
3.0 Lindane Yolk
2.5
20 Deposited lindane (µg)
Deposited yolk (g)
2.5
2.0 15 1.5 10 1.0 5
Deposited yolk (g)
Deposited B[a]P (ng)
50
0.5 0.0
0 0
2
4
6 Day (b)
8
10
12
Fig. 4.1 Relationship between yolk and benzo[a]pyrene (B[a]p) (a) or lindane (b) deposited in eggs of laying hens orally exposed to a single dose of 2 mg benzo[a] pyrene or 3 mg lindane/kg body weight (adapted from Fournier et al., 2010).
During the first period, the PCB content of the egg and abdominal fat increases, reaching a state of equilibrium in around 250 days. At this moment, the egg and the body fat show the same concentrations of PCB based on fat. Interrupting laying suppresses the evacuation of PCB via the egg and causes it to accumulate in the body fat. When laying resumes with this new bodily load, the content in the eggs is increased and progressively decreases towards a state of equilibrium.
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70 Improving the safety and quality of eggs and egg products 100
Abdominal fat Egg
PCB (ng/g fat)
80
60
40
20 One egg laid daily
Non-laying
One egg laid daily
0 0
100
200
300
Day
400
500
600
700
Fig. 4.2 Impact of non-laying on the kinetic of non-dioxin-like PCBs (NDL-PCBs) in eggs and abdominal fat of hens orally exposed to 450 ng NDL-PCB daily (from Fournier et al., 2009).
4.3.4 Transfer and bioaccumulation factors Pharmacokinetic studies are indispensable in calculating the necessary parameters to estimate the risk of contamination by xenobiotics and drugs in animal products, such as carry-over rates and the bioconcentration factor. Their over- or underestimation can lead to additional costs tied to taking precautionary measures that are either too drastic or are too risky for human health. The carry-over rates of antibiotics in eggs after being administered orally (Roudaut, 1997) is generally low (< 1%), or even very low (< 0.01%) for ampicillin and tylosin. On the other hand, for sulfamides and quinolones (Roudaut, 1998; Roudaut and Garnier 2002), the carry-over rate is around 1.5%. For persistent organic pollutants, carry-over rates are often over 30% and bioconcentration factors exceed the value of 1 (Table 4.3). According to Leeman et al. (2007), older pesticides (the most persistent) have a bioconcentration factor of around 1.4 as opposed to 0.01 for pesticides allowed for use today. Concerning veterinary drugs, this value would be around 0.03. For compounds found in congeneric mixtures (PCBs, dioxins/ furans) it is estimated that, depending on the congener, 5–50% of dioxins, furans and PCBs ingested are transferred towards the egg, 7–54% are stored in body fat and less than 1% in the liver (Stephens et al., 1995; Kan and Meijer, 2007). Twenty-five percent of excretion via the eggs is often the standard value used. However, these values are probably under-estimated as the experimentation times are often less than what is required to reach a state of equilibrium. The transfer of PCDD/Fs and most PCBs into the egg
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Chemical residues and contaminants in eggs 71 Table 4.3 Carry-over rate and bioconcentration factor of some persistent organic pollutants from feed to egg (from Kan and Meijer, 2007; Leeman et al., 2007; MacLachlan, 2008) Compound
Half-life (day)
Carry-over1 (%)
DDT/DDE 50–70 50 Lindane 5–14 10 Aldrin + dieldrin 42 60 Hexachlorobenzene 50–60 55 PCB 30–100 5–90 Heptachlor 35–45 35 Polychlorinated dioxins/furans 5–48
Bioconcentration factor2 1.0–1.6 0.13–0.2 1.5–1.5 1.5–1.9 0.09–2.2 0.5–0.7 0.2–1.0
1
Carry-over: amount excreted per day via eggs relative to the amount ingested per day (expressed as percentage). 2 Bioconcentration factor: ratio of concentration in egg (ng/g) to concentration in feed (ng/g). 5
BCF egg
4 3 2 1 0 6.5
7.0
7.5
Log Kow
8.0
8.5
9.0
Fig. 4.3 Bioconcentration factor (BCF; ratio of concentration in egg (ng/g fat) to concentration in feed (ng/g)) of polychlorinated dioxins/furans in eggs according to their hydrophobicity (log Kow) (from Stephens et al., 1995; Schuler et al., 1997).
drops when the degree of chlorination increases (or when log Kow rises) (Fig. 4.3). Transfer and bioconcentration factors are thus dependent on the nature of the mixture to which the animals have been exposed.
4.4 Monitoring strategies 4.4.1 Regulatory context In Europe, official testing is carried out in accordance to various European Community regulatory texts, among which feature Council Directive 96/23/ EC and Commission Decision 2002/657/EC. © Woodhead Publishing Limited, 2011
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72 Improving the safety and quality of eggs and egg products Council Directive 96/23/EC, regarding the monitoring of residues of chemical compounds in all animal-derived foodstuffs, codifies: ∑
national residue monitoring plans, with the role of National Reference Laboratories (NRL), and European Community Reference Laboratories (CRL); ∑ the list of contaminant groups to research and the types of products submitted for analysis (annexes I and II): it concerns substances that have an anabolic effect and other banned substances, veterinary drugs and environmental contaminants to be researched in meats for human consumption, poultry, aquaculture products, milk, eggs, rabbit meat, game and honey; ∑ sampling strategy and execution (annexes III and IV, complemented by the Commission Decision 97/747/EC); ∑ missions and operating conditions of the European Community Reference Laboratories (CRL) (annex V). Commission Decision 2002/657/EC concerns the performance of analytical methods and the interpretation of results in the framework of implementing Council Directive 96/23/EC. This text takes into account the evolution of analytical techniques and specifies performance criteria and other demands that analytical methods of confirmation must meet in distinguishing the research of authorized substances (with MRL) and banned substances. For substances according to Table 2 (Prohibited substances) of the Commission Regulation (EU) No. 37/2010, Commission Decision 2003/181/EC defines minimum required performance limits (MRPLs) for the determination of their residues in food of animal origin. This legislation for the field managing of the risks tied to these substances has led to the implementation of residue monitoring by qualified administrations. They carry out samplings during rearing, in the slaughterhouse and during importation so as to implement testing plans in accordance with Council Directive 96/23/EC, which are intended to estimate contamination levels in various animal-derived foodstuffs and detect eventual non-compliant results where the risk is greatest. The number of samples is established in accordance to annex IV of both Council Directive 96/23/ EC and Commission Decision 97/747/EC. For each branch, the European Community texts specify the number of samples to be taken according to national production and the residue groups to be researched. For poultry, the annual minimum number of animals to check is one per 200 tonnes of production, with a minimum of 100 samples per class if the production is over 5000 tonnes. 4.4.2 Methods of analysis Council Directive 96/23/EC makes provisions for the monitoring of all therapeutic classes used in veterinary medicine: antibacterials, anthelmintics,
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Chemical residues and contaminants in eggs 73 coccidiostats and also of organochlorine pesticides, heavy metals and PCBs. There is a need for sensitive, selective and reliable analytical methods to detect and monitor chemical contaminants and namely residues. Especially with regard to veterinary drug residues the scientific literature provides an overwhelming amount of information. Even in the field of sample preparation a multitude of methods is available, applicable depending on sample selection or matrix and the target residue (Kinsella et al., 2009). Antimicrobial residues and compounds with hormonal activity can be screened very cost-effectively using rapid immunochemical methods such as receptor assay systems, enzymelinked immunosorbent assays (ELISA), biosensors or microbial growth inhibition assays (Bovee and Pikkemaat, 2009). The recent developments in ultra-performance liquid chromatography (UPLC) with fast switching tandem mass spectrometry (MS/MS) and UPLC coupled with full-scan high resolution accurate mass analysers based on time-of-flight (TOF) or orbital trap technologies triggered the development of selective targeted approaches as well as multi-analyte and even multi-class detection methods (Peters et al., 2009). In the future, the ‘omic’ technologies such as transcriptomics, proteomics and metabolomics could be used for the screening for veterinary drug-treated or non-treated situations. For the determination of POPs such as PCDD, PCDF and dioxin-like PCBs (Menotta et al. 2010), non-dioxin-like PCBs (Arnich et al., 2009), PBDEs (Perelló et al, 2009) and PAHs (Ziegenhals et al., 2008) high resolution gas chromatography (HRGC) coupled with high resolution mass spectrometry (HRMS) is the method of choice. All of the methods described are applicable to various matrices, including eggs. Monitoring is also done on the feed and water that is consumed by the animals. For authorized substances, various techniques have been implemented, which most often involve a screening method and a confirmation method. They target molecule(s) belonging to one or several classes and make it possible to dose and identify these molecules on the basis of identification criteria proper to the technique being used. 4.4.3 Results of regulatory plans The number of samples planned to be taken from chicken and quail eggs in 2007 was 13,685 for the European Union. The most frequently tested matrix is the entire egg. Checking for banned substances makes up 42% of samples taken. Chloramphenicol and nitrofurans have not been detected for several years. The highest non-conformity rate is with coccidiostats and more particularly coccidiostatic additives, followed by antibiotics and organochlorine compounds (Efsa, 2010). In regards to antibiotics, the molecules most frequently found are sulfamides, tetracyclines and quinolones.
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74 Improving the safety and quality of eggs and egg products
4.5 Origin of non-conformities and preventing risk during rearing 4.5.1 Veterinary drugs and additives The origin of non-conformities in additives is essentially caused by crosscontamination, at different stages of the production chain, between unaltered feed and feed containing additives (Cannavan et al., 2000; Mortier et al., 2005). Nicarbazine, which is highly electrostatic, is the most prominent molecule (Dubreil-Chéneau et al., 2009). Establishing tolerance levels in eggs (Commission Regulation (EC) no. 124/2009), in effect since July 2009, together with the MRL for these substances in animal feed for nontarget animals established by the Commission Directive 2009/8/EC should reduce the non-conformity rate of these molecules. For veterinary drugs, the main causes of non-conformities are the intentional and unintentional use of an unauthorized drug on laying hens, not respecting the maximum age to administer drugs, not respecting the withdrawal times or dosages, and crosscontamination from supplemented feed during their preparation in the plant or at the farm (Cannavan et al., 2000). Another source of contamination that is rarer is when drugs are recycled by litter-raised poultry ingesting their bedding (Kan, 2005). The poultry farmer’s heeding veterinary prescriptions and the veterinarian’s understanding of ‘residue’ risks should guarantee the feed’s staying within the MRLs. The measure to certify production chains, combined with the management available information on drug use (breeder’s registry) should make it possible to trace products in treated animals, reduce the risk of residues being present and safeguard products from this risk. 4.5.2 Contaminants from the environment Serious excesses in the maximum regulatory limits of PCBs and PCDD/Fs were revealed at the moment of the Belgian crisis in 1999, with amounts in eggs reaching 46 mg NDL-PCB/g fat and 32 pg I-TEQ PCDD/F/g fat (Schoeters and Hoogenboom, 2006) and when contaminated clays were used to prevent caking in feed (Schmid et al., 2002). Outside these accidental situations, eggs coming from hens reared indoor without any outside contact had 1–22 ng NDL PCB/g fat and less than 1 pg I-TEQ PCDD/F/g fat (EFSA, 2005; Thébault, 2005; Le Bouquin et al., 2009). These data show the effectiveness of regulations on feed, which limit the transfer of these contaminants into food products. However, surveys carried out in different European countries (Belgium, Switzerland, Germany, Ireland, France, Sweden and the United Kingdom) pointed to possible contamination by these compounds in the eggs of hens which had outside access (Schoeters and Hoogenboom, 2006). Ingesting soil, plants and pedofauna has been put forth as the main source of contaminants coming from the environment. On clearly contaminated soils in urban and industrial areas, eggs were collected that considerably exceeded the 3 pg © Woodhead Publishing Limited, 2011
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Chemical residues and contaminants in eggs 75 I-TEQ PCDD/F/g fat (Schuler et al., 1997; Harnly et al., 2000; Pussemier et al., 2004; Kan, 2005). Thébault (2005) reported amounts ranging from 3 to 122 pg I-TEQ PCDD/F/g fat for home-produced eggs in areas known to be contaminated by an incinerator. Likewise, surveys on organic farms carried out in The Netherlands and Belgium showed that 13% of the eggs collected in 26% of the farms surveyed exceeded the regulatory limit of 3 pg I-TEQ PCDD/F/g fat, suggesting possible contamination including in rural areas, which a priori are not very contaminated (De Vries et al., 2006). More recently, surveys taken on family farms in Belgium revealed the fragility of home-produced eggs in regards to pollutants from the environment, whether it concerns heavy metals like lead, PCBs, dioxins-furans and organochlorine pesticides (Van Overmeire et al., 2009). The same was observed by Travel et al. (2008) in France, with amounts of PCBs, PCDD/Fs and PBDEs around five times higher in homeproduced eggs than in eggs produced in professional farms. Concerning contaminants coming from the soil, two elements must be taken into account to evaluate risk: the amount of contaminant ingested, which is the resultant of the amount of contaminant in the soil and of the quantity of soil ingested, and the availability of the contaminant present in the soil. Quantity of ingested soil In alternative rearing systems, the runs made available to animals allow them to express their innate behaviour to forage for their feed outdoors. The advantages are many; in addition to this opportunity for the animals to express their natural inclination to explore and seek their own feed, there is also the possible reduction in feed costs and the curbing of high risk elements (N, P, metallic trace elements) being introduced at rearing, thanks to the full use of local resources found on the runs. It is estimated that a hen on a run consumes 10 g of dry soil, 7 g of dry vegetation and 20 g of insects and earthworms a day (De Vries et al., 2006). Nevertheless, these amounts are subject to wide variations. Hens that have access outdoors ingest plants, even when they are given a balanced diet (Horsted et al., 2007). The volume of the ingested environmental matrix (especially soil) increases when the diet is unbalanced or has a coarse granulometry (whole wheat + grit + oyster shells). The authors also showed that the characteristics of the vegetation on the run influenced the eating habits of the hens. The amount of ash in the droppings, which is an indicator of soil ingestion, can reach from 25 to 80% in privately raised hens, indicating that the quantity of soil ingested can vary from 2 to 30 g per day (Waegeneers et al., 2009). In a study carried out in our laboratory, the droppings contained 40–60% of ash, depending on whether the animals received a standard or an unbalanced diet of only whole wheat and oyster shells. In the latter case, dry matter ingested was estimated to be made of 15% of soil (Fig. 4.4). Limiting the risk of transfer of contaminants from the environment into eggs thus requires knowing and evaluating rearing practices that are likely
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76 Improving the safety and quality of eggs and egg products
Contribution to dry matter ingested (%)
100
Stones Soil
90
Herbage
80
Feed
70 60 50 40 30 20 10 0 Complete feed
Whole wheat + oyster shell
Fig. 4.4 Proportion of feed items ingested by laying hens reared outdoors according to the type of feed (from Jondreville et al., unpublished data).
to favour soil ingestion. Foraging behaviours and prolonged periods of time spent outside are the necessary conditions to increase the ingestion of matrices found on the run. Favourable climatic conditions, reduced flock size, certain genetic types and the length of time the hatches are open, which promotes the animals’ contact with the outside, are thus risk factors (Hegelund et al., 2005; Kjaer and Isaksen, 1998). Nevertheless, their impact still needs to be assessed. Accessibility of contaminants found in the soil Studies made on feed cannot be directly used to estimate the transfer of contaminants ingested along with soil, plants or pedofauna. In particular, after being deposited, contaminants interact with particles in the soil and undergo a maturation process, which consists mainly of the phenomena of adsorption and trapping inside the micropores in the soil or in organic material (Reid et al., 2000). This maturation helps limit their accessibility in the digestive tract. Van Eijkeren et al. (2006) estimate that the accessibility of PCDD/ Fs and dioxin-like PCBs in the soil is 40% lower than contaminants found in oil. But this is an approximate value as the nature and the extent of the interactions between the soil and the contaminant depend on the properties of both the soil and the molecule.
4.6 Conclusion The results of the regulatory plans and surveys show that eggs marketed in European Union are for the most part free of regulated chemical contaminants. © Woodhead Publishing Limited, 2011
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Chemical residues and contaminants in eggs 77 This high sanitary quality is the result of strict regulations on animal feed and on the use of veterinary drugs and additives, as well the subsequent application of these regulations by farmers and other actors of the chain of egg production. The measure, establishing guidelines for good practices in response to the Hygiene Package, should allow for a reduction in nonconformity rates and a guarantee against risk for the products. Rearing systems that provide outside access to animals are more sensitive to contaminants in the environment for which exposure is difficult to control. Yet cases of non-conformity are still rare for professional farms, with the most worrying cases having been found on family farms. Nevertheless, to limit risk in these breeding environments, it is important to learn more about the impact of rearing practices on the ingestion of environmental matrices and on the accessibility of these contaminants once they are present in matrices such as the soil.
4.7 References anadon a, martinez-larranaga m r
and fernandez-cruz m l (1993), ‘Considérations physiologiques et pharmacologiques en thérapeutique aviaire’, Rev. Méd. Vét., 144, 745–757. arnich n, tard a, leblanc j c and le bizec b (2009), ‘Dietary intake of non-dioxinlike PCBs (NDL-PCBs) in France, impact of maximum levels in some foodstuffs’, Regulatory Toxicology and Pharmacology, 54, 287–293. arnold d and somogyi a (1986), Chloramphenicol residues in edible tissues of food animals, Proceedings of the 2nd World Congress on Foodborne Infection, Berlin, Germany, 832–836. bovee t f h and pikkemaat m g (2009), ‘Bioactivity-based screening of antibiotics and hormones’, J. Chromatogr. A, 1216, 8035–8050. brugère h (1992), ‘Pharmacologie chez les oiseaux’, Manuel de pathologie aviaire, Brugère-Picoux J and Silim A (Eds), ENVA, Maisons-Alfort, France, 355–363. cannavan a, ball g and kennedy g (2000), ‘Nicarbazin contamination in feeds as a cause of residues in eggs’, Food Add. Contam., 17, 829–836. Commision Decision 97/747/EC of 27 October 1997 fixing the levels and frequencies of sampling provided for by Council Directive 96/23/EC for the monitoring of certain substances and residues thereof in certain animal products. OJ L303, 06.11.1997, p. 12. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. OJ L221, 17.08.2002, p. 8. Commission Decision 2003/181/EC of 13 March 2003 amending Decision 2002/657/EC as regards the setting of minimum required performance limits (MRPLs) for certain residues in food of animal origin (Text with EEA relevance) (notified under document number C(2003) 764). OJ L71, 15.03.2003, p. 17. Commission Directive 2009/8/EC of 10 February 2009 amending Annex I to Directive 2002/32/EC of the European Parliament and of the Council as regards maximum levels of unavoidable carry-over of coccidiostats or histomonostats in non-target feed (Text with EEA relevance). OJ L40, p. 19. Commission Regulation (EC) no. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. OJ L364, 20.12.2006, p. 5.
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78 Improving the safety and quality of eggs and egg products Commission Regulation (EC) No. 124/2009 of 10 February 2009 setting maximum levels for the presence of coccidiostats or histomonostats in food resulting from the unavoidable carry-over of these substances in non-target feed. OJ L40, 11.02.2009, p. 7. Commission Regulation (EU) No. 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin Text with EEA relevance. OJ L015, 20.01.2010, p. 1. Council Directive 86/363/EEC of 24 July 1986 on the fixing of maximum levels for pesticide residues in and on foodstuffs of animal origin. OJ L363, 7.08.1986, p. 43. Council Directive 96/22/EC of 29 April 1996 concerning the prohibition on the use in stock farming of certain substances having a hormonal or thyrostatic action and of ß-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. OJ L125, 23.05.1996, p. 3. Council Directive 96/23/EC of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC. OJ L125, 23.05.1996, p. 10. Council Regulation (EEC) No. 2377/90 of 26 June 1990 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. OJ L224, 18.08.1990, p. 1. de vries m, kwakkel r p and kijlstra a (2006), ‘Dioxins in organic eggs: a review’, NJAS, 54, 207–2006. Directive 2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the community code relating to veterinary medicinal products. OJ L311, 28.11.2004, p. 1. Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. OJ L140, 30.05.2002, p. 10. Directive 2003/74/EC of the European Parliament and of the Council of 22 September 2003 amending Council Directive 96/22/EC concerning the prohibition on the use in stock farming of certain substances having a hormonal or thyrostatic action and of beta-agonists. OJ L28, 22.09.2003, p. 45. donoghue d j, schenck f j, hairston h and podhorniak l v (1997), ‘Modeling drug residue uptake by eggs: evidence of a consistent daily pattern of contaminant transfer into developing preovulatory yolks’, J. Food Protec., 60, 1251–1255. dubreil-chéneau . e, bessiral m, roudaut b, verdon e and sanders p (2009), ‘Validation of a multi-residue liquid chromatography-tandem mass spectrometry confirmatory method for 10 anticoccidials in eggs according to commission decision 2002/657/ EC’, J. Chromatogr. A, 1216, 8149–8157. efsa (2005), ‘Opinion of the scientific panel on contaminants in the food chain on a request from the Commission related to the presence on non dioxin-like polychlorinated biphenyls (PCB) in feed and food. Question N°EFSA-Q-2003-114’, European Food Safety Authority, EFSA Journal, 284, 1–137. efsa (2010), ‘Report for 2008 on the results from the monitoring of veterinary medicinal product residues and other substances in food of animal origin in the Member States‘, European Food Safety Authority, EFSA Journal, 8, 1–55. fournier a, feidt c, martin o, travel a and jondreville c (2009), ‘Elaboration d’un modèle de transfert des polluants organiques persistants du sol vers l’œuf de poule: un outil pour sécuriser les systèmes de production avicole’, Proc. 2èmes rencontres Nationales de la recherche sur les sites et sols pollués, 20–21 Octobre 2009, Paris, France. fournier a, feidt c, dziurla m a, grandclaudon c and jondreville c (2010), ‘Transfer kinetics to egg yolk and modeling residue recovered in yolk of readily metabolized molecules: polycyclic aromatic hydrocarbons orally administered to laying hens’, Chemosphere, 78, 1004–1010. harnly m e, petreas m x, flattery j and goldman lr (2000), ‘Polychlorinated dibenzop-dioxin and polychlorinated dibenzofuran contamination in soil and home-produced
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Chemical residues and contaminants in eggs 79 chicken eggs near pentachlorophenol sources’, Environ. Sci. Technol., 34, 1143– 1149. hegelund l, sørensen j t, kjær j b and kristensen i s (2005), ‘Use of the range area in organic egg production systems: effect of climatic factors, flock size, age and artificial cover’, Brit. Poult. Sci., 46, 1–8. horsted k, hermansen j e and ranvig h (2007), ‘Crop content in nutrient-restricted versus non-restricted organic laying hens with access to different forage vegetations’, Br. Poult. Sci., 48, 177–184. kan c a and meijer g a l (2007), ‘The risk of contamination of food with toxic substances present in animal feed’, Anim. Feed Sci. Technol., 133, 84–108. kan c a and petz m (2000), ‘Residues of veterinary drugs in eggs and their distribution between yolk and white’, J. Agric. Food Chem., 48, 6397–6403. kan k (2005), ‘Chemical residues in poultry and eggs produced in free-range or organic systems’, Proceedings of the XIth European Symposium on the quality of egg and egg Products, Doorwerth, 23–26 May 2005, The Netherlands, No. 210. kinsella b, o’mahony j, malone e, moloney, m, cantwell, h, furey a and danaher m (2009), ‘Current trends in sample preparation for growth promoter and veterinary drug residue analysis’, J. Chromatogr. A, 1216, 7977–8015. kjaer j b and isaksen p k (1998), ‘Individual use of the free range area by laying hens and effects of genetic strain’, Proc. 32nd congress of the International Society of Applied Ethology, Veissier I and Boissy A (Eds), 21–25 July, Clermond-Ferrand, France, p. 88. le bouquin s, allain v, chabault m and guinvarch j (2009), ‘Egg levels of persistent organic polluants in various laying hens housing systems in France’. Abstracts of the 19th European Symposium on the Quality of Poultry Meat and the 13th European Symposium on the Quality of Eggs and Eggs Products. 21–25 June, Turku, Finland, p. 78. leeman w r, van den berg k j and houben g f (2007), ‘Transfer of chemicals from feed to animal products: The use of transfer factors in risk assessment’, Food Add. Contam., 24, 1–13. maclachlan d j (2008), ‘Transfer of fat-soluble pesticides from contaminated feed to poultry tissues and eggs’, Br. Poult. Sci., 49, 290–298. menotta s, d’antonio m, diegoli g, montella l, raccanelli s and fedrizzi g (2010), ‘Depletion study of PCDD/Fs and dioxin-like PCBs concentrations in contaminated home-produced eggs: preliminary study’, Anal. Chim. Acta, 672, 50–54. mortier l, huet a c, daeseleire e, huyghebaert g, fodey t, elliott c, delahaut p and van peteghem c (2005), ‘Deposition and depletion of five anticoccidials in eggs’, J. Agric. Food Chem., 53, 7142–7149. opdycke j c and menzer r e (1984) ‘Pharmacokinetics of diflubenzuron in two types of chickens’, J. Toxicol. Environment Health, 134, 721–733. perelló g, roser m, castell v, llobet j m and domingo j l (2009), ‘Concentrations of polybrominated diphenyl ethers, hexachlorobenzene and polycyclic aromatic hydrocarbons in various foodstuffs before and after cooking’, Food Chem. Toxicol., 47, 709–715. peters r j b., bolck y j c, rutgers p, stolker a a m and nielen m w f (2009), ‘Multiresidue screening of veterinary drugs in egg, fish and meat using high-resolution liquid chromatography accurate mass time-of-flight mass spectrometry’, J. Chromatography A, 1216, 8035–8050. pussemier l, mohimont l, huyghebaert a and goeyens l (2004), ‘Enhanced levels of dioxins in eggs from free range hens; a fast evaluation approach’, Talanta, 63, 1273–1276. regulation (EC) No. 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L268, 18.10.2003, p. 29. regulation (EC) No. 396/2005 of the European Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed © Woodhead Publishing Limited, 2011
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80 Improving the safety and quality of eggs and egg products of plant and animal origin and amending Council Directive 91/414/EEC. OJ L70, 16.03.2005, p. 1. regulation (EC) No. 470/2009 of the European Parliament and of the Council of 6 May 2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin, repealing Council Regulation (EEC) No 2377/90 and amending Directive 2001/82/EC of the European Parliament and of the Council and Regulation (EC) No 726/2004 of the European Parliament and of the Council. OJ L152, 16.06.2009, p. 11. reid b j, jones k c and semple kt (2000), ‘Bioavailability of persistent organic pollutants in soils and sediments – a perspective on mechanisms, consequences and assessment’, Environ. Pollut., 108, 103–112. renwick a g (2001), ‘Toxicokinetics: pharmacokinetics in toxicology’. In: Principles and Methods of Toxicology, A. W. Hayes (Eds), Taylor Francis, Philadelphia, PA, 137–191. roudaut b (1997), ‘Antibiothérapie chez la poule pondeuse. Etude des résidus d’antibiotiques dans les œufs’, PhD Thesis, Institut Polytechnique de Toulouse. roudaut b (1998), ‘Elimination of oxolinic acid in eggs after oral treatment of laying hens’, Brit. Poult. Sci., 39, 47–52. roudaut b and garnier m (2002), ‘Sulphonamide residues in eggs following drug administration via the drinking water’, Food. Addit. Contam., 19, 373–378. samouris g, tsoukali-papadopoulou h, nathanael b and mirtsou-fidany v (1998), ‘Chloramphenicol residues in albumenn and yolk of hen’s eggs after experimental administration’, Arch. Gefluegelk., 62, 83–85. schmid p, gujer e, dengen s, zenneg m, kuchen a and wäthrich c (2002), ‘Levels of polychlorinated dibenzo-p-dioxins and dibenzofurans in food of animal origin. The Swiss dioxin monitoring program’, J. Agric. Food Chem., 50, 7482–7487. schoeters g and hoogenboom r (2006), ‘Contamination of free-range chicken eggs with dioxins and dioxin-like polychlorinated biphenyls’, Molecul. Nutr. Food Res., 50, 908–914. schuler f, schmid p and schlatter c (1997), ‘The transfer of polychlorinated dibenzop-dioxins and dibenzofurans from soil into eggs of foraging chicken’, Chemosphere, 34, 711–718. stephens r d, petreas m x and hayward dg (1995), ‘Biotransfer and bioaccumulation of dioxins and furans from soil: chickens as a model for foraging animals’, Sci. Total Environ., 175, 253–273. thébault a (2005), ‘Analyse des déterminants de la contamination en dioxines et furanes (PCB non compris) des oeufs issus d’élevages de volailles en plein air de particuliers’, Note technique AQR/ATH/2005–203, AFSSA, 27 September 2005p. http://www. editionsduboisbaudry.fr/docs/av/pdf/2006-06/2005-09-27_Afssa_dioxine.pdf travel a, jondreville c, guinvarch j, chabault m, lubac s, feidt c, marchand p, bonnard r, le nouquin-neveu s, allain v, thebault a, gonnier v and nys y (2008), ‘La filière fait le point sur le risque de transfert de polluants organiques persistants vers les œufs’ Tema, 6, 11–19. van eijkeren j c h, zeilmaker m j, kan c a, traag w a and hoogenboom l a p (2006), ‘A toxicokinetic model for the carry-over of dioxins and PCBs from feed and soil to eggs’, Food Addit. Contam., 23, 509–517. van overmeire i, pussemier l, waegeneers n, hanot v, windal i, boxus l, covaci a, eppe g, scippo m l, sioen i, bilau m, gellynck x, de steur h, tangni e k and goeyens l (2009), ‘Assessment of the chemical contamination in home-produced eggs in Belgium: General overview of the CONTEGG study’, Sci. Tot. Environ., 407, 4403–4410. waegeneers n, de steur h, de temmerman l, van steenwinkel s, gellynck x and viaene j (2009), ‘Transfer of soil contaminants to home-produced eggs and preventive measures to reduce contamination’, Sci. Tot. Environ., 407, 4438–4446. ziegenhals k, hübschmann h j, speer k and jira w (2008), ‘Fast-GC/HRMS to quantify the EU priority PAH’, J. Separation Sci., 31, 1779–1786. © Woodhead Publishing Limited, 2011
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5 Detection and monitoring of Salmonella in laying hen flocks R. Davies and J. J. Carrique-Mas, Veterinary Laboratories Agency, UK
Abstract: This chapter reviews published work and contributes the experience of the authors in the field of detection and monitoring of Salmonella in flocks of laying hens. Key words: Salmonella, Salmonella Enteritidis, laying hens, eggs, sampling, monitoring. Note: © Crown copyright 2011. Published with the permission of the Controller of Her Majesty’s Stationery Office. The views expressed are those of the author and do not necessarily reflect those of Her Majesty’s Stationery Office or the VLA or any other government department.
5.1 Introduction Sampling and testing are essential components of Salmonella control programmes, which are aimed at detecting infected flocks so that adequate control measures (restriction on the sale of eggs, depopulation, cleaning and disinfection between flocks, rodent control, etc.) can be implemented. Results from systematic testing can also generate useful population-based estimates of the prevalence of Salmonella at the flock/farm/geographical area level, provided that the methods used have a known sensitivity. The efficiency of any monitoring programme is highly dependent on the choice of an adequate sampling strategy (Fletcher 2006), combined with a sensitive culture method (Carrique-Mas and Davies 2008b). A standardised sampling strategy consists of the collection of a set number of samples at Published by Woodhead Publishing Limited, 2011
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84 Improving the safety and quality of eggs and egg products specific times during the life of a flock. Also the overall sensitivity of a monitoring programme is crucially dependent on the number, volume, timing and frequency of samples collected. There are important logistic, practical and economic considerations that condition the choice of a particular sampling/ testing strategy. In situations of high within-flock prevalence fewer samples are theoretically necessary, but this is normally not known a priori and in most situations a standardised protocol (with minor variations depending on the house type) is preferable in order to allow meaningful prevalence comparisons so that epidemiological analyses can be carried out. In the European Union (EU), the European Commission has played a major role in harmonising and coordinating monitoring and control programmes, with the ultimate aim of reducing the prevalence of Salmonella in poultry primary production across member states. Prevalence targets based on existing data and harmonised monitoring programmes were introduced for chicken breeding flocks in 2007. A baseline survey was carried out in 2004/2005 on a randomised selection of commercial scale (>1000 hens) egg laying farms producing table eggs across member states. This survey (the EU layer survey) was coordinated by DG SANCO and the European Food Safety Authority (EFSA). After results from this survey were known, national targets for reduction of the prevalence of S. Enteritidis and S. Typhimurium were established. The tools to achieve these targets were the national control programmes (NCP), which included the minimum sampling requirements and a ban of the use antimicrobials to control Salmonella serovars that are included in regulations that specify prevalence targets for EU member states as well as compulsory vaccination against S. Enteritidis of commercial laying flocks in countries with an S. Enteritidis prevalence greater than 10% (Carrique-Mas and Davies 2008a). Sampling for the NCPs is normally carried out by the producers, and consists of sampling/testing day-old chicks on arrival, as well as birds in the rearing and in the laying phase. Sampling of flocks during the laying phase is carried out on all flocks in a holding and consists of one sample at the age of 22–26 weeks, and then every 15 weeks during the life of the flock. In addition, in holdings with more than 1000 laying birds, one house is sampled by the competent authority each year (Regulation EC No. 1168/2006). A confirmed S. Enteritidis or S. Typhimurium positive laying flock is restricted so that no eggs from such flocks can be sold as fresh eggs.
5.2 What to sample In the egg industry many consider that testing eggs, and specifically egg contents, gives the best assessment of public health risk, but the prevalence of contaminated eggs, even from a flock known to be infected with S. Enteritidis, can be very low (Gast 1993; Gast and Holt 1998; Lapuz et al. 2008), making meaningful egg testing very laborious and expensive. Published by Woodhead Publishing Limited, 2011
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Detection and monitoring of Salmonella in laying hen flocks 85 Sampling caecal and/or ovary/oviduct samples from birds at post-mortem is considered to be a definitive test of infection in a flock (Nief and Hoop 1998), and is best accepted by the poultry industry for confirmatory testing but tissue samples are also subject to cross-contamination in the laboratory or collection point, and sampling a large number of birds is needed for detection of low within-flock prevalence (Barnhart et al. 1993). Another popular sampling method that has been favoured by the industry is cloacal swabs. This sample has the advantage of originating directly from the bird, so is less likely to be disputed by poultry flock operators, but a combination of low volume of material tested (Funk et al. 2000; Kotton et al. 2006) and intermittent excretion (Bichler et al. 1996; Van Immerseel et al. 2004) means that this method is relatively insensitive unless very large numbers of samples are taken, which has welfare implications for the birds. An intermediate stage between collection of cloacal swabs directly from the birds and environmental sampling (the collection of material from the birds’ ‘environment’) is collection of fresh faeces. This can be done by collecting naturally pooled fresh faecal material or by collecting and pooling individual voided faeces. The inclusion of larger volumes of mixed faecal material from a large number of birds enhances detection (Wales et al. 2006b), but there may be a ‘dilution effect ‘resulting from mixing positive with negative faecal material for culture (Harvey and Price 1974; Kivelä et al. 1999; Jarvis 2007; Arnold, et al. 2009b; Singer et al. 2009). However, it has been shown that in the case of laying flocks, as with other types of poultry, it is highly efficient to pool samples for Salmonella detection in spite of this dilution effect (Mark Arnold, personal communication), and therefore a single culture is maximised when faecal material from a large number of birds is included, since this increases the chance of including faeces originating from a high-level shedder (Hildebrandt and Bohmer 1998; Arnold et al. 2005, 2009b). It is recognised that the culture of faeces samples can provide a snapshot of the current prevalence of Salmonella excretion in the flock, particularly if faeces are fresh. Also when individual faeces are randomly allocated to pools, a reliable estimate of within-flock prevalence from pool cultures is possible (Cowling et al. 1999; Evers and Nauta 2001). Furthermore, it is also possible to quantify Salmonella in the samples (Wales et al. 2006a). However there is a risk that minority strains of Salmonella may fail to be detected in a mixed population of strains in the sample (Singer et al. 2009). In all cases the isolation of Salmonella from faecal material is likely to be compromised if antibacterial substances such as organic acids, disinfectants or antibiotics have been added to the feed ration or the water of the flock. The addition of lime to poultry litter may also compromise detection if litter samples or boot swabs are used. It has been recognised for some time that thorough environmental sampling is the most effective way to detect Salmonella in a poultry flock (Aho 1992; Johansson et al. 1996; Musgrove and Jones 2005; Carrique-Mas et al. 2009a),
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86 Improving the safety and quality of eggs and egg products although some have argued that Salmonella isolated from a sample from a poultry house internal environment may not originate from the birds, but from wildlife, feed or another source. This may be the case on rare occasions but normally the occurrence of Salmonella in any part of the environment does reflect infection in the flock, and inclusion of vectors such as mice in monitoring of laying flocks (Fig. 5.1) can increase the detection rate (Kinde et al. 2005; Wales et al. 2007). Litter has been traditionally collected from non-cage laying houses for Salmonella monitoring. It is a good sample when collected in a representative way from several locations throughout the house. This, however, is seldom thoroughly done by poultry flock operators and so other samples such as boot swabs or drag swabs may perform better (Kingston 1981). Certain types of litter, such as long straw, are bulky and difficult to sample in a representative way (Mueller-Doblies et al. 2009). Litter taken from deep litter systems is normally very biologically active (the ‘litter effect’) and Salmonella levels can drop rapidly if there is a significant delay between sampling and testing, especially in warm weather. Thin layers of litter from the scratching areas of barn and free-range laying houses are less likely to show a rapid reduction in positivity of samples. There has been a substantial amount of research aiming at comparing boot or sock swabs (Fig. 5.2) with drag swabs. Sock swabs, comprising a folded section of tubegrip bandage applied over the ball of the foot of the sampler and turned four times during sampling, were first described in Denmark (Skov et al. 1999). In the USA boot swabs utilising gauze surgical shoe covers were used and found to be superior to drag swabs (Caldwell et al. 1994; McCrea 2005; McCrea et al. 2006) even when the method of stepping on drag swabs was used (Buhr et al. 2007). The overshoe-type boot
Fig. 5.1 Mouse and mouse droppings samples.
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Detection and monitoring of Salmonella in laying hen flocks 87
Fig. 5.2 Sock and boot swab.
Fig. 5.3 Moistening boot swabs before sampling.
swabs can easily be worn during normal flock inspection visits by the farmer and do not need to be turned, as the surface area available for sampling is already quite large (Fig. 5.1). This means that this method of sampling is more likely to be carried out in a correct way than other more demanding procedures. Thorough moistening of the swab (Fig. 5.3) is important as litter in good condition can be rather dry and faecal material may fail to stick to the swabs. In any case, swabs should be taken before laying new litter. Drag swabs were developed in the USA (White et al. 1997; Castellan et al. 2004) to sample very large poultry houses. Each drag swab usually comprises at least three separate 100 cm2 moist surgical gauze swabs (Mallinson et Published by Woodhead Publishing Limited, 2011
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88 Improving the safety and quality of eggs and egg products al. 1989) that are dragged behind an operator who walks the length of the houses (Figs 5.4 and 5.5). This method proved more effective than limited litter sampling (Kingston 1981), but is subject to variability according to litter/manure moisture (Carr et al. 1995) or details of application and sample
Fig. 5.4 Swabbing poles with swabs.
Fig. 5.5 Swabbing pit.
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Detection and monitoring of Salmonella in laying hen flocks 89 processing (Opara et al. 1992; Byrd et al. 1997; Rolfe et al. 2000). Limited sensitivity is indicated by the identification of additional positive flocks when the number of drag-swab assemblies used is increased (Caldwell et al. 1994) which may be due to rapid overload of the available surface area of the swab (Rolfe et al. 2000). The performance of drag-swabs can be substantially improved by intermittently stepping on the swabs during sampling (Buhr et al. 2007), which may suggest that additional pressure may be beneficial, or that the sampler’s boot is actually what is being sampled. Collection and testing of dust has sometimes been included in Salmonella surveys and monitoring programmes. The superior resistance to desiccation and long-term survival of Salmonella compared with many other enteric bacteria (Davies and Wray 1996b; Davies and Breslin 2003b; Haysom and Sharp 2003) means that airborne Salmonella that is liberated from the dried faeces and body surface of birds (Kwon et al. 2000; Gast et al. 2004), particularly during periods of stress (Holt et al. 1998; Albrecht et al. 2003; Fallschissel et al. 2008) can settle on horizontal or sloping surfaces or extractor fan housings. This provides a ‘historic’ record of an earlier period of excretion; however, a significant dependence on the sensitivity of dust on the within-flock prevalence has been observed (Arnold et al. 2009a). The stage of infection may also condition the results since dust is likely to test negative in the very early stages of infection (Davies and Breslin 2001; Davies and Breslin 2004; EC 2004; EFSA 2007). Also the efficiency of dust sampling appears to depend on the type of house sampled, and it is suggested that it is more efficient in cage houses (Mahe et al. 2008; Huneau-Salaun et al. 2009). A plausible explanation for this is that in non-cage houses sufficient amounts of settled dust may not always be available for sampling, and this may compromise the efficiency of dust testing (Davies 2005). This is supported by a study in the UK where identical amounts of dust were collected from cage and non-cage houses, and the sensitivity of dust samples was only marginally lower for non-cage houses (Carrique-Mas et al. 2008). A further disadvantage of dust sampling is that operators often do not view a positive dust sample as a valid indicator of current flock infection. However, dust is generated rapidly in an occupied laying house, and positive dust soon follows the onset of excretion of Salmonella in faeces in laying flocks. Likewise, pooled faeces from S. Enteritidis positive flocks were soon followed by negative dust results when rodents are eliminated from laying houses (Carrique-Mas et al. 2009a). Compared with faeces, handling of dust in the laboratory requires extra attention to avoid cross-contamination from other samples. In practice, in order to increase the sensitivity of a sampling method, it is normally best to take both fresh faecal and dust samples (Carrique-Mas et al. 2008). Large hand-held gauze (or ‘chiffonette’) swabs (Fig. 5.6) can also be effective for sampling dust (Davies and Wray 1996a; Carrique-Mas et al. 2008; Zewde et al. 2009), but require more effort and dedication to achieve a representative sample, unless a larger number of samples from different
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90 Improving the safety and quality of eggs and egg products
Fig. 5.6 Sampling dust with a gauze fabric swab (chiffonette).
point locations are taken. In our laboratory we use this method extensively due to its great versatility since these swabs can be included as a sterile unit in plastic jars containing 225 ml buffered-peptone water (BPW) so that the sample is put directly back into the jar on site and returned to the laboratory for culture without the need for further manipulations. These samples can be used for all types of houses and to gather faeces/litter (25 g), dust (15 g) or residual organic matter on cleaned surfaces to evaluate the efficiency of cleaning and disinfection (C&D) procedures. This sampling method (Fig. 5.7) is particularly useful for assessing the efficacy of C&D of poultry houses because residual disinfectant that may still be present on the surfaces is diluted and buffered by excess BPW. The jars of BPW containing the samples are placed directly into incubation after arrival in the laboratory without the need of further processing. This ‘wet swabbing’ method is suitable only if samples can be quickly returned to the laboratory so that culture can begin soon after collection, and this usually requires trained staff to take the samples. A delay in the start of the culturing process may result in Salmonella being overgrown by psychotrophic competitor organisms in the BPW. In occupied laying houses, the collection of faeces/litter (25 g) and dust (15 g) from 10 different points in the house is more sensitive than the EU layer survey method, although on a ‘per sample’ basis each sample is less sensitive than EU survey samples (Carrique-Mas et al. 2008). By taking more samples from different locations the specific areas of contamination in the houses can be more precisely identified. Also, if each swab were to be used to sample multiple sites rather than used for a point sample, then the per-sample sensitivity would be expected to increase, allowing a reduction in the number of samples taken.
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Detection and monitoring of Salmonella in laying hen flocks 91
Fig. 5.7 Sampling cage house after cleaning and disinfection.
5.3 Recommended sampling regime of laying houses For flock sampling we recommend the collection of 10 samples of faeces and 10 samples of dust using large BPW-impregnated hand-held gauze (or ‘chiffonette’) swabs taken directly into 225 ml BPW jars as a sensitive method for detection of Salmonella. The sampling locations are largely dictated by the type of house. In cage houses with a scraper or belt manure removal system faeces can be collected by swabbing faeces from the discharge end or the belts or the scraper itself (Fig. 5.8). In step-cage houses sampling of the faeces present in the deep pit (Fig. 5.9) is required. In the case of non-cage houses we recommend the sampling of litter/faeces in scratching areas and slatted areas according to the design of the house. Dust is normally plentiful in air outlet vents, as well as ledges, tops of water tanks, feed corner units, beams, etc., but in cage houses it is often more easily gathered from the floor beneath the cages (Fig. 5.10), especially around cage support posts and egg elevators or trapped material between rollers of egg or droppings belts and the belts themselves. In houses with automatic egg collection dust can also be collected from belt spillage trays (Fig. 5.11) if they are present, or from beneath belt-cleaning brushes. In deep pit houses dust accumulates frequently on the cage support beams which are normally accessible for sampling. In non-cage houses dust is often more difficult to gather due to their structure, but it is normally possible to obtain sufficient dust from vents, ledges, beams, tops of nestboxes and partitions, especially above slatted areas of the house.
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Fig. 5.8 Sampling faeces from manure scrapers.
Fig. 5.9 Sampling manure in a deep pit house.
For the assessment of C&D we recommend the collection of at least 60 samples from each house. For cage houses, 10 samples from each of the following areas are collected: (1) cage interiors (8 cages per swab); (2) drinker cups/troughs (8 per swab); (3) feed troughs (0.5 m2 per swab); (4) Published by Woodhead Publishing Limited, 2011
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Detection and monitoring of Salmonella in laying hen flocks 93
Fig. 5.10 Sampling dust from beneath cage stacks.
Fig. 5.11 Dust from the ends of egg belts is an excellent sample in cage houses.
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94 Improving the safety and quality of eggs and egg products droppings boards/belts/flaps (1 m2 per swab); (5) house floor (0.5 m2 per swab); (6) egg belts (0.5 m2 per swab) (Fig. 5.4). From non-cage houses, samples are to be collected from: (1) drinkers (4 bell drinkers /10 nipple drinkers per swab); (2) feeders (0.5 m2 per swab); (3) scratching area (if present) (1 m2 per swab); (4) slats (0.5 m2 per swab); (5) nestbox interiors (5 per swab). In addition, in free-range houses, the soil from the paddocks close to the house can be sampled by scraping off 25 g of the topsoil (Fig. 5.12). Additional samples from other items may be taken according to the design of the house and equipment (Wales et al. 2006b; Carrique-Mas et al. 2009b).
5.4 Serology Serology can also be used to detect indirect evidence of likely exposure to Salmonella by detecting antibodies in serum or egg yolk (Davies et al. 1997; Feld et al. 2000). The use of serology together with bacteriology increases the sensitivity of detection of those serotypes included in the enzyme-linked immunosorbent assay (ELISA)-based test, normally S. Enteritidis and S. Typhimurium. A combined testing programme has been successfully used in Denmark for many years (Wegener et al. 2003). Serology is less commonly used now that vaccination of laying flocks has been implemented in many member states as even live oral vaccines can result in some false-positive reactions.
Fig. 5.12 The immediate surroundings of a Salmonella-positive free-range house are often highly contaminated.
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Detection and monitoring of Salmonella in laying hen flocks 95
5.5 Methods used for the 2004/2005 baseline survey and Salmonella control programmes in flocks of laying hens in the European Union The adoption of a harmonised method for sampling all laying flocks in laying hens for the EU 2004/2005 baseline survey of flocks of laying hens was not possible because of the different housing systems involved. The stated objective was to detect with 95% confidence flocks with a 1% withinflock prevalence (EC 2004) as such low levels of infection were expected, especially in vaccinated flocks of mature, healthy birds. The calculated required number of samples for this was 300 individual faeces, but this took no account of intermittent excretion; clustering of infection within houses; the limited sensitivity of the test and the effect of pooling faecal samples; or working with an undefined naturally pooled sample. Since it was impractical to collect 300 faeces from each flock in the survey this was extrapolated to taking at least 300 g of naturally mixed faecal material (Fig. 5.2), although little comparative data were available to support this at the time. In fact from cage flocks five 200 g samples were taken, thoroughly mixed and sub-sampled to provide a considerable margin for error. In non-cage flocks five pairs of boot or sock swabs (Fig. 5.13) were taken, based on the finding that in Danish broiler flocks this was equivalent to 300 individual faeces, each cultured as part of a 5 g pool involving 60 separate cultures in total (Skov et al. 1999). This took no account of the different housing systems used for laying flocks compared with broilers and the different age of the birds in egg production. It is likely that infected laying hens excrete lower numbers of Salmonella organisms than broilers because of their acquired immunity and the use of
Fig. 5.13 Boot swabbing in a free-range house.
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96 Improving the safety and quality of eggs and egg products vaccination in some cases. It has been shown in a study using cattle data that the sensitivity of test is largely dependent on number of organisms excreted (Jordan et al. 2004), or the use of vaccination. On the other hand the ISO 6579:2002 Annex D culture method used in the EU baseline survey is likely to have been more sensitive than the NMKL-71 method used in the Danish study (Voogt et al. 2001; Eriksson and Aspan 2007). The addition of two large samples of dust to the baseline survey method (Fig. 5.3) was considered to provide maximum detection potential given the constraints of a maximum limit of seven samples. In the EU, having defined methods for the 2004/2005 baseline survey, a repetition of the survey after approximately three years was considered to verify the extent of improvements made. This was not agreed and a decision was made to use routine monitoring results as the basis for assessing achievement of the Salmonella reduction target. Monitoring based on the use of seven samples every 15 weeks would have been a large economic burden on industry, so after debate in the EC Zoonoses Working Groups it was agreed that a single large faeces sample or two pairs of boot swabs cultured as one sample could be used for operator monitoring. Two pairs of boot swabs is considered to be equivalent to at least 60 faecal droppings samples cultured as one pool (Ellerbroek et al. 2002; Gradel et al. 2002) so reliably detecting a 5% prevalence on a single sampling occasion. Comparative studies of the various sampling options suggested that operator sampling carried out correctly could be more sensitive than this and cumulatively three sampling rounds could give equivalent sensitivity to the full baseline survey protocol (Carrique-Mas et al. 2008). The addition of dust in the official sampling should further enhance the sensitivity of detection but the influence of poor sampling technique by operators; removal of dust before official testing; delays between sampling and testing; and performance of individual testing laboratories is unclear. Farmers with laying flocks with a confirmed S. Enteritidis or S. Typhimurium can dispute the imposed restrictions by repeating testing of the flock using: (1) the EU baseline survey testing method; (2) the culture of ovaries/ oviduct and caecal contents from 300 birds; or (3) the culture of 4000 eggs. The sensitivity of (1) and (2) is related to detection of a 1% within-flock prevalence (300 bird test), which has equivalent sensitivity to that calculated for the baseline survey. The testing of 4000 eggs as 100 pools of 40 eggs is based on detection of prevalence of 0.06% positive eggs (including shells), a figure relating to findings of a retail egg survey carried out in the UK in 2003 (FSA 2004).
5.6 Factors affecting the detection of Salmonella infection 5.6.1 Flock housing/manure handling system Results from the EU baseline survey and other related studies (Methner et al. 2006; Snow et al. 2007; Much et al. 2007; EFSA 2007; Namata et al. Published by Woodhead Publishing Limited, 2011
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Detection and monitoring of Salmonella in laying hen flocks 97 2008) have shown an increased risk of Salmonella, mainly S. Enteritidis, in cage flocks, although in France a higher prevalence of S. Enteritidis in floor systems has been reported (Huneau-Salaun et al. 2009). It is not clear whether some of this reported difference might relate to the different sampling methods or the different sources of replacement birds. A study in France analysing EU layer survey data suggested a lower prevalence of positive environmental samples from flocks kept in non-cage houses (Mahe et al. 2008). In the UK there is some evidence of a higher within-flock prevalence in non-cage flocks (Arnold et al. 2009a). This is probably a reflection of easier transmission of infection within an open house, which can be enabled by the movement of birds and the sharing of open drinkers and feeders. A study comparing different sampling types suggests a lower sensitivity of EU baseline and NCP style samples in noncage houses compared with belt and scraper cage houses (Carrique-Mas et al. 2008). It is possible that in the case of litter/faeces this may be a reflection of the smaller amount of material collected by boot swabs compared with bulk faeces samples collected from scraper and belt houses, a greater die off due to desiccation or bacterial competition of Salmonella organisms on boot swabs or a litter effect leading to faster die-off and lower numbers of Salmonella in floor systems. Deep pit cage houses have been associated with increased persistence of S. Enteritidis infection (Carrique-Mas et al. 2009a), and this may be associated with greater rodent problems (Awad-Masalmeh and Thiemann 1993; Matsumoto et al. 2001; Garber et al. 2003; Carrique-Mas et al. 2009a). Under-detection of Salmonella using baseline survey style samples in step cage houses is thought to occur because of the difficulty in obtaining representative samples from rows of manure that can be difficult to access (Carrique-Mas et al. 2008). Dust samples are less efficient in non-cage systems and do not always increase detection compared with additional boot swabs (Mahe et al. 2008; (Namata et al. 2008). This is dependent on the type of house, the bedding used and the ventilation system. In smaller naturally ventilated floor houses, dust accumulation may be minimal because of the effect of wind or displacement of dust from surfaces by birds. Dust also often appears to be a less sensitive sample when straw bedding is used. 5.6.2 Flock size The baseline survey and various related studies cited above also detected an increased risk of identifying positive flocks as flock size increases (Mollenhorst et al. 2005; EFSA 2007; Namata et al. 2008; Huneau-Salaun et al. 2009). Flock size is often closely related to the type of housing system and only cage houses are likely to hold more than 30,000 birds. Large flocks are also more likely to be present on large holdings with several houses producing eggs at different stages, which is another risk factor (Huneau-Salaun et al.
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98 Improving the safety and quality of eggs and egg products 2009). Assuming that rodents are the main drivers of S. Enteritidis infection in most flocks (Carrique-Mas et al. 2009a), and that continuous challenge is necessary to maintain infection in vaccinated flocks, it could be expected that in typical large houses, although there may be a greater rodent burden, there may be a lower rodent : hen ratio because rodent harbourage is concentrated in the walls of the house. This could result in a lower challenge to birds in the centre of such houses. It is also possible that especially in large cage houses and sub-divided barn or free-range houses (often houses are divided longitudinally – with each half further sub-divided into several pens) infection may be highly clustered, making detection more difficult when taking a standard sample set. 5.6.3 Stage of lay Baseline surveys and other studies have shown an increased tendency for flocks to be identified as Salmonella-positive as the birds become older (Garber et al. 2003; van de Giessen et al. 2006; Wales et al. 2007) especially if birds have been molted (Garber et al. 2003; Castellan et al. 2004; Golden et al. 2008). In the case of S. Enteritidis, in most cases the initial infection in pullets occurs as a consequence of residual contamination of laying houses that have not been adequately cleaned and disinfected after the previous flock, or exposure to a high level of contamination due to the presence of infected rodents. Newly placed naive pullets are often suffering from transport, handling and relocation/remixing stress at a time when hormonal changes associated with the onset of lay may also increasing their susceptibility to infection (Line et al. 1997). This leads to a typical early peak of infection within three weeks of housing (Humbert et al. 1995; Gradel et al. 2002) but laying flocks are rarely sampled at this time (16–19 weeks of age). After this early peak of excretion Salmonella typically subsides, making detection more difficult (Li et al. 2007). In the case of S. Enteritidis there may be an increase of excretion towards the end of lay, but in the absence of rodents this is less likely to occur and infection may spontaneously resolve (Carrique-Mas et al. 2009a). 5.6.4 Vaccination Both live and inactivated vaccines are available for control of S. Enteritidis and/or S.Typhimurium. The protection conferred by vaccination is often not complete and although the likelihood of infection of eggs is reduced (Davies and Breslin 2003a; Toyota-Hanatani et al. 2009), detection of infected flocks may also be reduced as a result of reduction of the within-flock prevalence and number of organisms shed (Van Immerseel et al. 2004; Gantois et al. 2006; Inoue et al. 2008). Waning of vaccinal protection may be involved in the rise in excretion towards the end of lay, along with production stress and increases in potential Salmonella vectors and anaemia due to increasing numbers of red mites in the laying house.
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Detection and monitoring of Salmonella in laying hen flocks 99 There is also a possibility with certain live vaccines that the vaccine strain may be the only strain identified in mixed Salmonella populations. Avoiding sampling after recent vaccination and use of some selective agars can help avoid such problems.
5.7 Significance of under-detection The various factors mentioned above, as well as use of competitive exclusion products, organic acids and therapeutic antimicrobials can lead to failure to detect an infected flock (Seo et al. 2000; Davies and Breslin 2003a; Jarquin et al. 2007; Vila et al. 2009). Intermittent excretion and low within-flock prevalences (Desmidt et al. 1997) present the greatest challenge and more intensive sampling has demonstrated potential limitations of procedures used for baseline surveys and routine monitoring of laying flocks (Carrique-Mas et al. 2008; Carrique-Mas and Davies 2008b; Van Hoorebeke et al. 2009). Non-uniform distribution of infection and contamination of laying houses, and the difficulty of representative sampling in large complex housing systems, makes detection more problematic (Riemann et al. 1998; Hayes et al. 2000; Rolfe et al. 2000). It has been shown that infected non-cage egg flocks tend to produce eggs with a higher rate of Salmonella contamination (Kinde et al. 1996; CarriqueMas and Davies, unpublished data) despite the lower risk of such flocks being infected, which is likely to be a result of faecal soiling in the nest boxes. It has been claimed that some enriched cage systems have increased chance of producing faecally soiled eggs (Tactacan et al. 2009), which have been known to be a higher risk for Salmonella but this may be variable (De Reu et al. 2008) and the within-flock prevalence in infected flocks in such systems does not appear to be particularly high, possibly because enriched cage houses typically provide less harbourage for rodents unless refurbished old building structures such as cavity walls and roof spaces are already infested with mice. Diligence and experience of selection of the best sampling sites and sampling in a thorough and representative way are very important for maximising detection (Davies and Wray 1996a; Mallinson et al. 2000). Sample handling and testing methodology is also important (Fletcher 2006; Kawasaki et al. 2008) both to optimise detection and minimise the risk of false positives due to cross-contamination (Beatty et al. 2009; Pezzoli et al. 2009). Delays and extremes of temperature must be avoided in the period between sampling and testing (Himathongkham et al. 1999). Boot swabs and surface swabs are likely to be more affected by delays in testing than faeces samples (O’Carroll et al. 1999). Homogenisation of samples in which there is non-uniform distribution can be helpful but this must be done carefully to avoid mechanical damage to organisms and release of inhibitory substances from the sample matrix (Cannon and Nicholls 2002; Seo et al. 2003). Published by Woodhead Publishing Limited, 2011
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100 Improving the safety and quality of eggs and egg products During culture, competing organisms are the main limiting factor in detection (Arroyo and Arroyo 1995) and it is important to allow enough free enrichment media around the sample for Salmonella to migrate away from the sample matrix (McCrea et al. 2005). Low numbers of Salmonella organisms, e.g. < 10 cfu/g, can be especially difficult to identify against an overwhelming background of other organisms (Cox and Berrang 2000). It is clear that detection of Salmonella in laying flocks is far from straightforward and no practical sampling method will achieve 100% detection. The significance of this failure to detect in terms of public health is unclear, since it is the most highly infected flocks that are likely to be detected and eggs from flocks with low levels of infection are less likely to be contaminated (Van Hoorebeke et al. 2009). Reduction of the number of cases of S. Enteritidis in humans provides an indication of improvements in conditions on laying farms (Frank et al. 2009), but a repeat of the baseline survey would be the best way to verify the flock infection rates reported by current monitoring programmes. This is unlikely to be possible now that there are restrictions across the EU on sale of fresh eggs from flocks detected with S. Enteritidis or S. Typhimurium since more sensitive sampling could detect additional infected flocks and lead to severe financial consequences for egg producers.
5.8 References aho, m.
(1992). ‘Problems of Salmonella sampling.’ International Journal of Food Microbiology 15, 225–235. albrecht, a., t. redmann, et al. (2003). ‘Examination of airborne microorganisms in a rearing house for layers during vaccination.’ Deutsche Tierarztliche Wochenschrift 110(12), 487–493. arnold, m., j. carrique-mas, et al. (2009a). ‘Estimation of the sensitivity of environmental sampling methods for detecting Salmonella Enteritidis in commercial laying flocks relative to the within-flock prevalence.’ Epidemiology and Infection 138(3), 330– 339. arnold, m., d. mueller-doblies, et al. (2009b). ‘The estimation of pooled-sample sensitivity for detection of Salmonella in turkey flocks.’ Journal of Applied Microbiology 107, 936–943. arnold, m. e., a. cook, et al. (2005). ‘A modelling approach to estimate the sensitivity of pooled faecal samples for isolation of Salmonella in pigs.’ Journal of The Royal Society Interface 2, 365–372. arroyo , g., j. a. arroyo (1995). ‘Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal.’ Journal of Applied Bacteriology 79(4), 360–367. awad-masalmeh, m., thiemann, g. (1993). ‘Salmonella monitoring and related biological parameters in laying hen farms and hatcheries in Austria’. Tierarztliche Umschau 48(11), 706–713. barnhart, h. m., d. w. dreesen, et al. (1993). ‘Isolation of Salmonella from ovaries and oviducts from whole carcasses of spent hens.’ Avian Diseases 37(4), 977–980. beatty, m. e., g. shevick, et al. (2009). ‘Large Salmonella Enteritidis outbreak with prolonged transmission attributed to an infected food handler, Texas, 2002.’ Epidemiology and Infection 137(3), 417–427. Published by Woodhead Publishing Limited, 2011
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Detection and monitoring of Salmonella in laying hen flocks 101 bichler, l. a., k. v. nagaraja,
et al. (1996). ‘Salmonella enteritidis in eggs, cloacal swab specimens, and internal organs of experimentally infected White Leghorn chickens.’ American Journal of Veterinary Research 57(4), 489–495. buhr, r. j., l. j. richardson, et al. (2007). ‘Comparison of four sampling methods for the detection of Salmonella in broiler litter.’ Poultry Science 86(1), 21–25. byrd, j. a., d. e. corrier, et al. (1997). ‘Comparison of drag-swab environmental protocols for the isolation of Salmonella in poultry houses.’ Avian Diseases 41(3), 709–713. caldwell, d. j., b. m. hargis, et al. (1994). ‘Predictive value of multiple drag-swab sampling for the detection of Salmonella from occupied or vacant poultry houses.’ Avian Diseases 38, 461–466. cannon, r. m. and t. j. nicholls (2002). ‘Relationship between sample weight, homogeneity, and sensitivity of fecal culture for Salmonella enterica.’ Journal of Veterinary Diagnostic Investigation 14, 60–62. carr, l. e., e. t. mallinson, et al. (1995). ‘Prevalence of Salmonella in broiler flocks: effect of litter water activity, house construction, and watering devices.’ Avian Diseases 39(1), 39–44. carrique-mas, j. j. and r. davies (2008a). ‘Salmonella Enteritidis in commercial laying flocks in Europe: legislative background, on-farm sampling and main challenges.’ Brazilian Journal of Poultry Science 10(1), 1–9. carrique-mas, j. j. and r. h. davies (2008b). ‘Sampling and bacteriological detection of Salmonella in poultry and poultry premises: a review.’ OIE Scientific and Technical Review 27(3), 665–677. carrique-mas, j. j., m. breslin, et al. (2008). ‘Comparison of environmental sampling methods for detecting Salmonella in commercial laying flocks in the UK.’ Letters in Applied Microbiology 47(6), 514–519. carrique-mas, j. j., m. breslin, et al. (2009a). ‘Persistence and clearance of different Salmonella serovars in buildings housing laying hens.’ Epidemiology and Infection 137, 837–846. carrique-mas, j. j., c. marin, et al. (2009b). ‘A comparison of the efficacy of cleaning and disinfection methods in eliminating Salmonella spp. from commercial egg laying houses.’ Avian Pathology 38(5), 419–424. castellan, d. m., h. kinde, et al. (2004). ‘Descriptive study of california egg layer premises and analysis of risk factors for Salmonella enterica serotype Enteritidis as characterized by manure drag swabs.’ Avian Diseases 48(3), 550–561. cowling, d. w., i. a. gardner, et al. (1999). ‘Comparison of methods for estimation of individual-level prevalence based on pooled samples.’ Preventive Veterinary Medicine 39(3), 211–225. cox, n. a. and m. e. berrang (2000). ‘Inadequacy of selective plating media in field detection of Salmonella.’ Journal of Applied Poultry Research 9(3), 403–406. davies, r. and m. breslin (2004). ‘Observations of Salmonella contamination of eggs from infected commercial laying flocks where vaccination for Salmonella enterica serovar Enteritidis had been used.’ Avian Pathology 33(2), 133–144. davies, r. h. (2005) Pathogen populations on poultry farms. In: Food safety control in the poultry industry, (Ed.) G.C. Mead, Cambridge, Woodhead Publishing, 101–152. davies, r. h. and m. breslin (2001). ‘Environmental contamination and detection of Salmonella enterica serotype Enteritidis in laying flocks.’ Veterinary Record 149, 699–704. davies, r. h. and m. breslin (2003a). ‘Effects of vaccination and other preventive methods for Salmonella Enteritidis on commercial laying chicken farms.’ Veterinary Record 153, 673–677. davies, r. h. and m. breslin (2003b). ‘Persistence of Salmonella Enteritidis phage type 4 in the environment and arthropod vectors on an empty free-range chicken farm.’ Environmental Microbiology 5(2), 79–84. davies, r. h. and c. wray (1996a). ‘Persistence of Salmonella Enteritidis in poultry units and poultry feed.’ British Poultry Science 37, 589–596. Published by Woodhead Publishing Limited, 2011
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102 Improving the safety and quality of eggs and egg products davies, r. h. and c. wray (1996b). ‘Determination of an effective sampling regime to detect
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Detection and monitoring of Salmonella in laying hen flocks 103 the environment of experimentally infected laying hens by an electrostatic sampling device.’ Avian Diseases 48(1), 148–154. golden, n. j., h. h. marks, et al. (2008). ‘Review of induced molting by feed removal and contamination of eggs with Salmonella enterica serovar Enteritidis.’ Veterinary Microbiology 131(3–4), 215–228. gradel, k. o., j. andersen, et al. (2002). ‘Comparisons of sampling procedures and time of sampling for the detection of Salmonella in Danish infected chicken flocks raised in floor systems.’ Acta Veterinaria Scandinavica 43, 21–30. harvey, r. w. s. and t. h. price (1974). Isolation of Salmonellas. Public Health Laboratory Service, Monograph Series 8. London, UK, Her Majesty’s Stationery Office, 2–16. hayes, j. r., l. e. carr, e. t. mallinson, et al. (2000) ‘Characterization of the contribution of water activity and moisture content to the population distribution of Salmonella spp. in commercial poultry houses’. Poultry Science 79(11), 1557–1561. haysom, i. w. and k. sharp (2003). ‘The survival and recovery of bacteria in vacuum cleaner dust.’ Journal of the Royal Society of Health 123(1), 39–45. hildebrandt, g. and l. bohmer (1998). ‘Sampling plans for the assessment of Salmonella contamination.’ Fleischwirtschaft 78(4), 342–343. himathongkham, s., s. nuanualsuwan, et al. (1999). ‘Survival of Salmonella Enteritidis and Salmonella Typhimurium in chicken manure at different levels of water activity.’ FEMS Microbiology Letters 172(2), 159–163. holt, p. s., b. w. mitchell, et al. (1998). ‘Airborne horizontal transmission of Salmonella Enteritidis in molted laying chickens.’ Avian Diseases 42(1), 45–52. humbert, f., p. pommier, et al. (1995). ‘The evolution of microbial contamination of litter in a pen of broilers.’ Recueil de Medecine Veterinaire 171(8–9), 553–557. huneau-salaun, a., c. marianne, et al. (2009). ‘Risk factors for Salmonella enterica subsp. enterica contamination in 519 French laying hen flocks at the end of the laying period.’ Preventive Veterina Medicine 89(1–2), 51–58. inoue, a. y., a. berchieri, et al. (2008). ‘Passive immunity of progeny from broiler breeders vaccinated with oil-emulsion bacterin against Salmonella Enteritidis.’ Avian Diseases Medicine 52(4), 567–571. jarquin, r. l., g. m. nava, et al. (2007). ‘The evaluation of organic acids and probiotic cultures to reduce Salmonella enteritidis horizontal transmission and crop infection in broiler chickens.’ International Journal of Poultry Science 6(3), 182–186. jarvis, b. (2007). ‘On the compositing of samples for qualitative microbiological testing.’ Letters in Applied Microbiology 45, 592–598. johansson, t. m., r. schildt, et al. (1996). ‘The first Salmonella Enteritidis phage type 1 infection of a commercial layer flock in Finland.’ Acta Veterinaria Scandinavica 37(4), 471–479. jordan, d., t. vancov, et al. (2004). ‘The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture.’ Journal of Applied Microbiology 97(5), 1054–1062. kawasaki, t., m. t. musgrove, et al. (2008). ‘Comparative study of shell swab and shell crush methods for the recovery of Salmonella from shell eggs.’ Journal of Food Safety 28(4), 482–498. kinde, h., d. h. read, et al. (1996). ‘Salmonella Enteritidis, phase type 4 infection in a commercial layer flock in southern California: bacteriologic and epidemiologic findings.’ Avian Diseases 40(3), 665–671. kinde, h., d. m. castellan, et al. (2005). ‘Longitudinal monitoring of two commercial layer flocks and their environments for Salmonella enterica serovar Enteritidis and other salmonellae.’ Avian Diseases 49(2), 189–194. kingston, d. j. (1981). ‘A comparison of culturing drag swabs and litter for identification of infections with Salmonella spp. in commercial chicken flocks.’ Avian Diseases 25, 513–516.
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104 Improving the safety and quality of eggs and egg products kivelä, s.-l., o. ruoho,
et al. (1999). ‘Pooled faecal samples compared with individual samples for detection of Salmonella in cattle.’ The Bovine Practitioner 33(1), 74–75. kotton, c. n., a. j. lankowski, et al. (2006). ‘Comparison of rectal swabs with fecal cultures for detection of Salmonella Typhimurium in adult volunteers.’ Diagnostic Microbiology and Infectious Disease 56(2), 123–126. kwon, y. m., c. l. woodward, et al. (2000). ‘Litter and aerosol sampling of chicken houses for rapid detection of Salmonella Typhimurium contamination using gene amplification.’ Journal of Industrial Microbiology & Biotechnology 24, 379–382. lapuz, r., h. tani, et al. (2008). ‘The role of roof rats (Rattus rattus) in the spread of Salmonella Enteritidis and S. Infantis contamination in layer farms in eastern Japan.’ Epidemiology and Infection 136(9), 1235–43. li, x., j. b. payne, et al. (2007). ‘Salmonella populations and prevalence in layer feces from commercial high-rise houses and characterization of the Salmonella isolates by serotyping, antibiotic resistance analysis, and pulsed field gel electrophoresis.’ Poultry Science 86(3), 591–597. line, j. e., j. s. bailey, et al. (1997). ‘Yeast treatment to reduce Salmonella and Campylobacter populations associated with broiler chickens subjected to transport stress.’ Poultry Science 76(9), 1227–1231. mahe, a., s. bougeard, et al. (2008). ‘Bayesian estimation of flock-level sensitivity of detection of Salmonella spp., Enteritidis and Typhimurium according to the sampling procedure in French laying-hen houses.’ Preventive Veterinary Medicine 84(1–2), 11–26. mallinson, e. t., c. r. tate, et al. (1989). ‘Monitoring poultry farms for Salmonella by dragswab sampling and antigen-capture immunoassay.’ Avian Diseases 33, 684–690. mallinson, e. t., c. e. de rezende, n. l. tablante, et al. (2000) ‘A management technique to identify prime locations of Salmonella contamination on broiler and layer farms’. Journal of Applied Poultry Research 9(3), 364–370. matsumoto, a., m. miyama, s. murakami (2001) ‘Comparison of Salmonella isolation rates in different types of egg-layer hen houses in Chiba, Japan, Avian Diseases 45(1), 195–200. mccrea, b. a., norton, k.s., macklin, j.b., hess, j.b., bilgili, s.f. (2005). ‘Recovery and genetic similarity of Salmonella from broiler house drag swabs versus surgical shoe covers.’ Journal of Applied Poultry Research 14, 694–699. mccrea, b. a., k. s. macklin, et al. (2006). ‘A longitudinal study of Salmonella and Campylobacter jejuni isolates from day of hatch through processing by automated ribotyping.’ Journal of Food Protection 69(12), 2908–2914. methner, u., r. diller, r. reiche, and k. böhland (2006). ‘Occurrence of Salmonellae in laying hens in different housing systems and conclusion for the control. Berl. Munch. Tierarztl. Wochenschr. 119, 467–473. mollenhorst, h., c. j. van woudenbergh, et al. (2005). ‘Risk factors for Salmonella Enteritidis infections in laying hens.’ Poultry Science 84(8), 1308–1313. much, p., e. osterreicher, h. lassnig, et al. (2007). ‘Results of the EU–wide baseline study on the prevalence of Salmonella spp. in holdings of laying hens in Austria’. Archiv Fur Lebensmittelhygiene 58(6), 225–229. mueller-doblies, d., a. sayers, et al. (2009). ‘Comparison of sampling methods to detect Salmonella infection of turkey flocks.’ Journal of Applied Microbiology 107, 635–645. musgrove, m. t. and d. r. jones (2005). ‘Recovery of Salmonella from nest run egg cart shelves.’ Poultry Science 84, 77. namata, h., e. meroc, et al. (2008). ‘Salmonella in Belgian laying hens: An identification of risk factors.’ Preventive Veterinary Medicine 83(3–4): 323–336. nief, v. and r. k. hoop (1998). ‘Detection of Salmonella Enteritidis in suspected flocks of laying hens.’ Schweizer Archiv fur Tierheilkunde 140(2), 70–75.
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Detection and monitoring of Salmonella in laying hen flocks 105 o‘carroll, j. m., p. r. davies,
et al. (1999). ‘Effects of sample storage and delayed secondary enrichment on detection of Salmonella spp in swine feces.’ American Journal of Veterinary Research 60(3), 359–362. opara, o. o., e. t. mallinson, et al. (1992). ‘The effect of exposure, storage times, and types of holding media on the drag-swab monitoring technique for Salmonella.’ Avian Diseases 36, 63–68. pezzoli, l., c. campbell, et al. (2009). ‘A methodological approach to investigating a nationwide clinical specimen contamination problem in England.’ Eurosurveillance 14(23), Available at: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19234. (Accesed 6 August 2009). riemann, h., s. himathongkham, d. willoughby, r. tarbell and r. breitmeyer (1998) ‘A survey for Salmonella by drag swabbing manure piles in California egg ranches’. Avian Diseases 42(1), 67–71. rolfe, d. l., h. p. riemann, et al. (2000). ‘Drag swab efficiency factors when sampling chicken manure.’ Avian Diseases 44, 668–675. seo, k. h., p. s. holt, et al. (2000). ‘Combined effect of antibiotic and competitive exclusion treatment on Salmonella Enteritidis fecal shedding in molted laying hens.’ Journal of Food Protection 63(4), 545–548. seo, k. h., r. e. brackett, et al. (2003). ‘Comparison of homogenization methods for recovering Salmonella Enteritidis from eggs.’ Journal of Food Protection 66(9), 1666–1669. singer, r. s., a. e. mayer, et al. (2009). ‘Do microbial interactions and cultivation media decrease the accuracy of Salmonella surveillance systems and outbreak investigations?’ Journal of Food Protection 72(4), 707–713. skov, m. n., b. carstensen, et al. (1999). ‘Evaluation of sampling methods for the detection of Salmonella in broiler flocks.’ Journal of Applied Microbiology 86(4), 695–700. snow. l., davies, r. h., christiansen, k. h., carrique-mas, j. j., wales, a. d., o’connor, j.l., cook, a.j.c. and evans, s.j. (2007) ‘Survey of the prevalence of Salmonella species on commercial laying farms in the United Kingdom’. Veterinary Record, 161(14), 471–476. tactacan, g. b., w. guenter, et al. (2009). ‘Performance and welfare of laying hens in conventional and enriched cages.’ Poultry Science 88(4), 698–707. toyota-hanatani, y., t. ekawa, et al. (2009). ‘Public health assessment of Salmonella enterica serovar Enteritidis inactivated-vaccine treatment in layer flocks.’ Applied Environmental Microbiology 75(4), 1005–1010. van de giessen, a. w., m. bouwknegt, w. d. c. dam-deisz, et al. (2006). ‘Surveillance of Salmonella spp. and Campylobacter spp. in poultry production flocks in The Netherlands’. Epidemiology and Infection 134(6), 1266–1275. van hoorebeke, s., f. van immerseel, et al. (2009). ‘Faecal sampling underestimates the actual prevalence of Salmonella in laying hen flocks.’ Zoonoses Public Health 56(8), 471–476. van immerseel, f., j. debuck, et al. (2004). ‘Intermittent long-term shedding and induction of carrier birds after infection of chickens early posthatch with a low or high dose of Salmonella Enteritidis.’ Poultry Science 83(11), 1911–1916. vila, b., a. fontgibell, et al. (2009). ‘Reduction of Salmonella enterica var. Enteritidis colonization and invasion by Bacillus cereus var. Toyoi inclusion in poultry feeds.’ Poultry Science 88(5), 975–979. voogt, n., m. raes, et al. (2001). ‘Comparison of selective enrichment media for the detection of Salmonella in poultry faeces.’ Letters in Applied Microbiology 32, 89–92. wales, a., m. breslin, et al. (2006a). ‘Assessment of cleaning and disinfection in Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture techniques.’ Veterinary Microbiology 116(4), 283–293. wales, a., m. breslin, et al. (2006b). ‘Semiquantitative assessment of the distribution of Salmonella in the environment of caged layer flocks.’ Journal of Applied Microbiology 101, 309–318. Published by Woodhead Publishing Limited, 2011
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106 Improving the safety and quality of eggs and egg products wales, a., m. breslin,
et al. (2007). ‘A longitudinal study of environmental salmonella contamination in caged and free-range layer flocks.’ Avian Pathol 36(3), 187–197. wegener, h. c., t. hald, et al. (2003). ‘Salmonella control programs in Denmark.’ Emerging Infectious Diseases 9(7), 774–780. white, p. l., w. schlosser, et al. (1997). ‘Environmental survey of manure drag sampling for Salmonella Enteritidis in chicken layer houses.’ Journal of Food Protection 60, 1189–1193. zewde, b. m., r. robbins, et al. (2009). ‘Comparison of swiffer wipes and conventional drag swab methods for the recovery of Salmonella in swine production systems.’ Journal of Food Protection 72(1), 142–146.
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6 Epidemiology of Salmonella infections in laying hens with special emphasis on the influence of the housing system J. Dewulf, S. Van Hoorebeke and F. Van Immerseel, Ghent University, Belgium
Abstract: From 2012 onwards, housing of laying hens in conventional battery cages will be forbidden in the European Union and only enriched cages and non-cage housing systems such as aviaries, floor-raised, free-range and organic systems will be allowed. Although this ban aims at improving the welfare of laying hens, it has also initiated the discussion on whether there are any adverse consequences of this decision, especially with respect to the spread and/or persistence of zoonotic agents in a flock. A zoonotic agent that is traditionally associated with the consumption of eggs and egg products is Salmonella Enteritidis. This chapter provides a summary of the current knowledge regarding the direct and indirect effects of different housing systems on the occurrence and epidemiology of Salmonella in laying hen flocks. Key words: laying hens, Salmonella, housing system.
6.1 Introduction The European Union took a leading role in the debate on laying hens’ welfare by adopting Council Directive 1999/74/EC, stating that from 1 January 2012 onwards the housing of laying hens in conventional battery cages will be forbidden in all EU member states. This decision was inspired by a growing consumer aversion to eggs produced by laying hens housed in battery cages (Appleby, 2003). From 2012 onwards the housing of laying hens in the EU will be restricted to enriched cages and non-cage systems. The housing in enriched cages implies that the hens must have at least 750 cm2 of floor space per hen, a nest, perches and litter. A wide variety of group sizes exist © Woodhead Publishing Limited, 2011
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108 Improving the safety and quality of eggs and egg products in enriched cages (EFSA, 2005). The non-cage systems consist of an indoor area either or not combined with covered (‘wintergarden’) or uncovered (‘free-range’) outdoor facilities (EFSA, 2005; LayWel, 2006). Two main categories can be distinguished: single level systems where the ground floor is fully or partially covered with litter and aviaries with a ground floor area plus one or more platforms (EFSA, 2005; LayWel, 2006). The influences of these alternatives for conventional battery cages on laying hen welfare, productivity and usability have been extensively evaluated and discussed (Abrahamsson and Tauson, 1995; Tauson et al., 1999; Tauson, 2002; Rodenburg et al., 2005, 2008). One aspect of laying hen’s husbandry that needs also to be taken into account in this context is the influence of the housing system on the bacteriological integrity of eggs and egg products in relation to zoonotic pathogens. One of these pathogens is Salmonella, a bacterium that, worldwide, is still a very important cause of human disease (EFSA, 2009). In the EU, Salmonella Enteritidis and Salmonella Typhimurium are the two most commonly isolated serotypes in case of human salmonellosis (EFSA, 2009). Typically for S. Enteritidis, eggs and egg-related products are the main sources of infection for humans (Crespo et al., 2005; De Jong and Ekdahl, 2006; Vaillant et al., 2005). Recent data from Sweden and Switzerland show an increase in the incidence of bacterial diseases in laying hens since the Swedish and Swiss ban on conventional battery cages (Fossum et al., 2009; Kaufmann-Bart and Hoop, 2009). This is not surprising since one of the big advantages of conventional battery cages is that the risk of disease transmission through faeces can be minimized because hens are separated from their faeces (Duncan, 2000). This inevitably leads to the question whether the same effect is to be expected for zoonotic pathogens such as Salmonella. The aim of this chapter is to review the currently available information on the direct and indirect effects of the used housing system on the occurrence and epidemiology of Salmonella in laying hen flocks.
6.2 Effect of the housing system on Salmonella prevalence A number of observational and experimental studies evaluating the effect of the housing system on the prevalence of Salmonella in laying hens have been published in the past 15 years. As is often the case, there were large differences in sample size and methodology used and also the conclusions of these studies differed greatly, going from a preventive effect of the conventional battery cage system over no influence up to a higher risk of Salmonella in battery cages in comparison to non-cage systems (Table 6.1). In this chapter an overview of all published results originating from field surveys will be presented. The study of Mollenhorst and co-workers (2005) is the only study showing a significantly lower Salmonella Enteritidis prevalence in laying © Woodhead Publishing Limited, 2011
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Epidemiology of Salmonella infections in laying hens 109 Table 6.1 An overview of all published observational studies evaluating the effect of the housing system on the prevalence of Salmonella in laying hen flocks Comparison
No. of flocks
Odds ratio
95% CI
Comment
Reference
Cage v. deep litter 1642
0.48
NA*
Serology
Mollenhorst et al. (2005)
Cage v. free-range 34
0.61
0.15–2.34
–
Schaar et al. (1997)
Cage v. aviary
8
1.28
0.51–3.21
–
Pieskus et al. (2008)
Cage v. non-cage
329
2.34
1.42–3.85
EFSA baseline Methner et al. study (2006)
Cage v. non-cage
292
4.69
1.85–11.90
–
Cage v. non-cage
3768
5.12
4.07–6.45
EFSA baseline EFSA (2007) study
Van Hoorebeke et al. (2010)
Cage v. free-range 148 Cage v. floor-raised 148
10.27 2.13–49.57 EFSA baseline Namata et al. 20.11 2.52–160.49 study (2008) EFSA baseline Namata et al. study (2008)
Cage v. floor-raised 519
35.1
12.2–101.1
EFSA baseline Huneau-Salaün study et al. (2009)
*Could not be calculated due to lack of data.
hens housed in conventional battery cage systems in comparison with deep litter systems. However, a few considerations need to be made. First, this protective effect of battery cage systems was observed only on farms with hens of different ages. On farms where all hens were of the same age, the protective effect was restricted to battery cage systems with wet manure, whereas flocks housed in battery cage systems with dry manure had a higher chance of infection with S. Enteritidis compared with deep litter systems. A possible explanation could be that the process of drying manure using hot air enhances the airborne transmission of S. Enteritidis. Second, the categorization of the sampled flock into S. Enteritidis positive or negative was based on serology rather than on the bacteriological isolation of the pathogen. Prior to the above mentioned study, Garber et al. (2003) found that flocks that had been primarily floor reared as pullets were more likely to be positive for S. Enteritidis during their productive lifespan than were flocks that had been cage reared. A number of studies were not able to demonstrate any significant influence of the housing system on the prevalence of Salmonella. Based on the bacteriological analysis of faeces from 34 laying hen flocks, Schaar et al. (1997) did not detect any significant difference in Salmonella prevalence between battery cages and floor-raised systems. However, when testing the
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110 Improving the safety and quality of eggs and egg products flocks with a commercial enzyme-linked immunosorbent assay (ELISA), more positive flocks were found in the floor-raised systems. Hald et al. (2002) reported that the results of the serological monitoring of Danish laying hen flocks (from January 1998 through March 2000) suggested that the housing type had no influence on the prevalence of Salmonella in table eggs. In a more recent study of Pieskus et al. (2008) 47 laying hen flocks from eight different farms were bacteriologically tested. Also in this study no significant difference could be seen between hens housed in conventional battery cages, enriched cages and aviaries. In a retrospective epidemiological study in 2002, Mølbak and Neimann found that eggs from conventional battery cages yielded a higher risk for infection of humans with S. Enteritidis than eggs from non-cage housing systems. Later on, several other studies corroborated the theory that keeping laying hens in battery cages is a significant risk factor for the presence of Salmonella in laying hen flocks (Methner et al., 2006; EFSA, 2007; Namata et al., 2008; Huneau-Salaün et al., 2009; Van Hoorebeke et al., 2010). It has to be mentioned that the sampling of the first three publications were all performed in 2004–2005 in the framework of the European Baseline Study on the prevalence of Salmonella in laying hen flocks (EFSA, 2007). The aim of this baseline study was to determine the prevalence of Salmonella spp. in laying hen flocks in all European member states and Norway and to determine risk factors for the presence of Salmonella on laying hen farms. Both on the EU level and on the level of the individual member states the housing in conventional battery cages turned out to be a risk factor. The study of Van Hoorebeke et al. (2010) on the other hand was specifically designed to investigate the influence of the housing type on the prevalence of Salmonella on laying hen farms. For this purpose five main housing types – conventional battery cages, aviaries, floor-raised systems, free-range systems and organic systems – were sampled in equal proportions. In total, 292 laying hen flocks from 292 different laying hen farms in Belgium, Germany, Greece, Italy and Switzerland were sampled. A summary of the observational studies for which an estimation of the odds ratio was available or could be calculated based on the presented data is given in Table 6.1. The number of flocks included in the separate studies is mentioned to give an indication of the magnitude of the study. It needs to be stressed that the results in Table 6.1 are only indicative and one should be very cautious in comparing the results of the individual studies since both study designs and housing types compared change between studies. Because of the large heterogeneity in the study objectives and designs of the above data, it is difficult to draw one consistent conclusion on the influence of the housing system on the prevalence of Salmonella. Nevertheless, the majority of the studies indicate that housing of laying hens in conventional battery cages significantly increases the risk of detecting Salmonella compared to the housing in non-cage housing systems. Therefore it is reasonable to assume that it is highly unlikely that the move from conventional battery cages to
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Epidemiology of Salmonella infections in laying hens 111 enriched cages and non-cage systems will result in an increase in Salmonella infections and shedding in laying hen flocks. However, in most of the above cited studies it is also stated that the observed influence of the housing type does not necessarily mean that there is a causal relationship between the housing system and the level of Salmonella infection and excretion. On the contrary, it is more likely that the housing system is strongly entangled with several other production characteristics such as the farm and the flock size, the age of the building, previous Salmonella infections on the farm, etc. In the following section a number of laying hen husbandry characteristics which may be related to both the housing system and the probability of a Salmonella infection are discussed.
6.3 Factors related to housing system and Salmonella prevalence 6.3.1 Farm and flock size Independently from the housing system, number of flocks on the farm and number of hens in a flock are significant risk factors for Salmonella infections in laying hens (Mollenhorst et al., 2005; EFSA, 2007; Snow et al., 2007; Carrique-Mas et al., 2008; Huneau-Salaün et al., 2009). Several studies have shown that conventional battery cage farms are in general larger farms, not only with more hens per flock but also with more flocks on the farm (EFSA, 2007; Carrique-Mas et al., 2008; Van Hoorebeke et al., 2010). This could be one of the confounding factors of conventional battery cage farms being more frequently positive for Salmonella than non-cage housing systems. The presence of multiple flocks on one farm may enhance cross-contamination from one flock to another, especially when the different flocks and laying hen houses on the farm are linked through egg conveyor belts, feed pipes, passageways, etc. (Carrique-Mas et al., 2008). Furthermore, as is often the case on farms with multiple flocks, not all the hens are of the same age. Multistage production has also been identified as a risk factor for Salmonella in laying hens (Mollenhorst et al., 2005; Wales et al., 2007; Carrique-Mas et al., 2008; Huneau-Salaün et al., 2009). It is worth mentioning that the likelihood of persistence, in contrast to the probability of infection, does not seem to be significantly related to the number of hens in the flock (Carrique-Mas et al., 2009a), suggesting that a larger flock size especially increases the risk of introduction of the infection. 6.3.2 Stocking density The stocking density is often related to both the housing type and the flock size. For many infectious diseases in production animals it has been
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112 Improving the safety and quality of eggs and egg products demonstrated that a higher stocking density increases the prevalence of disease and the ease of spread (Dewulf et al., 2007). With reference to Salmonella, it has been shown in pigs that higher stocking densities increase the risk of Salmonella infections (Funk et al., 2001; Nollet et al., 2004). However, to our knowledge no results on this parameter are available regarding Salmonella infections in laying hens. Maybe this is due to the fact that this parameter was never evaluated or that it has been studied but never had been found to be significantly influential. Possibly the high density of laying hens in conventional battery cages and in connection with this the large volume of faeces and dust increases the incidence of Salmonella infections in this type of housing (Davies and Breslin, 2004). High stocking densities could also indirectly interact with Salmonella infections because of the stress caused. Yet, literature is not unequivocal on the influence of the stocking density on stress in laying hens and the interaction with the different housing types (Craig et al., 1986; Koeckelbeck et al., 1987; Davis et al., 2000). 6.3.3 Stress Stress is shown to have an immunosuppressive effect in laying hens (El-Lethey et al., 2003; Humphrey, 2006), which can have negative consequences with respect to Salmonella infection and shedding. There are several moments in the laying hen’s life where the bird is subjected to stress: re-housing from the rearing site to the egg producing plant (Hughes et al., 1989), the onset of lay (Jones and Ambali, 1987; Humphrey, 2006), final stages of the production period, thermal extremes (Thaxton et al., 1974; Marshally et al., 2004) or transportation to the slaughterhouse (Beuving and Vonder, 1978). Typical for laying hen husbandry is the practice of induced moulting. The effects on S. Enteritidis infections during moult are extensively studied: moulted hens shed more S. Enteritidis in their eggs and faeces (Holt, 2003; Golden et al., 2008), have higher levels of internal organ colonization (Holt et al., 1995) and cause the recurrence of previous S. Enteritidis infections (Holt and Porter, 1993). There are some contradictory data on the influence of the housing type on the stress levels in laying hens. Some studies suggest that laying hens have less stress in conventional battery cages (Koeckelbeck et al., 1987; Craig et al., 1986) whereas other authors state that hens housed in non-cage systems experience less stress (Hansen et al., 1993; Colson et al., 2008). With regard to the housing system, the age of the hens (Singh et al., 2009) and the breed of the hens could also play a role: certain hen breeds exhibit significantly higher stress responses when raised in deep litter versus free-range systems, compared to other breeds (Campo et al., 2008). Based on the few studies exploring the stress response of hens housed in different housing systems, no consistent conclusion on the influence of the housing system can be drawn.
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Epidemiology of Salmonella infections in laying hens 113 6.3.4 Carry-over infections and age of the infrastructure It is stated that the major part of Salmonella-infections on laying hen farms are not newly introduced on the farm but are the result of re-introduction of the pathogen from the farm’s environment (van de Giessen et al., 1994; Gradel et al., 2004; Carrique-Mas et al., 2009a). This observation underlines the importance of an adequate cleaning and disinfection policy. Nevertheless, because of their intrinsically complicated structures, laying hen houses are notoriously difficult to clean and disinfect (Wales et al., 2006). In particular conventional battery cage systems are thought to be extremely hard to clean and disinfect sufficiently because of the restricted access to cage interiors, feeders, egg belts, and so forth (Davies and Breslin, 2003a; Carrique-Mas et al., 2009b). Since it is believed that the challenge to newly introduced birds is dose dependent, the potential of a more efficient cleaning and disinfection process in non-cage housing system may result in a lower infection level, thus decreasing the risk of carry-over of infections between production rounds. Besides the specific difficulties in cleaning and disinfecting in the different housing systems, also the age of the current infrastructure might play a role. Owing to the wear of the materials and the inherent difficulties to thoroughly clean and disinfect them, older equipments increase the risk for Salmonella. In general, conventional battery cages are older than floor-raised, free-range and organic installations (Van Hoorebeke et al., 2010). This finding could also contribute to the fact that farms with conventional battery cages are more frequently found positive for Salmonella. 6.3.5 Pests As mentioned above, the prevention of re-introduction of Salmonella in a laying hens’ house after repopulation with a Salmonella-free replacement stock is one of the big challenges of modern laying hen husbandry. The role as vectors in the transfer of Salmonella has been extensively discussed for rodents, flies and beetles (Guard-Petter, 2001; Davies and Breslin, 2003a; Kinde et al., 2005; Carrique-Mas et al., 2009a). It has been suggested that non-cage housing systems present a less attractive environment to these pests because laying hens can interfere more with their movements since the birds are not restrained to cages (Carrique-Mas et al., 2009a). Another very important pest in laying hens’ houses is the poultry red mite (Dermanyssus gallinae). It has been shown under experimental conditions that mites could play a role in the persistence of Salmonella in laying hens, either by transferring the bacterium from hen to hen or by hens consuming contaminated mites, leading to a persisting infection (Valiente-Moro et al., 2007, 2009). Furthermore mass red mite infestations can lead to immunosuppression (Kowalski and Sokol, 2009), increasing the susceptibility for infections. This could also be the case with gastrointestinal helminths: it has been described that the prevalence of helminth infections in free-range and deep litter systems is much higher than in conventional battery cage systems (Permin et al., 1999;
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114 Improving the safety and quality of eggs and egg products Marcos-Atxutegi et al., 2009). The poor body condition the birds suffer from under such circumstances makes them in turn more susceptible to Salmonella infections. 6.3.6 Vaccination The use of vaccination against Salmonella has beyond doubt a significant protective influence on shedding of Salmonella in laying hen flocks since the currently available vaccines reduce both the shedding and colonization of the reproductive tract, leading to a decrease in the number of internally contaminated eggs (Gantois et al., 2006). Despite the decrease in colonization levels, Salmonella can still be found in the caeca and reproductive tract of a fairly large proportion of vaccinated hens in low numbers (Davies and Breslin, 2004; Van Hoorebeke et al., 2009) which implies the risk of a renewed shedding of the pathogen, especially at moments of stress, as mentioned above. Thus, even in vaccinated flocks there is still some contamination risk associated with the presence of S. Enteritidis in infected vaccinated laying hens (Davies and Breslin, 2004). Davies and Breslin (2003b) reported disappearance of Salmonella in freerange flocks vaccinated with Salmonella Enteritidis, but not among cage flocks, an observation that could be indicative of the lower challenge to the hens in free-range conditions. On the other hand, the move to non-cage housing systems is thought not to have any impact on the effectiveness of live and killed vaccines (P.S. Holt, personal communication).
6.4 Presence of Salmonella serotypes other than S. Enteritidis in outdoor production systems To date, the focus of Salmonella control has been mainly on S. Enteritidis and S. Typhimurium, because these two serotypes are responsible for the lion’s share of human salmonellosis cases in Europe and North America (Centers for Disease Control and Prevention, 2007; EFSA, 2009). Nevertheless some differences in epidemiology are reported between these two serotypes. Because S. Typhimurium is much more common in wildlife, pigs and cattle, it has been stated that free-range layer flocks will be at greater risk of becoming infected with S. Typhimurium than flocks housed in systems without an outdoor-run (Carrique-Mas and Davies, 2008). However, this could not be confirmed in the EFSA baseline study (EFSA, 2007) or in another large-scale study in Belgium, Germany, Greece, Italy and Switzerland (Van Hoorebeke et al., 2010).
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Epidemiology of Salmonella infections in laying hens 115
6.5
Conclusions
Based on the epidemiological data provided above it is highly unlikely that the move from conventional battery cages to enriched cages and non-cage systems will result in an increase in Salmonella infections and shedding in laying hen flocks. However, the true underlying mechanisms causing the prevalence of Salmonella to be generally lower in alternative housing systems remain unknown and hen breed, age of the infrastructure, disease status of the flock, rodent and insect load all contribute to the complexity of this issue. This indicates that, also in housing systems others than conventional battery cages, factors such as biosecurity, vaccination and professional skills of the farmer are of utmost importance to minimize the presence of Salmonella in laying hen flocks.
6.6 Acknowledgement This research was funded by the EU FP6, under the contract 035547 (Safehouse project).
6.7 References abrahamsson p.
and tauson r., 1995. Aviary systems and conventional cages for laying hens. Acta. Agric. Scand. 45: 191–203 appleby m.c., 2003. The European Union ban for conventional cages for laying hens: history and prospects. J. Appl. An. Welfare 6: 103–121 beuving g. and vonder g.ma., 1978. Effects of stressing factors on corticosterone levels in the plasma of laying hens. Gen. Comp. Endocrinol. 35: 153–159 campo j.l., prieto m.t. and davila s.g., 2008. Effects of housing system and cold stress on heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration of chickens. Poult. Sci. 87: 621–626 carrique-mas j.j. and davies r.h., 2008. Salmonella Enteritidis in commercial layer flocks in Europe: Legislative background, on-farm sampling and main challenges. Braz. J. Poult. Sci. 10(1): 1–9 carrique-mas j.j., breslin m., snow l., arnold m.e., wales a., laren i. and davies r.h., 2008. Observations related to the Salmonella EU layer baseline survey in the United Kingdom: follow-up of positive flocks and sensitivity issues. Epidemiol. Infect. 136: 1537–1546 carrique-mas j.j., breslin m., snow l., mclaren i, sayers a. and davies r.h., 2009a. Persistance and clearance of different Salmonella serovars in buildings housing laying hens. Epidemiol. Infect. 137: 837–846 carrique-mas j.j., marin c., breslin m., mclaren i. and davies r., 2009b. A comparision of the efficacy of cleaning and disinfection methods in eliminating Salmonella spp. from commercial egg laying houses. Avian Pathol. 38(5): 419–424 centers for disease control and prevention, 2007. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food – 10 states. MMWR 57(14): 366–370 colson s., arnould c. and michel v., 2008. Influence of rearing conditions of pullets of
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116 Improving the safety and quality of eggs and egg products space use and performance of hens placed in aviaries at the beginning of the laying period. Appl. Anim. Behav. Sci. 111: 286–300 council directive 1999/74/EC on 19 July 1999 laying down minimum standards for the protection of laying hens. Off. J. Eur. Communities L203: 53–57 craig j.v., craig j.a. and vargas j.v., 1986. Corticosteroids and other indicators of hens well-being in four laying-house environments. Poult. Sci. 65: 856–863 crespo p.s., hernandeze g., echeita a., torres a., ordonez p. and aladuena a., 2005. Surveillance of foodbourne disease outbreaks associated with consumption of eggs and egg products: Spain, 2002–2003. Eurosurveillance Weekly 10(6). Available online at http://www.eurosurveillance.org/ew/2005/050616.asp davies r. and m. breslin m. 2003a. Observations on Salmonella contamination of commercial laying farms before and after cleaning and disinfection. Vet. Rec. 152: 283–287 davies r. and breslin m., 2003b. Effects of vaccination and other preventive methods for Salmonella enteritidis on commercial laying chicken farms. Vet. Rec. 153: 673–677 davies r. and breslin m., 2004. Observations on Salmonella contamination of eggs from infected commercial laying flocks where vaccination for Salmonella enterica serovar Enteritidis had been used. Avian Path. 33(2): 133–144 davis g.s., anderson k.e. and carroll a.s., 2000. The effects of long-term caging and molt of single comb White Leghorn hens on heterophil to lymphocyte ratios, corticosteroid and thyroid hormones. Poult. Sci. 79: 514–518 de jong b. and ekdahl k., 2006. Human salmonellosis in travelers is highly correlated to the prevalence of Salmonella in laying hen flocks. Eurosurveillance 2006 11(7): E0607061 dewulf j., tuyttens f., lauwers l., van huylebroeck g. and maes d., 2007. Influence of pen density on pig meat production, health and welfare. Vlaams Diergen. Tijdschr. 76: 410–416 duncan i.j.h., 2000. The pros and cons of cages. Proceedings of the XXI World Poultry Congress, Montreal, Canada efsa, 2005. Welfare aspects of various systems for keeping laying hens. Scientific Report: p 143. Annex of the EFSA J. 197: 1–23 efsa, 2007. Report of the Task Force on Zoonoses Data Collection on the analysis of the baseline study on the prevalence of Salmonella in holdings of laying hen flocks of Gallus gallus. EFSA J. 97, 84 pp efsa, 2009. The Community Summary Report on trends and sources of zoonoses and zoonotic agents in the European Union in 2007. EFSA J., 223 el - lethey h ., huber - eicher b . and jungi t . w ., 2003. Exploration of stress-induced immunosuppression in chickens reveals both stress-resistant and stress-susceptible antigen responses. Vet. Immunol. Immunopathol. 95: 91–101 fossum o., jansson d.s., etterlin p.e. and vagsholm i., 2009. Causes of mortality in laying hens in different housing systems in 2001 to 2004. Acta Vet. Scan. 51: 3 funk j.a., davies p.r. and gebreyes w., 2001. Risk factors associated with Salmonella enteric prevalence in three-site swine production systems in North-Carolina, USA. Berl. Munch. Tierartzl. Wochenschr. 114: 335–338 gantois i., ducatelle r., timbermont l., boyen f., bohez l., haesebrouck f., pasmans f. and van immerseel f., 2006. Oral immunization of laying hens with the live vaccine strains of TAD Salmonella vac® E and TAD Salmonella vac® T reduces internal egg contamination with Salmonella Enteritidis. Vaccine 24: 6250–6255 garber l., smeltzer m., fedorka-cray p., ladely s. and ferris k., 2003. Salmonella entrica serotype enteritidis in table egg layer house environments and in mice in U.S. layer houses and associated risk factors. Avian Dis. 47: 134–142 golden n.j., marks h.m., coleman m.e., schroeder c.m., bauer jr. n.e. and schlosser w.d., 2008: Review of induced molting by feed removal and contamination of eggs with Salmonella enterica serovar Enteritidis. Vet. Microbiol. 131(3–4), 215–228. gradel k.o., sayers a.r. and davies r.h., 2004. Surface disinfection tests with Salmonella
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Epidemiology of Salmonella infections in laying hens 117 and a putative indicator bacterium, mimicking worst-case scenario’s in poultry houses. Poult. Sci. 83: 1636–1646 guard-petter j., 2001. The chicken, the egg and Salmonella enteritidis. App. Envir. Microbiol. 3: 421–430 hald t., kabell s. and madsen m., 2002. The influence of production on the occurrence of Salmonella in the Danish table-egg production. In Food Safety Assurance in the Pre-harvest Phase, Volume 1, edited by: Frans J.M. Smulders and John D. Collin Wageningen Academic Publishers, Wageningen hansen i., braastad b.o., storbraten j. and tofastrud m., 1993. Differences in fearfulness indicated by tonic immobility between laying hens in aviaries and cages. Anim. Welf. 2: 105–112 holt p.s., 2003. Molting and Salmonella Enterica serovar Enteritidis infection: The problem and some solutions. Poult. Sci. 82: 1008–1010 holt p.s. and porter r.e. jr., 1993. Effect of induced molting on the recurrence of a previous Salmonella enteritidis infection. Poult. Sci. 72: 2069–2078 holt p.s., macri n.p. and porter r.e. jr., 1995. Microbiological analysis of the early Salmonella enteritidis in molted and unmolted hens. Avian Dis. 39: 55–63 hughes c.s., gaskell r.m., jones r.c., bradbury j.m. and jordan f.t.w., 1989. Effects of certain stress factors on the re-excretion of infectious laryngotracheitis virus from latently infected carrier birds. Res. Vet. Sci. 46: 274–276 humphrey t., 2006. Are happy chickens safer chickens? Poultry welfare and disease susceptibility. Brit. Poult. Sci. 47(4): 379–391 huneau-salaün a., chemaly m., le bouquin s., lalande f., petetin i., rouxel s., michel v., fravallo p. and rose n., 2009. Risk factors for Salmonella enterica subsp. enterica contamination in 519 French laying hen flocks at the end of the laying period. Prev. Vet. Med. 89(1–2): 51–58 jones r.c. and ambali r.g., 1987. Re-excretion of an entero-tropic infectious-bronchitis virus by hens at point of lay after experimental-infection at day old. Vet. Rec. 120: 117–118 kaufmann-bart m. and hoop r.k., 2009. Diseases in chicks and laying hens during the first 12 years after battery cages were banned in Switzerland. Vet. Rec. 164: 203–207 kinde h., castellan d.m., kerr d., campbell j., breitmeyer r. and ardans a., 2005. Longitudinal monitoring of two commercial layer flocks and their environments for Salmonella enterica serovar Enteritidis and other salmonellae. Avian Dis. 49: 189–194 koelkebeck k.w., amoss m.s. and cain j.r., 1987. Production, physiological and behavioral responses of laying hens in different management environments. Poult. Sci. 63: 2123–2129 kowalski a. and sokol r., 2009. Influence of Dermanyssus gallinae (poultry red mite) invasion on the plasma levels of corticosterone, cathecholamines and proteins in layer hens. Pol. J. Vet. Sci. 12(2): 231–235 laywel, 2006. Welfare implications of changes in production systems for laying hens. Periodic Final Activity Report, LayWel, p 1–22 marcos-atxutegi c., gandolfi b., aranguena t., sepulveda r., arévalo m. and simon f., 2009. Antibody and inflammatory responses in laying hens with experimental primary infections of Ascaridia galli. Vet. Parasitol. 161: 69–75 marshally m.m., hendricks g.l., kalama 3rd m.a., gehad a.e., abbas a.o. and patterson p.h., 2004. Effect of heat stress on production parameters and immune responses of commercial laying hens. Poult. Sci. 83: 889–894 methner u., diller r., reiche r. and böhland k., 2006. Occurrence of Salmonellae in laying hens in different housing systems and conclusion for the control. Münch. Tierartz. Wochenschr. 119, 467–473 mølbak k. and neimann j., 2002. Risk factors for sporadic infection with Salmonella Enteritidis, Denmark 1997–1999. Am. J. Epidemiol. 156: 654–661
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118 Improving the safety and quality of eggs and egg products mollenhorst h., van woudenbergh c.j., bokkers e.g.m. and de boer i.j.m., 2005. Risk factors
for Salmonella Enteritidis infections in laying hens. Poult. Sci. 84: 1308–1313 and mintiens k., 2008. Salmonella in Belgian laying hens: an identification of risk factors. Prev. Vet. Med. 83: 323–336 nollet n., maes d., de zutter l., duchateau l., houf k., huysmans k., imberechts h., geers r., de kruif a. and van hoof j., 2004. Risk factors for the herd-level bacteriologic prevalence of Salmonella in Belgian slaughter pigs. Prev. Vet. Med. 65: 63–75 permin a., bisgaard m., frandsen f., pearman m., kold j. and nansen p., 1999. Prevalence of gastrointestinal helminths in different poultry production systems. Br. Poult. Sci. 40: 439–443 pieskus j., kazeniauskas e., butrimaite-ambrozeviciene c., stanevicius z. and mauricas m., 2008. Salmonella incidence in broiler and laying hens with the different housing systems. J. Poult. Sci. 45: 227–231. rodenburg t.b., tuyttens f., de reu k., herman l., zoons j. and sonck b., 2005. Welfare, health and hygiene of laying hens housed in furnished cages and in alternative housing systems. J. Appl. Anim. Welf. Sci. 8: 211–226 rodenburg t.b., tuyttens f., de reu k., herman l., zoons j. and sonck b., 2008. Welfare assessment of laying hens in furnished cages and non-cage systems: an on-farm comparison. Anim. Welf. 17: 363–373 schaar u., kaleta e.f. and baumbach b., 1997. Comparative studies on the prevalence of Salmonella enteritidis and Salmonella typhimurium in laying chickens maintained in batteries or on floor using bacteriological isolation techniques and two commercially available ELISA kits for serological monitoring. Tierarzlichen Praxis. 25: 451–459 singh r., cook n., cheng k.m. and silversides f.g., 2009. Invasive and noninvasive measurement of stress in laying hens kept in conventional cages and in floor pens. Poult. Sci. 88: 1346–1351 snow l.c., davies r.h., christiansen k.h., carrique-mas j.j., wales d., o’connor j.l., cook a.j.c. and evans s.j., 2007. Survey of the prevalence of Salmonella species on commercial laying farms in the United Kingdom. Vet. Rec. 161: 471–476 tauson r., 2002. Furnished cages and aviaries: production and health. World’s Poult. Sci. J. 58: 49–63 tauson r., wahlström a. and abrahamsson p., 1999. Effect of two floor housing systems and cages on health, production and fear response in layers. J. Appl. Poult. Res. 8: 152–159 thaxton p., wyatt r.d. and hamilton p.b., 1974. The effect of environmental temperature on paratyphoid infection in the neonatal chicken. Poult. Sci. 53: 88–94 valiente moro c., fravalo p., amelot m., chauve c., zenner l. and salvat g., 2007. Colonization and organ invasion in chicks experimentally infected with Dermanyssus gallinae contaminated by Salmonella Enteritidis. Avian Pathol. 36(4): 307–311 valiente moro c., de luna c.j., tod a., guy j.h., sparagano o.a.e. and zenner l., 2009. The poultry red mite (Dermanyssus gallinae): a potential vector of pathogenic agents. Exp. Appl. Acarol. 48: 93–104 vaillant v., de valk h., baron e., ancelle t., colin p., delmas m.c., dufour b., pouillot r., le strat y., weinbreck p., jougla e. and désenclos j.c., 2005. Foodborne infections in France. Foodborn. Path. and Disease. 2(3): 221–232 van de giessen a.w., ament a.j.h.a. and notermans s.h.w., 1994. Intervention strategies for Salmonella enteritidis in poultry flocks: a basic approach. Int. J. Food. Microbiol. 21: 145–154 van hoorebeke s., van immerseel f., de vylder j., ducatelle r., haesebrouck f., pasmans f., de kruif a. and dewulf j., 2009. Faecal sampling underestimates the actual prevalence of Salmonella in laying hen flocks. Zoon. Pub. Health 56: 471–476 van hoorebeke s., van immerseel f., schulz j., hartung j., harisberger m., barco l., ricci a., theodoropoulos g., xylouri e., de vylder j., ducatell r., haesebrouck f., pasmans namata h., méroc e., aerts m., faes c., cortinas abrahantes j., imberechts h.
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Epidemiology of Salmonella infections in laying hens 119 f., de kruif a. and dewulf j., 2010. Determination of the within and between flock prevalence and identification of risk factors for Salmonella infections in laying hen flocks housed in conventional and alternative systems. Prev. Vet. Med. 94: 94–100 wales a., breslin m. and davies r., 2006. Assessment of cleaning and disinfection in Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture techniques. Vet. Microbiol. 116: 283–293 wales a., breslin m., carter b., sayer r. and davies r., 2007. A longitudinal study of environmental Salmonella contamination in caged and free-range layer flocks. Avian Pathol. 36(3): 187–197.
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7 Pre-harvest measures to control Salmonella in laying hens R. K. Gast, United States Department of Agriculture, Agricultural Research Service, USA
Abstract: One of the principal recurring themes in the search for effective responses to the continuing public health and economic problems caused by Salmonella contamination of eggs concerns reducing the susceptibility of egg-laying chickens to infection. Decreasing the overall prevalence or level of Salmonella infection among commercial laying flocks has the potential to exert a corresponding influence on the likelihood that contaminated eggs will be produced and marketed. This chapter considers both the capabilities and limitations of three leading options for reducing the susceptibility of laying hens to Salmonella: vaccination to induce immunity, genetic selection for naturally occurring resistance to infection, and gastrointestinal colonization control. Key words: vaccination, genetic selection, gastrointestinal colonization control.
7.1 Introduction More than two decades have passed since an international surge in the reported incidence of human Salmonella enterica serovar Enteritidis (S. Enteritidis) infections brought the egg-borne transmission of this pathogen into prominence as a leading public health and economic issue. One of the most consistently prominent themes during the search for effective responses to this problem has concerned reducing the susceptibility of egg-laying chickens to S. Enteritidis infection. Any decreases that can be achieved in the overall prevalence of infection among commercial laying flocks, the within-flock incidence of infection, and the average pathogen load harbored or shed by © Woodhead Publishing Limited, 2011
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Pre-harvest measures to control Salmonella in laying hens 121 infected hens might have a corresponding influence on the likelihood that contaminated eggs will be produced and marketed. However, some intrinsic characteristics of Salmonella infections in poultry have posed very significant challenges to control efforts. Newly hatched chicks are highly susceptible to Salmonella colonization, but these infections are often unapparent and difficult to detect in mature hens. Many Salmonella serotypes have extremely wide host ranges and are highly persistent in the environment, creating diverse potential sources of pathogen introduction occurring throughout the productive life cycles of laying flocks. Two principal strategies for improving the resistance of egg-laying chickens to Salmonella infection have already been both extensively researched and widely implemented in commercial flocks. Vaccination to induce immunity has been of particular interest for protecting laying hens against S. Enteritidis infection because of its inherently serotype-specific orientation. Gastrointestinal colonization control, based on a presumably serotype-independent mechanism, has been more often used in broiler flocks but has nonetheless demonstrated its applicability to certain situations encountered in egg production as well. A third strategy, involving genetic selection for Salmonella resistance in egg-type chickens, has not yet reached the stage of widespread commercial implementation but could play an important role in long-term Salmonella control plans. The remainder of this chapter will consider each of these three options for reducing the susceptibility of laying hens to Salmonella infection, in terms of both their capabilities and their limitations, in order to assess their prospective contributions to overall risk reduction programs for protecting consumers against disease transmission from contaminated eggs.
7.2 Vaccination 7.2.1 The immune responses of poultry The naturally occurring immune response to Salmonella reduces the duration and severity of infections and helps to prevent subsequent re-infection. In poultry, this response is the basis for protection against infection by vaccination and for serological detection of infected flocks. High antibody titers have been observed within one week after the experimental inoculation of chickens (Gast and Holt, 2001), resulting in serological positivity that can persist for a full year (Skov et al., 2002). Poultry also mount a cellmediated immune response to Salmonella infection, although this has not been characterized as thoroughly as the antibody response. Avian heterophils are strongly phagocytic and bactericidal for salmonellae (Stabler et al., 1994) and can limit invasion to internal organs during the early stages of infection (Kogut et al., 1994). Both the humoral and cell mediated responses are important for protecting poultry against Salmonella infection, but their relative contributions have not been definitively resolved. The emergence © Woodhead Publishing Limited, 2011
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122 Improving the safety and quality of eggs and egg products of strong antigen-specific cellular or antibody responses has been correlated with the timing of S. Typhimurium clearance in infected chickens (Beal et al., 2004). Likewise, a decline in S. Enteritidis isolation from the reproductive tissues of laying hens was linked to the proliferation of both T and B cells (Withanage et al., 2003). The full expression of immunity may require both the opsonic function of antibodies and the phagocytic and lytic activity of cellular effectors (McSorley and Jenkins, 2000). 7.2.2 The goals and applications for Salmonella vaccination Vaccination of poultry against Salmonella infection has been extensively researched and applied in numerous locations around the world. The objective of vaccination is to reduce the principal manifestations and consequences of Salmonella infection, including the susceptibility of individual birds to infection, the horizontal transmission of infection within flocks, the pathogen load in poultry house environments (and the likelihood of transmission to subsequent flocks), the vertical transmission of infection to progeny of breeding flocks, and the frequency of product contamination and disease transmission to consumers. The vaccination of pullets or hens with either inactivated (killed) or attenuated (live) preparations has usually reduced – although rarely prevented entirely – fecal shedding, organ invasion, and egg contamination after experimental Salmonella challenge (Gast et al., 1992, 1993). Accordingly, both killed and live Salmonella vaccines have been used to immunize commercial flocks. The widespread implementation of a vaccination program for egg-laying hens in the United Kingdom was followed by a significant reduction in the incidence of human S. Enteritidis infections (Cogan and Humphrey, 2003). Vaccinated laying flocks were reported to produce eggs contaminated by S. Enteritidis at lower frequencies even when comparable progress could not be documented in lowering the levels of this pathogen in the environment of laying houses (Davies and Breslin, 2004). In Japanese commercial laying flocks, the frequency of isolation of S. Enteritidis from liquid egg samples declined sharply over time following the initiation of a vaccination program (Toyota-Hanatani et al., 2009a). The protective efficacy of vaccination can be especially significant for laying hens undergoing an induced molt via feed restriction, which can significantly increase their susceptibility to S. Enteritidis infection (Holt et al., 2003; Nakamura et al., 2004). 7.2.3 Killed (inactivated) and subunit vaccines Heightened interest in using killed Salmonella vaccines (bacterins) for poultry followed the emergence of egg-transmitted S. Enteritidis as a major public health issue in the 1980s. Inactivated vaccine preparations are commercially available for several economically and epidemiologically significant serotypes, including various phage types of S. Enteritidis (European Food Safety
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Pre-harvest measures to control Salmonella in laying hens 123 Authority, 2004). Killed vaccines have been shown to protect against diverse parameters of Salmonella infection in numerous laboratory and field studies. Subcutaneous or intramuscular administration of adjuvanted bacterins to laying hens has led to significant reductions in S. Enteritidis isolation from feces, internal tissues, and eggs of orally challenged laying hens (Gast et al., 1992, 1993; Clifton-Hadley et al., 2002). Similarly, chickens vaccinated with bacterins have exhibited reductions in mortality, lesions, clinical signs, organ invasion, and egg contamination for up to 12 weeks post-vaccination when challenged with S. Enteritidis by intravenous or intramuscular routes (Timms et al., 1994; Woodward et al., 2002). Bacterin administration also protected laying hens against increased fecal shedding of S. Enteritidis following the induction of molting by feed restriction (Nakamura et al., 2004). Field trials to evaluate the efficacy of bacterins have reported a reduced incidence of S. Enteritidis infection in Dutch broiler breeder flocks (Feberwee et al., 2000) and consistently negative S. Enteritidis testing results in vaccinated British laying flocks that were transferred into previously contaminated facilities (Davies and Breslin, 2003a). Several characteristics of killed vaccines are advantageous for application in commercial poultry flocks. Autogenous bacterins can be rapidly prepared from specific Salmonella strains and promptly made available to address urgent problems associated with particular locations or enterprises. Another benefit of using killed vaccines is that they do not generate concerns about introducing live strains of human pathogens into food-producing animals. The overall protective capabilities of killed vaccines are somewhat limited by their inability to effectively generate a substantial cell-mediated immune response (Muotiala et al., 1989), possibly due to adverse effects on important immunogenic antigens during bacterial inactivation procedures. High antibody titers do not necessarily ensure a correspondingly high degree of protection against infection (Mizumoto et al., 2006). Another significant limitation associated with killed vaccines is that they provide minimal cross-protection against antigenically unrelated Salmonella serotypes, possibly because bacterins elicit mainly a highly serotype-specific humoral response (Chambers and Lu, 2002). However, multivalent bacterins comprising a mixture of strains or serotypes can expand the spectrum of protection (Okamura et al., 2007). A multivalent killed vaccine stimulated more enduring serum and gut IgG responses than did a live S. Typhimurium vaccine (Bailey et al., 2007c). Vaccinating with a bacterin containing both S. Enteritidis and S. Typhimurium protected against fecal shedding and egg contamination after challenge with either serotype (Okamura et al., 2007). Treatment with a trivalent bacterin has been reported to reduce fecal and cecal recovery of Salmonella, even after heterologous challenge (Deguchi et al., 2009). Other ongoing concerns about bacterins include the local pathologic consequences that can follow parenteral injection of toxic bacterial cell constituents (especially lipopolysaccharides) and oil-emulsion adjuvants, and the labor costs incurred by injecting individual birds.
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124 Improving the safety and quality of eggs and egg products Considerable recent attention has also been focused on the development of vaccines consisting of or expressing very precisely defined antigens. Subunit vaccines composed of Salmonella outer-membrane proteins administered with adjuvants or incorporated into lipid-conjugated immunostimulating complexes offered protection against S. Enteritidis infection (Charles et al., 1994; Khan et al., 2003). Likewise, immunization of laying hens with purified S. Enteritidis fimbria protected against reproductive organ invasion and egg contamination (De Buck et al., 2005). A flagellar subunit vaccine reduced cecal colonization by S. Enteritidis (Toyota-Hanatani et al., 2009b). Vaccination of chickens with a siderophore receptor led to significantly lower mortality following intravenous challenge with S. Enteritidis (Kaneshige et al., 2009). Another relevant consideration for efforts to select or design vaccines (killed, live, and subunit) is that they should ideally present antigens to stimulate an immune response which is protective but which is also serologically distinguishable from the response to naturally occurring infections with wild-type strains (Mizumoto et al., 2004; Adriaensen et al., 2007). 7.2.4 Live (attenuated) vaccines Live (attenuated) vaccines must be able to persist in tissues long enough to induce a protective immune response, but must also be avirulent and eventually be cleared altogether from vaccinated birds. After immunization with a live attenuated S. Enteritidis vaccine and challenge with the wildtype strain, protected birds exhibited higher amounts of both IgA and CD8 + T cells (Carvajal et al., 2008). Live Salmonella vaccine strains, which are typically administered to poultry by an oral route, have been produced using an assortment of attenuation strategies. Administration of aroA mutant strains of S. Enteritidis, deficient in the ability to synthesize certain aromatic compounds required for prolific in vivo growth, has led to significantly lower fecal shedding, horizontal transmission, organ invasion, and egg contamination in chickens challenged with wild-type strains (Cooper et al., 1993, 1996). The observed protective effect continued for nearly six months after the vaccine was given (Cooper et al., 1994). A hilA mutant strain of S. Enteritidis reduced fecal shedding and internal organ invasion after wild-type challenge and also reduced horizontal transmission of infection (Bohez et al., 2008). Administration of a Dcya Dcrp mutant strain of S. Typhimurium (with deletions in genes for both adenylate cyclase and the cyclic AMP receptor protein) effectively protected against intestinal colonization and organ invasion by a wild-type strain (Hassan and Curtiss, 1994). A double deletion S. Enteritidis mutant (nonflagellated and deficient for guanine synthesis) diminished internal organ colonization of chickens after wild-type challenge, yet also allowed serological differentiation between vaccinated and infected birds (Adriaensen et al., 2007). Some other approaches to vaccine strain attenuation that have yielded significant protection against S. Enteritidis infection include using
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Pre-harvest measures to control Salmonella in laying hens 125 a temperature-sensitive mutant (Barrow et al., 1996) and a strain repeatedly passaged through chicken heterophils (Kramer, 1998). Documentation of cross-protection by live Salmonella vaccine strains against other epidemiologically important serotypes has been inconsistent, even for serotypes with some antigenic similarities. One investigation reported that primary infection with either S. Enteritidis or S. Typhimurium led to lymphocyte proliferation, the production of high levels of cross-reactive antibodies, and significant protection against heterologous secondary infection (Beal et al., 2006). However, in other studies, an avirulent S. Typhimurium vaccine reduced colonization, organ invasion, and egg contamination by S. Enteritidis (Hassan and Curtiss, 1997), but attenuated aroA-S. Enteritidis strains did not cross-protect against S. Typhimurium (Cooper et al., 1993). Another frequently discussed (and intensively investigated) live vaccine candidate is the S. Gallinarum 9R strain, which can induce cross-protective immunity against S. Enteritidis but does not interfere with serological detection of infected flocks (Feberwee et al., 2001). Even Salmonella strains sharing no common antigens have sometimes been observed to stimulate partial protection against S. Enteritidis challenge, perhaps via a combination of immune and competitive exclusion mechanisms (Holt and Gast, 2004). In both Europe and the United States, live S. Typhimurium vaccines have been licensed for use and are applied for controlling S. Enteritidis in laying flocks, often in conjunction with the subsequent administration of an S. Enteritidis bacterin to enhance the specificity of the immune response (European Food Safety Authority, 2004). The principal positive attributes of live attenuated vaccines are their induction of strong and persistent immunity and their ease of administration (they are often simply added to drinking water). Live Salmonella vaccines have sometimes been associated with protection of higher efficacy and duration than killed vaccines, perhaps as a result of more persistent presentation of relevant antigens to the host immune system (Babu et al., 2004). However, a few concerns have been raised about the safety of using live Salmonella vaccines, based on evidence that vaccine strains might sometimes be genetically unstable (Barbezange et al., 2000) and that these strains have been detected in vaccinated hens for unacceptably long periods of time after administration when highly sensitive culturing methods were used for testing (Tan et al., 1997). 7.2.5 Treatment with immune mediators Another important avenue of investigation for reducing the susceptibility of poultry to Salmonella involves prophylactic treatment with mediators of immunity. Preventive administration of lymphokines from immunized chickens has protected chicks against organ invasion after subsequent S. Enteritidis challenge (McGruder et al., 1995a). This effect is likely a result of increased phagocytosis and killing of S. Enteritidis by avian heterophils
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126 Improving the safety and quality of eggs and egg products (Desmidt et al., 1998). Subcutaneous, oral, nasal, and in ovo administration of immune lymphokines to poultry have all been found to provide some degree of protection against S. Enteritidis challenge (McGruder et al., 1995b; Kogut et al., 1998), although the duration of this effect can sometimes be relatively transient (Genovese et al., 2000). Cross-protection against other Salmonella serotypes has also been reported for lymphokine treatment (Ziprin and Kogut, 1997). In studies of another prophylactic approach, administering immunitystimulating oligodeoxynucleotides to chicks primed the innate immune response by increasing heterophil degranulation and thereby reduced colonization by S. Enteritidis (He et al., 2007; MacKinnon et al., 2009). Similarly, treatment of chicks with cationic peptides significantly reduced organ invasion after S. Enteritidis challenge by increasing heterophil phagocytosis, oxidative burst, and degranulation (Kogut et al., 2007). 7.2.6 Factors affecting the performance and efficacy of Salmonella vaccines Combined administration of both live and killed vaccines has been reported to reduce S. Enteritidis egg contamination to a greater degree than either individual type of vaccine (Gantois et al., 2006). One investigation found that a live S. Enteritidis vaccine induced higher post-challenge levels of anti-fimbrial antibodies in egg yolk whereas a killed vaccine induced higher levels of these antibodies in serum, although both vaccines were protective (Kassaify et al., 2008). Another study indicated that a live S. Enteritidis vaccine provided better protection against infection following a high-dose challenge in drinking water, but a bacterin generated the best reduction of S. Enteritidis numbers in the ceca (Atterbury et al., 2009). Hens immunized with either killed or live Salmonella vaccines have sometimes been shown to pass varying degrees of resistance to infection along to their progeny (Hassan and Curtiss, 1996; Chambers and Lu, 2002; Avila et al., 2006; Inoue et al., 2008), although in other instances vaccination protected breeders from colonization but not their chicks (Bailey et al., 2007b). For example, vaccinating breeding hens with an attenuated S. Typhimurium strain reduced intestinal colonization of their progeny when they were challenged with virulent wild-type strains (Hassan and Curtiss, 1996). In another study, vaccination of breeding hens with a trivalent bacterin resulted in passive transfer of immunity to their progeny against homologous, although not heterologous, Salmonella challenge (Young et al., 2007). Passively transferred maternal immunity may be further enhanced by treating the chicks with a live vaccine as well (Bailey et al., 2007a). Despite extensive documentation of the efficacy of both killed and live vaccines, some contrary evidence has also been recorded. Neither type of vaccine has consistently established an impenetrable barrier that altogether prevents the infection of poultry, especially when challenged by high doses of Salmonella (Gast et al., 1992; Roland et al., 2004). A microbiological
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Pre-harvest measures to control Salmonella in laying hens 127 survey of Irish eggs did not show any benefit from vaccination in comparison to a flock testing and culling approach to Salmonella control (Murchie et al., 2007). A field study in the United States reported that no significant protective effect against the presence of S. Enteritidis in either the laying house environment or internal organs of hens could be attributed to the vaccination of commercial flocks (Davison et al., 1999). Poor performance by vaccines has sometimes been associated with severe rodent control or sanitation problems in laying houses (Davies and Breslin, 2003a). Vaccine performance can also be compromised when birds are deprived of feed or water or subjected to environmental stresses such as heat (Nakamura et al., 1994). Because vaccination is characteristically less effective against Salmonella serotypes which are antigenically dissimilar to the immunizing strains, it may not be an especially dependable tool for preventing the emergence of new problems associated with previously infrequent or inconsequential serotypes. Nevertheless, vaccination can serve a critical role as an individual component within a comprehensive program of risk reduction practices, particularly when epidemiological evidence has led to heightened concerns about particular Salmonella serotypes. Effective vaccination can be especially valuable for protecting highly susceptible flocks or flocks exposed to severe challenges from environmental sources. In many nations, including the United Kingdom, vaccination has been a cornerstone of efforts to control S. Enteritidis infections in egglaying flocks, and has been associated with significant improvements in key public health parameters (Cogan and Humphrey, 2003). However, a recently published S. Enteritidis control regulation for egg-producing flocks in the United States (United States Food and Drug Administration, 2009) does not include a vaccination requirement. Instead, this program mandates specific management practices for risk reduction and a program of environmental and egg testing, with vaccination available to egg producers as a voluntary option for diminishing the probability that their flocks will yield positive testing results.
7.3 Genetic selection for naturally occurring resistance 7.3.1 Salmonella-resistant lines of chickens Immunization against Salmonella is intended to stimulate and enhance the natural ability of poultry to resist infection. Another option for decreasing the susceptibility of flocks over time involves genetic selection for the expression of resistance traits. Differences in the resistance to infection of genetically defined lines of chickens have been actively investigated in recent years as the potential basis for long-term progress in controlling the prevalence of Salmonella. Chicks from distinct lines have been reported to differ in their susceptibility to the lethal consequences of Salmonella
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128 Improving the safety and quality of eggs and egg products infection (Guillot et al., 1995). Lines of mature chickens infected with S. Enteritidis have exhibited different incidences of fecal shedding, organ invasion, and egg contamination (Beaumont et al., 1994; Lindell et al., 1994; Protais et al., 1996; Duchet-Suchaux, 1997; Girard-Santosuosso et al., 1998). Differences between lines of chickens have also been evident in resistance to the establishment of persistent intestinal colonization by S. Enteritidis (Beaumont et al., 1999; Berchieri et al., 2001). Relationships between different manifestations of genetically determined resistance to Salmonella infection are not always clear. For example, one study reported that inbred lines differing in susceptibility to systemic salmonellosis did not differ in S. Enteritidis localization in the reproductive tract or deposition in eggs (Berchieri et al., 2001). In another experiment, lines differed in organ invasion and egg contamination after S. Enteritidis infection, but not intestinal colonization (Beaumont et al., 1994). In a third instance, resistance to Salmonella organ carriage was demonstrably heritable for both chicks and adults, but genetic correlations between results obtained at these two ages were low and negative, suggesting that increasing the genetic resistance of hens would reduce that of chicks (Beaumont et al., 2009). 7.3.2 Mechanisms of heritable resistance Variations between lines of chickens in their susceptibility to Salmonella have been attributed to both innate and adaptive (immune) mechanisms. Macrophages from resistant lines of chickens have been observed to clear Salmonella faster than susceptible lines and this clearance was accompanied by a strong respiratory burst (Wigley et al., 2002). Macrophages from resistant lines expressed pro-inflammatory cytokines and chemokines more rapidly and at higher levels following Salmonella challenge (Wigley et al., 2006). In a broiler line that was highly susceptible to S. Enteritidis infection, recruitment of and phagocytosis by intraperitoneal macrophages was higher than for more resistant birds, but S. Enteritidis killing within macrophages was lower (Barbour et al., 2000). Resistance to S. Typhimurium infection has been found to correlate with an elevated T-cell response (Beal et al., 2005). Chickens selected for low heterophil : lymphocyte ratios mounted higher humoral and cellular immune responses (Al-Murani et al., 2002). Other investigators noted reproducible differences between lines of chickens in both the level and duration of fecal shedding of several Salmonella serotypes but could not associate these differences with either antibody titers or circulating heterophil numbers (Barrow et al., 2003a). Similar patterns of resistance of different chicken lines have been reported for various Salmonella serotypes, suggesting a common causal mechanism (Bumstead and Barrow, 1993). Diverse genetic and phenotypic mechanisms have been linked to Salmonella resistance, most prominently the expression of cytokines and defensins. Chicken lines which diverged in the intestinal carriage of S. Enteritidis were found to differ in the expression of two b-defensin genes (Derache et al., 2009). In
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Pre-harvest measures to control Salmonella in laying hens 129 another study, lines of chickens with lower susceptibility to cecal colonization by S. Enteritidis had higher expression of chemokine, anti-infectious cytokine, bacterial receptor, antimicrobial mediator, and (particularly) defensin genes (Sadeyen et al., 2006). A cluster of three gallinacin (defensin) genes has been associated with the Salmonella load in the cecal contents of infected chickens (Hasenstein and Lamont, 2007). Other investigators found that Salmonella infection induced genes that affect T-cell activation in one broiler line and genes involved in macrophage activation in another (Van Hemert et al., 2006). The levels of splenic expression of genes for toll-like receptors (which recognize conserved molecular motifs of pathogens and initiate immune responses) have been reported to differ between lines of chickens infected with S. Enteritidis (Abasht et al., 2009). 7.3.3 Factors affecting the performance and efficacy of genetic selection for resistance Genetic selection is an especially attractive Salmonella control option because it offers the possibility of permanent increases in resistance to infection with diverse serotypes without any ongoing need for investment in the administration of protective treatments. However, current efforts to select for resistance to Salmonella are progressing slowly and have not yet yielded levels of protection that are comparable to those which can be achieved by vaccination. Moreover, some studies have suggested that an inverse genetic relationship exists between resistance traits and production-related traits such as feed conversion and growth rate (Bolder et al., 2002). One study reported that commercial broiler and layer lines selected for increased growth or reproduction had lower cytokine gene expression levels after S. Enteritidis exposure than a native chicken line (Redmond et al., 2009). The genetic relationships between Salmonella resistance and economically relevant characteristics for laying hens (egg production and quality parameters) has not yet been clearly defined, but any interference with the optimized expression of critical production traits would severely diminish the cost-effectiveness of selecting for resistance. Accordingly, the development of genetically resistant stock remains a promising area for future research but does not appear likely to provide short-term solutions to the more immediate public health and economic problems due to Salmonella infections in poultry flocks.
7.4 Gastrointestinal colonization control 7.4.1 Principles and mechanisms of colonization control Newly hatched poultry are highly susceptible to infection by Salmonella, even when they are exposed to very low bacterial doses. However, chicks gradually become much more resistant to persistent infection during their first two weeks of life (Gast and Beard, 1989). This age-associated decrease © Woodhead Publishing Limited, 2011
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130 Improving the safety and quality of eggs and egg products in Salmonella susceptibility is principally due to the acquisition of protective intestinal microflora from the poultry house environment (Stavric et al., 1987). The ability of the normal bacterial flora of the gastrointestinal tract to inhibit or limit colonization by Salmonella and other enteric pathogens is the basis for a diverse group of preventive measures that are collectively referred to as gastrointestinal colonization control. This treatment strategy typically involves administering defined or undefined bacterial cultures, representing constituents of the normal flora, to recently hatched birds in order to establish these organisms in the gut prior to any exposure to Salmonella (Nurmi et al., 1992). The ‘probiotic’ activity by which these cultures exert protective effects is often described as competitive exclusion (CE). Diverse non-microbial manipulations of gastrointestinal biochemistry have also been proposed and assessed for their ability to control colonization by pathogens. The mechanisms by which CE cultures and other protective treatments reduce colonization by Salmonella and other enteric pathogens may include both direct steric interference with bacterial attachment to the gastrointestinal epithelium and indirect inhibition of bacterial growth within the gut via decreased pH and increased levels of undissociated volatile fatty acids (Schneitz and Mead, 2000). 7.4.2 Undefined CE preparations Colonization control preparations consisting of either the intestinal contents of mature birds or undefined anaerobic cultures derived from this material have consistently protected young poultry against both intestinal colonization and subsequent invasion to internal organs by Salmonella (Bailey, 1987; Bolder et al., 1992; Nuotio et al., 1992). Administration of a CE preparation to eggtype pullets before they were transferred into a contaminated laying house reduced the frequency of isolation of Salmonella from fecal and environmental samples (Davies and Breslin, 2003b). In field trials in commercial broiler chicken flocks, treatment with CE cultures has led to significant reductions in the incidence of salmonellae both in live birds and on carcasses (Bailey et al., 2000). Treatment with CE cultures has sometimes been reported to enhance the clearance of concurrent or pre-existing Salmonella infections (Corrier et al., 1998; Higgins et al., 2007; Vicente et al., 2008). 7.4.3 Defined CE preparations Although the majority of CE treatments or products of documented efficacy have been undefined materials presumed to contain a large proportion of the complete mature intestinal flora of poultry, various defined mixtures of microorganisms have also been evaluated for their protective capabilities. The advantages most often ascribed to treatment with defined CE cultures include their potential to achieve a greater consistency of performance than is likely for undefined preparations and greater assurances of safety than are provided
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Pre-harvest measures to control Salmonella in laying hens 131 by preparations of unspecified or unknown microbial composition. Very limited protection against pathogens is typically obtained when mixtures of small numbers of bacterial species are administered, but increasingly complex defined mixtures have been associated with corresponding improvements in the exclusion of Salmonella colonization (Stavric et al., 1985; Corrier et al., 1994c). Reductions in both intestinal colonization and horizontal transmission of S. Enteritidis were associated with a CE treatment consisting of seven strains of Enterobacteriaceae and two lactic acid bacteria (Wolfenden et al., 2007b). A mixture of 29 defined bacterial cultures provided highly significant protection to broiler chicks against S. Typhimurium colonization of both crops and ceca (Corrier et al., 1995). In another study, a commercial CE preparation containing 11 lactic acid bacteria was more effective than a combination of three Lactobacillus isolates in protecting against S. Enteritidis challenge (Higgins et al., 2010). Numerous specific microbial constituents of the mature intestinal flora have been evaluated in an effort to identify which members of colonization control preparations exert the strongest demonstrable protective effects. Among the most promising candidates are various Lactobacillus species (Van Coillie et al., 2007; Higgins et al., 2008), perhaps as a consequence of lactic acid production (Van Coillie et al., 2007). Other organisms associated with various degrees of probiotic activity include Bifidobacterium species (Fernandez et al., 2002), Escherichia coli (Wooley et al., 1999), Bacillus subtilis or B. cereus (La Ragione and Woodward, 2003; Vilá et al., 2009), Streptococcus cristatus (Zhang et al., 2007), Pediococcus acidilactici (AlZenki et al., 2009), and the yeasts Saccharomyces boulardii or S. cerevisiae (Line et al., 1998; Al-Zenki et al., 2009). One CE mixture, which was shown to reduce S. Enteritidis prevalence and levels in the ceca of challenged chicks, contained strains of Bifidobacterium, Enterococcus, Pediococcus, and two Lactobacillus species (Mountzouris et al., 2009). 7.4.4 Chemical feed and drinking water supplements A considerable assortment of feed additives have been examined as candidates for manipulating gastrointestinal biochemistry to either directly inhibit pathogen colonization or to support the survival and multiplication of protective microflora. Supplementation of feed or water with complex carbohydrates (including lactose, glucose, mannose, fructooligosaccharides, and arabinoxylooligosaccharides) has led to reduced Salmonella colonization of crops and ceca of chickens (Chambers et al., 1997; Corrier et al., 1997; Eeckhaut et al., 2008). The ‘prebiotic’ fermentation of these carbohydrates can enhance the protective action of administered CE cultures (Donalson et al., 2007). Combined treatment with b-glucan plus either a commercial CE product or an 18-strain defined CE preparation yielded better inhibition of cecal colonization and organ invasion by S. Typhimurium than was attained by any of the individual treatments (Revolledo et al., 2009). One of the
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132 Improving the safety and quality of eggs and egg products putative mechanisms by which dietary mannobiose reduced intestinal and internal organ colonization by S. Enteritidis was an improvement in the integrity of the gut mucosa (Agunos et al., 2007). Adding formic, propionic, caprylic, caproic, or butyric acids to feed has been associated with lower observed frequencies of Salmonella isolation from the feed itself and from challenged chicks (Thompson and Hinton, 1997; Van Immerseel et al., 2004; Fernández-Rubio et al., 2009; Johny et al., 2009). For example, an organic acid mixture was found to both lower S. Enteritidis numbers in feed and to reduce the horizontal transmission of S. Enteritidis among chicks when it was given in their drinking water (Jarquin et al., 2007). When administered along with a CE culture, this organic acid mixture also enhanced the protection of chicks against S. Enteritidis colonization of crops and ceca (Wolfenden et al., 2007c). Mixing an organic acid with vegetable fats to allow its slow release during digestion resulted in improved effectiveness against pathogen colonization (Fernández-Rubio et al., 2009). The addition of chlorate, lactic acid, sucrose, or acidified sodium chlorite to drinking water has been responsible for lower frequencies of Salmonella isolation from the crops of broiler chickens, which can become a focal point for infection after pre-slaughter feed withdrawal (Hinton et al., 2002; Byrd et al., 2003; Mohyla et al., 2007). Chlorate addition to feed has likewise demonstrated the ability to reduce S. Typhimurium levels in crops and ceca after challenge (Byrd et al., 2008). Another approach to controlling enteric pathogens does not fall within the classical biochemical paradigms for gastrointestinal colonization control, but has been a topic of some recent interest. The addition of cocktails of lytic bacteriophages to drinking water or delivered to chicks via aerosol spraying has been reported to reduce intestinal colonization by S. Enteritidis (Borie et al., 2008), although the administration of high bacteriophage titers may promote the emergence of phage-resistant bacteria (Atterbury et al., 2007). 7.4.5 Factors affecting the performance and efficacy of gastrointestinal colonization control The efficacy of CE cultures can be significantly influenced by the conditions under which they are prepared and handled. For example, turkey poults treated with fresh cecal material were better protected against Salmonella challenge than were poults given 1-day-old cecal material (Hofacre et al., 2000). The protective efficacy of CE cultures has been maintained over time by continuous flow culturing (Hollister et al., 1999). Flock management situations that might disrupt the normal intestinal microflora, such as antibiotic administration, feed deprivation, or water deprivation, can also interfere with the survival and efficacy of CE cultures (Weinack et al., 1985; Bailey et al., 1988). CE cultures have been reported to protect against Salmonella after administration by a wide variety of methods (Corrier et al., 1994a,b), including direct installation (gavage) into the crop, application to the vent lip, whole-body
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Pre-harvest measures to control Salmonella in laying hens 133 spraying, droplet application, addition to drinking water, or addition to feed (encapsulated in lyophilized alginate beads). Application of a CE culture by spraying was observed to provide a similar level of protection as addition to the drinking water (Wolfenden et al., 2007a). Combined administration by more than one route may increase the overall efficacy of treatment for preventing infection (Chen et al., 1998). The delivery of CE cultures to hatching eggs by fogging or by injection into the air cell can offer protection to newly hatched chicks when they are at their peak time of susceptibility to the initiation of infection (Cox et al., 1992; Primm et al., 1997). A number of contributing influences affect any assessment of the practical value of gastrointestinal colonization control as a tool for reducing the frequency and consequences of Salmonella infections in poultry flocks. Although CE treatment often leads to statistically significant reductions in both the incidence of Salmonella colonization and the numbers of colonizing Salmonella cells in poultry, total exclusion of the pathogen from the gastrointestinal tract is rarely observed. Moreover, the protective efficacy of CE treatment can sometimes be overcome by severe Salmonella challenges (Snoeyenbos et al., 1978). Because colonization control treatments are most effective when they are administered to newly hatched chicks prior to any opportunities for exposure to pathogens, the presence of salmonellae in hatcheries or brooding facilities can circumvent the useful application of CE cultures (Bailey et al., 1998). The optimal window of opportunity for CE cultures to exert a meaningful protective effect is during the first few days of life, when the susceptibility of chicks to infection is greatest. Infections established in newly hatched chicks can sometimes persist for many months (Gast and Holt, 1998), so controlling Salmonella in very young chicks can have beneficial consequences extending throughout the productive lives of flocks. However, the relevance of applying colonization control treatments to mature breeding or laying hens is far less certain. Laying flocks have been reported to be more often infected after transfer into contaminated laying houses than at any earlier rearing stages (Van de Giessen et al., 1994). Mature birds presumably already possess complete normal intestinal microflora, so the prophylactic provision of CE preparations would not seem likely to provide any additional protective advantage. Exceptions to this generalization may occur when the normal microflora have been compromised by antibiotic administration or feed restriction to induce molting, resulting in transiently increased susceptibility to pathogens such as salmonellae until the complete gut microbiota are restored. Like vaccination, gastrointestinal colonization control is a source of significant – but only partial – resistance to Salmonella and cannot be relied upon to create an impenetrable barrier against pathogen infection and subsequent product contamination. Accordingly, CE treatments can be valuable components within overall Salmonella risk reduction efforts, but they must be complemented by diligence in minimizing the opportunities for chicks to be exposed and infected.
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7.5 Future trends Control efforts for Salmonella in laying flocks have steadily evolved toward a reliance on comprehensive quality assurance programs which incorporate coordinated agendas of risk reduction practices extending throughout the production continuum. Targeted testing to detect pathogens of concern (principally S. Enteritidis) have been included to verify the effectiveness of risk reduction practices and to provide the basis for making decisions about the status and fate of flocks or eggs. Decreasing the susceptibility of laying hens to infection has been an another important objective within many control programs. Protective treatments such as vaccination are applied as adjuncts to risk reduction practices to account for any possible failures in preventing the introduction of pathogens into flocks. Immunization has also been useful for focusing the short-term responsiveness of control programs to address the emergence of specific serotypes of elevated public health significance. No single component of these programs constitutes an independently effective or unilateral solution to the problems caused by Salmonella in commercial flocks. Within the foreseeable future, research on strategies for increasing the resistance of poultry to Salmonella infection may improve the ability of these treatments to supplement and complement risk reduction practices, but are not likely to supplant reliance on those practices entirely. Accordingly, even modest incremental improvements in the efficacy of protective treatments can significantly enhance their contributions to the overall success of control programs. Vaccination is the method for increasing resistance to Salmonella infection that is currently used most widely in laying flocks around the world and it is also the approach with the most immediate opportunities for improved efficacy. Numerous killed and live vaccines have been reported to provide significant protection against diverse parameters of Salmonella infection, including intestinal colonization, invasion to internal organs, and deposition in eggs. However, this protection has been almost invariably only partial, reducing but not eliminating the consequences of exposure to the pathogen. Significant further progress in improving vaccine efficacy seems unlikely to come from continuing efforts to simply select optimal vaccine strains. Instead, fundamental research to build a deeper and more comprehensive understanding of the immune responses of poultry to Salmonella would allow the corresponding development of more precisely defined vaccines expressing the specific antigenic components which are critical for eliciting a strongly protective response. Gastrointestinal colonization control of infection in young chicks may continue to have only limited applications for egg-producing flocks as epidemiological evidence implicates laying houses as the most critical reservoirs for the persistence of S. Enteritidis, initiating cycles of infection and re-infection of successive flocks. Nevertheless, colonization control during highly pathogen-susceptible phases in the lives of laying hens can realize important protective benefits. Future refinements to this methodology
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Pre-harvest measures to control Salmonella in laying hens 135 are most likely to be provided by more precise identification of protective organisms in coordination with the utilization of dietary supplements that support their probiotic activity. Genetic selection for Salmonella-resistant lines of egg-laying hens does not seem especially likely to find a significant degree of application in commercial poultry in the immediate future. Although the heritability of resistance traits has been convincingly documented, selection for these traits will be useful only if it can be achieved without negatively affecting the retention of economically vital production-related genetic traits. Nevertheless, inherently disease-resistant laying stock will remain an attractive goal because of the possibility that the recurring expenses of administering other protective treatments could be lowered. However, like vaccination, genetic selection efforts may have an upper threshold of attainment which is limited by the extent to which the chicken’s innate and adaptive immune responses are capable of resisting Salmonella infection.
7.6 Sources of further information and advice In addition to the numerous research papers cited throughout this chapter, several pertinent review articles provide valuable perspective about the development and implementation of methods for increasing resistance to Salmonella infections in poultry. A concise overview of the immunobiology of Salmonella infections in poultry was provided by Chappel et al. (2009). The theoretical and practical opportunities and constraints associated with vaccinating poultry against Salmonella infections were discussed by Barrow (2007) and Barrow et al. (2003b). Van Immerseel et al. (2005) considered the potential for exploitation of the innate immune capabilities of young poultry. The use of competitive exclusion treatments for controlling pathogens in poultry was reviewed by Mead (2000), Schneitz (2005), and Revolledo et al. (2006). Enhancing colonization control by supplementation with feed additives and organic acids was summarized by Van Immerseel et al. (2002, 2006). A comparative overview of the strengths and weaknesses of various alternatives for controlling Salmonella infections in commercial poultry was offered by Gast (2008a). Several books also contain chapters with significant quantities of relevant information about controlling Salmonella infections in egg-laying poultry. The general topic of infections of poultry with foodtransmitted Salmonella serotypes was presented by Gast (2008b). Several chapters from Mead (2005) examined Salmonella control in poultry in terms of both research data and field applications. A more detailed and specific consideration of issues relating to the control of S. Enteritidis infection and egg contamination was made throughout many chapters from Saeed et al. (1999).
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136 Improving the safety and quality of eggs and egg products
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142 Improving the safety and quality of eggs and egg products (1998), ‘Effects of heterophil adaptation on Salmonella enteritidis fecal shedding and egg contamination’, Avian Dis 42, 6–13. la ragione r m and woodward m j (2003), ‘Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens’, Vet Microbiol, 94, 245–256. lindell k a, saeed a m and mccabe g p (1994), ‘Evaluation of resistance of four strains of commercial laying hens to experimental infection with Salmonella enteritidis phage type eight’, Poult Sci, 73, 757–762. line j e, bailey j s, cox n a, stern n j and tompkins t (1998), ‘Effect of yeast-supplemented feed on Salmonella and Campylobacter populations in broilers’, Poult Sci, 77, 405–410. mackinnon k m, he h, swaggerty c l, mcreynolds j l, genovese k j, duke s e, nerren j r and kogut m h (2009), ‘In ovo treatment with CpG oligodeoxynucleotides decreases colonization of Salmonella enteritidis in broiler chickens’, Vet Immunol Immunopathol, 127, 371–375. mcgruder e d, kogut m h, corrier d e, deloach j r and hargis b m (1995a), ‘Comparison of prophylactic and therapeutic efficacy of Salmonella enteritidis-immune lymphokines against Salmonella enteritidis organ invasion in neonatal leghorn chicks’, Avian Dis, 39, 21–27. mcgruder e d, ramirez g a, kogut m h, moore r w, corrier d e, deloach j r and hargis b m (1995b), ‘In ovo administration of Salmonella enteritidis-immune lymphokines confers protection to neonatal chicks against Salmonella enteritidis organ infectivity’, Poult Sci, 74, 18–25. mcsorley s j and jenkins m k (2000), ‘Antibody is required for protection against virulent but not attenuated Salmonella enterica serovar Typhimurium’, Infect Immun, 68, 3344–3348. mead g c (2000), ‘Prospects for ‘Competitive exclusion’ treatment to control salmonellas and other foodborne pathogens in poultry’, Vet J, 159, 111–123. mead g c (2005), Food Control in the Poultry Industry, Cambridge, England, Woodhead. mizumoto n, toyota-hanatani y, sasai k, tani h, ekawa t, ohta h and baba e (2004), ‘Detection of specific antibodies against deflagellated Salmonella Enteritidis and S. Enteritidis FliC-specific 9 kDa polypeptide’, Vet Microbiol, 99, 113–120. mizumoto n, toyota-hanatani y, sasai k, tani h, ekawa t, ohta h and baba e (2006), ‘Survey of Japanese layer farms for Salmonella Enteritidis with vaccination- and infection-specific antigens for egg yolk antibodies’, J Food Prot, 69, 17–21. mohyla p, bilgili s f, oyarzabal o a, warf c c and kemp g k (2007), ‘Application of acidified sodium chlorite in the drinking water to control Salmonella serotype Typhimurium and Campylobacter jejuni in commercial broilers’, J Appl Poult Res, 16, 45–51. mountzouris k c, balaskas c, xanthakos i, tzivinikou a and fegeros k (2009), ‘Effects of a multi-species probiotic on biomarkers of competitive exclusion efficacy in broilers challenged with Salmonella enteritidis’, Brit Poult Sci, 50, 467–478. muotiala a, hoi m and macalli p h (1989), ‘Protective immunity in mouse salmonellosis: Comparison of smooth and rough live and killed vaccines’, Microb Pathol, 6, 51–60. murchie l, whyte p, xie b, harridan s, kelly l and madden r h (2007), ‘Prevalence of Salmonella in Grade A whole shell eggs in the island of Ireland’, J Food Prot, 70, 1238–1240. nakamura m, nagami n, takahashi t, suzuki s and sato s (1994), ‘Evaluation of the efficacy of a bacterin against Salmonella enteritidis infection and the effect of stress after vaccination’, Avian Dis, 38, 717–724. nakamura m, nagata t, okamura s, takehara k and holt p s (2004), ‘The effect of killed Salmonella enteritidis vaccine prior to induced molting on the shedding of S. enteritidis in laying hens’, Avian Dis, 48, 183–188. kramer t t
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and nurmi e (1992), ‘Use of competitive exclusion to protect newly-hatched chicks against intestinal colonisation and invasion by Salmonella enteritidis PT4’, Brit Poult Sci, 33, 775–779. nurmi e, nuotio l and schneitz c (1992), ‘The competitive exclusion concept: development and future’, Int J Food Microbiol, 15, 237–240. okamura m, tachizaki h, kubo t, kikuchi s, suzuki a, takehara k and nakamura m (2007), ‘Comparative evaluation of a bivalent killed Salmonella vaccine to prevent egg contamination with Salmonella enterica serovars Enteritidis, Typhimurium, and Gallinarum biovar Pullorum using 4 different challenge models’, Vaccine, 25, 4837–4844. primm n d, vance k, wykle l and hofacre c l (1997), ‘Application of normal avian gut flora by prolonged aerosolization onto turkey hatching eggs naturally exposed to Salmonella’, Avian Dis, 41, 455–460. protais j, colin p, beaumont c, guillot j f, lantier f, pardon p and bennejean g (1996), ‘Line differences in resistance to Salmonella enteritidis PT4 infection’, Brit Poult Sci, 37, 329–339. redmond s b, chuammitri p, andreasen c b, palić d and lamont s j (2009), ‘Chicken heterophils from commercially selected and non-selected genetic lines express cytokines differently after in vitro exposure to Salmonella enteritidis’, Vet Immunol Immunopathol, 132, 129–134. revolledo l, ferreira a j p and mead g c (2006), ‘Prospects in Salmonella control: competitive exclusion, probiotics, and enhancement of avian intestinal immunity’, J Appl Poult Res, 15, 341–351. revolledo l, ferriera c s a and ferreira a j p (2009), ‘Prevention of Salmonella Typhimurium colonization and organ invasion by combination treatment in broiler chicks’, Poult Sci, 88, 734–743. roland k, tinge s, warner e and sizemore d (2004), ‘Comparison of different attenuation strategies in development of a Salmonella hadar vaccine’, Avian Dis, 48, 445–452. sadeyen j-r, trotereau j, protais j, beaumont c, sellier n, salvat g, velge p and lalmanach a-c (2006), ‘Salmonella carrier-state in hens: study of host resistance by a gene expression approach’, Microbes Infect, 8, 1308–1314. saeed a m, gast r k, potter m e and wall p g (1999), Salmonella enterica serovar Enteritidis in Humans and Animals: Epidemiology, Pathogenesis, and Control, Ames, Iowa, Iowa State University Press. schneitz c (2005), ‘Competitive exclusion in poultry – 30 years of research’, Food Control, 16, 657–667. schneitz c and mead g (2000), ‘Competitive exclusion’, in Wray C and Wray A, Salmonella in Domestic Animals, Wallingford, England, CABI, 301–322. skov m n, feld n c, carstensen b and madsen m (2002), ‘The serologic response to Salmonella enteritidis and Salmonella typhimurium in experimentally infected chickens, followed by an indirect lipopolysaccharide enzyme-linked immunosorbent assay and bacteriologic examinations through a one-year period’, Avian Dis, 46, 265–273. snoeyenbos g h, weinack o m and smyser c f (1978), ‘Protecting chicks and poults from Salmonellae by oral administration of ‘normal’ gut microflora’, Avian Dis, 22, 273–287. stabler j g, mccormick t w, powell k c and kogut m h (1994), ‘Avian heterophils and monocytes: phagocytic and bactericidal activities against Salmonella enteritidis’, Vet Microbiol, 38, 293–305. stavric s, gleeson t m, blanchfield b and pivnick h (1985), ‘Competitive exclusion of Salmonella from newly hatched chicks by mixtures of pure bacterial cultures isolated from fecal and cecal contents of adult birds’, J Food Prot, 48, 778–782. stavric s, gleeson t m, blanchfield b and pivnick h (1987), ‘Role of adhering microflora in competitive exclusion of Salmonella from young chicks’, J Food Prot, 50, 928–932.
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144 Improving the safety and quality of eggs and egg products tan s, gyles c l
and wilkie b n (1997), ‘Evaluation of an aroA mutant Salmonella typhimurium vaccine in chickens using modified semisolid Rappaport Vassiliadis medium to monitor faecal shedding’, Vet Microbiol, 54, 247–254. thompson j l and m hinton (1997), ‘Antibacterial activity of formic and propionic acids in the diet of hens on salmonellas in the crop’, Brit Poult Sci, 38, 59–65. timms l m, marshall r n and breslin m f (1994), ‘Laboratory and field trial assessment of protection given by a Salmonella enteritidis PT4 inactivated, adjuvant vaccine’, Brit Vet J, 150, 93–102. toyota-hanatani y, ekawa t, ohta h, igimi s, hara-kudo y, sasai k and baba e (2009a), ‘Public health assessment of Salmonella enterica serovar Enteritidis inactivated-vaccine treatment in layer flocks’, Appl Environ Microbiol, 75, 1005–1010. toyota-hanatani y, kyoumoto y, baba e, ekawa t, ohta h, tani h and sasai k (2009b), ‘Importance of subunit vaccine antigen of major Fli C antigenic site of Salmonella Enteritidis II: a challenge trial’, Vaccine, 27, 1680–1684. united states food and drug administration (2009), ‘Prevention of Salmonella Enteritidis in shell eggs during production, storage, and transportation; final rule’, Fed Reg, 74, 33030–33101. van coillie e, goris j, cleenwerck i, grijspeerdt k, botteldoorn n, van immerseel f, de buck j, vancanneyt m, swings j, herman l and heyndrickx m (2007), ‘Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis’, J Appl Microbiol, 102, 1095–1106. van de giessen a w, ament a j h a and notermans s h w (1994), ‘Intervention strategies for Salmonella enteritidis in poultry flocks: a basic approach’, Int J Food Microbiol, 21, 145–154. van hemert s, hoekman a j w, smits m a and rebel j m j (2006), ‘Gene expression responses to a Salmonella infection in the chicken intestine differ between lines’, Vet Immunol Immunopathol, 114, 247–258. van immerseel f, cauwerts k, devriese l a, haesebrouck f and ducatelle r (2002). Feed additives to control Salmonella in poultry’, World’s Poult Sci J, 58, 501–513. van immerseel f, de buck j, boyen f, bohez l, pasmans f, volf j, sevcik m, rychlik i, haesbrouck f and ducatelle r (2004), ‘Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with Salmonella enterica serovar Enteritidis’, Appl Environ Microbiol, 70, 3582–3587. van immerseel f, methner u, rychlik i, nagy b, velge p, martin g, foster n, ducatelle r and barrow p a (2005), ‘Vaccination and early protection against non-host-specific Salmonella serotypes in poultry: exploitation of innate immunity and microbial activity’, Epidemiol Infect 133, 959–978. van immerseel f, russell j b, flythe m d, gantois i, timbermont l, pasmans f, haesebrouck f and ducatelle r (2006), ‘The use of organic acids to combat Salmonella in poultry: a mechanistic explanation of the efficacy’, Avian Pathol, 35, 182–188. vicente j l, torres-rodriquez a, higgins s e, pixley c, tellez g, donoghue a m and hargis b m (2008), ‘Effect of a selected Lactobacillus spp.-based probiotic on Salmonella enterica serovar Enteritidis-infected chickens’, Avian Dis, 52, 143–146. vilá b, fontgibell a, badiola i, esteve-garcia e, jiménez g, castillo m and brufau j (2009), ‘Reduction of Salmonella enterica var. Enteritidis colonization and invasion by Bacillus cereus var. toyoi inclusion in poultry feeds’, Poult Sci, 88, 975–979. weinack o m, snoeyenbos g h, soerjadi-liem a s and smyser c f (1985), ‘Therapeutic trials with native intestinal microflora for Salmonella typhimurium infections in chickens’, Avian Dis, 29, 1230–1234. wigley p, hulme s d, bumstead n and barrow p a (2002), ‘In vivo and in vitro studies of genetic resistance to systemic salmonellosis in the chicken encoded by the SAL1 locus’, Microbes Infect, 4, 1111–1120. wigley p, hulme s d, rothwell l, bumstead n, kaiser p and barrow p a (2006), ‘Macrophages
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Pre-harvest measures to control Salmonella in laying hens 145 isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge’, Infect Immun, 74, 1425–1430. withanage g s k, sasai k, fukata t, miyamoto t, lillehoj h s and baba e (2003), ‘Increased lymphocyte subpopulations and macropohages in the ovaries and oviducts of laying hens infected with Salmonella enterica serovar Enteritidis’, Avian Pathol, 32, 583–590. wolfenden a d, pixley c m, higgins j p, higgins s e, vicente j l, torres-rodriguez a, hargis b m and tellez g (2007a), ‘Evaluation of spray application of a Lactobacillus-based probiotic on Salmonella enteritidis colonization in broiler chickens’, Int J Poult Sci, 6, 493–496. wolfenden a d, vicente j l, bielke l r, pixley c m, higgins s e, donoghue d j, donoghue a m, hargis b m and tellez g (2007b), ‘Effect of a defined competitive exclusion culture for prophylaxis and reduction of horizontal transmission of Salmonella enteritidis in broiler chickens’, Int J Poult Sci, 6, 489–492. wolfenden a d, vicente j l, higgins j p, filho r l a, higgins s e, hargis b m and tellez g (2007c), ‘Effect of organic acids and probiotics on Salmonella enteritidis infection in broiler chickens’, Int J Poult Sci, 6, 403–405. woodward m j, gettinby g, breslin m f, corkish j d and houghton s (2002), ‘The efficacy of Salenvac, a Salmonella enterica subsp. enterica serotype Enteritidis iron-restricted bacterin vaccine, in laying chickens’ Avian Pathol, 31, 383–392. wooley r e, gibbs p s and shotts e b jr (1999), ‘Inhibition of Salmonella typhimurium in the chicken intestinal tract by a transformed avirulent avian Escherichia coli’, Avian Dis, 43, 245–250. young s d, olusanya o, jones k h, liu t, liljebjelke k a and hofacre c l (2007), ‘Salmonella incidence in broilers from breeders vaccinated with live and killed Salmonella’, J Appl Poult Res, 16, 521–528. zhang g, ma l and doyle m p (2007), ‘Salmonellae reduction in poultry by competitive exclusion bacteria Lactobacillus salivarius and Streptococcus cristatus’, J Food Prot, 70, 874–878. ziprin r l and m h kogut (1997), ‘Efficacy of two avian Salmonella-immune lymphokines against liver invasion in chickens by Salmonella serovars with different O-group antigens’, Avian Dis, 41, 181–186.
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8 Management and sanitation procedures to control Salmonella in laying hen flocks R. Ducatelle and F. Van Immerseel, Ghent University, Belgium
Abstract: This chapter deals with a general hygienic plan for the layer farm. Such a plan requires a thorough knowledge of the different possible sources of contamination. The relative importance of each of these is discussed. Specific hygienic measures are proposed regarding rodent control, insect control, red mite control, etc. The hygienic plan of any layer farm should include a strategy of cleaning and disinfection of the poultry houses and the environment. In regions and conditions with high infection risk, decontamination of feed and drinking water may be indicated. Since eradication of Salmonella and completely Salmonella-free production will probably never be achieved, continuous vigilance and rigorous hygienic measures will still be needed for years to come. Key words: disinfection, cleaning, decontamination, rodents, insects, red mites, Salmonella.
8.1 Introduction The European baseline study on the prevalence of Salmonella in holdings of laying hen flocks, carried out between October 2004 and September 2005, has shown that Salmonella infections still are highly prevalent in laying hen flocks, even in regions where the hygienic standard is relatively high (Anonymous, 2007). More recently, it was shown that this European baseline study probably has underestimated the true prevalence of Salmonella in the layer flocks (Van Hoorebeke et al., 2009). The same European baseline study has confirmed that serotype Enteritidis is by far the most common © Woodhead Publishing Limited, 2011
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Management and sanitation procedures to control Salmonella 147 Salmonella serotype associated with laying hens (Anonymous, 2007). The specific ability of this serotype to preferentially colonize the reproductive organs of the laying hen and to survive at 42 °C in egg albumen probably explains why Salmonella enterica serovar Enteritidis is the predominant serotype in hens eggs (Gantois et al., 2008). Thus control of Salmonella in layer flocks should focus on the serotype Enteritidis, although other serotypes cannot be neglected. Taking into consideration the specificity of the serotype Enteritidis, one obvious strategy is vaccination. This approach is covered in the previous chapter. The protection afforded by vaccination is, however, greatly affected by the severity of the challenge. Indeed, under conditions of severe challenge, vaccination does not afford full protection but only reduces the internal egg contamination rate (Gantois et al., 2006). Therefore, vaccination should never stand alone and should always be accompanied by measures aiming to reduce the level of contamination of the environment of the birds. In this context, management and sanitation procedures have an essential role to play in any integrated control plan for Salmonella in layers. Management procedures should essentially aim at (1) preventing the introduction of Salmonella on the farm and in the poultry house, and (2) decreasing spread within a given animal population. These procedures include general biosecurity measures as well as specific measures to control potential active carriers of Salmonella, such as rodents, insects, red mites and wild birds. In this context one should realize that pet animals can also be carriers of Salmonella. Moreover, numerous goods can become mechanical vehicles of the microorganism, including feed and drinking water, but also clothing and shoes of visitors. Sanitation procedures include all sorts of management measures that can be taken once the infection is suspected or confirmed to be present on the farm. These range from eradication of a contaminated flock, through cleaning and disinfection of a contaminated house, to safe disposal of contaminated manure. When carrying out management and sanitation procedures to control Salmonella in layer flocks one needs to take into account the specific conditions of the different housing systems. Even though the shift from traditional battery cage housing to alternative housing systems does not seem to significantly affect either the susceptibility of layers under experimental conditions (De Vylder et al., 2009) or the prevalence of Salmonella in laying hen flocks under field conditions in Europe (Van Hoorebeke et al., 2010), floor rearing of pullets has been identified as a risk factor for the presence of Salmonella Enteritidis in layer houses in the US (Garber et al., 2003). Moreover, in a study in the UK, type of house was positively associated with persistence of Salmonella Enteritidis as well as non-Salmonella Enteritidis serotypes. Indeed, persistence of Salmonella Enteritidis was longest in step-cage and cage-scraper houses and lowest in non-cage and cage-belt houses (CarriqueMas et al., 2008). Even if the evidence seems to be somehow conflicting regarding the susceptibility of birds and persistence of the microorganisms in the different housing systems, it is clear that management and sanitation
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148 Improving the safety and quality of eggs and egg products procedures will be conceived and implemented differently depending on the type of house and on the type of cages if present.
8.2 Management procedures to prevent introduction of Salmonella on the farm or to suppress the infection pressure from the environment 8.2.1 General biosecurity In the EU, the Commission Regulation 2160/2003 requests tracing back in case of an outbreak of foodborne Salmonella infection. Thus, egg producers have a serious responsibility with respect to public health. They need to document that they have taken all the necessary precautions, specifically including hygiene and biosecurity measures, to avoid such problems. General hygiene and biosecurity on the layer farm must be based on three pillars: ∑
All activities on the farm should be planned and carried out in such a way as to minimize hygienic risks. ∑ A continuous monitoring plan should provide up to date information on the Salmonella status. ∑ A sanitation and decontamination plan should allow immediate action in case of detection of Salmonella. General hygienic and biosecurity measures should be integrated in the general management plan of the layer farm. The principles should be part of the rules of good management practice. All of these measures should aim at preventing the introduction of Salmonella on the farm and also at lowering infection pressure in general. In order to correctly set out the principles and rules, one needs a good insight in all possible routes by which Salmonella can be introduced, starting with the day-old chicks. Since many Salmonella serotypes may be transmitted vertically, the hatchery and all producers upstream in the production pyramid need to make the necessary efforts. Incoming birds should have a high health status and should be purchased from reliable suppliers that have quality assured breeding and hatching facilities. It should be emphasized that anything that is introduced voluntarily or involuntarily into the layer farm may act as a vector of Salmonella. This includes water, feed, bedding, rodents, insects, pets, wild birds, equipment, footwear, clothing, vehicles, people, etc. Some of the most critical factors are discussed in detail below. From a hygiene point of view, all critical points in the infrastructure and in the production cycle need to be identified. These are the points where samples can be taken for the monitoring. Moreover, in the EU, under Commission Regulation 2160/2003, minimum sampling requirements for laying hens have been laid down and monitoring is obligatory.
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Management and sanitation procedures to control Salmonella 149 8.2.2 Rodent control The environment around layer farms varies considerably but is usually rural. In this environment, wild rodents are attracted to spilled feedstuffs, the availability of water and the presence of shelter. Wild rodents are commonly associated with infrastructural damage and eating or spoilage of stored feed and products. It is well documented, however, that they also can be reservoirs and vectors of Salmonella (Gratz, 1994). The role of rodents in the transmission of Salmonella has been reviewed recently (Meerburg and Kijlstra, 2007). Wild rodents carry Salmonella without showing any clinical symptoms of disease. During a longitudinal monitoring of infected layer farms, mice on farms were found to be infected (Kinde et al., 2005). A high prevalence (24%) of Salmonella Enteritidis has been reported in mice on contaminated layer farms, while logically, Salmonella Enteritidis was not found in mice on Salmonella Enteritidis free layer farms (Henzler and Opitz, 1992). In a large-scale monitoring of mice, captured in henhouses in the US in two consecutive years, during which 621 and 526 mouse spleens were cultured for Salmonella, 25.0% and 17.9% respectively were positive for Salmonella Enteritidis (Guard-Petter et al., 1997). Mice and rats can acquire Salmonella infection from various sources. They may get infected through contact with faeces from infected pullets or hens, or else from infected wild birds. Since rodents tend to live closely together, transmission between individuals in the same group is likely (Shimi et al., 1979). There are indications also that Salmonella infections in mice can be transmitted vertically, since Salmonella has been isolated from fetal mouse tissues (Davies and Wray, 1995b). Only 15 colony forming units are sufficient to infect a mouse or a rat (Henzler and Opitz, 1992; Welch et al., 1941). Wild mice, infected naturally or experimentally, excrete Salmonella Enteritidis intermittently in their faeces, at a concentration of up to 10 exp 4 cfu per dropping (Davies and Wray, 1995b). The strains isolated from mice are capable of producing a large amount of high molecular weight lipopolysaccharide. This particular phenotype induces severe infections when experimentally inoculated to chicks. These strains would also have enhanced ability to contaminate eggs (Guard-Petter et al., 1997). In that same line of thought, passage of Salmonella Enteritidis in mice would select for more virulent and more egg-invasive strains (Humphrey et al., 1996). This would suggest that mice might play an important role in the contamination of the layer farms. Definitive evidence for the involvement of mice and rats in the maintenance of Salmonella Enteritidis infection on layer farms was provided by molecular fingerprinting of strains isolated from these rodents. Pulsed field gel electrophoresis and ribotyping identified the same Salmonella Enteritidis clones in the wild rodents and in the farm environment (Liebana et al., 2003). Experimental proof was already given by Davies and Wray in 1995, when they showed that 3-week-old chicks could be infected by contact with
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150 Improving the safety and quality of eggs and egg products droppings from mice which had been infected experimentally with Salmonella Enteritidis 2 and 5 months previously. Rodent droppings are actually sought out by the chickens when mixed in their feed or bedding (Davies and Wray, 1995b). Dead mice are even more of a problem, especially in layer houses where the hens are kept on the floor. Indeed, dead mice carcasses contain higher levels of Salmonella organisms than mouse droppings, and the dead mice carcasses may be pecked and consumed by the hens (Davies and Breslin, 2003a). In a recent large-scale epidemiologic study by Carrique-Mas et al. (2008), high levels of rodents were associated with a significantly longer persistence of Salmonella Enteritidis on the layer farms. Moreover, they showed that clearance of Salmonella Enteritidis incidents during the lay period was related to elimination of rodents, underlining once more the importance of rodent control as a valuable measure that needs to be integrated in a Salmonella control program. Thus mice (and rats) are a source of infection that plays an important role in the persistence of the infection on the layer farm. This is not only due to their capability of carrying the infection. The mice appear to further amplify the Salmonella, as these are more concentrated in the mice samples than in the environmental samples (Henzler and Optiz, 1992). Moreover, the presence of wild rodents appears to be associated with a reduced efficacy of cleaning and disinfection between two consecutive flocks (Davies and Breslin, 2003a), suggesting that they also may play a role in the carry-over of the infection from one flock to the next. Taking all these data together, rodent control should be an essential part of any Salmonella control programme on a laying hen farm. Most of the time, farmers do apply rodent control to prevent economic losses, as rodents can cause considerable damage to constructions and goods. However, they only do so when rodent densities exceed a certain threshold. This threshold is subjective. Rats are more often seen as a problem by the farmers than mice in Denmark (Leirs et al., 2004). Rodent control methods used on conventional layer farms usually are limited to rodenticides, while organic farmers use traps and cats. It has been documented however, that cats can carry and shed Salmonella as well (Davies and Breslin, 2003b). From 26 to 28 May 2004 an international seminar was held in Wageningen, the Netherlands, about the current knowledge on rodent management on organic pig and poultry farms. This has generated a series of recommendations (Meerburg et al., 2004). Management is based on: (1) prevention of rodent infestation through modifying the habitat and rodent proofing of buildings; (2) monitoring population density; and (3) the actual control measures. A series of different control methods are available (rodenticides, trapping, shooting, cats, domestic mustelids, etc.), but these need to be evaluated case by case with respect to their efficacy, cost and acceptability from social, environmental and sustainability points of view.
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Management and sanitation procedures to control Salmonella 151 8.2.3 Insect control One of the most abundant insect species found on the floor in poultry houses with deep litter is the darkling beetle, also known as lesser mealworm, Alphitobius diaperinus. These insects have high reproductive rates and are difficult to control. They feed on manure, spilled feed, cracked eggs and even chicken carcasses, since they are typically omnivorous. The mealworms in turn are eaten by the chickens (Axtell and Arends, 1990). These behavioural aspects should make the lesser mealworm a candidate risk factor for transmission of Salmonella. Delascas et al. (1968) probably were the first to study this topic. Salmonella has been isolated from lesser mealworms collected in poultry houses (Harein et al., 1970). In a turkey brooder house, 27% of the beetles collected were Salmonella positive (Harein et al., 1972). Experimental exposure of lesser mealworms to Salmonella contaminated fish meal however, suggested that the beetles would be relatively resistant to Salmonella uptake (Davies and Wray, 1995a). It was documented recently, however, that when exposed to Salmonella on an agar, these beetles can be contaminated not only externally but also internally, suggesting that Alphitobius diaperinus may not only be a mechanical vector, but also a reservoir of Salmonella (Crippen et al., 2009). The lesser mealworm is a difficult pest to control. Specific measures aiming at reducing the numbers or eradicating these beetles are not commonly included in Salmonella control programs on layer farms. If specific measures are to be taken, then a thorough knowledge of their biology and behaviour is required. These beetles are known to congregate in great numbers under and around feed and water stations (Axtell and Arends, 1990). They migrate into wall cracks and as such may survive routine cleaning and disinfection procedures. This propensity to hide also allows them to escape from routine insecticide applications, even though they are known to be highly susceptible to several chemical insecticides (Salin et al., 2003). Physical anti-beetle barriers have been developed with variable success. These tools, however, have not been commonly adopted by farmers (Geden and Carlson, 2001). Also a range of biological control tools have been tested. Predators of Alphitobius diaperinus have been described, but are not available for use in practice (Husband and Hassan, 1998). More recently, there is considerable interest in the use of diatomaceous earth (Alves et al., 2008) and entomopathogenic fungi (Gindin et al., 2009) for the control not only of the lesser mealworm, but also of red mites (see also below under red mite control). These tools offer great promise, although their efficacy can be influenced greatly by various factors. Indeed, diatomaceous earth is much more effective against lesser mealworms at high environmental temperature (32 °C) and when applied in feed rather than in litter. Moreover, the diatomaceous earth has a repellent effect on the beetles. Entomopathogenic fungi differ in their pathogenicity towards the beetles, and their efficacy is reduced in the presence of litter (Gindin et al., 2009). Next to the lesser mealworms, also houseflies (Musca domestica) are
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152 Improving the safety and quality of eggs and egg products considered to be a potential vector of Salmonella in laying hen houses. Davies and Breslin (2003a) found 8 out of 16 houseflies, collected in routinely disinfected laying hen houses, positive for Salmonella. Houseflies are commonly controlled by the use of insecticides. As in many other environments, house flies in laying hen houses are more and more resistant to the commonly used insecticides (Acevedo et al., 2009). Moreover, in many countries the use of insecticides is under pressure because of the fear for transfer of residues into the eggs. Currently attempts are made to use entomopathogenic fungi also for the control of house fly larvae and adults, sometimes in combination with entomotoxic bacteria, such as Bacillus thuringiensis (Mwanburi et al., 2009). 8.2.4 Red mite control The poultry red mite (Dermanyssus gallinae) is by far the most important ectoparasite of laying hens. Infestations with this mite cause increasing problems in the layers, particularly so because the use of classical insecticides is forbidden or currently being phased out. Although very common in laying hen houses, the role of this haematophagous mite in the epidemiology of Salmonella is a matter of controversy. It has been proven experimentally in vitro that red mites can become infected with Salmonella through cuticular contact or via a contaminated blood meal (Moro et al., 2007a). Moreover, experimental oral inoculation of day-old chicks with Salmonella infected red mites resulted in colonization of all inoculated birds (Moro et al., 2007c). Finally, Salmonella has been detected in pooled mite samples from 2 out of 16 sampled laying hen farms by PCR (Moro et al., 2007b). One of these samples was from a farm that had been contaminated according to routine testing, but was not contaminated at the moment of the red mite collection, thus suggesting that the red mites might be a reservoir of Salmonella. However, to our knowledge, Salmonella (other than Gallinarum) has not been isolated from red mites so far. Control measures are usually taken against red mites not because of their potential as a Salmonella vector, but rather because of the economic losses caused by this pest (irritation and anaemia of the birds, leading to reduced egg production). It is well documented that general hygienic measures help to control the poultry red mite population. Cleaning with water indeed can remove large numbers of mites (Nordenfors and Höglund, 2000). Control in the past has mainly relied on chemical pesticides. This has led to the development of resistance (Beugnet et al., 1997). Moreover, in most countries acaricides are approved for usage in empty poultry houses only in order to avoid chemical residues in eggs. All of this has seriously hampered red mite control in laying flocks in recent years. Therefore, the EU and various other authorities have encouraged research into the development of new strategies to control poultry red mites. The results of an international seminar on the subject have been summarized recently in an excellent review paper by Mul
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Management and sanitation procedures to control Salmonella 153 et al. (2009). This paper discusses the value and limitations of heating the hen houses to temperatures above 45 °C between the production cycles as a means to eliminate the red mite populations. Currently considerable efforts are made to develop new control strategies. Several of these strategies use oils, different plant extracts and so-called inert dusts (Maurer et al., 2009). The latter comprise a range of minerals, including diatomaceous earth and synthetic silica products. They mainly act as desiccants, absorbing the lipids of the cuticle surface of the mites, leading to death as a result of water loss. The formulation and mode of application of these inert dusts, however, are still subject to research (Kilpinen and Steenberg, 2009). It should also be stressed that, on the basis of epidemiologic studies, silica dust is considered a lung carcinogen for humans (Lacasse et al., 2009). A valuable new approach to red mite control is the use of entomopathogenic fungi, which are capable of infecting and killing the mites. Recently also efforts are being made to develop a vaccine (Arkle et al., 2008). The principle behind this approach is that hens should produce antibodies that, when taken up by the mites during their blood meal, make the blood coagulate. Finally, studies are ongoing to use predatory mites as a tool to control red mites (Lesna et al., 2009). 8.2.5 Wild birds In a recent study in Olsztyn, Poland, 74 wild birds, representing 17 different species, which were collected because of injuries or weakness, were examined for antibodies against Salmonella. High levels of Salmonella Typhimurium antibodies were detected in 34 individuals, and similar results were obtained with Salmonella Enteritidis (Stenzel et al., 2008). Salmonella Enteritidis contaminated wild bird droppings have been found on laying farms infected with Salmonella Enteritidis (Davies and Breslin, 2001). These results suggest that wild birds can act as vectors and possibly also reservoirs of Salmonella. Control measures for Salmonella thus should include measures to avoid contact with wild birds. For layers kept permanently indoors, this means that the layer house should be bird-proof. It is clear, however, that in the free-range system, opportunities for contact with wildlife in general will be much greater than when layers are kept permanently indoors.
8.3 Sanitation and decontamination 8.3.1 Decontamination of drinking water Next to the source of drinking water, the type of drinking water supply system will inevitably influence the risk of Salmonella colonization of the hens through the drinking water. With nipple drinkers, the risk of Salmonella (cross)-contamination is obviously much lower than with cups or with round © Woodhead Publishing Limited, 2011
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154 Improving the safety and quality of eggs and egg products drinkers. Moreover, the presence of organic material in the drinking water will adversely affect the efficacy of water decontamination. Therefore, as a preventive measure, drinkers need to be cleaned frequently. Decontamination of the drinking water can be done by chlorination. In a study using experimentally infected chickens, chlorination of the drinking water reduced the number of Salmonella bacteria in the water, but it had no significant effect on gut colonization in the chickens (Poppe et al., 1986). The most commonly used chemicals for decontamination of drinking water, however, are the short chain organic acids. A commercial drinking water disinfectant containing propionic acid successfully eliminated Salmonella from the drinking water in a naturally infected flock, but it did not have any effect on the presence of Salmonella in litter, cloacal swabs or caecal contents (Al-Chalaby et al., 1985). Several other organic acids also have been used for decontamination of drinking water. Details on the use of organic acids in drinking water have recently been reviewed by Wales et al. (2010). In theory, various other chemicals, including ozone, iodine, traces of silver, and copper ions, can be used to decontaminate drinking water. These are, however, not commonly used in daily practice. 8.3.2 Decontamination of feed Using conventional pre-enrichment and enrichment-based isolation procedures, Salmonella is regularly isolated from animal feed in general and from laying hen feed in particular. In a recent retrospective analysis of Salmonella isolates recovered from animal feed in the UK, Papadopoulou et al. (2009) found feed contamination rates ranging from 3.8% in 1993 to 1.1% in 2006. On average, they recovered serotype Enteritidis at a proportion of 0.5% from raw ingredients and 0.6% from finished feed. The contamination of feed ingredients and finished feed may come from various sources, including faeces of infected wild rodents, wild birds, pets or farm animals. For review, see Williams (1981). Different raw ingredients may differ in their degree of contamination. As an example, Veldeman et al. (1995) found 31% of 130 fish meal samples contaminated, as opposed to 4% of 83 meat and bone meal samples, 2% of 58 tapioca samples and 27% of 15 maize grit samples. Conventional isolation procedures most probably underestimate the true contamination rates of animal feedstuffs, since the sample size is inevitably very small with respect to the volume of the feedstuff. Moreover, it has been shown that Salmonella Enteritidis can survive for at least 26 months in artificially contaminated poultry feed (Davies and Wray, 1996). Furthermore, pullets and layers are highly susceptible to Salmonella contamination at certain time point during their life (immediately post-hatch, at point of lay, and at moulting). Thus, even though serotype Enteritidis represents only a small percentage and other serotypes are much more prevalent in feed, decontamination of poultry feed should be part of any integrated Salmonella control program in layers.
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Management and sanitation procedures to control Salmonella 155 Various techniques can be used to decontaminate poultry feed. Additives with antimicrobial activity exert their effect mainly after ingestion and moistening of the feed by the bird. These are discussed in the previous chapter. Here we shall discuss only those treatments that kill the Salmonella in the raw ingredients or in the finished feed before it is eaten by the bird. The most commonly used method probably is steam pelleting. The efficacy of this method appears to be highly variable. Indeed, while Cox et al. (1983) found 58% contamination in mash samples as opposed to 0% in pellets, Veldeman et al. (1995) found 21% in mash against 1.4% in pellets, and Jones and Richardson (2004) even found only a reduction from 8.8 to 4.2%. These authors investigated different pelleting temperatures and concluded that at least 85 °C should be reached. Both the temperature reached and the type of steam conditioner used may influence the efficacy of the process (Blank et al., 1996). Moreover, Salmonella containing dust, penetrating in the coolers, may abolish the favourable effect of the pelleting process (Jones and Richardson, 2004). The residual level of contamination after the pelleting process may be very low, though still high enough to initiate infection in highly susceptible chicks. Owing to the low concentration rates and the risk of false negatives, Escherichia coli is sometimes used as an indicator to evaluate the efficacy of the steam pelleting process. Poultry feed also can be decontaminated by irradiation, most often using a cobalt source producing gamma rays. At radiation doses of 5 kGy Salmonella appears to be eliminated, while no significant chemical changes are induced in the feed (Leeson and Marcotte, 1993a). For treatment of feed bags, depending on the initial contamination level, radiation doses of 5–25 kGy are needed to eliminate Salmonella (Leeson and Marcotte, 1993a). Doses of 25 kGy or more will lead to loss of potency of all fat-soluble vitamins, and to peroxidation of fats (Leeson and Marcotte, 1993b). Next to the above mentioned physical treatments, some chemicals can also be used to decontaminate poultry feed. In a laboratory set-up, treatment with anhydrous ammonia gas resulted in a >5.6 log10 cfu/g reduction of Salmonella on corn grain (Tajkarimi et al., 2008). This method has not yet been tested under practical (farm) conditions. The most commonly used chemicals to treat feed contaminated with Salmonella are organic acids. The original concept of incorporating organic acids in the feed was based on the notion that the acids would decontaminate the feed and thus avoid Salmonella uptake by the birds. Later on, it was realized that addition of acids in feed also exerts effects in the crop (Thompson and Hinton, 1997). Nevertheless, depending on the water activity and temperature of the feed, supplementation of organic acids at high concentrations (1–2%) can lead to a reduction in Salmonella contamination level following prolonged storage of the feed. Indeed, following experimental inoculation of feed with high doses of Salmonella Typhimurium, a 1000-fold reduction in Salmonella count was observed after 7 days when the feed was treated with a mixture of formic acid and propionic acid (Iba and Berchieri, 1995). When this same
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156 Improving the safety and quality of eggs and egg products mixture was applied to feed that was artificially inoculated with low levels of Salmonella Kedougou, significant reductions in contamination levels also were observed after several weeks of storage (Hinton and Linton, 1988). Formaldehyde is another chemical commonly used to decontaminate poultry feed either alone or in combination with organic acids. The decontaminating effects of formaldehyde and other added chemicals during storage of feed have recently been reviewed by Wales et al. (2010), who give more details on dosages and the resulting decontaminating effects. Whatever treatment has been used to decontaminate feed, it should be kept in mind that incorrect storage can lead to recontamination of the feed during storage. 8.3.3 Cleaning and disinfection of a layer house Salmonella Enteritidis has been shown to persist for at least a year in depopulated poultry houses (Davies and Wray, 1996). Salmonella also can survive for 2.5 years in dried bird faeces (Morse and Duncan, 1974). Thus cleaning and disinfection of the poultry house should be carried out immediately after depopulation. Cleaning should be started after all removable materials have been taken out of the poultry house. Cleaning should be thorough, including spots that are difficult to reach, such as ventilation tunnels. Disinfection should be carried out in a clean environment. In a study in the UK it has been clearly shown that Salmonella Enteritidis infection in laying flocks is predominantly a problem of persistent contamination of layer houses and associated wildlife vectors (Wales et al., 2006). In this study in 12 Salmonella-contaminated caged layer houses, standard cleaning and disinfection procedures did not achieve elimination of Salmonella Enteritidis in any of the premises examined. In another study comparing different disinfectants under field conditions, the recovery of Salmonella after cleaning and disinfection was variable. The use of 10% formalin led to a statistically greater reduction in the sample prevalence than any other disinfectant tested (Carrique-Mas et al., 2009). This concentration of formalin is much higher than the commonly used 1%, which was shown inadequate for Salmonella elimination in field conditions (Marin et al., 2009). It is clear that cleaning and disinfection will never result in a sterile (or even completely Salmonella-free) environment. Therefore the method proposed by Wales et al. (2006) for a semi-quantitative assessment may be a valuable tool to measure the success of decontamination and the level of infection pressure to which the next round of birds will be exposed. Disinfection aims to reduce the infection pressure to the lowest achievable level. Thus optimum performance of the disinfectants should be achieved. In a study under simulated field conditions, Stringfellow et al. (2009) recently evaluated the effect of temperature and time of storage, and presence or not of chicken litter, on the efficacy of a series of disinfectants commonly used in the poultry industry. Only after 6 weeks of storage at 43 °C or 16 weeks at 32 °C, a phenolic compound lost some of its activity against Salmonella
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Management and sanitation procedures to control Salmonella 157 Typhimurium. In the presence of chicken litter, however, quaternary ammonium compounds, chlorhexidine, binary ammonium-based as well as phenolic solutions lost considerable activity against Salmonella Typhimurium. These results emphasize again that all organic material must be removed if optimum activity of the disinfectant solutions is to be achieved. Moreover, it was shown under laboratory conditions that prolonged exposure of Salmonella Typhimurium to sublethal concentrations of commonly used disinfectants selects for decreased susceptibility to the biocides and for antibiotic resistance (Karatzas et al., 2007; Randall et al., 2007). As an alternative, or even following the use of chemical disinfectants, steam disinfection can be applied. In the recommended protocol, all openings of the poultry house must be sealed air tight. A hose attached to a steam generator is inserted through an opening. The steam generator heats the water to 160 °C and uses up to 1000 L/h of water. Inside the house temperature needs to be maintained at 60 °C in a relative humidity of 100% for 24 h. At the beginning of the process, 30 ppm formaldehyde may be added to the water. Under these conditions, it appears possible to eliminate high numbers of Salmonella from a persistently infected empty poultry house (Gradel et al., 2003, 2004). The problem of sanitation and decontamination is particularly difficult in the free-range system. Indeed, when Davies and Breslin (2003b) studied the persistence of Salmonella Enteritidis in the environment after depopulation of a free-range breeding chicken farm, they found that soil samples still contained viable Salmonella Enteritidis after 8 months.
8.4 Future trends: management and sanitation procedures for a Salmonella-free production chain The epidemiologic situation in the layers today shows that in most parts of the world, Salmonella in general and serotype Enteritidis in particular are still highly prevalent. Considering the successful efforts in several countries, it appears that host-adapted serotypes such as Pullorum/Gallinarum and possibly also Enteritidis (although the latter is not truly host adapted) may be eradicated from industrial poultry. Nevertheless, backyard poultry constitute a reservoir and thus a continuous threat for reappearance of Pullorum/ Gallinarum in industrial poultry. Regarding the serotype Enteritidis, not only backyard poultry, but also many different other animal species may constitute reservoirs from which reinfection may take place. We currently are only beginning to understand the virulence mechanisms that serotype Enteritidis acquired to cause the pandemic of egg-associated foodborne infections in humans that started in the 1980s and that still has not come to an end (Gantois et al., 2009). A wide variety of other Salmonella serotypes appears to colonize the intestinal tract of many different animal hosts, including both domestic and
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158 Improving the safety and quality of eggs and egg products wild animal species. Many of these serotypes are not host-specific and can easily pass from one animal host to another. These bacteria are shed in the faeces and can survive for a long time in the environment. Moreover, it is clear that, at any time, any serotype of Salmonella may acquire virulence traits that can make it more successful in colonizing a new niche. Such a serotype may arise from any unpredictable source or reservoir. One therefore should consider Salmonella as being part of the ecosystem and therefore a continuing threat to commercial egg production that will require continuous vigilance for years to come.
8.5
Sources of further information and advice
Information regarding general hygiene on poultry farms and a description of different types of chemical disinfectants can be found in Diseases of Poultry, Y.M. Saif (editor-in-chief), Iowa State Press, 2003 (ISBN 0-81380423-X). Measures taken in the EU for control of Salmonella have been laid down in a number of regulations published in the Official Journal of the European Union. The different member states all have national Salmonella control programmes for the production of table eggs, which all include a number of obligatory management and sanitation measures in the layer farms. The British Department for Environment, Food and Rural Affairs has published a ‘code of practice for the prevention and control of Salmonella in commercial egg laying flocks’ (PB2205, Defra Publications, Admail 6000, London SW1A, 2007). Further information also can be found in the book entitled Salmonella in Domestic Animals, C. Wray & A. Wray (eds), CABI Publishing, 2000 (ISBN 978-0851992617) The safe disposal of carcasses of dead birds and of litter and droppings collected during cleaning of the layer house are not covered in this chapter. Heinonen-Tanski et al. (2006) have reviewed the different methods to reduce pathogen microorganisms in animal manure in general. The different technologies available for carcass disposal have been reviewed in depth by Kastner et al. (2004).
8.6 References acevedo g, zapater m
& toloza a (2009), ‘Insecticide resistance of house fly, Musca domestica (L.) from Argentina’, Parasitol Res 105, 489–493. al-chalaby z, hinton m & linton a (1985), ‘Failure of drinking water sanitisation to reduce the incidence of natural Salmonella in broiler chickens’, Vet Rec 116, 364–365. alves l, oliveira d & neves p (2008), ‘Factors affecting diatomaceous earth effectiveness in the control of Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) adults’, Neotrop Entomol 37, 716–722.
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Management and sanitation procedures to control Salmonella 159 (2007), ‘Report of the task force on zoonoses data collection on the analysis of the baseline study on the prevalence of Salmonella in holdings of laying hen flocks of Gallus Gallus’, EFSA J, 97. arkle s, harrington d, de luna c, george d, guy j & sparagano o (2008), ‘Immunological control of poultry red mite: the use of whole mite antigens as a candidate vaccine’, Annals New-York Acad Sci, 1149, 36–40. axtell r & arends j (1990), ‘Ecology and management of arthropod pests of poultry’, Annu Rev Entomol, 35, 101–126. beugnet f, chauve c, gauthey m & beert l (1997), ‘Resistance of poultry red mite to pyrethroids in France’, Vet Rec, 140, 577–579. blank g, savoie s & campbell l (1996), ‘Microbiological decontamination of poultry feed – evaluation of steam conditioners’, J Sci Food Agric, 72, 299–305. carrique-mas j, breslin m, snow l, mclaren i, sayers a & davies r (2008), ‘Persistence and clearance of different Salmonella serovars in buildings housing laying hens’, Epidemiol Infect, 137, 837–846. carrique-mas j, marin c, breslin m, mclaren i & davies r (2009), ‘A comparison of the efficacy of cleaning and disinfection methods in eliminating Salmonella spp. from commercial egg laying houses’, Avian Pathol, 38, 419–424. commission regulation (EC) No. 2160/2003 of the European Parliament and of the Council of 17 November 2003, on the control of Salmonella and other specified foodborne zoonotic agents’, Official Journal of the European Union 12.12.2003 L325/1–15. cox n, bailey j, thomson j & juven b (1983), ‘Salmonella and other Enterobacteriaceae found in commercial poultry feed’, Poultry Sci, 62, 2169–2175. crippen t, sheffield c, esquivel s, droleskey r & esquivel j (2009), ‘The acquisition and internalization of Salmonella by the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae)’, Vector-borne Zoonotic Dis, 9, 65–71. davies r & breslin m (2001), ‘Environmental contamination and detection of Salmonella enterica serovar Enteritidis in laying flocks’, Vet Rec, 149, 699–704. davies r & breslin m (2003a), ‘Observations on Salmonella contamination of commercial laying farms before and after cleaning and disinfection’, Vet Rec, 152, 283–287. davies r & breslin m (2003b), ‘Persistence of Salmonella Enteritidis phage type 4 in the environment and arthropod vectors on an empty free-range chicken farm’, Environmental Microbiol, 5, 79–84. davies r & wray c (1995a), ‘Contribution of the lesser mealworm, Alphitobius diaperinus to carriage of Salmonella enteritidis in poultry’, Vet Rec, 137, 407–408. davies r & wray c (1995b), ‘Mice as carriers of Salmonella Enteritidis on persistently infected poultry units’, Vet Rec, 137, 337–341. davies r & wray c (1996), ‘Persistence of Salmonella Enteritidis in poultry units and poultry food’, Brit Poultry Sci, 37, 589–596. delascas e, pomeroy b & harein p (1968), ‘Infection and quantitative recovery of Salmonella Typhimurium and Escherichia coli from within lesser mealworm Alphitobius diaperinus (Panzer)’, Poultry Sci, 47, 1871–1875. de vylder j, van hoorebeke s, ducatelle r, pasmans f, haesebrouck f, dewulf j & van immerseel f (2009), ‘Effect of the housing system on shedding and colonization of gut and internal organs of laying hens with Salmonella Enteritidis’, Poultry Sci, 88, 2491–2495. gantois i, ducatelle r, timbermont l, boyen f, bohez l, haesebrouck f, pasmans f & van immerseel f (2006), ‘Oral immunisation of laying hens with the live vaccine strains of TAD Salmonella vacE and TAD Salmonella vacT reduces internal egg contamination with Salmonella Enteritidis’, Vaccine, 24, 6250–6255. gantois i, eeckhaut v, pasmans f, haesebrouck f, ducatelle r & van immerseel f (2008), ‘A comparative study on the pathogenesis of egg contamination by different serotypes of Salmonella’, Avian Pathol, 37, 399–406. gantois i, ducatelle r, pasmans f, haesebrouck f, gast r, humphrey t & van immerseel anonymous
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160 Improving the safety and quality of eggs and egg products (2009), ‘Mechanisms of egg contamination by Salmonella Enteritidis’, FEMS Microbiol Rev, 33, 718–738. garber l, smeltzer m, fedorka-cray p, ladely s & ferris k (2003), ‘Salmonella enterica serotype Enteritidis in table egg layer house environments and in mice in U.S. layer houses and associated risk factors’, Avian Dis, 47, 134–142. geden c & carlson d (2001), ‘Mechanical barrier for preventing climbing by the lesser mealworm (Coleoptera: Tenebrionidae) and hide beetle (Coleoptera: Dermestidae) larvae in poultry houses’, J Econ Entomol, 94, 1610–1616. gindin g, glazer i, mishoutchenko a & samish m (2009), ‘Entomopathogenic fungi as potential control agent against the lesser mealworm, Alphitobius diaperinus in broiler houses’, BioControl, 54, 549–558. gradel k, jorgensen j, andersen j & corry j (2003), ‘Laboratory heating studies with Salmonella spp. and Escherichia coli in organic matter, with a view to decontamination of poultry houses’, J Appl Microbiol, 94, 919–928. gradel k, jorgensen j, andersen j & corry j (2004), ‘Monitoring the efficacy of steam and formaldehyde treatment of naturally Salmonella-infected layer houses’, J Appl Microbiol, 96, 613–622. gratz n (1994), ‘Rodents as carriers of disease’, in: Buckle A & Smith R (Eds), Rodent pests and their control, Oxford, CAB International, 85–108. guard-petter j, henzler d, rahman m & carlson r (1997), ‘On-farm monitoring of mouseinvasive Salmonella enterica serovar Enteritidis and a model for its association with the production of contaminated eggs’, Appl Environ Microbiol, 63, 1588–1593. harein p, delascasas e, pomeroy b & york m (1970), ‘Salmonella spp and serotypes of Escherichia coli isolated from lesser mealworm collected in poultry brooder houses’, J Econom Entomol, 63, 80–85. harein p, delascasas e, larsen c & pomeroy b (1972), ‘Microbial relationship between the lesser mealworm and its associated environment in a turkey brooder house’, Environ Entomol, 1, 189–194. heinonen-tanski h, mohaibes m, karinen p & koivunen j (2006), ‘Methods to reduce pathogen microorganisms in manure’, Livestock Sci, 102, 248–255. henzler d & opitz h (1992), ‘The role of mice in the epizootiology of Salmonella Enteritidis infection on chicken layer farms’, Avian Dis, 36, 625–631. hinton m & linton a (1988), ‘Control of Salmonella infections in broiler chickens by the acid treatment of their feed’, Vet Rec, 123, 416–421. humphrey t, williams a, mcalpine k, lever m, guard-petter j & cox j (1996), ‘Isolates of Salmonella enterica Enteritidis PT4 with enhanced heat and acid tolerance are more virulent in mice and more invasive in chickens. Epidemiol Infect, 117, 79–88. husband r & hassan m (1998), ‘A new podapolipus (Acari: Podapolipidae) from Alphitobius sp. (Coleoptera: Tenebrionidae) from Bangladesh’, Int J Acarol, 24, 53–57. iba a & berchieri a (1995), ‘Studies on the use of a formic acid–propionic acid mixture (Bio-AddTM) to control experimental Salmonella infection in broiler chickens’, Avian Pathol, 24, 303–311. jones f & richardson k (2004), ‘Salmonella in commercially manufactured feed’, Poultry Sci, 83, 384–391. karatzas k, webber m, jorgensen f, woodward m, piddock l & humphrey t (2007), ‘Prolonged treatment of Salmonella enterica serovar Typhimurium with commercial disinfectants selects for multiple antibiotic resistance, increased efflux and reduced invasiveness’, J Antimicrob Chemother, 60, 947–955. kastner c et al. (2004), ‘Carcass disposal: a comprehensive review’, report prepared by the National Agricultural Biosecurity Center Consortium, Kansas State University. kilpinen o & steenberg t (2009), ‘Inert dusts and their effects on the poultry red mite (Dermanyssus gallinae)’, Exp Appl Acarol, 48, 51–62. kinde h, castellan d, kerr d, campbell j, breitmeyer r & ardans a (2005), ‘Longitudinal f
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Management and sanitation procedures to control Salmonella 161 monitoring of two commercial layer flocks and their environments for Salmonella enterica serovar Enteritidis and other Salmonellae’, Avian Dis, 49, 189–194. lacasse y, martin s & gagné d (2009), ‘Dose–response meta-analysis of silica and lung cancer’, Cancer Causes Control, 20, 925–933. leeson s & marcotte m (1993a), ‘Irradiation of poultry feed I. Microbial status and bird response’, World’s Poultry Sci J, 49, 19–33. leeson s & marcotte m (1993b), ‘Irradiation of poultry feed II. Effect on nutrient composition’, World’s Poultry Sci J, 49, 120–131. leirs h, lodal j and knorr m (2004), ‘Factors correlated with the presence of rodents on outdoor pig farms in Denmark and suggestions for management strategies’, NJASWageningen J Life Sci, 52, 133–144. lesna i, wolfs p, faranji f, roy l, komdeur j & sabelis m (2009), ‘Candidate predators for biological control of the poultry red mite Demanyssus gallinae’, Exp Appl Acarol, 48, 63–80. liebana e, garcia-migura l, clouting c, clifton-hadley f, breslin m & davies r (2003), ‘Molecular fingerprinting evidence of the contribution of wildlife vectors in the maintenance of Salmonella Enteritidis infection in layer farms’, J Appl Microbiol, 94, 1024–1029. marin c, hernandiz a & lainez m (2009), ‘Biofilm development capacity of Salmonella strains isolated in poutry, risk factors and their resistance against disinfectants’, Poultry Sci 88, 424–431. maurer v, perler e & heckendorn f (2009), ‘In vitro efficacies of oils, silicas and plant preparations against the poultry red mite Dermanyssus gallinae’, Exp Appl Acarol, 48, 31–41. meerburg b & kijlstra a (2007), ‘Role of rodents in transmission of Salmonella and Campylobacter’, J Sci Food Agric, 87, 2774–2781. meerburg b, bonde m, brom f, endepols s, jensen a, leirs h, lodal j, singleton g, pelz h-j, rodenburg t & kijlstra a (2004), ‘Towards sustainable management of rodents in organic animal husbandry’, NJAS-Wageningen J Life Sci, 52, 195–205. moro c, chauve c & zenner l (2007a), ‘Experimental infection of Salmonella Enteritidis by the poultry red mite, Dermanyssus gallinae’, Vet Parasitol, 146, 329–336. moro c, desloire s, chauve c & zenner l (2007b), ‘Detection of Salmonella sp. in Dermanyssus gallinae using an FTA filter-based polymerase chain reaction’, Med Vet Entomol, 21, 148–152. moro c, fravalo p, amelot m, chauve c, zenner l & salvat g (2007c), ‘Colonization and organ invasion in chicks experimentally infected with Dermanyssus gallinae contaminated by Salmonella Enteritidis’, Avian Pathol, 36, 307–312. morse e & duncan v (1974), ‘Salmonellosis – an environmental health problem’, J Am Vet Med Assoc, 165, 1015–1019. mul m, van niekerk t, chirico j, maurer v, kilpinen o, sparagano o, thind b, zoons j, moore d, bell b, gjevre a & chauve c (2009), ‘Control methods for Dermanyssus gallinae in systems for laying hens: results of an international seminar’, World’s Poultry Sci J, 65, 589–599. mwanburi l, laing m & miller r (2009), ‘Interaction of Beauveria bassiana and Bacillus thuringiensis var. israeliensis for the control of house fly larvae and adults in poultry houses’, Poultry Sci, 88, 2307–2314. nordenfors h & höglund j (2000), ‘Long term dynamics of Dermanyssus gallinae in relation to mite control measures in aviary systems’, Brit Poultry Sci, 41, 533–540. papadopoulou c, carrique-mas j, davies r & sayers a (2009), ‘Retrospective analysis of Salmonella isolates recovered from animal feed in Great Britain’, Vet Rec, 165, 681–688. poppe c, barnum d & mitchell w (1986), ‘Effect of chlorination of drinking water on experimental Salmonella infection in poultry’, Avian Dis, 30, 362–369. randall l, cooles s, coldham n, penuela e, mott a, woodward m, piddock l & webber m
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162 Improving the safety and quality of eggs and egg products (2007), ‘Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence’, J Antimicrob Chemother, 60, 1273–1280. salin c, delettre y, vernon p (2003), ‘Controlling the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) in broiler and turkey houses: field trials with a combined insecticide treatment: insect growth regulator and pyrethroid’, J Econ Entomol, 96, 126–130. shimi a, keyhani m & hedayati k (1979), ‘Studies on salmonellosis in the house mouse, Mus musculus. Lab Anim, 13, 33–34. stenzel t, tykalowski b, mazur-lech b & koncicki a (2008), ‘Infections in wildlife birds – results of a serological screening’, Bull Vet Inst Pulawy, 52, 63–66. stringfellow k, anderson p, caldwell d, lee j, byrd j, mcreynolds j, carey j, nisbet d & farnell m (2009), ‘Evaluation of disinfectants commonly used by the commercial poultry industry under simulated field conditions’, Poultry Sci, 88, 1151–1155. tajkarimi m, riemann h, hajmeer m, gomez e, razavilar v & cliver d (2008), ‘Ammonia disinfection of animal feeds – laboratory study’, Int J Food Microbiol, 122, 23–28. thompson j & hinton m (1997), ‘Antibacterial activity of formic and propionic acids in the diet of hens on Salmonellas in the crop’, Brit Poultry Sci, 38, 59–65. van hoorebeke s, van immerseel f, de vylder j, ducatelle r, haesebrouck f, pasmans f, de kruif a & dewulf j (2009), ‘Faecal sampling underestimates the actual prevalence of Salmonella in laying hen flocks’, Zoonoses Public Health, 56, 471–476. van hoorebeke s, van immerseel f, schulz j, hartung j, harisberger m, barco l, ricci a, theodoropoulos g, xylouri e, de vylder j, ducatelle r, haesebrouck f, pasmans f, de kruif a & de wulf j (2010), ‘Determination of the within and between flock prevalence and identification of risk factors for Salmonella infections in laying hen flocks housed in conventional and alternative systems’, Prevent Vet Med, 94, 94–100. veldeman a, vahl h, borggreve g & fuller d (1995), ‘A survey of the incidence of Salmonella species and enterobacteriaceae in poultry feeds and feed components’, Vet Rec, 136, 169–172. wales a, breslin m & davies r (2006), ‘Assessment of cleaning and disinfection in Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture techniques’, Vet Microbiol, 116, 283–293. wales a, allen v & davies r (2010), ‘Chemical treatment of animal feed and water for the control of Salmonella’, Foodborne Pathogens Dis, 7, 3–15. welch h, ostrolenk m & bartram m (1941), ‘Role of rats in the spread of food poisoning bacteria of the Salmonella group’, Am J Public Health, 31, 332–340. williams j (1981), ‘Salmonella in poultry feed – a worldwide review’, World’s Poultry J, 37, 6–25.
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9 Egg decontamination by washing W. Messens, Institute for Agricultural and Fisheries Research (ILVO), Belgium, J. Gittins, ADAS, UK, S. Leleu, Institute for Agricultural and Fisheries Research (ILVO), Belgium and N. Sparks, SAC, UK
Abstract: Although the practice of washing eggs was mainly developed to clean dirty eggs (Class B), this processing may also improve the visual appearance of eggs and their hygienic quality by decreasing the bacterial load on the shell surface (‘sanitising’). This chapter first reviews the historical and commercial perspectives of egg washing. It then gives an overview of the egg washing process. Next, factors that influence the microbiological quality of washed eggs are listed before describing postwashing treatments. Finally, the benefits and problems associated with egg washing are discussed. Key words: egg, washing process, quality, microbiology, benefits and problems.
9.1 Introduction Many spoilage organisms or foodborne pathogens can contaminate eggs. While the microbial flora on the eggshell is dominated by Gram-positive bacteria, Gram-negative bacteria are the principal contaminants of egg contents and rotten eggs (Board and Tranter 1995). Staphylococcus warneri, Acinetobacter baumannii, Alcaligenes spp., Serratia marcescens, Carnobacterium spp. and Pseudomonas spp., among others, have been isolated from the egg contents by De Reu et al. (2006). Staphylococcus spp. dominated the eggshell (De The views and findings in this chapter are solely those of the authors and do not necessarily reflect the views or position of the European Food Safety Authority.
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164 Improving the safety and quality of eggs and egg products Reu et al. 2008). Campylobacter jejuni can be present in the egg contents (Sahin et al. 2003), Listeria monocytogenes and Yersinia enterocolitica were isolated from eggshells (Nitcheva et al. 1990; Favier et al. 2005). However, Salmonella enterica serovar Enteritidis is the principal cause of egg-associated human infections (EFSA 2011). In 2009, salmonellosis was the second most often reported zoonotic disease in humans in the European Union with a notification rate of 23.7 per 100,000 population. S. Enteritidis is the serovar most frequently (52.3% of all reported serovars) associated with human illness and was the most frequently isolated serovar in table eggs in most member states (EFSA 2011). Apparently this serotype has intrinsic characteristics that allow an epidemiological association with hen eggs (Gantois et al. 2009), resulting in internally contaminated eggs, as well as eggshell contamination. The number per million of eggs contaminated internally and/or externally with S. Enteritidis was estimated to range between 14 and 150 in one member state and between 2 and 28 in another member state. Even though other serovars of Salmonella can be transmitted by eggshell contamination, the public health impact of this pathway is considered smaller compared with transmission by eggs internally contaminated with S. Enteritidis (EFSA 2010). Internal contamination of intact eggs with Salmonella can occur by vertical (or transovarian) or horizontal (or trans-shell) transmission. Vertical transmission is by direct contamination of the yolk, albumen, eggshell membranes or eggshells before oviposition originating from infection of the reproductive organs with S. Enteritidis for example. In the case of horizontal transmission, eggs can be contaminated by penetration through the eggshell from the colonised gut or from contaminated faeces during or after oviposition (Gantois et al. 2009; Messens et al. 2005). Following oviposition, the shell acquires contamination from all contact surfaces, and the extent of contamination is directly related to the cleanliness of these surfaces (Board and Tranter 1995). The practice of washing eggs was mainly developed to clean dirty eggs (Class B) (EFSA 2005) but washing may also improve the visual appearance of eggs and their hygienic quality, by decreasing the bacterial load on the shell surface (‘sanitising’). This chapter reviews first the historical and commercial perspectives of egg washing (Section 9.2). An overview of the egg washing process is given in Section 9.3. In Section 9.4, factors that influence the microbiological quality of washed eggs are listed. Post-washing treatments are discussed in Section 9.5. Benefits and problems associated with egg washing are described in Section 9.6.
9.2 Historical and commercial perspective Cleaning eggs by washing was at one time widely considered to be harmful to egg quality. Following a series of studies reported in the USA, Jenkins © Woodhead Publishing Limited, 2011
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Egg decontamination by washing 165 and Pennington (1919) found that washing adversely affected the keeping quality of eggs and they concluded that the practice of washing dirty eggs could not be condemned too strongly. From 1935, observations were made of the quality of shipments of chilled, washed eggs arriving in the UK from Australia. In a review of this and related studies, Brooks et al. (1952) concluded that the wetting or washing of dirty eggs caused progressive deterioration in the contents during subsequent handling and storage. They recommended that dirty eggs should be cleaned by a method other than washing. Several other studies, including those of Sayers (1943) and Cotterill and Hartman (1956), found a higher incidence of rots in machine-washed, as opposed to hand-washed eggs. Nevertheless, by the end of the 1940s, the washing of dirty eggs had become widespread in the USA (Hutchison et al. 2003b). According to Walters et al. (1966), the earliest machines used were single-line brush washers or tanks, capable of holding a basket of about 15 dozen eggs. These systems were later replaced by mechanised in-line machines which overcame previous problems such as high labour costs, high breakage rates and spoilage hazard. Walters et al. (1966) carried out series of field trials which were published in a United States Department of Agriculture (USDA) report, entitled ‘Improved Methods, Techniques and Equipment for Cleaning Eggs’. These studies sought to determine the time, temperature and bacteriological parameters for washing soiled eggs with minimum spoilage and loss in quality. Subsequently, a number of field trials were undertaken using experimental machinery and, together with the virtual elimination of spoilage problems, improved cleaning efficiency and reduced eggshell damage were reported. The apparent success of egg washing in the USA led to increased interest in the subject elsewhere. In Japan, egg washing became virtually universal in the 1990s in response to Salmonella concerns. However, egg marketing legislation in the European Community (Regulation (EC) No 557/2007 2007) has consistently stated that ‘Class A eggs shall not be washed or cleaned before or after grading’. When Sweden became a member state in 1995, most of its major egg packers were washing eggs and from 1998 this was only allowed to continue if the eggs were marked as Class B. From 2004, Regulation (EC) No. 1907/90, as amended by Regulation (EC) No. 5/2001, merged Classes B and C and the new Class B could no longer be sold as table eggs. Regulation (EC) No 2052/2005 amending Regulation (EC) No 1907/90 stated that ‘Packing centres which, on 1 June 2003, were authorised to wash eggs may, for a transitional period until 31 December 2006, be authorised to continue to wash eggs, under strict surveillance of the competent authority of the member state concerned’. Later, Regulation (EC) 557/2007 stated that ‘Member states which, on 1 June 2003, authorised packing centres to wash eggs may continue to authorise packing centres to wash eggs, provided that those centres operate in accordance with the national guides for egg-washing systems’. This possibility was adopted by a small number of premises, mainly in Sweden. The Swedish consumer generally
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166 Improving the safety and quality of eggs and egg products prefers washed eggs and according to EFSA (2005) more than 50% of table eggs in Sweden are washed. The latter regulation also stated that ‘Washed eggs may only be marketed in the member states in which such authorisations have been issued’. In 2005, the European Food Safety Authority (EFSA) published an opinion on the microbiological risks relating to the washing of table eggs (EFSA 2005). It concluded that if well done, there are clear advantages to egg washing because of the reduced microbial load. However, it also recognised that problems can arise if egg washing is not undertaken properly (see further), hence disadvantages and advantages must be assessed.
9.3 The egg washing process The experimental egg washing machinery developed in the USA by Walters et al. (1966) included a number of design features not found on previous machines. These included: (1) egg wetting prior to washing, (2) an egg spinning device to increase the treated eggshell surface by increasing contact between the egg and the brushes, and (3) a separate conveyor in the dryer unit. In a list of recommendations for cleaning eggs, they specified that attempts should not be made to clean excessively dirty eggs. These principles have since been incorporated into the design of commercial machinery and egg washing practices. A review of commercial equipment and procedures in the USA and Japan (two countries where virtually all eggs are washed) was undertaken by Hutchison et al. (2003b). Commercial egg washing precedes the normal grading and packing operations and is part of a continuous in-line process, with eggs being laid out in a regular pattern on conveyors. The egg washing process can be divided into four main stages, namely wetting, washing, rinsing and drying and these are successively discussed in the subsequent sections mainly based on the review by Hutchison et al. (2003a). 9.3.1 Wetting process Wetting the shell prior to the main washing operation is intended to soften any debris and is typically undertaken using a spray of warm water. Ideally, time should be allowed to enable this water to penetrate into any debris, before the main wash begins. However, commercial pressures often impose a limit on this interval. 9.3.2 Washing process The main washing process generally involves eggs being rubbed with brushes while being rotated on the conveyor and sprayed with warm water. However, some machines have been developed which use water jets alone, rather than © Woodhead Publishing Limited, 2011
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Egg decontamination by washing 167 in combination with brushes and this has been claimed to be more hygienic and to facilitate the cleaning of the machine. A typical in-line egg washing system with brushes is shown in Fig. 9.1. The brushes used usually consist of a series of soft, nylon bristles, arranged on parallel frames. To assist the washing process, the brushes oscillate on the frames, either parallel with or perpendicular to the movement of the eggs.
(a)
(b)
Fig. 9.1 Typical in-line egg washing system with brushes.
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168 Improving the safety and quality of eggs and egg products The brushes can normally be raised or lowered slightly for batches of very large or small eggs. Some machines also incorporate rotating cylindrical brushes at the start of the washing process, these are considered to ensure more effective cleaning. A section of tougher scalloped strip brushes may also be incorporated, specifically to remove any debris from near the poles (top and bottom) of the egg. To facilitate daily washing and inspection, some machines are designed so that the brush sections can be easily removed. Typically in commercial practice, eggs remain within the washing section of the machine for approximately 60 seconds. Some machines rely on large volumes of water during the washing process whilst others direct high pressure jets of water onto the eggs. The main wash may be carried out as a single stage or there may be up to three distinct zones, with separate tanks used to recirculate water. Fresh, potable water is introduced at the start of a work shift and more fresh water is then added for the final rinsing stage. This is then filtered and recirculated back through the machine so that eggs passing through are met by progressively cleaner water. Recirculated water which is used for the initial wetting is subsequently discarded. The use of separate zones also allows a slightly increasing temperature gradient to be maintained, which reduces the risk of thermal cracking and prevents the ingress of wash water due to a negative pressure in the egg, caused by the temperature differential. Wash water typically contains both a sanitiser and a defoamer. Chemicals used in the washing process are required to be food safe, should be compatible with the wash water and should not impart any odours or taints. Antimicrobials such as sodium hypochlorite (100–200 ppm) and ozone (2–5 ppm) are commonly used and these are automatically fed into the system at the start of and during the washing process. A disinfectant such as chlorine may be added to the rinse water. Most machines incorporate automated control systems for monitoring water temperature and quality and chemical levels. More information on sanitisers used is given in Section 9.4.5. 9.3.3 Rinsing process A spray rinse is designed to remove from eggs any loose debris, chemical residues or other dissolved matter which may have been picked up during the main washing process. The water for the rinse process should be warmer than for the main wash. This helps to maintain a positive pressure differential (see also Section 9.4) and in addition, the extra heat aids the subsequent drying of eggs (Hutchison et al. 2003a). 9.3.4 Drying process Eggs must be thoroughly dried after washing, to prevent mould growth and to ensure that moisture and bacteria are not subsequently drawn inside the shell (Hutchison et al. 2001). Drying must take place rapidly in order to maintain
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Egg decontamination by washing 169 throughput speeds. In general, eggs spend only about 10 to 20 seconds in the drying sections of commercial machines and assessments of commercial egg washing systems have frequently concluded that machines fail to dry eggs adequately (Hutchison et al. 2003a). This may be due to shortcomings either in design or in maintenance procedures (EFSA 2005). Drying is generally carried out in two or more stages and involves two physical processes. The first process involves mechanical drying which removes 70–80% of the surface water. This stage is usually assisted by the use of air jets. The air can either be supplied in large volume or as fine pressure jets, ‘cutting’ the free water from the shell (Hutchison et al. 2001). The second process is the evaporation of free water from the shell surface (Hutchison et al. 2001, 2003a). This evaporation process is enhanced by high speed air and can be aided if supplemented with a hot-air dryer or dehumidified air (Hutchison et al. 2001; Nield 1992). Although the eggs are generally conveyed on rollers as they move through the drying process, in some designs, the rotation is temporarily stopped and vacuum systems can be used to remove water which collects on the lower surface of the eggs. An alternative process uses soft brushes to gently ‘wipe’ the eggs. These brushes are kept dry by air movement (Hutchison et al. 2003a); however, concerns have been raised about the potential for re-contamination of eggshells because of the direct contact of the brushes (EFSA 2005).
9.4 Factors that influence the microbiological quality of washed eggs Several factors influence the microbiological quality of washed eggs. The most important risk factors are discussed in subsequent sections and include the temperature of both the wash and rinse water, the contact time of the egg with the water, the egg storage conditions prior to the washing process, the water quality and its sanitisers. Eggs also need to be dried immediately post-washing, a factor not covered in this section but referred to above. 9.4.1 Temperature of the wash and rinse water Of the risk factors that determine if the contents of an egg will become contaminated with bacteria during the washing process arguably the temperatures of the wash and rinse water are the most important. When placed in an environment that is cooler than itself the egg cools, causing the egg contents to contract. This contraction causes the shell membranes to pull away from the shell, creating a negative pressure that results in suction across the shell. So, if a warm egg is placed in cool air, the pressure differential results in air moving across the shell. In itself this may not threaten the microbiological status of the egg; however, if the surface of the
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170 Improving the safety and quality of eggs and egg products shell is covered with water or the pores contain water, then water will move across the shell into the egg contents, carrying with it any contaminants that may be in the water, on the shell or in the pores (Haines and Moran 1940; Lorenz and Starr 1952). When an egg emerges from a hen, the temperature of the egg will be that of the hen’s body temperature, some 41 °C. As the wash water should be warmer than the egg temperature the minimum temperature recommended for washing hen’s eggs tends therefore to be > 40 °C (Kinner and Moats 1981). In practice, to provide for a margin of error, commercial in-line egg washers may use wash water held at a temperature of 44 °C (MST 2007). The Egg Grading Manual (USDA 2000) states that eggs should be washed in water 11.1 °C warmer than the internal temperature of the egg and no cooler than 32.2 °C. Following the same logic, the manual states that rinse water should be ‘slightly warmer’ than the wash water. Thus in-line washers that wash at 44 °C may rinse at 48 °C (MST 2007). Some authors (Caudill et al. 2010; Jones et al. 2006) have expressed concerns, that washing eggs in warm water, which at 49 °C can increase the internal temperature of the egg by up to nearly 8 °C (Anderson et al. 1992), may encourage the growth of microorganisms that remain in or on the egg post-washing. This has led to studies that have compared the use of lower wash water temperatures than those recommended currently (Hutchison et al. 2003a). Caudill et al. (2010) compared four combinations of water temperature (49 °C wash with 49 °C rinse; 49 °C wash with 24 °C rinse; 24 °C wash with 24 °C rinse and 24 °C wash with 49 °C rinse). The authors reported that while the number of bacteria present in the egg contents in general increased by 39.5% during storage, the temperature of the wash water did not significantly affect the number of organisms in eggs stored post-washing for five weeks, or the eggs physical quality parameters (e.g. Haugh units (HU), vitelline membrane strength). It was concluded by the authors ‘that incorporating cool water into commercial shell egg processing, while maintaining a pH of 10 to 12 … allows for more rapid cooling, without causing a decline in egg quality or increasing the presence of aerobic microorganisms and fungi for approximately five weeks post-processing’. It could be argued that the lack of a significant improvement in the physical quality parameters of the egg would not justify the increased risk of drawing contaminants into the eggs using a cold water wash. However, Caudill et al. (2010) also noted that reducing the temperature of the wash water could significantly reduce energy use and increase cost savings – the former becoming an increasingly important consideration. This is not to argue that the generally held view that eggs should be washed in water that is significantly warmer than the egg, is wrong; it is after all based on scientific studies and many years of practical experience. Indeed in a practical test, Hutchison et al. (2004) noted that the egg contents of eggs that had been artificially contaminated on the shell with either S. Enteritidis or S. Typhimurium were more likely to become contaminated if the wash
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Egg decontamination by washing 171 water temperature was allowed to fall below that of the egg. However it may provide some evidence to indicate that if a trade-off is to be considered then, for eggs with a sound shell and cuticle, there may be scope to make energy/cost savings without necessarily compromising the microbial status of the egg. 9.4.2 Time that the egg is in contact with water The times stated for in-line washing can vary significantly and may include time for drying the egg but (as discussed in Section 9.3) a washing/rinsing time of around one minute would not be unusual (see for example MST 2007; Sanovo Engineering 2010). The use of high pressure wash water jets to cleave away organic contaminants and/or brushes enables the time required for water to be in contact with an egg in an in-line washer, to be relatively low. As was demonstrated by Hutchison et al. (2006), the time that the water is in contact with the shell, is a risk factor for water penetrating the shell (see previous section). In the USA immersion washing is not allowed, presumably in part because of the perceived enhanced risk of prolonged immersion leading to contaminants being taken up by the egg (USDA 2000). In essence, the aim, which is consistent with the commercial requirement of a high throughput, should be to minimise the time that the water is in contact with the shell while not compromising the efficacy of the sanitising agent. 9.4.3 Storage conditions prior to washing The time that elapses between the egg being laid and washed can vary considerably. It is feasible that some eggs may be laid and conveyed immediately out of the production unit to the packing centre via the egg washing machine. Other eggs may not be washed for many days post-lay. When an egg is laid, the cuticle is moist and has a structure that is open and porous (Sparks and Board 1984, 1985). As the moisture is lost from the cuticle, this changes in minutes into a dense layer that allows gaseous diffusion but is resistant to penetration by liquids (Sparks and Board 1984). Washing an egg before the cuticle has dried could result in gross contamination of the egg contents; however, in practice this is not likely to happen, the cuticle drying in minutes under normal production conditions. As the cuticle ages (i.e. in the days and weeks post-lay) so its structure flattens and loses some of its ability to resist the passage of liquids. This change over time accounts for the findings of Wang and Slavik (1998) who reported that there was a positive correlation between the time an egg was stored and the likelihood of subsequent washing leading to contamination of the egg contents with Salmonella spp. Such studies have led to the recommendation that, if eggs are to be washed, this should happen within 7 days of the egg being laid (EFSA 2005). Current commercial practice, however, aims to minimise the time between laying, grading, packing and delivery to the point of sale and
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172 Improving the safety and quality of eggs and egg products in countries where egg washing is permissible, companies aim to wash the majority of eggs within 24 hours of lay (Hutchison et al. 2003a). If eggs are to be stored prior to washing then the normal considerations would apply, and eggs would typically be stored at temperatures between 12 and 15 °C. However care should be taken if washing stored eggs to ensure that the temperature of the wash water does not cause thermal cracking of the shell (Upadhyaya et al. 1985). This thermal cracking results from the temperature differential between the egg and the wash water being excessive and to avoid this it has been recommended that the wash water temperature does not exceed 45 °C (Leclair et al. 1994). 9.4.4 Water quality Only potable water should be used for egg washing, although water is frequently recirculated (see Section 9.3). Care should also be taken to ensure that the iron content of the water is < 2 ppm (EFSA 2005; USDA 2000). This recommendation can be traced back to the work of Garibaldi and Bayne (1960; 1962), who linked an increase in the spoilage rate of washed eggs in the state of California with elevated levels of iron in the wash water. The concentration of iron in the wash waters was sufficient to saturate the iron chelating ovotransferrin, a key antimicrobial protein in egg albumen (Tranter 1994), allowing bacterial contaminants to survive and grow within the egg. Ovotransferrin has also been detected in eggshells and some studies have shown that antimicrobial proteins reside in the shell and cuticle (Wellman-Labadie et al. 2008) although, relative to those in the albumen, the antimicrobial efficacy of these proteins when incorporated into shell is less certain. Northcutt et al. (2005) analysed the wash water from a number of commercial egg washers and reported levels of soluble iron to be in the range of 0.3 to 1.6 ppm. 9.4.5 Sanitisers The application of sanitisers in the washing water has been reviewed previously (Hutchison et al. 2003a) although this review omits a recent addition to the list of possible sanitisers, slightly acidic electrolysed water (Cao et al. 2009). In practice, however, the majority of commercial egg washing systems will use either chlorine (100–200 ppm) or ammonium-based sanitisers (Hutchison et al. 2003a). Irrespective of which sanitiser is used, it is important that the efficacy is maintained by ensuring that the concentration of the active ingredient is monitored so that it is not allowed to fall to a level below which it ceases to be efficacious. Factors that may compromise the efficacy of the sanitiser, include the build up of organic material and the pH of the water. Because wash water is often recirculated in egg washing machines, many will include a filtration system to collect debris and organic material. Some countries limit the use
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Egg decontamination by washing 173 of recirculated water. So for example, Sweden only allows rinse water to be recirculated as wash water (EFSA 2005). Leclair et al. (1994) studied the interaction between pH, chlorine concentration, water temperature and egg solids. The authors concluded that, for the organisms used in their model system (L. monocytogenes and S. Typhimurium), the presence of egg solids required an increase in temperature (from 42 to 47.4 °C) and pH (from 10.5 to 10.8) to reduce the number of organisms at a rate similar to that achieved in the absence of egg solids. As the work of Leclair et al. (1994) indicates, the pH of the water is an important co-factor. Acid sanitisers are rarely used because of the detrimental effect on the shell (see for example Heath and Wallace 1978) and most egg washing operations would aim to have a wash water pH of 10 to 11 (Moats 1978). Thus following a survey of commercial egg washing units, Northcutt et al. (2005) reported that while the pH of the incoming water ranged from 6.1 to 6.7, the wash water had a pH between 10.0 and 11.4. The pH values in this range have been shown to be optimal for controlling Salmonella (Humphrey et al. 1993; Kinner and Moats 1981) including those that are heat resistant. At pH <9.5 S. Typhimurium was found to grow in egg wash waters at temperatures of between 38 and 42 °C but the organism died when the pH was increased to >10 (Holley and Proulx 1986). Similar results have been reported by others including Catalano and Knabel (1994) and Humphrey et al. (1993).
9.5 Post-washing treatments 9.5.1 Egg oiling As soon as the egg is laid, carbon dioxide and moisture begin to escape through the shell pores. This causes a gradual increase in pH as eggs get older and this is accompanied by thinning of the thick albumen. At the same time, the vitelline membrane becomes weaker and the egg loses weight due to the loss of moisture (Kirunda and Mckee 2000; Stadelman 1995). Oiling of eggs has been used to offset any cuticle damage which may have occurred during washing. Hence the need for it may also be related to the type of washing machinery used. Egg oiling helps to reduce the rate of internal quality decline by sealing the shell pores. As the rate of carbon dioxide loss is relatively rapid, oiling should preferably be carried out soon after lay (Goodwin et al. 1962). Sabrani and Payne (1978) have demonstrated that oiling of unwashed eggs using linseed oil slowed the rate of the decay of the albumen quality and markedly decreased the weight loss of eggs during storage at 28 °C for 24 days. Oiling of eggs using mineral oil during 12 weeks storage at 7 °C was also found to reduce the rate of decline of egg quality parameters compared to un-oiled eggs (Biladeau and Keener 2009). Using commercial egg washing and oiling machinery in the early 1980s, the effects of washing and oiling on the haugh unit (HU)s were assessed for three groups of eggs: © Woodhead Publishing Limited, 2011
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174 Improving the safety and quality of eggs and egg products eggs that had been washed and oiled, eggs that had been washed only and unwashed eggs. After 28 days of storage, quality was maintained best in eggs that had been both washed and oiled. Unwashed eggs were superior to those that had been washed but not oiled (Hutchison et al. 2003a). Egg oiling has also been shown to impede the movement of microbes through the shell (Waimaleongora-Ek et al. 2009). If undertaken, egg oiling is carried out immediately after drying and while the egg is still rotating on roller conveyors. A fine spray mist is applied, to ensure that the whole surface of the eggshell is covered. Mineral oils are normally used in commercial practice although studies have also demonstrated the successful use of silicone fluids (Knight et al. 1972). In the EU, oiling is generally not permitted for Class A eggs and is not universally practised on the small number of commercial sites where egg washing is undertaken. In the USA, oiling the eggs after washing is not universal, but it is recommended as it allows an increase in the shelf-life of the eggs (Hutchison et al. 2001). It is mainly used in warmer regions in the USA where there is a risk of inadequate refrigeration or if the eggs are destined for export. It is not considered necessary when eggs were distributed using refrigeration conditions (<12 °C) and likely to be consumed quickly (Hutchison et al. 2003a). 9.5.2 Storage conditions Cooling of table eggs controls the growth of pathogenic bacteria, especially S. Enteritidis, in both internally and externally contaminated eggs. It also helps to restrict eggshell and vitelline membrane penetration by Salmonella spp. (Chen et al. 1996; Gast et al. 2007). Additionally, it controls the growth of spoilage microorganisms and helps to maintain the chemical and physical properties of the egg components (yolk and albumen) and consequently to maintain its internal quality. However, if eggs are refrigerated, they need to be kept at refrigerator temperatures throughout the remainder of the distribution chain mainly because an increase in temperature (possibly accompanied by increased relative humidity) can lead to condensation forming on the eggshell. This in turn may lead to increased risk of growth and penetration of microorganisms, including Salmonella spp., present on the surface of the eggshell (EFSA 2009). The objective is to keep eggs cool and to deliver them quickly to the consumer. In the USA, the most recent regulation pertaining to egg processing applies to all shell eggs and requires them to be stored in a postprocessing environment of 7.2 °C or below (Caudill et al. 2010). Cooling of washed eggs and maintaining these products in this condition was also recommended by EFSA to prevent the growth of bacteria into the egg content (EFSA 2005).
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Egg decontamination by washing 175
9.6 Benefits and problems associated with egg washing 9.6.1 Benefits associated with egg washing The major benefit of washing Class A eggs is the reduction of the microbial load on the eggshell. Hutchison et al. (2004) have shown that S. Enteritidis and S. Typhimurium, artificially inoculated on the eggshell, were reduced by >5 log units by spray jet washing. In the study by Jones et al. (2004), Enterobacteriacea and pseudomonads were not affected by egg washing, while the total aerobes and yeast and mould counts on the shell were lowered by about 2–3 log and 1–2 log units, respectively, during extended storage at 4 °C. A decrease in Enterobacteriaceae counts on the shell surface of washed eggs was observed by Hutchison et al. (2001): no Enterobacteriaceae were counted on the washed eggs, while counts on the unwashed eggs were below 5000 CFU per egg during up to 14 days of storage. It can be expected that the hygienic improvement of the eggshell by washing will lead to a decrease in the potential for cross-contamination during food preparation. This sanitising of the shell might become more important with the increase in non-caged egg production (where laying hens are more in contact with litter, manure and microorganisms living in the environment) and this may even be true in enriched cage systems, where eggs are laid in nestboxes. Trans-shell contamination will be less likely to occur with this reduced microbial load on the eggshell, as it was shown before that eggs that became contaminated, had higher Salmonella counts on the eggshell (De Reu et al. 2006; Messens et al. 2005). On the other hand, egg washing will not prevent egg-related diseases that are vertically transmitted, or when the contaminant has already penetrated the shell. However, it should be noted that egg washing can be applied to cover up poor husbandry and hygiene standards on farms and in packing centres (EFSA 2005). It should also be acknowledged that at present some eggs may be wiped clean or even washed illegally and that controlled washing, properly regulated would be preferable to this. 9.6.2 Problems associated with egg washing If not done properly, washing eggs with brushes and, although possibly to a lesser extent, pressure sprays can cause damage to the cuticle, as shown before (Kuhl 1987; Overfield 1989). EU regulations state that ‘Class A eggs should have a normal, clean and undamaged shell and cuticle’, (Regulation (EC) No. 557/2007). Sparks (1992) found that pressure spray egg washers can cause a significant decrease in the resistance of the cuticle to water uptake due to the erosion of the cuticle. The cuticle can protect the egg against dehydration and offers a natural barrier to common microorganisms, and occasional pathogenic microorganisms, present in the flora that colonise the surface of an egg. As a result, such damage may favour moisture loss and trans-shell contamination with bacteria as studies have demonstrated that the level of cuticle deposition affects the trans-shell penetration: the mean cuticle deposition was lower for penetrated compared with unpenetrated eggshells © Woodhead Publishing Limited, 2011
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176 Improving the safety and quality of eggs and egg products (De Reu et al. 2006). Hence consumer risk can increase, in particular when drying and storage conditions are also sub-optimal. However, as noted at the beginning of this section, and reported recently (Leleu et al. 2011), when optimal conditions are used an egg can be washed without significant degradation of the cuticle. 9.6.3 Optimal conditions for washing eggs It is clear that a detailed blueprint specification for the washing of eggs prior to human consumption, as recommended by Hutchison et al. (2003a), should be strongly supported as the balance between the benefits and the risks of egg washing is highly dependent on the application of these specifications. This blueprint included a requirement that all excessively dirty eggs, as well as those which are cracked, broken or vulnerable, should be removed prior to washing. Eggs should ideally be washed less than 48 hours after being laid and definitely within 7 days (Hutchison et al. 2003a). Eggs must only be washed once, to prohibit excessively dirty eggs being re-washed. The blueprint also specified that the equipment must be easy to keep clean and must have monitoring systems in place to ensure that key washing parameters such as temperature and chemical concentrations are optimal, and that the water should contain less than 2 ppm of iron. Egg washing should be incorporated into an overall documented hazard analysis critical control point (HACCP) system.
9.7 Conclusions The greater use of non-cage systems (free range and barn) for egg laying in future and the end of the conventional cage system in the EU from 2012 could have an impact on the microbiological quality of eggs and a reduction in the number of ‘nest clean’ eggs produced. Egg washing improves the microbiological quality of the table egg at the point of use if the washing process is done properly and is combined with a cool chain. Egg washing is currently not generally allowed in the EU, because of some disadvantages. The major disadvantage of egg washing according to EFSA (2005) is ‘the potential damage to the physical barriers, such as the cuticle, that may favour trans-shell contamination with bacteria and moisture loss and thereby increase the risk to the consumer’. However, as a recent study (Leleu et al. 2011) showed, modern equipment allows eggs to be washed while minimising deleterious effects on the cuticle. Therefore it may be appropriate for the EU and individual member states to consider the use of egg washing as a means of improving the microbial quality of table eggs. It should then be clear that if egg washing is to be used though, the aim should be to produce nest-clean eggs for washing, not to use washing as a
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Egg decontamination by washing 177 panacea for poor production practices. However several other hazards can arise from washing eggs. Key risk factors include water quality and the use of washing water that is significantly below the temperature of the egg, the use of a sanitiser that becomes inactive during processing and failure to thoroughly dry the eggs prior to packing. It should be recognised that egg washing can degrade the cuticle if done under suboptimal conditions, and some methods may be more destructive than others. It should also be borne in mind that washing eggs that are heavily contaminated with organic material will remain a hazard to the consumer as it is likely that the contaminants will have penetrated the shell by the time that the egg is washed and that egg washing will not prevent egg related diseases that are vertically transmitted. On the basis of these factors, it is clear that if undertaken, the egg washing process needs to be properly monitored and controlled, not only by the operator but also through legislation and inspection of the process by appropriate bodies.
9.8 References anderson, k.e., jones, f.t.,
& curtis, p.a. 1992. Heat loss from commercially packed eggs in post-processing coolers, North Carolina Extension Service, Department of Poultry Science, Raleigh, North Carolina Extension Service Commercial Egg Special Report. 1: ER-1 biladeau, a.m. & keener, k.m. 2009. The effects of edible coatings on chicken egg quality under refrigerated storage. Poultry Science, 88, (6) 1266–1274 board, r.g. & tranter, h.s. 1995, ‘The microbiology of eggs,’ In Egg Science and Technology, W. J. Stadelman & O. J. Cotterill, eds., London, UK: The Haworth Press, pp. 81–104 brooks, j., coles, r., & holmes, n.e. 1952. The problem of dirty eggs. The Journal of the Ministry of Agriculture (UK), 59, 311–318 cao, w., zhu, z.w., shi, z.x., wang, c.y., & li, b.m. 2009. Efficiency of slightly acidic electrolyzed water for inactivation of Salmonella Enteritidis and its contaminated shell eggs. International Journal of Food Microbiology, 130, (2) 88–93 catalano, c.r. & knabel, s.j. 1994. Destruction of Salmonella Enteritidis by high pH and rapid chilling during simulated commercial egg processing. Journal of Food Protection, 57, (7) 592–595 caudill, a.b., curtis, p.a., anderson, k.e., kerth, l.k., oyarazabal, o., jones, d.r., & musgrove, m.t. 2010. The effects of commercial cool water washing of shell eggs on Haugh unit, vitelline membrane strength, aerobic microorganisms, and fungi. Poultry Science, 89, (1) 160–168 chen, j., clarke, r.c., & griffiths, m.w. 1996. Use of luminescent strains of Salmonella Enteritidis to monitor contamination and survival in eggs. Journal of Food Protection, 59, 915–921 cotterill, o.j. & hartman, p. 1956. Effect of antibiotic on the incidence of spoilage in shell eggs. Poultry Science, 32, 227–235 de reu, k., grijspeerdt, k., messens, w., heyndrickx, a., uyttendaele, m., debevere, j., & herman, l. 2006. Eggshell factors influencing eggshell penetration and whole egg contamination by different bacteria, including Salmonella enteritidis. International Journal of Food Microbiology, 112, (3) 253–260 de reu, k., messens, w., heyndrickx, m., rodenburg, t.b., uyttendaele, m., & herman, l.
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178 Improving the safety and quality of eggs and egg products 2008. Bacterial contamination of table eggs and the influence of housing systems. World’s Poultry Science Journal, 64, (1) 5–19 efsa. 2005. Opinion of the Scientific Panel on Biological Hazards on the request from the Commission related to the microbiological risks on washing of table eggs. The EFSA Journal, 269, (1) 39 efsa. 2009. Scientific Opinion – Special measures to reduce the risk for consumers through Salmonella in table eggs – e.g. cooling of table eggs – Scientific Opinion of the Panel on Biological Hazards. The EFSA Journal, 957, (1) 29 efsa. 2011. The European Union summary report on trends and sources of zoonoses, zoonotic agents and foodborne outbreaks in 2009. The EFSA Journal, 9, (3) 2090 efsa. 2010. Scientific Opinion on a quantitative estimation of the public health impact of setting a new target for the reduction of Salmonella in laying hens. The EFSA Journal, 8, (4) 1546 favier, g.i., escudero, m.e., & de guzmán, a.m.s. 2005. Genotypic and phenotypic characteristics of Yersinia enterocolitica isolated from the surface of chicken eggshells obtained in Argentina. Journal of Food Protection, 68, 1812–1815 gantois, i., ducatelle, r., pasmans, f., haesebrouck, f., gast, r., humphrey, t.j., & van immerseel, f. 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Reviews, 33, (4) 718–738 garibaldi, j.a. & bayne, h.g. 1960. The effect of iron on the Pseudomonas spoilage of experimentally infected shell eggs. Poultry Science, 39, (6) 1517–1520 garibaldi, j.a. & bayne, h.g. 1962. Effect of iron on Pseudomonas spoilage of farm washed eggs. Poultry Science, 41, (3) 850–853 gast, r.k., guraya, r., guard-bouldin, j., & holt, p.s. 2007. In vitro penetration of egg yolks by Salmonella Enteritidis and Salmonella Heidelberg strains during thirty-sixhour ambient temperature storage. Poultry Science, 86, (7) 1431–1435 goodwin, t.l., wilson, m.l., & stadelman, w.j. 1962. Effects of oiling time, storage position and storage time on the condition of shell eggs. Poultry Science, 41, 840–844 haines, r.b. & moran, t. 1940. Porosity of, and bacterial invasion through, the shell of the hen’s egg. Journal of Hygiene, 40, 453–461 heath, j.l. & wallace, j. 1978. Dilute acid immersion as a method of cleaning egg shells. Poultry Science, 57, (1) 149–155 holley, r.a. & proulx, m. 1986. Use of egg wash water pH to prevent survival of Salmonella at moderate temperatures. Poultry Science, 65, (5) 922–928 humphrey, t.j., wallis, m., hoad, m., richardson, n.p., & rowbury, r.j. 1993. Factors influencing alkali-induced heat-resistance in Salmonella Enteritidis phage type 4. Letters in Applied Microbiology, 16, (3) 147–149 hutchison, m.l., gittins, j., walker, a., sparks, n., & moore, a. 2001, A review of published literature concerning commercial egg washing with particular emphasis on the control of Salmonella, UK Food Standards Agency Project B03017 hutchison, m.l., gittins, j., walker, a., moore, a., burton, c., & sparks, n. 2003a. Washing table eggs: a review of the scientific and engineering issues. World’s Poultry Science Journal, 59, 233–248 hutchison, m.l., gittins, j., walker, a., sparks, n., & moore, a. 2003b, A review of commercial egg washing with particular emphasis on the control of Salmonella, UK Food Standards Agency Project B03017 hutchison, m.l., gittins, j., sparks, a.w., humphrey, t.j., burton, c., & moore, a. 2004. An assessment of the microbiological risks involved with egg washing under commercial conditions. Journal of Food Protection, 67, (1) 4–11 hutchison, m.l., walters, l.d., gittins, j., drysdale, l., & sparks, n. 2006. Egg washing using small-scale bucket washer. Worlds Poultry Science Journal, 62, (2) 259–267 jenkins, m.k. & pennington, m.e. 1919, Commercial preservation of eggs by cold storage USDA Bulletin 775 jones, d.r., musgrove, m.t., & northcutt, j.k. 2004. Variations in external and internal
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Egg decontamination by washing 179 microbial populations in shell eggs during extended storage. Journal of Food Protection, 67, (12) 2657–2660 jones, d.r., musgrove, m.t., caudill, a.b., & curtis, p.a. 2006. Frequency of Salmonella, Campylobacter, Listeria and Enterobacteriaceae detection in commercially cool water-washed shell eggs. Journal of Food Safety, 26, (4) 264–274 kinner, j.a. & moats, w.a. 1981. Effect of temperature, pH, and detergent on survival of bacteria associated with shell eggs. Poultry Science, 60, (4) 761–767 kirunda, d.f.k. & mckee, s.r. 2000. Relating quality characteristics of aged eggs and fresh eggs to vitelline membrane strength as determined by a texture analyzer. Poultry Science, 79, (8) 1189–1193 knight, d.w., bowrey, m., & cooke, d.j. 1972. The preservation of internal egg quality using silicone fluids. British Poultry Science, 13, 587–593 kuhl, h. 1987. Washing and sanitising hatching eggs. International Hatchery Practice, 2, (3) 20–21 leclair, k., heggart, h., oggel, m., bartlett, f.m., & mckellar, r.c. 1994. Modeling the inactivation of Listeria monocytogenes and Salmonella typhimurium in simulated egg wash water. Food Microbiology, 11, (4) 345–353 leleu, s., messens, w., de reu, k., de preter, s., herman, l., heyndrickx, m., de baerdemaeker, j., michiels, c.w. & bain, m. 2011. Effect of egg washing on the cuticle quality of brown and white table eggs. Journal of Food Protection (in press) lorenz, f.w., & starr, p.b. 1952. Spoilage of washed eggs: effect of sprayed versus static water under different washing temperatures. Poultry Science, 31, 204–213 messens, w., grijspeerdt, k., & herman, l. 2005. Eggshell penetration by Salmonella: a review. World’s Poultry Science Journal, 61, 71–85 moats, w.a. 1978. Egg washing – review. Journal of Food Protection, 41, (11) 919– 925 mst 2007. MST Gladiator. http://www.mstegg.com/tech/Tech-glad%20A4.pdf. 23-12010 nield, j.m. 1992. American approach to egg washing, In Cambridge Poultry Conference. Agricultural Developmental and Advising Service, Cambridge, UK, 25 March 1992 nitcheva, l., yonkova, v., popov, v., & manev, c. 1990. Listeria isolation from foods of animal origin. Acta Veterinaria Hungaria, 37, 223–225 northcutt, j.k., musgrove, m.t., & jones, d.r. 2005. Chemical analyses of commercial shell egg wash water. Journal of Applied Poultry Research, 14, (2) 289–295 overfield, n. 1989, Egg washing – time for re-assessment ADAS Report regulation (EC) No. 1907/90 1990. OJ, L173, 5–11 regulation (EC) No. 2052/2005 2005. OJ, L329, 18 regulation (EC) No. 5/2001 2001. OJ, L2, 1–3 regulation (EC) No. 557/2007 2007. OJ, L132, 5–20 sabrani, m. & payne, c.g. 1978. Effect of oiling on internal quality of eggs stored at 28 °C and 12 °C. British Poultry Science, 19, 567–571 sahin, o., kobalka, p., & zhang, q. 2003. Detection and survival of Campylobacter in chicken eggs. Journal of Applied Microbiology, 95, 1070–1079 sanovo engineering 2010. Egg washers: egg washing systems. http://www.briokft.hu/ new/csfiles/220_Egg%20washer.pdf . 23-1-2010 sayers, c.w. 1943. Rotting in eggs. The Agricultural Gazette of New South Wales, 54, 292–296 sparks, n.h.c. 1992, The effect of egg washing on cuticle quality. Report for Ross Breeders Ltd sparks, n.h.c. & board, r.g. 1984. Cuticle, shell porosity and water-uptake through hens eggshells. British Poultry Science, 25, (2) 267–276 sparks, n.h.c. & board, r.g. 1985. Bacterial penetration of the recently oviposited shell of hens eggs. Australian Veterinary Journal, 62, (5) 169–170 stadelman, w.j. 1995, ‘The preservation of quality in shell eggs,’ In Egg science and
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180 Improving the safety and quality of eggs and egg products technology, 4 ed. W. J. Stadelman & O. J. Cotterill, eds., New York, USA, The Howard Press, Inc., pp. 67–79 tranter, h.s. 1994, ‘Lysozyme, ovotransferrin and avidin,’ In Natural antimicrobial systems and food preservation, G. W. Gould et al., eds., Bath UK: Bath University Press, pp. 65–97 upadhyaya, s.k., cooke, j.r., & rand, r.h. 1985. A fluid-filled spherical shell model of the thermo-elastic behavior of avian eggs. Journal of Agricultural Engineering Research, 32, (2) 95–109 usda 2000. Egg grading manual. http://www.ams.usda.gov/AMSv1.0/ getfile?dDocName=STELDEV3004502 . 23-12-2009 waimaleongora-ek, p., garcia, k.m., no, h.k., prinyawiwatkul, w., & ingram, d.r. 2009. Selected quality and shelf life of eggs coated with mineral oil with different viscosities. Journal of Food Science, 74, (9) S423–S429 walters, r.e., robbins, r.o., bryant, a.w., & hamann, j.a. 1966, Improved methods, techniques, and equipment for cleaning eggs, US Department of Agriculture, Marketing Research Report No. 757 wang, h. & slavik, m.f. 1998. Bacterial penetration into eggs washed with various chemicals and stored at different temperatures and times. Journal of Food Protection, 61, (3) 276–279 wellman-labadie, o., picman, j., & hincke, m.t. 2008. Antimicrobial activity of cuticle and outer eggshell protein extracts from three species of domestic birds. British Poultry Science, 49, (2) 133–143
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10 Alternative egg decontamination techniques to washing A. Berardinelli, C. Cevoli, A. Fabbri, M. E. Guerzoni, G. Manfreda, F. Pasquali, L. Ragni and L. Vannini, University of Bologna, Italy
Abstract: This chapter discusses the alternative decontamination techniques to egg washing researched during the last ten years. Hot air or microwave pasteurization represents the most commonly evaluated methods to thermally inactivate the microbial cell, while gas plasma and pulsed light are some techniques where inactivation can be achieved at room temperature. The efficacy of the reviewed alternative methods against different microorganisms and the risks associated with the loss of egg quality such as the cuticle, the shell membranes, the albumen and the yolk are reported. Key words: shell egg decontamination, thermal treatments, non-thermal treatments.
10.1 Introduction To date, in several countries, shell eggs are washed, rinsed and sanitized with an approved egg sanitizer during the on-line egg grading process. As this process involves cuticle removal, with a consequent increase of the internal rate of the carbon dioxide and moisture loss, the eggs are coated with a food grade mineral oil and then refrigerated (USDA, 2000). Even if egg washing can reduce the bacterial load from 1 to 6 log units, this method is still under discussion in terms of advantages and disadvantages (EFSA, 2005). In order to reduce the disadvantages (such as cuticle loss and microbiological risks related to poor practices), disinfectant emissions and water use, and to minimize costs of the grading process and storage, alternative decontamination techniques to washing were analyzed and studied during the last ten years.
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182 Improving the safety and quality of eggs and egg products An alternative solution could also be an important tool in areas such as the European Union where shell eggs cannot be washed (EEC, 2003). The shell egg methods can be classified as thermal and non-thermal. Both thermal and non-thermal reviewed methods were set up taking into consideration not only the pathogen types but also the product physicalchemical characteristics in order to minimize the risks associated with the loss of egg quality, such as the cuticle, the shell membranes, the albumen and the yolk. Hot air or microwave pasteurization represent the most commonly evaluated methods to thermally inactivate the microbial cell, while gas plasma and pulsed light are some of the explored techniques where the inactivation can be achieved at room temperature.
10.2 Washing methods currently used in industry Washing of shell eggs is used commercially in the USA, Canada, Australia and, more recently, in Japan but is not permitted in the European Union for grade A eggs (Bartlett et al., 1993; Zeidler, 2001; Hutchison et al., 2003, 2004). Shell egg washing is usually a continuous process and can be divided into four stages: eggshell wetting, washing, rinsing, and drying (Hutchison et al., 2003). During the wetting step, the shell eggs, positioned on a conveyor belt, are sprayed with warm water to remove fecal matter on the shell. The washing step includes the use of water at least 32.2 °C to ensure proper shell cleaning (water should be at least 11 °C warmer than the egg). During rinsing, the shell eggs are sprayed with clean water, at approximately 60 °C, containing chlorine or quaternary ammonium compounds (USDA, 2000; Hutchison et al., 2003). The drying is the last step, including the mechanical removal of water from the egg surface using warm filtered air (USDA, 2000). Egg washing effectiveness depends on different factors such as the temperature and pH of the water, the equipment adjustment, the characteristics of the sanitizer, detergent and surface-active agents used during the process (Moats, 1978; Stadelman and Cotterill, 1995). In spite of its broad commercial application, egg washing does not eliminate the risk of salmonellosis transmission to humans. Even though egg washing improves the appearance of shell eggs, and reduces the microbial load on the shell, improper washing practices could lead to penetration of Salmonella Enteritidis into the egg contents with subsequent potential microbial proliferation (Kim and Slavik, 1996).
10.3 Hot air pasteurization Hot air pasteurization may represent a valuable alternative for the decontamination of shell eggs. Since the hot air treatment of the eggs involves © Woodhead Publishing Limited, 2011
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Alternative egg decontamination techniques to washing 183 a simple and inexpensive plant, it can be evaluated, before packaging, as an industrial process able to achieve a significant reduction of the bacterial population naturally infecting the surface of the eggs. Pasteurization techniques based on hot air reported in the literature may be classified in two categories substantially depending on the air speed value: forced (FC) and natural (NC) convection methods. The first method is particularly effective for surface decontamination while the natural convection methods are often able to be effective inside the egg. As regards the latter, the most documented experiences make use of convection ovens. In particular, Hou et al. (1996) observed a 5 log10 reduction of the Salmonella Enteritidis load on yolk of eggs treated with hot air oven at 55 °C for 180 min. In addition, these researchers reported that heating internally contaminated shell eggs in water at 57 °C for 25 min, followed by their treatment with hot air at 55 °C for 60 min resulted in microbial reductions by 7 log10. Stadelman et al. (1996) and Hou et al. (1996) reported that the Haugh unit, pH, yolk index, and color showed no significant difference between fresh and pasteurized eggs in oven, while albumen viscosity, turbidity and Hue value (Hue = tan–1 (b/a); a, b color parameters) showed significant differences that indicated partial protein denaturation. Cevoli (2010) observed a 2.5 log10 reduction of the Salmonella Enteritidis load on egg surfaces treated at 55 °C for 200 min without observing significant differences between fresh and pasteurized eggs as regards albumen turbidity, shell color and weight loss. Unfortunately, for an industrial application the oven seems not so promising since it cannot be easily applied on-line and the duration of the treatment might be too long. James et al. (2002) tested the applicability of a hot air gun treatment (FC) on egg surface pasteurization. They produced hot air using a hot air gun with two heat settings (300 and 500 °C). By different experiments they showed that the eggs had to be at least 150 mm from the heat nozzle and exposed no more than 8 s with the heat setting at 500 °C to prevent over-heating. Longer treatment times at 150 mm or shorter distances damaged the shell or partially cooked the egg contents. They investigated the temperatures of the interior and exterior of the egg and identified 180 °C for 8 s as the best treatment corresponding to the highest surface temperature that can be achieved without reaching coagulation temperature inside the egg. Unfortunately no quality and microbiological investigations were performed. Following the idea of a hot air gun, Pasquali et al. (2010) built and microbiologically tested a hot air prototype with the purpose of efficiently reducing the bacterial load on experimentally contaminated and naturally infected eggs without detrimental changes to egg quality traits. On the basis of a numerical model (Fig. 10.1) describing the thermal interaction between the air and the egg proposed by Cevoli (2010) and after some preliminary tests on experimentally inoculated eggs, Pasquali et al. (2010) selected a particular thermal cycle and built a specific apparatus to treat the eggs (Fig. 10.2). The thermal cycle was characterized by two shots of hot
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184 Improving the safety and quality of eggs and egg products Max: 113.356
Time = 8 Surface: Temperature (°C) 0.015
110
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Fig. 10.1 Particular of the numerical model describing the interaction between air and egg built by Cevoli (2010). X-axes: dimension (m). Picture property of University of Bologna.
Fig. 10.2 Prototype used for hot air treatments by Pasquali et al. (2010). Picture property of University of Bologna.
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Alternative egg decontamination techniques to washing 185 air for 8 s with an interval of 32 s of air at room temperature. The authors tested this hot air treatment on eggshell experimentally contaminated by Salmonella Enteritidis and indigenous microflora; Lucchi et al. (2009) and Fabbri et al. (2009) used Listeria monocytogenes and Escherichia coli. Pasquali et al. (2010), Fabbri et al. (2009) and Lucchi et al. (2009) evaluated surface decontamination feasibility at different days post-treatment after a period of shelf-life. Moreover these authors reported that, immediately after the treatment, the bacterial load on eggshell was about 1 log lower in treated eggs than in untreated ones for Salmonella Enteritidis and Listeria monocytogenes. In addition, there was a statistically significant difference in microbial load, with treated eggs being contaminated with the highest load. These data might be explained by a self-adapting metabolic pathway of heat-resistant bacteria, which showed an increased growth rate a few days after the treatment. The authors therefore assumed that this rate, associated with a reduced growth competition, might have induced a higher level of contamination on treated eggs in comparison to untreated ones. In the studies by Pasquali et al. (2010), Fabbri et al. (2009) and Lucchi et al. (2009) the effect of the treatment on the quality of eggs was also evaluated. In particular, the traits were evaluated immediately after the hot air treatment (pH and turbidity of albumen, shell color and cuticle assessment) and after a storage of 28 days at 20 °C (yolk index, eggshell breaking strength and the shell membranes assessment). All authors concluded that the analyzed quality indexes showed no statistically significant differences between treated and untreated eggs, assuming that the treatment had no negative effects on the egg quality. Since a reduction of 1 log10 corresponds to a bacterial population reduction of 90%, all the results presented by Pasquali et al. (2010), Fabbri et al. (2009) and Lucchi et al. (2009) confirm the effectiveness of this treatment in reducing the risk for human health due to Salmonella Enteritidis and Listeria monocytogenes contamination especially if we consider that the Salmonella load on eggshell is often reported to be approximately no more than 102, rising to 103 cfu/ml (or cfu/eggshell) only in rare cases (Humphrey et al., 1991) in eggs collected from hens reared in conventional cages. Other techniques might be coupled with hot air in order to improve its decontamination efficacy. Heat treatments have been already applied successfully on shell eggs or liquid whole egg in combination with other one-shot techniques such as irradiation (Alvarez et al., 2006), ultrasonication (Cabeza et al., 2005) and ozone (Perry et al., 2008). These one-shot techniques might be useful in order to increase the initial log reduction immediately after the treatment but they might not prevent the regrowth of surviving bacteria during storage. In order to minimize this regrowth, hot air pasteurization may be alternatively coupled with long-term techniques able to maintain their decontamination effect thoughout the chain from farm to fork. Two examples are refrigeration are modified atmosphere packaging.
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186 Improving the safety and quality of eggs and egg products
10.4 Microwave pasteurization Microwave heating is compared directly to conventional heating and is frequently found to be less effective owing to non-uniform heating effects, the unpredictability of cold spots (areas of less heat penetration leading to lower internal temperature and less destruction of target organisms), and changing product parameters (NACMCF, 2006). The kinetics of microwave inactivation of organisms should be the same (except for cold spot issues) as for conventional thermal inactivation (NACMCF, 2006). However, the conventional methods for thermal processing of foods result in peripheral over-heating before the food in the centre reaches the required temperature. This is potentially a great problem in pasteurization especially when it comes to shell eggs. Sivaramakrishnan (2007) suggested that microwave heating is an alternative to overcome the peripheral overheating problem during shell egg pasteurization. Different studies have been proposed about the use of microwave to reduce bacterial load in shell eggs (Jeng et al. 1987; Stadelman et al. 1996; Ferroni et al. 2003; Dev et al. 2007; Sivaramakrishnan 2007; Lakins et al. 2008). The effect of microwaves on pathogens can generally be expressed in two forms: thermal and non-thermal destruction of pathogens (Lakins et al. 2008). The first is caused by the heating of food during the microwave process, while the second can be caused by four different phenomena: selective heating, electroporation, cell membrane rupture and magnetic field coupling (Kozempel et al. 1998). The theory of selective heating suggests that microwaves heat solid microorganisms more successfully than by the surrounding medium, and this causes more rapid killing of the organism (Kozempel et al. 1998). Electroporation is related to leakage of cellular content. The electrical potential crosses the membrane of the bacteria causing the formation of pores in the membrane while the voltage across a membrane induces cell membrane rupture. Magnetic field coupling causes a disruption in the internal component of the cell, leading to cell lysis (Lakins et al. 2008). The studies about the use of microwaves to reduce bacterial load in shell eggs may be substantially classified in two categories depending on microwave technology: traditional microwave ovens (Fig. 10.3) and directional microwave. Stadelman et al. (1996) treated shell eggs, containing about 107 cfu/g Salmonella Enteritidis in the yolk, in a traditional microwave oven, and then placed contaminated eggs in a convection oven or in water at 56 °C for different periods of time. These authors observed 7 log10 reductions for the microwave–convection oven and the microwave–water treatments of 120 and 30 min, respectively. They also indicated that these treatments did not negatively affect egg quality.
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Alternative egg decontamination techniques to washing 187 Mode stirrer
Wave guide
Magnetron tube (source of radiation) Oven cavity
Front panel
Fig. 10.3 Traditional microwave oven used for eggshell decontamination. Picture property of University of Bologna.
Dev et al. (2007) studied the effects of microwave oven treatment (power density of 1 W/g for 7 min) on the different properties of albumen: enthalpy of protein denaturation, viscosity, foam density and foam stability, turbidity and dielectric properties. The authors reported that though there was a considerable change in all albumen properties of microwave treated eggs, the changes were much less when compared with those of water bath (60 °C, 20 min) treated eggs. Dev et al. (2008) and Sivaramakrishnan (2007) observed that the time to reach pasteurization temperatures during in-shell heating of eggs by traditional microwave oven ranged between 3.5 and 9 min. No microbiological investigations were performed in these studies. The pasteurization temperatures are defined by the Food Safety and Inspection Services (FSIS) that recommends heating the albumen and the yolk to 57.5 and 61.1 °C, respectively, for 2.5 min to ensure egg safety against Salmonella Enteritidis and other foodborne pathogens. Dev et al. (2009) evaluated microwave pasteurization efficiency on eggs containing about 107 cfu/ml Escherichia coli K12 in the yolk subjected to two different microwave treatments (regular domestic microwave oven and a laboratory microwave oven). The authors concluded that in the laboratory microwave pasteurized samples there were not colony forming units of Escherichia coli present, while the domestic microwave heated samples showed less than 10 cfu/ml. This difference may be due to non-uniformity of heating in the domestic microwave oven. As regards directional microwave technology, Lakins et al. (2008) and Ferroni et al. (2003) proposed treating eggs by an innovative technology very different from commercial or household microwaves using microwave equipment with horizontal and rotary movement and several sources of
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188 Improving the safety and quality of eggs and egg products microwaves (horizontal and vertical). Traditional microwave ovens only have rotary movement and a single source. Using directional microwave technology for 20 s, Lakins et al. (2008) observed a maximum of 2 log10 reduction of Salmonella Enteritidis using a 105 cfu/ml yolk inoculum level. The authors reported that the albumen pH showed no significant differences between fresh and treated egg, while the yolk pH showed significant differences that may be attributed to the formation of microscopic yolk spot, which caused the total solids of the yolk to become more concentrated. Lakins et al. (2009) developed a specific study on the quality traits of shell eggs subjected to directional microwave technology, concluding that the microwave technology can be applied to shell eggs without causing detrimental effects to quality. Using the same technology as Lakins et al. (2008), Ferroni et al. (2003) reported a reduction of up to 80% in Salmonella Enteritidis using a 103 cfu/ml yolk inoculum level. The authors reported that the pH, the weight and the sensory characteristics showed no significant differences between eggs treated by directional microwave technology and non-treated eggs. Pasteurization by directional microwave technology (20 s) is faster than by traditional microwave oven (3–9 min). This makes the directional microwave technology more appropriate for an in-line industrial application. However, there are no studies in the literature related to applicability of microwave technology on egg surface pasteurization. All the studies reported here refer to egg internal pasteurization, particularly of albumen and yolk.
10.5 Gas plasma Gas plasma used for food decontamination is a chemically active ionized gas and is characterized by electrons, ions and neutral species (atoms, molecules and radicals). After ionization, obtained by applying an electric field to the gas mixture, the excited electrons lose their acquired energy through collisions with the neutral species producing ions, and excited species can also be observed. According to their lifetime, these species reach their fundamental state by emitting radiations (especially UV photons) or by collisions with a surface (in this case the microorganisms) causing chemical reactions, mainly oxidations (Moisan et al., 2001). Images obtained by scanning electron microscope suggested an erosion of the outer cell membrane with a consequent exposure of the internal constituents to the reactive discharge (Laroussi et al., 1999). Depending on the power used to activate the electrons and the other species and consequently on their energy levels, plasma can be defined as thermal or non-thermal. Non-thermal or cold plasma is produced at low pressure, such as atmospheric pressure, and is characterized by low levels of electronic density and gas temperature (Moreau et al., 2008). These conditions, producing a non-equilibrium discharge (electronic and gas species are characterized by different temperature values), make the technique suitable for treatments © Woodhead Publishing Limited, 2011
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Alternative egg decontamination techniques to washing 189 where temperature and pressure must be controlled in order to preserve the product. Techniques able to generate gas plasma at atmospheric pressure and with a gas temperature close to ambient temperature were extensively researched and can be related to three main discharge generating modes: by means of AC (alternating current) or DC (direct current) power supply, by a RF (radio frequency) electromagnetic field and by microwave (Tendero et al., 2006). The plasma characteristics (electron and ion behavior) are influenced by the choice of the frequency exciting mode (Arefi-Konsari, 2003). The most industrialized atmospheric plasma source, typically operating at frequencies between 0.5 and 500 kHz, is the dielectric barrier discharge (DBD) method (Kogelschatz, 2003); it is based on two parallel conductive electrodes and a dielectric material covering at least one of them. The dielectric limits the discharge current and is responsible for the uniform distribution of the discharge flowing between the electrodes. Well-known industrial applications of the DBD method include ozone generation (Kamotani et al., 2010), pollution control, polymer surface modification and decontamination of medical devices. If, instead of the dielectric, a high resistivity material to prevent arc is placed in the DBD configuration, the discharge can be operated by using either DC or AC (60 Hz) power supplies (Laroussi, 2002). The method, named resistive barrier discharge (RBD), relatively new in the plasma panorama, can represent an economic way to generate the plasma. Figure 10.4 shows an RBD prototype working with air and set up for the decontamination of shell eggs (Ragni et al., 2010); the discharge is generated between three pairs of parallel plate electrodes made of brass (one of the two electrodes is covered by a 5 mm thick glass sheet) (Fig. 10.5) and the voltage
HV transformers (high voltage)
Pair of electrodes Egg samples
Fig. 10.4 RBD prototype cabinet and electronics set up by Ragni et al. (2010). Picture property of University of Bologna.
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190 Improving the safety and quality of eggs and egg products Electrodes
High voltage source
Air flow
High resistive material to prevent arc
Fig. 10.5 Configuration of a resistive barrier discharge (RBD). Picture property of University of Bologna.
at the electrodes is produced by three high voltage transformers and power switching transistors. Examples of investigated methods able to generate RF and microwave discharges are the atmospheric pressure plasma jet (APPJ) (Schütze et al., 1998) and the microwave plasma jet (MPJ) (Pau et al., 2000) methods, respectively. By using an APPJ device the plasma is produced by subjecting the gas, flowing between the inner and the outer conductors of a coaxial line, to an oscillating electromagnetic field; in MPJ systems the jet is generated by the interaction between the microwave electrical field, the waveguide aperture and the gas nozzle. Besides the plasma generation methods and the type of microorganism, decontamination efficacy is related to the gas mixture composition used (Laroussi, 2002; Moisan et al., 2002). In air plasma the bactericidal effect is mainly due to the presence of reactive oxygen and nitrogen species such as OH and NO radicals, atomic oxygen (O), ozone (O3) and NO2 (Laroussi and Leopold, 2004). OH and NO radicals are formed from the water, and from O2 and N2 dissociation respectively. These reactive species, after the adsorption onto the surface, attack the microorganism membrane, essentially characterized by lipids and proteins, by involving oxidative processes; the membrane consequently loses its role and exposes the genetic material to the plasma components (Moreau et al., 2007). An example of an RBD air glow emission is shown in Fig. 10.6 (Ragni et al., 2010). During the last five years, gas plasma has been investigated as a tool for the decontamination of food such as fruits and vegetables (Critzer et al., 2007; Perni et al., 2008), chilled poultry wash water (Rowan et al., 2007) and food packaging materials (Schneider et al., 2005; Deilmann et al., 2008). These studies showed that this technology is able to inactivate several microbial species, e.g. Escherichia coli, Listeria monocytogenes, Salmonella, Campylobacter jejuni, Campylobacter coli, Bacillus atrophaeus and Aspergillus niger, both on solid surfaces and in liquid media. Significant reductions up to 7 log10 units can be achieved in relation to the type of equipment, the
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191
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Fig. 10.6 Emission spectra of the air plasma glow obtained by means of the RBD prototype (Ragni et al., 2010) with a discharge voltage at electrodes of about 15 kV (RH = 35% at 25 °C). N2 (transition between C3Pu–B3Pg electronic states); N2+ (transition between B 2 S +u – X 2 S g+ electronic states); OH and NO g systems (transition between A2 ∑+ – X2P electronic states). Values in brackets refer to vibrational transition (v¢ Æ v≤). Picture property of University of Bologna.
operating voltage, the gas used in the plasma treatments and the exposure time. Although several studies have been carried out to assess the efficacy and applicability of this technology in different food sectors, only a few have been performed for shell egg decontamination. Higgins et al. (2005) evaluated the effect of ionized reactive oxygen species created using binary ionization technology (BIT) for disinfection of broiler carcasses, table eggs, and treatment of fertile eggs. Binary ionization technology (BIT), developed by Intecon (Sias and Sias, 2002; Sias et al., 2004), is the process of passing a cleaning and disinfecting mist through plasma, resulting in an effective and short disinfection and cleaning process. When BIT was used to disinfect table eggs that had been intentionally inoculated with 6.8 ¥ 108 cfu S. Enteritidis, recovery of the target pathogen from enrichment broths was reduced by 95%. Furthermore, significant reductions of 7.77 and 7.41 log10 units were detected from the BIT-treated eggs when compared with water treated ones (control samples). Davies and Breslin (2003) studied the efficacy of a gas plasma air ionizer (HSC Associates, London) as an alternative technique for reducing S. Enteritidis PT4 on egg surfaces. According to their results, a 5 min exposure to ionized air appeared to have no effect on the number of positive eggs (29.2%) compared with controls (27.1%). Even a longer treatment (20 min) appeared to be associated with only a marginal reduction in the number of contaminated eggs. The effectiveness of the RBD prototype Ragni et al. (2010) was evaluated with eggs deliberately contaminated with S. Enteritidis and S. Typhimurium.
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192 Improving the safety and quality of eggs and egg products The RDB device produced a low temperature after-glow gas mixture able to significantly reduce the populations of both pathogens on shell eggs by different extents, depending on the exposure time and relative humidity (RH) in the treatment chamber. The highest sensitivity values were observed for both microorganisms when the treatments were performed at the highest atmosphere RH value. In particular, maximum reductions of 3.8 and 3.5 log10 cfu/eggshell were observed for S. Enteritidis and S. Typhimurium, respectively. On the other hand, the efficacy of the RBD prototype was tested within the EU project Rescape (Food CT 2006-036018) also on shell eggs experimentally contaminated with Listeria monocytogenes (Vannini et al., 2009a), Escherichia coli, Bacillus cereus (both spores and vegetative cells) and on shell eggs containing only the indigenous microflora (Vannini et al., 2009b). By modulating the process parameters, i.e. exposure time to the gas plasma and the RH, significant cell reductions were observed both for all the target pathogens and the natural microflora immediately after the treatments. Moreover, the evolution of the surviving cells during a 50-day storage at 25 °C showed the inability of the survivors to recover the damage; on the contrary their cell loads decreased down to undetectable levels. A similar behaviour was observed for untreated eggs. However, such a viability reduction was faster in gas plasma treated eggs. According to the results showed by Ragni et al. (2010), gas plasma treatments conducted by the RBD prototype do not involve negative effects on cuticle, eggshell and membranes, albumen and yolk.
10.6 Pulsed light Pulsed light is a method approved by the Food and Drug Administration for food surface microorganism control (FDA, 1996). According to the cited Regulation, the pulsed light technique is based on the application of intense pulses characterized by short durations (no longer than 2 ms) in the range of wavelengths from UV to near-infrared and the limit of 12.0 J/cm2 is fixed (as total cumulative energy of the treatment). The high intensity pulses are obtained by means of a power unit able to generate high voltage powers generally used to charge a capacitor; the electromagnetic energy flows into the xenon gas chamber of the lamp unit (Fig. 10.7) and is then released in the form of emission of very intense flash light, about 20000 times the intensity of sunlight measured at the surface of the earth (Dunn et al., 1995). The pulsed light method is placed in the non-thermal treatment category because the amount of energy and the measured temperature on the food surface do not involve changes in nutritional constituents and in the organoleptic properties. Pulsed light treatments are more effective that the UV continuous disinfection systems because they are characterized by higher penetration depths, emission powers and lower exposure times (Dunn et al., 1995). © Woodhead Publishing Limited, 2011
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Alternative egg decontamination techniques to washing 193 Switch
~ +
~ AC power source
~ –
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DC rectifier
Fig. 10.7 Schematic representation of a pulsed light decontamination method. Picture property of University of Bologna.
The microbial inactivation mode is due to three main mechanisms: photochemical, photothermal and photophysical effects (Elmnasser et al., 2007; Keklik and Demerici, 2009; Oms-Oliu et al., 2010). The photochemical reactions, mainly due to the UV light spectrum composition, cause an inactivation of the cell replication through nucleic acid (DNA) destruction (Takeshita et al., 2003); in particular, the DNA cleavage is attributed to the formation of thymine–thymine dimers. The photothermal effect involves a vaporization of the water from the microorganism cell, leading to a final rupture of the cell as a consequence of temporary overheating imputable to a difference in the adsorption of the light between microorganism and medium (Fine and Gervais, 2004). Finally, structural changes in proteins, membranes and other cellular constituents can occur, following physical disturbance generated by pulsed light. Various systems for food and packaging industrial applications are currently available, such as PureBright® (PurePulse Technologies, San Diego, Ca) and the SteriPulse-XL®3000 (XENON Corporation, Wilmington, MA). The lethality of the method was widely tested against a great number of microorganisms and food typologies (Demerici and Panico, 2008; Oms-Oliu et al., 2010). Besides operating parameters such as the light characteristics (number, rate, wavelength and power of pulses) and the distance of the lamps from the material to be treated, decontamination efficacy is related to its transparency (Fine and Gervais, 2004). Since the efficacy of the method depends on the dose received by the microbial cell, the lethal effect is enhanced in the presence of substrates that limit the shadow effect. In liquid media, a slight penetration of the light can be observed and surface treatments are preferable. Studies have shown that Gram-positive bacteria are more resistant than Gram-negative ones owing to the difference in the cell wall composition (Rowan et al., 1999; Anderson et al., 2010). Studies conducted on shell eggs report the effect of the method against S. Enteritidis on the surface of eggs (Dunn, 1996; Hierro et al., 2009; © Woodhead Publishing Limited, 2011
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194 Improving the safety and quality of eggs and egg products Keklik et al., 2009). By means of treatments characterized by eight flashes at 0.5 J/cm2 for each pulse (4 J/cm2 in total), Dunn (1996) showed a surface microbial reduction up to 8 logs and demonstrated that, even if with lower intensity, the lethal effect is extended to the egg pores. The quality state of the eggshell in terms of cuticle damage greatly influences the efficacy of the treatment, which is suitable for applications on fresh and unwashed eggs (Hierro et al., 2009). To date, data concerning the effects of the technique on the shell egg quality traits have not been reported in the literature.
10.7 Conclusions and future trends Shell egg washing, currently adopted by law in several countries, is still under discussion because of the risk related to poor practice and to possible pathogen penetration due to cuticle loss. With different decontamination power levels, the alternative methods reviewed, which can be classified as thermal and non-thermal, appeared able to reduce the microbial presence on the eggshell. Most of the studies were conducted on Salmonella Enteritidis, Escherichia coli and Listeria monocytogenes. A comparison of methods is difficult because of different microbiological practical inoculations, dynamics of microbial inactivation, substrate typology and egg storage conditions. The selective adoption of emerging processing technologies to provide shelflife extension and enhanced levels of safety for food products requires a deeper knowledge of the mechanisms involved in microbial inactivation. In spite of the quite large amount of research on the efficacy of the above described alternative decontamination techniques on several foods, many questions about the mechanisms involved in microbial inactivation, and the most efficient way of practical application, remain unclear with many apparent contradictory studies. Therefore, for commercial scale-up, the selected equipment and conditions need to be validated with shell eggs and target organisms of interest. Such information is necessary for a quantitative microbial risk assessment which must be performed in order to evaluate the impact of any novel process on microbial risk for the consumers. More research effort must be undertaken to evaluate the projected cost of the treatment for large quantities of food commodities and also the safety of gases used (e.g. for gas plasma) before the proposed techniques become common in the food industry.
10.8 References alvarez i, niemira ba, fan x
and sommers ch (2006), ‘Inactivation of Salmonella serovars in liquid whole egg by heat following irradiation treatments’, J Food Prot, 69, 2066–2074. anderson jg, rowan nj, macgregor sj, fouracre ra and farish o (2010), ‘Inactivation
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Alternative egg decontamination techniques to washing 195 of foodborne enteropathogenic bacteria and spoilage fungi using pulsed-light’, IEEE Trans Plasma Sci, 28, 83–88. arefi-konsari f (2003), ‘Dépôt et traitement des polymères par procédés plasma’, Formation Continue INPG – 17ème session – Traitements de Surface par plasmas, 24–28 March, Grenoble, France. bartlett fm, laird jm, addison cl and mckellar rc (1993), ‘The analysis of egg wash water for the rapid assessment of microbial quality’, Poult Sci, 72, 1584–1591. cabeza mc, garcia ml, de la hoz l, cambero i and ordonez ja (2005), ‘Destruction of Salmonella Senftenberg on the shells of intact eggs by thermoultrasonication’, J Food Prot, 68, 841–844. cevoli c (2010), ‘Hot air treatments for the decontamination of table eggs surface’, PhD dissertation, University of Bologna (Italy). Available from: http://amsdottorato.cib. unibo.it/2517/ critzer fj, kelly-winterberg k, south sl and golden da (2007), ‘Atmospheric plasma inactivation of foodborne pathogens on fresh produce surfaces’, J Food Prot, 70, 2290–2296. davies rh and breslin m (2003), ‘Investigation into possible alternative decontamination methods for Salmonella enteritidis on the surface of table eggs’, J Vet Med Series B, 50, 38–41. deilmann m, halfmann h, bibinov n, wunderlich j and awakowicz p (2008), ‘Low pressure microwave plasma sterilization of polyethylene terephtalate bottles’, J Food Prot, 71, 2119–2123. demerici a and panico l (2008), ‘Pulsed ultraviolet light’, Food Sci Technol Int, 14, 443–446. dev srs, raghavan gsv and gariepy y (2007), ‘Physical properties of egg white after in-shell pasteurization by using microwave or by immersion in hot water’, ASABE Annual International Meeting, Minneapolis, Minnesota, 17–20 June 2007, Paper Number: 072326. dev srs, raghavan gsv and gariepy y (2008), ‘Dielectric properties of egg components and microwave heating for in-shell pasteurisation of eggs’, J Food Eng, 86, 207–214. dev srs, raghavan gsv and gariepy y (2009), ‘Microbial validation of microwave pasteurization of eggs’, ASABE Annual International Meeting, Reno, Nevada, 21–24 June 2009, Paper Number: 097463. dunn j (1996),‘Pulsed light and pulsed electric field for foods and eggs’, Poult Sci, 75, 1133–1136. dunn j, ott t and clark w (1995), ‘Pulsed light treatment of food and packaging’, Food Tech, 49, 95–98. eec (2003), ‘Commission Regulation (EC) No. 2295/2003’, Official J. European Union, 23 December 2003. efsa (2005), ‘Opinion of the scientific panel on biological hazards on the request from the Commission related to the microbiological risks on washing of table eggs’, EFSA J, 269, 1–39. elmnasser n, guillou s, leroi f, orange n, bakhrouf a and federighi m (2007), ‘Pulsedlight system as a novel decontamination technology: a review’, Can J Microbiol, 53, 813–821. fabbri a, cevoli c, pasquali f and manfreda g (2009), ‘Hot air technique for the decontamination of table eggs surface’ CIOSTA XXXIII: Technology and management to ensure sustainable agriculture, agro-systems, forestry and safety, 17–19 June, Reggio Calabria, Italy. fda (1996), Code of Federal Regulations, Title 21, Volume 3. ferroni c, coccoli g, baronio g, piazza s and paterlini f (2003), ‘Evaluation of new treatments for pasteurization-sterilization of shell eggs’, Large Anim Rev, 9, 83–84. fine f and gervais p (2004), ‘Efficiency of pulsed UV light for microbial decontamination of food powders’, J Food Prot, 67, 787–792.
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196 Improving the safety and quality of eggs and egg products hierro e, manzano s, ordóñez ja, de la hoz l
and fernández m (2009), ‘Inactivation of Salmonella enterica serovar Enteritidis on shell eggs by pulsed light technology’, Int J Food Microbiol, 135, 125–130. higgins se, wolfenden ad, bielke lr, pixley cm, torres-rodriguez a, vicente jl, bosseau d, neighbor n, hargis bm and tellez g (2005), ‘Application of ionized reactive oxygen species for disinfection of carcasses, table eggs, and fertile eggs’, J Appl Poultry Res, 14, 716–720. hou h, singh rk, muriana pm and stadelman wj (1996), ‘Pasteurization of intact shell eggs’, Food Micobiol, 13, 93–101. humphrey tj, whitehead a, gawler ah, henley a and rowe b (1991), ‘Numbers of Salmonella enteritidis in the contents of naturally contaminated hens’ eggs’, Epidemiol Infect, 106, 489–496. hutchison, ml, gittins j, walker a, moore a, burton c and sparks n (2003), ‘Washing table eggs: a review of the scientific and engineering issues’, World’s Poult Sci, 59, 233–248. hutchison ml, gittins j, walker a, sparks n, humphrey tj, burton c and moore a (2004), ‘An assessment of the microbiological risks involved with egg washing under commercial conditions’, J Food Prot, 67, 4–11. james c, lechevalier v and ketteringham l (2002), ‘Surface decontamination of shell eggs’, J Food Eng, 53, 193–197. jeng dkh, kaczmarek ka, woodworth ag and balasky g (1987), ‘Mechanism of microwave sterilization in the dry state’, Appl Environ Microbiol, 53, 2133–2137. kamotani s, hooker n, smith s and lee k (2010), ‘Consumer acceptance of ozone-treated whole shell eggs’, J Food Sci, 75, 103–107. keklik nm and demerici a (2009), ‘Pulsed UV-light, advantages for food decontamination’, Resource Magazine of American Society of Agricultural Engineers, July–August, 18–19. keklik nm, demerici a, patterson ph and puri vm (2009), ‘Decontamination of shell-egg with pulsed UV-light’, ASABE Annual International Meeting, 21–24 June, Reno, Nevada 2009, Paper Number: 095974. kim jw and slavik mf (1996), ‘Changes in eggshell surface microstructure after washing with cetylpyridinium chloride or trisodium phosphate’, J Food Prot, 59, 859–863. kogelschatz u (2003), ‘Dielectric-barrier discharges: their history, discharge physics, and industrial applications’, Plasma Chem Plasma Process, 43, 1–46 kozempel mf, annous ba, cook rd, scullen oj and whiting rc (1998), ‘Inactivation of microorganisms with microwaves at reduced temperatures’, J Food Prot, 61, 582–585. lakins dg, alvarado cz, thompson ld, brashears mt, brooks jc and brashears mm (2008), ‘Reduction of Salmonella Enteritidis in hell eggs using directional microwave technology’, Poult Sci, 87, 985–991 lakins dg, alvarado cz, luna am, o’keefe sf, boyce j, thompson ld, brashears mt, brooks jc and brashears mm (2009), ‘Comparison of quality attributes of shell eggs subjected to directional microwave technology’, Poult Sci, 88, 1257–1265 laroussi m (2002), ‘Nonthermal decontamination of biological media by atmosphericpressure plasmas: review, analysis, and prospects’, IEEE Trans Plasma Sci, 30, 1409–1415. laroussi m and leipold f (2004), ‘Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure’, Int J Mass Spectrom, 233, 81–86. laroussi m, sayler g, glascock b, mccurdy b, pearce m, bright n and malott c (1999), ‘Images of biological samples undergoing sterilization by a glow discharge at atmospheric pressure’, IEEE Trans Plasma Sci, 27, 34–35. lucchi a, pasquali f, cevoli c, fabbri a, manfreda g and franchini a (2009), ‘Hot air treatment for surface decontamination of table eggs’, Proceedings of the XIII
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Alternative egg decontamination techniques to washing 197 European Symposium on the Quality of Eggs and Egg Products, 21–25 June 2009, Turku, Finland, EP19. moats wa (1978), ‘Egg washing – a review’, J Food Prot, 41, 919–925. moisan m, barbeau j, moreau s, pelletier j, tabrizian m and yahia lh (2001), ‘Lowtemperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms’, Int J Pharm, 226, 1–21. moisan m, barbeau j, crevier mc, pelletier j, philip n and saoudi b (2002), ‘Plasma sterilization. Methods and mechanisms’, Pure Appl Chem, 74, 349–358. moreau m, feuilloley mgj, veron w, meylheuc i, chevalier s, brisset jl and orange n (2007), ‘Gliding arc discharge in the potato pathogen Erwinia carotovora subsp. atroseptica: mechanism of the lethal action and effect on membrane-associated molecules’, Appl Environ Microbiol, 73, 5904–5910. moreau mf, orange n and feuilloley mgj (2008), ‘Non-thermal plasma technologies: new tools for bio-decontamination’, Biotech Adv, 26, 610–617. nacmcf (2006), ‘Requisite scientific parameters for establishing the equivalence of alternative methods of pasteurization’, J Food Prot, 69, 1190–1216. oms-oliu g, martín-belloso o and soliva-fortuny r (2010), ‘Pulsed light treatments for food preservation. A review’, Food Bioprocess Tech, 3, 13–23. pasquali f, fabbri a, cevoli c, manfreda g and franchini a (2010), ‘Hot air treatment for surface decontamination of table eggs’, Food Control, 21, 431–435. pau cf, yan jd and wylie sr (2000), ‘The influence of the gaz flow rate and microwave source power on the behaviour of a microwave generated argon plasma jet’, XIII International Conference on Gas Discharges and their Applications, 3–8 September, Glasgow, UK, pp. 585–588. perni s, liu dw, shama g and kong m (2008), ‘Cold atmospheric plasma decontamination of the pericarps of fruit’, J Food Prot, 71, 302–308. perry jj, rodriguez-romo la and yousef ae (2008), ‘Inactivation of Salmonella enterica serovar Enteritidis in shell eggs by sequential application of heat and ozone’, Lett Appl Microbiol, 46, 620–625. ragni l, berardinelli a, vannini l, montanari c, sirri f, guerzoni me and guarnieri a (2010), ‘Non-thermal atmospheric gas plasma device for surface decontamination of shell eggs’, J Food Eng, 100, 125–132. rowan nj, macgregor sj, anderson jg, fouracre ra, mcilvaney l and farish o (1999), ‘Pulsed-light inactivation of food-related microorganisms’, Appl Environ Microbiol, 65, 1312–1315. rowan nj, espie s, harrower j, anderson jg, marsili l and macgregor sj (2007), ‘Pulsedplasma gas-discharge inactivation of microbial pathogens in chilled poultry wash water’, J Food Pro, 70 (12), 2805–2810. schneider j, baumgäertner km, feichtinger j, krüger j, muranyi p, schultz a, walker m, wunderlich j and schumacher u (2005), ‘Investigation of the practicability of lowpressure plasmas in the sterilization of food packaging materials at industrial level’, Surf Coat Tech, 200, 962–966. schütze a, jeong jy, babyan se, park j, selwyn gs and hicks rf (1998), ‘The atmospheric pressure plasma jet: a review and comparison to other plasma sources’, IEEE Trans Plasma Sci, 26, 1685–1694. sias rm and sias he (2002), Measurement and cleaning of elastomeric articles having particulate adhered thereto, US Patent 6, 343–425. sias rm, sias he, foster m and stewart t (2004), Apparatus and method for cleaning particulate matter and chemical contaminants from a hand, US Patent 6, 706–243. sivaramakrishnan sr (2007), ‘Microwave pasteurization of shell eggs – a prelude’, Thesis of Master of Science, McGill University Ste Anne de Bellevue, Quebec, Canada. Available online: http://digitool.library.mcgill.ca/R/?func=dbin-jump-full&object_ id=18270&local_base=GEN01-MCG02. stadelman wj and cotterill oj (1995), Egg science and technology, New York, Haworth Press Inc. © Woodhead Publishing Limited, 2011
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198 Improving the safety and quality of eggs and egg products stadelman wj, singh rk, muriana pm
and hou h (1996), ‘Pasteurization of eggs in shell’, Poult Sci, 75, 1122–1125. takeshita k, shibato j, sameshima t, fukunaga s, isobe s, arihara k and itoh m (2003), ‘Damage of yeast cells induced by pulsed light irradiation’, Int J Food Microbiol, 85, 151–158. tendero c, tixier c, tristan p, desmaison j and leprince p (2006), ‘Atmospheric pressure plasmas: a review’, Spectrochim Acta, Part B, 61, 2–30. usda (2000), Egg grading manual, Agricultural Handbook Number 75, Washington, DC. vannini l, montanari c, berardinelli a, ragni l, sirri f and guerzoni me (2009a), ‘Assessment of the efficacy of a low-temperature gas plasma prototype for superficial decontamination of table eggs’, Proceedings of the XIII European Symposium on the Quality of Eggs and Egg Products, 21–25 June, Turku, Finland, pp. 1–8. vannini l, montanari c, berardinelli a, ragni l and guerzoni me (2009b), ‘May lowtemperature gas plasma be considered an innovative technique to decontaminate table eggs?’, Proceedings of EFFOST 2009 Conference on New challenges in food preservation, 11–13 November, Budapest, Hungary. zeidler g (2001), ‘Processing and packaging shell eggs’, In Commercial chicken meat and egg production, Norwell, MA, Kluwer Academic Publishers.
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11 The nutritional quality of eggs I. Seuss-Baum, University of Applied Sciences Fulda, Germany and F. Nau and C. Guérin-Dubiard, AGROCAMPUS Ouest, France
Abstract: Scientific evidence and consumer awareness of the importance of diet in combination with a healthy lifestyle has led to an increased focus on the nutritional evaluation of certain foods such as eggs. Nutritional evaluation includes the nutrient quantification (per 100 g or per portion) of the food, which conveys the contribution of the nutrient content to an individual’s daily supply and the role played by the food as part of a balanced and healthy diet. With this in mind, this chapter describes the function of certain nutrients, taking into account beneficial or adverse effects of an average intake level. Based on this evaluation, the appropriate use of enrichment to improve the nutritional quality of eggs is discussed. Key words: nutrient concentration, nutrient bioavailability, nutrient profile, digestibility, protein, peptides, carbohydrates, lipids, vitamins, minerals, cholesterol, fatty acids.
11.1 Reputation of the egg For centuries, eggs have been a very important source of food owing to their high content of proteins and other essential nutrients, and this is still the case today. Eggs are an important part of the human diet both as shell eggs and as an ingredient in numerous prepared foods. The consumption of eggs in the European Union has remained consistently high (with regional variations; Elmadfa, 2009) over the last 20 years (Fig. 11.1) despite negative reporting in the media, and, as a consequence, something of a bad reputation (Fig. 11.2). © Woodhead Publishing Limited, 2011
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202 Improving the safety and quality of eggs and egg products 18 16 kg per capita
14
13.1
12.6
13.5
13.8
13.4
12 10 8 6 4 2 0
1990*
1995* 2000* 2005** *EU-15 **EU-25 ***EU-27
2008***
Fig. 11.1 Egg consumption per capita in kg in the EU since 1990 (source: MEG, 2009).
Fig. 11.2 Some headlines about eggs.
The negative publicity included reports that eggs were a source of cholesterol and that they constituted a safety risk due to contamination by pathogenic microorganisms (see also Part I, Chapters 1–3) and toxicological relevant residues (see also Part I Chapter 4). Because of their high cholesterol content, eggs are positioned near the top of the ‘food pyramid’ (Fig. 11.3). The ‘food pyramid’ helps to illustrate both the variety and the proportions of foods needed for a healthy diet. Only small amounts are needed from the upper part of the pyramid. From this perspective, eggs are not highly recommended for a healthy diet. In recent decades, the evaluation of eggs as a part of human diet was dominated by debates regarding ‘cholesterol’. However, eggs are also an important supply of various nutrients and substances with special functions and are therefore suitable as part of a balanced diet. A well-balanced diet
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The nutritional quality of eggs 203
Fig. 11.3 Foods of animal origin from the German food pyramid (DGE, 2005).
provides enough energy and nutrients for optimal growth and development of the body. That means that the diet must adequately meet the body’s nutritional needs, by including a wide variety of foods.
11.2 Nutritional evaluation of eggs: composition The nutritional value of foods can be illustrated in different ways. The first option is to list the content of important nutrients per 100 g or per single © Woodhead Publishing Limited, 2011
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204 Improving the safety and quality of eggs and egg products portion of a selected food in order to compare it with other foods. To show the contribution of foods to an individual’s nutrient supply, the nutrient content may be expressed as a percentage of the recommended daily intake (RDI) and as a ratio of nutrients to calories (nutrient density). Another method is ‘nutrient profiling’. The term ‘nutrient profile’ refers to the nutrient composition of a food or diet. ‘Nutrient profiling’ is the classification of foods for specific purposes or as a part of a balanced diet based on their nutrient composition. The nutrient profile of a ‘balanced’ diet is defined by science-based recommendations regarding energy and nutrient intake. Individual foods do not need to match the nutrient profile of a ‘balanced’ diet, but the nutrient profile of the overall diet might be influenced by the nutrient composition of individual foods and the quantity and frequency of intake (EFSA, 2008). 11.2.1 Composition of eggs and its variability Nutrient concentration (per 100 g or per portion) The content of the most important nutrients in 100 g or in one portion of egg is presented in food composition tables or databases. Table 11.1 presents the data from the official German database (BLS – Bundeslebensmittelschlüssel, 2009). A general problem with the evaluation of nutrient content lies in the identification of the best source of data. Various food composition databases and tables are maintained by different EU countries. A list of these databases can be found on the website of the European Food Information Resource Network (EuroFIR, 2005). EuroFIR is a European Network of Excellence on Food Composition Databank Systems established by the EU. In recent years EuroFIR has worked on developing and disseminating a comprehensive, coherent and validated databank to provide a single, authoritative source of food composition data in Europe both for nutrients and newly emerging bioactive compounds with potential health benefits. This network should provide researchers with more uniform data in the future. Tables 11.2–11.4 present the data on the nutrient content of whole egg from some European databases as well as that maintained by the USDA. Most of the values for nutrients vary between countries. Micronutrient (vitamin and mineral) values vary more widely than macronutrient values, with the exception of fat. The range of the values of the data from the different countries can be found in the last column in Table 11.2. These differences could be due to the varying age of the analytical data, the use of different analytical methods, or the normal range of the biological material. The last of these factors probably accounts for most of the variation in egg nutrient composition. It could be caused by variations among breeds or strains of hens or by differences in feeding and husbandry (Stadelman and Pratt, 1989; Baucells et al., 2000; Millet et al., 2006; Garcia-Rebollar et al., 2008). The age of the hens could also play a role in these variations
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5.8 7.5 7.0 30.8 3.2 15.0 14.0 3.1 7.3 44.5 35.0 58.0 16.7 64.0 9.1
Vitamins 0.15 0.28 1 15.0 0.8c 18.8 Vitamin Af (mg) Vitamin D (mg) 1.54 2.9 5 30.8 5c 30.8 Vitamin Eg (mg) 1.1 2.0 13 8.5 12c 9.2 Vitamin K (mg) 25 48 65 38.5 75c 33.3 Thiamine (mg) 0.05 0.1 1.2 4.2 1.1c 4.5
14 + 44 11 16.5
0.3 15.9
100ga as % of RDA-ECc
Minerals 3.3 Sodium (g) 0.08 0.14 0.55 14.5 2.4d Potassium (g) 0.08 0.15 3 2.6 2c 4.0 Calcium (mg) 30 56 900 3.3 800c 3.8 Phosphorus (mg) 115 216 700 16.4 700c 16.4 Magnesium (mg) 6.4 12.1 350 1.8 375c 1.7 Iron (mg) 1.1 2.1 12.5 8.8 14c 7.9 Zinc (mg) 0.74 1.4 8.5 8.7 10c 7.4 Fluoride (mg) 0.06 0.11 3.0 2.0 3.5c 1.7 Iodine (mg) 6 11 200 3.0 150 c 4.0 Selenium (mg) 13e 24.5e 45 28,8 55 c 23.6
12.9 55 0.7 245 11.1 85 1.3 6.5 0.27 + 0.11 1.6 1.1 ~ 7 3.3 –
12.4 0.2 230d 0.2 6.9 70d 8.4 10.8 12.5 2 + 0.25d 7 + 24 8.6 10c 6 – 20d 9.0
6.8 0.4 5.9 0.7 0.14 + 0.06 0.6 1.8
Protein (g) Carbohydrates (g) Fat (g) Essent. fatty acids (g) n-3 fatty acids (ALA+DHA) (g) n-6 fatty acids (g) Saturated fatty acids (g)
1 portion as % of c RDA-EC
one portion RDA-EC or RIVd as % of RDI or EDIb
Constituents per portion per RDI or (53 g)a 100 ga EDIb
c
Table 11.1 Nutrient and energy content of whole egg without shell in one portion (~ 60 g, edible portion 53 g) and 100 g and expressed as percentage of the recommended (or estimated) daily intake (RDI or EDI) for Germany or the recommended daily allowances (RDA-EC) or reference intake values (RIV) for the EU
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0.16 0.06 1.06 34 1.6 13.25 0.85
0.30 0.12 2.0 65 3.1 25.0 1.6
1.4 1.3 3 400 14.5 45 6
11.4 4.6 35.3 8.5 11.0 29.4 14.2
1.4c 1.4c 2.5c 200c 16c 50c 6c
one portion RDA-EC or RIVd as % of RDI or EDIb
c
11.4 4.3 42.4 17.0 10.0 26.5 14.2
82/342
154/646
2500/10450 3.3
2000/8360d
4.1
7.7
21.4 8.6 80.0 32.5 19.4 50.0 26.7
100ga as % of RDA-ECc
b
BLS (Bundeslebensmittelschlüssel – Federal foodstuffs database (Germany)) – version II.3 (2009). RDI Recommended dietary intake and EDI Estimated dietary intake for adults (mean of the values for men and women, 19–60 years) for Germany, Austria and Switzerland (D-A-CH 2000). c EC 2008 (COM Dir 2008/100/EC). d COM 2008/40/EC final, Annex XI, Part B – RIV for salt 6 g ª 2.4 g sodium; citation for n-3 fatty acids: EFSA (2009b). e Scherz et al. (2000). f Retinol equivalents (= 1 mg retinal = 6 mg all-trans-b-carotene = 12 mg other carotenoids). g Tocopherol equivalents (= mg a-tocopherol + mg b-tocopherol ¥ 0.5 + mg g-tocopherol ¥ 0.25 + a-tocotrienol ¥ 0.33). h Folate equivalents (1 mg food folate = 0.5 folic acid, 0.6 mg folic acid taken with meals). i Niacin equivalents (1 mg niacin = 60 mg tryptophan), ALA = a-linolenic acid, DHA = docosahexaenoic acid.
a
Energy content (kcal/kJ)
1 portion as% of c RDA-EC
Others Cholesterol (mg) 210 396 300 70.0
Riboflavin (mg) Vitamin B6 (mg) Vitamin B12 (mg) Folic acid/Folateh (mg) Niacini (mg) Biotin (mg) Pantothenic acid (mg)
Constituents per portion per RDI or (53 g)a 100 ga EDIb
Table 11.1 Continued.
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kcal 152 g 74 g 12.5 g 0.3 g 11.2 g 3.7 g 5.1 g 1.8 g 1.5 g – mg – mg – mg 352
142 75.8 12.6 0.8 9.9 2.6 3.8 1.8 1.25 0.09 0 216 423
Belgiuma Denmarkb 142 75.6 12.6 0.8 9.86 2.64 3.66 1.65 – – – – 378
143 – 12.5 0.3 10.3 2.1 3.3 1.2 0.89 0.06 0 107 360
154 74.09 12.9 0.7 11.2 3.33 4.46 1.51 – – – – 396
UKi
USAj
Range
141 147 143 128–154 76.9 75.2 75.84 74–77.1 12.7 12.6 12.57 12.4–12.9 0.68 – 0.78 0.3–0.8 9.7 10.9 9.94 8.7–11.2 2.8 3.1 3.1 2.1–3.7 3.6 4.7 3.81 2.58–5.1 1.6 1.2 1.36 1.2–1.8 1.34 1.15 0.89–1.50 0.04 – 0.03 0.03–0.09 7 – – 0–7.0 60 – – 60–216 410 – 423 352–423
Netherlandsg Spainh
128 138 77.1 75.4 12.4 12.6 – – 8.7 9.8 3.17 2.4 2.58 3.7 1.26 1.7 1.06 – 0.04 – – – – – 371 354
Francec Finlandd Germanye Italyf
SFA = saturated fatty acids, MUFA = monounsaturated fatty acids, PUFA = polyunsaturated fatty acids, EPA = eicosapentaenoic acid, DHA = docosahexaenoic acid. In some nutrient databases or tables not all nutrients have been listed or no value was presented. a Belgische Voedingsmiddelentabel, NUBEL, Version 3 (2005). b Danish Food Composition Databank, Version 7.01 (2009) – Danish Institute for Food and Veterinary Research. c Composition nutritionnelle des aliments (2008), CIQUAL, AFSSA France. d Fineli – Finnish Food Composition Database, Release 5 (2004) – KTL National Public Health Institute. e BLS [Bundeslebensmittelschlüssel – Federal foodstuffs database (Germany)], Version II.3 (2009). f Tabelle di composizione degli alimenti – Istituto Nazionale della Nutrizione, Edra, Milan (1997). g NEVO Nederlands Voedingsstoffenbestand (Dutch Food Composition Database) (2009). Netherlands Bureau for Nutrition Education, The Hague. h Tablas de composicion de alimentos españoles, Ministerio de Sanidad y Consumo España (1999). i The Composition of Foods, Sixth summary edition, Food Standards Agency (2002), McCane and Widdowson’s. Cambridge: Royal Society of Chemistry. j National Nutrient Database for Standard Reference – USDA – Release 22 (2009).
Energy content Water Protein Carbohydrates Fat SFA MUFA PUFA Linoleic acid Linolenic acid EPA DHA Cholesterol
Table 11.2 Nutrient content per 100 g of whole egg from nutrient tables of different EU countries and the USA
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125 104 72.4 181 11.1 1.7 38.3 0.06 1.01 13 – 125 104
Francec 110 130 57 210 13 2.5 44 – 1.4 22.5 – 110 130
Finlandd 144 147 56 216 12 2.1 10 0.14 1.35 – 0.11 144 147
Germanye 137 133 48 210 13 1.5 53 0.06 1.2 5.8 – 137 133
Italyf
In some nutrient databases or tables not all nutrients have been listed or no value was presented. See Table 11.2 for identifications.
a–j
–
140 130 40 210 13 2 21 0.07 1.4 23.3 – 140 130
116 125 91 312 9 0.9 – 0.1 2.3 – – 116 125
Sodium Potassium Calcium Phosphorus Magnesium Iron Iodine Copper Zinc Selenium Fluoride Sodium Potassium
mg mg mg mg mg mg mg mg mg mg mg mg mg
Belgiuma Denmarkb
125 131 50 179 11 1.8 32.4 0.08 1.33 11 – 125 131
144 147 56.2 216 12.1 2.2 12.7 0.01 2 10 0.11 144 147
Netherlandsg Spainh 140 130 56 200 12 1.9 52.3 0.1 1.4 11.6 – 140 130
UKi
USAj
Range 140 110–144 134 104–147 53 40–91 191 179–312 12 9–13.0 1.83 1.5–2.9 – 10.0–53 0.10 0.06–0.14 1.11 1.01–2.3 31.7 5.8–31.7 1.1 0.11–1.1 140 110–144 134 104–147
Table 11.3 Nutrient content (minerals) per 100 g of whole egg from nutrient tables of different EU countries and the USA
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mg mg mg mg mg mg mg mg mg mg mg mg mg
192 – – – 0.1 0.3 – 2.3 – – – – –
208 179 262 278 225 191 1.75 1.62 2.2 2.9 1.75 1.8 1.8 1.42 2 2 1.11 2.8 0.3 0.8 48 (8.9**) 0.07 0.08 0.12 0.1 0.09 0.1 0.45 0.46 0.37 0.3 0.3 0.3 0.12 0.13 0.14 0.12 0.12 0.17 2 1.36 2.5 2 2.5 2.3 21 45 58.3 65 50 57 0.05 0.08 3.4*** 3.1*** 0.1 0.1 25 – 25 20 1.6 1.58 1.6 1.77 – – 620
*
UKi
USAj
Range
227 190 140 140–278 1.8 1.7 1.2 1.2–2.9 1.9 1.1 1.51 1.1–2.8 8.9 0.3 0.3–48 0.11 0.1 0.07 0.07–0.12 0.37 0.5 0.48 0.3–0.5 0.12 0.12 0.14 0.12–0.17 2.1 2.5 1.29 1.29–2.5 51.2 50.4 47 21–65 3.3 3.8 0.07 0.05–3.8 20 19.4 19.4–25 1.8 1.8 1.44 1.44–1.8 331 331 331–620
Belgiuma Denmarkb Francec Finlandd Germanye Italyf Netherlandsg Spainh
See Table 11.2 for identifications. In some nutrient databases or tables not all nutrients have been listed or no value was presented. ** Souci Fachmann Kraut (2010); the value (48 mg) from the official German database (BLS) seems extremely high. *** Values are listed in the tables as niacin equivalents (means: niacin + tryptophan; 1 mg niacin = 60 mg tryptophan). 1 Retinol equivalents (= 1 mg retinal = 6 mg all-trans-b-carotene = 12 mg other carotenoids). 2 Tocopherol equivalents (= mg a-tocopherol + mg b-tocopherol ¥ 0.5 + mg g-tocopherol ¥ 0.25 + a-tocotrienol ¥ 0.33). 3 Folate equivalents (1 mg food folate = 0.5 folic acid, 0.6 mg folic acid taken with meals).
a–j
Vitamin A Vitamin D Vitamin E 2 Vitamin K Vitamin B1 Vitamin B2 Vitamin B6 Vitamin B12 Folic acid/folate3 Niacin Biotin Pantothenic acid Lutein
1
Table 11.4 Nutrient content (vitamins) per 100 g of whole egg from nutrient tables of different EU countries and the USA
210 Improving the safety and quality of eggs and egg products because the albumen to yolk ratio of whole egg changes with the age of the hen: the yolk percentage (as well as fat percentage) increases, while the total protein concentration decreases (Grashorn, 2007; Rossi et al., 2008). To exemplify the variation between the various sources of data, values from the Danish database were analysed. The values for seven nutrients out of about 30 originate from the USDA tables. The values for five minerals stem from an original paper from 1980, and the oldest reference is that for niacin from 1954. Other European databases present a similar picture. The problem seems to be the widespread origin of the analytical data. A Spanish research group (Aparicio et al., 2008) published new analytical data for Spanish eggs and the difference between the data from this study and that found in the official nutrient tables from Spain was remarkable, mainly with respect to the fat fraction (Table 11.5). As mentioned before this could be a result either of new (and more precise) analytical methods being adopted, or of variations in the composition of the analysed samples. In contrast to the data from the official database, the eggs analysed by Aparicio et al. (2008) were pooled, and the data are therefore based on a homogeneous sample. The obvious difference in the fat fraction (Table 11.2 – range 8.7–11.2 g – and Table 11.5) was not surprising, as manipulation by feeding was described early in the literature (Cruickshank, 1934). In Table 11.6, the composition of egg white and egg yolk is presented. The values are not consistent with the data from Table 11.1 and later tables. The reason is that outlined previously: the source material was not identical. Moreover, Table 11.1 does not mention egg white and egg yolk composition. For the data from the first tables no comparable differentiation in egg white and egg yolk was observed. The problem of varying nutrient content is not unique to egg data. The data for other foods indicate a similar variation. The difficulties encountered in using food composition databases and tables were described by Widdowson and McCance (1943). They wrote: Table 11.5 Comparison of nutrient content of eggs from the official Spanish database and new analytical data Nutrients
Official database1
New analytical data2
Energy content Water Protein Carbohydrates Fat SFA MUFA PUFA
160 – 12.1 0.7 12.1 3.3 4.9 1.8
141 76.9 12.7 0.68 9.7 2.8 3.6 1.6
kcal g g g g g g g
1
Tablas de composicion de alimentos españoles, Ministerio de Sanidad y Consumo España (1999). 2 Aparicio et al. (2008).
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The nutritional quality of eggs 211 Table 11.6 Composition of whole egg, egg yolk and white (in 100 g, without shell; adapted from Nys and Sauveur, 2004) Nutrients
Egg white
Proportionc
60
Energy (kcal) Water (g) Protein (g) Carbohydrates (g) Ash (g) Fat (g)
Egg yolk
Whole egga
CV (%)b
30.7
90.7
47 88.6 10.6 0.8 0.5 0.1
364 49 16.1 0.5 1.6 34.5
154 74.4 12.3 0.7 0.9 11.9
Triglycerides (g) Phospholipids (g)
– –
22.9 10.0
7.7 3.4
– –
Saturated fatty acids (g) 16:0 palmitic acid 18:0 stearic acid
– – –
13.0 7.3 2.5
4.4 2.5 0.86
– 21.4 23
Unsaturated fatty acids (g) 16:1 palmitoleic acid 18:1 oleic acid 18:2 linoleic acid (n-6) 18:3 linolenic acid (n-3) 20:4 arachidonic acid (n-6) 20:5 EPAd (n-3) 22:6 DHAe (n-3)
– – – – – – – –
20.7 1.1 12 3.6 0.12 0.6 0 0.4
7.0 0.4 4.1 1.25 0.04 0.2 0 0.15
– – – 30.4 18 40 – –
Cholesterol (g) Lecithin (g)
0 –
1.2 7.2
0.42 2.30
9.5 –
Minerals (mg) Sodium Chlorine Potassium Calcium Phosphorus Iron Magnesium Sulphur Zinc Copper Manganese Iodine
155 175 140 8 18 0.1 10 163 0.12 0.02 0.007 0.003
Vitamins (mg) Ascorbic acid Vitamin A, retinol equivalents Vitamin D Vitamin E Thiamine Riboflavin Vitamin B 6 Vitamin B12 Folic acid Niacin
0 0 0 0 10 430 10 0.1 12 90
50 162 100 133 530 4.8 15 165 3.9 0.14 0.11 0.14 0 450 4.5 3600 250 480 370 2.8 140 60
– – 1.2 4.7 – 4.6 6.9
120 8–11 172 – 125 8–11 50 8–11 193 8–11 1.7 12 12 – 164 – 1.4 28 0.06 40 0.04 28 0.05 – 0 150 1.5 1200 913 447 133 1 56 79
– 37 95 46 17 21 – 45 35 –
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212 Improving the safety and quality of eggs and egg products Table 11.6 Continued. Nutrients
Egg white
Egg yolk
Whole egga
CV (%)b
Biotin Pantothenic acid
7 250
60 4500
25 1700
32
a
Egg without shell. Coefficient of variation (Gittins and Overfield 1991). c proportion in shell eggs. d EPA = Eicosapentaenoic acid. e DHA= Docosahexaenoic acid. b
There are two schools of thought about food tables. One tends to regard the figures in them as having the accuracy of atomic weight measurements; the other dismisses them as valueless on the grounds that a foodstuff may be so modified by the soil, the season, or its rate of growth that no figure can be a reliable guide to its composition. The truth, of course, lies somewhere between these two points of view. Therefore nutrient databases can give only an approximate or typical indication of the levels of these nutrients. It would be helpful if they could provide average (or median) values for each nutrient, together with a statistical measure of variability which would give users some idea of the level of accuracy that can be expected from the database (EuroFIR, 2005). Why are these data so important? They have been used for analysing dietary intake in metabolic studies, for educational purposes or for evaluation and classification of foods as a part of a ‘healthy’ and balanced diet (nutrient profiling) or for special diets. Nutrient concentration in eggs in proportion to the RDI For the consumer the nutrient content (per 100 g or per portion) alone has no valuable information: what is required is a reference value for the evaluation of the nutrient content. The appropriate references are the RDI for single countries, or, with regard to labelling, the recommended daily allowances issued by the European Commission (RDA-EC; EC, 2008) and transposed into national laws. In Table 11.1 the nutrient content of eggs is demonstrated in terms of the RDI and the RDA-EC. According to the regulation (EC, 2008), as a rule, 15% of the recommended allowance supplied by 100 g or 100 ml or per package if the package contains only a single portion should be taken into consideration in deciding what constitutes a significant amount of nutrients. Per 100 g, then, eggs can be said to contain significant amounts of the following nutrients (Table 11.1): phosphorus, iron, selenium, vitamins A, D, E, K, B12, riboflavin, niacin, folic acid, biotin and pantothenic acid. One portion of egg (i.e. one egg) contains significant amounts of phosphorus, selenium, vitamins A, D, K, B12, folic acid and biotin. In the case of vitamin K, in comparison with other databases
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The nutritional quality of eggs 213 (see Table 11.4), it should be mentioned that the value (of 48 mg/100 g) specified by the official German database seems to be extremely high (see remark below Table 11.4). Using a value of 8.9 mg vitamin K/100 g whole egg (Souci Fachmann Kraut, 2010) for the calculation, the criterion for a ‘significant amount’ is not fulfilled. The particular nutrients are discussed below. Besides the variation in nutrient content, calculations based on different reference values that deviate from RDA-EC, e.g. national RDIs or the ‘guideline daily amounts’ (GDA) issued by the CIAA (Confederation des industries agro-alimentaires de l’UE, 2010), lead to varying percentages and therefore often to confusion (Seuss-Baum, 2007). For vitamins and minerals the former reference labelling values were revised (recommended daily allowances EC; EC, 2008) but for energy and the macronutrients there is currently only a proposal for labelling reference intakes (COM, 2008), which has been reviewed and accepted by the EFSA with the exception of the value for carbohydrates (EFSA, 2009a). Therefore in the future more consistency in nutritional evaluation and transparency in labelling in the EC is expected. Nutrient density and nutrient profile of eggs Nutrient density reflects the ratio of the nutrient content to the total energy content of the food. Therefore the nutrient density is expressed in terms of the amount of a specific nutrient (in weight) per 1000 calories or joules, e.g. the nutrient density of iron in eggs is 13.6 mg/1000 cal (according to Table 11.1) in comparison to 19.4 in meat and 150 in spinach. The only problem is that you have to eat about 500 g of spinach to get 13.6 mg of iron but only 100 g of egg. The contribution of 100 g of egg to the recommended daily energy intake (2000 kcal) is 7.7% but its contribution to most of the essential nutrients is substantially greater (Table 11.1), indicating a high nutrient density. The nutrient profile of a particular food provides information about the presence of nutrients known to have an effect on health, e.g. high concentrations of sodium, saturated fatty acids or sugars. Numerous methods are adopted to create nutrient profiles, e.g. scoring models (Seuss-Baum, 2007) or special indices (Drewnowski and Fulgoni, 2008), and these have been the subject of some debate. The use of nutrition and health claims on food labels could mask the overall nutritional value of a product and could mislead consumers in their choice in the context of a balanced diet. The EC made a proposal (EC, 2009) for the setting up of nutrient profiles, which food must comply with in order to bear nutrition or health claims (according to the Regulation No 1924/2006; EC, 2006). Generally for foods (including eggs) the proposed upper limits for sodium are 300 mg/100 g, for saturated fatty acids 2 g/100 g and for sugars 10 g/100 g. Certain foods or food categories (e.g. spreadable fats, fruits, breads) are exempted because of their role and importance in the diet of the population.
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214 Improving the safety and quality of eggs and egg products Table 11.1 shows that when applied to eggs, the upper limits for sodium and sugars would be unproblematic, but the limit for saturated fatty acids (3.3 g/100 g egg) would be exceeded. In this case, a special regulation would be in force (EC 1924/2006, Chapter II, Art.3 2.b). The discussion about the setting of nutrient profiles is ongoing, awaiting a decision by the EC in the near future. Nutrition claims Assuming that eggs can bear nutrition claims (depending on the nutrient profiles which are yet to be regulated) according to Regulation No 1924/2006 (EC, 2006), the average composition of eggs opens up the options shown in Table 11.7. 11.2.2 Composition versus bioavailability The nutritional value of food is a complicated concept that cannot be simply based on food composition alone. Nutrition, meaning the intake and use of food by the body system, aims to ensure individual maintenance or growth, homeothermy and energy production for physical activities. Nutritional science focuses on phenomena that happen after ingestion, meaning digestion, absorption, storage in tissues or use by the body, and finally excretion. For a long time, the aim was to determine nutritional requirements; today, however, nutrition is analysed with the aim of optimizing the function of nutrients, and in some cases even preventing metabolic disorders and/or pathologies. A nutrient is a food component that has nutritional and physiological effects. The RDI for a specific nutrient is calculated which is designed to ensure that individuals receive a sufficient quantity to meet their daily needs. The calculations are based on 97.5% of the population: more may be required at certain times of life (growth, pregnancy, lactation). The RDI is the average need plus twice standard deviation, which corresponds to the inter-individual variability. Digestibility expresses the degree to which major organic molecules (proteins, lipids, carbohydrates) are transformed by digestive enzymes. Bioavailability is a much more complicated concept, which can be defined in a number of ways. It can be the proportion of a nutrient that is digested, absorbed and metabolized; or the proportion that can be measured in the target tissues after absorption; or the proportion able to fill its biological role after absorption; or the proportion delivered in the bloodstream. The concept of bioavailability then brings together the absorbability (the nutrient’s capacity for absorption by cells of the intestinal epithelium) and the utilization of the nutrient by the organism (Guéguen and Pointillart, 2000). Bioavailability is dependent on physiological factors (human factors, e.g. digestive functions, health and physiological status, existent diseases), as well as on the composition of the food (e.g. presence of inhibitors, impact of food processing). Finally, both absorbability and bioavailability describe the ability of
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The nutritional quality of eggs 215 Table 11.7 Potential options for nutrition claims for eggs (according to EC 1924/2006 – Annex) Potential nutrition claim
conditions applying to them
condition in 100 g egg*
Source of protein
≥ 12% of the energy value provided by protein/100 g
33.5%
High protein
20% of the total energy value/100 g
33.5%
Low sugars
< 5 g/100 g sugar for solids
0.75 g/100 g
Source of … Vitamin A Vitamin D Vitamin E Vitamin K Riboflavin (B2) Niacin Folic acid Vitamin B12 Biotin Pantothenic acid
significant amount ≥ 15% of the RV** ≥ 15% ≥ 15% ≥ 15% ≥ 15% ≥ 15% ≥ 15% ≥ 15% ≥ 15% ≥ 15% ≥ 15%
35% 58% 17% 64% 21% 19% 33% 80% 50% 27%
Source of … Phosphorus Iron Selenium
significant amount ≥ 15% of the RV** ≥ 15% ≥ 15% ≥ 15%
31% 15% 45%
High in … Vitamin A Vitamin D Vitamin K Folic acid Vitamin B12 Biotin
at least twice the value of ‘source of’; ≥ 30% of RV** ≥ 30% ≥ 30% ≥ 30% ≥ 30% ≥ 30% ≥ 30%
35% 58% 64% 33% 80% 50%
High in … Phosphorus Selenium
at least twice the value of ‘source of’; ≥ 30% of RV** ≥ 30% ≥ 30%
31% 45%
*100 g whole egg – see Table 11.1. **RV = reference value (2008/100/EC Annex; EC, 2008).
nutrients to be absorbed and retained by the body system. In vitro methods allow the evaluation of absorbability, while in vivo experiments are needed to determine absorption and retention of nutrients in defined conditions.
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216 Improving the safety and quality of eggs and egg products Intestinal absorption is absolutely necessary for biological activity, but this alone does not mean that the nutrient will be bioavailable for the whole body system. The real bioavailability indeed depends on absorption and excretion. Then, while chemical analysis of a food enables its composition and potential richness in some beneficial components to be determined, it does not allow evaluation of the bioavailability of these nutrients. Indeed, all parameters liable to impact on nutrient accessibility can modify nutrient digestion, absorption and bioavailability. Moreover, parameters liable to impact on nutrient retention must also be taken into account. Finally, bioavailability depends on a number of factors, which include principally the kind of nutrient; the quantity that is ingested; the chemical structure, which can be modified by treatments before eating (processing and/or cooking); the composition of the meal (for example, whether the food matrix contains egg, and if the meal as a whole contains other nutrients which can have a positive or negative effect on absorption); and the status of the consumer (sex, age, health, hormonal and physiological factors, and nutritional status: the last of these refers to any deficiency or surplus of a specific nutrient). For example, egg has a high iron content and has consequently long been considered a good source of this mineral. However, it has now been established that the iron found in egg is poorly bioavailable because of its interaction with phosvitin; furthermore, iron in a complete meal is less bioavailable if egg forms part of this meal (Hallberg and Hulthén, 2000). On the other hand, cobalamin (vitamin B12) is retained in significantly higher quantities in humans when it is administered as egg yolk cobalamin than when administered in its crystalline form (Van Asselt et al., 1996). In addition, although egg is less rich in lutein and zeaxanthine than many vegetables, it is nevertheless considered to be a useful source of these xanthophylls, because they are highly absorbable when provided in egg (Chung et al., 2004). However, lutein bioavailability can be significantly reduced when the whole meal contains certain fibres (Riedl et al., 1999; Hoffmann et al., 1999) or ß-carotene (Kostic et al., 1995). This underlines the difficulty in evaluating the real nutritional value of foods, which can finally be estimated only thanks to in vivo experiments in humans. The difficulties and limitations faced by such studies explain why very few nutrients have been evaluated in conditions that simulate real consumption (essentially minerals, vitamins and pigments). Such studies have a number of disadvantages: they are generally highly expensive; the question of whether the group is truly representative is difficult to resolve; potential influencing external factors are difficult to control; and invasive methods are sometimes necessary for analysis. Finally, among all the published nutritional studies, very few have their main focus on egg, and fewer still have looked at egg using in vivo experiments in humans, despite these providing the most useful results. While a lot of research has been devoted to the enrichment of egg with minerals and vitamins, when these nutrients are provided by
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The nutritional quality of eggs 217 egg, their bioavailability has not yet been analysed. Moreover, owing to the interactions that exist between minerals, absorption and bioavailability should not be evaluated separately for each nutrient. Finally, it is not yet possible to establish whether egg enrichment is an efficient means of adding these elements to food.
11.3 Nutritional evaluation of eggs: macronutrients 11.3.1 Proteins Egg proteins are noteworthy because of their essential amino acid content, which is perfectly suited to human requirements, particularly due to the presence of a large quantity of lysine and sulphur-containing amino acids (Table 11.8). Digestibility and biological value of egg proteins The following criteria are typically used to define the nutritional value of proteins: chemical score, PD-CAAS, digestibility and biological value. The chemical score (CS) is obtained by comparing the amino acid profile of the protein being tested with that of a reference protein. PD-CAAS (proteindigestibility corrected amino acid score) is the chemical score corrected by real faecal digestibility of the protein being examined, measured in humans or in rats (Schaafsma, 2000). Digestibility is the proportion of an ingested amino acid that disappears from the digestive contents during digestion. Biological value (BV) is the proportion of digestible nitrogen (N) that is retained in the body system and used for maintenance or growth expenditures. In vitro digestibility (98%) and biological value measured in rats (94%) classify whole egg proteins among the best protein sources for humans Table 11.8 Essential amino acids content in egg proteins (from Nys and Sauveur, 2004; Ambroise, 2001)
Egg content (mg per 100 g)
Essential amino acids
Whole egg
Egg white
Egg yolk
RDI1 (mg/day)
% RDI covered by 100 g egg
Histidine Isoleucine Leucine Lysine Methionine + cysteine Phenylalanine + tyrosine Threonine Tryptophan Valine
290 660 1040 820 640 1150 590 190 790
240 560 880 660 670 1020 470 170 720
410 870 1390 1170 660 1420 850 240 960
840 1400 2400 2450 1400 2240 1120 280 1340
34.5 47 44 33.5 46 51 53 68 59
1
Amino acids RDI for an adult man (70 kg).
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218 Improving the safety and quality of eggs and egg products (Table 11.9). However, whole egg is no longer the protein reference source; this is an ideal (virtual) protein corresponding to the theoretical amino acid composition capable of covering all the needs of the human organism (‘Requirements’ column in Table 11.9). The PD-CAAS value is very high for egg, higher than for milk, as is the digestibility value. However, the digestibility value indicated here corresponds to faecal digestibility, as evaluated in rats. The first data for ileal digestibility of egg proteins were published by Evenepoel et al. in 1998: it was 51.3% (± 9.8) for raw egg and 90.9% for cooked egg; but it was measured on ileostomized patients. Further data obtained in healthy humans indicated that only 5.73% (± 0.5) of ingested proteins were neither digested nor absorbed when egg is cooked (Evenepoel et al., 1999). Protein assimilation would be lower when eggs are ingested in a complete meal containing proteins, carbohydrates and lipids, compared with when they are consumed in a meal containing purely protein (Geboes et al., 2004). The digestibility of raw egg differs substantially from that of cooked egg. This is usually attributed to the action of protease inhibitors contained in egg white (Matthews, 1990). But structural changes due to cooking would also facilitate the action of digestive enzymes. Moreover, raw egg white proteins weakly stimulate secretion of pancreatic enzymes and bile salts, thus reducing their digestibility (Thimister et al., 1996). In contrast, raw yolk proteins are highly digestible, while overcooking reduces the digestibility of lipoproteins (Nys and Sauveur, 2004). Refrigeration, freezing (Cook and Briggs, 1986), pasteurization (Allemeersch, 1983) or drying (Satyanarayama Rao, 1987) do not modify egg protein digestibility. But all these data were obtained from in vitro studies. Moreover, the dry-heating of egg white has yet to be evaluated; indeed dry-heating is a major technological step that leads to significant modifications in protein structure. Satiating effect of egg proteins In Western countries, the prevention of obesity has become a public health priority. In order to control body weight, it would be useful to identify foods able to give a sustainable sensation of satiety. Generally speaking, proteins seem more efficient in this respect than carbohydrates and lipids (Anderson and Moore, 2004). But very little is known about the satiety power of egg proteins, especially when compared with other protein sources. Moreover, the few studies carried out on humans are inconsistent: while some studies show that egg proteins (egg white proteins) would not be efficient (Anderson and Moore, 2004), some others demonstrate a significant decrease of food intake after a meal containing eggs, compared to a meal that is similar in terms of weight and energy but with no eggs (Vander Wal et al., 2005). Consumption of eggs affects glycaemic and hormonal responses as well as gastric draining kinetics (Pelletier et al., 1996). Yolk seems to be more efficient than egg white; it induces slower gastric draining and an increase
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Lys 61 91 55.8 65
28 50 85 70 28 88 42 14 53
38
Phe + Tyr 123 98 120.53 74
34 48 81 89 40 80 46 – 50
17.7
Met + Cys 132 95 125.4 84
34 63 123 71 33 131 44 – 73
3.5
Soya (seed)1 Meat (beef)1 Cow’s milk1
Met + Cys Met + Cys 81 113 – 95 – 107.7 – 73
23 43 68 75 20 73 41 – 47
22.5
Pea1
In italic, values have been calculated from bibliographic sources; biological value was mesasured in rats. 1 FAO (1985); 2Nys and Sauveur (2004); 3Young and Borgonha (2000); 4Schaafsma (2000); 5Dupin (1992).
Lys 76 – – 73
Limiting amino acid Leu Chemical score (%) 129 Digestibility 4 (%) 98 PD-CAAS (%) 126.45 Biological value5 (%) 94
12.2 25 35 71 31 43 80 31 – 47
12.4 6.7
Egg (hen)2 Rice1 Wheat1
Essential amino acids (mg/g protein) Histidine 23 25 Isoleucine 53 44 Leucine 84 87 Lysine 66 38 Met + Cys 52 39 Phe + Tyr 93 85 Threonine 48 35 Tryptophan 15 – Valine 64 61
Protein (%)
Phe + Tyr 103 – – 95
25 40 87 68 29 67 44 – 45
1.2 – 35 65 50 25 65 25 10 35
Human milk1 Requirements3
Table 11.9 Classification of different protein sources according to their essential amino acid compositions and to the four criteria for their nutritional value evaluation
220 Improving the safety and quality of eggs and egg products in the secretion of some satiety hormones. But it is not clear if yolk proteins are responsible for this. In conclusion, egg could play a useful role in a decreased food consumption strategy, but current knowledge is still insufficient to demonstrate its efficiency for the prevention of obesity. Proteins and peptides with biological activity which improve the nutritional value of egg Many biological activities of egg proteins have been described (Réhault et al., 2007). Among them are anti-hypertensive, anti-inflammatory and antioxidant properties, as well as improvement in the bioavailability of some minerals. All of these factors could be considered as nutritional advantages offered by egg consumption. The anti-hypertensive effect of egg proteins is a result of the inhibition of the angiotensin-converting enzyme (ACE). Ovokinin, a peptide produced by enzymatic digestion of ovalbumin, can then reduce blood pressure in hypertensive rats when administered by the oral route (Fujita et al., 1995). But other peptides obtained by egg white hydrolysis exhibit the same activity (Miguel et al., 2004), and one of them simultaneously exhibits an anti-oxidant effect through inactivation of free radicals that can damage DNA, proteins and lipids (Davalos et al., 2004). Such peptides, with dual activity, could prevent cardiovascular diseases, and especially hypertension. Moreover, because phosvitin is capable of interacting with iron(III) (Lu and Baker, 1986), this egg yolk protein could protect DNA from oxidative injuries due to iron and peroxides. This formed the basis for the suggestion of Ishikawa et al. (2004) that phosvitin could be used to prevent colorectal cancer, which is related to oxidative stress modulated by iron. Because of its strong interaction with iron, and because it is resistant to gastrointestinal proteolytic enzymes, phosvitin could be responsible for the poor availability of iron in eggs; additionally, the presence of eggs in a diet reduces the bioavailability of iron from other meal components (Hallberg and Hulthén, 2000). On the other hand, phosvitin can also bind calcium, and would consequently improve solubility of this element in ileal conditions (Choi et al., 2005); phosphopeptides produced by phosvitin hydrolysis would also increase calcium bioavailability (Jiang and Mine, 2000, 2001) and calcium deposit into bones in rats (Choi et al., 2005). Ovotransferrin, which is an egg white protein able to bind iron reversibly, could be used for iron supplementation (Rupa and Mine, 2006). 11.3.2 Lipids The lipid content in egg yolk does not significantly vary, but fatty acid composition is strongly dependent on the hen’s diet. The unsaturated fatty acid content is always very high in egg yolk in any case (see Chapter 1.6), as is the phospholipid content. Moreover, digestibility of egg triglycerides
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The nutritional quality of eggs 221 and that of phospholipids is excellent (98% and 90%, respectively) (Nys and Sauveur, 2004). Because long chain polyunsaturated fatty acids (LC-PUFA) are preferentially linked to sn-2 position on triglycerides, these fatty acids are essentially absorbed as 2-monoacylglycerol, i.e. by the most efficient means from a metabolic perspective. These characteristics contribute to the high nutritional value of egg lipids. Both linoleic acid and a-linolenic acid are known as essential fatty acids, and cannot be synthesized by animals, particularly humans. Furthermore, they initiate PUFA of n-6 (w6) and n-3 (w3) families, respectively. The high nutritional quality of these components has been clearly demonstrated. The n-6 PUFA can reduce cholesterol plasmatic level (Jacotot, 1988), and the n-3 PUFA can prevent cardiovascular diseases (Schmidt et al., 2006). Although egg is naturally rich in PUFA, many studies have been devoted to increasing its content. It is definitely possible to change the content of these unsaturated fatty acids in egg by simple modification of hen’s diet. Moreover, consumption of w3-enriched eggs by humans actually increases the w3-fatty acid content in the plasma of these consumers (Ferrier et al., 1995; Farrell, 1998). Many reviews exist on that topic (see Chapter 14). More recently, similar research and experiments have been performed with the aim of increasing the content of a special kind of unsaturated fatty acids in egg, specifically conjugated linoleic acids (CLA). These fatty acids have many biological properties, demonstrated either in vitro or in animal models, including: anti-cancer, anti-atherogenic, anti-diabetic, immunostimulating, and hypocholesterolaemic properties (Pariza, 2004). The supplementation of the hen’s diet with these fatty acids does indeed enable the enrichment of eggs. But a concomitant increase in saturated fatty acid content is observed, as well as a decrease in egg PUFA content (Schäfer et al., 2001; Raes et al., 2002; Szymczyk and Pisulewski, 2003; Suksombat et al., 2006). Moreover, the organoleptic and physical properties of eggs are affected by the presence of CLA; specifically, the yolk of hard-boiled eggs are harder, with a rubbery and elastic texture, when eggs are CLA enriched (Ahn et al., 1999); moreover, yolk size increases and some discoloration is observed (Devitt, 1999; Watkins et al., 2003). The enrichment of egg with CLA is then more complicated than enrichment with PUFA. In any case, the enrichment levels mentioned in literature (between 130 and 250 mg CLA per egg) would cover only 4–8% of the RDI of an adult, assuming a daily consumption of one egg (Raes et al., 2002). Cholesterol Despite many experiments and epidemiological studies, the cholesterol content of egg (about 250 mg per egg) remains a real concern for many consumers (see Chapter 12). Nevertheless, it is now well established that ingested cholesterol has no significant influence on cholesterolaemia (Kritchevsky, 2000; Kritchevsky and Kritchevsky, 2000; Herron et al., 2003). Many studies demonstrated that a daily intake of one to two eggs does not increase
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222 Improving the safety and quality of eggs and egg products cholesterolaemia or the risk of cardiovascular disease (Dawber et al., 1982; Hu et al., 1999). Even when hypersensitive human subjects are tested, the LDL-cholesterol increase observed after egg ingestion would not be harmful, because of the concomitant increase of the LDL particle size, which makes them less atherogenic (Herron et al., 2003). These results could be due to the cholesterol-lowering properties of egg white proteins, which have been demonstrated in rats (Yamamoto et al., 1993) and in humans (Asato et al., 1996); ovomucin is especially involved in this activity (Nagaoka et al., 2002). In addition, the phosphatidylcholine and sphingomyeline present in egg yolk inhibit cholesterol absorption in rats (Jiang et al., 2001; Noh and Koo, 2003). It is thus regrettable that the egg has retained its bad reputation/ image despite the large volume of information provided by several countries in an attempt to restore consumer interest in it as a source of nutrition (see also Chapters 12 and 15). 11.3.3 Carbohydrates Carbohydrates are minor components of eggs (<1%). Eggs contain no dietary fibre and the small amounts of carbohydrates have no significance for the carbohydrate and energy supply in the human diet. A few publications have investigated the sialyloligosaccharides found in egg yolk and the results indicate that they may play a role in the defence mechanism against pathogens and may also improve learning ability in infants (Juneja, 2000).
11.4 Nutritional evaluation of eggs: micronutrients 11.4.1 Vitamins Eggs are a good source of vitamins. As shown in Table 11.1 the contribution to the recommended intake values of the different vitamins varies considerably. One egg provides more than 15% of the recommended intake value of vitamins A (retinol-equivalents), D, K, B12, folic acid (folate equivalent) and biotin. Calculated for 100 g (~ two eggs) vitamin E (tocopherol-equivalents), B2 (riboflavin), niacin(-equivalent) and pantothenic acid exceed this percentage and eggs can therefore (according to EC, 2008) be said to contain a ‘significant amount’ of these vitamins. Egg is deficient in vitamin C, because there is no need for this vitamin during the development of the embryo. Fat-soluble vitamins are found almost exclusively in the egg yolk, as are most water-soluble vitamins (Seuss-Baum, 2007; Nys and Sauveur, 2004). Vitamin concentration in egg yolk and egg white is influenced by genetics and by the rate of egg production. Just as in the case of fatty acids and to a lesser extent of other macronutrients, levels of most of the vitamins in egg vary depending on the composition of the hen’s diet (Naber, 1993; Leeson and Caston, 2003). This is also a successful means of manipulating the vitamin content of eggs to improve the already high nutritional value. In © Woodhead Publishing Limited, 2011
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The nutritional quality of eggs 223 literature, examples of adding vitamin E (Flachowsky et al., 2000; Galobart et al., 2002; Jeroch et al., 2002), vitamin D (Mattila et al., 2003), folate and vitamin B12 (Herbert et al., 2005, Bunchasak and Kachana, 2009) to the hen’s diet are described. The efficiency of converting dietary vitamins to egg is variable and depends on the vitamin. As described above the nutritional value of foods should be expressed in terms of reference values outlining the significance of the food as a source of nutrients (Table 11.1). Another important factor in evaluating a food’s nutritional value is the overall supply of particular nutrients in the population, particularly taking into account any pre-existing deficiencies. With respect to the population of the European Union member countries, information about the nutrient supply is summarized in the European Nutrition and Health Report (for 25 member countries; Elmadfa, 2009). In adults the intake of vitamins on average is sufficient with the exception of vitamin D, a-tocopherol equivalents (vitamin E) and folate equivalents. These vitamins should therefore be the focus of further discussions. One egg provides about 30% of the recommended or reference value per person per day of vitamin D. In eggs, it is found almost exclusively as 25hydroxyvitamin D3 (Mattila et al., 2003; Ovesen et al. 2003), which has a greater biological activity than ergocalciferol (D2) and cholecalciferol (D3). Moreover gastrointestinal absorption is more efficient in this form. Vitamin D deficiency is characterized by a higher risk of osteoporosis; a relationship with the development of diabetes, multiple sclerosis, cancer and rheumatoid arthritis has also been discussed. The exposure of the skin to ultraviolet light catalyses the synthesis of vitamin D3: in the absence of exposure to sunlight, vitamin D becomes an essential nutrient. Surprisingly, the lowest mean vitamin D supplies were not found in the countries of northern Europe: the main reason for the relatively good vitamin D status in the Scandinavian countries is probably the fortification of food (EFSA, 2009c), for example the enrichment of eggs. The problem with fortification with vitamin D is the relatively low span between the recommended dietary allowances and the upper intake level (issued by the EFSA, 2009c; Seuss-Baum, 2007). The term ‘Vitamin E’ represents a group of eight naturally occurring homologues including tocopherols and tocotrienols which are nutritionally essential and exhibit antioxidant activity. In this context the main effect is the inhibition of lipid oxidation in foods and biological systems blocking the formation of free peroxide radicals and trapping any free radicals formed. The antioxidant efficacy of the homologues varies with the chemical structure (Elmadfa and Wagner, 2003). It is well recognized that a balanced system of antioxidant substances (b-carotene, vitamin C, vitamin E, some minerals, among others) reduces the susceptibility of cells to oxidative stress. Different forms of vitamin E also exhibit biological activity which is unrelated to their antioxidant properties, e.g. the anti-inflammatory properties which may contribute to a lower risk of development of rheumatoid arthritis, cancer and cardiovascular diseases. The role of tocopherol-equivalents in the prevention
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224 Improving the safety and quality of eggs and egg products of particular diseases is described as inconsistent (EFSA, 2006; Reiter et al., 2007). The contribution of one egg to the recommended reference value of vitamin E is only 9% but in 100 g ‘significant amounts’ (~17%) can be stated (Table 11.1). With respect to folic acid supply, one egg per day covers 8.5% of the RDI (D-A-CH, 2000), 17% of the RDA-EC (EC, 2008). This large variation can be explained by the differences in the reference values. Nevertheless, alongside green vegetables, eggs are a good source of this critical vitamin. High intake in folate may reduce the concentration of plasma homocysteine and so lower the risk of cardiovascular diseases. It has been shown that reduced maternal folate status is associated with the progressive increase in neural tube defects in infants (Scott et al., 2000). For folic acid EFSA accepted claims for the following effects: blood formation, homocysteine metabolism, function of the immune system and maternal tissue growth during pregnancy (EFSA, 2009c). Enzymatic digestibility studies were carried out with egg yolk and the result showed a high conversion of egg yolk folate (~70%) to absorbable folate in comparison to orange juice (~20%) or vegetables (<10%, Seyoum and Selhub, 1998). Feeding experiments showed the feasibility of producing folate-enriched eggs (House et al., 2002; Buchasak and Kachana, 2009). Also worth mentioning are the vitamins B12, K and biotin. The content of these vitamins is significantly high in eggs. B12 is found mainly in animal products but the absorption from eggs seems to be relatively poor compared with absorption from other animal products (Watanabe, 2007). For vitamin K, phylloquinone, EFSA (2006) made no recommendation for the daily intake and stated that an adequate intake would be provided by a normal diet; other good sources are green vegetables and vegetable oils. Biotin, a water-soluble vitamin, plays an important role in cell metabolism and the utilization of macronutrients. Comparable to vitamin K, the biotin requirement cannot be accurately estimated, but must be met by dietary uptake. One egg contributes significantly (~25%) to the RDA. The bioavailability of biotin is high in cooked or processed eggs but low in raw eggs. This is caused by the presence of the absorption inhibiting protein avidin in raw egg white (Mock, 1996). Vitamin retention in eggs during processing Nutritional evaluation depends on the nutrient content in the food products at time of consumption. This means that the effects of processing must be considered. The significance of nutrient retention is determined by the presence of a significant amount, the sensitivity to processing and the overall supply of the nutrient in the population. Vitamins are less stable than minerals during processing. In this context the main focus should be placed on the retention of vitamin D, E and folate. Normal household cooking of eggs was studied and the results show little effect on vitamin D retention (>90%, Mattila et al., 1999). Comparable losses (~10%) in folate and retinol (17–20%, Ramalho et al., 2006) were found in
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The nutritional quality of eggs 225 boiled eggs, but severe heating (fried, scrambled) resulted in higher folate losses (~30%, Ottaway and Ottaway, 2002). Vitamin E is quite stable during storage (Meluzzi et al., 2000) and boiling, but spray-drying decreased the tocopherol content of the egg product (Galobart et al., 2001). Xanthophylls Xanthophylls (except b-crypto-xanthine) are carotenoids with no vitamin A activity. The most common xanthophylls in eggs are lutein and its stereoisomer zeaxanthin; they contribute to the colour of eggs, together with other carotenoids. These substances are interesting because they accumulate in the macular region of the retina and are associated with a reduced incidence of age-related macular degeneration (ARMD). There are some reports which claim that there is a relationship between lutein and zeaxanthin intake via egg yolk and a higher macular pigment optical density (Moeller et al., 2000; Olmedilla et al., 2001; Granado et al., 2003) but the response to the treatment varies among individuals (Wenzel et al., 2006). Epidemiological studies show inconsistent results (Cho et al., 2008; Tan et al., 2008). Moreover, a review of the potential roles of xanthophylls in disease prevention suggests a protective effect against certain kinds of cancers, coronary heart disease and stroke (Ribaya-Mercado and Blumberg, 2004). Dietary lutein and zeaxanthin in egg yolk may be more bioavailable because of the lipid matrix, which may facilitate absorption (Handelman et al., 1999). Alongside dark-green leafy vegetables, egg yolk could contribute to a better supply of these xanthophylls. The concentration of carotenoids in the egg depends on husbandry type, genetic variations, processing and storage conditions (Schlatterer and Breithaupt, 2006; Wenzel et al., 2010). 11.4.2 Minerals Eggs are also a good source of minerals. According to the EC (2008), only the phosphorus (16.4%) and selenium (23.6%) content meets the definition of ‘significant amount’ for one egg; for 100 g, iron (15%) is sufficient and zinc (14%) just misses the mark. Besides these important elements, eggs contain almost all minerals and trace elements in small amounts (Table 11.1). Comparable to fatty acids and vitamins, the mineral content of eggs varies with their concentration in the hen’s diet, depending on the mineral and on various enhancing or inhibiting factors (Stadelman and Pratt, 1989). Following the procedure described for vitamins (see Section 11.4.1), the evaluation of critical minerals is based upon the supply of nutrients in the populations of the European Union. In most of the participating countries of the European Nutrition and Health Report (Elmadfa, 2009) the intake of calcium, magnesium and iron (women only) was below the respective recommendations for adults. Eggs play no important role in meeting the RDI of calcium and magnesium. On the other hand, egg yolk is rich in iron. As mentioned above in the section
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226 Improving the safety and quality of eggs and egg products dealing with bioavailability, iron in yolk is poorly bioavailable (~1–10%, Hallberg et al., 1997). The reason for this is that iron is present in ferric form and interacts with the phosvitin found in egg yolk. Additionally, the presence of eggs in the diet reduces the bioavailability of iron from other food sources. Combining eggs in the diet with enhancing factors such as vitamin C, citric acid, cysteine-containing peptides or ethanol results in significantly increased bioavailability of iron (Hallberg and Hulthen, 2000).
11.5 Improving the nutritional quality of eggs 11.5.1 Methods Improving the nutritional value of egg consists of either reducing or increasing the content of various nutrients. Reduction trials mainly concern cholesterol. However, while enrichment of egg is easily achieved through modification of the hen’s diet, it is much more difficult, or even impossible, to decrease the cholesterol content of egg by this way (Nys and Sauveur, 2004). Indeed, in laying hens, cholesterol is almost completely synthesized de novo in the liver, two-thirds of it being deposited in the yolk. The maximal reduction obtained through modification of the hen’s diet and selection programmes was less than 5% for cholesterol content in yolk. A greater reduction (45%) could be obtained through supplementation of the hen’s diet with atorvastatin (0.06%), one of the most efficient drugs for hypercholesterolaemia treatment in humans (Elkin et al., 1999). But treatment of this sort seems unacceptable for food production; moreover, it significantly lowers egg production. There are many more examples of the enrichment of egg with other nutrients: PUFA, some minerals, vitamins and xanthophylls. For all these kinds of component, enrichment is easily obtained by supplementation of the hen’s diet with the corresponding nutrient. Some enrichment levels can indeed be very high. Iodine and selenium contents can thus be up 60 times and 10 times higher than the initial content, respectively (Stadelman and Pratt, 1989). In addition, w3-fatty acid content can be increased up to 8 times (Lewis et al., 2000a), and w6/w3 ratio can be decreased to 1.8 (Bean and Leeson, 2003) or 1.9 (Botsoglou et al., 1998). Moreover, after ingestion of enriched eggs, w3 content of the plasma of the human volunteers increases, without significant modification of circulating cholesterol and triglycerides (Ferrier et al., 1995; Farrell, 1998; Lewis et al., 2000b). 11.5.2 Problems or consequences of nutritional improvement Firstly, it is not possible to enrich egg with all nutrients, essentially because of the physiological limits of the hen. Thus, enrichment will only concern nutrients that are directly deposited in the egg, without being metabolized by the hen. And even when enrichment is possible, nutrient transfer from the diet of the hen to the egg can be sometimes dependent on external factors. © Woodhead Publishing Limited, 2011
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The nutritional quality of eggs 227 For example, the response to diet enrichment is not always dose-dependent; a plateau has thus been observed for folate enrichment, suggesting the folate may be deposited in the egg in two different physiological ways, depending on supplementation level (House et al., 2002). The molecular way by which a nutrient is supplied to the hen can also influence its transfer into the egg; seleno-methionine (organic form) is thus the most efficient way to increase selenium content in egg (Payne et al., 2005), whereas inorganic selenium is metabolized as seleno-cystein in glutathione peroxydase (Forstrom et al., 1978), which protects the egg (shell and inner content) from free radical activity. Some conflicts also exist between nutrients to be absorbed by the hen and deposited in the yolk, for example between copper and zinc (Skrivan et al., 2005), between vitamins A and E (Surai et al., 1998; Grobas et al., 2002), and between on the one hand vitamins A and E, and on the other hand carotenoids (Surai et al., 1998), meaning that egg cannot be simultaneously enriched in liposoluble vitamins and carotenoids. While egg enrichment with antioxidant components could seem useful for PUFA enriched eggs, vitamin E deposit in yolk is reduced when high PUFA supplementations are provided to hens (Meluzzi et al., 2000). Moreover, the enrichment of egg can sometimes affect egg quality: when hens are supplemented with magnesium, this mineral is generally deposited in the egg shell, decreasing the calcium deposit and finally shell quality (Atteh and Leeson, 1983); as vitamin B12 content in egg increases, shell thickness and solidity decrease (Squires and Naber, 1992). But sometimes, positive side effects can be observed, due to interactions between nutrients. Thus, simultaneous supplementation of the hen’s diet with zinc, copper and iron increases iron content in egg in a more pronounced way than when simple iron supplementation is performed (Skrivan et al., 2005). Vitamin D supplementation enables egg content enrichment as well as improvement of shell egg quality (Frost et al., 1990; Mattila et al., 2003). Another question concerns nutrient stability after egg storage and/or processing. For example, eggs can be enriched in vitamin B1 (thiamin), but the thiamine content of the egg decreases during egg storage, all the more quickly as temperature increases (Hisil and Otles, 1997). While drying does not affect bioavailability or the biological value of egg proteins, powder storage in inappropriate conditions (high temperature and hygrometry) would promote a Maillard reaction which would have already begun during drying, with a significant resultant decrease in protein availability (Bergquist, 1977; Cotterill et al., 1978). Storage at room temperature would also result in an increase in cholesterol oxides and a decrease in PUFA in whole egg powder (Mazalli and Bragagnolo, 2007). Egg cooking, processing and storage can sometimes induce a decrease in nutrient content, as is the case for the xanthophylls found in yolk (Schlatterer and Breithaupt, 2006; Wenzel et al., 2010). Finally, the risk of oversupply of some nutrients must be considered, as high doses of some nutrients can also cause adverse effects. For example,
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228 Improving the safety and quality of eggs and egg products model calculations with milk and eggs show that the upper limit of iodine for adults and adolescents could be exceeded with the current approved maximum iodine level in feed for cows and laying hens (Seuss-Baum, 2007). Moreover, since the effects of interactions between nutrients on bioavailability in humans are not yet known, it is still impossible to say whether the multienrichment strategy (currently applied) is right for egg production.
11.6 Conclusions The nutritional value of eggs can be described by various methods. The result is: eggs are a good source of high quality protein and of certain vitamins and minerals. Most of the nutrients are highly bioavailable. The exact composition of the egg (especially the lipid fraction) is dependent on the diet of the hen. This is one reason for differences in the concentration of some nutrients in eggs found in European food databases. Moreover divergent recommended dietary allowances for individual European countries used as reference values lead to confusion. Nevertheless, based on their nutrient content, eggs are a suitable part of a balanced diet. A moderate enrichment of eggs with certain nutrients (e.g. PUFA and vitamins) in accordance with harmonized recommended dietary allowances and the regulations for nutrition and health claims could lead to reasonable concepts of ‘functional eggs’. The enrichment of food, however, must not cause a risk of oversupply of nutrients.
11.7 Sources of further information and advice: (food tables) BLS (2009), Bundeslebensmittelschlüssel – Federal foodstuffs database (Germany), Version II.3. CIQUAL (Centre informatique sur la QUalité des aliments) (2008), ‘Composition nutritionnelle des aliments – Table Ciqual’, AFSSA, http:// www.afssa.fr/TableCIQUAL/ Danish Institute for Food and Veterinary Research (2009) Danish Food Composition Databank, Version 7.01, Available from: www.foodcomp. dk [accessed February 2010] Fineli® - Finnish Food Composition Database, Release 5 (2004) - KTL National Public Health Institute. Available from: www.fineli.fi/food. php?foodid=858&lang=en [accessed February 2010] Food Standards Agency (2002) McCance and Widdowson’s The Composition of Foods, Sixth summary edition. Cambridge, Royal Society of Chemistry Instituto Nazionale della Nutrizione (1997): Tabelle di composizione degli alimenti, Edra Milano, Italia © Woodhead Publishing Limited, 2011
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The nutritional quality of eggs 229 Ministerio de Sanidad y Consumo (1999) Tablas de composicion de alimentos espanoles. Madrid NEVO (2009) Nederlands Voedingsstoffenbestand (Dutch Food Composition Database). Netherlands Bureau for Nutrition Education, The Hague NUBEL (2005) Belgische Voedingsmiddelentabel, Version 3. Available from: www.nubel.com [accessed February 2010] USDA (2009) USDA National Nutrient Database for Standard Reference – Release 22. Available from: http://www.nal.usda.gov/fnic/foodcomp/ search/ [accessed February 2010]
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and johnson e (2004), ‘Lutein bioavailability is higher from lutein-enriched eggs than from supplements and spinach in men’, J Nutr, 134, 1887–1893. ciaa (Confederation des industries agro-alimentaires del’UE, Brussels) (2010), Guideline Daily Amounts, Available from: http://gda.ciaa.eu, (accessed February 2010). com (2008), COM 2008/40 final: Proposal for a Regulation of the European Parliament and of the Council of 30 January 2008 on the provision of food information to consumers. cook f and briggs gm (1986), ‘The nutritive value of eggs’, in Stadelman WJ and Cotterill OJ, Egg science and technology, AVI Publishing Company, Westport, 141–163. cotterill oj, glauert j and froning gw (1978), ‘Nutrient composition of commercially spray-dried egg products’, Poultry Sci, 57(3), 439–442. cruickshank em (1934), ‘Studies in the fat metabolism in the fowl. I. The composition of the egg fat and depot fat of the fowl as affected by the ingestion of large amounts of different fats’. Biochemical Journal, 28, 965–977. d-a-ch (2000), Referenzwerte für die Nährstoffzufuhr [Reference values for nutrient intake], 1. Aufl., Frankfurt, Umschau Verlag. davalos a, miguel m, bartolome b and lopez-fandino r (2004), ‘Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis‘, J Food Protect, 67, 1939–1944. dawber tr, nickerson rj, brand fn and pool j (1982), ‘Eggs, serum cholesterol, and coronary heart disease’, Am J Clin Nutr, 36, 617–625. devitt aa (1999), ‘Nutraceutical fatty acid modification of chicken egg yolk‘, MS Thesis, Department of Food Science, Purdue University, West Lafeyette, IN. dge (Deutsche Gesellschaft für Ernährung) (2005), Dreidimensionale Lebensmittelpyramide. Available from: http://www.dge.de/modules.php?name=News&file=article&sid=481 [accessed February 2010]. drewnowski a and fulgoni 3rd v (2008), ‘Nutrient profiling of foods: creating a nutrientrich food index’, Nutr Rev, 66, 1, 21–22. dupin h (1992), ‘Besoins nutritionnels et apports conseillés pour la satisfaction de ces besoins’, in Alimentation et Nutrition Humaines, Paris, ESF Ed. ec (2006), Regulation (EC) No 1924/2006 of the European Parliament and of the council of 20 December 2006 on nutrition and health claims made on foods. ec (2008), Commission Directive 2008/100/EC amending Council Directive 90/496/EEC on nutrition labelling for foodstuffs as regards recommended daily allowances, energy conversion factors and definitions – OJ, L285, 28.10.2008, 9. ec (2009), European Commission Health and Consumers directorate-general, ‘Working document on the setting of nutrient profiles’, 13/02/2009. efsa (2006), ‘Tolerable Upper Intake Levels for Vitamins and Minerals’, Scientific Panel on Dietetic products, nutrition and allergies (NDA) and Scientific Committee on Food (SCF), Available from: http://www.efsa.europa.eu/en/home/oldsc/upper_level_ opinions_full-part33,0.pdf [accessed January 2010]. efsa (2008), ‘The setting of nutrient profiles for foods bearing nutrition and health claims pursant to article 4 of the regulation (EC) No. 1924/2006’, EFSA J., 644, 1–44. efsa (2009a), ‘Scientific Opinion of the Panel on Dietetic products, Nutrition and Allergies on a request from European commission on the review of labelling reference intake values’, EFSA J., 1008, 1–14. efsa (2009b), ‘Scientific Opinion of the Panel on Dietetic products, Nutrition and Allergies on a request from the Commission related to labelling reference intake values for n-3 and n-6 polyunsaturated fatty acids’, EFSA J, 1176, 1–11. efsa (2009c), ‘Scientific Opinion on the substantiation of health claims related to folate and blood formation (ID 79), homocysteine metabolism (ID 80), energy-yielding metabolism (ID 90), function of the immune system (ID 91), function of blood
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The nutritional quality of eggs 231 vessels (ID 94, 175, 192), cell division (ID 193), and maternal tissue growth during pregnancy (ID 2882) pursuant to Article 13(1) of Regulation (EC) No. 1924/20061’, EFSA J, 1213, 1–22. elkin rg, yan z, zhong y, donkin ss, buhman kk, story ja, turek jj, porter jrre, anderson m, homan r and newton rs (1999), ‘Select 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors vary in their ability to reduce egg yolk cholesterol levels in laying hens through alteration of hepatic cholesterol biosynthesis and plasma VLDL composition’, J Nutr, 129, 1010–1019. elmadfa i (2009), European nutrition and health report II, Basel, Karger. elmadfa i and wagner k h (2003), ‘Non-nutritive bioactive food constituents of plants: tocopherols (vitamin E)’, Int J Vitam Nutr Res, 73, 2, 89–94. eurofir (2005), European Food Information Resource Network, ‘Food composition databases’. Available from: www.eurofir.net [accessed January 2010]. evenepoel p, geypens b, luypaerts a, hiele m, ghoos y and rutgeerts p (1998), ‘Digestibility of cooked and raw egg protein in humans as assessed by stable isotope techniques’, J Nutr, 128, 1716–1722. evenepoel p, claus d, geypens b, hiele m, geboes kp, rutgeerts p and ghoos y (1999), ‘Amount and fate of egg protein escaping assimilation in the small intestine of humans’, Am J Physiol Gastrointest Liver Physiol, 277, 40, G935–G943. fao/who/unu (1985), ‘Energy and protein requirements: report of an FAO/WHO/UNU expert consultation’, WHO Tech Rept Ser No 724, World Health Organization, Geneva. farrell dj (1998), ‘Enrichment of hen eggs with n-3 long-chain fatty acids and evaluation of enriched eggs in humans’, Am J Clin Nutr, 68, 538–544. ferrier lk, caston lj, leeson s, squires j, weaver bj and holub bj (1995), ‘Alpha-linolenic acid- and docosahexaenoic acid-enriched eggs from hens fed flaxseed: influence on blood lipids and platelet phospholipid fatty acids in humans’, Am J Clin Nutr, 62, 1, 81–86. flachowsky g, engelmann d, sünder a, halle i and sallmann hp (2000), ‘Eggs and poultry meat as tocopherol sources in dependence on tocopherol supplementation of poultry diets’, Proceedings of 5th Karlsruhe Nutrition Congress, Session 3P, 102. forstrom jw, zakowski jj and tappel al (1978), ‘Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine’, Biochemistry, 17, 2639–2644. frost tj, roland da and untawale gg (1990), ‘Influence of vitamin D3, 1 alphahydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 on eggshell quality, tibia strength, and various production parameters in commercial laying hens’, Poultry Sci, 69 (11), 2008–2016. fujita h, sasaki r and yoshikawa m (1995), ‘Potentiation of the antihypertensive activity of orally administered ovokinin, a vasorelaxing peptide derived from ovalbumin, by emulsification in egg phosphatidylcholine‘, Biosci Biotechnol Biochem, 59, 2344–2345. galobart j, barroeta ac, baucells md, cortinas l and guardiola f (2001), ‘Alphatocopherol transfer efficiency and lipid oxidation in fresh and spray-dried eggs enriched with omega3-polyunsaturated fatty acids’, Poult Sci, 80, 10, 1496–1505. galobart j, barroeta ac, cortinas l, baucells md and codony r (2002), ‘Accumulation of alpha-tocopherol in eggs enriched with omega-3 and omega-6 polyunsaturated fatty acids’, Poult Sci, 81, 12, 1873–1876. garcıa-rebollar p, cachaldora p, alvarez c, de blas jc and mendez j. (2008), ‘Effect of the combined supplementation of diets with increasing levels of fish and linseed oils on yolk fat composition and sensorial quality of eggs in laying hens’, Animal Feed Sci Technol, 140, 337–348. geboes kp, bammens b, luypaerts a, malheiros r, buyse j, evenepoel p, rutgeerts p and verbeke k (2004), ‘Validation of a new test meal for a protein digestion breath test in humans‘, J Nutr, 134, 806–810.
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232 Improving the safety and quality of eggs and egg products and overfield nd (1991), ‘The nutrient content of eggs in Great Britain’, in: Oosterwod A and de Vries AW, Proceeding of the 4th European symposium on the quality of eggs and egg products, Beekbergen, Netherlands, 113–116. granado f, olmedilla b and blanco i (2003), ‘Nutritional and clinical relevance of lutein in human health’, Br J Nutr, 90, 487–502. grashorn ma (2007), ‘Faustzahlen zur Eiqualität’, in Zentralverband der deutschen Geflügelzüchter e.V., Jahrbuch für die Geflügelwirtschaft 2008, Stuttgart, Verlag Eugen Ulmer. grobas s, méndez j, lopez bote c, de blas c and mateos gg (2002), ‘Effect of vitamin E and A supplementation on egg yolk a-tocopherol concentration’, Poultry Sci, 81, 376–381. guéguen l and pointillart a (2000), ‘The bioavailability of dietary calcium’, J Am Coll Nutr, 19, 119S–136S. hallberg l and hulthén l (2000), ‘Prediction of dietary iron absorption: an algorithm for calculating absorption and bioavailability of dietary iron’, Am J Clin Nutr, 71, 1147–1160. hallberg l, hulthén l and gramatkovski e (1997), ‘Iron absorption from the whole diet in men: how effective is the regulation of iron absorption?’, Am J Clin Nutr, 66, 347–356. handelman gj, nightingale zd, lichtenstein ah, schaefer ej and blu jb (1999), ‘Lutein and zeaxanthin concentrations in plasma after dietary supplementation with egg yolk’, Am J Clin Nutr, 70, 2, 247–251. herbert k, house jd and guenter w (2005), ‘Effect of dietary folic acid supplementation on egg folate content and the performance and folate status of two strains of laying hens’, Poultry Sci, 84, 1533–1538. herron kl, vega-lopez s, conde k, ramjiganesh t, shachter ns and fernandez ml (2003), ‘Men classified as hypo- or hyper-responders to dietary cholesterol feeding exhibit differences in lipoprotein metabolism’, J Nutr, 133(4), 1036–1042. hisil y and ötles s (1997), ‘Changes of vitamin B1 concentrations during storage of hen eggs’, Lebensm-Wiss u-Technol, 30, 320–323. hoffmann j, linseisen j, reidl j and wolfram g (1999), ‘Dietary fiber reduces the antioxidative effect of a carotenoid and alpha-tocopherol mixture on LDL oxidation ex vivo in humans’, Eur J Nutr, 38, 278–285. house jd, braun k, balance dm, o’conno cp and guenter w (2002), ‘The enrichment of eggs with folic acid through supplementation of the laying hen diet’, Poult Sci, 81, 9, 1332–1337. hu fb, stampfer mj, manson je, rimm eb, wolk a, colditz ga, hennekens ch and willett wc (1999), ‘Dietary intake of alpha-linolenic acid and risk of fatal ischemic heart disease among women‘, Am J Clin Nutr, 69(5), 890–897. ishikawa s, yano y, arihara k and itoh m (2004), ‘Egg yolk phosvitine inhibits hydroxyl radical formation from the Fenton reaction’, Biosci Biotechnol Biochem, 68, 1324– 1331. jacotot b (1988), ‘Acides gras alimentaires pour la prévention du risque coronarien’, Cah Nutr Diét, 23, 211–214. jeroch h, eder k, schöne f, hirche f, böttcher w, sesekeviciene j and kluge h (2002), ‘Amounts of essentiel fatty acids, a-tocopherol, folic acid, selenium and iodine in designer eggs’, International Symposium on Physiology of Livestock, Lithuanian Veterinary Academy, 31–32. jiang b and mine y (2000), ‘Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin’, J Agric Food Chem, 48, 990–994. jiang b and mine y (2001), ‘Phosphopeptides derived from hen egg yolk phosvitin: effect of molecular size on the calcium binding properties’, Biosci Biotech Biochem, 65, 1187–1190. gittins je
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and koo si (2001), ‘Egg phosphatidylcholine decreases the lymphatic absorption of cholesterol in rats’, J Nutr, 131, 2358–2363. juneja lr (2000), ‘Biological characteristics of egg components, specifically sialyloligosaccharides in egg yolk’, in Sim JS, Nakai S and Guenter W, Egg Nutrition and Biotechnology, Wallingford, CAB International, 233–242. kostic d, white ws and olson ja (1995), ‘Intestinal absorption, serum clearance, and interactions between lutein and b-carotene when administered to human adults in separate and combined oral doses’, Am J Clin Nutr, 62, 604–610. kritchevsky d (2000), ‘Dietary fat and disease; what do we know and where do we stand’, in Sim JS, Nakai S and Guenter W, Egg Nutrition and Biotechnology, New York, CAB Int Publishing. kritchevsky sb and kritchevsky d (2000), ‘Egg consumption and coronary heart disease: an epidemiologic overview’, J Am Coll Nutr, 19(Suppl. 5), 549S–555S. leeson s and caston lj (2003), ‘Vitamin enrichment of eggs’, J Appl Poult Sci Res, 12, 24–26. lewis nm , seburg s and flanagan nl (2000a), ‘Enriched eggs as a source of n-3 polyunsaturarted fatty acids for humans’, Poultry Sci, 79, 971–974. lewis nm, schalch k and scheideler se (2000b), ‘Serum lipid response to n-3 fatty acid enriched eggs in persons with hypercholesterolemia’, J Am Diet Assoc, 100, 365–367. lu cl and baker r (1986), ‘Characteristics of egg yolk phosvitin as an antioxidant for inhibiting metal-catalyzed phospholipid oxidations’, Poultry Sci, 65, 2065–2070. matthews dm (1990), Protein absorption: development and present state of the subject, New York, Wiley-Liss. mattila p, ronkainen r, lehikoinen k and piironen v (1999), ‘Effect of household cooking on the vitamin D content in fish, eggs, and wild mushrooms’, J Fd Comp Anal, 12, 3, 153–160. mattila p, rokka t, könkö k, valaja j, rossow l and ryhänen el (2003), ‘Effect of cholecalciferol-enriched hen feed on egg quality’, J Agric Food Chem, 51, 283– 287. mazalli mr and bragagnolo n (2007), ‘Effect of storage on cholesterol oxide formation and fatty acid alterations in egg powder’, J Agric Food Chem, 55(7), 2743–2748. meg (2009), Marktbilanz – Eier und Geflügel 2009, Stuttgart, Eugen Ulmer Verlag. meluzzi a, sirri f, manfreda g, tallarico n and franchini a (2000), ‘Effects of dietary vitamin E on the quality of table eggs enriched with n-3 long-chain fatty acids’, Poultry Sci, 79, 539–545. miguel m, recio i, gomez-ruiz ja, ramos m and lopez-fandino r (2004), ‘Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis’, J Food Protect, 67, 1914–1920. millet s, de ceulaer k, van paemel m, raes k, de smet s and janssens gp (2006), ‘Lipid profile in eggs of Araucana hens compared with Lohmann selected leghorn and ISA Brown hens given diets with different fat sources’, Br Poult Sci, 47, 3, 294–300. mock dm (1996), ‘Biotin’, in: Luft F, Ekhard ZE and Filer LJ, Present knowledge in nutrition. 7th edn, Washington, DC, ILSI Press, 220–235. moeller sm, jacques pf and blumberg jb (2000), ‘The potential role of dietary xanthophylls in cataract and age-related macular degeneration’, J Am Coll Nutr, 19, 5, 522–527. naber ec (1993), ‘Modifying vitamin composition of eggs: a review’, J Appl Poultry Res, 2, 385–393. nagaoka s, masaoka m, zhang q, hasegawa m and watanabe k (2002), ‘Egg ovomucin attenuates hypercholesterolemia in rats and inhibits cholesterol absorption in Caco-2 cells’, Lipids, 37(3), 267–272. noh sk and koo si (2003), ‘Egg sphingomyelin lowers the lymphatic absorption of cholesterol and a-tocopherol in rats‘, J Nutr, 133, 3571–3576.
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234 Improving the safety and quality of eggs and egg products and sauveur b (2004), ‘Valeur nutritionnelle des oeufs’, INRA Prod Anim, 17, 5, 385–393. olmedilla b, granado f, blanco i, vaquero m and cajigal c (2001), ‘Lutein in patients with cataracts and age-related macular degeneration: a long-term supplementation study’, J Sci Food Agric, 81, 904–909. ottaway pb and ottaway b (2002), ‘The stability of vitamins during processing’, in: Henry CJK and Chapman C, The nutrition handbook for food processors, Boca Raton, Woodhead Publishing, 247–264. ovesen l, brot c and jakobsen j (2003), ’Food contents and biological activity of 25hydroxyvitamin D: a vitamin D metabolite to be reckoned with?’, Ann Nutr Metab, 47, 107–113. pariza mw (2004), ‘Perspective on the safety and effectiveness of conjugated linoleic acid‘, Am J Clin Nutr, 79, S1132–S1136. payne rl, lavergne tk and southern ll (2005), ‘Effect of inorganic versus organic selenium on hen production and egg selenium concentration’, Poultry Sci, 84, 232–237. pelletier x, thouvenot p, belbraouet s, chayvialle ja, hanesse b, mayeux d and debry g (1996), ‘Effect of egg consumption in healthy volunteers: influence of yolk, white or whole-egg on gastric emptying and on glycemic and hormonal responses‘, Ann Nutr Metab, 40(2), 109–115. raes k, huyghebaert g, de smet s, nollet l, arnouts s and demeyer d (2002), ‘The deposition of conjugated linoleic acids in eggs of laying hens fed diets varying in fat level and fatty acid profile’, J Nutr, 132, 182–189. ramalho hmm, santos vva, medeiros vpq, silva khd and dimenstein r (2006), ‘Effect of thermal processing on retinol levels of free-range and caged hen eggs’, Int J Fd Sci Nutr, 57, 3–4, 244–248. réhault s, anton m, nau f, gautron j and nys y (2007), ‘Les activités biologiques de l’œuf’, INRA Prod Anim, 20(4), 337–348. reiter e, jiang q and christen s (2007), ‘Anti-inflammatory properties of a- and g-tocopherol, Mol Aspects Med, 28, 5–6, 668–691. ribaya-mercado jd and blumberg jb (2004), ‘Lutein and zeaxanthin and their potential roles in disease prevention. J Am Coll Nutr, 23, 567S–587S. riedl j, linseisen j, hoffmann j and wolfram g (1999), ‘Some dietary fibers reduce the absorption of carotenoids in women’, J Nutr, 129, 2170–2176. rossi m, hidalgo a, clerici f (2008), ‘Whole egg whipping properties as affected by albumen and yolk fraction changes’, 1st Mediterranean Summit of WPSA – Advances and Challenges in Poultry Science, 7–10 May 2008. rupa p and mine y (2006), ‘Egg Proteins’, in Mine Y and Shahidi F, Nutraceutical science and technology (4), Nutraceutical protein and peptides in health and disease, Boca Raton, London, New York, CRC Press, Taylor & Francis Group, 445–459. satyanarayama rao ts (1987), ‘Studies on the spray-dried, foam-mat-dried and freezedried whole egg powder: changes in the nutritive qualities on storage’, Nutr Rep Int, 36(6), 1317–1323. schaafsma g (2000), ‘The protein digestibility-corrected amino acid score’, J Nutr, 130, 1865S–1867S. schäfer k, männer k, sagredos a, eder k and simon o (2001), ‘Incorporation of dietary linoleic and conjugated linoleic acids and related effects on eggs of laying hens’, Lipids, 36(11), 1217–1222. scherz h, senser f, souci sw, fachmann w and kraut h (2000), Souci/Fachmann/Kraut – Food composition and nutrition tables, 6th edn, Stuttgart, Medpharm Scientific Publishers. schlatterer j and breithaupt de (2006), ‘Xanthophylls in commercial egg yolks: quantification and identification by HPLC and LC-(APCI)MS using a C30 phase’, J Agric Food Chem, 54, 2267–2273. nys y
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The nutritional quality of eggs 235 schmidt eb, rasmussen lh, rasmussen jg, joensen am, madsen mb
and christensen jh (2006), ‘Fish, marine n-3 polyunsaturated fatty acids and coronary heart disease: a mini-review with focus on clinical trial data’, Prostaglandins Leukot Essent Fatty Acids, 75(3), 191–195. scott j, rebeille f and fletcher j (2000), ‘Review – folic acid and folates: the feasibility for nutritional enhancement in plant foods’, J Sci Food Agric, 80, 795–824. seuss-baum i (2007), ‘Nutritional evaluation of egg compounds’, in Huopalathi R, LopezFandino R, Anton M and Schade R, Bioactive Egg Compounds, Berlin, Heidelberg, Springer, 117–144. seyoum e and selhub j (1998), ‘Properties of food folates determined by stability and susceptibility to intestinal pteroylpolyglutamate hydrolase action’, J Nutr, 128, 11, 1956–1960. skřivan m, skřivanová v and marounek m (2005), ‘Effects of dietary zinc, iron, and copper in layer feed on distribution of these elements in eggs, liver, excreta, soil and herbage’, Poultry Sci, 84, 1570–1575. souci fachmann kraut (2010), ‘Die Zusammensetzung der Lebensmittel, NährwertTabellen – Hühnerei – Gesamteiinhalt’ (last update 2005), Medpharm Online Datenbank, Deutschen Forschungsanstalt für Lebensmittelchemie, Garching, Available from: www.sfk-online.net [accessed January 2010] squires mw and naber ec (1992), Vitamin profiles of eggs as indicators of nutritional status in the laying hen: vitamin B12 study, Poult Sci, 71(12), 2075–2082. stadelman wj and pratt de (1989), ‘Factor’s influencing composition of the hen’s egg‘, World Poultry Sci J, 45, 247–266. suksombat w, samitayotin s and lounglawan p (2006), ‘Effects of conjugated linoleic acid supplementation in layer diet on fatty acid compositions of egg yolk and layer performances’, Poultry Sci, 85(9), 1603–1609. surai pf, ionov ia, kuklenko tv, kostjuk ia, macpherson a, speake bk, noble rc and sparks nh (1998), ‘Effect of supplementing the hen’s diet with vitamin A on the accumulation of vitamins A and E, ascorbic acid and carotenoids in the egg yolk and in the embryonic liver’, Brit Poultry Sci, 39(2), 257–263. szymczyk b and pisulewski pm (2003), ‘Effects of dietary conjugated linoleic acid on fatty acid composition and cholesterol content of hen egg yolks’, Br J Nutr, 90(1), 93–99. tan js, wang jj, flood v, rochtchina e, smith w and mitchell p (2008), ‘Dietary antioxidants and the long-term incidence of age-related macular degeneration: the Blue Mountains Eye Study’, Ophthalmology, 115, 2, 334–341. thimister pwl, hopman wpw, sloots cej, rosenbusch g, willems hl, trijbels fjm and jansen jbmj (1996), ‘Role of intraduodenal proteases in plasma cholecystokinin and pancreaticobiliary responses to protein and amino acids’, Gastroenterology, 110, 567–575. van asselt dz, van den broek wj, lamers cb, corstens fh and hoefnagels wh (1996), ‘Free and protein-bound cobalamin absorption in healthy middle-aged and older subjects‘, J Am Geriatr Soc, 44(8), 949–953. vander wal js, marth jm, khosla p, jen c and dhurandhar nv (2005), ‘Short-term effect of eggs on satiety in overweight and obese subjects’, JACN, 24(6), 510–515. watanabe f (2007), ‘Vitamin B12 sources and bioavailability’, Exp Biol Med, 232, 1266–1274. watkins ba, feng s, strom ak, devitt aa, yu l and li y (2003), ‘Conjugated linoleic acids alter the fatty acid composition and physical properties of egg yolk and albumen’, J Agric Food Chem, 51, 6870–6876. wenzel aj, gerweck c, barbato d., nicolosi rj, handelman gj and curran-celentano j (2006), ‘A 12-wk egg intervention increases serum zeaxanthin and macular pigment optical density in women’, J Nutr, 136, 2568–2573.
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236 Improving the safety and quality of eggs and egg products wenzel m, seuss-baum i
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12 Eggs, dietary cholesterol and disease: facts and folklore B. A. Griffin, University of Surrey, UK
Abstract: The Oxford English Dictionary defines the word myth as; ‘a popular idea concerning natural or historical phenomena … that has no foundation in fact’. The popular idea in this context is that eating dietary cholesterol, typically from eggs, increases the risk of developing coronary heart disease (CHD), because it increases blood cholesterol. This contentious idea prevails, despite a lack of scientific foundation to support its existence, and almost global re-vamping of dietary recommendations to lift restrictions on the intake of cholesterol-rich foods. In an attempt to dispel the mythical status of dietary cholesterol and CHD, the following chapter will examine the role of dietary cholesterol in relation to what has been well established in terms of the relationships between blood cholesterol, diet and CHD. Key words: eggs, dietary cholesterol, serum cholesterol, LDL, coronary heart disease.
12.1 Egg nutrition: facts and folklore Eggs contain a high concentration of cholesterol and are the principal source of cholesterol in the diet of most populations. The cholesterol content of a yolk in a hen’s egg varies with the size of the egg and the amount of yolk but on average there is between 150 and 200 mg of cholesterol. Duck eggs have a higher content of cholesterol that is proportional to their greater size. Certain shellfish are another major source of dietary cholesterol, some of which contain similar amounts on a gram per gram basis to egg yolk. Historically, dietary recommendations to prevent cardiovascular disease © Woodhead Publishing Limited, 2011
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238 Improving the safety and quality of eggs and egg products (CVD) have restricted the intake of these foods on the grounds that dietary cholesterol increases serum (low density lipoprotein, LDL) cholesterol which increases risk. This placed eggs at the top of a food frequency pyramid for many years as a food that should be eaten only occasionally. From a nutritional standpoint, this advice is now deemed to be inappropriate for the prevention of CVD and for health in general, especially in view of the risk arising from a pandemic of obesity and associated metabolic disease. Eggs are relatively low in energy and fat compared with many other foods, giving them a low energy to nutrient density ratio (Gray and Griffin, 2009). This property makes eggs entirely appropriate for inclusion in balanced, energy-controlled diets that are designed to promote weight loss with the aim of reducing risk of obesity and its co-morbidities (Herron and Fernandez, 2004). Eggs provide the richest source of bioavailable essential amino acids (Layman and Rodriguez, 2009), and a combination of vitamins and minerals that is unmatched by any other single food (Table 12.1). Past recommendations to restrict their intake originate from the scientifically unfounded idea that dietary cholesterol is directly equivalent to blood cholesterol. What follows is a scientific perspective of our understanding of the relationship between dietary cholesterol, serum cholesterol and coronary heart disease (CHD) risk.
Table 12.1 Selected composition of raw hen’s egg. Source: Gray J and Griffin BA (2009), ‘Eggs and dietary cholesterol – dispelling the myth’, Nutr Bull, 34, 66–70. Per 100 g
Per medium egg (average 58 g)
Energy kJ/kcal Protein (g) Carbohydrate (g) Fat (g) Saturated fatty acids (g) Monounsaturated fatty acids (g) Polyunsaturated fatty acids (g) Cholesterol (mg) Retinol (mg) Vitamin D (mg) Riboflavin (mg) Folate (mg) Vitamin B12 (mg) Phosphorus (mg) Iron (mg) Zinc (mg) Iodine (mg) Selenium (mg)
324/78 6.5 trace 5.8 1.7 2.3 0.9 227 98 0.9 0.24 26 1.3 103 1.0 0.7 27 6
627/151 12.5 trace 11.2 3.2 4.4 1.7 391 190 1.3 0.47 50 1.8 200 1.9 1.3 53 11
Source: FSA (2002).
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Eggs, dietary cholesterol and disease: facts and folklore 239
12.2 Serum cholesterol and dietary cholesterol as risk factors for coronary heart disease The story of the long and controversial history of the ‘lipid’ or ‘cholesterol hypothesis’ that defines the relationship between serum cholesterol (LDL) and CHD has been told in an insightful set of reviews by Steinberg (2004, 2006). The first observation to link cholesterol with CHD was made by Windaus in 1910, who noted a higher concentration of cholesterol in the aortas of patients with CHD (Windaus, 1910). However, it was an earlier suggestion that high protein diets may be associated with ageing of tissues that led a young experimental pathologist from the Military Medical Academy in St. Petersburg, Nicolai Anitschkow, to feed rabbits a high protein diet of meat, eggs and milk. He discovered, somewhat unintentionally, that the rapid development of atherosclerosis in his rabbits had nothing to do with dietary protein but was linked instead to the cholesterol-raising effects of the dietary cholesterol from the eggs (Anitschkow and Chalatov (1913)). Anitschkow went on to confirm these findings by comparing the effects of pure cholesterol dissolved in sunflower oil against a sunflower oil control (Anitschkow, 1913). The dietary cholesterol increased the blood cholesterol of his rabbits to between 500 and 1000 mg/dl (12–26 mmol/l), that is, between 2 to 5 times higher than a desirable level in people, and as predicted, this accelerated the development of coronary atherosclerosis. Sadly, these landmark observations were dismissed on the grounds that the rabbit, as a species, was overly sensitive to dietary cholesterol and thus an inappropriate animal model in which to study such phenomenon. Although this conclusion is entirely correct, the non-acceptance of Anitschkow’s early findings meant that the world would remain in ignorance of the causal relationship between blood cholesterol and CHD until the late 1940s. At around this time, the identification of plasma lipoproteins, and specifically LDL, as the principal transporter of blood cholesterol, heralded a significant advance in our understanding of the causal role of blood cholesterol in CHD (Cohn et al., 1946; Gofman et al., 1950). Nevertheless, it would be another 50 years before the ‘cholesterol hypothesis’ would gain universal acceptance on the strength of a mass of irrefutable evidence from cross-cultural, prospective cohort and randomly controlled intervention studies with cholesterol-lowering drugs. The Seven Countries Study provides one of the most prolific examples of cross-cultural evidence to link raised blood cholesterol with CHD across a range of countries with the highest blood cholesterol and CHD risk such as Finland, to populations with lower cholesterol and CHD risk in the Mediterranean, to the lowest of all in Japan (Keys et al., 1966). The study was also one of the first to implicate dietary fat in the origin of CHD through an association between disease and the amount of energy derived from saturated fat and, to a lesser extent, dietary cholesterol. At about the same time, a number of large prospective cohort studies, such as the Framingham Study (Kannel et al., 1971) and the Multiple Risk Factor Intervention Trial
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240 Improving the safety and quality of eggs and egg products (MRFIT; Stamler et al., 1986; Martin et al., 1986) were producing evidence that would firmly establish a positive and continuous relationship between blood cholesterol and death from CHD over many years of follow-up (Fig. 12.1). This relationship was continuous across a wide spectrum of blood cholesterol values and showed statistical independence from the effects of other confounding co-variates such as age, gender, body weight, blood pressure and smoking. There was also no indication of any critical threshold at which CHD risk increased suddenly, indicating that the benefit, in terms of reducing absolute risk of dying from CHD by lowering serum cholesterol could, at least in theory, be attained on a population scale. This finding also provided rationale for what is now a common clinical practice of lowering blood (LDL) cholesterol to the greatest possible extent in order to attain the maximal therapeutic benefit. 12.2.1 Equations for predicting the blood cholesterol response to dietary fats and dietary cholesterol Keys et al. (1965), Hegsted et al. (1965) and Hegsted (1986) used the data from these prospective cohort studies and dietary intervention trials to formulate
Age-adjusted 6-year death rate per 1000 men
40 35 30
Total mortality
25 20 15 CHD mortality 10 5 0 140 160
200
240
280
4 5 6 7 Serum cholesterol concentration (mmol/l)
320 (mg/day) 8
Fig. 12.1 Relationship between serum cholesterol concentration and coronary heart disease and total mortality in the Multiple Risk Factor Intervention Trial (MRFIT) Source: Martin MJ, Hulley SB, Browner WS, Kuller LH and Wentworth D (1986), ‘Serum cholesterol, blood pressure, and mortality: implications from a cohort of 361,662 men’, Lancet, 2, 933–936.
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Eggs, dietary cholesterol and disease: facts and folklore 241 equations for predicting the effects of dietary fatty acids, and to a lesser extent dietary cholesterol, on serum cholesterol. These equations were originally designed to estimate the change in serum cholesterol in response to changes in the relative amounts of dietary saturated and polyunsaturated fatty acids (SFA and PUFA), that were well known to exert opposite effects on serum LDL cholesterol (LDL-raising effect of SFA being twice as potent as the LDLlowering effect of PUFA). Various permutations of these equations appeared in the literature, some of which included a more controversial contribution from dietary cholesterol. These equations have received criticism for not discriminating between different types of saturated and polyunsaturated fatty acids, and for not recognising other dietary constituents that can influence the concentration of LDL, as for example mono-unsaturated fatty acids. Despite these limitations, the equations have been used extensively and have proven dietetic and clinical utility. 12.2.2 LDL receptor pathway; a key to understanding cholesterol homeostasis and the inter-relationship between dietary and blood cholesterol Another major advance in our understanding of the inter-relationship between dietary and blood cholesterol came with the discovery of the LDL receptor pathway by Brown and Goldstein (Brown et al., 1975; Brown and Goldstein, 1986). This pathway provided a mechanism to explain how cells regulate their cholesterol metabolism and, inadvertently, how dietary factors and drugs control the concentration of LDL cholesterol circulating in the blood. While all cells can synthesise their own cholesterol, the LDL receptor is a cell surface receptor that can actively bind and internalise serum LDL. It thus provides a pathway by which cells can acquire cholesterol directly from the blood, a route that is cheaper in terms of energy, than cholesterol biosynthesis. The production of LDL receptors is governed by the rate of transcription of the LDL receptor gene, which in turn, is regulated by the amount of free cholesterol within the cell. A fall in intracellular free cholesterol increases the transcription of the LDL receptor and thus the number and activity of LDL receptors on the cell surface. This up-regulation of LDL receptors results in more LDL being bound and internalised into cells and effectively removed from the blood (Fig. 12.2). It follows that any dietary component or drug that alters the amount of intracellular cholesterol will either decrease or increase the concentration of serum (LDL) cholesterol via activation or suppression of this pathway respectively. Both dietary saturated fatty acids with a chain length of between 12 and 16 carbons and cholesterol are known to be effective at increasing free cholesterol within cells, suppressing LDL receptors and raising serum (LDL) cholesterol. Conversely, compounds that are capable of inhibiting either the synthesis or re-absorption of cholesterol in the gut, as for example the ‘statin’ drugs, or plant sterols respectively, lower blood cholesterol by reducing intracellular cholesterol, and up-regulating the
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242 Improving the safety and quality of eggs and egg products Serum LDL Liver cell
LDL
HMG-CoA reductase
Acetate
Free cholesterol
N-SREBP Transcription factor
LDL receptors +
LDL receptor protein
Transcription LDL receptor gene +
Fig. 12.2 Low density lipoprotein (LDL) receptor pathway. HMGCoA=3-hydroxy-3methyl-glutaryl-coenzyme A; N-SREBP = N terminal sequence of an sterol regulatory element binding protein. Source: Griffin BA and Cunnane SC (2008), ‘Nutrition and metabolism of lipids’, In: Introduction to human nutrition, 2nd edition, Nutrition Society Textbook Series, Edited by Gibney MJ, Lanham-New S, Cassidy A and Vorster HH, Published by Wiley-Blackwell, pp 86–121.
LDL receptor pathway. The implications of this discovery were far reaching, not only in terms of understanding the workings of the predictive equations of Keys et al. (1965), Hegsted et al. (1965) and Hegsted (1986), but also in allowing nutritional scientists to draw sensible conclusions about the relative impact of dietary saturated fat and cholesterol on LDL cholesterol. 12.2.3 Understanding how dietary cholesterol influences serum cholesterol: the limitations of human dietary intervention studies Despite irrefutable evidence to support the relationship between raised serum LDL cholesterol in the development of CHD (cholesterol hypothesis), and a clear understanding of how dietary fat influences serum LDL, there remains no definitive evidence to show that dietary modification in any form, including the removal of total and saturated fat, will reduce the risk of death from CHD or CVD. The reasons for this are multi-factorial and arise from problems that are inherent in human dietary intervention studies. They include the following: ∑ A general inadequacy of methods for assessing dietary intake. ∑ An inability to ensure dietary compliance in human interventions. ∑ Difficulty in blinding subjects and investigators, and producing a dietary placebo as a control and acquiring data on diet and clinical endpoints such as CHD death.
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Eggs, dietary cholesterol and disease: facts and folklore 243 ∑ ∑
Problems in distinguishing between biological effects that are ‘passive’ and ‘active’ as a result of dietary substitution. Considerable inter-individual variation in response to dietary modification.
Each one of these confounders, either alone or in combination, has the potential to compromise the scientific interpretation of results from dietary intervention studies, and, within the context of egg-feeding studies, has distorted the conclusions.
12.3 Impact of cholesterol perception on egg consumption The national success of a campaign in the United Kingdom to ‘go to work on an egg’ in the 1960s was reflected in the average intake of eggs at the time, which was 6.5 eggs per week (National Food Survey, 2001). This number was to decline significantly over the next 30 years, mainly in response to the obsession with the role of cholesterol in CHD, much of which emanated from the United States in the 1970s. Intakes in the UK continued to fall in the late 1980s in response to a government claim that all eggs may be infected with Salmonella (MAFF, 1993). Although the issue of infection has all but been eradicated through a successful vaccination programmes (Cogan and Humphrey, 2003; Food Standards Agency, 2004), the cholesterol debate continues to fuel an aversion to eggs for many people who would otherwise be egg consumers. Attempts to overcome this misconception by re-launching the ‘go to work on an egg’ campaign in 2005 were thwarted by a ban imposed by the Broadcast Advertising Clearance Centre on the grounds that a recommendation to eat an egg a day was failing to promote a varied diet (Bruxelles, 2007). A more realistic explanation for this decision would be that such advice contravened dietary recommendations, some of which advocated restricting the intake of eggs to no more than three per week, presumably on the grounds of food safety to avoid the health hazard of raising serum cholesterol.
12.4 Evidence from egg-feeding studies in humans Discovery of the LDL receptor pathway sparked great interest in its role as the mechanism to explain how dietary and lifestyle factors influence blood cholesterol. This prompted a spate of egg-feeding studies in humans throughout the 1970s and 1980s in which volunteers were fed unphysiologically high intakes of dietary cholesterol in the form of up to six eggs/day (≥ 1000 mg cholesterol) (Mann, 2000). As was expected, this extreme type of intervention resulted in significant increases in LDL cholesterol. Many of these eggfeeding studies took little account of the background diet, and were in effect © Woodhead Publishing Limited, 2011
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244 Improving the safety and quality of eggs and egg products examining the impact of dietary cholesterol as a supplement, superimposed on other dietary components. This made it impossible to disentangle the effects of cholesterol from the other dietary components and exclude the possibility of cholesterol having additive or even synergistic effects when consumed with saturated fat. Nevertheless, the outcome of these studies fostered the simple idea that eating dietary cholesterol increases blood cholesterol. This message was soon to be reinforced by the UK’s advisory Committee On Medical Aspects of Food (COMA) who stated that; ‘The weight of evidence supports the view that raising dietary cholesterol increases serum cholesterol, although there is considerable inter-individual variation’ (COMA, 1994). One notable study that did not contribute to this weight of evidence was undertaken in 1987 by a team in Oxford led by Professor Jim Mann (Edington et al., 1987). This study involved an egg intervention in 135 normo-lipidaemic subjects and 33 treated hyper-lipidaemic patients, all of whom were placed on a reduced fat, high fibre diet. In a cross-over design, two groups of subjects were randomly assigned to receive either two eggs/ week for 4 weeks followed by seven eggs/week for a further 4 weeks, or seven eggs/week for 4 weeks followed by two eggs for a further 4 weeks. After the first 4 weeks of intervention they reported a small but significant increase in total serum cholesterol but not in serum LDL cholesterol of normo-lipidaemic subjects after eating seven eggs a week. No other changes in total serum or LDL cholesterol were found after 8 weeks of intervention. The investigators concluded that when cholesterol intake has been reduced by reducing foods high in saturated fat, there was little to be gained by further emphasising reductions in the intake of food high in cholesterol such as eggs. It added the caveats that dietary cholesterol can exert greater effects on serum cholesterol under the following circumstances; when accompanied by a diet high in saturated fat; when extremely high, non-physiological intakes of cholesterol are studied; and due to the existence of hyper-responders. The existence of hyper-responders is a phenomenon that had been previously observed and documented by Katan and co-workers (Katan and Beynen, 1983; Beynen and Katan, 1985; Katan et al., 1986). While these studies suggested that between 20 and 30% of subjects may show an exaggerated LDL response to dietary cholesterol, this finding was not apparent in the Oxford study (Edington et al., 1989). Numerous well-controlled interventions followed throughout the mid-1990s that took account of the confounding influence and background diet, and tested a range of cholesterol intakes from 100 to 600 mg/day (Table 12.2). In general, these studies reported no significant effects of egg feeding on serum LDL cholesterol, though significant increases were found when egg feeding was accompanied by a high intake of saturated fat (Clifton et al., 1998; Sehayek et al., 1998). In spite of the evidence of considerable variation in the individual response of LDL cholesterol to increased dietary cholesterol, the mean serum lipid responses were shown to be linear in normal, healthy males (Ginsberg et al., 1994) and females (Ginsberg et al., 1995).
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Eggs, dietary cholesterol and disease: facts and folklore 245 Table 12.2 Change in plasma cholesterol in a selection of egg-feeding studies Reference Subjects Duration Dose DPlasma Chol (n) (weeks) (mg/day) (mmol/l/100 mg/ day) Ginsberg et al. (1994) Schnohr et al. (1994) Vuoristo and miettinen (1994) Kern (1994) Ginsberg et al. (1995) Knopp et al. (1997) Sehayek et al. (1998) Clifton et al. (1998) a
Male (24) Male/female (24) Vegetarian (5)
8 6 8
128–858 230 690
0.04 0.06 0.09
F galla (16) Female (13) Hyperlidaemics (75) Male/female (18) Male/female (105)
3 8 8 3 3
939 128–690 128–468 80–200 650–840
0.02 0.07 0.08 0.30 + SFAb 0.29 + SFAb
F gall – females with gallbladder disease. SFA – saturated fatty acids.
b
The concentration of serum cholesterol changes in accordance with not only the amount of added cholesterol but also the initial baseline intake of dietary cholesterol (Hopkins, 1992). It may also depend on other factors that influence serum cholesterol, such as energy-restriction and weight loss. In one of our own studies, the incorporation of two eggs per day in combination with an energy restricted, weight-losing diet of healthy, normo-lipidaemic males for 12 weeks was shown to produce no increase on serum LDL cholesterol (Harman et al., 2008). This finding suggested that even small increases seen in serum LDL cholesterol in other egg feeding studies can be negated by the effects of moderate weight loss (4 kg in 4 weeks). It also helped to remove doubt over the safety of eating eggs as part of an energy-restricted diet. Nevertheless, this study also showed a variable LDL response to dietary cholesterol, thus highlighting the need to consider the recommendation of a reduced intake of dietary cholesterol in hyper-responders.
12.5 The relative effects of dietary saturated fat and dietary cholesterol on serum cholesterol Metabolic ward studies indicate that the replacement of 5% of energy from saturated fat with polyunsaturated fat will reduce total serum cholesterol by 0.39 mmol/l, while removing 200 mg of cholesterol will reduce total serum cholesterol by 0.13 mmol/l (Fig. 12.3; Clarke et al., 1997). Similar values can be estimated from the equations of Keys et al. (1965) and Hegsted et al. (1965), Hegsted (1986), which predict that 100 mg of cholesterol (contained in approximately half an egg yolk) is equivalent to 1% of total energy derived from saturated fatty acids (approximately 2–3 g in weight). These quantities are calculated to produce a change in total serum cholesterol of 0.06 mmol/l (2 mg/100 ml). To demonstrate the relative effects of dietary cholesterol and © Woodhead Publishing Limited, 2011
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246 Improving the safety and quality of eggs and egg products Replacement of 5% calories as saturated fat (for example, 17% to 12%) by polyunsaturated fat Replacement of 5% calories as saturated fat (for example, 12% to 7%) by monounsaturated fat Reduction of dietary cholesterol by 200 mg (for example, 340 mg/day to 140 mg/day)
(–0.39)
(–0.24)
(–0.13)
Sum of above dietary changes 0
(–0.76) –0.25 –0.5 –0.76 –1.0 Change in total blood cholesterol (mmol/l)
Fig. 12.3 Mean (SE) changes in blood total cholesterol concentration associated with replacing dietary saturated fat by polyunsaturated and monounsaturated fats and with reducing dietary cholesterol. Source: Clarke R, Rrost C, Collins R, Appleby P and Peto R (1997) ‘Dietary lipids and blood cholesterol: quantitative meta-analysis of metabolic ward studies’, Brit Med J, 314, 112–117.
saturated fat on serum cholesterol within a real diet, these estimates of the relative cholesterol-raising capacity can be translated into a food that contains both components, as, for example, a beef burger. Since a burger contains on average about 10 g of saturated fat but less than 100 mg of cholesterol, the capacity of this food to raise serum cholesterol is primarily determined by its saturated fat content, which in quantitative terms, has approximately five times the capacity of its cholesterol to raise serum cholesterol. Once again, these calculations take no account of the differential effects of different types of saturated fatty acids on serum cholesterol. The calculations also relate to total serum cholesterol which consists, for the most part, of both LDL and high density lipoprotein (HDL). It is well known that dietary saturated fat and cholesterol increase both LDL and HDL, though metabolic ward studies suggest that up to 80% of the effect on total serum cholesterol may be due to changes in LDL cholesterol (Clarke et al., 1997). Nevertheless, since serum HDL is inversely associated with CVD risk, the increases in this lipoprotein that may occur with egg feeding should be considered beneficial to CHD risk, and thus counter the effects of any small increase in LDL.
12.6 Current consensus and recommendations The tide of opinion on the effects of cholesterol in eggs on cardiovascular health appeared to turn in 1997, when COMA updated its statement from 1994 on the impact of dietary cholesterol on blood cholesterol by stating that ‘Dietary cholesterol intake has a small effect on blood cholesterol levels…’. However, there was no further advice on dietary references values for the
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Eggs, dietary cholesterol and disease: facts and folklore 247 intake of dietary cholesterol above the previous recommendation not to exceed the ‘current level of intake of 245 mg/day’. The American Heart Association (AHA) was more candid in its approach when it published recommendations in 2000 to limit the intake of dietary cholesterol to 300 mg/ day for healthy, normo-lipidaemic people (Krauss et al., 2000). In addition to this information, it recommended that the intake should be achieved by limiting the consumption of foods high in saturated fat and not by the exclusion of eggs or shellfish. The AHA also recommended limiting intakes of dietary cholesterol to 200 mg/day for people with raised serum cholesterol (LDL >3.4 mmol/l), diabetes or existing CVD. While these guidelines did impose restrictions on dietary cholesterol and thus egg consumption, the upper limit of 300 mg/day meant that the majority of normal, healthy people could consume an egg a day with impunity. Re-appraisal of the scientific evidence to link dietary cholesterol with serum cholesterol and CHD has concluded that dietary cholesterol exerts a relatively minor and clinically insignificant effect on serum LDL cholesterol and CHD risk compared to saturated fats and other CHD risk factors (Hu et al., 1999; Mann, 2000; McNamara, 2002; Barraj et al., 2009). There is general consensus that recommendations for cardiovascular health should place emphasis on attaining an optimal body weight for your height through energy restriction and increased moderate physical activity. A priority is also to limit the intake of saturated fat and not dietary cholesterol from eggs (Krauss et al., 2000), advice that has been endorsed by the British Heart Foundation (2009), UK Food Standards Agency (2009) and British Dietetic Association (Griffin, 2009). In practice, it is important to appreciate the much greater impact of weight loss compared with dietary measures in lowering serum cholesterol, and the importance of combining these approaches in strategies to reduce CHD risk. Current guidelines for the UK population aim to reduce saturated fat intake from 13% to 11% of food energy (Mason et al., 2009). In terms of the impact on lowering serum cholesterol, the predictive equations indicate that an effect produced by the removal of approximately 2% of energy from saturated fat could also be achieved by excluding one egg a day (see above). However, this approach might be considered as nonsensical and contraindicated on the grounds of human nutrition, disease risk, and economics. Firstly, saturated fat has been implicated in other mechanisms associated with increased cardiovascular risk that are independent of serum cholesterol, as for example in promoting vascular dysfunction (Hall, 2009), insulin resistance and diabetes (Vessby et al., 2001). Secondly, an economic analysis has suggested that it would cost more, in monetary terms, to exclude eggs from our diet, as it would increase the disease risk to benefits ratio (Schmier et al., 2009). While the complex statistical analysis in this latter study must be taken on trust by most non-experts, the conclusion is consistent with the overall message from the scientific literature and is in accord with a growing consensus of opinion on the benefits rather than the adverse effects of eating eggs.
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248 Improving the safety and quality of eggs and egg products 12.6.1 Ongoing issues and controversies over dietary cholesterol and eggs: dietary cholesterol, eggs and the development of diabetes A re-analysis of data from two large prospective trials, the Physicians Health Study and Women’s Health Study in the United States, has generated concern over possible links between the consumption of dietary cholesterol in eggs and increase in all cause mortality, most notably in people with diabetes, and the risk of developing type 2 diabetes (Djousse and Gaziano, 2008; Djoussse et al., 2009). Unfortunately, the post hoc analyses in these studies were confounded on several fronts, not least of which was a lack of information on CVD risk factors such as LDL, and dietary intake data in the Physicians Health Study. The latter made it impossible to exclude confounding effects from other dietary constituents such as saturated fat. While the addition of the Women’s Health Study to this former analysis meant that there was now information on dietary intake in a fraction of the studied cohort, the conclusion that eating seven eggs a week increases the likelihood of developing diabetes must be interpreted in the light of how the groups were analysed. The cohort was stratified into groups based on weekly egg consumption, from no eggs to a group eating ≥seven eggs per week. The latter group contained a large number of subjects who consumed an egg a day and a smaller number of extremely high consumers. It is likely that the inclusion of extremely high egg consumers introduced bias that could influence the significance of an association with diabetes in favour of an erroneous conclusion on the effects of eating one egg a day. The general interpretation of the outcome of these studies could be seriously detrimental to the recommendation to include eggs, as a low energy, nutrient dense food, in energy-restricted diets designed to prevent the onset of type 2 diabetes in obesity. More well-controlled studies that have been specifically designed for the purpose of studying the relationship between egg consumption and diabetes are clearly required to resolve this issue. 12.6.2 Understanding the molecular basis of inter-individual variation and hyper-responsiveness to dietary cholesterol Cholesterol that circulates within LDL in the blood serum has two original sources; it can either be synthesised directly by the liver or be acquired from the diet. Dietary cholesterol arising from digested food, is absorbed with triglyceride in the intestine, and packaged into lipoproteins called chylomicrons in the intestinal epithelial cells. Chylomicrons pass into the blood via the lymphatic system and circulate after a meal in the postprandial phase until their triglyceride is rapidly hydrolysed, leaving a cholesterol-rich remnant. This remnant is removed from the blood by re-entry into the liver, thus providing an additional source of cholesterol. This cholesterol will increase the pool of free cholesterol, down-regulating LDL receptors and increasing serum LDL, and be available for the manufacture of lipoproteins (VLDL) that will eventually become LDL (Griffin and Cunnane, 2008). The rate at
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Eggs, dietary cholesterol and disease: facts and folklore 249 which humans synthesise cholesterol is known to be inversely related to the amount that they can absorb from their diet. Therefore, as the amount of dietary cholesterol increases and is absorbed in the intestine, the liver reduces its rate of synthesis and vice versa. Of these two mechanisms, the absorption of cholesterol in the intestine appears to be more critical in determining the impact of dietary cholesterol on serum LDL cholesterol, either directly through the production of VLDL or indirectly by down-regulating LDL receptors (Sehayek et al., 1998). Cholesterol absorption has been shown to increase in proportion to the intake of dietary cholesterol until intake reaches approximately 400 mg/day when cholesterol absorption effectively shuts down (Ostlund et al., 1999). The sensitivity of this mechanism and the interplay between absorption and cholesterol biosynthesis varies in different individuals, so while some individuals can decrease their absorption readily in response to an increased intake of cholesterol, others are less efficient at doing so, and express a hyper-serum LDL response to dietary cholesterol. As this is a real phenomenon, important questions must be; how many people are hyper-responsive to dietary cholesterol and how do we identify them? As previously mentioned, there is evidence to suggest 20–30% of subjects in some cohorts show a degree of exaggerated response to dietary cholesterol (Katan and Beynen, 1983; Katan et al., 1986), but this has not been substantiated in other studies or established in populations. One obvious marker is a raised concentration of serum LDL cholesterol, for which there is already a recommendation for a lower intake of dietary cholesterol. Another must be the ability to measure the rates of cholesterol absorption and synthesis or genetic or metabolic determinants of these pathways. Cholesterol absorption and synthesis can be measured in vivo using labelled forms of cholesterol and its precursors as metabolic tracers, and by the use of serum biomarkers such as plant sterols. Similarly, knowledge of the genes, regulatory proteins and nutrient–gene interactions that determine these pathways is growing rapidly, but these technologies have not yet progressed to a stage at which they could be used to predict responsiveness to dietary cholesterol and saturated fat. There is evidence to suggest that some individuals are naturally high absorbers and low synthesisers, while others are high absorbers and low synthesisers (Miettinen and Kesäniemi, 1989). An implication of such differences in metabolic phenotype could be that the serum LDL of high synthesisers is more responsive to agents that interrupt cholesterol synthesis such as the statins, whereas high absorbers may be more responsive to changes in dietary cholesterol and saturated fat, and compounds that impair the absorption of dietary cholesterol, such as plant sterols. There is also evidence that obesity and insulin resistance is associated with reduced cholesterol absorption and thus decreased sensitivity to dietary cholesterol (Knopp et al., 2003), providing further support for the inclusion of eggs in energy-restricted, weight-losing diets.
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250 Improving the safety and quality of eggs and egg products
12.7 Conclusion Reappraisal of the evidence to support a relationship between dietary cholesterol and serum cholesterol has helped to lift restrictions on the consumption of eggs. While this outcome has been aided by increased knowledge of the molecular mechanisms that explain how dietary cholesterol influences blood cholesterol, even scientific consensus can be slow in altering a fixed perception of how a food impacts on health. C.S. Lewis once wrote; ‘No clever arrangement of bad eggs ever made a good omelet’. The reappraisal of evidence in this case is not a ‘clever’ manipulation of information on a ‘bad’ food, but an examination of over 30 years of data that could find no conclusive grounds for restricting the intake of eggs.
12.8 References (1913), ‘Ueber die Veranderungen der Kaninchenaorta bei experimenteller Cholesterinsteatose’, Beitr Pathol Anat, 56, 379–404. anitschkow nn and chalatov s (1913), ‘Ueber experimentelle Choleserinsteatose und ihre Bedeutung fur die Entstehung einiger pathologischer Prozesse’, Zentralbl Allg Pathol, 24, 1–9. barraj l, tran n and mink p (2009), ‘A comparison of egg consumption with other modifiable coronary heart disease lifestyle risk factors: a relative risk apportionment study’, Risk Analysis, 29, 401–415. beynen ac and katan mb (1985), ‘Reproducibility of the variation between humans in the response of serum cholesterol to cessation of egg consumption’, Atherosclerosis, 57, 19–31. british heart foundation (2009), Reducing your blood cholesterol. Publication code: HIS3. brown ms and goldstein jl (1986), ‘A receptor-mediated pathway for cholesterol homeostasis’, Science, 232, 34–47. brown ms, faust jr and goldstein jl (1975), ‘Role of the low density receptor in regulating the content of free and esterified cholesterol in human fibroblasts’, J Clin Invest, 55, 783–793. bruxelles s (2007), ‘Go to work on an egg’ ad banned, www.timesonline.co.uk clarke r, frost c, collins r, appleby p and peto r (1997), ‘Dietary lipids and blood cholesterol: quantitative meta-analysis of metabolic ward studies’, Brit Med J, 314, 112–117. clifton pm, noakes m and nestel pj (1998), ‘LDL particle size and LDL and HDL cholesterol changes with dietary fat and cholesterol in healthy subjects’, J Lipid Res, 39, 1799–1804. cogan ta and humphrey tj (2003), ‘The rise and fall of Salmonella Enteritidis in the UK’, J Appl Microbiol, 94, 114S–119S. cohn ej, strong le, hughes wl, jr, mulford dj, ashworth jn, melin m and taylor hl (1946), ‘Preparation and properties of serum and plasma lipoproteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids’, J Am Chem Soc, 68, 459–475. coma (Committee on Medical Aspects of Food) (1994) Nutritional Aspects of Cardiovascular Disease, 46. HMSO: London. djousse l and gaziano jm (2008), ‘Egg consumption in relation to cardiovascular disease and mortality: the physicians’ health study’, Am J Clin Nutr, 87, 964–969. anitschkow n
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Eggs, dietary cholesterol and disease: facts and folklore 251 djousse l, gaziano jm, buring je
and lee im (2009), ‘Egg consumption and risk of type 2 diabetes in men and women’, Diabetes Care, 32, 295–300. edington j, geekie m, carter r, benfield l, fisher k, ball m and mann j (1987), ‘Effects of dietary cholesterol on plasma cholesterol concentration in subjects following a reduced fat diet, high fibre diet’, Brit Med J, 294, 333–336. edington j, geekie m, carter r benfield l, ball m and mann j (1989), ‘Serum lipid response to dietary cholesterol in subjects fed a low-fat, high fibre diet’, Am J Clin Nutr, 50, 58–62. food standards agency (2002), McCance and Widdowson’s the Composition of Foods, Sixth Summary Edition. Royal Society of Chemistry: Cambridge. food standards agency (2004), ‘Report of the survey of salmonella contamination of UK produced egg shells on retail sales’, www.food.gov.uk/multimedia/pdfs/fsis 5004 report. food standards agency (2009), ‘Eat wee, be well – Eggs’, www.food.gov.uk ginsberg hn, karmally w, siddiqui m, holleran s, tall ar, rumsey sc, deckelbaum rj, blaner ws and ramakrishnan r (1994), ‘A dose–response study of the effects of dietary cholesterol on fasting and postprandial lipid and lipoprotein metabolism in healthy young men’, Arterioscler Thromb Vasc Biol, 14, 576–586. ginsberg hn, karmally w, siddiqui m, holleran s, tall ar, blaner ws and ramakrishnan r (1995), ‘Increases in dietary cholesterol are associated with modest increases in both LDL and HDL cholesterol in healthy young women’, Arterioscler Thromb Vasc Biol, 15, 169–178. gofman jw, lindgren f, elliott h, mantz w, hewitt j and herring v (1950). ‘The role of lipids and lipoproteins in atherosclerosis’, Science, 111, 166–171. gray j and griffin ba (2009), ‘Eggs and dietary cholesterol – dispelling the myth’, Nutr Bull, 34, 66–70. griffin ba (2009), ‘Cracking the cholesterol myth’, Dietetics Today, 44, 44–47. griffin ba and cunnane sc (2008), ‘Nutrition and metabolism of lipids’, In: Introduction to human nutrition, 2nd edition, Nutrition Society Textbook Series, Edited by Gibney MJ, Lanham-New S, Cassidy A and Vorster HH, Wiley-Blackwell pp 86–121. hall wl (2009), ‘Dietary saturated and unsaturated fats as determinants of blood pressure and vascular function’, Nutr Res Rev, 22, 18–38. harman nl, leeds ar and griffin ba (2008), ‘Increased dietary cholesterol does not increase plasma low density lipoprotein when accompanied by an energy-restricted diet and weight loss’, Eur J Nutr, 47, 287–293. hegsted dm (1986), ‘Serum-cholesterol response to dietary cholesterol: a re-evaluation’, Am J Clin Nutr, 44, 299–305. hegsted dm, mcgandy rb, myers ml and stare fj (1965), ‘Quantitative effects of dietary fat on serum cholesterol in man’, Am J Clin Nutr, 17, 281–295. herron kl and fernandez ml (2004), ‘Are the dietary guidelines regarding egg consumption appropriate?’, J Nutr, 134, 187–190. hopkins pn (1992), Effects of dietary cholesterol on serum cholesterol: a meta-analysis and review, Am J Clin Nutr, 55, 1060–1070. hu fb, stampfer mj, rimm eb, manson je, ascherio a, colditz ga, rosner ba, spiegelman d, speizer fe, sacks fm, hennekens ch and willett wc (1999), ‘A prospective study of egg consumption and risk of cardiovascular disease in men and women’, J Am Med Assoc, 281, 1387–1394. kannel wb, castelli wp and gordon t (1971), ‘Serum cholesterol, lipoproteins, and the risk of coronary heart disease: The Framingham Study’, Ann Int Med, 74, 1–12. katan mb and beynen ac (1983), ‘Hyper-response to dietary cholesterol in man’, Lancet, 1, 1213. katan mb, beynen ac, de vries jh and nobels a (1986), ‘Existence of consistent hypo- and hyper-responders to dietary cholesterol in man’, Am J Epidemiol, 123, 221–234. kern f jr (1994), ‘Effects of dietary cholesterol on cholesterol and bile acid homeostasis in patients with cholesterol gallstones’, J Clin Invest, 93, 1186–1194. © Woodhead Publishing Limited, 2011
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252 Improving the safety and quality of eggs and egg products keys a, anderson jt
and grande f (1965), ‘Serum cholesterol response to changes in diet. II. The effects of cholesterol in the diet’, Metabolism, 14, 759–765. keys a, aravanis c, blackburn hw, van buchem fs, buzina r, djordjević bd, dontas as, fidanza f, karvonen mj, kimura n, lekos d, monti m, puddu v and taylor hl (1966), ‘Epidemiological studies related to coronary heart disease: characteristics of men aged 40–59 in seven countries’, Acta Med Scand, 460, 1–392. knopp rh, retzlaff bm and walden ce (1997), ‘A double-blind, randomized, controlled trial of two eggs per day in moderately hypercholesterolemic and combined hyperlipidemic subjects taught the NCEP step I diet’, J Am Coll Nutr, 16, 551–561. knopp rh, retzlaff bm and fish b (2003), ‘Effects of insulin resistance and obesity on lipoproteins and sensitivity to egg feeding’, Arterioscler Thromb Vasc Biol, 23, 1437–1443. krauss rm, eckel rh, howard b, appel lj, daniels sr, deckelbaum rj, erdman jw jr, krisetherton p, goldberg ij, kotchen ta, lichtenstein ah, mitch we, mullis r, robinson k, wylie-rosett j, st jeor s, suttie j, tribble dl and bazzarre tl (2000), ‘AHA Dietary Guidelines: revision 2000: A statement for healthcare professionals from the Nutrition Committee of the American Heart Association’, Circulation, 102, 2284–2299. layman dl and rodriguez nr (2009), ‘Egg protein as a source of power, strength and energy’, Nutr Today, 44, 1–6. maff (Ministry of Agriculture and Foods and Fisheries) (1993), Agriculture in the UK, HMSO, London. mann j (2000), ‘Dietary cholesterol: a review of research and practice over 30 years’, in Dietary Cholesterol as a Cardiac Risk Factor; Myth or Reality, Edited by Leeds AR & Gray J, Smith-Gordon: London, 11–16. martin mj, hulley sb, browner ws, kuller lh and wentworth d (1986), ‘Serum cholesterol, blood pressure, and mortality: implications from a cohort of 361,662 men’, Lancet, 2, 933–936. mason p, porter sc, berry se, stillman p, steele c, kirby a, griffin ba and minihane am (2009), ‘Saturated fatty acid consumption: outlining the scale of the problem and assessing the solutions’, Nutr Bull, 34, 74–84. mcnamara dj (2002), ‘Eggs and heart disease risk: perpetuating the misperception’, Am J Clin Nutr, 75, 333–335. miettinen ta and kesäniemi ya (1989), ‘Cholesterol absorption: regulation of cholesterol synthesis and elimination and within-population variations of serum cholesterol levels’, Am J Clin Nutr, 49, 629–635. national food survey (2001), ‘Consumption of selected household foods, 1942 to 1996’ https://statistics.defra.gov.uk/esg/publications/nfs/default.asp ostlund re jr, bosner ms and stenson wf (1999), ‘Cholesterol absorption efficiency declines at moderate dietary doses in normal human subjects’, J Lipid Res, 40, 1453–1458. schmier jk, barraj lm and tran nl (2009), ‘Single food focus dietary guidance: lessons learned from an economic analysis of egg consumption’, Cost Eff Resour Alloc, 14, 7. schnohr p, thomsen oo, riis hansen p, boberg-ans g, lawaetz h and weeke T (1994), ‘Egg consumption and high-density-lipoprotein cholesterol’, J Intern Med, 235, 249–51. sehayek e, nath c, heinemann t, mcgee m, seidman ce, samuel p and breslow jl (1998), ‘U-shape relationship between change in dietary cholesterol absorption and plasma lipoprotein responsiveness and evidence for extreme inter-individual variation in dietary cholesterol absorption in humans’, J Lipid Res, 39, 2415–2422. stamler j, wentworth d and neaton jd (1986), ‘Is relationship between serum cholesterol and risk of premature death from coronary heart disease continuous and graded? Findings in 356 222 primary screenees of the Multiple Risk Factor Intervention Trial (MRFIT)’, J Am Med Assoc, 256, 2823–2828. steinberg d (2004), ‘An interpretive history of the cholesterol controversy: Part I’, J. Lipid Res, 45, 1583–1593.
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Eggs, dietary cholesterol and disease: facts and folklore 253 (2006), ‘An interpretive history of the cholesterol controversy, part V: The discovery of the statins and the end of the controversy’, J. Lipid Res, 47, 1339–1351. vessby b, uusitupa m, hermansen k, riccardi g, rivellese aa, tapsell lc, nälsén c, berglund l, louheranta a, rasmussen bm, calvert gd, maffetone a, pedersen e, gustafsson ib and storlien lh (2001), ‘Substituting dietary saturated for monounsaturated fat impairs insulin sensitivity in healthy men and women: The KANWU Study’, Diabetologia, 44, 312–319. vuoristo m and miettinen ta (1994), ‘Absorption, metabolism, and serum concentrations of cholesterol in vegetarians: effects of cholesterol feeding’, Am J Clin Nutr, 59, 1325–1331. windaus a (1910),‘Uber den Gehalt nirmaler und atheromatoser Aorten an Cholsterin und Cholesterinestern’, Hoppe-Seyler’s Z Physiol Chem, 67, 174–176. steinberg d
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13 Egg allergy Y. Mine and M. Yang, University of Guelph, Canada
Abstract: In industrialized countries, egg allergy accounts for one of the most prevalent food hypersensitivities, especially in children. The dominant egg allergens are proteins, and are mainly present in the egg white, e.g. ovalbumin, ovomucoid, ovotransferrin and lysozyme. However, egg yolk also displays low-level allergenicity, e.g. a-livetin. Therefore, preventive approaches are being explored to protect consumers from inadvertent exposure and the ensuing adverse reactions. Attempts to produce hypoallergenic egg-containing products through food processing techniques have met with promising results, but the approach is limited owing to its potentially undesirable effects on the unique functional and sensory attributes of egg proteins. Therefore, the development of preventive or curative strategies for egg allergy remains strongly warranted. Key words: egg allergy, ovalbumin, ovomucoid, lysozyme, food processing, allergen stability, immunotherapy, immune tolerance induction.
13.1 Introduction The estimated prevalence of egg allergy varies between 1.6% and 3.2% and represents the second most common cause of food allergies in children (Sampson 2004). In several industrialized countries, egg allergy has in fact been reported as the most prevalent food hypersensitivities in the pediatric population, exceeding that of cow’s milk allergy (Gustafsson et al. 2003; Han et al. 2004). Currently, the most efficient approach for egg allergy is total avoidance of the offending compound. However, omnipresence of egg-derived
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Egg allergy 255 components in cooked or manufactured food products and vaccines which contain trace of egg proteins (e.g. influenza vaccination) renders the approach difficult (Allen et al. 2007) and inadvertent exposure may lead to life-threatening anaphylactic responses. Furthermore, when egg allergy occurs in association with other food allergies, such as cow’s milk, the risks of nutritional deficiency represent an additional hurdle for the patients (Grimshaw 2006). Therefore, the development of egg products safe for human consumption complemented with efficient forms of immunotherapy are highly warranted. This chapter provides the most recent information on egg protein allergenicity, and the effects of food processing on the allergenicity of egg proteins.
13.2 Egg allergy: an overview 13.2.1 Natural history of egg allergy Egg allergy usually develops within the first two years of life, and resolves by school age (Heine et al. 2006; Teuber et al. 2006). Clinical adverse reactions sometimes emerge prior to any egg ingestion (de Boissieu and Dupont 2006). In these cases, sensitization may have occurred in utero or may result from the transfer of small doses of antigen into breast milk (Vance et al. 2005). However, there is currently no evidence that strict egg avoidance during pregnancy or lactation can decrease the risk of sensitization in infants. A few studies suggested that early childhood sensitization to hen’s egg may favor the subsequent development of respiratory allergies (Tariq et al. 2000). In adults, occupational asthma has been associated with the inhalation of aerosolized dried egg powder, leading to the development of immunoglobulin E (IgE)-mediated food allergy upon egg ingestion (Escudero et al. 2003). Earlier studies documented that about two-thirds of children outgrew their allergy by the age of 5 (Boyano-Martínez et al. 2002). A very recent study, involving a cohort of more than 850 egg allergic patients aged 2–18 years old, reported that outgrowth of egg allergy may in fact occur at a later stage than previously reported (Savage et al. 2007). The study highlighted that the median time to develop tolerance was significantly increased in children suffering from other atopic diseases such as asthma or allergic rhinitis, and that specific IgE level ≥50 kU/L was a good marker of persistent egg allergy. It was also observed that concomitant sensitization with other food allergens (e.g. peanut or milk) tends to delay the outgrowth of egg allergy (Savage et al. 2007). Hen’s egg is an important cause of childhood allergy. Combined with peanut and cow’s milk allergies, they represent up to 80% of food allergy cases observed in infants (Rolinck-Werninghaus et al. 2005). At present, management of egg allergy focuses on strict avoidance of the offending food, and administration of drugs to suppress severe symptoms, e.g. epinephrine in cases of anaphylaxis. Currently, novel forms of specific immunotherapy are being investigated as detailed further. © Woodhead Publishing Limited, 2011
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256 Improving the safety and quality of eggs and egg products In a recent study examining thresholds of clinical reactivity to egg, it was identified that 16% of individuals (n = 124) with egg allergies reacted to 65 mg of egg as a solid food, equivalent to 6.5 mg of egg protein (Morisset et al. 2003). The study reported that 0.8% of individuals with egg allergies reacted to 10 mg or less of solid food and that the lowest reactive threshold was less than 2 mg of egg, i.e. 0.2 mg of hen’s egg protein. More recently, a separate study also involving oral food challenges, conducted in France, has reported an even lower threshold, equal to only 0.13 mg of hen’s egg protein (Taylor et al. 2004). 13.2.2 Mucosal immune response to egg allergens Immunoglobulin (Ig) E-mediated food allergy, also known as type I food allergy, accounts for the majority of food-induced allergic responses and is characterized by the presence of elevated titers of antigen-specific serum IgE antibodies. Currently, it remains unclear how the mucosal immune system is oriented towards sensitization vs. immune tolerance when exposed to innocuous dietary antigens. However, it is suggested that upon ingestion, food proteins are capable of crossing the intestinal epithelial barrier and are captured by the underlying immune system. Food proteins are then processed into peptidic fragments by a class of specialized immune cells, known as antigen presenting cells (APC). The peptidic fragments are displayed on their surface in association with major histocompatibility (MHC) class II molecules. The peptide-MHC II complexes can in turn be recognized by specific T-cell receptors or TCR (van Wijk and Knippels 2007). The usual non-pathogenic response to soluble dietary proteins is characterized as a status of immune hyporesponsiveness, also known as oral tolerance (Weiner 2000). An allergic immune response is believed to be orchestrated by a class of CD4+ T lymphocytes or helper T cells. Indeed cytokines produced by CD4+ T lymphocytes mediate a wide range of pro-inflammatory and antiinflammatory responses. Most CD4+ T cells belong to either a Th1 or a Th2 subgroup, producing type-1 or type-2 cytokines respectively. Interferon (IFN)-g is the archetypal type-1 cytokine, while type-2 cells typically produce a range of cytokines including IL-4, IL-5, IL-9 and IL-13, contributing to the differentiation of B cells into IgE-producing plasma cells, and the recruitment of effector cells such as eosinophils, basophils and mast cells (Fig. 13.1 (Prioult and Nagler-Anderson 2005). The mechanism of allergic sensitization is initiated by differentiation of naive antigen-specific T-helper cells into effector T-helper type-2 cells, leading to the differentiation of B cells into antibody-secreting plasma cells and the binding of allergen-specific IgE to high affinity Fc receptors (FcR I) present on the surface of mast cells and basophils. Upon subsequent exposure to the allergen, the cross-linking of IgE-receptors leads to their degranulation. The release of vasoactive amines, cytokines and synthesis of a variety of arachidonic acid-derived inflammatory mediators ensues
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Egg allergy 257 M
Allergen (subsequent ingestion)
Allergen
Th2 lgE Oral tolerance
T
IL-4, IL-13 IL-4 B
Th1
Eosinophils
Th2 Inflammatory mediators
IFN-g IL-4
lgM
IL-13 B
Mast cells
Clinical symptoms
Basophils
Fig. 13.1 Cellular and molecular mechanisms of food allergy (adapted from: Prioult and Nagler-Anderson 2005).
(Baral and O’B Hourihane 2005) and leads ultimately to the manifestations of clinical symptoms commonly associated with egg allergy (e.g. atopic dermatitis, urticaria, angiodema). A distinct population of CD4+ T cells, known as T regulatory (Treg) cells, is believed to control the balance between T-helper type-1 and type-2 responses. Regulatory T cells encompass ‘natural’ T regulatory cells (e.g. CD4+CD25+ T cells) and ‘induced’ or adaptive regulatory T cells such as IL-10-producing Tr1 cells and TGF-b-producing Th3 cells (Akdis et al. 2006). The manipulation of Treg cells functions in the prevention and treatment of (food) allergic diseases represent an attractive approach (Taams et al. 2006). A detailed discussion of the role of T lymphocytes in the immunopathology of food allergy is beyond the scope of this review; however, excellent reviews can be found (Akdis et al. 2006; Bohle 2004). 13.2.3 Allergenic components of the egg The chemical composition of hen eggs has been extensively investigated. Clinically relevant egg allergens have been identified in both the albumen (egg white) and the egg yolk fraction; however, reports have documented that the major egg allergens were mainly contained in the egg white (Anet
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258 Improving the safety and quality of eggs and egg products et al. 1985). The molecular and biological characteristics of egg allergens are presented in Table 13.1. Egg white allergens Early studies involving a cohort of 342 patients, reported that the major egg allergens were, in increasing order; lysozyme > ovomucin > ovalbumin > ovomucoid, based on skin tests (Miller and Campbell 1950). Using 13 egg allergic patients, further studies documented ovomucoid (OVM) as the dominant egg allergen (Bleumink and Young 1971). Based on radioallergosorbent test Table 13.1 Molecular and biological properties of identified egg allergens Protein name Mr Protein (kDa) family
Biological function(s) Allergen Selected epitopes references reported
Egg white proteins Ovomucoid (Gal d 1)
28
Kazal-type Serine protease inhibitor
Serine protease inhibition activity
Yes
Walsh et al. (1988, 2005), Mine and Rupa (2003)
Ovalbumin (Gal d 2)
45
Serine protease inhibitor
Storage protein?
Yes
Walsh et al. (1988, 2005)
Ovotransferrin 76–77 Transferrin (Gal d 3)
Iron-binding capacity No with antimicrobial activity
Langeland and Harbitz (1983), Walsh et al. (1988, 2005)
Egg lysozyme 14.3 (Gal d 4)
Glycoside hydrolase family 22
Antibacterial activity
No
Walsh et al. (1988, 2005), Escudero et al. (2003)
Ovomucin
165
Contains Heavily glycosylated trypsin protein with potent inhibitor-like antiviral activities domains
No
Walsh et al. (1988, 2005)
Phosvitin
35
Transferase? Metal chelating agent No
Walsh et al. (1988, 2005)
Egg yolk proteins Alpha-livetin 65–70 Serum (Gal d 5) albumin
Bind ions, fatty acids, hormones in physiological conditions
No
van Toorenenbergen et al. (1994), Quirce, et al. (2001)
YGP42 (Gal d 6)
Glycoprotein
No
Amo et al. (2010)
35
Serum albumin
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Egg allergy 259 (RAST) and crossed radio-immunoelectrophoresis (CRIE), studies completed in the 1980s established that ovalbumin (OVA), ovomucoid (OVM) and ovotransferrin (OVT) were the major egg allergens (Hoffman 1983; Langeland 1982b) and later, lysozyme (LYS) was also demonstrated to be a significant egg allergen (Holen and Elsayed 1990). Allergy to lysozyme has been described in sensitization cases after exposure via inhalation. Frequency of recognition in egg allergic patients was reported to range from 6 to 67% for lysozyme (Frémont et al. 1997) and to reach up to 22% for ovotransferrin (Djurtoft et al. 1991). The two major allergens ovomucoid and ovalbumin constitute about 11% and 54% of egg white proteins, respectively (Kovacs-Nolan et al. 2005). Both proteins are glycosylated, with as high as 25% of the mass of ovomucoid comprising carbohydrates. Using egg allergic patients’ sera, further studies confirmed that OVM was the dominant allergenic components in egg white (Cooke and Sampson 1997; Urisu et al. 1997; Zhang and Mine 1998, 1999). In an effort to establish which egg proteins were major allergens, a study investigated binding of specific IgE to eight purified egg white and yolk proteins by (RAST), using sera from 40 egg-sensitive children (Walsh et al. 2005). The study confirmed that the major egg allergens originated primarily from egg white, and include ovomucoid (Gal d 1), ovalbumin (Gal d 2), ovotransferrin (Gal d 3), and lysozyme (Gal d 4). The same report confirmed previous results that egg yolk also contains allergenic proteins, and were identified as apovitellenins I and VI and phosvitin (Walsh et al. 1988). In previous studies, other proteins such as ovoflavoprotein and ovoinhibitor were identified as antigenic, but lacked allergenic activity (Langeland 1982b; Hoffman 1983; Anet et al. 1985). Egg yolk allergens a-Livetin (GAL d5 ) is a minor allergen having a molecular weight of about 65–70 kDa and is identical to chicken serum albumin. It is believed to be significantly involved in the pathogenesis of bird-egg syndrome (development of respiratory and gastrointestinal symptoms upon exposure to bird antigens or upon egg yolk intake) (van Toorenenbergen et al., 1994). It has been shown that heating of a-livetin will reduce, but not totally eliminate, its allergenicity (Quirce et al., 2001). More recently, a Gal d 6 was newly identified as second egg yolk allergen characterized from egg yolk (Amo et al., 2010).
13.3 Egg proteins 13.3.1 Stability of egg proteins and their allergenicity The allergenicity of egg proteins depends a great deal, but not exclusively, on their resistance to heat and digestive enzymes (Astwood et al. 1996),
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260 Improving the safety and quality of eggs and egg products reflecting their capacity to stimulate a specific immune response, i.e. presence of B- and T-cell epitopes (Heine et al. 2006). A common approach to evaluating the stability of egg allergens is to examine alterations of their IgE/IgG-binding capacities upon physical, chemical or genetic manipulations. In one study, the four major egg allergens were either physically or chemically modified and their binding activity to egg allergic patients specific IgG/IgE and rabbit specific-IgG were examined (Mine and Zhang 2002). Thermal treatment was conducted for 15 min at 95 °C, and chemical modification with either urea 6M or carboxymethylation. Ureatreated OVA, LYS and OVT led to an increase in human IgG-binding activity, while carboxymethylation and thermal treatment of OVM and OVT led to a significant drop. Treatment with urea also enhanced the binding activity of rabbit IgG antibodies to OVT. Although carboxymethylation of OVM, OVT and LYS decreased their allergenicity, IgE binding activity to OVA was not affected. These findings suggest that OVA contains sequential IgE epitopes, while OVM and LYS may contain both sequential and conformational IgE epitopes (Mine and Zhang 2002). Ovomucoid (OVM) is characterized by its high heat stability and can also be resistant to other forms of denaturation (e.g. urea) (Gremmel and Paschke 2007), possibly related to the presence of its strong disulfide bonds (Djurtoft et al. 1991). In fact, in vitro stability of ovomucoid was reported in simulated intestinal fluid for at least 60 min (Takagi et al. 2003). A separate study reported on the other hand, that OVM can be easily denatured by trichloric acid-acetase or 8 M urea procedures (Bernhisel-Broadbent et al. 1994). The chemical stability of ovomucoid has been accounted for its dominance as a major egg allergen and its role in the prognosis of egg allergy. In a study involving 38 patients with allergic reactions to freeze-dried egg white, it was shown that most of them also developed a positive response to heated egg white (n = 21). On the other hand, 17 of these 21 subjects were tolerant to a challenge with heated-egg white depleted of ovomucoid (Urisu et al. 1997). Experiments pertaining to the allergenic stability of OVM led to contradictory results. While some studies reported that reduction of OVM did not affect its IgE-binding capacity (Djurtoft et al. 1991), other studies reported a decrease (Cooke and Sampson 1997) or an increase (Zhang and Mine 1998), using human patients’ sera. It was documented that heating of ovomucoid at 100 °C for duration of 30 min could lead to irreversible conformational changes, as detected by monoclonal antibodies (Hirose et al. 2004). However, the protein is non-coagulable upon heat application and human specific-IgE were shown to bind to both native and denatured forms (Hirose et al. 2004). Using egg allergic patients sera, an interesting study reported that the IC50 concentration for specific IgE inhibition was 1700-fold higher for denatured ovomucoid compared to intact or oxidized ovomucoid (Holen et al. 2001), suggesting that the allergenicity of OVM can significantly drop upon denaturation. Interestingly, the allergenic and antigenic properties of OVM were shown to be maintained after peptic digestion (Kovacs-Nolan et al. 2000; Takagi et
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Egg allergy 261 al. 2005). In vitro analyses showed that pepsin-digestion of ovomucoid did not alter its IgE-binding capacity, as measured by enzyme-linked immunosorbent assay (ELISA) using human egg allergic patients’ sera (Kovacs-Nolan et al. 2000). The study also documented that pepsin-digestion of OVM did not significantly affect its trypsin inhibitory activity. On the other hand, reduction of OVM enhanced its digestibility, indicating the importance of its disulfide bonds. These findings were supported by previous studies investigating IgE binding activity of OVM after pepsin, chymotrypsin and trypsin digestion using RAST inhibition and western blot tests, with serum samples obtained from egg allergic patients (Besler et al. 1999; Urisu et al. 1997). Finally, five genetic mutants of OVM Gal d 1.3 were analyzed and compared in vitro to their native counterpart. It was demonstrated that the genetic mutation of phenylalanine (F) at position 37 with alanine (A) residue caused a drastic loss of binding activity to human allergic patients IgE, attributed to a disruption of the a-helix. Furthermore, substitution of glycine with methionine at position 32 led to synergistic effect on decreasing its binding activity (Mine et al. 2003). Ovalbumin (OVA) is easily denatured by urea and guanidinium salts (Bernhisel-Broadbent et al. 1994). Furthermore, the protein is relatively heat labile, compared with OVM. Using egg-allergic patients’ sera, reports have shown that the antigenicity of OVA could however resist heat treatment under certain conditions (Elsayed et al. 1986). As mentioned above, studies found that OVA can retain its human IgE binding capacity after chemical treatment such as reduction and carboxymethylation, heat or urea, indicating that IgE epitopes are linear and thermostable (Mine and Zhang 2002). On the other hand, earlier report indicated that exposure of OVA to a temperature of 80 °C for 3 min only had the effect of decreasing its IgE-binding activity by 90% (Honma et al. 1994). The disparities observed from one study to another may be explained by variations inherent to patient cohort characteristics, an idea which was supported by subsequent reports (Walsh et al. 2005). Assessing resistance to enzymatic digestion represents an alternative manner to characterize egg allergen stability. It was documented that, in its native form, OVA is trypsin resistant but susceptible to pepsin digestion (Elsayed et al. 1986). Ovalbumin, along with phosvitin, was shown to be resistant to peptic digestion at pH 1.2 for more than 60 min (Astwood et al. 1996). Finally, it was reported that ovalbumin was relatively stable in simulated gastric and intestinal fluid, however preheating could lead to a decrease of its resistance to proteolysis (Takagi et al. 2003). Lysozyme (LYS) stability has been the object of a number of studies. A recent report reviews the data obtained on LYS thermal stability (Petersen et al. 2004) and demonstrates that polyols such as sorbitol exert a significant stabilizing effect on lysozyme in its native form. In adduct-free solutions, hen’s egg lysozyme was reported most stable in the pH range 3.5–5.0, with a denaturation temperature (Tm) between 75 and 80 °C, while at lower pH conditions, its stability decreases rapidly and its Tm ranges within 52–56 °C at © Woodhead Publishing Limited, 2011
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262 Improving the safety and quality of eggs and egg products pH 2 (Petersen et al. 2004). The mechanism of denaturation and unfolding of lysozyme has been finely characterized using the disulfide scrambling method (Chang and Li 2002). LYS has also been shown to be resistant to pepsin digestion and proteinase K after incubation at 37 °C for 60 min (Polverino de Laureto et al. 2002). In order to examine the relationship between the conformational stability of LYS and its allergenicity, BALB/c mice were sensitized to variants of lysozyme with different conformational stability, but similar three-dimensional structures (So et al. 2001). Surprisingly, the least stable forms were associated with the strongest Th2 associated responses, as measured by IL-4 and specific IgE production. Ovotransferrin (OVT) is a heat-labile allergen but it was reported that when coupled to bi- or trivalent metal ions, it could form heat-stable complexes (Gremmel and Paschke 2007; Yoshitada et al. 1980). With regard to egg yolk proteins, their allergenicity has not been extensively studied compared with egg white proteins. Ovomucin (egg white), riboflavin and other egg yolk proteins seems to be less reactive with human patient sera IgE and they remain to be fully characterized. Ovomucin represents a distinct egg allergen as it has been associated with an increased susceptibility to baker’s asthma, especially in central European population (Quirce et al. 2001). One study documented that heating of a-livetin can reduce, but does not eliminate its allergenicity (Quirce et al. 2001). 13.3.2 Role of egg protein glycosylation The primary role of protein glycosylation pertains to the functional properties of the protein. For instance, they notably contribute to the gelling properties of ovomucin present in the egg white. However, there has been evidence that glycosylation was not always essential to the function of egg proteins. For instance, deglycosylation of avidin does not affect its affinity for biotin. Furthermore, both glycosylated and unglycosylated forms of OVM domain III were shown to be equally inhibitory towards proteases (Stevens 1996). The presence of N-glycosylation usually has a rigidifying or a stabilizing effect on protein structure, thereby conferring an enhanced resistance to denaturation (Pedrosa et al. 2000). It was reported that human IgE-binding to ovomucoid domain III was greatly reduced by deglycosylation (Matsuda et al. 1985), but that mouse anti-ovomucoid IgG could still bind to deglycosylated ovomucoid domain III (Matsuda et al. 1985). The contribution of glycosylation to egg allergenicity has therefore been explored and yielded mixed results. On the one hand, some studies suggested that the carbohydrate moieties did not contribute to the IgE or IgG binding activity of human patients sera (Cooke and Sampson 1997; Besler 1999). Furthermore, carbohydrate moieties were found not to be important for antigen presentation of ovomucoid to T lymphocytes (Mizumachi and Kurisaki 2003), as assessed in murine models. On the other hand, subsequent reports provided evidence that OVM carbohydrate chains may in fact have inhibitory effects on specific IgE binding activity (Zhang and
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Egg allergy 263 Mine 1998). Recent in vitro studies using recombinant glycosylated variants of OVM domain III, indicate that a masking effect exerted by N-glycans could contribute to a reduced allergenicity of OVM (Rupa et al. 2007). This hypothesis was tested in vivo using a BALB/c mouse model (Rupa et al. 2007). A recombinant isoform of ovomucoid domain (P-Gly) was expressed in a Pichia Pastoris yeast system. Groups of mice were subcutaneously injected with either P-Gly or native OVM third domain (DIII). Results based on OVM-specific IgE levels, and cytokine secretion profiles showed that P-Gly is a hypoallergenic variant of DIII. Further structural analyses determined that P-Gly carbohydrate chains present in residue position 28, but not in position 45, may be involved in the hypoallergenicity of P-Gly. While the specific role and effect of carbohydrates on egg allergenicity remain an issue of debate, it has been suggested that the structure as well as the location of carbohydrate moieties present on a protein has immunomodulatory effect, and exert an adjuvant effect due to the existence of carbohydrate-specific innate receptors (Hsu et al. 2007). 13.3.3 Cross-reactivity of egg proteins Reports have determined that cross-reactivity could occur between egg white proteins and egg yolk proteins. Earlier studies showed cross-reactivity between ovotransferrin and ovalbumin, and between apovitellenin I and ovalbumin (Walsh et al. 1987). Although in Western countries, hen’s eggs are the most commonly consumed, eggs from duck, quail and goose are sometimes consumed in other countries (Laundry 2006). To this regard, serological cross-reactivity has been described between hen egg proteins and those of other bird eggs such as turkey, duck, goose and seagull (Langeland 1982a; Anet et al. 1985). Mouse monoclonal IgG raised against Japanese quail ovomucoid were capable to bind to both hen and duck ovomucoid (Takahashi et al. 1999). A case of duck and goose egg allergy has also been reported, but the patient was tolerant to hen’s egg (Añíbarro et al. 2000). A recent study also reported a case of quail egg anaphylaxis in a child with hen egg allergy. The bird-egg syndrome describes the cross-reactivity that can occur between egg and other components of chicken serum or chicken meat. Earlier studies provided evidence that allergic reactions to egg yolk were induced upon respiratory sensitization to bird serum proteins (de Maat-Bleeker et al. 1985; Hoffman and Guenther 1988). Sequence data are today available for egg proteins from many avian species and computational methods could further help predict IgE cross-reactivity (Aalberse and Stadler 2006; Breiteneder and Mills 2006).
13.4 Egg yolk allergenicity It was initially believed that egg yolk was void of allergenic components (Ankier 1969). However, as previously mentioned, subsequent studies © Woodhead Publishing Limited, 2011
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264 Improving the safety and quality of eggs and egg products reported the existence of significant IgE-binding activity against egg yolk components (Walsh et al. 2005). Clinical report of human specific IgE to egg yolk was first reported in the late 1980s (Carrillo Diaz et al. 1986). For example, a-livetin, also known as chicken serum albumin, has been identified as a minor allergen and is believed to be significantly involved in the pathogenesis of bird-egg syndrome (Quirce et al. 1998, 2001; Martorell Aragonés et al. 2001). Similarly, a recent study determined that apovitellenins, a class of apoproteins representing up to 37% of total egg yolk proteins (Kovacs-Nolan et al. 2005), also showed IgE-binding activity, as determined by RAST assays, and more specifically apovitellenins I and VI. Surprisingly, it has also been reported that egg-derived phospholipids (or lecithins) may have residual allergenic properties. However, their presence in manufactured food products was reported insufficient to elicit any adverse effects (Heine et al. 2006).
13.5 Effects of processing on the allergenicity of egg proteins The emergence of egg allergy has had various implications for the food industry, affecting their manufacturing and labeling practices. While no efficient therapy currently exists for egg allergy, strict avoidance of the offending food remains the treatment of choice. However, the omnipresence of eggderived components in prepackaged food products represents a significant hurdle for allergic consumers. Therefore regulatory bodies, food industrials and research scientists have made joint efforts to implement preventive measures encompassing the production of hypoallergenic egg-containing products using chemical or physical processing methods. Food processing methods may alter the allergenicity of food proteins in various ways. In general, food proteins present in a processed food will be in a denatured state, aggregated in protein networks, or interacting with carbohydrates (e.g. Maillard reactions) and lipids, leading either to the reduction of allergen content – and therefore potentially reducing its sensitizing potential – or to the formation of new epitopes (or neoepitopes) (Thomas et al. 2007). With the intention of developing approaches for reducing egg allergenicity in food products, a number of studies have investigated the use of food processing methods such as heat application, enzymatic fragmentation, irradiation or high pressure treatments. 13.5.1 Heat application Thermal processing is usually carried out to enhance texture and flavors, or to ensure microbiological safety, but is not primarily used to reduce allergenicity. However, the basis for this approach was demonstrated in a
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Egg allergy 265 clinical study during which patients allergic to freeze-dried egg white did not react to cooked egg white (Urisu et al. 1997). Another case report described two patients who developed anaphylactic reactions upon raw egg ingestion, while no reaction was observed after cooked egg consumption (Eigenmann 2000). Similarly, earlier in vitro studies showed that thermal treatment of egg white at 90 °C for 10 min led to more than 50% decrease of RAST binding intensity using 16 egg allergic patients sera (Anet et al. 1985). Recently, it was documented that heating of a-livetin at 90 °C for 30 min led to an 88% reduction of its IgE-reactivity (Quirce et al. 2001). On the other hand, other studies reported that residual IgG-binding activity against ovalbumin and ovomucoid could still be detected in both soft-boiled (100 °C, 3 min) and hard-boiled (100 °C, 20 min) eggs, as tested by radio-immunoelectrophoresis (Hoffman 1983). 13.5.2 Enzymatic fragmentation Enzymatic processing represents a more specific approach than thermal processing and was reported to be efficient in the production of milk-based hypoallergenic formulas (Coombs and McLaughlan 1984; Lee 1992). Stability of the major egg allergens to enzymatic digestion was discussed in Section 13.3.1. However, epitopes present on most food allergens are known to be sequential, so that reduction in egg allergenicity would be expected only when egg allergen epitopes are eliminated by the enzymatic fragmentation (Wal 2003). Enzymatic hydrolysis has not been documented for reduction of egg allergenicity in food products. Indeed, it may not represent a viable alternative since egg proteins are often incorporated into food products for their unique functional properties, e.g. foaming and gelling. Less denaturing approaches such as high pressure or irradiation should be examined instead. 13.5.3 Gamma-irradiation Radiation technology has been explored in a number of studies for the modification of egg and other food allergens, such as shrimp and milk allergens (Byun et al. 2000; Lee et al. 2001). In the food industry, treatment doses up to 3 kGy ensure a bacteriological quality of acceptable standard for liquid, frozen or dehydrated egg white preparations (Gremmel and Paschke 2007). One study has investigated the use of g-irradiation in the confection of hypoallergenic egg-based cakes (Seo et al. 2004). ELISA analysis using egg-allergic patients’ sera and OVA-specific rabbit IgG revealed a significant decrease in OVA content after g-irradiation treatment. The same authors reported that g-irradiation of OVA led to a hypoallergenic form of the protein (Seo et al. 2007). Results showed that g-irradiation superior to 10 kGy altered the structure of OVA and led to a significantly decreased immunogenicity. Immunization trials in BALB/c mouse revealed that the titers of OVAspecific immunoglobulins and OVA-specific T cell-mediated responses
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266 Improving the safety and quality of eggs and egg products were significantly lowered in groups of mice sensitized to irradiated-OVA. Furthermore, combination of g-irradiation and heat treatment has been shown efficient in reducing the IgE-binding properties of OVM, the immunodominant egg allergen (Kim et al. 2002; Lee et al. 2002). These findings suggest that structural alteration of protein allergens may offer great opportunities for development of desensitization approaches in the context of egg allergy. 13.5.4 Other food processing methods A main obstacle to the use of food processing in reducing egg allergenicity is the risk to alter the unique functional attributes of egg proteins. Therefore, other processing methods such as high pressure have been proposed as an alternative and milder approach. A recent review suggests that novel food processing methods, such as high pressure and pulse-electric field, combined to physical and biochemical treatments, hold great promises for the development of hypoallergenic food products (Wichers et al. 2003). No such treatment has yet been reported for reduction of egg allergenicity. On the other hand, the production of egg food products with low ovomucoidcontent by solvent extraction has been explored. Ethanol (20%) precipitation successfully removed 70% of the OVM content from egg white powder, without affecting the whipping ability and foam stability of the remaining egg white proteins (Tanabe et al. 2000).
13.6 Conclusion and future trends The emergence of egg allergy has both industrial and clinical implications. In industrialized countries, egg allergy accounts for one of the most prevalent food hypersensitivities, especially in children. Owing to the limitations of conventional OIT, novel forms of immunotherapy are sought based on information obtained from the molecular characterization of major egg allergens. Non-specific approaches have also been investigated, and preliminary trials with use of probiotic bacteria have yielded encouraging results. The current understanding of egg allergens offers novel approaches toward the making of food products safe for human consumption and the development of efficient immunotherapeutic strategies.
13.7 References & stadler bm. 2006. In silico predictability of allergenicity: from amino acid sequence via 3-D structure to allergenicity. Mol Nutr Food Res. 50:625–627. akdis m, blaser k & akdis ca. 2006. T regulatory cells in allergy. Chem Immunol Allergy. 91:159–173. allen cw, campbell de & kemp as. 2007. Egg allergy: are all childhood food allergies the same? J Paediatr Child Health. 43:214–218. aalberse rc
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Egg allergy 267 amo a, rodríguez-pérez r, blanco j, villota j, juste s, moneo i
& caballero ml. 2010. Gal d 6 is the second allergen characterized from egg yolk. J Agric Food Chem. 58:7453–7457. anet j, back jf, baker rs, barnett d, burley rw & howden me. 1985. Allergens in the white and yolk of hen’s egg. A study of IgE binding by egg proteins. Int Arch Allergy Appl Immunol. 77:364–371. añíbarro b, seoane fj, vila c & lombardero m. 2000. Allergy to eggs from duck and goose without sensitization to hen egg proteins. J Allergy Clin Immunol. 105:834–836. ankier si. 1969. Reactivity of rat and man to egg-white. Progress Drug Res. 13:340– 394. astwood jd, leach jn & fuchs rl. 1996. Stability of food allergens to digestion in vitro. Nature Biotechnol. 14:1269–1273. baral vr & o’b hourihane j. 2005. Food allergy in children. Postgrad Med J. 81:693– 701. bernhisel-broadbent j, dintzis hm, dintzis rz & sampson ha. 1994. Allergenicity and antigenicity of chicken egg ovomucoid (Gal d III) compared with ovalbumin (Gal d I) in children with egg allergy and in mice. J Allergy Clin Immunol. 93:1047–1059. besler m. 1999. Hen’s egg white. Internet Symp. Food Allergens. 1:13–33. besler m, petersen a, steinhart h & paschke a. 1999. Identification of IgE-binding peptides derived from chemical and enzymacti cleavage of ovomucoid (Gal d 1). Internet Symp Food Allergens. 1:1–12. bleumink e & young e. 1971. Studies on the atopic allergen in hen’s egg. II. Further characterization of the skin-reactive fraction in egg-white; immuno-electrophoretic studies. Int Arch Allergy Appl Immunol 40:72–88. bohle b. 2004. T lymphocytes and food allergy. Mol Nutr Food Res 48:424–433. boyano-martínez t, garcía-ara c, díaz-pena jm & martín-esteban m. 2002. Prediction of tolerance on the basis of quantification of egg white-specific IgE antibodies in children with egg allergy. J Allergy Clin Immunol. 110:304–309. breiteneder h & mills c. 2006. Structural bioinformatic approaches to understand crossreactivity. Mol Nutr Food Res. 50:628–632. byun mw, kim jh, lee jw, park jw, hong cs & kang ij 2000. Effects of gamma radiation on the conformational and antigenic properties of a heat-stable major allergen in brown shrimp. J Food Prot. 63:940–944. carrillo diaz t, cuevas agustin m, moneo goiri i, ibanez sandi md & urena vilardell v. 1986. Detection of IgE specific for egg yolk by enzyme immunoassay, description of a case. Allergo Immunopathol (Madr.). 14:9–14. chang jy & li l. 2002. The unfolding mechanism and the disulfide structures of denatured lysozyme. FEBS Lett. 511:73–78. cooke sk & sampson ha. 1997. Allergenic properties of ovomucoid in man. J Immunol. 159:2026–2032. coombs rra & mclaughlan p. 1984. Allergenicity of food proteins and its possible modification. Ann Allergy. 53:592–596. de boissieu d & dupont c. 2006. Natural course of sensitization to hen’s egg in children not previously exposed to egg ingestion. Allerg Immunol (Paris) 38:113–117. de maat-bleeker f, van dijk ag & berrens l. 1985. Allergy to egg yolk possibly induced by sensitization to bird serum antigens. Ann Allergy. 54:245–248. djurtoft r, redersen hs, aabin b & barkholt v. 1991. Studies of food allergens: soybean and egg proteins. Adv Exp Med Biol. 289:281–293. eigenmann pa. 2000. Anaphylactic reactions to raw eggs after negative challenges with cooked eggs. J Allergy Clin Immunol. 105:587–588. elsayed s, hammer as, kalvenes mb, florvaag e, apold j & vik h. 1986. Antigenic and allergenic determinants of ovalbumin. I. Peptide mapping, cleavage at the methionyl peptide bonds and enzymic hydrolysis of native and carboxymethyl OA. Int Arch Allergy Appl Immunol. 79:101–107.
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268 Improving the safety and quality of eggs and egg products escudero c, quirce s, fernandez-nieto m, miguel j, cuesta j
& sastre j. 2003. Egg white proteins as inhalant allergens associated with baker’s asthma. Allergy. 58:616– 620. frémont s, kanny g, nicolas jp & moneret-vautrin da. 1997. Prevalence of lysozyme sensitization in an egg-allergic population. Allergy. 52:224–228. gremmel s & paschke a. 2007. Reducing allergens in egg and egg products (Chap. 11). In: Mills, C., Wichers, H. & Hoffmann-Sommergruber, K., editors. Managing allergens in food. Cambridge: Woodhead Publishing Ltd. grimshaw ke. 2006. Dietary management of food allergy in children. Proc Nutr Soc. 65:412–417. gustafsson d, sjöberg o & foucard t. 2003. Sensitization to food and airborne allergens in children with atopic dermatitis followed up to 7 years of age. Pediatr Allergy Immunol. 14:448–452. han dk, kim mk, yoo je, choi sy, kwon bc, sohn mh, kim ke & lee sy. 2004. Food sensitization in infants and young children with atopic dermatitis. Yonsei Med J. 45:803–809. heine rg, laske n & hill dj 2006. The diagnosis and management of egg allergy. Curr Allergy Asthma Rep 6:145–152. hirose j, kitabatake n, kimura a & narita h. 2004. Recognition of native and/or thermally induced denatured forms of the major food allergen, ovomucoid, by human IgE and mouse monoclonal IgG antibodies. BioSci Biotechnol Biochem. 68:2490–2497. hoffman dr. 1983. Immunochemical identification of the allergens in egg white. J Allergy Clin Immunol. 71:481–486. hoffman dr & guenther dm. 1988. Occupational allergy to avian proteins presenting as allergy to ingestion of egg yolk. J Allergy Clin Immunol. 81:484–488. holen e & elsayed s. 1990. Characterization of four major allergens of hen egg-white by IEF/ SDS-PAGE combined with electrophoretic transfer and IgE-immunoautoradiography. Int Arch Allergy Appl Immunol. 91:136–141. holen e, bolann b & elsayed s. 2001. Novel B and T cell epitopes of chicken ovomucoid (Gal d 1) induce T cell secretion of IL-6, IL-13, and IFN-gamma. Clin Exp Allergy. 31:952–964. honma k, kohno y, saito k, shimojo n, tsunoo h & niimi h. 1994. Specificities of IgE, IgG and IgA antibodies to ovalbumin. Comparison of binding activities to denatured ovalbumin or ovalbumin fragments of IgE antibodies with those of IgG or IgA antibodies. Int ArchAllergy Immunol. 103:28–35. hsu sc, tsai th, kawasaki h, chen ch, plunkett b, lee rt, lee yc & huang sk. 2007. Antigen coupled with Lewis-x trisaccharides elicits potent immune responses in mice. J Allergy Clin Immunol. 119:1522–1528. kim mj, lee jw, yook hs, lee sy, kim mc & byun mw. 2002. Changes in the antigenic and immunoglobulin E-binding properties of hen’s egg albumin with the combination of heat and gamma irradiation treatment. J Food Protect. 65:1192–1195. kovacs-nolan j, zhang jw, hayakawa s & mine y. 2000. Immunochemical and structural analysis of pepsin-digested egg white ovomucoid. J Agric Food Chem. 48:6261– 6266. kovacs-nolan j, phillips m & mine y. 2005. Advances in the value of eggs and egg components for human health. J Agric Food Chem. 53:8421–8431. langeland t. 1982a. A clinical and immunological study of allergy to hen’s egg white. II. Antigens in hen’s egg white studied by crossed immunoelectrophoresis (CIE). Allergy. 37:323–333. langeland t. 1982b. A clinical and immunological study of allergy to hen’s egg white. III. Allergens in hen’s egg white studied by crossed radio-immunoelectrophoresis (CRIE). Allergy. 37:521–530. langeland t & harbitz o. 1983. A clinical and immunological study of allergy to hen’s egg white. V. Purification and identification of a major allergen (antigen 22) in hen’s egg white. Allergy. 38, 131–139.
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Egg allergy 269 2006. The relationship of T-cell epitope and allergen structure. In: Maleki, S. J, Burks, A. W. & Helm, R., editors. Food allergy. Washington, DC: ASM Press. p. 123–259. lee jw, kim jh, yook hs, kang ko, lee sy, hwang hj & byun mw. 2001. Effects of gamma radiation on the allergenic and antigenic properties of milk proteins. J Food Prot. 64:272–276. lee jw, lee ky, yook hs, lee sy, kim hy, jo c & byun mw. 2002. Allergenicity of hen’s egg ovomucoid gamma irradiated and heated under different pH conditions. J Food Prot. 65:1196–1199. lee yh. 1992. Food-processing approaches to altering allergenic potential of milk-based formula. J Pediatr. 121:47–50. martorell aragonés a, bone calvo j, garcia ara mc, nevot falco s & plaza martin am. 2001. Allergy to egg proteins. Food Allergy Committee of the Spanish Society of Pediatric Clinical Immunology and Allergy. Allergol Immunopathol (Madr). 29:72–95. matsuda t, nakamura r, nakashima i, hasegawa y & shimokata k. 1985. Human IgE antibody to the carbohydrate-containing third domain of chicken ovomucoid. Biochem Biophys Res Commun. 129–134. miller h & campbell dh. 1950. Skin test reactions to various chemical fractions of egg white and their possible clinical significance. J Allergy Clin Immunol. 21:522–524. mine y & rupa p 2003. Fine mapping and structural analysis of immunodominant IgE allergenic epitopes in chicken egg ovalbumin. Protein Eng. 16:747–752. mine y & zhang jw. 2002. Comparative studies on antigenicity and allergenicity of native and denatured egg white proteins. J Agric Food Chem. 50:2679–2683. mine y, sasaki e & zhang jw. 2003. Reduction of antigenicity and allergenicity of genetically modified egg white allergen, ovomucoid third domain. Biochem Biophys Res Commun. 302:133–137. mizumachi k & kurisaki j. 2003. Localization of T cell epitope regions of chicken ovomucoid recognized by mice. BioSci Biotechnol Biochem. 67:712–719. morisset m , moneret - vautrin da , kanny g , guenard l , beaudouin e , flabbee j & hatahet r. 2003. Thresholds of clinical reactivity to milk, egg, peanut and sesame in immunoglobulin E-dependent allergies: evaluation by double-blind or single-blind placebo-controlled oral challenges. Clin Exp Allergy. 33:1046–1051. pedrosa c, de felice fg, trisciuzzi c & ferreira st. 2000 Selective neoglycosylation increases the structural stability of vicilin, the 7S storage globulin from pea seeds. Arch Biochem Biophys. 382:203–210. petersen sb, jonson v, fojan p, wimmer r & pedersen s. 2004. Sorbitol prevents the selfaggregation of unfolded lysozyme leading to and up to 13 degrees C stabilisation of the folded form. J Biotechnol. 114:269–278. polverino de laureto p, frare e, gottardo r, van dael h & fontana a. 2002. Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis. Protein Sci. 11:2932–2946. prioult g & nagler-anderson c. 2005. Mucosal immunity and allergic responses: lack of regulation and/or lack of microbial stimulation? Immunol Rev. 206:204–218. quirce s, díez-gómez ml, eiras p, cuevas m, baz g & losada e. 1998. Inhalant allergy to egg yolk and egg white proteins. Clin Exp Allergy. 28:478–485. quirce s, maranon f, umpierrez a, de las heras m, fernandez-caldas e & sastre j 2001. Chicken serum albumin (Gal d 5) is a partially heat-labile inhalant and food allergen implicated in the bird-egg syndrome. Allergy. 56:754–762. rolinck-werninghaus c, staden u, mehl a, hamelmann e, beyer k & niggemann b. 2005. Specific oral tolerance induction with food in children: transient or persistent effect on food allergy? Allergy. 60:1320–1322. rupa p, nakamura s & mine y. 2007. Genetically glycosylated ovomucoid third domain can laundry sj
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270 Improving the safety and quality of eggs and egg products modulate Immunoglobulin E antibody production and cytokine response in BALB/c mice. Clin Exp Allergy. 37:918–928. sampson ha. 2004. Update on food allergy. J Allergy Clin Immunol. 113:805–819. savage jh, matsui ec, skripak jm & wood ra. 2007. The natural history of egg allergy. J Allergy Clin Immunol. 120:1413–1417. seo jh, lee jw, lee ys, lee sy, kim mr, yook hs & byun mw. 2004. Change of an egg allergen in a white layer cake containing gamma-irradiated egg white. J Food Prot. 67:1725–1730. seo jh, kim jh, lee jw, yoo yc, kim mr, park ks & byun mw. 2007. Ovalbumin modified by gamma irradiation alters its immunological functions and allergic responses. Int Immunopharmacol. 7:464–472. so t, ito h, hirata m, ueda t & imoto t. 2001. Contribution of conformational stability of hen lysozyme to induction of type 2 T-helper immune responses. Immunology. 104:259–268. stevens l. 1996. Egg proteins: what are their functions? Sci Prog. 79:65–87. taams ls, palmer db, akbar an, robinson ds, brown z & hawrylowicz cm. 2006. Regulatory T cells in human disease and their potential for therapeutic manipulation. Immunology. 118:1–9. takagi k, teshima r, okunuki h & sawada j 2003. Comparative study of in vitro digestibility of food proteins and effect of preheating on the digestion. Biol Pharm Bull. 26:969–973. takagi k, teshima r, okunuki h, itoh s, kawasaki n, kawanishi t, hayakawa t, kohno y, urisu a & sawada j 2005. Kinetic analysis of pepsin digestion of chicken egg white ovomucoid and allergenic potential of pepsin fragments. Int Arch Allergy Appl Immunol. 136:23–32. takahashi k, horiguchi m, bando n, tsuji h, ogawa t & asao t. 1999. Immunochemical characterization of ovomucoid from Japanese quail egg white using monoclonal antibodies. J Nutr Sci Vitaminol (Tokyo). 45:491–500. tanabe s, tesaki s & watanabe m. 2000. Producing a low ovomucoid egg white preparation by precipitation with aqueous ethanol. Biosci Biotechnol Biochem. 64:2005–2007. tariq sm, matthews sm, hakim ea & arshad sh. 2000. Egg allergy in infancy predicts respiratory allergic disease by 4 years of age. Pediatr Allergy Immunol. 11:162– 167. taylor sl, hefle sl, bindslev-jensen c, atkins fm, andre c, bruijnzeel-koomen c, burks aw, bush rk, ebisawa m, eigenmann pa, host a, hourihane jo, isolauri e, hill dj, knulst a, lack g, sampson ha, moneret-vautrin da, rance f, vadas pa, yunginger jw, zeiger rs, salminen jw, madsen c & abbott p. 2004. A consensus protocol for the determination of the threshold doses for allergenic foods: how much is too much? Clin Exp Allergy. 34:689–695. teuber ss, beyer k, comstock s & wallowitz m. 2006. The big eight foods: clinical and epidemiological overview. In: Maleki, S. J, Burks, A. W. & Helm, R. M., editors. Food Allergy. Washington DC: AMS Press. p. 49–79. thomas k, herouet-guicheney c, ladics g, bannon g, cockburn a, crevel r, fitzpatrick j, mills c, privalle l & vieths s. 2007. Evaluating the effect of food processing on the potential human allergenicity of novel proteins: international workshop report. Food Chem Toxicol. 45:1116–1122. urisu a, ando h, morita y, wada e, yasaki t, yamada k, komada k, torii s, goto m & wakamatsu t. 1997. Allergenic activity of heated and ovomucoid-depleted egg white. J Allergy Clin Immunol. 100:171–176. van toorenenbergen aw, huijskes-heins mi & gerth van wijk r. 1994. Different pattern of IgE binding to chicken egg yolk between patients with inhalant allergy to birds and food-allergic children. Int. Arch. Allergy Immunol. 104: 199–203. van wijk f & knippels l. 2007. Initiating mechanisms of food allergy: Oral tolerance versus allergic sensitization. Biomed Pharmacother. 61:8–20.
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Egg allergy 271 vance gh, lewis s, grimshaw ke, wood pj, briggs ra, thornton ca
& warner jo. 2005. Exposure of the fetus and infant to hens’ egg ovalbumin via the placenta and breast milk in relation to maternal intake of dietary egg. Clin Exp Allergy. 35:1318–1326. wal jm. 2003. Thermal processing and allergenicity of foods. Allergy. 58:727–729. walsh bj, elliott c, baker rs, barnett d, burley rw, hill dj & howden me. 1987. Allergenic cross-reactivity of egg-white and egg-yolk proteins. An in vitro study. Int Arch Allergy Appl Immunol. 84:228–232. walsh bj, barnett d, burley rw, elliott c, hill dj & howden me. 1988. New allergens from hen’s egg white and egg yolk. In vitro study of ovomucin, apovitellenin I and VI, and phosvitin. Int. Arch. Allergy Appl. Immunol. 87, 81–86. walsh bj, hill dj, macoun p, cairns d & howden me. 2005. Detection of four distinct groups of hen egg allergens binding IgE in the sera of children with egg allergy. Allergol Immunopathol (Madr). 33:183–191. weiner hl. 2000. Oral tolerance, an active immunologic process mediated by multiple mechanisms. J Clin Invest. 106:935–937. wichers h, matser a, van amerongen a, wickers j & soler-rivas c. 2003. Monitoring of and technological effects on allergenicity of proteins in the food industry. In: Mills, E.N.C. and Shewry, P.R. editors. Plant food allergens. Blackwell: Oxford; p 196–212. yoshitada n, yoshinobu f, noboru n & gtoshitaka y. 1980. Pasteurization and quality maintenance of eggs for industrial use. IX. Disc gel electrophoretic evaluation of heat-denatured egg proteins for detecting pasteurized liquid eggs. Rakanu Kagaku, Shokuhin no Kenkyu. 29:A85–A90. zhang jw & mine y. 1998. Characterization of IgE and IgG epitopes on ovomucoid using egg-white-allergic patients’ sera. Biochem Biophys Res Commun. 253:124–127. zhang jw & mine y. 1999. Characterization of residues in human IgE and IgG binding site by chemical modification of ovomucoid third domain. Biochem Biophys Res Commun. 261:610–613.
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14 Modifying egg lipids for human health F. Sirri and A. Meluzzi, University of Bologna, Italy
Abstract: In recent years, the lipid composition of food has received a great deal of consumer attention due to the correlation between human disease and characteristics of dietary fats. In addition the lipid composition of eggs has been viewed with suspicion due to its high cholesterol content. Several studies have demonstrated that it is possible to modify the fatty acid composition of the yolk by enriching the egg with n-3 polyunsaturated fatty acids (PUFA). This chapter describes the egg lipid fraction and fatty acid metabolism, along with the effect of supplementing the diet of hens with vegetable and marine sources of n-3 PUFA on the fatty acid composition of yolk and its sensory attributes. Key words: enriched egg, n-3 PUFA, conjugated linoleic acid, sensory properties.
14.1 Introduction As the means for the continuation of the avian species, the components of the egg are perfectly balanced for the creation of a living organism. Nevertheless the egg is, together with milk, one of the most important foods of animal origin for humans and it is consumed by different groups of people all over the world. Owing to its composition, some components may be modified in order to obtain a food tailored for human health or for industrial applications in both food and non food uses. The evidence correlating plasma cholesterol levels with coronary heart diseases was established on the basis of early observations that cholesterol is a major component of atherosclerotic plaques and that the induction of © Woodhead Publishing Limited, 2011
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Modifying egg lipids for human health 273 hypercholesterolaemia is a prerequisite for the production of these plaques in a variety of animal species (Hargis, 1988). Egg lipids have attracted attention both at the scientific and consumer level due to the link between high levels of dietary fat consumption and coronary heart diseases (CHD) (Cherian, 2005). One egg contains a large amount of lipids (about 6 g per 60 g of whole egg) exclusively concentrated in the yolk. It should be taken into account that the main function of yolk lipid is to provide metabolic energy for the embryo. For many years the egg has been viewed with suspicion due to both its high cholesterol content and to two significant nutritional recommendations: firstly, that dietary cholesterol should be restricted to less than 300 mg/day, and secondly, that those at high risk of developing heart disease should consume no more than three eggs per week. This issue is further examined in Chapter 12 of this volume. In recent decades several experiments have been carried out to find ways of reducing cholesterol levels and to obtain a lipid composition with advantages for human health by enhancing the concentrations of beneficial compounds. Moreover the consumer perception that eggs are principally a source of dietary cholesterol has changed in recent years and the evidence that cholesterol intake is related to cardiovascular disease is less consistent. Although evidence from various studies indicates that increasing dietary cholesterol intake can raise serum LDL cholesterol levels, leading to an increased risk of CHD, many results from earlier studies were complicated by the presence of saturated fatty acids (SFA) in the experimental diets (Gray and Griffin, 2009). The European Heart Network (2002) also emphasises the importance of dietary SFA and trans fatty acids in relation to the level of blood cholesterol and does not specifically recommend the restriction of dietary cholesterol intake from eggs. The American Council on Science and Health (2002) stated that cholesterol and the consumption of eggs had no significant implications for the risk of heart disease in healthy people. Numerous epidemiological and clinical studies have claimed that the onset of the pathologies typical of Western countries is related to the n-6/n-3 ratio (Simopoulos, 2000, 2008). A lower ratio of n-6/n-3 is more desirable in reducing the risk of chronic diseases of high prevalence both in Western society as well as in developing countries. The yolk of an ordinary egg contains a high proportion of n-6 polyunsaturated fatty acids (PUFA) and a low proportion of n-3 PUFA; however, it is easy to reduce the n-6/n-3 ratio of the egg through the enrichment of the diet of the hen with long chain n-3 PUFAs from both vegetable and animal ingredients. The n-3 PUFAs are essential fatty acids for a number of different physiological processes in humans (formation of brain tissue, cell membrane, etc.), and moreover the intake of eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA) exerts a higher potential cardio-protective effect than the intake of alpha-linolenic acid (He, 2009). A large body of evidence supports the theory that fish consumption or long chain n-3 PUFA intake may provide cardio-protective effects. It has been suggested that the beneficial effect of fish consumption or
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274 Improving the safety and quality of eggs and egg products long chain n-3 PUFA intake in preventing cardiovascular disease is related to their overall favourable effects on lipid profiles, the threshold for arrhythmias, platelet activity, inflammation and endothelial function, atherosclerosis and hypertension (He, 2009). In the following sections factors influencing the lipid composition of egg yolk and their impact on human health will be discussed.
14.2 Egg lipid fractions The lipids are entirely contained in the yolk, representing almost 33% of the total weight, and are mainly distributed in the form of triacylglycerols (66%), phospholipids (28%) and free cholesterol (5%). Other minor components are cholesterol esters and free fatty acids. Almost all the lipids are present as lipoprotein complexes with the overall lipid : protein ratio being approximately 2 : 1. Owing to a difference in physical properties, two major yolk lipoprotein fractions are identified: one of low and the other of high density. The low density fraction is similar to the very low density plasma lipoprotein of mammals and contains over 90% of the yolk lipids. It consists of a non-polar core of triacylglycerol surrounded by a coat of apoproteins, phospholipids and cholesterol. Owing to their amphipathic structure, egg phospholipids have recognised emulsifying properties. The concentration of triacylglycerol in the low density fraction is twice that of the high density fraction while in the high density fraction the amount of total phospholipids is twice that present in low density fraction (Noble, 1987; Leskanich and Noble, 1997). Phospholipids are mainly composed of phosphatidylcholine, phosphatidyethanolamine and phosphatidylserine. The proportion of each phospholipid is similar in low and high density fractions (Table 14.1). Moreover the proportion of triacylglycerol and phospholipids are about 63% and 30% of total lipids respectively; therefore one yolk contains about 4 and 2 g of these classes of lipids. Free cholesterol plus cholesterol esters account for 6% of total lipids (Fig. 14.1). Phosphatidylcholine and Table 14.1 Proportions of major lipid fraction in low and high density yolk fractions in percentage (from Noble, 1987)
Total lipid Triacylglycerola Total phospholipidsa Phosphatidylethanolammineb Phosphatidylcholineb Total cholesterola a
Low density fraction
High density fraction
93 69 27 19 72 4
7 35 61 18 75 4
% of total lipid in yolk; b % of total phospholipids.
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Modifying egg lipids for human health 275 4.9
1.3 0.9
29.7
63.1
Triacylglycerols
Phospholipids
Cholesterol esters
Free fatty acids
Free cholesterol
Fig. 14.1 Proportions of major lipids (% weight of total) in the yolk (from Noble, 1987). 3.2
2.7
1
23.9
69.1
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylserine
Sphingomyelin
Others
Fig. 14.2 Proportions of major phospholipids in the yolk (from Noble, 1987).
phosphatidylethanolamine make up more than 90% of total phospholipids (Fig. 14.2). Fatty acids are the most prevalent component of triacylglycerols and phospholipids. The fatty acids have different chain length, number and placement of double bonds, and isomerism around these bonds. The fatty acid categories of the yolk are saturated, monounsaturated and polyunsaturated, the latter being divided into n-3 and n-6. The predominant saturated fatty acids are palmitic (C16:0) and stearic (C18:0) which constitute between 22 and 26% and from 8 to 10% of the total saturated fatty acids respectively (Cherian, 2005) while myristic (C14:0) and arachic acids (C20:0) are present in small amounts. Of the monounsaturated fatty acids, which constitute 35–46% of the total fatty acids (Baucells et al., 2000; Meluzzi et al., 2001a), oleic acid (18:1) is the most abundant, making up between 38 and 44% depending on the dietary lipid source, followed by palmitoleic acid (C16:1)
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276 Improving the safety and quality of eggs and egg products (2–3%). An early study on the effect of dietary saturated, monounsaturated and polyunsaturated fatty acids on lipid profile in humans has reported that oleic acid has a hypolipidaemic effect, reducing both plasma cholesterol and triacylglycerols in humans without decreasing high density lipoprotein cholesterol (Mattson and Grundy, 1985). The PUFA content ranges from 14 to 25% in regular eggs (Baucells et al., 2000; Meluzzi et al., 2001a). The two PUFA families, n-3 and n-6, differ in the positioning of the last double bond in the chain of carbon atoms. The predominant n-6 PUFAs are linoleic acid (C18:2), ranging from 11 to 21% of total fatty acids, followed by arachidonic acid (C20:4) ranging from 1 to 2%. The n-3 PUFAs comprise alpha-linolenic (C18:3), eicosapentaenoic (C20:5), docosapentaenoic (C22:5) and docosahexaenoic acids (C22:6). The content of long chain PUFAs (> 20-carbon atoms) of both families in the yolk may vary greatly depending on the composition of the hen’s diet. The yolk contains a substantial amount of cholesterol ranging from 12 to 14 mg/g, depending on the size of the egg and on the breed and age of the hen. The younger the layer and the smaller the eggs, the greater the cholesterol content for each gram of yolk. This trend may be due to the more active metabolism of the younger birds. However, as the weight of yolk in larger eggs is greater, the overall cholesterol content is higher in eggs laid by older hens (Meluzzi et al., 1993). Commercial hybrids (Hy-Line brown and Hy-Line white) exhibited a lower cholesterol content than two dual-purpose breeds (Ermellinata di Rovigo and Robusta maculata) (215 and 222 mg/egg vs 256 and 252 mg/egg respectively) (Rizzi and Chiericato, 2010). Cholesterol is present in the yolk both as free cholesterol (4.9% of the total lipids) or as cholesterol esters (1.3%) (Leskanich and Noble, 1997). The hypothesis that dietary cholesterol raises blood cholesterol levels in humans, and that it is one of the main factors responsible for cardiovascular diseases, has limited the consumption of eggs for several decades. Later, many clinical epidemiological studies demonstrated that dietary cholesterol has a small and insignificant effect on blood cholesterol in healthy people, with other factors, such as levels of saturated fatty acids, proving much more dangerous. Despite the lifting of the restriction on egg consumption by major food and health advisory bodies in Europe, the public continues to view cholesterol from eggs with suspicion (Gray and Griffin, 2009).
14.3 Fatty acid metabolism in laying hens In poultry linoleic acid is the main essential fatty acid (EFA) although alpha-linolenic acid (ALA) also is required since a significant concentration of its derivatives (DHA) is found in the lipids of the retina and nervous tissues (Watkins, 1995). Linoleic acid belongs to the n-6 PUFAs whereas ALA is a member of the n-3 PUFA family. The linoleic acid requirement © Woodhead Publishing Limited, 2011
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Modifying egg lipids for human health 277 for physiological purposes is about 1% of the diet but an additional amount is necessary to achieve maximum egg size. Since linoleic acid is contained in several raw materials used in feed formulation, its presence in hen feed is normally adequate. Fatty acids are absorbed into the mucosal cells where resynthesis of triacylglycerols as well as packaging of lipids into portomicrons occurs for transport to the liver. The EFAs are the substrates for different metabolic pathways such as mitochondria oxidation to generate ATP, desaturation and elongation for their conversion into long chain PUFAs. As represented in Fig. 14.3, delta-5 and delta-6 desaturases are responsible for the conversion of EFAs to PUFAs of both the n-6 and n-3 families, as components of phospholipids and precursors of eicosanoids. The preferred substrates of delta-6 desaturase are, in order of importance, ALA, linoleic and oleic acids. Saturated fatty acids are converted to monounsaturated fatty acids by the delta-9 desaturase enzyme and, if EFA deficiency occurs, oleic acid is converted by delta-6 desaturase to eicosatrienoic acid (C20:3 n-9). Starting from linoleic acid, gamma-linolenic acid (C18:3 n-6) is formed and is elongated to C20:3 n-6 (dihomo-gamma linolenic acid) then desaturated to obtain arachidonic acid (C20:4 n-6). Concerning the n-3 PUFAs pathway, stearidonic acid (C18:4 n-3) is formed through the desaturation of alpha linolenic acid (C18:3n-3), then elongated to eicosatetraenoic acid (C20:4 n-3), which in turn is converted to eicosapentaenoic acid (C20:5 n-3) by the delta-5 desaturase. Following this, docosahexaenoic acid (C22:6 n-3) is formed through elongation and desaturation reactions (Fig. 14.3). Arachidonic acid, which takes part in the formation of membrane phospholipids, is the precursor of a range of highly biologically active derivatives called eicosanoides, including prostaglandins, thromboxanes, leukotrienes and lipoxins which act as autocrine hormones regulating many physiological processes such as haemostasis, reproduction and inflammatory responses. However, other C20 PUFAs, such as dihomo-gamma linolenic acid and EPA, are known to modulate eicosanoid metabolism by inhibiting the conversion of arachidonic acid to eicosanoids while simultaneously converting to eicosanoids with properties unlike those of their arachidonic acid homologues (Weber, 1990). Eicosanoids derived from EPA – prostaglandin E3, thromboxane B3 and leukotriene B5 – are less inflammatory and less potent than n-6 derived eicosanoids, while those derived from arachidonic acid – prostaglandin E2, thromboxane B2 and leukotriene B4 – are more pro-inflammatory and have been shown to increase vascular permeability, vasodilatation, edema, release of lysosomal enzymes, generation of reactive oxygen species, and production of inflammatory cytokines (Calder, 2006). The eicosanoid biosynthesis can be modulated in relation to the proportion of n-6 and n-3 PUFAs of the diet in order to produce the desired biological response in immunocompetent cells to enhance or depress the immune reaction in poultry.
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Lipoxygenase
Cyclooxygenase
Cyclooxygenase
w 1
Docosapentaenoic acid (DPA) C22:5
b-oxidation
Tetracosahexaenoic acid C24:5
D-6 desaturase
Tetracosapentaenoic acid C24:4
Elongase
Docosatetraenoic acid C22:4
Elongase
Arachidonic acid (ARA) C20:4
D-5 desaturase
Dihomo-y-linolenic (DGLA) C20:3
Elongase
y-linolenic acid (GLA) C18:3
D-6 desaturase
12 9 Linoleic acid (LA) C18:2
6
9
6 12
Elongase
Docosahexaenoic acid (DHA) C22:6
b-oxidation
Tetracosahexaenoic acid C24:6
D-6 desaturase
Tetracosapentaenoic acid C24:5
Elongase
Docosapentaenoic acid (DPA) C22:5
Elongase
Eicosapentaenoic acid (EPA) C20:5
D-5 desaturase
Eicosatetraenoic acid C20:4
Stearidonic acid C18:4
D-6 desaturase
HO 1 a 9 Linolenic acid (ALA) C 18:3
O
n-3 fatty acids biosynthesis w 1 18
PGI3 TXA3
Lipoxygenase
LTB6 LTC5 LTE5
Cyclooxygenase PGE3
15
3
Fig. 14.3 N-6 and n-3 PUFAs biosynthesis. LA and ALA are converted to long chain fatty acids (DPA n-6 and DHA n-3 respectively) by using elongases and delta-5 and delta-6 desaturases enzymes. DGLA, ARA and EPA are the substrate for eicosanid biosynthesis depending on the activities of cyclooxigenases and lipoxygenases (PGE, prostaglandins; PGI, prostacyclins; TXA, tromboxanes: PGF, prostaglandin F1a; LTB, Leukotriene B; LTC, Leukotriene C; LTE, Leukotriene E).
LTB4 LTC4 LTE4
PGE2 PGI2 TXA2
PGE1 PGF1a
HO 1
O
n-6 fatty acids biosynthesis
Modifying egg lipids for human health 279
14.4 Effect of hen’s diet on lipid components The possibility of manipulating the egg lipid composition through the hen’s diet was first studied in 1934 by Cruickshank, who observed that diet little influences the amount of fat in the yolk; the fatty acid composition of yolk is modulated by the diet with particular regard to unsaturated fatty acids, while saturated fatty acids have less influence on the yolk lipid profile. The lipid composition of the yolk is the result of a combination of de novo synthesis and incorporation from the hen’s diet. The first step in the modification of the fatty acid composition of the egg was focused on the increase in the proportion of unsaturated fatty acids, mainly linoleic acid, and on the U:S ratio (unsaturated/saturated fatty acids). In fact, during the 1980s, several reports regarding human nutrition and health recommended increasing the PUFAs intake to enhance the U:S ratio in the human diet. To achieve this objective several studies were also carried out specifically in the egg sector to evaluate the effect of different lipid supplements in the hen’s diet on the yolk composition. The addition of 3% of coconut butter or 2% of palm butter, both rich in saturated short chain fatty acids, produced eggs with a higher proportion of palmitoleic and oleic acid and lower proportions of stearic and linoleic acid compared with eggs obtained using corn or grapeseed oil, which both contain a high proportion of linoleic acid (Sirri et al., 1995; Meluzzi et al., 2001a), as the lipid source. Despite the high content of palmitic acid in the diet, the content of this fatty acid in ‘palm eggs’ was not significantly higher than in other groups (Meluzzi et al., 2001a). Moreover supplementation with corn and soybean oil increases the U:S ratio in the yolk. Grobas et al. (2001), in a detailed study on the use of different lipid sources (tallow, olive, soy and linseed oil) at the rate of 5 vs 10%, found that saturated fatty acid in the yolk decreased as the percentage of added fat increased, regardless the lipid source (36.5, 32.3 and 31.4 for diet containing 0%, 5% and 10% of added fat respectively). Olive oil increased the monounsaturated content of the yolk, with a particular increase in oleic acid content; soy oil increased the level of n-6 fatty acid, with a particular increase in linoleic acid. During the 1990s, the British Nutrition Foundation (BNF, 1992) recommended the abolition of the polyunsaturated : saturated fatty acid ratio, stressing that not all PUFAs have the same effect and that all saturated fatty acids are atherogenic. An increase in n-3 PUFAs instead of n-6 PUFAs was suggested, in order to reach a n-6 : n-3 ratio lower than 6:1. Following these recommendations as well as those of other health institutions (Department of Health, 1994) research on animal feeding was oriented towards the enrichment of eggs with n-3 fatty acids by feeding hens with vegetable or animal sources of these fatty acids. Table 14.2 summarises the n-3 PUFA concentrations in the yolk of eggs obtained from hens fed diets supplemented with different n-3 PUFA sources. The addition of rapeseed, flaxseed and evening primrose seeds to the
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1
Oil Oil Oil Oil Oil Oil Oil Oil Meal
Seeds Seeds Seeds Seeds Oil Oil
–
2.4 2 0.6 3
3 3 3 3 4 4 2 4
16 15 4 15 2 4
Inclusion (%)
n.r. 1.42 n.r. 1.22
23 26 37 n.r. 25.3 18.1 17.9 0.41 4.12
8.81 n.r. 35.2 420 226 4.91
ALA
Percent of total fatty acid;2 expressed as mg/g yolk; n.r.: not reported.
Algae Schizochytrium sp. Schizochytrium sp. Schizochytrium sp. Crypthecodinium coonii
Fish Mackerel Menhaden Sand eel Menhaden (ROPUFA 30 DHA oil) (ROPUFA 30 EPA oil) Fish Fish Fish
Flax
n-3PUFA source
n.r. 0.432 n.r. 0.392
40 30 26 n.r. 16.6 27.4 7.9 0.91 0.22
0.151 n.r. n.r. 8.5 5.92 0.31
EPA
8.82 132 103 10.92
251 185 148 n.r. 180 184 129 3.651 6.92
1.541 n.r. 35.0 91.8 117 1.851
DPA+DHA
9.52 14.72 123.8 12.62
343 241 207 232 227 230 156 5.061 11.32
10.81 242 70.2 520 350 7.071
Total n-3
Table 14.2 N-3 PUFA concentration in the yolk (mg/egg) in relation to the n-3 PUFA sources of the hen diet
Herber and Van Elswick (1996) Sirri et al. (2001) Wertelecki and Buttin (2009) Meluzzi et al. (2001b)
Farrell (2000) Hargis et al. (1991) Hammersoj (1995) Van Elswyk (1997) Meluzzi et al. (1997a) Meluzzi et al. (1997a) Meluzzi et al. (1997b) Baucells et al. (2000) Simopoulos (2000)
Niemiec et al. (1999) Jia et al. (2008) Meluzzi et al. (2001a) Baucells et al. (2000)
Cherian and Sim (1991)
Reference
Modifying egg lipids for human health 281 hen’s diet can increase the polyunsaturated fatty acid content by as much as 74% compared with the control group (Caston and Leeson, 1990; Cherian and Sim, 1991; Niemiec et al. 1999; Yannakopoulos et al., 1999; Gurbuz et al. 2010). Farrell (1994) demonstrated that the addition of vegetable oil, rapeseed or linseed to the hen’s diet provided a substantial increase in the n-3 PUFA content, mainly consisting of ALA in the egg yolk with a small increase in long chain n-3 PUFAs such as EPA and DHA. Jia et al. (2008) using a diet supplemented with 150 g/kg of diet of canola, flaxseed or Linpro (flaxseed:peas 1:1 wt/wt) found that hens fed flaxseed had the greatest n-3 fatty acid content (562 mg/60 g of egg) when compared with those from hens consuming canola seed (207 mg/60 g of egg) or Linpro (427 mg/60 g of egg). Deposition of ALA, EPA and DHA was significantly greater for diets containing flaxseed than for diets containing canola seed. As a result, the n-6 : n-3 ratio differed significantly. Amini and Ruiz-Feria (2007), using an increasing percentage of flaxseed (2, 4, 8 or 12%), found that 2% of dietary flaxseed had a limited effect on the increase in n-3 fatty acid content of the eggs and a supplement of more than 4% proved necessary in order to obtain eggs with a higher n-3 fatty acid content and a lower n-6 : n-3 ratio than control eggs. Hens given a diet based on 8% flaxseed produced eggs with a n-3 fatty acid content higher than that required for labelling eggs as ‘omega-3 enriched eggs’ (350 mg of n-3 per egg) in the Canadian market. The DHA content of the yolk was substantial, demonstrating that hens are able to synthesise DHA from linolenic acid. Among the n-3 fatty acid families, the long chain PUFAs EPA and DHA that are contained only in marine algae or fish products are the most efficient at preventing cardiovascular disease (Farrell, 2000; Degirolamo et al., 2010). Algae are the primary and unique synthesiser of long chain n-3 PUFAs; they produce these fatty acids to keep their membranes fluid in cold temperatures, enabling them to photosynthesise and grow. In the food chain, n-3 PUFAs are transferred to zooplankton, which consume algae and other small marine organisms, then to fish and finally to other animals, including humans, which consume fish and other marine products. Several studies have been carried out to evaluate the effect of fish oil on the proportion of n-3 PUFA in the yolk (Hargis et al., 1991; Farrell, 1994; Meluzzi et al., 1997a, 1997b; Sirri et al., 2001). The addition of 2, 3 and 4% of fish oils containing either 16.8% EPA and 9% DHA (ROPUFA-EPA oil) or 14.4% DHA and 9% EPA (ROPUFA-DHA oil) enhanced the proportions of EPA and DHA in the yolk, depending on the dose. When the same dose of dietary oil was administered, the EPA content of the yolk was always higher in Ropufa-EPA oil groups than in ropufa-DHA-oil groups. With the control eggs, the docosapentaenoic acid (C22:5 or DPA) concentration was higher by 50–200% and the DHA concentration by 200–370% according to the supplemented oil dose. With both refined fish oils, at any level of supplementation, a significant incorporation of n-3 fatty acids was achieved.
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282 Improving the safety and quality of eggs and egg products When ropufa-dha oil was added to the diet, eggs had statistically higher levels of DHA and ALA and lower levels of EPA and DPA compared to groups whose diet was enriched with ropufa-epa oil, closely reflecting the fatty acid composition of the diet. In all the enriched eggs the n-3 acid with the highest concentration was DHA, its level being six times higher than that of the other acids. In the diets, in contrast, the fatty acid distribution was quite different: in ROPUFA-DHA oil groups the DHA content was 1.5 and 3 times higher than that of EPA and ALA respectively, while in ROPUFA-EPA oil groups the EPA content was about 2 and 5–10 times higher than DHA and ALA respectively. This suggests that the DHA is synthesised by the hen, using LNA and EPA as the starting point, following the chain elongation and desaturation processes. When the yolk was enriched with n-3 PUFAs, the arachidonic acid, being synthesised from linoleic acid, decreased (25–50% less than in controls) due to the larger utilisation of delta-6-desaturase in the n-3 pathway rather than in the n-6 pathway (Fig. 14.3) (Meluzzi et al., 1997a, 1997b; Farrell, 1994). Van Elswyck (1997), feeding hens graded levels of dietary menhaden oil (0, 0.5, 1, 1.5, 2, 2.5, 3%), found the greatest n-3 PUFA deposition in yolk occurred with the highest levels of menhaden oil supplementation of up to 200 mg/yolk, comparable with that found in a 100 g serving of lean cold-water fish. Herber and Van Elswyck (1996) compared the efficiency of menhaden oil and marine microalgae (DHA Gold TM) rich in DHA and found that the incorporation of n-3 PUFAs into the yolk proved much higher with the latter source. However, doubling the amount of algae in the diet (from 2.4 to 4.8%) did not result in double the quantity of n-3 PUFAs deposited in the yolk, indicating that there is a limit to the utilization of the n-3 supplements. Moreover, the use of algae naturally rich in carotenoids provides a high level of protection against oxidative processes in both n-3 fatty acid enriched diets and n-3 enriched eggs (Herber and Van Elswyck, 1996). Loh et al. (2009), studying the effects of feeding layers with fermented fish on levels of PUFAs and cholesterol, found not only that EPA and DHA increased, but also that both yolk and plasma cholesterol decreased. A reduction of 20% in the cholesterol content of the yolk, and of 25% in its fat content other than the increase of n-3 PUFAs, was also reported by Yannakopoulos et al. (2004), who fed hens a diet rich in flaxseed, a herbal mix from Mediterranean plants, vitamin E, iodine and selenium. Sirri et al. (2001) comparing the dietary inclusion of extruded flaxseed with marine algae and with microincapsulated fish oil (control) found the highest concentration of DHA in marine algae group (12.50 vs 6.05 mg/g of yolk for flaxseed and 9.57 mg/g of yolk for the control). The EPA and DPA contents appeared negligible in all the groups, at around 1 mg/g or less. Combining the hen dietextrured flaxseed and marine algae, intermediate results were obtained.
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Modifying egg lipids for human health 283 The rate and efficiency of the elongation of ALA to DHA is affected by different factors such as the strain and age of the hen and the n-6 : n-3 ratio of the hen’s diet; accordingly, comparing these findings with those of different authors does not seem appropriate (Scheideler et al., 1998). The rate of transfer of the fatty acids from the diet to the yolk depends on the fatty acid proportion of the n-3 PUFA source and its rate of inclusion. In general, DHA has a better transfer efficiency than EPA, because the latter is partially converted into DHA by the hen’s metabolism. Moreover greater transfer efficiency is observed with low dietary supplementation. The rate of transfer ranges from 5 to 7% for EPA and from 40 to 70% for DHA (Hammershoj, 1995; Meluzzi et al., 1997a, 2000). Owing to the benefits of conjugated linoleic acid (CLA) for human health, such as reducing carcinogenesis, atherosclerosis, diabetes and body fat mass as well as modulating immune functions (Pariza and Hargraves, 1985; Chin et al., 1992; Ip et al., 1994; Lee et al., 1994; Banni and Martin, 1998; Du and Ahn, 2002) there has been some interest in fortifying animal products for human consumption, including eggs, with CLA. The results of different trials confirm that it is possible to deliver around 0.3–1 g of CLA to an egg (Chamruspollert and Sell, 1999; Du et al., 1999; Meluzzi et al., 2003). According to Yin et al. (2008) the concentration of total CLA and CLA isomers in the yolk lipids increased by increasing dietary CLA (0, 2.5, 5%). The most prevalent isomer was CLA cis 9–trans 11. Other fatty acids are modified by CLA supplementation: in particular, saturated fatty acid increased whereas monounsaturated fatty acids, particularly oleic acid, decreased (Cherian et al., 2002). This phenomenon is attributed to the inhibitory effect of CLA on the D-desaturase enzyme system in the liver, reducing the conversion of saturated fatty acids into MUFAs, and consequently promoting their accumulation in the yolk. For PUFAs, it emerged that both arachidonic acid and DHA decreased as a consequence of the CLA supplementation.
14.5 Sensory characteristics of enriched eggs The feeding of fish meal, fish oil or marine products to hens can cause problems in terms of the modification of the sensory characteristics of the egg, giving rise to fishy taints. The range of fishy odours and flavours are associated with volatile compounds and other substances originating from the oxidative deterioration processes of lipid substances. Panellists were able to distinguish between control and n-3 PUFAs enriched eggs obtained through a diet containing 3% menhaden oil if eggs were served scrambled but not if they were served hard boiled (Van Elswyck et al., 1992). The volatility of flavour compounds characteristic of fish oils was believed to be enhanced during the mixing of egg samples before scrambling as well as during exposure to high heat during scrambling. However, Meluzzi et © Woodhead Publishing Limited, 2011
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284 Improving the safety and quality of eggs and egg products al. (1997b), using 2–4% fish oil supplementation, did not detect significant differences between treated and control scrambled eggs. Moreover, when the fish oil supplementation of the diet exceeded 2% a fishy flavour was found in the yolk of boiled eggs. In a study to evaluate the effects of both animal (microencapsulated fish oil) and vegetable sources (extruded flaxseed and marine algae) of n-3 PUFAs included in the hen diet on the sensory properties of n-3 PUFA enriched eggs, it emerged that flaxseed gave better results both for taste and odour in boiled yolks (Tallarico et al., 2001). These data confirm the observations of Farrell (1994) who compared the effects of the addition of either flaxseed or fish or canola oils on sensory traits of eggs. Leeson et al. (1998), included flaxseed in the hen’s diet at levels (10, 20%) much higher than those used by Tallarico et al. (2001), found a fishy taint in the enriched hard boiled eggs when compared with the non-enriched eggs. Parpinello et al. (2006) found off-odour presence in eggs obtained from hens whose diets included fish oil supplements when compared with those from hens whose diets included algae or flaxseed. According to Tserveni-Gousi et al. (2004) the organoleptic acceptability of n-3 enriched eggs depends on the source of n-3 fatty acids and the contribution of vitamin E or natural antioxidants in the hen’s diet. Moreover the dietary enrichment of eggs with flaxseed with natural antioxidants resulted in better consumer acceptability (egg aroma and yolk colour).
14.6 Conclusion Attempts made over the last few decades to modify egg lipid fractions with the aim of enhancing their nutritional and functional values were carried out mainly to reduce the cholesterol content and increase the content of particular fatty acids. The cholesterol content of egg is relatively constant and depends mainly on the size of the egg and the breed and age of the hen. Modification of animal diet is one possible way to substantially alter the lipid composition of eggs and thereby benefit human health. In the last few decades it has been widely documented that long chain n-3 PUFAs, which have a noticeable benefit on human health, can be increased by including sources of n-3 PUFA, such as marine products, in the hen’s diet. In spite of the potential positive effect of the consumption of n-3 PUFAenriched eggs on human health, they have not been popular, probably because of the lack of evidence proving the health benefit for humans as well as the lack of promotion and nutritional information provided to the consumers regarding these products.
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14.7 References and health (2002). The role of eggs in the diet: update. Available on line at: www.acsh.org/docLib/20040405_eggs2002.pdf. amini k., ruiz-feria c.a. (2007). Evaluation of pearl millet and flaxseed effects on egg production and n-3 fatty acid content. British Poultry Science 48:661–668. banni s., martin j.c. (1998). Conjugated linoleic acid and metabolites. Pages 261–302 in Trans Fatty Acids in Human Nutrition. Oily Press, Bridgewater, UK. baucells m.d., crespo n., barroeta a.c., lopez-ferrer s., grashorn m.a. (2000). Incorporation of different polyunsaturated fatty acids into eggs. Poultry Science 79:51–59. bnf (1992). Unsaturated Fatty Acids: Nutritional and physiological significance. The report of the British Nutrition Foundation’s task force, Chapman and Hall, London. calder p . c . (2006). Polyunsaturated fatty acids and inflammation. Prostaglandins Leukotrienes Essential Fatty Acids 75:197–202. caston l., leeson s. (1990). Research Note: dietary flax and egg composition. Poultry Science 69: 1617–1620. chamruspollert m., sell j.l. (1999). Transfer of dietary conjugated linoleic acid to egg yolks of chickens. Poultry Science 78: 1138–1150. cherian g. (2005). Eggs: biology and nutrition. in: Hui Y.H. (ed.) Handbook of Food Science, Technology and Engineering Vol. IV, CRC Press, Taylor and Francis Group, Boca Raton, FL, USA. cherian g. sim j.s. (1991). Effect of feeding fulfat flax and canola seeds to laying hens on the fatty acids composition of eggs, embryos and newly hatched chicks. Poultry Science 70: 917–922. cherian g., holsonbake t.b., goeger m.p., bildfel r. (2002). Dietary conjugated linoleic acid alters yolk and tissue fatty acid composition and hepatic histopathology of laying hens. Lipids 37: 751–757. chin s.f., liu w., storkon j.m., ha y.l., pariza m.w. (1992). Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticancerogens. Journal of Food Comparative Analysis 5:185–197. cruikshank e.m. (1934). Stdies in fat metabolism in the fowl I. The composition of the egg fat and depot fat of the fowl has affected by the ingestion of large amounts of different fats. Biochemistry Journal 28:965–977. degirolamo c., kelley k.l., wilson m.d., rudel l.l. (2010). Dietary n-3 LCPUFA from fish oil but not {alpha}-linolenic acid-derived LCPUFA confers atheroprotection in mice. Journal of Lipid Research, 51:1897–1905. department of health (1994). Nutritional aspects of cardiovascular disease. Report of the Cardiovascular Review Group Committee on medical aspects of food policy. Report on health and social subjects, no. 46. Department of Health, London, pp 186. du m., ahn d.u. (2002). Effect of dietary conjugated linoleic acid on the growth rate of live birds and on the abdominal fat content and quality of broiler meat. Poultry Science 81:428–433. du m., ahn d.u., sell j.l. (1999). Effects of dietary conjugated linoleic acid on the composition of egg yolk lipids. Poultry Science 78:1639–1645. european heart network (2002). Food, Nutrition and Cardiovascular Disease Prevention in the European Region: Challenges for the New Millennium. Available at: http://www. ehnheart.org/ content/itemPublication.asp?docid=4518&level0= 1455&level1=1499 farrell d.j. (1994). The fortification of hen’s eggs with omega-3 long chain fatty acids and their effect in humans. In Sim J.S. and Nakai S. Egg Uses and Processing Technologies – New developments. CAB International, Wallingford. farrell d.j. (2000). Not all omega-3 enriched eggs are the same. In Sim J.S., Nakai S., Guenter W. Egg Nutrition and Biotechnology. CABI Publishing, Wallingford. gray j., griffin b. (2009). Eggs and dietary cholesterol – dispelling the myth. British Foundation Nutrition Bulletin 34:66–70. american council on science
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286 Improving the safety and quality of eggs and egg products grobas s., mendez j., lazaro r., de blas c., mateos g.g.
(2001). Influence of sources and percentage of fat added to diet on performance and fatty acid composition of egg yolks of two strains of laying hens. Poultry Science 80: 1171–1179. gurbuz e., balevi t., coskun b., baris c. (2010). Production of selenium and omega-3 fatty acid-enriched eggs. Proc. XIII European Poultry Conference, Tours, France. World’s Poultry Science Journal 66. Supplement. hammershoj m. (1995). Effects of dietary fish oil with natural content of carotenoids on fatty acid composition, n-3 fatty acid content, yolk colour and egg quality of hen eggs. Archiv für Geflügelkunde 59:189–197. hargis p.s. (1988). Modifying egg yolk cholesterol in the domestic fowl – a review. World’s Poultry Science Journal 44:17–26. hargis p.s., van elswyck m.e., hargis b.m. (1991). Dietary modification of yolk lipid with menhaden oil. Poultry Science 70:874–883. he k. (2009). Fish, long-chain omega-3 polyunsaturated fatty acids and prevention of cardiovascular disease – Eat fish or take fish oil supplement? Progress in Cardiovascular Diseases 52:95–114. herber s.m., van elswyck m.e. (1996). Dietary marine algae promotes efficient deposition of n-3 fatty acids for the production of enriched shell eggs. Poultry Science 75:1501– 1507. ip c., singh m., thompson h.j., scimeca j.a. (1994). Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Research 54:1212–1215. jia w., slominski b.a., guenter w., humphreys a., jones o. (2008). The effect of enzyme supplementation on egg production parameters and omega-3 fatty acid deposition in laying hens fed flaxseed and canola seed. Poultry Science 87:2005–2014. lee k.n., kritchevsky d., pariza m.w. (1994). Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19–25. leeson s., caston l., maclaurin t. (1998). Organoleptic evaluation of eggs produced by laying hens fed diets containing graded levels of flaxseed and vitamin E. Poultry Science 77:1436–1440. leskanich c.o., noble r.c. (1997). Manipulation of the n-3 polyunsaturated fatty acid composition of avian eggs and meat. World’s Poultry Science Journal 53:155–183. loh t.c., law f.l., goh y.m., foo h.l., zulkifli i. (2009). Effects of feeding fermented fish on egg cholesterol content in hens. Animal Science Journal 80:27–33. mattson f.h., grundy s.m. (1985). Comparison of effect of dietary saturated, monounsaturated and polyunsaturated fatty acids on plasma lipids and lipoproteins in man. Journal of Lipid Research 326:194–202. meluzzi a., giordani g., urrai g.f., sirri f., cristofori c. (1993). Cholesterol level and fatty acid composition of commercial eggs produced in Italy. Proc. 11th European Symposium on the Quality of Poultry Meat Tours, France pp. 427–432. meluzzi a., tallarico n., sirri f., cristofori c., giordani g. (1997a). Fortification of hen eggs with n-3 polyunsaturated fatty acids. VII Europ. Symp. on the Quality of Eggs and Egg Products, Poznan, Poland, pp. 270–277. meluzzi a., tallarico n., sirri f., franchini a. (1997b). Using dietary fish oil to enrich yolks with omega-3 polyunsaturated fatty acids. 11th Europ. Symp. on Poultry Nutrition, Faaborg, Denmark, pp. 283–285. meluzzi a., tallarico n., sirri f., franchini a., manfreda g. (2000). Effects of dietary vitamin E on the quality of table eggs enriched with omega-3 long chain fatty acids. Poultry Science 79: 539–545. meluzzi a., sirri f., tallarico n., franchini a. (2001a). Effect of different vegetable lipid sources on the fatty acid composition of egg yolk and on hen performance. Archiv für Geflügelkunde 65: 207–213. meluzzi a., sirri f., tallarico n., franchini a. (2001b). Replacing fish oil with marine algae to enrich chicken eggs with n-3 PUFA: effects on egg quality traits. Proceedings National Congress A.S.P.A, Firenze, Italy, pp. 433–435. © Woodhead Publishing Limited, 2011
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Modifying egg lipids for human health 287 meluzzi a., tallarico n., sirri f., minelli g., franchini a.
(2003). Dietary conjugated linoleic acid supplementation of laying hen: effects on egg fatty acids composition and quality. Italian Journal Animal Science Supplement 1: 456–458. niemiec j., stepinska m., swierczewska e., riedel j., kakowska r. (1999). Polyunsaturated fatty acid content in the egg yolk as influenced by rapeseed, flaxseed and evening primrose seed in the diet of layers. Proceedings of VIII European Symposium on the Quality of Eggs and Egg Products, Bologna, Italy, pp. 131–135. noble r.c. (1987). Egg lipids. In: Wells R.G. and Belyavin C.G. Egg Quality – Current problems and recent advances Poultry Science Symposium, no. 20, Butterworth, London. pp 159–177. pariza m.w., hargraves w.a. (1985). A beef derived mutagenesis modulator inhibits initiation of mouse epidermal tumors by 7, 12-dimethylbenz(a)anthracene. Carcinogenesis 6:591–593. parpinello g.p., meluzzi a., sirri f., tallarico n., versari a. (2006). Sensory evaluation of egg products and eggs laid from hens fed diets with different fatty acid composition and supplemented with antioxidants. Food Research International 39: 47–52. rizzi c., chiericato g.m. (2010). Chemical composition of meat and egg yolk of hybrids and Italian breed hens reared using an organic production system. Poultry Science 89:1239–1251. scheideler s.e., jaroni d., froning g. (1998). Strain and age effects on egg composition from hens fed diets rich in n-3 fatty acids. Poultry Science 77:192–196. simopoulos a.p. (2000). Human requirement for n-3 polyunsaturated fatty acids. Poultry Science 79:961–970. simopoulos a . p . (2008). The importance of omega-6/omega-3 fatty acid ratio in cardiovascular diseases and other chronic disease. Experimental Biology and Medicine 233:674–688. sirri f., meluzzi a., vandi l., pardo ramirez c., franchini a. (1995). Fatty acid composition of lipid fractions in eggs laid by hens fed diets supplemented with different fats – Proceedings VI European Symposium on the Quality of Egg and Egg Products, Saragoza, Spain, pp. 411–418. sirri f., meluzzi a., tallarico n., franchini a., iaffaldano n. (2001). Flaxseed and marine algae as substitutes of fish oil in fortifying chicken eggs with n-3 PUFA. Proceedings IX European Symposium on the Quality of Eggs and Egg Products, Kusadasi, Turkey, pp. 217–222. tallarico n., meluzzi a., sirri f., parpinello g., franchini a. (2001). Are the sensory and functional properties of n-3 PUFA enriched eggs affected by dietary lipid sources? Proceedings IX European Symposium on the Quality of Eggs and Egg Products, Kusadasi, Turkey, pp. 261–266. tserveni-gousi a., yannakopoulos a., botsoglou n., christaki e., florou-paneri p., yannakakis e. (2004). Sensory evaluation and oxidative stability n-3 fatty acids enriched eggs in Greece. Proceedings XXII World’s Poultry Congress, Brisbane, Australia, pp. 846. van elswyck m.e. (1997). Comparison of n-3 fatty acid sources in laying hen rations for improvement of whole egg nutritional quality: a review. British Journal of Nutrition, 78 Suppl.1:S61–S69. van elswyck m.e., sams a.r., hargis p.s. (1992). Composition, functionality and sensory evaluation of eggs from hens fed menhaden oil. Journal of Food Science 57: 342–349. watkins b.a. (1995). Biochemical and physiological aspects of polyunsaturates. Poultry and Avian Biology Reviews, 6(1):1–18. weber p.c. (1990). The modification of the arachidonic acid cascade by n-3 fatty acids. In: B. Samuelson, S.E. Dahlen, J. Fritsch and P. Hedqvist, Advances in Prostaglandin, Tromboxane and Leukotriene research, Vol 20, pp 232–240. Raven, New York. ® wertelecki t., buttin p. (2009). Influence of DHA-Gold enhances DHA Omega-3
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288 Improving the safety and quality of eggs and egg products enrichment of table eggs. Proceedings 2nd Mediterranean Summit of World’s Poultry Science Association, Antalya, Turkey, pp. 25–29. yannakopoulos a.l., tserveni-gousi a.s., yannakakis s. (1999). Effect of feeding flaxseed to laying hens on the performance and egg quality and fatty acid composition of egg yolk. Archiv für Geflügelkunde 63:260–263. yannakopoulos a.l., tserveni-gousi a.s., botsoglou n., valalis a. (2004). Bio-omega-3 agg: dietary enriched eggs with n-3 fatty acids vitamins and minerals. Proceedings of XXII World’s Poultry Congress, Brisbane, Australia, pp. 849. yin j.d., shang x.g., li d.f., wang f.l., guan y.f., wang z.y. (2008). Effects of dietary conjugated linoleic acid on the fatty acid profile and cholesterol content of egg yolks from different breeds of layers. Poultry Science 87:284–290.
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15 Egg enrichment with vitamins and trace minerals A. Schiavone, University of Torino, Italy and A. C. Barroeta, Universitat Autònoma de Barcelona, Spain
Abstract: The vitamin and trace mineral enrichment of eggs can be achieved by manipulating the diet of laying hens. Eggs enriched with certain trace minerals, vitamins and a variety of bioactive substances can be an excellent source of these substances in human diets. Several studies have confirmed that it is possible to produce novel eggs with an enhanced level of some important trace minerals (in particular Se, I, Fe and Zn) and vitamins, in particular fat-soluble ones (vitamin E, A and D), but also some water-soluble ones (especially vitamin B12 and folate). This review includes a description of the most relevant aspects of these compounds and special attention is paid to the different research results related to vitamin and trace mineral enrichment of eggs through nutrition. The vitamin and trace mineral levels reached in standard and enriched eggs are compared. Key words: egg, enrichment, vitamins, trace elements.
15.1 Introduction Eggs are a natural, low calorie food that contains a great number of essential nutrients, including vitamins, minerals and other bioactive compounds (as lutein, zeaxanthin, choline and antimicrobial molecules). Egg is widely used as whole food and food ingredient and represents an important part of human diet (Chapter 11). The nutritional value of eggs can be improved greatly through the dietary manipulation of hen diets. Eggs enriched with certain trace minerals, vitamins and variety of bioactive substances can be an excellent source of nutrients in © Woodhead Publishing Limited, 2011
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290 Improving the safety and quality of eggs and egg products the human diet. The potential health benefits of increasing the daily intake of some vitamins and trace minerals in humans are recognized, in particular to those compounds involved in specific biological functions or having a clear role in the prevention of the onset of some diseases. Thus, eggs can be considered a natural functional food. This review is divided into two sections regarding the different vitamins and trace minerals, including a description of the most relevant aspects of these compounds. Different research findings related to egg vitamin and trace mineral enrichment through nutrition are presented and special attention is paid to vitamin E, vitamin A, vitamin D, folate. In particular the vitamin and trace mineral levels reached in standard and enriched eggs are compared and the implications on recommended daily allowances (RDA) and consequently on consumer health are discussed. More information about this subject can be found in the review articles of Stadelman and Pratt (1989), Naber (1993), Hasler (2000), McDowell (2000), Barroeta et al. (2002), Surai (2002a), Sahlin and House (2006), Sirri and Barroeta (2007), Surai et al. (2007), Fisinin et al. (2008, 2009) and Weber (2009) among others. Table 15.1 shows the vitamin and trace mineral recommended daily allowances (RDA) established by European Union (European Commission Directive 90/496/EC and modifying Directive 2008/100/EC) and the RDA proportion supply of 100 g of standard egg (on the basis of Aparicio et al. (2008) data). Moreover, data relative to vitamin-enriched eggs found in the literature are indicated. In the European Commission Regulation 1924/2006 the allowed nutrition claims and the that conditions must be satisfied for their use are established. A claim that eggs are ‘source of’ or ‘high in’ a specific vitamin may be made only where a portion (100 g of egg) contains (should provide equal or more than) at least 15% or 30%, respectively, of the RDA for this vitamin fixed by European Commission.
15.2 Egg enrichment with vitamins Vitamins are organic substances which are not chemically related like other groups of nutrients (carbohydrates, proteins or lipids) and that are active at low levels, which is to say that they are required in very small amounts (mg or mg). Traditional vitamin classification is based on solubility, a property which determines its behaviour in the body. The fat-soluble group includes the vitamins A, D, E and K, while vitamins of the B complex (B1, B2, B6, B12, niacin, pantothenic acid, folate and biotin) as well as vitamin C are classified as water-soluble. Vitamin concentration in hen feed is the most important factor in determining vitamin content in egg. This is particularly true for fat-soluble vitamins. The lipid components of the egg, including fat-soluble vitamins, are present only in the yolk. During the last 10 days before hen ovulation, the lipids of © Woodhead Publishing Limited, 2011
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Egg enrichment with vitamins and trace minerals 291 Table 15.1 Recommended daily allowances (RDAs; Directive 2008/100/EC), vitamins and trace elements content and % RDA of 100 g egg and references related to vitamin and trace elements egg enrichment RDAs
100 g of standard egg Enriched egg founded in the literature Content % RDA
Vitamin A (mg) Vitamin D (mg)
800 5.0
227 1.8
28.4 36.0
Vitamin E (mg)
12.0
1.9
15.8
Vitamin K (mg)
75.0
8.9
11.9
Vitamin C (mg) Thiamin (mg) Riboflavin B2 (mg) Niacin B3 (mg) Vitamin B6 (mg) Folic acid B9 (mg) Vitamin B12 (mg)
80.0 1.1 1.4 16.0 1.4 200 2.5
0.0 0.11 0.37 3.3 0.12 51.2 2.1
0.0 10.0 26.4 20.6 8.6 25.6 84,0
Biotin B8 (mg) Pantothenic acid B5 (mg) Selenium (mg) Iodine (mg)
50.0 6.0
20.0 1.8
40.0 30.0
55.0 150
10.0 12.7
18.2 8.5
Iron (mg)
14.0
2.2
15.7
Zinc (mg) Copper (mg) Manganese (mg) Fluoride (mg) Chromium (mg) Molybdenum (mg)
10.0 1.0 2.0 3.5 40.0 50.0
2.0 0.014 0.071 0.11 2.5
20.0 1.4 3.5 3.1 6.3
1.5 times higher (Table 15.2) seven-fold increase in egg yolk Cholecalciferol content, 1.5-fold for 25OHD content (Table 15.3) Increases higher than five times (Table 15.4) Fifteen times. Suzuki and Okamoto (1997) No No No No No Two–three times (Table 15.5) Eleven times. Squires and Naber (1992), Leeson and Caston (2003) No Two times, Leeson and Caston (2003) Four–five times (Table 15.6) Three–four times, Yalçin et al. (2004) 1.1–1.2 times, Park et al. (2004, 2005) 1.4 times, Stahl et al. (1988) No No No No No
dietary origin are absorbed and deposited in the yolk in the last phase of its formation. As the fat-soluble vitamin concentration of the laying hen feed increases, so does the vitamin content of the egg yolk. Several researchers have confirmed that it is possible to produce novel eggs with an enhanced level of some important vitamins, in particular fat-soluble (vitamin E, A and D), but also water-soluble (especially vitamin B12 and folate). 15.2.1 Vitamin A enrichment Vitamin A is the collective term for compounds that show the biological properties of retinol, including maintenance of epithelial tissue and visual
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292 Improving the safety and quality of eggs and egg products function. This classification includes retinol, retinyl esters and retinal. Vitamin A compounds are not found as such in plant tissues, but rather are characteristic of the animal tissues. Plants contain some carotenoids which are vitamin A precursors (a- and b-carotenes, cryptoxanthin, citranaxanthin, b-apo 8 carotenal, zeaxanthin), of which b-carotene has the greatest biological potency. In the intestinal epithelium of the birds, some carotenoids are converted into vitamin A at different magnitudes, depending on the carotenoid and its dietary supply. One IU of vitamin A activity is equivalent to the activity of 0.3 mg of retinol, 0.334 mg vitamin A acetate or 0.6 mg of b-carotene; in other words 1 mg of b-carotene is equivalent to 1667 IU of vitamin A. The minimum requirement recommended by the National Research Council (1994) is 3000 IU/kg and is based on studies from 1961 and 1965. In the light of new studies, supplementation at a higher level (8000–10,000 IU vitamin A/kg feed) would result in benefits in production as well as improvements in the immune response of the birds in stress situations. Moreover, the ingestion of high quantities of vitamin A allows us to increase the vitamin A content of the egg, which is a positive trait for the quality of the eggs produced. An excess can lead to pigmentation problems and deficiencies in the other fatsoluble vitamins. For this reason, and because of its capacity to be stored, the maximum tolerance level is 20,000 IU/kg. Vitamin A is involved in several processes such as vision, reproduction, bone development, immune response, cell differentiation and proliferation. In fact, vitamin A plays an important role in the maintenance of integrity of epithelial tissue, particularly in the oviduct. Some studies indicate that variations in vitamin A dosage are reflected in parameters relating to the yolk and ovulation and underline its importance in embryo development (Surai, 2002a). Vitamin A is deposited in egg yolk and comes from the liver storage. The liver plays an important role in the metabolism and egg deposition of vitamin A. Squires and Naber (1993) note that in layers that ingest appropriate quantities of vitamin A, liver deposition is higher when they are forming eggs than during an unproductive phase. Also, different authors have demonstrated that vitamin A levels in the liver, and consequently in the egg yolk, are closely related to the level and administration time of vitamin A in hen feeding (Richter, 1995; Richter et al., 1996a, 1996b). For instance, Surai et al. (1998) point out that exclusion of added vitamin A from the hens’ diet for 3 months decreased its concentration in liver by 45%. In contrast, dramatic increases in the concentration of vitamin A in the hen’s liver and egg yolk were produced by increasing amounts of dietary vitamin A. Some studies have concentrated on increasing the levels of vitamin A in the egg (Table 15.2). In general, the results indicate that as the dietary dose of vitamin A increases, the levels in the egg and the liver also increase; however, the magnitude of increase is very variable from an author to the other one. Naber (1993) classified the vitamins in relation to the potential
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Egg enrichment with vitamins and trace minerals 293 Table 15.2 Effect of dietary retinyl acetate on retinol content of fresh egg yolk (adapted from Sirri and Barroeta, 2007) Authors
WFS
Retinyl acetate IU/kg diet
Squires and Naber (1993)
27–32
29 (BD) +4000 +8000 +16 000
Surai et al. (1998)
12
0 10 000 100 000 400 000
Mendoça et al. (2002)
15
9050 IU retinyl acetate (BD) +5000 +10 000 +15 000 +20 000 +25 000
Mori et al. (2003)
11
0 15 000 30 000
Retinol content IU/g yolk 2.4 10.3 16.9 24.0 13.4 23.5 68.4 283.7 23.9 25.9 29.8 30.2 34.7 35.9 25.7 35.0 38.3
WFS: weeks of feed supplementation.
transfer efficiency from hen diet to the egg and grouped the vitamins in four categories (low, medium, high, very high) with efficiency ranging from 5 to 80%. The vitamin A was classified as ‘very high’, with a transfer efficiency varying from 60 to 80%. Squires and Naber (1993), raising the retinyl acetate content of the diet from 4000 to 16,000 IU/kg, observed an enrichment in the egg vitamin A concentration from 10.3 to 24.0 IU/g respectively. Similar results were found by Mendonça et al. (2002) who showed a significant positive correlation between dietary retinyl acetate and egg retinol, indicating that egg retinol increased linearly as dietary vitamin A increased (increase of 50% when supplying 25,000 IU/kg). The regression equation was as follows: y = 0.0005x + 23.851(R2 = 0.8708; r = 0.9332), where y = egg retinol (IU/g), and x = dietary retinyl acetate (IU/kg). A higher supplementation level has been assayed (Surai et al., 1998) by feeding large amount of retinol/g feed (120 mg corresponding to 400,000 IU vitamin A/kg feed), resulting in a large increase in yolk content of 85 mg vitamin A/g (283 IU of vitamin A/g yolk) relative to that of 4 mg (13 IU) in the non-supplemented group. The yolk retinol content can therefore increase by 50% when less than 30,000 IU are supplemented in the diet but the increase can reach more than 20-fold when the dietary supply is very high (400,000, corresponding to 20-fold the authorized value).
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294 Improving the safety and quality of eggs and egg products Jiang et al. (1994) reported a linear increase in the b-carotene as well as slightly but significant increase in retinol in egg yolk, in response to increased dietary b-carotene. The authors concluded that some b-carotene was converted into retinol and deposited into the egg yolk. However, it is important to point out that there is a negative interaction, on the level of absorption, between vitamin A and the other fat-soluble vitamins, as well as possible interactions with other nutrients. This is dealt with different studies which demonstrate a clear interaction between vitamin E and vitamin A in the deposition of both (Jiang et al., 1994; Surai et al., 1998; Grobas et al., 2002; Mendoça et al., 2002; Mori et al., 2003). The yolk concentration of tocopherols was markedly reduced as supplemental dietary levels of either vitamin A or beta-carotene increased. The recommended daily allowance for vitamin A is 800 mg retinol equivalents. Then, ‘high in’ and ‘source of’ vitamin A nutritional label claims in eggs are possible when we achieved 240 mg retinol equivalents/100 g of egg (800 IU vitamin A/100 g egg) or 120 mg retinol equivalents/100 g of egg (400 IU vitamin A/100 g of egg), respectively. If we consider that regular eggs content about 756 IU vitamin A/100 g of egg, corresponding to 227 mg retinol equivalents, that meets 28.4% of the RDA and the ‘source of’ vitamin A nutritional claim is possible. Considering the yolk proportion average 30% of the total egg, this data correspond to 25.2 IU vitamin A/g of yolk. The data below point out that it is possible to achieve the required content of vitamin A that allows a ‘high in’ vitamin A nutritional label claim (up to 26.7 IU vitamin A/g of yolk) through dietary strategies. 15.2.2 Vitamin D enrichment Vitamin D is a generic term that refers to cholecalciferol (vitamin D 3) and ergocalciferol (vitamin D2), compounds which controls, principally, calcium and phosphorus metabolism, bone mineralization and egg formation. Vitamin D3 is the form which is most active in birds. There are two sources that supply vitamin D: the action of sunlight on the skin and the diet. Birds can synthesize cholecalciferol or vitamin D3 from the 7-dehydrocholesterol present in their skin when they receive ultraviolet light through exposure to the sun. In hen, which are generally housed, endogenous synthesis is insufficient to meet requirements and they must have a supplementary dietary intake. Cholecalciferol is stored in an inactive form, mainly in the liver and adipose tissue. This form is rapidly hydroxylated to 25-hydroxycholecalciferol (25OHD) in the liver. However, this metabolite needed an additional hydroxylation to issue to the active form of vitamin D and this last step if precisely regulated depending on the balance of the calcium metabolism in birds. This metabolite (25OHD) is carried to the kidneys where it is converted to 1,25-dihydroxycholecalciferol or calcitriol, which is the active form of vitamin D in animals. These successive hydroxylation reactions that convert
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Egg enrichment with vitamins and trace minerals 295 cholecalciferol to 1,25-hydroxycholecalciferol appear to be less efficient in older hens (Frost et al., 1990; Soares et al., 1988). It has also been shown that 25OHD has from one to five times higher activity than standard vitamin D, depending on the criteria and authors (Ovesen et al., 2003). An IU of vitamin D3 is defined as the activity of 0.025 mg vitamin D3 (cholecalciferol). The minimum requirements for layers established by the National Research Council (1994) are 300 IU/kg of vitamin D3. However, as Whitehead (1995) and Weber (2009) point out, vitamin D3 requirements of layers have not been re-evaluated in recent decades. Actual practical recommendations or allowances are in a range of 2000–3000 IU/kg of vitamin D3. In current practice vitamin D3 metabolites are starting to be used and great efforts are being made to observe their usefulness in the nutrition of layers. Regarding enrichment of the eggs with vitamin D, Mattila et al. (1999) found a high positive correlation between the cholecalciferol content of the feed and the cholecalciferol (r = 0.995) and 25OH (r = 0.941) content of the egg yolk. When the feed cholecalciferol content was raised from the regular level (62.4 mg; 2496 IU/kg feed) to a level of approximately 3.5 times higher (221.6 mg; 8649 IU/kg feed), the cholecalciferol content of egg yolk increased approximately 7-fold and the 25OHD content approximately 1.5-fold (Table 15.3). Furthermore, according to their results, the efficiency of vitamin D3 deposition in the yolk is around 2.2 times higher than D2 (Mattila et al., 2004). These authors also worked on the vitamin D deposition Table 15.3 Effect of dietary cholecalciferol dietary supplementation on cholecalciferol and 25OHD content of fresh egg yolk (adapted from Sirri and Barroeta, 2007) Authors Mattila et al. (1999)
WFS
Cholecalciferol IU (mg)/kg diet
4–6
1064 (26.6) 2496 (62.4) 8640 (216.0)
Cholecalciferol 25OHD mg/100 g yolk mg/100 g yolk 1.2–1.5 3.4–3.5 21–23
0.5 0.8–1.0 1.4–1.5
Mattila et al. (2003)
0 2 4 16 24
4280 (107) 12 000 (300) 12 000 (300) 12 000 (300) 12 000 (300)
4.2 30.2 31.1 26.6 21.7
– – – – –
Mattila et al. (2003)
0 1 2 3
1720 (43) 11 200 (280) 11 200 (280) 11 200 (280)
4.2 30.2 31.1 26.6
1.1 1.9 – 1.7
Mattila et al. (2004)
4–44
2500 (62.5) 6000 (150) 15 000 (375)
2.5–5.0 9.1–13.6 25.3–33.7
– – –
WFS: weeks of feed supplementation.
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296 Improving the safety and quality of eggs and egg products pattern in eggs (Mattila et al., 2003) and observed that 8–13 days of high dietary cholecalciferol supplementation (11,200 and 12,000 IU/kg feed) is enough to reach the top cholecalciferol content in egg (30 mg/100 g egg yolk). After 112 days feeding, the cholecalciferol content gradually decreased to approximately 22 mg/100 g yolk. However vitamin D can be toxic in high doses (the maximum safe dietary level of cholecalciferol is 1000 mg/ kg (40,000 IU)), it has been reported that 12,000 and 15,000 IU/kg feed of vitamin D were not toxic for hens (and no calcification of soft tissue as kidney, liver, heart, muscle and lung) and did not affect the sensory or functional properties, fatty acid composition, or the egg quality parameters such as egg shell strength (Mattila et al., 2003, 2004). Eggs are one of the limited foods contributing human dietary vitamin D and also contain substantial amounts of 25OHD (25% of the total vitamin D activity). 25OHD is absorbed better and faster from the diet than native vitamin D and has metabolic effects of its own in regulating cell growth and calcium metabolism (Ovesen et al., 2003). In some situations, dietary fortification could be necessary in people with restricted exposure to ultraviolet light. The RDA of vitamin D is from 5 mg/day in adults to 10 in elderly people (Scientific Committee on Food, 2003). There is evidence that vitamin D goes beyond mineral homeostasis and its consumption is associated with a reduced risk of other pathologies associated with age (some cancers, cardiovascular disease, blood pressure, arthritis and sclerosis). A regular egg contains around 1.8 mg/100 g of vitamin D that represents 36% of the RDA; thus a nutrition claim of egg ‘high in’ vitamin D is possible. Eggs enriched in vitamin D can potentially provide the total of the suggested requirement, and substantial quantities of 25OHD with greater biological activity than that of cholecalciferol. However, as Seuss-Baum (2007 and Chapter 11) notes, the problem with fortification of vitamin D is the relatively low span between the RDA and the upper intake level. 15.2.3 Vitamin E enrichment The term vitamin E includes a range of closely related active components. These components are divided into two groups: tocopherols and tocotrienols. Of the tocopherols the alpha form exhibits greatest biological activity and the broadest distribution, being very commonly found in the ingredients used in animal feeds. The natural isomer of alpha-tocopherol is RRR-alphatocopherol or d-alpha-tocopherol. This is the isomer with greatest biological activity, and is usually the reference compound when vitamin E is referred to. It is important to remember that most plants can synthesize vitamin E, although animals and human beings do not have this capacity. This means that meeting vitamin E requirements relies exclusively on food intake. The synthetic form of alpha-tocopherol (alpha-toc) is a racemic mix of eight stereoisomers, esterified to an acetate group. This form is designated dlalpha-tocopherol acetate (dl-alpha-TA) and is accepted as the standard for
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Egg enrichment with vitamins and trace minerals 297 establishing vitamin E activity. Thus, 1 mg of dl-alpha-TA is equivalent to 1 international unit (IU) of vitamin E, while 1 mg of the natural form (d-alphatoc) is 1.49 IU. Until alpha-TA is hydrolysed on an intestinal level it is not biologically active in the body (McDowell, 2000). The minimum requirements of vitamin E established for laying hens by the National Research Council (1994) are 10 mg/kg of feed. It is widely recognized that vitamin E plays a fundamental role in the normal functioning of the organism, because of its great antioxidant power, against the degenerative action of free radicals and cellular oxidation prevention. Moreover, there is increasing evidence that the inclusion of vitamin E in the human diet reduces the incidence rate of different high risk pathologies. Once the basic needs of the animal have been met, the possibility of using higher levels of vitamin E supplementation can be considered with a view to improve other aspects, in particular those related to the quality of the food reaching the consumer. Vitamin E can act on two levels: preventing food oxidation and as a source of fortification of the vitamin for the consumer. Different authors have investigated the possibility of increasing the vitamin E content of the egg (Frigg et al., 1992; Jiang et al., 1994; Surai et al., 1995; Chen et al., 1998; Meluzzi et al., 1999; Grobas et al. 2002; Mori et al., 2003; Qi and Sim 1998; Pita et al., 2004; among others). The alpha-toc content of the egg maintains a linear relationship with its level of supplementation in layer feed. It has been observed that alpha-toc content in fresh egg increased with dietary alpha-TA supplementation up to doses of 20 g alpha-toc/kg feed (Frigg et al., 1992; Surai et al., 1995; Grobas et al., 2002; Flachowsky et al., 2002). The alpha-toc transfer efficiency decreases with increasing levels of alpha-toc in the diet. Galobart et al. (2001a) showed that the alpha-toc transfer efficiency from feed to egg ranged from 41.8%, when 50 mg/kg alpha-TA was added to the diet, to 26% with 200 mg/kg alpha-TA supplementation. In this case, for every 100 mg/kg of alpha-toc increase in the diet the transfer efficiency was reduced by 8.4%. Similar observations have been described by other authors, calculating vitamin E transfer efficiency between 16 and 39% (Naber, 1993; Grobas et al., 2002). Another important aspect is the egg vitamin E deposition pattern. The most important studies in this field are those of Surai et al. (1995), Meluzzi et al. (1999, 2000) and Galobart et al. (2002). They studied the evolution of the egg alpha-toc content over an extended period of time. In general, it seems that the time needed to achieve the maximum level of alpha-toc in eggs is about 2 to 3 weeks, depending on the dietary supplementation. After reaching this maximum level, alpha-toc concentration remained constant for 2 weeks and then declined, in some cases, by approximately 50% over the following 4 weeks. Alpha-toc in egg then remained constant for as long as the dietary supplementation was maintained. It is important to supplement laying hens with vitamin E continuously. With each egg, a hen releases two times more vitamin E than what is stored in the liver, showing that the liver is not a good reservoir for the vitamin (Surai et al., 1998). Moreover, once
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298 Improving the safety and quality of eggs and egg products the dietary vitamin E is reduced, its content in egg is adjusted more quickly than when increasing dietary level is supplemented. In Table 15.4 values of alpha-toc concentration in egg relative to dietary alpha-toc doses obtained in different studies are reported. By comparing the same dietary inclusions of the vitamin, huge differences in the egg vitamin concentration emerge. For instance, by using 200 mg/kg of alphaTA supplementation, the alpha-toc concentration in egg ranges between 119 (Qi and Sim, 1998) and 1200 g egg yolk (Surai et al., 1995). This variability seems related to several factors, the most important being the laboratory methods and the experimental conditions adopted by the different authors but also to the hen diets. Indeed, differences in the alpha-toc content of the basal diets, the presence of antioxidants or particular feed ingredients, the interaction with nutrients during absorption (i.e. level of vitamin A – Grobas et al., 2002) and the type and amount of fat could be responsible of the differences observed in the egg vitamin E concentrations given in Table 15.4. Regarding the type of fat, several authors have found that alpha-toc deposition in eggs was lower when higher levels of unsaturated fats were administered (Frigg et al., 1992; Meluzzi et al., 2000; Galobart et al., 2001a). A higher polyunsaturated fatty acid (PUFA) content produces a higher oxidative susceptibility in the animal body, which contributes to a greater utilization of alpha-toc, leading to a reduction in the alpha-toc available for the egg yolk formation. Therefore, the vitamin E requirements increase as the unsaturation level of the diet grows and, consequently, the efficiency of alpha-toc deposition in eggs diminishes when dietary unsaturation level increases. As mentioned before in Section 15.2.1, the interrelation between the two vitamins A and E is very important, as they are involved in its absorption and subsequent deposition in the egg. Another important factor is the reduction in egg alpha-toc concentration during processing and storage. Some works have demonstrated that vitamin E content remained almost constant when shell eggs were stored (Cherian et al., 1996; Gebert et al., 1998; Meluzzi et al., 1999). In contrast, the vitamin E content of the egg quickly declined with hard-boiled, scrambled and spray-dry processing (Wahle et al., 1993; Galobart et al., 2001b; Cortinas et al., 2003; Murcia et al., 1999) or with high temperature during storage (Franchini et al., 2002). The RDA of vitamin E for humans is about 12 mg (Directive 2008/100/ EC), but as the dietary PUFA content grows, the vitamin E requirements increase by 0.4-0.6 mg/g PUFA (Dutta-Roy et al., 1994; Muggli, 1994). An egg presents an average of 1.9 mg/100 g of vitamin E that represents 16% of the RDA. On the other hand, an egg enriched with vitamin E can provide 60–80% of the RDA of this vitamin when 6–8 mg vitamin E/100 g are achieved
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Egg enrichment with vitamins and trace minerals 299 Table 15.4 Effect of dietary dl-alpha tocopheryl acetate supplementation on alphatocopherol content of fresh egg yolk (adapted from Sirri and Barroeta, 2007) Authors
WFS
Jiang et al. (1994)
3
Surai et al. (1995)
3
Chen et al. (1998)
7
Gebert et al. (1998)
4
Hossain et al. (1998)
30
Qi and Sim (1998)
1–4
Meluzzi et al. (2000)
4
Galobart et al. (2001a)
4
Grobas et al. (2002)
6
Flachowsky et al. (2002) 10
44
a-tocopherol diet supplementation (mg/kg diet)
a-tocopherol (mg/g of yolk)
27.5 (BD) +50 +100 +200 +400 14–18 (BD)+200 +2000 +20 000 11(BD) +15 +30 +60 +120 BD +100 +200 25 50 75 100 67 (BD) +200 +400 +800 33.2–43.88 (BD) +50 +100 +200 0 50 100 200 0 40 160 640 15(BD) +100 +1000 +10 000 +20 000 19(BD) +100 +1000 +10 000 +20 000
135.0 164.0 235.0 245.0 390.0 12001 70001 15 0001 25.0 35.0 45.0 50.0 75.0 120.0 378.0 606.0 99.0 169.0 207.0 203.0 83.6 119.0 175.4 234.9 90.9 136.1 227.5 313.8 50.0 149.0 237.0 397.0 28.1 109.2 353.9 967.5 7.8 25.1 139.6 250.2 313.3 10.4 34.3 131.6 250.6 236.9
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300 Improving the safety and quality of eggs and egg products Table 15.4 Continued Authors Franchini et al. (2002) Pita et al. (2004) Hayat et al. (2009)
Mohiti-Asli et al. (2008)
WFS 5 11 8.2
a-tocopherol diet supplementation (mg/kg diet) <10(BD) +100 +200 36–38(BD) +100 +200 12.65(BD) BD+10% flaxseed: FD FD+50 FD+100 FD+150 22.3(BD) +200
a-tocopherol (mg/g of yolk) 47.2 120.6 219.1 34.0 108.1 177.4 21.4 33.4 150.0 251.0 389.3 87.9 485.4
1
Mean estimated values. WFS weeks of feed supplementation; BD, basal diet.
15.2.4 Vitamin K enrichment Vitamin K includes a range of components called quinones. Vitamin K1 or phylloquinone is found in green plants and seeds, while K2 or menaquinone is the result of bacterial synthesis in the large intestine. Vitamin K3 or menadione is a compound of fat-soluble synthesis and with greater biological activity than the others; it is considered the standard of reference for vitamin K activity. The main function of vitamin K is to regulate the formation of various factors involved in blood clotting. Recently it has been suggested that vitamin K is also involved in the mineralization process during skeletal development and eggshell formation. Requirements of vitamin K for layers have not been sufficiently established, with the National Research Council giving a minimum requirement of 0.5 mg/ kg in 1994, based on a study from 1964. However, the Agricultural Research Council (ARC 1975) and other recognized nutritionists and institutions suggest a minimum of 1 mg/kg and recommend levels of 2–3 mg/kg. An average of 9 mg vitamin K is present in 100 g of egg. This quantity provides approximately 12% of the RDA (75 mg) vitamin K consumer need. Very few studies regarding the possibility of fortifying eggs with vitamin K have been carried out. The experiment by Suzuki and Okamoto (1997) demonstrates that vitamin K1 or phylloquinone supplementation between 10 and 100 mg/kg produces eggs with a content of 104–1908 mg vitamin K1 and 67–192 mg menaquinone K2 per 100 g yolk. Supplementation with vitamin K3 or menadione (10–1000 mg/kg) resulted in eggs fortified with menaquinone K2 at a level of 115–240 mg per 100 g yolk, and with menadione K3 at a level of only 1 mg per 100 g yolk. On the other hand, © Woodhead Publishing Limited, 2011
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Egg enrichment with vitamins and trace minerals 301 no differences were observed in egg production related to the dosage of vitamin K.
15.3 Water-soluble vitamin enrichment Egg contains all the vitamins with the exception of vitamin C. The B watersoluble vitamins are found in both, yolk and albumen. Biotin, pantothenic acid, thiamin B1, folate, B6, and B12 are concentrated in the yolk; 50% rivoflavin B2 is deposed in the albumen. The B vitamins have very important functions in metabolism, as most of them represent coenzymes, involved in protein, carbohydrate and fatty acid metabolic processes. A standard egg contributes up to 30% RDA of vitamin B12 (84%) and biotin (40%), between 15 and 30% of riboflavin B2 (26.4%), niacin (20.6%), folate (25.6%) and pantothenic acid (30%). It provides lower amounts of the other water-soluble vitamins. The literature about water-soluble vitamin enrichment in egg is scarce. We found some results related with vitamin B12 and folate egg content. Folate and vitamin B12 have attracted interest because of their functions as cofactors in the metabolism of homocysteine. Elevated concentrations of homocysteine in blood appear to be associated with increased risk of cardiovascular disease. Increased dietary intakes of the two vitamins may help prevent the accumulation of homocysteine in blood and tissues (Austic et al., 2008). 15.3.1 Folate enrichment Folacine and folate (the naturally occurring form) share compounds with pteroylglutamate, but differ with respect to oxidation state and number of attached glutamate residues. Both are forms of the water-soluble vitamin B9. Often the terms folic acid and folate are used interchangeably; however, technically they have different meanings. Folate refers to the forms found in food or in the body; whereas folic acid is the form found in supplements and fortified food. Principal food sources of folate include leafy green vegetables and offal, such as liver or kidneys. Much of the folate in foodstuffs is conjugated with varying numbers of glutamic acid molecules and then the absorption efficiency of folates could be compromised. Moreover it assumes a higher bioavailability of natural folate from food sources in relation to supplemented folic acid. On a practical level, dietary recommendations for layers are at a somewhat higher level (0.4–1.0 mg folic acid/kg feed) than the minimum established requirements (National Research Council, 1994: 0.25 mg/kg; ARC, 1975: 0.3 mg/kg). Folate metabolism is entirely related to the metabolism of many other methyl group donors (S-adenosylmethionine, serine, vitamin B12 and choline). © Woodhead Publishing Limited, 2011
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302 Improving the safety and quality of eggs and egg products These nutrients interact in such a way that their respective requirements may be changed. The achievement of optimal folate status in the population is an important concern because of the established role of folate in the prevention of neural tube defects and its potential role in the prevention of cardiovascular diseases, cognitive impairment among others. Recommended folic acid intake is especially important and high in pregnant women because its level shows a positive relationship with intrauterine growth and development. The egg provide 50 mg/100 g of folacin, that represent 25% of the RDA (200 mg), so it could be considered as a ‘source of’ folate for human consumption. Some 95% of egg folate is found in the yolk and more than 80% of the folate in eggs is 5-methyltetrahydrofolate (5-MTF), a form of folate that is rapidly absorbed. Hence, increasing folate in eggs through dietary supplementation may be a good strategy to increase the folate consumption of different population groups such as pregnant women and senior adults, promoting better health. Moreover, current concerns of some authorities exist regarding over-exposure to high dose of synthetic folic acid in humans but it considers ‘no health risk’ for natural sources of folate, such as egg. In relation to folate egg enrichment, it has been demonstrated that laying hens have the capacity to convert high doses of folic acid added to the feed into natural folates in their eggs (Table 15.5). Sherwood et al. (1993) Table 15.5 Effect of dietary folic acid on egg folate content Authors
WFS
Folic acid diet supplementation, mg/kg feed
Hebert et al. (2005) 2–3
BD +2 +4 +8 + 16 + 32 + 64 + 128
Hoey et al. (2009)
12
1(BD) +2 +4 +8 + 16 + 32
Bunchasak and Kachana (2009)
8
0.31 folate/ kg(BD) + 0.5 +4 + 10
Egg folate content mg/g yolk mg/egg 16.81 42.2 31.0 49.5 47.1 42.2 46.7 42.8
mg/100 g egg 29.42 73.12 54.02 83.52 76.82 72.42 81.12 68.82 60.0 96.8 90.7 122.2 144.6 131.9
3.42 4.85 5.12 5.44
WFS: weeks of feed supplementation. 1 Hy-Line W98. 2 Calculated from original data.
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Egg enrichment with vitamins and trace minerals 303 observed that by increasing the supplementation of synthetic crystalline folic acid, folate deposition in yolk and plasma reached saturation point. With 0.72 mg/kg folates in the diet the eggs produced contained somewhat less than half the maximum possible folate content in the yolk. Also, studies by House and co-workers (House et al., 2002; Hebert et al., 2005) focused on the enrichment of folate in the egg. They used levels between 0 and 128 mg of folic acid per kg of feed and showed how the content in the egg responds to increase dietary levels up to egg deposit saturation. First, a linear increase in egg folate content was described when crystalline folic acid was added from 0 to 2 mg/kg to hen diet. Then, egg folate levels reached a plateau at a concentration of approximately 30–50 mg/egg. The authors point out that higher levels of egg folate are not achieved due to the saturation of the precursor pool for egg folate deposition and so the efficiency of deposition declined significantly as supplementation rates increased. Another interesting finding of these authors is that egg yolk folate bioavailability is 100% if compared folic acid and that this folate content in enriched eggs is stable during storage at 4 °C for 4 weeks (House et al., 2002). Recent studies also describe a similar egg folate deposition model with significant increases on addition of a small dose of folic acid, which reaches a plateau (Bunchasak and Kachana, 2009; Hoey et al., 2009). In the Bunchasak and Kachana (2009) study, yolk folate content in the hens fed 10.31 mg folate/kg diet was higher by about 40% than that in the hens fed a basal diet (0.31 mg/kg diet). Also, no significant physiological interaction between folate and vitamin B12 was demonstrated. Nevertheless, in the Hoey et al. (2009) experiment, the optimal dose required to achieve a maximal egg total folate content was found to be 16 mg/kg, four-fold higher than previously reported. At week 0 when all hens received the basal feed (1 mg folate/kg feed), the mean egg folate content ranged from 53 to 66 mg/100 g egg. Across several levels of dietary crystalline folic acid in hen diets (from 0 to 32 mg/kg feed), the maximum folate content was 144.6 mg/100 g egg from hens fed 16 mg of folic acid per kg of feed. This is about 1.5 times higher than the level reported by Hebert et al. (2005). An important factor between the two studies is the use of different analytical procedures for the determination of egg folate content. Hoey et al. (2009) measured total folate in the whole egg whereas the methodology of previous studies was based on the measurement in egg yolk of the 5-MTF content only. Importantly, it was shown that the enhanced folate content of enriched eggs is predominantly (over 90%) natural folates, and that folic acid represents no more than 10% of the total folate content. Then, folate-enriched eggs could offer consumers a practical means of increasing folate intake (and potentially protecting against disease) without the safety concerns relating to folic acid fortified foods. We can conclude that it is possible to enhance the folate content of eggs by 2–3 fold (50 mg vs. 135 mg/100 g), which represents 50–75% of the RDA, by feeding hens a folic acid supplemented diet. Eggs are an important source
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304 Improving the safety and quality of eggs and egg products of dietary natural folate and can contribute to an improvement in consumer folate status. 15.3.2 Enrichment with other water-soluble vitamins Several years ago, Naber (1979) suggested that riboflavin B2 and vitamin B12 could be markedly influenced by dietary changes. Feeding breeding hens with graded amounts of dietary vitamin B12 (0.5, 4, 8 and 16 mg/kg) showed that after 12 weeks, egg concentrations of vitamin B12 stabilized and were proportional to the amount of vitamin added to the diet (0.4, 1.3, 2.6 and 4.8 mg/100 g), and decreased markedly within 2 weeks in hens fed the two lowest dietary levels (Squires and Naber, 1992). In contrast, Bunchasak and Kachana (2009) reported that supplementation with vitamin B12 did not significantly affect serum or egg yolk vitamin B12 levels. Some promising results have been highlighted by Leeson and Caston (2003) for the potential transfer efficiency of some vitamins from hen diet to the egg. The authors studied the effect of the supplementation of hen feeding with a vitamin premix containing supranutritional levels of the following vitamins: A (300% of the regular level), D3 (143%), E (670%), K (166%), B1 (500%), B2 (420%), B6 (200%), biotin (500%), folic acid (200%), niacin (1000%), pantothenic acid (100%) and B12 (1000%). Among the water-soluble vitamins, an interesting response was observed for vitamin B12, the level of which in the egg increased nearly four times by adding 11 times the vitamin B12 in the diet, and for panthotenic acid, which doubled its level in the egg in response to a doubling in the hen diet. Some modifications related to vitamins B2 (riboflavine), thiamine (B1), biotin (B8) and pantothenic (B5) in the egg through dietary manipulation have been described by Nys (2010). In all these vitamins, as the dietary level increased the egg tranference rate decreased. On the other hand, there is no evidence that pyridoxine (B6) and niacine (B3) egg content are modified by dietary supplementation.
15.4 Egg enrichment in trace minerals The relationship between diet and human health has received a great deal of attention in the last few years, due to the realization that unbalanced diets can cause serious health-related problems. The enrichment of hen eggs with additional trace elements could provide new niche markets and improve the nutritional status of particular cohorts of people at risk to trace minerals deficiency. In general, most trace minerals are deposited in the yolk, whereas their concentrations in the white portion of the egg is limited. Trace element enriched eggs may be achieved by supplementing the laying hens’ diets with trace elements in both inorganic and/or organic forms. © Woodhead Publishing Limited, 2011
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Egg enrichment with vitamins and trace minerals 305 Some reports have indicated that organic mineral sources, such as amino acid complexes or proteinates, have higher bioavailability than traditional inorganic forms and this justifies the increasing interest in the use of organic forms as sources of microelements for poultry. The higher bioavailability of organic minerals is probably related to the different absorption mechanism (peptide or amino acid uptake mechanism in the intestine) and to the better protection from binding to dietary constituents such as phytates, which forms indigestible complexes. Most of the studies that deal with trace element supplementation in laying hens’ diets have been aimed at studying the effect of these diets on birds’ performance and the functional properties of the eggs. Few studies have investigated the effect of microelement fortified diets on the egg content of these trace minerals. The trace minerals that have mainly been studied to obtain enriched-eggs are selenium, iodine, iron and zinc. 15.4.1 Selenium enrichment The average hen’s egg contains about 5 mg Se/egg, with a wide range of variation, depending on the selenium content in the feed, which is related to its availability from the soil. Selenium is necessary for growth and fertility in animals and for the prevention of a variety of diseases, which show a variable response to vitamin E. This complementary nature of selenium and vitamin E is the basis for their mutual sparing effect on each other (Combs and Combs, 1986); however, there are limits to the mutual substitution of selenium and vitamin E (McDonald et al., 2002). Selenium is an integral part of various selenoproteins, with glutathione peroxidase (GSH-Px) being the first selenoprotein to be identified (Rotruck et al., 1973). The participation of selenium in antioxidant defence and in the regulation of the redox status of the cell could explain its importance in various physiological functions, especially in reproduction. The other major role of selenium is in the production of thyroid hormones, for which it is a component of the enzyme type I iodothyronine deiodase, which converts thyroxine (T4) to the physiologically active triiodothyronine (T3) (Klasing, 2000; Underwood and Suttle, 2001). The biological effects of selenium in poultry have been extensively reviewed by Surai (2002a, 2002b). The dietary selenium requirement and selenium toxicity of laying hens have been recommended as 0.05–0.08 and 10 mg Se/kg diet, respectively (National Research Council, 1994). Selenium is the most toxic of the trace minerals and has the lowest margin between deficient and excess dietary levels (Klasing, 2000). The four common valence states of selenium are selenide (–2), elemental selenium (0), selenite (+4) and selenate (+6). The bioavailability, and thus the requirements and toxic levels, are highly dependent upon the dietary form. Selenite, selenate and organic selenides are absorbed with high efficiency in the small intestine, particularly in the duodenum. There is little homeostatic regulation of selenium absorption in chickens and uptake is high, even when the tissue levels are
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306 Improving the safety and quality of eggs and egg products repleted (Klasing, 2000). Most of the selenium in natural feeds is present as selenomethionine. Although well absorbed and retained, selenomethionine is slow in being converted into selenocysteine, which is needed for the synthesis of the functional proteins (Underwood and Suttle, 2001). Supplemental selenium is commonly added to diets as sodium selenite (Na2SeO3), sodium selenate (Na2SeO4), selenomethionine (Klasing, 2000) and selenium-enriched yeast (McDonald et al., 2002). A greater availability of organic over inorganic sources of selenium has been claimed (Hassan, 1990; Underwood and Suttle, 2001). The concentration and the form of selenium in the diet affect the selenium content in the egg and the selenium distribution in the egg white and yolk (Sirichakwal et al., 1984; Davis and Fear, 1996). Selenomethionine and selenized yeast are mainly deposited in egg white, while inorganic selenium and non-selenomethionine compounds are mainly deposited in the yolk. Selenoamino acids have different metabolism in animals. The selenomethionine metabolism follows the same route as methionine (Swanson, 1987) and is mainly deposited in the egg white, while selenocysteine, like cysteine, is mainly deposied in the egg yolk (Latshaw and Osman, 1975; Latshaw and Biggert, 1981). Se-enriched yeast has been used in a large number of experiments as an organic source of selenium supplement for laying hens (Swanson, 1987; Payne et al, 2005; Mohiti-Asli et al., 2008; Utterback et al., 2005; Vukasinovic et al., 2006). Other forms of Se-enriched organic supplements for laying hens have been proposed, such as Se-enriched barley (Kantee and Kurkela, 1980; Hassan, 1990), Se-enriched malt (Jiakui and Xiaolong, 2004), Se-enriched alga Chlorella (Skřivan et al., 2008), and Se-enriched bean sprout (Chinrasri et al., 2009). Selenium egg enrichment has recently been reviewed by Fisinin et al. (2008, 2009) and Surai et al. (2007). Evidence suggests that selenium is declining in the food chain in some regions and this may lead to adverse effects on human health (Fisinin et al., 2009). Se-enriched eggs could be used as an important delivery system of selenium for humans due to its safety, given that a toxic dose of Se from eggs would require the consumption of more than 25 eggs per day over a period of time (Fisinin et al., 2009; Surai et al., 2007). Many researchers have investigated dietary strategies, since the end of the 1970s, in order to improve the selenium egg content. Selenium concentration in the egg increases as a function of dietary supplementation (Combs and Scott, 1977; Swanson, 1987; Hassan, 1990; Davis and Fear, 1996; Surai and Sparks, 2001; Mohiti-Asli et al., 2008). It has been shown that the selenium content in eggs can easily be manipulated, especially when organic selenium is included in hen’s diets at a level that provides 0.3–0.5 mg/kg selenium in the feed, as reported in Table 15.6. Experimental studies that compare inorganic and organic Se sources have shown that both selenium forms increases the selenium egg content, but the organic form is more efficient (Kantee and Kurkela, 1980; Hassan, 1990; Vukasinovic et al., 2006; Fisinin et al., 2008, 2009). This effect has been
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Basal Sodium selenite Se-malt
Basal Sodium selenite Se-yeast
Basal Selenite Se-yeast Se-Chlorella
Basal Selenite Se-yeast Se-bean sprout
Sodium selenite Sodium selenite Se-yeast
Jiakui and Xiaolong (2004)
Utterback et al. (2005)
Skrˇivan et al. (2008)
Chinrasri et al. (2009)
Kralik et al. (2009)
0.2 0.4 0.4
0.3 0.3 0.3
0.3 0.3 0.3
0.3 0.3
0.51 0.51
0.2 0.4 0.8
Dietary supplementation (mg/kg)
on dry matter basis; 2on as-is basis, n.d.: not determined.
Basal Se-yeast Se-yeast Se-yeast
Surai and Sparks (2001)
1
Se diet
Autor
n.d. n.d. n.d.
0.40 0.78 0.72 0.82
0.07 0.40 0.41 0.37
0.11 0.38 0.34 n.d. n.d. n.d. n.d 1.31 2.28 3.28 2.90
1
2
2
2
2
1
1
1
2
2
n.d. n.d. n.d
0.065 0.182 0.311
2
1
2
2
2
2
n.d. n.d. n.d
1
1
2
2
2
n.d. n.d. n.d. n.d
n.d. n.d. n.d. n.d. 0.23 0.74 0.74
Whole egg (mg/kg)
Dietary amount (mg/kg)
0.57 0.76 0.78
1.25 2.57 2.60 2.97
0.33 0.62 1.32 1.35
n.d. n.d. n.d.
1.34 2.15 2.26
0.30 0.60 0.85 1.09
0.18 0.23 0.45
2 2
2
2
2
2
0.44 0.67 1.79 0.98
n.d. n.d. n.d. n.d
n.d. n.d. n.d.
0.75 1.67 1.59
0.05 0.19 0.40 0.62
2
2
2
2
2
2
2
1
1
1
Se white (mg/kg)
2
1
1
1
1
1
Se yolk (mg/kg)
n.d. n.d. n.d.
17.60 29.47 46.61 39.18
n.d. n.d. n.d. n.d
n.d. n.d. n.d.
12.60 22.15 22.55
7.10 18.40 30.67 43.35
mg Se/egg
Table 15.6 Effect of dietary selenium supplementation on the selenium concentration of egg yolk and egg white and on selenium amount of the whole egg
308 Improving the safety and quality of eggs and egg products attributed to the difference in the absorption mechanism of each source. Inorganic selenium is passively absorbed, while Se-amino acid is absorbed by active transport, which is consistent with the absorption process of amino acids (Surai 2002a, 2002b, 2002c). A beneficial influence of combined dietary iodine and selenium supplementation have been found and iodine enhances the selenium egg content (Bourre and Galea, 2006; Bargellini et al., 2008; Rizzi et al., 2009). Selenium may negatively affect zinc absorption and dietary selenium supplementation reduces the amount of zinc in the egg (Bargellini et al., 2007; Rizzi et al., 2009). The selenium that is delivered in a single enriched egg varies from 20 to 40 mg when hen’s supplemented diets of 0.3–0.5 mg/kg selenium from organic sources are used (Surai et al. 2007; Finisin et al., 2009) as reported in Table 15.6. A single egg can deliver up to 70% human Se RDA (Surai et al. 2007; Fisinin et al. 2009) and therefore Se-enriched eggs are suitable for the functional food category. In addition, it seems that the selenium form expressed in Se-enriched eggs is nutritionally highly available for humans (Surai et al., 2004). 15.4.2 Iodine enrichment The average hen’s egg contains 4–10 mg of iodine, most of which is in the yolk. This amount is reduced in goitre areas (Underwood and Suttle, 2001). Iodine has only one known, but very important, function as a constituent of the thyroid hormones, particularly T3, which control the oxidation rate and protein synthesis in all cells. Most iodine in feeds is presented in inorganic form, which is absorbed very efficiently (in form of iodide, I–) from the gastrointestinal tract (Underwood and Suttle, 2001). Organic forms of iodine, such as iodinated amino acids, are also efficiently absorbed. Following absorption, iodine is rapidly taken up by the thyroid gland and converted into organic iodine through a combination with tyrosine. Goitrogens in the feeds can impair the selective concentration of iodine in the thyroid and egg follicle (Larbier and Leclercq, 1992; Klasing, 2000; Underwood and Suttle, 2001). The iodine requirement for poultry is 0.3–0.4 mg/kg diet. However, the requirement is greatly affected by the nature of the diet and is increased by more than two-fold by the presence of goitrogenic feeds in the diet (Klasing, 2000). In addition, the remarkable ability of a hen to adjust the iodine concentration of eggs to the iodine content of the diet makes it difficult to related hen’s iodine requirements to the rate of egg production (Underwood and Suttle, 2001). Developing follicles in the ovary concentrate iodine, and the amount of iodine in the egg reflects that in the diet (Klasing, 2000). In areas where goitre is endemic, precautions are generally taken by supplementing the diet with the iodine in the form of iodized salts. Inorganic iodine is usually supplemented in birds as calcium iodate (Ca(IO3)2), potassium © Woodhead Publishing Limited, 2011
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Egg enrichment with vitamins and trace minerals 309 iodate (KIO) or potassium iodide (KI), which have all similar bioavailability (Klasing, 2000). Iodine becomes toxic in laying hens at over 300 mg I/kg diet (Underwood and Suttle, 2001). Several studies pointed out that iodine concentrations in the egg yolk, egg albumen and whole egg increase with increased iodine supplementation (Kaufmann et al., 1998; Feng et al., 1999; Kroupová et al., 1999; Ishikawa et al., 2008). Over the last decade, there have been limited studies on the iodine enrichment of eggs. Trckova et al. (2003) observed an increased concentration of iodine in egg yolk, following a single dose of oral potassium iodide (KI), oral iodized fatty acid esters (IFAE) or intramuscular IFAE at the rate of 10 mg I/kg body weight. Oral iodine administration induces a peak in egg iodine concentration 5 days after administration (12,863 and 14,037 mg I/ kg yolk for oral KI and IFAE respectively) and a drop after 10 days. Quite different results were recorded following the intramuscular application of IFAE; both the increase and drop in iodine concentrations were slow with maximum values measured from day 11 until day 35 of the experiment (from 769 to 1163 mg I/kg yolk). These data suggest that the intramuscular administration of IFAE probably induces the deposit of iodine at slow release rate. Yalçin et al. (2004) studied the effects of diets supplemented with 0, 3, 6, 12 and 24 mg iodine per kg diet (as Ca(IO3)2 ·H2O) in laying hens and observed increasing iodine concentrations in the egg yolk, egg albumen and whole egg. However, adverse effects were observed on feed consumption and egg production for 12 and 24 mg iodine/kg supplemented diets. Ishikawa et al. (2008) obtained I-enriched eggs (about 5 to 12-fold more than the control group) by supplementing laying hens diets with potassium iodide-enriched dried agar waste. A dietary iodine supplementation of up to 6 mg/kg is recommended to obtain iodine-enriched eggs containing up to 26 mg I/egg (or 20.5 mg I/egg yolk and 5.6 mg I/egg albumen, corresponding to 112.2 mg I/100 g yolk and 18.0 mg I/100 g albumen) (Yalçin et al., 2004). 15.4.3 Iron enrichment Iron is found in egg yolk in association with phosvitin. Very little iron is found in the albumen, where it forms complexes with ovotransferrin in order to protect the egg from bacterial infections (Klasing, 2000). Hen’s egg contains on average 1–2 mg iron and the daily demand for iron obviously rises with the onset of laying (Underwood and Suttle, 2001; McDonald et al. 2002). The iron requirement recommended by National Research Council (1994) for laying hens is 38–50 mg/kg diet. If the iron is present in available forms, a marginal range of 750–1250 mg Fe/kg dry matter can be considered to separate the nutritionally acceptable from the potentially harmful iron concentration for poultry (Underwood and Suttle, 2001). Dietary iron is available both in organic and inorganic forms and either
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310 Improving the safety and quality of eggs and egg products in the ferrous (Fe2+) or ferric (Fe3+) valence state. Dietary ferric iron is converted into the ferrous form in the proventriculus and gizzard by the action of hydrochloric acid prior to absorption. Iron absorption is regulated in order to avoid excess uptake. Vitamin C improves iron absorption (Klasing, 2000; Larbier and Leclercq, 1992). When iron body stores are adequate, most of the iron absorbed by the enterocyte remains bound to the ferritin until it is shed into the lumen of the intestinal tract. This mucosal block guarantees iron homeostasis (Klasing, 2000; Underwood and Suttle, 2001). Ferrous iron is oxidized to ferric form following transfer to the blood and is complexed by transferrin, the form that allows iron to be distributed to all the tissues. In addition to this mechanism, a large amount of vitellogenin is synthesized in the liver under oestrogen regulation, in egg-laying or oestrogenous-treated birds (Mullinix et al., 1976). Vitellogenin delivers iron to the developing ova, where the vitellogenin is split into phosvitin and lipovitellin. The iron transported to the ova in this way represents 30% of the plasma iron turnover and is therefore a quantitatively important process in the laying hen (Morgan, 1981). Iron is commonly supplemented as ferrous sulphate, ferric chloride, ferric citrate, ferric ammonium citrate, and iron amino acid complexes (Klasing, 2000). The enrichment of hen eggs with additional iron could provide new niche markets by improving the nutritional status of particular cohorts of people at risk to iron deficiency or anaemia. Several groups of people are considered to be at risk of iron-deficient anaemia: infants, growing children, pregnant women, and certain cultural and socio-economic groups. Enriching the iron content of eggs could be a particularly useful strategy because other iron-rich foods, such as meat, may not be suitable or available for all those people at risk to iron deficiency, and in many cases it may be a more viable option than fortifying staple foods with iron, which is often of low bioavailability (Revell et al., 2009). Park et al. (2004) found that the iron content of eggs could be increased by 5–18% through the dietary supplementation of iron in organic or inorganic form fed to 30-week-old ISA Brown laying hens. The dietary iron supplementation was at grade levels of 100, 200 and 300 mg/kg in the form of iron sulphate (FeSO4·7H2O), iron–methionine chelate (Fe-Met) or a commercial iron complex with amino acid (Fe–amino acid). The iron content in the egg increased significantly in all the supplemental Fe treatments and was maximized at 10–15 days after the supplemental iron feeding. Fe-Met was the most effective source of enriching iron in the eggs, followed by the commercial Fe–amino acid complex and FeSO4. Increasing supplementation of iron levels above 100 mg/kg was not effective in Fe-Met and Fe–amino acid complex treatments whereas ferrous sulphate needed 200 or 300 mg/ kg of iron to reach a maximum egg iron content. The iron egg amount during the 15–40 days of treatment was 2.26–2.37 mg/kg, 2.11–2.20 mg/kg and 2.19–2.25 mg/kg for Fe-Met, FeSO4 and Fe–amino acid supplemented groups, respectively. Park et al. (2005) confirmed the magnitude of iron egg
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Egg enrichment with vitamins and trace minerals 311 fortification by dietary Fe-Met supplementation at a level of 100 mg/kg. An iron content of 2.24–2.35 mg Fe/100 g egg was observed against a control group containing an average amount of 2.06 mg Fe/100 g egg. Paik et al. (2009) compared dietary iron–soy proteinate (Fe-SP) (100 and 200 mg/kg Fe) and Fe-Met (100 mg/kg Fe) supplemented on a basal diet containing 120 mg/kg of iron. The iron content of the egg yolk was maximized five weeks after feeding supplemented diets and no significant difference was found in the iron content of the egg yolk between Fe-SP and Fe-Met supplemented diets. However, the 100 mg/kg dietary Fe-SP produced the best result (267.1 mg/kg iron on a dry matter basis, fat free) being 16.6% higher than the control. The copper content in the egg yolk was not significantly affected by the treatments but the zinc content was significantly increased in the iron supplemented treatments at the fifth week after feeding. Revell et al. (2009) investigated different dietary strategies to manipulate the iron content of eggs: supplementation with organic (commercial products) or inorganic (ferrous sulphate) iron (equivalent to 450 mg Fe kg–1 diet) with or without a vitamin C supplement (0.2 g kg–1 diet); a supplement of blood meal to give haem iron (equivalent to 120 mg Fe kg–1 diet); enhancement of the provision of serine or methyl group donors in order to allow the synthesis of phosvitin; administration of phytoestrogens in order to stimulate the hepatic synthesis of vitellogenins. In this experiment, the basal diet contained 50 mg Fe/kg diet. The concentration (mg Fe/g egg yolk) and the total amount (mg Fe/egg) of iron in the egg yolk were not significantly affected by any of the treatments. The iron concentration in the yolk was within a very limited range (62–71 mg Fe/g egg yolk) and a similar trend was observed for the total amount of iron in the eggs (1.06–1.25 mg Fe/egg). The addition of blood meal or phytoestrogens increased the total iron content of the yolks (1.25–1.22 mg Fe/egg) by over 15% compared with the control diet (1.10 mg Fe/egg), although this increase was not statistically significant. However, the use of an ingredient of animal origin and the risk of the potential transfer of oestrogenic compounds to the eggs would reduce the acceptability of such enriched eggs by consumers. Manson et al. (1993), Revell and Hughes (2005) and Revell et al. (2009) observed, that some birds consistently produced eggs with a higher iron content than average, and that between egg iron concentration was consistent for individual birds. On the basis of these observations, Revell et al. (2009) suggest that a breeding programme that selects ‘high-iron’ birds, coupled with dietary strategies, would appear to be a worthwhile research objective, including the development of strategies to identify high iron birds for niche markets. The combined supplementation of dietary zinc, iron and copper on the carry-over of these elements in eggs has been investigated in depth by Skřivan et al. (2005). A basal diet containing 63.4 mg Zn/kg, 92.8 mg Fe/ kg and 9.0 mg Cu/kg was supplemented with ZnSO4·7H2O, FeSO4·7H2O and CuSO4·5H2O to increase concentrations of zinc, iron, and copper by 80, © Woodhead Publishing Limited, 2011
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312 Improving the safety and quality of eggs and egg products 120 and 25 mg/kg, respectively. The zinc, iron and copper supplementations were combined in seven experimental groups in order to include all the possibilities. A zinc–copper antagonism was observed when these minerals were at basal dietary levels and zinc and copper supplementation overcame this effect. The concentration of iron in the egg yolk and white reflected the iron concentration in the diet. The highest concentration of iron in the egg yolk (159.2 mg Fe/kg dry matter) and white (4.24 mg Fe/kg dry matter) was found when the hens were fed the diet supplemented with the three tested trace elements. Supplementation of the basal diet with iron, in combination with zinc and copper, increased the iron concentration in the yolk and white by 36.7 and 34.9%, respectively. The iron alone increased the iron concentration in the egg yolk and white by 6.3 and 2.2%, respectively. The enrichment of eggs with other trace elements was marginal (copper) or absent (zinc). No significant effect of iron on the deposition of zinc or copper was observed. Fe-enriched eggs may contain 10–20% more iron than standard eggs; this would provide up to 10% RDA for humans. However, the mixed results that have been obtained in iron egg enrichment in different experiments highlight the need to consider the source of iron supplementation, the dietary interaction with other minerals, the effect of the age and strain of birds and the differences between individual birds in the iron concentration in the eggs. 15.4.4 Zinc enrichment The total zinc content of eggs from hens consuming standard laying rations is generally 10–11 mg/g egg (Puwastien et al., 1999; Watson, 2002) and 99% of the total zinc is deposited in the yolk. The zinc requirement for laying hens is 29–44 mg/kg diet (National Research Council, 1994). Birds can tolerate relatively high levels of dietary zinc, probably because of a proficient regulation of the zinc absorption. A zinc dietary level of up to 1 g/kg diet is tolerated by adult chickens. In laying hens, a high dietary zinc level (above 2 g/kg diet) stops egg laying and induces a moult. Zinc is absorbed by passive diffusion throughout the small intestine, with the duodenum being the most important site. When body stores and dietary zinc are both high, intestinal cells synthesize large amounts of metallothionein, which tightly binds zinc and prevents its binding to cysteine-rich intestinal protein for subsequent transport to the blood (Klasing, 2000). Metallothionein is a small, cysteinerich protein which can bind Zn and other heavy metals with high affinity. The single isoform of metallothionein in the chicken has been found in liver, pancreas, kidney and intestinal mucosa (Oh et al., 1979; McCormick, 1984; Sandoval et al., 1998; Cao et al., 2000). Although, the concentration of tissue metallothionein in chickens was positively related to Zn intake (Fleet et al., 1988), long-term supplementation failed to continue to promote synthesis of metallothionein in avian species fed elevated Zn for 3 weeks
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Egg enrichment with vitamins and trace minerals 313 (Sandoval et al., 1998; Cao et al., 2000). A high dietary zinc level increases the requirement for selenium, iron and copper. Zinc absorption is decreased by high dietary levels of phytic acid, calcium, phosphorus, copper, cadmium or chromium (Klasing, 2000). Zinc is an essential trace element for physiologic and metabolic functions, such as physical growth neuro-behavioural development, the reproductive function, and the immune system function. Zinc is an important pigmenting metal in feathers and it is also an activator or cofactor of more than 200 enzymes. Zinc deficiencies impair all metabolic pathways, the regulation of gene expression, and the cell division (Larbier and Leclercq, 1992; Klasing, 2000; Underwood and Suttle, 2001). Zinc is usually supplemented in inorganic form (zinc sulphate, zinc oxide, zinc carbonate) or organic form (complexed to amino acid) (Klasing, 2000). A numbers of researchers have made efforts to produce Zn-enriched eggs by fortifying hens’ diets with various forms of zinc. An increase in yolk zinc content probably involves the production of vitellogenin, a trace mineral transporting protein. Vitellogenin may be increased when the zinc concentration increases, because of the transfer of zinc, which is stored in the liver and transferred to the egg yolk (Richards, 1997). Stahl et al. (1988) studied the effect of zinc supplementations at different levels: moderate (52 mg Zn/g diet), high (218–257 mg Zn/g diet) and very high (1762–1861 mg Zn/g diet); the control diets contained 28 or 26 mg Zn/g diet. Hens fed very high levels of zinc for 4 to 40 weeks produced eggs that contained 57–90% more zinc (up to 24 mg Zn/g egg) than eggs produced by hens fed control diets which contained around 10 mg Zn/g egg. The hens fed high levels of zinc produced eggs that contained as much as 25% more zinc (around 14 mg Zn/g egg) than the eggs produced by the hens fed the control diets. The ingestion of excess zinc did not have consistent effects on the iron or copper contents of the eggs or on the zinc content of the eggshells. Skrˇ ivan et al. (2005) studied the effect of a laying hen supplemented diet with zinc, iron and copper. Zinc supplemented diets containing about 140 mg Zn/kg diet against unsupplemented diets containing about 60 mg Zn/kg diet. The zinc amount of egg yolk, egg white and eggshell were not affected by dietary zinc supplementation with values of 62.1–72.5, 10.5–11.5 and 7.8–8.6 mg Zn/kg dry matter, respectively. The concentration of zinc in the yolk was reduced in the eggs of hens fed a copper-supplemented diet and vice versa, probably due to a zinc–copper antagonism; higher dietary zinc or copper could overcome the negative effect of the antagonizing element. The recovery of zinc in the eggs of hens fed the basal diet was around 10% of the alimentary feed intake, while, in the supplemented group, the recovery was around 5%, indicating a poor utilization of this element for egg production when supplied at higher concentrations in the feed. Plaimast et al. (2008) tested the effect of zinc-fortified diets at levels of 300 and 600 mg/g diet against a control diet (60 mg Zn/g diet). The zinc was supplemented in inorganic form (zinc sulphate) or organic form (zinc amino acid chelate). Increasing the zinc level
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314 Improving the safety and quality of eggs and egg products in the diet increased zinc deposition in the egg yolk but no difference was found between the organic and inorganic form. However, the zinc deposition in the egg yolk was at a weak magnitude as the zinc yolk content in the supplemented group ranged between 41.2 and 43.7 mg/g against the control group which showed an average zinc content of 39.5 mg/g. The zinc egg content increased linearly as dietary zinc levels increased, as described by Kim and Patterson (2005). These authors investigated the effect of very high zinc supplemented diets (1000, 2000 and 3000 mg/kg) on hen performance, ammonia volatilization and nitrogen retention in manure. The average zinc egg content resulted to be 112.5, 93.2 and 70.0 mg/kg zinc (on dry matter basis) for birds fed diets supplemented with 3000, 2000 and 1000 mg/kg zinc respectively; the control group produced eggs containing around 60.0 mg/kg zinc (on a dry matter basis). However, the hens fed the diets supplemented with 2000 and 3000 mg/kg zinc had reduced body weight, feed consumption, egg production, egg weight and shell thickness. Plaimast et al. (2009) found that no differences in zinc bioavailability were observed for humans for intrinsically Zn-enriched (either via organic or inorganic laying hen zinc dietary supplementation) was observed. These authors found that an enriched egg contains about 1 mg zinc and zinc availability (measured in vitro as percent dialysis) is 72–75% in the cooked (boiled or fried) intrinsically Zn-enriched eggs. However, the zinc content of eggs can be increased by dramatically increasing the level of zinc in the diets of laying hens, although the practical significance of this effect, in terms of human nutrition, is debatable.
15.5 References aparicio a, barroeta a m, lópez-sobaler a m, ortega r m
(2008), ‘Etiquetado Nutricional’, Guía de etiquetado del huevo, Instituto de Estudios del Huevo, Madrid, 38–57. arc (1975), ‘Poultry’ in Agricultural Research Council, The Nutrient Requirements of Farm Livestock, No 1, Agricultural Research Council, London. austic r e, hsu k, lartey f m (2008), ‘Functional food components in animal source foods: eggs from chickens’, Encyclopedia of Animal Science Eds: Wilson G, Pond:Alan W. Bell: Taylor&Francis, London, UK 1, 1, 1–4. bargellini a, marchesi i, rizzi l, cauteruccio l, masironi r, simioli m, borella p (2008), ‘Selenium interactions with essential and toxic elements in eg yolk from commercial and fortified eggs’, J Trace Elem Med Biol, 22, 234–241. barroeta a, calsamiglia s, cepero r, lópez-bote c j, hernández j m (2002), ‘Optima nutrición vitamínica de los animales para la producción de alimentos de calidad’, Pulso Ediciones, Barcelona, Spain. bourre j m, galea f (2006), ‘An important source of omega-3 fatty acids, vitamins D and E, carotenoids, iodine and selenium: a new natural multi-enriched egg’, J Nutr. Health Aging, 10, 371–376. bunchasak c, kachana s (2009), ‘Dietary folate and vitamin B12 supplementation and consequent vitamin deposition in chicken eggs’, Trop Anim Health Prod, 41, 1583–1589. cao j, henry p r, guo r, holwerda r a, toth j p, littell r c, miles r d, ammerman c b
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Egg enrichment with vitamins and trace minerals 315 (2000), ‘Chemical characteristics and relative bioavailability of supplemental organic zinc sources for poultry and ruminants’, J Anim Sci, 78, 2039–2054. chen j y, latshaw j d, lee h o, min d b (1998), ‘a-Tocopherol content and oxidative stability of egg yolk as related to dietary a-tocopherol’, J Food Sci, 63, 919–922. cherian g, wolfe f w, sim j s (1996), ‘Feeding dietary oils with tocopherols: effects on internal qualities of eggs during storage’, J Food Sci, 61, 15–18. chinrasri o, chantiratikul p, thosaikam w, atiwetin p, chumpawadee s, saenthaweesuk s, chantiratikul a (2009), ‘Effect of selenium-enriched bean spout and other selenium sources on productivity and selenium concentration in eggs of laying hens’, AsianAust J Anim Sci, 22, 1661–1666. combs g f, combs s b (1986), The role of selenium in nutrition, Academic Press, New York, USA. combs g f, scott m l (1977), ‘The selenium needs of laying and breeding hens’, Proceedings of the Cornell Nutrition Conference, pp 74–82. commission directive 2008/100/EC of 28 October 2008 amending Council Directive 90/496/ EC on nutrition labelling for foodstuffs as regards recommended daily allowances, energy conversion factors and definitions. OJ L285, 29.10.2008, p. 9–12. commission regulation 1924/2006/EC of 20 December 2006 relating to nutrition and health claims made on foods. OJ L404, 30.12.2006, p. 9–23. cortinas l, galobart j, barroeta a c, baucells m d, grashorn m a (2003), ‘Change in a-tocopherol contents, lipid oxidation and fatty acid profile in eggs enriched with linolenic acid or very long-chain w-3 polyunsaturated fatty acids after different processing methods’, J Sci Food Agric, 83, 820–829. davis r h, fear j (1996), ‘Incorporation of selenium into egg proteins from dietary selenite’, Br Poult Sci, 37, 197–211. dutta-roy a k, gordon m j, campbell f m, duthie g g, james w p t (1994), ‘Vitamin E requirements, transport, and metabolism: role of a-tocopherol-binding proteins’, J Nutr Biochem, 5, 562–570. feng c s, li’e y, rong c m, yong c l, qiang k c (1999), ‘Effects of feeding high-iron and high-iodine diet to hens on the iron and iodine content in eggs and egg quality’, J Shang Agr Coll, 17, 248–254. fisinin v i, papazyan t t, surai p f (2008), ‘Producing specialist poultry products to meet human nutrition requirements: selenium enriched eggs’, Worlds Poult Sc J, 64, 85–98. fisinin v i, papazyan t t, surai p f (2009), ‘Producing selenium-enriched eggs and meat to improve the selenium status of general populaton’, Crit Rev Biotechnol, 29, 18–28. flachowsky g, engelman d, sünder a, halle i, sallmann h p (2002), ‘Eggs and poultry meat as tocopherol sources in dependence on tocopherol supplementation of poultry diets’, Food Res Int, 35, 239–243. fleet j c, qureshi m a, dietert r r, mccormick c c (1988), ‘Tissue-specific accumulation of metallothionein in chickens as influenced by the route of zinc administration’, J Nutr, 118, 176–182. franchini a, sirri f, tallarico n, minelli g, iaffaldano n, meluzzi a (2002), ‘Oxidative stability and sensory and functional properties of eggs from laying hens fed supranutritional doses of vitamins E and C’, Poult Sci, 8, 1744–1750. frigg m, whitehead c c, weber s (1992), ‘Absence of effects of dietary a-tocopherol on egg yolk pigmentation’, Br Poult Sci, 33, 347–353. frost t j, roland d a, untawale g g (1990), ‘Influence of vitamin D3, 1a-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 on eggshell quality, tibia strength, and various production parameters in commercial laying hens’, Poultry Sci, 11, 2008–2016. galobart j, barroeta a c, baucells m d, cortinas l, guardiola f (2001a), ‘Alpha-tocopherol transfer efficiency and lipid oxidation in fresh and spray-dried eggs enriched with omega 3-polyunsaturated fatty acids’, Poult Sci, 80, 1496–1505. galobart j, barroeta a c, baucells m d, guardiola f (2001b), ‘Lipid oxidation in fresh
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316 Improving the safety and quality of eggs and egg products and spray-dried eggs enriched with omega 3 and omega 6 polyunsaturated fatty acids during storage as affected by dietary vitamin E and canthaxanthin supplementation’, Poult Sci, 80, 327–337. galobart j, barroeta a c, cortinas l, baucells m d, codony r (2002), ‘Accumulation of alpha-tocopherol in eggs enriched with omega 3 and omega 6 polyunsaturated fatty acids’, Poult Sci, 81, 1873–1876. gebert s, messikommer r, pfirter h p, bee g, wenk c (1998), ‘Dietary fats and vitamin E in diets for laying hens: effects on laying performance, storage stability and fatty acid composition of eggs’, Archiv Geflügelk, 62, 214–222. grobas s, mendez j, lopez bote c, de blas c, mateos g g (2002), ‘Effect of vitamin E and A supplementation on egg yolk a-tocopherol concentration’, Poult Sci, 81, 376–381. hasler c m (2000), ‘The changing face of functional foods’, J Am Coll Nutr, 19, 5, 499S–506S. hassan s (1990), ‘Selenium concentration in egg and body tissue as affected by the level and source of selenium in the diet’, Acta Agr Scand A-An, 40, 279–287. hayat z, cherian g, pasha t n, khattak f m, jabbar m a (2009), ‘Effect of feeding flax and two types of antioxidants on egg production, egg quality, and lipid composition of eggs’, J Appl Poult Res, 18, 541–551. hebert k, house j d, guenter w (2005), ‘Effect of dietary folic acid supplementation on egg folate content and the performance and folate status of two strains of laying hens’, Poult Sci, 84, 1533–1538. hoey l, mcnulty h, mccann e m e, mccracken k j, scott j m, marc b b, molloy a m, graham c, pentieva k (2009), ‘Laying hens can convert high doses of folic acid added to the feed into natural folates in eggs providing a novel source of food folate’, Br J Nutr, 101, 206–212. hossain s m, barreto s l, bertechini a g, rios a m, silva c g (1998), ‘Influence of dietary vitamin E level on egg production of broiler breeders, and on the growth and immune response of progeny in comparison with the progeny from eggs injected with vitamin E’, Anim Feed Sci Technol, 73, 307–317. house j d, braun k, balance d m, o’connor c p, guenter w (2002), ‘The enrichment of eggs with folic acid through supplementation of the laying hen diet’, Poult Sci, 81, 1332–1337. ishikawa s, ogawa m, sakai k (2008), ‘The effect of addition of dried agar waste to layer diet on iodine content of egg yolk and egg quality’, Japan J Poult Sci, 45, 87–92. jiakui l, xiaolong w (2004), ‘Effect of dietary organic versus inorganic selenium in laying hens on the productivity, selenium distribution in egg and selenium content in blood, liver and kidney’, J Trace Elem Med Biol, 18, 65–68. jiang y, mcgeachin r, bailey c (1994), ‘Alpha-tocopherol, beta-carotene, and retinol enrichment of chicken eggs’, Poult Sci, 73, 1137–1143. kantee e, kurkela p (1980), ‘Comparative effects of berley feed and sodium selenite on selenium levels in hen eggs and tissues’, J Scient Agr Soc Fin, 52, 357–367. kaufmann s, wolfram g, delange f, rambeck w a (1998), ‘Iodine supplementation of laying hen feed: a supplementary measure to eliminate iodine deficiency in humans?’, Zeitschrift Ernährungswissenschaft, 37, 288–293. kim w k, patterson p h (2005), ‘Effects of dietary zinc supplementation on hen performance, ammonia volatilization, and nitrogen retention in manure’, J Environ Sci Health B, 40, 675–686. klasing k c (2000), Comparative avian nutrition, CABI Publishing, New York, USA. kralik g, gacjevic z, suchy p, strakova e, hanzek d (2009), ‘Effects of dietary selenium source and storage on internal quality of eggs’, Acta Vet Brno, 78, 219–222. kroupová v, trávnic ˇ ek j, kursa j, kratochvil p, krabacˇ ová i (1999), ‘Iodine content in egg yolk during its excessive intake by laying hens’, Czech J Anim Sci, 44, 369–376. larbier m, leclercq b (1992), Nutrition et alimentation des vollailles, INRA editions, France.
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Egg enrichment with vitamins and trace minerals 317 latshaw j d, biggert m d (1981), ‘Incorporation of selenium into egg proteins after feeding
selenomethionine or sodium selenite’, Poult Sci, 60, 1309–1313. (1975), ‘Distribution of selenium in egg white and yolk after feeding natural and synthetic compounds’, Poult Sci, 54, 1244–1252. leeson s, caston l j (2003), ‘Vitamin enrichment of eggs’, J Appl Poult Res, 12, 24–26. manson j m, pcken k j, draper m h, thompson r (1993), ‘Variation among individual White-Leghorn hens in the concentration of minerals in the albumen and yolk content of their eggs’, Br Poultr Sci, 34, 899–909. mattila p , lehikoinen k , kiiskinen t , piironen v (1999), ‘Cholecalciferol and 25hydroxycholecalciferol content of chicken egg yolk as affected by the cholecalciferol content of feed’, J Agric Food Chem, 47, 4089–4092. mattila p, rokka t, konko k, valaja j, rossow l, ryhanen e l (2003), ‘Effect of cholecalciferolenriched hen feed on egg quality’, J Agric Food Chem, 51, 283–287. mattila p, valaja j, rossow l, venalainen e, tupasela t (2004), ‘Effect of vitamin D-2and D-3-enriched diets on egg vitamin D content, production, and bird condition during an entire production period’, Poult Sci, 83, 433–440. mccormick c c (1984), ‘Induction and accumulation of metallothionein in liver and pancreas of chicks given oral zinc: a tissue comparison’, J Nutr, 114, 191–203. mcdonald p, edwards r a, greenhalgh j f d, morgan c a (2002), Animal nutrition, Pearson Education Limited, Harlow, UK. mcdowell l (2000), Vitamins in animal and human nutrition, 2nd edition, Iowa State University Press, USA. meluzzi a, sirri f, tallarico n, vandi l (1999), ‘Dietary vitamin E in producing eggs enriched with n-3 fatty acids’, Proc. VIII Europ. Symp. on the Quality of Eggs and Egg Products. Bologna (Italy), September 19–23, 153–159. meluzzi a, sirri f, manfreda g, tallarico n, franchini a (2000), ‘Effects of dietary vitamin E on the quality of table eggs enriched with omega-3 long chain fatty acids’, Poult Sci, 79, 539–545. mendonça c x, almeida c r m, mori a v, watanabe c (2002), ‘Effect of dietary vitamin A on egg yolk retinol and tocopherol levels’, J App. Poult Res, 11, 373–378. mohiti-asli m, shariatmadari f, lotfollahian h, mazuji m t (2008), ‘Effects of supplementing layer hen diets with seleniumand vitamin E on egg quality, lipid oxidation and fatty acid composition during storage’ Can J An Sci, 88, 3, 475–483. morgan e h (1981), ‘Comparative iron metabolism’, in Jacobs A and Worwood M (Eds), Iron in Biochemistry and Medicine II, pp 641–687, Academic Press, London, UK. mori a v, mendoça c x, almeida c r m, pita c g (2003), ‘Supplementing hen diets with vitamins A and E affects egg yolk retinol and a-tocopherol levels’, J Appl Poult Res, 12, 106–114. muggli r (1994), ‘Physiological requirements of vitamin E as a function of the amount and type of polyunsaturated fatty acid. World Rev Nutr Diet, 75, 166–168. mullinix k, wetekam w, deeley r g, gordon j l, meyers m, kent k a, goldberger rf (1976), ‘Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin syntesis’, Proc Nat Acad Anim Sci, 73, 287–290. murcia m a, martínez-tomé m, del cerro i, sotillo f, ramírez a (1999), ‘Proximate composition and vitamin E levels in egg yolk: losses by cooking in a microwave oven’, J Sci Food Agric, 79, 1550–1556. naber e c (1979), ‘The effect of nutrition on the composition of eggs’, Poult Sci, 58, 518–528. naber e c (1993), ‘Modifying vitamin composition of eggs: a review’, J Appl Poult Res, 2, 385–393. national research council (1994), Nutrient requirements for poultry, 9th ed, National Academy Press, Washington, DC. latshaw j d, osman m
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318 Improving the safety and quality of eggs and egg products (2010), ‘Structure et formation de l’œuf’, Science et technologie de l’oef et des ovoproduits, Nau F, Guerin C, Thapon J L, (Coord), EdTech et Doc, Lavoisier, Paris, France, 1 (5), 161–249. oh s h, nakaue h, deagen j t, whanger p d, arscott g h (1979), ‘Accumulation and depletion of zinc in chick tissue metallothioneins’, J Nutr, 109, 1720–1729. ovesen l, brot c, jakobsen j (2003), ‘Food contents and biological activity of 25hydroxyvitamin D: A vitamin D metabolite to be reckoned with?’, Ann Nutr Metab, 47, 107–113. paik i k, lee h k, park s w (2009), ‘Effect of organic iron supplementation on the performance and iron content in the egg yolk of laying hens’, J Poult Sci, 46, 198–202. park s w, namkung h, ahn h j, paik i k (2004), ‘Production of iron enriched eggs of laying hens’, Asian-Aust J Anim Sci, 17, 1725–1728. park s w, namkung h, ahn h j, paik i k (2005), ‘Enrichment of vitamin D3, K and iron in eggs of laying hens’, Asian-Aust J Anim Sci, 18, 226–229. payne r l, lavergne t k, southern l l (2005), ‘Effect of inorganic versus organic selenium on hen production and selenium concentration’, Poult Sci, 84, 232–237. pita e, piber l n, de mendonça c x (2004), ‘Effect of dietary supplementation of unsaturated fatty acids and vitamin E upon yolk lipid composition and a-tocopherol incorportation into the egg yolk’, Brazilian J Vet Res Anim Sci, 41, 25–31. plaimast h, sirichakwal p, puwastien p, kijparkorn s (2008), ‘Effect of supplementary zinc from organic and inorganic sources on laying performance and zinc deposition in eggs’, Thai J Vet Med, 38, 47–53. plaimast h, sirichakwal p, puwastien p, judprasong k, wasantwisut e (2009), ‘In vitro bioaccessibility of intrinsically zinc-enriced egg and effect of cooking’, J Food Compos Anal, 22, 627–631. puwastien p, raroengwichit m, sungpuag p, judprasong k (1999), Thai food composition tables, first ed. Institute of Nutrition, Mahidol University, Thailand. qi g-h, sim j s (1998), ‘Natural tocopherol enrichment and its effect in n-3 fatty acid modified chicken eggs’, J Agric Food Chem, 46, 1920–1926. revell d k, hughes r j (2005), ‘Between-bird variation in the concentration of iron and other minerals in egg yolk across a laying cycle’, Proceedings of the Australian Poultry Science Symposium, 17, 287–290. revell d k, zarrinkalam m r, hughes r j (2009), ‘Iron content of eggs from hens given diets containing organic forms of iron, serine and methyl group donors, or phytoestrogens’, Br Poult Sci, 50, 536–542. richards m p (1997), ‘Trace mineral metabolism in the avian embryo’, Poult Sci, 76, 152–164. richter g (1995), ‘Incorporation and mobilization of vitamin A in laying hens’, Arch Tierernah, 48, 337–345. richter g, lemser a, ludke c, steinbach g, mockel p (1996a), ‘Studies on the vitamin A requirements and recommendations for laying hens’, Archiv Geflugelk, 60, 174–180. richter g, lemser a, jahreis g, wanka u, matthey m, schubert k, steinbach g (1996b), ‘Studies in vitamin A requirement and recommendations for chicken and pullets’, Archiv Geflugelk, 60, 193–202. rizzi l, bochicchio d, bargellini a, parazza p, simioli m (2009), ‘Effect of dietary microalgae,other lipid sources, inorganic selenium and iodine on yolk n-3 fatty acid composition, selenium content and quality of egs in laying hens’, J Sci Food Agric, 89, 1775–1781. rotruck j t, pope a l, ganther h e, swanson a b, hafeman d g, hoekstra w g (1973), ‘Selenium: biochemical role as a component of glutathione peroxidase’, Science, 179, 588–590. sahlin a, house j d (2006), ‘Enhancing the vitamin content of meat and eggs: Implications for the human diet’, Can J An Sci, 86, 2, 181–195. nys y
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Egg enrichment with vitamins and trace minerals 319 sandoval m, henry p r, luo x g, littell r c, miles r d, ammerman c b
(1998), ‘Performance and tissue zinc and metallothionein accumulation in chicks fed a high dietary level of zinc’, Poult Sci, 77, 1354–1363. scientific committee on food (2003), ‘Opinion of the Scientific Committee on Food on the revision of reference values for nutrition labeling’. SCF/CS/NUT/GEN/18 Final 6 March, 2003. seuss-baum i (2007), ‘Nutritional evaluation of egg compounds’, in Huopalathi R, LopezFandino R, Anton M and Schade R, Bioactive egg compounds, Springer-Verlag, Berlin, Heidelbergr, pp 117–144. sherwood t, alphin r, saylor w, white h (1993), ‘Folate metabolism and deposition in eggs by laying hens’, Arch Biochem Biophys, 15,307(1), 66–72. sirichakwal p p, newcomer c e, young v r, janghorbani m (1984), ‘Labelling hen’s egg with 74Se for use in human metabolic experiments’, J Nutr, 114, 1159–1168. sirri f, barroeta a c (2007), ‘Enrichment in Vitamin’, in Houpalahti R, Lopez-Fandino R, Anton M and Schade R, Bioactive egg compounds, Springer-Verlag, Berlin Heidelberg, pp 171–182. skr ˇ ivan m, skrˇ ivanová v, maurounek m (2005), ‘Effects of dietary zinc, iron, and copper in layer feed on distribution of these elements in egg, liver, excreta, soil, and herbage’, Poultry Sci, 84, 1570–1575. skr ˇ ivan m, marounek m, dlouhá g, ševcˇ iková s (2008), ‘Dietary selenium increases vitamin E contents of egg yolk and chicken meat’, Br Poult Sci, 49, 482–486. soares j h, ottinger m a, buss e g (1988), ‘Potential role of 1,25 dihydroxycholecalciferol in egg shell calcification’, Poultry Sci, 67, 1322–1328. squires m w, naber e c (1992), ‘Vitamin profiles of eggs as indicators of nutritional status in the laying hen: vitamin B12 study’, Poult Sci, 71, 2075–2082. squires m w, naber e c (1993), ‘Vitamin profiles of eggs as indicators of nutritional status in the laying hen: vitamin A study’, Poult Sci, 72, 154–164. stadelman w j, pratt d e (1989), ‘Factors influencing composition of the hens egg’, Worlds Poult Sci J, 45, 247–266. stahl j l, cook m e, greger j l (1988), ‘Zinc, iron, and copper contents of eggs from hens fed varying levels of zinc’, J Food Comps Anal, 1, 309–315. surai p f (2002a), Natural antioxidants in avian nutrition and reproduction, Nottingham University Press, Nottingham, UK. surai p f (2002b), ‘Selenium in poultry nutrition: a new look at an old element. 1. Antioxidant properties, deficiency and toxicity’, World Poultry Sci J, 3, 333–347. surai p f (2002c), ‘Selenium in poultry nutrition. 2. Reproduction, egg and meat quality and practical applications’, World Poultry Sci J, 58, 431–450. surai p f, sparks n h c (2001), ‘Designer eggs: from improvement of egg composition to functional food’, Trends Food Sci Tech, 12, 7–16. surai p f, ionov i a, buzhin a, buzhina n (1995), ‘Vitamin E and egg quality’, Proc. of the VI Europ. Symp. on the Quality of Eggs and Egg Products, Zaragoza (Spain), September 25–29, 387–394. surai p f, ionov i a, kuklenko v, kostjuk i a, macpherson a, speake b k, noble r c, sparks n h c (1998), ‘Effect of supplementing the hen’s diet with vitamin A on the accumulation of vitamin A and E, ascorbic acid and carotenoids in the egg yolk and in embryonic liver’, Brit Poult Sci, 39, 257–263. surai p f, yaroshenko f o, yaroshenko y f, karadas f, sparks n h c (2004) ‘Consumption of selenium-enriched eggs improves selenium status in human volunteers’, Proceedings of the XXII World’s Poultry Congress, Turkey. surai p f, papazynan t t, speake b k, sparks n h c (2007), ‘Enrichment in selenium and other trace elements’ in edited by Huopalahti R, López-Fandiño R, Anton M, Shade R (eds) Bioactive egg compounds, Springer, Berlin, Germany. suzuki y, okamoto m (1997), ‘Production of hen’s eggs rich in vitamin K’, Nutrition Research 17, 10, 1607–1615.
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320 Improving the safety and quality of eggs and egg products (1987), ‘Comparative utilization of selenite, selenomethionine, and selenized yeast by the laying hen’, Nutr Res, 7, 529–537. trckova m, pisarikova b, suchy p, herzig i (2003), ‘Effect of a single dose of iodized fatty acid ester Lipiodol® Ultra-Fluid on egg iodine concentrations and egg production’, Vet Med – Czech, 48, 293–300. underwood e j, suttle n f (2001), The mineral nutrition of livestock, CABI Publishing, New York, USA. utterback p l, parsons c m, yoon i, butler j (2005), ‘Effect of supplementing selenium yeast in diets of laying hens on egg selenium content’, Poult Sci, 84, 1900–1901. vukasinovic m, mihailovic r, sekler m, kaljevic m, kurcubic v (2006), ‘The impact of selenium content of laying hen feeds’, Arch Geflungelkd, 70, 91–96. wahle k w j, hoppe p p, mcintosh g (1993), ‘Effect of storage and various intrinsic vitamin E concentrations on lipid oxidation in dried egg powders’, J Sci Food Agric, 61, 463–469. watson r r (2002), Egg and health promotion, Iowa State Press, USA. weber g m (2009), ‘Improvement of flock productivity through supply of vitamins for higher laying performance and better egg quality’, World’s Poult Sci J, 65, 443–447. whitehead c c (1995), ‘Current knowledge on vitamin requirements of poultry’, World’s Poultry Science Association Proceedings, 10th European Symposium on Poultry, pp 64–73. yalçin s, kahraman z, yalçin s, yalçin s s, dedeoğlu h e (2004), ‘Effect of supplementary iodine on the performance and egg traits of laying hens’, Br Poult Sci, 45, 499– 503. swanson c a
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16 Bioactive fractions of eggs for human and animal health M. Anton, Institut National de la Recherche Agronomique (INRA), France and F. Nau and C. Guérin-Dubiard, AGROCAMPUS Ouest, France
Abstract: Hen’s eggs provide nutrients to the embryonic bird and protect it physically and microbiologically. Apart the physical barrier represented by shell, the constituents that eggs contain present biological functions vital for the protection of the embryonic bird. These constituents are consequently potential sources of bioactive fractions exploitable for medical, pharmaceutical, cosmetic, nutraceutical and biotechnological applications. Key words: egg, yolk, albumen, biological activities.
16.1 Introduction The hen’s egg plays a crucial role in the development of the embryonic bird as it delivers energy and highly digestible nutrients, and it protects against physical and chemical aggressions. An egg is formed sequentially in 21 days all along the hen’s reproductive system. Successively, different layers consisting chronologically of albumen, shell membrane and shell are deposited around the yolk containing the embryonic bird. The unique structure of an egg with yolk containing the embryo surrounded by albumen and shell represents the physical barrier against external attack. The chemical barrier consists of the specific constituents of yolk, albumen and shell which possess many molecules with elevated biological properties. A hen’s egg possesses an excellent nutritive value (important reserve of highly digestible proteins, lipids, vitamins and minerals) and constitutes a traditional food
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322 Improving the safety and quality of eggs and egg products used in many basic and formulated preparations. In 2010 it is estimated that there are around 5 billion egg laying hens in the world producing more than 60 million tonnes of eggs annually, corresponding to a consumption of 8 kg/capita/year. In addition, the constituents represent an important source of active principles that can be used for medical, pharmaceutical, cosmetic, nutraceutical and biotechnological applications. In this chapter we detail constitution and structure of hen egg and review some important potential bioactive applications.
16.2 Egg fractions 16.2.1 Egg yolk Chemical composition The yolk corresponds to 36% of whole hen egg weight. Its dry matter is about 50–52% according to the age of the laying hen and the duration of preservation (Kiosseoglou, 1989; Thapon and Bourgeois, 1994; Li-Chan et al., 1995). The compositions of fresh and dry yolks are presented in Table 16.1: the main components are lipids (about 65% of the dry matter) and the lipid to protein ratio is about 2:1. Lipids of yolk are exclusively associated with lipoprotein assemblies. They are made up of 62% triglycerides, 33% phospholipids, and less than 5% cholesterol. Carotenoids represent less than 1% of yolk lipids, and give it its colour. Proteins are present as free proteins or apoproteins (included in lipoprotein assemblies). The interactions between lipids and proteins result in the formation of lipoproteins (low and high density), which represent the main constituents of yolk. Macrostructure and main constituents Yolk is a complex system with different structuration levels consisting of aggregates (granules) in suspension in a clear yellow fluid (plasma) that contains lipoproteins and proteins. Granules consist of circular complexes ranging in diameter from 0.3 to 2 mm (Chang et al., 1977). Consequently, yolk can be easily separated into two fractions after a double dilution with 0.3 M NaCl and centrifuging at 10,000g (30 min) according to the method of Table 16.1 Composition of hen egg yolk
% of fresh yolk
% of dry yolk
Water Lipids Proteins Carbohydrates Minerals
51.1 3.6 16.0 0.6 1.7
– 62.5 33.0 1.2 3.5
Source: Powrie and Nakai (1986).
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Bioactive fractions of eggs for human and animal health 323 McBee and Cotterill (1979). The dark orange supernatant is called plasma and the pale pellet is named granules (Fig. 16.1). Granules represent 22% of yolk dry matter, accounting for about 50% of yolk proteins and 7% of yolk lipids. The dry matter content of granules is about 44%, with 64% proteins, 31% lipids and 5% ash (Dyer-Hurdon and Nnanna, 1993; Anton and Gandemer, 1997). They consist mainly of high density lipoproteins (HDL) (70%) and a protein – phosvitin (16%) – linked by phosphocalcic bridges between the phosphate groups of their phosphoseryl residues (Burley and Cook, 1961; Saari et al., 1964). Some low density lipoproteins (LDL: 12%) are included in the granular structure (Table 16.2). Recently Mann and Mann (2008) identified 119 proteins from yolk, of which 86 had not been previously identified, and estimated their distribution between plasma and granules. At low ionic strength, granules mainly form insoluble HDL–phosvitin aggregates linked by phosphocalcic bridges as HDL and phosvitin contain a high proportion of phosphoserin amino acids able to bind calcium (Causeret et al., 1991). The numerous phosphocalcic bridges make the granule structure very compact, poorly hydrated, weakly accessible to enzymes, and lead to an efficient protection against thermal denaturation and heat gelation. Yolk
Granules (HDL + phosvitin) LDL
10,000g/30 min
Plasma
Granules
Fig. 16.1 Fractionation of plasma and granules from hen egg yolk (LDL, low density lipoprotein; HDL, high density lipoprotein).
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324 Improving the safety and quality of eggs and egg products Table 16.2 Repartition of hen egg yolk constituents
Yolk
DM
lipids
Yolk
proteins
% of lipids
% of proteins
100
100
100
64
32
Plasma LDL livetins others
78 66 10 2
93 61 – –
53 22 30 1
73 88 – –
25 10 96 90
Granules HDL phosvitin LDLg
22 16 4 2
7 6 – 1
47 35 11 1
31 24 – 88
64 75 95 10
Source: Powrie and Nakai (1986) DM, dry matter.
At an ionic strength over 0.3 M NaCl, the phosphocalcic bridges are disrupted because monovalent sodium replaces divalent calcium. In such conditions, the solubility of granules reaches 80% because phosvitin is a soluble protein and HDL behave like soluble proteins (Cook and Martin, 1969; Anton and Gandemer, 1997). Complete disruption of granules occurs when ionic strength reaches 1.71 M NaCl. Acidification or alkalinization similarly causes the disruption of granules and the solubilization of these constituents by increasing the number of the positive (NH3+) or negative (COO–) charges, inducing electrostatic repulsions between granule constituents. A phase diagram drawing the different states of granules as function of pH and ionic strength was established by Sirvente (2007) (Fig. 16.2). Plasma comprises 78% of yolk dry matter and is composed of 85% LDL and 15% livetins (Burley and Cook, 1961; Table 16.2). It forms the aqueous phase where yolk particles are in suspension. It accounts for about 90% of yolk lipids (including nearly all the carotenoids), and 50% of yolk proteins. Plasma contains about 73% lipids, 25% proteins and 2% ash. Lipids of plasma are distributed thus: 70% triglycerides, 25% phospholipids, and 5% cholesterol. LDL are spherical particles (17–60 nm in diameter with a mean of about 35 nm) with a lipid core in a liquid state (triglycerides and cholesterol esters) surrounded by a monofilm of phospholipid and protein (Cook and Martin, 1969; Evans et al., 1973). LDL are soluble in aqueous solution (whatever the pH and ionic conditions) due to their low density (0.982) and small size. Phospholipids take an essential part in the stability of the LDL structure because association forces are essentially hydrophobic (Burley, 1975). Some cholesterol is included in the phospholipid film, increasing its rigidity. LDL are composed of 11–17% protein and 83–89% lipid, out of which latter 74% is neutral lipid and 26% phospholipid (Martin et al., 1964).
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Bioactive fractions of eggs for human and animal health 325
0.75 M Soluble micelles (100–200 nm) Intermediate: micelles (100–200 nm) + aggregates (1–8 µm) NaCl
0.1 M
Insoluble aggregates (1–8 µm) 4
pH
7
Fig. 16.2 Physical state of granules as function of pH and ionic strength. Table 16.3 Composition of hen egg white
% of hen egg white
Water Lipids Proteins Carbohydrates Minerals
88 – 10.6 0.8 0.6
Source: Thapon and Bourgeois (1994).
16.2.2 Egg white Egg white represents about 60% of the total egg weight. It consists of an aqueous protein solution, containing few minerals and carbohydrates (Table 16.3). Proteins represent more than 90% of the dry matter of egg white, but until very recently, only the major ones had been identified. However, the recent and powerful techniques for separation and analysis have enabled
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326 Improving the safety and quality of eggs and egg products the identification of many minor proteins (Table 16.4) (Guérin-Dubiard et al., 2006; Mann, 2007). The egg white proteins are predominantly globular proteins, and acidic or neutral, except for lysozyme and avidin, which are highly alkaline proteins. All are glycosylated, excepting cystatin and the major form of lysozyme. Some of them are very heat-sensitive and/or sensitive to surface denaturation, explaining their noteworthy functional properties. The major egg white protein (more than 50% of the total proteins) is ovalbumin, a 45 kDa globular and phosphorylated protein. Half of its amino acids are hydrophobic, and one-third are electrically charged, essentially negatively at physiologic pH. Ovalbumin possess six buried Cys residues, two being involved in a disulfide bridge (Cys73–Cys120). Ovalbumin is then the principal egg white protein with free thiol groups, capable of inducing some rearrangements with variations of storage conditions, pH and surface denaturation. Ovotransferrin (13% of total proteins) molecular weight is around 78 kDa. This protein consists of two lobes, each containing a specific binding site for iron (or copper, zinc, aluminum) (Kurakawa et al., 1995). It is the most heat-sensitive egg white protein, but the complexation of iron or aluminium significantly increases its heat stability (Lin et al., 1994). Ovomucoïde is a highly glycosylated protein (up to 25% carbohydrates, w/w) of 28 kDa. At pH 7, its denaturation temperature is around 77 °C, but Table 16.4 Composition and some physico-chemical and functional properties of egg white proteins Protein
%
MW (kDa)
pl
Major biological properites
Ovalbumin 54 45 5 Immunogenic phosphoprotein Ovalbumin Y 5 44 5.2 nd Ovalbumin X 0.5 56 6.5 nd Ovotransferrin 13 76 6.7 Iron binding, bacteriostatic activity Ovomucoid 11 28 4.8 Trypsin inhibitor Ovomucin 1.5–3.5 230–8300 4.5–5 Highly glycosylated, viral hemaglutination inhibition Lysozyme 3.5 14.4 10.7 Lysis of Gram+bacteria wall Ovoinhibitor 0.1–1.5 49 5.1 Serine protease inhibitor Ovoglycoprotein 0.5–1 24.4 3.9 nd Flavoprotein 0.8 32 4 Riboflavin (vitamin B2) binding Ovostatin 0.5 760–900 4.6 Serine protease inhibitor Cystatin 0.05 12.7 5.1 Cysteine protease inhibitor Avidin 0.05 68.3 10 Biotine binding Ex-FABP nd 18 5.5 Lipocaline family Cal gamma nd 20.8 6 Lipocaline family TENP nd 47.4 5.6 BPI (bactericidal permeability increasing protein) family Hep 21 nd 18 6.4 uPar/Ly6/Snake neurotoxin family Source: Li-Chan and Nakai (1989), Stevens (1991), Guérin-Dubiard et al. (2006).
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Bioactive fractions of eggs for human and animal health 327 this protein is much more heat resistant at acidic pH. Ovomucin is also a highly glycosylated protein, with a very high molecular weight (104 kDa). Electrostatic interactions can be observed between ovomucin and some of the other egg white proteins. In freshly laid eggs (pH 7.5), the carboxylic groups of the ovomucin sialic acids especially interact with the e-NH3+ of lysozyme lysine residues to form a lysozyme–ovomucin complex that should be responsible of the gel-like structure of egg white (Kato et al., 1975). Lysozyme is a small (14 kDa) globular, and strongly basic protein. Its structure is very rigid, stabilized by four disulfide bridges. The glucidic fraction of egg white consists of free glucose (0.5% w/w) and carbohydrates linked to proteins (0.5% w/w). Mineral fraction is predominantly composed by Na+, K+ and Cl–, as free minerals, whereas P and S are essentially constitutive elements of proteins. Egg white also contains CO2, in equilibrium with bicarbonate, which plays a major role in pH control (Thapon, 1994). 16.2.3 Eggshell Eggshell, made up from an organic matrix including membranes and some constituents surrounded by a layer of calcium carbonate (95%), represents about 4% of the total egg weight. Many reviews describe the polycrystalline organization throughout the calcified shell (Hamilton, 1986; Tullet, 1987; Nys et al., 1999, 2001, 2004; Gautron and Nys, 2007). The outer membrane is about 50 mm thick and is located between the inner membrane (in contact with the albumen) and the calcified part of the shell (70 mm). The most external layer is the cuticle. It is a 10 mm organic layer that contains the majority of the eggshell pigments (Nys et al., 1991) and a thin film of hydroxyapatite crystals in its inner part (Dennis et al., 1996). All the four different layers are important to operate as a physical barrier against microorganism penetration. The eggshell matrix is a mix of proteins (70%) and polysaccharides (11%) (Gautron and Nys, 2007). Since 1990, numerous studies have been focused on the identification of these proteins using combined biochemical and molecular biology methods, in relation with genomic tools for chicken genome identification. Globally three groups of proteins are identified: egg white proteins (ovalbumin, lysozyme, ovotransferrin), ubiquitous proteins (osteopontin and clusterin), and specific proteins (ovocleidin-17 and 116, and ovocalyxin-32, -36, -25 and -21) (Gautron and Nys, 2007).
16.3 Antibody applications The antibody activity of egg is due to g-livetins, named IgY: yolk immunoglobulin (Leslie and Clem, 1969) specifically contained in yolk. IgY
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328 Improving the safety and quality of eggs and egg products are synthesized in the hen serum and then transferred into yolk to immunize the embryonic chicken. g-livetin is a glycoprotein that contains heavy chains (60–70 kDa) and light chains (22 kDa) and represents about 3% of yolk dry matter (Fig. 16.3). The IgY technology is used for production of polyclonal antibody in chickens (Schade et al., 1996; Schade and Schwägele, 2004). One hen generally lays about 290 eggs per year, which represents 5000 g of yolk. The level of IgY in yolk ranges from 9 mg/ml to 25 mg/ml (Rose et al., 1974). Consequently, one hen can produce about 60 g of IgY per year (Nakai et al., 1994). Since this production is a non-sacrificial method, it belongs to the so-called alternative methods and thus follows a principal European guideline concerning animal protection. The production of IgY by hens is 30 times greater than that by rabbits. Egg yolk is then a potential source of antibodies for humans due to this possibility of large-scale fabrication. Furthermore, various types of IgY can be produced in large quantities in egg yolk by immunizing hens with specific antigens: proteins, bacteria, viruses, parasites, toxins. After yolk recovery, IgY must be extracted and isolated by conventional successive methods (salt centrifugation and affinity and/ or ion exchange chromatography). Consequently hen antibody production is a good alternative to conventional mammal antibody production because it respects the welfare of animals due to the non-invasive method of egg collection and because of the fast and simple subsequent IgY isolation from egg yolk (Tini et al., 2002). This important quantity of available IgY authorizes new fields of applications, such as immunotherapy and immunoprophylaxis applied to several viral and bacterial infections in veterinary and human medicine. In addition, IgY has no cross-reactivity with rheumatoid factors (Larsson et al., 1991) or human anti-mouse antibody (Carlander et al., 1999), and is unable to activate the mammalian complement system (Larsson et al., 1992). Furthermore, if chickens and rabbits are immunized with the same mammalian antigen, chickens often respond with an antibody specificity that can rarely be achieved in rabbits, as for instance, PIIINP (Gerl et al., 1996),
lgY Cv1 VH
Cv3 Cv4 CL
VL
Fig. 16.3 Schematic structure of IgY (IgY: yolk immunoglobulin; VH: heavy chain variable region; VL: low /**/chain variable region/*; CL: low chain variable region; Cv1: constant region 1; Cv3: constant region 3; Cv4: constant region 4).
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Bioactive fractions of eggs for human and animal health 329 parathyroid hormone-related protein, transforming growth factor b3 (TGF-b3; Danielpour and Roberts, 1995), and YKL-40 glycoprotein (De Ceuninck et al., 2001). The advantages of using chicken IgY have been recognized by many authors (Lösch et al., 1986; Carlander, 2002; Narat, 2003; Karlsson et al., 2004). Numerous publications have described the successful use of IgY in a variety of research fields (for a review, see Schade and Chacana, 2007). IgY-based immunoassays are being used to measure the concentration of proteins or peptides via enzyme-linkes immunosorbent assays (ELISAs), radii immunoassays (RIAs) or other assays in clinical chemistry and basic research. IgY are successfully used in immunohistochemistry for detection of antigens of viral, bacterial, plant and animal origin, to assess the incidence of intestinal parasites in domestic animals (Schniering et al., 1996) and the contamination of foods with toxins or drugs (Pichler et al., 1998). During the past decade, IgY antibodies have increasingly been used in therapy or prophylaxis of disease. Numerous applications of hen IgY antibodies have also been evaluated in humans against dental plaque formation or against rotavirus diarrhea in trout, to protect against Yersinia rucckeri, or in swine to control bacterial respiratory disease (Shin et al., 2001). Furthermore, hen IgY antibodies against specific proteins can be raised for biological quantification such as detection by ELISA of fungi.
16.4 Bioactive properties of eggs 16.4.1 Antibacterial activities An egg has to protect an embryonic chicken against microorganisms during the period of incubation. Some physical barriers exist such as eggshell and albumen but chemical protections are also present and are materialized by many antibacterial compounds. Some of these compounds have been well studied and are already being used as preservatives; some others are the subject of advanced research and represent promising leads for the treatment of various diseases (Baron and Réhault, 2007, review). Among egg white proteins, lysozyme and ovotransferrin have been the most studied for this type of application. However there are some results describing that the administration of non-immunized egg yolk powder can eliminate and prevent Salmonella colonization in laying hens with no adverse effects (Kassaify and Mine, 2004). Lysozyme from egg albumen catalyzes the hydrolysis of the glycosidic ß-(1–4) linkage between N-acetylmuramic acid and N-acetylglucosamine of the bacterial peptidoglycan, which is the structural component of Grampositive bacterial cell walls (Fig. 16.4). The peptidoglycan can be thought to be a strong, woven mesh that allows solutes to pass through but maintains the cell shape and protects cell from osmotic lysis. Without a wall or if it is © Woodhead Publishing Limited, 2011
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330 Improving the safety and quality of eggs and egg products
Fig. 16.4 Effect of lysozyme on Gram-positive cell walls.
attacked by lysozyme the cell would swell and burst. Cell death occurs by the lytic action of lysozyme on peptidoglycan only when in low osmotic-strength media, or when the rate of the synthesis and polymerization process for new peptidoglycan formation is slower than the lysozyme catalyzed degradation. Gram-negative bacteria are resistant to the lytic action of lysozyme because they possess an additional outer membrane. Their cell wall includes the outer membrane, lipoproteins, lipopolysaccharides and peptidoglycan layers, protected from the lytic action of lysozyme because the outermost surface acts as a permeability barrier. Lysozyme is particularly effective against oral affections, due to the cariogenic bacteria Streptococcus mutans or against periodontitis-associated bacteria (Tenovuo et al., 1991). Given orally, lysozyme alleviates the reduced endogenous production of saliva lysozyme during parodontopathy (Sava, 1996). Lysozyme is also used extensively as a food preservative. It shows high activity against mesophilic and thermophilic spore-forming bacteria such as Bacillus staerothermophilus, Clostridium thermosaccharolyticum and Clostridium tyrobutyricum (Johnson, 1994). It is used in cheese to
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Bioactive fractions of eggs for human and animal health 331 prevent contamination because it does not inhibit on starter and secondary cultures required for the ripening of cheeses. Lysozyme also prevents the growth of pathogenic bacteria on refrigerated foods: Listeria monocytogenes, Clostridium botulinum, Clostridium jejuni and Yersinia enterocolitica are susceptible to the lytic activity of lysozyme (Johnson, 1994). Given orally, after local application, or when combined with specific immunotherapy, lysozyme is effective in a wide range of viral skin diseases: herpetic lesions, verruca vulgaris and plantaris, aphtous stomatitis, polymorphous exudative erythema, molluscum contagiosum (De Douder and Morias, 1974; Asakura et al., 1990; Gavrilenko et al., 1992). Moreover, when combined with specific immunotherapy, lysozyme cures viral sinusitis or bronchitis (Gavrilenko et al., 1992). The antiviral action of lysozyme has partly been explained by its role on the precipitation of viral particles and by its immune-enhancing action on the host together with its interaction with the pathogens (Sava, 1996). Kijowski and Lesnierowski (1999) have proposed some protocols for thermal and/or chemical modification of the structure of lysozyme to increase its antiviral activity. Ovotransferrin from egg albumen inhibits Gram-negative bacteria by depriving bacteria of iron that is essential for their growth (Lock and Board, 1992; Baron et al., 1997). Iron participates in many major biological processes and some bacteria deposit intracellular reserves that can then be used to enhance growth when external iron supplies are restricted. Ovotransferrin belongs to the family of transferrins, a metal-binding transport protein family with an in vivo preference for iron, and widely distributed in physiological fluids. Valenti et al. (1986) proposed that ovotransferrin antimicrobial activity can result from a direct effect on the membranes: interaction of the cationic ovotransferrin with the anionic outer membrane of Gram-negative bacteria. Valenti et al. (1983, 1986) and Baron (1998) suggested that ovotransferrin antibacterial activity can result from a direct effect on the membranes of Candida albicans or Salmonella Enteritidis. This hypothesis was confirmed for OTAP-92, a cationic peptidic fragment of hen ovotransferrin which possesses a unique structural moiety similar to insect defensins. This domain is able to cross the bacterial membrane by a self-promoted uptake and working in synergy with the polycationic nature of OTAP-92, kills bacteria by damaging the biological function of its cytoplasmic membrane (Ibrahim et al., 2000). Its strong bactericidal activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains led the authors to envisage therapeutical applications for this natural peptide. More recently Aguilera et al. (2003) showed that transferrins are able to permeate the outer membrane of E. coli and to access the inner membrane where they cause permeation of ions in a selective manner. Ovotransferrin has therefore been proposed for infant nutrition and for the treatment of infants with acute diarrheas (del Giacco et al., 1985). Recently, the iron chelating activity of ovotransferrin has been shown to increase the stimulation (by inhibition of AMPc b-lactamase) (Syn 2190) of some
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332 Improving the safety and quality of eggs and egg products antibiotics which are efficient against most b-lactamase-producing bacteria (Babini and Livermore, 2000). Ovotransferrin therefore appears to be a key factor for drug associations able to overcome cephalosporin resistance. Interestingly, some proteins initially known for their contribution to the mechanical and microstructural properties of the shell also have antibacterial activities (Nys et al., 2004). These proteins are specifically and sequentially expressed in the uterus and are currently the subject of investigation. Furthermore other proteins belonging to egg white and possessing antiprotease activities could be involved in the antibacterial defence of eggs mainly by inhibiting bacterial proteases that are secreted by pathogens during host colonization process (Baron and Réhault, 2007). Among these proteins we can cite ovostatin, ovomucoid and cystatin. Finally other proteins binding vitamins such as riboflavin-binding protein, avidin and thiamin binding protein have antimicrobial properties by chelating riboflavin, biotin and thiamin respectively. All these proteins can have a synergistic effect on microbial development and strategies to purify them individually have to be linked for best efficiency for use in pharmaceutical, cosmetic or food industries. 16.4.2 Antihypertensive activities Eggs are generally recognized as a very important source of components possessing blood pressure lowering effects (López-Fandiño et al., 2007). In this area, a wide range of peptides derived from food proteins are useful in the prevention and/or treatment of hypertension (Li et al., 2004). Most of these peptides acts as inhibitors of angiotensin converting enzyme (ACE), an exopeptidase that cleaves dipeptides from the C-terminal side of various oligopeptides. ACE hydrolyzes angiotensin I to the potent vasoconstrictor angiotensin II. ACE also takes part in the kinin–kallikrein system, as it hydrolyzes bradykinin, which has a vasodilator action (Fitzgerald et al., 2004). The most common strategy used for the identification of novel antihypertensive food peptides is based on the preparation of protein hydrolysates and the search for fragments with in vitro ACE inhibitory activity coupled with in vivo tests in spontaneously hypertensive rats (SHR) (Fig. 16.5). The physiological effects of ACE-inhibitory peptides depend on their ability to reach their target sites intact, which may involve survival of gastrointestinal digestion and absorption through the intestinal epithelium to get to the peripheral organs (Vermeirssen et al., 2004). The release of ACEinhibitory peptides upon digestion of food proteins, as well as the resistance to digestion of known ACE-inhibitory sequences, have been tested in several in vitro studies that showed that proteolysis by gastrointestinal enzymes is an essential factor in determining ACE inhibitory activity (Vermeirssen et al., 2003; Gómez-Ruiz et al., 2004). Concerning egg proteins, two peptides derived from ovalbumin are effective in preventing hypertension. The first one, named ovokinin, was © Woodhead Publishing Limited, 2011
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Bioactive fractions of eggs for human and animal health 333 Angiotensin-I
Bradykinin Vasodilator
ACE Angiotensin-II
Bradykinin inactivation
Vasopressor ACE inhibitors (a) Hydrolysate of crude egg albumen with pepsin (3 h)
Nine different peptides < 3000 Da
Ultrafiltration membrane 3000 Da cut-off (b)
Fig. 16.5 (a) Schematic cascade of angiotensin converting enzyme (ACE); (b) fractionationation of lower albumen peptides.
purified from a peptic digest of ovalbumin (residues 358–365). It is a weak bradykinin B1 agonist peptide, which lowers the systolic blood pressure in spontaneously hypertensive rats when administered as an emulsion in egg yolk (Fujita et al., 1995). Maximum inhibition, expressed as the IC50, or concentration required to inhibit 50% of the enzyme activity, was observed after hydrolysis with pepsin (IC50 = 45.3 – 55.3 mg/ml) (Fujita et al., 2000; Miguel et al., 2004). Pepsin also exhibited the highest proteolytic activity towards ovalbumin, although there was not a direct relationship between the level of intact protein and the resultant ACE inhibition. Treatment with trypsin and chymotrypsin did not produce ACE inhibitory peptides after 3 h (Fujita et al., 2000). The second one, ovokinin (2–7) is obtained by chymotryptic digestion of ovalbumin (residues 359–364). This hexapeptide corresponds to the 2–7 fragment of ovokinin (RADHPF) was purified from a chymotrypsin digest of ovalbumin. When orally administered, this vasorelaxing peptide reduces the blood pressure of spontaneously hypertensive rats in a dose-dependent manner. Ovokinin (2–7) has also been used for designing peptide derivatives with activity comparable to that of synthetic anti-hypertensive drugs used clinically. The vasodilation due to ovokinin (2–7) is mediated by nitric oxide and an unknown receptor. Ovokinin (2–7) exerted a dose-dependent vasodilation in mesenteric arteries from hypertensive rates and exhibited hypotensive activities after the oral administration of 10 mg/kg to SHR. Miguel et al. (2004) have obtained and characterized peptides from egg albumen which inhibit the ACE responsible for hypertension. Pepsin is © Woodhead Publishing Limited, 2011
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334 Improving the safety and quality of eggs and egg products very efficient at obtaining active (ACE and antioxidant) peptides (molecular weight lower than 3000 Da) from egg albumen. These peptides significantly diminish the blood pressure of spontaneous hypertensive rats. A very innovative approach consists of selecting peptides in silico by specific software able to handle multiple peptide sequences, to screen for motifs and to quantify the theoretical total activity (Vermeissen et al., 2004). These authors have constructed a database containing more than 500 peptide sequences and their antihypertensive activities. In parallel, digestion simulations, and the inhibitory activity of the digests on ACE are determined in vitro. Strong experimental evidence indicates that oxidative stress and associated oxidative damage are mediators in cardiovascular pathologies and, thus, antioxidant-rich diets significantly reduce the arterial blood pressure in SHR (Rodriguez-Iturbe et al., 2003). The protective effect exerted by ACEinhibitors on the structure and functions of the cardiovascular system, the kidney and the brain also seems to be related to an antioxidant action and a reduced formation of reactive oxygen species (Basso et al., 2005), and it is noteworthy that the hydrolysate of egg white with pepsin and some of its peptides have shown antioxidant effects in vitro. The hydrolysates may be used as such, or after enrichment, in the manufacture of food products. The enrichment of fractions or the isolation of specific peptides from the total peptide mixture is a technological challenge in the production of ACE-inhibitory and antihypertensive peptides. Since a common feature of ACE-inhibitory peptides is their relatively small size, fractionation using ultrafiltration and size exclusion chromatography constitutes a useful step for pre-concentration (Fujita et al., 2001). In fact, when the hydrolysate obtained from crude egg white with pepsin for 3 h (IC50 = 55.3 mg/ml) was filtered through a 3000 Da cut-off membrane, the permeate presented approximately ten times more inhibitory activity than the retentate (IC50 = 34.5 and IC50 = 298.4 mg/ml, respectively) (Miguel et al., 2004). 16.4.3 Antioxidant properties Among egg proteins, two are known as excellent metal ion-binding proteins: ovotransferrin and phosvitin. The first one exists in egg white, the second one is a yolk protein. These binding properties allow biological properties, particularly antioxidant and antibacterial activities (Guérin-Dubiard et al., 2007). Concerning ovotransferrin it has been demonstrated that this protein binds iron very strongly, and in particular Fe(III), since its dissociation constant is 10–24 M (Stevens, 1991) and that it can bind two iron atoms per molecule. The order of iron binding is pH dependent: at pH 6.0 it binds first to the C-domain, whereas at pH 8.5 it first binds to the N-domain. The biological role of ovotransferrin is generally accepted as iron transport but it can bind other metal ions such as aluminum (Mizutani et al., 2005). Few
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Bioactive fractions of eggs for human and animal health 335 studies are concerned with ovotransferrin antioxidant activity although LiChan and Nakai (1989) reported that ovotransferrin iron-binding ability may have an indirect role in prevention of lipid oxidation. Concerning phosvitin, its capacity for metal chelation gives to this protein remarkable antioxidant potentialities. Phosvitin contains about 110 phosphoserine residues (50% of its amino acids), responsible for its iron binding capacity (Itoh et al., 1983). Ninety-five percent of the iron in egg is present in the yolk and all is bound to phosvitin (Greengard et al., 1964). Phosvitin contains 2–3 atoms of iron per molecule when isolated from hen egg, but its potential binding capacity is much higher (about 60 atoms per molecule) (Taborsky, 1983), with a constant of affinity (Ka) of 1018 M (Hegenauer et al., 1979). From a structural point of view, the iron ions are octahedrally coordinated by oxygen atoms of the serine-bound phosphate groups and by other ligands from either the protein or the solvent (Mangani et al., 1994). Phosvitin inhibits phospholipid oxidation catalyzed by Cu2+ and even more by Fe2+. It is efficient up to a Fe2+ to phosvitin ratio of 30:1, whereas this maximal ratio is 1:1 with Cu2+ (Lu and Baker, 1986). However, phosvitin loses its antioxidant capacity when oxidation is catalyzed by heminic iron. It is likely that heminic iron cannot be chelated by phosvitin because it is buried into a porphyrin structure which limits the interaction of iron and phosphoseryl residues of phosvitin. Castellani et al. (2004) have established that the optimal conditions for iron fixation (115 mg iron/mg protein) are at pH 6.5 and NaCl level of 0.15 M. When pH is fixed at 3.5, the chelation of iron is inhibited by protonation of the phosphate groups and by conformational changes in the protein. However, acidification of the solution after iron fixation does not promote the release of iron. Pasteurization does not affect the iron-binding capacity of phosvitin, or its antioxidant property (Lu and Baker, 1986). Furthermore, heating phosvitin at 110 °C for 20 and 40 min does not cause the release of iron bound to phosvitin. Consequently, phosvitin could be a very important natural food antioxidant. Lipid oxidation is of great concern to food industry because of the many undesirable flavors, colors and potentially toxic reaction products in many foodstuffs. It is essential to limit oxidation in food products, and this can be done by using antioxidants. Synthetic antioxidants exist, but the demand for natural molecules has recently increased. Phosvitin, with its strong metal ion-binding capacity, had already been tested as natural antioxidant. Lu and Baker (1986) reported that this protein can inhibit Fe2+-catalyzed phospholipid oxidation. Because food can have a wide range of pH varying from 2.5 to 9, Lu and Baker (1987) studied the effect of pH on phosvitin effectiveness as an antioxidant. The ionization of the phosphate group in phosvitin is affected by the pH of environment, and this could modify its conformation (Taborsky, 1974), and indeed its metal binding characteristic and so its antioxidant activity. Lu and Baker (1987) confirmed that at pH 6.1, phosvitin inhibits catalytic activity of Fe2+ but also the one of Cu2+. On
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336 Improving the safety and quality of eggs and egg products the other hand, since iron does not demonstrate any catalytic activity at pH 7.8, this is in accordance with Castellani et al. (2004), who reported that phosvitin still strongly binds iron at pH 7.1 at low ionic strength (0.15 M) (Fig. 16.6). Lu and Baker (1987) reported that copper conserve a catalysis activity at this alkaline pH. They followed copper capacity binding of phosvitin with increasing pH, and observed a considerable amount of free copper at pH 7.8, so they suggested that unbound copper could still catalyze lipid oxidation. Moreover, antioxidant activity of phosvitin can be improved by chemical modification. Nakamura et al. (1998) conjugated phosvitin with galactomannan through a controlled Maillard reaction at 60 °C in 79% relative humidity for one week. The conjugation reaction significantly enhanced the antioxidant activity of phosvitin and even improved emulsifying activity, emulsion and heat stability. Lastly, it is important to test phosvitin antioxidant activity under different pH but also under the various temperature and NaCl conditions expected in processed food. Lee et al. (2002) reported that phosvitin is capable of inhibiting lipid oxidation in phosphatidylcholine liposomes, muscle homogenates and ground pork under the pH, temperature and NaCl conditions expected in processed muscle foods. However, it is not able to completely inhibit lipid oxidation in salted uncooked and cooked ground pork; in these cases, it is necessary to use phosvitin in combination with other antioxidant technologies to effectively increase the shelf-life of muscle foods.
16.5 Cryoprotective activity For many years, hen egg yolk has been routinely used in media called extenders to cryopreserve the semen of domestic animals, as cows, sheep, Bound iron (µg/mg of phosvitin) 180 150 120 90 60 30 0
0
0.2
0.4 0.6 Ionic strength (M)
0.8 2.4
3.7
5
6.3
7.6
pH
Fig. 16.6 Iron fixation by phosvitin versus pH and ionic strength (M) values. The estimated response surface is represented in mg of bound iron per mg of phosvitin (from Castellani et al., 2004).
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Bioactive fractions of eggs for human and animal health 337 goats, dogs, horses, donkeys, rabbits, fishes, and wild animals (see BriandAmirat et al., 2007, for a review). The goal of any sperm conservation is the production of a bank of sperm cells to be used for artificial insemination (AI). However, various biochemical and anatomical compartments in the sperm cells (acrosome, nucleus, mitochondria, axoneme, plasma membrane) may be altered during freezing and thawing; known as cold shock. In practice, the freeze–thawing process results in various injuries to spermatozoa, either by the formation of intracellular ice crystals or by increasing the intracellular concentration of solutes while dehydration of cells occurs. Consequently, the first aim of sperm freezing protocols, including the use of extenders, is to prevent lethal intracellular ice crystal formation and to reduce spermatozoa damage during and after cryopreservation. After ejaculation, spermatozoa are rapidly mixed with a cryoprotective medium called extender before freezing or cooling. Yolk is used in semen dilutions in association with other components as glycerol, glutamine, Tris, citric acid and fructose. It is generally used at the concentration of 20% (w/v) in classical extenders (for example INRA 82 preservation medium). These last years, there have been increasing demands to replace whole egg yolk in semen extenders because the presence of some substances in yolk could inhibit respiration of spermatozoa, diminish their motility, introduce a possible sanitary risk and interfer with biochemical assays and metabolic investigations (Soderquist et al., 1991; Andersson et al., 1992). Consequently, it could be of great benefit to remove these detrimental substances in yolk, and add only the cryoprotectant agent of yolk, after extraction, to the extender, rather than complete egg yolk. This implies the need for a better understanding of the cryoprotection mechanisms and the ability to determine and isolate the constituents in hen egg yolk that provide cryoprotection. It has been proposed that the low-density fraction of yolk, mainly composed of LDL, could be responsible for the resistance against cold shock and for the improvement of motility of spermatozoa after storage. Foulkes (1977) and Graham and Foote (1987) suggested that LDL could adhere to cell membranes during the freeze–thaw process, thus preserving spermatozoa membranes. However, the respective roles of the protein and lipid components of LDL in interactions with the spermatozoa membrane have not been clearly established. Graham and Foote (1987) thought that phospholipids are implicated by merging with spermatozoa membranes and replacing some lipids, thus decreasing their phase transition temperature. Presently, questions exist about the respective role of lipids and apoproteins on the cryoprotective action of LDL. Semen motility was evaluated after freezing in a LDL solution in comparison with bull semen frozen in egg yolk extender. Semen motility was twofold higher after freezing with LDL than after freezing with egg yolk (Moussa et al., 2002; Amirat et al., 2004). Consequently, LDL could easily replace egg yolk in extenders for bull semen freezing. Different concentrations of LDL
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338 Improving the safety and quality of eggs and egg products were tested and 8% (w/v) was the concentration of LDL that offers the best protection for spermatozoa (Moussa et al., 2002). Over this concentration, a decrease in semen motility was observed. This could be related to the osmotic pressure of the extender which declined when the concentration of LDL increased. The effect of LDL on semen fertility has been also evaluated. The ability of spermatozoa to fertilize after cryopreservation is an important factor of high pregnancy rates in cattle after insemination (De Leeuw et al., 1993). Furthermore no correlation has been established between motility and fertility of mammalian semen. Consequently the measure of spermatozoa motility is an insufficient predictor of in vitro and in vivo fertility in many species (Comizzoli et al., 2001). Parameters such as in vitro and in vivo fertility could be associated with motility results. Amirat et al. (2004) have evaluated in vitro bull semen fertility after the freeze–thaw process with LDL compared with whole egg yolk extender (Fig. 16.7). The experiments showed that bull semen fertility was preserved after freezing in LDL extender. A high number of functional spermatozoa were available for artificial insemination, the number of spermatozoa in the straws were reduced and the number of straws available for sale was increased. Amirat et al. (2005) compared the morphological modifications induced to spermatozoa by egg yolk and LDL extenders after cooling to 4 °C and after freezing at –196°C. Alterations were visualized by electron microscopy. The ultrastructure of frozen spermatozoa were observed by electron microscopy of cryofixed cryosubstituted samples. The yolk extender was more deleterious for bull spermatozoa than LDL. The Spermatozoa 100
Acrosome lysis AR % Rupture PM %
In vitro fertilization 100
75
75
50
50
25
25
0
0 Control triladyl biociphos LDL
Control triladyl biociphos LDL
Fig. 16.7 Efficiency of LDL for spermatozoa defaults after freeze–thawing (acrosome lysis, acrosome reaction (AR%), rupture of plasma membrane (PM%) and for in vitro fertilization, in comparison with commercial extenders Triladyl (containing egg yolk) and Biociphos (containing soybean lecithin); control is without freeze– thawing (from Amirat et al., 2004).
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Bioactive fractions of eggs for human and animal health 339 electron microscopy indicated that the granular components of the egg yolk were rapidly adsorbed by the plasma membranes, first on the sperm heads, then on the flagella. The acrosomes were modified and destroyed. This could result from the intrusion of calcium which is present in high concentration in egg yolk (and rapidly enters the cells when the temperature is below 30 °C; Courtens et al., 1989). LDL were less aggressive to cells, there were no alterations of the plasma membranes and very few acrosome disruptions. The use of LDL for other species has now been intensively evaluated, particularly for horses (Khlifaoui, 2004) and dogs (Bencharif et al., 2006) in view of proposing new commercial extenders that are not presently available commercially for species presenting some difficulties to sperm cryoprotection. Hen egg yolk LDL could easily replace egg yolk in semen extenders as regards to all the advantages listed in this review. Moreover, it can be envisaged as extending the use of LDL in other cells such as embryos, tissues, etc. Moussa et al. (2002) developed an easy method to extract LDL from egg yolk. This extraction method reaches 97% purity and about 67% yield, and is easily reproducible on an industrial scale. This purified LDL had an improved efficiency on the spermatozoa motility and survival compared with commercial extenders when present at 8% in the extender for bull semen. A patent concerning the extraction of LDL and the constitution of a new extender for bovine spermatozoa has been deposited (Anton et al., 2001). Presently, other extenders for stallion semen are also developed using plasma from yolk (containing two-thirds of LDL) or phospholipids extracted from yolk and re-organized in liposomes to biomimic natural LDL (Pillet, 2009).
16.6 Conclusions Hen egg yolk is a powerful source of bioactive compounds due to its role of nutrition and protection of the embryonic bird. All these compounds are distributed in shell, albumen and yolk and can deliver many biological activities. Among them we have selected immune, antibacterial, antihypertensive, antioxidant and cryoprotective activities. These activities exist naturally in egg and can be maintained, after compound extraction and/or processes of food or product preparation, if we possess a sufficient knowledge of the molecules. This indicates that fundamental research on egg (minor or major in weight) molecules must be maintained and reinforced. An important question remains concerning the strategies of use of these compounds for the different biological applications: do we have to separate and use isolated and purified compounds or do we have to add some egg part obtained from mild extraction? The response depends on the activity and on the type of applications and all this area is presently in a phase of industrial development after many years of fundamental research in laboratories. A round trip © Woodhead Publishing Limited, 2011
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340 Improving the safety and quality of eggs and egg products between industrial and laboratory research is essential to exploit all these potentialities completely.
16.7 References aguilera o, quiros lm
& fierro jf (2003), ‘Transferrins selectively cause ion efflux through bacterial and artificial membranes’, FEBS Letters, 548, 5–10. amirat l, tainturier d, jeanneau l, thorin c, gérard o, courtens jl & anton m (2004), ‘Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl, a commercial egg yolk extender’, Theriogenology, 61(5), 895–907. amirat l, anton m, tainturier d, chatagnon g, battut i & courtens jl (2005), ‘Modifications of bull spermatozoa induced by three extenders: Biociphos, low density lipoproteins and Triladyl, before, during and after freezing and thawing’, Reproduction, 129, 535–543. andersson m, hellman t, holmstrom bg & jokinen l (1992), ‘Computerized and subjective assessments of post thaw motility of semen from Finnish Ayrshire AI bulls in relation to non return rates’, Acta Veterinaria Scandinavia, 33(1), 89–93. anton m & gandemer g (1997), ‘Composition, solubility and emulsifying properties of granules and plasma of hen egg yolk’, Journal of Food Science, 62(3), 484–487. anton m, martinet v, tainturier d & moussa m (2001), ‘Procédé de production d’une fraction purifiée de lipoprotéines de faible densité du jaune d’œuf et milieu de conservation incorporant de telles lipoprotéines’, French patent no. 0100292. asakura k, kojima t, shirasaki h & kataura a (1990), ‘Evaluation of the effects of antigen specific immunotherapy on chronic sinusitis in children with allergy’, Auris Nasus Larynx, 17, 33–38. babini gs & livermore dm (2000), ‘Effect of conalbumin on the activity of Syn2190, a 1,5 dihydroxy-4-pyridon monobactam inhibitor of AmpC b-lactamases’, Journal of Antimicrobial Chemotherapy, 45, 105–109. baron f (1998), ‘Etude du comportement de Salmonella enteritidis dans le blanc d’œuf’, PhD thesis, ENSA Rennes, France. baron f & réhault s (2007), ‘Compounds with antibacterial activity’, in Bioactive Egg Compounds, R. Huopalathi, R. Lopez-Fandino, M. Anton and R. Schade (Eds.), Springer, Berlin, Germany, pp 191–198. baron f, gautier m & brule g (1997), ‘Factors involved in the inhibition of growth of Salmonella enteritidis in liquid egg white’, Journal of Food Proteins, 60, 1318– 1323. basso n, paglia n, stella i, de cavanagh emv, ferder l, arnaiz mdl & inserra f (2005), ‘Protective effect of the inhibition of the renin–angiotensin system on aging’, Regulation of Peptides, 128, 247–252. bencharif d, tainturier d, briand-amirat l, becavin s & barrière jp (2006), ‘Development of a new diluent for freezing dog semen: preliminary results’, Abstract EVSSAR, 7–9 April 2006 Budapest, Hungary. briand-amirat l, tainturier d & anton m (2007), ‘Use of egg compounds for cryoprotection of spermatozoa’, in Bioactive Egg Compounds, R. Huopalathi, R. Lopez-Fandino, M. Anton and R. Schade (Eds.), Springer, Berlin, Germany, pp 259–264. burley rw (1975), ‘Recent advances in the chemistry of egg yolk’, CSIRO Food Res Quarterly, 35, 1–5. burley rw & cook wh (1961), ‘Isolation and composition of avian egg yolk granules and their constituents a- and b-lipovitellins’, Canadian Journal of Biochemistry and Physiology, 39, 1295–1307. carlander d (2002), ‘Avian IgY antibody. In vitro and in vivo. Acta Universitatis
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Bioactive fractions of eggs for human and animal health 341 Upsaliensis’, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1119, Uppsala Sweden, University of Uppsala, pp 1–153. carlander d, stalberg j & larsson a (1999), ‘Chicken antibodies: a clinical chemistry perspective’, Uppsala Journal of Medical Science, 104(3), 179–189. castellani o , guérin - dubiard c , david - briand e & anton m (2004), ‘Influence of physicochemical conditions and technological treatments on the iron binding capacity of egg yolk phosvitin’, Food Chemistry, 85, 569–577. causeret d, matringe e & lorient d (1991), ‘Ionic strength and pH effects on composition and microstructure of yolk granules’, Journal of Food Science, 56, 1532–1536. chang cm, powrie wd & fennema o (1977), ‘Microstructure of egg yolk’, Journal of Food Science, 42, 1193–1200. comizzoli p, mauget r & mermillod p (2001), ‘Assessment of in vitro fertility of deer spermatozoa by heterologous IVF with zona-free bovine oocytes’, Theriogenology, 56(2), 261–274. cook wh & martin wg (1969), ‘Egg lipoproteins’, in Structural and Functional Aspects of Lipoproteins in Living Systems, E. Tria and A.M. Scan (Eds), Academic Press, New York. courtens jl, ekwall h, paquignon m & plöen l (1989), ‘Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing’, Journal of Reproduction and Fertility, 87(2), 613–626. danielpour d & roberts ab (1995), ‘Specific and sensitive quantitation of transforming growth factor b3 by sandwich enzyme-linked immunosorbent assay’, Journal of Immunological Methods, 180, 265–272. de ceuninck f, pastoureau p, agnellet s, bonnet j & vanhoutte pm (2001), ‘Development of an enzyme-linked immunoassay for the quantification of YKL-40 (cartilage gp39) in guinea pig serum using hen egg yolk antibodies’, Journal of Immunological Methods, 152, 153–161. de douder c & morias j (1974), ‘On lysozyme therapy. II. Topical treatment of viral and non viral cutaneous lesions with lysozyme ointment’, Medickon, 3, 21–22. de leeuw fe, de leeuw am, den daas jhg, collenbrander b & verkleij aj (1993), ‘Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling and freezing’, Cryobiology, 30(1), 32–44. del giacco gs, leone al & ferlazzo a (1985), ‘Total IgE in newborns treated prophylactically with conalbumin’, International Journal of Tissue Reactivity, 7, 535–537. dennis je, xiao sq, agarval m, fink dj, heuer ah & caplan ai (1996), ‘Microstructure of matrix and mineral components of eggshells from white leghorn chicken (Gallus gallus)’, Journal of Morphology, 228, 287–306. dyer-hurdon jn & nnanna ia (1993), ‘Cholesterol content and functionality of plasma and granules fractionated from egg yolk’, Journal of Food Science, 58, 1277–1281. evans rj, bauer dh, bandemer sl, vaghefi sb & flegel cj (1973), ‘Structure of egg yolk very low density lipoprotein: polydispersity of the very low density lipoprotein and the role of lipovitellenin in the structure’, Archives of Biochemistry and Biophysics, 154, 493–500. fitzgerald rj, murray ba & walsh gj (2004), ‘Hypotensive peptides from milk proteins’, Journal of Nutrition, 134, 980S–988S. foulkes ja (1977), ‘The separation of lipoprotein from egg yolk and their effect on the motility and integrity of bovine spermatozoa’, Journal of Reproduction and Fertility, 49, 277–284. fujita h, sasaki r & yoshikawa m (1995), ‘Potentiation of the antihypertensive activity of orally administered ovokinin, a vasorelaxing peptide derived from ovalbumin, by emulsification in egg phosphatidylcholine’, Bioscience Biotechnology Biochemistry, 59, 2344–2345. fujita h, yokoyama k & yoshikawa m (2000), ‘Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins’, Journal of Food Science, 65, 564–569. © Woodhead Publishing Limited, 2011
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342 Improving the safety and quality of eggs and egg products fujita h, yamagami t & ohshima k (2001), ‘Effects of an ACE-inhibitory agent, katsuobushi
oligopeptide, in the spontaneously hypertensive rat and in borderline and mildly hypertensive subjects’, Nutrition Research, 21, 1149–1158. gautron j & nys y (2007), ‘Eggshell matrix proteins’, in Bioactive Egg Compounds, R. Huopalathi, R. Lopez-Fandino, M. Anton and R. Schade (Eds.), Springer, Berlin, Germany, pp 103–108. gavrilenko ti, siurin sa, lolaeva lt & sauchenko vm (1992), ‘The characteristics of lysozyme and carbenicillin action on the clinico-immunological status of patients with chronic bronchitis’, Vrachebnoe Delo, 8, 42–45. gerl m, steinert c, quint m, schade r & günzler v (1996), ‘Immunisation of chickens with the aminoterminal propeptide of bovine procollagen type III’, ALTEX 13, Suppl. 1, 51–56. gómez-ruiz ja, ramos m & recio i (2004), ‘Angiotensin converting enzyme-inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal digestion’, International Dairy Journal, 14, 1075–1080. graham jk & foote rh (1987), ‘Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing’, Cryobiology, 24(1), 42–52. greengard o, sentenac a & mendelsohn n (1964), ‘Phosvitin, the iron carrier of egg yolk’, Biochimicha Biophysica Acta, 90, 406–407. guérin-dubiard c, pasco m, mollé d, désert c, croguennec t & nau f (2006), ‘Proteomic analysis of hen egg white’, Journal of Agricultural and Food Chemistry, 54, 3901– 3910. guérin-dubiard c, castellani o & anton m (2007), ‘Egg compounds with antioxidant and mineral binding properties’, in Bioactive Egg Compounds, R. Huopalathi, R. LopezFandino, M. Anton and R. Schade (Eds.), Springer, Berlin, Germany, pp 223–228. hamilton rmg (1986), ‘The microstructure of the hen’s eggshell: a short review’, Food Microstructure, 5, 99–110. hegenauer j, saltman p & nace g (1979), ‘Iron (III)–phosphoprotein chelates: stoichiometric equilibrium constant for interaction of iron (III) and phosphorylserine residues of phosvitin and casein’, Biochemistry, 18, 3865–3879. ibrahim hr, sugimoto y & aoki t (2000), ‘Ovotransferrin antimicrobial peptide (OTAP–92) kills bacteria through a membrane damage mechanism’, Biochimica Biophysica Acta, 1523, 196–205. itoh t, abe y & adachi s (1983), ‘Comparative studies on the a- and b-phosvitin from hen’s egg yolk’, Journal of Food Science, 48, 1755–1757. johnson ea (1994), ‘Egg white lysozyme as a preservative for use in foods’, in Egg uses and processing technologies, new developments, J.S. Sim and S. Nakai (Eds.), CAB Int., Wallingford, UK, pp 177–191. karlsson m, kollberg h & larsson a (2004), ‘Chicken IgY: utilizing the evolutionary advantage’, World’s Poultry Science Journal, 60, 341–347. kassaify zg & mine y (2004), ‘Effect of food protein supplements on Salmonella Enteritidis infection and prevention in laying hens’, Poultry Science, 83, 753–760. kato a, imoto t & yagishita k (1975), ‘The binding groups in ovomucin–lysozyme interaction’, Agricultural and Biological Chemistry, 39, 541–544. khlifaoui m (2004), ‘Intérêts de la glutamine et des LDL dans la cryoconservation de la semence de l’étalon’, PhD Thesis, University of Rennes. kijowski j & lesnierowski g (1999), ‘Separation, polymer formation and antibacterial activity of lysozyme’, Polish Journal of Food and Nutrition Sciences, 8, 3–16. kiosseoglou vd (1989), ‘Egg yolk’, in Food Emulsifiers: Chemistry, Technology, Functional Properties and Applications, G. Charalambous and G. Doxastakis (Eds.), Elsevier, London. kurakawa h, mikami b & hirose m (1995), ‘Crystal structure of differic hen ovotransferrin at 2.4 Å resolution’, Journal of Molecular Biology, 254, 196–207.
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Bioactive fractions of eggs for human and animal health 343 larsson a, karlsson-parra a
& sjöquist j (1991), ‘Use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors’, Clinical Chemistry, 37, 411–414. larsson a, wejaker pe, forsberg po & lindahl t (1992), ‘Chicken antibodies: a tool to avoid interference by complement activation in ELISA’, Journal of Immunological Methods, 156, 79–83. lee sk, han jh & decker ea (2002), ‘Antioxidant activity of phosvitin in phosphotidylcholine liposomes and meat model systems’, Journal of Food Science, 67, 37–41. leslie ga & clem lw (1969), ‘Phylogeny of immunoglobulin structure and function. III. Immunoglobulin of the chicken’, Journal of Experimental Medicine, 130, 1337–1352. li gh, le gw, shi yh & sherstha s (2004), ‘Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects’, Nutrition Research, 24, 469–486. li-chan ecy & nakai s (1989), ‘Biochemical basis for the properties of egg white’, Critical Review in Poultry Biology, 2, 21–57. li-chan ecy, powrie wd & nakai s (1995), ‘The chemistry of eggs and egg products’, in Egg Science and Technology, 4th ed., W.J. Stadelman and O.J. Cotterill (Eds.), Food Product Press, New York, Chapter 6. lin ln, mason ab, woodworth rc & brandts jf (1994), ‘Calorimetric studies of serum transferring and ovotransferrin. Estimates of domain interactions and study of the kinetic complexities of ferric ion binding’, Biochemistry, 33, 1881–1888. lock jl & board rg (1992), ‘Persistence of contamination of hen’s egg albumen in vitro with Salmonella serotypes’, Epidemiology and Infection, 108, 389–396. lópez - fandiño r , recio i & ramos m (2007), ‘Egg-protein derived peptides with antihypertensive activity’, in Bioactive Egg Compounds, R. Huopalathi, R. LopezFandino, M. Anton and R. Schade (Eds.), Springer, Berlin, Germany, pp 199–212. lösch u, schranner i, wanke r & jürgens l (1986), ‘The chicken egg, an antibody source’, Journal of Veterinary Medicine B, 33, 609–619. lu cl & baker rc (1986), ‘Characteristic of egg yolk phosvitin as an antioxidant for inhibiting metal-catalyzed phospholipid oxidation’, Poultry Science, 65, 2065–2070. lu cl & baker rc (1987), ‘Effect of pH and food ingredients on the stability of egg yolk phospholipids and the metal-chelator antioxidant activity of phosvitin’, Journal of Food Science, 52, 613–616. mangani s, orioli pl, scozzafava a, messori l & carloni p (1994), ‘EXAFS studies of Fe(III)-phosvitin at high metal to protein ratios’, Biometals, 7, 104–108. mann k (2007), ‘The chicken egg white proteome’, Proteomics, 7, 3558–3568. mann k & mann m (2008), ‘The chicken egg yolk plasma and granule proteomes’, Proteomics, 8, 178–191. martin wg, augustyniak j & cook wh (1964), ‘Fractionation and characterization of the low-density lipoproteins of hen’s egg yolk’, Biochimica Biophysica Acta, 84, 714–720. mcbee le & cotterill oj (1979), ‘Ion-exchange chromatography and electrophoresis of egg yolk proteins’, Journal of Food Science, 44, 657–666. miguel m, recio ja, gomez-ruiz a, ramos m & lopez-fandino r (2004), ‘Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis’, Journal of Food Protection, 67, 1914–1920. mizutani k, mikami b, aibara s & hirose m (2005), ‘Structure of aluminium-bound ovotransferrine at 2.15 Å resolution’, Acta Crystallographica, 61, 1636–1642. moussa m, martinet v, trimeche a, tainturier d & anton m (2002), ‘Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen’, Theriogenology, 57(6), 1695–1706. nakai s, li-chan e & lo k v (1994), ‘Separation of yolk immunoglobulin’, in Egg Uses & Processing Technologies. New Developments, Sim J.S. and Nakai S. (Eds.), CAB International, Wallingford (UK), pp 94–105. © Woodhead Publishing Limited, 2011
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antibacterial effects on cariogenic bacteria and clinical applications in preventive denstistry’, Proceedings of the Finnish Dental Society, 87, 197–208. thapon jl (1994), ‘Evolution de l’œuf au cours de la conservation’, in L’œuf et les ovoprodutis, JL Thapon and CM Bourgeois (Eds.), Tec et Doc Lavoisier, Paris, France, pp 84–94. thapon jl & bourgeois cm (1994), ‘L’œuf et les ovoproduits’, Lavoisier, Tec & Doc, Paris, chapter 1. tini m, jewell ur, camanish g, chilov d & gassmann m (2002), ‘Generation and application of chicken egg-yolk antibodies, Comparative Biochemistry and Physiology A, 131, 569–574. tullet sg (1987), ‘Egg shell formation and quality’, in Egg quality current problems and recent advances, RG Wells and CG Belyavin (Eds.), Butterworth, London, pp 123–146. valenti p, antonini g, von hunolstein c, visca p, orsi n & antonini e (1983), ‘Studies on the antimicrobial activity of ovotransferrin’, International Journal of Tissue Reactivity, 5(1), 97–105. valenti p, visca p, antonini g & orsi n (1986), ‘Interaction between lactoferrin and ovotransferrin and Candida cells’, FEMS Microbiological Letters, 33, 271–275. vermeirssen v, van camp j, devos l & verstraete w (2003), ‘Release of angiotensin I converting enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: from batch experiment to semicontinuous model’, Journal of Agricultural and Food Chemistry, 51, 5680–5687. vermeirssen v, van camp j & verstraete w (2004), ‘Bioavailability of angiotensin I converting enzyme inhibitory peptides’, British Journal of Nutrition, 92, 357–366.
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17 Using egg IgY antibodies for health, diagnostic and other industrial applications J. Kovacs-Nolan and Y. Mine, University of Guelph, Canada
Abstract: Immunoglobulin Y (IgY) is the major antibody in chickens, and is transferred in large quantities to the egg yolk to confer passive immunity to the developing embryo. IgY can easily be harvested from the yolk, and is an ideal alternative to using mammalian IgG antibodies. The following chapter discusses the production and purification of IgY, as well as its many advantages and applications, including the use of IgY as a diagnostic and analytical tool and for passive immunotherapy to treat numerous health conditions. Key words: hen egg yolk antibodies (IgY), IgY purification, passive immunotherapy, diagnostics, immunoaffinity ligand.
17.1 Introduction It has been over 100 years since the existence of immunoglobulin (Ig) Y was first described by Klemperer (1893). It was found to be the major serum antibody in hens, and termed IgY due to its enrichment in egg yolk (Leslie and Clem, 1969). Since then, the avian immune system, and IgY, has been studied extensively, revealing many important characteristics and applications of IgY. The laying hen is an ethical alternative for the production of large quantities of specific antibodies at a low cost (Schade and Hlinak, 1996). Owing to its favorable immunochemical characteristics when compared to mammalian IgG, IgY has found many applications in the medical and research fields, including areas such as diagnostics and biomarker discovery. IgY has perhaps received the most attention for its immunotherapeutic potential. © Woodhead Publishing Limited, 2011
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Using egg IgY antibodies for health, diagnostic & other applications 347 Passive immunotherapy, the administration of pre-formed antibodies to prevent infectious diseases or treat existing conditions, may be one of the most valuable applications of antibodies, and is currently driven by the need to find alternatives to traditional antibiotic therapy (Reilly et al., 1997; Carlander et al., 2000). Antibody therapy is, however, often limited by the significant amount of antibody required for treatment (Hatta et al., 1997b). Since IgY is transferred in large quantities to the egg yolk, and can easily be extracted and isolated, eggs are an ideal source of specific antibodies for passive immunotherapy. The following chapter describes structural, physical and immunochemical properties of IgY, as well as an overview of IgY production and purification, and its applications in diagnostics, analytical applications and immunotherapy.
17.2 Overview of the avian immune system and IgY biosynthesis The avian immune system is made up of the bursa of Fabricius, bone marrow, spleen, thymus, the Harderian gland, lymph nodes, circulating lymphocytes and various lymphoid tissues. The thymus serves as the primary lymphoid organ for T-cell differentiation, while the antibody-synthesizing B-cells are produced in the bursa of Fabricius (Sharma, 1997; Carlander et al., 1999a). The spleen is the centre for plasma cell proliferation and memory B cells (Carlander et al., 1999a). The vertebrate immune system has the ability to produce an exceedingly high number of different antibody molecules, through the existence of multiple variable (V), diversity (D) and joining (J) elements in the genome (the germ line repertoire), as well as existence of somatic recombination processes and point mutations (Reynaud et al., 1985; Parvari et al., 1988). In the avian immune system, however, this combinatorial diversity is restricted, as the rearrangement of immunoglobulin genes is not an ongoing process, but rather takes place as a single wave during early embryogenesis. The total number of rearrangements from which the chicken B-cell repertoire may be generated is limited to the number of B-cell precursors in the bursa (Reynaud et al., 1989, 1991), estimated to be around 2–3 ¥ 104 cells (Pink et al., 1985). Despite the fact that chickens have an extremely limited number of immunoglobulin genes compared with mammals, they are capable of producing a wide range of immune responses and diverse antibody molecules (Sharma, 1997). Three classes of antibody are found in the chicken, IgY, IgA and IgM, and are present in the serum at concentrations of 5.0, 1.25 and 0.61 mg/mL, respectively (Leslie and Martin, 1973). IgY, the functional equivalent of mammalian IgG, is the major antibody produced by chickens, and makes up approximately 75% of the total antibody population (Carlander et al., 2000). During egg formation, serum IgY is selectively transferred to the
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348 Improving the safety and quality of eggs and egg products yolk via a receptor on the surface of the yolk membrane specific for IgY translocation, in order to confer passive immunity to the developing embryo (Fig. 17.1) (Loeken and Roth, 1983; Tressler and Roth, 1987; Morrison et al., 2002; West et al., 2004; Tesar et al., 2008). Egg yolk can contain between 5 to 25 mg/mL of IgY, whereas IgA and IgM are found in the egg white, at concentrations of around 0.15 and 0.7 mg/mL respectively (Rose et al., 1974; Schade et al., 1991; Li et al., 1997).
17.3 Production and purification of IgY 17.3.1 Production Several aspects of hen immunization, including the immunization route and vaccine adjuvant, have been studied in an effort to improve IgY production and yolk deposition (Levesque et al., 2007). Chang et al. (1999) demonstrated that intramuscular immunization resulted in higher levels of specific IgY when compared with immunization via the subcutaneous route. Oil-based adjuvants, such as Freund’s complete (FCA) and incomplete (FIA) adjuvant, continue to be the adjuvants of choice for IgY production (Trott et al., 2008); however, alternatives are continually being sought due to potentially severe injection site reactions (Wanke et al., 1996). The inclusion of bacterial components or immunostimulants in vaccine formulations has been shown to increase antigen-specific antibody levels in the yolk, and may require fewer injections and less antigen, and ultimately be more cost effective (Levesque et al., 2007; Trott et al., 2008; Herath et al., 2010). In fact, Levesque et al. (2007) found that adding synthetic oligonucleotides containing unmethylated CpG dinucleotides, characteristic of bacterial DNA (CpG ODNs), to FIA could IgY
Yolk
lgA
Ovary
Oviduct
lgM
Albumen lgM
lgA Yolk
Egg
lgY
Fig. 17.1 Distribution of antibodies in hen eggs. Adapted from Hatta et al. (1997a).
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Using egg IgY antibodies for health, diagnostic & other applications 349 increase specific IgY production by 480%. The use of DNA vaccines has also been described for IgY production (Romito et al., 2001; Cova, 2005; Witkowski et al., 2009). This process involves immunizing birds with plasmid DNA encoding the antigen of interest, and not only avoids the potentially costly and tedious preparation of purified antigens, but can also overcome interference by maternal antibodies (Elazab et al., 2009). 17.3.2 Purification Egg yolk is a liquid emulsion of water-soluble protein and a dispersed phase of lipoprotein particles. One of the major challenges in IgY purification is separating the water-soluble IgY from the yolk lipoproteins (Hatta et al., 2008). IgY purification typically involves isolation of the IgY-containing water-soluble fraction (WSF), followed by additional purification steps. Simple dilution of the yolk with water, which results in the aggregation of yolk lipoproteins at low ionic strength (Jensenius et al., 1981), followed by centrifugation or ultrafiltration (UF), has been reported for the isolation of WSF (Akita and Nakai, 1992; Kim and Nakai, 1996, 1998). Likewise, freezing and thawing of diluted yolk, producing lipid aggregates that are large enough to be removed by conventional low speed centrifugation (Jensenius and Koch, 1993), has also been employed, resulting in a purity of around 70% (Deignan et al., 2000). For these dilution methods, pH and extent of dilution are very important for optimal IgY recovery, and Nakai et al. (1994) found that the best results were obtained using a six-fold water dilution, at pH 5.0. Other methods of removing lipoproteins prior to IgY purification include separation by ultracentrifugation (McBee and Cotterill, 1979; Akita and Nakai, 1992; Akita and Li-Chan, 1998), organic solvent delipidation (Polson, 1990; Kwan et al., 1991; Horikoshi et al., 1993; McLaren et al., 1994), or use of lipoprotein coagulating agents such as polyethylene glycol (Akita and Nakai, 1993; Svendsen et al., 1995), dextran sodium sulfate (Jensenius et al., 1981), polyacrylic resin (Hamada et al., 1991) and dextran blue (Bizhanov and Vyshniauskis, 2000); however, application of these methods for largescale IgY production is limited by problems related to food safety as well as cost constraints (Hatta et al., 2008). To address this problem, natural polysaccharides, which have been found to act as effective yolk lipoprotein coagulating agents (Hatta et al., 2008), have been employed, including sodium alginate (Hatta et al., 1988, 1990), xanthan gum (Akita and Nakai, 1993) and a-carrageenan (Hatta et al., 1990). Chang et al. (2000) reported the precipitation of over 90% of lipoproteins from yolk using l-carrageenan, sodium alginate, carboxymethyl cellulose and pectin. A number of methods of purifying IgY following lipoprotein removal have been reported, and include ion exchange chromatography (McCannel and Nakai, 1990; Akita and Nakai, 1992; Fichtali et al., 1992, 1993; Ko and Ahn, 2007), ammonium sulfate precipitation (Ko and Ahn, 2007), hydrophobic
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350 Improving the safety and quality of eggs and egg products interaction chromatography (Hassl and Aspock, 1988), immobilized metal ion affinity chromatography (McCannel and Nakai, 1990; Greene and Holt, 1997), thiophilic interaction chromatography (Hansen et al., 1998), affinity chromatography using alkaline conditions (Kuronen et al., 1997) and synthetic peptide ligands, designed specifically for immobilizing IgY (Fassina et al., 1998; Verdoliva et al., 2000) which resulted in 92% purity and 78% recovery (Dong et al., 2008). Hernandez-Campos et al. (2010) recently demonstrated that UF purification of WSF using polyethersulphone (PES) or modified PES membranes increased purity and resulted in greater than 90% IgY recovery. Recent proteomic analysis and identification of the proteins in WSF prepared by the water dilution method revealed that preparations obtained by this method are highly reproducible with low levels of cholesterols and triglycerides, and provides information that may be valuable for the prediction of potential allergic reactions to WSF preparations (Nilsson et al., 2008).
17.4 Advantages of eggs as an alternative antibody source The use of chickens for antibody production provides several advantages over the conventional method of IgG production in mammals. Antibody production in chickens is much less invasive, requiring only the collection of eggs, rather than the collection of blood, and therefore is less stressful on the animal (Fig. 17.2) (Karlsson et al., 2004). Owing to the genetic distance between chickens and mammals, it is possible to produce antibodies Antigen
lgG Blood
Antigen
lgY Egg yolk
Fig. 17.2 Comparison of preparation of specific antibody from hen (Hatta et al., 1997a).
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Using egg IgY antibodies for health, diagnostic & other applications 351 against highly conserved mammalian proteins that otherwise would not be possible in mammals. In contrast to mammalian systems, much less antigen is required to produce an efficient immune response, and sustained high antibody titers reduce the need for frequent injections (Larsson et al., 1998). In fact, it has been estimated that the productivity of antibodies in hens is nearly 18 times greater than that in rabbits, based on the weight of antibody produced per animal (Nakai et al., 1994), which translates to an overall reduction in animal use for the same level of antibody production. A laying hen typically lays around 240–280 eggs per year (Sim et al., 2000), and an egg yolk contains 100–150 mg of IgY. Therefore, around 28–42 g of IgY per year may be obtained from a single chicken (Karlsson et al., 2004). In contrast to mammalian serum, egg yolk contains only a single class of antibody (IgY), which can easily be isolated from the yolk by precipitation techniques (Gassmann et al., 1990), as discussed in the previous section. Once purified, IgY has been found to be stable for years when stored at 4 °C (Larsson et al., 1993). 17.4.1 Structure and stability of IgY Although IgG and IgY share a similar biological function, there exist some profound differences in their chemical structures and resulting immunoreactivity (Camenisch et al., 1999; Zhang, 2003). In contrast to IgG, which has a molecular mass of 150 kDa, the IgY molecule has a molecular mass of 180 kDa, owing to its increased number of heavy-chain constant domains and an extra pair of carbohydrate chains (Hatta et al., 1993a; Sun et al., 2001). In addition, the hinge region of IgY is shorter and less flexible (Warr et al., 1995), and the content of b-sheet structure in the constant domains of IgY has been reported to be lower (Shimizu et al., 1992), which may account for the differences in stability observed between IgY and IgG. Both IgG and IgY contain Asn-linked oligosaccharides; however, the structure of oligosaccharides in IgY differ from those of any mammalian IgG, containing instead unusual monoglucosylated oligomannose-type oligosaccharides with Glc1Man7–9GlcNAc2 structure (Ohta et al., 1991; Matsuura et al., 1993). The isoelectric point of IgY is lower than that of IgG (Polson et al., 1980), IgY does not associate with mammalian complement, and the binding of IgY with human and bacterial Fc-receptors on cell surfaces is less than that of IgG (Gardner and Kaye, 1982). Furthermore, it has also been suggested that IgY may be more hydrophobic than IgG, which matches the lipid-rich environment of the yolk (Davalos-Pantoja et al., 2000). In general, when used as reagents for immunochemical or research purposes, where antibodies are stored and used under relatively mild conditions, stability does not warrant much attention. However, the stability of IgY under various processing and physiological conditions needs to be considered when they are to be used in industrial, food or medical applications (Shimizu et al., 1993b). IgY is stable in the pH range of 4–9, and up to temperatures of 65 °C
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352 Improving the safety and quality of eggs and egg products (Shimizu et al., 1992; K. A. Lee et al., 2002), however this range may be extended by the addition of stabilizers such as sugars, complex carbohydrates, or polyols (Shimizu et al., 1994; Chang et al., 2000; K. A. Lee et al., 2002). IgY is relatively resistant to treatment with trypsin and chymotrypsin, but sensitive to pepsin digestion (Shimizu et al., 1988). Hatta et al. (1993b) found that almost all of the IgY activity was lost following digestion with pepsin; however, activity remained even after 8 hours incubation with trypsin or chymotrypsin. Studies in humans have demonstrated that orally administered antibodies undergo a significant loss of activity upon passage through the stomach, and several reports have demonstrated around 4–20% survival of orally administered antibody, with similar results observed in both adults and infants (Blum et al., 1981; Hilpert et al., 1987; Roos et al., 1995). In order to circumvent the inactivation of IgY following oral administration, a number of encapsulation techniques have been examined. The microencapsulation of IgY using liposomes (Shimizu et al., 1993a) and multiple emulsions (Shimizu and Nakane, 1995) have been previously described; however, these have shown limited effectiveness, in some cases destroying antibody activity by the encapsulation procedure itself. The macroencapsulation of IgY using enteric-coated gelatin capsules has also been examined, and was found to significantly improve antibody stability (Akita and Nakai, 2000). The use of a pH-sensitive methacrylic acid copolymer and chitosan–alginate microcapsules (Kovacs-Nolan and Mine, 2005; Li et al., 2007, 2009a, b) have also been shown to increase IgY stability to gastric conditions both in vitro as well as in vivo.
17.5 Applications of IgY 17.5.1 Use of IgY as an immunoaffinity ligand Immunoaffinity chromatography, involving the specific interaction between an antigen and its corresponding antibody, has been widely used for the purification of proteins from complex starting materials. The use of this process on a large scale is limited by the high cost of the technique, and problems relating to the production of antibody and the efficiency of immobilization (Akita and Li-Chan, 1998). Specific IgY, which can easily be produced on a large scale required for industrial applications, would provide an ideal replacement for other polyclonal or monoclonal antibodies currently used in immunoaffinity chromatography (Li-Chan, 2000). Although IgY is more sensitive to low pH than IgG, it was found that an IgY immunoaffinity column was capable of retaining stability when subjected to standard affinity chromatography conditions, and could be reused over 50 times without a significant decrease in binding capacity (Akita and Li-Chan, 1998). More recently, the unique features of IgY have made it advantageous for use in proteomic applications (Huang et al., 2005). The depletion of abundant © Woodhead Publishing Limited, 2011
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Using egg IgY antibodies for health, diagnostic & other applications 353 proteins from complex biological samples containing a wide dynamic range of protein concentrations, such as plasma, serum and urine, prior to twodimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and mass spectrometry presents a significant challenge for proteomic analysis (Huang and Fang, 2008; Magagnotti et al., 2010). Immunoaffinity fractionation is one of the most effective methods used during sample preparation to improve the ability to detect low-abundance proteins (Huang and Fang, 2008). Owing to the ease of production of IgY and the ability to produce high affinity antibodies against conserved mammalian proteins, IgY is now widely used for immunoaffinity protein depletion in proteomic applications and biomarker discovery (Qian et al., 2008; Rajic et al., 2009; Polaskova et al., 2010). 17.5.2 Use of IgY in diagnostic and analytical applications Antibodies serve as essential components in a variety of diagnostic assays used for the qualitative and quantitative determination of a wide range of substances (Schade and Hlinak, 1996). While the antigen-binding specificity of IgY is comparable to that of IgG (Hatta et al., 1997b), IgY presents several advantages that make it well suited for applications in medical diagnostics. A number of proteins exist whose amino acid sequences are well preserved among mammals, and when used as an antigen in mammals have limited or no antigenicity (Hatta et al., 2008). Evolutionary differences allow the production of high avidity IgY against conserved mammalian proteins, with a more diversified antibody repertoire than is possible in mammals (Olovsson and Larsson, 1993; Woolley and Landon, 1995; Carlander et al., 1999a). The use of IgY as a substitute for IgG in clinical testing can potentially eliminate false positives and often results in low background and less interference (Xiao and Gao, 2010). Human serum samples often contain an active complement system, which would be activated by mammalian antibodies. IgY, which does not activate the human complement system, eliminates the interference that would otherwise be caused by IgG (Larsson and Mellstedt, 1992). Human serum can also contain rheumatoid factor (RF) and human anti-mouse IgG antibodies (HAMA), which are well-known causes of false-positive reactions in immunological assays (Carlander et al., 1999a). RF reacts with the Fc portion of IgG, and HAMA, which can occur naturally in human serum, will bind to any mouse antibodies being used in an immunoassay (Carlander et al., 1999a). Since IgY does not react with RF (Larsson and Sjoquist, 1988; Larsson et al., 1991) or HAMA (Larsson and Mellstedt, 1992), it has been suggested as a replacement for IgG in immunological assays of human serum. IgY has been used in numerous medical and veterinary diagnostic applications, including detection and quantitation of cancer biomarkers in breast and ovarian cancer and non-Hodgkin’s lymphoma patients (Grebenschikov et al., 1997; Al-Haddad et al., 1999; Lemamy et al., 1999; Pan et al., 2010;
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354 Improving the safety and quality of eggs and egg products Xiao and Gao, 2010), the diagnosis of gastric cancer (Noack et al., 1999), as well as for the detection of African horsesickness virus (Du Plessis et al., 1999), Mycobacterium avium subsp. paratuberculosis (Shin et al., 2009), Leptospira spp. in human blood (Vasconcellos et al., 2010), bovine leukemia virus (Juliarena et al., 2007), serotyping of foot-and-mouth disease (Veerasami et al., 2008), and detection of opiate drugs in urine (Gandhi et al., 2009). IgY against the SARS-CoV nucleocapsid protein was incorporated into an immunoswab assay for point of care detection of SARS-infected individuals (Kammila et al., 2008). Furthermore, IgY has been used in assays involving food safety concerns, including detection of Escherichia coli O157:H7 (Sunwoo et al., 2006), Listeria spp. (Kim et al., 2005), Campylobacter jejuni (Hochel et al., 2004), screening agricultural products for citrinin contamination (Duan et al., 2009), detection of ciguatoxin contamination in fish tissue (Empey Campora et al., 2008), the identification of allergens (Alessandro et al., 2009), and IgY against dichlorodiphenyltrichloroethane (DDT) was incorporated into a dipstick assay to test for pesticide contamination (Lisa et al., 2009).
17.6 Immunotherapeutic applications of IgY Increases in antibiotic resistant bacteria and pathogens that do not respond to antibiotics, such as viral pathogens, along with an escalating number of immunocompromised individuals, has prompted much research into the administration of specific antibodies as an alternative to antibiotics and antimicrobial chemotherapy to treat infections (Casadevall and Scharff, 1995; Reilly et al., 1997). IgY is well suited to passive immunotherapy applications since it does not react with rheumatoid factors, human Fcreceptors, or HAMA, which are all well-known cell activators and mediators of inflammation (Larsson et al., 1993; Larsson and Carlander, 2003). 17.6.1 Oral administration of IgY Prevention of Pseudomonas aeruginosa infection in cystic fibrosis patients Respiratory infection caused by colonization of the airways by Pseudomonas aeruginosa (PA) is the major cause of morbidity and mortality in cystic fibrosis (CF) patients (Shale and Elborn, 1996), and once a chronic infection has been established it is very difficult to eliminate, even with the use of antibiotics (Kollberg et al., 2003). An ongoing study in CF patients has found that an oral rinse containing anti-PA IgY given on a continuous basis could reduce or prevent PA colonization, thereby reducing the need for antibiotic treatment (Carlander et al., 2000; Kollberg et al., 2003; Nilsson et al., 2007b, 2008). In vitro, these antibodies were found to inhibit adhesion of the bacteria to epithelial cells, but did not inhibit bacterial growth, suggesting that the © Woodhead Publishing Limited, 2011
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Using egg IgY antibodies for health, diagnostic & other applications 355 IgY might be capable of interfering with the bacterial infection process and preventing colonization in CF patients (Carlander et al., 1999b). More recently, the immunoreactivity of these antibodies was examined in detail using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and PA flagellin was identified as the major antigen. The flagellin is important for establishing infections in hosts as well as being involved in PA chemotaxis, motility, adhesion and inflammation (Nilsson et al., 2007a). These results further support the ability of anti-PA IgY to prevent infection and host invasion. The stability of the anti-PA IgY in saliva from healthy individuals was also examined (Carlander et al., 2002), and antibody activity remained after 8 hours. After 24 hours, the antibody activity had decreased significantly, but was still detectable in some subjects, indicating that oral treatment with specific IgY for various local infections, including the common cold and tonsillitis, might also be possible. Prevention of Helicobacter pylori infection and gastric symptoms Helicobacter pylori is a common cause of gastritis and gastric ulcers, and has been implicated in the development of gastric carcinomas (Dunn et al., 1997). The development of antibiotic resistance has prompted the investigation into alternative methods of treating and preventing H. pylori infection (DeLoney and Schiller, 2000). IgY produced against H. pylori reduced bacterial adhesion, growth and urease activity in vitro and decreased H. pylori-induced gastric mucosal injury and inflammation in an animal model (Shin et al., 2002). Since antibodies produced against whole-cell H. pylori might cross-react with other bacteria, including normal human flora (Shin et al., 2003), the production and efficacy of IgY against immunodominant H. pylori proteins or epitopes has also been described, including IgY against urease (Nomura et al., 2005), urease-derived peptides (Shin et al., 2004) and a 58 kD highly reactive H. pylori antigen (Hp58) (Attallah et al., 2009). The effectiveness of anti-urease IgY has been demonstrated in humans. A functional drinking yogurt containing Lactobacillus acidophilus and Bifidobacterium spp. supplemented with 1% egg yolk IgY-urease was produced commercially, and given to volunteers testing positive for H. pylori (Horie et al., 2004). Volunteers consumed the IgY-containing yogurt three times daily for four weeks, following which urea breath test values and antigen detection in feces were significantly decreased, indicating suppression of H. pylori infection. Prevention of dental caries and periodontitis Porphyromonas gingivalis is one of the most important causative agents of periodontitis (Yokoyama et al., 2007b). In vitro, anti-P. gingivalis IgY dosedependently decreased bacterial adhesion and hydrolytic activity (Yokoyama et al., 2007a), and reduced levels of P. gingivalis when applied as a gel to the teeth of periodontitis patients (Yokoyama et al., 2007b). The production of
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356 Improving the safety and quality of eggs and egg products IgY against the P. gingivalis 40 kD outer membrane protein, which aggregates with other oral bacteria to form plaque (Hamajima et al., 2007), as well as P. gingivalis hemagglutinin (HagA),which allows the bacteria to adhere to gingival tissue cells (Tezuka et al., 2006), have also been described and found to inhibit aggregation and hemagglutination in vitro. Streptococcus mutans is believed to be the principal causative bacterium of dental caries in humans (Hamada and Slade, 1980), and IgY against S. mutans has also been shown to exert anti-cariogenic properties. IgY against S. mutans was examined for the passive prevention of dental caries (Hamada et al., 1991; Otake et al., 1991; Chang et al., 1999), and it was found that supplementation of 2% IgY-containing yolk powder to a cariogenic diet significantly lowered caries scores. Likewise, levels of S. mutans were decreased in volunteers who gargled with a mouth rinse containing anti-S. mutans IgY (Hatta et al., 1997c). IgY produced against the S. mutans glucan binding protein B (GBP-B), which is believed to be involved in S. mutans biofilm development, has also been examined. Using a rat model of dental caries, Smith et al. (2001) observed a decrease in S. mutans accumulation in rats treated with anti-GBP-B IgY, as well as a decrease in the overall amount of dental caries, as compared with control rats. Prevention of Escherichia coli infection Diarrheal diseases continue to be a health problem worldwide, especially in developing countries, where they are estimated to be responsible for 2.5 million infant deaths per year (Estrada-Garcia et al., 2009), as well as causing economic losses in livestock, especially neonatal calves and piglets (Morris and Sojka, 1985). One of the most prominent causes of diarrheal diseases remains Escherichia coli, and a number of strategies to prevent E. coli infection using IgY have been examined. The production of IgY against the fimbrial antigens K88, K99 and 987P of porcine enterotoxigenic E. coli (ETEC), has been described. Anti-K88, -K99 and -987P IgY inhibited the binding of E. coli K88+, K99+ and 987P+ strains to porcine epithelial cells (Yokoyama et al., 1992) and porcine intestinal mucus (Jin et al., 1998) in vitro. When given orally to piglets, these antibodies dose-dependently protected against E. coli infection (Yokoyama et al., 1992). Marquardt et al. (1999) also found that the oral administration of anti-K88 IgY resulted in 100% survival of ETEC-infected piglets, compared with a control group, and a reduction in the incidence and severity of diarrhea. The passive protective effect of anti-ETEC IgY, in neonatal calves has also been shown. Calves fed milk containing IgY had transient diarrhea, 100% survival and good body weight gain during the course of the study (Ikemori et al., 1992). More recently, IgY against recombinant adherence-associated proteins of E. coli O157:H7 was shown to reduce adherence in vitro in cultured cells, suggesting they may have the potential to reduce E. coli O157:H7 colonization in hosts such as cattle and humans (Cook et al., 2007). Similarly, Girard
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Using egg IgY antibodies for health, diagnostic & other applications 357 et al. (2006) demonstrated that IgY produced against virulence factors of attaching and effacing E. coli (AEEC) reduced bacterial adherence of porcine enteropathogenic E. coli, as well as heterologous AEEC strains, including human, bovine and canine enteropathogenic E. coli and O157:H7, and found that oral administration of these antibodies reduced E. coli-induced lesions in pigs challenged with the porcine enteropathogenic E. coli strain ECL1001. Furthermore, anti-E. coli O78:K80 IgY was found to reduce E. coli numbers and improve intestinal health in chickens challenged with E. coli (Mahdavi et al., 2010), and IgY against mastitis-causing E. coli (O111) dose-dependently inhibited E. coli growth and enhanced phagocytic activity in vitro (Zhen et al., 2008b). Amaral et al. (2008) demonstrated the in vitro anti-E. coli effects of IgY against EPEC O111, Shiga toxin-producing E. coli (STEC) O111 and STEC O157, suggesting possible future therapeutic use in humans. Prevention of Salmonella infection Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) are the main causes of salmonellosis outbreaks in humans and infections in chickens (E. N. Lee et al., 2002). Salmonella possesses several surface components which are virulence related, including outer membrane protein (OMP) (Isibasi et al., 1988), flagella (Fla) and, in some strains, fimbrial antigens (Thorns et al., 1990, 1992). The ability of IgY specific for OMP, LPS or flagella (Fla), to passively protect against experimental salmonellosis in mice has been demonstrated. In vitro using a cultured human intestinal cell line (Caco-2), Chalghoumi et al. (2009) found that IgY against OMP of SE and ST reduced the Salmonella-induced decrease in transepithelial electrical resistance (TER) of the infected Caco-2 cell monolayers and blocked Salmonella sp. adhesion in a concentration-dependent manner, suggesting that passive immunization with Salmonella OMP-specific IgY could be useful to prevent Salmonella colonization in broiler chickens and the subsequent carcass contamination during processing. In vivo in mice challenged with SE or ST, treatment with IgY against OMP, LPS or Fla resulted in a significantly higher survival rate when compared to control mice (Yokoyama et al., 1998b). Salmonella infection in calves is also a significant problem, with ST and S. dublin accounting for most cases of salmonellosis. Passive protection against ST and S. dublin was investigated by challenging calves with SE or S. dublin, and then orally administering anti-SE or anti-S. dublin IgY. All control calves died within 7–10 days, whereas only fever and diarrhea were observed in calves given the high titer IgY (Yokoyama et al., 1998a). Prevention and treatment of rotavirus gastroenteritis Rotavirus is a major cause of acute gastroenteritis in infants in both developed and developing countries (Clark et al., 1999). In vivo, IgY produced against murine, human and simian strains of rotavirus prevented rotavirus-induced
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358 Improving the safety and quality of eggs and egg products diarrhea in mice (Yolken et al., 1988; Ebina, 1996). The anti-rotavirus effect was seen both when administered before and after viral challenge, suggesting its use for both the prevention and treatment of rotavirus gastroenteritis (Hatta et al., 1993a). The production of IgY against recombinant rotavirus coat proteins, VP8 (Kovacs-Nolan et al., 2001) and VP7 (Zhang et al., 2009) has also been described, and anti-VP8 IgY exhibited significant neutralizing activity against human rotavirus (HRV) in vitro, suggesting its potential use for the prevention and treatment of rotavirus in humans. Neonatal calf diarrhea, caused by bovine rotavirus (BRV), is a significant cause of mortality in cattle (Lee et al., 1995). Using a mouse model of BRV infection, Kuroki et al. (1993) observed the protection against two strains of BRV using orally administered anti-BRV IgY. The passive protection of calves against BRV infection, using anti-BRV IgY, has also been demonstrated (Kuroki et al., 1994). Treatment of inflammatory bowel disease Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is characterized by chronic inflammation of the gastrointestinal tract (Podolsky, 2002). Treatment typically involves corticosteroids and immunosuppressive agents; however, these have shown limited therapeutic efficacy and have been associated with severe side effects and long-term toxicity (Atreya and Neurath, 2008). Tumor necrosis factor (TNF)-a is one of the key pro-inflammatory cytokines involved in the pathogenesis of IBD (Garside, 1999) and immunotherapy using monoclonal mouse antibodies against TNF-a has been approved for use; however, it can be costly and adverse side effects have been reported in patients receiving systemic anti-TNF therapy (Sandborn and Hanauer, 1999). Worledge et al. (2000) reported that orally administered anti-TNF-a IgY was capable of effectively treating acute and chronic phases of colitis in rats, as well as neutralizing human TNF-a in vitro, indicating its possible use for the treatment of IBD in humans. Use of IgY in aquaculture Yersinia ruckeri causes enteric redmouth disease, a systemic bacterial septicemia of salmonid fish (Stevenson et al., 1993), and the persistence of Y. ruckeri in carrier fish and shedding of bacteria in feces can present a continuing source of infection. Lower mortality rates and reduced infection rates were observed in rainbow trout fed anti-Y. ruckeri IgY when given before or after challenge with Y. ruckeri (Lee et al., 2000). White spot syndrome virus (WSSV) causes high mortality and large economic losses in cultured shrimp (Lu et al., 2008). IgY produced against WSSV was shown to passively protect shrimp (Lu et al., 2008) and crayfish (Lu et al., 2009) from WSSV infection. Edwardsiella tarda is a fish pathogen spread by infection through the intestinal mucosa, and Edwardsiellosis in Japanese eels is a serious problem for the eel farming industry, especially due to the appearance of antibiotic-
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Using egg IgY antibodies for health, diagnostic & other applications 359 resistant strains (Hatta et al., 1994). Eels challenged with E. tarda and then given anti-E. tarda IgY survived without any symptoms of infection, in contrast to the control eels which died within 15 days (Gutierrez et al., 1993; Hatta et al., 1994). This antibody is now in use commercially, following a field test on nearly 2,400,000 tails of cultured eels with confirmed disease prevention with anti-E. tarda IgY (Hatta et al., 2008). Prevention and treatment of other diseases Specific IgY has been shown to be effective at preventing and treating several other diseases, including for the passive protection of chicks against infectious bursal disease virus (IBDV) (Eterradossi et al., 1997; Yousif et al., 2006) and avian coccidiosis caused by Eimeria spp. (Lee et al., 2009a, b), for the protection of piglets against porcine epidemic virus (PEDV) (Kweon et al., 2000), for the protection of calves against bovine coronavirus (BCV) (Ikemori et al., 1997), and for the protection of dogs against canine parvovirus-2 (CPV-2) (Van Nguyen et al., 2006). IgY has also been shown to be effective against Candida albicans, dose-dependently preventing C. albicans growth, adherence and biofilm formation in vitro (Wang et al., 2008; Fujibayashi et al., 2009) and preventing colonization in mice (Ibrahim el et al., 2008). Finally, the production and testing of IgY against influenza virus H5N1 and H1N1 has also been described. When administered before or after infection, the IgY prevented mice from infection and significantly reduced viral replication resulting in complete recovery from the disease, suggesting that IgY may be a safe and affordable alternative method for the control of influenza outbreaks (Nguyen et al., 2010). 17.6.2 Systemic administration of IgY Neutralization of venom Envenoming resulting from the bites of venomous snakes, scorpions or spiders is an important public health hazard in many regions, particularly in tropical and subtropical countries. The most widely used treatment is the direct injection of specific anti-venoms, often antibodies produced in horses or sheep, to neutralize the toxic and potentially lethal effects of the venom (Gutierrez et al., 2006). There have been several reports of the production and neutralization of venom by chicken anti-venom IgY (Paul et al., 2007; Meenatchisundaram et al., 2008a, b; Araujo et al., 2010), including the production of polyvalent anti-African snake venom IgY to be used against bites of several different snakes (de Almeida et al., 2008). IgY was found to have a higher bioactivity than anti-venoms traditionally raised in horses (Thalley and Carroll, 1990; de Almeida et al., 2008), and also has a lower likelihood of producing side effects such as serum sickness and anaphylactic shock, which can occur upon administration of mammalian serum proteins (Thalley and Carroll, 1990).
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360 Improving the safety and quality of eggs and egg products Prevention of Staphylococcus aureus infection and toxic shock syndrome Staphylococcal enterotoxins are a family of bacterial superantigens produced by Staphylococcus aureus, and are associated with a number of serious diseases, including food poisoning and toxic shock syndrome (Fraser et al., 1976). Specific IgY has been shown to inhibit the production of S. aureus enterotoxin-A in vitro (Sugita-Konishi et al., 1996). It has also been reported that IgY against S. aureus enterotoxin-B (SEB) systemically administered both before and after challenge could protect rhesus monkeys from toxic shock syndrome (LeClaire et al., 2002), suggesting that it might be useful for both the prevention and treatment against lethal doses of S. aureus enterotoxins, and could be used to reduce or eliminate enterotoxin-mediated disorders. Anti-S. aureus IgY has also been used in veterinary applications, where it was found to inhibit S. aureus growth in vitro (Zhen et al., 2008a; Guimaraes et al., 2009) and prevented bovine mastitis caused by S. aureus in cattle when administered by intramammary infusion (Zhen et al., 2009). Prevention of rabies virus Rabies remains a major public health problem in developing countries, claiming the lives of an estimated 55,000 people each year (Ertl, 2009). Infection with rabies virus causes encephalitis in humans that has a case fatality rate of almost 100% (Johnson et al., 2010). Motoi et al. (2005a, b) reported the production of anti-rabies IgY, raised against a part of the G protein of rabies virus. In vitro, these antibodies bound virions and cells infected with the rabies virus, and neutralized rabies virus infectivity. Administration of anti-rabies IgY to mice infected with rabies virus reduced mortality caused by the virus, suggesting that IgY directed against the rabies virus G protein could serve as a possible alternative to currently available anti-rabies human or equine immunoglobulins (Motoi et al., 2005b). IgY for the prevention of hyperacute rejection in xenotransplantation Transgenic pigs are promising donor organisms for xenotransplantation as they share many anatomical and physiological characteristics with humans (Klymiuk et al., 2010); however one of the most important barriers with such xenografts is hyperacute rejection, mediated by natural antibodies in humans against pig antigens, complement fixation, and the rapid onset of intravascular coagulation (Sandrin and McKenzie, 1994). The major target of these natural antibodies is the carbohydrate epitope, Gala1-3Gal, which is expressed by all mammals except for humans, apes, and some monkeys. Besides humans and monkeys, chickens also lack Gala1-3Gal expression (Bouhours et al., 1998). Since IgY does not bind human complement or Fc-receptors, anti-a-Gal IgY may be a suitable candidate for use as blocking antibodies to inhibit the interactions that may contribute to xenograft rejection. Fryer et al. (1999) demonstrated that anti-a-Gal IgY blocked human xenoreactive antibody binding to both porcine and rat tissues in vitro, and inhibited lysis of porcine cells by human serum, suggesting that IgY could be of potential
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Using egg IgY antibodies for health, diagnostic & other applications 361 use in inhibiting pig-to-human xenograft rejection. Anti-a-Gal IgY was also found to significantly reduce the infectivity of porcine endogenous retrovirus (PERV), an a-Gal bearing virus which has emerged as a potential zoonotic agent, with possible pig-to-human transmission (Leventhal et al., 2001).
17.7 Conclusion and future trends The production of IgY in chickens and the extraction of specific antibodies from egg yolk is increasingly attracting the interest of the scientific community, as evidenced by the significant growth in the amount of IgY-related literature (Schade et al., 2005). Indeed, the immunization of hens represents an ideal alternative to efficiently generate large quantities of polyclonal antibodies since housing is inexpensive, egg collection is non-stressful and noninvasive, and the isolation and purification of IgY is relatively simple and high-yielding (Zhang, 2003). Recently, it has also been suggested that the selective transport of IgY from the blood into the egg yolk may also provide a new strategy for delivering substances into the egg yolk in an attempt to produce designer eggs (Bae et al., 2010), and may further expand the application of IgY. Immunization protocols and adjuvant systems continue to be modified in order to increase specific antibody production, and the identification and production IgY against of new antigens will continue to lead to new applications of egg yolk antibodies for human health and research applications.
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18 Strategic planning for the development of the egg nutraceutical industry V. Guyonnet, Burnbrae Farms Ltd., Canada
Abstract: Using the broad definition of nutraceuticals as foods and part of a food offering benefits beyond nutrition, this chapter analyzes the strengths, weaknesses, opportunities and threats (SWOT analysis) of the egg nutraceutical business. Some strategic goals are proposed and a plan of action is outlined to ensure that the egg nutraceutical business delivers on its promises. Key words: egg nutraceutical business, functional foods, SWOT analysis, strategic planning.
18.1 Introduction A review of the current literature shows that there is no commonly accepted definition for the term nutraceutical beyond the fact that nutraceuticals claim to provide some benefits other than nutritional. For the purpose of this chapter, we will used the broad definition outlined by Andlauer and Fürst (2002), where nutraceuticals refer to any substance that may be considered a food (often called functional or designer food) or part of a food and provides medical or health benefits. Therefore, the egg nutraceutical business will refer broadly to the industries dealing with the egg both as a functional food (eggs enriched with nutrients such as omega-3 fatty acids, lutein and/ or vitamins) and as a source of ingredient/component (such as lysozyme, IgY and biopeptides isolated from the egg), with activity above and beyond its typical nutritional value. For this chapter, we used the classic format of
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Strategic planning for development of egg nutraceutical industry 375 strategic planning taught in business schools: after a brainstorming exercise listing ideas which may be considered as strengths, weaknesses, opportunities and threats for the egg nutraceutical business (also referred to as a SWOT analysis), we will define some strategic goals and outline a possible plan of action for the egg nutraceutical business.
18.2 Egg nutraceutical business – strengths, weaknesses, opportunities and threats (SWOT) analysis As with every strategic plan, the first and most critical step consists in the assessment of the business itself with the identification of its strengths, weaknesses, opportunities and threats (SWOT). By definition, a SWOT analysis must allow for a free flow of ideas with an exhaustive list of points without an attempt to list them in order of importance or impact. Some of the points identified below will be well documented in the literature or accepted within the egg nutraceutical industry while others will be perceived as subjective. The overall goal of this SWOT analysis is to review the various factors which have the potential to influence the egg nutraceutical business. 18.2.1 Strengths Worldwide acceptance of eggs as a food We are extremely fortunate to be dealing with a food which has no interdiction from major religions, does not require a huge amount of space to produce, has a natural good shelf-life and is extremely nutritious. Eggs can be consumed as a main dish, incorporated in a dish or used as an ingredient in many home-made or processed food applications. Egg consumption per capita varies widely from country to country, with China (53 g/person/day), Japan (52 g/person/day) and Denmark (51 g/person/day) as the top nations for egg consumption (FAO, 2008). More detailed information is available in Volume 1, Chapter 1. Source of proteins, vitamins and minerals In spite of slight variations in the overall nutritional content of eggs from country to country (mostly related to the variations in diet fed to the hens), eggs are widely recognized as naturally rich in proteins and certain vitamins and minerals (see Chapter 11). The egg is often referred as one of the original and natural functional foods. Nutritional claims allowed for regular eggs (standard diet fed to the hens) vary from country to country mostly due to regulations (Table 18.1). Ease of production as a functional food Numerous studies have demonstrated that variations in the diet composition fed to laying hens can greatly impact on the level of nutrients such as vitamin © Woodhead Publishing Limited, 2011
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‘Good source’ for iron, phosphorus, folate, niacin, pantothenic acid and vitamin D ‘High’ for selenium, riboflavin and vitamin B12
‘Good source’, ‘contains’ or ‘provides’ if 10%-19% DV ‘High’, ‘rich in’ or ‘excellent source of’ if > 20% DV
‘Source’ if 5–14% DV ‘Good source’ if 15–24% DV ‘Excellent source’ if >25%
‘Source’ if 10–24% DV ‘Good source’ if >25% DV
‘Source’ if >15% RDA ‘Source’ for phosphorus, vitamin A, folate, ‘High’ if >30% RDA niacin, pantothenic acid and vitamin D ‘High’ for selenium, riboflavin, vitamin B12 and biotin
USA (2)
Canada (3)
Australia (4)
Europe (5)
‘Source’ for iron, phosphorus, vitamin A, folate, vitamin D and vitamin E ‘Good source’ for selenium, niacin, pantothenic acid, riboflavin and vitamin B12
‘Source’ for calcium, iron, magnesium, zinc, copper, niacin, vitamin A, thiamin, vitamin B6, vitamin E ‘Good source’ for phosphorus, folate, pantothenic acid and vitamin D ‘Excellent source’ for selenium, riboflavin, vitamin B12 and biotin
Nutritional claim rules Nutritional claims for regular egg per 100 g edible portion (1)
Country
Folate health claim which states: (a) That increased maternal folate consumption in at least the month before and 3 months following conception may reduce the risk of fetal neural tube defects; and (b) The recommendation that women consume a minimum of 400 micrograms folate per day in at least the month before and at least the first 3 months following conception
Other claim(s) allowed
Table 18.1 Nutritional and other claims allowed for regular eggs in the USA, Canada, Australia, the EU and Japan
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‘Contains’ or ‘enhanced’ – specific amount for each nutrient; ‘High’ – specific amount for each nutrient
‘Contains’ for zinc, iron, copper, pantothenic acid, vitamin D and folate ‘High’ for biotin, vitamin A, vitamin B2 and vitamin B12 Function claims: Vitamin A – helps to maintain vision in the dark, and helps to maintain skin and mucosa healthy/Warning indication: increased intake of this product will not result in curing diseases nor promoting health. Please comply with the advisable daily intake. Women within the third months of pregnancy or women planning to be pregnant should be careful of over consumption Vitamin B2 – helps to maintain skin and mucosa healthy/Warning indication: increased intake of this product will not result in curing diseases nor promoting health. Please comply with the advisable daily intake. Folic acid – aids in the red blood cell formation, and contributes the normal growth of the fetus/increased intake of this product will not result in curing diseases nor promoting health. Please comply with the advisable daily intake. This product helps normal development of fetus, but increased intake of this product will not result in better development of fetus
(1) For comparison, all claims are listed based on 100 g edible portion. Canada and the USA have a serving size equaled to one egg and not 100 g. (2) Based on 21CFR § 101.9 and USDA National nutrient database – Available from http://www.nal.usda.gov/fnic/foodcomp/search/ [accessed on 18 May 2010]. (3) Based on CFIA Guide to food labeling and advertising – Available from http://www.inspection.gc.ca/english/fssa/labeti/guide/toce.shtml [accessed on 30 January 2010] and Canadian nutrient file – Available from http://www.hc-sc.gc.ca/fn-an/nutrition/fiche-nutri-data/cnf_aboutus-aproposdenous_fcen-eng.php [accessed on 30 January 2010]. (4) Based on Australia New Zealand Food Standards Code – Standards 1.1.1, 1.1.A.2, and 1.2.8 – Available from http://www.foodstandards.gov.au/foodstandards/ foodstandardscode/ and NUTTAB 2006 Online version – Available from http://www.foodstandards.gov.au/consumerinformation/nuttab2006/onlineversionintroduction/ [accessed on 29 March 2010]. (5) Based on EU nutrition claims – Available from http://ec.europa.eu/food/food/labellingnutrition/claims/nutrition_claims_en.htm [accessed on 27 March 2010] and EU Directive 2008/100/EC Annex I (28 October 2008). (6) Based on Ministry of Health, Labour and Welfare – Available from http://www.mhlw.go.jp/english/topics/foodsafety/fhc/index.html [accessed on 1 May 2010].
Japan (6)
378 Improving the safety and quality of eggs and egg products A, vitamin D3, vitamin K, vitamins B1, B2 and B6, biotin, niacin, vitamin E, folic acid, pantothenic acid, choline, lutein, iron and selenium (see Chapter 15), total omega-3 and long chain polyunsaturated omega-3 (n-3 PUFA) like docosahexaenoic acid (DHA) (Chapter 14) (Caston and Leeson, 1990; Naber, 1993; Surai and Sparks, 2001; Flachowsky et al., 2002; House et al., 2002; Leeson and Caston, 2003; Leeson and Caston, 2004; Skřivan et al., 2005; Fisinin et al., 2008). Naber (1993) also defined the vitamin % transfer efficiency, predicting the amount of each vitamin in the eggs based on the vitamin level in the diet fed to the hens. Over the years, nutritionists have developed a well-established diet formula allowing some easy, predictable and repeatable ways to increase the level of certain nutrients in the eggs. The repeatability of this process has allowed the commercialization of functional eggs, as both consumers and regulatory agencies accept and require truthfulness in labeling. Multiple nutritional enhancements of eggs can also be achieved fairly easily. Bourre (2005a) reported the nutritional values of a functional egg in France with the following enhancements when compared with a regular egg: iodine ¥ 2.5, vitamin D ¥ 3, selenium ¥ 4, folic acid ¥ 4, vitamin E ¥ 6, lutein and zeaxanthin ¥ 6, alpha-linolenic acid (ALA) ¥ 6, DHA ¥ 3, total omega-3 ¥ 4, vitamin B12 ¥ 1.4. Leeson and Caston (2003) demonstrated simultaneous enrichment in vitamin B12 (¥ 3.8), vitamin D3 (¥ 2.9), vitamin E (¥ 2.8), niacin (¥ 1.6), pantothenic acid (¥ 1.6), thiamin (¥ 1.3), vitamin A (¥ 1.2). Egg products are also easily nutritionally – enhanced by the addition of ingredients such as vitamins and mineral premixes and functional ingredients such as omega-3 rich fish oil (Rose and Holub, 2006; Kassis et al., 2010). Currently, some commercial liquid egg products are enriched with omega-3 (flax oil and/or fish oil), calcium, vitamins and minerals and lutein. Natural way to produce functional foods Consumers have become more and more concerned about food safety and the origin of their food (Devcich et al., 2007). This reversion to ‘nature’ is demonstrated by the growth of numerous organic food products over the past decade. The egg industry has the clear advantage of being able to enhance the nutritional value of eggs in a very natural way. Hens act as natural ‘bioconverters and condensers’. For example, hens convert ALA from flax into DHA (Caston and Leeson, 1990) and synthetic folic acid into natural folates (Hoey et al., 2009), with both DHA and natural folate deposited in greater amount into the eggs. Source of bioactive ingredients Over the past decade, a tremendous amount of work has been done to identify a number of bioactive ingredients in the egg white, the egg yolk, the eggshell and shell membrane (Mine and Kovacs-Nolan, 2004, 2006; Kovacs-Nolan et al,. 2005; Anton et al., 2006). The recent development of high throughput technology has helped identify a large number of novel egg
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Strategic planning for development of egg nutraceutical industry 379 molecules with interesting potential activities predicted by bioinformatics (volume 1, Chapters 7 and 9). The most widely recognized and used bioactive component is the lysozyme, a small protein (3.5% of the total proteins in the egg white) with well-established antimicrobial activity. Lysozyme was the first component of the egg to be specifically isolated on a large scale with commercial applications in the food and packaging industries, wine and beer productions, and in the pharmaceutical industry as well. Overall, various bioactive components in the eggs have demonstrated either in vitro or in vivo the following activities: antimicrobial, antiviral, antiadhesive, immunomodulating, anticancer, antihypertensive, antioxidant, protease inhibition and biospecific ligand (Kovacs-Nolan et al., 2005; Anton et al., 2006; Mine and Kovacs-Nolan, 2006; Chapter 16). Immunoglobulins from the yolk (IgY) The technique of producing and harvesting immunoglobulins (Ig) from the yolk after immunization of hens against organisms or protein fragments has been widely documented and reviewed (Mine and Kovacs-Nolan, 2002; Karlsson et al., 2004; Kovacs-Nolan and Mine, 2004; Schade et al., 2005; Chapter 17). In recent years, IgY have shown some activities against various organisms and conditions such as Escherichia coli in piglets (Yokoyama et al., 1992), arthritis in human (Greenblatt et al., 1998), various enteric pathogens in humans and animals (Mine and Kovacs, 2002), dental caries in humans (Koga et al., 2002), parasites such as Cryptosporidium parvum in mice (Kobayashi et al., 2004), Helicobacter pylori in gerbils (Nomura et al., 2005), rabies-neutralizing antibodies in mice (Motoi et al., 2005), canine parvovirus (Nguyen et al., 2006), Porphyromonas gingivalis gingipains in periodontitis patients (Yokoyama et al., 2007b), Pseudomonas aeruginosa in cystic fibrosis patients (Nilsson et al., 2007) and Candida albicans in mice (Ibrahim et al., 2008). The production of IgY offers a number of advantages over conventional methods for polyclonal antibodies, both in terms of production (easier, cheaper and most importantly less traumatic for the animals) and in terms of biological potential (no interaction with mammalian IgG, with the rheumatoid factor and the mammalian complement) (Kovacs-Nolan and Mine, 2004). Health benefits from functional eggs Several clinical studies have demonstrated the benefits of functional eggs enriched with specific nutrients. We will focus here on eggs enriched in lutein and omega-3 to illustrate this point. Surai et al. (2000) showed that the daily consumption of one designer egg enriched in vitamin E (¥ 26.8), lutein (¥ 15.9), selenium (¥ 7.7) and DHA (¥ 6.4) for 8 weeks led to a significant increase in plasma concentration of a-tocopherol (19%) and lutein (87%) when compared with volunteers consuming a regular egg. Chung et al. (2004) also demonstrated that lutein bioavailability was higher from lutein-enriched eggs (6 mg per egg) than from lutein supplements and spinach, a well-known
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380 Improving the safety and quality of eggs and egg products source of lutein with about 12.2 mg per 100 g. Therefore, lutein-enriched eggs may be the ideal food to increase the macular pigment concentration in older people and contribute to the prevention of age-related macular degeneration (Handelman et al., 1999; Krinsky et al., 2003; Krinsky and Johnson, 2005; Goodrow et al., 2006; Thurnham, 2007; Vishwanathan et al., 2009). The benefits of omega-3 enriched eggs have also been studied under different conditions. Ferrier et al. (1995), using eggs from hens fed a diet with 10% or 20% ground flaxseed, reported a significant increase in total n-3 fatty acids and DHA content in platelet phospholipids of volunteers consuming these eggs, indicating an increased n-3 status of the body. Similar results were also observed by Farrell (1998) and Sindelar et al. (2004) in healthy volunteers. Gillingham et al. (2005) also demonstrated the benefits of consuming daily two DHA-enriched eggs for 21 days (217 mg DHA/day) in statin-treated hypercholesterolemic male patients. Total serum cholesterol levels remained unchanged with egg consumption in these patients at risk. They observed a significant increase in total n-3 PUFA (17%), DHA levels (22%), DHA + eicosa pentaenoic acid (EPA) levels (23%) and a decrease in the ratio of n-6 to n-3 PUFA (15%) which can be related to a reduced risk for fatal ischemic heart disease. A few studies have also looked at the benefits of n-3 PUFA enriched shell eggs during the last trimester of pregnancy and during nursing. Gestation was significantly increased by 6 days in women consuming 12 eggs enriched with DHA per week (133 mg/egg vs. 33 mg/egg) and birth weights were increased, although not significantly (Smuts et al., 2003). The consumption during lactation of two n-3 PUFA enriched eggs (690 mg/egg) per day during 6 weeks resulted in a significant (P < 0.05) increase of total n-3 PUFA in breast milk (3.6%) compared with pre-test milk (1.9%), thus increasing the overall intake of these PUFA critical for normal growth and development (Cherian and Sim, 1996). Makrides et al. (2002) demonstrated that yolks from omega-3 enriched eggs (315 mg DHA/100 g vs. 73 mg DHA/100 g) given four times per week in breast-fed and formula-fed infants contributed to an increase in DHA intake in infants without altering their plasma cholesterol concentrations. The benefits of omega-3 PUFA have also been demonstrated with a breakfast made of functional liquid eggs providing 1.3 g/ day of combined EPA + DHA. Rose and Holub (2006) demonstrated in a randomized crossover trial that the breakfast containing these liquid eggs significantly decreased the plasma triglycerides levels by 32% (P < 0.05), the triglyceride : high density lipoprotein (HDL)-cholesterol ratio by 37% (P < 0.05) and moderately reduced blood pressures whereas no such effects were observed with the control breakfast. In addition, they observed no effects on total cholesterol and low density lipoprotein (LDL)-cholesterol levels. Overall, the blood serum levels of DHA + EPA reached after consumption of the omega-3 enriched liquid eggs translated into a low risk status for fatal ischemic heart disease.
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Strategic planning for development of egg nutraceutical industry 381 18.2.2 Weaknesses Lack of visual product differentiation Nothing looks more like an egg than another egg and this is especially true for functional eggs! While packed with extra vitamins, minerals or omega 3 fatty acids, functional eggs look the same to the consumers. Therefore, functional eggs require the establishment of a three-way trust between the producers, the consumers and the regulatory agency overseeing labeling rules and truthfulness. Any lack of trust will greatly affect the market expansion of functional eggs. The cholesterol issue Eggs have had a negative image since the 1970s when physicians were taught that checking serum cholesterol and initiating early treatment if high was the best action to reduce the risk of coronary heart disease (CHD) (Constance, 2009). The public was advised to consume daily no more than 300 mg of cholesterol and to limit the consumption of eggs. More than 10 years ago, Hu et al. (1999) demonstrated by reviewing data from two massive prospective cohort studies (about 120,000 individuals) that the consumption of up to one egg per day is unlikely to have a substantial impact on the risk of CHD or stroke among healthy men and women. Unfortunately, this message has been slow to reach both physicians and patients This contentious idea that eating dietary cholesterol, typically from eggs, increases the risk of developing coronary heart disease by increasing blood cholesterol prevails, despite a lack of scientific proof to support its existence. The need for re-evaluation of dietary recommendations to lift the restrictions on the intake of cholesterol-rich foods are discussed in Chapter 12. The value of eggs as functional foods is still affected by this lasting cholesterol issue. Attitudes towards functional foods A number of studies have attempted to understand the complex attitudes of key groups of individuals towards functional foods, namely the consumers and the health professionals. ∑
Consumer general acceptance: Overall, consumers do not see functional foods as one homogeneous product group and different attitudes affect the willingness to use different functional foods (Urala and Lähteenmäki, 2007). Bech-Larsen and Grunert (2003), studying consumers in Finland, Denmark and the USA, noted that the consumers’ acceptance towards a functional food was dependent on their perception of the nutritional qualities of the base-product. The same observations were made by van Kleef et al. (2005) in the Netherlands and Williams et al. (2008) in Australia. Considering the ever-lasting negative image of eggs in the consumers’ mind (see above on the cholesterol issue), eggs may not be the ideal base-product for enrichment.
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382 Improving the safety and quality of eggs and egg products ∑
Role of price: Bowman (2005) in a study of shoppers’ attitudes in the US reported that food price was important to 40% of adults who considered nutrition very important. So, even with the consumers primed for functional foods, the premium price carried by functional eggs will likely affect their buying decision. ∑ Role of knowledge and disbelief of information provided: Overall, consumers have little knowledge about nutrition and the benefits brought by functional foods (Black and Campbell, 2006; Cox and Bastiaans, 2007; Cox, 2008; Verbeke, 2008; Siró et al., 2008; Tudoran et al., 2009; Landstöm et al., 2009). In addition, consumers are also very skeptical about the information provided, especially by food manufacturers (Childs and Poryzees, 1997; Patch et al., 2005; Sibbel, 2007; Verbeke, 2008; Hailu et al., 2009; Landström et al., 2009). In the USA, where health claims have been allowed, communication of these benefits has had limited success and may have in fact misled consumers (Hasler, 2008). ∑ Health professionals: Health professionals are in a great position to influence the consumption of functional foods by their patients. Therefore, the study reported by Landström et al. (2007) in Sweden does not bring good news for functional foods. The interviewed physicians and registered nurses expressed more skepticism and distrust about functional foods than dieticians. Lack of trust, interest and proficiency in functional foods were given as explanations for these health professionals being less willing to recommend functional foods to patients. Role of claims (nutritional, functional and/or health claims) Verbeke (2005) and Verbeke et al. (2009) in Belgium, Bech-Larsen and Grunert (2003) in Finland, Denmark and the USA, Patch et al. (2005) in Australia and Hailu et al. (2009) in Canada have shown that strong belief in health benefits for functional foods and government support of health claims were a major positive determinant in the acceptance of functional foods. The absence of a regulatory framework in a number of countries for claims made has certainly had a negative effect on the consumers’ attitude towards the functional foods. Novel food regulatory process A novel food is defined as a food whose composition differs from the ‘original’ food and has not been consumed by humans to a great extend (Kwak and Jules, 2001b). The difference in composition may be due to the addition of a new nutrient not currently present (i.e. lycopene in eggs) or to the enrichment of a natural nutrient present in the egg (i.e. lutein). In a number of countries, this novel food regulatory process will limit quick development of new functional eggs where the targets for enrichment are normal nutrients in the egg.
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Strategic planning for development of egg nutraceutical industry 383 Investment in R&D and marketing In spite of heavy investment in product development and marketing, the functional food segment has been characterized by a high rate of product failures. While the egg industry can likely develop a new functional egg well below the estimated US$ one to two million reported as an average for the food industry (Menrad, 2003), the cost of marketing functional eggs is often beyond the typical marketing expenditures in the egg industry. The branding and advertising efforts and the intense and difficult consumer education required come at a cost not typically seen in the egg industry. For the identification of new bioactive molecules and their potential values for human health, the cost of R&D is simply beyond the means of the egg industry. Skill sets required New opportunities for the egg nutraceutical business will require qualified personnel in fields such as biochemistry, biomolecular technology, immunology and human nutrition just to name a few. These qualifications are currently in short supply in the egg industry. Allergenicity of the egg This inherent characteristic of the egg affects its potential as a source of ingredients for the food industry as a number of countries have implemented strict allergen declaration regulations. The allergenicity of eggs will always be a source of concern for any biopharmaceutical application of molecules derived from the eggs as demonstrated by the typical contra-indication for individuals with severe egg allergy to use influenza vaccines produced in embryonated eggs. Chapter 13 updates the biological information in this area. 18.2.3 Opportunities Egg as a healthy source of nutrients The positive message about cholesterol and eggs, leading a number of medical associations and heart foundations to remove their cautionary statement about egg consumption and risk of CVD, must continue to be broadcast as it will further enhance the value of eggs as functional foods (Gray and Griffin, 2009). We must continue to work with physicians and other health professionals (Pelletier et al., 2002; Scarborough et al., 2007; Jones, 2009; Constance, 2009; Ward, 2009). Some progress has already been made as demonstrated by a recent survey conducted in the UK. Scarborough et al. (2007) showed that eggs ranked 23 out of 120 foods in terms of healthiness in the mind of nutrition professionals. Foods ahead of eggs were mostly fruit and vegetables, a few fish and a few bread items, soya milk (unsweetened) and stewed rabbit!
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384 Improving the safety and quality of eggs and egg products Expand the usage of current functional eggs With the reinforcement of the message that eggs are a healthy and nutritious food, consumers should more readily accept them as a base-product/vehicle for enrichment (Bech-Larsen and Grunert, 2003; Williams et al., 2008). Eggs have also two distinct advantages as functional foods: (i) they look like a very familiar food ‘on the outside’ and therefore are more likely to be accepted by older people, one of the fastest growing target segment for these products (Saher et al., 2004; Bowman, 2005; Patch et al., 2005) and (ii) with few exceptions, the enrichment of nutrients has in general no impact on the taste of the eggs. As pointed out by Verbeke (2006), consumers no longer consider good taste and health as attributes for which making a trade-off is expected or even obligatory. ‘Pandemy’ of the omega-3 nutritional gap In spite of current government programs recommending specific daily intake for various foods, our modern diets are often lacking vegetables, fruits and fish. The low consumption of fish and its impact on the intake of EPA + DHA has been well documented (Simopoulos, 1999; Hibbeln et al., 2006; Brenna et al., 2007; Vermunt and Zock, 2007; Givens and Gibbs, 2008; Madden et al., 2009). For example, Givens and Gibbs (2008) estimated in the UK that the mean daily intake for EPA + DHA was 244 mg or only about 54% of the target of 450 mg (daily recommended intakes varies from approximately 270 to 700 mg depending on the country). Omega-3 enriched eggs, along with other foods have the potential to play a great role in compensating this nutritional gap as noted by several authors (Lewis et al., 2000; Bourre, 2004; Bourre, 2005b; Patch et al., 2005; Sparks, 2006; Murphy et al., 2007; Givens and Gibbs, 2008; Ferguson, 2009; Madden et al., 2009). The consumption of one egg with 100 mg DHA per day would close about 50% of the current nutritional gap in the UK. Consumers’ education Functional foods are a novel and puzzling entity to consumers as their benefits (the reward for the consumers) can often be seen only later in life. As such, consumers education will play a critical role for their success (Lajolo, 2002; Roberfroid, 2002; Menrad, 2003; Gray et al., 2003; Cox et al., 2007; Sibbel, 2007; Siró et al., 2008; Verbeke, 2008; Tudoran et al., 2009). Pelletier et al. (2002) in the USA showed that educational programs with consumers have a significant effect on the intention to consume more functional foods (52–79% depending on the type of foods). This education will be complex, requiring the interaction between the consumers and the food industry, the retailers, the health sector and the government (McConnon et al., 2002). Health claims Back in 2004, Hawkes reviewed for the World Health Organization the nutrition labels and health claims from a global perspective: out of 74
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Strategic planning for development of egg nutraceutical industry 385 countries surveyed, 39 countries had no regulations specific to health claims, 30 countries had a prohibition on claims making reference to diseases, 23 countries allowed nutrient function claims, 7 countries allowed specific disease risk-reduction claims and 3 countries having a specific framework to permit product-specific health claims. Since that time, more countries now allow health claims or have initiated a regulatory review process, notably in Europe (Asp and Bryngelsson, 2008) and Australia (Tapsell, 2008). These claims will further help the market expansion of functional foods, although Pothoulaki and Chryssochoidis (2009) pointed out that health claims may not play as important a role as price and taste do. New opportunities for IgY Numerous advantages of IgY over other methods for producing polyclonal antibodies have been reported (Mine and Kovacs-Nolan, 2002; Karlsson et al., 2004; Schade et al., 2005). As pointed out by Tini et al. (2002), it is time to publicize the IgY technology and its inherent advantages. Work must continue on the potential of IgY to reduce enteric diseases in humans. IgY, available in yolk powder, could be stored for long periods of time at room temperature, and incorporated into foods for infants. This would be an ideal scenario for lesser developed countries. IgY have also been reported as a potential tool for drug target discovery and proteomics (Zhang, 2003; Fang et al., 2004; Lillico et al., 2005). More work in this area will be required to confirm this potential. Identification of new bioactive components As noted by Gautron et al. (2007), the composition of the eggs is still not completely understood with only the major proteins identified (see Chapters 7 and 9 in Volume 1). New molecular approaches such as high throughput methods and the availability of the chicken genome database have already allowed the identification of hundreds of additional components of the egg. Recent publications have identified the potential for peptides derived from egg proteins to play a role in reducing the risk of cardiovascular disease (Erdmann et al., 2008), in the prevention of hypertension (Miguel and Aleixandre, 2006; Miguel et al., 2007) and the treatment of intestinal inflammation (Lee et al., 2009). Peptides obtained from the egg white lysozyme have also shown some promising antimicrobial activities (Mine et al., 2004; Abdou et al., 2007; You et al., 2010). Another interesting potential for the egg nutraceutical business is the use of eggshell membrane as support for the immobilization of various enzymes and the application in diagnostics and therapeutics (Li et al., 2008; Pundir et al., 2009). The use of hens as ‘bioreactors’ and eggs as a vehicle for the recovery of therapeutic proteins has also been proposed (Lillico et al., 2005). Such applications would represent another impressive opportunity for the egg nutraceutical business.
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386 Improving the safety and quality of eggs and egg products New ‘blockbuster’ nutrient for functional eggs How will we identify the next nutrient likely to boost the consumption of functional eggs? Considering the entire concept of functional foods originated in Japan, we should likely take a look at their current list of functional foods and ingredients approved (Shimizu, 2003; Ohama et al., 2006; Yamada et al., 2008). Another country to consider would be China where traditional medicines have been used for centuries (Arai, 2002; Yang, 2008). Pennington (2002) reviewed food composition databases in an effort to identify new bioactive food components. Another key document would be the publication by Diplock et al. (1999) presenting the scientific concepts of functional foods in Europe. The authors presented the blueprints for future research, outlining the target function, the possible biological markers and the candidate food components for each major research opportunity. 18.2.4 Threats For threats, we will consider both existing threats as well as potential threats anticipated in the near future. Regulatory environment While ‘staple foods’ such as eggs may be considered the ideal food for enrichment in ‘classic’ nutrients like folate, vitamins A or even omega-3 DHA, regulators in some countries have expressed concerns that enrichment of ‘staple’ foods with new functional ingredients may lead to over consumption and potentially raise food safety issues (Hasler, 2002; Kruger and Mann, 2003; ADA, 2004; Spence, 2006; Sibbel, 2007; Beer-Borst et al., 2008; L’Abbé et al., 2008; Yamada et al., 2008). In Japan, the inclusion of nutrition functional claims for a nutrient must be accompanied by a mandatory warning indication (MHLW, 2010). Consider the case of folic acid, since eggs easily enriched to levels of 120 mg/100g will meet the requirements to qualify without further authorization for the following nutrient function claim: ‘aids in the red blood cell formation, and contributes the normal growth of the fetus’. The mandatory ‘Warning indication’ associated with the claim will read Increased intake of this product will not result in curing diseases nor promoting health. Please comply with the advisable daily intake. This product helps normal development of fetus, but increased intake of this product will not result in better development of fetus. Each of the 17 ingredients (12 vitamins and 5 minerals), for which a functional claim has been approved in Japan, will carry a warning indication. The inclusion of such warning statements would likely further confuse consumers in many countries where functional foods are not part of the culture as they have now been in Japan for almost 30 years. Such warning statements would make the marketing of functional eggs even more challenging.
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Strategic planning for development of egg nutraceutical industry 387 Competition from other animal products and plants Other food animal products are competing with the eggs for their share of the growing functional food market (Bourre, 2004, 2005b; Kues and Nienmann, 2004; Gebauer et al., 2006; Palmquist, 2009). Biofortification of crops, through plant breeding or genetic engineering strategies, enhance their nutritional values and these new varieties are now competing for their share of this lucrative functional food market (Zimmermann and Hurrell, 2002; Hasler, 2002; Pennington, 2002; Ursin, 2003; White and Broadley, 2005). New competitors With the attraction of lucrative sales, a number of new players have entered the functional food and dietary supplement business. The pharmaceutical industry has become more interested in this field, mainly for its potential as an adjunct to drug therapy for patients (Síró et al., 2008; Eussen et al., 2010). Known for its R&D and marketing expertise, the pharmaceutical industry will be a fierce rival for the egg nutraceutical industry. Lack of patent protection Most of the innovation in the field of functional eggs is based on what is often called ‘public knowledge’ information. Therefore, innovations and new product ideas cannot be protected by patents. Innovators will be quickly copied by others. ‘Mining of the egg’ for others While the egg is a great ‘mine’ for new bioactive molecules, there is the risk that scientists will develop ways to produce them by genetic engineering without any further need for the eggs and the laying hens. This will constitute another threat to the growth of the egg nutraceutical business. New epidemic diseases affecting poultry Each recent outbreak of influenza in birds has resulted in a reduction of egg consumption regardless of the expert advice given by health officials. Given the competitive environment of the food industry, each consumer lost will be harder to gain back, both in terms of time and expenses.
18.3 Strategic goals for the egg nutraceutical business With the SWOT analysis now completed (Table 18.2), we will briefly summarize the major trends observed. From a strategic standpoint, the industry must continue to play on the strengths of the business, act upon some of the weaknesses to convert them possibly into opportunities, and convert some opportunities into strengths. This is easily said but hard to do! Threats must also be dealt with, to eliminate them at best or minimize their impact on the business at worst. © Woodhead Publishing Limited, 2011
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388 Improving the safety and quality of eggs and egg products Table 18.2 SWOT analysis of the egg nutraceutical business Strenghts
Weakenesses
Opportunities
Threats
∑ Worldwide acceptance of eggs ∑ Source of proteins, vitamins and minerals ∑ Ease of production ∑ ‘Natural’ way of production ∑ Source of bioactive ingredients ∑ IgY ∑ Health benefits clinically proven
∑ Lack of visual differentiation ∑ Cholesterol issue ∑ Attitudes towards functional foods ∑ Role of claims ∑ Novel food process ∑ Investment in R&D and marketing ∑ Skill sets ∑ Allergenicity of eggs
∑ Healthy source of nutrients ∑ Increased consumption ∑ Omega-3 nutritional gap ‘pandemic’ ∑ Consumer education ∑ Health claims ∑ New opportunities for IgY ∑ New bioactive components ∑ New functional eggs
∑ Regulatory environment ∑ Competition from other foods ∑ New competitors ∑ Lack of patent protection ∑ ‘Mining’ the eggs for others ∑ New epidemic disease in poultry
The major trends observed are as follows: ∑
The usage of functional foods will continue to expand globally and functional eggs represent the major opportunity for the egg nutraceutical business. ∑ The expansion of the functional eggs business will be achieved using a new set of skills for the industry. Communication to consumers is complex because of the nature of the message and their lack of trust towards the food industry. This communication to consumers must also involve others players in a concerted approach, again making this process even more challenging. ∑ Governments and the health community can play a major role in the success of functional eggs but can also affect their success. ∑ The egg is still a ‘mine’ for bioactive molecules but more applications and usage of its current components must be developed commercially. New development will be costly in terms of the R&D. Novel food applications will need to cover a number of fields: human nutrition, animal nutrition, toxicology, chemistry and biochemistry to list a few. ‘Mining of the egg’ will become more and more high-tech and costly. The following strategic goals are therefore proposed: ∑ ∑
expand the usage of current functional eggs; identify new nutrient candidates for functional eggs and new bioactive egg components; ∑ develop strategic partnerships and alliances.
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Strategic planning for development of egg nutraceutical industry 389
18.4 Action plan for the egg nutraceutical business Having set three strategic objectives, we can now outline a possible plan of action. There are obviously a number of ways to address these strategic goals. Variations will be needed on a local basis to accommodate a number of factors inherent to each country/region: current status of the egg nutraceutical market, regulatory environment, socio-economic development, nutritional status of the population and availability of local expertise for research. 18.4.1 Expand the usage of current functional eggs The situation is almost perfect for functional eggs: ∑
There is a growing nutritional need for some of the nutrients present in the functional eggs (Simopoulos, 1999; Hibbeln et al., 2006; Brenna et al., 2007; Madden et al., 2009). ∑ This positive message is not coming only from egg producers and food manufacturers – the scientific community, health professionals and government officials have recognized the potential for these functional eggs (Bourre, 2004, 2005b; ADA, 2004; Murphy et al., 2007; Givens and Gibbs, 2008). ∑ Governments and health authorities have recognized the nutritional gaps and their potential cost to the health-care system, Japan being without contest the first country to have done so (Kwak and Jukes, 2001a; Arai, 2002; Hasler, 2002; Lajolo, 2002; Milner, 2002; Ohama et al., 2006; van Raaj et al., 2008). Marketing/branding of products Focused marketing efforts will communicate the attributes and benefits of specific products and ensure that consumers do not see eggs as a generic food. Strong branding will capture customers’ loyalty, so critical in an environment free of any type of patent protection; Need to gain the support of health officials Health officials have a goal of increasing the consumption of PUFA (Weststrate et al., 2002; van Raaj et al., 2008) and the egg industry has a viable and proven solution. There must be some increased interactions between all stakeholders to ensure functional eggs play their part in the closing of this ‘nutritional gap’ (McConnon et al., 2002; van Raaj et al., 2008). Omega-3 fatty acids have been often cited as an example but functional eggs can also address other nutritional gaps such as the ones for vitamin A, vitamin E and selenium. Recruit health professionals for trusted communication We will again take the example of the omega-3 shell eggs to illustrate the complexity of the message. In fact, the egg nutraceutical industry is faced with a real dilemma: © Woodhead Publishing Limited, 2011
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390 Improving the safety and quality of eggs and egg products ∑ ∑ ∑ ∑ ∑
∑
the average consumer lacks a proper understanding of omega-3 nutrition and needs; omega-3 is a family of fatty acids, some with short and some with long chain, and within the long chain, EPA and DHA are more important than alpha-linolenic acid (ALA) present mostly in plants; the required intake for EPA and DHA is also dependent on the consumption of omega-6 fatty acids, present in numerous processed foods, thus adding another level of complexity to the message to consumers; consumers have a limited trust in the information provided by the industry; health professionals are often reluctant to recommend functional foods, mostly due to a lack of confidence and knowledge in this field (Gebauer et al., 2006; Hibbeln et al., 2006; Landström et al., 2007; Givens and Gibbs, 2008; Sibbel, 2007; Verbeke, 2008). Clearly, the egg nutraceutical business needs health professionals but will have to be proactive in this interaction to ensure they receive the right information and tools to communicate targeted messages to their patients and consumers in general.
18.4.2 Identify new nutrient candidates for functional eggs and bioactive egg components The quest is to identify the new ‘DHA’ or the new ‘lysozyme’ of the egg nutraceutical business. This search is becoming more complex and now requires a concerted effort between plant scientists, chemists and biochemists, poultry scientists, immunologists, nutritionists, clinicians and physicians, toxicologists and regulatory experts to name only a few. The rising cost of R&D and the high rate of failure means that the egg nutraceutical industry must act wisely as it cannot afford to invest in a lot of pure fundamental research. Look at other industries Some large multinational companies have now embraced the functional food business. With their level of R&D investment estimated to be around 2% of sales, these companies will likely come up with a few ideas (Hasler, 2002; Menrad, 2003; Mark-Hebert, 2004). It will then be up to the egg industry to quickly adopt and adapt. Plant genetics and bio-fortifications of plants are also thriving fields which should be monitored routinely for new ideas. Look at traditional plant medicines The functional food concept originated in Japan and has been researched for almost 30 years (Ohama et al., 2006). Likewise, ancient civilizations like the Chinese or Native Americans have used plant medicines for centuries; would the next functional nutrient come from these sources?
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Strategic planning for development of egg nutraceutical industry 391 Search of current databases and development programs There is already many information in the public domain. The use of the Internet is a fantastic tool to review current R&D projects commissioned by various governments and research institutes around the world. A trend observed in most of the recent publications is the cooperation between scientists from various countries and continents. Develop specific research programs Owing to the complexity of the task and the cost associated, these programs will likely require some type of alliances (see below). 18.4.3 Develop strategic partnership and alliances Many of the opportunities identified will require a lot of work in R&D, both fundamental and applied research and in marketing to build awareness among targeted populations and in promotion to build a strong brand. Therefore, partnerships must be developed with research institutes, ingredient manufacturers, food manufacturers, retailers and government agencies to maximize these opportunities. R&D will require a well-coordinated multidisciplinary effort (Milner, 2002; Fogliano and Vitaglione, 2005) with food scientists, agronomists, poultry scientists, molecular biologists, geneticists, nutritionists, clinical researchers and statisticians working together. Likewise, the registration of new nutritional or functional claims will significantly increase the overall cost of innovation (Lucas, 2002; Roberfroid, 2002; Mark-Herbert, 2004). Some possible areas where strategic partnership would be beneficial to the industry are listed below: ∑ ∑
∑ ∑ ∑ ∑ ∑ ∑
identification of new functional and bioactive ingredients; identification of new bio-markers: these biomarkers are essential to demonstrate scientifically the efficacy of functional ingredients for specific health claims (Roberfroid, 2002; Milner, 2004; Elliott et al., 2007); clinical investigations in healthy and diseased subjects; government funding and incentives for research (Belem, 1999; Herath et al., 2010); campaigns to raise consumer awareness of nutritional gaps and available solutions; consultations with health professionals and government agencies; co-branding and co-marketing with large multinational companies involved in the food, supplement or pharmaceutical industries (MarkHerbert, 2004); co-development with small biotechnology companies or pharmaceutical companies (Mark-Hebert, 2004).
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392 Improving the safety and quality of eggs and egg products
18.5 Conclusion The egg nutraceutical business has been striving for the past 30 years and has still to realize all its potential. The SWOT analysis presented some fantastic opportunities to capture and some weaknesses to improve upon. The three strategic goals identified were: the expansion of the usage of functional eggs, the discovery of new nutrients for functional eggs and new bioactive components in the egg and finally the creation of strategic alliances and partnerships. This last strategic objective is considered as the most critical for the continuing success of the egg nutraceutical industry as it will be at the center of any new development and new opportunity.
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Strategic planning for development of egg nutraceutical industry 397 the prevention of Pseudomonas aeruginosa infections: a case report’, Journal of Medicinal Food, 10, 375–378 nomura s, suzuki h, masaoka t, kurabayashi k, ishii h, kitajima m, nomoto k and hibi t (2005), ‘Effect of dietary anti-urease immunoglobulin Y on Helicobacter pylori infection in Mongolian gerbils’, Helicobacter, 10, 43–52 ohama h, ikeda h and moriyama h (2006), ‘Health foods and foods with health claims in Japan’, Toxicology, 221, 95–111 palmquist d (2009), ‘Omega-3 fatty acids in metabolism, health, and nutrition and for modified animal product foods’, The Professional Animal Scientist, 25, 207–249 patch c, tapsell l and williams p (2005), ‘Overweight consumers’ salient beliefs on omega-3 enriched functional foods in Australia’s Illawarra region’, Journal of Nutrition Education and Behavior, 37, 83–89 pelletier s, kundrat s and hasler c (2002), ‘Effects of an educational program on intent to consumer functional foods’, Journal of the American Dietetic Association, 102, 1297–1300 pennington j (2002), ‘Food composition databases for bioactive food components’, Journal of Food Composition and Analysis, 15, 419–434 pothoulaki m and chryssochoidis g (2009), ‘Health claims: consumers’ matters’, Journal of Functional Foods, 1, 222–228 pundir c, bhambi m, chauhan n (2009), ‘Chemical activation of egg shell membrane for covalent immobilization of enzymes and its evaluation as inert support in urinary oxalate determination’, Talanta, 77, 1688–1693 roberfroid m (2002), ‘Global view on functional foods: European perspectives’, British Journal of Nutrition, 88, S133–S138 rose e and holub b (2006), ‘Effects of a liquid egg product containing fish oil on selected cardiovascular disease risk factors: a randomized crossover trial’, Food Research International, 39, 910–916 saher m, arvola a, linderman m and lähteenmäki l (2004), ‘Impressions of functional food consumers’, Appetite, 42, 79–89 scarborough p, rayner m, stockley l and black a (2007), ‘Nutrition professionals’ perception of the ‘healthiness’ of individual foods’, Public Health Nutrition, 10, 346–353 schade r, calzado e, sarmiento r, chacana p, porankiewicz-asplund j and terzolo h (2005), ‘Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine’, Alternatives to Laboratory Animals, 33, 129–154 shimizu t (2003), ‘Health claims on functional foods: the Japanese regulations and an international comparison’, Nutrition Research Reviews, 16, 241–252 sibbel a (2007), ‘The sustainability of functional foods’, Social Science & Medicine, 64, 554–561 simopoulos a (1999), ‘Essential fatty acids in health and chronic disease’, American Journal of Clinical Nutrition, 70, 560S–569S sindelar c, scheeger s, plugge s, eskridge k, wander r and lewis n (2004), ‘Serum lipids of physically active adults consuming omega-3 fatty acid-enriched eggs or conventional eggs’, Nutrition Research, 24, 731–739 siró i, kápolna e, kápolna b and lugasi a (2008), ‘Functional food. Product development, marketing and consumer acceptance – a review’, Appetite, 51, 456–467 skřivan m, skřvanová v and marounek m (2005), ‘Effects of dietary zinc, iron, and copper in layer feed on distribution of these elements in eggs, liver, excreta, soil, and herbage’, Poultry Science, 84, 1570–1575 smuts c, huang m, mundy d, plasse t, major s and carlson s (2003), ‘A randomized trial of docosahexaenoic acid supplement during the third trimester of pregnancy’, Obstetrics & Gynecology, 101, 469–479 sparks n (2006), ‘The hen’s egg – is its role in human nutrition changing?’, World’s Poultry Science Journal, 62, 308–315 © Woodhead Publishing Limited, 2011
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398 Improving the safety and quality of eggs and egg products (2006), ‘Challenges related to the composition of functional foods’, Journal of Food Composition and Analysis, 19, S4–S6 surai p and sparks n (2001), ‘Designer eggs: from improvement of egg composition to functional food’, Trends in Food Science & Technology, 12, 7–16 surai p, macpherson a, speake b and sparks n (2000), ‘Designer egg evaluation in a controlled trial’, European Journal of Clinical Nutrition, 54, 298–305 tapsell l (2008), ‘Evidence for health claims: a perspective from the Australia–New Zealand region’, Journal of Nutrition, 138, 1206S–1209S thurnham d (2007), Macular zeaxanthins and lutein – a review of dietary sources and bioavailability and some relationships with macular pigment optical density and agerelated macular disease’, Nutrition Research Reviews, 20, 163–179 tini m, jewell u, camenisch g, chilov d and gassmann m (2002), ‘Generation and application of chicken egg-yolk antibodies’, Comparative Biochemistry and Physiology Part A, 131, 569–574 tudoran a, olsen s and dopico d (2009), ‘The effect of health benefit information on consumers health value, attitudes and intentions’, Appetite, 52, 568–579 urala n and lähteenmäki l (2007), ‘Consumers’ changing attitudes towards functional foods’, Food Quality and Preference, 18, 1–12 ursin v (2003), ‘Modification of plant lipids for human health: development of functional land-based omega-3 fatty acids’, Journal of Nutrition, 133, 4271–4274 van kleef e, van trijp h and luning p (2005), ‘Functional foods: health claim-food product compatibility and the impact of health claim framing on consumer evaluation’, Appetite, 44, 299–308 van raaij j, hendriksen m and verhagen h (2008), ‘Potential for improvement of population diet through reformulation of commonly eaten foods’, Public Health Nutrition, 12, 325–330 verbeke w (2005), ‘Consumer acceptance of functional foods: socio-demographic, cognitive and attitudinal determinants’, Food Quality and Preference, 16, 45–57 verbeke w (2006), ‘Functional foods: consumer willingness to compromise on taste for health?’, Food Quality and Preference, 17, 126–131 verbeke w (2008), ‘Impact of communication on consumers’ food choices’, Proceedings of the Nutrition Society, 67, 281–288 verbeke w, scholderer j and lähteenmäki l (2009), ‘Consumer appeal of nutrition and health claims in three existing product concepts’, Appetite, 52, 684–692 vermunt s and zock p (2007), ‘Intake and recommendations of omega 3 fatty acids across the globe’, Annals of Nutrition and Metabolism, 51 (Suppl. 1), 133 vishwanathan r, goodrow-kotyla e, wooten b, wilson t and nicolosi r (2009), ‘Consumption of 2 and 4 egg yolks/d for 5 wk increases macular pigment concentrations in older adults with low macular pigment taking cholesterol-lowering statins’, American Journal of Clinical Nutrition, 90, 1272–1279 ward r (2009), ‘Talking with your patients about dietary cholesterol, diet and nutrition: best practices for family physicians’, International Journal of Clinical Practice, 63 (Suppl. 163), 22–26 weststrate j, van poppel g and verschuren p (2002), ‘Functional foods, trends and future’, British Journal of Nutrition, 88, S233–S235 white p and broadley m (2005), ‘Biofortifying crops with essential mineral elements’, Trends in Plant Science, 10, 586–593 williams p, ridges l, batterham m, ripper b and hung m (2008), ‘Australian consumer attitudes to health claim – food product compatibility’, Food Policy, 33, 640–643 yamada k, sato-mito n, nagata j and umegaki k (2008), ‘Health claims evidence requirements in Japan’, Journal of Nutrition, 138, 1192S–1198S yang y (2008), ‘Scientific substantiation of functional food health claims in China’, Journal of Nutrition, 138, 1199S–1205S yokoyama h, peralta r, diaz r, sendo s, ikemori y and kodama y (1992), ‘Passive spence j
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Strategic planning for development of egg nutraceutical industry 399 protective effect of chicken egg yolk immunoglobulins against experimental enterotoxigenic Escherichia coli infection in neonatal piglets’, Infection and Immunity, 60, 998–1007 yokoyama k, sugano n, rahman a, oshikawa m and ito k (2007a), ‘Activity of antiPorphyromonas gingivalis egg yolk antibody against gingipains in vitro’, Oral Microbiology Immunology, 22, 352–355 yokoyama k, sugano n, shimada t, shofiqur r, ibrahim e, isoda r, umeda k, nguyen s, kodama y and ito k (2007b), ‘Effects of egg yolk antibody against Porphyromonas gingivalis gingipains in periodontitis patients’, Journal of Oral Science, 49, 201–206 you s, udenigwe c, aluko r and wu j (2010), ‘Multifunctional peptides from egg white lysozyme’, Food Research International, 43, 848–855 zhang w (2003), ‘The use of gene-specific IgY antibodies for drug target discovery’, Drug Discovery Today, 8, 364–371 zimmermann m and hurrell r (2002), ‘Improving iron, zinc and vitamin A nutrition through plant biotechnology’, Current Opinion in Biotechnology, 13, 142–145
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Index
a-Livetin (GAL d5), 259 absorption, distribution, metabolism and excretion (ADME), 66–7 acceptable daily intake, 64 Acinetobacter baumanii, 48 adaptive (immune) mechanisms, 128 aerobic plating method, 7 Aerococcus, 7 affinity chromatography, 350 age-related macular degeneration, 225 Agricultural Marketing Service (AMS), 6 Alcaligenes, 6 Alcaligenes sp., 48 alternating current, 189 alternative egg decontamination techniques egg washing, 181–194 future trends, 195 gas plasma, 188–92 hot air pasteurisation, 182–5 microwave pasteurisation, 186–8 pulsed light, 192–4 washing methods in industry, 182 gas plasma, 188–92 air plasma glow emission spectra by RBD prototype, 191 RBD prototype cabinet and electronics set up, 189 resistive barrier discharge (RBD) configuration, 190 hot air pasteurisation, 182–5 hot air treatment prototype, 184 numerical model, air and egg interaction, 184 microwave pasteurisation, 186–8 traditional microwave oven, eggshell decontamination usage, 187
pulsed light, 192–4 schematic representation, pulsed light decontamination method, 193 American Heart Association, 247 ammonium sulfate precipitation, 349 angiotensin converting enzyme, 220, 332–4 animal feed additives, 63–5 antibacterial activity, 329–32 lysozyme effect on Gram-positive cell walls, 330 antibody activity, 327–9 IgY structure, 328 antigen presenting cells, 256 antihypertensive activity, 332–4 in vitro ACE inhibitory activity, 333 antioxidant activity, 334–6 iron fixation by phosvitin versus pH and ionic strength (M) values, 336 aquaculture, 358–9 arachidonic acids, 277 aroA, 124 atmospheric pressure plasma jet (APPJ), 190 avian immune system, 347–8 antibodies distribution in hen eggs, 348 bacterial shell contaminants, 19 bactericidal/permeability-increasing (BPI) domains, 53 bacterin, 122–3 basophils, 256 battery cage system, 108 Bayesian statistical techniques, 39 binary ionisation technology (BIT), 191 bioavailability, 214–16 biological value, 217 biosensors, 73
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Index 401 biotin, 224 boot swabs, 86–7 British Dietetic Association, 247 British Heart Foundation, 247 British Nutrition Foundation, 279 Broadcast Advertising Clearance Centre, 243 buffered-peptone water, 90 bursa of Fabricius, 347 caecal sampling, 84–5 Campylobacter, 35–6, 37, 38 carbohydrates, 222 carboxymethylation, 260 cardiovascular disease, 237–8 Carnobacterium sp., 48 carry-over rates, 70–1 CD4+ T lymphocytes, 256 cell membrane rupture, 186 Centres for Disease Control and Prevention, 21, 114 chemical contaminants and chemical residues in eggs, 62–77 animal-derived foodstuffs, 63–6 monitoring strategies, 71–3 non-conformities and rearing risk prevention, 74–6 transfer modes, 66–71 animal-derived foodstuffs, 63–6 regulated substances, 63–5 unintentional contaminants, 65–6 veterinary drugs in eggs, maximum residue limits (MRL), 65 chemical decontamination, 155–6 chemical residues and contaminants in eggs, 62–77 animal-derived foodstuffs, 63–6 monitoring strategies, 71–3 non-conformities and rearing risk prevention, 74–6 transfer modes, 66–71 chiffonette swabs see hand-held gauze swabs chlorination, 154 cholecalciferol, 294–5 cholesterol, 276 cholesterol esters, 276 Citrobacter, 6 cleaning and disinfection (C&D) procedures, 90 cloacal swabs, 85 Commission Decision 97/747/EC, 72 Commission Decision 2002/657/EC, 71–2 Commission Decision 2003/181/EC, 72 Commission Regulation 2160/2003, 148 Commission Regulation (EU) 37/2010, 72 Committee on Medical Aspects of Food, 244 competitive exclusion, 130 conjugated linoleic acid, 221, 283 conventional isolation procedures, 154 coronary heart disease, 238–43 Council Directive 96/23/EC, 71–2 cross-contamination, 37, 153–4 cross-reactivity, 263
crossed radio-immmunoelectrophoresis, 259 cryoprotective activity, 336–9 LDL efficiency for spermatozoa, 338 cystic fibrosis, 354–5 cytokines, 256 darkling beetle, 151 de novo synthesis, 279 dental caries, 355–6 Dermanyssus galinae, 152 DHA content, 281 diabetes, 248 dielectric barrier discharge (DBD) method, 189 dietary cholesterol consensus and recommendations, 246–9 dietary cholesterol, eggs, and diabetes development, 248 inter-individual variation molecular basis and dietary cholesterol hyperresponsiveness, 248–9 egg nutrition, 237–8 raw hen’s egg composition, 238 facts on disease and eggs, 237–50 cholesterol perception impact on egg consumption, 243 dietary saturated fat and cholesterol effects, 245–6 egg-feeding studies evidence in humans, 243–5 serum cholesterol and dietary cholesterol, coronary heart disease risk factors, 239–43 direct current, 189 diverse genetic mechanisms, 128–9 dl-alpha-TA, 296–7 DNA vaccines, 349 drag swabs, 86–9 dust collection and testing, 89 EC Directive 90/496/EC, 290 EC Regulation 1924/2006, 291 Edwardsiella tarda, 358–9 EFSA, 109 egg allergy, 254–66 components, 257–9 egg white allergens, 258–9 egg yolk allergens, 259 identified egg allergens, molecular and biological properties, 258 effects of processing on the allergenicity, 264–6 enzymatic fragmentation, 265 gamma-irradiation, 265–6 heat application, 264–5 other food processing methods, 266 egg proteins, 259–63 egg yolk allergenicity, 263–4 future trends, 266 mucosal immune response, 256–7 cellular and molecular mechanisms, 257
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402 Index natural history, 255–6 egg cleaning, 164–5 egg contamination reproductive tract development, 49–51 ovary colonisation, 51 upper oviduct colonisation, 50–1 vaginal colonisation, 50 Salmonella enteritidis, 46–57 contamination during reproductive tract development, 49–51 eggshell penetration, 47–9 eggshell surface contamination, 47 virulence factors, chicken reproductive tract colonisation, 51–7 egg decontamination egg washing, 163–77 benefits and problems, 175–6 historical and commercial perspective, 164–6 microbial quality factors, 169–73 post-washing treatments, 173–4 process, 166–9 egg foodborne disease hazard identification, 35–8 egg products, 37–8 eggshells, 36–7 farm, 35–6 microbial hazards and Salmonella enteritidis risk assessment, 34–41 quantitative risk assessment: Salmonella enteritidis in eggshell, 38–41 Egg Grading Manual, 170 egg lipid modification diet effect on lipid components, 279–83 n-3 PUFA concentration in yolk, 280 egg lipid fractions, 274–6 major lipid fraction distribution, low and high density yolk fractions, 274 major yolk lipids proportion, 275 major yolk phospholipids proportion, 275 fatty acid metabolism, laying hens, 276–8 n-6 and n-3 PUFAs biosynthesis, 278 human health, 272–84 enriched eggs sensory characteristics, 283–4 egg nutraceutical industry action plan, 389–91 expand usage of current functional eggs, 389–90 new nutrient candidates for functional eggs and bioactive egg components, 390–1 strategic partnership and alliances, 391 strategic development planning, 374–92 action plan, 389–91 strategic goals, 387–8 strengths, weaknesses, opportunities and threats (SWOT) analysis, 375–87 egg nutraceutical business, 388 opportunities, 383–6
strengths, 375–80 threats, 386–7 weaknesses, 381–3 egg-packing plant, 37 egg product processing, 38 egg proteins, 259–63 cross-reactivity, 263 glycosylation role, 262–3 stability and allergenicity, 259–62 egg spoilage, 20 egg washing alternative egg decontamination techniques, 181–194 future trends, 195 gas plasma, 188–92 microwave pasteurisation, 186–8 pulsed light, 192–4 washing methods in industry, 182 benefits and problems, 175–6 benefits, 175 optimal conditions, 176 problems, 175–6 egg decontamination, 163–77 historical and commercial perspective, 164–6 hot air pasteurisation, 182–5 hot air treatment prototype, 184 numerical model, air and egg interaction, 184 microbial quality factors, 169–73 sanitisers, 172–3 storage conditions prior to washing, 171–2 time, egg water contact, 171 wash and rinse water temperature, 169–71 water quality, 172 post-washing treatments, 173–4 egg oiling, 173–4 storage conditions, 174 process, 166–9 drying process, 168–9 rinsing process, 168 typical in-line egg washing system with brushes, 167 washing process, 166–8 wetting process, 166 egg white, 325–7 composition, physico-chemical and functional properties, 326 composition of hen egg white, 325 egg yolk, 322–5 chemical composition, 322 macrostructure and main constituents, 322–5 composition of hen egg yolk, 322 pH and ionic strength, 325 plasma and granules fractionation, 323 repartition of hen egg yolk constituents, 324 eggs allergenicity, 383
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Index 403 alternative antibody source, 350–2 comparison of specific antibody preparation, 350 biological activities, 321–40 antibody applications, 327–9 bioactive properties, 329–36 cryoprotective activity, 336–9 egg fractions, 322–7 chemical residues and contaminants, 62–77 animal-derived foodstuffs, 63–6 composition and variability, 204–14 comparison of content from official Spanish database and analytical data, 210 egg nutritional claims, potential options, 215 minerals content, 208 nutrient and energy content, 205 nutrient concentration (per 100g or per portion), 204–12 nutrient content per 100g of whole eggs, 207 nutrient density and nutrient profiles, 213–14 nutrition claims, 214 RDI, 212–13 vitamins content, 209 whole egg, egg yolk and white composition, 211 dietary saturated fat and dietary cholesterol effects, serum cholesterol, 245–6 blood total cholesterol concentration mean (SE) changes, 246 egg-feeding studies evidence in humans, 243–5 plasma cholesterol change in eggfeeding studies, 245 facts on dietary cholesterol and disease, 237–50 cholesterol perception impact on egg consumption, 243 consensus and recommendations, 246–9 egg nutrition, 237–8 healthy source of nutrients, 383 improving nutritional quality, 226–8 methods, 226 problems or nutritional improvement consequences, 226–8 macronutrients evaluation, 217–22 micronutrients evaluation, 222–6 monitoring strategies, 71–3 analysis methods, 72–3 regulation, 71–2 regulatory plans results, 73 non-conformities and rearing risk prevention, 74–6 environmental contaminants, 74–6 feed items proportion, ingested by laying hens, 76 veterinary drugs and additives, 74 nutritional evaluation, 203–17 composition and variability, 204–14
composition versus bioavailability, 214–17 nutritional quality, 201–28 egg reputation, 201–3 eggs composition nutritional evaluation, 203–17 serum cholesterol and dietary cholesterol, coronary heart disease risk factors, 238–43 blood cholesterol response equations, 240–1 human dietary intervention studies limitations, 242–3 LDL receptor pathway, 241–2 low density lipoprotein (LDL) receptor pathway, 242 serum cholesterol concentration and coronary heart disease relationship and MRFIT total mortality, 240 transfer modes, 66–71 animal transfer, 66–67 bioconcentration factor, polychlorinated dioxins/furans, 71 carry-over rate and bioconcentration factor, persistent organic pollutants, 71 contaminants and drugs distribution, egg’s component, 67 contaminants and drugs kinetics transfer, 67–70 non-laying impact, non-dioxin-like PCBs kinetics, 70 transfer and bioaccumulation factors, 70–1 veterinary drugs residues distribution, 68 yolk and benzo[a]pyrene or lindane relationship, 69 vitamins and trace mineral enrichment, 289–314 enrichment with vitamins, 290–301 trace minerals, 304–14 water soluble vitamins, 301–4 eggshell, 327 eggshell contamination, 37 eicosanoid biosynthesis, 277 electroporation, 186 Enterobacter, 6 envenomation, 359–60 environmental contaminants, 74–6 ingested soil quantity, 75–6 soil contaminants accessibility, 76 environmental sampling, 85 enzymatic fragmentation, 265 enzyme linked immunosorbent assay, 73, 94, 110, 261 eosinophils, 256 Escherichia coli, 6, 185 Escherichia coli infection, 356–7 essential fatty acids, 276–7 European Baseline Study, 110 European Community Reference Laboratories, 72
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404 Index European Food Information Resource Network, 204 European Food Safety Authority, 65, 122–3, 166 European Medicines Agency, 64 European Nutrition and Health Report, 223 European Union (EU), 46 evening primrose seeds, 279–80 faecal culture samples, 85 fast switching tandem mass spectrometry, 73 fat-soluble vitamins, 222–3 fatty acids, 275–6 fish oil, 281–2 fishy taints, 283–4 flagella, 56 Flavobacterium, 6 flaxseed, 279–80 flock prevalence, 39–40 floor-raised system, 109–10 folacin, 302 folate, 301–4 effect of dietary folic acid on egg folate content, 302 folic acid, 224 Food and Agriculture Organisation, 24, 39–40 Food and Drug Administration, 192 Food Chain Approach, 24 Food Composition Databank systems, 204 food processing methods, 264 food pyramid, 202 Food Safety and Inspection Services, 187 foodborne disease outbreak, 22 forced convection (FC) method, 183 Framingham study, 239–40 free cholesterol, 274, 276 gamma-irradiation, 265–6 gas plasma, 188–92 gastroenteritis, 357–8 gastrointestinal colonisation control, 129–33 Good Manufacturing Practices (GMPs), 15 granules, 322–3 guideline daily amounts, 213 hand-held gauze swabs, 89–90 haugh unit (HU), 173–4 hazard analysis critical control point system, 14, 176 health claims, 384–5 Helicobacter pylori infection, 355 helper T cells, 256 high density lipoprotein, 246 high resolution gas chromatography, 73 high resolution mass spectrometry, 73 hilA mutant strain, 124 horizontal transmission, 47, 122 hot air gun treatment, 183 hot air pasteurisation, 182–5 houseflies, 151–2 housing system epidemiology, Salmonella infection in laying hens, 107–115
housing system factors, Salmonella prevalence, 111–14 S. enteritidis and serotypes, outdoor production systems, 114 Salmonella prevalence, 108–11 human health modifying egg lipids, 272–84 egg lipid fractions, 274–6 enriched eggs sensory characteristics, 283–4 fatty acid metabolism, laying hens, 276–8 hen’s diet effect, lipid components, 279–83 hydrophobic interaction chromatography, 349–50 IgA, 347–8 IgG, 351 IgM, 347–8 IgY, 327–9, 379, 385 applications, 352–4 diagnostic and analytical applications, 353–4 immunoaffinity ligand, 352–3 health, diagnostic and other industrial applications, 346–61 advantages of eggs as alternative antibody source, 350–2 avian immune system, 347–8 future trends, 361 immunotherapeutic applications, 354–61 hyperacute rejection in xenotransplantation, 360–1 neutralisation of venom, 359 oral administration, 354–9 rabies virus prevention, 360 Staphylococcus aureus infection prevention, 360 synthetic administration, 359–61 production and purification, 348–50 production, 348–9 purification, 349–50 structure and stability, 351–2 IL-4, 256 IL-5, 256 IL-9, 256 IL-10, 257 IL-13, 256 immobilised metal ion affinity chromatography, 350 immune hyporesponsiveness, 256 immunoglobulin (Ig) E-mediated food allergy, 256–7 in-line (IL) commercial processing plant, 9 in vivo expression technology, 54 infectious bursal disease virus, 359 inflammatory bowel disease, 358 innate mechanisms, 128 inoculation, 11 interferon g, 256 intravaginal inoculation, 50
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Index 405 iodine, 308–9 ion exchange chromatography, 349 ionisation, 188 iron, 309–12 iron sulphate, 310 iron–methionine chelate, 310 iron–soy proteinate, 311 irradiation, 155, 185 isthmus contamination, 50–1 Klebsiella, 6 Lactobacillus, 7 laying hens pre-harvest measures, Salmonella control, 120–35 future trends, 134–5 gastrointestinal colonisation control, 129–33 genetic selection, naturally occurring resistance, 127–9 information source and advice, 135 vaccination, 121–7 Salmonella, detection and monitoring, 83–100 2004/2005 baseline survey methods and Salmonella control programs, European Union, 95–6 infection detection factors, 96–9 recommended sampling regime, 91–4 sampling, 84–91 serology, 94 under-detection significance, 99–100 Salmonella control, management and sanitation procedures, 146–58 future trend, 157–8 management procedures on Salmonella prevention on farm, 148–53 sanitation and decontamination, 153–7 Salmonella infections, housing system influence, epidemiology, 107–115 LDL, 324, 337–9 efficiency for spermatozoa after freezethaw process, 338 LDL cholesterol, 241 LDL receptor pathway, 241–2 lesser mealworm see darkling beetle lipid oxidation, 335–6 lipids, 220–2 cholesterol, 221–2 lipophilic molecules, 67 lipopolysaccharides (LPS), 53 Listeria monocytogenes, 7, 36, 37, 38, 185 litter collection, 86 long chain polyunsaturated fatty acids (LCPUFA), 221 lysozyme, 259, 260, 329–31 magnetic field coupling, 186 Maillard reactions, 264 major histocompatibility (MHC) class II molecules, 256
management procedures and sanitation, Salmonella control in laying hen flocks, 146–58 future trend, 157–8 management procedures on Salmonella prevention on farm, 148–53 sanitation and decontamination, 153–7 mast cells, 256 matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, 355 maximum required performance limits, 72 maximum residue limit, 63–4 metabolic ward studies, 245–6 5-methyltetrahydrofolate, 302 microbial growth inhibition assays, 73 microbial hazards and risk assessment, Salmonella enteritidis, egg foodborne disease, 34–41 hazard identification, 35–8 quantitative risk assessment, 38–41 microbial inactivation mode, 193 microbial recovery, 11 microencapsulated fish oil, 284 microwave pasteurisation, 186–8 microwave plasma jet (MPJ), 190 minerals, 225–6 monoclonal mouse antibodies, 358 Moraxella, 7 mucosal immune response, 256–7 Multiple Risk Factor Intervention Trial, 239–40 Musca domestica see houseflies National Reference Laboratories, 72 natural convection (NC) method, 183 NMKL-71 method, 96 no observable effect level, 64 non-cage system, 108 novel food, 382 numerical model, 183 nutrient profiling, 204 nutritional quality eggs, 201–28 consumption per capita in kg in EU since 1990, 202 egg headlines, 202 egg reputation, 201–3 eggs composition ntritional evaluation, 203–17 German food pyramid, 203 improving nutritional quality, 226–8 macronutrients evaluation, 217–22 micronutrients evaluation, 222–6 off-line (OL) commercial processing plant, 9 omega-3, 384 oral tolerance see immune hyporesponsiveness OTAP-92, 331 ovalbumin, 259, 261, 326 ovarian sampling, 84–5 oviduct contamination, 15
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406 Index oviduct sampling, 84–5 ovomucin, 262, 327 ovomucoid, 259, 260, 326–7 ovotransferrin, 172, 259, 262, 331–2, 334–5 oxidative deterioration process, 283 ozone, 185 passive immunotherapy, 347 Pasteurella haemolytica, 18 periodontitis, 355–6 persistent organic pollutants, 63 pesticide residue, 65 pharmacokinetics, 66–7 phenotypic mechanisms, 128–9 phospholipids, 274 phosvitin, 323, 335–6 Physicians Health Study, 248 phyto-pharmaceutical substances, 63 Pichia pastoris yeast system, 263 plasma, 323 polybrominated diphenyl ethers, 66 polychlorinated biphenyls, 66 polychlorinated dioxins, 66 polychlorinated furans, 66 polycyclic aromatic hydrocarbons, 66 polyunsaturated fatty acid, 241, 276 porcine epidemic virus, 359 portable water, 172 poultry red mite, 113 pre-harvest measures Salmonella control, laying hens, 120–35 future trends, 134–5 gastrointestinal colonisation control, 129–33 genetic selection, naturally occurring resistance, 127–9 information source and advice, 135 vaccination, 121–7 Propionibacterium, 7 protein digestibility corrected amino acid score (PD-CAAS), 217 protein glycosylation, 262–3 proteins, 217–20 digestibility and biological value, 217–18 different protein sources classification, 219 egg nutritional value improvement, 220 essential amino acids content, 217 satiating effects, 218–20 Proteus, 6 Pseudomonas, 6 Pseudomonas aeruginosa infection, 354–5 Pseudomonas sp., 48 pulsed field gel electrophoresis, 9, 149–50 pulsed light, 192–4 PureBright, 193 Quantitative Microbial Risk Assessment, 39 rabies virus, 360 radio frequency, 189 radioallergosorbent test, 259
rapeseed, 279–80 receptor assay systems, 73 recommended daily allowance, 212 recommended daily intake, 204 regions of difference, 56 relative humidity, 192 restrictive barrier discharge (RBD) method, 189 retinyl acetate, 293 riboflavin, 262 ribotyping, 149–50 risk assessment microbial hazards, Salmonella enteritidis, egg foodborne disease, 34–41 hazard identification, 35–8 quantitative risk assessment: Salmonella enteritidis in eggshell, 38–41 models, 39 rotavirus, 357–8 S. enteritidis, 7 S. typhimurium, 7 Safe Quality Food (SQF) certification, 14–15 Salmonella, 6, 8–9, 35–6, 37 boot swabbing in free-range house, 95 detection and monitoring in laying hen flocks, 83–100 2004/2005 baseline survey methods and Salmonella control programs, European Union, 95–6 moistening boot swabs before sampling, 87 mouse and mouse droppings sample, 86 sampling, 84–91 sampling cage after cleaning and disinfection, 91 sampling dust with gauze fabric swab (chiffonette), 90 serology, 94 swabbing pit, 88 swabbing poles with swabs, 88 under-detection significance, 99–100 gastrointestinal colonisation control, 129–33 chemical feed and drinking water supplements, 131–2 defined CE preparations, 130–1 performance and efficacy factors, 132–3 principles and mechanisms, 129–30 undefined CE preparations, 130 genetic selection, naturally occurring resistance, 127–9 heritable resistance mechanisms, 128–9 performance and efficacy factors, genetic selection resistance, 129 Salmonella-resistant lines, chickens, 127–8 infection detection factors, 96–9 flock housing/manure handling system, 96–7
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Index 407 flock size, 97–8 lay stage, 98 vaccination, 98–9 laying hen flocks, management and sanitation procedures, 146–58 future trend, 157–8 management procedures on Salmonella prevention on farm, 148–53 biosecurity, 148 insect control, 151–2 red mite control, 152–3 rodent control, 149–50 wild birds, 153 pre-harvest measures in laying hens, 120–35 future trends, 134–5 recommended sampling regime, 91–4 dust from egg belts ends, 93 Salmonella-positive free-range house surroundings, 94 sampling dust from beneath cage stacks, 93 sampling faeces from manure scrapers, 92 sampling manure in deep pit house, 92 sanitation and decontamination, 153–7 drinking water decontamination, 153–4 feed decontamination, 154–6 layer house cleaning and disinfection, 156–7 vaccination, 121–7 goals and applications, 122 killed (inactivated) and subunit vaccines, 122–4 live (attenuated) vaccines, 124–5 performance and efficacy factors, Salmonella vaccines, 126–7 poultry immune response, 121–2 treatment with immune mediators, 125–6 Salmonella enteritidis, 48, 108–11, 146–57, 185 eggs internal contamination, 46–57 eggshell penetration, 47–9 eggshell surface contamination, 47 reproductive tract development, 49–51 risk assessment and microbial hazards, egg foodborne disease, 34–41 hazard identification, 35–8 quantitative risk assessment: Salmonella enteritidis in eggshell, 38–41 virulence factors, chicken reproductive tract colonisation, 51–7 behaviour in laid eggs, 55–6 forming eggs survival and damage control mechanisms, 52–5 serotype enteritidis efficiency, 56–7 Salmonella enteritidis outbreak surveillance system, 21 Salmonella infection, 357 epidemiology, housing system influence, 107–115
observational studies, housing system effect on Salmonella prevalence, 109 S. enteritidis and serotypes, outdoor production systems, 114 housing system factors, Salmonella prevalence, 111–14 carry-over infections and infrastructure age, 113 farm and flock size, 111 pests, 113–14 stocking density, 111–12 stress, 112 vaccination, 114 Salmonella Pathogenicity Islands (SPIs), 51–2 Salmonella typhimurium, 108 Salmonella vaccine, 124–5 salmonellosis, 108, 182 sanitation procedures and management procedures, Salmonella control in laying hen flocks, 146–58 future trend, 157–8 information source and advice, 158 management procedures on Salmonella prevention on farm, 148–53 sanitation and decontamination, 153–7 Sanitation Standard Operating Procedures (SSOPs), 15 scanning electron microscope, 188 selective heating, 186 selenium, 305–8 effect of dietary selenium supplementation, 307 selenium-enriched yeast, 306 selenomethionine, 306 semi-quantitative assessment, 156–7 Serratia marcesens, 48 shell crushing and rubbing (CR) method, 12 shell rinse (SR) method, 12 shell thickness, 16 single nucleotide polymorphisms (SNPs), 56 sock swabs, 86–7 sodium selenate, 306 sodium selenite, 306 spleen, 347 sporadic egg-borne disease, 22 Staphylococcus aureus, 6 Staphylococcus aureus infection, 360 Staphylococcus warneri, 48 steam pelleting, 155 SteriPulse-XL, 193 strengths, weaknesses, opportunities and threats (SWOT) analysis, 375–87 egg nutraceutical business, 388 opportunities, 383–6 bioactive components identification, 385 consumer’s education, 384 egg as healthy source of nutrients, 383 expanded usage of functional eggs, 384 health claims, 384–5 new nutrient for functional eggs, 386
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408 Index new opportunities for IgY, 385 omega-3 nutritional gap, 384 strengths, 375–80 bioactive ingredient source, 378–9 functional food production, 375, 378 health benefits from functional eggs, 379–80 immunoglobulins from yolk, 379 natural way of functional food production, 378 nutritional and other claims allowed for regular eggs, 376–7 protein, vitamin and mineral source, 375 worldwide acceptance of eggs as food, 375 threats, 386–7 competition from other animal products and plants, 387 lack of patent protection, 387 ‘mining of the egg’ for others, 387 new competitors, 387 new poultry epidemic diseases, 387 regulatory environment, 386 weaknesses, 381–3 allergenicity of egg, 383 attitudes towards functional foods, 381–2 cholesterol issue, 381 investment in R&D and marketing, 383 lack of visual product differentiation, 381 novel food regulatory process, 382 role of claims, 382 skill sets required, 383 Streptococcus faecalis, 6 Streptococcus mutans, 356 surface rinse method, 12 T regulatory (Treg) cells, 257 table eggs eggs and foodborne disease, 22–4 concern levels, food borne salmonellosis exposure, 24 consumer and retailer’s role, 23–4 microbiology, 15–22 pathogens, 20–2 scanning electron micrograph, eggshell pores, 17 microbiology and safety, 3–24 Enterobacteriaceae prevalence, packer head brushes and table eggs, 13 regulations, 14–15 table egg facility sanitation, 13–14 United States table egg industry, 4–5 inline shell egg layer house and plant layout, 4 shell egg processing facility, 5 washing table eggs, 6–13 eggshell sampling methodology, 11–13 mean populations, microorganisms recovered from commercial shell egg surface, 10 washing effects, 6–11
thermal cracking, 172 thermal processing, 264–5 thiophilic interaction chromatography, 350 thymus, 347 Total Quality Assurance Food Safety Program, 21 toxic shock syndrome, 360 toxicokinetics, 66–7 trace minerals egg enrichment, 304–14 trans-ovarian infection, 15 trans-shell contamination, 15 triacylglycerols, 274 UK Food Standards Agency, 247 ultra-performance liquid chromatography, 73 ultrafiltration, 349 ultrasonication, 185 United States Department of Agriculture, 165 US Agricultural Marketing Act, 14–15 US Food and Drug Administration, 15 vertical transmission, 47, 122 veterinary drugs, 63–4 Veterinary Laboratories Agency, 40 virulence genes, 51–2 vitamin A, 291–4 effect of dietary retinyl acetate on retinol content, 293 vitamin B1, 304 vitamin B2, 304 vitamin B5, 304 vitamin B6, 304 vitamin B8, 304 vitamin B12, 224, 304 vitamin D, 223, 294–6 dietary cholecalciferol dietary supplementation on cholecalciferol and 25OHD content of fresh egg yolk, 295 vitamin E, 223–4, 296–300 dietary DL-alpha tocopheryl acetate supplementation, 299–300 vitamin K, 224, 300–1 vitamins, 222–5 egg enrichment, 290–301 RDA, 291 vitamin retention, processing eggs, 224–5 xanthophylls, 225 water dilution method, 350 water-soluble fraction, 349 water soluble vitamins, 222–3, 301–4 wet swabbing method, 90 Women’s Health Study, 248 World Health Organisation, 24, 39–40 xenobiotics, 70 Yersinia ruckeri, 358 zinc, 312–14 zoonotic agents, 35
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