Protective Cultures, Antimicrobial Metabolites and Bacteriophages for Food and Beverage Biopreservation (Woodhead Publishing Series in Food Science, Technology and Nutrition)

Protective cultures, antimicrobial metabolites and bacteriophages for food and beverage biopreservation

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Related titles: Foodborne pathogens: hazards, risk analysis and control Second edition (ISBN 978-1-84569-362-6) Effective control of pathogens continues to be of great importance to the food industry. The first edition of Foodborne pathogens quickly established itself as an essential guide for all those involved in the management of microbiological hazards at any stage in the food production chain. This major new edition strengthens that reputation, with extensively revised and expanded coverage, including more than ten new chapters. Part I focuses on risk assessment and management in the food chain. Chapters in this section cover pathogen detection, microbial modelling the risk assessment procedure, pathogen control in primary production, hygienic design and sanitation, among other topics. Parts II and III then review the management of key bacterial and non-bacterial foodborne pathogens. Natural antimicrobials for the minimal processing of foods (ISBN 978-1-85573-669-6) Consumers demand food products with fewer synthetic additives but increased safety and shelf-life. These demands have increased the importance of natural antimicrobials which prevent the growth of pathogenic and spoilage micro-organisms. Edited by a leading expert in the field, this important collection reviews the range of key antimicrobials together with their applications in food processing. There are chapters on antimicrobials such as nisin and chitosan, applications in such areas as postharvest storage of fruits and vegetables, and ways of combining antimicrobials with other preservation techniques to enhance the safety and quality of foods. Food preservation techniques (ISBN 978-1-85573-530-9) Extending the shelf-life of foods while maintaining safety and quality is a critical issue for the food industry. As a result there have been major developments in food preservation techniques, which are summarised in this authoritative collection. The first part of the book examines the key issue of maintaining safety as preservation methods become more varied and complex. The rest of the book looks both at individual technologies and how they are combined to achieve the right balance of safety, quality and shelf-life for particular products. 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: [email protected]; fax: +44 (0) 1223 893694; tel.: +44 (0) 1223 891358 ext. 130; address: Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK If you would like to receive information on forthcoming titles, please send your address details to: Francis Dodds (address, tel. and fax as above, e-mail: [email protected]). Please confirm which subject areas you are interested in.

ii © Woodhead Publishing Limited, 2011

Woodhead Publishing Series in Food Science, Technology and Nutrition: Number 201

Protective cultures, antimicrobial metabolites and bacteriophages for food and beverage biopreservation Edited by Christophe Lacroix

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Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK www.woodheadpublishing.com Woodhead Publishing, 525 South 4th Street #241, Philadelphia, PA 19147, 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 8 was prepared by United States government employees; that chapter is therefore in the public domain and cannot be copyrighted. 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 publisher cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, 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. ISBN 978-1-84569-669-6 (print) ISBN 978-0-85709-052-2 (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 RefineCatch Limited, Bungay, Suffolk Printed by TJI Digital, Padstow, Cornwall, UK © Cover image: Swiss National Science Foundation SNSF, Berne, Switzerland.

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Contents

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

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Preface.......................................................................................................... xxiii Part I  Food biopreservation 1 Identifying new protective cultures and culture components for food biopreservation.................................................. R. J. Jones, AgResearch Ltd, New Zealand, P. A. Wescombe, BLIS Technologies Ltd, New Zealand and J. R. Tagg, University of Otago, New Zealand 1.1 Introduction.................................................................................. 1.2 Historical perspectives................................................................. 1.3 Bacteriocins of Gram-positive bacteria and the nature of bacteriocin-like inhibitory substance (BLIS).............. 1.4 Characteristics of microbes and inhibitory products of relevance to their potential protective activity in food................ 1.5 Screening methodologies in food biopreservation...................... 1.6 Our procedure for inhibitor screening in food biopreservation............................................................................ 1.7 Molecular methods of screening in food biopreservation........... 1.8 Future considerations................................................................... 1.9 References....................................................................................

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2 Antifungal lactic acid bacteria and propionibacteria for food biopreservation............................................................................. S. Miescher Schwenninger, L. Meile and C. Lacroix, ETH Zurich, Switzerland 2.1 Introduction.................................................................................. 2.2 Spoilage fungi in food: undesired yeasts and moulds................. 2.3 Traditional control of spoilage fungi in food............................... 2.4 Antifungal lactic and propionic acid bacteria (LAB and PAB).... 2.5 Efficiency of antifungal LAB and PAB in food challenge tests: a first step from in vitro towards in vivo............. 2.6 Antifungal metabolites and further inhibitory mechanisms.................................................................................. 2.7 The long road from research to industry: commercial antifungal protective cultures...................................................... 2.8 Future trends................................................................................ 2.9 Summary...................................................................................... 2.10 References.................................................................................... 3 Nisin, natamycin and other commercial fermentates used in food biopreservation......................................................................... J. Delves-Broughton, Danisco Food Protection, UK and G. Weber, Danisco Food Protection, USA 3.1 Introduction.................................................................................. 3.2 Nisin used in food biopreservation.............................................. 3.3 Natamycin used in food biopreservation..................................... 3.4 Undefined fermentates used in food biopreservation.................. 3.5 Future trends................................................................................ 3.6 Sources of further information and advice.................................. 3.7 References.................................................................................... 4 The potential of lacticin 3147, enterocin AS-48, lacticin 481, variacin and sakacin P for food biopreservation................................ V. Fallico, O. McAuliffe, R. P. Ross, Teagasc Food Research Centre, Moorepark, Ireland and G. F. Fitzgerald and C. Hill, University College Cork, Ireland 4.1 Introduction.................................................................................. 4.2 The potential of lacticin 3147 for food biopreservation.............. 4.3 The potential of enterocin AS-48 for food biopreservation............................................................................ 4.4 The potential of lacticin 481 for food biopreservation................ 4.5 The potential of variacin for food biopreservation...................... 4.6 The potential of sakacin P for food biopreservation.................... 4.7 Future trends................................................................................ 4.8 Sources of further information and advice.................................. 4.9 References....................................................................................

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Contents 5 The potential of reuterin produced by Lactobacillus reuteri as a broad spectrum preservative in food.................................................. M. Stevens, S. Vollenweider and C. Lacroix, ETH Zurich, Switzerland 5.1 Introduction.................................................................................. 5.2 Lactobacillus reuteri, a probiotic bacterium with intestinal activity.......................................................................... 5.3 The reuterin-HPA system............................................................ 5.4 Antimicrobial activity of reuterin................................................ 5.5 Production of reuterin on a large scale........................................ 5.6 Reuterin as a food preservative.................................................... 5.7 Additional antimicrobial compounds produced by L. reuteri...... 5.8 Concluding remarks and future trends......................................... 5.9 References.................................................................................... 6 Bacteriophages and food safety........................................................... L. Fieseler and M. J. Loessner, ETH Zurich, Switzerland and S. Hagens, EBI Food Safety, The Netherlands 6.1 Introduction.................................................................................. 6.2 Bacteriophages............................................................................. 6.3 Pathogen detection using bacteriophages.................................... 6.4 Application of bacteriophages to control bacterial pathogens in foods: an overview................................................. 6.5 Phage therapy: on the way to safer food?.................................... 6.6 References....................................................................................

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Part II Applications of protective cultures, bacteriocins and bacteriophages in food animals and humans 7 Using antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry..... P. L. Connerton, A. R. Timms and I. F. Connerton, University of Nottingham, UK 7.1 Introduction.................................................................................. 7.2 Antimicrobial cultures to reduce carriage of food-borne bacterial pathogens in poultry...................................................... 7.3 Bacteriocins to reduce carriage of food-borne bacterial pathogens in poultry.................................................................... 7.4 Bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry.................................................................... 7.5 Regulatory issues in reduction of food-borne bacterial pathogens in poultry.................................................................... 7.6 Future trends................................................................................ 7.7 Sources of further information and advice.................................. 7.8 References....................................................................................

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8 Using antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of foodborne pathogens in cattle and swine....................................................................................................... T. R. Callaway, T. S. Edrington, R. C. Anderson, J. A. Byrd, M. H. Kogut, R. B. Harvey and D. J. Nisbet, United States Department of Agriculture, Agricultural Research Service (USDA-ARS), USA and C. W. Aiello, Carilion Medical Center, USA 8.1 Introduction.................................................................................. 8.2 Antimicrobial cultures: enhancing natural competition.................................................................................. 8.3 Direct assault: anti-pathogen intervention strategies................... 8.4 Conclusions.................................................................................. 8.5 Disclaimer.................................................................................... 8.6 References.................................................................................... 9 Controlling fungal growth and mycotoxins in animal feed............... M. Olstorpe, K. Jacobsson, V. Passoth and J. Schnürer, Swedish University of Agricultural Sciences, Sweden 9.1 Introduction.................................................................................. 9.2 Fungal growth and mycotoxins in animal feed............................ 9.3 Preservation techniques............................................................... 9.4 Biopreservation............................................................................ 9.5 From strain discovery to biopreservative starter culture............. 9.6 Concluding remarks..................................................................... 9.7 References.................................................................................... 10 Biological control of human digestive microbiota using antimicrobial cultures and bacteriocins.............................................. I. Fliss, R. Hammami and C. Le Lay, Laval University, Canada 10.1 Introduction.................................................................................. 10.2 Human gastrointestinal defenses................................................. 10.3 Gastrointestinal microbiota as partner for human defense............................................................................. 10.4 Antimicrobial cultures: lactic acid bacteria and probiotics..................................................................................... 10.5 Mechanisms of action in human digestive microbiota.................................................................................... 10.6 Antimicrobial cultures: prevention and treatment of gastrointestinal diseases............................................................... 10.7 Tools for studying biological activities of antimicrobial cultures.................................................................. 10.8 Conclusion and future trends....................................................... 10.9 References....................................................................................

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Part III Applications of protective cultures, bacteriocins and bacteriophages in foods and beverages 11 Applications of protective cultures, bacteriocins and bacteriophages in milk and dairy products........................................ M. Medina and M. Nuñez, INIA, Spain 11.1 Introduction.................................................................................. 11.2 Bacteriocins to improve the safety of dairy foods....................... 11.3 Bacteriocins in combined treatments........................................... 11.4 Bacteriocins to enhance the quality and flavour of cheese.......... 11.5 Bacteriophages to improve the safety and quality of milk and dairy products........................................................................ 11.6 Conclusions and future trends..................................................... 11.7 References.................................................................................... 12 Applications of protective cultures, bacteriocins and bacteriophages in fermented meat products...................................... T. Aymerich, M. Garriga and J. Monfort, IRTA, Spain 12.1 Introduction.................................................................................. 12.2 Food safety of fermented sausages.............................................. 12.3 Microbiota of fermented sausages............................................... 12.4 Bioprotective cultures for safety of fermented sausages............. 12.5 Application of bacteriocins in fermented sausages..................... 12.6 Use of bacteriophages to improve food safety and meat environment............................................................................. 12.7 Legislation aspects and constraints.............................................. 12.8 Future trends................................................................................ 12.9 Sources of further information and advice.................................. 12.10 Acknowledgement....................................................................... 12.11 References.................................................................................... 13 Applications of protective cultures, bacteriocins and bacteriophages in fresh seafood and seafood products...................... M.-F. Pilet, ONIRIS, Nantes, France and F. Leroi, Ifremer, Nantes, France 13.1 Introduction.................................................................................. 13.2 Microbial risk in seafood............................................................. 13.3 Lactic acid bacteria in seafood products...................................... 13.4 Bioprotective lactic acid bacteria, bacteriocins and bacteriophages for bacteria control.............................................. 13.5 Industrial application................................................................... 13.6 Future trends................................................................................ 13.7 Sources of further information and advice.................................. 13.8 References....................................................................................

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14 Microbial applications in the biopreservation of cereal products...... G. Font de Valdez, G. Rollán, C. L. Gerez and M. I. Torino, Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina 14.1 Introduction.................................................................................. 14.2 Cereals in human nutrition and animal feed................................ 14.3 Major contaminant agents in cereal-based products.................... 14.4 Fermentative technologies as a tool for microbial biopreservation............................................................................ 14.5 Production in situ of antimicrobial compounds........................... 14.6 Microbial metabolites used as additives in cereal biopreservation............................................................................ 14.7 Phage-based strategies................................................................. 14.8 Conclusion................................................................................... 14.9 References.................................................................................... 15 Biological control of postharvest diseases in fruit and vegetables....................................................................................... N. Teixidó and R. Torres, IRTA, Catalonia, Spain, I. Viñas, University of Lleida, Catalonia, Spain and M. Abadias and J. Usall, IRTA, Catalonia, Spain 15.1 Introduction.................................................................................. 15.2 Development programme of a biocontrol agent (BCA)................................................................................ 15.3 The search for biocontrol agents of postharvest diseases............ 15.4 Mechanisms of action.................................................................. 15.5 Production and formulation of biocontrol agents........................ 15.6 Improvement of biocontrol agents............................................... 15.7 Integration of biocontrol agents with other alternative methods...................................................................... 15.8 Hurdles for biocontrol commercial application........................... 15.9 Future trends................................................................................ 15.10 Sources of further information and advice.................................. 15.11 Acknowledgements...................................................................... 15.12 References.................................................................................... 16 Biological control of pathogens and post-processing spoilage microorganisms in fresh and processed fruit and vegetables....................................................................................... A. Gálvez, H. Abriouel, R. L. López and N. Ben Omar, University of Jaén, Spain 16.1 Introduction.................................................................................. 16.2 Biocontrol of bacterial pathogens in fresh-cut produce............... 16.3 Biocontrol strategies for minimal processed fruits...................... 16.4 Application of bacteriocins in fruit juices and vegetable drinks...........................................................................

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Application of bacteriocins in ready-to-eat and canned vegetable foods................................................................ Application of bacteriocins or their producer strains in fermented vegetables............................................................... General conclusions and perspectives......................................... References....................................................................................

17 Applications of protective cultures and bacteriocins in wine making........................................................................................... F. Ruiz-Larrea, University of La Rioja, Spain 17.1 Introduction.................................................................................. 17.2 Wine fermentation....................................................................... 17.3 Lactic acid bacteria in wine making............................................ 17.4 Wine spoilage by bacteria............................................................ 17.5 Sulphur dioxide: the classical antimicrobial agent in wine making................................................................................. 17.6 Bacteriocins................................................................................. 17.7 Bacteriocins produced by enological bacteria............................. 17.8 Bacteriocins with antimicrobial activity against wine spoilage bacteria.......................................................................... 17.9 Future trends................................................................................ 17.10 References.................................................................................... 18 Control of mycotoxin contamination in foods using lactic acid bacteria................................................................................ H. S. El-Nezami, University of Hong Kong, China and S. Gratz, University of Aberdeen, UK 18.1 Introduction.................................................................................. 18.2 Control of the mycotoxin problem.............................................. 18.3 Reduction of toxic effects in vitro............................................... 18.4 Effectiveness under physiological conditions.............................. 18.5 References....................................................................................

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19 Active packaging for food biopreservation......................................... S. Yildirim, ZHAW, Zurich University of Applied Sciences, Switzerland 19.1 Introduction.................................................................................. 19.2 Food and active packaging.......................................................... 19.3 Antimicrobial packaging for food biopreservation...................... 19.4 Natural antimicrobial agents and polymers................................. 19.5 Other antimicrobial packaging systems....................................... 19.6 Design of antimicrobial packaging systems................................ 19.7 Future trends................................................................................ 19.8 References....................................................................................

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

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Contributor contact details PO Box 56 Dunedin 9016 New Zealand

(* = main contact)

Editor C. Lacroix ETH Zurich Institute of Food, Nutrition and   Health Schmelzbergstrasse 7, LFV C20 CH-8092 Zürich Switzerland Email: [email protected]. ethz.ch

Chapter 1

Email: [email protected]; [email protected]

Chapter 2 S. Miescher Schwenninger*,   L. Meile and C. Lacroix ETH Zurich Institute of Food, Nutrition and   Health Schmelzbergstrasse 7, LFV C20 CH-8092 Zurich Switzerland Email: [email protected]. ethz.ch

R. J. Jones* Food, Metabolism and   Microbiology AgResearch Ltd Private Bag 3123 Hamilton 3240 New Zealand

Chapter 3

Email: [email protected] P. A. Wescombe and J. R. Tagg BLIS Technologies Ltd Centre for Innovation University of Otago

J. Delves-Broughton Danisco 6 North Street Beaminster Dorset DT8 3DZ UK Email: joss.delves-broughton@ danisco.com

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Contributor contact details G. Weber Danisco Four New Century Parkway New Century, KS 66031 USA

Chapter 4 V. Fallico, O. McAuliffe   and P. Ross* Teagasc Moorepark Food Research Centre Fermoy County Cork Ireland Email: [email protected] G. F. Fitzgerald and C. Hill Department of Microbiology University College Cork Ireland

Chapter 5 M. Stevens*, S. Vollenweider   and C. Lacroix ETH Zurich Institute of Food, Nutrition   and Health Schmelzbergstrasse 7, LFV C20 CH-8092 Zurich Switzerland Email: [email protected]. ethz.ch

Chapter 6

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CH-8092 Zurich Switzerland Email: [email protected] S. Hagens EBI Food Safety Nieuwe Kanaal 7P 6709 PA Wageningen The Netherlands

Chapter 7 P. L. Connerton, A. R. Timms and   I. F. Connerton* Division of Food Sciences School of Biosciences University of Nottingham, Sutton   Bonington Campus Loughborough Leicestershire LE12 5RD UK Email: ian.connerton@nottingham. ac.uk

Chapter 8 T. R. Callaway*, T. S. Edrington,   R. C. Anderson, J. A. Byrd,   M. H. Kogut, R. B. Harvey   and D. J. Nisbet USDA/Agricultural Research Service Southern Plains Agricultural Research   Center Food and Feed Safety Research Unit 2881 F&B Road College Station, TX 77845 USA Email: [email protected]

L. Fieseler and M. J. Loessner* ETH Zurich Institute of Food, Nutrition and Health Schmelzbergstrasse 7, LFV C20

C. W. Aiello Carilion Medical Center Roanoke, VA 24033 USA

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Contributor contact details

Chapter 9 M. Olstorpe*, K. Jacobsson,   V. Passoth and J. Schnürer Swedish University of Agricultural   Sciences Department of Microbiology Box 7025 SE-750 07 Uppsala Sweden Email: [email protected]

Chapter 10 I. Fliss*, R. Hammami and   C. Le Lay Nutraceuticals and Functional Foods   Institute (INAF) Université Laval Quebec City, PQ Canada G1K 7P4 Email: [email protected]

Chapter 11 M. Medina* and M. Nuñez Departamento de Tecnología de   Alimentos INIA Ctra. La Coruña km 7 28040 Madrid Spain Email: [email protected]

Chapter 12 T. Aymerich*, M. Garriga   and J. Monfort Food Safety IRTA-Food Technology 18121 Monells

Girona Spain Email: [email protected];

Chapter 13 M. F. Pilet* UMR INRA 1014 Sécurité des   Aliments et Microbiologie   (SECALIM) ONIRIS Site de la Géraudière BP 82225 44322 Nantes Cedex 03 France Email: marie-france.pilet@ oniris-nantes.fr F. Leroi Laboratoire de Science et Technologie   de la Biomasse Marine Ifremer Rue de l’Ile d’Yeu BP 21105 44311 Nantes Cedex 03 France

Chapter 14 G. Font de Valdez*, G. Rollán, C. L.   Gerez and M. I. Torino Centro de Referencia para   Lactobacilos (CERELA-CONICET) Facultad de Bioquímica, Química y   Farmacia Universisidad Nacional de Tucumán San Miguel de Tucumán T4000ILC Argentina Email: [email protected]

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Contributor contact details

Chapter 15 N. Teixidó*, R. Torres, M. Abadias   and J. Usall Postharvest Pathology IRTA Centre UdL-IRTA 191 Rovira Roure Avenue 25198 Lleida Catalonia Spain Email: [email protected] I. Viñas University of Lleida Centre UdL-IRTA 191 Rovira Roure Avenue 25198 Lleida Catalonia Spain

Chapter 16 A. Gálvez*, H. Abriouel, R. L. López   and N. Ben Omar Área de Microbiología Departamento de Ciencias de la Salud Facultad de Ciencias Experimentales Edif. B3 Universidad de Jaén Campus Las Lagunillas s/n 23071-Jaén Spain Email: [email protected]

Chapter 17 F. Ruiz-Larrea University of La Rioja Instituto de Ciencias de la Vid y del   Vino Av. Madre de Dios 51

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26006 Logroño Spain Email: [email protected]

Chapter 18 H. S. El-Nezami* School of Biological Sciences University of Hong Kong S5-13 Kadoorie Biological Sciences   Building Pokfulam Hong Kong SAR China Email: [email protected] S. Gratz Rowett Institute of Nutrition and   Health University of Aberdeen Greenburn Road Aberdeen AB21 9SB UK

Chapter 19 S. Yildirim Zurich University of Applied Sciences School of Life Sciences and Facility   Management Institute of Food and Beverage   Innovation Campus Reidbach, Postfach CH-8820 Wädenswil Switzerland Email: [email protected]

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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 Edited by C. Doona, K. Kustin and F. Feeherry 198 Freeze-drying of pharmaceutical and food products Tse-Chao Hua, Bao-Lin Liu and Hua 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 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

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Preface

Biopreservation refers to the enhancement of food safety and stability using microorganisms and/or their metabolites. Spontaneous fermentation is one of the oldest biopreservation technologies and has been used empirically for millennia. At the end of the nineteenth century, the role of lactic acid bacteria in the fermentation of dairy and meat products was discovered. Lactic acid bacteria have since been used to control acid production which is today achieved by applying selected starter cultures. Lactic acid bacteria and other food microorganisms produce a wide range of metabolites that can inhibit growth of spoilage and pathogenic bacteria and act as multiple hurdles. These metabolites are active during food fermentation and/or subsequent ripening and storage. The production of weak organic acids, such as acetic and lactic acids, inhibits microbial growth through multiple actions, including membrane disruption, inhibition of metabolic reactions, disturbance of pH homeostasis, and accumulation of toxic anions in the cell. Other antimicrobials produced by protective cultures include hydrogen peroxide, bacteriocins, and several other low molecular weight antimicrobial compounds often acting in synergy. The search for new natural antimicrobial compounds and mechanisms is an active area for research in response to consumers’ demands for high quality, safer, and healthier foods containing less or no chemical preservatives. The combination of protective cultures harboring different antimicrobial mechanisms and the application of complex natural microflora with high barrier properties may further enhance protective effects but also represents even greater challenges with regard to the search for defined mechanisms. Future knowledge on microorganisms and the mechanisms involved in naturally occurring antagonisms should enable the tailored application of new biopreservation strategies. Biopreservation is nowadays achieved by: (a) application of antimicrobial metabolites without the producing strain (e.g. fermented and bacteriocin extracts); (b) application of an adjunct culture producing antimicrobial metabolites in situ or ex situ that does not influence food quality; or (c) application of a technological xxiii © Woodhead Publishing Limited, 2011

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flora harboring protective effects. In this book, state of the art information is presented on protective cultures and antimicrobial metabolites and their broad application in food, feed, and intestinal health. Initial chapters review central aspects in food biopreservation, including the identification of new protective cultures and antimicrobial culture components, existing commercial fermentates including nisin and natamycin, and the potential of novel antifungal bacterial mixtures, antimicrobial peptides and other low molecular weight compounds, and bacteriophages to improve food quality and food safety. Part II highlights the potential and use of antimicrobial probiotics and complex microflora with barrier properties to control the carriage of pathogenic microorganisms in food animals and to modulate human gut microbiota. Chapters in the final section of the book review biopreservation of different types of foods, including milk and dairy products, fermented meats, fresh seafood, and fruit. A review of active packaging for food biopreservation completes the volume. The chapters are written by renowned experts and comprise a summary of the most up to date scientific and technical developments and applications of biopreservation strategies. The information collected in this book covers different scientific areas and viewpoints and will be useful to food and feed scientists and developers involved in the work on food, nutrition, and health. I wish to thank sincerely all the authors who contributed to this book and all the staff at Woodhead Publishing Limited who supported me tremendously in my role as editor. Christophe Lacroix

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To my wife, Janice, and three children, Mélanie, Fabrice and Anna, for their constant help and support

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Part I Food biopreservation

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1 Identifying new protective cultures and culture components for food biopreservation R. J. Jones, AgResearch Ltd, New Zealand, P. A. Wescombe and J. R. Tagg, BLIS Technologies Ltd, University of Otago, New Zealand

Abstract: Lactic acid bacteria (LAB) produce a range of mechanisms, notably bacteriocins, that restrict the development of competing bacterial populations. As such, LAB and their products are increasingly viewed as natural preservatives for a range of foods. In this chapter we discuss the nature and detection of inhibitory activities in a range of producer strains. Key words: bacteriocins, lactic acid bacteria, deferred and simultaneous antagonism.

1.1  Introduction The bio-preservation of food, especially utilizing lactic acid bacteria (LAB) that are inhibitory to food spoilage microbes, has been practiced since antiquity, at first instinctively but now with an increasingly robust scientific foundation. There are a wide variety of mechanisms whereby one microorganism can interfere with the growth of others. Much of the preservative effect conferred on fermented food materials is attributable to its content of acids (especially lactic and acetic), resulting in a reduction of pH and the antimicrobial activity of the un-dissociated acid molecules (de Vuyst and Vandamme 1994; Ammor et al. 2006). A wide variety of small inhibitory molecules including hydrogen peroxide, diacetyl, hypothiocyanate, reuterin and bacteriocins, sometimes powerfully active against pathogens and food spoilage organisms, can also be produced during the growth of fermentative microbes. Other mechanisms of microbial interference potentially operative within the food matrix include competition for space and essential 3 © Woodhead Publishing Limited, 2011

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nutrients, as well as the action of bacteriophages (Holzapfel et al. 1995; Chen and Hoover 2003; Jones 2004; Chaillou et al. 2005). In the present chapter, we introduce the bacteriocins, the bacteria that produce them and the broad subject of strategies available for the identification of protective cultures and culture components. We have especially focused attention upon the LAB and their production of bacteriocinlike inhibitory substances (BLIS), because the vast majority of contemporary research in this field is concentrated on these microbes and their inhibitory products. Nevertheless, similar principles are generally applicable to other microbes as well as for the detection of non-BLIS inhibitory mechanisms.

1.2  Historical perspectives The origins of the laboratory-based study of inter-bacterial inhibition can be traced to Louis Pasteur’s studies of the interference with growth of the anthrax bacillus by common bacteria (probably Escherichia coli) when these bacteria were co-inoculated in urine (Pasteur and Joubert 1877). The basic characteristics of antimicrobials of the bacteriocin class were first elucidated by the systematic investigations of interstrain E. coli antagonism by Gratia and Fredericq in the first half of the twentieth century (Gratia 1925; Fredericq 1946). The first described bacteriocins, the colicins, were so-named by Gratia because of their killing action against E. coli. Bacteriocins are ribosomally-synthesized antimicrobial peptides, apparently produced by strains of all bacterial species and indeed it has been speculated by most (if not all) bacteria growing in natural ecosystems (Riley and Wertz 2002). Unlike classical therapeutic antibiotics, bacteriocins tend to have a relatively narrow killing spectrum and this is typically centred upon members of species closely-related to the producing cell (Riley and Wertz 2002). It is presumed that bacteriocins provide the producer bacterium with a growth advantage in complex highly-competitive microbial populations. Since there is a metabolic cost as well as a significant genetic investment associated with bacteriocin production, the survival value of retention and expression of bacteriocins must outweigh the burden that they impose on the host bacterium in order for bacteriocinogenicity to persist in natural populations. Fredericq used specific (receptor-deficient) colicin-resistant mutants to classify the colicins (Fredericq 1946). Key defining characteristics included: (i) a plasmidencoded, large domain-structured protein composition; (ii) bacteriocidal activity via specific receptors; and (iii) lethal SOS-inducible biosynthesis. By comparison, the study of the bacteriocins of Gram-positive bacteria had a relatively-faltering start. The initial focus was on the staphylococci and attempts were made to apply similar classification criteria to those that had been previously established for the colicins. However, it soon became apparent that relatively few of the protein antibiotics of Gram-positive bacteria fit the classical colicin criteria. Substantial differences included their relatively broad activity spectra, somewhat less stringent producer cell self-protection (immunity) and the absence of SOS-inducibility. In the past three decades, studies of the bacteriocins of Gram-positive bacteria and especially those of LAB have come to dominate the bacteriocin literature, and

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this change in emphasis has largely been driven by commercial imperatives, especially in the nascent field of biopreservation (Deegan et al. 2006).

1.3 Bacteriocins of Gram-positive bacteria and the nature of bacteriocin-like inhibitory substance (BLIS) In this laboratory we first proposed use of the acronym BLIS (bacteriocin-like inhibitory substance) as a term of convenience to denote inter-bacterial inhibition that appears likely to be due to the production of bacteriocin(s), but prior to confirmation of the genetic and molecular identity of the inhibitory agent(s). Bacteriocins of Gram-positive bacteria have recently been classified into four major divisions: (a) Class I: post-translationally modified small (<10 kDa) peptides (the lantibiotics) (b) Class II: non-modified small peptides (c) Class III: large (> 10 kDa) proteins and (d) Class IV: cyclic peptides (Heng et al. 2007). Examples of bacteriocins found in Classes I–IV and their sub-divisions are presented in Figure 1.1. It seems prudent to regard bacteriocin classification schema as works in progress since the range of molecular entities potentially classifiable as bacteriocins is continuing to expand both in numbers and in compositional heterogeneity. Bacteriocins are composed of peptides or peptidecomplexes, typically comprise between 30 and 60 amino acid residues, and are released in bioactive forms extracellularly. Many act on the bacterial cytoplasmic membrane, disrupting the proton motive force by forming pores in the phospholipid bi-layer (Cintas et al. 2001; Ammor et al. 2006). Other modes of action described include the inhibition of protein synthesis, peptidoglycan formation and spore germination; and interference with sodium and potassium transport (Upreti 1994; Chatterjee et al. 2005). The bacteriocins of LAB are generally ineffective against Gram-negative bacteria due to the possession by such organisms of an outer membrane (Gänzle et al. 1999). Exposure to certain sub-lethal stresses may however render the outer membrane permeable to bacteriocins such as nisin and pediocin and under these conditions killing activity has been demonstrated (Kalchayanand et al. 1992). Some bacteriocinogenic LAB have also been found to have limited direct inhibitory activity against Gram-negative bacteria. For example, in a study that used simple agar diffusion assays to screen over 10,000 LAB from poultry production environments for activity against Campylobacter jejuni, 2% of tested isolates were found to be inhibitory (Stern et al. 2005). Similarly, propionin PLG-1, a heat-labile 10 kDa bacteriocin produced by the dairy bacterium Propionibacterium thoenii, has been reported to be inhibitory toward C. jejuni (Barefoot and Nettles 1993) and bacteriocin-like inhibitory activity against both Campylobacter and Helicobacter pylori has been reported in lactobacilli from the human gut (Strus et al. 2001).

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Fig. 1.1  Gram-positive bacteriocin classes and sub-divisions. Based on Cotter et al. (2005) with modifications by Heng et al. (2007).

Some bacteria can produce more than one bacteriocin and multiplybacteriocinogenic strains are, for example, especially common in the species Streptococcus salivarius, Streptococcus uberis and Streptococcus mutans (Table 1.1). Bacteriocin-producing S. salivarius harbour megaplasmids (160–220 kb), some of which have been shown to encode as many as five different bacteriocins. Streptococcus uberis 42 produces both nisin U (a class I [lantibiotic] bacteriocin) and uberolysin (a class IV [cyclic] bacteriocin). Streptococcus mutans UA140 produces the lantibiotic mutacin I and a class II bacteriocin (mutacin IV). Conversely, the same bacteriocin can sometimes be produced by strains of different LAB species (Table 1.2). For example, the bioactive forms of the lantibiotics SA-FF22 (Tagg and Wannamaker 1978) and macedocin (Georgalaki et al. 2002) are identical peptides, initially shown to be produced by Streptococcus pyogenes and more recently by Streptococcus macedonicus respectively. Highly-homologous SA-FF22-like peptides are also known to be

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Table 1.1  Examples of LAB that produce more than one bacteriocin Producer

Bacteriocin

C. piscicola LV17 Carnobacteriocin A, B2, BM1 E. faecium CTC492 Enterocin A, B L. plantarum C11 Plantaricin EF, JK L. sakei 5 Sakacin 5X, P, T S. uberis 42 Nisin U, uberolysin S. mutans UA140 Mutacin I, IV S. mutans K8 Mutacin K8, IV S. salivarius 9 Salivaricin 9, A4 S. salivarius K12 Salivaricin A2, B

Reference Quadri et al. (1994) Worobo et al. (1994) Nilsen et al. (1998) Anderssen et al. (1998) Vaughan et al. (2001) Wirawan et al. (2007) Qi et al. (2001) Robson et al. (2007) Wescombe et al. (2009) Hyink et al. (2007)

Table 1.2  Examples of the same bacteriocin produced by different LAB species Bacteriocin

Producer species

Reference

SAFF22a

S. pyogenes S. macedonicus

Jack et al. (1994) Georgalaki et al. (2002)

Sakacin-A (curvacin-A)

L. sakei L. curvatus

Axelsson and Holck (1995)

Salivaricin A1a

S. pyogenes S. dysgalactiae subsp. equisimilis S. agalactiae

Wescombe et al. (2006b)

Pediocin PA-1

Pediococcus (several spp.) L. plantarum

Miller et al. (2005)



(macedocin)

a Similar

peptides are also known to be produced by strains of S. salivarius and S. dysgalactiae subsp. equisimilis.

produced by strains of Streptococcus salivarius and Streptococcus equisimilis (Wescombe 2002). Similarly, sakacin A and curvacin A are the same molecule produced by Lactobacillus sakei and Lactobacillus curvatus respectively (Axelsson and Holck 1995; Axelsson 2007 – pers. comm.). The kinetics of production of a particular bacteriocin may also differ according to the host strain. For example, sakacin A is produced throughout the growth of L. sakei, but curvacin A is only produced in the late logarithmic growth-phase by L. curvatus (Holck et al. 1992; Vogel et al. 1993). The naming of bacteriocins lacks formal guidelines but is generally based upon either the species or generic designation of the original source bacterium. Examples of bacteriocins named for their species of origin are the salivaricins, ubericins, and curvacin, whereas the staphylococcins and lactocins display their generic heritage. Since a variety of bacteriocins may be produced by bacteria belonging to a single species, additional designations are required in order to more precisely specify each particular bacteriocin molecule. Once again, a variety

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of conventions have been adopted, but we favour the allocation of successive letters of the alphabet (e.g., salivaricin A and salivaricin B were the first bacteriocins characterized from the species S. salivarius). For even more precise specification of a particular bacteriocin the strain designation of the producer bacterium can be included within the bacteriocin name (e.g., streptococcin A-FF22 is a bacteriocin produced by S. pyogenes strain FF22 – Tagg and Wannamaker 1978). Bacteriocins that have only minor conservative differences in the amino acid sequences of their propeptide components resulting in no significant change to their (a) secondary structure, (b) activity spectrum and (c) the specific crossimmunities of their respective producer strains are more appropriately referred to as natural variants (Heng et al. 2007). For example, nisin Z, nisin Q and nisin U are natural variants of the first-described nisin A (Wirawan et al. 2006). Most small bacteriocins are active over a wide pH range and their high isoelectric points allow them to interact, under physiological pH conditions, with the anionic surface of bacterial cells (Oscáriz and Pisabarro 2001). This feature, combined with their generally highly hydrophobic nature, has enabled purification procedures to be developed based on hydrophobic interaction, cation exchange and reversed phase chromatography resins (Oscáriz and Pisabarro 2001). Small bacteriocins tend also to be heat-stable due to their content of di-sulphide and thioether bonds, which limit the potential for un-folding under heat stress conditions. Consequently, small bacteriocins tend to retain their activity after autoclaving, whereas larger bacteriocins such as helveticin J (Joerger and Klaenhammer 1986) and zoocin A (Simmonds et al. 1996) are inactivated by 10–30 min at temperatures ranging between 60 and 100 °C. A landmark observation in the field of LAB bacteriocin research was the confirmation in 1947 that the inhibitory activity of some lactococci (then referred to as group N streptococci) toward other LAB was at least in part attributable to an antimicrobial substance called nisin (for group N inhibitory substance) (Mattick et al. 1947). Nisin, now approved for use as a food additive in more than 50 countries, is regarded as the prototype of the bacteriocins of Gram-positive bacteria and more specifically of those belonging to the lantibiotic class. Interestingly, the original discovery of nisin (Rogers 1928), the progenitor of the commercially-applicable peptide antibiotics, was one year earlier than the much more celebrated discovery of penicillin (Fleming 1929), still the benchmark of the clinically-significant non-proteinaceous antibiotics. The success of nisin as a food preservative adjunct stimulated frenetic prospecting for alternative inhibitory agents that might find comparable application to food preservation. In 1976, the first review of the then burgeoning studies of the bacteriocinogenicity of Gram-positive bacteria (Tagg et al. 1976) predicted that this field would continue to flourish and that it would be largely motivated by the perceived potential for applications of these bacteriocins to bacterial interference and food preservation. Indeed, several groups of enthusiasts continued to explore the potential application of bacterial interference through the early days of the antibiotic era, mostly targeting Staphlyococcus aureus, due to its predilection for

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antibiotic resistance development. More recently however, bacterial interference research has become more focused on modulation of the microflora of the human oral cavity in an attempt to control a variety of ailments ranging from halitosis to dental caries and streptococcal pharyngitis (Tagg and Dierksen 2003). Bacteriocin-producing probiotic strains to gain commercial traction for the control of oral infections are S. salivarius K12 (producer of the lantibiotics salivaricin A and salivaricin B) (Power et al. 2008) and the genetically-modified S. mutans JH1140 (producer of mutacin 1140 and mutacin IV) (Hillman et al. 2007). Studies of the bacteriocins of LAB now dominate the literature in this field, most of the reports however containing only relatively superficial descriptions of bacteriocin activity spectra against randomly-selected collections of indicator bacteria and ending with optimistic predictions of the potential of these bacteriocins for commercial application. Few bacteriocins have actually lived up to these aspirations, among the more successful being nisin and the pediocins (class 2 bacteriocins of various Pediococcus species) (Schillinger et al. 1996; Paul Ross et al. 2002; Parada et al. 2007). An account of bacteriocinogenic LAB viewed as useful to the food industry has been prepared by Schillinger et al. (1996) and some examples that have progressed into commercial applications are presented in Table 1.3. More recent examples include the Lactococcus lactis producer of the two-component lantibiotic lacticin 3147, which has been used to control Listeria on the surface of smear-ripened cheese (O’Sullivan et al. 2006) and a leucocinproducing strain of Leuconostoc carnosum (Budde et al. 2003) which has been Table 1.3  Examples of bacteriocins that may potentially be useful in the food industry Bacteriocin Producer

Active against

Food applications

Example

Variety of Gram-positive strains Listeria

Dairy, bakery, Nisaplin® vegetable products Processed Alta 2341® meats

Reference

Nisin A

L. lactis

Pediocin PA-1

P. acidilactici

Leucocin A/B

L. carnosum Listeria, LAB 4010

Processed meats

Lacticin 3147

L. lactis

Broad range of Gram-positive strains

No Dairy, commercial fermented product meats, biomedical applications in humans and animals

O’Sullivan et al. (2006)

LAB, clostridia

Cheese, meat products, vegetables

O’Sullivan et al. (2003)

Lacticin 481 L. lactis

SafePro® B-SF-43

No commercial product

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Paul Ross et al. (2002) Rodríguez et al. (2002) Budde et al. (2003)

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incorporated into a commercial bio-preservation product for the control of Listeria in stored meat. One of the more recent strains to enter the commercial arena is Streptococcus macedonicus ACA-DC 198, a producer of the SA-FF22 look-alike lantibiotic macedocin (Georgalaki et al. 2002). It has been suggested that highly-competitive strains such as this that can be found as part of the natural microbiota of foodstuffs can perhaps be considered to mediate a rudimentary form of ‘immunity’ in food (Cotter et al. 2005). As such, the directed modification of the natural food microbiota by supplementation with safe but highly-competitive LAB is perhaps the equivalent of the microbiota-modification strategies adopted for the implementation of microbial interference/colonization resistance to prevent infections of the human host.

1.4 Characteristics of microbes and inhibitory products of relevance to their potential protective activity in food Bio-preservation has been explored as a means of increasing the storage life and enhancing the safety of stored meat as a result of seeding the product with ‘naturally’ associated microorganisms or their products (Holzapfel 1998). While a few LAB species have been associated with negative attributes, such as spoilage and pathogenicity, most can be viewed as relatively innocuous and in some cases may even contribute to the safety and durability of the stored meat (Holzapfel et al. 1995). Many LAB are now permitted as food additives and lists of GRAS (generally recognized as safe) strains are maintained by several countries. For example, a range of Lactobacillus and Carnobacterium species are permitted as food additives in the U.S. (Tarantino 2005a,b,c, 2008) and the New Zealand Food Safety Authority also maintains a list of GRAS strains that contains several species of Lactobacillus (Anonymous 2009). Some of the desirable characteristics of biopreservative agents are listed in Table 1.4, reflecting a need for agents to be (i) safe (i.e., do not contribute to health risk of the food), (ii) stable (i.e., maintains inhibitory activity during storage), (iii) effective (i.e., broadly active against all major infective/spoilage bacteria and fungi),

Table 1.4  Desirable criteria for bio-preservative agents 1. Non-toxic 2. Regulatory approved (GRAS) 3. Low cost 4. No negative organoleptic effects 5. Effective in low concentrations 6. Stable at storage conditions 7. No medical application

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(iv) resistant to selection (i.e., resistant strains of target bacteria not readily selected for), (v) complementary (i.e., not significantly neutralized by antagonistic activity of the food environment (pH, fats, etc.) and compatible with the physiological and chemical characteristics of the food material), and (vi) lethal (i.e., bacteri-/fungi-/spori-cidal in preference to static). 1.4.1  Strain sources It tends to be fermentative organisms that are most typically evaluated for their potential in food bio-preservation. For the preservation of foods where minimal flavour and textural changes are desired, homofermentative LAB strains may provide a greater range of seeding candidates than heterofermentative strains due to their production of relatively innocuous (from a sensory point of view) antimicrobial compounds, such as lactic acid, as a dominant proportion of their fermentation end-products. Culture collections are a source of well-characterized strains, although most of these have probably by now been thoroughly tested for antimicrobial activity. Nevertheless, they still provide a potential source of inhibitory agents targeting newly-emerging pathogens and spoilage organisms or of indicator strains for the evaluation of new screening methods. Alternatively, natural environments provide rich sources of bio-preservation strains, particularly the surfaces of living plant material or strains adapted to growth on materials similar to the envisaged environment of application (e.g., the surfaces of chill-stored packaged meat). The indigenous microbiota of humans and other animals also provide an abundant resource, especially if it is the antimicrobial product and not the microbe itself which is to be utilized, as this allows the opportunity for inhibitory products to be exploited from organisms that may otherwise be associated with spoilage or pathogenicity. There are also prospects for genetic engineering of strains as has been the case with mutacin 1140-producing Streptococcus mutans – engineered to reduce acid formation without compromising bacteriocin-related competitive mechanisms against dental caries-inducing strains of S. mutans (Hillman et al. 2007). 1.4.2  Inhibitory products The inhibition of one strain by another can be due to simple or complex mechanisms, some of which may be growth medium-dependent. It is therefore important to examine potential bio-preservation strains in media and environments reflecting those intended for their application. The production of a bacteriocin is a relatively simple inhibitory mechanism whereby the proliferation of one organism is restricted by the generation by another of a proteinaceous inhibitory molecule. For example, in some stored meat and broth environments L. monocytogenes cells are inhibited by sakacin A, produced by L. sakei Lb706 (Schillinger et al. 1991; Jones et al. 2009). On the other hand, inhibitory mechanisms may involve a combination of more general factors. For example, LAB generate large amounts

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of organic acids during growth. Un-dissociated acid molecules are inhibitory to a range of bacteria due to such factors as disrupting membrane permeability (BairdParker 1980; Ammor et al. 2006). The associated reduction of pH in the environment is also inhibitory to less acid tolerant bacteria such as meat spoiling Enterobacteriaceae and Brochothrix thermosphacta. Depending on the bacterial species involved, inhibitory products may also include small molecules such as alcohols, hydrogen peroxide, hypothiocyanate and diacetyl. Nisin has been reported to be poorly effective against target strains in the raw meat environment (Leisner et al. 1996; Stergiou et al. 2006) and hence is not widely used in the meat industry. The reduction of activity is thought to be due to the formation of nisin-glutathione complexes in the meat matrix and this may be an enzyme-mediated process because neutralization is more pronounced on raw meat than cooked (Stergiou et al. 2006). Furthermore, nisin binds to fats and proteins and its efficacy against L. monocytogenes has been observed to reduce with increasing fat content (Schillinger et al. 1996). Such factors further demonstrate the importance of screening producer strains using conditions that resemble the intended application environment as closely as possible.

1.5  Screening methodologies in food biopreservation Although potential food bio-preservative strains should ideally be screened for inhibitory activities using food substrates and storage conditions relevant to their intended use, both cost and time considerations may preclude the rapid screening of large numbers of strains using simulations of typical food storage conditions. Agar-substrate-based deferred and simultaneous antagonism methods (and variations thereof ) have formed the mainstay of screening methods used to detect antibiosis in vitro. Indeed, influenced by mechanisms such as quorum sensing (Riley and Wertz 2002), the activity of many bacteriocins is better demonstrated in agar culture systems than in liquid cultures. On the other hand, some bacteriocins such as streptocins STH 1 and STH 2 appear only to be produced in liquid media (Schlegel and Slade 1973; Tompkins et al. 1997). Screening methodologies should ideally eliminate previously-characterized compounds early in the characterization process. They also should not preclude antimicrobials produced in small quantities under the specific in vitro assay conditions because the levels (and efficacy) of inhibitor production may be more substantial in situ or under different incubation conditions. Similarly, inhibitor production in the food environment may not be detectable using agar-based methods, representing a possible drawback that needs to be considered when designing an experimental approach. 1.5.1  Variations of agar diffusion tests Screening tests for inter-bacterial inhibition on agar media do not, of course, distinguish the activities of bacteriocins from inhibition due to non-bacteriocin

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agents such as bacteriophage, primary metabolites such as H2O2 and lactic acid or non-ribosomally-encoded antibiotics such as bacitracin. Nor can such tests discriminate inhibition attributable to nutrient depletion or to the combined activities of multiple bacteriocins and/or other inhibitory agents. Direct (simultaneous) antagonism In simultaneous antagonism the test and indicator bacteria are typically grown together on an agar surface and detection of bacteriocin production is dependent on release of the inhibitory agent(s) relatively early in the growth of the test culture (i.e., before overgrowth of the indicator bacterium) (Fig. 1.2a). The culture medium and incubation parameters must provide conditions for simultaneous growth of the inhibitor-producing and indicator strains. The indicator

Fig. 1.2  (a) Simultaneous antagonism test. (b) Deferred antagonism.

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is seeded into the agar growth medium, swabbed on the surface or applied as a seeded overlay agar. The potential inhibitor-producer strains are applied as ‘spots’ onto or stab inocula into the surface of the seeded plates or plates that will be subsequently overlaid with indicator culture. Other methods include application of the test strain into wells cut into the agar or as paper discs saturated with culture or perpendicular cross-streaks of indicator and antagonist cultures on the agar surface. Deferred antagonism By contrast, the deferred antagonism test, which is most commonly used in bacteriocin typing procedures, allows for independent variation of the incubation parameters (time, temperature, atmosphere) of the test and indicator bacteria (Fig. 1.2b). This is a variation in which the inhibitor-producer cells are either killed after producing inhibitor or are physically separated from the indicator. Exposure to chloroform or UV irradiation is commonly used to kill the producer cells. Otherwise ‘flipping’ the agar surface on which producer cultures have grown with indicator application on the reverse side provides physical separation of the producer and indicator strains. 1.5.2  Medium composition Media components can impact on the ability of a strain to produce detectable quantities of inhibitory substances, as well as affecting the stability of the inhibitory substances and the sensitivity of the indicator strains. In our experience, for example, culturing LAB in Brain Heart Infusion typically results in poor bacteriocin production, whereas higher yields may be obtained using Todd Hewitt broth-based media. A trial and error approach should be used to determine which medium gives maximum inhibitor production. Luria agar has been used for screening meat LAB, reflecting the low sugar levels found in such substrates. Typical modifications include increasing or decreasing the levels of sugars and buffers or the addition of the emulsifier Tween 80, providing indicator strain sensitivity is not significantly affected. Some examples of the influence of the growth medium on bacteriocin production are listed in Table 1.5. Another factor indirectly influencing bacteriocin yield can be the culture pH. For example, the release of the S. pyogenes lantibiotic SA-FF22 from the producer cells is increased as the culture pH falls below 6.5. At more neutral pH values the bacteriocin largely remains surface-associated. Another effect of pH decrease in growing cultures of S. pyogenes can be the activation of SpeB (streptococcal proteinase) and this can impact on the yield of the proteinase-susceptible lantibiotic. Bacteriocin production is typically enhanced when the producer cells are relatively stressed (nutritionally or environmentally) and so it may be helpful to slow the growth of cultures by utilizing diluted sources of nutrients and also to expose the growing culture to a wide variety of incubation conditions.

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Table 1.5  Examples of medium composition influence on bacteriocin production 1. Produced only in solid media in vitro, e.g. mutacin Ia, streptinb 2. Susceptible to catabolite repression, e.g. nisinc 3. Enhanced by yeast extract, e.g. mutacinsd 4. Dependent on blood supplementation, e.g. streptine 5. Repressed by magnesium ions, e.g. SA-FF22f a Qui

et al. (1985). and Tagg (2003). c Tagg et al. (1975). d Rogers (1972). e Hynes and Tagg (1985). f Jack and Tagg (1992). b Wescombe

1.5.3  Incubation parameters Temperature Incubation temperature can affect production of antimicrobials as well as having a direct effect on bacterial growth rate. Incubation at higher temperatures can also result in curing of plasmids containing genetic information required for the production and externalization of bacteriocins. Most bacteriocins are sufficiently thermostable to remain active well beyond the time when viable cells can no longer be recovered from the producer culture. Time Inhibitors can accumulate to maximum levels during the late log or early stationary growth phase accompanied by diminishing levels of detectable activity thereafter. The reduction of bacteriocin activity can be due to: (i) the action of proteinases or of immunity substances appearing later in the growth of the culture, (ii) destabilization of the bacteriocin due to acid accumulation, or (iii) adsorption of bacteriocins to producer cells or components of the growth medium. Atmosphere The effect of atmospheric conditions on production of inhibitors by fermentative bacteria has not been systematically investigated. Static conditions in normal atmosphere are typically used in bioassays though anaerobic conditions are substituted when using more oxygen-sensitive strains and 5% CO 2 supplemented air for oral bacteria. Hydrogen peroxide can be generated in oxygen-containing environments and this can cause inhibitory activity even though some LAB possess mechanisms to repair such ‘self-harm’ (Chaillou et al. 2005). The potential to generate H2O2 can be reduced using anaerobic incubation (or by addition of catalase or fresh blood).

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1.5.4  Indicator strains Indicator strain sensitivity can vary within a species and can be influenced by the growth conditions. Although various chemical and physical methods are available to detect and quantitate bacterial antagonism, the most commonly used methods are microbiological assays (bioassays) that directly measure the key property of interest – antagonism. The main methods of bioassay are agar diffusion and turbidimetric tube assays which measure the response of indicator organisms to introduced potentially inhibitory substance(s). The assay outcome is usually recorded visually in terms of the extent of the growth response (inhibition). In practice, it is important to test by both simultaneous and deferred antagonism when screening bacteria for bacteriocinogenicity. Optimal conditions for test strain growth do not necessarily coincide with optimal bacteriocin production conditions. In fact, some strains produce their highest levels of bacteriocins under stressful environments, such as when growth factors are limiting. For screening purposes a set of carefully chosen relevant indicators should be used. For example, if screening for LAB that inhibit L. monocytogenes growth on chilled stored meat it is appropriate to use indicator strains of L. monocytogenes that have been associated with human disease and that are capable of growth on stored meat. It is also recommended that representatives of the same species as the potential inhibitor producer be included as well as some of related species or of species co-isolated from the same habitat. It is our experience that Micrococcus luteus is a particularly sensitive indicator of cationic peptides and so this is routinely included in our set of primary indicators for bacteriocin screening. Use of known bacteriocin-producers as indicator strains can be used to exclude from consideration strains producing the same inhibitor. The assessment of inhibition patterns against sets of standard indicators can also be used to help eliminate previously known inhibitors.

1.6 Our procedure for inhibitor screening in food biopreservation As standard practice in this laboratory, we first test LAB for their production of bacteriocins by use of a three-step screening process: (i) deferred antagonism bacteriocin ‘fingerprinting’ using a set of nine standard indicator strains (Tagg and Bannister 1979), (ii) repeating the bacteriocin fingerprinting procedure, but incorporating a heating step (80 °C for 45 min) prior to application of the indicator (detector) bacteria, and (iii) polymerase chain reaction (PCR)-based detection in the case of lantibiotic processing genes (lanM, lanB and lanC). This process can sometimes provide preliminary evidence for the production of multiple bacteriocins by the test strain and also may hint to the possible class of inhibitory molecule(s) being produced. For example, the lantibiotics (Class I)

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typically produce heat-stable inhibition of the M. luteus indicator strain, whereas inhibitory activity due to Class III (large) bacteriocins is usually eliminated by the heating step. Similarly, if involvement of Class II bacteriocins is suspected, such as may be the case with strains of Lactobacillus, additional information on molecule composition has been obtained using well-diffusion assays and cell-free filtrates treated with a range of temperatures and proteases (Jones et al. 2008). Our application of such procedures to many different species of LAB has shown that even use of just a single set of nine indicator strains can demonstrate a very high frequency of BLIS detection. S. mutans, S. salivarius and S. uberis exhibit a particularly high incidence of bacteriocinogenicity, with some strains producing combinations of bacteriocins belonging to different classes. Some notable examples include: (i) bacteriocin-producing S. salivarius harbour mega-plasmids (typically 160– 220 kb), some of which encode no less than five different bacteriocins (Wescombe and co-workers, unpublished observations), (ii) S. uberis 42 produces the lantibiotic nisin U and uberolysin, a circular (cyclic) bacteriocin (Wirawan et al. 2007), and (iii) S. mutans UA140 elaborates a lantibiotic (mutacin I) and a Class II inhibitory agent (mutacin IV) (Qi et al. 2001).

1.7  Molecular methods of screening in food biopreservation More recently, with the advent of molecular techniques capable of sequencing whole bacterial genomes within 24 hours, molecular screening methods have become feasible as a way of searching for desirable properties in candidate bacterial strains for food preservation. There are now approximately 950 complete bacterial genomes available through the NCBI database, with the elucidation of a further 2100 genomes in progress and additional projects being added continually. This presents a huge resource for those interested in identifying new bacteriocins, antibiotics and other factors of potential value for the food industry. Each genome of a species can be searched for genes or gene clusters of interest such as those having homology to known bacteriocins, or those encoding for non-ribosomally synthesized peptides (NRSP) such as bacitracin. Once such genes are identified, the gene products can be further characterized for their actual function and strains naturally producing them can then be searched for either in culture collections or in foods of interest with the intention of identifying a strain useful for food production or preservation purposes. In many genomes however, small open reading frames (ORFs) have not been adequately identified or annotated and often many bacteriocin-encoding genes can be missed. No doubt this issue will be resolved in the future but for now it is important that researchers should not simply rely on the publicly annotated version, but should look closely at small ORFs for their potential to be bacteriocins. An example of this approach was by Dirix and colleagues (Dirix et al. 2004a;

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Dirix et al. 2004b) who screened for the presence of potential bacteriocins/signal pheromones having double glycine leader sequences in both Gram-positive and Gram-negative genomes. They determined that 33% of genomes from Gramnegative bacteria contain one or more transporters carrying a Peptidase C39 domain, compared to 44% of the genomes of Gram-positive bacteria. In addition, more than 40% of the identified peptide genes were either un-annotated or had not yet been recognized as secreted peptides in the genome-sequencing projects. A second web-based peptide bacteriocin search engine has been developed which has been given the acronym BAGEL (de Jong et al. 2006). BAGEL has been designed to apply a number of ORF prediction tools that take into account genes involved in bacteriocin biosynthesis machinery, regulation, transport function and immunity increasing the likelihood of identifying these loci in genomes. For example, a BAGEL evaluation of the Streptococcus pneumoniae TIGR 4 genome identified 11 significant peptide bacteriocin genes – including all seven originally annotated bacteriocins (Tettelin et al. 2001) and four additional ones. In addition, a further 18 potential bacteriocin genes and 44 ORFs having some homology to bacteriocin genes were identified (Nes et al. 2007). It is important to note, however, that the mere presence of a bacteriocin structural gene in a genome does not mean that it is produced. For example, in S. pyogenes most M-serotypes encode the salivaricin A structural gene but only M-type 4 S. pyogenes have so far been found to produce the active peptide (Johnson et al. 1979; Simpson et al. 1995). This has been determined to be due to deletions in other genes within the locus such as the transporter and modification genes (Upton et al. 2001; Wescombe et al. 2006a). Similarly, sakacin A structural genes are present within an 8.7 kb sequence on a 60 kb plasmid in L. sakei Lb706 and also in its non-bacteriocinogenic analogue L. sakei Lb706-B (Schillinger and Lücke 1989; Axelsson and Holck 1995). In the case of L. sakei Lb706-B, original plasmid curing attempts using acriflavin caused a mutation in the HPK (sapK) gene region responsible for transport functions resulting in the loss of ability to externalize the active bacteriocin (Axelsson et al. 1993) (pers comm. Urlich Schillinger, May 2007). Additionally, it can be difficult to detect the action of some of the bacteriocins since many are extremely limited in their inhibitory spectrum and this can make the identification of the susceptible species also difficult. Furthermore, bacteriocins can be highly regulated and only expressed in certain circumstances which may not be easily replicated in vitro (Nes and Eijsink 1999; Rawlinson et al. 2002). Such factors mean that phenotypic screening is probably the preferred option for initially identifying strains of interest while molecular methods can be used to further narrow the search down and provide information around the useful aspects of each chosen strain. Genetic engineering of strains either to disable virulence genes or to increase the efficacy of a particular strain is yet another way to use the power of molecular biology to select or create new strains useful for food preservation. Many bacteriocins are encoded for on plasmids or transposable elements which can be transferred either naturally, or through standard molecular procedures to another

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strain or even species (Jack et al. 1995; Wescombe et al. 2006b). This allows for the introduction of useful characteristics to species of interest either for the prevention of food spoilage in fermented foods or for other downstream properties such as useful probiotic qualities or survival mechanisms. An example of such an engineered strain is S. mutans BCS3-L1, in which recombinant DNA methods have been used to delete essentially the entire open reading frame (ORF) for lactic acid dehydrogenase (LDH). This mutation created a metabolic blockade that was lethal when exchanged for the wild-type allele, but it was found that replacing the ORF for LDH with the ORF for alcohol dehydrogenase B from Zymomonas mobilis overcame this blockade to yield a viable strain called BCS3-L1 that produced wild-type levels of mutacin 1140 and no lactic acid (Chen et al. 1994; Hillman et al. 1994; Hillman et al. 2000). Due to the modifications, the strain has significantly reduced cariogenicity, but excellent colonization potential through the production of a natural antibiotic called mutacin 1140. Further modifications were able to be introduced for use in human clinical trials to enable rapid elimination of the strain in case of adverse side effects and to increase genetic stability (Hillman et al. 2007). The genetic locus for the production of nisin has been used as the basis for a Gram-positive expression system capable of specifically over-expressing genes of interest at controlled time points due to the ability to induce gene expression in response to the addition of exogenous nisin (Kuipers et al. 1995). This system has been named the ‘NIsin Controlled gene Expression system’ or NICE. This powerful system has been able to be transferred into other species including Leuconostoc lactis, Lactobacillus sp., Streptococcus sp., Enterococcus sp. and Bacillus subtilis enabling advances in understanding the pathogenicity of some of the bacteria and helping facilitate dose-response studies for live vaccine work (Kleerebezem et al. 1997; Mierau and Kleerebezem 2005). One of the major problems with this approach is codon usage of the gene to be expressed. Genes from genera closely related to Lactococcus generally have little trouble with expression while those from other organisms depend largely on their use of rare codons for successful expression (Mierau and Kleerebezem 2005). To get around this issue, alternative systems also based on two-component regulatory systems of bacteriocins have been developed such as the SURE system for B. subtilis (Kleerebezem et al. 2004), L. plantarum (Mathiesen et al. 2004), L. sakei (Axelsson et al. 2003) and Enterococcus sp. (Hickey et al. 2003), all of which help to improve the range of Gram-positive organisms that can be genetically manipulated to express proteins of interest. Some of the applications of these systems have included the expression of enzymes for use in food applications such as phage lysins or peptidases and esterases to influence flavour formation in dairy fermentations (de Ruyter et al. 1997; Wegmann et al. 1999; Hickey et al. 2004; Berlec and Strukelj 2009). Given time and a wider acceptance of genetically modified organisms by the public it would be envisioned that the majority of strains used in food preservation will be engineered derivatives combining traits of stability, bacteriocin production and flavour or texture enhancing properties from different species.

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1.8  Future considerations The currently-favoured candidates for screening as protective cultures or as sources of biopreservative antimicrobials are microbes that either have been accorded GRAS status or that are recognized as being of low virulence, as in the case of the producers of lacticin 3147 and macedocin. It is interesting to note however that both lacticin 3147 and macedocin are homologs of the bioactive lantibiotics, staphylococcin C55 and SA-FF22, originally characterized in two of the classic Gram-positive pathogens of humans, S. aureus and S. pyogenes respectively. That a number of bacteriocin loci have been detected in both pathogens and commensals indicates that natural intra-specific transmission of bacteriocin determinants occurs commonly in nature and this observation raises potential concerns about bacteriocin-promiscuity, which could potentially render target pathogens present in foods specifically insensitive (immune) to seeded cultures of putative bioprotective bacteriocin-producing bacteria. One way to help alleviate this concern may be to select for biopreservative strains producing multiple bacteriocins (having different modes of action) which should reduce the likelihood of pathogenic species simultaneously acquiring immunity to all of these bacteriocins. Alternatively, combinations of protective bacterial strains, each expressing different bacteriocins, could be adopted as food preservation ‘cocktails’. Given time and a wider acceptance of genetically modified organisms by the public it can also be envisioned that many of the strains finding successful application in food preservation will ultimately be engineered derivatives combining optimized traits of stability, bacteriocin production and flavour or texture enhancing properties from different species.

1.9  References ammor s , tauveron g , dufour e

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et al. (2006a), ‘Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius.’ Antonie Van Leeuwenhoek 90(3): 269–280. wescombe p a and tagg j r (2003), ‘Purification and characterization of streptin, a type A1 lantibiotic produced by Streptococcus pyogenes.’ Applied and Environmental Microbiology 69: 2737–2747. wescombe p a , upton m , dierksen k p , ragland n l , sivabalan s et al. (2006b), ‘Production of the lantibiotic salivaricin A and its variants by oral streptococci and use of a specific induction assay to detect their presence in human saliva.’ Applied and Environmental Microbiology 72(2): 1459–1466. wirawan r e , klesse n a , jack r w and tagg j r (2006), ‘Molecular and genetic characterization of a novel nisin variant produced by Streptococcus uberis.’ Applied and Environmental Microbiology 72(2): 1148–1156. wirawan r e , swanson k m , kleffmann t , jack r w and tagg j r (2007), ‘Uberolysin: A novel cyclic bacteriocin produced by Streptococcus uberis.’ Microbiology 153(5): 1619–1630. worobo r w, henkel t , sailer m , roy k l , vederas j c and stiles m e (1994), ‘Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A.’ Microbiology 140(3): 517–526. wescombe p a , burton j p , cadieux p a , klesse n a , hyink o

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2 Antifungal lactic acid bacteria and propionibacteria for food biopreservation S. Miescher Schwenninger, L. Meile and C. Lacroix, ETH Zurich, Switzerland

Abstract: Foodborne fungi, i.e. yeasts and moulds, cause serious spoilage of stored food leading to enormous economic losses. Moulds can also produce mycotoxins that are associated with several acute and chronic diseases in humans. Although many bacteriocin-producing cultures have been described and proposed as biopreservatives in the past few years, research carried out with fungus suppressors concerning their role in food spoilage is still very limited. We discuss here the potential of antifungal lactic acid bacteria (LAB), propionic acid bacteria (PAB), and combinations thereof in food biopreservation highlighting recent achievements in the study of antifungal metabolites and further inhibitory mechanisms. Key words: antifungal, protective culture, low-molecular-weight metabolites, Lactobacillus, Propionibacterium.

2.1  Introduction Yeasts and moulds are recognized worldwide as serious food spoilage microorganisms that resist many food processing steps not only in small artisanal production but also in highly sophisticated industrial-scale sites where they lead to enormous economic losses. Fungal mycotoxins affect food quality and may provoke a toxic response after consumption of the spoiled food. In addition to their important role in food fermentation, the antimicrobial activities of lactic acid bacteria (LAB) and propionic acid bacteria (PAB) make them especially appropriate for industrial biopreservation applications. This reflects the general public demand of reduced use of chemical preservatives and food additives and a concomitant reduction of E-numbers in food labelling. LAB and PAB are highly suited for a broadened application beyond classical food fermentation due to their status as food grade organisms and the consumer’s safe 27 © Woodhead Publishing Limited, 2011

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association of these bacteria with fermented foods (Bernardeau et al., 2008; Meile et al., 2008). The antimicrobial activities of LAB and PAB rely on a variety of antimicrobial compounds, such as the pH-reducing lactic, propionic and acetic acids, hydrogen peroxide, diacetyl, and other low-molecular metabolites as well as bacteriocins (Glatz, 1992; Daeschel, 1993). Antibacterial effects due to bacterial peptides, i.e. bacteriocins, have been studied extensively and many bacteriocinproducing cultures mainly of LAB but also from PAB have been described and studied as biopreservatives (Holo et al., 2002; Deegan et al., 2006). The range of antifungal LAB–PAB cultures is in contrast still very limited and research in this field has just started over the last 20 years (Miescher Schwenninger and Meile, 2004; Schnürer and Magnusson, 2005).

2.2  Spoilage fungi in food: undesired yeasts and moulds 2.2.1  Yeasts in food spoilage Yeasts play a central role in food and beverage spoilage, mainly those products with high acidity and reduced water activity (aw). A few species are even able to survive and grow under stress conditions where other microorganisms are not competitive. The ability to resist extreme conditions allows their presence in low pH products and products containing preservatives to such extent that bacteria cannot grow (Loureiro, 2000). But despite a high incidence, yeast food spoilage still receives little attention, even in foods highly sensitive to this group of microorganisms (Loureiro and Malfeito-Ferreira, 2003). Infections arising from the few known pathogenic yeasts, e.g. Candida albicans, are not transmitted through food and consequently, the public health significance of yeasts in foods has been considered by most health authorities to be minimal, if not negligible (Fleet, 1990). Allergic reactions of consumers to foods and their contaminants are however of increasing concern and yeasts have been recognized in this context (Airola et al., 2006). Changes in the sensory properties of foods do not become apparent to the consumer until yeasts have grown to populations of 105–106 cells/g and are most evident at populations of 107–108 cells/g. Carbon dioxide, one of the major products of yeast growth in foods, typically leads to swollen packages. Alcohols, organic acids, and esters, which are other major fermentation products, are in contrast often positively associated with their aromas if present at low concentrations (Fleet, 1992). Dairy products are especially sensitive to yeast spoilage. The following properties of yeasts support their growth and predominance in dairy products: • fermentation and assimilation of lactose due to the production of β-galactosidase • production of extracellular proteolytic enzymes • production of extracellular lipolytic enzymes • assimilation of lactic acid • assimilation of citric acid

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• growth at low temperatures • tolerance of elevated salt concentrations (Fleet, 1990). Typical spoilage yeasts isolated from dairy products were identified as Kluyveromyces marxianus, Zygosaccharomyes bailli, Candida spp., Pichia spp., or Rhodotorula spp. (Fleet, 1990; Miescher, 1999). In milk, raw or pasteurized, yeast growth was only reported at insignificant populations below 103 to occasionally 104 cells/ml that were quickly overgrown by psychroptrophic bacteria whereas spoilage of cream is largely known due to lipolytic species such as Rhodotorula (Fleet, 1990). Yoghurt is one of the most critical dairy products with respect to yeast spoilage where they are a major cause of spoilage of the final product. The freshly fermented yoghurt mass is usually free of yeasts since the ingredients are heat-treated (high pasteurisation) prior to fermentation and yeasts are heat-sensitive organisms destroyed by this treatment. Spoilage yeasts in yoghurt may originate either from contaminated ingredients such as fruits and nuts added to the fermented yoghurt immediately before packaging or from unsanitary equipment. We determined a predominance of Candida pulcherrima, Candida parapsilosis, Candida magnoliae, and Candida krusei in 128 isolates from yoghurt with untreated natural berries that rapidly increased to 106–107 cells/g during refrigerated storage for four weeks (Miescher, 1999; Miescher Schwenninger and Meile, 2004). Unripened fresh cheeses including curd or cottage cheese are likewise prone to yeast spoilage. Yeast populations of 106–107 cells/g frequently develop during refrigerated storage of the final product, leading to flavour defects, gas formation, and appearance of surface colonies (Fleet, 1990). Yeasts will likely spoil soft brined cheeses of the pasta filata group (e.g. Mozzarella) or Feta where they appear on the surface leading to musty off-flavours and undesirable appearance (Aly, 1996). In a survey of yeast species and populations in unripened dairy products including Mozzarella and Ricotta from southern Italy, yeasts were isolated with an incidence of 71% and 57% from cow and buffalo products, respectively (Minervini et al., 2001). Candida inconspicua and Candida famata were the predominant species and total yeast populations up to 105 cells/g were observed. Kluyveromyces lactis and Dekkera anomala were likely the predominant species in swelling samples of Sardinian Feta with counts of 106 cells/g for the latter (Fadda et al., 2001). Yeasts are common contaminants of fruits and represent a major problem in fruit processing industries due to their ability to grow at low pH and high sugar contents. They can be isolated from fresh and processed fruit such as ready-to-eat slices, fruit juices and soft drinks (Restuccia et al., 2006). Total yeast populations in a range of below ten to 105 cells/ml were determined in fruit juices re-diluted from concentrates with Saccharomyces cerevisiae, Candida stellata, and Zygosaccharomyces rouxii most frequently isolated (Deak and Beuchat, 1993). Fresh and processed vegetables are similarly prone to yeast spoilage. Ready-to-eat vegetable salads, such as coleslaws or potato salad, present high-risk examples of yeast spoilage in this group of food. Retail samples of these salads frequently have yeast populations of

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106 cells/g or higher and can show evidence of spoilage through development of off-flavours, gassiness, and surface colonies of yeasts. Mayonnaise that is used as a base for many of these products lowers the pH and makes them a selective environment for growth of yeasts (Smittle, 1977; Fleet, 1992). Retail samples of processed delicatessen-type meats are frequently contaminated with significant populations of 105–107 cells/g that is in contrast to fresh meat, where yeast spoilage is insignificant when compared with bacteria (Fleet, 1992). Recent studies have shown that these high numbers might be related to products that were produced under insufficient hygienic standards or were recontaminated (Nielsen et al., 2008). 2.2.2  Moulds in food spoilage Moulds are widely distributed. They are natural inhabitants in soil and contaminants in air and water and are responsible for many cases of food spoilage (De Ruiter et al., 1993). They can grow on all kinds of food such as cereals, fruits, vegetables, nuts, fats, meat, and products thereof (Filtenborg et al., 1996). Contamination is ubiquitous, i.e. in the field before harvesting, during harvesting, or during storage and processing (Filtenborg et al., 1996; Kabak et al., 2006). Mould growth on foodstuffs is determined mainly by pH, temperature, oxygen, water activity (aw), and other microorganisms. They are able to grow at low pH and tolerate low aw, where they have less prokaryotic competitors. Mould growth may result in several kinds of food-spoilage, e.g. off-flavours, toxin production, discoloration, rotting, and formation of pathogenic or allergenic extracellular compounds. Off-flavours and complete disintegration of the food structure are mostly due to fungal enzymes such as lipases, proteases, and carbohydrases that are active in or on the food even after removal or destruction of the mycelium (Filtenborg et al., 1996). Fungal metabolites are produced during primary and secondary metabolism from a wide variety of substrates, e.g. acetate, amino acids, fatty acids, and keto fatty acids and enable moulds to colonize a wide range of ecosystems (Schnürer et al., 1999). Many moulds produce secondary metabolites of pharmaceutical importance, e.g. antibiotics such as penicillin and cyclosporine. This may also give them a competitive advantage over prokaryotic bacteria in normally unfavourable environments, e.g. at neutral pH and high aw (Frisvad et al., 2007a). Moulds are also responsible for the formation of undesired mycotoxins that are small (molecular weight of ~700 DA) secondary metabolites produced by several fungi belonging mainly to the genera Aspergillus, Penicillium, Fusarium, and Alternaria (Kabak et al., 2006; Turner et al., 2009). Some mycotoxins appear to be produced in response to environmental changes, usually due to the onset of stress conditions (Magan and Aldred, 2007). The most significant mycotoxins, from both public health and agronomic perspectives, include aflatoxins, trichothesenes, fumonisins, ochratoxin A, patulin, tremogenic toxins, and ergot alkolids. The Food and Agriculture Organization (FAO) estimated that as much as 25% of the world’s agricultural commodities are contaminated with mycotoxins leading to significant economic losses (Kabak et al., 2006). In the European Union

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(EU), a significant amount (20%) of mycotoxin-contaminated crops was detected (Logrieco and Visconti, 2004). Mycotoxins occur primarily in the mycelium of filamentous fungi but can also be found in the spores of these organisms. They are either ingested, absorbed through the skin, or inhaled and rapidly induce a toxic response, termed mycotoxicosis, that can lead to teratogenic, carcinogenic, oestrogenic, neurotoxic, and immunosuppressive effects (Kabak et al., 2006). Some mycotoxins even act synergistically or additively (Frisvad et al., 2007b). Most mycotoxins are heat stable in the range of food processing temperatures (80–121 °C), denaturing at 237–306 °C (aflatoxins; Rustom, 1997), 169 °C (ochratoxin A; Kabak et al., 2006), or 111 °C (patulin; Trucksess and Tang, 1999). However, baking, frying, roasting, microwave heating, and extrusion reduce mycotoxin levels in food, despite their relatively high heat stability. The amount of reduction is however highly dependent on cooking conditions, i.e. temperature, time, water, and pH, as well as the type of mycotoxin and its concentration in the food matrix. Further detailed toxicity studies of putative mycotoxin degradation by-products would be required prior to evaluating an overall reduction of toxicity (Kabak et al., 2006). Fungal volatiles can affect the quality of food and beverages even when present in small amounts (Filtenborg et al., 1996). Typical key volatiles were determined with 1-octen-3-ol as the predominant compound accompanied by 3-octanone, 3-octanal, 3-methyl-butanol, 1-octene, and limonene (Jelen and Grabarkiewicz-Szczesna, 2005). 2-Methyl-propanol, 3-methylfurane, ethyl acetate, 2-methyl-isoborneol, and geosmin (the latter two leading to a strong malodour) are further typical fungal volatiles indicating fungal food and feed spoilage (Schnürer et al., 1999). Production of volatiles and patterns thereof have been studied extensively for determining the presence of mycotoxigenic moulds in food raw materials and commercial sensory arrays, known as ‘electronic noses’, have been successfully examined even in the discrimination between mycotoxigenic and non-mycotoxigenic strains (Sahgal et al., 2007). Fungal spores bear a remarkable risk of rapid environmental contamination and it is believed that inhalation of fungal spores damages the human respiratory system (Schnürer et al., 1999). Alternaria and Cladiosporum, as well as typical food-borne fungi such as Aspergillus and Penicillium and their spores have been recognized as significant airborne allergens and were associated with respiratory allergic symptoms and allergen sensitization (Fischer and Dott, 2003).

2.3  Traditional control of spoilage fungi in food 2.3.1  Weak acid preservatives Traditional food preservation is achieved by either physical or chemical treatments. The most common chemical food preservatives are weak organic acids, such as acetic, lactic, benzoic, and sorbic acids inhibiting bacterial and fungal growth (Brul and Coote, 1999). These can be produced either by chemical syntheses or by biological fermentation. Propionic acid is the preservative of choice to prevent

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mould spoilage in bakery products (Suhr and Nielsen, 2004). Minimal inhibitory concentrations (MIC) of propionic acid were determined from eight to over 500 mM (0.6 to over 37.0 g/l) varying pH from 3.0–7.0 for strains of Aspergillus fumigatus, Penicillium roqueforti, Penicillium commune, Aspergillus nidulans, and Fusarium sporotrichioides (Lind et al., 2005) and in a similar range of 10–500 mM (0.7–37.0 g/l) varying pH from 4.0–6.0 for strains of Candida parapsilosis, Candida pulcherrima, and Rhodotorula mucilaginosa (Miescher Schwenninger et al., 2008). Sorbic acid is more effective than propionic acid in inhibiting fungi and used in a broad variety of food products whereas benzoic acid is used in many types of acidic foods and often applied in combination with sorbic acid for confectionery and other types of products (Sofos, 2000; Suhr and Nielsen, 2004). Propionic (E280) and sorbic acid (E200) may be added to bakery wares in concentrations up to 3000 and 2000 mg/kg, respectively, and benzoic acid (E210) up to 1500 mg/kg is permissible according to Directive No. 95/2/EC (European Union, 1995). Weak acid preservatives have optimal inhibitory activity at low pH where they are favourably in their uncharged and undissociated state enabling free diffusion across the plasma membrane into the cell (Brul and Coote, 1999). The effectiveness of the preservative is dependent on its pKa value and the pH of the food. The pKa values of propionic, sorbic, and acetic acids are 4.85, 4.76, and 4.18, respectively, and their maximum pH for activity around 6.0–6.5 for sorbate, 5.0–5.5 for propionate, and 4.0–4.5 for benzoate (Suhr and Nielsen, 2004). Use of weak acid preservatives is usually applied in the form of a salt of the acid that is more soluble in aqueous solutions. In a study of inhibitory effects of weak acid preservatives on growth of bakery product spoilage fungi, propionate applied at legal concentrations of 0.3% (w/v) totally inhibited fungal growth for a two-week period, with the exception of Penicillium roqueforti, Penicillium commune, and Eurotium rubrum (Suhr and Nielsen, 2004). The main spoiler of rye bread, Penicillium roqueforti, was even stimulated by propionate and stimulation significantly enhanced at high water activity levels (aw 0.97). Sorbate and benzoate were more effective than propionate in the same study (Suhr and Nielsen, 2004). Potassium sorbate was the most effective preservative to prevent mould spoilage of intermediate moisture bakery products (aw ranging from 0.80–0.90) of relative low pH (4.5–5.5) when applied at 0.3% and calcium propionate and sodium benzoate were effective only at low aw when applied at 0.3% (Guynot et al., 2005). Minimal inhibition of fungal species such as Aspergillus flavus, Eurotium repens, Endomyces fibuliger, Penicillium corylophilum, and Monilia sitophila by calcium propionate was observed when tested with 3 g/l in a disc assay whereas sodium benzoate inhibition was slightly higher at this concentration (Lavermicocca et al., 2000). 2.3.2  Natamycin – an antifungal antibiotic Natamycin (E235) is an antibiotic widely used as food preservative to prevent mould, mainly on cheese (Jay, 1995). It was approved in 1976 by the Joint Food

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and Agricultural Organization/World Health Organization Expert Committee on Food Additives (JECFA) and permitted for use in surface treatment of hard, semihard, and semi-soft cheese as well as dried, cured sausages in concentrations up to 1 mg/dm2 surface but should not be present at a depth of 5 mm (European Union, 1995). It is effective at lower concentrations than most common antifungal agents. Natamycin, also known as pimaricin, is a polyene antibiotic with the empirical formula C33H47NO 13 and a molecular weight of 665.75 that is produced by certain strains of Streptomyces spp. It is light-sensitive but otherwise stable in the dry state (Jay, 1995). It is insoluble in water and most organic solvents and not absorbed from the gastrointestinal tract (Vanden Bossche et al., 2003). Most moulds are inhibited at natamycin concentrations from 0.5–6 mg/l, but some require 10–25 mg/l whereas most yeasts are inhibited at concentrations from 1–5 mg/l (Davidson and Doan, 1993). The development and evaluation of the antimicrobial efficiency of natamycin-incorporated films in the production process of Gorgonzola cheese revealed that films with two and four per cent natamycin yielded satisfactory results for fungus inhibition and the amount of natamycin released into the cheese were below permissible levels (De Oliveira et al., 2007). Almost complete growth inhibition was observed with 5–10 μg/l incubated at 15 °C and different water availabilities (aw of 0.98, 0.96, and 0.94) in an efficacy study of natamycin against strains of Aspergillus carbonarius (Medina et al., 2007). Complete inhibition of growth and ochratoxin A production over a range of environmental conditions was observed with 50–100 μg/l natamycin. 2.3.3  Development of resistance to antifungal preservatives Despite the considerable history of antimicrobials in the food industry, there is little data about the development of microbial resistance to these compounds. This may indicate that resistance development is not a major problem. Tolerance to antimicrobials may however be generated within microorganisms exposed to certain stresses (Davidson and Harrison, 2002). Adaption mechanisms of yeasts and moulds to weak acids include enzymatic degradation, H+-pumping P-type membrane ATPase activity, induction of the integral membrane protein Hsp30, and efflux systems removing accumulated anions from inside the cell (Brul and Coote, 1999). Yeasts grown in the presence of benzoic acid were observed to tolerate 40–100% higher benzoic acid concentrations than did those grown in the absence of weak-acid preservatives (Warth, 1988). The export of hydrogen ions arising from dissociation of benzoic acid continuously entering the cell required an additional energy source such as glucose. Yeast species tolerant to one preservative such as benzoic and sorbic acid were also observed to be tolerant to the other, but significant differences in the relative effectiveness were described (Warth, 1985). Natamycin was shown to kill yeasts by specifically binding to ergosterol without permeabilizing the plasma membrane. Resistance against natamycin and generally polyene antibiotics is still rare although antibiotic resistances have increased dramatically over the last 30 years (Te Welscher et al., 2007). No resistant fungi

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were observed in a study of cheese warehouses performed in the 1970s where natamycin was used for periods up to several years. It was even impossible to decrease sensitivity to natamycin under laboratory conditions in 26 strains isolated in cheese warehouses (De Boer and Stolk-Horsthuis, 1977).

2.4 Antifungal lactic and propionic acid bacteria (LAB and PAB) 2.4.1  Lactic acid bacteria and their long history in food fermentation Lactic acid bacteria (LAB) have a long documented history of use in food and the number and diversity of applications has increased considerably. LAB are Gram-positive, low-GC-content, catalase-negative bacteria found in nutrient-rich environments like milk, meat, decomposing plant material, and in the mammalian gastrointestinal tract (Teusink and Smid, 2006). They classically comprise a group of morphological different, non-motile, and non-spore-forming bacteria which produce lactic acid as one of their main fermentation products (Teuber, 1993). Mainly members of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Pediococcus are involved in food fermentation (Soomro et al., 2002). LAB glucose degradation is species-dependent, either homofermentatively via the fructose-bisphosphate-pathway to lactic acid or heterofermentatively via the pentosephosphate-pathway to lactic acid, acetic acid/ethanol, and CO 2 (Teuber, 1993). Their ability to degrade lactose is a typical characteristic of LAB that is enabled by a housekeeping β-galactosidase. LAB are commercially important microorganisms, used as starter cultures in various food-fermentation processes. Their traditional application in artisanal spontaneous food fermentation has been expanded over centuries to well-controlled defined starter cultures developed for distinct dairy, meat, and further complex food fermentations. Global production of cheese starter cultures, for example, already exceeds 1.5 × 106 tons per year (Teusink and Smid, 2006). Furthermore, LAB are used in many biotechnological processes and industrial applications for the production of various metabolites, including lactic acid as a substrate for the chemical and biological production of other organic compounds, e.g. propionic acid, acrylic acid, acetic acid, propylene glycol, ethanol, acetaldehyde, flavour compounds, (e.g. diacetyl, acetoin), acetaldehyde, acetic acid, exopolysaccharides (EPS), and B vitamins (Teusink and Smid, 2006). 2.4.2  Dairy propionic acid bacteria and typical applications Propionic acid bacteria (PAB) also have a long application history in Swiss type cheeses and vitamin B12 production. The genus Propionibacterium describes two principal groups of organisms distinguished on the basis of their habitat: the ‘dairy’ or ‘classical propionibacteria’ and the ‘cutaneous’ or ‘acnes’ group. The dairy propionibacteria are commercially important cultures and considered safe whereas the cutaneous species are pathogens (Meile et al., 2008). Both groups © Woodhead Publishing Limited, 2011

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are Gram-positive, high-GC-content, catalase-positive, non-spore-forming, non-motile, pleomorphic rod-shaped bacteria predominantly preferring microaerophilic to anaerobic growth conditions. Dairy PAB have been traditionally isolated from dairy products, especially Swiss-type cheeses and raw milk, but have also been found in other natural fermentation environments such as silage and fermenting olives, and also in soil (Cummins and Johnson, 1986). The starter cultures used in cheese manufacture are usually described as Propionibacterium freudenreichii with its subspecies freudenreichii and shermanii (Glatz, 1992). PAB degrade lactic acid to propionic acid, acetic acid, and CO 2. These endproducts are key-components of Swiss-type cheeses with propionic and acetic acids as flavour compounds and CO 2 responsible for the characteristic gas vacuoles or ‘holes’ of these cheeses. The central enzyme of propionic acid fermentation is methylmalonyl-coenzyme A that requires coenzyme B12 for its activity. PAB are therefore one of the most important groups of bacteria for biosynthesis of vitamin B12. All dairy PAB except P. freudenreichii subsp. freudenreichii can metabolise lactose but utilise lactate faster than sugars when lactate and carbohydrates are both available (Piveteau, 1999). 2.4.3 Potential of antifungal lactic and propionic acid bacteria in food applications: antifungal screenings of a broad range of biodiversities The discovery of high antifungal strains is often a very tedious process involving broad screening of often overwhelming numbers of isolates from different habitats and biodiversities. Although robot-based high-throughput screening clearly supersedes laborious manual screening, the latter still yields high potential strains through careful work. Detection is largely influenced by screening conditions and antimicrobial test sensitivity, i.e. a highly sensitive screening test may reveal many positive strains but may not directly discriminate potential strains for food applications. One must find the optimal compromise between sensitivity and the power of discrimination. Test optimization is thus a very important step prior to extensive screenings. Antifungal screening of lactic acid bacteria The study of antifungal activities is still a novel research field. Screening LAB for antifungal activity has however increased markedly over the last decade yielding various antagonistic strains. More than 1200 isolates of LAB collected from a variety of natural environments with the majority of plant material were screened in a dual-culture agar plate assay system against the mould Aspergillus fumigatus (Magnusson et al., 2003). Approximately ten per cent of the isolates showed antifungal activity, of which four per cent exhibited strong antifungal activity. A majority of 15 isolates out of 37 with strong or moderate activity were identified by 16S rDNA sequencing as Lactobacillus coryniformis, followed by Pediococcus pentosaceus (ten isolates), and Lactobacillus plantarum (six isolates). We selected 82 strains using an agar-spot assay inhibiting Candida spp., Zygosaccharomyces bailii, and Penicillium spp. in a similar screening of 1424 presumptive lactobacilli

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isolated from different food and feed samples, i.e. raw milk, cheese, yoghurt, olives, sourdough, as well as corn and grass silage (Miescher Schwenninger et al., 2005). Predominant species were within the Lactobacillus casei group with Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus using API 50 CHL. These were followed by Lactobacillus plantarum (14%). Strains SM20, SM29, and SM63 revealed particularly high antifungal activities and were finally identified as Lactobacillus paracasei subsp. paracasei using molecular typing such as 16S rDNA sequencing analyses, specific PCR, and RAPD genotyping. Antifungal activity of strains belonging to the Lactobacillus casei group (Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus) and Lactobacillus plantarum were likewise determined by Suzuki et al. (1991) using an agar plate assay and Penicillium spp., Aspergillus spp., and Fusarium spp. as indicator organisms. Lactobacillus paracasei ST68 was selected based on its inhibitory effect against Fusarium proliferatum. It was isolated in a dual overlay agar plate assay from 322 lactobacilli strains isolated from Edam cheese at different stages of the ripening process (Tuma et al., 2007). Lactobacillus paracasei subsp. tolerans L17 was selected from 116 lactic acid bacteria from four sourdough bread cultures due to its high activity against strains of Fusarium proliferatum and Fusarium graminearium observed in a dual agar plate assay (Hassan and Bullermann, 2008). A predominance of an antifungal Lactobacillus plantarum within 359 lactic acid bacteria isolated from fresh vegetables was determined in an agar plate assay (Sathe et al., 2007) and similar, antifungal strains of Lactobacillus plantarum and Lactobacillus pentosus strains were observed in a set of 65 strains of lactobacilli isolated from salami using an agar overlay assay with Penicillium and Aspergillus as indicator organisms (Coloretti et al., 2007). Antifungal activities were observed in the presence of live or even growing cells in agar cultures in all the preceding assays as opposed to Gourama (1997) who described a broad screening for antifungal and antimycotoxigenic activities in cell-free supernatants of liquid cultures. They observed four isolates that excreted antifungal compounds active against four Penicillium species out of 420 LAB isolated from dairy products, vegetables, and fruits. The antimould activity of 232 sourdough Lactobacillus strains was similarly determined in cell-free culture supernatants by a well-diffusion agar plate assay revealing 46 mainly obligate fermentative strains with inhibitory activity against bread spoilage related fungi (Corsetti et al., 1998). A single strain, Lactobacillus sanfrancisco CB1, had the broadest spectrum and inhibited moulds belonging to Fusarium, Penicillium, Aspergillus, and Monilia. Inhibition of Aspergillus, Fusarium, and Penicillium was likewise evaluated testing cell-free supernatants of 95 strains in a microtiter plate assay revealing four strains that displayed antifungal activity, i.e. Lactobacillus plantarum CRL778, Lactobacillus reuteri CRL1000, and Lactobacillus brevis CRL772 and CRL796 (De Muynck et al., 2004; Gerez et al., 2009). A pH-dependent antifungal activity was found in culture supernatants of Lactobacillus acidophilus LMG9433, Lactobacillus amylovorus DSM20532, Lactobacillus brevis LMG6906, and Lactobacillus coryniformis LMG9196 selected from 17

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lactic acid bacterial strains as well as of three commercial probiotic cultures (De Muynck et al., 2004). Antifungal screening of propionic acid bacteria The study of antifungal activities in the genus Propionibacterium just started recently and a limited number of published data are available to date. To the best of our knowledge, one broad screening protocol of dairy propionibacteria is still the only study in this regard (Miescher Schwenninger and Meile, 2004). 197 presumptive propionibacteria from cheese, raw milk, maize- and grass-silage were isolated and a total of 13 isolates were selected showing high inhibitory activities against a set of yoghurt-spoilage yeasts and moulds in an agar spot assay. A predominance of Propionibacterium jensenii (nine isolates) was identified using 16S rDNA sequencing followed by Propionibacterium thoenii (two isolates), and Propionibacterium acidipropionici (two isolates). The antifungal activity of five type strains of dairy propionibacteria, i.e. Propionibacterium acidipropionici, Propionibacterium jensenii, Propionibacterium thoenii, and Propionibacterium freudenreichii subsp. freudenreichii and shermanii were evaluated in a dual agar culture assay against eight food- and feedborne moulds and yeasts (Lind et al., 2005). Propionibacterium thoenii was the most potent antifungal inhibitor. In contrast to the preceding studies in which antifungal effects of propionibacteria were detected in agar cultures, Gwiazdowska et al. (2008) observed antifungal activity of extracellular metabolites in liquid cultures of two strains of Propionibacterium freudenreichii. The strongest effects were found in propionibacteria cultures containing viable cells and not in cell-free culture supernatants.

2.5 Efficiency of antifungal LAB and PAB in food challenge tests: a first step from in vitro towards in vivo 2.5.1 Single cultures of lactobacilli and propionibacteria in challenge studies Antifungal bacteria and their antagonistic metabolites are preferably applied in the form of protective cultures. Challenge tests studying the inhibitory capacity of antifungal strains in food models or food products against a defined and artificial contamination are needed to close the gap between in vitro tests performed under controlled laboratory conditions and in vivo studies. Antimicrobial cultures also behave differently in agar plate assays under optimal culturing conditions than in food or food models where metabolic activity may be suppressed due to a suboptimal matrix or the original microflora present. Several challenge tests with antagonistic cultures in bread were carried out targeting suppression of fungal spoilage in bakery products. A seven-day delay of artificial fungal growth in wheat bread started with Saccharomyces cerevisiae and the antifungal strain Lactobacillus plantarum 21B was observed by Lavermicocca et al. (2000). The addition of antifungal Lactobacillus plantarum strains FST1.7

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and FST1.9 to sourdough resulted in wheat bread with delayed growth of Fusarium culmorum and Fusarium graminearum (Ryan et al., 2008). Chemically acidified bread containing a mixture of lactic and acetic acids (4:1, v/v) had an inhibitory effect against Fusarium culmorum comparable to that of strain FST1.7 until day six but the rate of mould growth markedly increased from day seven compared to control breads. Ryan et al. (2008) even observed strong synergistic effects when calcium propionate (0.3%) and sourdoughs fermented with antifungal Lactobacillus plantarum strains FST1.7 or FST1.9 were combined into wheat bread formulations and growth of Penicillium roqueforti was inhibited. Reducing the level of calcium propionate to 0.1% in combination with antifungal sourdoughs still ensured an acceptable shelf life of the bread. Strain FST1.7 was also successfully evaluated to produce gluten-free bread with increased quality and shelf life (Moore et al., 2008). The inclusion of four antifungal strains of Lactobacillus plantarum (1), Lactobacillus reuteri (1), and Lactobacillus brevis (2) in a mixed starter culture combined with Saccharomyces cerevisiae allowed a reduction in the concentration of calcium propionate by 50% while still attaining a maximum shelf life of eight days that was similar to that of traditional wheat bread containing 0.4% calcium propionate without antifungal LAB (Gerez et al., 2009). Sathe et al. (2007) showed a significant delay of spoilage fungi after eight-day storage of cucumber inoculated with Lactobacillus plantarum CUK501. Antifungal effects of LAB were also observed in malting and brewing applications. Laitilia et al. (2002) investigated the antifungal effect of Lactobacillus plantarum E76 during laboratory-scale malting of naturally contaminated barley. The addition of strain E76 in the early stage of malting reduced natural Fusarium contamination of barley by over 20%, corresponding to 5–17% in the final malts. Large variations observed were obviously due to the differences in the composition of Fusarium flora and the contamination level of barley. Pilot scale malting experiments confirmed strain E76 in combination with a Pediococcus pentosaceus as potential starter culture for malting in order to balance the microbial community and to enhance malt processability (Laitila et al., 2006). Reviews by Lowe and Arendt (2004) and Rouse and van Sinderen (2008) provide detailed information on the application and antifungal effects of LAB in malting and brewing. Antifungal cultures were not only proposed for preventing fungal spoilage and mycotoxin formation in food but also in animal feed. Ström et al. (2002) isolated high antifungal Lactobacillus plantarum MiLAB 393 from a silo without chemical or biological additives revealing total growth inhibition of Fusarium sporotrichioides, Aspergillus fumigatus, and Kluyveromyces marxianus. A strain of Lactobacillus plantarum as well as Lactobacillus acidophilus ATCC20552 were likewise proposed for biocontrol of grain storage due to their inhibitor effects against Aspergillus spp. (Elsanhoty, 2008). Apart from the application of antifungal protective cultures, metabolites produced by Propionibacterium thoenii P-127 were successfully tested in challenge tests with Domiati cheese, a white Egyptian cheese, against yeasts and moulds (Tawfik et al., 2004). The addition of 1.5% (w/v) pasteurized and lyophilized P-127 culture to the cheese milk obviously prolonged shelf life of this

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soft cheese in absence of viable antifungal cells. Whey permeate supplemented with casein hydrolysate yielded maximum production of antifungal compounds. 2.5.2  Co-cultures of lactobacilli and propionibacteria in challenge studies LAB and PAB have close and even synergistic metabolic pathways and are often isolated from the same environments, such as raw milk, dairy products, or silage (Cummins and Johnson, 1992; Teuber, 1993). LAB classically produce lactic acid from lactose that is then degraded to propionic and acetic acids by PAB (Fig. 2.1). Additional metabolites of LAB and PAB might support synergistic growth effects of these two genera. Baer (1995) described such an example of PAB-supported growth in Swiss-type cheeses. This effect was due to protease activity of LAB and production of free amino acids. Stimulating interactions of PAB and LAB were further exploited in a combination of Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii subsp. shermanii JS that exhibited a stronger antimicrobial effect than either culture alone (Suomalainen and Mäyrä-Mäkinen, 1999). A concentration of 2–4 × 107 cfu/g of both strains was sufficient to inhibit Rhodotorula rubra RHO in quark and yoghurt and cell numbers were held at a constant level of ca. 102 cfu/g during five-week storage at 6 °C. Yeasts levels increased to almost 107 cfu/g in a control sample without protective culture. The strains of the protective culture did not grow but had a minimal metabolism evidenced by a slight increase in propionic acid, acetic acid, and diacetyl at concentrations which did not explain antifungal activity. Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii JS also improved the shelf life and sensorial properties of wheat bread by total inhibition of ropy Bacillus spp. (Suomalainen and Mäyrä-Mäkinen, 1999). Sourdough with initial levels of

Fig. 2.1  Complementary metabolic pathways of LAB (production of lactic acid from lactose) and PAB (production of propionic and acetic acids from lactic acid). All PAB, except P. freudenreichii subsp. freudenreichii, can metabolise lactose but use preferentially lactic acid when both substrates are present.

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1 × 108 and 3 × 108 cells/g, respectively, was added to the wheat bread formulation after a fermentation time of ten hours. This anti-Bacillus effect was partially explained by lower pH and higher amounts of lactic acid in test bread compared to control bread. We likewise observed increased antifungal activity of Lactobacillus paracasei strains SM20, SM29, and SM63 when combined with Propionibacterium jensenii SM11 in a model assay in agar plates as illustrated in Fig. 2.2 (Miescher

Fig. 2.2  Growth behaviour of indicator yeasts on yeast mould agar (YM) plates with and without embedded protective culture, during storage of 21 days at 6 °C. (a) Plate with protective culture (Propionibacterium jensenii SM11 [3.8 × 108 cfu/ml agar] and Lactobacillus paracasei subsp. paracasei SM20 [1.1 × 108 cfu/ml agar]). (b) Control plate without protective culture. Yeasts (spot inoculated, ranging from 104 cells/spot in column a to 100 cells/spot in column e): 1, Candida pulcherrima 1–50/13; 2, Candida reukaufii 4–73/4; 3, Candida sp. 1–50/15; 4, Sporobolomyces salmonicolor 2–46/2 (Image: Swiss National Science Foundation SNSF, Berne, Switzerland).

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Schwenninger and Meile, 2004). These antagonistic effects were confirmed in yoghurt fermentations with a commercial yoghurt starter culture that were challenged with a mixture of Candia pulcherrima, Candida magnoliae, Candida parapsilosis, and Zygosaccharomyces bailii (Fig. 2.3). We observed no increase of viable yeasts in samples containing a minimal concentration of 1.7 × 108 cfu/ml of Lactobacillus paracasei SM20 and 5.5 × 107 cfu/ml of Propionibacterium jensenii SM11 over four-week storage at 6 °C whereas yeasts counts increased from initially 102 cfu/ml to 107 cfu/ml in control samples lacking a protective culture (Miescher Schwenninger and Meile, 2004). The protective strains did not grow throughout storage, but an increased acetic acid concentration up to non-inhibiting concentrations of 0.061–0.069% in samples with additional Propionibacterium–Lactobacillus culture in contrast to 0.004% in the control batches, suggested a minimal in situ metabolism. Figure 2.4 shows a scanning electron micrograph of a culture cocktail of Propionibacterium jensenii SM11 and Lactobacillus paracasei subsp. paracasei SM20 that was prepared for use in yoghurt challenge trials. Similar total inhibition of yeasts by Lactobacillus paracasei SM20, SM29, and SM63, each in combination with Propionibacterium jensenii SM11 during three-week storage at 6 °C, was determined on cheese surface models when applied at a minimal concentration of 1.0 × 106 cfu/g surface of PAB and 3.0 × 106 cfu/g surface of LAB. Protective cultures SM20/SM11, SM29/SM11, and SM63/SM11 were also successful in challenge tests in Mozzarella brine and ready-to-eat salad for suppression of fungi and Gram-negative bacteria suggesting a broad application potential (unpublished data). With respect to future food applications, novel strains should preferably exhibit supplementary characteristics

Fig. 2.3  Yoghurt with untreated berries produced with a classical yoghurt starter culture (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) without (a) and with (b) additional protective culture Lactobacillus paracasei subsp. paracasei SM20 (1 × 108 cfu/g) and Propionibacterium jensenii SM11 (5 × 107 cfu/g) after four-weeks storage at 6 °C (Image: Alexander Sauer for ETHGlobe, Corporate Communications, ETH Zurich, Zurich, Switzerland).

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Fig. 2.4  Scanning electron micrograph of a protective co-culture of (A) rod-shaped Lactobacillus paracasei subsp. paracasei SM20 and (B) pleomorphic rod-shaped Propionibacterium jensenii SM11.

in addition to antifungal properties, e.g. texture-enhancing properties in dairy products or retrogradation-delaying (anti-staling) properties in bakery products. A slimy growth suggested exopolysaccharide (EPS) production by protective cultures composed of Propionibacterium jensenii SM11 and Lactobacillus paracasei strains SM20, SM29, or SM63 that increased the viscosity of yoghurt samples produced with these antifungal strains and thus led to an improvement of texture (Miescher Schwenninger and Meile, 2004).

2.6 Antifungal metabolites and further inhibitory mechanisms 2.6.1  Purification and identification of antifungal metabolites Contrary to the antibacterial bacteriocins that classically act as single substances, antifungal inhibitory mechanisms are assumed to be related mainly to a complex pool of mostly low-molecular-mass compounds with putative synergistic effects (Miescher Schwenninger et al., 2008). These novel low-molecular-mass

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compounds are generally produced at very low levels by antifungal cultures, in contrast to the high MIC values determined for pure compounds in antagonistic tests. The precise mechanism of antifungal activity is very complex and its elucidation difficult. Several methods were developed to purify and identify antifungal metabolites but, as of the date of this publication, the complete secret of antifungal activity has not been solved for any strain or strain combination. A prerequisite for the study of antifungal metabolites is to determine their activity in liquids, e.g. cell-free culture supernatant that will facilitate their further characterization, purification, and identification. Antifungal compounds that were identified from LAB and PAB are summarized in Table 2.1, while their molecular structures are depicted in Figure 2.5. Low-molecular-mass antifungal compounds A mixture of low-molecular-mass compounds including acetic, caproic, formic, propionic, butyric, and valeric acids was identified in cell-free supernatants of antifungal sourdough strain Lactobacillus sanfrancisco CB1 with gas chromatography-mass spectrometry (GC-MS) (Corsetti et al., 1998). The compounds appeared to act synergistically with caproic acid playing a key role. Strain CB1 produced 13.75 mM (0.8 g/l) acetic acid, 0.88 mM (0.1 g/l) caproic acid, 1.43 mM (65.8 mg/l) formic acid, 0.14 mM (10.4 mg/l) propionic acid, and about 0.10 mM (8.8 mg/l) butyric acid and 0.10 mM (10.2 mg/l) valeric acid after 48 hours of growth in wheat flour hydrolysate (WHF) broth. Acetic acid was responsible for about one-half of the inhibitory activity of mixtures with pure compounds. Antifungal activity decreased markedly to about one-third when caproic acid was excluded. Niku-Paavola et al. (1999) observed 37% growth inhibition of Fusarium avenaceum by a Lactobacillus plantarum strain. The low molecular mass fraction collected after gel chromatography of cell-free supernatant revealed only 27% inhibition. Characteristic compounds from this fraction were identified by GC-MS and included benzoic acid, methylhydantoin, mevalonolactone, and cyclo(Gly-L-Leu). Pure compounds in concentrations of 10 ppm (10 mg/l) inhibited growth of test organisms by 10–15% increasing to 20% when applied in mixtures. Ten-fold concentrated cell-free supernatant of the sourdough strain Lactobacillus plantarum 21B grown in wheat flour hydrolysate exhibited fungicidal activity towards strains of Eurotium spp., Penicillium spp., Endomyces fibuliger, Aspergillus spp., Monilia sitophila, and Fusarium graminearum (Lavermicocca et al., 2000). Extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and GC-MS identified 3-phenyllactic and 4-hydroxyphenyllactic acids in active culture filtrates of Lacobacillus plantarum 21B. These acids are involved in phenylalanine metabolism and known antimicrobial compounds of LAB (Sato et al., 1986). 3-Phenyllactic acid has been recognized as the major component of antifungal activity in strain 21B (Lavermicocca et al., 2000) and has been shown to inhibit fungal test organisms at high concentration of about 50 g/l. Detailed microdilution tests with 23 fungal strains belonging to 14 species of bread and cereal spoilage Aspergillus, Penicillium, and Fusarium showed that less than

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Fig. 2.5  Chemical structures of antifungal compounds (synonyms of the original publications are listed; additional names corresponding to IUPAC (International Union of Pure and Applied Chemistry, USA) are included in square brackets in case of differentiations). (a), Benzoic acid, molecular weight (MW): 122.12; (b), mevalonolactone[4-hydroxy-4methyloxan-2-one], MW: 130.14; (c), cyclo(Gly-L-Leu) [3-(2-methylpropyl)piperazine2,5-dione], MW: 170.21; (d), methylhydantoin [1-methylimidazolidine-2,4-dione], MW: 114.10; (e), 3-phenyllactic acid [2-hydroxy-3-phenylpropanoic acid], MW: 166.17; (f), 4-hydroxyphenyllactic acid [2-hydroxy-3-(4-hydroxyphenyl)propanoic acid], MW: 182.17; (g), 2-pyrrolidone-5-carboxylic acid [(2S)-5-oxopyrrolidine-2-carboxylic acid], MW: 129.11; (h), cyclo(L-Phe-L-Pro) [(3R,8aS)-3-benzyl-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a] pyrazine-1,4-dione], MW: 244.29; (i), cyclo(L-Phe-trans-4-OH-L-Pro) [no IUPAC name­ © Woodhead Publishing Limited, 2011

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available], MW: 260.29; (j), (R)-3-hydroxydecanoic acid, MW: 188.26; (k), (R)-3hydroxydodecanoic acid, MW: 216.32; (l), (R)-3-hydroxytetradecanoic acid, MW: 244.37; (m), 3-hydroxy-5-cis-dodecenoic acid [no IUPAC name available], MW: 214.30; (n), propionic acid [propanoic acid], MW: 74.08; (o), acetic acid, MW: 60.05; (p), lactic acid [2-hydroxypropanoic acid], MW: 90.08; (q), succinic acid [butanedioic acid], MW: 118.09; (r), caproic acid [hexanoic acid], MW: 116.16; (s), butyric acid [butanoic acid], MW: 88.11; (t), valeric acid [pentanoic acid], MW: 102.13; and (u), formic acid, MW: 46.03.

7.5 g/l of 3-phenyllactic acid was required to obtain 90% (MIC 90) growth inhibition for all strains (Lavermicocca et al., 2003). As with other weak acid preservatives, e.g. propionic, benzoic, and sorbic acids, antifungal activity of 3-phenyllactic acid is pH dependent and due to its rather low pKa (3.46) activity increased at lower pH. Addition of lactic acid (15.8 g/l) increased 3-phenyllactic acid inhibitory activity about 30% (Lavermicocca et al., 2003). The active compounds identified from salami originating strain Lactobacillus plantarum VLT01 were likewise 3-phenyllactic (46.6 mg/l) and 4-hydroxyphenyllactic acids (67.6 mg/l) (Coloretti et al., 2007). Production of 3-phenyllactic acid and 4-hydroxyphenyllactic acid was also determined for 29 LAB belonging to 12 species widely used in the

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Aspergillus fumigatus J9 Kluyveromyces marxianus J137

L. plantarum MiLAB393 Fusarium sprotrichioides J304

Eurotium repens IBT18000 Endomyces fibuliger IDM3812 Penicillium corylophilum IBT18687 Monilia sitophila IDM/ FS5

L. plantarum 21B

3-Phenyllactic acid Cyclo(L-Phe-L-Pro) Cyclo(L-Phe-trans-4OH-L-Pro)

3-Phenyllactic acid 4-Hydroxyphenyllactic acid

Fusarium avenacum VTT Benzoic acid D-80147 Methylhydantoin Mevalonolactone Cyclo(Gly-L-Leu)

Acetic acid Caproic acid Formic acid Propionic acid Butyric acid Valeric acid

Antifungal compound(s)

L. plantarum

Low molecular mass compounds L. sanfrancisco CB1 Fusarium graminearum 623

Antifungal culture

n.d. n.d. n.d.

Ström et al., 2002; Broberg et al., 2007

7.5 g/lc n.d.

7.5 g/l 20 g/l n.d.

Lavermicocca et al., 2003

10 ppmb 10 ppmb 10 ppmb

n.d. n.d. n.d. n.d. n.d.

Niku-Paavola et al., 1999

Corsetti et al., 1998

Reference

10 ppmb

8.33 mM (0.5 g/l) 4.30 mM (0.5 g/l) 19.50 mM (0.9 g/l) 8.10 mM (0.6 g/l) 9.08 mM (0.8 g/l) 7.83 mM (0.8 g/l)

MIC a

n.d.

13.75 mM (825.7 mg/l) 0.88 mM (102.2 mg/l) 1.43 mM (65.8 mg/l) 0.14 mM (10.4 mg/ml) 0.10 mM (8.8 mg/ml) 0.10 mM (10.2 mg/ml)

Production level

Table 2.1  Antifungal Lactobacillus spp. (L.) and Propionibacterium spp. (P.) and their inhibitory spectrum, antifungal compounds and MIC (minimal inhibitory concentrations)

46

Candida pulcherrima 1-50/13

P. jensenii SM11

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L. sp. B3

Penicillium spp.

Proteinaceous compounds L. pentosus TV35b Candida albicans

P. spp. type strainse Bacteriocin-like peptide (pentocin TV35b) Possibly proteinaceoush

3-Phenyllactic acid

n.d.

n.d.

1.0–15.1 mg/l

168 mM (10.1 g/l)e

362 mM (26.8 g/l)e

0.2 mM (36.4 mg/l)e 29 mM (3.4 g/l)e

4-Hydroxyphenyllactic acid Succinic acid

n.d.

n.d.

n.d.

10–200 mM (0.7–14.8 g/l)f 50–500 mM (3.0–30.0 g/l)f

n.d.e

Acetic acid 1 mM (166.2 mg/l)e

n.d.e

Lactic acid

3-Phenyllactic acid

> 500 mM (> 64.5 g/l)f > 500 mM (> 45.0 g/l)f 50–500 mM (3.0–30.0 g/l)f 50–500 mM (8.3–83.1 g/l)f n.d. 200–>500 mM (23.6–>59.1 g/l)f

7 mM (903.8 mg/l)e

>100 mg/l

0.2 mg/l

2-Pyrrolidone5-carboxylic acid

25 mg/l

0.5 mg/l

n.d.

100 mg/l n.d.

1.6 mg/l 1.0 mg/l

n.d.

2-Pyrrolidone5-carboxylic acid

3-(R)-Hydroxydecanoic acid 3-Hydroxy-5-cis-dodecenoic acid 3-(R)-Hydroxydodecanoic acid 3-(R)-Hydroxytetradecanoic acid

Rhodotorula mucilaginosa Propionic acid FSQE63 Acetic acid

(Enterobacter cloaceae Pseudomonas fluorescens)d

L. rhamnosus LC705

L. paracasei subsp. paracasei SM20

Aspergillus fumigatus J9

L. plantarum MiLAB14

(Continued )

Gourama, 1997

Okkers et al., 1999

Lind et al., 2007

Miescher Schwenninger et al., 2008

Miescher Schwenninger et al., 2008

Yang et al., 1997

Sjögren et al., 2003

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Peptidic compoundsh,i 3-Phenyllactic acid 4-Hydroxyphenyllactic acid n.d. 46.6 mg/l 67.6 mg/l

n.d.

Production level

n.d. n.d. n.d.

n.d.

MIC a

Coloretti et al., 2007

Magnusson and Schnürer, 2001

Reference

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b 10–15%

inhibitory concentration. inhibition by separate compounds applied at 10 ppm (10 mg/l) and maximally 20% in combinations. c MIC corresonding to 90% growth inhibition. 90 d Antibacterial activity as main activity determined. e Produced with immobilized cells in a co-culture of L. paracasei subsp. paracasei SM20 with P. jensenii SM11. f MIC dependent on pH (pH 4.0, 5.0, and 6.0) and yeast. g P. jensenii DSMZ20535, P. thoenii DSMZ20276, P. acidipropionici DSMZ4900, P. freudenreichii subsp. freudenreichii DSMZ20271, P. freudenreichii subsp. shermanii DSMZ4902. h Sensitive to proteolytic enzymes (trypsin and pepsin). i Determined after autolysis of 30-day-old cultures. n.d.: not determined.

a Minimal

Aspergillus spp. Penicillium spp. Geotrichum candidum Moniliella spp. Mucor racemosus Wallemia sebi Eurotium herbariorum

3-kDa compound Aspergillus fumigatus J9 Aspergillus nidulans J10 Penicillium commune J238 Mucor hiemalis J42 Talaromyces flavus J37 Fusarium poae J24 Fusarium graminearum J114 Fusarium culmuorum J300 Fusarium sporotrichoides J319

L. coryniformis subsp. coryniformis Si3

L. plantarum VLT01

Inhibitory spectrum

Antifungal culture

Antifungal compound(s)

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production of fermented foods (Valerio et al., 2004). According to analysis of variance, strains were divided into three groups comprising 15 strains that produced both metabolites (0.16–0.46 mM corresponding to 26.6–76.4 mg/l 3-phenyllactic acid and 0.07–0.29 mM corresponding to 12.8–52.8 mg/l 4-hydroxyphenyllactic acid), five strains accumulating only 3-phenyllactic acid (0.17–0.57 mM corresponding to 28.3–94.7 mg/l), and nine non-producer strains (≤0.10 mM corresponding to ≤16.6 mg/l 3-phenyllactic acid and ≤0.02 mM corresponding to ≤3.6 mg/l 4-hydroxyphenyllactic acid). 3-Phenyllactic acid production was increased in Lactobacillus plantarum ITM21B (identical to Lactobacillus plantarum 21B) by increasing the concentration of phenylalanine in culture and using low amounts of tyrosine. A direct correlation between phenylalanine and 3-phenyllactic acid, tyrosine and 4-hydroxyphenyllactic acid was suggested for Lactobacillus plantarum ITM21B, as it was described in the conversion of amino acids to cheese flavor compounds by Lactococcus lactis subsp. cremoris (Yvon et al., 1998). Ström et al. (2002) identified 3-phenyllactic acid as the key antifungal compound of Lactobacillus plantarum MiLAB 393 isolated from grass silage. 3-Phenyllactic acid was also found in grass silage inoculated with strain MiLAB 393 (Broberg et al., 2007). Fractionation of cell-free supernatant of Lactobacillus plantarum MiLAB 393 on a C18 column followed by further separation on a preparative high-performance liquid chromatography (HPLC) C18 and a porous graphitic carbon column, as well as structure determination by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS), and gas chromatography (GC) revealed the presence of antifungal cyclo(L-Phe-L-Pro) and cyclo(L-Phetrans-4-OH-L-Pro) in addition to 3-phenyllactic acid. Minimal inhibitory concentrations (MIC) against Aspergillus fumigatus and Penicillium roqueforti were 20 g/l and 7.5 g/l for cyclo(L-Phe-L-Pro) and 3-phenyllactic acid, respectively, and weak synergistic effects were proposed. Synergistic effects of cyclo(L-Leu-L-Pro) and cyclo(L-Phe-L-Pro) were similarly described as inhibitors of pathogenic microorganisms including Candida albicans as well as anti-mutagenic effects in Salmonella strains (Rhee, 2004). We identified a pool of low-molecular-mass compounds including 3-phenyllactic acid, 4-hydroxyphenyllactic acid, 2-pyrrolidone5-carboxylic acid, succinic acid as well as propionic, acetic, and lactic acids in cell-free culture supernatants of the protective co-culture Lactobacillus paracasei subsp. paracasei SM20 and Propionibacterium jensenii SM11 (Miescher Schwenninger et al., 2008). Purification was achieved with a microplate bioassay controlled procedure with solid-phase extraction (C18) followed by either gel filtration chromatography or semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC) and identification by LC-MS. A fermentation process with separate cell immobilization of the two strains was developed to produce high antagonistic activity expression, as observed on semisolid or solid matrices. Only low concentrations of 2-pyrrolidone-5-carboxylic acid (7 mM corresponding to 0.9 g/l), 3-phenyllactic acid (1 mM corresponding to 0.2 g/l), and 4-hydroxyphenyllactic acid (0.2 mM corresponding to 36.4 mg/l) were produced during fermentation

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which were in contrast to relatively high MIC values of 50 (e.g. corresponding to 8.3 g/l for 3-phenyllactic acid) to more than 500 mM (83.1 g/l) determined with increasing pH from 4.0–6.0 for strains of Candida pulcherrima and Rhodotorula mucilaginosa (Table 2.1). Succinic acid was present at higher concentrations (29 mM corresponding to 3.4 g/l) but with comparable high MICs of 200 (23.6 g/l) to more than 500 mM (59.1 g/l) for pH 4.0–6.0. We therefore assumed synergistic effects between several low-molecular-mass compounds that were heat resistant (121 °C for 15 min) and resistant to protein degrading enzymes. 3-Phenyllactic acid, 4-hydroxyphenyllactic acid, and succinic acid production were associated with Propionibacterium jensenii SM11 and 2-pyrrolidone-5-carboxylic acid with Lactobacillus paracasei SM20 (Miescher Schwenninger et al., 2008). Lind et al. (2007) likewise observed the production of 3-phenyllactic acid in five type strains of dairy propionibacteria, at extremely low concentrations ranging from 1.0 mg/l (Propionibacterium freudenreichii subsp. shermanii) to 15.1 mg/l (Propionibacterium thoenii). 2-Pyrrolidone-5-carboxylic acid is a widespread pyroglutamic acid and can be synthesized from glutamic acid by a heating process (Airaudo et al., 1987; Mijin et al., 1989). LAB are known producers of 2-pyrrolidone-5-carboxylic acid which has antibacterial activity against Enterobacter cloacae, Pseudomonas fluorescens, Pseudomonas pudida, and Bacillus subtilis (Huttunen et al., 1995; Yang et al., 1997). Purification of 2-pyrrolidone-5-carboxylic acid from cell-free supernatants was achieved by ethanol precipitation, gel filtration, anion exchange, RP-HPLC, NMR, and MS. Yang et al. (1997) observed complete inhibition of the bacterial indicator strains in a concentration range of 2-pyrrolidone-5carboxylic acid from 6–23 mM (corresponding to 0.8–3.0 g/l) for pH 5.0 and 5.5. Although most of the preceding studies have suggested that antifungal activity is mainly based on 3-phenyllactic acid in combination with organic acid, e.g. lactic and acetic acids, Yang and Clausen (2005) isolated high antifungal strains of Lactobacillus casei and Lactobacillus acidophilus that did not produce 3-phenyllactic acid but instead, at least four unknown heat resistant antifungal metabolites were recognized. Using the isolation procedure described by Ström et al. (2002), Sjögren et al. (2003) identified extremely low amounts of four hydroxylated fatty acids with antifungal activity after 78 h of growth of Lactobacillus plantarum MiLAB 14, i.e. 3-(R)-hydroxydecanoic acid (1.6 mg/l), 3-hydroxy-5-cis-dodecenoic acid (1.0 mg/l), 3-(R)-hydroxydodecanoic acid (0.5 mg/l), and 3-(R)-hydroxytetradecanoic acid (0.2 mg/l). MICs for total growth inhibition of yeasts and moulds were 10–100 mg/l for the racemic forms. None of the above low-molecular-mass compounds was found to be solely responsible for high antifungal activity of its producer. These substances may however be important for more target-oriented screenings and also for the development and optimization of fermentation processes aimed to produce highly active protective cultures for food applications. Further research in this field is thus of the highest importance.

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Proteinaceous antifungal compounds In addition to these characteristic low molecular mass antifungal metabolites, often observed in LAB and PAB, a few studies have described the production of proteinaceous antifungal compounds. The Propionibacterium bacteriocin propionicin PLG-1 was isolated by freezing and centrifugation of soft-agar (0.4%, w/v) cultures since its activity was never determined in cell-free supernatants (Lyon and Glatz, 1991). Propionicin PLG-1 showed broad antagonistic activities including yeast, moulds, Gram-positive, and Gram-negative bacteria. Lactobacillus pentosus TV35b was shown to produce a 3.9-kDa bacteriocin-like peptide with fungistatic effects against Candida albicans (Okkers et al., 1999). Lactobacillus coryniformis subsp. coryniformis was observed to produce heat stable (121 °C for 15 min) proteinaceous compound(s) of about 3 kDa, which were inactivated after treatment with proteolytic enzymes (Magnusson and Schnürer, 2001). Gourama (1997) similarly described antifungal compounds that were sensitive to proteolytic enzymes such as trypsin and pepsin suggesting proteinaceous molecules. Lactobacillus plantarum strain VLT32 isolated from salami did not show any antifungal activity after 48 h of growth at 30 °C, but an inhibitory activity was determined in 30-day cultures which suggested the release of peptidic compounds after autolysis (Coloretti et al., 2007). 2.6.2  Search for further antifungal mechanisms Research has mainly been focused towards the identification of single antifungal compounds in cell-free culture supernatants, which were often only detected in traces, but there is still little known about the overall and putative complex antifungal mechanisms. Important characteristics in antifungal mechanisms are competition for nutrients and cell-to-cell communication, particularly when microorganisms are in close contact and defence mechanisms are activated. The presence of live cells was needed for certain antifungal systems and supported the hypothesis of cell interactions. Tuma et al. (2007) only observed inhibitory effects of various Lactobacillus paracasei and Lactobacillus fermentum strains in the presence of live cells and not in cell-free supernatants suggesting further inhibitory antifungal mechanisms. We likewise showed that close contact of antifungal strains, as exists when they are immobilized in gel beads, was necessary to yield antagonistic activities in culture supernatant enabling identification of antifungal compounds (Miescher Schwenninger et al., 2008). Production of the antifungal compound pyrrolnitrin by certain strains of Burkholderia spp. was even shown to be dependent on N-acylhomoserine lactone (AHL) signal molecules which are utilized by the bacteria to monitor their own population densities in a process known as ‘quorum sensing’ (Schmidt et al., 2009). In order to study interactions between organisms able to grow in the same substrate, Ström et al. (2005) developed a co-cultivation system where different microorganisms were separated by a transparent PET membrane with a pore size of 4 μm. Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 strongly affected the morphology of Aspergillus nidulans mycelium and decreased the biomass to 36% of the control. Higher concentrations of several Aspergillus

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nidulans proteins were observed during co-cultivation and following the addition of inhibiting metabolites of strain MiLAB 393 as pure substances, i.e. cyclo(LPhe-L-Pro), lactic acid, and 3-phenyllactic acid.

2.7 The long road from research to industry: commercial antifungal protective cultures Various biopreservatives mainly based on LAB alone or LAB in combination with PAB have been proposed for applications in food and feed as discussed above. But only a few biopreservatives have followed the long road from research to industry to finally reach commercialization (Table 2.2). To the best of our knowledge only the following four antifungal protective cultures/biological systems have met all the requirements for commercialization. HOLDBAC YM-B, formerly BioProfit, and HOLDBAC YM-C (Danisco A/S; Denmark) are commercial combinations of Lactobacillus rhamnosus LC705/ Propionibacterium freudenreichii JS (Suomalainen and Mäyrä-Mäkinen, 1999) and Lactobacillus paracasei subsp. paracasei SM20/Propionibacterium freudenreichii JS (Miescher Schwenninger and Meile, 2004), respectively, and can be used in sour milk (yoghurt), quark, and cottage cheese to suppress yeasts and moulds. Strains Lactobacillus rhamnosus LC705/Propionibacterium freudenreichii JS, and Lactobacillus paracasei subsp. paracasei SM20 were protected by European Patents (Mäyrä-Mäkinen and Suomalainen, 1993; Miescher Schwenninger and Meile, 2001), and the safety of Lactobacillus paracasei subsp. paracasei SM20 was also shown by careful identification using molecular approaches (Miescher Schwenninger et al., 2005). HOLDBAC YM-B and HOLDBAC YM-C were launched in 1996 and 2004, respectively, with a continuously expanding market supporting applications of LAB–PAB antifungal protective cultures. The antifungal strain Lactobacillus plantarum MiLAB 393 (Ström et al., 2002) has been commercialized as a biological silage additive in co-culture with Pediococcus acidilactici, Enterococcus faecium, and Lactococcus lactis, the latter showing inhibition of Clostridium due to the production of the bacteriocin nisin. This culture has been introduced to the European Market in 2005 (DeLaval; Sweden). In addition to these quite recent antifungal protective cultures, the antifungal biopreservative Microgard™ has a long tradition on the market (Danisco A/S; Denmark). In the 1980s, about 30% of cottage cheese produced in the United States was preserved by Microgard™ (Daeschel, 1989). Microgard™ is a heat treated ferment of Propionibacterium freudenreichii subsp. shermanii that does not contain living antifungal cells (Salih and Yayres, 1990; Al-Zoreky et al., 1991). It was shown to inhibit Gram-negative bacteria such as Pseudomonas, Salmonella, and Yersinia as well as yeasts and moulds. Microgard™ is approved for use by the Food and Drug Administration (FDA) and can be used in cottage cheese, yoghurt, sour cream, ricotta, refrigerated salad sauces and sauces, soups, fresh pasta and fillings, as well as marinated meats. Further information on MicroGARD® is presented in Chapter 3.

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L. plantarum MiLab 393 Yeasts, moulds Pediococcus acidilactici Clostridium spp. Enterococcus faecium Lactococcus lactis

Yeasts, moulds   (Candida spp., Rhodotorula   mucilaginosa)

Yeasts, moulds   (Rhodotorula, rubra, Pichia   quilermondii) Bacillus spp.

Inhibitory spectrum

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b DeLaval

(Denmark). (Sweden).

a Danisco A/S

Heat treated ferment (inactivated bacteria) Microgard™a P. freudenreichii subsp. Yeasts and moulds Gram-negative bacteria   shermanii   (Pseudomonas, Salmonella,   Yersinia)

Feedtech® Silage F300b

HOLDBAC™ L. paracasei SM20 YM-Ca P. freudenreichii subsp.   shermanii JS

Protective cultures (live bacteria) HOLDBAC™ L. rhamnosus LC705 YM-Ba P. freudenreichii subsp.   shermanii JS

Biopreservative Strain(s)

Organic acids   (propionic, acetic,   and lactic acids)

3-Phenyllactic acid Cyclic dipeptides Nisin (bacteriocin)

Propionic and acetic   acids Succinic acid 2-Pyrrolidone-5  carboxylic acid 3-Phenyllactic acid

Propionic and acetic   acids Diacetyl 2-Pyrrolidone-5  carboxylic acid

Bakery products and fillings; dairy products   such as cheese; low pH dressings and   sauces; processed meat products; chilled,   pasteurised ready-to-eat meals; soups

Silage

Fresh fermented dairy products (yoghurt,   sour cream, quark, cottage cheese)

Fresh fermented dairy products (yoghurt,   sour cream, quark, cottage cheese)

Inhibitory mechanism Application range

Table 2.2  Commercialized antifungal biopreservatives based on Lactobacillus spp. (L.) and Propionibacterium spp. (P.)

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The application of new preservatives requires approval by government authorities, a process that often delays commercialization. This might be avoided by developing applications of protective cultures with in situ production of antimicrobials. The following main factors often disrupt the commercialization process: • loss of antimicrobial activity during food production • interactions between food ingredients, food matrix or microbiota of a food, and therefore influence on the efficacy of protective cultures during storage • changes in organoleptic and textural properties of food caused by the protective culture • problematic or unsuccessful scaled-up fermentation and down-stream processes at culture producing companies • failed verification of strain status as safe and food grade organisms (Hansen, 2002; Devlieghere et al., 2004; Melin et al., 2007). Figure 2.6 summarizes the many steps from a selected antifungal strain to commercialization of a protective culture including safety and technological aspects and with particular emphasis on the many hurdles to overcome.

2.8  Future trends Consumers are demanding that chemical additives be reduced or even eliminated and this has directed both the food industry and food research towards natural antimicrobial compounds and their producing strains with the aim of producing ‘green label’ food. High throughput screenings covering broad biodiversities and a broad range of habitats including unidentified microbiota, e.g. from traditional spontaneous artisanal fermentations, will support the detection of high-potential antifungal strains. Research on inhibitory mechanisms and the identification of antifungal metabolites is needed to identify ‘key substances’ which could be used for a target-oriented selection of potential strains. Following this strategy, Lactobacillus rhamnosus LC705 was selected as a potential protective strain due to the production of antimicrobial compound 2-pyrrolidone-5-carboxylic acid (Yang et al., 1997). Production of 3-phenyllactic acid and 4-hydroxphenyllactic acid by LAB was likewise successfully evaluated to support the selection of strains contributing to food quality and safety (Valerio et al., 2004). Increased knowledge of antifungal compounds will be a helpful tool not only for selecting potential strains but also for improving antifungal production by optimizing culture conditions and media (Miescher Schwenninger et al., 2008; Valerio et al., 2008). Favouring increased metabolic activity (Lacroix and Yildirim, 2007; Miescher Schwenninger et al., 2008), immobilized cell technology has high potential for the production of highly active protective cultures and for direct applications of immobilized antagonistic cultures in food. The application of multi-strain antagonistic cultures with synergistic properties might further

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Fig. 2.6  Schematic overview of the development of protective cultures including safety (left) and technological (right) aspects. Modified from Melin et al. (2007).

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increase antifungal activities and broaden antifungal spectra by production of various inhibitory compounds and mechanisms. Promising in vitro results in laboratory experiments are not a guarantee for efficacy of antagonistic cultures in vivo in a food matrix (Devlieghere et al., 2004). Application of the protective culture in or on food is often difficult if the raw material can not be inoculated, e.g. inoculation of milk in yoghurt or cheese production. Spraying or dipping must then be applied which might be a further critical step with respect to sufficient concentrations reaching surfaces as well as hygiene during food processing. Only a few resistances to antifungal compounds have been reported to date, however this should still be carefully checked in the future. The application of various antifungal biopreservatives following the hurdle principle could be one way to avoid future developments of resistance. The application of safe strains in biopreservation is of the highest importance and should be treated as for starter cultures. Cultures should therefore be clearly identified to confirm their food grade status. Information on inhibitory mechanisms including synergistic actions between the strains will further support applications of acceptable strains leading to increased and more ‘natural’ food safety.

2.9  Summary Fungal spoilage by yeasts and moulds is gaining more and more attention not only due to enormous economic losses caused by these ubiquitous microorganisms but also due to the ability of moulds to produce mycotoxins and allergenic spores. Antimicrobial metabolites and their general status as food grade and safe strains make LAB and PAB especially suitable for applications in biopreservation. Natural synergic effects between these two genera were even shown to enhance antimicrobial activities. Limited research has been done on protective LAB and PAB antifungal cultures. Various low-molecular-mass antifungal compounds, e.g. organic acids, 3-phenyllactic acid, 2-pyrrolidone-5-carboxylic acid, cyclic dipeptides, or hydroxy fatty acids, were described in addition to a few proteinaceous and further (unidentified) substances. A general characteristic of the group of low-molecular-mass antifungal compounds was their low concentrations in cell-free culture supernatants versus high minimal inhibitory concentrations (MIC) values making their study very complex and at the same time suggesting further antifungal mechanisms such as completion for nutrients or cell-to-cell communication, known as ‘quorum sensing’. Antifungal activities mainly derived from protective cultures were proposed for applications in dairy and bakery products, malting and brewing processes, and also animal feed such as silage. Despite high potential for food biopreservation, few protective cultures have reached commercialization, which is largely due to the many hurdles along the long road from laboratory tests in vitro in agar plates and challenge studies in vivo in food/food models to industrial applications. When considering the development of antifungal cultures for food biopreservation, several important points should

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therefore be verified as follows: antifungal activities are maintained during food processing and not negatively affected by food ingredients and natural or functional added microbiota; antagonistic strains are successfully produced in scaled-up fermentation and downstream processes; their application does not alter food product’s quality with respect to organoleptic properties and texture; and their status as safe and food grade strains is confirmed.

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and magan n (2007), ‘Potential for detection and discrimination between mycotoxigenic and non-toxigenic spoilage moulds using volatile production patterns: a review’, Food Addit Contam 24, 1161–1168. salih m a and yayres j w (1990), ‘Inhibitory effects of Microgard on yogurt and cottage cheese spoilage organisms’, J Dairy Sci 73, 887–893. sathe s j , nwani n n , dhakephalkar p k and kapadnis b p (2007), ‘Antifungal lactic acid bacteria with potential to prolong shelf-life of fresh vegetables’, J Appl Microbiol 103, 2622–2628. sato k , ito h , ei h and rao g r (1986), ‘Microbial conversion of phenyllactic acid to L-phenylalanine’, Japan Patent JP 86212293. schmidt s , blom j f , pernthaler j , berg g , baldwin a et al. (2009), ‘Production of the antifungal compound pyrrolnitrin is quorum sensing-regulated in members of the Burkholderia cepacia complex’, Environ Microbiol 11, 1422–1437. schnürer j and magnusson j (2005), ‘Antifungal lactic acid bacteria as biopreservatives’, Trends Food Sci Technol 16, 70–78. schnürer j , olsson j and borjesson t (1999), ‘Fungal volatiles as indicators of food and feeds spoilage’, Fungal Genet Biol 27, 209–217. sjögren j , magnusson j , broberg a , schnürer j and kenne l (2003), ‘Antifungal 3-hydroxy fatty acids from Lactobacillus plantarum MiLAB 14’, Appl Environ Microbiol 69, 7554–7557. smittle r (1977), ‘Microbiology of mayonnaise and salad dressing – review’, J Food Prot 40, 415–422. sofos j n (2000), ‘Sorbic acid’, in Naidu A S, Natural Food Antimicrobial Systems, Boca Raton, CRC Press, Taylor and Francis Group, 637–659. soomro a h , masud t and anwaar k (2002), ‘Role of lactic acid bacteria (LAB) in food preservation and human health – a review’, Pak J Nutr 1, 20–24. ström k , schnürer j and melin p (2005), ‘Co-cultivation of antifungal Lactobacillus plantarum MiLAB 393 and Aspergillus nidulans, evaluation of effects on fungal growth and protein expression’, FEMS Microbiol Lett 246, 119–124. ström k , sjögren j , broberg a and schnürer j (2002), ‘Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(LPhe-trans-4-OH-L-Pro) and 3-phenyllactic acid’, Appl Environ Microbiol 68, 4322–4327. suhr k i and nielsen p v (2004), ‘Effect of weak acid preservatives on growth of bakery product spoilage fungi at different water activities and pH values’, Int J Food Microbiol 95, 67–78. suomalainen t h and mäyrä - mäkinen a m (1999), ‘Propionic acid bacteria as protective cultures in fermented milks and breads’, Lait 79, 165–174. suzuki b i , nomura m and morichi t (1991), ‘Isolation of lactic acid bacteria which suppress mold growth and show antifungal action’, Milchwissenschaft 46, 635–639. tawfik n f , sharaf o m , effat b a and mahanna n s (2004), ‘Preserving domiati cheese using metabolites of Propionibacterium thoenii P-127’, Pol J Food Nutr Sci 13, 269–272. te welscher y, ten napel h h , masia balague m , souza c m , riezman h et al. (2007), ‘Natamycin blocks fungal growth by binding specifically to ergosterol woithout permeabilizing the membrane’, J Biol Chem 283, 6393–6401. teuber m (1993), ‘Lactic acid bacteria’, in Rehm H J and Reed G, Biotechnology, Weinheim, Wiley-VCH, Vol. 1, 325–366. teusink b and smid e j (2006), ‘Modelling strategies for the industrial exploitation of lactic acid bacteria’, Nature Rev Microbiol 4, 46–56. trucksess m w and tang y f (1999), ‘Solid-phase extraction method for patulin in apple juice and unfiltered apple juice’, J AOAC Int 82, 1109–1113. tuma s , vogensen f k , plockova m and chumchalova j (2007), ‘Isolation of antifungally active lactobacilli from Edam cheese’, Acta Alimentaria 36, 405–414.

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and piletsky s a (2009), ‘Analytical methods for determination of mycotoxins: a review’, Anal Chim Acta 632, 168–180. valerio f , de bellis p , lonigro s l , visconti a and lavermicocca p (2008), ‘Use of Lactobacillus plantarum fermentation products in bread-making to prevent Bacillus subtilis ropy spoilage’, Int J Food Microbiol 122, 328–332. valerio f , lavermicocca p , pascale m and visconti a (2004), ‘Production of phenyllactic acid by lactic acid bacteria: an approach to the selection of strains contributing to food quality and preservation’, FEMS Microbiol Lett 233, 289–295. vanden bossche h , engelen m and rochette f (2003), ‘Antifungal agents of use in animal health – chemical, biochemical and pharmacological aspects’, J Vet Pharmacol Ther 26, 5–29. warth a d (1985), ‘Resistance of yeast species to benzoic and sorbic acids and to sulfur oxide’, J Food Prot 48, 564–569. warth a d (1988), ‘Effect of benzoic-acid on growth-yield of yeasts differing in their resistance to preservatives’, Appl Environ Microbiol 54, 2091–2095. yang v w and clausen c a (2005), ‘Determining the suitability of lactobacilli antifungal metabolites for inhibiting mould growth’, World J Microbiol Biotechnol 21, 977–981. yang z , suomalainen t , mäyrä - mäkinen a and huttunen e (1997), ‘Antimicrobial activity of 2-pyrrolidone-5-carboxylic acid produced by lactic acid bacteria’, J Food Prot 60, 786–790. yvon m , berthelot s and gripon j (1998), ‘Adding α-ketoglutarate to semi-hard cheese curd highly enhances the conversion of amino acids to aroma compounds’, Int Dairy J 8, 889–898.

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3 Nisin, natamycin and other commercial fermentates used in food biopreservation J. Delves-Broughton, Danisco Food Protection, UK and G. Weber, Danisco Food Protection, USA

Abstract: The chapter reviews the history, physical and chemical properties, antimicrobial spectrum, mode of action, assay, safety, legislation, and current and potential uses as natural biological preservatives of nisin, natamycin, and undefined fermentates. Key words: food preservation, natural preservation, spoilage, nisin, Nisaplin®, bacteriocins, natamycin, Natamax®, polyene macrolide, fermentates, MicroGARD®.

3.1  Introduction Preservatives made by fermentation processes that are available commercially and have approval for use as food additives fall into three categories. These are nisin preparations effective against Gram positive bacteria, particularly spore formers, natamycin preparations effective against yeasts and moulds, and undefined cultured milk and cultured dextrose preparations which, depending on the culture used, can be effective against Gram negative bacteria, Gram positive bacteria, or yeasts and moulds. Use of such preparations made by fermentation is often preferred by food processors as they are considered to be more natural and label friendly methods of food preservation compared to the use of chemicals such as sorbate, benzoates, nitrites and sulphates. This chapter reviews the history, physical and chemical properties, antimicrobial spectrum, modes of action, assay and current and potential applications of nisin, natamycin, and undefined fermentates.

3.2  Nisin used in food biopreservation Nisin is a polypeptide antibacterial substance or bacteriocin produced by the fermentation of a suitable substrate by certain strains of Lactococcus lactis subsp. 63 © Woodhead Publishing Limited, 2011

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lactis (hereafter referred to as L. lactis). Nisin is active against Gram positive bacteria but has little or no effect against Gram negative bacteria, yeasts and moulds. 3.2.1  History Nisin was discovered in 1928 when inhibitory streptococci were causing problems in the production of cheese due to the inhibition of starter cultures (Rogers, 1928; Rogers and Whittier, 1928). Initially the presence of bacteriophage was suspected but investigations indicated that an inhibitory polypeptide produced by certain strains of L. lactis was responsible. Mattick and Hirsch (1947) characterised the compound and called it ‘nisin’ deriving the name from ‘Group N Inhibitory Substance’, N being the serotype group determined by the Lancefield serotyping group of streptococci. Early research into nisin and its properties was based on its potential therapeutic effect for veterinary and clinical uses. Due mainly to its relatively narrow antibacterial spectrum, its low solubility in body liquids, and its instability at physiological pH, it has never been developed for such purposes, but interest is still apparent. Development as a food preservative began in the 1950s. The first report of nisin used as a food preservative was the use of a nisin producing starter to prevent clostridial spoilage of Swiss cheese (Hirsch et al., 1951). McClintock et al. (1952) successfully used nisin-producing cultures to inhibit the development of clostridial spores in Gruyere cheese, but problems with inhibition of cheese starter cultures hampered such use (Winkler and Fröhlich, 1957). The development of a dry powder nisin preparation was pioneered by Aplin and Barrett Ltd. in the UK and this resulted in the introduction in 1953 of the first nisin preparation with the trade name of Nisaplin® (Hawley, 1955, 1957). Early uses of nisin were for prevention of clostridial spoilage of processed cheese but since then numerous other applications have been identified and its use is now approved in over 50 countries for a variety of applications (Turtell and Delves-Broughton, 1998). The Nisaplin® product is still in use today, but is now manufactured by Danisco who acquired Aplin and Barrett in 1999. Early preparations were made using a modified milk based medium as substrate and concentrated by foam extraction, but this has now been changed to a sugar-based medium and concentration using membrane technology. The change to a sugar-based medium prevents problems of allergy associated with consumption of dairy products. Other nisin preparations apart from Nisaplin® are now commercially available: brand names include Chrisin® (Chr. Hansens, Denmark), Delvoplus® (DSM, Holland) and Silver Elephant Nisin made by Zheijiang Silver Elephant Bio-Engineering in China. There are also four or five other smaller manufacturers in China. All these preparations have a similar potency and contain 1,000,000 international units (IU) per gram or approximately 2.5% nisin. A difference between Chinese nisin preparations and European produced nisin preparations is that all Chinese preparations are based on nisin Z, whereas nisin manufactured in Europe is all based on nisin A. This difference will be explained later. Due to the fact that all toxicological studies have been carried out on nisin A preparations some countries such as Australia, © Woodhead Publishing Limited, 2011

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New Zealand and Japan specifically state that only nisin A preparations can be used. In other countries that approve the use of nisin either nisin A or nisin Z preparations can be used. Units of nisin can be confusing. In this chapter nisin concentrations are expressed as levels of pure nisin, i.e. μg/ml or μg/g. Multiplication by 40 will convert these levels to IU (International Unit) /ml or g or level of commercial preparations (mg/kg). For example 1 μg/g of nisin is equivalent to 40 IU nisin/g or 40 mg Nisaplin®/kg. 3.2.2  Physical and chemical properties Nisin A is a polypeptide consisting of 34 amino acids with a molecular weight of 3510 Daltons. Its unusual structure was solved in 1971 by Gross and Morrell (1971) (Fig. 3.1). It is an atypical protein in that it contains unusual amino acids and lanthionine rings. The presence of lanthionine is now known to be characteristic of a larger group of bacteriocins produced by different Gram positive bacteria and collectively known as ‘lantibiotics’. Various natural nisin variants have been discovered. Nisin Z has a substitution of Asn27 for His27 (Mulders et al., 1991), nisin F has substitutions of Asn27 for His27 and Val30 for Ileu30 (de Kwaadsteniet et al., 2008) and nisin Q has substitutions of Val15 for Ala15, Leu21 for Met21, Asn27 for His27, and Val30 for Ileu30 (Zendo et al., 2003). Nisin potency and spectrum for nisin A and nisin Z are similar, but nisin Z diffuses more readily through agar gel and has a positive charge of 2 compared to a positive charge of 3 for nisin A. Only nisin A and Z are used in commercial preparations. Most published scientific information pertains to nisin A. Solubility of nisin A is pH dependent (Liu and Hansen, 1990). Thus for pure nisin A solubility at pH 2.2 is approximately 56,000 μg/ml, at pH 5 is 3000 μg/ ml and pH11 is 1000 μg/ml. Solubility is not a problem in food products as nisin levels used are less than 250 μg/ml.

Fig. 3.1  The structure of nisin A. ABA: aminobutryic acid; DHA: dehydroalanine; DHB: dehydrobutyrine (β-methyldehydroalanine); ALA-S-ALA: lanthionine; ABA-SALA: β-methyllanthionine.

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In the dry state nisin preparations show excellent stability when protected from direct sunlight, moisture uptake, and at temperatures below 22 °C. Nisin A stability of solutions is optimum between pH 3.0 and 3.5. Thus autoclaving nisin A solution in buffer (25 μg/ml) at 121 °C for 15 min resulted in the retention of less than 5% activity at pH1, 42.5% at pH 2, 87.5% at pH 3, 84% at pH 3.5, 80% at pH 4 and 25% at pH 5 (Davies et al., 1998). Even greater losses would be expected at higher pHs near neutrality and above. Pasteurisation temperatures are less damaging to nisin and various components in foods can protect the nisin molecule to an extent from heat. 3.2.3  Antimicrobial spectrum Nisin has a broad spectrum of activity against Gram positive bacteria and the most significant species associated with food spoilage and safety are shown in Table 3.1. It is important to remember that the sensitivity of nisin to bacteria varies between genera, species and even strains of the same species (Gupta and Prasad, 1989). In normal circumstances nisin does not significantly inhibit Gram negative bacteria, yeasts and moulds. Among Gram positive bacteria that are sensitive to nisin are members of the mesophilic spore forming genera Bacillus, Alicyclobacillus, Clostridium, Desulfomaculum, and the thermophilic spore-forming genera Geobacillus and Themoanaerobacterium. Both vegetative and spores are sensitive, with levels of nisin required to inhibit spore outgrowth generally less than those required to inhibit vegetative cells. Such an action against spores has resulted in nisin preparations being used as a preservative in products which, by their nature, cannot be fully sterilised but only pasteurised during their production. Nisin also shows activity against many types of lactic acid bacteria. As such bacteria are capable of growth at low pH, nisin can be used as a preservative in low pH foods and beverages that are not heat processed, such as salad dressings, acidified cheese, and alcoholic beverages. The fact that yeasts are insensitive to nisin means that nisin can be used in fermentations alongside yeasts to control the growth of lactic acid bacteria with no effect on the yeast. 3.2.4  Mode of action Nisin like other preservatives works in a concentration dependent manner in terms of the amount of nisin applied and the level of contamination in the food. Condition of test can dictate whether nisin action against vegetative cells will be predominantly bactericidal or bacteriostatic. The more energised the bacterial cells, the more bactericidal effect the nisin will have, whereas if the cells are in non-energised state because they are in the lag or stationary phase of growth or are in a medium or food of non-optimum pH, water activity, low nutrient availability, and/or at a non-optimum temperature of growth, the nisin effect will be predominantly bacteriostatic (Sahl, 1991; Maisnier-Patin et al., 1995). The use of nisin as a food preservative in combination with other factors is the basis of © Woodhead Publishing Limited, 2011

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Table 3.1  Nisin-sensitive bacterial species associated with food Genus

Species

Alicyclobacilius acidoterrestris Bacillus brevis, cereus, coagulans, licheniformis, megaterium, pumilis, subtilis, stearothermophilus Brochothrix thermosphacta Clostridium bifermentans, botulinum, butyricum, cochlearium, histolyticum, pasteurianum, perfringens, putificum, sordelli, sporogenes, tertium, tyrobutyricum Desulfotomaculum nigrificans Enterococcus faecalis, faecium Geobacillus stearothermophilus Lactobacillus bulgaricus, brevis, buchneri, casei, curvatus, helveticus, fermentum, lactis, plantarum Leuconostoc oenos, mesenteroides Listeria innocua, monocytogenes Sporolactobacillus inulinus Staphylococcus aureus Thermoanaerobacterium thermosaccharolyticum

Description Heat-resistant spore former. Growth at pH 2.5–6, 25°–60 °C. Spoilage organism of fresh/ pasteurised fruit juice stored at ambient temperature. Heat-resistant aerobic and facultative anaerobic spore formers. Includes psychotrophs, acid-resistant, spoilage organisms, and food poisoning pathogens. Heat-sensitive spoilage organism of meat. Growth between 0 °–30 °C. Often associated with modified atmosphere packing. Heat-resistant spore-forming obligate anaerobes. Causes spoilage and food poisoning.

Heat-resistant spore-forming obligate anaerobe. Causes blackening of canned food. Aerobes/anaerobes. Spoilage organism. Varied nisin sensitivity. Thermophilic spore former causes flat-sour spoilage of canned vegetables. Spores very heat resistant. Causes spoilage of acid products, salad dressings, cured meat products, soft drinks, wine, beer, cider. Can grow at low pH. Aerobes/anaerobes. Causes spoilage of wine and beer. Slime producing. L. monocytogenes – psychroduric food poisoning organism. Aerobe/anaerobe. Spore forming. Growth at low pH. Aerobe/anaerobe. Varied sensitivity. Causes food poisoning. Thermophilic spore-former causes can swelling/blowing spoilage of canned vegetables. Spores very heat resistant.

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multifactorial preservation otherwise known as ‘hurdle technology’ (Leistner and Gorris, 1995). The target for nisin action against vegetative cells is the cytoplasmic membrane. A major breakthrough on the mode of action of nisin against vegetative cells was the discovery that the cell wall peptidoglycan precursor lipid II acts as a docking molecule for nisin, and it is the nisin-lipid II complex that inserts itself into the cytoplasmic membrane forming transient pores that cause leakage of essential cellular material (Breukink et al., 1999; Wiedemann et al., 2001). A further mode of action of nisin is that it also inhibits peptidoglycan synthesis, a component of bacteria cell walls. The outer membrane of Gram negative bacteria effectively prevents nisin from making contact with the cytoplasmic membrane (Kordel et al., 1989). In combination with a chelating agent such as disodium ethylene-diamine-tetraacetic acid (EDTA), nisin can be effective against a variety of Gram negative bacteria (Stevens et al., 1991; Delves-Broughton, 1993; Cutters and Siragusa, 1995). Chelating agents remove divalent ions from Gram negative cell walls, releasing phospholipids and lipoproteins thus increasing cell outer membrane permeability. Unfortunately, chelating agents are much less effective in food compared to in buffer solutions due to their preferential binding to free divalent ions within the food. Any treatment such as sub-lethal heat, hydrostatic pressure, pulsed electric field, or freezing which disrupt the outer membrane may render Gram negative bacteria sensitive to nisin. Mode of action against bacterial spores has not been so intensively studied and it is still uncertain as to its precise mode of action, and even whether it is sporostatic or sporicidal. Thorpe (1960) showed that when nisin was applied to spores of Geobacillus stearothermophilus, the reduction in heat resistance observed was apparent rather than real and was due to adsorption of nisin onto the spores and that the nisin could be removed and viability restored if the nisin was removed using the enzyme trypsin. However, more recent research by Gut et al. (2008) demonstrated that spores of B. anthracis lost their heat resistance when nisin was applied and the spores became hydrated. Previously Morris et al. (1984) showed that nisin bound on to sulphydryl groups on protein residues on the spore surface. It is clear that the more spores are heat damaged the more they are sensitive to nisin and that thermophilic spores belonging to Geobacillus stearothermophilus and Thermoanaerobacterium thermosaccharolyticum are extremely sensitive. 3.2.5  Assay Basically nisin can be measured in two ways – either directly by chemical, immunological, or genetic measurement of the nisin molecule; or indirectly by measuring its biological activity or potency by turbidometry, agar diffusion assay, or measurement of efflux of cellular material. The various methods with their limit of detection are shown in Table 3.2. The preferred method of quantitative assay of nisin in foods is the Micrococcus luteus plate diffusion assay.

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Table 3.2  Methods for assay of nisin with approximate minimum levels of detection Method

Detection limit

Biological activity measurement Turbidometric assay 0.025 μg/ml Agar diffusion assay 0.025 μg/ml Efflux and assay of adenosine triphosphate 0.025 μg/ml Efflux and assay of potassium 0.018 μg/ml Impedance 0.05 μg/ml Chemical measurement High performance liquid chromatography 0.25 μg/ml Enzyme linked absorption assay 5–10 ng/ml Genetic-based bioluminescence 0.0125–0.75 ng/ml 0.02–10 pg/ml

Reference

Barreteau et al. (2004), Turcotte et al. (2004) Tramer and Fowler (1964), Fowler et al. (1975) Waites and Ogden (1987), Valat et al. (2003) White et al. (1992), Mugochi et al. (2001) Giraffa et al. (1990), Čurda et al. (1995), Kozáková et al. (2005) Delves-Broughton and Friis (1998), Matusaki et al. (1995) Falahee et al. (1990), Leung et al. (2002), Suárez et al. (1996) Wahlström and Sarris (1999), Reuanen and Saris (2003) Hakovirta et al. (2006), Hanan et al. (2009)

3.2.6  Current applications of nisin in foods Use of nisin in foods is dependent on regulatory approval which varies from country to country. Nisin is often used as a preservative in foods which are pasteurised but not fully sterilised during production thus protecting the food from outgrowth of spores which survive the pasteurisation process. Nisin insensitive organisms such as Gram negative bacteria, yeasts, and moulds are sensitive to heat and will be killed by the pasteurisation. Nisin can also be used to control contaminant lactic acid bacteria in the brewing and wine making process where its lack of effect against yeasts is a benefit. Applications are shown in Table 3.3. The outcome of nisin activity within a food system will depend on numerous factors. Other preservative hurdles such as severity of heat treatment, low water activity, modified atmosphere, low temperature, low pH, and the presence of other natural or chemical preservatives can enhance activity. Nisin works better in liquid or homogenous foods compared to solid or heterogenous products because the bacteriocin can be better or more evenly distributed in the former. Nisin is hydrophobic in nature so fat in food may hinder its distribution or render it unavailable for activity (Jung et al., 1992). Certain food additives have been

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Table 3.3  Examples of nisin applications, typical addition levels, and supporting references Food

Nisin (μg/g)

Typical target organism

Processed 2.5–15 Bacillus, Clostridium cheese Milk and milk 0.25–1.25 Bacillus, Clostridium products Pasteurised 1.88–5 Bacillus, Clostridium chilled dairy desserts Liquid egg 1.25–5 Bacillus Pasteurised 2.5–6.25 Bacillus soups Crumpets 3.75–6.25 Bacillus cereus Fruit juice 0.75–1.5 Alicyclobacillus acidoterrestris Canned food 2.5–5 Geobacillus stearothermophilus, Thermoanaerobacterium thermosaccharolyticum Dressings and 1.25–5 Lactic acid bacteria, sauces Bacillus Processed meats 5–10 Lactic acid bacteria, such as bologna, Brocothrix frankfurter sausages thermosphacta Ricotta cheese 2.5–5 L. monocytogenes, Bacillus Beer Lactic acid bacteria Reduced 0.25–1.25 pasteurisation During fermentation 0.63–2.5 Post fermentation 0.25–1.25

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Reference Somers and Taylor, (1987), DelvesBroughton (1998) Maisnier-Patin et al. (1995), Wirjantaro and Lewis (1996), Wirjantaro et al. (2001) Sukumar et al. (1976), Anonymous (1985) Delves-Broughton et al. (1992)

Jenson et al. (1994) Komitopoulou et al. (1999), Yamazaki et al. (2000), Peña and de Massaguer (2006), Walker and Phillips (2008) Gillespy (1953), O’Brien et al. (1956), Duran et al. (1964), Hernandez et al. (1964), Nekhotenova (1961), Vas et al. (1967) Muriana and Kanach (1995), Beuchat et al. (1997) Davies et al. (1999), Gill and Holley (2000) Davies et al. (1997)

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shown in our laboratories to be antagonistic to nisin and these include sodium metabisulphite (antioxidant, bleaching agent and broad spectrum preservative) and titanium dioxide (whitener). In foods that are not heat treated, or that have been minimally processed, nisin may be degraded by proteolytic enzymes. During heat processing a certain amount of the nisin will be degraded. This will depend on the severity of the heat treatment, pH of the food, and the degree of protection the food may give the nisin. For example, in processed cheese manufacture about 20–25% can be lost during a typical melt process; and in retorting of canning of vegetables where nisin is used to protect against thermophilic spoilage, up to 95% can be lost. The low residual levels in canned vegetables are still very effective reflecting the extreme sensitivity of thermophilic spore formers to nisin. Similarly, nisin retention in foods will be dependent on the food itself, pH and the length and temperature of storage. Low pH is beneficial on two counts: first, nisin retention during heat processing is optimum at low pH; and second, low pH is often a further hurdle in itself inhibiting bacterial growth. The use of nisin in beer and wine production makes use of the fact that nisin has no effect on yeast viability and vitality but is active against many of the Gram positive lactic acid bacteria that can spoil beer and wine. Uses of nisin in beer especially for washing pitching yeast have been proposed (Ogden, 1986, 1987; Ogden and Tubb, 1995; Ogden et al., 1988). Uses of nisin in wine have also been proposed (Radler, 1990a, 1990b; Daeschel et al., 1991; Knoll et al., 2008). Nisin may also have potential in fuel alcohol production by inhibiting lactic acid bacteria competing with yeast for substrate (Mawson and Costar, 1993; Franchi et al., 2003a, b). 3.2.7  Potential applications of nisin in foods New combinations of nisin with other preservatives The use of nisin in combination with other preservatives and food ingredients with the objective of finding combinations that demonstrate additive or synergistic effect has been the subject of much research and many successful combinations have been identified. Space does not allow all to be described, but some examples are shown in Table 3.4. Synergies usually occur with nisin in combination with other preservatives that have the cytoplasmic membrane as target (Adams and Smid, 1983). Much of the research has been carried out with L. monocytogenes as the chosen target bacteria which reflects the concern in the USA to the problem of listeriosis and their zero tolerance policy on the presence of the pathogen in foods that are not heated sufficiently to kill the bacteria prior to consumption. With the increased development of chilled long-shelf-life, ready to eat meals, concern is now being directed at the need to ensure against botulism. Powerful synergies that both increase the effectiveness and broaden the antimicrobial spectrum of nisin may be required to be subjected to toxicological review to ensure they are safe. Nisin in combination with novel food processing technology Increasing consumer demand for minimally processed, shelf-stable foods has prompted food technologists and scientists to explore other physical preservation © Woodhead Publishing Limited, 2011

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Table 3.4  Examples of published papers demonstrating nisin synergy with other antimicrobials Antimicrobial substance

Target organism(s) Substrate

Reference

Organic acids Potassium sorbate L. monocytogenes CO 2 and vacuum Avery and Buncic (1997)   packed beef Acetic acid E. coli Ground beef Fang and Hseuh (2000) Potassium sorbate S. aureus B. cereus Vegetarian food Fang et al. (1997) Sodium benzoate Sodium lactate L. monocytogenes Vacuum packed Neetoo et al. (2008) smoked salmon Monoglycerides Monolaurin Spoilage bacteria Model meat system Bell and de Lacy (1987) Monolaurin L. lactis subsp. Milk Blackburn et al. (1989)   agalactiae Sucrose fatty acid esters Sucrose palmitate Various Gram Buffer and agar Thomas et al. (1998) Sucrose stearate positive bacteria   medium Chelating agents EDTA Gram negative Buffer Stevens et al. (1992a,b)   bacteria EDTA L. monocytogenes Vacuum packed Zhang and Mustapha   beef   (1999) EDTA Pseudomonas Whole and cut Ukuku and Fett (2002)   melon Maltol E. coli Buffer Shved et al. (1996) Lactoperoxidase system L. monocytogenes Skimmed milk Zapico et al. (1998) L. monocytogenes Milk Boussouel et al.   (1999, 2000) Various bacteria Sardines Elotmani and Assobhei (2003) Lysozyme Listeria spp. Processed cheese, Ter Steeg (1993)   paté L. monocytogenes, Hot dogs Proctor and   S. aureus Cunningham (1993) Various lactic acid Broth Chung and Hancock   bacteria   (2000) Other bacteriocins Pediocin Various Gram Buffer Hanlin et al. (1993)   positive bacteria Pediocin ACH, Various Gram Broth and agar Mulet-Powell et al. Lacticin 481 positive bacteria medium   (1998) ɛ-Poly-L-lysine L. monocytogenes, Buffer Najar et al. (2007)   Bacillus

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Reuterin L. monocytogenes, Milk Arqués et al. (2008)   S. aureus Lactoferrin L. monocytogenes Broth Cleveland and   Tchikindas (2001) Bacterial flora Meat balls Colak et al. (2008) Essential oils Cavacrol B. cereus, Broth, potato puree Rajkovic et al. (2005)   B. circulans Grape seed extract, L. monocytogenes Soy protein film on Theivendran et al. Green tea extract   frankfurters   (2006) Rosemary extract L. monocytogenes, Bolognese sauce, Thomas and Isak   B. cereus   carbonara sauce   (2006)

methods as alternatives to traditional treatments such as freezing, canning, or drying. Although these traditional technologies have helped to ensure a high level of safety, the heating and cooling of foods can contribute to deterioration of various quality attributes such as colour, nutritional content and flavour (DelvesBroughton, 2008). Promising novel methods of preservation of food and beverages include the use of ultra high pressure (UHP), pulsed electric field (PEF), edible coatings and active packaging. Nisin as an adjunct to all four of these novel methods of preservation has been the subject of considerable research. Nisin in combination with UHP UHP shows considerable promise as a novel means of food preservation and a number of commercial foods are now processed using the technology (Black et al., 2005; Yaldagard et al., 2008). Examples are ready-to-eat chicken meat, sliced ham, fresh whole oysters, jams, fruit juices and guacamole. Methods and equipment used for UHP treatment are outlined in review articles by Cheftel (1995) and Yaldagard et al. (2008). The commercial success of UHP will depend upon the effective destruction and/or control of food pathogenic and spoilage microorganisms. The vegetative cells of bacteria, moulds and yeasts and spores of moulds can be reduced by 6 log cycles at or below 690 Megapascals (MPa) at ambient temperature or by a combination of less pressure but increased temperature. Bacterial spores are far more resistant and a 5–6 log reduction of Bacillus and Clostridium spores requires a combination of very high pressure and high temperature (Farkas and Hoover, 2000; Ray et al., 2001). Such a drastic treatment can be costly in terms of equipment design and operating costs (Gao and Ju, 2008) and may adversely effect the quality of many foods. This has prompted the evaluation of nisin as an adjunct to UHP treatment of foods as a means of reducing the level of pressure required to ensure required shelf life and safety. The mode of action of UHP against microorganisms is that it causes aggregation of proteins and disruption of the cytoplasmic membrane (Rovere et al., 1998). It

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has been demonstrated that nisin and UHP not only are synergistic in the killing of Gram positive bacteria but can also widen the spectrum to kill Gram negative bacteria and to a lesser extent yeasts. The sensitisation of Gram negative bacteria by UHP to nisin is considered to be due to outer membrane damage allowing nisin to reach the target site, the cytoplasmic membrane (Kalachayanand et al., 1994; Hauben et al., 1996; Masschalck et al., 2001; Black et al., 2008). An alternative approach to destruction of spores using UHP is to to induce germination by low to medium range hydrostatic pressure to cause germination and outgrowth and then expose the outgrown vegetative cells to nisin, thus preventing their multiplication (Stewart et al., 2000; Kalachayand et al., 2004). Nisin in combination with PEF The mode of action of PEF is that it produces structural changes in the cytoplasmic membrane resulting in pore formation, efflux of essential cellular material and a loss of selective permeability. Since nisin and PEF both act on cytoplasmic membranes it is logical to predict that their combination or use in sequence would have additive or synergistic bactericidal effects. PEF treatment of foods is restricted to pumpable liquid foods and research has been carried out in milk, soups, fruit juice and liquid egg products. Detailed information on PEF theory, and equipment and technology, can be found in a review by Vega-Mecardo et al. (1999). Several investigations have demonstrated that nisin in combination with PEF is effective in buffer and various liquid foods and beverages (Table 3.5). Exposure of Listeria innocua to nisin in liquid whole egg following PEF treatment exhibited an additive effect on the inactivation of the bacteria (Calderón-Miranda et al., 1999a). A synergistic effect was observed as the electric field intensity (30–50 kV/ cm), number of pulses and nisin concentration (0.25–2.5 μg/ml) increased both in liquid egg and in skimmed milk (Calderón-Miranda et al., 1999a,b). Transmission electron microscopy reveals that L. innocua treated by PEF alone in skimmed milk exhibited an increase in the cell wall roughness, cytoplasmic clumping, leakage of cellular material, and rupture of cell walls and cell membranes, whereas treatment with nisin and PEF in combination exhibited an increase in cell wall width (Calderón-Miranda et al., 1999c). Thus the combination may cause damage of the cell wall rather than the cell membrane. Interestingly it has been observed that the efficiency of a combined treatment of nisin and PEF in liquid whey protein was strongly dependent on the sequence of application, since exposure to nisin after PEF produced a lower effect on L. innocua inactivation. Studies have demonstrated the efficacy of PEF against Gram negative bacteria can be enhanced by nisin. When PEF treatment was applied to Salmonella cells in the presence of nisin (2.5 μg/ml), lysozyme (2,400 units/ml) or a mixture of nisin (0.688 μg/ml) and lysozyme (690 units/ml), cell inactivation by the combination was increased by an additional 0.04 to 2.75 log units. Furthermore, the combination of nisin and lysozyme had a more pronounced bactericidal effect (by at least 1.37 log cycles) than either nisin or lysozyme alone (Liang et al., 2006).

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Table 3.5  Reported effects of the application of nisin and pulsed electric field for microbial inactivation References

Observed effects

Dutreux et al. (2000) Santi et al. (2003) Sobrino-López and Martín-Belloso (2006, 2008) Calderón- Miranda et al. (1999 a,b,c), Gallo et al. (2007), Miranda et al. (2001) Terebiznik et al. (2000, 2001,   2002) Pol et al. (2000, 2001a,b) Liang et al. (2006) Hodgins et al. (2002) Iu et al. (2001) Ulmer et al. (2002) Nguyen and Mittal, (2007)

Increased activation of M. luteus in phosphate buffer Increased activation of P. aeruginosa Increased activation of S. aureus in skimmed milk Increased inactivation of L. innocua in liquid whole egg, skimmed milk, and liquid protein concentrate Increased inactivation of E. coli in simulated milk ultrafiltrate media Observed synergism with reduced water activity Increased inactivation of B. cereus vegetative cells (more efficient in buffer than skimmed milk) Observed synergism with cavacrol Inactivation of Salmonella in orange juice. Observed synergism with lysozyme Increased inactivation of microorganisms in orange juice. Observed synergism with lysozyme Inactivation of E. coli 0157:H7 in fresh apple cider. Observed synergism with cinnamon Inactivation of L. plantarum in model beer Increased inactivation of microorganisms in tomato juice

Modified and expanded from Gálvez et al. (2007).

It should be noted that bacterial spores are resistant to PEF treatments. Incorporation of nisin into food may provide an additional hurdle if the nisin survived the PEF treatment against surviving spores. However, limited evidence to date suggests that nisin is destroyed by PEF treatment (Terebiznik et al., 2000). To make use of nisin as a means of preventing spore outgrowth, the nisin would have to be added aseptically to the food post PEF treatment or possibly protected during the PEF treatment by encapsulation. Use of nisin in active antimicrobial packaging Antimicrobial active packaging acts by inhibiting or killing the growth of undesirable microorganisms on the surface of foods. Of all the antimicrobials studied for their effectiveness in both edible and non-edible films, nisin has been the most extensively studied. It has been studied alone or in combination with other antimicrobial agents such as EDTA, lysozyme, organic acids, grape seed extract and green tea extract. Joeger (2007) carried out an extensive review of the literature and found that the majority of results reported around a log 2

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reduction of target vegetative cells although at times it was significantly higher. Most studies use as test organism L. monocytogenes which again reflects the concern for this psychroduric pathogen, particularly in the USA. Joeger concludes that active antimicrobial packaging still faces limitations and is best viewed as a part of a hurdle strategy to provide safe foods or as a method of increasing shelf life. 3.2.8  Safety and tolerance Nisin-producing L. lactis occur not only in raw cow’s milk and cheese but have been found in a variety of other foods and even human breast milk (Table 3.6). Inadvertently and apparently harmlessly, humans and animals probably have consumed nisin, albeit in small amounts, for centuries. Numerous toxicological studies have been carried out and it should be noted that all these have been confined to nisin A preparation. No toxicological study has been carried out with Table 3.6  Foods and other sources from which nisin producing L. lactis have been isolated Food or other sources

Reference

Cow’s milk Bovine milk, Grana cheese Sauerkraut (fermented cabbage) Mixed salad, fermented carrots Buffalo market milk Various cheese, bovine milk, and meats Dry fermented sausages Various ready to eat meats, fish, cheeses, vegetables Soil, effluent water, cattle skin Bean sprouts Kimchi (fermented cabbage) Bovine milk, goat milk, Chinese radish seed, soil, saliva of cow River water Human breast milk Rigouta cheese (Tunisia) Freshwater catfish Tsuda–turnip pickles Tunisian cheeses Slovenian cheese

Rogers (1928), Rogers and Whittier (1928), Delves–Broughton (1990), Rodríguez et al. (2000), Şanilibaba et al. (2009) Carini and Baldini (1969) Harris et al. (1992), Tolonen et al. (2004) Uhlman et al. (1992) Gupta et al. (1993) Vaughan et al. (1994) Rodríguez et al. (1995) Kelly et al. (1996, 1998) Klijn et al. (1995) Cai et al. (1997) Choi et al. (2000) Ayad et al. (2002) Zendo et al. (2003) Beasley and Saris (2004) Ghrairi et al. (2004) De Kwaadsteniet et al. (2008) Aso et al. (2008) Ouzari et al. (2008) Trmčič et al. (2008)

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nisin Z or any other nisin variant. The studies carried out with nisin A preparation confirm that nisin A is non toxic at levels much higher than those used in food (Frazer et al., 1962; Hara et al., 1962; Bogorditskaya et al., 1970; Shtenberg and Igant’ev, 1970). Digestive enzymes rapidly inactivate nisin and consequently do not alter the microflora in the intestinal tract (Barber et al., 1952; Heinemann and Williams, 1966; Jarvis and Mahoney, 1969). The LD 50 value is about 7g/kg body weight, similar to that of common salt. As the preparation tested contained 75% salt, the toxicity can be attributed to that component alone (Hara et al., 1962). No ill effects were observed in pigs and poultry from feeding experiments (Barber et al., 1952; Coates et al., 1951). There is no evidence of any cross resistance with antibiotics used in medicine (Szybalski, 1953; Carlson and Bauer, 1957; Hossack et al., 1983; Chikindas et al., 2000). In 1969 the FAO/WHO Expert Committee decided from the available evidence that a suitable acceptable daily intake (ADI) was 33,000 IU (0.825 mg nisin A)/kg of body weight/day. In 1988, the US Food and Drug Administration (FDA) affirmed nisin as GRAS (generally recognised as safe) for direct use as a food ingredient (FDA, 1988). The EU Expert Scientific Panel (EFSA) reviewed nisin as a food additive in 2006 and concluded that it was a safe and useful preservative (EFSA, 2006). Various expert opinions outline the reasons as to how nisin is different from antibiotics and to why it is a safe food preservative and should be considered for wider use (Hurst, 1981; Wessels et al., 1998; Cleveland et al., 2001).

3.3  Natamycin used in food biopreservation Natamycin, previously sometimes known as pimaracin or tennectin, is a polyene macrolide antimycotic produced by the actinomycete Streptomyces natalensis and other closely related Streptomyces spp. Natamycin is active against yeasts and moulds, and shows no activity against bacteria. 3.3.1  History Natamycin was first produced in 1955 from a culture filtrate of a Streptomycetes isolated from a soil sample in South Africa (Struyk et al., 1959; Brik, 1981). It is produced by fermentation of S. natalensis in a medium containing a carbon source (e.g., starch or molasses) and a fermentable nitrogen source (e.g., corn steep liquor, casein, soya bean meal). Fermentation is aerobic and mechanical agitation and antifoaming agents can aid the process. The temperature range is 26–30°C and the pH range is 6–8. Due to its low solubility, natamycin will accumulate mainly as crystals and these can be extracted following separation of the biomass by solvent extraction (Struyk and Waivisz, 1975). Natamycin preparations have been used for several years as a preservative protecting foods and beverages against yeast and mould spoilage. Many applications are in bacteria fermented foods prone to yeast or mould spoilage as

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the preservative has a selective action against yeasts and moulds with no action against bacteria. Commercial preparations available are Natamax® (Danisco, Denmark), Delvocid® (DSM, Holland) and Silver Elephant Natamycin (Zheijiang Silver Elephant Bio-Engineering, China). The natamycin content of most preparations is 50% with the incipient being lactose, glucose, or salt. Preparations are also available that contain food grade polymers that aid the adherence of natamycin for surface treatments of foods (Delves-Broughton et al., 2006). 3.3.2  Physical and chemical properties Natamycin belongs to a group of antifungals known as polyene macrolides. The structure (Fig. 3.2) was first determined by Ceder (1964) and the stereo structure by Lancelin and Beau (1995). It has a molecular weight of 665.7 Daltons, is amphoteric and has an isoelectric point of 6.5. Natamycin is a white/creamcoloured crystalline powder with no taste and little odour. It is stable in powder form if stored at room temperature but in aqueous solutions is less stable particularly if exposed to acidic conditions, light, certain oxidants and heavy metals (Raab, 1972). Natamycin has low solubility in water (approximately 40 μg/ml), but this low solubility is an advantage in the surface treatment of foods because it ensures that the preservative remains on the surface of the food where it is needed, rather than migrating into the foods. Increased solubility occurs with a range of solvents (Delves-Broughton et al., 2005). Raab (1972) reports on the effect of pH on stability of natamycin solutions: it is more stable in the pH range 4.5 to 9, and at pHs above and below this it is significantly less stable. 3.3.3  Antimicrobial spectrum Natamycin is effective against a wide range of yeasts and moulds and the preservative is usually effective at concentrations between 1 and 10 μg/ml. In

Fig. 3.2  The structure of natamycin.

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general yeasts are more sensitive than moulds, the minimum inhibitory concentrations (MIC) of yeasts are usually less than 5 μg/ml whereas that of moulds can be 10 μg/ml or higher. Examples of yeasts and moulds sensitive to natamycin are shown in Table 3.7. 3.3.4  Mode of action The mode of action of natamycin involves an interaction between natamycin and ergosterol, an essential component of membranes of yeasts and moulds. Originally it was proposed that this interaction resulted in increased membrane permeability efflux of cellular material. However, recent research by Te Welscher et al. (2008) and van Leeuwen et al. (2009) has shown that the action of natamycin does not increase permeability of the cytoplasmic but more likely prevents cell growth, spore germination, and inhibits membrane associated enzyme activity. Penicillium discolor, Verticillium cinnabarinum and Botrytis cinerea, three moulds with reduced ergosterol content in their cell membrane and ergosterol deficient mutants of Aspergillus nidulans, have much reduced natamycin sensitivity (Ziogas et al., 1993). De Boer and Stolk-Horsthuis (1977) and De Boer et al. (1979) compared the sensitivity of yeasts and moulds from cheese and sausage factories where natamycin had been used for several years and where it had never been used. Table 3.7  Examples of yeasts and moulds that are sensitive to natamycin Absidia Alternaria Aspergillus chevalieri A. flavus A. niger A. ochraceus A. oryzae A. penicilloides A. roqueforti A. versicolor Botrytis cinerea Brettanomyces bruxellensis Bassochlymas fulva Candica albicans C. guillermondii C. vini Cladosporium cladosporiodes Fusarium Gloeosporium album Hansenula polymorpha Koeckera apiculata Mucor mucedo M. raceosus Penicillium camemberti

P. commune P. chysogenum P. cyclopodium P. digitatum P. expansum P. islandicum P. notatum P. roqueforti Rhizopus oryzae Rhodotolura gracilis Saccharomyces bailii S. bayanus S. cerevisiae S. exiguus S. florentinus S. ludwigii S. rouxii S. sake Sclerotina fructicola Scopulariopsis saperula Toluropsis candida T. lactis-condensi Wallensis sebii Zygosaccharomyces barkeri

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There was no difference in the sensitivity to natamycin of yeasts and moulds between sites. 3.3.5  Method of assay Shirk et al. (1962) developed an agar diffusion bioassay using Saccharomyces cerevisiae as indicator organism. HPLC is however the preferred method of assay (Anon., 2008). Surface natamycin can be extracted from the surface of foods using methanol and the limit of detection for the HPLC assay is 0.5 μg/g. Various other methods have been described, such as ultraviolet spectrophotometry (Capitán-Vallvey et al., 2000) and enzyme immunoassay (Maertlbauer et al., 1990). 3.3.6  Natamycin uses in foods The uses of natamycin as a preservative in foods and beverages are shown in Table 3.8. The main applications are for the surface treatment of cheeses and Table 3.8  Applications of natamycin in foods and beverages, levels, method of addition and supporting references Food application

Natamycin level Method (μg/g)

References

Hard/semi-hard 1250–2000 Surface treatment by Delves-Broughton et al. cheese spray or immersion (2006) 500 Direct addition to De Ruig and van den coating emulsion Berg (1985) Grated cheese 15–20 Surface treatment by Berry (1999) spray or direct addition Meat products: 1250–2000 Surface treatment by Cattaneo et al. (1978), dry sausage spray or immersion Caserio et al. (1974), Delves-Broughton et al. (2006) Yoghurt 5–10 Direct addition to Şahan et al. (2004), yoghurt mix El- Diasty et al. (2009) Bakery products 1250–2000 Surface treatment by Williams et al. (2005) spray Tomato puree/paste 7.5 Direct addition Olives Direct addition Gourama et al. (1998) Fruit juice, malt 2.5–10 Direct addition Shirk and Clark (1963) beverage Wine 30–40 Direct addition to stop Thomas et al. (2005) fermentation 3–10 Added prior to bottling to prevent secondary fermentation

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fermented sausages to prevent the growth of yeasts and moulds that are unsightly and can produce carcinogenic mycotoxins, and these two applications have wide regulatory approval. The three main methods of surface treatment of cheese are by spraying, dipping, or by applying the natamycin in a polyvinyl acetate (PVA) suspension coating. Fermented sausages are prone to mould spoilage during the ripening process as the pH drops and reduces the water holding capacity of the sausages, resulting in a decrease in moisture content and providing ideal conditions for the growth of yeasts and moulds. Use of natamycin for the surface treatment of cheeses and sausages is allowed in the EU and many other countries at a maximum level of 1 mg natamycin/dm2 with a penetration depth of no more than 5 mm. In the USA, natamycin is not approved in meats but is approved in cheese at a maximum level of 20 μg/g, and also in other foods such as non-standardised yoghurt, cottage cheese, sour cream, non-standardised dressing, and marinades and sauces (Delves-Broughton et al., 2005). Other existing or potential applications that have more limited authorisation are use on the surface of baked goods and in fruit juice, malt drinks, and wine. The application in wine is mainly in wines sweetened at the end of fermentation to prevent secondary fermentations from occurring (Thomas et al., 2005). 3.3.7  Safety and tolerance Natamycin was last extensively reviewed in 2003 by JECFA who confirmed that the previously established ADI of 0–0.3 mg/kg body weight was satisfactory and that consumption of treated cheese and meats would not exceed this ADI (www. inchem.org/documents/jecfa/jecmono/v48je06.htm). The EU have not set an ADI, hence use in the EU is restricted to the surface of cheeses and dried fermented sausages. The intravenous route is the path by which polyene macrolide antimicrobials are most toxic and oral administration is less toxic (HamiltonMiller, 1973). There is apparently no adsorption of up to 500 mg/ day natamycin from the human intestinal tract after 7 days administration (Brik, 1981). Laboratory feeding studies to determine the above ADI were carried out by Levinskas et al. (1966) and are summarised by Delves-Broughton et al. (2005). Natamycin is used in the pharmaceutical industry for topical treatment of fungal infections of the eye and ring worm in horses and cattle.

3.4  Undefined fermentates used in food biopreservation The use of spray-dried undefined fermentates produced by GRAS status lactic acid bacteria as culture organisms as a means of food preservation occurred in the USA with the introduction of MicroGARD ® in the late 1980s and early 1990s (Weber and Broich, 1986; Ayres et al., 1987, 1992, 1993). Since the original MicroGARD™ product was introduced various types aimed at specific target organisms have been marketed (Table 3.9). The important difference between these undefined fermentates and nisin and natamycin preparations are that they

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Table 3.9  The MicroGARD® range of undefined microbial fermentates MicroGARD® Composition Target brand microorganism number

Typical Application use level (%)

100 Skim milk, Yeasts, moulds, 0.1–1.5 cultured skim milk Gram negative bacteria 200 Maltodextrin, Yeasts, moulds, 0.1–1.5 cultured dextrose Gram negative bacteria 300 Skim milk, Lactic acid bacteria, 0.3–1.5 cultured skim milk Gram positive spore formers, Listeria 400 Skim milk, Yeasts, moulds, 0.5–1.5 cultured skim milk Gram negative bacteria, lactic acid bacteria 520 maltodextrin, Lactic acid bacteria, 0.25–1.5 Cultured dextrose Gram positive spore formers, Listeria 730 Cultured dextrose, Yeasts, moulds, 0.5–0.75 maltodextrin Gram negative bacteria, lactic acid bacteria, Gram positive spore formers, Listeria CM1–50 Cultured skim milk, Gram positive 0.1–0.5 maltodextrin bacteria CS1–50 Cultured dextrose, Gram positive 0.1–0.5 maltodextrin bacteria

Cottage cheese, sour cream, yoghurt, cultured dairy products, chocolate confections Sauces, dressings, pasta Some flavoured drinks Various dairy products Soups, salad dressings Cooked meat and poultry, refrigerated delicatessen salads

Dairy based products, dressings, prepared meals Non-dairy based products, soups, sauces, dressings, prepared meals

are not purified by downstream processing so can be simply labelled as cultured milk or dextrose powder dependent on the fermentation substrate. As they are undefined their active ingredients are not declared. This in some countries, notably the USA, results in extremely friendly labelling when used in processed foods. They are simply declared as ‘cultured skim milk’ or ‘cultured dextrose’. The EU, however, has decided not to adopt this approach and requires the labelling to declare the active ingredients contained. For this reason undefined fermentates are not used in the EU. Various media can be cultured to produce the optimal concentration of antimicrobial metabolites. Also the media chosen can be similar to the final

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application, such as ‘cultured skim milk’ for the dairy industry, ‘cultured wheat’ for the baking industry and ‘cultured dextrose’ for unrelated foods. The starters used in fermentate production are selected for their antimetabolite producing characteristics and frequently include lactic acid bacteria. Common within this group are the genera Lactobacillus, Pediococcus, Propionibacterium, Leuconostoc and Lactococcus. It should be noted that unpurified fermentates are not as active as purified fermenates such as nisin and natamycin preparations, so therefore end users usually need to use them at levels from 0.1% to as high as 1.5–2%. Any additional ingredient, particularly fermented products such as these, can impart an off-flavour. Antimicrobial activity must be balanced with organoleptic profiles when fermentates are used. 3.4.1  Physical, chemical and antimicrobial properties of fermentates The physical, chemical and antimicrobial properties of microbial fermentates are as diverse as the cultures and media used to generate them. All are invariably combinations of mixed fermentation end products. Some of the most common commercially available fermentates available today, particularly with respect to total usage within the food industries, are based on the metabolites generated from the genera Propionibacterium and Lactococcus with either milk or dextrose used as the base starting media. Organic acids, obviously very common in lactic acid bacteria fermentates, usually contribute significantly to the chemical properties of end products. It is because of this that many of the fermentates are inherently very hygroscopic and will absorb moisture quickly in humid conditions. Consequently, they should always be kept in a cool dry environment. In addition to rather high organic acid composition, there are always a number of known and unknown metabolites usually including, but not limited to, bacteriocins, enzymes, alcohols and small molecules that contribute to the overall physical and antimicrobial characteristics of the fermentate. Fermentates, as their purified counterparts, are generally classified by which class of organism(s) they are designed to control, be they Gram negative bacteria, Gram positive bacteria, yeast and/or moulds. In some instances they can be multifunctional in having the ability to affect the outgrowth of more than one group of organisms. Likewise, blends of fermentates can be made which have a single label declaration (e.g. ‘cultured dextrose’), but provide wide antimicrobial properties. Propionibacteria are used in the manufacture of Swiss cheeses and also in the production of fermentates that are used frequently in the dairy and baking industries. Known as a source of organic acids, propionibacterial fermentates are able to supply these naturally generated, very heat stable antimycotics (Ray and Sandine, 1992). In general, propionibacterial metabolites have very little, if any, activity against Gram positive bacteria but do exert an inhibitory effect on many Gram negative bacteria. The modes of action against the latter are unclear but a number of published reports suggest that propionibacteria are capable of producing a variety of additional antimicrobial compounds against Gram negative

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bacteria (Holo et al., 2002; Van der Merwe, 2004; Grindsted and Barefoot, 1992; Gwiazdowska and Trojanowska, 2006; Ayres et al., 1987; Al-Zoreky et al., 1991). Because of activity against this class of organism, propionibacterial fermentates are widely used within the North American dairy industry to control the outgrowth of common spoilage organisms in fresh, cultured dairy products such as cottage cheese (Weber and Broich,1986; Ayres et al., 1987, 1992, 1993). Lactococci and pediococci form the bases of other commercially available fermentates. These have been formulated to interfere with the outgrowth of Gram positive bacteria. As with the propionibacteria, fermentates from these lactic acid bacteria contain significant amounts of organic acids in addition to small molecules and defined bacteriocins. Each specific fermentate possess its own antimicrobial characteristics. Nevertheless, it should be kept in mind that all fermentates, because they are unpurified, possess antimicrobial activity that cannot be ascribed to a single molecule such as nisin, natamycin, pediocin or sakacin. Rather the activity is due to the cumulative effects of combinations of extracts, organic acids and various proteins and peptides. Assays for specific, single ingredients are invariably misleading as to the total activity present in the product and, consequently in the finished food. 3.4.2  Assay protocols and mode of action It is imperative to reiterate that antimicrobial activity of fermentates cannot be ascribed to a single molecule. Consequently using biochemical analytical analyses (e.g. HPLC, GC, etc.) to determine the concentration of single components invariably generates misleading determinations. Optimal in-vitro inhibition assays are best done measuring total antimicrobial activity in the entire fermentate. On a routine basis, the agar diffusion methods of Tramer and Fowler (1964) and Fowler et al. (1975) are still used today. More recently turbidometric methods of Barreteau et al. (2004) and Turcotte et al. (2004) have been adopted for a more accurate and reproducible estimation of antimicrobial activity of fermentates. Directly comparing in-vitro specific activity to that which would be expected in-situ is a common misconception with inexperienced users (Davidson and Branen, 2005). In-vitro assays are meant to monitor inhibition against specific organisms under precise growth conditions (medium composition, pH, temperature, etc.). In reality, the final results represent the net effects of microbial growth and antimicrobial inhibition. Results must be viewed as a careful balance between the two. Because organic acids and their salts are routinely present in many commercially available fermentates, they invariably play a part in the overall inhibition spectra seen both in-vitro and in the finished foods. The modes of action of each of the organic acids present are unclear, but are commonly thought to be a function of the diffusion of a protonised (or undissociated) form of the molecule into the cell where the internal pH is lowered. In addition other factors may also be involved such as a disruption in active transport, nucleic acid replication and enzyme system integrity (Bogaert and Naidu, 2000).

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The activity spectrum of a fermentate containing multiple organic acids can change dramatically depending on the environmental pH and individual dissociation constants. Understandably, it is extremely difficult to separate the antimicrobial contributions due to the contribution of organic acids mixtures from that of microbially generated bacteriocins. In essence all fermentates are component blends of known compounds together with those molecules we have some evidence do exist, but may be present in minute amounts. 3.4.3  Existing and potential uses in foods Fermentates are used in a wide variety of refrigerated and ready-to-eat, minimally processed foods. In North America, simple propionibacterial based fermentates were first introduced into the dairy industry over 25 years ago to control the outgrowth of Gram negative bacteria, yeast and moulds in products such as cottage cheese, yogurt and sour cream (Salih et al., 1990; Weber and Broich, 1986). Soon afterwards non-dairy versions (‘cultured dextrose’) found their way into products such as refrigerated soups, salad dressings, culinary items such as pasta fillings, prepared meals, side dishes and various cooked meat products. Likewise, propionibacterial fermentates were marketed heavily into the baking industry to ‘naturally’ control mould and rope spoilage. Available MicroGARD ® products (Danisco) are shown in Table 3.9. With the introduction of additional lactic acid based fermentates that target Gram positive bacteria, product applications for fermentate usage were expanded greatly. In addition to controlling spoilage contaminants, label friendly fermentates were also shown to be effective in controlling the outgrowth of certain pathogens such as L. monocytogenes in or on processed meat and poultry. Consequently ‘all purpose’ fermentates have been formulated to include additional ingredients such as rosemary extract, lysozyme and sodium diacetate, which act as antimicrobial potentiators (Bender et al., 2001; Ming et al., 1997). Refrigerated deli salads, various cooked meat and poultry products and prepared meals are typical users of fermentate blends. Manufacturers have an impetus to utilise fermentates as ingredients as they can very often provide an alternative to chemical preservatives, afford a friendly ‘natural’ label declaration, reduce returns and possibly even protect from pathogen outgrowth. Most recently there has been a significant interest in pathogen control in minimally processed foods, and currently Salmonella outbreaks seem to be in the forefront. However there are few ‘natural’ solutions to Gram negative bacteria and coliform control, and available ‘natural’ Gram negative fermentates are static in nature. 3.4.4  Safety and regulatory status The regulatory status of fermentates differs with each country and can vary significantly. Consequently it should be emphasised that regional and local authorities should be consulted prior to considering the use of fermentates as

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antimicrobial hurdles in foods. For example in Canada fermentates are generally regarded as food ingredients. However they may also fall under the Canadian Food Inspection Agency’s (CFIA) definition of a novel food which includes considerations such as its composition, history of safe use, whether it causes the food to undergo a major change or whether it was manufactured using genetically modified organisms (http://www.inspection.gc.ca/english/fssa/fispoi/product/ novbroche.shtml). A number of cultured milk or dextrose fermentates can be used, not as novel foods, but as CFIA approved ingredients. In the EU there are some who feel that the antimicrobial inhibitors present in any fermentate must be identified. However, because of the inherent, complex composition of fermentates, not all of the antimicrobial components can be defined. Consequently, they are currently classified under existing legislation. By default they are most often classified as food additives with their known antimicrobial components associated with preservatives, many of them chemical, bearing an ‘E’ number. As a consequence many countries limit the usage of fermentates to application areas where the preservatives are permitted. In the United States there are a number of factors that must be considered in order for a fermentate to be Generally Recognized As Safe (GRAS) by the Food and Drug Administration (FDA). Currently the most common method of FDA acceptance of a fermentate for food use is through the use of scientific procedures that utilise a panel of experts testifying both that the fermentate ingredients, cultures and their associated by-products have a history of safe consumption and that the naturally produced antimicrobial components have not been selectively purified or concentrated (http://www.fda.gov). The FDA and the United States Department of Agriculture’s (USDA) Food Safety and Inspection Service (FSIS) have recognised a number of fermentates, including cultured skim milk or cultured dextrose, to be acceptable in a variety of foods products including meat and poultry products (http://www. fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasSafeGRAS/ GRASListings/default.htm and http://www.fsis.usda.gov/Regulations_&_Policies/ 7000_Series Processed_Products/index.asp).

3.5  Future trends There continues to be customer demand for minimally processed foods with a long shelf life that contain few, if any, chemical preservatives. At the same time there are also concerns about the high level of salt in our diet, with recommendations being made to reduce our intake. In many instances salt can be a major microbiological hurdle and reducing its level will have microbiological consequences both in terms of product safety and shelf life. Consequently, technology based on non-thermal treatment methods such as high pressure, pulsed electric field technology and active packaging systems will ensure that research and development into novel preservation systems will continue. Likewise, specific antimicrobials such as purified and undefined fermentates as outlined in this chapter added to various non-thermal treatments are likely to play

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an increased role. In this regard, control, regulation and harmonisation by foods safety authorities will be an important factor. The emergence of new microbiological problems and development of resistance will ensure that food technologists, molecular biologists and microbiologists will continue to search for new solutions.

3.6  Sources of further information and advice delves - broughton j

(2008). ‘Use of the natural food preservatives, nisin and natamycin, to reduce detrimental thermal impact on food quality.’ In In-pack Processed Food. Improving Quality, Ed. Richardson, P, Woodhead Publishing Limited, Cambridge, England, pp. 319–337. delves - broughton j , thomas l v, doan c h and davidson p m (2005). ‘Natamycin.’ In Antimicrobials in Food, Eds Davidson P M, Sofos J N and Branen A L (3rd edition). CRC Press, Boca Raton, Florida, pp. 275–288. ray b and daeschel m (1992). Food Biopreservatives of Microbial Origin. CRC Press, Boca Raton, Florida, 386 pp. stark j and tan h s (2003). ‘Natamycin.’ In Food Preservatives, Eds Russell N J and Gould G W, Kluwer Academic, London, pp. 179–195. thomas l v, clarkson m r, and delves - broughton j (2000). ‘Nisin.’ In Natural Food Antimicrobial Systems, Ed. Naidu A S, CRC Press, Boca Raton, Florida, pp. 463–524. weber g , steenson l and delves - broughton j (2008) ‘Antimicrobial Fermentate Technology.’ Proc. II IS of Natural Preservatives in Food, Feed, and Cosmetics, Eds Havkin-Frenkel D et al. Acta. Hort, ISHS, 79–83.

3.7  References and smid e (1983). ‘Nisin in multifactorial food preservation.’ In Natural Antimicrobials for the Minimal Processing of Foods, Ed. Roller S, Woodhead Publishing, Cambridge, England, pp. 11–33. al - zoreky n , ayres j w and sandine w e (1991). ‘Antimicrobial activity of MicroGARD ® against food spoilage and pathogenic organisms’. Journal of Dairy Science 74, 758–763. anonymous (1985). ‘Nisin preservation of chilled desserts’. Dairy Industries International 50, 41–43. anonymous (2008). ‘Cheese and cheese rind-determination of natamycin content – Method by molecular absorption spectrophotometry and by high-performance liquid chromatography’. International Standard ISO 9233. arquès j l , nuňez m , rodríguez e and medina m (2008). ‘Inactivation of Gram-negative pathogens in refrigerated milk by reuterin in combination with nisin or the lactoperoxidase system’. European Food Research and Technology 227, 77–82. aso y, takeda a , sato m , takahashi t , yamamoto t and yoshikiyo , k (2008). ‘Characterization of lactic acid bacteria coexisting with a Nisin Z producer in Tsuda – turnip pickles’. Current Microbiology 57, 89–94. avery s m and buncic s (1997). ‘Antilisterial effects of a sorbate-nisin combination in vitro and on packaged beef at refrigeration temperature’. Journal of Food Protection 60, 1075–1080. ayad e h e , verheul a , wouters j t m and smit g (2002). ‘Antimicrobial-producing wild lactococci isolated from artisanal and non-dairy origins’. International Dairy Journal 12, 145–150. adams m

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and weber g h (1987). ‘Propionates and metabolites of propionibacteria affecting microbial growth’. Canadian Patent No. 1,218,894. ayres j w, sandine w e and weber g h (1992). ‘Preserving foods using metabolites of propionibacteria other than propionic acid’. U.S. Patent No. 5,096,718. ayres j w, sandine w e and weber g h (1993). ‘Propionibacteria metabolites inhibit spoilage yeasts in foods’. U.S. Patent No. 5,260,061. barber r s , braude r and hirsch a (1952). ‘Growth of pigs given skimmed milk soured with nisin-producing streptococci’. Nature 169, 200. barreteau h , mandoukou l , adt i , gaillard b , courtois b and courtois j (2004). ‘Rapid method for determining the antimicrobial activity of novel natural molecules’. Journal of Food Protection 67, 1961–1964. beasley s s and saris p e j (2004). ‘Nisin-producing Lactococcus lactis strains isolated from human milk.’ Applied and Environmental Microbiology 70, 5051–5053. bell r g and de lacy k m (1987). ‘The efficacy of nisin, sorbic acid and monolaurin as preservatives in pasteurized cured meat products.’ Food Microbiology 4, 277–283. bender f g , king w, ming x and weber g (2001). ‘Broad-range antibacterial composition and process of applying to food surfaces.’ U.S. Patent 6,207,210. berry d (1999). ‘Natamycin for shredded cheese.’ Dairy Foods 100, 45. beuchat l r, clavero m r and jaquette c b (1997). ‘Effects of nisin and temperature on survival, growth, and enterotoxin production chracteristics of psychrotrophic Bacillus cereus in beef gravy.’ Applied and Environmental Microbiology 63, 1953–1958. black e p , kelly a l and fitzgerald g f (2005). ‘The combined effect of high pressure and nisin on inactivation of microorganisms in milk.’ Innovative Food Science and Emerging Technologies 6, 286–292. black e p , linton m , mccall r d , fitzgerald g f , kelly a l and patterson m f (2008). ‘The combined effects of high pressure and inactivation of Bacillus spores in milk.’ Journal of Applied Microbiology 105, 75–87. blackburn p , polak j , gusik s and rubino s d (1989). ‘Nisin combinations for use as enhanced, broad range bacteriocins.’ International Patent Application PCT/US89/02625; International Publication WO89/12399 Applied Microbiology, New York. bogaert j - c , and naidu a s (2000). ‘Lactic acid.’ In Natural Food Antimicrobial Systems, Ed. Naidu A S. CRC Press, Boca Raton, Florida, pp. 613–636. bogorditskaya p , scillinger y i and osipova i n (1990). ‘Hygienic study of food products preserved with nisin.’ Gigiena i Sanitariya 35, 37–40. boussoeul n , mathieu f , benoit v, linder m , revol - junelles a - m and millière j b (1999). ‘Response surface methodology, an approach to predict the effects of the lactoperoxidase system, nisin, alone or in combination, on Listeria monocytogenes in skim milk.’ Journal of Applied Microbiology 86, 642–652. boussoeul n , mathieu f , revol - junelles a - m and millière j b (2000). ‘Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC15313 in skim milk.’ International Journal of Food Microbiology 61, 169–175. breukink e , wiedemann i , van kraaij c , kuipers o p , sahl h - g and de kruijff b (1999). ‘Use of the cell wall precursor lipid II by a pore forming peptide antibiotic.’ Science 286, 2361–2364. brik h (1981). ‘Natamycin.’ In Analytical Profiles of Drug Substances, Ed. Flory K. Academic Press, New York, 513 pp. cai y, ng l - k and farber j m (1997). ‘Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts.’ Journal of Applied Microbiology 83, 499–507. calderón - miranda m l , barbosa - cánovas g v and swanson b g (1999a). ‘Inactivation of Listeria innocua in liquid whole egg by pulsed electric fields and nisin.’ International Journal of Food Microbiology 51, 7–17.

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and swanson b g (1999b). ‘Inactivation of Listeria innocua in skim milk by pulsed electric fields and nisin.’ International Journal of Food Microbiology 51, 19–30. calderón - miranda m l , barbosa - cánovas g v and swanson b g (1999c). ‘Transmission electron microscopy of Listeria innocua treated by pulsed electric fields and nisin in skimmed milk.’ International Journal of Food Microbiology 51, 31–38. capitán - vallvey l f , checa - moreno r and navas n (2000). ‘Rapid ultraviolet spectrophotometric and liquid chromatographic methods for the determination of natamycin in lactoserum matrix.’ Journal of AOAC International 83, 802–808. carini s and baldini r (1969). ‘La presenza di Streptococchi produttori di nisina nel latte destinato alla produzione di formaggio grana e sua influenza sulla microflora lattice.’ Annali di Microbiologia ed Enzimologia, xix, 9–17. carlson s and bauer h m (1957). ‘A study of problems associated with resistance to nisin.’ Arch. Hyg. Bakteriol. 141, 445. caserio g , gronchi c , marini c , gennari m , falo g and panizzi a (1974). ‘Recherché Sull’utilizazione della pimaracina nel trattmento superficiale di morttadelle.’ Archivo Veterrinarioo Italiano 25, 155–160. cattaneo p , d ’ aubert s and rigahetti a (1978). ‘Attivita antifungina della pimaracina in salami crudi stagionati.’ Industrie Alimentari 17, 658–664. ceder o (1964). ‘Pimaracin. VI. Complete structure of the antibiotic.’ Acta Chemica Scadinavica 18, 126–134. cheftel j c (1995). ‘Review: high pressure, microbial inactivation and food preservation.’ Food Science and Technology International 1, 75–90. chikindas m , cleveland j , li j and montville t j (2000). ‘Unrelatedness of nisin resistance and antibiotic resistance in Listeria monocytogenes.’ Abstract No: P054. Poster presentation at the 2000 Annual Meeting of the Association for Food Protection, Atlanta. choi h , cheigh c - i , kim s - b and pyun y - r (2000). ‘Production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis 164 isolated from Kimchi.’ Journal of Applied Microbiology 88, 563–571. chung w and hancock r e w (2000). ‘Action of lysozyme and nisin mixtures against lactic acid bacteria.’ International Journal of Food Microbiology 60, 25–32. cleveland j , montville t , nes i g and chikindas m l (2001). ‘Bacteriocins: safe, natural antimicrobials for food preservation.’ International Journal of Food Microbiology 71, 1–20. cleveland j e and tchikindas m l (2001). ‘Inhibition of Escherichia coli 0157:H7 and Listeria monocytogenes Scott A by synergistic action of lactoferrin and nisin. 59E-9.’ Abstract of paper presented at 2001 Institute of Food Technologists Annual Conference. coates m e , harrison g f , kon s k , mann m e and rose c d (1951). ‘Effects of antibiotics and vitamin B12 on the growth of normal and “animal protein factor” deficient chicks.’ Proceedings Biochemical Society, xii–xiii. colak h , hampikyan h , bingol e b and aksu h (2008). ‘The effect of nisin and bovine lactoferrin on the microbiological quality of Turkish-style meatball (Tekirdag köfte).’ Journal of Food Safety 28, 355–375. čurda l , plocková m and sviráková e (1995). ‘Growth of Lactococcus lactis in the presence of nisin evaluated by impedance method.’ Chem. Mikrobiol. Technol. Lebensm. 17, 53–57. cutters c n and siragusa g r (1995). ‘Population reduction of Gram-negative pathogens following treatments with nisin and chelators under various conditions.’ Journal of Food Protection 58, 977–983. daeschel m a , jung d - s and watson b t (1991). ‘Controlling wine malolactic fermentation with nisin and nisin-resistant strains of Leuconostoc oenos.’ Applied Environmental Microbiology 57, 601–603.

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4 The potential of lacticin 3147, enterocin AS-48, lacticin 481, variacin and sakacin P for food biopreservation V. Fallico, O. McAuliffe and R. P. Ross, Teagasc Food Research Centre, Moorepark, Ireland and G. F. Fitzgerald and C. Hill, University College Cork, Ireland

Abstract: In the last few decades, much research has been undertaken to characterize the antimicrobial and preservative qualities of many bacteriocins produced by lactic acid bacteria (LAB). To date, only nisin and pediocin PA-1/AcH have gained wide commercial use as natural food biopreservatives. However, many other bacteriocins also offer promising perspectives in terms of preservation and shelf-life extension of food products. Some of them exhibit narrow-spectrum activity and therefore may be used in applications requiring the selective inhibition of certain food pathogens (i.e. Listeria monocytogenes) without affecting the natural beneficial microflora. Others with broadspectrum activity potentially present wider uses. Additionally, when used in combination with selected hurdles (physico-chemical treatments, antimicrobial agents or peptides), these bacteriocins have proved a highly effective form of preservation and should find commercial application as food preservatives in the near future. Key words: lacticin 3147, enterocin AS-48, lacticin 481, variacin, sakacin P, biopreservatives.

4.1  Introduction Many microrganisms, including lactic acid bacteria (LAB), produce the ribosomally-synthesized peptides known as bacteriocins. These peptides are considered to be natural preservatives and their potential application in the food industry has attracted the interest of both researchers and consumers, in search of foods which are minimally processed, naturally preserved and richer in organoleptic and nutritional properties. Among LAB bacteriocins, only nisin and 100 © Woodhead Publishing Limited, 2011

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pediocin PA-1/AcH are extensively used commercially and use of their powder preparations for food preservation is now largely established. However, other bacteriocins have recently emerged that also hold great potential for biopreservation and shelf-life extension. Some of them exhibit narrow-spectrum activity and therefore may be used in applications requiring the selective inhibition of certain food pathogens (i.e., Listeria monocytogenes) without affecting the natural beneficial microflora. Others with broad-spectrum activity potentially present wider uses. This chapter will review the studies detailing the characterization and biopreservative applications of five of the most promising bacteriocins: lacticin 3147, enterocin AS-48, lacticin 481, variacin, and sakacin P.

4.2  The potential of lacticin 3147 for food biopreservation 4.2.1 History, isolation and generally recognized as safe (GRAS) status of the producing strain Lacticin 3147 is a plasmid-encoded bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147, a strain isolated from an Irish Kefir grain during a screening of natural sources for food-grade producers of antimicrobial compounds (Rea and Cogan, 1994). Other lacticin 3147 producers have been successively isolated such as the strain L. lactis IFPL105 (Martinez-Cuesta et al., 2000). These, and other lactococci, are considered GRAS organisms, since they have been isolated from natural food sources, and, more importantly, because lactococci from dairy products have a long history of use and consumption by humans (Casalta and Montel, 2008). 4.2.2  Characterization, structure and genetics Lacticin 3147 is a heat-stable proteinaceous compound produced during the exponential phase of bacterial growth (Ryan et al., 1996). FPLC purification of the bacteriocin from the supernatant of L. lactis DPC3147 showed that lacticin 3147 is composed of two peptides (LtnA1 and LtnA2) whose synergistic activity is required for full antimicrobial activity (McAuliffe et al., 1998). LtnA1 is a 30-amino acid peptide with a mass of 3,306 Da, whereas LtnA2 is a 29-amino acid peptide with a mass of 2,847 Da. They are encoded as precursor peptides of 59 (LtnA1) and 64 (LtnA2) amino-acids that are subsequently processed to form the biologically active peptides. Maturation of LtnA1 and LtnA2 involves a series of complex post-translational modifications, which includes serine to D-alanine conversion, dehydration of serines and threonines, lanthionine bridge formation, and leader peptide cleavage (Ryan et al., 1999; Morgan et al., 2005). Lacticin 3147 is therefore classified as a member of Class I lantibiotics (‘lanthioninecontaining antibiotic’), a unique group of small (<5 kDa) bacteriocins containing the unusual thioether amino-acids lanthionine (Lan) and β-methyllanthionine (MeLan), which form characteristic intramolecular rings, in addition to a number of dehydrated amino-acids (McAuliffe et al., 2001b). Both lacticin 3147 peptides contain Lan residues (Ryan et al., 1999), but a study of their three-dimensional

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structures by Nuclear Magnetic Resonance (NMR) revealed that the Lan bridging pattern of LtnA1 closely resembles that of the globular type-B lantibiotic mersacidin, whereas LtnA2 has a more elongated structure similar to type-A lantibiotics (Fig. 4.1a) (Martin et al., 2004). Both peptides also contain D-alanine residues that derive from conversion of L-serine in a two-step reaction having dehydroalanine (Dha) as intermediate (Ryan et al., 1999). It has been demonstrated that this post-translational conversion is vital for optimal production and activity of the lantibiotic lacticin 3147 (Cotter et al., 2005). LtnA1 was found to exhibit independent inhibitory activity (MIC 50 = 200 nM), which was greatly enhanced by the presence of LtnA2, whereas LtnA2 on its own possessed no activity (Morgan et al., 2005). The genetic determinants for lacticin 3147 production and immunity are encoded on a 60.2 kb conjugative plasmid, pMRC01, the sequence of which has been fully determined (GenBank accession n. AE001272) (Dougherty et al., 1998). Biosynthesis and immunity to lacticin 3147 is encoded by ten genes organized in two divergently transcribed operons, ltnRIFE and ltnA1 A2 M1TM2 J (Fig. 4.1b), stretching over 12.6 kb (Dougherty et al., 1998; McAuliffe et al., 2000a). The larger operon, ltnA1 A2 M1TM2 J, is responsible for bacteriocin production, modification and export. The ltnA1 and ltnA2 genes encode the precursors that will be processed to give rise to the mature LtnA1 and LtnA2 peptides (Ryan et al., 1999). The products of ltnM1 and ltnM2 act as modification enzymes by catalysing the dehydration and thioether-forming reactions, which result in lanthionine bridge formation. Mutagenesis experiments, where the genes were individually inactivated by frameshift mutations, demonstrated that both modification enzymes are necessary for lacticin 3147 activity, with LtnM1 being required to produce mature LtnA1, and LtnM2 required to produce mature LtnA2 (McAuliffe et al., 2000a). LtnT encodes a putative ABC-transporter implicated in the secretion of lacticin 3147 (Dougherty et al. 1998); it also contains a proteolytic domain which is probably involved in the cleavage of the leader peptides during export (Ryan et al., 1999). Finally, ltnJ encodes a protein sharing significant homology to zinc-containing alcohol dehydrogenases and shown to be responsible for the conversion of Dha to D-alanine (Cotter et al., 2005). Lacticin 3147 immunity is regulated by the second operon, ltnRIFE, divergently located upstream of the biosynthesis operon. LtnE and ltnF encode proteins with significant sequence homologies to multicomponent ABC transporters involved in immunity to staphylococcal and lactococcal lantibiotics; this supported the initial idea that they might play a similar role in lacticin 3147 too (Dougherty et al., 1998). Surprisingly, deletion analysis excluded their involvement in bacteriocin immunity, and indicated instead the product of ltnI as the sole protein responsible for conferring the host with protection to lacticin 3147 (McAuliffe et al., 2000b). Finally, ltnR encodes a 79-residue protein sharing homology with a number of transcriptional repressors of the PBSX (Xre) family that are known to auto-regulate their own production. Interesting observations were made on studying the regulation of lacticin 3147 biosynthesis and immunity. While the promoter controlling lacticin 3147 biosynthesis appears to be constitutive, a stem

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Fig. 4.1  (a) Primary structure of the lacticin 3147 peptides, LtnA1 and LtnA2. Thioether aminoacids are shown in dark (Ala-S-Ala, lanthionine) and light grey (Abu-S-Ala, β-methyllanthionine). Dehydrated amino acids (Dhb, 2,3-didehydrobutyrine) and D-alanine residues are shown in bold and italics (adapted from Wiedemann et al., 2006). (b) Organization of the gene cluster involved in the production of and immunity to lacticin 3147 (adapted from McAuliffe et al., 2001a). (c) Three-step model for the action of the two-peptide lantibiotic lacticin 3147 (I) membrane association of the A1 peptide and binding to lipid II; (II) binding of the A2 peptide to A1:lipid II and formation of a highaffinity three-component complex; (III) translocation of the C-terminus of A2 and formation of a defined pore (adapted from Wiedemann et al., 2006).

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loop structure within the ltnM1 gene acts as a rho-independent attenuator that controls the level of transcription of downstream genes in order to maintain the correct stoichiometry between the structural peptides and the biosynthetic machinery (McAuliffe et al., 2001a). The authors also demonstrated that LtnR regulates producer immunity to lacticin 3147 by binding to a region that overlaps the promoter of the ltnRIFE operon and thus repressing its own transcription and that of the downstream immunity genes, ltnIFE. 4.2.3  Spectrum of inhibition and mode of action The inhibitory spectrum of lacticin 3147 was defined using a panel of 54 indicator strains chosen from a number of gram-positive and gram-negative genera. On agar plate assays, a very broad spectrum of inhibition was observed, closely resembling that of nisin. All gram-positive bacteria tested were inhibited, including the pathogens Listeria, Clostridium, Staphylococcus and Streptococcus species. Moreover, a number of gram-positive strains which exhibited reduced sensitivity to lacticin 3147 in agar plate assays, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), penicillinresistant Pneumococcus (PRP), Propionibacterium acne and Streptococcus mutans, were found to be efficiently killed in broth (Galvin et al., 1999). No activity was detected against gram-negative bacteria (Ryan et al., 1996). The mode of action of lacticin 3147 has been the subject of extensive studies leading to the first detailed analysis of the synergistic activity of a two-peptide bacteriocin at the molecular level (McAuliffe et al., 1998; Martin et al., 2004; Morgan et al., 2005; Wiedemann et al., 2006). In a three-step model proposed by Wiedemann et al. (2006) (Fig. 4.1c), the membrane-bound cell wall precursor, lipid II, is used as a docking molecule for the formation of defined and stable pores. LtnA1 first binds to lipid II and this binding induces a conformational change in LtnA1 which unveils a previously inaccessible binding site for LtnA2. Thus, the LtnA1:lipid II complex is able to recruit LtnA2. Interaction of LtnA2 with LtnA1:lipid II results in a stable three-component complex with high-affinity for the membrane that allows LtnA2 to insert deeper into the membrane assuming a trans-bi-layer conformation and consequently forming a defined pore. According to these authors, the affinity of LtnA1 for lipid II is low and strongly increases after addition of LtnA2. The synergistic action of LtnA1 and LtnA2 is therefore essential for stabilizing the interaction with the target membrane and to provide lacticin 3147 with dual mode of action: pore formation and inhibition of cell wall biosynthesis by sequestration of the precursor. By using planar bi-layer model membranes supplemented with lipid II, the same authors observed that lacticin 3147 was able to form defined pores of a diameter of 0.6 nm. McAuliffe et al. (1998) found these small pores to be selective for potassium ions and inorganic phosphate but not for larger compounds such as amino-acids and ATP; the leakage of ions from sensitive cells is accompanied by a change in electrical charge across the membrane that causes immediate and selective dissipation of the membrane potential (∆Ψ). In a futile attempt to recover these ions by use of ATP-dependent

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uptake systems, the cells rapidly consume the available internal ATP leading to the eventual collapse of the pH gradient (∆pH) and, ultimately, to cell death. 4.2.4  Applications The ability of lacticin 3147 to inhibit a wide range of food pathogens prompted a series of studies to evaluate its preservative potential in a variety of food systems, either in the form of ex-situ produced bacteriocin or of in-situ production by bacteriocinogenic strains. Preparation of a bioactive powdered ingredient containing lacticin 3147 was optimized by growing L. lactis DPC3147 in 10% reconstituted demineralized whey powder at a constant pH of 6.5 and spraydrying conditions that maintained optimal bacteriocin activity (10,240 AU/ml). Initial results were promising, as the lacticin 3147-enriched powder was able to reduce numbers of L. moncytogenes Scott A and Staphylococcus aureus (Morgan et al., 1999). This led to further investigations in more complex food systems. Addition of 10% lacticin 3147 powder to natural yogurt resulted in a 98% reduction of L. monocytogenes Scott A (104 cfu/ml) within 5 min at 30 °C, and no viable cells were detected after 60 min. When the same challenging parameters were tested in cottage cheese, viable cells of Listeria were reduced by 40% within 5 min and by 85% within 120 min at 30 °C. Even greater efficacy was observed in powdered soup contaminated with Bacillus cereus (105 cfu/ml); a 5% bacteriocin powder was sufficient to completely eliminate the pathogen within 1 h, whereas a 1% preparation reduced the bacilli population by 80% within 3 h (Morgan et al., 2001). In minced pork-meat, addition of 1.5% powdered lacticin 3147 decreased the population of Listeria innocua by 50% (Soriano et al., 2004). Moreover, the efficacy of lacticin 3147 powder can be increased in combination with selected hurdles. A doubling in bacteriocin activity was observed following treatment of lacticin 3147 preparations with high hydrostatic pressure (HHP) greater than 400 MPa. The combined use of lacticin 3147 concentrates (1-log reduction) and 250 MPa HHP (2.2-logs reduction) demonstrated greater than an additive effect (6 logs reduction) against S. aureus ATCC6538 and L. innocua DPC1770 in milk and whey (Morgan et al., 2000). Addition of organic acids (sodium citrate or sodium lactate) also enhanced the activity of lacticin 3147 against food-borne pathogens (Salmonella kentucky and Clostridium perfringens) and spoilage bacteria in fresh pork sausage (Scannell et al., 2000b). In situ production by bacteriocinogenic strains represents another means of exploiting the preservative qualities of bacteriocins via incorporation of bacteriocinproducing cultures into foods. This approach harbours several advantages over the addition of concentrated bacteriocin preparations as it lowers the cost of the biopreservation process and, unlike bacteriocin concentrates which may be considered as additives, no regulatory barrier exists since the bacteriocin is not added to the food but produced during its manufacture (Ross et al., 1999; Galvez et al., 2007). Lacticin 3147-producing cultures are very promising for such applications. The presence of the lacticin 3147 genetic machinery on a conjugative plasmid (pMRC01) has allowed the creation of a vast and heterogeneous array of

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commercial starter cultures with improved technological features, namely bacteriocin production and phage resistance, by using a food-grade approach. These specialized starters retained acidification properties sufficient for use in Cheddar cheese manufacture, can generate significant levels of bacteriocin throughout the cheese ripening process, and have proved to be very effective in increasing the safety of fermented dairy foods (Ryan et al., 1996; Coakley et al., 1997; Ross et al., 1999). As protective cultures on the surface of mould-ripened (Ross et al., 2000) and smear-ripened cheeses (O’Sullivan et al., 2006), lacticin 3147-producing cultures were capable of controlling deliberately inoculated L. monocytogenes Scott A. In both cases, spraying the lacticin 3147 producer on the cheese surface resulted in a 1000-fold reduction of the initial pathogen counts (104 cfu/ml). In cottage cheese produced with a lacticin 3147-producing transconjugant (L. lactis DPC4275), a 3-log reduction in counts of L. monocytogenes Scott A (104 cfu/g cheese) was observed after 5 days of storage at 4 °C (McAuliffe et al., 1999). In this case, the protective effect was most likely to be associated with residual lacticin seeded into the cheese as the starter does not survive the heating step in cottage cheese manufacture. L. lactis DPC4275 was also found to significantly reduce the levels of L. innocua and S. aureus in beaker sausage (Scannell et al., 2001), in addition to performing satisfactorily as a single starter for manufacture of salami (Coffey et al., 1998) and fermented sausage (Scannell et al., 2001). In a further step to obtain more stable and long-term production of lacticin 3147 in such systems, L. lactis DPC3147 was efficiently immobilized on double-layered calcium alginate beads. Results showed that bacteriocin production from the immobilized cells remained constant for 180 h, compared to production by free cells which had declined after 80 h (Scannell et al., 2000a). This technique may improve the protective efficacy of lacticin 3147 on food surfaces as the immobilizing substrates act as reservoirs in which bacteriocin molecules are protected from inactivation by food components (Galvez et al., 2007). It is interesting to note that lacticin 3147-producing starters have also been shown to contribute to enhanced cheese quality and aroma while protecting its safety. Cheddar cheese manufactured with L. lactis DPC4275 was shown to contain 100-fold less adventitious nonstarter lactic acid bacteria than control cheese after 6 months of ripening, while maintaining unaltered physicochemical and organoleptic qualities (Ryan et al., 1996; Fenelon et al., 1999; Ryan et al., 2001). In addition, lacticin 3147-producing starters were found to be more susceptible to permeabilization and autolysis (Fallico et al., 2009) and this has been shown to be associated with improved cheese flavour due to increased proteolysis and enhanced amino-acid transamination and α-keto acids formation (Martinez-Cuesta et al., 1998, 2002, 2006).

4.3  The potential of enterocin AS-48 for food biopreservation 4.3.1  History, isolation and GRAS status of the producing strain Enterocin AS-48 (AS-48) is a plasmid-encoded bacteriocin whose activity was first recognized in Enterococcus faecalis subsp. liquefaciens S-48, a strain isolated

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during a screening for the production of bacteriocin-like substances by enterococci (Galvez et al., 1986). A PCR-based study showed that the ability to produce peptides identical or similar to AS-48 is very widespread among enterococci (Joosten et al., 1997). Though some of these variants have been designated differently, e.g. AS-48RJ (Abriouel et al., 2005), enterococcin EFS2 (MaisnierPatin et al., 1996), enterocin 4 (Joosten et al., 1996), and bacteriocin 21 (Tomita et al., 1997), they all share significant homology, if not identity, with AS-48 at the genetic and/or structural level (Maqueda et al., 1998). However, none of the producing strains concerned have been accorded GRAS status, as enterococci are organisms considered at the crossroads of food safety. They have been ascribed beneficial roles such as producing antilisterial bacteriocins, contributing to ripening and flavour development in some artisanal cheeses, and as probiotics to improve the microbial balance of the intestine and for treating gastroenteritis in humans and animals. However, in contrast to this, there is serious concern about the safety of enterococci as a result of their implication in outbreaks of food-borne illness and their recognized role in bacteraemia, endocarditis, and urinary tract infections (Franz et al., 1999). 4.3.2  Characterization, structure and genetics AS-48 is a proteinaceous substance that is extremely thermo-stable (Cobos et al., 2001, 2002). It is active at temperatures of up to 80 °C or below freezing throughout pH values ranging from 3 to 8 (Maqueda et al., 2004). Optimal production of AS-48 may be obtained by growing the producer strain at 37 °C in a complex medium broth supplemented with brain-heart infusion, glucose and magnesium sulphate (Galvez et al., 1986). A simple two-step procedure, consisting of cation exchange followed by reversed phase chromatography, guarantees a recovery of up to 9 mg of highly purified bacteriocin AS-48 directly from a pH-controlled 25-liter culture broth (Galvez et al., 1989a; Abriouel et al., 2003). AS-48 is a 70-amino acid peptide with a mass of 7,149.17 Da and represents the very first example of a cyclic bacteriocin to be described in literature, with the cyclic structure originating from a post-translational ‘tail-to-head’ peptide bond formation (Samyn et al., 1994). AS-48 is a strongly basic peptide (pI close to 10.5) and lacks any cysteine or modified residues, such as Lan or MeLan. AS-48 has therefore been included in the class IId of thermostable non-lantibiotics circular bacteriocins (Nes et al., 2002). NMR determination of the 3-D structure of AS-48 in aqueous solution showed a globular arrangement of five α-helices (α1 to α5) enclosing a compact hydrophobic core (Fig. 4.2a). AS-48 may adopt two different dimeric forms in crystals. The molecules in dimeric form I (DF-I) interact through the hydrophobic helices α1 and α2, suggesting a DF-I for the peptide when in solution. In contrast, dimeric form II (DF-II) involves the interaction of the hydrophilic helices α4 and α5, with the hydrophobic moiety buried within the membrane and the hydrophilic moiety exposed to the solvent. AS-48 may adopt the DF-II structure when inserting itself into the membrane (Sanchez-Barrena et al., 2003).

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Fig. 4.2  (a) Three-dimensional structures of enterocin AS-48, with α-helices represented as cylinders. (b) Organization of the gene cluster involved in the production of and immunity to AS-48. (c) Schematic representation of the mechanism for the molecular function of AS-48. The model includes the approach of DF-I to the membrane and the transition from DF-I to DF-II at the membrane surface. Hydrophobic and polar helices are depicted in light grey and dark grey respectively. The arrow represents the direction of the intrinsic dipolar moment of DF-I (adapted from Maqueda et al., 2004; permission to reproduce material granted by Bentham Science Publishers Ltd).

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The genetic determinants for AS-48 biosynthesis and immunity were first detected on the 68-kb conjugative and pheromone-responsive plasmid pMB2 carried by E. faecalis S-48 (Martinez-Bueno et al., 1990). The gene cluster of AS-48 extends over 10.4 kb of sequence and is composed of ten genes, transcribed in the same direction and organized in two operons, as-48ABCC1DD1 and as-48EFGH (Fig. 4.2b). The bacteriocin structural gene, as-48A, encodes a precursor peptide of 105 amino-acids (Martinez-Bueno et al., 1994) whose 70 C-terminal residues coincide with the mature AS-48 peptide (Samyn et al., 1994). The gene products of as-48B, as-48C1 and as-48D are thought to form a putative multi-component system able to carry out the simultaneous maturation (involving removal of the leader peptide and head-to-tail cyclization) and secretion of AS-48. As-48D1 encodes a small cationic hydrophobic peptide and studies of functional analysis demonstrated that it confers some degree of bacteriocin resistance to the producer (Martinez-Bueno et al., 1998). However, appropriate levels of resistance against AS-48 are reached only when the expression of the as-48D1 immunity gene is combined with that of the as-48EFGH operon, which has been proposed to encode a multi-component ABC system: As-48G would be the ATP-binding domain, As-48E and As48-H the trans-membrane subunits, and As-48F an accessory protein. This second transporter would be mainly responsible for higher levels of producer self-protection against AS-48, whereas the As-48D1 immunity protein would operate as a second resistance mechanism (Diaz et al., 2003). 4.3.3  Spectrum of inhibition and mode of action The inhibitory spectrum of AS-48 is remarkably broad, being highly active against most of gram-positive and some gram-negative bacteria tested (Galvez et al., 1989b). A low concentration (3 and 4 µg/ml) of AS-48 exerted a rapid bactericidal activity followed by gradual bacteriolysis against all species of Bacillus, Streptococcus and Enterococcus tested, and in most Corynebacterium strains. In contrast, 10 µg AS-48/ml induced no bacteriolytic effect in Mycobacterium phlei, M. smegmatis and Nocardia corallina, although these acid-fast actinomycetes were among the most sensitive gram-positive bacteria, and the same occurred in Micrococcus and Staphylococcus species. Clostridium species were also strongly inhibited by AS-48 (Maqueda et al., 2004), but L. monocytogenes emerged as the most sensitive bacterium, with a minimum inhibitory concentration (MIC) of 0.1 µg/ml at 37 °C (Mendoza et al., 1999). AS-48 also inhibits some gramnegative species, but at much higher concentrations. These include Myxococcus strains, Rhizobium, E. coli, Agrobacterium, Salmonella, Shigella, Pseudomonas and Klebsiella. Finally, no effect of AS-48 was detected against the eukaryotic organisms Saccharomyces cerevisiae, Naegleria fowleri and Acanthamoeba, even at concentrations as high as 100 µg/ml (Galvez et al., 1989b), nor against HeLa and MCDK cell lines nor erythrocytes (Maqueda et al., 2004). On the basis of the two dimeric forms found during the crystallographic study (Sanchez-Barrena et al., 2003), a mechanism for the molecular action of AS-48 has been proposed that does not depend upon membrane potential, but implies an

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effective insertion into the membrane consequent to a structural reorganization of a hydrosoluble dimeric form of AS-48 at the membrane surface (Fig. 4.2c). The strong dipolar moment of DF-I in solution is suggested to drive the approach of AS-48 to the membrane. The low pH provided by the membrane interface would destabilize DF-I due to the protonation of the glutamic side chains. This process would, in turn, allow the interaction between the carboxylate side chains and the phospholipid polar heads, and the stabilization of the hydrophobic moiety of AS-48 via the interaction with the phospholipid aliphatic chains. The transition from the water soluble DF-I to the membrane bound DF-II would allow molecules of AS-48 to insert itself into the bacterial membrane via an accumulation of positive charges at the membrane surface that would destabilize the membrane potential leading to pore formation and cell leakage (Maqueda et al., 2004). 4.3.4  Applications Enterocin AS-48 has significant potential as a biopreservative in a large variety of food systems, although its efficacy is noticeably decreased in the food environment compared with laboratory media. This has been attributed to the interaction of AS-48 molecules with food components which may result in a higher retention, or slower diffusion, or irregular distribution of the bacteriocin molecules in the food (Munoz et al., 2007; Galvez et al., 2008). Ex-situ application of a semi-purified preparation of AS-48 (40 µg/g) was shown to reduce viable counts of S. aureus by 5 logs in a meat sausage model system (Ananou et al., 2005c). In the same food system, a concentration of 225 AU/g AS-48 reduced L. monocytogenes counts below the detection level at 3 days of incubation, but did not prevent listeria re-growth after 9 days. Increasing the AS-48 concentration to 450 AU/g resulted in complete kill of listeria over the same incubation time (Ananou et al., 2005b). In yogurt-type soy-based desserts and in gelatin pudding, AS-48 proved more effective against L. monocytogenes with bacteriocin concentrations of 87.5 AU/g sufficient to reduce viable counts below detection levels and avoid regrowth of survivors, whereas a two-fold amount of AS-48 (175 AU/g) reduced viable counts of S. aureus by only 1.8 log units (Martinez-Viedma et al., 2009). AS-48 also proved effective in decontaminating raw and processed vegetables and avoiding listeria proliferation during storage. Application of immersion treatments (5 min at room temperature) with AS-48 solutions (25 µg/ml) reduced L. monocytogenes counts by 2.0 to 2.4 logs in sprouts. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 µg of AS-48/ml, listeria counts were reduced below detection limits in sprouts and green asparagus over 7 days at 15 °C (Molinos et al., 2005). Addition of 30–60 µg/g AS-48 in Russian-type salad significantly reduced L. monocytogenes counts during 1-week storage at 10 °C (Molinos et al., 2009a), whereas, in lettuce juice, AS-48 caused strong inhibition of S. aureus and complete inactivation of L. monocytogenes and B. cereus (Grande et al., 2005b). In tomato paste supplemented with 6 µg AS-48/ml and stored at different temperatures, vegetative cells of Bacillus coagulans were reduced by 2.4 (4 °C),

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4.3 (22 °C) and 3.0 (37 °C) logs within 24 h storage, and no viable cells were detected in any sample after 15-days storage. AS-48 was also very active against the same pathogen in juice from canned pineapple stored at 22 °C, and slightly less active in syrup from canned peaches (Lucas et al., 2006). In apple juice and in commercial apple ciders, 3 µg/ml AS-48 completely inhibited rope-forming B. licheniformis as well as exopolysaccharide- and acrolein-producing LAB (Grande et al., 2006a; Martinez-Viedma et al., 2008a), whereas vegetative cells of Alicyclobacillus acidoterrestris were inactivated by 2.5 µg/ml AS-48 in several types of fruit juices for up to 3 months (Grande et al., 2005a). Interestingly, AS-48 completely eliminated vegetative cells of B. cereus in boiled rice, in a rice gruel, and in a rice-based infant formula dissolved in whole milk where it also prevented the pathogen re-growth for at least 15 days at 37 °C (Grande et al., 2006b). In gelatin and soy pudding, AS-48 (175 AU/g) reduced viable cell counts of B. cereus below detection levels after 8 h at 10 °C or after 48 h at 22 °C (MartinezViedma et al., 2009). When used in combination with other antimicrobial hurdles, AS-48 showed increased bactericidal activity or the ability to enhance the efficacy of the selected hurdle. In skimmed milk, a moderate heat treatment (65 °C for 5 min) and 20 µg AS-48/ml eliminated staphylococci after 6 h of incubation (Munoz et al., 2007). Sub-lethally heat-injured cells of E. coli O157:H7 were inhibited significantly by AS-48 in apple juice, providing a means to lower the intensity of juice processing treatments (Ananou et al., 2005a). AS-48 also significantly increased the heat sensitivity of B. licheniformis and B. coagulans spores in cider reducing the time for complete inactivation of intact spores (Lucas et al., 2006; Grande et al., 2006a). In Russian-type salad, the antilisterial activity of AS-48 (30 µg/g) was strongly enhanced by essential oils, and slightly less in combination with bioactive components from essential oils and plant extracts, with other natural or synthetic antimicrobials (Molinos et al., 2009a). In the same food matrix, AS-48 (30 µg/g) acted synergistically with lactic acid, PHBME and Nisaplin™ in reducing below the detection limit for 7 days a Salmonella enterica cocktail of strains (Molinos et al., 2009b). Food biopreservation via application of bacteriocinogenic strains producing AS-48 in situ has been tested in dairy and meat systems with satisfactory results. In Taleggio cheese, the activity of AS-48, produced by strain E. faecium 7C5 during cheese manufacture, remained stable for at least 40 days (Giraffa et al., 1995). In a non-fat hard cheese, E. faecalis A-48-32 strain produced enough AS-48 to inhibit B. cereus and reduce the cell count of bacilli by 5.6 logs after 30 days of ripening (Munoz et al., 2004), while inhibition of S. aureus proved less effective (Munoz et al., 2007). Noticeably, growth of starter cultures used in cheese making was not affected by the bacteriocin-producing strain. In Manchego and Hispano cheeses, growth of L. monocytogenes was successfully controlled by enterocin 4, a bacteriocin produced by strain E. faecalis INIA 4 and analogue to AS-48 (Nunez et al., 1997). In skimmed milk, AS-48 released by E. faecalis A-48-32 effectively inhibited B. cereus (Munoz et al., 2004) and S. aureus (Munoz et al., 2007) provided that a population of at least 106 enterococci was used as

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inoculum. A 107 cfu/g inoculum of AS-48-producing strains proved also effective in controlling growth of L. monocytogenes (Ananou et al., 2005b) and S. aureus (Ananou et al., 2005c) in a meat sausage model system. The antimicrobial efficacy of in situ produced AS-48 is also enhanced by combination with other hurdles. In apple juice, high-intensity pulsed-electric field (HIPEF) treatment was shown to enhance bactericidal effect of enterocin AS-48 against S. enterica (MartinezViedma et al., 2008b). In raw-milk cheese, the combination of 300 MPa HHP and AS-48-producing adjunct strain was extremely effective in tackling E. coli O157:H7, with results varying according to the time of treatment application (Rodriguez et al., 2005).

4.4  The potential of lacticin 481 for food biopreservation 4.4.1  History, isolation and GRAS status of the producing strain Lacticin 481 is a narrow-spectrum lantibiotic bacteriocin produced by strains of L. lactis (Piard et al., 1990; O’Sullivan et al., 2002a). Also named lactococcin (Dufour et al., 1991) and lactococcin DR (Rince et al., 1994) in some early reports, the bacteriocin was first isolated from L. lactis subsp. lactis CNRZ 481 during a screening for bacteriocin producers (Piard et al., 1990). Considering the GRAS status of L. lactis strains, lacticin 481 may be regarded as food-grade and therefore the use of ex situ or in situ produced lacticin 481 for food preservation do not pose any legislative issues. 4.4.2  Characterization, structure and genetics Optimal purification of lacticin 481 from broth culture was obtained by sequential ammonium sulfate precipitation, gel filtration chromatography, and reversed-phase HPLC leading to a 107,506-fold increase in its specific activity. A preliminary amino-acid analysis of purified lacticin 481 revealed its lantibiotic nature (Piard et al., 1992), while a combination of sequencing of the structural gene, amino-acid analysis, and bidimensional NMR spectroscopy determined the complete aminoacid sequence and the positions of Lan residues (Piard et al., 1993; van den Hooven et al., 1996). Lacticin 481 has a molecular mass of 2,901 Da and is ribosomally synthesized as a prepropeptide containing an N-terminal leader peptide of 24 residues followed by a 27-residue C-terminal propeptide (Fig. 4.3). The mature lacticin 481 contains the unusual amino-acid dehydrobutyrine, two Lan and one MeLan residues. Overlapping thioether bridges in the molecule result in a globular structure at the the C-terminal end, whereas the N-terminal part remains linear. As a result of this conformation, lacticin 481 cannot be strictly defined as a Type A (linear peptides) or a Type B (globular peptides) lantibiotic, though the C-terminal bicyclic ring structure makes lacticin 481 more similar to the globular Type B peptides. A lacticin 481 subgroup was therefore proposed which also includes other structurally distinct lantibiotics such as streptococcin A-FF22, salivaricin A and variacin (Sahl and Bierbaum, 1998; Dufour et al., 2007).

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Fig. 4.3  Sequence comparison of precursor peptides (prepeptides) between lacticin 481 and variacin. Identical residues are shown in bold (adapted from Dufour et al., 2007; permission to reproduce material granted by Wiley-Blackwell).

The genetic determinants for biosynthesis and immunity to lacticin 481 are encoded on a 70-kb plasmid and are organized as an operon, lctAMTFEG, flanked by two insertion sequence elements to form a putative transposon (Dufour et al., 1991, 2000). The first three genes, lctA, lctM, and lctT, are involved in the bacteriocin biosynthesis and export (Rince et al., 1994). LctA is the lacticin 481 structural gene encoding the prepropeptide of 51 amino-acids. The gene product of LctM was shown to interact directly with prelacticin 481, thus suggesting an essential role for LctM in Lan residue formation in lacticin 481 (Rince et al., 1997; Uguen et al., 2000). LctT encodes an ABC transporter, containing an N-terminal protease domain, that has the dual function of cleaving the leader peptide and exporting the mature bacteriocin (Rince et al., 1994); inactivation of this gene resulted in production of a truncated form of lacticin 481 (Uguen et al., 2005). The proteins encoded by lctFEG genes have hydrophobicity profiles and sequence similarities that strongly suggest their association into an ABC transporter protecting the lacticin 481 producer strain from its own lantibiotic. Co-expression of the three genes into a lacticin 481-sensitive L. lactis strain provided the host with immunity to the bacteriocin; deletion of any of the three genes restored strain sensitivity to lacticin 481 (Rince et al., 1997). No regulatory genes have been identified in the lacticin 481 operon suggesting that this lantibiotic may lack a specific regulator (Dufour et al., 2000). The observation that increasing osmolarity stimulates lacticin 481 production suggests that the bacteriocin may be under the influence of global regulation (Uguen et al., 1999). 4.4.3  Spectrum of inhibition and mode of action The inhibitory capacity of lacticin 481 was determined on agar plates using selected strains of lactic acid and food spoilage bacteria. Lacticin 481 exerted a bactericidal effect on all species of Lactococcus, some lactobacilli and leuconostocs. Of particular interest is the sensitivity of Clostridium tyrobutyricum to lacticin 481, as this spoilage organism is responsible for butyric acid formation and late swelling in Emmental-type cheese (Piard et al., 1990). During trials for food preservation, in-situ produced lacticin 481 was also found to control growth of L. monocytogenes (Rodriguez et al., 2001; O’Sullivan et al., 2003b). Few studies have addressed the mode of action of bacteriocins of the lacticin 481 group. Recently, Dufour et al. (2007) postulated that these lantibiotics might

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share a related mode of action due to their close similarity in terms of sequence and structure; their self-protection system is thought to expel bacteriocins from the membrane to the extracellular medium and this supports the idea of a membrane-targeted action. Lacticin 481 was shown to be membrane-active as judged by interaction with artificial lipid monolayers and to have higher affinity for zwitterionic lipids than for anionic lipids (Demel et al., 1996). In another member of the group, nukacin ISK-1, interaction with artificial lipid monolayers and the role of membrane binding in the bacteriocin mechanism of action have been demonstrated. Based on structural similarity, it is conceivable that lacticin 481 acts in a similar manner. However, the N-terminal lysine of lacticin 481 was shown to be not essential for antimicrobial activity (Xie et al., 2004), and even the absence of the five N-terminal residues simply reduced but did not abolish its biological activity (Uguen et al., 2005). These observations suggest that although lantibiotics of the lacticin 481 group are likely to share a similar mechanism by acting at the membrane level, their mode of interaction with membranes is probably different. 4.4.4  Applications A limited number of studies are available in which lacticin 481 has been applied in food systems. In semi-hard raw milk cheese made with the lacticin 481-producing strain L. lactis spp. lactis TAB 24, inhibition of L. monocytogenes growth was observed; the pathogen counts were reduced by 1.6 logs after 8 h from milk contamination (Rodriguez et al., 2001). The efficacy of lacticin 481 antimicrobial activity is enhanced in combination with other hurdle treatments. In raw-milk cheeses artificially inoculated with E. coli O157:H7 (105 cfu/ml), combining HHP at 500 MPa with the addition of a lacticin 481-producing adjunct strain completely eliminated the pathogen in 60-day-old cheeses. When pressurization was reduced at 300 MPa, results varied according to the time of treatment application; a higher reduction in E. coli O157:H7 counts was obtained when the treatment was applied on day 50 (3.8 logs) than if applied on day 2 (1.3 logs) (Rodriguez et al., 2005). The inhibitory effect of lacticin 481 was also shown to work synergistically with that of other bacteriocins. A lactococcal starter derivative producing both lacticins 3147 and 481 reduced the growth rate of L. monocytogenes by almost fourfold and showed an increased antilisterial activity compared to the single bacteriocin-producers (O’Sullivan et al., 2003b). A recent trend is the application of bacteriocins to enhance the release of intracellular enzymes from starter culture cells in order to accelerate ripening and/or improve cheese flavour. Lacticin 481 produced by L. lactis subsp. lactis DPC5552 was shown to increase lysis and release of proteolytic enzymes by starter L. lactis HP without severely compromising its acid-producing capabilities during a cheddar cheese-making trial (O’Sullivan et al., 2002a, 2003a). Similarly, lacticin 481-producing cultures promoted early lysis of Lactobacillus helveticus cells in Hispanico cheese and increased the proteolytic activity (Garde et al., 2006).

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4.5  The potential of variacin for food biopreservation 4.5.1  History, isolation and GRAS status of the producing strain Variacin is a lanthionine-containing bacteriocin produced by Kocuria varians NCC 1482 (formerly referred to as Micrococcus varians). The bacteriocin was identified in two bacterial strains isolated as part of the natural meat flora from laboratory production trials of Italian-type raw salami fermentations. Purification and characterization of the antimicrobial compound produced by both strains revealed the same bacteriocin, though the strains were not identical and had different plasmid profiles. Considering the traditional use of K. varians strains as starters in sausage fermentations, these organisms and/or their metabolites may be applied in food preservation without posing any safety or regulatory issues (Pridmore et al., 1996). 4.5.2  Characterization, structure and genetics Variacin was purified from cell-free supernatants by hydrophobic interaction followed by fast protein liquid chromatography (FPLC) (Pridmore et al., 1996). The bacteriocin is completely inactivated by proteinases pronase E, proteinase K, and ficin, but retains its activity after incubation with catalase. Variacin is resistant to heat (100 °C for 15, 30, and 45 min) and pH values from 2 to 10. Total aminoacid composition of variacin was determined by means of peptide sequencing and mass spectrometry analysis of the FPLC active fractions, returning a peptide molecular weight of 2,658.61 Da. Computer analysis revealed a primary structure sharing significant homology with that of lacticin 481, especially at the amino-terminal sequence, and containing the same number of Lan and MeLan residues. At DNA level, variacin and lacticin 481 share 58.7% overall similarity, basically restricted to the 144-bp bacteriocin-encoding segment. In contrast, different degrees of homology exist at the peptide level (Fig. 4.3). The 22-residue leader peptide of variacin is only 60% identical (75% similarity) to that of lacticin 481 (Pridmore et al., 1996) but it contains the ‘double-glycine’ motif that is typically conserved in bacteriocins of the lacticin 481 group (Piard et al., 1993; Dufour et al., 2007). These observations led to postulate that different processing and transport enzymes may have evolved for a similar bacteriocin in different species (M. varians and L. lactis). In contrast, the 25-residues pro-peptide of variacin is 84% identical (92% similarity) to that of lacticin 481 and shows the conserved residues (three cysteine, two serines and a threonine) potentially involved in the Lan ring formation; mature variacin differs from lacticin 481 for being two amino-acids shorter and having three conservative amino-acid substitutions (Pridmore et al., 1996). No data has been reported to date regarding the immunity genes for variacin; however, the observation that a lacticin 481-producing strain is sensitive to variacin, while natural producers of variacin are both resistant to lacticin 481, suggests that significant differences probably exist between the immunity mechanisms of the two bacteriocins (Pridmore et al., 1996).

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4.5.3  Spectrum of inhibition and mode of action The inhibitory capacity of variacin was determined by the agar well diffusion method against selected strains of lactic acid and food spoilage bacteria. Variacin exhibited a wide spectrum of activity inhibiting all gram-positive bacteria tested, including pathogenic and spoilage organisms such as listeriae, staphylococci, and the vegetative cells and spores of clostridia and bacilli. Common to most known bacteriocins produced by gram-positives, variacin did not exhibit inhibitory activity against gram-negative bacteria (Pridmore et al., 1996). No data is available to date regarding the mode of action of variacin. However, considering its sequence and structural similarity with lacticin 481 (Fig. 4.3), the mechanism of action of variacin most likely resembles that of lacticin 481 (described in paragraph 4.4.3 of this chapter) and related bacteriocins (i.e., nukacin ISK-1) (Dufour et al., 2007). 4.5.4  Applications The biopreservative potential of variacin has only been tested in dairy systems so far (O’Mahony et al., 2001). The authors evaluated the feasibility of applying a spray-dried, fermented preparation of variacin to control the growth of a cocktail of three Bacillus cereus strains in chilled dairy foods. These authors obtained a powder preparation of variacin by growing K. varians in reconstituted skim milk and yeast extract broth at 30 °C for 18 h, and then spray-drying the cell-free fermentate; the amount of active variacin in the fermented milk powder was 3.1 µg/g (20,000 AU/ml). Subsequently, they produced a range of chilled dairy food formulations containing different percentages of fat, carbohydrate and protein to resemble the composition of commercially available products. These dairy models along with two commercial products (chocolate mousse and vanilla dessert), included to validate the experiments, were added with varying amounts of the variacin ingredient and then challenged with the B. cereus spore cocktail over a range of abuse temperatures. Addition of 1% variacin inhibited the growth of B. cereus at 8 °C in all products and models. At 12 °C, addition of 3% variacin was required to inhibit outgrowth of the spores, whereas a 1% concentration was no longer sufficient to exert long-term inhibition of the pathogen cocktail. When stored at 30 °C, the use of 3% variacin inhibited spoilage in only two of eight chilled food models.

4.6  The potential of sakacin P for food biopreservation 4.6.1  History, isolation and GRAS status of the producing strain Sakacin P is a small, class IIa bacteriocin produced by certain strains of the GRAS organism, Lactobacillus sakei. Production of sakacin P was first reported in Lb. sakei LTH 673 (Tichaczek et al., 1992) and subsequently in several other producers (Urso et al., 2006), including Lb. sakei Lb674 where it was initially named sakacin 674 (Holck et al., 1994). Lactobacillus species have a long history

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of safe use as food adjuncts, and some species are also part of the human gut microflora. Their addition to fermented foods is known to improve the storage stability, flavour, and texture of the products and, following food ingestion, they exert several beneficial ‘probiotic’ effects at the intestinal and immune-system levels. Lb. sakei strains are regularly used as starters in salami fermentations and, recently, a patent application proposed their use in pharmaceutical, feed, food, and cosmetic compositions (Park et al., 2009). 4.6.2  Characterization, structure and genetics Maximal production of sakacin P may be obtained by growing the producer strain in a completely defined medium at low temperature without pH control (Moretro et al., 2000). Amino-acid composition analysis and protein sequencing of purified sakacin P revealed a prepeptide of 61 amino-acids containing a typical ‘doubleglycine’ leader peptide (Havarstein et al., 1995), which is enzymatically removed during bacteriocin maturation. Mature sakacin P is a 43 amino-acids peptide containing no unusual amino-acids but an N-terminal YGNGVXC consensus motif, typical of class IIa bacteriocins (Tichaczek et al., 1994; Eijsink et al., 1998). The three-dimensional structure of sakacin P has been determined by NMR spectroscopy. The cationic N-terminal region (residues 1–17) has an S-shaped conformation resembling a three-stranded antiparallel β-sheet and contains four positively charged residues pointing in the same direction. The C-terminal tail (residues 34–43) lacks any apparent common secondary structural motif, but it is able to fold back onto a central amphiphilic α-helix (residues 18–33), thereby creating a hairpin-like structure (Uteng et al., 2003). Biosynthesis and immunity to sakacin P are regulated by seven genes, sppIP, sspK, sspR, sspA, spiA, sppT and sppE, with chromosomal (Tichaczek et al., 1992; Holck et al., 1994) or plasmid (Vaughan et al., 2003) location. Unidirectionally transcribed, these genes are functionally organized in three operons, each one preceded by typical inducible promoters, PsppIP, PsppA, and PsppT. The first operon, sppIPKR, contains a three-component regulatory system coupling sakacin P production to its cell density. SppIP encodes a 19-residue peptide pheromone responsible for inducing bacteriocin production in non-producing strains. SppK and sppR encode products homologous to proteins of bacterial two-component signal transduction systems of the AgrB/AgrA family: the 448-residues SppK is 59% similar to histidine kinase protein of the plantaricin A operon, whereas the 248-residues SppR shows 65–68% similarity to response regulator proteins of the same operon (Huhne et al., 1996; Brurberg et al., 1997). The three-component regulatory system acts as a quorum-sensing device (Nes and Eijsink, 1999); the increase in extracellular pheromone consequent to low cell density is registered by the histidine kinase, which activates the response regulator that in its turn induces the spp promoters. The second operon consists of the sakacin P structural gene (sppA) and a cognate immunity gene (spiA) encoding a protein of 98 aminoacids. Finally, the genes of the third operon are involved in transport and processing of the bacteriocin and pheromone precursors. SppT encodes a 718-residues

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peptide homologous to ATP-dependent transporters of the HlyB family, which are involved in the signal-sequence independent transport of proteins across the bacterial membrane. SppE encodes a 458 amino-acids peptide that probably acts as accessory protein for the SppT ABC-transporter; SppE is indeed homologous to HlyD-like proteins, which are implicated in the signal-sequence independent export of haemolysin A in E. coli. SppT and sppE are not only essential for sakacin P production but also for immunity; the transcription of the immunity gene spiA is indeed regulated by the extracellular pheromone, whose secretion is associated with the activity of SppT and SppE (Huhne et al., 1996; Brurberg et al., 1997). The efficiency of the pheromone-regulated promoter system of sakacin P has been successfully exploited to obtain high-level inducible gene expression in Lb. sakei and Lb. plantarum. Plasmid vectors or gene expression systems based on sakacin P promoters and regulatory genes have been shown to produce some of the highest levels of regulated gene expression reported so far in lactobacilli (Sorvig et al., 2003; Mathiesen et al., 2004a, 2004b). 4.6.3  Spectrum of inhibition and mode of action The antimicrobial activity of sakacin P was found to inhibit the growth of several Gram-positive bacteria, including food pathogen (L. monocytogenes and E. faecalis) and spoilage (Carnobacterium) species (Tichaczek et al., 1992). The mode of action of this and other pediocin-like bacteriocins has not been fully elucidated to date. According to NMR studies, the primary structure of pediocinlike bacteriocins is formed by a hydrophilic, cationic and highly conserved N-terminal region that forms a three-stranded antiparallel β-sheet supported by a conserved disulphide bridge and an amphiphilic α-helix. The less conserved, hydrophobic/amphiphilic C-terminal stretch has instead a rather extended structure that folds back onto the helical part when it inserts into the target-cell membrane (Fimland et al., 1996, 2000; Nes et al., 2002). The N-terminal region is thought to mediate the initial binding of pediocin-like bacteriocins to target cells via electrostatic interactions. Mutational analysis of charged residues in the N-terminus of sakacin P showed indeed reduced binding to target cells and bacteriocin potency (Kazazic et al., 2002; Fimland et al., 2006). A mutagenesis approach also highlighted the essential role played by tryptophan residues in determining membrane interaction and antimicrobial activity in sakacin P (Fimland et al., 2002). The main role of the C-terminal region would be to penetrate into the hydrophobic part of the bacterial membrane, thereby mediating membrane leakage (Fimland et al., 1996, 1998). However, in some pediocin-like bacteriocins (i.e., pediocin PA-1), it also contains a disulfide bridge capable to influence the target-cell specificity. This bridge is missing in sakacin P, but its introduction by site-directed mutagenesis made sakacin P mutants 10 to 20 times more potent than the wild-type towards certain indicator strains (Fimland et al., 2000). Finally, hydrophobic residues located on one side of the C-terminal amphipathic helix were also suggested to be important for receptor recognition and specificity toward particular organisms (Kaur et al., 2004).

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4.6.4  Applications Because of the combination of high anti-listerial activity and a narrow inhibitory spectrum, sakacin P is one of the most promising bacteriocins for preservation of foods in which contamination with listeria is a problem (Eijsink et al., 1998). Addition of purified sakacin P completely eliminated L. monocytogenes in coldsmoked salmon, despite the bacteriocin being subject to proteolytic degradation in salmon tissue (Aasen et al., 2003). Sakacin P alone is ineffective against E. coli but it was shown to enhance synergistically the potency of the fish antimicrobial peptide pleurocidin against the pathogen. A combination of 32 µg/ml sakacin P with 2 µg/ml of pleurocidin resulted in complete inhibition of E. coli growth after incubation for about 18 h (Luders et al., 2003). Effective inhibition of bacterial pathogens in food products was also achieved by using sakacin P-producing lactobacilli. A bacteriocinogenic L. sakei strain significantly decreased listeria, fecal enterococci and total bacterial counts in sausages, in addition to performing satisfactorily as a starter during meat fermentation (Urso et al., 2006). In raw beef, the sakacin P producer Lb. curvatus CWBI-B28 successfully inhibited L. monocytogenes. The same strain proved ineffective against the pathogen in raw chicken meat, but it showed a clear anti-listerial effect when inoculated together with a sakacin G-producing strain (Dortu et al., 2008). In cold-smoked salmon, sakacin P released by L. sakei L6790 only had a bacteriostatic activity on L. monocytogenes; in this case, addition of a sublethal concentration of purified sakacin P in combination with the bacteriocinogenic culture was necessary to obtain partial inactivation of the pathogen (Katla et al., 2001). Similarly, combined use of a sakacin P preparation and a sakacin P-producing Lb. sakei strain resulted in good inhibitory activity of L. monocytogenes in vacuum-packaged chicken cuts over a 4-week period (Katla et al., 2002).

4.7  Future trends In response to the growing demand for safer foods free from chemical additives, research into the application of bacteriocins as biopreservatives in food matrices has been underway for more than 20 years. Since then, numerous bacteriocins have been reported to inhibit spoilage and/or pathogen bacteria in several food systems, but only nisin has been licensed as a food preservative (E234) to date. Currently used in almost 50 countries, nisin is also the only bacteriocin to have received the GRAS status for food in the United States. However, several deficiencies of nisin, such as the instability at neutral to alkaline pH and a spectrum of activity restricted to gram-positive bacteria, call for other bacteriocins to be examined (Settanni and Corsetti, 2008). Some of the bacteriocins described in this chapter, particularly lacticin 3147 and enterocin AS-48, possess qualities which make them ideal candidates as alternatives to nisin. Since the effectiveness of bacteriocins in foods is known to decrease in response to a number of food-related factors (pH, temperature, food composition, structure, microbiota) (Schillinger et al., 1996), a

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single bacteriocin-based technique is unlikely to alleviate the safety issues associated with a large variety of food products. Therefore, the feasibility of these agents as preservatives should be tested on a ‘product by product’ basis using specific bacteriocins for specific tasks. This explains why the use of bacteriocins as part of hurdle technology has received great attention in recent years. Combination of physico-chemical treatments (i.e., heat, HHP, HIPEF), antimicrobial agents (i.e., chelators, essential oils, phenolic compounds) or other bacteriocins with several bacteriocins has been shown to result in additive or synergistic antibacterial effects (Galvez et al., 2007). Ex situ-produced bacteriocins may also be exploited to prepare bioactive food inserts that represent one of the most interesting preservation technologies for foods stored in modified atmosphere packaging (MAP) at refrigeration temperatures. Many of the examples presented in this chapter have shown how the combination of bacteriocins with selected hurdles often results in a more effective form of preservation, providing an additional barrier even to the more refractive forms like gram-negative bacteria endospores. While the search for new bacteriocins, exhibiting different antimicrobial properties, will certainly continue in the near future, one of the most attractive directions of future research on food protection remains the exploitation of bacteriocinogenic cultures or their pure bacteriocins within a hurdle technology programme. In particular, the combination of bacteriocin-activated packaging films and traditional hurdles holds promise for the extension of shelf-life and improvement of microbiological safety of food products.

4.8  Sources of further information and advice A number of public databases are now available for the detection and structural/ functional analysis of bacteriocins during the de novo sequencing of bacterial genomes. BAGEL is a web-based software tool which enables the identification of putative bacteriocin gene clusters in novel DNA sequences using a number of ORF prediction tools and knowledge-based bacteriocin and motif databases (de Jong et al., 2006). A typical BAGEL search on a genome sequence results in a set of putative bacteriocin gene clusters ranked according to the presence of significant features in the amino-acid sequences and their genomic context. BAGEL is freely accessible at: http://bioinformatics.biol.rug.nl/websoftware/bagel. A complementary tool to BAGEL for bacteriocin characterization is BACTIBASE, a web-based database containing the calculated or predicted physicochemical properties of 123 bacteriocins produced by both Gram-positive and Gram-negative bacteria. The information in this database allows rapid prediction of relationships structure/ function and target organisms of these antimicrobial peptides. BACTIBASE is freely accessible at: http://bactibase.pfba-lab-tun.org. Finally, the manually annotated UniProtKB/Swiss-Prot database stores relevant data regarding the majority of sequenced bacteriocins that include mode of action, 3D-structure, posttranslational modification of the precursor protein, and interactions with other proteins, other than cross-references to external databases.

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4.9  References aasen i m , markussen s , moretro t , katla t , axelsson l

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and holzapfel w h (1996). ‘Potential of antagonistic microorganisms and bacteriocins for the biological preservation of foods.’ Trends in Food Science & Technology 71, 58–64. settanni l and corsetti a (2008). ‘Application of bacteriocins in vegetable food biopreservation.’ International Journal of Food Microbiology 121, 123–138. soriano a , ulmer h m , scannell a g m , ross r p , hill c et al. (2004). ‘Control of food spoiling bacteria in cooked meat products with nisin, lacticin 3147, and a lacticin 3147-producing starter culture.’ European Food Research and Technology 219, 6–13. sorvig e , gronqvist s , naterstad k , mathiesen g , eijsink v g h and axelsson l (2003). ‘Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum.’ Fems Microbiology Letters 229, 119–126. tichaczek p s , nissen - meyer j , nes i f , vogel r f and hammes w p (1992). ‘Characterization of the bacteriocins curvacin A from Lactobacillus curvatus LTH1174 and Sakacin P from L sake LTH673.’ Systematic and Applied Microbiology 15, 460–468. tichaczek p s , vogel r f and hammes w p (1994). ‘Cloning and sequencing of sakp encoding sakacin-p, the bacteriocin produced by Lactobacillus sake LTH-673.’ Microbiology-UK 140, 361–367. tomita h , fujimoto s , tanimoto k and ike y (1997). ‘Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1.’ Journal of Bacteriology 179, 7843–7855. uguen p , hamelin j , le pennec j p and blanco c (1999). ‘Influence of osmolarity and the presence of an osmoprotectant on lactococcus lactis growth and bacteriocin production.’ Applied and Environmental Microbiology 65, 291–293. uguen p , hindre t , didelot s , marty c , haras d et al. (2005). ‘Maturation by LctT is required for biosynthesis of full-length lantibiotic lacticin 481.’ Applied and Environmental Microbiology 71, 562–565. uguen p , le pennec j p and dufour a (2000). ‘Lantibiotic biosynthesis: Interactions between prelacticin 481 and its putative modification enzyme, LctM.’ Journal of Bacteriology 182, 5262–5266. urso r, rantsiou k , cantoni c , comi g and cocolin l (2006). ‘Technological characterization of a bacteriocin-producing Lactobacillus sakei and its use in fermented sausages production.’ International Journal of Food Microbiology 110, 232–239. uteng m , hauge h h , markwick p r l , fimland g , mantzilas d et al. (2003). ‘Three-dimensional structure in lipid micelles of the pediocin-like antimicrobial peptide sakacin P and a sakacin P variant that is structurally stabilized by an inserted C-terminal disulfide bridges.’ Biochemistry 42, 11417–11426. van den hooven h w, lagerwerf f m , heerma w, haverkamp j , piard j c et al. (1996). ‘The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges.’ Febs Letters 391, 317–322. vaughan a , eijsink v g and van sinderen d (2003). ‘Functional characterization of a composite bacteriocin locus from malt isolate Lactobacillus sakei 5.’ Applied and Environmental Microbiology 69, 7194–7203. wiedemann i , bottiger t , bonelli r r, wiese a , hagge s o et al. (2006). ‘The mode of action of the lantibiotic lacticin 3147 – a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II.’ Molecular Microbiology 61, 285–296. xie l , miller l m , chatterjee c , averin o , kelleher n l and van der donk w a (2004). ‘Lacticin 481: in vitro reconstitution of lantibiotic synthetase activity.’ Science 303, 679–681.

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5 The potential of reuterin produced by Lactobacillus reuteri as a broad spectrum preservative in food M. Stevens, S. Vollenweider and C. Lacroix, ETH Zurich, Switzerland

Abstract: Reuterin is an antimicrobial compound that consists of hydrated, non-hydrated, and dimeric forms of 3-hydroxypropionaldehyde (3-HPA). It is produced and excreted by the food grade lactic acid bacterium Lactobacillus reuteri and it is thought to play a pivotal role in the persistence and activity of this bacterium in the gastro-intestinal tract and food fermentation. Reuterin can be produced out of glycerol in relatively large amounts, is active against prokaryotic and eukaryotic organisms, and is resistant to proteases and lipases. Therefore reuterin has a high potential as a broad spectrum food preservative. However, before reuterin can be applied as a preservative, additional functionality and toxicology studies have to be performed. Key words: reuterin, Lactobacillus reuteri, 3-hydroxypropionaldehyde.

5.1  Introduction Reuterin is an antimicrobial compound consisting of hydrated, non-hydrated, and dimeric forms of 3-hydroxypropionaldehyde (3-HPA) (Hall & Stern, 1950). In nature 3-HPA is produced out of glycerol in a coenzyme B12-dependent reaction catalysed by the enzyme glycerol dehydratase (Fig. 5.1) (Talarico & Dobrogosz, 1990; Smiley & Sobolov, 1962; Sriramulu et al., 2008). The capability to produce reuterin is relatively uncommon in nature and Lactobacilllus reuteri is the only lactic acid bacterium frequently supplied in foods that is capable of production and excretion of large amounts of reuterin (Talarico & Dobrogosz, 1989). The first patent for the use of the dimer form of reuterin as an antimicrobial compound was filed as early as 1988 (Dobrogosz & Lindgren, 1988). 129 © Woodhead Publishing Limited, 2011

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The first recognition of the compound 3-HPA was in the wine production process. During wine fermentation the formation of small amounts of glycerol is desired because glycerol contributes to the sweetness and fullness of the wine. Afterwards, during wine ripening lactic acid bacteria (LAB) contribute to wine flavour by degrading malic acid to the less acidic lactic acid. However, the capability of some lactobacilli to convert glycerol to 3-HPA (reuterin) can have an undesired side-effect; the acidic conditions in wine enable the conversion of 3-HPA to acrolein (Fig. 5.2), which in combination with polyphenols causes a bitter tasting of the wine (Lonvaud-Funel, 2002). Although undesired in wine, reuterin is of industrial interest because it exhibits strong anti-microbial activity against a large range of micro-organisms including Gram-positive and Gram-negative bacteria, yeasts, moulds, and protozoa (reviewed by Vollenweider & Lacroix, 2004). Furthermore, 3-HPA is a precursor for the production of chemicals such as acrylic acid and 3-hydroxypropionic acid (Vollenweider & Lacroix, 2004). Reuterin produced by food-grade lactic acid bacteria is a potential broad spectrum preservative for the (food) industry, but until now it has not been applied in foods. This chapter focuses on reuterin as antimicrobial compound, on reuterin production, and on its potential and limits as broad spectrum food preservative. Additionally, the modes of action of reuterin and its in situ role are briefly addressed. L. reuteri, a lactic acid bacterium producing reuterin is generally regarded as safe and has been used for over 20 years as a probiotic bacterium (Casas & Dobrogosz, 2000). Therefore a major part of this chapter addresses the potential of this bacterium in food fermentations, its application as reuterin producer, and its probiotic properties.

5.2 Lactobacillus reuteri, a probiotic bacterium with intestinal activity 5.2.1  Origin and characteristics In 1980 Kandler et al. proposed to classify the Lactobacillus fermentum biotype II strains identified by Reuter and Lerche (Lerche & Reuter, 1965; Reuter, 1965) as the new species Lactobacillus reuteri (Kandler et al., 1980). L. reuteri is a heterofermentative lactic acid bacterium (LAB) found in a variety of ecological niches like food fermentations (e.g. sourdough, meat, and dairy products (Vogel et al., 1994; Lerche & Reuter, 1965; Reuter, 1965)) and the gastro-intestinal (GI) as well as the urogenital tract of humans and other animals (Lindgren & Dobrogosz, 1990; Molin et al., 1992b; Naito et al., 1995; Rodriguez et al., 2003; Jin et al., 2007; Van Coillie et al., 2007). The animal GI-tract appears to be the species main habitat (Casas & Dobrogosz, 2000) and because L. reuteri was found to be the most predominant Lactobacillus in the intestine of a large number of animals, including 40 human individuals, it has been suggested that L. reuteri is the ‘universal enterolactobacillus’ (Mitsuoka, 1992; Fujisawa et al., 1996; Casas & Dobrogosz, 2000). © Woodhead Publishing Limited, 2011

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The species L. reuteri and L. fermentum cannot be distinguished solely on physiological properties and additional identification using molecular tools is necessary. Nowadays L. reuteri is frequently identified using phenotypic profiles, genetic profiles, and its ability to produce reuterin out of glycerol (Casas & Dobrogosz, 2000). However, the identification of other lactobacilli species that produce reuterin (Sauvageot et al. 2002; Tanaka et al., 2009) and of L. reuteri strains unable to produce reuterin (Cadieux et al., 2008) complicates correct classification of isolates. The use of L. reuteri as a food supplement is accepted and consumption of 10exp11 living L. reuteri cells per day is generally regarded as safe, even in immune deficient individuals (Wolf et al., 1995, 1998). L. reuteri is capable of fermenting a number of carbohydrates including pre-biotic dietary fibres like dextrin and inulin (Stewart et al., 2008). Additionally, many strains produce exopolysaccharides, for example the highly branched glucan reuteran (Kralj et al., 2002), which have potential use as food thickeners in the food industry (Arskold et al., 2007). L. reuteri is frequently found in sourdough fermentation (Vogel et al., 1994; De Vuyst & Vancanneyt, 2007) and can be used in sourdough starter culture (Gerez et al., 2008). The species can also be used to ferment fruit juices, and high levels of folate and vitamin B12 are present in L. reuteri fermented melon juice (Santos et al., 2008b). An interesting feature of L. reuteri is its capability to produce vitamin B12 (Taranto et al., 2003). This is a rare feature among lactic acid bacteria, the only other species in this group reported to produce vitamin B12 being Lactobacillus coryniformis (Martin et al., 2005). However, the reported vitamin levels in the latter species are 700 times lower compared to L. reuteri under comparable conditions and there is no genetic evidence for vitamin B12-synthesis (Santos, 2008). The bulk form of vitamin B12 produced by L. reuteri differs slightly from cobalamin, the best studied form of vitamin B12, and was therefore designated pseudovitamin B12 (Santos et al., 2007). Nevertheless, the pseudovitamin could fulfil auxotrophic B12 requirements in bacterial indicator strains and prevented vitamin B12 deficiency in mice (Taranto et al., 2003; Molina et al., 2009), indicating that the produced form is biologically active. Even in the post-genomic era L. reuteri is the only LAB in which a vitamin B12 biosynthesis gene cluster is found so far. Interestingly, another gene cluster in L. reuteri relatively unique for LAB is the propanediol utilization (pdu) operon, an operon that encodes, among others, the function to produce the antimicrobial compound reuterin. As mentioned above, reuterin is produced out of glycerol in a vitamin B12 dependent step (Fig. 5.1). The pdu- and the vitamin B12 (cob)-operon in L. reuteri form a genomic island (a cluster of genes that is not found in closely related organisms and has divergent features compared to other gene clusters on the chromosome) and the genetic and phenotypic linkage of the two features (Fig. 5.3) suggests a common evolutionary origin (Morita et al., 2008; Sriramulu et al., 2008). Genomic islands are often involved in pathogenesis or symbiosis and are believed to be significant in the evolution of a bacterium in a specific niche. Similarly, the genomic island in L. reuteri is proposed to be important in the evolution of health promoting strains in the human gut (Morita et al., 2008).

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Fig. 5.1  Conversion of glycerol to 3-HPA and further to 1,3-propanediol. The first step is catalysed by glycerol dehydratase and is coenzyme B12 dependent. The second is catalysed by a 1,3-propanediol dehydrogenase at the expense of one NADH.

5.2.2  Probiotic activity Like many species from the genus Lactobacillus, L. reuteri has probiotic properties. Furthermore, it is one of the most studied probiotic bacteria, and research is still blooming (exemplified by the fact more than 50% of all papers on L. reuteri in the literature database PubMed were published in the last five years). Probiotics are live-microorganisms which when administered in adequate amounts confer health benefits to the host (FAO/WHO, 2002). Probiotic bacteria must be safe for the host, genetically stable, and be able to reside in the GI-tract (Goossens et al., 2003). L. reuteri is a commensal bacterium that can survive and persist in mammalian GI-tract and there are numerous reports about its beneficial effects on the host (as presented below). Furthermore, the genetic accessibility of lactobacilli is relatively high compared to the main other probiotic genus, Bifidobacterium, and the rising number of complete sequenced genomes, the availability and development of genetic tools, and the reconstruction and modelling of metabolic pathways enables research to confirm phenotypic characteristics and biological traits (Fang & O’Toole, 2009). The main beneficial effects of probiotic bacteria include modulation of the composition of the intestinal flora, adherence to the mucosa, competition with pathogenic microbes, modulation of enzymatic activities in the colon, stimulation of the immune system, and production of short chain fatty acids (SCFA) and other metabolic products (Fuller, 1991; Holzapfel et al., 2001). L. reuteri is able to survive and persist in the human GI-tract, the first property of a probiotic species. A number of L. reuteri isolates can survive low pH (3.0) and subsequent bile salt treatment, showing that L. reuteri can survive stomach and intestine passing (Rodriguez et al., 2003). It is even able to grow in physiologically relevant concentrations of bile (0.5%) and a number of genes involved in bile survival have been identified (Whitehead et al., 2008). Interaction of probiotic bacteria with mucosa cells via adhesion is considered to be an important factor in health-promoting effects (Mattila-Sandholm et al., 1999). It was shown that L. reuteri can interact with and adhere to mucosa cells (Valeur et al., 2004) and adhesion has been linked to the presence of a 29 kDa protein at the cell surface (Wang et al., 2008). Activity of L. reuteri in the GI-tract prevents allergic responses and its oral consumption might even prevent atopic responses such as (asthmatic) allergic airway responses (Forsythe

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et al., 2007). Additionally, consumption of L. reuteri by mothers resulted in decreased levels of the transforming growth factor TGF-β2 in breast milk which is associated with low sensitization and less eczema in breast-fed infants (Böttcher et al., 2008). Apart from preventing allergic responses, L. reuteri reduces visceral pains (Kamiya et al., 2006), probably by enhancing the excitability of colonic sensory neurons by decreasing Ca2+/K+-channel openings (Kunze et al., 2009). A number of cases have been reported on the effect of L. reuteri against pathogenic microorganisms and inflammation. It is capable of competing with Streptococcus mutants in oral cavity thereby preventing caries (Caglar et al., 2006). Additionally, a reduction of pro-inflammatory cytokines in gingival crevicular fluid is triggered by L. reuteri (provided in chewing gum) leading to prevention of oral cavity inflammation (Twetman et al., 2009). Another competitive property of L. reuteri is its lowering effect on the enterobacteriaceae population in the GI-tract of pigs (De Angelis et al., 2007) and in a colonic fermentation model (Cleusix et al., 2008). L. reuteri was also shown to inhibit Helicobacter pylori in the human stomach, thus providing new opportunities in the treatment of chronic stomach inflammation (Francavilla et al., 2008). Oral administered L. reuteri in combination with L. rhamnosus enhanced vaginal flora quality and was effective against bacterial vaginosis (Petricevic et al., 2008, Anukam et al., 2006). Other anti-inflammatory properties of L. reuteri include prevention of rat colitis in model studies (Peran et al., 2007), reducing the incidence and severity of virus-type diarrhoea (Shornikova et al., 1997b), reducing the duration of acute gastroenteritis of 0.5 to 3-year-old children (Shornikova et al., 1997a), prevention of infantile colic (Savino et al., 2007), inhibition of bacterial translocation from the gut in acute liver failure rats (Molin et al., 1992a), and formation of an anti-inflammatory environment in the peripheral blood of inflammatory bowel disease patients (Lorea Baroja et al., 2007; Schreiber et al., 2009). The anti-inflammatory effect of L. reuteri is thought to be due to stimulation of the anti-inflammatory nerve growth factor (NGF) and to inhibition of nuclear translocation of NF-κB, the key player in regulation of the immune response to infection (Ma et al., 2004). Additionally, L. reuteri secretes factors that stimulate apoptosis of myeloid cells, which potentially prevents colonic cancer and inflammatory bowel disease (Iyer et al., 2008). A remarkable study was performed with healthy employees of the Swedish Tetrapack company, where daily administration of L. reuteri reduced the employees’ sick-days caused by respiratory or gastrointestinal diseases by more than 50%, when translated to the complete workforce in Sweden, this would lead to an improved productivity of 4.3 million working days per year (Tubelius et al., 2005). The capability of L. reuteri to ferment a whole range of different carbon sources, its widely described probiotic properties, its capability to produce vitamin B12 plus a whole range of other favourable compounds, and its safe status makes it a promising organism in the food industry. Furthermore, L. reuteri has the capability to produce reuterin, a compound with numerous potential

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applications in the food, medical, and chemical industries (Vollenweider & Lacroix, 2004).

5.3  The reuterin-HPA system 5.3.1  Synthesis of reuterin The capability of L. reuteri to produce vitamin B12 seems directly related to the capability of this bacterium to produce and excrete reuterin (Talarico et al., 1988; Morita et al., 2008). At first glance cellular production of reuterin is a relative simple biological process; glycerol is converted to 3-HPA, which subsequently can be converted to 1,3-propanediol at the expense of one NADH (Fig. 5.1). In fact it is much more complicated (see Fig. 5.3). The enzyme involved in glycerol conversion, the glycerol dehydratase, is encoded by three genes within the same operon, the pduoperon, as so-called microcompartment proteins. These microcompartment proteins build intracellular structures called metabolosomes that contain the glycerol dehydratase enzyme, indicating that glycerol to 3-HPA conversion occurs in these structures (Sriramulu et al., 2008). The aldehyde 3-HPA reacts with sulfhydryl groups and probably disturbs the redox balance of the cell (Vollenweider et al., in prep) and the function of the metabolosomes seems therefore to provide a separated reaction chamber inside the cell to protect proteins and DNA in the cytosol against reactive molecules (Sampson & Bobik, 2008). The pdu-operon encoding the microcompartment proteins and the glycerol dehydratase consists of 21 genes including all genes necessary for diol and glycerol utilization. Additional proteins involved in the production of 3-HPA include the enzymes involved in vitamin B12synthesis and specific transporters for glycerol and cobalt (Fig. 5.3a) and intriguingly the genes encoding these proteins are found on the same region of the chromosome (Fig. 5.3b). The production of the anti-bacterial compound reuterin gives L. reuteri a competitive advantage in its ecological niches, such as the mammalian GI-tract. Apart from the competitive advantage it also allows the bacterium to close its NADH-NAD balance by converting one 3-HPA further to one 1,3-propanediol (1,3-PDO) at the expense of one NADH (Fig. 5.3a). Glucose grown L. reuteri cells are limited in their energy production due to an NADH-NAD imbalance (Arskold et al., 2008) and addition of glycerol leads to increased biomass production and growth, indicating more efficient energy production and suggesting that glycerol to 1,3-PDO conversion indeed is used to restore the redox balance in the cell (Santos, 2008). The role of reuterin in the ecology of the GI-tract has not yet been fully understood. It has been shown that L. reuteri produces small amounts of reuterin in the GI-tract (Morita et al., 2008), suggesting reuterin is indeed a competitive compound in situ. Furthermore, addition of glycerol alone or of glycerol plus live L. reuteri cells to a colonic fermentation decreased the number of E. coli counts and increased the counts of the Lactobacillus–Enterococcus group, an effect likely due to in situ production of reuterin (Cleusix et al., 2008). These studies

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suggest that reuterin has a role in GI-tract maintenance and probiotic properties of L. reuteri. 5.3.2  Composition of the reuterin system The antimicrobial activity of reuterin is well-established (see below), but the exact mode of action is still not clarified. The reuterin system consists of a number of molecules (Fig. 5.2) and the question of which molecule is most active as an anti-microbial might already give a lead to identify the modes of action. NMRanalysis revealed the composition of the reuterin system at different 3-HPAconcentrations: above 1M the dimeric form was most abundant; whereas at lower, physiological concentrations of approximately 30 mM the hydrated (69%) and non-hydrated (27%) molecules were predominant (Vollenweider et al., 2003). The anti-microbial activity of reuterin occurs at concentrations below 30 mM (Cleusix et al., 2007) and therefore the hydrated and aldehyde form are most likely the active molecules of HPA. In some studies in which the anti-microbial activity of 3-HPA was established, molar concentrations were calculated using the molecular mass of the dimeric form (Dobrogosz et al., 1989; Yunmbam & Roberts, 1992). However, if 3-HPA occurs mainly in its monomeric form at applied concentrations, the concentrations in these studies were actually twice as high as those reported. The composition of the reuterin system is pH-dependent (Fig. 5.2). In a basic environment, mainly monomer and aldol dimers and trimers are formed, whereas under acidic conditions the monomer, the hydrated monomer, the cyclic dimer, and acetal forms are found (Chen et al., 2002; Sung et al., 2003). The dimeric and polymeric forms seem to be predominant in organic solutions where H2O is absent (Talarico & Dobrogosz, 1989; Vollenweider et al., 2003). With exception of the hydrated form, all forms still contain aldehyde groups that react with amino groups in biological tissues (Sung et al., 2003). However, it is assumable that the highly dynamic nature of the reuterin system affects its biological activity under different circumstances. 5.3.3  Modes of action Reuterin inhibits the substrate binding subunit B1 of ribonucleotide reductase, an enzyme that catalyses the reduction of ribonucleotides to the DNA building blocks deoxyribonucleotides and is essential for growth (Dobrogosz & Lindgren, 1988). The first proposed structure for reuterin was a carbohydrate-like structure and a three-dimensional molecular model of the hemiacetal revealed an analogue of D-ribose (Dobrogosz & Lindgren, 1988). Therefore it was postulated that reuterin acts as an inhibitor by competing for the ribose recognition site of ribonucleotide reductase. However, this hypothesis does not explain the observed inhibition of thioredoxin by reuterin (Dobrogosz et al., 1989) and it contradicts with the NMR-analysis on the composition of the reuterin system, which revealed predominantly monomers in physiological concentrations (Vollenweider et al.,

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Fig. 5.2  Different forms of 3-HPA under acidic and basic conditions as described by Sung et al. (2003) and Vollenweider et al. (2003).

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Fig. 5.3  Reuterin synthesis in L. reuteri. (a) Pathways involved in reuterin production; (b) genetic organization of genes.

2003). Therefore another hypothesis stating that the aldehyde group of 3-HPA reacts with unstable sulfhydryl groups of ribonucleotide reductases and thioredoxin seems a more plausible explanation (Schauenstein et al., 1977; Dobrogosz & Lindgren, 1988). Indeed it was shown that reuterin reacts with sulfhydryl groups of amino acids, eventually leading to a disturbance of the redox status of the cell and to activation of the oxidative stress response in E. coli (Schäfer et al. 2010; Vollenweider et al., in press). 5.3.4  Activity of reuterin in situ One of the first questions addressed in L. reuteri and reuterin research is the function of reuterin in the ecological role of L. reuteri in the GI-tract. A possible link between reuterin production and probiotic activity would provide a strong

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tool for manipulation of the intestinal microbiota composition. Reuterin is synthesized by L. reuteri cells residing in the GI-tract (Morita et al., 2008), suggesting a role for reuterin in providing a selective advantage for L. reuteri. However, as already mentioned before, L. reuteri has a number of probiotic properties and some of them are clearly not linked to reuterin production; for example adherence of L. reuteri to enterocytes is unlikely to be due to reuterin production. The easiest way to evaluate the contribution of reuterin to the probiotic properties of L. reuteri is by studying strains that are unable to synthesize reuterin, a number of which have been reported (McCoy & Gilliland, 2007), although strain 100–103 (Tannock & Savage, 1985) and strain RC-14 are the only ones reported so far in which the reuterin genes (pdu-operon) are absent (Tannock, 2007; Cadieux et al., 2008). The absence of reuterin production in some strains raises the question whether a subspecies should be classified within the species L. reuteri. The fact that strain RC-14 has been shown to confer beneficial health effects in both the GI-tract and the vagina (Reid et al., 2001; Anukam et al., 2008), indicates no direct relation between reuterin and probiotic activity (Cadieux et al., 2008). Additionally, reuterin production occurs only in few lactobacilli, whereas many species in this genus have health promoting effects, apparently confirming the minor role of reuterin in situ. However, it cannot be excluded that strains that do produce reuterin in the GI-tract benefit from this activity and that reuterin does play an important role for these strains in their functionality and maintenance.

5.4  Antimicrobial activity of reuterin Reuterin has potential as a food protective agent against microbial spoilage. One of the first reports about the antimicrobial properties of reuterin describes an effect of a not yet identified molecule against a whole range of microorganisms, including Escherichia, Salmonella, Shigella, Proteus, Pseudomonas, Clostridium, Bacillus, and Staphylococcus species (Axelsson et al., 1989). Also species belonging to the group of LAB (Streptococcus, Pediococcus, Leuconostoc, and Lactobacillus) were affected, but to a fourfold less extent compared to other bacteria (Axelsson et al., 1989). Because the inhibiting molecule was not yet chemically characterized, the activity in this study was based on dilutions of culture supernatants of L. reuteri grown in the presence of glycerol. Several studies on the activity of reuterin do not provide exact effective concentrations but report activity units, whereas other studies provide concentrations calculated for the dimer form. Furthermore, the test-conditions vary between studies in parameters like growth phase, growth medium, temperature, and purity of the reuterin and comparison of data from different studies is therefore arbitrary. Here a review of studies on reuterin activity is presented and when possible the concentration was recalculated based on the concentration of the aldehydic monomer (see Table 5.1, 5.2, and 5.3).

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Since the first study, a broad range of organisms has been reported to be sensitive to reuterin including Gram-positives, Gram-negatives, yeasts, and protozoa. Reuterin has not only an inhibiting effect on microbial growth but also a bactericidal effect at higher concentration, allowing disinfection beside preservation as, for example, in biological tissues fixation (Chen et al., 2002). 5.4.1  Activity against Gram-positive bacteria Reuterin is active against a range of Gram-positive bacteria (Table 5.1). Dominant species in the GI-tract, such as genus of Bifidobacterium, Enterococcus, and Eubacterium, displayed minimal inhibitory concentrations (MIC) of less than 7.5 mM (Cleusix et al., 2007). The minimal bactericidal concentration (MBC) of these species was just slightly higher, i.e. 15 mM. The species Listeria innocua and Listeria ivanovii were affected at similar concentrations, indicating a potential use of reuterin against the food-borne pathogen Listeria monocytogenes.

Table 5.1  Activity of reuterin against Gram-positive bacteria Strain

Concentrationa

Reference

Bifidobacterium catenulatum

LMG 11043

MIC: 1.9–3.8 mM; MBC: 7.5–15.0 mM

Cleusix et al., 2007

Bifidobacterium longum

DSM 20219

MIC: 1.9–3.8 mM; MBC: 3.8–7.5 mM

Cleusix et al., 2007

Bifidobacterium longum infantis

DSM 20088

MIC: 1.9–3.8 mM; MBC: 1.9–3.8 mM

Cleusix et al., 2007

Bifidobacterium adolescentis

DSM 20083

MIC: 3.8–7.5 mM

Cleusix et al., 2007

Bifidobacterium bifidum

DSM 20456

MIC: 3.8–7.5 mM; MBC: 7.5–15.0 mM

Cleusix et al., 2007

Bifidobacterium breve

DSM 20213

MIC: 7.5–15 mM; MBC: 7.5–15.0 mM

Cleusix et al., 2007

Lactobacillus acidophilus

ATCC 4356

MIC: 15.0–40.0 mM; MBC: 15.0–40.0 mM

Cleusix et al., 2007

Lactobacillus casei

ATCC 334

MIC: 15.0–40.0 mM; MBC: 40.0–80.0 mM

Cleusix et al., 2007

Lactobacillus fermentum

ETH

MIC: 15.0–40.0 mM; MBC: 15.0–40.0 mM

Cleusix et al., 2007

Organism In vitro activity

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Table 5.1  Continued Organism

Strain

Concentrationa

Reference

Lactobacillus salivarius

ETH

MIC: 15.0–40.0 mM; MBC: 40.0–80.0 mM

Cleusix et al., 2007

Lactobacillus reuteri

DSM 20016

MIC: 30.0–50.0 Cleusix et al., 2007 mM; MBC: 60–120 mM

Lactobacillus reuteri

ATCC 55730

MIC: 30.0–50.0 Cleusix et al., 2007 mM; MBC: 60–120 mM

Lactobacillus acidophilus

Six strains

MIC: 2–5 AU b (0.2–0.5 mM)

Axelsson et al., 1989

Lactobacillus plantarum

Two strains

MIC: 5 AU b (0.5 mM)

Axelsson et al., 1989

Pediococcus cerevisiae

–c

MIC: 5 AU b (0.5 mM)

Axelsson et al., 1989

Leuconostoc mesenteroides

–c

MIC: 4 AU b (0.4 mM)

Axelsson et al., 1989

Enterococcus faecium

DSM 20477

MIC: 3.8–7.5 mM; MBC: 30.0–50.0 mM

Cleusix et al., 2007

Eubacterium biforme DSM 3989

MIC: 1.9–3.8 mM; MBC: 3.8–7.5 mM

Cleusix et al., 2007

Eubacterium eligens

DSM 3376

MIC: 1.9–3.8 mM; MBC: 1.9–3.8 mM

Cleusix et al., 2007

Colinsella aerofaciens

DSM 3979

MIC: 3.8–7.5 mM; MBC: 7.5–15.0 mM

Cleusix et al., 2007

Streptococcus salivarius

DSM 20560

MIC: 3.8–7.5 mM; MBC: 15.0–30.0 mM

Cleusix et al., 2007

Streptococcus lactis

Two strains

MIC: 5 AU b (0.5 mM)

Axelsson et al., 1989

Streptococcus cremoris

–c

–c; very sensitive

Dobrogosz & Lindgren, 1994

Clostridium difficile

ETH

MIC: < 1.9 mM; MBC: 3.8–7.5 mM

Cleusix et al., 2007

Clostridium clostridioforme

DSM 933

MIC: 15.0–30.0 mM; MBC: 15.0–30.0 mM

Cleusix et al., 2007

Clostridium sporogenes

–c

–c; sensitive

Dobrogosz & Lindgren, 1994

Ruminococcus productus

DSM 2950

MIC: 7.5–15.0 Cleusix et al., 2007 mM; MBC: 3.8–7.5 mM

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Table 5.1  Continued Organism

Strain

Concentrationa

Reference

Listeria innocua

HPB 13

MIC: 7.5–15.0 mM; MBC: 7.5–15.0 mM

Cleusix et al., 2007

Listeria ivanovii

HPB 28

MIC: 3.8–7.5 mM; MBC: 7.5–15.0 mM

Cleusix et al., 2007

Staphylococcus epidermidis

–c

MIC: 15 AU b (1.6 mM)

Axelsson et al., 1989

Staphylococcus aureus

ATCC 25923

MIC: 23.0±2.9 ppm; MBC: 41.0±2.5 ppm

Chen et al., 2002

Bacillus megaterium

–c

MIC: 20 AU b (2.2 mM)

Axelsson et al., 1989

Bacillus subtilis

ATCC 6633

MIC: 35.0±0.0 ppm; MBC: 30.0±0.0 ppm

Chen et al., 2002

Listeria monocytogenes

LMG 10470, LMG 13305, Ohio serotype 4b, Scott A, V7, 121

MIC: 8 Ud (0.5 mM)

El-Ziney & Debevere, 1998

Listeria innocua

LMG 11387

MIC: 8 Ud (0.5 mM)

El-Ziney & Debevere, 1998

Listeria ivanovii

LMG 11388

MIC: 8 Ud (0.5 mM)

El-Ziney & Debevere, 1998

Listeria seeligeri

LMG 11383

MIC: 8 Ud (0.5 mM)

El-Ziney & Debevere, 1998

Listeria welshimeri

LMG 11389

MIC: 8 Ud (0.5 mM)

El-Ziney & Debevere, 1998

MBC: 90.0±0.0 ppm (1.2 mM)

Chen et al., 2002

MBC: 30.0±0.0 ppm (0.4 mM)

Chen et al., 2002

50–250 Ud g–1 cheese and 150 Ud milk; all concentrations bactericidal at 7 °C

El-Ziney & Debevere, 1998

Activity on biological tissue ATCC 25923 Staphylococcus aureus Bacillus subtilis

ATCC 6633

UHT milk and cottage cheese Ohio serotype 4b Listeria monocytogenes

Surface of pork meat Ohio serotype 4b Listeria monocytogenes

500 Ud: reduction El-Ziney et al., of log 0.63 after 1999 dipping for 15 s and storage 24 h at 7 °C (Continued )

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Table 5.1  Continued Organism

Strain

Concentrationa

Ground pork Listeria monocytogenes

Ohio serotype 4b

50–250 Ud g–1: El-Ziney et al., bactericidal for 1999  150 U g–1 at 7 °C

Milk Listeria monocytogenes

Ohio serotype 4b

8 Ud (0.5 mM): bacteristatic for at least 24 h at 37 °C

Arques et al., 2004

CECT 4013

8 Ud (0.5 mM): bacteristatic for at least 24 h at 37 °C

Arques et al., 2004

2 Ud (0.1 mM): no activity at 10 °C

Arques et al., 2004

2 Ud (0.1 mM): slower growth at 10 °C

Arques et al., 2004

Staphylococcus aureus

Cujada – semisolid dairy product Scott A Listeria monocytogenes Staphylococcus aureus

CECT 976

Reference

a MIC

= minimal inhibiting concentration, MBC = minimal bactericidal concentration. equals 8 µg reuterin ml–1 (0.11 mM 3-HPA monomer) (Dobrogosz & Lindgren, 1994; Cleusix et al., 2007). c No data available, not stated in reference. d 1 U equals 5 µg reuterin ml–1 (0.07 mM 3-HPA monomer) (Yunmbam & Roberts, 1993; Chung et al., 1989). b 1 AU

Interestingly, all tested species belonging to the genus Lactobacillus could grow at concentrations of up to 15 mM reuterin and L. reuteri even at concentrations of 30 mM (Cleusix et al., 2007). The relatively high resistance of these species enables reuterin to prevent microbiological contamination in lactobacilli fermentations; moreover, it opens opportunities to use reuterin for modulation of the GI-tract microflora composition towards higher lactobacilli counts. It is unclear if this high resistance of lactobacilli to reuterin is due to a reuterin specific defence mechanism in lactobacilli or to a general stress-response of the cells. Reuterin is still active at low pH (<6) and can therefore be used in acidified environments. Furthermore, it is also active at higher NaCl concentrations (2–4%) and these features allow a combined use of reuterin, acidic pH and salt in food preservation. Indeed, a combination of high salt, low pH and reuterin inhibits growth of L. innocua even more efficiently than the sole use of reuterin at neutral pH and physiological salt concentrations (Rasch et al., 2007). Remarkably, this effect was not seen using E. coli as indicator strain; inhibition by reuterin was similar at all tested pHs and salt concentrations (Rasch, 2002).

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5.4.2  Activity against Gram-negative bacteria The described effect of reuterin on E. coli shows that reuterin is also active against Gram-negative bacteria (Table 5.2). Species of the genus Bacteroides, one of the most predominant species in the human GI-tract, were inhibited at minimal concentrations as low as 7.5 mM (Cleusix et al., 2007). L. reuteri derived reuterin was also highly effective against the common infectious bacterium Pseudomonas aeruginosa (Liang et al., 2003), against the enterohemorrhagic E. coli (EHEC) strain O157:H7 (Arques et al., 2004) and colonic E. coli strains (Cleusix et al., 2008). Other Gram-negative microorganisms showed to be affected by reuterin are Salmonella typhimurium, Salmonella enterica, Pseudomonas fluorescens, Shigella sp., Shewanella putrefaciens, Vibrio cholera, and Yersinia enterocolitica (Chung et al., 1989; Dobrogosz et al., 1989; El-Ziney et al., 2000; Spinler et al., 2008). This list of bacteria contains a number of (food)-pathogens, exemplifying again the potential of reuterin for food preservation. Table 5.2  Activity of reuterin against Gram-negative bacteria Strain

Concentrationa

Reference

Escherichia coli

DSM 5698

MIC: 7.5–15.0 mM; MBC: 15.0–30.0 mM

Cleusix et al., 2007

Escherichia coli

ATCC 25922

MIC: 35.0±0.0 ppm; MBC: 43.0±2.9 ppm

Chen et al., 2002

Escherichia coli

K12 (wt), 431, 73, P55, 263, P159, PII-C7

MIC: 20–25 AU (2.2–2.7 mM)b

Chung et al., 1989; Dobrogosz & Lindgren, 1994

Salmonella typhimurium

–c

MIC: 20 AU b (2.2 mM)

Axelsson et al., 1989

Pseudomonas aeruginosa

2 strains

MIC: 25 AU b (2.7 mM)

Axelsson et al., 1989

Pseudomonas fluorescens

–c

–c; very sensitive

Dobrogosz & Lindgren, 1994

Pseudomonas aeruginosa

ATCC 27853

MIC: 33.0±2.9 ppm; MBC: 50.0±0.0 ppm

Chen et al., 2002

Bacteroides vulgatus

DSM 1447

MIC: < 1.9 mM; MBC: 1.9–3.8 mM

Cleusix et al., 2007

Bacteroides thetaiotaomicron

DSM 2079

MIC: 1.9–3.8 mM; MBC: 1.9–3.8 mM

Cleusix et al., 2007

Bacteroides fragilis

LMG 10263

MIC: 3.8–7.5 mM; MBC: 3.8–7.5 mM

Cleusix et al., 2007

Organism/ test conditions In vitro activity

(Continued )

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Table 5.2  Continued Organism/ test conditions

Strain

Concentrationa

Reference

Shigella species

–c

–c; very sensitive

Dobrogosz & Lindgren, 1994

Proteus species

–c

–c; very sensitive

Dobrogosz & Lindgren, 1994

Klebsiella pneumoniae

ATCC 25955

20 mM: no inhibition

Barbirato et al., 1996

Citrobacter freundii

ATCC 8090

20 mM: no inhibition

Barbirato et al., 1996

Enterobacter agglomerans

CNCM1210

30 mM: inhibition of growth

Barbirato et al., 1996

Escherichia coli VTEC

LMG 8223

MIC: 4 Ud (0.3 mM)

El-Ziney & Debevere, 1998

Escherichia coli K12 LMG 2578

MIC: 4 Ud (0.3 mM)

El-Ziney & Debevere, 1998

Escherichia coli K12 LMG 2579

MIC: 4 Ud (0.3 mM)

El-Ziney & Debevere, 1998

Escherichia coli ETEC

CIP 81.86

MIC: 4 Ud (0.3 mM)

El-Ziney & Debevere, 1998

Escherichia coli O157:H7

932

MIC: 4 Ud (0.3 mM)

El-Ziney & Debevere, 1998

Escherichia coli O157:H7

MRK 1542

MIC: 4 Ud (0.3 mM)

El-Ziney & Debevere, 1998

Activity on biological tissue Escherichia coli

ATCC 25922

MBC: 90.0±0.0 ppm (1.2 mM)

Chen et al., 2002

Pseudomonas aeruginosa

ATCC 27853

MBC: 50.0±0.0 ppm (0.7 mM)

Chen et al., 2002

50–250 Ud g–1 cheese and 150 Uc milk; all concentrations bacteriocidal at 7 °C

El-Ziney & Debevere, 1998

50–100 U g–1: bactericidal at 4 °C, 6 days

Daeschel, 1989

UHT milk and cottage cheese Escherichia coli O157:H7

932

Ground beef Coliform bacteria

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Table 5.2  Continued Strain

Concentrationa

MRK 1542

500 Ud: reduction of El-Ziney et al., log 2.8 after dipping 1999 for 15 s and storage for 24 h at 7 °C

MRK 1542

50–150 Ud g–1: all El-Ziney et al., concentrations 1999 bacteriocidal at 7 °C

Escherichia coli O157:H7

ATCC 43894

8 Ud: reduction of log 1 after 24 h at 37 °C

Arques et al., 2004

Salmonella choleraesuis subsp. choleraesuis

CECT 409

8 Ud: reduction of log 1.5 after 24 h at 37 °C

Arques et al., 2004

Yersinia enterocolitica

CECT 559

8 Ud: reduction of log 1.7 after 24 h at 37 °C

Arques et al., 2004

Aeromonas hydrophila subsp. hydrophila

CECT 398

8 Ud: elimination of log 4 after 24 h at 37 °C

Arques et al., 2004

Campylobacter jejuni

LMG 6629

8 Ud: elimination of log 4 after 24 h at 37 °C

Arques et al., 2004

Organism/ test conditions

Reference

Surface of pork meat Escherichia coli O157:H7

Ground pork Escherichia coli O157:H7 Milk

In vitro model of colonic fermentation Escherichia coli

–c

Lactobacillus reuteri Cleusix et al., 2008 ATCC 55730 produced 3-HPA that decreased E. coli population

a MIC

= minimal inhibiting concentration, MBC = minimal bactericidal concentration. equals 8 µg reuterin ml–1 (0.11 mM 3-HPA monomer) (Dobrogosz & Lindgren, 1994; Cleusix et al., 2007). c No data available, not stated in reference. d 1 U equals 5 µg reuterin ml–1 (0.07 mM 3-HPA monomer) (Yunmbam & Roberts, 1993; Chung et al., 1989). b 1 AU

5.4.3  Activity against other microorganisms Reuterin also inhibits eukaryotic organisms (Table 5.3) such as the pathogenic yeasts Candida albicans and Candida glabrata (formerly known as Torulopsis glabarta) with MIC values of below 1 mM (Chung et al., 1989). The baker’s

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Table 5.3  Activity of reuterin against eukaryotics Strain

Concentrationa

Reference

Candida albicans

–c

MIC: 2Ub (0.15 mM)

Chung et al., 1989

Candida glabrata

–c

MIC: 4Ub (0.28 mM)

Chung et al., 1989

Saccharomyces cerevisiae

–c

MIC: 12Ub (0.98 mM)

Chung et al., 1989

MIC: 14Ub (1.12 mM)

Chung et al., 1989

Organism/ test conditions Yeast

Saccharomycopsis fibuligera Filamentous fungi Fusarium samfucienum

–c

MIC: 36Ub (3 mM)

Chung et al., 1989

Aspergillus flavus

–c

MIC: 8Ub (0.56 mM)

Chung et al., 1989

Trypanosoma cruzi

–c

MIC: 5U (0.35 mM)

Chung et al., 1989

Trypanosoma brucei brucei

367H

100 % growth inhibition > 35 µM after 12 h

Yunmbam & Roberts, 1992

Protozoa

a MIC

= minimal inhibiting concentration. U equals 5 µg reuterin ml–1 (0.07 mM 3-HPA monomer) (Yunmbam & Roberts, 1993; Chung et al., 1989). c No data available, not stated in reference. b 1

yeast Saccharomyces cerevisiae, the major amylolytic yeast in indigenous food fermentation Saccharomycopsis fibuligera, and the filamentous fungi Fusarium samfucienum and Aspergillus flavus were less affected (Table 5.3). Interestingly, also the pathogenic protozoa Trypanosoma cruzi (Chagas disease), was sensitive to reuterin at low concentrations (MIC < 1mM, Table 5.3). Further, it was shown that the closely related pathogenic protozoa Trypanosoma brucei brucei (sleeping sickness) was sensitive to reuterin in vivo in mice in similar concentrations (Yunmbam & Roberts, 1992, 1993). This sensitivity provides new leads for the control of both diseases that plague world countries. The activity of reuterin against both prokaryotic as well as eukaryotic species enables a broad use of the compound beyond food application. Additionally, the occurrence of relative resistant species in both prokaryotics and eukaryotics suggests sophisticated protection mechanisms, which would be interesting research subjects.

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5.5  Production of reuterin on a large scale 5.5.1  Chemical production Chemical synthesis of 3-HPA is performed via dehydration of the α,β-unsaturated aldehyde acrolein (2-propenal, Fig. 5.2). Such chemical synthesis involves high reaction temperatures and high pressures, making the process relatively expensive (Vollenweider & Lacroix, 2004). Furthermore, a number of side products are formed during synthesis which necessitates additional purification steps. The chemical processes are relatively complex and biotechnological one-step enzymatic conversion of glycerol to 3-HPA provides a promising alternative for industrial scale production (Vollenweider & Lacroix, 2004). Biotechnological production can be performed at moderate temperatures (15–37 °C) under standard pressure using the relatively cheap raw material glycerol. Furthermore, the yields of biotechnological production are shown to be higher than those obtained by chemical synthesis (Vollenweider & Lacroix, 2004). 5.5.2  Biotechnological production of reuterin using L. reuteri Glycerol is a promising raw material for industrial fermentations for both biomass production and enzymatic conversions (da Silva et al., 2009). Being a by-product of biodiesel production, the production of glycerol has increased significantly in the past decade whereas its price has fallen (da Silva et al., 2009). Conversion of glycerol to reuterin and further to 1,3-propanediol at industrial scale is proposed to be possible in a number of microorganisms (Biebl et al., 1999). The capability to convert glycerol to 3-HPA and further to 1,3-propanediol has been found so far in six bacterial genera: Bacillus, Klebsiella, Citrobacter, Enterobacter, Clostridium, and Lactobacillus (reviewed by Vollenweider & Lacroix, 2004). This capability is always linked to the presence of genes encoding a glycerol dehydratase. Furthermore, the genetic organization of the pdu-operons is comparable in all these species (Sriramulu et al., 2008). Remarkably, the genes of the vitamin B12 gene cluster are frequently linked to the pdu-operon (Santos et al., 2008a), indicating horizontal gene transfer events and a common evolutionary origin. There are different biotechnological ways to produce reuterin out of glycerol. Production using isolated enzymes would eliminate any reductase activity, thus preventing the conversion of 3-HPA to 1,3-PDO, potentially resulting in high yields and limited by-products. However, this method did not yield high amounts of 3-HPA (Slininger et al., 1983) due to the complexity of the conversion which involves two enzymes (glycerol dehydratase and its activator), subcellular structures (microcompartments), and vitamin B12. Therefore conversion using intact cells is a more suitable method. The easiest way for whole cell conversion would be addition of glycerol to a full grown bacterial culture. However, a two step process in which biomass is formed in the first step and production takes place in the second step with washed cells in glycerol solution, has the advantage that minimal side products are formed during production (Talarico et al., 1988). Optimization of this production process resulted in a 3-HPA yield of 235 mM

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out of 400 mM glycerol by approximately 1×1010 L. reuteri cells per ml (Doleyres et al., 2005). The use of L. reuteri as a 3-HPA producer has a number of advantages above other bacteria. As mentioned before, L. reuteri is encountered in the human GI-tract and in food fermentation and is therefore generally regarded as safe (Vogel et al., 1994; Lerche & Reuter, 1965; Reuter, 1965). Metabolites produced by L. reuteri are therefore relatively easily accepted as food additives. Furthermore, lactic acid bacteria are highly suitable for metabolic engineering and their genetic accessibility as well as the availability of a range of genetic tools makes them good candidates for strain optimization (de Vos & Hugenholtz, 2004). Such optimization can be performed via engineering of metabolic pathways, potentially leading to high and pure production of 3-HPA. Two targets are interesting in this respect: engineering of 1,3-PDO oxidoreductase resulting in decreased formation of 1,3-PDO; and of glycerol dehydrogenase, the first enzyme of the oxidative glycerol conversion pathway, leading to less side product formation of dihydroxyaceton (Vollenweider & Lacroix, 2004). A major drawback in the biotechnological production of 3-HPA is the toxicity of the product to the producer itself leading to a production blockage. Remarkably, under some circumstances the production stop of the cells leads to a stress relief and subsequently to renewed production resulting in oscillatory behaviour of reuterin synthesis (Rasch et al., 2002). Therefore another strategy to optimize production is engineering the stress tolerance of cells, thereby creating efficient producer strains. This could not only result in higher 3-HPA production, but possibly also in successful re-use of the expensive biomass. Stress tolerant strains can be produced via directed mutagenesis or by classical undirected mutagenesis. The latter option might be more useful for the food industry as the optimized strain is not considered a genetic modified organism and therefore can still be applied in food and feed products.

5.6  Reuterin as a food preservative The antimicrobial activity of reuterin and the possibility to produce high amounts of it in a relatively cheap biotechnological process open opportunities for the use of reuterin as a food preservative. Alternatively, supplementation of food with production strains plus glycerol to produce reuterin in situ might also be suitable as a preservation technique. However, before reuterin can be applied as a preservative in food, its toxicity to humans should be studied extensively. Furthermore, the stability of reuterin and the toxicity of reuterin derivatives, such as acrolein, that might accumulate in food, should be established. Legislation in most countries demands such additional knowledge before the use of reuterin in food can be considered. 5.6.1  Stability of reuterin in food The activity of preservatives depends on a number of parameters, such as pH, salt concentration, and temperature. Reuterin keeps its activity in acidic environments

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(pH < 6) and at higher NaCl concentrations (2–4%) providing possibilities to apply reuterin in fermented and salted foods (Rasch et al., 2007). Reuterin is chemically stable at 4 °C and the use of small amounts in cold stored foods such as dairy products is therefore considered to be safe in terms of anti-microbial activity (El-Ziney et al., 1999; Vollenweider et al., 2003). However, at higher temperatures above 20 °C, reuterin can react with other components in the food or dehydrate to acrolein (Lüthi-Peng et al., 2002; Sung et al., 2003). Acrolein is a toxic α,β-unsaturated aldehyde that easily reacts with polyphenol compounds such as tannins in wine, leading to a bitter taste (Rentschler & Tanner, 1951; Noble, 1999; Lonvaud-Funel, 2002; Garai-Ibabe et al., 2008). There are a number of potential advantages for the use of reuterin as a food preservative above other preservatives. The reuterin form 3-HPA is a metabolite and not sensitive to proteases or lipases. Further, reuterin is water soluble, stable and active over a wide pH range (Axelsson et al., 1989). It has a broad spectrum of antimicrobial activity and is relatively cheap to produce biotechnologically by the safe organism L. reuteri. Finally, no natural resistance mechanism has been described and consequently no resistance genes have been identified, suggesting stable activity of reuterin as anti-microbial when applied at different scales over long periods of time. 5.6.2  Toxicity of reuterin and its derivatives The impact of aldehydes on human health concerning their mutagenic properties and cytotoxicity has been studied thoroughly (O’Brien et al., 2005). Unfortunately there is little knowledge about the toxicity of reuterin and therefore additional research is still required (Ghilarducci & Tjeerdema, 1995; Kehrer & Biswal, 2000; Lüthi-Peng et al., 2002; Sung et al., 2003). Cytotoxicity of reuterin was tested using an in vitro test on mouse fibroblasts and it was shown that reuterin was significantly less toxic than glutaraldehyde (Chen et al., 2002). However, glutaraldehyde itself is one of the most mutagenic aldehydes (Marnett et al., 1985; Kuykendall & Bogdanffy, 1992) and therefore such comparison provides only very limited information about the toxicity of reuterin. The toxicity of reuterin in mice was also established and the median lethal dose for 50% of subjects (LD 50) was estimated to be 7.5 mg (Yunmbam & Roberts, 1993). The weight of the mice was 24–29 g and hence the median lethal concentration for 50% of subjects (LC 50) would be 236–287 mg/kg which equals 3.1–3.9 mmol/kg. However, irritation of mucosal, for example, might already occur at lower concentrations. Additionally, the metabolic fate of reuterin in humans is completely unknown and long-term effects of reuterin on human health have still to be elucidated. A major problem in toxicity of the reuterin system is acrolein. As already mentioned, reuterin can be easily converted to acrolein at moderate temperatures (Vollenweider et al., 2003). Acrolein reacts with sulfhydryl groups leading to a disturbance of the sulphur balance in the cell and to inhibition of sulphurcontaining enzymes. The toxicity of acrolein has been well studied; mucosal

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irritation occurs after intake of 1–2 mg/kg, the median lethal concentration for 50% of subjects (LC 50) is 46 mg/kg in rats, which is approximately 10 times lower than for reuterin (Ghilarducci & Tjeerdema, 1995; Faroon et al., 2008). Fortunately, the odour perception of acrolein is very low (0.07 mg/m3), thereby preventing intake of high doses (Ghilarducci & Tjeerdema, 1995). 5.6.3  Legislation The reuterin producer organism L. reuteri is considered ‘Generally Recognized As Safe’ (GRAS) and this GRAS status simplifies the acceptation of reuterin as a food preservative by the authorities. Therefore, in the USA the use of L. reuteri cells and glycerol to produce reuterin in situ should be acceptable according to the Code of Federal Regulations (U.S.G.P. Office, 1990), although the Food and Drug Administration (FDA) may require justification of the GRAS status. However, for extracted reuterin the guidelines for the safety assessment of a new preservative in the USA will be applicable (U.S. Food and Drug Administration, 1993). According to these guidelines, a new preservative must be chemically identified and characterized. Additionally, its use and efficacy must be shown and its manufacturing process must be described. Assays for quantification and standardization must have been developed and toxicological data on the molecule and its fate after ingestion are also required. Only the latter directions are not established for reuterin. The fate of reuterin after ingestion and its toxic effect are still poorly understood and therefore additional research is needed before application in foods can be considered in the USA. In the European Union, authorization demands for food additives are evaluated for their safety by the EU Scientific Committee on Food. Authorization is only given if there is a technological need for their use, they do not mislead the consumer, and they present no hazard to the health of the consumer (EU Directive, 1988). Reuterin is active against a wide range of organisms and there is always technical need for such substances, so again the poorly understood toxic effects of reuterin will hamper permission by the authorities. Finally, the first patent about the use of the dimer form of reuterin as an antibiotic compound was filed in 1988 (Dobrogosz & Lindgren, 1988), and as legislation in most countries regulates patent-protection for only a limited time, free use of reuterin can be possible within the near future. 5.6.4  Potential use of reuterin in food: some examples The inhibitory and antibiotic activities of reuterin against microorganisms were mostly established under laboratory conditions in standard growth media. Because these conditions do not necessarily reflect conditions in food, the antimicrobial activity of reuterin was also tested in food products. Unfortunately, the number of such studies is limited. It should be emphasized that in all studies on the activity of reuterin in food presented below, the reuterin was produced by L. reuteri.

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Reuterin applied as a preservative in milk has bacteriostatic activity against L. monocytogenes and bactericidal activity against S. aureus, E. coli, Salmonella choleraesuis, Yersinia enterocolitica, Aeromonoas hydrophila, and Campylobacter jejuni at a concentration as low as 0.5 mM at 37 °C (Arques et al., 2004). A combination of reuterin and the lactococcal bacteriocin nisin in milk also resulted in a strong bactericidal effect against Listeria monocytogenes (Arques et al., 2004; Table 5.1 and 5.2). It was concluded that under the tested conditions reuterin was effective against Gram negative bacteria in milk, whereas the combination of reuterin and nisin was effective against Gram positive bacteria. In another study, higher concentrations of reuterin (10 mM) at 7 °C had a bactericidal effect against L. monocytogenes and the antimicrobial activity of reuterin activity was not affected by up to 3% milk fat (El-Ziney & Debevere, 1998; Table 5.1 and 5.2). In cuajada, a traditional Spanish semisolid dairy product, both L. monocytogenes and S. aureus were relatively resistant to reuterin and only a combination of reuterin with nisin and lactoperoxidase resulted in significant bactericidal activity (Arques et al., 2008; Table 5.1). These studies showed that reuterin can be used as a biopreservative in the dairy industry, although its effectiveness seems species specific and the best effect is reached in combination with other inhibitors. Reuterin was also tested in cottage cheese at a typical lower pH of approximately 5.4. Reuterin strongly reduced the cell count of the enterohemorrhagic E. coli O157:H7, and to a lesser extent that of L. monocytogenes (El-Ziney & Debevere, 1998). Also, totally viable counts of mesophilic aerobic microbes were reduced due to the effect of reuterin. A similar effect was seen on the viable cell counts on the surface of cooked pork inoculated with E. coli O157:H7 and L. monocytogenes (El-Ziney et al., 1999). In the past decade, E. coli O157:H7 and L. monocytogenes outbreaks have become of high concern in public health and the food industry (Cossart, 2007; Franz & van Bruggen, 2008), and a reuterin-triggered decrease of cell counts of these pathogens exemplifies the potential of reuterin as a food preservative. Activity of reuterin against L. monocytogenes was also shown on the surface of sausages (Kuleasan & Cakmakci, 2002). Sausages were submerged for a short time in a reuterin solution and subsequently inoculated with L. monocytogenes or Salmonella spec. on the surface. Reuterin treatment resulted in a significant reduction of viable L. monocytogenes cell numbers, but antimicrobial effect against Salmonella spec. was not observed (Kuleasan & Cakmakci, 2002). Instead of the addition of reuterin itself, the addition of the producer organism plus glycerol might increase the shell life of the food. An advantage would be that a probiotic bacterium is added to the food, additionally providing potential health benefits to the consumer. The addition of L. reuteri cells and glycerol during dry fermented sausage manufacture lead to a reduction in E. coli O157:H7 numbers within 30 d of manufacturing (including 25 d of ripening), but the reduction was only moderate and no reuterin production could be detected in the sausage (Muthukumarasamy & Holley, 2007), probably due to the low survival of L. reuteri cells during fermentation. Microencapsulation of L. reuteri significantly increased the number of surviving cells during ripening (Muthukumarasamy &

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Holley, 2006), but also reduced the antimicrobial activity (Muthukumarasamy & Holley, 2007). Taken together, reuterin has potential as a food preservative, especially due to its activity against the food spoilage organism L. monocytogenes and E. coli O157:H7. However, its antimicrobial effect can differ depending on the target species and the food matrix. The potential of the simultaneous use of L. reuteri cells (alive or attenuated) with the substrate glycerol, providing continious relief of reuterin by minimal metabolism of the producer strain, is an unexplored area of food preservation that needs further research.

5.7  Additional antimicrobial compounds produced by L. reuteri Production of small ribosomal synthesized peptides (bacteriocins) by lactic acid bacteria is an important feature for fermentation stability, probiotic activity, and food preservation (reviewed by Cotter et al., 2005). The best-known example for an LAB bacteriocin is the lactococcal peptide nisin, marketed as a foodpreservative since the 1950s (Cotter et al., 2005). So far a bacteriocin produced by a L. reuteri strain isolated from infant faeces has been identified and designated reutericin 6 (Toba et al., 1991). Reutericin 6 is a 2.7 kDa peptide, heat stable, and mainly active against Lactobacillus delbrueckii and Lactobacillus acidophilus, but not against E. coli, Bacillus subtilis, and Staphylococcus aureus (Toba et al., 1991; Kabuki et al., 1997). The plasmid harbouring the genes encoding for reutericin 6 production is genetically indistinguishable from a Lactobacillus gasseri plasmid harbouring genes for production of the bacteriocin gassericin A and it is likely that the plasmid conjugated from L. gasseri to L. reuteri (Ito et al., 2009). Remarkably, gassericin A has a broader antimicrobial spectrum compared to reutericin 6 due to different post-translational modifications (Kawai et al., 2004). Another interesting antimicrobial compound produced by a L. reuteri strain isolated from cereal fermentation is reutericyclin, a tetramic acid produced from Tween 80 or from oleic acid. It displays a broad inhibitory spectrum against Gram-positive bacteria including Lactobacillus spec., Bacillus spec., Enterococcus faecalis, L. innocua, and S. aureus (Gänzle et al., 2000). Reutericyclin was also active against other L. reuteri strains, indicating that production and resistance are linked (Gänzle et al., 2000). All reutericyclin-producing strains isolated so far originated from cereal fermentation and therefore reutericyclin might play a role in the persistence of L. reuteri in cereal fermentation (Gänzle, 2004). Since reutericyclin is produced by food isolates, this antimicrobial compound also has potential as a food preservative. The presence of genes encoding for antimicrobial compounds like reutericyclin or reutericin 6 is strain specific, which parallels observations in Lactobacillus plantarum strains (Molenaar et al., 2005). The availability of the complete genome sequences of more L. reuteri strains in the future will therefore probably lead to the discovery of new antimicrobial compounds deriving from L. reuteri.

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5.8  Concluding remarks and future trends The antimicrobial activity of reuterin is beyond any doubt and this activity against both prokaryotic and eukaryotic organisms gives reuterin potential for food preservation. Another advantage of reuterin is its activity at wide pH-range, salt concentration, and temperatures that are relevant for foods. However, additional studies on the stability of reuterin over storage time in different food matrices are still necessary to ensure activity against spoilage bacteria at sufficient rates. Therefore, the application of L. reuteri plus the substrate glycerol for controlled production of reuterin in situ during food storage could be a powerful alternative for reuterin addition in foods. Reuterin can be produced in high amounts in a biotechnological process using food-grade producer strains as L. reuteri. These features give reuterin an advance above bacteriocins with low productivity. However, toxicity studies on humans and animals and probably ecological studies on the fate of reuterin are required before authorization for use will be given by the responsible organizations. Although application of reuterin as a food preservative may not be legislated in the short term, biotechnological production could increase just because of the demand of 3-HPA by the chemical industry. The use of reuterin as an antimicrobial agent for sanitation and non-food applications was not discussed in this chapter, but reuterin also has high potential for such purposes. Since the production of 3-HPA is expected to increase in the near future (Vollenweider & Lacroix, 2004) it is likely that more applications for the molecule will be developed, including disinfection and food biopreservation.

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and b bjorn budde (2007) ‘The effect of reuterin on the lag time of single cells of Listeria innocua grown on a solid agar surface at different pH and NaCl concentrations.’ Int J Food Microbiol 113: 35–40. reid g , a w bruce , n fraser, c heinemann , j owen and b henning (2001) ‘Oral probiotics can resolve urogenital infections.’ FEMS Immunol Med Microbiol 30: 49–52. rentschler h and h tanner (1951) ‘Das Bitterwerden der Rotweine.’ Mitt. Lebensm. Unters. Hygiene 42: 463–475. reuter g (1965) ‘Das Vorkommen von Laktobazillen in Lebensmitteln und ihr Verhalten in menschlichen Intestinaltrakt.’ Zbl Bak Parasit Infec Hyg I Orig 197: 468–487. rodriguez e , j l arques , r rodriguez , m nunez and m medina (2003) ‘Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits.’ Lett Appl Microbiol 37: 259–263. sampson e m and t a bobik (2008) ‘Microcompartments for B12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate.’ J Bacteriol 190: 2966–2971. santos f (2008) ‘Vitamin B12 synthesis in Lactobacillus reuteri.’ PhD Thesis. Microbiology Department, Wageningen University, Wageningen, The Netherlands. santos f , j l vera , p lamosa , g f de valdez , w m de vos et al. (2007) ‘Pseudovitamin B12 is the corrinoid produced by Lactobacillus reuteri CRL1098 under anaerobic conditions.’ FEBS Lett 581: 4865–4870. santos f , j l vera , r van der heijden , g valdez , w m de vos et al. (2008a) ‘The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098.’ Microbiology 154: 81–93. santos f , a wegkamp , w m de vos , e j smid and j hugenholtz (2008b) ‘High-level folate production in fermented foods by the B12 producer Lactobacillus reuteri JCM1112.’ Appl Environ Microbiol 74: 3291–3294. sauvageot n , c muller, a hartke , y auffray and j m laplace (2002) ‘Characterisation of the diol dehydratase pdu operon of Lactobacillus collinoides.’ FEMS Microbiol Lett 209: 69–74. savino f , e pelle , e palumeri , r oggero and r miniero (2007) ‘Lactobacillus reuteri ATTC 55730 versus simethicone in the treatment of infantile colic: a prospective randomized study.’ Pediatrics 119: e124–130. schäfer l , t a auchtung , k e hermans , d whitehead , b borhan and r a britton (2010) ‘The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups.’ Microbiology 156: 1589–1599. schauenstein e , h esterbauer and h zollner (1977) ‘Saturated aldehydes.’ In E Schauenstein, H Esterbauer and H Zollner (eds) Aldehydes in Biological Systems: Their Natural Occurrence and Biological Activities, Pion, London, pp. 9–24. schreiber o , j petersson , m phillipson , m perry, s roos and l holm (2009) ‘Lactobacillus reuteri prevents colitis by reducing P-selectin-associated leukocyte- and platelet-endothelial cell interactions.’ Am J Physiol Gastrointest Liver Physiol 296: G534–542. shornikova a v, i a casas , e isolauri , h mykkanen and t vesikari (1997a) ‘Lacto­ bacillus reuteri as a therapeutic agent in acute diarrhea in young children.’ J Pediatr Gastroenterol Nutr 24: 399–404. shornikova a v, i a casas , h mykkanen , e salo and t vesikari (1997b) ‘Bacterio­ therapy with Lactobacillus reuteri in rotavirus gastroenteritis.’ Pediatr Infect Dis J 16: 1103–1107. slininger p j , r j bothast and k l smiley (1983) ‘Production of 3-hydroxy­ propionaldehyde from glycerol.’ Appl Environ Microbiol 46: 62–67. smiley k l and m sobolov (1962) ‘A cobamide-requiring glycerol dehydrase from an acrolein-forming Lactobacillus.’ Arch Biochem Biophys 97: 538–543.

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antimicrobial activities targeting diverse enteric bacterial pathogens.’ Anaerobe 14: 166–171. sriramulu d d , m liang , d hernandez - romero , e raux - deery, h lunsdorf et al. (2008) ‘Lactobacillus reuteri DSM 20016 produces cobalamin-dependent diol dehydratase in metabolosomes and metabolizes 1,2-propanediol by disproportionation.’ J Bacteriol 190: 4559–4567. stewart m l , v savarino and j l slavin (2008) ‘Assessment of dietary fiber fermentation: Effect of Lactobacillus reuteri and reproducibility of short-chain fatty acid concentrations.’ Mol Nutr Food Res 53(Suppl 1): 5114–120. sung h w, c n chen , h f liang and m h hong (2003) ‘A natural compound (reuterin) produced by Lactobacillus reuteri for biological-tissue fixation.’ Biomaterials 24: 1335–1347. talarico t l , i a casas , t c chung and w j dobrogosz (1988) ‘Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri.’ Antimicrob Agents Chemother 32: 1854–1858. talarico t l and w j dobrogosz (1989) ‘Chemical characterization of an antimicrobial substance produced by Lactobacillus reuteri.’ Antimicrob Agents Chemother 33: 674–679. talarico t l and w j dobrogosz (1990) ‘Purification and characterization of glycerol dehydratase from Lactobacillus reuteri.’ Appl Environ Microbiol 56: 1195–1197. tanaka o , t komatsu , a oshibe , y cai , s miyazaki and k nakanishi (2009) ‘Production of 3-hydroxypropionaldehyde in silage inoculated with Lactobacillus coryniformis plus glycerol.’ Biosci Biotechnol Biochem 73: 1494–1499. tannock g w and d savage (1985) ‘Detection of plasmids in gastrointestinal strains of lactobacilli.’ Proc Univ Otago Med Sch 63: 29–30. tannock g w (2007) ‘Complete genome sequence Lactobacillus reuteri 100–103.’ Computational Biology and Bioinformatics Group, Oak Ridge National Laboratory. Available from: http://genome.ornl.gov/microbial/lreu_23/ (accessed 30 September 2009). taranto m p , j l vera , j hugenholtz , g f de valdez and f sesma (2003) ‘Lactobacillus reuteri CRL1098 produces cobalamin.’ J Bacteriol 185: 5643–5647. toba t , s k samant , e yoshioka and t itoh (1991) ‘Reutericin 6, a new bacteriocin produced by Lactobacillus reuteri.’ Letters in Applied Microbiology 13: 281–286. tubelius p , v stan and a zachrisson (2005) ‘Increasing work-place healthiness with the probiotic Lactobacillus reuteri: a randomised, double-blind placebo-controlled study.’ Environ Health 4: 25. twetman s , b derawi , m keller, k ekstrand , t yucel - lindberg and c stecksen blicks (2009) ‘Short-term effect of chewing gums containing probiotic Lactobacillus reuteri on the levels of inflammatory mediators in gingival crevicular fluid.’ Acta Odontol Scand 67: 19–24. u . s . food and drug administration (1993) ‘Toxilogical principles for the safety assessment of direct food additives and color additives used in food.’ U.S. Food and Drug Administration, Washington, D.C. u . s . g . p . office (1990) Food Additives. U.S. Governmental Printing Office, Washington D.C. valeur n , p engel , n carbajal , e connolly and k ladefoged (2004) ‘Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract.’ Appl Environ Microbiol 70: 1176–1181. van coillie e , j goris , i cleenwerck , k grijspeerdt , n botteldoorn et al. (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. vogel r f , g bocker, p stolz , m ehrmann , d fanta et al. (1994) ‘Identification of lactobacilli from sourdough and description of Lactobacillus pontis sp. nov.’ Int J Syst Bacteriol 44: 223–229.

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and c lacroix (in press) ‘Unraveling the HPA System: An Active Antimicrobial Agent Against Human Pathogens.’ J Agric Food Chem in press, DOI: 10.1021/jf1010897. vollenweider s , g grassi , i könig and z puhan (2003) ‘Purification and structural characterization of 3-hydroxypropionaldehyde and its derivatives.’ J Agric Food Chem 51: 3287–3293. vollenweider s and c lacroix (2004) ‘3-Hydroxypropionaldehyde: applications and perspectives of biotechnological production.’ Appl Microbiol Biotechnol 64: 16–27. wang b , h wei , j yuan , q li , y li et al. (2008) ‘Identification of a surface protein from Lactobacillus reuteri JCM1081 that adheres to porcine gastric mucin and human enterocyte-like HT-29 cells.’ Curr Microbiol 57: 33–38. whitehead k , j versalovic , s roos and r a britton (2008) ‘Genomic and genetic characterization of the bile stress response of probiotic Lactobacillus reuteri ATCC 55730.’ Appl Environ Microbiol 74: 1812–1819. wolf b w, k a garleb , d g ataya and i a casas (1995) ‘Safety and tolerance of Lactobacillus reuteri in healthy adult male subjects.’ Microbial Ecol Health Dis 8: 41–50. wolf b w, k b wheeler, d g ataya and k a garleb (1998) ‘Safety and tolerance of Lactobacillus reuteri supplementation to a population infected with the human immunodeficiency virus.’ Food Chem Toxicol 36: 1085–1094. yunmbam m k and j f roberts (1992) ‘The in vitro efficacy of reuterin on the culture and bloodstream forms of Trypanosoma brucei brucei.’ Comp Biochem Physiol C 101: 235–238. yunmbam m k and j f roberts (1993) ‘In vivo evaluation of reuterin and its combinations with suramin, melarsoprol, DL-alpha-difluoromethylornithine and bleomycin in mice infected with Trypanosoma brucei brucei.’ Comp Biochem Physiol C 105: 521–524.

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6 Bacteriophages and food safety L. Fieseler and M. J. Loessner, ETH Zurich, Switzerland and S. Hagens, EBI Food Safety, The Netherlands

Abstract: Bacteriophages and phage encoded endolysins exhibit valuable properties to specifically target and control unwanted bacteria in foods. Here, the construction of reporterphages for pathogen detection, characteristics of phage encoded endolysins, and application of phages for biocontrol purposes are summarized. Moreover, the standards required for use of phages in foods are discussed. Key words: bacteriophage, food-borne pathogen, reporterphage, endolysins.

6.1  Introduction Bacteriophages represent a powerful tool for biocontrol of bacterial pathogens and for food safety. The advantage of using virulent bacteriophages is their great specificity which distinguishes phages from any other available antibacterial treatment. Phages will not harm bacteria which are desired in foods, e.g. starter cultures or protective cultures, and consumption of phage treated food will not be harmful to the commensal microflora of the human gastrointestinal tract. In addition, bacteriophages are not known to cause allergic reactions in humans, do not leave ecological footprints, and are organic. Recently, a bacteriophage preparation received the Generally Recognized As Safe (GRAS) status from the U.S. Food and Drug Administration. Moreover, the application of phages on foods is very simple and does not change structure, flavor or smell. Likewise, application of phages could become a valuable option for treatment of bacterial infections in medicine and preharvest agriculture. This chapter provides an overview of how phages and phage encoded proteins can be used for detection and diagnosis of pathogenic bacteria. After briefly summarizing key information about phages, construction and properties of reporterphages are described. Then the potential of phage therapy is illustrated 161 © Woodhead Publishing Limited, 2011

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and explored and finally we discuss requirements of bacteriophages for biocontrol.

6.2  Bacteriophages Bacteriophages (Greek for ‘bacteria eater’) or simply phages are viruses which infect bacteria. The majority of all bacteriophages known exhibit a double stranded DNA genome inside the virion capsid and belong to the order of tailed phages (Caudovirales). The tailed phages can be further separated into three families: Podoviridae are characterized by very short tails, Myoviridae exhibit longer, straight and contractile tails, and Siphoviridae can be identified due to their long and flexible tails. Another well studied group of phages with many applications (although minor in terms of species diversity) is represented by filamentous phages which exhibit a single stranded DNA genome decorated by a helical protein layer surrounding the DNA molecule. A detailed overview about phage classification is provided by Ackermann (2007). Phages are ubiquitously distributed in nature and can also be isolated from human or animal associated microflora. They outnumber their bacterial host species by a factor of ten representing the most abundant self-replicating entities on earth with an estimated 1031 phages in total (Brüssow and Kutter, 2005). Among many different environments, a wide range of foods contain diverse bacteriophages at relatively high numbers and therefore phages also resemble a general part of the natural food microflora. Accordingly, phages have been isolated from ground beef, pork, chicken and other meat products, chilled and frozen crabs, fermented dairy products like cheese and yoghurt, fresh produce like lettuce and vegetables, and mushrooms (Whitman and Marshall, 1970, 1971; DiGirolamo et al., 1972; Kennedy and Bitton, 1987; Kilic et al., 1996; Hsu et al., 2002). Likewise, some of the bacteriophages targeting food-contaminating bacteria, such as Escherichia coli, Salmonella, and Campylobacter jejuni have also been isolated from foods (Kennedy et al., 1986; Hansen et al., 2007). Like all viruses, bacteriophages lack an own metabolism and rely on a host to reproduce. The first step in infection by tailed phages is adsorption of the phage particle to the bacterial surface. The attachment is extremely specific and mediated by recognition of the primary host cell receptor followed by binding to a secondary receptor. Thereafter, the bacteriophage injects its genome into the target cell. From now on, two different main strategies of reproduction can be distinguished. The virulent (or strictly lytic) phages immediately start gene expression and replication to assemble newly synthesized genomes and structural proteins into progeny virions. At the end of this process, the host cell is lysed through action of phage encoded pore-forming holins and cell wall degrading murein hydrolases. On the other hand, temperate phages are able to lysogenize the host cell after infection (Fig. 6.1). During lysogenization, a site-specific integrase inserts the phage genome into the chromosome of the host bacterium. This prophage is replicated along with the chromosome by the host cell replication machinery and

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Fig. 6.1  Life cycles of bacteriophages.

further passed on to the daughter cells. After lysogenization, daughter cells are generally resistant against superinfection by the same phage, a phenomenon referred to as homoimmunity suppression. However, it is well known that lysogenic conversion can enhance bacterial pathogenicity (Reidl and Mekalanos, 1995; Datz et al., 1996). Therefore, temperate phages are not considered as suitable tools for control of bacterial pathogens. A second drawback with temperate phages is that they often exhibit rather narrow host ranges and are capable of either specific or generalized transduction, thereby possibly altering genotypes, fitness or virulence of next generation lysogenized host cells. In contrast the strictly lytic, non-transducing phages exhibiting broad host ranges perfectly meet the requirements of a biocontrol or pathogen detection agent.

6.3  Pathogen detection using bacteriophages 6.3.1  Phage typing Bacteriophages exhibit striking host specificity because they coevolved with their bacterial prey. Accordingly, phages combat cellular defense mechanisms and circumvent DNA restriction the particular host. On the other hand, phages rely upon the host metabolism to reproduce. Hence, precise host cell recognition must be assured, otherwise phages cannot multiply. Specificity of phages towards their host bacteria is impressive and can be used to distinguish bacterial isolates and single serovars, a procedure referred to as phage typing. While some phages can exhibit a very narrow host range infecting a particular serovar group only, others can infect 95% of all available strains of the given host species (Loessner et al., 1996). Basically, different phages recognize a variety of molecules on the host cell surface by receptor binding proteins. For Gram-positive bacteria, targets can

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be teichoic and lipoteichoic acids, cell wall associated proteins or peptidoglycan. In the case of Gram-negative bacteria, lipopolysaccharide components, capsule antigens, cellular appendages such as pili and flagella or surface associated proteins like porins or transporters are recognized by phages. In comparison to alternative and advanced typing methods, such as restriction fragment length polymorphism analyses of chromosomal DNA via pulsed field gel electrophoresis or 16S rRNA-based approaches, phages typing still offers several advantages: it is based upon classical microbiological methods which are easily established for any organism of interest, it is relatively cheap, and different phage typing sets exist for important food-borne pathogens such as E. coli, Salmonella, Campylobacter, and Listeria. 6.3.2  Reporterphages Phage amplification assay Application of reporterphages is meant for specific detection of bacteria in a given sample, e.g. in food. A very simple approach to use phages as reporters is the phage amplification assay (Stewart et al., 1998). Here, a test sample is subjected to a defined titer of a native non-modified phage ideally exhibiting a broad host range. Then growth of the phage is monitored over time by determining plaque counts on a suitable laboratory indicator strain. Before plaques can be determined, remaining extracellular phages are removed from the test sample by a virucide that does not harm the bacterium. The assay offers the advantages of classical microbiological methods which can be easily adapted to laboratory needs. In in vitro experiments, forty Pseudomonas aeruginosa cells per ml and 600 Salmonella Typhimurium cells per ml were detectable by phage amplification after four hours of incubation. On chicken breast, phage amplification was performed to detect Salmonella qualitatively (de Siquera et al., 2003). Wilson et al. (1997) established an amplification assay for detection of slow growing Mycobacteria, using the lytic bacteriophage D29 (Siphoviridae). D29 exhibits a very broad host range and can infect different species of the M. tuberculosis complex. As an advantage, both slow and fast growing Mycobacteria are infected. Using the fast growing indicator M. smegmatis, detection of M. tuberculosis can be performed within 12–48 hours. Normally, slow growing M. tuberculosis would need up to eight weeks to form visible colonies (McNerney et al., 1998). Other approaches require genetically modified phages which carry a reporter gene that is heterologously expressed by the infected host cell. Luciferase reporter phages (LRP) A very useful reporter gene is bacterial luciferase (from Vibrio and other marine bacteria), catalyzing the oxidation of a long chain aldehyde by molecular oxygen; a reaction which emits light at 490 nm wavelength. The luciferase (lux) gene cluster comprises a small operon consisting of luxRICDABE genes. While luxR and luxI regulate luciferase expression via a quorum sensing mechanism, luxCDE

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are required for synthesis of a long chain n-decyl fatty acid aldehyde substrate. For construction of a reporterphage, only luxAB are used due to the limited space that is available for a packaged phage genome in the corresponding phage capsid. Thus, aldehyde substrate is added externally to the test samples for reporter assays. Reduced flavin mononucleotide (FMNH) and molecular oxygen are supplied by the host cell and the aldehyde is then oxidized by the luciferase (Hastings and Nelson, 1977). The first reporterphage being constructed this way was lambda::luxAB (Ulitzur and Kuhn, 1987). Insertion of luxAB was mediated by random transposon mutagenesis. Lambda is a temperate virus infecting E. coli, and one of the best studied bacteriophages. It served as a model for investigating DNA replication, control of lysogeny by a genetic switch, virus morphogenesis, phage assembly, DNA packaging mechanisms, and timed lysis of infected host cells, respectively. Advantageously, direct cloning systems are available to construct recombinant lambda phages today. If such convenient phage vectors do not exist (the majority of cases), reporterphages can also be constructed by homologous recombination, which however can be a quite challenging task. Using the engineered lambda::lux reporterphage, approximately 10 E. coli cells could be detected in milk within one hour (Ulitzur and Kuhn, 1987). Because lambda is a temperate phage, application of this reporterphage is limited due to homoimmunity suppression and resistance to superinfection by lysogenized cells. Thus, application of temperate reporterphages might lead to false negative results. Kodikara et al. (1991) used other Enterobacteria lux-phages and described bacterial detection limits of 104 per gram without prior enrichment after 50 min, whereas four hours of enrichment could enhance the detection limit to 10 cells per gram. For detection of Salmonella bacteriophages, P22::lux and Felix O1::luxAB were constructed, and P22 was successfully applied to enumerate Salmonella from spiked sewage sludge and soil samples. Using this phage the authors managed to determine one Salmonella Typhimurium cell per 100 ml within 24 hours (Turpin et al., 1993). However, P22 is a temperate phage with only a limited host range, while Felix O1 is a lytic, broad host range phage infecting most Salmonella serovars. The corresponding reporterphage was constructed by transposon mutagenesis and is non-replicative, because transposon insertion disrupted an essential gene. If this gene is provided in trans, Felix O1:luxAB can be propagated on an engineered production strain. Unfortunately the authors reported that bioluminescence was inconsistent (Kuhn et al., 2002). Detection of Listeria monocytogenes can be achieved by A511::luxAB. Phage A511 is a strictly lytic, virulent Myovirus with a broad host range, infecting 95% of all relevant L. monocytogenes serovars. Here, a luxAB fusion from Vibrio harveyi was inserted into the phage genome by homologous recombination and placed under control of the strong major capsid protein (cps) promoter yielding a bicistronic cps-luxAB transcript. The reporterphage remained fully functional and its suitability for Listeria detection was shown in contaminated food samples. A511::luxAB provides for high sensitivity; in cabbage samples, detection of a single Listeria cell per gram was possible after 24 hours (including selective

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pre-enrichment of the samples). In more complex samples such as soft cheese and minced meat, detection limits were in the range of 10–100 cells per gram (Loessner et al., 1997). Mycobacterium tuberculosis can also be detected by luciferase reporterphage. Sarkis et al. (1995) constructed the temperate phage L5 carrying a firefly (Photinus pyralis) luciferase gene (luc) inserted into a tRNA region of the phage genome. Insect luciferase represents an alternative to the bacterial luciferase. For reporter phage construction, a cDNA version of luc lacking intron sequences is used. In contrast to bacterial luciferase (lux), Luc reaction is ATP dependent and requires a large substrate molecule, luciferin. Using L5::luc, approximately 100 M. tuberculosis cells could be detected after 40 hours of incubation and other studies proved the suitability of the lytic bacteriophage TM4 for reporterphage construction (Jacobs et al., 1993). The drawback of using Luc is that the luciferin substrate does not easily cross membranes and, therefore, complete lysis of target cells is necessary before the assay can be started. Ripp et al. (2006) developed a binary reporter assay using the luxI gene as the reporter, as LuxI produces 3-oxo-hexanoyl homoserine lactone (OHHL), a diffusible autoinducer. In a growing bacterial culture, increasing OHHL levels cause induction of the lux operon, due to binding to the LuxR repressor protein. Together LuxI and LuxR resemble a typical quorum sensing system. Accordingly, bacteriophage mediated luxI expression by infected target cells can be monitored applying a reporter bacterium carrying luxRCDABE but lacking luxI. Then, light emission from the reporter bacterium can be determined. The recombinant lambda::luxI phage detected a single E. coli cell per ml in vitro after ten hours of incubation, and 130 CFU per ml in artificially contaminated lettuce leaf washings after 22 hours of incubation. A disadvantage of this system might be presence of autoinducers produced by the natural microflora of the corresponding food sample causing false positive results. Reporterphages using other systems The green fluorescent protein (gfp) of the jellyfish Aequorea victoria can also be used as a reporter when fused to bacteriophage small outer capsid (soc) protein thereby generating a fluorescent fusionprotein. Bacteriophage PP01, a T-even like phage, was qualitatively used for this purpose. PP01 is a virulent phage specifically infecting pathogenic E. coli O157:H7 strains (Oda et al., 2004). The gfprecombinant phage adsorbs to its target cells which are then fluorescently labeled. Unfortunately, the phage can also adsorb to dead cells, which limits the usefulness of the system. Phage-labeling can also be performed with fluorescent nucleic acid stains such as YOYO-1. The stain was used to label DNA of intact bacteriophage LG1, an E. coli O157:H7 specific bacteriophage. Application of fluorescent LG1 was combined with flow cytometry and enabled detection of 2.2 CFU per gram of artificially contaminated ground beef after six hours enrichment and 10–100 CFU per ml of artificially contaminated raw milk after ten hours enrichment (Goodridge et al., 1999).

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Yet another possibility is to measure the release of adenylate kinase from lysed cells after bacteriophage infection. The enzyme synthesizes ATP from ADP which has been added to the sample. ATP can then be monitored by using firefly luciferase, however this approach is quite laborious. Infected cells should be removed and washed before addition of luciferase, because ATP available in the sample would cause false positive results. As an alternative to bioluminescence or fluorescence, reporters such as the ice nucleation protein (inaZ ) from Pseudomonas syringea were used for the construction of reporterphages. InaZ is a membrane located protein which mimics the lattice of ice crystals. Two water molecules are arranged in such a way that ice nucleation starts at temperatures of –3 °C. InaZ activity can be measured very sensitively by a simple droplet freezing assay, measured by a phase-sensitive dye which changes color upon freezing. Wolber and Green (1990) developed P22::ina phage, and could detect approximately 10 Salmonella cells per ml in vitro. While all of the reporter assays described above are based upon the specificity of bacteriophage to their bacterial host, the dual phage assay uses phage to detect binding of an antibody to a single specific antigen in a given sample. The assay is similar to the phage display technique. In phage display, a library of different proteins is displayed on the surface of filamentous phage. The phage mixture is then added to a protein of interest to specifically identify the corresponding binding partner by several rounds of biopanning. Antigen binding phages can be selected and propagated, leading to identification of specific protein-binding partners. In the dual phage assay two filamentous phages are used for maximum specificity simultaneously. Each phage contains a selection marker, usually an antibiotic resistance gene. It is important to note that filamentous phages usually do not lyse, but transduce their host cell after infection. Moreover, both phages display a chemically crosslinked antibody on their surface each targeting a single specific, but different, epitope of the desired antigen. The assay is performed by applying both phages to the test sample. In case the targeted antigen is present, it will be bound specifically by both phages. Then, an indicator host strain is added to the sample and later on plated onto selective agar plates containing both selection markers. After successful transduction, arising colonies prove presence of the antigen in the sample. Moreover, simple colony counting reveals the titer of the antigen in the studied sample. Using bacteriophage M13 the dual phage assay can be nicely performed because cloning systems are readily available. 6.3.3  Phage endolysins and cell wall binding domains The life cycle of a bacteriophage can be divided into two distinct stages. On the one hand, the intact, non-replicating, but infectious particle is freely distributed in the environment by passive diffusion or external factors such as wind, current or through vectors such as insects, etc. On the other hand, phages pass through an intracellular stage of multiplication. Both entry and exit of host cells require

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interaction with the bacterial cell wall and membrane and, in each case, specific phage-encoded proteins mediate crossing the barriers. After multiplication, the host cell is lysed to release phage progeny. Here, a combinatorial mechanism consisting of two proteins comes into play. First, phage-encoded holin proteins insert into the bacterial cytoplasmic membrane where they form pores (Young, 1992). Then the endolysin passes through the pore by diffusion and binds to the peptidoglycan through its C-terminal cell wall binding domain (CBD). Simultaneously, the N-terminal enzymatic active domain (EAD) cleaves the peptidoglycan. Both domains are connected to each other by a flexible linker (Korndoerfer et al., 2006). Endolysins generally exhibit unusual high affinity to target cell surface (Loessner et al., 2002) and can be grouped into different protein classes which resemble endopeptidases, amidases, glycosamidases, muramidases and transglycosylases, targeting every possible bond of the peptidoglycan structure. It is evident that CBDs targeted against Gram-positive cells can also recognize their ligand when applied from without. Gaeng et al. (2000) genetically fused the endolysin ply511 to the signal peptide of the S-layer protein SlpA from Lactobacillus brevis to functionally express and secrete Ply511 from growing Lactococcus lactis cells. The enzyme was successfully secreted into the growth medium where it led to rapid lysis and death of L. monocytogenes cells. The use of such recombinant strains as starter cultures in cheese production currently remains an attractive option for the future. Kretzer et al. (2007) coated paramagnetic beads with CBDs of endolysins Ply118 and Ply500 for immobilization and separation of Listeria. While CBD118 binds to Listeria serovars 1/2 and 3, CBD500 binds to serovars 4, 5, and 6, therefore, these two CBDs feature non overlapping binding ranges and cover the full diversity of different Listeria serovars. Proof of concept for generalization of CDB approaches was provided by developing specific CDBs for Bacillus cereus and Clostridium perfringens. In magnetic separation experiments, CBD-coated beads exhibited high sensitivity compared to standard antibody-coated beads (Kretzer et al., 2007).

6.4 Application of bacteriophages to control bacterial pathogens in foods: an overview The idea of using phages as an agent against unwanted bacteria developed shortly after their discovery. However, due to the improvements in organic chemistry during the 1950s, exploration and development of broad spectrum antibiotics displaced interest in bacteriophage research in industrialized countries. Several laboratories have been testing suitability of bacteriophage isolates to control certain bacterial pathogens. A significant body of experience was gained at the Bacteriophage Institute in Tbilisi, Georgia, where phage therapy is routinely applied in medicine. Today treatment of antibiotic resistant bacteria is a challenging task. Because medicine faces severe problems in treatment of infectious diseases caused by (multiple) antibiotic resistant pathogens, the application of antibiotics © Woodhead Publishing Limited, 2011

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Table 6.1  Approved bacteriophage preparations for control of bacterial pathogens in food Target organism

Product

L. monocytogenes Listex™ P100 L. monocytogenes ListShield™ E. coli O157:H7 EcoShield™ Salmonella SalmShield™ X. campestris AgriPhage™ and P. syringae

Remarks Food safety product, virulent myovirus P100 received GRAS status in the USA and is approved for all foods susceptible to Listeria. Food safety product, phage cocktail consisting of six different bacteriophages applied to meat and poultry. Food safety product, phage cocktail consisting of three different bacteriophages applied on livestock prior to further processing. Food safety product, in development. ‘Pesticide’, preharvest control of bacterial spot on tomato and capsicum.

in mast and agriculture is prohibited, therefore research on the application of bacteriophages is again constantly increasing. 6.4.1  Treatment of Enterobacteria and Campylobacter Generally speaking, E. coli, Salmonella, and C. jejuni contaminations are associated with either cattle, pigs, fish, or poultry, respectively. With the exception of E. coli O157:H7, the bacteria can be asymptomatically present in the animal gastrointestinal tract (GIT) and, after slaughter, raw meat can become contaminated. Therefore, bacteriophages could be administered orally provided that they remain functional during GIT passage. Barrow and coworkers (1998) determined use of the K1-antigen-specific lytic bacteriophage to cure E. coli infection in chickens and calves. They found that protection was obtained even when administration of the phage was delayed until signs of disease appeared, and that the phage multiplied during the treatment period. Bach et al. (2003) applied bacteriophage DC22 for control of E. coli O157:H7 in an artificial rumen system and reported complete eradication after four hours incubation. Sheng et al. (2006) treated mice and cattle with lytic bacteriophages KH1 and SH1. Orally applied phages terminated E. coli O157:H7 from mice feces after two to six days. In a second experimental setup, E. coli O157:H7 was rectally injected to steers. After seven days phages were applied directly to the rectoanal junction mucosa and reduced the average number of E. coli O157:H7, but did not eliminate the bacteria completely. In the gut of experimentally inoculated sheep, Callaway et al. (2008) showed significant reduction of E. coli O157:H7 when a phage cocktail was given 24 hours after the bacteria. Recently, Rozema et al. (2009) reported oral administration of a bacteriophage cocktail to steers, but they found that E. coli O157:H7 counts were only nominally lower compared to the control. Interestingly, the authors noted

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that bacteriophages could also be isolated from the non-treated control animals and suggested that cattle might acquire phages from the feedlot environment. Goode et al. (2003) reported complete removal of Salmonella Enteritidis from contaminated chicken skin after application of lytic bacteriophages. When applied at high doses, even non-host Salmonella strains were eliminated, probably due to a phenomenon referred to as ‘lysis from without’. Here the phage specifically attaches to the host cell surface and penetrates both the cell wall and membrane, but is unable to replicate in the cytosol. Although the phage does not produce any offspring or lytic enzymes, a massive cell perforation leads to cell death because the membrane potential is disrupted. Another phage cocktail was used by Fiorentin et al. (2005) to treat artificially caeca-infected broilers. The cocktail was applied orally seven days after infection. After five days, a 3.5 log unit reduction was evident per gram caecal content compared to the controls. Cecal colonization of Ross broiler chickens by Salmonella Enteritidis and Thyphimurium could be effectively treated with newly isolated bacteriophages φ151 (Myoviridae) and φ10 (Siphoviridae), respectively. Both bacteriophages were chosen because they exhibit broad host ranges in in vitro studies. In vivo colonization by S. Enteritidis was reduced by four log units and colonization of S. Thyphimurium by two log units after 24 hours. However, a third bacteriophage which exhibited good performance in vitro failed to control Salmonella in vivo (Atterbury et al., 2007). Colonization of chicken by Campylobacter jejuni is also common, widespread and difficult to prevent. Loc Carrillo et al. (2005) employed orally administered bacteriophages CP8 and CP34 (both Myoviridae) and monitored up to five log unit reduction of C. jejuni in cecal content after five days. Goode et al. (2003) could successfully treat C. jejuni on chicken skin preparation and achieved 95% reduction. Suitability of bacteriophages for control purposes was also shown on food products. O’Flynn et al. (2004) evaluated three distinct lytic bacteriophages, e4/1c (Siphoviridae), e11/2, and PP01 (Myoviridae), either separately or as a phage cocktail for their ability to lyse E. coli O157:H7 both in vitro and on beef meat. In vitro treatment using phage cocktail or e11/2 and PP01 alone each resulted in a five log reduction of CFU after one hour at 37 °C. Similarly, phage e4/1c reduced cell counts by three logs within two hours at both 30 and 37 °C. However, the authors noted regrowth of the E. coli culture after phage treatment regardless of which phage or combination thereof was used, and the regrown cells exhibited an altered cell shape: they were smaller and coccoid shaped. On beef meat the phage cocktail completely eliminated E. coli in seven out of nine cases. Salmonella Enteritidis contamination of honeydew melon slices could be reduced by 3.5 log units under variable conditions. However, no significant effect was evident on apple slices probably because of the acidic pH (Leverentz et al., 2001). Modi et al. (2001) treated artificially contaminated milk containing a starter culture with bacteriophage SJ2, thereby reducing Salmonella counts by up to two logs. The milk was later used to produce cheddar cheese, and, even after several month of storage, reduced Salmonella counts were still evident. On

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Frankfurters the virulent bacteriophage Felix O1 (Myoviridae) reduced Salmonella cell counts efficiently by two log units (Whichard et al., 2003) and on sliced raw beef, application of the T-even like bacteriophage P7 (Myoviridae) resulted in an up to five log unit reduction (Bigwood et al., 2008). Recently, Kocharunchitt and coworkers (2009) described treatment of Salmonella Oranienburg on sprout seeds using bacteriophages SSP5 and SSP6; both were able to infect 65% of all tested strains. While SSP5 (Myoviridae) was only effective in vitro, phage SSP6 (Siphoviridae) could be applied to alfalfa seeds where it reduced Salmonella by one log after sixty minutes. Enterobacter sakazakii can grow in reconstituted infant milk formula which has been implicated in outbreaks of the pathogen. Kim and Loessner, (2008) isolated bacteriophages ESP1-3 (Siphoviridae) and ESP 732-1 (Myoviridae) and showed that phages were able to effectively inhibit growth of E. sakazakii in infant formula at 24 °C and 37 °C. Using 109 PFU per ml the organism could be completely eradicated. 6.4.2  Treatment of Listeria monocytogenes Infection by Listeria monocytogenes is submitted to humans exclusively via contaminated food. In many cases contamination occurs during food production and, therefore, reduction of L. monocytogenes should be performed in processing, e.g. during cheese ripening or salmon fillet packaging, etc. Application of Listeria bacteriophages on contaminated honeydew melons revealed a reduction of viable counts by up to four logs. On apple slices, the phage was found to be inactivated – similar to the situation with Salmonella phages (Leverentz et al., 2003). When applied in combination with nisin (bacteriocin), an additive effect was evident leading to 5.7 log reduction. Carlton et al. (2005) studied the biocontrol potential of the lytic bacteriophage P100 during surface ripening of red-smear soft cheese. They reported a frequency- and dose-dependent reduction of 3.5 log units or complete eradication of Listeria, respectively. Bacteriophage A511, a relative of P100, can also infect 95% of relevant L. monocytogenes serovars. Recently, Guenther et al. (2009) examined control of L. monocytogenes in several ready-to-eat foods. When applied to liquids such as chocolate milk and mozzarella cheese brine, Listeria counts rapidly dropped below detection limit. On solid food (hot dogs, sliced turkey meat, smoked salmon, mixed seafood, cabbage, and lettuce) a maximum of five log unit reduction was achieved. Generally, phage titers remained stable on animal products, while application on plant material resulted in inactivation by one log unit or more. 6.4.3  Control of spoilage bacteria by phages In addition to pathogenic bacteria, spoilage-causing bacteria can also be controlled by bacteriophages in foods. Greer (1983) isolated phages for the control of Brochothrix thermosphacta and treatment of adipose tissue discs revealed a two log reduction of B. thermosphacta counts and a three log increase in phage

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Fig. 6.2  Major discoveries and developments in bacteriophage research and application during the last century.

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numbers. Accordingly, off-odor development caused by the organism was suppressed and the storage life of adipose tissue could be doubled from four days in controls to eight days in treated samples. However, 68% of the surviving cells exhibited resistance to phage infection (Greer and Dilts, 2002). The same authors also intended to control beef spoilage caused by Pseudomonas, but unfortunately spoilage could not be prevented. Most likely the phages applied were not able to specifically target the indigenous spoilage microflora, which is comprised of other non-target pseudomonades. In fact the phages host ranges were shown to be narrow and the authors claimed a lack of specificity (Greer and Dilts, 1990).

6.5  Phage therapy: on the way to safer food? While bacteriophages have been shown to be specific and sensitive tools for detection of food-borne pathogens, there are still some concerns about their application in bacterial control. These can be summarized as follows. (i) Upon sudden and massive cell lysis, bacterial membrane-bound endotoxins might be released into treated samples. To circumvent this issue, Hagens et al. (2004) designed a non-replicating filamentous Pseudomonas phage Pf3R by replacing a phage export protein gene with a restriction endonuclease. The recombinant phage destroyed its target cells after infection due to action of the restriction enzyme, but was not able to lyse the cell and to produce any progeny particles. Importantly, infection by Pf3R significantly reduced endotoxin release. While bacterial lysis is a serious problem in medical phage therapy, it is of less importance for phage application in food production. (ii) Bacteria can become resistant against bacteriophage infection by diverse mechanisms, such as altering structure of surface components, DNA restriction and modification systems, plasmid-borne abortive systems, or clustered, regularly interspaced short palindromic repeats (CRISPR) in the bacterial genome sequences (Sturino and Klaenhammer, 2004; Nechaev and Severinov, 2008, and references therein). However, phage resistance after spontaneous mutation does not necessarily mean advantages for the bacterium in the absence of phage, but are likely to decrease the fitness of the organism or be detrimental. This phenomenon was described during phage treatment of E. coli O157:H7 contaminated beef (O’Flynn et al., 2004) and during phage therapy of broiler chickens colonized with C. jejuni (Loc Carrillo et al., 2005). Moreover, phages are continuously co-evolving with their hosts and adapt to bacterial defense strategies. In fact, phages can mutate at much higher frequency than bacteria and therefore maintain the efficacy of phage therapy (Parisien et al., 2008) and novel phage variants can be easily selected for in case phage resistant bacteria emerge (Smith et al., 1987). Provided that the host ranges are non-overlapping, e.g. that different phage receptors are required for phage adsorption, application of phage cocktails containing a set of different bacteriophages in a combination therapy (Sulakvelidze et al.,

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In general, the application of bacteriophages on foods requires high standards for the phage itself: bacteriophages should be strictly lytic, non-transducing, and covering a broad host range. Moreover, phages must not encode bacterial virulence factors, cause allergic reactions and should ideally receive GRAS status. They should be easy to propagate, ideally on a non-pathogenic or highly attenuated production strain in relatively high yields. On the other hand, foods treated with phages have to meet requirements, e.g. pH, concentration of salt and other osmolytes, and temperature should be in a specific range so that the phage is not inactivated. In order to achieve a sufficient coverage, particularly on solid or semi-solid foods, a relatively high dose of phage should be applied. While bacteriophage preparations can be easily added to liquid food, their distribution on solid food can be achieved by spraying or nebulizing. Usually 108 PFU per ml, gram or cm2 reduces bacterial counts very efficiently (Guenther et al., 2009). However, optimal coverage of specific food items depends on their particular surface texture, and it is necessary to adapt the individual conditions for use of phage for best results (Hagens and Loessner, 2010). Phage therapy appears as a very effective tool for food preservation. Accordingly, some bacteriophage preparations were already approved by the U.S. Food and Drug Administration and by the U.S. Department for Agriculture. The preparations are commercially available and can be applied to control pathogenic bacteria, e.g. Listeria monocytogenes (approved for control in all foods susceptible to Listeria), E. coli O157:H7 (approved for application on livestock) or Xanthomonas campestris and Pseudomonas syringae (preharvest control of bacterial spot on tomato and capsicum). Implemented in industrial production processes, phage preparations can be used either prophylactic or to remove a nascent contamination. In practice, phages can be applied either on working surfaces of production facilities or directly on food products. In the long term more phage preparations might be considered as disinfectants, processing aids or food additives to further enhance safety and quality of food.

6.6  References and eisenstark a (1974). ‘The present state of phage taxonomy.’ Intervirol 3: 201–19. ackermann h w (2007). ‘5500 phages examined in the electron microscope.’ Arch Virol 152: 227–43. atterbury r j , van bergen m a , ortiz f , lovell m a , harris j a et al. (2007). ‘Bacteriophage therapy to reduce salmonella colonization of broiler chickens.’ Appl Environ Microbiol 73: 4543–9. bach s j , mcallister t a , veira d m , gannon v p j and holley r a (2003). ‘Effect of bacteriophage DC22 on Escherichia coli O157:H7 in an artificial rumen system (Rusitec) and inoculated sheep.’ Anim Res 52: 89–101. ackermann h w

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and berchieri a jr. (1998). ‘Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and meningitis in chickens and calves.’ Clin Diagn Lab Immunol. 5: 294–8. bigwood t , hudson j a , billington c , carey - smith g v and heinemann j a (2008). ‘Phage inactivation of foodborne pathogens on cooked and raw meat.’ Food Microbiol 25: 400–6. brüssow h and kutter e (2005). ‘Phage ecology.’ In Kutter E and Sulakvelidze A (eds) Bacteriophages – Biology and Application, New York, CRC Press, 131 pp. callaway t r, edrington t s , brabban a d , anderson r c , rossman m l et al. (2008). ‘Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts.’ Foodborne Pathog Dis 5: 183–91. carlton r m , noordman w h , biswas b , de meester e d and loessner m j (2005). ‘Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxicity study, and application.’ Regul Toxicol Pharmacol 43: 301–12. datz m , janetzki - mittmann c , franke s , gunzer f , schmidt h and karch h (1996). ‘Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes.’ Appl Environ Microbiol 62: 791–797. de siqueira r s , dodd c e r and rees e d r (2003). ‘Phage amplification assay as rapid method for Salmonella detection.’ Brazilian Journal of Microbiology 34: 118–120. digirolamo r, wiczynski l , daley m , miranda f and viehweger c (1972). ‘Uptake of bacteriophage and their subsequent survival in edible West Coast crabs after processing.’ Appl Microbiol 23: 1073–6. fiers w, contreras r, duerinck f , haegeman g , iserentant d et al. (1976). ‘Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene.’ Nature 260: 500–7. fiorentin l , vieira n d and baroni w jr. (2005). ‘Oral treatment with bacteriophages reduces the concentration of Salmonella Enteritidis PT4 in caecal contents of broilers.’ Avian Pathol 34: 258–63. gaeng s , scherer s , neve h and loessner m j (2000). ‘Gene cloning and expression and secretion of Listeria monocytogenes bacteriophage-lytic enzymes in Lactococcus lactis.’ Appl Environ Microbiol 66: 2951–8. goode d , allen v m and barrow p a (2003). ‘Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages.’ Appl Environ Microbiol 69: 5032–6. goodridge l , chen j and griffiths m (1999). ‘The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk.’ Int J Food Microbiol 47: 43–50. greer g g (1983). ‘Psychrotrophic Brochothrix thermospacta bacteriophages isolated from beef.’ Appl Environ Microbiol 46: 245–51. greer g g and dilts b d (1990). ‘Inability of a bacteriophage pool to control beef spoilage.’ Int J Food Microbiol 10: 331–42. greer g g and dilts b d (2002). ‘Control of Brochothrix thermosphacta spoilage of pork adipose tissue using bacteriophages.’ J Food Prot 65: 861–3. guenther s , huwyler d , richard s and loessner m j (2009). ‘Virulent bacteriophage for efficient biocontrol of Listeria monocytogenes in ready-to-eat foods.’ Appl Environ Microbiol 75: 93–100. hagens s , habel a , von ahsen u , von gabain a and bläsi u (2004). ‘Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage.’ Antimicrob Agents Chemother 48: 3817–22. hagens s and loessner m j (2010). ‘Bacteriophage for biocontrol of foodborne pathogens: calculations and considerations.’ Curr Pharm Biotechnol 11: 58–68.

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and kuhn j (1987). ‘Introduction of lux genes into bacteria, a new approach for specific determination of bacteria and their antibiotic susceptibility.’ In J Sclomerich, R Andreesen, A Kapp, M Ernst, and WG Woods (eds), Bioluminescence and Chemiluminescence: New Perspectives, Wiley Interscience, Bristol, United Kingdom, pp. 463–72. whichard j m , sriranganathan n and pierson f w (2003). ‘Suppression of Salmonella growth by wild-type and large-plaque variants of bacteriophage Felix O1 in liquid culture and on chicken frankfurters.’ J Food Prot 66: 220–5. whitman p a and marshall r t (1970). ‘Characterization of two psychrophilic Pseudomonas bacteriophages isolated from ground beef.’ Appl Microbiol 22: 463–8. whitman p a and marshall r t (1971) ‘Isolation of psychrophilic bacteriophage-host systems from refrigerated food products.’ Appl Microbiol 22: 220–3. wilson s m , al - suwaidi z , mcnerney r, porter j and drobniewski f (1997). ‘Evaluation of a new rapid bacteriophage-based method for the drug susceptibility testing of Mycobacterium tuberculosis’. Nat Med 3: 465–8. wolber p k and green r l (1990). ‘Detection of bacteria by transduction of ice nucleation genes.’ Trends Biotechnol 8: 276–9. young r (1992). ‘Bacteriophage lysis: mechanism and regulation.’ Microbiol Rev 56: 430–81. ulitzur s

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Part II Applications of protective cultures, bacteriocins and bacteriophages in food animals and humans

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7 Using antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry P. L. Connerton, A. R. Timms and I. F. Connerton, University of Nottingham, UK

Abstract: This chapter will focus on the use of antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry. The characteristics, practical considerations together with potential shortcomings of each type of treatment will be described, along with specific examples of their application. Pathogenic bacteria from the genera Campylobacter and Salmonella constitute a common challenge to the poultry industry world wide in terms of reducing human food-borne disease. Because of the pre-eminence of these genera, this chapter will focus on research aimed at controlling these food-borne pathogens. Key words: antimicrobial cultures, bacteriocins, bacteriophages, food-borne bacterial pathogens, Campylobacter jejuni, Salmonella.

7.1  Introduction This chapter will focus on the use of antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry. The characteristics and practical considerations, together with potential shortcomings of each type of treatment, will be described along with specific examples of their application. Pathogenic bacteria from the genera Campylobacter and Salmonella constitute a common challenge to the poultry industry world wide in terms of reducing human food-borne disease. Because of the pre-eminence of these genera this chapter will focus on research aimed to control these food-borne pathogens. The 181 © Woodhead Publishing Limited, 2011

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composition of the normal microbial flora of the poultry intestine is governed by many factors including: bird age, diet, disease status, environmental factors and the immune response (Mead, 2005). Food-borne pathogens may form part of this flora but this is not an inevitable outcome. This chapter will centre on the use of antimicrobial cultures, bacteriophages and bacteriocins to manipulate the normal flora to reduce carriage of pathogens and/or prevent them becoming established. It is unrealistic to expect that these treatments would provide a magic bullet to eradicate pathogens from poultry altogether, but combined with other methods (hurdle technology) could prove to be effective and sustainable. The most important criteria for a treatment involving the administration of antimicrobial cultures, bacteriocins or bacteriophages to poultry is that their effects are benign on the health of the birds, and the eventual consumer. One way to ensure this level of safety is to employ microbes that are already present in the intestine of poultry, either directly or indirectly for example to produce bacteriocins. Thus, nothing is being added that is not already present and the risk of ill effects, including allergies, is minimised. The bacteria or phage populations taken from the normal flora of birds are simply manipulated to achieve the best outcome in reducing pathogens when returned to their natural host. The use of bacteriophages and other microbial treatments have been recognised for many years but the advent of the antibiotic era resulted in these types of treatments being left undeveloped. Recently, there has been renewed interest in more natural, sustainable types of treatments to reduce pathogens which stems from the increase in antibiotic resistance among food-borne pathogens and a general desire to use fewer chemicals in food production.

7.2 Antimicrobial cultures to reduce carriage of food-borne bacterial pathogens in poultry 7.2.1  Overview of antimicrobial cultures Antimicrobial cultures administered to live birds via the oral route are usually known as ‘probiotics’. The definition of a probiotic is ‘a live microbial feed supplement which beneficially affects the host animal by improving its microbial balance’ (Fuller, 1989). In the context of pathogen reduction, use of the term ‘probiotic’ is slightly at variance with the usual definition as the improvement in microbial balance does not necessarily benefit the bird but will benefit the consumer. Probiotic species may be overtly antagonistic to the growth and persistence of target pathogenic bacteria or be able to effectively out-compete the pathogen in its preferred intestinal niche. The latter types of microorganisms are said to work by competitive exclusion (CE). Probiotics that are administered to poultry usually contain one or more defined microorganisms with potentially different effects and often include lactobacilli. However, the beneficial effects in either treatment type can be through a number of means, although all the actual mechanisms are not fully understood. The suggested mechanisms include: competition between © Woodhead Publishing Limited, 2011

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pathogen and normal flora either for adhesion sites or for nutrients, or by production of inhibitory substances or conditions that affect the pathogen. Other means include stimulation of immune response or sequestration of the pathogen by co-aggregation (reviewed by Mead, 2005). 7.2.2  Probiotics The selection of defined probiotic organisms, for administration to poultry to prevent colonisation by food-borne pathogens, has been made largely on an empirical basis. Not only do such organisms have to be effective against their target, but they must also be able to withstand acid pH in the proventriculus of poultry, withstand the potentially hostile intestinal environment, be able to colonise effectively when they reach their colonisation site, must be amenable to commercial culture and remain stable during storage. They must also be shown to be harmless to the animals and to consumers and have no adverse effects on the feed conversion rates of the birds. These exacting requirements make the search for effective probiotics challenging. Studies have generally been carried out using relatively small-scale laboratory trials with little information available regarding the commercial scale of production required for application. When carrying out trials with probiotic organisms it is important to consider the effects of the environment and the stress status of the birds, particularly feed withdrawal, as these factors may greatly affect the results (Patterson and Burkholder, 2003). Early trials of probiotic microorganisms proved fairly ineffective against prevention of salmonellas (Mead, 2005). However, in recent years promising results have been obtained using various lactobacilli, for example L. acidophilus and L. salivarius, Enterococcus faecium and some fungi such as Saccharomyces cerevisiae, to reduce colonisation principally by salmonellas and Campylobacter. Examples of commercially available products include: PrimaLac, containing Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium and Bifidobacterium bifidium (Grimes et al., 2008); Calsporin, containing Bacillus subtilis C-3102 (Fritts et al., 2000); Avian Pac Plus, which contains Lactobacillus acidophilus, Streptococcus faecium, together with S. typhimurium-specific antibodies (Promsopone et al., 1998). From a practical point of view probiotics can be relatively easily administered to poultry either in drinking water or mixed with food or even through spraying the bird’s feathers leading to ingestion through preening. They are relatively cheap to produce but stability on long term storage of live cultures may be an issue. For a review of probiotics relating to poultry in general see Nava et al. (2005). 7.2.3  Competitive exclusion The concept and use of competitive exclusion (CE) was pioneered by Nurmi and Rantala (1973) who put forward the idea that attempting to keep newly hatched birds in abnormally hygienic conditions caused them to be more vulnerable to

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colonisation by pathogens, in particular by salmonellae. This could be remedied by administering protective microorganisms, predominantly anaerobes, obtained from older birds whose established flora provides a greater resistance to colonisation by salmonellae. A large number of studies have followed (reviewed by Mead, 2000; Wagner, 2006), that involved administering various preparations of microorganisms, collected from the caeca, the caecal mucosa, excreta or even spent litter from adult birds, to very young chicks. One of the problems of using CE preparations is that, because of the unknown bacterial composition, they are not acceptable to regulatory agencies in some countries. The drawback of having an undefined preparation of microorganisms is that they may include transferable antimicrobial drug resistance and virulence genes that would obviously be undesirable in a food product for consumption by humans (Wagner, 2006). To solve this problem researchers have attempted to produce defined products that mimic the natural protective flora of poultry (reviewed by Stavric, 1992; Nisbet, 2002). Examples of commercially available CE products (Schneitz, 2005) include defined cultures such as: Broilact, which is composed of 32 identified bacteria able to adhere to the gut wall of the bird; and Preempt, consisting of 15 facultative anaerobic bacteria and 14 obligate anaerobic bacteria. Examples of CE products that rely on undefined cultures include Aviguard, Avifree and MSC. Despite much progress in the development of CE as a viable treatment, one of the greatest challenges to overcome is the fact that treatments that give protection against Salmonella colonisation do not necessarily prevent colonisation by Campylobacter and other pathogens. For the latter this is probably because campylobacters have evolved to exploit a different niche from most intestinal bacteria being closely associated with the intestinal mucosa (Beery et al., 1988). However, bacteria that inhibit Campylobacter can be isolated from the caeca of chickens under anaerobic conditions (Aho et al., 1992; Zhang et al., 2007) but the effect on their target may be dependent on the particular type of bird (Laisney et al., 2004). Therefore, combinations of treatments aimed at specific pathogens will most probably be the most effective strategy. 7.2.4  Prebiotics Prebiotics are defined as ‘non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon and thus attempt to improve host health’ (Gibson and Roberfroid, 1995). The potential to stimulate beneficial microbes that may reduce or inhibit colonisation by food-borne pathogens in food animals is an obvious, but attractive, departure from this original idea. Prebiotics are generally sugars which provide a substrate for the growth of a limited range of bacteria and are not metabolised by the host, for example fructo-oligosaccharides and mannose-oligosaccharides (reviewed by Patterson and Burkholder, 2003). These have been shown to have the potential to reduce colonisation by Salmonella and Campylobacter (Schoeni and Wong, 1994; Fukata et al., 1999; Spring et al., 2000). While the effect is thought largely to be

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due to the boost in beneficial microorganisms resulting in competition, it is also suggested that prebiotic oligosaccharides may have a direct anti-adhesive activity by inhibiting the adherence of pathogens to the host epithelial cell surface (Shoaf et al., 2006). 7.2.5  Combination therapies By applying a competitive exclusion flora to one-day-old chicks then feeding them with a combination of a probiotic organism together with a prebiotic, Revelledo et al. (2009) demonstrated that this combination of treatments was more effective against Salmonella colonisation than any of the individual treatments alone. It is likely that combinational treatments will be the focus of more studies in the future to optimise treatments, and that these will include considerations relating to nutrient requirements and feed conversion rates in broiler chickens and turkeys.

7.3 Bacteriocins to reduce carriage of food-borne bacterial pathogens in poultry 7.3.1  Overview of bacteriocins Bacteriocins are defined as small peptide or protein toxins that are produced by bacteria and are active against other, often closely related, bacteria. While their use as food preservatives is well established, the use of bacteriocins in live farm animals is at an early stage of development (reviewed by Diez-Gonzalez, 2007). However, models of the digestive system have indicated the potential for bacteriocins to survive and remain active in the intestine (reviewed by Joerger, 2003). 7.3.2  Characteristics of bacteriocins Bacteriocins were first described by Gratia in 1925 as filtrates of E. coli that inhibited the growth of another strain of the same species. Bacteriocins are produced by both Gram-positive and Gram-negative bacteria, and were the focus of a great deal of pioneering research during the 1940s and 1950s (reviewed by Gratia, 2000). Bacteriocins produced by the lactic acid bacteria (LAB) as constituents of fermented milk products have been consumed since ancient times, and in the context of their historic use they are perceived to be natural and assumed to be safe (Cleveland et al., 2001). Perhaps the best known of these is nisin, which is in common use as a preservative in the food industry (Abee et al., 1995). Nisin has a long history of use since its discovery in 1928 and recovery from Lactococcus lactis (Hurst, 1967). The activity of bacteriocins produced by LAB is usually, but not exclusively, confined to Gram-positive species (Cotter et al., 2005), but the fact that bacteriocins form such a heterogenous group of peptides and proteins has led to difficulties in their classification. A proposed classification scheme (Cotter et al., 2005), suggests assigning them to one of two groups, class I and class II.

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The class I bacteriocins include the lanthionine-containing antibiotics, which are post-translationally modified peptides that contain unusual amino acids such as lanthionine. Class II bacteriocins include the non-lanthionine-containing bacteriocins, which can be further sub-divided into groups such as the class IIa pediocin-like bacteriocins. It is the class II bacteriocins that are more common and probably more useful in terms of food safety applications. The structural and functional characteristics of the different sub-groups within this class have been reviewed by Cotter et al. (2005) and Drider et al. (2006). Bacteriocins are often described using the genus or species designation of the bacterium that produces them (e.g. staphylococcins, colicins etc), and are classified according to their molecular weight. Some are small (< 40 amino acids), whereas others are large, with molecular weights exceeding 90 000 Da. The genes encoding bacteriocin production can be chromosomally located or associated with mobile genetic elements, such as plasmids or transposons, reviewed by Cleveland et al. (2001) and Drider et al. (2006). The modes of action of the bacteriocins are variable; they may inhibit cell-wall formation, possess nuclease activity or they may cause pores to form in cell membranes (Héchard and Sahl, 2002; Drider et al., 2006). Bacteriocins are often, but by no means universally, heat-stable (15 minutes at 100 °C) but as proteins they can be sensitive to hydrolysis upon exposure to proteolytic enzymes. Proteolysis can of course be an obstacle if their intended use is as a feed component in order to reduce intestinal bacteria in live animals. Unless protected, the bacteriocin can be degraded through proteolysis before affecting the target bacteria. Bacteria that produce bacteriocins are always immune to the bacteriocin they produce, often through the production of an associated immunity protein (reviewed by Drider et al., 2006). Genes that encode these proteins are as a consequence generally located in close proximity to those responsible for bacteriocin synthesis (Siegers and Entian, 1995). However, the exact mechanisms of immunity are complex and, as yet, poorly understood. Resistance of the target species to bacteriocin exposure has also been reported, with research focused on the class II bacteriocins (reviewed by Cotter et al., 2005; Draper et al., 2008) and those active against Listeria monocytogenes (Gravesen et al., 2002). It is however anticipated that, by using a combination of strategies that include bacteriocins to control pathogens (hurdle technology), development of resistance to bacteriocins should not be an insurmountable problem, although it is clear that further research is required into the causes of resistance to ensure the sustainability of this approach. 7.3.3  Use of bacteriocins to inhibit Campylobacter and Salmonella Antagonistic activities of several bacteria have been demonstrated against the growth of campylobacters (Humphrey et al., 1989; Schoeni and Doyle, 1992; Chaveerach et al., 2004; Nazef et al., 2008; Shin et al., 2008). Similarly, antagonistic activities against Salmonella have also been demonstrated (Svetoch et al., 2008) but reports seem to be far less numerous perhaps due to commercial sensitivities of the findings. The Campylobacter-antagonistic bacteriocins produced by Bacillus

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circulans and Paenibacillus polymixa have been characterised (Svetoch et al., 2005). Purified preparations of the bacteriocin from Paenibacillus polymixa have been microencapsulated in polyvinylpyrrolidone and incorporated into chicken feed. Feeding chickens these medicated feeds has proven to be successful in reducing or even preventing the colonisation of chickens by C. jejuni (Stern et al., 2005). The same combination was also shown to significantly reduce C. coli colonisation to undetectable levels in turkey poults (Cole et al., 2006). Two important recent developments are the demonstration of the efficacy in chicken treatment trials, of bacteriocins produced by Lactobacillus salivarius OR-7 and Enterococcus faecium E 50–52 (Stern et al., 2006; Svetoch et al., 2008). Both are class IIa bacteriocins and have broad activity against C. jejuni types but E50–52 also shows activity against Yersinia spp., Salmonella spp., Escherichia coli O157:H7, Shigella dysenteriae, Morganella morganii, Staphylococcus spp., and Listeria spp. E50–52 produced a 6 log10 decline in numbers of C. jejuni and Salmonella enteritidis in the caeca of broiler chickens and the wide range of antibacterial activity exhibited by purified bacteriocin E50–52 against pathogens is obviously an exciting development. Similarly, Line et al. (2008) reported the isolation and purification of enterocin E-760, also with a broad antimicrobial activity that resulted in an impressive 8 log10 decline in Campylobacter counts in broiler chicken trials. A summary of bacteriocins that have been shown to be effective in reducing pathogens in chickens is given in Table 7.1.

7.4 Bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry 7.4.1  Discovery and taxonomy Bacteriophages, often simply called phages, are defined as viruses that can infect and replicate on susceptible bacteria. They are ubiquitous in the environment, with recent estimates placing the number of phages in the biosphere at around 1031 phage particles, making them the most abundant biological entities on the planet (Hendrix et al., 1999). Based on work credited to their co-discoverers, Frederick Twort and Félix d’Hérelle (Twort 1915; d’Hérelle 1917), they were first identified almost 100 years ago. Seventeen families of phage are now recognised, based principally on their morphological characteristics and nucleic acid content (Ackermann, 2007). However, by far the most frequently encountered bacteriophages are the tailed phages with genomes of double-stranded DNA, these account for approximately 96% of phages so far characterised using electron miscroscopy (Ackermann, 2001). Less frequently encountered bacteriophages may have genomes comprising singlestranded DNA or RNA and include a variety of morphological forms, for example polyhedral, filamentous and pleomorphic phages, but these forms currently only account for a small minority (3–4%). Bacteriophages that infect Campylobacter generally belong to either the Myoviridae or Siphoviridae tailed phage families. An electron micrograph of a Campylobacter bacteriophage with typical Myoviridae morphology is shown in

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Table 7.1  Summary of bacteriocins that have been shown to be effective in reducing pathogens when administered to chickens Bacteriocin Bacterial producer

Species of bacteria shown to be inhibited

Reference

B602 Paenibacillus polymixa OR-7 Lactobacillus salivarius E50–52 Enterococcus faecium E-760 Enterococcus spp.

C. jejuni C. coli C. jejuni C. coli C. jejuni Yersinia spp. Salmonella spp. E. coli O157:H7 Shigella dysenteriae Morganella morganii Staphylococcus spp. Listeria spp. C. jejuni Salmonella spp. E. coli O157:H7 Y. enterocolitica Citrobacter freundii Klebsiella pneumoniae S. dysenteriae Pseudomonas aeruginosa Proteus mirabilis M. morganii Staphylococcus spp. L. monocytogenes

Svetoch et al. (2005) Cole et al. (2006) Stern et al. (2006) Cole et al. (2006) Svetoch et al. (2008)

Line et al. (2008)

Fig. 7.1. In common with the prototype phage T4 of Escherichia coli, Campylobacter phages of the Myoviridae have DNA base modifications that make them difficult to clone and sequence, nevertheless the first genomic sequence has recently been completed at the Sanger Institute (www.sanger.ac.uk). The essential characteristics of bacteriophages that infect Campylobacter are reviewed by Connerton et al. (2008). Campylobacter-specific bacteriophages can be readily isolated from poultry excreta regardless of their mode of production (Connerton et al., 2004; Atterbury et al., 2005; El-Shibiny et al., 2005; Loc Carrillo et al., 2007). Additionally, at least a proportion of the phages associated with broiler chickens remain viable in processing plants and can be isolated from retail chicken portions (Atterbury et al., 2003a; Tsuei et al., 2007). Similarly Salmonella-specific phages can be isolated from poultry farms, abattoirs and waste water (Andreatti Filho et al., 2007; Atterbury et al., 2007) and were also shown to be from the Myoviridae or Siphoviridae tailed phage families (Atterbury et al., 2007). A T7-like phage with a short non-contractile tail typical of the family Podoviridae has also been shown to infect Salmonella enterica serovar Gallinarum biovar Gallinarum (Kwon et al., 2008).

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Fig. 7.1  Electron micrograph of Campylobacter bacteriophage CP8.

7.4.2  Phage lifecycles Two major types of phage lifestyle are generally distinguished, these being termed the lytic and the lysogenic life cycles, with the phage adopting these routes termed virulent (lytic) phage or temperate (lysogenic) phage respectively. A few examples exist of alternative lifecycles, for example the filamentous phages that do not fit neatly into either category and may form a third grouping. The virulent and temperate phages share certain characteristics; they always infect from the outside, requiring specific receptors on the external surfaces of bacteria. Upon infection they use the host cell to produce more phage particles and release these as a burst of phages through cellular lysis. Phage amplification then occurs via successive rounds of infection and replication. However, while temperate phages usually replicate using a lytic pathway occasionally rather than lyse the host, they will integrate their DNA into the bacterial genome thus rendering the bacterium resistant to further infection through the production of a phage-encoded repressor. The repressor regulates expression of both the phage’s own genes and those of other related phages that may subsequently infect, termed homo- and hetero-immunity respectively.

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Furthermore, lysogenic phages are prone to transduction (the phage-mediated transfer of genetic material from one bacterial host to another), and this form of DNA transfer may include the dissemination of pathogenic traits among their hosts (Cheetham and Katz, 1995; Boyd and Brussow, 2002). Phages themselves are known to carry toxinogenic genes, for example shiga-like toxin-producing strains of E. coli that include O157:H7 carry the toxin-encoding genes on lamboid prophages that are integrated into the bacterial genome (Scotland et al., 1983; O’Brien et al., 1984). The toxin-encoding genes are under the genetic control of the integrated phage genome, with the consequence that any factor that will commit the prophage to excise and initiate replication and lysis will result in an increased expression of the toxin (Smith et al., 1984). These factors can include stress, such as antibiotic therapy or even the presence of a susceptible host bacterial population that allows amplification of spontaneously released phages (Gamage et al., 2003). Hence, the choice of phage is critical to the success of potential phage treatments and the phage must be virulent and demonstrate high potency against the target bacteria, although it should be noted that not all phages that are able to lyse the target bacteria in the laboratory are suitable for practical application (Berchieri et al., 1991; Reynaud et al., 1992). Thus, temperate phages should not be used to reduce the risk of disseminating unfavourable genetic traits through lysogeny and transduction (Schicklmaier and Schmieger, 1995). In contrast, obligatory virulent phages, such as the T-even phages of Escherichia coli that adopt a lytic lifestyle that results in lysis and death of the host, make them ideal candidates for antibacterial phage intervention. 7.4.3  Bacteriophage therapy The potential of applying bacterial viruses for the treatment of bacterial infections (phage therapy) was recognised not long after their discovery (d’Herelle, 1922), although their use in many countries was eclipsed by the development and commercial production of antibiotics. In contrast, former Warsaw Pact countries have exploited the use of bacteriophages for therapeutic, prophylactic and disinfection purposes for many years, reviewed by Alisky et al. (1998). It is only since the dramatic rise in multi-drug resistant bacteria that Western scientists have re-examined phage therapy as an alternative to combat infection; see the reviews by Merril et al. (2003), Sulakvelidze and Morris (2001) and Summers (2001). Modelling phage treatment Successful phage therapy depends on various parameters, including dosage size, treatment timing, phage absorption-rate and fecundity (Levin and Bull, 1996; Payne and Jansen, 2001; Weld et al., 2004). Phage infection is critically dependent on the density of the susceptible host population and early therapeutic failures may have been due to a general lack of understanding of the kinetics of phage– host interaction. There is thought to be a distinct threshold, termed the ‘phage proliferation threshold’ (Wiggins and Alexander, 1985; Payne and Jansen, 2003), above which phage numbers increase by replication and below which they

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decrease due to ‘natural wastage’. This threshold may appear to be higher in vivo due to the potentially greater rate of phage loss and non-homogeneous nature of the milieu (Payne and Jansen, 2002). Reduction in numbers of the target bacteria may also be dependent on the phage numbers exceeding the ‘inundation threshold’, which is defined as the concentration of phages required to effect a reduction in bacterial numbers. It is difficult to translate information gained from a model system, using a single strain of bacteria and phages in a homogeneous and controlled environment, to the situation in vivo. The intestinal environment is extremely complex, where various factors can affect phage efficacy including: host defences, proteolytic enzymes and physical factors such as constant flowthrough of material, absorption to food particles or non-host bacteria that may reduce the phage titre significantly (Rabinovitch et al., 2003). In addition, the kinetics of phage absorption in the intestine may be quite different from that in laboratory media, due to the viscosity of the mucus layer (Weld et al., 2004) and spatial distribution of the target organism. The choice of strategy to achieve the desired therapeutic outcome depends on whether a ‘passive’ or an ‘active’ mode of treatment is deemed desirable. The two thresholds defined above can be used to help define these modes of phage action (Cairns et al., 2009). When phages are mixed with susceptible bacteria at ratios where phages greatly outnumber the bacteria and exceed the inundation threshold (i.e. high multiplicity of infection, MOI) bacteria may be ‘lysed from without’, due largely to the destabilisation of bacterial membranes. This may lead to an initial drop in numbers of bacteria but also of phages, as free phages may adhere to the large amounts of bacterial-cell debris rather than to healthy cells (Rabinovitch et al., 2003). This strategy is known as ‘passive inundation’, where the phages simply overwhelm the bacteria and do not replicate (Payne and Jansen, 2001). In contrast an alternative strategy, known as ‘active proliferation’ (Payne and Jansen, 2001), involves the provision of a low initial dose of phages that then actively replicates or proliferates on target bacteria, provided the hosts are above the proliferation threshold. The result is an eventual decline in numbers of bacteria, when phages have replicated sufficiently to exceed the inundation threshold. This has the advantage that less starting material is required, but may allow time for phage insensitive bacteria to dominate, hence timing of treatment is critical if this method is to be adopted. Phage resistance The selection of resistant bacteria has always been perceived as a potential obstacle to phage therapy (Barrow, 2001), and has been reported following experimental phage treatments (Smith and Huggins, 1982; Smith et al., 1987a; Sklar and Joerger, 2001). However, phage resistance is usually acquired at a price, such as a reduction in the colonisation potential or virulence of an organism. Selecting phages that target a virulence factor, such as the capsular antigen (K) of E. coli, has proved to be of particular value, since the number of resistant variants isolated following phage treatment can be low (Smith and Huggins, 1983; Smith et al., 1987a; Levin and Bull, 1996). Evidence against the dominance of phage-resistant populations can be

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gained from the examination of natural phage infections. We have previously conducted a longitudinal study of a broiler chicken house naturally infected with Campylobacter and phages over three successive rearing cycles (Connerton et al., 2004). Occasionally, phage-resistant Campylobacter strains could be isolated, but these did not dominate or outgrow the sensitive types; instead, they co-existed. In this case, the parental, phage-sensitive Campylobacter strain and its phages were maintained from the first flock to the next. However, in this second flock, the phagesensitive Campylobacter had largely, but not completely, been replaced by several genotypically unrelated phage-insensitive strains, probably by succession rather than de novo development of resistance (Scott et al., 2007b). In this case, the broilerhouse was selected specifically because of the carry-over of strains from the first to the second flock. In practice, however, Campylobacter strains do not always persist from one flock to the next within a broiler-house (Petersen and Wedderkopp, 2001; Shreeve et al., 2002). Moreover, the experimental transfer of litter contaminated with excreta from a Campylobacter-colonised flock to a new broiler-house did not result in colonisation by genotypically-related strains in chickens reared in the new house, indicating that the incomplete clearance of litter is probably not a critical source for the transfer of infection between subsequent flocks inhabiting the same broiler-house (Payne et al., 1999). In the UK, it is common practice to remove all the litter between flocks and the resulting litter slurries are often negative for the culture of campylobacters, despite positive isolation from birds that were reared on the same litter. The observation that phage-resistant campylobacters do not emerge as dominant populations, despite their obvious advantage in the presence of phages, and the observation that the majority of infected flocks do not lead to Campylobacter strain carry-over, would indicate that campylobacters are acquired from the environment and that phage treatment is unlikely to be selected for the persistence of specific resistant types in the broiler-house environment. 7.4.4  Use of bacteriophages to reduce the presence of pathogens in poultry Bacteria such as Campylobacter or certain Salmonella serovars are commensal organisms of poultry and cause no obvious pathogenesis in the birds, but are obvious pathogens of man. It is unlikely that bacteriophages could be used to completely eradicate these target organisms, since predators seldom totally eliminate their hosts in nature (van den Ende, 1973; Alexander, 1981). However, mathematical models for the risks associated with Campylobacter infection in Denmark, for instance, indicate that reductions of 2 log10 or greater in number of viable organisms on chicken carcasses could result in a significant reduction (30 times fewer) in the incidence of campylobacteriosis associated with consumption of chicken meals (Rosenquist et al., 2003; Lindqvist and Lindblad, 2008). Therefore, even treatments that do not eliminate, but reduce the numbers below critical thresholds, may have beneficial effects on public health. Synergistic strategies that combine microbial treatments with physical and hygiene-control measures could bring about significant reductions in the exposure of the human population to pathogens.

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Practical considerations Phages have the advantage that they are fairly robust in nature and therefore can simply be added to drinking water and feed, provided that the intended targets are intestinal bacteria. However, some phages may be sensitive to the low pH encountered in the stomach or proventriculus (Leverentz et al., 2003). This problem can be overcome through the use of antacid or by selection of appropriate low-pH-tolerant phages. Antacids, such as Maalox (aluminium and magnesium hydroxide) or calcium carbonate, have been used to improve the ability of phages to survive low acidity in digestive systems (Smith et al., 1987b; Koo et al., 2001). The point in the poultry rearing cycle at which phages are applied may be critical to success. If the strategy is to overwhelm the bacteria with phages, then administration two to three days before slaughter would reduce the chance of resistance developing. If, however, active phage replication is required, then there will be a prerequisite delay to allow phages to proliferate before the impact on bacterial host numbers. The lead-time must account for phage absorption rates, phage replication rates, the inherent dilution factors associated with the intestinal contents and the transit time of the gut. These processes may be estimated from model data, but the estimates will require validation in practice. Quality control may be necessary to ensure that the treatment phages can be distinguished from wild type phages and that the phages recoverable from treated birds are the same as those administered. Phages are mutable and can evolve with their host, so the efficacy of stocks must be checked. They are also frequent and ubiquitous in the environment, so that contamination of stocks can easily occur. The frequency at which Campylobacter phages are isolated from conventional broilerchicken caecal contents (those that can be propagated on a universal propagating strain C. jejuni PT14) has been estimated to be 17% in the UK (Atterbury, 2003b). The frequency observed in extensively reared birds (organic and free-range flocks) that are exposed to the environment is significantly higher at 50% (El-Shibiny et al., 2005). The frequency of phages that are specific to other pathogens in poultry intestines is completely unknown, although one report aiming to evaluate phages as faecal indicators showed the incidence of F+ RNA coliphage, somatic coliphage and Salmonella phages from chicken breast meat to be 100, 69 and 65% respectively (Hsu et al., 2002). F+ RNA coliphage are particularly prevalent in chickens, with one of the highest titres recorded in the survey published by Calci et al. (1998). Being able to track particular phages, in order to evaluate treatment success, may not be trivial. However, PCR primers designed to amplify diagnostic genomic sequences present in specific Campylobacter-phages have been developed, as a simple assay to be used in experimental systems. Another important quality-control issue is that older phage stocks may become less effective, despite retaining high titres in laboratory tests (Weld et al., 2004). Due to the highly specific nature of phages, it has been suggested that they be applied as a mixture or ‘cocktail’ to cover a broader range of hosts (Kudva et al., 1999; Sklar and Joerger, 2001). This tactic will assist in the efficacy of the phage preparation against the broadest range of target strains, but will require that the individual components are produced and tested individually, to ensure their contribution to the host range coverage of the target bacterium.

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Phage treatment to reduce the presence of Campylobacter Campylobacter is an obvious target for phage therapy, because of the magnitude of the problem, with more than 80% of birds in the UK harbouring these organisms as a part of their intestinal flora (Newell and Wagenaar, 2000; Corry and Atabay, 2001). The presence of these bacteria in the intestines of poultry at very high densities, ranging between 4 log10 and 8 log10 colony-forming units (CFU)/g (Rudi et al., 2004), is another factor that makes phage treatment feasible as many hosts are susceptible to phage infection and amplification. To test the efficacy of phage therapy against Campylobacter, it is first necessary to design and evaluate experimental models of Campylobacter infection in chickens (Newell and Wagenaar, 2000). We have, in our laboratory, tested candidate bacteriophage isolates from broiler chickens for their efficacy in vitro prior to use in experimental birds (Loc Carrillo et al., 2005). Bacteriophages were administered to the colonised birds at three different doses in an antacid suspension. The reduction in caecal numbers of C. jejuni varied from phage to phage but ranged from 2 log10 to 5 log10 per g of caecal content, compared to controls. By way of comparison, a Campylobacter bacteriophage isolated from poultry meat was found to be ineffective in a similar trial. Phage-resistant campylobacters were isolated at a relatively low frequency (less than four per cent) following treatment, and these resistant strains were compromised in their ability to colonise experimental birds, rapidly reverting back to the sensitive phenotype in the absence of phages (Scott et al., 2007a). In contrast, phage resistance was maintained as a stable phenotype in vitro. Optimisation of dose and selection of appropriate phages were found to be the key elements in the use of phage therapy to reduce campylobacters in broiler chickens. Wagenaar et al. (2005) reported the use of the Campylobacter type phages to prevent as well as reduce Campylobacter colonisation of broiler chickens. The administration of phages resulted in a 3 log10 decline in caecal counts of C. jejuni. Preventative phage treatment delayed the onset of C. jejuni colonisation, and the peak titres remained 2 log10 lower than the controls. In both applications, the colony-forming units and phage-forming-units rose and fell over time, and were out of phase with each other, which is typical of a predator–prey population in nature. Phage therapy treatments to reduce C. jejuni and C. coli in experimental birds were reported by El-Shibiny et al. (2009). Phage treatment to reduce the presence of Salmonella Early phage treatment trials to reduce levels of Salmonella in chickens were carried out by Sklar and Joerger (2001) and Fiorentin et al. (2005). Mixtures of bacteriophages were administered either orally or in feed. Hurley et al. (2008) reported on the use of a well characterised model phage SP6, in an attempt to reduce colonisation of chickens by Salmonella. Although this phage did not reduce the numbers of Salmonella shed by the birds, it was able to replicate in the intestine and importantly, it did not result in the emergence of resistance among the recovered Salmonella. A significant reduction in the incidence of Salmonella in experimental birds, treated with three different lytic bacteriophages, was reported by Borie et al.

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(2008); in this case the phages were applied using an aerosol spray. Salmonellaspecific bacteriophages have also been used successfully in combination with competitive exclusion (Toro et al., 2005). However, as noted above (Hurley et al., 2008), even if phages do replicate, it is no guarantee that they will always affect the desired reductions in the enteric bacterial population. For example, phages active against S. Typhimurium could be shown to multiply in chicks challenged by the bacterium, but phage replication did not affect the numbers of Salmonella recoverable from the caeca (Berchieri et al., 1991). In a study by Andreatti Filho et al. (2007) significant reductions in Salmonella colonisation were observed, but in this case the reduction was not sustained after the first 24 hours. However, if treatments were administered in the 24 h immediately prior to slaughter this could be effective. Atterbury et al. (2007) reported significant reductions in both S. Enteritidis and S. Typhimurium when virulent phages selected from a large collection of 232 were used to treat experimentally colonised birds. The effects were greatest when high titres, 11 log10 plaque forming units (PFU), were administered. The role of development of phage resistance was examined and found to be high after the first 24 h (approximately 80–90% of the recovered isolates) but phage resistance was not maintained for long periods either in vitro or in vivo. It is clear that phage therapy can reduce both Salmonella and Campylobacter but there are fundamental differences in the optimal dose, duration of effect, and development of resistance that probably reflect the fact that the bacteria occupy different niches within the gut and colonise to different degrees. Phage treatment to reduce the presence of Escherichia coli The genus Escherichia contains both commensal and pathogenic members affecting both avian and human species. In terms of frequency, human infection by poultry-borne pathogenic Escherichia is not as significant as for Salmonella and Campylobacter. Escherichia is, however, responsible for a number of pathogenic conditions in poultry and is therefore of economic interest. Phage therapies have been attempted for various types of infections; including respiratory and septicaemic colibacillosis (Barrow et al., 1998; Huff et al., 2002, 2003, 2005; Xie et al., 2005) with success in decreasing mortality or delaying the progress of disease, providing the phages were administered rapidly after experimental infection with the pathogenic Escherichia strains.

7.5 Regulatory issues in reduction of food-borne bacterial pathogens in poultry There are stringent regulatory requirements for the use of naturally-occurring antimicrobial substances, such as bacteriocins, in food preservation. The toxicology data must be acceptable to the recognised regulatory authorities and the bacteriocins must not have any deleterious effect on any of the organoleptic properties of the foods on which they are to be used. The form in which the bacteriocin is used must be economic, since the cost of using purified bacteriocins

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can be prohibitory. The bacteriocin must be stable during storage and, if the activity depends upon residual concentrations, it must be sufficiently stable to cover the shelf-life of the food at effective, and probably lower concentrations. For reasons that have become obvious with the over use of antibiotics, the bacteriocin should have no medical use. Nisin is the only bacteriocin to have ‘generally regarded as safe’ (GRAS) status, since it has been approved in 40 countries with a history of use of more than 50 years (Cleveland et al., 2001). For a new bacteriocin to obtain GRAS status, it must be chemically identified and characterised, and its efficacy proved. Details of the manufacturing process, quality control and toxicological data are required. In practice, this has meant that bacteriocins with good food-preservation potential, such as pediocin AcH, cannot be used in food at present, although pediocin has been found to control Listeria on raw chicken (Goff et al., 1996). Regulations regarding the use of bacteriophage have not yet been fully formulated in relation to their therapeutic potential. However, in 2006 the FDA had already proclaimed GRAS status for LISTEX(™) against a phage active against Listeria in cheese. This was later extended to all food products. The Dutch designated inspection office, SKAL, confirmed the ‘organic’ status of LISTEX(™) under EU law in 2007, as a result of which it can be used in the EU in regular and organic products.

7.6  Future trends In addition to antimicrobial treatments using microbes, antimicrobial peptides that are not of microbial origin could be exploited by genetic manipulation to enable their production in microbes. Furthermore, the use of bacteriophage-derived enzymes (lysins), produced by genetically modified bacteria, may also be possible, but technically challenging. A successful example of this is the production of murein hydrolase, an endolysin from bacteriophage φ3626 that attacks Clostridium perfringens. Cl. perfringens produces an enterotoxin that can cause food-borne disease and is responsible for severe economic losses in chicken production, as is the aetiological agent responsible for necrotic enteritis. The φ3626 endolysin was expressed in E. coli and shown to be active against 48 different strains of Cl. perfringens (Zimmer et al., 2002). The structures and actions of phage enzymes may provide data allowing the development of synthetic therapeutics (Bernhardt et al., 2002), and phages may also be modified to deliver specific toxins to infecting bacteria (Westwater et al., 2003). Genetic modification of strains to produce bacteriocin is one area where preliminary reports are encouraging. The inhibition of S. Typhimurium in the chicken intestinal tract by a transformed avirulent avian E. coli, with a plasmid coding for the production of microcin 24, was demonstrated by Wooley et al. (1999). Similarly, it has been proposed to engineer avirulent bacteria to produce the antimicrobial peptides produced by many eukaryotic organisms, called defensins. However, it is becoming apparent that the role of defensins is not restricted to antibacterial activity. These proteins have wider antimicrobial properties and can interact with immune regulatory components.

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Another possibility is the bioengineering of bacteriocins, particularly the Lantibiotic group, to generate enhanced forms of these peptides (Piper et al., 2009). The development of new microbial treatments for poultry is beginning to result in feasible alternatives to conventional antimicrobials. The use of biotechnological tools may accelerate their development, but the public desire for more ‘natural’ food should not be ignored. However, both bacteriophage and bacteriocins provide the possibility of novel, acceptable solutions to the problems of microbiological safety in the poultry industry.

7.7  Sources of further information and advice A comprehensive review of the range of alternative treatments to antibiotics to reduce pathogens in poultry including microbial, dietary, enzymic, physical and chemical treatments and also selective breeding, vaccines and other potential reduction methods are given by Huyghebaert (2005) and by Doyle and Erickson (2006). Most fundamental aspects of bacteriocins are covered by Cleveland et al. (2001) and Drider et al. (2006) and their use in livestock by Diez-Gonzalez (2007). For more information about the history, biology and types of bacteriophage, the book by Adams (1959) is probably an excellent starting point. For up-to-date information, the web site of The Evergreen State College, USA (http://www. evergreen.edu/phage/home.html) is an excellent resource and provides many useful links. The ASM phage group (http://www.asm.org) is another useful resource for the latest phage research.

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‘Paenibacillus polymyxa purified bacteriocin to control Campylobacter jejuni in chickens’, J Food Prot 68, 1450–1453. stern n j , svetoch e a , eruslanov b v, perelygin v v, mitsevich e v et al. (2006) ‘Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system’, Antimicrob Agents Chemother 50, 3111–3116. sulakvelidze a and morris j g (2001), ‘Bacteriophages as therapeutic agents’, Ann Med 33, 507–509. summers w c (2001), ‘Bacteriophage therapy’, Annu Rev of Microbiol 55, 437–451. svetoch e a , stern n j , eruslanov b v, kovalev y n , volodina l i et al. (2005) ‘Isolation of Bacillus circulans and Paenibacillus polymyxa strains inhibitory to Campylobacter jejuni and characterization of associated bacteriocins’, J Food Prot 68, 11–17. svetoch e a , eruslanov b v, perelygin v v, mitsevich e v, mitsevich i p et al. (2008) ‘Diverse antimicrobial killing by Enterococcus faecium E 50–52 bacteriocin’, J Agric Food Chem 56, 1942–1948. toro h , price s b , mckee a s , hoerr f j , krehling j et al. (2005), ‘Use of bacteriophages in combination with competitive exclusion to reduce Salmonella from infected chickens’, Avian Dis 49, 118–124. tsuei a c , carey - smith g v, hudson j a , billington c and heinemann j a (2007), ‘Prevalence and numbers of coliphages and Campylobacter jejuni bacteriophages in New Zealand foods’, Int J Food Microbiol 116, 121–125. twort fw (1915), ‘An investigation on the nature of ultramicroscopic viruses’, Lancet 2, 1241. van den ende p (1973), ‘Predator prey interactions in continuous culture’, Science 181, 562–564. wagenaar j a , van bergen m a , mueller m a , wassenaar t m and carlton r m (2005), ‘Phage therapy reduces Campylobacter jejuni colonization in broilers’, Vet Microbiol 109, 275–283. wagner r d (2006), ‘Efficacy and food safety considerations of poultry competitive exclusion products’, Mol Nutr Food Res 50, 1061–1071. weld r j , butts c and heinemann j a (2004), ‘Models of phage growth and their applicability to phage therapy’, J Theoret Biol 227, 1–11. westwater c , kasman l m , schofield d a , werner p a , dolan j w et al. (2003), ‘Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections’, Antimicrob Agents Chemother 47, 1301–1307. wiggins b a and alexander m (1985), ‘Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems’, Appl Environ Microbiol 49, 19–23. wooley r e , gibbs p s and shotts e b (1999), ‘Inhibition of Salmonella typhimurium in the chicken intestinal tract by a transformed avirulent avian Escherichia coli’, Avian Dis 43, 245–250. xie h , zhuang x , kong j , ma g and zhang h (2005), ‘Bacteriophage Esc-A is an efficient therapy for Escherichia coli 3–1 caused diarrhea in chickens’, J Gen Appl Microbiol 51, 159–163. zhang g , ma l and doyle m p (2007), ‘Potential competitive exclusion bacteria from poultry inhibitory to Campylobacter jejuni and Salmonella’, J Food Prot 70, 867–873. zimmer m , vukov n , scherer s and loessner m j (2002), ‘The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains’, Appl Environ Microbiol 68, 5311–5317.

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8 Using antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of foodborne pathogens in cattle and swine T. R. Callaway, T. S. Edrington, R. C. Anderson, J. A. Byrd, M. H. Kogut, R. B. Harvey and D. J. Nisbet, United States Department of Agriculture, Agricultural Research Service (USDA-ARS), USA and C. W. Aiello, Carilion Medical Center, USA

Abstract: The intestinal microbial ecosystem is a dense and diverse population that can be utilized to reduce pathogenic bacterial populations that affect animal production efficiency and the safety of food products. Strategies to capture and utilize this complex natural resource have been developed that reduce the populations of foodborne pathogenic bacteria and eliminate pathogens that negatively impact animal production or food safety on the farm. Products used in animals to reduce pathogens in the food supply include probiotics, prebiotics and competitive exclusion cultures, as well as bacteriocins and bacteriophage (bacterial viruses). The individual efficacy of any of these compounds is due to specific microbial ecological factors within the gut of the food animal and its native microflora that alter the competitive pressures of the gut. This review explores the ecology behind the efficacy of these products against foodborne pathogens that inhabit food animals. Key words: probiotics, antimicrobial proteins, food safety, microbial ecology.

8.1  Introduction Far too many human illnesses are associated with consumption of animal-derived foods (Mead et al., 1999). Each year a conservative estimate suggests more than 76 million people in the U.S. are made ill by foodborne pathogens (Mead et al., 1999). The indirect and direct cost each year of the five most common foodborne pathogenic bacteria in the U.S. totals more than $7 billion US and more than 1600 deaths (Mead et al., 1999; USDA-ERS, 2001). 204

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Foodborne pathogenic bacteria can be harbored asymptomatically in the gut of food animals, or on the animal’s hide (Arthur et al., 2007b; Doyle and Erickson, 2006; Porter et al., 1997; Reid et al., 2002). Enterohemorrhagic E. coli (including E. coli O157:H7), Salmonella, Campylobacter, and Listeria have all been isolated from cattle, swine and poultry (Borland, 1975; Callaway et al., 2006; Oliver et al., 2005). Several foodborne pathogens, such as Salmonella, can be a shared problem both from a food safety and animal health perspective and are found in multiple animal species (Coburn et al., 2007). Thus asymptomatic carriage of pathogenic bacteria represents a threat to the integrity as well as the efficiency and profitability of the food supply. It has been a focus of the animal industry for many years to develop strategies that reduce foodborne pathogenic bacteria in the food supply (Koohmaraie et al., 2005). Reducing populations of pathogens in live animals can aid the effectiveness of in-plant interventions (Sargeant et al., 2007), but perhaps more importantly because recent human foodborne illness outbreaks have been linked to indirect human contact with animal feces (Anonymous, 2000) it is critical for public health to reduce these pathogens on the farm (Steinmuller et al., 2006). As a result, it has been stated that pre-harvest intervention strategies can produce the greatest improvement in human health (Hynes and Wachsmuth, 2000). Consequently, many new intervention strategies have been developed to reduce pathogens in live animals prior to slaughter, and many of these are dependent on an understanding of the microbial ecology of the gastrointestinal tract. The gastrointestinal tract of food animals is a fully-mature ecosystem that occupies all environmental niches and utilizes nearly all available nutrients, which generally prevents pathogenic bacteria from obtaining a foothold in the gastrointestinal tract. The symbiotic relationship between the host animal and its resident gastrointestinal microbial ecosystem is critical to animal health and production efficiency (Jayne-Williams and Fuller, 1971; Savelkoul and Tijhaar, 2007). Recent studies have demonstrated that certain intestinal microbial populations can cause obesity and may be linked to conditions such as autism (DiBaise et al., 2008; Finegold, 2008; Ley et al., 2006). As our understanding of the members and activities of the gastrointestinal microbial ecosystem has grown, so has interest in using various facets of this ecosystem as an anti-pathogen mechanism to improve animal and human health. Pathogen reduction strategies can be loosely categorized into two groups: ‘competitive enhancement’ or probiotics, and ‘directly anti-pathogen’ strategies. Each of these categories is useful in various phases of animal production for each species, and no single category will eliminate all pathogens from all food animals, although the erection of multiple hurdles will increase the chances of successfully reducing pathogens in food animals. It is important to understand how the microbial population interacts with pathogens in order to best utilize the current methods available, as well as developing new pre-harvest intervention strategies. In this chapter, we will discuss the theory behind these competitive enhancement and anti-pathogen pre-harvest intervention strategies, as well as their benefits, and challenges for future implementation (see Table 8.1).

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Table 8.1  Examples of some preharvest intervention strategies and their effects on pathogenic bacteria in food animals Preharvest Culture or source strategy

Food animal species

Effect

Reference

Competitive Mixed cecal Chickens Reduced Salmonella Nisbet et al., exclusion bacteria colonization 1996 Mixed bacteria Swine Reduced Salmonella Fedorka-Cray from pig mucus colonization et al., 1999 Mixed cecal Swine Reduced E. coli Genovese bacteria from diarrhea and mortality/ et al., 2003; swine morbidity and Harvey Salmonella et al., 2005 choleraesuis E. coli strains Cattle Reduced E. coli Zhao et al., from cattle O157:H7 colonization 1998 in cattle Probiotic Enterococcus Swine Reduced diarrhea and Zeyner and (DFM) faecium reduced Entero. Boldt, 2006; faecalis Vahjen et al., 2007 Bifidobacterium Swine Reduced adherence of Collado et al., animalis and Salmonella, E. coli 2007 Lactobacillus and Clostridum rhamnosus Lactobacillus Cattle Reduced E. coli Brashears acidophilus O157:H7 colonization et al., in cattle and on their 2003a, b hides Prebiotics Maltodextrins and Swine Reduced E. coli Nemcova fructooligosaccharides O8:K88 intestinal et al., 2007 adherence Galactooligosaccharides In vitro Reduced adherence of Shoaf et al., enteropathogenic 2006 E. coli (EPEC) to intestinal cells Phage T-even phage from Sheep Reduced E. coli Callaway cattle O157:H7 colonization et al., 2008 in sheep Antimicrobial Colicins Cattle Reduced E. coli Schamberger proteins O157:H7 populations and Diez in cattle Gonzalez, 2005 Synbiotics Lactobacillus Reduced Bomba et al., plantarum and enterotoxigenic 1999 maltodextrins E. coli (ETEC) strain populations

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8.2  Antimicrobial cultures: enhancing natural competition Utilization of a native or artificially-introduced microbial population to improve animal health and productivity, and/or to reduce pathogenic organisms, has been termed a ‘probiotic’ or competitive enhancement approach (Fuller, 1989). Competitive enhancement strategies that have been developed include: 1. Introduction of a normal microbial population to the gastrointestinal tract (competitive exclusion, or CE). 2. Addition of a microbial supplement (probiotic) that improves gastrointestinal health and the diversity of the intestinal microbial ecology (Collins and Gibson, 1999). 3. Adding a limiting, non-host digestible nutrient (prebiotic) that provides an existing (or introduced) commensal microbial population a competitive advantage in the gastrointestinal tract. Each of these approaches utilizes the activities of the native microbial ecosystem against pathogens by capitalizing on the natural microbial competition. Generally speaking, competitive enhancement strategies offer a natural ‘green’ method to reduce pathogens in the gut of food animals. Historically, probiotic studies in food animals have been characterized by inconsistency, primarily due to a lack of understanding of the microbial ecology of the gastrointestinal tract, and of conditions that affect the growth of pathogens as well as the probiotic organisms. Some variation can be attributed to the fact that mature animals (to whom probiotics are generally fed) contain a stable, relatively individualistic intestinal microbial population with which the probiotic must come to equilibrium; when probiotics are applied to neonates that are equipped with a sparse or poorly established intestinal flora, results are more consistent. All of these factors have contributed to difficulties in reproducing effects of some probiotics in animals beyond the neonatal stage. Competitive enhancement products have had somewhat limited applications commercially as pathogen-reduction strategies, in part due to the availability of cheap antibiotics which can counteract the effectiveness of competitive enhancement strategies (Steer et al., 2000). Given increasing fears over the dissemination of antimicrobial resistance (Taylor, 2001), it is expected that prophylactic antibiotic usage in food animals will become more closely regulated and expensive, causing probiotic/competitive enhancement strategies to become more economically feasible and widely used. Recently, the advent of molecular methodologies has allowed a more precise monitoring of specific changes caused by individual probiotic cultures, and has allowed a better understanding of the ‘normal’ intestinal microbial ecosystem. These advances can potentially lead to the development of highly tailored probiotic products for use in specific production situations. 8.2.1  Competitive exclusion Competitive exclusion (CE) as a technology involves the addition of a nonpathogenic bacterial culture of a single or multiple strains, to the intestinal tract of

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food animals in order to reduce populations of pathogenic bacteria (Fuller, 1989; Nisbet et al., 1993; Nurmi et al., 1992). By definition, CE cultures are isolated from the same animal species that they will be used in, in order to best utilize the symbiotic relationship between the host animal and its native microbial ecosystem that developed during co-evolution. Because a mature gastrointestinal microbial population fills all available environmental niches, it makes an animal more resistant to pathogen colonization (Fuller, 1989). This natural anti-pathogen activity has been called ‘bacterial antagonism’, ‘bacterial interference’, or ‘competitive exclusion’ (Lloyd et al., 1974; Nurmi et al., 1992). The early addition of a microbial consortium to the naïve gut can allow the early establishment of a normal microbial population that can competitively prevent the establishment of a pathogenic bacterial population (Nurmi et al., 1992; Crittenden, 1999; Steer et al., 2000). This is especially critical in the production of poultry and eggs because eggs and newly-hatched chicks can be quickly colonized by pathogens such as Salmonella and Campylobacter (Cox et al., 1990, 1991). However, when CE is used in older animals, it must often compete against the established native population that must be displaced. Therefore, the mixture of bacteria chosen for use as a CE culture must be specific for the animal, production stage, and scenario in which it will be utilized. Several modes of action have been proposed for how CE technologies eliminate pathogens, including: 1. Direct and indirect competition for nutrients. 2. Competition for physical attachment sites. 3. Production of antimicrobial compounds (including Volatile Fatty Acids [VFA]). 4. Enhancement of host immune system activity. 5. A synergistic interaction of two or more of the above activities. If bacteria (including pathogens) cannot grow at least as fast as the digesta passage rate then the pathogen will ‘wash out’ of the environment. If the physical binding sites of pathogens are blocked by this added bacterial population then the pathogenic bacteria that are dependent on epithelial adherence would be subject to wash out. After a CE culture (or the animal’s natural flora) is established within the gut, bacteria bind to the surface of the intestinal epithelium preventing opportunistic pathogens from attaching and thus obtaining a colonization foothold (Collins and Gibson, 1999; Lloyd et al., 1977). Volatile fatty acids produced by the normal microbial fermentation in the gut are toxic to some pathogenic bacteria, and may reduce the competitive fitness of these pathogens in the gut environment (Prohaszka and Baron, 1983; Wolin, 1969). Furthermore, some bacteria produce antimicrobial protein compounds, such as bacteriocins (including colicins), that can inhibit or eliminate species competing within the same niche, and specific use of these antimicrobial proteins will be discussed further below (Al-Qumber and Tagg, 2006; Jack et al., 1995; Schamberger et al., 2004; Walsh et al., 2008). In food animals, most CE research has focused on poultry (Nava et al., 2005). This can be attributed to the need to control Salmonella colonization in chicks, as well as production diseases. This has prompted CE cultures to be used in many

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countries (Bielke et al., 2003; Stavric, 1992; Stavric and D’Aoust, 1993; Stavric and Kornegay, 1995). In the U.S. a mixed, defined (exact species identified) commercial CE product, comprised of several defined species of bacteria, was developed and used to reduce Salmonella colonization of chicks (Nisbet et al., 1994; 1996). Subsequently, undefined CE products have also been relatively widely adopted in the poultry industry (Schneitz, 2005, Zhang et al., 2007). Future trade regulations within the European Union (EU 1003/2005) are expected to increase the use of CE in poultry as a non-antibiotic method to reduce Salmonella on eggs and in chicks shipped into the EU. Further studies have found that Campylobacter colonization of poultry can be inhibited by the use of specific CE cultures (Zhang et al., 2007). This topic is further discussed in detail in the excellent Chapter 7. In swine, CE cultures have also found usage to reduce foodborne pathogenic bacteria as well as animal health threats (Francisco, 1999). Supplementation of a CE culture (mixed bacterial population) derived from healthy pig mucosa reduced Salmonella populations in the intestinal tract of young pigs (Fedorka-Cray et al., 1999). A swine CE culture derived from the cecal contents of healthy pigs was reported to reduce the incidence of Salmonella choleraesuis (Genovese et al., 2003; Nisbet, 2002). This CE culture also reduced post-weaning diarrhea, morbidity and mortality caused by ETEC, an economically important infection for the swine industry (Harvey et al., 2003, 2005). A threat to human health that has been recently associated with food animals is Clostridium difficile, and a CE culture comprised of spores of a non-toxigenic strain of C. difficile given to piglets reduced colonization, diarrhea and growth depression caused by a toxigenic C. difficile (Songer et al., 2007). Because many pathogenic bacterial species are killed by high concentrations of volatile fatty acids (VFA), it was assumed that pathogenic E. coli would have limited opportunities to colonize cattle intestinal tracts (Hollowell and Wolin, 1965; Wolin, 1969). However, it is now apparent that the foodborne pathogen E. coli O157:H7 and other EHEC strains are found primarily at the recto-anal junction of cattle (Cobbold, 2007; Lim et al., 2007; Low et al., 2005; Naylor et al., 2003). Researchers have sought to utilize the complex microbial ecosystem in the ruminant intestinal tract as a CE culture to eliminate E. coli O157:H7 and Salmonella from cattle (Zhao et al., 1998). Researchers have isolated and defined several E. coli strains (non enterohemorrhagic) from cattle, and discovered that this generic E. coli culture could displace an established E. coli O157:H7 population from the gastrointestinal tract of calves (Zhao et al., 1998). While this is the only true CE culture for cattle that has been shown to reduce E. coli O157:H7, there are other probiotic/DFM cultures that target this pathogen in cattle. 8.2.2  Probiotics Probiotics are a general category of dietary products that can be included in animal rations (called Direct Fed Microbials [DFM] in the U.S.) to enhance performance and/or reduce pathogenic bacteria (Collins and Gibson, 1999; Fuller, 1989). A

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proposed definition for probiotics is ‘a preparation or a product containing viable, defined microorganisms . . . which alter the micro-flora . . . and exerts beneficial health effects in this host’ (Schrezenmeir and De Vrese, 2001). In fact, some of the probiotic products used have directly affected immune parameters increasing CD8 production, as well as IgG and IgM concentrations in the serum and gut of swine (Duncker et al., 2006; Walsh et al., 2008; Zhang et al., 2008). Probiotics/DFM in animals are typically comprised of lactic acid bacteria (LAB), yeasts, or their end-products and are not species-specific, or even necessarily originally isolated from animals (Wiemann, 2003). Regulations in this field have allowed a wide variety of claims to be made about the improvements in growth efficiency and other potential benefits, and the consistency of results in the field has not always been demonstrated (Barroga et al., 2007; LeJeune et al., 2006). However, the most commonly used probiotic bacterial strains in animals remain Bifidobacteria and Lactobacillus and are primarily targeted for improving animal production and efficiency (Gomes and Malcata, 1999; Midilli et al., 2008). Some probiotics reduce foodborne pathogens and other pathogenic bacteria that affect growth and production in food animals (Stephens et al., 2007; Tkalcic et al., 2003). In order to prevent post-weaning E. coli diarrhea and Salmonella colonization in pigs (Bertschinger, 1999) early-weaning procedures and antibiotics are often used (Fedorka-Cray et al., 1997); yet these pathogens still pose a significant problem for the swine industry. As a result, researchers have investigated several probiotic/DFM approaches to reducing these important production problems. A mixture of Lactobacillus casei cultures and maltodextrins resulted in a reduction of adherence of 1 log10 to 2.5 log10 in pigs by an Enterotoxigenic E. coli (ETEC) strain O8:K88 (Bomba et al., 1999). Another Lactobacillus plantarum DFM product reduced counts of E. coli O8:K88 in the jejunum and colon of piglets, and was associated with increased acetate concentrations in the ileum and colon (Nemcova et al., 2007). Daily oral administration of E. coli strain Nissle 1917 for 10 days was reported to abolish hypersecretion by the intestine associated with experimental infection of weaned pigs with an O149:K88 strain of ETEC (Schroeder et al., 2006). A culture of Enterococcus faecium given from birth to weaning reduced the frequency of diarrhea and improved weight gain in weaned pigs, however the cause of the diarrhea was undetermined (Zeyner and Boldt, 2006). Another E. faecium DFM was found to reduce populations of the potential human pathogen Enterococcus faecalis in the colon of weaned pigs (Vahjen et al., 2007). The inclusion of a Bacillus subtilis DFM in the diet resulted in a reduction in K88 ETEC scours in pigs (Bhandari et al., 2008). Probiotics comprised of Bifidobacterium animalis ssp. lactis and Lactobacillus rhamnosus individually reduced adherence of Salmonella, E. coli and Clostridum spp. to the intestinal mucosa in swine; together the two organisms acted synergistically (Collado et al., 2007). Reduced mucosal adhesion by pathogens is thought to lead to reduced severity of clinical disease in pigs, though this has not been conclusively demonstrated. The cattle industry has used various types of probiotics for many years primarily to increase growth rate, milk production, or production efficiency

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(Lehloenya et al., 2008); however, recent years have seen the development of probiotic preparations to address other concerns related to cattle production. Researchers found that commercial probiotics provided neither benefit nor detriment in regards to E. coli O157:H7 populations in cattle (Keen and Elder 2000). Another probiotic examined did reduce fecal shedding of E. coli O157:H7 in sheep (Lema et al., 2001). Because of the U.S. Food Safety Inspection Service’s declaration of E. coli O157:H7 an adulterant in ground beef, there has been intensified interest in probiotic research aimed at reducing E. coli O157:H7 in cattle. A L. acidophilus culture reduced E. coli O157:H7 shedding by more than 50% in finishing cattle (Brashears et al., 2003a, b). Further research indicated that this commercial DFM reduced fecal shedding of E. coli O157:H7 in cattle from 46% of animals to 13% (Ransom et al., 2003). Other research demonstrated this DFM reduced E. coli O157:H7 populations on the hides of cattle by up to 75%; furthermore the highest DFM dosage reduced Salmonella shedding in the feces by 50% (Stephens et al., 2007; Younts-Dahl et al., 2004). This product is currently used widely in feedlots across the U.S. and Canada because the enhanced growth performance economically balances the cost of its inclusion in cattle rations, thus making a food safety enhancement pay for itself. In another study, a different DFM using L. acidophilus also significantly reduced fecal shedding of E. coli O157:H7; fecal shedding of Salmonella was not reduced but new Salmonella infections in cattle were reduced (Tabe et al., 2008). 8.2.3  Prebiotics Prebiotics are organic compounds that are unused by the host animal, but are available to members of the microbial population and are often described as ‘functional foods’ or ‘nutraceuticals’ (Schrezenmeir and De Vrese, 2001). Some carbohydrates, as well as other organic compounds, are not enzymatically degraded in the stomach or intestine and pass to the cecum and colon where they become ‘colonic food’ (Crittenden, 1999; Houdijk et al., 1998; Kontula, 1999). Prebiotics can provide limiting nutrients to the intestinal bacteria for fermentation, yielding increased B vitamin production by the microbial consortium (Branner and RothMaier, 2006). Some prebiotics provide a competitive advantage to specific members of the native microflora (e.g., Bifidobacteria, Butyrivibrio) (Willard et al., 2000) that can act as a natural, in-place CE culture against pathogens. Coupling the use of CE or probiotics/DFM with prebiotics is a technique known as ‘synbiotics’ which can synergistically reduce pathogen populations (Branner and Roth-Maier, 2006; Collins and Gibson, 1999; Schrezenmeir and De Vrese, 2001). Recent research has indicated that the use of prebiotics, such as inulin and oligofructans, can directly modulate immune activity (Seifert and Watz, 2007). Further evidence for the role of prebiotics in modulating human health through the intestinal microbial ecosystem is the reduction in inflammatory bowel disease and colitis in humans (Leenen and Dieleman, 2007; Winkler et al., 2007). While much of the research into prebiotics has focused on the use in humans due to the expense of these products, prebiotics have been used in the animal feed

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industry to improve the health of animals (Respondek et al., 2008; TorresRodriguez et al., 2007; Willard et al., 2000). The use of maltodextrins and fructooligosaccharides in combination with L. plantarum has been shown to reduce adherence of E. coli O8:K88 to the jejunum and colon of weaned pigs as discussed previously (Nemcova et al., 2007). Galactooligosaccharides, another prebiotic, reduced the adherence of a human enteropathogenic E. coli (EPEC) to the human cell lines HEp-2 and Caco-2 (Shoaf et al., 2006). The use of prebiotics in cattle has been limited due to the expense and the capacity of the ruminal microbial population to degrade most prebiotics; in future, enhancements in rumen-protective technologies may allow these compounds to be used in cattle.

8.3  Direct assault: anti-pathogen intervention strategies In contrast to competitive enhancement strategies, anti-pathogen strategies directly target killing pathogenic bacteria within food animals. Several anti-pathogen strategies have been investigated in recent years, including the use of medicallyimportant antibiotics. However, the use of traditional medically important antibiotics is not recommended due to concerns over antibiotic resistance. Therefore in this section we will focus on other types of anti-pathogen treatments such as: (a)  the use of antimicrobial proteins produced by bacteria (b)  the use of bacteriophages (c) the use of alternative antimicrobial compounds that specifically target the physiology of pathogenic bacteria.

8.3.1  Antimicrobial proteins: bacteriocins (including colicins) Some bacteria produce antimicrobial proteins that wipe out bacteria that compete for the same nutrients (Jack et al., 1995; Klaenhammer, 1988). These antimicrobial proteins are classified as bacteriocins, and members of this category of protein that specifically affect E. coli are colicins (Konisky, 1982). Bacteriocins are small proteins (bacteriocins range from approximately 3 to 20 kDa in size; colicins range from 29 to 75 kDa in size) that exhibit antimicrobial activity against bacteria that occupy the same or a similar ecological niche as their producers (Konisky, 1982; Lakey and Slatin, 2001). Bacteriocins have been extensively studied over the years as potential antimicrobial agents to alter the intestinal microflora in many ways (Hugas, 1998; Kalmokoff et al., 1996; Wells et al., 1997), but have not been widely used in animals as interventions against foodborne infections to date. However, bacteriocins such as nisin are widely used around the world in processed foods and will rightfully receive little public concern over their inclusion in animal rations due to their proteinaceous nature. Antimicrobial proteins are commonly produced by ruminal and intestinal bacteria (Iverson and Millis, 1976; Laukova and Marckova, 1993). It has been suggested that these natural antimicrobials could be used to reduce foodborne and

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animal health pathogens in the animal and to improve the efficiency of the intestinal fermentation (Cutler et al., 2007; Patton et al., 2007). Some of these antimicrobial protein-producing strains have been incorporated (intentionally and inadvertently) as members of CE and probiotic cultures over the years (Schamberger and Diez-Gonzalez, 2005; Walsh et al., 2008). Colicins bind to specific outer membrane receptors of sensitive E. coli cells, where they are translocated across the outer membrane and span the periplasmic space to insert into the inner membrane (Lazdunski et al., 2000). Bacteriocins are generally active against gram-positive bacteria, thus their mode of action does not require the complex gymnastics exhibited by colicins, instead these hydrophobic proteins insert directly into the cell membrane (Konisky, 1982). Following membrane insertion, these proteins form a voltage-dependent pore that allows ions to ‘leak’ out of the cell, destroying the electrochemical gradients and the critical protonmotive force (Guihard et al., 1993, Klenker et al., 2002). Cellular death results from a loss of K+ gradients, as well as a depletion of intracellular ATP (Stroud et al., 1998). Bacteriocins have been used by some researchers to alter the ruminal fermentation (Morovsky et al., 1998; Russell and Mantovani, 2002); however due to the small amounts of proteins produced it has been difficult to obtain enough protein to utilize in feeding trials. The ease of cloning has allowed for antimicrobial protein production to be transferred and hyperexpressed in yeasts to obtain useable quantities of these proteins. Using large quantities of purified colicins, researchers have demonstrated that colicin E1 can reduce intestinal populations of E. coli responsible for post-weaning swine diarrhea and can also inhibit the growth of Listeria (Callaway et al., 2004c; Cutler et al., 2007; Patton et al., 2007; Stahl et al., 2004). Other researchers have used colicins to reduce populations of E. coli O157:H7 in cattle (Schamberger and Diez-Gonzalez, 2002, 2005; Schamberger et al., 2004). In order for antimicrobial proteins to be widely implemented as a pathogen reduction strategy in food animals, they must be protected from gastric and intestinal degradation and released at the appropriate site of intestinal pathogen colonization. 8.3.2  Bacteriophages Bacteria can be infected by bacterial viruses known as bacteriophages, which typically have fairly narrow prey spectra (Lederberg, 1996; Summers, 2001). Bacteriophages have been called the most common form of life on the planet, and have been frequently isolated from the gastrointestinal tracts of food animals (Klieve and Bauchop, 1988; Orpin and Munn, 1973; Rogers and Sarles, 1963). This specificity allows phage to be used to kill specific microorganisms in a mixed microbial population without perturbing the overall ecosystem (Ho, 2001). Phages have been used instead of antibiotics to treat human diseases in many parts of the world (Barrow and Soothill, 1997; Lederberg, 1996) and have been used for many years to treat animal diseases experimentally (d’Herelle, 1919; Huff et al., 2002; Smith and Huggins, 1982; 1983, 1987). Phages are commonly isolated from food

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animals, and in recent years interest in the natural ecology of these predators has grown in order to understand how they may best be used as anti-pathogen interventions in food animals (Callaway et al., 2006, 2007; Connerton et al., 2004; Oot et al., 2007). Interestingly, a survey found naturally-occuring antiCampylobacter phages are commonly isolated from retail poultry products in the U.K., which illustrates the frequency of natural human consumption of these bacterial predators, indicating that phages are already part of the food chain (Atterbury et al., 2003b). Because of their ubiquity in the environment and human diets, as well as the specificity of action against pathogens, phages have been suggested to be an excellent preharvest foodborne pathogenic bacteria control method for use in live animals (Barrow et al., 2003). Phages bind to receptors on bacteria and inject their genetic material into the bacterium and hijack its biosynthetic machinery to produce daughter phages that are released in a ‘burst’, releasing daughter phages to repeat the process. An exponential increase in the number of phages continues as long as prey bacteria are present. Thus phages can persist in the gut when ‘prey’ bacteria are present, but when the prey disappears, so do the phages, making phages a self-limiting treatment. Administration of selected bacteriophage to food animals and in meat products has been suggested as an intervention strategy to specifically eliminate gastrointestinal foodborne pathogenic bacteria (Bigwood et al., 2008; Greer, 2005; Hudson et al., 2006). Bacteriophage treatment reduced enterotoxigenic E. coli (ETEC)-induced diarrhea as well as splenic ETEC colonization in calves (Smith and Huggins, 1983, 1987). Other research has found that phages can be used to reduce Campylobacter and Salmonella in poultry (Desmidt et al., 1997; Loc Carrillo et al., 2005; Sklar and Joerger, 2001; Toro et al., 2005; Wagenaar et al., 2005) as well as on meat products (Atterbury et al., 2003a; Goode et al., 2003; Higgins et al., 2005). Phages have also been used in studies with ruminants to reduce E. coli O157:H7 in ruminants, albeit with variable success (Bach et al., 2003; Callaway et al., 2008; Kudva et al., 1999). Studies using phages to control Salmonella in live animals have been hampered by the broad diversity of serotypes, but development of phages cocktails against many serotypes will likely alleviate this problem. However, much research into the ecology and specificity of phages is needed to be able to effectively use phages to control foodborne pathogens in live animals on a large scale. 8.3.3  Sodium chlorate: inhibitor of bacterial physiology It is possible to target certain bacteria based upon their physiology or metabolic pathways. One pathway that can be so targeted is dissimilatory nitrate reduction in Enterobacteriaceae. Salmonella and E. coli strains can respire under anaerobic conditions by reducing nitrate to nitrite via the dissimilatory nitrate reductase enzyme (Stewart, 1988; Stouthamer, 1969). This intracellular enzyme does not differentiate between nitrate and its analog chlorate which is reduced to chlorite in the cytoplasm; and the resultant accumulation of intracellular chlorite kills bacteria (Stewart, 1988). Chlorate quickly reduced populations of E. coli O157:H7 and Salmonella in vitro by more than 5 log10 (Anderson et al., 2000a). Chlorate addition to animal

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rations reduced Salmonella and E. coli O157:H7 populations in swine and sheep intestinal tracts (Anderson et al., 2001a, b; Callaway et al., 2003; Edrington et al., 2003) and poultry crop and intestinal contents (Byrd et al., 2003; Jung et al., 2003; Moore et al., 2006). Other studies indicated that chlorate administered via drinking water significantly reduced E. coli O157:H7 ruminal, cecal and fecal populations in both cattle and sheep (Anderson et al., 2002; Callaway et al., 2002; Edrington et al., 2003). Hide populations of E. coli O157:H7 play a key role in carcass/ product contamination (Arthur et al., 2007a; Barkocy-Gallagher et al., 2004; Keen and Elder, 2002; Mather et al., 2007), and chlorate treatment reduced both fecal and hide populations of E. coli (Anderson et al., 2005). Further studies demonstrated that chlorate treatment did not adversely affect the endproducts or efficiency of the ruminal or the cecal/colonic fermentation in ruminant or monogastric animals (Anderson et al., 2000b, 2002). Additional studies have demonstrated that chlorate alters neither the antibiotic resistance, nor toxin production by E. coli O157:H7 (Callaway et al., 2004a, b). Chlorate-resistant mutants do not compete and survive in mixed microbial populations or in vivo (Callaway et al., 2001).

8.4  Conclusions Improving human food safety begins on the farm because people can be made sick from direct animal contact and from run-off from farms. Reducing pathogen loads that enter the abattoir will also reduce human illnesses by allowing in-plant interventions a smaller pathogen load to address, directly improving human foodborne illness levels. The microbial population of the gut of food animals is a weapon against pathogens that is yet to be fully utilized in our war on foodborne disease. The addition of microbial populations from healthy animals or stimulation of an existing normal flora prevents the colonization of the gastrointestinal tract by pathogenic bacteria. The primary weapons in this arsenal that stimulate the competitive nature of the native microflora include: competitive exclusion, probiotics and prebiotics. However, the use of one concept is not going to defeat foodborne pathogens, therefore we also include antimicrobial proteins, bacteriophage and bacterial metabolic inhibitors in our arsenal. No ‘silver bullet’ will be found that will completely eliminate all foodborne pathogens from food animals. However, the erection of multiple, complimentary hurdles that reduce human exposure to pathogens will produce the greatest improvement in human health because it creates overlapping spheres of pathogen control during animal production.

8.5  Disclaimer Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and/or exclusion of others that may be suitable.

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8.6  References al - qumber

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9 Controlling fungal growth and mycotoxins in animal feed M. Olstorpe, K. Jacobsson, V. Passoth and J. Schnürer, Swedish University of Agricultural Sciences, Sweden

Abstract: This chapter will focus on the control of fungal growth in animal feed of plant origin, i.e. forage and cereal grain, and primarily on the use of biopreservation to improve feed hygiene. Microbes may interfere with the hygiene and storage stability of feed and different by-products utilised in the feed industry; reduce palatability of the feed; and reduce bioavailability of minerals and proteins. It is therefore of great interest to manage/ control the microbial species present in animal feed. Fungi (yeasts and moulds) belong to the natural microbial flora on plants and thus also occur in plant-derived feed during storage. The impact of yeasts on storage is not well defined, but they may degrade organic compounds and thus decrease the nutritional value of the feed, as well as degrade lactic acid in silage, decreasing the preservation capacity. Moulds can produce mycotoxins and spores. Spores can promote allergic reactions while mycotoxins can persist in the feed and food chain, having acute or long term toxic effects on animals and humans. Traditional methods of preservation include drying or the addition of acids, both of which are very energy consuming. Inappropriate processing may even increase the risk of mould contamination. Biopreservation based on microbial activities may therefore provide a sustainable alternative to traditional conservation methods. Key words: feed preservation, mycotoxins, moulds, yeasts, lactic acid bacteria, biocontrol.

9.1  Introduction This chapter will focus on the control of fungal growth in animal feed of plant origin, i.e. forage and cereal grain, and primarily on the use of biopreservation to improve feed hygiene. Microbes may interfere with the hygiene and storage stability of feed and different by-products utilised in the feed industry; reduce palatability of the feed; and reduce bioavailability of minerals and proteins. It is therefore of great interest to manage/control the microbial species present in animal feed. The hygienic quality of 225 © Woodhead Publishing Limited, 2011

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feeds is estimated by evaluating their general microbial status and the microbial population of a feed or feed component may negatively influence both production and performance of animals. Different feeds may pose various risks, depending on management, composition, production site, weather conditions, etc. Grain entering initial storage contains a wide range of potential spoilage microorganisms and this population depends for the most part on field conditions and may be modified during storage. Thus, poor pre- and postharvest management can result in rapid quality loss as well as hazards from mycotoxins (Aldred and Magan 2004).

9.2  Fungal growth and mycotoxins in animal feed 9.2.1  Fungal growth in animal feed Fungi seldom occur in isolation on crops, but usually as a mixed consortium of bacteria, yeasts and filamentous fungi (Magan et al. 2003; Olstorpe et al. 2010a, b). Yeasts are well known for their contribution to society, in the production of bread, alcoholic beverages, and other fermented foods, but there are also many studies published on the spoilage of food and feed by yeasts (Middelhoven and van Balen 1988; Fleet 1992; Loureiro and Malfeito-Ferreira 2003). Yeasts of different genera such as Candida, Cryptococcus, Pichia, Rhodotorula and Sporobolomyces have been isolated from grains at harvest (Flannigan 1987; Olstorpe et al. 2010a, b). However, the significance of their presence has not been examined in cereal grains, as filamentous fungi are usually considered to be the main agents of pre- and postharvest spoilage of grain (Lacey 1989; Lacey and Magan 1991). Yeasts associated with deterioration need further investigation, as they play a significant role in both the production and spoilage of fermented feed (Fleet 1990). Feed spoilage postharvest is initiated due to insufficient drying or by subsequent moisture increases due to inadequate storage facilities, as the water availability is the main factor limiting the growth of fungi. Prevailing temperatures during storage will also have an effect on fungal growth and activity. Temperature and gaseous composition as well as interactions with other microorganisms may also affect fungal growth. Moulds cause undesirable effects such as loss of dry matter, discoloration, reduced nutritional value and digestibility, and production of off-flavours, and can result in the production of mycotoxins (Lacey 1989; Magan et al. 2003). Mould invasion also results in an increased dust fraction, containing substantial numbers of fungal conidia, and this dust fraction has been associated with chronic and recurrent airway disease in horses. There are no comparable investigations in cattle, however inhalation of mould spores could be assumed to comprise a continuous pro-inflammatory challenge to the upper airways of cattle, as well as horses (Fink-Gremmels 2008). 9.2.2  Mycotoxins in animal feed In forage and cereal grains, mould growth does not necessarily indicate the presence of mycotoxins. Similarly, mycotoxins may be present in feed even in the

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absence of visible mould growth, if the crop was infected in the field, e.g. toxins produced by Alternaria spp. and Fusarium spp. A. alternata, a common airborne fungus present in most parts of the world, is known to produce several toxins such as tenuazonic acid, alternariol and altertoxins (Lacey 1989). F. culmorum and F. graminearum infect various cereals as well as maize, and can produce deoxynivalenol and zearalenone. Both these toxins, and metabolic products thereof, can be detected in milk from cows fed with mycotoxin-contaminated feed (Galtier 1998). Storage fungi, such as Penicillium spp., are present in low numbers at harvest and increase during storage. For example, Penicillium roqueforti, also used in cheese manufacture, is an important spoilage fungus in airtight storage systems (Lacey and Magan 1991). and has been found in both acid-preserved cereals and in airtight stored grain with insufficient oxygen exclusion (Kaspersson et al. 1988). After ingestion, ruminants displayed symptoms such as lack of appetite, ketosis, paralysis and spontaneous abortions (Häggblom 1990). Müller and Amend (1997) reported that although mycophenolic acid, patulin, penicillic acid and PR toxin were produced in maize silage with visible growth of P. roqueforti, concentrations decreased during prolonged storage. In addition to acute or long term toxic effects, the presence of moulds and mycotoxins in feed has been reported to reduce palatability, resulting in decreased feed intake. This, in turn, leads to production losses (lower weight gain or milk production), which also may be the consequence of mycotoxins affecting the immune system. Furthermore, although much of the ingested mycotoxin is excreted in the faeces and urine, mycotoxins or their metabolic derivatives may end up in food intended for human consumption. One example is aflatoxin B1,which in cattle is metabolised into aflatoxin M1 and then secreted into milk (Al-Hilli and Smith 1979). The fate of ingested mycotoxins will vary depending on the properties of the toxin but also on the animal species (Galtier 1998; Yiannikouris and Jouany 2002). Different toxins can accumulate in different organs or tissues, but concentrations decrease once animals are fed uncontaminated feed. In general, ruminal metabolism makes ruminants more tolerant to mycotoxins than monogastric animals, but aflatoxins are only poorly degraded in ruminal fluid (Westlake et al. 1989). Although some reports on the removal of mycotoxins by microorganisms have been published (reviewed in Styriak and Conková 2002), minimising fungal growth in feed materials is likely to remain the major preventive measure to reduce the risk of feeding mycotoxins to food producing animals.

9.3  Preservation techniques Generally, grain stored at a moisture content (MC) equivalent to less than water activity (aw) 0.70 will not be subjected to fungal spoilage and mycotoxin production (Aldred and Magan 2004). However, cereals at harvest normally have aw 0.86 to 0.97, and are often traded on a wet weigh basis. Certain technological © Woodhead Publishing Limited, 2011

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challenges associated with bulk drying and storage of grain, and instances of poor practices and negligence, result in a significant risk for mycotoxin production in the postharvest situation. Harvested crops are also inhabited by bacteria that may produce toxin or spoil feed. Prior to harvest, cereals normally contain bacteria at approximately 6.5 log units g–1 fresh material. However, these numbers vary substantially between different microbial groups and production regions (Olstorpe et al. 2010a). Generally, control measures during grain storage do not focus on bacteria, as they are not regarded to be problem organisms. The minimum aw that supports active growth of most Gram-negative and Gram-positive bacteria is 0.97 is 0.90, respectively (Adams and Moss 2000). However, in a study by Olstorpe et al. (2010a), growth of bacteria was detected in grain of much lower aw. 9.3.1  Drying Traditionally, feeds were preserved by drying, i.e. reducing aw. Forage was mainly preserved as hay, and after cutting the crop was left in the field to dry, a practice very much relying on weather conditions and usually requiring additional drying in the barn. Similarly, most of the harvested cereal grain is preserved by drying, either with cold air, cold air with additional heating, or with hot air. Cold air drying often results in uneven water content in the cereal grain, indicating that the drying zone had not passed through the entire batch in the dryer. Drying without heat may also be ineffective due to high moisture levels in the air, so the safest storage method is hot air drying with air temperatures of 60–100 °C. However, this method demands a substantial input of energy as up to 60% of the total energy consumption during grain production may be expended during drying alone (Pick et al. 1989). Today, drying is the only technique available for grain intended for human consumption, whereas several alternatives exist for grains used as animal feed. 9.3.2  Acid treatment Acid treatment, i.e. addition of inorganic or organic acids, is used both for forage and cereal grains and results in a rapid decrease in pH, which efficiently prevents the growth of most microorganisms. Acid treatment inactivates the sprout and interferes with the baking process, and thus is not appropriate for storage of grain intended for baking or malting (Jonsson 1997). Acid application is a delicate process and needs to be monitored accurately as addition of the correct concentrations of acid depends on the water content of the crop (Lacey and Magan 1991). Uneven distribution of acid over the kernel surface may permit mould growth during storage as inaccurate dosage of formic acid has, for example, been shown to increase the risk of aflatoxin production (Clevström et al. 1989). Low concentrations of propionate may also stimulate the production of aflatoxins (Al-Hilli and Smith 1979). Balanced concentrations of propionic acid may be sufficient to inhibit the normal spoilage moulds associated with cereals in temperate climates, but not Aspergillus flavus. Even though growth of this fungus has been partially

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inhibited, it can still produce aflatoxin B1 at enhanced levels in these conditions. The production of acids is very energy consuming and has been estimated to represent 15–20% of the energy consumed during silage production (Strid and Flysjö 2007). 9.3.3  Airtight storage Airtight storage of high moisture feed grain requires only ~ 2% of the energy consumed in high-temperature drying (Pick et al. 1989). Safe storage of grains relies on a perfectly airtight silo with a modified atmosphere, enabling storage of the cereal grain at higher MC. Respiration of both the grain and endogenous microflora reduces levels of oxygen and increases levels of carbon dioxide (Lacey and Magan 1991; Magan et al. 2003). However, the control of spoilage microorganisms depends on maintaining the modified atmosphere. Temperature fluctuations may, in turn, generate pressure fluctuations in the silo (Druvefors et al. 2002). Also, imperfect sealing and feed outtake lead to gas leakage. Feed outtakes also result in a continuously diminishing grain bulk, making it difficult for the microbial and grain respiration to sustain the modified atmosphere needed for safe storage. Deteriorative microbial development and spontaneous heating may then occur (Lacey and Magan 1991). Airtight storage is not suitable for grain intended for baking, as the gluten protein is adversely affected, and the germination capacity impaired.

9.4  Biopreservation Bacterial and mould growth during storage can be minimised by introducing biocontrol organisms into the storage system. For example, the yeast Debaromyces hansenii has been used for postharvest control of citrus-rot (Wilson and Wisniewski 1989), and several species of Cryptococcus for the control of postharvest rot on apples, pears, strawberries, tomatoes, cucumbers, etc. (Passoth and Schnürer 2003). The yeast Candida oleophila is available commercially and used on pomme and citrus fruit (Janisiewicz and Korsten 2002). A traditional way of controlling pathogenic fungi in feed is to use lactic acid bacteria (LAB). These are used in a variety of fermented foods, including sauerkraut and a multitude of dairy products, as well as in silage for use as animal feed (Stiles 1996). Both endogenous and artificially introduced antagonists have been proposed as promising alternatives to fungicide-based control of postharvest diseases (Wilson and Wisniewski 1989; Wisniewski and Wilson 1992). 9.4.1  Fermentation – silage and moist crimped grains Whole crops, including grasses, legumes and whole cereal plants, especially wheat and maize, can be conserved by ensiling. Ensiling is a technique that relies on the production of lactic acid from fermentable sugars in the material by LAB. One

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prerequisite for preserving crops by this method is the generation of anaerobic conditions to initiate fermentation. Imperfect sealing may lead to re-entry and circulation of air during storage, which in turn, supports aerobic microbial activity, resulting in decay of the material to a useless, inedible and frequently toxic product (Weinberg 2008). Another essential problem is to inhibit proliferation of undesirable microorganisms, which may produce objectionable fermentation products, and this can be done by encouraging the growth of LAB or by using chemical additives (McDonald et al. 1991; Schnürer and Magnusson 2005). Aerobic fungi and bacteria are the dominant microorganisms on fresh herbage, but when anaerobic conditions develop in the storage system, they are replaced by bacteria able to grow in the absence of oxygen. These include LAB, Clostridia and Enterobacteriaceae. LAB, which are facultative anaerobes (able to grow in the presence or absence of oxygen) are generally present on growing crops in small numbers, but usually multiply rapidly after harvest. They differ in their ability to ferment carbohydrates and can be divided into three groups: obligate homofermentative; facultative heterofermentative; and obligate heterofermentative. Obligate homofermentative LAB convert hexoses into lactic acid, whereas heterofermentative metabolism of hexoses yields equimolar amounts of lactic acid, carbon dioxide and ethanol (or in certain conditions, acetic acid). Distinct from the first group, the latter two groups of heterofermentative LAB can also ferment pentoses. The facultative heterofermentative LAB include species important for ensiling such as Lactobacillus plantarum and Lactococcus pentosus. These typically ferment hexoses using the homofermentative pathway but can, in certain conditions, switch to heterofermentative metabolism. They can also ferment pentoses into lactic and acetic acids. Due to these differences in end products, the composition of the LAB population naturally present in the plant material will influence the outcome of the fermentation. If lactic acid is the major end product, there will be a rapid decrease in pH to a level that inhibits other anaerobic bacteria. On the other hand, the presence of lactic acid-utilising yeasts in the feed, which are also tolerant to low pH, such as Candida lambica and Geotrichum candidum, can lead to low aerobic stability – degradation of the lactic acid raises the pH and the feed deteriorates rapidly on exposure to air due to the growth of moulds and aerobic bacteria. A predominance of heterofermentative metabolism will yield higher fermentation losses due to production of carbon dioxide and ethanol, but usually the presence of acetic acid leads to better aerobic stability. LAB are also know to produce a variety of antibacterial and antifungal compounds, such as hydrogen peroxide, bacteriocins, proteinaceous compounds, phenyl lactic acid, diacetyl and cyclic dipeptides, reviewed in Schnürer and Magnusson 2005. Studies on antifungal compounds have mainly been performed in artificial media and little is known about their contribution to fungal inhibition in animal feed. Broberg et al. (2007) have shown that several antifungal compounds are produced by LAB in silage but at low concentrations. The use of natural fermentation permits harvesting of the crop at higher water contents, which protects the crop from prolonged exposure to inclement weather that might otherwise lead to

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weathering and mould infections of the grain in the field (Lacey and Magan 1991). However, the success of natural fermentation depends on a number of factors, for example: the strains of indigenous LAB and yeasts present and their population density; cultivation; crop management; and conditions of harvest and storage. Cereal grains may also be preserved by lactic acid fermentation comparable to that in silage production (Finch et al. 2002), and this is termed moist crimped cereal grain. Harvest of cereals intended for moist crimping should preferentially occur during grain yellow ripeness and while the kernels have a MC of 30–45%. Prior to storage, the cereals are preferably rolled to facilitate packing and thereby reduce air-space between the kernels. At sufficiently high MC, a spontaneous fermentation is proposed to start, resulting in ensiled cereal grain (www. kelvincave.com). However, Olstorpe et al. (2010a) noted only a slight reduction in pH during storage of moist cereal grain. It is likely that the concentrations of accessible and easily degradable sugars in the grain are too low to support substantial acid production by the LAB and the concomitant decrease in pH during the storage period. Thus, moist grain storage is very different from established grass silage systems, where storage stability is primarily achieved by the decrease in pH. Moist crimped cereal grain cannot be stored in silos, as a MC above 25% impedes the feed outtake system (Jonsson 1996). However, other structures could be used, such as permanent clamps or bunkers, or large plastic tubes. The use of plastic tubes has increased in the last few years, probably because capital and maintenance costs for permanent storage space are replaced by mobile costs when using plastic tubes for storage (Sundberg 2007). 9.4.2  Improving airtight storage using Pichia anomala Harvesting and packing moist crimped grain at optimal moisture content can prove difficult, as once the kernel reaches yellow ripening, the moisture content can change rapidly with weather conditions (Sundberg 2007). If the grain is poorly fermented, the preservative effect solely depends on the absence of oxygen in the system. This creates a substantial risk for mould growth when the plastic tube is opened, or if the plastic is damaged. However, yeasts possess several important characteristics that make them well suited to the biopreservation of cereal grain. In contrast to filamentous fungi, they do not produce allergenic spores or mycotoxins and the production of antibiotic metabolites as described for several bacterial antagonists has not been observed for yeasts. Yeasts generally have simple nutritional requirements, can grow rapidly on cheap substrates in fermenters, and are, therefore, easy to produce in large quantities. Various by-products from the food or ethanol industry can be utilised as growth substrates for yeasts, avoiding wasteful disposal, and thus decreasing costs and burden to the environment (Scholten and Verdoes 1997; Brooks et al. 2001). Yeasts are able to colonise dry surfaces for long periods of time, can rapidly utilise available nutrients and sustain many pesticides used in the postharvest environment. Furthermore, yeast cells contain high amounts of vitamins, minerals and essential amino acids, and the beneficial effects of yeast addition in food and feed have been reported several times, including positive

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effects in the gastrointestinal tract (Stringer 1982; Bui and Galzy 1990; Schroeder et al. 2004). However, sometimes yeasts are considered undesirable in feed, as they may confer off-flavours and taints that would affect palatability of the feed (Brooks et al. 2003). In fermented feeds, they are assumed to compete with LAB for the same substrates. Some yeast species may metabolise lactic acid resulting in an increased pH endangering the hygienic quality (Middelhoven and van Balen 1988). The yeast Pichia anomala has been shown to inhibit mould growth in malfunctioning airtight storage systems. P. anomala tolerates a wide range of temperatures (3–37 °C) and pH values (2.0–12.4), and can grow in anaerobic environments and at low water activity (aw 0.85) (Fredlund et al. 2002). The mouldinhibitory properties of P. anomala have been confirmed in studies using small to large scale silos containing moist grain (Petersson and Schnürer 1995, 1998; Petersson et al. 1999; Druvefors et al. 2002; Druvefors and Schnürer 2005; Olstorpe et al. 2010b). Several different mechanisms of fungal inhibition have been suggested for P. anomala, including competition for nutrients and space (Janisiewicz and Korsten 2002), production of killer toxins (Walker et al. 1995) and cell wall degrading enzymes (Jijakli and Lepoivre 1998). However, these mechanisms are probably not the primary basis of the anti-fungal activity of P. anomala in moist cereal grain storage. Ethyl acetate, a product of glucose metabolism in P. anomala, is likely to be involved in the inhibition of fungi on grain. It is a highly volatile compound with production increasing tenfold under oxygen limitation, and it has been shown to reduce growth of P. roqueforti at headspace concentrations of ≥ 2µg ml–1 (Fredlund et al. 2004; Druvefors and Schnürer 2005). Ethyl acetate is easily dispersed throughout the crimped grain and evaporates quickly once the fed is taken out of the tube. P. anomala inoculated moist grain has been fed to both bulls (Olstorpe et al. 2010b) and chickens (J. Schnürer, personal communication) without any observable negative effects on animal performance. During a field trial of moist grain storage, the number of Enterobacteriaceae, surprisingly, decreased in the P. anomala-inoculated grain to below detection level (10 cfu/g grains) (Olstorpe et al. 2010b). This finding is of great importance for feed hygiene, as reducing the number of Enterobacteriaceae in feed, in turn decreases their number later in the food chain (Brooks et al. 2001). It has earlier been shown in agar plate assays that P. anomala strains can inhibit E. coli and other Gram-negative bacteria, which was probably due to the production of killer toxins (Polonelli and Morace 1986). Whether these killer toxins also play a role in this grain storage system still needs further investigation. 9.4.3  Biopreservation and bioprocessing of liquid feed Liquid feed is attained by mixing dry formulations with some type of liquid, yielding a feed that is more or less fermented at feed out. In the literature, there is no clear distinction between liquid and fermented feed – fermented feed is sometimes categorised as liquid feed, and soaking vs fermentation processes are not defined. However, when a feed is mixed with liquid, fermentation is rapidly initiated (Canibe and Jensen 2003). Thus, most liquid diets are affected by

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microbial activity to varying extents. Mould growth is seldom a problem in these liquid feeding systems, as they do not proliferate during the biopreservation process (Lyberg et al. 2008; Olstorpe et al. 2010c). Thus, any mycotoxins present in bioprocessed liquid feed most certainly entered with the raw feed material. However, toxins from different Enterobacteriaceae potentially growing in the liquid feed may be detrimental to animal health. The microbial and chemical characteristics of fermented liquid feed differ among fermentations (Scholten et al. 2001a, b; Beal et al. 2005; Moran et al. 2006; Lyberg et al. 2008; Olstorpe et al. 2008, 2010c). The microorganisms involved in fermentation produce organic acids, which may reduce the pH to approximately 3.5–4.5. A low pH and high concentrations of lactic and acetic acids in liquid fermented feed can prevent proliferation of Enterobacteriaceae both in the feed and along the animal gastrointestinal tract (Geary et al. 1996, 1999; Mikkelsen and Jensen 1998; Lyberg et al. 2008). The varying microbial flora in fermented feed may influence the organic acid profile and, in turn, affect palatability and feed hygiene. Molecular species identification of microbial isolates demonstrated that the microbial population changes substantially during fermentation (Olstorpe et al. 2008), even though traditional plate counting methods suggested that the population was fairly stable (Lyberg et al. 2008). It is therefore important to influence the species composition in the feed, preferably by adding a starter culture to the bioprocessing system. In this way, feed hygiene and safe biopreservation can contribute to decreased Enterobacteriaceae later in the food chain.

9.5  From strain discovery to biopreservative starter culture Yeasts, LAB and propionic acid bacteria can all be used to prevent spoilage and mycotoxin formation in silage and feed grain (Weinberg and Muck 1996; Passoth et al. 2006). At present, two major challenges severely restrict the development of novel biopreservative products for the market: (i) issues relating to microbial safety and regulatory aspects, and (ii) formulation of microorganism, i.e. the stabilisation of microbial cells as a dry stable powder, with high survival and activity upon rehydration (Melin et al. 2007b). 9.5.1  Safety and regulatory requirements Risks to human health during production, manufacture, storage and application of new microorganisms must always be minimised. In particular, pathogenicity and production of toxic compounds and allergens must be considered. We have characterised antifungal metabolites from biopreservative LAB (Ström et al. 2002) and also detected these in the LAB-inoculated grass silage (Broberg et al. 2007). Standard biosafety protocols designed for general protection from pathogens do not suffice for biopreservation purposes. Moreover, existing tests for production of toxins and sensitising agents are poorly adapted for testing

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microorganisms. New assessment systems/packages encompassing a survey of the current body of knowledge, in vitro testing of potential toxicity, determination of antibiotic resistance pattern, and temperature range for growth will thus have to be developed. Adoption of such systems early within investigations will reveal whether any particular safety precautions are needed for handling, and hence, whether strains are suitable for product development. When organisms are subsequently applied to the environment in large numbers, it is also important to evaluate whether their introduction results in harmful side-effects to the environment (Doblhoff-Dier et al. 1999). Possible concerns include harm to non-target organisms and interference with essential ecosystem functioning (Goettel et al. 2001; Winding et al. 2004). However, in general, environmental safety assessments are hampered by lack of fundamental knowledge of the organisms’ ecology and metabolite formation patterns. Regulations and policies influence the use of microorganisms in beneficial environmental and biotechnological applications, but requirements for authorisation, and hence the scope of safety assessments, vary depending on intended use. In the EU, work with microorganisms is regulated by Council Directive 2000/54/EC, while in Sweden, the Work Environment Authority is responsible for control (guideline AFS2005:1). Swedish regulation of ‘bio-technical’ organisms is legislated by Environmental Code 1998:808, valid for biopreservation strains. Specific EU legislation regulates new microbiological plant protection products, biocides and feed biopreservatives, but development of European biopesticides in particular has been slow, hampered by extensive data requirements and long authorisation processes (Chandler et al. 2008). 9.5.2  Formulation technology and biology Practical use of microorganisms in environmental application requires stable dry formulations of microbial agents, as drying and subsequent storage and rewetting impose severe stresses on the organisms. The biological basis for surviving drought and rehydration stress is not well understood, but both intrinsic biological factors of microorganisms and extrinsic physical factors influence the final formulation outcome (Melin et al. 2007a; Schoug et al. 2006, 2008). Development of novel dry microbial products remains a difficult, timeconsuming process of trial and error and the challenge to stabilise dry products in ambient conditions for long time periods remains. Loss of viability and dry-state quality result from both physical breakdown of the surrounding matrix and cellular degradation (Higl et al. 2007; Santivarangkna et al. 2008). Microbial responses to mild stress improve their ability to survive drastic environmental fluctuations through physiological adaptive changes (Prasad et al. 2003). Recombinant introduction of genes originating from organisms tolerating low water activity or complete desiccation, into microbes sensitive to drying is one possible route to improve drying tolerance, known as anhydrobiotic engineering (anhydrobiosis = life without water). Current thinking on anhydrobiotic engineering is that mechanisms needed for the full protective effect rely on both microbiological and

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physical factors (Iturriaga 2008; Caramelo and Iusem 2009). Combining general formulation science (pharmaceutics and galenics), chemistry, and molecular microbiology, may improve microbial products for different applications.

9.6  Concluding remarks The formation of mycotoxins is a recalcitrant problem both in feed and food – these toxins represent a substantial health risk for humans and animals and a risk for economic losses. Prevention of mould growth in all parts of the feed and food chains can be regarded as the best method of decreasing the mycotoxin burden. In principle, fungal growth can be diminished by the use of fungicides; however, their utilisation is increasingly restricted, due to environmental and health concerns. Chemical conservation such as acid treatment may be an environmental burden due to potential leakage of the conservation agent, and furthermore, reducing the risk for mould contamination is highly dependent on operator competence. Physical treatment (drying) currently provides the safest method for feed conservation, but increasing energy prices generate a demand for alternatives. Bioconservation may provide such an alternative as the potential of a variety of microorganisms to prevent mould growth has frequently been shown. Moreover, fermentation-based conservation of feed and food material has been used extensively in agriculture and is well-established. Microbes that grow during fermentation may even have potential to improve the nutritional value of the feed. However, the microbial ecology in feed bioconservation is often only poorly understood. Most of the established processes build on spontaneous microbial developments, and recent investigations in which microbial species were identified have shown that microbial populations differ substantially between different batches produced with the same preservation technology. This uncertain output of the spontaneous bioconservation processes represents a risk not only for increased mycotoxin production, but also for the introduction of pathogenic organisms into the food chain. It is, therefore, necessary to study microbial interactions in the different storage systems, and to generate appropriate starter cultures to ensure a predictable storage flora.

9.7  References and m o moss (2000). Food Microbiology. Cambridge, UK., The Royal Society of Chemistry. al - hilli a l and j e smith (1979). ‘Influence of propionic acid on growth and aflatoxin production by Aspergillus flavus.’ FEMS Microbiology Letters 6: 367–370. aldred d and n magan (2004). ‘Prevention strategies for trichothecenes.’ Toxicology Letters 153: 165–171. beal j d , s j niven et al. (2005). ‘Variation in short chain fatty acids and ethanol concentration resulting from the natural fermentation of wheat and barley for inclusion in liquid diets for pigs.’ J. Sci. Food Agric. 85: 433–440. adams m r

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broberg a , jacobsson k , ström k

and schnürer j (2007). ‘Metabolite profiling of lactic acid bacteria in silage.’ Appl. Envl. Microbiol. 73: 5547–5552. brooks p h , j d beal et al. (2001). ‘Liquid feeding of pigs: potential for reducing environmental impact and for improving productivity and food safety.’ Recent Advances in Animal Nutrition in Australia 13: 49–63. brooks p h , j d beal et al. (2003). ‘Liquid feeding of pigs. II. Potential for improving pig health and food safety.’ Anim. Sci. Pap. Rep. 21 (suppl. 1): 23–29. bui k and p galzy (1990). ‘Food yeast.’ In yeast technology (Spencer J F T and Spencer D M, eds), p. 407. Springer, Berlin. canibe n and b b jensen (2003). ‘Fermented and nonfermented liquid feed to growing pigs: Effect on aspects of gastrointestinal ecology and growth performance.’ J. Anim. Sci. 81: 2019–2031. caramelo j j and iusem n d (2009). ‘When cells lose water: Lessons from biophysics and molecular biology.’ Prog. Biophys. Mol. Biol. 99: 1–6. chandler d , davidson g , grant w p et al. (2008). ‘Microbial biopesticides for integrated crop management: an assessment of environmental and regulatory sustainability.’ Food Sci. Tech. 19: 275–283. clevström g , t möller et al. (1989). ‘Influence of formic acid on fungal flora of barley and on aflatoxin production in Aspergillus flavus.’ Journal Mycopathologia 107: 101–109. doblhoff - dier o h , bachmayer a , bennett g et al. (1999). ‘Safe biotechnology 9: Values in risk assessment for the environmental application of microorganisms.’ Trends Biotech. 17: 307–311. druvefors u , n jonsson et al. (2002). ‘Efficacy of the biocontrol yeast Pichia anomala during long-term storage of moist feed grain under different oxygen and carbon dioxide regimens.’ Yeast Research 2: 289–394. druvefors u ä and j schnürer (2005). ‘Mold-inhibitory activity of different yeast species during airtight storage of wheat grain.’ FEMS Yeast Research 5: 373–378. finch h j s , a m samuel et al. (2002). Cereals. Lockhart and Wiseman’s Crop Husbandry Including Grasslands. Cambridge, England, Woodhead Publishing Limited: 259–302. fink - gremmels j (2008). ‘The role of mycotoxins in the health and performancce of dairy cows.’ The Veterinary Journal 176: 84–92. flannigan b (1987). The Microflora in Barley and Malt. London, Elsevier. fleet g (1990). ‘Yeasts in dairy products.’ Journal of Applied Bacteriology 68: 199–211. fleet g (1992). ‘Spoilage yeasts.’ Critical Reviews in Biotechnology 12: 1–44. fredlund e , u ädel druvefors et al. (2004). ‘Influence of ethyl acetate production and ploidy on the anti-mould activity of Pichia anomala.’ FEMS Microbiol. Letters 238 (2): 475–478. fredlund e , u druvefors et al. (2002). ‘Physiological characteristics of the biocontrol yeast Pichia anomala J121.’ FEMS Yeast Resarch 2: 395–402. galtier p (1998). ‘Biological fate of mycotoxins in animals.’ Revue Méd. Vét. 149: 549–554. geary t m , p h brooks et al. (1999). ‘Effect on weaner pig performance and diet microbiology of feeding a liquid diet acidified to pH 4 with either lactic acid or through fermentation withPediococcus acidilactici.’ Journal of the Science of Food and Agriculture 79(4): 633–640. geary t m , p h brooks et al. (1996). ‘Performance of weaner pigs fed ad libitum with liquid feed at different dry matter concentrations.’ Journal of the Science of Food and Agriculture 72(1): 17–24. goettel m s hajek a e et al. (2001). ‘Safety of fungal biocontrol agents.’ In TM Butt C W Jackson, and N Magan (eds), Fungi as Biocontrol Agents – Progress, Problems and Potential. CABI Publishing, Wallingford, UK. häggblom p (1990). ‘Isolation of Roquefortine C from feed grain.’ Appl. Microbiol. Biotechnol. 56(9): 2924–2926.

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et al. (2007). ‘Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix.’ Biotechn. Prog. 23: 794–800. iturriaga g (2008). ‘The LEA proteins and trehalose loving couple: a step forward in anhydrobiotic engineering.’ Biochem. J. 410: 1–2. janisiewicz w j and l korsten (2002). ‘Biological control of postharvest diseases of fruits.’ Annu. Rev. Phytopathol. 40: 411–441. jijakli m h and p lepoivre (1998). ‘Characterization of an Exo-beta.1,3-glucanase produced by Pichia anomala strain K, antagonist of Botrytis cinerea on apples.’ Phytopathology 88(4): 335–343. jonsson n (1996). ‘Konservera och lagra spannmål rätt.’ Uppsala, Teknik för lantbruket, JTI – Jordbrukstekniska institutet: 1–11. jonsson n (1997). ‘Syrabehandla spannmål rätt.’ Uppsala, Teknik för lantbruket, JTI – Jordbrukstekniska institutet: 1–11. kaspersson a , s lindgren et al. (1988). ‘Microbial Dynamics in Barley Grain Stored Under Controlled Atmosphere.’ Animal Feed Science and Technology 19: 299–312. lacey j (1989). ‘Pre- and post-harvest ecology of fungi causing spoilage of foods and other stored products.’ Journal of Applied Bacteriology 67: 11–25. lacey j and n magan (1991). ‘Fungi in cereal grains: Their occurence and water and temperatur relationships.’ In Cereal Grain Mycotoxins, Fungi and Quality in Drying and Storage. (ed. J Chelkowski). Amsterdam, The Netherlands, Elsevier Science Publishers B.V. 26: 77–118. loureiro v and m malfeito - ferreira (2003). ‘Spoilage yeasts in the wine industry.’ International Journal of Food Microbiology 86(1): 23–50. lyberg k , m olstorpe et al. (2008). ‘Biochemical and microbiological properties of a cereal mix fermented with whey, wet wheat distillers’ grain or water at different temperatures.’ Anim. Feed Sci. Technol. 144: 137–148. magan n , r hope et al. (2003). ‘Post-harvest fungal ecology: Impact of fungal growth and mycotoxin accumulation in stored grain.’ European Journal of Plant Pathology 109: 723–730. mcdonald p , a henderson et al. (1991). The Biochemistry of Silage. Marlow, Chalcombe Publications. middelhoven w j and a h m van balen (1988). ‘Development of the yeast flora of whole-crop maize during ensiling and during subsequent aerobiosis.’ J. Sci. Food Agric. 42: 199–207. mikkelsen l l and b b jensen (1998). ‘Performance and microbial activity in the gastrointestinal tract of piglets fed fermented liquid feed at weaning.’ Journal of Animal Feed Sciences 7: 211–215. melin p , håkansson s , schnürer j (2007a). ‘Optimisation and comparison of liquid and dry formulations of the biocontrol yeast Pichia anomala J121.’ Appl. Microbiol. Biotechnol. 73: 1008–1016. melin p , sundh i , håkansson s and schnürer j (2007b). ‘Biological preservation of plant derived animal feed with antifungal microorganisms – Safety and formulation aspects.’ Biotechnology Letters 73:1008–1016. moran c a , r h j scholten et al. (2006). ‘Fermentation of wheat: Effects of backslopping different proportions of pre-fermented wheat on the microbial and chemical composition.’ Archives of Animal Nutrition 60(2): 158–169. müller h - m and r amend (1997). ‘Formation and disappearance of mycophenolic acid, patulin, penicllic acid and PR toxin in maize silage inoculated with Penicillium roqueforti.’ Arch. Anim. Nutr. 50: 213–225. olstorpe m , k lyberg et al. (2008). ‘Population diversity of yeasts and lactic acid bacteria in pig feed fermented with whey, wet wheat distillers’ grains or water at different temperatures.’ Appl. Environ. Microbiol. 74(6): 1696–1703. higl b , kurtmann l , carlsen c

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et al. (2010a). ‘Microbial changes during storage of moist crimped cereal barley grain under Swedish farm conditions.’ Anim. Feed Sci. Technol. 156: 37–46. olstorpe m , j borling et al. (2010b). ‘Pichia anomala yeast improves feed hygiene during storage of moist crimped barley grain under Swedish farm conditions.’ Anim. Feed Sci. Technol. 156: 47–56. olstorpe m , l axelsson et al. (2010c). ‘Effect of starter culture inoculation on feed hygiene and microbial population development in fermented pig feed composed of a cereal grain mix with wet wheat distillers grain.’ Journal of Applied Microbiology 108: 129–138. passoth v and j schnürer (2003). ‘Non-conventional yeast in antifungal application.’ In de Winde J H (ed.) Functional Genetics of Industrial Yeast, Springer-Verlag, Berlin, Heidelberg, Germany, pp. 297–319. passoth v, fredlund e , druvefors u and schnürer j (2006). ‘Biotechnology, physiology and genetics of the yeast Pichia anomala’. FEMS Yeast Res 6: 3–13. petersson a , n jonsson et al. (1999). ‘Pichia anomala as a biocontrol agent during storage of high-moisture feed grain under airtight conditions.’ Postharvest. Biol. Technol. 15: 175–184. petersson s and j schnürer (1995). ‘Biocontrol of mould growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.’ Appl. Environ. Microbiol. 61(3): 1027–1032. petersson s and j schnürer (1998). ‘Pichia anomala as a biocontrol agent of Penicillium roqueforti in high-moisture wheat, rye, barley and oats stored under airtight conditions.’ Can. J. Microbiol. 44: 471–476. pick e , o noren et al. (1989). Energy Consumtion and Input Output Relations in Field Operations. Food and Agricultural Organization of the United Nations, Rome, Italy. polonelli l and g morace (1986). ‘Reevaluation of the yeast killer phenomenon.’ Journal of Clinical Microbiology 24(5): 866–869. prasad j , mcjarrow p and gopal p (2003). ‘Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying.’ Appl. Env. Microbiol. 69: 917–25. santivarangkna c , kulozik u and foerst p (2008). ‘Inactivation mechanisms of lactic acid starter cultures preserved by drying processes.’ J. Appl. Microbiol. 105: 1–13. schnürer j and j magnusson (2005). ‘Antifungal lactic acid bacteria as biopreservatives.’ Trends in Food Science & Technology 16: 70–78. scholten r, m j a rijnen et al. (2001a). ‘Fermentation of liquid coproducts and liquid compound diets: Part 1. Effects on chemical composition during 6-day storage period.’ Journal of Animal Physiology and Animal Nutrition 85: 111–123. scholten r, m j a rijnen et al. (2001b). ‘Fermentation of liquid coproducts and liquid compound diets: part 2. Effects on pH, acid-binding capacity, organic acids and ethanol during 6-day storage period.’ Journal of Animal Physiology and Animal Nutrition 85: 124–134. scholten r h j and n verdoes (1997). ‘The dutch benefit of a recycling role.’ Pigs 13(2): 16–17. schoug å , fischer j , heipieper h j , schnürer j , håkansson s (2008). ‘Impact of fermentation pH and temperature on freeze-drying survival and membrane lipid composition of Lactobacillus coryneformis Si3.’ J. Ind. Microbiol. Biotechnol. 35, 175–181. schoug å , olsson j , carlfors j , schnürer j and håkansson s (2006). ‘Freezedrying of Lactobacillus coryniformis Si3 – effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties.’ Cryobiol 53: 119–127. schroeder b , c winckler et al. (2004). ‘Studies on the time course of the effects of the probiotic yeast Saccharomyces boulardii on electrolyte transport in pig jejunum.’ Digestive Diseases and Sciences 49: 1311–1317. olstorpe m , j schnürer

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(1996). ‘Biopreservation by lactic acid bacteria.’ Antonie van Leeuwenhoek 70: 331–345. strid i and a flysjö (2007). Livscykelanalys (LCA) av ensilage – jämförelse av tornsilo, plansilo och rundbal, 3. stringer d a (1982). ‘Industrial development and evaluation of new protein sources: microorganisms.’ Proc. Nutr. Soc. 41: 289–300. ström k , sjögren j , broberg a and schnürer j (2002). ‘Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro), cyclo(LPhe-trans-4-OH-L-Pro) and 3-phenyllactic acid.’ Appl. Env. Microbiol. 68: 4322–4327. styriak i and e conková (2002). ‘Microbial binding and degradation of mycotoxins.’ Vet. Human Toxicol. 44: 358–361. sundberg m (2007). ‘Foderkonservering i slang. JTI – Institutet för jordbruks- och miljöteknik.’ Uppsala, JTI – Istitutet för jordbruks- och miljöteknik, 116: 1–11. walker g m , a h mcleod et al. (1995). ‘Interactions between killer yeasts and pathogenic fungi.’ FEMS Microbiol. Letters 127: 213–222. weinberg z g and muck r e (1996). ‘New trends and opportunities in the development and use of inoculants for silage.’ FEMS Microbiol. Rev. 19: 53–68. weinberg z g (2008). Current Developments in Solid-state Fermentation Preservation of Forage Crops by Solid-state Lactic Acid Fermentation-Ensiling C. R. S. a. C. L. Ashok Pandey, Springer New York, pp. 443–467. westlake , k , r i mackie et al. (1989). ‘In vitro metabolism of mycotoxins by bacterial, protozoal and ovine ruminal fluid preparations.’ Anim. Feed. Sci. Technol. 25: 169–178. wilson c l and m e wisniewski (1989). ‘Biological control of postharvest diseases of fruits and vegetables: an emerging technology.’ Annual Review of Phytopathology 27: 425–441. winding a , binnerup s j and pritchard h (2004). ‘Non-target effects of bacterial biological control agents suppressing root pathogenic fungi.’ FEMS Microbiol. Ecol. 47: 129–141. wisniewski m e and c l wilson (1992). ‘Biological control of postharvest diseases of fruits and vegetables – recent advances.’ Hortscience 27: 94–98. yiannikouris a and j - p jouany (2002). ‘Mycotoxins in feeds and their fate in animals: a review.’ Anim. Res. 51: 81–99. stiles m e

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10 Biological control of human digestive microbiota using antimicrobial cultures and bacteriocins I. Fliss, R. Hammami and C. Le Lay, Laval University, Canada

Abstract:  Over the past decade, several studies have evaluated probiotic treatments of various gastrointestinal diseases. As molecular data have accumulated, a debate has arisen around how much we really know about probiotics diversity, benefits and mechanisms of action in the human gastrointestinal tract (GIT). Interactions between bacterial species are considered essential for maintaining the equilibrium of the intestinal microflora. The lactic acid bacteria (LAB) population, including bifidobacteria, in particular plays an important role in the regulation of the diversity of gut microbiota and in the defense of the human GIT. These bacteria colonize the GIT and form a barrier against the proliferation of exogenous pathogens and also inhibit pathogen growth. This chapter summarizes the most significant studies dealing with use of antimicrobial cultures in controlling human digestive microbiota, along with the different mechanisms involved. Key words:  human gastrointestinal defense, gastrointestinal microbiota, antimicrobial cultures, lactic acid bacteria, probiotics.

10.1  Introduction The gastrointestinal tract (GIT) is one of the largest systems of the body and consists of a series of tubes that begin at the mouth and end at the rectum. The main organs of the digestive system are mouth, esophagus, stomach, small and large intestines, rectum, gallbladder, liver and pancreas. The anatomy and physiology of the gut are organized to serve many important functions, the first of which relates to the assimilation of nutrients and elimination of waste. The GIT also plays a major protective role against deleterious factors including medications, toxins, and infectious organisms. This protective role may be divided into immunological and non-immunological mechanisms. The former include 240 © Woodhead Publishing Limited, 2011

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both specific and non-specific responses to the presence of foreign agents, while the latter include non-specific barriers to invasion in which intestinal microbiota seems to play a major role. The factors involved in the success of an organism as an intestinal colonizer are associated primarily with nutrition, that is, the ability to utilize the available carbon sources faster than the competitors. Successful utilization of substrates generally results in rapid production of organic acids and many other inhibitory compounds and therefore inhibition of many other bacteria. In the case of what we call ‘protective cultures’, their protective effects are associated to at least five mechanisms: • They produce lactic and acetic acids, which are inhibitory to many undesirable bacteria. • They utilize the available carbon sources faster than the undesirable competitors. • They attach to the intestinal wall and thereby prevent pathogens from doing so, and possibly displace attached pathogens. • They appear to produce and excrete bacteriocins or bacteriocin-like substances, which exert more specific and more potent antibacterial effects than organic acids. • They are believed to produce or shed substances that bring about local stimulation of the immune system, making it more responsive to invasion by pathogens. This chapter summarizes the most significant in vitro and in vivo studies dealing with the potential of antimicrobial cultures in controlling human digestive microbiota. The different mechanisms involved in this antimicrobial are also discussed.

10.2  Human gastrointestinal defenses The gastrointestinal (GI) defenses of an animal host may be divided into immunological and non-immunological mechanisms. The former include both specific and non-specific responses to the presence of foreign agents, while the latter include non-specific barriers to invasion (for review see Israel, 1994). Gastric acid secretion (Udall, 1981), proteolytic pancreatic enzymes, mucin gel (Laboisse et al., 1996), the colonizing microbiota (O’Hara and Shanahan, 2006; O’Toole and Cooney, 2008), lysozyme (Porter et al., 2002), hepatic bile acids (Bertók, 2004) and intestinal peristalsis (Berseth, 1989) are the major contributors to the non-immunological defense mechanisms. The immune system contains an array of specialized cells that interact with non-immune cells and other mediators to generate complex, overlapping, specific immune and non-specific inflammatory responses (Mason et al., 2008). Their coordinated effect is to generate immediate inflammatory responses to contain invading pathogens, generate specific cellular and antibody responses and promote long-term immunological memory. Specific and non-specific immune mechanisms function in concert with non-immunological mechanisms to protect the host.

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10.3 Gastrointestinal microbiota as partner for human defense The human GI tract harbors trillions of microorganisms (approximately 1011 per g feces) that carry out vital processes for normal digestive functions of the host and play an important, albeit not yet not fully understood, role in the maturation of human immunity and defense against pathogens (Verberkmoes et al., 2008). Despite the efforts made in this field, only a few commensal microbial species have been characterized and much information on the human microbiota is still missing. The GI microbiota is incredibly diverse and dynamic and each human possesses a unique community of bacteria, archaea and eukarya (see RajiliStojanovi et al., 2007 for a review). Unless disrupted by external factors such as antibiotic treatment, the microbiota composition is quite stable. The causes and consequences of temporal and inter-individual variation in microbiota community composition remain questions to be resolved. The intestinal microbiota of humans is a complex mixture of microbial organisms, mostly bacteria, comprising at least 80 genera and around 1800 phylotypes (Rajilic-Stojanovic et al., 2009). Their numbers in the large intestine are typically in the range of 1013–1014 cells, which corresponds to a wet mass of less than 1 kg, or up to half of the fecal mass. Less than 20% of the intestinal flora has been characterized taxonomically to any significant degree. Flora metabolism is fermentative, with or without gas production and the vast majority of the organisms are either anaerobic or have limited tolerance to oxygen, although this latter trait is variable, even within species of anaerobes. What is known for sure is that intestinal floral equilibrium may be altered by several factors, by either infection or antibiotic therapy resulting in diarrhea and hence exposure of the subject to additional and often more dangerous infections (e.g. Clostridium difficile). One efficient way to restore this equilibrium is the use of probiotics which are defined as live microbial organisms that when fed to an animal are benign and colonize the large intestine, where they may persist for some unspecified but prolonged period of time and in doing so, ‘stabilize’ perturbed flora or make the intestinal environment refractory to invasion by pathogenic organisms. They thus confer a health benefit to the animal or human consumer of the probiotic.

10.4 Antimicrobial cultures: lactic acid bacteria and probiotics Probiotics are defined as ‘living microbial organisms, which upon ingestion in sufficient numbers exert health benefits beyond inherent basic nutrition’ (FAO). One of the most significant groups of probiotic organisms are the lactic acid bacteria (LAB), commonly used in fermented dairy products. These bacteria are widespread in nature – in soil, vegetables, meat, milk and the human body. The qualifier ‘lactic acid’ refers to the property of fermenting sugars primarily to lactic

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acid. Many members of the LAB group have been isolated from food-related sources and are therefore generally recognized as safe (GRAS) for food use (Salminen et al., 1998). Since the first LAB (Bacterium lactis, now known as Lactococcus lactis) was isolated by Joseph Lister in 1873, many genera have been thoroughly characterized and used in the food industry. A typical LAB is a Grampositive rod or coccus, non-spore-forming, catalase negative in the absence of porphorinoids, aerotolerant, acid tolerant, organotrophic, strictly fermentative and producing lactic acid as a major end product. It lacks cytochromes and is unable to synthesize porphyrins. The genera Lactobacillus, Leuconostoc, Pediococcus and Streptococcus form the core of the group. However, the genus Bifidobacterium, often considered in the same context as genuine LAB and sharing some of their typical features, is phylogenetically unrelated and has a unique mode of sugar fermentation. The essential feature of LAB metabolism is efficient carbohydrate fermentation coupled to substrate-level phosphorylation. LAB as a group exhibits an enormous capacity to utilize different carbohydrates and related compounds with the predominant end product being lactic acid (>50% of sugar carbon). Interactions between various bacterial species are considered essential to maintaining the equilibrium of the intestinal microflora. The LAB population in particular plays an important role in the regulation of the diversity of gut microbiota and the defense of the human GI tract. These bacteria colonize the GI tract and form a barrier against the proliferation of exogenous pathogens by preventing their adhesion to the intestinal lining. They also inhibit pathogen growth by producing and releasing organic acids, volatile fatty acids, antibiotic compounds, bacteriocins and/or host immune response-stimulating factors (Lomax and Calder, 2009). Salminen et al., 2005 have reviewed this subject. Many species of microorganisms have been used as probiotics, including lactic acid bacteria (e.g. lactobacilli, streptococci, enterococci, lactococci) and bifidobacteria, but also Escherichia coli and species of Bacillus, yeasts such as Saccharomyces and molds such as Aspergillus. However, the most common probiotics for human consumption belong to the genera Lactobacillus (e.g. L. casei, L. acidophilus, L. rhamnosus, L. johnsonii, L. reuteri) and Bifidobacterium (e.g. B. bifidum, B. longum, B. breve) (Gibson, 2008). The science of the intestinal flora and probiotics remains dogged by confusion surrounding at least one question regarding whether there is a relationship between the aptitude of a bacterial species to colonize the intestine and its aptitude to confer beneficial effects, in particular protection against pathogens. The persistence of an organism in the large intestine is not a criterion for meeting the definition of probiotic. In fact, probiotics owe their commercial viability to frequent consumption by healthy persons whose intestinal flora is normal and presumably stable, that is, well established and perhaps even forming a bio-film. In the case of the therapeutic use of a probiotic following a perturbation of the intestinal flora, for example by antibiotic therapy, it is quite likely that the probiotic strain establishes itself and persists as a component of the flora for a considerable time. It is generally acknowledged that the microbes that have become the most commercially successful probiotics (Saccharomyces boulardii, strains of

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Lactobacillus and especially Bifidobacterium) do not necessarily become permanent intestinal residents, making it necessary to ingest them on a regular basis as additives in food products (primarily dairy) if one hopes to enjoy the prophylactic benefits attributed to them. The ability of probiotic organisms to colonize the intestine for a long enough time to confer a benefit (e.g. protection against pathogens) varies considerably among strains within a specific species.

10.5  Mechanisms of action in human digestive microbiota Many mechanisms of action have been proposed to explain the stabilizing effects of LAB and probiotics on the intestinal microbiota. These include interfering with the attachment of pathogens to adhesion sites, out-competing pathogens for nutrients, degradation or other alterations of toxin receptors, production of inhibitory substances and stimulation of immunity/immunomodulation (Fig. 10.1) (see Corr et al., 2009 for review). 10.5.1  Blocking of adhesion sites The ability of probiotic bacteria to compete with pathogens for adhesion sites on the intestinal epithelial surface is one of these mechanisms of action (Kleeman

Fig. 10.1  Schematic representation of potential or known mechanisms by which probiotic bacteria might have an impact on the stability of intestinal microbiota (O’Toole and Cooney, 2008). IEC: epithelial cells; DC: dendritic cells: T: T-cells; B: B-cells; IL-10: interleukin-10; TGF-β: transforming growth factor beta.

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and Klaenhammer, 1982; Conway et al., 1987; Goldin et al., 1992). The key characteristic of intestinal pathogens is the ability to adhere to the surface of epithelial cells (Levine, 1987; Alam et al., 1996; Weinstein et al., 1998; Scaletsky et al., 2002). Adherence to mucosal surfaces can be mediated by different mechanisms, such as electrostatic, hydrophobic and hydrophilic attractions via capsular and fimbrial structures, or by a wide range of mammalian cell surface constituents such as glycoproteins and glycolipids acting as receptors for attachment (Lu and Walker, 2001). Competition for specific carbohydrate receptors or steric hindrance can inhibit the adhesion of pathogens, as shown in a study of E. coli and Salmonella adhesion to Caco-2 cells in the presence of Lactobacillus rhamnosus GG (Chauviere et al., 1992). All lactobacilli are able to attach to Caco-2 cells, although the degree of adhesion is dependent on bacterial strain (Ostad et al., 2009). Likewise, lactobacilli are able to compete with, exclude and displace GI bacteria, with the degree of inhibition of adhesion being strain dependent (Lee et al., 2003). For example, the probiotic agents Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG quantitatively inhibit the adherence of an attaching and effacing pathogenic Escherichia coli to HT-29 intestinal epithelial cells (Mack et al., 1999). 10.5.2  Pathogen exclusion via indirect mechanisms Another mode of action of antimicrobial cultures is pathogen exclusion by mechanisms such as maintaining the barrier function of intestinal cells. In a recent study, premature infants fed bifidobacteria displayed decreased gut permeability (Stratiki et al., 2007). Gupta and co-workers (2000) earlier reported a similar result with lactobacilli. Another study has demonstrated that L. rhamnosus GG can protect against E. coli O157:H7-induced decreases in whole-cell expression of ZO-1 that lead to decreased barrier function, by preventing changes in host cell morphology, monolayer formation and resistance and macromolecular permeability (Zareie et al., 2005). Moreover, pretreatment with L. rhamnosus GG has been found to prevent E. coli O157:H7-induced morphological redistribution of intercellular tight junction proteins as well as decreases in ZO-1 expression (Johnson-Henry et al., 2008). 10.5.3  Competition for nutrients Competition for nutrients has also been proposed frequently as a mechanism by which probiotics exert their effects as they may utilize more rapidly some of the nutrients that are required by pathogenic microorganisms (Sonnenburg et al., 2006). 10.5.4  Degradation of toxin receptors Another suggested mechanism is degradation of toxin receptors on the intestinal mucosa. This mode of action has been proposed for S. boulardii, which protects

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animals against C. difficile intestinal disease (Pothoulakis et al., 1993; Castagliuolo et al., 1996). 10.5.5  Stimulation of immunity/immunomodulation Accumulating evidence suggests that stimulation of adaptive and innate nonspecific immunity may be another mechanism by which probiotics exert protective effects against intestinal diseases (Kaila et al., 1992; Linkamster et al., 1994; Saavedra et al., 1994; Pouwels et al., 1996; Fukushima et al., 1998). Effects on adaptive immunity include increased proliferation of cells secreting IgA, IgG and IgM (Kaila et al., 1992; Rinne et al., 2005), increased total and specific secretory IgA in the serum and intestinal lumen (Rautava et al., 2006) and modulation of gut inflammatory immune responses. Increased levels of IFN-γ, IFN-α and IL-2 have been reported in numerous studies of healthy subjects ingesting probiotics (De Simone et al., 1991; Halpern et al., 1991; Solis Pereyra and Lemonnier, 1991; Kishi et al., 1996; Aattouri and Lemonnier, 1997; Wheeler et al., 1997; Arunachalam et al., 2000). Another study has shown that consumption of Lactobacillus acidophilus La1 and bifidobacteria increased specific and total secretory anti-Salmonella IgA after S. typhi oral vaccination (Linkamster et al., 1994). Effects of probiotic use on innate non-specific immunity include stimulation of mucin production and enhancement of natural killer cell and macrophage activities. Consumption of L. johnsonii La1, B. lactis Bb12, L. rhamnosus HN001, or B. lactis HN019 has been shown to enhance phagocytic capacity of peripheral blood leukocytes in healthy subjects. These effects were maintained for several weeks after ceasing consumption (Schiffrin et al., 1995) and were dose-dependent (Donnet-Hughes et al., 1999). The proportions of helper (CD4+), activated (CD25+) and total T-lymphocytes as well as natural killer cells measured in the blood have been shown to increase in subjects consuming B. lactis HN019 (Gill et al., 2001). Similar results were demonstrated earlier with a strain of B. lactis (Schiffrin et al., 1997). Meanwhile, Mack and co-workers (1999) demonstrated that incubating L. plantarum 299v with HT-29 cells increased MUC2 and MUC3 mRNA expression levels. 10.5.6  Production of inhibitory substances Probiotic bacteria produce a variety of substances that are inhibitory to both Gram-positive and Gram-negative bacteria. These inhibitory substances include organic acids, hydrogen peroxide and bacteriocins. Production of organic acids A large number of lactobacilli produce and excrete metabolites such as acetic and lactic acids, which lower the pH. The growth of bacterial pathogens may be inhibited by these products (Vandenbergh, 1993). One study has shown that antimicrobial activities of L. casei Shirota, L. acidophilus IBB 801, L. rhamnosus

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GG and L. amylovorus DCE 471 are likely due to the production of lactic acid and that antimicrobial activity of L. plantarum ACA-DC 287 and L. johnsonii La1 are apparently due to the production of lactic acid combined with an unknown inhibitory substance that is active only in the presence of lactic acid (Makras et al., 2006). Moreover, significant inhibition of the invasion of Caco-2/TC7 cells by Salmonella has been attributed to lactic acid (Makras et al., 2006). The lactic, acetic and hydrochloric acids produced by L. acidophilus, L. casei subsp. rhamnosus, L. bulgaricus and B. bifidus have been shown to inhibit the growth of H. pylori (Midolo et al., 1995). Production of hydrogen peroxide Resting cells of many strains of L. johnsonii and L. gasseri are able to produce H2O2 when incubated in the presence of oxygen and supernatants of cultures of these strains have been shown effective in killing Salmonella enterica serovar Typhimurium SL1344 (Pridmore et al., 2008). Production of bacteriocins Recent reports have revealed that some intestinal lactobacilli and bifidobacteria produce antimicrobial substances that are active against enterovirulent microorganisms. Bacteriocins are bactericidal proteinaceous molecules that have a relatively narrow killing spectrum, being toxic only to bacteria closely related to the producing strain (Hatakka and Saxelin, 2008). Bacteriocins were first identified almost 100 years ago and have been found among most families of bacteria and many actinomycetes and described as universally produced, including by some members of the Archaea (Riley and Wertz, 2002; Shand and Leyva, 2008). It has been speculated that almost all bacterial species produce some kind of bacteriocin or bacteriocin-like inhibitory substance waiting to be discovered. Bacteriocins make up a highly diverse family of proteins in terms of size, microbial target, mode of action and release and mechanism of immunity and can be divided into two broad groups: those produced by Gram-negative bacteria and those produced by Gram-positive bacteria (Gordon et al., 2007; Heng et al., 2007). Bacteriocins of Gram-positive bacteria are more abundant and more diverse than those found in Gram-negative bacteria (Hammami et al., 2009). According to their biochemical and genetic properties, bacteriocins are categorized into three different classes (see Cintas et al., 2001 for review): Class I bacteriocins are the lantibiotics (Willey and van der Donk, 2007); class II bacteriocins are subdivided into three subclasses, namely, class IIa (pediocin-like), class IIb (two-peptide), and IIc (other) (Drider et al., 2006); and class III bacteriocins are large (>30 kDa) and heat-labile proteins. We have developed BACTIBASE, a database dedicated to bacteriocins produced by both Gram-positive and Gram-negative bacteria (Hammami et al., 2007) (http://bactibase.pfba-lab-tun. org). This database provides physicochemical, structural, microbiological, and taxonomic information about bacteriocins, which would allow better and more comprehensive structural and functional analysis of this special group of antimicrobial peptides.

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The inhibitory mechanism of action of bacteriocins is also well studied. Bacteriocin action starts with entry into the target cell by recognizing specific cell surface receptors. Then, microbial cell killing occurs through various mechanisms: formation of ion-permeable channels in the cytoplasmic membrane, non-specific degradation of cellular DNA, inhibition of protein synthesis through the specific cleavage of 16s rRNA, or by cell lysis resulting from inhibition of peptidoglycan synthesis (Vriezen et al., 2009). The efficacy of bacteriocin as well as their producing strains for inhibiting several bacterial pathogens has been shown in different food matrices including cheese, meat and vegetables. However implication of bacteriocins as a mechanism of action in the inhibitory activity of probiotics remains unclear and needs more investigation. Only a few well known probiotic strains are shown to produce bacteriocins: P. acidilactici UL5, a pediocin PA-1 producing strain, has shown a very interesting probiotic potential with a high survival in the GI condition (Kheadr et al., 2010). L. salivarius UCC118, a probiotic strain of human origin, produces a bacteriocin (ABP-118) in vivo that can significantly protect mice against infection with the invasive foodborne pathogen L. monocytogenes (Corr et al., 2007). L. acidophilus La-5, a widely used probiotic strain in fermented milk manufacture, produces lactacin B (Tabasco et al., 2009). E. coli strain Nissle 1917, a producer of microcins H47 and M (Patzer et al., 2003), is a well characterized probiotic for use in humans and livestock. Its potential to protect from infectious gastroenteritis and for treatment of inflammatory bowel diseases is well documented (Schultz, 2008). Compared to other genera very little is known about the production of bacteriocins by Bifidobacterium. The bacteriocin bifidin, produced by Bifidobacterium bifidum NCDO 1452, was identified in 1984 but not characterized further (Anand et al., 1984). A second bacteriocin, identified by Yildirim and Johnson (1998) and called bifidocin B, is produced by B. bifidum NCFB 1454. This bacteriocin was purified and partially characterized by Yildirim et al. (1999). Three bacteriocin-producing strains of Bifidobacteria were isolated from baby feces by Touré et al. (2003) and were shown to be effective in inactivating adhesion and invasion of foodborne L. monocytogenes (Moroni et al., 2006). One of these three strains identified as B. thermophilum RBL67 was found to produce proteinaceous antilisterial activity against L. monocytogenes (von Ah et al., 2007).

10.6 Antimicrobial cultures: prevention and treatment of gastrointestinal diseases Many studies have been performed to evaluate the effect of antimicrobial cultures in the prevention and treatment of gastrointestinal diseases, such as antibiotic associated diarrhea (AAD), Clostridium difficile associated diarrrhea (CDAD),

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enterohemorragic E. coli infection, rotavirus diarrhea, traveler’s diarrhea, Helicobacter pylori gastroenteritis, HIV/AIDS diarrhea and other diarrhea-related infections. 10.6.1  Antibiotic-induced diarrheal disease Diarrhea occurs in about 5–25% of patients treated with antibiotics (Turck et al., 2003; Surawicz, 2005). AAD is due to an imbalance of intestinal bacterial flora (Nomoto, 2005) and its impact varies with patient-associated risk factors and with the class of antibiotic (Sullivan and Nord, 2005). In a double blind, placebocontrolled study, Surawicz et al. (1989) treated 180 patients receiving antibiotic therapy with either a placebo or Saccharomyces boulardii. Twenty-six percent of the patients developed diarrhea, compared to 22% of the patients treated with the placebo and 9% of those treated with S. boulardii, which was a statistically significant difference. In different studies, S. boulardii reduced the prevalence of AAD to 4.5% compared to 17.5% in the placebo group (P < 0.001) (Adam et al., 1977), to 9.5% compared to 22% in the placebo group (Surawicz et al., 1989) and to 7.2% compared to 14.6% (Mcfarland et al., 1995). The incidence of AAD can be reduced also by drinking a preparation containing L. casei, L. bulgaricus and Streptococcus thermophilus (Hickson et al., 2007). In this study, the probiotic group received a 100 g (97 ml) of the drink twice a day during the antibiotic treatment and for one week after. Twelve percent of the probiotic group developed AAD, compared to 34% of the placebo group (P = 0.007). Bifidobacteria are also known to reduce the incidence of AAD (Correa et al., 2005), while one recent study failed to reveal any reduction in antibiotic-related symptoms in association with consumption of L. rhamnosus GG (Thomas et al., 2001). 10.6.2  Clostridium difficile-associated diarrhea Clostridium difficile is responsible for 15–25% of cases of AAD. Antimicrobial agents induce the majority of C. difficile infections, due to their extensive destructive impact on the normal intestinal microbiota composition (Barbut and Petit, 2001). In a study of the effect of standard antibiotic therapy (vancomycin or metronidazole) combined with complementary S. boulardii or placebo treatment, patients with a history of at least one prior episode of C. difficile disease responded to S. boulardii with significant reductions in further recurrence of the disease, while those experiencing their first episode did not (Mcfarland et al., 1994). These results have been confirmed in two other studies. In a study of antimicrobial treatment dose and duration (Surawicz et al., 2000) in patients with recurrent CDAD, combined vancomycin (2 g/day) and S. boulardii treatment decreased recurrence to 17% from 50% (Sullivan and Nord, 2005). Another study also demonstrated that S. boulardii reduced the likelihood of further recurrence in patients with recurrent C. difficile disease (34.6% vs. 64.7% with the placebo, P = 0.04), but not in those who had had no previous episodes (19.3% vs. 24.2%, P = 0.86) (Mcfarland et al., 1994).

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10.6.3  Enterohemorrhagic E. coli Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a highly infectious pathogen that causes gastrointestinal illness with potentially serious consequences in humans worldwide (Mead and Griffin, 1998). The organism is known to produce one or more shiga toxins, which may produce diarrhea, hemorrhagic colitis and life-threatening hemolytic uremic syndrome in humans and animals (O’Brien and Kaper, 1998; LeBlanc, 2003). An in vitro murine study has shown the effectiveness of probiotic use of Bifidobacterium thermacidophilum RBL 71 at reducing the severity of E. coli O157:H7 infection and suggests that this strain could help prevent enteric infections in humans (Gagnon et al., 2006). 10.6.4  Rotavirus Enteric rotavirus represents a major cause of gastroenteritis, especially in young children. Rotavirus infection results in acute gastroenteritis with accompanying dehydration and vomiting mainly in children 3–24 months of age (Britton and Versalovic, 2008). Many double-blind placebo-controlled randomized studies have shown the effects of various probiotics on diarrhea caused by rotavirus diarrhea (Isolauri et al., 1991; Guandalini et al., 2000). In one study, the duration of symptoms was reduced in 75% of the patients (aged 6–36 months) after ingestion of L. reuteri strain SD 2222 (1010–1011 CFU) for 5 days, which was a greater reduction than in the placebo group (Shornikova et al., 1997). Another study has shown shortened duration of symptoms following administration of L. rhamnosus GG strain (1010 CFU). This study was performed on neonatal patients aged 1–3 months treated first for dehydration (Guandalini et al., 2000). 10.6.5  Traveler’s diarrhea Since about 80% of the pathogens identified in cases of acute diarrhea in travelers are enterotoxinogenic E. coli, shigellae and salmonellae (Sanders and Tribble, 2001), probiotics provide an attractive approach to preventing this ailment. Many probiotics, including Lactobacillus, Bifidobacterium, Streptococcus and Saccharomyces, have been evaluated for their ability to prevent traveler’s diarrhea (Takahashi et al., 2007). These studies have involved groups such as Finnish travelers to Turkey, American travelers to Mexico, British soldiers to Belize, Austrian travelers to three regions and European travelers to Egypt. The findings of these studies vary widely. A placebo-controlled double-blind study of 1016 travelers divided into three groups treated for five days before as well as during the entire trip showed 39.1% infection in the placebo group, 34.4% in the group receiving 250 mg/day of S. boulardii (P = 0.019 vs. placebo) and 28.7% in the group receiving 1000 mg/day of S. boulardii (P = 0.005). A tendency was noted for the effect of S. boulardii to vary with region (Kollaritsch et al., 1993). A study of Finnish travelers at two different destinations in Turkey showed that L. rhamnosus GG protected against traveler’s diarrhea at one destination but failed to do so at the other destination (Oksanen et al., 1990). In summary, the

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prevention of traveler’s diarrhea seems to depend generally on the strains used and the locality of the travel (for review see DuPont et al., 2009). 10.6.6  Helicobacter pylori gastroenteritis The stomach can be colonized by a pathogenic bacterium, Helicobacter pylori, which is the main cause of gastritis and gastric ulcers and may increase the risk of gastric cancer. Many in vitro and animal data have demonstrated that lactic acid bacteria can inhibit the growth of this pathogen and decrease its urease activity, which is necessary for its survival in the acidic environment of the stomach (Lesbros-Pantoflickova et al., 2007). A study on the treatment of H. pylori-positive patients with L. johnsonii La1-acidified milk (LC-1) and clarithromycin showed decreased H. pylori density, inflammation and gastritis activity without increasing eradication (Felley et al., 2001). Inhibition of H. pylori infection has also been shown in humans consuming L. johnsonii (Michetti et al., 1999; Marteau et al., 2001). The use of antibiotics with certain probiotics or combinations of probiotics has been shown to improve the eradication of H. pylori (Tursi et al., 2004; Myllyluoma et al., 2005; Sykora et al., 2005). In summary, several Lactobacillus species appear efficacious at decreasing the bacterial load of H. pylori in controlled trials, although their effect on eradication remains unclear. Probiotics may have a role as an adjunct in reducing side effects associated with conventional eradication therapy (Huebner and Surawicz, 2006). 10.6.7  HIV/AIDS diarrhea Acquired immune deficiency syndrome (AIDS) develops as a result of infection with the human immunodeficiency virus (HIV). It is characterized by immune cell dysfunction and subsequent immunodeficiency, as well as intestinal disorder (Kotler et al., 1984). Diarrhea is a very serious consequence of AIDS. The etiology of this diarrhea is frequently unknown and there are no effective treatment modalities. However, S. boulardii has been used to treat HIV-positive patients with chronic diarrhea. In a randomized, double blind, placebo-controlled study conducted with 35 patients, it was shown that administering S. boulardii (3 g/day) for one week decreased the incidence of diarrhea. After seven days of treatment, 61% of patients were diarrhea-free, compared to 12% in the placebo group (P < 0.002) (Saint-Marc et al., 1991). 10.6.8  Other diarrhea-related infections (Campylobacter) The anti-diarrhea effect of granules of B. breve Yakult strain has been reported in a study of patients 6 months to 15 years old with Campylobacter jejuniinduced enteritis (Tojo et al., 1987). Fecal Campylobacter was decreased more significantly in the B. breve Yakult treatment group than in the control group, but B. breve Yakult did not have a significant effect on diarrhea symptoms (Tojo et al., 1987).

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10.7 Tools for studying biological activities of antimicrobial cultures 10.7.1  In vitro studies Human intestinal cell lines Differentiated human intestinal cell lines including HT-29, Caco-2, and HT29-MTX are widely used to study the in vitro adhesion and colonization properties of probiotic strains. The effect of a number of probiotics such as Lactobacillus rhamnosus DR20, L. acidophilus HN017, Bifidobacterium lactis DR10, L. acidophilus LA-1 and L. rhamnosus GG on the adhesion and colonization of human epithelial cells by pathogenic organisms has been studied (Gopal et al., 2001; Burkholder and Bhunia, 2009; Miyauchi et al., 2009). In vitro models of the human colon Since the human colon is difficult to access for in vivo research purposes, in vitro modeling represents an alternative way of studying gut microbiota and microbial activities (Macfarlane and Macfarlane, 2007). In vitro models allow fast and reproducible experiments under standardized conditions. However, the degree to which the inoculum represents the human colonic microbiota (Drasar, 1988) and how well colonic conditions are mimicked (Edwards and Rowland, 1992) are recurring points of discussion. Various in vitro models have been developed, including SHIME (Molly et al., 1993), a continuous three-stage system (Macfarlane et al., 1998), the TIM (TNO) intestinal model (Minekus et al., 1995, 1999), a continuous two-stage system (Bruck et al., 2003), the three-stage culture system with immobilized fecal microbiota (Cinquin et al., 2006) and a human proximal colon system (Jimenez-Vera et al., 2008). (For a review of these systems, see Rohwer, 2007.) In vitro modeling systems have been used successfully to evaluate the ability of human GI microbiota to colonize mucus and to establish bio-film communities (Macfarlane et al., 2005). In vitro models of the stomach and small intestine (TIM-1) and the large intestine (TIM-2) have been used to investigate respectively the survival of bifidobacteria during passage through the GI tract and the effect of lactulose as a prebiotic on colonic microflora (especially bifidobacteria) (van der Werf and Venema, 2001). The continuous colonic fermentation model with immobilized fecal bacteria is an in vitro tool for mimicking the colonic environment and investigating bacterial composition and activity of the fecal microbiota (Cinquin et al., 2004). This model was developed with pediatric fecal microbiota (Cinquin et al., 2004) and has been used to study Salmonella infection (Le Blay et al., 2009). The effects of the probiotic strain Lactobacillus reuteri on the intestinal microbiota and its capacity to secrete reuterin from glycerol have been investigated in vitro using a colonic fermentation model (Cleusix et al., 2008), while another dynamic model that simulates the human upper gastrointestinal tract has been developed for the study of probiotics (Mainville et al., 2005). These in vitro models provide precious information about probiotics, such as factors affecting their viability as they pass through the human GI tract.

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10.7.2  In vivo studies Gnotobiotic mice harboring human fecal flora are excellent for investigating human GI microbiota. Ovalbumin (OVA)-induced allergic mice are used widely as a model for studying the effect of administering probiotics such as Bifidobacterium pseudocatenulatum JCM 7041 (Tsuda et al., 2009), L. acidophilus AD031 and B. lactis AD011 (Kim et al., 2008) on the prevention or attenuation of this allergy. In other research, the effects of lactobacilli and bifidobacteria on impaired intestinal barrier function and paracellular permeability have been evaluated in mice with colitis induced by dextran sodium sulfate (Nanda Kumar et al., 2008; Miyauchi et al., 2009). The effect of administering Saccharomyces boulardii on the composition of the fecal microbiota during and after antibiotic treatment has been evaluated in a human-microbiota-associated mouse model by Barc et al., 2008. They obtained quicker recovery of normal intestinal microbiota equilibrium and a preventive effect on antibiotic-associated diarrhea in humans by administering S. boulardii. The NC/NgaTnd mouse is a well-known animal model of atopic dermatitis (AD) (Matsuda et al., 1997; Vestergaard et al., 1999), and the preventive effect of L. rhamnosus GG on the onset of AD has also been shown using this model (Ka et al., 2005). The exact pathways that mediate the barrier-preserving effect of probiotic bacteria in vivo are not yet completely clarified (Mennigen and Bruewer, 2009), but some studies have shown that the protective effects of probiotics are mediated by their own DNA rather than by their metabolites or ability to colonize the colon (Grabig et al., 2006; Bai and Ouyang, 2006; Cario et al., 2007) and that nonviable probiotics are equally effective (Rachmilewitz et al., 2004). 10.7.3  Clinical studies Over the past decade, several studies have evaluated probiotic treatments of various gastrointestinal diseases. Four children with Crohn’s disease (CD) displayed significantly improved intestinal permeability (measured as reduced paracellular cellobiose uptake) after 12 weeks of a treatment with L. rhamnosus GG lasting six months (Gupta et al., 2000). The probiotic S. boulardii was found to decrease the lactulose/mannitol ratio (normally increased in CD patients) in a study involving 34 adult patients with CD in remission and 15 healthy volunteers (Vilela et al., 2008). Despite these promising results, probiotic treatment of CD has not so far met with clinical success (Mennigen and Bruewer, 2009). Whorwell et al. (2006) have demonstrated the efficacy of an encapsulated probiotic B. infantis 35624 in women with irritable bowel syndrome (IBS) and this trial, conducted for four weeks on 263 randomized patients, demonstrated that administering B. infantis 35624 in a 1 × 108 cfu/ml dose provided effective treatment of IBS symptoms. A study conducted on 45 patients with acute pancreatitis has demonstrated that L. plantarum 299, together with an oat fiber substrate, prevents colonization of the gut by potential pathogens and thus reduces pancreatic sepsis and the number of surgical interventions (Oláh et al., 2002). In studies of pancreatoduodenectomy (pylorus-preserving surgery) (Rayes et al.,

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2007) and liver transplant patients (Rayes et al., 2005), a significant reduction of bacterial infections in the groups receiving probiotics and fibers (prebiotics) was demonstrated. Although a reduction of infectious complications has been reported in several clinical studies with probiotics in patients, administration of probiotics to critically ill patients or patients at risk for non-occlusive mesenteric ischaemia cannot be considered to be harmless adjuncts to enteral nutrition (Besselink et al., 2008). This randomized, double-blind, placebo-controlled trial in patients with predicted severe acute pancreatitis showed no beneficial effect of probiotic prophylaxis on the occurrence of infectious complications. This result suggests that administration of probiotics in patients with predicted severe acute pancreatitis must be handled with great care and in some cases be considered as unsafe.

10.8  Conclusion and future trends Scientific evidence has accumulated during recent years to support the direct inhibition of pathogens as a major contributor to the efficacy of probiotic bacteria. However, much uncertainty continues to surround the significance of the various mechanisms proposed. In particular, the subject of bacteriocin involvement in this antimicrobial activity has inspired more speculation than research. It appears more likely that potential probiotic bacteria owe their inhibitory effects against pathogens to a combination of effects, including in particular the ability to compete for attachment sites on intestinal epithelial cells. Furthermore, if they do secrete large molecules that contribute to their value as probiotics, these too could act by interfering with pathogen attachment to epithelial cells. The lack of knowledge about the bacteriocin production by probiotics as well as in involvement of bacteriocins in their inhibitory activity is being addressed through research, but results are emerging very slowly, especially in the area of purification and characterization.

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et al. (1998). ‘Demonstration of safety of probiotics – a review.’ International Journal of Food Microbiology 44, 93–106. salminen s , von wright a and ouwehand a (2005). Lactic Acid Bacteria: Microbiological and Functional Aspects (Third Edition, Revised and Expanded), Taylor & Francis Group. sanders j and tribble d (2001). ‘Diarrhea in the returned traveler.’ Current Gastroenterology Reports 3, 304–314. scaletsky i c a , fabbricotti s h , aranda k r, morais m b and fagundes - neto u (2002). ‘Comparison of DNA hybridization and PCR assays for detection of putative pathogenic enteroadherent Escherichia coli.’ Journal of Clinical Microbiology 40, 1254–1258. schiffrin e j , brassart d , servin a l , rochat f and donnethughes a (1997). ‘Immune modulation of blood leukocytes in humans by lactic acid bacteria: Criteria for strain selection.’ American Journal of Clinical Nutrition 66, S515–S520. schiffrin e j , rochat f , linkamster h , aeschlimann j m and donnethughes a (1995). ‘Immunomodulation of human blood-cells following the ingestion of lactic-acid bacteria.’ Journal of Dairy Science 78, 491–497. schultz m (2008). ‘Clinical use of E. coli Nissle 1917 in inflammatory bowel disease.’ Inflammatory Bowel Diseases 14, 1012–1018. shand r f and leyva k j (2008). ‘Archaeal antimicrobials: an undiscovered country.’ In Norfolk B P (ed.) Archaea: New Models for Prokaryotic Biology, Caister Academic. shornikova a v, casas i a , isolauri e , mykkanen h and vesikari t (1997). ‘Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children.’ Journal of Pediatric Gastroenterology and Nutrition 24, 399–404. solis pereyra b and lemonnier d (1991). ‘Induction of 2´–5´ A synthetase activity and interferon in humans by bacteria used in dairy products.’ Eur Cytokine Netw 2, 137–40. sonnenburg j l , chen c t l and gordon j i (2006). ‘Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host.’ Plos Biology 4, 2213–2226. stratiki z , costalos c , sevastiadou s , kastanidou o , skouroliakuo m (2007). ‘The effect of a bifidobacter supplemented bovine milk on intestinal permeability of preterm infants.’ Early Human Development 83, 575–579. sullivan a and nord c e (2005). ‘Probiotics and gastrointestinal diseases.’ Journal of Internal Medicine 257, 78–92. surawicz c m (2005). ‘Antibiotic-associated diarrhea and pseudomembranous colitis: Are they less common with poorly absorbed antimicrobials?’ Chemotherapy 51, 81–89. surawicz c m , elmer g w, speelman p , mcfarland l v, chinn j and vanbelle g (1989). ‘Prevention of antibiotic-associated diarrhea by Saccharomyces-Boulardii – a prospective study.’ Gastroenterology 96, 981–988. surawicz c m , mcfarland l v, greenberg r n , rubin m , fekety r et al. (2000). ‘The search for a better treatment for recurrent Clostridium difficile disease: Use of high-dose vancomycin combined with Saccharomyces boulardii.’ Clinical Infectious Diseases 31, 1012–1017. sykora j , valeckova k n , amlerova j , siala k , dedek p et al. (2005). ‘Effects of a specially designed fermented milk product containing probiotic Lactobacillus casei DN-114 001 and the eradication of H-pylori in children – A prospective randomized double-blind study.’ Journal of Clinical Gastroenterology 39, 692–698. tabasco r, garcía - cayuela t , peláez c and requena t (2009). ‘Lactobacillus acidophilus La-5 increases lactacin B production when it senses live target bacteria.’ International Journal of Food Microbiology 132, 109–116. takahashi o , noguchi y, omata f , tokuda y and fukui t (2007). ‘Probiotics in the prevention of traveler’s diarrhea: meta-analysis.’ J Clin Gastroenterol 41, 336–7. salminen s , von wright a , morelli l , marteau p , brassart d

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thomas m r, litin s c , osmon d r, corr a p , weaver a l

and lohse c m (2001). ‘Lack of effect of Lactobacillus GG on antibiotic-associated diarrhea: A randomized, placebo-controlled trial.’ Mayo Clinic Proceedings 76, 883–889. tojo m , oikawa t , morikawa y, yamashita n , iwata s et al. (1987). ‘The effects of Bifidobacterium breve administration on Campylobacter Enteritis.’ Pediatrics International 29, 160–167. touré r, kheadr e , lacroix c , moroni o and fliss i (2003). ‘Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes.’ Journal of Applied Microbiology 95, 1058–1069. tsuda m , hosono a , yanagibashi t , hachimura s , hirayama k et al. (2009). ‘Intestinal Bifidobacterium association in germ-free T cell receptor transgenic mice down-regulates dietary antigen-specific immune responses of the small intestine but enhances those of the large intestine.’ Immunobiology 214, 279–289. turck d , bernet j p , marx j , kempf h , giard p et al. (2003). ‘Incidence and risk factors of oral antibiotic-associated diarrhea in an outpatient pediatric population.’ Journal of Pediatric Gastroenterology and Nutrition 37, 22–26. tursi a , brandimarte g , giorgetti g m and modeo m e (2004). ‘Effect of Lactobacillus casei supplementation on the effectiveness and tolerability of a new second-line 10-day quadruple therapy after failure of a first attempt to cure Helicobacter pylori infection.’ Medical Science Monitor 10, Cr662–Cr666. udall j n (1981). ‘Maturation of intestinal host defense: An update.’ Nutrition Research 1, 399–418. van der werf m j and venema k (2001). ‘Bifidobacteria: genetic modification and the study of their role in the colon.’ J Agric Food Chem 49, 378–383. vandenbergh p a (1993). ‘Lactic-acid bacteria, their metabolic products and interference with microbial growth.’ Fems Microbiology Reviews 12, 221–238. verberkmoes n c , russell a l , shah m , godzik a , rosenquist m (2008). ‘Shotgun metaproteomics of the human distal gut microbiota.’ ISME J 3, 179–189. vestergaard c , yoneyama h , murai m , nakamura k , tamaki k (1999). ‘Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis-like lesions.’ Journal of Clinical Investigation 104, 1097–1105. vilela e g , ferrari m d d , torres h o d , pinto a g , aguirre a c c et al. (2008). ‘Influence of Saccharomyces boulardii on the intestinal permeability of patients with Crohn’s disease in remission.’ Scandinavian Journal of Gastroenterology 43, 842–848. von ah u , mozzetti v, lacroix c , kheadr e , fliss i and meile l (2007). ‘Classification of a moderately oxygen-tolerant isolate from baby faeces as Bifidobacterium thermophilum.’ BMC Microbiology 7, 79. vriezen j a c , valliere m and riley m a (2009). ‘The evolution of reduced microbial killing.’ Genome Biol Evol 2009, 400–408. weinstein d l , o ’ neill b l , hone d m and metcalf e s (1998). ‘Differential early interactions between Salmonella enterica serovar typhi and two other pathogenic Salmonella serovars with intestinal epithelial cells.’ Infection and Immunity 66, 2310–2318. wheeler j g , shema s j , bogle m l , shirrell m a , burks a w et al. (1997). ‘Immune and clinical impact off Lactobacillus acidophilus on asthma.’ Annals of Allergy Asthma & Immunology 79, 229–233. whorwell p j , altringer l , morel j , bond y, charbonneau d et al. (2006). ‘Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable bowel syndrome.’ American Journal of Gastroenterology 101, 1581–1590. willey j m and van der donk w a (2007). ‘Lantibiotics: Peptides of diverse structure and function.’ Annual Review of Microbiology 61, 477–501. yildirim z and johnson m g (1998). ‘Characterization and antimicrobial spectrum of Bifidocin B, a bacteriocin produced by Bifidobacterium bifidum NCFB 1454’. Journal of Food Protection 61, 47–51.

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and johnson m g (1999). ‘Purification, amino acid sequence and mode of action of bifidocin B produced Bifidobacterium bifidum NCFB 1454.’ Journal of Applied Microbiology 86, 45–54. zareie m , riff j , donato k , mckay d m , perdue m h (2005). ‘Novel effects of the prototype translocating Escherichia coli, strain C25 on intestinal epithelial structure and barrier function.’ Cellular Microbiology 7, 1782–1797.

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Part III Applications of protective cultures, bacteriocins and bacteriophages in foods and beverages

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11 Applications of protective cultures, bacteriocins and bacteriophages in milk and dairy products M. Medina and M. Nuñez, INIA, Spain

Abstract: Many strains of lactic acid bacteria produce bacteriocins useful for improving the safety and quality of milk and dairy products. Nisin is applied worldwide by the cheese industry in the prevention of the late-blowing defect. Other bacteriocins have a great potential to inhibit pathogenic and spoilage bacteria and to enhance quality and flavour development in cheese. The combination of bacteriocins with non-thermal or other biological treatments opens new strategies in dairy safety and quality. More research should be devoted to the development of bacteriocin-producing starter cultures and new methods for bacteriocin incorporation in dairy foods. Key words: bacteriocins, dairy products, safety, quality, flavour.

11.1  Introduction Bacteriocins of lactic acid bacteria, substances of proteinaceous nature produced by food-grade organisms, can inhibit many pathogenic and spoilage microorganisms that contaminate foods. In 1928 it was observed that certain lactococcal strains had an antimicrobial effect on the growth of other lactic acid bacteria. The responsible compound, the bacteriocin named nisin, was described in 1947 and first marketed in England in 1953 (Cotter et al., 2005). The utility of nisin in dairy technology was reported in 1951 by Hirsch et al., in the control of gas-blowing in Swiss-type cheese. Nisin has been approved by the FDA and the EU, and is widespread in food in more then 50 countries, particularly in processed cheese and cheese spreads, other dairy products and canned foods (Delves-Broughton, 1990). Bacteria susceptible to nisin include the genera Clostridium, Bacillus and Listeria. In the absence of other preservation methods, nisin and other bacteriocins of lactic acid bacteria 267 © Woodhead Publishing Limited, 2011

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are generally not active against Gram-negative bacteria, yeasts or moulds. Their antimicrobial spectra might be extended to Gram-negatives by combination with strategies that affect the integrity of the outer membrane. Bacteriocins are often used in combination with other preservation methods in the concept of hurdle technology to enhance individual antimicrobial activity. Bacteriocins represent a valuable approach to satisfy consumer demands of safe foods with a long shelf-life and minimally processed, without chemical preservatives. Their potential in biopreservation has been demonstrated on different foods, either alone or in combination with other methods, or incorporated into packaging films. Bacteriocins should be used to complement, and not to replace, good manufacturing practices in the food industry. Many strains of lactic acid bacteria produce bacteriocins useful for improving the safety and quality of milk and dairy products. The majority of bacteriocinogenic cultures used in dairy foods are milk or cheese isolates. This chapter will focus on the inhibition of pathogens by protective cultures, bacteriocins and other antimicrobials such as bacteriophages. The strategies reviewed to incorporate these compounds into dairy foods include the addition of bacteriocins directly to milk and the addition of bacteriocin-producing cultures. The combined use of bacteriocins with physical treatments and other biopreservatives has been proposed to improve the safety of dairy foods. Bacteriocin-producing cultures might participate in the acceleration of cheese ripening and the enhancement of cheese flavour by means of the lysis and the controlled release of intracellular enzymes from the starter culture. Applications of bacteriocins to improve dairy products quality have also been developed. Besides the extended use of nisin to prevent late-blowing of cheese by Clostridium, alterations caused by the uncontrolled growth of non-starter lactic acid bacteria (NSLAB) in cheese or the formation of biogenic amines have been reduced by bacteriocin-producing starter or adjunct cultures. Finally, the potential use of bacteriophages or products derived from them for the inactivation of pathogens in milk and dairy products, a subject which is receiving increased interest, is reviewed.

11.2  Bacteriocins to improve the safety of dairy foods Milk is an ideal medium for the growth of both pathogenic and spoilage microorganisms. Excretion from animals and contamination during milking collection and storage are the origin of foodborne pathogens in raw milk. The reduction of the incidence of foodborne diseases associated to milk and dairy products has been related to the decreased prevalence of zoonoses at the farm, to the improved hygiene, and to the implementation of HACCP plans and other preventive measures in the dairy industry. Although pasteurization destroys potential pathogenic microorganisms, post-pasteurization processing can lead to the recontamination of dairy products. The pathogens of major concern to the dairy industry are Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella. L. monocytogenes

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is a ubiquitous microorganism that grows at low temperatures and can increase to hazardous levels and survive for long periods in dairy products. While pasteurization destroys L. monocytogenes in milk, recalls of dairy products contaminated with the pathogen mainly soft cheeses are relatively frequent. S. aureus is a causative agent of bovine mastitis capable of producing thermostable enterotoxins. Dairy products may contain low levels of enterotoxigenic staphylococci, however temperature abuse above 10 °C and poor starter culture activity during fermentation are factors involved in dairy related outbreaks of staphylococcal intoxication. Enterohemorrhagic E. coli (EHEC) O157:H7 has been detected in raw milk, and milk and other dairy products have been implicated in several outbreaks. The pathogen is destroyed by pasteurization and is unable to grow in pasteurized refrigerated milk, although growth can occur under temperature abuse. Salmonellosis associated to milk and dairy products have been related to inadequate pasteurization and post-process contamination, but most cheeses, including raw or pasteurized milk cheeses, properly manufactured and aged, appear to pose no significant health risk of Salmonella infection. The potential of bacteriocins to control the growth of pathogens in foodstuffs has been extensively reviewed (Holzapfel et al., 1995; Cleveland et al., 2001; O’Sullivan et al., 2002a; Ross et al., 2002; Guinane et al., 2005; Deegan et al., 2006; De Vuyst and Leroy, 2007; Gálvez et al., 2007, 2008; Sobrino-López and Martín-Belloso, 2008; Grattepanche et al., 2008). 11.2.1  Application of bacteriocin-producing lactic acid bacteria Starter cultures produce a wide range of metabolites with antimicrobial activity, organic acids, diacetyl, acetoin, hydrogen peroxide and bacteriocins. The use of live cultures to produce bacteriocins in situ is based on the incorporation of bacteriocin-producing strains as starters or adjunct cultures in a fermented product or their application as protective cultures to improve the safety of the product (Table 11.1). The use of nisin in cheese can result in the inhibition of acidifying or aromaproducing starter cultures, and decrease growth or acidification. Nisin-producing strains during fermentation processes have been proposed as an alternative to the addition of nisin in commercial form. Usually, these strains exhibited low rates of acidification, limited proteolytic activity and high sensitivity to bacteriophages, decreasing the interest in their use as starter cultures. The optimization of the composition of nisin-producing starter cultures has been achieved by using bacteriocin-producing strains in combination with other nisin resistant or tolerant cultures with desirable properties. Roberts et al. (1992) developed a nisinproducing starter culture system with a high rate of acid production for the manufacture of Cheddar cheese, consisting of naturally occurring lactose and proteinase positive, nisin-producing Lactococcus lactis subsp. lactis NCDO 1404 and the lactose and proteinase positive, nisin-producing transconjugant L. lactis subsp. cremoris JS102. The starter culture system constructed by these authors using a nisin-producing starter with the nisin-resistant plasmid pFG010 was not

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Pathogen L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes S. aureus S. aureus S. aureus

BP culture

L. lactis 1881 L. lactis CNRZ 150 L. lactis TAB 50 L. lactis DPC 4275 L. lactis DPC 4275 L. lactis TAB 24 E. faecium 7C5 E. faecalis INIA 4 E. faecalis TAB 28 E. faecium DPC 1146 E. faecium RZS C5 L. lactis MM217 Lb. plantarum WHE 92 Lb. plantarum ALC01 L. lactis CL1 L. lactis TAB 50 L. lactis IPLA 729 L. lactis CL1

Bacteriocin

Nisin Nisin Nisin Lacticin 3147 Lacticin 3147 Lacticin 481 Enterocin 7C5 Enterocin AS-48 Enterocin AS-48 Enterocin 1146 Enterocin RZS C5 Pediocin Pediocin Pediocin Pediocin Nisin Nisin Pediocin

Starter culture Starter culture Starter culture Starter culture Surface sprayed Starter culture Surface sprayed Starter or adjunct Starter culture Adjunct culture Adjunct culture Starter culture Surface sprayed With smear culture Adjunct culture Starter culture Adjunct culture Adjunct culture

Application

Table 11.1  Bacteriocin-producing (BP) lactic acid bacteria for cheese safety

Camembert cheese Camembert cheese Semi-hard cheese Cottage cheese Smear-ripened cheese Semi-hard cheese Taleggio cheese Manchego cheese Semi-hard cheese Cheddar cheese Cheddar cheese Cheddar cheese Munster cheese Red smear cheese Semi-hard cheese Semi-hard cheese Afuega’l Pitu cheese Semi-hard cheese

Product

Sulzer and Busse, 1991 Maisnier-Patin et al., 1992 Rodríguez et al., 2001 McAuliffe et al., 1999 O’Sullivan et al., 2006 Rodríguez et al., 2001 Giraffa and Carminati, 1997 Nuñez et al., 1997 Rodríguez et al., 2001 Foulquié Moreno et al., 2003 Foulquié Moreno et al., 2003 Buyong et al., 1998 Ennahar et al., 1998 Loessner et al., 2003 Rodríguez et al., 2005a Rodríguez et al., 2000 Rilla et al., 2004 Rodríguez et al., 2005b

Reference

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successful due to the nisin inactivation caused by the mechanism of nisin resistance encoded by the plasmid. Selected mixed starter cultures with a nisin Z-producing L. lactis subsp. lactis biovar diacetylactis strain and a commercial starter were successfully developed by Bouksaim et al. (2000). According to these authors, the selection and control of the proportions of bacteriocin-producing cultures and commercial starters is fundamental for the application of bacteriocin-producing starters in cheese making. On the other hand, nisin-producing strains exhibiting technological properties suitable for cheese making have been isolated from natural environments as raw milk and raw-milk cheese (Martínez et al., 1995; Rodríguez et al., 1998). Many studies have examined the effect of bacteriocins when bacteriocinogenic cultures were added for the inhibition of L. monocytogenes in various types of cheese. L. monocytogenes was not inhibited in Camembert cheese made with a nisin-producing L. lactis strain inoculated into milk together with the starter culture, whereas the pathogen was suppressed when the nisin-producing strain was used as starter culture (Sulzer and Busse, 1991). The antilisterial activity of a nisin-producing starter culture in Camembert cheese was also demonstrated by Maisnier-Patin et al. (1992). L. monocytogenes numbers decreased rapidly during the first 24 hours and the inhibitory activity continued until the end of the second week of ripening, but regrowth of the pathogen in the interior and the surface of cheese was observed later on. L. lactis subsp. lactis ESI 515 and TAB 50 (Rodríguez et al., 1998, 2001) used as single-starter cultures in the manufacture of raw milk cheese decreased Listeria levels throughout the 60 days of ripening. Lactococcal strains producing other lantibiotics as lacticin 3147 and lacticin 481 have shown their suitability as starters in cheese making. Lacticin 3147 is a twocomponent lantibiotic produced by L. lactis DPC 3147 isolated from a kefir grain (Ryan et al., 1996) with a broad spectrum of activity and potential uses in food safety, and was stable in Cheddar cheese over the the 6-month ripening studied (Ryan et al., 1996). Genetic determinants of lacticin 3147 have been transferred to different hosts, many of them derivatives of commercial starter strains (Ryan et al., 1996; Coakley et al., 1997). Lacticin 3147-producing transconjugant strain used as a starter culture in the manufacture of cottage cheese reduced numbers of L. monocytogenes to <10 cells/g within five days at 4 °C (McAuliffe et al., 1999). However, the application of the lacticin 3147-producing transconjugant to the surface of smear-ripened cheese did not eliminate L. monocytogenes (O’Sullivan et al., 2006), although pathogen numbers in the bacteriocin-treated cheeses were 3 log units lower than in control cheeses made without the bioprotectant culture. Lacticin 481 is a single-peptide lantibiotic produced by some strains of L. lactis (Piard et al., 1990), mainly active against other lactic acid bacteria. The antilisterial activity of lacticin 481-producing lactococci isolated from milk was described by Rodriguez et al. (2000). Levels of L. monocytogenes in 60-day rawmilk cheeses manufactured with lacticin-481 producing L. lactis subsp. cremoris TAB 24 as single-starter were 2.5 log units lower than in cheese made with a commercial starter (Rodríguez et al., 2001).

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Pediocin PA1/AcH is a broad spectrum bacteriocin produced by strains of Pediococcus acidilacti with high antilisterial activity. A limitation that restricts the use of pediocin-producing pediococci in dairy products is their inability to ferment lactose which results in slow growth in milk (Caldwell et al., 1996). Cell suspensions of pediocin-producing Lactobacillus plantarum WHE 92 (Ennahar et al., 1996) sprayed on the surface of Munster cheese inhibited L. monocytogenes growth for 21 days (Ennahar et al., 1998). The appearance of resistant strains of L. monocytogenes was reported by Loessner et al. (2003) when pediocin or the bacteriocin-producing strain was used in red-smear cheeses. The production of pediocin in heterologous hosts is considered an alternative to extend the application of this bacteriocin in milk and dairy products. Pediocin-producing L. lactis MM217 starter culture containing a plasmid coding the pediocin PA1 operon reduced L. monocytogenes levels in Cheddar cheese by 3 log units after 92 days of ripening (Buyong et al., 1998). Similarly, the use of pediocin-producing transformant L. lactis CL1 added at 1% as adjunct to the starter culture in cheese manufacture resulted in a 3 log reduction of L. monocytogenes and reduced S. aureus population by 1 log after 30 days of ripening (Rodríguez et al., 2005a). Food-grade pediocin-producing lactococcal strains developed by Reviriego et al. (2007) used as starter cultures also reduced L. innocua counts in a cheese model system. Enterococci are widely distributed in the environment and contribute to the organoleptic properties of fermented foods, mainly traditional European cheeses manufactured in the Mediterranean countries (Foulquié-Moreno et al., 2006). Bacteriocin production is a common trait among strains of the genus Enterococcus (Giraffa, 1995) and many enterococcal bacteriocins are class II pediocin-like bacteriocins with strong antilisterial activity. This characteristic has led to their application as starter or adjunct cultures in cheese making. As the safety assessment for enterococci remains controversial, a case-by-case evaluation of each potential strain has been recommended (Ogier and Serror, 2008). Enterocin AS-48 is a cyclic bacteriocin produced by Enterococcus faecalis INIA 4 with antilisterial activity in milk (Rodríguez et al., 1997a). Used at 1% as a starter or co-culture with a commercial lactic starter in the manufacture of rawmilk Manchego cheese, it decreased L. monocytogenes counts by 3 log units after 8 hours and by 6 log units after 7 days, although the inhibition was dependent on the strain of L. monocytogenes investigated (Nuñez et al., 1997). The pathogen was completely inactivated during the manufacture and ripening of raw-milk cheese manufactured without starter culture and with enterocin AS-48-producing E. faecalis TAB 28 (Rodríguez et al., 2001). Enterocin 1146 from E. faecium DPC 1146 (Parente and Hill, 1992) exhibited a rapid bactericidal effect on L. monocytogenes in milk and was stable throughout the ripening of Cheddar cheese. When E. faecium RZS C5 (Foulquié-Moreno et al., 2003) was used in Cheddar cheese manufacture, enterocin was detected from the beginning of cheese production, remaining stable during cheese ripening. The use of other enterocins active against L. monocytogenes has been reported for some cheese varieties. In Taleggio cheese, Giraffa et al. (1995) observed

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bacteriocin-production by E. faecium 7C5 during cheese making that was stable until the end of the ripening and inactivated L. monocytogenes on the cheese surface (Giraffa and Carminati, 1997). E. faecium M241 and M249 isolated from goat milk produced bacteriocin active against L. monocytogenes (Cocolin et al., 2007). On the contrary, E. faecium FAIR-E 198 did not produce bacteriocin during the manufacture of Feta cheese (Sarantinopoulos et al., 2002) when used as adjunct culture to the starter. Bacteriocins produced by streptococci isolated from food-related environments have also been characterized. Streptococcus salivarius subsp. thermophilus is present in yogurt and thermophilic cheese starters, while thermophilin-producing S. salivarius subsp. thermophilus B used in the manufacture of yogurt exhibited higher activity against L. monocytogenes than against S. aureus (Benkerroum et al., 2002). Isolates from yogurt produced thermophilin 13, a two peptide class IIb bacteriocin (Marciset et al., 1997) and thermophilin 347 (Villani et al., 1995). S. macedonicus ACA-DC 198 isolated from Kasseri cheese produces, when used as starter or adjunct culture, the lantibiotic macedocin, active during the 90 days of ripening of this variety (Van den Berghe et al., 2006; Anastasiou et al., 2009). The efficacy of bacteriocins to control S. aureus in cheese is less well known. The population of S. aureus was reduced in cheese spreads made from Cheddar cheese manufactured with nisin-producing lactococci (Zottola et al., 1994), and in cheese made with other nisin-producing starters (Rodríguez et al., 2000; Rilla et al., 2004) or a pediocin-producing L. lactis as adjunct culture (Rodríguez et al., 2005a), although the inhibition was lower than in cheese spreads. Although a large number of bacteriocins have been characterized, their use in cheese making requires the selection of compatible combinations of lactic starters and bacteriocin-producing strains permitting correct growth and acidification during incubation in milk, cheese making and ripening. 11.2.2  Application of bacteriocins as additives Although several bacteriocins have demonstrated their efficacy in foods, only nisin is utilised as additive and has received regulatory approval. The earliest application of nisin in dairy products was the prevention of spoilage by clostridial species responsible for the late-blowing defect in cheese (Delves-Broughton et al., 1996). Nisin is also effective in the control of C. botulinum outgrowth and toxin formation in processed cheese spreads with reduced sodium content and/or high moisture levels. Complete antibotulinal protection was achieved with 10 000 IU nisin/g in the formulation of the blend (Sommers and Taylor, 1987). Interest in applying bacteriocins in foods was directed against L. monocytogenes since the first observations on the antimicrobial activity of nisin on this pathogen. Nisin was bactericidal against different strains of L. monocytogenes, and the effect was enhanced by addition of NaCl or reduction of pH (Harris et al., 2001). The addition of nisin to milk in the manufacture of cheeses made without starter culture was effective to control the pathogen. L. monocytogenes was able to grow

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at refrigeration temperatures in cottage cheese, but was completely inhibited by the addition of 2550 IU nisin/g (Benkerroum and Sandine, 1988), while the levels of L. monocytogenes decreased by 3 log units after 7 days at 20 °C with the addition of 2000 IU nisin/g (Ferreira and Lund, 1996). In ricotta-type cheese (Davies et al., 1997), nisin completely inhibited the growth of L. monocytogenes during at least 8 weeks at 6–8 °C. Lacticin 3147 has been applied in the form of a powder in yogurt and in cottage cheese and L. monocytogenes was eliminated with 10% of the powdered product in yogurt and cottage cheese (Morgan et al., 2001). A fermented milk-based concentrate containing the lantibiotic variacin, produced by Kocuria varians NCC 1482, is used as food ingredient inhibited B. cereus in chilled dairy food models and commercial dairy desserts (O´Mahony et al., 2001). Pediocin is commercialized in the form of crude fermentates (ALTA™, Kerry Group plc) from pediocin-producing strains of P. acidilactici and is used in the inhibition of pathogens, mainly in ready-to-eat (RTE) meat products (Rodríguez et al., 2002). Due to its strong antilisterial activity and to its characteristics of stability and activity in a wide range of pH values, its application has been extended to dairy products. A dried preparation of pediocin decreased L. monocytogenes counts in various dairy products including cottage cheese, cream and cheese sauce systems (Pucci et al., 1988), although the pathogen restarted growth in the mildy acidic and neutral food systems. The class IIa piscicolin 126 produced by Carnobacterium piscicola JG126 inhibited L. monocytogenes in milk and in Camembert cheese, although regrowth of the pathogen occurred (Wan et al., 1997a). According to the authors, the application was limited by the emergence of resistant isolates and the inactivation of the bacteriocin by proteolytic enzymes from starter bacteria and moulds. Various enterocins have been applied directly as cell-free preparations in the manufacture of cheese. Enterocin CRL 35 reduced Listeria up to 9 log units in goat cheese at the end of the ripening period (Farias et al., 1999), although bacteriocin activity was not detected in cheese during ripening. Also enterocin 226 NWC in Mozzarella (Villani et al., 1993) and enterocin CCM 4231 in Saint-Paulin cheese (Lauková et al., 2001) exhibited antilisterial activity. Nisin used in free form in cheese manufacture may interfere with the cheese making process by inhibiting the starter culture or the NSLAB involved in ripening and flavour development. Microencapsulation of bacteriocins in liposomes has been proposed as an alternative to the direct addition of free bacteriocin to milk to improve stability and distribution in cheese, while preventing the antimicrobial action of nisin on the cheese starter during the first hours of manufacture (Benech et al., 2002a). L. innocua viability was reduced in Cheddar cheese by nisin encapsulated in liposomes, with higher reductions than in cheese made with a nisinogenic strain (Benech et al., 2002a). Nisin activity after 6 months was 90% of the initial in cheeses made with encapsulated nisin and only 12% in cheeses made with the nisinogenic starter. The encapsulation of nisin Z in liposomes was optimized by Laridi et al. (2003). The protection of nisin in the encapsulated form would reduce its diffusion to the fat phase and the accessibility

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of proteolytic enzymes to the bacteriocin. Controlled delivery of nisin from liposomes would be effective against spoilage and pathogenic bacteria in cheeses with a high pH, in which nisin activity is highly reduced (Benech et al., 2002b). Nisin has also been incorporated into microparticles of calcium alginate (Wan et al., 1997b), with incorporation efficiency up to 87–93%. The bacteriocin was 100% active against an indicator culture in reconstituted skim milk. Also, nisin immobilized in calcium alginate gels has been assayed in other food systems. Different packaging systems from a variety of biopolymers appear to be good vehicles for incorporating different antimicrobials against spoilage and pathogenic microorganisms. Nisin-coated films exhibited antimicrobial activity during storage of milk (Mauriello et al., 2005). Cellulose-based bioactive inserts with nisin adsorbed to the surface reduced levels of L. innocua and S. aureus in sliced Cheddar cheese stored in modified atmosphere packaging (MAP) at refrigeration temperatures (Scannell et al., 2000). On the contrary, cellulose films containing nisin were not effective against S. aureus, although they delayed psychrotrophs growth in sliced Mozzarella cheese (Pires et al., 2008). Although many bacteriocins are potentially useful for the dairy industry, only nisin is permitted as a biopreservative, but other bacteriocins can be added to foods as culture fermentates. Different systems to incorporate bacteriocins to dairy foods include their encapsulation or immobilization. The development of bioactive packaging systems with bacteriocins as antimicrobials is currently in progress.

11.3  Bacteriocins in combined treatments On the basis of the hurdle approach, a bacteriocin and a physical or biological treatment may be combined to allow the use of lower concentrations of bacteriocins and other inhibitors, or a lower severity of the physical treatment, while achieving a higher lethality than when the bacteriocin is used by itself (Table 11.2). Combinations may act synergistically providing higher protection than single preservation methods. In addition, non-thermal physical treatments have little effect on functional and nutritional characteristics of food. 11.3.1  Combination of bacteriocins with physical treatments Bacteriocins have been combined for use in milk and dairy products with physical treatments such as heating, high pressure, and pulsed electric fields. Combinations of bacteriocins with thermal treatments generally increase the lethal effect of heating. Thus, the time to achieve a 3 log unit reduction of L. monocytogenes counts when heated at 54 °C in milk containing 25 IU nisin/ml was only 16 min, while it was 77 min in the absence of nisin, times from which D values of 5.3 and 25.7 min, respectively, could be calculated (Maisnier-Patin et al., 1995). The addition of nisin to skim milk before heating lowered D values of Bacillus cereus and B. stearothermophilus (Wandling et al., 1999), suggesting the enhanced

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Treatment

Pathogen

Product

Nisin None Heat (54 °C) L. monocytogenes Milk Nisin None Heat (103 °C) B. cereus Milk AS-48 E. faecalis A-48-32 Heat (65 °C) S. aureus Milk and fresh cheese E. faecium UJA32-81 Lacticin 3147 None High pressure (HP) S. aureus Skim milk and whey Nisin + pediocin None HP S aureus Milk L. monocyogenes Nisin None HP B. cereus Model cheese Nisin A L. lactis TAB 50 HP S. aureus Semi-hard cheese Nisin Z L. lactis TAB 26 L. monocytogenes Lacticin 481 L. lactis TAB 24 E. coli O157:H7 TAB 57 L. lactis TAB 57 TAB 7 E. faecium TAB 7 Enterocin I E. faecalis TAB 52 Enterocin AS-48 E. faecalis INIA 4 Nisin None Pulsed electric fields (PEF) S. aureus Milk Nisin None Lactoperoxidase system (LPS) L. monocytogenes Milk Nisin L. lactis ESI 515 LPS L. monocytogenes Milk Nisin None Reuterin L. monocytogenes Milk S. aureus Nisin None LPS L. monocytogenes Curdled milk

Bacteriocin

Table 11.2  Combined treatments to improve the safety of milk and dairy products

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Arqués et al., 2008b

Sobrino-López and Martín-Belloso, 2006 Gaya et al., 1991 Zapico et al., 1998 Boussouel et al., 2000 Rodríguez et al., 1997b Arqués et al., 2004a,b

Morgan et al., 2000 Alpas and Bozoglu, 2000 Alpas and Bozoglu, 2002 López-Pedemonte et al., 2003 Arqués et al., 2005a Arqués et al., 2005b Rodríguez et al., 2005b

Maisnier-Patin et al., 1995 Wandling et al., 1999 Muñoz et al., 2007

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sensitivity of spores to heat by prior nisin exposure and the prevention of the growth of survivors due to residual nisin in the milk. However, the combination of heating at 65 °C for 5 min and adding enterocin AS-48 to skim milk had scarce additional effect on S. aureus counts with respect to only heating, but in the presence of enterocin AS-48 S. aureus growth during milk incubation following heat treatment was retarded (Muñoz et al., 2007). If a bacteriocin is combined with high pressure processing, a high bactericidal effect may be reached at lower pressures, with a lesser impact on food characteristics. Synergism usually occurs because of the combined effect of high pressure and bacteriocins on bacteria, but it may also happen that the bacteriocin antibacterial activity as such is increased by the high pressure treatment. Thus, lacticin 3147 activity increased considerably after treatments of the bacteriocin in skim milk or whey at pressures of 400–800 MPa for 30 min (Morgan et al., 2000). According to the same authors, pressurization at 250 MPa (2.2 log unit reduction) combined with lacticin 3147 (1 log unit reduction) lowered S. aureus counts in milk by more than 6 log units (no survivors), which shows a clear synergy of the combined treatment. This synergistic effect was also observed against L. innocua. In milk treated at 345 MPa for 5 min at 50 °C, counts of a S. aureus strain were lowered by 5.5 log units, whereas the reduction exceeded 8.3 log units (no survivors) if the same treatment was combined with the addition of a bacteriocin preparation containing nisin and pediocin AcH. Under the same conditions, counts of two L. monocytogenes strains were lowered by 8.3 log units (no survivors), independently of the addition of the bacteriocin preparation (Alpas and Bozoglu, 2000). These authors observed that S. aureus and L. monocytogenes recovered after high pressure treatment, but there was no recovery of pathogens after the high pressure treatment if the bacteriocin preparation had been added to milk (Alpas and Bozoglu, 2002). In fresh cheese treated at 500 MPa for 5 min at 25 °C in the presence of nisin, the effect of the combined treatment was additive on aerobic mesophilic bacteria (Capellas et al., 2000), whereas results on spore counts excluded even an additive effect. Similarly, in model cheeses inoculated with B. cereus spores, the reduction in counts by pressurization was low and increased slightly with nisin (LópezPedemonte et al., 2003). The combined effect of bacteriocin-producing lactic acid bacteria and high pressure treatments on the inactivation of pathogens inoculated in a semi-hard raw-milk cheese has been investigated. When cheese was inoculated with S. aureus, strains producing nisin A, nisin Z, lacticin 481, bacteriocin TAB 57, bacteriocin TAB 7, enterocin I and enterocin AS-48 achieved reductions in 3-days-old experimental cheeses from 0.2 to 0.5 log units. Pressurization at 500 MPa for 10 min lowered by itself S. aureus counts by 2.4 log units. High pressure combined with bacteriocin-producing strains achieved reductions ranging from 3.3 to 4.0 log units, which show a synergistic effect of all bacteriocins with the high pressure treatment (Arqués et al., 2005a). In the case of L. monocytogenes, the same bacteriocin-producing strains achieved reductions in 3-day-old experimental cheeses ranging from 0.3 to 1.0 log units. A 10 min treatment at

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300 MPa lowered L. monocytogenes counts by 0.9 log units. A synergistic effect with high pressure and all bacteriocins resulted in reductions from 1.6 to 3.2 log units, with the only exception nisin A, for which the effect was only additive (Arqués et al., 2005b). When E. coli O157:H7 was inoculated into milk, the above bacteriocin-producing strains achieved count reductions ranging from 0.4 to 0.7 log units. Pressurization at 300 MPa for 10 min lowered E. coli O157:H7 counts by 0.9 log units, whereas combined treatments achieved reductions ranging from 2.0 to 3.4 log units. The combined effect of high pressure treatment and bacteriocins against E. coli O157:H7 in cheese was synergistic for all the bacteriocin-producing strains (Rodríguez et al., 2005b). Sublethal damage of the outer membrane in the case of Gram-negatives or changes in membrane fluidity by pressurization could facilitate the access of bacteriocins to the cytoplasmic membrane. The combination of bacteriocins with pulsed electric fields in milk and dairy products has also been investigated, in particular for the inactivation of S. aureus in skim milk (Sobrino-López and Martín-Belloso, 2006). Pulsed electric fields and nisin acted synergistically, probably due to sublethal injury caused by the treatment that enhanced nisin activity. Maximum microbial inactivation (more than 6 log units) was achieved with 20 ppm nisin, 35 kV/cm, and 2400 µs at a pH of 6.8. Moderate pressures combined with bacteriocins, within the hurdle concept of food preservation, are feasible procedures to control the growth of pathogens from postpasteurization contamination or present in the milk used to manufacture raw-milk cheeses. Synergism between nisin and pulsed electric fields opens new possibilities for research on this combination. 11.3.2  Combination of bacteriocins with other biological antimicrobials Bacteriocins may be combined in milk and dairy products with other biological inhibitors, some of dairy origin. This is the case for the lactoperoxidase system, which had been reported to inhibit by itself L. monocytogenes in raw milk at refrigeration temperatures (Gaya et al., 1991). Lactic acid bacteria producing nisin or enterocin AS-48 were combined with the lactoperoxidase system to inhibit L. monocytogenes in refrigerated raw milk held at 4 and 8 °C. The effect of bacteriocins, lactoperoxidase system or their combination at 4 °C on L. monocytogenes was close to null. However, an additive effect of nisin and lactoperoxidase system was observed at 8 °C with the nisin-producing strain, whereas no additive effect at all of enterocin AS-48 and lactoperoxidase system was observed (Rodríguez et al., 1997b). A different behaviour of L. monocytogenes was recorded when UHT skim milk with added nisin and lactoperoxidase was held at higher temperatures (Zapico et al., 1998). Nisin by itself had no effect on L. monocytogenes Ohio counts after 24 hours at 30 °C, lactoperoxidase system by itself achieved counts 3 log units lower than control, and their combination counts were 5.7–6.5 log units lower than control if added simultaneously or 6.8–7.4 log units lower than control if added in two steps, with a clear synergistic effect. These results were confirmed on L. monocytogenes ATCC 15313 in skim milk at

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25 °C (Boussouel et al., 2000). Nisin effect was bactericidal and lasted 1 day, whereas lactoperoxidase system was bacteriostatic. The combined addition resulted in an early bacteriostatic effect followed by a strong bactericidal effect, with no survivors from day 5 onwards. Bacteriocins have also been combined with reuterin, an antimicrobial compound produced by some strains of Lb. reuteri, on the aim of inhibiting pathogens such as L. monocytogenes and S. aureus in milk (Arqués et al., 2004a). The combination of nisin and reuterin in milk at 37 °C exhibited a clear synergistic effect on L. monocytogenes, with counts more than 9 log units lower than control (no survivors) after 24 hours, compared to 1.6 log units lower in the presence of nisin and 4.6 log units lower in the presence of reuterin. This combination also had a synergistic effect on S. aureus. Addition of nisin and reuterin in two steps may be more effective than their simultaneous addition (Arqués et al., 2004b). Nisin had null or very scarce effect on five selected Gram-negative pathogens inoculated into UHT skim milk after 5 days at 8 °C (Arqués et al., 2008a), whereas reuterin by itself reduced counts of E. coli O157:H7, S. Enteritidis, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica by 0.9, 0.2, 5.1, 4.9, and 5.1 log units, respectively. The combination of nisin and reuterin achieved reductions close to those obtained with only reuterin, whereas reuterin and lactoperoxidase system were strongly synergistic against these Gram negative pathogens. Inhibitory compounds in combination have been assayed for the inhibition of L. monocytogenes in dairy products, such as ‘cuajada’, curdled milk made with no added cultures in Northern Spain, which shows a high pH favourable for growth of contaminating bacteria. After 12 days at 10 °C, the synergistic effect of the combination nisin and lactoperoxidase system was demonstrated (Arqués et al., 2008b). Combinations of biopreservatives resulted in greater inhibitory effects, with the primary target of nisin and lactoperoxidase system being the cytoplasmic membrane. The increase in membrane permeability might facilitate the antimicrobial action of reuterin (for general aspects including the mechanism of action of reuterin see Chapter 5). Reuterin combined with bacteriocins appears to be effective for the inhibition of bacteriocin-resistant Gram-positive pathogens, although the combined action of reuterin and nisin did not enhance the antimicrobial activity of reuterin on Gram-negative pathogens in milk.

11.4  Bacteriocins to enhance the quality and flavour of cheese The first application of bacteriocins in cheese ripening was focused on the elimination of C. tyrobutyricum, the spore-forming bacterium responsible for the late-blowing defect. In addition to the reduction of viability of pathogenic and spoilage microorganisms in cheese, bacteriocin-producing cultures participate in the enhancement of cheese flavour and the acceleration of cheese ripening by means of the lysis and controlled release of intracellular enzymes from the starter

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culture (Table 11.3). Other applications of bacteriocins to improve cheese quality include the prevention of alterations caused by the uncontrolled growth of NSLAB and the formation of biogenic amines in cheese. 11.4.1  Bacteriocin-producing strains to accelerate cheese ripening The first proposal for the use of bacteriocin-producing strains in the acceleration of cheese ripening was, to our knowledge, for L. lactis subsp. cremoris LMG 2130, the producer of a new bacteriocin with high lytic activity, which was the subject of a patent (Holo and Nes, 1992). Among its applications, the authors included its use in cheese manufacture, as a lytic agent for the acceleration of ripening. The first requirement for the accelerated ripening to occur is the production of bacteriocin by the bacteriocin producer in co-culture with the starter culture. Bacteriocin-sensitive strains of lactic acid bacteria are lysed by bacteriocins, sometimes through the concerted action of more than one bacteriocin, and their intracellular enzymes and metabolites are subsequently released into the surrounding substrate (Morgan et al., 1995). On the basis of their lytic effect on lactococci, these authors suggested that bacteriocin-producing strains had potential applications as accelerators of cheese ripening. The above hypothesis was confirmed by manufacturing Cheddar cheese with L. lactis subsp. cremoris HP as starter culture and L. lactis subsp. lactis DPC 3286, producing lactococcins A, B, and M, as adjunct. Time to reach the milling pH of 5.2 took 10–30 min longer, cell viability of strain HP was reduced during overnight pressing, and release of intracellular enzymes during the first hours of manufacture was favoured, leading to an increase in the concentration of free amino acids in cheese (Morgan et al., 1997). Acceleration of cheese ripening was also demonstrated by using a multiple-strain mesophilic LD-type culture as starter and an enterocin AS-48 producer as adjunct in the manufacture of a semi-hard cheese, which showed a higher extracellular aminopeptidase activity and a more pronounced proteolysis than control cheese made without the enterocin producer (Garde et al., 1997). A second condition to be fulfilled is the extensive lysis of starter lactic acid bacteria, which may be monitored using markers such as lactate dehydrogenase. Thus, a 16-fold increase in LDH concentration was recorded when L. lactis MG 1614 was cultured in the presence of the lacticin 481-producer L. lactis DPC 5552 (O’Sullivan et al., 2002b). Other enzymes leak from the cell interior into the surrounding dairy matrix, some of which may be relevant to cheese ripening, as is the case for aminopeptidases. Extracellular aminopeptidase activity can be increased up to 22-, 9- or 25-fold when starters consisting of lactococci, thermophilic streptococci or thermophilic lactobacilli, respectively, are co-cultured with bacteriocin-producers, depending on starter composition and the bacteriocinproducing strain (Oumer et al., 2001a; O’Sullivan et al., 2002b; Ávila et al., 2005b). During early cheese ripening, up to 10-fold increases in extracellular aminopeptidase activity were recorded when bacteriocin-producing strains were

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BP culture

Application

Product

Lactococcins A, B and M L. lactis DPC 3286 Adjunct culture Cheddar cheese Lacticin 481 L. lactis DPC 5552 Adjunct culture Cheddar cheese Lacticin 481 L. lactis CNRZ 481 Adjunct culture Cheddar cheese Lacticin 481 L. lactis INIA 639 Adjunct culture Semi-hard cheese Lacticin 481 L. lactis INIA 639 Adjunct culture Hispánico cheese Nisin Z and lacticin 481 L. lactis INIA 415 Adjunct culture Hispánico cheese Nisin Z and lacticin 481 L. lactis INIA 415 Adjunct culture Manchego cheese Lacticin 3147 L. lactis DPC 4275 Starter culture Reduced fat Cheddar cheese Lacticin 3147 L. lactis IFLP 3593 Starter culture Semi-hard goat milk cheese Lacticin 3147 L. lactis IFLP 3593 Starter culture Semi-hard cheese Enterocin AS-48 E. faecalis INIA 4 Adjunct culture Semi-hard cheese Enterocin AS-48 E. faecalis INIA 4 Adjunct culture Hispánico cheese

Bacteriocin

Table 11.3  Bacteriocin-producing lactic acid bacteria to enhance the flavour of cheese

Morgan et al., 1997 Morgan et al., 2002 O’Sullivan et al., 2002b O’Sullivan et al., 2003 Ávila et al., 2007b Garde et al., 2006 Garde et al., 2002a,b Garde et al., 2005 Ávila et al., 2005a Ávila et al., 2007c Fenelon et al., 1999 Martínez-Cuesta et al., 2001 Fernández de Palencia et al., 2004 Garde et al., 1997 Oumer et al., 2001b

Reference

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used as adjuncts of mesophilic starters (Oumer et al., 2001b; Garde et al., 2002a), and up to 3-fold if they were used as adjuncts to a combination of mesophilic and thermophilic starters (Garde et al., 2002a, 2006). Also, extracellular aminopeptidase activity increased in cheese when nisin in microparticles was added to milk inoculated with L. lactis and S. thermophilus cultures (Garde et al., 2003). A fact to be remarked is that starter lysis can be hindered by the appearance of mutants resistant to the bacteriocin, which may happen after just a two hour exposure to the bacteriocin (Garde et al., 2004). Thirdly, phenomena related to cheese ripening such as proteolysis, lipolysis and formation of volatile compounds must be enhanced by the accelerated lysis of starter cultures. Slight increases in cheese primary proteolysis when milk was inoculated with a bacteriocin producer have been recorded, in particular for αs-casein (Garde et al., 2002a, 2006), although retarded casein degradation in the presence of a bacteriocin-producer has also been observed (Ávila et al., 2005a, 2006b). Small differences between the pH value of experimental and control cheeses might be partly responsible for this variable effect on caseins. In reduced fat Cheddar cheese, no difference in caseinolysis between cheeses made with and without a bacteriocin producer was detected (Fenelon et al., 1999). Regarding the influence of bacteriocin producers on peptide accumulation, these authors did not find any effect on Cheddar cheese pH 4.6-soluble N until day 240. However, significantly higher levels of hydrophilic peptides, up to 1.6-fold, were detected in Hispánico cheese made from a 80:20 mixture of cows’ and ewes’ milk, from day 25 onwards, when a nisin Z- and lacticin 481-producing L. lactis strain was added to milk inoculated with L. lactis and S. thermophilus cultures (Garde et al., 2002a). An additional benefit for cheese flavour derives from the lower hydrophobic peptides to hydrophilic peptides ratio, an indicator of lower bitterness, recorded by these authors in cheese made with the bacteriocin producer. However, the levels of hydrophilic peptides were lower in experimental than in control cheese when a lacticin 481-producing L. lactis strain was added to milk inoculated with a Lb. helveticus culture (Garde et al., 2006). Secondary proteolysis is clearly accelerated by bacteriocin-producing adjuncts. Thus, 1.3- or 1.5-fold increases in the overall concentration of free amino acids of Cheddar cheese were achieved by using 0.03% or 0.125% inocula of the bacteriocin producer (Morgan et al., 1997). Overall, proteolysis of a semi-hard cheese, as determined by the o-phthaldialdehyde method, exhibited a 2.2-fold increase on day 15 when 0.003% inoculum of the bacteriocin-producer was added (Garde et al., 1997). The levels of non-protein nitrogen and amino nitrogen were increased in a semi-hard goat milk cheese when a bacteriocin-producing transconjugant was used as adjunct (Martínez-Cuesta et al., 2001). Total free amino acids increased in Hispánico cheese by an enterocin AS-48 producer as adjunct (Oumer et al., 2001b), by a nisin Z- and lacticin 481-producing L. lactis strain as adjunct to milk inoculated with a L. lactis or with L. lactis and S. thermophilus starter cultures (Garde et al., 2002a; Ávila et al., 2005a), or when a lacticin 481-producing L. lactis strain was added to milk inoculated with a Lb. helveticus culture (Garde et al., 2006). In all the above reported increases in

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cheese, free amino acid concentrations closely correlated with the release of intracellular aminopeptidases brought about by bacteriocins. Information on the effect of bacteriocin-mediated starter cell lysis on cheese lipolysis is scarce. Esterase activity increased in Hispánico cheese made with L. lactis and S. thermophilus as starter cultures and a nisin Z- and lacticin 481-producing L. lactis adjunct (Ávila et al., 2007a). In spite of this increase, total free fatty acids were lower in the experimental cheese, a decrease which was attributed by the authors to its low pH value. On the contrary, a higher level of total free fatty acids was detected in Hispánico cheese made with a Lb. helveticus culture and a lacticin 481-producing L. lactis strain (Ávila et al. 2007b), the pH value being very similar in experimental and control cheeses throughout ripening. From these results, it seems evident that cell lysis and esterase release do not suffice to increase cheese lipolysis. Substrate conditions seem critical for the liberation of free fatty acids, since both esterase stability and activity may be affected by lower than normal cheese pH values. Addition of bacteriocin-producing cultures modifies the volatile profile of cheeses, but changes are dependent on the starter cultures used and the bacteriocinproducer added (Garde et al., 2002b, 2005; Ávila et al., 2006a). Transamination of branched-chain amino acids, a key reaction in the formation of some volatile branched-chain compounds, is favoured by the cell membrane permeabilization caused by bacteriocins (Martinez-Cuesta et al., 2002, 2003). A two-fold increase in the 2-methyl-butanal concentration in cheese was achieved by using a lacticin 3147-producing L. lactis strain as adjunct culture (Fernández de Palencia et al., 2004). The effect of bacteriocin-producing strains on cheese texture has been investigated. A softening of cheese was observed when a bacteriocin producer was added to milk previously inoculated with a Lb. helveticus culture, as a consequence of the higher proteolysis and the more pronounced degradation of the casein network in the experimental cheese (Garde et al., 2006). But the opposite effect was recorded in cheese made from milk inoculated with L. lactis and S. thermophilus cultures and a bacteriocin producer as adjunct, which showed a firmer texture, ascribed by the authors to its higher αs-casein content (Ávila et al., 2005a, 2006b). For a real acceleration of cheese ripening by bacteriocin-producing strains, its flavour characteristics must be modified above a certain threshold of change, perceivable by consumers. Results obtained so far vary depend on the cheese variety, the starter cultures and the bacteriocin-producing strains. Cheddar cheese flavour quality score was improved, though slightly, with respect to that of control cheese when a L. lactis strain producing lactococcins A, B, and M was added as an adjunct culture (Morgan et al., 1997), but the flavour quality score of reduced fat Cheddar cheese was not affected by milk inoculation with a bacteriocin producer (Fenelon et al., 1999). Considerably higher flavour intensity scores were achieved with 0.003% inoculum of an enterocin AS-48 producer in the manufacture of semi-hard cheese (Garde et al., 1997). Moreover, both flavour quality and intensity of Hispánico cheese were improved by the addition of a nisin

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Z- and lacticin 481-producer (Garde et al., 2002a), with a considerable reduction in the ripening period needed to reach a certain intensity score, without impairment of cheese quality. Reduction of Cheddar cheese bitterness was achieved by milk inoculation with L. lactis strains producing either lactococcins A, B and M (Morgan et al., 2002) or lacticin 481 (O’Sullivan et al., 2003). Addition of a lacticin 481-producer to milk inoculated with L. lactis and Lb. helveticus enhanced flavour intensity of Hispánico cheese, without significant changes in its flavour quality (Ávila et al., 2006b). Also, flavour quality and intensity of raw-milk Manchego cheese were higher when a nisin Z- and lacticin 481-producer was added to milk (Ávila et al., 2007c). 11.4.2  Bacteriocin-producing strains to improve cheese quality Besides the extended use of nisin commercial preparations to prevent the lateblowing defect of cheese, strains of bacteriocin-producing lactic acid bacteria belonging to the species L. lactis, Lb. gasseri, and S. macedonicus have been assayed as tools on the prevention of this defect. Counts of C. tyrobutyricum were lowered by 6 log units in 30-day-old cheese in the presence of the nisin Z producer (Rilla et al., 2003), by 1 log unit in cheese made using the macedocin producer (Anastasiou et al., 2009), and by only 0.5 log units when Lb. gasseri was added (Bogovic Matijasic et al., 2007). Butyric acid concentrations in cheese were reduced in all cases. Some adventitious NSLAB, mostly belonging to the genus Lactobacillus, have been described as responsible for the appearance of off-flavours and calcium lactate crystals during cheese ripening. Their population in cheese can be reduced by milk inoculation with bacteriocin-producing strains. Counts of NSLAB, which exceeded 107 cfu/g in 120-day-old control Cheddar cheese, were lowered to undetectable levels in experimental cheese by adding a lacticin 3147-producing L. lactis strain (Ryan et al., 1996). Similar results were obtained for reduced fat Cheddar cheese ripened at 7 °C using another lacticin 3147-producing strain (Fenelon et al., 1999). Variants of lactobacilli selected for their flavour characteristics, resistant to lacticin 3147, can be used as adjuncts, in combination with the lacticin 3147 producer, in order to control the adventitious microbiota without impairing cheese sensory characteristics (Ryan et al., 2001). A lacticin 481-producing L. lactis strain reduced levels of NSLAB in 120-day-old Cheddar cheese by 4 log units (O’Sullivan et al., 2003). Lower reductions were obtained in Hispánico cheese with a nisin Z- and lacticin 481-producing L. lactis strain (Ávila et al., 2005a), and when a bacteriocin-producing Lb. gasseri strain was used in the manufacture of semi-hard cheese (Bogovic Matijasic et al., 2007). Among cheese adventitious microbiota, biogenic amine formers deserve particular attention. For biogenic amine accumulation in cheese to occur, precursor free amino acids and bacteria with decarboxylase activity must be present. Histamine is mostly formed, sometimes at concentrations of public health significance, by particular strains of lactobacilli (Joosten, 1988). All 13 biogenic amine-forming lactobacilli tested were sensitive to nisin and to five bacteriocins

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of enterococcal origin (Joosten and Nuñez, 1996). When cheese was made from milk inoculated with a histamine-forming Lb. buchneri strain, inhibition in the presence of lactic acid bacteria producing nisin, enterocin AS-48 or enterocin EFS 2 was complete, with null histamine formation, whereas in 120-day-old control cheese counts of the Lb. buchneri strain increased by 6 log units and histamine concentration reached 200 mg/kg (Joosten and Nuñez, 1996). Enterococci are the main formers of tyramine in cheese, but enterococci inhibition is not an easy task, since many strains are resistant to one or several bacteriocins, although tyrosine decarboxylaseless mutants of bacteriocin-producing enterococci may be obtained and used to control undesirable bacteria without the concomitant formation of tyramine (Joosten et al., 1995).

11.5 Bacteriophages to improve the safety and quality of milk and dairy products Bacteriophages naturally present in raw milk have been largely studied and identified due to their potential role in lysing starter cultures used in dairy fermentations. Bacteriophages as antibacterials to control foodborne pathogens and spoilage organisms are receiving increasing attention as preservation strategy in food safety (for general aspects of bacteriophages and their application see Chapter 6). Phage specifity results in the elimination of only the target organisms without compromising the viability of other bacteria. This property is specially desired in the case of fermented dairy products. Initial studies in the control of undesirable bacteria in milk and dairy products by bacteriophages showed that their application could reduce the numbers of Pseudomonas fragi WY in refrigerated milk, although the need of a relatively high population of the host bacteria and the presence of its homologous phage limited its application (Ellis et al., 1973). Other studies have evaluated the efficacy of the addition of bacteriophages to milk in the elimination of pathogenic microorganisms in dairy products. Bacteriophage P100 has received GRAS status for application to foods (Guenther et al., 2009) and a product based in this phage was approved by the FDA as a food preservative. Broad-host-range bacteriophage P100 applied to the surface of a Munster-type soft cheese during the rind-washing eradicated L. monocytogenes (Carlton et al., 2005) without interfering with the lactic acid bacteria (Schellekens et al., 2007). Phage A511, which can infect about 95% of L. monocytogenes strains of the major serovar groups 1/2 and 4 (Loessner and Busse, 1990), suppressed or prevented the growth of two L. monocytogenes strains in chocolate milk and Mozzarella cheese brine (Guenther et al., 2009). Resistance against these bacteriophages was not detected, suggesting that development of insensitivity of Listeria cells against strictly virulent phages appears to be a rare event. The potential of bacteriophages to eliminate S. aureus has been described. Investigations of phage K activity against S. aureus in raw milk showed that © Woodhead Publishing Limited, 2011

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heat-labile immunoglobulins, which recognize and bind to the bacterial cell surface, would agglutinate S. aureus rendering them inaccessible to the phage K activity (O’Flaherty et al., 2005). The adsorption of whey proteins to the cell surface and the tendence of S. aureus to agglutinate following exposure to raw whey also inhibited phage binding (Gill et al., 2006). The use of two lytic phages of dairy origin to control enterotoxin production in cheese was assayed by García et al. (2007) and the lytic phage derivatives added to milk prior to acid and enzymatic coagulation were able to rapidly decrease S. aureus counts, being more effective during enzymatic manufacture of curd. Partial inactivation of phages by low pH values occurred in acid manufacturing conditions. Two bacteriophages isolated from sewage prevented the development of Enterobacter sakazakii in reconstituted infant milk formula at various temperatures and eradicated this pathogen at 109 pfu/ml, the highest concentration assayed (Kim et al., 2007). In Cheddar cheese manufactured from raw or pasteurized milk contaminated with S. Enteritidis at 104 cfu/g, the addition of SJ2 phage reduced the ability of S. Enteritidis to survive (Modi et al., 2001). In the presence of phages, Salmonella was not detected in pasteurized milk cheese after 89 days of ripening and presented counts of 50 cfu/g in raw-milk cheese, whereas counts were 103 cfu/g after 99 days of ripening in cheese without phages. Phage endolysins are synthesized at the end of the phage lytic cycle to lyse the host cell and to release the newly produced virions. They are potential antimicrobials due to their ability to lyse the bacteria when they are applied exogenously. Endolysin-encoding genes from L. monocytogenes bacteriophages were expressed in a L. lactis starter that secreted endolysin and lysed L. monocytogenes in the culture medium (Gaeng et al., 2000). Moreover, Obeso et al. (2008) cloned and over-expressed the endolysin gene of S. aureus bacteriophage φH5 in E. coli, which eliminated S. aureus growth in milk after four hours at 37 °C. The application of bacteriophages in cheese ripening to increase lysis of the starter culture to release intracellular enzymes into the cheese curd has also been investigated (Crow et al., 1995), while dairy starters associated with autolytic properties could harbour prophage determinants (O’Sullivan et al., 2000). According to these authors, the cooking step in cheese manufacture induced prophage cell lysis, and release of bacteriophages, into the cheese curd. Cheddar cheese made with L. lactis strain ML8 as starter, two levels of rennet and three levels of homologous phage in milk resulted in various degrees of starter lysis early in the ripening process. The levels of activity of two starter cytoplasmic enzymes, lysylaminopeptidase and FBP-aldolase in the cheese matrix increased with the level of phage added. Elevated starter lysis was associated with an increased rate of formation of free amino acids and ammonia, but the rate of lactose removal in the cheese decreased. Bitter flavour was prominent in cheese with high rennet concentration, but not when there was also high starter lysis. The results suggest that a balance of lysed and intact cells is important for an adequate cheese ripening.

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Safety and technological issues would need to be addressed while expanding bacteriophage antimicrobial abilities. Lytic (virulent) phages are especially suitable for bacteriophage-based food biocontrol (EFSA, 2009). They cannot integrate their genome in the bacterial chromosome and will always lyse and kill infected target cells. Other limitations of the use of phages in dairy systems include the reduced diffusion rates that decrease the chance of host-phage collisions, the interactions with microbiota in dairy products that could act as mechanical barrier providing phage-binding sites, and factors such as temperature, pH, and presence of other inhibitory compounds (García et al., 2008).

11.6  Conclusions and future trends Environmental conditions in food systems influence the production and the antimicrobial activity of bacteriocins. Also, their effectiveness may be hindered by the proteolytic activity of food or microbial enzymes, their adsorption to fat and the appearance of resistant variants in sensitive strains. In the particular case of cheese, bacteriocin production and activity is affected by competition with the starter culture, interactions with the cheese matrix, proteolytic activities and conditions of pH, temperature, salt and water content. The activity of combined starters including both technological strains and bacteriocin-producing cultures is rather difficult to control for correct acidification, bacteriocin production and quality of cheese. The plausible inhibition of lactic starters surged as one of the main limitations of bacteriocins in industrial cheese production. Compatible combinations of lactic starters and bacteriocin-producing strains permitting a correct acidification of the dairy product had to be found in order to prevent the appearance of defects caused by abnormally high pH values. The screening of commercial lactic starters compatible with bacteriocin producers, with none or just a slight retard in curd acidification when combined with a bacteriocin-producing strain, may help to solve the problem. By selecting the adequate ratio of bacteriocin producer and starter culture inocula, a balanced growth of the starter culture and a suitable production of lactic acid and bacteriocin in curd and cheese can be achieved. Bacteriophage sensitivity and low proteolytic activity of bacteriocin-producing cultures are also responsible for the limited application of bacteriocin-producing cultures at industrial scale. Commercial culture suppliers use natural conjugation to transfer nisin production to functionally suitable lactic acid bacteria selected for their resistance to bacteriophages and their organoleptic and metabolic characteristics (Dairy Safe™ cultures, CSK). A range of adjunct cultures to accelerate cheese flavour production and enhance flavour intensity based on bacteriocins are also commercially available, but more research is needed for the optimization of bacteriocin production and activity in dairy products. The simultaneous application of more than one bacteriocin or multiple bacteriocin producers may reduce the emergence of resistances in target strains, although cross-resistance to class I and class II bacteriocins has been described.

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Future work should focus on the better understanding of factors favouring the appearance of bacteriocin-resistant pathogens in foods and on the development of strategies to mitigate their emergence. Multiple bacteriocin production has been reported in some lactic acid bacteria and practical applications in the inhibition of pathogens associated with cheese have been successful. In addition, the development of bacterial strains exhibiting over-expression of bacteriocins or multiple heterologous bacteriocin producers has received particular attention, although their industrial use would be limited by the restrictive legal regulations and the lack of acceptance by consumers. On the other hand, combined treatments of bacteriocins with physical processes or other biopreservatives offer a wide scenario of practical future applications. While nisin is a bacteriocin of widespread industrial use, the numerous bacteriocins characterized during the last three decades, some of which have successfully performed in dairy products at pilot plant scale, have not been applied industrially. Milk inoculation with bacteriocin-producing strains, as starter or adjunct cultures in the manufacture of dairy products, has no regulatory constraints. Nevertheless, the use of bacteriocins and bacteriocin-producing cultures by dairy food manufacturers should be considered only as a complement of good manufacturing practices.

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o ’sullivan l , morgan s m , ross r p

and hill c (2002b) ‘Elevated enzyme release from lactococcal starter cultures on exposure to the lantibiotic lacticin 481, produced by Lactococcus lactis DPC5552’, J Dairy Sci 85, 2130–2140. o ’sullivan l , o ’ connor e b , ross r p and hill c (2006) ‘Evaluation of live-cultureproducing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese’, J Appl Microbiol 100, 135–143. o ’sullivan l , ross r p and hill c (2003) ‘A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening’, J Appl Microbiol 95, 1235–1241. o ’sullivan l , ross r p and hill c (2002a) ‘Potential of bacteriocin-producing lactic acid bacteria for improvements in food safety and quality’, Biochimie 84, 593–604. obeso j m , martínez b and rodríguez a (2008) ‘Lytic activity of recombinant staphylococcal bacteriophage Phi H5 endolysis active against Staphylococcus aureus in milk’, Int J Food Microbiol 128, 212–218. ogier j c and serror p (2008) ‘Safety assessment of dairy microorganisms: The Enterococcus genus’, Int J Food Microbiol 126, 291–301. oumer a , garde s , gaya p , medina m and nuñez m (2001a) ‘The effects of cultivating lactic starter cultures with bacteriocin-producing lactic acid bacteria’, J Food Prot 64, 81–86. oumer a , gaya p , fernández - garcía e , mariaca r, garde s et al. (2001b) ‘Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture’, J Dairy Res 68, 117–129. parente e and hill c (1992) ‘Characterization of enterocin 1146, a bacteriocin from Enterococcus faecium inhibitory to Listeria monocytogenes’, J Food Prot 55, 497–508. piard j c , delorme f , giraffa g , commissaire j and desmazeaud m (1990) ‘Evidence for a bacteriocin produced by Lactococcus lactis CNRZ-481’, Neth Milk Dairy J 44, 143–158. pires a c s , ferreira n f , de andrade n j , mendes da silva l h , camilloto g p and campos p (2008) ‘Development and evaluation of active packaging for sliced mozzarella preservation’, Packag Technol Sci 21, 375–383. pucci m j , vedamuthu e r, sunka b s and vanderbergh p a (1988) ‘Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0’, Appl Environ Microbiol 54, 2349–2353. reviriego c , fernández l and rodríguez j m (2007) ‘A food-grade system for production of pediocin PA-1 in nisin-producing and non-nisin-producing Lactococcus lactis strains: application to inhibit Listeria growth in a cheese model system’, J Food Prot 70, 2512–2517. rilla n , martínez b , delgado t and rodríguez a (2003) ‘Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer’, Int. J. Food Microbiol 85, 23–33. rilla n , martínez b and rodríguez a (2004) ‘Inhibition of a methicilin-resistant Staphylococcus aureus in Afuega’l Pitu cheese by the nisin-Z-producing strain Lactococcus lactis subsp. lactis IPLA 729’, J Food Prot 67, 928–933. roberts r f , zottola e a and mckay l l (1992) ‘Use of a nisin-producing starter culture suitable for Cheddar cheese manufacture’, J Dairy Sci 75, 2353–2363. rodríguez e , arqués j l , gaya p , nuñez m and medina m (2000) ‘Behaviour of Staphylococcus aureus in semi-hard cheese made from raw milk with nisin-producing starter cultures’, Milchwissenschaft 55, 633–635. rodríguez e , arqués j l , gaya p , nuñez m and medina m (2001) ‘Control of Listeria monocytogenes by bacteriocins and monitoring of bacteriocin-producing lactic acid bacteria by colony hybridization in semi-hard raw milk cheese’, J. Dairy Res 68, 131–137. rodríguez e , arqués j l , nuñez m , gaya p and medina m (2005b) ‘Combined effect of high-pressure treatments and bacteriocin-producing lactic acid bacteria on inactivation of Escherichia coli O157-H7 in raw- milk cheese’, Appl Environ Microbiol 71, 3399–3404.

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and medina m (2005a) ‘Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 in cheese’, Int Dairy J 15, 51–57. rodríguez e , gaya p , nuñez m and medina m (1998) ‘Inhibitory activity of a nisinproducing starter culture on Listeria innocua in raw ewes milk Manchego cheese’, Int J Food Microbiol 39, 129–132. rodríguez j l , gaya p , medina m and nuñez m (1997a) ‘Bactericidal effect of enterocin 4 on Listeria monocytogenes in a model dairy system’, J Food Prot 60, 28–32. rodríguez e , tomillo j , nuñez m and medina m (1997b) ‘Combined effect of bacteriocin-producing lactic acid bacteria and lactoperoxidase system activation on Listeria monocytogenes in refrigerated raw milk’, J Appl Microbiol 83, 389–395. rodríguez j m , martinez m i and kok j (2002) ‘Pediocin PA-1, a wide-spectrum bacteriocin from lactic acid bacteria’, Crit Rev Food Sci Nutr 42, 91–121. ross r p , morgan s and hill c (2002) ‘Preservation and fermentation: past, present and future’, Int J Food Microbiol 79, 3–16. ryan m p , rea m c , hill c and ross r p (1996) ‘An application in Cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin’, lacticin 3147, Appl Environ Microbiol 62, 612–619. ryan m p , ross r p and hill c (2001) ‘Strategy for manipulation of cheese flora using combinations of lacticin 3147-producing and -resistant cultures’, Appl Environ Microbiol 67, 2699–2704. sarantinopoulos p , leroy f , leontopoulou e , georgalaki m d , kalantzopoulos g , tsakalidou e and de vuyst l (2002) ‘Bacteriocin production by Enterococcus faecium FAIR-E 198 in view of its application as adjunct starter in Greek Feta cheese making’. Int J Food Microbiol 72, 125–136. scannell a g m , hill c , ross r p , marx s , hartmeier w and arendt e k (2000) ‘Development of bioactive food packaging materials using immobilised bacteriocins Lacticin 3147 and Nisaplin’, Int J Food Microbiol 60, 241–249. schellekens m m , wouters j , hagens s and hugenholtz j (2007) ‘Bacteriophage P100 application to control Listeria monocytogenes on smeared cheese’, Milchwissenschaft 62, 284–287. sobrino - lópez a and martín - belloso o (2006) ‘Enhancing inactivation of Staphylococcus aureus in skim milk by combining high-intensity pulsed electric fields and nisin’, J Food Prot 69, 345–353. sobrino - lópez a and martín - belloso o (2008) ‘Use of nisin and other bacteriocins fror preservation of dairy products’, Int Dairy J 18, 329–343. sommers e b and taylor s l (1987) ‘Antibotulinal effectiveness of nisin in pasteurized process cheese spreads’, J Food Prot 50, 842–848. sulzer g and busse m (1991) ‘Growth inhibition of Listeria spp. on Camembert cheese by bacteria producing inhibitory substances’, Int J Food Microbiol 14, 287–296. van den berghe e , skourtas g , tsakalidou e and de vuyst l (2006) ‘Streptococcus macedonicus ACA-DC 198 produces the lantibiotic, macedocin, at temperature and pH conditions that prevail during cheese manufacture’, Int J Food Microbiol 107, 138–147. villani f , pepe o , mauriello g , salzano g , moschetti g and coppola s (1995) ‘Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus’, Int J Food Microbiol 25, 179–190. villani f , salzano g , sorrentino e , pepe o , marino p and coppola s (1993) ‘Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes’, J Appl Bacteriol 74, 380–387. wan j , gordon j b , muirhead k , hickey m w and coventry m j (1997b) ‘Incorporation of nisin in micro-particles of calcium alginate’, Lett Appl Microbiol 24, 153–158.

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et al. (1997a) ‘Inhibition of Listeria monocytogenes by piscicolin 126 in milk and Camembert cheese manufactured with a thermophilic starter’, J Appl Microbiol 82, 273–280. wandling l r, sheldon b w and foegeding p m (1999) ‘Nisin in milk sensitizes Bacillus spores to heat and prevents recovery of survivors’, J Food Prot 62, 492–498. zapico p , medina m , gaya p and nuñez m (1998) ‘Synergistic effect of nisin and the lactoperoxidase system on Listeria monocytogenes in skim milk’, Int J Food Microbiol 40, 35–42. zottola e a , yezzi t l , ajao d b and roberts r f (1994) ‘Utilization of a cheddar cheese containing nisin as an antimicrobial agent in other foods’, Int J Food Microbiol 24, 227–238. wan j , harmack k , davidson b e , hillier a j , gordon j b

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12 Applications of protective cultures, bacteriocins and bacteriophages in fermented meat products T. Aymerich, M. Garriga and J. Monfort, IRTA, Spain

Abstract: Fermented sausages are considered self-stable products; nevertheless they have been implicated in several outbreaks. To increase food safety of fermented sausages and to cope with consumers’ demands for products with less chemical additives and less salt and fat content, biopreservation, understood to provide increased food safety and extension of shelf life by natural microbiota and/or their antimicrobial products, is considered an excellent alternative. Two major topics for biopreservation are considered in this chapter, lactic acid bacteria and their antimicrobial products and bacteriophages. Lactic acid bacteria have a long history of safe use, they are accepted by consumers as natural and health promoting and are the dominant natural microbiota of fermented meat products. Moreover, GRAS (Generally Recognized as Safe) bacteriophages used against specific food-borne pathogens do not disturb technological microbiota and could also be applied to the food environment. Key words: biopreservation, fermented sausages, lactic acid bacteria, bioprotective cultures, bacteriocins, bacteriophages, food safety.

12.1  Introduction Fermentation and drying of foodstuffs was probably the first step in food preservation methods, thus contributing to the development of civilization. One of the most common types of sausage is salami, which appears to have originated in the ancient Greek town Salamis on the Cyprian East coast (Pederson, 1979). A long time before Christ, a Chinese type of sausage, Lup Cheong, has been described. Sausages were well known in the Roman Empire before the technology spread to the rest of Europe. Fermented meat sausages are a mixture of minced meat with variable contents of lean, fat and ingredients (salt, spices, nitrate and or nitrite) that are afterwards 297 © Woodhead Publishing Limited, 2011

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stuffed, then fermented and dried. The variable composition and technology determine the developing microbiota and the final flavour of the product, producing a high diversity of fermented sausages, even with geographical specification (Lebert et al., 2007). However, two main types of sausages are distinguished. The Northern type is acid with pH under 5.0 and generally smoked and the Southern type is less acid, spiced, not smoked and exhibits low water activity as the main hurdle (Lücke and Hechelmann, 1987; Demeyer et al., 2000). Fermented sausages are considered stable and safe products. Nevertheless, several outbreaks have been related to the consumption of this type of ready-to-eat (RTE) product. Selected lactic acid bacteria (LAB) from the endogenous meat environment, added as biopreservation cultures, and/or their antimicrobial products are efficient extra hurdles to increase food safety of these products without increasing the content of chemical additives. Their application in fermented meat products together with bacteriophage technology will be discussed in this chapter.

12.2  Food safety of fermented sausages Fermented sausage technology converts highly perishable meat products into stable and safe products due to a sequence of hurdles applied during the fermentation and ripening process. Nevertheless, the preparation of fermented sausages involves cutting up a mixture of meat (often pork) into small pieces. The process allows the uniform distribution of salt and other ingredients, but meat comminution also allows distribution of microbes (desirable, spoilage or pathogens). Contamination leads to faulty production (blowing and discoloration, health problems, infection and toxi-infections) depending on the efficiency of the different hurdles applied during processing. Indeed the initial contamination of meat batter and meat environment by several food-borne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. or Staphylococcus aureus) has been reported. In the EU, 1.1% of fresh pig meat samples were on average found Salmonella-positive (0% to 19.4%), although a very low proportion (<1.0%) were reported positive for bovine meat. In Germany, from 517 samples of fermented sausages at retail, 8.5% were positive for L. monocytogenes but only 0.2% were higher than 100 CFU/g (European Food Safety Authority, 2009b). Moreover, Salvat et al. (1995) reported that 68% of environmental samples in different curing plants were positive for L. monocytogenes and as many as 17% of the samples from raw product areas and 7% from finished product areas were still found positive after cleaning. The residual microflora and food-borne pathogens contaminating surfaces and equipment of 54 Southern and Eastern European small-scale processing units (PUs) manufacturing traditional dry fermented sausages were analysed after cleaning and disinfection procedures (314 environmental samples). All PU environments were colonized at various levels by spoilage and technological microflora with excessive contamination levels in

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some of the PUs. Sporadic contamination by pathogenic microflora was recorded. Salmonella and L. monocytogenes were detected in 4.8% and 6.7% of the samples, respectively, and S. aureus was enumerated in 6.1% of the samples. No E. coli O157 was reported. Several critical points were identified, such as machines for S. aureus and tables and knives for L. monocytogenes (Talon et al., 2007). Food-borne pathogens have also been reported to be able to survive during processing in different types of fermented meat sausages (bologna, pepperoni, salami, sucuk, soudjouk and others) (Farber et al., 1993; Hinkens et al., 1996; Faith et al., 1997, 1998; Incze, 1998; Nissen and Holck, 1998; Chickthimmah et al., 2001; Calicioglu et al., 2001, 2002; Benkerroum et al., 2003; Erkmen, 2008) or even to grow (L. monocytogenes and S. aureus) in low-acid fermented sausages under certain processing conditions (Garriga et al., 2005; Jofré et al., 2009). Further storage of the finished products, in general, does not provide a favourable environment for survival and fate of pathogens in Spanish low-acid fermented sausage (Jofré et al., 2009), in teewurst and salami (Gounadaki et al., 2007; Dourou et al., 2009) have been reported to be greater at high storage temperature. Although the risk of human listeriosis from fermented ready-to-eat meat is considered very low, an outbreak due to the consumption of salami was reported in Philadelphia (Schwartz et al., 1989). Outbreaks related to fermented sausages were also associated with Salmonella in bologna sausage (van Netten et al., 1986), salami stick (Cowden et al., 1989) and fermented sausage (Bremer et al., 2004). Outbreaks of E. coli O157:H7 were associated to a mettwurst in Australia (Paton et al., 1996), a dry-fermented salami in Washington and California (Tilden et al., 1996), a Genoa salami in Canada in 1998 (Williams et al., 2000) and a salami from British Columbia (Canada) in 1999 (MacDonald et al., 2004). Staphylococcal food poisoning was also associated to fermented sausages (US Department of Health Education and Welfare, 1979). In Europe, no outbreaks were associated with this kind of product during 2007 (European Food Safety Authority, 2009a).

12.3  Microbiota of fermented sausages In traditional meat fermented sausages, fermentation is driven by strains from the indigenous microbiota derived from raw materials, mainly meat and from the environment. Indigenous homofermentative lactobacilli and coagulase negative staphylococci, selected by the physicochemical conditions imposed by production technology, dominate the fermentation process. In general, sausage fermentation is characterized by an increase in the number of LAB from 103–105 CFU/g to 106–109 CFU/g within the first days of fermentation (1–3 days) or maturation/ripening (up to 14 days) which becomes the dominant microbiota and generally remains stable. The initial number of GCC+ (Gram-positive catalase-positive cocci), between 103–105 CFU/g may be kept stable or increase to 105–108 CFU/g during the first 14–20 days of fermentation and ripening (Schillinger and Lücke, 1987; Torriani et al., 1990; Hugas et al.,

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1993; Samelis et al., 1994; Cocolin et al., 2001; Metaxopoulos et al., 2001; Aymerich et al., 2003; Fontana et al., 2005a). Short ripening time and higher fermentation temperature lead to higher numbers of lactobacilli from the early stages of fermentation and produce a final product with acid flavour and little aroma. In contrast, sausages with longer maturation times and low fermentation temperatures contain higher numbers of GCC+ in the early stages and produce a less tangy more flavoured product (Demeyer et al., 2000). Yeast showed slow growth during the first half-ripening and then decreased at different rates (Mauriello et al., 2004). They confer a characteristic surface appearance and flavours due to their proteolytic and lipolytic activity, especially in Mediterranean countries. Although meat batter composition, together with final pH and process technology, may influence the microbiota, the species of LAB most commonly found in different kinds of fermented sausages (processed with different technologies in Germany, Hungary, Greece, Spain, Italy and Argentina) were Lactobacillus sakei and Lactobacillus curvatus. In the majority of studies, L. sakei was the dominant species in the final products exceeding 55% of the total LAB population. High intraspecies diversity and relevant genomic characteristics for successful adaptation were observed (Chaillou et al., 2009). Some other species as Lactobacillus plantarum, L. alimentarius, L. casei, L. paraplantarum, L. pentosus, L. paracasei, Pediococcus spp. Lactococcus lactis, Lactococcus garviae, Leuconostoc citreum, Leuconostoc mesenteroides, Weisella paramesenteroides/hellenica, Weissella viridescens, and Enterococcus faecium, E. faecalis, E. durans and E. pseudoavium were also described (Torriani et al., 1990; Montel et al., 1991; Hugas et al., 1993; Samelis et al., 1994; Santos et al., 1998; Cocolin et al., 2000; Parente et al., 2001; Aymerich et al., 2003, 2006a; Rantsiou et al., 2005a; Bonomo et al., 2008). In some Italian slightly fermented sausages, Enterococcus and lactobacilli population were reported to be highly equilibrated (Dellapina et al., 1994). In natural fermentation of Urutan, a sequential growth of LAB was reported: at the early stage L. plantarum was dominant followed by P. acidilactici, which started to grow on the second day of fermentation; then Lactobacillus farciminis grew on the third day, and became dominant by the end of fermentation (Antara et al., 2004). Concerning GCC+, Staphylococci (mainly Staphylococcus xylosus) were described as the predominant species in Spanish, German and Italian fermented sausages (Simonetti and Cantoni, 1983; Seager et al., 1986; Coppola et al., 1996; García-Varona et al., 2000; Aymerich et al., 2003; Martín et al., 2006). In some fermented Greek and Argentinian sausages a dominance of Staphylococcus saprophyticus was reported (Samelis et al., 1998; Papamanoli et al., 2002; Fontana et al., 2005b). A co-dominance of S. saprophyticus, S. xylosus and Staphylococcus equorum has been reported in three different types of Italian fermented sausages by Mauriello et al. (2004). Other species such as Staphylococcus carnosus, S. succinus, S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epidermidis, S. haemolyticus, S. intermedius, S. pulverei, S. sciuri, S. cohnii, S. hyicus, Kocuria varians, Kocuria kristiniae, Micrococcus luteus and Macrococcus caseolyticus were described (Seager et al., 1986; Montel et al., 1992; Cocolin et al., 2001; Aymerich et al., 2003; Rantsiou et al., 2005b; Martín et al., 2006). A high biodiversity at strain level was

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described by genotyping and/or biochemical methods (Rossi et al., 2001; Martín et al., 2006; Fontana et al., 2005b).

12.4  Bioprotective cultures for safety of fermented sausages Biopreservation, which refers to the extended storage life and enhanced safety of foods using their natural or controlled microbiota and (or) their antibacterial products, is an efficient alternative hurdle considering consumer trends: demanding products with less chemical additives, salt, acid and fat but with minimal changes in the sensory properties. In fermented meat products, bioprotective lactic acid bacteria (LAB) have a high potential because they have a long record of being generally recognized as safe (GRAS) for consumption and they are very competitive as they naturally dominate in these products (Stiles, 1996). Moreover their use is generally accepted by consumers as natural and health promoting (Deegan et al., 2006) and they could be applied to the meat batter as bioprotective starter culture or as adjunct of the starter culture. LAB may exert their antimicrobial properties by producing organic (lactic and acetic) acids, hydrogen peroxide, carbon dioxide, diacetyl, acetaldehyde, bacteriocins or by competitive exclusion. While the first generation of cultures, Lactobacillus plantarum and Pediococcus strains, from vegetable products, were applied with the aim to accelerate acidification and meat fermentation (Jensen and Paddock, 1940; Ninivaara, 1955; Deibel and Niven, 1957; Nurmi, 1966), the second generation was based on the selection of dominant lactic acid bacteria from the meat fermentation environment carrying important technological characteristics for biological safety, final flavour of the product and with the ability to compete with the indigenous microbiota thus leading the fermentation process (Demeyer et al., 2000; Lücke, 2000). Isolation and selection of safe bioprotective bacteriocin-producing LAB from endogenous microbiota, offering extra hurdles for food protection without antibiotic resistance and no biogenic amine producers, seems the appropriate way to select new bioprotective cultures for food fermentation purposes (Lücke, 2000; Aymerich et al., 2006b). Bioprotective cultures isolated from different styles of fermented sausages made from pork, beef, horse meat and other meats (Spanish, Thai, Balinese, Croatian, Turkish, Italian, Norwegian, German, Argentinian, Greek, American) and belonging to different species (Lactococcus lactis, Lactobacillus brevis, L. curvatus, L. plantarum, L. sakei, Leuconostoc mesenteroides, P. acidilactici, Enterococcus faecium and E. casseliflavus) were reported (Berry et al., 1990; Campanini et al., 1993; Winkowski et al., 1993; Hugas et al., 1995; Stiles, 1996; Foegeding, 1997). Identification of the specific strain by molecular typing methodologies such as PFGE and RAPD-PCR facilitates the assessment of colonization and the sensorial effect of the inoculated protective strain (Fontana et al., 2005a; Aymerich et al., 2006b; Cocolin et al., 2006). Table 12.1 summarizes the successful use of several bioprotective cultures used in fermented meat products. Antimicrobial activity against other LAB, L. monocytogenes, Brochrothix thermosphacta, Bacillus cereus, Clostridium botulinum, E. faecalis/E. faecium,

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Sakacin P Sakacin P

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L. curvatus LTH1174 L. plantarum MCS L. plantarum ALC01 DDEN 2205 (WHE92) 423

L. curvatus Lb3 L. curvatus DF38

7

Pediocin AcH Pediocin AcH Bacteriocin 423 6

6 7

5 g/kg lyophilized culture – 6

6

Curvacin A –

Curvacin DF38

5–6 5

–

L. sakei 2a L. sakei Lb674 I151

L. curvatus LBPE

5

Sakacin K

L. sakei CTC494

6

Sakacin A

L. sakei Lb706

Inocula (log CFU/g)

Bacteriocin

Bacteriocinogenic starter culture

Nordic fermented sausages Dry sausage model Salami from beef, horse, mutton, blesbok (Damaliscus dorcas phillipsi) and springbok (Antidorcas marsupialis)

Finnish fermented sausages Salami from beef, horse, mutton, blesbok (Damaliscus dorcas phillipsi) and springbok (Antidorcas marsupialis) Spanish, German fermented sausages Salami

Vacuum-packaged sliced bologna-type sausages Croatian fermented sausages Dry fermented sausages

Brazilian sausages

Spanish, German fermented sausages Turkish fermented sausages Spanish, Belgian, Italian fermented sausages

Product

Työppönen et al., 2003 Tolvanen et al., 2008a Todorov et al., 2007

Hugas et al., 1997 Campanini et al., 1993

Lahti et al., 2001 Todorov et al., 2007

Zdolec et al., 2007 Benkerroum et al., 2005a,b

Kröckel, 1997

Hugas et al., 1997 Erol et al., 1999 Hugas et al., 1995; Hugas et al, 1997; Ravyts et al., 2008 Liserre et al., 2002

References

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Table 12.1  Bacteriocinogenic cultures successfully applied for biopreservation of meat products

302

7 Commercial culture

–

Enterocins A, B and P 6 Enterocin 416K1 5 Pediocin AcH 7

HA-6111–2 P. acidilactici PA-2

L. bavaricus MI-401 E. faecium RZS C5 E. casseliflavus IM416K1 L. rhamnosus LC-705

7

Pediocin AcH 7

5–6

–

PAC 1.0

Lc. lactis LMG21206 P. acidilactici JBL1095

Spanish fermented sausages Italian sausages (cacciatore) Nordic fermented sausages

Chicken summer sausages American style sausages Turkish fermented sausages Finnish dry fermented sausages

Turkey summer sausages

Dry fermented sausages

Callewaert et al., 2000 Sabia et al., 2003 Työppönen et al., 2003

Erol et al., 1999 Lahti et al., 2001

Luchansky et al., 1992; Baccus-Taylor et al., 1993 Foegeding et al., 1992

Benkerroum et al., 2005a

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Propionibacteria and S. aureus, depending on the isolated strain, have been observed. Nevertheless, strain growth and bacteriocin production in laboratory media cannot be directly transposed to foodstuffs. When evaluating a bacteriocinogenic culture, we have to take into account that meat and meat products are complex systems with a number of factors influencing microbial growth and metabolite production. Therefore, the influence of formula and technology on the performance of the bacteriocinogenic cultures needs to be assayed. The growth of E. faecium CTC492 was not affected by the concentration of the ingredients used in the sausage manufacture; though sodium chloride, sodium nitrite and pepper, important ingredients for the salt–spice taste of this kind of sausage, were detrimental for bacteriocin production (Aymerich et al., 2000a). The addition of black pepper, a spice containing manganese that was shown to stimulate lactobacilli starter culture growth (Hagen and Holck, 1998) did not increase the production of sakacin K but enhanced the inhibitory activity of sakacin K against L. monocytogenes. Leroy and De Vuyst (1999) reported that the temperature and the acidity conditions that prevail during the fermentation process of dry fermented sausages was optimal for the production of sakacin K by L. sakei CTC494. A synergistic antilisterial effect between pepper, salt and nitrite and L. sakei CTC494 Bac+ was observed (Hugas et al., 2002). Several authors have reported the antilisterial effect of bacteriocin-producing strains in meat products. L. sakei CTC494, proved sensorially to be a suitable bioprotective starter culture, was able to suppress the growth of Listeria, initially spiked at 9×103 CFU/g, and to diminish their number by 2.64 log in dry Spanish sausages when compared to the initial counts. The inhibition observed was shown to be due to the bacteriocin produced by L. sakei CTC494, since the nonbacteriocinogenic starter strain alone led to lower pH and higher production of lactic acid and was unable to diminish Listeria to the same extent as the Bac+ strain (Hugas et al., 1995). When different strains producing the same bacteriocin were assayed in fermented sausages manufactured under different formula/ technology, their activity against Listeria varied. In batches of series A (Spanish nitrate-nitrite formulation including abundant addition of ingredients), L. curvatus LTH1174 (producing curvacin A) and L. sakei CTC494 (producing sakacin K), Listeria counts were reduced by more than 1.5–2 log cycles when compared to the non-bacteriocinogenic control batch. In series B (German formulation with only glucose, nitrate and sodium chloride), 1 log reduction of Listeria count was observed in batches with L. sakei Lb706 (sakacin A producer) and L. curvatus LTH1174 (Hugas et al., 1997). L. sakei CTC494, applied as a co-culture in fermented sausages with commercial fermentative starters inoculated with 3.5 log CFU/g of L. monocytogenes, was able to reduce up to 1.4 and 0.6 log CFU/g L. monocytogenes counts in Belgian-type sausage and Italian salami, respectively. In the control sausage, containing only the commercial fermentative starter, the reduction was limited to 0.8 log CFU/g for the Belgian-type recipe (where pH decreased from 5.9 to 4.9) while an increase of 0.2 log CFU/g was observed for Italian salami (where pH rose from 5.7 to 5.9 after an initial decrease to pH 5.3). In a Cacciatore recipe inoculated with 5.5 log CFU/g of L. monocytogenes, a

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pathogen reduction of 1.8 log CFU/g was observed in the presence of L. sakei CTC494, while in the control sausage only a 0.8 log CFU/g reduction was observed. Two commercial antilisterial cultures yielded reductions of 1.2 and 1.5 log CFU/g. Moreover, repetitive DNA sequence-based PCR fingerprinting demonstrated the superior competitiveness of L. sakei CTC494 (Ravyts et al., 2008). Other L. sakei strains (1151, 1154 and 1155) isolated from Italian fermented sausages were assayed in the production of Croatian traditionally fermented sausages in order to evaluate the protective effect against experimentally inoculated L. monocytogenes (4–5 log CFU/g). While a batch with strain 1155 was already Listeria negative after 14 days, control sausages and sausages with L. sakei 1151 and 1154 were negative after 28 days of ripening (Zdolec et al., 2007). Drosinos et al. (2006) also reported the effectiveness against L. monocytogenes NCTC10527 of a bacteriocin-producer L. sakei during fermentation, ripening and storage of fermented sausages from four European countries (Serbia-Montenegro, Hungary, Croatia and Bosnia-Herzegovina). The presence of the bacteriocinogenic strain reduced the time for a 4 log reduction of L. monocytogenes and the risk of ready-to-eat fermented sausages at the time of consumption when storage temperature, packaging methods, pH and water activity were considered. The addition of bacteriocinogenic L. sakei in Croatian sausages was also reported to decrease free fatty acid content and enhance sensory properties (Zdolec et al., 2008). The use of an endogenous non-bacteriocinogenic starter culture composed of L. sakei, S. equorum and S. succinus, isolated from a traditional French fermented sausage, was reported to improve safety by inhibiting L. monocytogenes, decreasing the level of biogenic amines and by limiting free fatty acid and cholesterol oxidation without affecting the typical sensory quality of the traditional sausages (Talon et al., 2008). The application of several bioprotective strains belonging to L. plantarum either applied alone or in combination with other strains have been reported in different types of fermented sausages. In Urutan (a Balinese traditional dry fermented sausage prepared from lean pork and various spices and fermented under warm conditions), the use of L. plantarum U201 (the dominant LAB), and P. acidilactici U318 (a bacteriocin producer), was able to eliminate Enterobacteriaceae in 24 hours (Antara et al., 2004). Counts of L. monocytogenes, artificially contaminated in salami, tended to decrease and no significant differences were observed between samples inoculated with L. plantarum MCS or with the bacteriocin negative mutant strain (Campanini et al., 1993). Dicks et al. (2004) reported a limited antilisterial effect of bacteriocinogenic strains, L. plantarum 423 and L. curvatus DF126 during the first 9 hours of fermentation, but they could not inhibit the re-growth in the ostrich meat salami. L. plantarum DDEN 2205 (previously WHE92) was proved to be an effective antilisterial agent when tested together with two composed commercial starter cultures in a dry fermented sausage producing L. monocytogenes-negative sausages after 17 days of ripening, either with low or high initial level inocula (Tolvanen et al., 2008b). Todorov et al. (2007) reported the superior antimicrobial and sensorial

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suitability of L. plantarum 423, producer of bacteriocin 423 when compared to L. curvatus DF38, producer of curvacin DF38, and a bacteriocin-negative mutant of L. plantarum 423 (423m) as starter culture in the production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis). Other species of lactobacilli have also been tested. In dry fermented sausages from Morocco, Benkerroum et al. (2005b) reported the application of two bacteriocin-producing strains: Lact. lactis subsp. lactis LMG21206 and L. curvatus LBPE together with a commercial starter culture (Bel’meat(TM) SL-25) not inhibitory to L. monocytogenes. This study showed that L. monocytogenes did not grow in any of the contaminated batches without Bac(+) cultures. However, in the batch with Bac(+) starter, L. curvatus LBPE, cell counts of L. monocytogenes decreased below the detection limit (<10 CFU/g) after only four hours of fermentation (initial inocula 102–103 CFU/g) and no survivors could be recovered by enrichment beyond day 8 of drying. When the Bac(+) starter culture containing Lact. lactis LMG21206 was used, a decrease in Listeria counts below the detection limit was achieved after 15 days of drying. In American-style dry fermented sausages several bioprotective strains of Pediococcus have been tested. With the use of pediocin producer P. acidilactici PAC1.0, absence of L. monocytogenes at the end of the drying process could only be achieved when pH was under 4.9; although a 10–100-fold reduction of a five strain cocktail of Listeria was observed when compared to its non-bacteriocin producer isogenic strain (Foegeding et al., 1992). Pediocins produced by P. acidilactici during fermentation also provided an additional hurdle against Listeria proliferation in turkey summer sausages, in chicken summer sausages and in pork/beef fermented sausages (Luchansky et al., 1992; Baccus-Taylor et al., 1993). Lahti et al. (2001) observed in fermented, dry, smoked sausage inoculated with high, medium and low inocula of two food-borne pathogens that starter culture A (composed of S. xylosus DD-34 and the bacteriocinogenic strains, P. acidilactici PA-2 and L. bavaricus MI-401) produced a rapid decrease of L. monocytogenes. Nevertheless, starter B (composed of S. carnosus MIII and a non-bacteriocinogenic L. curvatus Lb3) had a higher efficiency for E. coli O157:H7. Erol et al. (1999) reported the use of several bacteriocinogenic starter cultures (P. acidilactici PAC 1.0 and L. sakei Lb706) together with a non-bacteriocinogenic L. curvatus Lb3 in turkey fermented sausage. While L. monocytogenes counts of 2.4 MPN/g (Most Probable Number per gram) were obtained in the final product with the non-bacteriocinogenic culture, a reduction to only 0.0360-3 MPN/g was achieved with the bacteriocinogenic cultures. P. acidilactici HA-6111-2 (PA-1 producer) was tested for its suitability to be used as bioprotective culture against Listeria population in ‘Alheira’ (Portuguese garlic fermented sausage). The pathogen population decreased below the detection limit (1.5 log CFU/g) and the trained panel considered the protected product to be sensorially acceptable (Albano et al., 2009). Some strains, belonging to the more controversial LAB genera, Enterococcus, have also been tested for bioprotective purposes. The bacteriocinogenic E.

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casseliflavus IM 416KI (Bac+), isolated from Italian sausages or its bacteriocin Enterocin 416K1, with strong anti-listerial activity, was efficient to eliminate L. monocytogenes in artificially inoculated Italian sausages (‘cacciatore’) (Sabia et al., 2003). When E. faecalis A-48-32 was used as inoculum at approximately 107 CFU/g, listeria counts decreased progressively and were below detection level at day 9. Best results were obtained with E. faecium S-32-81, since listeria was undetectable at six days of incubation. Bacteriocin concentrations in samples reached concentrations of 60 and 80 AU/g for strains A-48-32 and S-32-81, respectively. These results clearly indicate that AS-48 can be used in the control of L. monocytogenes in sausages (Ananou et al., 2005a). Isolation and application of safe bioprotective culture belonging to Enterococcus have also been tested. Sparo et al. (2008) tested E. faecalis CECT7121, a strain deprived of haemolysin, gelatinase, biogenic amines production and capsule formation genes, sensitive to almost all the antibiotics tested and with a high LD50 (1011 CFU/ml in mice) for the manufacture of craft dry-fermented sausages. Sausages inoculated with E. faecalis CECT7121 had lower viable counts of Enterobacteriaceae, S. aureus and other Gram-positive cocci at the end of fermentation and no viable Enterobacteriaceae and S. aureus were recovered at the end of drying. E. faecalis CECT7121 did not affect the growth of Lactobacillus spp. but displaced the autochthonous populations of enterococci and E. faecalis CECT7121 was recovered at each time point. No statistical differences were found between control and sausages inoculated with E. faecalis CECT7121 regarding the production of lactic acid, pH development and sensory analysis in both series. A new generation of bioprotective culture with added probiotic properties has also been tested in fermented sausages and its efficiency against L. monocytogenes inoculated at 3 log CFU/g assayed in Nordic dry fermented sausages. The comparative study included two probiotic and bioprotective cultures, L. rhamnosus E-97800 and L. rhamnosus LC-705 (pediocin AcH producer), a bioprotective culture, L. plantarum ALC01, as well as P. pentosaceus RM2000 (commercial control). In the first trial, both experimental and control sausages (pH 5.0–5.2) were L. monocytogenes negative after 21 days of ripening. However, in the second and third trials L. monocytogenes was not detected after seven days of fermentation while for the control sausages L. monocytogenes was negative only after 28 days of ripening. The pH values were typical for North European type dry sausages (pH 4.7–4.9) (Työppönen et al., 2003).

12.5  Application of bacteriocins in fermented sausages Bacteriocins from LAB are antimicrobial peptides or proteins which have raised considerable interest for its suitability for application in food preservation, thus providing an additional barrier within the hurdle technology approach and enhancing preservation and wholesomeness of meat. Bacteriocins are ribosomally synthesized by GRAS strains, lack action to eukaryotic cells, are inactivated through digestive proteases, preserving gut microbiota and showing no cross-resistance with antibiotics,

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tolerate pH and heat and mainly show bactericidal mode of action to closely related bacteria through destabilization of the cell membrane (Gálvez et al., 2007). Bacteriocins are mainly classified into 3 groups (Deegan et al., 2006; Drider et al., 2006; Naghmouchi et al., 2007). Bacteriocins of interest to the meat industry mainly belong to Class IIa except for nisin, which belongs to Class Ia. Class Ia bacteriocins are small, cationic, elongated lantibiotic peptides (<5 KDa) containing unusual amino acids lanthionine (Lan), α-methyllanthionine (MeLan), dehydroalanine, and dehydrobutyrine. Class IIa bacteriocins are the pediocin-like group, they are small (<10 KDa), heat-stable, cationic, non-lantibiotic peptides with pediocin PA-1 as the reference bacteriocin. They are one-peptide bacteriocins with a double-glycine leader peptide and a dedicated secretion and processing machinery. This subgroup has attracted much interest due to the inhibition capacity of these bacteriocins against Listeria (Ennahar et al., 2000b; Chen and Hoover, 2003). The activity of bacteriocins is exerted through the formation of pores in the bacterial membrane, the anionic lipids of the cytoplasmic membrane being their primary receptors. Nevertheless, while Class I bacteriocins may induce pore formation according to a wedge-like model, Class II bacteriocins may function by creating barrel stave-like pores or carpet mechanisms whereby peptides orient parallel to the membrane surface and interfere with the membrane structure (Moll et al., 1999). Class I bacteriocins have a broader inhibitory spectrum when compared to Class II. Calculated or predicted physicochemical properties of diverse bacteriocins are web-accessible in BACTIBASE (http.//bactibase.pfba-ab.org) (Hammami et al., 2007). Bacteriocins against several food-spoilage organisms and food-borne pathogens (e.g. some LAB, B. cereus, B. thermosphacta, Cl. botulinum, Cl. perfringens, E. faecalis/faecium, L. monocytogenes, Propionibacteria, S. aureus) have been reported. Considerable effort has been made to develop food applications for many different bacteriocins. Bacteriocins can be added as ingredient (crude or purified extract) or by inoculation of the producer strain, either in the meat batter, sprayed onto the surface or forming part of an active interleaver or packaging system. The application modus will depend on the food matrix, interactions with the food system affecting activity and/or the desired content of the antimicrobial in the food system. The successful application of bacteriocins requires careful testing in food systems for which they are intended to be applied and against the selected target bacteria, in order to assess the cooperative or synergistic effect with the global sequence of hurdles present during sausage manufacture and storage. Their effectiveness may be affected by many factors acting simultaneously, like adsorption to food macromolecules such as fat and degradation by proteolytic enzymes. The presence of ethanol and emulsifiers may partially prevent adsorption losses. Nisin was the first bacteriocin isolated from LAB and considered for potential application as food biopreservative (Delves-Broughton, 1990). Thomas and Wimpenny (1996) reported increased antimicrobial activity of nisin against L. monocytogenes and S. aureus by NaCl and pH down to 5.0. Yang et al. (1992) also showed an increased antilisterial activity of sakacin P at low pH. At acidic pH (5.5) the addition of salt increased the activity of sakacin P while divalent and

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trivalent cations, such as magnesium and calcium, may decrease their activity (Gänzle et al., 1996). Moreover, natural resistances to Class IIa bacteriocins and to nisin were reported (Larsen and Norrung, 1993; Rasch and Knochel, 1998; Ukuku and Shelef, 1997; Ennahar et al., 2000a). Resistance, although not always stable, of L. monocytogenes to bacteriocins have been reported at frequencies ranging from <10–9 to 10–5 (Ming and Daeschel, 1993; Dykes and Hastings, 1998; Bouttefroy and Millière, 2000). Successful applications of several bacteriocins (nisin, pediocin, plantaricin, sakacins, enterocins and leucocins) as biopreservatives in fermented meat products are summarized in Table 12.2. The effect of nisin in Turkish fermented sausage (‘sucuk’) has been reported to increase with the concentration of nisin (in the range from 0 to 100 µg/g). No survival of L. monocytogenes ATCC 7644, spiked at a concentration of 106 CFU/g in the dough, was measured in sausages containing 50 and 100 µg/g of nisin after 20 and 25 days (Hampikyan and Ugur, 2007). Application of enterocins A and B (648 AU/g) from E. faecium CTC492 in the meat batter of low acid fermented sausages spiked with 3 log CFU/g of Listeria decreased the numbers of Listeria to 6 MPN/g from the third day of fermentation until the end of the drying period. The higher efficiency of semi-purified enterocins compared with the Bac+ strain (E. faecium CTC492) may be explained by the inhibition of bacteriocin production due to salt, pepper and low acid content of this type of dry fermented sausages (Aymerich et al., 2000b). In another trial, also with low acid fermented sausages of small caliber, the addition of enterocins A and B (2,450 AU/g) to raw meat batter spiked with 3 log CFU/g of Salmonella, L. monocytogenes and S. aureus showed an immediate reduction in the counts of L. monocytogenes due to the enterocins, while Salmonella was more affected by the endogenous hurdles associated with the ripening process (Fig. 12.1). Neither the ripening process nor the enterocins could control the levels of S. aureus (Jofré et al., 2009). Ananou et al. (2009) reported that enterocin AS-48 at 148 AU/g was able to cause a significant reduction of 5.5 log CFU/g for L. monocytogenes and 1.79 log CFU/g for Salmonella at the end of the ripening process of low acid Spanish fermented sausages. In a model meat sausage system, enterocin AS-48 (E. faecalis A-48-32) applied at concentrations of 30 or 40 μg/g, was able to achieve a significant viable count reduction of 2 and 5.31 log units, respectively, for S. aureus compared with the untreated control. Nevertheless the presence of bacteriocin also had a moderate negative effect on total lactic acid bacteria (Ananou et al., 2005b). A concentration dependent effect of bacteriocin AS-48 on L. monocytogenes was also shown in model sausages. A concentration of 225 AU/g reduced pathogen counts below the detection level (1.99 CFU/g) in meat at 3 days of incubation, but regrowth was observed after 9 days, whereas with 450 AU/g, no viable listeria were detected after 6 and 9 days (Ananou et al., 2005a). Other enterocins also showed efficiency in other types of fermented sausages. Lauková et al. (1999) reported the effectiveness of enterocin CCM4231 in Hornad sausages at 12,800 AU/g to immediately decrease the number of L. monocytogenes inoculated at 106 CFU/g

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L. sakei CTC494 L. sakei I154, L. mesenteroides E131 L. mesenteroides E131 E. faecium CCM4231 E. casseliflavus IM416K1

E. faecium CTC492

E. faecalis AS48 Lc. lactis ssp. lactis

Sakacin A/K Sakacin, leucocin Mesenterocin Y Enterocin CCM4231 Enterocin 416K1 Enterocin M Enterocin A and B

Enterocin AS48 Nisin

2,560 AU/g 12,800 AU/g 10 AU/g 3,200 AU/g 1,600–4,800 AU/g 221–648 AU/g 2000 AU/g 30 or 40 µg/g nisin (0, 5, 10, 25, 50 and 100 µg/g)

Dosage

Producer

Bacteriocin

Model sausage system Turkish fermented sausage (‘sucuk’)

Fermented sausages Bosnian, Hungarian, Serbian and Croatian fermented sausages Croatian fermented sausages Dry fermented hornád Italian sausage ‘cacciatore’ Gombasek sausage Fermented sausages

Product

Ananou et al., 2005a, 2005b Hampikyan and Ugur, 2007

Zdolec et al., 2007, 2008 Lauková et al., 1999 Sabia et al., 2003 Lauková et al., 2003 Aymerich et al., 2000b Jofré et al., 2009

Hugas et al., 1995 Zdolec et al., 2007

References

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Table 12.2  Bacteriocins successfully applied as natural biopreservatives for meat batter of fermented sausages

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Fig. 12.1 Growth of L. monocytogenes and survival of Salmonella in worst-case low-acid fermented sausage with or without addition of enterocins A and B (EntAB).

and the ability to reduce re-growth of the bacteria after day 2, assessing a 3 log count difference when compared to the control batch with no bacteriocin added. In Gombasek sausages, enterocin M at 3,200 AU/g, achieved a high immediate reduction of 2.7 log which was kept for one week, but no significant difference with the control batch was observed after ripening (Lauková and Mareková, 2002; Lauková et al., 2003). Enterocin 416K1 also showed its efficiency in Italian cacciatore (Sabia et al., 2003). Drosinos et al. (2006) examined the behaviour of L. monocytogenes NCTC10527 with respect to inactivation kinetics during fermentation, ripening and storage of fermented sausages from four European countries (SerbiaMontenegro, Hungary, Croatia and Bosnia-Herzegovina) produced with or without sakacin P and mesenterocin Y. Bacteriocins were able to reduce the time for a 4 log reduction of L. monocytogenes only in Serbian products. Other bacteriocins such as sakacin from L. sakei I154 and leucocin from Lc. mesenteroides E131 were applied to traditional Bosnian, Hungarian, Serbian and Croatian fermented sausages, with leucocin exhibiting higher antilisterial activity than sakacin (Zdolec et al., 2008).

12.6 Use of bacteriophages to improve food safety and meat environment In recent years, bacteriophages have become widely recognized for several potential applications in the food industry. They have been proposed as natural, non-toxic, safe and effective alternatives to antibiotics in animal health, as

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biopreservatives in food (products, animal carcasses, food-contact surfaces) and as tools for detecting pathogenic bacteria and as indicators of virus water contamination through the food chain. They display two unique features relevant and suitable for food safety, being harmless to mammalian cells and they are host bacteria specific. Thus proper performance in fermented meat products could be assessed without disturbing natural microbiota (Garcia et al., 2008). Moreover, phage-mediated biocontrol has been considered as more economic when compared with other carcass surface decontamination techniques (Gellynck et al., 2008). Bacteriophage efficacy to control Campylobacter, Salmonella, E. coli and L. monocytogenes, as well as spoilage organisms, on fruits, vegetables, dairy products and meat was shown (Hagens and Offerhaus, 2008). Aplications of bacteriophages have been mainly for decontamination of meat and processing environment, targeting L. monocytogenes and Salmonella. Indeed, rigorous assessment must be done before considering phage applications (Hudson et al., 2006). Carlton et al. (2005) proposed the safe use of strictly virulent phage P100 of L. monocytogenes to be used as a food decontaminant. The bacteriophage did not carry any of the putative protein associated with toxins, pathogenicity factors, antibiotic resistance determinants, or any known allergens, while the dose oral toxicity study did not produce any abnormal histological changes, morbidity or mortality. Moreover, it is able to infect and kill a majority of L. monocytogenes strains. The first testings in cheese and fish were promising and its use was proposed to extend to other products. The phage was considered GRAS level by FDA in 2006 and accepted in the EU for its use in food at levels not exceeding 1×109 pfu/ gram. Its is commercially available as Listex™ P-100 (EBI, Netherlands). Guenther et al. (2009) applied the broad-host-range phages A511 and P100 in different kinds of liquid and solid foods. The reduction of Listeria increased with greater number of phages being applied. In products of animal origin, the phages were stable during storage. Other bacteriophages such as CP220 of Campylobacter have also been succesfully applied in poultry (El-Shibiny et al., 2009). Sharma et al. (2005) reported the use of bacteriophages to control E. coli O157:H7 biofilms on stainless steel with limited efficiency when compared with other treatments (1 log reduction in surface coupons and no reduction on biofilms). Nevertheless, (Sillankorva et al., 2008) reported a proper efficiency of bacteriophage phi IBB-PF7A to control P. fluorescens biofilms. Between 63% and 91% of biomass removal was assessed depending on the conditions of biofilm formation. The integration of phages into the biofilm matrix and their entrapment to the surface may have further beneficial effects when phage treatment is considered alone or in addition to chemical biocides in industrial environments. A preparation that combines six-naturally different phages isolated from the environment has been shown to be effective against 170 different strains of L. monocytogenes and proved to significantly reduce (100–1000-fold) the contamination of L. monocytogenes when sprayed onto RTE foods without changing general composition, taste, odour or colour of foods. The preparation developed by Intralytix INC (LMP-102™, now ListShield™) was the first bacteriophage to be accepted by the FDA as a food additive (Food and Drug Administration US, 2006). The product

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was also approved by the U.S. Environmental Protection Agency (EPA) to prevent or significantly reduce bacterial contamination of food processing plants (EPA registration number 74234-1). The Intralytix company is also performing validation assays for the approval of the commercially available bacteriophage preparation ECP-100™ (now EcoShield™) against E. coli O157:H7. The company is also developing new products for other pathogens such as Salmonella. Other bacteriophage preparations have been proposed to be used to reduce the microbial load of animals prior to slaughter and are commercially available from Omnilytics (USA) as the BacWash product line, either against E. coli O157:H7 (FSIS Case No. 06-NT-0239-N-A December 2006) or Salmonella (FSIS Case No. 07-NT-0253-N-A December 2006). No reported scientific papers on the specific application of bacteriophages on fermented sausages have yet been published.

12.7  Legislation aspects and constraints Food products with LAB are perceived as being traditional, safe and even healthy. They are generally recognized as safe (GRAS) substances based on the experience of common usage by the FDA. The European Commission proposed the term ‘qualified presumption of safety’ (QPS) for microorganisms with a history of safe use (European Food Safety Authority, 2007) and only Denmark and France have legislation that explicitly regulates the addition of microbial cultures to food. The only qualification that has to be attached is evidence of the absence of acquired antibiotic resistance and virulence factors, mainly regarding enterococci. In this sense, food-grade cultures, regarded as a processing aid, do not need special labelling (Holzapfel et al., 1995). Nevertheless, purified substances as new purified bacteriocins must fulfil the criteria laid down in legislation concerning novel food ingredients (European Parliament and the Council, 1997) and safety information should be provided according to specific guidelines. Nisin (E234) is the only bacteriocin accepted by EU legislation as a preservative, although with use restricted to some dairy products, tapioca, semolina puddings and mascarpone (European Parliament and of the Council, 1995). In 1988, nisin was granted GRAS status by the U.S. Food and Drug Administration (Food and Drug Administration (U.S.), 1988) and the Scientific Committee for Food of the European Community decided to allocate an acceptable daily intake of 0.13 mg/kg body weight based on a nisin product of 40,000 IU/mg. Today the use of nisin is allowed in more than 50 countries. Nisaplin™, the commercial preparation of nisin A, has been approved for Food Standards Australia New Zealand (FSANZ) to be used in processed meat, poultry and game products. In 2006, a new bacteriophage preparation from Intralytix INC (LMP-102™, Listshield™) with potential antimicrobial use against L. monocytogenes was approved by the FDA and received EPA approval (Food and Drug Administration U.S., 2006; EPA registration #74234-1) to be applied as a spray at a level not to exceed 1 ml of the additive per 500 cm2 product surface area. In October of the same year, another bacteriophage preparation, Listex™ P100 from EBI Food

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Safety (Netherlands) was also accepted as a GRAS product at a level of 1×107 to 1×109 plaque forming units (pfu) per gram of product to be applied first to cheese and then extended to cover all food products in 2007 (United States Department of Agriculture Food Safety and Inspection Service 2008). In the EU, the product is available for use in certified organic foods. The European Food and Feed Law Review (EFFL) in March 2007 published an article that clarifies that the use of LISTEX™ as a processing aid was exempted from EU regulation and free of labelling requirements, provided the manufacturer observes the mandatory safety requirements pursuant to Art 14 Regulation EC/178/2002 (European Parliament and of the Council, 2002) and that is according to organic production EC/834/2007 (European Parliament and the Council, 2007). The United States Department of Agriculture (USDA) issued separate ‘no objection’ letters for use of bacteriophage targeting Salmonella and E. coli O157:H7, also known as Omnilytics BacWash product line (FSIS Case No. 07-NT-0253-N-A December 2006) on animal hides prior to slaughter (Omnilytics 2007a,b). These ‘no objection’ letters open the door for livestock processors who wish to reduce the microbial load of the animals prior to slaughter. Use of BacWash targeting E. coli O157:H7 (FSIS Case No. 06-NT-0239-N-A December 2006) may reduce the number of positive samples on trim and save slaughter and processing facilities extra traceability paperwork that is required when selling or processing trim that is positive or presumptive for E. coli O157:H7.

12.8  Future trends The combination of bioprotective cultures and their antimicrobial substances together with bacteriophages, probiotic strains, other functional microbes and the so-called ‘pharma-food microbes’ could enhance further research to develop excellent alternative hurdles to assure food safety of fermented sausages while promoting consumers’ health and add extra value to the product. The application of appropriate dosage, either at the meat batter or to the final products or their combination with other post-processing alternative technologies such as high hydrostatic technology that do not interfer with the technological microbiota and sensorial characteristics of the product must be considered. Moreover, the interest of the scientific community and producers to cope with consumers’ demands of functional safe products with typical and traditional flavour and taste will enhance further research for natural antimicrobials and functional microbes thus increasing the number of commercially available products.

12.9  Sources of further information and advice Books and top review papers Handbook of Fermented Meat and Poultry (2007), editor Fidel Toldrà. Blackwell Publishing. ISBN 13:978–0–8138–1477–3.

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Major research groups on the topic Regine Talon. Microbiologie, INRA, UR 454 F-63122 Saint-Genès Champanelle, France. Margarita Garriga. Food Safety. IRTA-Monells. 17121 Monells, Girona, Spain. Luc de Vuyst. Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium. George Nychas. Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece. Marc Offerhaus. EBI Food Safety BV, NL-2582 NE The Hague, Netherlands.

Commercial sources A range of bioprotective starter cultures for fermented sausages and other products are produced by two large companies: Chr Hansen (Hoersholm, Denmark, http:// www.chr-hansen.com/) and Danisco (Copenhagen, Denmark, http://www. danisco.com). Bactoferm F-LC (Chr Hansen) has been patented as a culture capable of preventing growth of Listeria in fermented sausages. It is a mixed culture of P. acidilactici and L. curvatus producing pediocin and sakacin A, respectively. B-LC-20 is also an antilisteria bioprotective culture to be applied in fermented sausages and does not call for any changes to the recipe or processing procedure. Alternatively, B-LC-35 has the same anti-listerial properties as F-LC, but gives the fermented sausage less acidification. Also nisin preparation is available as Chrisin™. Danisco produces MicroGARD™, a fermentate based antimicrobials, HOLDBAC™, a line of protective cultures and NISAPLIN®, a crude natural antimicrobial containing nisin. For bacteriophages three companies should be mentioned. Omnilytics (http:// www.omnilytics.com/home5.html) with the BacWash product line, Intralytix (http://www.intralytix.com/) with ListShield™ and EcoShield™ and EBI (http:// www.ebifoodsafety) with Listex™ P-100.

12.10  Acknowledgement CSD2007-00016 Consolider project Carnisenusa.

12.11  References et al. (2005a) ‘Control of Listeria monocytogenes in model sausages by enterocin AS-48’, Int J Food Microbiol 103, 179–190. ananou s , maqueda m , martínez - bueno m , gálvez a and valdivia e (2005b) ‘Control of Staphylococcus aureus in sausages by enterocin AS-48’, Meat Sci 71, 549–556. ananou s , garriga m , jofré a , aymerich t , gálvez a et al. (2009) ‘Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages’, Meat Sci (in press). ananou s , garriga m , hugas m , maqueda m , martínez - bueno m

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catalase-positive cocci from slightly fermented sausages’, Int J Food Microbiol 107, 147–158. mauriello g , casaburi a , blaiotta g and villani f (2004) ‘Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy’, Meat Sci 67, 149–158. metaxopoulos j , samelis j and papadelli m (2001) ‘Technological and microbiological evaluation of traditional processes as modified for the industrial manufacturing of dry fermented sausage in Greece’, Ital J Food Sci 1, 3–18. ming x and daeschel m a (1993) ‘Nisin resistance of foodborne bacteria and the specific resistance responses of Listeria monocytogenes Scott A’, J Food Protect 56, 944–948. moll g n , konings w n and driessen a j (1999) ‘Bacteriocins: mechanism of membrane insertion and pore formation’, Antonie Van Leeuwenhoek 76, 185–198. montel m c , talon r, cantonnet m and fournaud j (1992) ‘Identification of Staphylococcus from french dry sausage’, Lett Appl Microbiol 15, 73–77. montel m c , talon r, fournaud j and champomier m c (1991) ‘A simplified key for identifying homofermentative Lactobacillus and Carnobacterium spp. from meat’, J Appl Bacteriol 70, 469–472. naghmouchi k , kheadr e , lacroix c and fliss i (2007) ‘Class I/Class IIa bacteriocin cross-resistance phenomenon in Listeria monocytogenes’, Food Microbiol 24, 718–727. ninivaara f (1955) ‘Über den Einfluss von bacterienreinkulturen auf die reifung und umrötung der Rohwurst’, Acta Agralia Fennica 84, 1–128. nissen h and holck a (1998) ‘Survival of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella kentucky in norwegian fermented, dry sausage’, Food Microbiol 15, 273–279. nurmi e (1966) ‘Effect of bacterial inoculation on characteristics and microbial flora of dry sausage’, Acta Agralia Fennica 108, 1–77. papamanoli e , kotzekidou p , tzanetakis n and litopoulou - tzanetaki e (2002) ‘Characterization of Micrococcaceae isolated from dry fermented sausage’, Food Microbiol 19, 441–449. parente e , griego s and crudele m a (2001) ‘Phenotypic diversity of lactic acid bacteria isolated from fermented sausages produced in Basilicata (Southern Italy)’, J Appl Microbiol 90, 943–952. paton a w, ratcliff r m , doyle r m , seymour - murray j , davos d et al. (1996) ‘Molecular microbiological investigation of an outbreak of hemolytic-uremic syndrome caused by dry fermented sausage contaminated with Shiga-like toxin-producing Escherichia coli’, J Clin Microbiol 34, 1622–1627. pederson c s (ed.) (1979) Microbiology of Food Fermentations. AVI Publishing Company I W, Connecticut, pp. 153–172. rantsiou k , drosinos e h , gialitaki m , urso r, krommer j et al. (2005a) ‘Molecular characterization of Lactobacillus species isolated from naturally fermented sausages produced in Greece, Hungary and Italy’, Food Microbiol 22, 19–28. rantsiou k , urso r, iacumin l , cantoni c , cattaneo p et al. (2005b) ‘Culturedependent and -independent methods to investigate the microbial ecology of Italian fermented sausages’, Appl Environ Microbiol 71, 1977–1986. rasch m and knochel s (1998) ‘Variations in tolerance of Listeria monocytogenes to nisin, pediocin PA-1 and bavaricin A’, Lett Appl Microbiol 27, 275–278. ravyts f , barbuti s , frustoli m a , parolari g , saccani g et al. (2008) ‘Competitiveness and antibacterial potential of bacteriocin-producing starter cultures in different types of fermented sausages’, J Food Protect 71, 1817–1827. rossi f , tofalo r, torriani s and suzzi g (2001) ‘Identification by 16S-23S rDNA intergenic region amplification, genotypic and phenotypic clustering of Staphylococcus xylosus strains from dry sausages’, J Appl Microbiol 90, 365–371.

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and bondi m (2003) ‘Bacterocinproducing Enterococcus casseliflavus IM 416K1, a natural antagonist for control of Listeria monocytogenes in Italian sausages (“cacciatore”)’, Int J Food Microbiol 87, 173–179. salvat g , toquin m t , michel y and colin p (1995) ‘Control of listeria-monocytogenes in the delicatessen industries – the lesson of a listeriosis outbreak in France’, Int J Food Microbiol 25, 75–81. samelis j , maurogenakis f and metaxopoulos j (1994) ‘Characterisation of lactic acid bacteria isolated from naturally fermented Greek dry salami’, Int J Food Microbiol 23, 179–196. samelis j , metaxopoulos j , vlassi m and pappa a (1998) ‘Stability and safety of traditional Greek salami – a microbiological ecology study’, Int J Food Microbiol 44, 69–82. santos e m , gonzález - fernández c , jaime i and rovira j (1998) ‘Comparative study of lactic acid bacteria house flora isolated in different varieties of “chorizo” ’, Int J Food Microbiol 39, 123–128. schillinger u and lücke f k (1987) ‘Identification of lactobacilli from meat and meat products’, Food Microbiol 4, 199–208. schwartz b , hexter d , broome c v, hightower a w, hirschhorn r b et al. (1989) ‘Investigation of an outbreak of listeriosis: new hypotheses for the etiology of epidemic Listeria monocytogenes infections’, J Infect Dis 159, 680–685. seager m s , banks j g , blackburn c and board r g (1986) ‘A taxonomic study of Staphylococcus spp. isolated from fermented sausages’, J Food Sci 51, 295–297. sharma m , ryu j h and beuchat l r (2005) ‘Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage’, J Appl Microbiol 99, 449–459. sillankorva s , neubauer p and azeredo j (2008) ‘Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A’, BMC Biotechnology 8, 12. simonetti p and cantoni c (1983) ‘Coagulase negative staphylococci for dry sausage ripening’, Ind Alim 22, 262–264. sparo m , nuñez g g , castro m , calcagno m l , garcía allende m a et al. (2008) ‘Characteristics of an environmental strain, Enterococcus faecalis CECT7121, and its effects as additive on craft dry-fermented sausages’, Food Microbiol 25, 607–615. stiles m e (1996) ‘Biopreservation by lactic acid bacteria’, Antonie Van Leeuwenhoek 70, 331–345. talon r, lebert i , lebert a , leroy s , garriga m et al. (2007) ‘Traditional dry fermented sausages produced in small-scale processing units in Mediterranian countries and Slovakia. 1; Microbial ecosystems of processing environments’, Meat Sci 77, 570–579. talon r, leroy s , lebert i , giammarinaro p , chacornac j - p et al. (2008) ‘Safety improvement and preservation of typical sensory qualities of traditional dry fermented sausages using autochthonous starter cultures’, Int J Food Microbiol 126, 227–234. thomas l v and wimpenny j w t (1996) ‘Investigation of the effect of combined variations in temperature, pH, and NaCl concentration on nisin inhibition of Listeria monocytogenes and Staphylococcus aureus’, Appl Environ Microbiol 62, 2006–2012. tilden j , young w, mcnamara a m , custer c , boesel b et al. (1996) ‘A new route of transmission for Escherichia coli: Infection from dry fermented salami’, American Journal of Public Health 86, 1142–1145. todorov s d , koep k s c , van reenen c a , hoffman l c , slinde e and dicks l m t (2007) ‘Production of salami from beef, horse, mutton, Blesbok (Damaliscus dorcas phillipsi) and Springbok (Antidorcas marsupialis) with bacteriocino genic strains of Lactobacillus plantarum and Lactobacillus curvatus’, Meat Sci 77, 405–412. tolvanen r, hellstrom s , elsser d , morgenstern h , bjorkroth j and korkeala h (2008a) ‘Survival of Listeria monocytogenes strains in a dry sausage model’, J Food Protect 71, 1550–1555.

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Protect 71, 1550–1555.

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and palummeri m (1990) ‘Characterization of Lactobacilli Isolated from Italian Salami’, Ann Microbiol 40, 225–233. työppönen s , markkula a , petäjä e , suihko m l and mattila - sandholm t (2003) ‘Survival of Listeria monocytogenes in North European type dry sausages fermented by bioprotective meat starter cultures’, Food Contr 14, 181–185. ukuku d o and shelef l a (1997) ‘Sensitivity of six strains of Listeria monocytogenes to nisin’, J Food Protect 60, 867–869. u . s . department of health education and welfare (1979) ‘Staphylococcal food poisoning associated with Genoa and hard salami’, Morbidity and Mortality Weekly Report 28, 179–180. van netten p , leenaerts j , heikant g m and mossel d a (1986) ‘A small outbreak of salmonellosis caused by Bologna sausage’, Tijdschr Dieegeneeskd 24, 1271–1275. williams r c , isaacs s , decou m l , richardson e a , buffett m c et al . (2000) ‘Illness outbreak associated with Escherichia coli O157:H7 in Genoa salami’, Can Med Assoc J 162, 1409–1413. winkowski k , crandall a d and montville t j (1993) ‘Inhibition of Listeria monocytogenes by Lactobacillus bavaricus MN in beef systems at refrigeration temperatures’, Appl Environ Microbiol 59, 2552–2557. yang r, johnson m c and ray b (1992) ‘Novel method to extract large amounts of bacteriocins from lactic acid bacteria’, Appl Environ Microbiol 58, 3355–3359. zdolec n , hadziosmanovic m , kozacinski l , cvrtila z , filipovic i et al. (2007) ‘Protective effect of Lactobacillus sakei in fermented sausages’, Archiv fur Lebensmittelhygiene 58, 152–155. zdolec n , hadziosmanovic m , kozacinski l , cvrtila z , filipovic i et al. (2008) ‘Microbial and physicochemical succession in fermented sausages produced with bacteriocinogenic culture of Lactobacillus sakei and semi-purified bacteriocin mesenterocin Y’, Meat Sci 80, 480–487.

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13 Applications of protective cultures, bacteriocins and bacteriophages in fresh seafood and seafood products M.-F. Pilet, ONIRIS, Nantes, France and F. Leroi, Ifremer, Nantes, France

Abstract: Microbial seafood-borne disease represents 10 to 20% of the total food-borne outbreaks. Most of these are from bacterial origin and involve seafood products that have been contaminated by pathogenic Vibrio, Listeria monocytogenes and histaminogen bacteria. On the other hand, seafood products are very sensitive to the development of spoiling bacteria producing off-odours. Pathogenic and spoiling microorganisms are not always reduced or limited by the processing steps that are currently used in these foodstuffs, and the interest in alternative techniques such as bioprotection to improve the quality and safety of seafood has increased in recent years. Among the microbiota of lightly preserved seafood products, lactic acid bacteria usually become dominant during storage under vacuum or modified atmosphere. In some cases these bacteria are responsible for spoilage but some of them have demonstrated potential for pathogenic or spoiling microorganisms inhibition, mainly for the control of Listeria monocytogenes in cold smoked salmon and to a lesser extent in other products to enhance sensory shelf-life. Many successful results have been obtained at the laboratory scale, nevertheless, the application in the seafood industry is still limited. Key words: biopreservation of seafood, seafood safety and spoilage, lactic acid bacteria, sensory quality, Listeria monocytogenes.

13.1  Introduction Fishery products contribute to a huge source of valuable nutrients such as proteins, vitamins, minerals, omega-3 fatty acid, taurine, etc. However, they are also responsible for human intoxication and infection, and 10 to 20% of food-borne illnesses are attributed to fish consumption. The aetiology of seafood is not always known but it is clear that indigenous bacteria present in the marine environment 324 © Woodhead Publishing Limited, 2011

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as well as the result of post contamination during process are responsible for many cases of illness. Recently, the traditional processes applied to seafood like salting, smoking and canning have decreased in favour of mild technologies involving lower salt content, lower cooking temperature and vacuum packing (VP) or modified atmosphere packing (MAP). These products, designed as lightly preserved fish products (LPFP), are usually produced from fresh seafood and further processing increases risk of cross contamination. The treatments are usually not sufficient to destroy microorganisms and in some cases psychrotolerant pathogenic and spoiling bacteria can develop during the extended shelf-life of LPFP (usually up to 10 days). As several of these products are eaten raw, minimising the presence and preventing growth of microorganisms is essential for the food quality and safety. The microbial safety and stability of food are based on an application of preservative factors called hurdles. Most of the decontamination technologies such as the oldest one, cooking, and more recent mild technologies, i.e. pulsedlight, high pressure, ozone, ultrasound . . . are not efficient or not compatible with the delicate texture and flavour of seafood. Chemical preservative have also been used but consumers require more natural products with lower chemical treatment. An alternative solution that is gaining more and more attention is biopreservation technology (Dortu and Thonart, 2009; Calo-Mata et al., 2008; Rodgers, 2001). It consists in inoculating food with microorganisms, or their metabolites, selected for their antibacterial properties. Lactic acid bacteria (LAB) are generally good candidates as some of them show natural capacities to inhibit growth of microorganisms and because they are naturally present in many food products and have been eaten for years by human without any safety risk. LAB have complex nutritional requirements and in order to obtain a good implantation it is generally the strategy to use bacteria isolated from the food that has to be preserved. LAB in fish flesh have long been disregarded because they are not currently present in seafood. However, in some environmental conditions, for instance in lightly processed seafood with salt, smoke, vacuum or modified atmosphere packaging, LAB can become dominant. Their occurrence and role vary according to fish and bacterial species, and in some cases they may be responsible for strong off-flavours and degradation of texture that prevents their use as a food preservative (Leroi, 2010). In many other cases they do not change the organoleptic characteristics of the products and their use as a protective culture could offer an alternative to the use of chemical compounds. However, the knowledge of LAB from seafood is still in its infancy, explaining why their use by humans for preservation or transformation of marine products is still limited. After a description of the microbial risks associated with seafood, this chapter presents the particular position of LAB among the microbiota of processed seafood and the bioprotective solutions that have so far been proposed to ensure quality and safety of fisheries.

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13.2  Microbial risk in seafood Fish constitutes a major part of protein consumption in many places in the world. In 2006 the total fisheries production, including fish, crustaceans and molluscs, was 143 million tons, about 35% of which was of aquaculture origin (http://www. fao.org/fishery/statistics/en, accessed 25 January 2010). Only 110 million tons were for human consumption, out of which 48% were marketed as fresh products, 25% frozen, 15% canned and 11% processed (salted, dried, smoked, etc.). 13.2.1  Microbial seafood-borne disease statistics The number of outbreaks attributed to seafood consumption is generally high (10–20% of the total food-borne outbreaks), but varies according to the quality of the surveillance system, the level of consumption (from 5–6 kg in some countries to 180 kg/person/year in the Maldives) and the consumers’ habits (higher risk associated to raw fish and mollusc consumption). During the period between 1988–1992, the percentage of total number of outbreaks was 7.4% in the USA (Bean et al., 1996) compared with 21.7% during the years 1981–1990 in Japan where people consume a larger amount of raw seafood (Lee et al., 1996). Some of the largest food poisoning outbreaks have been associated with seafood: in 1991 in Shanghai, a hepatitis A outbreak due to consumption of clams involved 300 000 cases with nine deaths (Tang et al., 1991). Around the same time, cholera caused more than 400 000 illnesses and more than 4000 deaths in Peru, with the primary source probably Ceviche, a typical raw and lightly marinated fish. Fish consumption is responsible for more outbreaks than shellfish (Huss et al., 2000) but the number of cases per outbreak is often much more higher with shellfish. In finfish, most of the diseases are from bacterial origin, the highest number being attributed to histamine, a biogenic amine from bacterial origin, accounting for 30–40% of fish intoxication. Histamine fish poisoning is principally due to scombroid fish consumption such as tuna and mackerel which contain high level of histidine, the precursor of histamine, although other species have also been involved (Dalgaard and Emborg, 2008). In molluscs, virus generally accounts for more than 50% of outbreaks but the lack of routine sensitive detection methods does not allow a precise estimation. Vibrio parahaemolyticus, V. vulnificus, and V. cholerae are also important causes of illness in molluscan bivalves that concentrate different particles during their filter feeding. Of the 2500 reported cases of illness due to bivalve molluscs, 50% were due to Vibrio, with 95 deaths during the period 1984–1993 in the USA (Wittman and Flick, 1995). As biopreservation is not used at the moment for live molluscs, this chapter will focus on microbial risk in finfish. 13.2.2  Microbial pathogens in seafood Microbial seafood pathogens can be classified in two categories: 1.

Indigenous bacteria that are naturally present in the marine environment, i.e. Vibrio vulnificus, V. parahaemolyticus and V. cholerae, Listeria monocytogenes,

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Clostridium botulinum and Aeromonas hydrophila. The presence of indigenous microorganisms is normally not a safety concern since they are present at too low a level to cause disease. Moreover, adequate cooking eliminates these bacteria or their toxin (toxin of C. botulinum is thermolabile). Therefore, the hazard concerns: (a) products in which the growth of these bacteria is possible during the storage period and which are eaten raw or insufficiently cooked, as in the case of Vibrio in raw fish or tropical shrimp preparation. Vibrio are mesophilic bacteria found in tropical water or in temperate water at the end of summer and their growth is rapid if the products are kept for some hours at room temperature. L. monocytogenes is also a problem in LPFP such as cold-smoked, lightly marinated fish or insufficiently cooked seafood stored under VP or MAP. During the extended shelf-life of those products, L. monocytogenes can still develop and reach unacceptable concentration. Insufficiently salted seafood stored in anaerobic conditions or traditional fermented fish can also support growth of C. botulinum and production of the botulinic toxin (b) Scombroid and Clupeid fish with high histamine content kept for some hours at abuse temperature (> 5 °C). The origin of histamine-producing bacteria is not completely well established although there is evidence that some of them are present in the gut, gills and skin of the fish. Most of the histamine producers are mesophilic bacteria (Morganella morganii, Hafnia alvei, Raoultella planticola) that produce histamine when fish is stored at abuse temperature, for instance during storage on the vessels or during the thawing stage before processing. More recently, psychrotolerant bacteria (Photobacterium phosphoreum, Morganella psychrotolerans) that grow at 2 °C have been associated with histamine fish poisoning in cold-smoked tuna (Emborg and Dalgaard, 2006). Once produced, histamine is not destroyed during the canning process and may cause serious problems in those products.   All those indigeneous bacteria can also post contaminate products during the processing stage, either by cross-contamination in industry or because some of them (L. monocytogenes) are ubiquitous bacteria naturally present in many food industrial environments or in human skin. 2.

Exogenous bacteria due to post contamination during fish processing: these bacteria are the same as those that can be found in other food products, i.e. Staphylococcus aureus, Salmonella, Shigella, Clostridium perfringens, Bacillus cereus, Yersinia enterocolitica or enterohaemorrhagic Escherichia coli. Some of these bacteria can also be present in coastal and estuarine marine water or in aquaculture ponds, due to human activities. They constitute a serious problem since low dose can cause illness. Normal cooking eliminates the risk but a lot of ready-to-eat food are raw or insufficiently cooked (shellfish salads, shrimps, soup, etc.). Moreover, the toxin of S. aureus is heat stable.

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The different pathogenic bacteria, symptoms, minimal infectious doses and seafood responsible for infection are summarized by Feldhusen (2000) and Lee and Rangdale (2008). 13.2.3  Microbial seafood safety risk assessment Different qualitative and quantitative risk assessment strategies have been used to categorize risk from seafood. Risk categories and associated microorganisms are described in Table 13.1 In a semi quantitative seafood safety risk assessment performed on statistics of seafood-borne illness during the period 1990–2000 in Australia, Sumner and Ross (2002) have shown that very high risks were due to V. parahaemolyticus and V. cholerae in cooked prawns, V. vulnificus in oysters, L. monocytogenes in cold-smoked seafoods, enteric bacteria in imported cooked shrimp eaten by vulnerable consumers and scombrotoxicosis. Almost all the hazard/ product pairs in this category have caused the outbreaks of food poisoning in Australasia. In developed countries, changes in consumers’ habits have led to an increase of ready-to-eat and convenient food, concepts that include both the easy-touse aspect and an extended shelf-life of the products. The nutritional aspects are also increasingly taken into consideration by the consumers who want natural products, with technological treatment and level of preservatives as low as possible. Lightly preserved fish products, like carpaccio-type marinated fish, cold-smoked fish, peeled and lightly cooked shrimp, desalted cod packed under VP or MAP, etc., meet those requirements and their production has increased dramatically in the last Table 13.1  Risk categories for seafood products and associated microorganisms Risk

Seafood products

Agent

High

Mollusc (fresh or frozen)

Virus, bacteria, toxin from microalgae (heat stable)

Raw fish: Ceviche, Sushi, etc.

Indigenous bacteria (Vibrio)

Lightly preserved fish (NaCl < 6% WP, pH > 5): carpaccio, cold-smoked fish, marinated products, gravads, etc.

Growth of indigenous bacteria (Listeria monocytogenes, production of toxin from Clostridium botulinum)

Mildly heat processed: cooked and peeled shrimp, salads, soup, etc.

Recontamination with enteric bacteria, growth of Listeria monocytogenes, Vibrio

Scombroid fish

Histamine production

Cooked fish and crustacean

Ciguatera in tropical areas

Semi-preserved fish (NaCl > 6% WP, pH < 5): salted, dried, marinated, hot-smoked fish, etc.

Recontamination with enteric bacteria

Low

Heat processed: sterilized, canned, etc. Clostridium botulinum toxin Note: WP = water phase

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few years. The major safety risk associated with LPFP is L. monocytogenes with a quite elevated prevalence, varying from 2 to 60% depending on the studies (Beaufort et al., 2007; Hu et al., 2006; Gudmundsdöttir et al., 2005; Nakamura et al., 2004; Jorgensen and Huss, 1998; Valdimarsson et al., 1998). L. monocytogenes may be present in the raw material in low numbers but contamination mainly occurs during processing. A strict hygienic manufacturing practice has been emphasized to reduce the cross contamination with L. monocytogenes, with daily cleaning and disinfection of production lines and special attention to hygiene by the employees. However, production of LPFP consistently free of the bacterium seems impossible as L. monocytogenes is not destroyed by the different processing steps. The risk associated with consumption of LPFP is due to the possible growth of L. monocytogenes rather than to the initial contamination of freshly processed products, which are commonly inferior to 1 CFU g–1. L. monocytogenes can multiply at low temperatures, in a wide range of pH, in aero and anaerobic conditions in the presence of salt or smoke and it can sometimes exceed the European tolerated limit of 100 CFU g–1 (Commission Regulation 1441/2007/EC). In these kinds of products with an extended shelf-life, psychrotrophic LAB have time to develop their use as a protective culture to prevent L. monocytogenes, and spoiling microorganisms are a subject of increasing investigation.

13.3  Lactic acid bacteria in seafood products 13.3.1  Lactic acid bacteria in living fish The skin, mucus, gills and gut of fish contain high numbers of bacteria, whose composition and quantity vary according to the fish species and many environmental parameters. The microbiota of marine fish from temperate waters is usually composed of Gram-negative psychrotrophic bacteria from the genera Pseudomonas, Shewanella, Acinetobacter, Aeromonas, Vibrio, Moraxella, Psychrobacter and Photobacterium. Nevertheless, Gram-positive bacteria in Micrococcus, Corynebacterium, Bacillus, Lactobacillus and Clostridium may also be present in variable proportions. In tropical fish the microbiotica has the same composition overall, but with a predominance of Gram-positive bacteria, Enterobacteriaceae and Vibrionaceae. It is generally accepted that LAB occur among the normal intestinal microbiota of fish from the first few days onward, and many genera and species have been reported: Lactobacillus plantarum, Carnobacterium maltaromaticum (previously C. piscicola), C. divergens, C. gallinarum and C. inhibens, Streptococcus spp., Leuconostoc spp., Lactococcus lactis and Lc. piscium, Vagococcus salmoninarum, Weissella spp., etc. (Yang et al., 2007; Huber et al., 2004; Ringo et al., 2001; Jöborn et al., 1999; Ringo and Gatesoupe, 1998). Lactic acid bacteria are generally recognized as non-pathogenic for humans but virulence of some species such as Lactococcus garvieae, C. maltaromaticum and Weissella sp. has been clearly established for farmed fish (Liu et al., 2009; Eldar et al., 1996; Toranzo et al., 1993).

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13.3.2  Lactic acid bacteria in fresh fish stored in ice or under packaging At fish death and during evisceration and filleting, microorganisms may contaminate the flesh and this often occurs all along the production line as far as the finished product. Lactic acid bacteria are not naturally dominant in the microbiota of fresh fish stored in ice. The low temperature, high post-mortem pH (>6), low percentage of sugars (0.2 to 1.5% depending on the species) and high content of non-protein, low molecular weight nitrogenous compounds are more favourable for the Gramnegative psychrotrophic bacteria naturally present in living fish, like Pseudomonas and Shewanella. Vacuum packing does not slow their growth as many of them, notably Shewanella putrefaciens, Photobacterium phosphoreum and the Vibrionaceae, are able to use trimethylamine oxide (OTMA), a common marine molecule, as a terminal electron acceptor in anaerobic respiration. These bacteria produce strong off-odours typical of rotten fish due to the reduction of OTMA to trimethylamine, and also sulphurous odours resulting from the breakdown of cysteine and methionine (Gram and Huss, 1996). Modified atmosphere packing decreases the number of respiratory microorganisms like Pseudomonas and Shewanella but P. phosphoreum is resistant to CO 2. It therefore multiplies quickly in this type of product and is recognized as the main spoilage bacterium of fresh MAP fish (Dalgaard et al., 1997). This explains why this type of packaging only slightly increases the use-by date of fish compared to meat. However, numerous studies carried out on fatty or low-fat fish have shown that more LAB are found in products preserved under MAP than under air (Lalitha et al., 2005; Fletcher et al., 2004). MAP selects both P. phosphoreum and LAB but the latter are less competitive and so often play a minor role in the spoilage. When P. phosphoreum is eliminated by a frozen stage, LAB becomes the dominant group during the MAP storage of thawed fish (Dalgaard et al., 2006; Emborg et al., 2002). 13.3.3  Lactic acid bacteria in lightly preserved fish Lightly preserved fish products are highly perishable and are often stored at chilled temperature and under VP or MAP to extend shelf-life. The initial microbiota depends strongly on the hygiene conditions in the company but is often dominated by Gram-negative bacteria typical of fresh fish (GonzalezRodriguez et al., 2002; Leroi et al., 1998; Paludan-Müller et al., 1998). During storage, Gram-positive bacteria, particularly LAB, become predominant, sometimes associated with Enterobacteria and Brochothrix thermosphacta (Jaffrès et al., 2008; Cardinal et al., 2004). LAB can easily reach 107–8 CFU g–1 and such amounts have been found in cold-smoked salmon (CSS) (Leroi et al., 1998 , 2000), smoked trout (Lyhs et al., 1998), smoked herring (Gancel et al., 1997), salted lumpfish roe (Basby et al., 1998), cooked cold-water shrimp (Dalgaard et al., 2003) and warm-water shrimp (Jaffrès et al., 2008; Mejlholm et al., 2005). The cause of LAB predominance in LPFP has not been extensively studied but it is clear that they are well adapted to the conditions prevailing in those products. Most of the LAB strains isolated from LPFP are psychrotrophic, able to catabolize arginine with low glucose concentration and known to grow with up

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to 8–10% of salt. VP and MAP are probably other factors promoting LAB development since they are aero-anaerobic bacteria. It has been demonstrated, by challenge tests performed in CSS, that Lactobacillus sakei, Lb. alimentarius and Lb. farciminis grew faster than S. putrefaciens, P. phophoreum, B. thermosphacta and S. liquefaciens (Joffraud et al., 2006). 13.3.4  Spoilage potential of lactic acid bacteria (LAB) The use of LAB as a protective culture in seafood implies that they do not have any spoiling capacity. LAB have often been thought to play a minor role in the spoilage of marine products. They are not very competitive in refrigerated fresh fish and they produce fewer unpleasant odours compared to Gram-negative bacteria like S. putrefaciens, P. phosphoreum and Pseudomonas sp. (Leisner, 1992). Although dominant in LPFP, their role is not very clear and several authors have found no correlation between LAB and sensory spoilage (Leroi et al., 2001; Hildebrandt and Erol, 1988). However, Paludan-Müller et al. (1998) succeeded in increasing the shelf-life of CSS by inhibiting LAB with nisin, suggesting a possible spoiling effect of this bacterial group (for a review of the different characteristics of spoilage, the compounds responsible and their associated precursors, see Huss et al., 1995). Stohr et al. (2001) clearly showed that some Lactobacillus species found in CSS were very spoiling (Lb. sakei) while others had no effect (Lb. alimentarius). Lb. sakei generally produces sulphurous and acidic odours (Stohr et al., 2001; Nilsson et al., 1999), associated with the production of H2S, acetic acid and ethyl and n-propyl acetate (Joffraud et al., 2001), but some Lb. sakei strains do not affect the organoleptic quality of this product (Weiss and Hammes, 2006). Lb. alimentarius which does not spoil CSS has been identified as the bacterium responsible for the sensory deterioration of marinated herring (Lyhs et al., 2001). Carnobacteria are microorganisms resistant to freezing that grow very well at refrigerated temperatures, in all packaging conditions and in the presence of many preservatives (Laursen et al., 2005; Leroi et al., 2000), explaining why this genus is often found in refrigerated meat or fish products, but its role is still under discussion (Leisner et al., 2007; Laursen et al., 2005). Many studies show that the inoculation of CSS by various strains of C. maltaromaticum and C. divergens leads to few or no changes in organoleptic quality (Brillet et al., 2005; Nilsson et al., 1999). When the carnobacteria reach a high enough level, flavours of butter and plastic may be detected, associated with the production of 2,3-butanedione (diacetyl) and 2,3-pentanedione (Joffraud et al., 2001; Stohr et al., 2001) but are not sufficient for a trained panel to reject the product (Brillet et al., 2005). In contrast, strains of C. maltaromaticum and C. divergens inoculated into Arctic shrimp generated strong chlorine, malt, nuts and sour odours and the samples were judged unfit for consumption (Laursen et al., 2006). Ammonia and numerous alcohols, aldehydes and ketones were produced. Nevertheless, here again, there was variability depending on the strain. The interaction with other microorganisms should not be disregarded. In a sterile CSS model, Joffraud et al. (2006) have shown that the spoilage observed

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with Lb. sakei was weakened in the presence of S. liquefaciens even though the latter also had a spoiling effect in monoculture. On the other hand, some associations appear to be much more spoiling than in pure culture (Carnobacterium with Vibrio or B. thermosphacta) due to de novo synthesis of total volatile basic nitrogen (Brillet et al., 2005). Similarly, Laursen et al. (2006) showed that the unpleasant odours generated in cooked shrimp by an association of Carnobacterium sp. and B. thermosphacta were different from those due to these two bacteria in pure culture.

13.4 Bioprotective lactic acid bacteria, bacteriocins and bacteriophages for bacteria control The application of protective microorganisms, bacteriophages or bacteriocins on seafood products for biopreservation is generally less documented than in dairy or meat products. The main reason is probably that the early stages of biopreservation have occurred mainly in fermented foodstuffs that are not so developed among seafood products. Moreover, the selection of potential protective bacteria in seafood products remains a challenge since they must be adapted to the seafood matrix (poor in sugar) and their metabolic activities should not change the initial characteristic of the product, i.e. by acidification, and not induce spoilage that could lead to a sensory rejection. Among the microbiota identified in fresh or processed seafood, LAB remains the category that offers the highest potential for direct application as a bioprotective culture or for bacteriocin production. Most of the studies concern LPFP such as CSS and focus on the inhibition of L. monocytogenes (Table 13.2), considered as explained before as the main bacterial risk associated with the consumption of these products. The increase of knowledge about the microbial spoilage bacteria of those foodstuffs has also recently highlighted the new interest of bioprotective culture or biopreservatives to extend the sensory shelf-life of several LPFP. 13.4.1  Control of pathogenic bacteria Many studies concerning pathogenic bacteria inhibition have been performed in a liquid model medium, but the effects are not often confirmed in real products. In the following part of this chapter, we will focus on studies that have given successful results on seafood products. Control with protective cultures Among the LAB that were described before, strains belonging to the genus Carnobacterium have been particularly studied for their role as protective bacteria in CSS, probably because they are not acidic bacteria. They belong to the major LAB of such products at the end of storage and although their presence can sometimes be associated with spoilage activities on seafood products, in many cases they are not directly responsible for undesirable odours or flavours.

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Bacteriocinogenic Y/N (bacteriocin name) Y (carnobacteriocin B2) N Y (divercin V41)

Y (piscicocin V1a and V1b) Y (unknown) Y (piscicocin CS526) N

Protective strains (inoculum level)

Carnobacterium maltaromaticum A9b (106 CFU g–1)

Carnobacterium maltaromaticum A10a (106 CFU g–1)

Carnobacterium divergens V41 (105 CFU g–1)

Carnobacterium divergens V1 (105 CFU g–1)

Carnobacterium divergens SF668 (105 CFU g–1)

Carnobacterium maltaromaticum CS526 (104 or 106 CFU g–1)

Carnobacterium maltaromaticum JCM5348 (104 or 106 CFU g–1)

Products

Cold smoked salmon

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Initial level maintained during 24 days

Decrease until 102 CFU g–1 L. monocytogenes Vacuum packed 21 days after 12 days maintained (103 CFU g–1) during 24 days at 4 °C

Growth limitation of 1 to 3 log CFU g–1 at the end of storage period

<102 CFU g–1 maintained during 28 days

(Continued )

Yamazaki et al., 2003

Brillet et al., 2004

20 CFU g–1 maintained during 28 days

L. monocytogenes Vacuum packed 9 days (20 CFU g–1) at 4 °C and 19 days at 8 °C

Nilsson et al., 1999

Reference

103 CFU g–1 maintained during 31 days

Effect

Nilsson et al., 1999, 2004

Storage conditions

Initial level until 25 days L. monocytogenes Vacuum packed 32 days and decrease below (2 × 102 at 5 °C 1 CFU g–1 CFU g–1)

Target microorganisms (inoculation level)

Table 13.2  Applications of protective cultures for pathogenic bacteria control in seafood

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Cooked shrimp

Effect

L. innocua (104 CFU g–1)

Y (unknown)

N

Y (unknown)

Enterococcus faecium ET05 (ns)

Lactococcus piscium EU2241 (105 CFU g–1)

Leuconostoc gelidum EU2247 (105 CFU g–1)

S. aureus (103 CFU g–1)

Weiss and Hammes, 2006

Katla et al., 2001

Tahiri et al., 2009

Reference

Growth limitation of 1.5 log CFU g–1 after 21 days

Matamoros et al., 2009a

Vacuum Growth limitation of 2.5 log Tomé et al., packed 21 days CFU g–1 after 21 days 2008 at 5 °C

Vacuum Growth limitation of 1.5 log Vescovo et al., packed 30 days CFU g–1 after 30 days 2006 at 4 °C

Growth limitation of 2 log L. monocytogenes Vacuum packed 28 days CFU g–1 after 7 days and (103 CFU g–1) at 8 °C, then 7 during the storage period days at 20 °C

L. innocua (104 CFU g–1)

Lactobacillus casei T3 and Y (unknown) Lactobacillus plantarum Pe2 (106 CFU g–1)

Vacuum Growth limitation of 4 log packed 14 days CFU g–1 after 14 days at 4 °C

Decrease until 102 CFU g–1 in 28 days

Initial level maintained L. monocytogenes Vacuum packed 28 days during 28 days (103 CFU g–1) at 10 °C

L. innocua (104 CFU g–1)

Lactobacillus sakei 5754 (107 CFU g–1)

Storage conditions

Growth limitation of 3 log L. monocytogenes Vacuum packed 21 days CFU g–1 after 21 days (102 CFU g–1) at 4 °C

Y (unknown)

Lactobacillus sakei Lb790 (103 CFU g–1) + sakacine P (1.1 µg g–1)

Y (sakacin P)

Y (divergicin M35)

Carnobacterium divergens M35 (106 CFU g–1)

Cold smoked salmon

Lactobacillus sakei Lb790 (103 CFU g–1)

Bacteriocinogenic Y/N (bacteriocin name)

Protective strains (inoculum level)

Products

Target microorganisms (inoculation level)

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Moreover, several Carnobacterium strains are known to produce anti-listerial bacteriocins (Drider et al., 2006). Two strains of C. maltaromaticum isolated from CSS were evaluated for the inhibition of L. monocytogenes in VP CSS. Both strains demonstrated their efficiency to limit the growth of this pathogenic bacteria during 31 days of storage at 5 °C (Nilsson, 1999), but the growth of the protective Carnobacterium strains did not modify the sensory characteristic of the product. One of these strains showing the strongest inhibition activity produces a bacteriocin, named carnobacteriocin B2 that was involved in the anti-listerial activity (Nilsson et al., 2004). Three other strains of bacteriocin-producing Carnobacterium have been tested with the agar diffusion test method against a wide collection of L. monocytogenes (51 strains) isolated from seafood. All of the Listeria strains were sensitive. The inhibition was confirmed in co-culture with a mix of L. monocytogenes strains in sterile CSS (Brillet et al., 2004), and one of these strains, C. divergens V41 showed its ability to maintain L. monocytogenes at the initial inoculating level of 20 CFU g–1 during 28 days of storage at 4 °C and 8 °C. The effect of this strain on sensory characteristics and physico-chemical parameters revealed that it did not spoil the product (Brillet et al., 2005). In this case also, the inhibitory activity could be linked to the bacteriocin divercin V41, since a bacteriocin negative mutant failed to limit the growth of L. monocytogenes in the same conditions (Richard et al., 2003). In the presence of the bacteriocinogenic strain C. maltaromaticum CS526 isolated from surimi, the population of L. monocytogenes in CSS decreased from 103 to 50 CFU g–1 after 7 days at 4 °C (Yamazaki et al., 2003). This activity could be linked to the production of the bacteriocin piscicocin CS526, since a nonbacteriocin-producing strain had a lower effect on the growth of the pathogenic bacteria (Yamazaki et al., 2005, 2003). In another study, the application of C. divergens M35 towards L. monocytogenes in CSS resulted in a maximal decrease of 3.1 log CFU g–1 of the pathogenic bacteria after 21 days of storage at 4 °C whereas a non-bacteriocinogenic strain had no effect (Tahiri et al., 2009). Among the other LAB, Lb. sakei has also been used as a protective culture for L. monocytogenes inhibition on CSS and the strain Lb790 producing sakacin P was compared to a non bacteriocinogenic strain for the inhibition of L. monocytogenes. In both cases, no bactericidal effect was obtained but the growth of the pathogenic bacteria was stopped during 28 days at 10 °C (Katla et al., 2001). Another bacteriocinogenic strain of Lb. sakei isolated from CSS allowed a 4 log reduction of Listeria innocua after 14 days of storage at 4 °C. A reduction of 2 log units after 24 hours at 5 °C was also demonstrated with that strain in CSS juice towards L. monocytogenes (Weiss and Hammes, 2006). A mix of bacteriocin-producing LAB like Lb. casei, Lb. plantarum and C. maltaromaticum was successfully used to limit the growth of L. innocua in CSS (Vescovo et al., 2006). In their study Tomé et al. (2008) have also selected a strain of Enterococcus faecium among five bacteriocinogenic LAB strains for its ability to induce a decrease of the population of L. innocua inoculated in CSS. However, in these studies the inhibition activities were not confirmed on L. monocytogenes.

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Protective cultures have not been applied in many other seafood products except for CSS and L. monocytogenes control. Matamoros et al. (2009a) have performed challenge-tests in cooked shrimp stored under VP using protective LAB and L. monocytogenes and S. aureus as target pathogens. Two LAB strains, Lactococcus piscium EU2241 and Leuconostoc gelidum EU2247, were efficient to limit the growth of both pathogenic bacteria from 2 to 3 log CFU g–1 units after 4 weeks at 8 °C followed by 1 week at 20 °C. The strain of Leuconostoc produced a bacteriocin-like compound but its activity was slightly lower than the Lactococcus strain that was non-bacteriocinogenic. Control with bacteriocins Most of the applications of protective LAB for the control of pathogens have been conducted with bacteriocinogenic strains isolated from seafood products. Some of these bacteriocins have been purified and characterized, in particular those produced by carnobacteria, and they are listed in Table 13.3. Divercin V41 is produced by C. divergens V41 (Métivier et al., 1998) and piscicocin V1a and V1b are two bacteriocins produced by the same strain, C. maltaromaticum V1 (Bhugaloo-Vial et al., 1996). Divercin V41 is closed to divergicin M35, another class IIa bacteriocin that is produced by a C. divergens strain isolated from frozen smoked mussels (Tahiri et al., 2004). Piscicocin V1a, piscicocin V1b and the bacteriocin produced by C. maltaromaticum A9b (Nilsson et al., 2004) are peptides that have also been characterized from other C. maltaromaticum strains isolated from meat or cheese (Leisner et al., 2007). All these bacteriocins belong to class IIa of antilisterial bacteriocins that are heat stable peptides with low molecular weight (<10 KDa) (Drider et al., 2006). Piscicocin CS526 produced by C. maltaromaticum CS526 isolated from surimi is also considered as a class IIa bacteriocin although it possesses an alternate residue in the N-terminal consensus motif shared by these peptides (Yamazaki et al., 2005). Carnocin UI49 is the only class I bacteriocin that has been characterized from LAB isolated from seafood products (Stoffels et al., 1992). Recently, Pinto et al. (2009) have described two bacteriocins produced by strains of Enterococcus faecium and Pediococcus pentosaceus isolated from non fermented shellfish. These peptides were similar to the well-known class II bacteriocins enterocin B and pediocin PA-1. These last studies suggest that the specificity of bacteriocins is not linked to LAB origin but more likely connected to the bacterial species. A new peptide showing no similarity with other known bacteriocins has lately been characterized. It is called Weissellicin 110, produced by a strain of Weissella cibaria that comes from a traditional fermented fish product from Thailand, however its amino acid sequence has not been totally determined yet (Srionnual et al., 2007). Among the peptides that have been described above, very few have been applied directly on seafood products for pathogenic bacteria control. Since bacteriocin purification techniques allowing the recovery of high amounts of peptides in water or salt solution are usually not available, most of the studies use bacteriocin containing supernatant or semi-purified fractions. Crude extract of culture

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Smoked salmon Fish viscera

C. maltaromaticum A9b

C. maltaromaticum V1

C. divergens V41 C. divergens M35 Carnobacterium sp. Weissella cibaria 110

Bacteriocin A9b

Piscicocin V1b

Divercin V41 Divergicin M35 Carnocin UI49 Weissellicin 110

Bac ALP7 Bac ALP57

Surimi Fish viscera

C. maltaromaticum CS526 C. maltaromaticum V1

Piscicocin CS526 Piscicocin V1a

Fish viscera Frozen smoked mussels Fish Plaa-som, fermented fish product Shellfish Enterococcus faecium ALP7 Pediococcus pentosaceus ALP57 Shellfish

Strain origin

Producing strain

Bacteriocin name

Table 13.3  Bacteriocins from lactic acid bacteria isolated from seafood

Enterocin B Pediocin PA-1

Piscicolin 126 (Leisner et al. 2007) Carnobacteriocin B2 (Leisner et al. 2007) Carnobacteriocin BM1 (Leisner et al. 2007)

Synonyms

Pinto et al., 2009 Pinto et al., 2009

Metivier et al., 1998 Tahiri et al., 2004 Stoffels et al., 1992 Srionnual et al., 2007

Bhugaloo-Vial et al., 1996

Nilsson et al., 1999, 2004

Yamazaki et al., 2003, 2005 Bhugaloo-Vial et al., 1996

Reference

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supernatant containing piscicocins or divercin V41 was added on CSS to limit the growth of L. monocytogenes during 21 days of storage at 4 °C and 8 °C (Duffes et al., 1999). In both cases, the bacteriocins showed a rapid bactericidal effect after three days and during the first week of storage. However this effect tended to disappear after ten days on the opposite with the constant bacteriostatic effect obtained with the protective strain. Nilsson et al., 1999 made the same observations with semi-purified carnobacteriocin A9b in CSS inoculated with L. monocytogenes. Recently, Tahiri et al. (2009) have also shown that the application of non-purified or purified divergicin M35 on CSS inoculated with L. monocytogenes resulted in more rapid but less pronounced reduction of the pathogenic bacteria counts compared to the producing strain. In the case of Lb. sakei Lb790, addition of nearly pure sakacin P to the protective culture carried out an immediate bactericidal effect with no re-growth of the pathogenic bacteria during storage, leading to a more efficient effect than with the protective culture alone (Katla et al., 2001). Some attempts with commercial bacteriocins like nisin were made to limit the development of L. monocytogenes in CSS (Nilsson et al., 1997). However the growth was only delayed and the final population was similar to that obtained in the control at the end of the storage. In the same way, the results obtained with nisin or pediocin ACCEL to control the growth of L. monocytogenes on cooked fish showed a limited and short effect (Yin et al., 2007). It is likely that high and buffered pH usually encountered in fish products is not suitable for nisin solubility and activity. Control with bacteriophages Although bacteriophages were proposed for several applications in food safety to control the major pathogenic bacteria (Garcia et al., 2008) the only application in seafood is reported by Guenther et al. (2009) in smoked salmon and mixed seafood contaminated with two different L. monocytogenes strains at a level of 103 CFU g–1. The best results were an inhibition by 2 log CFU g–1 during 6 days of storage in mixed seafood but this effect was variable considering the product and the L. monocytogenes strain used (Guenther et al., 2009).

13.4.2  Control of spoiling microorganisms Control with protective culture Less information is available in this field, since the microbiotica involved in the spoilage activity of seafood product is complex and in most of the cases has not been characterized. The activity of protective culture or bacteriocin is thus directed on the increase of sensory shelf-life, or the inhibition of common microbial indicators such as total viable count or LAB. The Carnobacterium species that were described above for the inhibition of L. monocytogenes do not seem to offer great potential in extending the shelf-life of seafood products. Leroi et al. (1996) succeeded in increasing the sensory use-by-date of CSS slices by inoculating them with strains of Carnobacterium sp. However the results varied depending on the batch treated. For Brillet et al. (2004) no effect of C. divergens V41 was

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recorded on the spoilage bacteria of CSS and this strain did not improve its organoleptic properties. Using a strain of C. maltaromaticum, Paludan-Müller et al. (1998) only slightly extended the shelf-life of smoked salmon. The application of the protective strain C. divergens M35 had no significant effects on the total microorganisms of CSS during storage at 4 °C for 21 days (Tahiri et al., 2009). Similarly, C. maltaromaticum had no effect on the inhibition of the Gram positive spoilage bacteria B. thermosphacta in cooked shrimps (Laursen et al., 2006). These studies suggest that selection of protective strains to improve the sensory quality of seafood products should focus on specific spoilage microorganism’s inhibition. This approach was chosen by Matamoros et al. (2009b) who have isolated seven strains from various marine products on the basis of their activity against many spoiling and pathogenic Gram-positive and Gram-negative marine bacteria. Among these strains, two Le. gelidum, and two Lc. piscium demonstrated promising effect in delaying the spoilage of tropical shrimp and of VP CSS. A recent study demonstrated that this protective effect could be due to the inhibition of B. thermosphacta identified as one of the major spoiler organisms in cooked shrimp stored under MAP (Fall et al., 2010). One of the strain of Lc. piscium was able to limit by 4.1 log CFU g–1 the growth of this target bacteria and thus to avoid the apparition of undesirable odours in the products. Altieri et al. (2005) also succeeded in inhibiting Pseudomonas sp. and P. phosphoreum in VP fresh plaice fillets at low temperatures with a strain of Bifidobacterium bifidum. In the same field, some attempts have been made to select microorganisms that are able to limit the growth of histaminogen bacteria or to induce biogenic amine degradation. In seafood, biogenic amines are usually produced by spoiling microorganisms like P. phosphoreum or enterobacteria like Morganella morganii or M. psychrotolerans (Emborg and Dalgaard, 2006). They are often used as indirect spoilage indicators, signalling the presence of these bacteria, and have thus been included in some models proposed to predict the spoilage of marine products (Jorgensen et al., 2000; Veciana-Nogues et al., 1997). Moreover, histamine is also responsible for food poisoning often linked to seafood consumption (Emborg and Dalgaard, 2006). The studies concerning biogenic amine degrading bacteria mainly focus on fermented fish. A strain of Staphylococcus xylosus showed its ability to degrade histamine and tyramine in salted and fermented anchovy (Mah and Hwang, 2009). In the same way, mixed starter cultures of Lb. plantarum, Lb. casei, Pediococcus pentosaceus and S. xylosus were efficient to limit accumulation of histamine, tyramine, cadaverine, putrescine and tryptamine in silver carp sausages (Yongjin et al., 2007). Control with bacteriocins As most of the bacteriocins produced by LAB isolated from seafood products are active against Gram-positive bacteria only, their relevance in preventing spoilage activities that are usually linked to various Gram-negative and Gram-positive bacteria is limited. The effect of nisin and pediocin ACCEL was observed on total viable counts of fresh fish fillets at 0 °C and 4 °C (Yin et al., 2007). The growth of the total microbiota was slightly delayed when the bacteriocin were present in the samples

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stored at 0 °C but the bacterial counts reached the same level in the non-treated control after 5 days of storage. In brined shrimp stored at 4.5 °C, nisin Z was more efficient to extend the microbial shelf-life than other tested bacteriocins (Einarsson and Lauzon, 1995). However, this effect was limited compared to the use of food additives such as benzoic or sorbic acids that are usually found in such products.

13.5  Industrial application Unlike meat or dairy products, seafood products are mainly non-fermented. Therefore the addition of the bacterial cultures concept, even with protective effects, is new and probably not totally yet accepted by seafood producers, for which the main goal is to avoid bacterial contamination by the use of good hygienic practices. However, in LPFP, the use of protective culture is gradually considered as an alternative to the use of food additives and it is gaining interest in the seafood industry. Industrial starters like SafePro® (CHR Hansen, DK), Bovamine Meat Culture™ (NPC, US), HOLDBAC™ (Danisco, DK) have been developed for L. monocytogenes control for the meat industry, but to our knowledge they are not used in seafood products. Some patents claimed the usefulness of LAB for the treatment of food without seafood specification (Nauth and Zheng, 2006; Stiles et al., 2005; Fliss et al., 2004). However, one of them mentions the application of protective culture for seafood (Daniel and Lorre, 2001). This starter, named LLO, is applied in France for extending the shelf-life of cooked shrimp stored under MAP (Meyer, 2005) and has also showed limitation of histamine production in tuna stored at 5 °C (http://www.bioceane.com/uk/pdf/ ferment_histamine.pdf, accessed 25 January 2010). Concerning bacteriophages, the preparation LISTEX™ P100 was approved by the FDA for all food products (http://www.ebifoodsafety.com/en/news-2007. aspx, accessed 25 January 2010), but no studies are available concerning its efficiency in seafood.

13.6  Future trends The presence of LAB in many processed seafood products is now well established and although some strains are sometimes involved in spoilage, the bioprotective potential of many strains has been highlighted in recent years. The results obtained with protective culture, bacteriocins and bacteriophages for improving safety and quality of seafood products are at this moment in favour of the use of live cultures that seem to be more efficient than bacteriocin during the long storage period on these foodstuffs. Nevertheless, some fields such as bacteriophages application or control of spoilage microorganisms still have to be more fully explored in marine products. Control of pathogenic bacteria has widely focused on L. monocytogenes, considered as the main risk in ready-to-eat seafood. However, in these minimally

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processed products, the new combination of hurdles like low salt content, coupled to MAP can give selective advantages to other pathogenic bacteria like clostridia, vibrio or staphylococci that should also be addressed in biopreservation studies. It is also important to note that most of the studies are done in challenge-test where sometimes only one strain of the pathogenic bacteria is used as the target. The applications of protective cultures, bacteriocins or bacteriophages in naturally contaminated products or using mixed strains of target pathogenic microorganisms isolated form seafood should improve the certainty of reproducing the results in real industrial products. Concerning control of sensory quality, identification of the specific spoilage microorganisms for the different products is required to select appropriate bioprotective solutions. LPFP spoilage is often due to a complex association of Gram-positive and negative bacteria that still needs to be explored. The use of combinations of protective cultures with different antimicrobial spectra to master both pathogenic and spoiling bacteria is an exciting challenge for the future. Although many protective strains have shown their efficiency to control L. monocytogenes in CSS without any sensory modification, industrial applications delay development. A brake on expansion is that CSS is a traditional product that benefits from a high quality image by the consumers, so producers are not yet ready to communicate on other ingredients than salt and smokewood. For the moment the QPS status is not required for these strains as far as the European regulation concerning addition of protective cultures in unfermented ready-to-eat food is still under discussion. In 2007, the European Food Safety Authority (EFSA) adopted guidelines for Qualified Presumption of Safety (QPS) that can be referred to as the European equivalent of the American GRAS status in terms of risk assessment (EFSA, 2007). QPS is a generic approach to the safety assessment of microorganisms used in food or feed. A list of 47 LAB species has been published in the EFSA journal (EFSA, 2008), but LAB species that gave promising results in seafood are not included in this list, which is supposed to be updated annually. Proof of their beneficial effect, precise taxonomic data and strong evidence of safety will probably be needed for obtaining the QPS status if notified to EFSA. However, with the increasing market of LPFP, the request for alternative preservation solutions and the intensification of research, there is no doubt that biopreservation of fisheries products will expand in the future. Additional research work is therefore needed in the selection of appropriate strains and their combinations to limit the growth of both pathogenic and spoilage flora; understanding of the inhibition mechanism to optimise their activity in the products; and characterization and safety aspects of the cultures or their metabolites to obtain the QPS status.

13.7  Sources of further information and advice Borresen, T. (ed.) (2008), Improving Seafood Products for the Consumer. Cambridge, Woodhead Publishing Limited.

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Information on QPS status: http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812 _1178620763335.htm (accessed 25 January 2010). Information on French application of protective culture for seafood products: http://www. bioceane.com/uk/index.htm.

13.8  References altieri c , speranza b , del nobile m a

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14 Microbial applications in the biopreservation of cereal products G. Font de Valdez, G. Rollán, C. L. Gerez and M. I. Torino, Centro de Referencia para Lactobacilos (CERELA)-CONICET, Argentina

Abstract: Microorganisms play a central role in the manufacture of nutritious fermented foods and beverages. They are also of importance in the maintenance of food storage, food safety, and quality. Cereals and cereal-based products are usually spoiled by bacteria and moulds resulting either in unpleasant texture, smell and odour, or in toxic or poisoning products. The food preservation mediated by microorganisms and fermentation technologies has been attributed to the production of metabolites such as organic acids, hydrogen peroxide, alcohols and antibiotics. Antibiosis by bacteriocins offers potential applications in food biopreservation, while the use of enzymes derived from phages (i.e. enzybiotics) remains as a promising option. Key words: bioconservation, starter culture for fermentation, bioactive compounds as additives, enzybiotics in bakery.

14.1  Introduction Cereals and cereal foods have great significance in human progress because of their impact on world agriculture, economy and nutrition. In industrialized countries, where animal proteins are mainly consumed, cereals are increasingly used as animal feed. Hence the ensilage of crops (maize, wheat, sorghum and barley) offers an attractive method of preservation for ensuring a regular supply of high quality forage to animals (Wasaya et al., 2008). Cereal fermentation represents the major technological process applied to cereals for human nutrition; the volume of fermented cereals surpasses those obtained from milk, meat, fish, soy, olives, or cabbage (Hammes et al., 2005). Traditionally, the primary purpose of fermenting cereals was to achieve a preservation effect through the conditions generated by the fermentation to ensure the shelf-life and microbiological safety of the products. Nowadays, most cereal 348 © Woodhead Publishing Limited, 2011

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fermentations involve controlled bio-processes with selected starter cultures and adjusted environmental conditions. Nevertheless, traditional foods and beverages from cereal fermentation (e.g. bread and beer) are still produced in an artisanal way in many parts of the world (Hammes et al., 2005). In several countries, the production of regional cereal fermented foods (e.g. ogi, cassava, bogobe, kisra, burukutu) results in sustainable activities for the local population as well as revalorization of their culture and indigenous microflora whose contribution in the corresponding fermentative processes has not yet been elucidated. The quality and safety of cereal grains and cereal-based food are affected by fungi and bacteria contamination, causing economic losses and health risks to consumers. The biopreservation – defined as the extension of food shelf life and/or food safety by using biological systems and/or their antimicrobial compounds – is an interesting tool to avoid these harmful effects, either alone or together with traditional, physical and chemical preservation methods (Stiles, 1996; Brijesh et al., 2008). For beverage and cereal-based foods, bio-preservative strategies are mainly based on the use of microorganisms (e.g., lactic acid bacteria) as starter or adjunct cultures that produce inhibitory metabolites in situ during fermentative processes, and on the employment ex situ of purified antimicrobial compounds that are added as a food additive (e.g. nisin). The novel phage-based methods developed for some food systems have not yet been evaluated in cereal foods. This approach could be promising for fresh pasta and regional fermented cereals.

14.2  Cereals in human nutrition and animal feed The Food and Agriculture Organization (FAO) reported a world cereal production of 2.192 million tons in 2008, wheat, rice, maize and barley being the major cereal crops produced. Accordingly, the usage of all cereals is about 4% for seed, 81% for human food and animal feed, and 15% for other industrial applications (FAO, 2008; Hammes et al., 2005). Nutritionally, cereals are an important source of dietary protein, carbohydrate, fat and fiber (macronutrients) that are required for growth and maintenance. They also supply important minerals (iron), vitamins (E and B-complex) and other micronutrients, essential for optimal health. Since the 1970s, cereals have provided about 50–60% and 30–35% of the daily calories consumed per capita in developing and industrialized countries, respectively (FAO, 2003). Cereals have multiple beneficial effects that can be exploited to design novel cereal-based foods addressed to target specific populations such as children and the elderly, e.g. ready-to-eat breakfast cereals, cereals with freeze-dried fruits for lowering the sugar content in the diet, and whole-grain cereals plus non-fat milk to replace energetic sport drinks. Cereals play a significant role as energy and protein source in animal diets (pigs, chickens, bovine livestock, etc). As nearly half of the world’s cereal production is used to produce animal feed, the dietary proportion of meat (but also

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milk and eggs) has a great influence on food demand. With meat consumption projected to be over 52 kg/person/year by 2050, cereal requirements for more intensive animal feed and meat production may increase substantially to more than 50% of total cereal production (Nellemann et al., 2009).

14.3  Major contaminant agents in cereal-based products Cereal grains, factories and manufacturing utensils are hosts to a large undesirable microflora that includes spoilage and pathogenic organisms, which affect the quality and safety of cereal-based products. The most important contaminants are fungi, e.g. species of Aspergillus (A.), Penicillium (P.) and Fusarium (F.) (Keshri et al., 2002). These fungi are ubiquitous spore-formers and their widespread spores germinate under suitable environmental conditions. In baked goods, spores in the factory environment are one of the major sources of contamination during the post-baking cooling stage (Knight and Menlove, 2006). In addition to the great economic losses due to the presence of moulds, another concern is the potential production of mycotoxins that may cause public health problems (Legan, 1993). Filamentous moulds and yeasts are common spoilage organisms of fermented cereal products such as bread, stored crops and feed such as silage (Bonestroo et al., 1993). Richard et al. (2007) confirmed in maize silage the presence of six Fusaria (F. culmorum, F. equiseti, F. graminearum, F. oxysporum, F. solani and F. verticillioides) and one Aspergillus (A. fumigatus) species, which produced mycotoxins in vitro. Three mycotoxins (citrinin, gliotoxin and deoxynivalenol) were detected in the silage, the strongly immunosuppressive gliotoxin produced by A. fumigatus being detected at a particularly high concentration (877 ppm). Bacillus (B.) subtilis is a well known spoilage bacteria causing ropiness in bread that deteriorates the bread texture due to slime formation (Valerio et al., 2008) and spores can survive the baking process where the temperature in the centre of the crumb reaches 97–101 °C. The presence of B. subtilis can be detected early by the development of odour similar to pineapple; later, the crumb becomes discoloured, soft and sticky, making the bread inedible. Ropiness can develop very rapidly under warm and humid conditions, so it is a common problem in Mediterranean countries, Africa and Australia (Voysey and Hammond, 1993). Another bacterium giving health concern is B. cereus, a food-borne spore-forming pathogen that often contaminates fermented and fresh cereal-based products, especially pasta and rice (Fang et al., 2003). Escherichia (E.) coli (a normal inhabitant of the gut), Salmonella (S.) typhi (a bacteria that is transmitted through contaminated poultry, eggs and other foods), and Staphylococcus (Staph.) aureus are other frequent contaminants in these products (Frazier and Westhoff, 1986). Raw ingredients and their manipulation during processing are the primary sources of contamination in fresh pasta, and due to their high water content (30% maximum) fresh pasta are susceptible to bacteria, yeast, and mould contamination. The shelf-life of fresh pasta is essentially influenced by both the microbial counts

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in products at the end of the process, and the efficacy of methods used to control their proliferation (modified atmosphere of packaging, cooling). Indeed, a high number of four spoilage microbial groups (mesophilic and psychotropic bacteria, coliforms and staphylococci) was found in amaranth-based homemade fresh pasta after 5 days storage at 4 °C (Del Nobile et al., 2009). The health concern is the production of heat-resistant toxins, e.g. those produced by B. cereus and Staph. aureus, which are not destroyed by the pasta cooking and are the main cause of the reported outbreaks attributed to fresh and fresh-filled pasta consumption (Giannuzzi, 1998). This kind of food has also been associated with outbreaks of salmonellosis (Bryant et al., 2001). In Argentina, a high proportion (88%) of fresh spaghetti samples, elaborated either with liquid or dried eggs, was contaminated with Salmonella spp. (Cortinez et al., 1988) and 239 episodes of food-borne diseases due to S. enteritidis were registered between 1986 and 1988 due to raw egg contamination (Eiguer et al., 1990).

14.4 Fermentative technologies as a tool for microbial biopreservation To improve the hygienic and healthy state of foods, several methods of sanitation and decontamination have been developed. In the past decade, the concept of biopreservation has been revalorized since it offers several benefits such as extended shelf-life, decreased risk for transmission of food-borne contaminants, less economic losses, and reduced application of chemical preservatives, thus responding to the consumer’s demands for minimally-processed foods and for ready-to-eat foods (Thomas et al., 2000; Robertson et al., 2004). Fermentation is one of the oldest and most economic methods for producing and preserving food (Motarjemi, 2001). Most cereal-based foods are subjected to at least one fermentation step involving endogenous enzymes, bacteria, yeast and moulds, which play key roles (either alone or in combination) for the creation of a great variety of products. Organic acids, peroxides, peptides (e.g. bacteriocins), volatile compounds and alcohols are some of the major inhibitory metabolites produced by the complex microflora developed during the manufacturing process of fermented cereal foods. According to fermentation parameters such as temperature and presence of alternative electron acceptors, e.g. fructose or citrate, some microbial groups are favoured, as also is the compound produced (Van der Meulen et al., 2007). Lactic acid bacteria (LAB) are of utmost importance in food fermentations including cereal products; the end products produced from carbohydrate catabolism contribute not only to the preservation of the raw matter but also to the development of flavour and texture of the food which acquires unique characteristics and properties. Lactic acid fermentation is an ancient method for food and feed preservation during which spoilage and pathogenic organisms such as yeasts, moulds, enterobacteria, bacilli or clostridia are inhibited by the conditions developed throughout fermentation, i.e. decrease in pH, low oxidation reduction potential, and competition for essential nutrients (Bonestroo et al., 1993).

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14.4.1  Natural fermentations occurring in cereals Fermentation is probably the best method to improve the nutritional quality of most common cereals such as rice, wheat, maize and sorghum. The process leads to a general improvement of the shelf-life, texture, taste and aroma of the final product. Some fermented cereal-based products are used as beverages, breakfast and light meal foods, but a few of them are also used as main foods in the diet. In most of these products the fermentation is natural and involves a mix of yeasts, bacteria and fungi, usually present in raw materials such as flour. The common fermenting bacteria are species of Leuconostoc, Lactobacillus, Streptococcus, Pediococcus, Micrococcus and Bacillus while among fungi, Aspergillus, Paecilomyces, Cladosporium, Fusarium, Penicillium and Trichothecium are the most frequent genera found. Species of Saccharomyces are the main fermenting yeasts used in alcoholic fermentation of cereal based beverages (Steinkraus, 1998). Bacterial flora developed during food fermentation depend on the water activity, pH, salt concentration, temperature and the composition of the food matrix. Most fermented foods (including those commonly consumed in the Western world as well as less characterized indigenous fermented foods) rely on LAB to mediate the fermentation process. Major naturally fermented cereal-based products are classified according to the main raw constituent and described in detail by Blandino et al. (2003). Sourdough Originally, sourdough was produced by fermentation of flour and water with natural occurring flora, yeasts and LAB representing the prevailing microorganisms in a 1:100 ratio approximately (Gobbetti et al., 1994). The main genera of LAB isolated from sourdough are Lactobacillus, Leuconostoc, Pediococcus and Weissella, and the majority of the strains belong to the genus Lactobacillus (Ehrmann and Vogel, 2005; De Vuyst and Neysens, 2005), while yeasts found in sourdoughs belong to more than 20 species (Gullo et al., 2002). The frequently dominant one, Saccharomyces (S.) cerevisiae (Vernocchi et al., 2004) is often introduced through the addition of baker’s yeast (Corsetti et al., 2001). Sourdoughs, on the basis of the technology applied, have been grouped into three types (Böcker et al., 1995). Type I sourdoughs are produced by traditional techniques with a daily dough refreshment to keep the microorganisms in an active state; Type II are often used as dough-souring supplements during bread preparation (these kind of sourdoughs are semi-fluid and characterized by long fermentation periods); and Type III sourdoughs are industrialized dried preparations (Hammes and Gänzle, 1998). Unlike Type I, the Types II and III sourdough require the addition of baker’s yeast (S. cerevisiae) as a leavening agent. Fermentation by LAB improves shelf-life of sourdough-based products (Hammes and Gänzle, 1998; Gänzle et al., 2007; Gerez et al., 2009) since during sourdough fermentation, the prevailing LAB produce organic acids (mainly lactic acid and acetic acid but also phenyllactic, formic and butyric acids), ethanol, volatile compounds and peptides (e.g., bacteriocins) (Corsetti et al., 1998; Leroy and De Vuyst, 2004; Gänzle et al., 2007). These metabolites exhibit variable

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degrees of activity against contaminant bacteria, moulds and yeasts. The selection of antimicrobial lactic starter cultures from natural sourdough, the identification and characterization of the inhibitory metabolites and their demonstration in food systems will allow developing defined preservative cultures either as starter or adjunct cultures. Regional products Some features linked to indigenous cereal fermentations that resulted in a traditional activity sustainable for local population, are: integration of production and consumption processes into familiar and village life, utilization of inexpensive locally produced raw materials, and manufactured products with improved sensorial properties (Haard et al., 1999). Nevertheless, this traditional practice affects to a certain extent the nutritional value and safety of foods due to uncontrolled and unhygienic conditions. Most of these fermented cereal-based foods are produced in Africa and Asia, e.g. soy sauce and miso in Southeast Asia; idli and dosa in India; kishk in the Middle East (Campbell-Platt, 1987). Bouza and kishk (derived of wheat); busa and sake (rice); kenkey (maize); kwunu-zaki (millet); pito and ogi (maize, sorghum); and bogobe, kisra, burukutu, which derive from sorghum, are examples of important indigenous fermented foods produced in African countries (Caplice and Fitzgerald, 1999; Blandino et al., 2003). In America, maize is the principal raw source used for fermented cereal food such as tesgüino (alcoholic beverage from Mexico), chicha (spongy solid from Perú) and jamin-bang (bread from Brazil) (Blandino et al., 2003). The microbiology of many of these fermentations is complex and many indigenous cereal fermentations involve the contribution of bacteria, yeasts and fungi. LAB are believed to have a key role, and they are inoculated to cereals mix in the form of a homemade starter (e.g. yogurt, fermented milk, curdled milk, cheese, etc.) and left to ferment. Thus, a lactic fermentation takes place resulting in organic acid production with preservative effect. Nevertheless, two LAB strains (Lb. plantarum KKY12 and Lb. casei OGM12) isolated from ogi (Nigeria), produce bacteriocins (antimicrobial compounds) that inhibit pathogen bacteria in vitro (Olasupo et al., 1995). Tarhana and kishk which are two important families of fermented cereal food produced in Turkey, Egypt, Bulgaria, Greece, Iran, etc., can be used as typical examples. Tarhana includes dried acid doughs commonly consumed as soup, with a characteristic sour taste containing lactic acid and other compounds. The combined organic acids in tarhana fermentation ensure the microbiological safety of the product (Erbas et al., 2006). Besides the low pH (3.4–4.2) reached, the drying step reduces the moisture content to 6–10%, resulting in a protective environment to spoilage and pathogenic microrganisms such as E. coli and Staph. aureus. Silage In animal feed, silage of maize, wheat and sorghum is the best method for forage conservation keeping it in a fresh condition and with a high nutrient value

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(Filya et al., 2004; Nishino et al., 2004; Steidlova and Kalac, 2004). One of the technological strategies applied to enhance this practice includes compression of forage during silage followed by airtight sealing. In this way, the natural lactic microflora present on crops ferment free sugars with production of mainly lactic and acetic acids by homo and heterofermentative lactobacilli. This results in pH reduction and preservation of ensiled forage against undesirable microorganisms growing under anaerobic conditions, e.g. coliforms and Clostridium tyrobutyricum. To control the growth of yeasts and moulds in the opened silo at feeding time, high amounts of selected lactic cultures are inoculated at the beginning of the fermentation process in the silage (Holzer et al., 2003).

14.5  Production in situ of antimicrobial compounds 14.5.1  Antifungal metabolites Lactic acid bacteria have been exploited for decades for their antibacterial activity. More recently they have also received scientific attention because of their antifungal potential in food preservation (Magnusson et al., 2003; Sjögren et al., 2003; Hassan and Bullerman, 2008) since LAB strains isolated from cereals with antifungal properties have been reported (Corsetti et al., 1998; Lavermicocca et al., 2003; Rouse et al., 2008; Gerez et al., 2009). However, the application of these particular LAB cultures in cereal-based food is still limited despite the advances made on the characterization of their antifungal metabolites regarding molecular weight, heat-resistance, spectrum of action and effectiveness. The inhibition of the outgrowth of Fusarium spp. by strains of Lb. plantarum added into wheat bread was reported (Dal Bello et al., 2007). The metabolites responsible for the antifungal activity were lactic acid, phenyllactic acid, and two cyclic dipeptides: cyclo (L-Leu–L-Pro) and cyclo (L-Phe–trans-4-OH-L-Pro). The combination of these Lb. plantarum strains together with 0.3% calcium propionate, a chemical preservative frequently used in bakery, showed a strong synergistic antifungal effect increasing the shelf-life of bread (Ryan et al., 2008). Gerez et al. (2009) reported that the inclusion of antifungal strains of Lb. plantarum allowed reducing by 50% the concentration of calcium propionate (2 g/kg of flour) in packed wheat bread to attain a shelf-life of nine days. The addition of the starter culture also improved the fermentation quotient (lactate/ acetate molar ratio = 2.0) and the leaving volume (80 cm3) of the wheat doughs. The authors claimed that the major antifungal compounds were acetic and phenyllactic acids; however, the effectiveness against Aspergillus, Fusarium and Penicillium was dependent both on the LAB strain and the pH reached after wheat dough fermentation. The effect of antifungal LAB (as a mixed starter culture LAB-yeast) has also been demonstrated in sour-maize bread, but it was mainly attributed to the presence of antifungal peptides rather than to the decrease in pH after dough fermentation (Edema and Sanni, 2008). Recently, Gerez et al. (2010) developed a ready-to-use antifungal starter culture that improved the shelf-life of packed bread due to the production of

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acetic, phenyl lactic acid as well as high levels of lactic acid, which lowered the pH of the dough, favouring the un-dissociated fraction of the organic acids. The use of antifungal LAB has been investigated not only for bakery-products, several studies have been conducted to examine the antifungal properties of LAB against fungal contaminations in malting (production of beer) as an effective and natural option to reduce the deleterious effects of some microorganisms during steeping. LAB reduce the fungal contamination and the formation of mycotoxins leading to a higher quality malt regardless of the natural variations of the barley flora (Laitila et al., 2002; Rouse et al., 2008). 14.5.2  Bacteriocins Biochemical and genetic characteristics of bacteriocins as well as applications of bacteriocin-producing LAB either as starter or adjunct cultures in fermented food preservation, have been extensively reviewed (Cotter et al., 2005; Fimland et al., 2005; Deegan et al., 2006; Drider et al., 2006; Gálvez et al., 2007). Bacteriocins produced by sourdough LAB have been purified and well characterized with regards to their in vitro activity, for example plantaricin ST31 produced by Lb. plantarum ST31 (Todorov et al., 1999), bavaricin A produced by Lb. bavaricus MI401 (Larsen et al., 1993) and amylovorin L471 produced by Lb. amylovorus LMG P-13139 (De Vuyst et al., 2004). The in situ bacteriocin activity of amylovorin L471 was detected in rye dough fermented with Lb. amylovorus LMG P-13139 (an obligate homofermentative strain) fermentation being apparently dominated by the starter lactic strain (De Vuyst et al., 2004). This strain contributed to a controlled growth of the cereal microflora during cereal fermentation. Similar results were reported for Lb. plantarum ST31, a strain producing plantaricin ST31, when used as starter culture in wheat sourdough (Todorov et al., 2003). Corsetti et al. (2004) reported antimicrobial compounds produced by rye sourdough lactobacilli, which displayed characteristics similar to bacteriocins and were hence designated as bacteriocin-like inhibitory substances (BLIS). Lactococcus lactis subsp. lactis M30, isolated from unmalted barley, produced the BLIS lacticin 3147 which showed activity in the wheat sourdough ecosystem during a 20-day period of sourdough propagation. Both the stability of L. lactis M30 and the absence of interference with the starter strain Lb. sanfranciscensis CB1 were demonstrated (Settanni et. al., 2005). Bacteriocin-producing Lactobacillus strains were also used as starter culture in African fermented cereal-based foods (ogi and its solid form agidi), allowing control of spoilage and safety, and waste reduction (Olasupo et al., 1997). Lb. plantarum KKY12 and Lb. casei OGM12, both strains isolated from ogi (a Nigerian fermented product), produced plantacin N and caseicin A, respectively. These compounds were active against Pseudomonas (Ps.), Aeromonas (A.) sobria and A. cavice (plantacin N), and enterotoxigenic E. coli and Vibrio cholerae (caseicin A) in assays in vitro (Olasupo et al., 1995). The inhibition of E. coli and a shelf-life of 11 days were obtained in African fermented cereal-based products

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containing these bacteriocin-producing strains. Inhibition of an enterotoxigenic E. coli strain was observed within two hours of incubation in ogi fermented with starter bacteriocin-producing Lb. casei OGM12 (Olasupo et al., 1997). These results showed the potential of bacteriocin-producing cultures in the control and retardation of spoilage and food-borne infections in some African fermented foods.

14.6 Microbial metabolites used as additives in cereal biopreservation The Codex of General Standard for Food Additives (GSFA, 2008) sets forth the conditions under which permitted food additives may be used ex situ in foods. Additives listed as preservatives by the Codex Alimentarius Commission to ensure safety of cereal-based food manufacture include propionic, sorbic, acetic and benzoic acids and its salts (mainly of calcium, sodium and/or potassium), pimaricin (natamycin) and nisin. The first three additives are mainly obtained by chemical synthesis although some ex situ production by microorganisms is registered. Benzoic acid, which performs ideally in fine bakery wares and mixes (fruit fillings, cakes, cookies and scones) is exclusively synthesized by partial oxidation of toluene with oxygen (Namie et al., 1975) while natamycin and nisin (bacteriocin) are produced ex situ by microbial fermentation followed by an adequate recovery-purification process. Calcium and sodium propionates inhibit a broad spectrum of moulds (genera Aspergillus Penicillium, Mucor and Rhizopus) and ropy bacteria (B. subtilis, B. licheniformis, B. cereus and B. mesentericus) increasing the shelf-life of bakery products (packed bread, Christmas puddings, cakes and pastries and part-baked bread) (Gerdes, 2004; Valerio et al., 2008). Compared to other preservatives, propionate minimally impacts yeast growth and activity, making it the ingredient of choice for yeast-raised products and tortillas. However calcium propionate, which is more effective at pH 5.5 or lower, may adversely affect baking powder activity at high concentrations and also produce a bitter flavour in the dough (Ingredient Wizard Ltd, 2008). To counteract these effects, the yeast level in the formulation can be increased and proof times extended. Recently, a preparation for bakery containing microencapsulated calcium propionate combined with emulsifiers was developed by Danisco (Frost & Sullivan, 2008). The commercial product GRINDSTED ® PRO 4 reduces the fermentation time from 60 to 50 minutes and secures shelf-life of baked products for up to ten days. To minimize the inhibitory effect of calcium propionate, bakers usually add this preservative to the dough at the mixing stage rather than at the pre-fermentation stage. The sodium and potassium salts are recommended for using in chemically leavened products because the calcium (in calcium propionate) may interfere with some chemical leavening agents. Potassium sorbate proves to be effective against a broad range of bacteria and moulds. However, as sorbate inhibits yeast fermentation it is used in non-leavened

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products such as pie fillings and for spray application after baking (e.g. in packaged products) to prevent surface mould. Sorbates have been microencapsulated in a lipid matrix that melts during the cooking stage. By this technology, the additive may be added in leavened products since it remains inactive during the leavening stage without affecting the yeast activity. Natamycin is a well-known polyene macrolide antifungal that is naturally produced by Streptomyces natalensis, Streptomyces chatanoogen and Streptomyces gilvosporeus under submerged aerobic fermentations. It acts as inhibitor of mould and yeast and it is mainly used to prevent the synthesis of mycotoxin by strains of Aspergillus, Fusarium and Penicillium (Brothers and Wyatt, 2000). Natamycin is available on the market as highly concentrated solutions that require water miscible organic solvent and appropriate pH range to ensure activity (Van Rijn and Stark, 2006). Natamax® SF containing natamycin against yeasts and moulds is marketed for bakery products (and cheeses) (Danisco, 2005). Recently, a new method was proposed involving spraying a solution or suspension of natamycin on the surface of the product and then removing the solvent by heat, blowing, microwaves or any combination thereof (Wisler et al., 2008). A suspension of natamycin including a thickener has been tested to increase the shelf-life of fine bakery products with intermediate or high residual moisture (aw > 0.8) such as partially-baked and unbaked products, sliced or non-sliced bread, fine bakery and bakery-based food products (cakes, muffins, waffles, pancakes, tortillas, pizza bases, dough, pastry, toastbread, rolls, hamburger buns, baguettes and the like) (Williams et al., 2006). The thickener (agar, alginates, carrageenan, cellulose, gums, gelatin, pectins, starches, etc.) produces a gel which prevents or reduces the sedimentation of natamycin in the suspension, ensuring a good distribution when spraying the surface of the product. This additive was successfully tested in surface-sprayed sandwich bread with mould growth observed after 16 days of storage compared to growth after 5 days for the control (Williams et al., 2006). Nisin A is currently the only bacteriocin licensed as a food preservative (E234) and products are mainly commercialized by Danisco (Nisaplin® and Novasin™) as dried concentrates containing approximately 2.5% nisin. In cereal-based products, nisin is used to control spoilage in semolina pudding, pies, cakes and fine bakery products, and beer. The heat-protected nisin in Nisaplin® BS is useful against Gram-positive bacteria; particularly B. cereus, in hot-plate flour products (crumpets, muffins, etc.). Crumpets are high-moisture, flour-based products that are popular as breakfast or snack food in Canada, the U.K. and Australia. They are produced on a hot-plate or griddle from flour batter that contains yeast, or an aerating agent, or both, to give a final product with a spongy and open texture. Commercially produced crumpets have a broad range of pH (6–9), moisture content (48–56%) and water activity (aw) (0.95–0.99), i.e. conditions that are conducive to microbiological spoilage (Koukoutsis et al., 2005). Flour used in the manufacture of crumpets contains a low number of B. cereus spores that are not killed during the cooking process. During the shelf-life of the product (3–5 days) at room temperature, B. cereus can grow and cause food poisoning.

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During beer manufacture, the addition of nisin into fermenters at levels of 0.25–2.5 mg/L proved to be effective in preventing spoilage by certain LAB strains of the genera Lactobacillus, Pediococcus, and Leuconostoc. Furthermore, nisin can also be used in the pitching yeast wash at 25.0–37.8 mg/L as an alternative to acid washing for the control of LAB (Delves-Broughton, 2005). Technologies that bind bacteriocins to carriers (e.g. films of cellulose, polyethylene, polysaccharides, PVC, nylon, etc.) were successfully applied for food preservation and exhaustively reviewed by Gálvez et al. (2007). Nevertheless, only films containing natural preservatives derived from plants (as the essential oil of Cinnamomum zeylanicum) were tested for baked goods at packaging. Cinnamon-essential oil in combination with micro-perforated polypropylene increased the shelf-life of handmade bakery products (a complex mixture containing yolk, almonds, nuts and raisins swilled in rum) from 3 to 10 days with maximum quality and safety (Gutiérrez et al., 2009).

14.7  Phage-based strategies This novel biopreservative strategy in food takes advantage of the lytic enzymes (endolysins) from bacteriophages of specific Gram-positive and, to a lesser extent, Gram-negative bacteria hosts. Endolysins are mureolytic enzymes that directly target unique and highly conserved bonds in the peptidoglycan (Strauch et al., 2007). Up to now, there is no report concerning the antimicrobial activity of phages in cereal-based foods. Nevertheless, this approach is promising in dairy products and fruits to control Salmonella sp. and Staph. aureus (Modi et al., 2001; Leverentz et al., 2001; Obeso et al., 2008), two pathogen bacteria commonly found in indigenous fermented cereal food and fresh pasta.

14.8  Conclusion The benefits of cereals in nutrition and the current demands for high quality and minimally processed foods renewed the scientific interest toward biopreservation technologies. Nowadays, these strategies are widely extended by taking advantage of fermentative processes carried out by ubiquitous microrganisms growing in cereals. Although natural fermentations are useful for preserving indigenous foods, the process is rather difficult to control. Starter cultures and selected strains with antimicrobial properties and/or their inhibitory metabolites proved to be efficient in cereal-based food for extending shelf-life and enhancing the quality of products. However, for certain food preservation, e.g. bread, biological strategies do not match up to ideal standards and they have been combined with chemical additives in lesser amounts. In the future, biopreservation might be exploited as hurdle technology in order to decrease the survival of contaminant microorganisms, thus obtaining more natural cereal-based foods.

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14.9  References blandino a , al - aseeri m e , pandiella s s , cantero d

and webb c (2003), ‘Cerealbased fermented foods and beverages’, Food Res Int 36, 527–543. böcker g , stolz p and hammes w p (1995), ‘Neue erkenntnisse zum ökosystem sauerteig und zur physiologie des sauerteig-typischen stämme Lactobacillus sanfrancisco und Lactobacillus pontis’, Getreide Mehl und Brot 49, 370–374. bonestroo m h , de wit j c , kusters b j and rombouts f m (1993), ‘Inhibition of the growth of yeasts in fermented salads’, Int J Food Microbiol 17, 311–320. tiwari b k , valdramidis v p , o ’ donnell c p , muthukumarappan k , bourke p and cullen p j (2008), ‘Application of natural antimicrobials for food preservation’, J Agric Food Chem 57, 5587–5600. brothers a m and wyatt r d (2000), ‘The antifungal activity of natamycin toward molds isolated from commercially manufactured Poultry Feed’, Avian Dis 44, 490–497. bryant r j , kadan r s , champagne t e , vinyard b t and boykin d (2001), ‘Functional and digestive characteristics of extruded rice flour’, Cereal Chem 78, 131–137. campbell - platt g (1987), Fermented Foods of the World, London, Butterworths. caplice e and fitzgerald g f (1999), ‘Food fermentations: role of microorganisms in food production and preservation’, Int J Food Microbiol 50, 131–149. codex general standard for food additives ( gsfa ) online database (2008), ‘Food categories’, Codex Alimentarius Commission. Available from: http://www. codexalimentarius.net/gsfaonline/foods/index.html (accessed 31 July 2009). corsetti a , gobbetti m and damiani p (1998), ‘Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1’, Appl Microbiol Biotechnol 50, 253–256. corsetti a , lavermicocca p , morea m , baruzzi f , tosti n and gobbetti m (2001) ‘Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy’, Int J Food Microbiol 64, 95–104. corsetti a , settanni l and van sinderen d (2004), ‘Characterization of bacteriocinlike inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity’, J Appl Microbiol 96, 521–534. cortinez y j m , velazquez l , eiguer t , caffer m i and guaman a m s (1988), ‘Determinación de la calidad higiénico sanitaria de fideos frescos’, Rev Arg Microbiol 20, 195–200. cotter p d , hill c and ross r p (2005), ‘Bacteriocins: developing innate immunity for food’, Nat Rev Microbiol 3, 777–788. dal bello f , clarke c i , ryan l a m , ulmer h , schober t j et al. (2007), ‘Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7’, J Cereal Sci 45, 309–318. danisco , products & services (2005), ‘Natamax™ antimicrobials’. Available from: http://www.danisco.com/wps/wcm/connect/danisco/corporate/products%20and%20 services/product%20range/antimicrobials/antimicrobial%20ingredients/natamax%20 antimicrobials/ (accessed 30 June 2009). de vuyst l , avonts l , neysens p , hoste b , vancanneyt m et al. (2004), ‘The lactobin A and amylovorin L471 encoding genes are identical, and their distribution seems to be restricted to the species Lactobacillus amylovorus that is of interest for cereal fermentations’, Int J Food Microbiol 90, 93–106. de vuyst l and neysens p (2005), ‘The sourdough microflora: Biodiversity and metabolic interactions’, Tren Food Sci Technol 16, 43–56. deegan l h , cotter p d , hill c and ross p (2006), ‘Bacteriocins: biological tools for bio-preservation and shelf-life extension’, Int Dairy J 16, 1058–1071.

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15 Biological control of postharvest diseases in fruit and vegetables N. Teixidó and R. Torres, IRTA, Catalonia, Spain, I. Viñas, University of Lleida, Catalonia, Spain and M. Abadias and J. Usall, IRTA, Catalonia, Spain

Abstract: This chapter describes the situation on the use of microorganisms for controlling postharvest diseases in fruit and vegetables after more than 20 years of research. The most remarkable results in selection, mode of action, production and formulation of biocontrol agents (BCAs), possible approaches to improve their viability and efficacy and the integration with other alternative methods have been described. However, all the effort conducted until now to implement the use of biocontrol agents as a practical control strategy has not been enough to bring in a commonly used method at a commercial level. More governmental and industrial support is necessary and new areas of research should be developed to make biological control a real substitute for chemicals. Key words: formulation, improvement, integration, mode of action, production.

15.1  Introduction Postharvest fruit and vegetable diseases continue causing significant world-wide losses, estimated to be around 20% in industrialized regions and more than 50% in regions with storage and transport limitations. Infection by pathogens can occur either prior to harvest, during harvesting and subsequent handling, or in storage. Development of a fungal disease during the postharvest phase depends on storage conditions, and physiological age and defence mechanisms of the host. Postharvest decay can be reduced by minimizing fruit injuries, by maintaining the natural resistance of the host, and by delaying senescence (Shewfelt, 1986). However, these beneficial practices are usually not sufficient to protect the product from fungal infection. Application of synthetic fungicides has been the primary means of controlling postharvest diseases (Eckert and Ogawa, 1988), although their use is becoming increasing restricted because of concerns for the environment and human 364 © Woodhead Publishing Limited, 2011

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health, as well as the cost of developing new pesticides to overcome resistance developed by pathogens. Some consumers are demanding fruit and vegetables free of fungicide residues and regulatory agencies have caused effective fungicides to be withdrawn from use because of possible negative health and environmental impacts. Biological control using microbial antagonists has attracted much interest as an alternative to chemical products (Chanchaichaovivat et al., 2007; Droby et al., 1992; Janisiewicz and Marchi, 1992; Pusey and Wilson, 1984; Teixidó et al., 2001; Usall et al., 2001; Viñas et al., 1998). The ‘first generation’ of biological control organisms relied on the use of single antagonists (Droby et al., 2003). However, these and other BCAs cannot provide by themselves the consistent or broadspectrum control of synthetic fungicides. In general, microbial antagonists show great variability in their efficacy, only confer a protective effect that diminishes with ripening, and usually cannot eradicate incipient or pre-existing infections or prevent fungal sporulation (El Ghaouth et al., 2002). Therefore, considerable research efforts are currently being devoted to develop a ‘next generation’ of biologically based control methods (Droby et al., 2003). According to Janisiewicz and Korsten (2002), the primary approaches used to improve biocontrol of postharvest diseases are manipulation of the environment, mixtures of antagonists, physiological and genetic manipulation of antagonists, combining field and postharvest applications, manipulation of formulations, and integration with other non-biological methods. Spadaro and Gullino (2004) stated that biological means must be currently considered instruments to be used in combination with other methods in an integrated vision of postharvest disease management. It has also been demonstrated that it is possible to improve BCAs’ stress tolerance by manipulating the growth medium, thus enhancing its behaviour in practical conditions, both in formulation process (Usall et al., 2009) and preharvest applications (Teixidó et al., 2009). However, there is still need for more research supported by government and industry to accelerate the development and commercial use of BCAs. This chapter is a review of the situation of biocontrol use to control postharvest diseases in fruit and vegetables, describing the different steps included in a development biocontrol programme and going deeply into production, formulation and enhancing BCAs, which are the less reviewed approaches in the past.

15.2  Development programme of a biocontrol agent (BCA) The first work of postharvest biocontrol was published by Tronsmo and Denis (1977) who reported the control of Botrytis rot of strawberry with Trichoderma spp. A few years later, Wilson and Pusey (1985) published the most featured work on the potential of postharvest biocontrol and showed the control of brown rot on peaches by Bacillus subtilis. Many postharvest research laboratories have been engaged in research of effective antagonists that control postharvest diseases. However, not all have devised successful antagonists mainly due to some aspects lacking in the research programme. Indeed the successful development of a biocontrol product should include the following steps:

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Isolation of BCAs from the environment. Studies of efficacy: from laboratory to practical conditions. Mode of action. Enhancement of efficacy. Economical mass production. Formulation, packaging and shelf life. Increase range of activity. Toxicological studies. Technologies for their applications. Registration and commercialization.

In the next section most of these steps will be described in more detail. However, for a successful control of postharvest diseases, a thorough understanding of other components should be considered: 1. Biology and epidemiology of fungal pathogens: a profound knowledge of strategies of infection, survival, and pathogen division on the basis of the nutritional relationship that they maintain with the host (e.g. necrotrophic and biotrophic pathogens) is a pre-requisite for the proper choice of biocontrol strategies (Köhl and Fokkema, 1998). In general, the more internal the pathogen during the host-pathogen interaction, the less vulnerable the pathogen to control by antagonists. 2. Role of host plant as a passive and/or an active component in the biological control of plant pathogens. In the passive role, the host is seen as an environment of absorptive and adsorptive surface and of fluctuating temperature, water potential, and pH mainly (Cook and Baker, 1983). However, it is important to consider the fruit as a responsive entity. In this active role, the host has genetically programmed defence mechanisms that recognize and/or reject microorganisms or their products. The challenge lies in knowing how these defence mechanisms operate in the postharvest environment, and in elucidating how BCAs interact to induce, support or replace such mechanisms (Wilson and Wisniewski, 1989). 3. Physical environment including temperature, relative humidity and partial pressures of gases (controlled atmosphere) must be recognized as a dynamic component of biological control in fruits and vegetables. A manipulation of physical environment is one of the disease management strategies developed to control postharvest diseases. In general, for an optimal decay control, two or more environmental factors should be modified simultaneously (Spotts and Sanderson, 1994).

15.3  The search for biocontrol agents of postharvest diseases The primary concern in obtaining BCAs is its isolation material or environment. It is generally accepted that antagonists should be selected from the environment where they will be applied rather than from other systems. Many bacteria and

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yeasts with significant biocontrol potential have been isolated by direct screening on fruit, indicating the importance of in situ tests on fruits and vegetables (Narayanasamy, 2006). For postharvest antagonists, one should look for those that are well adapted to survive and grow on fruit and vegetable surfaces and wounds under storage conditions and have an ‘adaptive advantage’ over specific pathogens (Wilson and Wisniewski, 1989). Moreover, and from a commercial point of view, BCAs that naturally occur on fruit or vegetable surfaces will gain public acceptance before those isolated from other sources such as soil. Wilson and Wisniewski (1989) indicated the following characteristics for an ideal antagonist: • • • • • • • • • • • •

Genetic stability. Efficacy at low concentrations. Simple nutritional requirements. Survival in adverse environmental conditions. Effective against a wide range of pathogens on various fruit and vegetables. Growth on cheap substrates in fermenters. Amenable to formulation with a long shelf life. Easy to dispense. No production of metabolites potentially toxic to humans. Resistance to the most frequently used pesticides. Compatible with other physical and chemical treatments. Lack of pathogenicity for the host plant.

Some of these key requirements are intuitive (e.g. efficacy at low concentrations) but others require a more intimate knowledge of commercialization of microbial pesticides (e.g. production, formulation and distribution). Therefore a close collaboration between scientists working on developing a biocontrol system and industry that can commercialize the BCA is recommended (Janisiewicz and Korsten, 2002). As a first step to evaluate the effectiveness of the antagonist against pathogens, in vitro tests have been used, but only when this antagonist produces antimicrobial products against pathogen/s is a usefulness test. From the beginning of the 1990s until now, the strategy described by Wilson et al. (1993) to utilize fruit wounds to screen for potential yeast antagonists against postharvest pathogens is the most widely used. It is usually performed through primary and secondary screenings using a pathogen conidial concentration encountered under commercial conditions in packing houses. One to three percent of the original isolates generally pass through the secondary screening when they should be effective at concentrations reasonable for commercial development (Janisiewicz, 1998). However, a shortcoming of this strategy is that it favours the selection of antagonists that are generally fast growers with the ability to colonize specific surface wounds rich in nutrients, therefore exhibiting protective rather than curative activity, and rarely having an effect on latent infections (Droby et al., 1989; El Ghaouth et al., 2000b). In the next step, the most efficient isolates are tested for their survival on fruits and vegetables at the wound site or on the surface under different storage conditions, and for their

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compatibility with other postharvest treatments and additives generally used in packing houses (Janisiewicz, 1998). Then microorganisms with potential biocontrol activity have to be accurately identified by using traditional combined with molecular methods. A potential antagonist against postharvest rot pathogens should not be plant-pathogen or taxonomically related to pathogens to avoid special problems in the registration process for commercialization. In the past decades, screening for antagonists has been made by many research groups and a broad range of BCAs have been tested to control postharvest diseases of fruit and vegetables (Table 15.1). The potential biocontrol activity of both bacteria and fungi for controlling postharvest diseases of fruit has been clearly demonstrated, although most of works have been conducted only at laboratory scale and have not been tested in different packing-house environments and different commodities. An important number of antagonists have been shown to control main postharvest diseases of hosts such as apples, peaches, citrus fruit, pears and nectarines. In contrast, few antagonists for biological control of postharvest diseases of vegetables have been isolated. It is important to note that only BCAs from a very narrow range of genera have been isolated (mainly Aureobasidium, Bacillus, Candida, Cryptococcus, Metschnikowia, Pantoea, Pichia, Pseudomonas, Rhodotorula and Trichoderma) since the method of screening used by most researchers is similar. From all isolates, only a small number of BCAs has moved from the laboratory to commercial application; moreover, the majority of BCA products commercially available for postharvest applications are based on yeasts (Metschnikowia fructicola, Candida oleophila and Candida sake). To overcome this shortcoming, Droby et al. (2009) suggested the idea of using a new variety of screening procedures to increase the range of new microbial species that could exhibit higher commercial potential either for fruit or vegetables.

15.4  Mechanisms of action The initial driving force in the commercial development of BCAs for postharvest disease of fruit and vegetables was studies regarding applied microbial ecology, but further progress in improving biocontrol will largely depend on the basic understanding of the mode of action (Janisiewicz and Korsten, 2002). Droby et al. (2009) proposed that full information on these mechanisms of action will lead to the selection of more effective antagonists, and also new methods for optimising their activity. This knowledge could enhance BCAs activity through the development of appropriate formulation and methods of application, and also lead to facilitated registration (Wilson and Wisniewski, 1989). Antagonists can control postharvest pathogens by several mechanisms of action, including antibiosis or other antimicrobial substances, parasitism, competition by nutrients and space, induction of resistance and resistance to oxidative stress. In general, BCAs show multiple interactions with pathogens, hosts and also other resident microorganisms to control fungal diseases.

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Table 15.1  Antagonists for biological control of postharvest diseases of fruit and vegetables Fruit/ vegetable

Antagonist

Pathogen

Reference

Apple

Acremonium brevae Aureobasidium pullulans

Botrytis cinerea Penicillium expansum; B. cinerea

Candida oleophila

P. expansum

Candida sake

P. expansum; B. cinerea; Rhizopus stolonifer P. expansum

Janisiewicz (1988) Castoria et al. (2001); Ippolito et al. (2000); Vero et al. (2009) El Neshawy and Wilson (1997) Viñas et al. (1998)

Cryptococcus spp. Cryptococcus albidus Cryptococcus laurentii

P. expansum; B. cinerea B. cinerea Glomerella cingulata P. expansum

Metschnikowia fructicola

P. expansum

Metschnikowia pulcherrina Muscodor albus

P. expansum; B. cinerea

Pantoea agglomerans

P. expansum; B. cinerea; R. stolonifer P. expansum P. expansum B. cinerea

Pantoea ananatis Pichia anomala Pichia angusta

P. expansum; B. cinerea

Pichia guilliermondii Pseudomonas cepacia

B. cinerea; Monilinia fructicola P. expansum; B. cinerea P. expansum; B. cinerea

Pseudomonas fluorescens Pseudomonas syringae

Botrytis mali P. expansum; B. cinerea

Rahnella aquatilis Rhodotorula glutinis

P. expansum; B. cinerea P. expansum; B. cinerea

Chand-Goyal and Spotts (1997) Fan and Tian (2001) Roberts (1990a) Blum et al. (2004) Lima et al. (1998); Vero et al. (2002); Li and Tian (2006) Kurtzman and Droby (2001) Janisiewicz et al. (2001); Spadaro et al. (2002) Mercier and Jiménez (2004) Nunes et al. (2002a) Manso et al. (2010) Torres et al. (2005) Jijakli and Lepoivre (1998) Fiori et al. (2008)

P. expansum

McLaughlin et al. (1990) Janisiewicz and Roitman (1988) Mikani et al. (2008) Janisiewicz (1987); Nunes et al. (2007); Zhou et al. (2001) Calvo et al. (2007) Lima et al. (1998); Zhang et al. (2009) Li et al. (2008)

Apricot

Bacillus subtilis Pestalotiopsis frequentans

M. fructicola Colletotrichum gloeosporioides

Pusey and Wilson (1984) Adikaram and Karunaratne (1998)

Avocado

Bacillus subtilis

Botryodiplodia theobromae

Demoz and Korsten (2006) (Continued )

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Table 15.1  Continued Fruit/ vegetable

Antagonist

Pathogen

Reference

Banana

Burkholderia cepacia

Colletotrichum musae

Candida oleophila Pichia anomala Pseudomonas sp.

C. musae C. musae C. musae

Trichoderma harzianum

B. cinerea

De Costa and Erabadupitiya (2005) Lassois et al. (2008) Lassois et al. (2008) De Costa and Subasinghe (1998) Devi and Arumugam (2005)

Cabbage

Pseudomonas aeruginosa

Erwinia carotovora

Adeline and Sijam (1999)

Citrus fruit

Aureobasidium pullulans

Penicillium spp.

Bacillus subtilis

Penicillum digitatum; Geotrichum candidum; B. theobromae; Phomopsis citri; Alternaria citri P. digitatum; Penicillium italicum; G. candidum

Wilson and Chalutz (1989) Singh and Deverall (1984)

Debaryomyces hansenii

Cherry

Candida famata Candida oleophila

P. digitatum P. digitatum; P. italicum

Cryptococcus laurentii Kloeckera apiculata Metschnikowia fructicola

P. italicum Penicillium spp. P. digitatum; P. italicum

Muscodor albus

P. digitatum

Pantoea agglomerans Pichia anomala Pichia guilliermondii

P. digitatum; P. italicum P. digitatum Penicillium spp. P. digitatum

Pseudomonas cepacia Pseudomonas glathei Pseudomonas syringae

P. digitatum P. digitatum P. digitatum; P. italicum

Rhodotorula glutinis

P. digitatum

Aureobasidium pullulans Bacillus subtilis

B. cinerea Monilinia laxa B. cinerea

Cryptococcus laurentii

M. fructicola

Enterobacter aerogenes

B. cinerea

Kloeckera apiculata Pseudomonas syringae

B. cinerea B. cinerea

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Singh (2002); Chalutz and Wilson (1990); Droby et al. (1989) Arras (1996) Droby et al. (1998); Lahlali et al. (2004) Zhang et al. (2005) Long et al. (2006) Kurtzman and Droby (2001) Mercier and Smilanick (2005) Teixidó et al. (2001) Manso et al. (2010) Lahlali et al. (2004) Arras et al. (1996); Droby et al. (1993); Wilson and Chalutz (1989) Huang et al. (1993) Huang et al. (1995) Wilson and Chalutz (1989) Arras et al. (1996) Ippolito et al. (2005) Wittig et al. (1997) Utkhede and Sholberg (1986) Karabulut et al. (2005); Qin et al. (2004) Utkhede and Sholberg (1986) Karabulut et al. (2005) Janisiewicz (1987)

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Table 15.1  Continued Fruit/ vegetable

Antagonist

Pathogen

Reference

Chilli fruits

Pichia guilliermondii

Colletotrichum capsici

Chanchaichaovivat et al. (2007)

Grapes

Aphanocladium album Aureobasidium pullulans Cryptococcus laurentii Metschnikowia fructicola

Ciccarese et al. (2007) Schena et al. (2003) Liu et al. (2002) Karabulut et al. (2003) Kurtzman and Droby (2001)

Muscodor albus

B. cinerea B. cinerea B. cinerea B. cinerea B. cinerea; R. stolonifer; Aspergillus niger B. cinerea

Pichia guilliermondii Trichoderma harzianum

P. expansum; B. cinerea B. cinerea

Pichia anomala

B. theobromae

Trichoderma spp.

Lasiobasidium theobromae; Phomopsis psidi; Rhizopus spp.

Cryptococcus laurentii

Alternaria alternata; P. expansum A. alternata; P. expansum A. alternata

Qin and Tian (2004)

Guava

Jujube fruit

Rhodotorula glutinis Rhodosporium paludigenum

Mlikota-Gabler et al. (2006) Chalutz et al. (1988) Batta (2007) Hashem and Alamri (2009) Majumdar and Pathak (1995)

Tian et al. (2005) Wang et al. (2009)

Kiwi fruit

Bacillus licheniformis Trichoderma harzianum

C. gloeosporoides B. cinerea

Govender et al. (2005) Batta (2007)

Loquat

Pichia membranaefaciens

Colletotrichum acutatum

Cao et al. (2009)

Lychee

Bacillus subtilis

A. alternata

Jiang et al. (2001)

Mango

Candida membranifaciens Rhodotorula minuta Trichoderma viride

C. gloeosporoides

Kefialew and Ayalew (2008) Patiño-Vera et al. (2005) Kota et al. (2006)

Trichoderma spp.

C. gloeosporoides Botryodiplodia theobromae Lasiobasidium theobromae; Rhizopus spp.

Pathak (1997)

Muskmelon

Bacillus subtilis

A. alternata

Yang et al. (2006)

Nectarine

Bacillus subtilis Candida guilliermondii Candida oleophila

M. fructicola B. cinerea P. expansum

Pichia membranaefaciens Pseudomonas cepacia Pseudomonas corrugata

Rhizopus sp. M. fructicola M. fructicola

Pusey and Wilson (1984) Tian et al. (2002) Lurie et al. (1995); Karabulut and Baykal (2003) Qing and Shiping (2000) Smilanik et al. (1993) Smilanik et al. (1993)

Candida oleophila

C. gloeosporioides

Gamagae et al. (2003)

Papaya

(Continued )

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Table 15.1  Continued Fruit/ vegetable

Antagonist

Pathogen

Reference

Peach

Bacillus subtilis Candida guilliermondii Candida oleophila Cryptococcus laurentii

M. frunticola B. cinerea B. cinerea E. stolonifer; B. cinerea; P. expansum; M. fructicola R. stolonifer R. stolonifer Monilinia spp. P. expansum M. fructicola M. laxa; R. stolonifer Monilinia sp. M. fructicola M. fructicola M. laxa

Pusey and Wilson (1984) Tian et al. (2002) Karabulut and Baykal (2003) Zhang et al. (2007c); Yao and Tian (2005)

Mari et al. (1996) Viñas et al. (1998)

Pseudomonas cepacia

B. cinerea P. expansum; B. cinerea; R. stolonifer Mucor piriformis M. piriformis M. piriformis B. cinerea; P. expansum P. expansum; B. cinerea; R. stolonifer B. cinerea; P. expansum

Rhodotorula glutinis Trichoderma harzianum

P. expansum; B. cinerea B. cinerea

Pineapple

Penicillium funiculosum

Penicillium spp.

Tong and Rohrbock (1980)

Plum

Bacillus subtilis

L. theobromae

Pusey and Wilson (1984)

Potato

Pseudomonas putida

Erwinia carotovora

Colyer and Mount (1984)

Rambutan

Trichoderma harzianum

C. gloeosporioides; Gliocephalotrichum microchlamydosporum; Botryodiplodia theobromae

Sivakumar et al. (2000)

Strawberry

Aureobasidium pullulans Bacillus subtilis Candida oleophila Cryptococcus laurentii Metschnikowia fructicola

B. cinerea B. cinerea B. cinerea R. stolonifer B. cinerea; R. stolonifer

Rhodotorula glutinis Trichoderma harzianum

B. cinerea B. cinerea

Lima et al (1997) Zhao et al. (2007) Lima et al. (1997) Zhang et al. (2007b) Kurtzman and Droby (2001) Zhang et al. (2007a) Batta (2007)

Candida guilliermondii Candida oleophila Cryptococcus laurentii Pichia guilliermondii

B. cinerea B. cinerea B. cinerea A. alternata; B. cinerea; R. stolonifer

Saligkarias et al. (2002) Saligkarias et al. (2002) Xi and Tian (2005) Chalutz et al. (1988); Zhao et al. (2008)

Denaryomyces hansenii Enterobacter cloacae Epicoccum nigrum Metschnikowia fructicola Muscodor albus Pantoea agglomerans Penicillium frequentans Pseudomonas cepacia Pseudomonas corrugata Pseudomonas syringae Pear

Bacillus pumilus Candida sake Cryptococcus albidus Cryptococcus flavus Cryptococcus laurentii Pantoea agglomerans

Tomato

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Mandal et al. (2007) Wilson et al. (1987) Larena et al. (2005) Kurtzman and Droby (2001) Mercier and Jiménez (2004) Bonaterra et al. (2003) Guijarro et al. (2007) Smilanick et al. (1993) Smilanick et al. (1993) Zhou et al. (1999)

Roberts (1990b) Roberts (1990b) Roberts (1990b) Zhang et al. (2003) Nunes et al. (2001b) Janisiewicz and Roitman (1988) Zhang et al. (2008) Batta (2007)

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Information on the complex mechanism of action is still incomplete for most antagonists because not all potential mechanisms of action and their interaction have been thoroughly studied. 15.4.1  Competition for space and nutrients Nutrient competition is considered as the major mode of action by which BCAs could control disease-causing pathogens, particularly for yeasts and bacteria antagonists such as Aureobasidium pullulans (Bencheqroun et al., 2007), Candida famata (Arras, 1996), Cryptococcus laurentii (Roberts, 1990a), Metschnikowia pulcherrima (Spadaro et al., 2002), Pantoea agglomerans (Poppe et al., 2003) and Pichia guilliermondii (Droby et al., 1992). First studies demonstrated through in vitro tests that microbial antagonists take up nutrients more rapidly than pathogens, get established and inhibit spore germination of the pathogens at the wound site, which however stay alive (Wisniewski et al., 1989; Droby and Chalutz, 1994). To compete successfully with pathogens at the wound site, BCAs should be established faster than pathogens in the wounds or on the fruit surface mainly due to a better adaptation to various environmental and nutritional conditions compared with postharvest pathogens. Although in most cases nutrient competition was reported to play an important role, it was rather difficult to separate this mode of action from other mechanisms. To solve this problem, Janisiewicz et al. (2000) and Lindow et al. (2001) described methodologies using natural substrates in in vitro cylinder-well tests and biological sensors, respectively, for evaluating only microbial competition for nutrients in fruit. 15.4.2  Production of antimicrobial substances Production of antibiotics could be considered as the main mechanism by which bacteria suppress postharvest pathogens of fruits and vegetables, but in many works the role of antibiotics produced by these antagonists is uncertain (Janisiewicz, 1998). The first works describing this antagonistic mechanism were with B. subtilis synthesizing iturins against Monilinia spp. on peaches (Gueldner et al., 1988) and Pseudomonas cepacia producing pyrrolnitrin against Botrytis cinerea and Penicillium expansum on pome fruits (Janisiewicz et al., 1991). However, although antibiosis might be an effective mechanism to control postharvest diseases, the development of non-antibiotic-producing antagonists for food products has high potential to avoid human pathogen resistance (El Ghaouth et al., 2004; Sharma et al., 2009). The production of volatile antibiotics is rare among microorganisms. However, Mercier and Jiménez (2004) demonstrated the potential control of Muscodor albus applied by fumigation for controlling postharvest fungal diseases on apples and peaches. It was shown that this antagonist synthesizes volatiles as 2-methyl-1-butanol, isobutyric acid and ethyl propionate which were produced at sub-micromolar concentrations, and did not lead to significant residues in the treated fruit.

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15.4.3  Direct interaction The attachment of antagonists to pathogen hyphae has been described for Enterobacter cloacae against Rhizopus stolonifer on peach (Wisniewski et al., 1989) and P. guilliermondii against Penicillium italicum on citrus fruit (Arras et al., 1998). Production of lytic enzymes as gluconase degrading fungal cell walls has been reported in Pichia anomala (Jijakli and Lepoivre, 1998). Another work through ultrastructural and cytochemical studies showed that Candida saitoana cells were associated with B. cinerea hyphae showing cytological damage and degeneration of the cytoplasma (El Ghaouth et al., 1998). It seems that due to this direct attachment, antagonist microbes compete more efficiently for nutrients than pathogen at the invasion site, and then prevent spore germination and growth of pathogens. 15.4.4  Induced resistance Microbial antagonists could induce disease resistance by increasing the healing process, resulting in a significant reduction in decay development. The increased amount of enzymes as gluconase, peroxidase and chitinase in apple wounds treated with A. pullulans was attributed to higher production of this antagonist and to the induction of these enzymes in the fruit itself against P. expansum (Castoria et al., 2001; Ippolito et al., 2000). More recently, molecular approaches have been used to examine the role of glucanase in the biocontrol activity of C. oleophila (Yehuda et al., 2003) and to enhance biocontrol activity by overexpression of antimicrobial peptides (Janisiewicz et al., 2008). Furthermore, using molecular tools, the identification of 50 differentially expressed genes (such as encoding chitinase, glucanase, some PR-proteins, mannitol dehydrogenase, mitogenactivated protein kinase and ethylene receptors) after applications of C. laurentii to cherry tomato fruit showed the potential role of these proteins in increasing fruit resistance to postharvest pathogen infection (Jiang et al., 2009). Production of antifungal compounds by Colletotrichum magna in avocado fruit (Prusky et al., 1994) and accumulation of phytoalexins in citrus fruit (Rodov et al., 1994) were also described as other inducing defence mechanisms. 15.4.5  Tolerance to oxidative stress Castoria et al. (2003) demonstrated that the ability to tolerate high levels of reactive oxygen species (ROS) produced by apple tissue could be another mode of action enhancing the efficacy of yeast antagonists against B. cinerea and P. expansum. Other authors have shown changes in the activities of pro- and antioxidant enzymes after inoculation of C. laurentii in peach fruit (Wang et al., 2004), and some defence-related proteins when sweet cherry fruit were treated with Pichia membranaefaciens (Xu and Tian, 2008) which could be involved in controlling or reducing fungal diseases.

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15.5  Production and formulation of biocontrol agents One of the most critical aspects for the success of a biocontrol product is the commercial applicability: mass production of large amounts and formulation with a reasonable shelf life that maintains efficacy under practical conditions. There is a lack of scientific information because usually production and formulation are conducted in association or directly by private companies and all this research is developed under secrecy agreements. It is essential that a formulated product retains its species purity (no contamination) and microbial cells must retain their genetic stability, viability, attributes as colonizers on fruit surfaces, as well as other physiological properties relating to their activity. Industrial fermentation and formulation is accomplished under conditions quite different from those in the laboratory and scaling-up is a difficult process. 15.5.1  Production Production of BCAs is an important step in the commercial use of the product. The development of an economic culture medium and growth conditions optimization are necessary to obtain a large quantity of high quality biomass. Medium constituents must satisfy basic requirements for biomass and metabolite production, providing an adequate supply of nutrients and energy for biosynthesis and cell maintenance. In general, and depending on the BCA’s nature (filamentous fungi, yeast or bacteria), the production methods used in industry are solid- or liquid-phase fermentations. Bacteria and yeasts are usually produced by liquid fermentation using continuously stirred tank bioreactors, although many fungi have problems with these systems and are produced in solid media. Independently of the method used, the aim is to achieve high cell yield and high quality at the lowest possible cost. The use of commercial products or by-products from food industries, such as nitrogen and carbon sources, has tended to meet the above criteria for production media because they are cheap substrates (Zabriskie et al., 1980). However, by-products are not as standardized and may contain impurities that will need to be removed before fermentation (Stanbury et al., 1995); moreover, their composition may vary with the season and origin. All these aspects have limited their use in industrial processes. An optimized mass production system has been developed for P. agglomerans CPA-2, an effective BCA to control the major postharvest diseases on pome fruit and citrus. High BCA yields have been reported with yeast extract plus sucrose (Costa et al., 2002a). To reduce the cost of media, commercial products such as dry beer yeast, soy powder, sucrose, fructose, skimmed milk, malt extract and apple concentrate and by-products such as fine flour, fish, meat and bone meals, lactoserum and molasses were tested. CPA-2 can be produced using a combination of nitrogen sources such as yeast extract and dry beer yeast with inexpensive carbohydrates such as sucrose and molasses, respectively, maintaining biocontrol efficacy (Costa

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et al., 2001). Production conditions were also optimized for another strain of P. agglomerans (PBC-1) using fed-batch technology by Manso et al. (2010). A low cost medium, which could be used at industrial scale, was developed for the yeast Candida sake CPA-1, an effective BCA to control postharvest diseases on pome fruit. This medium is based on cane molasses, a by-product from the sugar industry (Abadias et al., 2000). Operating conditions (aeration, agitation, pH and temperature) as well as initial inoculum may affect the final quantity and quality of microorganism, and optimizing these parameters is essential for success in the production process. Abadias et al. (2003a) optimized CPA-1 growth conditions and studied the potentiality of fed-batch technology adding glucose or molasses. The fermentation process was successfully scaled-up using a low-cost culture medium, based on a mineral balanced broth with industrial grade yeast extract, for the yeast Rhodotorula minuta, an antagonistic micro-organism against mango anthracnose (Patiño-Vera et al., 2005). Conidial production of Penicillium frequentans, a BCA of brown rot of stone fruits, was tested in liquid and solid-state fermentation being solid production in specially designed plastic bags (VALMIC®) containing peat : vermiculite (1 : 1 w/w), lentil meal addition and an initial moisture content of about 30–40%, the process which showed the best conidial production and germinability maintaining biocontrol efficacy (De Cal et al., 2002). Similar results were obtained with Epicoccum nigrum, another BCA of Monilinia laxa on stone fruits (Larena et al., 2004). Concluding, production process development includes selection of media components, selecting the best fermentation system (solid or liquid), optimizing growth conditions (temperature, pH, aeration, agitation, initial inoculum, moisture content) and measuring biocontrol efficacy of the final product. 15.5.2  Downstream processing and formulation Fermentation biomass can be separated from spent medium by various types of filtration (pressure filtration, rotary vacuum drum filtration), centrifugation, or in some cases, flocculation. Formulation of microorganisms for biocontrol of plant pathogens is undeveloped compared with other applications of microorganisms, although this step largely impacts on BCA shelf life, ability to grow and survive after application, effectiveness in disease control, handling and distribution, ease of operation and application, and cost (Fravel et al., 1998). The process aims to maintain and stabilize the viability of the microorganism. This can be achieved in liquid state and maintained by refrigeration, by freezing in the presence of cryoprotectant substances, or by keeping it as a dehydrated product (solid state). Dehydration is one of the best ways to formulate microbial agents that can be handled using normal distribution and storage channels (Rhodes, 1993). Unfortunately, not all microorganisms are amenable to drying and many tend to lose viability during both the drying process and storage. Classical dehydration processes includes freeze-drying, spray-drying and fluidized bed-drying.

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Freeze-drying is the method that best maintains viability, but its cost is high in relation to other dehydration systems. However, commercial products such as Bio-Save 10 and 11 are freeze-dried. The feasibility of preserving the BCA C. sake CPA-1 as a solid formulation using freeze-drying was studied (Abadias et al., 2001a, 2001b). Although high viability of 85% was achieved, the efficacy of the product against P. expansum infection of apples was lower than for fresh cells and decreased up to 10% after two months storage at 4 °C. In contrast 100% cells viability was reported for freeze-dried P. agglomerans CPA-2 with sucrose as a protectant and non-fat skimmed milk as a rehydration medium (Costa et al., 2000). Freeze-drying formulations have also been studied with the yeast C. laurentii and an increased content of trehalose in the yeast was associated with enhanced survival of Cryptococcus strain after freeze-drying (Li and Tian, 2006). Later studies demonstrated that citric acid used as carbon source could induce accumulation of intracellular trehalose in the yeast. This effect together with adding exogenous protectants (sugars such as glucose, galactose, sucrose and trehalose + skimmed milk) could improve viability and shelf life after freeze-drying and maintain biocontrol effect against blue mould in apples (Li and Tian, 2007). Spray-drying may be an alternative method for preserving BCAs in a dry state because it allows large quantities of cultures to be dried at low cost. The main disadvantage of spray-drying is the extent of the destruction of microbial cells during the drying process caused by high temperatures used with this system. Low survival of BCAs P. agglomerans CPA-2 (Costa et al., 2002b) and C. sake CPA-1 (Abadias et al., 2005) in a spray-drying process were reported, with <8% and 10% respectively. Furthermore, a low recovery of powder < 55% was measured, concluding that this method is not a suitable dehydration system for both BCAs. Fluidized bed-drying is used extensively to dry heat sensitive biological material as lower inlet air temperatures are used and some fungal BCAs have been successfully formulated with this system. E. nigrum conidia dried twice, for 20 minutes at the highest air flow rate and at a temperature range of 30–40 °C, maintained 100% viability after 90 days at room temperature (Larena et al., 2003). Similar results were obtained with P. frequentans, demonstrating that fluidized bed-drying is the most suitable drying system for this BCA compared with freezeand spray-drying (Guijarro et al., 2006). The BCA like-yeast A. pullulans was also dried in a fluidized bed-dryer with a final viability of 62%. However, after seven months at 4 °C, viability was only 28% of the initial value (Mounir et al., 2007). Granular formulations may also be used to protect BCAs from environmental conditions. For example Kinay and Yildiz (2008) developed granular formulations of M. pulcherrima (M1/1) and P. guilliermondii (P1/3) containing talc, sodium alginate, sucrose, and yeast extract with high stability for up to six months for both viability and efficacy to control postharvest decay of citrus fruit. Isotonic liquid formulations of C. sake CPA-1 have been found as an alternative to solid formulations. Abadias et al. (2003b) described an improved liquid formulation of C. sake by growing cells in several media and preserved in a

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liquid with the same water potential of cells (isotonic solutions) using different compounds to adjust water activity (aw). Best results were for cells grown in a sorbitol-modified medium and preserved in an isotonic solution of trehalose (0.96 M), with stable viability after seven months at 4 °C and biocontrol efficacy. To reduce costs, sugars such as lactose and trehalose without sorbitol addition in the growth medium could be used (Torres et al., 2003). Liquid formulation has also been tested with R. minuta, using glycerol as aw reducer and xanthan gum as viscosity-enhancer, although yeast viability loss was observed after six months storage (Patiño-Vera et al., 2005). Trehalose was a suitable protectant for liquid formulations of C. laurentii, while galactose was better for P. membranaefaciens during storage at 4 and 25 °C. Combining L-Ascorbic acid with sugars further improved the protective efficiency, but formulation stability was only obtained with cold conditions (Liu et al., 2009). Independently of the method used for the preservation of microbial cells, additives are often used to improve the final product. They are added to a formulation for a specific purpose, normally to assist or modify the action of the active ingredient, and can be incorporated into biological products during mass production, formulation and storage, or added later to spray tank mixes (Burges, 1998). Additives can be used as stickers, diluents, suppressants, dispersants, emulsifiers, wetters, gelants, humectants, brighteners, spreaders, stabilizers, sunscreens, synergists, thickeners, nutrients, binders, or protectors, depending on their function in the formulation (Burges and Jones, 1998). P. frequentans conidial adhesion to peach fruit surfaces was improved when sodium alginate or carboxymethyl cellulose were added to the conidial mass before the fluidized bed-drying process, or the above additives and gelatine were added to conidia after fluidized bed-drying, improving brown rot control (Guijarro et al., 2008). Among different additives tested to improve the formulation of freeze-dried P. agglomerans cells, the addition of 5% Fungicover in the bacterial suspension improved adherence and persistence of freeze-dried P. agglomerans on oranges exposed to unfavourable conditions (Cañamás et al., 2008a). Fungicover is an edible film-forming compound for fruit and vegetables to reduce weight loss, delay senescence, improve natural brightness and reduce physiological disorders. It also reduces droplet size and improves uniformity of distribution on the surface to be protected and did not show any fungicidal effect on Penicillium digitatum (Cañamás et al., 2008a). Under experimental conditions reflecting practical conditions, skimmed milk applied in combination with C. oleophila (strain O) resulted in improved biocontrol efficacy by 74.65% (Lahlali and Jijakli, 2009). 15.5.3  Packaging and shelf life Another issue involved in the commercial development of biocontrol products is shelf life, which should be as long as possible. A biofungicide should be effective for at least six months and preferably for two years (Pusey, 1994). The BCA must be packaged and stored in such a way as to maintain the product in a suitable state and this may involve careful selection of the packaging material to control gas

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exchange, prevent the loss or gain of moisture and avoid contamination of the product (Powell, 1992). Few studies have been published on this topic as product shelf life is usually assessed by private industry, but the effect of storage temperature, packaging and atmosphere conditions on the stability and biocontrol efficacy of freeze-dried P. agglomerans cells was evaluated, concluding that freeze-dried cells could be stored in glass vials or in high barrier plastic bags at 4 °C while maintaining high viabilities and efficacy against P. digitatum (Costa et al., 2002c). To enhance shelf life of E. nigrum fluidized bed-dried conidia, nontoxic stabilizers were added at different stages of the production and drying processes. The highest viability was obtained when conidia were produced with 1% KCl or 50% PEG 8000, and dried with 2.5% methylcellulose or 1% KCl + silica powder, to produce a wettable powder with a shelf life of 365 days (Larena et al., 2007).

15.6  Improvement of biocontrol agents 15.6.1  Physiological manipulations Biological control of postharvest diseases of fruits has advanced greatly during the past decade and several microbial antagonists, based on either yeast or bacteria, have been developed. However, the success of these products remains limited and just a few microorganisms are commercially available to control postharvest decays. There are several reasons for the limited number of commercially available BCAs, such as the limited tolerance to fluctuating environmental conditions, the difficulties in developing a shelf-stable formulated product that is as effective as fresh cells due to stressing conditions such as low aw (dehydration) and high temperatures. Thus, improvement in physiological quality of the BCAs, such as stress adaptation, during production which can lead to better survival and activity under such environmental conditions is an important challenge for exploitation and suitability in commercial conditions. Research studies in improving BCAs’ (C. sake and P. agglomerans) behaviour in front stress conditions have been conducted achieving interesting results that allow enhanced biocontrol treatments at field conditions, improve BCAs behaviour during the formulation process and broaden their spectrum of action. Specific or general protection mechanisms are induced when microorganisms are grown under stressing conditions. Stress responses are characterized by a transient induction of general and specific proteins and by physiological changes that generally enhance the particular organism’s ability to withstand more adverse environmental conditions (Ang et al., 1991). In the case of osmotic stress, the significant physiological changes reported in bacteria include the induction of stress proteins, as well as the accumulation of compatible solutes such as K+ ions, the amino acids glutamate, glutamine, proline and alanine, the quaternary amines glycine betaine, the tetrahydropyrimidine ectoine and sugars such as sucrose and trehalose (Csonka, 1989; Ko et al., 1994). In yeasts and filamentous fungi the

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accumulated compatible solutes are mainly low- (glycerol and erythritol) and high- (arabitol and mannitol) molecular weight sugar alcohols (Beever and Laracy, 1986; Ellis et al., 1991; Van Eck et al., 1993). These compatible solutes allow equilibration of the cytoplasmic aw with the surrounding environment, thereby retaining water in the cell and thus maintaining turgor pressure, and helping to preserve protein function within cells (Yancey et al., 1982; Csonka, 1989; Van Eck et al., 1993). Subjection to a mild stress can make cells resistant to a lethal challenge with the same stress condition. Preadaptation to one particular stress condition can also render cells resistant to other stress conditions, a phenomenon known as cross-protection (Sanders et al., 1999). It is possible to improve BCAs’ stress tolerance by controlling growth medium and conditions, thus enhancing survival and efficacy both in formulation process (Usall et al., 2009) and preharvest applications (Teixidó et al., 2009). Formulation process enhancement The improvement of tolerance to low aw and desiccation in P. agglomerans cells subjected to mild osmotic stress during growth was studied using different solutes to change aw of growth media. It was shown that cells grown in media at low aw using NaCl exhibited osmotic adaptation in solid media at low aw, obtaining a high production level and maintaining biocontrol efficacy (Teixidó et al., 2006). Osmotic-adapted cells also demonstrated thermotolerance (Teixidó et al., 2005) and better desiccation tolerance after spray-drying (Teixidó et al., 2006), freezedrying (unpublished data) and fluidized bed-drying (Usall et al., 2009). P. agglomerans adapted cells not only showed better survival after the freezedrying process but also had longer shelf life and good biocontrol efficacy. The role of different compatible solutes in adaptation of the bacterium to osmotic stress was determined and data suggest that glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement (Teixidó et al., 2005; Cañamás et al., 2007). Osmoadaptation of another strain (EPS125) of P. agglomerans has also been described by Bonaterra et al. (2005). Significant improvements in low aw tolerance of C. sake cells were achieved by modifying both aw and a nutrient concentration of growth media. The best results were obtained with glucose and glycerol solutes, and the intracellular accumulation of polyols, glucose and trehalose in aw stress-improved cells was significantly different than with unmodified control cells (Teixidó et al. 1998b, 1998c). Another way to improve the tolerance of BCAs to desiccation at high temperature during spray-drying is to expose the microorganisms to mild temperatures during growth. C. sake cells grown in unmodified molasses medium exposed to mild heat treatments at 30 °C or 33 °C during mid or late-exponential or early or mid-stationary growth phases showed increased survival when exposed to lethal shock at 40 °C, but only a very small improvement after spray-drying (Cañamás et al., 2008b). In the case of P. agglomerans, the highest thermotolerance was obtained when thermal stress treatment (40 °C) was applied in late exponential or early stationary phase of growth (Usall et al., 2009).

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Finally, the combination of thermal and osmotic stresses was studied in order to improve fluidized bed-drying formulations of P. agglomerans. Using NaCl to adjust aw to 0.988 in the growth medium and increasing the temperature to 35 °C for one hour during early stationary growth phase reduced viability loss to only 0.5 log reductions during fluidized bed-drying (Usall et al., 2009). Preharvest treatments enhancement When BCAs are applied at postharvest, they often show reduced efficacy to control previously established or incipient infections, which originated in the field. Indeed, the effectiveness of the antagonist decreases when the time period between pathogen infection and application of the antagonist increases (Ippolito and Nigro, 2000). Biocontrol of fruit decay during storage with microbial antagonists has so far been mainly studied under controlled environmental conditions at postharvest: few studies have attempted to apply BCAs to fruit under field conditions with the purpose of controlling postharvest decay (Benbow and Sugar, 1999; Leibinger et al., 1997; Teixidó et al., 1998a). Infection of fruit by postharvest pathogens often occurs in the field prior to harvest as the pathogens P. digitatum and P. italicum infect fruit through wounds produced by mechanical injuries during the growing season and harvest handling operations (Obagwu and Korsten, 2003). An antagonist applied in the field, prior to harvest, can interact longer with the pathogen than when applied after harvest (Ippolito and Nigro, 2000). It would therefore be advantageous to apply antagonists before harvest, which would reduce initial infection, and keep them active to suppress pathogens during storage (Elad and Kirshner, 1992; Teixidó et al., 1998a). However, an important consideration for the application of BCAs at preharvest is their ability to colonize the surface of fruit both in the field and during storage and to persist, for as long as possible, in sufficient numbers on the fruit surface to maintain an efficient decay control. However, under field conditions, rapid fluctuations in water availability and temperature are both characteristic of this environment and constitute the main factors limiting the development of microbial populations (Köhl and Fokkema, 1998). Different formulation strategies of P. agglomerans cells were tested to improve survival under field conditions and efficacy in controlling postharvest rots in citrus, including lyophilised cells, osmotic adaptation by NaCl treatments (the above osmoadapted cells described) and different additives. In general, osmotic adapted and lyophilised P. agglomerans cells showed greater survival rates than non-osmotic adapted or fresh cells when these bacterial treatments were sprayed at field conditions and when the additive Fungicover was added to suspension treatments. The improved formulation of P. agglomerans provided an effective control for oranges against natural postharvest pathogen infections and P. digitatum induced infections (Cañamás et al., 2008a, 2008c). Therefore it is possible to improve environmental stress tolerance and ecological competence of P. agglomerans by integrating certain formulation strategies. Enhancing stress tolerance and formulation strategies could be appropriate approaches to obtain consistency and broaden the spectrum of use of BCAs.

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15.6.2  Genetic manipulations Genetic manipulation of antagonists to improve biocontrol of postharvest decays is another way to improve their consistency and regularity under commercial conditions. Current efforts are focused on developing efficient transformation procedures for bacterial and yeast antagonists and inserting genes for tracking the antagonist in the environment rather than enhancing biocontrol (Bassett and Janisiewicz, 2001; Nigro et al., 1999; Yehuda et al., 2001). However, it is possible to increase ecological competence and the capability to control the pathogens by manipulating antagonists with genetic tools as this offers tremendous potential for improving biocontrol of postharvest decays (Janisiewicz et al., 2008). For example, antagonists can be manipulated to over-express mechanisms of biocontrol, or foreign genes can be transferred to antagonists to increase tolerance to environmental stresses or to produce antifungal substances. Jones and Prusky (2002) used this approach to make Saccharomyces cerevisiae a BCA by cloning the gene for cecropin production, an antifungal defensin from insects. This genetically modified yeast expressed the cloned gene and controlled postharvest decay caused by Colletotrichum coccodes on tomatoes. Wisniewski et al. (2005) cloned and expressed in Pichia pastoris a defensin gene originally isolated from peach bark that had antifungal activity against B. cinerea and P. expansum in vitro. The production of defensin peptides is a common defence strategy present in a variety of organisms including plants, insects, humans and other mammals (Raj and Dentino, 2002). Defensins are not toxic to plants and are inhibitory to a variety of fungi in micromolar concentrations. These peptides are encoded by single genes and can be synthesized by the host with a minimal expenditure of energy (Thomma et al., 2002). Recently, a binary vector encoding the constitutive expression of the gene for the pea defensin Psd1 was used to transform the yeast P. pastoris, and transformed strains were evaluated for enhancing biocontrol potential by Psd1 with promising results (Janisiewicz et al., 2008). Since mycoparasitism is one mechanism of action involved in the biocontrol of postharvest fruit pathogens, another approach in genetic manipulations could be evaluated; cell wall degrading enzymes, such as chitinases, proteases and glucanases, produced by bacterial and fungal microorganisms, could be inserted into the potential antagonists to improve the degradation of the pathogen cell walls, resulting in death or growth inhibition of the antagonized fungus (Spadaro and Gullino, 2004). This area of research is still in its infancy although the application potential is high. Regulation limits of the release and acceptance of GMOs for field spreading or product treatment should be stated, because they certainly are a serious hurdle for developing applications of GM BCAs.

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15.7 Integration of biocontrol agents with other alternative methods The use of microorganisms as an alternative to fungicides is not the only method studied to control postharvest diseases, as many attempts have been made using physical and chemical treatments. They include heat treatments (Casals et al., 2010; Lurie, 1998), UV-C irradiation (Arcas et al., 2000), ozone (Palou et al., 2003), salt additives (Smilanick et al., 1999; Palou et al., 2002), plant extracts (Bautista-Baños et al., 2000; Wilson et al., 1997), volatile compounds (Mari et al., 2002); essential oils (Plaza et al., 2004a) and elicitors (El Ghaouth et al., 2000a). Generally, the efficacy of these alternative treatments is limited and influenced by many environmental factors (Janisiewicz and Korsten, 2002), sometimes the mode of action is fungistatic instead of fungicidal (Smilanick et al., 1999) or they do not provide protection on fruit after the treatment. Typically, the antagonistic activity of BCAs fails to control established infections. For these reasons, in recent years research has been focused on enhancing the efficacy of the postharvest alternative treatments managing the combination of two or more postharvest treatments. Efficacy and consistency of the BCAs can be enhanced by applying the microorganisms and other alternative treatments in a cascade, similar to the hurdle technology strategy used in the food industry. 15.7.1  Use of microorganisms in combination with chemical products Carbonic acid salts, such as sodium carbonate (SC, Na2CO 3, soda ash) or sodium bicarbonate (SBC, NaHCO 3), are generally considered as good candidates to be used in combination with biological methods for the integrated control of postharvest diseases, including citrus (Teixidó et al., 2001; Smilanick et al., 2005; Narayanasamy, 2006; Palou et al., 2007; Usall et al., 2008), apples (Conway et al., 2007), stone fruits (Droby et al., 2003; Karabulut et al., 2005), papaya (Gamagae et al., 2003) and tomatoes (Xi and Tian, 2005). These products are very attractive alternatives because they are readily available, inexpensive, and have low risk of phytotoxicity at the low concentrations (1–4%) used. These compounds are common food additives allowed with no restrictions for many applications by European and North American regulations and they are classified as generally recognized as safe (GRAS) by the United States Food and Drug Administration (US FDA) and listed as approved ingredients on products labelled ‘organic’ proposed by the United States Department of Agriculture (USDA), but are still not approved in the EU. Palou et al. (2002, 2009) evaluated a list of food additives as alternative or complementary chemicals to conventional fungicides for the control of major postharvest diseases of citrus and stone fruit. The best products could be combined with BCAs, as was done by Karabulut et al. (2001), with potassium sorbate and yeast antagonists to control postharvest decay in sweet cherries. The combination of C. sake at 106 CFU/ml and ammonium molybdate at 5 mM on apples and pears (Nunes et al., 2001a, 2002b) showed similar efficiency

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to control main postharvest diseases as C. sake at 107 CFU/ml. A similar strategy was developed to control P. expansum and M. fructicola in jujube fruits and sweet cherries, respectively, with the combination of C. laurentii and ammonium molybdate (Wan et al., 2003; Qin et al., 2006). Peracetic acid is another studied product for several crops such as peaches (Mari et al., 2004). A combination of this chemical low-residue product at sublethal dosage with a yeast commercial product (Shemer™) against Thielaviopsis basicola in carrots showed a synergistic effect (Eshel et al., 2009). Several products such as methyl jasmonate (Cao et al., 2009), salicylic acid (Yu et al., 2007a), giberelic acid (Yu et al., 2006) and chitosan (Yu et al., 2007b) have been shown to activate defence mechanisms associated with resistance to fungal decay. Chitosan and derivates such as glycolchitosan and carboxymethylchitosan have been studied in combination with BCAs. Chitosan is a molecular polymer, nontoxic, could act directly on the morphology of the microorganisms as a fungistatic or fungicidal treatment, can induce resistance in hosts, and has shown efficacy for controlling diseases in postharvest including brown rot in peaches (Li and Yu, 2001) and sweet cherries (Romanazzi et al., 2003). It has also been reported to control B. cinerea in grapes (Elmer and Reglinski, 2006). Several formulations with different efficacy exist on the market, from different origins and obtained following different procedures. A combination of C. saitoana with 0.2% of glycolchitosan was studied under semi-commercial conditions in apples and citrus, and was shown to be more effective in controlling their main pathogens than either treatment alone (El Ghaouth et al., 2000a). 15.7.2  Use of microorganisms in combination with physical treatments Many attempts to combine physical treatments with BCAs have been carried out, but the most successful were combinations with heat treatments. Hot water (HW) is a simple technique that can be easily used in packing houses to reduce postharvest diseases of several crops. A previous hot water treatment at 55 °C for 10 seconds could improve the later application of C. oleophila to control B. cinerea and P. expansum on peaches (Karabulut and Baykal, 2004). The combination of a preharvest application of E. nigrum and a postharvest sodium bicarbonate hot treatment (60 °C for 20 seconds and 1% SBC) significantly reduced the incidence of Monilinia spp. on stone fruits (Mari et al., 2007). The combination of the strain BIO126 of M. pulcherrima with hot water at 50 °C for 3 min and/or with some chemical treatments to control P. expansum and B. cinerea in apple improved the efficacy of the strain BIO126 alone (Spadaro et al., 2004). An application of P. agglomerans CPA-2 at 2×108 CFU/ml effectively reduced green mould incidence on recently inoculated lemons. However, it failed to control established infections of P. digitatum of more than 24 hours. A combination of P. agglomerans plus storage at 33 °C for 65 hours completely controlled 24-hour-old infections on artificially inoculated lemons stored at 20 °C for 14 days and on naturally infected lemons stored at 10 °C for 3 weeks plus 7 additional

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days at 20 °C (Plaza et al., 2004b). The combination of M. pulcherrima FMB24H-2 with storage at 38 °C for 4 days completely eliminated Colletotrichum acutatum in apple fruit (Conway et al., 2005). In the near future, the preferred alternative to chemical treatments will probably be a combination of different methods by incorporating several treatments along the packing line. Usall et al. (2008) considered the simulation of commercial situations in which reinfection of the same fruit may occur during handling and processing within the packing house. For such simulation, P. digitatum (at either the same or different rind infection sites) was reinoculated in fruit that had been previously inoculated or just wounded, and treated with sodium carbonates, the BCA P. agglomerans CPA-2, or the combination of these treatments. With this combination it is possible to protect both existing and new infections.

15.8  Hurdles for biocontrol commercial application Biological control of postharvest diseases of fruits and vegetables has advanced greatly during the past decade and several microbial antagonists, based on either yeast or bacteria were developed and commercially tested. However the success of these products remains limited and only a few microorganisms are commercially available to control the postharvest decay of fruit (Table 15.2). There are several reasons for this limited success, such as the inconsistency, variability of the efficacy under commercial conditions, limited tolerance to fluctuating environmental conditions and the difficulties in developing a shelf-stable formulated product that retains biocontrol activity similar to that of fresh cells. Also, not all pathosystems are likely to be successful. Diseases with a limited window of opportunity for infection, monocyclic cycles and low rates of progress as well as diseases affecting crops in some degree of environmental control (as postharvest storage), are logical targets (Fravel, 2005). However, there are also economic, political and social reasons that limit the introduction of new BCAs onto the market. Postharvest bioprotection of fruit and vegetables is a niche market, with a relatively small profit potential and the packing houses still have some fungicides available to use. The success of implementing biological control products will depend on product knowledge and a thorough understanding of the complexity of the disease and the postharvest environment and these requirements are often overlooked in the commercialization. Until now, small companies have been the leaders in developing biocontrol products for postharvest use, however the lack of financial resources and an established marketing network have been important obstacles for these small companies trying to commercialize their products. But something is now changing and big companies have started to show interest in biocontrol products. Legal authorization (phytosanitary registration) must be obtained before commercial application of BCAs. The registration process for a biofungicide in Europe is more difficult than elsewhere. Registration of a BCA by the EPA (Environmental Protection Agency, in the USA) requires an average of two years,

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Shemer

Pantovital

Metschnikowia fructicola

Candida oleophila strain O Pantoea agglomerans CPA-2

Nexy

Candifruit

Aureobasidium pullulans DSM 14941 DSM 14940 Candida sake CPA-1

Boni Protect®

Citrus fruit, Pome fruit, Grapes, Stone fruit, Strawberries, Sweet potatoes

Citrus fruit, Pome fruit

Pome fruit

Pome fruit

Pome fruit

Pome fruit, Potatoes, Sweet potatoes

B. cinerea, P. expansum B. cinerea, P. expansum, P. digitatum, Penicillium italicum, R. stolonifer Aspergillus niger, B. cinerea, P. expansum, P. digitatum, P. italicum, R. stolonifer

Fluidized bed-dried formulation Water dispersible granules (WDG)

Freeze-dried formulation Wettable powder (WP)

Dilutable powder

Liquid formulation Soluble concentrate (SL)

Water dispersible powder (WDP)

Freeze-dried formulation

Bayer Cropscience

BioDURCAL S.L.

BioNext sprl

SIPCAM-INAGRA S.A.

BioFerm GmbH

JET Harvest Solutions

Pseudomonas syringae ESC11

Bio-Save® 11LP

Freeze-dried formulation

JET Harvest Solutions

Botrytis cinerea, Fusarium sambucinum, Geotrichum candidum, Mucor piriformis, Penicillium expansum, Penicillium digitatum, Penicillium italicum B. cinerea, Fusarium sambucinum, M. piriformis, P. expansum, Rhizopus stolonifer B. cinerea, Monilinia fructigena, P. expansum, Pezicula malicortici B. cinerea, P. expansum, R. stolonife

Pseudomonas syringae ESC10

Bio-Save® 10LP

Citrus fruit, Cherries, Pome fruit, Potatoes

Manufacturer/ distributor

Product BCA Fruit/vegetables Pathogen Formulation

Ambient

Refrigeration or freezing

–

Refrigeration

Ambient

Refrigeration

Refrigeration

Storage

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Table 15.2  Biological products for controlling postharvest disease of fruit and vegetables

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while in Europe registration of the same products takes almost seven years. To accelerate the registration process, the European Community has supported a policy action, called REBECA (www.rebeca-net.de), that reviews the possible risks of BCAs, compares regulations in the EU and the USA and proposes less bureaucratic alternative and more efficient regulation procedures maintaining the same level of safety for human health and the environment but accelerating market access and lowering registration costs (Mari et al., 2009). Another problem is that non-target effects may also be possible, as pathogenicity to plants, and as allergenicity and toxicogenicity to animals and humans. In order to evaluate all these risks it is necessary to carry out additional studies such as hypersensitivity in tobacco and the effect on auxiliary organisms, but more important are toxicological studies in mammals. The most common test is the oral acute toxicity in rats to determine the median and limit of lethal dose, but other tests will be requested by administration for registration, increasing the final cost of commercialization. Concern has been raised about the health and safety of introducing antagonists into our diet. Although this may represent an obstacle to public acceptance of this technology, the majority of postharvest BCAs were originally isolated from fruit and vegetables and are indigenous to agricultural commodities. Therefore humans are exposed to them daily when consuming fresh vegetables and fruit. Even though these antagonists are added at high concentrations on the surface of a commodity, they survive and grow only in very restricted sites on the fruit surfaces (e.g. surface wounds) and after their introduction on intact fruit surfaces, populations usually diminish to the level of natural epiphytic microflora within a very short period of time (Droby et al., 2009).

15.9  Future trends It is likely that in the future more BCAs will become available in the market and their use will increase. However, it will be necessary to increase the level of familiarity with these new methods by all involved agents (regulatory agencies, packing houses, retailers, pesticide companies) and the pressure of good distributors and consumers for healthier fruits and environmentally friendly treatments would have to increase to generate a real positive ambient for their common use. The selection of new BCA microorganisms with enhanced host and environment spectrum of hosts is expected. Postharvest stable conditions provide an ideal niche for the BCAs, but preharvest application could become more widespread with the aim of controlling latent or preharvest infections or improving the colonization of BCAs in the injuries produced before storage. More ecophysiological research will be needed to understand and improve stress response behaviour and stimulate mechanisms of environmental survival of BCAs, in order to optimise their efficacy and suitability for practical conditions. It is also necessary to adapt the formulations of the BCAs to the current application strategies in production systems, to increase the shelf life and reduce the storage requirements. A deeper understanding of the interactions of host-pathogen-environment-BCA

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is needed to achieve the full potential of postharvest biocontrol as a viable commercial technology. Molecular, biochemical and pathological approaches simultaneously are necessary to better exploit defence responses of fruit and vegetables, to choose the targets to manipulate the capability of pathogens to cope with the host environment and to improve BCAs action in order to enhance biocontrol of diseases. An integrated strategy which includes the BCA and other alternative methods, combined with predictive models, host defence mechanisms and good agricultural practices that provide a suitable environment for antagonists and limit pathogen development, is necessary to achieve comparable efficacy results to chemicals with the well-known environmental advantages. But in the end, the expansion of BCAs’ commercial use will depend on the demand and the market. The organic production is an important growing market, still very small, that could benefit from the use of BCAs as one possible tool to reduce losses. In the same way the Integrated Pest Management (IPM) production where biological control should be an essential component for diseases control in order to minimize chemical residues, could also be a potential target. Finally, the conventional agriculture could also be an opportunity if consumers, consumer associations, retailers and Non-Governmental Organizations push the distributors to be stricter with the residue levels or the legislation becomes more restrictive. Scientific knowledge on biocontrol should be developed to ensure that alternatives are available when new strategies are required because of consumer pressure or just because the fruit and vegetables industry cannot use the current methods any longer.

15.10  Sources of further information and advice A large number of publications have been written on biocontrol in general during the last 20 years. However there is less information specifically on postharvest diseases. Some interesting books, chapters and reviews are listed below in order to provide a starting point in the search. boland g j and kuykendal l d (1998), Plant–Microbe Interactions and Biological Control, New York, Marcel Dekker Inc. • prusky d and gullino m l (2009), Postharvest Pathology, Vol 2, The Netherlands, Springer. • wilson c l and wisniewski m e (1994), Biological Control of Postharvest Diseases. Theory and Practice, Boca Raton, CRC Press.

•

Some reviews listed in the References section are also noteworthy: • • • •

Droby et al. (2009) Fravel (2005) Janisiewicz and Korsten (2002) Sharma et al. (2009)

There are also interesting papers in the IOBC/WPRS Bulletin published by the © Woodhead Publishing Limited, 2011

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International Organization for Biological and Integrated Control of Noxious Animals and Plants. Web pages: IOBC/WPRS: http://www.iobc-wprs.org and REBECA: www.rebeca-net.de.

15.11  Acknowledgements The authors thank M. Sisquella, C. Casals and C. Calvo for their valuable assistance in literature searches. Mention of trade names or commercial products in this chapter is solely for the purpose of providing scientific information and does not imply recommendation by the authors or their institution.

15.12  References abadias m , teixidó n , usall j , viñas i

and magan n (2000), ‘Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake’, J Appl Microbiol 89, 1009–1017. abadias m , benabarre a , teixidó n , usall j and viñas i (2001a), ‘Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake’, Int J Food Microbiol 65, 173–182. abadias m , teixidó n , usall j , benabarre a and viñas i (2001b), ‘Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media’, J Food Protect 64, 856–861. abadias m , teixidó n , usall j and viñas i (2003a), ‘Optimization of growth conditions of the postharvest biocontrol agent Candida sake CPA-1 in a lab-scale fermenter’, J Appl Microbiol 95, 301–309. abadias m, usall j, teixidó n and viñas i (2003b), ‘Liquid formulation of postharvest biocontrol agent Candida sake CPA-1 in isotonic solutions’, Phytopathology 93, 436–442. abadias m , teixidó n , usall j , solsona c and viñas i (2005), ‘Survival of the postharvest biocontrol yeast Candida sake CPA-1 after dehydration by spray-drying’, Biocontrol Sci Techn 15, 835–846. adeline t s y and sijam k (1999), ‘Biological control of bacterial soft rot of cabbage’, in Hong L W, Sastroutomo S, Caunter I G, Ali J, Yeang L K, Vijaysegaran S and Sen Y H, Biological Control in the Tropics: Towards Efficient Biodiversity and Bioresource Management for Effective Biological Control: Proceedings of the Symposium on Biological Control in the Tropics, Wallingford, CABI Publishing, 133–134. adikaram n k b and karunaratne a (1998), ‘Suppression of avocado anthracnose and stem-end rot pathogens by endogenous antifungal substances and a surface inhabiting Pestalotiopsis sp.’, ACIAR Proceedings Series 80, 72–77. ang d , libereck k , skowyra d , zylicz m and georgopoulos c (1991), ‘Biological role and regulation of the universally conserved heat shock proteins’, J Biol Chem 266, 24233–24236. arcas m c , botía j m , ortuño a m and del río j a (2000), ‘UV irradition alters the levels of flavonoids involved in the defence mechanism of Citrus aurantium fruits against Penicillium digitatum’, Eur J Plant Pathol 106, 617–622. arras g (1996), ‘Mode of action of an isolate of Candida famata in biological control of Penicillium digitatum in orange fruit’, Postharvest Biol Technol 8, 191–198. arras g , de montis s and sussarellu l (1996), ‘Characterization of yeasts (Pichia guilliermondii and Rhodotorula glutinis) antagonistic to Penicillium digitatum’, Ann Microbiol Enzim 46, 285–298.

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and chen j k (2007), ‘Inhibitory effect of Bacillus subtilis B10 on the diseases of postharvest strawberry’, J Fruit Sci 24, 339–343. zhao y, tu k , shao x , jing w and su z (2008), ‘Effects of the yeast Pichia guilliermondii against Rhizopus nigricans on tomato fruit’, Postharvest Biol Technol 49, 113–120. zhou t , northover j and schneider k e (1999), ‘Biological control of postharvest diseases of peach with phyllosphere isolates of Pseudomonas syringae’, Can J Plant Pathol 21, 375–381. zhou t , chu c l , liu w t and schneider k e (2001), ‘Postharvest control of blue mold and gray mold on apples using isolates of Pseudomonas syringae’, Can J Plant Pathol 23, 246–252. zhao y, shao x f , tu k

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16 Biological control of pathogens and post-processing spoilage microorganisms in fresh and processed fruit and vegetables A. Gálvez, H. Abriouel, R. L. López and N. Ben Omar, University of Jaén, Spain

Abstract: This chapter discusses the application of biopreservation methods in raw and processed vegetable foods and fruits, fruit juices, canned vegetables, and fermented vegetables, and the efficacy of treatments on the survival and proliferation of pathogenic and spoilage microorganisms. Different approaches are discussed, such as competitive exclusion, treatment with bacteriophages, and application of bacteriocins and lytic enzymes as washing treatments or added to the foods alone or in combination with physico-chemical treatments. Application of bacteriocin-producing cultures to improve vegetable fermentations is also considered. Key words: biopreservation, fruits and vegetables, fruit juices, processed vegetable foods, fermented vegetables, competitive exclusion, bacteriocins, antimicrobial peptides, bacteriophages.

16.1

Introduction

The fruit and vegetable food industry is permanently facing new issues regarding preservation, economic losses and safety aspects of raw materials and products. On one side, the types of food-processing treatments that can be applied to certain categories of food products (such as the raw or lightly processed fruits and vegetables) is strongly limited by the high vulnerability of the food organoleptic properties and physical aspect. On the other hand, consumers demand vegetable foods that are fresh-tasting, ready to eat, more naturally preserved, minimally processed and available on the market all year around. The globalization of food 403 © Woodhead Publishing Limited, 2011

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markets creates new needs to prolong the shelf life of raw materials (and sometimes processed products as well), given the longer periods of time required to take the product from farm to fork, but also creates new opportunities for pathogen growth. For example, specific pathogenic strains previously endemic to a geographic region can now spread easily and infect new hosts that were previously unexposed and therefore may be even more susceptible. Also, the extensive use of cold preservation and the application of chemical disinfectants selects for adapted or resistant bacterial strains that tend to persist in the food chain and can be pathogenic or produce toxins in food products. Within the global food market, it is much more difficult to control the sanitary conditions along the food chain, starting with observation of sound agricultural practices, quality of water used for irrigation and postharvest processing, or correct handling of food products. The issues stated above highlight the potential of biopreservation for the vegetable food industry. Biopreservation refers to the use of living cells and/or their antimicrobial products to control foodborne pathogenic, toxinogenic, or spoilage microbes (Ross et al., 2002). So far, lactic acid bacteria (LAB) are the best known microbial group for their capacity to displace harmful bacterial populations in food systems thanks to their capacity to compete for the nutrients and create a selective environment due to the production of an array of antimicrobial substances such as organic acids, diacetyl, acetoin, hydrogen peroxide, reuterin, reutericidin, as well as antifungal and antibacterial peptides or bacteriocins (Holzapfel et al., 1995; Cleveland et al., 2001). The bacteriocins can be defined as ribosomally synthesized antimicrobial peptides or proteins produced by bacteria (Jack et al., 1995). Microbes can also be applied as cell factories to produce a broader variety of antimicrobials, such as lytic enzymes, genetically modified antimicrobial peptides of diverse origin, and for the massive propagation of lytic bacteriophages. Many of these strategies can be applied (or have already been tested) for biopreservation of vegetable foods. Interestingly, the potency (as well as the spectrum) of these natural antimicrobials can be increased in combination with other antimicrobials or physico-chemical treatments, an approach currently known as hurdle technology (Leistner, 2000). The vegetable food and drink industries use a wide variety of raw materials, processing conditions and technologies leading to a broad range of products: raw fruit and vegetables, ready-to-eat vegetable foods, canned products, fermented vegetables, fruit juices and drinks, as well as beverages. They also vary greatly in their general microbial composition, ranging from the complex microbiota of raw vegetables to the more specific groups (such as spore formers) that can be found in processed foods such as canned vegetables. Therefore, strategies for biopreservation (as well as their efficacy) will depend greatly on the type of food product, target microbiota, and combination with other treatments compatible with food processing (Gálvez et al., 2007). For example, living microbial cells and bacteriophages could be applied in raw vegetable foods for the competitive exclusion or infective suppression of target bacteria, although they could not be used in processed food products such as fruit juices or canned vegetables. In this chapter the potential and limits of biopreservation strategies for different vegetable food categories are reviewed.

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16.2

405

Biocontrol of bacterial pathogens in fresh-cut produce

Fresh-cut produce represents one of the largest food industry sectors all over the world, and the microbiological quality of the produce relies largely on good hygiene practices during farming and processing. Recent findings indicating that bacterial pathogens can not only survive but also multiply on vegetables (Brackett, 1999; Palmai and Buchanan, 2002; Castillejo Rodríguez et al., 2000; Cobo Molinos et al., 2005, 2008a, 2008b) are rapidly changing our perception on the safety of vegetable foods. Raw vegetables have been shown to be the vehicle for transmission in a number of outbreaks of foodborne illness (Beuchat, 2002). Since fresh-cut produce is most often consumed without cooking, the risk for transmission of pathogens is greater compared to cooked foods. An additional concern is the population susceptibility (such as the elderly and immunocompromised people). Consequently the FDA has warned against consumption of raw vegetables which are known to act as vehicles for bacterial pathogens. In order to control foodborne pathogens in fresh produce, different approaches have been proposed such as inoculation with microbial strains producing antagonistic substances, treatment with specific bacteriophages, or addition of bacteriocin preparations alone or in combination with other treatments (Table 16.1). 16.2.1 Microbial strains Microorganisms originally isolated from vegetables are probably the best candidates for improving the microbiological safety of fresh produce, because they are better adapted to the environmental conditions in vegetables and should therefore be more competitive than protective cultures from other sources. However, the capacity of microbes to grow and produce antimicrobial substances at low temperature, the influence of the vegetable substrate on growth and bacteriocin production, and inhibition caused by other food and postharvest related factors must be taken into consideration. Several studies have confirmed that bacterial as well as yeast strains from vegetables can produce antimicrobial substances active against foodborne pathogens, highlighting their potential for biopreservation (Carlin et al., 1996; Buchanan and Bagi, 1999; Liao and Fett, 2001; Randazzo et al., 2004; Wilderdyke et al., 2004; Allende et al., 2007; Ponce et al., 2008). Nisin-producing lactococci from bean sprouts reduced the levels of L. monocytogenes in Caesar salad during refrigeration storage (Cai et al., 1997), while Pseudomonas fluorescens A3 and yeast strain D1 reduced the growth of Salmonella Chester and L. monocytogenes on green pepper disks by 1 and 2 logs, respectively (Liao and Fett, 2001). Strains A3 and D1 were suggested as potential biopreservatives for enhancing the quality and safety of fresh produce. Another study reported that leuconostocs and other LAB strains isolated from fresh vegetables and fresh fruit were able to inhibit foodborne bacterial pathogens (Trias et al., 2008a). The antagonistic strains grew on Iceberg lettuce leaf cuts without negative effects on the general aspect of tissues, reduced the cell counts of S. Typhimurium and E. coli by 1 to 2 log cycles,

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Washing treatments with enterocin AS-48 in combination with several other antimicrobials

Bacteriophage cocktail containing three Myoviridae phages lytic for E. coli O157:H7

Combined treatments

Bacteriophages

Soaking in semi-crude colicin Hu194 preparation

Washing treatments with enterocin AS-48 (alfalfa, soybean sprouts and green asparagus)

Washing treatment with bioactive preparations from LAB strains (nisin Z, coagulin and a nisin:coagulin cocktail) Treatment with BLIS (RUC9) from L. lactis RUC 9 Addition of enterocin AS-48

Washing treatments with nisin and pediocin

Culture supernatant of Lb. casei Mundticin preparation

Bacteriocin preparations

Significant reduction of viable E. coli O157:H7 cells on tomato, spinach, and broccoli surfaces

Increased inactivation of L. monocytogenes, B. cereus and B. weihenstephanensis on sprouts, and protection during storage Reduction or complete inactivation of S. enterica, E. coli O157:H7, Shigella spp., E. aerogenes, Y. enterocolitica, A. hydrophila and P. fluorescens populations on sprouts stored at 6 °C and 15 °C

Reduced coliform counts in salads Delayed growth of L. monocytogenes on modified atmosphere-stored mung bean sprouts Reduction of L. monocytogenes populations on cabbage, broccoli, and mung bean sprouts Reduction of L. monocytogenes cell counts on fresh-cut iceberg lettuce stored in microperforated plastic bags; lack of protection during storage Reduction of L. monocytogenes cell counts on iceberg lettuce Strong inhibition of S. aureus and complete inactivation of L. monocytogenes and B. cereus in lettuce juice Reduced viable cell counts of L. monocytogenes, B. cereus and B. weihenstephanensis, but lack of protection during storage at abuse temperature Variable inactivation of E. coli strains on alfalfa seeds

Nisin-producing lactococci isolated from bean sprouts Reduced levels of L. monocytogenes after refrigeration storage Microbial isolates (Bacillus spp. strains, P. Strains A3 and D1 inhibited S. Chester and L. aeruginosa, P. fluorescens A3, and yeast strain D1) monocytogenes on green pepper disks LAB strains isolated from fresh vegetables and fresh Complete inhibition of L. monocytogenes and reduction fruit (mainly Leuconostoc spp. and Lb. plantarum of S. Typhimurium and E. coli counts on lettuce cuts strains; Weissella spp. and L. lactis strains)

Microbial strains

Treatment

Effect

Abuladze et al., 2008

Cobo Molinos et al., 2008b

Cobo Molinos et al., 2005, 2008a

Nandiwada et al., 2004

Cobo Molinos et al., 2005, 2008a

Grande et al., 2005

Randazzo et al., 2009

Allende et al., 2007

Bari et al., 2005

Torriani et al., 1997 Bennik et al., 1999

Trias et al., 2008a,b

Liao and Fett, 2001

Cai et al., 1997

Reference(s)

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Table 16.1  Biocontrol of bacterial pathogens in fresh-cut produce

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407

and inhibited growth of L. monocytogenes completely (Trias et al., 2008a). Leuconostoc strains produced organic acids, hydrogen peroxide and the bacteriocin mesentericin Y105 as main inhibitors (Trias et al., 2008b). Ln. mesenteroides CM160 was the most effective strain against L. monocytogenes, and the authors stressed its high potential for prevention of L. monocytogenes in vegetables (Trias et al., 2008b). 16.2.2 Bacteriocin preparations Bacteriocins and BLIS solutions can be applied as crude, partially purified or purified preparations. Torriani et al. (1997) reported that salads treated with culture supernatant of a Lactobacillus casei strain had reduced coliform counts. A mundticin preparation retarded growth of L. monocytogenes in modified atmosphere-stored mung bean sprouts when used in a washing step or a coating procedure (Bennik et al., 1999). Nisin and pediocin solutions were tested as possible sanitizer treatments on cabbage, broccoli, and mung bean sprouts, resulting in 1.11 to 1.94 (for pediocin) or 1.31 to 2.77 (for nisin) log reductions of the inoculated five-strain cocktail of L. monocytogenes (Bari et al., 2005). Bacteriocin preparations (nisin Z, coagulin and a nisin:coagulin cocktail) produced by cultivation of selected LAB strains on a lettuce extract reduced viable cell counts of L. monocytogenes on fresh-cut Iceberg lettuce stored in microperforated plastic bags, but did not prevent further growth of survivors during refrigeration storage of samples (Allende et al., 2007). Treatment of minimally processed Iceberg lettuce with a BLIS from strain L. lactis RUC 9 (previously isolated from minimally processed mixed salads) reduced L. monocytogenes viable counts by 2.7 logs after 7 days of storage at 4 °C, suggesting that this treatment could be used to improve microbial safety and reduce the chemical treatment in vegetable processing (Randazzo et al., 2009). One main limitation of studies in vegetable foods is the large amounts of bacteriocin preparation required for the challenge tests. Most of the bacteriocins currently produced on a large scale (such as nisin or pediocins) are obtained as milkbased concentrates, which may impart undesirable taste to the treated product. By contrast, partially-purified preparations of enterocin AS-48 (a cyclic peptide from E. faecalis; Gálvez et al., 1989) are easily produced in semi-synthetic media (Abriouel et al., 2003), which makes this bacteriocin an amenable antimicrobial for testing on vegetable foods. In lettuce juice, added enterocin AS-48 had a strong inhibitory effect on S. aureus and completely inactivated L. monocytogenes and B. cereus (Grande et al., 2005), and when tested in washing treatments for decontamination of alfalfa, soybean sprouts and green asparagus spiked with L. monocytogenes, counts were reduced by up to 2.4 log cycles (Cobo Molinos et al., 2005). No viable listeria were detected in stored samples at 6 to 15 °C for 7 days, but regrowth was often observed at 22 °C (Cobo Molinos et al., 2005). Similar trends were observed for samples spiked with Bacillus cereus and Bacillus weihenstephanensis during storage at 6 °C, although bacteriocin treatment failed to prevent growth of the bacilli in samples stored at higher temperatures (15 or 22 °C; Cobo Molinos et al., 2008a).

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Transmission of enteric bacteria through fresh-cut produce is one of the main concerns in this food industry sector. Because of the permeability barrier imposed by the cell outer membrane, Gram-negative bacteria are in general resistant to bacteriocins from Gram-positive bacteria. Exceptions are some bacteriocins described recently like bacteriocin OR-7 from Lactobacillus salivarius (Stern et al., 2006), acidocin 1B from Lactobacillus acidophilus (Han et al., 2007), bacteriocin L23 from Lactobacillus fermentum (Pascual et al., 2008), plantaricin from Lactobacillus plantarum LP31 (Müller et al., 2009), enterocin E760 from Enterococcus sp. NRRL B-30745 (Line et al., 2008), and enterocin E50-52 from E. faecium (Svetoch et al., 2008), together with the colicins and microcins produced by Gram-negatives. Among them, only colicin Hu194 has been tested so far on fresh produce (Nandiwada et al., 2004). Semi-crude colicin Hu194 showed variable effects on E. coli O157:H7 strains 43890, 43895 and 3081 in alfalfa seeds. Strain 43890 was successfully inactivated (5 log CFU/g) from inoculated alfalfa seeds after soaking in a colicin Hu194 suspension (10,000 AU/g), while strains 43895 and 3081 required 20-fold higher colicin concentrations to achieve a reduction of 3 log cycles (Nandiwada et al., 2004). Variations in strain sensitivity to colicins (and other bacteriocins as well) need to be further investigated. 16.2.3 Combined treatments with bacteriocins In order to increase the efficacy of treatments and decrease the risk of proliferation of survivors during storage, bacteriocins have been tested in combination with several other types of antimicrobials. Antilisterial activity of nisin and pediocin washing treatments increased remarkably in combination with citric acid, sodium lactate, potassium sorbate, and phytic acid (Bari et al., 2005). Best results (4.25 log cycles reduction) were reported for nisin-phytic acid on cabbage; however, lowest reductions were always reported for mung bean sprouts, indicating the influence of the food substrate on the efficacy of treatments. The effect of enterocin AS-48 against L. monocytogenes on sprouts could also be potentiated by several antimicrobial agents (Cobo Molinos et al., 2005). Treatment with solutions containing combinations of AS-48 and chemical preservatives or sanitizers (including lactic acid, sodium lactate, sodium nitrate, tri-sodium phosphate, tri-sodium tri-metaphosphate, n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid or sodium hypochlorite) yielded no detectable listeria on sprouts and avoided regrowth during storage, providing an effective method for inactivation of L. monocytogenes on sprouts (Cobo Molinos et al., 2005). Similarly, combined treatments of enterocin AS-48 and other antimicrobials (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the bactericidal effects against B. cereus and B. weihenstephanensis on sprouts (Cobo Molinos et al., 2008a). The combinations of AS-48 and sodium hypochlorite, peracetic acid, or hexadecylpyridinium chloride prevented growth of the bacilli for one week at 15 °C (Cobo Molinos et al., 2008a).

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The effects of bacteriocins against Gram-negative bacteria can be enhanced by cell-damaging treatments, especially those that destabilize the bacterial outer membrane. For example, inactivation of Salmonella enterica cells inoculated on soybean sprouts increased greatly when sprouts were heated for 5 min at 65 °C in an alkaline solution (25 µg/ml, pH 9.0) of enterocin AS-48 (Cobo Molinos et al., 2008b). Increased microbial inactivation was also reported for the combinations of enterocin AS-48 and EDTA, lactic acid, peracetic acid, polyphosphoric acid, sodium hypochlorite, hexadecylpyridinium chloride, propyl-p-hydroxybenzoate, or hydrocinnamic acid. Combinations of enterocin AS-48 (25 µg/ml) and polyphosphoric acid (0.1 to 2.0%) significantly reduced the populations of S. enterica, E. coli O157:H7, Shigella spp., Enterobacter aerogenes, Yersinia enterocolitica, Aeromonas hydrophila and P. fluorescens in sprout samples stored at temperatures of 6 °C and 15 °C. 16.2.4 Bacteriophages Application of bacteriophages as biocontrol agents in foods has been proposed in several studies (reviewed by Greer, 2005 and by Hudson et al., 2005). In 2007, the United States Food and Drug Administration (FDA) approved a Listeria-specific bacteriophage preparation, ‘Listex P100’, for food preservation on ready-to-eat meat and deli products (U.S. Food and Drug Administration, 2007). A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for E. coli O157:H7 was examined for its ability to reduce experimental contamination of inert surfaces and foods including tomato, spinach, and broccoli (Abuladze et al., 2008). Treatment with a phage suspension (109 PFU/ml) significantly reduced E. coli O157:H7 counts for all three vegetables, with effectiveness ranging from 94% in tomato to 100% in spinach. Pao et al. (2004) tested the potential of lytic phages (the Myoviridae Phage-A, specific for Salmonella Typhimurium and Salmonella Enteritidis, and the Siphoviridae Phage-B, specific for Salmonella Montevideo) in experimentally contaminated broccoli and mustard seed with promising results. A 1.37 log suppression of Salmonella growth was achieved by applying Phage-A on mustard seeds, while the mixture of Phage-A and Phage-B caused a 1.50 log suppression of Salmonella growth in the soaking water of broccoli seeds. Because of the host specificity of phages, the authors stressed the importance of developing phage mixtures that can control a broad range of potential contaminants. By contrast, contradictory results were reported when two Salmonella bacteriophages (SSP5 and SSP6, also belonging to the Myoviridae and Siphoviridae families, respectively) were evaluated against Salmonella Oranienburg in vitro and on experimentally contaminated alfalfa seeds, suggesting the existence of a temporary, acquired, non-specific phage resistance phenomenon (Kocharunchitt et al., 2009). These factors may complicate the use of phages for biocontrol. In conclusion, several biocontrol strategies can improve the control of foodborne pathogenic bacteria on fresh produce. Application of protective cultures based on antagonistic bacterial strains isolated from vegetables may be a promising

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approach, but more research is needed to solve critical issues like the narrow spectrum and low antagonistic activity of protective cultures during refrigeration storage of vegetable products. Treatment with bacteriocin preparations may solve the problems associated with low in situ bacteriocin production and since bacteriocins have not been reported to elicit adverse effects on vegetable cells or tissues, they could be applied for decontamination of fresh produce. Some of the recently-described bacteriocins active against Gram-negative bacteria as well as combinations of different bacteriocins should be tested for inactivation of enteric pathogens. Nevertheless, the contact time of washing treatments may not be sufficient to kill the bacteria located on the vegetable surface or even inside plant tissue or associated with dirt particles. Furthermore, the residual bacteriocin concentration on the treated vegetable matter may be too low to kill the remaining viable or sublethally injured bacteria during storage. For these reasons, additional hurdles (such as refrigeration storage) should be applied to avoid regrowth of survivors during storage. Antimicrobial activity of bacteriocins can be increased by combination with other antimicrobials such as sanitizers, while the effects of treatments with cocktails of different bacteriocins should also be investigated. Bacteriophages may be an interesting approach as biocontrol agents on fresh produce, especially against Gram-negative bacteria and given the host specificity of phages, biocontrol strategies should be based on broad spectrum phage mixtures. Phages could also be applied in combination with bacteriocins or with bacteriocin-producing, phage-resistant strains. However, this is still an unexplored field where more research needs to be carried out.

16.3

Biocontrol strategies for minimal processed fruits

Sliced fruits may be contaminated with bacterial pathogens from the fruit surface (as well as other cross-contamination sources). Although survival of bacterial pathogens on sliced fruits is strongly limited by the organic acid content and low pH, others (such as melon or watermelon or pear) have pH values sufficiently high to allow bacterial growth and survival. Several biocontrol strategies have been tested to avoid transmission of pathogenic bacteria through minimal processed fruits (Table 16.2). 16.3.1 Microbial strains Biocontrol of postharvest decay of fruits has been widely investigated (reviewed by Droby et al., 2009 and by Sharma et al., 2009). Some microbial strains responsible for antagonism of fungi and bacteria causing postharvest decay of fruits can also displace human pathogenic bacteria from fruit surfaces and tissues. A saprophytic Pseudomonas syringae (sold in BioSave 110, a frozen liquid formulation for controlling postharvest decays caused by molds on apples and pears) was shown to prevent the growth of E. coli on apple wounds (Janisiewicz et al., 1999). Other microbial strains used to control postharvest decay caused by © Woodhead Publishing Limited, 2011

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Bacteriophages

Bacteriocins alone or in combined treatments

Microbial strains

Effect Effective in preventing the growth of E. coli in apple wounds Effective in preventing the growth or survival of L. monocytogenes and S. enterica on fresh-cut apple tissue

Reduction of S. Typhimurium and E. coli viable cell counts; complete inhibition of L. monocytogenes on wounds of Golden Delicious apples Reduction of L. monocytogenes counts on honeydew melon and apple slices. Increased listericidal effect in combination with a phage mixture Nisin combination with hydrogen Decontamination of whole cantaloupe and honeydew melon peroxide, sodium lactate and surfaces; prevented further transfer of the inoculated L. citric acid monocytogenes and E. coli to fresh-cut pieces during processing Nisin combinations with sodium Reduction of Salmonella counts on whole and fresh-cut cantaloupe lactate, potassium sorbate or both Nisin-EDTA sanitation treatment Reduced microbial growth on fresh-cut Galia melon Washing treatments with Partial or complete inactivation of L. monocytogenes on strawberries, enterocin AS-48 raspberries and blackberries at 15 and 22 °C for up to 2 days, and on blackberries and strawberries at 6 °C for up to 7 days. Reduction of L. monocytogenes viable counts in sliced melon, watermelon, pear and kiwi; regrowth observed during storage at 15 and 22 °C Enterocin AS-48 combination Complete inactivation and regrowth inhibition of L. monocytogenes with carvacrol or with n-propyl on sliced melon stored at 22 °C p-hydroxybenzoate Addition of nisin, or bovicin HC5 Prevention of gas formation caused by C. tyrobutyricum in mango pulp Mixture of four distinct lytic Significant reduction in the numbers of salmonellae (ca. 3.5 logs) on phages specific for Salmonella honeydew melon slices stored at 5 and 10 °C (ca. 3.5 logs) or Enteritidis 20 °C (ca. 2.5 logs). No significant effects on apple slices due to phage inactivation Mixture of listeriophages Reduction of L. monocytogenes counts by 2.0 to 4.6 logs on honeydew melon slices stored at 10 °C. No significant effect on Golden Delicious apples

Treatment P. syringae (BioSave 110) G. asaii (T1-D1), Candida sp. (T4-E4), D. fagi (ST1-C9), M. pulcherrima (T1-E2) Antagonistic LAB strains isolated from fruits and vegetables Nisin

Table 16.2  Biocontrol strategies for minimal processed fruits

Leverentz et al., 2003

De Carvalho et al., 2007 Leverentz et al., 2001

Cobo Molinos et al., 2008c

Silveira et al., 2008 Cobo Molinos et al., 2008c

Ukuku and Fett, 2004

Ukuku et al., 2005

Leverentz et al., 2003

Trias et al., 2008a,b

Reference(s) Janisiewicz et al., 1999 Leverentz et al., 2006

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fungi on fruits were tested against L. monocytogenes and S. enterica serovar Poona on fresh-cut apples (Leverentz et al., 2006). Four of the antagonists, including Gluconobacter asaii (T1-D1), a Candida sp. (T4-E4), Discosphaerina fagi (ST1-C9), and Metschnikowia pulcherrima (T1-E2), were effective in preventing the growth or survival of L. monocytogenes and S. enterica. However, most of the remaining antagonist strains did in fact increase the growth of foodborne pathogens, which raises concerns about the influence of postharvest biocontrol on the survival of human pathogens (Leverentz et al., 2006). Antagonistic LAB strains isolated from fruits and vegetables reduced cell counts of Salmonella Typhimurium and E. coli and completely inhibited growth of L. monocytogenes on wounds of Golden Delicious apples (Trias et al., 2008a). Among them, strain Ln. mesenteroides CM160 was the most effective against L. monocytogenes (Trias et al., 2008b). 16.3.2 Bacteriocin preparations and combined treatments Bacteriocins can be applied alone or in combination with other antimicrobials as sanitation treatments on fruit surfaces and sliced fruits, as exemplified by nisin. The single application of nisin reduced L. monocytogenes populations on honeydew melon slices and apple slices, and its listericidal effect was enhanced in combination with a phage mixture (Leverentz et al., 2003). Decontamination of whole cantaloupe and honeydew melon surfaces with a combination of hydrogen peroxide, nisin, sodium lactate and citric acid prevented further transfer of L. monocytogenes and E. coli to fresh-cut pieces during processing (Ukuku et al., 2005). The combination treatments nisin and sodium lactate, nisin and potassium sorbate or both gave significant reductions of Salmonella inoculated onto whole and fresh-cut cantaloupe (Ukuku and Fett, 2004). Application of a nisin-EDTA sanitation treatment (250 mg/l nisin plus 100 mg/l EDTA) in fresh-cut ‘Galia’ melon reduced microbial growth more efficiently than chlorine treatment (Silveira et al., 2008). Washing treatments with enterocin AS-48 (25 µg/ml) significantly inhibited or completely inactivated L. monocytogenes on whole or sliced fruits (strawberries, raspberries, blackberries, melon, watermelon, pear and kiwi) but did not avoid proliferation of survivors during storage at 15 or 22 °C (Cobo Molinos et al., 2008c). The combined treatments of enterocin AS-48 and 12 mM carvacrol or 100 mM n-propyl p-hydroxybenzoate avoided regrowth of Listeria during storage at 22 °C and the authors concluded that enterocin AS-48 alone or in combination with other preservatives could serve as an additional hurdle against L. monocytogenes in fruits. Minimal-processed fruits are used in the manufacture of several food products, such as fruit yoghurt, acting as vehicles for contaminating microbes. Nisin was ineffective in controlling fruit yoghurt spoilage (Penney et al., 2004), while in mango pulp, nisin and bovicin HC5 (a bacteriocin from Streptococcus bovis HC5) prevented gas production by Clostridium tyrobutyricum (De Carvalho et al., 2007). But this is still an unexplored field for bacteriocin application where more research needs to be done. © Woodhead Publishing Limited, 2011

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16.3.3 Bacteriophages Lytic bacteriophages may provide an attractive alternative for decontaminating fresh-cut fruits that may contain various types of bacterial pathogens. A mixture of four lytic phages specific for Salmonella Enteritidis produced a significant inactivation of salmonellae (2.5 to 3.5 logs) on honeydew melon slices (Leverentz et al., 2001). However, the phages did not significantly reduce Salmonella populations on apple slices as the phages could not persist, and phage numbers declined to below detectable levels within 24 hours (Leverentz et al., 2001). Similarly, a mixture of listeriophages reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melon slices, but it had no effect on Golden Delicious apples (Leverentz et al., 2003). The listeriophage titre was stable on melon slices, but declined rapidly on apple slices (Leverentz et al., 2003). These results were attributed to the increased sensitivity of phages to the more acid environment of the apple slices (pH 4.37) compared with that of the melon slices (pH 5.77), and clearly suggest that the food pH may be an important limiting factor for application of phages on sliced fruits. In conclusion, the health benefits attributed to fresh fruits and the growing trend to consume sliced fruit products strengthens the needs for application of novel preservation methods including biopreservation. Application of biocontrol strategies on whole and sliced fruits may help to reduce the microbial load and avoid transmission of pathogenic bacteria. Inoculation with antagonistic microbial strains to avoid postharvest decay of fruits and at the same time inhibit foodborne human pathogens on fruit surfaces seems a promising alternative, although further research needs to be carried out on the microbial ecology of fruit surfaces in order to determine the influence of environmental factors on synergistic and antagonistic interactions between microbial communities. Bacteriocin preparations (either alone or in combination with other antimicrobials) can also be applied on the surfaces of intact fruits as well as on sliced fruits in order to reduce microbial contamination levels and increase the product safety during further storage under refrigeration. However, application of phage mixtures should be restricted to intact fruit surfaces, due to phage inactivation under the acidic conditions of most sliced fruits.

16.4 Application of bacteriocins in fruit juices and vegetable drinks Freshly-made fruit juices have been implicated in food poisoning episodes, mainly due to contamination of fruits, and juices are also prone to bacterial spoilage, especially during long-distance transportation in large containers. Since the organoleptic properties and vitamin content are key parameters of fruit juices, application of efficient heat treatments is not always possible. For this reason, application of alternative mild preservation technologies, including bacteriocins, may be useful to avoid many bacterial problems in fruit juices (Table 16.3). © Woodhead Publishing Limited, 2011

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Bacteriocins

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BLIS from E. faecium CCM 4231

Enterocin EJ97

Enterocin AS-48

Enterocin AS-48

Enterocin AS-48

Nisin

Nisin

Nisin

Treatment

(Continued)

Martínez Viedma et al., 2009a Martínez Viedma et al., 2010 Laukova and Czikkova, 1999

Grande et al., 2006b Martínez-Viedma et al., 2008a

Grande et al., 2005

Thomas et al., 2000

Reference(s) Yamazaki et al., 2000; Pena and de Massaguer, 2006 Walker and Phillips, 2008

Effect Inactivation of Alicyclobacillus in fruit juices. Inhibition of endospore germination Reduction of viable counts of P. cyclohexanicum in fruit juices for up to 15 days Inhibition of G. stearothermophilus in soy milk, as well as thermophilic clostridia and bacilli in coconut milk/water Inactivation of A. acidoterrestris cells in freshly-made and commercial fruit juices. Inhibition of endospore germination and alteration of endospore structure Inactivation of rope-forming B. licheniformis LMG 19409 cells, EPS-producing LAB (Lb. collinoides, Lb. diolivorans, P. parvulus) and 3-hydroxypropionaldehyde-producing Lb. collinoides strains in apple juice and apple ciders Inactivation of G. stearothermophilus in coconut milk and in coconut water Inactivation of G. stearothermophilus in coconut milk and in coconut water Inactivation of L. monocytogenes and reduction of S. aureus viable counts in soy milk

Table 16.3  Application of bacteriocins in fruit juices and drinks

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Combined treatments

Table 16.3  Continued Effect

Nisin and cinnamon

Accelerated death of S. Typhimurium and E. coli O157:H7 in apple juice Populations decline (E. coli O157:H7, Salmonella, and L. Nisin and EDTA monocytogenes) in apple cider Nisin and PEF (80 kV/cm, 42 °C) Increased inactivation of E. coli O157:H7 in apple juice Nisin and PEF (80 kV/cm, 50 °C) Increased inactivation of naturally occurring microorganisms in tomato juices Increased inactivation of S. Typhimurium in orange juices Nisin, lysozyme, and PEF treatment (90 kV/cm, 45 °C) Nisin or nisin/lysozyme and PEF Increased inactivation of naturally occurring microorganisms (yeast and moulds) in freshly squeezed treatment (27–33 kV/cm, apple cider and in grape juices 50 °C) Increased inactivation of L. innocua cells in apple or Nisin and HPH (0 to 350 MPa) carrot juice Increased inactivation of E. coli O157:H7 in apple juice Enterocin AS-48 and EDTA, sodium tri-polyphosphate, pH 5.0, sublethal heat Increased inactivation of exopolysaccharide-producing Enterocin AS-48 and PEF Lb. diolivorans in apple juice treatment (35 kV/cm, 22 °C) Increased inactivation of S. enterica in apple juice Enterocin AS-48 and PEF treatment (35 kV/cm, 40 °C)

Treatment

Martínez-Viedma et al., 2008b Martínez Viedma et al., 2009b

Ananou et al., 2005

Pathanibul et al., 2009

Wu et al., 2005 Liang et al., 2006

Liang et al., 2002

Iu et al., 2001 Wu et al., 2005

Ukuku et al., 2009

Yuste and Fung, 2004

Reference(s)

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16.4.1 Bacteriocin preparations Species of genus Alicyclobacillus may cause serious problems in fruit juices as this thermophilic bacterium produces endospores that survive mild heat treatments, and can germinate in fruit juices (Walls and Chuyate, 1998). Alicyclobacillus spp. may produce guaiacol in fruit juices, conferring an unpleasant medicinal taste (Orr et al., 2000). The effectiveness of nisin (1.25 to 100 IU/ml) against this depended on the pH of the media, the type of juice and the solid content (Komitopoulou et al., 1999; Yamazaki et al., 2000; Pena and de Massaguer, 2006). Nisin was also able to inhibit spore germination at 25–50 IU/ml in orange and mixed fruit drinks, but not by 600 IU/ml in clear apple juice, probably because of the competitive effect of phenols (Yamazaki et al., 2000). Enterocin AS-48 (2.5 µg/ml) inactivated A. acidoterrestris cells and endospores in various types of freshly-made fruit juices for at least 15 days and in commercial fruit juices for up to three months, suggesting its application as a nisin substitute for preservation of fruit juices (Grande et al., 2005). Propionibacterium cyclohexanicum is a heat-resistant, non-spore forming bacterium, implicated in the spoilage of orange juice (Kusano et al., 1997). Treatment with nisin (500 and 1000 IU/ml) significantly reduced the viable population of P. cyclohexanicum in orange juice for up to 15 days, but did not prevent regrowth of the bacterium during higher storage periods (Walker and Phillips, 2008). Further research needs to be carried out to achieve a satisfactory biocontrol of this bacterium. Fruit juices (specially unpasteurized juices) may also be spoiled by exopolysaccharide (EPS)-producing bacteria. In apple juice and apple ciders, added enterocin AS-48 (2.5 to 5 µg/ml) was very effective against EPSproducing strains (including Bacillus licheniformis LMG 19409, Lactobacillus collinoides, Lactobacillus diolivorans and Pediococcus parvulus) as well as 3-hydroxypropionaldehyde-producing Lb. collinoides strains (Grande et al., 2006b; Martínez-Viedma et al., 2008a). Bacteriocins have also been tested against spoilage or pathogenic bacteria in other vegetable-based drinks. In soy milk, a BLIS preparation from E. faecium CCM 4231 completely eliminated L. monocytogenes and markedly reduced the counts of S. aureus (Laukova and Czikkova, 1999). Nisin addition inhibited proliferation of Geobacillus stearothermophilus in soy milk, as well as thermophilic clostridia and bacilli in coconut milk/water (Thomas et al., 2000). In a similar way, enterocins AS-48 and EJ97 completely inactivated G. stearothermophilus in coconut milk and coconut water (Martínez-Viedma et al., 2009a; Martínez Viedma et al., 2010). 16.4.2 Combined treatments with bacteriocins Since unpasteurized fruit juices may act as vehicles for transmission of enteric pathogens, bacteriocins have been tested against E. coli and Salmonella in combination with outer membrane-permeabilizing treatments and other antimicrobials in order to increase microbial inactivation. In apple juice, a

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combination of nisin and cinnamon accelerated death of Salmonella Typhimurium and E. coli O157:H7, enhancing the safety of the product (Yuste and Fung, 2004). In apple cider, addition of a nisin (300 IU) and EDTA (20 mM) combination caused a decline in the populations of E. coli O157:H7, Salmonella, and L. monocytogenes, suggesting possible addition of this preparation to freshly prepared apple cider to enhance its microbial safety and prevent costly recalls (Ukuku et al., 2009). Similarly, E. coli O157:H7 cells sub-lethally injured by other treatments (EDTA, sodium tri-polyphosphate, pH 5.0, or sublethal heat) were inhibited significantly (3.8 to 8.1 log cycles inactivation) by enterocin AS-48 in apple juice, providing a means to lower the intensity of juice processing treatments (Ananou et al., 2005). The efficacy of bacteriocins in food systems can be improved in combination with novel food processing technologies such as high-intensity pulsed electric fields (PEF). PEF treatments seem very promising in the liquid food industry since they have little or no effect on organoleptic and nutritional properties (Martín-Belloso and Elez-Martínez, 2005; Mittal and Griffiths, 2005). In freshly squeezed, unpasteurized, and preservative-free apple juice, the addition of nisin (2%, wt/vol) together with a PEF treatment (80 kV/cm, 10 pulses, 42 °C) caused a greater reduction in E. coli O157:H7 cell counts (more than 3 log cycles) compared to PEF treatment alone (Iu et al., 2001). Liang et al. (2002) studied the effect of PEF treatments against Salmonella Typhimurium in pasteurized and freshly squeezed orange juice in combination with nisin and/or lysozyme and found increasing the treatment temperature to 45 °C or above was critical for inactivation of Salmonella by PEF. When PEF treatment (90 kV/cm, 30 pulses, 45 °C) was carried out in the presence of nisin (100 U/ml of orange juice), lysozyme (2,400 U/ml), or a mixture of nisin (27.5 U/ml) and lysozyme (690 U/ml), cell viability losses increased compared with the single treatments. The combination of nisin and lysozyme was the most effective, resulting in a Salmonella count reduction of ca. 6.5 log cycles in the juice (Liang et al., 2002). In apple juice, the combination of enterocin AS-48 (60 µg/ml) and PEF (35 kV/ cm, 1000 µs) at 40 °C decreased the survival of S. enterica cells by 4.5-log cycles while treatment with bacteriocin alone had no effect (Martínez-Viedma et al., 2008b). In apple juice, PEF treatment with enterocin AS-48 (at a subinhibitory concentration of 2 µg/ml) showed higher efficacy against the exopolysaccharideproducing strain Lb. diolivorans 29 compared to PEF and enterocin alone (Martínez-Viedma et al., 2009b). Furthermore, the added bacteriocin prevented regrowth of survivors during storage of treated samples for at least 15 days at 4 and 22 °C (Martínez-Viedma et al., 2009b). Combined bacteriocin-PEF treatments have been tested to reduce total microbial counts and to prevent or retard spoilage. In freshly squeezed apple cider, inactivation of naturally occurring microorganisms (yeast and molds) was increased by 1.1 to 1.8 logs when PEF treatment (27–33 kV/cm, 200 pulses/s, 50 °C, at a continuous flow of 10 l/h) was applied in the presence of a nisin/ lysozyme mixture (27.5 U/ml nisin, 690 U/ml lysozyme) (Liang et al., 2006). A

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similar treatment was tested for inactivation of naturally occurring spoilage microorganisms in red and white grape juice, increasing bacterial reductions up to 5.9 logs (Wu et al., 2005). In tomato juice, application of a PEF treatment (80 kV/cm, 20 pulses, 50 °C) in the presence of nisin (100 U/ml) stably reduced cell counts by about 4.4 log units during 28 days of storage at 4 °C without affecting the vitamin C content (Nguyen and Mittal, 2007). High pressure homogenization (HPH) is another emerging nonthermal technology that has demonstrated capability to inactivate various bacterial and fungal microorganisms without significant loss of product quality. Interestingly, HPH treatment sensitized E. coli cells to antibacterial peptides and enzymes (Diels et al., 2004, 2005). Pathanibul et al. (2009) tested the effect of HPH (0 to 350 MPa) on E. coli and Listeria innocua cells in apple or carrot juice. Addition of nisin (10 IU) increased the bactericidal effect of HPH treatment against L. innocua in juices, reducing to some extent the intensity of the HPH treatment (Pathanibul et al., 2009). However, addition of nisin did not increase the effect of HPH treatment on E. coli cells. In conclusion, the bacteriocins nisin and enterocin AS-48 perform satisfactorily as biopreservatives in fruit juices and therefore can be highly useful to prolong the shelf life of unpasteurized fruit juice. However, there are no reports on the effects of other bacteriocins (such as pediocins) in fruit juice. Bacteriocins could also be applied in combination with emerging food processing technologies such as pulsed electric fields in order to increase the efficacy of treatments and protect against proliferation of survivors during storage. Interestingly, the combined treatments greatly increase the efficacy of bacteriocins against Gram-negative bacteria, but further research is needed to determine the effects of bacteriocins in combination with other technologies that are currently being considered in fruit juice processing such as UV light or high hydrostatic pressure treatment.

16.5 Application of bacteriocins in ready-to-eat and canned vegetable foods Ready-to-eat (RTE) foods have become increasingly popular in parallel with changing consumer habits. There prepared with different vegetables are produced with different ingredients and technologies, and can be pasteurized, partially cooked, mixed, or slightly preserved. In general, the microbial load of these processed food products is much lower compared to other vegetable foods, although the efficacy of bacteriocin treatments varies depending on the food product and type of contamination (Table 16.4). 16.5.1 Salads and sauces Deli-type salads usually contain a mixture of cooked and/or uncooked vegetables (e.g. potatoes, tomatoes, olives, peas, carrots, lettuce or cabbage) and other ingredients (e.g. ham, chicken, tuna, egg, or seafood) blended with mayonnaise or

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Bacteriocins

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Enterocin AS-48 and phenolic compounds (carvacrol, eugenol, geraniol and hydrocinnamic acid)

Enterocin AS-48 and PHBME or 2-nitropropanol Enterocin AS-48 and heat

Nisin–pediocin combination Enterocin AS-48 combinations with other antimicrobials

Enterocin AS-48

Enterocin AS-48

Enterocin AS-48 Enterocin AS-48 Enterocin AS-48

Nisin

Nisin Nisin

Nisin

Treatment

Outgrowth inhibition of B. subtilis spores in sous vide mushrooms Inactivation of L. monocytogenes in Russian salad at reduced bacteriocin concentration of 30 µg/g. Increased protection during storage at 10 °C Inactivation of a cocktail of Salmonella strains in Russian salad, and protection during storage at 10 °C Increased inactivation of B. cereus and B. coagulans endospores in rice and in canned foods Increased inactivation of a cocktail of Bacillus and Paenibacillus cells in purees. Increased inactivation of S. aureus in vegetable sauces (carvacrol, hydrocinnamic acid)

Schillinger, 2001

Grande et al., 2007a

Cobo Molinos et al., 2009b Grande et al., 2006a; Lucas et al., 2006 Grande et al., 2007b

Cabo et al., 2009 Cobo Molinos et al., 2009a

Martínez-Viedma et al., 2009a, b

Lucas et al., 2006

Grande et al., 2006a Grande et al., 2007b

Thomas et al., 2000

Thomas et al., 2000 Thomas et al., 2002

Reference(s)

Effect Inactivation of Listeria in deli-type salads; regrowth observed during storage Preservation of fresh pasteurized ‘home-made’-type soups Control of Bacillus spp. and Clostridium spp. in cooked potato products Inactivation of non-aciduric (G. stearothermophilus, C. thermosaccharolyticum) and aciduric (C. pasteurianum, B. macerans, B. coagulans) spore formers in canned foods Inactivation of L. monocytogenes in Russian salad (60 µg/g) Inactivation of B. cereus in rice gruel and boiled rice Inactivation of B. cereus, B. macroides, Paenibacillus sp., P. polymyxa and P. amylolyticus in soups and purees Inactivation of B. coagulans cells in tomato paste, syrup from canned peaches, and juice from canned pineapple Inactivation of G. stearothermophilus in canned corn and peas

Table 16.4  Application of bacteriocins in canned and other pre-cooked vegetables

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salad dressing. The microbial safety of deli salads may be compromised due to extensive handling during preparation by foodservice personnel, crosscontamination, potential abusive temperatures during storage, and also the lack of heat-treatment before consumption. In addition, due to the low numbers of competing microbiota as a result of cooking steps foodborne pathogens can easily proliferate in salads (Jay et al., 2005). Transmission of bacterial pathogens such as S. enterica and L. monocytogenes through deli salads is a concern (FDA/ USDA/CDC, 2001; Unicomb et al., 2003; Mokhtari et al., 2006). Nisin was reported to reduce viable cell counts of Listeria in deli-type salads, but did not prevent growth of survivors during storage (Schillinger, 2001), while Enterocin AS-48 (30 to 60 µg/g) significantly reduced viable counts of L. monocytogenes in Russian-type salad during one week storage at 10 °C (Cobo Molinos et al., 2009a). Antilisterial activity was strongly enhanced by several other antimicrobials (such as essential oils, bioactive components from essential oils and plant extracts as well as other related antimicrobials of natural origin or derived from chemical synthesis, and food preservatives) while reducing bacteriocin concentration to 30 µg/g. The combinations of AS-48 with lactic acid, p-hydroxybenzoic acid methyl esther (PHBME) or Nisaplin reduced listeria counts in salad to basal levels for at least one week storage. Furthermore, the combinations of PHBME and AS-48 (80 µg/g) or 2-nitropropanol and AS-48 (40 µg/g) achieved complete inactivation (ca. 4.5 log cycles) on a cocktail of S. enterica strains in Russian-type salad stored at 10 °C for one week (Cobo Molinos et al., 2009b). The efficacy of bacteriocins in food systems has been shown to depend greatly on the food substrate, the target bacterium, and the storage conditions. This also concerns vegetable foods as inactivation of S. aureus in vegetable sauces required enterocin AS-48 concentrations ranging from 25 µg/ml to 80 µg/ml (Grande et al., 2007a). This was due to bacteriocin interaction with food components together with the higher bacteriocin resistance of staphylococci. Anti-staphylococcal activity in sauces was enhanced significantly by addition of phenolic compounds and best results were reported for hydrocinnamic and carvacrol bacteriocin combinations. 16.5.2 Canned and other pre-cooked vegetables In canned as well as in other cooked vegetables, endospore-forming bacteria represent the main risk for spoilage due to the frequent contamination of raw materials and the high thermal resistance of endospores. Several studies have suggested the application of bacteriocins to inhibit endospore outgrowth and also to increase the efficacy of thermal treatments. Nisin has been proven to be an effective preservative in fresh pasteurized ‘home-made’ -type soups (Thomas et al., 2000) and in the control of Bacillus and Clostridium in cooked potato products (Thomas et al., 2002). In sous vide mushrooms, addition of a nisin-pediocin mixture prevented outgrowth of B. subtilis spores (Cabo et al., 2009). In boiled rice and in rice gruel inoculated with vegetative cells and endospores of B. cereus,

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enterocin AS-48 (20 to 35 µg/ml) completely inactivated the bacilli and prevented enterotoxin production during storage under refrigeration as well as ambient temperature (Grande et al., 2006a). Although the bacterial endospores were resistant, application of AS-48 in combination with heat treatments decreased the thermal death D values for endospore inactivation (Grande et al., 2006a; Lucas et al., 2006). In vegetable soups and purees, enterocin AS-48 (10 µg/ml) was active against aerobic mesophilic endospore-forming bacteria (B. cereus, B. macroides, and Paenibacillus strains from spoiled purees) for up to 30 days at 6, 15 and 22 °C (Grande et al., 2007b). However, cocktails of 8 strains (composed of three B. cereus and two B. macroides strains, Paenibacillus sp., P. polymyxa, and P. amylolyticus) showed a higher bacteriocin resistance. These results stress the importance of the complexity of food flora on the efficacy of bacteriocin treatments. Bacteriocin activity against the eight-strain cocktail increased greatly in combination with phenolic compounds (carvacrol, eugenol, geraniol and hydrocinnamic acid), and to a less extent with nisin (Grande et al., 2007b). In canned vegetables, incorporation of nisin can prevent spoilage caused by non-aciduric (G. stearothermophilus and Clostridium thermosaccharolyticum) as well as by aciduric (Clostridium pasteurianum, Bacillus macerans, Bacillus coagulans) spore formers (Thomas et al., 2000). Enterocin AS-48 was able to suppress B. coagulans vegetative cells in tomato paste, syrup from canned peaches, and juice from canned pineapple for at least 15 days of storage at 37 °C (Lucas et al., 2006). Although enterocin AS-48 had no significant effect on B. coagulans CECT 12 spores, the combined treatment of AS-48 and heat (80–95 °C for 5 min) significantly increased spore inactivation and afforded an increased protection during storage. Therefore, this bacteriocin could be of great relevance for application of less intense heat treatments in canned fruit and vegetable foods. In canned corn and peas, addition of enterocin AS-48 (7 µg/g) inactivated G. stearothermophilus cells for at least 30 days at a temperature of 45 °C simulating tropical conditions (Martínez-Viedma et al., 2009a). The same effect was observed for intact endospores at even lower bacteriocin concentration (1.75 µg/g) and was due to strong bacteriocin adsorption on endospores. This phenomenon is of great interest to prevent spoilage of canned foods by G. stearothermophilus endospores surviving heat treatments (Martínez-Viedma et al., 2009a). Enterocin EJ97 could also inactivate G. stearothermophilus vegetative cells in canned vegetables. Although this bacteriocin had no activity on intact endospores, it increased the efficacy of heat treatments (Martínez-Viedma et al., 2010). Altogether, results from these previous studies clearly indicate that bacteriocins are promising biopreservatives for processed vegetable foods, since most bacteriocins show anti-listeria activity and some of them are also active against endospore formers. Since vegetable foods usually contain a much lower fat content compared to meat or dairy products, residual bacteriocin activity is comparatively much higher, which is an additional advantage. Being small peptides, many bacteriocins can also withstand the processing conditions applied

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to vegetable foods such as salting, acidification, or heat pasteurization. Although intact endospores are usually bacteriocin-resistant, they become sensitive during the process of germination and outgowth and for this reason bacteriocins could be applied as an extra hurdle against endospores surviving heat treatments. Since bacteriocins increase the heat sensitivity of endospores, they could also be applied to increase the efficacy of treatments or decrease heat intensity. Bacterial endospores are also resistant to a variety of other treatments such as high hydrostatic pressure, but the synergistic effects of bacteriocins and high hydrostatic pressure in processed vegetable foods still need to be determined.

16.6 Application of bacteriocins or their producer strains in fermented vegetables Most vegetable fermentations rely on the selective growth of the microbiota that occurs naturally in the raw materials, water and equipment. Bacteriocin production could be exploited to enhance the competitiveness of starter cultures and yield a more homogeneous fermentation, but also to avoid proliferation of inadequate LAB (which due to their homofermentative or heterofermentative traits may have desirable or undesirable effects depending on the fermented product) as well as other spoilage or pathogenic bacteria. There are many examples of bacteriocin or BLIS-producing LAB isolated from fermented vegetables, such as L. lactis 23 from fermented carrots (Uhlman et al., 1992), Lb. plantarum strains C-11 and C19 from cucumber fermentations (Daeschel et al., 1990; Atrih et al., 1993), Lactobacillus sake C2 from traditional Chinese fermented cabbage (Gao et al., 2009), Pediococcus pentosaceus 05–10 isolated from Sichuan Pickle, a traditionally fermented vegetable product from China (Huang et al., 2009), Lb. plantarum LPCO10 from fermented table olives (Jimenez-Diaz et al., 1993), Lb. plantarum strains ST23LD and ST341LD from spoiled olive brine (Todorov and Dicks, 2005), Lactobacillus pentosus B96 from fermenting green olives (Delgado et al., 2005), and E. faecium BFE 900 from fermented black olives (Franz et al., 1996). However, there are only a few examples where bacteriocins or their producer strains have been tested in vegetable fermentations (Table 16.5). Inoculation with a paired culture consisting of a nisin-producer L. lactis and a nisin-resistant Ln. mesenteroides was tested to enhance the fermentation of cabbage (Harris et al., 1992a, 1992b). Furthermore, addition of a nisin preparation to cabbage inoculated with nisin-resistant Ln. mesenteroides improved control of the fermentation and delayed growth of the homofermentative LAB (Breidt et al., 1995), while in kimchi, nisin was added to control lactobacilli responsible for over-ripening. Nisin addition showed higher growth inhibition of Lactobacillus spp. than Leuconostoc spp. (Choi and Park, 2000). Inoculation of kimchi with a pediocin-producing strain of P. acidilactici achieved a successful inhibition of L. monocytogenes (Choi and Beuchat, 1994). Bacteriocin-producing strains have been tested successfully in table olive fermentations. In the Spanish-style process, green olives are first treated with lye,

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Table 16.5  Application of bacteriocins and bacteriocin-producing strains in fermented vegetables Bacteriocins

Treatment

Effect

Reference(s)

Nisin Nisin

Improved cabbage fermentation by a nisin-resistant Ln. mesenteroides strain Control of kimchi over-ripening by lactobacilli

Breidt et al., 1995

Bacteriocin or Nisin-producer L. lactis BLISproducing strains Pediocin-producing strain of P. acidilactici Plantaricin-producing Lb. plantarum LPCO10

Choi and Park, 2000

Improved cabbage Harris et al., fermentation by a 1992a, b nisin-resistant Ln. mesenteroides strain Control of L. Choi and Beuchat, monocytogenes in 1994 kimchi Patented as starter culture Jiménez-Díaz for fermentation of et al., 1993; table olives and other Ruiz-Barba et vegetable foods al., 1994; Vega Leal-Sánchez et al., 2003

which destroys most of the epiphytic microbiota. The lactic fermentation that occurs afterwards is often a slow process that relies mostly on the resident microbiota from the tanks and manufacturing plant environment. In some cases, sufficient lactic acid is not produced to warrant product preservation, and spoilage may occur if exogenous acid is not supplied. A starter culture based on the plantaricin S and T producer strain Lb. plantarum LPCO10 has been patented for fermentation of table olives and other vegetable foods (Jiménez-Díaz et al., 1993; Ruiz-Barba et al., 1994; Vega Leal-Sánchez et al., 2003). Inoculation with the plantaricin-producing culture improved the microbiological control of the fermentation process, increased the lactic acid yield and provided a consistent high quality product (Ruiz-Barba et al., 1994; Vega Leal-Sánchez et al., 2003). The starter culture is also particularly useful to speed up fermentation and ensure a homogeneous fermentation process in newly-operating plants that still lack the appropriate resident LAB microbiota. Plantaricin production is widely distributed among Lb. plantarum strains (Maldonado et al., 2002). This bacterium has been detected as one of the main components in other fermentations such as capers and Almagro eggplants (Seseña et al., 2004; Pérez Pulido et al., 2005). Plantaricin-producing strains could also find applications in these (and probably other) fermented vegetables. In summary, while the preparation of fermented vegetables relies strongly on the spontaneous selection of resident microbiota, this food sector could benefit greatly from application of starter cultures with selected beneficial properties. Application of bacteriocin-producing strains or addition of bacteriocins could enhance the predominance of starter cultures, improve product quality and uniformity, decrease

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the risks for spoilage and improve microbiological safety against foodborne pathogens. Control of toxin-producing microorganisms (such as moulds) in fermented vegetables is still a matter of debate, and further research is needed on application of bacterial starter cultures with antifungal properties as well as on the development of novel antifungal peptides suitable for addition in vegetable fermentations.

16.7 General conclusions and perspectives A great effort of research on food biopreservation has focused on meat and dairy products, while comparatively much less input has been made on vegetable foods. The increasing awareness of the risks for transmission of foodborne pathogens through vegetable foods added to the increasing demands of consumers for foods that are fresh-tasting and lightly preserved has fuelled a growing interest in recent years to apply the principles of biopreservation in this field. Economic losses caused by bacterial spoilage are also a driving force to apply biopreservation in vegetable foods. The risks associated with fruit juices, raw fruits and vegetable food products vary greatly depending on the food category, as do the possibilities for application of biopreservation technologies. In the case of raw or lightly processed products, biopreservation seems an attractive alternative because of the restrictions for application of other antimicrobial treatments such as heat and the limited efficacy and lower consumer acceptance of chemical treatments or additives. Application of bacteriocin treatments in fruit juices and on raw fruits and vegetables has been a matter of only limited studies, encompassing no more than two or three bacteriocins. Results obtained for enterocin AS-48 are highly promising, and indicate that this bacteriocin could be applied to avoid spoilage of fruit juices and for decontamination of fruits and vegetables in combination with other antimicrobials in order to increase the efficacy of treatments and to broaden the inhibitory spectrum against Gram-negative bacteria. Other bacteriocins (including the colicins and microcins) and antimicrobial peptides (or cocktails of these), such as those from non-bacterial sources or developed through combinatory peptide design, should also be tested on raw vegetable foods and drinks. This could be an interesting way to broaden the antimicrobial spectrum of treatments and to decrease the risks for selection of bacteriocin-resistant or bacteriocinadapted strains. At present, extensive research is carried out on antimicrobial peptides, providing highly relevant insights into their modes of action, structure– function relationships, and also on the effects of peptide modification on the selectivity and potency of their antimicrobial activity. The usefulness of these new developments for preservation of vegetable foods needs to be investigated. Furthermore, the spectrum of antimicrobial activity of antimicrobial peptides intended to increase the safety and shelf life of raw vegetable foods also needs to be broadened to include yeasts, moulds, viruses and parasites. Lytic bacteriophages have been proposed for biopreservation of fruits and raw vegetable foods. However, because of the high species and strain-specificity of bacteriophages, it would be necessary to apply complex phage mixtures in order

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to achieve a broader protection. The stability of phages under the environmental conditions found in foods and the transient resistance to phage infection reported in some case also need to be taken into consideration. However, application of phage technology on vegetable foods is still a largely unexplored field. The effects of phages on spoilage fungi and on foodborne parasitic protozoa should be further investigated. Phages may be amenable for genetic manipulation and also as factories for novel antimicrobial products such as phage lysins, which seem highly attractive for biopreservation. Food processing has strong effects on the food microbiota, but may also create new growth opportunities for endospore formers and cross-contaminating bacteria such as L. monocytogenes, which at present are one of the main concerns in the food processing industries. The frequent contamination with bacterial endospores found in vegetable raw materials and the lack of competitive microbiota in the processed food products are also aggravating factors. Most bacteriocins are highly active against endospore-forming bacteria and L. monocytogenes, however further studies are needed to make new bacteriocins available to the food market and to investigate synergistic effects with novel food processing technologies such as ohmic heating, microwave heating, high hydrostatic pressure, or low dose irradiation. Last, but not least, bacteriocin production is nowadays being considered as a probiotic trait, and the use of bacteriocin-producing strains is being considered as an attractive approach to control foodborne pathogens in the gastrointestinal tract. Vegetable fermented foods (such as table olives) have now been proposed as vehicles for administration of probiotic LAB (Lavermicocca et al., 2005; Prado et al., 2008). Similar probiotic preparations based on bacteriocin-producing strains are to be expected in a near future.

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17 Applications of protective cultures and bacteriocins in wine making F. Ruiz-Larrea, University of La Rioja, Spain

Abstract: Wine making is a series of operations from harvesting of grapes to bottling wine ready to be consumed, and aspects of major importance are those related to microbiological control throughout the whole process and during wine ageing. This chapter will focus on those microbiological aspects related to wine quality, spoilage and preservation, and the possibility of applying either protective cultures of lactic acid bacteria (LAB), or protective fermentates, or those antimicrobial metabolites named bacteriocins, for wine microbiological control. Bacteriocins are peptides with antimicrobial activity that are naturally produced by some bacteria strains, which thus are able to inhibit the growth of other competing bacteria. Bacteriocins produced by food-grade LAB have high potential for wine biopreservation, with the additional advantage of allowing a decrease of sulphur dioxide levels, which is the major preservative that is currently used in wine making. Key words: lactic acid bacteria, wine malolactic fermentation, wine spoilage, wine preservation, bacteriocins.

17.1  Introduction The main themes of this chapter deal with the wine making process and the major events that take place during wine elaboration and ageing, with special attention to three different aspects: beneficial effects of lactic acid bacteria on wine quality, wine spoilage by bacteria, and wine biopreservation. In all cases microbiological control of bacterial growth is the key issue for premium wine quality. Bacteriocins can find a novel field of applications for microbiological control in wine making, they could constitute new and efficient tools for enologists, and they present the advantage of allowing a decrease of the current levels of sulphur dioxide, which is the chemical preservative that is widely used in wine making. Some of the most recent studies on bacteriocin activities produced by enological bacteria and on the 433 © Woodhead Publishing Limited, 2011

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effect of both bacteriocins (nisin and pediocin) on the growth of wine bacteria are presented in the final sections of this chapter, as well as some final considerations on potential future trends in bacteriocin applications for wine biopreservation.

17.2  Wine fermentation The first transformation that grape musts undergo during wine making is the well known alcoholic fermentation, conducted by yeasts that convert sugars into ethanol. This is the first and main fermentation, nevertheless yeasts are not the unique microorganisms responsible for the production of premium wines. Wines, and especially those elaborated from red grapes that still retain their content in L-malic acid, require a secondary fermentation, named malolactic fermentation (MLF), which transforms L-malic acid into L-lactic acid (Boulton et al., 1996). Malic acid is a dicarboxylic acid that constitutes about half the titrable acidity of musts and contributes to grape and wine sour taste. In contrast, lactic acid is a monocarboxylic acid, and consequently, weaker than malic acid. Normal contents of malic acid in red grapes vary from 2 to 10 gl–1. Malolactic fermentation converts the harsher tasting malic acid into the smoother tasting lactic acid and this fermentation is carried out by lactic acid bacteria (LAB) that are natural constituents of the endogenous microbiota of grapes and musts in fermentation. LAB possess a unique enzyme: the malolactic enzyme that stoechiometrically decarboxylates malic acid to lactic acid with the concomitant production of one molecule of CO 2 and the disappearance of one hydrogen ion (Fig. 17.1). Therefore, from the biochemical point of view, MLF is a biological deacidification of wine, and both aspects of deacidification, the decrease in titrable acidity and the slight increase in pH, have important consequences for wine quality: wine becomes smoother and loses part of its sour aggressiveness, the pH increase (0.1–0.2 units) favours equilibrium shifts in anthocyanin pigment configurations, and the wine full red colour acquires a bluish hue (Boulton et al., 1996). Moreover, MLF provokes a decrease in free anthocyan levels and reactions between tannins COO– HO

CH

HO

CH2 COO– L-Malic acid

COO–

H+

H+

CH CH3 +

H+

CO2

L-Lactic acid

Fig. 17.1  Decarboxylation of L-malic acid into L-lactic acid catalysed by the malolactic enzyme.

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(molecules responsible for the astringent taste of wine) and anthocyans (responsible for wine red colour). Large aggregates of wine pigments precipitate during MLF, and all these effects have as a consequence a decrease in wine astringency and sour taste, as well as a better stabilisation of wine red colour (Lonvaud-Funel, 1999). LAB growth during MLF produces secondary products that have important consequences in wine sensorial properties: volatile acidity increases slightly (around 0.2 gl–1 expressed as H2SO 4) (Ribereau-Gayon et al., 1998), wine acquires lactic bouquets and develops the aroma complexity necessary for normal ageing premium red wines (Baldy, 1997). One of the most relevant taste descriptors for wines after MLF is ‘buttery’. This character has been associated with the production of diacetyl and its related acetoinic compounds: acetoin and 2,3 butanediol. These metabolites are mainly produced as secondary products of the degradation of citric acid by LAB during MLF (Lonvaud-Funel, 1999) and their production should be maintained under threshold as higher concentrations are not appreciated by wine tasters. The influence of acetoinic compounds on wine aroma is determined by technological factors, such as early racking or clarification that enhance their effect (Lonvaud-Funel, 1999), and by LAB growth. In fast-growing conditions, less acetoinic compounds are synthesised, whereas under slow-growing conditions, more acetoinic compounds are produced. Also, an increase of these acetoinic secondary metabolic products occurs with an overgrowth of LAB after malic acid has been consumed, i.e. after MLF has been completed. With regard to aroma complexity, aroma studies (Henick-Kling, 1993) for both white and red wines revealed that, after MLF, fruity aromas are enhanced while vegetal and herbaceous aromas are reduced, and that the whole complexity in the flavour of wine after MLF improves the body and mouthfeel of wine and gives a longer after-taste (Henick-Kling et al., 1994; Liu, 2002). MLF also brings microbiological stability to wines as LAB consume residual sugars (hexoses and pentoses) left by yeast and malic acid, which constitute excellent carbon sources for the growth of wine spoilage microorganisms. Summarising, from the point of view of wine making, MLF conducted by LAB is a desirable transformation for high quality red wines and a mandatory transformation for wines that are going to be submitted to ageing, and this transformation has to be strictly controlled to obtain the desired results.

17.3  Lactic acid bacteria in wine making Lactic acid bacteria constitute a heterogeneous group of bacteria that present some common features: they are gram positive, catalase negative, non-sporulated, facultative anaerobic bacteria with a fermentative metabolism (Konings, 2002), and they are capable of transforming malic acid into lactic acid, as mentioned above. Many LAB possess the status of qualified presumption of safety (QPS), i.e. they are regarded as safe microorganisms for human consumption because

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they and their metabolites have been consumed in fermented foods for countless generations without adverse effects on the population. LAB genera that have been identified in wine are Lactobacillus, Pediococcus, Leuconostoc and Oenococcus (Ribereau-Gayon et al., 1998). Depending on the species, on the strain, on their population, and on the moment they multiply, LAB growth may be beneficial or spoilage bacteria for wine quality, and therefore the control of bacterial growth during wine making is imperative. On grape berries LAB populations are low, and when grape musts are poured into fermentation tanks, cell enumeration can vary from 102 cfu ml–1 to 104 cfu ml–1 (Lonvaud-Funel, 1999). During alcoholic fermentation LAB population decreases to around 102 cfu ml–1, and the major reason is the production of ethanol by yeast. Ethanol is regarded as one of the major inhibitors of bacterial growth and ethanol resistance varies with a number of conditions in the medium (e.g. pH and temperature) and oenococci have long been reported as the most resistant LAB to ethanol presence in wine (Mills et al., 2005) and in most cases oenococci predominate at the end and after alcoholic fermentation. However, other strains of different LAB genera (Lactobacillus, Pediococcus, Leuconostoc) can also survive and have been identified in finished wines. The lag phase of MLF is very variable, and depends mainly on wine temperature, ethanol content and pH (the main factors affecting LAB growth in wine), and MLF starts when the LAB population reaches around 106 cfu ml–1, and increases up to 107–108 cfu ml–1 (López et al., 2008). Once malic acid has been degraded by LAB, the enologist should stop LAB growth to prevent wine spoilage, and this is achieved by racking or clearing and subsequent sulfiting. Most of the bacteria, and to a lesser extent the remaining yeast, are sensitive to sulphur dioxide, which in this way stabilises wine microbiologically. However, the efficacy of sulphur dioxide is directly related to pH, and in high pH wines (> 3.8) the efficacy of sulphur dioxide is highly reduced, and the lesser stringency of the medium allows LAB to survive more easily. Levels of viable cells of 105 and 106 cfu ml–1 can be found some months after the end of MLF in this type of high pH wine (Lonvaud-Funel, 1999). Regarding the timing for LAB growth, early growth (i.e. they multiply before alcoholic fermentation ends) may increase lactic fermentation of sugars that have not yet been utilised by yeasts. The major products of sugar degradation by LAB are acetic acid and lactic acid, and consequently volatile acidity increases and wine is depreciated. Moreover, when lactic acid concentration reaches values above 4.5 gl–l the wine is said to suffer a lactic alteration (Flanzy, 1998) and it is unmarketable. Similarly, when LAB are allowed to continue growing without control after MLF is completed (i.e. after malic acid has been totally degraded), the same lactic alteration of wine arises. The four enological genera of LAB are: Lactobacillus, Pediococcus, Leuconostoc and Oenococcus, and as mentioned above, the latter is the most abundant genus, probably as a consequence of its high tolerance to the stressing conditions that wine offers to bacterial growth. It is generally accepted that all Oenococcus strains grow in a medium containing 10% ethanol at pH 4.7. Oenococci optimum pH for growing is between 4.3 and 4.8 (Britz and Tracey, 1990), they grow normally in wine in the

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pH range 3–4, and small quantities of ethanol (< 7 %) can stimulate their growth (G-Alegria et al., 2004). All these reasons explain the natural selection of Oenococcus during spontaneous MFL. In the late 1970s the use of selected malolactic starters began, and Oenococcus has since then been the preferred starter genus.

17.4  Wine spoilage by bacteria When wine making is well controlled, LAB growth and metabolism improve wine quality and stability, as shown above. Wines named as fortified wines, which are elaborated adding brandies or wine spirits to grape musts in fermentation that still retain high concentrations of sugars, offer suitable conditions for certain LAB strains to grow in spite of the high ethanol content (16–20%), and this is due to the high content of fermentable sugars and low acidity of some of these fortified wines. Under these conditions certain strains, mainly of the species L. hilgardii and L. fructivorans, can proliferate and cause the lactic alteration (Lonvaud-Funel, 1999). Another type of wine altering bacteria belongs to the species P. damnosus. Some strains of this species are able to synthesise exoplysaccharides, form biofilms, and can cause an increase in the viscosity of the spoiled wine (DolsLafargue et al., 2008) or cider (Ibarburu et al., 2007), described as ‘ropiness’ or ‘oiliness’. These strains have been reported to be very resistant to the stress conditions of wine and to sulphur dioxide (Lonvaud-Funel, 1999), and their elimination from the cellar is arduous. Wine may be altered either by an increase in its sweet and sour taste, or an increase in bitterness. In the first case, some LAB strains transform fructose into mannitol, the corresponding six-carbon sugar alcohol, and this pathway is accompanied by a high production of acetic acid, lactic acid and fusidic alcohols. On the other hand, the wine alteration named bitterness is due to the formation of acrolein, a metabolite produced by the degradation of glycerol by certain LAB strains. Acrolein combines with wine polyphenols and increases its bitter taste. LAB strains responsible for this alteration belong mainly to Lactobacillus sp. (Bartowsky, 2009). Both types of alterations, the mannitol taint and wine bitterness, can occur in wines with high content of residual sugars, low alcohol content, and high pH. LAB can spoil wine as well by producing off-flavours. The molecules directly responsible for this are not easily identified, and thus descriptors related to ‘mousy’ or ‘animal’ off-flavours have been associated with volatile ethyl-phenols and/or nitrogen heterocyclic compounds (esters of tetrahydropyridine, or 2-acetyl1-pirroline) (Bartowsky, 2009). Nevertheless, the microorganisms responsible for the formation of these molecules are not clearly identified, and the wine spoiling yeast Brettanomyces/Dekkera and some heterofermentative LAB strains have been described as responsible for this type of wine alteration (Bartowsky, 2009; Lonvaud-Funel, 1999). Acetic acid bacteria (AAB) belong to a different group of bacteria from that of LAB. AAB are classified in the family Acetobacteriaceae, they are gram-negative,

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they have an obligated aerobic metabolism, which is their main characteristic, and oxygen availability limits their growth (Guillamon and Mas, 2009). AAB species that have been described in wines are Gluconacetobacter xylinus, Ga. europaeus (Gonzalez et al., 2004), Ga. hansenii (Du Toit and Lambrechts, 2002; González et al., 2005), Gluconobacter oxydans, Ga. liquefaciens and Acetobacter pasteurianus (González et al., 2005). AAB ability to efficiently convert ethanol to acetic acid constitutes the major threat to wine quality as AAB are in all cases wine-spoiling bacteria. They confer to wine high volatile acidity, a vinegar-like sourness and a number of off-flavours that depreciate wine, even to make it unmarketable. Wine is at high risk of spoilage by AAB during the period of ageing if wine is not topped up and monitored regularly to prevent oxygen promoting AAB growth.

17.5 Sulphur dioxide: the classical antimicrobial agent in wine making Sulphur dioxide has been used in wine making at least since the 18th century (Ribereau-Gayon et al., 1998) as it possesses a number of properties that make it indispensable in wine making. Moreover, in the case that the enologist does not add any sulphur dioxide at all during the vinification process, yeast strains themselves produce small quantities of sulphur dioxide during alcoholic fermentation (Ribereau-Gayon et al., 1998). Once sulphur dioxide is dissolved in wine, it undergoes a number of ionisation equilibria and all the forms are referred to as sulphites. Sulphur dioxide is also a fruit and vegetable preservative, widely used for dried fruits and fermented vegetables, and it can be used as well for bleaching food starches (Foulke, 1993). Those properties of sulphur dioxide that have been appreciated by enologists for such a long time are the following. • It is a potent antimicrobial agent, as it enters microbial cells and binds to a number of cellular structures and crucial enzymes so that microbial death eventually occurs. Bacteria (both LAB and AAB) are more sensitive to sulphur dioxide than yeast or filamentous fungi, and it is efficient in inhibiting flor yeasts (a particular kind of Saccharomyces cerevisiae strains responsible for wine re-fermentation) that are dangerous spoilage yeasts in sweet wines. Moreover, sulphur dioxide increases its LAB antimicrobial activity in the presence of ethanol (Rojo-Bezares et al., 2007a,b). The microbiologically most active form of sulphur dioxide is the free molecule SO 2, and just a slight concentration of 1–10 mg l–1 of this molecular form is able to provoke bacteria cell death (Boulton et al., 1996). • Sulphur dioxide is an antioxidant and it efficiently binds oxygen molecules that are dissolved in wine. Thus it prevents wine and must oxidation and preserves polyphenols (responsible for wine red colour) and aroma compounds from browning or alteration by oxidation (Ribereau-Gayon et al., 1998).

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• It is an efficient inhibitor of oxidoreductases (tyrosinases) that are present in musts. The origin of these oxidoreductases is the vegetable grape cell, and when this enzyme activity is not inhibited, it leads to wine browning and loss of quality. • Sulphur dioxide combines with aldehydes, mainly acetaldehyde, and thus protects wine from reduction taint aromas (Ribereau-Gayon et al., 1998). Nevertheless, sulphur dioxide addition is limited by regulation and the maximum amounts allowed are in the range of 160 mg l–1 of total sulphur dioxide. Consumption of food and beverages preserved with sulphur dioxide is generally not a problem except for hypersensitive individuals who apparently could suffer some type of allergic or asthmatic responses (Valley and Thompson, 2001; Armentia, 2008). Currently there is a general trend towards reduction of sulphite levels, not only in wines but also in all sorts of foods and beverages, mainly for public health concerns and due to consumers’ preferences. In the case of wine, filtration can be used to remove microorganisms before bottling, but it is not a methodology that can be implemented in earlier stages of wine making. Moreover, complete removal of microbes by filtration impacts negatively on wine flavour. Alternative methods using emerging technologies have been successfully applied for food and beverage preservation and these include non-thermal treatments such as ultrasound processing, ultraviolet irradiation, pulsed electric fields or high pressure treatments. The major limitation of these methods is that they can affect wine colour and aroma; the molecules responsible for both sensorial properties of wine are extremely labile, they are in most cases photo-sensitive and photo-reactive, and some of them can precipitate (as in the case of large pigment macromolecules or pigment aggregates) or evaporate (in the case of volatile aroma compounds). Therefore, the search for alternative methodologies for microbiological control of wine during the whole process of wine making is a topic of current interest in enology.

17.6  Bacteriocins Bacteriocins are peptides with antimicrobial activity that are secreted by some bacteria to inhibit the growth of other competing microorganisms. Currently, bacteriocins produced by LAB arouse most interest because, as mentioned above, some LAB possess the status of QPS and bacteriocins produced by these QPS LAB species have found important applications as natural food preservatives. The first description of some activity related to bacteriocins was published more than eighty years ago, when an antagonism between Escherichia coli strains was reported, and these substances were originally named ‘colicins’ (Gratia, 1925). The first reports on LAB bacteriocins started in 1928, when it was reported that some Lactococcus strains, which were used in cheese making, inhibited the growth of other LAB strains (Rogers and Whittier, 1928) and potentially they could inhibit the growth of pathogenic and cheese spoilage bacteria. In 1933 the description of a proteinaceous molecule with antimicrobial activity produced by some strains of the

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species Lactococcus lactis subsp. lactis was reported, which was later named as nisin (or group N inhibitory substance) (Mattick and Hirsh, 1947). Therefore, nisin is the most studied bacteriocin, with the longest history of safe use in the food industry. In 1953 it was first marketed in England, in 1969 it was assessed to be safe for food use by the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives, and in 1983 it was included in the European food additive list with the number E234. Later on, in 1988, it was approved by the U.S. Food and Drug Agency (Cotter et al., 2005). Nisin is available on food grade as Nisaplin (Danisco), which is a preparation that contains 2.5% nisin with NaCl (77.5 %) and non-fat dried milk (12% protein and 6% carbohydrate). The product Nisaplin ND (non-dairy nisin) does not contain milk proteins, which facilitates its solubility in aqueous solutions, and contains 6.25% nisin, NaCl (90%), carbohydrates (2%) and proteins (approximately 2%). Nisin has a broad inhibitory spectrum among Gram-positive bacteria (Cintas et al., 2001; O’Sullivan et al., 2002). It is used as a biopreservative in dairy products (especially cheeses), meat products, liquid egg products, fruit juices and canned foods. Another LAB bacteriocin that has found a wide field of applications in the food industry as a biopreservative is pediocin. Pediocin PA-1 (formerly also named AcH) is produced by some strains of the species Pediococcus acidilactici of meat origin, P. parvulus of vegetable origin and one Lactobacillus plantarum strain isolated from cheese (Cintas et al., 2001). Pediocin PA-1 has a narrow inhibitory spectrum compared to nisin, and shows no activity on many LAB strains. Currently, pediocin is commercially exploited and is covered by several U.S. and European patents (Cheng and Hoover, 2003). A LAB fermentate that contains pediocin PA-1, named Alta™ (Kerry Bio-Science) is commercially available and is used as a food preservative for ready-to-eat meat products, salads and sauces. The proposed mode of action for bacteriocins is an initial binding to the bacterial membrane of the susceptible strain by electrostatic forces between the negatively charged membrane lipids and the positive charges of bacteriocins, which are basically localised in one of their extreme segments (C-terminal region of nisin, and N-terminal region of pediocin). Subsequently, bacteriocin insertion in the lipid bilayer of the attacked bacteria cell takes place, and pores are formed through the bacteria membrane, which becomes permeabilised (Moll et al., 1999). The cell loses ions and metabolites which are essential for its survival, and eventually bacteria death supervenes. Bacteriocins for use in the food industry should fulfil a number of requisites: QPS status of the producing strain, heat stability, inhibitory activity against pathogenic or food spoilage bacteria but not against beneficial biota, no associated health risks, and beneficial effects in food biopreservation (such as improved safety and quality when used as food additives in the range of concentrations that are naturally produced by strains in their natural source). Currently, nisin and pediocin have proved their efficiency and they are biopreservatives widely used in the food industry, either individually or in combination with other preservation techniques such as heat treatment, high pressures or modified atmosphere packaging (Allende et al., 2006).

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17.7  Bacteriocins produced by enological bacteria As mentioned earlier, Oenococcus oeni is the species reported to lead wine MLF and the most abundant during this period of wine making. A number of efforts have been carried out to investigate the possibility of bacteriocin production by O. oeni strains. Nevertheless, to date no report exists on the production of such molecules by this species. One paper reported the presence of putative bacteriocin encoding genes in the genome of some O. oeni strains (Knoll et al., 2008), but no evidence was reported on the antimicrobial activity of those O. oeni strains. Lactobacillus plantarum is a species that has been reported in grapes and at different stages of the wine making process, and bacteriocin activity (named plantaricin) has been reported on strains isolated from grapes and wine (Navarro et al., 2000; Rojo-Bezares et al., 2007a; Saenz et al., 2009). This LAB species is highly versatile and able to survive in an ample range of ecosystems. Among the different bacteriocin systems described in L. plantarum species (Diep et al., 1996, 2009; Remiger et al., 1999; Holo et al., 2001; Maldonado et al., 2002), pln loci are the best known, and they are organised into different operons. The first pln structure and sequence that was reported was in the strain L. plantarum C11, which was isolated from a vegetable source (Diep et al., 1996), and the same sequence was found in the whole genome sequence published for L. plantarum strain WCFS1, which was isolated from human saliva (Kleerebezem et al., 2003). In this structure, a regulatory operon encodes an inducing peptide (PlnA), a histidine protein kinase (PlnB) and some response regulators (PlnC and PlnD). A number of additional genes encode two-peptide bacteriocins and a highly conserved operon encodes proteins of an ABC transport system, which secretes and processes the bacteriocin precursors. The inducing peptide PlnA is responsible for the transcription of the other operons (Diep et al., 2003). A different pln locus sequence was reported for L. plantarum NC8 strain isolated from grass silage, which produces the inducible two-peptide plantaricin NC8βα (Aukrust and Blom, 1992; Maldonado et al., 2003; Maldonado et al., 2004), and two different pln loci of L. plantarum were described in strains isolated from grape and wine, those of strains J23 and J51 (Rojo-Bezares et al., 2008; Navarro et al., 2008). The common features of all these pln loci are the presence of: • a two-component regulatory system, which consists of a membrane-located histidine protein kinase and some cytoplasmatic response regulator • an inducing peptide • a dedicated ABC transport system • a number of adjacent bacteriocin-related peptides. A study by Knoll et al. (2008) showed that isolates from musts and wine contained one or several genes of the pln locus. A more extensive study by Saenz et al. (2009), who used a PCR approach to screen for pln genes in a wide collection of L. plantarum isolates from grape musts and wines, showed that the majority of the strains were positive for several pln genes and together with the known pln loci of type strains C11, WCFS1, J23, J51 and NC8 (Fig. 17.2) they constitute 18

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Fig. 17.2  Plantaricin pln loci of type strains C11, WCFS1, J23, J51 and NC8. Abbreviations A–Y: different pln genes related to plantaricin synthesis, secretion and regulation.

different pln gene compositions, which could be clustered into 7 groups, named plantaritypes. The most frequent pln composition among the enological L. plantarum isolates was shown to be that of C11-pln and WCFS1-pln, and the second most frequent pln composition was that of J51-pln. Both groups gathered 74% of the total enological L. plantarum strains, they shared the common feature of the plnABCD regulatory system (for a more detailed description of mechanisms of bacteriocin production and regulation see Diep et al., 2009), and showed slight differences in the presence of the bacteriocin peptide genes (Saenz et al., 2009). Not all the enological strains harbouring plantaricin pln genes show antimicrobial activity (33% of the enological LAB strains harbouring plantaricin genes showed antimicrobial activity according to the study of Saenz et al., 2009), and this would suggest that pln genes could be involved not only in growth inhibition of competing bacteria by secretion of antimicrobial peptides, but also in other bacterial responses. It is worth noting that the genes encoding integral membrane proteins and the genes that encode the ABC transport protein and its accessory protein were present in nearly all the studied strains (94%). Similarly, two pln genes that encode a two-peptide bacteriocin (named EF) were present in 88% of the studied strains (Saenz et al., 2009). These results suggest the prevalence of certain genes, and thus the importance for bacteria survival and adaptation of genes encoding extrusion systems and of genes encoding bacteriocins. It should be pointed out that the base composition (C+G content) of the fully sequenced pln loci was between 36.1 and 39.0% (Navarro et al., 2008), lower than the overall C+G content reported for the whole chromosome of L. plantarum (44.5%)

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(Kleerebezem et al., 2003), which suggests that the pln locus could have been acquired by L. plantarum species by horizontal gene transfer, and could constitute an islet of functional genes for bacterial adaptation, competition and survival in harsh environments, such as wine. Wine isolates of other species have also been reported to produce some type of inhibitory effect on the growth of competing bacteria, and thus inhibitory effects between P. pentosaceus, L. plantarum and O. oeni were first described by Lonvaud-Funel and Joyeux in 1993. A P. pentosaceus strain isolated from Argentinian wines was reported to produce pediocin N5p (Strasser de Saad and Manca de Nadra, 1993). This bacteriocin showed high heat stability, optimum pH for activity in the acid range and resistance to ethanol and sulphur dioxide. Additionally, bacteriocin-like inhibitory activities have been reported for Le. pseudomesenter­oides and Lc. lactis isolates from koshu vineyards (Yanagida et al., 2008) and for a Le. mesenteroides subsp. cremoris strain isolated from a white wine (Yurdugu and Bozoglu, 2002, 2008).

17.8 Bacteriocins with antimicrobial activity against wine spoilage bacteria Nisin has been tested as an inhibitor of enological bacteria, alone and in combination with ethanol and sulphur dioxide, which are the most potent antimicrobial agents of wine (Rojo-Bezares et al., 2007b). These studies have been carried out at laboratory scale and results showed that the minimal inhibitory concentrations (MIC) of sulphur dioxide in the presence of nisin were lower than MIC values in the absence of nisin, both in the case of O. oeni strains and the other LAB enological strains. These results indicate a synergic effect of nisin and sulphur dioxide in inhibiting LAB growth. In the presence of a subinhibitory concentration of nisin (0.01 mg l–1 for O. oeni and 0.39 mg l–1 for the other LAB), the inhibitory activity of sulphur dioxide significantly increased (from two-fold to four-fold) for O.oeni and for the other LAB enological strains (Rojo-Bezares et al., 2007b). An earlier study used nisin to prevent MLF in Pinot noir wines (Daeschel et al., 1991) and suggested as well the possibility of using nisin to control MLF in wines. In the case of AAB, Rojo-Bezares et al. (2007b) showed that there were no differences between inhibitory concentrations of sulphur dioxide in the presence or absence of a subinhibitory concentration of nisin (1.5 mg l–1), and in these cases nisin proved not to be an efficient antimicrobial agent. Pediocin PD-1 from P. damnosus was shown to be active against one O. oeni strain (Bauer et al., 2005) and against the formation of biofilms of O. oeni on stainless steel surfaces in Chardonnay must (Nel et al., 2002). Although the use of both nisin and pediocin to control LAB growth has great potential, their use has not been approved in wine making. The enzyme lysozyme is a similar antimicrobial product, in that it is a small protein with lytic activity against Gram-positive bacteria cell walls, it has a very limited activity against

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AAB cells, and no effect on yeasts. This antimicrobial enzyme has been proved to be efficient in controlling MLF and its use in wine making has been approved (maximum addition amount: 500 mg l–1) (Bartowsky, 2009; OIV, 1997). Lysozyme can be added during alcoholic fermentation to inhibit early LAB growth, and this is recommended in cases of risk of sluggish or stuck alcoholic fermentations, as lysozyme can prevent the increase of volatile acidity due to LAB growth at this stage (Bartowsky, 2009). Lysozyme is also advised to be used at the end of wine elaboration, before bottling, to inhibit the growth of residual LAB cells after MLF. The aroma of wine is not affected by the addition of lysozyme, but one of the major inconveniences of its use is its high cost and the consequent rise in price of the final product. Other peptides that have been shown to inhibit enological LAB growth are milk lactoferrin-derived peptides. Enrique et al. (2009) showed that these lactoferrin-derived peptides do not affect the growth rate and alcoholic fermentation of a commercial wine Saccharomyces cerevisiae strain, although the organoleptic effect of these peptides on wine sensorial analysis was not evaluated in this work. Wine and grape polyphenolic compounds have been shown to possess antimicrobial activity against wine spoilage bacteria (Cushnie and Lamb, 2005; Rodríguez et al., 2007). García-Ruiz et al. (2009) reported the inactivation of wine spoilage LAB by wine phenols, and Figueiredo et al. (2008) showed the effect of phenolic aldehydes, flavonoids and tannins inhibiting the growth of two enological LAB strains (O. oeni and L. hilgardii). Nevertheless, these compounds are naturally included in wine composition and their concentration is not a variable susceptible of being increased by the enologist.

17.9  Future trends Bacteriocins could be used for wine and beverage elaboration in three different ways: • adding living starter cultures for obtaining the fermented wine or beverage, and thus bacteriocins would be produced ‘in situ’ by the starter culture or by one of the strains included as part of the starter culture • purified or semipurified as an additive for biopreservation • as a food-quality ingredient for wine elaboration, based on a grape juice fermentate of a bacteriocin-producing strain. Nevertheless, bacteriocin use involves some inconveniences, such as requirement of a number of specific validations and approvals for their use in wine making in their purified or semipurified form; technical skills are required to prepare and use them correctly, and this fact increases the budget. The technological potential of bacteriocin-producing LAB bacteria is very high, and bacteriocin production is a field where basic research on the cellular mechanisms involved in the response to external signals must continue. More studies are required on the effect of well characterised bacteriocins (such as nisin,

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pediocin or plantaricin) in wine and on the growth of enological bacteria strains, alone and in combination with sulphur dioxide, to preserve wine during elaboration, ageing and storage. Other important issues are searching for bacteriocin-producing LAB fermentates with grape must composition, and characterising wine-indigenous LAB strains showing bacteriocin activity and able to grow in wines and to carry out MLF so that they can be used as starter cultures in the future. It could be naïve to believe that bacteriocins are going to solve all wine preservation problems. However, they are effective antimicrobials, they can constitute additional and efficient tools for wine making and preservation, their production and use in food and beverage industries is economically viable, and consumers’ desire for organic and premium wines impels the continued search for innovative procedures in wine biopreservation. Bacteriocins alone or in combination with other agents offer an excellent means to decrease sulphur dioxide levels in wine.

17.10  References allende a , tomás - barberán f a

and gil m i (2006), ‘Minimal processing for healthy traditional foods’, Trends in Food Sc Technol 17, 513–519. armentia a (2008), ‘Adverse reactions to wine: think outside the bottle’, Curr Opin Allergy Clin Immunol 8(3), 266–269. baldy m w (1997), The University Wine Course, San Francisco, Van Stenberg and Kushner. bartowsky e j (2009), ‘Bacterial spoilage of wine and approaches to minimize it’, Lett Appl Microbiol 48(2), 149–156. bauer r, chikindas m l and dicks l m (2005), ‘Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832’, Int J Food Microbiol 101(1), 17–27. beneduce l , spano g , vernile a , tarantino d and massa s (2004), ‘Molecular characterization of lactic acid populations associated with wine spoilage’, J Basic Microbiol 44(1), 10–16. boulton r b , singleton v l and bisson l f (1996), Principles and Practices of Winemaking, New York, Chapman and Hall. britz t j and tracey r p (1990), ‘The combination effect of pH, SO2, ethanol and temperature on the growth of Leuconostoc oenos’, J Appl. Bacteriol 68, 23–31. chen h and hoover d g (2003), ‘Bacteriocins and their food applications’, Comprehensive Reviews in Food Science and Food Safety 2, 82–100. cintas l m , casaus m p , herranz c , nes i f and hernández p e (2001), ‘Review: Bacteriocins of lactic acid bacteria’, Food Sci Tech Int 7, 281–305. cotter p d , hill c and ross r p (2005), ‘Bacteriocins: developing innate immunity for food’, Nat Rev Microbiol 3, 777–788. cushnie t t p and lamb a j (2005), ‘Antimicrobial activity of flavonoids. Review’, Int J Antimicrob Ag 26, 343–356. daeschel m a , dong - sun j and watson b t (1991), ‘Controlling wine malolactic fermentation with nisin and nisin-resistant strains of Leuconostoc oenos’, Appl Environ Microbiol 57(2), 601–603. diep d b , straume d , kjos m , torres c and nes i f (2009), ‘An overview of the mosaic bacteriocin pln loci from Lactobacillus plantarum’, Peptides 30(8), 1562–1574. diep d b , håvarstein l s and nes i f (1996), ‘Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11’, J Bacteriol 178, 4472–4483.

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and nes i f (2003), ‘Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators, the activity of which is enhanced upon phosphorylation’, Mol Microbiol 47, 483–494. dols - lafargue m , hyo young lee h y, le marrec c , heyraud a , chambat g and lonvaud - funel a (2008), ‘Characterization of gtf, a glucosyltransferase gene in the genomes of Pediococcus parvulus and Oenococcus oeni, two bacterial species commonly found in wine’, Appl Environ Microbiol 74(13), 4079–4090. du toit w j and lambrechts m g (2002), ‘The enumeration and identification of acetic acid bacteria from South African red wine fermentations’, Int J Food Microbiol 74, 57–64. enrique m , manzanares p , yuste m , martínez m , vallés s and marcos j f (2009), ‘Selectivity and antimicrobial action of bovine lactoferrin derived peptides against wine lactic acid bacteria’, Food Microbiol 26, 340–346. figueiredo a r, campos f , de freitas v, hogg t and couto j a (2008), ‘Effect of phenolic aldehides and flavonoids on growth and inactivation of Oenoccoccus oeni and Lactobacillus hilgardii’, Food Microbiol 25, 105–112. flanzy c (1998), Oenologie. Fondaments scientifiques et technologiques, Paris, Lavoisier. foulke j e (1993), U.S. Food and Drug Administration (FDA Consumer), ‘A Fresh Look at Food Preservatives.’ Available from: http://vm.cfsan.fda.gov/~dms/fdpreser.html g - alegría e , lópez i , ruiz j i , sáenz j , fernández e et al. (2004), ‘High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol’, FEMS Microbiol Lett 230, 53–61. garcía - ruiz a , bartolomé b , cueva c , martín - alvarez p j and moreno - arribas m v (2009), ‘Inactivation of oenological lactic acid bacteria (Lactobacillus hilgardii and Pediococcus pentosaceus) by wine phenolic compounds’, J Appl Microbiol 107, 1042–1053. gonzález a , hierro n , poblet m , mas a and guillamón j m (2005), ‘Application of molecular methods to demonstrate species and strain evolution of acetic acid bacteria population during wine production’, Int J Food Microbiol 102, 295–304. gonzález a , hierro n , poblet m , rozés n , mas a and guillamón , j m (2004), ‘Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation’, J Appl Microbiol 96, 853–860. gratia a (1925), ‘Sur un remarquable exemple d’antagonisme entre deux souches de colibacille’, C R Soc Biol 93, 1040–1041. guillamon j m and mas a (2009), ‘Acetic acid bacteria’, in König H, Unden G and Fröhlich J, Biology of Microorganisms on Grapes, in Must and in Wine, Berlin, Springer, 31–46. henick - kling t (1993), ‘Malolactic fermentation’, in Fleet G H, Wine Microbiology and Biotechnology, Amsterdam, Harwood A P. henick - kling t , acree t e , gavit b k , kreiger s a , laurent m h and edinger (1994), ‘Modifications of wine flavour by malolactic fermentation’, Wine East 4, 8–15 and 29–30. holo h , jeknic z , daeschel m , stevanovic s and nes i f (2001), ‘Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics’, Microbiol 147, 643–651. ibarburu i, soria diaz m e, rodriguez-carvajal m a, velasco s e, tejero mateo p et al. (2007), ‘Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs’, J Appl Microbiol 103, 477–486. izquierdo p m , ruiz p , seseña s and palop m l (2009), ‘Ecological study of lactic acid microbiota isolated from Tempranillo wines of Castilla-La Mancha’, J Biosci Bioeng 108(3), 220–224.

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kleerebezem m , boekhorst j , van kranenburg r, molenaar d , kuipers o p

et al. (2003), ‘Complete genome sequence of Lactobacillus plantarum WCFS1’, P Natl Acad Sci USA 100, 1990–1995. knoll c , divol b and du toit m (2008), ‘Genetic screening of lactic acid bacteria of oenological origin for bacteriocin-encoding genes’, Food Microbiol 25(8), 983–991. konings w n (2002), ‘The cell membrane and the struggle for life of lactic acid bacteria. Review’, Antonie Van Leeuwenhoek 82, 3–27. liu s q (2002), ‘A review. Malolactic fermentation in wine-beyond deacidification’, J Appl Microbiol 92, 589–601. lonvaud - funel a (1999), ‘Lactic acid bacteria in the quality improvement and depreciation of wine’, Antonie Van Leeuwenhoek 76, 317–331. lópez i , lópez r, santamaría p , torres c and ruiz - larrea f (2008), ‘Performance of malolactic fermentation by inoculation of selected Lactobacillus plantarum and Oenococcus oeni strains isolated from Rioja red wines’, Vitis 47(2), 123–129. maldonado a , ruiz - barba j l , floriano b and jiménez - díaz r (2002), ‘The locus responsible for production of plantaricin S, a class IIb bacteriocin produced by Lactobacillus plantarum LPCO10, is widely distributed among wild-type Lactobacillus plantarum strains isolated from olive fermentations’, Int J Food Microbiol 77, 117–124. mattick a t r and hirsh a (1947) ‘Further observations on an inhibitory substance (nisin) from lactic streptococci’, Lancet 2, 5–7. mills d a , rawsthorne h , parker c , tamir d and makarova k (2005), ‘Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking’, FEMS Microbiol Rev 29(3), 465–475. moll g n , konings n and driessen a j (1999), ‘Bacteriocins: mechanism of membrane insertion and pore formation’, Antonie van Leeuwenhoek 76, 185–198. navarro l , rojo - bezares b , sáenz y, díez l , zarazaga m et al. (2008), ‘Comparative study of the pln locus of the quorum-sensing regulated bacteriocinproducing L. plantarum J51 strain’, Int J Food Microbiol 128(2), 390–394. navarro l , zarazaga m , sáenz j , ruiz - larrea f and torres c (2000), ‘Bacteriocin production by lactic acid bacteria of Rioja red wines’, J Appl Microbiol 88, 44–51. nel h a , bauer r, wolfaardt g m and dicks l m t (2002), ‘Effect of bacteriocins pediocin PD-1, plantaricin 423 and nisin on biofilms of Oenococcus oeni on a stainless steel surface’, Am J Enol Vitic 53, 191–196. o ’sullivan l , ross r p and hill c (2002), ‘Potential of bacteriocin-producing lactic acid bacteria for improvements in food safety and quality’, Biochimie 84, 593–604. oiv (1997). Available from: http://news.reseau-concept.net/images/oiv/client/Resolution_ Oeno_FR_1997_10.pdf remiger a , eijsink v g h , ehrmann m a , sletten k , nes i f and vogel r f (1999), ‘Purification and partial amino acid sequence of plantaricin 1.25a and b, two bacteriocins produced by Lactobacillus plantarum TMW1.25’, J Appl Microbiol 86, 1053–1058. ribereau - gayon p , donèche b , dubourdieu d and lonvaud a (1998), Handbook of Enology: The microbiology of wine and vinifications, Australia, Wiley and Sons. rodríguez m j , alberto m r and manca de nadra m c (2007), ‘Antibacterial effect of phenolic compounds from different wines’, Food Control 18, 93–101. rogers l a and whittier e d (1928), ‘Limiting factors in lactic fermentation’, J Bacteriol 16, 211–229. rojo - bezares b , sáenz y, navarro l , jiménez - díaz r, zarazaga m et al. (2008), ‘Characterization of a new organization of the plantaricin locus in the inducible bacteriocin-producing Lactobacillus plantarum J23 of grape must origin’, Arch Microbiol 89(5), 491–499. rojo - bezares b , saenz y, navarro l , zarazaga m , ruiz - larrea f and torres c (2007a), ‘Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23 isolated from grape must’, Food Microbiol 24(5), 482–491.

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rojo - bezares b , sáenz y, zarazaga m , torres c

and ruiz - larrea f (2007b), ‘Antimicrobial activity of nisin against Oenococcus oeni and other wine bacteria’, Int J Food Microbiol 116(1), 32–36. sáenz y, rojo - bezares b , navarro l , díez l , somalo s et al. (2009), ‘Genetic diversity of the pln locus among oenological Lactobacillus plantarum strains’, Int J Food Microbiol 134(3), 176–183. strasser de saad a m and manca de nadra m c (1993), ‘Characterization of bacteriocin produced by Pediococcus pentosaceus from wine’, J Appl Bacteriol 74, 406–410. valley h and thompson p j (2001), ‘Role of sulfite additives in wine induced asthma: single dose and cumulative dose studies’, Thorax 56, 763–769. yanagida f , srionnual s and chen y s (2008), ‘Isolation and characteristics of lactic acid bacteria from koshu vineyards in Japan’, Lett Appl Microbiol 47(2), 134–139. yurduguel s and bozoglu f (2002), ‘Studies on an inhibitor produced by lactic acid bacteria of wines on the control of malolactic fermentation’, Eur Food Res Technol 215(1), 38–41. yurduguel s and bozoglu f (2008), ‘Effects of a bacteriocin-like substance produced by Leuconostoc mesenteroides subsp. cremoris on spoilage strain Lactobacillus fructivorans and various pathogens’, Int J Food Sci Tech 43, 76–81.

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18 Control of mycotoxin contamination in foods using lactic acid bacteria H. S. El-Nezami, University of Hong Kong, China and S. Gratz, University of Aberdeen, UK

Abstract: Mycotoxins pose an unavoidable problem in agricultural crop production and methods to control contamination are constantly sought. In this chapter we focus on the use of lactic acid bacteria (LAB) as a potential means to minimize mycotoxin contamination and toxicity. Experimental evidence is reviewed on the antifungal and antimycotoxigenic properties of LAB and on their potential to destroy or deactivate mycotoxins. Furthermore, the potential of LAB to reduce mycotoxin absorption and toxic effects in animals and humans is discussed. Key words: mycotoxins, probiotic bacteria, bioavailability, binding, physiological conditions.

18.1  Introduction Food is the fuel of life, and we are all concerned about the quality and safety of our food. Harmful components in plant-derived foods can be either produced by the plant itself, or are contaminants deriving from manmade sources or from microorganisms. Among these microorganisms, toxin producing fungi are ubiquitous in the environment and can invade our crops and produce toxic secondary metabolites known as mycotoxins. Worldwide, millions of tons of crops are destroyed every year due to fungal growth and spoilage, in order to reduce human exposure to mycotoxins.1 Technologies are available to minimize fungal growth and contamination during harvest, processing and storing of crops, but these methods are only available in developed countries, resulting in a divided prevalence of mycotoxin exposure. Low level mycotoxin exposure occurs in parts of the world where food is available in higher quality and variety, whereas high-level exposure causes 449 © Woodhead Publishing Limited, 2011

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acute disease which may result in death and is prevalent in areas where populations depend on a single staple food commodity. The major mycotoxin-producing fungi are found in the genera Aspergillus, Penicillium, Fusarium and Alternaria, where the first two commonly contaminate drops during drying and storage whereas the later two occur before or immediately after harvest.2 Among the mycotoxins, Fusarium mycotoxins, aflatoxins and ochratoxin A and patulin are common problems. Fusarium mycotoxins are a large group of toxins, containing trichothecens (deoxynivalenol DON, nivalenol NIV, 3-Acetyldeoxynivalenol 3-AcDON, T-2 Toxin, HT-2 Toxin) that commonly contaminate corn, wheat and oats, zeralenone and fumonisins B1 and B2, all major contaminants in corn and corn products.3 Aflatoxins are a group of mycotoxins, commonly contaminating maize and groundnuts, and are categorized as class 1A human carcinogens by the International Agency for Research on Cancer.4 Low level chronic aflatoxin exposure is linked to the development of ‘occult’ conditions such as impaired growth and immune function and chronic diseases such as liver cancer in areas where the aflatoxin-producing Aspergillus fungi are prevalent. It is therefore of major interest to prevent formation of mycotoxins in the first place, or to reduce their bioavailability from foods to prevent harmful effects. Microorganisms, especially bacteria, have been studied for their potential to either degrade mycotoxins or reduce their bioavailability. Among these bacteria, probiotic lactic acid bacteria have been identified as a safe means to reduce availability of aflatoxins in vitro. Furthermore, probiotic bacteria exert a number of other beneficial health effects, which make them even more suitable additives to food and feed. A recent outbreak of aflatoxicosis in May 2004 in Kenya5 has reminded us that the aflatoxin problem, although being known for decades, has not been solved.

18.2  Control of the mycotoxin problem Due to the increasing number of reports on the toxic nature of various mycotoxins, there is a need to control their levels in food and feed. Methods of control can be classified in three categories: 1 prevention of mould contamination and growth, 2 detoxification of contaminated products, and 3 prevention of mycotoxin absorption and toxicity6,7 (Fig. 18.1). 18.2.1  Prevention of mould growth The prevention of mould growth can be achieved by pre- or post-harvest strategies. Potential pre-harvest approaches include measures to reduce crop stress and associated fungal colonization, the use of non-aflatoxigenic strains of Aspergillus flavus to out-compete the toxigenic strains, and genetic engineering to produce more resistant crops.2 Among post-harvest methods, improved drying and storage

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Fig. 18.1  Commonly used approaches for the control of mycotoxins.

have been successful in reducing aflatoxin exposure in rural West African communities.8 Antagonistic microorganisms including yeasts, fungi and bacteria, have been studied to reduce mould growth and mycotoxin production, while lactic acid-producing bacteria are of special interest as preservation organisms due to their long history of use in food products. Studies mainly in the 1990s show that various strains of Lactobacillus casei, acidophilus, bulgaricus, plantarum and alimentarus are capable of inhibiting both growth and aflatoxin production of various strains of Aspergillus flavus and parasiticus (recently summarized in reference9). The mechanisms by which LAB can exert their antifungal and antimycotoxigenic effects are still unknown, but several mechanisms are suggested: microbial competition, depletion of nutrients, lowering of pH, and the production of heat-stable low molecular weight metabolites including lactic acid, benzoic acid and others.10 The main effect of LAB appears to be antifungal. A recent study found that L. plantarum inhibited the growth of A. flavus in vitro, but had no effect on aflatoxin production.11 Alternatively, Gourama and Bullerman12 reported earlier that various Lactobacillus species slightly reduced mould growth and aflatoxin production. Interestingly, when they grew LAB in a dialysis sack submerged in broth containing Aspergillus, they found that culture supernatants of lactobacilli greatly inhibited aflatoxin B1 and G1 production without affecting mould growth. Viable Propionibacterium shermanii strains and their culture supernatant have recently been shown to inhibit growth of Fusarium and Alternaria fungi and reduced production of the Fusarium toxins NIV, DON, fumonisin B1 and zeralenone.13 It appears that antifungal and antimycotoxigenic effects of LAB are mainly due to low molecular weight compounds such as short chain fatty acids (capoic acid, lactic acid benzoic acid) and antifungal cyclic dipeptides.9 However, experimental evidence suggests that these interactions strongly differ with different strains of LAB and fungi, and no firm conclusion can be drawn about the most potent strains to be used as antifungal or antimycotoxigenic agents. 18.2.2  Decontamination Once a commodity is contaminated with mycotoxins, methods to remove or destroy the toxins are sought to make the crop fit for animal or human consumption.

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In animal feed, chemical degraders such as ammonia are successfully used to reduce aflatoxin exposure of livestock.1 This procedure uses ammonium hydroxide or gaseous ammonia (ideally under high temperature/high pressure conditions) to degrade up to 99% of aflatoxins in maize and peanut products and is approved by the US FDA and EU to be used for decontaminating animal feeds.9 Microorganisms including yeasts, moulds and bacteria have also been tested on their ability to modify or inactivate mycotoxins. The soil bacterium Flavobacterium aurantiacum has been shown to remove aflatoxin B1 from liquid media14 and is used as biodegrader in the production of peanut milk and peanut based mild products,15 while non-aflatoxigenic Aspergillus strains have been shown to convert AFB 1 into its less toxic metabolite aflatoxicol.16 A recent publication reports a strain of Eubacterium can degrade Trichothecens. The same group also found a new yeast that can degrade ochratoxin A and zeralenone and therefore named it Trichosporon mycotoxinivorans.17 Studies in dairy products showed a transformation of aflatoxin B1 into the less toxic aflatoxin B2a during the fermentation process,16 and Streptococcus lactis was found to perform this transformation and produce aflatoxin B2a and aflatoxicol which were not present in the fermented milk before.18 Similarly, aflatoxin M1 has been shown to be degraded during the fermentation of milk, and some strains of Lactobacillus, Streptococcus and Bifidobacterium have been shown to degrade ochratoxin A and aflatoxin B1 during milk fermentation.9 One recent study investigated the direct effect of lactic acid on aflatoxin in vitro and found that AFB 1 and AFG 1 get reduced to less toxic products AFB 2 and AFG 2 by lactic acid.19 However, each of these approaches is limited in applicability to certain products and complete elimination of contamination is not achieved. Evidence is scarce on the ability of fermentation cultures that can degrade mycotoxins and is limited to experimental studies, but it is evident that fermentation alone cannot be relied on to reduce mycotoxin levels20 and to date no cultures have been developed into commercial products used in industry. 18.2.3  Prevention of absorption As a last resort, toxic effects of mycotoxin ingestion can be counteracted by reducing their bioavailability and absorption inside the gastrointestinal tract. For this purpose, a variety of non-nutrient feed additives are in common use, including activated carbons, hydrates sodium calcium aluminosilicate, zeolites, bentonites and certain clays. These prove suitable in feed production and have a very high affinity and specificity for certain mycotoxins.9 However, in food for human consumption, the use of non-nutrients is less appealing. In recent years, the potential use of microorganisms present in food systems to bind mycotoxins has been studied extensively, mainly in in vitro experiments, and yeast cell wall components have been shown to effectively bind mycotoxins.9 Numerous investigators focused their interest on the interaction of dairy strains of bacteria with mycotoxins and several bacterial strains, of food or human origin, have been tested for their ability to bind aflatoxins and other mycotoxins21–23 to

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their surface. Gram-positive bacteria (several lactobacilli and propionibacteria) appear to be more efficient in removing aflatoxin from liquid medium than gramnegative E. coli.24 Among the 5 strains of lactobacilli, Lactobacillus rhamnosus strain GG and strain LC-705 appeared to be most efficient binders for aflatoxin B1, removing approximately 80% of AFB 1 from liquid media within 0 hours of incubation, which implies that the binding is a very rapid process. This fast removal has been confirmed by other investigators.25, 26 The two strains Lactobacillus rhamnosus GG and LC-705 were later confirmed as most efficient AFB 1 binders27 among nine stains of lactobacilli. Peltonen et al.28 also studied a range of lactobacilli and bifidobacteria and found remarkable differences in AFB 1-binding abilities, even in strains very closely related. Besides the specificity of the bacterial strain, also the bacterial concentration influences the AFB 1 removal, but different minimum concentrations have been reported. Bolognani et al.29 used 5 × 109 CFU/mL of either Lactobacillus acidophilus or Bifidobacterium longum and only 13% of the AFB 1 could be removed within one hour. A minimum concentration of 2 × 109 CFU/ml appears to be required to remove 50% of free AFB 1 and higher binding occurs at 1010 CFU/ml.24 The rate of AFB 1 binding is a direct function of AFB 1 concentration added for a wide range of AFB 1 levels (1.7ng/ml–13ug/ml), suggesting no specific AFB 1 receptor involved in AFB 1 binding.30 This concentration range covers AFB 1 naturally occurring in food items. When the bacteria are subjected to various chemical and physical treatments, their ability to remove AFB 1 can be increased significantly. Autoclaved cells of L. casei remove significantly more AFB 1 from phosphate buffered saline (PBS) compared to viable bacteria.31 Heat treatment (boiling for 1 hour) and acid treatment also significantly enhanced AFB 1 binding by Lactobacillus rhamnosus strain GG and LC-705.24,32 Peltonen et al.28 compared the binding ability of various strains of viable and heat treated bifidobacteria and found that the viable bacteria bound 4–56% while heat treated bacteria bound 12–82% of the AFB 1. In contrast, Lankaputra and coworkers33 found that viable bacteria bound more dietary mutagens (including AFB 1) than heat treated bacteria. As heat treated bacteria are more efficient to remove AFB 1 than viable cells, its metabolic degradation can be excluded as the potential mechanism responsible for removal. Furthermore, HPLC analysis of the culture supernatant did not show appearance of any aflatoxin metabolites. It seems to be more likely that the toxin is bound to the bacterial surface. To elucidate this mechanism of surface binding, several experiments with the aflatoxin-bacteria-complex were carried out. Oatley et al.34 tested the stability of the AFB 1-bifidobacteria-complex by washing it six times with water, and found that the toxin is released from the bacteria and therefore only reversibly bound. These findings were confirmed for lactobacilli.28,30 In two studies Haskard et al.27,35 found that cell wall polysaccharides and peptidoglycans are mainly responsible for the binding of AFB 1 to the surface of Lactobacillus rhamnosus strain GG and LC-705. This was further confirmed by Lahtinen et al.36 who studied different cell wall components (exopolysaccharides, cell wall isolates and peptidoglycans) of Lactobacillus

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rhamnosus strain GG and concluded that peptidoglycans are the most likely binding sites for AFB 1. Heat treatment may denaturate proteins and lead to the formation of maillardproducts while acid treatment may break glycosidic linkages in polysaccharides and amine linkages in peptides and proteins and therefore increase the pore size in the peptidoglycan layer of the bacterial surface.27 This process could allow the binding of AFB 1 to the cell wall and would explain the increased binding ability of heat- and acid-killed bacteria compared to viable bacteria. Although most of the early work in this area has been done with aflatoxin, more recent in vitro studies also demonstrate the efficiency of LAB to remove other mycotoxins including ochrotoxin A (37, 38), Fusarium toxins (39) or patulin (38).

18.3  Reduction of toxic effects in vitro Following on in vitro binding studies, Lankaputhra33 found that binding of several food mutagens (including AFB 1) by lactobacilli also reduced the mutagenicity of the liquid in the Ames assay. This was also observed when lactobacilli were cultured in milk.40 Reduced mycotoxin-induced genotoxicity in the presence of lactobacilli was also observed in the hepatocyte Micronucleus test, and in vitro assay for DNA damage in liver cells.17 In colonocyte cell cultures, Lactobacillus rhamnosus strain GG was found to reduce the toxic effect of aflatoxin and DON on the function and integrity of the intestinal cell layer.41,42

18.4  Effectiveness under physiological conditions Before applying this approach to humans, further studies on the effect of physiological conditions on binding capacity are needed. In vitro binding assays can be modified to include gastric pH, digestive enzymes or intestinal mucus. Addition of HCl-enhanced bacterial AFB 1 binding by LAB,24,32,35 and that digestive enzymes like lipase35 or proteases such as pronase E,35 trypsin or α-chymotrypsin36 had no effect. A recent study screened nine human isolates of Lactobacillus, and found that the presence of bile acids had no effect on aflatoxin binding.43 Intestinal mucus, present in the gastrointestinal tract, significantly reduced aflatoxin binding by Lactobacillus rhamnosus strain LC-705 but not by strain GG, and similar strain differences in aflatoxin binding ability were observed in duodenal loops of chicks.44,45 This implies that the situation in the intestinal tract of the animal is more complex than can be explained, and intestinal mucus and other factors may influence AFB 1 binding. In a pilot clinical study, the effect of a mixture of Lactobacillus rhamnosus LC-705 and Propionibacterium freudenreichii ssp. shermanii JS on the AFB 1 levels in human faeces samples was investigated.46 The investigators found a significant reduction in the aflatoxin levels in the faeces of subjects who received the probiotic mixture for two weeks compared to the subjects receiving a placebo,

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but expected to find higher levels of AFB 1 in the faeces of the test group as a result of binding the toxin by the bacteria and reducing the uptake into the body. A possible explanation for the observed results could be that the faecal samples were taken one week after commencing the bacteria preparation, when the major part of AFB 1 in the body was already excreted in the faeces. To confirm that the reduction of aflatoxin levels in the faeces were not a result of accumulation of AFB1 into tissues, Kankaanpaa et al.47 investigated the adhesion capability of the AFB1-probiotic-complex in vitro. They report that the Caco-2 cell adhesion properties of a complex formed by Lactobacillus rhamnosus GG and AFB1 are reduced compared to Lactobacillus rhamnosus GG alone. Similar results were obtained from Hwan,48 where AFB1 binding by Lactobacillus casei reduced its subsequent adhesion to HT29 intestinal cells. These results are important, because the adhesion of the probiotic to the epithelium of the intestine is the first step in its colonization of the gastrointestinal tract. Therefore, the adhesion of the AFB1-probiotic-complex would increase the time the gastrointestinal tract is exposed to AFB1. Under in vivo conditions, one would expect probiotic bacteria to bind AFB 1 as soon as they interact with each other inside the intestinal tract. Thereafter bacteria should travel through the intestinal tube and be excreted eventually into faeces, taking bound AFB 1 with them (Fig. 18.2). Consequently, faecal levels of AFB 1 should allow us to estimate AFB 1 binding occurring in vivo, even though a percentage of binding calculated from results obtained from rat faecal samples lies well below results in vitro or ex vivo. As seen from the pilot clinical trial,46 faecal collections have to be conducted soon after probiotic intervention has

Fig. 18.2  Aflatoxin absorption from the gastrointestinal tract may be prevented by selected strains of probiotic bacteria. Such prevention may lead to a reduced circulation of aflatoxin metabolites in systemic circulation and an increased fecal excretion.

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started to assess the effect of probiotics. This is in agreement with in vivo findings from a rat trial,49 suggesting that probiotic administration only increased faecal excretion of AFB 1 within 24 hours of AFB 1 dosage, but not at later time points. However, the ultimate goal is to reduce AFB 1 bioavailability in the intestinal tract, and an increase in fecal AFB 1 excretion in the presence of probiotics is a promising result and warrants further investigation. The first human intervention trial used probiotic supplementation (Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp shermanii JS) in subjects naturally exposed to aflatoxin in their diet, to reduce AFB 1 exposure. The subjects (n=45 in each group) were randomized into two groups, either receiving a probiotic capsule or a placebo capsule twice a day for five weeks. Probiotic administration led to a significant decrease in the mean level of urinary aflatoxin B1-N7-guanine, an effective biomarker of biologically active dose of aflatoxin in humans. Thus this study showed that it is possible to reduce the biologically effective dose of aflatoxin by giving subjects with detectable aflatoxin exposure a regular dose of probiotics on a daily basis. These pioneering findings are demonstrating that the binding of mycotoxins by LAB might be used in the future in humans and animals to reduce adverse health effects. Clearly, exciting future research will further explore the potential of LAB to reduce mycotoxin exposure. In this chapter we have summarized research findings on the potential use of LAB to reduce mycotoxin contamination and exposure of animals and humans. To date, most of the evidence on antifungal and antimycotoxigenic effects of LAB is based on in vitro culture experiments, and no field trials have been conducted to test whether LAB would be able to hinder mould growth and mycotoxin formation during crop storage. Findings of mycotoxin transformation in dairy products during fermentation could be of great importance to reduce exposure to AFM 1 through dairy products, but again evidence is limited to some experimental studies. The interesting results on mycotoxin binding by LAB also need to be taken further, to explain the mechanism of toxin removal in more detail and to produce more evidence on the potential use of this approach in animals and humans. Altogether, several relevant lines of research indicate that LAB could play a major role in tackling the mycotoxin problem, but more extensive studies need to be conducted before these approaches can be translated into products to benefit agricultural production and human health.

18.5  References   1. cast coasat . Mycotoxins: Risks in Plant, Animal and Human Systems. Task force report No. 139. Ames, Iowa, USA, 2003.   2. williams j h , phillips t d , jolly p e , stiles j k , jolly c m , aggarwal d (2004). ‘Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions.’ American Journal of Clinical Nutrition 80: 1106–1122.

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  3. schothorst r c , van egmond h p (2004). ‘Report from SCOOP task 3.2.10 “collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states”: Subtask: trichothecenes.’ Toxicology Letters 153: 133–143.   4. iarc (2002). ‘IARC monographs on the evaluation of carcinogenic risks to humans: Some traditional herbal medicines, some mycotoxinsm naphtalene and styrene.’ IARC Monographs 82: 171.   5. cdc codcap (2004). ‘Outbreak of aflatoxin poisoning–eastern and central provinces, Kenya, January–July 2004.’ MMWR Morb Mortal Wkly Rep 53: 790–793.   6. riley r t , norred w r (1999). ‘Mycotoxin prevention and decontamination – a case study on maize.’ FNA/ANA 23: 25–30.   7. mishra h n , das c (2003). ‘A review on biological control and metabolism of aflatoxin.’ Crit Rev Food Sci Nutr 43: 245–264.   8. turner p c , sylla a , gong y y et al. (2005). ‘Reduction in exposure to carcinogenic aflatoxins by postharvest intervention measures in west Africa: a community-based intervention study.’ Lancet 365: 1950–1956.   9. kabak b , dobson a d , var i (2006). ‘Strategies to prevent mycotoxin contamination of food and animal feed: a review.’ Crit Rev Food Sci Nutr 46: 593–619. 10. gourama h , bullerman l b (1995). ‘Inhibition of growth and aflatoxin prodiction of Aspergillus flavus by Lactobacillus species.’ J Food Prot 58: 1249–1256. 11. xu j , wang h , ji r, luo x (2003). ‘Study on the effect of the growth and aflatoxin production by Aspergillus flavus parasiticus NRRL 2999 in the present of Lactobacillus plantarum ATCC 8014.’ Wei Sheng Yan Jiu 32: 334–338. 12. gourama h , bullerman l b (1995). ‘Inhibition of growth and aflatoxin production of Aspergillus flavus by Lactobacillus species.’ Journal of Food Protection 58: 1249–1256. 13. gwiazdowska d , czaczyk k , filipiak m , gwiazdowski r (2008). ‘Effects of Propionibacterium on the growth and mycotoxin production by some species of Fusarium and Alternaria.’ Pol J Microbiol 57: 205–212. 14. phillips t d (1994). ‘Approaches to reduction of aflatoxins in foods and feeds,’ in Eaton D L, Groopman J (eds). The Toxicology of Aflatoxins: Human Health, Veterinary and Agricultural Significance, New York: Academic Press. 15. diarra k , nong z g , jie c (2005). ‘Peanut milk and peanut milk based products production: a review.’ Crit Rev Food Sci Nutr 45: 405–423. 16. wu q , jezkova a , yuan z , pavlikova l , dohnal v, kuca k (2009). ‘Biological degradation of aflatoxins.’ Drug Metab Rev 41: 1–7. 17. schatzmayr g , zehner f , taubel m , et al. (2006). ‘Microbiologicals for deactivating mycotoxins.’ Mol Nutr Food Res 50: 543–551. 18. megalla s e , mohran m a (1984). ‘Fate of aflatoxin B-1 in fermented dairy products.’ Mycopathologia 88: 27–29. 19. shukla r s , verma r j , mehta d n (2002). ‘Kinetic and mechanistic investigations on reductions of aflatoxins by lactic acid.’ Bioorg Med Chem Lett 12: 2737–2741. 20. motarjemi y, nout m j (1996). ‘Food fermentation: a safety and nutritional assessment.’ Joint FAO/WHO Workshop on Assessment of Fermentation as a Household Technology for Improving Food Safety. Bull World Health Organ 74: 553–559. 21. styriak i , conkova e (2002). ‘Microbial binding and biodegradation of mycotoxins.’ Vet Hum Toxicol 44: 358–361. 22. el - nezami h s , chrevatidis a , auriola s , salminen s , mykkänen h (2002). ‘Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium.’ Food Addit Contam 19: 680–686. 23. el - nezami h , polychronaki n , salminen s , mykkänen h (2002). ‘Binding rather than metabolism may explain the interaction of two food-grade Lactobacillus strains with zearalenone and its derivative (´)alpha-earalenol.’ Appl Environ Microbiol 68: 3545–3549.

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24. el - nezami h , kankaanpää p , salminen s , ahokas j (1998). ‘Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin B1.’ Food Chem Toxicol 36: 321–326. 25. bueno d j , casale c h , pizzolitto r p , salvano m a , oliver g (2007). ‘Physical adsorption of aflatoxin B1 by lactic acid bacteria and Saccharomyces cerevisiae: a theoretical model.’ J Food Prot 70: 2148–2154. 26. khanafari a , soudi h , miraboulfathi m , osboo r k (2007). ‘An in vitro investigation of aflatoxin B1 biological control by Lactobacillus plantarum.’ Pak J Biol Sci 10: 2553–2556. 27. haskard c a , el - nezami h s , kankaanpää p e , salminen s , ahokas j t (2001). ‘Surface binding of aflatoxin B-1 by lactic acid bacteria.’ Applied and Environmental Microbiology 67: 3086–3091. 28. peltonen k , el - nezami h , haskard c , ahokas j , salminen s (2001). ‘Aflatoxin B1 binding by dairy strains of lactic acid bacteria and bifidobacteria.’ J Dairy Sci 84: 2152–2156. 29. bolognani f , rumney c j , rowland i r (1997). ‘Influence of carcinogen binding by lactic acid-producing bacteria on tissue distribution and in vivo mutagenicity of dietary carcinogens.’ Food Chem Toxicol 35: 535–545. 30. lee y k , el - nezami h , haskard c a , et al. (2003). ‘Kinetics of adsorption and desorption of aflatoxin B1 by viable and nonviable bacteria.’ J Food Prot 66: 426–430. 31. thyagaraja n , hosono a (1994). ‘Binding properties of lactic acid bacteria from “Idly” towards food-borne mutagens.’ Food Chem Toxicol 32: 805–809. 32. el - nezami h , kankaanpää p , salminen s , ahokas j (1998). ‘Physicochemical alterations enhance the ability of dairy strains of lactic acid bacteria to remove aflatoxin from contaminated media.’ J Food Prot 61: 466–468. 33. lankaputhra w e , shah n p (1998). ‘Antimutagenic properties of probiotic bacteria and of organic acids.’ Mutat Res 397: 169–182. 34. oatley j t , rarick m d , ji g e , linz j e (2000). ‘Binding of aflatoxin B1 to bifidobacteria in vitro.’ J Food Prot 63: 1133–1136. 35. haskard c , binnion c , ahokas j (2000). ‘Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG.’ Chem Biol Interact 128: 39–49. 36. lahtinen s j , haskard c a , ouwehand a c , salminen s j , ahokas j t (2004). ‘Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG.’ Food Addit Contam 21: 158–164. 37. piotrowska m , zakowska z (2005). ‘The elimination of ochratoxin A by lactic acid bacteria strains.’ Pol J Microbiol 54: 279–286. 38. fuchs s , sontag g , stidl r, ehrlich v, kundi m , knasmuller s (2008). ‘Detoxification of patulin and ochratoxin A, two abundant mycotoxins, by lactic acid bacteria.’ Food Chem Toxicol 46: 1398–1407. 39. niderkorn v, boudra h , morgavi d p (2006). ‘Binding of Fusarium mycotoxins by fermentative bacteria in vitro.’ J Appl Microbiol 101: 849–856. 40. hosoda m , hashimoto h , he f , yamazaki k , hosono a (1997). ‘Inhibitory effect of milk cultured with Lactobacillus strains on the aflatoxin mutagenicity.’ Anim Sci Technol 68: 555–562. 41. gratz s , wu q k , el - nezami h , juvonen r o , mykkanen h , turner p c (2007). ‘Lactobacillus rhamnosus strain GG reduces aflatoxin B1 transport, metabolism, and toxicity in Caco-2 cells.’ Appl Environ Microbiol 73: 3958–3964. 42. turner p c , wu q k , piekkola s , gratz s , mykkanen h , el - nezami h (2008). ‘Lactobacillus rhamnosus strain GG restores alkaline phosphatase activity in differentiating Caco-2 cells dosed with the potent mycotoxin deoxynivalenol.’ Food Chem Toxicol 46: 2118–2123. 43. hernandez - mendoza a , garcia h s , steele j l (2009). ‘Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1.’ Food Chem Toxicol 47: 1064–1068.

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44. gratz s , mykkanen h , ouwehand a c , juvonen r, salminen s , el - nezami h (2004). ‘Intestinal mucus alters the ability of probiotic bacteria to bind aflatoxin B1 in vitro.’ Appl Environ Microbiol 70: 6306–6308. 45. gratz s , mykkanen h , el - nezami h (2005). ‘Aflatoxin B1 binding by a mixture of Lactobacillus and Propionibacterium: in vitro versus ex vivo.’ J Food Prot 68: 2470–2474. 46. el - nezami h , mykkänen h , kankaanpää p , suomalainen t , salminen s , ahokas j (2000). ‘Ability of a mixture of lactobacillus and propionibacterium to influence the faecal aflatoxin content in healthy Egyptian volunteers: a pilot clinical study.’ Bioscience Microflora 19: 41–45. 47. kankaanpaa p , tuomola e , el - nezami h , ahokas j , salminen s j (2000). ‘Binding of aflatoxin B1 alters the adhesion properties of Lactobacillus rhamnosus strain GG in a Caco-2 model.’ J Food Prot 63: 412–414. 48. hwang k - t , lee w, kim g - y, lee j , jun w (2005). ‘The binding of aflatoxin B1 modulates the adhesion properties of Lactobacillus casei KCTC 3260 to a HT29 colon cancer cell line.’ Food Sci Biotechnol 14: 866–870. 49. gratz s , taubel m , juvonen r o , et al. (2006). ‘Lactobacillus rhamnosus strain G G modulates intestinal absorption, fecal excretion, and toxicity of aflatoxin B(1) in rats.’ Appl Environ Microbiol 72: 7398–7400.

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19 Active packaging for food biopreservation S. Yildirim, ZHAW, Zurich University of Applied Sciences, Switzerland

Abstract: There has been an increasing interest in antimicrobial food packaging during the last decades. This chapter presents an overview of the recent research and developments in antimicrobial packaging systems containing natural antimicrobial agents including bacteriocins, organic acids, plant extracts and enzymes, and natural antimicrobial polymers such as chitosan. Specific emphasis is given to non-edible films that have the potential to be used as an antimicrobial packaging for the biopreservation of food. The chapter also discusses different design considerations required for a successful introduction of antimicrobial packaging systems into the market. Key words: antimicrobial packaging, natural antimicrobial agents, active packaging, biopreservation of food.

19.1  Introduction In this chapter, an overview of antimicrobial packaging systems containing natural antimicrobial agents or natural antimicrobial polymers is given and antimicrobial activities of such systems with growth media or food are presented. Organic acids which are present naturally in certain foods but are mostly manufactured for commercial applications by chemical synthesis are also included in this chapter. Specific emphasis is given to the non-edible antimicrobial films which have potential to be used as packaging material for biopreservation of food. Other types of packaging systems showing antimicrobial functions but not containing natural antimicrobial agents are also briefly discussed. Furthermore, different technical and industrial requirements for successful applications of antimicrobial packaging are discussed.

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19.2  Food and active packaging 19.2.1  Packaging and its functions Packaging can be described as a coordinated system of preparing goods for transport, distribution, storage, retailing and use. It has a significant role in the food supply chain and performs several tasks (for basic functions of packaging see Table 19.1). Food packaging has to fulfil both technical and marketing needs. It should serve as a containment for the food, enabling the efficient transport within the whole supply chain and preventing any physical damage during the transport, and protect against tampering and theft. Another very important function of packaging is to Table 19.1  Packaging functions Technical functions Containment Transportation Protection Preservation

• Contain product; for example, a free-flowing powder, corrosive product or an aseptic product • Effective movement of goods from production to consumption • Prevent physical damage during the whole supply chain including transportation and storage • Prevent tampering and theft • Prevent spoilage of product by being a barrier to gases, moisture, light and volatiles • Prevent chemical or biological contamination of the product

Marketing functions Information

Display Communication Promotion

Convenience Wastage reduction

• Product identification and quantity • List of ingredients • Product preparation and use • Storage data and expiry date • Legal and safety requirements • Address of the responsible body • Opening instructions • Point of sale display • Branding • Appeal to the potential customer by the use of typography, symbols, icons, colour, illustrations, etc. • Promotional information • Free extra product or free token • New product • Product features and benefits • Product handling and serving for the packaging handlers and users • Portioning • Information about the use, reuse, or recycling and proper disposal of the packaging material • End of life

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maintain the quality and safety of the food from the production to the final consumption by preventing any chemical and biological changes. Packaging should be a good barrier to protect the food from environmental conditions such as oxygen, moisture, light, dust, pests, and volatiles, to prevent the deterioration of food by oxidation of the components, discoloration, loose texture, nutrient loss, etc. It should also be a barrier against chemical and microbial contamination which could lead to spoilage of the products. Besides the technical functions, packaging also has marketing functions. Packaging should inform about the product and its ingredients, preparation and use, storage conditions and expiry date, etc., depending on the regulatory requirements. It should also enable the efficient display of the product and communication of the packaging ‘persona’ to the potential customers. It can help to improve the selling of the product by informing about the specific promotions of the producers and providing convenience to the handlers and the end consumers. Also, environmental issues are increasingly important. In this regard, packaging can communicate to the consumers information about the resources of the packaging and reuse, recycle or compostability options of the packaging materials. 19.2.2  Active packaging As previously described, one of the main functions of food packaging is to protect the food from microbial and chemical contamination, oxygen, water vapour and light. Mostly, this role is a rather passive, with packaging as a barrier between the food and the gas surrounding the food, and the external environment. Such packaging systems have limits with regard to further extension of the shelf life of packaged food. During the last decades, consumer trends for better quality of food, fresh and convenient food products have intensified and this led to developing new packaging technologies to extend the shelf life of food, minimize the food lost and provide safe food products to the customers. Among such new technologies, active packaging is an innovative approach to enhance the shelf life of food stuffs while improving their quality, safety and integrity. Active packaging can be defined as a packaging system that interacts with the package components and the food to extend the shelf life or to improve the safety or sensory properties of the food, while maintaining the quality of the packaged product. Active packaging systems can be classified into active-releasing systems (emitters) which add compounds to the packaged food or into the headspace, or active scavenging systems (absorbers), which remove undesired compounds from the food or its environment (Fig. 19.1). Examples for main active packaging systems and their benefits are listed in Table 19.2. Some of the active packaging systems can have other functions than emitting or scavenging, such as shelf heating cans, microwave susceptors, etc. Several active packaging technologies have been developed in the form of sachets, where the active agent containing sachets is placed in the package headspace. However, since the content of the sachet can be accidentally ingested by the end consumer or the sachet can be broken inside the package, use of sachets

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Fig. 19.1  Active packaging systems.

Table 19.2  Active packaging systems, their benefits and possible applications Active packaging

Example of benefits

Possible food applications

Antimicrobial releasers

Inhibition of growth of pathogens and spoilage microorganisms on food Inhibition of growth of aerobic bacteria and moulds Inhibition of oxidation of fats and oils Enhancing the flavour of food Accelerated ripening

Fresh meat, processed meat, fish, bread, cheese, cakes

Inhibition of oxidation of food components. Inhibition of growth of aerobic bacteria, yeast and moulds Remove the excess moisture

Bread, snack foods, dried foods, wine, cakes, tea, nuts, milk powder

Carbon dioxide releasers Antioxidant releasers Flavour releasers Ethylene releasers Oxygen absorbers

Moisture absorbers Ethylene absorbers Carbon dioxide absorbers Odour absorbers

Reduce the rate of ripening Prevention of bursting of the package Improving the flavour of the food

Fresh meat, fish, cakes Snack foods, dried foods, meat Cereals, dried foods Fruits, vegetables

Snack foods, cereals, dried foods, sandwiches Fruits, vegetables Coffee Fried snacks

has not always been the preferred way to incorporate the active agent into the packaging. Alternatively, active packaging functions can be incorporated into the label or film. This is more difficult to achieve concerning the capacity, process ability and stability of the active agents and several researchers have been working to overcome such hurdles to develop successful active packaging film applications (Lopez-Rubio et al., 2004).

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19.3  Antimicrobial packaging for food biopreservation Among the active packaging technologies, interest in antimicrobial food packaging has increased significantly in recent decades. It has been considered as a complementary method to the existing preservation methods to control undesirable microorganisms on foods by means of the incorporation of antimicrobial substances in the packaging films or application as a coating onto the packaging materials. Direct addition of antimicrobials such as bacteriocins, enzymes, organic acids, essential oils, natural extracts, etc., could result in some loss of their activities because of their diffusion into the food matrix, and interaction with other food components such as lipids or proteins (Han and Floros, 1997; Davies et al., 1999; Rose et al., 1999; Hoffman et al., 2001). Use of packaging films containing antimicrobial agents could be more efficient than the direct addition of these compounds into the food. Controlled migration of the active compound from the packaging material into the food not only enables the initial inhibition of undesirable microorganisms present in food, but also creates a residual activity over time, during transport, storage and distribution of food (Cutter, 2002; Quintavalla and Vicini, 2002). Antimicrobial activity is possible by several methods, including the addition of sachets or pads containing antimicrobial volatiles, incorporation of antimicrobial agents into the matrix or the surface of the packaging material by co-extrusion, coating, adsorption or immobilization methods, or using inherently antimicrobial substances as packaging raw materials. Microbial contamination of most foods occurs primarily at the surface due to post-processing handling. Therefore, it is appropriate to design antimicrobial systems that can act by direct contact or, more desirably, that can undergo positive migration from the package structure into the foods to reach potential inner contamination. Antimicrobial packaging systems can be divided into three types (Fig. 19.2). The first class includes systems that are intentionally releasing the anti­ microbial agents, such as organic acids from the packaging directly to the food (Fig. 19.2(a)). These systems require direct contact of the packaging material with the food surface. Diffusivity of the active agent in the packaging material, and its solubility, stability and diffusivity in the food are important characteristics determining the concentration of the antimicrobial agent in the food. It should be higher than the minimum inhibitory concentration (the lowest concentration of the antimicrobials that inhibits the growth of specific microorganisms) to provide an improvement in safety or extension of the shelf life. A second class of antimicrobial packaging systems implies the release of volatile antimicrobial agents, such as herbs and spice extracts, from the packaging material to the headspace of the packaging (Fig. 19.2(b)). Antimicrobial agents diffuse through the headspace and are absorbed by the food. These systems are very suitable for the packaging of porous, powdered, shredded or irregularly shaped food such as ground beef, fruit pieces, mixed vegetables, etc., or when the packaging film is not in direct contact with the food.

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Fig. 19.2  Antimicrobial packaging systems. (a) Packaging releasing antimicrobial agent to the food; (b) packaging releasing antimicrobial agent into the headspace; (c) non-migratory antimicrobial polymers.

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The third class involves non migratory antimicrobial polymers where the antimicrobial agent is attached to the polymer backbone and does not intentionally migrate out (Fig. 19.2(c)). Antimicrobial activities of these systems are limited to the contact of the packaging with the food surface. Since the antimicrobial agent does not migrate, these systems require use of smaller amounts of antimicrobial agents and are especially suitable for antimicrobial agents that are not permitted as food ingredients. Polymer films that are antimicrobial themselves or surface modified films with antimicrobial activities belong to this group.

19.4  Natural antimicrobial agents and polymers Active packagings for biopreservation of food use antimicrobial agents or polymers occurring in nature or isolated from microbial, plant or animal sources. Antimicrobial agents produced by microorganisms include bacteriocins such as nisin or pediocin, antibiotics such as natamycin, organic acids such as sorbic and benzoic acids, or enzymes such as lysozyme. Plant origin antimicrobial agents include extracts of herbs such as thyme, oregano, etc. Chitosan is an example of a natural antimicrobial polymer obtained by deacetylation of chitin obtained commercially from shrimp and crabshell. 19.4.1  Bacteriocins Food-borne outbreaks resulting from the consumption of foods contaminated with pathogens continue to be a major concern for food safety. Consequently, considerable effort has been made to control pathogens in food. In this regard, natural antimicrobials, especially bacteriocins, have considerable potential for biopreservation of food (Dawson and Sheldon, 2000). Bacteriocins are antimicrobial peptides produced by bacteria which inhibit other closely related bacteria although some bacteriocins exhibit a broader inhibition spectrum. Nisin is the most common bacteriocin, tested for many applications (see Chapter 3). It has been approved for use as a food preservative by the Joint FAO/WHO Expert Committee on Food Additives and was affirmed generally recognized as safe (GRAS) by the U.S. Food and Drug Administration. Additionally, it is non-toxic, heat stable, commercially available and already used for specific applications in a variety of foods (Adams and Smid, 2000). It has a broad spectrum of antimicrobial activity, which includes spoilage bacteria and food-borne pathogens. Such properties increased the interest in the incorporation of bacteriocins, especially nisin, into the packaging (Table 19.3) to develop antimicrobial packaging systems. Mainly, nisin has been incorporated into coatings which can release the compound when in contact with food. Studies have focused on establishing methods for coating polyolefin-based films or barrier films with methylcellulose (MC), hydroxypropylmethylcellulose (HPMC) (Cha et al., 2003a; Franklin et al., 2004; Grower et al., 2004), or protein coatings such as whey protein isolates as a carrier for nisin (Lee et al., 2008). Polyethylene glycol, glycerol,

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Table 19.3  Antimicrobial packaging systems containing bacteriocins as antimicrobial agents Bacteriocin

Packaging material

Food/media

Nisin

PE/MC/ Tofu HPMC PE, BHI agar κ-carrageenan, chitosan/MC, HPMC/MC PE Beef tissue

Microorganism

Reference

Listeria monocytogenes Micrococcus luteus

Cha et al., 2003a Cha et al., 2003b

Brochothrix thermosphacta LAB, Brochothrix thermosphacta, Carnobacterium spp., Enterobacteriaceae Listeria monocytogenes Listeria monocytogenes TAB

Cutter et al., 2001 Ercolini et al., 2010

PE/PE oxide LDPE

Beef chops

Plastic with MC/HPMC MC

Hot dogs

Cellophane

Veal meat

PP/κcarrageenan/ glycerol PLA

Agar medium

Lactobacillus plantarum

Liquid egg white, orange juice

Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli Listeria monocytogenes Listeria monocytogenes

TSA

Pectin/PLA Orange juice, composite film liquid egg Liquid egg Glass/PLA white, skim milk Paper board Pasteurized with AP or milk, orange VAE juice co-polymer LDPE Oyster, ground beef SC film TSA/NaCl Agar Paper board Water, NB with VAE co-polymer

Franklin et al., 2004 Grower et al., 2004 Guerra et al., 2005 Hong et al., 2000 Jin and Zhang, 2008

Jin et al., 2009 Jin, 2010

TAB, yeast

Kim et al., 2002a

TAB Coliform bacteria Listeria monocytogenes Listeria monocytogenes, Escherichia coli, Micrococcus flavus

Kim et al., 2002b Kristo et al., 2008 Lee et al., 2003

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Table 19.3  Continued Bacteriocin

Packaging material

Food/media

Microorganism

Reference

Paper board with VAE co-polymer Paper board with VAE co-polymer WPI coated PP EAA copolymer PVDC

Milk, orange juice

TAB, yeast

Lee et al., 2004b

Cellulose film LDPE

Milk cream, TAB, Micrococcus water/paraffin flavus oil emulsion Culture media Lactobacillus plantarum MRS Lactobacillus leichmanii TSA Listeria monocytogenes Frankfurter Listeria monocytogenes Milk, TSA Micrococcus luteus Broiler skin Salmonella and drumstick typhimurium

PVC, LLDPE, Nylon LDPE Cellulose film Polyolefin

Smoked salmon Frankfurter

PE/PA

Ham, beef, turkey breast Cheese, processed ham GM17 agar

HPMC

Nutrient agar

LDPE

Beef tissue, MRS

Cellulosebased inserts

Pediocin

ClearTite™ plastic bag

Enterocin

Alginate, zein, PVA

Lee et al., 2004a Lee et al., 2008 Leung et al., 2003 Limjaroen et al., 2003 Luchansky and Call, 2004 Mauriello et al., 2005 Natrajan and Sheldon, 2000

Listeria monocytogenes Listeria monocytogenes, TAB Listeria monocytogenes Listeria innocua, Staphylococcus aureus Listeria innocua, Staphylococcus aureus, LAB, Lactobacillus lactis

Neetoo et al., 2008 Nguyen et al., 2008 Nicholson, 1998 Scannell et al., 2000

Micrococcus luteus

Sebti et al., 2003 Siragusa et al., 1999

Brochothrix thermophacta, Lactobacillus helveticus Turkey breast, Listeria ham, beef, monocytogenes BHI soft agar Ham Listeria monocytogenes

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Table 19.3  Continued Bacteriocin

Packaging material

Food/media

Bacteriocin from Lactobacillus curvatus PE Frankfurters, TSB PE Smoked salmon PE-OPA Pork steak, ground beef Lacticin LDPE/PA Agar NK24 Buffer

Microorganism

Reference

Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Micrococcus flavus, Listeria monocytogenes

Ercolini et al., 2006 Ghalfi et al., 2006 Mauriello, et al. 2004 Scannell et al., 2000

Notes: AP, acrylic polymer; BHI, brain heart infusion; EAA, ethylene acrylic acid; HPMC, hydroxypropylmethylcellulose; LAB, lactic acid bacteria; LDPE, low density polyethylene; LLDPE, linear low density polyethylene; MC, methylcellulose; MRS, de Man-Rugosa-Sharpe; NB, nutrient broth; PA, polyamide; PLA, polylactic acid; PE, polyethylene; OPA, oriented polyamide; PP, polypropylene; PVA, polyvinyl alcohol; PVC, polyvinyl chloride; PVDC; polyvinylidene chloride copolymer; SC, sodium caseinate; TAB, total aerobic bacteria; TSA, tryptose soy agar; TSB, tryptose soy broth; VAE, vinyl acetate-ethylene; WPI, whey protein isolate.

sorbitol and sucrose were used as a plasticizer for the coating solutions (Shaw et al., 2002; Kristo et al., 2008; Lee et al., 2008). Other methods of incorporation include spraying, or immobilization where the packaging films were sprayed with or soaked into bacteriocin-containing solution. Rarely, nisin was blended with polymers and extruded (Siragusa et al., 1999; Cutter et al., 2001). Antimicrobial activities of nisin-containing films have been tested with different foods. Table 19.3 shows successful applications where the antimicrobial films retarded the growth of pathogenic and/or spoilage bacteria. Polyethylene films coated with nisin-containing solution inhibited the growth of pathogenic Listeria monocytogenes strains on tofu and also contributed to the protection of the product even after the original packing solution has been discarded and replaced with tap water (Cha et al., 2003a). Nisin-incorporated polymer films reduced numbers of Brochothrix thermosphacta inoculated on beef tissue (Cutter et al., 2001). Antimicrobial protection of beef was also achieved by using nisincontaining low density polyethylene films where antimicrobial films reduced the growth of several microorganisms on beef (Siragusa et al., 1999; Ercolini et al., 2010). Paper boards coated with nisin-containing solutions were tested mainly for liquid products and effectively reduced the growth of different bacteria and yeast in milk and orange juice (Kim et al., 2002a, b; Lee et al., 2004b), milk cream (Lee et al., 2004b), and water (Lee et al., 2003). Nisin was also tested with packaging films obtained from renewable resources such as polylactic acid (PLA) or cellulose films. Nisin was loaded into pectin/PLA films by the diffusion coating method after extrusion and the resulting composite films were effective in reducing L. monocytogenes in orange juice and liquid egg (Jin et al., 2009). In

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another study, a glass jar was coated with a mixture of PLA and nisin and such coating treatments effectively inactivated L. monocytogenes in liquid egg and skim milk (Jin, 2010). Cellophane films containing nisin reduced the total aerobic bacteria on veal meat (Guerra et al., 2005), Listeria monocytogenes on frankfurters (Nguyen et al., 2008), and Listeria innocua and Staphylococcus aureus on sliced cheese and ham (Scannell et al., 2000). Other applications of nisin-containing antimicrobial films include packaging of milk (Mauriello et al., 2005), ham (Nicholson, 1998; Scannell et al., 2000), smoked salmon (Neetoo et al., 2008), and broiler skin and drumstick (Natrajan and Sheldon, 2000). Bacteriocins were sometimes combined with chelator EDTA (Cutter et al., 2001; Natrajan and Sheldon, 2000) to improve the antimicrobial spectrum especially on Gram-negative bacteria. Pediocin, enterocin, lacticin and bacteriocin from Lactobacillus curvatus are other bacteriocins that have been studied for the development of antimicrobial packaging systems (Table 19.3). They have been successfully applied for the packaging of foods such as ham, beef, frankfurters, and smoked salmon and showed antimicrobial activities, especially against the food-borne pathogen Listeria monocytogenes. 19.4.2  Organic acids, their salts and anhydrides Organic acids are natural constituents of many foods and they have been used for a long time as additives in food preservation as they show none-specific antimicrobial properties against bacteria and fungi. Organic acids, their salts or anhydrides can be added into food product formulations, coated onto the surface of food or incorporated into food packaging materials. Direct surface application by spraying or dipping may result in the reduction in or loss of antimicrobial activity due to a possible reaction with food components, diffusion into the food, and evaporation or instability during food processes. A more effective way could be the incorporation of organic acids into the packaging followed by release to food, which may result in longer-term protection of the food. Organic acids, their salts or anhydrides have been incorporated into the packaging as a coating or were mixed with the film-forming solutions prior to casting or extrusion. Anhydrides were found to be more compatible with the polyethylene due to their lower polarity and higher molecular weight compared to organic acids and salts. Packaging films containing organic acids, their salts or anhydrides have been tested with defined media as well as with food systems (Table 19.4). Sorbic acid, potassium sorbate and sorbic anhydride have been incorporated into different polymer based packaging films and the resulting films were tested with media against fungi such as Aspergillus niger, Penicillium sp., or Saccharomyces cerevisiae, or against bacteria including pathogens such as Listeria monocytogenes. Polyvinylidene chloride films containing sorbic acid placed between slices of beef prevented the growth of inoculated Listeria monocytogenes (Limjaroen et al., 2005). Benzoic acid sodium benzoate and benzoic anhydride-containing films were mainly tested against yeast and moulds with agar media. Low density polyethylene films incorporated with benzoic anhydride delayed the growth of moulds on cheese (Weng and Hotchkiss,

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Table 19.4  Antimicrobial packaging systems containing organic acids, their salts and anhydrides as antimicrobial agents Antimicrobial agent

Packaging material

Food/media

Sorbic acid

PVDC

LDPE

Beef bologna Listeria monocytogenes, MAB, LAB TSA Listeria monocytogenes Agar Aspergillus niger, medium Penicillium sp. PDA Penicillium notatum, Rhodotorula rubra YM agar S. cerevisiae

SC film

TSA/NaCl

PVDC

TSA

Sorbic anhydride

PE

PDA

Benzoic acid

PEMA

Sodium benzoate

Chitosan MC

Benzoic anhydride

LDPE

Sodium lactate

SC film

Aspergillus niger, Penicillium sp. PDA Penicillium notatum, Rhodotorula rubra PDA, cheese Penicillium sp., Aspergillus toxicarius, moulds Agar Listeria medium monocytogenes Smoked Listeria salmon monocytogenes

PVDC PEMA Potassium sorbate

Chitosan MC

Microorganism

Listeria monocytogenes Listeria monocytogenes Aspergillus niger, Penicillium sp.

PDA

Chitosan/ HPMC/E/ MAA Chitosan/ HPMC/E/ MAA

Reference Limjaroen et al., 2005 Limjaroen et al., 2003 Weng et al., 1999 Chen et al., 1996 Han and Floros, 1997 Kristo et al., 2008 Limjaroen et al., 2003 Weng and Chen, 1997 Weng et al., 1999 Chen et al., 1996 Weng and Hotchkiss, 1993b Kristo et al., 2008 Ye et al., 2008b

Ham, steak

Listeria monocytogenes

Ye et al., 2008a

Serratia liquefaciens, L. sakei Enterobacteriaceae Serratia liquefaciens, Lactobacillus sakei

Ouattara et al., 2000b

Acetic acid

Chitosan

Ham, pastrami, bologna

Propionic acid

Chitosan

Ham

Ouattara et al., 2000b

Notes: E/MAA, ethylene methacrylic acid copolymer, HPMC, hydroxypropylmethylcellulose; LAB, lactic acid bacteria; LDPE, low density polyethylene; MAB, mesophilic aerobic bacteria; MC, methyl cellulose; PE, polyethylene; PDA, potato dextrose agar; PEMA, poly(ethyleneco-methacrylic acid); PVDC, polyvinylidene chloride copolymer; SC, sodium caseinate; TSA, tryptose soy agar; YM, yeast mannitol. © Woodhead Publishing Limited, 2011

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1993b). Other organic acids and salts, such as acetic acid, propionic acid and sodium lactate incorporated in sodium caseinate or chitosan showed significant antibacterial activities in foods such as smoked salmon (Ye et al., 2008b), ham (Ye et al., 2008a; Ouattara et al., 2000b), and pastrami and bologna (Ouattara et al., 2000b). The design of packaging and properties of packaged food are important to develop effective systems. Packaging films containing organic acids should have direct contact with the food in order to release the active compounds to the food surface. Antimicrobial activity of organic acids depends on the dissociation constant pKa (corresponding to the pH at which 50% of the total acid is dissociated), and the pKa of most organic acids lies between pH 3 and 5. Because the undissociated portion of the molecule has been shown to be responsible for the antimicrobial effect, protection of organic acids is essential for food with pH near or lower to pKa. Therefore, an appropriate organic acid system has to be carefully chosen according to the packaged food. 19.4.3  Plant extracts Natural plant extracts have high potential for the development of new products and nutraceuticals, as many plant-derived antimicrobial compounds have a wide spectrum of activity against bacteria and fungi. Although several plants have been reported to be a source of antimicrobial compounds, not all the compounds have been tested as food preservatives. Among these plants, spices and herbs are important sources of antimicrobial agents which are effective against several Gram-positive and Gram-negative bacteria as well as yeast and fungi (Nychas, 2003). The composition, structure, as well as functional groups of the oils, play an important role in determining their antimicrobial activity. Among these, the oils of caraway, cinnamon, clove, coriander, cumin, garlic, mint, onion, oregano, pepper, rosemary, thyme and sage have been commonly used for food preservation. Essential plant oils are found in edible, medicinal or herbal plants, which minimize questions regarding their safe use in food products. Majority of the natural extracts such as essential oils are approved as GRAS by the U.S. Food and Drug Administration, and some such as clove extract, cinnamon oil, carvacrol and mustard oil have been incorporated into the packaging materials via coating or the co-extrusion process. Their antimicrobial activities have been tested on agar medium against different bacteria, yeast and moulds (Table 19.5). Grapefruit seed extract has been incorporated into multilayer polyethylene film by coating with polyamide binder or by the co-extrusion process (Ha et al., 2001). Coated films showed enhanced antimicrobial activities and a broader antimicrobial spectrum on laboratory media than co-extruded films, reducing the growth of aerobic and coliform bacteria on ground beef without affecting quality. Beef was also packaged with whey protein isolate films containing oregano oil (Zinoviadou et al., 2009) and the maximum specific growth rate of total flora as well as pseudomonads was reduced significantly, whereas the growth of lactic acid bacteria was completely inhibited. Another study showed a significant reduction of the total microbial counts on sprouts (alfalfa, broccoli and radish) packaged with oriented polypropylene/polyethylene films

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Table 19.5  Antimicrobial packaging systems containing natural extracts as antimicrobial agents Antimicrobial agent

Packaging material

Food/ media

Microorganism

Reference

Transcinnamaldehyde, garlic oil, allyl isothiocyanate, rosemary oil Grapefruit seed extract

SPI coated OPP/PE

Sprouts

Escherichia coli, Samonella typhimurium, Enterobacter sakazaki, Bacillus cereus

Gamage et al., 2009

PE

Agar medium, ground beef

Ha et al., 2001

Clove extract

LDPE

Essential oil of cinnamon, oregano oil, clove, cinnamon fortified with cinnamaldehyde Carvacrol

PP PE/EVOH

Culture media Agar medium

Escherichia coli, Staphylococcus aureus, P. aeruginosa, Bacillus cereus, Bacillus subtilis, Leuconostoc mesenteroides, Aspergillus flavus, Saccharomyces cerevisiae, Aspergillus niger, Penicillium. Chrysogenum, TAB, coliforms Lactobacillus plantarum and Fusarium oxysporum Yeasts, moulds, bacteria

LDPE

BHI agar

Brochothrix thermosphacta, Listeria innocua, Carnobacterium sp.

Mustard oil Linaolol, methylchavicol

LDPE

Eugenol, thymol, menthol Oregano oil

OPP

Agar, Cheddar cheese Grapes

WPI

Beef

Hong et al., 2000 Lopez et al., 2007

Persico et al., 2009

Escherichia coli, Listeria inocua

Suhr and Nielsen, 2005 Suppakul et al., 2008

Mesophilic aerobic yeast and moulds TVC, pseudomonads, LAB

Valverde et al., 2005 Zinoviadou et al., 2009

Notes: BHI, brain heart infusion; EVOH, ethylene vinyl alcohol copolymer; LAB, lactic acid bacteria; LDPE, low density polyethylene; OPP, oriented polypropylene; PE, polyethylene; PP, polypropylene; SPI, soy protein isolate; TAB, total aerobic bacteria; TVC, total viable count, WPI, whey protein isolate.

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coated with different extracts, especially with allyl isothiocyanate (Gamage et al., 2009). Linalool and methylchavicol-containing low density polyethylene films used to wrap cheddar cheese suppressed the growth of E. coli and L. innocua while not affecting the flavour of the cheese (Suppakul et al., 2008). Antimicrobial films were also combined with other preservation technologies such as modified atmosphere packaging (MAP). Eugenol, tymol or menthol-containing oriented polypropylene films reduced the total viable counts and prolonged shelf life of table grapes compared to application of MAP alone (Valverde et al., 2005). Although essential oils have broad antimicrobial spectra their application in packaging for food can be limited owing to their strong flavours. 19.4.4  Enzymes Antimicrobial enzymes play an important role in the defence mechanism of living organisms against infection by bacteria and fungi. Lysozyme is an antimicrobial enzyme active on beta 1–4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine of the bacterial peptidoglycan. It is one of the most frequently used biopreservatives in antimicrobial packaging (Han, 2000; Quintavalla and Vicini, 2002). This enzyme shows antimicrobial activity on Gram-positive bacteria but not on Gram-negative bacteria because of their protective outer membrane surrounding the peptidoglycan layer. However, susceptibility of Gram-negative bacteria could be enhanced by the addition of chelating agents such as EDTA (Gemili et al., 2009). Lysozyme has been immobilized on polyvinyl alcohol (PVOH) or cellulose triacetate films (Appendini and Hotchkiss, 1997) or incorporated into PVOH (Buonocore et al., 2005; Conte et al., 2006b, 2007) or PE (Conte et al., 2006a, 2008) matrix. Resulting films showed significant antimicrobial activities against Micrococcus lysodeikticus in buffer or agar media (Table 19.6). Lysozyme immobilized in PVOH films inhibited viable spores of Alicyclobacillus acidoterrestris in apple juice (Conte et al., 2006b) and was also successfully incorporated into natural polymers such as cellulose acetate (Gemili et al., 2009), zein films (Mecitoglu et al., 2006) or chitosan (Park et al., 2004) and the resulting films showed antimicrobial activities with laboratory media. Glucose oxidase is another enzyme that can be used as an active agent for antimicrobial packaging. It does not possess antimicrobial activity, but the products from the reaction exhibit antimicrobial power. Glucose oxidases produced by moulds such as Aspergillus niger and Penicillium spp. catalyse the formation of H2O2 and D-glucono-δ-lactone, which then reacts with H2O to form D-gluconic acid. The antimicrobial activity of the system is due to the cytotoxicity of the H2O2 formed, although the lowering of pH by the production of D-gluconic acid may also influence the growth of some microorganisms (Fuglsang et al., 1995). Glucose oxidase was immobilized onto plasma-activated bioriented polypropylene films and the resulting films inhibited the growth of Escherichia coli and Bacillus subtilis in liquid medium (Vartiainen et al., 2005). Application of this enzyme in packaging is limited by the cost of enzyme and the requirement of glucose, which is not present

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Table 19.6  Antimicrobial packaging systems containing enzymes as antimicrobial agents Antimicrobial agent

Packaging material

Food/media

Microorganism

Reference

Lysozyme

TSB

Lysozyme

PVOH, CTA PVOH

Lysozyme

PE

Buffer

Lysozyme

PVOH

Buffer

Appendini and Hotchkiss, 1997 Buonocore et al., 2005 Conte et al., 2006a, 2008 Conte et al., 2007

Lysozyme

PVOH

AME broth, apple juice

Lysozyme

CA

Lysozyme and Na2EDTA Lysozyme

Agar medium

Micrococcus lysodeikticus Micrococcus lysodeikticus Micrococcus lysodeikticus Micrococcus lysodeikticus Alicyclobacillus acidoterrestris cells and spores Bacillus amyloliquefaciens Escherichia coli

Zein

NA

Bacillus subtilis

MRS

Lactobacillus plantarum Escherichia coli, Entorococcus faecalis Escherichia coli, Bacillus subtilis

Mecitoglu et al., 2006

Buffer

Lysozyme

Chitosan

BHI

Glucose oxidase

BOPP

Liquid medium

Conte et al., 2006b Gemili et al., 2009

Park et al., 2004 Vartiainen et al., 2005

Notes: AME, acidified malt extract; BHI, brain heart infusion; BOPP, bioriented polypropylene; CA, cellulose acetate; CTA, cellulose triacetate; MRS, de Man-Rugosa-Sharpe; NA, nutrient agar; PE, polyethylene; PVOH, polyvinyl alcohol; SP, soy protein; TSB, tryptic soy broth.

in many foods in sufficient concentrations. In order to apply the antimicrobial properties of enzymes, it is necessary to preserve the enzyme activity during the processing and storage. The low tolerance of enzymes to high temperatures restricts the application of these compounds for antimicrobial packaging by extrusion and casting from solutions, but immobilization is an alternative way to incorporate enzymes into packaging films. Activity of the enzymes that are released from the packaging to the food will depend on temperature, pH, presence of salts etc. Until now, antimicrobial activities of the enzyme-containing packaging films were mainly tested with laboratory media. However, stability of the enzymatic activity has to be proven under real application conditions. 19.4.5  Chitosan Some polymers such as chitosan are inherently antimicrobial. Chitosan is a linear β-1,4-D-glucosamine obtained by deacetylation of chitin, which is one of the world’s most abundant biopolymers obtained commercially from shrimp and © Woodhead Publishing Limited, 2011

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crabshell as a by-product in the seafood industry. It has been shown to be nontoxic, biodegradable, biofunctional, biocompatible and to possess antimicrobial properties (Rabea et al., 2003). Antibacterial and antifungal activity of chitosan depends on the molecular weight of chitosan, degree of deacetylation, its concentration, and pH of the medium (Dutta et al., 2009). Chitosan has also been reported to have no impact on food quality. Additionally, it has been approved as a food ingredient from the FDA. Therefore, chitosan films show promise for application in food preservation as antimicrobial film and have been tested as a packaging material for the preservation of different foods (Dutta et al., 2009). Chitosan films were produced by the casting technique (Fernandez-Saiz et al., 2009) or by incorporation into polyethylene film before extrusion (Hong et al., 2000). Alternatively, chitosan-based antimicrobial films were obtained by immobilization on poly(vinyl alcohol) (Cho et al., 2000) or plasma-activated biaxially oriented polypropylene (Vartiainen et al., 2005), and by coating on poly(acrylic acid)/poly(ethylene glycol) diacrylate (Don et al., 2005), on paper (Lee et al., 2003, 2004b), or on polyvinylidene chloride polyamide film (Yingyuad et al., 2006). Chitosan was also blended with poly(vinyl alcohol) (PVA) using glutaraldehyde as cross-linker (Tripathi et al., 2009). Chitosan-containing films were effective in suppressing the growth of different bacteria including pathogens such as Escherichia coli and Listeria monocytogenes, as well as yeasts and moulds (Table 19.7). Antimicrobial activity of chitosan-based films was tested with growth media and with different food systems, and chitosonium acetate films showed reduction of bacterial growth in fish soup, without affecting sensorial properties and pH (Fernandez-Saiz et al., 2009). Antimicrobial activities of chitosan coated paper boards with other liquid foods such as milk, orange juice and water have been shown (Lee et al., 2003). Bacterial growth was suppressed in minimally processed tomato (Tripathi et al., 2009) and grilled pork (Yingyuad et al., 2006) when chitosanbased antimicrobial films were used. Besides having antimicrobial properties, chitosan enables the incorporation and release of antimicrobial agents while chitosan-based films were used to release acetic and propionic acid (Ouattara et al., 2000a), sodium benzoate and potassium sorbate (Chen et al., 1996), and lysozyme (Park et al., 2004).

19.5  Other antimicrobial packaging systems In addition to natural antimicrobial agents, several chemical agents have been incorporated into packaging to develop antimicrobial packaging systems. According to the EU regulations (Commission regulation (EC) No. 450/2009) active agents that are incorporated into the packaging material to be released into the food should comply with the legislation on food additives. Therefore, chemical antimicrobial agents that are released from the packaging into the food or its environment should be food grade chemicals. Non-food grade chemicals can be incorporated into packaging if they are not released into the food. In this case specific migration of the chemical antimicrobial agents to the food should be checked to comply with the specifications. © Woodhead Publishing Limited, 2011

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Table 19.7  Antimicrobial packaging systems containing chitosan Packaging material

Food/media

Microorganism

Reference

MC/chitosan

PDA

Chen et al., 1996

Chitooligosaccharide immobilized on PVA Chitosan/PAA/ PEGDA Chitosonium acetate

Agar

Penicillium notatum, Rhodotorula rubra Staphylococus aureus

PE/chitosan

Paper board with VAE co-polymer

Paper board with VAE co-polymer Chitosan/PVA

Chitosan immobilized on BOPP Chitosan coating on PVDC/Nylon

Staphylococus aureus, Escherichia coli Salmonella spp., Staphylococus aureus, Listeria monocytogenes Buffer Lactobacillus plantarum Escherichia coli, Saccharomyces cerevisiae, Fusarium oxysporum Milk, TAB orange Yeast juice, water, Listeria monocytogenes TSB, BHI Escherichia coli O157:H7 Listeria monocytogenes Escherichia coli O157:H7 Milk, TAB orange juice Yeast Processed Escherichia coli tomato Staphylococus aureus Bacillus subtilis PW Escherichia coli, Bacillus subtillis Nutrient broth Fish soup, TSB

Grilled pork TVC

Cho et al., 2000 Don et al., 2005 Fernandez-Saiz et al., 2009 Hong et al., 2000

Lee et al., 2003

Lee et al., 2004b Tripathi et al., 2009

Vartiainen et al., 2005 Yingyuad et al., 2006

Notes: BHI, brain heart infusion; BOPP, bioxially oriented polypropylene films; MC, methyl cellulose; PAA, polyacrylic acid; PDA, potato dextrose agar; PE, polyethylene; PEGDA, poylethylene glycol diacrylate; PVA, polyvinyl alcohol; PVDC, polyvinylidene chloride copolymer; PW; peptone water; TAB, total aerobic bacteria; TSB, tryptose soy broth; TVC, total viable count; VAE, vinyl acetate-ethylene.

Initially, fungicides such as imazalil and benomyl have been incorporated into films to develop antimicrobial packaging (Weng and Hotchkiss, 1993a; Halek and Garg, 1989). However, since such fungicides are not listed as food preservatives, they are not allowed to be released from the packaging into the food. A potential chemical agent is silver, whose antimicrobial activity has been known for a long time, and in recent years there has been an increasing interest in using it in polymers as an antimicrobial agent. Metallic silver is considered to be non reactive but in

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aqueous environments it can release silver ions that possess antimicrobial activity (Silver, 2003). Several silver-ion-containing zeolite or glass systems have been incorporated into many polymers, such as polyethylene, polypropylene, and polyamide and become commercially available, and such polymers can be incorporated into food contact layers of a laminate structure in order to reduce the amount of silver used. Antimicrobial activities of the silver-containing films have been shown mainly using laboratory media; however, studies with real food systems are required because antimicrobial activity can be weakened by food components such as sulphur-containing amino acids which are present in many food products (Brody et al., 2001). Another interesting chemical agent is triclosan, an antimicrobial aromatic chloroorganic compound that is used in soaps, deodorants and shower gels (Bhargava and Leonard, 1996), and recently, polyethylene films containing triclosan were developed by extrusion (Camilloto et al., 2009). The films showed antimicrobial effect in vitro and with ham against Escherichia coli and Staphylococcus aureus. Many volatile compounds are known to exhibit antimicrobial properties, including gases, such as SO 2 or ClO2 , and several studies have been focused on the incorporation of these substances into the packaging system. Chlorine dioxide is generated using sodium chlorite and acid precursors which are embedded in hydrophobic and hydrophilic phases of a copolymer. When moisture from the food contacts the hydrophobic phase, acid is released, which in turn reacts with the sodium chlorite releasing chlorine dioxide (Appendini and Hotchkiss, 2002). Release of antimicrobial gases from the packaging to the headspace enables antimicrobial effects on food without direct contact. Therefore, such systems are very appropriate for the packaging of porous, powdered, or irregularly shaped foods. High levels of oxygen present in food packages may facilitate microbial growth, thereby causing significant reduction in the shelf life of foods. Therefore, control of oxygen levels in food packages can help to limit the growth of aerobic spoilage microorganisms in foods. Vacuum and modified atmosphere packaging technologies are well established in the industry to lower the concentration of oxygen in the headspace; however, such technologies cannot remove the oxygen totally from the headspace, or oxygen diffusing through packaging during storage, or trapped within the food. Oxygen absorbing systems can be used as a complementary technology to remove the oxygen from the packaging and until now oxygen scavenger systems have mainly been used as sachets. However, new technologies are being developed to incorporate oxygen scavengers into the packaging films. Such technologies include the incorporation of inorganic scavengers such as oxidizable iron, organic scavengers such as cyclohexenyl covalently bound to the polymer backbone, polyamide copolymers, vitamin C, and catalytic systems such as palladium or glucose oxidase in combination with high oxygen barrier films such as EVOH, PVDC, metallized films or aluminium foil in order to decrease oxygen diffusion into the packaging. Another possibility to obtain antimicrobial polymers is by modifying their surfaces by introducing active functional groups. Some synthetic polymers such as ultraviolet or excimer laser irradiated nylon films possess antimicrobial activity (James et al., 1998). Antimicrobial activity of nylon (6,6) films irradiated using a

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UV excimer laser at 193 nm in air results from the conversion of amide groups at the nylon surface to amines (with bactericidal properties). Antimicrobial activity of such films have been shown against Gram-positive and Gram-negative bacteria (Shearer et al., 2000).

19.6  Design of antimicrobial packaging systems Development and design of an efficient antimicrobial packaging requires an interdisciplinary approach which involves advances in food technology, microbiology, biotechnology, chemistry and packaging and material sciences. Antimicrobial agents should be selected carefully according to several issues listed in Table 19.8. An appropriate incorporation method should be developed according to the selected antimicrobial agent and food application, and regulatory requirements, cost and quality control issues should be taken into consideration for a successful introduction of the antimicrobial films on the market. 19.6.1  Appropriate antimicrobial agents Selection of the most appropriate antimicrobial agent depends on several factors. The source of the agent is an important factor for consumer acceptance; in general, Table 19.8  Packaging design considerations Appropriate antimicrobial agent     Source of antimicrobial agent         • Natural         • Chemical     Antimicrobial spectrum and target microorganisms         • Gram-positive, Gram-negative         • Yeasts, moulds         • Specific spoilage or pathogenic bacteria     Antimicrobial activity         • Required antimicrobial activity         • Microbial load on food         • Shelf life of food     Stability of activity of antimicrobial agents         • Inactivation of antimicrobial agents by food components         • Dilution of antimicrobial agent in food         • Loss of antimicrobial activity due to storage conditions     Effect of antimicrobial packaging on the organoleptic properties of food         • Colour         • Texture         • Flavour     Impacts of antimicrobial packaging on environment         • Environmental impact of production of antimicrobial agent         • Environmental impact of incorporation process         • Disposal of packaging         • Effect on recyclability

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Incorporation of antimicrobial agents     Incompatibility of antimicrobial agent         • Incompatibility with the packaging material     Possible incorporation processes         • Extrusion         • Coating         • Immobilization     Stability of the antimicrobial agent during processing         • Heat         • Chemical reactions         • Mechanical energy input     Contamination of process environment         • Contamination of machines and materials         • Cleaning possibilities     Mass transfer of antimicrobial agent         • Diffusion of antimicrobial agent through the film         • Release rate of antimicrobial agent from the packaging Effects of antimicrobial agents on the properties of the packaging film     Effects on the physical and mechanical properties of the films         • Barrier properties         • Stiffness         • Tensile strength         • Coefficient of friction         • Sealing and peeling properties Regulatory requirements     Legislation on packaging and food         • Active packaging regulations         • Legislation of food additives         • Specific migration limitations Quality control     Stability of the antimicrobial activity during the supply chain         • Storage         • Transportation         • Retailer     Quality control systems         • Quality control of antimicrobial films after production         • Quality control after converting processes (lamination, printing, lacquering)         • Quality control at food packers Cost     Material cost         • Cost of antimicrobial agents         • Other packaging and processing material costs     Production cost         • Cost of production of antimicrobial agents         • Economy of scale         • Requirements of investments

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natural additives are more easily accepted than chemically synthesized products. Therefore, the use of natural antimicrobial agents will be preferable for the acceptance of the final packaging by the consumers. Antimicrobial spectrum is another important factor in selecting the agent. There is no antimicrobial agent active against all the spoilage and pathogenic microorganisms, but different natural antimicrobial agents have different antimicrobial spectrum due to the antimicrobial mechanism of the agents and the physiology of the microorganisms. Some are only active against Gram-negative or Gram-positive bacteria or fungi whereas some have a broader spectrum including several bacteria, yeast and moulds. Effective antimicrobial agents should be chosen according to the target microorganisms in food. Different agents can be combined in order to cover a broader range of target microorganisms. Besides the antimicrobial spectrum, antimicrobial activity is an important issue to effectively prevent the growth of microorganisms in food. It is difficult to compare applications and efficiencies of different antimicrobial systems because different methods were used. Joerger (2007) has analysed quantitative results of microbiological assays conducted over the last decade involving antimicrobial films intended for use in food packaging. Antimicrobial activity of the chosen antimicrobial agent should allow reducing the undesired microorganisms on food to an acceptable level and prevent any microbial spoilage of food during shelf life. Antimicrobial agents that are released from the packaging film to the food can be inactivated or their efficacy can be decreased by some components in the food matrix, e.g. proteins, fats, saturated fatty acids or NaCl. Also, they may lose their activity due to dilution effect or storage conditions of the food. Antimicrobial activity of organic acids may decrease significantly in a food with high pH, or activity of enzymes can be affected by the storage temperature. Until now, antimicrobial films containing natural antimicrobial agents have been tested mainly with culture media, which provides good nutrition and environment for microbial growth. In order to broaden the application, possible interactions between antimicrobial agents and the food matrix should be better understood and the activities of the antimicrobial films should be tested with the specific food products under real conditions. Also, one important factor in choosing the antimicrobial agent is its possible effect on organoleptic properties such as the colour, texture or flavour of food. Sensorial properties of the food should remain acceptable by the consumer until the end of shelf life. Some of the antimicrobial agents, especially plant extracts, can have a strong flavour which may affect the sensorial properties and therefore limit the applications for food packaging, but migration of the antimicrobial agent can be adjusted in order to overcome any adverse effects. However, the concentration of the antimicrobial agent should not fall below its minimum inhibitory concentration. Introduction of a new antimicrobial functionality to the packaging may have additional negative impacts on the environment. On the other hand, antimicrobial films can help to reduce food losses by preventing microbial spoilage or increasing the shelf life, and thus have a positive effect on the environment. Therefore the whole product, i.e. packaging and food, should be evaluated together to determine the global effect on the environment. Disposal of the antimicrobial films and the

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effect of antimicrobial agents on the recycling process should also be taken into consideration, depending on the end life of the packaging. 19.6.2  Incorporation of antimicrobial agents In active packaging systems, the choice of components is often limited by the compatibility of the component with the packaging material or the stability of the active agent during the packaging process. Therefore, it is important to carefully choose the active agent, polymer matrix, and other ingredients, as well as a suitable incorporation technique. The polymer films that have been used to develop antimicrobial food packaging are polyethylene (PE), polyvinyl chloride (PVC), polyvinyl alcohol (PVA), polylactic acid (PLA), nylon, and others. Antimicrobial agents have been directly incorporated into the film, or bound on the polymer surfaces via coating or immobilization techniques. Direct incorporation of antimicrobial additives in packaging films is a convenient means by which antimicrobial activity can be achieved. The agents can be mixed with the polymers before they are extruded into films and, in general, such a process is straightforward to apply at an industrial scale and does not require large investments or changes in the film production processes, which could have a significant effect on production cost. Co-extrusion or extrusion coating technologies enable the incorporation of antimicrobial agents into the food contact layer only, which decreases the amount of active product for antimicrobial activity. The agents, however, should be stable through the extrusion process conditions, such as high extrusion temperatures or specific mechanical energy input. Volatilization of the active compound results in decrease or loss of activity and also pollution of the atmosphere in the production plant and the extruders. An alternative way is binding antimicrobials to polymeric surfaces which have been achieved by different means, ranging from simply spreading antimicrobial solutions onto the polymer surface, combining the antimicrobials with binders, or via immobilization of the active agent on a polymer. Binders can be cellulosic, or acrylic co-polymers. Sometimes the antimicrobials were covalently attached, with natural and synthetic cross-linkers like genipine, glutaraldehyde, and formaldehyde (Dutta et al., 2009). Immobilization can be carried out via ionic or covalent linkages, thus requiring suitable functional groups on both the antimicrobial substance and the packaging surface (Appendini and Hotchkiss, 2002). Examples of antimicrobial substances with functional groups are bacteriocins, peptides, enzymes, polyamines and organic acids. Additionally, some spacers such as dextrans, polyethylene glycol, ethylenediamine and polyethyleneimine can be used to link the polymer surface with the antimicrobial agent, such as bacteriocins (An et al., 2000). The method of incorporation of antimicrobial agents into the packaging films affects its transfer rate to the food as this transfer rate depends on the concentration of the active agent in the packaging, release rate from the packaging, absorption by the food surface and diffusion through the food. Release rate, absorption and diffusion may depend on various conditions such as storage temperature and time (Guerra et al., 2005). An appropriate method of incorporation

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of an antimicrobial agent should be chosen and the stability and mass transfer of the antimicrobial agent should be known to design an appropriate antimicrobial packaging system. 19.6.3 Effects of antimicrobial agents on the properties of the packaging film Physical and mechanical properties of the packaging films are very important to ensure efficient packaging and preservation of food. Antimicrobial agents that are incorporated into the packaging should not significantly influence the properties of the packaging films such as its transparency, barrier properties, tensile strength, stiffness, and coefficient of friction. When antimicrobial agents are incorporated into the food contact layer the effect of the agents on sealing and/ or peeling functions should be particularly evaluated. 19.6.4  Regulatory issues In the European Union, active and intelligent materials that are intended to be used in contact with foodstuffs must comply with Framework Regulation (EC) No. 1935/2004 and the specific Regulation (EC) No. 450/2009. This regulation stipulates that every substance that is intentionally released into food from packaging should comply with the requirements of the authorization under the specific legislation on food, such as legislation on food additives. Therefore, the use of natural antimicrobial agents approved as food ingredient or categorized as GRAS is promising for commercial applications. 19.6.5  Quality control Antimicrobial activity of the packaging films should be stable during the whole supply chain of the product. For an easy commercialization, antimicrobial packaging systems should not require totally new storage, transportation, distribution, and retailing systems. Appropriate quality control systems should be developed and be available to the packaging converters and food packers to monitor the antimicrobial activity of the packaging films. The effectiveness and the integrity of the antimicrobial packaging systems can be controlled by intelligent packaging systems which monitor the condition of packaged foods to give information about the quality of the food during the whole supply chain. Time temperature indicators can be used to monitor the chill chain from the point of packaging to consumption, thereby ensuring the freshness of the food products. It enables detection of any temperature abuse of food by producers, retailers and consumers, which is known to be the major cause of spoilage of the chilled perishable foods and beverages such as meat, poultry, fish, dairy products, fresh produce, juices and convenience meals. Such systems have already been developed and are available on the market. Another intelligent packaging system used to indicate the quality of the product is freshness indicators, giving

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information on the microbiological quality based on the reaction between the indicator and metabolites produced by microorganisms during the growth in the product. Pathogen indicators work in a similar way, but only indicate the growth of specific pathogenic bacteria such as E. coli O157. Several concepts of such indicators are being developed. However, validation and quality control of such systems are required to avoid false-true or true-false cases. Many perishable foods are packaged in modified atmospheres to hinder the growth of spoilage or pathogenic microorganisms and oxygen or carbon dioxide indicators can detect changes of oxygen or carbon dioxide content in the headspace, respectively. Therefore, they can indicate any leakage in the packaging leading to the growth of spoilage or pathogenic microorganisms. 19.6.6  Cost Cost is a crucial point for commercialization of the active packaging films. Initially, the antimicrobial packaging will find niche markets. The benefits of using antimicrobial films such as extension of shelf life, improvement of food quality and safety should be beyond the additional cost of introduction of the antimicrobial properties to the packaging. Therefore, production processes for antimicrobial films should be developed which preferably do not require extensive investments or cause significant changes in the production. Additionally, the cost of antimicrobial agents should be reduced by improving their production processes or reducing the amount of antimicrobial agent required. The latter can be achieved by increasing the activity of antimicrobial agents or through optimizing the design of the packaging and packaging process. Incorporation of antimicrobial agents only in the food contact layer instead of the whole packaging film or applied as a coating on the food contact surface will reduce the total amount of additive required for good antimicrobial activity.

19.7  Future trends Incorporation of antimicrobial agents directly into packaging is an interesting development, which allows the industry to combine the preservative function of antimicrobials with the protective function of packaging. Over the last few decades numerous antimicrobial agents have been evaluated for incorporation into the packaging. Among them, natural antimicrobial agents have high potential for commercial food packaging applications and would be preferred by the consumers to produce safer food. However, there is still a big gap between research and commercial applications. Antimicrobial activities of packaging systems have been mainly tested with laboratory media, with only a few studies carried out with food systems. To demonstrate the real potential of natural antimicrobial agents for packaging applications, their antimicrobial activities should be proven with food systems under real storage and distribution conditions. Additional to the antimicrobial functionality, antimicrobial packaging should

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fulfil several technical, economical, marketing and regulatory requirements. Therefore an interdisciplinary approach is necessary to support further developments in this area. Collaborative research activities between research institutes and food and packaging companies will pave the way for future commercial applications.

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and dawson p l (2001), ‘Antimicrobial effects of corn zein films impregnated with nisin, lauric acid, and EDTA’, J. Food Prot. 64, 885–889. hong s i , park j d and kim d m (2000), ‘Antimicrobial and physical properties of food packaging films incorporated with some natural compounds’, Food Sci. Biotechnol. 9, 38–42. james s p , muthukumar d and michael j k (1998), ‘Antimicrobial activity of UV-irradiated nylon film for packaging applications’, Packag. Technol. Sci. 11, 179–187. jin t (2010), ‘Inactivation of Listeria monocytogenes in skim milk and liquid egg white by antimicrobial bottle coating with polylactic acid and nisin’, J. Food Sci. 75, M83–M88. jin t , liu l s , zhang h and hicks k (2009), ‘Antimicrobial activity of nisin incorporated in pectin and polylactic acid composite films against Listeria monocytogenes’, Int. J. Food Sci. Technol. 44, 322–329. jin t and zhang h (2008), ‘Biodegradable polylactic acid polymer with nisin for use in antimicrobial food packaging’, J. Food Sci. 73, M127–M134. joerger r d (2007), ‘Antimicrobial films for food applications: A quantitative analysis of their effectiveness’, Packag. Technol. Sci. 20, 231–273. kim y m , an d s , park h j , park j m and lee d s (2002a), ‘Properties of nisinincorporated polymer coatings as antimicrobial packaging materials’, Packag. Technol. Sci. 15, 247–254. kim y m , paik h d and lee d s (2002b), ‘Shelf-life characteristics of fresh oysters and ground beef as affected by bacteriocin-coated plastic packaging film’, J. Sci. Food Agric. 82, 998–1002. kristo e , koutsoumanis k p and biliaderis c g (2008), ‘Thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on Listeria monocytogenes’, Food Hydrocolloids 22, 373–386. lee c h , an d s , lee s c , park h j and lee d s (2004a), ‘A coating for use as an antimicrobial and antioxidative packaging material incorporating nisin and alphatocopherol’, J. Food Eng. 62, 323–329. lee c h , park h j and lee d s (2004b), ‘Influence of antimicrobial packaging on kinetics of spoilage microbial growth in milk and orange juice’, J. Food Eng. 65, 527–531. lee c h , an d s , park h f and lee d s (2003), ‘Wide-spectrum antimicrobial packaging materials incorporating nisin and chitosan in the coating’, Packag. Technol. Sci. 16, 99–106. lee j w, son s m and hong s i (2008), ‘Characterization of protein-coated polypropylene films as a novel composite structure for active food packaging application’, J. Food Eng. 86, 484–493. leung p p , yousef a e and shellhammer t h (2003), ‘Antimicrobial properties of nisin-coated polymeric films as influenced by film type and coating conditions’, J. Food Saf. 23, 1–12. limjaroen p , ryser e , lockhart h and harte b (2005), ‘Inactivation of Listeria monocytogenes on beef bologna and cheddar cheese using polyvinylidene chloride films containing sorbic acid’, J. Food Sci. 70, M267–M271. limjaroen p , ryser e , lockhart h and harte b (2003), ‘Development of a food packaging coating material with antimicrobial properties’, J. Plast. Film & Sheeting 19, 95–109. lopez p , sanchez c , batlle r and nerin c (2007), ‘Development of flexible antimicrobial films using essential oils as active agents’, J. Agric. Food Chem. 55, 8814–8824. lopez - rubio a , almenar e , hernandez - munoz p , lagaron j m , catala r and gavara r (2004), ‘Overview of active polymer-based packaging technologies for food applications’, Food Rev. Int. 20, 357–387.

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and call j e (2004), ‘Evaluation of nisin-coated cellulose casings for the control of Listeria monocytogenes inoculated onto the surface of commercially prepared frankfurters’, J. Food Prot. 67, 1017–1021. marcos b , aymerich t , monfort j m and garriga m (2007), ‘Use of antimicrobial biodegradable packaging to control Listeria monocytogenes during storage of cooked ham’, Int. J. Food Microbiol. 120, 152–158. mauriello g , de luca e , la storia a , villani f and ercolini d (2005), ‘Antimicrobial activity of a nisin-activated plastic film for food packaging’, Lett. Appl. Microbiol. 41, 464–469. mauriello g , ercolini d , la storia a , casaburi a and villani f (2004), ‘Development of polythene films for food packaging activated with an antilisterial bacteriocin from Lactobacillus curvatus 32Y’, J. Appl. Microbiol. 97, 314–322. mecitoglu c , yemenicioglu a , arslanoglu a , elmaci z s , korel f and cetin a e (2006), ‘Incorporation of partially purified hen egg white lysozyme into zein films for antimicrobial food packaging’, Food Res. Int. 39, 12–21. ming x t , weber g h , ayres j w and sandine w e (1997), ‘Bacteriocins applied to food packaging materials to inhibit Listeria monocytogenes on meats’, J. Food Sci. 62, 413–415. natrajan n and sheldon b w (2000), ‘Efficacy of nisin-coated polymer films to inactivate Salmonella typhimurium on fresh broiler skin’, J. Food Prot. 63, 1189–1196. neetoo h , ye m , chen h q , joerger r d , hicks d t and hoover d g (2008), ‘Use of nisin-coated plastic films to control Listeria monocytogenes on vacuum-packaged cold-smoked salmon’, Int. J. Food Microbiol. 122, 8–15. nguyen v t , gidley m j and dykes g a (2008), ‘Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats’, Food Microbiol. 25, 471–478. nicholson m d (1998), ‘The role of natural antimicrobials in food/packaging biopreservation’, J. Plast. Film & Sheeting 14, 234–241. nychas g j e and skandamis p n (2003), ‘Antimicrobials from herbs and spices’, in Roller S, Natural Antimicrobials for the Minimal Processing of Foods, Cambridge, Woodhead, 176–200. ouattara b , simard r e , piette g , begin a and holley r a (2000a), ‘Diffusion of acetic and propionic acids from chitosan-based antimicrobial packaging films’, J. Food Sci. 65, 768–773. ouattara b , simard r e , piette g , begin a and holley r a (2000b), ‘Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan’, Int. J. Food Microbiol. 62, 139–148. park s i , daeschel m a and zhao y (2004), ‘Functional properties of antimicrobial lysozyme-chitosan composite films’, J. Food Sci. 69, M215–M221. persico p , ambrogi v, carfagna c , cerruti p , ferrocino i and mauriello g (2009), ‘Nanocomposite polymer films containing carvacrol for antimicrobial active packaging’, Polym. Eng. Sci. 49, 1447–1455. quintavalla s and vicini l (2002), ‘Antimicrobial food packaging in meat industry’, Meat Sci. 62, 373–380. rabea e i , badawy m e t , stevens c v, smagghe g and steurbaut w (2003), ‘Chitosan as antimicrobial agent: Applications and mode of action’, Biomacromolecules 4, 1457–1465. rose n l , sporns p , stiles m e and mcmullen l m (1999), ‘Inactivation of nisin by glutathione in fresh meat’, J. Food Sci. 64, 759–762. scannell a g m , hill c , ross r p , marx s , hartmeier w and arendt e k (2000), ‘Development of bioactive food packaging materials using immobilised bacteriocins Lacticin 3147 and Nisaplin (R)’, Int. J. Food Microbiol. 60, 241–249. sebti i , delves - broughton j and coma v (2003), ‘Physicochemical properties and bioactivity of nisin-containing cross-linked hydroxypropylmethylcellulose films’, J. Agric. Food Chem. 51, 6468–6474. luchansky j b

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Active packaging for food biopreservation shaw n b , monahan f j , o ’ riordan e d ,

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and o ’sullivan , m (2002), ‘Physical properties of WPI films plasticized with glycerol, xylitol, or sorbitol’, J. Food Sci. 67, 164–167. shearer a e h , paik j s , hoover d g , haynie s l and kelley m j (2000), ‘Potential of an antibacterial ultraviolet-irradiated nylon film’, Biotechnol. Bioeng. 67, 141–146. silver s (2003), ‘Bacterial silver resistance: molecular biology and uses and misuses of silver compounds’, FEMS Microbiol. Rev. 27, 341–353. siragusa g r, cutter c n and willett j l (1999), ‘Incorporation of bacteriocin in plastic retains activity and inhibits surface growth of bacteria on meat’, Food Microbiol. 16, 229–235. suhr k i and nielsen p v (2005), ‘Inhibition of fungal growth on wheat and rye bread by modified atmosphere packaging and active packaging using volatile mustard essential oil’, J. Food Sci. 70, M37–M44. suppakul p , sonneveld k , bigger s w and miltz j (2008), ‘Efficacy of polyethylenebased antimicrobial films containing principal constituents of basil’, Lebensm.-Wiss. Technol. 41, 779–788. tripathi s , mehrotra g k and dutta p k (2009), ‘Physicochemical and bioactivity of cross-linked chitosan-PVA film for food packaging applications’, Int. J. Biol. Macromol. 45, 372–376. valverde j m , guillen f , martinez - romero d , castillo s , serrano m and valero d (2005), ‘Improvement of table grapes quality and safety by the combination of modified atmosphere packaging (MAP) and eugenol, menthol, or thymol’, J. Agric. Food Chem. 53, 7458–7464. vartiainen j , ratto m , tapper u , paulussen s and hurme e (2005), ‘Surface modification of atmospheric plasma activated BOPP by immobilizing chitosan’, Polym. Bull. 54, 343–352. weng y m , chen m j and chen w (1999), ‘Antimicrobial food packaging materials from poly(ethylene-co-methacrylic acid)’, Food Sci. and Technol.-Lebensmittel-Wissenschaft & Technologie 32, 191–195. weng y m and chen m j (1997), ‘Sorbic anhydride as antimycotic additive in polyethylene food packaging films’, Lebensm.-Wiss. Technol. 30, 485–487. weng y m and hotchkiss j h (1993a), ‘Anhydrides as antimycotic agents added to polyethylene films for food packaging’, Packag. Technol. Sci. 6, 123–128. weng y m and hotchkiss j h (1993b), ‘Inhibition of surface molds on cheese by polyethylene containing the antimycotic imazalil’, J. Food Prot. 55, 367–369. ye m , neetoo h and chen h (2008a), ‘Control of Listeria monocytogenes on ham steaks by antimicrobials incorporated into chitosan-coated plastic films’, Food Microbiol. 25, 260–268. ye m , neetoo h and chen h q (2008b), ‘Effectiveness of chitosan-coated plastic films incorporating antimicrobials in inhibition of Listeria monocytogenes on cold-smoked salmon’, Int. J. Food Microbiol. 127, 235–240. yingyuad s , ruamsin s , reekprkhon d , douglas s , pongamphai s and siripatrawan u (2006), ‘Effect of chitosan coating and vacuum packaging on the quality of refrigerated grilled pork’, Packag. Technol. Sci. 19, 149–157. zinoviadou k g , koutsoumanis k p and biliaderis c g (2009), ‘Physico-chemical properties of whey protein isolate films containing oregano oil and their antimicrobial action against spoilage flora of fresh beef’, Meat Sci. 82, 338–345.

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Index

1-octen-3-ol, 31 1-octene, 31 2-methyl-isoborneol, 31 2-methyl-propanol, 31 3-acetyldeoxynivalenol 3-AcDON, 450 3-hydroxypropionaldehyde (3-HPA), 129, 136, 147–8 3-hydroxypropionaldehyde, 416 3-methyl-butanol, 31 3-methylfurane, 31 3-octanal, 31 3-octanone, 31 3-oxo-hexanoyl homoserine lactone (OHHL), 166 α-methyllanthionine (MeLan), 308 Acanthamoeba, 109 acetic acid bacteria (AAB), 437, 438 acetic acid, 352 Acetobacteriaceae, 437 acetoin, 269 Acinetobacter, 329 active packaging, 73, 460–85 Actobacillus rhamnosus, 36 Actobacillus rhamnosus DR20, 252 Actobacillus rhamnosus GG, 245, 453 Actobacillus rhamnosus LC705, 39, 52, 210, 453 Aeromonas hydrophila, 279, 327, 355, 409 aflatoxin absorption from the GI tract, 455 aflatoxins, 31, 450 agar diffusion tests, 12–14, 16, 116 variations of, 12–14 deferred antagonism, 13, 14 direct (simultaneous) antagonism, 13–14

agar well diffusion method, 116 agar, 357 alanine, 379 alcohols, 28 alginates, 357 Alicyclobacillus, 416 Alicyclobacillus acidoterrestris, 111 Alternaria, 29, 450 amino acids, 379 amylovorin L471, 355 anhydrides, 470–2 animal feed, 225–35 acid treatment, 228–9 airtight storage, 229 improving using Pichia anomala, 231 biopreservation, 229–33, 232–3 drying, 228 fermentation of, 229–31 formulation of, 234–5 fungal growth in, 226 mycotoxins in, 226–7 preservation techniques, 227–9 anti-Bacillus effect, 40 antibiosis, 368 antibiotic associated diarrhea (AAD), 249 antifungal LAB and PAB, 34–7 commercialised antifungal biopreservatives based on, 53 complementary metabolic pathways, 39 efficiency of in food challenge tests, 37–42 single cultures of lactobacilli and propionibacteria, 37–9 co-cultures of lactobacilli and propionibacteria, 39–42

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492 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 2 3 4 5 6 7 8 9 40 1 2 43 44 45X

Index

for food biopreservation, 27–57 future trends, 54 inhibitory spectrum and MIC, 46–8 potential in food applications, 35–7 antifungal metabolites anti-staling properties in bakery products, 43 chemical structures of antifungal compounds, 44–5 low-molecular-mass antifungal compounds, 43–51 proteinaceous antifungal compounds, 51 purification and identification of, 42–51 search for further antifungal mechanisms, 51–2 antilisterial agent, 305 antimicrobial agents, 120 antimicrobial cultures, 182–5, 242–4 clinical studies, 253 combination therapies, 185 competitive exclusion (CE), 183–4, 207–9 future trends, 196–7 overview of, 182–3 prebiotics, 184–5, 211–12 prevention and treatment of gastrointestinal diseases, 248–51 probiotics, 183, 209–11, 242–4 regulatory issues, 195–6 to reduce carriage of foodborne bacterial pathogens in poultry, 182–5 to reduce carriage of foodborne pathogens in cattle and swine, 204–15 tools for studying biological activities of, 252–4 antimicrobial packaging, 464–6, 467–9, 473, 476–84 antimicrobial proteins, 212–15 antifungal metabolites, 354–5 bacteriocins, 4, 100, 120, 185–7, 212–13, 297–315, 355–6, 407–9, 412, 466–70, 482 bacteriophages, 161–74, 187–95, 213–14, 311–13, 409–10, 413 colicins, 4, 212–13 sodium chlorate, 214–15 apples, 373, 374 arabitol, 380 Aspergillus, 29, 450 Aspergillus flavus, 32, 450 Aspergillus fumigatus, 32 Aspergillus nidulans, 32, 79 Aureobasidium pullulans, 373 B. infantis 35624, 253 Bacillus cereus, 105, 116, 168, 274, 301, 327, 350, 408, 420 Bacillus licheniformis LMG 19409, 416 Bacillus subtilis, 19, 50, 152, 210, 350, 365, 373, 420, 474 Bacillus subtilis C-3102, 183 Bacillus weihenstephanensis, 407

bacitracin, 17 bacteria antagonists, 208, 373 bacterial interference, 208 bacteriocin-like inhibitory substances (BLIS), 4, 17, 355, 407 bacteriocin-mediated starter cell lysis, 283 bacteriocinogenic cultures for biopreservation of meat products, 302–3 bacteriocins, 4, 100, 120, 185–7, 212–13, 297–315, 355–6, 407–9, 412, 466–70, 482 as additives, 273–5 bacteriocin-producing lactic acid bacteria, 269–73 characteristics of, 185–6 cultures for biopreservation of meat products, 302–3 for bacteria control, 332–40 for cheese safety, 270 future trends, 196–7 in cheese ripening, 280–4 in combined treatments, 275–9 with physical treatments, 275–8 with other biological antimicrobials, 278 in fermented vegetables, 422–4 in fruit juices and vegetable drinks, 413–18 in milk and dairy products, 267–88 in packaging, 465–9 in ready-to-eat and canned vegetable foods, 418–22 in wine making, 324–44, 439–44 isolated from LAB in seafood, 337 naming of, 7 overview of, 185 production of, 247 regulatory issues, 195–6 that may potentially be useful in the food industry, 9 to enhance the quality and flavour of cheese, 279–85 to improve the safety of dairy foods, 268–75 use of to inhibit Campylobacter and Salmonella, 186–7 use of to reduce carriage of food-borne bacterial pathogens in poultry, 185–7 bacteriophages, 161–74, 187–95, 213–14, 311–13, 409–10, 413 and food safety, 161–74 application of to control bacterial pathogens in foods, 168–73 bacteriophage therapy, 190 control of spoilage bacteria by phages, 171 discovery and taxonomy, 187–8 for bacteria control, 332–40 future trends, 196–7 life cycles, 163, 189–90 luciferase reporter phages (LRP), 164–6 major discoveries and developments, 172 pathogen detection using bacteriophages, 163 phage amplification assay, 164

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Index phage endolysins, 167–8 phage resistance, 191–2 phage therapy and food safety, 173–4 phage treatment to reduce the presence of Escherichia coli, 195 phage treatment to reduce the presence of salmonella, 194–5 phage typing, 163–4 regulatory issues, 195–6 reporterphages, 163–7 reporterphages using other systems, 166–7 use of to improve food safety and meat environment, 311–13 use of to improve the safety and quality of milk and dairy products, 285–7 use of to reduce carriage of food-borne bacterial pathogens in poultry, 187–95 BACTIBASE, 120, 308 BacWash, 313, 314, 315 BAGEL, 18, 120 bakery-based food products, 357 barley, 349 beer, 349 Bel’meat™, 306 benzoic acid, 43, 356 Bifidobacterium, 132, 243, 248, 452 Bifidobacterium animalis, 210 Bifidobacterium bifidium, 183, 248, 339 Bifidobacterium lactis DR10, 252 Bifidobacterium longum, 453 Bifidobacterium pseudocatenulatum JCM 7041, 253 Bifidobacterium thermacidophilum, 250 biocontrol agents (BCA), 365–88 development programme, 365–6 future trends, 387–8 genetic manipulations, 382 hurdles for commercial application, 385–7 improvement of, 379–82 in fresh-cut produce, 406 integration with other alternative methods, 383–5 mechanisms of action, 368–72 competition for space and nutrients, 373 direct interaction, 374 induced resistance, 374 production of antimicrobial substances, 373 tolerance to oxidative stress, 374 packaging and shelf life, 378–9 physiological manipulations, 379–81 production and formulation, 375–9 downstream processing and formulation, 376–8 production, 375–6 strategies for minimal processed fruits, 410–13 the search for biocontrol agents of postharvest diseases, 366–8 biopreservative starter culture, 233 Bio-Save 10, 377 Bio-Save 11, 377

493

BioSave 110, 410 black pepper, 304 BLIS see bacteriocin-like inhibitory substances bogobe, 353 Botrytis cinerea, 79, 373 bouza, 353 Bovamine Meat Culture™, 340 bovicin HC5, 412 brain heart infusion, 14 bread, 349, 357 Brochothrix thermosphacta, 11, 301 Burkholderia, 51 burukutu, 353 busa, 353 butyric acid, 352 Caco-2, 252 calcium propionate, 354, 356 Campylobacter, 181, 186–7, 205, 208, 251, 312 Campylobacter diarrhea-related infections, 251 Campylobacter jejuni, 5, 151, 162, 169, 181–97, 279 Canadian Food Inspection Agency (CFIA), 86 Candida, 226, 368 Candida albicans, 28, 145 Candida famata, 29, 373 Candida glabrata, 145 Candida inconspicua, 29 Candida krusei, 29 Candida lambica, 230 Candida magnoliae, 29, 41 Candida oleophila, 229, 368 Candida parapsilosis, 29, 41 Candida pulcherrima, 29, 32, 41 Candida sake CPA-1, 376 Candida sake, 368 Candida stellata, 29 carbon dioxide, 28 carboxymethyl cellulose, 378 carboxymethylchitosan, 384 carnobacteria, 331 Carnobacterium maltaromaticum, 329 carnocin UI49, 336 carrageenan, 357 carvacrol, 408 cellulose, 357 centrifugation, 376 cereals in human nutrition and animal feed, 349–50 major contaminant agents in cereal-based products, 350–1 natural fermentations occurring in cereals, 352–4 regional products, 353 silage, 353–4 microbial metabolites used as additives in cereal biopreservation, 356 CFIA see Canadian Food Inspection Agency cheese 268–88

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494 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 2 3 4 5 6 7 8 9 40 1 2 43 44 45X

Index

chemical structures of antifungal compounds, 44–5 cherry tomato, 374 chitosan, 384, 475–6 Chrisin®, 64 cinnamic acid, 408 citrinin, 350 citrus fruit, 374 citrus-rot, 229 Clostridium, 52, 66, 104, 138, 147, 267, 420 Clostridium botulinum, 301, 327 Clostridium difficile, 209, 242 Clostridium difficile-associated diarrrhea (CDAD), 249 Clostridium perfringens, 105, 168, 196, 327 Clostridium tyrobutyricum, 113, 412 coagulase negative staphylococci, 299 Codex Alimentarius Commission, 356 Codex of General Standard for Food Additives, 356 colicin E1, 213 colicin Hu194, 408 colicins, 4, 212–13 coliforms, 351 Colletotrichum coccodes, 382 colonic food, 211 commercial antifungal protective cultures, 52–54 commercialised antifungal biopreservatives, 53 HOLDBAC YM-B, 52 HOLDBAC YM-C, 52 Lactobacillus plantarum MiLAB 393, 52 Lactobacillus rhamnosus LC705, 54 MicroGARD®, 52, 81–2 safety and technological aspects of development, 55 competitive exclusion (CE), 183–4, 207–9 continuous colonic fermentation model, 252 continuous three-stage system, 252 continuous two-stage system, 252 Corynebacterium, 329 Crohn’s disease (CD), 253 crumpets, 357 Cryptococcus, 229 cultured dextrose, 82 cultured skim milk, 82 cultured wheat, 82 curvacin A, 7, 304 cyclo (L-Leu–L-Pro), 354 cyclo (L-Phe–trans-4-OH-L-Pro), 354 cyclo (Gly-L-Leu), 43 Dairy Safe™ cultures, 287 Debaromyces hansenii, 229 defensin peptides, 382 dehydration, 376 dehydroalanine, 308 dehydrobutyrine, 308 Dekkera anomala, 29

Delvocid®, 78 Delvoplus®, 64 deoxynivalenol DON, 450 deoxynivalenol, 350 dextrans, 482 dextrin, 131 diacetyl, 269 Discosphaerina fagi, 412 divercin V41, 336 divergicin M35, 336 DNA damage in liver cells, 453 dosa, 353 dual phage assay, 167 dual-culture agar plate assay system, 35 E. casseliflavus IM 416KI (Bac+), 307 E. faecalis A-48–32, 307 E. faecalis CECT7121, 307 E. faecium DPC 1146, 272 E. faecium S-32–81, 307 EcoShield™, 313, 315 ectoine, 379 edible and non-edible films, 75 edible coatings, 73 edible film-forming compound, 378 EDTA, 75 elicitors, 383 Endomyces fibulgier, 32 ensilage of crops, 348 Enterobacteriaceae, 11, 169 enterocin 1146, 272 enterocin 416K1, 307 enterocin AS-48, 100–120, 272, 407, 418–22 applications of, 110–12 characterization, structure and genetics, 107–9 history and GRAS status, 106–12 spectrum of inhibition and mode of action, 109–10 structure, 108 enterocin CCM4231, 309 enterocins, 272 enterococci, 272 Enterococcus, 19, 139, 272, 306 Enterococcus faecalis, 106, 152, 210, 272 Enterococcus faecalis INIA 4, 272 Enterococcus faecium, 183, 210, 300, 336 enterohemorrhagic E. coli infection, 250 enterohemorrhagic E. coli (EHEC) O157:H7, 143, 205, 269, 417 enzymes, 473–4 EPA see US Environmental Protection Agency Escherichia, 138 Escherichia coli O157:H7, 111–14, 151, 169, 190, 245, 268, 298 Escherichia coli, 109, 142, 151, 164, 185, 195, 232, 353, 439 ethanol, 352 ethyl acetate, 31 ethylenediamine, 482

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Index European Food and Feed Law Review (EFFL), 314 European Food Safety Authority (EFSA), 77, 341 Eurotium repens, 32 Eurotium rubrum, 32 exogenous bacteria, 328 exopolysaccharide (EPS), 42, 416 exopolysaccharides, 437 FAO see Food and Agriculture Organization fast protein liquid chromatography (FPLC), 115 FDA see US Food and Drug Administration fermentates, 63–87 assay protocols and mode of action, 84–5 existing and potential uses in foods, 85 MicroGARD®, 52, 82 physical, chemical and antimicrobial properties, 83–4 safety and regulatory status, 85 fermentation, 299, 348–9, 375, 376 fermentative technologies as a tool for microbial biopreservation, 351–4 natural fermentations occurring in cereals, 351–4 silage, 353–4 lactic fermentation, 353 of wine, 43–5 fermented sausages, 297–315 application of bacteriocins in, 307–11 bioprotective cultures for safety of, 301–7 food safety of, 298–9 legislation aspects and constraints, 313–14 micriobiota of, 299–301 filtration, 376 flavin mononucleotide (FMNH), 165 Flavobacterium aurantiacum, 452 flocculation, 376 fluidized bed-drying, 377 Food and Agriculture Organization (FAO), 30, 349 food biopreservation, 3–20 active packaging for, 460–85 antifungal lactic acid bacteria and propionibacteria, 27–57 as a means of increasing storage life, 10 desirable criteria for bio-preservative agents, 10 future considerations, 20 nisin, natamycin and other commercial fermentates used in, 63–87 of cereal products, 348–58 of seafood, 324–42 phage-based strategies, 358 potential for enterocin AS-48 in, 100–20 potential for lacticin 3147 in, 100–20 potential for lacticin 481 in, 100–20 potential for sakacin P in, 100–20 potential for variacin in, 100–20 procedure for inhibitor screening, 16–17

495

screening methodologies, 12–16 agar diffusion methods, 12–14 medium composition, 14–15 undefined fermentates used in, 81–6 food packaging, 460–85 active packaging systems, 462–3 antimicrobial packaging systems, 464–6, 473, 476–84 appropriate agents, 479–82 cost, 484 design considerations, 479–80 effects of agents on packaging film, 483 incorporation of agents, 482–3 quality control, 483–4 regulatory issues, 483 functions, 461 future trends, 484–5 Food Safety and Inspection Service (FSIS), 86 Food Standards Australia New Zealand (FSANZ), 313 formaldehyde, 482 formic acid, 352 freeze-drying, 376 fructooligosaccharides, 184, 212 fructose, 375 fruit juices, 413–18 fruit, 364–89, 403–25 FSIS see Food Safety and Inspection Service functional foods, 211 fungal pathogens, 366 fungi, 225–35, 352 in animal feed, 226 Fungicover, 378 Fusarium, 29, 450 Fusarium sporotrichioides, 32 galactooligosaccharides, 212 gas chromatography (GC), 49 gas chromatography-mass spectrometry (GC-MS), 43 gastrointestinal tract (GIT), 240 gel filtration chromatography, 43, 49, 112 gelatin, 357 Generally Recognized As Safe (GRAS), 10, 20, 77, 81, 86, 101, 150, 161, 196, 243, 285, 301, 313, 383, 483 genetic engineering, 450 genipine, 482 geosmin, 31 Geotrichum candidum, 230 gliotoxin, 350 Gluconacetobacter, 438 Gluconobacter asaii, 412 glutamate, 379 glutamine, 379 glutaraldehyde, 482 glycine betadine, 379 glycolchitosan, 384 GMOs, 382 gnotobiotic mice, 253

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496 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 2 3 4 5 6 7 8 9 40 1 2 43 44 45X

Index

Gram-negative bacteria, 63, 68, 143–5, 185, 228, 268, 408 Gram-negative psychrotrophic bacteria, 329 Gram-positive bacteria, 4, 51, 63, 66, 118, 139–42, 185, 228, 329, 408 and the nature of bacteriocin-like inhibitory substance, 5–10 classes, 5 subdivisions, 6 Gram-positive catalase-positive cocci (GCC+), 299–300 grape seed extract, 75 grapes, 384 GRAS see Generally Recognized As Safe green fluorescent protein (gfp), 166 green tea extract, 75 GRINDSTED® PRO 4, 356 gums, 357 heat treatments, 383, 384 Helicobacter pylori, 5, 133, 249, 251 Helicobacter pylori gastroenteritis, 251 helveticin J, 8 heterofermentative lactobacilli, 354 hexadecylpyridinium chloride, 408 high hydrostatic pressure (HHP), 105 high pressure homogenization (HPH), 418 high-intensity pulsed-electric field (HIPEF) treatment, 112 high-performance liquid chromatography (HPLC), 49, 80 histamine fish poisoning, 326 HIV/AIDS diarrhea, 251 HOLDBAC YM-B, 52 HOLDBAC YM-C, 52 HOLDBAC, 315, 340 homofermentative lactobacilli, 299, 354 HT-2 Toxin, 450 HT-29, 252 HT29-MTX, 252 human colon, 252 models of, 252 continuous colonic fermentation model, 252 continuous three-stage system, 252 continuous two-stage system, 252 human proximal colon system, 252 SHIME, 252 three-stage culture system with immobilized fecal microbiota, 252 TIM (TNO) intestinal model, 252 human digestive microbiota as partner for human defense, 242 biological control of using antimicrobial cultures and bacteriocins, 240–54 competition for nutrients, 245 degradation of toxin receptors, 245–6 mechanisms of action, 244–8 production of inhibitory substances, 246–8

stimulation of immunity/immunomodulation, 246 human epithelial cells, 252 human gastrointestinal defences, 241 human intestinal cell lines, 252 Caco-2, 252 HT-29, 252 HT29-MTX, 252 human proximal colon system, 252 human-microbiota-associated mouse model, 253 hurdle technology, 68 hydrocinnamic acid, 408 hydrogen peroxide, 247, 269 ice nucleation protein (inaZ), 167 idli, 353 immobilized cell technology, 54 Integrated Pest Management (IPM), 388 International Agency for Research on Cancer, 450 inulin, 131, 211 irritable bowel syndrome (IBS), 253 jamin-bang, 353 Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives (JECFA), 33, 81 kenkey, 353 kishk, 353 kisra, 353 Kluyveromyces lactis, 29 Kluyveromyces marxianus, 29 Kocuria varians NCC 1482, 115 kwunu-zaki, 353 L. acidophilus HN017, 252 L. acidophilus LA-1, 252 L. curvatus LTH1174, 304 L. monocytogenes NCTC10527, 311 L. monocytogenes, 268 L. plantarum 299, 253 L. rhamnosus GG, 252 L. sakei CTC494 Bac+, 304 LAB see lactic acid bacteria Lact. lactis LMG21206, 306 lactate dehydrogenase, 280 lactic acid, 352 lactic acid bacteria (LAB), 3, 27–57, 100, 138, 229–31, 234, 242–4, 269–73, 298–315, 325, 351, 405 acrolein-producing, 111 antifungal effects in malting and brewing, 38 antifungal screening of, 35–7 bacteriocin produced by different LAB species, 7 bioprotective LAB for bacteria control, 332–40 exopolysaccharide-producing, 111

© Woodhead Publishing Limited, 2011

Index history in food fermentation, 34 in fresh fish stored in ice or under packaging, 330 in lightly preserved fish, 330–1 in living fish, 329 in seafood products, 329–32 in wine making, 433–45 spoilage potential of, 331–2 that produce more than one bacteriocin, 7 to control mycotoxin contamination in foods, 449–56 lactic fermentation, 353 lacticin 3147, 9, 100–120, 274, 355 applications of, 105–6 characterization, structure and genetics, 101–4 history and GRAS status, 101 primary structure, 103 spectrum of inhibition and mode of action, 104–5 lacticin 481, 100–20, 271 applications of, 114 characterization, structure and genetics, 112–13 history and GRAS status, 112 sequence comparison of precursor peptides between lacticin 481 and variacin, 113 spectrum of inhibition and mode of action, 113–4 lactobacilli, 306 Lactobacillus, 4–20, 34, 243, 436, 451 Lactobacillus acidophilus, 152, 183 Lactobacillus casei, 407 Lactobacillus collinoides, 416 Lactobacillus coryniformis, 35 Lactobacillus curvatus, 7 Lactobacillus delbrueckii, 152 Lactobacillus diolivorans, 416 Lactobacillus helveticus, 114 Lactobacillus plantarum MiLAB 393, 52 Lactobacillus plantarum WHE 92, 272 Lactobacillus plantarum, 35 Lactobacillus reuteri, 129–53 additional antimicrobial compounds produced by L. reuteri, 152 origin and characteristics, 130–1 probiotic activity, 132–4 Lactobacillus rhamnosus DR20, 252 Lactobacillus rhamnosus GG, 453 Lactobacillus rhamnosus LC705, 54 Lactobacillus sakei Lb706, 18 Lactobacillus sakei Lb706-B, 18 Lactobacillus sakei, 7, 116 Lactobacillus sanfrancisco CB1, 43 Lactococcus lactis, 9, 63, 101 lactoperoxidase system, 278 lanthionine (Lan), 308 lantibiotics, 65 lacticin 3147, 9

497

macedocin, 6, 10 nisin U, 17 nisin Z, 340, 407 SA-FF22, 6, 14 salivaricin A, 9 salivaricin B, 9 L-ascorbic acid, 378 LC-705, 453 Leuconostoc, 243, 406, 435 Leuconostoc carnosum, 9 lightly preserved fish products (LPFP), 325, 327, 328, 329, 332 limonene, 31 Listeria, 9, 104, 119, 164, 196, 205, 267 Listeria innocua, 74, 105, 139 Listeria monocytogenes, 151, 165, 171, 186, 268, 298 listeriosis, 299 LISTEX™, 196, 312, 314 LISTEX™ P-100, 312, 340 ListShield™, 312, 313, 315 liver transplant patients, 254 L-lactic acid, 434 LLO, 340 L-malic acid, 434 luciferase reporter phages (LRP), 164–6 luria agar, 14 lysogenization, 162–3 lysozyme, 74, 443 Maalox, 193 macedocin, 6, 10 maize, 349 malolactic fermentation (MLF), 434–5, 441 malting, 355 maltodextrins, 212 mannitol, 380 mannose-oligosaccharides, 184 mass spectrometry (MS), 49 mesophilic bacteria, 351 methicillin-resistant Staphylococcus aureus (MRSA), 104 methylhydantoin, 43 Metschnikowia fructicola, 368 Metschnikowia pulcherrima, 373, 411 mevalonolactone, 43 MIC see minimum inhibitory concentrations microbes antimicrobial agents, 466–70, 478–83 antimicrobial packaging systems, 464–6, 473, 476–84 characteristics of and inhibitory products of relevance to their potential protective activity in food, 10–12 fermentative technologies as a tool for microbial biopreservation, 351–4 inhibitory products, 11 microbial antagonists, 373 microbial applications in the biopreservation of cereal products, 348–58

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498 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 20 1 2 3 4 5 6 7 8 9 30 1 2 3 4 5 6 7 8 9 40 1 2 43 44 45X

Index

microbial metabolites used as additives in cereal biopreservation, 356–8 microbial pathogens in seafood, 326–8 microbial risk in seafood, 326–9 microbial seafood safety risk assessment, 328–9 microbial seafood-borne disease statistics, 326 microbial strains, 405–7, 410–12 risk categories for seafood products, 328 strain sources, 11 microbial antagonists, 373 microbial strains, 405–7, 410–12 microbiological assays (bioassays), 16 Micrococcus, 329 Micrococcus luteus, 16 MicroGARD®, 52, 81–2, 315 microorganisms control of in fresh and processed fruit and vegetables, 403–25 microbial strains, 405–7, 410–12 use of in combination with chemical products, 383–4 use of in combination with physical treatments, 384–5 microplate bioassay controlled procedure, 49 milk inoculation, 288 milk, 268–88 millet, 353 minimum inhibitory concentrations (MIC), 49, 464 of L. monocytogenes, 109 of moulds, 79 of yeasts, 79 miso, 353 modified atmosphere packaging (MAP), 120 modified atmosphere packing (MAP), 325, 327, 328, 330 molecular typing methodologies, 301-PFGE, 301-RAPD-PCR, 301 Monilia sitophila, 32 Monilinia laxa, 376 moulds, 27, 63, 225–35, 268 in food spoilage, 30–1, prevention of growth, 450–1 sensitive to natamycin, 79 spoilage in bakery products, 32 traditional control of spoilage fungi in food, 31–4 natamycin, 32–3 resistance to antifungal preservatives, 33–4 weak acid preservatives, 31–2 MRSA see methicillin-resistant Staphylococcus aureus mutacins, 6–17 Muscodor albus, 373 mutacin I, 17 mutagenesis experiments, 102 Mycobacterium phlei, 109

Mycobacterium smegmatis, 109 mycoparasitism, 382 mycotoxicosis, 31 mycotoxins, 31, 350 citrinin, 350 control of contamination in foods using LAB, 449–56 decontamination, 451–2 deoxynivalenol, 350 gliotoxin, 350 in animal feed, 226–7 prevention of absorption, 452–4 reduction of toxic effects, 454 Naegleria fowleri, 109 Natamax®, 78 Natamax® SF, 357 natamycin (E235), 32–3, 63–87, 356, 357 antimicrobial spectrum, 78–9 examples of yeasts and moulds that are sensitive to natamycin, 79 history, 77 in food biopreservation, 77–81 method of assay, 80 mode of action, 79–80 physical and chemical properties, 78 safety and tolerance, 81 structure of, 78 uses in foods and beverages, 80 NCBI database, 17 New Zealand Food Safety Authority, 10 NICE see NIsin Controlled gene Expression system Nisaplin®, 64, 111, 357, 420, 440 nisin, 63–87, 100, 267, 278–9, 271, 273–5, 308, 313, 356, 357, 416–18, 440 antimicrobial spectrum, 66 assay, 68–9 current applications in foods, 69–71 future trends, 86 history, 64–5 in active antimicrobial packaging, 75–6 in combination with novel food processing technology, 71–3 in combination with PEF, 74–5 in combination with UHP, 74 in food biopreservation, 63–77 mode of action, 66–8 new combinations with other preservatives, 71 nisin-sensitive bacterial species associated with food, 67 physical and chemical properties, 65–6 potential applications in foods, 71–6 published papers demonstrating synergy with other antimicrobials, 72–3 safety and tolerance, 76–7 sources of nisin producing L. lactis, 76 structure of nisin A, 65 nisin A, 64, 65, 357

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Index NIsin Controlled gene Expression system (NICE), 19 nisin Q, 8 nisin U, 8, 17 nisin Z, 8, 64, 65 nitrogen heterocyclic compounds, 437 nivalenol NIV, 450 Nocardia coralline, 109 non-occlusive mesenteric ischaemia, 254 non-ribosomally synthesized peptides (NRSP), 17 non-starter lactic acid bacteria (NSLAB), 268, 284 Novasin™, 357 nuclear magnetic resonance spectroscopy (NMR), 49, 102 nutraceuticals, 211 nylon, 482 ochratoxin A, 31, 452 Oenococcus, 436 ogi, 353 oligofructans, 211 open reading frames (ORFs), 17 organic acids, 75, 246–7, 269, 352, 470–72 ‘organic’ labelling, 383 osmoadaptation, 380 ostrich meat salami, 305 ovalbumin (OVA)-induced allergic mice, 253 oxidoreductases, 439 ozone, 383 P. acidilactici HA-6111-2, 307 P. agglomerans CPA-2, 375 P. pentosaceus RM2000, 307 packaging and shelf life, 378–9 pancreatoduodenectomy, 253 Pantoea agglomerans, 373 parasitism, 368 pasta, 350 pathogens control of in fresh and processed fruit and vegetables, 403–25 patulin, 31 peaches, 373 pectins, 357 pediocin PA-1/AcH, 101, 272, 308, 440 pediocin PD-1, 443 Pediococcus, 243, 306, 436 Pediococcus acidilacti, 272 Pediococcus parvulus, 416 Pediococcus pentosaceus, 35 PEF see pulsed electric field penicillin, 8 penicillin-resistant Pneumococcus (PRP), 104 Penicillium, 29, 450 Penicillium commune, 32 Penicillium corylophilum, 32 Penicillium discolor, 79

499

Penicillium expansum, 373 Penicillium frequentans, 376 Penicillium roqueforti, 32 peracetic acid, 384, 408, 409 PFGE, 301 phage amplification assay, 164 phage display technique, 167 phage endolysins, 167–8 phage typing, 163–4 phage-mediated biocontrol, 312 PHBME, 111 phenyllactic acid, 352 p-hydroxybenzoic acid methyl esther (PHBME), 420 physico-chemical treatments, 120 phytosanitary registration, 385 Pichia anomala, 231–2 Pichia guilliermondii, 373 Pichia pastoris, 382 piscicocin CS526, 336 piscicocin V1a, 336 piscicocin V1b, 336 pito, 353 plant extracts, 383, 472–4 plantaricin loci, 442 plantaricin ST31, 355 plate diffusion assay, 68 polyene macrolide antimicrobials, 77, 78, 81 polyethylene (PE), 482 polyethylene glycol, 482 polyethyleneimine, 482 polylactic acid (PLA), 482 polymer films, 482 polyphosphoric acid, 408 polyvinyl acetate (PVA) suspension coating, 81 polyvinyl alcohol (PVA), 482 polyvinyl chloride (PVC), 482 postharvest diseases of fruit and vegetables antagonists for biological control, 369–72 biocontrol agents (BCA), 365–88 biological products for controlling, 386 potassium sorbate, 356 prebiotics, 184–5, 211–12 predicted severe acute pancreatitis, 254 preharvest intervention strategies, 206 preservation of food and beverages, 73 preservatives made by fermentation, 63 preservatives, 356 probiotics, 183, 209–11, 456 proline, 379 Propionibacterium acidipropionici, 37 Propionibacterium acne, 104 Propionibacterium cyclohexanicum, 416 Propionibacterium freudenreichii JS, 39, 454 Propionibacterium freudenreichii, 37 Propionibacterium jensenii, 37 Propionibacterium shermanii, 451 Propionibacterium thoenii, 37 Propionibacterium thoenii, 5 propionic acid, 31, 32

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Index

propionic acid bacteria (PAB), 27–57 antifungal screening of, 37 typical applications, 34–5 propionicin PLG-1, 5, 51 protective cultures, 241 for bacteria control, 332–6 for control of spoiling microorganisms, 338–9 in fermented meat products, 297–315 in fresh seafood and seafood products, 324–42 in milk and dairy products, 267–88 in wine making, 433–45 industrial application, 340 Pseudomonas cepacia, 373 Pseudomonas fragi WY, 285 Pseudomonas syringae, 410 psychotropic bacteria, 351 public databases, 120 pulsed electric field (PEF), 73, 74–5, 278, 417 Qualified presumption of safety (QPS), 313, 341, 439 RAPD-PCR, 301 reactive oxygen species (ROS), 374 ready-to-eat (RTE) foods, 298, 418 REBECA, 387 reporteorphages, 163–7 reuterin, 129–53, 279 activity against gram-negative bacteria, 143–5 activity against gram-positive bacteria, 139–42 activity against other microorganisms, 145–6 activity of reuterin in situ, 137 antimicrobial activity of, 138–46 as a food preservative, 148–51 future trends, 153 legislation, 150 potential use in food, 150–2 production of on a large scale, 147–8 biotechnological production of, 147 chemical production, 147 stability in food, 148–9 synthesis of reuterin, 134–5 toxicity of, 149–50 reuterin-HPA system, 134–8 activity of reuterin in situ, 137 composition of the reuterin system, 135 modes of action, 135–7 reuterin synthesis in L. reuteri, 137 synthesis of reuterin, 134–5 reversed-phase high-performance liquid chromatography (RP-HPLC), 49, 112 Rhodotorula, 29 Rhodotorula minuta, 376 Rhodotorula mucilaginosa, 32 rice, 349 ropy bacteria, 356

Saccharomyces boulardii, 243 Saccharomyces cerevisiae, 29, 37, 109 SafePro®, 340 sakacin A, 7, 11 sakacin K, 304 sakacin P, 100–20 applications of, 119 characterization, structure and genetics, 117–18 history and GRAS status, 116–17 spectrum of inhibition and mode of action, 118 sake, 353 salami, 299 Salmonella, 49, 74, 109, 138, 143, 151, 162, 169, 181–97, 247, 268, 286, 298, 309, 314, 327, 351, 409 Salmonella Chester 404 Salmonella enterica, 111, 143 Salmonella Kentucky, 105 salmonella outbreaks, 85 salmonellosis, 351 seafood, 324–42 screening methodologies in food biopreservation, 12–16 agar diffusion methods, 12–14 incubation parameters, 15 atmosphere, 15 temperature, 15 time, 15 indicator strains, 16 agar diffusion, 16 microbiological assays (bioassays), 16 turbidimetric tube assays, 16 medium composition, 14–15 brain heart infusion, 14 influence on bacteriocin production, 15 luria agar, 14 Todd Hewitt broth-based media, 14 Tween 80, 14 Shewanella, 329 SHIME, 252 silage, 350, 353–4 Silver Elephant Natamycin, 78 Silver Elephant Nisin, 64 SKAL, 196 soda ash, 383 sodium alginate, 377, 378 sodium bicarbonate, 383 sodium chlorate, 214–15 sodium hypochlorite, 407 sodium metabisulphite, 71 sodium propionate, 356 sorbic acid, 32 sorghum, 348 sourdough, 352–3 soy sauce, 353 spray drying, 377 staphylococci, 300, 351 Staphylococcus, 104, 138, 187, 350

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Index Staphylococcus aureus, 105, 152, 268, 298, 470, 478 Staphylococcus xylosus, 339 starches, 357 starter cultures, 349 starter lactic acid bacteria, 280 streptocins, 12 Streptococcus, 34, 104, 138, 243, 250, 329, 352 Streptococcus equisimilis, 7 Streptococcus faecium, 183 Streptococcus macedonicus ACA-DC 198, 10 Streptococcus macedonicus, 6 Streptococcus mutans BCS3-L1, 19 Streptococcus mutans JH1140, 9 Streptococcus mutans, 6, 104, 133 Streptococcus pneumoniae TIGR 4 genome, 18 Streptococcus pyogenes, 6 Streptococcus salivarius K12, 9 Streptococcus salivarius, 6, 273 Streptococcus uberis, 6 Streptomyces natalensis, 77 sucrose, 375 sucuk, 309 sulphur dioxide, 438–9 SURE system, 19 synbiotics, 211 T-2 Toxin, 450 tesgüino, 353 The Evergreen State College, USA, 197 thickeners, 357 three-stage culture system with immobilized fecal microbiota, 252 TIM (TNO) intestinal model, 252 TIM-1, 252 TIM-2, 252 titanium dioxide (whitener), 71 tobacco, 387 Todd Hewitt broth-based media, 14 tomatoes, 382 traveler’s diarrhea, 250–1 trehalose, 377 trichothecens, 450 trimethylamine oxide (OTMA), 330 turbidimetric tube assays, 16 Tween 80, 14 uberolysin, 17 ultra high pressure (UHP) treatment, 73–4 undefined fermentates used in food biopreservation, 81–6

501

US Department of Agriculture (USDA), 314, 383 US Environmental Protection Agency (EPA), 313, 385 US Food and Drug Administration (FDA), 52, 77, 150, 161, 312, 383, 409 UV-C irradiation, 382 V. cholerae, 326 V. vulnificus, 326 vacuum packing (VP), 325, 327, 328, 330 Vagococcus salmoninarum, 329 VALMIC®, 376 vancomycin-resistant Enterococcus faecalis (VRE), 104 variacin, 100–20 applications, 116 characterization, structure and genetics, 115 history and GRAS status, 115 spectrum of inhibition and mode of action, 116 vegetables, 364–89, 403–25 Verticillium cinnabarinum, 79 Vibrio parahaemolyticus, 326 volatile compounds, 382 VRE see vancomycin-resistant Enterococcus faecalis Weissella cibaria, 336 wheat, 348 wine making, 324–44 bacteriocins in, 439–44 fermentation, 434–5 future trends, 444 spoilage by bacteria, 437–8 sulphur dioxide in, 438–9 World Health Organization Expert Committee on Food Additives, 440 yeast extract, 375 yeasts, 27, 63, 233–4, 268, 352, 353, 368 in food spoilage, 28–30 growth behaviour of indicator yeasts, 40 properties of, 28–9 sensitive to natamycin, 79 yoghurt, 29, 353 zoocin A, 8 zoonoses, 268 Zygosaccharomyces bailli, 29, 41 Zygosaccharomyces rouxii, 29, 41

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