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

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

i © Woodhead Publishing Limited, 2011

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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

iii © Woodhead Publishing Limited, 2011

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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.

iv © Woodhead Publishing Limited, 2011

Contents

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

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Woodhead Publishing Series in Food Science, Technology and Nutrition.............................................................................................

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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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Contents

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....................................................................................

© Woodhead Publishing Limited, 2011

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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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Contents

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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Contents

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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Contents 16.5 16.6 16.7 16.8

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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460 461 464 466 476 479 484 485

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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

xii © Woodhead Publishing Limited, 2011

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]


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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57 Food chemical safety Volume 1: contaminants Edited by D. Watson 58 Making the most of HACCP: learning from others’ experience Edited by T. Mayes and S. Mortimore 59 Food process modelling Edited by L. M. M. Tijskens, M. L. A. T. M. Hertog and B. M. Nicolaï 60 EU food law: a practical guide Edited by K. Goodburn 61 Extrusion cooking: technologies and applications Edited by R. Guy 62 Auditing in the food industry: from safety and quality to environmental and other audits Edited by M. Dillon and C. Griffith 63 Handbook of herbs and spices Volume 1 Edited by K. V. Peter 64 Food product development: maximising success M. Earle, R. Earle and A. Anderson 65 Instrumentation and sensors for the food industry Second edition Edited by E. Kress-Rogers and C. J. B. Brimelow 66 Food chemical safety Volume 2: additives Edited by D. Watson 67 Fruit and vegetable biotechnology Edited by V. Valpuesta 68 Foodborne pathogens: hazards, risk analysis and control Edited by C. de W. Blackburn and P. J. McClure 69 Meat refrigeration S. J. James and C. James 70 Lockhart and Wiseman’s crop husbandry Eighth edition H. J. S. Finch, A. M. Samuel and G. P. F. Lane 71 Safety and quality issues in fish processing Edited by H. A. Bremner 72 Minimal processing technologies in the food industries Edited by T. Ohlsson and N. Bengtsson 73 Fruit and vegetable processing: improving quality Edited by W. Jongen 74 The nutrition handbook for food processors Edited by C. J. K. Henry and C. Chapman 75 Colour in food: improving quality Edited by D. MacDougall 76 Meat processing: improving quality Edited by J. P. Kerry, J. F. Kerry and D. A. Ledward 77 Microbiological risk assessment in food processing Edited by M. Brown and M. Stringer 78 Performance functional foods Edited by D. Watson 79 Functional dairy products Volume 1 Edited by T. Mattila-Sandholm and M. Saarela 80 Taints and off-flavours in foods Edited by B. Baigrie 81 Yeasts in food Edited by T. Boekhout and V. Robert 82 Phytochemical functional foods Edited by I. T. Johnson and G. Williamson 83 Novel food packaging techniques Edited by R. Ahvenainen 84 Detecting pathogens in food Edited by T. A. McMeekin 85 Natural antimicrobials for the minimal processing of foods Edited by S. Roller 86 Texture in food Volume 1: semi-solid foods Edited by B. M. McKenna 87 Dairy processing: improving quality Edited by G. Smit 88 Hygiene in food processing: principles and practice Edited by H. L. M. Lelieveld, M. A. Mostert, B. White and J. Holah 89 Rapid and on-line instrumentation for food quality assurance Edited by I. Tothill 90 Sausage manufacture: principles and practice E. Essien 91 Environmentally-friendly food processing Edited by B. Mattsson and U. Sonesson 92 Bread making: improving quality Edited by S. P. Cauvain



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93 Food preservation techniques Edited by P. Zeuthen and L. Bøgh-Sørensen 94 Food authenticity and traceability Edited by M. Lees 95 Analytical methods for food additives R. Wood, L. Foster, A. Damant and P. Key 96 Handbook of herbs and spices Volume 2 Edited by K. V. Peter 97 Texture in food Volume 2: solid foods Edited by D. Kilcast 98 Proteins in food processing Edited by R. Yada 99 Detecting foreign bodies in food Edited by M. Edwards 100 Understanding and measuring the shelf-life of food Edited by R. Steele 101 Poultry meat processing and quality Edited by G. Mead 102 Functional foods, ageing and degenerative disease Edited by C. Remacle and B. Reusens 103 Mycotoxins in food: detection and control Edited by N. Magan and M. Olsen 104 Improving the thermal processing of foods Edited by P. Richardson 105 Pesticide, veterinary and other residues in food Edited by D. Watson 106 Starch in food: structure, functions and applications Edited by A-C Eliasson 107 Functional foods, cardiovascular disease and diabetes Edited by A. Arnoldi 108 Brewing: science and practice D. E. Briggs, P. A. Brookes, R. Stevens and C. A. Boulton 109 Using cereal science and technology for the benefit of consumers: proceedings of the 12th International ICC Cereal and Bread Congress, 24–26th May, 2004, Harrogate, UK Edited by S. P. Cauvain, L. S. Young and S. Salmon 110 Improving the safety of fresh meat Edited by J. Sofos 111 Understanding pathogen behaviour in food: virulence, stress response and resistance Edited by M. Griffiths 112 The microwave processing of foods Edited by H. Schubert and M. Regier 113 Food safety control in the poultry industry Edited by G. Mead 114 Improving the safety of fresh fruit and vegetables Edited by W. Jongen 115 Food, diet and obesity Edited by D. Mela 116 Handbook of hygiene control in the food industry Edited by H. L. M. Lelieveld, M. A. Mostert and J. Holah 117 Detecting allergens in food Edited by S. Koppelman and S. Hefle 118 Improving the fat content of foods Edited by C. Williams and J. Buttriss 119 Improving traceability in food processing and distribution Edited by I. Smith and A. Furness 120 Flavour in food Edited by A. Voilley and P. Etievant 121 The Chorleywood bread process S. P. Cauvain and L. S. Young 122 Food spoilage microorganisms Edited by C. de W. Blackburn 123 Emerging foodborne pathogens Edited by Y. Motarjemi and M. Adams 124 Benders’ dictionary of nutrition and food technology Eighth edition D. A. Bender 125 Optimising sweet taste in foods Edited by W. J. Spillane 126 Brewing: new technologies Edited by C. Bamforth 127 Handbook of herbs and spices Volume 3 Edited by K. V. Peter 128 Lawrie’s meat science Seventh edition R. A. Lawrie in collaboration with D. A. Ledward 129 Modifying lipids for use in food Edited by F. Gunstone 130 Meat products handbook: practical science and technology G. Feiner 131 Food consumption and disease risk: consumer-pathogen interactions Edited by M. Potter


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132 Acrylamide and other hazardous compounds in heat-treated foods Edited by K. Skog and J. Alexander 133 Managing allergens in food Edited by C. Mills, H. Wichers and K. HoffmanSommergruber 134 Microbiological analysis of red meat, poultry and eggs Edited by G. Mead 135 Maximising the value of marine by-products Edited by F. Shahidi 136 Chemical migration and food contact materials Edited by K. Barnes, R. Sinclair and D. Watson 137 Understanding consumers of food products Edited by L. Frewer and H. van Trijp 138 Reducing salt in foods: practical strategies Edited by D. Kilcast and F. Angus 139 Modelling microrganisms in food Edited by S. Brul, S. Van Gerwen and M. Zwietering 140 Tamime and Robinson’s Yoghurt: science and technology Third edition A. Y. Tamime and R. K. Robinson 141 Handbook of waste management and co-product recovery in food processing: Volume 1 Edited by K. W. Waldron 142 Improving the flavour of cheese Edited by B. Weimer 143 Novel food ingredients for weight control Edited by C. J. K. Henry 144 Consumer-led food product development Edited by H. MacFie 145 Functional dairy products Volume 2 Edited by M. Saarela 146 Modifying flavour in food Edited by A. J. Taylor and J. Hort 147 Cheese problems solved Edited by P. L. H. McSweeney 148 Handbook of organic food safety and quality Edited by J. Cooper, C. Leifert and U. Niggli 149 Understanding and controlling the microstructure of complex foods Edited by D. J. McClements 150 Novel enzyme technology for food applications Edited by R. Rastall 151 Food preservation by pulsed electric fields: from research to application Edited by H. L. M. Lelieveld and S. W. H. de Haan 152 Technology of functional cereal products Edited by B. R. Hamaker 153 Case studies in food product development Edited by M. Earle and R. Earle 154 Delivery and controlled release of bioactives in foods and nutraceuticals Edited by N. Garti 155 Fruit and vegetable flavour: recent advances and future prospects Edited by B. Brückner and S. G. Wyllie 156 Food fortification and supplementation: technological, safety and regulatory aspects Edited by P. Berry Ottaway 157 Improving the health-promoting properties of fruit and vegetable products Edited by F. A. Tomás-Barberán and M. I. Gil 158 Improving seafood products for the consumer Edited by T. Børresen 159 In-pack processed foods: improving quality Edited by P. Richardson 160 Handbook of water and energy management in food processing Edited by J. Klemeš, R. Smith and J-K Kim 161 Environmentally compatible food packaging Edited by E. Chiellini 162 Improving farmed fish quality and safety Edited by Ø. Lie 163 Carbohydrate-active enzymes Edited by K-H Park 164 Chilled foods: a comprehensive guide Third edition Edited by M. Brown 165 Food for the ageing population Edited by M. M. Raats, C. P. G. M. de Groot and W. A. Van Staveren



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166 Improving the sensory and nutritional quality of fresh meat Edited by J. P. Kerry and D. A. Ledward 167 Shellfish safety and quality Edited by S. E. Shumway and G. E. Rodrick 168 Functional and speciality beverage technology Edited by P. Paquin 169 Functional foods: principles and technology M. Guo 170 Endocrine-disrupting chemicals in food Edited by I. Shaw 171 Meals in science and practice: interdisciplinary research and business applications Edited by H. L. Meiselman 172 Food constituents and oral health: current status and future prospects Edited by M. Wilson 173 Handbook of hydrocolloids Second edition Edited by G. O. Phillips and P. A. Williams 174 Food processing technology: principles and practice Third edition P. J. Fellows 175 Science and technology of enrobed and filled chocolate, confectionery and bakery products Edited by G. Talbot 176 Foodborne pathogens: hazards, risk analysis and control Second edition Edited by C. de W. Blackburn and P. J. McClure 177 Designing functional foods: measuring and controlling food structure breakdown and absorption Edited by D. J. McClements and E. A. Decker 178 New technologies in aquaculture: improving production efficiency, quality and environmental management Edited by G. Burnell and G. Allan 179 More baking problems solved S. P. Cauvain and L. S. Young 180 Soft drink and fruit juice problems solved P. Ashurst and R. Hargitt 181 Biofilms in the food and beverage industries Edited by P. M. Fratamico, B. A. Annous and N. W. Gunther 182 Dairy-derived ingredients: food and neutraceutical uses Edited by M. Corredig 183 Handbook of waste management and co-product recovery in food processing Volume 2 Edited by K. W. Waldron 184 Innovations in food labelling Edited by J. Albert 185 Delivering performance in food supply chains Edited by C. Mena and G. Stevens 186 Chemical deterioration and physical instability of food and beverages Edited by L. H. Skibsted, J. Risbo and M. L. Andersen 187 Managing wine quality Volume 1: viticulture and wine quality Edited by A. G. Reynolds 188 Improving the safety and quality of milk Volume 1: milk production and processing Edited by M. Griffiths 189 Improving the safety and quality of milk Volume 2: improving quality in milk products Edited by M. Griffiths 190 Cereal grains: assessing and managing quality Edited by C. Wrigley and I. Batey 191 Sensory analysis for food and beverage control: a practical guide Edited by D. Kilcast 192 Managing wine quality Volume 2: oenology and wine quality Edited by A. G. Reynolds 193 Winemaking problems solved Edited by C. E. Butzke 194 Environmental assessment and management in the food industry Edited by U. Sonesson, J. Berlin and F. Ziegler 195 Consumer-driven innovation in food and personal care products Edited by S. R. Jaeger and H. MacFie


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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


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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Preface

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


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

xxv © Woodhead Publishing Limited, 2011

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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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Food and beverage biopreservation

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


Identifying new protective cultures and culture components

5

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) 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



7

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



9

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


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



13

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.



15

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)



17

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



19

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

and chevallier i (2006), ‘Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility. 1 – Screening and characterization of the antibacterial compounds.’ Food Control 17(6): 454–461. anderssen e l , diep d b , nes i f , eijsink v g h and nissen - meyer j (1998), ‘Antagonistic activity of Lactobacillus plantarum C11: Two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A.’ Applied and Environmental Microbiology 64(6): 2269–2272. anonymous (2009), ‘Substances Generally Recognized as Safe (GRAS)’, New Zealand Food Safety Authority. Available from: http://www.nzfsa.govt.nz/acvm/registers-lists/ index.htm, retrieved 25 March 2009. axelsson l and holck a (1995), ‘The genes involved in production of and immunity to sakacin A, a bacteriocin from Lactobacillus sake Lb706.’ Journal of Bacteriology 177(8): 2125–2137. axelsson l , holck a , birkeland s e , aukrust t and blom h (1993), ‘Cloning and nucleotide sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and immunity.’ Applied and Environmental Microbiology 59(9): 2868–2875. axelsson l , lindstad g and naterstad k (2003), ‘Development of an inducible gene expression system for Lactobacillus sakei.’ Letters in Applied Microbiology 37(2): 115–120.



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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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40 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


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).



41

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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42 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


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



43

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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44 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


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


45

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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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

Inhibitory spectrum


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


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

47

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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


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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Table 2.1 Continued

48


49

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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50 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


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.


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


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



55

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), ‘Efficacy of natamycin for control of growth and ochratoxin A production by Aspergillus carbonarius strains under different environmental conditions’, J Appl Microbiol 103, 2234–2239. meile l , le blay g and thierry a (2008), ‘Safety assessment of dairy microorganisms: Propionibacterium and Bifidobacterium’, Int J Food Microbiol 126, 316–320. melin p , sundh i , hakansson s and schnürer j (2007), ‘Biological preservation of plant derived animal feed with antifungal microorganisms: safety and formulation aspects’, Biotechnol Lett 29, 1147–1154. miescher s (1999), ‘Antimicrobial and autolytic systems of dairy propionibacteria’, PhD Thesis No. 13486, ETH Zurich, Zurich, Switzerland. miescher schwenninger s , lacroix c , truttmann s , jans c , bommer d et al. (2008), ‘Characterization of low-molecular weight antiyeast metabolites produced by a food-protective Lactobacillus/Propionibacterium co-culture’, J Food Prot 71, 2481–2487. miescher schwenninger s and meile l (2001), ‘Novel biological system with antimicrobial activities for the use as natural preservation system’, European Patent EP 1 442 113. miescher schwenninger s and meile l (2004), ‘A mixed culture of Propionibacterium jensenii and Lactobacillus paracasei subsp. paracasei inhibits food spoilage yeasts’, Syst Appl Microbiol 27, 229–237. miescher schwenninger s , von ah u , niederer b , teuber m and meile l (2005), ‘Detection of antifungal properties in Lactobacillus paracasei subsp. paracasei SM20, SM29, and SM63 and molecular typing of the strains’, J Food Prot 68, 111–119. mijin d , petrovic s and stojanovic o (1989), ‘Intramolecular dehydration of L-glutamic acid’, J Serb Chem Soc 54, 11–15. minervini f , montagna m t , spilotros g , monaci l , santacroce m p and visconti a (2001), ‘Survey on mycoflora of cow and buffalo dairy products from Southern Italy’, Int J Food Microbiol 69, 141–146. moore m m , dal bello f and arendt e (2008), ‘Sourdough fermented by Lactobacillus plantarum FST1.7 improves the quality and shelf life of gluten-free bread’, Eur Food Res Technol 226, 1309–1316. nielsen d s , jacobsen t , jespersen l , koch a g and arneborg n (2008), ‘Occurrence and growth of yeasts in processed meat products – implications for potential spoilage’, Meat Sci 80, 919–926. niku - paavola m l , laitila a , mattila - sandholm t and haikara a (1999), ‘New types of antimicrobial compounds produced by Lactobacillus plantarum’, J Appl Microbiol 86, 29–35. okkers d , dicks l m t , silvester m , joubert j and odendaal h (1999), ‘Characterization of pentocin TV35b, a bacteriocin-like peptide ioslated from Lactobacillus pentosus with fungistatic effect on Candida albicans’, J Appl Microbiol 87, 726–734. piveteau p (1999), ‘Metabolism of lactate and sugars by dairy propionibacteria: a review’, Lait 79, 23–41. restuccia c , randazzo c and caggia c (2006), ‘Influence of packaging on spoilage yeast population in minimally processed orange slices’, Int J Food Microbiol 109, 146–150. rhee k - h (2004), ‘Cyclic dipeptides exhibit synergistic, broad spectrum antimicrobial effects and have anti-mutagenic properties’, Int J Antimicro Ag 24, 423–427. rouse s and van sinderen d (2008), ‘Bioprotective potential of lactic acid bacteria in malting and brewing’, J Food Prot 71, 1724–1733. rustom i y s (1997), ‘Aflatoxin in food and feed: occurrence, legislation and inactivation by physical methods’, Food Chem 59, 57–67. ryan l a m , dal bello f and arendt e k (2008), ‘The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread’, Int J Food Microbiol 125, 274–278.


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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.


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


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)

Ogden (1986), Ogden et al. (1988)


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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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ayres j w, sandine w e

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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and branen a l (2005). ‘Food Antimicrobials – An Introduction.’ In Antimicrobials in Foods, eds Davidson P M, Sofos J and Branen A L. Taylor & Francis, Boca Raton, FL, pp. 1–10. davies e a , bevis h e and delves - broughton j (1997). ‘The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes.’ Letters in Applied Microbiology 24, 343–346. davies e a , bevis h , potter r, harris j , williams g c and delves - broughton j (1998). ‘The effect of pH on the stability of nisin solutions during autoclaving.’ Letters in Applied Microbiology 27, 186–187. davies e a , milne c f , bevis h e , potter r w, harris j m et al. (1999). ‘Effective use of nisin to control lactic acid bacteria spoilage in vacuum packed Bologna-type sausage.’ Journal of Food Protection 62, 1004–1010. de boer e , labots h , stolk - horsthuis m and visser j n (1979) ‘Sensitivity to natamycin of fungi in factories producing dry sausage.’ Fleishwirtsh 59, 1868. de boer e and stolk - horsthuis m (1977). ‘Sensitivity to natamycin (pimaricin) of fungi isolated in cheese warehouses.’ Journal of Food Protection 40, 533–536. de kwaadstenient m k , ten doeschate k and dicks l t m (2008). ‘Characterization of the structural gene encoding Nisin F, a new lantibiotic produced by a Lactococcus lactis subsp. lactis isolate from freshwater catfish (Clarias gariepinus).’ Applied and Environmental Microbiology 74, 547–549. de ruig w g and van den berg g (1985). ‘Influence of the fungicides sorbate and natamycin in cheese coatings on the quality of cheese.’ Netherlands Milk Dairy Research Journal 39, 165–172. delves - broughton j (1990). ‘Nisin and its uses as a food preservative.’ Food Technology 44, 100–117. delves - broughton j (1993). ‘The use of EDTA to enhance the efficacy of nisin towards Gram-negative bacteria.’ International Biodeterioration Biodegradation 32, 87–97. delves - broughton j (1998). ‘Use of nisin in processed and natural cheese.’ Bulletin of the International Dairy Federation 329, 13–17. 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 and friss m (1998) ‘Nisin preparations – production, specifications, and assay procedures.’ Bulletin of International Dairy Federation 32, 18–19. 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. delves - broughton j , thomas l v and williams g (2006). ‘Natamycin as an antimycotic preservative on cheese and fermented sausages.’ Food Australia 58, 19–21. delves - broughton j , williams g c and wilkinson s (1992). ‘The use of the bacteriocin, nisin, as a preservative in pasteurized liquid whole egg.’ Letters in Applied Microbiology 15, 133–136. duran l , hernandez e and flores j (1964). ‘Empleo de nisina en la esterilizacion de conservas de pimientos.’ Agroquimica y Technologia de Alimentos 4, 87–92. dutreaux n , notermans s , góngora - nieto m m and swanson b g (2000). ‘Effects of combined exposure of Micrococcus luteus to nisin and pulsed electric fields.’ International Journal of Food Microbiology 60, 147–152. efsa ( european food safety authority ) (2006). ‘Opinion of the Scientific Panel on Food additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission related to “The use of nisin (E234) as a food additive”.’ The EFSA Journal 314, 1–16. davidson p m


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and salem r m (2009). ‘The effect of natamycin on keeping quality and organoleptic quality of yogurt.’ Arab Journal of Biotechnology 12, 41–48. elotmani f and assobhei o (2003). ‘In vitro inhibition of microbial flora of fish by nisin and lactoperoxidase system.’ Letters in Applied Microbiology 38, 60–65. falahee m b , adams m r, dale j w and morris b a (1990). ‘An enzyme immunosaay for nisin.’ International Journal of Food Science and Technology 25, 590–595. fang t j , chen c - y and chen h l (1997). ‘Inhibition of Staphylococcus aureus and Bacillus cereus on a vegetarian food treated with nisin combined with either potassium sorbate or sodium benzoate.’ Journal of Food Safety 17, 69–87. fang t j and hseuh y - t (2000). ‘Effect of chelators, organic acid and storage temperature on growth of Escherichia coli O157:H7 in ground beef treated with nisin, using response surface methodology.’ Journal of Food and Drug Analysis 8, 187–194. farkas d f and hoover d g (2000). ‘High pressure processing.’ Journal of Food Science 65, 47–64. fda ( food and drug administration ) (1988). ‘Nisin preparation: Affirmation of GRAS status as a direct human food ingredient.’ Federal Register 53: 11247. fowler g g , jarvis b and tramer j (1975). ‘The assay of nisin in foods.’ In Some Methods of Microbiological Assay, Eds R G Board and D W Lovelock. Academic Press, London, pp. 91–105. franchi m a , serra g e and cristianini m (2003a). ‘The use of biopreservatives in the control of bacterial contaminants of sugarcane alcohol fermentation.’ Journal of Food Microbiology and Safety 68, 2310–2315. franchi m a , serra g e , svilosen j and cristianini m (2003b). ‘Thermal death kinetics of bacterial contaminants during cane sugar and alcohol production.’ International Sugar Journal 105, 527–530. frazer a c , sharratt m , and hickman j r (1962). ‘The biological effects of food additives. Nisin.’ Journal of Food Science and Agriculture 13, 32–42. gallo l i , pilosof a m r and jagus r j (2007). ‘Effect of the sequence of nisin and pulse electric fields treatments involved in the inactivation of Listeria innocua in whey.’ Journal of Food Engineering 79, 188–193. gálvez a , abriouel h , lópez r l and omar n b (2007). ‘Bacteriocin-based strategies for food biopreservation.’ International Journal of Food Microbiology 120, 51–70. gao y - l and ju x - r (2008). ‘Exploiting the combined effects of high pressure and moderate heat on inactivation of Clostridium botulinum spores.’ Journal of Microbiological Methods 72, 20–28. ghrairi t , manai m , berjeaud j m and frére j (2004). ‘Antilisterial activity of lactic acid bacteria isolated from Rigouta, a traditional Tunisian cheese.’ Journal of Applied Microbiology 97, 621–628. gill a o and holley r a (2000). ‘Surface application of lysozyme, nisin and EDTA to inhibit spolage and pathogenic bacteria on ham and bologna.’ Journal of Food Protection 63, 1338–1346. gillespy t g (1953). ‘Nisin trials with canned beans in tomato sauce and canned garden peas.’ Research Association Leaflet No. 3, Fruit and Vegetable Canning and Quick Freezing Research Association, Chipping Campden, UK. giraffa g , neviani e and verernoni a (1990) ‘Use of conductance to detect bacteriocin activity.’ Journal of Food Protection 53, 772–776. gourama h and bullermann l b (1988). ‘Effects of potassium sorbate and natamycin on growth and penicillic acid production by Aspergillus ochraceus.’ Journal of Food Protection 51, 139–144. grindstead d a and barefoot s f (1992). ‘Jenseniin G, a heat stable bacteriocin produced by Propionibacterium jensenii P126.’ Applied and Environmental Microbiology 58, 215–220.


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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 (4) and alpha(1–>6) glucosidic bonds.’ Appl Environ Microbiol 68: 4283–4291. kuleasan h and m l cakmakci (2002) ‘Effect of reuterin produced by Lactobacillus reuteri on the surface of sausages to inhibit the growth of Listeria monocytogenes and Salmonella spp.’ Nahrung 46: 408–410. kunze w a , y k mao , b wang , j d huizinga , x ma et al. (2009) ‘Lactobacillus reuteri enhances excitability of colonic AH neurons by inhibiting calcium dependent potassium channel opening.’ J Cell Mol Med. 13 (8B): 2261–2270. kuykendall j r and m s bogdanffy (1992) ‘Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro.’ Mutat Res 283: 131–136. lerche m and g reuter (1965) ‘Das Vorkommen von aerob wachsender Gram positiver Stäbchen des Genus Lactobacillus beijerinck im Darminhalt erwachsen Menschen.’ Zbl Bak Parasit Infec Hyg I Orig 185: 446–481. liang h f , c n chen , y chang and h w sung (2003) ‘Natural antimicrobial agent (reuterin) produced by Lactobacillus reuteri for sanitization of biological tissues inoculated with pseudomonas aeruginosa.’ Biotechnol Bioeng 84: 233–239. lindgren s e and w j dobrogosz (1990) ‘Antagonistic activities of lactic acid bacteria in food and feed fermentations.’ FEMS Microbiol Rev 7: 149–163. lonvaud - funel a (2002) ‘Lactic acid bacteria in winemaking: Influence on sensorial and hygienic quality.’ Progress in Industrial Microbiology 36: 231–262. lorea baroja m , p v kirjavainen , s hekmat and g reid (2007) ‘Anti-inflammatory effects of probiotic yogurt in inflammatory bowel disease patients.’ Clin Exp Immunol 149: 470–479. lüthi - peng q , s schärer and z puhan (2002) ‘Production and stability of 3-hydroxypropionaldehyde in Lactobacillus reuteri.’ Appl Microbiol Biotechnol 60: 73–80. ma d , p forsythe and j bienenstock (2004) ‘Live Lactobacillus reuteri is essential for the inhibitory effect on tumor necrosis factor alpha-induced interleukin-8 expression.’ Infect Immun 72: 5308–5314.


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and b n ames (1985) ‘Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104.’ Mutat Res 148: 25–34. martin r, m olivares , m l marin , j xaus , l fernandez and j m rodriguez (2005) ‘Characterization of a reuterin-producing Lactobacillus coryniformis strain isolated from a goat’s milk cheese.’ Int J Food Microbiol 104: 267–277. mattila - sandholm t , j mättö and m saarela (1999) ‘Lactic acid bacteria with health claims: interactions and interference with gastrointestinal flora.’ Int Dairy J 9: 25–35. mccoy s and s e gilliland (2007) ‘Isolation and characterization of Lactobacillus species having potential for use as probiotic cultures for dogs.’ J Food Sci 72: M94–97. mitsuoka t (1992) ‘Intestinal flora and aging.’ Nutr Rev 50: 438–446. molenaar d , f bringel , f h schuren , w m de vos , r j siezen and m kleerebezem (2005) ‘Exploring Lactobacillus plantarum genome diversity by using microarrays.’ J Bacteriol 187: 6119–6127. molin g , r andersson , s ahrne , c lonner, i marklinder et al. (1992a) ‘Effect of fermented oatmeal soup on the cholesterol level and the Lactobacillus colonization of rat intestinal mucosa.’ Antonie Van Leeuwenhoek 61: 167–173. molin g , m l johansson , m stahl , s ahrne , r andersson et al. (1992b) ‘Systematics of the Lactobacillus population on rat intestinal mucosa with special reference to Lactobacillus reuteri.’ Antonie Van Leeuwenhoek 61: 175–183. molina v c , m medici , m p taranto and g font de valdez (2009) ‘Lactobacillus reuteri CRL 1098 prevents side effects produced by a nutritional vitamin B12 deficiency.’ J Appl Microbiol 106: 467–473. morita h , h toh , s fukuda , h horikawa , k oshima et al. (2008) ‘Comparative genome analysis of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic island for reuterin and cobalamin production.’ DNA Res 15: 151–161. muthukumarasamy p and r a holley (2006) ‘Microbiological and sensory quality of dry fermented sausages containing alginate-microencapsulated Lactobacillus reuteri.’ Int J Food Microbiol 111: 164–169. muthukumarasamy p and r a holley (2007) ‘Survival of Escherichia coli O157:H7 in dry fermented sausages containing micro-encapsulated probiotic lactic acid bacteria.’ Food Microbiol 24: 82–88. naito s , h hayashidani , k kaneko , m ogawa and y benno (1995) ‘Development of intestinal lactobacilli in normal piglets.’ J Appl Bacteriol 79: 230–236. noble a (1999) ‘Why do wines taste bitter and feel astrigent.’ In A L Waterhouse and S E Ebeler (eds) Chemistry of Wine Flavor, American Chemical Society, Washington D.C., pp. 156–165. o ’ brien p j , a g siraki and n shangari (2005) ‘Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health.’ Crit Rev Toxicol 35: 609–662. peran l , s sierra , m comalada , f lara - villoslada , e bailon et al. (2007) ‘A comparative study of the preventative effects exerted by two probiotics, Lactobacillus reuteri and Lactobacillus fermentum, in the trinitrobenzenesulfonic acid model of rat colitis.’ Br J Nutr 97: 96–103. petricevic l , f m unger, h viernstein and h kiss (2008) ‘Randomized, double-blind, placebo-controlled study of oral lactobacilli to improve the vaginal flora of postmenopausal women.’ Eur J Obstet Gynecol Reprod Biol 141: 54–57. rasch m (2002) ‘The influence of temperature, salt and pH on the inhibitory effect of reuterin on Escherichia coli.’ Int J Food Microbiol 72: 225–231. rasch m , g c barker, k sachau , m jakobsen and n arneborg (2002) ‘Characterisation and modelling of oscillatory behaviour related to reuterin production by Lactobacillus reuteri.’ Int J Food Microbiol 73: 383–394.


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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.


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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Food and beverage biopreservation 2001) or use of a rotation scheme, respectively, reduces the probability of phage resistance development.

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 christensen b b (2007). ‘Characterization of Campylobacter phages including analysis of host range by selected Campylobacter Penner serotypes.’ BMC Microbiol 7: 90–9. hastings j w and nelson k h (1977). ‘Bacterial bioluminescence.’ Ann Rev Microbiol 31: 549–95. hershey a d and chase m (1952). ‘Independent functions of viral protein and nucleic acid in growth of bacteriophage.’ J Gen Physiol 36: 39–56 hsu f c , shieh y s c and sobsey md (2002). ‘Enteric bacteriophages as potential fecal indicators in ground beef and poultry meat.’ J Food Prot 65: 93–9. jacobs w r jr., barletta r g , udani r, chan j , kalkut g et al. (1993). ‘Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages.’ Science 260: 819–22. kennedy j e and bitton g (1987). ‘Bacteriophages in foods.’ In Goyal S M, Gerba C P and Bitton G (eds) Phage Ecology, John Wiley and Sons, New York, pp. 289–316. kennedy j e , wie c i and oblinger j l (1986). ‘Distribution of coliphages in various foods.’ J Food Prot 49: 944–51. kilic a o , pavlova s i , ma w g and tao l (1996). ‘Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt.’ Appl Environ Microbiol 62: 2111–16. kim k p and loessner m j (2008). ‘Enterobacter sakazakii invasion in human intestinal Caco-2 cells requires the host cell cytoskeleton and is enhanced by disruption of tight junction.’ Infect Immun 76: 562–70. kocharunchitt c , ross t and mcneil d l (2009). ‘Use of bacteriophages as biocontrol agents to control Salmonella associated with seed sprouts.’ Int J Food Microbiol 128: 453–9. kodikara c p , crew h h and stewart g s (1991). ‘Near on-line detection of enteric bacteria using lux recombinant bacteriophage.’ FEMS Microbiol Lett 67: 261–5. korndoerfer i p , danzer j , schmelcher m , zimmer m , skerra a and loessner m j (2006). ‘The crystal structure of the bacteriophage PSA endolysin reveals a unique fold responsible for specific recognition of Listeria cell walls.’ J Mol Biol 364: 678–89. kretzer j w, lehmann r, schmelcher m , banz m , kim k p et al. (2007). ‘Use of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization and separation of bacterial cells.’ Appl Environ Microbiol 73: 1992–2000. kuhn j , suissa m , wyse j , cohen i , weiser i et al. (2002). ‘Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella.’ Int J Food Microbiol 74: 229–38. leverentz b , conway w s , alavidze z , janisiewicz w j , fuchs y et al. (2001). ‘Examination of bacteriophage as a biocontrol method for Salmonella on fresh-cut fruit: a model study.’ J Food Prot 64: 1116–21. leverentz b , conway w s , camp m j , janisiewicz w j , abuladze t et al. (2003). ‘Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin.’ Appl Environ Microbiol 69: 4519–26. loc carrillo c , atterbury r j , el - shibiny a , connerton p l , dillon e et al. (2005). ‘Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens.’ Appl Environ Microbiol 71: 6554–63. loessner m j , rees c e , stewart g s and scherer s (1996). ‘Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells.’ Appl Environ Microbiol 62: 1133–40. loessner m j , rudof m and scherer s (1997). ‘Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods.’ Appl Environ Microbiol 63: 2961–5. loessner m j , kramer k , ebel f and scherer s (2002). ‘C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition



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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.

7.8 References abee t , krockel l

and hill c (1995), ‘Bacteriocins: modes of action and potentials in food preservation and control of food poisoning’, Int J Food Microbiol 28, 169–185. ackermann , h w (2001), ‘Frequency of morphological phage descriptions’, Arch Virol 146, 843–857. ackermann , h w (2007), ‘5500 Phages examined in the electron microscope’, Arch Virol 152, 227–243. adams m h (1959), Bacteriophages, New York, USA, Interscience Publishers Inc. aho m , nuotio l , nurmi e and kiiskinen t (1992) ‘Competitive exclusion of campylobacters from poultry with K-bacteria and Broilact’, Int J Food Microbiol 15, 265–275. alexander m (1981), ‘Why microbial predators and parasites do not eliminate their prey and hosts’, Annu Rev Microbiol 35, 113–133. alisky j , iczkowski k , rapoport a and troitsky n (1998), ‘Bacteriophages show promise as antimicrobial agents’, J Infect 36, 5–15. andreatti filho r l , higgins j p , higgins s e , gaona g , wolfenden a d et al. (2007), ‘Ability of bacteriophages isolated from different sources to reduce Salmonella enterica serovar enteritidis in vitro and in vivo’, Poult Sci 86, 1904–1909. atterbury r j , connerton p l , dodd c e , rees c e and connerton i f (2003a), ‘Isolation and characterization of Campylobacter bacteriophages from retail poultry’, Appl Environ Microbiol 69, 4511–4518.


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atterbury r j , connerton p l , dodd c e , rees c e

and connerton i f (2003b), ‘Application of host-specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of Campylobacter jejuni’, Appl Environ Microbiol 69, 6302–6306. atterbury r j , dillon e , swift c , connerton p l , frost j a et al. (2005), ‘Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca’, Appl Environ Microbiol 71, 4885–4887. 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, 4543–4549. barrow p a (2001), ‘The use of bacteriophages for treatment and prevention of bacterial disease in animals and animal models of human infection’, J Chem Technol Biotechnol 76, 677–682. barrow p , lovell m and berchieri , a (1998), ‘Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and meningitis in chickens and calves’, Clin Diagn Lab Immunol 5, 294–298. beery j t , hugdahl m b and doyle m p (1988), ‘Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni’, Appl Environ Microbiol 54, 2365–2370. berchieri a , lovell m a and barrow p a (1991), ‘The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella typhimurium’, Res Microbiol 142, 541–549. bernhardt t g , wang i n , struck d k and young r (2002), ‘Breaking free: “protein antibiotics” and phage lysis’, Res Microbiol 153, 493–501. borie c , albala i , sánchez p , sánchez m l , ramírez s et al. (2008), ‘Bacteriophage treatment reduces Salmonella colonization of infected chickens’, Avian Dis 52, 64–67. boyd e f and brussow h (2002), ‘Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved’, Trends Microbiol 10, 521–529. cairns b j , timms a r, jansen v a , connerton i f and payne r j (2009), ‘Quantitative models of in vitro bacteriophage-host dynamics and their application to phage therapy’, PLoS Pathog 5, e1000253. calci , k r, burkhardt w, watkin w d and rippey s r (1998), ‘Occurrence of malespecific bacteriophage in feral and domestic animal wastes, human feces, and humanassociated wastewaters’, Appl Environ Microbiol 64, 5027–5029. chaveerach p , lipman l j a and van knapen f (2004), ‘Antagonistic activities of several bacteria on in vitro growth of 10 strains of Campylobacter jejuni/coli’, Int J Food Microbiol 90, 43–50. cheetham b f and katz m e (1995), ‘A role for bacteriophages in the evolution and transfer of bacterial virulence determinants’, Mol Microbiol 18, 201–208. cleveland j , montville t j , nes i f and chikindas m l (2001), ‘Bacteriocins: safe, natural antimicrobials for food preservation’, Int J Food Microbiol 71, 1–20. cole k , farnell m b , donoghue a m , stern n j , svetoch e a et al. (2006), ‘Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults’, Poult Sci 85, 1570–1575. connerton p l , loc carrillo c m , swift c , dillon e , scott a et al. (2004), ‘A longitudinal study of Campylobacter jejuni bacteriophage and their hosts from broiler chickens’, Appl Environ Microbiol 70, 3877–3883. connerton i f , connerton p l , barrow p , seal b s and atterbury r j (2008), Bacteriophage therapy and Campylobacter. In I Nachamkin, C M Szymanski and M J Blaser, Campylobacter (3rd edn), Washington, ASM Press, 679–693. corry j e l and atabay h i (2001), ‘Poultry as a source of Campylobacter and related organisms’, J Appl Microbiol 90, 96S–114S. cotter p d , hill c and ross r p (2005), ‘Bacteriocins: developing innate immunity for food’, Nat Rev Microbiol 3, 777–788.


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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

Reducing carriage of foodborne pathogens in cattle and swine

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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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et al. (2008). ‘Predominance of a bacteriocin-producing Lactobacillus salivarius component of a five-strain probiotic in the porcine ileum and effects on host immune phenotype’. FEMS Microbiol. Ecol. 64: 317–327. wells j e , krause d o , callaway t r and russell j b (1997). ‘A bacteriocin-mediated antagonism by ruminal lactobacilli against Streptococcus bovis’. FEMS Microbiol. Ecol. 22: 237–243. wiemann m (2003). ‘How do probiotic feed additives work?’ Int. Poultry Prod. 11: 7–9. willard m d , simpson r b , cohen n d and clancy j s (2000). ‘Effects of dietary fructooligosaccharide on selected bacterial populations in feces of dogs’. Amer. J. Vet. Res. 61: 820–825. winkler j , butler r and symonds e (2007). ‘Fructo-oligosaccharide reduces inflammation in a dextran sodium sulphate mouse model of colitis’. Dig. Dis. Sci. 52: 52–58. wolin m j (1969). ‘Volatile fatty acids and the inhibition of Escherichia coli growth by rumen fluid’. Appl. Microbiol. 17: 83–87. younts - dahl s m , galyean m l , loneragan g h , elam n a and brashears m m (2004). ‘Dietary supplementation with Lactobacillus- and Propionibacterium-based direct-fed microbials and prevalence of Escherichia coli O157 in beef feedlot cattle and on hides at harvest’. J. Food Prot. 67: 889–893. zeyner a and boldt e (2006). ‘Effects of a probiotic Enterococcus faecium strain supplemented from birth to weaning on diarrhoea patterns and performance of piglets’. J. Anim. Physiol. Anim. Nutr. 90: 25–31. 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. zhang w, azevedo m s p , gonzalez a m , saif l j , van nguyen t et al. (2008). ‘Influence of probiotic Lactobacilli colonization on neonatal B cell responses in a gnotobiotic pig model of human rotavirus infection and disease’. Vet. Immunol. Immunopath. 122: 175–181. zhao t , doyle m p , harmon b g , brown c a , mueller p o e and parks a h (1998). ‘Reduction of carriage of enterohemorrhagic Escherichia coli O157:H7 in cattle by inoculation with probiotic bacteria’. J. Clin. Microbiol. 36: 641–647. walsh m c , gardiner g e , hart o m , lawlor p g , daly m

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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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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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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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


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

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