Encyclopedia of Food Microbiology, Volumes 1-3-Elsevier (2000)

ENCYCLOPEDIA OF FOOD MICROBIOLOGY Editor-in-Chief

RICHARD K. ROBINSON Editors

CARL A. BATT PRADIP D. PATEL

u ACADEMIC PRESS A Harcourt Science and Technology Company

San Diego San Francisco New York Boston London Sydney Tokyo

This book is printed on acid-free paper Copyright 02000 by ACADEMIC PRESS The following articles are US government works in the public domain and are not subject to copyright: Aspergillus: Introduction; Aspergillus: Aspergillus flaucrs; Cyclospovu; Helminths and Nematodes All Rights Reserved. N o part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press A Harcourt Science and Technology Company 24-28 Oval Road, London NW1 7DX, UK http://www.hbuk.co,uk/ap/ Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com ISBN 0-12-227070-3 A catalogue for this encyclopedia is available from the British Library Library of Congress Catalog Card Number: 98-87954 Access for a limited period to an on-line version of the Encyclopedia o f Food Microbiology is included in the purchase price of the print edition. This on-line version has been uniquely and persistently identified by the Digital Object Identifier (DOI) 10.1006/rwfm.2000 By following the link

http://dx.doi.org/l0.l006/rwfm.2000 from any Web Browser, buyers of the Encyclopedia of Food Microbiology will find instructions on how to register for access. Typeset by Selwood Systems, Midsomer Norton, Bath Printed in Great Britain by The Bath Press, Bath

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EDITORIAL ADVISORY BOARD

R G Board South Lodge Northleigh Bradford-on-Avo? Wi Its hi re BA152RG UK

R L Buchanan US Food and Drug Admiiistration Center for F3od Safety a i d Applied Nutritior: 200 C-Street, SW Washington 20204 DC USA D 0 Cliver Department of PopLlation Health and Reproduction School of Veterinary Medicine University of California, Davis Davis CA 956 16-8743 USA B Colonna Ceccaldi Industrial Microbiology Pernod Ricard Centre de Recherche 120 Avenue du Marechal Foch 94015 Creteil Cedex, France

M A Cousin DepartTent of Food Science 1160 Smth Hall Purdue University W Lafayette IN 47907 USA C 0 Gill Agriculture and Agri-Food Canada Researcn Centre 6000 C & E Trail Lacombe, Alberta T4L 1W 1 Canada

G W Gould 17 Dove Road Bedford MK41 7AA UK M Griffiths Department of Food Science University of Guelph Guelph, Ontaro N1G 2W1 Canaca G Kalantzopoulos Department of Food Science and Technology Agricultural University of Athens Botanikos 118 55 Athens Greece T Keshavarz Schoo of Biological and Health Sciences University of Westminster 115 New Cavendish Street London W1 M 83s UK P Kulkarni University of Murnbai Department of Chemical Technology Nathalal Parikh Marg Matunga Mumbai-400 019 India

S Notermans TNO Nutrition and Food Research Institute PO Box 360 3700 AJ Zeist The Netherlands

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Editorial Advisory Board

Y Oda

F M Rombouts

Department of Applied Biological Science Fukuyarna University Fukuyarna, Hiroshima 729-0292 Japan

LUW PO Box 8129 6700 EV Wageningen The Netherlands

B Ozer

A N Sharpe PO Box 1224 Alrnonte Ontario KOA 1AO Canada

Faculty of Agriculture Department of Food Science and Technology University of Harran Sanliurfa Turkey T A Roberts Food Safety Consultant 59 Edenharn Crescent Reading RGl 6HU UK

D Roberts Food Hygiene Laboratories Central Public Health Laboratory 61 Colindale Avenue London NW9 5HT UK

K Steinkraus Department of Food Microbiology Cornell University 15 Cornell Street, lthaca N Y 14850, USA

M-L Tortorello National Center for Food Safety and Technology US Food and Drug Administration 6502 South Archer Road Summit-Argo IL 60501 USA

FOREWORD

Public concern about food safety has never been greater. In part this is due to the ever increasing demand from consumers for higher and higher standards. But new food-borne pathogens like E. coli 0157 have emerged in recent years to become important public health problems, and changes in production and manufacturing sometimes reopen doors of opportunity for old ones. A powerful reminder that food scientists have much unfinished business to attend to is provided by the succession of food scares that generate strong stories for the media. Experience tells us that science must underpin all approaches to food safety, whether through the application and implementation of well-tried approaches or the development of new or improved methods. Microbiologists have had a central role in this since the high quality work of pioneers like van Ermengem on botulism and Gaffky on typhoid more than a century ago. The large amount of important data that has accumulated since then joins with the current rapid rates of technological and scientific advance to make the need for a structured and authoritative source of information a very pressing one. It is provided by this encyclopedia. These are exciting times for food microbiologists. Expectations are high that as scientists we will soon provide answers to the many problems still posed by microbes - from spoilage to food poisoning. Approaches like HACCP are making everyone think hard about how best to apply the data we have to develop better ways for reducing and eliminating food-borne pathogens. The pace of scientific developments continues to accelerate and more and better methods are available for the detection and enumeration of microbes than ever before. The microbes themselves continue to evolve and so present moving targets. The solid foundation presented by the mass of information in this encyclopedia provides the launching pad and guide for meeting these challenges. It could be said that a penalty of working in food microbiology is that because the subject is broad-ranging, mature and dynamic, its practitioners, teachers and students have to know about many things in breadth and depth. For most of us, of course, this is not a penalty but an attractive bonus because of its intellectual challenge. I am particularly pleased to be associated with the encyclopedia because it will help us all to meet this test with confidence. I wish it every success. Professor H Pennington Department of Medical Microbiology University of Aberdeen

INTRODUCTION

The advent of antibiotics gave the general public, and many professional microbiologists as well, the feeling that bacterial diseases were under control, and the elimination of smallpox and the control of polio suggested that even viruses posed few problems. However, this complacency has received a nasty jolt over the last decade, and the emergence of HIV and multiple-drug-resistant strains of bacteria has become a major concern for the medical profession. The food industry has been similarly shaken by the appearance of new, and potentially fatal, strains of Escherichia coli, a species that for over 100 years was regarded as little more than a nuisance. Equally unexpected was the devastating impact of BSE, and fresh reports of the activities of socalled ‘emerging food-borne pathogens’ are appearing with alarming regularity. In some cases, it has been possible to understand, with the advantages of hindsight, why a particular species of bacterium, fungus or protozoan has become a major risk to human health while, on other occasions, the vagaries of nature have left the ‘experts’ totally bemused. However, even in these latter situations, control over the threat posed to food supplies has to be instituted, but the ability of the food industry, in conjunction with Public Health and other bodies, to develop effective responses can only be as good as the scientific knowledge available. In the case of food microbiology, this background has to be derived from a wide range of sources. Thus, agricultural practices may alter the biochemistry of a crop and, perhaps, its microflora as well; the microflora of any given foodstuff and/or processing facility will have specific characteristics that need to be understood before control is possible; techniques must be available to monitor a retail food for microorganisms that would pose a risk to the consumer. As the procedures necessary to monitor these various facets become ever more sophisticated, so fewer microbiologists can claim total competence, and the need for a specialist source of outside knowledge increases. It is this latter need that the Encyclopedia of Food Microbiology seeks to satisfy for, within this work, a busy microbiologist can find details of all the important genera of food-borne bacteria and fungi, how the same genera may react in different foods and under different environmental conditions, and how to detect the growth and/or metabolism of the same organisms in foods using classical o r modern techniques. In order to place this information into a broader context, the reader can explore the latest advice concerning food standards/specifications, or the role of monitoring systems like HACCP in achieving product targets for specific microorganisms; potential concerns over viruses and protozoa are also evaluated in the light of current knowledge. Readers interested in fermented foods will find the pertinent information in a similarly accessible form; indeed, purchasers of the print version of the encyclopedia will be entitled to register for access to the on-line version as well. This form allows the user the benefit of extensive hypertext linking and advanced search tools, adding value to the encyclopedia as a reference source, teaching aid and text for general interest. It is inevitable, of course, that short articles written to a tight deadline may have omissions, but it is to be hoped that such faults are minimal and, in any event, more than compensated for through the careful selections of further reading. If this optimism is justified, then the major credit rests with the authors of each article. They are all recognized as experts in their fields, and their willing participation has been much appreciated by the editors. The role of the Editorial Advisory Board merits a special mention as well, for their constructive

xii Introduction

criticisms of the list of articles, their suggestions for authors and their expert refereeing of the manuscripts has provided a solid foundation for the entire enterprise. However, the finest manuscripts are of little value to the scientific community until they have been published, and the editorial team at Academic Press - Carey Chapman (Editor-in-Chief),Tina Holland (Associate Editor), Nick Fallon (Commissioning Editor), Laura O’Neill (Editorial Assistant), Tamsin Cousins (Production Project Manager), Richard Willis (Freelance Project Manager), Emma Parkinson (Electronic Publishing Developer), Peter Lord (Publishing Services Manager), Emma Krikler (Picture Researcher) - have been outstanding in their support of the project. Obviously, each member of the team has made an important contribution, but it must be recorded that the role of Tina Holland has been absolutely invaluable. Thus, not only has Tina coordinated the numerous inputs from the editors, referees and authors, but even found time to help the editors with the location of authors; the editors acknowledge this unstinting assistance with much gratitude. R.K. Robinson, C.A. Batt, P.D. Pate1 Editors

PREFACE

Although food microbiology and food safety have, in recent times, become major concerns for governments around the world, equally importts the fact that, without yeasts and bacteria, popular meals like bread and cheese would not exist. Consequently, a knowledge of the relationship between foodstuffs and the activities of bacteria, yeasts and mycelial fungi has become a top priority for everyone associated with food and its production. Farmers have concerns related to produce harvesting and storage, food processors have to generate wholesome retail products that are both free from pathogenic organisms and have a satisfactory shelf life and, last but not least, food handlers and consumers need to be aware of the procedures necessary to ensure that food is safely prepared and stored. In order for these disparate groups to operate successfully, accurate and objective information about the microbiology of foods is essential, and this encyclopedia seeks to provide a source of such information. In some areas, introductory articles are provided to guide readers who may be less familiar with the subject but, in general, superficiality has been avoided. Thus, the coverage has been developed to include details of all the important groups of bacteria, fungi, viruses and parasites, the various methods that can be employed for their detection in foods, the factors that govern the behaviour of the same organisms, together with an analysis of likely outcomes of microbial growth/metabolism in terms of disease and/or spoilage. A further series of articles describes the contribution of microorganisms to industrial fermentations, to traditional food fermentations from the Middle or Far East, as well as during the production of the fermented foods like bread, cheese or yoghurt that are so familiar in industrialized societies. The division of these topics into 358 articles of approximately 4000 words, has meant that the contributing authors have been able to handle their specialist subject(s) in real depth. Obviously, another group of editors might have approached the project in a different manner, but we feel confident that this encyclopedia will provide readers at all levels of expertise with the data being sought. A point enhanced, perhaps, by the inclusion at the end of each article of a list for further reading, comprising a selection of review articles and key research papers that should encourage further exploration of any selected topic. If this confidence is borne out in practice, then the efforts of the contributors, the members of the Editorial Board and the editorial team from Academic Press will be well rewarded, for raising the scientific profile of food microbiology is long overdue. R.K. Robinson, C.A. Batt, P.D. Patel Editors

CONTENTS

VOLUME 1 A ACCREDITATION SCHEMES see LABORATORY MANAGEMENT Accreditation Schemes ACETOBACTER R K Hommel, P Ahnert ACINETOBACTER P Kampfer ADENYLATE KINASE M J Murphy, D J Squirrel1 AEROBIC METABOLISM see METABOLIC PATHWAYS: Release of Energy (Aerobic); Release of Energy (Anaerobic) AEROMONAS Introduction IS Blair, M A S McMahon, D A McDowell Detection by Cultural and Modern Techniques B Austin AFLATOXIN see MYCOTOXlNS: Classification ALCALIGENES T J Klem ALE see LAGER ALGAE see SINGLE-CELL PROTEIN: The Algae ALTERNARIA S E Lopez, D Cabral ANAEROBIC METABOLISMsee METABOLIC PATHWAYS: Release of Energy (Anaerobic) ANTIMICROBIAL PACKAGING see CHILLED STORAGE OF FOODS: Packaging with Antimicrobial Properties ANTIMICROBIAL SYSTEMS see NATURAL ANTIMICROBIAL SYSTEMS: Preservative Effects During Storage; Antimicrobial Compounds in Plants; Lysozyme and Other Proteins in Eggs; Lactoperoxidase and Lactoferrin ARCOBACTER IV Wesley ARTHROBACTER M Gobbetti, E Smacchi ASPERGILLUS Introduction P-K Chang, D Bhatnagar, T E Cleveland Aspergillus oryzae K Gomi Aspergillus flaws D Bhatnagar; T E Cleveland, G A Payne ATOMIC FORCE MICROSCOPY see MICROSCOPY Atomic Force Microscopy ATP BIOLUMINESCENCE Application in Meat Industry D A Bautista

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Application in Dairy Industry W Reybroeck Application in Hygiene Monitoring M W Griffiths Application in Beverage Microbiology A Thompson AUREOBASIDIUM T Roukas

B BACILLUS Introduction M K Dah/ Bacillus cereus C A Batt Bacillus stearothermophilus P Kotzekidou Bacillus anthracis L Baillie Bacillus subtilis M K Dah1 Detection of Toxins S H Beattie, A G Williams Detection by Classical Cultural Techniques I Jenson BACTERIA The Bacterial Cell R W Lovitt, C J Wright Bacterial Endospores G W Gould Classification of the Bacteria - Traditional V M de Ambrosini, C H Gusils, S N Gonzalez, G Oliver Classification of the Bacteria - Phylogenetic Approach E Stackebrandt BACTERIAL ADHESION see POLYMER TECHNOLOGIES FOR CONTROL OF BACTERIAL ADHESION BACTERlOClNS Potential in Food Preservation T O’Keeffe, C Hill Nisin E A Davies, J Delves-Broughton BACTEROIDES AND PREVOTELLA H J Flint, C S Stewart BACTERIOPHAGE-BASED TECHNIQUES FOR DETECTION OF FOOD-BORNE PATHOGENS R J Mole, V K Dhir, S P Denyer, G S A B Stewart (dec) BEER see LAGER BENZOIC ACID see PRESERVATIVES: Permitted Preservatives- Benzoic Acid BEVERAGE MICROBIOLOGY see ATP BIOLUMINESCENCE:Application in Beverage Microbiology BIFIDOBACTERIUM D G Hoover BIOCHEMICAL and MODERN IDENTIFICATION TECHNIQUES Introduction D Y C Fung Food Spoilage Flora (Le. Yeasts and Moulds) G G Khachatourians, D K Arora Food-poisoning Organisms D Y C Fung Enterobacteriaceae, Coliforms and E. coli R R Beumer, M C te Giffel, AGE Microfloras of Fermented Foods J P Tamang, W H Holzapfel BlOFlLMS B Carpentier, 0 Cerf BIOPHYSICAL TECHNIQUES FOR ENHANCING MICROBIOLOGICALANALYSIS A D Goater, R Pethig BIOSENSORS Scope in Microbiological Analysis M C Goldschmidt BIO-YOGHURTsee FERMENTED MILKS: Yoghurt BOTRYTIS M D Alur BOVINE SPONGIFORM ENCEPHALOPATHY (BSE) D M Taylor, R A Somerville

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Contents xlv

BREAD Bread from Wheat Flour R S Singhal, P R Kulkarni Sourdough Bread B J B Wood BRETANOMYCES J Jimenez, M Fidalgo, M Alguacil BREVIBACTERIUM B Weimer BREWER’S YEAST see SACCHAROMYCES: Brewer’s Yeast BROCHOTHRIX R H Holley BRUCELLA Characteristics J Theron, T E Cloete Problems with Dairy Products P Papademas BURKHOLDERIA COCOVENENANSsee PSEUDOMONAS: Burkholderia cocovenenans BUTTER see MILK AND MILK PRODUCTS: Microbiology of Cream and Butter BYSSOCHLAMYS P Kotzekidou C CAKES see CONFECTIONERY PRODUCTS: Cakes and Pastries CAMPYLOBACTER Introduction M T Rowe, R H Madden Detection by Cultural and Modern Techniques J E L Corry Detection by Latex Agglutination Techniques W C Hazeleger, R R Beumer CANDIDA Introduction R K Hommel Yarrowia (Candida) lipolytica G M Heard, G H Fleet CANNING see HEAT TREATMENT OF FOODS: Principle of Canning; Spoilage Problems Associated with Canning CATERING INDUSTRY see PROCESS HYGIENE: Hygiene in the Catering Industry CELLULOMONAS M I Rajoka, K A Malik CEREALS see SPOILAGE OF PLANT PRODUCTS: Cereals and Cereal Flours CENTRIFUGATION see PHYSICAL REMOVAL OF MICROFLORAS: Centrifugation CHEESE In the Marketplace A Y Tamime Microbiology of Cheese-making and Maturation N Y Farkye Mould-ripened Varieties A W Nichol Role of Specific Groups of Bacteria M El Soda Microflora of White-brined Cheeses B H Ozer CHEMILUMINESCENT DNA HYBRIDIZATION see LISTERIA: Listeria monocytogenes - Detection by Chemiluminescent DNA Hybridization CHILLED STORAGE OF FOODS Principles B P F Day Use of Modified-atmosphere Packaging R E O’Connor-Shaw, V G Reyes Packaging with Antimicrobial Properties D Collins-Thompson, Cheng-An Hwang CIDER (HARD CIDER) B Jarvis CITRIC ACID see FERMENTATION (INDUSTRIAL): Production of Organic Acids CITROBACTER see SALMONELLA: Detection by Enzyme Immunoassays CLOSTRIDIUM Introduction H P Blaschek Clostridium perfringens H P Blaschek

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Detection of Enterotoxins of C. perfringens L Petit, M Gibert, M R Popoff Clostridium acetobutylicum H Biebl Clostridium tyrobutyricum M Wiedmann, K J Boor, H Eisgruber, K-J Zaadhof Clostridium botulinum E A Johnson Detection of Neurotoxins of Clostridium botulinum S Notermans COCOA AND COFFEE FERMENTATIONS P Nigam COFFEE see COCOA AND COFFEE FERMENTATIONS COLORIMETRIC DNA HYBRIDIZATION see LlSTERlA: Detection by Colorimetric DNA Hybridization; SALMONELLA: Detection by Colorimetric DNA Hybridization COLOURS see FERMENTATION (INDUSTRIAL): Production of Colours/Flavours CONFECTIONERY PRODUCTS - CAKES AND PASTRIES P A Voysey, J D Legan CONFOCAL LASER MICROSCOPY see MICROSCOPY: Confocal Laser Scanning Microscopy COSTS/BENEFITS OF MICROBIAL ORIGIN J E Hobbs, W A Kerr CREAM see MILK AND MILK PRODUCTS: Microbiology of Cream and Butter CRITICAL CONTROL POINTS see HAZARD APPRAISAL (HACCP): Critical Control Points CRUSTACEA see SHELLFISH (MOLLUSCS AND CRUSTACEA): Characteristics of the Groups; Contamination and Spoilage CRYPTOSPORlDlUM R W A Girdwood, H V Smith CULTURAL TECHNIQUES see AEROMONAS: Detection by Cultural and Modern Techniques; BACILLUS: Detection by Classical Cultural Techniques; CAMPYLOBACTER: Detection by Cultural and Modern Techniques; ENRICHMENT SEROLOGY An Enhanced Cultural Technique for Detection of Food-borne Pathogens; FUNGI: Food-borne Fungi - Estimation by Classical Cultural Techniques; LISTERlA: Detection by Classical Cultural Techniques; SALMONELLA: Detection by Classical Cultural Techniques; SHIGELLA: Introduction and Detection by Classical Cultural Techniques; STAPHYLOCOCCUS: Detection by Cultural and Modern Techniques; VEROTOXIGENIC E. COLI AND SHIGELLA SPP: Detection by Cultural Methods; VIBRIO: Detection by Cultural and Modern Techniques CULTURE COLLECTIONS F M Dugan, J. S Tang CURING see MEAT AND POULTRY: Curing of Meat CYCLOSPORA A M Adams, K C Jinneman, Y R Ortega CYTOMETRY see FLOW CYTOMETRY

D DAIRY PRODUCTS see BRUCELLA: Problems with Dairy Products; CHEESE: In the Market Place; Microbiology of Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific Groups of Bacteria; Microflora of White-brined Cheeses; FERMENTED MILKS: Yoghurt; Products from Northern Europe; Products of Eastern Europe and Asia; PROBIOTIC BACTERIA: Detection and Estimation in Fermented and Non-fermented Dairy Products DEBARYOMYCES W Praphailong, G H Fleet DESULFOVIBRIO M D Alur DEUTEROMYCETES see FUNGI: Classification of the Deuteromycetes DIRECT (AND INDIRECT) CON DUCT1M ETR IC/I M PEDI M ETRIC TECHNIQUES Food-borne Pathogens D Blivet DIRECT EPIFLUORESCENT FILTER TECHNIQUES (DEFT) 5 H Pyle DISINFECTANTS see PROCESS HYGIENE: Testing of Disinfectants DRIED FOODS M D Alur, V Venugopal

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E ECOLOGY OF BACTERIA AND FUNGI IN FOODS Influence of Available Water K Krist, D S Nichols, T Ross Influence of Temperature T Ross, D S Nichols Influence of Redox Potential and pH A Rompf, D Jahn EGGS Microbiology of Fresh Eggs N H C Sparks Microbiology of Egg Products J Delves-Broughton, R G Board ELECTRICAL TECHNIQUES Introduction D Blivet Food Spoilage Flora and Total Viable Count (TVC) G Salvat, D Blivet Lactics and other Bacteria L Curda ELECTRON MICROSCOPY see MICROSCOPY: Scanning Electron Microscopy; Transmission Electron Microscopy ELECTROPORATIONsee MINIMAL METHODS OF PROCESSING: Electroporation - Pulsed Electric Fields ENDOSPORES see BACTERIA: Bacterial Endospores ENRICHMENT SEROLOGY An Enhanced Cultural Technique for Detection of Food-borne Pathogens C de W Blackburn ENTAMOEBA see WATERBORNE PARASITES: Entamoeba ENTEROBACTER T W Huber ENTEROBACTERIACEAE, COLIFORMS AND E, COLI Introduction A Pandey, V K Joshi, P Nigam, C R Soccol Classical and Modern Methods for Detection/Enumeration E de Boer ENTEROCOCCUS G Giraffa ENTEROTOXINS see BAClLLUS: Detection of Enterotoxins; STAPHYLOCOCCUS: Detection of Staphylococcal Enterotoxins ENTEROVIRUSES see VIRUSES ENZYME IMMUNOASSAYS: OVERVlEW A Sharma ESCHERlCHlA COLI Escherichia coli C A Batt Detection of Enterotoxins of E. coli H - Y Tsen ESCHERlCHlA COLI 0157 Escherichia coli 0 1 57:H7 M L Tortorello Detection by Latex Agglutination Techniques E W Rice Detection by Commercial lmmunomagnetic Particle-based Assays P M Fratamico, C G Crawford EUKARYOTIC ASCOMYCETES see FUNGI: Classification of the Eukaryotic Ascomycetes (Ascomycotina)

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VOLUME 2 F FATTY ACIDS see FERMENTATION (INDUSTRIAL): Production of Oils and Fatty Acids FERMENTATlON (I NDUSTR IAL) Basic Considerations Y Chisti Media for Industrial Fermentations G M Walker

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Control of Fermentation Conditions T Keshavarz Recovery of Metabolites P A Pawar, M C Misra, N P Ghildyal, N G Karanth Production of Xanthan Gum M K Gowthaman, M S Prasad, N G Karanth Production of Organic Acids M Moresi, E Parente Production of Oils and Fatty Acids P Nigam Colours/Flavours Derived by Fermentation R G Berger FERMENTED FOODS Origins and Applications G Campbell-Platt Fermented Vegetable Products G Oliver, M Nuhez, S Gonzalez Fermented Meat Products M C Montel Fermented Fish Products J H Al-Jedah, M Z Ali Beverages from Sorghum and Millet J Dewar, J R N Taylor Fermentations of the Far East IGandjar FERMENTED MILKS Range of Products E Litopoulou, N Tzanetakis Yoghurt R K Robinson Products from Northern Europe H Roginski Products of Eastern Europe and Asia D Ozer, B H Ozer FILTRATION see PHYSICAL REMOVAL OF MICROFLORAS: Filtration FISH Catching and Handling A Chattopadhyay Spoilage of Fish J J Leisner, L Gram FLAVOBACTERlUM M-L Garcia-Lopez, J-A Santos, A Otero FLAVOURS see FERMENTATION (INDUSTRIAL): Production of Colours/Flavours FLOURS see SPOILAGE OF PLANT PRODUCTS: Cereals and Cereal Flours FLOW CYTOMETRY C N Jacobsen, J Jakobsen FOOD POISONING OUTBREAKS S Notermans FOOD PRESERVATION see BACTERIOCINS: Potential in Food Preservation; HEAT TREATMENT OF FOODS; HIGH PRESSURE TREATMENT OF FOODS; LASERS: Inactivation Techniques; MICROBIOLOGY OF SOUS-VIDE PRODUCTS; MINIMAL METHODS OF PROCESSING: Electroporation - Pulsed Electric Fields; ULTRASONIC STANDING WAVES; ULTRA-VIOLET LIGHT FREEZING OF FOODS Damage to Microbial Cells R S Singhal, P R Kulkarni Growth and Survival of Microorganisms P Chattopadhyay FUNGI The Fungal Hypha J Silva, S Gonzalez, J Palacios, G Oliver Food-borne Fungi - Estimation by Classical Cultural Techniques A K Sarbhoy, M Kulshreshtha Overview of Classification of the Fungi B C Sutton Classification of the Peronosporomycetes M W Dick Classification of the Zygomycetes P M Kirk Classification of the Eukaryotic Ascomycetes M A Cousin Classification of the Deuteromycetes B C Sutton Classification of the Hemiascomycetes A K Sarbhoy FUSARIUM U Thrane

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G GASTRIC ULCERS see HELICOBACTER GENET1C ENGINEERING Modification of Yeast and Moulds R Sandhir, S K Garg, D R Modi Modification of Bacteria E Johansen GENETICS OF MICROORGANISMS Fungi R Sandhir, S K Garg, D R Modi Bacteria S K Garg, R Sandhir GEOTRICHUM A Botha GIARDIA R W A Girdwood, H V Smith GLUCONOBACTER R K Hommel. P Ahnert GOOD MANUFACTURING PRACTICE B Jarvis GUIDELINES GOVERNING MICROBIOLOGY see NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY: Canada; European Union; Japan

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H HAFNIA ALVEI J Ridell HANSENULA G Gellissen, C P Hollenberg HARD CIDER see CIDER (HARD CIDER) HAZARD APPRAISAL (HACCP) The Overall Concept F Untermann Critical Control Points S Leaper Establishment of Performance Criteria T Mahmutoglu, f Bozoglu Involvement of Regulatory Bodies 0 P Snyder, V K Juneja HEAT TREATMENT OF FOODS Thermal Processing Required for Canning A Azizi Spoilage Problems Associated with Canning L Ababouch Ultra-high Temperature (UHT) Treatments M J Lewis Principles of Pasteurization R A Wilbey Action of Microwaves A Stolle, B Schalch Synergy Between Treatments E A Murano HELICOBACTER I V Wesley HELMINTHS AND NEMATODES K D Murre11 HEMIASCOMYCETES- 1 AND 2 see FUNGI: Classification of the Hemiascomycetes HEPATITIS VIRUSES see VIRUSES: Hepatitis Viruses HIGH-PRESSURE TREATMENT OF FOODS M Patterson HISTORY OF FOOD MICROBIOLOGY N D Cowell HURDLE TECHNOLOGY L G M Gorris HYDROPHOBIC GRID MEMBRANE FILTER TECHNIQUES (HGMF) P Entis HYDROXYBENZOIC ACID see PRESERVATIVES: Permitted Preservatives - Hydroxybenzoic Acid HYGIENE MONITORING see ATP BIOLUMINESCENCE: Application in Hygiene Monitoring HYGIENIC PROCESSING see PROCESS HYGIENE: Overall Approach to Hygienic Processing

I ICE CREAM A Kambamanoli-Dimou IMMUNOLOGICAL TECHNIQUES see MYCOTOXINS: Immunological Techniques for Detection and Analysis IMMUNOMAGNETIC PARTICLE-BASEDASSAYS see ESCHERICHIA COLI 0757: Detection by

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Commercial lmmunomagnetic Particle-based Assays; LISTERIA: Detection by Commercial lmmunomagnetic Particle-based Assays; SALMONELLA: Detection by lmmunomagnetic Particle-based Assays IMMUNOMAGNETIC PARTICLE-BASED TECHNIQUES: OVERVIEW K S Cudjoe INACTIVATION TECHNIQUES see LASERS: Inactivation Techniques INDIRECT CONDUCTIMETRIC/IMPEDlMETRlCTECHNIQUES see DIRECT (AND INDIRECT) CONDUCTIMETRIC/IMPEDlMETRlC TECHNIQUES: Food-borne Pathogens; Enterobacteriaceae, Coliforms and E, coli INDUSTRIAL FERMENTATIONsee FERMENTATION (INDUSTRIAL): Basic Considerations; Media for Industrial Fermentations; Control of Fermentation Conditions; Recovery of Metabolites; Production of Xanthan Gum; Production of Organic Acids; Production of Oils and Fatty Acids; Colours/Flavours Derived by Fermentation INTERMEDIATE MOISTURE FOODS K Prabhakar INTERNATIONAL CONTROL OF MICROBIOLOGY B Pourkomailian

K KLEBSIELLA P T Vanhooren, S De Baets, G Bruggeman, E J Vandamme KLUYVEROMYCES C A Batt L LABORATORY DESIGN M Ahmed LABORATORY MANAGEMENT- ACCREDITATION SCHEMES C Bowles LACTIC ACID BACTERIA see LACTOBACILLUS: Introduction; Lactobacillus bulgaricus; Lactobacillus brevis; Lactobacillus acidophilus; Lactobacillus casei; LACTOCOCCUS: Introduction; Lactococcus lactis Sub-species lactis and cremoris; PEDIOCOCCUS LACTOBACILLUS Introduction C A Batt Lactobacillus bulgaricus P C M Teixeira Lactobacillus brevis P C M Teixeira Lactobacillus acidophilus T R Klaenhammer; W M Russell Lactobaciius casei M Gobbetti LACTOCOCCUS Introduction C A Batt Lactococcus lactis Sub-species lactis and cremoris P D Courtney LACTOFERRIN see NATURAL ANTIMICROBIAL SYSTEMS: Lactoperoxidase and Lactoferrin LACTOPEROXIDASE see NATURAL ANTIMICROBIAL SYSTEMS: Lactoperoxidase and Lactoferrin LAGER ICampbell LASERS: INACTIVATION TECHNIQUES IA Watson, D E S Stewart-Tu11 LATEX AGGLUTINATION TECHNIQUES see CAMPYLOBACTER: Detection by Latex Agglutination Techniques; ESCHERICHIA COLI 0 157: Detection by Latex Agglutination Techniques; SALMONELLA: Detection by Latex Agglutination Techniques LEGISLATION see NATIONAL LEGISLATION, GUIDELINES 8, STANDARDS GOVERNING MICROBIOLOGY Canada; European Union; Japan LEUCONOSTOC A Lonvaud-Funel LIGHT MICROSCOPYsee MICROSCOPY Light Microscopy LIPID METABOLISM see METABOLIC PATHWAYS: Lipid Metabolism

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Contents Ii

LlSTERlA Introduction C A Batt Detection by Classical Cultural Techniques G D W Curtis Detection by Commercial Enzyme Immunoassays M Wagner, A Bubert Detection by Colorimetric DNA Hybridization A D Hitchins Detection by Commercial lmmunomagnetic Particle-based Assays B Kohn Listeria monocytogenes S E Martin, C W Fisher Listeria monocytogenes - Detection by Chemiluminescent DNA Hybridization A D Hitchins Listeria monocytogenes - Detection using NASBA (an Isothermal Nucleic Acid Amplification System) M R Uyttendaele, J M Debevere LYSINS see MINIMAL METHODS OF PROCESSING: Potential use of Phages and/or Lysins LYSOZYME see NATURAL ANTIMICROBIAL SYSTEMS: Lysozyme and other Proteins in Eggs

M MALOLACTIC FERMENTATION see WINES: The Malolactic Fermentation MANOTHERMOSONICATION see MINIMAL METHODS OF PROCESSING: Manothermosonication MANUFACTURING PRACTICE see GOOD MANUFACTURING PRACTICE MATHEMATICAL MODELLING see PREDICTIVE MICROBIOLOGY AND FOOD SAFETY MEAT AND POULTRY Spoilage of Meat G-J E Nychas, E H Drosinos Curing of Meat K Prabhakar Spoilage of Cooked Meats and Meat Products I Guerrero, L P Chabela METABOLIC ACTIVITY TESTS see TOTAL VIABLE COUNTS: Metabolic Activity Tests METABOLIC PATHWAYS Release of Energy (Aerobic) A Brandis-Heep Release of Energy (Anaerobic) M D Alur Nitrogen Metabolism M D Alur Lipid Metabolism R Sandhir Metabolism of Minerals and Vitamins C Umezawa, M Shin Production of Secondary Metabolites - Fungi P Nigam, D Singh Production of Secondary Metabolites - Bacteria M D Alur METABOLITE RECOVERY see FERMENTATION (INDUSTRIAL): Recovery of Metabolites METHANOGENS W Kim, W B Whitman MICROBIOLOGY OF SOUS-VIDE PRODUCTS F Cadin MlCROCOCCUS M-L Garcia-Lopez, J-A Santos, A Otero MICROFLORA OF THE INTESTINE The Natural Microflora of Humans C L Willis, G R Gibson Biology of Bifidobacteria A Y Tamime Biology of Lactobacillus acidophilus W R Aimutis Biology of the Enterococcus spp. N Tunail Detection and Enumeration of Probiotic Cultures G Kalantzopoulos MICROSCOPY Light Microscopy R W Lovitt, C J Wright Confocal Laser Scanning Microscopy D F Lewis Scanning Electron Microscopy U J Potter, G Love Transmission Electron Microscopy U J Potter, G Love

1195 1199 1207 1214 1222 1228 1238 1244

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1272 1279 1288 1298 1313 1319 1328 1334 1338 1344 1351 1355 1361 1365 1373 1379 1389 1397 1407

lii Contents

Atomic Force Microscopy W R Bowen, M Hila/, R W Lovitt, C J Wright Sensing Microscopy M Nakao MICROWAVES see HEAT TREATMENT OF FOODS: Action of Microwaves MILK AND MILK PRODUCTS Microbiology of Liquid Milk B H Ozer Microbiology of Dried Milk Products D Muir Microbiology of Cream and Butter R S Singhal, P R Kulkarni MILLET see FERMENTED FOODS: Beverages from Sorghum and Millet MINERAL METABOLISM see METABOLIC PATHWAYS: Metabolism of Minerals and Vitamins MINIMAL METHODS OF PROCESSING Electroporation - Pulsed Electric Fields M L Calderdn-Miranda, G V Barbosa-Canovas, B G Swanson Manothermosonication J Burgos Potential use of Phages and/or Lysins J Jofre, M Munisa MODIFIED ATMOSPHERE PACKAGING see CHILLED STORAGE OF FOODS: Use of Modified Atmosphere Packaging MOLECULAR BIOLOGY IN MICROBIOLOGICAL ANALYSIS - DNA-BASED METHODS FOR THE DETECTION OF FOOD-BORNE PATHOGENS L O’Connor, M Maher MOLLUSCS see SHELLFISH (MOLLUSCS AND CRUSTACEA): Characteristics of the Groups; Contamination and Spoilage MONASCUS L Martinkova, P Patakova MORAXELLA J-A Santos, M-L Garcia-Ldpez, A Otero MOULDS see BIOCHEMICAL IDENTIFICATION TECHNIQUES - MODERN TECHNIQUES: Food Spoilage Flora (Le. Yeasts and Moulds); FUNGI; GENETIC ENGINEERING: Modification of Yeast and Moulds; STARTER CULTURES: Moulds Employed in Food Processing MPN see TOTAL VIABLE COUNTS: MPN MUCOR A Botha, J C du Preez MYCELIAL FUNGI see SINGLE-CELL PROTEIN: Mycelial Fungi MYCOBACTERIUM J B Payeur MYCOTOXINS Classification L B Bullerman Occurrence M de Nijs, S H W Nofermans Detection and Analysis by Classical Techniques IA Ahmed Immunological Techniques for Detection and Analysis A Sharma, M R A Pillai Toxicology D Abramson

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1436 1441 1445

1456 1462 1469

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1481 1487

1493 1500 1512 1520 1526 1532 1539

VOLUME 3 N NASBA see LISTERIA: Listeria monocytogenes - Detection using NASBA (an Isothermal Nucleic Acid Amplification System) NATAMYCIN see PRESERVATIVES: Permitted Preservatives- Natamycin NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY Canada B E Brown European Union B Schalch, H Beck Japan S Kumagai NATURAL ANTIMICROBIAL SYSTEMS Preservative Effects During Storage V M Dillon

1549 1561 1564 1570

Contents liii

Antimicrobial Compounds in Plants C Umezawa, M Shin Lysozyme and Other Proteins in Eggs E A Chartec G Lagarde Lactoperoxidase and Lactoferrin B H Ozer NEISSERIA S A S Hanna NEMATODES see HELMINTHES AND NEMATODES NISIN see BACTERIOCINS: Nisin NITRATE see PRESERVATIVES: Permitted Preservatives - Nitrate and Nitrite NITRITE see PRESERVATIVES: Permitted Preservatives - Nitrate and Nitrite NITROGEN METABOLISM see METABOLIC PATHWAYS: Nitrogen Metabolism NUCLEIC ACID-BASED ASSAYS Overview M W Griffiths

1576 1582 1587 1591

1599

0 OENOLOGY see WINES: Specific Aspects of Oenology OILS see FERMENTATION (INDUSTRIAL): Production of Oils and Fatty Acids; PRESERVATIVES: Traditional Preservatives - Oils and Spices ORGANIC ACIDS see FERMENTATION (INDUSTRIAL): Production of Organic Acids, e.g. Citric, Propionic; PRESERVATIVES:Traditional Preservatives - Organic Acids P PACKAGING OF FOODS A L Brody PANARY FERMENTATION see BREAD: Bread from Wheat Flour PANTOEA A Morin, Z Parveen PARASITES see CRYPTOSPORIDIUM; CYCLOSPORA; GIARDIA; HELMINTHES AND NEMATODES: TRICHINELLA: WATERBORNE PARASITES: Entamoeba; Detection by Classic and Modern Techniques PASTEURIZATIONsee HEAT TREATMENT OF FOODS: Principles of Pasteurization PASTRY see CONFECTIONERY PRODUCTS: Cakes and Pastries PCR-BASED COMMERCIAL TESTS FOR PATHOGENS P A Bertram-Drogatz, F Wilborn, P Scheu, A Pardigol, C Koob, C Gronewald, M Fandke, A Gasch, K Berghof PEDIOCOCCUS M Raccach PENlClLLIUM Introduction J IPitt Penicillium in Food Production G Blank PERONOSPOROMYCETES see FUNGI: Classification of the Peronosporomycetes PETRIFILM -AN ENHANCED CULTURAL TECHNIQUE R Jordano, L M Medina PHAGES see BACTERIOPHAGE-BASED TECHNIQUES FOR DETECTION OF FOOD-BORNE PATHOGENS; MINIMAL METHODS OF PROCESSING: Potential Use of Phages and/or Lysins PHYCOTOXINS A Sharma PHYLOGENETIC APPROACH TO BACTERIAL CLASSIFICATION see BACTERIA: Classification of the Bacteria - Phylogenetic Approach PHYSICAL REMOVAL OF MICROFLORAS Filtration P Boyaval Centrifugation V V Mistry PlCHlA PASTORIS C Kalidas POLYMER TECHNOLOGIES FOR CONTROL OF BACTERIAL ADHESION D Cunliffe, C A Smart, C Alexander POLYSACCHARIDES see FERMENTATION (INDUSTRIAL): Production of Xanthan Gum

1611 1623

1630 1641 1647 1655 1662

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1675 1681 1686 1692

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Contents

POULTRY see MEAT AND POULTRY: Spoilage of Meat; Curing of Meat; Spoilage of Cooked Meats and Meat Products POUR PLATE TECHNIQUE see TOTAL VIABLE COUNTS: Pour Plate Technique PREDICTIVE MICROBIOLOGY AND FOOD SAFETY T Ross, T A McMeekin, J Baranyi PR ESERVATIVES Classification and Properties M Surekha, S M Reddy Traditional Preservatives - Oils and Spices G-J E Nychas, C C Tassou Traditional Preservatives - Sodium Chloride M S Brewer Traditional Preservatives - Organic Acids M Stratford Traditional Preservatives - Wood Smoke L J Ogbadu Traditional Preservatives - Vegetable Oils V Venugopal Permitted Preservatives - Sulphur Dioxide K Prabhakar, K S Reddy Permitted Preservatives - Benzoic Acid L J Ogbadu Permitted Preservatives - Hydroxybenzoic Acid R S Singhal, P R Kulkarni Permitted Preservatives - Nitrate and Nitrite R S Singhal, P R Kulkarni Permitted Preservatives - Sorbic Acid L V Thomas Permitted Preservatives - Natamycin J Stark Permitted Preservatives - Propionic Acid R S Singhal, P R Kulkarni PROB IOTIC BACTERIA Detection and Estimation in Fermented and Non-fermented Dairy Products W Kneifel, T Mattila-Sandholm, A von Wright PROBlOTlCS see BIFIDOBACTERIUM; MICROFLORA OF THE INTESTINE: The Natural Microflora of Humans PROCESS HYGIENE Designing for Hygienic Operation G C Gurakan, T F Bozoglu Types of Biocides J F Williams, S D Worley Overall Approach to Hygienic Processing M A Mostert, H L M Lelieveld Modern Systems of Plant Cleaning Y Chisti Risk and Control of Airborne Contamination G J Curie/, H M J van Eijk, H L M Lelieveld Testing of Disinfectants J F Williams, J R Bickert Involvement of Regulatory and Advisory Bodies R Cocker, H L M Lelieveld Hygiene in the Catering Industry.. N Johns PROPlONlBACTERlUM M Gautier PROPIONIC ACID see FERMENTATION (INDUSTRIAL): Production of Organic Acids, e.g. Citric, Propionic ; PRESERVATIVES : Permitted Preservatives - Propio nic Acid PROTEUS B W Senior PSEUDOMONAS Introduction M A Cousin Pseudomonas aeruginosa M H J Bennik Burkholderia cocovenenans J Cox, E Kartadarma, K Buckle PSYCHROBACTER M-L Garcia-Lopez, M P Maradona

1699 1710 1717 1723 1729 1737 1743 1750 1754 1757 1762 1769 1776 1781 1783

1790 1794 1802 1806 1816 1822 1830 1845 1850

1857 1864 1867 1871 1875

Q QUALITY ASSURANCE AND MANAGEMENT see HAZARD APPRAISAL (HACCP): The Overall Concept QUANTITATIVE RISK ANALYSIS S H W Notermans

1883

Contents Iv

R RAPID METHODS FOR FOOD HYGIENE INSPECTION M Upmann, C Bonaparte REDOX POTENTIAL see ECOLOGY OF BACTERIA AND FUNGI IN FOODS: Influence of Redox Potential and pH REFERENCE MATERIALS P H In’t Veld REGULATORY BODIES see HAZARD APPRAISAL (HACCP): Involvement of Regulatory Bodies [1340] RHODOTORULA Y Yeeh RISK ANALYSIS see QUANTITATIVE RISK ANALYSIS S SACCHAROMYCES Introduction Y Oda, K Ouchi Saccharomyces sake Y limura Saccharomyces cerevisiae 5 C Viljoen, G M Heard Saccharomyces: Brewer’s Yeast G G Stewart SAKE see SACCHAROMYCES: Saccharomyces sake SALMONELLA Introduction J Cox Salmonella enteritidis T S Hammack, W H Andrews Salmonella typhi J Cox Detection by Classical Cultural Techniques R M ArnaguaAa, W H Andrews Detection by Latex Agglutination Techniques J Cox Detection by Enzyme Immunoassays P Patel Detection by Colorimetric DNA Hybridization H-Y Tsen Detection by lmmunomagnetic Particle-based Assay K S Cudjoe SALT see PRESERVATIVES: Traditional Preservatives - Sodium Chloride SAMPLING REGIMES & STATISTICAL EVALUATION OF MICROBIOLOGICAL RESULTS G Hildebrandt SCANNING ELECTRON MICROSCOPY see MICROSCOPY: Scanning Electron Microscopy SCHIZOSACCHAROMYCES G H Fleet SECONDARY METABOLITES see METABOLIC PATHWAYS: Production of Secondary Metabolites - Fungi; Production of Secondary Metabolites - Bacteria SENSING MICROSCOPY see MICROSCOPY: Sensing microscopy SERRATIA F Rafii SHELLFISH (MOLLUSCSAND CRUSTACEA) Characteristics of the Groups L Le Vay, B Egan Contamination and Spoilage C A Kaysner SHEWANELLA L Gram, B F Vogel SHIGELLA: Introduction and Detection by Classical Cultural Techniques K A Lampel, R C Sandlin, S Formal SINGLE-CELL PROTEIN The Algae M Garcia-Garibay, L Gdmez-Ruiz, E Barzana Yeasts and Bacteria M Garcia-Garibay, L Gomez-Ruiz, E Barzana Mycelial Fungi P Nigam SODIUM CHLORIDE see PRESERVATIVES: Traditional Preservatives - Sodium Chloride SORBIC ACID see PRESERVATIVES: Permitted Preservatives- Sorbic Acid SORGHUM see FERMENTED FOODS: Beverages from Sorghum and Millet

1887

1895 1900

1907 1913 1918 1925

1928 1937 1943 1948 1952 1956 1964 1968

1976 1984

1989 1993 2001 2008 201 5 2021 2027 2034

Ivi Contents

SOURDOUGH BREAD see BREAD: Sourdough Bread SOUS-VIDE PRODUCTS see MICROBIOLOGY OF SOUS-VIDE PRODUCTS SPICES see PRESERVATIVES: Traditional Preservatives- Oils and Spices SPIRAL PLATER see TOTAL VIABLE COUNTS: Specific Techniques SPOILAGE OF PLANT PRODUCTS Cereals and Cereal Flours D R Twiddy, P Wareing SPOILAGE PROBLEMS Problems Caused by Bacteria K M J Hansen, D A Bautista Problems Caused by Fungi M 0 Moss STAPHYLOCOCCUS Introduction S E Martin, J J landolo Staphylococcus aureus J Harvey, A Gilmour Detection by Cultural and Modern Techniques S R Tatini, R Bennett Detection of Staphylococcal Enterotoxins M S Bergdoll (dec) STARTER CULTURES Uses in the Food Industry R C Wigley Importance of Selected Genera C R Dass Cultures Employed in Cheese-making T M Cogan Moulds Employed in Food Processing T Uraz, B H Ozer STATISTICAL EVALUATION OF MICROBIOLOGICAL RESULTS see SAMPLING REGIMES & STAT1STICAL EVALUAT I0N 0F MICROBI0LOGICAL RESULTS STERILANTS see PROCESS HYGIENE: Types of Sterilant STREPTOCOCCUS Introduction M Gobbetti, A Corsetti Streptococcus thermophilus G Zirnstein, R Hutkins STREPTOMYCES A Sharma SULPHUR DIOXIDE see PRESERVATIVES: Permitted Preservatives - Sulphur Dioxide

T THERMUS AQUATICUS C K K Nair TORULOPSIS R K Hommel, H-P Kleber TOTAL COUNTS Microscopy S R Tatini, K L Kauppi TOTAL VIABLE COUNTS Pour Plate Technique J W Messer, C H Johnson Spread Plate Technique J W MeSser, E W Rice, C H Johnson Specific Techniques M G Williams, F F Busta Most Probable Number (MPN) M G Williams, F F Busta Metabolic Activity Tests A F Mendonca, V K Juneja Microscopy C D Zook, F F Busta TOXICOLOGY see MYCOTOXINS: Toxicology TRANSMISSION ELECTRON MICROSCOPY see MICROSCOPY: Transmission Electron Microscopy TRICHINELLA H R Gamble TRICHODERMA D E Eveleigh TRICHOTHECIUM A Sharma

2045 2051 2056 2062 2066 2071 2076 2084 2095 2100 2109

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Contents lvii

U UHT TREATMENTS see HEAT TREATMENT OF FOODS: Ultra-high Temperature (UHT) Treatments ULTRASONIC IMAGING Non-destructive Methods to Detect Sterility of Aseptic Packages L Raaska, T MattilaSandholm ULTRASONIC STANDING WAVES Inactivation of Food-borne Microorganisms using Power Ultrasound G D Betts, A Williams, R M Oakley ULTRA-VIOLET LIGHT G Shama

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2202 2208

v VAGOCOCCUS L M Teixeira, M da G S Carvalho, R R Facklam VEGETABLE OILS see PRESERVATIVES: Traditional Preservatives- Vegetable Oils VEROTOXIGENIC E. COLl Detection by Commercial Enzyme Immunoassays D W Pimbley VEROTOXIGENIC E. COLl AND SHlGELLA SPP. Detection by Cultural Methods C Vernozy-Rozand VIBRIO Introduction, Including Vibrio vulnificus, and Vibrio parahaemolyticus P M Desmarchelier Vibrio cholerae F Y K Wong, P M Desmarchelier Standard Cultural Methods and Molecular Detection Techniques in Foods K Venkateswaran VINEGAR M R Adams VIRUSES Introduction D 0 Cliver EnvironmentallyTransmissible Enteric Hepatitis Viruses: A and E T L Crorneans, M D Sobsey Detection D 0 Cliver VITAMIN METABOLISM see METABOLIC PATHWAYS: Metabolism of Minerals and Vitamins W WATER QUALITY ASSESSMENT Routine Techniques for Monitoring Bacterial and Viral Contaminants J Watkins, L Straszynski, D Sartory, P Wyn-Jones Modern Microbiological Techniques C Fricker WATERBORNE PARASITES Entamoeba W A Petri Jc J M Schaenman Detection by Conventional and Developing Techniques H V Smith, R W A Girdwood WINES Microbiology of Wine-making G M Walker Malolactic Fermentation T F Bozoglu, S Yurdugul Specific Aspects of Oenology P Nigam WOOD SMOKE see PRESERVATIVES: Traditional Preservatives- Wood Smoke

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2221 2232 2237 2242 2248 2258 2264 2267 2274

2281 2287 2292 2295 2306 231 1 2316

X XANTHAN GUM see FERMENTATION (INDUSTRIAL):Production of Xanthan Gum XANTHOMONAS A Sharma XEROMYCES BlOSPORUS FRASER A D Hocking, J I Pitt

2323 2329

Y YEASTS: Production and Commercial Uses R Joseph

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YERSlNlA Introduction P Kampfer Yersinia enterocolitica S Bhaduri YOGHURT see FERMENTED MILKS: Yoghurt

2342 2350

z ZYGOMYCETES see FUNGI: Classification of the Zygomycetes ZYGOSACCHAROMYCES J P Erickson, D N McKenna ZYMOMONAS H Yanase COLOUR PLATE SECTIONS: Volume 1 Volume 2 Volume 3 APPENDICES: Appendix 1: Bacteria and Fungi Appendix 2: List of Suppliers INDEX

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between pages 358 and 359 between pages 1116 and 1117 between pages 1970 and 1971

Ai Aiii li

ACETOBACTER

I Accreditation Schemes

1

see Laboratory Management:Accreditation Schemes.

ACETOBACTER Rolf K Hommel, Cell Technologie Leipzig, Germany Peter Ahnert, Department of Biochemistry, Ohio State University, Columbus, USA Copyright 0 1999 Academic Press

Acetic acid bacteria have been used for making vinegar, their best-known product, since Babylonian times. For most of this time, vinegar was obtained by fermentation from alcoholic solutions without understanding of the natural process. A number of researchers established the microbial basis of this process in the beginning of the nineteenth century, such as Kiitzing, Lafare and Boerhaave. In 1822 Persoon performed the first biological study of surface films of wine and beer and proposed the name Mycoderma. Later Kiitzing (1837) isolated bacteria from naturally fermented vinegar for the first time. Considering them to be a kind of algae, he named them Ulvina aceti. Pasteur established the causal connection between the presence of Mycoderma aceti and vinegar formation in the first systematic studies on acetic acid fermentation. These discoveries and following studies resulted in better understanding and new methods (Pasteur method) of vinegar formation.

Characteristics of the Genus Acetobacter The classification of protobacteria by DNA-rRNA hybridization studies places acetic acid bacteria in the rRNA superfamily IV (synonymous: alpha group). Acetic acid bacteria, formerly classified into the family Pseudomonadaceae, constitute the family Acetobacteraceae consisting of only two closely related genera, Acetobacter and Gluconobacter, each of which is a separate rRNA branch. The family Acetobacteraceae represents strictly aerobic chemoorganotrophic bacteria, able to carry out a great variety of incomplete oxidations and living in or on plant materials, such as fruits and flowers. Some members of this family are plant pathogens. None display any pathogenic effect toward mammals, including humans. Based on physiological criteria the present nomen-

clature of the genus Acetobacter subdivides it into four species: A. aceti, A. liquefaciens, A. pasteurianus and A. hansenii. DNA-rRNA hybridization studies indicate the presence of three additional species: A. diazotrophicus, A. methanolicus and A. xylinum. Based on DNA-DNA hybridization a new species, A. europaeus, has been proposed; strains of this species show very low similarity to other species of the genus. Acetobacter are Gram-negative rods. Old cells may become Gram-variable. Cells appear singly, in pairs, or in chains and they are motile by peritrichous flagella or non-motile. There is no endospore formation. Acetobacter spp. are obligate aerobes except for A. diaztrophicus and A. nitrocaptans which belong to the diverse group of free-living aerobic or microaerophilic diazotrophs. The metabolism is respiratory and never fermentative. Single amino acids do not serve as sole source of nitrogen and carbon. Essential amino acids are not known. Depending on growth substrates, some strains may require p-aminobenzoic acid, niacin, thiamin, or pantothenic acid as growth factors. The temperature range is 5-42°C with optima between 25 and 30°C. Acetobacter strains show a moderate to high acid tolerance. The pH range is between pH 4 and pH 7, with optima between pH 5.4 and pH 6.3. Strains used in making vinegar are more resistant toward acidic pH. The minimum accepted by A. acidophilus is pH2.6. The maximum is pH4.2. The internal pH closely follows the external (A. aceti). At or below pH 5.0 the membrane potential of a cell is normally uncoupled, resulting in free proton exchange across the cytoplasmic membrane, thus depriving ATP synthesis of its driving force. However, the formation of acetic acid (or other acids) proceeds via membranebound dehydrogenases. These processes are closely

2 ACETOBACTER

connected to irreversible ATP-yielding reactions, sufficient to keep the energy metabolism alive. In A. aceti, a gene encoding citrate synthase is involved in acetic acid tolerance. This enzyme is assumed to play a central role in supplying sufficient ATP to protect the cell against accumulation of acetic acid. Ethanol concentrations higher than 8% and 10% inhibit strains A. aceti and A. xylinum, respectively. Some strains, for example spoilers of sakC, tolerate a higher ethanol content. In general, the ethanol tolerance in Acetobacter is higher than in Gluconobacter. The high direct oxidative capacity for sugars, alcohols and steroids is a special feature of Acetobacter. This ability is used in vinegar fermentation, food processing, chemical synthesis, and even in enantioselective oxidations, for example with A. pasteurianus. Examples of other reactions are the formation of 2,5-dioxogluconic acid by A. melanogenum and A. carinus, the oxidations of ethanediol to glycolic acid, of lactate to acetoin, of glycerol to dihydroxyacetone, for example polyols in which two secondary cis-arranged hydroxyl groups in Dconfiguration may be oxidized to ketoses. Two strains, A. rancens and A. peroxidans, are reported to oxidize n-alkanes, mainly by monoterminal attack yielding the corresponding fatty alcohols and fatty acids. Acetobacter are equipped with two sets of enzymes, catalysing the same oxidation reactions. Enzymes in the first set are bound in the cytoplasmic membrane, the active site facing the periplasm. Enzymes in the second set are located in the cytoplasm and are NADP-dependent. The latter enzymes display neutral or alkaline pH optima. Membrane-bound enzymes show acidic optima. The high oxidative capacity of Acetobacter is attributed to membrane-bound proteins such as alcohol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase and sorbitol dehydrogenase. The specific activities of these enzymes are up to three orders of magnitude higher than those of their cytoplasmic counterparts. Most membrane-bound enzymes share the prosthetic group pyrroloquinoline quinone (PQQ; Fig. 1). The substrates do not need to be transported into the cell for oxidation. Electrons generated are transferred by the reduced form of PQQ either directly to a ubiquinone ( Q S ) of the respiratory chain or via a cytochrome COOH I

HOOC

0

Figure 1 Structure of pyrroloquinoline quinone (PQQ).

c (subunit of some alcohol dehydrogenases) to the terminal ubiquinol oxidase which is either cytochrome al or cytochrome 0 . Energy is gained by these oxidations but they are not contributing to carbon metabolism. For instance, the oxidation of one mole ethanol to one mole acetic acid yields six moles of ATP. It is assumed that these systems function as ancillary energy-generating pathways when the energy demand of the cell is high. Nz-fixing cells of A. diazotrophicus contain three-times higher enzyme levels of quinoprotein glucose dehydrogenase than under non-N2-fixing conditions. Flavin (FAD) is an additional covalently bound prosthetic group present in the membrane-bound gluconate dehydrogenase. It is also linked directly to the respiratory chain. For intracellular sugar metabolism, Acetobacter possesses the hexose monophosphate pathway and a complete tricarboxylic acid cycle. Glycolysis is absent or rudimentary. In A. xylinum sugar metabolism proceeds (asin Gluconobacter)via the Entner-Doudoroff pathway. In addition to the typical growth substrates, such as sugars or ethanol, A. methanolicus also accepts methanol. The major assimilatory pathway of this facultative methylotrophic bacterium proceeds via the ribulose monophosphate cycle. In contrast, most of the Gram-negative methanol-utilizing bacteria that contain the ribulose monophosphate cycle are obligate methylotrophs. Some Acetobacter species, e.g. A. xylinum, synthesize bacterial cellulose. The fibres may be regarded as part of the glycocalyx and serve to maintain these highly aerobic organisms at the liquid-air interface. This exopolysaccharide, (f3-glul+4P-glu)n, is excreted into the medium and then rapidly aggregates as microfibrils yielding a surface pellicle. Bacterial cellulose is produced either in static cultures, or in submerged, fed-batch cultures with low share conditions. Yields up to 28 g 1-' of dry polysaccharide may be obtained after selection of high yielding strains. The product, cellulose I form, is very pure. It does not contain hemicelluloses, lignins or pectic substances, and is therefore used mainly in medicine as wound dressings for patients with burns, extended loss of tissue, etc. The majority of Acetobacter species have 1-8 plasmids varying in size from 1.5 to 95 kb. Isolates from some German submerse vinegar processes have 3-1 1 plasmids, isolates from surface fermentation processes 3-7 plasmids (2-70 kb). Plasmid profile analysis has become a powerful tool for controlling homogeneity, stability, identity etc. of the microbial populations in production processes. However, many strains used industrially are highly variable, changing their phenotypic and other properties in just a few generations.

ACETOBACTER 3

This phenomenon could not be correlated with plasmid profiles. Acetobacter contains four ribosomal RNA operons on the chromosome. Recombinant DNA techniques have been adapted to Acetobacter. Host-vector systems and transformation methods have been developed. Transformation systems are available for A. aceti and A. xylinum. For instance, bacteriophages specific for Acetobacter lead to a complete stop of submerged fermentation. Morphologically different phage types are described which were isolated from vinegar fermentations in Europe. They belong to the Bradley's group A and to the Myoviridae. The high number of phages in disturbed acetic acid fermentations suggests their responsibility for production problems. The classical, well known and well-studied, niches of Acetobacter are in making vinegar and spoilage of beer and wine (see below). Acetobacter spp. were originally associated with plants and soils. Preferred habitats, such as fruits and flowers, are rich in sugars, alcohols and/or acids. Fermenting fruits, in particular, are excellent sources of sugar and ethanol. Various Acetobacter spp. have been isolated from apricots, almonds, beets, bananas, figs, guavas, grapes, mandarins, mangoes, oranges, pomegranates, pears, peaches, persimmons, pineapples, plums, strawberries and tomatoes. A. aceti, A. xylinum and A. pasteurianus were predominantly associated with ripe grapes. Local priorities have been found: A. pasteurianus accounted for 75% of the strains in isolates from Southern France. Especially high numbers were found on damaged grapes. Acetobacter spp. have been isolated from the immature spadix of the palm tree. A. xylinum was present on the leaflets of the palm tree and in the surrounding air. Acidic acid bacteria, such as A. aceti and A. pasteurianus, hibernate in dried and injured apples and in spring they are able to spread to flowers. The nitrogen fixing A. diazotrophicus has settled the stem and roots of sugarcane in Brazil. Different Acetobacter spp. have been isolated from cocoa bean flora. Acetobacter is the causal agent of bacterial rot in pears and apples, resulting in different shades of browning and tissue degradation. Pears are more susceptible to bacterial brown rot. Acetobacter spp. have been isolated from decaying apple tissue and from the larvae and adults of apple maggots. Artificial inoculation of 100 cells may induce apple brown rot. Although the optimum temperature is about 25"C, rotting also proceeds at 4°C. The pink disease of pineapple fruit is caused by A. liquefaciens.Through the open flowers the bacterium enters the internal nectary and placental regions, invades the ovaries and starts to grow in the ripening tissue. The dilution of nectar by rain during flowering

is a prerequisite, because undiluted nectar is not a growth substrate.

Methods of Detection Strains of both Acetobacter and Gluconobacter are present in the same habitat. Members of the latter genus are normally co-isolated. For routine isolation of Acetobacter from natural or artificial habitats, culture media of low pH, containing 2-4% ethanol as energy source, are recommended. Aerobic growth is optimal between 25°C and 30°C. As low cell counts are expected, enrichment cultures become necessary. For such purposes beer has been recommended. However, preservatives added to the beer may limit success. Many specific enrichment cultures adapted to individual sources are described in older literature. Yeast water-glucose medium is recommended for isolation and purification. It contains yeast water (supernatant of autoclaved bakers' or brewers' yeast, 200 g I-'), and glucose (20 g I-'), p H 5.5-6.0. This composition is also very useful for the enrichment of solid media (agar concentration: 15-30 g I-'). Wort medium comprises malt powder diluted with tap water to 8% soluble solids; for solid medium the pH should be 5.5-6. Peptone glucose agar comprises bacto-peptone or bacto-tryptone ( 5 g I-'), glucose (20 g I-'), primary potassium phosphate (1g I-') in tap water; agar concentration: 15-20 gl-'. To enhance the growth of some strains, the addition of yeast extract (3-Sg1-') may be useful. Further enhancement may be achieved by the addition of 100ml of filtered and freshly prepared tomato juice to 1 litre of culture medium. An isolation procedure to differentiate between A. pasteurianus, A. aceti and Gluconobacter oxydans has been developed by Frateur, which uses three to five different culture media for each species. It includes enrichment in liquid media (30°C) and subsequent agar plating. Isolation of production strains from vinegar tends to be difficult due to the strains being highly adapted to the production conditions (see below). A mixture ( 4 ml) of vinegar to be tested and pasteurized vinegar is added to tubes with 15ml of solid medium (yeast water, 100 g I-' glucose, 30 g I-' CaC03, 20 g IF' agar). Bacterial growth proceeds in the interphase. Alternatively, yeast extract-calcium lactate agar or wort agar containing 1.5% ethanol or 5gl-l yeast extract, and 25 g I-' agar may be used. Acetobacter settling on flowers or fruits may be efficiently enriched in a broth containing 50 g I-' glucose, 10 g IF' yeast extract, and 0.1 mg I-' cyclohe--imide at 30°C. The ring or pellicle formed after 2-8

4 ACETOBACTER

days is plated out on a solid medium which may Table 1 Common media for maintenance and cultivation of Acetobacter also servc for further purification of the acid-forming Medium Component Amount colonies: 50 g I-'glucose, 10 g IF' yeast extract, 30 g I-' C a C 0 3 and 25 g IF1 agar. Glucose Glucose-yeast extract agar In cider making, various culture media are re('Acefobacter/G/uconobacter Yeast extract CaCO, agar') commended for successful isolation of acetic acid bacAgar pH 7.5 f 0.2, 25°C teria from orchard soil, apples, pomace, juice, Distilled/deionized fermenting juice, cider or from the factory equipment. water The media are based on apple juice-yeast extract Glucose made from apple juice with low tannin content, 'Acetobacter agar' Yeast extract pH4.8 and 3Ogl-l agar. The addition of 0.1 mgl-' CaCO, actidione is recommended to suppress yeasts and Agar Tap water moulds. Incubation is carried out at 28°C for 3-5 days. Alternatively, a broth composed of 500ml of 'Acetobacter medium' Glucose Autolysed yeast sweet cider, 500 ml deionized water, 12 g I-' yeast pH 7.0 f 0.2, 25°C CaC03 extract, and 2 g (NH4)#04( p H 5),yields good results. Agar Strains of A. diazotrophicus can be isolated by Distilled/deionized stepwise enrichment in different media, including water semisolid ones. Acetobacter strains may be held in or Mannitol Mannitol agar on a variety of media, such as beer (without preYeast extract (YPM agar: 12 g I-' agar) servation agents) or wort. Recommended conPeptone servation media are summarized in Table 1. Optimum Agar DistiIled/deionized growth is obtained at 25-30°C. Agar cultures should water be kept at 4°C and transferred monthly. Most strains Glucose stay alive lyophilized for several years and some for Yeast-glucose agar Yeast extract pH 7.0 f 0.2, 25°C longer than 10 years. Agar Phenotypic identification of Acetobacteraceae is Distilled/deionized based on general properties which are partially shared water with Gluconobacter and some members of the genus Glycerol Potato glycerol agar Frateuria (superfamily I1 syn. gamma subclass). One Glucose 25°C of the properties used to further identify Acetobacter Yeast extract Agar is the oxidation of acetate and lactate to COZ and Supernatant of H z O (overoxidation) at neutral and acidic pH. This freshly sliced and is detected on medium composed of ethanol ( 3 % ) , boiled potatoes C a C 0 3 (20 g I-'), and agar (20 g I-'). The appearance (200 g 1-1) of acetic acid reveals clear zones around the colonies and overoxidation results in (re-)precipitation of C a C 0 3 . Alternatively, bromcresol green (0.022 g I-') oxidation of acetic acid and lactic acid to C 0 2 and may be added to a medium composed of yeast extract HZO (30gl-'), ethanol (2%) and agar (2OgI-I). The colour n o H2S formation shifts from green to yellow as acid is formed. Overoxgrowth factors may or may not be required idation results in a change of the indicator to blue. specific ubiquinone types. Bacteria belonging to Acetobacteraceae may be The ubiquinones are Q9 and Q S with minor comGram-negative or Gram-variable (namely older cells), ponent of Q8( A . aceti, A. pasteurianus);some strains are strictly aerobic and oxidize ethanol to acetic acid have Qloor Qlowith minor component Q9 (A. methin neutral or acidic media. Cells are ellipsoidal t o rodanolicus, A. diazotrophicus, A. xylinum, A. shaped (0.6-0.8 x 1-4 pm), have a respiratory type of liquefaciens). Acetobacter strains grow at p H 5 and metabolism, are oxidase negative and acidify glucose prefer ethanol or lactate over glucose for growth. below p H 4.5. They d o not form endospores, liquefy Further differentiation among Acetobacter species can gelatin, reduce nitrate or form indole. The rapid be achieved as shown in Table 2. phenotypic idcntification of Acetobacter is based on The phenotypic identification may be affected by the following features: spontaneously occurring mutations even in taxonomically important properties. Mutants of A. aceti 0 peritrichous flagella exist that are unable to oxidize ethanol. In these cases

ACETOBACTER 5

Table 2 Phenotypical differences among Acetobacter species (Reproduced with permission from Swings 1992) Feature

Formation of Water-soluble brown pigments on GYC" medium y-Pyrones from D-fructose 5-Oxogluconic acid from o-glucose 2, 5-Dioxogluconic acid from o-glucose Ketogenesis from glycerol Growth factor required Growth on carbon sources Ethanol Methanol Sodium acetate Growth on L-amino acids in the presence of D-mannitol as carbon source L-Asparagine L-Glutamine Formation of H2S Growth in presence of 30% D-glucose Ubiqinone type G+C content (mol%)

A. aceti

A. liquefaciens

A. Pasteurianus

A. hansenii

A. A. A. xylinum methano- diazolicus trophicus

+ + -

+ d

-

+ + d d

-

Qg

56-60

+ + -

-

+ + -

-

Qio 62-65

Qs

~~

53-63

-

-

-

-

-

+ +

ND 58-63

Qio 55-63

Qio 62

Qio 61-63

~~~~~

-

~~~~

~

Symbols: +, 90% or more of the strains positive; (+), weakly positive reaction; d, 11-89% of the strains positive; -, 90% or more of the strains negative; ND, not determined. Glucose-yeast extract cycloheximide.

DNA-rRNA hybridization studies and polymerase chain reaction (PCR) studies will be more useful for exact taxonomic classification.

Importance to the Food Industry Food Processes

Acetobacter spp. are used in different processes of making foods and food additives. Well established are the fermentations to produce one special product, such as acetic acid or gluconic acid. These oxidation reactions are backed by the high oxidative capacity of enzymes bound in the cytoplasmic membrane with the active centre directed into the periplasm. Other processes also use such enzymes but are more complex with regard to the microbial population and the substrates used. Vinegar is the most popular product of Acetobacter. This process is discussed in detail in a separate article. In acetic acid fermentation, mixtures of highly adapted predominantly Acetobactsr spp. are used, which are not derived from pure cultures. These strains display an extremely strong tolerance to acetic acid. The most important detectable species are A . pasteurianus, A . lovaniensis, A . ascendens, A. paradox, A . aceti, A . xylinum and A . orleanensis.

Difficulties may arise during isolation, subsequent cultivation under artificial conditions, and preservation due to the high adaptation as demonstrated with A . polyoxogenes isolated in Japan and with A . acidophilus. Both could not be maintained in strain collections and were lost. A . europaeus isolated from production facilities in Switzerland requires acetic acid for growth on agar plates and tolerates 4-8'30. Specialized industrial strains tolerate p H values down to 2.6. DNA-DNA hybridization studies shows nearly no difference between isolates from different commercial processes (9O-lOO% hybridization). However, a comparison of highly productive strains from German plants and those from strain collections showed large differences. Values below 45% were obtained with definite strains ( A . pasteurianus, A . aceti, G . oxydans).The DNA-DNA similarities of A . europaeus strains isolated from industrial processes versus strains from collections are below 22%. Membrane-bound quinoproteins, i.e. alcohol and aldehyde dehydrogenases, are the enzymatic basis of acetic acid formation (Fig. 2). These enzymes are more active and stable under acidic conditions than those of Gluconobacter. Prevention of overoxidation of acetic acid to COz and H20 requires a constant high concentration of ethanol. Lack of ethanol and oxygen damage acetic acid bacteria populations. Even

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6 ACETOBACTER

naturally fermented by a high supply of air in up to 13 days. Yeasts, moulds and bacteria are involved. Ethanol produced by yeasts is oxidized by Acetobacter spp. Seeds are killed in the presence of acetic acid and by a temperature of 50°C. The fruit mass is then degraded and the typical flavour and brownish colour of the bean are developed. Nata is a dessert delicacy in Southeast Asia. This gelatin-like, firm, creamy-yellow to pinkish substance is composed of a form of cellulose formed by bacteria Ethanol +--;?--- 104cfu)per processed carcass, perhaps the best approach is to concentrate on devising methods which will ensure that the birds arrive at the processing plant with significantly reduced Campylobacter contamination. This focuses attention on the rearing conditions. Campylobacter is rarely found in poultry feed or the hatchery environment and generally colonizes the chicks only after the second or third week. The most likely vectors are flies, wild birds, rodents or contaminated water. Three main strategies are currently being employed: drinking water quality, vaccination and competitive exclusion. Certainly some authors contend that disinfection of drinking water is likely to have the greatest impact on the prevalence of Campylobacter spp. Passive immunization of chicks has resulted in reduced colonization by the organism but the cost-effectiveness of this approach still has to be determined. Competitive exclusion involves the administration early in the chick’s life of a cocktail of organisms that prevents subsequent colonization of the bird when challenged with Campylobacter spp. A

three-strain mixture comprising Klebsiella pnew moniae, Citrobacter diversus and Escherichia coli (013:H-) provided 43-100% (average 78%) protection.

Viable but Nan-culturable Forms Campylobacter cells, in common with other genera such as Vibrio, Salmonella and Shigella, have been shown to metamorphose into a viable but non-culturable (VNC) state when subjected to unfavourable conditions such as would be experienced in water, which generally has a low nutrient status. With Campylobacter the cells transform from a motile spiral form to a coccoid VNC form which is incapable of cell division in normal media entirely suitable for the normal culturable form. If the VNC form of Campylobacter is capable of initiating an infection in humans or colonizing the gut of domestic animals and poultry or indirectly via food contact surfaces, then contaminated water must pose a risk. There is still much controversy over the infectivity of VNC Campylobacter cells. It must be stated that such a phenomenon has been found with Vibrio cholerae and other related enteric water-borne pathogenic bacteria. Such authors suggest that the VNC form is a degenerative state and that there is a continuum of physiological states, with one extreme being highly culturable and the other dead cells. The VNC state is between these but tending towards the latter state. Certainly more research is needed to elucidate the role, if any, of the VNC formof Campylobacter in the transmission of disease and colonization of domestic animals and birds. See also: Campylobacter: Detection by Cultural and Modern Techniques: Detection by Latex Agglutination Techniques. Food Poisoning Outbreaks. Milk and Milk Products: Microbiology of Liquid Milk.

DEBARYOMYCES 515

D Dairy Products see Brucella: Problems with Dairy Products; Cheese: In the Market Place; Microbiology of Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific Groups of Bacteria; Microflora of White-brined Cheeses; Fermented Milks: Yoghurt; Products from Northern Europe; Products of Eastern Europe and Asia; Probiotic Bacteria: Detection and estimation in fermented and non-fermented dairy products.

DEBARYOMYCES W Praphailong, National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand G H Fleet, Department of Food Science and Technology, The University of New South Wales, Sydney, Australia Copyright 0 1999 Academic Press

Characteristics of the Genus and Relevant Species Species of Debaryomyces are commonly found in soils, waters, plants, foods and clinical specimens. Present taxonomic classification accepts 15 species although an additional within the genus (Table l), species Debaryomyces prosopidis has been proposed. Debaryomyces hansenii (imperfect form Candida famata) is, by far, the most significant species found in foods. Of the other species, there are only occasional reports on the isolation of D. polymorphus, D. etchellsii, D. maramus and D. carsonii from foods. Species of the genus undergo asexual reproduction by multilateral budding, with cells occurring singly, in pairs, short chains or small clusters. Pseudomycelium is usually lacking, but primitive or even well-developed pseudohyphae may be produced in some species. Sexual reproduction characteristically occurs by conjugation between a mother cell and its bud, but occasionally conjugation between separated cells is observed. Variation in the morphology and number of ascospores per ascus provides a good criterion for differentiation between the species. Ascospores are usually spheroidal to ovoidal in shape and are often distinguished by a warty or roughened surface. The ascospores of some species (e.g. D. occidentalis)have a distinct equatorial ledge. The number of ascospores per ascus varies from one to four depending on the species and, with the exception of three species, they are not usually liberated from the ascus. The ability to ferment sugars varies from an absent, weak to vigorous reaction, nitrate is not assimilated but strains of some species assimilate nitrite. Ubiquinone Q-9 is present and the diazonium blue B reaction

is negative. Lipid composition is characterized by the presence of linoleic (C18 : 2) and linolenic (C18 :3 ) fatty acids. The mol% G+C content is in the range of 33-43%. Karyotyping of the genus is not complete, but three to seven chromosomes arc generally present within the species. Inclusion of species within the genus has undergone significant revision over the years. The first major description of the genus by Lodder and Kreger-van Rij in 1952 included only five species, namely D. hansenii, D. kloeckeri, D. subglobosus, D. nicotianae and D. vini. Lodder’s 1970 classification, based largely on fermentation and assimilation tests, combined the first four of these species into one species D. hansenii, and introduced seven new species, D. castellii, D. coudertii, D. marama, D. phaffii, D. cantarellii, D. tamarii and D. vanriji. Price and coworkers in 1978 proposed a major revision of species within the genus after a detailed study of DNA sequence similarity by reassociation/hybridization kinetics. In particular, they showed that several species of Pichia were related to some Debaryomyces species. Consequently, D. cantarellii and D. phaffii were merged with Pichia polymorpha to become D. polymorphus. Pichia pseudopolymorpha became D. pseudopolymorphus. Using data from partial sequencing of ribosomal RNA subunits, Kurtzman and coworkers and Yamada and co-workers have further refined the description of the genus, and this has given the current recognition of 15 species (Table 1). A notable outcome from these studies was the close similarity of Schwanniomyces occidentalis with Debaryomyces species and its redefinition as D. occidentalis with two varieties. However, some authors do not agree with classification of Schwanniomyces

Table 1 Key properties" of species within the genus Debaryornyces Species

Mol% G+C

Fermentation 37°C

NaCl

Vit

G

su

Assimilation Tr

su

La

Ascospores Me

Ra

XY

V

V

+

GI

Er

Shape

NurnbeP LibC

Spheroidal

1-4

Spheroidal Spheroidal Spheroidal

1-3(1)

(1 0%) D. carsonii D. castellii D. coudertii D. etchellsii

36.839.7 37.1 37.4 38.5-

+

+

+

-

+

+ + +

-

+

-

wl-

wl-

wl-

+

V

V

+

+

X

V

Spheroidal

1-2(1)

+

+

-

wl-

wl-

wl-

+

V

V

+

+

X

V

Spheroidal

1-2(1)

-

+

-

wl-

V

V

S

+ +

+

+

-

-

-

+

-

-

-

+

-

wl-

+

V

+

+

+

+

+

Ovoidal Spheroidal Spheroidal

142) 1-4 1

+

-

-

+

+

+

V

V

+

+

+

+

+

+

V

+

-

+

V

V

+

+

+

+

Spheroidal (L) 1-2(1) 1-2(1) Spheroidal

+

+

+

+

+

+

Spheroidal

1-4

-

+

+

+

+

+

+

+

+

Spheroidal Spheroidal

1-4 1-4

+

+

+

+

Spheroidal

1-4

+

+ +

W

V -

+

+

Spheroidal Spheroidal Globose

14 1-4 1-2

-

-

+

-

-

-

+

+

-

-

X

wl-

+

+

+

+

-

+

+

+

1 1-4

40.6 D. hansenii var. hansenii var. fabryi D. rnararnus D. melissophilus D. nepalensis

37.338.6 36.436.8 39.1 39.8 37.638.0

D. occidentalis var. 35.2 occidentalis var.persoonii 35.4 D.polyrnotphus 35.735.9 D. 35.7 pseudopolyrnorphus D. robertsiae 42.7 D. udenii 35.8 D. vanrijiae var. vanrijiae 33.233.3 var. yarrowii 33.0 D. yarnadae 34.5 D. prosopidis 37-38

+ +

-

-

+

+

+

+

+

S

S

+

-

+

-

+

S

+

-

+

+

+

+

+

-

ws

wl-

wl-

wl-

-

+

+

+

+

wl-

-

+

+

wl-

-

S

-

+

+ +

-

+ +

+

+

+

+ + +

+ +

+

+

+

Spheroidal (L) 1-2(1) -

Table adapted from Nakase et al(1998) with permission from Elsevier Science. "Abbreviations: 37"C, growth at 37°C; NaCl (lo%), growth in 10% NaC1+5% glucose; Vit, growth in vitamin-free medium; G, glucose; Su, sucrose; Tr, trehalose; La, lactose; Me, melibiose; Ra, raffinose; Xy, D-xylose; GI, gluconate; Er, erythritol; +, positive; s, positive but slow; x, positive or weak; w, weak; ws, weak and slow; wl-, weak or negative; v, variable; -, negative. bNumbersof ascospores per ascus; numbers in parentheses refer to the number of ascospores most frequently observed. (L) indicates ascospores with equatorial ledge. "Ascospores liberated by lysis of asci.

-

+

DEBARYOMYCES 51 7

occidentalis within the genus Debaryomyces because

membrane bound ATPase that accomplishes an effective extrusion of Na- ions. K'ingea robertsii was described as D. robertsiae. With the exception of D.hansenii, little is known Other changes were the description of two former about the environmental factors which limit the species of Pichia as D. carsonii and D. etschellsii and growth of species listed in Table 1. Other than D . the removal of D. tamarii. The key properties that occidentalis, all grow in the presence of 10% NaCI. differentiate species within the genus are shown in D. hansenii and D. etchellsii, at least, are also tolerant Table 1. of very high concentrations of sugars and grow in the sucrose. Growth of D. hansenii presence of 60% (wh) is very weak at p H 2 . 5 but strong in the p H range 3.0-8.0. Many authors have made the qualitative Physiological and Biochemical Properties observation that D. hansenii exhibits faster growth at Of the 15 species in the genus, only Debaryomyces l-5OC compared with other yeast species, and there hansenii and D. occidentalis have attracted significant is a report of growth at -12.5"C. D.hansenii is not study of their physiological, biochemical and molecu- particularly tolerant of preservatives or heat treatlar properties. These studies reflect the substantial ment. It is inhibited at p H 5 . 5 by 250-500mg1-' of diversity in growth and metabolic behaviour of yeasts benzoic or sorbic acids and has a D value of 1 2 m i n within the genus. at 48". Some strains have a strong tendency to flocDebaryomyces hansenii is considered to be non- culate and this could be a potential survival mechfermentative. It metabolizes sugars to pyruvate by the anism in hostile environments. In contrast t o D. hansenii, D. occidentalis is a Embden-Meyerhof-Parnas (EMP) pathway and then oxidizes pyruvate through the tricarboxylic acid vigorous fermenter of sugars under non-aerated con(TCA) cycle. Organic acids such as citric, lactic and ditions. It is a Crabtree negative yeast and, under succinic are assimilated through the TCA cycle. The aerated conditions, it channels the sugars into the pentose phosphate pathway also operates in this TCA cycle. Unlike D.hansenii, this species is not yeast. Contrary to the general view, there are reports particularly tolerant of high s;lt or high sugar envirof some strains of D . hansenii and C. famata that onments. The most distinctive property of D. occiferment glucose and other hexoses. Extracellular pro- dentalis is its efficient degradation of starch by the tease and lipase production have been reported in production of extracellular a-amylases and a glucosome but not all strains. These enzymes have not been amylase that can by-pass the a-(l+b)-linked branch isolated and characterized. Amylolytic and pec- points in amylopectin. Because of this property, there tinolytic activities are absent. The most distinguishing has been substantial scientific and industrial interest feature of D. hansenii is its ability to grow in the in this yeast. The kinetics of production and properties presence of extremely high concentrations of salt of these amylolytic enzymes have been well char(KaC1).Although the growth response to NaCl varies acterized and their genes have been cloned and with the strain, most grow in the presence of 15% sequenced. Techniques for manipulating the expres(wiv) NaCl and there are some strains that grow at sion of these genes and for transferring them to other 20-24% (wiv) SaC1. High salt tolerance has also yeast species have been developed. been reported for D. etchellsii. Salt tolerance of D. hansenii is greatest at p H values near 5.0 and Significance in Foods decreases at p H 3.0 and p H 7.0. The molecular basis of salt tolerance in D.hansenii has been extensively Literature on the occurrence of Debaryomyces species studied and is related to the ability of this yeast to in foods is largely unfocused and scattered over many accumulate high intracellular concentrations of gly- years. It is difficult to track because of the numerous cerol as an osmo-protectant or compatible solute. changes of name of the species. Most studies concern Substantial amounts of this glycerol are excreted into D . hansenii and there are only occasional reports on the extracellular medium, especially during the sta- the occurrence and significance of other species, such tionary phase, but it is re-utilized when glucose sub- as D. etschellsii, D. polymorphus, D . maramus and strate is exhausted. The pathway of glycerol D. carsonii in foods (Table 2). There is no reason to production has been studied and it originates from explain why the other species listed in Table 1 (e.g. glucose by the EMP pathway. Intracellular arabitol is D. occidentalis) are not found in food ecosystems, also accumulated and excreted, but its production (via but more systematic and focused study will probably the pentose phosphate pathway) appears constitutive reveal their presence. The early literature reveals the frequent isolation of and occurs in the absence of salt stress. It has been suggested that D. hansenii also has an appropriate D. hansenii from meat products, especially processed it has some quite distinct phenotypic properties. Also

51 8 DEBARYOMYCES

Table 2

Significance of Debaryomyces species in the food and beverage industries

Speoes

Significance

Debaryomyces hansenii (Candida famata)

Occurrencelspoiiage: delicatessen, cured, fermented, minced meats; seafoods, fish sauces; yoghurts, cheeses; brined vegetables; mayonnaise-based salads; silage Biotechnological: starter cultures for meat sausage fermentation; starter cultures for maturation of cheeses; biocontrol agent of bacterial and fungal spoilage; xylitol production Occurrencelspoiiage: carbonated soft drinks; sugar syrups; brined vegetables: mayonnaisebased salads, soy sauce; fermented meat products Occurrencelspoiiage: carbonated soft drinks; fruit products delicatessen, cured and fermented meats Occurrencelspoiiage: meat products; cheese Spoilage: salted fish paste 6iotechno/ogicai: amylase production; waste utilization; single-cell protein

Debaryomyces etscheilsii Debaryomyces polymorphus Debaryomyces maramus Debaryomyces carsonii Debaryomyces occidentaiis

products, such as frankfurters, bacon, hams and fermented and unfermented sausages. In some cases, presence of the yeast was associated with the development of a slimy surface layer on the product. Recent, more extensive studies have confirmed the predominance of D. hansenii in meat products compared with other yeasts, and these conclusions have been extended to include seafoods such as fresh fish. Populations in the range 104-10hcfu g-' (or even higher in fermented salami) are often reported. D. etschellsii, D. polymorphus and D . maramus are also found in these products, but less frequently. The impact of this yeast growth on the flavour of meat products is not clear, but cannot be assumed to be negative. Indeed, there is a positive correlation between the desired flavour of some Italian salami sausages and the presence of D. hansenii. Some, but not all, of the strains of D.hansenii isolated from meat products produce extracellular proteases and lipases that could contribute to flavour development by the breakdown of meat proteins and fats. The ability of these enzymes to operate well at low p H may be an appealing property. Consequently, consideration has been given to the use of selected strains of D. hansenii as starter cultures in the production of fermented sausages. Factors thought to select for the growth of D. hansenii in meat products include its tolerance of salt, utilization of organic acids (e.g. lactic), protease and lipase production, good growth at low temperatures, and the ability of some strains to utilize sodium nitrite which is added as a curing agent in some products. D. hanseniilC. famata have now emerged as the most important yeasts in the dairy industry. Weakly fermenting species have been linked to the spoilage of yoghurts, but their greatest significance is in cheese production, especially with the mould-ripened soft cheeses such as Camembert, Brie and blue-veined varieties. Many surveys of these and other types of cheeses have revealed a consistently high incidence of D. hansenii, often at populations of 106-10'cfug~' or

higher. The yeast originates as a natural contaminant of the cheese brine and grows at both the outer and inner parts of the cheese curd during the maturation stage. Again, the ability of the yeast to tolerate the high salt environment of the cheese, utilization of lactic acid, protease and lipase production, growth at low temperature and, possibly, production of polyols such as glycerol are key factors that favour its growth and contribution to the biochemistry of cheese maturation. A clear link between such activity and a sensory outcome remains to be established, but the relationship is assumed to be positive since commercial starter cultures of D . hansenii are available for encouraging the maturation process. An important property of these strains might be the ability of their proteases and lipases to operate at high salt concentrations and low temperatures. Debaryomyces species, especially D. etschellsii, are frequently isolated from brines used to ferment products such as olives and cucumbers, and they are also associated with traditional Japanese fermented products such as soy sauce and miso. Curiously, a high proportion of killer strains of D. hansenii with broadspectrum killer activity has been isolated from the latter ecosystems. Presumably, these yeasts grow on the surface of brine solutions, utilizing lactic acid produced by the lactic acid bacteria involved in these fermentations. However, some strains of D . etschellsii also ferment sugars. This latter property may also explain the occasional association of D. etschellsii with the spoilage of high sugar syrups. There are occasional reports of the isolation of Debavyomyces species from soft drinks, beer, wine and vegetable salads but, generally they are not significant spoilers of these products. An unusual but significant form of spoilage has been reported for D . cavsonii, which grew in a Japanese chickuwa fish paste, transforming trans-cinnamic acid to styrene which gave the product an unacceptable petroleum-like aroma. 4 s noted already, the association of D. hansenii with foods does not necessarily have negative impli-

Next Page DEBARYOMYCES 519

cations and there is significant interest in exploiting this species as a starter culture in meat and cheese production. In the case of cheese production, it has been reported to have good biocontrol over spoilage species of Clostridium. Also in the context of biocontrol, several papers in the late 1980s suggested that D.hansenii was an effective natural antagonist for controlling the fungal spoilage of various fruits by species of Penicillium, Botrytis and Rhizopus. However, later study revealed that the yeast is an unusual strain of Candida guilliermondii and not D. hansenii. Nevertheless, this work has stimulated interest in the yeast as a potential novcl biocontrol agent. In another industrial application, D. hansenii has potential value in bioconversion of xylose into the sweetener, xylitol. The enzymes, xylose reductase and xylitol dehydrogenase, associated with this process have been examined. The amylases of D . occidentalis have potential application in the production of sugar syrups for food and beverage processing. The genes for these amylases have been incorporated into brewing strains of S. cerevisiae for the purpose of using these strains in the production of low calorie or dextrin-free beers. D. occidentalis could be used to process starchy waste material into single-cell protein. Debaryomyces spp. are not generally regarded as pathogenic to humans and no food-borne disease outbreaks have been attributed to these organisms. However, D. hanseniilC. famata have been implicated in isolated cases of septicaemia and skin and mucosal surface infections where they are considered as weak opportunistic pathogens, especially for immunocompromised patients.

fore, it will be necessary to isolate and identify individual colonies. The identification of Debaryomyces spp. follows standard morphological biochemical and physiological tests and keys as outlined in The Yeasts, a Taxonomic Study, 4th edition, edited by CP Kurtzman and JW Fell, Elsevier Science (1998) (Table 1). D.hanseniilc. famata, a t least, identifies very well in the rapid computer-based Biolog (Biolog Inc California) and ATB 32C (bioMtrieux) systems that incorporate a range of these tests in kit form. To avoid potential osmotic shock and stress, it has been suggested that 5-10% NaCl be included in the dilucnt and plating medium when isolating these yeasts from high salt foods. However, wc and others have not found these steps to offer any benefit. No selective or differential media have been reported for these yeasts. However, inclusion of 101 5 % (wlv) NaCl into the medium formulation could assist in selecting for the growth of these species, except D. occidentalis. A differential medium based on the hydrolysis of starch could be developed for the isolation of D. occidentalis. A PCR method that differentiates D.hanseniilC. famata from Candida guilliermondii has been reported and is based on amplification of the large subunit rDNA between base positions 402 and 669. A D. hansenii nucleic acid probe based on sequences in the 18s rRNA has been reported. As yet, neither of those molecular methods has been developed for routine use. See also: Fermented Foods: Fermentations of the Far East. Fermented Milks: Yoghurt. Meat and Poultry: Spoilage of Cooked Meats and Meat Products.

Enumeration and Identification Food sample ( l o g ) is suspended in 90ml of 0.1% peptone water, homogenized for approximately 1min and then diluted 10-fold, as necessary, in 0.1% peptone water. Aliquots (0.1ml) of the dilution are then spread inoculated over the surface of plates of media such as malt extract agar, glucose-yeast extract agar or tryptone glucose yeast extract agar. Bacterial antibiotics, such as chloramphenicol, oxytetracycline, chlorotetracycline, gentamicin and streptomycin can be added to these media at concentrations up to 100 pgml-' to suppress bacterial growth. For the isolation of Debaryomyces from products like cheese, overgrowth of moulds (Penicilliumspp.) on the plates can occur. Incorporation of the mould inhibitor, biphenyl (50 mg 1-') into the medium can overcome this problem. Plates are incubated a t 25°C for 4-7 days and yeast colonies counted. Virtually all yeast species will grow on the media just described. There-

Further Reading Andrews S, de Graaf H and Stamation H (1997) Optimisation of methodology for enumeration of xerophilic yeasts from foods. International Journal of Food Microbiology 35: 109-1 16. Dillon VM and Board KG (1991) Yeasts associated with red meats. Journal of Applied Bacteriology 71: 93-108. Dohmen RJ and Hollenberg CP (1996) Schwanniomyces occidentalis. In: Wolf K (ed.) Nonconventional Yeasts in Biotechnology. A Handbook. P. 117. Berlin: SpringerVerlag. Girio FM, Pelica F and Amaral-Collae MT (1996) Characterisation of xylitol dehydrogenase from Debaryomyces hansenii. Applied Biochemistry and Biotechnology 56: 79-87. Kosse D, Ostenrieder I, Seiler H and Scherer S (1998) Rapid

detection and identification of yeasts in yogurt using fluorescently labelled oligonucleotide probes In: Jakobsen M, Narvhus J and Viljoen BC (eds) Yeasts in the

ECOLOGY OF BACTERIA AND FUNGI IN FOODWlnfluence of Available Water

539

ECOLOGY OF BACTERIA AND FUNGI IN FOODS Contents

Influence of Available Water Influence of Temperature Influence of Redox Potential and pH

Influence of Available Water K Krist, Meat and Livestock Australia, Sydney, Australia

D S Nichols and T Ross, School of Agricultural Science, University of Tasmania, Hobart, Australia Copyright C 1999 .Academic Press

Introduction

Although there is a variety of resting or survival stages of microorganisms that are resistant to drying, all organisms need water to remain metabolically active. The availability of water to a n organism in an environment is not simply a function of how much water is present, but the degree to which it is adsorbed to the insoluble components of the environment or chemically associated with solutes in that environment. For this reason, the concept of water activity ( a & ) ,a measure of the availability of water to participate in chemical reactions, was devised. Though a,, is not a perfect predictor of the behaviour of microorganisms in a specified environment iknowledge of the solutes and factors that contribute to the a , is also required), it is widely used to describe the relationship between the water in a n environment and its microbial ecology. The reduction of a,+to increase the microbiological stability of foods has probably been used since antiquity. The drying effect of the air and the sun required no special technology and is still used today. Similarly, the addition of salt or sugars requires no special technology and has been used for centuries to preserve food. Those techniques are still in use in many parts of the world, using free energy and providing safe products. More recently, technology ie.g. hurdle technology) has sought to maximize the potential of drying techniques while minimizing the severity of treatments to develop shelf-stable products that are less altered from the fresh state.

This article considers the microbial ecology of bacteria and fungi in relation to a,. a, and related terms, are defined. Methods for manipulating a,, in foods are discussed, and the effects of a,, on growth rate, lagphase duration, jield and death rate of microorganisms described. The physiology of the response of microbial cells to a,, stress is also discussed.

Concept of Water Activity/Available Water Water activity can be affected by both solutes and adsorption. The solutes effect is called osmotic potential. The adsorption effect is called matric water potential but it is not widely considered in food microbiology. None the less, insoluble materials such as wood, paper, metal and glass, and including foods, adsorb water. The strength of the attachment is a function of the physical and chemical properties of the material. Those materials will tend to take water up from, or release water to, the atmosphere until a n equilibrium is reached between the atmosphere and the material. Foods will tend to equilibrate with the relative humidity of the container or environment they are stored in. Thus, dry foods can take up water from humid environments, o r moist foods will tend to dry out in dry environments. If a food is allowed to equilibrate with the humidity of the storage atmosphere, the matric a,. will affect the organism just as if the osmotic a,. had been altered to the same relative humidity. The terms water activity, water potential, osmotic pressure and solute concentration are often used interchangeably by microbiologists to refer to the availability of water to microorganisms. Although each of these concepts is related, they are different. Solute concentration is self-explanatory, although it may be expressed in different ways (e.g. w h , w/v, molarity, molality, etc.). High solute concentrations result in decreased a,, , and less water available to micro-

540

ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water

Increased osmotic pressure literally means that the cell is subjected to an increased external pressure, or alternatively, a decreased internal pressure. Increased Water Activity extracellular osmotic pressure refers to a situation A,%is a fundamental property of aqueous solutions. It where the availability of water to bacteria is decreased. is defined as: The term water potential, widely used by soil microP (Equation 1) biologists, also expresses the availability of water, a ,-but is defined as the difference in free energy of the Po environment being considered, and a pool of pure where p = vapour pressure of the solution; po = vapour water at the same temperature: the terms water activpressure of the pure water under the same conditions ity and water potential are measures of the energy of of temperature, etc. And where: water. Water potential may be expressed in a number of units, of which the most widely used is the bar P = relative humidity ( IO6dyn cm-2). Water potential is always a negative Po value or zero. As shown in Table 1, a,$ and water potential are The a,b of most solutions is temperature-dependent. Equilibrium relative humidity, a measure widely used not directly proportional, however, a 0.01 decrease in in meteorology and building environmental control, a , corresponds to a decrease of approximately 15 bar water potential in the range of a , typical of foods. is related to a, by the simple expression: Tables of a, for various solutes and solute mixtures Equilibrium relative humidity ( % ) a, = (Equation 2) are available in the literature. The effect on a, of 100 solutions containing several solutes can be estimated When solutes are dissolved in water, some of the from the concentration of each solute, using the folwater molecules become more ordered as they become lowing formula: oriented on the surfaces of the solute molecule. This x ...............X awn (Equation 5 ) reduces the vapour pressure of the solution, since on a,tota, = a , l x aw2x average the water molecules then have less entropy. In turn, a,v is reduced. The a,v of a solution decreases where a,,l, a,$2,a,,3, a,,, are the a, calculated from the with increasing solute concentration. The effect of concentration of each solute independently. solute concentration on a., is expressed mathThis equation can readily be applied to liquid foods, ematically: e.g. broths, juices and syrups and can also be used for solid foods by determining the concentration of [Equation 3 ) solutes in the aqueous phase. Water potential, y ~ ,is related to water activity by the equation: where v = the number of ions generated by each molorganisms for metabolism. Solutes that alter a, are termed humectants.

ecule of solute (e.g. for non-electrolytes, v = l; for NaC1, v = 2 ; for HzS04, v = 3 ) ; m=molaI concentration of the solute; cp = molal osmotic coefficient. Equation 3 reveals that the a , at a given solute concentration is dependent on the specific solute, because each solute has its own osmotic coefficient and will dissociate into a different number of ions.

(Equation 6 )

where M = t h e molecular weight of water (0.018 kgmol-l) and all other terms are as previously defined.

Osmotic Pressure

The osmotic pressure of a solution is related to its a, and includes this term in its definition: Osmotic pressure =

-RT In a, V

[Equation 4)

where R = the universal gas constant (8.314Jk-lmol-'); T =absolute temperature (K); V = partial molar volume of water and all other terms are as previously defined.

Factors Affecting Water Activity Addition of water or removal of solutes can increase a, In food microbiology, however, one is usually interested in reducing a,%, to improve the microbiological stability of the product. The a,$ of an environment can be reduced by the addition of solutes, or water binding substances that decrease matric water potential, or by the removal of liquid water.

ECOLOGY OF BACTERIA AND FUNGI IN FOODS/Influence of Available Water

541

Table 1 Comparison of water activity (a,) and water potential ( y )values and concentration of solutes required to achieve them a /:

Water potential (bar)'

NaCl concentration (g /-'I

Sucrose (9 i-')

concentration

0.995 0.980 0.850 0.843 0.753 0.577 0.328 0.113 0.1 00

-7 -28 -224 -235 -390 -757 -1534 -3000 -31 68

8.7 35 190

92 342 2050 (saturated)

Other solutes (a,,/ at 25°C) (9 I-')

KCI (saturated. 357) 260 (saturated) NaBr (saturated, 909) MgClz (saturated,l667) LiCl (saturated. 769)

al bar = -1 00 J kg-'

Freezing

Liquid water can be removed, in effect, by freezing. The preservative effects of freezing are due not only to temperature depression, but also to the effect of decreasing in the remaining liquid water. As the water in the food freezes it increases the effective concentration of solutes in the remaining liquid water. Those organisms remaining in the liquid phase are exposed to increasingly severe osmotic challenge as freezing proceeds. The same ecological challenges apply to bacteria naturally present in Arctic and Antarctic environments. The physiology of the organisms naturally present in those extreme environments is instructive for understanding the effects on microorganisms present in frozen foods and is discussed briefly later. Drying

The removal of water by evaporation also increases the concentration of the solutes in the remaining water. As described below, the effect on a, of the remaining free water will depend on the level and type of solutes initially present. Specific Solutes

The a,-modifying effects of several different solutes are shown in Table 1. Addition of solutes increases the osmotic potential of the water. As suggested by Equation 3, the effect of specific ionic solutes is related to their concentration, the number of ions that the molecule dissociates into, its dissociation constant, and also specific properties of the solute. Son-ionic solutes also reduce water activity. Generally, IiaC1, KCI, glucose and sucrose shon7 similar patterns of effect on microbial responses while glycerol usually permits growth at lower a,$, although there are specific exceptions, e.g. Staphylococcus auyeus is more inhibited by glycerol than NaCI. Glpcero1 differs from other solutes in that it is able to permeate the cell freely. NaCl is somewhat unique in terms of humectants

in that the ionic species Na' is also a primary ion in cell function. Symporters are proteins that transport selected substances across the cell membrane, in a manner dependent on the co-transport of a second substrate in the same direction. A number of symporter systems are Na+-driven. Cytoplasmic levels of Na' are also tightly regulated in most species, and fluctuating external Na' levels challenge microbial cells beyond the osmotic effect of a,,. Much of the research in this area has been conducted using bacteria; however, the general principlcs also hold for fungi. Within Escherichia coli, an active extrusion mechanism is responsible for the regulation of intracellular Na' concentration which enters via symporter systems. The primary mechanism consists of a series of membrane-associated transport proteins known as antiporters. As protons flow into the cell (through the antiporter channel) along the concentration gradient established by respiratory chains, Na' is extruded from the cytoplasm. Many marine and anaerobic bacteria rely heavily on Na' cycling, with additional Na*translocating respiratory chains and ATPases responsible for Na' removal from the cell interior. Most, if not all, symporters in these bacteria are coupled to Iia' influx. The linkage between Na'/H- antiporters results in an increased interaction between p H and NaC1 in marine and anaerobic bacteria, so that their growth tends to be increasingly inhibited by S a C l as the p H of the medium increases. This is an example of specific effects of the humectant itself other than its direct effect on a,.

Levels in np i c a l Foods Representative a, of food5 are shown in Table 2. Foods range from those with very little free water (freeze-dried products, cereals, powdered products) to almost completely free water ie.g. fresh meat and produce, bottled water products). hlost fresh produce has a,, close to 1.00 if the tissues are cut but may have

542

ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water

Table 2 Representative water activity of foods Food

Typical water activity

Milk, fruit, vegetables Fresh meat, fish Cooked meat, cold smoked salmon Liverwurst Cheese spread Caviar Bread Salami (dry) Soft, moist pet food; chocolate syrup Fruit cakes, preserves, soy sauce Salted fish, honey Dried fruit Dried milk (8% moisture) Cereals, confectionery,dried fruit, peanut butter Ice at -40°C Dried pasta, spices, milk powder Freeze-dried foods

0.995-0.998 0.990-0.995 0.965-0.980 0.96 0.95 0.92 0.90-0.95 0.85-0.90 0.83 0.80 0.75 0.60-0.75 0.70 0.70-0.80 0.68 0.20-0.60 0.10-0.25

significantly lower surface water activity, e.g. on intact fruits and vegetables due to the presence of the cuticle. Meat carcass surfaces can also dry during processing, lowering the a, sufficiently to inhibit microbial activity greatly. Thus, it is important to know not only the type of food but also the form and packaging that it is in to understand the microbial ecology.

inhibitory effect on microbial metabolism than nonionic solutes (e+ sugars). Range of Growth

Each microorganism has a minimum and maximum a, for growth. For many species, the maximum a, for growth is effectively 1.000. Although growth could not occur in pure water, some organisms are able to grow in the presence of very low levels of nutrients. Pseudomonads, and even algae, are able to grow in some types of bottled water, indicating the need for techniques to eliminate viable organisms from these products during production. A range of terms used to describe the response and tolerance of microorganisms to a, and specific solutes is shown in Table 3. Table 3 Classification of microorganisms according to their preferred water activity range for growth Nomenclature

Water activity range for growth

Haloduric

Able to withstand, but not grow at, high concentrations of salt Requiring salt for growth Requiring 1 5 2 0 % salt for growth

Halophile Extreme halophile Osmotolerant Osmophile

Able to withstand, but not grow at, high concentrations of sugar Organisms that grow best, or only, under high osmotic pressure, due to sugars Requiring reduced water activity (as distinct from requiring high osmotic pressure)

General Reactions of Bacteria, Yeasts and Mycelial Fungi

Xerophilic

Most microorganisms are active over only a relatively narrow range of a, and a," differences in the order of 0.001-2 are significant on the microbial ecology of an environment. Thus, a, values in food microbiology arc normally quoted to three significant figures. Gram-negative bacteria, typically, are only able to grow in environments of a, greater than about 0.95. Many Gram-positive bacteria can withstand a, as low as about 0.9, but few can grow at a, lower than 0.8. Some, specifically adapted to life in hyper-saline environments, are active at a, as low as 0.75 and might be found, e.g. in dried salted fish. Fungi are generally more tolerant of reduced a, than are bacteria. Some yeasts and moulds are able to withstand a, as low as 0.60. Growth rates of bacteria are typically faster than those of eukaryotes. Thus, despite the fact that many yeasts and moulds are able to grow on foods of high a, such foods are usually rapidly dominated and spoiled by bacterial contaminants. Fungi have a selective advantage at lower a, and are more usually associated with the spoilage of reduced a, products, e.g. bread, cheese, jams, syrups, fruit juice concentrates, grains, etc. As indicated above, the effect of a, depends on the major solutes responsible for the reduced a,. Ionic solutes (salts) have a greater

The a, range of growth is solute-dependent. Many bacteria, for example, are more tolerant of reduced a, if the solute is glycerol. This characteristic is not, however, universal. Tolerance to a, is greatest when all other factors in the environment are optimal for growth. As other environmental factors become less optimal the range of a, that supports growth is reduced. Examples are presented in Figures 3 and 5 of the related entry 'Predictive microbiology'. The effects, however, are not always intuitive. Representative tolerance ranges under otherwise optimal conditions for various microbial groups are shown in Table 4. Combinations of Factors

It is common in some foods for a variety of factors to be used to control microbial growth. This approach exploits the interaction of a, and other physicochemical parameters such as temperature and pH in food environments. Such interactions form the basis of the hurdle concept. The physico-chemical factors of NaCl and temperature have a close interaction, with the temperature range for growth of most organisms displaying a dependence on salinity. In general,

ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water 543

0.012

Table 4 Representative tolerance ranges for various microbial groups and species

0.010 Organism or group

Lower a, limit (Solute)

(Most) Gram-negative rods Escherichia coli Pseudomonas fluorescens Pseudomonas fluorescens Vibrio parahaemolyticus Vibrio parahaemolyticus

0.95-0.96 (NaCI) 0.95-0.955 (NaCI) 0.97 (Sucrose) 0.96 (NaCI) 0.96 (Glucose) 0.93 (NaCI)

(Most) Gram-positive bacteria Listeria monocytogenes Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Bacillus cereus Bacillus cereus Bacillus cereus

0.90-0.94 (NaCI) 0.92-0.93 (NaCI) 0.89 (Glycerol) 0.87 (Sucrose) 0.86 (NaCI) 0.95 (Glucose) 0.94 (NaCI) 0.92 (Glycerol)

Yeasts Zygosaccharomyces rouxii Saccharomyces cerevisiae

0.65-0.92 (NaCI) 0.65 (Sucrose) 0.90 (Sucrose)

Moulds Penicillium chrysogenum Wallemia sebi Eurotium spp.

0.65-0.90 (NaCI) 0.80 (KCI, glucose) 0.75 (Glycerol) 0.66 (Glucose and fructose)

Algae Most groups Dunaliella

0.75-0.90 0.90-0.95 (NaCI) 0.75 (NaCI)

reduced a, confers enhanced heat resistance on microbial cells. The basis for this behaviour is perhaps due to the cross-protection that osmotic stress affords against temperature stress, believed to be mediated by a general stress response under the control of the Rpos gene. (Interestingly, if grown at suboptimal salinities, a number of marine bacteria exhibit a lowered maximal temperature for growth compared to growth at the optimal salinity.) The minimum temperature for growth for many food-borne organisms is, however, increased by decreasing a,.,. This raises the intriguing possibility that the basis of these effects lies in the energy of the water itself, i.e. if the kinetic energy of water molecules mediates the lethal effect of temperature, then the reduction of water energy by solutes may have the same effect as reducing temperature. The growth rate response of microorganisms to water activity is illustrated in Figure 1. Growth rate increases in proportion, approximately, with increasing a,, above the minimum a, for growth, and up to an optimum growth rate value. Beyond this value the growth rate declines, usually rapidly as a function of increasing a, until the maximum a, is reached. Growth rate is a characteristic of the environment, and is not affected by the previous history of the cell, unlike lag time. The effect of a, on growth rate is affected by the specific humectant.

a,

I

e

0.008

2 0.006 2 (3 0.004

0.002

0.000 .... 0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1.00 Water activity

Figure 1 Effect of water activity on the growth rate of bacteria. Curves A and D represent two organisms, each adapted to a different water activity range for growth. Curve B represents the effect of a second suboptimal environmentalfactor on the growth rate of organism A. The water activity range is unaltered, the relative response remains the same, but the absolute growth rate is reduced at all water activities. Curve C represents the effect of a different, non-ionic solute (or humectant) on the growth response of organism A. That humectant permits A to grow over a wider range of water activities. After Ross T (1999) Predictive Food Microbiology Models in the Meat Industry. Sydney: Meat and Livestock Australia.

There is no specific correlation between a, tolerance and tolerance to other environmental factors. Thus, the manipulation of a, in a product could have different consequences for the microbial ecology of the foods at different temperatures. An illustration of the selective effect of temperature and a, on different organisms is presented in Figure 2. Lag, Germination and Sporulation, Toxin Production

The lag time is generally considered to be a period of adjustment to a new environment, requiring the synthesis of new enzymes and cell components to enable the maximum rate of growth possible in that environment. As indicated above, the growth rate and, by inference the metabolic rate, is a function of the environment. As such, the lag time observed upon transfer of a cell to a new environment could be expected to result from both the amount of adjustment to that new environment and the rate at which those adjustments could be made. In general, lag times are longer at a, that are less optimal for growth and where the difference between the old and new growth environment is larger, especially when the new environment is less favourable for growth than the old. Generally the limits for microbial sporulation are the same as the limits for growth, although sporulation may occur at a, slightly lower than that required for growth. Spores can sometimes also germinate at a, below those which permit growth. Toxin production does not occur at a, below those which permit growth, and is often prevented at a, con-

544 ECOLOGY OF BACTERIA AND FUNGI IN FOODShfluence of Available Water

h h

f. 4 -

...........S.aureus

-_---L.monocytogenes

//\

,

h h

5.

3r

!i

...........S.aureus

----- L. monocytogenes

-Pseudomonads --- E. coli

.....

.... .... ...:

.'

.

Temperature ("C)

Figure 2 Comparison of the combined effect of environmental factors on growth rate of psychrotrophic spoilage pseudomonads, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus. (A) The predicted effect of temperature on rates of aerobic growth at aw=0.990. (8)The predicted effect of temperature on rates of growth at aw=0.960.(C)Interactive effects of temperature and water activity on the microbial ecology of foods. Dominance domains for selected microorganisms potentially present on raw foods were estimated from predictive models for the aerobic growth of psychrotrophic spoilage pseudomonads, L. monocytogenes, E. coli and S. aureus at many combinations of water activity and temperature. The shaded areas represent that combination of factors in which the indicated organism would be expected to limit the acceptability of the product. The limits imposed for acceptability were that the predicted increase in the pathogen should not exceed a factor of 10 after 7 days storage. The limits for pseudomonads were that the increase in 7 days should be not more than 1000-fold, assuming an initial level of l000cfu cm-'. All organisms were assumed to experience a lag time equivalent to one generation time at the nominated conditions. The part of the plot to the left of the bold line shows those sets of conditions under which the required bacterial growth limits are exceeded. For all conditions the organism closest to attaining the tolerance limit, and hence posing the greatest risk, is indicated. Note: The growth rate of pseudomonadswas scaled to reflect the greater tolerance of this organism on the product, i.e. approx. 10 doublings of pseudomonads but only approx. 3 doublings of pathogens are tolerable by the criteria described. After McMeekin and Ross (1996) with permission from Elsevier Science.

siderably higher than those required to prevent growth.

as a function of a,,, until the lower a, limit for growth, approximately 0.95.5, is reached.

Yield Inactivation

At a, less than the optimum, cell yield declines. The decline is not always a direct function of the a,,,stress A t a, lower than the minimum for growth, the cell applied and it appears that some bacteria, at least, either remains dormant or dies. Compounding this can tolerate a range of a, without a change in yield. action, however, is the effect of a, on the cell and the In E. coli, for example, in the a, range from approxi- environment itself. Reduced a, usually correlates with mately 0.970 to 0.997 (using NaCl as the humectant), decreased chemical activity, with the result that the yield declined slightly (620%) with decreasing a, preservative effect of low a, on foods may also precompared to the optimum a, (ca. 0.995). At a, lower serve microorganisms present in the foods. This is than approximately 0.970, yield declines dramatically particularly true for low a, (i.e. cO.7) products, in

ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water 545

which microbial survival may be enhanced in comparison to that at higher a , . Mechanisms

While the changes in cell physiology that accompany osmotic stress are known in some detail, the physicochemical mechanisms that underlie the effects of those responses are not well understood. One interpretation of the effects of a, on the ecology of microorganisms considers that a, creates a homeostatic burden. To maintain homeostasis, the cell must expend energy, whether to import or synthesize compatible solutes, modify membrane components, etc. This energy is unavailable for synthesis of new biomass and leads to reduced yield. This hypothesis further proposes that the cells' homeostatic demands ultimately consume all the available energy and the cell is able only to survive. Extending this paradigm, cell death could be interpreted to result when the homeostatic demands are unable to be met and the cell is unable to maintain the functional integrity of those enzymes and pathways necessary for continued viability.

Effect of Water Activity on Intracellular Structures and Chemical Composition of Cells To remain viable, microorganisms, like plant cells, need to maintain a positive turgor pressure, possibly to provide a stimulus for cell elongation and growth. When a cell experiences an osmotic 'upshock' (i.e. transfer to lower a,), the cell loses water due to osmosis because the microbial cell membrane is permeable to water and relatively impermeable to solutes. Water moves out of the cell to restore osmotic equilibrium, resulting in shrinkage of the cells. In extreme cases the cell membrane shrinks away from the cell wall, a process termed plasmolysis. Microbial cells must counteract the osmotic stress to restore the turgid, pre-stress state and have evolved a number of physiological responses to reduced a, including changes in: cell membrane composition protein synthesis adjustment of cytoplasmic a,,,. The cell membrane is the main barrier to water and solute exchange between the cytoplasm and the external environment. It plays an important role in the physiological response to osmotic stress, responding with changes to both its lipid and protein components. The synthesis of some proteins is induced by osmotic stress. Increased levels of solute transport proteins (porins) are likely during the osmoregulatory response. Like porins, many other osmotically

induced proteins form the cellular machinery to facilitate a change in cytoplasmic a,". Macromolecular conformation, and therefore function and activity, is affected by intracellular a, due, in part, to the effects of humectants on the physical structure of water. Some changes to membrane structure in response to a, stress appear to enable membrane-bound enzymes to retain the conformation required for catalytic activity. The role of compatible solutes in optimizing molecular conformation is discussed below. Cell Membrane Composition

The chemical composition of microbial cell membranes is described clsewhere in this volume. In response to high salinity there is an increase in the proportion of negatively charged phospholipids, often phosphatidylglycerol and/or glycolipids. This alteration is needed to maintain the membrane in the proper lipid bilayer phase for normal function. Apart from the extreme halophiles of the Archaea there does not appear to be a correlation between microbial membrane composition and intrinsic a, tolerance. However, the effect of a, on a given membrane composition does depend to a large extent on the type of membrane (correlated with chemotaxonomic grouping, e.g. Bacteria, Archaea, yeast, fungi) and to a lesser extent, the nature of the humectant. There are several elements common to cell membrane responses to changing a,. The first of these is membrane surface charge. The head groups of the major microbial membrane lipids (phospholipids and phosphoglycolipids) are negatively charged from the associated phosphate residue. Certain phospholipid classes also contain positively charged head-group moieties, resulting in all polar lipid classes being either anionic or zwitterionic. The membrane surface of all microbes therefore possesses a net surface charge dependent on the phospholipid classes present. Ionic humectants may disrupt the membrane surface charge by interaction with phospholipid groups, requiring a n alteration in membrane composition. Many halotolerant and modcrately halophilic bacteria respond to reduced a , by increasing the proportion of anionic phospholipids in the membrane at the expense of zwitterionic components, believed to aid the membrane in maintaining a functional bilayer phase. The fatty acid composition of the cellular membrane also influences functionality and is actively modified in response to changing environmental factors. In general, in response to decreasing a, most hacteria increase fatty acid chain length and/or decrease fatty acid unsaturation. Again, this is thought to maintain the membrane in a functional bilayer phase. In certain cases, the mechanism may involve

Next Page 546 ECOLOGY OF BACTERIA AND FUNGI IN FOODSAnfluence of Available Water

direct inhibition of fatty acid biosynthetic enzymes by increased levels of NaCl. Archaeal membranes possess phosphorus-containing lipid species as in other microorganisms but consisting of a glycerol backbone with two etherlinked C2"prenyl chains. This Domain contains all the extremely halophilic bacteria, with their membranes characterized by diphytanylglycerol diethers. Dephosphorylated derivatives may be present with a significant proportion of glycolipids. Extreme halophiles are characterized (but not exclusively) by the presence of neutral lipid Components, mostly isoprenoid hydrocarbons, such as squalene. The resulting membrane bilayer is more ordered and less flexible than those formed from other lipid types. The C20 phytanyl residues may be present as branched or ringcontaining structures which act as similar adaptive responses to fatty acid structure within other microorganisms. It is believed that the close packing exhibited by phytanyl residues in Archaeal membranes is the basis for their resistance to extreme environmental conditions. While yeasts and fungi, as eukaryotes, contain many additional lipid types as storage and intracellular membrane components, their cellular membrane is dominated by phospholipid species as for the Bacteria. Thus, the common changes in fungal cell membrane composition to changing a, are similar to those of the Bacteria, both in terms of polar lipid class manipulation and adaptation of fatty acid composition.

make additional physiological adjustments, especially in regard to enzyme function. The enzymes of prokaryotes that use the salt-in-cytoplasm strategy have additional negative charge that makes them stable at high solute concentrations but unstable at low concentrations.

Compatible Solutes

The activity of water is significantly influenced by the molecular structure of the solution. Water as a liquid is characterized by a (relatively) high degree of molecular motion resulting in a dynamic random distribution of molecular orientation. The potential degree of hydrogen bonding between water molecules is therefore not fully realized, allowing water molecules to pack together in a relatively tight manner or higher density. As the degree of molecular motion decreases (e.g. with lower temperature), a higher degree of hydrogen bonding between water molecules becomes possible and molecules adopt a more ordered array with a decreased density. With decreased temperature this process continues until the ordered molecular array of ice is achieved. Solute molecules decrease the activity of water by the same process. The organic compounds synthesized or accumulated by microorganisms to balance their intracellular osmotic potential to that of their cnvironmcnt share the property that they do not affect the function of normal salt-sensitive enzymes. The use of compatible solutes to counter osmotic stress is not limited to microorganisms. Plants and animals also use the Cytoplasmic Water Activity organic-solute-in-cytoplasm strategy and employ the Moulds and yeasts accomplish the restoration and same compounds as compatible solutes, suggesting maintenance of turgor pressure by accumulation from that these compounds share fundamental properties the environment, or by de novo synthesis, of intra- that make them suitable for this role. cellular polyols to establish equivalent osmotic presThe compatible solutes have low molecular weights sure intracellularly as exists extracellularly. Bacteria and polar functional groups, properties which make also accumulate or synthesize a range of compounds them highly soluble and facilitate their accumulation for the same purpose. Compounds used in this way to high intracellular concentration. They are share the property that they do not interfere with uncharged at normal cytoplasmic pH - an important metabolic processes. As such, they have been termed property because high cytoplasmic ionic strength compatible solutes. would be detrimental to enzyme function. These Microorganisms adjust their cytoplasmic a, using requirements limit the range of compounds that can one of two strategies: the salt-in-cytoplasm type and be utilized as compatible solutes. Classes of comthe organic-osmolyte-in-cytoplasm type. Most, like pounds that are known to perform this function and the yeasts and moulds, use the organic-osmolyte-in- specific examples are presented in Table 5. cytoplasm strategy for osmoadaptation. In this stratCompatible solutes do not hinder the function of egy salts are excluded, while organic solutes are syn- normal (salt-sensitive)enzymes and, in fact, protect thesized or accumulated from the environment. Some proteins from the denaturation that would otherwise bacteria can also adjust their cytoplasmic water by occur in solutions of high ionic strength. That proaccumulating KC1 to high intracellular concentration. tection also extends to the denaturing effects of freezThis is considered a primitive strategy because it does ing, heating and drying. not provide a normal cytoplasmic environment. This The mechanism of this protective effect is unknown. salt-in-cytoplasmstrategy requires that the cell should One observation, fundamental to attempts to resolve

FERMENTATION (INDUSTRIAL)/Basic Considerations 663

1

Fatty Acids see Fermentation (Industrial): Production of Oils and Fatty Acids.

1

FERMENTATION (INDUSTRIAL) Contents

Basic Considerations Media for Industrial Fermentations Control of Fermentation Conditions Recovery of Metabolites Production of Xanthan Gum Production of Organic Acids Production of Oils and Fatty Acids Colours/Flavours Derived by Fermentation

Basic Considerations Yusuf Chisti, Department of Chemical Engineering, University of Almeria, Spain Copyright 0 1999 Academic Press

Introduction Fermentation processes utilize microorganisms to convert solid or liquid substrates into various products. The substrates used vary widely, any material that supports microbial growth being a potential substrate. Similarly, fermentation-derived products show tremendous variety. Commonly consumed fermented products include bread, cheese, sausage, pickled vegetables, cocoa, beer, wine, citric acid, glutamic acid and soy sauce. Types of Fermentation

Most commercially useful fermentations may be classified as either solid-state or submerged cultures. In solid-state fermentations, the microorganisms grow on a moist solid with little or no ‘free’ water, although capillary water may be present. Examples of this type of fermentation are seen in mushroom cultivation, bread-making and the processing of cocoa, and in the manufacture of some traditional foods, e.g. miso (soy paste), sakk, soy sauce, tempeh (soybean cake) and gari (cassava), which are now produced in large industrial operations. Submerged fermentations may use a

dissolved substrate, e.g. sugar solution, or a solid substrate, suspended in a large amount of water to form a slurry. Submerged fermentations are used for pickling vegetables, producing yoghurt, brewing beer and producing wine and soy sauce. Solid-state and submerged fermentations may each be subdivided - into oxygen-requiring aerobic processes, and anaerobic processes that must be conducted in the absence of oxygen. Examples of aerobic fermentations include submerged-culture citric acid production by Aspergillus niger and solid-state koji fermentations (used in the production of soy sauce). Fermented meat products such as bologna sausage (polony), dry sausage, pepperoni and salami are produced by solid-state anaerobic fermentations utilizing acid-forming bacteria, particularly Lactobacillus, Pediococcus and Micrococcus species. A submergedculture anaerobic fermentation occurs in yoghurtmaking. Fermentations may require only a single species of microorganism to effect the desired chemical change. In this case the substrate may be sterilized, to kill unwanted species prior to inoculation with the desired microorganism. However, most food fermentations are non-sterile. Typically fermentations used in food processing require the participation of several microbial species, acting simultaneously and/or sequentially, to give a product with the desired properties, including appearance, aroma, texture and taste. In non-sterile fermentations, the culture environment

664 FERMENTATION (INDUSTRIAL)/Basic Considerations

fermentation batch. The volume of the fermenting broth increases with each addition of the medium, and the fermenter is harvested after the batch time. In continuous fermentations, sterile medium is fed continuously into a fermenter and the fermented Factors Influencing Fermentations product is continuously withdrawn, so the ferA fermentation is influenced by numerous factors, mentation volume remains unchanged. Typically, conincluding temperature, pH, nature and composition tinuous fermentations are started as batch cultures of the medium, dissolved 0 2 , dissolved COZ, oper- and feeding begins after the microbial population has ational system (e.g. batch, fed-batch, continuous), reached a certain concentration. In some continuous feeding with precursors, mixing (cycling through fermentations, a small part of the harvested culture varying environments), and shear rates in the fer- may be recycled, to continuously inoculate the sterile menter. Variations in these factors may affect: the rate feed medium entering the fermenter (Fig. l ( D ) ) . of fermentation; the product spectrum and yield; the Whether continuous inoculation is necessary depends organoleptic properties of the product (appearance, on the type of mixing in the fermenter. ‘Plug flow’ taste, smell and texture); the generation of toxins; fermentation devices (Fig. l ( D ) ) ,such as long tubes nutritional quality; and other physico-chemical that do not allow back mixing, must be inoculated properties. continuously. Elements of fluid moving along in a The formulation of the fermentation medium plug flow device behave like tiny batch fermenters. affects the yield, rate and product profile. The medium Hence, true batch fermentation processes are relamust provide the necessary amounts of carbon, nitro- tively easily transformed into continuous operations gen, trace elements and micronutrients (e.g. vitamins). in plug flow fermenters, especially if pH control and Specific types of carbon and nitrogen sources may be aeration are not required. Continuous cultures are required, and the carbon : nitrogen ratio may have particularly susceptible to microbial contamination, to be controlled. An understanding of fermentation but in some cases the fermentation conditions may be biochemistry is essential for developing a medium selected (e.g. low pH, high alcohol or salt content) with an appropriate formulation. Concentrations of to favour the desired microorganisms compared to certain nutrients may have to be varied in a specific potential contaminants. way during a fermentation to achieve the desired In a ‘well-mixed’ continuous fermenter (Fig. 1(C)), result. Some trace elements may have to be avoided - the feed rate of the medium should be such that the for example, minute amounts of iron reduce yields in dilution rate, i.e. the ratio of the volumetric feed rate citric acid production by Aspergillus niger. Additional to the constant culture volume, remains less than the factors, such as cost, availability, and batch-to-batch maximum specific growth rate of the microorganism variability also affect the choice of medium. in the particular medium and at the particular fermentation conditions. If the dilution rate exceeds the Submerged Fermentations maximum specific growth rate, the microorganism will be washed out of the fermenter. Fermentation Systems Industrial fermentations are mostly batch operations. Typically, a pure starter culture (or seed),mainIndustrial fermentations may be carried out either tained under carefully controlled conditions, is used batchwise, as fed-batch operations, or as continuous to inoculate sterile Petri dishes or liquid medium in the cultures (Fig. 1). Batch and fed-batch operations are shake flasks. After sufficient growth, the pre-culture is quite common, continuous fermentations being relaused to inoculate the ‘seed’ fermenter. Because industively rare. For example, continuous brewing is used trial fermentations tend to be large (typically 150commercially, but most beer breweries use batch 250m3), the inoculum is built up through several processes. successively larger stages, to 5-10% of the working In batch processing, a batch of culture medium in volume of the production fermenter. A culture in rapid a fermenter is inoculated with a microorganism (the ‘starter culture’). The fermentation proceeds for a exponential growth is normally used for inoculation. certain duration (the ‘fermentation time’ or ‘batch Slower-growing microorganisms require larger time’), and the product is harvested. Batch fer- inocula, to reduce the total duration of the fermentations typically extend over 4-5 days, but some mentation. An excessively long fermentation time (or traditional food fermentations may last months. In fed- batch time) reduces productivity (amount of product batch fermentations, sterile culture medium is added produced per unit time per unit volume of fermenter), either continuously or periodically to the inoculated and increases costs. Sometimes inoculation spores,

may be tailored specifically to favour the desired microorganisms. For example, the salt content may be high, the pH may be low, or the water activity may be reduced by additives such as salt or sugar.

FERMENTATION (INDUSTRIAL)/Basic Considerations 665

Feed

- Final volume

- Initial volume

(6)

Feed Feed

Harvest

Recycle inocuium

-

Feed

Harvest

(C)

Harvest

Inoculum from separate source (D)

Figure 1 Fermentation methodologies. (A) Batch fermentation. (B) Fed-batch culture. (C) Continuous-flow well-mixed fermentation. (D) Continuous plug flow fermentation, with and without recycling of inoculum.

produced as seeds, are blown directly into large fermentation vessels with the ingoing air. Microbial Growth

Microbial growth in a newly inoculated batch fermenter typically follows the pattern shown in Figure 2. Initially, in the lag phase, the cell concentration does not increase very much. The length of the lag phase depends on the growth history of the inoculum, the composition of the medium, and the amount of culture used for inoculation. An excessively long lag phase ties up the fermenter unproductively - hence the duration of the lag phase should be minimized. Short lag phases occur when: the composition of the medium and the environmental conditions in the seed culture and the production vessel are identical (hence less time is needed for adaptation); the dilution shock is small (i.e. a large amount of inoculum is used); and the cells in the inoculum are in the late exponential phase of growth. The lag phase is essentially an adaptation period in a new environment. The lag phase is followed by exponential growth, during which the cell mass increases exponentially. Eventually, as the

~

/ ~ ~ ~ ~ ~ ~ Stationary n t i a l ' qrowth Phase ~

~

~

~

Death phase

nutrients are exhausted and inhibitory products of metabolism build up, the culture enters a stationary phase. Ultimately, starvation causes cell death and lysis, and hence the biomass concentration declines. Exponential growth can be described by Equation 1:

dX ,Ux- k d x (Equation 1) dt where: X is the biomass concentration at time t; p is the specific growth rate (i.e. growth rate per unit cell mass); and k d is the specific death rate. During exponential growth, the specific death rate is negligible and Equation 1 reduces to Equation 2: dX -=px (Equation 2) dt For a cell mass concentration Xo at the beginning of the exponential growth ( X , usually equalling the concentration of inoculum in the fermenter), and taking the time at which exponential growth commences as zero, Equation 2 can be integrated to produce Equation 3: X In- = p t (Equation 3) XO Using Equation 3 , the biomass doubling time, t d , can be derived (Equation 4): ln2 -=

t d =-

(Equation 4)

iu

0

Fermentation time

Figure 2 Typical growth profile of microorganisms in a submerged culture.

Doubling times typically range over 45-160 min. Bacteria generally grow faster than yeasts, and yeasts multiply faster than moulds. The maximum biomass concentration in submerged microbial fermentations is typically 40-50 kg m-3. The specific growth rate p depends on the concentration S of the growth-limiting substrate, until

666 FERMENTATION (INDUSTRIAL)/Basic Considerations

the concentration is increased to a non-limiting level O2 demand, or the fermentation will be 02-limited. and p attains its maximum value pmax.The dependence O2 demand is especially difficult to meet in viscous of the growth rate on substrate concentration typ- fermentation broths and in broths containing a large ically follows Monod kinetics. Thus the specific concentration of 02-consuming cells. As a general guide, the capability of a fermenter in terms of 0 2 growth rate is given as Equation 5: supply depends on the aeration rate, the agitation S (Equation 5 ) intensity and the properties of the culture broth. In P = Pmax k, + S large fermenters, supplying 0 2 becomes difficult when where k, is the saturation constant. Numerically, k, is demand exceeds 4-5 kg m-3h-’. the concentration of the growth-limiting substrate At concentrations of dissolved 0 2 below a critical when the specific growth rate is half its maximum level, the amount of O2 limits microbial growth. The value. critical dissolved 0 2 level depends on the microAn excessively high substrate concentration may organism, the culture temperature and the substrate also limit growth, for instance by lowering water being oxidized. The higher the critical dissolved 0 2 activity. Moreover, certain substrates inhibit product value, the greater the likelihood that 0 2 transfer will formation, and in yet other cases, a fermentation become limiting. Under typical culture conditions, product may inhibit biomass growth. For example, fungi such as Penicillium chrysogenum and Asperethanol produced in the fermentation of sugar by gillus oryzae have a critical dissolved 0 2 value of yeast can be inhibitory to cells. Multiple lag phases about 3.2 x kgm-3. For bakers’ yeast and Esch(or diauxic growth) are sometimes seen when two or erichia coli, the critical dissolved 0 2 values are more growth-supporting substrates are available. As 6.4 x 10-’ kgm-3 and 12.8 x 10-jkg m-3 respectively. the preferentially-utilized substrate is exhausted, the The aeration of fermentation broths generates cells enter a lag phase while the biochemical machin- foam. Typically, 20-30% of the fermenter volume ery needed for metabolizing the second substrate is must be left empty to accommodate the foam and developed. Growth then resumes. Details of the kin- allow for gas disengagement. In addition, mechanical etics of continuous culture, fed-batch fermentation, ‘foam breakers’ and chemical antifoaming agents are product formation and more complex phenomena, commonly used. Typical antifoams are silicone oils, such as the inhibition of growth by substrates and vegetable oils and substances based on low-molecularproducts, are given in the references listed under weight polypropylene glycol or polyethylene glycol. Further Reading. Emulsified antifoams are more effective, because they disperse better in the fermenter. Antifoams are added Aeration and Oxygen Demand in response to signals from a foam sensor. The excesSubmerged cultures are most commonly aerated by sive use of antifoams may interfere with some downbubbling with sterile air. Typically, in small fer- stream separations, such as membrane filtrations menters, the maximum aeration rate does not exceed hydrophobic silicone antifoams are particularly 1 volume of air per unit volume of culture broth. In troublesome. large bubble columns and stirred vessels, the Heat Generation and Removal maximum superficial aeration velocity tends to be c 0.1 m s-’. Superficial aeration velocity is the volume All fermentations generate heat. In submerged culflow rate of air divided by the cross-sectional area tures, typically 3-15 kW m-3 comes from microbial of fermenter. Significantly higher aeration rates are activity. In addition, mechanical agitation of the broth achievable in airlift fermenters. In these, aeration gas produces up to 15 kW m-3. Consequently, a fermenter is forced through perforated plates, perforated pipes must be cooled to prevent a rise in temperature and or single-hole spargers located near the bottom of damage to the culture. Heat removal tends to be the fermenter. Because 0 2 is only slightly soluble in difficult, because typically the temperature of the aqueous culture broths, even a short interruption of cooling water is only a few degrees lower than that aeration results in the available 0 2 becoming quickly of the fermentation broth. Therefore industrial ferexhausted, causing irreversible damage to the culture. mentations are commonly limited by their heat-transThus uninterrupted aeration is necessary. Prior to use fer capability. The ability to remove heat depends for aeration, any suspended particles, microorganisms on: the surface area available for heat exchange; the and spores in the gas are removed by filtering through temperature difference between the broth and the cooling water; the properties of the broth and the microporous membrane filters. The 0 2 requirements of a fermentation depend on coolant; and the turbulence in these fluids. The geomthe microbial species, the concentration of cells, and etry of the fermenter determines the surface area that the type of substrate. 0 2 supply must at least equal can be provided for heat exchange. Heat generation

FERMENTATION (INDUSTRIAL)/Basic Considerations 667

due to metabolism depends on the rate of 0 2 consumption, and heat removal in large vessels becomes difficult as the rate of 0 2 consumption approaches 5 kg m-3h-'. A fermenter must provide for heat transfer during sterilization and subsequent cooling, as well as removing metabolic heat. Liquid medium, or a slurry, for a batch fermentation may be sterilized using batch or continuous processes. In batch processes, the medium or some of its components and the fermenter itself are commonly sterilized together in a single step, by heating the medium inside the fermenter. Steam may be injected directly into the medium, or heating may take place through the fermenter wall. Heating to high temperatures (typically 121°C) during sterilization often leads to undesirable reactions between components of the medium. Such reactions reduce the yield, by destroying nutrients or by generating compounds which inhibit growth. This thermal damage can be prevented or reduced by sterilizing only certain components of the medium in the fermenter and adding other, separately-sterilized components, later. Sugars and nitrogen-containing components are often sterilized separately. Dissolved nutrients that are especially susceptible to thermal degradation may be sterilized by passage through hydrophilic polymer filters, which retain particles of 0.45pm or more. Even finer filters (e.g. retaining particles of 0.2 pm) are also available. The heating and cooling of a large fermentation batch takes time, and ties up a fermenter unproductively. In addition, the longer a medium remains at a high temperature, the greater the thermal degradation or loss of nutrients. Therefore, continuous sterilization of the culture medium en route to a presterilized fermenter is preferable, even for batch fermentations. Continuous sterilization is rapid and it limits nutrient loss - however, the initial capital expense is greater, because a separate sterilizer is necessary. Photosynthetic Microorganisms

Photosynthetic cultures of microalgae and cyanobacteria require light and COZ as nutrients. Microalgae such as Chlorella and the cyanobacterium Spirulina are produced commercially as health foods in Asia. Algae are also cultivated as aquaculture feeds for shellfish. Typically, open ponds or shallow channels are used for the outdoor photosynthetic culture of microalgae. Culture may be limited by the availability of light, but under intense sunlight, photoinhibition limits productivity. Temperature variations also affect performance. More controlled production is achieved in outdoor

tubular photobioreactors, bubble columns and airlift systems. Tubular bioreactors use a 'solar receiver', consisting of either a continuous tube looped into several U-shapes to fit a compact area, or several parallel tubes connected to common headers at either end. The continuous looped-tube arrangement is less adaptable, because the length of the tube cannot exceed a certain value: photosynthetically-produced 0 2 builds up along the tube, and high levels of dissolved 0 2 inhibit photosynthesis. The parallel-tube arrangement can be readily scaled up by increasing the number of tubes. Typically, the tubes are 0.050.08 m in diameter and the continuous-run length of any tube does not exceed 5 0 m . However, greater lengths may be feasible, depending on the flow velocity in the tube. The tubular solar receivers may be mounted horizontally, or horizontal tubes may be stacked in a ladder configuration, forming the rungs of the ladder. The latter arrangement reduces the area of land required. The culture is circulated through the tubes by an airlift pump or other suitable low-shear mechanism. The maximum flow rate is limited by the tolerance of the algae to hydrodynamic stress. The flow velocity is usually 0.3-0.5 m c'.The tube diameter is limited by the need to achieve adequate penetration of light. This declines as the cell concentration increases, due to self-shading. Closed, temperature-controlled outdoor tubular systems attain significantly higher productivity than open channels. The protein content of the algal biomass, and the adequacy of the development of colour (chlorophyll)affect the acceptability of the product. Among other types of culture system, airlift devices tend to perform better than bubble columns because only part of the airlift system is aerated and hence the penetration of light is less affected by air bubbles. Conventional external-loop airlift devices may not be suitable because of the relatively high hydrodynamic shear rates they generate. However, concentric-tube airlift devices, with gas forced into the draft tube (zone of poor light penetration), are likely to perform well. Also, split-cylinder types of airlift system may be suitable. However, the volume of the aerated zone in any airlift device for microalgal culture should not exceed approximately 40% of the total volume of the circulating zones. This way the light blocking effect of bubbles remains confined to a small zone. Submerged-culture Fermenters

Types The major types of submerged-culture bioreactor are: 0 stirred-tank fermenter bubble column 0 airlift fermenter

668 FERMENTATION (INDUSTRIAL)/Basic Considerations

-

Liquid Liquid overflow

Recycle

(F)

Product

Figure 3 Types of submerged-culture fermenter. (A) Stirred-tank fermenter. (B) Bubble column. (C) Internal-loop airlift fermenter. (D) External-loop airlift fermenter. (E) Fluidized-bed fermenter. (F) Trickle-bed fermenter.

fluidized-bed fermenter trickle-bed fermenter. These are shown in Figure 3.

of its poor performance relative to other systems. It is not suitable for very viscous broths or those containing large amounts of solids.

Stirred-tank Fermenter (See Fig. 3(A).) This is a cylindrical vessel with a working height-to-diameter ratio (aspect ratio) of 3-4. A central shaft supports three to four impellers, placed about 1 impeller-diameter apart. Various types of impeller, that direct the flow axially (parallel to the shaft) or radially (outwards from the shaft) may be used (Fig. 4). Sometimes axial- and radial-flow impellers are used on the same shaft. The vessel is provided with four equally spaced vertical baffles, that extend from near the walls into the vessel. Typically, the baffle width is 8-10% of the vessel diameter.

Airlift Fermenters (See Figs. 3(C) and 3(D).)These come in internal-loop and external-loop designs. In the internal-loop design, the aerated riser and the unaerated downcomer are contained in the same shell. In the external-loop configuration, the riser and the downcomer are separate tubes that are linked near the top and the bottom. Liquid circulates between the riser (upward flow) and the downcomer (downward flow). The working aspect ratio of airlift fermenters is 6 or greater. Generally, these are very capable fermenters, except for handling the most viscous broths. Their ability to suspend solids and transfer 0 2 and heat is good. The hydrodynamic shear is low. The external-loop design is relatively little-used in industry.

0

0

Bubble Column (See Fig. 3(B).)This is a cylindrical vessel with a working aspect ratio of 4-6. It is sparged at the bottom, and the compressed gas provides agitation. Although simple, it is not widely used because

Fluidized-bed Fermenter

(See Fig. 3(E).) These are

FERMENTATION (INDUSTRIAL)/Basic Considerations 669

(D)

(E)

(F)

Figure 4 Impellers for stirred-tank fermenters. (A) Rushton disc turbine (radial flow). (B) Marine propeller (axial flow). (C)Lightnin’ hydrofoil (axial flow). (D)Prochem hydrofoil (axial flow). (E)lntermig (axial flow). (F)Chemineer hydrofoil (axial flow).

similar to bubble columns with an expanded cross section near the top. Fresh or recirculated liquid is continuously pumped into the bottom of the vessel, at a velocity that is sufficient to fluidize the solids or maintain them in suspension. These fermenters need an external pump. The expanded top section slows the local velocity of the upward flow, such that the solids are not washed out of the bioreactor.

Trickle-bed Fermenter (See Fig. 3(F).)These consist of a cylindrical vessel packed with support material (e.g. woodchips, rocks, plastic structures). The support has large open spaces, for the flow of liquid and gas and the growth of microorganisms on the solid support. A liquid nutrient broth is sprayed onto the top of the support material, and trickles down the bed. Air may flow up the bed, countercurrent to the liquid flow. These fermenters are used in vinegar production, as well as in other processes. They are suitable for liquids with low viscosity and few suspended solids. Design Irrespective of their configuration, industrial bioreactors for sterile operations are designed as pressure vessels, capable of being sterilized in situ with saturated steam at a minimum guage pressure of 0.11 MPa. Typically, the bioreactor is designed for a maximum allowable working pressure of 0.280.31 MPa (guage) and a temperature of 150-180°C. The vessels are designed to withstand a full vacuum. Modern commercial fermenters are predominantly made of stainless steel. Type 316L stainless steel is

preferred, but the less expensive Type 304L (or 304) may be used in less corrosive situations. Fermenters are typically designed with clean-in-place capability. A typical submerged-culture vessel has the features shown in Figure 5. Sight glasses in the side and top of the vessel allow for easy viewing. The top sight glass can be cleaned during fermentation, using a short-duration spray of sterile water derived from condensed steam. An external lamp is provided, to light the vessel through the sight glass or a separate window. The vessel has ports for sensors of pH, temperature and dissolved 0 2 . A steam-sterilizable sampling valve is provided. Connections for the introduction of acid and alkali (for p H control), antifoam agents, substrate and inoculum are located above the liquid level in the bioreactor vessel. Additional ports on the top support a foamsensing electrode, a pressure sensor and sometimes other instruments. Filter-sterilized gas for aeration is supplied through a submerged sparger. Sometimes COZ or ammonia may be added to the aeration gas, for p H control. A harvest valve is located at the lowest point on the fermenter. A mechanical agitator, entering from either the top or the bottom, may be used. The agitator shaft supports one or more impellers, of various designs (Fig. 4). A high-speed mechanical foam breaker may be provided at the top of the vessel, and waste gas may exit through the foam breaker. Commonly, the exhaust gas line also has a heat exchanger, to condense and return water in the gas to the fermenter. The top

670

FERMENTATION (INDUSTRIAL)/Basic Considerations

Selection Considerations in selecting industrial fermenters are:

11

-23

3 4

1. Nature of substrate solid, liquid, suspended slurry, water-immiscible oils). 2. Flow behaviour (rheology), broth viscosity and type of fluid (e.g. Newtonian, viscoelastic, pseudoplastic, Bingham plastic). 3. Nature and amount of suspended solids in broth. 4. Whether fermentation is aerobic or anaerobic, and O2 demand. 5. Mixing requirements. 6. Heat-transfer needs. 7. Shear tolerance of microorganism, substrate and product. 8. Sterility requirements. 9. Process kinetics, batch or continuous operation, single-stage or multistage fermentation. 10. Desired process flexibility. 11. Capital and operational costs. 12. Local technological capability and potential for technology transfer.

Solid-state Fermentations Figure 4 A typical submerged-culture fermenter. (1) Reactor vessel. (2) Jacket. (3) Insulation. (4) Protective shroud. (5) Inoculum connection. (6) Ports for sensors of pH, temperature and dissolved 02.(7) Agitator. (8) Gas sparger. (9) Mechanical seal. (10) Reducing gearbox. (11) Motor. (12) Harvest nozzle. (13) Jacket connections. (14) Sample valve with steam connection. (15)Sight glass. (16)Connections for acids, alkalis and antifoam agents. (17) Air inlet. (18) Removable top. (19) Medium feed nozzle. (20) Air exhaust nozzle (connects to condenser, not shown). (21) Instrumentation ports for foam sensor, pressure gauge and other devices. (22) Centrifugal foam breaker. (23) Sight glass with light (not shown) and steam connection. (24) Rupture disc nozzle. Vertical baffles are not shown. Baffles are mounted on brackets attached to the wall. A small clearance remains between the wall and the closest vertical edge of the baffle.

of the fermenter is either removable or provided with a manhole. A port on the top supports a ‘rupture disc’ that is piped to a drain. The disc is intended to protect the vessel in the event of a pressure build-up. The fermentation vessel is jacketed for heat exchange, and the jacket may be covered with fibreglass insulation and a protective metal shroud. Additional surfaces for heat exchange, typically coils, may be located inside the vessel. The equipment for fermenting slurries containing undissolved solid substrates is identical to that used in submerged-culture processes. Commonly-used slurry fermenters include stirred tanks, bubble columns, and airlift vessels.

Substrate Characteristics

Water Activity Typically, solid-state fermentations are carried out with little or no free water. Excessive moisture tends to aggregate the substrate particles, and hence aeration is made difficult. For example steamed rice, a common substrate, becomes sticky when the moisture level exceeds 30-35%w/w. Percentage moisture by itself is unreliable for predicting growth: for a given microorganism growing on different substrates, the optimum moisture level may differ widely. This water activity correlates with microbial growth. The water activity of the substrate is the ratio of the vapour pressure of water in the substrate to the saturated vapour pressure of pure water at the temperature of the substrate. Water activity equals 1/100th of the relative humidity (RH%) of the air in equilibrium with the substrate. Typically, water activities of 115 species and subspecies) in the family Enterobacteriaceae, about 50 differential biochemical characteristics can be verified; these can also be found in Bergey’s Manual. Due to the large number of data to be compared in such an identification attempt, one tries to identify members of this family directly to the genus or species level using the above battery of about 50 tests. Additional tests are then available to differentiate between certain species and subspecies; for Klebsiella specieshbspecies, these are summarized in

Serological tests based on the K-antigen can be used to confirm the identification results. A total of 62 serotypes among 72 different serological Klebsiella strains could be distinguished, based on a unique agglutination pattern with plant lectins. Based on an extensive survey of about 160 strains of Bacillus, lactic acid bacteria, Enterobacteriaceae and Staphylococcus, it can be concluded that gas chromatographic analysis of cellular fatty acid composition was not sufficiently specific to the species level in several cases; characterization of food-borne bacteria, including Klebsiella sp. by the analysis of their cellular fatty acids should thus only be used to complement other taxonomical methods. Recently, several molecular identification techniques have been proposed for a wide range of medically and food-related bacteria, including Klebsiella SP. A fluorescence-based polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) analysis of the 16s rRNA gene has been described to identify a broad range of Gram-positive and Gram-negative bacteria: 178 bacterial strains, Table 1. representing 51 species in 21 genera were examined. The usefulness of the pyrrolidonyl-arylamidase All strains gave species-specific patterns, except Shiactivity test to differentiate among Enterobacteriaceae gella which resembled E . coli. This sensitive technique and non-fermentative Gram-negative rods has also can be applied on very low numbers of bacteria, i.e. been studied. Positive results were uniformly obtained IO colony forming units (cfu). Two 16s DNA targeted oligonucleotides were used with Citrobacter, Klebsiella, Enterobacter and Serratia species. Negative results were displayed by as PCR primers for the specific detection of Salmonella Escherichia coli, Proteus, Salmonella, Shigella, serotypes in food. Some of the primers (16s 111) also Pseudomonas and Flavobacterium species, indicating hybridized with Klebsiella and Serratia sp., however. its value as a complementary differentiation test. The Biolog System has been evaluated for the idem- Recommended Methods for Detection tification of 55 Gram-negative taxa (789 strains), and Enumeration of Klebsiella likely to be encountered commonly in clinical laboratories. It performed best with oxidase-positive ferDetection and Enumeration menters, but although for 39 of the 55 taxa an identification rate of 70 % was achieved, problems Conventionally, eventual resuscitation (2 h at 17were encountered, particularly with the identification 25°C) in tryptic soy broth and (subsequent) plating on violet red bile glucose agar (VRBG) allows an of capsulated strains of Klebsiella. The new BBL CrystalEnterichIon Fermenter efficient presumptive enumeration of Entero(Crystal, Becton Dickinson Microbiology Systems) bacteriaceae, in foods or in other substrates. Other identification system for Gram-negative rods has been isolation media commonly used in this respect are: compared with the well-known API 20 E or API 20 NE Simmons citrate agar, MacConkey agar and eosin (Bio-Merieux) system. More than 100 clinical isolates methylene blue agar. Incubation is at 35°C (clinical were studied, including six K . oxytoca strains and ’12 samples) and 10°C (environmental samples) for 24K. pneumoniae strains; it was concluded that the 48 h. Raised mucoid colonies are selected and further Becton Dickinson Crystal test allowed a quicker, differentiation and confirmation of Klebsiella is then easier and more accurate identification of Gram-nega- based on the battery of tests mentioned above. The detection and isolation from sources such as tive clinical isolates compared to the API system. A remaining problem is to distinguish K. pneu- faeces, soil, water and food can be facilitated by use moniae strains from non-motile Enterobacter aero- of the standard selective media (see Table 2). Specific genes strains; however, the latter liquefy gelatin very Klebsiella enumeration is also of great importance to environmental microbiologists investigating the slowly and are urease negative.

1110 KLEBSIELLA

Table 1 Differential biochemical characterization of Klebsiella spp. Test

Klebsiella oxytoca

Gram stain (24 h) Oxidase (24 h) Indole production Methyl red Voges-Proskauer Citrate (Simmons) Hydrogen sulphide production Urea hydrolysis Phenylalanine deaminase Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase Motility Gelatin hydrolysis, 22°C KCN, growth Acid production D-Adonitol L-Arabinose Cellobiose Dulcitol p-Gentibiose D-Glucose Glycerol myo-Inositol Lactose Maltose D-Mannitol o-Mannose D-Melezitose Melibiose a-Methyl-o-glucoside Mucate Raffinose L-Rhamnose Salicin D-Sorbitol Sucrose Trehalose D-Xylose Tartrate, Jordans Aesculin hydrolysis Pectate hydrolysis Utilization Acetate Gentisate m-Hydroxybenzoate Malonate D-Glucose, gas production Growth 10°C Lactose, gas production 44°C Nitrate reduction Deoxyribonuclease, 25°C Lipase ONPG" Pigment

Klebsiella Klebsiella planticola pneumoniae subsp. ozaenae

-

-

Klebsiella pneumoniae subsp. rhinoscleromatis

Klebsiella terrigena

-

-

+ + +

[-I + +

d

+

+

-

-

+

+

+

-

[+I + +

Klebsiella pneumoniae subsp. pneumoniae

[-I

-

-

-

+

d

-

-

[-I

+

+

+

+

+

+ +

+ + +

d

[-I

+ + [-I + + + + + + + + -

+

+ + + + + + + + + + + d

-

+ + +

+ + + + + + + + -

+ + +

+ + + + + + + + + [+I

-

+ + -

+

+ +

+

+

-

-

-

-

+ + + [+I +

+ + + + + + + + + + + + + + + + -

[-I + + + [+I + -

+ -

+ -

KLEBSlELLA

1111

Table 1 contd Test

Klebsiella oxytoca

Flagella arrangementb Catalase production (24 h) Oxidation-fermentationc

P

-

+

+

F

F

Klebsiella Klebsiella planticola pneumoniae subsp. ozaenae

Klebsiella pneumoniae subsp. pneumoniae

Klebsiella pneumoniae subsp. rhinoscleromatis

Klebsiella terrigena

-

-

+

+

-

+ F

F

F

F

+

-, 0-10% positive; [-I, 11-25% positive; d, 26-75% positive; [+I, 76-89% positive: +, 90-100% positive. Results are for 48 h incubation. “ONPG, 0-nitrophenyl-p-D-galactopyranoside. bP, peritrichous. ‘F, fermentative.

effects of pulp and paper mill and cannery effluents in receiving waters. The search for improved selective media and diagnostic tests goes on and is outlined below. A synthetic medium, based on myo-inositol as the sole carbon source has also been proposed for selection of Klebsiella (and Serratia). As a further elaboration, a MacConkey-inositol-carbenicillin agar medium (MCIC) was proposed, the selectivity of this medium being based on the high resistance of the capsulated Klebsiella cells towards carbenicillin. Based on its oligotrophic characteristics, the development of a synthetic medium was claimed for the detection of K . pneumoniae; it only contains, apart from agar, I g l - l KN03,2 g I-’ KH2P04, 20 g 1-l sucrose, but it is supplemented with 1 0 pg I-’ carbenicillin. A highly selective, differential medium for the enumeration and isolation of Klebsiella species has been tellurite devised: MacConkey-inositol-potassium (MCIK) agar. With pure cultures, 100% recovery of Klebsiella was observed, and with environmental samples recovery of Klebsiella was as good as or better than on MCIC agar. MCIK agar was subsequently field tested for its ability to selectively enumerate Klebsiella species from the cold waters (1-6°C) of the Saint John River Basin (New Brunswick, Canada) which include fresh and marine waters. Results of this study indicate that 77% of the typical colonies on MCIK agar were Klebsiella species, but the total Klebsiella population enumerated was greatly underestimated; the MICK medium seems to be more specific for its target organisms but appears to lack sensitivity. Various selective media have been assessed as to their ability to detect and differentiate K. oxytoca and E. coli in drinking-water samples. Only two media, membrane lauryl sulphate agar and deoxycholate agar allowed differentiation, with K. oxytoca only able to grow at 37°C and not at 44°C. The CPS ID2 medium (Bio-Merieux) enabled the

presumptive identification of urinary tract bacterial isolates, including Klebsiella, in specimens from a rehabilitation centre. Recently, a new chromogenic plate medium (CHROMagar Orientation) for the visual differentiation and presumptive identification of Gram-negative bacterial species and enterococcal isolates was evaluated. Similarity in colour resulted in failure to discriminate between Klebsiella, Enterobacter and Citrobacter species, but these species could be readily differentiated from other members of the Enterobacteriaceae. These data indicate an urgent need for the development of a simple, reliable and specific detection and enumeration methodology for Klebsiella sp. Media Composition Suited for Cultivation of Klebsiella Strains

The composition of media suitable for cultivation of Klebsiella strains is listed in Table 2. The media are routinely sterilized by autoclaving (20min at 121°C and 2.1 atm). The carbon source is Table 2 Composition of media for cultivation of Klebsiella strains Concn. (g 1”)

Nutrient broth (Oxoid) - pH 7.4 ‘Lab-Lemco’ beef broth Yeast extract Peptone NaCl Klebsiella medium - pH 7.0 Glucose Soya peptone Yeast extract MgSO,. 7H20 K2HPO4 NaH2P04

1.o 2.0 5.0 5.0

100.0 10.0 0.5 0.5 0.7 0.7

Orskov and Orskov medium - pH 7.0 Glucose Bacteriologicalpeptone NaCl

15.0 7.0 5.0

1112 KLEBSIELLA

thereby separated from the nitrogen source to prevent Maillard reactions. Solidified media are obtained by adding 20 g 1-1 of agar before sterilization. Culture Maintenance

Klebsiella strains can be easily maintained in meat extract agar stabs at room temperature. They can also be preserved either by storage in broth, containing 10% glycerol at -80°C or by lyophilization.

Procedures Specified in National/lnternational Regulations or Guidelines No official guidelines/regulations seem to exist at national or international level with regard to specific Klebsiella detection, enumeration or threshold numbers. The reader is referred to water and food quality guidelines, related to Enterobacteriaceae or coliforms.

Medical Aspects of Klebsiella Bacteria As indicated above, Klebsiella bacteria are present in the respiratory tract and faeces of about 5 % of normal individuals. They cause a minor proportion (about 3 %) of bacterial pneumonias and can cause extensive haemorrhagic necrotizing consolidation of the lung. They occasionally produce urinary tract infection, septicaemia, bacteraemia with focal lesions and meningitis in debilitated patients. K. pneumoniae and K. oxytoca, especially, cause hospital-acquired infections. Two other Klebsiella subspecies are associated with inflammatory conditions of the upper respiratory tract:

K. pneumoniae subsp. ozaenae has been isolated from the nasal mucosa in ozena, a fetid, progressive atrophy of mucous membranes; K. pneumoniae subsp. rhinoscleromatis has been isolated from rhinoscleroma, a destructive granuloma of the nose and pharynx. K. pneumoniae is resistant to penicillin and ampicillin; resistant strains usually produce R-plasmid encoded p-lactamase. Broad-spectrum third generation cephalosporins such as cefotaxime, or aminoglycosides are used to combat normal strains (community-acquired), whereas hospital-acquired strains are multiple antibiotic resistant. Often, K. pneumoniae infections commonly occur following antibiotic treatment.

Environmental Relevance of Klebsiella Bacteria Since Klebsiella species are widely distributed in the environment and in water systems, and since often little or no differences can be detected between environmental and clinical strains, there is increasing concern about the potential health hazard related to Klebsiella. There is, therefore, a growing need to monitor these organisms in the environment, especially in (drinking) water systems, soils, aerosols, cooling waters, biofilms and industrial effluents.

Relevance of Klebsiella in the Food Sector General Aspects

The relevance of Klebsiella in foods as a contaminant or spoilage organism is only recently being addressed. Even in standard texts on food microbiology, there is little or no mention of problems related to Klebsiella food spoilage, contamination or transmission to humans. This is in sharp contrast with its ubiquitous presence in the daily human environment, and the pathogenic character of the clinical Klebsiella isolates, which are taxonomically very similar to the environmental strains. As indicated already, Klebsiella species are opportunistic pathogens, that can give rise to bacteraemia, pneumonia, urinary tract and several other types of human infection. The origin and transfer of the infection is not always clear, since Klebsiella spp. are widely distributed in nature, occurring in soil, water, grain, fruits, vegetables, biofilm etc. Many of these environmental strains belong to the species K. terrigena and K . planticola. Since they also occur, albeit in low numbers, in the human respiratory and intestinal tracts, these seem to form the main reservoir for human-to-human infection (via food or otherwise), particularly in hospitals, where the hands of personnel and aerosol formation are the main factors in transmission. Outbreaks particularly occur in urological patients and in neonatal and intensive care units. Enterotoxin-producing Klebsiella strains have also been described. K. pneumoniae strains are mainly isolated in association with several pathological processes in humans (respiratory and urinary tract infections) and in animals (metritis in mares and mastitis in cows) and as such can also enter the food chain. Equally, the importance of the occurrence of Klebsiella species in the food sector is difficult to judge. Klebsiella species are usually not selectively cultured from foodstuffs, but are present when total counts or

KLEBSIELLA 1113

the presence of Enterobacteriaceae are tested. It is assumed that K. terrigena, isolated mainly from aquatic and soil environments, and K. planticola, mainly isolated from botanical, aquatic and soil environments, are saprophytic strains, that are easily transmitted to food. K. oxytoca is present in the intestinal tract of humans and animals, and has also been isolated from botanical and aquatic environments and as such it can also contaminate food and infect humans, indicating again an urgent need for specific Klebsiella detection. In this respect, a bioluminescence-based detection of lux’ K. oxytoca strains was developed and their survival in the barley rhizosphere was studied; it was found that K. oxytoca specifically survived on the plant roots during the whole vegetative period and that it could not be isolated from the soil. Drinking Water Quality

Although it has been claimed that the attachment of bacteria, i.e. Klebsiella sp., to surfaces via capsule formation is a cause of increased bacterial disinfection resistance (for instance survival in chlorinated water supplies), it has also been found that resistance to chlorine was not related to the presence of polysaccharide, but to the formation of cell aggregates. Indeed, K. pneumoniae and K. oxytoca grown in lownutrient media were found to be more resistant to chloramine than cells grown in rich media, and to form large aggregates/flocs of 10-io3 cells per millilitre. This formation of flocs and aggregates allows the cells to survive chlorination and to enter the water distribution system. Indeed, Klebsiella sp. is one of the principal microorganisms involved in bacterial regrowth within chlorinated drinking water systems. The regrowth of this organism is of particular importance since, as a coliform, it will make compliance to water quality guidelines difficult, and it may be involved in opportunistic infections. The growth kinetics of coliform bacteria, including K. pneumoniae, have been studied under conditions relevant to drinking water distribution systems. It was found that most of them - and Klebsiella in particular - could develop in unsupplemented mineral salts medium and in the unsupplemented distribution water. This proves that environmental coliforms, and equally K. pneumoniae, can develop under the conditions found in operating municipal drinking water systems. In this context, the ability of K. pneumoniae, Entero bacter aerogenes, Agro bacterium tumefaciens, Bacillus subtilis and Pseudomonas strains to grow and maintain motility and viability in drinking water has been studied. Plate counts dropped below the detection limit within 7 days for all strains mentioned, except for Bacillus and Pseudomonas strains.

The drinking water quality in a major South African metropolitan area, in collecting water samples from private houses, apartments and public places was assessed over a period of two years. Enterobacteriaceae bacteria were found in 33% of the samples, as well as Bacillus sp. Klebsiella was also frequently found. The age of the plumbing system was clearly correlated with poorer microbiological quality of the potable water. Among 62 trademarks of bottled drinking water, a sampling of 158 bottles revealed the presence of K . oxytoca, along with other coliforms, in three bottles. The quality of packaged ice, sold in retail establishments in Iowa, USA has also been studied. A total of 18 samples were analysed in relation to the drinking water standards of the US-EPA. Only one sample exceeded the health standard and contained K. pneumoniae. Several samples had heterotrophic plate counts, which exceeded the recommendation (< 500 cfu ml-l) of the Packed Ice Association. Although such ice consumption does not represent an immediate threat to personal or public health, the potential for disease transmission exists in a sector, which is in this respect self-regulated. It is clear that Klebsiella species comprise a large part of the coliforms, usually detected as indicators of water quality; in most instances however, they are not differentiated. Food Quality

As to their occurrence in foods, the microbiology of contaminated foods in health-care facilities in the USA was surveyed and the importance of microbial surveillance, quality assurance and employee education was stressed. K. pneumoniae is mentioned as one of the encountered contaminants, together with E. coli, Yersinia intermedia, Aeromonas hydrophila, Enterobacter agglomerans, E. cloaca, Campylobacter jejuni, Acinetobacter anitraturn, Streptococcus viridans, Serratia liquefaciens, Staphylococcus sp., Salmonella sp., Corynebacterium sp., Lactobacillus s ~ . , Listeria sp. and others. High numbers of Klebsiella species were found in samples of the local food, pap ‘akamu’, prepared in Nigeria from cereals (maize, guinea corn and millet); Klebsiella sp., Enterobacter sp. and Staphylococcus sp. were the most common bacteria found. These data indicate the widespread occurrence of Klebsiella sp. in these indigenous foods, in combination with other opportunistic pathogens. K. pneumoniae subsp. pneumoniae (using the API 20 E system) was also found to be present in the industrial fermentation process of ‘Saccharina’ production (fermented fodder) from sugar cane. ‘Coliforms’ were enumerated in fresh and processed

Next Page 1114 KLEBSIELLA

mangoes (puree and cheeks) in order to establish the source of the organisms in the production chain, to determine whether they have any public health significance, and to devise methods for their control. Products from four processors were tested on two occasions, The retail packs of cheeks-in-puree having the highest coliform counts were those in which raw puree was added to the cheeks. Coliform counts in these samples ranged between 1.4 x 1O3 and 5.4 x IO4cfu g-'. Pasteurization reduced the coliform count of raw puree to 5 cfug-'. Around 47% of the 73 colonies, isolated as coliforms on the basis of their colony morphology on violet red bile agar, were identified as K. pneumoniae, using the ATB 23 E Identification System. Klebsiella strains were tested for growth at 10°C, faecal coliform response and fermentation of D-melezitose; these tests are used commonly to differentiate the three phenotypically similar strains K. pneumoniae, K. terrigena and K. planticola. Results indicated that 41% of the isolates gave reactions typical of K. pneumoniae. A further 44% of strains gave an atypical reaction pattern and were designated 'psychrotrophic' K. pneumoniae. K. pneumoniae counts of 2.1 x lo3-4.9 x lo4cfu g-' were predicted to occur in the retail packs of mango cheeksin-puree produced by the processors, who constituted this product with raw puree. In view of the opportunistic pathogenic nature of K. pneumoniae, its presence in these products is considered undesirable and steps, such as pasteurization of puree, should be taken in order to inactivate it. Recently attempts have been made to correlate the presence of selected pathogens (Campylobacter jejuni, C. coli, Salmonella, K. pneumoniae and E. coli 0157:H7) in fresh hand-picked blue crab meat and general microbial quality to sanitation practices by the processors (Chesapeake Bay region, USA). K. pneumoniae was isolated from 51 samples out of the 240 (21%) (0.3-4.3 most probable number (MPN)g-I), followed by C. jejuni (36 out of 240), C. coli (14 out of 240). Salmonella and E . coli 0157:H7 were not detected in any of the 240 samples analysed. The foregoing data indicate again that Klebsiella sp. is frequently present as a contaminant in water and food, often in high numbers. They are commonly lumped within the group of the Enterobacteriaceae or coliforms, with most attention always focused on the well known food pathogen members of the group. It is only recently that Klebsiella is being selectively searched for and 'looked after' as a genushpecies, relevant to food microbiologists too.

Industrial Aspects of Klebsiella Bacteria Production of 2J-butanediol

Most Klebsiella species are saprophytic, some are pathogenic and only a few are of industrial use. Under controlled fermentation conditions, K. oxytoca strains produce high levels of 2,3-butanediol, an interesting chemical feedstock or liquid fuel, from sugary substrates such as glucose, xylose and whey permeate. Due to its toxicity to the producer cells, only moderate concentrations (approximately 100 g 1-I) can be obtained in even optimized fermentation processes. This, together with the high boiling point and hygroscopicity of 2,3-butanediol, makes recovery costs high. 2,3-Butanediol can be chemically converted into butadiene, the raw feedstock for synthetic rubber, or into other derivatives such as ethylmethylketone (used as a solvent, fuel additive) and tetramethylether (antifreeze) or into polyester plastics. Biofilm Formation by Klebsiella sp.

As a result of its capacity to form capsules, Klebsiella species are often a main cause of (undesirable) biofilm formation and fouling in cooling water systems, piping and other industrial equipment. The biofilm-forming capacity of several Klebsiella species, isolated from pulp and paper mill water, and of Klebsiella terrigena BCCM strains has been studied in vitro by the authors in 2 litre lab fermenters. The capsular polysaccharide from one isolate was recovered (up to 4.6gl-'), its rheological properties were identified as pseudoplastic and its sugar composition was identified as: L-fucose, Lrhamnose, D-galactose, D-glucose, D-mannose and D-glucuronic acid. Enzymes which can efficiently hydrolyse and remove biofilm have been looked for. The Klebsiella Capsule as a Source of Unusual Sugars

The Klebsiella capsule, as described above, often contains unusual sugar moieties such as L-fucose and L-rhamnose, and the authors have cultivated such capsular bacteria on a large scale, as a source of these specialty sugars, which are otherwise difficult to obtain. Klebsiella as a Vitamin Producer in Fermented Foods

Recently, the formation of vitamin B12 was demonstrated by strains of K. pneumoniae, isolated from Indonesian tempeh samples, during controlled solidstate tempeh fermentation. The absence of enterotoxins in these strains was confirmed by using PCR techniques, and it was even suggested that these safe

LABORATORY DESIGN 1119

LABORATORY DESIGN M Ahmed, Food Control Laboratory, Dubai, United Arab Emirates Copyright 0 1999 Academic Press

Introduction Food microbiology laboratories play an important role in the control of food hygiene, quality and safety. The potential hazards associated with pathogenic microorganisms in these laboratories together with the development of strict legislation to promote health and safety at work have led to higher standards of laboratory design. Contamination of samples within the laboratory through air and other sources has been a major problem associated with microbiological analysis. The laboratory design must meet the requirements to avoid contamination. Cleanliness, ventilation, accessibility, storage, waste disposal, security, fire protection and emergency precautions must all be considered at the initial stage of design. Even though the final design of the laboratory is made by architects and engineers, involvement of microbiologists is essential when taking important decisions that affect the working environment and conditions. Microbiologists should work in close association with the architect and explain all the technical and safety requirements of each room. They should also follow the building through the different stages of construction to ensure that all the requirements included in the design are fulfilled.

Study Report Microbiological laboratories can be broadly classified into three categories: 1. Hygiene control laboratories performing limited microbiological tests to evaluate sanitation and hygiene procedures followed in food production plants, restaurants and catering establishments. 2. Quality control laboratories involved in the testing of imported and locally produced foods as well as hygiene control, which perform a wide range of analyses and carry out work-related research and investigations. 3 . Research laboratories involved in carrying out

research and development (R&D) but not involved in quality control. Before designing a laboratory, a study report should be prepared by a consultant with good knowledge and experience of designing laboratories. The technical experts of the consultant should meet the management, microbiologists and other technical staff and discuss in detail their requirements. Due consideration should be given to their views and recommendations while preparing the study report, which should consist of the following: scope and objectives of the laboratory organization chart indicating the various functions of the laboratory number of technical, administrative and support staff expected number of samples to be analysed details of technical facilities required service requirements the interrelationships, if any, between the functions of the laboratory and other disciplines (chemistry, biochemistry, nutrition, etc.) budget requirements. The study report should also address the scientific and technical developments in the area and make provisions for future expansion of the laboratory. The recommended organization of a food microbiology laboratory suitable for routine quality control analysis is shown in Figure 1. The laboratory consists of a general microbiology unit, with culture techniques and media preparation sub-units, and an advanced microbiology unit, with rapid diagnostic techniques and instrumental techniques sub-units. The administration sample management, quality management, R&D and training management, and calibration and maintenance constitute other functions. These may be common to a laboratory consisting of multiple disciplines such as chemistry, biochemistry and nutrition. The major activities of different functions in the laboratory are listed in Table I.

1120 LABORATORY DESIGN

FOOD MICROBIOLOGY LABORATORY (Head of Laboratory)

DISCIPLINES

1

Quality Management

kl

Administration

I

'ifResearch and Training Management

Sample Management

Calibration and Maintenance

1

Advanced Microbiology Unit (Head of Unit)

I

I

Techniques (In-Charge) Isolation and identification Food poisoning 1analysis I

Certification and monitoring

- Standardization

Techniques (in-Charge)

Media preparation and sterilization lmpedimetry

preparation Sterilization of glasswareiutensils

DNA hybridization

Bioluminescence

Decontamination of materials Other techniques

Figure 1 Food laboratory organization chart.

Building Layout Many types of laboratory layouts are possible, depending on scope of work, space and budget. The building layout for a food microbiology laboratory

I

Turbidimetry

Polymerase chain reaction

carrying out routine quality control analysis of a wide range of samples in addition to conducting a limited number of applied research projects is shown in Figure 2*

Table 1 Major activities of a food microbiology laboratory Unitlfunction

Sub-unit

Major activity

General microbiology

Culture techniques

Advanced microbiology

Media preparation and sterilization Rapid diagnostic techniques

Certification and monitoring programmes, food poisoning -emergency analysis, standardization Preparation and sterilization of media, glassware, sample utensils, decontamination and washing of used materials Application development and implementation of immunoassay, DNA hybridization, API, etc. Application development and implementation of impedimetry, turbidimetry, bioluminescence, PCR, etc. Implementation of quality assurance system (IS0 9002/ISO Guide 25), internal quality control, proficiency testing, audits, etc. Equipment and building maintenance, calibration of equipment, maintenance of services Planning, budgeting of R&D work, coordination with different units, training requirements and their planning and scheduling, management of external training programmes Receipt, identification, registration, preparation of composite samples, assigning code numbers, distribution of samples to different functions Secretariat, personnel management, budget/accounts, purchase/stores, library and housekeeping

Instrumental techniques Quality management

Quality management

Calibration and maintenance

Calibration and maintenance

R&D, training management

R&, training management

Sample management

Sample management

Administration

Administration

LABORATORY DESIGN 1121

‘ 8 ’ 28

5

29

-

6

2

A

3

n

M

/I/I

4 26 27 25

23

f-

w

3

General Considerations

The microbiology laboratories have a unique contamination problem and should have a central air conditioning system if possible. This system should be divided into zones depending on the type of work carried out in different rooms to facilitate exchange of fresh air and to take necessary precautions against environmental contamination. The incoming air is filtered through 0.2ym filters to reduce the risk of environmental contamination of the laboratory. The humidity must be kept low to reduce problems with hygroscopic materials such as media and chemicals, and to avoid growth of moulds on laboratory surfaces. Air conditioning also stabilizes room temperatures, enabling incubators to function more efficiently. Temperatures and relative humidity should be comfortable for workers and suitable to the requirements of the laboratory equipment. Normally an ambient temperature of 21-23°C and a relative humidity of 40-50% are recommended.

12

M

w

E

3 15

ia

3

As, W

16L

- 19 21

11

’

d 22

10

M

20

U

7

M

7

24

u

L

7

9

14

13

17

Future Expansion Future expansion of activities, increases in workload and staff should be considered when designing a laboratory. The design should include provision for a minimum of 25% of expansion. The design should also be flexible to allow room functional changes and allocation of new activities. Allocation of Space The design should allow maximum utilization of laboratory space. The subunit of media preparation and filling, decontamination of used material and cleaning of glassware should be separated from the analytical area. Within the analytical area, isolation and identification of pathogenic materials should be carried out in a separate room if possible. Safety The safety of the laboratory personnel should be taken care of in the design. The laboratory should be equipped with fire extinguishers and alarms, a sprinkler system, eye-wash stations and safety showers. Fire and smoke detectors are also recommended. A comprehensive safety programme should be a vital part of all laboratory procedures.

Lighting It is recommended that laboratory lighting be maintained at an average intensity of 0.5-1 klx (50-1 00 footcandles). Dependence on natural sunlight during the day is discouraged: direct exposure to sunlight is known to alter the performance of media, chemicals and reagents. Likewise, analytical work must not be performed in direct sunlight since final results are affected.

Access Two exits should be provided for the building for prompt exit in the event of fire or other emergencies. Entrances should be designed to minimize pedestrian traffic.

Storage Sufficient storage space should be provided for equipment, materials and samples. The laboratory wall space should be utilized for additional shelving, protected by glass-enclosed cabinets to provide a dustfree environment for storage of media, chemicals and other materials. Samples should be stored in refrigerators, freezers or at room temperature according to the procedures outlined in the operational manual.

Security A security system must be provided to restrict entry into the laboratory building. Laboratory rooms should be separated from offices by another security system, apart from the general security system, to restrict unauthorized entry into the laboratory rooms, to avoid contamination and for effective operation.

1122 LABORATORY DESIGN

The Building Programme

The building programme is a written document that describes and quantifies the design goals for a building. The goal of a good programme is to define a building that will have ample space, meet the technical requirements of the user, function safely and meet the owners’ budget. The design team with the assistance of the laboratory management and technical staff will develop the building programme from the analysis of data collected on the following: 0 0

0

the range of analyses to be carried out the number and types of personnel who will occupy the building the interrelationships of functions and personnel the expected workload.

appropriate places in each room (Figs 3, 4). Centralized services for gases, deionized or distilled water, etc., are preferred. Electrical Connections

It is essential to determine the total electrical load of each room. In order to achieve this, the equipment to be placed in each room must be decided upon and its power requirements (voltage, current rating, etc.) listed and supplied to the consultant or contractor. Equipment such as autoclaves and washing machines may require three-phase connections. These have to be identified and separated from equipment of lowenergy consumption. It is recommended that items of high electrical rating are placed in different rooms to balance the power consumption. Proper earth ---

The programme should describe the architectural, mechanical, electrical, plumbing and fire protection criteria for the functions to be accommodated. It should also identify areas of special concern for safety such as high hazard areas containing flammable, toxic or pathogenic materials, and should also address the problem of waste removal. The main tasks and sequence of a building programme are as follows: 0 0 0

analyse the study report interview management, technical staff and other users establish space standards list various activities and room types required for such activities draw a layout diagram for different room types determine the number and area of each room type develop room data sheets specifying details of functions establish building net and gross areas describe basic mechanical, electrical, electromechanical and plumbing systems describe the services estimate the cost of construction.

1

r

3

m

Bench

ic TA

Telephone connection

C=

Data communication

c-t

Supply of Services Proper supply of services such as electrical connections, gases, hot water, demineralized or distilled water, compressed air, vacuum, telephone and data networks, fire protection systems, smoke detection system and alarms, emergency showers, sprinklers, eye-wash stations, etc. is essential for efficient running of a laboratory. The services should be installed in

Double electric point

E

-

Cold water tap Medical mixing tap Demineralizedidistilled water

- G

LPG

0- CA

Compressed air

0- N

Nitrogen

Figure 3 Room data sheet - culture techniques. General laboratory furniture: 1, window bench, 75cm high; 2, island bench, 90 cm high; 3, stool; 4, slab sink; 5, safety storage cabinet; 6, biohazard safety cupboard.

LABORATORY DESIGN 1123

5

for the purpose. The piped supply runs along the corridors, with branches into the laboratory rooms. Each branch should be equipped with a valve enabling the supply to be shut off in an emergency. Stainless steel tubing with Swagelock@ fittings is recommended for piping the gases. Welding should be avoided. Pressure checks and certification from the contractor duly approved by the consultant and approval from the civil defence authority are required prior to the actual supply of gas. All the lines must be accessible for future leak checks. Gases such as nitrogen and COz may be required only for anaerobic work stations. If the use of such gases is limited to one or two rooms, the cylinders may be housed in a purpose-built cabinet near to the point of use or within the laboratory, as they are not inflammable or hazardous. Compressed Air and Vacuum

i (

Data communication

C c

3--t

-it

0-

G

0- CA

3-

Double electric point Telephone connection

TA

Cold water tap Medical mixing tap Demineralizedidistilled water LPG

Compressed air

N Nitrogen

Figure 4 Room data sheet - instrumental techniques. General laboratory furniture: 1, window bench, 75 cm high; 2, wall bench, 90cm high; 3, wall bench with sink, 90cm high; 4, island bench, 90cm high; 5, stool; 6, slab sink; 7, safety storage cabinet; 8, adjustable chair.

connections must be provided with bonding resistance per earth of less than 1ohm. The minimum resistance of the earthing net should be 1.2 ohm. Circuit breakers must be installed at each workbench. Gases

Most microbiological laboratories require the following gases:

0

liquefied petroleum gas (LPG) nitrogen carbon dioxide oxygen.

The rooms and the locations in each room requiring supply of different gases should be identified and listed. It is possible to provide all laboratory rooms with a supply of piped gases; the gases are supplied in bulk cylinders and stored in an outhouse built

Laboratories requiring compressed air may be supplied from a centrally located compressor connected to the laboratory by a system o n copper or highpressure plastic pipes. The air should be dried to a dewpoint of - 15°C and freed from oil droplets with the aid of filters. The pressure in the system as far as the branches into the laboratories should be 7 bar, which in the laboratories should be reduced to a working pressure of 3 bar. Vacuum may be supplied through a central system if it is required in many rooms; otherwise, small vacuum pumps may be used. Hot and Cold Water

The building should be provided with a supply of process water and also drinking water if necessary. The process water should be equipped, downstream of the meter, with a break installation. The pressure measured at the highest tap should be 2.5 bar. The pipes should be laid in such a way that water nowhere becomes stagnant. Wherever necessary, hot water should be provided from closed-in boilers. The minimum temperature of the water should be 60°C. Medical mixing taps with a lever should be provided for mixing cold and hot water, in order to avoid contamination from the hands of the microbiologist. Demineralized and Distilled Water

A supply of demineralized or distilled water should be available in all the laboratories. Demineralized water can be prepared with the aid of an automatic double-column demineralization system housed in a centrally located room. Distilled water can be prepared with the aid of electrically operated distillation equipment. In both cases, the water should be transported through plastic tubing to the laboratories. The demineralized water should have specific conductance less than 5 pa cm-'.

1124 LABORATORY DESIGN

Telephone and Data Network Connections

Eye-washes and Emergency Showers

Rooms and laboratories requiring telephones must be identified and the appropriate connections provided. Most modern laboratories use client-server technology to manage sample information and analytical data. Several laboratory information management systems (LIMS)are commercially available, and could be customized. Data network connections are provided in the laboratories to facilitate data entry and necessary approvals. They should be located preferably on the side workbenches at a height of 75cm or near the desks, slightly away from the working area. Network connections are also required on island benches where analytical instruments with data stations are located, to hook up with LIMS for direct transport of instrumental data.

All laboratory rooms should be provided with eyewash stations if possible. Emergency showers are required in laboratories where hazardous chemicals or other materials are being used, and must be easily accessible.

Fire Protection, Smoke Detectors and Alarms

For the purpose of preventing fires, and quelling any fires that break out, it is necessary to draw up plans for fire prevention and firefighting systems. The laboratory building should be divided into compartments separated by fireproof walls and ceilings. The floor surface area of a compartment may vary from 500m’ to 750m2, or as necessary to achieve a logical arrangement of the compartments. In accordance with internationally accepted test methods, the fire retardance of floors and ceilings should not be less than 1h. All electric and other cables should be passed through fireproof blocks. All spots within the building should be within reach of the jet of a fire hose connected to the process water mains. The reels should be hung on the walls of the corridors, and the hoses should not exceed 30 m in length. In addition to the fire hoses, fire-extinguishers should be distributed throughout the building. Their filling should be in accordance with the type of fires they are likely to be used against. The building should be equipped with an automatic fire alarm system. Ionization detectors should be mounted in all rooms or spaces where fires may start, and are mandatory in rooms where people are at work. A (repeater)fire alarm should accompany each fire-hose reel. The system should be combined with an acoustic alarm system (hooters or sirens) and should be fully independent of the building control system. An emergency power supply system is needed to illuminate and mark escape routes, enabling people to leave the building in the shortest possible time in emergencies. It should be equipped with a no-break unit. The emergency power supply must serve all the electrical equipment that must be kept in operation in emergencies.

Design of Furniture and Choice of Finishes Laboratory furniture normally consists of workbenches, cupboards, wall units, desks and drawers. Prefabricated furniture units are available. Benches

Workbenches can be wall-mounted or island type. The framework should comprise a mild steel tubular framework based on a modular construction with an epoxy-based plastic coating, and should incorporate adjustable levelling jacks, pipe clips and cableways. The bench top should be set at a height of 90-95 cm for normal work in a standing position. The desk tops or ‘sit down’ benches can be at a height of 65-79 cm as needed to accommodate microscopes, plate counting, computer usage or paperwork. The low-level benches should be mounted on the window side walls to accommodate microscopes, network computers, etc. It is also necessary to keep instruments on low-level island benches for easy access to the reverse of the instrument. Services such as electrical sockets and gas connections on island benches meant for installing instruments should run at the side of the bench for optimum utilization of the bench space. The storage cabinets and drawers should be suspended from the bench connections, and there should be a combination of cabinets and drawers on each bench. Cabinets may be built with WBP grade plywood with an inert and corrosion-resistant finish with minimum seams (e.g. seamless melamine). Drawers may be constructed with corrosion-resistant faced plywood. The cabinets and drawers on the workbench should be fixed in such a way that adequate legroom remains. Ample space should be allowed for refrigerators and writing desks when installing wall-mounted workbenches. Bench Tops

The bench tops may be constructed from solid melamine, WBP plywood with a seamless melamine finish, WBP plywood with stainless steel top and edges, or solid hardwood with a laminate finish. The bench tops should have a smooth surface and be easily disinfected. Cracks and crevices should be minimal as they provide an opportunity for the build-up of debris which may contribute to cross contamination of

LABORATORY DESIGN 1125

samples. Stainless steel tops must be provided on the benches in the washing room. Sinks

At either end of the benches (apart from benches meant for installing instruments) stainless steel sinks should be mounted with 60 cm side adjoining them, a 50 cm side jutting out, and a depth of 25 cm. Medical mixing taps (cold and hot water) and a deionized or distilled water tap should be mounted above the sinks, as required. Seating

Laboratory stools and chairs of adjustable or fixed heights should be provided. Stools should be used at the workbenches, and chairs may be used at computer desks. Wall-mounted Cupboards

Cupboards with sliding glass doors may be mounted on the walls for storing reagents, media, etc. Other cupboards may be used for books, catalogues and instrument files. Laminar Flow and Biohazard Safety Cabinets

Safety cabinets should comply with standards set by organizations such as the British Standards Institution, the Standards Association of Australia and the National Sanitation Foundation of the USA. Care must be taken in siting equipment that might generate air currents, e.g. fans and heaters. The safety cabinets should be installed in proper sites in the laboratory. Safety cabinets are intended to protect the worker from airborne infection. Work should be done in the middle to the rear of the cabinet, not near the front and workers should not remove their arms from the cabinet until the procedure is completed. After each set of manipulations, aerosols should be swept into the filters. The operator’s hands and arms may be contaminated and should be washed immediately after ceasing work. Bunsen burners and micro-incinerators should not be used as they disturb airflow.

Facilities for Incubation and Refrigeration lncubators

Incubators and incubator rooms must be properly constructed and controlled. It is best to obtain the largest possible models to prevent crowding of the interior. Small incubators suffer wider temperature fluctuations when their doors are open than do larger models. Incubator rooms, if used, must be well insulated, equipped with properly distributed heating

units and have appropriate air circulation. They should be installed by specialist suppliers. The rooms should be supplied with stainless steel shelves suitable for holding Petri dishes, flasks, etc. Wooden shelves are not recommended because of the problem of mould growth in a humid atmosphere. The recommended temperatures for incubators in food laboratories are 15-20°C, 30-37°C and 55°C. Cooled incubators must be fitted with a refrigeration system and heating and cooling controls, which must be correctly balanced. Incubators should be kept in rooms where temperatures are within the range 16-27°C. The incubator temperature must not vary by more than 2 1°C. Chamber temperature must be checked twice daily (morning and afternoon). The thermometer bulbs and stem must be submerged in water or glycerol to the stem mark. For best results use a recording thermometer. Water Baths

Water baths should be of an appropriate size for the required workload with a suitable water level maintained. When the level of water in the bath is at half to two-thirds the level of the column of liquid in the tube, convection currents keep the constituents of the tube well mixed and hasten reactions such as agglutination. Water baths should be equipped with electrical stirrers to prevent temperature stratification. They must also be lagged to prevent heat loss, although the walls are fitted with sloping lids to prevent heat loss and dripping of condensed water on materials. To avoid choke deposits on tubes and internal surfaces, distilled or deionized water should be used. Only racks made with stainless steel, heatresistant rubber, plastic, plastic-coated substances or corrosion-proof materials should be used. The temperature of the water bath must be monitored and recorded daily using a certified thermometer. Refrigerators

A refrigerator maintained at 0-4°C for storing untested food samples is required. Another refrigerator to cool and maintain the temperature of media and reagents may also be used. The temperature of the refrigerator should be checked and recorded daily, and it should be cleaned monthly or more often when required. Refrigerated rooms, if used, must be well insulated and equipped with a distributed cooling system. A continuous temperature monitoring and recording system equipped with an alarm must be used. The temperature at different points should be recorded daily. Stainless steel shelves should be installed for storing samples. Stored materials should be identified and dated, and stored in such a way that

1126 LABORATORY DESIGN

cross contamination does not occur. Expired materials should be discarded at regular intervals, e.g. quarterly. Freezers

A freezer or a freezer room to maintain the temperature of frozen food items at - 18°C is required. The temperature should be recorded daily. A recording thermometer with an alarm system is highly desirable. The freezer should be defrosted and cleaned twice a year. Materials should be identified and dated, and outdated materials should be discarded quarterly. A separate freezing space should be identified for storing freeze-dried bacterial cultures.

Clean/Dirty Sterilization Facilities Sterilization facilities are required for sterilizing prepared media, diluents, etc., and used glassware, Petri dishes, flasks, tubes, etc. prior to washing or disposal. The use of heat, particularly moist heat, is the most desirable and widely used method of sterilization in the microbiology laboratory. When using heat sterilizing techniques, it is necessary to know the difference between dry and moist heat and the limitations of each. Moist heat leads to the destruction of microorganisms through the irreversible denaturation of enzymes and structured proteins. The temperature at which denaturation occurs varies with the latent heat of steam. With dry heating, the primary lethal process is considered to be oxidation of cell constituents. Thus, sterilization methods involving dry heat require higher temperatures and longer exposure time than are required with moist heat. Hot-air Oven

Sterilization by hot-air oven is achieved by the slow penetration of heat into the materials. The efficiency of this process can be increased by the use of circulating fans. Modern equipment has electronic controls which can be set to raise the temperature to the required level, heat for a specified time and switch off automatically. These ovens are fitted with solenoid locks to prevent the oven being opened before the cycle is completed. This protects the staff from accidental burns and safeguards the sterility of the materials. The load should be packed in the oven chamber in such a way that sufficient space remains between the articles for circulation of hot air. The high temperature needed to achieve dry heat sterilization has a damaging effect on many materials. This method should therefore be used only for thermostable materials that cannot be sterilized by steam owing to deleterious effects or failure to penetrate. Materials that can be sterilized by this method include heat-resistant articles such as glass Petri dishes, flasks, pipettes, metallic objects and coated materials.

The performance of a hot-air oven should be tested quarterly with commercially available spore strips or spore suspension. The temperature should be monitored with a certified thermometer, accurate in the temperature range of 160-180°C. Autoclaves

The minimum recommended standard for sterilization by autoclaves is the exposure to steam at approximately 1 bar pressure, equivalent to 121"C, for 15 min. Saturated steam is a much more efficient means of destroying microorganisms than either boiling water or dry heat. Air has an important influence on the efficiency of autoclaving. If about 50% of the air remains in the autoclave, the temperature of the steam-air mixture at 1 bar is only 112°C. As successful autoclaving depends on the removal of all the air from the chamber, the materials to be sterilized should be packed loosely. There are two types of laboratory autoclaves: 0

pressure cooker models gravity displacement models.

The pressure cooker is a simple benchtop autoclave consisting of a vertical metal chamber with a strong metal lid which can be fastened down and sealed with a suitable gasket. The lid is fitted with an aidsteam discharge trap, a pressure gauge and a safety valve (Fig. 5). Steam is generated from the water in the bottom of the autoclave by an external immersion heater or a steam coil. The gravity displacement autoclave, widely used in microbiological laboratories, consists of a chamber surrounded with a jacket containing steam under pressure, which heats the chamber wall. The steam enters the jacket from the main supply which is at high pressure, thus forcing the air and condensate to flow out of the drain by gravity displacement (Fig. 6). In modern autoclaves, air and steam are removed by

*

Safety valve ~

iPressure /gauge

Airisteam discharge valve

Chamber

Figure 5

Pressure cooker autoclave.

Next Page LABORATORY DESIGN 1127

Combined pressure and vacuum gauge

Pressure gauge Jacket

\

Valve

Safety valve

Cotton-wool filter

Q 1 ,

,

f .-2 3

Tochamber Chamber

Vave or steam ejector

Strainers

11 7Nr-;-ptee ,,

lireFigure 6

Door

I I /’

,/ Thermometer Valve

Gravity displacement autoclave.

vacuum pumps and flexible thermocouple probes are fitted in the chamber so that the temperatures at various parts of the load may be recorded. The performance of the autoclave should be checked monthly using spore strips or suspension. Log books and other records should be maintained for each run, listing the items sterilized, temperature, pressure and time. Washing Machines

A washing machine may be used for cleaning and drying glassware and other heat-resistant articles. The machine should be capable of washing, rinsing and drying cycles. A log book should be maintained with the details of the programmes used and the materials washed.

Safety Glasses or Goggles

Safety goggles are essential for viewing ultraviolet cabinets and other equipment that may emit UV radiation. Masks

Face masks with various filters are available for use in laboratories. Appropriate filters are required for working with pathogenic microorganisms and spores, acid fumes, solvent vapours, etc. Clothing for Entering Freezers or Cold Rooms

Special clothing is available to protect staff entering freezers or cold rooms, and must be worn if staff intend to work for long periods in such rooms. Gloves

Personnel Requirements Lockers

Lockers are needed to hold the personal belongings of the staff. They should be spacious enough to hold laboratory coats, etc. They may be kept in a staff room. Laboratory Coats

Laboratory coats must be composed of 100% cotton materials. Polyester or polyester blends must not be used as they easily catch fire. Coats should be longsleeved and knee-length. They should be washed and decontaminated at least once a week.

Latex, rubber, leather and heat-resistant gloves are available for use. Gloves, Hot Hand@ Protector pads must be used when handling hot beakers, conical flasks, etc. First Aid

A first-aid box and fireproof blankets must be kept in a conspicuous place near the door for use in an emergency. See also: Good Manufacturing Practice. Laboratory Management: Accreditation Schemes. Process Hygiene: Designing for Hygienic Operation.

MEAT AND POULTRY/Spoilage of Meat 1253

M I

Malolactic Fermentation see Wines: The Malolactic Fermentation.

I

I

Manothermosonication see Minimal Methods of Processing: Manothermosonication.

I

~~

Manufacturing Practice see Good Manufacturing Practice.

1

Mathematical Modelling see Predictive Microbiology and Food Safety.

I

MEAT AND POULTRY Contents Spoilage of Meat

Curing of Meat Spoilage of Cooked Meats and Meat Products

I Spoilage of Meat George-John E Nychas and Eleftherios H Drosinos, Department of Food Science and Technology, Laboratory of Microbiology and Biotechnology of Foods, Agricultural University of Athens, Greece Copyright 0 1999 Academic Press

Introduction Spoilage of meat is an ecological phenomenon, encompassing the changes of the meat ecosystem during the development of its microbial association. The establishment of a particular microbial association of meat depends on the ecological factors that persist during processing, storage, transportation and in the market. In meat, five categories of ecological determinants influence the development of the initial and transient microbial associations and determine the rate of attainment of a climax population by the ephemeral spoilage microorganisms (those that fill the niche available by adopting R-ecological strategy as a result of enrichment disturbance of an ecosystem).

These are (1)the intrinsic factors associated with the physico-chemical attributes and structure of meat (e.g. pH, water activity, buffering power, the presence of naturally occurring or added antimicrobial components, Eh and redox poising capacity, and nutrient composition - carbohydrate content and, in particular, the concentration of glucose); (2) the processing factors; ( 3 ) extrinsic parameters that have selective influences, such as temperature, relative humidity and the composition of the gaseous atmosphere obtaining during distribution and storage; (4) the implicit factors (intrinsic biotic parameters) that play an important role in the genesis of spoilage associations; and ( 5 ) the emergent effects due to those factors that interact to produce effects greater than would be expected from their action in isolation. In essence all of the above determinants constitute the dimensions of a particular ecological niche - an ndimensional hypervolume. Indeed, the ecosystem approach is pertinent to an analysis of changes occurring in meat or meat products. In practice, therefore, meat technologists attempt to modify some or all of

1254 MEAT AND POULTRY/Spoilage of Meat

the dimensions noted above in order to extend the shelf life of meat or to create new products.

Table 1. The genera of bacteria and yeasts most frequently found on meats and poultry ~~

Genus

Typical Microflora of Fresh or Frozen Meat Contamination and Spoilage

The microbiology of carcass meats is greatly dependent on the conditions under which animals are reared, slaughtered and processed. Thus the physiological status of the animal at slaughter, the spread of contamination during slaughter and processing, the temperature, and other conditions of storage and distribution are the most important factors that determine the microbiological quality of meat. The characteristic microbial associations developing on meat and in meat products are the result of the determinants noted above on the growth of microbes initially present in the fresh meat or, more generally, introduced during processing. As the inherent antimicrobial defence mechanisms of the live animal are destroyed at slaughter, the resultant meat is liable to rapid microbial decay. Unless effectively controlled, the slaughtering process may cause extensive contamination of the cut face of muscle tissue with a vast range of both Gram-negative and Gram-positive bacteria as well as yeasts (Table 1). Some of these microorganisms will be derived from the animal’s intestinal tract, and others from the environment with which the animal had contact at some time before or during slaughter. Studies on the origin of the contaminants have shown that the source of Enterobacteriaceae on meats is associated with work surfaces and not with direct faecal contamination. Moreover, psychrotrophic bacteria are recovered from hides and work surfaces within an abattoir as well as from carcasses and butchered meat at all stages of processing. Microorganisms of the Spoilage Association

Although a range of microbial taxa are found in meat (Table l ) ,its spoilage in developed countries is caused by the selection of relatively few of these organisms (Table 2). It is evident from Table 3 that chill storage and the gaseous composition around meat packed in vacuum or in modified atmospheres exert strong selectivity on its microflora. Selective factors favour the growth of particular organisms and, as a consequence, a characteristic microbial association is present at the time of spoilage and it will manifest characteristic spoilage features. For example, with the advent of supermarkets in the late 1950s, storage of meat aerobically at chill temperatures and high relative humidity became a major selective factor and

Fresh meat

Processed meat

VP/MAP Poultry

xx xx

X

X

xx

X

X

Bacteria Acinetobacter Aeromonas Alcaligenes Alteromonas Arthrobacter Bacillus Bacteroides Brochothrix Campylobacter Carnobacterium Chromobacterium Citrobacter Clostridium Corynebacterium Enterobacter Enterococcus Escherichia Flavobacterium Hafnia Janthinobacterium Klebsiella Kluyvera Kurthia Lactobacillus Leuconostoc Listeria Micrococcus Moraxella Neisseria Pantoea Pediococcus Planococcus Plesiomonas Providencia Proteus Pseudomonas Psychrobacter Serratia Shewanella Streptococcus Streptomyces Staphylococcus Vibrio Weissella

X

X X

X

X

X

X

X

X

X

X

X

X

X X

X

xx

X

X

xx

X

xx X

X X

X X

X

X

X

X

X

X

X

X

X

X

xx X

X

xx

X X

X

X

xx xx X

X X

xx

xx

X

X

X

X

X

X X

xx X

X X

X

X X

X

X

X X X X

X

xx

X X

X

xx

X

X

X

X X

X

X

xx

X

X

X

X X X

X

Yeasts Candida Debaryomyces Rhodotorula Saccharomyces Torulaspora Trichosporon

xx

xx

X X

X X

xx X

xx X

X

Key: x, known to occur; xx, most frequently reported. VP/MAP, meat stored under vacuum or modified-atmosphere packaging.

MEAT AND POULTRY/Spoilage of Meat 1255

Table 2 Psychrotrophic bacteria associated with chilled meats and meat products

Table 3 Specific spoilage flora on fresh meat stored at 0-4°C in different gas atmospheres

Gram-negative bacteria

Gas composition

Gram-positive bacteria

Aerobes Catalase reaction weak Pseudomonas spp. Brochothrix thermosphacta rRNA homology, group I: P fluorescens biovars I, (I, 111, IV, V (includes 7 clusters) I? lundensis, I? fragi Shewanella putrefaciens Alteromonas spp. Alcaligenes spp., Achromobacter spp. Flavobacterium spp. Moraxella spp. Psychrobacter spp. I? immobilis, P phenylpyruvica Acinetobacter spp. A. lwoffi, A. johnsonii Facultative anaerobes Catalase reaction Photobacterium spp. negative Vibrio spp. Lactobacillus spp. Aeromonas spp. L. sake Plesiomonas spp. L. curvatus L. bavaricus Serratia spp. Carnobacterium spp. S. liquefaciens S. marcescens C. divergens C. piscicola Citrobacter spp. C. freundii, C. koseri Leuconostoc spp. L. carnosum Providencia spp. L. gelidum I? alcalifaciens, F! stuarti/, L. amelibiosum P rettgeri L. mesenteroides subsp Hafnia spp. mesenteroides Hafnia alvei Weissella spp. Pantoea agglomerans W. hellenica Enterobacter spp. W paramesenteroides E. cloacae, Lactococcus raffinolactis E. aerogenes Clostridium estertheticum E. agglomeransl Erwinia herbicola complex Klebsiella spp. K. pneumoniae Kluyvera spp. Proteus spp. I? vulgaris, I? mirabilis

Specific spoilage flora

Air Pseudomonas spp. > 50% C02 mixed with O2 Brochothrix thermosphacta > 50% COP Enterobacteriaceae < 50% C o n mixed with O2 Brochothrix thermosphacta, lactic acid bacteria > 50% CO? Lactic acid bacteria Lactic acid bacteria 100% con Vacuum pack Lactic acid bacteria, Brochothrix thermosphacta ~~

Under Aerobic Conditions Although the Gramnegative aerobic psychrotrophic bacteria of meat include a number of well-defined species (see Table 2), it is now well established that under aerobic storage three species of Pseudomonas - P. fragi, P. fluorescens and P. lundensis - are the most important. Off odours are present when the population of pseudomonads is of the order of l o 7per square centimetre and slime when these organisms reach 10' per square centimetre. In practice off odours become evident when the pseudomonads have exhausted the glucose and lactate present in meat and begin to metabolize the amino acids. Although rarely, if ever, contributing significantly to the spoilage flora on meat and meat products, the Enterobacteriaceae have been considered as indicators of food safety. With ground beef, Pantoea agglomerans, Escherichia coli and Serratia liquefaciens were the major representatives of this family (see Table 2). Brochothrix thermosphacta has been detected in the aerobic spoilage flora of chilled meat but it is not considered to be important in spoilage except possibly of lamb. This organism has been isolated from beef carcasses during boning, dressing and chilling. Moreover, lairage slurry, cattle hair, rumen contents, soil from the walls of slaughterhouses, the hands of workers, the air in the chill room, the neck and skin of the animal as well as the cut muscle surfaces have been shown to be contaminated with this organism. Brochothrix thermosphacta is one of the main - if not the most important - cause of spoilage which can be recognized as souring rather than putrefaction. This type of spoilage is commonly associated with meat packed under modified atmospheres.

Pseudomonas spp. are considered to be the main spoilage organisms. Gram-positive bacteria (lactic acid bacteria and Brochothrix thermosphacta) are the main spoilage organisms in chill meat stored in a modified atmosphere. To date studies on the con- Under Vacuum or Modified-atmosphere Packaging tribution of yeasts to the spoilage of meat, whole or Conditions The atmosphere may be modified by minced, has attracted little attention even though they vacuum packaging or storage of meat in atmospheres are common contaminants. Yeasts do not outgrow containing a mixture of gases (Nz, COz and 0 2 ) . Meat bacteria on meat or meat products unless a bac- in a vacuum pack or modified atmosphere (protective teriostatic agent is included, such as sulphite in British atmosphere) has an extensive shelf life when compared with meat stored aerobically. Shelf life is fresh sausages, or the water activity is reduced.

1256 MEAT AND POULTRY/Spoilage of Meat

determined by the choice of atmosphere, storage temperature and meat type. As the bacterial population of meat (particularly the aerobes, e.g. pseudomonads) is restricted by the relative high concentration of COZ and the oxygen limitation, the spoilage of meat stored under vacuum or modified atmosphere occurs later than that of meat stored aerobically. In meat samples stored under vacuum or modified-atmosphere packaging lactic acid bacteria are recognized as important members of the spoilage association (Table 3). Many of the isolates could not be identified with existing species of Lactobacillus (see Table 2 ) . It is now recognized that many of these isolates belong to a recently defined genus, Carnobacterium. It needs to be stressed that each of the atmospheres in Table 3 selects a microbial flora dominated by Gram-positive bacteria (principally Brochothrix thermosphacta and lactic acid bacteria) rather than the Gram-negative ones that develop on meat stored aerobically at chill temperatures. As the former grow much more slowly than the latter, the shelf life of meat is extended. It needs to be stressed also that there are differences in the metabolic attributes of these two groups of spoilage organisms. These are manifested at different times and in different ways as judged by odours coming from the meat. Another cold-tolerant microorganism, Clostridium estertheticum, causes distension or explosion of packs of vacuum-packaged meat. The optimum growth temperature of these organisms is 20°C. It is tempting to speculate that the production of a spore protects this organism from those factors in meat processing that kills psychrophiles lacking this means of protection. Spoilage in Frozen Meat Studies of microbial growth at subfreezing temperatures clearly indicate that microbial growth does not occur in meat ecosystems with a temperature below -8°C. Thus, the main determinant for the storage period of a properly frozen meat ecosystem is the physical, chemical or biochemical changes which are unrelated to microbiological proliferation. Therefore, frozen storage life is limited by changes in other qualities such as appearance or taste which are unrelated to microbiological activity. The key problem with frozen meat is the enumeration of the microbial populations of such ecosystems. Microorganisms are injured by exposure to reduced temperatures leading to sublethal damage, the effects of which in microbial populations include (1)increased lag times and (2)the inability to develop quantitatively on selective media that do not exert any inhibitory effect on undamaged populations of the same taxon. These effects - especially the pro-

longed lag phase - are less noticeable when the meat ecosystem is refrozen and analysed again after a short period of storage. The appropriate resuscitation of frozen meat flora prior to its enumeration is crucial; resuscitation of the injured flora may take place in the meat ecosystem during thawing, or in nonselective culture media. Studies on the effect of different environmental stresses on the enumeration and the recovery of microorganisms are focused on pathogenic microorganisms; in which case the important feature is to ascertain the presence or absence of the pathogenic bacterium. The results obtained have a cardinal role in the evaluation of microbiological hazards.

Roles of Microbes and Enzymes in Spoilage The role of the microbial flora is cardinal for the spoilage of meat. The metabolic activity of the organisms selected in a meat ecosystem leads to the manifestation of changes or spoilage of meat. This manifestation is related to the level of (1)the population and (2) the substrates in meat. Under both aerobic and vacuum or modified-atmosphere packaging the corresponding flora catabolize glucose for growth. By the end of this phase changes and subsequently overt spoilage are due to catabolism of nitrogenous compounds and amino acids as well as secondary metabolic reactions. The contribution of indigenous meat enzymes to spoilage is negligible compared with the action of the microbial flora. Postmortem glycolysis ceases after the death of the animal when ultimate p H reaches a value of 5.4-5.5. During storage, however, there is a proteolytic activity by indigenous enzymes. The activity of these enzymes has a role in the conditioning (ageing) of meat. Added enzymes in meat may be used to artificially ameliorate its organoleptic properties. Enzymes used for their tenderizing effects are proteolytic and of bacterial, fungal or plant origin. Chemistry of Spoilage

The critical physico-chemical changes occurring during spoilage take place in the aqueous phase of meat. This phase contains glucose, lactic acid, certain amino acids, nucleotides, urea and water-soluble proteins which are utilized by most of the bacteria of the meat microflora. The concentration of these lowmolecular-mass compounds is sufficient to support massive microbial growth. Glucose is the prime nutrient in a meat ecosystem and it is catabolized initially during microbial growth. This substrate is attacked by almost all groups of spoilage bacteria, under aerobic and anaerobic conditions (Table 4). Until spoilage is evident organoleptically, the major detectable

MEAT AND POULTRY/Spoilage of Meat

1257

Table 4 Substrates used for growth of major meat spoilage microorganisms Substrates used for growth Microorganism

Aerobic

Anaerobic

Pseudomonas spp.

Glucose, giucose 6-phosphate, lactic acid, pyruvate, gluconate, 6-phosphogluconate, amino acids, creatine, creatinine, citrate, aspartate, glutamate Amino acids, lactic acid, glucose Glucose, lactic acid, pyruvate, gluconate, propionic acid, ethanol, acetate, amino acids Glucose, amino acids, ribose, glycerol Glucose, glucose 6-phosphate, amino acids, lactic acid Glucose

Glucose, lactic acid, pyruvate, gluconate, amino acids

AcinetobacterlMoraxella Shewanella putrefaciens Brochothrix fhermosphacfa Enferobacter spp. Lactobacillus spp.

effect of bacterial growth is a reduction of the glucose concentration. This does not alter the organoleptic properties of meat. When glucose or its oxidative products are reduced to non-substrate levels, lactic acid is catabolized. It needs to be stressed that when this second major carbon and energy source is exhausted the microbial association is at its climax stage. Under Aerobic Conditions The relative potential of bacteria depends upon which species predominate, and upon their ability to form malodorous compounds such as H2S, volatile amines, esters and acetoin. Pseudomonas spp. are important because of their dominance in the aerobic climax associations at chill temperatures. The key chemical changes associated with the metabolic attributes of pseudomonads have been studied extensively in broth and in model systems such as meat juice (Table 5). Among these major attributes are (1)the sequential catabolism of D-glucose and L- and D-lactic acid with D-glucose used in preference to lactate, and (2)the oxidization of glucose and glucose 6-phosphate via the extracellular pathway causing a transient accumulation of D-glu-

Glucose, amino acids Formate Glucose Glucose, glucose 6-phosphate, amino acids Glucose, lactic acid, amino acids

conate and an increase in the concentration of 6phosphogluconate. The increase in the concentration of D-gluconate led investigators to propose a method for controlling the microbial activity in meat by the addition of glucose to meat and its transformation to gluconate. The rationale for this suggestion is the fall in pH due to the accumulation of oxidative products. The transient pool of gluconate and its inability to be catabolized by all the taxa of the association may offer a selective determinant on the meat ecosystem. Another important feature is the catabolism of creatine and creatinine by Pseudomonas fragi under aerobic conditions. The phenomenal release of ammonia and the increase in pH are inextricably linked with the catabolism of these substrates. Ammonia, which is the major cause of the increase of pH, can be produced by many microbes, including pseudomonads during their amino acid metabolism. A list of other volatile compounds found in spoiled meat is given in Table 6. Pseudomonad species growing on the surface of meat will preferentially consume glucose until the rate of diffusion of glucose from underlying tissues becomes inadequate to meet

Table 5 Metabolic activity of pseudomonads in meat juice at 0-4°C Substrate

Pseudomonas fragi

Pseudomonas lundensis

Pseudomonas fluorescens

D-Glucose D-Glucose 6-phosphate D-Gluconate 6-Phospho-~-gluconate L-lactic acid D-lactic acid Pyruvate Acetic acid Amino acids Creatine Creatinine Proteolysis Ammonia

C

C

C

C

C

f f

f f

f

C

C

C

C

C

C

flc C

flc nd

flc nd

C

C

C

-

C

C

+ f

nd f

+ f

Key: The substrate was catabolized (c) or formed (f) during growth; - neither catabolized nor formed; +, positive; nd, no available data.

1258 MEAT AND POULTRY/Spoilage of Meat

Table 6 End product formation of Gram-negative bacteria (e.g. Pseudomonas spp., Shewanella putrefaciens, Moraxella etc) when grown in broth, sterile meat model system and in naturally spoiled meat Sulphur compounds: sulphides, dimethylsulphide, dimethyldisulphite, methylmercaptan, methanethiol, hydrogen sulphide, dimethyltrisulphide Esters: methyl esters (acetate), ethyl esters (acetate) Ketones: acetone, 2-butanone, acetoin/diacetyl Aromatic hydrocarbons: diethylbenzene, trimethylbenzene, toluene Aliphatic hydrocarbons: hexane, 2,4-dimethylhexane, methyl heptone Aldehydes: 2-methyl butanal Alcohols: methanol, ethanol, 2-methylpropanol, 2-methylbutanol, 3-methylbutanol Biogenic amines - other compounds: cadaverine, ammonia, putrescine, methylamine, trimethylamine

thermosphacta) usually occurs in meat during its storage under modified atmosphere packaging. Among these, the physiological attributes of the lactic acid bacteria and B. thermosphacta have been studied extensively. Environmental determinants such as the oxygen tension, glucose concentration and the initial pH have a major influence on the physiology of these organisms, and hence on the end products formed. Brochothrix thermosphacta has a much greater spoilage potential than the lactobacilli and can be important in both aerobic and anaerobic spoilage of meat. This organism utilizes glucose and glutamate but no other amino acid during aerobic incubation. It produces a mixture of end products including acetoin, acetic, iso-butyric and iso-valeric acids, 2,3-butanediol, diacetyl, 3-methylbutanal, 2-methylpropanol and 3-methylbutanol during its aerobic metabolism in media containing glucose, ribose or glycerol as the main carbon and energy source (Table 7). The precise proportion of these end products is affected by the concentration of glucose, pH and temperature. Lactobacillus spp. constitute only a small proportion of the initial spoilage bacterial population of meat. When oxygen is in low concentration, as in vacuum-packed meats, the developing microflora is usually dominated by Lactobacillus spp. These fermentative organisms probably grow faster than would-be competitors because they are unaffected by p H and antimicrobial products such as lactic acid, Hz02 and antibiotics. These organisms utilize glucose for growth and produce lactic acid. When carbohydrates are exhausted, amino acids are utilized with

their demand; when high numbers ( l o 8 per cm2) are reached and glucose becomes depleted at the meat surface, pseudomonads start proteolysis and use nitrogenous compounds and free amino acids as their growth substrate with production of malodorous sulphides and esters (Table 6). The Enterobacteriaceae can be important in spoilage if the meat ecosystem favours their growth. This group utilize mainly glucose and glucose 6-phosphate as the main carbon sources; the exhaustion of these substances allows amino acid degradation. Moreover, some members of this family produce ammonia, vola- Table 7 End products of homofermentative lactic acid bacteria tile sulphides including H2S and malodorous amines (HO), heterofermentative lactic acid bacteria (HT) and from amino acid metabolism (Table 6). Brochothrix thermosphacta (BT) when grown in broth, sterile Acinetobacter and Moraxella constitute a major meat model system and in naturally spoiled meat part of the aerobic spoilage population. These organ- Aerobic In different gaseous isms are of low spoilage potential. They utilize amino atmospheres acids as their growth substrate but do not form mal- Acetoin - HO, HT, BT Acetoin - HO odorous by-products from amino acid degradation; Acetic acid - HO, HT, BT Acetic acid - HO, HT, BT they rather enhance the spoilage activities of pseudo- L-Lactic acid - HO, HT, BT L-Lactic acid - HO, HT, BT D-Lactic acid - HO, HT monads and Shewanella putrefaciens by restricting D-Lactic acid - HO, HT HO, HT, BT Formic acid Formic acid - HO, HT, BT the availability of 0 2 to these organisms. When O2 Ethanol - HO, HT, BT Ethanol - HO, HT, BT limits growth, pseudomonads attack amino acids, COP - HO, HT, BT even when glucose is present, with the subsequent HzOZ - HO, HT production of malodorous substances. Under anaer- iso-Butyric acid - BT obic conditions S. putrefaciens will generate H2S, iso-Valeric acid - BT resulting in the greening of meat due to sulph- 2-Methylbutyric acid - BT 3-Methylbutanol - BT myoglobin formation. 2-Methylbutanol - BT Under Vacuum or Modified-atmosphere Packaging Conditions A shift from a diverse initial flora to one dominated by Gram-positive facultative anaerobic microflora (lactic acid bacteria and Brochothrix

2,3-Butanediol- BT Diacetyl - HO, HT, BT 2-Methylpropanol - BT 2-Methylpropanal -BT Free fatty acids - BT

Next Page MEAT AND POULTRY/Spoilage of Meat

1259

the consequent production of volatile fatty acids given metabolites with the microbial spoilage of meat. which impart a ‘dairy’ or ‘cheesy’ odour to the The idea for these methods is that as the bacteria vacuum-packaged meat. Because with meat stored grow on meat they utilize nutrients and create byunder modified atmosphere increased concentration products. The quantitative determination of these of COz inhibits growth of aerobic flora - and glucose metabolites could provide information about the assimilation by pseudomonads - the cheesy odours degree of spoilage. The identification of the ideal are found mainly in samples stored in gas mixtures metabolite, fulfilling the criteria noted above, has containing COZ,where they are probably produced by proved a difficult task for the following reasons: Brochothrix thermosphacta and lactic acid bacteria. 1. Most metabolites are specific to certain organisms These also form diacetyl, acetoin and alcohols from (e.g. gluconate to pseudomonads). glucose under aerobic conditions or low partial pres2. Although the metabolites are the product of the sure of oxygen (Poz).In addition, alcohols (ethanol metabolism of a specific substrate, the absence of and propanol) are present at only trace levels at the the given substrate or its presence in low quantities beginning of storage but their concentrations increase does not preclude spoilage. significantly before the onset of spoilage, making them 3. The rate of microbial metabolite production and promising compounds as indicators of spoilage (Table the metabolic pathways of spoilage bacteria are 7). affected by the environmental conditions (e.g. pH, oxygen tension, temperature). Evaluation of Spoilage 4. The accurate detection and measurement of metaEnumeration of bacterial population by culture techbolites require sophisticated procedures. niques (agar media) and rapid methods (malthusian) 5 . Many of them provide retrospective information. in food is used as indicator of its hygiene. As the spoilage of meat is caused by specific spoilage bacteria, different selective media should be applied. Role of Cooking in Susceptibility to Because the correlation between the population of Spoilage specific spoilage bacteria and the sensorial manifestation of spoilage is imprecise, it is difficult to use Cooking raw meat results in the death of its microbial bacterial levels as an estimate of spoilage. association. Subsequent recontamination of the The time-consuming microbiological analyses can cooked meat and temperature abuse lead to the develbe replaced by assessment of the chemical, enzymatic opment of a new spoilage association. As the antagand physico-chemical changes associated with micro- onists belonging to the initial microflora of raw meat bial growth on meat. For this reason a number of are absent, pathogens that contaminate cooked meat chemical and physical methods have been proposed have a rich substratum for their proliferation. The for the estimation of bacterial spoilage in meats. microbiological stability of cooked meat products However, there is as yet no single test available to depends on extrinsic factors, mainly the packaging assess meat quality. Spoilage is a subjective evaluation method and storage temperature, as well as on intrinand therefore a sound definition is required to develop sic ones, e.g. product composition. a suitable method. The lack of a general agreement on the signs of incipient spoilage in meat and the Special Problems Associated with Meat changes in the technology of meat preservation (e.g. vacuum or modified-atmosphere packaging) make it Production of biogenic amines by microbial flora is a problem in stored meat. Amines have been detected in difficult to identify spoilage indicators. The spoilage indicators or microbial metabolite fresh meat stored under aerobic or vacuum/modifiedatmosphere packaging conditions. Among them, should meet the following criteria: putrescine and cadaverine show a constant increase 1. The indicator should be absent or initially at low during storage. Concentrations of spermine, spermlevels in meat. idine and tryptamine remain steady, and a small 2. It should increase proportionally with the storage is usually observed increase in tyramine concentration period. As lactic acid bacteria and after long storage periods. 3. It should be produced by the dominant flora and Brochothrix thermosphacta do not produce amines, have a good correlation with sensory evaluation. the formation of these compounds has been attributed As noted earlier, physico-chemical analyses of meat to Enterobacteriaceae. However, tyramine could also can be used instead of microbiological ones for the be formed by some strains of the genus Lactobacillus. The limiting factors of meat stored under modifiedevaluation of spoilage. For this reason, numerous attempts have been made since the 1970s to associate atmosphere packaging is another issue. Concerns have

NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1549

NASBA

see Listeria: Listeria monocytogenes - Detection using NASBA (an Isothermal Nucleic Acid

Amplification System).

1

Natamycin see Preservatives:Permitted Preservatives - Natamycin.

NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY Contents

Canada European Union Japan

Canada Bruce E Brown, B. E. Brown Associates, Ottawa, Ontario, Canada Copyright 0 1999 Academic Press

three departments may well influence regulations, guidelines, etc. However, the mandate of each of the three departments to develop legislation and regulations and to designate enforcement policy remains unchanged.

Introduction

Health Canada

The regulation of the microbiological safety and quality of foods in Canada operates in a complex jurisdictional context involving federal, provincial and municipal authorities and division of responsibilities between the federal departments of agriculture, fisheries and health. In addition, each of the 10 provinces have departments of agriculture and health that also regulate the microbiological safety and quality of food. In recent years under an initiative entitled ‘The Canadian Food Inspection System’ efforts are being made to harmonize legislation, regulations and guidelines and integrate inspectional services at the federal, provincial and municipal levels. The Federal Acts and regulations involving the hygienic practices for production and manufacturing premises as well as the microbiological safety and quality of food products are administered by the departments of Health Canada, Agriculture and AgriFood Canada, and Fisheries and Oceans Canada. The recent creation of the Canadian Food Inspection Agency which united the inspection resources of the

Sections 4 , 5 , 6 and 7 of Part I of the Food and Drugs Act are the primary national legislation governing the overall safety and quality of food. The microbiological safety and quality regulations fall under Sections 4, 6 and 7. Section 4 states that no person shall sell any article of food that: (a) has in or on it any poisonous or harmful substance; (b) is unfit for human consumption; (c) consists in whole or in part of any filthy, putrid, disgusting, rotten, decomposed or diseased animal or vegetable substance; (d) is adulterated; or (e)was manufactured, prepared, preserved, packaged or stored under unsanitary conditions. Foods containing pathogens in numbers that would constitute a direct hazard to health or their toxins would be considered not to be in compliance with Subsections 4(a) and 4(b) and possible 4(e). Spoilage (i.e. microbiological quality) can be considered to contravene Subsection 4(c). Subsection 4(e) and Section 7 deal with the hygienic conditions in which foods are processed, and opens the door for the sanitary inspection

1550 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

of premises where food is manufactured, prepared, preserved, packaged or stored. Subsection 6.1 permits the establishment of regulatory microbiological standards as being necessary to prevent injury to the health of the consumer or purchaser of the food. The term ‘sell’ is defined to include offer for sale, expose for sale, have in possession for sale and distribute, whether or not the distribution is made for consideration. ‘Unsanitary conditions’ means such conditions or circumstances as might contaminate with dirt or filth, or render injurious to health, a food, drug or cosmetic. The Act also permits the establishment of regulations for carrying out the purposes and provisions of the Act, and examples of subject matter for regulations include: setting the sale or conditions of any food, drug, cosmetic or device prescribing standards of composition, strength, potency, purity, quality or other property of any article of food, drug, cosmetic or device respecting the importation of foods, drugs, cosmetics and devices in order to ensure compliance with the Act and regulations respecting the method of manufacture, preparation, preserving, packing, storing and testing of any food, drug, cosmetic or device in the interest of, or for the prevention of injury to, the health of the purchaser or consumer the keeping of books and records by persons who sell food, drugs, cosmetics or devices that are considered necessary for the proper enforcement and administration of the Act and regulations. Microbiological Standards

The Food and Drug Regulations currently contain a number of regulatory microbiological standards. The standards have been developed on the basis of data gathered over the years as an aid to the administration of Sections 4 to 7 (inclusive) of the Act and relate to the microbiological safety and general cleanliness of food. Most of the standards are specific to a microorganism or a group of microorganisms, while in others the organism is not specified but implied. There are two types of standards specific to microorganisms. One requires prohibition, that is zero tolerance, while the other permits some acceptable level. An example of a prohibition standard can be found in regulation B.08.014A which states that no person shall sell milk powder, whole milk powder, dry whole milk, powdered whole milk, skim milk powder or dry skim milk unless it is free from bacteria of the genus Salmonella, as determined by official method MFO-121, Microbiological Examination of Milk Powder, November

30,198 1. The official method is part of the regulation and specifies the method that must be used to establish compliance with the regulation, the sampling plan and compliance criteria (Table 1). Regulation B.08.016 which states that flavoured milks may contain not more than 50000 total aerobic bacteria per cubic centimetre, is determined by official method MFO-7, Microbiological Examination of Milk, November 30, 1981 is an example of a standard in which an acceptable level is permitted. The standards are classified with respect to three degrees of risk, referred to as Health 1, 2 and Sanitation. The degree of risk is reflected in the sampling plan and compliance criteria part of the official method. Two-class plans are used where there is a Health 1 risk (Table 1) and three-class plans for Health 2 and Sanitation risks (Tables 2 and 3). The sample size ( n )designates the number of sample units to be taken and examined from a lot. The acceptance number (c) is the maximum allowable number of sample units that may exceed the level or concentration designated as acceptable, the m value. The lot is unacceptable and can be considered to be in violation of the respective regulatory standard when this number is exceeded. The acceptable con, per gram centration of microorganisms ( m ) expressed or millilitre in a three-class plan separates sample units of acceptable microbiological quality from those classed as marginally acceptable, and in a two-class plan separates acceptable sample units from unacceptable. In a three-class plan unacceptable concentrations of microorganisms are represented by M which separates sample units of marginally acceptable quality from those of defective quality. The lot is unacceptable and in violation of the regulatory standard if one or more sample units exceed the M value. Health 1 indicates that there is a direct risk to human health and appropriate action, for example a product recall, should be taken to limit exposure in the population. Follow-up action should ensure that the cause has been determined and appropriate corrective action has been taken. For Health 2 the hazard Table 1 Microbiological standards for Salmonella considered as a Health 1 risk Product

Chocolate Cocoa Milk powder Egg products Frog legsb

Regulation

8.04.010 8.04.01 1 B.08.014 8.22.033 6.22.033

Method

MFO-11 MFO-11 MFO-12 MFO-6 MFO-10

Criteria” n

c

m

10 10 20 6 10

0 0

0 0 0 0 0

0

0 0

a n , sample size; c, acceptance number; m, acceptable concentration of microorganisms per gram. This regulation and hence standard is due to be repealed.

NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1551

Table 2 Health 2 risk microbiological standards Product

Cheese made from pasteurized milk Cheese made from unpasteurized milk

Microorganism

Escherichia coli Staphylococcus aureus Escherichia coli Staphylococcus aureus

Regulation

6.08.048 6.08.048

Method

MFO-14 MFO-14

Criteria" n

C

m

M

5 5 5 5

2 2

io2

2x103

2 2

io2

io4

5 x 1 0 ~2 x 1 0 3 103 io4

n, sample size; c, acceptance number; m, acceptable concentration of microorganisms per gram; M , unacceptable concentration of microorganisms per gram. a

identified represents a risk to human health only if present in sufficient numbers and appropriate action, such as product recall, should be taken to limit exposure in the population to the product if the unacceptable level (M value) is exceeded. If the acceptance number ( c )for levels between the acceptable level ( m ) and the unacceptable level ( M )is exceeded, corrective action should be taken to bring about compliance. In Sanitation the hazard identified is an indication of a breakdown in hygienic practice. A review of the manufacturer's good hygienic practices (GHP) and/or the hazard analysis critical control point (HACCP) system is appropriate where M or c/m values are exceeded. The sampling plans for Salmonella in Table 1, considered a Health 1 risk, are two-class plans. The lot from which the sample units were drawn to be classed is judged not to be in compliance with the specific regulation if Salmonella is found in any of the sample unit. Lots in violation of Health 1 risk standards are generally ordered to be destroyed and the legal owner could be prosecuted. Salvage operations may be permitted if it can be established that the treatment would decrease the hazard to acceptable levels. In such cases, the verification sampling and acceptance criteria may well exceed that of the particular regulation. In an investigation of a suspected outbreak of a food-borne illness, for example salmonellosis, sample numbers may well exceed the values designated in the regulation. There are regulatory standards in which the microorganisms of concern are implied rather than stated. Clostridium botulinum is the microorganism of concern in regulation B.27.002 which requires a lowacid food packaged in a hermetically sealed container to be commercially sterile, unless it is kept refrigerated at a temperature not exceeding 4°C or frozen and is so labelled. Commercial sterility is defined in regulation B.27.001 as 'the condition obtained in a food that has been processed by the application of heat, alone or in combination with other treatments, to render the food free from viable forms of microorganisms, including spores, capable of growing in the food at temperatures at which the food is designed normally to be held

during distribution or storage'. Under the regulation, a hermetically sealed container means a container designed and intended to be secure against the entry of microorganisms, including spores. A low-acid food is a food, other than an alcoholic beverage, where any component of the food has a p H greater than 4.6 and a water activity greater than 0.85. Brucella and Mycobacterium bovis, and more recently Salmonella and Listeria are the organisms of concern in B.08.002.2. This regulation requires the normal lacteal secretion obtained from the mammary gland of the cow, genus Bos, or of any other animal, or a dairy product made with any such secretion, to be pasteurized by being held at a temperature and for a period that ensure the reduction of the alkaline phosphatase activity so as to meet the tolerances specified in official method MFO-3, Determination of Phosphatase Activity in Dairy Products, dated November 30, 1981. Regulation B.21.025 deals with marine and freshwater animals, or marine and freshwater animal products, that are packed in a container that has been sealed to exclude air and that are smoked or to which liquid smoke flavour or liquid smoke flavour concentrate has been added. Under the regulation these products must be heat processed after sealing at a temperature and for a time sufficient to destroy all spores of the species Clostridium botulinum (i.e. commercially sterile). The only exception to the heat processing requirements are where the contents of the container comprise not less than 9% salt, the contents of the container are customarily cooked before eating, or the contents of the container are frozen and the product so labelled. The specific organism of concern is C. botulinum type E, which can grow - albeit slowly - at normal refrigeration temperatures. Microbiological Guidelines

Guidelines take three forms - microbiological guidelines, codes of hygienic practice and field compliance guides. These have been developed after consultation with the Canadian food industry, are published and copies are available upon request. Microbiological guidelines were developed from surveys conducted

1552 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 3 Sanitary risk microbiological standards Product

Standard

Regulation

Method

Criteria" n

Flavoured milks

ACCb

Milk for manufacture Cottage cheese Ice cream

ACC Coliforms ACC Coliforms ACC Coliforms Coliforms Coliforms ACC Coliforms

Ice milk Mineral or spring water Prepackaged ice Water in sealed containers

C

mC

M"

B.08.016 B.08.018 8.08.026 B.08.024 8.08.054 8.08.062

MFO-7

5

2

5x104

1o6

MFO-7 MFO-4 MFO-2 MFO-2

B.12.001 B.12.005 8.12.004

MFO-9 MFO-15 MFO-15

0 1 2 2 2 2 1 1 2 1

2 x loE 10 105 10 105 10 0 per 1000 ml < 1.8 per 100 ml 10' < 1.8 per 100 ml

-

B.08.072

5 5 5 5 5 5 10 10 5 10

103 1OE 1o3 1O6 103 10 per 100 ml 10 per 100 ml 1o4 10 per 100 ml

n, sample size; c, acceptance number; m, acceptable concentration of microorganisms; M, unacceptable concentration of microorganisms. ACC, Aerobic Colony Count. Per millimetre unless otherwise stated.

a

Volume 1 of the compendium is devoted to the official microbiological methods (MFO). These are cited in the respective Food and Drug Regulations, are an integral part of the standard and must be used by the regulatory agencies to determine compliance. Health Protection Branch methods (MFHPB),used in the guidelines, are found in volume 2 of the compendium. Both the official and HPB methods have been fully validated by interlaboratory studies. Laboratory procedures (MFLP) are given in volume 3 . These have been validated in at least one HPB laboratory, apart from the laboratory that originated the method. These methods include those undergoing development, newly developed rapid methods or methods for newly emerging pathogens. As for a regulatory standard, the sampling plans and compliance criteria are an integral part of each method and form C. jejuni, Yersinia enterocolitica, Pseudomonas aeyu- part of the respective guideline. ginosa, and Aeromonas hydrophila. The microThe Code of Practice for the General Principles of organisms considered to be a Health 2 risk are Food Hygiene developed by the Codex Alimentarius Escherichia coli, Staphylococcus aureus, Bacillus Commission has been modified to reflect current Cancereus, and Clostvidium pevfringens. Aerobic colony adian good hygienic practices. The Canadian version count, coliforms and yeast and mould counts form of this code of practice is intended to provide guidance the basis for a sanitation or hygiene hazard. to the Canadian food industry on hygienic food handAll the methods cited in the standards and ling practices in order to comply with Sections 4 and guidelines are contained in the Compendium of 7 of the Food and Drugs Act. The Code provides to Analytical Methods, published by Polyscience Pub- both the regulatory inspector and the food industry a lications for Health Canada. The compendium pro- template for the sanitary or hygienic inspection of vides a ready reference of the food microbiological food processing and manufacturing premises. methods used by the Health Protection Branch (HPB) The recommended Canadian Code of Hygienic of Health Canada to determine compliance of the Practice for Low-acid and Acidified Low-acid Foods food industry with standards and guidelines, to assess in Hermetically Sealed Containers (Canned Foods) the quality of foods with respect to their micro- was adapted directly from the Codex Alirnentarius biological content, and to support investigations of Recommended International Code of Hygienic Pracfood-borne diseases and consumer complaints. tice for Low-acid and Acidified Low-acid Canned on specific products or groups of products across Canada. While guidelines are not regulatory standards, they are used in judging compliance with Sections 4 and 7 of the Act. Even though a given guideline may embody the same limiting criteria that would be employed in a standard, it is generally based on fewer data than are used in developing a standard. However, guidelines serve as useful indicators of levels that should be achievable using GHP. Guidelines can be readily modified, if necessary, as additional data become available. The microbiological guidelines that are currently in force are given in Table 4. The same three levels of concern or risk (Health 1, 2 and Sanitation) are applied in the guidelines. In addition to Salmonella, the microorganisms considered to be a Health 1 risk are Campylobacter coli,

NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1553

Foods. The Canadian Code is a guideline for com- foods is included as an appendix to the Field Commercial processors who thermally process these prod- pliance Guide. ucts for compliance with the regulations B.27.001 to The definition for recall under the Food and Drugs B.27.006, inclusively. While Clostridium botulinum Act with respect to a product, other than a medical is the primary microorganism of concern, the code device, means a firm’s removal from further sale or also addresses microbiological spoilage. use, or correction, of a marketed product that violates A good example of a field compliance guide is legislation administered by the Health Protection that for ready-to-eat (RTE) foods contaminated with Branch. Three types or classes of recalls are desListeria monocytogenes. An RTE food is defined as ignated. Class I is a situation in which there is a one requiring no further processing before con- reasonable probability that the use of, or exposure to, sumption. Of primary concern are RTE foods that a non-compliant product will cause serious adverse have been subjected to some form of processing not health consequences or death. Class I1 is a situation only to render them ready-to-eat but also to extend in which the use of, or exposure to, such a product their shelf life. Such RTE foods can support the may cause temporary adverse health consequences growth of L. monocytogenes even when maintained or where the probability of serious adverse health under conditions of commercial refrigeration. The consequences is remote. Class I11 is a situation in field compliance guide combines inspection, envir- which the use of, or exposure to, a product is not onmental sampling and end product testing. The likely to cause any adverse health consequences. results of the inspection should show the inspector whether or not GHPs in place are adequate to control Agriculture and Agri-Food Canada the potential for contamination of the product with L. monocytogenes. However, if the inspector does not This department administers a number of acts and consider them to be adequate, environmental sam- associated regulations. Only the Canadian Agripling should be conducted. If the environmental sam- cultural Products Act, Health of Animals Act and the pling indicates that there is a probability of finished Meat Inspection Act and their associated regulations product becoming contaminated with pathogenic have microbiological standards or specifications dirmicroorganisms, then the finished product should be ectly applicable to foods. It should be noted that the sampled in accordance with Table 5 and analysed. administration of many of the Acts by this department The results of that analysis will determine the choice is limited to foods that are imported, exported or of enforcement action as set out in Table 6. If envir- traded interprovincially. The Food and Drugs Act and onmental sampling indicates little or no probability regulations have no such limitation. for product contamination no further action is taken Canadian Agricultural Products Act by the inspector other than to encourage strict implementation of GHPs, i.e. compliance with the Code of The relevant regulations under the Canadian AgriPractice for the General Principles of Food Hygiene. cultural Products Act are: For the purposes of this guide, an RTE food is Livestock and Poultry Carcass Grading Regulations considered capable of supporting growth of L. mono- 0 Egg Regulations (upgraded 18/03/98) cytogenes if, in a naturally contaminated lot of the 0 Processed Egg Regulations RTE food under consideration, L. monocytogenes can 0 Dairy Regulations (upgraded 15/04/98) be detected by direct plating after the food has been 0 Fresh Fruit and Vegetable Regulations (updated stored at 4°C or less until the end of its stated shelf 0 1/04/98) life; OY if, in an inoculated batch representative of the 0 Honey Regulations (updated 01/04/98) RTE food, L. monocytogenes increases in number by 0 Maple Products Regulations (updated 01/04/98) at least 1 log after it has been stored at 4°C or less 0 Processed Products Regulations (updated until the end of its stated shelf life, as determined 15/04/98) by the direct plating method. The guide encourages 0 Licensing and Arbitration Regulations (updated manufacturers to consider performing challenge tests 04/03/9 8). not only under normal conditions of storage and distribution, but also under conditions of mild tem- The Act stipulates that no person shall market a food perature abuse (e.g. 7-10°C). A challenge test involves product in import, export or interprovincial trade as the incubation of samples of the RTE food inoculated food unless the food product, including every subwith a known concentration of a cocktail of at least stance used as a component or ingredient thereof, five strains of L. monocytogenes for periods of time ( a ) is not adulterated to reflect the desired product shelf life. A guide for the (b) is not contaminated challenge testing of L. monocytogenes on refrigerated (c) is sound, wholesome and edible

1554 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 4

Microbiological guidelines

Food

Method

Guideline

Risk

Criteria" n 5

2

Sanitation Sanitation Sanitation

5 5 5

2 2 2

MFHPB-19 MFHPB-18

Sanitation Sanitation

5 5

2 2

< 1.8

MFHPB-19 MFHPB-20 M FHPB-2 1 MFHPB-42 MFHPB-23 MFHPB-18 MFHPB-22 MFHPB-19 M FHPB-21 MFHPB-20 MFHPB-18 MFHPB-19 MFHPB-19 MFHPB-22 M FHPB-21 MFHPB-20 MFHPB-19

Escherichia coli" Salmonella Staphylococcus aureus' Bacillus cereusc Clostridium perfringens" ACC Yeasts and moulds" Escherichia coli" Staphylococcus aureus Salmonella ACC Coliforms E. coli Yeasts and moulds S. aureus Salmonella E. coli'

Health 2 Health 1 Health 2 Health 2 Health 2 Sanitation Sanitation Health 2 Health 2 Health 1 Sanitation Sanitation Health 2 Sanitation Health 2 Health 1 Health 2

10 20 10 10 10 5 5 5 5 5 5 5 5 5 5 5 5

1 0 1 1 1 2 2 2 2 0 2 2 1 2 2 0 1

< 1.8

MFHPB-21 MFHPB-19 MFHPB-21 MFHPB-20

S. aureuse E. coli" S. aureus Salmonella

Health 2 Health 2 Health 2 Health 1

5 5 5 5

1 1 1 0

50 io4 10' 103 2.5~10' lo4 0 -

MFLP-46 MFLP-48 MFHPB-19

Campylobacter coli or C. jejunig Health 1 Yersinia enterocoliticag Health 1 Escherichia coli Health 2

5 5 5

0 0 2

0 0

Staphylococcus aureus Salmonella ACC

Health 2 Health 1 Sanitation

5 5 5

2 0 3

E. coli' Health 2 S. aureus Health 2 Salmonella Health 1 Campylobacterjejuni or C. colig Health 1 Yersinia enteroco/iticag Health 1 ACC Sanitation

5 5 5 5 5 5

2 1 0 0 0 3

Coliforms' Yeasts and moulds E. coli' S. aureus Clostridium perfringens Salmonella Psychrotrophic bacteria

Sanitation Sanitation Health 2 Health 2 Health 2 Health 2 Sanitation

5 5 5 5 5 5 5

Escherichia colik Staphylococcus aureus Salmonella Yersinia enterocolitica'

Health 2 Health 2 Health 1 Health 1

5 5 5 5

Chocolate

MFHPB-22 MFHPB-19 MFHPB-18

Bakery products'

Heat-treated fermented sausage Raw fermented sausage Heat-treated and raw fermented sausage

Non-fermented meat products (ready-to-eat)h

M

Sanitation

MFHPB-18

Fresh and dry pasta

m

ACCb includes aerobic sporeform ers Yeasts and mouldsb,d Coliformsb ACC' includes aerobic sporeformers Coliforms' ACC'

Cocoa

Instant infant cereal and powdered infant formula

C

MFHPB-21 MFHPB-20 Deboned poultry products MFHPB-18 (precooked) MFHPB-19 MFHPB-21 MFHPB-20 MFLP-46 MFLP-48 MFHPB-18 Dry mixes (gravy, sauce, soup) heat and serve MFHPB-19 MFHPB-22 MFHPB-19 MFHPB-21 MFHPB-23 MFHPB-20 Soya bean products MFHPB-18 (ready-to-eat) MFHPB-19 MFHPB-21 MFHPB-20 MFLP-48

105 2x10~

< 1.8 3x104

103

0 10

106

io4 10 IO6 10' 104 10 0

lo2 io2 io4 i o 2 103 5x104 l o 6 2x103 < 1.8

sXio 0 5x104 50 < 1.8 5xio2

ioz 0

io

loo

104 103 104 106 104 103 104 104 103

103

loo io4 0 104 106

io

103

100 0 0 0 104

106

3 3 2 2 2 0 2

10 500

io4

2 2 0 0

100 100 0 0

io 100 100 0

io4

103

io4 103 -

io5 io7 103

io4 -

NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1555

Table 4

Microbiological guidelines (Continued) ~

Food

Spices (ready-to-eat only)

Bottled water and ice"

Alfalfa and bean sprouts"

Method

Guideline

Risk

Criteria" n

C

MFHPB-23 MFHPB-42 MFHPB-19 MFHPB-21 MFHPB-20 MFHPB-22 MFLP-61B

Clostridium petfringens Bacillus cereus E. coli S. aureusm Salmonella Yeasts and moulds" Pseudomonas aeruginosa

Health 2 Health 2 Health 2 Health 2 Health 1 Sanitation Health 1

5 5 5 5 5 5 5

2 2 2 2 0 2 0

MFLP-58B

A. hydrophila

Health 1

5

0

MFHPB-19 MFHPB-20

ColiformsP Salmonella

Sanitation Health 1

5 5

2 0

m

M 104

106

io4

106 100 103 100 i o 4 0 100 104 OperlOO ml 0per100 ml

io3 io5 0

-

For definitions of n, c, m and M see previous tables. From HPB Data-Gathering Survey Results. From Microorganisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. International Commission on Microbiological Specifications for Foods (ICMSF). M adjusted for uniformity. e M value adjusted for uniformity. For E. coli M = 103,for S. aureus, M = 104,and for yeasts and moulds M = 104. Products that are microbiologically sensitive, i.e. containing eggs or dairy products. This food category consisted previously of only cream pies but has been extended to other bakery products. Cream pies probably represent the worst case of this product type. Designates an optional analysis. It is not expected that these determinations will be done routinely. Guidelines for this food category are new. Only organisms indicating a health concern are provided. The limiting values are consistent with those for other products. ' M value adjusted for uniformity. For E. coli M = 103, for S. aureus M = 104. ' Values of m and M modified according to Health Canada monitoring results. M value adjusted for uniformity. For E. coli M = lo3,for S. aureus M = lo4. Designates an optional analysis. It is not expected that these determinations will be done routinely. Values are proposed by the International Commission on Microbiological Specifications for Foods (ICMSF). Includes mineral or spring water, or water in sealed containers, or pre-packaged ice. The microbiological standards for ACC and coliforms in these products (see Table 3) are under review. Based on data-gathering survey results. High coliform counts that do not confirm as faecal coliforms or E. coli should be investigated to determine if Klebsiella pneumoniae is present. Take action appropriate for a Health 2 risk if K. pneumoniae levels exceed those for coliforms. a

'

(d) is prepared in a sanitary manner (e) where irradiated, is irradiated in accordance with Division 26 of Part B of the Food and Drugs Act and the Food and Drug Regulations (f) meets all other requirements of the Food and Drugs Act and the Food and Drug Regulations. For the purposes of this Act, the term 'contaminated' means containing a chemical, drug, food additive, heavy metal, industrial pollutant, ingredient, medicament, microbe, pesticide, poison, toxin or any other substance not permitted by, or in an amount in excess of limits prescribed under, the Canadian Environmental Protection Act, the Food and Drugs Act or the Pest Control Products Act, or containing any substance that renders the food inedible. Paragraphs (b),(c), (d) and (f) address the microbiological safety and quality of foods. These general provisions are repeated in the regulations for each specific food group. The regulations for the various food groups

may contain microbiological standards as well as directions with respect to sanitary preparation. Processed Eggs Regulations The regulations contain a general stipulation that no processed egg shall be marked with a departmental inspection legend unless the processed egg tests negative for salmonellae and other pathogenic organisms of human health significance as determined by a method approved by the Minister. All establishments involved in the handling and processing of eggs and egg product for import, export or interprovincial trade are subject to inspection by Agriculture and Agri-Food Canada and the product packaging must bear the inspection legend. Unlike the microbiological standards under the Food and Drug Regulations the specifics of the method and sampling plan to be used are not given. In addition to this general stipulation, there are a number of microbiological standards for specific product types. Frozen egg, frozen egg mix, liquid egg, liquid egg

1556 NATIONAL LEGISLATION, GUIDELINES 81STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 5 Sampling plans for analysing ready-to-eat (RTE) foods for Listeria monocytogenes (LM) Food product category

Sampling

Analysis

Type of analysis

1. RTE foods causally linked to listeriosis (this list includes soft cheese, liver pate, coleslaw mix with shelf life > 10 days, jellied pork tongue) 2. All other RTE foods supporting growth of LM with refrigerated shelf life > 10 days (e.g. vacuum-packaged meats, modified atmosphere-packaged sandwiches, cooked seafood, packaged salads, refrigerated sauces) 3. RTE foods supporting growth of LM with refrigerated shelf-life 610 days and all foods not supporting growthb (e.g. cooked seafood, packaged salads, ice cream, hard cheese, dry salami, salted fish, breakfast and other cereal products)

Five sample units (100 g or ml each) taken at random from each lot

5 x 10 g or 2 x 25 g analytical unitsa are either analysed separately or composited

ENRICHMENT ONLY

Five sample units (100 g or ml each) taken at random from each lot

5 x 5 analytical unitsa are either analysed separately or cornposited

ENRICHMENT ONLY

Five sample units (100 g or ml each) taken at random from each lot

5 x 10 g analytical unitsaare analysed separately Where enrichment is necessary‘ 5 x 5 g analytical unitsa are analysed separately or composited

DIRECT PLATING ENRICHMENT

The designated analytical unit is taken from each sample unit. Foods not supporting growth of LM include the following: pH 5.0-5.5 and a, < 0.95 pH < 5.0 regardless of a, a, 60.92 regardless of pH frozen foods The pH and a, determinations should be done on three out of five analytical units. The food is presumed to support the growth of L. monocytogenes if any one of the analytical units falls into the range of pH and a, values which are thought to support the growth of the organism. For the last category, if GMP is inadequate and L. monocytogenes has been found in the environment of the finished product area, or where examination of GMP status is not possible, the method to isolate L. monocytogenes from foods and environmental samples (MFHPB-30) and the method for enumeration of L. monocytogenes (MFLP-74) may be used as appropriate.

a

mix, frozen egg product or liquid egg product that is marked with an inspection legend shall, in addition to meeting the general requirements for salmonellae, have a coliform count of no more than 1 0 per gram, and a total viable bacteria count of no more than 50 000 per gram. Dried egg, dried egg mix or dried egg product that is marked with an inspection legend shall, in addition to meeting the requirements for salmonellae, have a coliform count of no more than 1 0 per gram, and a total viable bacteria count of no more than 50000 per gram in the case of whole egg, whole egg mix and yolk mix. In the case of albumen, the total viable bacteria count standard is reduced to 100000 per gram. The pasteurization of liquid egg products, while initially directed to reduce salmonellae to levels that do not represent a health hazard, will also have the same beneficial effect for other pathogens having the same or lower thermal resistance that may be present. The heating requirements are given in Table 7. Spraydried albumen shall be pasteurized at 54°C (130°F) for 7 days, and pan-dried albumen at 52°C (125°F) for 5 days.

Dairy Products Regulations In addition to the general requirement as stipulated in the Act, the regulations specify the compositional standards (e.g. percentage of butterfat in various milk categories). The regulations reference the microbiological standards in the Food and Drug Regulations. As the production, processing, sale and distribution of milk and associated products for the most part are intraprovincial, they are also subject to regulation by each province. Each province has specific pasteurization requirements with respect to time and temperatures. Processed Products Regulations The regulations require that a low-acid food product packed in a hermetically sealed container be thermally processed, until at least commercial sterility is achieved. A lowacid food product packed in a hermetically sealed container is exempt, if it is stored continuously under refrigeration and if the container in which it is packed, as well as the shipping container, is marked ‘Keep Refrigerated’, or kept continuously frozen and the container in which it is packed, as well as the shipping container, is marked ‘Keep Frozen’. This duplicates the same requirement under B.27.002 of the Food and Drug Regulations.

NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1557

Table 6

Compliance criteria for Listeria monocytogenes in ready-to-eat foods

Food product category

RTE foods causally linked to listeriosis (see Table 5) All other RTE foods supporting growth of LM with a refrigerated shelf life > 10 days RTE foods supporting growth of LM with a refrigerated shelf life 5 10 days and all RTE foods not supporting growth (see Table 5)

Action level

GMP status

Immediate action

> 0 cfu per 50 g"

n/a

> 0 cfu per 25 ga

n/a

Class I recall to retain level, consideration of public alert Class II recall to retain level, health alert consideration

5 100 cfu gb

Adequate GMP

Allow sale

5 100 cfu/gb

Inadequate or no GMP'

> 100 cfu/gb

n/a

Consideration of class II recall or stop sale Class II recall or stop sale ~

~~

Enumeration by enrichment only (MFHPB-30) Enumeration to be done by direct plating onto LPM and Oxford agar (MFLP-74) No information on GMP is considered as no GMP and the burden of proof remains with the legal agent for the product. In all of the above cases where L. monocytogenes is detected, the processing establishment should be inspected to determine the source of the contamination and to ensure that corrective measures are taken. cfu, colony forming unit; GMP, Good Manufacturing Practice: LM, Listeria monocytogenes; n/a, not applicable; RTE, ready-to-eat. a

There is also a requirement that the water used to cool the containers after thermal processing shall be of an acceptable microbiological quality. The regulation does not specify what is an acceptable quality. Water used in a cooling canal system must contain a residual amount of a bactericide at the discharge end of the canal and records must be kept of all bactericidal treatments. The specific hygiene requirements detailed in the regulations generally follow those in the Canadian Code of Practice for the General Principles of Food Hygiene for use by the food industry in Canada. Meat Inspection Regulations All establishments involved in the slaughter, preparation, manufacture, storage, distribution and sale of meat and meat products in import, export and interprovincial trade must be registered by Agriculture and Agri-Food Canada. The regulations contain specific requirements:

0

0

governing the design, construction and maintenance of registered establishments and of the equipment and facilities therein prescribing the equipment and facilities to be used, the procedures to be followed and the standards to be maintained in registered establishments to ensure humane treatment and slaughter of animals and hygienic processing and handling of meat products prescribing standards for meat products that are prepared or stored in registered establishments, for meat products that enter into interprovincial or international trade and for meat products in connection with which the meat inspection legend is applied or used.

Registered establishments have resident federal inspectors to ensure compliance with the regulations and to conduct product inspection sampling when required. All processes must meet departmental requirements. Premortem and postmortem inspections are carried out routinely in all registered slaughtering plants. The requirements for low-acid meat products packaged in hermetically sealed containers duplicates the requirements found in Section B.27.002 of the Food and Drug Regulations The container cooling water must be of an acceptable microbiological quality and, in the case of water used in a cooling canal system, contains a residual amount of a bactericide at the discharge end of the canal, and the container be handled in a manner that ensures that the container remains hermetically sealed.

Fisheries and Oceans Canada The microbial safety and quality of fish and fish products for export, import or interprovincial trade are regulated under the Fish Inspection Act and the Fish Inspection Regulations. The regulations: 0 0

0

prescribe grades, quality and standards set the quality and specifications for containers and the marking and inspection of containers require the registration of establishments and the licensing of persons engaged as principals or agents in the export or import of fish or containers prescribe the requirements for the equipment and sanitary operation of establishments, of premises operated by an importer for the purpose of importing fish, and of any boats, vehicles or other equipment used in connection with an establishment or

1558 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 7 Pasteurization of liquid processed egg (see Processed Eggs Regulations - Schedule, Part I) Minimum temperature of the processed egg at the automatic diversion valve

Liquid processed egg product

Whole egg with less than 24% milk solids Whole egg with no less than 24% and no more than 38% egg solids Whole egg mix with less than 2% added salt or sweetening agent, or both Whole egg mix with no less than 2% and no more than 12% added sweetening agent Whole egg mix with no less than 2% and more than 12% added salt Yolk Yolk mix with less than 2 % added salt or sweetening agent, or both Yolk mix with no less than 2% and no more than 12% added sweetening agent Yolk mix with no less than 2% and no more than 12% added salt Ova Egg product with less than 24% total solids" Egg product with no less than 24% and no more than 38% total solids" Egg products with more than 38% solids"

"C

"F

60 61 60 61 60 61 60 63 62 61 60 61 60 63 62 63 62 63 62 61 60 62 61 63 62

140 142 140 142 140 142 140 146 144 142 140 142 140 146 144 146 144 146 144 142 140 144 142 146 144

Minimum heating time (mins)

3.5 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2 3.5 6.2

Regardless of the total solids content, egg product must be heated to 63°C (146°F) for 3.5 min or to 62°C (144°F) for 6.2 min if there is no less than 2% and no more than 12% added sweetening agent or salt, or both. The director, at the request of an operator, may designate a lower minimum temperature depending on the composition of the egg product.

a

in connection with fishing or the import or export of fish prescribe the manner in which samples of any fish may be taken. The general requirement that no person shall import, export, sell for export or have in his or her possession for export any fish intended for human consumption that is tainted, decomposed or unwholesome has microbiological significance: 'decomposed' means fish that has an offensive or objectionable odour, flavour, colour, texture or contains a substance associated with spoilage; 'tainted' means fish that is rancid or has an abnormal odour or flavour; 'unwholesome' means fish that has in or upon it bacteria of public health significance or substances toxic or aesthetically offensive to humans. Any of these conditions can be the result of microbial growth, i.e. spoilage. There are a number of regulations that are specific to the requirements for the equipment and sanitary operation of the fish processing establishments, for vessels used for fishing or transporting fish for processing, and for the storage of frozen fish. Some microbiological guidelines have been

Table 8 Bacteriological guidelines for fish and fish products Product

Microorganism

Cooked or RTE products All other types All types

Escherichia coli

5

2

4

E. coli Staphylococcus aureus Salmonella Vibrio cholerae

5 5

2 1

4

4

lo3

lo4

5b 5b

0

0

0

0

All types Cooked or RTE products

Criteria"

40

n , sample size; c, acceptance number; m, acceptable concentration of microorganisms; M, unacceptable concentration of microorganisms. The analytical unit is 25 g. Each analytical unit must be negative for the microorganism. The analytical units may be pooled. RTE, ready-to-eat.

a

designed to meet specific product risks (Table 8).The interpretation of the sampling plans and acceptance criteria is the same as that described for the microbiological standards and guidelines under the Food and Drug Regulations.

NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1559

The Canadian Shellfish Sanitation Program

Canada are the basis for the classification of coastal harvesting areas for clams, oysters, mussels and whole The Canadian Shellfish Sanitation Program (CCSP) scallops. Classifications are based on the sanitary conwas developed in 1925 under the Fish Inspection Act ditions of the area as defined by the shoreline survey as a result of a typhoid fever outbreak in the USA in with supporting information from the microbiological 1924-25 involving 1500 cases and 150 deaths as a evaluation of the area. result of consuming contaminated oysters. The CSSP is jointly administered by the Canadian Food Inspec- 1. Approved: the area is not contaminated with faecal tion Agency (CFIA), Department of Fisheries and material, poisonous or deleterious substances or Oceans (DFO) and Environment Canada (EC) in marine biotoxins to the extent that consumption cooperation with Health Canada. The parameters of of the shellfish might be hazardous. In these areas interdepartmental cooperation between EC and DFO the median or geometric mean faecal coliform level are established by a Memorandum of Understanding must be less than 1 4 Most Probable Number which is under revision to reflect the responsibilities (MPN) per 100ml with no more than 10% of the of the Canadian Food Inspection Agency. samples in excess of 43 MPN per 100 ml. Environment Canada is responsible for carrying out 2. Conditionally approved: this area must meet the shoreline sanitary and bacteriological water quality same sanitary quality criteria as an approved area. surveys of the shellfish growing areas according to the However, under certain conditions which are preprocedures, standards and protocols of the Canadian dictable and verifiable, water quality can exceed Shellfish Sanitation Program Manual of Operations. approved area criteria. The quality can vary with: This includes the continuing evaluation of the level of (a) the effectiveness of sewage treatment at a comfaecal contamination in the water overlying shellfish munity, (b) rainfall or river flow, (c) seasonal growing areas, the identification of point and nonchanges in sanitary conditions (i.e. tourist or point pollution sources that have a negative impact summer cottage activity, vessel traffic, seasonal on these areas, and classification of these areas based industrial operation). Management plans which on sanitary quality and general sanitary conditions. detail the criteria for opening and closing such In order for a shellfish area to be recommended for areas, and the responsibilities of all parties are approval, the overlying waters must be free from required for conditionally approved classifications. hazardous concentrations of pathogenic micro- 3 . Closed: direct harvesting from this area is prohibited owing to chemical or bacteriological conorganisms, poisonous or deleterious substances (or tamination. Shellfish can be used only under marine biotoxins and monitored by the CFIA) as specified permit conditions for depuration, relayoutlined in the Canadian Shellfish Sanitation Program ing, experimental purposes or other approved proManual of Operations. cessing. Depending on the level of contamination, The 1948 Canada-United States Bilateral Agreeharvesting may be prohibited for any purpose. The ment on Shellfish governing trade in shellfish between Closed classification includes the subclassifications the two countries required agreement on practices for of Restricted for Controlled Purification, sanitary control of the shellfish industry. The CanRestricted for Relaying and Prohibited Area. adian Manual of Operations is based on the protocols and procedures of the American National Shellfish Regional Shellfish Classification Committees, comSanitation Program Manual of Operations, now posed of DFO, EC and provincial government repcalled the NSSP Guide. The Agreement also required resentatives, are responsible for: each country to facilitate inspections of each other’s shellfish handling facilities and shellfish growing areas 0 the technical reviews of the sanitary and bacif requested. The United States Food and Drug Adminteriological surveys and evaluation of growing area istration (USFDA) has routinely audited the CSSP classification recommendations (about every 2 years - most recently in 1996 on both 0 reviewing the policies, procedures, criteria and Atlantic and Pacific coasts). Growing area clasregulations affecting the implementation and applisification based on water quality is the basis of the cation of shellfish growing area classification NSSP as it is for the CSSP. According to the NSSP ‘the 0 recommendations to DFO (for closures under the first critical control point in the sanitary control of Management of Contaminated Fisheries Regulations under the Fisheries Act) and CFIA (as shellfish is identifying harvesting areas of acceptable required for approved areas under the Fish Inspecsanitary quality’. Water quality requirements in both tion Regulations under the Fish Inspection Act programmes are the same for shellfish aquaculture as 0 implementation of the classification decisions, and they are for wild harvest. recommending survey priorities. The sanitary surveys completed by Environment

Next Page 1560 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

It is the policy of Environment Canada either to use its own laboratories and personnel, or to audit laboratories and personnel under contract, so as to ensure impartiality of sample collection and integrity of the data. For example, Environment Canada currently contracts out sampling and analyses under Q N Q C (Quality AssurancelQuality Control) controls to private laboratories in Quebec. In addition, EC has a cooperative agreement with the Department of the Environment of the Province of Prince Edward Island to carry out sampling and analysis for its growing areas. In all cases, the data are interpreted by Environment Canada which then makes classification recommendations to DFO at the regional classification committees. With reducing resources and increasing demand from the aquaculture industry for classified areas, EC distributed a discussion paper in May 1997 outlining cost-sharing approaches including stakeholder sampling and the use of third-party laboratories. The programme is routinely evaluated by both internal and USFDA auditors to determine compliance with CSSP and NSSP protocols. The EC laboratories are evaluated by USFDA. In addition, EC participates in USFDA’s Laboratory Evaluation Officer training programme. All laboratories participating in the CSSP must be evaluated by a Laboratory Evaluation Officer, have an internal QA/QC programme and participate in a split sample programme.

The Future The preceding has summarized the present status of national legislation, standards and guidelines concerned with the microbiological safety and quality of food in Canada. There are five recent initiatives, the Canadian Food Inspection Agency Act and the Canadian Food Inspection System, that may well have a profound effect on the status quo. Under the Canadian Food Inspection Agency Act, the Canadian Food Inspection Agency (CFIA) was created to consolidate all federally mandated food inspection and animal and plant health services. The CFIA legislation sets out its responsibilities, accountability, regimes, powers and reporting framework. The legislation also amended the enforcement provisions and penalty structures of the federal statutes relating to food and animal and plant health that are enforced and/or administered by the Agency. To meet its mandate, the Agency administers and/or enforces the following Acts: 0

Canadian Agricultural Products Act Consumer Packaging and Labelling Act as it relates to food

Feeds Act Fertilizers Act 0 Fish Inspection Act 0 Food and Drugs Act as it relates to food 0 Health of Animals Act Meat Inspection Act Plant Breeders’ Rights Act Plant Protection Act 0 Seeds Act. The Agency is a departmental corporation, reporting to Parliament through the Minister of Agriculture and Agri-Food. While the setting of standards and guidelines remains the prerogative of the mother departments that administer the underpinning legislation, these as well as the enforcement policy will certainly be affected by the feedback information from the inspectional services and associated laboratories. A blueprint for the Canadian Food Inspection System (CFIS) was prepared in 1993 by the Joint Steering Committee of CFIS, the Federal/Provincial Agri-Food Inspection Committee and the Federal/Provincial/TerritorialFood Safety Committee and revised in 1995. The goal of the system is to integrate the activities of all food inspection departments at all levels of government where appropriate. Priority will be placed on developing regulatory standards which will be outcome-based, and supported by nationally accepted guidelines. Effective, ongoing communication among stakeholders and government agencies is a requirement of integration. Specifically, the process of integration will include one set of food safety standards which are nationally recognized and a common legislative base reflective of international developments. This will be extended to include the establishment of common standards for product identity, including grade, composition, net quantity and product description. There will be a movement from prescriptive standards to standards which are outcome- or performancebased, where practical. Commonality will extend to the manner and environment under which food is produced, processed and distributed. Standard methods for laboratory testing and reporting that are reflective of the Canadian Food Inspection System and international developments will be established by the Joint Steering Committee as will an accreditation (certification) programme for government and private laboratories. Research into new methodologies is also critical to the success of this process. 0 0

See also: Brucella: Characteristics. Clostridium: C/ostridium botulinum. Eggs: Microbiology of Egg Products. Fish: Spoilage of Fish. Food Poisoning Outbreaks. Listeria: Introduction; Listeria monocytogenes. Milk and Milk Products: Microbiology of Liquid Milk. Myco-

OENOLOGYIsee Wines

1609

Oenology see Wines: Specific Aspects of Oenology.

oils

see Fermentation (Industrial): Production of Oils and Fatty Acids; Preservatives: Traditional Pre-

servatives - Oils and Spices.

Organic Acids

see Fermentation (Industrial): Production of Organic Acids, e.g. Citric, Propionic;

Preservatives: Traditional Preservatives - Organic Acids

PACKAGING OF FOODS 1611

PACKAGING OF FOODS Aaron L Brody Rubbright Brody Inc., Duluth, Georgia, USA Copyright 0 1999 Academic Press

Packaging is intended to protect foods against environmental invasion. Among the many external variables that may adversely affect foods are an excess or deficiency of moisture, oxygen, dirt, humans (through tampering), dust, animals, insects and microorganisms. Packaging and processing are increasingly becoming integrated with each other; an example is canning, which is really a packaging and thermal preservation operation in which the can, its product contents, the filling temperature, air removal, closure, heating, cooling and distribution must be an uninterrupted continuum or else preservation is not effected. More traditional preservation processes such as drying and freezing do not necessarily require close relationships between the product, process and packaging; the process and the packaging may be separate and the preservation effect will still be achieved. In contrast, in preservation processes such as thermal pasteurization, modified-atmosphere packaging, aseptic packaging, retort pouch and tray packaging, it is necessary to integrate all the elements to ensure the optimum preservation of the contained foods. For example, in aseptic packaging, preservation is achieved by sterilization of the product independently of the package, and the packaging equipment and assembly environment must therefore be sterile to exclude microorganisms from the ultimately hermetically sealed package. It is essential that the operations be connected by sterile linkages and that no microorganisms are permitted to contaminate any element. For these reasons it has become increasingly important that the packaging be incorporated into the system if the objectives of delivering safe and highquality food are to be achieved. To understand fully the role of packaging in food preservation it is perhaps instructive to offer a few definitions. ‘Packaging’ is a term describing the totality of containment for the purpose of protecting the food contents and includes the package material, its structure and the equipment that marries the package structure to the food. Package materials are the com-

ponents that constitute the structures usually known as packages or containers. Package materials are no longer single elements but rather composites of several different materials. In addition, new forms of packaging are increasingly replacing the traditional cans, bottles, jars, cartons and cases.

Preservation Requirements of Common Food Categories Meats

Fresh Meat Most meat offered to consumers is freshly cut, with little further processing to suppress the normal microbiological flora present from the contamination received during the killing and breaking operations required to reduce carcass meat to edible cuts. Fresh meat is highly vulnerable to microbiological deterioration from indigenous microorganisms. These microorganisms can range from benign forms such as lactic acid bacteria or slime-formers, to proteolytic producers of undesirable odours and pathogens such as Escherichia coli 0157:H7. The major mechanisms that retard fresh meat spoilage are temperature reduction to (or near) the freezing point and a reduced oxygen atmosphere during distribution to retard microbial growth. Reduced oxygen levels could provide conditions for the expression of pathogenic anaerobic microorganisms, a situation usually obviated by the presence of competitive spoilage organisms. Reduced oxygen levels also lead to the colour of fresh meat being the purple of myoglobin; exposure to air converts the natural meat pigment to the bright cherryred oxymyoglobin characteristic of most fresh meat offered to and accepted by consumers in industrialized societies. Reduced oxygen packaging is achieved through the mechanical removal of air from the interiors of gas-impermeable multilayer flexible material pouches closed by heat-sealing the end after filling. Ground Meat About 40% of fresh beef is offered in ground or minced form to enable the preparation of

1612

PACKAGING OF FOODS

hamburger sandwiches and related foods. Ground beef was originally a by-product, that is, the trimmings from reducing muscle to edible portion size. The demand for ground beef is now so great that some muscle cuts are specifically ground to meet the demand. Grinding the beef further distributes the surface and below-surface microflora and thus provides a rich substrate for microbial growth even under refrigerated conditions. Relatively little pork is reduced to ground fresh form; however, increasing quantities of poultry meat are being comminuted and offered fresh to consumers, both on its own and as a cheaper substitute for ground beef. The major portion of ground beef is coarsely ground at abattoir level and packaged under reduced 0 2 levels for distribution at refrigeration temperatures to help retard microbiological growth. The most common packaging technique is pressure-stuffing into chubs, which are tubes of flexible gas-impermeable materials closed at each end by tight-fitting metal clips. Pressure-stuffing the pliable contents forces most of the air out of the ground beef, and since there is no head-space within the package, little air is present to support the growth of aerobic spoilage microorganisms such as Lactobacillus and Leuconostoc spp. At retail level the coarsely ground beef is finely ground to restore the desirable oxymyoglobin red colour and to provide the consumer with the desired product. In almost all instances, the retail cuts and portions are placed in expanded polystyrene (EPS) trays which are overwrapped with plasticized polyvinyl chloride (PVC) film. The tray materials are resistant to fat and moisture to the extent that many trays are internally lined with absorbent pads to absorb the purge from the meat as it ages and/or deteriorates in the retail packages. Because of the prognosis the PVC materials are not sealed but rather tacked so that the somewhat water vapour-impermeable structure does not permit loss of significant moisture during short refrigerated distribution. Being a poor gas barrier, PVC film permits the access of air and hence the oxymyoglobin red colour is retained for the short duration of retail distribution. Case-ready Meat For many years, attempts have been made to shift the retail cutting of beef and pork away from the retailer’s back room and into centralized factories. This movement has been stronger in Europe than in the USA, but some action has been detected in the latter country in the wake of the E. coli 0157:H7 incidents. Case-ready retail packaging in the UK where the practice is relatively common, involves cutting and packaging meat under extremely hygienic conditions to reduce the probability of microbiological contamination beyond that of the

indigenous microflora. Packaging is usually in a gas barrier structure, typically gas/moisture barrier foam polystyrene trays heat-sealed with polyester gas barrier film. The internal gas composition is altered to a high content of O2 (up to 80%) and of CO2 (up to 3 0 % ) , with the remainder (if any) being nitrogen as a filler gas to ensure against package collapse arising from internal vacuum formation. The high 0 2 concentration fosters the retention of the oxymyoglobin red colour preferred by consumers, while the elevated CO, level suppresses the growth of aerobic spoilage microorganisms. Using this or similar technologies, refrigerated microbiological shelf lives of retail cuts may be extended from a few days to as much as a few weeks, permitting long-distance distribution, e.g. from a central factory to a multiplicity of retail establishments. One thesis favouring the centralized packaging of ground beef is that the probability of the presence of E . coli 0157:H7 is reduced. On the other hand, if the pathogen is present at the central location, the probability of its being spread among a number of retailers is greatly increased. Nevertheless, the use of central factories, which would probably be under federal government supervision in the USA, and certainly under technical supervision, would increases the probability of the emerging packaged meat being microbiologically safe. Alternative packaging systems for case-ready beef and pork include the ‘master bag’ system used widely for freshly cut poultry (see below) in which retail cuts are placed in conventional PVC film overwrapped EPS trays and the trays are multipacked in gas barrier pouches whose internal atmospheres are enhanced with COZ to retard the growth of aerobic spoilage microorganisms. Another popular system involves the use of gas barrier trays with heat-seal closure using flexible gas and moisture barrier materials. Conventional non-gas-barrier trays such as EPS may be overwrapped with gadmoisture barrier flexible films subsequently shrunk tightly around the tray to impart an attractive appearance. Other systems, all of which involve removal of 0 2 , include vacuum skin packaging in which a film is heated and draped over the meat on a gas/moisture barrier tray. The film clings to the meat so that no head-space remains, with the result that the meat retains the purple colour of myoglobin. In one such system the drape film is a multilayer whose outer gas barrier layer may be removed by the retailer, exposing a gas-permeable film that permits the entry of air, which reblooms the pigment and restores the desired colour. Variations on this double film system include packaging systems in which the film is not multilayer but is composed of two independent flexible layers, the outer being impermeable to gas and moisture and the inner gas-

PACKAGING OF FOODS 1613

permeable to permit air entry to restore the red colour. In all instances the microbiological shelf life is extended by reduced temperature plus reduced O2 levels, which may incidentally or intentionally be enhanced by elevated CO1 concentration. Processed Meat Longer-term preservation of meats may be achieved by curing, using agents such as salt, sodium nitrite, sugar, seasonings, spices and smoke, and by processing methods such as cooking and drying; these treatments alter the water activity, add antimicrobial agents, provide a more stable red colour, and generally enhance the flavour and mouth feel of the cured meats. Cured meats are often offered in tubular or sausage form which means that the shape is dictated by the traditional process and consumer demand. Because of the added preservatives, the refrigerated shelf life of processed meat is generally several times longer than that of the fresh meat. Because cured meats are not nearly so sensitive to oxygen variations as fresh meat, the use of reduced 0 2 atmospheres to enhance the refrigerated shelf life is quite common. The O2 reduction may be achieved by mechanical vacuum, inert gas flushing or a combination of methods. Since the conditions have been changed to obviate the growth of anaerobic pathogenic microorganisms, reduced oxygen conditions are generally effective in retarding the growth of aerobic spoilage microorganisms. The containers for reduced O2 packaging of cured meats are selected from a multiplicity of materials and structures depending on the protection required and the marketing needs: frankfurters are generally sold in twin web vacuum packages in which the base tray is an in-line thermoformed nylon/polyvinylidene chloride (PVDCj web and the closure is a heat-sealed polyester (PET)/PVDCflexible material. Sliced luncheon meats and similar products are packed in thermoformed unplasticized PVC or polyacrylonitrile (PAN) trays, heat-seal closed with PET/PVDC. Sliced bacon packaging employs one of several variations of PVDC skin packaging (in contact with the surface of the product) to achieve the oxygen barrier. Ham may be fresh, cured or cooked, with the cooking often performed in the package. The oxygen barrier materials employed are usually a variation of nylon/PVDC in pouch form. Poultry Poultry meat is most commonly chicken, but turkey is becoming an increasingly significant category of protein. Further, chicken is increasingly penetrating the cured meat market as a less expensive but nutritionally and functionally similar substitute for beef or pork. Since the 1970s poultry processing in westernized societies has shifted into large-scale,

almost entirely automated killing and dressing operations. In such facilities the dressed birds are chilled in water to near the freezing point after which they are usually cut into retail parts and packaged in caseready form: expanded polystyrene trays overwrapped with printed PVC or polyethylene film. The package is intended to appear as if it has been prepared in the retailer’s back room, but in reality it is only a moisture and microorganism barrier. Individual retail packages, however, may be multipacked in gas-impermeable flexible materials to permit gas flush packaging, thus extending the refrigerated shelf life of the fresh poultry products. Poultry is especially susceptible to infection with Salmonella spp., which are pathogenic in large quantities. Such organisms are not removed or destroyed by the extensive washing and chemical sanitation of current poultry processing plants, merely reduced in numbers. Modified-atmosphere packaging has relatively little effect on Salmonella and so refrigeration during distribution is critical in the drive to avoid increasing populations of this bacterium. All meat products may be preserved by thermal sterilization in metal cans or, less frequently, glass jars. The product is filled into the container which is hermetically sealed, usually by double-seam metal end closure (see Fig. 2). After sealing, the cans are retorted to destroy all microorganisms present and cooled to arrest further cooking. The metal (or glass) serves as a barrier to gas, moisture, microbes etc. to ensure indefinite microbiological preservation. Cans or jars do not, however, ensure against further biochemical deterioration of the contents. Fish

Fish is among the most difficult of all foods to preserve in its fresh state because of its inherent microbiological population, many organisms of which are psychrophilic, i.e. capable of growth at refrigerated temperatures. Further, seafoods may harbour a nonproteolytic, quasi-psychrophilic anaerobic pathogen, Clostridium botulinum type E. The need to prolong the refrigerated shelf life of fresh fish suggests the application of modified-atmosphere packaging in which reduced 0 2 levels and elevated C02 levels are present (Table 1j. However, a reduced 0 2 atmosphere can permit the expression of type E . botulinum, and for this reason reduced O2 packaging for seafood is discouraged in the USA. This is not the situation in Europe, where gas barrier flexible and semirigid plastic packaging similar to that described above for case-ready fresh beef is often applied. Packaging for fresh seafood is generally moistureresistant but not necessarily proof against microbial contamination. Simple polyethylene film is employed

1614 PACKAGING OF FOODS

Table 1 Pathogens of concern in modified atmospherepackaged andvacuum-packagedfoods

-

Psychrotrophs growth at 3-4°C Listeria monocytogenes Yersinia enterocolitica Bacillus cereus Non-proteolytic Clostridium botulinum

-

Pseudopsychrotrophs growth at 7-8°C Escherichia coli 0157:H7 Salmonella sp.

-

Mesophiles growth at ~ 1 0 ° C Proteolytic Clostridium botulinum

often as liners in corrugated fibreboard cases. The polyethylene serves not only to retain product moisture but also protects the structural case against internal moisture. Seafood may be frozen, in which case the packaging is usually a form of moisture-resistant material plus structure such as polyethylene pouches or polyethylene-coated paperboard cartons. Canning of seafood is much like that of meats since all seafoods have a p H above 4.6 and so require highpressure cooking or retorting to effect sterility in metal cans (Table 2). One variation unique to seafood is thermal pasteurization, in which the product is packed into plastic cans under reasonably clean conditions, achievable in contemporary commercial seafood factories. The filled and hermetically sealed cans are heated to temperatures of up to 80°C to effect pasteurization to permit several weeks of refrigerated shelf life. The system is usually effective because Clostridium botulinum type E spores are thermally sensitive and may be destroyed by temperatures of 80°C. To ensure against growth of other pathogens which may grow at ambient temperatures, however, distribution at refrigerated temperatures is dictated. Dairy Products

Milk Milk and its derivatives are generally excellent microbiological growth substrates and therefore potential sources of pathogens. For these reasons, almost all milk is thermally pasteurized as an integral element of processing. Refrigerated distribution is generally dictated for all products that are pasteurized to minimize the probability of spoilage. Milk is generally pasteurized and packaged in relatively simple polyethylene-coated paperboard gabletop cartons or extrusion blow-moulded polyethylene bottles for refrigerated short-term (several days to 2 weeks) distribution. Such packages offer little beyond containment and avoidance of contamination as protection benefits; they retard the loss of moisture and resist fat intrusion. Newer forms of milk packaging

Table 2

Ranges for bacterial growth

Organism

pH range

Gram-negative bacteria Escherichia coli Pseudomonas fluorescens Salmonella typhimurium

4.4-9.0 6.0-8.5 5.6-8.0

Gram-positive bacteria Bacillus subtilis Clostridium botulinum Lactobacillus sp. Staphylococcus aureus

4.5-8.5 4.7-8.5 3.8-7.2 4.3-9.2

incorporate reclosure, a feature that was missing from the traditional gable-top cartons. Further, modern packaging environmental conditions have been upgraded microbiologically to enhance refrigerated shelf life by the use of pre-sterilization of the equipment, shrouding and use of clean air. An alternative, popular in Canada, employs polyethylene pouches formed on vertical form/fill/seal machines and heat-sealed after filling. This variant has been enhanced by re-engineering into aseptic format, a system that has not become widely accepted. Pouch systems are generally less expensive than paperboard and semirigid bottles, but are less convenient for consumers. Little difference exists between the three packaging systems from a microbiological perspective. In some countries, aseptic packaging is employed to deliver fluid dairy products that are shelf-stable at ambient temperatures. The most common processing technology is ultra-high temperature short time thermal treatment to sterilize the product followed by aseptic transfer into the packaging equipment. Three general types of aseptic packaging equipment are employed commercially: vertical form/fill/seal in which the paperboard composite material is sterilized by high temperature/high concentration hydrogen peroxide (removed by mechanics plus heat); erected preformed paperboard composite cartons which are sterilized by hydrogen peroxide spray (removed by heat); and bag-in-box, in which the plastic pouch is pre-sterilized by ionizing radiation. The former two are generally employed for consumer sizes while the last is applied to hotel, restaurant or institutional sizes, largely for ice cream mixes. Fluid milk is generally pasteurized, cooled and filled into bag-in-box pouches for refrigerated distribution.

Cheese Fresh cheeses such as cottage cheese fabricated from pasteurized milk are generally packaged in polystyrene tubs or polyethylene pouches for refrigerated distribution. Such packages afford little microbiological protection beyond acting as a barrier against recontamination, i.e. they are little more than

PACKAGING OF FOODS 1615

rudimentary moisture loss and dust protectors, but are adequate because the distribution time is so short. Enhancement of refrigerated shelf life may be achieved by clean filling and/or the use of a low 0 2 , high CO2 atmosphere, all of which retard the growth of lactic acid spoilage microorganisms. Fermented Milks Fermented milks such as yoghurts fall into the category of fresh cheeses from a packaging perspective, i.e. they are packaged in polystyrene or polypropylene cups or tubs to contain and to protect minimally against moisture loss and microbial recontamination. Their closures are not hermetic and so gas passes through both the closures and the plastic walls, and microorganisms could enter after the package is opened. Because the refrigerated shelf life is short, however, few measures are taken from a packaging standpoint to lengthen the shelf life. Clean packaging is often used to achieve several weeks of refrigerated shelf life. Aseptic packaging is occasionally used to extend the ambient temperature shelf life of these products. Two basic systems are employed; one uses preformed cups, and the other is thermoform/fill/seal. In the former, the cups are sterilized by spraying with H20z and heating to remove the residue prior to filling and heat-sealing a flexible closure to the flanges of the cups, which are impermeable to gas and water vapour. In the thermoform/fill/seal method, a sheet of multilayer barrier plastic sheet (usually polystyrene plus PVDC) is immersed in HL02 to sterilize it, air-knifed to remove the residual sterilant, heated to softening, and formed into cups by pressure. The web containing the connected cups is within a sterile environment under positive pressure of sterile air. The cavities are filled with sterile product and a flexible barrier material web, usually an aluminium foil lamination (also sterilized by H2O2immersion), is heat-sealed to the cup flanges. Filled and sealed cups then pass through a sterile air lock. These aseptic dairy packaging systems may also be employed for juices and soft cheeses. Recently, aseptic packaging of dairy products has been complemented by ultra-clean packaging on both preformed cup deposit/fill/seal and thermoform/ fillheal systems. In these systems, intended to offer extended refrigerated shelf life for low-acid dairy products, the microbicidal treatment is with hot water to achieve a 4D kill (i.e. four times the decimal reduction time) on the package material surfaces. The same systems may be employed to achieve ambient temperature shelf stability for high-acid products such as juices and related beverages. Cured cheeses are subject to surface mould spoilage as well as to further fermentation by the natural microflora. These microbiological growths may be

retarded by packaging under reduced O2 atmospheres which may or may not be complemented by the addition of C02. To retain the internal environmental condition, the use of gas barrier package materials is commercial. Generally, flexible barrier materials such as nylon plus PVDC are employed on horizontal flow wrapping machines or on twin web thermoform/vacuum/seal machines. On twin web machines, the flat sealing web is usually a variant of polyester plus PVDC. One problem is that some cured cheeses continue to produce COz as a result of fermentation, and so the excess gas must be able to escape from the package or else the package might bulge or even burst. Somewhat less gas-impermeable materials are suggested for such cheeses. In recent years, shredded cheeses have been popularized. Shredded cheeses have increased surface areas which increase the probability of microbiological growth. Gas packaging under C 0 2 in gas-impermeable pouches is mandatory. One feature of all shredded cheese packages today is the zipper reclosure which does not represent an outstanding microbiological barrier after the package has first been opened. Ice Cream Ice cream and similar frozen desserts are distributed under frozen conditions and so are not subject to microbiological deterioration, but the product must be pasteurized prior to freezing and packaging. The packaging needs to be moisture resistant because of the presence of liquid water prior to freezing and sometimes during removal from refrigeration for consumption. Water-resistant paperboard, polyethylene-coated paperboard and polyethylene structures are usually sufficient for containment of other frozen desserts. Fruit and Vegetables

In the commercial context, fruits are generally highacid foods and vegetables are generally low-acid. Major exceptions are tomatoes, which commercially (not botanically) are regarded as vegetables, and melons and avocados, which are low-acid. The most popular produce form is fresh, and increasingly fresh cut or minimally processed. Fresh produce is a living, ‘breathing’ entity with active enzyme systems fostering the physiological consumption of O2 and production of COz and water vapour. From a spoilage standpoint, fresh produce is more subject to physiological than to microbiological spoilage, and measures to extend the shelf life are designed to retard enzyme-driven reactions and water loss. The simplest means of retarding fresh produce deterioration is temperature reduction, ideally to near

1616

PACKAGING OF FOODS

freezing point but more commonly to about 4 4 ° C . Temperature reduction also reduces the rate of microbiological growth, which is usually secondary to physiological deterioration. Since the 1960s, alteration of the atmospheric environment in the form of modified or controlled atmosphere preservation and packaging has been used commercially to extend the refrigerated shelf life of fresh produce items, such as apples, pears, strawberries, lettuce and now fresh cut vegetables. Controlled atmosphere preservation has been largely confined to warehouses and transportation vehicles such as trucks and seaboard containers. In this form of preservation, the 0 2 , CO2, ethylene and water vapour levels are under constant control to optimize refrigerated shelf life. For each class of produce a separate set of environmental conditions is required for optimum preservation effect. In modified-atmosphere packaging, the produce is placed in a package structure and an initial atmosphere is introduced. The normal produce respiration plus the permeation of gas and water vapour through the package material and structure drive the interior environment towards an equilibrium gas environment that extends the produce quality retention under refrigeration. In some instances the initial gas may be air (passive atmosphere establishment). Produce respiration rapidly consumes most of the oxygen within the package and produces CO2 and water vapour to replace it, generating the desired modified atmosphere. The target internal atmosphere is to retard respiration rate and microbiological growth. Reduced O2 and elevated C 0 2 levels independently or in concert retard the usual microbiological growth on fruit and vegetable surfaces. One major problem is that produce may enter into respiratory anaerobiosis if the O2 concentration is reduced to near extinction. In respiratory anaerobiosis, the pathways produce undesirable compounds such as alcohols, aldehydes and ketones instead of the aerobic end products such as C 0 2 . To minimize the production of these undesirable end products, elaborate packaging systems are being developed. Most of these involve mechanisms to permit air into the package to compensate for the oxygen consumed by the respiring produce. High-gas-permeability plastic films, microperforated plastic films, plastic films disrupted with mineral fill, and films fabricated from polymers with temperature-sensitive side chains have all been proposed or used commercially. The need for reduced temperature is emphasized in modified-atmosphere packaging because the dissolution rate of CO2 in water is greater at lower temperatures than at higher temperatures. Carbon dioxide is one of the two major gases involved in

reducing the rate of respiration and the growth of microorganisms. Since the late 1980s, fresh cut vegetables, especially lettuce, cabbage, carrots, etc., have been a major product in both the retail trade and the hotel, restaurant and institutional markets. Cleaning, trimming and size reduction lead to a greater surface area to volume ratio and expression of fluids from the interior, increasing the respiration rate and offering a better substrate for microbiological growth than the whole fruit or vegetable. On the other hand, commercial fresh cutting operations generally are far superior to mainstream fresh produce handling in cleanliness, speed through the operations, temperature reduction and application of microbicides such as chlorine. Although some would argue, on the basis of microbial counts found in fresh cut produce in distribution channels, that uncut produce is safer, the paucity of its cleaning coupled with the rarity of adverse incidents related to fresh cut produce lead to the opposite conclusion - that fresh cut is significantly safer microbiologically. Another argument is that the low O2 environment within most fresh cut produce packages plus the risk of soil contamination lead to ideal conditions for the proliferation of Clostvidium botulinum. Further, distribution temperatures are often in excess of 1O"C, well within the range of growth and production of spores. However, extensive testing has demonstrated that after responsible fresh cut processing, pathogenic spores are present in relatively small numbers, distribution temperatures prior to retail level are significantly lower than for uncut produce, and times are too short for pathogenic expression. These data indicate that while anaerobic pathogenic problems may occur, they are significantly less likely in fresh cut than in uncut fruit and vegetables. Uncut produce packaging comprises a multitude of materials, structures and forms, ranging from traditional containers such as wooden crates, to inexpensive ones such as injection-moulded polypropylene baskets, to polyethylene liners within waxed, corrugated fibreboard cases. Much of the packaging is designed to help retard moisture loss from the fresh produce or to resist the moisture evaporating or dripping from the produce (or occasionally its associated ice), to ensure the maintenance of the structure throughout distribution. Some packaging designs recognize the issue of anaerobic respiration and incorporate openings to allow passage of air into the package, for example perforated polyethylene pouches for apples or potatoes. Almost none of the contemporary packaging for fresh uncut produce encompasses any specific microbiological barriers or countermeasures. That result is a direct extension of

PACKAGING OF FOODS 1617

the observation that uncut produce ‘processing’ is virtually nonexistent. Packing house operations include collection and the removal of debris and gross dirt, and packaging is usually the least expensive structure that will contain the contents during distribution, often at sub-optimum temperatures. For freezing, vegetables are cleaned, trimmed, cut and blanched, prior to freezing and then packaging (or packaging and then freezing). Blanching and the other processing operations reduce the numbers of microorganisms. Fruit may be treated with sugar to help retard enzymatic browning and other undesirable oxidations. Produce may be individually quick frozen (IQF) using cold air or cryogenic liquids prior to packaging, or frozen after packaging as in folding paperboard cartons. Frozen food packages are generally relatively simple monolayer polyethylene pouches or polyethylene-coated paperboard to retard moisture loss. No special effort is engineered to obviate further microbiological contamination after freezing, although the polyethylene pouches are generally heat-sealed. Canning of low-acid vegetables to achieve longterm ambient temperature microbiological stability is the same as for other low-acid foods, with blanching prior to placement in steel cans (today all welded side seam tin-free steel, with some two-piece cans replacing the traditional three-piece type), hermetic sealing by double seaming, and retorting and cooling. Canned fruit is generally placed into lined three-piece steel cans using hot filling coupled with post-fill thermal treatment. Increasingly, one end is ‘easy open’ for consumer convenience. Newer techniques involve placing fruit hot into multilayer gas- and moistureimpermeable tubs and cups prior to heat-sealing with flexible barrier materials and subsequent thermal processing to achieve ambient temperature shelf-stability or extended refrigerated temperature shelf life. These plastic packages are intended to provide greater convenience for the consumer as well as to communicate that the contained product is not ‘overprocessed’ like canned food.

Tomato Products The highly popular tomato-based sauces and pizza toppings must be treated as lowacid foods if they contain meat, as so many do. For marketing purposes, tomato-based products for retail sale are commonly packed in glass jars with reclosable metal lids. The glass jars are often retorted after filling and hermetic sealing; major differences from the technique using metal cans include counterpressured retorting and longer times for heating and cooling, since the thick-walled glass is a thermal insulator.

Juices and Juice Drinks Juices and fruit beverages may be hot-filled or aseptically packaged. Traditional packaging has been hot-filling into steel cans and glass bottles and jars. Aseptic packaging, described above for paperboard composite cartons, is being applied for polyester bottles using various chemical sterilants to effect the sterility of the package and closure interiors. Much fruit beverage is currently hot-filled into heat-set polyester bottles capable of resisting temperatures of up to 80°C without distortion. Hermetic sealing of the bottles provides a microbiological barrier, but the polyester is a modest oxygen barrier and so the ambient temperature shelf life from a biochemical perspective is somewhat limited. Since the 1970s high-acid fluid foods such as tomato pastes and non-meat-containing sauces have been hot-filled into flexible pouches, usually on vertical form/fill/seal machines. The hot filling generates an internal vacuum within the pouch after cooling so that the contents are generally shelf-stable at ambient temperature. Package materials are usually laminations of polyester and aluminium foil with linear low-density polyethylene (LLDPE) internal sealant; this resists the relatively lengthy exposure to the high heat of the contents during and immediately following filling. The heat seal is hermetic. Some efforts have been made to employ transparent gadwater vapour barrier films in the structures: polyester/ethylene vinyl alcohol laminations with the same LLDPE sealant. Transparent flexible pouches offer the opportunity for the consumer to see the contents, and for the hotel, restaurant or institutional worker to identify the contents without needing to read the label. Other Products

A variety of food products that do not fall clearly into the meat, dairy, fruit or vegetable categories may be described as ‘prepared foods’, a rapidly increasing segment of the industrialized society food market during the 1990s. Prepared foods are those that combine several different ingredient components into dishes that are ready to eat, or simply require heating. If the food is canned, the thermal process must be suitable for the slowest heating component, meaning that much of the product is overcooked to ensure microbiological stability. If it is frozen, the components are separate but the freezing process reduces the eating quality. The preferred preservation technology from a quality retention or consumer preference perspective is refrigeration. Incorporation of several ingredients from a variety of sources correctly implies many sources for microorganisms - aerobic, anaerobic, spoilage, benign and pathogenic. Where refrigeration is the sole barrier, microbial problems are minimized by reducing the

1618 PACKAGING OF FOODS

time between preparation and consumption to less than 1 day (under refrigeration at temperatures above freezing) plus a nodding acknowledgment of cleanliness during preparation. As commercial operations attempt to prolong the quality retention periods beyond same-day or next-day consumption, enhanced preservation ‘hurdles’ have been introduced. These microbiological growth retardant factors include elevated salt or sugar concentrations, reduced water activity, reduced pH to minimize the probability of pathogenic microbiological growth, selection of ingredients from reduced microbial count sources, and modified-atmosphere packaging. The last is often suggested as a potential stimulus for the growth of pathogenic anaerobic microorganisms, since the multiple ingredient sources can almost assure the presence of Clostridium spores, and the reduced 0 2 low-acid conditions are common to the types of products such as potato salad, pasta dishes, etc. Further, distribution temperatures may often be in the 5°C range or higher. Packaging for air-packaged prepared dish products is generally oriented thermoformed polystyrene trays with oriented polystyrene dome closures snap-locked into position i.e. no gas, moisture or microbiological barriers of consequence. Refrigerated shelf life is measured in days. When the product is intended to be heated for consumption, the base tray packaging may be thermoformed polypropylene or crystallized polyester with no particular barrier closure. For modified-atmosphere packaging the tray material is a thermoformed, coextruded polypropylene/ethylene vinyl alcohol with a flexible gas/moisture barrier lamination closure heat-sealed to the tray flanges. Refrigerated shelf life for such products may be measured in weeks. For several years, the concept of pasteurizing the contents, vacuum packaging and distribution under refrigeration has been debated and commercially developed in both the USA and Europe. The ‘sous vide’ technique is the most publicized process of this type. In sous vide processing the product is packaged under vacuum and heat-sealed in an appropriate gadwater vapour barrier flexible package structure such as aluminium foil lamination. The packaged product is thermally processed at less than 100°C to destroy spoilage microorganisms and then chilled for distribution under refrigerated or (in the USA) frozen conditions. The US option is to ensure against the growth of pathogenic anaerobic microorganisms. A similar technology is cook-chill in which pumpable products such as chili, chicken a la King and cheese sauce are hot-filled at 80°C or more into nylon pouches which are immediately chilled (in cold water) to 2°C and then distributed at temperatures of 1°C. The hot filling generates a partial vacuum within the

package to virtually eliminate the growth of any spoilage microorganisms that might be present. This listing is only a sampling of the many alternative packaging forms offered and employed commercially for foods subject to immediate microbiological deterioration. An entire encyclopedia would be required to enumerate all of the known options available to the food packaging technologist with the advantages and issues associated with each.

Package Materials and Structures Package Materials

In describing package materials, different conventions are employed depending on the materials and their origins. The commercial conventions are used with some common indicator of quantitative meaning to establish relative values. Paper The most widely used package material in the world is paper and paperboard derived from cellulose sources such as trees. Paper is used less in packaging because its protective properties are almost non-existent and its usefulness is almost solely as decoration and dust cover. Paper is cellulose fibre mat in gauges of less than 250 microns. When the gauge is 250 microns to perhaps as much as 1000 microns the material is known as paperboard, which in various forms can be an effective structural material to protect contents against impact, compression and vibration. Only when coated with plastic is paper or paperboard any sort of protection against other environmental variables such as moisture. For this reason, despite their long history as packaging materials, paper and paperboard are only infrequently used as protective packaging against moisture, gas, odours or microorganisms. Paper and paperboard may be manufactured from trees or from recycled paper and paperboard. Virgin paper and paperboard, derived from trees, has greater strength than recycled materials whose fibres have been reduced in length by multiple processing. Therefore, increased gauges or calipers of recycled paper or paperboard are required to achieve the same structural properties. On the other hand, because of the short fibre lengths, the printing and coating surfaces are smoother. Paper and paperboard are moisturesensitive, changing their properties significantly and thus often requiring internal and external treatments to ensure suitability.

Metals Two metals are commonly employed for package materials: steel and aluminium. The former is traditional for cans and glass bottle closures, but is

PACKAGING OF FOODS 1619

Canner’s end

Canner’s end ,component

component /

Canner’s end seam

Seaming wall radius

Bod

Body hookradius

Seaming panel radius

/

Lining compound

Seaming wall

-Chuck

End hook

End hook radius

\

(4

Maker’s end component

(6)

Figure 1 Metal can construction. (A) Three-piece steel can. (6)Two-piece steel or aluminium can. From Soroka (1995) with permission. Lining compound

Can body

7: I!

Can end resting on body

First curl

Finished

double seam

Figure 2 Operation of affixing or double-seaming a metal closure to a metal can body. From Soroka (1995) with permission.

subject to corrosion in the presence of air and moisture and so is almost always protected by other materials. Until the 1980s, the most widely used steel protection was tin, which also acted as a base for lead soldering of the side seams of ‘tin’ cans. When lead was declared toxic and removed from cans during the 1980s in the USA, tin was also found to be superfluous and its use as a steel can liner declined. The tin in ‘tin-free’ cans was chrome and chrome oxide. The construction and closure techniques of metal cans are shown in Figures 1-3. In almost every instance the coated steel is further protected by organic coatings such as vinyls and epoxies which are really the principal protection. Steel is rigid, a perfect microbial, gas and water vapour barrier, and resistant to every temperature to whicha food may be subjected. Because steel-steel or steel-glass interfaces are not necessarily perfect, the metal is often complemented by resilient plastic to compensate for the minute irregularities. Aluminium is lighter in weight than steel and easier to fabricate; it has therefore become the metal of choice for beverage containers in the USA and is favoured in other countries. As with steel, the alu-

wall

Chuck wall radius

Body wall

Figure 3 Double-seam closure on a metal can. From Soroka (1995) with permission.

minium must be coated with plastic to protect it from corrosion. It is the most commonly used material for can-making in the USA. However, aluminium cans must have internal pressure from COz or Nz to maintain their structure, and so aluminium is not widely used for food canning applications in which internal vacuums and pressures change as a result of retorting. Aluminium may be rolled to very thin gauges (825 microns) to produce foil, a flexible material with excellent microbial, gas and water vapour barrier properties when it is protected by plastic film. Aluminium foil is generally regarded as the only ‘perfect’ barrier flexible package material. Its deficiencies include a tendency to pinholing, especially in thinner gauges, and to cracking when flexed. In recent years, some applications of aluminium foil have been replaced by vacuum metallization of plastic films such as polyester or polypropylene.

Glass The oldest and least expensive package material is glass, derived from sand. Furthermore, glass is a perfect barrier material against gas, water vapour, microorganisms, odours, etc. The transparency of glass is often regarded by marketers and consumers as a desirable property. Technologists may view the transparency as less than desirable because visible and ultraviolet radiation accelerates biochemical (particularly oxidative) reactions. Glass is energy-intensive to produce; it is heavy and vulnerable to impact and vibration even though it has excellent vertical compressive strength. For these reasons, glass is being displaced by plastic materials in industrialized societies. Plastics The term ‘plastics’ describes a number of families of polymeric materials (Table 3), each with different properties. Most plastics are not suitable as package materials because they are too expensive or toxic in contact with food, or do not possess properties desired in packaging applications. The most commonly used plastic package materials are poly-

1620 PACKAGING OF FOODS

Table 3 Packase plastic structures Plastic

Structure

Oualities

~

Polyethylene (PE)

H

H

H

I I I -c - c - c - c I I I I Polypropylene (PP)

H

H

H

H

CHS

H

CHJ

I

I

I

I c- cI I

H

H

H

Ethylene vinyl alcohol (EVOH)

H

H

I

I

H

O

H

O

I

l

l

1

I

I

I

I

H

H

H

H

H

CI

H

CI

I

I

I

I

-c-c-c-c-

Polyvinyl chloride (PVC)

I

I

I

I

H

CI

H

CI

H

CI

H

CI

I

I

I

I

-c-c-c-c-

Polyamide (PA) (Nylon)

Higher temperature than polyethylene Low density high yield Very good moisture barrier

H

-c-c-c-c-

Polyvinylidene chloride (PVDC)

-

H

-c-cI I

Three basic types: high-density linear low-density lowdensity Moisture barrier

H

I

I

l

l

1

H

H

H

H

Excellent O2 barrier resin Moisture sensitive, poor water barrier Used in coextrusion; expensive

Excellent 0 2 , moisture, flavour and fat barrier Dense

Stiff, clear - without plasticizer Soft with plasticizer No barrier

0

O

H

H

I1

I/

I

I

- C - ( C H Z )~ C - N - (CH2)s - N Polyethylene terephthalate (PET)

0

0

H

H

(polyester)

I/

II

I

I

-0-

Polyacrylonitrile (PAN)

H

H

H

I I I I -c-c-c-c-cl l l l

I

H

Polystyrene (PS)

c - (-J- c - 0 - c -

H

H

C

H

H

C

111

111

N

N

H

H

I

c I

H

H

l

Temperature resistant Very good 0 2 barrier Thermoformabie

High temperature after orientation

Very good O2 barrier Not processable in extrusion unless copolymer

H

H

H

1 1 1 1 -c-c-c-c-cI l l l

1

l

H c l H o H

Stiff, brittle, clear Very little barrier

PACKAGING OF FOODS 1621

Table 4

Properties of plastic package materials

Material

Specific gravity

Clarity or colour

Water vapour transmission‘

Gas transmissionb

Resistance to grease ~

Polyethylene high density medium density low density Polypropylene Polystyrene Plasticized vinyl chloride Nylon

0.941-0.965 0.926-0.940 0.910-0.926 0.900-0.91 5 1.04-1.08 1.16-1.35 1.13-1.1 6

Semi-opaque Hazy to clear Hazy to clear Transparent Clear Clear to hazy Clear to translucent

Low Medium Good Good High High to low Varies

High High High High High High Low

~~

Excellent Good Good Excellent Fair to good Good Excellent

aWatervapour transmission rate is measured in gm-’ for 24h at 38°C and 90% relative humidity. bGas transmission is measured in crn3ml-’m-*for 24h at latmosphere, 30°C and 0% relative humidity.

ethylene, polypropylene, polyester, polystyrene and nylon. Each has quite different properties (Table 4). Plastics may be combined with each other and with other materials to deliver the desired properties.

Polyethylene Polyethylene is the most used plastic in the world for both packaging and non-packaging applications. It is manufactured in a variety of densities ranging from 0.89 g cm-3 (very low density) to 0.96 g ~ m (high - ~ density), and is lightweight, inexpensive, impact-resistant, relatively easily fabricated, and forgiving. Polyethylene is not a good gas barrier and is generally not transparent, but rather translucent. It may be extruded into film with excellent water vapour and liquid containment properties. Low-density polyethylene film is more commonly used as a flexible package material. Low-density polyethylene is also extrusion-coated onto other substrates such as paper, paperboard, plastic or even metal to impart water and water vapour resistance or heat sealability. Although used for flexible packaging, high-density polyethylene is more often seen in the form of extrusion blow-moulded bottles with impact resistance, good water and water vapour barrier, but poor gas barrier properties. Any of the polyethylenes in proper structure functions as an effective microbial barrier. Polypropylene Like polyethylene, polypropylene is a polyolefin, but it has better water vapour barrier properties and greater transparency and stiffness. Although more difficult to fabricate, polypropylene may be extruded into films that are widely used for making pouches particularly on vertical form/fill/seal machines. In cast film form, polypropylene is the heat sealant of choice on retort pouches because of its fusion sealing properties, and because in this form it is a good microbial barrier. Polypropylene’s heat resistance up to about 133°C permits it to be employed for microwave-only heating trays. Unfortunately microwave heating alone is insufficiently uniform to be a reliable mechanism for

reducing microbiological counts or destroying heatlabile microbial toxins in foods.

Polyester A cyclical polymer that is relatively difficult to fabricate, polyethylene terephthalate polyester is increasingly the plastic of choice as a glass replacement in making food and beverage bottles. Polyester plastic is a fairly good gas and moisture barrier ;in bottle, tray or film form it is dimensionally quite stable and strong. Its heat resistance in amorphous form is sufficient to permit its use in hot-fillable bottles. When polyester is partially crystallized the heat resistance increases to the level of being able to resist conventional oven heating temperatures. For this reason crystallized polyester is employed to manufacture ‘dual ovenable’ trays for heat-and-eat foods (‘dual ovenable’ means that the plastic is capable of being heated in either conventional or microwave ovens). The transparency of polyester makes it highly desirable from a marketing standpoint for foods that are not light sensitive. Nylon Polyamide or nylon is a family of nitrogencontaining polymers noted for their excellent gas barrier properties. Moisture permeability tends to be less than in the polyolefin polymers and nylon is somewhat hygroscopic, meaning that the gas barrier may be reduced in the presence of moisture. Gas and water vapour barriers are enhanced by multilayering with polyolefins and high-gas-barrier polymers. Nylons are thermoformable and both soft and tough, and so are often used for thermoformed processed meat package structures in which the oxygen within the package is reduced to extend the refrigerated shelf life. Polystyrene Polystyrene is a poor barrier to moisture or gas. It is, however, very machinable and usually highly transparent. Its structural strength is not good unless the plastic is oriented or admixed with a rubber modifier which reduces the transparency. Polystyrene

Next Page 1622 PACKAGING OF FOODS

is often used as an easy and inexpensive tray material

for prepared refrigerated foods.

Polyvinyl Chloride Polyvinyl chloride is a polymer capable of being modified by chemical additives into plastics with a wide range of properties. The final materials may be soft films with high gas permeabilities, such as used for overwrapping fresh meat in retail stores; stiff films with only modest gas barrier properties; readily blow-mouldable semirigid bottles; or easily thermoformed sheet for trays. Gas and moisture impermeability is fairly good but must be enhanced to achieve ‘barrier’ status. This material falls into a category of halogenated polymers which are regarded by some environmentalists as less than desirable. For this reason, in Europe and to a lesser extent in the USA, PVC has been resisted as a package material. Polyvinylidene Chloride Polyvinylidene chloride (PVDC) is an excellent barrier to gas, moisture, fat and flavours, but is so difficult to fabricate on its own that it is almost always used as a coating on other substrates to gain the advantages of its properties. Ethylene Vinyl Alcohol Ethylene vinyl alcohol (EVOH) is an outstanding gas and flavour barrier polymer which is highly moisture sensitive and so must be combined with polyolefin to render it an effective package material. Often EVOH is sandwiched between layers of polypropylene which act as water vapour barriers and thus protect the EVOH from moisture. Package Structures

Currently, rigid and semirigid forms are the most common commercial structures used to contain foods. Paperboard is most common, in the form of corrugated fibreboard cases engineered for distribution packaging. In corrugated fibreboard three webs of paperboard are adhered to each other with the central or fluted section imparting the major impact and compression resistance to the structure. Folding cartons constitute the second most significant structure fabricated from paperboard. Folding cartons are generally rectangular in shape and often are lined with flexible films to impart the desired barrier. Metal cans have traditionally been cylindrical (Figures 1 , 2 and 3 ) , probably because of the need to minimize problems with heat transfer into the contents during retorting. Recently, metal - and particularly aluminium - has been fabricated into tray, tub and cup shapes for greater consumer appeal, with consequential problems with measuring and computing the thermal inputs to achieve sterilization.

Thread

\

\ /

Sealing surface (land)

Neck ring Neck (bead) ring 7 \ 7 : ; i s h parting line I

Bottom plate parting line Heel Push-up/

’

Base

I

~ 7r ~ - , ~

Figure 4 Glass bottle nomenclature. From Soroka (1995) with permission.

During the 1990s shaped cylinders entered the market again in efforts to increase consumer market share. Few have been applied for cans requiring thermal sterilization, but barrel and distorted body cans are not rare in France for retorted low-acid foods. Analogous regular-shaped cans are being used for hot filling of high-acid beverages. Noted for its formability, glass has traditionally been offered in a very wide range of shapes and sizes including narrow-neck bottles (Fig. 4) and widemouth jars. Each represents its own singular problems in terms of fabrication, closure and - when applicable - thermal sterilization. Plastics are noteworthy for their ability to be formed into the widest variety of shapes. Thin films can be extruded for fabrication into flexible package materials. These flexible materials may then be employed as pouch or bag stock or as overwraps on cartons or other structures, or as inner protective liners in cartons, drums, cases, etc. Thicker films (sheets) may be thermoformed into cups, tubs and trays for containment. Plastic resins may be injectionor extrusion-moulded into bottles or jars by melting the thermoplastic material and forcing it, under pressure, into moulds that constitute the shape of the hollow object, e.g. the bottle or jar. See also: Cheese: In the Market Place. Chilled Storage of Foods: Use of Modified Atmosphere Packaging; Packaging with Antimicrobial Properties. Fermented Milks: Range of Products. Fish: Spoilage of Fish. Heat Treatment of Foods: Thermal Processing Required for Canning; Principles of Pasteurization. Ice Cream. Meat and Poultry: Spoilage of Meat: Curing of Meat; Spoilage

QUANTITATIVE RISK ANALYSIS 1883

1

Quality Assurance and Management see Hazard Appraisal (HACCP): The Overall Concept

1

QUANTITATIVE RISK ANALYSIS S H W Notermans, TNO Nutrition and Food Research Institute, Zeist, The Netherlands Copyright 0 1999 Academic Press

Introduction Risk analysis is a structured, multidisciplinary approach to the identification and reduction of risk. Interest in risk analysis in the context of food-borne pathogens, contaminants and additives has increased due to the Sanitary and Phyto-Sanitary (SPS) Agreement of the World Trade Organization (WTO). The aim of the SPS Agreement is to endorse food safety objectives, such as microbial standards and guidelines, that are based on the application of risk analysis to sound scientific knowledge. Figure 1 illustrates the use of risk analysis in the development of food safety objectives (e.g. end product specifications) from the food safety policy of the WHO/FAO Codex Alimentarius Commission. Risk analysis can also be used to determine criteria at the critical control points in HACCP (hazard analysis critical control point) processes. Risk analysis involves the evaluation of risk in the context of science, an understanding of all the activities involved and a structured approach. The process of risk analysis consists of three essential components.

I-I

Food safety policy :

1

I Food safeti objectives I

I

I

I

I

I I

I

I

I Food producers

Good Manut;ng

Quantitative risk analvsis

Practices

Hazard analvsis critical control point process (HACCP) Own responsibility

k l I

Quantitative

risk analysis

Evaluationiverification

Figure 1 The use of risk analysis in the context of food safety.

These were recommended following F A O N H O expert consultations, and later adopted by its Codex Alimentarius Commission. They are: 1. Risk assessment: the evaluation of known or potential adverse health effects resulting from human exposure to food-borne hazards. The outcome of the risk assessment is called the risk estimate. 2. Risk management: the control of risks associated with food-borne pathogens and contaminants, in order to protect consumers. Risks are controlled as effectively as possible through the selection and implementation of appropriate measures, as formulated by the World Health Organization and the Food and Agriculture Organization (FAONHO). 3 . Risk communication: an interactive process of exchange of information and opinion between risk assessors, risk managers and other interested parties, such as consumers. The concept of risk analysis as adopted by F A O N H O and several Codex Committees is primarily aimed at consumer protection, and involves the establishment of safety objectives for foods that are based on science. Risk analysis may also be used in selecting the most appropriate food processing and preservation methods for compliance with the food safety objectives set. In addition, it is possible to use risk analysis to set (sub)criteriaat critical control points, as defined by the hazard analysis critical control point (HACCP) concept (see below). Thus risk analysis is used both in compliance with the food safety objectives set by the regulating bodies, and in meeting any additional objectives set by the producers themselves.

1884 QUANTITATIVE RISK ANALYSIS

Components of Quantitative Risk Analysis Risk Assessment

Risk assessment is the evaluation of known or potential adverse health effects resulting from human exposure to food-borne factors such as additives, contaminants and pathogenic microorganisms. Risk assessment involves the documentation and analysis of scientific evidence, the measurement of risk and the identification of factors that influence it. This information is used to produce the risk estimate. The process of risk assessment consists of four steps:

used to plot dose-response curves directly applicable to humans. Hazard characterization also involves consideration of the characteristics of a pathogenic microorganism in relation to factors such as the nature of the product, the processing conditions, and the storage conditions. This information is necessary, for example, to estimate the outgrowth of the organism in the food product of interest. There are a number of uncertainties in hazard characterization, and so the introduction of an uncertainty factor must be considered.

Exposure Assessment This is the qualitative and/or quantitative estimation of the likely intake of biological, chemical and physical agents via food. The 1. Hazard identification ultimate goal of exposure assessment is the estimation 2. Hazard characterization of the hazardous agents in food at the time of con3. Exposure assessment sumption. This requires specific expertise and infor4. Risk characterization. mation, about food consumption (e.g. from intake There are several strategies for obtaining information surveys) and about the concentration and distribution about the factors which contribute to risk and their of particular hazardous agents in foods. In the case impact. One approach is a case-control study, in of food-borne microbiological hazards, the estimated which unacceptable products are compared with concentration of microorganisms may be based on acceptable ones. product surveillance and testing, storage conditions Hazard Identification This is the identification of and the use of mathematical models which predict the potential adverse health effects associated with expos- growth and death of microorganisms. There are many ure to, inter alza, additives, contaminants and patho- sources of uncertainty involved in exposure assessgenic microorganisms. It is a qualitative approach. ment, resulting in either underestimates or overFor example, the microbiological hazards present in estimates. These uncertainties should be reflected in food may be identified with reference to a list (based the risk characterization. Although it is seldom poson published data) of pathogenic microorganisms able sible to provide fully quantified assessments of uncerto cause food-borne disease. The likelihood is deter- tainties, the introduction of a negative or a positive mined of each listed organism being present in the bias should be made clear. raw materials used and/or entering the food proRisk Characterization This is the quantitative cessing area. Organisms that have never been found in either location can be deleted from the list. Any and/or qualitative estimation of the probabilities of organisms which are completely destroyed during occurrence and severity of known or potential adverse processing can also be deleted from the list. The pos- health effects in a given population, taking into sibility of recontamination must then be considered. account attendant uncertainties. It is the last step in risk assessment, and from it a risk management stratAny organisms which are not known to cause a foodborne disease involving either an identical or a related egy can be formulated. Although the Codex Alimentarius document does not suggest that the food product can be deleted from the list. identification and quantification of the factors conHazard Characterization This is the qualitative tributing to a risk is part of the risk characterization, and/or quantitative consideration of the nature of the it is logical to include them. adverse health effects associated with the biological, Risk Management chemical and physical agents which may be present in food. If practicable, dose-response relationships Risk management is the process of evaluating altershould be assessed for all the adverse effects produced native policies in the light of the risk estimate and, by the agents being considered, e.g. changes in organ if required, selecting and implementing appropriate function and clinical symptoms. In the case of addi- controls, including regulation. The purpose of risk tives and contaminants, epidemiological data are of management is the identification of acceptable risk value in verifying the dose-response relationships levels and the development and implementation of obtained in experimental animals. In the case of control measures within the framework of public pathogenic microorganisms, such data may also be health policy. Risk management takes into account

QUANTITATIVE RISK ANALYSIS 1885

the factors contributing to a risk and their quantitative effect, and also a cost-benefit analysis of options. The outcome of risk management is the derivation of food safety objectives, for example banning additives or reducing their usage; setting maximum levels for contaminants and pathogenic microorganisms; and the obligatory use of Good Manufacturing Practices (GMP) and controls at national level. In setting food safety objectives, risk managers should take into account the difficulties of control, the feasibility of monitoring, the availability of suitable methods of analysis and the economic importance of the food. The F A O N H O document on risk management formulates some general principles covering a structured approach embracing risk evaluation, the assessment of risk management options, decision implementation and monitoring and review. The document also emphasizes that the primary consideration in risk management should be the protection of human health, and that the decisions and practices associated with risk management should be clear. Risk Communication

Risk communication is defined as an interactive process of exchange of information and opinion between risk assessors, risk managers, and other interested parties. communication starts with the provision of information about food safety policy to all parties involved in the process of risk analysis, as the basis for the purpose and scope of risk assessment and risk management. Clear, interactive communication is necessary between all involved, including consumers, and at all stages of the processes, and is likely to assume increasing importance.

Framework for the Establishment of Food Safety Objectives Food safety objectives reflect the food safety policy, which should present a general outline of what is acceptable or not acceptable, and quantitative risk analysis is used in the derivation process. There is increasing consensus that food safety policy is an international issue, and F A 0 and W H O are the international bodies responsible for setting this policy. They have delegated this task to the F A O N H O Codex Alimentarius Commission (CAC). This was established in 1962 as an intergovernmental organization for developing food-related standards, guidelines and recommendations in order to protect the health of consumers and facilitate international trade. These standards are recognized by WTO, and provide a reference point for the safety of foodstuffs traded internationally.

Risk assessment in relation to additives and contaminants is carried out by the Joint FAOPWHO Expert Committee on Food Additives (JECFA). The outcome of its risk assessment, i.e. the risk characterization, is the starting point for the Codex Alimentarius Committee on Food Additives and Contaminants (CCFAC). This Committee, in which all member states are represented, is responsible for risk management and sets standards which are subsequently adopted by CAC. Risk assessment in the context of additives and contaminants is a well-established activity. It involves, firstly, the determination of dose-response relationships for additives and contaminants which can cause an adverse health effect. This involves experiments on animals and the use of a data package obtained by testing several health parameters. From the dose-response relationship, the so-called ‘no observed effect’ level is estimated. This is used as the basis for determining acceptable daily intakes for additives, and provisional tolerable daily/weekly intakes for contaminants, through the application of uncertainty factors. Risk analysis in relation to food-borne pathogens is a newly emerging activity. Criteria and guidelines are set by the Committee for Food Hygiene (CCFH), and are based on results obtained from the analysis of outbreaks of food-borne disease. At present, CCFH lacks the assistance of a JECFA-like body for risk assessment studies. WHO/FAO have recommended that such a body be established, because it is unacceptable that a single body should carry responsibility for both risk assessment and risk management. Both CCFH and the JECFA equivalent should then elaborate the criteria and guidelines regarding the risk assessment of food-borne pathogens. In addition, CCFH should clarify the criteria for the selection of pathogens for referral to the JECFA equivalent and should clearly identify the factors to be taken into account in its decision making, particularly in relation to the evaluation of risk management options.

The Use of Quantitative Risk Analysis in Food Production The principles of quantitative risk analysis can also be applied to food production. Internationally established legal food safety objectives, together with safety objectives set by individual food production companies, focus on safe food production. In adhering to these objectives, food producers make use of general guidelines such as GMP. In addition, the use of hazard analysis critical control points (HACCP)is mandatory in most countries. The use of HACCP entails a systematic approach to the identification, assessment and

1886 QUANTITATIVE RISK ANALYSIS

control of hazards in a particular food operation. This approach aims to identify problems before they occur, and to establish measures for the control of stages in production that are critical in terms of food safety. The controls are thus preventive, remedial action being taken in advance of problems developing. The critical control points are defined as steps, points and procedures where control can be exercised. In relation to each point, criteria are specified such that if met, the food produced will be safe. The traditional, largely qualitative HACCP system can be converted into a quantitative system using elements of quantitative risk analysis, as indicated in Figure 2. In HACCP, a hazard is defined as it is in risk analysis: an agent with the potential to cause an adverse health effect. International standards have been established for most agents, which means that a risk assessment is not necessary. Critical control points may be defined as factors that contribute to the risk that a standard is not met. The effect of such a factor should preferably be quantified. In relation to each critical control point, risk managers set criteria, based on this quantification. In most cases, the actual risk is the result of a

I Hazard Analysis Critical Control Point system I 1

I

combination of several factors. A knowledge of the effect of each individual factor enables optimization of the process, taking into account economic factors. This can be illustrated with a simple example described by Notermans, Zwietering and Mead in 1994. One of the legal standards for pasteurized milk is that Bacillus cereus must number < l o 4 organisms per millilitre at the time of consumption. The factors which determine the B. cereus count at the time of consumption are the spore load of B. cereus after pasteurization and the storage time and temperature. The effect of each factor can be calculated, as can the cost of control of each factor. Clearly, the wishes of the consumer, especially in relation to storage conditions, must be taken into consideration in reaching a final managerial decision. See also: Bacillus: Bacillus cereus. Good Manufacturing Practice. Hazard Appraisal (HACCP): The Overall Concept; Critical Control Points; Involvement of Regulatory Bodies; Establishment of Performance Criteria. International Control of Microbiology. National Legislation, Guidelines 81 Standards Governing Microbiology: Canada; European Union; Japan. Predictive Microbiology 81 Food Safety. Process Hygiene: Involvement of Regulatory Bodies

I

Hazard analysis Determination of critical control points

I

~

Risk assessment identification and quantification of factors contributing to the risk Risk management: setting of criteria matching food safety objectives

Y

I System for monitoring Corrective actions

Documentation

Figure 2 The hazard analysis critical control point (HACCP) concept and the possible use of elements of risk analysis.

Further Reading Codex Alimentarius Commission (1996) Terms and Definitions used in Risk Analysis. Doc. CXIEXEC 9614316. Annex 1.

FAOAVHO (1995) Applications of Risk Analysis to Food Standavd Issues. Repovt of a Joint FAOIVIIHO Consultation. FAOAVHO (1997) Risk Management and Food Safety. Report of a Joint FAO/WHO Consultation. Notermans S , Zwietering MH and Mead GC (1994) The HACCP concept: identification of potentially hazardous micro-organisms. Food Microbiology 11: 203-214. World Trade Organization (1994) The Results of the Uruguay Round of Multilateval Trade Negotiations: the Legal Texts. Agveement on the Application of Sanitary and Phytosanitary Measures. MTN/FA 11-A1A-4.

RAPID METHODS FOR FOOD HYGIENE INSPECTION 1887

R RAPID METHODS FOR FOOD HYGIENE INSPECTION Matthias Upmann, Institute of Meat Hygiene, Veterinary University of Vienna, Austria Christine Bonaparte, Department of Dairy Research and Bacteriology, Agricultural University, Vienna, Austria Copyright 0 1999 Academic Press

Introduction Supplying consumers with microbiologically safe products is a high priority with regulatory authorities worldwide. But, since recognizing that governmental supervision cannot assure absolute food safety, strong emphasis is placed on the manufacturer’s responsibility for the hygienic and toxicological quality of foods, limiting the state’s task to the ‘control of the control’. To meet these product liability demands, the food industry increasingly relies on process control systems and longitudinally integrated quality and safety assurance programmes. The underlying idea is that safety and quality of the products are controlled best through effective management of those processing areas where hazards may arise. After assessing the risks associated with the food, processing steps are selected where preventive measures will lead to the elimination of the hazard. Establishing critical limits within these processing steps and monitoring relevant parameters will result in its control. But, with respect to microbial hazards such a systematic approach known as hazard analysis critical control point (HACCP) system suffers from slow and cumbersome conventional methods in microbiology which neither allow rapid evaluation of raw materials on delivery nor ‘on-line’ control measures during processing. Even with end-product testing, they often permit only a retrospective assessment of the food’s microbiological condition, since many foods are highly perishable. Therefore, much effort has been made to develop methods which enable a more rapid estimation of the microbiological quality of foods.

Microbiological Examination of Foods General Considerations

To get reliable results from microbiological examination of foods many factors must be taken into

consideration. Firstly, ‘food’ is an extremely varied matrix which contains infinite arrays of ingredients, shows a high variability in physical composition, is subjected to multifold processing technologies and is stored under many different conditions. Furthermore, its intrinsic flora may consist of high numbers of typical quality indicating microorganisms as in the case of fermented products. Also, they may contain varying amounts of shelf-life limiting or even hazardous microorganisms. O n the other hand there are numerous sterilized products. In contrast to chemical and physical contaminants, microorganisms are mostly heterogeneously distributed in foods and their concentration seldom remains constant. Additionally, microbial cells may be injured sublethally due to food manufacturing processes or food ingredients, thus escaping detection if no preventive measures are taken. The same problem may occur when a high background flora prevents selective isolation of specific bacteria.

Methodological Requirements

Three main categories of analytical procedures can be distinguished. Firstly, analysis may be directed towards qualitative detection of specific microorganisms (presence/absence tests). Secondly, analysis may be performed in order to quantify the total microbial number, special indicative groups or specific microorganisms. Thirdly, characterization of isolated microorganisms may be desired. Considering the broad range of analytical procedures available particular requirements were defined which an optimum method should meet. High sensitivity, which is defined as the lowest amount of microorganisms detectable, should be of primary importance. Likewise, high accuracy is essential. The analytical result should meet the true value and repetitions of the analytical procedure should ideally give the same results (Le. high precision). As explained

1888 RAPID METHODS FOR FOOD HYGIENE INSPECTION

above, rapidity is another important factor. Under practical conditions economic considerations favour the use of simple, inexpensive, universally applicable and less laborious methods. Furthermore, the testing system must operate at a high level of hygienic safety, as for instance provided by self-contained units, especially if the user group consists of non-specialists. Unfortunately, an optimum technique covering all requirements does not exist. In particular, the accuracy of different analytical techniques is quite different, hence validations by in-laboratory and/or interlaboratory comparisons against commonly agreed standard methods are necessary. European standards for validation and official acknowledgement of alternative microbiological methods are now dealt with at the technical committee of the European Committee for Standardization (CEN/TC) in Brussels. Improving Methodological Rapidity

Ideally, rapid methods should enable such a quick estimation of microbiological parameters that food manufacturers are able to take corrective actions immediately in the course of the manufacturing process. However, the majority of methods characterized as ‘rapid’ do not meet this demand. Nevertheless, they offer a more or less pronounced advantage in analytical time compared to their conventional equivalent by eliminating laborious and/or subjective elements through mechanization and automation. Improved rapidity can be applied at each step of the analysis, i.e. the sampling process, sample treatment and detection/enumeration procedure. Although labour-saving and automated methods speed up the processes of sampling and sample treatment, thus improving the laboratory’s output, the influence on the total analysis time is usually negligible due to the incubation time required for traditional culture-based methods. A real shortening of the analytical time can only be obtained if alternatives to the traditional incubation methods are developed.

Training of Inspection Staff New inspection techniques make great demands on the qualifications of the inspection staff. The numerous analytical options can be confusing and overwhelming to the user. It is the user who decides whether a microbiological test is reasonable -the fact that it is applicable does not mean that it is necessary or useful - and which technique should be applied. Because new analytical procedures are based on various technologies and designs, their performances are highly variable. Moreover, many automated instruments exhibit a so-called ‘black-box’ phe-

nomenon. The utmost caution is advised with such instruments; reliable results are only feasible when they are properly maintained and calibrated. Test results must never be accepted in an uncritical manner. Therefore, incorporation of accelerated methods into the microbiological analytical repertoire must be accompanied by training of the inspection staff. By following the literature or attending occasional meetings, one is not likely to be able to keep abreast of the rapidly changing and developing field of inspection techniques.

Methods with Improved Rapidity Sampling

The sampling method depends on the material (processing environment, solid, semisolid or liquid foods), the surface structure (smooth/rough, horizontal/vertical, fladcurved), and the expected microbial contamination level. Additionally, the practicability on the spot should be considered: the use of electrical sampling devices, for example in the processing areas, may be a problem due to lacking plug sockets. With a few exceptions, such as ultrasonic sterility testing of heat-treated milk, food samples are taken destructively (excision, scraping) which destroys the integrity of the food. Sampling of foods is almost exclusively performed manually. On the other hand, surfaces of the food processing environment are sampled by non-destructive methods. Mostly, contact slides or swabbing techniques are employed. The former is often regarded as ‘rapid’ as there is no necessity for further sample treatment and its simple application, although the incubation time remains unchanged. Other methods, such as manual or mechanical rinsing, do not have any practical importance. Sample Treatment

During sample treatment, the sample is comminuted, liquefied and homogenized. Subsequently either dilution or enrichment steps may be necessary according to the expected level of microorganisms. Several semi- or fully automated dilution procedures which reduce the laboratory work have been developed (e.g. stomacher, pipetting instruments, gravimetric diluters). Substituting for microbial enrichment procedures and enabling quantitative results at low microbial concentrations, several physical techniques for extraction and concentration of microorganisms are employed. Techniques such as filtration, centrifugation, ion exchange resins and the very promising area of magnetic separations are men-

RAPID METHODS FOR FOOD HYGIENE INSPECTION

tioned. Furthermore, the polymerase chain reaction (PCR), has become more applicable as a non-cultural means of target amplification for food analysis nowadays. Microbial Detection, Enumeration and Characterization

1889

Table 1 Rapid methods for microbial detection, enumeration and characterization in food microbiology: overview Direct methods Microcolony and single cell detection Conventional microscopy Epifluorescent techniques Direct epifluorescent filter technique (DEFT) Antibody direct epifluorescent filter technique (Ab-DEFT) Membrane filter microcolony fluorescence technique (MMCF) Flow cytometry

New time-saving detection methods utilize principles originally belonging to disciplines such as chemistry, biochemistry, physics or immunology. This development was rendered possible because of major tech- Indirect methods Methods based on growth and metabolic activity nological advances in data-processors, which allow Optical methods rapid collection and interpretation of vast amounts of Colorimetry and fluorometry data. Since these methods often measure parameters Turbidimetry which are different from the traditional ones, the Pyruvate determination Thermal methods correlation with traditional methods may be Microcalorimetry problematic. Electrical methods The methods can be placed in two categories: (1) Direct conductimetry/impedimetry direct methods based on the detection of whole cells Indirect conductimetry/impedimetry (single cells or colonies), and (2) indirect methods Radiometry which measure cell components, metabolites, metaImmunological methods bolic activities or changes caused by cell growth. Table Agglutination tests 1 gives a survey of rapid methods used for microbial lmmunodiffusion tests Immunoassays based on labelled antibodies detection, enumeration and characterization. Direct Methods

Usually, colony-based techniques cannot be characterized as rapid due to the continuing necessity for incubation, although several devices (dehydrated nutrient pads, spiralplater, laser or image analyser etc.) can help to reduce the total laboratory work. Therefore, rapid direct methods are microcolony or single-cell based. Microcolony and Single-cell Detection Microscopical Techniques In order to visualize objects for microscopical examination, colouring agents are used to provide information on the total levels of microorganisms (e.g. methylene blue, acridine orange staining), special bacterial groups (e.g. Gram stains) or specific types of microorganisms (e.g. fluorescently labelled antibodies). In combination with different pre-treatments (e.g. membrane filtration, pre-incubation) and detecting principles (microscope, image analyser), microscopy has developed into a commonly used technique.

Epifluorescent techniques The direct epifluorescent filter technique (DEFT) was originally developed for rapid assessment of bacterial numbers in raw milk. However, the introduction of several pre-treatment techniques has considerably enlarged the range of successful applications. Homogenized, prefiltered and subsequently enzyme-surfactant-treated food samples are passed

Immunofluorescent assays (IF) Radioimmunoassays (RIA) Enzyme immunoassays (EIA) Immunomagnetic separation

Methods based on microbial cell components Luminometry ATP-bioluminescence Bacterial bioluminescence ('in-vivo bioluminescence') Limulus amoebocyte lysate test Ergosterol de termination Nucleic acid-based methods DNA probe hybridization Polymerase chain reaction Fingerprinting-like methods Combined methods Biosensors

through membrane filters and are stained, most commonly with acridine orange which binds to nucleic acids. O n epifluorescence-microscopical examination, aggregates of orange fluorescing cells are counted. Another technique uses tetrazolium chloride, which is reduced to purple-coloured formazan by an active cellular respiration apparatus. By using fluorescently labelled antibodies, specific types of microorganisms can be detected. This antibody-direct epifluorescent filter technique ( Ab-DEFT) is especially useful in detecting pathogens. Since pathogens usually occur in foods at low numbers and microbial cell surface antigenicity has to be preserved,

1890 RAPID METHODS FOR FOOD HYGIENE INSPECTION

special product preparation steps (enrichment, immunocapture, pre-incubation) are necessary. Short-term incubation of the membrane filters before the staining process, results in the growth of microcolonies. Hence, only viable microorganisms are detected by this ‘membrane filter microcolony fluorescence (MMCF)’technique. DEFT and related techniques have been used for counting bacteria in milk, milk products, water, beverages, raw meat, fish, poultry and food contact surfaces. If large numbers of samples have to be analysed daily, an automated counting procedure linking the microscope to an image-analysing system is advisable. Due to its rapidity and broad applicability DEFT is recommended for quality control, shelf-life prediction, irradiation control, as well as hygiene monitoring. Further details are given in Table 2.

Flow Cytometry Flow cytometry enables both qualitative and quantitative analysis of microbial cells in liquids. The sample is injected in a thin, rapidly moving carrier fluid which passes through a light beam. The previously fluorescently labelled cells are detected one by one with a photoelectric unit. By using nonspecific and specific fluorochromes, different wavelengths and measuring at different angles, it is feasible to discriminate between bacteria in mixed populations. The practical use of flow cytometry is still limited to few examples. However, since the possible applications are numerous, it should be considered as a promising technology in the future. Some methodological properties are given in Table 2. Indirect Methods

Methods Based on Growth and Metabolic Activity Several promising analytical procedures are based on the detection of microbial growth during incubation. Detection times ranging from a few minutes to 30 h depend on many factors, including inoculum density, microbial growth rate and type of metabolic activity. Generally, detection time is related inversely to the bacterial number: the lower the initial bacterial content, the longer the detection time. According to the physico-chemical properties considered, optical, thermal, electrical, and radiometric methods are distinguished.

Optical Methods Colorimetry and fluorometry: Specific physical or chemical changes (pH, oxidation/reduction potential, enzymatic transformations) associated with microbial metabolic activity can be indicated by changes in colour, fluorescence or colour intensity of an added reagent dye during sample incubation. Many

chromogenic and fluorogenic dyes are used depending on the metabolic change to be shown. A multitude of miniaturized and computer-aided or even fully automated identification systems for pure cultures are based on this principle. Colorimetry and fluorometry can also be used for quantitative purposes by measuring the required incubation time in order to produce a colour reaction or fluorochrome formation. Broadly known indicators are litmus and bromocresol purple for detecting pH shifts or resazurin, methylene blue, and triphenyltetrazolium chloride as oxidation/reduction indicators. Fully automated procedures are now available which use reflectance colorimeters or fluorometers. These techniques are applicable for rapid estimation of total microbial numbers or specific microorganisms, for product shelf-life stability, starter culture activity, and antibiotic testing. Fur further details see Table 2. Turbidimetry: Increasing cell numbers lead to an increase in optical density of liquid growth media. Therefore, a light beam will increasingly we weakened on transillumination when a liquid sample is incubated. By varying the sample dilution, the growth medium and the incubation temperature the result can be narrowed to specific bacterial species or numbers. Some methodological properties are given in Table 2. Turbidimetry is widely applied in vitamin bioassays and disinfectant testing. It has been used for sterility testing in food quality control. Its application may be limited by background turbidity of foods (fat globules, blood cells, food particles). Pyruvate determination: Pyruvate is a key compound in bacterial lactose metabolism and can serve as an indicator for milk quality monitoring. Pyruvate is measured indirectly by spectrophotometric detection of reduced nicotinamide-adenine dinucleotide (NADH) which is a cofactor in the enzymatic breakdown of pyruvate. Since somatic cells contribute to the pyruvate content of milk and not all bacteria produce pyruvate, the relation between this metabolite and total microbial count is limited (see Table 2).

Thermal Methods Bacterial growth is accompanied by heat production, which can be used for microcalorimetric estimation of the bacterial content. Highly sensitive calorimeters are necessary to detect the heat generated. Due to multiple interfering factors, microcalorimetry has thus far not assumed any practical importance. Electrical Methods

Measurement of electrical con-

RAPID METHODS FOR FOOD HYGIENE INSPECTION 1891

Table 2

Methodological properties of selected rapid methods in food microbiology Purpose

Method qual. Direct methods Epifluorescence microscopy DEFT

Ab-DEFT MMCF Flow cytometry

-

+ -

+

quant. chal:

Detection limit (cells per ml or Per 91

Rapidity

Selected instruments and suppliers

1000 p.p.m. of potassium sorbate. Intrinsic preservative resistance mechanisms are extremely adaptable and robust. Their functionality and effectiveness are unaffected or marginally suppressed by physiochemical environmental conditions such as low pH, low a , high osmotic pressure and sparse nutrients. Interestingly, there is strong evidence that Z. bailii preservative resistance is stimulated by the presence of multiple antimicrobial constituents. Cellular acetic acid uptake was inhibited when sorbic acid, benzoic acid or ethanol was incorporated into yeast culture medium. Similarly, ethanol levels up to 10% did not adversely alter Z . bailii intrinsic sorbic acid and benzoic acid resistance at p H 4.0-5.0. Sugar substrate investigations also revealed negligible effects on preservative resistance. Comparable sorbic and benzoic acid resistance was observed regardless of whether Z. bailii cells were grown in culture medium containing glucose, fructose or sucrose as the fermentable substrate. Conversely, synergistic interaction between salt and acetic acid generated antagonistic effects against Z . bailii. As salt levels increased, the yeast was inactivated by lower amounts of acetic acid. Sugar fermentation within the Zygosaccharomyces genus is unique. Unlike the vast majority of yeast genera, fructose is metabolized more rapidly than glucose. For species like Z. bailii and Z . rouxii this produces a phenomenon known as fructophily, in which yeast growth rates are greatly accelerated when a food’s fructose level approaches and exceeds 1% of product composition. The slow and delayed fermentation of sucrose is directly linked to fructose metabolism: sucrose, a disaccharide composed of glucose and fructose, is hydrolysed by food acids, i.e. low p H conditions. Hence, in acidic processed foods and beverages there is a steady accumulation of glucose and fructose during storage. If sucrose is the primary carbohydrate ingredient and Z . bailii contamination is present, cell growth can be impeded for several weeks until sufficient quantities of fructose and glucose are available to support reproduction and proliferation. This is usually preceded by a 2-4-week lag before visible spoilage defects are noticeable. In toto, overt product quality deterioration does not surface until 2-3 months after manufacturing. Z . bailii’s key preservative, physiological and metabolic resistance properties are summarized in Table 2. 2. rouxii and 2.bisporus

These two species differ from Z . bailii in their inferior resistance to acetic acid and chemical antimycotic

ZYGOSACCHAROMYCES 2361

Table 2 Key preservative and physiological resistance factors of Zygosaccharomyces bailii factor

Resistance properties

Acetic acid (pH 4.0) Benzoic acid (pH 4.0) Sorbic acid (pH 4.0) Sulphur dioxide Ethanol Sodium chloride Sugar (glucose) PH Water activity (a,) Temperature range Atmosphere

33% 31000 p.p.m. 31000 p.p.m. 3500 p . p m 2 10-1 5%

75" Brix and salt concentrations close to 20%, corresponding to 0.75 and 0.85 a,\,respectively. With the exception of a few rare strains, the growth of Z . rouxii and Z . bisporus is completely inhibited by 3 500 p.p.m. of sorbic and benzoic acids within a 4.04.5pH range. They are especially sensitive to acetic acid, rapidly dying off at 2 1% levels. Physiochemical environmental effects on sugar and salt-tolerant yeast have not been studied as comprehensively as Z . bailii. This is most likely due to the latter's broader economic impact regarding food and beverage categories vulnerable to spoilage. Comparative studies of sugar- and salt-tolerant 2. rouxii strains indicated that distinct physiological differences existed. The sugar-tolerant strains grew over a 1.8-8.0 pH range in high sugar media, whereas salt-tolerant strains were more sensitive to pH conditions. At 1mol sodium chloride concentration, growth was detected over a 3.0-6.6 pH range. When sodium chloride molarity was doubled, growth was restricted to a narrow 4.0-5.0 range. Z. rouxii optimum growth temperature increased as a,v decreased. Surprisingly, the optimum temperature reached 35°C at 60.96 a, levels, which is more typical of mesophilic bacteria incubation requirements.

Zygosaccharomyces Heat Resistance The heat resistance profiles of Z . bailii, Z . rouxii and Z . bisporus are comparable to other ascosporeforming yeasts. Z . bailii asci were significantly more heat-resistant than Z . rouxii at 0.963 and 0.858 a, in liquid medium adjusted to pH4.5. Z . bailii vegetative cells were also more heat-resistant than Z . rouxii.

As expected, asci and vegetative cell heat resistance increased as a,v decreased. Six log reductions (D64sC) were calculated as 1.2mid0.963 a,,, and 5.4 min/0.858 a,,,. Commercially processed fruit drinks and sugar syrups are normally pasteurized at 75-85°C. This provides a large quality assurance margin with respect to Zygosaccharomyces thermal destruction efficacy.

Zygosaccharomyces Preservative and Environmental Resistance Mechanisms Due to the pioneering and elegant research efforts of Dr A.D. Warth the Z . bailti preservative resistance mechanism has been elucidated and thoroughly understood. The organism utilizes an inducible, active transport pump to counteract the toxic effects produced by undissociated preservative molecule buildup inside individual cells. The pump provides two levels of protection. First, it physically expels preservative molecules from the cell, which assists in maintaining low, non-injurious preservative levels within the cell. Second, the rapid and efficient purging of undissociated molecules prevents deleterious cytoplasm pH changes that could disrupt or shut down critical metabolic pathways. Because the pump requires energy to function optimally, high sugar levels enhance Z. bailii preservative resistance. It is equally effective in excreting monocarboxylic organic acids and lipophilic straight chain fatty acid preservatives such as sorbic acid and benzoic acid. Additionally, the active transport successfully operates across a wide p H and a , range, and broad nutritional conditions. The resistance to osmotic stress of Z. rouxii is primarily associated with internal synthesis of polyols, mainly glycerol and arabitol, which raises intracellular pressure, bringing it in balance with the external osmotic gradient. Cell membrane and wall composition as well as ATPase enzyme activity may be important in augmenting poly01 formation, thus regulating osmotolerance.

Zygosaccharomyces Spoilage in Food and Beverages Z . bailii is the most troublesome and persistent spoilage yeast confronting acidified food and beverage manufacturers. Early reports of inexplicable fermentation spoilage in mayonnaise and salad dressing date back to the 1920s. Several incidents described violent fermentation coupled with the recovery of a few yeasts. More detailed investigations in the 1940s and 1950s confirmed Z. bailii spoilage in cucumber pickles, sundry pickled vegetable mixes, acidified

2362 ZYGOSACCHAROMYCES

Table 3 Food and Drug Administration yeast fermentation spoilage recalls in dressings and related acidified processed foods (1978-1 996)

-

Year

Product

1978 1981 1984 1985 1986 1987 1988 1990

Imitation mayonnaise Mayonnaise (single serving pouch) Low-calorie Roquefort dressing Homestyle salad dressing, real mayonnaise Real mayonnaise Carbonated beverages (soda and fruit concentrate) Ketchup Reduced-calorie mayonnaise, fat-free French dressing Light mayonnaise Lite Caesar dressing, lite creamy Parmesan dressing, olive oil vinaigrette dressing Salad dressing, tartar sauce, coleslaw dressing, burger tartar sauce, thousand island dressing, Parmesan pepper dressing, lite Parmesan pepper dressing White salad dressing Salad dressing

1991 1992 1993

1995 1996

sauces, mayonnaise and salad dressings. Spoilage invariably occurred in acidic shelf-stable foods which relied upon acetic acid (vinegar) to negate microbiological growth risks. Around the same time, sporadic gaseous fermentation spoilage incidents suddenly appeared in high-acid/sugar fruit syrups and beverages preserved with moderate benzoic acid levels of 400-500 p.p.m. Again, Z . bailii was indisputably identified as the spoilage culprit. The near simultaneous emergence of Z . bailii spoilage in two divergent processed food categories was probably due to improved laboratory and field evaluation techniques, better communication channels, movement towards consolidated, mass-production manufacturing facilities and large-scale complex distribution networks. Remarkably, 50 years later, Z. bailii spoilage risks and root causes closely parallel the processed food industry’s situation in the late 1940s to early 1950s. Despite quantum leaps in formulation control, food process equipment design and construction, and sanitation technologies (automated clean-in-place, sanitary transfer valves, etc.), Z. bailii remains problematic in mayonnaise, salad dressings, tomato ketchup, pickled/brined vegetables, low to moderate Brix fruit concentrates, and various non-carbonated fruit drinks. This is mute testimony to the organism’s adaptability, resilience and overall hardiness. Furthermore, Z . bailii spoilage is expanding into new food categories. Two recent examples are spoilage incidents in prepared mustards and fruit-flavoured carbonated soft drinks containing citrus, apple and grape juice concentrates. The specialized but persistent nature of Z . bailii spoilage problems is exemplified in Table 3,which summarizes the US Food and

Drug Administration yeast fermentation - spoilage recalls in high acetic acid and/or chemically preserved foods and beverages covering an 18-year period (1978-1996). Most likely, Table 3 represents a small fraction of economic losses produced by Z . bailii contamination. Obviously, company rejections, prolonged holding times and spoilage risks caught before finished production lots reached retail distribution channels are not accounted for. In most cases it is difficult to pinpoint the specific cause of spoilage. This is because the problem does not show up until 2-4 months after production and the finished product conformed to applicable formulation, processing and microbiological specifications at the time of manufacture. Z . bailii spoilage manifestations are readily recognized by both customers and consumers. Overt physical and organoleptic decomposition signs include product oozing from jars or bottles, emission of pungent yeast and alcoholic odours, occasional emulsion breakage (dressings), sediment formation (beverages) and brown surface film development on product surfaces. Z . bailii and Z . rouxii spoilage must never be taken lightly. Under extreme circumstances, internal COz pressure increases inside glass jars or bottles to the level where on-the-shelf explosions may take place. Although the possibility is remote, personal injury could result from flying debris. Plastic containers and polyfilm pouches burst open rather than explode. If slip and fall injuries were caused by spilled product residue, the company is exposed to contentious liability issues. A plausible scenario is that individuals become angry after being soiled by high-velocity product expelled from jars or bottles immediately upon opening. More often than not, these types of incidents result in unwanted government involvement, or costly consumer complaint investigations. Z. rouxii and Z. bisporus exclusively spoil highsugar/low a, foods and food ingredients. The most prevalent types are sugar syrups, fruit syrups, molasses, honey, fruit concentrates, sweetened wines and cordials and confectionaries such as marzipan and candy fillings. These yeasts ferment the food product, leading to effervescence, alcohol odour or taste, and turbidity which is more easily noted in clear sugar syrups. However, Z. rouxii and Z . bisporus ferment slower and less aggressively than Z. bailii. Thus, the potentially serious ramifications generated by vigorous Z. bailii fermentative metabolism are not a concern with osmotolerant yeast. As mentioned previously, it is generally impossible to ascertain the exact reason why Z. bailii or Z . rouxii contamination occurred in processed food lots made 2-3 months earlier. Circumstantial and inferential evidence is readily attainable from additional finished

ZYGOSACCHAROMYCES 2363

product and environmental sample testing, but accurate and concrete information gathering is highly suspect when the investigation focuses on reconstructing and interpreting events that happened weeks ago. However, trend analysis of past spoilage incidents suggests that certain commonalties exist among diverse Zygosaccharomyces contamination failures. They include undetected introduction of the offending Z. bailii or Z. rouxii strain into the plant environment from a low-level heterogeneous contaminated ingredient lot. This is followed by yeast build-ups inside key processing equipment because of inadequate or poorly executed sanitation procedures. In tandem, routine quality assurance/quality control (QA/QC) monitoring protocols missed or overlooked contamination risks. Eventually, finished product cross contamination occurred during production runs, and QA/QC standard operating procedures (SOPS)lacked appropriate detection sensitivity, discrimination abilities and sampling discipline/focus to discover the problem. These shortcomings and deficiencies are compounded in product formulations which are excessively sensitive to yeast growth. Certain fruit beverages can be spoiled by Z. bailii contamination as low as one viable cell in 3 10 1 of finished product. No sanitation or microbiological QA/QC programme can cope with this degree of risk. The only viable alternatives would be reformulation to increase stability and/or application of high-lethality thermalprocessing parameters.

-

Zygosaccharomyces Pract i caI Food/Beverage Industry Aspects Recovery and Enumeration

- Q N Q C Microbiology

0

0

0

Rich nutrient content and absence of acidulants concomitantly resuscitate debilitated cells and maximize growth rates. Use of antibiotics to eliminate bacterial growth. Plating or streaking on selective media to separate spoilage from innocuous yeast strains.

The detection and enumeration of sugar- and salttolerant yeast ( Z . rouxiij rely upon high solute concentrations in plating media and enrichment broths. Typically, 40-60% glucose is added to the basal medium to lower a , and establish a high osmotic pressure environment, The most popular basal plating medium is unacidified total plate count agar, which is often supplemented with antibiotics that obviate bacterial growth. Incubation conditions and length are identical to preservative-resistant yeast. One controversial aspect is whether plating diluent must contain 40-60% glucose in order to prevent osmotic shock (transfer from low to high a , environment) that dramatically decreases yeast recovery rates. Recent observations suggest that 30°C is the best incubation temperature for yeast detection. Faster growth rates and larger surface colonies occurred at 30°C vs 25°C incubation for 3-5 days. It is also widely accepted that all yeast and mould plating assays should employ surface plating techniques which simplify counting procedures, improve discrimination between food particles and yeast colonies, and enhance recovery rates due to high oxygen tension. Surface plating effectiveness is further optimized when the sample aliquot is analysed by membrane filtration methods (for example, hydrophobic grid membrane filtration j. Membrane filtration physically separates viable yeast cells from food and beverage ingredients that may inhibit or slow yeast growth. The filtration step is superior to manual spreading and streaking regarding depositing and distributing individual yeast cells over the entire agar surface, which increases enumeration accuracy and improves analytical reliability and simplicity. Recommended preservative-resistant and sugar-tolerant Zygosaccharomyces microbiological Q C plating and enrichment media are summarized in Table 4.

Several media were developed for the selective detection and quantification of preservative-resistant and sugadsalt-tolerant yeast in susceptible foods and beverages. As regards the former yeast group, standard non-differential yeast and mould plating media (malt extract or potato dextrose agars) were supplemented with 20.5% glacial acetic acid and/or 0.05% sodium benzoate. Recommended incubation times ranged from 5 to 14 days. Common incubation conditions were 20-25"C, aerobic atmosphere and acidifying Zygosaccharomyces Identification media to 4.0-4.5 p H range to ensure proper selectivity. The addition of 0.5% glacial acetic acid was As discussed earlier, Zygosaccharomyces colonies on non-selective and semi-selective media are morusually sufficient to produce a final p H of 4.0-4.5. phologically similar to many common yeasts, and Specialized yeast enrichment broths were co-develtraditional biochemical and physiological tests used oped to increase recovery sensitivity below the typical to identify Z. bailii and Z . rouxii do not lend themb 10 per gram or millilitre detection threshold of direct microbiological plating methods. Up to 1000-fold selves to routine microbiological Q C applications. sensitivity increases were reported. The enrichment Yeast identification kits have been available for over 10 years. However, they have limited Q C benefits broths operate on three principles:

Next Page 2364 ZYGOSACCHAROMYCES

Table 4

Recommended enumeration and recovery media for preservative-resistant and sugarhalt-tolerant Zygosaccharomyces

Medium

Composition (per litre)

Preparation

lncubation timeltempera ture

Special comments

Acidified tryptone glucose yeast extract agar (ATGYE)

100 g Glucose monohydrate 5 g Tryptone 5 g Yeast extract 15 g Agar 5 ml Glacial acetic acid

5 days 25-30°C

1. 0.5% glacial acetic acid in final medium: pH ca. 3.9 2. Best total recovery of preservativeresistant yeast 3. Not selective for Z. bailii

Z. bailii selective agar medium (ZBM)"

30 g Sabouraud's dextrose broth 30 g Fructose 25 g Sodium chloride 15 g Agar 5 g Tryptone 2.5 g yeast extract 0.1 g Trypan blue dye (optional) 5 ml Glacial acetic acid 1 ml Potassium sorbate 10% solution

1. Mix and boil dry ingredients 2. Autoclave at 121"C for 15minat 15Ibsteam 3. Cool to 45-52°C 4. Add 5 ml glacial acetic acid and mix thoroughly 5. Pour plates, let solidify. Store at 4-6°C for up to 2 weeks 1. Same as ATGY E except autoclave 121"C for 5 min at 15 Ib steam 2. Add potassium sorbate and glacial acetic acid after cooling to 45-52°C

3-5 days 30°C

Dichloran-18% glycerol agar (DGl8)

Commercially available

As directed by manufacturer

5 days 25°C

Malt-yeast extractdo% glucose agar (MY50G)

500 g Glucose 10 g Malt extract 10 g Agar 2.5 g Yeast extract

5 days 25°C

Plate count 30% sugar agar (PCAS)

200 g Glucose 100 g Fructose 23 g Plate count agar 0.1 g Trypan blue dye (optional) 20 ml Sterile antibiotic solution

1. Mix and boil dry ingredients 2. Autoclave at 121"C for 15 min at 15 Ib steam 3. Cool to 45-52°C 4. Pour plates, let solidify. Store at 4-6°C for up to 2 weeks 1. Mix and boil dry ingredients 2. Cool to 45-50°C 3. Add 20 ml antibiotic solution and mix thoroughly 4. Pour plates, let solidify. Store at 4-6°C for up to 2 weeks

1. 0.5% glacial acetic acid in final medium: pH 3.7-4.0 2. Filter sterilize 10% potassium sorbate solution before use: 100 p.p.m. in final medium 3. Highly specific for Z. bailii 4. Targeted to pickled vegetables, fruit concentrates and salad dressings 5. Requires validation in alcoholic and acidified beverages 1. Recovered Z. bailii and Z.rouxii 2. Best yeast recovery when mixed with xerophilic mould 1. Does not recover Z. bailii 2. Colonies small at 5 days

3 days 30°C

1. Used with hydrophobic membrane filtration method 2. Leverages fructophily response of Z. rouxii to speed up growth rate

~~

aModified from Erickson JP (1993) Hydrophobic membrane filtration method for the selective recovery and differentiation of Zygosaccharomyces bailii in acidified ingredients. J. Food Prot. 56: 234-238.

because of labour requirements, complexity and length of testing, which can take 37 days for a final result. A trained laboratory staff is needed to reduce human error. These kits are useful plant investigation, trouble-shooting and problem-solving tools. Various genetic DNA probe techniques have been successfully applied to identify spoilage yeast, includ-

ing Z.bailii and Z.rouxii. Three technologies mentioned were random amplified polymorphic DNA (RAPD), RAPD-polymerase chain reaction, and microsatellite polymerase chain reaction assays. All have been described as extremely precise and faster than traditional cultural identification methods. Whether there is enough industry demand and volume

APPENDIX I: BACTERIA AND FUNGI

The genera listed here are those associated with food, agricultural products and environments in which food is prepared or handled. Abiotrophia Acinetobacter Actinobacillus Actinomyces Aerococcus Aeromonas Agrobacterium Alcaligenes Alloiococcus Anaerobiospirillum Arcanobacterium Arcobacter Arthrobacter Aureobacterium Bacillus Bacteroides Bergeyella Bifidobacterium Blastoschizomyces Bordetella Branhamella Brevibacillus Brevibacterium Brevundimonas Brochothrix Brucella Budvicia Burkholderia Buttiauxella Campylobacter Candida Capnocytophaga Cardiobacterium Carnobacterium CDC Cedecea Cellulomonas

Chromobacterium Chryseobacterium Chryseomonas Citrobacter Clostridium Comamonas Corynebacterium Cryptococcus Debaryomyces Dermabacter Dermacoccus Dietzia Ed wardsiella Eikenella Empedobacter Enterobacter Enterococcus Erwinia Erysipelothrix Escherichia Eubacterium Ewingella Flavimonas Flavobacterium Fusobacterium Gardnerella Gemella Geotrichum Gordona Haemophilus Hafnia Hansenula Helicobacter King ella Klebsiella Kloeckera Kluyvera

Kocuria Kytococcus Lactobacillus Lactococcus Leclercia Leptotrichia Leuconostoc Listeria Malassezia Methylobacterium Microbacterium Micrococcus Mobiluncus Moellerella Moraxella Morganella Myroides Neisseria Nocardia Ochrobactrum Oerskovia Oligella Paenibacillus Pantoea Pasteurella Pediococcus Peptococcus Peptostreptococcus Photobacterium Pichia Plesiomonas Porphyromonas Prevotela Propionibacterium Proteus Prototheca Providencia

Aii APPENDIX I: BACTERIA AND FUNGI

Pseudomonas Psychrobacter Rahnella Rals tonia Rhodococcus Rhodotorula Rothia Saccharomyces Salmonella Serratia Shewanella

Shigella Sphingobacterium Sphingomonas Sporobolomyces Staphylococcus Stenotrophomonas Stomatococcus Streptococcus Suttonella Tatumella Tetragenococcus

Trichosporon Turicella Veillonella Vibrio Weeksella Weissella Xanthomonas Yarrowia Yersinia Yokenella Zygosaccharomyces

APPENDIX II: LIST OF SUPPLIERS

The suppliers below are mentioned in the text as main sources of specialist equipment, culture media or diagnostic materials. This list is not intended to be comprehensive. 3 M Microbiology Products 3M Center Building 260-6B-01 St Paul MN 55144-1 000 USA ABC Research Corporation 3437 SW 24th Avenue Gainesville FL 32607 USA Adgen Ltd Nellies Gate Auchincruive Ayr KA6 5HW UK Agi-Diagnostics Associates Cinnaminson New Jersey USA ANI Biotech OY Temppelikatu 3-5, 00100 Helsinki Finland Applied Biosystems The Perkin-Elmer Corporation 12855 Flushing Meadow Drive St Louis MO 63131 1824 USA

Becton Dickinson Microbiology Systems 7 Loveton Circle Sparks M D 21 152-0999 USA bio resources 9304 Canterbury Leawood KS 66206 USA BioControl Systems 19805 North Creek Parkway Bothwell WA 98011 USA BioControl Systems, Inc 12822 SE 32nd Street Bellevue WA 98005 USA Bioenterprises Pty Ltd 28 Barcoo Street PO Box 20 Roseville NSW 2069 Australia Biolog, Inc 3938 Trust way Hayward CA 94545 USA

Aiv APPENDIX II: LIST OF SUPPLIERS

Bioman Products, Inc 400 Matheson Blvd Unit 4 Mississauga Ontario LAZ 1N8 Canada

Celsis-Lumac Ltd Cambridge Science Park Milton Road Cambridge CB4 4FX UK

bioMerieux Chemin de I'Orme 69280 Marcy L'Etoile France

Charm Sciences Inc 36 Franklin Street Malden MA02148 3141 USA

bioMerieux (UK) Grafton House Grafton Way Basingsto ke Hants RG22 6HY UK

Chemunex Corporation St John's Innovation Centre Cowley Road Cambridge CB4 4WS UK

bioMerieux Vitek, Inc 595 Anglum Drive Hazelwood MO 63042 2320 USA

Crescent Chemical Co, Inc 1324 Motor Parkway Hauppauge NY 11788 USA

Bioscience International 11607 Mcgruder Lane RockviI le MD 20852 4365 USA Biosynth AG PO Box 125 9422 Staad Switzerland Biotecon Hermannswerder haus 17 14473 Potsdam Germany Biotrace 666 Plainsboro Road Suite 1116 Plainsboro NJ 08536 USA Celsis 2948 Old Britain Circle Chattanooga TN 37421 USA Celsis International plc Cambridge Science Park Milton Road Cambridge CB4 4FX UK

diAgnostix, Inc 1238 Anthony Road Burlington NC 27215 USA DIFCO PO Box 331058 Detroit MI 48232 USA Diffchamb (UK) 1 Unit 12 Block 2/3 Old Mill Trading Estate Mansfield Woodhouse Nottingham NG19 9BG UK Diffchamb SA 8 Rue St Jean de Dieu 69007 Lyons France Digen Ltd 65 High Street Wheatley Oxford OX33 1UL UK DiverseyLever Weston Favell Centre Northampton NN3 8PD UK

APPENDIX II: LIST OF SUPPLIERS Av

Diversy Ltd Technical Lane Greenhill Lane Riddings DE55 4BA UK

Don Whitley Scientific Ltd 14 Otley Road Shipley West Yorkshire BD17 7SE UK

DuPonVQualicon E35711001A Rouote 141 & Henry Clay Road PO Box 80357 Wilmington DE 19880 0357 USA

Dynal PO Box 158 Skoyen 0212 Oslo Norway

Dynal (UK) Ltd Station House 26 Grove Street New Ferry Wirral Merseyside L62 5AZ UK

Dynal (USA) 5 Delaware Drive Lake Success NY 11042 USA

Dynatech Laboratories Inc 14340 Sulleyfield Circle Chantilly VA 22021 USA

Ecolab Ltd David Murray John Building Swindon Wiltshire SNl 1NH UK

Envirotrace (BioProbe) 675 Potomac River Road McLean VA 22100 USA

Foss Electric (UK) Parkway House Station Road Didcot Oxon OX11 7NN UK Fluorochem Ltd Wesley Street Old Glossop Derbyshire SK13 9RY UK

Foss Electric AIS 69 Slangerupgade PO Box 260 DK-3400 Hillerod Denmark GENE-TRAK Systems 94 South Street Hopkinton MA 01748 USA Gist-Brocades Australia PO Box 83 Mooreban k NSW 2170 Australia Gist-Brocades BV PO Box 1345 2600 M A Delft The Netherlands I.U.L. 1670 Dolwick Drive Suite 8 Erlanger KY 41 01 8 IDEXX Laboratories, Inc One IDEXX Drive Westbrook ME 04092 USA

Avi APPENDIX II: LIST OF SUPPLIERS

Industrial Municipal Equipment Inc (ime, Inc) 1430 Progress Way Suite 105 Ridersburg MD 21784 USA

Innovative Diagnostic Systems 2797 Peterson Place Norcross GA 30071 USA

International BioProducts Tecra Diagnostics 14780 NE 95th Street Redmond WA 98052 USA

Lab M Ltd Topley House 52 Wash Lane Bury Lancashire BL9 6AU UK

Launch Diagnostics Ltd Ash House Ash Road New Ash Green Longfield Kent DA3 8JD UK

Malthus Instruments Ltd Topley House 52 Wash Lane Bury Lancashire BL9 6AU UK Merck (UK) Ltd Merck House Poole Dorset BH15 1TD UK Meridian Diagnostics Inc 3741 River Hills Drive Cincinnati OH 45244 USA MicroBioLogics 217 Osseo Ave N St Cloud MN 56303 4455 USA Microbiology International 10242 Little Rock Lane Fredrick MD 21702 USA Microgen Bioproducts 1 Admiralty Way Camberley Surrey GU15 3DT UK MicroSys, Inc 2210 Brockman Ann Arbor MI 48104 USA

Lionheart Diagnostics Bio-lek Instruments, Inc Highland Park Box 998 Winooski VT 05404 0998 USA

Minitek-BBL BD Microbiology Systems 7 Loveton Circle Sparks MD 21152 USA

M. 1. Biol BioPharma Technology Ltd BioPharma House Winnall Valley Road Winchester SO23 OLD UK

Mitsubishi Gas Chemical America, Inc 520 Madison Avenue 25th Floor New York NY 10022 USA

APPENDIX II: LIST OF SUPPLIERS Avii

M-Tech Diagnostics 49 Barley Road Thelwall Warrington Cheshire WA4 2EZ UK

Pharmacia Biotech 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA

National Food Processors Assoc 1401 New York NW Washington DC 20005 USA

Prolab Diagnostics Unit 7 Westwood Court Clayhill Industrial Estate Neston Cheshire L64 3UJ UK

Neogen Corporation 620 Lesher Place Lansing MI 48912 USA

QA Life Sciences Inc 6645 Nancy Ridge Drive San Diego CA 92121 USA

New Horizons Diagnostic Corp 91 10 Red Branch Road Suite B Columbis MD 21045 2014 USA

Radiometer Ltd Manor Court Manor Royal Crawley West Sussex RHlO 2PY UK

Olympus Precision Instruments Division 10551 Barkley Suite 140 Overland Park KS 66212 USA

R-Biopharm GmbH Dolivostr 10 D-64293 Darmstadt Germany

Organon Teknika AKZO NOBEL 100 Akzo Avenue Durham NC 27712 USA

RCR Scientific Inc 206 West Lincoln PO Box 340 Goshen IN 46526 0340 USA

Oxoid, Inc 21 7 Colonnade Road Nepean Ontario K2E 7K3 Canada

Remel 12076 Santa Fe Drive Lenexa KS 66215 USA

Oxyrase Inc PO Box 1345 Mansfield OH 44901 USA

Rhone-Poulenc Diagnostics Ltd 3.06 Kelvin Campus West of Scotland Science Park Maryhill Road Glasgow G20 OSP UK

Perkin Elmer Corporation 50 Tanbury Road Mail Station 251 Wilton CT 06897 0251 USA

SciLog, Inc 14 Ellis Potter Ct Madison WI 5371 1-2478 USA

Aviii APPENDIX II: LIST OF SUPPLIERS

Silliker Laboratory Inc 1304 Halstead Street Chicago Heights IL 6041 1 USA Spiral Biotech 7830 Old Georgetown Road Bethesda MD 20814 USA

Unipath, Oxoid Division Wade Road Basingstoke Hampshire RG24 8PW UK Unipath, Oxoid Division (USA) 800 Proctor Avenue Ogdensburg NY 13669 USA

Tecra Diagnostics 28 Barcoo Street PO Box 20 Roseville NSW Australia

Vicam 29 Mystic Avenue Somerville MA 02145 USA

Tecra Diagnostics (UK) Batley Business Centre Technology Drive Batley W Yorkshire WF17 6ER UK

Wescor, Inc 1220 E 1220 N Logan UT 84321 USA

GUIDE TO USE OF THE ENCYCLOPEDIA

Structure of the Encyclopedia The material in the Encyclopedia is arranged as a series of entries in alphabetical order. Some entries comprise a single article, whilst entries on more diverse subjects consist of several articles that deal with various aspects of the topic. In the latter case the articles are arranged in a logical sequence within an entry. To help you realize the full potential of the material in the Encyclopedia we have provided three features to help you find the topic of your choice.

1. Contents Lists Your first point of reference will probably be the contents list. The complete contents list appearing in each volume will provide you with both the volume number and the page number of the entry. On the opening page of an entry a contents list is provided so that the full details of the articles within the entry are immediately available. Alternatively you may choose to browse through a volume using the alphabetical order of the entries as your guide. To assist you in identifying your location within the Encyclopedia a running headline indicates the current entry and the current article within that entry. You will find ‘dummy entries’ where obvious synonyms exist for entries or where we have grouped together related topics. Dummy entries appear in both the contents list and the body of the text. For example, a dummy entry appears for Butter which directs you to Milk and Milk Products: Microbiology of Cream and Butter, where the material is located.

Example If you were attempting to locate material on Dairy Products via the contents list. DAIRY PRODUCTS see BRUCELLA: Problems with Dairy Products; CHEESE: In the Market Place; Microbiology of Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific Groups of Bacteria; Microflora of Whitebrined Cheeses; FERMENTED MILKS: Yoghurt; Products from Northern Europe; Products of Eastern Europe and Asia; PROBIOTIC BACTERIA: Detection and Estimation in Fermented and Non-fermented Dairy Products

At the appropriate location in the contents list, the page numbers for articles under Brucella, etc. are given.

If you were trying to locate the material by browsing through the text and you looked up Dairy Products then the following information would be provided.

xiv Guide to use of the Encyclopedia

DAIRY PRODUCTS see BRUCELLA: Problems with Dairy Products; CHEESE: In the Market Place; Microbiology of Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific Groups of Bacteria; Microflora of White-brined Cheeses; FERMENTED MILKS: Yoghurt; Products from Northern Europe; Products of Eastern Europe and Asia; PROBIOTIC BACTERIA: Detection and Estimation in Fermented and Nonfermented Dairv Products.

Alternatively, if you were looking up Brucella the following information would be provided.

BRUCELLA Contents Characteristics Problems with Dairy Products

2. Cross References All of the articles in the Encyclopedia have an extensive list of cross references which appear a t the end of each article, e.g.: ATP BlOLUMlNESCENCElApplication in Dairy Industry. See also: Acetobacter. ATP Bioluminescence: Application in Meat Industry; Application in Hygiene Monitoring; Application in Beverage Microbiology. Bacteriophage-based Techniques for Detection of Food-borne Pathogens. Biophysical Techniques for Enhancing Microbiological Analysis: Future Developments. Electrical Techniques: Food Spoilage Flora and Total Viable Count (TVC). Immunomagnetic Particle-based Techniques: Overview. Rapid Methods for Food Hygiene Inspection. Total Viable Counts: Pour Plate Technique: Spread Plate Technique: Specific Techniques; MPN; Metabolic Activity Tests; Microscopy. Ultrasonic Imaging: Non-destructive Methods to Detect Sterility of Aseptic Packages. Ultrasonic Standing Waves.

3. Index The index will provide you with the volume number and page number of where the material is to be located, and the index entries differentiate between material that is a whole article, is part of a n article or is data presented in a table. O n the opening page of the index, detailed notes are provided.

4. Colour Plates The colour figures for each volume have been grouped together in a plate section. The location of this section is cited both in the contents list and before the See also list of the pertinent articles.

5. Contributors A full list of contributors appears at the beginning of each volume.

CONTRIBUTORS

Lahsen Ababouch Department of Food Microbiology and Quality Control lnstitut Agronomique et Veterinaire Hassan II Rabat Morocco

lmad Ali Ahmed Central Food Control Laboratory, Ajman Municipality PO Box 3717 Ajman UAE

D Abramson Agriculture & Agri-Food Canada Cereal Research Centre 195 Dafoe Road Winnipeg Manitoba R3T 2M9 Canada

Peter Ahnert Department of Biochemistry Ohio State University Columbus OH 4321 0 USA

Ann M Adams Seafood Products Research Center US Food and Drug Administration PO Box 3012 22201 23rdDrive SE Bothell WA 98041-301 2 USA Martin R Adams School of Biological Sciences University of Surrey Guildford GU2 5XH UK G E Age PO Box 553 Wageningen The Netherlands M Ahmed Food Control Laboratory PO Box 7463 Dubai United Arab Emirates

William R Aimutis Land 0' Lakes Inc. PO Box 674101 St Paul Minnesota 55164-01 01 USA J H AI-Jedah Central Laboratories Ministry of Public Health Qatar

Cameron Alexander Macromolecular Science Department Institute of Food Research, Reading Laboratory Earley Gate Whiteknights Road Reading RG6 6BZ UK Marcos Alguacil Departmento de Genetica Facultad de Ciencias, Universidad de Malaga Spain

xvi

Contributors

M Z Ali Central Laboratories Ministry of Public Health Qatar M D Alur Food Technology Division Bhabha Atomic Research Centre Mumbai 400085 India R Miguel Amaguatia US Food and Drug Administration Washington, DC USA Vilma Moratade de Ambrosini Centro de Referencia para Lactobacilos and Universidad Nacional de Tucuman Casilla de Correo 21 1 (4000)-Tuc uman Argentina Wallace H Andrews US Food and Drug Administration Washington, DC 20204 USA Dilip K Arora Department of Botany Banaras Hindu University Varanasi 221 005 India B Austin Department of Biological Sciences Heriot-Watt University Riccarton Edinburgh EH14 4AS Scotland, UK Aslan Azizi Iranian Agricultural Engineering Research Institute Agricultural Research Organization Evin Tehran Iran

S De Baets Laboratory of Industrial Microbiology and Biocatalysis Department of Biochemical and Microbial Technology Faculty of Agricultural and Applied Biological Sciences University of Gent Coupure links 653 B-9000 Gent Belgium

Les Baillie Biomedical Sciences DERA CBD Porton Down Salisbury W i Its hire UK Gustavo V Barbosa-Canovas Biological Systems Engineering Washington State University Pullman Washington 99164-6120 USA

J Baranyi Institute for Food Research Reading UK Eduardo Barzana Departamento de Alimentos y Biotecnologia Facuitad de Quimica Universidad Nacional Autonoma de MBxico Mexico City 0451 0 Mexico Carl A Batt Department of Food Science Cornell University lthaca NY 14853 USA Derrick A Bautista Saskatchewan Food Product Innovation Program Department of Applied Microbiology and Food Science University of Saskatchewan Canada S H Beattie Hannah Research Institute Ayr KA6 5HL UK H Beck Department for Health Service South Bavaria Veterinarstrasse 2 85764 Oberschleissheim Germany Reginald Bennett FDA Center for Food Safety and Applied Nutrition Washington, DC USA

Contributors

Marjon H J Bennik Agrotechnological Research Institution (ATO-DLO) Bornsesteeg 59 6709 PD Wagen i ngen The Netherlands Merlin S Bergdoll (dec) Food Research Institute University of Wisconsin-Madison Madison, WI USA R G Berger Food Chemistry University of Hannover Germany K Berghof BioteCon Gesellschaft fur Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany P A Bertram-Drogatz Mediport VC Management GmbH Wiesenweg 10 12247 Berlin Germany

Gail D Betts Campden and Chorleywood Food Research Association Chipping Campden Gloucestershire GL55 6LD UK

R R Beumer Wageningen Agricultural University Laboratory of Food Microbiology Bomenweg 2 NL 6703 HD Wageningen The Netherlands Rijkelt R Beumer Wageningen University and Research Centre Department of Food Technology and Nutritional Sciences Bomenweg 2 NL 6703 HD Wageningen The Netherlands Saumya Bhaduri Microbial Food Safety Research Unit Eastern Regional Research Center Agricultural Research Service US Department of Agriculture 600 East Mermaid Lane Wyndmoor PA 19038 USA

Deepak Bhatnagar Southern Regional Research Center Agricultural Research Service US Department of Agriculture LA USA

J R Bickert Halosource Corporation First Avenue South Seattle WA 98104 USA Hanno Biebl GBF - National Research Centre for Biotechnology Braunschweig Germany Clive de W Blackburn Microbiology Unit Unilever Research Colworth Colworth House Sharnbrook Bedford UK I S Blair Food Studies Research Unit University of Ulster at Jordanstown Shore Road Newtownabbey Go. Antrim Northern Ireland BT37 9QB

G Blank Department of Food Science University of Manitoba Winnipeg MB Canada Hans P Blaschek Department of Food Science and Human Nutrition University of Illinois 488 Animal Science Lab 1207 West Gregory Drive Urbana IL 61801 USA D Blivet AFSSA Ploufragan France

xvii

xviii Contributors

R G Board South Lodge Northleigh Bradford-on-Avon Wiltshire UK

Astrid Brandis-Heep Philipps Universitat Fachbereich Biologie Laboratorium fur Mikrobiologie D-35032 Marburg Germany

Enne de Boer Inspectorate for Health Protection PO Box 9012 7200 GN Zutphen The Netherlands

Susan Brewer Department of Food Science and Human Nutrition University of Illinois Urbana Illinois USA

Christine Bonaparte Department of Dairy Research and Bacteriology Agricultural University Gregor Mendel-Str. 33 A-1 180 Vienna Austria Kathryn J Boor Department of Food Science Cornell University lthaca NY 14853 USA A Botha Department of Microbiology University of Stellenbosch Stellenbosch 7600 South Africa W Richard Bowen Biochemical Engineering Group Centre for Complex Fluids Processing University of Wales Swansea Singleton Park Swansea SA2 8PP UK

Catherine Bowles Leatherhead Food Research Association Leatherhead Surrey UK Patrick Boyaval INRA Laboratoire de Recherches de Technologie Laitiere 65 rue de Saint-Brieuc 35042 Rennes Cedex France F Bozoglu Department of Food Engineering Middle East Technical University Ankara Turkey

Aaron L Brody Rubbright Brody Inc. PO Box 9561 87 Duluth Georgia 30095-9504 USA Bruce E Brown B. E. Brown Associates 328 Stone Quarry Priv. Ottawa Ontario K1K 3Y2 Canada G Bruggeman Laboratory of Industrial Microbiology and Biocatalysis Department of Biochemical and Microbial Technology Faculty of Agricultural and Applied Biological Sciences University of Gent Coupure links 653 8-9000 Gent Belgium

Andreas Bubert Department for Microbiology Theodor-Boveri Institute for Biosciences University of Wurzburg Am Hubland 97074 Wurzburg Germany Ken Buckle Department of Food Science and Technology The University of New South Wales Sydney Australia Lloyd B Bullerman Department of Food Science and Technology University of Nebraska PO Box 830919 Lincoln NE 68583-0919 USA

Contributors

Justin0 Burgos Food Technology Section Department of Animal Production and Food Science University of Zaragoza SDain Frank F Busta Department of Food Science and Nutrition University of Minnesota St Paul Minnesota 55108 USA Daniel Cabral Departmento de Ciencias Biologicas Facultad de Ciencias Exactas y Naturales Pabellon II 4to piso - Ciudad Universitaria 1428 Buenos Aires Argentina Maria Luisa Calderon-Miranda Biological Systems Engineering Washington State University Pullman Washington 99164-61 20 USA Geoffrey Campbell-Platt Gyosei Liaison Office Gyosei College London Road Reading Berks RGl 5AQ UK lain Campbell International Centre for Brewing and Distilling Heriot-Watt University Edinburgh -EHl4 4AS Scotland Frederic Carlin lnstitut National de la Recherche Agronomique Unite de Technologie des Produits Vegetaux Site Agroparc 84914 Avignon Cedex 9 France Brigitte Carpentier National Veterinary and Food Research Centre 22 rue Pierre Curie F-94709 Maisons-Alfort Cedex France

Maria da Gloria S Carvalho Departamento de Microbiologia Medica lnstituto de Microbiologia Universidade Federal do Rio de Janeiro Rio de Janeiro 21941 Brazil

0 Cerf Alfort Veterinary School 7 Avenue du General de Gaulle F-94704 Maisons-Alfort Cedex France Lourdes Perez Chabela Universidad Autonoma Metropolitana-lztapalapa Mexico Apartado Postal 55-535 CP 09340 Mexico DF Mexico Perng-Kuang Chang Southern Regional Research Center Agricultural Research Service US Department of Agriculture LA USA E A Charter Canadian lnovatech Inc. 31212 Peardonville Road Abbotsford BC V2T 6K8 Canada

Parimal Chattopadhyay Department of Food Technology and Biochemical Engineering Jadavpur University Calcutta-700 032 India Yusuf Chisti Department of Chemical Engineering University of Almeria E-04071 Almeria Spain Thomas E Cleveland Southern Regional Research Center Agricultural Research Service US Department of Agriculture LA USA Dean 0 Cliver University of California, Davis, School of Veterinary Medicine Department of Population Health and Reproduction One Shields Avenue Davis California 95616-8743 USA

xix

xx

Contributors

T E Cloete Department of Microbiology and Plant Pathology Faculty of Biological and Agricultural Sciences University of Pretoria Pretoria 0002 South Africa

N D Cowell Elstead Godalming Surrey GU8 6HT UK

Roland Cocker Cocker Consulting Bergeendlaan 16 1343 AR Almere The Netherlands

Julian Cox Department of Food Science and Technology The University of New South Wales Sydney Australia

Timothy M Cogan Dairy Products Research Centre Teagasc Fermoy Ireland

C Gerald Crawford US Department of Agriculture Agricultural Research Service Eastern Regional Research Center 600 E. Mermaid Lane Wyndmoor PA 19038 USA

David Collins-Thompson Nestle Research and Development Center 210 Housatonic Avenue New Milford Connecticut USA Janet E L Corry Division of Food Animal Science Department of Clinical Veterinary Science University of Bristol Langford Bristol BS40 5DT UK Aldo Corsetti Institute of Dairy Microbiology Faculty of Agriculture of Perugia 06126 S. Costanzo Perugia Italy Polly D Courtney Department of Food Science and Technology Ohio State University 2121 Fyffe Road Columbus OH 43210 USA M A Cousin Department of Food Science Purdue University West Lafayette Indiana 47907-1 160 USA

Theresa L Cromeans Department of Environmental Sciences and Engineering School of Public Health University of North Carolina North Carolina USA Kofitsyo S Cudjoe Department of Pharmacology Microbiology and Food Hygiene Norwegian College of Veterinary Medicine PO Box 8146 Dep 0033 Oslo Norway David Cunliffe Macromolecular Science Department Institute of Food Research Reading Laboratory Earley Gate, Whiteknights Road Reading RG6 6BZ UK Ladislav Curda Department of Dairy and Fat Technology Prague Institute of Chemical Technology Czech Republic G J Curie1 Unilever Research Vlaardingen PO Box 114 3130 AC Vlaardingen The Netherlands

Contributors

G D W Curtis Bacteriology Department John Radcliffe Hospital Oxford UK

Michael K Dah1 Department of Microbiology University of Erlangen Staudtstrasse 5 91058 Erlangen Germany

Crispin R Dass The Heart Research Institute Ltd 145 Missenden Road Camperdown Sydney NSW 2050 Australia

E Alison Davies Technical Services & Research Department Aplin & Barrett Ltd (Cultor Food Science) 15 North Street Beaminster Dorset DT8 3DZ UK

Brian P F Day Campden and Chorleywood Food Research Association Chipping Campden Gloucestershire GL55 6LD UK

J M Debevere Laboratory of Food Microbiology and Food Preservation Faculty of Agricultural and Applied Biological Sciences University of Ghent Coupure Links 654 9000 Ghent Belgium

Joss Delves-Broughton Technical Services and Research Department Aplin & Barrett Ltd (Cultor Food Science) 15 North Street Beaminster Dorset DT8 3DZ UK

xxi

Stephen P Denyer Department of Pharmacy The University of Brighton Coc kcroft BuiIding Moulescoomb Brighton BN2 4GJ UK P M Desmarchelier Food Safety and Quality Food Science Australia PO Box 3312 Tingalpo DC Queensland 41 73 Australia

Janice Dewar CSlR Food Science and Technology PO Box 395 Pretoria 001 South Africa Vinod K Dhir Biotec Laboratories Ltd 32 Anson Road Martlesham Heath lpswich Suffolk IP5 3RD UK

M W Dick Department of Botany University of Reading Reading RG6 6AU UK Vivian M Dillon Department of Biology and Biochemistry University of Bath Bath UK Eleftherios H Drosinos Department of Food Science and Technology Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens lera Odos 75 Athens Greece

F M Dugan USDA-ARS Western Regional Plant Introduction Station Washington State University Washington USA

xxii Contributors

B Egan Marine Biological and Chemical Consultants Ltd Bangor UK H M J van Elijk Unilever Research Vlaardingen PO Box 114 3130 AC Vlaardingen The Netherlands Hartmut Eisgruber Institute for Hygiene and Technology of Foods of Animal Origin, Veterinary Faculty Ludwig-Maximilians University 80539 Munich Germany Phyllis Entis QA Life Sciences, Inc. 6645 Nancy Ridge Drive San Diego CA 92121 USA John P Erickson Microbiology - Research and Development Bestfoods Technical Center Somerset New Jersey USA Douglas E Eveleigh Department of Microbiology Rutgers University Cook College 76 Lipman Drive New Brunswick NJ 08901-8525 USA Richard R Facklam Streptococcus Laboratory Respiratory Diseases Branch Division of Bacterial and Mycotic Diseases Centres for Disease Control and Prevention Mail Stop CO-2 Atlanta GA 30333 USA M Fandke BioteCon Gesellschaft fur Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany

Nana Y Farkye Dairy Products Technology Center Dairy Science Department California Polytechnic State University San Luis Obispo CA 93407 USA Manuel Fidalgo Departmento de Genetica Facultad de Ciencias, Universidad de Malaga Spain Christopher W Fisher Department of Food Science and Human Nutrition University of Illinois Urbana IL 61801 USA G H Fleet CRC for Food Industry Innovation Department of Food Science and Technology The University of New South Wales Sydney New South Wales 2052 Australia Harry J Flint Rowett Research Institute Greenburn Road Bucksburn Aberdeen UK Samuel Formal Department of Microbiology and Immunology Uniformed Services University of the Health Sciences F Edward Hebert School of Medicine 4301 Jones Bridge Road Bethesda MD 20814 USA Pina M Fratamico US Department of Agriculture Agricultural Research Service Eastern Regional Research Center 600 E. Mermaid Lane Wyndmoor PA 19038 USA Colin Fricker Thames Water Utilities Manor Farm Road Reading RG2 OJN UK

Contributors xxiii

Daniel Y C Fung Department of Animal Sciences and Industry Kansas State University Manhattan Kansas66506 USA

N P Ghildyal Fermentation Technology and Bioengineering Department Central Food Technological Research Institute Mysore 570013 India

H Ray Gamble United States Department of Agriculture Agricultural Research Service Parasite Biology and Epidemiology Laboratory Building 1040, Room 103, BARC-East Beltsville MD 20705 USA

M Gibert lnstitut Pasteur Unite Interactions Bacteries Cellules 28 rue du Dr Roux 75724 Paris Cedex 15 France

lndrawati Gandjar Department of Biology Faculty of Science and Mathematics University of Indonesia Jakarta Indonesia Mariano Garcia-Garibay Departamento de Biotechnolog/a Universidad Autonoma Metropolitana Iztapalapa, Apartado Postal 55-535 Mexico City 09340 Mexico Maria-Luisa Garcia-Lopez Department of Food Hygiene and Food Technology University of Leon 24071-Leon Spain S K Garg Department of Microbiology Dr Ram Manohar Lohia Avadh University Faizabad 224 001 India A Gasch BioteCon Gesellschaft fur Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany Michel Gautier Ecole Nationale Superieure d’Agronomie lnstitut National de la Recherche Agronomique 65 rue de SrBrieuc 35042 Rennescedex France Gerd Gellissen Rhein Biotech GmbH EichsFelder Str. 11 40595 Dusseldorf Germany

Glenn R Gibson Microbiology Department Institute of Food Research Reading UK

M C te Giffel Wageningen Agricultural University Laboratory of Food Microbiology Bomenweg 2 NL 6703 HD Wageningen The Netherlands A Gilmour Food Science Division (Food Microbiology) Department of Agriculture for Northern Ireland Agriculture and Food Science Centre Newforge Lane Belfast BT9 5PX Northern Ireland, UK Giorgio Giraffa lstituto Sperimentale Lattiero Caseario Via A. Lombard0 11 - 26900 Lodi Italy R W A Girdwood Scottish Parasite Diagnostic Laboratory Stobhill Hospital Glasgow GL21 3UW UK Andrew D Goater Institute of Molecular and Biomolecular Electronics University of Wales Dean St Bangor Gwynedd LL57 1UT UK

xxiv

Contributors

Marco Gobbetti lnstituto di Produzioni e Preparazioni Alimentari Facolta di Agraria di Foggia Via Napoli 25 71 100 Foggia Italy Millicent C Goldschmidt Department of Basic Sciences Dental Branch The University of Texas Health Center at Houston 6516 John Freeman Avenue Houston Texas 77030 USA Lorena Gomez-Ruiz Departamento de Biotechnologia Universidad Autonoma Metropolitana Iztapalapa, Apartado Postal 55-535 Mexico City 09340 Mexico Katsuya Gomi Division of Life Science Graduate School of Agricultural Science Tohoku University Japan M Marcela Gongora-Nieto Biological Systems Engineering Washington State University Pullman Washington 99164-61 20 USA S Gonzalez Universidad Nacional de Tucuman, Argentina Cerela-Conicet San Miguel de Tucuman Argentina

M K Gowthaman Fermentation Technology and Bioengineering Department Mysore 57001 3 India Lone Gram Danish Institute for Fisheries Research Department of Seafood Research Technical University of Denmark Bldg 221 DK-2800 Lyngby Denmark AGE Griff ioen Stichting EFFl PO Box 553 Wageningen The Netherlands Mansel W Griffiths Department of Food Science University of Guelph Guelph Ontario N1G 2W1 Canada C Gronewald BioteCon Geseilschaft fur Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany

Isabel Guerrero Universidad Autonoma Metropolitana-lztapalapa Mexico Apartado Postal 55-535 CP 09340 Mexico DF Mexico

Silvia N Gonzaler Centro de Referencia para Lactobacilos (Cerela) and Universidad Nacional de Tucuman Chacabuco 145 (4000) Tu c uman Argentina

G C Giirakan Middle East Technical University Ankara Turkey

Leon G M Gorris Unit Microbiology and Preservation Unilever Research Vlaardingen PO Box 114 3130 AC Vlaardingen The Netherlands

Carlos Horacio Gusils Centro de Referencia para Lactobacilos and Universidad Nacional de Tucuman Casilia de Correo 21 1 (4000)-Tucuman Argentina

Grahame W Gould 17 Dove Road Bedford MK41 7AA UK

Thomas S Hammack US Food and Drug Administration Washington, DC 20204 USA

Contributors

S A S Hanna, 48 Kensington Street Newton MA 02460 USA

A D Hitchins Center for Food Safety and Applied Nutrition Food and Drug Administration Washington, DC USA

Karen M J Hansen Saskatchewan Food Product Innovation Program University of Saskatchewan Saskatoon SK S7N 5A8 Canada

Jill E Hobbs George Morris Centre 345, 21 16 27th Avenue NE Calgary Alberta T2E 7A6 Canada

J Harvey Food Science Division (Food Microbiology) Department of Agriculture for Northern Ireland Agriculture and Food Science Centre Newforge Lane Belfast BT9 5PX Northern Ireland, UK

Ailsa D Hocking, CSlRO Food Science Australia Riverside Corporate Park North Ryde New South Wales 21 13 Australia

Wilma C Hazeleger Wageningen University and Research Centre Department of Food Technology and Nutritional Sciences Bomenweg 2 NL 6703 HD Wageningen The Netherlands

G M Heard CRC for Food Industry Innovation Department of Food Science and Technology University of New South Wales Sydney New South Wales 2052 Australia Nidal Hila1 Biochemical Engineering Group Centre for Complex Fluids Processing Department of Chemical and Biological Process Engineering University of Wales Swansea Singleton Park Swansea SA2 8PP UK G Hildebrandt Institute for Food Hygiene Free University of Berlin Germany

Colin Hill Department of Microbiology and National Food Biotechnology Centre University College Cork Ireland

Cornelis P Hollenberg lnstitut fur Microbiology Heinrich-Heine-Universitat Dusseldorf 40225 Dusseldorf Germany Richard A Holley Department of Food Science University of Manitoba Winnipeg Manitoba R3T 2N2 Canada Wilhelm H Holzapfel Institute of Hygiene and Toxicology Federal Research Centre for Nutrition Bundesforschungsanstalt Haid-und-Neu-Str. 9 D-7613 Karlsruhe Germany Rolf K Hommel Cell Technologie Leipzig Fontanestr. 21 Leipzig D-04289 Germany Dallas G Hoover Department of Animal and Food Sciences University of Delaware Newark DE 19717-1 303 USA

xxv

xxvi Contributors

Thomas W Huber Medical Microbiology and Immunology Texas A&M College of Medicine Temple Texas USA Robert Hutkins Department of Food Science and Technology University of Nebraska 338 FIC Lincoln NE 68583-0919 USA Cheng-An Hwang Nestle Research and Development Center 210 Housatonic Avenue New Milford Connecticut USA John J landolo Department of Microbiology and Immunology University of Oklahoma Health Sciences Center Oklahoma City OK 73190 USA Y limura Department of Applied Chemistry and Biotechnology Yamanashi University Kofu Japan

Charlotte Nexrnann Jacobsen Department of Dairy and Food Research Royal Veterinary and Agricultural University Rolighedsvej 3,O 1958 Frederiksberg C Denmark Mogens Jakobsen Department of Dairy and Food Research Royal Veterinary and Agricultural University Rolighedsvej 3,O 1958 Frederiksberg C Denmark Dieter Jahn Institute for Organic Chemistry and Biochemistry Albert Ludwigs University Freiburg Albertstr. 21 79104 Freiburg Germany

B Jarvis Ross Biosciences Ltd Daubies Farm Upton Bishop Ross-on-Wye Herefordshire HR9 7UR UK

Ian Jenson Gist-brocades Australia Pty, Ltd Moorebank NSW Australia Juan Jimenez Departmento de Genetica Facultad de Ciencias, Universidad de Malaga Spain Karen C Jinneman Department of Veterinary Science and Microbiology University of Arizona Tucson AZ 85721 USA Juan Jofre Department of Microbiology University of Barcelona Spain Eric Johansen Department of Genetics and Microbiology Chr. Hansen N S 10-1 2 B0ge Alle DK-2970 H~rsholm Denmark Nick Johns Independent Research Consultant 15 Collingwood Close Steepletower Hethersett Norwich NR9 3QE UK Eric A Johnson Department of Food Microbiology Food Research Institute, University of Wisconsin Madison WI USA Clifford H Johnson US Environmental Protection Agency Cincinatti Ohio USA

Contributors

Rafael Jordan0 Department of Food Science and Technology Campus Rabanales, University of Cordoba E-I4071 Cordoba Spain

Embit Kartadarma Department of Food Science and Technology The University of New South Wales Sydney Australia

Richard Joseph Department of Food Microbiology Central Food Technological Research Institute Mysore 570 013 India

K L Kauppi University of Minnesota Department of Food Science and Nutrition St Paul USA

Vinod K Joshi Department of Post-harvest Technology Dr YSP University of Horticulture and Foresty Nauni Solan-173 230 India Vijay K Juneja United States Department of Agriculture Eastern Regional Research Center 600 East Mermaid Lane Wyndmoor Pennsylvania USA G Kalantzopoulos Department of Food Science and Technology Agricultural University of Athens Greece Chitkala Kalidas Field of Microbiology Department of Food Science Cornell University lthaca NY 14853 USA

A Kambamanoli-Dimou Department of Animal Production Technological Education Institute Larissa Greece Peter Kampfer lnstitut fur Angewandte Mikrobiologie Justus-Liebig-Universitat Giessen Senckenbergstr. 3 D-35390 Giessen Germany N G Karanth Fermentation Technology and Bioengineering Department Mysore 570013 India

xxvii

C A Kaysner US Food and Drug Administration 22201 23rd Drive SE Bothell Washington 98021 USA William A Kerr Department of Economics University of Calgary 2500 University Drive NW Calgary Alberta T2N I N 4 Canada Tajalli Keshavarz Department of Biotechnology University of Westminster 115 New Cavendish Street London W I M 8JS UK George G Khachatourians Department of Applied Microbiology and Food Science University of Saskatchewan Saskatoon Canada

W Kim Department of Microbiology University of Georgia Athens Georgia USA

P M Kirk CAB1 Bioscience UK Centre (Egham) Bakeham Lane Egham Surrey TW20 9TY

xxviii

Contributors

Todd R Klaenhammer Departments of Food Science and Microbiology Southeast Dairy Foods Research Center Box 7624 North Carolina State University Raleigh NC 27695-7624 USA Hans-Peter Kleber lnstitut fur Biochemie Fakultot fur Biowissenschaften Pharmazie und Psychologie Universitat Leipzig Talstr. 33 Leipzig D-04103 Germany Thomas J Klem Department of Food Science Cornell University USA Wolfgang Kneifel Department of Dairy Research and Bacteriology Agricultural University Gregor Mendel-Str. 33 A-1 180 Vienna Austria Barb Kohn VICAM LP 313 Pleasant Street Watertown MA 02172 USA

C Koob BioteCon Gesellschaft fur Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany P Kotzekidou Department of Food Science and Technology Faculty of Agriculture Aristotle University of Thessaloniki PB 250 GR 540 06 Thessaloniki Greece K Krist Meat and Livestock Australia Sydney Australia

Pushpa R Kulkarni University Department of Chemical Technology University of Mumbai Matunga Mumbai 400 019 India Madhu Kulshreshtha Division of Plant Pathology Indian Agricultural Research Institute New Delhi 11012 India Susumu Kumagai Department of Biomedical Food Research National Institute of Infectious Diseases Toyama 1-23-1 Shinjuku-ku Tokyo 162-8640 Japan

G Lagarde lnovatech Europe B.V. Landbouwweg The Netherlands Keith A Lampel US Food and Drug Administration Center for Food Safety and Applied Nutrition HFS-327 200 c St sw Washington DC 20204 USA S Leaper Campden and Chorleywood Food Research Association Chipping Campden Gloucestershire GL55 6LD UK J David Legan Microbiology Department Nabisco Research PO Box 1944 DeForest Avenue East Hanover NJ 017871 USA J J Leisner Department of Veterinary Microbiology Royal Veterinary and Agricultural University Stigb~jlen4 DK-1870 Frederiksberg C Denmark

H L M Lelieveld Unilever Research Vlaardingen PO Box 114 3130 AC Vlaardingen The Netherlands

Contributors

D F Lewis Food Systems Division SAC Auchincruive Ayr KA6 5HW Scotland UK

M J Lewis Department of Food Science and Technology University of Reading UK E Litopoulou-Tzanetaki Department of Food Science, Faculty of Agriculture Aristotle University of Thessaloniki 54006 Thessaloniki Greece Aline Lonvaud-Funel Faculty of Enology University Victor Segalen Bordeaux 2 351, Cours de la Liberation 33405 Talence Cedex France

S E Lopez Departamento de Ciencias Biologicas Facultad de Ciencias Exactas y Naturales Pabellon I1 4to piso - Ciudad Universitaria 1428 Buenos Aires Argentina G Love Centre for Electron Optical Studies University of Bath Claverton Down Bath BA2 7AY UK Robert W Lovitt Biochemical Engineering Group Centre for Complex Fluids Processing Department of Chemical and Biological Process Engineering University of Wales Swansea Singleton Park Swansea SA2 8PP UK Majella Maher National Diagnostics Centre National University of Ireland Galway Ireland

R H Madden Food Microbiology Food Science Department Department of Agriculture for Northern Ireland and Queen’s University of Belfast Newforge Lane Belfast BT9 5PX Northern Ireland

T Mahmutoglu TATKO TAS Gayrettepe Istanbul Turkey

K A Malik Chairman Pakistan Agricultural Research Council Islamabad Pakistan

Miguel Prieto Maradona Department of Food Hygiene and Food Technology University of Leon 24071-Leon Spain

Scott E Martin Department of Food Science and Human Nutrition University of Illinois 486 Animal Sciences Laboratory 1207 West Gregory Drive Urbana IL 61 801 USA

L Martinkova Laboratory of Biotransformation Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

Tina Mattila-Sandholm VTT Biotechnology and Food Research Tietotie 2 Espoo PO Box 1501 FIN-02044 v1T Finland

xxix

xxx

Contributors

D A McDowell Food Studies Research Unit University of Ulster at Jordanstown Shore Road Newtownabbey Co. Antrim Northern Ireland BT37 9QB

Denise N McKenna Microbiology - Research and Development Bestfoods Technical Center Somerset New Jersey USA M A S McMahon Food Studies Research Unit University of Ulster at Jordanstown Shore Road Newtownabbey Co. Antrim Northern Ireland BT37 9QB T A McMeekin School of Agricultural Science University of Tasmania Hobart Australia

Luis M Medina Department of Food Science and Technology Campus Rabanales University of Cordoba E-14071 Cordoba Spain Aubrey F Mendonca Iowa State University Department of Food Science and Human Nutrition Ames Iowa USA James W Messer US Environmental Protection Agency Cincinnati Ohio USA M C Misra Fermentation Technology and Bioengineering Department Central Food Technological Research Institute Mysore 570013 India

Vikram V Mistry Dairy Science Department South Dakota State University Brookings South Dakota 57007 USA D R Modi Department of Microbiology Dr Ram Manohar Lohia Avadh University Faizabad 224 001 India Richard J Mole Biotec Laboratories Ltd. 32 Anson Road Martlesham Heath lpswich Suffolk IP5 3RD UK M C Monte1 Station de Recherches sur la Viande INRA 63122 Saint Genes Champanelle France M Moresi lstituto di Tecnologie Agroalimentari Universita della Tuscia Via S C de Lellis 01 100 Viterbo Italy Andre Morin Imperial Tobacco Limited 3810 rue St-Antoine Montreal Quebec H4C 1B5 Canada Maurice 0 Moss School of Biological Sciences, University of Surrey Guildford GU2 5XH UK M A Mostert Unilever Research Vlaardingen PO Box 114 3130 AC Vlaardingen The Netherlands Donald Muir Hannah Research Institute AYr KA6 5HL Scotland, UK

Contributors

Maite Muniesa Department of Microbiology University of Barcelona Spain E A Murano Center for Food Safety and Department of Animal Science Texas A&M University Texas USA

M J Murphy CBD Porton Down Salisbury SP4 OJQ UK K Darwin Murrell Agricultural Research Service US Department of Agriculture Beltsville Maryland 20705 USA C K K Nair Radiation Biology Division Bhabha Atomic Research Centre Mumbai 400 085 India

M de Nijs TNO Nutrition and Food Research Institute Division of Microbiology and Quality Management PO Box 360 3700 AJ Zeist The Netherlands S H W Notermans TNO Nutrition and Food Research Institute PO Box 360 3700 AJ Zeist The Netherlands

Martha Nufiez Centro de Referencia par Lactobacilos (Cerela) Chacabuco 145 (4000) Tucuman Argentina George-John E Nychas Department of Food Science and Technology Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens lera Odos 75 Athens 11855 Greece R E O’Connor-Shaw Food Microbiology Consultant Birkdale Queensland Australia

Motoi Nakao Horiba Ltd Miyanohigashimachi Kisshoin Minami-ku Kyoto Japan 601-8510

A W Nichol Charles Sturt University NSW Australia

Louise O’Connor National Diagnostics Centre National University of Ireland Galway Ireland Triona O’Keeffe Department of Microbiology and National Food Biotechnology Centre University College Cork Ireland

School of Agricultural Science University of Tasmania Hobart Australia

Rachel M Oakley United Biscuits (UK Ltd) High Wycombe Buckinghamshire HP12 4JX UK

Poonam Nigam Biotechnology Research Group School of Applied Biological and Chemical Sciences University of Ulster Coleraine BT52 1SA UK

Yuji Oda Department of Applied Biological Science Fukuyama University Fukuyama Hiroshima 729-0292 Japan

D S Nichols

xxxi

xxxii Contributors

Lucy J Ogbadu Department of Biological Sciences Benue State University Makurdi Nigeria Guillermo Oliver Centro Referencia para Lactobacilos and Universidad Nacional de Tucuman Casilla de Correo 21 1 (4000)-Tucuman Argentina Ynes R Ortega Seafood Products Research Center US Food and Drug Administration PO Box 3012 22201 23rdDrive SE Bothell WA 98041-301 2 USA Andres Otero Department of Food Hygiene and Food Technology University of Leon 24071-Leon Spain Kozo Ouchi Kyowa Hakko Kogyo Co. Ltd 1-6-1 Ohtemachi Chiyoda-ku Tokyo 100-81 85 Japan

Barbaros H Ozer Department of Food Science and Technology Faculty of Agriculture University of Harran 63040 Qanliurfa Turkey Dilek drer GAP Regional Development Administration Sanliurfa Turkey

J Palacios Universidad Nacional de Tucuman, Argentina Cerela-Conicet San Miguel de Tucuman Argentina Ashok Pandey Laboratorio de Processos Biotecnologicos Universidade Federal do Parana Departmento de Engenharia Quimica CEP 81531-970 Curitiba-PR Brazil

Photis Papademas Department of Food Science and Technology University of Reading Whiteknights Reading Berkshire RG6 6AP UK A Pardigol BioteCon Gesellschaft fur Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany E Parente Dipartimento di Biologia, Difesa e Biotecnologie AgroForestali Universita della Basilicata Via N Sauro 85 85100 Potenza Italy

Zahida Parveen University of Huddersfield Department of Chemical and Biological Sciences Queensgate Huddersfield HD1 3DH UK P Patakova Faculty of Food and Biochemical Technology Institute of Chemical Technology Prague Czech Republic Pradip Patel Science and Technology Group Leatherhead Food Research Association Randalls Road Leatherhead Surrey KT22 7RY UK Margaret Patterson Food Science Division Department of Agriculture for Northern Ireland and The Queen's University of Belfast Agriculture and Food Science Centre Newforge Lane Belfast BT9 5PX UK

Contributors xxxiii ~~~

~~

~

P A Pawar Fermentation Technology and Bioengineering Department Central Food Technological Research Institute Mysore 570013 India

J I Pitt CSlRO Food Science Australia Riverside Corporate Park North Ryde New South Wales 21 13 Australia

Janet B Payeur National Veterinary Services Laboratories Veterinary Services Animal and Plant Health Inspection Service Department of Agriculture 1800 Dayton Road Ames IA 50010 USA

M R Popoff lnstitut Pasteur Unite Interactions Bacteries Cellules 28 rue du Dr Roux 75724 Paris Cedex 15 France

Gary A Payne Department of Plant Pathology North Carolina State University Raleigh North Carolina USA Ron Pethig Institute of Molecular and Biomolecular Electronics University of Wales Dean St Bangor Gwynedd LL57 1UT UK

L Petit Unite Interactions Bacteries Cellules lnstitut Pasteur 28 rue du Dr Roux 75724 Paris Cedex 15 France William A Petri Jr Department of Medicine, Division of Infectious Diseases University of Virginia Health Sciences Center MR4, Room 21 15,300 Park Place Charlottesville VA 22908 USA M R A Pillai Isotope Division Bhabha Atomic Rsearch Centre Mumbai 400 085 India D W Pimbley Leatherhead Food Research Association Randalls Road Leatherhead Surrey KT22 7RY UK

U J P,otter Centre for Electron Optical Studies University of Bath Claverton Down Bath BA2 7AY UK

B Pourkomailian Department of Food Safety and Preservation Leatherhead Food RA Randalls Road Surrey UK

K Prabhakar Department of Meat Science and Technology College of Veterinary Science Tirupati 517 502 India W Praphailong National Center for Genetic Engineering and Biotechnology Rajdhevee Bangkok Thailand

M S Prasad FermentationTechnology and Bioengineering Department Mysore 570013 India J. C du Preez Department of Microbiology and Biochemistry University of the Orange Free State PO Box 339 Bloemfontein 9300 South Africa Barry H Pyle Montana State University Bozeman Montana USA

xxxiv

Contributors

Laura Raaska VTT Biotechnology and Food Research PO Box 1501 FIN-02044 VTT Finland Moshe Raccach Food Science Program School of Agribusiness and Resource Management Arizona State University East Mesa Arizona 85206-01 80 USA Fatemeh Rafii, Division of Microbiology National Center for Toxicological Research, US FDA Jefferson AR USA

M I Rajoka, National Institute for Biotechnology and Genetic Engineering (NIBGE) PO Box 577 Faisalabad Pakistan Javier Raso Biological Systems Engineering Washington State University Pullman Washington 99164-61 20 USA

K S Reddy Department of Meat Science and Technology College of Veterinary Science Tirupati 517 502 India

E W Rice US Environmental Protection Agency Cincinnati Ohio 45268 USA

Jouko Ridell Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine University of Helsinki Finland R K Robinson Department of Food Science University of Reading Whiteknights Reading Berkshire RG6 6AP UK Hubert Roginski Gilbert Chandler College The University of Melbourne Sneydes Road Werri bee Victoria 3030 Australia Alexandra Rompf Institute for Organic Chemistry and Biochemistry Albert Ludwigs University Freiburg Albertstr. 21 79104 Freiburg Germany

S M Reddy Department of Botany Kakatiya University Warangal 506 009 India

T Ross School of Agricultural Science University of Tasmania Hobart Australia

Wim Reybroeck Department for Animal Product Quality and Transformation Technology Agricultural Research Centre CLO-Ghent Melle Belgium

T Roukas Department of Food Science and Technology Aristotle University of Thessaloniki Greece

U G Reyes Food Science Australia Private Bag 16 Sneydes Road Werri bee Victoria VIC 3030 Australia

M T Rowe Food Microbiology Food Science Department Department of Agriculture for Northern Ireland and Queen’s University of Belfast Newforge Lane Belfast BT9 5PX Northern Ireland

Contributors

xxxv

W Michael Russell Departments of Food Science and Microbiology Southeast Dairy Foods Research Center Box 7624 North Carolina State University Raleigh NC 27695-7624 USA

Barbara Schalch Institute of Hygiene and Technology of Food of Animal Origin Ludwig-Maximilians-University Munich Veterinary Faculty Veterinarstr. 13 81369 Munich Germany

G Salvat AFSSA Ploufragan France

P Scheu BioteCon Gesellschaft fur Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany

R Sandhir Department of Biochemistry Dr Ram Manohar Lohia Avadh University Faizabad 224 001 India

Robi C Sandlin Department of Microbiology and Immunology Uniformed Services University of the Health Sciences F Edward Hebert School of Medicine 4301 Jones Bridge Road Bethesda MD 20814 USA Jesus-Angel Santos Department of Food Hygiene and Food Technology University of Leon 24071-Leon Spain A K Sarbhoy Division of Plant Pathology Indian Agricultural Research Institute New Delhi 110012 India

David Sartory Severn Trent Water Shrewsbury UK Joanna M Schaenman Department of Medicine Division of Infectious Diseases University of Virginia Health Sciences Center MR4, Room 21 15,300 Park Place Charlottesville VA 22908 USA

Bernard W Senior Department of Medical Microbiology University of Dundee Medical School Ninewells Hospital Dundee DDl 9SY UK Gilbert Shama Department of Chemical Engineering Loughborough University UK Arun Sharma Food Technology Division Bhabha Atomic Research Centre Mumbai 400 085 India M Shin Faculty of Pharmaceutical Sciences Kobe Gakuin University Kobe Japan

J Silva Universidad Nacional de Tucuman, Argentina Cerela-Conicet San Miguel de Tucuman Argentina Dale1 Singh Microbiology Department CCS Haryana Agricultural University Hisar 125 004 India Rekha S Singhal University Department of Chemical Technology University of Mumbai Matunga Mumbai 400 01 9 India

xxxvi Contributors

Emanuele Smacchi Institute of lndustrie Agranie (Microbiologia) Faculty of Agriculture of Perugia 06126 S. Constanzo Perguia Italy Christopher A Smart Macromolecular Science Department Institute of Food Research Reading Laboratory Earley Gate Whiteknights Road Reading R66 6BZ UK H V Smith Scottish Parasite Diagnostic Laboratory Stobhill Hospital Glasgow G21 3UW Scotland. UK

0 Peter Snyder Hospitality Institute of Technology and Management 670 Transfer Road Suite 21A St Paul MN 55114 USA

Mark D Sobsey Department of Environmental Sciences and Engineering School of Public Health University of North Carolina North Carolina USA Carlos R Soccol Laboratorio de Processos Biotecnologicos Departamento de Engenharia Quimica Universidade Federal do Parana CEP 81531-970 Curitiba-PR Brazil M El Soda Department of Dairy Science and Technology Faculty of Agriculture Alexandria University Alexandria Egypt R A Somenrille Neuropathogenesis Unit Institute for Animal Health West Mains Road Edinburgh EH9 3JF UK

N H C Sparks Department of Biochemistry and Nutrition Scottish Agricultural College Auchincruive AYr Scotland M Van Speybroeck Laboratory of Industrial Microbiology and Biocatalysis Department of Biochemical and Microbial Technology Faculty of Agricultural and Applied Biological Sciences University of Gent Coupure links 653

8-9000 Gent Belgium

D J Squirrel1 CBD Porton Down Salisbury SP4 OJQ UK E Stackebrandt DSMZ - German Collection of Microorganisms and Cell Cultures Brunswick Gerrnany Deutsche Sammlung von Mikroorganisem und Mascheroder Weg 1 B 38124, Braunschweig Germany Jacques Stark Gist-brocades Food Specialties R&D Delft The Netherlands Colin S Stewart Rowett Research Institute Greenburn Road Bucksburn Aberdeen UK G G Stewart International Centre for Brewing and Distilling Heriot-Watt University Riccarton Edinburgh Scotland EH14 4AS UK

Contributors xxxvii

Gordon S A B Stewart (dec) Department of PharmaceuticalSciences The University of Nottingham University Park Nottingham NG7 2RD UK

Jyoti Prakash Tamang Microbiology Research Laboratory Department of Botany Sikkim Government College Gangtok Sikkim 737 102 India

Duncan E S Stewart-Tu11 University of Glasgow Glasgow G12 8QQ UK

A Y Tamime Scottish Agricultural College Auchincruive AYr UK

A Stolle Institute of Hygiene and Technology of Food of Animal Origin Ludwig-Maximilians-UniversityMunich Veterinary Faculty Veterinarstr. 13 81369 Munich Germany

J S Tang American Type Culture Collection 10801 University Blvd Manassas VA 201 10-2209 USA

Liz Straszynski Alcontrol Laboratories Bradford UK

Chrysoula C Tassou National Agricultural Research Foundation Institute of Technology of Agricultural Products S. Venizelou 1 Lycovrisi 14123 Athens Greece

M Stratford Microbiology Section Unilever Research Colworth House Sharnbrook Bedfordshire MK44 1LQ UK

S R Tatini University of Minnesota Department of Food Science and Nutrition 1334 Eckles Ave St Paul MN 55108 USA

M Surekha Department of Botany Kakatiya University Warangal 506 009 India

D M Taylor Neuropathogenesis Unit Institute for Animal Health West Mains Road Edinburgh EH9 3JF UK

B C Sutton Apple Tree Cottage Blackheath Wenhaston Suffolk IP19 9HD UK

John R N Taylor Cereal Foods Research Unit Department of Food Science University of Pretoria Pretoria 0002 South Africa

Barry G Swanson Food Science and Human Nutrition Washington State University Pullman Washington 99164-6376 USA

Lucia Martins Teixeira Departamento de Microbiologia Medica lnstituto de Microbiologia Universidade Federal do Rio de Janeiro Rio de Janeiro 21941 Brazil

xxxviii Contributors

Paula C M Teixeira Escola Superior de Biotecnologia Rua Dr Antonio Benardino de Almeida 4200 Port0 Portugal J Theron Department of Microbiology and Plant Pathology Faculty of Biological and Agricultural Sciences University of Pretoria Pretoria 0002 South Africa

Linda V Thomas Aplin & Barrett Ltd 15 North Street Beaminster Dorset DT8 3DZ UK Angus Thompson Technical Centre Scottish Courage Brewing Ltd Sugarhouse Close 160 Canongate Edinburgh EH8 8DD UK

Ulf Thrane c/o Eastern Cereal and Oilseed Research Centre K.W. Neatby Building, FM 1006, Agriculture and Agri-Food Canada Ottowa Ontario K1A OC6 Canada Mary Lou Tortorello National Center for Food Safety and Technology US Food and Drug Administration 6502 South Archer Road Summit-Argo Illinois 60501 USA Hau-Yang Tsen Department of Food Science National Chung Hsing University Taichung Taiwan Republic of China Nezihe Tunail Department of Food Engineering Faculty of Agriculture University of Ankara Diskapi Ankara Turkey

D R Twiddy Consultant Microbiologist 27 Guildford Road Horsham West Sussex RH12 1LU UK N Tzanetakis Department of Food Science Faculty of Agriculture Aristotle University of Thessaloniki 54006 Thessaloniki Greece

C Umezawa Faculty of Pharmaceutical Sciences Kobe Gakuin University Kobe Japan F Untermann Institute for Food Safety and Hygiene University of Zurich Switzerland

Matthias Upmann, Institute of Meat Hygiene Meat Technology and Food Science Veterinary University of Vienna Veterinhrplatz 1 A-1 210 Vienna Austria Turner Uraz Ankara University Faculty of Agriculture Department of Dairy Technology Ankara Turkey M R Uyttendaele Laboratory of Food Microbiology and Food Preservation Faculty of Agricultural and Applied Biological Sciences University of Ghent Coupure Links 654 9000 Ghent Belgium E J Vandamme Laboratory of Industrial Microbiology and Biocatalysis Department of Biochemical and Microbial Technology Faculty of Agricultural and Applied Biological Sciences University of Gent Coupure links 653 8-9000 Gent Belgium

Contributors xxxix

P T Vanhooren Laboratory of Industrial Microbiology and Biocatalysis Department of Biochemical and Microbial Technology Faculty of Agricultural and Applied Biological Sciences University of Gent Coupure links 653 8-9000 Gent Belgium L Le Vay School of Ocean Sciences University of Wales Bangor UK P H In’t Veld National Institute of Public Health and the Environment Microbiological Laboratory for Health Protection PO Box 1 3720 BA Bilthoven The Netherlands Kasthuri Venkateswaran Jet Propulsion Laboratory National Aeronautics and Space Administration Planetary Protection and Exobiology, M/S 89-2, 4800 Oakgrove Dr. Pasadena CA 91 109 USA V Venugopal Food Technology Division Bhabha Atomic Research Centre Mumbai 400 085 India Christine Vernozy-Rozand Food Research Unit National Veterinary School Lyon France Ecole Nationale Vetenaire de Lyon France

Philip A Voysey Microbiology Department Campden and Chorleywood Food Research Association Chipping Campden Gloucestershire GL55 6LD UK Martin Wagner Institute for Milk Hygiene Milk Technology and Food Science University for Veterinary Medicine Veterinarplatz 1 1210 Vienna Austria Graeme M Walker Reader of Biotechnology Division of Biological Sciences School of Science and Engineering University of Abertay Dundee Dundee DDI I H G Scotland P Wareing Natural Resources Institute Chatham Maritime Kent ME4 4TB UK John Watkins CREH Analytical Leeds UK Ian A Watson University of Glasgow Glasgow G I 2 8QQ UK

B C Viljoen Department of Microbiology and Biochemistry University of the Orange Free State Bloemfontein South Africa

Bart Weimer Center for Microbe Detection and Physiology Utah State University Nutrition and Food Sciences Logan UT 84322-8700 USA

Birte Fonnesbech Vogel Danish Institute for Fisheries Research Department of Seafood Research Technical University of Denmark Bldg 221 DK-2800 Lyngby Denmark

Irene V Wesley Enteric Diseases and Food Safety Research USDA, ARS, National Animal Disease Center Ames IA 50010 USA

XI

Contributors

W B Whitman Department of Microbiology University of Georgia Athens Georgia USA Martin Wiedmann Department of Food Science Cornell University lthaca NY 14853 USA R C Wigley Boghall House Linlithgow West Lothian EH49 7LR Scotland

R Andrew Wilbey Department of Food Science University of Reading Whiteknights Reading UK F Wilborn BioteCon Gesellschaft fiir Biotechnologische Entwicklung und Consulting Hermannswerder Haus 17 14473 Potsdam Germany

A G Williams Hannah Research Institute AYr KA6 5HL UK Alan Williams Campden and Chorleywood Food Research Association Chipping Campden Gloucestershire GL55 6LD UK

J F Williams Department of Microbiology Michigan State University East Lansing MI 48824 USA Michael G Williams 3M Center 260-68-0 1 St Paul MN55144-1000 USA

Caroline L Willis Public Health Laboratory Service Southampton, UK F Y K Wong Food Science Australia Cannon Hill Queensland Australia

Brian J B Wood Reader in Applied Microbiology Dept. of Bioscience and Biotechnology University of Strathclyde Royal College Building George Street Glasgow G1 1XW Scotland

S D Worley Department of Chemistry Auburn University Auburn AL 36849

us

Atte von Wright Department of Biochemistry and Biotechnology University of Kuopio PO Box 1627 FIN-70211 Kuopio Finland Chris J Wright Biochemical Engineering Group Centre for Complex Fluids Processing Department of Chemical and Biological Process Engineering University of Wales Swansea Singleton Park Swansea SA2 8PP UK Peter Wyn-Jones Sunderland University UK Hideshi Yanase Department of Biotechnology Faculty of Engineering Tottori University 4-1 01 Koyama-cho-minami Tottori Tottori 680-0945 Japan

Contributors xli

Yeehn Yeeh Institute of Basic Science lnje University Obang-dong Kimhae 621-749 South Korea Seyhun Yurdugul Middle East Technical University Department of Biochemistry Ankara Turkey Klaus-Jurgen Zaadhof Institute for Hygiene and Technology of Foods of Animal Origin Veterinary Faculty Ludwig-Maximilians University 80539 Munich Germany

Gerald Zirnstein Centers for Disease Control GA USA

Cynthia Zook Department of Food Science and University of Minnesota St Paul MN 55108 USA

INDEX

NOTE Page numbers in bold refer to major discussions. Page numbers suffixed by T refer to Tables; page numbers suffixed by F refer to Figures. vs denotes comparisons. This index is in letter-by-letter order, whereby hyphens and spaces within index headings are ignored in the alphabetization. Terms in parentheses are excluded from the initial alphabetization. Cross-reference terms in italics are general cross-references, or refer to subentry terms within the same main entry (the main entry is not repeated to save space). Readers are also advised to refer to the end of each article for additional cross-references cross-references have been included in the index cross-references.

A

14-3-3 protein 287 AAL toxin 1518 ABC-STD 105 1979 ABC-transporters, Bacillus subtilis producing 138 AB milk products 780-781 abortion brucellosis 320-321 listeriosis 1198 abrasives, cell disruption by 698 Absidia 861 characteristic appearance on cultural media 857 accreditation of HACCP 1831 accreditation of laboratories 1128 advantagesidisadvantages 1129-1 130 assessment preparations 1132-1 133 auditing 1132, 1133 documentation 1132, 1133 future prospects 1133-1134 implementation of schemes 1131-1133 maintenance of schemes 1133-1134 organizations 1128-1129, 1130 quality manager 113 1 Quality Manual 1131 standards 1128-1130 trace-ability 1128 AccuProbe system 1238-1244 advantages/limitations 1241-1242 comparison with other methods 12437 Listeria monocytogeizes detection 12381239 protocol 1239-1241, 1239T, 1240T

AccuProbe system (coiztinued) microorganisms detection 1 2 4 2 1 principle 1238-1239 results 1242-1243, 1243T, 1243T RNA extraction from cultures 1242F sampling points and application 1241F acetaldehyde 184 accumulation in yoghurt manufacture 787 production lactic acid bacteria 184, 2105 Streptococcus thermophdus 213 1 acetate carbon dioxide reduction to 1338 formation cheese 384-385 lactic acid bacteria 2104-2105 metabolism in gut 1354 acetic acid 184, 705, 706T, 1292, 2258, 2263 applications 562 chemical properties 1731F in cider 426 effectiveness for microbial control 562 effect on cytoplasmic pH 1733 effect on food 1730 formation 1-2 butanol-acetone fermentation 447448 quinoproteins 5-6 as preservative 1715, 1730 spectrum of activity 1711 production 2258 Brettanomyces 303

- not all of these

acetic acid (cotztinued) Brevibacterium 312 lactic acid bacteria 184 see also acetic acid bacteria recovery in industrial fermentations 691 in sausages 1267 in sourdough bread 301 structure and chemical properties 1732T in vinegar 2259 acetic acid bacteria 1, 2260 classification 1 cocoa fermentation 468 ethanol oxidation 6F isolation 4 tolerance to acid 1735 vinegar production 2259 wine-making 2306 see also Acetobacter acetification 2260 composition (GK) value 2260 exothermic reaction 2261 microorganisms uses 2262 processes 2260-2262 quick vinegar process 2261-2262, 2261F reactions 2260 submerged culture method 2262 surface culture technique 2261 see also vinegar Acetobactev A1 characteristics of genus 1-3 classification 181-182 culture media for 4T dendrogram of 16s rDNA 181, 181F

I ii INDEX Acetobacter (continued) detection 3-5 differentiation between species 3 differentiation from other genera 958, 958T enrichment cultures 3 enzymes 2 ethanol oxidation 2260.2261F on grapes 960 growth and requirements for 1 habitats 3 identification features 4 phenotypic 4-5, 5T importance to food industry 5-7 food processes 5-6 food spoiling see below inhibition by ethanol 2 isolation 3 metabolism 1, 4 nomenclature 1 phenotypic features 4-5, 5T, 958T pH range tolerance 1 plasmids 2 species 1 importance 5 spoilage of foods 6-7 fruit spoilage 3 sugar metabolism 2 vinegar production 2260 see also acetic acid bacteria Acetobacteraceae 955 16s rDNA signature nucleotides 182T phenotypic identification 4 Acetobacter aceti 2 Acetobacter carinus 2 Acetobacter diazotrophicus 2 Acetobacter eiwopaeus, acetification process 2262 Acetobacter hansenii 6 Acetobacter melanogenum 2 Acetobacter methanoliczis 2 Acetobacter pasteurianus 2, 7 Acetobacter peroxidans 2 Acetobacter rancens 2 Acetobacter xylinum 2, 6, 7 acetoin 1271 a-acetolactate synthase 920 acetone aqueous, aflatoxin extraction 1528, 1527T precipitation of metabolites in fermentations 693 acetone-butanol-ethanol (ABE) fermentation 431 Clostridium acetobutylicum 429 see also butanol-acetone fermentation acetyl-coA (acetyl coenzyme A) 718, 719 b-oxidation of fatty acids 1306 pyruvate oxidation to 1276 acetyl-coA carboxylase 1308 acetyl-coA dehydrogenase 1306, 1307F acetyl-coA synthetase 1306, 1307F 3-acet);l-DON, oral toxicity 1546T 15-acetyl-DON, oral toxicity 1546T Achromobacter 1875 Achromobacter piechaudii 38 acidis) 1729 antimicrobial actions 1729 dissociation 1730-1731 in fermented meat products 749-750 organic see organic acids pK 562 production, Cellulomonas 369 taste in foods 1731, 1733F weak 2263, 1731, 1733, 1733F diffusion through membrane 1735 from wood smoke 1739, 1740T see also specific acids acid cleaners 1810 acid-fast bacteria 1500, 1504 cell wall structure 163, 176

acid-fast stains 1384, 1385T, 1504 acidic foods microbial growth control 561-562 spoilage microorganisms and 1009 acidification 1730-1731, 1731 acidulants power 1732F effect on Candida 359 of media 1731-1732 milk 382 see also acidulants acidified tryptone glucose yeast extract agar (ATGYE) 2364T acidocin B 188. 1153 Acidomonas, classification 181-182 acidophiles 174 acidophilus milk 779, 2086 acidophilus yogburt 781 acid phosphatase stain method, Saccharomyces sake 1915 acid-resistant bacteria 1735 acid shock proteins 561 acid tolerance, fermentation 1571 acid tolerance response (ATR), by adaptation 1736, 1736F acid-tolerant bacteria 1735 acidulants 1730, 1757 acidification power 1732F amount added 1730 bread-making 291T buffering ranges 1732F chemical properties 1730-1731, 1732T effect on cytoplasmic pH 1732-1734 effect on foods 1731 food 705,706T function 1731-1732 interactions and factors affecting 17361737 minimum inhibitory concentrations 1733F taste in foods 1731, 1733F temperature effect 1737 acid washes 1810 Acinetobacter 7-8, 1875 antibiotic resistance 15 biotechnological applications 15-16 biotransformation 16 characteristics of genus 8 classification 8 culture media, acidification 11 detection in foods 14F methods 9-13 differentiating characteristics 1876T ecology 9 epidemiological typing 13 extracellular polymer production 16 genomic species 8-9, 9T distribution 9 growth in food 13 habitats 9 identification biochemical tests 11 chemical analyses of cellular components 12 commercial systems 11-12 at genomic species level 11 at genus level and metabolic characters 11 molecular methods 12-13 PCR 12-13 physiologic tests 11-12, 12T ribotyping 1 3 importance of genus in food industry 1315 dairy products 15 eggs 15 meats and poultry 13-14 seafood 14-15 importance of genus in other environments 15 clinical 15 water and soil 15

Acinetobacter (continued) isolation 10-1 1 enrichment procedure 10 media 11 National/International Regulations procedures 1 3 nomenclature 8-9 optimum growth temperature 11 pathogenicity 9 selective growth 10-1 1 species 8-9, 8-9 spoilage of foods 9 fish 1489 meat 1258,2052 taxonomy 1487,1876 transformation assay 11 virulence of strains 15 whole cell hybridization 10F Acinetobacter baumanii 9 in intensive care units 15 Acinetobacter calcoaceticus-A. baumanii complex 1 3 Acinetobacter johnsonii 7 distribution in food 9 Acinetobacter lwoffii 7, 8-9 distribution in food 9 acoustic wave transducers 274 Acremonium 863-864, 865, 868-870, 894 acridine orange 1388, 1387T Botrytis detection 281 Lactobacillus bulgaricns staining 1137F, 1137F acridine orange direct count (AODC) 527 actagardine 192 Actrnomucor 861 Actinomvcetales 211 Actinomicetes 211 cellular differentiation 167, 167F metabolism 1298 actinomycetomas 2137 actinomycin 1334 actinorhodin 1334 active dry yeast (ADY)291-292, 291F, 2340 active packaging see antimicrobial packaging; modified-atmosphere packaging (hfAP) active transport 1273, 1273, 1280, 1290, 1291F acyl carrier protein 1308F adaptation, microbial to acids 1735-1736. 1736F to environmental stress, biofilm bacteria 255-256 by fungi, to redox potential and pH 559561.559F to pH changes 559-561 to redox potential and pH changes 559561,559F to stress, hurdle technology 1075 yeasts, to acids 1736, 1736F added value, microorganisms to industry 485 additives see food additives additive tree methods 179 ADE2 gene 911 adenine auxotrophic mutants, sake yeasts 1917 adenosine diphosphate (ADP) 17 bioluminescent assays, adenylate kinase assay us 24T preparations 18 adenosine monophosphate (AMP), adenylate kinase role 17. 17 adenosine iriphosphate (L4TP)’80,88, 94, 220,2170 in adenylate kinase assay 17, 18, 19 analysis, rationale for 95 assay 2170 applications 2170 limitations 2170 schemes 88-89 technique 2170

INDEX I iii

adenosine triphosphate (ATP) (continued) assay (continued) see also ATP bioluminescence background levels 81, 82 bioluminescence see ATP bioluminescence biomass measurement 80 brewing yeast vitality analysis 103 content of microorganisms 81 energy conversion pathways 1272-1279 see also metabolic pathways formation 97 anaerobic 556 electrochemical p H gradient 556 Embden-Meyerhof-Parnas pathway 1274-1275,1274F fermentation 556-557, 1284 Zymomonas 2367 see also specific metabolic pathways free 89 kits for monitoring 220 malolactic fermentation 2312 microbial (bacterial) 89 microorganism number 80, 88 in milk and dairy products 89 quantification 80 reasons for measuring 88 recycling reaction 97 somatic 89 sources 95 structure 80F, 88F synthesis, protons and energy-transducing systems 1286-1287,1288F adenylate kinase (AK) 16-24, 97, 107 advantages 18 amplification reaction 17 ATP bioluminescence sensitivity increase by 97 beverage industry 107 commercial systems available 107 concerns over dead cell detection 107 mechanism 107F bioluminescent assay 17-18 applications 21 assav cauabilitv 19-20 ATP-baied assay us 17-18, 18F, 19F, 20F, 24T detergent effects 18-19, 1 8 7 extraction step 18-19 factors affecting correlation with colony forming units 19 hygiene monitoring 21, 2 1 incubation time effect 19, 19F materials and methods 18-19 matrix effects 20 sensitivities 21, 21T, 21T typical procedure 21 as cell marker 17 chemicals affecting 20 distribution/sources 1 7 in immunoassays 21-22, 22F microbial 17-18 molecular mass 1 7 molecule number in bacteria 1 7 nonmicrobial, detection 20, 20T reaction catalysed 1 7 specific assays using 21-22, 22F bacteriophage-mediated 22-23,22F, 23F magnetic beads-mediated 21-22, 22F, 22F for Staphylococcus aureus 23,23F, 23T adenyl cyclase, edema factor (EF) as 130 adenylic acid, biosynthetic pathway 1297F adherence Brettanomyces to insects 307 Escherichia coli O157:Hi 647 lactobacilli 1370 phage to bacteria 1471 probiotics 1370 Salmonella enterrtidrs 1938 Staphylococcus aureus 2066

adherence (continued) see also bacterial adhesion adhesins 164 adhesion bacteria see bacterial adhesion biofilm bacteria 253-254, 255 strength 256 adipic acid, as preservative 1730 adjunct cultures cheese-making 382-383 Lactobacillus bulgaricus 1140 meat curing and 1261, 1261T adjuvants 625 adsorbents commercial 691T properties 691 adsorption, metabolite recovery in industrial fermentations 691-692 column operation 691-692, 692F concentrations of solutes 692F adsorption chromatography 691 adsorption curve 532 adsorption effect 539 adsorptionielution methods, viruses 22842285 adsorption isotherm 692 adulterants, detection by enzyme immunoassays 632 adverse food reactions 1004 Advisory Committee on Dangerous Pathogens (ACDP)2221 aeration solid-state fermentation 671 submerged fermentation 666 aerobes facultative 173 on meatimeat products 1255, 1255T microaerophilic 173 obligate 173 strict 556. 1284 transport systems 1273T types 173, 1 7 4 1 aerobic metabolism, ATP formation 12721279 see also metabolic pathways aerobic plate count (APC)method 2160, 2161 aerobiosis 557F Aerococcus, characteristics 1 1 6 5 1 aerolysin, Aeromonas expression 28 Aeromonas 25-30,30-37 biochemical tests 26, 26T capsular layer 28 carrier rates 29 chemo-organotrophic facultative anaerobes 25 classification 25, 26F clinical laboratories 26 detection 30-37 rapid methods 36 detection by culturing 31-35 common media 31-32 differential media 3 5 enrichment techniques 32 from fish 35 from food 34-35 media 3 3 1 methods 32-33 most probable number method 33-34 nonselective approach 32 selection isolation techniques 34-35 from water 35 detection using modern methods 35-37 molecular methods 3 7 serology 35-37 distribution 30 endotoxin 28 exotoxins 28 extracellular protein expression 28 fermentation and 34 fimbriae and adhesins 28 general characteristics 25-27, 25T ~~~~~

~

Aeromonas (continued) growthisurvival at low temperature 27, 27-28,27T atmosphere 29 background microflora effect 29 factors affecting 28-29 salt and pH affecting 28-29 growth temperatures 25, 27-28 haemolytic and proteolytic activities 28 hybridization groups 3 1 identification 3 5 importance to food industry 27-30, 3 7 isolation from food, stages 31, 31F meat spoilage 1266 0-antigen LPS from smooth strains 28 pathogenicity 3 1 pathogenic serotypes 29 phenons 2 7 phenospecies 31 Plesiomonas differences 2 5 1 prevalence in foods 27-28, 27T psychrotrophic nature 27, 27-28, 2 7 1 in shellfish 2003-2004 significance in foods 37 S-layer 28 speciation 35 methods 26-27 species 25-26, 31 biochemical characteristics 26, 26T new 26 pathogenicity 29-30 virulence factors 28 Aeromonas caviae 26 Pathogenicity 29, 30T prevalence in foods 2 7 , 2 7 1 Aeromonas hydrophila 26, 30 capsular layer 28 prevalence in foods 27, 27T psychrotrophic nature 27, 27-28, 27T spices and essential oils effect 1721 Aeromonas (Ryans)agar 31 Aeromonas salmonicida detection 3 5 rapid diagnostic test 36 Aeromonas sobria 26 prevalence in foods 27T Aeromonas veronti biotype sobvia 28 aerosols, microorganisms in 1816 aesculin, hydrolysis 618, 620 Cellulomonas 368 affinity sensor 274 aflatoxin(s) 1325T, 1512, 1514 adverse effects 1540 in butter and cream 1454 carcinogenicity 1514, 1519, 1533, 1540 concerns 1512 detection 75 using biosensors 270 disease associated 1325 effect on animal health 1519 in fermented foods 1326 meat products 747-748 funei oroducine 1514. 1540 .&pergillus ?0-71,72, 1512 Aspergillus flavus 71, 72, 77-78, 74F Aspernillus parasiticus 72, 74F dekccon 70-71 fungal characteristics 77T genes involved 71 in koji moulds 71 mechanisms 78 reduction methods 78 levels in seeds 77 maximum level allowed 77 occurrence 1520 in foods 1540 food types and geographic distribution 1523T production inhibition by Lactobacillus 2100 regulations affecting 77 solvents for extraction 1527T

I iv INDEX

aflatoxinis) (continued) solvents for separation 1529T structures 74F, 1515F synthesis and functions 77-78 tblerance limits 1533T toxicity 1514, 1519, 1540 types 1454,1514, 1515F, 1533, 1540 see also mycotoxins aflatoxin B,63, 1540 actions 77 Aspergillus flavus producing 7 2 , 7 7 Aspergillus producing 6 3 as carcinogen 1514 hepatocellular carcinoma 77 mass spectrum 1531F radioactive labelling 1536, 1536F, l536F species producing 77T structure 74F, 1541F toxicity 77T, 1540 aflatoxin Bz Aspergillus flavus producing 72 species producing and toxic effects 77T structure 74F aflatoxin G, Aspergillus parasrticus producing 72-73 mass spectrum 1531F species producing and toxic effects 77T structure 74F aflatoxin Gz Aspergillus parasiticus producing 72-73 species producing and toxic effects 77T structure 74F aflatrem 78, 76F XFNOR, PCR commercial test validation 1638-1639 Africa fermented fish products 757 fermented foods 736-737 agar standard methods, formulation 2 1 5 5 1 see also specific types agar-based kits food-poisoning organisms 238-239 miniaturized techniques 222-223 Agaricus bisporus 909, 1401F agglutination tests 627 Brucella 323 latex see latex agglutination rapid detection of microbes 1892 air compressed, laboratory supply 1123 contamination risks from 1793, 1804 microorganism concentration 1816, 1817 microorganism transport 1816-1 817 milk contamination from 1437 particle removal 1820-1821, 1820F particles 1680 sizesiclasses 1820F quality 1680, 1820 UV treatment 2212 air-blast coolers 407 airborne contamination 1816-1822 controlireduction 1818-1819 heat inactivation 1819-1821 need for 181 6 particle removal 1820-1821, 1820F measurement methods 1817-1818 Andersen perforated disc sampler 1817-18 18, 1818F centrifugation 1818, 1818F filtration 1818, 1818F impinger 1818, 1818F slit sampler 1817, 1818F see also air filtration milk 1437 recontamination prevention 1821 risks 1793, 1804, 1817 risks from 1793, 1804 sources 1816-1817 validationiassurance and maintenance 1821-1822 air conditioning 1121, 1804

air conditioning (continued) airborne contamination 1816 air filters 1680-1681 \.alidationiassurance and maintenance 1821-1822 air filtration 1680-1681, 1820-1821, 1820F airlift fermenters 2339-2340, 2339F air sampler Andersen perforated disc 1817-1818, 1818F centrifugal 1818, 1818F slit sampler 1817, 1818F alanine 1292 b-alanine method, Saccharomyces sake detection 1915 ‘alarm water content’ 534, 535T albicidins, detoxification by Pantoea agglomerans 1628 Albuginaceae 880 albumen p H effect on Salmonella growth 565, 566F see also eggs Alcaligenes 38-42 antibiotic resistance 4 1 biosensor for heavy metals 40 biotechnological applications 38, 38-40 biodegradable plastics 38-39 bioremediation 39-40 Bordetella relationshipidifferentiation 4 1, 41T catabolism of PCBs 39 classification 38, 39 curdlan synthesis 40 detection methods 4 1 enzymatic production of amino acids 40 food microbiology 40 heavy metal removal 39-40 heavy metal toxicity resistance 39-40 location/distriburion 38 psychrotrophic nature 40 relevance to food industry 38, 40 species 38 xenobiotic-metabolizing isolates 39 Alcaligenes defragrans 38 Alcaligenes eutrophus glucose-utilising mutant 39 polyhydroxbutyrate production 38-39 Alcaligenes latus, polyhydroxbutyrate production 38-39 alcohol dehydrogenase 787 alcohol (ethanol) Acetobacter inhibition 2 as biocide 1799, 1801 cellulose as source 2371 formationiproduction Cellulomonas 3 70-3 71 from lactose 2371 from starch in bread fermentation 293, 294F in wine 2307 see also alcohol fermentation measurement by biosensors 686 metabolite from kefir fermentation 802 oxidation by acetic acid bacteria 6F Acetobacter 2260, 2261F precipitation of metabolites in fermentations 693 Saccharomyces cereuisiae tolerance 1919 Saccharomyces sake tolerance 1914,1917 sake yeasts tolerance 1917 utilization, Breuibacterium 31 1, 31 1 T yeast single-cell protein production 2030 Zymomonas tolerance to high levels 2368 alcohol fermentation 1285 beet molasses 2371 sorghum beer 763vinegar production 2259-2260 Zymomonas 2367 see also alcoholic beverages; Saccharomyces cereuisiae alcoholic beverages

alcoholic beverages (continued) Candida role 357 fermentation 2098 nisin application 197 Saccharomyces carlsbergensis role 1926 Saccharomyces cerevisiae role 19211922,1925, 1926 Saccharomyces role 1925 Schizosaccharomyces significance 1987 smoke-processed 1738 from sorghum see sorghum beer spoilage Gluconobacter 960 Saccharomyces cerevisiae 1922 Zymomonas fermentation 2369-2370 see also alcohol fermentation; beer; fermented beverages; wine; specific beverages alcohol oxidase AOXl uromoter 1688 production in Pichia expression system 1687-1688 alcohols assimilation and transformation by Rhodotorula 1904-1905 flavour in fermented meat products 751T secondary, in cheeses 1660T in wine 2308 alcohol vapour, antimicrobial packaging 419 aldehydes flavour in fermented meat products 7 5 l T from wood smoke 1739 ale, production 1 9 2 7 Alexandrium 1672 algae biomass 2024,2026 blue-green, toxigenic 1674 see also cyanobacteria classification 2021 cultivation 2021-2024 closed systems 2024 contamination avoidance 2023 harvesting 2024 mass-culture open systems 2023-2024 photobioreactors 2024 productivities 2023 substrate requirements 2022-2023 water types and quality 2026 doubling time 2035T as food 2021 types 2022T see also single-cell protein (SCP) heavy metals 2026 macroalgae, cultivation 2021-2022 nucleic acid consumption from 2026 phycotoxin production 1672 single-cell protein see single-cell protein (SCP) toxins see phycotoxins unicellular cultivation 2022 as food 2021 see also dinoflagellates yellow-brown, toxigenic 1673 algal blooms 1672 Alicyclobacillus acidoterrestris 149, 150 detection 153, 1 5 4 alimentary toxic aleukia (ATA) 1542 aliphatic esters 733-734 aliphatics, flal-ours 734 alkaline cleaners 1810, 1811 alkaline peptone water (XPW)31 Vzbrro cholerae enrichment 2244 alkaline phosphatase calf, in enzyme immunoassays 629 milk pasteurization indicator 1035 alkalinophiles 174 alkanes flavour in fermented meat products 751T oxidation and assimilation 721, 723F 4-alkanolides 734 5-alkanolides 734

INDEX I v

alkenes, flavour in fermented meat products 751T allergic skin test (AST) 326, 326 allergy sulphur dioxide 1753 treatment, Lactobacillus acidothilus role 1364 allicin, antimicrobial compound 1577 allozymes 229 all purpose Tween 80 (APT) medium 1145, 1145T ALTA 187 altenuene, Alternaria producing 49T Alternaria 42-50, 862, 868-869, 894 appearance on cultural media 857 characteristics 42-46 conidia 45T detection in foods 48-49 culture methods 48-49 direct methods 48 endophytic 48 phyiloplane detection 48-49 platingidiluting methods 49 seeds 48 ecological aspects 46-47 enzymes 46 on fresh foods 46 grouping by macroscopic appearance 45T growthidispersal, environmental factors affecting 46 growth requirementsiconditions 46T metabolites 47-48 mycotoxins 46,47-48, 47-48,49T, 1512 optimal temperature and water activity 46T as pathogen 47 physiological aspects 46 phytotoxins 47-48 host-specific 47-48,48T nonspecific 47 as saprophyte 42, 46 species 42-46, 45T identification 44 important 44, 4 5 1 spores 43 on harvested crops 47 sporulation 47, 46T patterns 45 taxonomy 4 2 , 4 2 , 4 3 conidia number 43 toxins 42, 857, 1518 in foodstuffs 1518 see also phytotoxins (above) type species 43-44 see also Alternaria alternata Alternaria alternata 42 characteristics 43-44 colonization 46 ecological aspects 46-47 infection appearance 43F metabolites 47-48 mycotoxins 47-48 as saprophyte 46 structure and conidia 44F type species 42 Alternaria brassicae 44, 45T Alternaria brassicicola 44, 45T Alternaria cheiranthi 45T Alternaria citri 44, 4 5 1 Alternaria cucumerina 4 5 1 Alternaria dauci 45T Alternaria gaisen 45T Alternaria helianthi 45T Alternaria helianthinfciens 45T Alternaria infectoria 45T Alternaria longipes 48, 4 5 1 Alternaria longissima 45T Akernaria peponicola 45T A!ternaria petroselini 45T Alternaria porri 45T Alternaria radicina 4 5 1 Alternaria raphani 45T

Alternaria solani 47, 4 5 1 Alternaria sonchi 45T Alternaria tenuis 43 Alternaria tenuissima 44, 4 5 1 Alternaria triticina 45T Alternaria zinniae 4 5 1 alternariol, Alternaria producing 49T alternariol monomethyl ether (AWE), Alternaria producing 49T Alteromonas, in fish 807-808 Alteromonas putrefaciens 2008 cells as biosensor 270 altertoxin I (ATX-I),Alternaria producing 49T altertoxin I1 (ATX-II),Alternarra producing 49T altertoxin 111 (ATX-1111, Alternaria producing 49T aluminium cans 1619 Amadori compounds 1742 ambrox 734,734F America fermented foods 737 see also USA American National Standards Institute 1841 American Organization of Analytical Chemists (AOAC), HGMF methods 1079-1081 amines biogenic see biogenic amines in colon 1354 in fermented meat products 748-749 N-nitrosopyrrolidine formation 1767 production 748-749 enterococci 1369 Pediococcus 1646 amino acidis) aromatic 1296 synthesis 1295 aromatic compounds from, in fermented meat products 750 aspartate, synthesis 1296, 1296F deamination Brevibacterium 31 1 Clostridium 1292 Proteus 1857-1 85 8 see also deamination; specific amino acids decarboxylation Brevibacterium 311 in cheese maturation 391 degradationicata holism bacteria in fish 815 Brevibacterium 308 fermented meat products 750 energy release (aerobic) 1277-1278 essential, mycoprotein products 2042T fermentation 1291 in fermented meat products 750 in fermented milks 783 free .~ cheeses 1660T fish 807 Droteolvsis of cheese 385-386 grdups 1235 metabolic pathways 1291 metabolism, Lactococcus lactis role 1168-1169 mycoprotein products 20421 as nitrogen source 1289 Pediococcus requirement 1643 single-cell Drotein content algal 20'24,2025T yeast 2030T svnthesisiproduction 1295 . Alcaligenes, enzymatic 40 Brevibacterium 311 pathways 1295-1296 transamination, Brevibacterium 3 11 transport 1290 uptake and accumulation 1290 utilization, Brevibacterium 31 1

amino acid auxotrophs, Bacillus subtilis 136 amino acid complexes, hazardous 1743 D-amino acid oxidase, Rhodotorula 1901 g-aminobutyric acid, production, by Lactobacillus brevis 1150 aminoglycosides 2138 synthesis 1330 6-aminopenicillanic acid, structure 1320F aminopeptidase, Brevibacterium linens 313 aminopterin 626 ammonia formation in gut 1354 neoplastic growth in colon 1354 productionisynthesis 677 Brevibacteriurn linens 396 cheese maturation 391 ammonium ions, conversion to organic nitrogen 1296, 1296F ammonium salts, utilization by fungi 12971298 ammonium sulphate, salting-out of proteins 693 amnesic shellfish poisoning 2000-2001, 1673 amoebiasis 2292 pathogenesis 2293 treatment 2294 see also Entamoeba hrstolytica amoebic colitis 2293 amoebic liver abscesses 2293-2294 amoebic ulcers 2293 amoebopores 2293 amperometric biosensors 277 amperometric transducers 272-273 organic conductors 273 ampicillin-dextrin agar (ADA) 31 amplified fragment length polymorphism (AFLP), Clostridium beijerinckii 43 1 AmpliSensor system, polymerase chain reaction 1604F amylase Debaryomyces occidentalis 519 fungal, synthesis by recombinant DNA technology 915 production, Aspergillus 907 a-amylase Aspergillus oryzae 68 egg pasteurization indicator 1036 Flavobacterium 825 genes 68 production, by moulds 21 11 recovery from fermented broth 693, 693F test, egg yolk 571 vinegar production and 2259 Anabaena spp. 1674 anaerobes 173, 1284 aerotolerant 173 facultative 200, 557 on meatimeat products 1255T phosphotransferase system 1290 growth, yields 560 growth rates 559 heat-treated fish product spoilage 820 normal gut flora 198 obligate (strict) 173, 556 methanogens 1334 sulphate-reducers 520 transoort svstems 12737 types'173, i 7 4 1 anaerobic bioreactor 1336-1337 anaerobic conditions, E . coli regulatory systems 560 anaerobic digestion 1336 anaerobic metabolism carbohydrate catabolism 1281-1282 catabolic pathways 1281-1286 carbohydrate breakdown 1281-1282 Embden-Meyerhof 1282 Entner-Doudoroff pathway 12821283 fermentation 1283-1286 monophosphate shunt 1282

I vi

INDEX

anaerobic metabolism (continued) carbohydrate catabolism (continued) see also fermentation; specific

pathways energy release 1279-1288 sites 1286-1287 substrates utilized 1280 substrate uptake mechanisms 1280-1281 anaerobic respiration 1284 Shewanella putrefaciens 2010, 2010F anaerobiosis 557F, 1189 analytes 625, 627 anamorphic state 62, 861, 887 anatase 2213 anatoxin a 1674 anatoxin ajs) 1674 Andersen perforated disc sampler 18171818,1818F aneuploidy, in fungi 928-929 ang-khak 2115 animal bioassays botulinum toxin see botulinum toxin (BoNT) emetic toxin from Bacillus cereus 120121 rabbit ileal loop assay 120 animal feeds antibiotic bans 1836 bovine spongiform encephalopathy association 284 ochratoxin A in 1541 single-cell proteins 2035 nucleic acid content 2032 animals anthrax 129 carcasses, anthrax 130, 134, 134 doublinev time 2035T methanogenesis in gastrointestinal tract 133 7-1 3 3 8 monitorin# for contamination 837 mycotoxins effect on health 1518-1519 Trichinella prevalence 2182-2183 see also specific animalslinfections anion removal 562 Anisakiasis. symptoms of human disease 1054 Anisakis simplex, life cycle 1054 Anoxyphotobacteria 177 anthocyanins 732 anthraquinone 73 1 anthrax 118, 129, 129-130 animals 129 cutaneous 130, 134, 130F gastrointestinal 130, 142 intestinal and oropharyngeal types 130 pulmonary 130

see also Bacillus anthracis anthrax toxin 130 antibacterial effects, fermented milks 798 antibacterial substances detection in food, by electrical techniques 581-582 see also antibiotic(s); antimicrobial compounds antibiotic(s) 1320 in animal feeds 1836 bacteria producing 1320, 1330, 1332, 1331F beta-lactam 1320F Bifidobacterium selection 216-217 biosynthesis gene control 1334 induction 1332 inhibition of enzymes 1332 recombinant DNA technology 13331334 Brevibacterium linens sensitivity 309 definition 1330, 1711 depletion of intestinal lactobacilli 1362 diarrhoea associated 1363 drug residue hazards 1003 effect on colonic microflora 1353

antibioticis) (contiizued) enterococci sensitivity 1371 Enterococcus cultivationiderection 619620 fungal synthesis 1320-1324, 1320T kefir microflora 802 Lactobacillus brevis sensitivity 1145T Lactobacillus bulgaricus sensitivity 1141, 1142T markers for Bacillus subtilis 137, 137T in media Botrytzs detection 280, 280

see also specific media types in milk, effect on Lactobacillus bulgaricus 1141 mode of action 2138 novel 1334, 1902 Pedzococctcs sensitivity 1643. 1643T polypeptide, synthesis' 1330 as preservatives 1711, 1712T uses 1715-1716 production, in fermented meat products 1326 resistance see antibiotic resistance below Rhodotorula sensitivitv 1902 virev of action 1330 ~~~. Staphylococcus sensitivity 2073T Streptomyces 21 37-21 3 8, 21 37F, 21 38T mode of action 2138 susceptibility testing, using flow cytometry 832 types 1320, 1320, 1321T Yersinia susceptibility 2347 Zymomonas mobilis sensitivityiresistance 2366T antibiotic properties, Monascus pigments 1485 antibiotic resistance Acinetobacter 15 Alcaligenes xylosoxidans subsp. xylosoxzdans 41 Bacteroides 198 Enterobacter 603 enterococci 624 gene transfer, Acinetobacter 15 Gram-negative bacteria 163 Klebsiella 1107-1108, 1112 Lactobacillus brevis 1144 Rhodotorula 1902 Salmonella 1948 Salmonella typhi 1945-1946 Staphylococcus 2073T Zymomonas mobilis 2366T see also multidrug resistance antibodies 625, 625-626 binding to antigens 627 characterization 627 conjugate fluorescent 1388, 1387T immobilization blocking and 1090 for immunomagnetic separation see immunomagnetic separation (IMS) labelled 1892 monoclonal see monoclonal antibodies to moulds 281 polyclonal 626 comparison with monoclonal Abs 627T production 626-627 radioimmunoassay (RIA) 628 synthetic, as biosensors 269 use in direct epifluorescent filter technique 529 antibody-antigen reaction analytical use 627-628 biospecific interactions analysis 275 types 627 antibody-microcolony epifluorescence microscopy (MEM) 2179 antifungal agents, Botrytrs control 282 antigenis) 625, 625 antibody binding 627 ~

~~

~

~

~~~~

antigenis) (continued) antibody reaction see antibody-antigen reaction as biosensors 269 coupling with enzymes 629 food spoilage fungi detection 231 Geotrichum candidum 945 polyclonal antibody production 626 radioimmunoassay (RIA) 628 antigenic variation, Giardia trophozoites 954 antimicrobial actions, acids 1729 antimicrobial compounds 1577 bacteriocins 1573-1574 see also bacteriocins; nisin biofilm resistance 256-257, 257F, 257T ecology of natural systems 1570-1572 electroporation treatment medium 1461 enzymes 1575 future developments 1575-1576 GRAS status 417 lactic acid bacteria 1574 lactoferrin 1589-1590 lysozyme 1582-1587 milk Droteins 1587-1591 in mddified atmosphere packaging 412 natural s)stems 1570, 1572-1575 nisin 1573-1574 see also nisin plant-derived 190, 1718T essential oils 1718-1720 sources 1576-1582,1574 see also essential oils; spices production, recombinant DNA technology 939 spices as 1717 storage and natural systems 1570 transferrins 1575 antimicrobial effects, low pH 562 antimicrobial herbal extracts 420 antimicrobial packaging 416-420 applications 419-420 edible films/coatings 418-419, 4 1 8 1 materials 417-41 8 new developments 417-418 Microban 417, 1692 polymers/films 41 7 principles 417 regulations and control 420 sachet technology 419 antimicrobial soaps 1799 contamination 1799, 1799F antimutagenic activity, of fermented milks 797 antioxidants acids 1730 importance of fermented foods and 738 sulphur dioxide 1752 aoules 782 API 20E system 128, 2 2 5 2 239 advantages and disadvantages 239 biochemical reactions used in 248T comparisons with Enterotube and BBL Crystal systems 248-249, 2487 method and evaluation 239 principle 245 protocol 245-246 MI-50 CHL 251 API system 223 fungi 23 1 microflora in fermented foods 251-252 API YEAST-IDENT 231 API-ZYIM system, rapid 252 appertization, preservation methods 15711572 apple juice centrifugation, microbes removed 1684, 1684T for cider 421 preparation 421 composition 426 contaminants 422T

INDEX I vii

apple juice (continued) Escherichia coli 0 1 5 7 : H 7 650, 651 microbiology 422-423 nitrogenous compounds 426 sulphur dioxide role 422-423 sulphur dioxide treatment 426 apple juice-gelatin medium 2369T apple juice-yeast extract medium 2369T appressoria 853 Approved Quality Arrangement (AQA) 1842 aquatic animals see fish; seafood; shellfish aqueous acetone, aflatoxin extraction 1528, l527T ARBOR database 178 Arc AIB system 560 Archaea (archaebacreriai 178 cell membranes, water activity effect 546 lipids 1335-1336 methanogens 1335 Arcobacter 336, 341, 50-54 aerotolerance 50 biochemical tests 52 Campylobacter differences 50, 50 Campylobacter jejuni similarities 5 3 characteristics of genus 50-51 classification 50 detection methods 50 DNA-based fingerprinting 5 2 polymerase chain reaction 52, 52, 51F distributionisources 50, 51T foods 52,52F gastroenteritis 341 human infections 52-53 importance in foods, chicken and poultry 53 importance in livestockifoods 52-53 cattle and beef 52-53 swine and pork 5 3 inactivation methods 5 3 isolation methods 51 serotyping 52 species 5 1 identification 5 2 taxonomy 50 rhermotolerance 5 3 Arcobacter bzttzleri 50 identification 52 inactivation methods 5 3 isolation 345-346 in poultry 53 rRNA 5 2 Arcobacter cryaerphilus 50 Arcobacter nitrofigilis 50 Arcobacter skrrrowii 50, 52 arginine deamination, Vagococcz~sidentification 2219 metabolism 1293, 1294F arginine deaminase pathway 1291 aromatics 732-733 aromatic substrates, energy release (aerobic) 1278-1279 Arrhenius plot 552, 552F arterial pumping, meat curing 1263 arthritis, reactive 336 Arthrobacter 54-61, 308 bacteriophage 58 biotechnological potentialities 60-61 in clinical specimens 6 1 culture media 56-57, 56T ecology 56 electrotransformation 5 8 general characteristics 54-56, 55T genes and genetics 58 glycogen storage 5 7 heterotrophic nitrification 5 7 isolation 56-57 from oil spills 6 1 by selective media 56T metabolic properties 56T metabolism and enzymes 57-58

Arthrobacter (continued) Micrococcus separation 56 nutritional versatility 56 pcd plasmid 58 polysaccharides 5 7 proteolytic actions 5 7 psychrotrophic strains, in milk 60 remediation of ground water 5 9 role in foods 58-61 biodegradation of pesticides 5 9 meat, eggs and fish 59-60 milk and cheese 60 vegetables 5 9 species 54, 54T groups 54 new 61 sensu stricto 541, 55T strain Q36, genes 58 Arthrobacter agilis Characteristics 1345, 1346T differentiation 1347T Arthrobacter citreus 54 Arthrobacter cumininsit sp. noti 61 Arthrobacter globiformis 54 metabolic properties 56T Arthrobacter nicotianae 54 as biosensor 270 inhibition of Listeria 60 metabolic properties 56T Arthrobacter sulfztreus 54 Arthrobacter ureafacieiis, proteinase 5 7 Arthrobacter woluwensis sp. nov 6 1 arrhrofactin 61 arplsulphatase, mycobacteria 1507 asci 890F Ascodesinis sphaerospora 1403F Ascomycetes 861, 855 classification 899 basis 889-891 commercial importance 891-893, 8927, 892T defining teatures 887-889 Deuteromycete relationship 887, 888F eukaryotic 887-893 general features 889 reproduction 887-889, 888F sexual 888F see also yeast(sj Ascomycota 862-868 Ascomycotina 887-893 ascorbates, as curing agents 1263 L-ascorbic acid as antioxidant 960 nitrous acid reaction 1767 production 960 Gluconobacter 960 ascospores 855, 887, 888F,890F Aspergillus 62 characteristics of fungi producing 889T species 890T ascus 862 aseptic packaging 2195 filling procedures 1029-1030 sterility detection by ultrasound see sterility testing aseptic processing, UHT 1024 Asia fermented fish products 756-757 fermented milk oroducts 798-805.799T oriental foods, moulds application'21142116,2114T origin of fermented foods 736 aspartate amino acids, synthesis 1296, 1296F aspartate aminotransferase, Rhodotorula 1902 aspartate protease, in cheese maturation 391 aspartic acid 1292, 1292 deamination 1293 synthesis 1295 Xspergillaceae, antibiotics produced 1320 aspergillic acid 78, 76F

aspergillic acid (continued) Aspergilloides 856, 1647 importance in food 1651 aspergillosis 65, 66 Aspergillus 62-66, 863, 864, 869 aflatoxin production 70-71, 1512, 1540 amylase production 907 antigens, ELISA 281 appearance on cultural media 856 in cereals during storage 2046 characteristics 889T, 894-895 classification 894-895 conidial structures 891F, 891F detection methods 62 diseases associated 63, 65 enzymes and organic acids from 65, 65T in foods and feeds 63, 64T genetic engineering 907 identification fatty acids for 229 media 62-63 of species 63 identification keys 6 3 isolation methods 62-63 morphological characteristics 62, 62F mycotoxin production 63, 65, 64T, 1520 see also aflatoxin production (above) parasexual hybridization 909-910 species 62, 64T teleomorphic genera 62, 63, 63, 64T see also individual Aspergillus species Aspergillus awamori ? ? ? ? ? , mammalian protein synthesis 915 Aspergillus differential medium (ADM) 75 Aspergillus fischeri, cultivation medium 724T AsberPihs flavus 69. 72-79 'afl&xin'production 71,72,77-78,1325, 1514, I 5 4 0 detection 75 types 74F as allergen 78-79 a s animal pathogen 78-79 biology 73-74 changes to resemble A . oryzae 69-70 chromosomes 6 7 colonization of seeds after inlur, 74 conidia 76 conidiophores 76, 73F detection in foodsifeeds 75-77 DNA methods 76-77 ELISA 75 growth media 75 differentiation from A. oryzae 70 differentiation from other species 75-77 diversity 74-75 ecological benefits 79 economic significance 77-79 in fermented meat products 747-748 freezing effect 843 growth, appearance 72F growth media 75 habitat 73-74 insects relationship 79 interactions with hosts 74 morphological characteristics 75-76, 73F, 77T mycotoxin production 1325 see also aflatoxin production (above) phialides 76 as plant pathogen 73-74 post-harvest contamination 74 pre-harvest contamination 74 as saprophyte 73-74 sclerotia 74, 76 strains (5. and L.j and isolates 75 summary 79 toxins produced 77, 76F vegetative compatibility groups (VCGs) 74-75

Aspergillus flavus and parasrticus agar (AFPA) 75

I viii

INDEX

Aspergtllirs f l m u s (continued) Aspergillus f l a w s group 66 Aspergillus fumigatits 79 Aspergillus nidulans A,$fAl gene 914 chromosome 6 7 cultivation medium 724T homologous recombination 913 nuclei 922 pyrC gene 913 sterigmatocystin production 71 transformation by recombinant D S A 914 Aspergillus niger 710F chromosome 67 citric acid synthesis 706-707, 707-714, 708F enzy-mes, applications 915 glucoamylase 68 gluconic acid synthesis 6, 715 importance in food industr) 2057 Asheivillus nomius 72 'afl&Jxins73, 1514 morphological characteristics 77T Asmrnilirrs o c h r ~ c e u sochratoxin , 15 14 Aspergillus oryzae 66-72, 1327 aflatoxin not produced 71, 1326, 1327 applicationsiuses 66 Characteristics 66-67 chromosome 67 conidia 67, 69 cyclopiazonic acid 1514 detection methods 69-71 differenriation from A. flavzts 70 distribution 66 enzymes produced 68 applications 915 genes 68, 69T genetics 67 GRAS status 66 growth 67 hl-phae (mycelia) 67 identification methods 69-71 molecular biologJ- 70 importance in food industry 67-69 fermented foods 66, 67-69 morphological characteristics 67, 69, 67F mycotoxins 68-69 non-aflatoxigenicit!; molecular characterization 71 seed cultures 68 so>-sauce production 65 taxonomy 66, 69-70, 70T Aspergillits parmiticus 69, 72 aflatoxins 72-73, 74F,1325, 1514 biology and habitat 73 groarh media 75 identitication 70 interactions with hosts 74 morphological characteristics 77T mycotoxin production 1325 as plant pathogen 73-74 as saprophyte 73-74 Aspergillus pentcilliotdes in cereals during storage 2046 cereal spoilage 2046 Aspergillus restrictus, cereal spoilage 2046, 2046 Aspergillus section Flaui 66 Aspergdhs s o p 65, 69, 1327 identification 70 inability to produce aflatoxins 1326, 1327 aspertoxin 78 asphyxiation 1002-1003 assays, nucleic acid-based 1599-1609 Association of Official Analytical Chemists (-\O.\C) 1102 Escherichia colt 0 157 immunoassays 2227,22281,2228T evaluations of Bacillus detection methods I55 mycotoxin detection 1527, 1528 PCR commercial t e i t validation 1639

Association of Official Analytical Chemists (AOAC) (continued) Salinone/la detection method 1967 Assurance Pol\ clonal Enzi me Immunoassa\ for E. coh 0 1 5 7 2223-2225,2231, 2228T astaxanthin 730, 730F astro\ iruses 2265, 2274 morphology 2275 ATB system 240 atomic force microscope 1419, 1419F atomic force microscopy (AFhI) 1418-1425 colloid probe technique 1421 contact mode 1419-1420 in food microbiology 1421-1424 bacterial, yeast and animal cells 14221423 forces of interaction measurement 1424-1425, 1424F, 1424F macromolecular components of cells 1423-1424 surfaces 1421-1422. 1422F viruses 1423 force-distance curves 1420-1421, 1421F future prospects 1425 image analysis 1420 imaging in liquids (double layer mode) 1420 intermittent contact mode 1420 non-contact mode 1420 principles 1419-1421. 1419F tip geometry 1420 uses 1418-1419 ATP see adenosine triphosphate (ATPj ATP bioluminescence l6-17,209, 220, 1475,2170 advantages 80, 86-87, 97, 101 disadvantages 108 summary 108 applications 16-17, 2170 in food processing plants 98-101 ATP from non-microbial sources 220 in beverages 104 differentiation (from microbial ATP) 98,220 minimization 81, 82 bacteriophage I! sin-release 208-209, 210,209F, 209F in beverage microbiology 101-109 accelerated forcing tests 107-108 adenylate kinase methods 107 background ATP measurement I05 benefits 101, 108 brewing yeast vitalit)- analysis 102-104 contamination analysis 105 conramination prevention 104-108 detection limits 105-106 disadvantages 108 fail and pass I05 filtered samples 104, 105 need for rapid testing 101 quality assurance I 0 1 reagents and instruments 101-102 reagent storage 102 sample preparation 104-105 test kits 102, 102T use of Microstar system 107 comparison with XK-based assay 18F, 19F, 20F concentration-dependent transition phenomenon 98 correlation with plate counts 82, 83F, 83F, 84F,84F, 83T, 2170 advantages of ATP bioluminescence 101 beef and poultry 87T hygiene monitoring 97-98 hygiene monitoring of milk transporters 99F cutoff limits 87, 87F in dairy industry 88-94 assay for psychrotroph proteases 91

.ATP bioluminescence (continued) in dairy industry (continued) detection of inhibitory substances 93 interpretation of results 90, 91 monitoring of starter culture activity 92-93 other applications 93-94 r a a milk quality assessment 89-91, 907 screening of hygiene of farms and milk tankers 89 shelf-life prediction of dairy products 91, 92T somatic cell count and mastitis control 93 sterility testing of UHT dairy products 91-92, 92T tests 90-91, 90T, 90T, 90T definition 80 disadvantages 86-87, 97-98 environmental effects 98 sampling problems in hJ-giene monitoring 97 effects of chemical sanitizers on 98, 98T in hygiene monitoring 94-101, 220 adequacy of monitoring kits 96-97 advantages 9 7 cleaning procedures in institutions 95, 95F cutoff value criteria 96 data expressioniinterpretation 96 dry cleaning methods effect 100 evaluations of commercial kits 96 in food processing plants 98-101 limitations 97-98 manufacturers of kits 95T, 220 methods 95-98 reagents and instruments 96 sensitivity 96-97, 9 7 1 theoretical limits of detection (TDL) 96,97T inadequate for current regulations 8 7 limitations 17, 97-98, 2170 luminometers see luminomerer manufacturers of kits 931, 95T, 102T in meat industry 80-88 assays 81-85 BactoFoss (automation) 83 €scherichia colt O l 5 7 : H 7 detection

84-85 finished meat products 82-83 hygiene monitoring 99-100 meat homogenization problems 82 poultrl- hygiene monitoring 99 raw meat materials 8 1-82 'rinse-bag' method 82 role in meat processing 85-86, 85 sterile sponge method 82 total viable counts on meat 82 principle 80-81, 88-89, 9 5 procedure 82F aseptic technique 86 rapid detection of microbes in food 1893 rapidishort turnover time 80, 86. 95, 101 reaction 80-81, 81F. 88-89: 94 reagent checking 102, 103T reagent storage 102 real-time testing 85-86, 9 7 sensitivity 82, 96-97, 97T. 209 methods to increase 97, 107 technique 2170 total viable microbial count relationship 86-87 use in hygiene monitoring 1 7 ATP:citrate lyase 1300, 719 Rhodotorula 1901 ATP synthetase system 1287 audit laboratory 1129, 1132 maintenance of accreditation schemes 1133 Aureobusidtum 109-112, 869, 895

INDEX I ix

Aureobasidium (continued) characteristics of genus 109-110, 1 1 0 1 colony appearance 109 conidiogenesis 109 detection methods 110 immunological 110 plating 1IO, 112F enzymes 111 T in foods 109-110 fruits and vegetables 109 survival in reduced water activity 110 unacceptable levels 110 Aureobasidium pullulans characteristics 109-110, 11OT conidiation 109 control 112 enzymes 1IO food additives produced 111 T fruit spoilage l i 2 importance to consumer 112 importance to food industry 111-112 merabolismhutrition 110 opportunistic mycosis 112 pullulan production 110 Aureobasidium pullulans var. melanogenum 109 Australia food hygiene regulations 1841-1842 regulatory systems for process hygiene 1833T autoclaves 1126-1127 gravity displacement 1126, 1127F pressure cooker 1126, 1126F autolysin 448, 933-934 autolysis bacteria 1474 fish 813, 814 yeast 2032 autolytic genes 1474 autonomously replicating sequences (ARSj 912 autoradiography, microautoradiography 2180 hutotrack system 107 auxotrophic markers 91 1 auxotrophic microorganisms 173, 1280 avenacins antimicrobial compounds 1577 spoilage reduction 1577-1578 avidin 629 antimicrobial chelating agent 1574-1575 food application 1586 mode of action 1584 occurrence 1582-1583 properties I584 structure 1583 avocado, antimicrobial compounds 15761577 a,> see water activity Azobacter vrvelandii 1289 azo compounds 202 azoreductases 202

B

BAhX Pathogen Detection svstem 16361637 Bach process, microwaves 1039 bacilli 159-160, 159F Bacillus 113-119 aerobic endospore-forming 113 carbon catabolite repression 117, 117F cell wall composition 114-115 central regulitory component (CcpA) 117. 117F characteristics of genus 113-114 colonial morphology 149 detection by cultural techniques 149-158 aciduric flat sour spore-formers 152, 155,ljlT advantages and limitations 155

Bacillus (continued) detection by cultural techniques jcont.) collaborative evaluations/validations 155 diluenrsisolutions used 156 flat sour spore-formers 155 formulations of media 151 heatine of samules 151-152 incubation of samples 152 media 149-150,156, l j O T media for confirmation 157-158 media for enumeration 156-157 mesouhilic aerobic soore-formers 152. li5, l5lT procedures 151-154 procedures in food samples 151T roue suores 150, 152-153,155,15lT sample size 151 sample type 150 see also specific Bacillus species detection of environmental changes 117 enzymes 113, 116T reactions 116-117 food-borne illness 141-143 characteristics 144T see also Bacillus cereus food spoilage I50 acid foods 1009 flat sour 128, 150 see also Bacillus stearothertnophilus; canned foods gene regulation 117 genetic diversit). 113, 114 gene transfer 115-116 genome 114, 115T identification of species of public health interest 154T mesophilic aerobic spore-formers 149 detection 152, 155, 1 5 l T in milk 1444 non-pathogenic strains 113 pathogenicity 117-1 18 phylogenetic tree 114, 114F products 113, 116-117, 116T species characteristics 116T importance in food industry 149T spores, in food samples 150 sporulating, isolation 115 sporulation 113, 115 thermophilic flat sour spore-formers 149 toxins 146 detection 141-149, 148 see also Bacillus cereus Bacillus aerogenes 598 see also Enterobacter aeropenes Bacillus anthracis 129-135 capsule 130, 133, 132F characteristics of species 129-131 classification 129 control 129 detection 131-134, 134, 1 3 3 1 antigen-based methods 132 preliminary tests 132 presumptive tests 132-133 gamma phage sensitivity 132 gene, capsule s)-nthesis 130 haemolysis absence 132 herbivore infections 129 human infections meningitis 129-130 see also anthrax importance to consumers 134-135 importance to food industry 134 isolation, WHO protocol 132, 133FT motility absence 132 as obligate pathogen 129 penicillin sensitivity 132 regulations relating to 134 5-layer proteins (Ea1 and Sap) 131 spores 129 germination requirements 129 u

Bacillus anthracis (continued) toxin 130 edema factor (EF) 130-131 genes 131 lethal factor (LFj 130, 131 protective antigen (PA) 130, 131, 132 toxins 118 virulence, detection 133 virulence factors 130 coordinate regulation 131F Bacillus brevis food-borne illness 142, 143. 144T gramicidin synthetase synthesis 1332 Bacillus cereus 119-124 Bacillus thtiringiensis toxin gene transfer 118 cereolysin 120, 131 certified reference materials (CRMsi 18981. 1 8 9 8 1 characteristics of species 119-121, 121122, 1340T colony morphology 165F detection methods 121-123, 153 confirmatory tests 121-122 cultural techniques 155, 155 direct plating 121, 153 enrichment 153 in foods 121 most probable number 121, 155 presumptive tests 153 specific tests 122-123 differentiation from other species 121 enzymes 120 food-borne illness 119, 119-120, 123. 123, 136, 141-142, 143, 837 characteristics 144T diarrhoeal syndrome 120, 141, 143 emetic syndrome 119-120, 141, 146 foods associated 119, 123 incidence 142 risk factors 124, 142 symptoms and signs 123, 141, 146 food-borne pathogen 150 genes 119 haemolysin 120, 122, 146 structure 146 haemolysis 154 importance to consumers 124-125 importance to food industry 124 infant formulae contamination 123 infectious dose 142 in liquid egg products 571 milk contamination 123, 124 counts in pasteurized milk 1886 most probable number (MPN)technique 121, 155 motility 122, 154 pathogenicity to humans 118 to insects 118 phylogenetic relationships 119 protein toxin crystal formation 121, 122 regulations relating to 123 rhizoid growth 122, 154 shellfish contamination 2004-2005 in sous-vide products 1341, 1342 spores 142, 143 germination 119 survival at low remueratures 123 structure, haemolysin 145 toxin crystal production 154 toxin detection 122, 145, 146-148, 147T diagnostic kits 148 various methods 148 in wtYo 147-148 in vivo 146, 147T toxins 119-120, 120, 120T, 145T amount produced 143 bce7 gene 145 diarrhoeal enterotoxin 143 emetic 120-121, 146 enterotoxin T 1 4 5

I x INDEX Bacillus cereus (continued) toxins (continued) factors affecting 146 genes 119 mechanism of action 143 non-haemolytic enterotoxin complex 145 production 122-123 structure 145-146 various 146 virulence, assessment 122 virulence factors 119, 119-120 Bacillus cloacae see Enterobacter cloacae Bacillus cocovenenans see Burkholderia cocovenennns Bacillus Genetic Stock Center 135 Bacillus licheniformis amylase gene in Zymomonas mobilis 2372 detection 154 food-borne illness 142, 142-143, 144T growth in bakery products 150 Bacillus megaterium, spores, germination 172 Bacillus mesentericus, propionic acid action 1781,1782 Bacillus mycoides 121 Bacillus popilliae, pathogenicity 118 Bacillus pumilus, food-borne illness 142, 143, 144T Bacillus sporothermodurans heat resistance data 1027T UHT processes and milk contamination 1027 Bacillus stearothermophilus 124-129 aerobic thermophilic spore-former 126 characteristics of species 124-126, 127, 124T detection methods 126-128 specific tests 127-128 distribution and sources 125 enzymes 126, 127T in foods 125 food spoilage fish 811 flat-sour spoilage 1010 growth requirements 124 heat resistance 126 importance to consumer 128 importance to food industrl- 128, 150 regulations relating to 128 spores in canned foods in tropical areas 128 in canneries 128 germination 125-126 heat-resistance 125, 124T heat-shocked 125-126 immobilized, uses 126 inactivation 125 as indicator of sterilization 126 ‘process-resistant’ 126 prolonged heating effect 128 regulations 128 risk factors associated 128 sodium chloride effect 1726 strains 126 transmission electron microscopy 1417F Bacillus subtilis 113, 135-141 ABC-transporters 138 bread spoilage 2050 capsule 135 characteristics of species 135-136 chromosome 135 detection 136-137, 154 DNA uptake, natural competence 138, 139 exoproteins 138 food-borne illness 136, 142, 142, 144T foreign proteins in 139 genes, categories 114 genome 113, 1157, 135, 135 growth 136-137

Bacillus subtilis (continued) growth conditions 136 supplements 136 growth in bakery products 150 as host for genes 116 importance to food industry 138-139 logarithmic growth, end (To)138, 138F Marburg strain (168) 135, 136 as model organism 114 non-pathogenicity 135, 136 plasmids 140, 140 copy number 140 instability 140 proteins secreted 138-139 secretion process 139 proteome 114, 136 recombinant strains 136 regulatory aspects 117 safety aspects 136 selective markers 137, 137T spores culture recovery from 138 heat-resistance 137 long-term storage 137-138, 137F sporulation 135, 137, 137F stages 169F strain construction 139-140 homologous recombination 140, 140F transformation 139-140 subtilisin 135 toxin, heat-stable 136 tryptophan auxotrophy 136 Bacillus subtilis var. niger, detection, adenylate kinase assay 22, 22F Bacillus thuringiensis 118 Bacillus cereus relationship 119, 143 Bt toxin 118 characteristics 121 concerns over virulence 119 food-borne illness 142, 143 characteristics 144T pathogenicity and insect control 118 toxin genes from Bacillus cereus 119 bacon laser radiation 1181-1182, 1182F storage, Arthrobacter role 59 vacuum packaged 1264 bacteraemia, Proteus 1859-1860 bacteria 158-183, 580 165 rDNX 178-179 acid resistance 1735 adaptation to acids 1736 to redox potential and p H changes 559-561.559F adenvlate kinase molecules 1 7 adheiion see bacterial adhesion age of culture, effect of freezing 848 antigens 1 7 7 in apple juice 422T atomic force microscopy 1422-1423 autolytic 1474 bioluminescence, rapid detection in food 1893 biomass estimation, instruments 220-221 as biosensors 270-271, 271T capsule 164 characteristics 792-793 formation and functions 164 Nordic fermented milks 792, 792F staining 1388 see also individual bacterial species carotenes production 730 cell concentration measurement 686 cell division, structural changes 166 cell membrane 161-162, 161F acid diffusion 1733-1734 functions 161-162 permeability increase by essential oilsiphenolics 1718-1719 protein binding 1719 zones 162, 161F

bacteria (continued) cell membrane (continued) see also cell membrane cell organization 160-166 cell sorting using flow cytometry 828-829 cellular contents and inclusions 164-166 cellular differentiation 1 6 7 cell wall 162 acid-fast 163, 176 Bacillus 114-115 composition and taxonomy 176-177 disruption by sorbate 1773 freezing effect 843 Gram-negative bacteria 162-163, 163F, 176, 176F Gram-positive bacteria 162, 163F, 176, 177, 176F ion-exchange system 162 phenolics and essential oils effects 1718-1719 Staphylococcus 2063-2064 strength 162 see also specific bacterial geneva chemotaxonomy 176-177 chromosome 166 see also specific genera classification, phylogenetic 178-183 1 6 s rDNA 178-179 advantageiobjective 1 8 0 application of results 179-183 laboratory procedures 179 limitations 182-183 link with traditional classification 180182 classification, traditional 173-178 groups based on energy sources 173 groups based on oxygen need 173, 174T groups based on temperature 173-174 nucleic acids 175 objectives 175 phylogenetic method links 180-182 clumping 934 colony formation and characteristics 166167 surface topology 167, 165F composition 159T, 160T conductance changes 575 conjugation 934-935, 934F crystalline surface layers 163-164 cultivation conditions, optimization by electrical techniques 582 cultures, turbidity measurement 685 cytosol 164-165 damage due to freezing see freezing death growth phase-dependence 1735, 1735F kinetics 1735, 1735F timescale of preservative action 1713 death curves, non-thermal 1704, 1704F death rate heat killed bacteria 1012 modelling 1704 definition 173 destruction kinetics 1464 manothermosonication 1463-1464, 1465 see also microbial inactivation detection time ( D T ) 576-577, 585 diversity 158 doubling time 2035T D values 1340, 1341 ecology in food see ecology effect of freezing on 842-843 effect of rehydration 535-536 encapsulated, Nordic fermented milks 792 endospores see endospores envelope 160 methanogens 1336

INDEX I xi

bacteria (continued) envelope (continued) S-la) er 1336 structure 161-164 F’ and F strains 934 fat and lipid composition 718,720T in fermented foods 249T fish 807 flagella see flagella flavours produced by 7331 food poisoning due to 835 see also food poisoning food spoilage by see spoilage of food G+C values see DNA, G+C content generation time, calibration of impedimetric technique 587 genetic engineering see genetic engineering genetics 929-940 gene transfer 934-938 genus 175 glycocalyx, fermented milk products 792 growth 205 after rehydration 536 environment-dependence 1709-1710 factors influencing 542-543 freezing effect 846-847 as function of environment and models

1708-1710 lag phase 543-544,550,665 limits 550-551 low p H foods 561-562 at low temperatures 845,845T minimumimaximum p H 5581,1729F minimum temperatures 846T minimum water activity 841T modified atmosphere packaging effect

414-415 normal profiles 665F optimization 548 phases and effect of freezing 842 reaction rates and 551-552 redox dependence 557F requirements 542-545 sous-vide products 1341-1342 submerged fermentations 665-666,

665F suppression by salt 1724-1725 temperature control 548 temperature effect 575,845 temperature interaction with other factors 550 temperatures for 840 tolerance of low water activity 542,

bacteria (continued) high-frequency recombination (hfr) strains 934 identification 174,175 electrical techniques 582 inactivation see bacteria, destruction; microbial inactivation inhibition of undesirable microbes by fermentation 1726 by salt 1725,1726-1727 in intestine see gastrointestinal flora intracellular structures, water activity effect 545-546 lipids 1299 lysis by bacteriophage 203-204 PCR sample preparation 1479 by phage lysins 1473 marine, tetrodotoxin production 1674 metabolism, temperature effect 553-555 mineral uptake 1313-1314 morphology 159-160,l59F environmental influences 160 variations and flow cytometry 828 nomenclature 174,174 nucleoid 166 organelles 165 origin of term 173 outer membrane in Gram-negative cells

163 pathogen detection by phage-based techniques see bacteriophage-based techniques periplasm 163 phage adherence 1471 phage as viability indicator 205 phage interactions see bacteriophage phage-resistant mutants 1472 phage typing see phage typing oili 164.934 polysomes 165 preservatives active against 1712T protective cultures, meat preservation

1271 replication 205,933-934 replication rate, phage rate comparison

208,208F reproduction, binary fission 933-934 riboflavin production 730 secondary metabolites 1328-1334 see also secondary metabolites selective adsorption 1696-1699 single-cell protein see single-cell protein

543T water activity levels 542,542T growth curve 548,548F,665F,1709F maximum carrying capacity 548,548F growth limit models 1706,1706F growth rate 1723 absolute and specific 1709F Arrhenius plot 552,552F carbon dioxide effect 559 comparison of food-borne bacteria

55OF effect of freezing 848 effect of water activity 543,543F environmental factors effect 543,544F fastest-growing organisms 549-550 in food 548 interactions of factors 551,551F modelling 1704,1709F models 1707T optimum temperature 549 solute tolerances 174F temperature effect 549-550,552-553,

549F see also temperature harvesting, centrifugation application

1685-1686 heat resistance 1340,1340,1341 higher taxa 175

S-layer 163-164 atomic force microscopy 1423 slime 792 composition 793T determinants affecting production 793 production process 793-794 production rate 794 sourdough bread 300 spore-forming 168 see also endospores; spore-formers spores see endospores, bacterial sporulation 543-544 starter cultures see starter cultures storage granules 166 structure 158,1591 sublethal injury due to freezing 844 recovery 844 surface-ripening, as starter culture 20851 survival at low temperatures 847-848 see also psychrophiles taxonomy 174 chemical 176-177 classical and numerical 175 genetic methods 175 major taxa 177-178 serology 176 toxin production 543-544

bacteria (continued) transduction see transduction transformation 935-936,935F competence 935 ‘type strains’ 174 viability freezing effect 847,847F freezing rates and 841 staining for 830-832 viable cell counts alternative methods 219-220 method 219 virulence factors 1472 viruses see bacteriophage water activity inhibitory levels 1724T requirements 542,542T,1723 tolerance of low levels 542,543T yield, water activity effect 544 bacterial adhesion conditioning layer of organic materials

1693 control by polymer technologies 1692-

1699 free energy change 1693 importance 1692 inhibition 1692-1696 hydrophilic surface polymers 1693-

1695 low surface energy polymers 1695 mobile surface polymers 1695-1696 polymers for retardation 1692-1693 polymer structures 1694T thermodynamic treatments 1693 selective adsorption of bacteria 1696-

1699 see also adherence; biofilms; polymers bacteria-specific adsorbents 1696-1698 bactericidal barriers, bacteriocins as 190 bactericides, ultrasound 1463-1464 bacteriocins 183-191,1711 advantages/disadvantages 188-189 applications 185 as bactericidal barriers 190 Bacteroides 202 Brevibacterium /mens 309 cost-effectiveness issues 190 definition and description 184-186 detection 184,184F effect in fermented meat products 746 effect on Bifidobacterium 1356 enterococci producing 623-624 Enterococcus faecalis 1367 Enterococcus faecrum 1367 fast- and slow-acting 190 future prospects 190 genetics 189 GRAS status 184,190 harvesting 189-190 hurdle technology 1074 hydrophobicity 189 lactic acid bacteria 2103-2104 lactobacilli 1136,1136T Lactobacillus acidophilus 1153-1154,

1153T Lactobacillus brevis 1149,1 l5OT Lactococcus lactis 1169 Leuconostoc 1193-1194 as markers for food-grade cloning vectors

919 meat preservation 1271

Moraxella 1491 mutants resistant to 189 natural antimicrobials 1573-1574 as natural food preservatives 185 as natural products 188 origin of term 184 pediocin-like 187-188 potential uses 188 production 189-190 Proteus 1863 safety aspects 189

I xii INDEX

bacteriocins (continued) in starter cultures 2103-2104 strains producing 184 ‘super producers’ 1573-1574 thermostability 188-189 toxicity trials 189 types and classes 185, 1 8 5 1 see also nisin: other specific bacteriocms Bacteriological Analytical Manual (BAM) Salmonella detection 1967 Salmonella enteritidis detection 1939 bacterioohaee 203., 936.2264. 1469-1471 in adenylate kinase assay for bacteria 2223,22F, 23F adsorption 2091.2105. 1471. 1473 ampli’fication technique see phage amplification appearance 1470F applicability for bacterial pathogen detection 204-205 Arthrobacter 58 bacterial interactions in food 1469-1470, 1472 concentrations for contact 1472 bacrerial resistance see bacteriophage, resistance bacterial taxonomy 175 as bacterial viability indicator 205 Bacterordes 202 Bacteroides fragilis 2278 burst size 2091-2092, 2105 characteristics 203-204, 1470, 1471F concentrations in foods 1472 defective 936 discovery 1469 as disinfectants 1473 distributionisources 1469 DNA packaging 936 faecal contamination indicator 2284 fermented milks from Northern Europe 794 gene product detection 205 generalized transduction by 936-937, 936F harmful effects 1470 helper 937 host specificity 1471 induction of temperate phage 1471 infecting food pathogens 1470 infection butanol-acetone fermentation 447 detection by electrical techniques 582 infection cycle 203-204 infection process 1471 lactic acid bacteria 2105-2107 Lactobacillus bulgaricus 1141 Lactobacillus casei 1159-1160, 1161T Lactococcus lactis 1169-1170, 2106F lux see lux-bacteriophage lysins see lysins lysogenic cycle 1471 lysogenic phage 1170, 2106-2107 lytic cycle 2092, 2105-2107, 1471 lytic phage 1169-1170 Propionibacterium 1853 receptors 1471 recombinant 205, 205F replication 204F, 2091-2092, 1470-1471 rate, bacterial rate us 208, 208F rates 205 requirements 205 in water 1472 resistance 1472 genetic engineering 2099 lactic acid bacteria 920 Lactococcus lacris 1169-1170 starter cultures 939, 2106-2107 in starter cultures 2090, 2091-2092 contraliprevention 2094, 2107 management 2092T testing 2094-2095 I

bacteriophage (continued) in starter cultures (cotztiuued) test kits 2093 yoghurt culture contamination 788 Streptococctts thermophilus 21 32 Streptomyces 2138 structure 203, 1470 survival wirhout hosts 2092 taxonomy 1470, 1471F temperate 1471 toxin synthesis 1333 transduction by 936-937 typing see phage typing use to control pathogens in foods 14721473 psychrotrophic pathogens 1473 yoghurt starter culture contamination 788 bacteriophage A511 206 bacteriophage-based techniques 203-210 advantagesidisadvantages 204T bacterial pathogen detection 205 ltrx-bacteriophage see lux-bacteriophage lysin-release ATP bioluminescence 208209,209F, 209F Listeria detection 210 phage amplification 207-208 see also phage amplification bacteriophage fFSW 1160 bacteriophage J1 1160 bacteriophage 1 9 3 7 bacteriophage P22 1853F transduction 936-937 bacteriophage PL1 1160 bacteriophage T4 1472 transmission electron microscopy 1417F ‘bacteriophage test,’ Salmonella 526 bacteriostatic compounds, meat preservation 1271 Bacterium monocytogenes see Listeria nzonocytogenes Bacteroides 198-203 anaerobe 1 9 8 , 2 0 0 , 2 0 0 antibiotic resistance 198 bacteriocins and bacteriophage 202 bile salt metabolism 202 capsules 200 classification and characteristics 198, 200 cultivation 200 media 200,200T effect on foods in gastroinrestinal tract 200-202 glycosidases 201 polysaccharide breakdown 200-201 protein metabolism 201 xenobiotic and carcinogen metabolism 201-202 Flavobacterium relationship 821 glycosidases 201 importance in agriculture and food production 202 lipopolysaccharide 198, 200 nitroreductases 202 pathogenicity 202 proteinases 201 species 198 characteristics 199T Bacteroides distasonis 199T Bacteroides eggertii 199T Bacteroides fragilis catalase and superoxide dismutase production 200 characteristics 199T enterotoxin 202 faecal contamination indicator 2284 pathogenicity 202 phage 2278 protein metabolism 201 Bacteroides uodosus 1418F Bacteroides ovatus characteristics 199T polysaccharide fermentation 201

Bacteroides thetaiotaomicron characteristics 199T polysaccharide fermentation 201, 201T Bacteroides uniformis 199T Bacteroides vtilgatus 199T BactimediaO 2094 BactoFoss system 83, 83F procedure 83F raw milk assessment 90 bactofugation 1685 Bacillus cereus spore removal 123 Clostridium tyrobutyricum spores 457 Bacrofuge 1682, 1683, 1684F Bactometer 220, 574T, 584T bactoprenol 1303 Bacto Rogosa SL Broth 1156, 1156T BactoScan 529 Bactotherm 1683 BACTRXC 5741, 584T bagoong756-757 Baird-Parker (BP) agar 2067, 2073-2074 bakery products Bacillus growth 150 modified-atmosphere packaging 4 1 1 effect on spoilage 414 nisin addition 195 propionic acid addition 1781 shelf-life 195-196 sorbate addition 1772 s p oi I a g e bacterial 2055 fungal 2060 staphylococcal food poisoning 2078 see-alio bread baking bread 289 confectionery products 474 Saccharomyces cerevisiae role 1920-1 922 yeast see Saccharomyces cerevisiae see also bread-making baking industry, nisin application 187 balao-balao 757 Balkan endemic nephropathy 1519, 15411542, 1654 ballistoconidia 855, 899 Bantu beer 1921 barrier technology see hurdle technology bases, nucleic acid 930, 930F Basidiobolus ranarum 883 Basidiomycetes 868 edible species 868T Basidiomycota 868 basil, effects 1721 Basipetospora 863, 895 characteristics 8 8 9 7 conidial structures 891F batch processes fermentation see fermentation microbial ecology of foods and 548, 548 pasteurization 1033-1034, 1033F BAX screening system 226 Bayes theorem 2167 BB factors 1356-1358 BBL agar 1156, 1156T BBL Campyslide 348, 348T, 350T detection limits and sensitivity 351T, 351T protocols 348-349 BBL Crystal system 240 biochemical reactions used in 248T comparison with other systems 248-249, 248T principle 245 protocol 247 BBL Enterotube system biochemical reactions used in 248T comparison with other systems 248-249, 248T principle 245 protocol 246-247 BBMB-lactate medium 455T B cells, mitogen, from fermented milks 797

INDEX I xiii

BC motilitj medium. composition I 5 8 BDE VIA kit 148 beach peas, Pantoen infection 1629 bead mill, high-speed 698, 699F bean curd 2098 beef Arcobacter importance 52-53 microbiological analysis using BactoFoss 83 see also meat beer accelerated forcing tests 107-108 acidification, Acetobacter role 7 ATP bioluminescence. sample preparation 104 Bantu 1921 brewing see brewing fermentation 1926-1927 Saccharomyces rereuisiae role 1921 yeasts see Saccharomyces cerevisiae; yeastis), brewer's see aiso brewing filtration 1679 lager see lager Iambic, Brettanomyces role in 305 from millet 766 nisin application 187, 197 nutrient composition 766T production 1921 brewing yeast energy levels 103 Lvort, constituents 677T see also brewing; wort sorghum see sorghum beer spoilage Lactobncillits brevis 1149, 1149T Fedtococcus 1646 Torulopsis 2 1 48 Zymomonas 2370 starter cultures 2089 sterility analysis 106F, 106F beer-glucose medium 2369T bees, Gluconobacter in 957 beet molasses, ethanol fermentation 2371 benches, for laboratories 1124 bench tops, in laboratories 1124-1125 benomyl-containing medium 1 4 9 7 1 benzaldehyde 732-733 oroduction 732-733 be&o(a)pyrene 1742 benzoic acid 961, 1754-1757 antimicrobial action 1756-1757 mechanisms 1756-1757 p H effect 1756, 1756 speciesistrain tolerance 1756-1757 appiicationsiuses 562 assimilation and transformation by Rhodotoritla 1904 behariour in foods 1755-1756 chemical properties 1755-1756, 1756 enzymes inhibited by 1756 GRAS status 1755 ionized and non-ionized 1756 maximum levels used 1756 as preservative 1754-1757 foods preserved 1755-1756, 1755T important criteria 1755T interaction with other preservatives 1757 limitations 1754 mechanism of action 1713 spectrum of activity 1712T temperature effect 1757 regulations affecting 1755 spectrum of activity 171 1 toxicology and regulatory aspects 1716 uptake by microbes 1756 benzylpenicillin 1321, 1321T Bergeyella 821 Berlin process 298 Best Manufacturing Practices (BMP) 972 beta-lactam antibiotics 1320F

betalains 732 betuloside 735 beverages alcoholic see alcoholic be\-erages Bifidobacteriitm in 216 Brettariomyces contaminationieffects 305 clarification 1679 contamination prevention. ATP bioluminescence role 104-108 Debaryomyces significance 5 1 8 1 fermented 2098 Brettanomyces contaminationieffects 305 Lactobacillus casei role 116 I nutritional aspects 765-766 Saccharomyces cerevisiae role 19201922 from sorghum and millet 759-767 see also alcoholic beverages; beer; sorghum beer; wine microbial stability, assessment 101 microbiology. ATP bioluminescence role 101-109 see also ATP bioluminescence natamvcin application 1779-1780 smoke-processed 1738 from sorghum and millet 759-767 spoilage Saccharomyces ceret'iszae 1922-1 923 sorghum beer 764, 765T see also beer; wine sterility testing 106, 106F, 106F thermoduric spoilage organisms 101 see also beer; soft drinks; wine BIXcore 275 instrument 277 bias, models 1705 Bzfidobacteritim 2 10-2 17, 1 35 5-1 360 acid production 1356 actions 215 anaerobic growth conditions 216 antibiotics for selection 216-217 bacteriocins effect on 1356 breast-feeding and intestinal levels 214 cell morphology 1357F characteristics of species 780T for fermented milks 1377T colonization of intestine 214, 1356 factors affecting 1356 culture stock/isolates characterization 1359-1360 development after birth 1358 discovery 211, 1355 enumeration 216-217 enzymatic characteristics 1375T fermented milks using 7 7 7 2 780T, 1374T in foodsibeverages 216 in gastrointestinal tract 1367 colon 212 counrs and supplementation 13581359 depletion and effects of 1356 ecology 212, 214 role 1355-1356 genus description 2 l l T growth media for 216 growth promoting factors 1356, 1358, 1358T health benefits (implied) 215-216, 215T, 1374 health-promoting activities 1358-1360 historical perspective 211-212 inhibitory effects on pathogenic bacteria 216 isolation methods 216-217 lactobacilli similarity 212 metabolism 1356 as probiotic 1138, 1374,2085 effects 212 products containing 1359 rapid identification 217

Bifidobactertum (coiztintted) species 211, 212, 1355-1356, 1357F descriptions 213T survival after consumption 1359 taxonomy 211-212 actinomycetes differences 21 1 vitamin production 1358 Btfidobactertzrm acidophilus, ice-cream making 1359 Bifidobactetkm adolescentis 213T, 780T, 1356, 1358 Bifidobacterium urzgulatum 213T Bifidobactertum antmalzs 213T. 1357F Bi$dobacterium asteroides 213T Bifidobactertum hifidum 211. 213T. 1356 'characteristics hOT, 1377T in fermented milks 1359, 1359, 1360F characteristics 1 3 7 7 1 preparation 779 ice-cream making 1359 Bifidobacterium bourn 21 3T Bifidobacterizim breve 213T, 780T Bzfidobacterium cateiiulatirm 213T Bifidobacterium choerimtm 213T Bifidobacterium coryrzeforme 213T Bzfidobacteriutn cuniculi 2 1 3 1 Bifidobacterium dentiuni 213T pathogenicity 212 Bifidobacteriirm globosum 213T Bifidobacterium iiidicum 213T Bifidobacterium infantis 213T, 1357F characteristics 780T, 1377T in fermented milks 1359 Bifidobacterium lactis 1356 Bzfidobactertum lactzs BB 12 1786 Bifidobacterium longunt 213T, 1358. 1357F characteristics 780T, 1377T in fermented milks 1359 characteristics 1 377T probiotic product 1786 supplementation with 1359-1360 Bifidobacterittm magnum 213T Bifidobacterium minimum 213T Bifidobacterium pseudocatenulatum 213T Bifidobacterizim pseudolongum 213T, 1357F Bifidobacterium puliortrm 214T Bifidobacterium subtzle 214T Bifidobacterium suis 214T Bifidobacterium thermophilum 214T bifidogenic factors 1356-1358, 1374 bifidus factor 1374 Bifidus milk 781 bifidus yoghurr 781 Bifighurt 780 bile, conjugated xenobiotic secretion via 202 bile acid conjugates, metabolism by Lactobaczllus acidophilus 1362 bile-aesculin test, Vagoroccus identification 221 8 bile salts, metabolism, Bacterozdes action 202 bile salts-brilliant green agar (BBG) 31-32 bile salts-brilliant green-starch agar 32 binary fission 933-934 bacteria 166 binomial names 174 biocatalysts Ptchin pastoris 1691 Schizosaccharomyces pombe 1988 Zymomonas mobzlis 2372 biochemical identification techniques 218228,228 alternative methods for viable cell counts 219-220 areas of recent developments 218 Enterobacteriaceae, coliforms and E . coli 244-249 comparisons 248-249 principles and types of tests 245 protocols 245-247 wet and dry systems 244

I xiv

INDEX

biochemical identification techniques (contrnued) food-poisoning organisms 237-244 agar-based kits 238-239 application range 241-244 dehydrated media kits 239-241 diagnostics kits 238-241 miniaturization 238 food spoilage yeastsimoulds 228-237 advantagesilimitarions 235-236 molecular methods vs 234-235 see also fungi, identification instruments for microbial biomass 22022 1 microflora of fermented foods 249-252 miniaturized techniques 221-224 see also miniaturized microbiological techniques prediction of developments 227T refinements of novel methods 224-227 genetic methods 226-227 immunology 224-226 relative interest in 218F sample preparation improvements 218219 usual biochemical tests 221 see also indrvidual microorgarzisms/techtzrques biochips, D S A arrays 1603 biocides 1794-1801, 1826 chemical classification 1794-1795 effects on microorganisms 1800-1801 effluent and waste stream issues 1800 food rinses 1828 hand-sensitizers 1826 ideal characteristics 1 7 9 5 7 microbial resistance 1800 physicalichemical properties 1795-1 800 testing systems 1823-1824, 1824F types used 1 7 9 5 1 see also disinfectants biocontrol see biological control biodegradation, Flavobacterium applications 825 biodispersans 16 bioenergy 2187 biofilm bacteria 255T adaptation to environmental stress 255256 adhesion 253-254,255 characteristics 253 resistance to antimicrobials, deep-lying cells 257 surface properties 254 viable but non-culturable (VBNC) 256, 256F biofilms 252-259,253F, 1692, 1828 architecrure 253, 254 coliform 608 composition on floors 255,255T definition 252-253 dense confluenr 253 disinfectant resting and 1828 elimination by cleaningidisinfecrion 258259 formation 253,253-255, 1791-1792 adhesion 253-254,255 chlorine effectiveness 1798 colonization 254-255 conditioning 254 forces involved 254 Pseudomonas aeruginosa 1870 Shewanella putrefaciens 2013-2014 surfaces 1870 time required 254-255 Klebsiella production 1114 locationsisurfaces with 253 preventionireducrion 258, 1828 design and surface modificarion 258 dryness 258 surface texture 258 see also bacterial adhesion, inhibirion

biofilms (contrnnedi properties 255-257 active but non-culturable cells 256, 256F adaptation to environmental stresses 255-256 adhesion strength 256 linked to exrracellular oolvmeric subsrances 255 resistance to anrimicrobials 256-257 resistance to cleaning agents 256 Pseudomonas aerziginosa 1870 removal 1692, 1870 testing by electrical techniques 582 substratum effect on biocide efficacy 257 BIOgardeB 780,2085 biogenic amines 748 enterococci forming 1369-1370 Lactobacillus brevis forming 1150 Leucouostoc forming 1193 in malolactic fermentation 2312 production meat spoilage 1266-1267 organiims 1366-1267 stored mear 1259 wine 2312 see also amines Bioghurt 780 biohazard safety cabinets, in laboratories 1125 Biokys 78 1 biological conrrol Bacillus role 117-1 18 Bacillus thuriizgiensrs 119 Trichoderma for fungal plant pathogens 2 188-2 189 ‘biological ennoblement’ 737 biological enrichment of nutritional value 250 biological remediation see bioremediation biological value (BVj, single-cell protein 2040 Biolog system 223, 232, 240-241 advantages 240 fungi 231-232 microflora in fermented foods 252 bioluminescence adenylate kinase assay see adenylate kinase (XKj ATP see ATP bioluminescence bacterial 1893 comparison with PCR 1631T definition 8 0 , 9 4 Lactobacilltrs brevis detection 1146-1 1 4 7 Leuconostoc 1191, 1 1 9 1 1 shellfish 2007 biomass algae 2024, 2026 ATP as cell marker for 1 6 control in industrial fermenrations 685686 estimarion, instruments 220-221 fungi 2036T measurement 80, 685-686 calorimetry 685 instruments 220-221 production, Yurrowia lrpolytica 364 single-cell prorein 2030 veact 291 , bioparticles 259 application of AC field 261, 261F application of D C field 260-261, 260F distribution of charge 260F innare electrical properries 260, 260F investigated by non-uniform XC electric fields 262T levitation by dielectrophoresis 264 morion in inhomogeneous AC electric field see dielecrrophoresis (DEP) motion in rorating electric fields see electrorotarion (ROT) I

~~~

.

bioparticles (contrnued) motion in travelling wave electric fields 266-267 orienration (induced motion) 260 oscillations 261 polarizability variations 263, 263F polarization in AC electric field 261, 261F rotation 265 separation by dielectrophoresis 262-263 surface charge 260 torque generation 265, 265F biopesticides Bacillus thuringiensts 119 see also biological control biopharmaceutical industry, clean-in-place in 1815 biophysical techniques 259-267 basic concepts 260-261 see also dielectrophoresis (DEPj; electrorotation ( R O T ) biopolymers 1693 fractionation 264 biopreservative, Pedrococcns role 1646 BioProbe 9 7 beer sterility analysis 106F bioprocess 683 bioprotectire species, in meats 1271 bioreactors Alcaligenes in 4 0 continuous high-cell-density, filtration use 1680 membrane 1680, 1680F methanogenic 1336-1337, 1337F bioremediaGon Acinetobacter 1 6 Alcaligenes 39-40 biosensors 268-278, 1894 acceptance by regulatory agencies and users 278 advantages 278 Alcaligenes, for heavy metals 40 alcohol measurement 686 Alteromonas ptitrefacreus 270 amperometric on-line 277 applications 269T Arthrobacter nicotianae 270 bacreria 270-271, 2 7 1 7 citations 268 Clostridrum acidrririci 270 Clostrrdrunz botnlinnm 275 cyanide 270 definitions 268-276 development and sales projections 268 enzymes as 269-270,272, 686 fish freshness 270, 270 flow injection analysis and on-line systems 276-278,273F flow-through and on-line future prospects 278 microprocessor-conrrolled 277 future prospects 278 Gluconobacter role 957 glucose oxidase 270 see also glucose Hansenula unomala 270 hydrogen peroxide detection 277 industrial fermentation 686 integrated multi-biosensors 686 limitationsiproblems 278 method of operation 686F optical detection of D S X 274 optical flow cells in 277 peptides as 269 Pseudomonas 270 regeneration of activity 278 Rhodococcus 270 sensors 269T affinity (IXsys) 274 enzyme 272 with potentiometric transducers 272 shelf life 278 stability problem 278

INDEX I xv

biosensors (continued) Synechococcus 270 thermistor-based 276 tissue 270 transducers 268, 271-276, 272T acoustic wave 274 amperometric 272-273 automated optical 274 conductance and capacitative 273-274 electrochemical 271-274, 272T evanescent wave 275-276 with fibre optics 274-275 hybrid 274-275 optical 274-275, 272T piezoelectric 274 potentiometric 271-272 sensitivity 268 surface plasmon resonance 275-276 thermal 276, 277 without fibre optics 274 typical sensors 268-271 antigens and other compounds 269 enzymes 269-270 immunoglobulin 268-269 microbial cells 270-271, 271T whole cells 270-271, 271T urea measurement 686 biospecific interactions analysis 275 biosynthesis, definition 1279 biotechnology Acinetobacter applications 15-16 Avthrobacter applications 60-61 European Union safety standards 18371840 fungi use 2036 Thevmus aquaticus value 2140-2141 wine-making 2310, 2309T wine yeasts 2308T see also genetic engineering; recombinant DKA techniques BioteCon Diaenostis FoodProof Kit svstems 1633-1g35, 1636T, 1638T ‘ biotin 629 biosvnthesis and uptake 1315-1316 enzymes 1315 function 1315 operon regulation 1315-1316, 1316F in PCR amplification product 1480, 1480F requirements 1308 biotin protein ligase 1316 Biotrace Dairy Kit assay 92, 92T biotransformation, by Acinetobacter 1 6 biotyping Campylobactev 339 Clostridium perfririgens 4 3 9 1 Pseudomonas aeruginosa 1869 Staphylococcus aureus 2067 Vibrio cholerae 2245 bio-yoghurt 785 birds, control in manufacturing facilities 968

2’,7’-bis-(2-carboxyethyl)5[6)carboxyfluorescein acetoxymethylester (BCECF-AM) 831 bismuth sulphite agar (BSX) 1947 bisulphites 422F, 1750, 1753 ‘bitty (broken) cream’ 123, 1444, 150 Biverticillium 856, 1649 importance in food 1652-1653 blanching of foods 846 blastospores 855, 900 bleach, effect on ATP bioluminescence 98 bleomycin, media containing, Aspergillus parasiticus detection 75 blood agar base medium 6 4 2 7 blood films, Bacillus anthvacis detection 131 blood pressure control, role of fermented milks 784 reduction, Lactobacillus casei role 1163 Boerhaave, Hermann, fermentation process classification 1068 bone charcoal 134

bone sour 2052-2053 bone taint 2052-2053 bongkrek acid 1871, 1872, 1872F actions 1873 production 1873, 1873T ‘bootstrap’ method, phvlogenetic trees 179 Bordetella Alcaligenes relationshipidifferentiation 41,411 rapid diagnostic tests 4 1 Bordet-Gengou (BG) agar 4 1 bornanes 734 ’bot cook‘ 459 Botrvotmia 865. 895 Botryotrichum, characteristics 8 8 9 1 Botrytis 279-283, 865, 869, 895 characteristics of genus 279 control 282 cultural characteristics 279 detectionienumeration methods 280 acidified media 280 preferred antibiotic method 280, 280 staining method 280-281 distinguishing characteristics 279-280, 2807 fluorescence microscopy 281 growth characteristics 279 immunological assays 28 1 importance to food industry 281-282 species 279 zones of growth 279 Botrytis aclada 282 Botrytis cinerea 2317-2318 advantages in food industry 282 citric acid formation 282 detectioniindicators 23 17-231 8 effect on winesisherry 282 enumeration methods 281, 282T grape infection 2316-2317, 2317-2318 characteristics of juice 2 3 1 8 1 grey mould (disease)due to 282 metabolism 23 17-231 8 by-products 2318 noble rot due to 282 sweet white wine production 2316-2317 Botrytis fabae 282 botrytized wines 2318-2319, 2 3 1 9 1 ’botulinum cook’ canning process 1017 UHT 1027 botulinum toxin IBoNT) 429, 458 antiserum production 465 detection 430-431,461-462, 463-465 bioassays 463-464 biosensors 275 ELISA 462,464-465,463T immunoassays 464-465 immunoassay sensitivity 465 mouse lethality assay 430, 461-462, 463-464,462F nonspecific reactions 465 in dried foods 534 in fermented meat products 746-748 haemagglutinin activity 430 mechanism of action 430,430, 461, 463 progenitor toxin 430 properties 461-462 in sous-vide foods 1343 synthesis inhibition by nitrite 1765 by parabens 1760 use as pharmaceutical 462 see also Clostridium botulinum, toxins botulism 429, 432, 458,463, 1333 clinical features 460-461, 460F, 10201021 diagnosis 461 foods associated 429, 4 6 1 1 fishifish products 809, 812 sous-vide products 1339 underprocessed food 1020-1021 geographic distribution 460

botulism (continued) infant 460-461 outbreaks 4 6 0 , 4 6 1 T pathogenesis 460 prevention 461 see also Clostridium botulinum bovine spongiform encephalopathy (BSE) 283-288 in Britain 283-284 in cattle, case numbers 285T clinical signs 286 diagnosis 2 8 6-2 8 7 in Europe 285-286,285T exotic animal species infected 284T infectivity of agent 284. 287 notifiable disease 285 pathogenesis 285 pathology 284, 286,283F protection of humanianimal health 285 regulations after 285,285 related diseases 284-285 animals 284-285 humans 285 see also Creutzfeldt-Jakob disease (CJD) brain-heart infusion agar (BHIA) 32 brain-heart infusion broth and agar 6 4 2 1 branched DNA (bDNA) signal amplification 1478, 1477F Branhamella 1487 pathogenic species 1492 taxonomy 1876 bread 288-301 ‘black’ 297 frozen 843 ingredients 288, 289T leavened 288 quality effect of ingredients 288, 2 8 9 1 role of wheat flour constituents 290T rising and yeast causing 2097-2098 rope in 150 ropiness 293,294, 294T rye 289 sourdough see sourdough spoilage 293-294,294T, 2050 mould 293-294,294T prevention 293-294 susceptibility 293-294 staling 1749 prevention 1749 types 289-290 from wheat flour 288-301 bread-making 288-290, 290T baking 289 biochemical actions of yeast 293 comparison of procedures 2 8 9 7 continuous process 288, 289F, 2 8 9 1 effect of temperature changes 291F fermentation 289 historical aspects 295 Lactobacillus importance 1135-1136 mixing and dough development 289 overmixing 289 role of additives 291T starch fermentation 293, 294F steps 288-289,289F yeast forms see Saccharomyces cerevisiae; yeast, bakers’ see also baking breast-feedine. intestinal bifidobacterial levels 274 brem 770 brem cake 770 brem wine 770 brem wonogiri 770 Brettanomyces 302-308, 867, 869, 895 adherence to insects 307 appearance 302F aroma production 303 assimilation 3 0 3 1 characteristics of genus 302, 303T

I xvi INDEX Brettanomyces (coiztinued) cider contamination 425 Custer effect 303 detection methods 306-307, 307F nested PCR 306-307 selective media 307 fermentation 305, 303T fluorescent microscopy 303F genomic analysis 304 genomic properties 304F importance in fermented beveragesifoods 305 isolation 306 media 302 methods of control 307 mitochondrial genomes 304 nutritional requirements 303 petite mutants 303 physiologicalinutritional properties 302304 relationship to Dekkera 302 RFLP analysis of miDNA 304-305 sensitivity to sulphur dioxide 307 species 302 characteristics 303T Brevibacteriuni 308-3 14 alcohol usage 311, 311T applications 313 biochemical characteristics 310-312 metabolic end products 311-312 substrate utillization 310-31 1 carbonienergy sources 310T carotenoid pigments 309, 310, 309F catabolism of amino acids 308, 311 cellular morpho1og)- 309F cheese surface growth characteristics 309-3 10 classification 308 control 309 enzymes for cheese ripening 308 for proteolysis and lipolysis 312-313 genetic engineering 313 growth characteristics 308-309 pure culture characteristics 308-309 species 308 Brevibacterium lactofermentum 3 13 Brevibacteriuin h e n s 308, 310 amino acid utilization 311 antibiotic sensirivity 309 characteristics 309, 395 cheese Brie preparation 1658-1659 growth on and effects 309-310, 311 isolation from 310 maturation 392 ripening 60, 379,2102 role in 395-396, 396F colour formation 309 control 309 esters produced 733 th conditions 2102 linecin production 309 metabolism 396 in microcapsules 312 pH range for growth 309 pigment production 309, 310 proteinases 396 proteolytic enzymes 313 vitamin requirements 310 breweries cylindroconical fermentation vessels (CCFVs) 1175, 1176, 1175F fermentation 1175 lager 1174F brewing hops and wort boiling 1175 lautering 1174-1175 milling and mashing 1173-1 174 process 1172-1175, 1 1 7 3 1

brewing (continued) sorghum beer industrial methods 761-763, 761F, 761F traditional methods 760-761, 761F see also sorghum beer yeast see yeast, breLvers’ see also beer Brie 387,2103T defects 392 history 388 manufacture 379 bright green yellow fluorescence test (BGYF), for mycotoxins 153 1 brilliant green bile broth (BGBB), Escherichia coli detection 637 brine 1741 Debaryomyces etschellsii in 518 effect on microorganisms 402 hams in 1728 microflora and spoilage of foods 1264 strength 1741 vegetables 1726-1727 brine curing 1264 cucumber 741 intermediate moisture foods 1100 brined foods, spoilage bacteria 1741 British Calibration Service (BCS) 1130 British Standards 1128 BS57501130 Brochothrrx 3 14-3 18, 3 17-3 18 bacteriophage specificity 316 characteristics 3 14-3 15 distinguishing Characteristics 314T food spoilage, oxygen-modified atmosphere 317, 317-318 growth, on meats 317 importance to food industry 317-318 isolation and enumeration 316 from foodienvironment samples 316317 international guideline 317 rapid detection 317 species 314 comparisons 31 6 Brochothrix catnpestris 314 Brochothrix thermosphacta 314,314,12681269 characteristics 315 cooked cured meats 1268-1269 distribution 314 enzymes 315 as facultative anaerobe 315 food spoilage 314-315, 317-318 meat see meat spoilage (below) prevention 317 glycerol esterase 315 in meat 316 meat spoilage 1255, 1256, 1258 fermented products 745 meat products 1266 modified atmosphere packaging 1270 substrates and end products 1258, 1258T metabolism 315 pH range for growth 315 ‘broken cream’ 123, 1444, 150 bromothymol blue (BTB) teepol agar 2251 browning of foods enzymatic 1752 non-enzymatic 1752 browning reaction, cheese 392 Brucella 319-324, 324-328 characteristics 319, 320T in culture 322 chromosomes 319 classification 319 controliprevention 325-326 on farms 326 pasteurization 325-326 detection methods 322-323 commercial kits 322

Brucella (corztinuedi detection methods (continued) cultivation 322 serological identification 322-323 discovery 324 importance to food industry 320-321 disease epidemiology 320 entry into food and transmission 320321 fate during processingistorage 321 lipopolysaccharide 322 morphology and physiology 320 0 chain in LPS 322 pathogenicity and symptomatology 321322 p H range 325 rough strains 322 smooth strains 322-323 species 319-320, 324 survival and growth in milkimilk products 325 temperature range 325 temperature sensitivity 321 see also brucellosis Brttcella abortus 319, 321, 1441 behaviour during cheese manufactureistorage 3 2 5 1 epidemiology 3 2 7 in milkimilk producrs 1441 Brucella blood agar 200, 322 Brucella canis 319, 320 Brucella maris 319-320 Brucella melitensis 319, 324 epidemiology 327 Brucella neotomae 319 B r z ~ e l l aovis 319, 320 Brucella suis 319, 320, 321 brucellosis 319, 326-328 in animals 320, 320-321 control 21 1, 326 from butter and cream 1454 clinical features 327-328 complications 322, 328 control and prevention 323-324 epidemiology 320, 326-327 outbreaks due to milk products 328T incidence 320-321 incubation period 321-322, 327 occupations associated 321 onset and clinical features 321-322 pathogenesis 321-322 relapses, chronicity and recurrence 322 transmission 320, 320-321. 327 aerosol inhalation 321 dietary practices associated 321 direct contact 321, 327 foods 327 treatment 323, 328 vaccines 323-324 see also Brttcella brucellosis-free herds 326 brushes, cleaning 1848 BSE see bovine spongiform encephalopathy iBSEi bubbles manothermosonication 1464-1465 ultrasonic waves causing 1463 buckets 1848 budu 756 buffered peptone water 1199-1200 modified 2230 pre-enrichment for Salmonella 19481950 buffers bread-making 291T for immunomagnetic separation of Salmonella 1 9707 for pour plate technique 2155, 2155T buildings construction and design 1803 contamination risks 1793 design for hygienic operation 1791

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INDEX I xvii buildings (continued) exterior 1803 good manufacturing practice and 964 hygienic processing and 1803-1 804 Bulgarian buttermilk 779 Burkholderia cepacia, phenotypicigenotypic characteristics 1872T Bttrkholdevia cocouenenans 1871-8175 characteristics 1871 control 1874-1875 detectioniisolation 1875 phenotypicigenotypic characteristics 1872T significance in foods 1873-1874 taxonomy 1871 toxins 1871-1873 actions and symptoms 1873 biochemistry 1871-1 873 control 1874-1875, 1874T control in fermented foods 1874 detecrion 1872-1873 effect on onion extract 1874, 1874T production 1873, 1873T Burkholderia cocot’enenans biovar farinofermentans 825, 1871 phenotypicigenotypic characteristics 1872T toxins 1872, 1872F 2,3-butanediol, Klebsiella production 1114 2,3-butanedione see diacetyl but an oI genetically engineered C. acetobcrtylicunz producing 450-451 mutant C. acetobcitylicuin producing 450, 45lT toxicity 448 butanol-acetone fermentation bacteriophage infection of clostridia 447 butano1:acetone ratio 448 cell recycling 449 Clostridium spp. 446T continuous 449 development and product recovery 449450 factors affecting 447 genetic strain improvement for 450-451 history 445-446 industrial process 446-447, 447F industrial production 449 new substrates 449 physiology 447-449 recent progress in research 449-451 spore formation and 448 time course 447, 447F see also acetone-butanol-ethanol (ABE) fermentation butter 1445,1450-1453 aflatoxins 1454 brucellosis from 1454 defects 1452T enterococci as indicators of poor hygiene 623 fat content 1450 food poisoning outbreaks 1453-1454 herb 1451-1452 in ice cream 1083 lactic 2103T manufacture 1450-1452, 1450F, 1451T NIZO method 1451 microbiological standards 1453, 1453T microflora 1450-1452 public health concerns 1453-1455 rancidity 1453 salt 1451 soft spreadable 1452 sorbate additioniuse 1772 spoilage 1452-1453, 1452T starter cultures used 2084-2085 storage 1452-1453 problems 1452 whey cream 1451 Butterfield’s phosphate 156

buttermilk 776, 1451 Bulgarian 779 preparation 776 in Northern Europe 795 butyl parabens 1759T, 1761T butyrate formation in intestine 1786 metabolism in gut 1354 butyric acid fermentation 1285, 1287F formationiorodnction 1281 Breuibacteri~m312 butanol-acetone fermentation 447448 Clostridium tyrobtityrictrm 453 in intestine 1786 butyric-butanol fermentation 1286, 1287F butyric esterase, Lactobacdltcs casei 1159 butyric fermentation 1285, 1287F ’butyric late blowing,’ prevention by lysozyme 1585-1586 byssochlamic acid 329 Byssochlanzys 328-333, 863, 896 ascocarp 328 ascospores acceptable levels 332 heat inactivation 330, 330T heat resistance 329, 332, 330T inactivation methods 329, 330T soil as reservoir 333 characteristics of genus 328-329, 330T, 889T commercial importance 892 detection methods 329-332 impedimetry and conductimetry 332, 332F plating techniques 329-332, 331F distribution 328-329 food spoilage 328-329, 892 fruits 333 products at risk 332 importance to food industry 333 mycotcjxins 329, 332-333, 331T patulin production 329 pectolytic e n q m e s 329 species 328 unacceptable levels 332 Byssochlamys fulva 328 characteristics 330T fruit spoilage 333 growth at low oxygen tensions 329 heat resistance of spores 329, 330T Byssochlamys niuea 328 ascospores In milk 333 characteristics 330T fruit spoilage 333 growth at low oxygen tensions 329 heat resistance of spores 330T

C

cabbage, acid see sauerkraut cacao fruit 467 fermentation 467-469 fermentation procedures 467 harvesting 467 pod 467F see a h cocoa cadaverine Hafnia alvei production 975 in stored meat 1259 ‘caged Fenton reaction’ 1465 cakes 474-479 see also bakery products calcium, Pediococcns requirement 1643 calcium citrate 712 calcium dipicolnate (CaDPA) 168 calcium gluconate 714 isolation 715 calcium hypochlorite 1797 calcium propionate 1781

calcofluor white 831, 1388 calcofluor white Primulin 1387T caldo-de-cana-picado, Zymomonas fermentation 2370 calf rennin (calf chymosin), genetically engineered 939 caliciviruses 2271-2272 calories, mycoprotein content 2042T calorimetry biomass measurement 685 non-destructive sterility testing 21982199 camalexin, mode of action 1581 Camembert cheese 387, 2103T defects 392 history 388 manufacture 379 volatile compounds 2113T CAMP test 1196 Listeria identification 1206, 1206F, 1206T Listeria inonocytogenes 1236 Campylobactev 335-352 adhesion method 338 Avcobacter diiferences 50, 50 bacteriophage 204 characteristics 342, 50 chemo-organotroph 336 detection by latex agglutination 347-352 advantages and limitations 347-348, 351-352 background 347-348 comparison of protocols of tests 350, 35OT detection limits 351, 3 5 l T enrichment serology 591 points of application 350 principle and test types 348, 348F protocols 348-350 regulations/guide~inesidirectjves 350 test comparisons 348T detection methods 341-347 comparisons of media 345, 345T conductimerriciimpedimetric technique 524 enrichment media 34, 344T enrichment serology 591 from environmental samples 345 FBP media 342 inhibitors for plating media 342-343, 3431 membrane filtration 343-344 phage amplification 208, 208, 208F, 208F rapid 346 selective media 342-343, 3 4 3 1 ecology 337 enterotoxins 338-339 fimbriae 338 flagella 338 general physiology 336-337 growth requirements 336-337 habitats 3 3 7 historical aspects 33.5 identification 205 infections 340, 341-342, 341-342 causative species 341 enteritis, incubation period 335 gastroenteritis 34 1-342 Guillain-Barr6 syndrome 335-336 reactive arthritides 336 sources 341-342 iron requirements 338 isolation from faeces 345 isolation of less common species 345-346 isolation of thermophilic strains from foods 344-345 methods of control 340 morphology 336 oxygen requirements 336 oxygen sensitivity 336 pathogenicity 338-339